Recombinant microorganisms and uses therefor

Abstract

Terpenes are valuable commercial products used in a diverse number of industries. Terpenes may be produced from petrochemical sources and from terpene feed-stocks, such as turpentine. However, these production methods are expensive, unsustainable and often cause environmental problems including contributing to climate change. Microbial fermentation provides an alternative option for the production of terpenes. One or more terpenes and/or precursors can be produced by microbial fermentation of a substrate comprising CO. Recombinant microorganisms may be used in such methods. Carboxydotrophic, acetogenic, recombinant microorganisms can be used in such methods. The recombinant microorgnsims may contain exogenous mevalonate (MVA) pathway enzymes and/or DXS pathway enzymes, for example.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to recombinant microorganisms and methods for the production of terpenes and/or precursors thereof by microbial fermentation of a substrate comprising CO.

BACKGROUND OF THE INVENTION

[0002] Terpenes are a diverse class of naturally occurring chemicals composed of five-carbon isoprene units. Terpene derivatives include terpenoids (also known as isoprenoids) which may be formed by oxidation or rearrangement of the carbon backbone or a number of functional group additions or rearrangements.

[0003] Examples of terpenes include: isoprene (C5 hemiterpene), farnesene (C15 Sesquiterpenes), artemisinin (C15 Sesquiterpenes), citral (C10 Monoterpenes), carotenoids (C40 Tetraterpenes), menthol (C10 Monoterpenes), Camphor (C10 Monoterpenes), and cannabinoids.

[0004] Terpenes are valuable commercial products used in a diverse number of industries. The highest tonnage uses of terpenes are as resins, solvents, fragrances and vitamins. For example, isoprene is used in the production of synthetic rubber (cis-1,4-polyisoprene) for example in the tyre industry; farnesene is used as an energy dense drop-in fuel used for transportation or as jet-fuel; artemisinin is used as a malaria drug; and citral, carotenoids, menthol, camphor, and cannabinoids are used in the manufacture of pharmaceuticals, butadiene, and as aromatic ingredients.

[0005] Terpenes may be produced from petrochemical sources and from terpene feed-stocks, such as turpentine. For example, isoprene is produced petrochemically as a by-product of naphtha or oil cracking in the production of ethylene. Many terpenes are also extracted in relatively small quantities from natural sources. However, these production methods are expensive, unsustainable and often cause environmental problems including contributing to climate change.

[0006] Due to the extremely flammable nature of isoprene, known methods of production require extensive safeguards to limit potential for fire and explosions.

[0007] It is an object of the invention to overcome one or more of the disadvantages of the prior art, or at least to provide the public with an alternative means for producing terpenes and other related products.

SUMMARY OF INVENTION

[0008] Microbial fermentation provides an alternative option for the production of terpenes. Terpenes are ubiquitous in nature, for example they are involved in bacterial cell wall biosynthesis, and they are produced by some trees (for example poplar) to protect leaves from UV light exposure. However, not all bacteria comprise the necessary cellular machinery to produce terpenes and/or their precursors as metabolic products. For example, carboxydotrophic acetogens, such as C. autoethanogenum or C. ljungdahlii, which are able to ferment substrates comprising carbon monoxide to produce products such as ethanol, are not known to produce and emit any terpenes and/or their precursors as metabolic products. In addition, most bacteria are not known to produce any terpenes which are of commercial value.

[0009] The invention generally provides, inter alia, methods for the production of one or more terpenes and/or precursors thereof by microbial fermentation of a substrate comprising CO, and recombinant microorganisms of use in such methods.

[0010] In a first aspect, the invention provides a carboxydotrophic acetogenic recombinant microorganism capable of producing one or more terpenes and/or precursors thereof and optionally one or more other products by fermentation of a substrate comprising CO.

[0011] In one particular embodiment, the microorganism is adapted to express one or more enzymes in the mevalonate (MVA) pathway not present in a parental microorganism from which the recombinant microorganism is derived (may be referred to herein as an exogenous enzyme). In another embodiment, the microorganism is adapted to over-express one or more enzymes in the mevalonate (MVA) pathway which are present in a parental microorganism from which the recombinant microorganism is derived (may be referred to herein as an endogenous enzyme).

[0012] In a further embodiment, the microorganism is adapted to:

a) express one or more exogenous enzymes in the mevalonate (MVA) pathway and/or overexpress one or more endogenous enzyme in the mevalonate (MVA) pathway; and b) express one or more exogenous enzymes in the DXS pathway and/or overexpress one or more endogenous enzymes in the DXS pathway.

[0013] In one embodiment, the one or more enzymes from the mevalonate (MVA) pathway is selected from the group consisting of:

a) thiolase (EC 2.3.1.9), b) HMG-CoA synthase (EC 2.3.3.10), c) HMG-CoA reductase (EC 1.1.1.88), d) Mevalonate kinase (EC 2.7.1.36), e) Phosphomevalonate kinase (EC 2.7.4.2), f) Mevalonate Diphosphate decarboxylase (EC 4.1.1.33), and g) a functionally equivalent variant of any one thereof.

[0014] In a further embodiment, the one or more enzymes from the DXS pathway is selected from the group consisting of:

[0015] a) 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC:2.2.1.7),

[0016] b) 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC:1.1.1.267),

[0017] c) 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC:2.7.7.60),

[0018] d) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC:2.7.1.148),

[0019] e) 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase IspF (EC:4.6.1.12),

[0020] f) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC:1.17.7.1),

[0021] g) 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC:1.17.1.2), and

[0022] h) a functionally equivalent variant of any one thereof.

[0023] In a further embodiment, one or more further exogenous or endogenous enzymes are expressed or over-expressed to result in the production of a terpene compound or a precursor thereof wherein the exogenous enzyme that is expressed, or the endogenous enzyme that is overexpressed, is selected from the group consisting of:

a) geranyltranstransferase Fps (EC:2.5.1.10), b) heptaprenyl diphosphate synthase (EC:2.5.1.10), c) octaprenyl-diphosphate synthase (EC:2.5.1.90), d) isoprene synthase (EC 4.2.3.27), e) isopentenyl-diphosphate delta-isomerase (EC 5.3.3.2), f) farnesene synthase (EC 4.2.3.46/EC 4.2.3.47), and g) a functionally equivalent variant of any one thereof.

[0024] In one embodiment, the parental microorganism is capable of fermenting a substrate comprising CO to produce Acetyl CoA, but not of converting Acetyl CoA to mevalonic acid or isopentenyl pyrophosphate (IPP) and the recombinant microorganism is adapted to express one or more enzymes involved in the mevalonate pathway.

[0025] In one embodiment, the one or more terpene and/or precursor thereof is chosen from mevalonic acid, IPP, dimethylallyl pyrophosphate (DMAPP), isoprene, geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) and farnesene.

[0026] In one embodiment, the microorganism comprises one or more exogenous nucleic acids adapted to increase expression of one or more endogenous nucleic acids and which one or more endogenous nucleic acids encode one or more of the enzymes referred to herein before.

[0027] In one embodiment, the one or more exogenous nucleic acids adapted to increase expression is a regulatory element. In one embodiment, the regulatory element is a promoter.

[0028] In one embodiment, the promoter is a constitutive promoter. In one embodiment, the promoter is selected from the group comprising Wood-Ljungdahl gene cluster or Phosphotransacetylase/Acetate kinase operon promoters.

[0029] In one embodiment, the microorganism comprises one or more exogenous nucleic acids encoding and adapted to express one or more of the enzymes referred to hereinbefore. In one embodiment, the microorganisms comprise one or more exogenous nucleic acids encoding and adapted to express at least two of the enzymes. In other embodiments, the microorganism comprises one or more exogenous nucleic acids encoding and adapted to express at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or more of the enzymes.

[0030] In one embodiment, the one or more exogenous nucleic acid is a nucleic acid construct or vector, in one particular embodiment a plasmid, encoding one or more of the enzymes referred to hereinbefore in any combination.

[0031] In one embodiment, the exogenous nucleic acid is an expression plasmid.

[0032] In one particular embodiment, the parental microorganism is selected from the group of carboxydotrophic acetogenic bacteria. In certain embodiments the microorganism is selected from the group comprising Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Clostridium aceticum, Clostridium formicoaceticum, Clostridium magnum, Butyribacterium methylotrophicum, Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorella thermautotrophica, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, Oxobacter pfennigii, and Thermoanaerobacter kiuvi.

[0033] In one embodiment the parental microorganism is Clostridium autoethanogenum or Clostridium ljungdahlii. In one particular embodiment, the microorganism is Clostridium autoethanogenum DSM23693. In another particular embodiment, the microorganism is Clostridium ljungdahlii DSM13528 (or ATCC55383).

[0034] In one embodiment, the parental microorganism lacks one or more genes in the DXS pathway and/or the mevalonate (MVA) pathway. In one embodiment, the parental microorganism lacks one or more genes encoding an enzyme selected from the group consisting of:

[0035] a) thiolase (EC 2.3.1.9),

[0036] b) HMG-CoA synthase (EC 2.3.3.10),

[0037] c) HMG-CoA reductase (EC 1.1.1.88),

[0038] d) Mevalonate kinase (EC 2.7.1.36),

[0039] e) Phosphomevalonate kinase (EC 2.7.4.2),

[0040] f) Mevalonate Diphosphate decarboxylase (EC 4.1.1.33),

[0041] g) 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC:2.2.1.7),

[0042] h) 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC:1.1.1.267),

[0043] i) 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC:2.7.7.60),

[0044] j) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC:2.7.1.148),

[0045] k) 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase IspF (EC:4.6.1.12),

[0046] l) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC:1.17.7.1),

[0047] m) 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC:1.17.1.2), and

[0048] n) a functionally equivalent variant of any one thereof.

[0049] In a second aspect, the invention provides a nucleic acid encoding one or more enzymes which when expressed in a microorganism allows the microorganism to produce one or more terpenes and/or precursors thereof by fermentation of a substrate comprising CO.

[0050] In one embodiment, the nucleic acid encodes two or more enzymes which when expressed in a microorganism allows the microorganism to produce one or more terpenes and/or precursors thereof by fermentation of a substrate comprising CO. In one embodiment, a nucleic acid of the invention encodes at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or more of such enzymes.

[0051] In one embodiment, the nucleic acid encodes one or more enzymes in the mevalonate

[0052] (MVA) pathway. In one embodiment, the one or more enzymes is chosen from the group consisting of:

a) thiolase (EC 2.3.1.9), b) HMG-CoA synthase (EC 2.3.3.10), c) HMG-CoA reductase (EC 1.1.1.88), d) Mevalonate kinase (EC 2.7.1.36), e) Phosphomevalonate kinase (EC 2.7.4.2), f) Mevalonate Diphosphate decarboxylase (EC 4.1.1.33), and

[0053] g) a functionally equivalent variant of any one thereof.

[0054] In a particular embodiment, the nucleic acid encodes thiolase (which may be an acetyl CoA c-acetyltransferase), HMG-CoA synthase and HMG-CoA reductase,

[0055] In a further embodiment, the nucleic acid encodes one or more enzymes in the mevalonate (MVA) pathway and one or more further nucleic acids in the DXS pathway pathway. In one embodiment, the one or more enzymes from the DXS pathway is selected from the group consisting of:

[0056] a) 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC:2.2.1.7),

[0057] b) 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC:1.1.1.267),

[0058] c) 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC:2.7.7.60),

[0059] d) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC:2.7.1.148),

[0060] e) 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase IspF (EC:4.6.1.12),

[0061] f) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC:1.17.7.1),

[0062] g) 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC:1.17.1.2), and

[0063] h) a functionally equivalent variant of any one thereof.

[0064] In a further embodiment, the nucleic acid encodes one or more further exogenous or endogenous enzymes are expressed or over-expressed to result in the production of a terpene compound or a precursor thereof wherein the exogenous nucleic acid that is expressed, or the endogenous enzyme that is overexpressed, encodes and enzyme selected from the group consisting of:

[0065] a) geranyltranstransferase Fps (EC:2.5.1.10),

b) heptaprenyl diphosphate synthase (EC:2.5.1.10), c) octaprenyl-diphosphate synthase (EC:2.5.1.90), d) isoprene synthase (EC 4.2.3.27), e) isopentenyl-diphosphate delta-isomerase (EC 5.3.3.2), f) farnesene synthase (EC 4.2.3.46/EC 4.2.3.47), and g) a functionally equivalent variant of any one thereof.

[0066] In one embodiment, the nucleic acid encoding thiolase (EC 2.3.1.9) has the sequence SEQ ID NO: 40 or is a functionally equivalent variant thereof.

[0067] In one embodiment, the nucleic acid encoding thiolase (EC 2.3.1.9) is acetyl CoA c-acetyl transferase that has the sequence SEQ ID NO: 41 or is a functionally equivalent variant thereof.

[0068] In one embodiment, the nucleic acid encoding HMG-CoA synthase (EC 2.3.3.10) has the sequence SEQ ID NO: 42 or is a functionally equivalent variant thereof.

[0069] In one embodiment, the nucleic acid encoding HMG-CoA reductase (EC 1.1.1.88) has the sequence SEQ ID NO: 43 or is a functionally equivalent variant thereof.

[0070] In one embodiment, the nucleic acid encoding Mevalonate kinase (EC 2.7.1.36) has the sequence SEQ ID NO: 51 or is a functionally equivalent variant thereof.

[0071] In one embodiment, the nucleic acid encoding Phosphomevalonate kinase (EC 2.7.4.2) has the sequence SEQ ID NO: 52 or is a functionally equivalent variant thereof.

[0072] In one embodiment, the nucleic acid encoding Mevalonate Diphosphate decarboxylase (EC 4.1.1.33) has the sequence SEQ ID NO: 53 or is a functionally equivalent variant thereof.

[0073] In one embodiment, the nucleic acid encoding 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC:2.2.1.7) has the sequence SEQ ID NO: 1 or is a functionally equivalent variant thereof.

[0074] In one embodiment, the nucleic acid encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC:1.1.1.267) has the sequence SEQ ID NO: 3 or is a functionally equivalent variant thereof.

[0075] In one embodiment, the nucleic acid encoding 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC:2.7.7.60) has the sequence SEQ ID NO: 5 or is a functionally equivalent variant thereof.

[0076] In one embodiment, the nucleic acid encoding 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC:2.7.1.148) has the sequence SEQ ID NO: 7 or is a functionally equivalent variant thereof.

[0077] In one embodiment, the nucleic acid encoding 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase IspF (EC:4.6.1.12) has the sequence SEQ ID NO: 9 or is a functionally equivalent variant thereof.

[0078] In one embodiment, the nucleic acid encoding 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC:1.17.7.1) has the sequence SEQ ID NO: 11 or is a functionally equivalent variant thereof.

[0079] In one embodiment, the nucleic acid encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC:1.17.1.2) has the sequence SEQ ID NO: 13 or is a functionally equivalent variant thereof.

[0080] In one embodiment, the nucleic acid encoding geranyltranstransferase Fps has the sequence SEQ ID NO: 15, or it is a functionally equivalent variant thereof.

[0081] In one embodiment, the nucleic acid encoding heptaprenyl diphosphate synthase has the sequence SEQ ID NO: 17, or it is a functionally equivalent variant thereof.

[0082] In one embodiment, the nucleic acid encoding octaprenyl-diphosphate synthase (EC:2.5.1.90) wherein the octaprenyl-diphosphate synthase is polyprenyl synthetase is encoded by sequence SEQ ID NO: 19, or it is a functionally equivalent variant thereof.

[0083] In one embodiment, the nucleic acid encoding isoprene synthase (ispS) has the sequence SEQ ID NO: 21, or it is a functionally equivalent variant thereof.

[0084] In one embodiment, the nucleic acid encoding Isopentenyl-diphosphate delta-isomerase (idi) has the sequence SEQ ID NO: 54, or it is a functionally equivalent variant thereof.

[0085] In one embodiment, the nucleic acid encoding farnesene synthase has the sequence SEQ ID NO: 57, or it is a functionally equivalent variant thereof.

[0086] In one embodiment, the nucleic acid encodes the following enzymes:

a) isoprene synthase; b) Isopentenyl-diphosphate delta-isomerase (idi); and c) 1-deoxy-D-xylulose-5-phosphate synthase DXS; or functionally equivalent variants thereof.

[0087] In one embodiment, the nucleic acid encodes the following enzymes:

a) Thiolase;

[0088] b) HMG-CoA synthase; c) HMG-CoA reductase; d) Mevalonate kinase; e) Phosphomevalonate kinase; f) Mevalonate Diphosphate decarboxylase; g) Isopentenyl-diphosphate delta-isomerase (idi); and h) isoprene synthase; or functionally equivalent variants thereof.

[0089] In one embodiment, the nucleic acid encodes the following enzymes:

a) geranyltranstransferase Fps; and b) farnesene synthase or functionally equivalent variants thereof.

[0090] In one embodiment, the nucleic acids of the invention further comprise a promoter. In one embodiment, the promoter allows for constitutive expression of the genes under its control. In a particular embodiment a Wood-Ljungdahl cluster promoter is used. In another particular embodiment, a Phosphotransacetylase/Acetate kinase operon promoter is used. In one particular embodiment, the promoter is from C. autoethanogenum.

[0091] In a third aspect, the invention provides a nucleic acid construct or vector comprising one or more nucleic acid of the second aspect.

[0092] In one particular embodiment, the nucleic acid construct or vector is an expression construct or vector. In one particular embodiment, the expression construct or vector is a plasmid.

[0093] In a fourth aspect, the invention provides host organisms comprising any one or more of the nucleic acids of the second aspect or vectors or constructs of the third aspect.

[0094] In a fifth aspect, the invention provides a composition comprising an expression construct or vector as referred to in the third aspect of the invention and a methylation construct or vector.

[0095] Preferably, the composition is able to produce a recombinant microorganism according to the first aspect of the invention.

[0096] In one particular embodiment, the expression construct/vector and/or the methylation construct/vector is a plasmid.

[0097] In a sixth aspect, the invention provides a method for the production of one or more terpenes and/or precursors thereof and optionally one or more other products by microbial fermentation comprising fermenting a substrate comprising CO using a recombinant microorganism of the first aspect of the invention.

[0098] In one embodiment the method comprises the steps of:

(a) providing a substrate comprising CO to a bioreactor containing a culture of one or more microorganisms of the first aspect of the invention; and (b) anaerobically fermenting the culture in the bioreactor to produce at least one terpene and/or precursor thereof.

[0099] In one embodiment the method comprises the steps of:

(a) capturing CO-containing gas produced as a result of the industrial process; (b) anaerobic fermentation of the CO-containing gas to produce at least one terpene and/or precursor thereof by a culture containing one or more microorganism of the first aspect of the invention.

[0100] In particular embodiments of the method aspects, the microorganism is maintained in an aqueous culture medium.

[0101] In particular embodiments of the method aspects, the fermentation of the substrate takes place in a bioreactor.

[0102] In one embodiment, the one or more terpene and/or precursor thereof is chosen from mevalonic acid, IPP, dimethylallyl pyrophosphate (DMAPP), isoprene, geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) and farnesene.

[0103] Preferably, the substrate comprising CO is a gaseous substrate comprising CO. In one embodiment, the substrate comprises an industrial waste gas. In certain embodiments, the gas is steel mill waste gas or syngas.

[0104] In one embodiment, the substrate will typically contain a major proportion of CO, such as at least about 20% to about 100% CO by volume, from 20% to 70% CO by volume, from 30% to 60% CO by volume, and from 40% to 55% CO by volume. In particular embodiments, the substrate comprises about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50% CO, or about 55% CO, or about 60% CO by volume.

[0105] In certain embodiments the methods further comprise the step of recovering a terpene and/or precursor thereof and optionally one or more other products from the fermentation broth.

[0106] In a seventh aspect, the invention provides one or more terpene and/or precursor thereof when produced by the method of the sixth aspect. In one embodiment, the one or more terpene and/or precursor thereof is chosen from the group consisting of mevalonic acid, IPP, dimethylallyl pyrophosphate (DMAPP), isoprene, geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) and farnesene.

[0107] In another aspect, the invention provides a method for the production of a microorganism of the first aspect of the invention comprising transforming a carboxydotrophic acetogenic parental microorganism by introduction of one or more nucleic acids such that the microorganism is capable of producing, or increasing the production of, one or more terpenes and/or precursors thereof and optionally one or more other products by fermentation of a substrate comprising CO, wherein the parental microorganism is not capable of producing, or produces at a lower level, the one or more terpene and/or precursor thereof by fermentation of a substrate comprising CO.

[0108] In one particular embodiment, a parental microorganism is transformed by introducing one or more exogenous nucleic acids adapted to express one or more enzymes in the mevalonate (MVA) pathway and optionally the DXS pathway. In another embodiment, a parental microorganism is transformed with one or more nucleic acids adapted to over-express one or more enzymes in the mevalonate (MVA) pathway and optionally the DXS pathway which are naturally present in the parental microorganism.

[0109] In certain embodiments, the one or more enzymes are as herein before described.

[0110] In one embodiment an isolated, genetically engineered, carboxydotrophic, acetogenic bacteria are provided which comprise an exogenous nucleic acid encoding an enzyme in a mevalonate pathway or in a DXS pathway or in a terpene biosynthesis pathway, whereby the bacteria express the enzyme. The enzyme is selected from the group consisting of:

[0111] a) thiolase (EC 2.3.1.9);

[0112] b) HMG-CoA synthase (EC 2.3.3.10);

[0113] c) HMG-CoA reductase (EC 1.1.1.88);

[0114] d) Mevalonate kinase (EC 2.7.1.36);

[0115] e) Phosphomevalonate kinase (EC 2.7.4.2);

[0116] f) Mevalonate Diphosphate decarboxylase (EC 4.1.1.33); 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC:2.2.1.7);

[0117] g) 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC:1.1.1.267);

[0118] h) 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC:2.7.7.60);

[0119] i) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC:2.7.1.148);

[0120] j) 2-C-methyl-D-erythritol 2;4-cyclodiphosphate synthase IspF (EC:4.6.1.12);

[0121] k) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC:1.17.7.1);

[0122] l) 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC:1.17.1.2); geranyltranstransferase Fps (EC:2.5.1.10);

[0123] m) heptaprenyl diphosphate synthase (EC:2.5.1.10);

[0124] n) octaprenyl-diphosphate synthase (EC:2.5.1.90);

[0125] o) isoprene synthase (EC 4.2.3.27);

[0126] p) isopentenyl-diphosphate delta-isomerase (EC 5.3.3.2); and

[0127] q) farnesene synthase (EC 4.2.3.46/EC 4.2.3.47).

[0128] In some aspects the bacteria do not express the enzyme in the absence of said nucleic acid. In some aspects the bacteria which express the enzyme under anaerobic conditions.

[0129] One embodiment provides a plasmid which can replicate in a carboxydotrophic, acetogenic bacteria. The plasmid comprises a nucleic acid encoding an enzyme in a mevalonate pathway or in a DXS pathway or in a terpene biosynthesis pathway, whereby when the plasmid is in the bacteria, the enzyme is expressed by said bacteria. The enzyme is selected from the group consisting of:

[0130] a) thiolase (EC 2.3.1.9);

[0131] b) HMG-CoA synthase (EC 2.3.3.10);

[0132] c) HMG-CoA reductase (EC 1.1.1.88);

[0133] d) Mevalonate kinase (EC 2.7.1.36);

[0134] e) Phosphomevalonate kinase (EC 2.7.4.2);

[0135] f) Mevalonate Diphosphate decarboxylase (EC 4.1.1.33); 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC:2.2.1.7);

[0136] g) 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC:1.1.1.267);

[0137] h) 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC:2.7.7.60);

[0138] i) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC:2.7.1.148);

[0139] j) 2-C-methyl-D-erythritol 2;4-cyclodiphosphate synthase IspF (EC:4.6.1.12);

[0140] k) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC:1.17.7.1);

[0141] l) 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC:1.17.1.2); geranyltranstransferase Fps (EC:2.5.1.10);

[0142] m) heptaprenyl diphosphate synthase (EC:2.5.1.10);

[0143] n) octaprenyl-diphosphate synthase (EC:2.5.1.90);

[0144] o) isoprene synthase (EC 4.2.3.27);

[0145] p) isopentenyl-diphosphate delta-isomerase (EC 5.3.3.2); and

[0146] q) farnesene synthase (EC 4.2.3.46/EC 4.2.3.47).

[0147] A process is provided in another embodiment for converting CO and/or CO₂ into isoprene. The process comprises: passing a gaseous CO-containing and/or CO₂-containing substrate to a bioreactor containing a culture of carboxydotrophic, acetogenic bacteria in a culture medium such that the bacteria convert the CO and/or CO₂ to isoprene, and recovering the isoprene from the bioreactor. The carboxydotrophic acetogenic bacteria are genetically engineered to express a isoprene synthase.

[0148] Another embodiment provides an isolated, genetically engineered, carboxydotrophic, acetogenic bacteria which comprise a nucleic acid encoding an isoprene synthase. The bacteria express the isoprene synthase and the bacteria are able to convert imethylallyldiphosphate to isoprene. In one aspect the isoprene synthase is a Populus tremuloides enzyme. In another aspect the nucleic acid is codon optimized. In still another aspect, expression of the isoprene synthase is under the transcriptional control of a promoter for a pyruvate: ferredoxin oxidoreductase gene from Clostridium autoethanogenum.

[0149] Another embodiment provides a process for converting CO and/or CO₂ into isopentyldiphosphate (IPP). The process comprises: passing a gaseous CO-containing and/or CO₂-containing substrate to a bioreactor containing a culture of carboxydotrophic, acetogenic bacteria in a culture medium such that the bacteria convert the CO and/or CO₂ to isopentyldiphosphate (IPP), and recovering the IPP from the bioreactor. The carboxydotrophic acetogenic bacteria are genetically engineered to express a isopentyldiphosphate delta isomerase.

[0150] Still another embodiment provides isolated, genetically engineered, carboxydotrophic, acetogenic bacteria which comprise a nucleic acid encoding an isopentyldiphosphate delta isomerase. The bacteria express the isopentyldiphosphate delta isomerase and the bacteria are able to convert dimethylallyldiphosphate to isopentyldiphosphate. In some aspects the nucleic acid encodes a Clostridium beijerinckii isopentyldiphosphate delta isomerase. In other aspects, the nucleic acid is under the transcriptional control of a promoter for a pyruvate: ferredoxin oxidoreductase gene from Clostridium autoethanogenum. In still other aspects, the nucleic acid is under the transcriptional control of a promoter for a pyruvate: ferredoxin oxidoreductase gene from Clostridium autoethanogenum and downstream of a second nucleic acid encoding an isoprene synthase.

[0151] Still another embodiment provides a process for converting CO and/or CO₂ into isopentyldiphosphate (IPP) and/or isoprene. The process comprises: passing a gaseous CO-containing and/or CO₂-containing substrate to a bioreactor containing a culture of carboxydotrophic, acetogenic bacteria in a culture medium such that the bacteria convert the CO and/or CO₂ to isopentyldiphosphate (IPP) and/or isoprene, and recovering the IPP and/or isoprene from the bioreactor. The carboxydotrophic acetogenic bacteria are genetically engineered to have an increased copy number of a nucleic acid encoding a deoxyxylulose 5-phosphate synthase (DXS) enzyme, wherein the increased copy number is greater than 1 per genome.

[0152] Yet another embodiment provides isolated, genetically engineered, carboxydotrophic, acetogenic bacteria which comprise a copy number of greater than 1 per genome of a nucleic acid encoding a deoxyxylulose 5-phosphate synthase (DXS) enzyme. In some aspects, the isolated, genetically engineered, carboxydotrophic, acetogenic bacteria may further comprise a nucleic acid encoding an isoprene synthase. In other aspects, the isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of may further comprise a nucleic acid encoding an isopentyldiphosphate delta isomerase. In still other aspects the isolated, genetically engineered, carboxydotrophic, acetogenic bacteria may further comprise a nucleic acid encoding an isopentyldiphosphate delta isomerase and a nucleic acid encoding an isoprene synthase.

[0153] Another embodiment provides isolated, genetically engineered, carboxydotrophic, acetogenic bacteria which comprise a nucleic acid encoding a phosphomevalonate kinase (PMK). The bacteria express the encoded enzyme and the enzyme is not native to the bacteria. In some aspects the enzymes are Staphylococcus aureus enzymes. In some aspects the enzyme is expressed under the control of one or more C. autoethanogenum promoters. In some aspects the bacteria further comprise a nucleic acid encoding thiolase (thlA/vraB), a nucleic acid encoding a HMG-CoA synthase (HMGS), and a nucleic acid encoding an HMG-CoA reductase (HMGR). In some aspects the thiolase is Clostridium acetobutylicum thiolase. In some aspects the bacteria further comprise a nucleic acid encoding a mevalonate disphosphate decarboxylase (PMD).

[0154] Still another embodiment provides isolated, genetically engineered, carboxydotrophic, acetogenic bacteria which comprise an exogenous nucleic acid encoding alpha-farnesene synthase. In some aspects the nucleic acid is codon optimized for expression in C. autoethanogenum. In some aspects the alpha-farnesene synthase is a Malus×domestica alpha-farnesene synthase. In some aspects the bacteria further comprise a nucleic acid segment encoding geranyltranstransferase. In some aspects the gernayltranstransferase is an E. coli geranyltranstransferase.

[0155] Suitable isolated, genetically engineered, carboxydotrophic, acetogenic bacteria for any of the aspects or embodiments of the invention may be selected from the group consisting of Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Clostridium aceticum, Clostridium formicoaceticum, Clostridium magnum, Butyribacterium methylotrophicum, Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorella thermautotrophica, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, Oxobacter pfennigii, and Thermoanaerobacter kiuvi.

[0156] The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

BRIEF DESCRIPTION OF THE FIGURES

[0157] These and other aspects of the present invention, which should be considered in all its novel aspects, will become apparent from the following description, which is given by way of example only, with reference to the accompanying figures.

[0158] FIG. 1: Pathway diagram for production of terpenes, gene targets described in this application are highlighted with bold arrows.

[0159] FIG. 2: Genetic map of plasmid pMTL 85146-ispS

[0160] FIG. 3: Genetic map of plasmid pMTL 85246-ispS-idi

[0161] FIG. 4: Genetic map of plasmid pMTL 85246-ispS-idi-dxs

[0162] FIG. 5: Sequencing results for plasmid pMTL 85246-ispS-idi-dxs

[0163] FIG. 6: Comparison of energetics for production of terpenes from CO via DXS and mevalonate pathway

[0164] FIG. 7: Mevalonate pathway

[0165] FIG. 8: Agarose gel electrophoresis image confirming presence of isoprene expression plasmid pMTL 85246-ispS-idi in C. autoethanogenum transformants. Lanes 1, and 20 show 100 bp Plus DNA Ladder. Lane 3-6, 9-12, 15-18 show PCR with isolated plasmids from 4 different clones as template, each in the following order: colE1, ermB, and idi. Lanes 2, 8, and 14 show PCR without template as negative control, each in the following order: colE1, ermB, and idi. Lanes 7, 13, and 19 show PCR with pMTL 85246-ispS-idi from E. coli as positive control, each in the following order: colE1, ermB, and idi.

[0166] FIG. 9--Mevalonate expression plasmid pMTL8215-Pptaack-thlA-HMGS-Patp-HMGR

[0167] FIG. 10--Isoprene expression plasmid pMTL 8314-Pptaack-thlA-HMGS-Patp-HMGR-Pmf-MK-PMK-PMD-Pfor-idi-ispS

[0168] FIG. 11--Farnesene expression plasmid pMTL8314-Pptaack-thlA-HMGS-Patp-HMGR-Prnf-MK-PMK-PMD-Pfor-idi-ispA-FS

[0169] FIG. 12--Genetic map of plasmid pMTL 85246-ispS-idi-dxs

[0170] FIG. 13--Amplification chart for gene expression experiment with C. autoethanogenum carrying plasmid pMTL 85146-ispS

[0171] FIG. 14--Amplification chart for gene expression experiment with C. autoethanogenum carrying plasmid pMTL 85246-ispS-idi

[0172] FIG. 15--Amplification chart for gene expression experiment with C. autoethanogenum carrying plasmid pMTL 85246-ispS-idi-dxs

[0173] FIG. 16--PCR check for the presence of the plasmid pMTL8314Prnf-MK-PMK-PMD-Pfor-idi-ispA-FS. Expected band size 1584 bp. The DNA marker Fermentas 1 kb DNA ladder.

[0174] FIG. 17--Growth curve for transformed C. autoethanogemun carrying plasmid pMTL8314Prnf-MK-PMK-PMD-Pfor-idi-ispA-FS

[0175] FIG. 18--RT-PRC data showing the expression of the genes Mevalonate kinase (MK SEQ ID NO: 51), Phosphomevalonate Kinase (PMK SEQ ID NO: 52), Mevalonate Diphosphate Decarboxylase (PMD SEQ ID NO: 53), Isopentyl-diphosphate Delta-isomerase (idi SEQ ID NO: 54), Geranyltranstransferase (ispA SEQ ID NO: 56) and Farnesene synthase (FS SEQ ID NO: 57).

[0176] FIG. 19--GC-MS detection and conformation of the presence of farnesene in 1 mM mevalonate spiked cultures carrying pMTL8314Prnf-MK-PMK-PMD-Pfor-idi-ispA-FS. GC-MS chromatogram scanned for peaks containing ions with a mass of 93. Chromatogram 1 and 2 are transformed C. autoethanogenum, 3 is beta-farnesene standard run at the same time as the C. autoethanogenum samples. 4 is E. coli carrying the plasmids pMTL8314Prnf-MK-PMK-PMD-Pfor-idi-ispA-FS grown on M9 Glucose showing alpha-farnesene production and 5 is beta-farnesene standard run at the time of the E. coli samples. The difference in retention time between the E. coli and the C. autoethanogenum samples are due to minor changes to the instrument. However the difference in retention time between the beta-farnesene standard and the produced alpha-farnesene are the exact same in both cases, which together with the match in mass spectra's confirm the production of alpha-farnesene in C. autoethanogenum.

[0177] FIG. 20--MS spectrums for peaks labeled 1A and 2A in FIG. 19. The MS spectra's matches up with the NIST database spectra (FIG. 21) confirming the peak is alpha-farnesene.

[0178] FIG. 21--MS spectrum for alpha-farnesen from the NIST Mass Spectral Database.

DETAILED DESCRIPTION OF THE INVENTION

[0179] The following is a description of the present invention, including preferred embodiments thereof, given in general terms. The invention is further elucidated from the disclosure given under the heading "Examples" herein below, which provides experimental data supporting the invention, specific examples of various aspects of the invention, and means of performing the invention.

[0180] The inventors have surprisingly been able to engineer a carboxydotrophic acetogenic microorganism to produce terpene and precursors thereof including isoprene and farnesene by fermentation of a substrate comprising CO. This offers an alternative means for the production of these products which may have benefits over the current methods for their production. In addition, it offers a means of using carbon monoxide from industrial processes which would otherwise be released into the atmosphere and pollute the environment.

[0181] As referred to herein, a "fermentation broth" is a culture medium comprising at least a nutrient media and bacterial cells.

[0182] As referred to herein, a "shuttle microorganism" is a microorganism in which a methyltransferase enzyme is expressed and is distinct from the destination microorganism.

[0183] As referred to herein, a "destination microorganism" is a microorganism in which the genes included on an expression construct/vector are expressed and is distinct from the shuttle microorganism.

[0184] The term "main fermentation product" is intended to mean the one fermentation product which is produced in the highest concentration and/or yield.

[0185] The terms "increasing the efficiency", "increased efficiency" and the like, when used in relation to a fermentation process, include, but are not limited to, increasing one or more of the rate of growth of microorganisms catalysing the fermentation, the growth and/or product production rate at elevated product concentrations, the volume of desired product produced per volume of substrate consumed, the rate of production or level of production of the desired product, and the relative proportion of the desired product produced compared with other by-products of the fermentation.

[0186] The phrase "substrate comprising carbon monoxide" and like terms should be understood to include any substrate in which carbon monoxide is available to one or more strains of bacteria for growth and/or fermentation, for example.

[0187] The phrase "gaseous substrate comprising carbon monoxide" and like phrases and terms includes any gas which contains a level of carbon monoxide. In certain embodiments the substrate contains at least about 20% to about 100% CO by volume, from 20% to 70% CO by volume, from 30% to 60% CO by volume, and from 40% to 55% CO by volume. In particular embodiments, the substrate comprises about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50% CO, or about 55% CO, or about 60% CO by volume.

[0188] While it is not necessary for the substrate to contain any hydrogen, the presence of H₂ should not be detrimental to product formation in accordance with methods of the invention. In particular embodiments, the presence of hydrogen results in an improved overall efficiency of alcohol production. For example, in particular embodiments, the substrate may comprise an approx 2:1, or 1:1, or 1:2 ratio of H₂:CO. In one embodiment the substrate comprises about 30% or less H₂ by volume, 20% or less H₂ by volume, about 15% or less H₂ by volume or about 10% or less H₂ by volume. In other embodiments, the substrate stream comprises low concentrations of H₂, for example, less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%, or is substantially hydrogen free. The substrate may also contain some CO₂ for example, such as about 1% to about 80% CO₂ by volume, or 1% to about 30% CO₂ by volume. In one embodiment the substrate comprises less than or equal to about 20% CO₂ by volume. In particular embodiments the substrate comprises less than or equal to about 15% CO₂ by volume, less than or equal to about 10% CO₂ by volume, less than or equal to about 5% CO₂ by volume or substantially no CO₂.

[0189] In the description which follows, embodiments of the invention are described in terms of delivering and fermenting a "gaseous substrate containing CO". However, it should be appreciated that the gaseous substrate may be provided in alternative forms. For example, the gaseous substrate containing CO may be provided dissolved in a liquid. Essentially, a liquid is saturated with a carbon monoxide containing gas and then that liquid is added to the bioreactor. This may be achieved using standard methodology. By way of example, a microbubble dispersion generator (Hensirisak et. al. Scale-up of microbubble dispersion generator for aerobic fermentation; Applied Biochemistry and Biotechnology Volume 101, Number 3/October, 2002) could be used. By way of further example, the gaseous substrate containing CO may be adsorbed onto a solid support. Such alternative methods are encompassed by use of the term "substrate containing CO" and the like.

[0190] In particular embodiments of the invention, the CO-containing gaseous substrate is an industrial off or waste gas. "Industrial waste or off gases" should be taken broadly to include any gases comprising CO produced by an industrial process and include gases produced as a result of ferrous metal products manufacturing, non-ferrous products manufacturing, petroleum refining processes, gasification of coal, gasification of biomass, electric power production, carbon black production, and coke manufacturing. Further examples may be provided elsewhere herein.

[0191] Unless the context requires otherwise, the phrases "fermenting", "fermentation process" or "fermentation reaction" and the like, as used herein, are intended to encompass both the growth phase and product biosynthesis phase of the process. As will be described further herein, in some embodiments the bioreactor may comprise a first growth reactor and a second fermentation reactor. As such, the addition of metals or compositions to a fermentation reaction should be understood to include addition to either or both of these reactors.

[0192] The term "bioreactor" includes a fermentation device consisting of one or more vessels and/or towers or piping arrangement, which includes the Continuous Stirred Tank Reactor (CSTR), Immobilized Cell Reactor (ICR), Trickle Bed Reactor (TBR), Bubble Column, Gas Lift Fermenter, Static Mixer, or other vessel or other device suitable for gas-liquid contact. In some embodiments the bioreactor may comprise a first growth reactor and a second fermentation reactor. As such, when referring to the addition of substrate to the bioreactor or fermentation reaction it should be understood to include addition to either or both of these reactors where appropriate.

[0193] "Exogenous nucleic acids" are nucleic acids which originate outside of the microorganism to which they are introduced. Exogenous nucleic acids may be derived from any appropriate source, including, but not limited to, the microorganism to which they are to be introduced (for example in a parental microorganism from which the recombinant microorganism is derived), strains or species of microorganisms which differ from the organism to which they are to be introduced, or they may be artificially or recombinantly created. In one embodiment, the exogenous nucleic acids represent nucleic acid sequences naturally present within the microorganism to which they are to be introduced, and they are introduced to increase expression of or over-express a particular gene (for example, by increasing the copy number of the sequence (for example a gene), or introducing a strong or constitutive promoter to increase expression). In another embodiment, the exogenous nucleic acids represent nucleic acid sequences not naturally present within the microorganism to which they are to be introduced and allow for the expression of a product not naturally present within the microorganism or increased expression of a gene native to the microorganism (for example in the case of introduction of a regulatory element such as a promoter). The exogenous nucleic acid may be adapted to integrate into the genome of the microorganism to which it is to be introduced or to remain in an extra-chromosomal state.

[0194] "Exogenous" may also be used to refer to proteins. This refers to a protein that is not present in the parental microorganism from which the recombinant microorganism is derived.

[0195] The term "endogenous" as used herein in relation to a recombinant microorganism and a nucleic acid or protein refers to any nucleic acid or protein that is present in a parental microorganism from which the recombinant microorganism is derived.

[0196] It should be appreciated that the invention may be practised using nucleic acids whose sequence varies from the sequences specifically exemplified herein provided they perform substantially the same function. For nucleic acid sequences that encode a protein or peptide this means that the encoded protein or peptide has substantially the same function. For nucleic acid sequences that represent promoter sequences, the variant sequence will have the ability to promote expression of one or more genes. Such nucleic acids may be referred to herein as "functionally equivalent variants". By way of example, functionally equivalent variants of a nucleic acid include allelic variants, fragments of a gene, genes which include mutations (deletion, insertion, nucleotide substitutions and the like) and/or polymorphisms and the like. Homologous genes from other microorganisms may also be considered as examples of functionally equivalent variants of the sequences specifically exemplified herein. These include homologous genes in species such as Clostridium acetobutylicum, Clostridium beijerinckii, C. saccharobutylicum and C. saccharoperbutylacetonicum, details of which are publicly available on websites such as Genbank or NCBI. The phrase "functionally equivalent variants" should also be taken to include nucleic acids whose sequence varies as a result of codon optimisation for a particular organism. "Functionally equivalent variants" of a nucleic acid herein will preferably have at least approximately 70%, preferably approximately 80%, more preferably approximately 85%, preferably approximately 90%, preferably approximately 95% or greater nucleic acid sequence identity with the nucleic acid identified.

[0197] It should also be appreciated that the invention may be practised using polypeptides whose sequence varies from the amino acid sequences specifically exemplified herein. These variants may be referred to herein as "functionally equivalent variants". A functionally equivalent variant of a protein or a peptide includes those proteins or peptides that share at least 40%, preferably 50%, preferably 60%, preferably 70%, preferably 75%, preferably 80%, preferably 85%, preferably 90%, preferably 95% or greater amino acid identity with the protein or peptide identified and has substantially the same function as the peptide or protein of interest. Such variants include within their scope fragments of a protein or peptide wherein the fragment comprises a truncated form of the polypeptide wherein deletions may be from 1 to 5, to 10, to 15, to 20, to 25 amino acids, and may extend from residue 1 through 25 at either terminus of the polypeptide, and wherein deletions may be of any length within the region; or may be at an internal location. Functionally equivalent variants of the specific polypeptides herein should also be taken to include polypeptides expressed by homologous genes in other species of bacteria, for example as exemplified in the previous paragraph.

[0198] "Substantially the same function" as used herein is intended to mean that the nucleic acid or polypeptide is able to perform the function of the nucleic acid or polypeptide of which it is a variant. For example, a variant of an enzyme of the invention will be able to catalyse the same reaction as that enzyme. However, it should not be taken to mean that the variant has the same level of activity as the polypeptide or nucleic acid of which it is a variant.

[0199] One may assess whether a functionally equivalent variant has substantially the same function as the nucleic acid or polypeptide of which it is a variant using any number of known methods. However, by way of example, the methods described by Silver et al. (1991, Plant Physiol. 97: 1588-1591) or Zhao et al. (2011, Appl Microbiol Biotechnol, 90:1915-1922) for the isoprene synthase enzyme, by Green et al. (2007, Phytochemistry; 68:176-188) for the farnesene synthase enzyme, by Kuzuyama et al. (2000, J. Bacteriol. 182, 891-897) for the 1-deoxy-D-xylulose 5-phosphate synthase Dxs, by Berndt and Schlegel (1975, Arch. Microbiol. 103, 21-30) or by Stim-Herndon et al. (1995, Gene 154: 81-85) for the thiolase, by Cabano et al. (1997, Insect Biochem. Mol. Biol. 27: 499-505) for the HMG-CoA synthase, by Ma et al. (2011, Metab. Engin., 13:588-597) for the HMG-CoA reductase and mevalonate kinase enzyme, by Herdendorf and Miziorko (2007, Biochemistry, 46: 11780-8) for the phosphomevalonate kinase, and by Krepkiy et al. (2004, Protein Sci. 13: 1875-1881) for the mevalonate diphosphate decarboxylase. It is also possible to identify genes of DXS and mevalonate pathway using inhibitors like fosmidomycin or mevinoline as described by Trutko et al. (2005, Microbiology 74: 153-158).

[0200] "Over-express", "over expression" and like terms and phrases when used in relation to the invention should be taken broadly to include any increase in expression of one or more proteins (including expression of one or more nucleic acids encoding same) as compared to the expression level of the protein (including nucleic acids) of a parental microorganism under the same conditions. It should not be taken to mean that the protein (or nucleic acid) is expressed at any particular level.

[0201] A "parental microorganism" is a microorganism used to generate a recombinant microorganism of the invention. The parental microorganism may be one that occurs in nature (ie a wild type microorganism) or one that has been previously modified but which does not express or over-express one or more of the enzymes that are the subject of the present invention. Accordingly, the recombinant microorganisms of the invention may have been modified to express or over-express one or more enzymes that were not expressed or over-expressed in the parental microorganism.

[0202] The terms nucleic acid "constructs" or "vectors" and like terms should be taken broadly to include any nucleic acid (including DNA and RNA) suitable for use as a vehicle to transfer genetic material into a cell. The terms should be taken to include plasmids, viruses (including bacteriophage), cosmids and artificial chromosomes. Constructs or vectors may include one or more regulatory elements, an origin of replication, a multicloning site and/or a selectable marker. In one particular embodiment, the constructs or vectors are adapted to allow expression of one or more genes encoded by the construct or vector. Nucleic acid constructs or vectors include naked nucleic acids as well as nucleic acids formulated with one or more agents to facilitate delivery to a cell (for example, liposome-conjugated nucleic acid, an organism in which the nucleic acid is contained).

[0203] A "terpene" as referred to herein should be taken broadly to include any compound made up of C₅ isoprene units joined together including simple and complex terpenes and oxygen-containing terpene compounds such as alcohols, aldehydes and ketones. Simple terpenes are found in the essential oils and resins of plants such as conifers. More complex terpenes include the terpenoids and vitamin A, carotenoid pigments (such as lycopene), squalene, and rubber. Examples of monoterpenes include, but are not limited to isoprene, pinene, nerol, citral, camphor, menthol, limonene. Examples of sesquiterpenes include but are not limited to nerolidol, farnesol. Examples of diterpenes include but are not limited to phytol, vitamin A₁. Squalene is an example of a triterpene, and carotene (provitamin A₁) is a tetraterpene.

[0204] A "terpene precursor" is a compound or intermediate produced during the reaction to form a terpene starting from Acetyl CoA and optionally pyruvate. The term refers to a precursor compound or intermediate found in the mevalonate (MVA) pathway and optionally the DXS pathway as well as downstream precursors of longer chain terpenes, such as FPP and GPP. In particular embodiments, it includes but is not limited to mevalonic acid, IPP, dimethylallyl pyrophosphate (DMAPP), geranyl pyrophosphate (GPP) and farnesyl pyrophosphate (FPP).

[0205] The "DXS pathway" is the enzymatic pathway from pyruvate and D-glyceraldehyde-3-phosphate to DMAPP or IPP. It is also known as the deoxyxylulose 5-phosphate (DXP/DXPS/DOXP or DXS)/methylerythritol phosphate (MEP) pathway.

[0206] The "mevalonate (MVA) pathway" is the enzymatic pathway from acetyl-CoA to IPP.

Microorganisms

[0207] Two pathways for production of terpenes are known, the deoxyxylulose 5-phosphate (DXP/DXPS/DOXP or DXS)/methylerythritol phosphate (MEP) pathway (Hunter et al., 2007, J. Biol. chem. 282: 21573-77) starting from pyruvate and D-glyceraldehyde-3-phosphate (G3P), the two key intermediates in the glycolysis, and the mevalonate (MVA) pathway (Miziorko, 2011, Arch Biochem Biophys, 505: 131-143) starting from acetyl-CoA. Many different classes of microorganisms have been investigated for presence of either of these pathways (Lange et al., 2000, PNAS, 97: 13172-77; Trutko et al., 2005, Microbiology, 74: 153-158; Julsing et al., 2007, Appl Microbiol Biotechnol, 75: 1377-84), but not carboxydotrophic acetogens. The DXS pathway for example was found to be present in E. coli, Bacillus, or Mycobacterium, while the mevalonate pathway is present in yeast Saccharomyces, Cloroflexus, or Myxococcus.

[0208] Genomes of carboxydotrophic acetogens C. autoethanogenum, C. ljungdahlii were analysed by the inventors for presence of either of the two pathways. All genes of the DXS pathway were identified in C. autoethanogenum and C. ljungdahlii (Table 1), while the mevalonate pathway is absent. Additionally, carboxydotrophic acetogens such as C. autoethanogenum or C. ljungdahlii are not known to produce any terpenes as metabolic end products.

TABLE-US-00001 TABLE 1 Terpene biosynthesis genes of the DXS pathway identified in C. autoethanogenum and C. ljungdahlii: C. Gene/Enzyme autoethanogenum C. ljungdahlii 1-deoxy-D-xylulose-5- SEQ ID NO: 1-2 YP_003779286.1; phosphate synthase DXS GI: 300854302, (EC: 2.2.1.7) CLJU_c11160 1-deoxy-D-xylulose 5- SEQ ID NO: 3-4 YP_003779478.1; phosphate reductoisomerase GI: 300854494, DXR (EC: 1.1.1.267) CLJU_c13080 2-C-methyl-D-erythritol 4- SEQ ID NO: 5-6 YP_003782252.1 phosphate GI: 300857268, cytidylyltransferase IspD CLJU_c41280 (EC: 2.7.7.60) 4-diphosphocytidyl-2-C- SEQ ID NO: 7-8 YP_003778403.1; methyl-D-erythritol kinase GI: 300853419, IspE (EC: 2.7.1.148) CLJU_c02110 2-C-methyl-D-erythritol 2,4- SEQ ID NO: 9-10 YP_003778349.1; cyclodiphosphate synthase GI: 300853365, IspF (EC: 4.6.1.12) CLJU_c01570 4-hydroxy-3-methylbut-2-en- SEQ ID NO: 11-12 YP_003779480.1; 1-yl diphosphate synthase GI: 300854496, IspG (EC: 1.17.7.1) CLJU_c13100 4-hydroxy-3-methylbut-2- SEQ ID NO: 13-14 YP_003780294.1; enyl diphosphate reductase GI: 300855310, (EC: 1.17.1.2) CLJU_c21320

[0209] Genes for downstream synthesis of terpenes from isoprene units were also identified in both organisms (Table 2).

TABLE-US-00002 Gene/Enzyme C. autoethanogenum C. ljungdahlii geranyltranstransferase Fps SEQ ID NO: 15-16 YP_003779285.1; (EC: 2.5.1.10) GI: 300854301, CLJU_c11150 heptaprenyl diphosphate SEQ ID NO: 17-18 YP_003779312.1; synthase (EC: 2.5.1.10) GI: 300854328, CLJU_c11420 octaprenyl-diphosphate SEQ ID NO: 19-20 YP_003782157.1; synthase [EC: 2.5.1.90] GI: 300857173, CLJU_c40310

[0210] Terpenes are energy dense compounds, and their synthesis requires the cell to invest energy in the form of nucleoside triposphates such as ATP. Using sugar as a substrate requires sufficient energy to be supplied from glycolysis to yield several molecules of ATP. The production of terpenes and/or their precursors via the DXS pathway using sugar as a substrate proceeds in a relatively straightforward manner due to the availability of pyruvate and D-glyceraldehyde-3-phosphate (G3P), G3P being derived from C5 pentose and C6 hexose sugars. These C5 and C6 molecules are thus relatively easily converted into C5 isoprene units from which terpenes are composed.

[0211] For anaerobic acetogens using a C1 substrate like CO or CO2, it is more difficult to synthesise long molecules such as hemiterpenoids from C1 units. This is especially true for longer chain terpenes like C10 monoterpenes, C15 sesquiterpenes, or C40 tetraterpenes. To date the product with most carbon atoms reported in acetogens (both native and recombinant organisms) are C4 compounds butanol (Kopke et al., 2011, Curr. Opin. Biotechnol. 22: 320-325; Schiel-Bengelsdorf and Durre, 2012, FEBS Letters: 10.1016/j.febslet.2012.04.043; Kopke et al., 2011, Proc. Nat. Sci. U.S.A. 107: 13087-92; US patent 2011/0236941) and 2,3-butanediol (Kopke et al., 2011, Appl. Environ. Microbiol. 77:5467-75). The inventors have shown that it is surprisingly possible to anaerobically produce these longer chain terpene molecules using the C1 feedstock CO via the acetyl CoA intermediate.

[0212] Energetics of the Wood-Ljungdahl pathway of anaerobic acetogens are just emerging, but unlike under aerobic growth conditions or glycolysis of sugar fermenting organisms no ATP is gained in the Wood-Ljungdahl pathway by substrate level phosphorylation, in fact activation of CO₂ to formate actually requires one molecule of ATP and a membrane gradient is required. The inventors note that it is important that a pathway for product formation is energy efficient. The inventors note that in acetogens the substrate CO or CO₂ is channeled directly into acetyl-CoA, which represents the most direct route to terpenes and/or their precursors, especially when compared to sugar based systems, with only six reactions required (FIG. 1). Though less ATP is available in carboxydotrophic acetogens, the inventors believe that this more direct pathway may sustain a higher metabolic flux (owing to higher chemical motive force of intermediate reactions). A highly effective metabolic flux is important as several intermediates in the terpene biosynthesis pathway, such as key intermediates Mevalonate and FPP, are toxic to most bacteria when not turned over efficiently.

[0213] Despite having a higher ATP availability, this problem of intermediate toxicity can be a a bottleneck in production of terpenes from sugar.

[0214] When comparing the energetics of terpene precursor IPP and DMAPP production from CO (FIG. 6) via the mevalonate pathway versus the DXS pathway, the inventors noted that the mevalonate pathway requires less nucleoside triposphates as ATP, less reducing equivalents, and is also more direct when compared to the DXS pathway with only six necessary reaction steps from acetyl-CoA. This provides advantages in the speed of the reactions and metabolic fluxes and increases overall energy efficiency. Additionally, the lower number of enzymes required simplifies the recombination method required to produce a recombinant microorganism.

[0215] No acetogens with a mevalonate pathway have been identified, but the inventors have shown that it is possible to introduce the mevalonate pathway and optionally the DXS pathway into a carboxydotrophic acetogen such as Clostridium autoethanogenum or C. ljungdahlii to efficiently produce terpenes and/or precursors thereof from the C1 carbon substrate CO. They contemplate that this is applicable to all carboxydotrophic acetogenic microorganisms.

[0216] Additionally, the production of terpenes and/or precursors thereof has never been shown to be possible using recombinant microorganisms under anaerobic conditions. Anaerobic production of isoprene has the advantage of providing a safer operating environment because isoprene is extremely flammable in the presence of oxygen and has a lower flammable limit (LFL) of 1.5-2.0% and an upper flammable (UFL) limit of 2.0-12% at room temperature and atmospheric pressure. As flames cannot occur in the absence of oxygen, the inventors believe that an anaerobic fermentation process is desirable as it would be safer across all product concentrations, gas compositions, temperature and pressure ranges.

[0217] As discussed hereinbefore, the invention provides a recombinant microorganism capable of producing one or more terpenes and/or precursors thereof, and optionally one or more other products, by fermentation of a substrate comprising CO.

[0218] In a further embodiment, the microorganism is adapted to: express one or more exogenous enzymes from the mevalonate (MVA) pathway and/or overexpress one or more endogenous enzyme from the mevalonate (MVA) pathway; and

a) express one or more exogenous enzymes from the DXS pathway and/or overexpress one or more endogenous enzymes from the DXS pathway.

[0219] In one embodiment, the parental microorganism from which the recombinant microorganism is derived is capable of fermenting a substrate comprising CO to produce Acetyl CoA, but not of converting Acetyl CoA to mevalonic acid or isopentenyl pyrophosphate (IPP) and the recombinant microorganism is adapted to express one or more enzymes involved in the mevalonate pathway.

[0220] The microorganism may be adapted to express or over-express the one or more enzymes by any number of recombinant methods including, for example, increasing expression of native genes within the microorganism (for example, by introducing a stronger or constitutive promoter to drive expression of a gene), increasing the copy number of a gene encoding a particular enzyme by introducing exogenous nucleic acids encoding and adapted to express the enzyme, introducing an exogenous nucleic acid encoding and adapted to express an enzyme not naturally present within the parental microorganism.

[0221] In one embodiment, the one or more enzymes are from the mevalonate (MVA) pathway and are selected from the group consisting of:

a) thiolase (EC 2.3.1.9), b) HMG-CoA synthase (EC 2.3.3.10), c) HMG-CoA reductase (EC 1.1.1.88), d) Mevalonate kinase (EC 2.7.1.36), e) Phosphomevalonate kinase (EC 2.7.4.2), f) Mevalonate Diphosphate decarboxylase (EC 4.1.1.33), and g) a functionally equivalent variant of any one thereof.

[0222] In a further embodiment, the optional one or more enzymes are from the DXS pathway is selected from the group consisting of:

[0223] a) 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC:2.2.1.7),

[0224] b) 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC:1.1.1.267),

[0225] c) 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC:2.7.7.60),

[0226] d) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC:2.7.1.148),

[0227] e) 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase IspF (EC:4.6.1.12),

[0228] f) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC:1.17.7.1),

[0229] g) 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC:1.17.1.2), and

[0230] h) a functionally equivalent variant of any one thereof.

[0231] In a further embodiment, one or more exogenous or endogenous further enzymes are expressed or over-expressed to result in the production of a terpene compound and/or precursor thereof wherein the exogenous enzyme that is expressed, or the endogenous enzyme that is overexpressed is selected from the group consisting of:

a) geranyltranstransferase Fps (EC:2.5.1.10), b) heptaprenyl diphosphate synthase (EC:2.5.1.10), c) octaprenyl-diphosphate synthase (EC:2.5.1.90), d) isoprene synthase (EC 4.2.3.27), e) isopentenyl-diphosphate delta-isomerase (EC EC 5.3.3.2), f) farnesene synthase (EC 4.2.3.46/EC 4.2.3.47), and g) a functionally equivalent variant of any one thereof.

[0232] By way of example only, sequence information for each of the enzymes is listed in the figures herein.

[0233] The enzymes of use in the microorganisms of the invention may be derived from any appropriate source, including different genera and species of bacteria, or other organisms. However, in one embodiment, the enzymes are derived from Staphylococcus aureus.

[0234] In one embodiment, the enzyme isoprene synthase (ispS) is derived from Poplar tremuloides. In a further embodiment, it has the nucleic acid sequence exemplified in SEQ ID NO: 21 hereinafter, or it is a functionally equivalent variant thereof.

[0235] In one embodiment, the enzyme deoxyxylulose 5-phosphate synthase is derived from C. autoethanogenum, encoded by the nucleic acid sequence exemplified in SEQ ID NO: 1 and/or with the amino acid sequence exemplified in SEQ ID NO: 2 hereinafter, or it is a functionally equivalent variant thereof.

[0236] In one embodiment, the enzyme 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR is derived from C. autoethanogenum and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 3 or is a functionally equivalent variant thereof.

[0237] In one embodiment, the enzyme 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD is derived from C. autoethanogenum and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 5 or is a functionally equivalent variant thereof.

[0238] In one embodiment, the enzyme 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE is derived from C. autoethanogenum and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 7 or is a functionally equivalent variant thereof.

[0239] In one embodiment, the enzyme 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase IspF is derived from C. autoethanogenum and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 9 or is a functionally equivalent variant thereof.

[0240] In one embodiment, the enzyme 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG is derived from C. autoethanogenum and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 11 or is a functionally equivalent variant thereof.

[0241] In one embodiment, the enzyme 4-hydroxy-3-methylbut-2-enyl diphosphate reductase is derived from C. autoethanogenum and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 13 or is a functionally equivalent variant thereof.

[0242] In one embodiment, the enzyme mevalonate kinase (MK) is derived from Staphylococcus aureus subsp. aureus Mu50 and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 51 hereinafter, or it is a functionally equivalent variant thereof.

[0243] In one embodiment, the enzyme phosphomevalonate kinase (PMK) is derived from Staphylococcus aureus subsp. aureus Mu50 and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 52 hereinafter, or it is a functionally equivalent variant thereof.

[0244] In one embodiment, the enzyme mevalonate diphosphate decarboxylase (PMD) is derived from Staphylococcus aureus subsp. aureus Mu50 and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 53 hereinafter, or it is a functionally equivalent variant thereof.

[0245] In one embodiment, the enzyme Isopentenyl-diphosphate delta-isomerase (idi) is derived from Clostridium beijerinckii and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 54 hereinafter, or it is a functionally equivalent variant thereof.

[0246] In one embodiment, the enzyme thiolase (thIA) is derived from Clostridium acetobutylicum ATCC824 and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 40 hereinafter, or it is a functionally equivalent variant thereof.

[0247] In one embodiment, the enzyme is a thiolase enzyme, and is an acetyl-CoA c-acetyltransferase (vraB) derived from Staphylococcus aureus subsp. aureus Mu50 and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 41 hereinafter, or it is a functionally equivalent variant thereof.

[0248] In one embodiment, the enzyme 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) is derived from Staphylococcus aureus subsp. aureus Mu50 and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 42 hereinafter, or it is a functionally equivalent variant thereof.

[0249] In one embodiment, the enzyme Hydroxymethylglutaryl-CoA reductase (HMGR) is derived from Staphylococcus aureus subsp. aureus Mu50 and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 43 hereinafter, or it is a functionally equivalent variant thereof.

[0250] In one embodiment, the enzyme Geranyltranstransferase (ispA) is derived from Escherichia coli str. K-12 substr. MG1655 is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 56 hereinafter, or it is a functionally equivalent variant thereof.

[0251] In one embodiment, the enzyme heptaprenyl diphosphate synthase is derived from C. autoethanogenum and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 17 or is a functionally equivalent variant thereof.

[0252] In one embodiment, the enzyme polyprenyl synthetase is derived from C. autoethanogenum and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 19 or is a functionally equivalent variant thereof.

[0253] In one embodiment, the enzyme Alpha-farnesene synthase (FS) is derived from Malus×domestica and is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 57 hereinafter, or it is a functionally equivalent variant thereof.

[0254] The enzymes and functional variants of use in the microorganisms may be identified by assays known to one of skill in the art. In particular embodiments, the enzyme isoprene synthase may be identified by the method outlined Silver et al. (1991, Plant Physiol. 97: 1588-1591) or Zhao et al. (2011, Appl Microbiol Biotechnol, 90:1915-1922). In a further particular embodiment, the enzyme farnesene synthase may be identified by the method outlined in Green et al., 2007, Phytochemistry; 68:176-188. In further particular embodiments, enzymes from the mevalonate pathway may be identified by the method outlined in Cabano et al. (1997, Insect Biochem. Mol. Biol. 27: 499-505) for the HMG-CoA synthase, Ma et al. (2011, Metab. Engin., 13:588-597) for the HMG-CoA reductase and mevalonate kinase enzyme, Herdendorf and Miziorko (2007, Biochemistry, 46: 11780-8) for the phosphomevalonate kinase, and Krepkiy et al. (2004, Protein Sci. 13: 1875-1881) for the mevalonate diphosphate decarboxylase. Ma et al., 2011, Metab. Engin., 13:588-597. The 1-deoxy-D-xylulose 5-phosphate synthase of the DXS pathway can be assayed using the method outlined in Kuzuyama et al. (2000, J. Bacteriol. 182, 891-897). It is also possible to identify genes of DXS and mevalonate pathway using inhibitors like fosmidomycin or mevinoline as described by Trutko et al. (2005, Microbiology 74: 153-158).

[0255] In one embodiment, the microorganism comprises one or more exogenous nucleic acids adapted to increase expression of one or more endogenous nucleic acids and which one or more endogenous nucleic acids encode one or more of the enzymes referred to herein before. In one embodiment, the one or more exogenous nucleic acid adapted to increase expression is a regulatory element. In one embodiment, the regulatory element is a promoter. In one embodiment, the promoter is a constitutive promoter that is preferably highly active under appropriate fermentation conditions. Inducible promoters could also be used. In preferred embodiments, the promoter is selected from the group comprising Wood-Ljungdahl gene cluster or Phosphotransacetylase/Acetate kinase operon promoters. It will be appreciated by those of skill in the art that other promoters which can direct expression, preferably a high level of expression under appropriate fermentation conditions, would be effective as alternatives to the exemplified embodiments.

[0256] In one embodiment, the microorganism comprises one or more exogenous nucleic acids encoding and adapted to express one or more of the enzymes referred to herein before. In one embodiment, the microorganisms comprise one or more exogenous nucleic acid encoding and adapted to express at least two, at least of the enzymes. In other embodiments, the microorganism comprises one or more exogenous nucleic acid encoding and adapted to express at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or more of the enzymes.

[0257] In one particular embodiment, the microorganism comprises one or more exogenous nucleic acid encoding an enzyme of the invention or a functionally equivalent variant thereof.

[0258] The microorganism may comprise one or more exogenous nucleic acids. Where it is desirable to transform the parental microorganism with two or more genetic elements (such as genes or regulatory elements (for example a promoter)) they may be contained on one or more exogenous nucleic acids.

[0259] In one embodiment, the one or more exogenous nucleic acid is a nucleic acid construct or vector, in one particular embodiment a plasmid, encoding one or more of the enzymes referred to hereinbefore in any combination.

[0260] The exogenous nucleic acids may remain extra-chromosomal upon transformation of the parental microorganism or may intergrate into the genome of the parental microorganism. Accordingly, they may include additional nucleotide sequences adapted to assist integration (for example, a region which allows for homologous recombination and targeted integration into the host genome) or expression and replication of an extrachromosomal construct (for example, origin of replication, promoter and other regulatory elements or sequences).

[0261] In one embodiment, the exogenous nucleic acids encoding one or enzymes as mentioned herein before will further comprise a promoter adapted to promote expression of the one or more enzymes encoded by the exogenous nucleic acids. In one embodiment, the promoter is a constitutive promoter that is preferably highly active under appropriate fermentation conditions. Inducible promoters could also be used. In preferred embodiments, the promoter is selected from the group comprising Wood-Ljungdahl gene cluster and Phosphotransacetylase/Acetate kinase promoters. It will be appreciated by those of skill in the art that other promoters which can direct expression, preferably a high level of expression under appropriate fermentation conditions, would be effective as alternatives to the exemplified embodiments.

[0262] In one embodiment, the exogenous nucleic acid is an expression plasmid.

[0263] In one particular embodiment, the parental microorganism is selected from the group of carboxydotrophic acetogenic bacteria. In certain embodiments the microorganism is selected from the group comprising Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Clostridium aceticum, Clostridium formicoaceticum, Clostridium magnum, Butyribacterium methylotrophicum, Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorella thermautotrophica, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, Oxobacter pfennigii, and Thermoanaerobacter kiuvi.

[0264] In one particular embodiment, the parental microorganism is selected from the cluster of ethanologenic, acetogenic Clostridia comprising the species C. autoethanogenum, C. ljungdahlii, and C. ragsdalei and related isolates. These include but are not limited to strains C. autoethanogenum JAI-1T (DSM10061) [Abrini J, Naveau H, Nyns E- J: Clostridium autoethanogenum, sp. nov., an anaerobic bacterium that produces ethanol from carbon monoxide. Arch Microbiol 1994, 4: 345-351], C. autoethanogenum LBS1560 (DSM19630) [Simpson S D, Forster R L, Tran P T, Rowe M J, Warner I L: Novel bacteria and methods thereof. International patent 2009, WO/2009/064200], C. autoethanogenum LBS1561 (DSM23693), C. ljungdahlii PETC^T (DSM13528=ATCC 55383) [Tanner R S, Miller L M, Yang D: Clostridium ljungdahlii sp. nov., an Acetogenic Species in Clostridial rRNA Homology Group I. Int J Syst Bacteriol 1993, 43: 232-236], C. ljungdahlii ERI-2 (ATCC 55380) [Gaddy J L: Clostridium stain which produces acetic acid from waste gases. US patent 1997, U.S. Pat. No. 5,593,886], C. ljungdahlii C-01 (ATCC 55988) [Gaddy J L, Clausen E C, Ko C- W: Microbial process for the preparation of acetic acid as well as solvent for its extraction from the fermentation broth. US patent, 2002, U.S. Pat. No. 6,368,819], C. ljungdahlii O-52 (ATCC 55989) [Gaddy J L, Clausen E C, Ko C- W: Microbial process for the preparation of acetic acid as well as solvent for its extraction from the fermentation broth. US patent, 2002, U.S. Pat. No. 6,368,819], C. ragsdalei P11^T (ATCC BAA-622) [Huhnke R L, Lewis R S, Tanner R S: Isolation and Characterization of novel Clostridial Species. International patent 2008, WO 2008/028055], related isolates such as "C. coskatii" [Zahn et at--Novel ethanologenic species Clostridium coskatii (US Patent Application number US20110229947)] and "Clostridium sp." (Tyurin et al., 2012, J. Biotech Res. 4: 1-12), or mutated strains such as C. ljungdahlii OTA-1 (Tirado-Acevedo O. Production of Bioethanol from Synthesis Gas Using Clostridium ljungdahlii. PhD thesis, North Carolina State University, 2010). These strains form a subcluster within the Clostridial rRNA cluster I, and their 16S rRNA gene is more than 99% identical with a similar low GC content of around 30%. However, DNA-DNA reassociation and DNA fingerprinting experiments showed that these strains belong to distinct species [Huhnke R L, Lewis R S, Tanner R S: Isolation and Characterization of novel Clostridial Species. International patent 2008, WO 2008/028055].

[0265] All species of this cluster have a similar morphology and size (logarithmic growing cells are between 0.5-0.7×3-5 μm), are mesophilic (optimal growth temperature between 30-37° C.) and strictly anaerobe [Tanner R S, Miller L M, Yang D: Clostridium ljungdahlii sp. nov., an Acetogenic Species in Clostridial rRNA Homology Group I. Int J Syst Bacteriol 1993, 43: 232-236; Abrini J, Naveau H, Nyns E- J: Clostridium autoethanogenum, sp. nov., an anaerobic bacterium that produces ethanol from carbon monoxide. Arch Microbiol 1994, 4: 345-351; Huhnke R L, Lewis R S, Tanner R S: Isolation and Characterization of novel Clostridial Species. International patent 2008, WO 2008/028055]. Moreover, they all share the same major phylogenetic traits, such as same pH range (pH 4-7.5, with an optimal initial pH of 5.5-6), strong autotrophic growth on CO containing gases with similar growth rates, and a similar metabolic profile with ethanol and acetic acid as main fermentation end product, and small amounts of 2,3-butanediol and lactic acid formed under certain conditions. [Tanner R S, Miller L M, Yang D: Clostridium ljungdahlii sp. nov., an Acetogenic Species in Clostridial rRNA Homology Group I. Int J Syst Bacteriol 1993, 43: 232-236; Abrini J, Naveau H, Nyns E- J: Clostridium autoethanogenum, sp. nov., an anaerobic bacterium that produces ethanol from carbon monoxide. Arch Microbiol 1994, 4: 345-351; Huhnke R L, Lewis R S, Tanner R S: Isolation and Characterization of novel Clostridial Species. International patent 2008, WO 2008/028055]. Indole production was observed with all three species as well. However, the species differentiate in substrate utilization of various sugars (e.g. rhamnose, arabinose), acids (e.g. gluconate, citrate), amino acids (e.g. arginine, histidine), or other substrates (e.g. betaine, butanol). Moreover some of the species were found to be auxotroph to certain vitamins (e.g. thiamine, biotin) while others were not.

[0266] In one embodiment, the parental carboxydotrophic acetogenic microorganism is selected from the group consisting of Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Butyribacterium limosum, Butyribacterium methylotrophicum, Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorella thermautotrophica, Oxobacter pfennigii, and Thermoanaerobacter kiuvi.

[0267] In one particular embodiment of the first or second aspects, the parental microorganism is selected from the group of carboxydotrophic Clostridia comprising Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Clostridium aceticum, Clostridium formicoaceticum, Clostridium magnum.

[0268] In a one embodiment, the microorganism is selected from a cluster of carboxydotrophic Clostridia comprising the species C. autoethanogenum, C. ljungdahlii, and "C. ragsdalei" and related isolates. These include but are not limited to strains C. autoethanogenum JAI-1^T (DSM10061) (Abrini, Naveau, & Nyns, 1994), C. autoethanogenum LBS1560 (DSM19630) (WO/2009/064200), C. autoethanogenum LBS1561 (DSM23693), C. ljungdahlii PETC^T (DSM13528=ATCC 55383) (Tanner, Miller, & Yang, 1993), C. ljungdahlii ERI-2 (ATCC 55380) (U.S. Pat. No. 5,593,886), C. ljungdahlii C-01 (ATCC 55988) (U.S. Pat. No. 6,368,819), C. ljungdahlii O-52 (ATCC 55989) (U.S. Pat. No. 6,368,819), or "C. ragsdalei P11^T" (ATCC BAA-622) (WO 2008/028055), and related isolates such as "C. coskatii" (US patent 2011/0229947), "Clostridium sp. MT351" (Michael Tyurin & Kiriukhin, 2012) and mutant strains thereof such as C. ljungdahlii OTA-1 (Tirado-Acevedo O. Production of Bioethanol from Synthesis Gas Using Clostridium ljungdahlii. PhD thesis, North Carolina State University, 2010).

[0269] These strains form a subcluster within the Clostridial rRNA cluster I (Collins et al., 1994), having at least 99% identity on 16S rRNA gene level, although being distinct species as determined by DNA-DNA reassociation and DNA fingerprinting experiments (WO 2008/028055, US patent 2011/0229947).

[0270] The strains of this cluster are defined by common characteristics, having both a similar genotype and phenotype, and they all share the same mode of energy conservation and fermentative metabolism. The strains of this cluster lack cytochromes and conserve energy via an Rnf complex.

[0271] All strains of this cluster have a genome size of around 4.2 MBp (Kopke et al., 2010) and a GC composition of around 32% mol (Abrini et al., 1994; Kopke et al., 2010; Tanner et al., 1993) (WO 2008/028055; US patent 2011/0229947), and conserved essential key gene operons encoding for enzymes of Wood-Ljungdahl pathway (Carbon monoxide dehydrogenase, Formyl-tetrahydrofolate synthetase, Methylene-tetrahydrofolate dehydrogenase, Formyl-tetrahydrofolate cyclohydrolase, Methylene-tetrahydrofolate reductase, and Carbon monoxide dehydrogenase/Acetyl-CoA synthase), hydrogenase, formate dehydrogenase, Rnf complex (rnfCDGEAB), pyruvate:ferredoxin oxidoreductase, aldehyde:ferredoxin oxidoreductase (Kopke et al., 2010, 2011). The organization and number of Wood-Ljungdahl pathway genes, responsible for gas uptake, has been found to be the same in all species, despite differences in nucleic and amino acid sequences (Kopke et al., 2011).

[0272] The strains all have a similar morphology and size (logarithmic growing cells are between 0.5-0.7×3-5 μm), are mesophilic (optimal growth temperature between 30-37° C.) and strictly anaerobe (Abrini et al., 1994; Tanner et al., 1993) (WO 2008/028055). Moreover, they all share the same major phylogenetic traits, such as same pH range (pH 4-7.5, with an optimal initial pH of 5.5-6), strong autotrophic growth on CO containing gases with similar growth rates, and a metabolic profile with ethanol and acetic acid as main fermentation end product, with small amounts of 2,3-butanediol and lactic acid formed under certain conditions (Abrini et al., 1994; Kopke et al., 2011; Tanner et al., 1993) However, the species differentiate in substrate utilization of various sugars (e.g. rhamnose, arabinose), acids (e.g. gluconate, citrate), amino acids (e.g. arginine, histidine), or other substrates (e.g. betaine, butanol). Some of the species were found to be auxotroph to certain vitamins (e.g. thiamine, biotin) while others were not. Reduction of carboxylic acids into their corresponding alcohols has been shown in a range of these organisms (Perez, Richter, Loftus, & Angenent, 2012).

[0273] The traits described are therefore not specific to one organism like C. autoethanogenum or C. ljungdahlii, but rather general traits for carboxydotrophic, ethanol-synthesizing Clostridia. Thus, the invention can be anticipated to work across these strains, although there may be differences in performance.

[0274] The recombinant carboxydotrophic acetogenic microorganisms of the invention may be prepared from a parental carboxydotrophic acetogenic microorganism and one or more exogenous nucleic acids using any number of techniques known in the art for producing recombinant microorganisms. By way of example only, transformation (including transduction or transfection) may be achieved by electroporation, electrofusion, ultrasonication, polyethylene glycol-mediated transformation, conjugation, or chemical and natural competence. Suitable transformation techniques are described for example in Sambrook J, Fritsch E F, Maniatis T: Molecular Cloning: A laboratory Manual, Cold Spring Harbour Labrotary Press, Cold Spring Harbour, 1989.

[0275] Electroporation has been described for several carboxydotrophic acetogens as C. ljungdahlii (Kopke et al., 2010; Leang, Ueki, Nevin, & Lovley, 2012) (PCT/NZ2011/000203; WO2012/053905), C. autoethanogenum (PCT/NZ2011/000203; WO2012/053905), Acetobacterium woodii (Stratz, Sauer, Kuhn, & Dune, 1994) or Moorella thermoacetica (Kita et al., 2012) and is a standard method used in many Clostridia such as C. acetobutylicum (Mermelstein, Welker, Bennett, & Papoutsakis, 1992), C. cellulolyticum (Jennert, Tardif, Young, & Young, 2000) or C. thermocellum (MV Tyurin, Desai, & Lynd, 2004).

[0276] Electrofusion has been described for acetogenic Clostridium sp. MT351 (Tyurin and Kiriukhin, 2012).

[0277] Prophage induction has been described for carboxydotrophic acetogen as well in case of C. scatologenes (Prasanna Tamarapu Parthasarathy, 2010, Development of a Genetic Modification System in Clostridium scatologenes ATCC 25775 for Generation of Mutants, Masters Project Western Kentucky University).

[0278] Conjugation has been described as method of choice for acetogen Clostridium difficile (Herbert, O'Keeffe, Purdy, Elmore, & Minton, 2003) and many other Clostridia including C. acetobuylicum (Williams, Young, & Young, 1990).

[0279] In one embodiment, the parental strain uses CO as its sole carbon and energy source.

[0280] In one embodiment the parental microorganism is Clostridium autoethanogenum or Clostridium ljungdahlii. In one particular embodiment, the microorganism is Clostridium autoethanogenum DSM23693. In another particular embodiment, the microorganism is Clostridium ljungdahlii DSM13528 (or ATCC55383).

Nucleic Acids

[0281] The invention also provides one or more nucleic acids or nucleic acid constructs of use in generating a recombinant microorganism of the invention.

[0282] In one embodiment, the nucleic acid comprises sequences encoding one or more of the enzymes in the mevalonate (MVA) pathway and optionally the DXS pathway which when expressed in a microorganism allows the microorganism to produce one or more terpenes and/or precursors thereof by fermentation of a substrate comprising CO. In one particular embodiment, the invention provides a nucleic acid encoding two or more enzymes which when expressed in a microorganism allows the microorganism to produce one or more terpene and/or precursor thereof by fermentation of substrate comprising CO. In one embodiment, a nucleic acid of the invention encodes three, four, five or more of such enzymes.

[0283] In one embodiment, the one or more enzymes encoded by the nucleic acid are from the mevalonate (MVA) pathway and are selected from the group consisting of:

a) thiolase (EC 2.3.1.9), b) HMG-CoA synthase (EC 2.3.3.10), c) HMG-CoA reductase (EC 1.1.1.88), d) Mevalonate kinase (EC 2.7.1.36), e) Phosphomevalonate kinase (EC 2.7.4.2), f) Mevalonate Diphosphate decarboxylase (EC 4.1.1.33), and g) a functionally equivalent variant of any one thereof.

[0284] In a further embodiment, the one or more optional enzymes encoded by the nucleic acid are from the DXS pathway are selected from the group consisting of:

[0285] a) 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC:2.2.1.7),

[0286] b) 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC:1.1.1.267),

[0287] c) 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC:2.7.7.60),

[0288] d) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC:2.7.1.148),

[0289] e) 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase IspF (EC:4.6.1.12),

[0290] f) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC:1.17.7.1),

[0291] g) 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC:1.17.1.2), and

[0292] h) a functionally equivalent variant of any one thereof.

[0293] In a further embodiment, the nucleic acid encodes one or more further enzymes that are expressed or over-expressed to result in the production of a terpene compound and/or precursor thereof wherein the exogenous enzyme that is expressed, or the endogenous enzyme that is overexpressed is selected from the group consisting of:

a) geranyltranstransferase Fps (EC:2.5.1.10), b) heptaprenyl diphosphate synthase (EC:2.5.1.10), c) octaprenyl-diphosphate synthase (EC:2.5.1.90), d) isoprene synthase (EC 4.2.3.27), e) isopentenyl-diphosphate delta-isomerase (EC EC 5.3.3.2), f) farnesene synthase (EC 4.2.3.46/EC 4.2.3.47), and g) a functionally equivalent variant of any one thereof.

[0294] Exemplary amino acid sequences and nucleic acid sequences encoding each of the above enzymes are provided herein or can be obtained from GenBank as mentioned hereinbefore. However, skilled persons will readily appreciate alternative nucleic acid sequences encoding the enzymes or functionally equivalent variants thereof, having regard to the information contained herein, in GenBank and other databases, and the genetic code.

[0295] In a further embodiment, the nucleic acid encoding thiolase (thIA) derived from Clostridium acetobutylicum ATCC824 is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 40 hereinafter, or it is a functionally equivalent variant thereof.

[0296] In a further embodiment, the nucleic acid encoding thiolase wherein the thiolase is acetyl-CoA c-acetyltransferase (vraB) derived from Staphylococcus aureus subsp. aureus Mu50 is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 41 hereinafter, or it is a functionally equivalent variant thereof.

[0297] In a further embodiment, the nucleic acid encoding 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) derived from Staphylococcus aureus subsp. aureus Mu50 is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 42 hereinafter, or it is a functionally equivalent variant thereof.

[0298] In a further embodiment, the nucleic acid encoding Hydroxymethylglutaryl-CoA reductase (HMGR) derived from Staphylococcus aureus subsp. aureus Mu50 is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 43 hereinafter, or it is a functionally equivalent variant thereof.

[0299] In a further embodiment, the nucleic acid encoding mevalonate kinase (MK) derived from Staphylococcus aureus subsp. aureus Mu50 is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 51 hereinafter, or it is a functionally equivalent variant thereof.

[0300] In a further embodiment, the nucleic acid encoding phosphomevalonate kinase (PMK) derived from Staphylococcus aureus subsp. aureus Mu50 is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 52 hereinafter, or it is a functionally equivalent variant thereof.

[0301] In a further embodiment, the nucleic acid encoding mevalonate diphosphate decarboxylase (PMD) derived from Staphylococcus aureus subsp. aureus Mu50 is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 53 hereinafter, or it is a functionally equivalent variant thereof.

[0302] In a further embodiment, the nucleic acid encoding deoxyxylulose 5-phosphate synthase derived from C. autoethanogenum, is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 1 and/or with the amino acid sequence exemplified in SEQ ID NO: 2 hereinafter, or it is a functionally equivalent variant thereof.

[0303] In one embodiment, the nucleic acid encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC:1.1.1.267) has the sequence SEQ ID NO: 3 or is a functionally equivalent variant thereof.

[0304] In one embodiment, the nucleic acid encoding 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC:2.7.7.60) has the sequence SEQ ID NO: 5 or is a functionally equivalent variant thereof.

[0305] In one embodiment, the nucleic acid encoding 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC:2.7.1.148) has the sequence SEQ ID NO: 7 or is a functionally equivalent variant thereof.

[0306] In one embodiment, the nucleic acid encoding 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase IspF (EC:4.6.1.12) has the sequence SEQ ID NO: 9 or is a functionally equivalent variant thereof.

[0307] In one embodiment, the nucleic acid encoding 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC:1.17.7.1) has the sequence SEQ ID NO: 11 or is a functionally equivalent variant thereof.

[0308] In one embodiment, the nucleic acid encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC:1.17.1.2) has the sequence SEQ ID NO: 13 or is a functionally equivalent variant thereof.

[0309] In a further embodiment, the nucleic acid encoding Geranyltranstransferase (ispA) derived from Escherichia coli str. K-12 substr. MG1655 is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 56 hereinafter, or it is a functionally equivalent variant thereof.

[0310] In one embodiment, the nucleic acid encoding heptaprenyl diphosphate synthase has the sequence SEQ ID NO: 17, or it is a functionally equivalent variant thereof.

[0311] In one embodiment, the nucleic acid encoding octaprenyl-diphosphate synthase (EC:2.5.1.90) wherein the octaprenyl-diphosphate synthase is polyprenyl synthetase is encoded by sequence SEQ ID NO: 19, or it is a functionally equivalent variant thereof.

[0312] In one embodiment, the nucleic acid encoding isoprene synthase (ispS) derived from Poplar tremuloides is exemplified in SEQ ID NO: 21 hereinafter, or it is a functionally equivalent variant thereof.

[0313] In a further embodiment, the nucleic acid encoding Isopentenyl-diphosphate delta-isomerase (idi) derived from Clostridium beijerinckii is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 54 hereinafter, or it is a functionally equivalent variant thereof.

[0314] In a further embodiment, the nucleic acid encoding Alpha-farnesene synthase (FS) derived from Malus×domestica is encoded by the nucleic acid sequence exemplified in SEQ ID NO: 57 hereinafter, or it is a functionally equivalent variant thereof.

[0315] In one embodiment, the nucleic acids of the invention will further comprise a promoter. In one embodiment, the promoter allows for constitutive expression of the genes under its control. However, inducible promoters may also be employed. Persons of skill in the art will readily appreciate promoters of use in the invention. Preferably, the promoter can direct a high level of expression under appropriate fermentation conditions. In a particular embodiment a Wood-Ljungdahl cluster promoter is used. In another embodiment, a Phosphotransacetylase/Acetate kindase promoter is used. In another embodiment a pyruvate:ferredoxin oxidoreductase promoter, an Rnf complex operon promoter or an ATP synthase operon promoter. In one particular embodiment, the promoter is from C. autoethanogenum.

[0316] The nucleic acids of the invention may remain extra-chromosomal upon transformation of a parental microorganism or may be adapted for intergration into the genome of the microorganism. Accordingly, nucleic acids of the invention may include additional nucleotide sequences adapted to assist integration (for example, a region which allows for homologous recombination and targeted integration into the host genome) or stable expression and replication of an extrachromosomal construct (for example, origin of replication, promoter and other regulatory sequences).

[0317] In one embodiment, the nucleic acid is nucleic acid construct or vector. In one particular embodiment, the nucleic acid construct or vector is an expression construct or vector, however other constructs and vectors, such as those used for cloning are encompassed by the invention. In one particular embodiment, the expression construct or vector is a plasmid.

[0318] It will be appreciated that an expression construct/vector of the present invention may contain any number of regulatory elements in addition to the promoter as well as additional genes suitable for expression of further proteins if desired. In one embodiment the expression construct/vector includes one promoter. In another embodiment, the expression construct/vector includes two or more promoters. In one particular embodiment, the expression construct/vector includes one promoter for each gene to be expressed. In one embodiment, the expression construct/vector includes one or more ribosomal binding sites, preferably a ribosomal binding site for each gene to be expressed.

[0319] It will be appreciated by those of skill in the art that the nucleic acid sequences and construct/vector sequences described herein may contain standard linker nucleotides such as those required for ribosome binding sites and/or restriction sites. Such linker sequences should not be interpreted as being required and do not provide a limitation on the sequences defined.

[0320] Nucleic acids and nucleic acid constructs, including expression constructs/vectors of the invention may be constructed using any number of techniques standard in the art. For example, chemical synthesis or recombinant techniques may be used. Such techniques are described, for example, in Sambrook et al (Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Further exemplary techniques are described in the Examples section herein after. Essentially, the individual genes and regulatory elements will be operably linked to one another such that the genes can be expressed to form the desired proteins. Suitable vectors for use in the invention will be appreciated by those of ordinary skill in the art. However, by way of example, the following vectors may be suitable: pMTL80000 vectors, pIMP1, pJIR750, and the plasmids exemplified in the Examples section herein after.

[0321] It should be appreciated that nucleic acids of the invention may be in any appropriate form, including RNA, DNA, or cDNA.

[0322] The invention also provides host organisms, particularly microorganisms, and including viruses, bacteria, and yeast, comprising any one or more of the nucleic acids described herein.

Methods of Producing Organisms

[0323] The one or more exogenous nucleic acids may be delivered to a parental microorganism as naked nucleic acids or may be formulated with one or more agents to facilitate the transformation process (for example, liposome-conjugated nucleic acid, an organism in which the nucleic acid is contained). The one or more nucleic acids may be DNA, RNA, or combinations thereof, as is appropriate. Restriction inhibitors may be used in certain embodiments; see, for example Murray, N. E. et al. (2000) Microbial. Molec. Biol. Rev. 64, 412.)

[0324] The microorganisms of the invention may be prepared from a parental microorganism and one or more exogenous nucleic acids using any number of techniques known in the art for producing recombinant microorganisms. By way of example only, transformation (including transduction or transfection) may be achieved by electroporation, ultrasonication, polyethylene glycol-mediated transformation, chemical or natural competence, or conjugation. Suitable transformation techniques are described for example in, Sambrook J, Fritsch E F, Maniatis T: Molecular Cloning: A laboratory Manual, Cold Spring Harbour Labrotary Press, Cold Spring Harbour, 1989.

[0325] In certain embodiments, due to the restriction systems which are active in the microorganism to be transformed, it is necessary to methylate the nucleic acid to be introduced into the microorganism. This can be done using a variety of techniques, including those described below, and further exemplified in the Examples section herein after.

[0326] By way of example, in one embodiment, a recombinant microorganism of the invention is produced by a method comprises the following steps:

[0327] b) introduction into a shuttle microorganism of (i) of an expression construct/vector as described herein and (ii) a methylation construct/vector comprising a methyltransferase gene;

[0328] c) expression of the methyltransferase gene;

[0329] d) isolation of one or more constructs/vectors from the shuttle microorganism; and,

[0330] e) introduction of the one or more construct/vector into a destination microorganism.

[0331] In one embodiment, the methyltransferase gene of step B is expressed constitutively.

[0332] In another embodiment, expression of the methyltransferase gene of step B is induced.

[0333] The shuttle microorganism is a microorganism, preferably a restriction negative microorganism, that facilitates the methylation of the nucleic acid sequences that make up the expression construct/vector. In a particular embodiment, the shuttle microorganism is a restriction negative E. coli, Bacillus subtillis, or Lactococcus lactis.

[0334] The methylation construct/vector comprises a nucleic acid sequence encoding a methyltransferase.

[0335] Once the expression construct/vector and the methylation construct/vector are introduced into the shuttle microorganism, the methyltransferase gene present on the methylation construct/vector is induced. Induction may be by any suitable promoter system although in one particular embodiment of the invention, the methylation construct/vector comprises an inducible lac promoter and is induced by addition of lactose or an analogue thereof, more preferably isopropyl-β-D-thio-galactoside (IPTG). Other suitable promoters include the ara, tet, or T7 system. In a further embodiment of the invention, the methylation construct/vector promoter is a constitutive promoter.

[0336] In a particular embodiment, the methylation construct/vector has an origin of replication specific to the identity of the shuttle microorganism so that any genes present on the methylation construct/vector are expressed in the shuttle microorganism. Preferably, the expression construct/vector has an origin of replication specific to the identity of the destination microorganism so that any genes present on the expression construct/vector are expressed in the destination microorganism.

[0337] Expression of the methyltransferase enzyme results in methylation of the genes present on the expression construct/vector. The expression construct/vector may then be isolated from the shuttle microorganism according to any one of a number of known methods. By way of example only, the methodology described in the Examples section described hereinafter may be used to isolate the expression construct/vector.

[0338] In one particular embodiment, both construct/vector are concurrently isolated.

[0339] The expression construct/vector may be introduced into the destination microorganism using any number of known methods. However, by way of example, the methodology described in the Examples section hereinafter may be used. Since the expression construct/vector is methylated, the nucleic acid sequences present on the expression construct/vector are able to be incorporated into the destination microorganism and successfully expressed.

[0340] It is envisaged that a methyltransferase gene may be introduced into a shuttle microorganism and over-expressed. Thus, in one embodiment, the resulting methyltransferase enzyme may be collected using known methods and used in vitro to methylate an expression plasmid. The expression construct/vector may then be introduced into the destination microorganism for expression. In another embodiment, the methyltransferase gene is introduced into the genome of the shuttle microorganism followed by introduction of the expression construct/vector into the shuttle microorganism, isolation of one or more constructs/vectors from the shuttle microorganism and then introduction of the expression construct/vector into the destination microorganism.

[0341] It is envisaged that the expression construct/vector and the methylation construct/vector as defined above may be combined to provide a composition of matter. Such a composition has particular utility in circumventing restriction barrier mechanisms to produce the recombinant microorganisms of the invention.

[0342] In one particular embodiment, the expression construct/vector and/or the methylation construct/vector are plasmids.

[0343] Persons of ordinary skill in the art will appreciate a number of suitable methyltransferases of use in producing the microorganisms of the invention. However, by way of example the Bacillus subtilis phage ΦT1 methyltransferase and the methyltransferase described in the Examples herein after may be used. In one embodiment, the methyltransferase has the amino acid sequence of SEQ ID NO: 60 or is a functionally equivalent variant thereof. Nucleic acids encoding suitable methyltransferases will be readily appreciated having regard to the sequence of the desired methyltransferase and the genetic code. In one embodiment, the nucleic acid encoding a methyltransferase is as described in the Examples herein after (for example the nucleic acid of SEQ ID NO: 63, or it is a functionally equivalent variant thereof).

[0344] Any number of constructs/vectors adapted to allow expression of a methyltransferase gene may be used to generate the methylation construct/vector. However, by way of example, the plasmid described in the Examples section hereinafter may be used.

Methods of Production

[0345] The invention provides a method for the production of one or more terpenes and/or precursors thereof, and optionally one or more other products, by microbial fermentation comprising fermenting a substrate comprising CO using a recombinant microorganism of the invention. Preferably, the one or more terpene and/or precursor thereof is the main fermentation product. The methods of the invention may be used to reduce the total atmospheric carbon emissions from an industrial process.

[0346] Preferably, the fermentation comprises the steps of anaerobically fermenting a substrate in a bioreactor to produce at least one or more terpenes and/or a precursor thereof using a recombinant microorganism of the invention.

[0347] In one embodiment, the one or more terpene and/or precursor thereof is chosen from mevalonic acid, IPP, dimethylallyl pyrophosphate (DMAPP), isoprene, geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) and farnesene.

[0348] Instead of producing isoprene directly from terpenoid key intermediates IPP and DMAPP then using this to synthesise longer chain terpenes, it is also possible to synthesise longer chain terpenes, such as C10 Monoterpenoids or C15 Sesquiterpenoids, directly via a geranyltransferase (see Table 6). From C15 Sesquiterpenoid building block farnesyl-PP it is possible to produce farnesene, which, similarly to ethanol, can be used as a transportation fuel.

[0349] In one embodiment the method comprises the steps of:

(a) providing a substrate comprising CO to a bioreactor containing a culture of one or more microorganism of the invention; and (b) anaerobically fermenting the culture in the bioreactor to produce at least one or more terpene and/or precursor thereof.

[0350] In one embodiment the method comprises the steps of:

[0351] a) capturing CO-containing gas produced as a result of the industrial process;

[0352] b) anaerobic fermentation of the CO-containing gas to produce the at least one or more terpene and/or precursor thereof by a culture containing one or more microorganism of the invention.

[0353] In an embodiment of the invention, the gaseous substrate fermented by the microorganism is a gaseous substrate containing CO. The gaseous substrate may be a CO-containing waste gas obtained as a by-product of an industrial process, or from some other source such as from automobile exhaust fumes. In certain embodiments, the industrial process is selected from the group consisting of ferrous metal products manufacturing, such as a steel mill, non-ferrous products manufacturing, petroleum refining processes, gasification of coal, electric power production, carbon black production, ammonia production, methanol production and coke manufacturing. In these embodiments, the CO-containing gas may be captured from the industrial process before it is emitted into the atmosphere, using any convenient method. The CO may be a component of syngas (gas comprising carbon monoxide and hydrogen). The CO produced from industrial processes is normally flared off to produce CO₂ and therefore the invention has particular utility in reducing CO₂ greenhouse gas emissions and producing a terpene for use as a biofuel. Depending on the composition of the gaseous CO-containing substrate, it may also be desirable to treat it to remove any undesired impurities, such as dust particles before introducing it to the fermentation. For example, the gaseous substrate may be filtered or scrubbed using known methods.

[0354] It will be appreciated that for growth of the bacteria and CO-to-at least one or more terpene and/or precursor thereof to occur, in addition to the CO-containing substrate gas, a suitable liquid nutrient medium will need to be fed to the bioreactor. The substrate and media may be fed to the bioreactor in a continuous, batch or batch fed fashion. A nutrient medium will contain vitamins and minerals sufficient to permit growth of the micro-organism used. Anaerobic media suitable for fermentation to produce a terpene and/or a prescursor thereof using CO are known in the art. For example, suitable media are described Biebel (2001). In one embodiment of the invention the media is as described in the Examples section herein after.

[0355] The fermentation should desirably be carried out under appropriate conditions for the CO-to-the at least one or more terpene and/or precursor thereof fermentation to occur. Reaction conditions that should be considered include pressure, temperature, gas flow rate, liquid flow rate, media pH, media redox potential, agitation rate (if using a continuous stirred tank reactor), inoculum level, maximum gas substrate concentrations to ensure that CO in the liquid phase does not become limiting, and maximum product concentrations to avoid product inhibition.

[0356] In addition, it is often desirable to increase the CO concentration of a substrate stream (or CO partial pressure in a gaseous substrate) and thus increase the efficiency of fermentation reactions where CO is a substrate. Operating at increased pressures allows a significant increase in the rate of CO transfer from the gas phase to the liquid phase where it can be taken up by the micro-organism as a carbon source for the production of at least one or more terpene and/or precursor thereof. This in turn means that the retention time (defined as the liquid volume in the bioreactor divided by the input gas flow rate) can be reduced when bioreactors are maintained at elevated pressure rather than atmospheric pressure. The optimum reaction conditions will depend partly on the particular micro-organism of the invention used. However, in general, it is preferred that the fermentation be performed at pressure higher than ambient pressure. Also, since a given CO-to-at least one or more terpene and/or precursor thereof conversion rate is in part a function of the substrate retention time, and achieving a desired retention time in turn dictates the required volume of a bioreactor, the use of pressurized systems can greatly reduce the volume of the bioreactor required, and consequently the capital cost of the fermentation equipment. According to examples given in U.S. Pat. No. 5,593,886, reactor volume can be reduced in linear proportion to increases in reactor operating pressure, i.e. bioreactors operated at 10 atmospheres of pressure need only be one tenth the volume of those operated at 1 atmosphere of pressure.

[0357] By way of example, the benefits of conducting a gas-to-ethanol fermentation at elevated pressures has been described. For example, WO 02/08438 describes gas-to-ethanol fermentations performed under pressures of 30 psig and 75 psig, giving ethanol productivities of 150 g/l/day and 369 g/l/day respectively. However, example fermentations performed using similar media and input gas compositions at atmospheric pressure were found to produce between 10 and 20 times less ethanol per litre per day.

[0358] It is also desirable that the rate of introduction of the CO-containing gaseous substrate is such as to ensure that the concentration of CO in the liquid phase does not become limiting. This is because a consequence of CO-limited conditions may be that one or more product is consumed by the culture.

[0359] The composition of gas streams used to feed a fermentation reaction can have a significant impact on the efficiency and/or costs of that reaction. For example, O2 may reduce the efficiency of an anaerobic fermentation process. Processing of unwanted or unnecessary gases in stages of a fermentation process before or after fermentation can increase the burden on such stages (e.g. where the gas stream is compressed before entering a bioreactor, unnecessary energy may be used to compress gases that are not needed in the fermentation). Accordingly, it may be desirable to treat substrate streams, particularly substrate streams derived from industrial sources, to remove unwanted components and increase the concentration of desirable components.

[0360] In certain embodiments a culture of a bacterium of the invention is maintained in an aqueous culture medium. Preferably the aqueous culture medium is a minimal anaerobic microbial growth medium. Suitable media are known in the art and described for example in U.S. Pat. Nos. 5,173,429 and 5,593,886 and WO 02/08438, and as described in the Examples section herein after.

[0361] Terpenes and/or precursors thereof, or a mixed stream containing one or more terpenes, precursors thereof and/or one or more other products, may be recovered from the fermentation broth by methods known in the art, such as fractional distillation or evaporation, pervaporation, gas stripping and extractive fermentation, including for example, liquid-liquid extraction.

[0362] In certain preferred embodiments of the invention, the one or more terpene and/or precursor thereof and one or more products are recovered from the fermentation broth by continuously removing a portion of the broth from the bioreactor, separating microbial cells from the broth (conveniently by filtration), and recovering one or more products from the broth. Alcohols may conveniently be recovered for example by distillation. Acetone may be recovered for example by distillation. Any acids produced may be recovered for example by adsorption on activated charcoal. The separated microbial cells are preferably returned to the fermentation bioreactor. The cell free permeate remaining after any alcohol(s) and acid(s) have been removed is also preferably returned to the fermentation bioreactor. Additional nutrients (such as B vitamins) may be added to the cell free permeate to replenish the nutrient medium before it is returned to the bioreactor.

[0363] Also, if the pH of the broth was adjusted as described above to enhance adsorption of acetic acid to the activated charcoal, the pH should be re-adjusted to a similar pH to that of the broth in the fermentation bioreactor, before being returned to the bioreactor.

EXAMPLES

[0364] The invention will now be described in more detail with reference to the following non-limiting examples.

Example 1

Expression of Isoprene Synthase in C. autoethanogenum for Production of Isoprene from CO

[0365] The inventors have identified terpene biosynthesis genes in carboxydotrophic acetogens such as C. autoethanogenum and C. ljungdahlii. A recombinant organism was engineered to produce isoprene. Isoprene is naturally emitted by some plant such as poplar to protect its leave from UV radiation. Isoprene synthase (EC 4.2.3.27) gene of Poplar was codon optimized and introduced into a carboxydotrophic acetogen C. autoethanogenum to produce isoprene from CO. The enzyme takes key intermediate DMAPP (Dimethylallyl diphosphate) of terpenoid biosynthesis to isoprene in an irreversible reaction (FIG. 1).

Strains and Growth Conditions:

[0366] All subcloning steps were performed in E. coli using standard strains and growth conditions as described earlier (Sambrook et al, Molecular Cloning: A laboratory Manual, Cold Spring Harbour Labrotary Press, Cold Spring Harbour, 1989; Ausubel et al, Current protocols in molecular biology, John Wiley & Sons, Ltd., Hoboken, 1987).

[0367] C. autoethanogenum DSM10061 and DSM23693 (a derivative of DSM10061) were obtained from DSMZ (The German Collection of Microorganisms and Cell Cultures, Inhoffenstraβe 7 B, 38124 Braunschweig, Germany). Growth was carried out at 37° C. using strictly anaerobic conditions and techniques (Hungate, 1969, Methods in Microbiology, vol. 3B. Academic Press, New York: 117-132; Wolfe, 1971, Adv. Microb. Physiol., 6: 107-146). Chemically defined PETC media without yeast extract (Table 1) and 30 psi carbon monoxide containing steel mill waste gas (collected from New Zealand Steel site in Glenbrook, NZ; composition: 44% CO, 32% N₂, 22% CO₂, 2% H₂) as sole carbon and energy source was used.

TABLE-US-00003 TABLE 1 Media component Concentration per 1.0 L of media NH₄Cl 1 g KCl 0.1 g MgSO₄•7H₂O 0.2 g NaCl 0.8 g KH₂PO₄ 0.1 g CaCl₂ 0.02 g Trace metal solution 10 ml Wolfe's vitamin solution 10 ml Resazurin (2 g/L stock) 0.5 ml NaHCO₃ 2 g Reducing agent 0.006-0.008% (v/v) Distilled water Up to 1 L, pH 5.5 (adjusted with HCl) Wolfe's vitamin solution per L of Stock Biotin 2 mg Folic acid 2 mg Pyridoxine hydrochloride 10 mg Riboflavin 5 mg Nicotinic acid 5 mg Calcium D-(+)-pantothenate 5 mg Vitamin B₁₂ 0.1 mg p-Aminobenzoic acid 5 mg Lipoic acid 5 mg Thiamine 5 mg Distilled water To 1 L Trace metal solution per L of stock Nitrilotriacetic Acid 2 g MnSO₄•H₂O 1 g Fe (SO₄)₂(NH₄)₂•6H₂O 0.8 g CoCl₂•6H₂O 0.2 g ZnSO₄•7H₂O 0.2 mg CuCl₂•2H₂O 0.02 g NaMoO₄•2H₂O 0.02 g Na₂SeO₃ 0.02 g NiCl₂•6H₂O 0.02 g Na₂WO₄•2H₂O 0.02 g Distilled water To 1 L Reducing agent stock per 100 mL of stock NaOH 0.9 g Cystein•HCl 4 g Na₂S 4 g Distilled water To 100 mL

Construction of Expression Plasmid:

[0368] Standard Recombinant DNA and molecular cloning techniques were used in this invention (Sambrook J, Fritsch E F, Maniatis T: Molecular Cloning: A laboratory Manual, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, 1989; Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K: Current protocols in molecular biology. John Wiley & Sons, Ltd., Hoboken, 1987). The isoprene synthase of Poplar tremuloides (AAQ16588.1; GI:33358229) was codon-optimized (SEQ ID NO: 21) and synthesized. A promoter region of the Pyruvate:ferredoxin oxidoreductase of C. autoethanogenum (SEQ ID NO: 22) was used to express the gene.

[0369] Genomic DNA from Clostridium autoethanogenum DSM23693 was isolated using a modified method by Bertram and Dune (1989). A 100-ml overnight culture was harvested (6,000×g, 15 min, 4° C.), washed with potassium phosphate buffer (10 mM, pH 7.5) and suspended in 1.9 ml STE buffer (50 mM Tris-HCl, 1 mM EDTA, 200 mM sucrose; pH 8.0). 300 μl lysozyme (˜100,000 U) was added and the mixture was incubated at 37° C. for 30 min, followed by addition of 280 μl of a 10% (w/v) SDS solution and another incubation for 10 min. RNA was digested at room temperature by addition of 240 μl of an EDTA solution (0.5 M, pH 8), 20 μl Tris-HCl (1 M, pH 7.5), and 10 μl RNase A (Fermentas Life Sciences). Then, 100 μl Proteinase K (0.5 U) was added and proteolysis took place for 1-3 h at 37° C. Finally, 600 μl of sodium perchlorate (5 M) was added, followed by a phenol-chloroform extraction and an isopropanol precipitation. DNA quantity and quality was inspected spectrophotometrically. The Pyruvate:ferredoxin oxidoreductase promoter sequence was amplified by PCR using oligonucleotides Ppfor-NotI-F (SEQ ID NO: 23: AAGCGGCCGCAAAATAGTTGATAATAATGC) and Ppfor-NdeI-R (SEQ ID NO: 24: TACGCATATGAATTCCTCTCCTTTTCAAGC) using iProof High Fidelity DNA Polymerase (Bio-Rad Laboratories) and the following program: initial denaturation at 98° C. for 30 seconds, followed by 32 cycles of denaturation (98° C. for 10 seconds), annealing (50-62° C. for 30-120 seconds) and elongation (72° C. for 30-90 seconds), before a final extension step (72° C. for 10 minutes).

Construction of Isoprene Synthase Expression Plasmid:

[0370] Construction of an expression plasmid was performed in E. coli DH5α-T1^R (Invitrogen) and XL 1-Blue MRF' Kan (Stratagene). In a first step, the amplified P_pfor promoter region was cloned into the E. coli-Clostridium shuttle vector pMTL85141 (FJ797651.1; Nigel Minton, University of Nottingham; Heap et al., 2009) using NotI and NdeI restriction sites, generating plasmid pMTL85146. As a second step, ispS was cloned into pMTL85146 using restriction sites NdeI and EcoRI, resulting in plasmid pMTL 85146-ispS (FIG. 2, SEQ ID NO: 25).

Transformation and Expression in C. autoethanogenum

[0371] Prior to transformation, DNA was methylated in vivo in E. coli using a synthesized hybrid Type II methyltransferase (SEQ ID NO: 63) co-expressed on a methylation plasmid (SEQ ID NO: 64) designed from methyltransferase genes from C. autoethanogenum, C. ragsdalei and C. ljungdahlii as described in US patent 2011/0236941.

[0372] Both expression plasmid and methylation plasmid were transformed into same cells of restriction negative E. coli XL1-Blue MRF' Kan (Stratagene), which is possible due to their compatible Gram-(-) origins of replication (high copy ColE1 in expression plasmid and low copy p15A in methylation plasmid). In vivo methylation was induced by addition of 1 mM IPTG, and methylated plasmids were isolated using QIAGEN Plasmid Midi Kit (QIAGEN). The resulting mixture was used for transformation experiments with C. autoethanogenum DSM23693, but only the abundant (high-copy) expression plasmid has a Gram-(+) replication origin (repL) allowing it to replicate in Clostridia.

Transformation into C. autoethanogenum:

[0373] During the complete transformation experiment, C. autoethanogenum DSM23693 was grown in PETC media (Table 1) supplemented with 1 g/L yeast extract and 10 g/l fructose as well as 30 psi steel mill waste gas (collected from New Zealand Steel site in Glenbrook, NZ; composition: 44% CO, 32% N₂, 22% CO₂, 2% H₂) as carbon source.

[0374] To make competent cells, a 50 ml culture of C. autoethanogenum DSM23693 was subcultured to fresh media for 3 consecutive days. These cells were used to inoculate 50 ml PETC media containing 40 mM DL-threonine at an OD.sub.600nm of 0.05. When the culture reached an OD.sub.600nm of 0.4, the cells were transferred into an anaerobic chamber and harvested at 4,700×g and 4° C. The culture was twice washed with ice-cold electroporation buffer (270 mM sucrose, 1 mM MgCl₂, 7 mM sodium phosphate, pH 7.4) and finally suspended in a volume of 600 μl fresh electroporation buffer. This mixture was transferred into a pre-cooled electroporation cuvette with a 0.4 cm electrode gap containing 1 μg of the methylated plasmid mixture and immediately pulsed using the Gene pulser Xcell electroporation system (Bio-Rad) with the following settings: 2.5 kV, 600Ω, and 25 μF. Time constants of 3.7-4.0 ms were achieved. The culture was transferred into 5 ml fresh media. Regeneration of the cells was monitored at a wavelength of 600 nm using a Spectronic Helios Epsilon Spectrophotometer (Thermo) equipped with a tube holder. After an initial drop in biomass, the cells started growing again. Once the biomass has doubled from that point, the cells were harvested, suspended in 200 μl fresh media and plated on selective PETC plates (containing 1.2% Bacto® Agar (BD)) with appropriate antibiotics 4 μg/ml Clarithromycin or 15 μg/ml thiamphenicol. After 4-5 days of inoculation with 30 psi steel mill gas at 37° C., colonies were visible.

[0375] The colonies were used to inoculate 2 ml PETC media with antibiotics. When growth occurred, the culture was scaled up into a volume of 5 ml and later 50 ml with 30 psi steel mill gas as sole carbon source.

Confirmation of the Successful Transformation:

[0376] To verify the DNA transfer, a plasmid mini prep was performed from 10 ml culture volume using Zyppy plasmid miniprep kit (Zymo). Since the quality of the isolated plasmid was not sufficient for a restriction digest due to Clostridial exonuclease activity [Burchhardt and Dune, 1990], a PCR was performed with the isolated plasmid with oligonucleotide pairs colE1-F (SEQ ID NO: 65: CGTCAGACCCCGTAGAAA) plus colE1-R (SEQ ID NO: 66: CTCTCCTGTTCCGACCCT). PCR was carried out using iNtRON Maximise Premix PCR kit (Intron Bio Technologies) with the following conditions: initial denaturation at 94° C. for 2 minutes, followed by 35 cycles of denaturation (94° C. for 20 seconds), annealing (55° C. for 20 seconds) and elongation (72° C. for 60 seconds), before a final extension step (72° C. for 5 minutes).

[0377] To confirm the identity of the clones, genomic DNA was isolated (see above) from 50 ml cultures of C. autoethanogenum DSM23693. A PCR was performed against the 16s rRNA gene using oligonucleotides fD1 (SEQ ID NO: 67: CCGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG) and rP2 (SEQ ID NO: 68: CCCGGGATCCAAGCTTACGGCTACCTTGTTACGACTT) [Weisberg et al., 1991] and iNtRON Maximise Premix PCR kit (Intron Bio Technologies) with the following conditions: initial denaturation at 94° C. for 2 minutes, followed by 35 cycles of denaturation (94° C. for 20 seconds), annealing (55° C. for 20 seconds) and elongation (72° C. for 60 seconds), before a final extension step (72° C. for 5 minutes). Sequencing results were at least 99.9% identity against the 16s rRNA gene (rrsA) of C. autoethanogenum (Y18178, GI:7271109).

Expression of Isoprene Synthase Gene

[0378] qRT-PCR experiments were performed to confirm successful expression of introduced isoprene synthase gene in C. autoethanogenum.

[0379] A culture harboring isoprene synthase plasmid pMTL 85146-ispS and a control culture without plasmid was grown in 50 mL serum bottles and PETC media (Table 1) with 30 psi steel mill waste gas (collected from New Zealand Steel site in Glenbrook, NZ; composition: 44% CO, 32% N₂, 22% CO₂, 2% H₂) as sole energy and carbon source. 0.8 mL samples were taken during logarithmic growth phase at an OD.sub.600nm of around 0.5 and mixed with 1.6 mL RNA protect reagent (Qiagen). The mixture was centrifuged (6,000×g, 5 min, 4° C.), and the cell sediment snap frozen in liquid nitrogen and stored at -80° C. until RNA extraction. Total RNA was isolated using RNeasy Mini Kit (Qiagen) according to protocol 5 of the manual. Disruption of the cells was carried out by passing the mixture through a syringe 10 times, and eluted in 50 μL of RNase/DNase-free water. After DNase I treatment using DNA-free® Kit (Ambion), the reverse transcription step was then carried out using SuperScript III Reverse Transcriptase Kit (Invitrogen, Carlsbad, Calif., USA). RNA was checked using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, Calif., USA), Qubit Fluorometer (Invitrogen, Carlsbad, Calif., USA) and by gel electrophoresis. A non-RT control was performed for every oligonucleotide pair. All qRT-PCR reactions were performed in duplicate using a MyiQ® Single Colour Detection System (Bio-Rad Labratories, Carlsbad, Calif., USA) in a total reaction volume of 15 μL with 25 ng of cDNA template, 67 nM of each oligonucleotide (Table 2), and 1×iQ® SYBR® Green Supermix (Bio-Rad Labratories, Carlsbad, Calif., USA). The reaction conditions were 95° C. for 3 min, followed by 40 cycles of 95° C. for 15 s, 55° C. for 15 s and 72° C. for 30 s. For detection of oligonucleotide dimerisation or other artifacts of amplification, a melting-curve analysis was performed immediately after completion of the qPCR (38 cycles of 58° C. to 95° C. at 1° C./s). Two housekeeping genes (guanylate kinase and formate tetrahydrofolate ligase) were included for each cDNA sample for normalization. Determination of relative gene expression was conducted using Relative Expression Software Tool (REST©) 2008 V2.0.7 (38). Dilution series of cDNA spanning 4 log units were used to generate standard curves and the resulting amplification efficiencies to calculate concentration of mRNA.

TABLE-US-00004 TABLE 2 Oligonucleotides for qRT-PCR Oligo- SEQ Target nucleotide DNA Sequence ID Name (5' to 3') NO: Guanylate kinase (gnk) GnK-F TCAGGACCTTCTGGAACTGG 108 GnK-R ACCTCCCCTTTTCTTGGAGA 109 Formate tetrahydrofolate FoT4L-F CAGGTTTCGGTGCTGACCTA 110 ligase (FoT4L) FoT4L-F AACTCCGCCGTTGTATTTCA 111 Isoprene Synthase ispS-F AGG CTG AAT TTC TTA CAC 69 TTC TTG A ispS-R GTA ACT CCA TCA AAT CCT 70 CCA CTA C

[0380] While no amplification was observed with the wild-type strain using oligonucleotide pair ispS, a signal with the ispS oligonucleotide pair was measured for the strain carrying plasmid pMTL 85146-ispS, confirming successful expression of the ispS gene.

Example 2

Expression of Isopentenyl-Diphosphate Delta-Isomerase to Convert Between Key Terpene Precursors DMAPP (Dimethylallyl Diphosphate) and IPP (Isopentenyl Diphosphate)

[0381] Availability and balance of precursors DMAPP (Dimethylallyl diphosphate) and IPP (Isopentenyl diphosphate) is crucial for production of terpenes. While the DXS pathway synthesizes both IPP and DMAPP equally, in the mevalonate pathway the only product is IPP. Production of isoprene requires only the precursor DMAPP to be present in conjunction with an isoprene synthase, while for production of higher terpenes and terpenoids, it is required to have equal amounts of IPP and DMAPP available to produce Geranyl-PP by a geranyltransferase.

Construction of Isopentenyl-Diphosphate Delta-Isomerase Expression Plasmid:

[0382] An Isopentenyl-diphosphate delta-isomerase gene idi from C. beijerinckii (Gene ID:5294264), encoding an Isopentenyl-diphosphate delta-isomerase (YP_--001310174.1), was cloned downstream of ispS. The gene was amplified using oligonucleotide Idi-Cbei-SacI-F (SEQ ID NO: 26: GTGAGCTCGAAAGGGGAAATTAAATG) and Idi-Cbei-KpnI-R (SEQ ID NO: 27: ATGGTACCCCAAATCTTTATTTAGACG) from genomic DNA of C. beijerinckii NCIMB8052, obtained using the same method as described above for C. autoethanogenum. The PCR product was cloned into vector pMTL 85146-ispS using Sad and KpnI restriction sites to yield plasmid pMTL85146-ispS-idi (SEQ ID NO: 28). The antibiotic resistance marker was exchanged from catP to ermB (released from vector pMTL82254 (FJ797646.1; Nigel Minton, University of Nottingham; Heap et al., 2009) using restriction enzymes PmeI and FseI to form plasmid pMTL85246-ispS-idi (FIG. 3).

[0383] Transformation and expression in C. autoethanogenum was carried out as described for plasmid pMTL 85146-ispS. After successful transformation, growth experiment was carried out in 50 mL 50 mL serum bottles and PETC media (Table 1) with 30 psi steel mill waste gas (collected from New Zealand Steel site in Glenbrook, NZ; composition: 44% CO, 32% N₂, 22% CO₂, 2% H₂) as sole energy and carbon source. To confirm that the plasmid has been successfully introduced, plasmid mini prep DNA was carried out from transformants as described previously. PCR against the isolated plasmid using oligonucleotide pairs that target colE1 (colE1-F: SEQ ID NO: 65: CGTCAGACCCCGTAGAAA and colE1-R: SEQ ID NO: 66: CTCTCCTGTTCCGACCCT), ermB (ermB-F: SEQ ID NO: 106: TTTGTAATTAAGAAGGAG and ermB-R: SEQ ID NO: 107: GTAGAATCCTTCTTCAAC) and idi (Idi-Cbei-SacI-F: SEQ ID NO: 26: GTGAGCTCGAAAGGGGAAATTAAATG and Idi-Cbei-KpnI-R: SEQ ID NO: 27: ATGGTACCCCAAATCTTTATTTAGACG) confirmed transformation success (FIG. 8). Similarly, genomic DNA from these transformants were extracted, and the resulting 16s rRNA amplicon using oligonucleotides fD1 and rP2 (see above) confirmed 99.9% identity against the 16S rRNA gene of C. autoethanogenum (Y18178, GI:7271109).

[0384] Successful confirmation of gene expression was carried out as described above using a oligonucleotide pair against Isopentenyl-diphosphate delta-isomerase gene idi (idi-F, SEQ ID NO: 71: ATA CGT GCT GTA GTC ATC CAA GAT A and idiR, SEQ ID NO: 72: TCT TCA AGT TCA CAT GTA AAA CCC A) and a sample from a serum bottle growth experiment with C. autoethanogenum carrying plasmid pMTL 85146-ispS-idi. A signal for the isoprene synthase gene ispS was also observed (FIG. 14).

Example 3

Overexpression of DXS Pathway

[0385] To improve flow through the DXS pathway, genes of the pathway were overexpressed. The initial step of the pathway, converting pyruvate and D-glyceraldehyde-3-phosphate (G3P) into deoxyxylulose 5-phosphate (DXP/DXPS/DOXP), is catalyzed by an deoxyxylulose 5-phosphate synthase (DXS).

Construction of DXS Overexpression Expression Plasmid:

[0386] The dxs gene of C. autoethanogenum was amplified from genomic DNA with oligonucleotides Dxs-SalI-F (SEQ ID NO: 29: GCAGTCGACTTTATTAAAGGGATAGATAA) and Dxs-XhoI-R (SEQ ID NO: 30: TGCTCGAGTTAAAATATATGACTTACCTCTG) as described for other genes above. The amplified gene was then cloned into plasmid pMTL85246-ispS-idi with SalI and XhoI to produce plasmid pMTL85246-ispS-idi-dxs (SEQ ID NO: 31 and FIG. 4). DNA sequencing using oligonucleotides given in Table 3 confirmed successful cloning of ispS, idi, and dxs without mutations (FIG. 5). The ispS and idi genes are as described in example 1 and 2 respectively.

TABLE-US-00005 TABLE 3 Oligonucleotides for sequencing SEQ Oligonucleotide ID Name DNA Sequence (5' to 3') NO: M13R CAGGAAACAGCTATGAC 32 Isoprene-seq1 GTTATTCAAGCTACACCTTT 33 Isoprene-seq2 GATTGGTAAAGAATTAGCTG 34 Isoprene-seq3 TCAAGAAGCTAAGTGGCT 35 Isoprene-seq4 CTCACCGTAAAGGAACA 36 Isoprene-seq5 GCTAGCTAGAGAAATTAGAA 37 Isoprene-seq6 GGAATGGCAAAATATCTTGA 38 Isoprene-seq7 GAAACACATCAGGGAATATT 39

Transformation and Expression in C. autoethanogenum

[0387] Transformation and expression in C. autoethanogenum was carried out as described for plasmid pMTL 85146-ispS. After successful transformation, a growth experiment was carried out in 50 mL 50 mL serum bottles and PETC media (Table 1) with 30 psi steel mill waste gas (collected from New Zealand Steel site in Glenbrook, NZ; composition: 44% CO, 32% N₂, 22% CO₂, 2% H₂) as sole energy and carbon source. Confirmation of gene expression was carried out as described above from a sample collected at OD.sub.600nm=0.75. Oligonucleotide pair dxs-F (SEQ ID NO: 73: ACAAAGTATCTAAGACAGGAGGTCA) and dxs-R (SEQ ID NO: 74: GATGTCCCACATCCCATATAAGTTT) was used to measure expression of gene dxs in both wild-type strain and strain carrying plasmid pMTL 85146-ispS-idi-dxs. mRNA levels in the strain carrying the plasmid were found to be over 3 times increased compared to the wild-type (FIG. 15). Biomass was normalized before RNA extraction.

Example 4

Introduction and Expression of Mevalonate Pathway

[0388] The first step of the mevalonate pathway (FIG. 7) is catalyzed by a thiolase that converts two molecules of acetyl-CoA into acetoacetyl-CoA (and HS-CoA). This enzyme has been successfully expressed in carboxydotrophic acetogens Clostridium autoethanogenum and C. ljungdahlii by the same inventors (US patent 2011/0236941). Constructs for the remaining genes of the mevalonate pathway have been designed.

Construction of Mevalonate Expression Plasmid:

[0389] Standard recombinant DNA and molecular cloning techniques were used (Sambrook, J., and Russell, D., Molecular cloning: A Laboratory Manual 3rd Ed., Cold Spring Harbour Lab Press, Cold Spring Harbour, N.Y., 2001). The three genes required for mevalonate synthesis via the upper part of the mavalonate pathway, i.e., thiolase (thlA/vraB), HMG-CoA synthase (HMGS) and HMG-CoA reductase (HMGR), were codon-optimised as an operon (P.sub.ptaack-thlA/vraB-HMGS-P_atp-HMGR).

[0390] The Phosphotransacetylase/Acetate kinase operon promoter (P.sub.pta-ack) of C.

[0391] autoethanogenum (SEQ ID NO: 61) was used for expression of the thiolase and HMG-CoA synthase while a promoter region of the ATP synthase (P_atp) of C. autoethanogenum was used for expression of the HMG-CoA reductase. Two variants of thiolase, thlA from Clostridium acetobutylicum and vraB from Staphylococcus aureus, were synthesised and flanked by NdeI and EcoRI restriction sites for further sub-cloning. Both HMG-CoA synthase (HMGS) and HMG-CoA reductase (HMGR) were synthesised from Staphylococcus aureus and flanked by EcoRI-SacI and KpnI-XbaI restriction sites respectively for further sub-cloning. All optimized DNA sequences used are given in Table 4.

TABLE-US-00006 TABLE 4 Sequences of mevalonate expression plasmid SEQ ID Description Source NO: Thiolase (thlA) Clostridium acetobutylicum ATCC 824; 40 NC_003030.1; GI: 1119056 Acetyl-CoA c- Staphylococcus aureus subsp. aureus 41 acetyltransferase Mu50; (vraB) NC_002758.2; region: 652965..654104; including GI: 15923566 3-hydroxy-3- Staphylococcus aureus subsp. aureus 42 methylglutaryl-CoA Mu50; NC_002758.2; region: synthase (HMGS) 2689180..2690346; including GI: 15925536 Hydroxymethyl- Staphylococcus aureus subsp. aureus 43 glutaryl-CoA Mu50; NC_002758.2; region: reductase (HMGR) complement(2687648..2688925); including GI: 15925535 Phospho- Clostridium autoethanogenum DSM10061 44 transacetylase- acetate kinase operon (P.sub.pta-ack) ATP synthase Clostridium autoethanogenum DSM10061 45 promoter (P_atp)

[0392] The ATP synthase promoter (P_atp) together with the hydroxymethylglutaryl-CoA reductase (HMGR) was amplified using oligonucleotides pUC57-F (SEQ ID NO: 46: AGCAGATTGTACTGAGAGTGC) and pUC57-R (SEQ ID NO: 47: ACAGCTATGACCATGATTACG) and pUC57-Patp-HMGR as a template. The 2033 bp amplified fragment was digested with Sad and XbaI and ligated into the E. coli-Clostridium shuttle vector pMTL 82151 (FJ7976; Nigel Minton, University of Nottingham, UK; Heap et al., 2009, J Microbiol Methods. 78: 79-85) resulting in plasmid pMTL 82151-Patp-HMGR (SEQ ID NO: 76).

[0393] 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) was amplified from the codon-synthesised plasmid pGH-seq3.2 using oligonucleotides EcoRI-HMGS_F (SEQ ID NO: 77: AGCCGTGAATTCGAGGCTTTTACTAAAAACA) and EcoRI-HMGS_R (SEQ ID NO: 78: AGGCGTCTAGATGTTCGTCTCTACAAATAATT). The 1391 bp amplified fragment was digested with Sad and EcoRI and ligated into the previously created plasmid pMTL 82151-Patp-HMGR to give pMTL 82151-HMGS-Patp-HMGR (SEQ ID NO: 79). The created plasmid pMTL 82151-HMGS-Patp-HMGR (SEQ ID NO: 79) and the 1768 bp codon-optimised operon of P.sub.ptaack-thlA/vraB were both cut with NotI and EcoRI. A ligation was performed and subsequently transformed into E. coli XL1-Blue MRF' Kan resulting in plasmid pMTL8215-P.sub.ptaack-thlA/vraB-HMGS-P_atp-HMGR (SEQ ID NO: 50).

[0394] The five genes required for synthesis of terpenoid key intermediates from mevalonate via the bottom part of the mevalonate pathway, i.e., mevalonate kinase (MK), phosphomevalonate kinase (PMK), mevalonate diphosphate decarboxylase (PMD), isopentenyl-diphosphate delta-isomerase (idi) and isoprene synthase (ispS) were codon-optimised by ATG:Biosynthetics GmbH (Merzhausen, Germany). Mevalonate kinase (MK), phosphomevalonate kinase (PMK) and mevalonate diphosphate decarboxylase (PMD) were obtained from Staphylococcus aureus.

[0395] The promoter region of the RNF Complex (P_rnf) of C. autoethanogenum (SEQ ID NO: 62) was used for expression of mevalonate kinase (MK), phosphomevalonate kinase (PMK) and mevalonate diphosphate decarboxylase (PMD), while the promoter region of the Pyruvate:ferredoxin oxidoreductase (P_for) of C. autoethanogenum (SEQ ID NO: 22) was used for expression of isopentenyl-diphosphate delta-isomerase (idi) and isoprene synthase (ispS). All DNA sequences used are given in Table 5. The codon-optimised Prnf-MK was amplified from the synthesised plasmid pGH-Prnf-MK-PMK-PMD with oligonucleotides NotI-XbaI-Prnf-MK F (SEQ ID NO: 80: ATGCGCGGCCGCTAGGTCTAGAATATCGATACAGATAAAAAAATATATAATACA G) and SalI-Prnf-MK R (SEQ ID NO: 81: TGGTTCTGTAACAGCGTATTCACCTGC). The amplified gene was then cloned into plasmid pMTL83145 (SEQ ID NO: 49) with NotI and SalI to produce plasmid pMTL8314-Prnf-MK (SEQ ID NO: 82). This resulting plasmid and the 2165 bp codon optimised fragment PMK-PMD was subsequently digested with SalI and HindIII. A ligation was performed resulting in plasmid pMTL 8314-Prnf-MK-PMK-PMD (SEQ ID NO: 83).

[0396] The isoprene expression plasmid without the mevalonate pathway was created by ligating the isoprene synthase (ispS) flanked by restriction sites AgeI and NheI to the previously created farnesene plasmid, pMTL 8314-Prnf-MK-PMK-PMD-Pfor-idi-ispA-FS (SEQ ID NO:91) to result in plasmid pMTL8314-Prnf-MK-PMK-PMD-Pfor-idi-ispS (SEQ ID NO:84). The final isoprene expression plasmid, pMTL 8314-Pptaack-thlA-HMGS-Patp-HMGR-Prnf-MK-PMK-PMD-Pfor-idi-ispS (SEQ ID NO.: 58, FIG. 10) is created by ligating the 4630 bp fragment of Pptaack-thlA-HMGS-Patp-HMGR from pMTL8215-Pptaack-thlA-HMGS-Patp-HMGR (SEQ ID NO: 50) with pMTL 8314-Prnf-MK-PMK-PMD-Pfor-idi-ispS (SEQ ID NO: 84) using restriction sites NotI and XbaI.

TABLE-US-00007 TABLE 5 Sequences of isoprene expression plasmid from mevalonate pathway SEQ ID Description Source NO: Mevalonate kinase Staphylococcus aureus subsp. aureus 51 (MK) Mu50; NC_002758.2; region: 665080..665919; including GI: 15923580 Phosphomevalonate Staphylococcus aureus subsp. aureus 52 kinase (PMK) Mu50; NC_002758.2; region: 666920..667996; including GI: 15923582 Mevalonate Staphylococcus aureus subsp. aureus 53 diphosphate Mu50; NC_002758.2; region: decarboxylase 665924..666907; including GI: 15923581 (PMD) Isoprene synthase isoprene synthase of Poplar tremuloides 21 (isIS) (AAQ16588.1; GI: 33358229) Isopentenyl- Clostridium beijerinckii NCIMB 8052; 54 diphosphate YP_001310174.1; region: delta-isomerase (idi) complement(3597793..3598308); including GI: 150017920 RNF Complex Clostridium autoethanogenum DSM10061 55 promoter (P_rnf)

Example 5

Introduction of Farnesene Synthase in C. autoethanogenum for Production of Farnesene from CO Via the Mevalonate Pathway

[0397] Instead of producing isoprene directly from terpenoid key intermediates IPP and DMAPP then using this to synthesise longer chain terpenes, it is also possible to synthesise longer chain terpenes, such as C10 Monoterpenoids or C15 Sesquiterpenoids, directly via a geranyltransferase (see Table 6). From C15 Sesquiterpenoid building block farnesyl-PP it is possible to produce farnesene, which, similarly to ethanol, can be used as a transportation fuel.

Construction of Farnesene Expression Plasmid

[0398] The two genes required for farnesene synthesis from IPP and DMAPP via the mevalonate pathway, i.e., geranyltranstransferase (ispA) and alpha-farnesene synthase (FS) were codon-optimised. Geranyltranstransferase (ispA) was obtained from Escherichia coli str. K-12 substr. MG1655 and alpha-farnesene synthase (FS) was obtained from Malus×domestica. All DNA sequences used are given in Table 6. The codon-optimised idi was amplified from the synthesised plasmid pMTL83245-Pfor-FS-idi (SEQ ID NO: 85) with via the mevalonate pathways idi_F (SEQ ID NO: 86: AGGCACTCGAGATGGCAGAGTATATAATAGCAGTAG) and idi R2 (SEQ ID NO:87: AGGCGCAAGCTTGGCGCACCGGTTTATTTAAATATCTTATTTTCAGC). The amplified gene was then cloned into plasmid pMTL83245-Pfor with XhoI and HindIII to produce plasmid pMTL83245-Pfor-idi (SEQ ID NO: 88). This resulting plasmid and the 1754 bp codon optimised fragment of farnesene synthase (FS) was subsequently digested with HindIII and NheI. A ligation was performed resulting in plasmid pMTL83245-Pfor-idi-FS (SEQ ID NO: 89). The 946 bp fragment of ispA and pMTL83245-Pfor-idi-FS was subsequently digested with AgeI and HindIII and ligated to create the resulting plasmid pMTL83245-Pfor-idi-ispA-FS (SEQ ID NO: 90). The farnesene expression plasmid without the upper mevalonate pathway was created by ligating the 2516 bp fragment of Pfor-idi-ispA-FS from pMTL83245-Pfor-idi-ispA-FS to pMTL 8314-Prnf-MK-PMK-PMD to result in plasmid pMTL 8314-Prnf-MK-PMK-PMD-Pfor-idi-ispA-FS (SEQ ID NO: 91). The final farnesene expression plasmid pMTL83145-thlA-HMGS-Patp-HMGR-Prnf-MK-PMK-PMD-Pfor-idi-ispA-FS (SEQ ID NO: 59 and FIG. 18) is created by ligating the 4630 bp fragment of Pptaack-thlA-HMGS-Patp-HMGR from pMTL8215-Pptaack-thlA-HMGS-Patp-HMGR (SEQ ID NO: 50) with pMTL 8314-Prnf-MK-PMK-PMD-Pfor-idi-ispA-FS (SEQ ID NO: 91) using restriction sites NotI and XbaI.

TABLE-US-00008 TABLE 6 Sequences of farnesene expression plasmid from mevalonate pathway SEQ ID Description Source NO: Geranyltranstransferase Escherichia coli str. K-12 substr. 56 (ispA) MG1655; NC_000913.2; region: complement(439426..440325); including GI: 16128406 Alpha-farnesene synthase Malus x domestica; 57 (FS) AY787633.1; GI: 60418690

Transformation into C. autoethanogenum

[0399] Transformation and expression in C. autoethanogenum was carried out as described in example 1.

Confirmation of Successful Transformation

[0400] The presence of pMTL8314-Prnf-MK-PMK-PMD-Pfor-idi-ispA-FS (SEQ ID NO: 59) was confirmed by colony PCR using oligonucleotides repHF (SEQ ID NO: 92:AAGAAGGGCGTATATGAAAACTTGT) andcatR (SEQ ID NO: 93: TTCGTTTACAAAACGGCAAATGTGA) which selectively amplifies a portion of the garm+ve perplicon and most of the cat gene on the pMTL831xxx series palsmids. Yielding a band of 1584 bp (FIG. 16).

Expression of Lower Mevalonate Pathway in C. autoethanogenum

[0401] Confirmation of expression of the lower mevalonate pathway genes Mevalonate kinase (MK SEQ ID NO: 51), Phosphomevalonate Kinase (PMK SEQ ID NO: 52), Mevalonate Diphosphate Decarboxylase (PMD SEQ ID NO: 53), Isopentyl-diphosphate

[0402] Delta-isomerase (idi; SEQ ID NO: 54), Geranyltranstransferase (ispA; SEQ ID NO: 56) and Farnesene synthase (FS SEQ ID NO: 57) was done as described above in example 1. Using oligonucleotides listed in table 7.

TABLE-US-00009 TABLE 7 List of oligonucleotides used for the detection of expression of the genes in the lower mevalonate pathway carried on plasmid pMTL8314Prnf-MK-PMK-PMD-Pfor-idi-ispA-FS (SEQ ID NO: 91) SEQ ID Oligonucleotide NO: Target Name DNA Sequence (5' to 3') NO. Mevalonate kinase MK-RTPCR-F GTGCTGGTAGAGGTGGTTCA 94 MK-RTPCR-R CCAAGTATGTGCTGCACCAG 95 Phosphomevalonate PMK-RTPCR-F ATATCAGACCCACACGCAGC 96 Kinase PMK-RTPCR-R AATGCTTCATTGCTATGTCACATG 97 Mevalonate PMD-RTPCR- GCAGAAGCAAAGGCAGCAAT 98 Diphosphate F Decarboxylase PMD-RTPCR- TTGATCCAAGATTTGTAGCATGC 99 R Isopentyl-diphosphate idi-RTPCR-F GGACAAACACTTGTTGTAGTCACC 100 Delta-isomerase idi-RTPCR-R TCAAGTTCGCAAGTAAATCCCA 101 Geranyltranstransferase ispA-RTPCR-F ACCAGCAATGGATGACGATG 102 ispA-RTPCR-R AGTTTGTAAAGCGTCACCTGC 103 Farnesene synthase FS-RTPCR-F AAGCTAGTAGATGGTGGGCT 104 FS-RTPCR-R AATGCTACACCTACTGCGCA 105

[0403] Rt-PCR data confirming expression of all genes in the lower mevalonate pathway is shown in FIG. 18, this data is also summarised in Table 8.

TABLE-US-00010 TABLE 8 Avarage CT values for the genes genes Mevalonate kinase (MK SEQ ID NO: 51), Phosphomevalonate Kinase (PMK SEQ ID NO: 52), Mevalonate Diphosphate Decarboxylase (PMD SEQ ID NO: 53), Isopentyl-diphosphate Delta-isomerase (idi SEQ ID NO: 54), Geranyltranstransferase (ispA SEQ ID NO: 56) and Farnesene synthase (FS SEQ ID NO: 57). for two independent samples taken from the two starter cultures for the mevalonate feeding experiment (see below). Gene Sample 1 (Ct Mean) Sample 2 (Ct Mean) MK 21.9 20.82 PMK 23.64 22.81 PMD 24 22.83 Idi 24.23 27.54 ispA 23.92 23.22 FS 21.28 (single Ct) 21.95 (single Ct) HK (rho) 31.5 28.88

Production of Alpha-Farnesene from Mevalonate

[0404] After conformation of successfully transformed of the plasmid pMTL8314-Prnf-MK-PMK-PMD-Pfor-idi-ispA-FS, a growth experiment was carried out in 50 ml PETC media (Table 1) in 250 ml serum bottles with 30 psi Real Mill Gas (collected from New Zealand Steel site in Glenbrook, NZ; composition: 44% CO, 32% N₂, 22% CO₂, 2% H₂) as sole energy and carbon source. All cultures were incubated at 37° C. on an orbital shaker adapted to hold serum bottles. Transformants were first grown up to an OD600 of ˜0.4 before being subcultured into fresh media supplemented with 1 mM mevalonic acid. Controls without mevalonic acid were set up at the same time from the same culture. Samples for GC-MS (Gas Chromatography--Mass Spectroscopy) were taken at each time point. FIG. 17 shows a representative growth curve for 2 control cultures and two cultures fed 1 mM mevalonate. Farnesene was detected in the samples taken at 66 h and 90 h after start of experiment (FIG. 19-21).

Detection of Alpha-Farnesene by Gas Chromatography--Mass Spectroscopy

[0405] For GC-MS detection of alpha-farnesene hexane extraction was performed on 5 ml of culture by adding 2 ml hexane and shaking vigorously to mix in a sealed glass balch tube. The tubes were then incubated in a sonicating water bath for 5 min to encourage phase separation. 400 μl hexane extract were transferred to a GC vail and loaded on to the auto loader. The samples was analysed on a VARIAN GC3800 MS4000 iontrap GC/MS (Varian Inc, CA, USA. Now Agilent Technologies) with a EC-1000 column 0.25 μm film thickness (Grace Davidson, OR, USA) Varian MS workstation (Varian Inc, Ca. Now Agilent Technologies, CA, USA) and NIST MS Search 2.0 (Agilent Technologies, CA, USA). Injection volume of 1 μl with Helium carrier gas flow rate of 1 ml per min.

[0406] The invention has been described herein, with reference to certain preferred embodiments, in order to enable the reader to practice the invention without undue experimentation. However, a person having ordinary skill in the art will readily recognise that many of the components and parameters may be varied or modified to a certain extent or substituted for known equivalents without departing from the scope of the invention. It should be appreciated that such modifications and equivalents are herein incorporated as if individually set forth. Titles, headings, or the like are provided to enhance the reader's comprehension of this document, and should not be read as limiting the scope of the present invention.

[0407] The entire disclosures of all applications, patents and publications, cited above and below, if any, are hereby incorporated by reference. However, the reference to any applications, patents and publications in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0408] Throughout this specification and any claims which follow, unless the context requires otherwise, the words "comprise", "comprising" and the like, are to be construed in an inclusive sense as opposed to an exclusive sense, that is to say, in the sense of "including, but not limited to."

Sequence CWU 1

1

11111875DNAClostridium autoethanogenum 1atgagtaatt tattagataa ttataaagat ataaatgacg taaagaagat gtcgttaaat 60gataaaaaaa agctagctag agaaattaga aaatttttaa tagacaaagt atctaagaca 120ggaggtcatt tggcgtctaa cttaggggtt gtggagctca ctttgagttt atttagtgta 180tttgatctaa attatgataa acttatatgg gatgtgggac atcaggctta tgtgcataaa 240atcctcacgg gaagaaagga taaatttgat actttaaggc aatttggagg attaagtgga 300tttcctaaaa ggtgcgaaag tatatatgat tttttcgaaa cagggcatag tagtacttca 360atatctgcag cacttggaat ggctagggct agagatttaa agcatgagaa atataatgtt 420gttgcagtta taggagatgg agcacttact ggaggtatgg cactagaggc cctaaatgat 480gtaggttata gaaaaactaa gcttataata atattaaatg ataatcaaat gtctatagga 540aaaaatgtag gtggagtatc taaatattta aataaactta gagtggaccc taagtataat 600aaatttaaag cggatgtaga agctaaatta aaaaagatac ctaatatagg aaaaggaatg 660gcaaaatatc ttgaaaaggt aaaaaatgga ataaaacaaa tggtagttcc tggaatgttt 720tttgaagata tgggaattaa atatttagga ccaatagatg gtcataatat aaaagaactt 780acagacgtac tcgcttctgc aaaagacata caaggtccag ttattataca tataataact 840aagaaaggaa aaggatatga atttgcagaa aaaaatccag gtaaattcca tggaataggg 900ccttttaatt gcgccaatgg tgaactggat gctggatctt caaatactta ttccaaggcc 960tttggaaatg aaatggtaaa gctagcagaa aaagacgata gaatagtggc tataactgca 1020gccatgaggg atggaacagg tcttaaaagt ttttctcaaa agtttcctga aaggtttttt 1080gatgtgggaa tagcagaaca gcatgctgta accctggcag ctggaatggc acaggcaaat 1140ttaaaacctg tatttgcagt ttactctact tttcttcaaa gagcttatga tcaacttatt 1200catgatgtat gtatgcaaaa acttccagta gtttttgctg tagatagggc cggcattgta 1260ggagaagatg gtgaaacaca tcagggaata tttgatttat cttacttaac ggaaatgcca 1320catatgacgc ttatgtctcc taaatgtata gatgaacttc catatatgtt aaaatgggca 1380ttaggccaga gttttcctgt agctataagg tatccaaggg gaggagatag tgtatgtctc 1440aatcccgtag aaaattttaa acttggaaag tgggactgta tttcaaatga aggcagtgta 1500gcaataattg ctcagggtaa aatggtacaa aatgcagtgt tagcaggaaa aaaacttaaa 1560gaaaagggta tagatgtaag gattataagt gcatgtttta ttaagccgct ggacaaggaa 1620atgttaaaca ggttagttga agaaagtgta actatcgtta ctgttgaaga caatgtaata 1680agaggaggat taggatccta tatattagaa tatgtaaata aattaaataa aaaagtaaaa 1740ataataaact tagggtttga tgataagttt gtacagcatg gaaaatccga tattttgtat 1800aagctgtatg gtttggatcc taaaggtatc gtaaatagtg tacttgaagc agcagaggta 1860agtcatatat tttaa 18752627PRTClostridium autoethanogenum 2Met Ser Asn Leu Leu Asp Asn Tyr Lys Asp Ile Asn Asp Val Lys Lys 1 5 10 15 Met Ser Leu Asn Asp Lys Lys Lys Leu Ala Arg Glu Ile Arg Lys Phe 20 25 30 Leu Ile Asp Lys Val Ser Lys Thr Gly Gly His Leu Ala Ser Asn Leu 35 40 45 Gly Val Val Glu Leu Thr Leu Ser Leu Phe Ser Val Phe Asp Leu Asn 50 55 60 Tyr Asp Lys Leu Ile Trp Asp Val Gly His Gln Ala Tyr Val His Lys 65 70 75 80 Ile Leu Thr Gly Arg Lys Asp Lys Phe Asp Thr Leu Arg Gln Phe Gly 85 90 95 Gly Leu Ser Gly Phe Pro Lys Arg Cys Glu Ser Ile Tyr Asp Phe Phe 100 105 110 Glu Thr Gly His Ser Ser Thr Ser Ile Ser Ala Ala Leu Gly Met Ala 115 120 125 Arg Ala Arg Asp Leu Lys His Glu Lys Tyr Asn Val Val Ala Val Ile 130 135 140 Gly Asp Gly Ala Leu Thr Gly Gly Met Ala Leu Glu Ala Leu Asn Asp 145 150 155 160 Val Gly Tyr Arg Lys Thr Lys Leu Ile Ile Ile Leu Asn Asp Asn Gln 165 170 175 Met Ser Ile Gly Lys Asn Val Gly Gly Val Ser Lys Tyr Leu Asn Lys 180 185 190 Leu Arg Val Asp Pro Lys Tyr Asn Lys Phe Lys Ala Asp Val Glu Ala 195 200 205 Lys Leu Lys Lys Ile Pro Asn Ile Gly Lys Gly Met Ala Lys Tyr Leu 210 215 220 Glu Lys Val Lys Asn Gly Ile Lys Gln Met Val Val Pro Gly Met Phe 225 230 235 240 Phe Glu Asp Met Gly Ile Lys Tyr Leu Gly Pro Ile Asp Gly His Asn 245 250 255 Ile Lys Glu Leu Thr Asp Val Leu Ala Ser Ala Lys Asp Ile Gln Gly 260 265 270 Pro Val Ile Ile His Ile Ile Thr Lys Lys Gly Lys Gly Tyr Glu Phe 275 280 285 Ala Glu Lys Asn Pro Gly Lys Phe His Gly Ile Gly Pro Phe Asn Cys 290 295 300 Ala Asn Gly Glu Leu Asp Ala Gly Ser Ser Asn Thr Tyr Ser Lys Ala 305 310 315 320 Phe Gly Asn Glu Met Val Lys Leu Ala Glu Lys Asp Asp Arg Ile Val 325 330 335 Ala Ile Thr Ala Ala Met Arg Asp Gly Thr Gly Leu Lys Ser Phe Ser 340 345 350 Gln Lys Phe Pro Glu Arg Phe Phe Asp Val Gly Ile Ala Glu Gln His 355 360 365 Ala Val Thr Leu Ala Ala Gly Met Ala Gln Ala Asn Leu Lys Pro Val 370 375 380 Phe Ala Val Tyr Ser Thr Phe Leu Gln Arg Ala Tyr Asp Gln Leu Ile 385 390 395 400 His Asp Val Cys Met Gln Lys Leu Pro Val Val Phe Ala Val Asp Arg 405 410 415 Ala Gly Ile Val Gly Glu Asp Gly Glu Thr His Gln Gly Ile Phe Asp 420 425 430 Leu Ser Tyr Leu Thr Glu Met Pro His Met Thr Leu Met Ser Pro Lys 435 440 445 Cys Ile Asp Glu Leu Pro Tyr Met Leu Lys Trp Ala Leu Gly Gln Ser 450 455 460 Phe Pro Val Ala Ile Arg Tyr Pro Arg Gly Gly Asp Ser Val Cys Leu 465 470 475 480 Asn Pro Val Glu Asn Phe Lys Leu Gly Lys Trp Asp Cys Ile Ser Asn 485 490 495 Glu Gly Ser Val Ala Ile Ile Ala Gln Gly Lys Met Val Gln Asn Ala 500 505 510 Val Leu Ala Gly Lys Lys Leu Lys Glu Lys Gly Ile Asp Val Arg Ile 515 520 525 Ile Ser Ala Cys Phe Ile Lys Pro Leu Asp Lys Glu Met Leu Asn Arg 530 535 540 Leu Val Glu Glu Ser Val Thr Ile Val Thr Val Glu Asp Asn Val Ile 545 550 555 560 Arg Gly Gly Leu Gly Ser Tyr Ile Leu Glu Tyr Val Asn Lys Leu Asn 565 570 575 Lys Lys Val Lys Ile Ile Asn Leu Gly Phe Asp Asp Lys Phe Val Gln 580 585 590 His Gly Lys Ser Asp Ile Leu Tyr Lys Leu Tyr Gly Leu Asp Pro Lys 595 600 605 Gly Ile Val Asn Ser Val Leu Glu Ala Ala Glu Val Ser His Ile Phe 610 615 620 Arg Glu Phe 625 31158DNAClostridium autoethanogenum 3atgaagagaa tttcaataat tggagccaca ggttctatag gaacccaaac tcttgatgta 60cttagaaaac aaaaaggaga ttttcagctt ataggtgtat ctgcaaatag tagtgtagat 120aaacttttac atataataga tgaatttaac cccaaatatg cggtgctaac cgaaaaagaa 180tcttatttaa agataaaaga tatttttagt aataaaaaat caaatacaaa aatattattt 240ggagtagatg gattaaatac tatagctagt cttcctgaag ttgatatggt tgtaacatct 300gtagttggaa tgatagggct tgtaccaact ataaaagcaa ttaaagcgaa gaaagacata 360gctttagcta ataaggagac attagttgta ggaggagaac tggttacaaa attatcgaaa 420gaaaataata taaaaatatt tcctgtagat tcagagcata gtgctgtttt tcaatgcctt 480cagggaaata attttgacga agttgctaat ttgattttaa ccgcttcagg tggacctttt 540aggggaaaaa caaaagatca actctcaaaa gtaactgtaa aagaggcgtt gaatcatcca 600aattggagta tgggaaaaaa gctcacaata gattctgcta ctcttatgaa taagggactt 660gaagttatag aagctcactt cttatttaac ttaccttatg aaaatataaa ggttgtagtt 720catccacaaa gtatagtaca ttctatggtg gaatataggg atggaagtgt tatggcacag 780cttgccactg cagatatgag attacctata caatatgcac tgaattatcc gaaaagaaag 840gaagctgtaa tagataaatt ggacttctat agcgtaggaa atttaagttt tgaaaagcct 900gatacagata cattcagacc acttaaatta gcttatgaag cagggaggat aggaggcaca 960atgccagcta tactaaattg tgcaaatgag gaagcagtaa gtttattcct tgctaataaa 1020ataaattttt tggatatagg caacatatta gaagagtgta tgaataaatt tacttcacaa 1080agtacgtata ctctggatga tttacttgac ctagaaataa aagttaagaa atatgtaaaa 1140gataaattta tcaaataa 11584385PRTClostridium autoethanogenum 4Met Lys Arg Ile Ser Ile Ile Gly Ala Thr Gly Ser Ile Gly Thr Gln 1 5 10 15 Thr Leu Asp Val Leu Arg Lys Gln Lys Gly Asp Phe Gln Leu Ile Gly 20 25 30 Val Ser Ala Asn Ser Ser Val Asp Lys Leu Leu His Ile Ile Asp Glu 35 40 45 Phe Asn Pro Lys Tyr Ala Val Leu Thr Glu Lys Glu Ser Tyr Leu Lys 50 55 60 Ile Lys Asp Ile Phe Ser Asn Lys Lys Ser Asn Thr Lys Ile Leu Phe 65 70 75 80 Gly Val Asp Gly Leu Asn Thr Ile Ala Ser Leu Pro Glu Val Asp Met 85 90 95 Val Val Thr Ser Val Val Gly Met Ile Gly Leu Val Pro Thr Ile Lys 100 105 110 Ala Ile Lys Ala Lys Lys Asp Ile Ala Leu Ala Asn Lys Glu Thr Leu 115 120 125 Val Val Gly Gly Glu Leu Val Thr Lys Leu Ser Lys Glu Asn Asn Ile 130 135 140 Lys Ile Phe Pro Val Asp Ser Glu His Ser Ala Val Phe Gln Cys Leu 145 150 155 160 Gln Gly Asn Asn Phe Asp Glu Val Ala Asn Leu Ile Leu Thr Ala Ser 165 170 175 Gly Gly Pro Phe Arg Gly Lys Thr Lys Asp Gln Leu Ser Lys Val Thr 180 185 190 Val Lys Glu Ala Leu Asn His Pro Asn Trp Ser Met Gly Lys Lys Leu 195 200 205 Thr Ile Asp Ser Ala Thr Leu Met Asn Lys Gly Leu Glu Val Ile Glu 210 215 220 Ala His Phe Leu Phe Asn Leu Pro Tyr Glu Asn Ile Lys Val Val Val 225 230 235 240 His Pro Gln Ser Ile Val His Ser Met Val Glu Tyr Arg Asp Gly Ser 245 250 255 Val Met Ala Gln Leu Ala Thr Ala Asp Met Arg Leu Pro Ile Gln Tyr 260 265 270 Ala Leu Asn Tyr Pro Lys Arg Lys Glu Ala Val Ile Asp Lys Leu Asp 275 280 285 Phe Tyr Ser Val Gly Asn Leu Ser Phe Glu Lys Pro Asp Thr Asp Thr 290 295 300 Phe Arg Pro Leu Lys Leu Ala Tyr Glu Ala Gly Arg Ile Gly Gly Thr 305 310 315 320 Met Pro Ala Ile Leu Asn Cys Ala Asn Glu Glu Ala Val Ser Leu Phe 325 330 335 Leu Ala Asn Lys Ile Asn Phe Leu Asp Ile Gly Asn Ile Leu Glu Glu 340 345 350 Cys Met Asn Lys Phe Thr Ser Gln Ser Thr Tyr Thr Leu Asp Asp Leu 355 360 365 Leu Asp Leu Glu Ile Lys Val Lys Lys Tyr Val Lys Asp Lys Phe Ile 370 375 380 Lys 385 5693DNAClostridium autoethanogenum 5atgaatggta attatgctat tattgtagct gccggcaagg gaaaaagaat gggaactact 60attaataagc aatttattaa aattaagggt aagcctatat tatattattc cataagggca 120ttttccataa atcctcttat agatggaatt atactggtat gtgcagaaac tgagatagaa 180tattgtaaaa gagaagtagt agataaatat gggcttcaga aggtaattaa attagttgct 240gggggtaaag aacgtcagga ttcggtattt aatggactag gagttttaga aaaagaaaac 300tgtagtgttg ttctaattca cgatggggct agaccttttg tcactagtaa aattattgat 360gatggaataa aatattctaa taggtatggg gcttgtgctt gtggagttag gcctaaggat 420acactaaaag ttagggaaga aagtggattt tcttcttcta cattagagag aaaaagttta 480tttgcagttc aaactccgca gtgttttaaa tatgatttaa tttatgactg tcataaaaaa 540ttaatgaatg aaaaaatgtg tgttactgat gatactatgg tagtagagcg ttatggaaat 600aaggtttatt tgtatgaagg taactatgaa aacataaaag tgaccacacc agaagattta 660aatatagctg aaagtatagt tgaaaaatat taa 6936230PRTClostridium autoethanogenum 6Met Asn Gly Asn Tyr Ala Ile Ile Val Ala Ala Gly Lys Gly Lys Arg 1 5 10 15 Met Gly Thr Thr Ile Asn Lys Gln Phe Ile Lys Ile Lys Gly Lys Pro 20 25 30 Ile Leu Tyr Tyr Ser Ile Arg Ala Phe Ser Ile Asn Pro Leu Ile Asp 35 40 45 Gly Ile Ile Leu Val Cys Ala Glu Thr Glu Ile Glu Tyr Cys Lys Arg 50 55 60 Glu Val Val Asp Lys Tyr Gly Leu Gln Lys Val Ile Lys Leu Val Ala 65 70 75 80 Gly Gly Lys Glu Arg Gln Asp Ser Val Phe Asn Gly Leu Gly Val Leu 85 90 95 Glu Lys Glu Asn Cys Ser Val Val Leu Ile His Asp Gly Ala Arg Pro 100 105 110 Phe Val Thr Ser Lys Ile Ile Asp Asp Gly Ile Lys Tyr Ser Asn Arg 115 120 125 Tyr Gly Ala Cys Ala Cys Gly Val Arg Pro Lys Asp Thr Leu Lys Val 130 135 140 Arg Glu Glu Ser Gly Phe Ser Ser Ser Thr Leu Glu Arg Lys Ser Leu 145 150 155 160 Phe Ala Val Gln Thr Pro Gln Cys Phe Lys Tyr Asp Leu Ile Tyr Asp 165 170 175 Cys His Lys Lys Leu Met Asn Glu Lys Met Cys Val Thr Asp Asp Thr 180 185 190 Met Val Val Glu Arg Tyr Gly Asn Lys Val Tyr Leu Tyr Glu Gly Asn 195 200 205 Tyr Glu Asn Ile Lys Val Thr Thr Pro Glu Asp Leu Asn Ile Ala Glu 210 215 220 Ser Ile Val Glu Lys Tyr 225 230 7798DNAClostridium autoethanogenum 7gtgggaaaaa gaaaagatgg gtatcatctt ttgaaaatga taatgcagaa tatagactta 60tatgatgttt taaaaataga tgagatcaaa actggaatac agatatgctc taataataga 120tatattccct gtgacaggag aaatttggtt tacagagcag caaaattatt tattgataaa 180tataatataa agaatggaat tagtataaac ataggtaaaa atatacctgt atcagctgga 240cttgctggtg gaagtgcgga tgctgcagct atactaaaga ctatgagaaa tatttatact 300cctgaagtaa gtgataaaga attgagcgaa ttaggcttaa atataggggc agatgttcct 360tattgtataa taggaggtac agccttgtgc gaggggatag gagagaaggt tacaccactc 420atgccgttta gaaaccatat actcatatta attaaaccac cttttggagt gagcacagca 480gaggtatata agagtttaga cataagtaaa ataaaaaggc atcctaatac agaaatttta 540atagatgcgg ttaatgaatc aaaattggag atgctgagta aaaacatgaa aaatgttttg 600gaaaatgtaa ctttaaaaaa atatcccgtg cttagaaaaa taaaaactga tttgatagat 660tttggagcag ttggttcact tatgagtgga agcggtccaa gcatttttgc tttttttgat 720gatatgctaa aagcacagaa atgttatgat aatatgaaaa ctaggtatag agaggtgttt 780attacaagaa ccatttaa 7988265PRTClostridium autoethanogenum 8Met Gly Lys Arg Lys Asp Gly Tyr His Leu Leu Lys Met Ile Met Gln 1 5 10 15 Asn Ile Asp Leu Tyr Asp Val Leu Lys Ile Asp Glu Ile Lys Thr Gly 20 25 30 Ile Gln Ile Cys Ser Asn Asn Arg Tyr Ile Pro Cys Asp Arg Arg Asn 35 40 45 Leu Val Tyr Arg Ala Ala Lys Leu Phe Ile Asp Lys Tyr Asn Ile Lys 50 55 60 Asn Gly Ile Ser Ile Asn Ile Gly Lys Asn Ile Pro Val Ser Ala Gly 65 70 75 80 Leu Ala Gly Gly Ser Ala Asp Ala Ala Ala Ile Leu Lys Thr Met Arg 85 90 95 Asn Ile Tyr Thr Pro Glu Val Ser Asp Lys Glu Leu Ser Glu Leu Gly 100 105 110 Leu Asn Ile Gly Ala Asp Val Pro Tyr Cys Ile Ile Gly Gly Thr Ala 115 120 125 Leu Cys Glu Gly Ile Gly Glu Lys Val Thr Pro Leu Met Pro Phe Arg 130 135 140 Asn His Ile Leu Ile Leu Ile Lys Pro Pro Phe Gly Val Ser Thr Ala 145 150 155 160 Glu Val Tyr Lys Ser Leu Asp Ile Ser Lys Ile Lys Arg His Pro Asn 165 170 175 Thr Glu Ile Leu Ile Asp Ala Val Asn Glu Ser Lys Leu Glu Met Leu 180 185 190 Ser Lys Asn Met Lys Asn Val Leu Glu Asn Val Thr Leu Lys Lys Tyr 195 200 205 Pro Val Leu Arg Lys Ile Lys Thr Asp Leu Ile Asp Phe Gly Ala Val 210 215 220 Gly Ser Leu Met Ser Gly Ser Gly Pro Ser Ile Phe Ala Phe Phe Asp 225 230 235 240 Asp Met Leu Lys Ala Gln Lys Cys Tyr Asp Asn Met Lys Thr Arg Tyr 245 250 255 Arg Glu Val Phe Ile Thr Arg Thr Ile 260 265 9480DNAClostridium autoethanogenum 9gtgaaaatcg ggcttggtta tgatgtccat aaattagttt ataatagacc tcttatttta 60ggaggcgtaa atatcccttt tgaaaaaggt cttatgggac attcagatgc agatgtactt 120cttcatgcaa taatggatag tctccttgga gccttgtgtc taggtgatat cggcaagcat 180ttccctgata atgataataa atataagaac atatgtagtc ttaaattgct gtcacatgta

240tcagctttga ttaatgaaaa aggatatact atagggaaca tagattctat tataatagcc 300gaaaagccta aactttcttc atacatacaa gatatgaggg taaatatagc taaaactcta 360aatgtaacta cagccgtaat aagtgtaaaa gccactacag aggaaggtct tggctttacc 420ggcaaaggag aaggcatagc cgctcaaagc atctgtttgt taacagctaa ttcaaaataa 48010159PRTClostridium autoethanogenum 10Met Lys Ile Gly Leu Gly Tyr Asp Val His Lys Leu Val Tyr Asn Arg 1 5 10 15 Pro Leu Ile Leu Gly Gly Val Asn Ile Pro Phe Glu Lys Gly Leu Met 20 25 30 Gly His Ser Asp Ala Asp Val Leu Leu His Ala Ile Met Asp Ser Leu 35 40 45 Leu Gly Ala Leu Cys Leu Gly Asp Ile Gly Lys His Phe Pro Asp Asn 50 55 60 Asp Asn Lys Tyr Lys Asn Ile Cys Ser Leu Lys Leu Leu Ser His Val 65 70 75 80 Ser Ala Leu Ile Asn Glu Lys Gly Tyr Thr Ile Gly Asn Ile Asp Ser 85 90 95 Ile Ile Ile Ala Glu Lys Pro Lys Leu Ser Ser Tyr Ile Gln Asp Met 100 105 110 Arg Val Asn Ile Ala Lys Thr Leu Asn Val Thr Thr Ala Val Ile Ser 115 120 125 Val Lys Ala Thr Thr Glu Glu Gly Leu Gly Phe Thr Gly Lys Gly Glu 130 135 140 Gly Ile Ala Ala Gln Ser Ile Cys Leu Leu Thr Ala Asn Ser Lys 145 150 155 111051DNAClostridium autoethanogenum 11ttgaatagag taaaaaagaa aacagtaaag gtaggcaata tatttttagg tggagatttt 60ccagtagccg tacaatctat gacaaatacg gatactaggg atgtagaagc cactacagct 120cagatatttc agctaaaaga agcaggttgt gatatcgtca gatgtgcggt gcctgatgat 180atagcttgca attccatgaa aaaaatcata gaaagagtag atattccact tgtagcagat 240atacattttg attataagtt ggcgcttaaa tctatagaaa atgggatatc tgcacttaga 300ataaatcctg gaaatattgg aagcatagaa agagtacgag aagtggcaag agcagcaaaa 360gaagctaata ttccaattag aataggggta aactctggat cattaaaaaa agatatttta 420aataaatatg gtagagtttg ttcggatgca ctagtagaga gtgctctaga acatgtaaaa 480attttggaaa acgtaggatt ttatgatata gttatatcca taaaatcttc aaatgtaaat 540cagatgatag aaagttatag aaaaatatct gaaattgtag attatccact tcaccttgga 600gtaacagaag caggaactat ttggcgagga actataaaat caagcatagg cataggtact 660cttttgatgg aaggtatagg agacactata agagtatctc ttacaggaaa tccagtggaa 720gaagtaagag tgggaaaaga aatattaaaa tcctgtggaa ttataaaaga aggtgtggaa 780tttatatcat gtcccacctg tggtagaact gaaattgatt taattaaaat agctgagcaa 840gtggaaaaaa gacttttaaa tatgcataaa aacataaagg ttgctgttat gggatgtgta 900gtaaatggac caggtgaggc tcgggaagca gatattggta tagcaggcgg caaaggtgaa 960ggcattatat ttaaaaaagg aaaaatagta aaaaaggtaa gtgaagaaag tttagtagaa 1020tcacttatag aagaaataga aaacatttga r 105112349PRTClostridium autoethanogenum 12Met Asn Arg Val Lys Lys Lys Thr Val Lys Val Gly Asn Ile Phe Leu 1 5 10 15 Gly Gly Asp Phe Pro Val Ala Val Gln Ser Met Thr Asn Thr Asp Thr 20 25 30 Arg Asp Val Glu Ala Thr Thr Ala Gln Ile Phe Gln Leu Lys Glu Ala 35 40 45 Gly Cys Asp Ile Val Arg Cys Ala Val Pro Asp Asp Ile Ala Cys Asn 50 55 60 Ser Met Lys Lys Ile Ile Glu Arg Val Asp Ile Pro Leu Val Ala Asp 65 70 75 80 Ile His Phe Asp Tyr Lys Leu Ala Leu Lys Ser Ile Glu Asn Gly Ile 85 90 95 Ser Ala Leu Arg Ile Asn Pro Gly Asn Ile Gly Ser Ile Glu Arg Val 100 105 110 Arg Glu Val Ala Arg Ala Ala Lys Glu Ala Asn Ile Pro Ile Arg Ile 115 120 125 Gly Val Asn Ser Gly Ser Leu Lys Lys Asp Ile Leu Asn Lys Tyr Gly 130 135 140 Arg Val Cys Ser Asp Ala Leu Val Glu Ser Ala Leu Glu His Val Lys 145 150 155 160 Ile Leu Glu Asn Val Gly Phe Tyr Asp Ile Val Ile Ser Ile Lys Ser 165 170 175 Ser Asn Val Asn Gln Met Ile Glu Ser Tyr Arg Lys Ile Ser Glu Ile 180 185 190 Val Asp Tyr Pro Leu His Leu Gly Val Thr Glu Ala Gly Thr Ile Trp 195 200 205 Arg Gly Thr Ile Lys Ser Ser Ile Gly Ile Gly Thr Leu Leu Met Glu 210 215 220 Gly Ile Gly Asp Thr Ile Arg Val Ser Leu Thr Gly Asn Pro Val Glu 225 230 235 240 Glu Val Arg Val Gly Lys Glu Ile Leu Lys Ser Cys Gly Ile Ile Lys 245 250 255 Glu Gly Val Glu Phe Ile Ser Cys Pro Thr Cys Gly Arg Thr Glu Ile 260 265 270 Asp Leu Ile Lys Ile Ala Glu Gln Val Glu Lys Arg Leu Leu Asn Met 275 280 285 His Lys Asn Ile Lys Val Ala Val Met Gly Cys Val Val Asn Gly Pro 290 295 300 Gly Glu Ala Arg Glu Ala Asp Ile Gly Ile Ala Gly Gly Lys Gly Glu 305 310 315 320 Gly Ile Ile Phe Lys Lys Gly Lys Ile Val Lys Lys Val Ser Glu Glu 325 330 335 Ser Leu Val Glu Ser Leu Ile Glu Glu Ile Glu Asn Ile 340 345 131923DNAClostridium autoethanogenum 13gtgataaaat tgaacattat tttagcagac aaatccggat tttgctttgg agtaaaaaga 60gctgtagacg aatctttaaa ggttcaaaaa aaatttaata aaaaaatata tactttaggt 120cctttgattc ataatagtga tgtagtaaat aaattaaagg aaaaaggtat atatcctata 180gaaatagata atatagataa tctaagggaa gatgatgtgg ttataatacg ttctcatggt 240gttcccgaaa aaatattttt tactttaaaa aataaaaaaa taaacatagt aaatgcaact 300tgcccatatg ttttaaatat acaaagaaaa gtacaagaat attataaatt agggtattct 360atattaatag taggagataa aaatcatcct gaagtaattg gaataaatgg atggtgtgaa 420aataaagctt taatatctaa agatggcacc aatttagaaa agttaccatc aaaactgtgt 480atagtttctc aaactacaga aaaacaatct aactgggaaa aagtgcttag tatagtggct 540aaaaattgta aagaatttat tgcttttaat actatatgca gtgccacaga atttcgtcag 600aaggcagcag cagatatttc taaagaagta gatatgatgg tagtaatagg tggtaaaaac 660agctctaata ctactaaact ttatgaaata tgtaaagata actgcaataa tactatttat 720gttgaaaatt caggagaaat acctgatgat ataagtaatt gtaataaaat taaaactata 780ggtgttacag caggagcttc aacaccagat tggataataa aggaggcaat tttaaaaatg 840agtgatgaca aaaatttaga actaaatgag caactatctt atatggacaa aaatgatacc 900caaataatat taggtgaaaa aattaagggt acagtaatat ctgtaaatcc aaaagaggtt 960tttttaaata taggatataa atcagaaggt gtacttccaa aacgtgaaat aacaaaaaat 1020gaaagtgaca acttagaaga attaattcat tgtggagatg aattatatgt taaagtaata 1080agaagacaaa atgaagatgg atatgtggta ttatctaaga tagaattaga aagagaaaat 1140gcttataaag aattaaagga agctaatgga aatagtcagg tattaaaggt tattgtaaaa 1200gaagctgtaa atggaggtct tgttgccaat tacaaaggtg ctagggtatt tatacctgct 1260tctcatgtag aattatatca tgtagatgat ctttcacaat atgtagataa agagcttgat 1320gtaactataa ttgaatttaa agaagaaaag aaaggtacca gaatagtagc ttcaagaaga 1380gaccttttga gaatggaaag agaaaaaatg gaagaacaga cttggaatgt gcttgaaaaa 1440gatactgtag tagatggtga agttagaaga ttgactgatt ttggcgcatt tgttgatgta 1500caaggagttg acgggcttct acatgtatct gaactttcct ggggaagagt tggaaaacca 1560agtgatgttt taaaaatcgg agatacgatt aaggtttata tcttagacat tgataaagaa 1620aaaaagaagt tatctttatc tttaaaaaag ctcatggaag atccatggat caacgtagac 1680ataaaatatc ctgttggcaa tgtagttctt ggtaaagtag ttaggtttgc aaattttggt 1740gcatttgttg aattagagcc aggtgtagat gcattagttc atatatcaca aataagccat 1800aagagaatag ataaaccaga agatgtactt aaaataggtc aggaaataaa ggctaagatc 1860cttgaagtaa acaaagatag cgaaaaaata gctttaagta taaaagaagt agatgaaatc 1920taa 192314640PRTClostridium autoethanogenum 14Met Ile Lys Leu Asn Ile Ile Leu Ala Asp Lys Ser Gly Phe Cys Phe 1 5 10 15 Gly Val Lys Arg Ala Val Asp Glu Ser Leu Lys Val Gln Lys Lys Phe 20 25 30 Asn Lys Lys Ile Tyr Thr Leu Gly Pro Leu Ile His Asn Ser Asp Val 35 40 45 Val Asn Lys Leu Lys Glu Lys Gly Ile Tyr Pro Ile Glu Ile Asp Asn 50 55 60 Ile Asp Asn Leu Arg Glu Asp Asp Val Val Ile Ile Arg Ser His Gly 65 70 75 80 Val Pro Glu Lys Ile Phe Phe Thr Leu Lys Asn Lys Lys Ile Asn Ile 85 90 95 Val Asn Ala Thr Cys Pro Tyr Val Leu Asn Ile Gln Arg Lys Val Gln 100 105 110 Glu Tyr Tyr Lys Leu Gly Tyr Ser Ile Leu Ile Val Gly Asp Lys Asn 115 120 125 His Pro Glu Val Ile Gly Ile Asn Gly Trp Cys Glu Asn Lys Ala Leu 130 135 140 Ile Ser Lys Asp Gly Thr Asn Leu Glu Lys Leu Pro Ser Lys Leu Cys 145 150 155 160 Ile Val Ser Gln Thr Thr Glu Lys Gln Ser Asn Trp Glu Lys Val Leu 165 170 175 Ser Ile Val Ala Lys Asn Cys Lys Glu Phe Ile Ala Phe Asn Thr Ile 180 185 190 Cys Ser Ala Thr Glu Phe Arg Gln Lys Ala Ala Ala Asp Ile Ser Lys 195 200 205 Glu Val Asp Met Met Val Val Ile Gly Gly Lys Asn Ser Ser Asn Thr 210 215 220 Thr Lys Leu Tyr Glu Ile Cys Lys Asp Asn Cys Asn Asn Thr Ile Tyr 225 230 235 240 Val Glu Asn Ser Gly Glu Ile Pro Asp Asp Ile Ser Asn Cys Asn Lys 245 250 255 Ile Lys Thr Ile Gly Val Thr Ala Gly Ala Ser Thr Pro Asp Trp Ile 260 265 270 Ile Lys Glu Ala Ile Leu Lys Met Ser Asp Asp Lys Asn Leu Glu Leu 275 280 285 Asn Glu Gln Leu Ser Tyr Met Asp Lys Asn Asp Thr Gln Ile Ile Leu 290 295 300 Gly Glu Lys Ile Lys Gly Thr Val Ile Ser Val Asn Pro Lys Glu Val 305 310 315 320 Phe Leu Asn Ile Gly Tyr Lys Ser Glu Gly Val Leu Pro Lys Arg Glu 325 330 335 Ile Thr Lys Asn Glu Ser Asp Asn Leu Glu Glu Leu Ile His Cys Gly 340 345 350 Asp Glu Leu Tyr Val Lys Val Ile Arg Arg Gln Asn Glu Asp Gly Tyr 355 360 365 Val Val Leu Ser Lys Ile Glu Leu Glu Arg Glu Asn Ala Tyr Lys Glu 370 375 380 Leu Lys Glu Ala Asn Gly Asn Ser Gln Val Leu Lys Val Ile Val Lys 385 390 395 400 Glu Ala Val Asn Gly Gly Leu Val Ala Asn Tyr Lys Gly Ala Arg Val 405 410 415 Phe Ile Pro Ala Ser His Val Glu Leu Tyr His Val Asp Asp Leu Ser 420 425 430 Gln Tyr Val Asp Lys Glu Leu Asp Val Thr Ile Ile Glu Phe Lys Glu 435 440 445 Glu Lys Lys Gly Thr Arg Ile Val Ala Ser Arg Arg Asp Leu Leu Arg 450 455 460 Met Glu Arg Glu Lys Met Glu Glu Gln Thr Trp Asn Val Leu Glu Lys 465 470 475 480 Asp Thr Val Val Asp Gly Glu Val Arg Arg Leu Thr Asp Phe Gly Ala 485 490 495 Phe Val Asp Val Gln Gly Val Asp Gly Leu Leu His Val Ser Glu Leu 500 505 510 Ser Trp Gly Arg Val Gly Lys Pro Ser Asp Val Leu Lys Ile Gly Asp 515 520 525 Thr Ile Lys Val Tyr Ile Leu Asp Ile Asp Lys Glu Lys Lys Lys Leu 530 535 540 Ser Leu Ser Leu Lys Lys Leu Met Glu Asp Pro Trp Ile Asn Val Asp 545 550 555 560 Ile Lys Tyr Pro Val Gly Asn Val Val Leu Gly Lys Val Val Arg Phe 565 570 575 Ala Asn Phe Gly Ala Phe Val Glu Leu Glu Pro Gly Val Asp Ala Leu 580 585 590 Val His Ile Ser Gln Ile Ser His Lys Arg Ile Asp Lys Pro Glu Asp 595 600 605 Val Leu Lys Ile Gly Gln Glu Ile Lys Ala Lys Ile Leu Glu Val Asn 610 615 620 Lys Asp Ser Glu Lys Ile Ala Leu Ser Ile Lys Glu Val Asp Glu Ile 625 630 635 640 15882DNAClostridium autoethanogenum 15atggaaatta aaggtgtaat tgaaacatta agagaggaat tgaataaata cctctatgac 60tatatggagg gaaaaggatc ttataataag agagtatatg aagctatgca gtatagctta 120gatgcaggag gaaagagaat aagacctcta ctatttcttt tgacatataa actttataag 180acagattgca atgaggttat ggatatagca gcagctatag aaatgataca cacttattcc 240ttaattcatg atgatttacc tgctatggac aatgatgatt taagaagggg caaacctaca 300aatcataagg tatttggaga agctattgct gtacttgcgg gagatggact tttaaatgaa 360gcaatgagtc tgatgtttag acactgtatt gggaaaaagg ataacgctat aagggcttgt 420agcattattt ctgaaagtgc aggagctgat gggatggttg gcggacagac agtggatatt 480ttaagtgaaa acactaagat acctatagat cagctctatt acatgcacag taaaaaaacg 540ggagcgctca taaaaggatc tataatatct gcagcagtat atgcgggagc aagtaaagct 600gaaatagata aattaagcta ttatggagaa aagttaggat tggcatttca aataaaggat 660gatatattgg atttaacagg agatactgct cttttaggta aaaagataaa aagtgatcta 720aataataaca aaactacatt tataagtact tatggaataa ataaatgcaa agaaatgtgc 780aattcaatta caagtgaatg tataggagta ctgaatggga tgagtgtaga tacttcttat 840ctaaaagatt taacatcatt tttattaaat agagaaaagt ga 88216293PRTClostridium autoethanogenum 16Met Glu Ile Lys Gly Val Ile Glu Thr Leu Arg Glu Glu Leu Asn Lys 1 5 10 15 Tyr Leu Tyr Asp Tyr Met Glu Gly Lys Gly Ser Tyr Asn Lys Arg Val 20 25 30 Tyr Glu Ala Met Gln Tyr Ser Leu Asp Ala Gly Gly Lys Arg Ile Arg 35 40 45 Pro Leu Leu Phe Leu Leu Thr Tyr Lys Leu Tyr Lys Thr Asp Cys Asn 50 55 60 Glu Val Met Asp Ile Ala Ala Ala Ile Glu Met Ile His Thr Tyr Ser 65 70 75 80 Leu Ile His Asp Asp Leu Pro Ala Met Asp Asn Asp Asp Leu Arg Arg 85 90 95 Gly Lys Pro Thr Asn His Lys Val Phe Gly Glu Ala Ile Ala Val Leu 100 105 110 Ala Gly Asp Gly Leu Leu Asn Glu Ala Met Ser Leu Met Phe Arg His 115 120 125 Cys Ile Gly Lys Lys Asp Asn Ala Ile Arg Ala Cys Ser Ile Ile Ser 130 135 140 Glu Ser Ala Gly Ala Asp Gly Met Val Gly Gly Gln Thr Val Asp Ile 145 150 155 160 Leu Ser Glu Asn Thr Lys Ile Pro Ile Asp Gln Leu Tyr Tyr Met His 165 170 175 Ser Lys Lys Thr Gly Ala Leu Ile Lys Gly Ser Ile Ile Ser Ala Ala 180 185 190 Val Tyr Ala Gly Ala Ser Lys Ala Glu Ile Asp Lys Leu Ser Tyr Tyr 195 200 205 Gly Glu Lys Leu Gly Leu Ala Phe Gln Ile Lys Asp Asp Ile Leu Asp 210 215 220 Leu Thr Gly Asp Thr Ala Leu Leu Gly Lys Lys Ile Lys Ser Asp Leu 225 230 235 240 Asn Asn Asn Lys Thr Thr Phe Ile Ser Thr Tyr Gly Ile Asn Lys Cys 245 250 255 Lys Glu Met Cys Asn Ser Ile Thr Ser Glu Cys Ile Gly Val Leu Asn 260 265 270 Gly Met Ser Val Asp Thr Ser Tyr Leu Lys Asp Leu Thr Ser Phe Leu 275 280 285 Leu Asn Arg Glu Lys 290 17519DNAClostridium autoethanogenum 17atgaataaaa caaggaaaat ggttttttta agctttctaa caagtatggc tttagtcata 60tacataatag aaactcaagt tccggtttta tttcccggaa taaaattagg acttgcaaat 120acaatttccc tagctgcact tatacttata ggatggaaag aagccttact aattatgttt 180ttaaggacgc ttctaggatc tatgtttggt gggacaatgt ctacctttat gttcagcata 240gccggaggaa ttttaagtaa cattgttatg atccttctat acaaatattt taaaaattcc 300ttaagtctat ggactataag catatgcggg gcaatatttc acaacatagg ccaactttta 360gtagcttcta tagtaattca agattttagg atatacatat atctaccggt gcttttaatc 420tctgctataa tcacaggata ctttataggt tggtgcgtga aattcctaac taataactta 480tataaaattc ctatgtttaa agaattaaaa aataagtaa 51918172PRTClostridium autoethanogenum 18Met Asn Lys Thr Arg Lys Met Val Phe Leu Ser Phe Leu Thr Ser Met 1 5 10 15 Ala Leu Val Ile Tyr Ile Ile Glu Thr Gln Val Pro Val Leu Phe Pro 20 25 30 Gly Ile Lys Leu Gly Leu Ala Asn Thr Ile Ser Leu Ala Ala Leu Ile 35 40 45 Leu Ile Gly Trp Lys Glu Ala Leu Leu Ile Met Phe Leu Arg Thr Leu 50 55 60 Leu Gly Ser Met Phe Gly Gly Thr Met Ser Thr Phe Met Phe Ser Ile 65 70 75 80 Ala Gly Gly Ile Leu Ser Asn Ile Val Met Ile Leu Leu Tyr Lys Tyr 85 90

95 Phe Lys Asn Ser Leu Ser Leu Trp Thr Ile Ser Ile Cys Gly Ala Ile 100 105 110 Phe His Asn Ile Gly Gln Leu Leu Val Ala Ser Ile Val Ile Gln Asp 115 120 125 Phe Arg Ile Tyr Ile Tyr Leu Pro Val Leu Leu Ile Ser Ala Ile Ile 130 135 140 Thr Gly Tyr Phe Ile Gly Trp Cys Val Lys Phe Leu Thr Asn Asn Leu 145 150 155 160 Tyr Lys Ile Pro Met Phe Lys Glu Leu Lys Asn Lys 165 170 19825DNAClostridium autoethanogenum 19atgaacttcg atggaatttc aattccaata ataaaagaac ttaatcaact tgagttagag 60ttaaaaaata ttgcatcaaa attagattct actgttacac aagatatttt tacctacttt 120ttttcaattc caggtaaaag actaagacct acattaacat ttttatctgc aggtgctatt 180agtagcgagc ttacttcatc tgcaaaacac aacttaattc agttgtcaat aagcttagag 240cttattcaca gcgctagtct aattcatgat gatatcatag atggtgactt actaagacgt 300ggtcagaaaa ccttaaataa gacctttgga aataaaatag cagtacttgc cggtgatgct 360ttgtactcaa gggcctttac tattttctca gatactctgc caagagaatt tgcgcaggta 420atgggcagag ttactgaatc aatgtctgta gctgaaatat taaatgctaa caatccctct 480cccgatcgtg aaacctattt taaaatcatc ttaggaaaaa cagcatcttt catgagcgct 540tgttgtaggc ttggtggcag catagcttat gccccttacg aagagtctaa tatgctttct 600aaatacggtg aaaaccttgg tatggcatat caaatactgg atgattatat cgatgaggat 660cccgttgcaa tgaaaaatgt aactattgaa gagggatttg aatttgcata taatgccaaa 720gcttctattg aaaatttaaa agactcagca tacaaacaaa gcttaataat gttagtagac 780tatgttttag atttttatag tcctaaggta gagaatacat tatag 82520274PRTClostridium autoethanogenum 20Met Asn Phe Asp Gly Ile Ser Ile Pro Ile Ile Lys Glu Leu Asn Gln 1 5 10 15 Leu Glu Leu Glu Leu Lys Asn Ile Ala Ser Lys Leu Asp Ser Thr Val 20 25 30 Thr Gln Asp Ile Phe Thr Tyr Phe Phe Ser Ile Pro Gly Lys Arg Leu 35 40 45 Arg Pro Thr Leu Thr Phe Leu Ser Ala Gly Ala Ile Ser Ser Glu Leu 50 55 60 Thr Ser Ser Ala Lys His Asn Leu Ile Gln Leu Ser Ile Ser Leu Glu 65 70 75 80 Leu Ile His Ser Ala Ser Leu Ile His Asp Asp Ile Ile Asp Gly Asp 85 90 95 Leu Leu Arg Arg Gly Gln Lys Thr Leu Asn Lys Thr Phe Gly Asn Lys 100 105 110 Ile Ala Val Leu Ala Gly Asp Ala Leu Tyr Ser Arg Ala Phe Thr Ile 115 120 125 Phe Ser Asp Thr Leu Pro Arg Glu Phe Ala Gln Val Met Gly Arg Val 130 135 140 Thr Glu Ser Met Ser Val Ala Glu Ile Leu Asn Ala Asn Asn Pro Ser 145 150 155 160 Pro Asp Arg Glu Thr Tyr Phe Lys Ile Ile Leu Gly Lys Thr Ala Ser 165 170 175 Phe Met Ser Ala Cys Cys Arg Leu Gly Gly Ser Ile Ala Tyr Ala Pro 180 185 190 Tyr Glu Glu Ser Asn Met Leu Ser Lys Tyr Gly Glu Asn Leu Gly Met 195 200 205 Ala Tyr Gln Ile Leu Asp Asp Tyr Ile Asp Glu Asp Pro Val Ala Met 210 215 220 Lys Asn Val Thr Ile Glu Glu Gly Phe Glu Phe Ala Tyr Asn Ala Lys 225 230 235 240 Ala Ser Ile Glu Asn Leu Lys Asp Ser Ala Tyr Lys Gln Ser Leu Ile 245 250 255 Met Leu Val Asp Tyr Val Leu Asp Phe Tyr Ser Pro Lys Val Glu Asn 260 265 270 Thr Leu 211797DNAPopulus tremuloides 21catatggcaa cagaattatt atgtttacac agacctatat cacttactca caaacttttt 60aggaatccat tacctaaagt tattcaagct acacctttaa cattaaaact taggtgtagt 120gtttctacag aaaatgtatc atttagtgag acagaaactg aaacaagaag atcagcaaat 180tatgaaccaa attcttggga ttatgattat cttctttctt ctgatactga tgagtcaata 240gaagtacata aagataaggc taagaaatta gaagctgaag ttaggagaga aataaataat 300gagaaggctg aatttcttac acttcttgaa cttattgata atgtacaaag acttggatta 360ggatatagat ttgagtctga tataagaaga gcattagata gatttgtaag tagtggagga 420tttgatggag ttactaaaac ttcattacat ggaacagcat tatcatttag gttattaagg 480caacatggtt ttgaagtatc tcaagaagct tttagtggat ttaaagatca gaatggaaac 540tttcttgaga atttaaagga agacataaaa gcaattcttt ctctttatga agcatcattt 600ttagcattag aaggtgagaa tatattagat gaggctaaag tatttgcaat atctcatctt 660aaagaactta gtgaagaaaa gattggtaaa gaattagctg aacaagtttc acatgcttta 720gaattaccat tacatagaag aacacaaaga ttagaagcag tttggtcaat agaagcatat 780agaaagaaag aagacgcaaa tcaagtactt ttagaacttg caatacttga ctacaatatg 840attcaaagtg tatatcagag ggatttaaga gaaacatcaa gatggtggag aagagtagga 900ttagcaacta aattacattt tgctagagat aggcttattg aaagttttta ttgggctgtt 960ggagttgctt ttgaaccaca atattctgat tgcagaaata gtgtagcaaa gatgttttca 1020tttgttacta taattgacga tatttacgat gtatatggaa ctttagatga acttgaactt 1080tttactgatg cagttgaaag atgggatgta aatgctatta atgatcttcc tgattatatg 1140aagttatgtt ttcttgcact ttacaatact attaacgaga tagcttacga taacttaaaa 1200gataaaggtg agaacatact tccttattta acaaaagcat gggcagattt atgtaatgca 1260tttcttcaag aagctaagtg gctttataat aaatcaacac ctacatttga tgattatttt 1320ggaaatgcat ggaaaagttc tagtggacct ttacagctta tttttgctta ttttgctgta 1380gtacagaaca ttaaaaagga agagattgag aatcttcaga aatatcatga cataatatca 1440agacctagtc acatttttag gctttgtaat gatttagcat ctgcttcagc agaaatagca 1500agaggtgaaa ctgctaattc tgtaagttgt tatatgagaa caaaaggtat atctgaagaa 1560ttagctactg aaagtgttat gaatcttata gacgaaactt ggaagaaaat gaacaaagaa 1620aaacttggtg gatctttatt tgcaaaacct tttgttgaga ctgctataaa tttagctaga 1680cagtctcatt gcacatatca taatggtgat gcacatacta gtccagatga attaactagg 1740aaaagagtac ttagtgtaat aactgaacca atattaccat ttgaaagata agaattc 179722617DNAClostridium autoethanogenum 22ggttaatgtt aaaaatttat agtataactt taaaaaactg tcttaaaaag ttgttatata 60aaaaatgttg acaattaaac agctatttag tgcaaaacaa ccataaaaat ttaaaaaata 120ccataaatta cttgaaaaat agttgataat aatgtagagt tataaacaaa ggtgaaaagc 180attacttgta ttctttttta tatattatta taaattaaaa tgaagctgta ttagaaaaaa 240tacacacctg taatataaaa ttttaaatta atttttaatt ttttcaaaat gtattttaca 300tgtttagaat tttgatgtat attaaaatag tagaatacat aagatactta atttaattaa 360agatagttaa gtacttttca atgtgctttt ttagatgttt aatacaaatc tttaattgta 420aaagaaatgc tgtactattt actgtactag tgacgggatt aaactgtatt aattataaat 480aaaaaataag tacagttgtt taaaattata ttttgtatta aatctaatag tacgatgtaa 540gttattttat actattgcta gtttaataaa aagatttaat tatatacttg aaaaggagag 600gaatttttat gcgtaaa 6172330DNAArtificial Sequenceoligonucleotide Ppfor-NotI-F 23aagcggccgc aaaatagttg ataataatgc 302430DNAArtificial Sequenceoligonucleotide Ppfor-NdeI-R 24tacgcatatg aattcctctc cttttcaagc 30255345DNAArtificial Sequenceplasmid pMTL 85146-ispS 25aattcgagct cggtacccgg ggatcctcta gagtcgacgt cacgcgtcca tggagatctc 60gaggcctgca gacatgcaag cttggcactg gccgtcgttt tacaacgtcg tgactgggaa 120aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt 180aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa 240tggcgctagc ataaaaataa gaagcctgca tttgcaggct tcttattttt atggcgcgcc 300gcattcactt cttttctata taaatatgag cgaagcgaat aagcgtcgga aaagcagcaa 360aaagtttcct ttttgctgtt ggagcatggg ggttcagggg gtgcagtatc tgacgtcaat 420gccgagcgaa agcgagccga agggtagcat ttacgttaga taaccccctg atatgctccg 480acgctttata tagaaaagaa gattcaacta ggtaaaatct taatataggt tgagatgata 540aggtttataa ggaatttgtt tgttctaatt tttcactcat tttgttctaa tttcttttaa 600caaatgttct ttttttttta gaacagttat gatatagtta gaatagttta aaataaggag 660tgagaaaaag atgaaagaaa gatatggaac agtctataaa ggctctcaga ggctcataga 720cgaagaaagt ggagaagtca tagaggtaga caagttatac cgtaaacaaa cgtctggtaa 780cttcgtaaag gcatatatag tgcaattaat aagtatgtta gatatgattg gcggaaaaaa 840acttaaaatc gttaactata tcctagataa tgtccactta agtaacaata caatgatagc 900tacaacaaga gaaatagcaa aagctacagg aacaagtcta caaacagtaa taacaacact 960taaaatctta gaagaaggaa atattataaa aagaaaaact ggagtattaa tgttaaaccc 1020tgaactacta atgagaggcg acgaccaaaa acaaaaatac ctcttactcg aatttgggaa 1080ctttgagcaa gaggcaaatg aaatagattg acctcccaat aacaccacgt agttattggg 1140aggtcaatct atgaaatgcg attaagggcc ggccagtggg caagttgaaa aattcacaaa 1200aatgtggtat aatatctttg ttcattagag cgataaactt gaatttgaga gggaacttag 1260atggtatttg aaaaaattga taaaaatagt tggaacagaa aagagtattt tgaccactac 1320tttgcaagtg taccttgtac ctacagcatg accgttaaag tggatatcac acaaataaag 1380gaaaagggaa tgaaactata tcctgcaatg ctttattata ttgcaatgat tgtaaaccgc 1440cattcagagt ttaggacggc aatcaatcaa gatggtgaat tggggatata tgatgagatg 1500ataccaagct atacaatatt tcacaatgat actgaaacat tttccagcct ttggactgag 1560tgtaagtctg actttaaatc atttttagca gattatgaaa gtgatacgca acggtatgga 1620aacaatcata gaatggaagg aaagccaaat gctccggaaa acatttttaa tgtatctatg 1680ataccgtggt caaccttcga tggctttaat ctgaatttgc agaaaggata tgattatttg 1740attcctattt ttactatggg gaaatattat aaagaagata acaaaattat acttcctttg 1800gcaattcaag ttcatcacgc agtatgtgac ggatttcaca tttgccgttt tgtaaacgaa 1860ttgcaggaat tgataaatag ttaacttcag gtttgtctgt aactaaaaac aagtatttaa 1920gcaaaaacat cgtagaaata cggtgttttt tgttacccta agtttaaact cctttttgat 1980aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 2040gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 2100acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 2160tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag 2220ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 2280atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 2340agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 2400cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 2460agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 2520acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 2580gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 2640ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 2700gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt 2760gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag 2820gaagcggaag agcgcccaat acgcagggcc ccctgcagga taaaaaaatt gtagataaat 2880tttataaaat agttttatct acaatttttt tatcaggaaa cagctatgac cgcggccgcg 2940gttaatgtta aaaatttata gtataacttt aaaaaactgt cttaaaaagt tgttatataa 3000aaaatgttga caattaaaca gctatttagt gcaaaacaac cataaaaatt taaaaaatac 3060cataaattac ttgaaaaata gttgataata atgtagagtt ataaacaaag gtgaaaagca 3120ttacttgtat tcttttttat atattattat aaattaaaat gaagctgtat tagaaaaaat 3180acacacctgt aatataaaat tttaaattaa tttttaattt tttcaaaatg tattttacat 3240gtttagaatt ttgatgtata ttaaaatagt agaatacata agatacttaa tttaattaaa 3300gatagttaag tacttttcaa tgtgcttttt tagatgttta atacaaatct ttaattgtaa 3360aagaaatgct gtactattta ctgtactagt gacgggatta aactgtatta attataaata 3420aaaaataagt acagttgttt aaaattatat tttgtattaa atctaatagt acgatgtaag 3480ttattttata ctattgctag tttaataaaa agatttaatt atatacttga aaaggagagg 3540aatttttatg cgtcatatgg caacagaatt attatgttta cacagaccta tatcacttac 3600tcacaaactt tttaggaatc cattacctaa agttattcaa gctacacctt taacattaaa 3660acttaggtgt agtgtttcta cagaaaatgt atcatttagt gagacagaaa ctgaaacaag 3720aagatcagca aattatgaac caaattcttg ggattatgat tatcttcttt cttctgatac 3780tgatgagtca atagaagtac ataaagataa ggctaagaaa ttagaagctg aagttaggag 3840agaaataaat aatgagaagg ctgaatttct tacacttctt gaacttattg ataatgtaca 3900aagacttgga ttaggatata gatttgagtc tgatataaga agagcattag atagatttgt 3960aagtagtgga ggatttgatg gagttactaa aacttcatta catggaacag cattatcatt 4020taggttatta aggcaacatg gttttgaagt atctcaagaa gcttttagtg gatttaaaga 4080tcagaatgga aactttcttg agaatttaaa ggaagacata aaagcaattc tttctcttta 4140tgaagcatca tttttagcat tagaaggtga gaatatatta gatgaggcta aagtatttgc 4200aatatctcat cttaaagaac ttagtgaaga aaagattggt aaagaattag ctgaacaagt 4260ttcacatgct ttagaattac cattacatag aagaacacaa agattagaag cagtttggtc 4320aatagaagca tatagaaaga aagaagacgc aaatcaagta cttttagaac ttgcaatact 4380tgactacaat atgattcaaa gtgtatatca gagggattta agagaaacat caagatggtg 4440gagaagagta ggattagcaa ctaaattaca ttttgctaga gataggctta ttgaaagttt 4500ttattgggct gttggagttg cttttgaacc acaatattct gattgcagaa atagtgtagc 4560aaagatgttt tcatttgtta ctataattga cgatatttac gatgtatatg gaactttaga 4620tgaacttgaa ctttttactg atgcagttga aagatgggat gtaaatgcta ttaatgatct 4680tcctgattat atgaagttat gttttcttgc actttacaat actattaacg agatagctta 4740cgataactta aaagataaag gtgagaacat acttccttat ttaacaaaag catgggcaga 4800tttatgtaat gcatttcttc aagaagctaa gtggctttat aataaatcaa cacctacatt 4860tgatgattat tttggaaatg catggaaaag ttctagtgga cctttacagc ttatttttgc 4920ttattttgct gtagtacaga acattaaaaa ggaagagatt gagaatcttc agaaatatca 4980tgacataata tcaagaccta gtcacatttt taggctttgt aatgatttag catctgcttc 5040agcagaaata gcaagaggtg aaactgctaa ttctgtaagt tgttatatga gaacaaaagg 5100tatatctgaa gaattagcta ctgaaagtgt tatgaatctt atagacgaaa cttggaagaa 5160aatgaacaaa gaaaaacttg gtggatcttt atttgcaaaa ccttttgttg agactgctat 5220aaatttagct agacagtctc attgcacata tcataatggt gatgcacata ctagtccaga 5280tgaattaact aggaaaagag tacttagtgt aataactgaa ccaatattac catttgaaag 5340ataag 53452626DNAArtificial SequenceOligonucleotide Idi-Cbei-SacI-F 26gtgagctcga aaggggaaat taaatg 262727DNAArtificial SequenceOligonucleotide Idi-Cbei-KpnI-R 27atggtacccc aaatctttat ttagacg 27285905DNAArtificial Sequenceplasmid pMTL85246-ispS-idi 28ccggggatcc tctagagtcg acgtcacgcg tccatggaga tctcgaggcc tgcagacatg 60caagcttggc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc 120aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc 180gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc tagcataaaa 240ataagaagcc tgcatttgca ggcttcttat ttttatggcg cgccgcattc acttcttttc 300tatataaata tgagcgaagc gaataagcgt cggaaaagca gcaaaaagtt tcctttttgc 360tgttggagca tgggggttca gggggtgcag tatctgacgt caatgccgag cgaaagcgag 420ccgaagggta gcatttacgt tagataaccc cctgatatgc tccgacgctt tatatagaaa 480agaagattca actaggtaaa atcttaatat aggttgagat gataaggttt ataaggaatt 540tgtttgttct aatttttcac tcattttgtt ctaatttctt ttaacaaatg ttcttttttt 600tttagaacag ttatgatata gttagaatag tttaaaataa ggagtgagaa aaagatgaaa 660gaaagatatg gaacagtcta taaaggctct cagaggctca tagacgaaga aagtggagaa 720gtcatagagg tagacaagtt ataccgtaaa caaacgtctg gtaacttcgt aaaggcatat 780atagtgcaat taataagtat gttagatatg attggcggaa aaaaacttaa aatcgttaac 840tatatcctag ataatgtcca cttaagtaac aatacaatga tagctacaac aagagaaata 900gcaaaagcta caggaacaag tctacaaaca gtaataacaa cacttaaaat cttagaagaa 960ggaaatatta taaaaagaaa aactggagta ttaatgttaa accctgaact actaatgaga 1020ggcgacgacc aaaaacaaaa atacctctta ctcgaatttg ggaactttga gcaagaggca 1080aatgaaatag attgacctcc caataacacc acgtagttat tgggaggtca atctatgaaa 1140tgcgattaag ggccggccag tgggcaagtt gaaaaattca caaaaatgtg gtataatatc 1200tttgttcatt agagcgataa acttgaattt gagagggaac ttagatggta tttgaaaaaa 1260ttgataaaaa tagttggaac agaaaagagt attttgacca ctactttgca agtgtacctt 1320gtacctacag catgaccgtt aaagtggata tcacacaaat aaaggaaaag ggaatgaaac 1380tatatcctgc aatgctttat tatattgcaa tgattgtaaa ccgccattca gagtttagga 1440cggcaatcaa tcaagatggt gaattgggga tatatgatga gatgatacca agctatacaa 1500tatttcacaa tgatactgaa acattttcca gcctttggac tgagtgtaag tctgacttta 1560aatcattttt agcagattat gaaagtgata cgcaacggta tggaaacaat catagaatgg 1620aaggaaagcc aaatgctccg gaaaacattt ttaatgtatc tatgataccg tggtcaacct 1680tcgatggctt taatctgaat ttgcagaaag gatatgatta tttgattcct atttttacta 1740tggggaaata ttataaagaa gataacaaaa ttatacttcc tttggcaatt caagttcatc 1800acgcagtatg tgacggattt cacatttgcc gttttgtaaa cgaattgcag gaattgataa 1860atagttaact tcaggtttgt ctgtaactaa aaacaagtat ttaagcaaaa acatcgtaga 1920aatacggtgt tttttgttac cctaagttta aactcctttt tgataatctc atgaccaaaa 1980tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 2040cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 2100taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 2160gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc 2220acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 2280ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 2340ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 2400cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 2460aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 2520gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 2580gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 2640gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc 2700ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg 2760ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc 2820caatacgcag ggccccctgc aggataaaaa aattgtagat aaattttata aaatagtttt 2880atctacaatt tttttatcag gaaacagcta tgaccgcggc cgcggttaat gttaaaaatt 2940tatagtataa ctttaaaaaa ctgtcttaaa aagttgttat ataaaaaatg ttgacaatta 3000aacagctatt tagtgcaaaa caaccataaa aatttaaaaa ataccataaa ttacttgaaa 3060aatagttgat aataatgtag agttataaac aaaggtgaaa agcattactt gtattctttt 3120ttatatatta ttataaatta aaatgaagct gtattagaaa aaatacacac ctgtaatata 3180aaattttaaa ttaattttta attttttcaa aatgtatttt acatgtttag aattttgatg 3240tatattaaaa tagtagaata cataagatac ttaatttaat taaagatagt taagtacttt 3300tcaatgtgct tttttagatg tttaatacaa atctttaatt gtaaaagaaa tgctgtacta 3360tttactgtac

tagtgacggg attaaactgt attaattata aataaaaaat aagtacagtt 3420gtttaaaatt atattttgta ttaaatctaa tagtacgatg taagttattt tatactattg 3480ctagtttaat aaaaagattt aattatatac ttgaaaagga gaggaatttt tatgcgtcat 3540atggcaacag aattattatg tttacacaga cctatatcac ttactcacaa actttttagg 3600aatccattac ctaaagttat tcaagctaca cctttaacat taaaacttag gtgtagtgtt 3660tctacagaaa atgtatcatt tagtgagaca gaaactgaaa caagaagatc agcaaattat 3720gaaccaaatt cttgggatta tgattatctt ctttcttctg atactgatga gtcaatagaa 3780gtacataaag ataaggctaa gaaattagaa gctgaagtta ggagagaaat aaataatgag 3840aaggctgaat ttcttacact tcttgaactt attgataatg tacaaagact tggattagga 3900tatagatttg agtctgatat aagaagagca ttagatagat ttgtaagtag tggaggattt 3960gatggagtta ctaaaacttc attacatgga acagcattat catttaggtt attaaggcaa 4020catggttttg aagtatctca agaagctttt agtggattta aagatcagaa tggaaacttt 4080cttgagaatt taaaggaaga cataaaagca attctttctc tttatgaagc atcattttta 4140gcattagaag gtgagaatat attagatgag gctaaagtat ttgcaatatc tcatcttaaa 4200gaacttagtg aagaaaagat tggtaaagaa ttagctgaac aagtttcaca tgctttagaa 4260ttaccattac atagaagaac acaaagatta gaagcagttt ggtcaataga agcatataga 4320aagaaagaag acgcaaatca agtactttta gaacttgcaa tacttgacta caatatgatt 4380caaagtgtat atcagaggga tttaagagaa acatcaagat ggtggagaag agtaggatta 4440gcaactaaat tacattttgc tagagatagg cttattgaaa gtttttattg ggctgttgga 4500gttgcttttg aaccacaata ttctgattgc agaaatagtg tagcaaagat gttttcattt 4560gttactataa ttgacgatat ttacgatgta tatggaactt tagatgaact tgaacttttt 4620actgatgcag ttgaaagatg ggatgtaaat gctattaatg atcttcctga ttatatgaag 4680ttatgttttc ttgcacttta caatactatt aacgagatag cttacgataa cttaaaagat 4740aaaggtgaga acatacttcc ttatttaaca aaagcatggg cagatttatg taatgcattt 4800cttcaagaag ctaagtggct ttataataaa tcaacaccta catttgatga ttattttgga 4860aatgcatgga aaagttctag tggaccttta cagcttattt ttgcttattt tgctgtagta 4920cagaacatta aaaaggaaga gattgagaat cttcagaaat atcatgacat aatatcaaga 4980cctagtcaca tttttaggct ttgtaatgat ttagcatctg cttcagcaga aatagcaaga 5040ggtgaaactg ctaattctgt aagttgttat atgagaacaa aaggtatatc tgaagaatta 5100gctactgaaa gtgttatgaa tcttatagac gaaacttgga agaaaatgaa caaagaaaaa 5160cttggtggat ctttatttgc aaaacctttt gttgagactg ctataaattt agctagacag 5220tctcattgca catatcataa tggtgatgca catactagtc cagatgaatt aactaggaaa 5280agagtactta gtgtaataac tgaaccaata ttaccatttg aaagataaga attcgagctc 5340gaaaggggaa attaaatggc agaatatata atagctgtag atgaatttga taacgaaata 5400ggttcaattg aaaaaatgga ggctcaccgt aaaggaacat tacatagagc tttttctata 5460ttagtattta attctaaaaa tcaattgtta ttacagaaaa gaaatgtaaa aaaatatcat 5520tcgcctggtc tctggacaaa tacgtgctgt agtcatccaa gatacggtga aagtttacat 5580gatgcgattt atagaaggct taaggaagaa atgggtttta catgtgaact tgaagaagta 5640tttagtttta tttataaagt aaaacttgaa gataatcttt ttgaaaatga atatgatcat 5700gtattcattg ggaaatatga tggagaaata attgtaaaca aagatgaagt agatgatttt 5760aagtgggttg atattaatga ggttaagaag gatattatag aaaggccaga agcatacact 5820tattggttca agtatttagt taataaggca gaaaacaaaa tatttaaata agtaagaatt 5880tcgtctaaat aaagatttgg ggtac 59052929DNAArtificial SequenceOligonucleotide Dxs-SalI-F 29gcagtcgact ttattaaagg gatagataa 293031DNAArtificial SequenceOligonucleotide Dxs-XhoI-R 30tgctcgagtt aaaatatatg acttacctct g 31317784DNAArtificial Sequenceplasmid pMTL85246-ispS-idi-dxs 31tcgaggcctg cagacatgca agcttggcac tggccgtcgt tttacaacgt cgtgactggg 60aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc 120gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg 180aatggcgcta gcataaaaat aagaagcctg catttgcagg cttcttattt ttatggcgcg 240ccgcattcac ttcttttcta tataaatatg agcgaagcga ataagcgtcg gaaaagcagc 300aaaaagtttc ctttttgctg ttggagcatg ggggttcagg gggtgcagta tctgacgtca 360atgccgagcg aaagcgagcc gaagggtagc atttacgtta gataaccccc tgatatgctc 420cgacgcttta tatagaaaag aagattcaac taggtaaaat cttaatatag gttgagatga 480taaggtttat aaggaatttg tttgttctaa tttttcactc attttgttct aatttctttt 540aacaaatgtt cttttttttt tagaacagtt atgatatagt tagaatagtt taaaataagg 600agtgagaaaa agatgaaaga aagatatgga acagtctata aaggctctca gaggctcata 660gacgaagaaa gtggagaagt catagaggta gacaagttat accgtaaaca aacgtctggt 720aacttcgtaa aggcatatat agtgcaatta ataagtatgt tagatatgat tggcggaaaa 780aaacttaaaa tcgttaacta tatcctagat aatgtccact taagtaacaa tacaatgata 840gctacaacaa gagaaatagc aaaagctaca ggaacaagtc tacaaacagt aataacaaca 900cttaaaatct tagaagaagg aaatattata aaaagaaaaa ctggagtatt aatgttaaac 960cctgaactac taatgagagg cgacgaccaa aaacaaaaat acctcttact cgaatttggg 1020aactttgagc aagaggcaaa tgaaatagat tgacctccca ataacaccac gtagttattg 1080ggaggtcaat ctatgaaatg cgattaaggg ccggccagtg ggcaagttga aaaattcaca 1140aaaatgtggt ataatatctt tgttcattag agcgataaac ttgaatttga gagggaactt 1200agatggtatt tgaaaaaatt gataaaaata gttggaacag aaaagagtat tttgaccact 1260actttgcaag tgtaccttgt acctacagca tgaccgttaa agtggatatc acacaaataa 1320aggaaaaggg aatgaaacta tatcctgcaa tgctttatta tattgcaatg attgtaaacc 1380gccattcaga gtttaggacg gcaatcaatc aagatggtga attggggata tatgatgaga 1440tgataccaag ctatacaata tttcacaatg atactgaaac attttccagc ctttggactg 1500agtgtaagtc tgactttaaa tcatttttag cagattatga aagtgatacg caacggtatg 1560gaaacaatca tagaatggaa ggaaagccaa atgctccgga aaacattttt aatgtatcta 1620tgataccgtg gtcaaccttc gatggcttta atctgaattt gcagaaagga tatgattatt 1680tgattcctat ttttactatg gggaaatatt ataaagaaga taacaaaatt atacttcctt 1740tggcaattca agttcatcac gcagtatgtg acggatttca catttgccgt tttgtaaacg 1800aattgcagga attgataaat agttaacttc aggtttgtct gtaactaaaa acaagtattt 1860aagcaaaaac atcgtagaaa tacggtgttt tttgttaccc taagtttaaa ctcctttttg 1920ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 1980tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 2040aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 2100tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt 2160agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 2220taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 2280caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 2340agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 2400aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 2460gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 2520tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 2580gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 2640ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 2700ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 2760aggaagcgga agagcgccca atacgcaggg ccccctgcag gataaaaaaa ttgtagataa 2820attttataaa atagttttat ctacaatttt tttatcagga aacagctatg accgcggccg 2880cggttaatgt taaaaattta tagtataact ttaaaaaact gtcttaaaaa gttgttatat 2940aaaaaatgtt gacaattaaa cagctattta gtgcaaaaca accataaaaa tttaaaaaat 3000accataaatt acttgaaaaa tagttgataa taatgtagag ttataaacaa aggtgaaaag 3060cattacttgt attctttttt atatattatt ataaattaaa atgaagctgt attagaaaaa 3120atacacacct gtaatataaa attttaaatt aatttttaat tttttcaaaa tgtattttac 3180atgtttagaa ttttgatgta tattaaaata gtagaataca taagatactt aatttaatta 3240aagatagtta agtacttttc aatgtgcttt tttagatgtt taatacaaat ctttaattgt 3300aaaagaaatg ctgtactatt tactgtacta gtgacgggat taaactgtat taattataaa 3360taaaaaataa gtacagttgt ttaaaattat attttgtatt aaatctaata gtacgatgta 3420agttatttta tactattgct agtttaataa aaagatttaa ttatatactt gaaaaggaga 3480ggaattttta tgcgtcatat ggcaacagaa ttattatgtt tacacagacc tatatcactt 3540actcacaaac tttttaggaa tccattacct aaagttattc aagctacacc tttaacatta 3600aaacttaggt gtagtgtttc tacagaaaat gtatcattta gtgagacaga aactgaaaca 3660agaagatcag caaattatga accaaattct tgggattatg attatcttct ttcttctgat 3720actgatgagt caatagaagt acataaagat aaggctaaga aattagaagc tgaagttagg 3780agagaaataa ataatgagaa ggctgaattt cttacacttc ttgaacttat tgataatgta 3840caaagacttg gattaggata tagatttgag tctgatataa gaagagcatt agatagattt 3900gtaagtagtg gaggatttga tggagttact aaaacttcat tacatggaac agcattatca 3960tttaggttat taaggcaaca tggttttgaa gtatctcaag aagcttttag tggatttaaa 4020gatcagaatg gaaactttct tgagaattta aaggaagaca taaaagcaat tctttctctt 4080tatgaagcat catttttagc attagaaggt gagaatatat tagatgaggc taaagtattt 4140gcaatatctc atcttaaaga acttagtgaa gaaaagattg gtaaagaatt agctgaacaa 4200gtttcacatg ctttagaatt accattacat agaagaacac aaagattaga agcagtttgg 4260tcaatagaag catatagaaa gaaagaagac gcaaatcaag tacttttaga acttgcaata 4320cttgactaca atatgattca aagtgtatat cagagggatt taagagaaac atcaagatgg 4380tggagaagag taggattagc aactaaatta cattttgcta gagataggct tattgaaagt 4440ttttattggg ctgttggagt tgcttttgaa ccacaatatt ctgattgcag aaatagtgta 4500gcaaagatgt tttcatttgt tactataatt gacgatattt acgatgtata tggaacttta 4560gatgaacttg aactttttac tgatgcagtt gaaagatggg atgtaaatgc tattaatgat 4620cttcctgatt atatgaagtt atgttttctt gcactttaca atactattaa cgagatagct 4680tacgataact taaaagataa aggtgagaac atacttcctt atttaacaaa agcatgggca 4740gatttatgta atgcatttct tcaagaagct aagtggcttt ataataaatc aacacctaca 4800tttgatgatt attttggaaa tgcatggaaa agttctagtg gacctttaca gcttattttt 4860gcttattttg ctgtagtaca gaacattaaa aaggaagaga ttgagaatct tcagaaatat 4920catgacataa tatcaagacc tagtcacatt tttaggcttt gtaatgattt agcatctgct 4980tcagcagaaa tagcaagagg tgaaactgct aattctgtaa gttgttatat gagaacaaaa 5040ggtatatctg aagaattagc tactgaaagt gttatgaatc ttatagacga aacttggaag 5100aaaatgaaca aagaaaaact tggtggatct ttatttgcaa aaccttttgt tgagactgct 5160ataaatttag ctagacagtc tcattgcaca tatcataatg gtgatgcaca tactagtcca 5220gatgaattaa ctaggaaaag agtacttagt gtaataactg aaccaatatt accatttgaa 5280agataagaat tcgagctcga aaggggaaat taaatggcag aatatataat agctgtagat 5340gaatttgata acgaaatagg ttcaattgaa aaaatggagg ctcaccgtaa aggaacatta 5400catagagctt tttctatatt agtatttaat tctaaaaatc aattgttatt acagaaaaga 5460aatgtaaaaa aatatcattc gcctggtctc tggacaaata cgtgctgtag tcatccaaga 5520tacggtgaaa gtttacatga tgcgatttat agaaggctta aggaagaaat gggttttaca 5580tgtgaacttg aagaagtatt tagttttatt tataaagtaa aacttgaaga taatcttttt 5640gaaaatgaat atgatcatgt attcattggg aaatatgatg gagaaataat tgtaaacaaa 5700gatgaagtag atgattttaa gtgggttgat attaatgagg ttaagaagga tattatagaa 5760aggccagaag catacactta ttggttcaag tatttagtta ataaggcaga aaacaaaata 5820tttaaataag taagaatttc gtctaaataa agatttgggg tacccgggga tcctctagag 5880tcgactttat taaagggata gataaggatg agtaatttat tagataatta taaagatata 5940aatgacgtaa agaagatgtc gttaaatgat aaaaaaaagc tagctagaga aattagaaaa 6000tttttaatag acaaagtatc taagacagga ggtcatttgg cgtctaactt aggggttgtg 6060gagctcactt tgagtttatt tagtgtattt gatctaaatt atgataaact tatatgggat 6120gtgggacatc aggcttatgt gcataaaatc ctcacgggaa gaaaggataa atttgatact 6180ttaaggcaat ttggaggatt aagtggattt cctaaaaggt gcgaaagtat atatgatttt 6240ttcgaaacag ggcatagtag tacttcaata tctgcagcac ttggaatggc tagggctaga 6300gatttaaagc atgagaaata taatgttgtt gcagttatag gagatggagc acttactgga 6360ggtatggcac tagaggccct aaatgatgta ggttatagaa aaactaagct tataataata 6420ttaaatgata atcaaatgtc tataggaaaa aatgtaggtg gagtatctaa atatttaaat 6480aaacttagag tggaccctaa gtataataaa tttaaagcgg atgtagaagc taaattaaaa 6540aagataccta atataggaaa aggaatggca aaatatcttg aaaaggtaaa aaatggaata 6600aaacaaatgg tagttcctgg aatgtttttt gaagatatgg gaattaaata tttaggacca 6660atagatggtc ataatataaa agaacttaca gacgtactcg cttctgcaaa agacatacaa 6720ggtccagtta ttatacatat aataactaag aaaggaaaag gatatgaatt tgcaagaaaa 6780aaatccaggt aaattccatg gaatagggcc ttttaattgc gccaatggtg aactggatgc 6840tggatcttca aatacttatt ccaaggcctt tggaaatgaa atggtaaagc tagcagaaaa 6900agacgataga atagtggcta taactgcagc catgagggat ggaacaggtc ttaaaagttt 6960ttctcaaaag tttcctgaaa ggttttttga tgtgggaata gcagaacagc atgctgtaac 7020cctggcagct ggaatggcac aggcaaattt aaaacctgta tttgcagttt actctacttt 7080tcttcaaaga gcttatgatc aacttattca tgatgtatgt atgcaaaaac ttccagtagt 7140ttttgctgta gatagggccg gcattgtagg agaagatggt gaaacacatc agggaatatt 7200tgatttatct tacttaacgg aaatgccaca tatgacgctt atgtctccta aatgtataga 7260tgaacttcca tatatgttaa aatgggcatt aggccagagt tttcctgtag ctataaggta 7320tccaagggga ggagatagtg tatgtctcaa tcccgtagaa aattttaaac ttggaaagtg 7380ggactgtatt tcaaatgaag gcagtgtagc aataattgct cagggtaaaa tggtacaaaa 7440tgcagtgtta gcaggaaaaa aacttaaaga aaagggtata gatgtaagga ttataagtgc 7500atgttttatt aagccgctgg acaaggaaat gttaaacagg ttagttgaag aaagtgtaac 7560tatcgttact gttgaagaca atgtaataag aggaggatta ggatcctata tattagaata 7620tgtaaataaa ttaaataaaa aagtaaaaat aataaactta gggtttgatg ataagtttgt 7680acagcatgga aaatccgata ttttgtataa gctgtatggt ttggatccta aaggtatcgt 7740aaatagtgta cttgaagcag cagaggtaag tcatatattt taac 77843217DNAArtificial SequenceOligonucleotide M13R 32caggaaacag ctatgac 173320DNAArtificial SequenceOligonucleotides Isoprene-seq1 33gttattcaag ctacaccttt 203420DNAArtificial SequenceOligonucleotide Isoprene-seq2 34gattggtaaa gaattagctg 203518DNAArtificial SequenceOligonucleotide Isoprene-seq3 35tcaagaagct aagtggct 183617DNAArtificial SequenceOligonucleotide Isoprene-seq4 36ctcaccgtaa aggaaca 173720DNAArtificial SequenceOligonucleotide Isoprene-seq5 37gctagctaga gaaattagaa 203820DNAArtificial SequenceOligonucleotide Isoprene-seq6 38ggaatggcaa aatatcttga 203920DNAArtificial SequenceOligonucleotide Isoprene-seq7 39gaaacacatc agggaatatt 20401179DNAClostridium acetobutylicum 40atgaaagagg ttgttattgc atcagctgtt agaactgcaa taggatctta tggaaaaagt 60cttaaagatg taccagcagt agacttaggt gcaactgcaa taaaggaagc agtaaagaaa 120gcaggtataa aacctgaaga tgttaatgaa gttattttag gaaacgtatt acaagctgga 180cttggacaga atccagctag acaggcatca ttcaaagcag gattaccagt agagatacct 240gctatgacta ttaataaagt ttgtggttca ggattaagaa cagtttcttt agctgctcaa 300attataaaag ctggtgacgc agatgtaata atagcaggtg gtatggaaaa tatgtcaaga 360gcaccatacc ttgctaataa tgctagatgg ggttatagaa tgggaaacgc taaatttgta 420gacgaaatga taactgatgg actttgggat gcatttaacg attatcacat gggaattact 480gctgaaaata tagctgagag atggaatata agtagagaag aacaagatga gtttgcactt 540gcatctcaga aaaaggcaga agaagctatt aaatcaggac aatttaaaga tgaaattgtt 600ccagtagtaa ttaaaggtag aaaaggtgaa acagttgtag acactgatga acatcctaga 660tttggatcta caatagaagg tttagctaaa ttaaagcctg cttttaagaa agacggaaca 720gtaactgctg gaaacgcatc aggtttaaat gattgtgcag ctgttttagt tattatgtct 780gctgaaaagg caaaggaatt aggtgttaaa ccacttgcta agatagttag ttatggttca 840gcaggtgtag atcctgctat tatgggatat ggaccttttt atgctacaaa ggcagctatt 900gaaaaggctg gttggacagt tgatgaactt gatcttatag agtcaaatga ggcatttgca 960gcacaaagtc ttgctgttgc taaggatctt aaattcgata tgaataaagt aaatgtaaac 1020ggtggtgcta tagcacttgg tcatccaata ggtgctagtg gtgctagaat tttagttaca 1080ttagttcatg caatgcaaaa gagagacgct aaaaagggac ttgcaacttt atgcataggt 1140ggtggtcaag gaacagcaat acttcttgaa aaatgttaa 1179411140DNAStaphylococcus aureus subsp. aureus 41atgaatcagg ctgttatagt tgctgcaaag agaactgcat ttggaaaata tggtggtact 60ttaaaacacc ttgaaccaga acaattactt aagccattat ttcagcactt taaagaaaaa 120tatcctgaag ttatatctaa aatagatgat gtagttttag gaaacgtagt tggaaatggt 180ggtaatattg ctagaaaagc acttcttgaa gcaggattaa aagatagtat accaggtgta 240actatagaca gacaatgcgg atctggatta gaatcagtac aatacgcatg tagaatgata 300caagcaggtg ctggaaaagt ttatattgca ggtggtgttg aatctacatc aagagcacct 360tggaaaataa agagaccaca ttctgtttat gaaactgcat taccagagtt ctatgaaaga 420gcatcattcg cacctgaaat gtcagatcca agtatgatac aaggtgctga gaatgtagct 480aaaatgtatg atgttagtag agaacttcaa gatgagtttg catacagatc acatcaactt 540acagctgaaa atgtaaagaa tggaaatatt tcacaagaaa ttcttccaat aacagtaaag 600ggtgaaatat tcaatactga tgaaagttta aaatctcata ttccaaaaga taatttcggt 660agatttaaac ctgtaataaa aggtggtact gtaacagctg ctaatagttg tatgaagaac 720gatggtgcag tattattact tattatggaa aaggatatgg cttatgaact tggatttgag 780catggattat tatttaaaga cggtgtaact gtaggtgtag atagtaactt tccaggaata 840ggacctgttc ctgctatatc aaatctttta aagagaaacc aacttacaat agaaaatatt 900gaagtaattg agataaatga agcattttct gctcaggtag ttgcttgtca gcaagctctt 960aatataagta atactcagtt aaacatttgg ggtggtgcat tagcaagtgg tcatccttat 1020ggtgcatcag gtgcacagtt agttacaaga ttattttata tgttcgataa agagacaatg 1080attgcttcta tgggaatagg tggtggttta ggaaatgcag ctctttttac tagattttaa 1140421167DNAStaphylococcus aureus subsp. aureus 42atgactatag gaattgacaa aataaacttt tacgtaccaa aatattatgt agatatggca 60aaattagcag aagcaagaca agtagaccca aataaatttc ttattggaat aggacagact 120gaaatggcag ttagtccagt aaaccaagat atagtatcaa tgggtgctaa tgctgctaaa 180gatataataa ctgatgaaga caaaaagaaa ataggaatgg taatagtagc aactgagtca 240gcagtagatg cagcaaaggc agcagcagta cagattcata atttattagg tattcaacca 300tttgcaagat gtttcgaaat gaaagaagca tgttatgctg ctactcctgc aattcagtta 360gctaaggatt atttagctac aagaccaaat gagaaagttt tagttatagc tacagataca 420gctagatatg gacttaattc aggtggtgaa cctactcaag gtgctggtgc tgttgctatg 480gttatagctc ataatcctag tatacttgca ttaaatgaag acgctgttgc ttatacagaa 540gatgtttatg atttctggag accaacagga cataagtatc cattagtaga tggtgcttta 600tcaaaagacg catatattag atcttttcaa caatcttgga atgaatatgc taagagacaa 660ggaaagagtt tagctgattt tgctagtctt tgctttcatg ttccttttac taaaatgggt 720aaaaaggctt tagaatctat aatagataac gcagatgaaa caactcaaga gagattaaga 780tctggatatg aagatgcagt tgattacaat agatatgttg gaaatatata cacaggaagt 840ctttatcttt ctcttataag tcttcttgaa aatagagatt tacaggctgg tgaaactatt 900ggattatttt catacggatc aggttctgtt ggtgaatttt attcagctac acttgtagaa 960ggatataaag atcaccttga tcaggcagca cacaaagcac

ttttaaacaa tagaactgaa 1020gtatcagtag atgcatacga aacatttttc aagagatttg atgatgtaga atttgatgaa 1080gagcaggatg cagttcatga agatagacat atattctatc tttcaaacat agagaataat 1140gtaagagaat atcatagacc tgaataa 1167431278DNAStaphylococcus aureus subsp. aureus 43atgcaatcat tagacaaaaa tttcagacat ttatcaagac aacaaaagtt acaacaatta 60gttgataaac agtggctttc agaagatcag tttgatattt tacttaatca tcctcttata 120gatgaagaag ttgctaatag tcttatagaa aatgtaattg cacagggtgc attaccagtt 180ggacttcttc ctaatataat agttgatgat aaggcttatg ttgtaccaat gatggttgaa 240gaacctagtg ttgttgcagc tgcatcttat ggtgctaaat tagtaaatca gacaggtgga 300tttaaaactg tatcatcaga aagaataatg attggacaga tagtatttga tggtgtagat 360gacactgaaa aattaagtgc agatattaaa gcattagaaa aacaaataca taagattgca 420gatgaagcat atcctagtat aaaagcaaga ggtggtggtt atcaaagaat agcaatagat 480acatttccag agcaacaact tttaagtctt aaggtatttg tagatacaaa agatgctatg 540ggtgctaata tgcttaatac tatacttgag gcaataactg cattccttaa aaatgaatct 600cctcaatcag atatattaat gtctatactt tcaaaccatg caactgctag tgtagtaaaa 660gtacaaggtg agatagatgt aaaagatctt gctagaggtg aaagaacagg tgaagaagta 720gctaagagaa tggaaagagc ttctgtatta gctcaggttg atattcatag agctgcaaca 780cataacaaag gtgttatgaa tggaatacat gctgttgttt tagctacagg aaatgatact 840agaggtgctg aagcatctgc acatgcatac gcatcaagag acggacaata tagaggtata 900gcaacttgga gatatgatca gaagagacaa agacttattg gaactattga agttccaatg 960acacttgcta tagtaggtgg tggtactaaa gtattaccaa tagctaaggc atcattagag 1020ttattaaatg ttgattctgc acaagaactt ggacacgtag ttgctgctgt tggattagca 1080caaaactttg ctgcttgtag agcacttgtt tctgaaggta ttcaacaagg acacatgtca 1140ttacaatata aaagtttagc aatagtagta ggtgcaaaag gtgacgagat agcacaagta 1200gcagaagctc ttaaacagga accaagagct aatacacagg ttgctgaaag aattttacag 1260gaaattagac agcaataa 127844552DNAClostridium autoethanogenum 44agatagtcat aatagttcca gaatagttca atttagaaat tagactaaac ttcaaaatgt 60ttgttaaata tataccaatc tagtatagat attttttaaa tactggactt aaacagtagt 120aatttgccta aaaaattttt tcaatttttt ttaaaaaatc cttttcaagt tgtacattgt 180tatggtaata tgtaattgaa gaagttatgt agtaatattg taaacgtttc ttgatttttt 240tacatccatg tagtgcttaa aaaaccaaaa tatgtcacat gcacttgtat atttcaaata 300acaatattta ttttctcgtt aaattcacaa ataatttatt aataatatca ataaccaaga 360ttatacttaa atggatgttt attttttaac acttttatag taaatatatt tattttatgt 420agtaaaaagg ttataattat aattgtattt attacaatta attaaaataa aaaatagggt 480tttaggtaaa attaagttat tttaagaagt aattacaata aaaattgaag ttatttcttt 540aaggaggaaa tt 55245386DNAClostridium autoethanogenum 45gttataattt tcaattttca ttctttttaa aggagattag catacatttt atcataatta 60tacagacaat atagtaatat atgatgttaa aatatcaata tatggttaaa aatctgtata 120ttttttccca ttttaattat ttgtactata atattacact gagtgtattg catatttaaa 180aaatatttgg tacaattagt tagttaaata aattctaaat tgtaaattat cagaatcctt 240attaaggaaa tacatagatt taaggagaaa tcataaaaag gtgtaatata aactggctaa 300aattgagcaa aaattgagca attaagactt tttgattgta tctttttata tatttaaggt 360atataatctt atttatattg ggggaa 3864621DNAArtificial Sequenceoligonucleotide pUC57-F 46agcagattgt actgagagtg c 214721DNAArtificial Sequenceoligonucleotide pUC57-R 47acagctatga ccatgattac g 21483552DNAArtificial Sequenceplasmid pMTL 85245 48aaactccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 60gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 120atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 180gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 240gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 300tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 360accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 420ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 480cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 540agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 600ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 660tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 720ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 780cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 840gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca gggccccctg caggataaaa 900aaattgtaga taaattttat aaaatagttt tatctacaat ttttttatca ggaaacagct 960atgaccgcgg ccgcaatatg atatttatgt ccattgtgaa agggattata ttcaactatt 1020attccagtta cgttcataga aattttcctt tctaaaatat tttattccat gtcaagaact 1080ctgtttattt cattaaagaa ctataagtac aaagtataag gcatttgaaa aaataggcta 1140gtatattgat tgattattta ttttaaaatg cctaagtgaa atatatacat attataacaa 1200taaaataagt attagtgtag gatttttaaa tagagtatct attttcagat taaatttttg 1260attatttgat ttacattata taatattgag taaagtattg actagcaaaa ttttttgata 1320ctttaatttg tgaaatttct tatcaaaagt tatatttttg aataattttt attgaaaaat 1380acaactaaaa aggattatag tataagtgtg tgtaattttg tgttaaattt aaagggagga 1440aatgaacatg aaacatatgg tgaccatgat tacgaattcg agctcggtac ccggggatcc 1500tctagagtcg acgtcacgcg tccatggaga tctcgaggcc tgcagacatg caagcttggc 1560actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg 1620ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg 1680cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc tagcataaaa ataagaagcc 1740tgcatttgca ggcttcttat ttttatggcg cgccgcattc acttcttttc tatataaata 1800tgagcgaagc gaataagcgt cggaaaagca gcaaaaagtt tcctttttgc tgttggagca 1860tgggggttca gggggtgcag tatctgacgt caatgccgag cgaaagcgag ccgaagggta 1920gcatttacgt tagataaccc cctgatatgc tccgacgctt tatatagaaa agaagattca 1980actaggtaaa atcttaatat aggttgagat gataaggttt ataaggaatt tgtttgttct 2040aatttttcac tcattttgtt ctaatttctt ttaacaaatg ttcttttttt tttagaacag 2100ttatgatata gttagaatag tttaaaataa ggagtgagaa aaagatgaaa gaaagatatg 2160gaacagtcta taaaggctct cagaggctca tagacgaaga aagtggagaa gtcatagagg 2220tagacaagtt ataccgtaaa caaacgtctg gtaacttcgt aaaggcatat atagtgcaat 2280taataagtat gttagatatg attggcggaa aaaaacttaa aatcgttaac tatatcctag 2340ataatgtcca cttaagtaac aatacaatga tagctacaac aagagaaata gcaaaagcta 2400caggaacaag tctacaaaca gtaataacaa cacttaaaat cttagaagaa ggaaatatta 2460taaaaagaaa aactggagta ttaatgttaa accctgaact actaatgaga ggcgacgacc 2520aaaaacaaaa atacctctta ctcgaatttg ggaactttga gcaagaggca aatgaaatag 2580attgacctcc caataacacc acgtagttat tgggaggtca atctatgaaa tgcgattaag 2640ggccggccga agcaaactta agagtgtgtt gatagtgcag tatcttaaaa ttttgtataa 2700taggaattga agttaaatta gatgctaaaa atttgtaatt aagaaggagt gattacatga 2760acaaaaatat aaaatattct caaaactttt taacgagtga aaaagtactc aaccaaataa 2820taaaacaatt gaatttaaaa gaaaccgata ccgtttacga aattggaaca ggtaaagggc 2880atttaacgac gaaactggct aaaataagta aacaggtaac gtctattgaa ttagacagtc 2940atctattcaa cttatcgtca gaaaaattaa aactgaatac tcgtgtcact ttaattcacc 3000aagatattct acagtttcaa ttccctaaca aacagaggta taaaattgtt gggagtattc 3060cttaccattt aagcacacaa attattaaaa aagtggtttt tgaaagccat gcgtctgaca 3120tctatctgat tgttgaagaa ggattctaca agcgtacctt ggatattcac cgaacactag 3180ggttgctctt gcacactcaa gtctcgattc agcaattgct taagctgcca gcggaatgct 3240ttcatcctaa accaaaagta aacagtgtct taataaaact tacccgccat accacagatg 3300ttccagataa atattggaag ctatatacgt actttgtttc aaaatgggtc aatcgagaat 3360atcgtcaact gtttactaaa aatcagtttc atcaagcaat gaaacacgcc aaagtaaaca 3420atttaagtac cgttacttat gagcaagtat tgtctatttt taatagttat ctattattta 3480acgggaggaa ataattctat gagtcgcttt tgtaaatttg gaaagttaca cgttactaaa 3540gggaatgtgt tt 3552494186DNAArtificial Sequenceplasmid pMTL 83145 49aaactccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 60gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 120atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 180gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 240gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 300tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 360accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 420ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 480cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 540agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 600ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 660tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 720ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 780cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 840gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca gggccccctg caggataaaa 900aaattgtaga taaattttat aaaatagttt tatctacaat ttttttatca ggaaacagct 960atgaccgcgg ccgcaatatg atatttatgt ccattgtgaa agggattata ttcaactatt 1020attccagtta cgttcataga aattttcctt tctaaaatat tttattccat gtcaagaact 1080ctgtttattt cattaaagaa ctataagtac aaagtataag gcatttgaaa aaataggcta 1140gtatattgat tgattattta ttttaaaatg cctaagtgaa atatatacat attataacaa 1200taaaataagt attagtgtag gatttttaaa tagagtatct attttcagat taaatttttg 1260attatttgat ttacattata taatattgag taaagtattg actagcaaaa ttttttgata 1320ctttaatttg tgaaatttct tatcaaaagt tatatttttg aataattttt attgaaaaat 1380acaactaaaa aggattatag tataagtgtg tgtaattttg tgttaaattt aaagggagga 1440aatgaacatg aaacatatgg tgaccatgat tacgaattcg agctcggtac ccggggatcc 1500tctagagtcg acgtcacgcg tccatggaga tctcgaggcc tgcagacatg caagcttggc 1560actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg 1620ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg 1680cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc tagcataaaa ataagaagcc 1740tgcatttgca ggcttcttat ttttatggcg cgccgccatt atttttttga acaattgaca 1800attcatttct tattttttat taagtgatag tcaaaaggca taacagtgct gaatagaaag 1860aaatttacag aaaagaaaat tatagaattt agtatgatta attatactca tttatgaatg 1920tttaattgaa tacaaaaaaa aatacttgtt atgtattcaa ttacgggtta aaatatagac 1980aagttgaaaa atttaataaa aaaataagtc ctcagctctt atatattaag ctaccaactt 2040agtatataag ccaaaactta aatgtgctac caacacatca agccgttaga gaactctatc 2100tatagcaata tttcaaatgt accgacatac aagagaaaca ttaactatat atattcaatt 2160tatgagatta tcttaacaga tataaatgta aattgcaata agtaagattt agaagtttat 2220agcctttgtg tattggaagc agtacgcaaa ggctttttta tttgataaaa attagaagta 2280tatttatttt ttcataatta atttatgaaa atgaaagggg gtgagcaaag tgacagagga 2340aagcagtatc ttatcaaata acaaggtatt agcaatatca ttattgactt tagcagtaaa 2400cattatgact tttatagtgc ttgtagctaa gtagtacgaa agggggagct ttaaaaagct 2460ccttggaata catagaattc ataaattaat ttatgaaaag aagggcgtat atgaaaactt 2520gtaaaaattg caaagagttt attaaagata ctgaaatatg caaaatacat tcgttgatga 2580ttcatgataa aacagtagca acctattgca gtaaatacaa tgagtcaaga tgtttacata 2640aagggaaagt ccaatgtatt aattgttcaa agatgaaccg atatggatgg tgtgccataa 2700aaatgagatg ttttacagag gaagaacaga aaaaagaacg tacatgcatt aaatattatg 2760caaggagctt taaaaaagct catgtaaaga agagtaaaaa gaaaaaataa tttatttatt 2820aatttaatat tgagagtgcc gacacagtat gcactaaaaa atatatctgt ggtgtagtga 2880gccgatacaa aaggatagtc actcgcattt tcataataca tcttatgtta tgattatgtg 2940tcggtgggac ttcacgacga aaacccacaa taaaaaaaga gttcggggta gggttaagca 3000tagttgaggc aactaaacaa tcaagctagg atatgcagta gcagaccgta aggtcgttgt 3060ttaggtgtgt tgtaatacat acgctattaa gatgtaaaaa tacggatacc aatgaaggga 3120aaagtataat ttttggatgt agtttgtttg ttcatctatg ggcaaactac gtccaaagcc 3180gtttccaaat ctgctaaaaa gtatatcctt tctaaaatca aagtcaagta tgaaatcata 3240aataaagttt aattttgaag ttattatgat attatgtttt tctattaaaa taaattaagt 3300atatagaata gtttaataat agtatatact taatgtgata agtgtctgac agtgtcacag 3360aaaggatgat tgttatggat tataagcggc cggccagtgg gcaagttgaa aaattcacaa 3420aaatgtggta taatatcttt gttcattaga gcgataaact tgaatttgag agggaactta 3480gatggtattt gaaaaaattg ataaaaatag ttggaacaga aaagagtatt ttgaccacta 3540ctttgcaagt gtaccttgta cctacagcat gaccgttaaa gtggatatca cacaaataaa 3600ggaaaaggga atgaaactat atcctgcaat gctttattat attgcaatga ttgtaaaccg 3660ccattcagag tttaggacgg caatcaatca agatggtgaa ttggggatat atgatgagat 3720gataccaagc tatacaatat ttcacaatga tactgaaaca ttttccagcc tttggactga 3780gtgtaagtct gactttaaat catttttagc agattatgaa agtgatacgc aacggtatgg 3840aaacaatcat agaatggaag gaaagccaaa tgctccggaa aacattttta atgtatctat 3900gataccgtgg tcaaccttcg atggctttaa tctgaatttg cagaaaggat atgattattt 3960gattcctatt tttactatgg ggaaatatta taaagaagat aacaaaatta tacttccttt 4020ggcaattcaa gttcatcacg cagtatgtga cggatttcac atttgccgtt ttgtaaacga 4080attgcaggaa ttgataaata gttaacttca ggtttgtctg taactaaaaa caagtattta 4140agcaaaaaca tcgtagaaat acggtgtttt ttgttaccct aagttt 4186509827DNAArtificial Sequenceplasmid pMTL8215-Pptaack-thlA-HMGS-Patp-HMGR 50cctgcaggat aaaaaaattg tagataaatt ttataaaata gttttatcta caattttttt 60atcaggaaac agctatgacc gcggccgcag atagtcataa tagttccaga atagttcaat 120ttagaaatta gactaaactt caaaatgttt gttaaatata taccaatcta gtatagatat 180tttttaaata ctggacttaa acagtagtaa tttgcctaaa aaattttttc aatttttttt 240aaaaaatcct tttcaagttg tacattgtta tggtaatatg taattgaaga agttatgtag 300taatattgta aacgtttctt gattttttta catccatgta gtgcttaaaa aaccaaaata 360tgtcacatgc acttgtatat ttcaaataac aatatttatt ttctcgttaa attcacaaat 420aatttattaa taatatcaat aaccaagatt atacttaaat ggatgtttat tttttaacac 480ttttatagta aatatattta ttttatgtag taaaaaggtt ataattataa ttgtatttat 540tacaattaat taaaataaaa aatagggttt taggtaaaat taagttattt taagaagtaa 600ttacaataaa aattgaagtt atttctttaa ggaggaaatt catatgaaag aggttgttat 660tgcatcagct gttagaactg caataggatc ttatggaaaa agtcttaaag atgtaccagc 720agtagactta ggtgcaactg caataaagga agcagtaaag aaagcaggta taaaacctga 780agatgttaat gaagttattt taggaaacgt attacaagct ggacttggac agaatccagc 840tagacaggca tcattcaaag caggattacc agtagagata cctgctatga ctattaataa 900agtttgtggt tcaggattaa gaacagtttc tttagctgct caaattataa aagctggtga 960cgcagatgta ataatagcag gtggtatgga aaatatgtca agagcaccat accttgctaa 1020taatgctaga tggggttata gaatgggaaa cgctaaattt gtagacgaaa tgataactga 1080tggactttgg gatgcattta acgattatca catgggaatt actgctgaaa atatagctga 1140gagatggaat ataagtagag aagaacaaga tgagtttgca cttgcatctc agaaaaaggc 1200agaagaagct attaaatcag gacaatttaa agatgaaatt gttccagtag taattaaagg 1260tagaaaaggt gaaacagttg tagacactga tgaacatcct agatttggat ctacaataga 1320aggtttagct aaattaaagc ctgcttttaa gaaagacgga acagtaactg ctggaaacgc 1380atcaggttta aatgattgtg cagctgtttt agttattatg tctgctgaaa aggcaaagga 1440attaggtgtt aaaccacttg ctaagatagt tagttatggt tcagcaggtg tagatcctgc 1500tattatggga tatggacctt tttatgctac aaaggcagct attgaaaagg ctggttggac 1560agttgatgaa cttgatctta tagagtcaaa tgaggcattt gcagcacaaa gtcttgctgt 1620tgctaaggat cttaaattcg atatgaataa agtaaatgta aacggtggtg ctatagcact 1680tggtcatcca ataggtgcta gtggtgctag aattttagtt acattagttc atgcaatgca 1740aaagagagac gctaaaaagg gacttgcaac tttatgcata ggtggtggtc aaggaacagc 1800aatacttctt gaaaaatgtt aagaattcga ggcttttact aaaaacaata aaaacaggag 1860gaaataatat gactatagga attgacaaaa taaactttta cgtaccaaaa tattatgtag 1920atatggcaaa attagcagaa gcaagacaag tagacccaaa taaatttctt attggaatag 1980gacagactga aatggcagtt agtccagtaa accaagatat agtatcaatg ggtgctaatg 2040ctgctaaaga tataataact gatgaagaca aaaagaaaat aggaatggta atagtagcaa 2100ctgagtcagc agtagatgca gcaaaggcag cagcagtaca gattcataat ttattaggta 2160ttcaaccatt tgcaagatgt ttcgaaatga aagaagcatg ttatgctgct actcctgcaa 2220ttcagttagc taaggattat ttagctacaa gaccaaatga gaaagtttta gttatagcta 2280cagatacagc tagatatgga cttaattcag gtggtgaacc tactcaaggt gctggtgctg 2340ttgctatggt tatagctcat aatcctagta tacttgcatt aaatgaagac gctgttgctt 2400atacagaaga tgtttatgat ttctggagac caacaggaca taagtatcca ttagtagatg 2460gtgctttatc aaaagacgca tatattagat cttttcaaca atcttggaat gaatatgcta 2520agagacaagg aaagagttta gctgattttg ctagtctttg ctttcatgtt ccttttacta 2580aaatgggtaa aaaggcttta gaatctataa tagataacgc agatgaaaca actcaagaga 2640gattaagatc tggatatgaa gatgcagttg attacaatag atatgttgga aatatataca 2700caggaagtct ttatctttct cttataagtc ttcttgaaaa tagagattta caggctggtg 2760aaactattgg attattttca tacggatcag gttctgttgg tgaattttat tcagctacac 2820ttgtagaagg atataaagat caccttgatc aggcagcaca caaagcactt ttaaacaata 2880gaactgaagt atcagtagat gcatacgaaa catttttcaa gagatttgat gatgtagaat 2940ttgatgaaga gcaggatgca gttcatgaag atagacatat attctatctt tcaaacatag 3000agaataatgt aagagaatat catagacctg aataagagct cgttataatt ttcaattttc 3060attcttttta aaggagatta gcatacattt tatcataatt atacagacaa tatagtaata 3120tatgatgtta aaatatcaat atatggttaa aaatctgtat attttttccc attttaatta 3180tttgtactat aatattacac tgagtgtatt gcatatttaa aaaatatttg gtacaattag 3240ttagttaaat aaattctaaa ttgtaaatta tcagaatcct tattaaggaa atacatagat 3300ttaaggagaa atcataaaaa ggtgtaatat aaactggcta aaattgagca aaaattgagc 3360aattaagact ttttgattgt atctttttat atatttaagg tatataatct tatttatatt 3420gggggaaggt accatgcaat cattagacaa aaatttcaga catttatcaa gacaacaaaa 3480gttacaacaa ttagttgata aacagtggct ttcagaagat cagtttgata ttttacttaa 3540tcatcctctt atagatgaag aagttgctaa tagtcttata gaaaatgtaa ttgcacaggg 3600tgcattacca gttggacttc ttcctaatat aatagttgat gataaggctt atgttgtacc 3660aatgatggtt gaagaaccta gtgttgttgc agctgcatct tatggtgcta aattagtaaa 3720tcagacaggt ggatttaaaa ctgtatcatc agaaagaata atgattggac agatagtatt 3780tgatggtgta gatgacactg aaaaattaag tgcagatatt aaagcattag aaaaacaaat 3840acataagatt gcagatgaag catatcctag tataaaagca agaggtggtg gttatcaaag 3900aatagcaata gatacatttc cagagcaaca acttttaagt cttaaggtat ttgtagatac 3960aaaagatgct atgggtgcta atatgcttaa tactatactt gaggcaataa ctgcattcct 4020taaaaatgaa tctcctcaat cagatatatt aatgtctata ctttcaaacc atgcaactgc 4080tagtgtagta aaagtacaag gtgagataga tgtaaaagat cttgctagag gtgaaagaac 4140aggtgaagaa gtagctaaga gaatggaaag agcttctgta ttagctcagg ttgatattca 4200tagagctgca acacataaca aaggtgttat

gaatggaata catgctgttg ttttagctac 4260aggaaatgat actagaggtg ctgaagcatc tgcacatgca tacgcatcaa gagacggaca 4320atatagaggt atagcaactt ggagatatga tcagaagaga caaagactta ttggaactat 4380tgaagttcca atgacacttg ctatagtagg tggtggtact aaagtattac caatagctaa 4440ggcatcatta gagttattaa atgttgattc tgcacaagaa cttggacacg tagttgctgc 4500tgttggatta gcacaaaact ttgctgcttg tagagcactt gtttctgaag gtattcaaca 4560aggacacatg tcattacaat ataaaagttt agcaatagta gtaggtgcaa aaggtgacga 4620gatagcacaa gtagcagaag ctcttaaaca ggaaccaaga gctaatacac aggttgctga 4680aagaatttta caggaaatta gacagcaata atctagagtc gacgtcacgc gtccatggag 4740atctcgaggc ctgcagacat gcaagcttgg cactggccgt cgttttacaa cgtcgtgact 4800gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct 4860ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg 4920gcgaatggcg ctagcataaa aataagaagc ctgcatttgc aggcttctta tttttatggc 4980gcgccgttct gaatccttag ctaatggttc aacaggtaac tatgacgaag atagcaccct 5040ggataagtct gtaatggatt ctaaggcatt taatgaagac gtgtatataa aatgtgctaa 5100tgaaaaagaa aatgcgttaa aagagcctaa aatgagttca aatggttttg aaattgattg 5160gtagtttaat ttaatatatt ttttctattg gctatctcga tacctataga atcttctgtt 5220cacttttgtt tttgaaatat aaaaaggggc tttttagccc ctttttttta aaactccgga 5280ggagtttctt cattcttgat actatacgta actattttcg atttgacttc attgtcaatt 5340aagctagtaa aatcaatggt taaaaaacaa aaaacttgca tttttctacc tagtaattta 5400taattttaag tgtcgagttt aaaagtataa tttaccagga aaggagcaag ttttttaata 5460aggaaaaatt tttcctttta aaattctatt tcgttatatg actaattata atcaaaaaaa 5520tgaaaataaa caagaggtaa aaactgcttt agagaaatgt actgataaaa aaagaaaaaa 5580tcctagattt acgtcataca tagcaccttt aactactaag aaaaatattg aaaggacttc 5640cacttgtgga gattatttgt ttatgttgag tgatgcagac ttagaacatt ttaaattaca 5700taaaggtaat ttttgcggta atagattttg tccaatgtgt agttggcgac ttgcttgtaa 5760ggatagttta gaaatatcta ttcttatgga gcatttaaga aaagaagaaa ataaagagtt 5820tatattttta actcttacaa ctccaaatgt aaaaagttat gatcttaatt attctattaa 5880acaatataat aaatctttta aaaaattaat ggagcgtaag gaagttaagg atataactaa 5940aggttatata agaaaattag aagtaactta ccaaaaggaa aaatacataa caaaggattt 6000atggaaaata aaaaaagatt attatcaaaa aaaaggactt gaaattggtg atttagaacc 6060taattttgat acttataatc ctcattttca tgtagttatt gcagttaata aaagttattt 6120tacagataaa aattattata taaatcgaga aagatggttg gaattatgga agtttgctac 6180taaggatgat tctataactc aagttgatgt tagaaaagca aaaattaatg attataaaga 6240ggtttacgaa cttgcgaaat attcagctaa agacactgat tatttaatat cgaggccagt 6300atttgaaatt ttttataaag cattaaaagg caagcaggta ttagttttta gtggattttt 6360taaagatgca cacaaattgt acaagcaagg aaaacttgat gtttataaaa agaaagatga 6420aattaaatat gtctatatag tttattataa ttggtgcaaa aaacaatatg aaaaaactag 6480aataagggaa cttacggaag atgaaaaaga agaattaaat caagatttaa tagatgaaat 6540agaaatagat taaagtgtaa ctatacttta tatatatatg attaaaaaaa taaaaaacaa 6600cagcctatta ggttgttgtt ttttattttc tttattaatt tttttaattt ttagttttta 6660gttctttttt aaaataagtt tcagcctctt tttcaatatt ttttaaagaa ggagtatttg 6720catgaattgc cttttttcta acagacttag gaaatatttt aacagtatct tcttgcgccg 6780gtgattttgg aacttcataa cttactaatt tataattatt attttctttt ttaattgtaa 6840cagttgcaaa agaagctgaa cctgttcctt caactagttt atcatcttca atataatatt 6900cttgacctat atagtataaa tatattttta ttatattttt acttttttct gaatctatta 6960ttttataatc ataaaaagtt ttaccaccaa aagaaggttg tactccttct ggtccaacat 7020atttttttac tatattatct aaataatttt tgggaactgg tgttgtaatt tgattaatcg 7080aacaaccagt tatacttaaa ggaattataa ctataaaaat atataggatt atctttttaa 7140atttcattat tggcctcctt tttattaaat ttatgttacc ataaaaagga cataacggga 7200atatgtagaa tatttttaat gtagacaaaa ttttacataa atataaagaa aggaagtgtt 7260tgtttaaatt ttatagcaaa ctatcaaaaa ttagggggat aaaaatttat gaaaaaaagg 7320ttttcgatgt tatttttatg tttaacttta atagtttgtg gtttatttac aaattcggcc 7380ggccagtggg caagttgaaa aattcacaaa aatgtggtat aatatctttg ttcattagag 7440cgataaactt gaatttgaga gggaacttag atggtatttg aaaaaattga taaaaatagt 7500tggaacagaa aagagtattt tgaccactac tttgcaagtg taccttgtac ctacagcatg 7560accgttaaag tggatatcac acaaataaag gaaaagggaa tgaaactata tcctgcaatg 7620ctttattata ttgcaatgat tgtaaaccgc cattcagagt ttaggacggc aatcaatcaa 7680gatggtgaat tggggatata tgatgagatg ataccaagct atacaatatt tcacaatgat 7740actgaaacat tttccagcct ttggactgag tgtaagtctg actttaaatc atttttagca 7800gattatgaaa gtgatacgca acggtatgga aacaatcata gaatggaagg aaagccaaat 7860gctccggaaa acatttttaa tgtatctatg ataccgtggt caaccttcga tggctttaat 7920ctgaatttgc agaaaggata tgattatttg attcctattt ttactatggg gaaatattat 7980aaagaagata acaaaattat acttcctttg gcaattcaag ttcatcacgc agtatgtgac 8040ggatttcaca tttgccgttt tgtaaacgaa ttgcaggaat tgataaatag ttaacttcag 8100gtttgtctgt aactaaaaac aagtatttaa gcaaaaacat cgtagaaata cggtgttttt 8160tgttacccta agtttaaact cctttttgat aatctcatga ccaaaatccc ttaacgtgag 8220ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 8280ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 8340tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 8400cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct 8460gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 8520gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 8580tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 8640ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 8700gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 8760ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 8820tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 8880ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct 8940gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga 9000acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcagggcc 9060ccctgcttcg gggtcattat agcgattttt tcggtatatc catccttttt cgcacgatat 9120acaggatttt gccaaagggt tcgtgtagac tttccttggt gtatccaacg gcgtcagccg 9180ggcaggatag gtgaagtagg cccacccgcg agcgggtgtt ccttcttcac tgtcccttat 9240tcgcacctgg cggtgctcaa cgggaatcct gctctgcgag gctggccggc taccgccggc 9300gtaacagatg agggcaagcg gatggctgat gaaaccaagc caaccaggaa gggcagccca 9360cctatcaagg tgtactgcct tccagacgaa cgaagagcga ttgaggaaaa ggcggcggcg 9420gccggcatga gcctgtcggc ctacctgctg gccgtcggcc agggctacaa aatcacgggc 9480gtcgtggact atgagcacgt ccgcgagctg gcccgcatca atggcgacct gggccgcctg 9540ggcggcctgc tgaaactctg gctcaccgac gacccgcgca cggcgcggtt cggtgatgcc 9600acgatcctcg ccctgctggc gaagatcgaa gagaagcagg acgagcttgg caaggtcatg 9660atgggcgtgg tccgcccgag ggcagagcca tgactttttt agccgctaaa acggccgggg 9720ggtgcgcgtg attgccaagc acgtccccat gcgctccatc aagaagagcg acttcgcgga 9780gctggtgaag tacatcaccg acgagcaagg caagaccgat cgggccc 982751843DNAStaphylococcus aureus subsp. aureus 51atgatagctg ttccatttaa cgctggaaaa ataaaagttt taattgaggc attagaatct 60ggaaattatt catcaataaa atcagatgta tatgacggaa tgttatatga tgcaccagat 120caccttaaat cattagtaaa cagatttgta gaacttaata atataactga gccattagca 180gtaactatac agacaaatct tcctccttca agaggtcttg gatctagtgc agctgttgct 240gttgcttttg taagagcaag ttatgatttc ttaggaaaaa gtttaactaa agaagagctt 300atagaaaagg ctaattgggc tgaacaaata gctcatggaa agccatctgg aatagataca 360caaacaatag tatctggaaa gcctgtttgg tttcaaaagg gacatgcaga aacacttaaa 420actctttcac ttgatggata catggtagta attgatacag gtgttaaagg aagtacaaga 480caggctgtag aagatgttca taaactttgc gaagatcctc aatatatgag tcacgtaaaa 540cacataggaa aacttgtact tagagcatct gatgttattg aacatcataa ctttgaagca 600cttgctgata tattcaatga atgtcatgct gatttaaagg ctcttacagt aagtcatgac 660aaaatagaac agttaatgaa gataggaaaa gaaaatggtg ctatagctgg taaattaact 720ggtgctggta gaggtggttc aatgttatta cttgcaaaag acttaccaac tgcaaagaat 780atagttaaag cagtagagaa agctggtgca gcacatactt ggattgaaaa tttaggtggt 840taa 843521077DNAStaphylococcus aureus subsp. aureus 52atgatacaag taaaggcacc aggaaaatta tatatagcag gtgaatacgc tgttacagaa 60ccaggatata aatctgttct tatagctctt gatagatttg ttacagctac tattgaggaa 120gctgatcaat acaaaggaac aatacattca aaggcattac atcacaatcc agtaactttt 180agtagagatg aagattctat tgttatatca gacccacacg cagcaaaaca acttaattat 240gtagtaactg ctatagaaat atttgagcaa tatgcaaaat catgtgacat agcaatgaag 300cattttcatt taactataga ttctaactta gatgatagta atggacataa gtatggactt 360ggatcttctg ctgctgtttt agtttcagta attaaagtac ttaacgaatt ttatgatatg 420aaactttcaa acctttatat atataagtta gcagtaattg ctaatatgaa attacagagt 480ttatcttcat gcggtgatat agcagtaagt gtttattcag gttggttagc ttattctaca 540tttgaccatg aatgggtaaa acaccagata gaagatacaa cagttgaaga agtacttatt 600aaaaattggc ctggattaca catagagcca cttcaagctc ctgaaaatat ggaagttctt 660ataggttgga caggtagtcc agctagtagt cctcattttg tttctgaagt taaaagactt 720aagtcagatc cttcatttta cggtgatttc ttagaagatt cacatagatg tgtagaaaaa 780ttaattcatg cattcaaaac taataatatt aagggtgttc agaaaatggt aagacagaat 840agaactatta tacaaagaat ggataaggaa gcaacagttg atatagagac tgagaagtta 900aaatatttat gtgatattgc tgaaaaatat catggtgcaa gtaaaacttc aggtgctggt 960ggtggtgatt gcggaataac tataataaat aaggatgtag acaaagagaa aatatatgat 1020gaatggacta aacatggaat aaagcctctt aagtttaata tttatcatgg acaataa 107753984DNAStaphylococcus aureus subsp. aureus 53atgataaaat ctggaaaagc aagagcacac actaatatag cacttataaa atattggggt 60aagaaagatg aggcattaat aataccaatg aataactcaa tatcagtaac tttagaaaag 120ttttatactg aaacaaaagt tacatttaac gatcagctta ctcaagatca attttggctt 180aatggtgaaa aagtttctgg aaaagaatta gaaaagattt caaagtatat ggatattgtt 240agaaatagag ctggaataga ttggtatgct gagatagaat ctgataattt tgttcctaca 300gctgctggtc ttgctagttc tgctagtgct tatgcagcat tagctgctgc atgtaaccaa 360gcacttgatt tacagttaag tgataaagac ttaagtagat tagctagaat tggatcagga 420tcagcatcaa gatcaatata cggtggtttt gcagaatggg aaaaaggata taatgacgaa 480acttcttatg ctgttccatt agaaagtaat cactttgaag atgatcttgc tatgattttt 540gtagtaataa accaacattc taaaaaggtt ccttcaagat atggaatgtc tcttacaaga 600aatacaagta gattctatca atattggtta gaccatattg atgaagatct tgcagaagca 660aaggcagcaa tacaagataa ggattttaag agattaggtg aagttattga agagaatgga 720cttagaatgc atgctacaaa tcttggatca actccacctt ttacttactt agtacaagag 780tcatacgatg taatggcatt agtacatgag tgtagagaag caggatatcc atgctatttc 840actatggatg ctggacctaa tgtaaaaata cttgtagaga agaaaaacaa acaacagata 900atagataaac ttttaactca gttcgataat aatcagataa tagatagtga tattatagct 960acaggtattg aaattataga ataa 98454516DNAClostridium beijerinckii 54atggcagagt atataatagc agtagatgag ttcgataacg aaataggatc aatagaaaag 60atggaagctc atagaaaagg aacacttcat agagcattca gtattttagt ttttaactca 120aagaatcaac ttttattaca gaaaagaaat gtaaagaaat atcactctcc aggattatgg 180acaaacactt gttgtagtca cccaagatat ggtgaatctc ttcatgatgc tatatacaga 240agattaaaag aagagatggg atttacttgc gaacttgaag aagtattctc attcatatat 300aaggtaaaac ttgaagataa tttatttgag aatgaatatg accatgtatt tattggtaaa 360tatgatggtg agataattgt taataaagat gaagttgatg attttaaatg ggtagacatt 420aatgaagtta aaaaggacat aatagaaaga cctgaggcat atacttactg gtttaagtat 480cttgtaaata aagctgaaaa taagatattt aaataa 51655120DNAClostridium autoethanogenum 55atatcgatac agataaaaaa atatataata cagaagaaaa aattataaat ttgtggtata 60atataaagta tagtaattta agtttaaacc tcgtgaaaac gctaacaaat aataggaggt 12056900DNAEscherichia coli 56atggatttcc cacaacaatt agaagcatgt gtaaaacagg ctaatcaggc acttagtaga 60tttattgctc ctcttccttt tcaaaataca ccagtagtag aaactatgca atacggtgca 120cttttaggtg gtaaaagatt aagaccattc ttagtatatg ctacaggaca tatgtttggt 180gtatcaacta atactttaga cgctccagct gctgctgttg aatgtattca tgcatattct 240ttaatacatg atgacttacc agcaatggat gacgatgatt taagaagagg tttacctaca 300tgtcatgtta aatttggtga agctaatgca attttagcag gtgacgcttt acaaacttta 360gctttttcta tactttcaga tgcagacatg cctgaagttt cagatagaga tagaatttct 420atgatatcag agcttgcatc tgcatcagga atagctggaa tgtgcggtgg tcaagcactt 480gatttagatg cagaaggtaa acacgtacca cttgatgcat tagagagaat tcatagacat 540aaaacaggtg ctcttataag agcagcagta agattaggtg ctttaagtgc tggtgacaag 600ggtagaagag cacttccagt acttgataag tatgcagaaa gtataggatt agcttttcaa 660gttcaagatg acatacttga cgttgttggt gatactgcta ctttaggaaa aagacagggt 720gcagatcagc aattaggaaa atctacatac cctgctttac ttggattaga acaggctaga 780aagaaagcaa gagacttaat agatgacgca agacaaagtc ttaaacagtt agctgaacaa 840tcacttgaca caagtgcact tgaagcactt gcagattata ttatacagag aaacaagtaa 90057975DNAMalus x domestica 57atggaattta gagtacattt acaggcagac aacgaacaga aaatatttca aaatcaaatg 60aaaccagagc cagaagcatc atatcttata aatcaaagaa gaagtgctaa ttataaacca 120aacatttgga aaaacgattt tcttgatcag tctttaatat caaaatatga tggtgatgaa 180tatagaaaac tttcagaaaa gttaatagaa gaagtaaaga tatacatatc agcagagact 240atggatttag ttgctaaatt agaacttata gattctgtta gaaaacttgg acttgctaat 300ctttttgaga aagaaataaa ggaagcatta gacagtatag cagcaataga atcagataat 360ttaggaacta gagacgatct ttatggaaca gctcttcatt ttaagattct tagacagcat 420ggatataagg taagtcaaga tatatttggt agatttatgg atgagaaagg aacattagaa 480aatcatcact ttgcacactt aaaaggaatg ttagaattat ttgaggcaag taatcttgga 540tttgaaggtg aagacatatt agatgaagct aaagcatctc ttacacttgc tcttagagat 600tcaggacata tttgttatcc agactcaaac ttaagtagag atgtagttca tagtttagaa 660ttacctagtc atagaagagt tcaatggttc gatgtaaaat ggcagattaa tgcatacgaa 720aaagatattt gtagagtaaa tgcaacttta ttagagttag caaagttaaa ttttaatgtt 780gttcaagctc agcttcagaa gaatcttaga gaagctagta gatggtgggc taatcttggt 840ttcgcagata atttaaagtt tgctagagat agacttgtag agtgtttttc atgcgcagta 900ggtgtagcat ttgaaccaga gcattcatct tttagaatat gtttaactaa ggtaattaat 960cttgttctta ttata 9755813817DNAArtificial SequencepMTL8314-Pptaack-thlA-HMGS-Patp-HMGR-Prnf-MK- PMK-PMD-Pfor-idi-isp S 58aaactccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 60gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 120atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 180gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 240gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 300tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 360accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 420ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 480cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 540agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 600ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 660tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 720ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 780cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 840gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca gggccccctg caggataaaa 900aaattgtaga taaattttat aaaatagttt tatctacaat ttttttatca ggaaacagct 960atgaccgcgg ccgcagatag tcataatagt tccagaatag ttcaatttag aaattagact 1020aaacttcaaa atgtttgtta aatatatacc aatctagtat agatattttt taaatactgg 1080acttaaacag tagtaatttg cctaaaaaat tttttcaatt ttttttaaaa aatccttttc 1140aagttgtaca ttgttatggt aatatgtaat tgaagaagtt atgtagtaat attgtaaacg 1200tttcttgatt tttttacatc catgtagtgc ttaaaaaacc aaaatatgtc acatgcactt 1260gtatatttca aataacaata tttattttct cgttaaattc acaaataatt tattaataat 1320atcaataacc aagattatac ttaaatggat gtttattttt taacactttt atagtaaata 1380tatttatttt atgtagtaaa aaggttataa ttataattgt atttattaca attaattaaa 1440ataaaaaata gggttttagg taaaattaag ttattttaag aagtaattac aataaaaatt 1500gaagttattt ctttaaggag gaaattcata tgaaagaggt tgttattgca tcagctgtta 1560gaactgcaat aggatcttat ggaaaaagtc ttaaagatgt accagcagta gacttaggtg 1620caactgcaat aaaggaagca gtaaagaaag caggtataaa acctgaagat gttaatgaag 1680ttattttagg aaacgtatta caagctggac ttggacagaa tccagctaga caggcatcat 1740tcaaagcagg attaccagta gagatacctg ctatgactat taataaagtt tgtggttcag 1800gattaagaac agtttcttta gctgctcaaa ttataaaagc tggtgacgca gatgtaataa 1860tagcaggtgg tatggaaaat atgtcaagag caccatacct tgctaataat gctagatggg 1920gttatagaat gggaaacgct aaatttgtag acgaaatgat aactgatgga ctttgggatg 1980catttaacga ttatcacatg ggaattactg ctgaaaatat agctgagaga tggaatataa 2040gtagagaaga acaagatgag tttgcacttg catctcagaa aaaggcagaa gaagctatta 2100aatcaggaca atttaaagat gaaattgttc cagtagtaat taaaggtaga aaaggtgaaa 2160cagttgtaga cactgatgaa catcctagat ttggatctac aatagaaggt ttagctaaat 2220taaagcctgc ttttaagaaa gacggaacag taactgctgg aaacgcatca ggtttaaatg 2280attgtgcagc tgttttagtt attatgtctg ctgaaaaggc aaaggaatta ggtgttaaac 2340cacttgctaa gatagttagt tatggttcag caggtgtaga tcctgctatt atgggatatg 2400gaccttttta tgctacaaag gcagctattg aaaaggctgg ttggacagtt gatgaacttg 2460atcttataga gtcaaatgag gcatttgcag cacaaagtct tgctgttgct aaggatctta 2520aattcgatat gaataaagta aatgtaaacg gtggtgctat agcacttggt catccaatag 2580gtgctagtgg tgctagaatt ttagttacat tagttcatgc aatgcaaaag agagacgcta 2640aaaagggact tgcaacttta tgcataggtg gtggtcaagg aacagcaata cttcttgaaa 2700aatgttaaga attcgaggct tttactaaaa acaataaaaa caggaggaaa taatatgact 2760ataggaattg acaaaataaa cttttacgta ccaaaatatt atgtagatat ggcaaaatta 2820gcagaagcaa gacaagtaga cccaaataaa tttcttattg gaataggaca gactgaaatg 2880gcagttagtc cagtaaacca agatatagta tcaatgggtg ctaatgctgc taaagatata 2940ataactgatg aagacaaaaa gaaaatagga atggtaatag tagcaactga gtcagcagta 3000gatgcagcaa aggcagcagc agtacagatt cataatttat taggtattca accatttgca 3060agatgtttcg aaatgaaaga agcatgttat gctgctactc ctgcaattca gttagctaag 3120gattatttag ctacaagacc aaatgagaaa gttttagtta tagctacaga tacagctaga 3180tatggactta attcaggtgg tgaacctact caaggtgctg gtgctgttgc tatggttata 3240gctcataatc ctagtatact tgcattaaat gaagacgctg ttgcttatac agaagatgtt 3300tatgatttct ggagaccaac aggacataag tatccattag tagatggtgc tttatcaaaa 3360gacgcatata ttagatcttt tcaacaatct tggaatgaat atgctaagag acaaggaaag 3420agtttagctg attttgctag tctttgcttt catgttcctt

ttactaaaat gggtaaaaag 3480gctttagaat ctataataga taacgcagat gaaacaactc aagagagatt aagatctgga 3540tatgaagatg cagttgatta caatagatat gttggaaata tatacacagg aagtctttat 3600ctttctctta taagtcttct tgaaaataga gatttacagg ctggtgaaac tattggatta 3660ttttcatacg gatcaggttc tgttggtgaa ttttattcag ctacacttgt agaaggatat 3720aaagatcacc ttgatcaggc agcacacaaa gcacttttaa acaatagaac tgaagtatca 3780gtagatgcat acgaaacatt tttcaagaga tttgatgatg tagaatttga tgaagagcag 3840gatgcagttc atgaagatag acatatattc tatctttcaa acatagagaa taatgtaaga 3900gaatatcata gacctgaata agagctcgtt ataattttca attttcattc tttttaaagg 3960agattagcat acattttatc ataattatac agacaatata gtaatatatg atgttaaaat 4020atcaatatat ggttaaaaat ctgtatattt tttcccattt taattatttg tactataata 4080ttacactgag tgtattgcat atttaaaaaa tatttggtac aattagttag ttaaataaat 4140tctaaattgt aaattatcag aatccttatt aaggaaatac atagatttaa ggagaaatca 4200taaaaaggtg taatataaac tggctaaaat tgagcaaaaa ttgagcaatt aagacttttt 4260gattgtatct ttttatatat ttaaggtata taatcttatt tatattgggg gaaggtacca 4320tgcaatcatt agacaaaaat ttcagacatt tatcaagaca acaaaagtta caacaattag 4380ttgataaaca gtggctttca gaagatcagt ttgatatttt acttaatcat cctcttatag 4440atgaagaagt tgctaatagt cttatagaaa atgtaattgc acagggtgca ttaccagttg 4500gacttcttcc taatataata gttgatgata aggcttatgt tgtaccaatg atggttgaag 4560aacctagtgt tgttgcagct gcatcttatg gtgctaaatt agtaaatcag acaggtggat 4620ttaaaactgt atcatcagaa agaataatga ttggacagat agtatttgat ggtgtagatg 4680acactgaaaa attaagtgca gatattaaag cattagaaaa acaaatacat aagattgcag 4740atgaagcata tcctagtata aaagcaagag gtggtggtta tcaaagaata gcaatagata 4800catttccaga gcaacaactt ttaagtctta aggtatttgt agatacaaaa gatgctatgg 4860gtgctaatat gcttaatact atacttgagg caataactgc attccttaaa aatgaatctc 4920ctcaatcaga tatattaatg tctatacttt caaaccatgc aactgctagt gtagtaaaag 4980tacaaggtga gatagatgta aaagatcttg ctagaggtga aagaacaggt gaagaagtag 5040ctaagagaat ggaaagagct tctgtattag ctcaggttga tattcataga gctgcaacac 5100ataacaaagg tgttatgaat ggaatacatg ctgttgtttt agctacagga aatgatacta 5160gaggtgctga agcatctgca catgcatacg catcaagaga cggacaatat agaggtatag 5220caacttggag atatgatcag aagagacaaa gacttattgg aactattgaa gttccaatga 5280cacttgctat agtaggtggt ggtactaaag tattaccaat agctaaggca tcattagagt 5340tattaaatgt tgattctgca caagaacttg gacacgtagt tgctgctgtt ggattagcac 5400aaaactttgc tgcttgtaga gcacttgttt ctgaaggtat tcaacaagga cacatgtcat 5460tacaatataa aagtttagca atagtagtag gtgcaaaagg tgacgagata gcacaagtag 5520cagaagctct taaacaggaa ccaagagcta atacacaggt tgctgaaaga attttacagg 5580aaattagaca gcaataatct agaatatcga tacagataaa aaaatatata atacagaaga 5640aaaaattata aatttgtggt ataatataaa gtatagtaat ttaagtttaa acctcgtgaa 5700aacgctaaca aataatagga ggtcaattga tgatagctgt tccatttaac gctggaaaaa 5760taaaagtttt aattgaggca ttagaatctg gaaattattc atcaataaaa tcagatgtat 5820atgacggaat gttatatgat gcaccagatc accttaaatc attagtaaac agatttgtag 5880aacttaataa tataactgag ccattagcag taactataca gacaaatctt cctccttcaa 5940gaggtcttgg atctagtgca gctgttgctg ttgcttttgt aagagcaagt tatgatttct 6000taggaaaaag tttaactaaa gaagagctta tagaaaaggc taattgggct gaacaaatag 6060ctcatggaaa gccatctgga atagatacac aaacaatagt atctggaaag cctgtttggt 6120ttcaaaaggg acatgcagaa acacttaaaa ctctttcact tgatggatac atggtagtaa 6180ttgatacagg tgttaaagga agtacaagac aggctgtaga agatgttcat aaactttgcg 6240aagatcctca atatatgagt cacgtaaaac acataggaaa acttgtactt agagcatctg 6300atgttattga acatcataac tttgaagcac ttgctgatat attcaatgaa tgtcatgctg 6360atttaaaggc tcttacagta agtcatgaca aaatagaaca gttaatgaag ataggaaaag 6420aaaatggtgc tatagctggt aaattaactg gtgctggtag aggtggttca atgttattac 6480ttgcaaaaga cttaccaact gcaaagaata tagttaaagc agtagagaaa gctggtgcag 6540cacatacttg gattgaaaat ttaggtggtt aagtcgacaa agacactaaa aaattataaa 6600agtaaaggag gacattaaat gatacaagta aaggcaccag gaaaattata tatagcaggt 6660gaatacgctg ttacagaacc aggatataaa tctgttctta tagctcttga tagatttgtt 6720acagctacta ttgaggaagc tgatcaatac aaaggaacaa tacattcaaa ggcattacat 6780cacaatccag taacttttag tagagatgaa gattctattg ttatatcaga cccacacgca 6840gcaaaacaac ttaattatgt agtaactgct atagaaatat ttgagcaata tgcaaaatca 6900tgtgacatag caatgaagca ttttcattta actatagatt ctaacttaga tgatagtaat 6960ggacataagt atggacttgg atcttctgct gctgttttag tttcagtaat taaagtactt 7020aacgaatttt atgatatgaa actttcaaac ctttatatat ataagttagc agtaattgct 7080aatatgaaat tacagagttt atcttcatgc ggtgatatag cagtaagtgt ttattcaggt 7140tggttagctt attctacatt tgaccatgaa tgggtaaaac accagataga agatacaaca 7200gttgaagaag tacttattaa aaattggcct ggattacaca tagagccact tcaagctcct 7260gaaaatatgg aagttcttat aggttggaca ggtagtccag ctagtagtcc tcattttgtt 7320tctgaagtta aaagacttaa gtcagatcct tcattttacg gtgatttctt agaagattca 7380catagatgtg tagaaaaatt aattcatgca ttcaaaacta ataatattaa gggtgttcag 7440aaaatggtaa gacagaatag aactattata caaagaatgg ataaggaagc aacagttgat 7500atagagactg agaagttaaa atatttatgt gatattgctg aaaaatatca tggtgcaagt 7560aaaacttcag gtgctggtgg tggtgattgc ggaataacta taataaataa ggatgtagac 7620aaagagaaaa tatatgatga atggactaaa catggaataa agcctcttaa gtttaatatt 7680tatcatggac aataaccatg gtcaataatc ttacaataaa taaaagaaag gaggcaaaaa 7740tatgataaaa tctggaaaag caagagcaca cactaatata gcacttataa aatattgggg 7800taagaaagat gaggcattaa taataccaat gaataactca atatcagtaa ctttagaaaa 7860gttttatact gaaacaaaag ttacatttaa cgatcagctt actcaagatc aattttggct 7920taatggtgaa aaagtttctg gaaaagaatt agaaaagatt tcaaagtata tggatattgt 7980tagaaataga gctggaatag attggtatgc tgagatagaa tctgataatt ttgttcctac 8040agctgctggt cttgctagtt ctgctagtgc ttatgcagca ttagctgctg catgtaacca 8100agcacttgat ttacagttaa gtgataaaga cttaagtaga ttagctagaa ttggatcagg 8160atcagcatca agatcaatat acggtggttt tgcagaatgg gaaaaaggat ataatgacga 8220aacttcttat gctgttccat tagaaagtaa tcactttgaa gatgatcttg ctatgatttt 8280tgtagtaata aaccaacatt ctaaaaaggt tccttcaaga tatggaatgt ctcttacaag 8340aaatacaagt agattctatc aatattggtt agaccatatt gatgaagatc ttgcagaagc 8400aaaggcagca atacaagata aggattttaa gagattaggt gaagttattg aagagaatgg 8460acttagaatg catgctacaa atcttggatc aactccacct tttacttact tagtacaaga 8520gtcatacgat gtaatggcat tagtacatga gtgtagagaa gcaggatatc catgctattt 8580cactatggat gctggaccta atgtaaaaat acttgtagag aagaaaaaca aacaacagat 8640aatagataaa cttttaactc agttcgataa taatcagata atagatagtg atattatagc 8700tacaggtatt gaaattatag aataaactag ttgtatatta aaatagtaga atacataaga 8760tacttaattt aattaaagat agttaagtac ttttcaatgt gcttttttag atgtttaata 8820caaatcttta attgtaaaag aaatgctgta ctatttactg ttctagtgac gggattaaac 8880tgtattaatt ataaataaaa aataagtaca gttgtttaaa attatatttt gtattaaatc 8940taatagtacg atgtaagtta ttttatacta ttgctagttt aataaaaaga tttaattata 9000tacttgaaaa ggagaggaac tcgagatggc agagtatata atagcagtag atgagttcga 9060taacgaaata ggatcaatag aaaagatgga agctcataga aaaggaacac ttcatagagc 9120attcagtatt ttagttttta actcaaagaa tcaactttta ttacagaaaa gaaatgtaaa 9180gaaatatcac tctccaggat tatggacaaa cacttgttgt agtcacccaa gatatggtga 9240atctcttcat gatgctatat acagaagatt aaaagaagag atgggattta cttgcgaact 9300tgaagaagta ttctcattca tatataaggt aaaacttgaa gataatttat ttgagaatga 9360atatgaccat gtatttattg gtaaatatga tggtgagata attgttaata aagatgaagt 9420tgatgatttt aaatgggtag acattaatga agttaaaaag gacataatag aaagacctga 9480ggcatatact tactggttta agtatcttgt aaataaagct gaaaataaga tatttaaata 9540aaccggtggg aggaaatgaa catggcaaca gaattattat gtttacacag acctatatca 9600cttactcaca aactttttag gaatccatta cctaaagtta ttcaagctac acctttaaca 9660ttaaaactta ggtgtagtgt ttctacagaa aatgtatcat ttagtgagac agaaactgaa 9720acaagaagat cagcaaatta tgaaccaaat tcttgggatt atgattatct tctttcttct 9780gatactgatg agtcaataga agtacataaa gataaggcta agaaattaga agctgaagtt 9840aggagagaaa taaataatga gaaggctgaa tttcttacac ttcttgaact tattgataat 9900gtacaaagac ttggattagg atatagattt gagtctgata taagaagagc attagataga 9960tttgtaagta gtggaggatt tgatggagtt actaaaactt cattacatgg aacagcatta 10020tcatttaggt tattaaggca acatggtttt gaagtatctc aagaagcttt tagtggattt 10080aaagatcaga atggaaactt tcttgagaat ttaaaggaag acataaaagc aattctttct 10140ctttatgaag catcattttt agcattagaa ggtgagaata tattagatga ggctaaagta 10200tttgcaatat ctcatcttaa agaacttagt gaagaaaaga ttggtaaaga attagctgaa 10260caagtttcac atgctttaga attaccatta catagaagaa cacaaagatt agaagcagtt 10320tggtcaatag aagcatatag aaagaaagaa gacgcaaatc aagtactttt agaacttgca 10380atacttgact acaatatgat tcaaagtgta tatcagaggg atttaagaga aacatcaaga 10440tggtggagaa gagtaggatt agcaactaaa ttacattttg ctagagatag gcttattgaa 10500agtttttatt gggctgttgg agttgctttt gaaccacaat attctgattg cagaaatagt 10560gtagcaaaga tgttttcatt tgttactata attgacgata tttacgatgt atatggaact 10620ttagatgaac ttgaactttt tactgatgca gttgaaagat gggatgtaaa tgctattaat 10680gatcttcctg attatatgaa gttatgtttt cttgcacttt acaatactat taacgagata 10740gcttacgata acttaaaaga taaaggtgag aacatacttc cttatttaac aaaagcatgg 10800gcagatttat gtaatgcatt tcttcaagaa gctaagtggc tttataataa atcaacacct 10860acatttgatg attattttgg aaatgcatgg aaaagttcta gtggaccttt acagcttatt 10920tttgcttatt ttgctgtagt acagaacatt aaaaaggaag agattgagaa tcttcagaaa 10980tatcatgaca taatatcaag acctagtcac atttttaggc tttgtaatga tttagcatct 11040gcttcagcag aaatagcaag aggtgaaact gctaattctg taagttgtta tatgagaaca 11100aaaggtatat ctgaagaatt agctactgaa agtgttatga atcttataga cgaaacttgg 11160aagaaaatga acaaagaaaa acttggtgga tctttatttg caaaaccttt tgttgagact 11220gctataaatt tagctagaca gtctcattgc acatatcata atggtgatgc acatactagt 11280ccagatgaat taactaggaa aagagtactt agtgtaataa ctgaaccaat attaccattt 11340gaaagataag ctagcataaa aataagaagc ctgcatttgc aggcttctta tttttatggc 11400gcgccgccat tatttttttg aacaattgac aattcatttc ttatttttta ttaagtgata 11460gtcaaaaggc ataacagtgc tgaatagaaa gaaatttaca gaaaagaaaa ttatagaatt 11520tagtatgatt aattatactc atttatgaat gtttaattga atacaaaaaa aaatacttgt 11580tatgtattca attacgggtt aaaatataga caagttgaaa aatttaataa aaaaataagt 11640cctcagctct tatatattaa gctaccaact tagtatataa gccaaaactt aaatgtgcta 11700ccaacacatc aagccgttag agaactctat ctatagcaat atttcaaatg taccgacata 11760caagagaaac attaactata tatattcaat ttatgagatt atcttaacag atataaatgt 11820aaattgcaat aagtaagatt tagaagttta tagcctttgt gtattggaag cagtacgcaa 11880aggctttttt atttgataaa aattagaagt atatttattt tttcataatt aatttatgaa 11940aatgaaaggg ggtgagcaaa gtgacagagg aaagcagtat cttatcaaat aacaaggtat 12000tagcaatatc attattgact ttagcagtaa acattatgac ttttatagtg cttgtagcta 12060agtagtacga aagggggagc tttaaaaagc tccttggaat acatagaatt cataaattaa 12120tttatgaaaa gaagggcgta tatgaaaact tgtaaaaatt gcaaagagtt tattaaagat 12180actgaaatat gcaaaataca ttcgttgatg attcatgata aaacagtagc aacctattgc 12240agtaaataca atgagtcaag atgtttacat aaagggaaag tccaatgtat taattgttca 12300aagatgaacc gatatggatg gtgtgccata aaaatgagat gttttacaga ggaagaacag 12360aaaaaagaac gtacatgcat taaatattat gcaaggagct ttaaaaaagc tcatgtaaag 12420aagagtaaaa agaaaaaata atttatttat taatttaata ttgagagtgc cgacacagta 12480tgcactaaaa aatatatctg tggtgtagtg agccgataca aaaggatagt cactcgcatt 12540ttcataatac atcttatgtt atgattatgt gtcggtggga cttcacgacg aaaacccaca 12600ataaaaaaag agttcggggt agggttaagc atagttgagg caactaaaca atcaagctag 12660gatatgcagt agcagaccgt aaggtcgttg tttaggtgtg ttgtaataca tacgctatta 12720agatgtaaaa atacggatac caatgaaggg aaaagtataa tttttggatg tagtttgttt 12780gttcatctat gggcaaacta cgtccaaagc cgtttccaaa tctgctaaaa agtatatcct 12840ttctaaaatc aaagtcaagt atgaaatcat aaataaagtt taattttgaa gttattatga 12900tattatgttt ttctattaaa ataaattaag tatatagaat agtttaataa tagtatatac 12960ttaatgtgat aagtgtctga cagtgtcaca gaaaggatga ttgttatgga ttataagcgg 13020ccggccagtg ggcaagttga aaaattcaca aaaatgtggt ataatatctt tgttcattag 13080agcgataaac ttgaatttga gagggaactt agatggtatt tgaaaaaatt gataaaaata 13140gttggaacag aaaagagtat tttgaccact actttgcaag tgtaccttgt acctacagca 13200tgaccgttaa agtggatatc acacaaataa aggaaaaggg aatgaaacta tatcctgcaa 13260tgctttatta tattgcaatg attgtaaacc gccattcaga gtttaggacg gcaatcaatc 13320aagatggtga attggggata tatgatgaga tgataccaag ctatacaata tttcacaatg 13380atactgaaac attttccagc ctttggactg agtgtaagtc tgactttaaa tcatttttag 13440cagattatga aagtgatacg caacggtatg gaaacaatca tagaatggaa ggaaagccaa 13500atgctccgga aaacattttt aatgtatcta tgataccgtg gtcaaccttc gatggcttta 13560atctgaattt gcagaaagga tatgattatt tgattcctat ttttactatg gggaaatatt 13620ataaagaaga taacaaaatt atacttcctt tggcaattca agttcatcac gcagtatgtg 13680acggatttca catttgccgt tttgtaaacg aattgcagga attgataaat agttaacttc 13740aggtttgtct gtaactaaaa acaagtattt aagcaaaaac atcgtagaaa tacggtgttt 13800tttgttaccc taagttt 138175914709DNAArtificial Sequenceplasmid pMTL8314-Pptaack-thlA-HMGS-Patp-HMGR-Prnf-MK-PMK-PMD-Pfor-idi-isp A-FS 59aaactccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 60gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 120atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 180gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 240gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 300tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 360accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 420ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 480cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 540agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 600ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 660tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 720ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 780cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 840gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca gggccccctg caggataaaa 900aaattgtaga taaattttat aaaatagttt tatctacaat ttttttatca ggaaacagct 960atgaccgcgg ccgcagatag tcataatagt tccagaatag ttcaatttag aaattagact 1020aaacttcaaa atgtttgtta aatatatacc aatctagtat agatattttt taaatactgg 1080acttaaacag tagtaatttg cctaaaaaat tttttcaatt ttttttaaaa aatccttttc 1140aagttgtaca ttgttatggt aatatgtaat tgaagaagtt atgtagtaat attgtaaacg 1200tttcttgatt tttttacatc catgtagtgc ttaaaaaacc aaaatatgtc acatgcactt 1260gtatatttca aataacaata tttattttct cgttaaattc acaaataatt tattaataat 1320atcaataacc aagattatac ttaaatggat gtttattttt taacactttt atagtaaata 1380tatttatttt atgtagtaaa aaggttataa ttataattgt atttattaca attaattaaa 1440ataaaaaata gggttttagg taaaattaag ttattttaag aagtaattac aataaaaatt 1500gaagttattt ctttaaggag gaaattcata tgaaagaggt tgttattgca tcagctgtta 1560gaactgcaat aggatcttat ggaaaaagtc ttaaagatgt accagcagta gacttaggtg 1620caactgcaat aaaggaagca gtaaagaaag caggtataaa acctgaagat gttaatgaag 1680ttattttagg aaacgtatta caagctggac ttggacagaa tccagctaga caggcatcat 1740tcaaagcagg attaccagta gagatacctg ctatgactat taataaagtt tgtggttcag 1800gattaagaac agtttcttta gctgctcaaa ttataaaagc tggtgacgca gatgtaataa 1860tagcaggtgg tatggaaaat atgtcaagag caccatacct tgctaataat gctagatggg 1920gttatagaat gggaaacgct aaatttgtag acgaaatgat aactgatgga ctttgggatg 1980catttaacga ttatcacatg ggaattactg ctgaaaatat agctgagaga tggaatataa 2040gtagagaaga acaagatgag tttgcacttg catctcagaa aaaggcagaa gaagctatta 2100aatcaggaca atttaaagat gaaattgttc cagtagtaat taaaggtaga aaaggtgaaa 2160cagttgtaga cactgatgaa catcctagat ttggatctac aatagaaggt ttagctaaat 2220taaagcctgc ttttaagaaa gacggaacag taactgctgg aaacgcatca ggtttaaatg 2280attgtgcagc tgttttagtt attatgtctg ctgaaaaggc aaaggaatta ggtgttaaac 2340cacttgctaa gatagttagt tatggttcag caggtgtaga tcctgctatt atgggatatg 2400gaccttttta tgctacaaag gcagctattg aaaaggctgg ttggacagtt gatgaacttg 2460atcttataga gtcaaatgag gcatttgcag cacaaagtct tgctgttgct aaggatctta 2520aattcgatat gaataaagta aatgtaaacg gtggtgctat agcacttggt catccaatag 2580gtgctagtgg tgctagaatt ttagttacat tagttcatgc aatgcaaaag agagacgcta 2640aaaagggact tgcaacttta tgcataggtg gtggtcaagg aacagcaata cttcttgaaa 2700aatgttaaga attcgaggct tttactaaaa acaataaaaa caggaggaaa taatatgact 2760ataggaattg acaaaataaa cttttacgta ccaaaatatt atgtagatat ggcaaaatta 2820gcagaagcaa gacaagtaga cccaaataaa tttcttattg gaataggaca gactgaaatg 2880gcagttagtc cagtaaacca agatatagta tcaatgggtg ctaatgctgc taaagatata 2940ataactgatg aagacaaaaa gaaaatagga atggtaatag tagcaactga gtcagcagta 3000gatgcagcaa aggcagcagc agtacagatt cataatttat taggtattca accatttgca 3060agatgtttcg aaatgaaaga agcatgttat gctgctactc ctgcaattca gttagctaag 3120gattatttag ctacaagacc aaatgagaaa gttttagtta tagctacaga tacagctaga 3180tatggactta attcaggtgg tgaacctact caaggtgctg gtgctgttgc tatggttata 3240gctcataatc ctagtatact tgcattaaat gaagacgctg ttgcttatac agaagatgtt 3300tatgatttct ggagaccaac aggacataag tatccattag tagatggtgc tttatcaaaa 3360gacgcatata ttagatcttt tcaacaatct tggaatgaat atgctaagag acaaggaaag 3420agtttagctg attttgctag tctttgcttt catgttcctt ttactaaaat gggtaaaaag 3480gctttagaat ctataataga taacgcagat gaaacaactc aagagagatt aagatctgga 3540tatgaagatg cagttgatta caatagatat gttggaaata tatacacagg aagtctttat 3600ctttctctta taagtcttct tgaaaataga gatttacagg ctggtgaaac tattggatta 3660ttttcatacg gatcaggttc tgttggtgaa ttttattcag ctacacttgt agaaggatat 3720aaagatcacc ttgatcaggc agcacacaaa gcacttttaa acaatagaac tgaagtatca 3780gtagatgcat acgaaacatt tttcaagaga tttgatgatg tagaatttga tgaagagcag 3840gatgcagttc atgaagatag acatatattc tatctttcaa acatagagaa taatgtaaga 3900gaatatcata gacctgaata agagctcgtt ataattttca attttcattc tttttaaagg 3960agattagcat acattttatc ataattatac agacaatata gtaatatatg atgttaaaat 4020atcaatatat ggttaaaaat ctgtatattt tttcccattt taattatttg tactataata 4080ttacactgag tgtattgcat atttaaaaaa tatttggtac aattagttag ttaaataaat 4140tctaaattgt aaattatcag aatccttatt aaggaaatac atagatttaa ggagaaatca 4200taaaaaggtg taatataaac tggctaaaat tgagcaaaaa ttgagcaatt aagacttttt 4260gattgtatct ttttatatat ttaaggtata taatcttatt tatattgggg gaaggtacca 4320tgcaatcatt agacaaaaat ttcagacatt tatcaagaca acaaaagtta caacaattag 4380ttgataaaca gtggctttca gaagatcagt ttgatatttt acttaatcat cctcttatag 4440atgaagaagt tgctaatagt cttatagaaa atgtaattgc acagggtgca ttaccagttg 4500gacttcttcc taatataata gttgatgata aggcttatgt tgtaccaatg atggttgaag

4560aacctagtgt tgttgcagct gcatcttatg gtgctaaatt agtaaatcag acaggtggat 4620ttaaaactgt atcatcagaa agaataatga ttggacagat agtatttgat ggtgtagatg 4680acactgaaaa attaagtgca gatattaaag cattagaaaa acaaatacat aagattgcag 4740atgaagcata tcctagtata aaagcaagag gtggtggtta tcaaagaata gcaatagata 4800catttccaga gcaacaactt ttaagtctta aggtatttgt agatacaaaa gatgctatgg 4860gtgctaatat gcttaatact atacttgagg caataactgc attccttaaa aatgaatctc 4920ctcaatcaga tatattaatg tctatacttt caaaccatgc aactgctagt gtagtaaaag 4980tacaaggtga gatagatgta aaagatcttg ctagaggtga aagaacaggt gaagaagtag 5040ctaagagaat ggaaagagct tctgtattag ctcaggttga tattcataga gctgcaacac 5100ataacaaagg tgttatgaat ggaatacatg ctgttgtttt agctacagga aatgatacta 5160gaggtgctga agcatctgca catgcatacg catcaagaga cggacaatat agaggtatag 5220caacttggag atatgatcag aagagacaaa gacttattgg aactattgaa gttccaatga 5280cacttgctat agtaggtggt ggtactaaag tattaccaat agctaaggca tcattagagt 5340tattaaatgt tgattctgca caagaacttg gacacgtagt tgctgctgtt ggattagcac 5400aaaactttgc tgcttgtaga gcacttgttt ctgaaggtat tcaacaagga cacatgtcat 5460tacaatataa aagtttagca atagtagtag gtgcaaaagg tgacgagata gcacaagtag 5520cagaagctct taaacaggaa ccaagagcta atacacaggt tgctgaaaga attttacagg 5580aaattagaca gcaataatct agaatatcga tacagataaa aaaatatata atacagaaga 5640aaaaattata aatttgtggt ataatataaa gtatagtaat ttaagtttaa acctcgtgaa 5700aacgctaaca aataatagga ggtcaattga tgatagctgt tccatttaac gctggaaaaa 5760taaaagtttt aattgaggca ttagaatctg gaaattattc atcaataaaa tcagatgtat 5820atgacggaat gttatatgat gcaccagatc accttaaatc attagtaaac agatttgtag 5880aacttaataa tataactgag ccattagcag taactataca gacaaatctt cctccttcaa 5940gaggtcttgg atctagtgca gctgttgctg ttgcttttgt aagagcaagt tatgatttct 6000taggaaaaag tttaactaaa gaagagctta tagaaaaggc taattgggct gaacaaatag 6060ctcatggaaa gccatctgga atagatacac aaacaatagt atctggaaag cctgtttggt 6120ttcaaaaggg acatgcagaa acacttaaaa ctctttcact tgatggatac atggtagtaa 6180ttgatacagg tgttaaagga agtacaagac aggctgtaga agatgttcat aaactttgcg 6240aagatcctca atatatgagt cacgtaaaac acataggaaa acttgtactt agagcatctg 6300atgttattga acatcataac tttgaagcac ttgctgatat attcaatgaa tgtcatgctg 6360atttaaaggc tcttacagta agtcatgaca aaatagaaca gttaatgaag ataggaaaag 6420aaaatggtgc tatagctggt aaattaactg gtgctggtag aggtggttca atgttattac 6480ttgcaaaaga cttaccaact gcaaagaata tagttaaagc agtagagaaa gctggtgcag 6540cacatacttg gattgaaaat ttaggtggtt aagtcgacaa agacactaaa aaattataaa 6600agtaaaggag gacattaaat gatacaagta aaggcaccag gaaaattata tatagcaggt 6660gaatacgctg ttacagaacc aggatataaa tctgttctta tagctcttga tagatttgtt 6720acagctacta ttgaggaagc tgatcaatac aaaggaacaa tacattcaaa ggcattacat 6780cacaatccag taacttttag tagagatgaa gattctattg ttatatcaga cccacacgca 6840gcaaaacaac ttaattatgt agtaactgct atagaaatat ttgagcaata tgcaaaatca 6900tgtgacatag caatgaagca ttttcattta actatagatt ctaacttaga tgatagtaat 6960ggacataagt atggacttgg atcttctgct gctgttttag tttcagtaat taaagtactt 7020aacgaatttt atgatatgaa actttcaaac ctttatatat ataagttagc agtaattgct 7080aatatgaaat tacagagttt atcttcatgc ggtgatatag cagtaagtgt ttattcaggt 7140tggttagctt attctacatt tgaccatgaa tgggtaaaac accagataga agatacaaca 7200gttgaagaag tacttattaa aaattggcct ggattacaca tagagccact tcaagctcct 7260gaaaatatgg aagttcttat aggttggaca ggtagtccag ctagtagtcc tcattttgtt 7320tctgaagtta aaagacttaa gtcagatcct tcattttacg gtgatttctt agaagattca 7380catagatgtg tagaaaaatt aattcatgca ttcaaaacta ataatattaa gggtgttcag 7440aaaatggtaa gacagaatag aactattata caaagaatgg ataaggaagc aacagttgat 7500atagagactg agaagttaaa atatttatgt gatattgctg aaaaatatca tggtgcaagt 7560aaaacttcag gtgctggtgg tggtgattgc ggaataacta taataaataa ggatgtagac 7620aaagagaaaa tatatgatga atggactaaa catggaataa agcctcttaa gtttaatatt 7680tatcatggac aataaccatg gtcaataatc ttacaataaa taaaagaaag gaggcaaaaa 7740tatgataaaa tctggaaaag caagagcaca cactaatata gcacttataa aatattgggg 7800taagaaagat gaggcattaa taataccaat gaataactca atatcagtaa ctttagaaaa 7860gttttatact gaaacaaaag ttacatttaa cgatcagctt actcaagatc aattttggct 7920taatggtgaa aaagtttctg gaaaagaatt agaaaagatt tcaaagtata tggatattgt 7980tagaaataga gctggaatag attggtatgc tgagatagaa tctgataatt ttgttcctac 8040agctgctggt cttgctagtt ctgctagtgc ttatgcagca ttagctgctg catgtaacca 8100agcacttgat ttacagttaa gtgataaaga cttaagtaga ttagctagaa ttggatcagg 8160atcagcatca agatcaatat acggtggttt tgcagaatgg gaaaaaggat ataatgacga 8220aacttcttat gctgttccat tagaaagtaa tcactttgaa gatgatcttg ctatgatttt 8280tgtagtaata aaccaacatt ctaaaaaggt tccttcaaga tatggaatgt ctcttacaag 8340aaatacaagt agattctatc aatattggtt agaccatatt gatgaagatc ttgcagaagc 8400aaaggcagca atacaagata aggattttaa gagattaggt gaagttattg aagagaatgg 8460acttagaatg catgctacaa atcttggatc aactccacct tttacttact tagtacaaga 8520gtcatacgat gtaatggcat tagtacatga gtgtagagaa gcaggatatc catgctattt 8580cactatggat gctggaccta atgtaaaaat acttgtagag aagaaaaaca aacaacagat 8640aatagataaa cttttaactc agttcgataa taatcagata atagatagtg atattatagc 8700tacaggtatt gaaattatag aataaactag ttgtatatta aaatagtaga atacataaga 8760tacttaattt aattaaagat agttaagtac ttttcaatgt gcttttttag atgtttaata 8820caaatcttta attgtaaaag aaatgctgta ctatttactg ttctagtgac gggattaaac 8880tgtattaatt ataaataaaa aataagtaca gttgtttaaa attatatttt gtattaaatc 8940taatagtacg atgtaagtta ttttatacta ttgctagttt aataaaaaga tttaattata 9000tacttgaaaa ggagaggaac tcgagatggc agagtatata atagcagtag atgagttcga 9060taacgaaata ggatcaatag aaaagatgga agctcataga aaaggaacac ttcatagagc 9120attcagtatt ttagttttta actcaaagaa tcaactttta ttacagaaaa gaaatgtaaa 9180gaaatatcac tctccaggat tatggacaaa cacttgttgt agtcacccaa gatatggtga 9240atctcttcat gatgctatat acagaagatt aaaagaagag atgggattta cttgcgaact 9300tgaagaagta ttctcattca tatataaggt aaaacttgaa gataatttat ttgagaatga 9360atatgaccat gtatttattg gtaaatatga tggtgagata attgttaata aagatgaagt 9420tgatgatttt aaatgggtag acattaatga agttaaaaag gacataatag aaagacctga 9480ggcatatact tactggttta agtatcttgt aaataaagct gaaaataaga tatttaaata 9540aaccggtcag taacgaatag aattagaaaa acaaaggagg caagacaatg gatttcccac 9600aacaattaga agcatgtgta aaacaggcta atcaggcact tagtagattt attgctcctc 9660ttccttttca aaatacacca gtagtagaaa ctatgcaata cggtgcactt ttaggtggta 9720aaagattaag accattctta gtatatgcta caggacacat gtttggtgta tcaactaata 9780ctttagacgc tccagctgct gctgttgaat gtattcatgc ttattcttta atacatgatg 9840acttaccagc aatggatgac gatgatttaa gaagaggttt acctacatgt catgttaaat 9900ttggtgaagc taatgcaatt ttagcaggtg acgctttaca aactttagct ttttctatac 9960tttcagatgc agacatgcct gaagtttcag atagagatag aatttctatg atatcagagc 10020ttgcatctgc atcaggaata gctggaatgt gcggtggtca agcacttgat ttagatgcag 10080aaggtaaaca cgtaccactt gatgctttag agagaataca tagacataaa acaggtgctc 10140ttataagagc agcagtaaga ttaggtgctt taagtgctgg tgacaagggt agaagagcac 10200ttccagtact tgataagtat gcagaaagta taggattagc ttttcaagtt caagatgaca 10260tacttgacgt tgttggtgat actgctactt taggaaaaag acagggtgca gatcagcaat 10320taggaaaatc tacataccct gctttacttg gattagaaca ggctagaaag aaagcaagag 10380acttaataga tgacgcaaga caaagtctta aacagttagc tgaacaatca cttgacacaa 10440gtgcacttga agcacttgca gattatatta tacagagaaa caagtaaaag cttttaaagg 10500aggggaaaaa atggaattta gagtacattt acaggcagac aacgaacaga aaatatttca 10560aaatcaaatg aaaccagagc cagaagcatc atatcttata aatcaaagaa gaagtgctaa 10620ttataaacca aacatttgga aaaacgattt tcttgatcag tctttaatat caaaatatga 10680tggtgatgaa tatagaaaac tttcagaaaa gttaatagaa gaagtaaaga tatacatatc 10740agcagagact atggatttag ttgctaaatt agaacttata gattctgtta gaaaacttgg 10800acttgctaat ctttttgaga aagaaataaa ggaagcatta gacagtatag cagcaataga 10860atcagataat ttaggaacta gagacgatct ttatggaaca gctcttcatt ttaagattct 10920tagacagcat ggatataagg taagtcaaga tatatttggt agatttatgg atgagaaagg 10980aacattagaa aatcatcact ttgcacactt aaaaggaatg ttagaattat ttgaggcaag 11040taatcttgga tttgaaggtg aagacatatt agatgaagct aaagcatctc ttacacttgc 11100tcttagagat tcaggacata tttgttatcc agactcaaac ttaagtagag atgtagttca 11160tagtttagaa ttacctagtc atagaagagt tcaatggttc gatgtaaaat ggcagattaa 11220tgcatacgaa aaagatattt gtagagtaaa tgcaacttta ttagagttag caaagttaaa 11280ttttaatgtt gttcaagctc agcttcagaa gaatcttaga gaagctagta gatggtgggc 11340taatcttggt ttcgcagata atttaaagtt tgctagagat agacttgtag agtgtttttc 11400atgcgcagta ggtgtagcat ttgaaccaga gcattcatct tttagaatat gtttaactaa 11460ggtaattaat cttgttctta ttatagatga tgtatacgat atatatggat ctgaagaaga 11520gttaaaacat tttacaaatg ctgttgatag atgggacagt agagaaacag aacagcttcc 11580tgaatgcatg aaaatgtgtt ttcaagtatt atataacact acttgcgaaa tagcaagaga 11640gatagaagaa gaaaacggtt ggaatcaagt attacctcaa cttactaagg tttgggctga 11700tttttgtaag gctcttttag ttgaagcaga gtggtacaat aaatcacata ttccaacatt 11760agaagaatat cttagaaacg gatgtatatc aagtagtgta tctgtacttt tagttcactc 11820tttcttttca ataactcatg aaggtacaaa agaaatggct gatttcttac ataaaaatga 11880agatctttta tacaacataa gtcttatagt aagattaaac aatgatttag gtacatcagc 11940tgctgaacag gaaagaggtg attctccttc ttctatagtt tgctatatga gagaagttaa 12000tgcttctgaa gagactgcaa gaaagaatat aaagggaatg attgataatg cttggaaaaa 12060ggttaatgga aaatgtttca caactaacca agttccattt ctttcatcat tcatgaataa 12120tgcaactaac atggcaagag tagcacactc attatataaa gacggtgatg gttttggtga 12180tcaagaaaaa ggacctagaa cacatattct tagtttatta ttccaacctt tagtaaatta 12240agctagcata aaaataagaa gcctgcattt gcaggcttct tatttttatg gcgcgccgcc 12300attatttttt tgaacaattg acaattcatt tcttattttt tattaagtga tagtcaaaag 12360gcataacagt gctgaataga aagaaattta cagaaaagaa aattatagaa tttagtatga 12420ttaattatac tcatttatga atgtttaatt gaatacaaaa aaaaatactt gttatgtatt 12480caattacggg ttaaaatata gacaagttga aaaatttaat aaaaaaataa gtcctcagct 12540cttatatatt aagctaccaa cttagtatat aagccaaaac ttaaatgtgc taccaacaca 12600tcaagccgtt agagaactct atctatagca atatttcaaa tgtaccgaca tacaagagaa 12660acattaacta tatatattca atttatgaga ttatcttaac agatataaat gtaaattgca 12720ataagtaaga tttagaagtt tatagccttt gtgtattgga agcagtacgc aaaggctttt 12780ttatttgata aaaattagaa gtatatttat tttttcataa ttaatttatg aaaatgaaag 12840ggggtgagca aagtgacaga ggaaagcagt atcttatcaa ataacaaggt attagcaata 12900tcattattga ctttagcagt aaacattatg acttttatag tgcttgtagc taagtagtac 12960gaaaggggga gctttaaaaa gctccttgga atacatagaa ttcataaatt aatttatgaa 13020aagaagggcg tatatgaaaa cttgtaaaaa ttgcaaagag tttattaaag atactgaaat 13080atgcaaaata cattcgttga tgattcatga taaaacagta gcaacctatt gcagtaaata 13140caatgagtca agatgtttac ataaagggaa agtccaatgt attaattgtt caaagatgaa 13200ccgatatgga tggtgtgcca taaaaatgag atgttttaca gaggaagaac agaaaaaaga 13260acgtacatgc attaaatatt atgcaaggag ctttaaaaaa gctcatgtaa agaagagtaa 13320aaagaaaaaa taatttattt attaatttaa tattgagagt gccgacacag tatgcactaa 13380aaaatatatc tgtggtgtag tgagccgata caaaaggata gtcactcgca ttttcataat 13440acatcttatg ttatgattat gtgtcggtgg gacttcacga cgaaaaccca caataaaaaa 13500agagttcggg gtagggttaa gcatagttga ggcaactaaa caatcaagct aggatatgca 13560gtagcagacc gtaaggtcgt tgtttaggtg tgttgtaata catacgctat taagatgtaa 13620aaatacggat accaatgaag ggaaaagtat aatttttgga tgtagtttgt ttgttcatct 13680atgggcaaac tacgtccaaa gccgtttcca aatctgctaa aaagtatatc ctttctaaaa 13740tcaaagtcaa gtatgaaatc ataaataaag tttaattttg aagttattat gatattatgt 13800ttttctatta aaataaatta agtatataga atagtttaat aatagtatat acttaatgtg 13860ataagtgtct gacagtgtca cagaaaggat gattgttatg gattataagc ggccggccag 13920tgggcaagtt gaaaaattca caaaaatgtg gtataatatc tttgttcatt agagcgataa 13980acttgaattt gagagggaac ttagatggta tttgaaaaaa ttgataaaaa tagttggaac 14040agaaaagagt attttgacca ctactttgca agtgtacctt gtacctacag catgaccgtt 14100aaagtggata tcacacaaat aaaggaaaag ggaatgaaac tatatcctgc aatgctttat 14160tatattgcaa tgattgtaaa ccgccattca gagtttagga cggcaatcaa tcaagatggt 14220gaattgggga tatatgatga gatgatacca agctatacaa tatttcacaa tgatactgaa 14280acattttcca gcctttggac tgagtgtaag tctgacttta aatcattttt agcagattat 14340gaaagtgata cgcaacggta tggaaacaat catagaatgg aaggaaagcc aaatgctccg 14400gaaaacattt ttaatgtatc tatgataccg tggtcaacct tcgatggctt taatctgaat 14460ttgcagaaag gatatgatta tttgattcct atttttacta tggggaaata ttataaagaa 14520gataacaaaa ttatacttcc tttggcaatt caagttcatc acgcagtatg tgacggattt 14580cacatttgcc gttttgtaaa cgaattgcag gaattgataa atagttaact tcaggtttgt 14640ctgtaactaa aaacaagtat ttaagcaaaa acatcgtaga aatacggtgt tttttgttac 14700cctaagttt 1470960601PRTArtificial Sequencehybrid Type II methyltransferase 60Met Phe Pro Cys Asn Ala Tyr Ile Glu Tyr Gly Asp Lys Asn Met Asn 1 5 10 15 Ser Phe Ile Glu Asp Val Glu Gln Ile Tyr Asn Phe Ile Lys Lys Asn 20 25 30 Ile Asp Val Glu Glu Lys Met His Phe Ile Glu Thr Tyr Lys Gln Lys 35 40 45 Ser Asn Met Lys Lys Glu Ile Ser Phe Ser Glu Glu Tyr Tyr Lys Gln 50 55 60 Lys Ile Met Asn Gly Lys Asn Gly Val Val Tyr Thr Pro Pro Glu Met 65 70 75 80 Ala Ala Phe Met Val Lys Asn Leu Ile Asn Val Asn Asp Val Ile Gly 85 90 95 Asn Pro Phe Ile Lys Ile Ile Asp Pro Ser Cys Gly Ser Gly Asn Leu 100 105 110 Ile Cys Lys Cys Phe Leu Tyr Leu Asn Arg Ile Phe Ile Lys Asn Ile 115 120 125 Glu Val Ile Asn Ser Lys Asn Asn Leu Asn Leu Lys Leu Glu Asp Ile 130 135 140 Ser Tyr His Ile Val Arg Asn Asn Leu Phe Gly Phe Asp Ile Asp Glu 145 150 155 160 Thr Ala Ile Lys Val Leu Lys Ile Asp Leu Phe Leu Ile Ser Asn Gln 165 170 175 Phe Ser Glu Lys Asn Phe Gln Val Lys Asp Phe Leu Val Glu Asn Ile 180 185 190 Asp Arg Lys Tyr Asp Val Phe Ile Gly Asn Pro Pro Tyr Ile Gly His 195 200 205 Lys Ser Val Asp Ser Ser Tyr Ser Tyr Val Leu Arg Lys Ile Tyr Gly 210 215 220 Ser Ile Tyr Arg Asp Lys Gly Asp Ile Ser Tyr Cys Phe Phe Gln Lys 225 230 235 240 Ser Leu Lys Cys Leu Lys Glu Gly Gly Lys Leu Val Phe Val Thr Ser 245 250 255 Arg Tyr Phe Cys Glu Ser Cys Ser Gly Lys Glu Leu Arg Lys Phe Leu 260 265 270 Ile Glu Asn Thr Ser Ile Tyr Lys Ile Ile Asp Phe Tyr Gly Ile Arg 275 280 285 Pro Phe Lys Arg Val Gly Ile Asp Pro Met Ile Ile Phe Leu Val Arg 290 295 300 Thr Lys Asn Trp Asn Asn Asn Ile Glu Ile Ile Arg Pro Asn Lys Ile 305 310 315 320 Glu Lys Asn Glu Lys Asn Lys Phe Leu Asp Ser Leu Phe Leu Asp Lys 325 330 335 Ser Glu Lys Cys Lys Lys Phe Ser Ile Ser Gln Lys Ser Ile Asn Asn 340 345 350 Asp Gly Trp Val Phe Val Asp Glu Val Glu Lys Asn Ile Ile Asp Lys 355 360 365 Ile Lys Glu Lys Ser Lys Phe Ile Leu Lys Asp Ile Cys His Ser Cys 370 375 380 Gln Gly Ile Ile Thr Gly Cys Asp Arg Ala Phe Ile Val Asp Arg Asp 385 390 395 400 Ile Ile Asn Ser Arg Lys Ile Glu Leu Arg Leu Ile Lys Pro Trp Ile 405 410 415 Lys Ser Ser His Ile Arg Lys Asn Glu Val Ile Lys Gly Glu Lys Phe 420 425 430 Ile Ile Tyr Ser Asn Leu Ile Glu Asn Glu Thr Glu Cys Pro Asn Ala 435 440 445 Ile Lys Tyr Ile Glu Gln Tyr Lys Lys Arg Leu Met Glu Arg Arg Glu 450 455 460 Cys Lys Lys Gly Thr Arg Lys Trp Tyr Glu Leu Gln Trp Gly Arg Lys 465 470 475 480 Pro Glu Ile Phe Glu Glu Lys Lys Ile Val Phe Pro Tyr Lys Ser Cys 485 490 495 Asp Asn Arg Phe Ala Leu Asp Lys Gly Ser Tyr Phe Ser Ala Asp Ile 500 505 510 Tyr Ser Leu Val Leu Lys Lys Asn Val Pro Phe Thr Tyr Glu Ile Leu 515 520 525 Leu Asn Ile Leu Asn Ser Pro Leu Tyr Glu Phe Tyr Phe Lys Thr Phe 530 535 540 Ala Lys Lys Leu Gly Glu Asn Leu Tyr Glu Tyr Tyr Pro Asn Asn Leu 545 550 555 560 Met Lys Leu Cys Ile Pro Ser Ile Asp Phe Gly Gly Glu Asn Asn Ile 565 570 575 Glu Lys Lys Leu Tyr Asp Phe Phe Gly Leu Thr Asp Lys Glu Ile Glu 580 585 590 Ile Val Glu Lys Ile Lys Asp Asn Cys 595 600 61419DNAClostridium autoethanogenum 61agaaattttc ctttctaaaa tattttattc catgtcaaga actctgttta tttcattaaa 60gaactataag tacaaagtat aaggcatttg aaaaaatagg ctagtatatt gattgattat 120ttattttaaa atgcctaagt gaaatatata catattataa caataaaata agtattagtg 180taggattttt aaatagagta tctattttca gattaaattt ttgattattt gatttacatt 240atataatatt gagtaaagta ttgactagca aaattttttg atactttaat ttgtgaaatt 300tcttatcaaa agttatattt ttgaataatt tttattgaaa aatacaacta aaaaggatta 360tagtataagt gtgtgtaatt ttgtgttaaa tttaaaggga ggaaatgaac atgaaattg 41962567DNAClostridium autoethanogenum 62ctcctaattt tgaaatctaa tatatctatt aaatcatatt ttcatatgta aataaataag 60tttttatgca attttgaaaa aggtatttgc ataaaacggc ttgaaatcaa tagttaacgc 120aatagttatt cttttagcat acattaagtc aacaaaatta gcatgtaata attatgaata 180attattacat atattcaata ttatattaaa aaaaatactt tgttttaagt ataaagtaaa 240aaaataggca taaatgtaac aaaaactgtt aattttttgt gtcaataatt tttgttatat 300tattttaatt aaatttttca catgtataat taaaagtaag atagatattc taatgtactt 360acttaggtag aaaaacatgt atacaaaatt

aaaaaactat tataacacat agtatcaata 420ttgaaggtaa tactgttcaa tatcgataca gataaaaaaa atatataata cagaagaaaa 480aattataaat ttgtggtata atataaagta tagtaattta agtttaaacc tcgtgaaaac 540gctaacaaat aataggaggt gtattat 567631806DNAArtificial Sequencehybrid Type II methyltransferase 63atgtttccgt gcaatgccta tatcgaatat ggtgataaaa atatgaacag ctttatcgaa 60gatgtggaac agatctacaa cttcattaaa aagaacattg atgtggaaga aaagatgcat 120ttcattgaaa cctataaaca gaaaagcaac atgaagaaag agattagctt tagcgaagaa 180tactataaac agaagattat gaacggcaaa aatggcgttg tgtacacccc gccggaaatg 240gcggccttta tggttaaaaa tctgatcaac gttaacgatg ttattggcaa tccgtttatt 300aaaatcattg acccgagctg cggtagcggc aatctgattt gcaaatgttt tctgtatctg 360aatcgcatct ttattaagaa cattgaggtg attaacagca aaaataacct gaatctgaaa 420ctggaagaca tcagctacca catcgttcgc aacaatctgt ttggcttcga tattgacgaa 480accgcgatca aagtgctgaa aattgatctg tttctgatca gcaaccaatt tagcgagaaa 540aatttccagg ttaaagactt tctggtggaa aatattgatc gcaaatatga cgtgttcatt 600ggtaatccgc cgtatatcgg tcacaaaagc gtggacagca gctacagcta cgtgctgcgc 660aaaatctacg gcagcatcta ccgcgacaaa ggcgatatca gctattgttt ctttcagaag 720agcctgaaat gtctgaagga aggtggcaaa ctggtgtttg tgaccagccg ctacttctgc 780gagagctgca gcggtaaaga actgcgtaaa ttcctgatcg aaaacacgag catttacaag 840atcattgatt tttacggcat ccgcccgttc aaacgcgtgg gtatcgatcc gatgattatt 900tttctggttc gtacgaagaa ctggaacaat aacattgaaa ttattcgccc gaacaagatt 960gaaaagaacg aaaagaacaa attcctggat agcctgttcc tggacaaaag cgaaaagtgt 1020aaaaagttta gcattagcca gaaaagcatt aataacgatg gctgggtttt cgtggacgaa 1080gtggagaaaa acattatcga caaaatcaaa gagaaaagca agttcattct gaaagatatt 1140tgccatagct gtcaaggcat tatcaccggt tgtgatcgcg cctttattgt ggaccgtgat 1200atcatcaata gccgtaagat cgaactgcgt ctgattaaac cgtggattaa aagcagccat 1260atccgtaaga atgaagttat taagggcgaa aaattcatca tctatagcaa cctgattgag 1320aatgaaaccg agtgtccgaa tgcgattaaa tatatcgaac agtacaagaa acgtctgatg 1380gagcgccgcg aatgcaaaaa gggcacgcgt aagtggtatg aactgcaatg gggccgtaaa 1440ccggaaatct tcgaagaaaa gaaaattgtt ttcccgtata aaagctgtga caatcgtttt 1500gcactggata agggtagcta ttttagcgca gacatttata gcctggttct gaagaaaaat 1560gtgccgttca cctatgagat cctgctgaat atcctgaata gcccgctgta cgagttttac 1620tttaagacct tcgcgaaaaa gctgggcgag aatctgtacg agtactatcc gaacaacctg 1680atgaagctgt gcatcccgag catcgatttc ggcggtgaga acaatattga gaaaaagctg 1740tatgatttct ttggtctgac ggataaagaa attgagattg tggagaagat caaagataac 1800tgctaa 1806644709DNAArtificial Sequencedesigned methylation plasmid 64gtttgccacc tgacgtctaa gaaaaggaat attcagcaat ttgcccgtgc cgaagaaagg 60cccacccgtg aaggtgagcc agtgagttga ttgctacgta attagttagt tagcccttag 120tgactcgtaa tacgactcac tatagggctc gaggcggccg cgcaacgcaa ttaatgtgag 180ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg 240tggaattgtg agcggataac aatttcacac aggaaacaca tatgtttccg tgcaatgcct 300atatcgaata tggtgataaa aatatgaaca gctttatcga agatgtggaa cagatctaca 360acttcattaa aaagaacatt gatgtggaag aaaagatgca tttcattgaa acctataaac 420agaaaagcaa catgaagaaa gagattagct ttagcgaaga atactataaa cagaagatta 480tgaacggcaa aaatggcgtt gtgtacaccc cgccggaaat ggcggccttt atggttaaaa 540atctgatcaa cgttaacgat gttattggca atccgtttat taaaatcatt gacccgagct 600gcggtagcgg caatctgatt tgcaaatgtt ttctgtatct gaatcgcatc tttattaaga 660acattgaggt gattaacagc aaaaataacc tgaatctgaa actggaagac atcagctacc 720acatcgttcg caacaatctg tttggcttcg atattgacga aaccgcgatc aaagtgctga 780aaattgatct gtttctgatc agcaaccaat ttagcgagaa aaatttccag gttaaagact 840ttctggtgga aaatattgat cgcaaatatg acgtgttcat tggtaatccg ccgtatatcg 900gtcacaaaag cgtggacagc agctacagct acgtgctgcg caaaatctac ggcagcatct 960accgcgacaa aggcgatatc agctattgtt tctttcagaa gagcctgaaa tgtctgaagg 1020aaggtggcaa actggtgttt gtgaccagcc gctacttctg cgagagctgc agcggtaaag 1080aactgcgtaa attcctgatc gaaaacacga gcatttacaa gatcattgat ttttacggca 1140tccgcccgtt caaacgcgtg ggtatcgatc cgatgattat ttttctggtt cgtacgaaga 1200actggaacaa taacattgaa attattcgcc cgaacaagat tgaaaagaac gaaaagaaca 1260aattcctgga tagcctgttc ctggacaaaa gcgaaaagtg taaaaagttt agcattagcc 1320agaaaagcat taataacgat ggctgggttt tcgtggacga agtggagaaa aacattatcg 1380acaaaatcaa agagaaaagc aagttcattc tgaaagatat ttgccatagc tgtcaaggca 1440ttatcaccgg ttgtgatcgc gcctttattg tggaccgtga tatcatcaat agccgtaaga 1500tcgaactgcg tctgattaaa ccgtggatta aaagcagcca tatccgtaag aatgaagtta 1560ttaagggcga aaaattcatc atctatagca acctgattga gaatgaaacc gagtgtccga 1620atgcgattaa atatatcgaa cagtacaaga aacgtctgat ggagcgccgc gaatgcaaaa 1680agggcacgcg taagtggtat gaactgcaat ggggccgtaa accggaaatc ttcgaagaaa 1740agaaaattgt tttcccgtat aaaagctgtg acaatcgttt tgcactggat aagggtagct 1800attttagcgc agacatttat agcctggttc tgaagaaaaa tgtgccgttc acctatgaga 1860tcctgctgaa tatcctgaat agcccgctgt acgagtttta ctttaagacc ttcgcgaaaa 1920agctgggcga gaatctgtac gagtactatc cgaacaacct gatgaagctg tgcatcccga 1980gcatcgattt cggcggtgag aacaatattg agaaaaagct gtatgatttc tttggtctga 2040cggataaaga aattgagatt gtggagaaga tcaaagataa ctgctaagaa ttcgatatca 2100cccgggaact agtctgcagc cctttagtga gggttaattg gagtcactaa gggttagtta 2160gttagattag cagaaagtca aaagcctccg accggaggct tttgactaaa acttcccttg 2220gggttatcat tggggctcac tcaaaggcgg taatcagata aaaaaaatcc ttagctttcg 2280ctaaggatga tttctgctag agatggaata gactggatgg aggcggataa agttgcagga 2340ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 2400gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 2460gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 2520gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata 2580ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 2640gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 2700ttaataagat gatcttcttg agatcgtttt ggtctgcgcg taatctcttg ctctgaaaac 2760gaaaaaaccg ccttgcaggg cggtttttcg aaggttctct gagctaccaa ctctttgaac 2820cgaggtaact ggcttggagg agcgcagtca ccaaaacttg tcctttcagt ttagccttaa 2880ccggcgcatg acttcaagac taactcctct aaatcaatta ccagtggctg ctgccagtgg 2940tgcttttgca tgtctttccg ggttggactc aagacgatag ttaccggata aggcgcagcg 3000gtcggactga acggggggtt cgtgcataca gtccagcttg gagcgaactg cctacccgga 3060actgagtgtc aggcgtggaa tgagacaaac gcggccataa cagcggaatg acaccggtaa 3120accgaaaggc aggaacagga gagcgcacga gggagccgcc aggggaaacg cctggtatct 3180ttatagtcct gtcgggtttc gccaccactg atttgagcgt cagatttcgt gatgcttgtc 3240aggggggcgg agcctatgga aaaacggctt tgccgcggcc ctctcacttc cctgttaagt 3300atcttcctgg catcttccag gaaatctccg ccccgttcgt aagccatttc cgctcgccgc 3360agtcgaacga ccgagcgtag cgagtcagtg agcgaggaag cggaatatat cctgtatcac 3420atattctgct gacgcaccgg tgcagccttt tttctcctgc cacatgaagc acttcactga 3480caccctcatc agtgccaaca tagtaagcca gtatacactc cgctagcgct gaggtctgcc 3540tcgtgaagaa ggtgttgctg actcatacca ggcctgaatc gccccatcat ccagccagaa 3600agtgagggag ccacggttga tgagagcttt gttgtaggtg gaccagttgg tgattttgaa 3660cttttgcttt gccacggaac ggtctgcgtt gtcgggaaga tgcgtgatct gatccttcaa 3720ctcagcaaaa gttcgattta ttcaacaaag ccacgttgtg tctcaaaatc tctgatgtta 3780cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag 3840taatacaagg ggtgtttact agaggttgat cgggcacgta agaggttcca actttcacca 3900taatgaaata agatcactac cgggcgtatt ttttgagtta tcgagatttt caggagctaa 3960ggaagctaaa atggagaaaa aaatcacggg atataccacc gttgatatat cccaatggca 4020tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt 4080tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc 4140ggcctttatt cacattcttg cccgcctgat gaacgctcac ccggagtttc gtatggccat 4200gaaagacggt gagctggtga tctgggatag tgttcaccct tgttacaccg ttttccatga 4260gcaaactgaa acgttttcgt ccctctggag tgaataccac gacgatttcc ggcagtttct 4320ccacatatat tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg 4380gtttattgag aatatgtttt ttgtctcagc caatccctgg gtgagtttca ccagttttga 4440tttaaacgtg gccaatatgg acaacttctt cgcccccgtt ttcacgatgg gcaaatatta 4500tacgcaaggc gacaaggtgc tgatgccgct ggcgatccag gttcatcatg ccgtttgtga 4560tggcttccat gtcggccgca tgcttaatga attacaacag tactgtgatg agtggcaggg 4620cggggcgtaa taatactagc tccggcaaaa aaacgggcaa ggtgtcacca ccctgccctt 4680tttctttaaa accgaaaaga ttacttcgc 47096518DNAArtificial SequenceOligonucleotide colE1-F 65cgtcagaccc cgtagaaa 186618DNAArtificial SequenceOligonucleotide colE1-R 66ctctcctgtt ccgaccct 186737DNAArtificial SequenceOligonucleotide fD1 67ccgaattcgt cgacaacaga gtttgatcct ggctcag 376837DNAArtificial SequenceOligonucleotide Rp2 68cccgggatcc aagcttacgg ctaccttgtt acgactt 376925DNAArtificial SequenceOligonucleotide ispS-F 69aggctgaatt tcttacactt cttga 257025DNAArtificial SequenceOligonucleotide ispS-R 70gtaactccat caaatcctcc actac 257125DNAArtificial SequenceOligonucleotide idi-F 71atacgtgctg tagtcatcca agata 257225DNAArtificial SequenceOligonucleotide idiR 72tcttcaagtt cacatgtaaa accca 257325DNAArtificial Sequenceoligonucleotide dxs-F 73acaaagtatc taagacagga ggtca 257425DNAArtificial Sequenceoligonucleotide dxs-R 74gatgtcccac atcccatata agttt 25756018DNAArtificial Sequenceplasmid pMTL85246-IspS-Idi 75ccggggatcc tctagagtcg acgtcacgcg tccatggaga tctcgaggcc tgcagacatg 60caagcttggc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc 120aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc 180gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc tagcataaaa 240ataagaagcc tgcatttgca ggcttcttat ttttatggcg cgccgcattc acttcttttc 300tatataaata tgagcgaagc gaataagcgt cggaaaagca gcaaaaagtt tcctttttgc 360tgttggagca tgggggttca gggggtgcag tatctgacgt caatgccgag cgaaagcgag 420ccgaagggta gcatttacgt tagataaccc cctgatatgc tccgacgctt tatatagaaa 480agaagattca actaggtaaa atcttaatat aggttgagat gataaggttt ataaggaatt 540tgtttgttct aatttttcac tcattttgtt ctaatttctt ttaacaaatg ttcttttttt 600tttagaacag ttatgatata gttagaatag tttaaaataa ggagtgagaa aaagatgaaa 660gaaagatatg gaacagtcta taaaggctct cagaggctca tagacgaaga aagtggagaa 720gtcatagagg tagacaagtt ataccgtaaa caaacgtctg gtaacttcgt aaaggcatat 780atagtgcaat taataagtat gttagatatg attggcggaa aaaaacttaa aatcgttaac 840tatatcctag ataatgtcca cttaagtaac aatacaatga tagctacaac aagagaaata 900gcaaaagcta caggaacaag tctacaaaca gtaataacaa cacttaaaat cttagaagaa 960ggaaatatta taaaaagaaa aactggagta ttaatgttaa accctgaact actaatgaga 1020ggcgacgacc aaaaacaaaa atacctctta ctcgaatttg ggaactttga gcaagaggca 1080aatgaaatag attgacctcc caataacacc acgtagttat tgggaggtca atctatgaaa 1140tgcgattaag ggccggccga agcaaactta agagtgtgtt gatagtgcag tatcttaaaa 1200ttttgtataa taggaattga agttaaatta gatgctaaaa atttgtaatt aagaaggagt 1260gattacatga acaaaaatat aaaatattct caaaactttt taacgagtga aaaagtactc 1320aaccaaataa taaaacaatt gaatttaaaa gaaaccgata ccgtttacga aattggaaca 1380ggtaaagggc atttaacgac gaaactggct aaaataagta aacaggtaac gtctattgaa 1440ttagacagtc atctattcaa cttatcgtca gaaaaattaa aactgaatac tcgtgtcact 1500ttaattcacc aagatattct acagtttcaa ttccctaaca aacagaggta taaaattgtt 1560gggagtattc cttaccattt aagcacacaa attattaaaa aagtggtttt tgaaagccat 1620gcgtctgaca tctatctgat tgttgaagaa ggattctaca agcgtacctt ggatattcac 1680cgaacactag ggttgctctt gcacactcaa gtctcgattc agcaattgct taagctgcca 1740gcggaatgct ttcatcctaa accaaaagta aacagtgtct taataaaact tacccgccat 1800accacagatg ttccagataa atattggaag ctatatacgt actttgtttc aaaatgggtc 1860aatcgagaat atcgtcaact gtttactaaa aatcagtttc atcaagcaat gaaacacgcc 1920aaagtaaaca atttaagtac cgttacttat gagcaagtat tgtctatttt taatagttat 1980ctattattta acgggaggaa ataattctat gagtcgcttt tgtaaatttg gaaagttaca 2040cgttactaaa gggaatgtgt ttaaactcct ttttgataat ctcatgacca aaatccctta 2100acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 2160agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 2220ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 2280cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa 2340gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 2400cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 2460gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 2520caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 2580aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 2640tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 2700gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 2760ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt 2820atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg 2880cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg 2940cagggccccc tgcaggataa aaaaattgta gataaatttt ataaaatagt tttatctaca 3000atttttttat caggaaacag ctatgaccgc ggccgcggtt aatgttaaaa atttatagta 3060taactttaaa aaactgtctt aaaaagttgt tatataaaaa atgttgacaa ttaaacagct 3120atttagtgca aaacaaccat aaaaatttaa aaaataccat aaattacttg aaaaatagtt 3180gataataatg tagagttata aacaaaggtg aaaagcatta cttgtattct tttttatata 3240ttattataaa ttaaaatgaa gctgtattag aaaaaataca cacctgtaat ataaaatttt 3300aaattaattt ttaatttttt caaaatgtat tttacatgtt tagaattttg atgtatatta 3360aaatagtaga atacataaga tacttaattt aattaaagat agttaagtac ttttcaatgt 3420gcttttttag atgtttaata caaatcttta attgtaaaag aaatgctgta ctatttactg 3480tactagtgac gggattaaac tgtattaatt ataaataaaa aataagtaca gttgtttaaa 3540attatatttt gtattaaatc taatagtacg atgtaagtta ttttatacta ttgctagttt 3600aataaaaaga tttaattata tacttgaaaa ggagaggaat ttttatgcgt catatggcaa 3660cagaattatt atgtttacac agacctatat cacttactca caaacttttt aggaatccat 3720tacctaaagt tattcaagct acacctttaa cattaaaact taggtgtagt gtttctacag 3780aaaatgtatc atttagtgag acagaaactg aaacaagaag atcagcaaat tatgaaccaa 3840attcttggga ttatgattat cttctttctt ctgatactga tgagtcaata gaagtacata 3900aagataaggc taagaaatta gaagctgaag ttaggagaga aataaataat gagaaggctg 3960aatttcttac acttcttgaa cttattgata atgtacaaag acttggatta ggatatagat 4020ttgagtctga tataagaaga gcattagata gatttgtaag tagtggagga tttgatggag 4080ttactaaaac ttcattacat ggaacagcat tatcatttag gttattaagg caacatggtt 4140ttgaagtatc tcaagaagct tttagtggat ttaaagatca gaatggaaac tttcttgaga 4200atttaaagga agacataaaa gcaattcttt ctctttatga agcatcattt ttagcattag 4260aaggtgagaa tatattagat gaggctaaag tatttgcaat atctcatctt aaagaactta 4320gtgaagaaaa gattggtaaa gaattagctg aacaagtttc acatgcttta gaattaccat 4380tacatagaag aacacaaaga ttagaagcag tttggtcaat agaagcatat agaaagaaag 4440aagacgcaaa tcaagtactt ttagaacttg caatacttga ctacaatatg attcaaagtg 4500tatatcagag ggatttaaga gaaacatcaa gatggtggag aagagtagga ttagcaacta 4560aattacattt tgctagagat aggcttattg aaagttttta ttgggctgtt ggagttgctt 4620ttgaaccaca atattctgat tgcagaaata gtgtagcaaa gatgttttca tttgttacta 4680taattgacga tatttacgat gtatatggaa ctttagatga acttgaactt tttactgatg 4740cagttgaaag atgggatgta aatgctatta atgatcttcc tgattatatg aagttatgtt 4800ttcttgcact ttacaatact attaacgaga tagcttacga taacttaaaa gataaaggtg 4860agaacatact tccttattta acaaaagcat gggcagattt atgtaatgca tttcttcaag 4920aagctaagtg gctttataat aaatcaacac ctacatttga tgattatttt ggaaatgcat 4980ggaaaagttc tagtggacct ttacagctta tttttgctta ttttgctgta gtacagaaca 5040ttaaaaagga agagattgag aatcttcaga aatatcatga cataatatca agacctagtc 5100acatttttag gctttgtaat gatttagcat ctgcttcagc agaaatagca agaggtgaaa 5160ctgctaattc tgtaagttgt tatatgagaa caaaaggtat atctgaagaa ttagctactg 5220aaagtgttat gaatcttata gacgaaactt ggaagaaaat gaacaaagaa aaacttggtg 5280gatctttatt tgcaaaacct tttgttgaga ctgctataaa tttagctaga cagtctcatt 5340gcacatatca taatggtgat gcacatacta gtccagatga attaactagg aaaagagtac 5400ttagtgtaat aactgaacca atattaccat ttgaaagata agaattcgag ctcgaaaggg 5460gaaattaaat ggcagaatat ataatagctg tagatgaatt tgataacgaa ataggttcaa 5520ttgaaaaaat ggaggctcac cgtaaaggaa cattacatag agctttttct atattagtat 5580ttaattctaa aaatcaattg ttattacaga aaagaaatgt aaaaaaatat cattcgcctg 5640gtctctggac aaatacgtgc tgtagtcatc caagatacgg tgaaagttta catgatgcga 5700tttatagaag gcttaaggaa gaaatgggtt ttacatgtga acttgaagaa gtatttagtt 5760ttatttataa agtaaaactt gaagataatc tttttgaaaa tgaatatgat catgtattca 5820ttgggaaata tgatggagaa ataattgtaa acaaagatga agtagatgat tttaagtggg 5880ttgatattaa tgaggttaag aaggatatta tagaaaggcc agaagcatac acttattggt 5940tcaagtattt agttaataag gcagaaaaca aaatatttaa ataagtaaga atttcgtcta 6000aataaagatt tggggtac 6018766909DNAArtificial Sequenceplasmid pMTL 82151-Patp-HMGR 76cctgcaggat aaaaaaattg tagataaatt ttataaaata gttttatcta caattttttt 60atcaggaaac agctatgacc gcggccgctg tatccatatg accatgatta cgaattcgag 120ctcgttataa ttttcaattt tcattctttt taaaggagat tagcatacat tttatcataa 180ttatacagac aatatagtaa tatatgatgt taaaatatca atatatggtt aaaaatctgt 240atattttttc ccattttaat tatttgtact ataatattac actgagtgta ttgcatattt 300aaaaaatatt tggtacaatt agttagttaa ataaattcta aattgtaaat tatcagaatc 360cttattaagg aaatacatag atttaaggag aaatcataaa aaggtgtaat ataaactggc 420taaaattgag caaaaattga gcaattaaga ctttttgatt gtatcttttt atatatttaa 480ggtatataat cttatttata ttgggggaag gtaccatgca atcattagac aaaaatttca 540gacatttatc aagacaacaa aagttacaac aattagttga taaacagtgg ctttcagaag 600atcagtttga tattttactt aatcatcctc ttatagatga agaagttgct aatagtctta 660tagaaaatgt aattgcacag ggtgcattac cagttggact tcttcctaat ataatagttg 720atgataaggc ttatgttgta ccaatgatgg ttgaagaacc tagtgttgtt gcagctgcat 780cttatggtgc taaattagta aatcagacag gtggatttaa aactgtatca tcagaaagaa 840taatgattgg acagatagta tttgatggtg tagatgacac tgaaaaatta agtgcagata 900ttaaagcatt

agaaaaacaa atacataaga ttgcagatga agcatatcct agtataaaag 960caagaggtgg tggttatcaa agaatagcaa tagatacatt tccagagcaa caacttttaa 1020gtcttaaggt atttgtagat acaaaagatg ctatgggtgc taatatgctt aatactatac 1080ttgaggcaat aactgcattc cttaaaaatg aatctcctca atcagatata ttaatgtcta 1140tactttcaaa ccatgcaact gctagtgtag taaaagtaca aggtgagata gatgtaaaag 1200atcttgctag aggtgaaaga acaggtgaag aagtagctaa gagaatggaa agagcttctg 1260tattagctca ggttgatatt catagagctg caacacataa caaaggtgtt atgaatggaa 1320tacatgctgt tgttttagct acaggaaatg atactagagg tgctgaagca tctgcacatg 1380catacgcatc aagagacgga caatatagag gtatagcaac ttggagatat gatcagaaga 1440gacaaagact tattggaact attgaagttc caatgacact tgctatagta ggtggtggta 1500ctaaagtatt accaatagct aaggcatcat tagagttatt aaatgttgat tctgcacaag 1560aacttggaca cgtagttgct gctgttggat tagcacaaaa ctttgctgct tgtagagcac 1620ttgtttctga aggtattcaa caaggacaca tgtcattaca atataaaagt ttagcaatag 1680tagtaggtgc aaaaggtgac gagatagcac aagtagcaga agctcttaaa caggaaccaa 1740gagctaatac acaggttgct gaaagaattt tacaggaaat tagacagcaa taatctagag 1800tcgacgtcac gcgtccatgg agatctcgag gcctgcagac atgcaagctt ggcactggcc 1860gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca 1920gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc 1980caacagttgc gcagcctgaa tggcgaatgg cgctagcata aaaataagaa gcctgcattt 2040gcaggcttct tatttttatg gcgcgccgtt ctgaatcctt agctaatggt tcaacaggta 2100actatgacga agatagcacc ctggataagt ctgtaatgga ttctaaggca tttaatgaag 2160acgtgtatat aaaatgtgct aatgaaaaag aaaatgcgtt aaaagagcct aaaatgagtt 2220caaatggttt tgaaattgat tggtagttta atttaatata ttttttctat tggctatctc 2280gatacctata gaatcttctg ttcacttttg tttttgaaat ataaaaaggg gctttttagc 2340cccttttttt taaaactccg gaggagtttc ttcattcttg atactatacg taactatttt 2400cgatttgact tcattgtcaa ttaagctagt aaaatcaatg gttaaaaaac aaaaaacttg 2460catttttcta cctagtaatt tataatttta agtgtcgagt ttaaaagtat aatttaccag 2520gaaaggagca agttttttaa taaggaaaaa tttttccttt taaaattcta tttcgttata 2580tgactaatta taatcaaaaa aatgaaaata aacaagaggt aaaaactgct ttagagaaat 2640gtactgataa aaaaagaaaa aatcctagat ttacgtcata catagcacct ttaactacta 2700agaaaaatat tgaaaggact tccacttgtg gagattattt gtttatgttg agtgatgcag 2760acttagaaca ttttaaatta cataaaggta atttttgcgg taatagattt tgtccaatgt 2820gtagttggcg acttgcttgt aaggatagtt tagaaatatc tattcttatg gagcatttaa 2880gaaaagaaga aaataaagag tttatatttt taactcttac aactccaaat gtaaaaagtt 2940atgatcttaa ttattctatt aaacaatata ataaatcttt taaaaaatta atggagcgta 3000aggaagttaa ggatataact aaaggttata taagaaaatt agaagtaact taccaaaagg 3060aaaaatacat aacaaaggat ttatggaaaa taaaaaaaga ttattatcaa aaaaaaggac 3120ttgaaattgg tgatttagaa cctaattttg atacttataa tcctcatttt catgtagtta 3180ttgcagttaa taaaagttat tttacagata aaaattatta tataaatcga gaaagatggt 3240tggaattatg gaagtttgct actaaggatg attctataac tcaagttgat gttagaaaag 3300caaaaattaa tgattataaa gaggtttacg aacttgcgaa atattcagct aaagacactg 3360attatttaat atcgaggcca gtatttgaaa ttttttataa agcattaaaa ggcaagcagg 3420tattagtttt tagtggattt tttaaagatg cacacaaatt gtacaagcaa ggaaaacttg 3480atgtttataa aaagaaagat gaaattaaat atgtctatat agtttattat aattggtgca 3540aaaaacaata tgaaaaaact agaataaggg aacttacgga agatgaaaaa gaagaattaa 3600atcaagattt aatagatgaa atagaaatag attaaagtgt aactatactt tatatatata 3660tgattaaaaa aataaaaaac aacagcctat taggttgttg ttttttattt tctttattaa 3720tttttttaat ttttagtttt tagttctttt ttaaaataag tttcagcctc tttttcaata 3780ttttttaaag aaggagtatt tgcatgaatt gccttttttc taacagactt aggaaatatt 3840ttaacagtat cttcttgcgc cggtgatttt ggaacttcat aacttactaa tttataatta 3900ttattttctt ttttaattgt aacagttgca aaagaagctg aacctgttcc ttcaactagt 3960ttatcatctt caatataata ttcttgacct atatagtata aatatatttt tattatattt 4020ttactttttt ctgaatctat tattttataa tcataaaaag ttttaccacc aaaagaaggt 4080tgtactcctt ctggtccaac atattttttt actatattat ctaaataatt tttgggaact 4140ggtgttgtaa tttgattaat cgaacaacca gttatactta aaggaattat aactataaaa 4200atatatagga ttatcttttt aaatttcatt attggcctcc tttttattaa atttatgtta 4260ccataaaaag gacataacgg gaatatgtag aatattttta atgtagacaa aattttacat 4320aaatataaag aaaggaagtg tttgtttaaa ttttatagca aactatcaaa aattaggggg 4380ataaaaattt atgaaaaaaa ggttttcgat gttattttta tgtttaactt taatagtttg 4440tggtttattt acaaattcgg ccggccagtg ggcaagttga aaaattcaca aaaatgtggt 4500ataatatctt tgttcattag agcgataaac ttgaatttga gagggaactt agatggtatt 4560tgaaaaaatt gataaaaata gttggaacag aaaagagtat tttgaccact actttgcaag 4620tgtaccttgt acctacagca tgaccgttaa agtggatatc acacaaataa aggaaaaggg 4680aatgaaacta tatcctgcaa tgctttatta tattgcaatg attgtaaacc gccattcaga 4740gtttaggacg gcaatcaatc aagatggtga attggggata tatgatgaga tgataccaag 4800ctatacaata tttcacaatg atactgaaac attttccagc ctttggactg agtgtaagtc 4860tgactttaaa tcatttttag cagattatga aagtgatacg caacggtatg gaaacaatca 4920tagaatggaa ggaaagccaa atgctccgga aaacattttt aatgtatcta tgataccgtg 4980gtcaaccttc gatggcttta atctgaattt gcagaaagga tatgattatt tgattcctat 5040ttttactatg gggaaatatt ataaagaaga taacaaaatt atacttcctt tggcaattca 5100agttcatcac gcagtatgtg acggatttca catttgccgt tttgtaaacg aattgcagga 5160attgataaat agttaacttc aggtttgtct gtaactaaaa acaagtattt aagcaaaaac 5220atcgtagaaa tacggtgttt tttgttaccc taagtttaaa ctcctttttg ataatctcat 5280gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat 5340caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa 5400accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa 5460ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt 5520aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt 5580accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata 5640gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt 5700ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac 5760gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga 5820gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg 5880ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa 5940aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 6000gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc 6060tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga 6120agagcgccca atacgcaggg ccccctgctt cggggtcatt atagcgattt tttcggtata 6180tccatccttt ttcgcacgat atacaggatt ttgccaaagg gttcgtgtag actttccttg 6240gtgtatccaa cggcgtcagc cgggcaggat aggtgaagta ggcccacccg cgagcgggtg 6300ttccttcttc actgtccctt attcgcacct ggcggtgctc aacgggaatc ctgctctgcg 6360aggctggccg gctaccgccg gcgtaacaga tgagggcaag cggatggctg atgaaaccaa 6420gccaaccagg aagggcagcc cacctatcaa ggtgtactgc cttccagacg aacgaagagc 6480gattgaggaa aaggcggcgg cggccggcat gagcctgtcg gcctacctgc tggccgtcgg 6540ccagggctac aaaatcacgg gcgtcgtgga ctatgagcac gtccgcgagc tggcccgcat 6600caatggcgac ctgggccgcc tgggcggcct gctgaaactc tggctcaccg acgacccgcg 6660cacggcgcgg ttcggtgatg ccacgatcct cgccctgctg gcgaagatcg aagagaagca 6720ggacgagctt ggcaaggtca tgatgggcgt ggtccgcccg agggcagagc catgactttt 6780ttagccgcta aaacggccgg ggggtgcgcg tgattgccaa gcacgtcccc atgcgctcca 6840tcaagaagag cgacttcgcg gagctggtga agtacatcac cgacgagcaa ggcaagaccg 6900atcgggccc 69097731DNAArtificial Sequenceoligonucleotide EcoRI-HMGS_F 77agccgtgaat tcgaggcttt tactaaaaac a 317832DNAArtificial Sequenceoligonucleotide EcoRI-HMGS_R 78aggcgtctag atgttcgtct ctacaaataa tt 32798116DNAArtificial Sequenceplasmid pMTL 82151-HMGS-Patp-HMGR 79cctgcaggat aaaaaaattg tagataaatt ttataaaata gttttatcta caattttttt 60atcaggaaac agctatgacc gcggccgctg tatccatatg accatgatta cgaattcgag 120gcttttacta aaaacaataa aaacaggagg aaataatatg actataggaa ttgacaaaat 180aaacttttac gtaccaaaat attatgtaga tatggcaaaa ttagcagaag caagacaagt 240agacccaaat aaatttctta ttggaatagg acagactgaa atggcagtta gtccagtaaa 300ccaagatata gtatcaatgg gtgctaatgc tgctaaagat ataataactg atgaagacaa 360aaagaaaata ggaatggtaa tagtagcaac tgagtcagca gtagatgcag caaaggcagc 420agcagtacag attcataatt tattaggtat tcaaccattt gcaagatgtt tcgaaatgaa 480agaagcatgt tatgctgcta ctcctgcaat tcagttagct aaggattatt tagctacaag 540accaaatgag aaagttttag ttatagctac agatacagct agatatggac ttaattcagg 600tggtgaacct actcaaggtg ctggtgctgt tgctatggtt atagctcata atcctagtat 660acttgcatta aatgaagacg ctgttgctta tacagaagat gtttatgatt tctggagacc 720aacaggacat aagtatccat tagtagatgg tgctttatca aaagacgcat atattagatc 780ttttcaacaa tcttggaatg aatatgctaa gagacaagga aagagtttag ctgattttgc 840tagtctttgc tttcatgttc cttttactaa aatgggtaaa aaggctttag aatctataat 900agataacgca gatgaaacaa ctcaagagag attaagatct ggatatgaag atgcagttga 960ttacaataga tatgttggaa atatatacac aggaagtctt tatctttctc ttataagtct 1020tcttgaaaat agagatttac aggctggtga aactattgga ttattttcat acggatcagg 1080ttctgttggt gaattttatt cagctacact tgtagaagga tataaagatc accttgatca 1140ggcagcacac aaagcacttt taaacaatag aactgaagta tcagtagatg catacgaaac 1200atttttcaag agatttgatg atgtagaatt tgatgaagag caggatgcag ttcatgaaga 1260tagacatata ttctatcttt caaacataga gaataatgta agagaatatc atagacctga 1320ataagagctc gttataattt tcaattttca ttctttttaa aggagattag catacatttt 1380atcataatta tacagacaat atagtaatat atgatgttaa aatatcaata tatggttaaa 1440aatctgtata ttttttccca ttttaattat ttgtactata atattacact gagtgtattg 1500catatttaaa aaatatttgg tacaattagt tagttaaata aattctaaat tgtaaattat 1560cagaatcctt attaaggaaa tacatagatt taaggagaaa tcataaaaag gtgtaatata 1620aactggctaa aattgagcaa aaattgagca attaagactt tttgattgta tctttttata 1680tatttaaggt atataatctt atttatattg ggggaaggta ccatgcaatc attagacaaa 1740aatttcagac atttatcaag acaacaaaag ttacaacaat tagttgataa acagtggctt 1800tcagaagatc agtttgatat tttacttaat catcctctta tagatgaaga agttgctaat 1860agtcttatag aaaatgtaat tgcacagggt gcattaccag ttggacttct tcctaatata 1920atagttgatg ataaggctta tgttgtacca atgatggttg aagaacctag tgttgttgca 1980gctgcatctt atggtgctaa attagtaaat cagacaggtg gatttaaaac tgtatcatca 2040gaaagaataa tgattggaca gatagtattt gatggtgtag atgacactga aaaattaagt 2100gcagatatta aagcattaga aaaacaaata cataagattg cagatgaagc atatcctagt 2160ataaaagcaa gaggtggtgg ttatcaaaga atagcaatag atacatttcc agagcaacaa 2220cttttaagtc ttaaggtatt tgtagataca aaagatgcta tgggtgctaa tatgcttaat 2280actatacttg aggcaataac tgcattcctt aaaaatgaat ctcctcaatc agatatatta 2340atgtctatac tttcaaacca tgcaactgct agtgtagtaa aagtacaagg tgagatagat 2400gtaaaagatc ttgctagagg tgaaagaaca ggtgaagaag tagctaagag aatggaaaga 2460gcttctgtat tagctcaggt tgatattcat agagctgcaa cacataacaa aggtgttatg 2520aatggaatac atgctgttgt tttagctaca ggaaatgata ctagaggtgc tgaagcatct 2580gcacatgcat acgcatcaag agacggacaa tatagaggta tagcaacttg gagatatgat 2640cagaagagac aaagacttat tggaactatt gaagttccaa tgacacttgc tatagtaggt 2700ggtggtacta aagtattacc aatagctaag gcatcattag agttattaaa tgttgattct 2760gcacaagaac ttggacacgt agttgctgct gttggattag cacaaaactt tgctgcttgt 2820agagcacttg tttctgaagg tattcaacaa ggacacatgt cattacaata taaaagttta 2880gcaatagtag taggtgcaaa aggtgacgag atagcacaag tagcagaagc tcttaaacag 2940gaaccaagag ctaatacaca ggttgctgaa agaattttac aggaaattag acagcaataa 3000tctagagtcg acgtcacgcg tccatggaga tctcgaggcc tgcagacatg caagcttggc 3060actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg 3120ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg 3180cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc tagcataaaa ataagaagcc 3240tgcatttgca ggcttcttat ttttatggcg cgccgttctg aatccttagc taatggttca 3300acaggtaact atgacgaaga tagcaccctg gataagtctg taatggattc taaggcattt 3360aatgaagacg tgtatataaa atgtgctaat gaaaaagaaa atgcgttaaa agagcctaaa 3420atgagttcaa atggttttga aattgattgg tagtttaatt taatatattt tttctattgg 3480ctatctcgat acctatagaa tcttctgttc acttttgttt ttgaaatata aaaaggggct 3540ttttagcccc ttttttttaa aactccggag gagtttcttc attcttgata ctatacgtaa 3600ctattttcga tttgacttca ttgtcaatta agctagtaaa atcaatggtt aaaaaacaaa 3660aaacttgcat ttttctacct agtaatttat aattttaagt gtcgagttta aaagtataat 3720ttaccaggaa aggagcaagt tttttaataa ggaaaaattt ttccttttaa aattctattt 3780cgttatatga ctaattataa tcaaaaaaat gaaaataaac aagaggtaaa aactgcttta 3840gagaaatgta ctgataaaaa aagaaaaaat cctagattta cgtcatacat agcaccttta 3900actactaaga aaaatattga aaggacttcc acttgtggag attatttgtt tatgttgagt 3960gatgcagact tagaacattt taaattacat aaaggtaatt tttgcggtaa tagattttgt 4020ccaatgtgta gttggcgact tgcttgtaag gatagtttag aaatatctat tcttatggag 4080catttaagaa aagaagaaaa taaagagttt atatttttaa ctcttacaac tccaaatgta 4140aaaagttatg atcttaatta ttctattaaa caatataata aatcttttaa aaaattaatg 4200gagcgtaagg aagttaagga tataactaaa ggttatataa gaaaattaga agtaacttac 4260caaaaggaaa aatacataac aaaggattta tggaaaataa aaaaagatta ttatcaaaaa 4320aaaggacttg aaattggtga tttagaacct aattttgata cttataatcc tcattttcat 4380gtagttattg cagttaataa aagttatttt acagataaaa attattatat aaatcgagaa 4440agatggttgg aattatggaa gtttgctact aaggatgatt ctataactca agttgatgtt 4500agaaaagcaa aaattaatga ttataaagag gtttacgaac ttgcgaaata ttcagctaaa 4560gacactgatt atttaatatc gaggccagta tttgaaattt tttataaagc attaaaaggc 4620aagcaggtat tagtttttag tggatttttt aaagatgcac acaaattgta caagcaagga 4680aaacttgatg tttataaaaa gaaagatgaa attaaatatg tctatatagt ttattataat 4740tggtgcaaaa aacaatatga aaaaactaga ataagggaac ttacggaaga tgaaaaagaa 4800gaattaaatc aagatttaat agatgaaata gaaatagatt aaagtgtaac tatactttat 4860atatatatga ttaaaaaaat aaaaaacaac agcctattag gttgttgttt tttattttct 4920ttattaattt ttttaatttt tagtttttag ttctttttta aaataagttt cagcctcttt 4980ttcaatattt tttaaagaag gagtatttgc atgaattgcc ttttttctaa cagacttagg 5040aaatatttta acagtatctt cttgcgccgg tgattttgga acttcataac ttactaattt 5100ataattatta ttttcttttt taattgtaac agttgcaaaa gaagctgaac ctgttccttc 5160aactagttta tcatcttcaa tataatattc ttgacctata tagtataaat atatttttat 5220tatattttta cttttttctg aatctattat tttataatca taaaaagttt taccaccaaa 5280agaaggttgt actccttctg gtccaacata tttttttact atattatcta aataattttt 5340gggaactggt gttgtaattt gattaatcga acaaccagtt atacttaaag gaattataac 5400tataaaaata tataggatta tctttttaaa tttcattatt ggcctccttt ttattaaatt 5460tatgttacca taaaaaggac ataacgggaa tatgtagaat atttttaatg tagacaaaat 5520tttacataaa tataaagaaa ggaagtgttt gtttaaattt tatagcaaac tatcaaaaat 5580tagggggata aaaatttatg aaaaaaaggt tttcgatgtt atttttatgt ttaactttaa 5640tagtttgtgg tttatttaca aattcggccg gccagtgggc aagttgaaaa attcacaaaa 5700atgtggtata atatctttgt tcattagagc gataaacttg aatttgagag ggaacttaga 5760tggtatttga aaaaattgat aaaaatagtt ggaacagaaa agagtatttt gaccactact 5820ttgcaagtgt accttgtacc tacagcatga ccgttaaagt ggatatcaca caaataaagg 5880aaaagggaat gaaactatat cctgcaatgc tttattatat tgcaatgatt gtaaaccgcc 5940attcagagtt taggacggca atcaatcaag atggtgaatt ggggatatat gatgagatga 6000taccaagcta tacaatattt cacaatgata ctgaaacatt ttccagcctt tggactgagt 6060gtaagtctga ctttaaatca tttttagcag attatgaaag tgatacgcaa cggtatggaa 6120acaatcatag aatggaagga aagccaaatg ctccggaaaa catttttaat gtatctatga 6180taccgtggtc aaccttcgat ggctttaatc tgaatttgca gaaaggatat gattatttga 6240ttcctatttt tactatgggg aaatattata aagaagataa caaaattata cttcctttgg 6300caattcaagt tcatcacgca gtatgtgacg gatttcacat ttgccgtttt gtaaacgaat 6360tgcaggaatt gataaatagt taacttcagg tttgtctgta actaaaaaca agtatttaag 6420caaaaacatc gtagaaatac ggtgtttttt gttaccctaa gtttaaactc ctttttgata 6480atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 6540aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 6600caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 6660ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc 6720cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 6780tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 6840gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 6900ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 6960gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 7020caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 7080ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 7140tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 7200ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 7260agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 7320aagcggaaga gcgcccaata cgcagggccc cctgcttcgg ggtcattata gcgatttttt 7380cggtatatcc atcctttttc gcacgatata caggattttg ccaaagggtt cgtgtagact 7440ttccttggtg tatccaacgg cgtcagccgg gcaggatagg tgaagtaggc ccacccgcga 7500gcgggtgttc cttcttcact gtcccttatt cgcacctggc ggtgctcaac gggaatcctg 7560ctctgcgagg ctggccggct accgccggcg taacagatga gggcaagcgg atggctgatg 7620aaaccaagcc aaccaggaag ggcagcccac ctatcaaggt gtactgcctt ccagacgaac 7680gaagagcgat tgaggaaaag gcggcggcgg ccggcatgag cctgtcggcc tacctgctgg 7740ccgtcggcca gggctacaaa atcacgggcg tcgtggacta tgagcacgtc cgcgagctgg 7800cccgcatcaa tggcgacctg ggccgcctgg gcggcctgct gaaactctgg ctcaccgacg 7860acccgcgcac ggcgcggttc ggtgatgcca cgatcctcgc cctgctggcg aagatcgaag 7920agaagcagga cgagcttggc aaggtcatga tgggcgtggt ccgcccgagg gcagagccat 7980gactttttta gccgctaaaa cggccggggg gtgcgcgtga ttgccaagca cgtccccatg 8040cgctccatca agaagagcga cttcgcggag ctggtgaagt acatcaccga cgagcaaggc 8100aagaccgatc gggccc 81168055DNAArtificial SequenceOligonucleotide NotI-XbaI-Prnf-MK_F 80atgcgcggcc gctaggtcta gaatatcgat acagataaaa aaatatataa tacag 558127DNAArtificial SequenceOligonucleotide SalI-Prnf-MK_R 81tggttctgta acagcgtatt cacctgc 27824633DNAArtificial Sequenceplasmid pMTL8314-Prnf-MK 82aaactccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 60gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 120atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 180gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 240gttcttctag

tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 300tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 360accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 420ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 480cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 540agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 600ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 660tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 720ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 780cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 840gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca gggccccctg caggataaaa 900aaattgtaga taaattttat aaaatagttt tatctacaat ttttttatca ggaaacagct 960atgaccgcgg ccgctaggtc tagaatatcg atacagataa aaaaatatat aatacagaag 1020aaaaaattat aaatttgtgg tataatataa agtatagtaa tttaagttta aacctcgtga 1080aaacgctaac aaataatagg aggtcaattg atgatagctg ttccatttaa cgctggaaaa 1140ataaaagttt taattgaggc attagaatct ggaaattatt catcaataaa atcagatgta 1200tatgacggaa tgttatatga tgcaccagat caccttaaat cattagtaaa cagatttgta 1260gaacttaata atataactga gccattagca gtaactatac agacaaatct tcctccttca 1320agaggtcttg gatctagtgc agctgttgct gttgcttttg taagagcaag ttatgatttc 1380ttaggaaaaa gtttaactaa agaagagctt atagaaaagg ctaattgggc tgaacaaata 1440gctcatggaa agccatctgg aatagataca caaacaatag tatctggaaa gcctgtttgg 1500tttcaaaagg gacatgcaga aacacttaaa actctttcac ttgatggata catggtagta 1560attgatacag gtgttaaagg aagtacaaga caggctgtag aagatgttca taaactttgc 1620gaagatcctc aatatatgag tcacgtaaaa cacataggaa aacttgtact tagagcatct 1680gatgttattg aacatcataa ctttgaagca cttgctgata tattcaatga atgtcatgct 1740gatttaaagg ctcttacagt aagtcatgac aaaatagaac agttaatgaa gataggaaaa 1800gaaaatggtg ctatagctgg taaattaact ggtgctggta gaggtggttc aatgttatta 1860cttgcaaaag acttaccaac tgcaaagaat atagttaaag cagtagagaa agctggtgca 1920gcacatactt ggattgaaaa tttaggtggt taagtcgacg tcacgcgtcc atggagatct 1980cgaggcctgc agacatgcaa gcttggcact ggccgtcgtt ttacaacgtc gtgactggga 2040aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg 2100taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga 2160atggcgctag cataaaaata agaagcctgc atttgcaggc ttcttatttt tatggcgcgc 2220cgccattatt tttttgaaca attgacaatt catttcttat tttttattaa gtgatagtca 2280aaaggcataa cagtgctgaa tagaaagaaa tttacagaaa agaaaattat agaatttagt 2340atgattaatt atactcattt atgaatgttt aattgaatac aaaaaaaaat acttgttatg 2400tattcaatta cgggttaaaa tatagacaag ttgaaaaatt taataaaaaa ataagtcctc 2460agctcttata tattaagcta ccaacttagt atataagcca aaacttaaat gtgctaccaa 2520cacatcaagc cgttagagaa ctctatctat agcaatattt caaatgtacc gacatacaag 2580agaaacatta actatatata ttcaatttat gagattatct taacagatat aaatgtaaat 2640tgcaataagt aagatttaga agtttatagc ctttgtgtat tggaagcagt acgcaaaggc 2700ttttttattt gataaaaatt agaagtatat ttattttttc ataattaatt tatgaaaatg 2760aaagggggtg agcaaagtga cagaggaaag cagtatctta tcaaataaca aggtattagc 2820aatatcatta ttgactttag cagtaaacat tatgactttt atagtgcttg tagctaagta 2880gtacgaaagg gggagcttta aaaagctcct tggaatacat agaattcata aattaattta 2940tgaaaagaag ggcgtatatg aaaacttgta aaaattgcaa agagtttatt aaagatactg 3000aaatatgcaa aatacattcg ttgatgattc atgataaaac agtagcaacc tattgcagta 3060aatacaatga gtcaagatgt ttacataaag ggaaagtcca atgtattaat tgttcaaaga 3120tgaaccgata tggatggtgt gccataaaaa tgagatgttt tacagaggaa gaacagaaaa 3180aagaacgtac atgcattaaa tattatgcaa ggagctttaa aaaagctcat gtaaagaaga 3240gtaaaaagaa aaaataattt atttattaat ttaatattga gagtgccgac acagtatgca 3300ctaaaaaata tatctgtggt gtagtgagcc gatacaaaag gatagtcact cgcattttca 3360taatacatct tatgttatga ttatgtgtcg gtgggacttc acgacgaaaa cccacaataa 3420aaaaagagtt cggggtaggg ttaagcatag ttgaggcaac taaacaatca agctaggata 3480tgcagtagca gaccgtaagg tcgttgttta ggtgtgttgt aatacatacg ctattaagat 3540gtaaaaatac ggataccaat gaagggaaaa gtataatttt tggatgtagt ttgtttgttc 3600atctatgggc aaactacgtc caaagccgtt tccaaatctg ctaaaaagta tatcctttct 3660aaaatcaaag tcaagtatga aatcataaat aaagtttaat tttgaagtta ttatgatatt 3720atgtttttct attaaaataa attaagtata tagaatagtt taataatagt atatacttaa 3780tgtgataagt gtctgacagt gtcacagaaa ggatgattgt tatggattat aagcggccgg 3840ccagtgggca agttgaaaaa ttcacaaaaa tgtggtataa tatctttgtt cattagagcg 3900ataaacttga atttgagagg gaacttagat ggtatttgaa aaaattgata aaaatagttg 3960gaacagaaaa gagtattttg accactactt tgcaagtgta ccttgtacct acagcatgac 4020cgttaaagtg gatatcacac aaataaagga aaagggaatg aaactatatc ctgcaatgct 4080ttattatatt gcaatgattg taaaccgcca ttcagagttt aggacggcaa tcaatcaaga 4140tggtgaattg gggatatatg atgagatgat accaagctat acaatatttc acaatgatac 4200tgaaacattt tccagccttt ggactgagtg taagtctgac tttaaatcat ttttagcaga 4260ttatgaaagt gatacgcaac ggtatggaaa caatcataga atggaaggaa agccaaatgc 4320tccggaaaac atttttaatg tatctatgat accgtggtca accttcgatg gctttaatct 4380gaatttgcag aaaggatatg attatttgat tcctattttt actatgggga aatattataa 4440agaagataac aaaattatac ttcctttggc aattcaagtt catcacgcag tatgtgacgg 4500atttcacatt tgccgttttg taaacgaatt gcaggaattg ataaatagtt aacttcaggt 4560ttgtctgtaa ctaaaaacaa gtatttaagc aaaaacatcg tagaaatacg gtgttttttg 4620ttaccctaag ttt 4633836753DNAArtificial Sequenceplasmid pMTL 8314-Prnf-MK-PMK-PMD 83aaactccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 60gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 120atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 180gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 240gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 300tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 360accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 420ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 480cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 540agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 600ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 660tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 720ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 780cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 840gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca gggccccctg caggataaaa 900aaattgtaga taaattttat aaaatagttt tatctacaat ttttttatca ggaaacagct 960atgaccgcgg ccgctaggtc tagaatatcg atacagataa aaaaatatat aatacagaag 1020aaaaaattat aaatttgtgg tataatataa agtatagtaa tttaagttta aacctcgtga 1080aaacgctaac aaataatagg aggtcaattg atgatagctg ttccatttaa cgctggaaaa 1140ataaaagttt taattgaggc attagaatct ggaaattatt catcaataaa atcagatgta 1200tatgacggaa tgttatatga tgcaccagat caccttaaat cattagtaaa cagatttgta 1260gaacttaata atataactga gccattagca gtaactatac agacaaatct tcctccttca 1320agaggtcttg gatctagtgc agctgttgct gttgcttttg taagagcaag ttatgatttc 1380ttaggaaaaa gtttaactaa agaagagctt atagaaaagg ctaattgggc tgaacaaata 1440gctcatggaa agccatctgg aatagataca caaacaatag tatctggaaa gcctgtttgg 1500tttcaaaagg gacatgcaga aacacttaaa actctttcac ttgatggata catggtagta 1560attgatacag gtgttaaagg aagtacaaga caggctgtag aagatgttca taaactttgc 1620gaagatcctc aatatatgag tcacgtaaaa cacataggaa aacttgtact tagagcatct 1680gatgttattg aacatcataa ctttgaagca cttgctgata tattcaatga atgtcatgct 1740gatttaaagg ctcttacagt aagtcatgac aaaatagaac agttaatgaa gataggaaaa 1800gaaaatggtg ctatagctgg taaattaact ggtgctggta gaggtggttc aatgttatta 1860cttgcaaaag acttaccaac tgcaaagaat atagttaaag cagtagagaa agctggtgca 1920gcacatactt ggattgaaaa tttaggtggt taagtcgaca aagacactaa aaaattataa 1980aagtaaagga ggacattaaa tgatacaagt aaaggcacca ggaaaattat atatagcagg 2040tgaatacgct gttacagaac caggatataa atctgttctt atagctcttg atagatttgt 2100tacagctact attgaggaag ctgatcaata caaaggaaca atacattcaa aggcattaca 2160tcacaatcca gtaactttta gtagagatga agattctatt gttatatcag acccacacgc 2220agcaaaacaa cttaattatg tagtaactgc tatagaaata tttgagcaat atgcaaaatc 2280atgtgacata gcaatgaagc attttcattt aactatagat tctaacttag atgatagtaa 2340tggacataag tatggacttg gatcttctgc tgctgtttta gtttcagtaa ttaaagtact 2400taacgaattt tatgatatga aactttcaaa cctttatata tataagttag cagtaattgc 2460taatatgaaa ttacagagtt tatcttcatg cggtgatata gcagtaagtg tttattcagg 2520ttggttagct tattctacat ttgaccatga atgggtaaaa caccagatag aagatacaac 2580agttgaagaa gtacttatta aaaattggcc tggattacac atagagccac ttcaagctcc 2640tgaaaatatg gaagttctta taggttggac aggtagtcca gctagtagtc ctcattttgt 2700ttctgaagtt aaaagactta agtcagatcc ttcattttac ggtgatttct tagaagattc 2760acatagatgt gtagaaaaat taattcatgc attcaaaact aataatatta agggtgttca 2820gaaaatggta agacagaata gaactattat acaaagaatg gataaggaag caacagttga 2880tatagagact gagaagttaa aatatttatg tgatattgct gaaaaatatc atggtgcaag 2940taaaacttca ggtgctggtg gtggtgattg cggaataact ataataaata aggatgtaga 3000caaagagaaa atatatgatg aatggactaa acatggaata aagcctctta agtttaatat 3060ttatcatgga caataaccat ggtcaataat cttacaataa ataaaagaaa ggaggcaaaa 3120atatgataaa atctggaaaa gcaagagcac acactaatat agcacttata aaatattggg 3180gtaagaaaga tgaggcatta ataataccaa tgaataactc aatatcagta actttagaaa 3240agttttatac tgaaacaaaa gttacattta acgatcagct tactcaagat caattttggc 3300ttaatggtga aaaagtttct ggaaaagaat tagaaaagat ttcaaagtat atggatattg 3360ttagaaatag agctggaata gattggtatg ctgagataga atctgataat tttgttccta 3420cagctgctgg tcttgctagt tctgctagtg cttatgcagc attagctgct gcatgtaacc 3480aagcacttga tttacagtta agtgataaag acttaagtag attagctaga attggatcag 3540gatcagcatc aagatcaata tacggtggtt ttgcagaatg ggaaaaagga tataatgacg 3600aaacttctta tgctgttcca ttagaaagta atcactttga agatgatctt gctatgattt 3660ttgtagtaat aaaccaacat tctaaaaagg ttccttcaag atatggaatg tctcttacaa 3720gaaatacaag tagattctat caatattggt tagaccatat tgatgaagat cttgcagaag 3780caaaggcagc aatacaagat aaggatttta agagattagg tgaagttatt gaagagaatg 3840gacttagaat gcatgctaca aatcttggat caactccacc ttttacttac ttagtacaag 3900agtcatacga tgtaatggca ttagtacatg agtgtagaga agcaggatat ccatgctatt 3960tcactatgga tgctggacct aatgtaaaaa tacttgtaga gaagaaaaac aaacaacaga 4020taatagataa acttttaact cagttcgata ataatcagat aatagatagt gatattatag 4080ctacaggtat tgaaattata gaataaacta gttccgctaa gcttggcact ggccgtcgtt 4140ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat 4200ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag 4260ttgcgcagcc tgaatggcga atggcgctag cataaaaata agaagcctgc atttgcaggc 4320ttcttatttt tatggcgcgc cgccattatt tttttgaaca attgacaatt catttcttat 4380tttttattaa gtgatagtca aaaggcataa cagtgctgaa tagaaagaaa tttacagaaa 4440agaaaattat agaatttagt atgattaatt atactcattt atgaatgttt aattgaatac 4500aaaaaaaaat acttgttatg tattcaatta cgggttaaaa tatagacaag ttgaaaaatt 4560taataaaaaa ataagtcctc agctcttata tattaagcta ccaacttagt atataagcca 4620aaacttaaat gtgctaccaa cacatcaagc cgttagagaa ctctatctat agcaatattt 4680caaatgtacc gacatacaag agaaacatta actatatata ttcaatttat gagattatct 4740taacagatat aaatgtaaat tgcaataagt aagatttaga agtttatagc ctttgtgtat 4800tggaagcagt acgcaaaggc ttttttattt gataaaaatt agaagtatat ttattttttc 4860ataattaatt tatgaaaatg aaagggggtg agcaaagtga cagaggaaag cagtatctta 4920tcaaataaca aggtattagc aatatcatta ttgactttag cagtaaacat tatgactttt 4980atagtgcttg tagctaagta gtacgaaagg gggagcttta aaaagctcct tggaatacat 5040agaattcata aattaattta tgaaaagaag ggcgtatatg aaaacttgta aaaattgcaa 5100agagtttatt aaagatactg aaatatgcaa aatacattcg ttgatgattc atgataaaac 5160agtagcaacc tattgcagta aatacaatga gtcaagatgt ttacataaag ggaaagtcca 5220atgtattaat tgttcaaaga tgaaccgata tggatggtgt gccataaaaa tgagatgttt 5280tacagaggaa gaacagaaaa aagaacgtac atgcattaaa tattatgcaa ggagctttaa 5340aaaagctcat gtaaagaaga gtaaaaagaa aaaataattt atttattaat ttaatattga 5400gagtgccgac acagtatgca ctaaaaaata tatctgtggt gtagtgagcc gatacaaaag 5460gatagtcact cgcattttca taatacatct tatgttatga ttatgtgtcg gtgggacttc 5520acgacgaaaa cccacaataa aaaaagagtt cggggtaggg ttaagcatag ttgaggcaac 5580taaacaatca agctaggata tgcagtagca gaccgtaagg tcgttgttta ggtgtgttgt 5640aatacatacg ctattaagat gtaaaaatac ggataccaat gaagggaaaa gtataatttt 5700tggatgtagt ttgtttgttc atctatgggc aaactacgtc caaagccgtt tccaaatctg 5760ctaaaaagta tatcctttct aaaatcaaag tcaagtatga aatcataaat aaagtttaat 5820tttgaagtta ttatgatatt atgtttttct attaaaataa attaagtata tagaatagtt 5880taataatagt atatacttaa tgtgataagt gtctgacagt gtcacagaaa ggatgattgt 5940tatggattat aagcggccgg ccagtgggca agttgaaaaa ttcacaaaaa tgtggtataa 6000tatctttgtt cattagagcg ataaacttga atttgagagg gaacttagat ggtatttgaa 6060aaaattgata aaaatagttg gaacagaaaa gagtattttg accactactt tgcaagtgta 6120ccttgtacct acagcatgac cgttaaagtg gatatcacac aaataaagga aaagggaatg 6180aaactatatc ctgcaatgct ttattatatt gcaatgattg taaaccgcca ttcagagttt 6240aggacggcaa tcaatcaaga tggtgaattg gggatatatg atgagatgat accaagctat 6300acaatatttc acaatgatac tgaaacattt tccagccttt ggactgagtg taagtctgac 6360tttaaatcat ttttagcaga ttatgaaagt gatacgcaac ggtatggaaa caatcataga 6420atggaaggaa agccaaatgc tccggaaaac atttttaatg tatctatgat accgtggtca 6480accttcgatg gctttaatct gaatttgcag aaaggatatg attatttgat tcctattttt 6540actatgggga aatattataa agaagataac aaaattatac ttcctttggc aattcaagtt 6600catcacgcag tatgtgacgg atttcacatt tgccgttttg taaacgaatt gcaggaattg 6660ataaatagtt aacttcaggt ttgtctgtaa ctaaaaacaa gtatttaagc aaaaacatcg 6720tagaaatacg gtgttttttg ttaccctaag ttt 6753849198DNAArtificial Sequenceplasmid pMTL8314-Prnf-MK-PMK-PMD-Pfor-idi-ispS 84aaactccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 60gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 120atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 180gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 240gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 300tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 360accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 420ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 480cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 540agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 600ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 660tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 720ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 780cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 840gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca gggccccctg caggataaaa 900aaattgtaga taaattttat aaaatagttt tatctacaat ttttttatca ggaaacagct 960atgaccgcgg ccgctaggtc tagaatatcg atacagataa aaaaatatat aatacagaag 1020aaaaaattat aaatttgtgg tataatataa agtatagtaa tttaagttta aacctcgtga 1080aaacgctaac aaataatagg aggtcaattg atgatagctg ttccatttaa cgctggaaaa 1140ataaaagttt taattgaggc attagaatct ggaaattatt catcaataaa atcagatgta 1200tatgacggaa tgttatatga tgcaccagat caccttaaat cattagtaaa cagatttgta 1260gaacttaata atataactga gccattagca gtaactatac agacaaatct tcctccttca 1320agaggtcttg gatctagtgc agctgttgct gttgcttttg taagagcaag ttatgatttc 1380ttaggaaaaa gtttaactaa agaagagctt atagaaaagg ctaattgggc tgaacaaata 1440gctcatggaa agccatctgg aatagataca caaacaatag tatctggaaa gcctgtttgg 1500tttcaaaagg gacatgcaga aacacttaaa actctttcac ttgatggata catggtagta 1560attgatacag gtgttaaagg aagtacaaga caggctgtag aagatgttca taaactttgc 1620gaagatcctc aatatatgag tcacgtaaaa cacataggaa aacttgtact tagagcatct 1680gatgttattg aacatcataa ctttgaagca cttgctgata tattcaatga atgtcatgct 1740gatttaaagg ctcttacagt aagtcatgac aaaatagaac agttaatgaa gataggaaaa 1800gaaaatggtg ctatagctgg taaattaact ggtgctggta gaggtggttc aatgttatta 1860cttgcaaaag acttaccaac tgcaaagaat atagttaaag cagtagagaa agctggtgca 1920gcacatactt ggattgaaaa tttaggtggt taagtcgaca aagacactaa aaaattataa 1980aagtaaagga ggacattaaa tgatacaagt aaaggcacca ggaaaattat atatagcagg 2040tgaatacgct gttacagaac caggatataa atctgttctt atagctcttg atagatttgt 2100tacagctact attgaggaag ctgatcaata caaaggaaca atacattcaa aggcattaca 2160tcacaatcca gtaactttta gtagagatga agattctatt gttatatcag acccacacgc 2220agcaaaacaa cttaattatg tagtaactgc tatagaaata tttgagcaat atgcaaaatc 2280atgtgacata gcaatgaagc attttcattt aactatagat tctaacttag atgatagtaa 2340tggacataag tatggacttg gatcttctgc tgctgtttta gtttcagtaa ttaaagtact 2400taacgaattt tatgatatga aactttcaaa cctttatata tataagttag cagtaattgc 2460taatatgaaa ttacagagtt tatcttcatg cggtgatata gcagtaagtg tttattcagg 2520ttggttagct tattctacat ttgaccatga atgggtaaaa caccagatag aagatacaac 2580agttgaagaa gtacttatta aaaattggcc tggattacac atagagccac ttcaagctcc 2640tgaaaatatg gaagttctta taggttggac aggtagtcca gctagtagtc ctcattttgt 2700ttctgaagtt aaaagactta agtcagatcc ttcattttac ggtgatttct tagaagattc 2760acatagatgt gtagaaaaat taattcatgc attcaaaact aataatatta agggtgttca 2820gaaaatggta agacagaata gaactattat acaaagaatg gataaggaag caacagttga 2880tatagagact gagaagttaa aatatttatg tgatattgct gaaaaatatc atggtgcaag 2940taaaacttca ggtgctggtg gtggtgattg cggaataact ataataaata aggatgtaga 3000caaagagaaa atatatgatg aatggactaa acatggaata aagcctctta agtttaatat 3060ttatcatgga caataaccat ggtcaataat cttacaataa ataaaagaaa ggaggcaaaa 3120atatgataaa atctggaaaa gcaagagcac acactaatat agcacttata aaatattggg 3180gtaagaaaga tgaggcatta ataataccaa tgaataactc aatatcagta actttagaaa 3240agttttatac tgaaacaaaa gttacattta acgatcagct tactcaagat caattttggc 3300ttaatggtga aaaagtttct ggaaaagaat tagaaaagat ttcaaagtat atggatattg 3360ttagaaatag agctggaata gattggtatg ctgagataga atctgataat tttgttccta 3420cagctgctgg tcttgctagt tctgctagtg cttatgcagc attagctgct gcatgtaacc 3480aagcacttga tttacagtta agtgataaag acttaagtag attagctaga attggatcag 3540gatcagcatc aagatcaata tacggtggtt ttgcagaatg ggaaaaagga tataatgacg 3600aaacttctta tgctgttcca ttagaaagta atcactttga agatgatctt gctatgattt 3660ttgtagtaat aaaccaacat tctaaaaagg

ttccttcaag atatggaatg tctcttacaa 3720gaaatacaag tagattctat caatattggt tagaccatat tgatgaagat cttgcagaag 3780caaaggcagc aatacaagat aaggatttta agagattagg tgaagttatt gaagagaatg 3840gacttagaat gcatgctaca aatcttggat caactccacc ttttacttac ttagtacaag 3900agtcatacga tgtaatggca ttagtacatg agtgtagaga agcaggatat ccatgctatt 3960tcactatgga tgctggacct aatgtaaaaa tacttgtaga gaagaaaaac aaacaacaga 4020taatagataa acttttaact cagttcgata ataatcagat aatagatagt gatattatag 4080ctacaggtat tgaaattata gaataaacta gttgtatatt aaaatagtag aatacataag 4140atacttaatt taattaaaga tagttaagta cttttcaatg tgctttttta gatgtttaat 4200acaaatcttt aattgtaaaa gaaatgctgt actatttact gttctagtga cgggattaaa 4260ctgtattaat tataaataaa aaataagtac agttgtttaa aattatattt tgtattaaat 4320ctaatagtac gatgtaagtt attttatact attgctagtt taataaaaag atttaattat 4380atacttgaaa aggagaggaa ctcgagatgg cagagtatat aatagcagta gatgagttcg 4440ataacgaaat aggatcaata gaaaagatgg aagctcatag aaaaggaaca cttcatagag 4500cattcagtat tttagttttt aactcaaaga atcaactttt attacagaaa agaaatgtaa 4560agaaatatca ctctccagga ttatggacaa acacttgttg tagtcaccca agatatggtg 4620aatctcttca tgatgctata tacagaagat taaaagaaga gatgggattt acttgcgaac 4680ttgaagaagt attctcattc atatataagg taaaacttga agataattta tttgagaatg 4740aatatgacca tgtatttatt ggtaaatatg atggtgagat aattgttaat aaagatgaag 4800ttgatgattt taaatgggta gacattaatg aagttaaaaa ggacataata gaaagacctg 4860aggcatatac ttactggttt aagtatcttg taaataaagc tgaaaataag atatttaaat 4920aaaccggtgg gaggaaatga acatggcaac agaattatta tgtttacaca gacctatatc 4980acttactcac aaacttttta ggaatccatt acctaaagtt attcaagcta cacctttaac 5040attaaaactt aggtgtagtg tttctacaga aaatgtatca tttagtgaga cagaaactga 5100aacaagaaga tcagcaaatt atgaaccaaa ttcttgggat tatgattatc ttctttcttc 5160tgatactgat gagtcaatag aagtacataa agataaggct aagaaattag aagctgaagt 5220taggagagaa ataaataatg agaaggctga atttcttaca cttcttgaac ttattgataa 5280tgtacaaaga cttggattag gatatagatt tgagtctgat ataagaagag cattagatag 5340atttgtaagt agtggaggat ttgatggagt tactaaaact tcattacatg gaacagcatt 5400atcatttagg ttattaaggc aacatggttt tgaagtatct caagaagctt ttagtggatt 5460taaagatcag aatggaaact ttcttgagaa tttaaaggaa gacataaaag caattctttc 5520tctttatgaa gcatcatttt tagcattaga aggtgagaat atattagatg aggctaaagt 5580atttgcaata tctcatctta aagaacttag tgaagaaaag attggtaaag aattagctga 5640acaagtttca catgctttag aattaccatt acatagaaga acacaaagat tagaagcagt 5700ttggtcaata gaagcatata gaaagaaaga agacgcaaat caagtacttt tagaacttgc 5760aatacttgac tacaatatga ttcaaagtgt atatcagagg gatttaagag aaacatcaag 5820atggtggaga agagtaggat tagcaactaa attacatttt gctagagata ggcttattga 5880aagtttttat tgggctgttg gagttgcttt tgaaccacaa tattctgatt gcagaaatag 5940tgtagcaaag atgttttcat ttgttactat aattgacgat atttacgatg tatatggaac 6000tttagatgaa cttgaacttt ttactgatgc agttgaaaga tgggatgtaa atgctattaa 6060tgatcttcct gattatatga agttatgttt tcttgcactt tacaatacta ttaacgagat 6120agcttacgat aacttaaaag ataaaggtga gaacatactt ccttatttaa caaaagcatg 6180ggcagattta tgtaatgcat ttcttcaaga agctaagtgg ctttataata aatcaacacc 6240tacatttgat gattattttg gaaatgcatg gaaaagttct agtggacctt tacagcttat 6300ttttgcttat tttgctgtag tacagaacat taaaaaggaa gagattgaga atcttcagaa 6360atatcatgac ataatatcaa gacctagtca catttttagg ctttgtaatg atttagcatc 6420tgcttcagca gaaatagcaa gaggtgaaac tgctaattct gtaagttgtt atatgagaac 6480aaaaggtata tctgaagaat tagctactga aagtgttatg aatcttatag acgaaacttg 6540gaagaaaatg aacaaagaaa aacttggtgg atctttattt gcaaaacctt ttgttgagac 6600tgctataaat ttagctagac agtctcattg cacatatcat aatggtgatg cacatactag 6660tccagatgaa ttaactagga aaagagtact tagtgtaata actgaaccaa tattaccatt 6720tgaaagataa gctagcataa aaataagaag cctgcatttg caggcttctt atttttatgg 6780cgcgccgcca ttattttttt gaacaattga caattcattt cttatttttt attaagtgat 6840agtcaaaagg cataacagtg ctgaatagaa agaaatttac agaaaagaaa attatagaat 6900ttagtatgat taattatact catttatgaa tgtttaattg aatacaaaaa aaaatacttg 6960ttatgtattc aattacgggt taaaatatag acaagttgaa aaatttaata aaaaaataag 7020tcctcagctc ttatatatta agctaccaac ttagtatata agccaaaact taaatgtgct 7080accaacacat caagccgtta gagaactcta tctatagcaa tatttcaaat gtaccgacat 7140acaagagaaa cattaactat atatattcaa tttatgagat tatcttaaca gatataaatg 7200taaattgcaa taagtaagat ttagaagttt atagcctttg tgtattggaa gcagtacgca 7260aaggcttttt tatttgataa aaattagaag tatatttatt ttttcataat taatttatga 7320aaatgaaagg gggtgagcaa agtgacagag gaaagcagta tcttatcaaa taacaaggta 7380ttagcaatat cattattgac tttagcagta aacattatga cttttatagt gcttgtagct 7440aagtagtacg aaagggggag ctttaaaaag ctccttggaa tacatagaat tcataaatta 7500atttatgaaa agaagggcgt atatgaaaac ttgtaaaaat tgcaaagagt ttattaaaga 7560tactgaaata tgcaaaatac attcgttgat gattcatgat aaaacagtag caacctattg 7620cagtaaatac aatgagtcaa gatgtttaca taaagggaaa gtccaatgta ttaattgttc 7680aaagatgaac cgatatggat ggtgtgccat aaaaatgaga tgttttacag aggaagaaca 7740gaaaaaagaa cgtacatgca ttaaatatta tgcaaggagc tttaaaaaag ctcatgtaaa 7800gaagagtaaa aagaaaaaat aatttattta ttaatttaat attgagagtg ccgacacagt 7860atgcactaaa aaatatatct gtggtgtagt gagccgatac aaaaggatag tcactcgcat 7920tttcataata catcttatgt tatgattatg tgtcggtggg acttcacgac gaaaacccac 7980aataaaaaaa gagttcgggg tagggttaag catagttgag gcaactaaac aatcaagcta 8040ggatatgcag tagcagaccg taaggtcgtt gtttaggtgt gttgtaatac atacgctatt 8100aagatgtaaa aatacggata ccaatgaagg gaaaagtata atttttggat gtagtttgtt 8160tgttcatcta tgggcaaact acgtccaaag ccgtttccaa atctgctaaa aagtatatcc 8220tttctaaaat caaagtcaag tatgaaatca taaataaagt ttaattttga agttattatg 8280atattatgtt tttctattaa aataaattaa gtatatagaa tagtttaata atagtatata 8340cttaatgtga taagtgtctg acagtgtcac agaaaggatg attgttatgg attataagcg 8400gccggccagt gggcaagttg aaaaattcac aaaaatgtgg tataatatct ttgttcatta 8460gagcgataaa cttgaatttg agagggaact tagatggtat ttgaaaaaat tgataaaaat 8520agttggaaca gaaaagagta ttttgaccac tactttgcaa gtgtaccttg tacctacagc 8580atgaccgtta aagtggatat cacacaaata aaggaaaagg gaatgaaact atatcctgca 8640atgctttatt atattgcaat gattgtaaac cgccattcag agtttaggac ggcaatcaat 8700caagatggtg aattggggat atatgatgag atgataccaa gctatacaat atttcacaat 8760gatactgaaa cattttccag cctttggact gagtgtaagt ctgactttaa atcattttta 8820gcagattatg aaagtgatac gcaacggtat ggaaacaatc atagaatgga aggaaagcca 8880aatgctccgg aaaacatttt taatgtatct atgataccgt ggtcaacctt cgatggcttt 8940aatctgaatt tgcagaaagg atatgattat ttgattccta tttttactat ggggaaatat 9000tataaagaag ataacaaaat tatacttcct ttggcaattc aagttcatca cgcagtatgt 9060gacggatttc acatttgccg ttttgtaaac gaattgcagg aattgataaa tagttaactt 9120caggtttgtc tgtaactaaa aacaagtatt taagcaaaaa catcgtagaa atacggtgtt 9180ttttgttacc ctaagttt 9198856841DNAArtificial Sequenceplasmid pMTL83245-Pfor-FS-idi 85aaactccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 60gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 120atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 180gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 240gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 300tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 360accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 420ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 480cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 540agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 600ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 660tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 720ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 780cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 840gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca gggccccctg caggataaaa 900aaattgtaga taaattttat aaaatagttt tatctacaat ttttttatca ggaaacagct 960atgaccgcgg ccgcaatatg atatttatgt ccattgtgaa agggattata ttcaactatt 1020attccagtta cgttcataga aattttcctt tctaaaatat tttattccat gtcaagaact 1080ctgtttattt cattaaagaa ctataagtac aaagtataag gcatttgaaa aaataggcta 1140gtatattgat tgattattta ttttaaaatg cctaagtgaa atatatacat attataacaa 1200taaaataagt attagtgtag gatttttaaa tagagtatct attttcagat taaatttttg 1260attatttgat ttacattata taatattgag taaagtattg actagcaaaa ttttttgata 1320ctttaatttg tgaaatttct tatcaaaagt tatatttttg aataattttt attgaaaaat 1380acaactaaaa aggattatag tataagtgtg tgtaattttg tgttaaattt aaagggagga 1440aatgaacatg aaacatatgg tgaccatgat tacgaattcg agctcggtac ccggggatcc 1500tctagttgta tattaaaata gtagaataca taagatactt aatttaatta aagatagtta 1560agtacttttc aatgtgcttt tttagatgtt taatacaaat ctttaattgt aaaagaaatg 1620ctgtactatt tactgttcta gtgacgggat taaactgtat taattataaa taaaaaataa 1680gtacagttgt ttaaaattat attttgtatt aaatctaata gtacgatgta agttatttta 1740tactattgct agtttaataa aaagatttaa ttatatactt gaaaaggaga ggaactcgag 1800atggaattta gagtacattt acaggcagac aacgaacaga aaatatttca aaatcaaatg 1860aaaccagagc cagaagcatc atatcttata aatcaaagaa gaagtgctaa ttataaacca 1920aacatttgga aaaacgattt tcttgatcag tctttaatat caaaatatga tggtgatgaa 1980tatagaaaac tttcagaaaa gttaatagaa gaagtaaaga tatacatatc agcagagact 2040atggatttag ttgctaaatt agaacttata gattctgtta gaaaacttgg acttgctaat 2100ctttttgaga aagaaataaa ggaagcatta gacagtatag cagcaataga atcagataat 2160ttaggaacta gagacgatct ttatggaaca gctcttcatt ttaagattct tagacagcat 2220ggatataagg taagtcaaga tatatttggt agatttatgg atgagaaagg aacattagaa 2280aatcatcact ttgcacactt aaaaggaatg ttagaattat ttgaggcaag taatcttgga 2340tttgaaggtg aagacatatt agatgaagct aaagcatctc ttacacttgc tcttagagat 2400tcaggacata tttgttatcc agactcaaac ttaagtagag atgtagttca tagtttagaa 2460ttacctagtc atagaagagt tcaatggttc gatgtaaaat ggcagattaa tgcatacgaa 2520aaagatattt gtagagtaaa tgcaacttta ttagagttag caaagttaaa ttttaatgtt 2580gttcaagctc agcttcagaa gaatcttaga gaagctagta gatggtgggc taatcttggt 2640ttcgcagata atttaaagtt tgctagagat agacttgtag agtgtttttc atgcgcagta 2700ggtgtagcat ttgaaccaga gcattcatct tttagaatat gtttaactaa ggtaattaat 2760cttgttctta ttatagatga tgtatacgat atatatggat ctgaagaaga gttaaaacat 2820tttacaaatg ctgttgatag atgggacagt agagaaacag aacagcttcc tgaatgcatg 2880aaaatgtgtt ttcaagtatt atataacact acttgcgaaa tagcaagaga gatagaagaa 2940gaaaacggtt ggaatcaagt attacctcaa cttactaagg tttgggctga tttttgtaag 3000gctcttttag ttgaagcaga gtggtacaat aaatcacata ttccaacatt agaagaatat 3060cttagaaacg gatgtatatc aagtagtgta tctgtacttt tagttcactc tttcttttca 3120ataactcatg aaggtacaaa agaaatggct gatttcttac ataaaaatga agatctttta 3180tacaacataa gtcttatagt aagattaaac aatgatttag gtacatcagc tgctgaacag 3240gaaagaggtg attctccttc ttctatagtt tgctatatga gagaagttaa tgcttctgaa 3300gagactgcaa gaaagaatat aaagggaatg attgataatg cttggaaaaa ggttaatgga 3360aaatgtttca caactaacca agttccattt ctttcatcat tcatgaataa tgcaactaac 3420atggcaagag tagcacactc attatataaa gacggtgatg gttttggtga tcaagaaaaa 3480ggacctagaa cacatattct tagtttatta ttccaacctt tagtaaatta actgcagggt 3540tcaaaacata gattaaaaaa ttaaaggagg ggaaaaaatg gcagagtata taatagcagt 3600agatgagttc gataacgaaa taggatcaat agaaaagatg gaagctcata gaaaaggaac 3660acttcataga gcattcagta ttttagtttt taactcaaag aatcaacttt tattacagaa 3720aagaaatgta aagaaatatc actctccagg attatggaca aacacttgtt gtagtcaccc 3780aagatatggt gaatctcttc atgatgctat atacagaaga ttaaaagaag agatgggatt 3840tacttgcgaa cttgaagaag tattctcatt catatataag gtaaaacttg aagataattt 3900atttgagaat gaatatgacc atgtatttat tggtaaatat gatggtgaga taattgttaa 3960taaagatgaa gttgatgatt ttaaatgggt agacattaat gaagttaaaa aggacataat 4020agaaagacct gaggcatata cttactggtt taagtatctt gtaaataaag ctgaaaataa 4080gatatttaaa taaaagcttg gcactggccg tcgttttaca acgtcgtgac tgggaaaacc 4140ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 4200gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc 4260gctagcataa aaataagaag cctgcatttg caggcttctt atttttatgg cgcgccgcca 4320ttattttttt gaacaattga caattcattt cttatttttt attaagtgat agtcaaaagg 4380cataacagtg ctgaatagaa agaaatttac agaaaagaaa attatagaat ttagtatgat 4440taattatact catttatgaa tgtttaattg aatacaaaaa aaaatacttg ttatgtattc 4500aattacgggt taaaatatag acaagttgaa aaatttaata aaaaaataag tcctcagctc 4560ttatatatta agctaccaac ttagtatata agccaaaact taaatgtgct accaacacat 4620caagccgtta gagaactcta tctatagcaa tatttcaaat gtaccgacat acaagagaaa 4680cattaactat atatattcaa tttatgagat tatcttaaca gatataaatg taaattgcaa 4740taagtaagat ttagaagttt atagcctttg tgtattggaa gcagtacgca aaggcttttt 4800tatttgataa aaattagaag tatatttatt ttttcataat taatttatga aaatgaaagg 4860gggtgagcaa agtgacagag gaaagcagta tcttatcaaa taacaaggta ttagcaatat 4920cattattgac tttagcagta aacattatga cttttatagt gcttgtagct aagtagtacg 4980aaagggggag ctttaaaaag ctccttggaa tacatagaat tcataaatta atttatgaaa 5040agaagggcgt atatgaaaac ttgtaaaaat tgcaaagagt ttattaaaga tactgaaata 5100tgcaaaatac attcgttgat gattcatgat aaaacagtag caacctattg cagtaaatac 5160aatgagtcaa gatgtttaca taaagggaaa gtccaatgta ttaattgttc aaagatgaac 5220cgatatggat ggtgtgccat aaaaatgaga tgttttacag aggaagaaca gaaaaaagaa 5280cgtacatgca ttaaatatta tgcaaggagc tttaaaaaag ctcatgtaaa gaagagtaaa 5340aagaaaaaat aatttattta ttaatttaat attgagagtg ccgacacagt atgcactaaa 5400aaatatatct gtggtgtagt gagccgatac aaaaggatag tcactcgcat tttcataata 5460catcttatgt tatgattatg tgtcggtggg acttcacgac gaaaacccac aataaaaaaa 5520gagttcgggg tagggttaag catagttgag gcaactaaac aatcaagcta ggatatgcag 5580tagcagaccg taaggtcgtt gtttaggtgt gttgtaatac atacgctatt aagatgtaaa 5640aatacggata ccaatgaagg gaaaagtata atttttggat gtagtttgtt tgttcatcta 5700tgggcaaact acgtccaaag ccgtttccaa atctgctaaa aagtatatcc tttctaaaat 5760caaagtcaag tatgaaatca taaataaagt ttaattttga agttattatg atattatgtt 5820tttctattaa aataaattaa gtatatagaa tagtttaata atagtatata cttaatgtga 5880taagtgtctg acagtgtcac agaaaggatg attgttatgg attataagcg gccggccgaa 5940gcaaacttaa gagtgtgttg atagtgcagt atcttaaaat tttgtataat aggaattgaa 6000gttaaattag atgctaaaaa tttgtaatta agaaggagtg attacatgaa caaaaatata 6060aaatattctc aaaacttttt aacgagtgaa aaagtactca accaaataat aaaacaattg 6120aatttaaaag aaaccgatac cgtttacgaa attggaacag gtaaagggca tttaacgacg 6180aaactggcta aaataagtaa acaggtaacg tctattgaat tagacagtca tctattcaac 6240ttatcgtcag aaaaattaaa actgaatact cgtgtcactt taattcacca agatattcta 6300cagtttcaat tccctaacaa acagaggtat aaaattgttg ggagtattcc ttaccattta 6360agcacacaaa ttattaaaaa agtggttttt gaaagccatg cgtctgacat ctatctgatt 6420gttgaagaag gattctacaa gcgtaccttg gatattcacc gaacactagg gttgctcttg 6480cacactcaag tctcgattca gcaattgctt aagctgccag cggaatgctt tcatcctaaa 6540ccaaaagtaa acagtgtctt aataaaactt acccgccata ccacagatgt tccagataaa 6600tattggaagc tatatacgta ctttgtttca aaatgggtca atcgagaata tcgtcaactg 6660tttactaaaa atcagtttca tcaagcaatg aaacacgcca aagtaaacaa tttaagtacc 6720gttacttatg agcaagtatt gtctattttt aatagttatc tattatttaa cgggaggaaa 6780taattctatg agtcgctttt gtaaatttgg aaagttacac gttactaaag ggaatgtgtt 6840t 68418636DNAArtificial SequenceOligonucleotide idi_F2 86aggcactcga gatggcagag tatataatag cagtag 368747DNAArtificial SequenceOligonucleotide idi_R2 87aggcgcaagc ttggcgcacc ggtttattta aatatcttat tttcagc 47885075DNAArtificial Sequenceplasmid pMTL83245-Pfor-idi 88aaactccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 60gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 120atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 180gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 240gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 300tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 360accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 420ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 480cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 540agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 600ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 660tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 720ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 780cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 840gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca gggccccctg caggataaaa 900aaattgtaga taaattttat aaaatagttt tatctacaat ttttttatca ggaaacagct 960atgaccgcgg ccgcaatatg atatttatgt ccattgtgaa agggattata ttcaactatt 1020attccagtta cgttcataga aattttcctt tctaaaatat tttattccat gtcaagaact 1080ctgtttattt cattaaagaa ctataagtac aaagtataag gcatttgaaa aaataggcta 1140gtatattgat tgattattta ttttaaaatg cctaagtgaa atatatacat attataacaa 1200taaaataagt attagtgtag gatttttaaa tagagtatct attttcagat taaatttttg 1260attatttgat ttacattata taatattgag taaagtattg actagcaaaa ttttttgata 1320ctttaatttg tgaaatttct tatcaaaagt tatatttttg aataattttt attgaaaaat 1380acaactaaaa aggattatag tataagtgtg tgtaattttg tgttaaattt aaagggagga 1440aatgaacatg aaacatatgg tgaccatgat tacgaattcg agctcggtac ccggggatcc 1500tctagttgta tattaaaata gtagaataca taagatactt aatttaatta aagatagtta 1560agtacttttc aatgtgcttt tttagatgtt taatacaaat ctttaattgt aaaagaaatg 1620ctgtactatt tactgttcta gtgacgggat taaactgtat taattataaa taaaaaataa 1680gtacagttgt ttaaaattat attttgtatt aaatctaata gtacgatgta agttatttta 1740tactattgct agtttaataa aaagatttaa ttatatactt gaaaaggaga ggaactcgag 1800atggcagagt atataatagc agtagatgag ttcgataacg aaataggatc aatagaaaag 1860atggaagctc atagaaaagg aacacttcat agagcattca gtattttagt ttttaactca 1920aagaatcaac ttttattaca gaaaagaaat gtaaagaaat atcactctcc aggattatgg 1980acaaacactt gttgtagtca cccaagatat ggtgaatctc ttcatgatgc tatatacaga 2040agattaaaag aagagatggg atttacttgc gaacttgaag aagtattctc attcatatat 2100aaggtaaaac ttgaagataa tttatttgag aatgaatatg accatgtatt tattggtaaa 2160tatgatggtg agataattgt taataaagat gaagttgatg attttaaatg ggtagacatt 2220aatgaagtta aaaaggacat aatagaaaga cctgaggcat atacttactg gtttaagtat 2280cttgtaaata

aagctgaaaa taagatattt aaataaaccg gtgcgccaag cttggcactg 2340gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt 2400gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 2460tcccaacagt tgcgcagcct gaatggcgaa tggcgctagc ataaaaataa gaagcctgca 2520tttgcaggct tcttattttt atggcgcgcc gccattattt ttttgaacaa ttgacaattc 2580atttcttatt ttttattaag tgatagtcaa aaggcataac agtgctgaat agaaagaaat 2640ttacagaaaa gaaaattata gaatttagta tgattaatta tactcattta tgaatgttta 2700attgaataca aaaaaaaata cttgttatgt attcaattac gggttaaaat atagacaagt 2760tgaaaaattt aataaaaaaa taagtcctca gctcttatat attaagctac caacttagta 2820tataagccaa aacttaaatg tgctaccaac acatcaagcc gttagagaac tctatctata 2880gcaatatttc aaatgtaccg acatacaaga gaaacattaa ctatatatat tcaatttatg 2940agattatctt aacagatata aatgtaaatt gcaataagta agatttagaa gtttatagcc 3000tttgtgtatt ggaagcagta cgcaaaggct tttttatttg ataaaaatta gaagtatatt 3060tattttttca taattaattt atgaaaatga aagggggtga gcaaagtgac agaggaaagc 3120agtatcttat caaataacaa ggtattagca atatcattat tgactttagc agtaaacatt 3180atgactttta tagtgcttgt agctaagtag tacgaaaggg ggagctttaa aaagctcctt 3240ggaatacata gaattcataa attaatttat gaaaagaagg gcgtatatga aaacttgtaa 3300aaattgcaaa gagtttatta aagatactga aatatgcaaa atacattcgt tgatgattca 3360tgataaaaca gtagcaacct attgcagtaa atacaatgag tcaagatgtt tacataaagg 3420gaaagtccaa tgtattaatt gttcaaagat gaaccgatat ggatggtgtg ccataaaaat 3480gagatgtttt acagaggaag aacagaaaaa agaacgtaca tgcattaaat attatgcaag 3540gagctttaaa aaagctcatg taaagaagag taaaaagaaa aaataattta tttattaatt 3600taatattgag agtgccgaca cagtatgcac taaaaaatat atctgtggtg tagtgagccg 3660atacaaaagg atagtcactc gcattttcat aatacatctt atgttatgat tatgtgtcgg 3720tgggacttca cgacgaaaac ccacaataaa aaaagagttc ggggtagggt taagcatagt 3780tgaggcaact aaacaatcaa gctaggatat gcagtagcag accgtaaggt cgttgtttag 3840gtgtgttgta atacatacgc tattaagatg taaaaatacg gataccaatg aagggaaaag 3900tataattttt ggatgtagtt tgtttgttca tctatgggca aactacgtcc aaagccgttt 3960ccaaatctgc taaaaagtat atcctttcta aaatcaaagt caagtatgaa atcataaata 4020aagtttaatt ttgaagttat tatgatatta tgtttttcta ttaaaataaa ttaagtatat 4080agaatagttt aataatagta tatacttaat gtgataagtg tctgacagtg tcacagaaag 4140gatgattgtt atggattata agcggccggc cgaagcaaac ttaagagtgt gttgatagtg 4200cagtatctta aaattttgta taataggaat tgaagttaaa ttagatgcta aaaatttgta 4260attaagaagg agtgattaca tgaacaaaaa tataaaatat tctcaaaact ttttaacgag 4320tgaaaaagta ctcaaccaaa taataaaaca attgaattta aaagaaaccg ataccgttta 4380cgaaattgga acaggtaaag ggcatttaac gacgaaactg gctaaaataa gtaaacaggt 4440aacgtctatt gaattagaca gtcatctatt caacttatcg tcagaaaaat taaaactgaa 4500tactcgtgtc actttaattc accaagatat tctacagttt caattcccta acaaacagag 4560gtataaaatt gttgggagta ttccttacca tttaagcaca caaattatta aaaaagtggt 4620ttttgaaagc catgcgtctg acatctatct gattgttgaa gaaggattct acaagcgtac 4680cttggatatt caccgaacac tagggttgct cttgcacact caagtctcga ttcagcaatt 4740gcttaagctg ccagcggaat gctttcatcc taaaccaaaa gtaaacagtg tcttaataaa 4800acttacccgc cataccacag atgttccaga taaatattgg aagctatata cgtactttgt 4860ttcaaaatgg gtcaatcgag aatatcgtca actgtttact aaaaatcagt ttcatcaagc 4920aatgaaacac gccaaagtaa acaatttaag taccgttact tatgagcaag tattgtctat 4980ttttaatagt tatctattat ttaacgggag gaaataattc tatgagtcgc ttttgtaaat 5040ttggaaagtt acacgttact aaagggaatg tgttt 5075896662DNAArtificial Sequenceplasmid pMTL83245-Pfor-idi-FS 89aaactccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 60gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 120atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 180gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 240gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 300tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 360accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 420ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 480cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 540agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 600ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 660tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 720ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 780cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 840gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca gggccccctg caggataaaa 900aaattgtaga taaattttat aaaatagttt tatctacaat ttttttatca ggaaacagct 960atgaccgcgg ccgcaatatg atatttatgt ccattgtgaa agggattata ttcaactatt 1020attccagtta cgttcataga aattttcctt tctaaaatat tttattccat gtcaagaact 1080ctgtttattt cattaaagaa ctataagtac aaagtataag gcatttgaaa aaataggcta 1140gtatattgat tgattattta ttttaaaatg cctaagtgaa atatatacat attataacaa 1200taaaataagt attagtgtag gatttttaaa tagagtatct attttcagat taaatttttg 1260attatttgat ttacattata taatattgag taaagtattg actagcaaaa ttttttgata 1320ctttaatttg tgaaatttct tatcaaaagt tatatttttg aataattttt attgaaaaat 1380acaactaaaa aggattatag tataagtgtg tgtaattttg tgttaaattt aaagggagga 1440aatgaacatg aaacatatgg tgaccatgat tacgaattcg agctcggtac ccggggatcc 1500tctagttgta tattaaaata gtagaataca taagatactt aatttaatta aagatagtta 1560agtacttttc aatgtgcttt tttagatgtt taatacaaat ctttaattgt aaaagaaatg 1620ctgtactatt tactgttcta gtgacgggat taaactgtat taattataaa taaaaaataa 1680gtacagttgt ttaaaattat attttgtatt aaatctaata gtacgatgta agttatttta 1740tactattgct agtttaataa aaagatttaa ttatatactt gaaaaggaga ggaactcgag 1800atggcagagt atataatagc agtagatgag ttcgataacg aaataggatc aatagaaaag 1860atggaagctc atagaaaagg aacacttcat agagcattca gtattttagt ttttaactca 1920aagaatcaac ttttattaca gaaaagaaat gtaaagaaat atcactctcc aggattatgg 1980acaaacactt gttgtagtca cccaagatat ggtgaatctc ttcatgatgc tatatacaga 2040agattaaaag aagagatggg atttacttgc gaacttgaag aagtattctc attcatatat 2100aaggtaaaac ttgaagataa tttatttgag aatgaatatg accatgtatt tattggtaaa 2160tatgatggtg agataattgt taataaagat gaagttgatg attttaaatg ggtagacatt 2220aatgaagtta aaaaggacat aatagaaaga cctgaggcat atacttactg gtttaagtat 2280cttgtaaata aagctgaaaa taagatattt aaataaaccg gtgcgccaag cttttaaagg 2340aggggaaaaa atggaattta gagtacattt acaggcagac aacgaacaga aaatatttca 2400aaatcaaatg aaaccagagc cagaagcatc atatcttata aatcaaagaa gaagtgctaa 2460ttataaacca aacatttgga aaaacgattt tcttgatcag tctttaatat caaaatatga 2520tggtgatgaa tatagaaaac tttcagaaaa gttaatagaa gaagtaaaga tatacatatc 2580agcagagact atggatttag ttgctaaatt agaacttata gattctgtta gaaaacttgg 2640acttgctaat ctttttgaga aagaaataaa ggaagcatta gacagtatag cagcaataga 2700atcagataat ttaggaacta gagacgatct ttatggaaca gctcttcatt ttaagattct 2760tagacagcat ggatataagg taagtcaaga tatatttggt agatttatgg atgagaaagg 2820aacattagaa aatcatcact ttgcacactt aaaaggaatg ttagaattat ttgaggcaag 2880taatcttgga tttgaaggtg aagacatatt agatgaagct aaagcatctc ttacacttgc 2940tcttagagat tcaggacata tttgttatcc agactcaaac ttaagtagag atgtagttca 3000tagtttagaa ttacctagtc atagaagagt tcaatggttc gatgtaaaat ggcagattaa 3060tgcatacgaa aaagatattt gtagagtaaa tgcaacttta ttagagttag caaagttaaa 3120ttttaatgtt gttcaagctc agcttcagaa gaatcttaga gaagctagta gatggtgggc 3180taatcttggt ttcgcagata atttaaagtt tgctagagat agacttgtag agtgtttttc 3240atgcgcagta ggtgtagcat ttgaaccaga gcattcatct tttagaatat gtttaactaa 3300ggtaattaat cttgttctta ttatagatga tgtatacgat atatatggat ctgaagaaga 3360gttaaaacat tttacaaatg ctgttgatag atgggacagt agagaaacag aacagcttcc 3420tgaatgcatg aaaatgtgtt ttcaagtatt atataacact acttgcgaaa tagcaagaga 3480gatagaagaa gaaaacggtt ggaatcaagt attacctcaa cttactaagg tttgggctga 3540tttttgtaag gctcttttag ttgaagcaga gtggtacaat aaatcacata ttccaacatt 3600agaagaatat cttagaaacg gatgtatatc aagtagtgta tctgtacttt tagttcactc 3660tttcttttca ataactcatg aaggtacaaa agaaatggct gatttcttac ataaaaatga 3720agatctttta tacaacataa gtcttatagt aagattaaac aatgatttag gtacatcagc 3780tgctgaacag gaaagaggtg attctccttc ttctatagtt tgctatatga gagaagttaa 3840tgcttctgaa gagactgcaa gaaagaatat aaagggaatg attgataatg cttggaaaaa 3900ggttaatgga aaatgtttca caactaacca agttccattt ctttcatcat tcatgaataa 3960tgcaactaac atggcaagag tagcacactc attatataaa gacggtgatg gttttggtga 4020tcaagaaaaa ggacctagaa cacatattct tagtttatta ttccaacctt tagtaaatta 4080agctagcata aaaataagaa gcctgcattt gcaggcttct tatttttatg gcgcgccgcc 4140attatttttt tgaacaattg acaattcatt tcttattttt tattaagtga tagtcaaaag 4200gcataacagt gctgaataga aagaaattta cagaaaagaa aattatagaa tttagtatga 4260ttaattatac tcatttatga atgtttaatt gaatacaaaa aaaaatactt gttatgtatt 4320caattacggg ttaaaatata gacaagttga aaaatttaat aaaaaaataa gtcctcagct 4380cttatatatt aagctaccaa cttagtatat aagccaaaac ttaaatgtgc taccaacaca 4440tcaagccgtt agagaactct atctatagca atatttcaaa tgtaccgaca tacaagagaa 4500acattaacta tatatattca atttatgaga ttatcttaac agatataaat gtaaattgca 4560ataagtaaga tttagaagtt tatagccttt gtgtattgga agcagtacgc aaaggctttt 4620ttatttgata aaaattagaa gtatatttat tttttcataa ttaatttatg aaaatgaaag 4680ggggtgagca aagtgacaga ggaaagcagt atcttatcaa ataacaaggt attagcaata 4740tcattattga ctttagcagt aaacattatg acttttatag tgcttgtagc taagtagtac 4800gaaaggggga gctttaaaaa gctccttgga atacatagaa ttcataaatt aatttatgaa 4860aagaagggcg tatatgaaaa cttgtaaaaa ttgcaaagag tttattaaag atactgaaat 4920atgcaaaata cattcgttga tgattcatga taaaacagta gcaacctatt gcagtaaata 4980caatgagtca agatgtttac ataaagggaa agtccaatgt attaattgtt caaagatgaa 5040ccgatatgga tggtgtgcca taaaaatgag atgttttaca gaggaagaac agaaaaaaga 5100acgtacatgc attaaatatt atgcaaggag ctttaaaaaa gctcatgtaa agaagagtaa 5160aaagaaaaaa taatttattt attaatttaa tattgagagt gccgacacag tatgcactaa 5220aaaatatatc tgtggtgtag tgagccgata caaaaggata gtcactcgca ttttcataat 5280acatcttatg ttatgattat gtgtcggtgg gacttcacga cgaaaaccca caataaaaaa 5340agagttcggg gtagggttaa gcatagttga ggcaactaaa caatcaagct aggatatgca 5400gtagcagacc gtaaggtcgt tgtttaggtg tgttgtaata catacgctat taagatgtaa 5460aaatacggat accaatgaag ggaaaagtat aatttttgga tgtagtttgt ttgttcatct 5520atgggcaaac tacgtccaaa gccgtttcca aatctgctaa aaagtatatc ctttctaaaa 5580tcaaagtcaa gtatgaaatc ataaataaag tttaattttg aagttattat gatattatgt 5640ttttctatta aaataaatta agtatataga atagtttaat aatagtatat acttaatgtg 5700ataagtgtct gacagtgtca cagaaaggat gattgttatg gattataagc ggccggccga 5760agcaaactta agagtgtgtt gatagtgcag tatcttaaaa ttttgtataa taggaattga 5820agttaaatta gatgctaaaa atttgtaatt aagaaggagt gattacatga acaaaaatat 5880aaaatattct caaaactttt taacgagtga aaaagtactc aaccaaataa taaaacaatt 5940gaatttaaaa gaaaccgata ccgtttacga aattggaaca ggtaaagggc atttaacgac 6000gaaactggct aaaataagta aacaggtaac gtctattgaa ttagacagtc atctattcaa 6060cttatcgtca gaaaaattaa aactgaatac tcgtgtcact ttaattcacc aagatattct 6120acagtttcaa ttccctaaca aacagaggta taaaattgtt gggagtattc cttaccattt 6180aagcacacaa attattaaaa aagtggtttt tgaaagccat gcgtctgaca tctatctgat 6240tgttgaagaa ggattctaca agcgtacctt ggatattcac cgaacactag ggttgctctt 6300gcacactcaa gtctcgattc agcaattgct taagctgcca gcggaatgct ttcatcctaa 6360accaaaagta aacagtgtct taataaaact tacccgccat accacagatg ttccagataa 6420atattggaag ctatatacgt actttgtttc aaaatgggtc aatcgagaat atcgtcaact 6480gtttactaaa aatcagtttc atcaagcaat gaaacacgcc aaagtaaaca atttaagtac 6540cgttacttat gagcaagtat tgtctatttt taatagttat ctattattta acgggaggaa 6600ataattctat gagtcgcttt tgtaaatttg gaaagttaca cgttactaaa gggaatgtgt 6660tt 6662907077DNAArtificial Sequenceplasmid pMTL83245-Pfor-idi-ispA-FS 90aaactccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 60gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 120atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 180gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 240gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 300tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 360accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 420ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 480cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 540agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 600ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 660tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 720ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 780cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 840gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca gggccccctg caggataaaa 900aaattgtaga taaattttat aaaatagttt tatctacaat ttttttatca ggaaacagct 960atgaccgcgg ccgcaccgag actagttgta tattaaaata gtagaataca taagatactt 1020aatttaatta aagatagtta agtacttttc aatgtgcttt tttagatgtt taatacaaat 1080ctttaattgt aaaagaaatg ctgtactatt tactgttcta gtgacgggat taaactgtat 1140taattataaa taaaaaataa gtacagttgt ttaaaattat attttgtatt aaatctaata 1200gtacgatgta agttatttta tactattgct agtttaataa aaagatttaa ttatatactt 1260gaaaaggaga ggaactcgag atggcagagt atataatagc agtagatgag ttcgataacg 1320aaataggatc aatagaaaag atggaagctc atagaaaagg aacacttcat agagcattca 1380gtattttagt ttttaactca aagaatcaac ttttattaca gaaaagaaat gtaaagaaat 1440atcactctcc aggattatgg acaaacactt gttgtagtca cccaagatat ggtgaatctc 1500ttcatgatgc tatatacaga agattaaaag aagagatggg atttacttgc gaacttgaag 1560aagtattctc attcatatat aaggtaaaac ttgaagataa tttatttgag aatgaatatg 1620accatgtatt tattggtaaa tatgatggtg agataattgt taataaagat gaagttgatg 1680attttaaatg ggtagacatt aatgaagtta aaaaggacat aatagaaaga cctgaggcat 1740atacttactg gtttaagtat cttgtaaata aagctgaaaa taagatattt aaataaaccg 1800gtcagtaacg aatagaatta gaaaaacaaa ggaggcaaga caatggattt cccacaacaa 1860ttagaagcat gtgtaaaaca ggctaatcag gcacttagta gatttattgc tcctcttcct 1920tttcaaaata caccagtagt agaaactatg caatacggtg cacttttagg tggtaaaaga 1980ttaagaccat tcttagtata tgctacagga cacatgtttg gtgtatcaac taatacttta 2040gacgctccag ctgctgctgt tgaatgtatt catgcttatt ctttaataca tgatgactta 2100ccagcaatgg atgacgatga tttaagaaga ggtttaccta catgtcatgt taaatttggt 2160gaagctaatg caattttagc aggtgacgct ttacaaactt tagctttttc tatactttca 2220gatgcagaca tgcctgaagt ttcagataga gatagaattt ctatgatatc agagcttgca 2280tctgcatcag gaatagctgg aatgtgcggt ggtcaagcac ttgatttaga tgcagaaggt 2340aaacacgtac cacttgatgc tttagagaga atacatagac ataaaacagg tgctcttata 2400agagcagcag taagattagg tgctttaagt gctggtgaca agggtagaag agcacttcca 2460gtacttgata agtatgcaga aagtatagga ttagcttttc aagttcaaga tgacatactt 2520gacgttgttg gtgatactgc tactttagga aaaagacagg gtgcagatca gcaattagga 2580aaatctacat accctgcttt acttggatta gaacaggcta gaaagaaagc aagagactta 2640atagatgacg caagacaaag tcttaaacag ttagctgaac aatcacttga cacaagtgca 2700cttgaagcac ttgcagatta tattatacag agaaacaagt aaaagctttt aaaggagggg 2760aaaaaatgga atttagagta catttacagg cagacaacga acagaaaata tttcaaaatc 2820aaatgaaacc agagccagaa gcatcatatc ttataaatca aagaagaagt gctaattata 2880aaccaaacat ttggaaaaac gattttcttg atcagtcttt aatatcaaaa tatgatggtg 2940atgaatatag aaaactttca gaaaagttaa tagaagaagt aaagatatac atatcagcag 3000agactatgga tttagttgct aaattagaac ttatagattc tgttagaaaa cttggacttg 3060ctaatctttt tgagaaagaa ataaaggaag cattagacag tatagcagca atagaatcag 3120ataatttagg aactagagac gatctttatg gaacagctct tcattttaag attcttagac 3180agcatggata taaggtaagt caagatatat ttggtagatt tatggatgag aaaggaacat 3240tagaaaatca tcactttgca cacttaaaag gaatgttaga attatttgag gcaagtaatc 3300ttggatttga aggtgaagac atattagatg aagctaaagc atctcttaca cttgctctta 3360gagattcagg acatatttgt tatccagact caaacttaag tagagatgta gttcatagtt 3420tagaattacc tagtcataga agagttcaat ggttcgatgt aaaatggcag attaatgcat 3480acgaaaaaga tatttgtaga gtaaatgcaa ctttattaga gttagcaaag ttaaatttta 3540atgttgttca agctcagctt cagaagaatc ttagagaagc tagtagatgg tgggctaatc 3600ttggtttcgc agataattta aagtttgcta gagatagact tgtagagtgt ttttcatgcg 3660cagtaggtgt agcatttgaa ccagagcatt catcttttag aatatgttta actaaggtaa 3720ttaatcttgt tcttattata gatgatgtat acgatatata tggatctgaa gaagagttaa 3780aacattttac aaatgctgtt gatagatggg acagtagaga aacagaacag cttcctgaat 3840gcatgaaaat gtgttttcaa gtattatata acactacttg cgaaatagca agagagatag 3900aagaagaaaa cggttggaat caagtattac ctcaacttac taaggtttgg gctgattttt 3960gtaaggctct tttagttgaa gcagagtggt acaataaatc acatattcca acattagaag 4020aatatcttag aaacggatgt atatcaagta gtgtatctgt acttttagtt cactctttct 4080tttcaataac tcatgaaggt acaaaagaaa tggctgattt cttacataaa aatgaagatc 4140ttttatacaa cataagtctt atagtaagat taaacaatga tttaggtaca tcagctgctg 4200aacaggaaag aggtgattct ccttcttcta tagtttgcta tatgagagaa gttaatgctt 4260ctgaagagac tgcaagaaag aatataaagg gaatgattga taatgcttgg aaaaaggtta 4320atggaaaatg tttcacaact aaccaagttc catttctttc atcattcatg aataatgcaa 4380ctaacatggc aagagtagca cactcattat ataaagacgg tgatggtttt ggtgatcaag 4440aaaaaggacc tagaacacat attcttagtt tattattcca acctttagta aattaagcta 4500gcataaaaat aagaagcctg catttgcagg cttcttattt ttatggcgcg ccgccattat 4560ttttttgaac aattgacaat tcatttctta ttttttatta agtgatagtc aaaaggcata 4620acagtgctga atagaaagaa atttacagaa aagaaaatta tagaatttag tatgattaat 4680tatactcatt tatgaatgtt taattgaata caaaaaaaaa tacttgttat gtattcaatt 4740acgggttaaa atatagacaa gttgaaaaat ttaataaaaa aataagtcct cagctcttat 4800atattaagct accaacttag tatataagcc aaaacttaaa tgtgctacca acacatcaag 4860ccgttagaga actctatcta tagcaatatt tcaaatgtac cgacatacaa gagaaacatt 4920aactatatat attcaattta tgagattatc ttaacagata taaatgtaaa ttgcaataag 4980taagatttag aagtttatag cctttgtgta ttggaagcag tacgcaaagg cttttttatt 5040tgataaaaat tagaagtata tttatttttt cataattaat ttatgaaaat gaaagggggt 5100gagcaaagtg acagaggaaa gcagtatctt atcaaataac aaggtattag caatatcatt 5160attgacttta gcagtaaaca ttatgacttt tatagtgctt gtagctaagt agtacgaaag 5220ggggagcttt aaaaagctcc ttggaataca tagaattcat aaattaattt atgaaaagaa 5280gggcgtatat gaaaacttgt aaaaattgca aagagtttat taaagatact gaaatatgca 5340aaatacattc gttgatgatt catgataaaa cagtagcaac ctattgcagt aaatacaatg

5400agtcaagatg tttacataaa gggaaagtcc aatgtattaa ttgttcaaag atgaaccgat 5460atggatggtg tgccataaaa atgagatgtt ttacagagga agaacagaaa aaagaacgta 5520catgcattaa atattatgca aggagcttta aaaaagctca tgtaaagaag agtaaaaaga 5580aaaaataatt tatttattaa tttaatattg agagtgccga cacagtatgc actaaaaaat 5640atatctgtgg tgtagtgagc cgatacaaaa ggatagtcac tcgcattttc ataatacatc 5700ttatgttatg attatgtgtc ggtgggactt cacgacgaaa acccacaata aaaaaagagt 5760tcggggtagg gttaagcata gttgaggcaa ctaaacaatc aagctaggat atgcagtagc 5820agaccgtaag gtcgttgttt aggtgtgttg taatacatac gctattaaga tgtaaaaata 5880cggataccaa tgaagggaaa agtataattt ttggatgtag tttgtttgtt catctatggg 5940caaactacgt ccaaagccgt ttccaaatct gctaaaaagt atatcctttc taaaatcaaa 6000gtcaagtatg aaatcataaa taaagtttaa ttttgaagtt attatgatat tatgtttttc 6060tattaaaata aattaagtat atagaatagt ttaataatag tatatactta atgtgataag 6120tgtctgacag tgtcacagaa aggatgattg ttatggatta taagcggccg gccgaagcaa 6180acttaagagt gtgttgatag tgcagtatct taaaattttg tataatagga attgaagtta 6240aattagatgc taaaaatttg taattaagaa ggagtgatta catgaacaaa aatataaaat 6300attctcaaaa ctttttaacg agtgaaaaag tactcaacca aataataaaa caattgaatt 6360taaaagaaac cgataccgtt tacgaaattg gaacaggtaa agggcattta acgacgaaac 6420tggctaaaat aagtaaacag gtaacgtcta ttgaattaga cagtcatcta ttcaacttat 6480cgtcagaaaa attaaaactg aatactcgtg tcactttaat tcaccaagat attctacagt 6540ttcaattccc taacaaacag aggtataaaa ttgttgggag tattccttac catttaagca 6600cacaaattat taaaaaagtg gtttttgaaa gccatgcgtc tgacatctat ctgattgttg 6660aagaaggatt ctacaagcgt accttggata ttcaccgaac actagggttg ctcttgcaca 6720ctcaagtctc gattcagcaa ttgcttaagc tgccagcgga atgctttcat cctaaaccaa 6780aagtaaacag tgtcttaata aaacttaccc gccataccac agatgttcca gataaatatt 6840ggaagctata tacgtacttt gtttcaaaat gggtcaatcg agaatatcgt caactgttta 6900ctaaaaatca gtttcatcaa gcaatgaaac acgccaaagt aaacaattta agtaccgtta 6960cttatgagca agtattgtct atttttaata gttatctatt atttaacggg aggaaataat 7020tctatgagtc gcttttgtaa atttggaaag ttacacgtta ctaaagggaa tgtgttt 70779110090DNAArtificial Sequenceplasmid pMTL 8314-Prnf-MK-PMK-PMD-Pfor-idi- ispA-FS 91aaactccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 60gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 120atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 180gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 240gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 300tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 360accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 420ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 480cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 540agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 600ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 660tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 720ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 780cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 840gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca gggccccctg caggataaaa 900aaattgtaga taaattttat aaaatagttt tatctacaat ttttttatca ggaaacagct 960atgaccgcgg ccgctaggtc tagaatatcg atacagataa aaaaatatat aatacagaag 1020aaaaaattat aaatttgtgg tataatataa agtatagtaa tttaagttta aacctcgtga 1080aaacgctaac aaataatagg aggtcaattg atgatagctg ttccatttaa cgctggaaaa 1140ataaaagttt taattgaggc attagaatct ggaaattatt catcaataaa atcagatgta 1200tatgacggaa tgttatatga tgcaccagat caccttaaat cattagtaaa cagatttgta 1260gaacttaata atataactga gccattagca gtaactatac agacaaatct tcctccttca 1320agaggtcttg gatctagtgc agctgttgct gttgcttttg taagagcaag ttatgatttc 1380ttaggaaaaa gtttaactaa agaagagctt atagaaaagg ctaattgggc tgaacaaata 1440gctcatggaa agccatctgg aatagataca caaacaatag tatctggaaa gcctgtttgg 1500tttcaaaagg gacatgcaga aacacttaaa actctttcac ttgatggata catggtagta 1560attgatacag gtgttaaagg aagtacaaga caggctgtag aagatgttca taaactttgc 1620gaagatcctc aatatatgag tcacgtaaaa cacataggaa aacttgtact tagagcatct 1680gatgttattg aacatcataa ctttgaagca cttgctgata tattcaatga atgtcatgct 1740gatttaaagg ctcttacagt aagtcatgac aaaatagaac agttaatgaa gataggaaaa 1800gaaaatggtg ctatagctgg taaattaact ggtgctggta gaggtggttc aatgttatta 1860cttgcaaaag acttaccaac tgcaaagaat atagttaaag cagtagagaa agctggtgca 1920gcacatactt ggattgaaaa tttaggtggt taagtcgaca aagacactaa aaaattataa 1980aagtaaagga ggacattaaa tgatacaagt aaaggcacca ggaaaattat atatagcagg 2040tgaatacgct gttacagaac caggatataa atctgttctt atagctcttg atagatttgt 2100tacagctact attgaggaag ctgatcaata caaaggaaca atacattcaa aggcattaca 2160tcacaatcca gtaactttta gtagagatga agattctatt gttatatcag acccacacgc 2220agcaaaacaa cttaattatg tagtaactgc tatagaaata tttgagcaat atgcaaaatc 2280atgtgacata gcaatgaagc attttcattt aactatagat tctaacttag atgatagtaa 2340tggacataag tatggacttg gatcttctgc tgctgtttta gtttcagtaa ttaaagtact 2400taacgaattt tatgatatga aactttcaaa cctttatata tataagttag cagtaattgc 2460taatatgaaa ttacagagtt tatcttcatg cggtgatata gcagtaagtg tttattcagg 2520ttggttagct tattctacat ttgaccatga atgggtaaaa caccagatag aagatacaac 2580agttgaagaa gtacttatta aaaattggcc tggattacac atagagccac ttcaagctcc 2640tgaaaatatg gaagttctta taggttggac aggtagtcca gctagtagtc ctcattttgt 2700ttctgaagtt aaaagactta agtcagatcc ttcattttac ggtgatttct tagaagattc 2760acatagatgt gtagaaaaat taattcatgc attcaaaact aataatatta agggtgttca 2820gaaaatggta agacagaata gaactattat acaaagaatg gataaggaag caacagttga 2880tatagagact gagaagttaa aatatttatg tgatattgct gaaaaatatc atggtgcaag 2940taaaacttca ggtgctggtg gtggtgattg cggaataact ataataaata aggatgtaga 3000caaagagaaa atatatgatg aatggactaa acatggaata aagcctctta agtttaatat 3060ttatcatgga caataaccat ggtcaataat cttacaataa ataaaagaaa ggaggcaaaa 3120atatgataaa atctggaaaa gcaagagcac acactaatat agcacttata aaatattggg 3180gtaagaaaga tgaggcatta ataataccaa tgaataactc aatatcagta actttagaaa 3240agttttatac tgaaacaaaa gttacattta acgatcagct tactcaagat caattttggc 3300ttaatggtga aaaagtttct ggaaaagaat tagaaaagat ttcaaagtat atggatattg 3360ttagaaatag agctggaata gattggtatg ctgagataga atctgataat tttgttccta 3420cagctgctgg tcttgctagt tctgctagtg cttatgcagc attagctgct gcatgtaacc 3480aagcacttga tttacagtta agtgataaag acttaagtag attagctaga attggatcag 3540gatcagcatc aagatcaata tacggtggtt ttgcagaatg ggaaaaagga tataatgacg 3600aaacttctta tgctgttcca ttagaaagta atcactttga agatgatctt gctatgattt 3660ttgtagtaat aaaccaacat tctaaaaagg ttccttcaag atatggaatg tctcttacaa 3720gaaatacaag tagattctat caatattggt tagaccatat tgatgaagat cttgcagaag 3780caaaggcagc aatacaagat aaggatttta agagattagg tgaagttatt gaagagaatg 3840gacttagaat gcatgctaca aatcttggat caactccacc ttttacttac ttagtacaag 3900agtcatacga tgtaatggca ttagtacatg agtgtagaga agcaggatat ccatgctatt 3960tcactatgga tgctggacct aatgtaaaaa tacttgtaga gaagaaaaac aaacaacaga 4020taatagataa acttttaact cagttcgata ataatcagat aatagatagt gatattatag 4080ctacaggtat tgaaattata gaataaacta gttgtatatt aaaatagtag aatacataag 4140atacttaatt taattaaaga tagttaagta cttttcaatg tgctttttta gatgtttaat 4200acaaatcttt aattgtaaaa gaaatgctgt actatttact gttctagtga cgggattaaa 4260ctgtattaat tataaataaa aaataagtac agttgtttaa aattatattt tgtattaaat 4320ctaatagtac gatgtaagtt attttatact attgctagtt taataaaaag atttaattat 4380atacttgaaa aggagaggaa ctcgagatgg cagagtatat aatagcagta gatgagttcg 4440ataacgaaat aggatcaata gaaaagatgg aagctcatag aaaaggaaca cttcatagag 4500cattcagtat tttagttttt aactcaaaga atcaactttt attacagaaa agaaatgtaa 4560agaaatatca ctctccagga ttatggacaa acacttgttg tagtcaccca agatatggtg 4620aatctcttca tgatgctata tacagaagat taaaagaaga gatgggattt acttgcgaac 4680ttgaagaagt attctcattc atatataagg taaaacttga agataattta tttgagaatg 4740aatatgacca tgtatttatt ggtaaatatg atggtgagat aattgttaat aaagatgaag 4800ttgatgattt taaatgggta gacattaatg aagttaaaaa ggacataata gaaagacctg 4860aggcatatac ttactggttt aagtatcttg taaataaagc tgaaaataag atatttaaat 4920aaaccggtca gtaacgaata gaattagaaa aacaaaggag gcaagacaat ggatttccca 4980caacaattag aagcatgtgt aaaacaggct aatcaggcac ttagtagatt tattgctcct 5040cttccttttc aaaatacacc agtagtagaa actatgcaat acggtgcact tttaggtggt 5100aaaagattaa gaccattctt agtatatgct acaggacaca tgtttggtgt atcaactaat 5160actttagacg ctccagctgc tgctgttgaa tgtattcatg cttattcttt aatacatgat 5220gacttaccag caatggatga cgatgattta agaagaggtt tacctacatg tcatgttaaa 5280tttggtgaag ctaatgcaat tttagcaggt gacgctttac aaactttagc tttttctata 5340ctttcagatg cagacatgcc tgaagtttca gatagagata gaatttctat gatatcagag 5400cttgcatctg catcaggaat agctggaatg tgcggtggtc aagcacttga tttagatgca 5460gaaggtaaac acgtaccact tgatgcttta gagagaatac atagacataa aacaggtgct 5520cttataagag cagcagtaag attaggtgct ttaagtgctg gtgacaaggg tagaagagca 5580cttccagtac ttgataagta tgcagaaagt ataggattag cttttcaagt tcaagatgac 5640atacttgacg ttgttggtga tactgctact ttaggaaaaa gacagggtgc agatcagcaa 5700ttaggaaaat ctacataccc tgctttactt ggattagaac aggctagaaa gaaagcaaga 5760gacttaatag atgacgcaag acaaagtctt aaacagttag ctgaacaatc acttgacaca 5820agtgcacttg aagcacttgc agattatatt atacagagaa acaagtaaaa gcttttaaag 5880gaggggaaaa aatggaattt agagtacatt tacaggcaga caacgaacag aaaatatttc 5940aaaatcaaat gaaaccagag ccagaagcat catatcttat aaatcaaaga agaagtgcta 6000attataaacc aaacatttgg aaaaacgatt ttcttgatca gtctttaata tcaaaatatg 6060atggtgatga atatagaaaa ctttcagaaa agttaataga agaagtaaag atatacatat 6120cagcagagac tatggattta gttgctaaat tagaacttat agattctgtt agaaaacttg 6180gacttgctaa tctttttgag aaagaaataa aggaagcatt agacagtata gcagcaatag 6240aatcagataa tttaggaact agagacgatc tttatggaac agctcttcat tttaagattc 6300ttagacagca tggatataag gtaagtcaag atatatttgg tagatttatg gatgagaaag 6360gaacattaga aaatcatcac tttgcacact taaaaggaat gttagaatta tttgaggcaa 6420gtaatcttgg atttgaaggt gaagacatat tagatgaagc taaagcatct cttacacttg 6480ctcttagaga ttcaggacat atttgttatc cagactcaaa cttaagtaga gatgtagttc 6540atagtttaga attacctagt catagaagag ttcaatggtt cgatgtaaaa tggcagatta 6600atgcatacga aaaagatatt tgtagagtaa atgcaacttt attagagtta gcaaagttaa 6660attttaatgt tgttcaagct cagcttcaga agaatcttag agaagctagt agatggtggg 6720ctaatcttgg tttcgcagat aatttaaagt ttgctagaga tagacttgta gagtgttttt 6780catgcgcagt aggtgtagca tttgaaccag agcattcatc ttttagaata tgtttaacta 6840aggtaattaa tcttgttctt attatagatg atgtatacga tatatatgga tctgaagaag 6900agttaaaaca ttttacaaat gctgttgata gatgggacag tagagaaaca gaacagcttc 6960ctgaatgcat gaaaatgtgt tttcaagtat tatataacac tacttgcgaa atagcaagag 7020agatagaaga agaaaacggt tggaatcaag tattacctca acttactaag gtttgggctg 7080atttttgtaa ggctctttta gttgaagcag agtggtacaa taaatcacat attccaacat 7140tagaagaata tcttagaaac ggatgtatat caagtagtgt atctgtactt ttagttcact 7200ctttcttttc aataactcat gaaggtacaa aagaaatggc tgatttctta cataaaaatg 7260aagatctttt atacaacata agtcttatag taagattaaa caatgattta ggtacatcag 7320ctgctgaaca ggaaagaggt gattctcctt cttctatagt ttgctatatg agagaagtta 7380atgcttctga agagactgca agaaagaata taaagggaat gattgataat gcttggaaaa 7440aggttaatgg aaaatgtttc acaactaacc aagttccatt tctttcatca ttcatgaata 7500atgcaactaa catggcaaga gtagcacact cattatataa agacggtgat ggttttggtg 7560atcaagaaaa aggacctaga acacatattc ttagtttatt attccaacct ttagtaaatt 7620aagctagcat aaaaataaga agcctgcatt tgcaggcttc ttatttttat ggcgcgccgc 7680cattattttt ttgaacaatt gacaattcat ttcttatttt ttattaagtg atagtcaaaa 7740ggcataacag tgctgaatag aaagaaattt acagaaaaga aaattataga atttagtatg 7800attaattata ctcatttatg aatgtttaat tgaatacaaa aaaaaatact tgttatgtat 7860tcaattacgg gttaaaatat agacaagttg aaaaatttaa taaaaaaata agtcctcagc 7920tcttatatat taagctacca acttagtata taagccaaaa cttaaatgtg ctaccaacac 7980atcaagccgt tagagaactc tatctatagc aatatttcaa atgtaccgac atacaagaga 8040aacattaact atatatattc aatttatgag attatcttaa cagatataaa tgtaaattgc 8100aataagtaag atttagaagt ttatagcctt tgtgtattgg aagcagtacg caaaggcttt 8160tttatttgat aaaaattaga agtatattta ttttttcata attaatttat gaaaatgaaa 8220gggggtgagc aaagtgacag aggaaagcag tatcttatca aataacaagg tattagcaat 8280atcattattg actttagcag taaacattat gacttttata gtgcttgtag ctaagtagta 8340cgaaaggggg agctttaaaa agctccttgg aatacataga attcataaat taatttatga 8400aaagaagggc gtatatgaaa acttgtaaaa attgcaaaga gtttattaaa gatactgaaa 8460tatgcaaaat acattcgttg atgattcatg ataaaacagt agcaacctat tgcagtaaat 8520acaatgagtc aagatgttta cataaaggga aagtccaatg tattaattgt tcaaagatga 8580accgatatgg atggtgtgcc ataaaaatga gatgttttac agaggaagaa cagaaaaaag 8640aacgtacatg cattaaatat tatgcaagga gctttaaaaa agctcatgta aagaagagta 8700aaaagaaaaa ataatttatt tattaattta atattgagag tgccgacaca gtatgcacta 8760aaaaatatat ctgtggtgta gtgagccgat acaaaaggat agtcactcgc attttcataa 8820tacatcttat gttatgatta tgtgtcggtg ggacttcacg acgaaaaccc acaataaaaa 8880aagagttcgg ggtagggtta agcatagttg aggcaactaa acaatcaagc taggatatgc 8940agtagcagac cgtaaggtcg ttgtttaggt gtgttgtaat acatacgcta ttaagatgta 9000aaaatacgga taccaatgaa gggaaaagta taatttttgg atgtagtttg tttgttcatc 9060tatgggcaaa ctacgtccaa agccgtttcc aaatctgcta aaaagtatat cctttctaaa 9120atcaaagtca agtatgaaat cataaataaa gtttaatttt gaagttatta tgatattatg 9180tttttctatt aaaataaatt aagtatatag aatagtttaa taatagtata tacttaatgt 9240gataagtgtc tgacagtgtc acagaaagga tgattgttat ggattataag cggccggcca 9300gtgggcaagt tgaaaaattc acaaaaatgt ggtataatat ctttgttcat tagagcgata 9360aacttgaatt tgagagggaa cttagatggt atttgaaaaa attgataaaa atagttggaa 9420cagaaaagag tattttgacc actactttgc aagtgtacct tgtacctaca gcatgaccgt 9480taaagtggat atcacacaaa taaaggaaaa gggaatgaaa ctatatcctg caatgcttta 9540ttatattgca atgattgtaa accgccattc agagtttagg acggcaatca atcaagatgg 9600tgaattgggg atatatgatg agatgatacc aagctataca atatttcaca atgatactga 9660aacattttcc agcctttgga ctgagtgtaa gtctgacttt aaatcatttt tagcagatta 9720tgaaagtgat acgcaacggt atggaaacaa tcatagaatg gaaggaaagc caaatgctcc 9780ggaaaacatt tttaatgtat ctatgatacc gtggtcaacc ttcgatggct ttaatctgaa 9840tttgcagaaa ggatatgatt atttgattcc tatttttact atggggaaat attataaaga 9900agataacaaa attatacttc ctttggcaat tcaagttcat cacgcagtat gtgacggatt 9960tcacatttgc cgttttgtaa acgaattgca ggaattgata aatagttaac ttcaggtttg 10020tctgtaacta aaaacaagta tttaagcaaa aacatcgtag aaatacggtg ttttttgtta 10080ccctaagttt 100909225DNAArtificial Sequenceoligonucleotide repHF 92aagaagggcg tatatgaaaa cttgt 259325DNAArtificial Sequenceoligonucleotide catR 93ttcgtttaca aaacggcaaa tgtga 259420DNAArtificial Sequenceoligonucleotide MK-RTPCR-F 94gtgctggtag aggtggttca 209520DNAArtificial Sequenceoligonucleotide MK-RTPCR-R 95ccaagtatgt gctgcaccag 209620DNAArtificial Sequenceoligonucleotide PMK-RTPCR-F 96atatcagacc cacacgcagc 209724DNAArtificial Sequenceoligonucleotide PMK-RTPCR-R 97aatgcttcat tgctatgtca catg 249820DNAArtificial Sequenceoligonucleotide PMD-RTPCR-F 98gcagaagcaa aggcagcaat 209923DNAArtificial Sequenceoligonucleotide PMD-RTPCR-R 99ttgatccaag atttgtagca tgc 2310024DNAArtificial Sequenceoligonucleotide idi-RTPCR-F 100ggacaaacac ttgttgtagt cacc 2410122DNAArtificial Sequenceoligonucleotide idi-RTPCR-R 101tcaagttcgc aagtaaatcc ca 2210220DNAArtificial Sequenceoligonucleotide ispA-RTPCR-F 102accagcaatg gatgacgatg 2010321DNAArtificial Sequenceoligonucleotide ispA-RTPCR-R 103agtttgtaaa gcgtcacctg c 2110420DNAArtificial Sequenceoligonucleotide FS-RTPCR-F 104aagctagtag atggtgggct 2010520DNAArtificial Sequenceoligonucleotide FS-RTPCR-R 105aatgctacac ctactgcgca 2010618DNAArtificial Sequenceoligonucleotide ermB-F 106tttgtaatta agaaggag 1810718DNAArtificial Sequenceoligonucleotide ermB-R 107gtagaatcct tcttcaac 1810820DNAArtificial Sequenceoligonucleotide GnK-F 108tcaggacctt ctggaactgg 2010920DNAArtificial Sequenceoligonucleotide GnK-R 109acctcccctt ttcttggaga 2011020DNAArtificial Sequenceoligonucleotide FoT4L-F 110caggtttcgg tgctgaccta 2011120DNAArtificial Sequenceoligonucleotide FoT4L-F 111aactccgccg ttgtatttca 20

Claims

1. Isolated, genetically engineered, carboxydotrophic, acetogenic bacteria which comprise an exogenous nucleic acid encoding an enzyme in a mevalonate pathway or in a DXS pathway or in a terpene biosynthesis pathway, whereby the bacteria express the enzyme, said enzyme selected from the group consisting of: a) thiolase (EC 2.3.1.9); b) HMG-CoA synthase (EC 2.3.3.10); c) HMG-CoA reductase (EC 1.1.1.88); d) Mevalonate kinase (EC 2.7.1.36); e) Phosphomevalonate kinase (EC 2.7.4.2); f) Mevalonate Diphosphate decarboxylase (EC 4.1.1.33); 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC:2.2.1.7); g) 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC:1.1.1.267); h) 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC:2.7.7.60); i) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC:2.7.1.148); j) 2-C-methyl-D-erythritol 2;4-cyclodiphosphate synthase IspF (EC:4.6.1.12); k) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC:1.17.7.1); l) 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC:1.17.1.2); geranyltranstransferase Fps (EC:2.5.1.10); m) heptaprenyl diphosphate synthase (EC:2.5.1.10); n) octaprenyl-diphosphate synthase (EC:2.5.1.90); o) isoprene synthase (EC 4.2.3.27); p) isopentenyl-diphosphate delta-isomerase (EC 5.3.3.2); and q) farnesene synthase (EC 4.2.3.46/EC 4.2.3.47).

2. The bacteria of claim 1 wherein in the absence of said nucleic acid, the bacteria do not express the enzyme.

3. The bacteria of claim 1 which express the enzyme under anaerobic conditions.

4. The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of claim 1 where the bacteria express the isoprene synthase enzyme and the bacteria are able to convert dimethylallyldiphosphate to isoprene.

5. The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of claim 4 wherein the isoprene synthase is a Populus tremuloides enzyme.

6. The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of claim 4 wherein the nucleic acid is codon optimized.

7. The isolated, genetically engineered, carboxydrotrophic, acetogenic bacteria of claim 4 wherein expression of the isoprene synthase is under the transcriptional control of a promoter for a pyruvate: ferredoxin oxidoreductase gene from Clostridium autoethanogenum.

8. The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of claim 1 where the bacteria express the isopentyldiphosphate delta isomerase enzyme and the bacteria are able to convert dimethylallyldiphosphate to isopentyldiphosphate.

9. The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of claim 8 wherein the nucleic acid encodes a Clostridium beijerinckii isopentyldiphosphate delta isomerase.

10. The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of claim 8 wherein the nucleic acid is under the transcriptional control of a promoter for a pyruvate: ferredoxin oxidoreductase gene from Clostridium autoethanogenum.

11. The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of claim 8 wherein the nucleic acid is under the transcriptional control of a promoter for a pyruvate: ferredoxin oxidoreductase gene from Clostridium autoethanogenum and downstream of a second nucleic acid encoding an isoprene synthase.

12. The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of claim 1 where the bacteria has a copy number of greater than 1 per genome of a nucleic acid encoding a1-deoxy-D-xylulose 5-phosphate synthase (DXS) enzyme.

13. The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of claim 12 further comprising a nucleic acid encoding an isoprene synthase.

14. The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of claim 12 further comprising a nucleic acid encoding an isopentyldiphosphate delta isomerase.

15. The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of claim 12 further comprising a nucleic acid encoding an isopentyldiphosphate delta isomerase and a nucleic acid encoding an isoprene synthase.

16. The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of claim 1 where the bacteria encodes a phosphomevalonate kinase (PMK enzyme, wherein the enzyme is not native to the bacteria.

17. The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of claim 16 wherein the enzymes are Staphylococcus aureus enzymes.

18. The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of claim 16 wherein the enzyme is expressed under the control of one or more C. autoethanogenum promoters.

19. The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of claim 16 further comprising a nucleic acid encoding thiolase (thlA/vraB), a nucleic acid encoding a HMG-CoA synthase (HMGS), and a nucleic acid encoding an HMG-CoA reductase (HMGR).

20. The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of claim 19 wherein the thiolase is Clostridium acetobutylicum thiolase.

21. The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of claim 16 further comprising a nucleic acid encoding a mevalonate disphosphate decarboxylase (PMD).

22. The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of claim 1 where the bacteria encodes alpha-farnesene synthase.

23. The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of claim 22 wherein the nucleic acid is codon optimized for expression in C. autoethanogenum.

24. The bacteria of claim 22 wherein the alpha-farnesene synthase is a Malus×domestica alpha-farnesene synthase.

25. The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of claim 22 further comprising a nucleic acid segment encoding geranyltranstransferase.

26. The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of claim 25 wherein the gernayltranstransferase is an E. coli geranyltranstransferase.

27. The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of claim 1, which is selected from the group consisting of Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Clostridium aceticum, Clostridium formicoaceticum, Clostridium magnum, Butyribacterium methylotrophicum, Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorella thermautotrophica, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, Oxobacter pfennigii, and Thermoanaerobacter kiuvi.

28. A plasmid which can replicate in a carboxydotrophic, acetogenic bacteria which comprises a nucleic acid encoding an enzyme in a mevalonate pathway or in a DXS pathway or in a terpene biosynthesis pathway, whereby when the plasmid is in said bacteria the enzyme is expressed by said bacteria, said enzyme selected from the group consisting of: thiolase (EC 2.3.1.9); a) HMG-CoA synthase (EC 2.3.3.10); b) HMG-CoA reductase (EC 1.1.1.88); c) Mevalonate kinase (EC 2.7.1.36); d) Phosphomevalonate kinase (EC 2.7.4.2); e) Mevalonate Diphosphate decarboxylase (EC 4.1.1.33); 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC:2.2.1.7); f) 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC:1.1.1.267); g) 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC:2.7.7.60); h) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC:2.7.1.148); i) 2-C-methyl-D-erythritol 2;4-cyclodiphosphate synthase IspF (EC:4.6.1.12); j) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC:1.17.7.1); k) 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC:1.17.1.2); geranyltranstransferase Fps (EC:2.5.1.10); l) heptaprenyl diphosphate synthase (EC:2.5.1.10); m) octaprenyl-diphosphate synthase (EC:2.5.1.90); n) isoprene synthase (EC 4.2.3.27); o) isopentenyl-diphosphate delta-isomerase (EC 5.3.3.2); and p) farnesene synthase (EC 4.2.3.46/EC 4.2.3.47).