Patent application title: RECOMBINANT MICROORGANISM AND METHODS OF PRODUCTION THEREOF
Inventors:
Michael Koepke (Parnell, NZ)
Fungmin Liew (Parnell, NZ)
Assignees:
Lanzatech New Zealand Limited
IPC8 Class: AC12P716FI
USPC Class:
435160
Class name: Containing hydroxy group acyclic butanol
Publication date: 2011-09-29
Patent application number: 20110236941
Abstract:
A novel genetically modified microorganisms capable of using CO to
produce 1-butanol and/or a precursor thereof, novel methyltransferases
and nucleic acids encoding same, methods for producing genetically
modified microorganisms using said novel methyltransferases, and methods
of producing 1-butanol and/or a precursor thereof by microbial
fermentation.Claims:
1.-65. (canceled)
66. An acetogenic recombinant microorganism which one or a combination of produces 1-butanol and a precursor thereof as the main fermentation product.
67. An acetogenic recombinant microorganism as claimed in claim 66, wherein the microorganism is capable of producing one or both 1-butanol and a precursor thereof by fermentation from a substrate comprising CO at a concentration of greater than approximately 1 mM or 0.075 grams per liter of fermentation broth.
68. An acetogenic recombinant microorganism as claimed in claim 66, wherein the microorganism further comprises exogenous nucleic acids adapted to express one or more enzymes in the butanol biosynthesis pathway.
69. An acetogenic recombinant microorganism as claimed in claim 68, wherein the microorganism further comprises one or more exogenous nucleic acids adapted to express one or more of enzymes chosen from the group consisting of: Thiolase 3-hydroxybutyryl-CoA dehydrogenase Crotonase/crotonyl-CoA hydratase Butyryl-CoA dehydrogenase Electron transport flavoprotein Phosphotransbutyrylase; butyrate kinase; ferredoxin dependent aldehyde oxidoreductase; butyraldehyde dehydrogenase; butanol dehydrogenase; and, a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase.
70. An acetogenic recombinant microorganism as claimed in claim 69, wherein the microorganism comprises one or more exogenous nucleic acids encoding each of Thiolase, 3-hydroxybutyryl-CoA dehydrogenase, Crotonase, and Butyryl-CoA dehydrogenase.
71. An acetogenic recombinant microorganism as claimed in claim 70, wherein the microorganism further comprises one or more exogenous nucleic acids encoding at least one electron transport flavoprotein chosen from the group consisting of Electron Transfer Flavoprotein A and Electron Transfer Flavoprotein B.
72. An acetogenic recombinant microorganism as claimed in claim 70, wherein the microorganism further comprises one or more exogenous nucleic acids encoding one or more of butyraldehyde dehydrogenase, butanol dehydrogenase and a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase.
73. An acetogenic recombinant microorganism as claimed in claim 70, wherein the microorganism further comprises one or more exogenous nucleic acids encoding one or more of Phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase and butanol dehydrogenase.
74. An acetogenic recombinant microorganism as claimed in claim 66, wherein the microorganism is selected from the group comprising Clostridium autoethanogenum, Clostridium ljungdahlii Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatalogenes, Clostridium aceticum, Clostridium formicoaceticum, Butyribacterium limosum, Acetobacterium woodii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorella thermautotrophica, Oxobacter pfennigii, and Thermoanaerobacter kiuvi.
75. An acetogenic recombinant microorganism as claimed in claim 74, wherein the microorganism is Clostridium autoethanogenum DSM23693.
76. A method for the production of one or both of 1-butanol and a precursor thereof by microbial fermentation comprising fermenting a substrate using a recombinant microorganism as claimed in claim 66.
77. A method as claimed in claim 76, wherein one or both of 1-butanol and a precursor thereof is the main fermentation product.
78. A method as claimed in claim 76, wherein at least one or both 1-butanol and a precursor thereof is produced in a yield of from approximately 0.075 grams per liter of fermentation broth to approximately 20 grams per liter.
79. A method as claimed in claim 78, wherein the yield is from approximately 0.15 grams per liter to approximately 1.54 grams per liter.
80. A method as claimed in claim 78, wherein the yield is approximately 10 grams per liter, approximately 5 grams per liter, or approximately 2 grams per liter.
81. A method as claimed in claim 78, wherein the yield of 1-butanol is up to the limit at which butanol becomes toxic.
82. A method as claimed in claim 76, wherein the substrate comprises CO.
83. A method as claimed in claim 82, wherein the substrate comprises at least about 20% to about 100% CO by volume.
84. A recombinant microorganism having the defining characteristics of the microorganism deposited at the DSMZ under the accession number DSM24138.
85. (canceled)
86. The method of claim 76, wherein the substrate comprises an industrial waste gas.
Description:
FIELD OF DISCLOSURE
[0001] The present disclosure relates to methods for the production of biofuels by microbial fermentation and genetically modified micro-organisms suitable for use in such methods.
BACKGROUND
[0002] Butanol is an important bulk chemical with a wide range of industrial uses that has worldwide production of 4.5-5.5 million tonnes per annum. It is used as a precursor for the production of acrylate and methacrylate esters (used in coatings, plastics, textiles, adhesives, etc), glycol ethers (coatings, electronics) and butyl acetate (paints, ink, coatings, synthetic fruit flavoring) as well as butylamines (production of pesticides and pharmaceuticals) and amine resins. It also has direct use as a solvent (in ink, dyes, etc), an extractant (for the production of drugs and natural substances such as alkaloids, antibiotics, hormones, and vitamins), and in deicing fluids, cosmetics and chromatography.
[0003] Butanol also has potential as a second generation biofuel, and in this context is referred to as Biobutanol (Kopke & Durre, 2010). It has similar properties to gasoline and superior properties to ethanol. Specifically, it has increased mileage due to higher energy density, it can be mixed with gasoline in any concentration (while ethanol can only be blended up to 85%) and is not hygroscopic or corrosive.
[0004] Biofuels for transportation are attractive replacements for gasoline and are rapidly penetrating fuel markets as low concentration blends. Biofuels, derived from natural plant sources, are more environmentally sustainable than those derived from fossil resources (such as gasoline), their use allowing a reduction in the levels of so-called fossil carbon dioxide (CO2) gas that is released into the atmosphere as a result of fuel combustion. In addition, biofuels can be produced locally in many geographies, and can act to reduce dependence on imported fossil energy resources.
[0005] The vast majority of biofuels are produced via traditional yeast-based fermentation processes that use crop derived carbohydrates as the main carbon source and are known as first generation biofuels. However, these crops are required for food and many crops also require high agricultural inputs in the form of fertilizers. These limitations mean that first generation biofuels are considered unsustainable and the greenhouse gas reductions that can be achieved are limited. The aim of second generation biofuels is the sustainable use of non-food parts of current crops or other industrial waste to reduce greenhouse gas emissions and reduce dependency on fossil fuels.
[0006] Recent 1-butanol production has been mainly by oxo synthesis (Weiβermel & Arpe, 2003). Petrochemicals including crude oil are cracked to form propylene which is used during oxo synthesis. However the synthesis process requires use of non-renewable resources as well as suffering from being expensive and non-specific in the products formed.
[0007] Butanol can also be produced through biological production methods, the most common being the Acetone-Butanol-Ethanol (ABE) fermentation which has been used industrially since 1913 (Kopke & Durre, 2010). This method has the unwanted by-product of acetone which is usually produced at about half the volume of butanol which therefore substantially reduces the yield. Additionally, this method of fermentation is limited by the toxicity of butanol to the micro-organism which results in growth being almost completely inhibited at such low butanol concentrations as 1.5% (Kopke and Durre 2010). Furthermore ABE fermentation uses sugar from corn, starch, cassaya and sugar cane as a feedstock. This results in the undesirable use of arable land to produce fuel rather than food. It can also exacerbate problems related to deforestation and desertification.
[0008] Only a few organisms are known to naturally produce butanol and none of these produce butanol at a high yield from abundant sources (such as carbon monoxide --CO). Two organisms known to naturally produce butanol from CO are Butyribacterium methylotrophicum (which synthesises only traces of butanol (Heiskanen et al, 2007)), and Clostridium carboxidivorans (which produces low yields of 1-butanol as a by-product to the main fermentation products ethanol and acetate (Liou et al, 2005)).
[0009] A number of organisms have been genetically modified to produce 1-butanol including E. coli, Bacillus subtilis, Saccharomyces cerevisiae, Pseudomonas putida, or Lactobacillus brevis. However all of these organisms still rely on sugar as feedstock (Kopke & Durre, 2010). Despite over 250 Clostridium species being known, only a few are genetically accessible. There is no natural competence (uptake of extracellular DNA from the cell's environment) known in Clostridia and electrotransformation or conjugation are the only methods available for transformation. These issues present significant difficulties in effectively transforming Clostridia species. Most Clostridia have one or more restriction/methylation systems to protect against foreign and phage DNA which means that transformation is particularly difficult and unpredictable.
SUMMARY OF INVENTION
[0010] It is an object of the invention to overcome one or more disadvantages of the prior art, or to at least provide the public with a useful alternative to known technologies.
[0011] In accordance with the invention, it has been discovered that a genetically modified microorganism is capable of using CO to produce 1-butanol or a precursor thereof as the main fermentation product.
[0012] In a first aspect, the invention provides an acetogenic recombinant microorganism which produces 1-butanol and/or a precursor thereof as the main fermentation product.
[0013] In a related aspect, the invention provides an acetogenic recombinant microorganism which is capable of producing 1-butanol and/or a precursor thereof by fermentation from a substrate comprising CO at a concentration of greater than approximately 1 mM or 0.075 g/l per litre of fermentation broth.
[0014] Preferably, the microorganism comprises exogenous nucleic acids adapted to express one or more enzymes in the butanol biosynthesis pathway.
[0015] In one embodiment, the one or more enzymes are chosen from the group consisting of: Thiolase 3-hydroxybutyryl-CoA dehydrogenase, Crotonase/crotonyl-CoA hydratase, Butyryl-CoA dehydrogenase, Electron Transfer Flavoprotein A, and Electron Transfer Flavoprotein B
[0016] Preferably, the microorganism comprises one or more exogenous nucleic acids encoding one or more of the enzymes.
[0017] Preferably, the one or more nucleic acids encoding the one or more enzymes is chosen from the nucleic acids SEQ ID NO. 1 to SEQ ID NO. 6 or functionally equivalent variants thereof.
[0018] Preferably, the microorganism comprises one or more exogenous nucleic acids encoding each of Thiolase, 3-hydroxybutyryl-CoA dehydrogenase, Crotonase, Butyryl-CoA dehydrogenase, Electron Transfer Flavoprotein A and Electron Transfer Flavoprotein B.
[0019] Preferably, the microorganism comprises a plasmid encoding one or more of, or preferably each of, Thiolase, 3-hydroxybutyryl-CoA dehydrogenase, Crotonase, Butyryl-CoA dehydrogenase, Electron Transfer Flavoprotein A and Electron Transfer Flavoprotein B.
[0020] In one embodiment, the microorganism comprises one or more exogenous nucleic acids encoding each of the enzymes thiolase 3-hydroxybutyryl-CoA dehydrogenase, crotonase/crotonyl-CoA hydratase and butyryl-CoA dehydrogenase.
[0021] Preferably, the microorganism further comprises an exogenous phosphotransacetylase/acetate kinase promoter. Preferably, the promoter corresponds to SEQ_ID No. 7 or a functionally equivalent variant thereof.
[0022] Preferably, the promoter is contained on a construct encoding one or more of the enzymes referred to herein before.
[0023] In one embodiment, the microorganism comprises exogenous nucleic acids adapted to express one or more of the enzymes chosen from the group consisting of: Phosphotransbutyrylase; butyrate kinase; ferredoxin dependent aldehyde oxidoreductase; butyraldehyde dehydrogenase; butanol dehydrogenase; a bifunctional butyraldehyde dehydrogenase and butanol dehydrogenase.
[0024] In one embodiment, the microorganism comprises exogenous nucleic acids adapted to express one or more of butyraldehyde dehydrogenase, butanol dehydrogenase and a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase. Preferably, the microorganism comprises one or more exogenous nucleic acids encoding one or more of butyraldehyde dehydrogenase, butanol dehydrogenase and a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase.
[0025] In one embodiment, the microorganism comprises exogenous nucleic acids adapted to express one or more of Phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase, and butanol dehydrogenase. Preferably, the microorganism comprises one or more exogenous nucleic acids encoding one or more of Phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase, and butanol dehydrogenase. In particular embodiments, the microorganism comprises exogenous nucleic acids adapted to express each of Phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase, and butanol dehydrogenase.
[0026] In one embodiment, the one or more nucleic acids encoding the one or more enzymes is chosen from the nucleic acids outlined in tables 7 to 10 herein after and functionally equivalent variants thereof.
[0027] In one embodiment, the microorganism comprises one or more nucleic acid adapted to express at least two of the enzymes in the butanol biosynthesis pathway, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 of the enzymes. Preferably, the microorganism is selected from the group of acetogenic bacteria. In certain embodiments the microorganism is selected from the group comprising Clostridium autoethanogenum, Clostridium ljungdahlii Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatalogenes, Clostridium aceticum, Clostridium formicoaceticum, Butyribacterium limosum, Acetobacterium woodii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorella thermautotrophica, Oxobacter pfennigii, and Thermoanaerobacter kiuvi.
[0028] Preferably, the microorganism is Clostridium autoethanogenum DSM23693.
[0029] In one embodiment, the recombinant microorganism of the invention has the defining characteristics of the microorganism deposited at the DSMZ (Deutsche Sammlung fur Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany) under the accession number DSM24138.
[0030] In a second aspect, the invention provides a recombinant methyltransferase gene according to nucleotide SEQ_ID NO 27 or a functionally equivalent variant thereof.
[0031] In a third aspect, the invention provides a methyltransferase according to SEQ_ID NO 28 or a functionally equivalent amino acid variant thereof.
[0032] In a related aspect the invention provides a recombinant microorganism comprising a methyltransferase gene according to the second aspect. The methyltransferase gene may be present on a nucleic acid construct or integrated into the genome of the microorganism.
[0033] In a fourth aspect, the invention provides a nucleic acid comprising SEQ_ID No 1 to 6, or functionally equivalent variants thereof, in any order.
[0034] Preferably, the nucleic acid comprises SEQ_ID No 1 to 6 in the order shown in FIG. 2.
[0035] Preferably, the nucleic acid further comprises a phosphotransacetylase/acetate kinase promoter. Preferably, the promoter corresponds to SEQ_ID No. 7 or a functionally equivalent variant thereof.
[0036] In a fifth aspect, the invention provides an expression construct comprising one or more nucleic acid sequences wherein the construct, when expressed in an acetogenic microorganism, results in 1-butanol and/or a precursor thereof being produced as the main fermentation product.
[0037] Preferably, the one or more nucleic acid sequences encode one or more enzymes that are part of the 1-butanol biosynthesis pathway.
[0038] Preferably, the nucleic acids are selected from nucleic acids encoding thiolase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, electron transfer flavoprotein A and/or electron transfer flavoprotein B.
[0039] Preferably, the one or more nucleic acid sequences are selected from SEQ_ID NO. 1 to SEQ_ID NO. 6 or functionally equivalent variants thereof.
[0040] In one embodiment, the nucleic acids are further selected from nucleic acids encoding Phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase, butyraldehyde dehydrogenase, butanol dehydrogenase, and a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase.
[0041] In one embodiment, the nucleic acids are selected from the group of nucleic acids outlined in tables 7 to 10 herein after and functionally equivalent variants thereof.
[0042] In one embodiment, the expression construct encodes at least 2 enzymes in the butanol biosynthesis pathway, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 or at least 12 of the enzymes.
[0043] Preferably, the expression construct further comprises a phosphotransacetylase/acetate kinase operon promoter. In another embodiment, the expression construct comprises another highly active promoter such as the promoter of the pyruvate:ferredoxin oxidoreductase (SEQ_ID No. 48), the Wood-Ljungdahl gene cluster (SEQ_ID No 47), Rnf operon (SEQ_ID No 49) or the ATP synthase operon (SEQ_ID No 50). Preferably, the phosphotransacetylase/acetate kinase operon promoter corresponds to SEQ_ID No. 7 or a functionally equivalent variant thereof.
[0044] In a sixth aspect, the invention provides a methylation construct comprising a methyltransferase gene as described herein.
[0045] In a seventh aspect, the invention provides a composition comprising the expression construct of the fifth aspect and the methylation construct of the sixth aspect.
[0046] Preferably, the composition is able to produce a recombinant microorganism which produces 1-butanol and/or a precursor thereof as the main fermentation product.
[0047] In an eighth aspect, the invention provides a method of producing a recombinant microorganism comprising: [0048] a. introduction into a shuttle microorganism of (i) an expression construct and (ii) a methylation construct according to the sixth aspect comprising a methyltransferase gene; [0049] b. expression of the methyltransferase gene; [0050] c. isolation of one or more constructs from the shuttle microorganism; and, [0051] d. introduction of at least the expression construct into a destination microorganism; [0052] wherein the expression construct comprises one or more genes encoding enzymes to be expressed in the destination microorganism.
[0053] In one embodiment, expression of the methyltransferase gene in step b. is constitutive. In another embodiment, expression of the methyltransferase gene in step b. is induced.
[0054] In one embodiment, both the methylation construct and the expression construct are isolated in step C. In another embodiment, the expression construct is isolated in step C.
[0055] In one embodiment, only the expression construct is introduced into the destination microorganism. In another embodiment, both the expression construct and the methylation construct are introduced into the destination microorganism.
[0056] Preferably, the expression construct is as defined in the fifth aspect.
[0057] Preferably, the recombinant microorganism produces 1-butanol and/or a precursor thereof as the main fermentation product.
[0058] In a related aspect, the invention provides a method of producing a recombinant microorganism comprising: [0059] a. methylation of an expression construct in vitro by a methyltransferase according to SEQ_ID No 28 or a functionally equivalent variant thereof [0060] b. introduction of an expression construct into a destination microorganism; [0061] wherein the expression construct comprises one or more genes encoding enzymes to be expressed in the destination microorganism.
[0062] Preferably, the expression construct is as defined in the fifth aspect.
[0063] Preferably, the recombinant microorganism produces 1-butanol and/or a precursor thereof as the main fermentation product.
[0064] Preferably, the methyltransferase is produced by expressing a methyltransferase gene, preferably according to SEQ_ID No 27 or a functionally equivalent variant thereof, in a microorganism and isolating the methyltransferase enzyme.
[0065] In a further related aspect, the invention provides a method of producing a recombinant microorganism comprising: [0066] a. introduction into the genome of a shuttle microorganism of a methyltransferase gene, preferably according to SEQ_ID No 27 or a functionally equivalent variant thereof [0067] b. introduction of an expression construct into the shuttle microorganism [0068] c. isolation of one or more constructs from the shuttle microorganism; and, [0069] d. introduction of at least the expression construct into a destination microorganism; [0070] wherein the expression construct comprises one or more genes encoding enzymes to be expressed in the destination microorganism.
[0071] Preferably, the expression construct is as defined in the fifth aspect.
[0072] Preferably, the recombinant microorganism produces 1-butanol and/or a precursor thereof as the main fermentation product.
[0073] In a further related aspect, the invention provides a method of producing a recombinant microorganism comprising: [0074] a. methylation of an expression construct in accordance with the fifth aspect in vitro by a methyltransferase [0075] b. introduction of the expression construct into a destination microorganism.
[0076] Preferably, the methyltransferase is encoded by a methyltransferase gene as defined in the second aspect or a methyltransferase as defined in the third aspect.
[0077] Preferably, the recombinant microorganism produces 1-butanol and/or a precursor thereof as the main fermentation product.
[0078] In a ninth aspect, the invention provides a method of producing a recombinant microorganism comprising: [0079] a. introduction of (i) an expression construct according to the fifth aspect and (ii) a methylation construct comprising a methyltransferase gene into a shuttle microorganism; [0080] b. expression of the methyltransferase gene; [0081] c. isolation of one or more constructs from the shuttle microorganism; and [0082] d. introduction of at least the expression construct into a destination microorganism; wherein the expression construct comprises one or more genes encoding enzymes to be expressed in the destination microorganism.
[0083] In one embodiment, expression of the methyltransferase gene in step b. is constitutive. In another embodiment, expression of the methyltransferase gene in step b. is induced.
[0084] In one embodiment, both the methylation construct and the expression construct are isolated in step C. In another embodiment, the expression construct is isolated in step C.
[0085] In one embodiment, only the expression construct is introduced into the destination microorganism. In another embodiment, both the expression construct and the methylation construct are introduced into the destination microorganism.
[0086] Preferably, the recombinant microorganism produces 1-butanol and/or a precursor thereof as the main fermentation product.
[0087] In a tenth aspect, the invention provides a method of producing a recombinant microorganism that produces 1-butanol or a precursor thereof as the main fermentation product comprising: [0088] a. Introduction of (i) an expression construct and (ii) a methylation construct comprising a methyltransferase gene into a shuttle microorganism; [0089] b. expression of the methyltransferase gene; [0090] c. isolation of one or more constructs from the shuttle microorganism; and, [0091] d. introduction of at least the expression construct into a destination microorganism; wherein the expression construct comprises one or more genes encoding enzymes to be expressed in the destination microorganism.
[0092] In one embodiment, expression of the methyltransferase gene in step b. is constitutive. In another embodiment, expression of the methyltransferase gene in step b. is induced.
[0093] In one embodiment, both the methylation construct and the expression construct are isolated in step C. In another embodiment, the expression construct is isolated in step C.
[0094] In one embodiment, only the expression construct is introduced into the destination microorganism. In another embodiment, both the expression construct and the methylation construct are introduced into the destination microorganism.
[0095] Preferably, the expression construct is as defined in the fifth aspect.
[0096] Preferably, the methylation construct is as defined in the sixth aspect.
[0097] In an eleventh aspect, the invention provides a method of production of 1-butanol and/or a precursor thereof by microbial fermentation comprising fermenting a substrate using a recombinant microorganism.
[0098] Preferably, 1-butanol and/or a precursor thereof is the main fermentation product.
[0099] Preferably, the recombinant microorganism is as described in any one of the eighth to the tenth aspects.
[0100] Preferably, 1-butanol and/or a precursor thereof is produced in a yield of from approximately 0.075 grams per litre of fermentation broth (g/l) to approximately 20 g/l. In one embodiment, the yield is from approximately 0.15 g/l to approximately 1.54 g11. In other embodiments, the yield is approximately 10 g/l, approximately 5 g/l, or approximately 2 g/l. Preferably, the yield of 1-butanol is up to the limit at which butanol becomes toxic to the surrounding media.
[0101] Preferably, the substrate comprises CO. Preferably, the substrate 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.
[0102] 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.
[0103] 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.
[0104] Preferably, the precursor produced by the method of any of the preceding aspects is converted to 1-butanol in the presence of phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase, and butanol dehydrogenase.
[0105] Preferably, the microorganism produces phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase, and butanol dehydrogenase both before and after introduction of an exogenous nucleic acid.
[0106] Preferably, the precursor produced by the method of any of the preceding aspects is converted to 1-butanol in the presence of butyraldehyde dehydrogenase, butanol dehydrogenase and/or a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase.
[0107] Preferably, the microorganism produces butyraldehyde dehydrogenase, butanol dehydrogenase and/or a bifunctional butyraldehyde dehyrogenase/butanol deydrogenase before and after introduction of an exogenous nucleic acid.
[0108] In a twelfth aspect, the invention provides 1-butanol or a precursor thereof when produced by the method of the eleventh aspect.
[0109] In a thirteenth aspect, the invention provides a shuttle microorganism comprising a methylation construct as defined herein.
[0110] Preferably, the shuttle microorganism further comprises an expression construct as defined herein.
[0111] Preferably, the shuttle microorganism is E. coli or Bacillus subtillis.
[0112] Preferably, the methylation construct of any of the previous aspects comprises a lac promoter and the methyltransferase gene and is induced by Isopropyl-β-D-thio-galactoside (IPTG). Expression of the methyltransferase could also be controlled by other inducible promoter systems such as ara, tet, or T7.
[0113] In a fourteenth aspect, the invention provides a nucleic acid having a sequence chosen from the group consisting of SEQ_ID NOs 8 to 13.
[0114] In a fifteenth aspect, the invention provides a nucleic acid having a sequence chosen from the group consisting of SEQ_ID NOs 16 to 23.
[0115] In a sixteenth aspect, the invention provides a nucleic acid comprising at least the nucleic acid sequence of SEQ ID No. 7 or a functionally equivalent variant thereof, a nucleic acid construct or vector comprising same, and microorganisms comprising said nucleic acid or nucleic acid construct or vector.
[0116] In a seventeenth aspect, the invention provides a nucleic acid which encodes a methyltransferase according to SEQ_ID No 28.
[0117] In an eighteenth aspect, the invention provides a nucleic acid comprising a nucleic acid encoding a polypeptide having the amino acid sequence of a polypeptide chosen from the group listed in tables 7 to 10 herein after and functionally equivalent variants of any one or more thereof.
[0118] In a nineteenth aspect, the invention provides a nucleic acid comprising a nucleic acid chosen from the group listed in tables 7 to 10 herein after and functionally equivalent variants of any one or more thereof.
[0119] In a twentieth aspect, the invention provides constructs and microorganisms comprising a nucleic acid of the eighteenth or nineteenth aspects of the invention.
[0120] In a twenty first aspect, the invention provides a nucleic acid having a sequence chosen from the group consisting of SEQ_ID NOs 32 to 38 and 123 to 135.
[0121] In a twenty second aspect, the invention provides a polypeptide comprising the amino acid sequence of a polypeptide chosen from the group listed in tables 7 to 10 herein after and functionally equivalent variants of any one or more thereof.
[0122] 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
[0123] 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, in which:
[0124] FIG. 1 shows the butanol biosynthesis pathway from CO.
[0125] FIG. 2 shows an exemplary expression plasmid encoding genes involved in 1-butanol biosynthesis.
[0126] FIG. 3 shows sequencing results of pMTL85245-thlA-crt-hbd which demonstrate that the 1-butanol biosynthesis genes found on the expression plasmid were free of mutations.
[0127] FIGS. 4a, 4b and 4c show a nucleotide alignment of the C. autoethanogenum (CAU), C. ljungdahlii (CLJ), C. ragsdalei (CRA) and the designed methyltransferase (DMT) genes.
[0128] FIG. 4d shows an amino acid alignment of the methyltransferases from C. autoethanogenum (CAU1+2), C. ljungdahlii (CLJ), C. ragsdalei (CRA1+2) and the designed methyltransferase (DMT).
[0129] FIG. 5 shows an exemplary methylation plasmid of the invention
[0130] FIG. 6 shows an agarose gel electrophoresis image of isolated plasmid DNA. Lane 1, 6, 11, 16, 21 and 26 show 100 by Plus DNA Ladder. Lane 2-5 shows PCR with original methylated plasmid mix as template in the following order: ermB, ColE1, thlA, crt. Lane 7-10, 12-15, 17-20, 22-25 and 27-30 show PCR with isolated plasmids from 4 different clones as template, each in the following order ermB, ColE1, thlA, crt. Lane 32-35 shows plasmid prep from 4 different clones. Lane 36 shows plasmid prep from original C. autoethanogenum DSM23693.
[0131] FIG. 7 shows HPLC results showing 1-butanol production with C. autoethanogenum harboring butanol plasmid pMTL85245-thlA-crt-hbd.
[0132] FIG. 8 shows an analysis of expression of over 200 genes during a typical fermentation with Clostridium autoethanogenum at standard conditions using real-time PCR to identify appropriate promoter regions for the expression of heterologous genes.
[0133] FIG. 9 shows the sequence for SEQ_ID No 1, 2 and 3.
[0134] FIG. 10 shows the sequence for SEQ_ID No 4, 5 and 6.
[0135] FIG. 11 shows the sequence for promoter regions encoded by SEQ_ID No 7, 47, 48, 49 and 50.
[0136] FIG. 12 shows the sequence for SEQ_ID No 14
[0137] FIG. 13 shows the sequence for SEQ_ID No 15
[0138] FIG. 14 shows the sequence for SEQ_ID No 24 and 25
[0139] FIG. 15 shows the sequence for SEQ_ID No 26
[0140] FIG. 16 shows the sequence for SEQ_ID No 27
[0141] FIG. 17 shows the sequence for SEQ_ID No 28
[0142] FIG. 18 shows the sequence for SEQ_ID No 29
[0143] FIG. 19 shows the 16s rRNA gene of C. autoethanogenum (Y18178, GI:7271109)
[0144] FIGS. 20 and 21 show the sequence for SEQ_ID No 31
[0145] FIG. 22 shows Seq. ID 39: Nucleotide acid sequence of bifunctional butanol/butyraldehyde dehydrogenase of C. autoethanogenum
[0146] FIG. 23 shows Seq. ID 40: Nucleotide acid sequence of bifunctional butanol/butyraldehyde dehydrogenase of C. autoethanogenum
[0147] FIG. 24 shows Seq. ID 41: Nucleotide acid sequence of butyraldehyde dehydrogenase of C. autoethanogenum; and, Seq. ID 42: Amino acid sequence of butyraldehyde dehydrogenase of C. autoethanogenum
[0148] FIG. 25 shows Seq. ID 43: Nucleotide acid sequence of butyraldehyde dehydrogenase of C. autoethanogenum; and, Seq. ID 44: Amino acid sequence of butyraldehyde dehydrogenase of C. autoethanogenum
[0149] FIG. 26 shows Seq. ID 45: Nucleotide acid sequence of butyraldehyde dehydrogenase of C. autoethanogenum
[0150] FIG. 27 shows Seq. ID 46: Amino acid sequence of butyraldehyde dehydrogenase of C. autoethanogenum; and, Seq. ID 119: Nucleotide acid sequence of butanol dehydrogenase of C. autoethanogenum
[0151] FIG. 28 shows Seq. ID 120: Amino acid sequence of butanol dehydrogenase of C. autoethanogenum; and Seq. ID 121: Nucleotide acid sequence of butanol dehydrogenase of C. autoethanogenum.
[0152] FIG. 29 shows Seq. ID 122: Amino acid sequence of butanol dehydrogenase of C. autoethanogenum; and, Seq. ID 51: Nucleotide acid sequence of butanol dehydrogenase of C. autoethanogenum.
[0153] FIG. 30 shows Seq. ID 52: Amino acid sequence of butanol dehydrogenase of C. autoethanogenum; and, Seq. ID 53: Nucleotide acid sequence of butanol dehydrogenase of C. autoethanogenum
[0154] FIG. 31 shows Seq. ID 54: Amino acid sequence of butanol dehydrogenase of C. autoethanogenum; and, Seq. ID 55: Nucleotide acid sequence of butanol dehydrogenase of C. autoethanogenum
[0155] FIG. 32 shows Seq. ID 56: Amino acid sequence of butanol dehydrogenase of C. autoethanogenum; and, Seq. ID 57: Nucleotide acid sequence of butanol dehydrogenase of C. autoethanogenum.
[0156] FIG. 33 shows Seq. ID 58: Amino acid sequence of butanol dehydrogenase of C. autoethanogenum; and Seq. ID 59: Nucleotide sequence of phosphate acetyl/butyryl transferase from C. autoethanogenum; and Seq. ID 60: Amino acid sequence of phosphate acetyl/butyryl transferase from C. autoethanogenum.
[0157] FIG. 34 shows Seq. ID 61: Nucleotide sequence of acetate/butyrate kinase from C. autoethanogenum; and Seq. ID 62: Amino acid sequence of acetate/butyrate kinase from C. autoethanogenum.
[0158] FIG. 35 shows Seq. ID 63: Nucleotide sequence of aldehyde:ferredoxin oxidoreductase from C. autoethanogenum; and Seq. ID 64: Amino acid sequence of aldehyde:ferredoxin oxidoreductase from C. autoethanogenum.
[0159] FIG. 36 shows Seq. ID 65: Nucleotide sequence of aldehyde:ferredoxin oxidoreductase from C. autoethanogenum; and Seq. ID 66: Amino acid sequence of aldehyde:ferredoxin oxidoreductase from C. autoethanogenum.
[0160] FIG. 37 shows Seq. ID 67: Nucleotide acid sequence of bifunctional butanol/butyraldehyde dehydrogenase of C. ljungdahlii
[0161] FIG. 38 shows Seq. ID 68: Amino acid sequence of bifunctional butanol/butyraldehyde dehydrogenase of C. ljungdahlii
[0162] FIG. 39 shows Seq. ID 69: Nucleotide acid sequence of bifunctional butanol/butyraldehyde dehydrogenase of C. ljungdahlii
[0163] FIG. 40 shows Seq. ID 70: Amino acid sequence of bifunctional butanol/butyraldehyde dehydrogenase of C. ljungdahlii; and Seq. ID 71: Nucleotide acid sequence of butyraldehyde dehydrogenase of C. ljungdahlii.
[0164] FIG. 41 shows Seq. ID 72: Amino acid sequence of butyraldehyde dehydrogenase of C. ljungdahlii; and Seq. ID 73: Nucleotide acid sequence of butyraldehyde dehydrogenase of C. ljungdahlii; and Seq. ID 74: Amino acid sequence of butyraldehyde dehydrogenase of C. ljungdahlii.
[0165] FIG. 42 shows Seq. ID 75: Nucleotide acid sequence of butanol dehydrogenase of C. ljungdahlii; and Seq. ID 76: Amino acid sequence of butanol dehydrogenase of C. ljungdahlii; and Seq. ID 77: Nucleotide acid sequence of butanol dehydrogenase of C. ljungdahlii.
[0166] FIG. 43 shows Seq. ID 78: Amino acid sequence of butanol dehydrogenase of C. ljungdahlii; and Seq. ID 79: Nucleotide acid sequence of butanol dehydrogenase of C. ljungdahlii; and Seq. ID 80: Amino acid sequence of butanol dehydrogenase of C. ljungdahlii.
[0167] FIG. 44 shows Seq. ID 81: Nucleotide acid sequence of butanol dehydrogenase of C. ljungdahlii; and Seq. ID 82: Amino acid sequence of butanol dehydrogenase of C. ljungdahlii; and Seq. ID 83: Nucleotide acid sequence of butanol dehydrogenase of C. ljungdahlii.
[0168] FIG. 45 shows Seq. ID 84: Amino acid sequence of butanol dehydrogenase of C. ljungdahlii; and Seq. ID 85: Nucleotide sequence of phosphate acetyl/butyryl transferase from C. ljungdahlii; and Seq. ID 86: Amino acid sequence of phosphate acetyl/butyryl transferase from C. ljungdahlii; and Seq. ID 87: Nucleotide sequence of acetate/butyrate kinase from C. ljungdahlii.
[0169] FIG. 46 shows Seq. ID 88: Amino acid sequence of acetate/butyrate kinase from C. ljungdahlii; and Seq. ID 89: Nucleotide sequence of aldehyde:ferredoxin oxidoreductase from C. ljungdahlii; and Seq. ID 90: Amino acid sequence of aldehyde:ferredoxin oxidoreductase from C. ljungdahlii.
[0170] FIG. 47 shows Seq. ID 91: Nucleotide sequence of aldehyde:ferredoxin oxidoreductase from C. ljungdahlii; and Seq. ID 92: Amino acid sequence of aldehyde:ferredoxin oxidoreductase from C. ljungdahlii.
[0171] FIG. 48 shows Seq. ID 93: Nucleotide Acid sequence of bifunctional butanol/butyraldehyde dehydrogenase from C. ragsdalei
[0172] FIG. 49 shows Seq. ID 94: Amino Acid sequence of bifunctional butanol/butyraldehyde dehydrogenase from C. ragsdalei
[0173] FIG. 50 shows Seq. ID 95: Nucleotide Acid sequence of bifunctional butanol/butyraldehyde dehydrogenase from C. ragsdalei.
[0174] FIG. 51 shows Seq. ID 96: Amino Acid sequence of bifunctional butanol/butyraldehyde dehydrogenase from C. ragsdalei; and Seq. ID 97: Nucleotide Acid sequence of butyraldehyde dehydrogenase from C. ragsdalei.
[0175] FIG. 52 shows Seq. ID 98: Amino Acid sequence of butyraldehyde dehydrogenase from C. ragsdalei; Seq. ID 99: Nucleotide Acid sequence of butyraldehyde dehydrogenase from C. ragsdalei; and Seq. ID 100: Amino Acid sequence of butyraldehyde dehydrogenase from C. ragsdalei.
[0176] FIG. 53 shows Seq. ID 101: Nucleotide Acid sequence of butanol dehydrogenase from C. ragsdalei; and Seq. ID 102: Amino Acid sequence of butanol dehydrogenase from C. ragsdalei; and Seq. ID 103: Nucleotide Acid sequence of butanol dehydrogenase from C. ragsdalei.
[0177] FIG. 54 shows Seq. ID 104: Amino Acid sequence of butanol dehydrogenase from C. ragsdalei; and Seq. ID 105: Nucleotide Acid sequence of butanol dehydrogenase from C. ragsdalei; and Seq. ID 106: Amino Acid sequence of butanol dehydrogenase from C. ragsdalei:
[0178] FIG. 55 shows Seq. ID 107: Nucleotide Acid sequence of butanol dehydrogenase from C. ragsdalei; and Seq. ID 108: Amino Acid sequence of butanol dehydrogenase from C. ragsdalei; and Seq. ID 109: Nucleotide Acid sequence of butanol dehydrogenase from C. ragsdalei.
[0179] FIG. 56 shows Seq. ID 110: Amino Acid sequence of butanol dehydrogenase from C. ragsdalei; and Seq. ID 111: Nucleotide sequence of phosphate acetyl/butyryl transferase from C. ragsdalei; and Seq. ID 112: Amino acid sequence of phosphate acetyl/butyryl transferase from C. ragsdalei; and Seq. ID 113: Nucleotide sequence of acetate/butyrate kinase from C. ragsdalei.
[0180] FIG. 57 shows Seq. ID 114: Amino acid sequence of acetate/butyrate kinase from C. ragsdalei; and Seq. ID 115: Nucleotide sequence of aldehyde:ferredoxin oxidoreductase from C. ragsdalei; and Seq. ID 116: Amino acid sequence of aldehyde:ferredoxin oxidoreductase from C. ragsdalei.
[0181] FIG. 58 shows Seq. ID 117: Nucleotide sequence of aldehyde:ferredoxin oxidoreductase from C. ragsdalei; and Seq. ID 118: Amino acid sequence of aldehyde:ferredoxin oxidoreductase from C. ragsdalei.
DETAILED DESCRIPTION OF THE INVENTION
[0182] 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.
[0183] Among others, the closely related microorganisms C. autoethanogenum, C. ljungdahlii, and C. ragsdalei are known to be useful for production of ethanol as biofuel from carbon monoxide. In order to produce 1-butanol as a biofuel from a gaseous substrate, a universal transformation system for these organisms has been developed and production of 1-butanol as the main fermentation product from CO has been demonstrated.
[0184] The inventors have found that when particular genes encoding proteins in the 1-butanol biosynthesis pathway (FIG. 1) were introduced into acetogenic microorganisms, such microorganisms were able to use a gaseous substrate to produce 1-butanol or a precursor thereof as the main fermentation product. Although some unmodified microorganisms are known to produce 1-butanol, the yield of 1-butanol from CO produced by such unmodified microorganisms is very low. As a result, their utility for production of biofuels from gaseous substrates is extremely limited due to their low efficiency and a subsequent lack of commercial viability. Clostridium autoethanogenum naturally produces ethanol, acetate, 2,3-butandiol and lactic acid but is not known to produce 1-butanol.
[0185] As shown in FIG. 1, the Wood-Ljungdahl pathway converts CO to acetyl-CoA. This compound may be further converted to 1-butanol in acetogenic microorganisms by the action of the enzymes thiolase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase/crotonyl-CoA hydratase, butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase and butanol dehydrogenase. In a particular embodiment of the invention, the microorganism expresses the first four enzymes which may be encoded by the nucleic acid SEQ_ID Nos 1 to 4 or functionally equivalent variants thereof. The present invention provides a microorganism that facilitates the conversion of acetyl-CoA to 1-butanol by the action of enzymes encoded by recombinant nucleic acids as well as naturally occurring enzymes. The invention also provides for the use of microorganisms expressing other recombinant nucleic acid sequences which encode enzymes at other stages in the Wood-Ljungdahl or butanol biosynthesis pathways. The inventors have also identified a number of novel enzymes and nucleic acids.
[0186] Since there is no natural competence (uptake of extracellular DNA from the cell's environment) known in Clostridia and electrotransformation or conjugation are the only methods available for transformation. These issues present significant difficulties in effectively transforming Clostridium species. Additionally, the restriction/methylation systems found in Clostridia protect against foreign and phage DNA and result in their genetic transformation being particularly troublesome. Transformation of several Clostridium strains (C. acetobutylicum ATCC824, C. cellulolyticum ATCC35319, C. botulinum ATCC25765, and C. difficile CD3 and CD6) was shown to be only possible if DNA is methylated in vivo in E. coli or methylated in vitro in a specific pattern prior to transformation (Mermelstein et al, 1993; Herbert et al, 2003; Jennert et al, 2000; Davis et al, 2000). However, the determination of the correct methylation pattern is often not possible due to unspecific exonucleases, etc. Additionally, many Clostridium species also possess restriction systems which digest DNA that is methylated at a specific ("wrong") position.
[0187] The abovementioned major hurdles have been overcome by the inventors in developing the recombinant microorganisms of the present invention. A novel methylation system comprising a novel methyltransferase gene was developed to circumvent the naturally occurring restriction barriers present in native acetogenic microorganisms. Accordingly, the methylation method and methyltransferase gene of the present invention may be applied to a number of compatible microorganisms that have restriction barriers preventing effective introduction and expression of desirable recombinant nucleic acids in microorganisms.
DEFINITIONS
[0188] As referred to herein, "precursors of 1-butanol" include butyryl CoA, butyryl-phosphate, butyrate, and butyraldehyde.
[0189] As referred to herein, a "fermentation broth" is a culture medium comprising at least a nutrient media and bacterial cells.
[0190] As referred to herein, a "shuttle microorganism" is a microorganism in which a methyltransferase enzyme is expressed and is distinct from the destination microorganism.
[0191] As referred to herein, a "destination microorganism" is a microorganism in which the genes included on the expression construct are expressed and is distinct from the shuttle microorganism.
[0192] As referred to herein, the term "main fermentation product" is intended to mean the one fermentation product which is produced in the highest concentration and/or yield.
[0193] 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 volume of desired product (such as alcohols) produced per volume of substrate (such as sugar) 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.
[0194] 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.
[0195] 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.
[0196] 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.
[0197] 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.
[0198] 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.
[0199] 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.
[0200] 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. As is described herein after, 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.
[0201] "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, 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)). 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.
[0202] 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 bacteria capable of butyric acid or butanol fermentation 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, Clostridium tetani, Clostridium pasteurianum, Clostridium kluyveri, Clostridium cellulovorans, Clostridium perfringens, Clostridium botulinum, Clostridium butyricum strain DSM10702, Clostridium tyrobutyricum strain ATCC 25755, Anaerococcus prevotii DSM 20548, Thermoanaerobacter tengcongensis, Brachyspira pilosicoli, Bacillus megaterium, Streptococcus pyogenes and Clostridium 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. In a particular embodiment, the functionally equivalent variant of the thiolase gene as defined herein may be the atoAB gene in E. coli (NC--000913.2; atoA=GeneID: 946719; atoB=GeneID: 946727). Functionally equivalent variants of the eftAB gene as defined herein may be found in Tsai and Saier (1995).
[0203] 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 Ito 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.
[0204] "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.
[0205] 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 outlined in Inui et al (2008) may be used to assess enzyme activity.
[0206] "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 protein as compared to the expression level of the protein of a parental microorganism under the same conditions. It should not be taken to mean that the protein is expressed at any particular level.
[0207] 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 the subject of the present invention. Accordingly, the recombinant microorganisms of the invention have been modified to express or over-express one or more enzymes that were not expressed or over-expressed in the parental microorganism.
[0208] 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, among other elements, sites and markers. 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).
[0209] It should be appreciated that nucleic acids of the invention may be in any appropriate form, including RNA, DNA, or cDNA, including double-stranded and single-stranded nucleic acids.
[0210] In one aspect the invention provides genetically modified microorganisms capable of using CO to produce 1-butanol and/or a precursor thereof as the main fermentation product. The microorganism is preferably an acetogenic recombinant microorganism which produces 1-butanol and/or a precursor thereof as the main fermentation product. In one particular embodiment, the acetogenic recombinant microorganism is capable of producing 1-butanol or a precursor thereof by fermentation from a substrate comprising CO at a concentration of greater than approximately 1 mM or 0.075 g/l of butanol per litre of fermentation broth.
[0211] In one particular embodiment, the microorganism comprises one or more exogenous nucleic acid adapted to express or over-express one or more enzymes in the butanol biosynthesis pathway. In one embodiment, the microorganism is adapted to express one or more enzyme in the butanol biosynthesis pathway which is not naturally present in the parental microorganism from which it is derived, or to over-express one or more enzyme in the butanol biosynthesis pathway which are naturally present in the parental microorganism.
[0212] 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.
[0213] In certain embodiments, the parental microorganism may be transformed to provide a combination of increased or over-expression of one or more genes native to the parental microorganism and introduction of one or more genes not native to the parental microorganism.
[0214] Preferably, the microorganism comprises one or more exogenous nucleic acids encoding one or more of the enzymes chosen from the group consisting: Thiolase; 3-hydroxybutyryl-CoA dehydrogenase; Crotonase/crotonyl-CoA hydratase; Butyryl-CoA dehydrogenase; Electron Transfer Flavoprotein A; and, Electron Transfer Flavoprotein B. In one embodiment, the one or more nucleic acids encoding the one or more enzymes is chosen from the nucleic acids SEQ ID NO. 1 to SEQ ID NO. 6 or functionally equivalent variants thereof.
[0215] In one embodiment the recombinant microorganism is adapted to express one or more of the genes which encode the enzymes thiolase (IUBMB enzyme nomenclature EC:2.3.1.9) (thlA), 3-hydroxybutyryl-CoA dehydrogenase (EC:1.1.1.157) (hbd), crotonase/crotonyl-CoA hydratase (EC:1.1.1.157) (crt or cch) and/or butyryl-CoA dehydrogenase (EC4.2.1.55) (bcd). In one embodiment, the microorganism is adapted to express all of these enzymes. In a further embodiment, the genes correspond to one or more of the nucleic acid sequences selected from SEQ_ID Nos 1 to 4 or functionally equivalent variants thereof. The recombinant microorganism of the invention may also contain two electron transferring proteins. In one embodiment, the electron transferring proteins are electron transferring flavoproteins (EC1.3.99.2) (etfAB) encoded by SEQ_ID Nos 5 and 6, or functionally equivalent variants thereof. The use of these electron-transferring flavoproteins enhances the efficiency of the microorganism in producing 1-butanol. The flavoproteins provide a stable complex that is required for the activity of Bcd.
[0216] In one particular embodiment, the microorganism comprises one or more exogenous nucleic acids encoding each of Thiolase, 3-hydroxybutyryl-CoA dehydrogenase, Crotonase, Butyryl-CoA dehydrogenase, Electron Transfer Flavoprotein A and Electron Transfer Flavoprotein B.
[0217] In one embodiment, the microorganism comprises a plasmid encoding one or more of, or preferably each of, Thiolase, 3-hydroxybutyryl-CoA dehydrogenase, Crotonase, Butyryl-CoA dehydrogenase, Electron Transfer Flavoprotein A and Electron Transfer Flavoprotein B.
[0218] In one embodiment, the microorganism alternatively or further comprises exogenous nucleic acids adapted to express one or more of the enzymes chosen from the group consisting of: Phosphotransbutyrylase; butyrate kinase; ferredoxin dependent aldehyde oxidoreductase (or in other words aledhyde:ferredoxin oxidoreductase); butyraldehyde dehydrogenase; butanol dehydrogenase; a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase.
[0219] In one embodiment, the microorganism comprises exogenous nucleic acids adapted to express one or more of butyraldehyde dehydrogenase, butanol dehydrogenase and a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase. Preferably, the microorganism comprises one or more exogenous nucleic acids encoding one or more of butyraldehyde dehydrogenase, butanol dehydrogenase and a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase.
[0220] In one embodiment, the microorganism comprises exogenous nucleic acids adapted to express one or more of Phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase, and butanol dehydrogenase. Preferably, the microorganism comprises one or more exogenous nucleic acids encoding one or more of Phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase, and butanol dehydrogenase. In particular embodiments, the microorganism comprises exogenous nucleic acids adapted to express each of Phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase, and butanol dehydrogenase.
[0221] In one embodiment, the microorganism comprises one or more nucleic acid adapted to express at least two of the enzymes in the 1-butanol biosynthesis pathway, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 of the enzymes.
[0222] In one embodiment, the microorganism further comprises an exogenous phosphotransacetylase/acetate kinase promoter, although other promoters may be used. Preferably, the promoter corresponds to SEQ_ID No. 7 or a functionally equivalent variant thereof. Preferably, the promoter is contained on a construct encoding one or more of the enzymes referred to herein before.
[0223] Preferably, the parental microorganism is selected from the group of acetogenic bacteria. In certain embodiments the microorganism is selected from the group comprising Clostridium autoethanogenum, Clostridium ljungdahlii Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatalogenes, Clostridium aceticum, Clostridium formicoaceticum, Butyribacterium limosum, Acetobacterium woodii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorella thermautotrophica, Oxobacter pfennigii, and Thermoanaerobacter kiuvi.
[0224] 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 LB S1561 (DSM23693), C. ljungdahlii PETCT (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. 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. 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. 2002, U.S. Pat. No. 6,368,819], C. ragsdalei P11T (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 J A, Saxena J, Do Y, Patel M, Fishein S, Datta R, Tobey R: Clostridium coskatii, sp. nov., an Anaerobic Bacterium that Produces Ethanol from Synthesis Gas. Poster SIM Annual Meeting and Exhibition, San Francisco, 2010], 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].
[0225] All species of this cluster have a similar morphology and size (logarithmic growing cells are between 0.5-0.7×3-5 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.
[0226] In one embodiment, the microorganism produces phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase, and butanol dehydrogenase both before and after introduction of an exogenous nucleic acid.
[0227] In one embodiment, the microorganism produces butyraldehyde dehydrogenase and/or butanol dehydrogenase both before and after introduction of an exogenous nucleic acid.
[0228] In one particular embodiment, the microorganism is Clostridium autoethanogenum DSM23693.
[0229] In one embodiment, the recombinant microorganism of the invention has the defining characteristics of the microorganism deposited at the DSMZ (Deutsche Sammlung fur Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany) under the accession number DSM24138.
[0230] 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 tranformation 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.
[0231] 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, conjugation, or chemical and natural competence. Suitable transformation techniques are described for example in Sambrook et al, 1989.
[0232] 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.
[0233] In another aspect, the invention provides a method of producing a recombinant microorganism comprising the following steps:
a. introduction into a shuttle microorganism of (i) an expression construct and (ii) a methylation construct comprising a methyltransferase gene; b. expression of the methyltransferase gene; c. isolation of one or more constructs from the shuttle microorganism; and, d. introduction of the one or more constructs into a destination microorganism; wherein the expression construct comprises one or more genes encoding enzymes to be expressed in the destination organism.
[0234] In one embodiment, the methyltransferase gene of step B is expressed constitutively. In another embodiment, expression of the methyltransferase gene of step B is induced.
[0235] 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. In a particular embodiment, the shuttle microorganism is a restriction negative E. coli or Bacillus subtillis.
[0236] Once the expression construct and the methylation construct are introduced into the shuttle microorganism, the methyltransferase gene present on the methylation construct is expressed. In one embodiment, where expression must be induced, induction may be by any suitable promoter system although in one particular embodiment of the invention, the methylation construct comprises an inducible lac promoter (preferably encoded by SEQ_ID NO 28) 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 an alternative embodiment of the invention, the methylation construct promoter is a constitutive promoter.
[0237] In one embodiment the expression construct promoter is a constitutive promoter that is preferably highly active under appropriate fermentation conditions. However, an inducible promoter could be used. In preferred embodiments, the expression construct promoter is selected from the group comprising phosphotransacetylase/acetate kinase operon promoter, pyruvate:ferredoxin oxidoreductase (SEQ_ID No. 48), the Wood-Ljungdahl gene cluster (SEQ_ID No 47), Rnf operon (SEQ_ID No 49) or the ATP synthase operon ((SEQ_ID No 50). Preferably, the phosphotransacetylase/acetate kinase operon promoter corresponds to SEQ_ID No. 7 or a functionally equivalent variant thereof FIG. 8 shows that expression of genes operably linked to these promoters have a high level of expression in Clostridium autoethanogenum under standard conditions.
[0238] In a particular embodiment, the methylation construct has an origin of replication specific to the identity of the shuttle microorganism so that any genes present on the methylation construct are expressed in the shuttle microorganism. Preferably, the expression construct has an origin of replication specific to the identity of the destination microorganism so that any genes present on the expression construct are expressed in the destination microorganism.
[0239] Expression of the methyltransferase enzyme results in methylation of the genes present on the expression construct. The expression construct 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.
[0240] In one particular embodiment, both constructs are concurrently isolated. The expression construct 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 is methylated, the nucleic acid sequences present on the expression construct are able to be incorporated into the destination microorganism and successfully expressed.
[0241] In a further embodiment, the invention provides a method of producing a recombinant microorganism comprising: [0242] a. methylation of an expression construct in vitro by a methyltransferase, preferably according to SEQ_ID No 28 or a functionally equivalent variant thereof; and, [0243] b. introduction of an expression construct, preferably according to the fifth aspect, into a destination microorganism; wherein the expression construct comprises one or more genes encoding enzymes to be expressed in the destination microorganism.
[0244] It is envisaged that a methyltransferase gene of the invention, preferably according to SEQ_ID No 27 or a functionally equivalent variant thereof, may be introduced into a shuttle microorganism and over-expressed. The resulting methyltransferase enzyme may be collected using known methods and used in vitro to methylate an expression construct, preferably, the expression construct is as defined in the fifth aspect. The expression construct may then be introduced into the destination microorganism for expression. Preferably, the recombinant microorganism produces 1-butanol and/or a precursor thereof as the main fermentation product.
[0245] In a further embodiment, the invention provides a method of producing a recombinant microorganism comprising: [0246] a. introduction into the genome of a shuttle microorganism of a methyltransferase gene, preferably according to SEQ_ID No 27 or a functionally equivalent variant thereof; [0247] b. introduction of an expression construct into the shuttle microorganism; [0248] c. isolation of one or more constructs from the shuttle microorganism; and, [0249] d. introduction of at least the expression construct into a destination microorganism; [0250] wherein the expression construct comprises one or more genes encoding enzymes to be expressed in the destination microorganism.
[0251] Standard methods are used for the introduction of a methyltransferase gene, preferably according to SEQ_ID No 27, into the genome of the shuttle microorganism. The methyltransferase may be constitutively expressed by the microorganism and result in the production of a methyltransferase enzyme, preferably according to SEQ_ID No 28 or a functionally equivalent variant thereof. An expression construct is methylated, isolated and introduced into the destination microorganism which preferably, produces 1-butanol and/or a precursor thereof as the main fermentation product.
[0252] The invention also includes microorganisms comprising a recombinant methyltransferase gene or methylation construct as herein described.
[0253] The present invention also provides a hybrid methyltransferase gene (SEQ_ID NO 28) developed following analysis of methyltransferase nucleic acid sequences and restriction barrier systems from C. autoethanogenum, C. ljungdahlii, and C. ragsdalei.
[0254] The methyltransferase gene is expressed in a shuttle microorganism which results in the production of a methyltransferase enzyme which methylates the sequence of the expression construct. The methyltransferase gene may be present on a construct or integrated into the genome of the shuttle microorganism. The hybrid methyltransferase gene is codon optimised for E. coli and may be incorporated into a methylation construct (FIG. 5). The methyltransferase gene may be codon optimised for use in another species of microorganism where appropriate, for example Bacillus subtillus. Methods for codon optimisation are standard and would be known to one of skill in the art (Carbone et al, 2003). Also incorporated within the scope of the invention are methyltransferase genes that have at least 70%, preferably 75%, preferably 80%, preferably 85%, preferably 90%, preferably 95% or greater nucleic acid sequence identity to SEQ_ID NO 28 and express a polypeptide which is able to methylate DNA.
[0255] It will be appreciated by one of skill in the art that the methylation method and methyltransferase gene will have utility across a range of microorganisms. In one embodiment, the destination microorganism is selected from the group comprising Clostridium autoethanogenum, Clostridium ljungdahlii and Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatalogenes, Clostridium aceticum, Clostridium formicoaceticum, Butyribacterium limosum, Acetobacterium woodii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorella thermautotrophica, Oxobacter pfennigii, and Thermoanaerobacter kiuvi. In one particular embodiment, the destination microorganism is selected from the group consisting Clostridium autoethanogenum, Clostridium ljungdahlii and Clostridium ragsdalei. In one particular embodiment the destination microorganism is Clostridium autoethanogenum DSM23693.
[0256] The invention also provides various nucleic acids or nucleic acid constructs as outlined in aspects 4, 5, 14, 15, 16, 18, 19 and 21 of the invention herein before described.
[0257] In another embodiment of the invention, there is an expression construct comprising one or more nucleic acids encoding one or more enzymes chosen from Thiolase, 3-hydroxybutyryl-CoA dehydrogenase, Crotonase, Butyryl-CoA dehydrogenase and an electron transfer protein or a functionally equivalent variant thereof. Preferably, the electron transfer protein is Electron Transfer Flavoprotein A or Electron Transfer Flavoprotein B. In a particular embodiment, both Electron Transfer Flavoprotein A and Electron Transfer Flavoprotein B are included on the expression construct.
[0258] Exemplary sequence information for each gene and equivalent enzyme is provided on GenBank as detailed in Table 1 herein after. Skilled persons will readily appreciate alternative genes and enzymes which may be used. In one embodiment, the enzymes are encoded by the nucleic acid SEQ_ID No 1 to 6 which may be present in any order on the construct or in the order shown in FIG. 2. SEQ_ID Nos 8 to 13 and SEQ_ID Nos 16 to 23 are novel sequences used to clone and sequence the genes referred to in the immediately preceding paragraph.
[0259] In order to obtain 1-butanol from a precursor the activity of one or more of butyraldehyde dehydrogenase (EC1.2.1.10), alcohol dehydrogenase (EC 1.1.1.1), phosphotransbutyrylase (EC 2.3.1.19), butyrate kinase (EC 2.7.2.7), aldehyde:ferredoxin oxidoreductase (EC1.2.7.5) and alcohol dehydrogenase (EC 1.1.1.1) may be required. The alcohol dehydrogenase of the invention is a butanol dehydrogenase. In certain embodiments, butyraldehyde dehydrogenase (EC1.2.1.10) and alcohol dehydrogenase (EC 1.1.1.1), or phosphotransbutyrylase (EC 2.3.1.19), butyrate kinase (EC 2.7.2.7), aldehyde:ferredoxin oxidoreductase (EC1.2.7.5) and alcohol dehydrogenase (EC 1.1.1.1), or a combination of both sets of enzymes is required. In one embodiment, the butyraldehyde dehydrogenase and butanol dehydrogenase activity is supplied by a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase. These various enzymes are shown in the butanol biosynthesis pathway depicted in FIG. 1. In some microorganisms butyraldehyde dehydrogenase, butanol dehydrogenase, phosphotransbutyrylase, butyrate kinase, and/or aldehyde:ferredoxin oxidoreductase are naturally expressed by the microorganism and therefore catalyse the conversion of butyryl-CoA to 1-butanol.
[0260] Accordingly, in one embodiment, the expression construct comprises nucleic acids encoding one or more of phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase, butyraldehyde dehydrogenase, butanol dehydrogenase, and a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase in addition to or in the alternative to one or more of Thiolase, 3-hydroxybutyryl-CoA dehydrogenase, Crotonase, Butyryl-CoA dehydrogenase and an electron transfer protein.
[0261] Examples of appropriate enzymes and amino acid and nucleic acid sequence information include, but are not limited to: butyraldehyde dehydrogenase, such as Ald from C. beijerinckii (ABR35947, GI:149905114), C. saccharobutylicum (CAQ57983, GI:189310620), or Clostridium saccharoperbutylacetonium (AAP42563, GI:31075383); butanol dehydrogenase, such as BdhB from C. acetobutylicum (NP--349891, GI:15896542); bifunctional butyraldehyde/butanol dehydrogenase enzyme, such as AdhE1 from C. acetobutylicum (NP--149325, GI:15004865) or AdhE2 from C. acetobutylicum (NP--149199, GI:15004739), C. beijerinckii. YP--001307449, GI:150015195); a phosphotransbutyrylase such as Ptb from C. acetobutylicum (NP--348368); butyrate kinase such as Buk from C. acetobutylicum (AAK81015.1); aldehyde:ferredoxin oxidoreductase AOR from C. acetobutylicum (NP--348637). Persons of ordinary skill in the art to which the invention relates may readily appreciate alternative examples of appropriate enzymes of use in the invention. The inventors have also identified a number of novel enzymes and genes which may be used in the invention, the details of which are provided herein after in the Examples section (in particular see tables 7 to 10). The invention also encompasses functionally equivalent variants of these enzymes and genes and their use in methods of the invention.
[0262] The inclusion of one or more of these genes may help avoid co-production of butyrate completely, increasing the efficiency of 1-butanol production. The invention also provides recombinant microorganisms comprising one or more nucleic acids adapted to express or increase expression of one or more of these enzymes.
[0263] In one embodiment, the nucleic acid(s) encode an enzyme chosen from the group of enzymes listed in tables 7 to 10 herein after and functional equivalents of any one or more thereof. In a particular embodiment, the nucleic acids are chosen from the group of nucleic acids listed in tables 7 to 10 herein after and functional equivalents of any one or more thereof.
[0264] In one embodiment, the expression construct encodes at least 2 enzymes in the butanol biosynthesis pathway, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 or at least 12 of the enzymes.
[0265] Preferably, the expression construct further comprises a suitable promoter as hereinbefore described. In one embodiment the promoter is a phosphotransacetylase/acetate kinase promoter. Preferably, the promoter corresponds to SEQ_ID No. 7 or a functionally equivalent variant thereof.
[0266] In a preferred embodiment, the expression construct comprises a nucleic acid encoding all of said enzymes. It will be appreciated by one of skill in the art that the expression construct may comprise nucleic acids encoding alternative electron transferring proteins.
[0267] The genes to be expressed in the recombinant microorganism may be assembled in the expression construct under the control of any appropriate promoter. In a particular embodiment, the promoter allows for substantially constitutive expression of the genes under its control. In a particular embodiment, the promoter is a phosphotransacetylase/acetate kinase (SEQ_ID NO 7) promoter. Other promoters which may find use in the invention include those from C. autoethanogenum (or C. ljungdahlii). The inventors have also identified a number of other promoters that are operably linked to genes that were highly expressed under typical fermentation conditions in Clostridium autoethanogenum (FIG. 8). Analysis of expression of over 200 genes during typical fermentation conditions using real-time PCR identified a number of appropriate promoters. These include pyruvate:ferredoxin oxidoreductase (SEQ_ID No. 48), the Wood-Ljungdahl gene cluster (SEQ_ID No 47), Rnf operon (SEQ_ID No 49) and the ATP synthase operon (SEQ_ID No 50). 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 presently preferred embodiments.
[0268] In one embodiment, the invention comprises a construct, recombinant microorganism or a nucleic acid sequence comprising nucleic acid SEQ_ID NOs 1 to 6 in the order shown in FIG. 2. However, it will be appreciated by one of skill in the art that the invention may still have the desired utility when the nucleic acid sequences are presented in any order and with one or more of the sequences absent.
[0269] In another embodiment, the invention comprises a nucleic acid comprising the promoter sequence represented by Seq ID No. 7, or a functionally equivalent variant thereof, construct comprising said promoter and recombinant microorganisms comprising same.
[0270] It will be appreciated that an expression construct 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 construct includes one promoter. In another embodiment, the construct includes two or more promoters. In one particular embodiment, the construct includes one promoter for with each gene to be expressed. In one embodiment, the construct includes one or more ribosomal binding sites, preferably a ribosomal binding site for each gene to be expressed.
[0271] It will be appreciated by those of skill in the art that the nucleic acid sequences and construct sequences defined 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.
[0272] When the expression construct of the invention is expressed in an acetogenic microorganism, the microorganism produces 1-butanol or a precursor thereof as the main fermentation product. It is envisaged that other genes which encode enzymes catalyzing different steps of the Wood-Ljungdahl or butanol biosynthesis pathways may also be incorporated in the expression construct in order to produce 1-butanol as the main fermentation product.
[0273] It is envisaged that the expression construct and the methylation construct as defined above may be combined to provide a composition of matter. Such a composition has particular utility in circumventing restriction barrier mechanisms in a wide variety of microorganisms but in a preferred embodiment, the recombinant microorganism produced by use of the composition produces 1-butanol or a precursor thereof as the main fermentation product.
[0274] Nucleic acids and nucleic acid constructs, including expression constructs 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 (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 shuttle vectors, pIMP1, pJIR750 and the plasmids exemplified in the Examples section herein after.
[0275] To the extent that the invention provides novel nucleic acids and nucleic acid vectors, it also provides nucleic acids which are capable of hybridising to at least a portion of a nucleic acid herein described, a nucleic acid complementary to any one thereof, or a functionally equivalent variant of any one thereof. Such nucleic acids will preferably hybridise to such nucleic acids, a nucleic acid complementary to any one thereof, or a functionally equivalent variant of any one thereof, under stringent hybridisation conditions. "Stringent hybridisation conditions" means that the nucleic acid is capable of hybridising to a target template under standard hybridisation conditions such as those described in Sambrook et al (1989). It will be appreciated that the minimal size of such nucleic acids is a size which is capable of forming a stable hybrid between a given nucleic acid and the complementary sequence to which it is designed to hybridise. Accordingly, the size is dependent on the nucleic acid composition and percent homology between the nucleic acid and its complementary sequence, as well as the hybridisation conditions which are utilised (for example, temperature and salt concentrations). In one embodiment, the nucleic acid is at least 10 nucleotides in length, at least 15 nucleotides in length, at least, 20 nucleotides in length, at least 25 nucleotides in length, or at least 30 nucleotides in length.
[0276] It should be appreciated that nucleic acids of the invention may be in any appropriate form, including RNA, DNA, or cDNA, including double-stranded and single-stranded nucleic acids.
[0277] 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.
[0278] The invention provides a method of production of 1-butanol and/or a precursor thereof by microbial fermentation comprising fermenting a gaseous substrate comprising CO using a recombinant microorganism. In certain embodiments, 1-butanol or a precursor thereof is co-produced with another fermentation product (for example, ethanol). In one embodiment, the 1-butanol or a precursor thereof is the main fermentation product. In one, embodiment, the recombinant microorganism is as herein before described.
[0279] In one embodiment, 1-butanol and/or a precursor thereof is produced in a yield of from approximately 0.075 grams per litre of fermentation broth (g/l) to approximately 20 g/l. In one embodiment, the yield is from approximately 0.15 g/l to approximately 1.54 g11. In other embodiments, the yield is approximately 10 g/l, approximately 5 g/l, or approximately 2 g/l. Preferably, the yield of 1-butanol is up to the limit at which butanol becomes toxic to the bacteria.
[0280] Preferably, the fermentation comprises the steps of anaerobically fermenting a substrate in a bioreactor to produce 1-butanol and/or a precursor thereof using recombinant microorganisms as described herein.
[0281] Where the precursor of 1-butanol is referred to herein it is envisaged that it may be optionally converted to 1-butanol in the presence of butyraldehyde dehydrogenase, butanol dehydrogenase, a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase, phosphotransbutyrylase, butyrate kinase, and/or ferredoxin dependent aldehyde oxidoreductase. Preferably, the microorganism produces one or more of these enzymes both before and after introduction of a recombinant nucleic acid.
[0282] 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 butanol 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.
[0283] It will be appreciated that for growth of the bacteria and CO-to-lbutanol fermentation to occur, in addition to the CO-containing substrate gas, a suitable liquid nutrient medium will need to be fed to the bioreactor. A nutrient medium will contain vitamins and minerals sufficient to permit growth of the micro-organism used. Anaerobic media suitable for fermentation to produce butanol 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.
[0284] The fermentation should desirably be carried out under appropriate conditions for the CO-to-butanol 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.
[0285] 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 butanol. 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-butanol 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.
[0286] The benefits of conducting a gas-to-ethanol fermentation at elevated pressures has been described elsewhere. 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.
[0287] 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.
[0288] 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.
[0289] Butanol, or a mixed alcohol stream containing butanol and one or more other alcohols, may be recovered from the fermentation broth by methods known in the art, such as fractional distillation or evaporation, pervaporation, and extractive fermentation, including for example, liquid-liquid extraction. By-products such as acids including butyrate may also be recovered from the fermentation broth using methods known in the art. For example, an adsorption system involving an activated charcoal filter or electrodialysis may be used. Alternatively, continuous gas stripping may also be used.
[0290] In certain preferred embodiments of the invention, butanol and by-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 butanol and optionally acid from the broth. Alcohols may conveniently be recovered for example by distillation, and acids 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 the 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.
[0291] 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.
[0292] In one embodiment of the invention, butanol is recovered from the fermentation reaction using extractive fermentation procedures in which butanol is recovered into an oil phase in the reactor. Skilled persons would readily appreciate techniques for achieving this
EXAMPLES
[0293] The invention will now be described in more detail with reference to the following non-limiting examples.
[0294] Genetic modifications were carried out using a plasmid containing a synthetic operon consisting of a strong, native C. autoethanogenum promoter controlling a thiolase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, and 2 electron transferring flavoproteins genes from C. acetobutylicum (FIG. 1-2). This plasmid was methylated in vivo using a novel methyltransferase and then transformed into C. autoethanogenum DSM23693. Production of 1-butanol as the main fermentation product was shown on different industrial gas streams (steel mill waste gas, syngas).
Construction of Expression Plasmid:
[0295] Standard Recombinant DNA and molecular cloning techniques were used in this invention and are described by Sambrook et al, 1989 and Ausubel et al, 1987. DNA sequences of butanol biosynthetic genes of Clostridium acetobutylicum ATCC824 used were obtained from NCBI (Table 1). The phosphotransacetylase/acetate kinase operon promoter of C. autoethanogenum DSM10061 were sequenced and used for expression of target genes (Table 1). RT-PCR experiments showed that this promoter is constitutively expressed at a high level (FIG. 8).
TABLE-US-00001 TABLE 1 Sources of 1-butanol pathway genes SEQ_ID Gene/Promoter GenBank Citation NO. Thiolase (thlA) NC_003030 Clostridium acetobutylicum ATCC 824, 1 complete genome; GI: 15896127; GeneID: 1119056 3-hydroxybutyryl-CoA dehydrogenase NC_003030 Clostridium acetobutylicum ATCC 824, 2 (hbd) complete genome; GI: 15895965; GeneID: 1118891 Crotonase (crt) NC_003030 Clostridium acetobutylicum ATCC 824, 3 complete genome; GI: 15895969; GeneID: 1118895 butyryl-CoA dehydrogenase (bcd) NC_003030 Clostridium acetobutylicum ATCC 824, 4 complete genome; GI: 15895968; GeneID: 1118894 Electron Transfer Flavoprotein A NC_003030 Clostridium acetobutylicum ATCC 824, 5 (etfA) complete genome; GI: 15895966; GeneID: 1118892 Electron Transfer Flavoprotein B NC_003030 Clostridium acetobutylicum ATCC 824, 6 (etfB) complete genome; GI: 15895967; GeneID: 1118893 phosphotransacetylase/acetate Clostridium autoethanogenum DSM10061 7 kinase promoter (P.sub.pta-ack)
[0296] Genomic DNA from Clostridium acetobutylicum ATCC824 and Clostridum autoethanogenum DSM10061 was isolated using a modified method by Bertram and Durre (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 (400,000 U) were 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). Then, 100 μl Proteinase K (0.5 U) were added and proteolysis took place for 1-3 h at 37° C. Finally, 600 μl of sodium perchlorate (5 M) were added, followed by a phenol-chloroform extraction and an isopropanol precipitation. DNA quantity and quality was inspected spectrophotometrically.
[0297] Butanol biosynthesis genes and the phosphotransacetylase/acetate kinase promoter were amplified by PCR with oligonucleotides in table 2 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 45 seconds), before a final extension step (72° C. for 10 minutes).
TABLE-US-00002 TABLE 2 Oligonucleotides for cloning Oligonucleotide SEQ_ID Target Name DNA Sequence (5' to 3') NO. Ppta-ack Ppta-ack-NotI-F GAGCGGCCGCAATATGATATTTATGTCC 8 Ppta-ack Ppta-ack-NdeI-R TTCCATATGTTTCATGTTCATTTCCTCC 9 ThlA ThlA-Cac-NdeI-F GTTCATATGAAAGAAGTTGTAATAGC 10 ThlA ThlA-Cac-EcoRI-R CAAGAATTCCTAGCACTTTTCTAGC 11 crt-bcd-etfB- Crt-Cac-KpnI-F AAGGTACCTTAGGAGGATTAGTCATGG 12 etfA-hbd operon crt-bcd-etfB- Crt-hbd-Cac- GAGGATCCGGATTCTTGTAAACTTATTTTG 13 etfA-hbd operon BamHI-R
[0298] The amplified 498 by promoter region of the phosphotransacetylase/acetate kinase operon (P.sub.pta-ack) was cloned into the E. coli--Clostridium shuttle vector pMTL 85141 (Seq. ID 14; FJ797651.1; Nigel Minton, University of Nottingham; Heap et al., 2009) using NotI and NdeI restriction sites and strain DH5α-T1R (Invitrogen). The created plasmid pMTL85145 and the 1,194 by PCR product of the thiolase gene were both cut with NdeI and EcoRI. A ligation was transformed into E. coli XL1-Blue MRF' Kan (Stratagene) resulting in plasmid pMTL85145-thlA. Subsequently, the amplified 4,764 by PCR fragment of the crt-bcd-etfB-etfA-hbd operon from C. acetobutylicum ATCC 824 was cloned into this vector using KpnI and BamHI and E. coli ABLE K (Stratagene), creating plasmid pMTL85145-thlA-crt-hbd. Finally, the antibiotic resistance cassette was changed from chloramphenicol to clarithromycin. Therefore, an ermB cassette was released from vector pMTL82254 (Seq. ID 15; FJ797646.1; Nigel Minton, University of Nottingham; Heap et al., 2009) using restriction enzymes Pmel and FseI and exchanged with the catP cassette of plasmid pMTL85145-thlA-crt-hbd. The insert of the resulting expression plasmid pMTL85245-thlA-crt-hbd (SEQ_ID No. 31 was completely sequenced using oligonucleotides given in table 3 and results confirmed that the butanol biosynthesis genes were free of mutations (FIG. 3).
TABLE-US-00003 TABLE 3 Oligonucleotides for sequencing Oligonucleotide SEQ_ID Name DNA Sequence (5' to 3') NO. seq-ThlA-hbd- CAGAGGATGTTAATGAAGTC 16 3562-4162 seq-ThlA-hbd- GCATCAGGATTAAATGACTG 17 4163-4763 seq-ThlA-hbd- ATAGCGAAGTACTTG 18 4764-5364 seq-ThlA-hbd- GATGCAATGACAGCTTTC 19 5365-5965 seq-ThlA-hbd- GGAACAAAAGGTATATCAGC 20 5966-6566 seq-ThlA-hbd- CGGAGCATTTGATAAAGAA 21 7168-7768 seq-ThlA-hbd- GCTGATTGTACATCACTTGA 22 7769-8369 seq-ThlA-hbd- CCAGAATTAATAGCTCAAGT 23 8370-8870
Methylation of DNA:
[0299] A hybrid methyltransferase gene fused to an inducible lac promoter was designed (Seq. ID 28), by alignment of methyltransferase genes from C. autoethanogenum (SEQ_ID No. 24), C. ljungdahlii (SEQ_ID No. 25), and C. ragsdalei (SEQ_ID No. 26) (FIGS. 4a, 4b and 4c). Expression of the methyltransferase gene resulted in production of a methyltransferase enzyme according to SEQ_ID No. 28. Methyltransferase amino acid sequence alignment data is shown in FIG. 4d. The hybrid methyltransferase gene (SEQ_ID No. 27) was chemically synthesized and cloned into vector pGS20 (Seq. ID 29; ATG:biosynthetics GmbH, Merzhausen, Germany) using EcoRI (FIG. 5). The resulting methylation plasmid pGS20-methyltransferase was double transformed with the expression plasmid pMTL85245-thlA-crt-hbd into the restriction negative E. coli XL1-Blue MRF' Kan (Stratagene). In vivo methylation was induced by addition of 1 mM IPTG, and methylated plasmids were isolated using the PureLink® HiPure Plasmid Maxiprep Kit (Invitrogen). The resulting methylated plasmid composition was used for transformation of C. autoethanogenum DSM23693.
Transformation:
[0300] During the complete transformation experiment, C. autoethanogenum DSM23693 was grown in PETC media (Tab. 4) with 10 g/l fructose and 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 at 37° C. using standard anaerobic techniques described by Hungate (1969) and Wolfe (1971).
TABLE-US-00004 TABLE 4 PETC media (ATCC media 1754; http://www.atcc.org/Attachments/2940.pdf) 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 Yeast Extract 1 g 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 Thiamine•HCl 5 mg Riboflavin 5 mg Nicotinic acid 5 mg Calcium D-(+)-pantothenate 5 mg Vitamin B12 0.1 mg p-Aminobenzoic acid 5 mg Thioctic acid 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
[0301] 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 mix and immediately pulsed using the Gene pulser Xcell electroporation system (Bio-Rad) with the following settings: 2.5 kV, 600 μl, 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 start 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 4 μg/μl Clarithromycin. After 4-5 days of inoculation with 30 psi steel mill gas at 37° C., 15-80 colonies per plate were clearly visible.
[0302] The colonies were used to inoculate 2 ml PETC media containing 4 μg/μl Clarithromycin. When growth occurred, the culture was upscaled into 5 ml and later 50 ml PETC media containing 4 μg/μ1 Clarithromycin and 30 psi steel mill gas as sole carbon source.
Conformation of the Successful Transformation:
[0303] To verify the DNA transfer, a plasmid mini prep was performed from 10 ml culture volume using the QIAprep Spin Miniprep Kit (Qiagen). Due to Clostridial exonuclease activity (Burchhardt and Durre, 1990), the isolated plasmid DNA from 4 analyzed clones were partly degraded and only resulted in a smear on an agarose gel, while a plasmid isolation from the original C. autoethanogenum DSM23693 strain didn't result in a signal at all (FIG. 6). However, the quality of the isolated plasmid DNA was sufficient to run a control PCR using 4 sets of primers, covering all relevant different regions of the plasmid (Table 5). The PCR was performed with illustra PuReTaq Ready-To-Go® PCR Beads (GE Healthcare) using a standard conditions (95° C. for 5 min; 32 cycles of 95° C. for 30 s, 50° C. for 30 s, and 72° C. for 1 min; 72° C. for 10 min). PCR of all 4 analyzed transformants resulted in the same signals as with the original methylated plasmid mix as template (FIG. 6). As a further control, 1 μl of each of the partly degraded isolated plasmids were re-transformed in E. coli XL1-Blue MRF' Kan (Stratagene), from where the plasmids could be isolated cleanly and verified by restriction digests.
[0304] To confirm the identity of the 4 clones, genomic DNA was isolated (see above) from 40 ml of each culture and a PCR was performed against the 16s rRNA gene (Tab. 5; Weisberg et al., 1991) using illustra PuReTaq Ready-To-Go® PCR Beads (GE Healthcare) and standard conditions (95° C. for 5 min; 32 cycles of 95° C. for 30 s, 50° C. for 30 s, and 72° C. for 1 min; 72° C. for 10 min). The respective PCR products were purified and sequenced. Sequences of all clones showed at least 99.9% identity against the 16s rRNA gene of C. autoethanogenum (Seq. ID 30; Y18178, GI:7271109).
[0305] A respective strain was deposited at DSMZ (Deutsche Sammlung fur Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany) under the accession number DSM24138 on 26 Oct. 2010.
TABLE-US-00005 TABLE 5 Oligonucleotides for PCR confirmation of plasmid and species Oligonucleotide Seq ID Target region Name DNA Sequence (5' to 3') No. 16s rRNA gene fD1 CCGAATTCGTCGACAACAGAGTTTGATCCTGGC 135 TCAG 16s rRNA gene rP2 CCGGGATCCAAGCTTACGGCTACCTTGTTACGA 32 CTT Antibiotic resistance ermB-F TTTGTAATTAAGAAGGAG 33 cassette (ermB) Antibiotic resistance ermB-R GTAGAATCCTTCTTCAAC 34 cassette (ermB) Insert 1 (thlA) ThlA-Cac-NdeI-F GTTCATATGAAAGAAGTTGTAATAGC 10 Insert 1 (thlA) ThlA-Cac-EcoRI-R CAAGAATTCCTAGCACTTTTCTAGC 11 Insert 2 (crt-bcd- Crt-conserved-F GCTGGAGCAGATAT 35 etfAB-hbd) Insert 2 (crt-bcd- Crt-conserved-R GCTGTCATTCCTTC 36 etfAB-hbd) Replication origin ColE1-F CGTCAGACCCCGTAGAAA 37 (ColE1) Replication origin ColE1-R CTCTCCTGTTCCGACCCT 38 (ColE1)
1-Butanol Production:
[0306] To demonstrate 1-butanol production from CO as sole energy and carbon source, PETC media without yeast extract and fructose were prepared and inoculated with the novel C. autoethanogenum strain harboring butanol plasmid pMTL85245-thlA-crt-hbd. Bottles were pressurized with 30 psi of a CO containing gas stream from two industrial sources, steel mill waste gas (collected from New Zealand Steel site in Glenbrook, NZ; composition: 44% CO, 32% N2, 22% CO2, 2% H2) and syngas (Range Fuels Inc., Broomfield, Colo.; composition: 29% CO, 45% H2, 13% CH4, 12% CO2, 1% N2). 1-Butanol production could be demonstrated on both gas mixes over several subculturing periods and co-production of butyrate was observed as well.
[0307] Analysis of metabolites were performed by HPLC using an Agilent 1100 Series HPLC system equipped with a RID operated at 35° C. (Refractive Index Detector) and an Alltech IOA-2000 Organic acid column (150×6.5 mm, particle size 5 μm) kept at 60° C. Slightly acidified water was used (0.005 M H2SO4) as mobile phase with a flow rate of 0.7 ml/min. To remove proteins and other cell residues, 400 μl samples were mixed with 100 μl of a 2% (w/v) 5-Sulfosalicylic acid and centrifuged at 14,000×g for 3 min to separate precipitated residues. 10 μl of the supernatant were then injected into the HPLC for analyses.
[0308] The highest 1-butanol production measured in two cultures was 1.54 g/l (25.66 mM) with 1-butanol as main fermentation end product (Table 6, FIG. 7). The production of the other metabolites was reduced compared to the original strain C. autoethanogenum DSM23693, which only produces ethanol, acetate, and 2,3-butandiol. Although the carbon flux was shifted towards 1-butanol production, the amount of total carbon incorporated into metabolic end products remain almost the same (Table 6). The slight increase of 20% is likely to be the result of an extra reducing equivalents offload by producing 1-butanol and butyrate compared to ethanol and respectively acetate. The production of 2,3-butandiol which usually acts as electron sink, is completely diminished.
TABLE-US-00006 TABLE 6 Metabolite production and carbon balance of C. autoethanogenum harboring butanol plasmid pMTL85245-thlA-crt-hbd compared to C. autoethanogenum DSM23693 Original C. autoethanogenum C. autoethanogenum DSM23693 + DSM23693 pMTL85245-thlA-crt-bcd M P Carbon Product Product Carbon Product Product Carbon Product [g/mol] [g/cm3] atoms [g/l] [mmol/l] [mmol/l] [g/l] [mmol/l] [mmol/l] Ethanol 46.08 0.789 2 1.02 28.06 56.11 0.37 10.18 20.35 Acetate 60.05 1.049 2 1.87 29.69 59.37 0.30 4.76 9.52 2,3-butandiol 90.12 0.987 4 0.18 2.02 8.09 0 0 0 1-butanol 74.12 0.810 4 0 0 0 1.54 25.66 102.63 Butyrate 88.11 0.960 4 0 0 0 0.31 3.67 14.67 Total 123.58 147.17
Conversion of Butyryl-CoA to Butanol:
[0309] The expression plasmid only contains the genes necessary for production of butyryl-CoA from acetyl-CoA. Butyryl-CoA can then be converted directly to butanol by action of a butyraldehyde dehydrogenase and butanol dehydrogenase (FIG. 1). A second possibility is that butyryl-CoA is converted to butyrate via a phosphotransbutyrylase and butyrate kinase (FIG. 1), in which case ATP is gained via substrate level phosphorylation (SLP). Since operation of the Wood-Ljungdahl pathway requires ATP, acetogenic cells rely on ATP from SLP, which is also reflected in the fact that every acetogenic bacteria known produces acetate (Drake et al., 2006). However, the recombinant cell can now also generate ATP via SLP also by producing butyrate. Butyrate can then be further reduced to butyraldehyde via a aldehyde:ferredoxinoxidoreductase (AOR) (FIG. 1). This reaction could be driven by reduced ferredoxin, provided by oxidation of CO via the carbon monoxide dehydrogenase (CO+Fdred->CO2+Fdox), the initial step in the Wood-Ljungdahl pathway. Butyraldehyde can then be converted to butanol via a butanol dehydrogenase (FIG. 1). Conversion of externally added butyrate to butanol by a culture of C. autoethanogenum has been demonstrated (WO2009/113878).
[0310] Respective genes/enzymes with butyraldehyde dehydrogenase, butanol dehydrogenase, phophotransbutyrylase, butyrate kinase, and aldehyde:ferredoxin oxidoreductase activity have been identified by the inventors in C. autoethanogenum, C. ljungdahlii, and C. ragsdalei (Tab. 7-10). Potential genes and enzymes were predicted by comparison with characterized genes and enzymes using BLAST (Altschul et al, 1990), COG (Tatusov et al, 2003), and TIGRFAM (Haft et al, 2002) databases. Motif scans were performed against PROSITE (Hulo et al., 2008) Pfam (Finn et al., 2010) databases. Genomes of C. autoethanogenum, C. ljungdahlii, and C. ragsdalei contain several genes encoding enzymes with alcohol and aldehyde dehydrogenase activity. As indicated in tables 7 to 10, some of these were found to have high homology of over 70% to characterized butyraldehyde and butanol dehydrogenases from C. acetobutylicum, C. beijerinckii, or C. saccharobutylicum, while others have at least in some 40% identity to these enzymes. All three genomes encode exactly one enzyme with Phosphate acetyl/butyryl transferase activity and one with Acetate/butyrate kinase activity. C. autoethanogenum, C. ljungdahlii, and C. ragsdalei each possess 2 aldehyde:ferredoxin oxidoreductase genes.
TABLE-US-00007 TABLE 7 Genes of C. autoethanogenum potentially conferring butyraldehyde and butanol dehydrogenase activity Sequence Description Identity (protein) to characterized enzymes Seq. ID 39-40 Bifunctional butanol/ bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. beijerinckii NCIMB butyraldehyde dehydrogenase 8052 (Identities = 644/861 (75%), Positives = 748/861 (87%), e-value = 0.0) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (Identities = 594/858 (70%), Positives = 730/858 (86%), e-value = 0.0) Seq. ID 41-42 Butyraldehyde dehydrogenase bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. beijerinckii NCIMB 8052 (Identities = 367/504 (73%), Positives = 437/504 (87%), e-value = 0.0) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (354/504 (71%), Positives = 440/504 (88%), e-value = 0.0) Seq. ID 43-44 Butyraldehyde dehydrogenase bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (Identities = 173/352 (50%), Positives = 236/352 (68%), e-value = 1e-91) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. beijerinckii NCIMB 8052 (Identities = 160/374 (43%), Positives = 234/374 (63%), e-value = 5e-87) bifunctional aldehyde/alcohol dehydrogenase AdhE1 from C. acetobutylicum ATCC824 (Identities = 158/366 (44%), Positives = 235/366 (65%), e-value = 5e-82) butyraldehyde dehydrogenase Ald from C. beijerinckii NCIMB8052 (Identities = 110/354 (32%), Positives = 184/354 (52%), e-value = 9e-44) butyraldehyde dehydrogenase from C. saccharoperbutylacetonicum (111/354 (32%), Positives = 182/354 (52%), e-value = 2e-44) Seq. ID 45-46 Butyraldehyde dehydrogenase bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. beijerinckii NCIMB 8052 (Identities = 188/477 (40%), Positives = 270/477 (57%), e-value = 9e-84) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (Identities = 164/428 (39%), Positives = 256/428 (60%), e-value = 1e-79) Seq. ID 119-120 Butanol dehydrogenase NADPH-dependet butanol dehydrogenase from C. saccharobutylicum (Identities = 285/388 (74%), Positives = 334/388 (87%), e-value = 7e-177) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (Identities = 163/396 (42%), Positives = 237/396 (60%), e-value = 4e-80) Seq. ID 121-122 Butanol dehydrogenase NADPH-dependet butanol dehydrogenase from C. saccharobutylicum (Identities = 271/388 (70%), Positives = 328/388 (85%), e-value = 3e-168) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (Identities = 169/403 (42%), Positives = 240/403 (60%), e-value = 3e-83) Seq. ID 51-52 Butanol dehydrogenase bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. beijerinckii NCIMB 8052 (246/315 (79%), Positives = 287/315 (92%), e-value = 1e-153) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (208/312 (67%), Positives = 260/312 (84%), e-value = 4e-128) Seq. ID 53-54 Butanol dehydrogenase NADPH-dependet butanol dehydrogenase from C. saccharobutylicum (Identities = 264/388 (69%), Positives = 326/388 (85%), e-value = 5e-163) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. beijerinckii NCIMB8052 (Identities = 169/410 (42%), Positives = 246/410 (60%), e-value = 5e-82) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (Identities = 162/402 (41%), Positives = 240/402 (60%), e-value = 2e-78) Seq. ID 55-56 Butanol dehydrogenase NADH-dependent butanol dehydrogenase BdhA from C. acetobutylicum ATCC824 (Identities = 161/388 (42%), Positives = 243/388 (63%), e-value = 7e-92) NADH-dependent butanol dehydrogenase BdhB from C. acetobutylicum ATCC824 (Identities = 155/389 (40%), Positives = 242/389 (63%), e-value = 4e-85) Seq. ID 57-58 Butanol dehydrogenase NADPH-dependet butanol dehydrogenase AdhE2 from C. saccharobutylicum (Identities = 156/385 (41%), Positives = 236/385 (62%), e-value = 1e-72) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (Identities = 154/412 (38%), Positives = 233/412 (57%), e-value = 8e-70) Seq. ID 59-60 Phosphate acetyl/butyryl phosphate butyryltransferase from C. acetobutylicum ATCC 824 (Identities = transferase 85/338 (26%), Positives = 146/338 (44%), e-value = 2e-12) Seq ID 61-62 Acetate/butyrate kinase butyrate kinase from C. acetobutylicum ATCC 824 (Identities = 49/175 (28%), Positives = 78/175 (45%), e-value 5e-08) Seq ID 63-64 Aldehyde: ferredoxin aldehyde: ferredoxin oxidoreductase from C. acetobutylicum ATCC 824 oxidoreductase (Identities = 183/618 (30%), Positives = 311/618 (51%), e-value = 6e-72) Seq ID 65-66 Aldehyde: ferredoxin aldehyde: ferredoxin oxidoreductase from C. acetobutylicum ATCC 824 oxidoreductase (Identities = 191/633 (31%), Positives = 308/633 (49%), e-value = 2e-70)
TABLE-US-00008 TABLE 8 Genes of C. ljungdahlii potentially conferring butyraldehyde and butanol dehydrogenase activity Sequence Description Identity to characterized enzymes Seq. ID 67-68 Bifunctional butanol/ bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. beijerinckii NCIMB butyraldehyde dehydrogenase 8052 (Identities = 644/862 (75%), Positives = 751/862 (88%), e-value = 0.0) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (Identities = 592/858 (69%), Positives = 729/858 (85%), e-value = 0.0) Seq. ID 69-70 Bifunctional butanol/ bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. beijerinckii NCIMB butyraldehyde dehydrogenase 8052 (Identities = 636/860 (74%), Positives = 752/860 (88%), e-value = 0.0) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (Identities = 585/858 (69%), Positives = 733/858 (86%), e-value = 0.0) Seq. ID 71-72 Butyraldehyde dehydrogenase bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (Identities = 209/429 (49%), Positives = 286/429 (67%), e-value = 4e-111) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. beijerinckii NCIMB 8052 (Identities = 196/467 (42%), Positives = 286/467 (62%), e-value = 1e-102) bifunctional aldehyde/alcohol dehydrogenase AdhE1 from C. acetobutylicum ATCC824 (Identities = 193/443 (44%), Positives = 283/443 (64%), e-value = 7e-100) butyraldehyde dehydrogenase Ald from C. beijerinckii NCIMB8052 (Identities = 125/409 (31%), Positives = 206/409 (51%), e-value = 3e-49) butyraldehyde dehydrogenase from C. saccharoperbutylacetonicum (124/409 (31%), Positives = 204/409 (50%), e-value = 2e-48) Seq. ID 73-74 Butyraldehyde dehydrogenase bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. beijerinckii NCIMB 8052 (Identities = 188/477 (40%), Positives = 270/477 (57%), e-value = 9e-84) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (Identities = 164/428 (39%), Positives = 256/428 (60%), e-value = 1e-79) Seq. ID 75-76 Butanol dehydrogenase NADPH-dependet butanol dehydrogenase from C. saccharobutylicum (Identities = 285/388 (74%), Positives = 335/388 (87%), e-value = 9e-177) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (Identities = 164/396 (42%), Positives = 238/396 (61%), e-value = 1e-80) Seq. ID 77-78 Butanol dehydrogenase NADPH-dependet butanol dehydrogenase from C. saccharobutylicum (Identities = 281/388 (73%), Positives = 327/388 (85%), e-value = 2e-173) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (Identities = 169/403 (42%), Positives = 240/403 (60%), e-value = 3e-83) Seq. ID 79-80 Butanol dehydrogenase NADPH-dependet butanol dehydrogenase from C. saccharobutylicum (Identities = 264/388 (69%), Positives = 326/388 (85%), e-value = 5e-163) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. beijerinckii NCIMB8052 (Identities = 169/410 (42%), Positives = 246/410 (60%), e-value = 4e-82) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (Identities = 162/402 (41%), Positives = 240/402 (60%), e-value = 2e-78) Seq. ID 81-82 Butanol dehydrogenase NADH-dependent butanol dehydrogenase BdhA from C. acetobutylicum ATCC824 (Identities = 161/388 (42%), Positives = 243/388 (63%), e-value = 7e-92) NADH-dependent butanol dehydrogenase BdhB from C. acetobutylicum ATCC824 (Identities = 155/389 (40%), Positives = 242/389 (63%), e-value = 4e-85) Seq. ID 83-84 Butanol dehydrogenase NADPH-dependet butanol dehydrogenase from C. saccharobutylicum (Identities = 150/389 (39%), Positives = 233/389 (60%), e-value = 7e-73) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (Identities = 154/412 (38%), Positives = 233/412 (57%), e-value = 8e-70) Seq. ID 85-86 Phosphate acetyl/butyryl phosphate butyryltransferase from C. acetobutylicum ATCC 824 (91/340 (27%), transferase Positives = 156/340 (46%), e-value = 1e-16) Seq ID 87-88 Acetate/butyrate kinase butyrate kinase from C. acetobutylicum ATCC 824 (49/162 (31%), Positives = 77/162 (48%), e-value 5e-08) Seq ID 89-90 Aldehyde: ferredoxin aldehyde: ferredoxin oxidoreductase from C. acetobutylicum ATCC 824 (188/631 oxidoreductase (30%), Positives = 318/631 (51%), e-value = 3e-11) Seq ID 91-92 Aldehyde: ferredoxin aldehyde: ferredoxin oxidoreductase from C. acetobutylicum ATCC 824 oxidoreductase (Identities = 191/633 (31%), Positives = 308/633 (49%), e-value = 2e-70)
TABLE-US-00009 TABLE 10 Genes of C. ragsdalei potentially conferring butyraldehyde and butanol dehydrogenase activity Sequence Description Identity to characterized enzymes Seq. ID 93-94 Bifunctional butanol/ bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. beijerinckii NCIMB butyraldehyde dehydrogenase 8052 (Identities = 645/861 (75%), Positives = 751/861 (88%), e-value = 0.0) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (Identities = 591/858 (69%), Positives = 731/858 (86%), e-value = 0.0) Seq. ID 95-96 Bifunctional butanol/ bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. beijerinckii NCIMB butyraldehyde dehydrogenase 8052 (Identities = 639/860 (75%), Positives = 752/860 (88%), e-value = 0.0) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (Identities = 591/858 (69%), Positives = 735/858 (86%), e-value = 0.0) Seq. ID 97-98 Butyraldehyde dehydrogenase bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (Identities = 214/457 (47%), Positives = 294/457 (65%), e-value = 5e-111) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. beijerinckii NCIMB 8052 (Identities = 200/457 (44%), Positives = 283/457 (62%), e-value = 1e-103) bifunctional aldehyde/alcohol dehydrogenase AdhE1 from C. acetobutylicum ATCC824 (Identities = 198/457 (44%), Positives = 289/457 (64%), e-value = 4e-101) butyraldehyde dehydrogenase Ald from C. beijerinckii NCIMB8052 (Identities = 125/409 (31%), Positives = 206/409 (51%), e-value = 3e-49) butyraldehyde dehydrogenase from C. saccharoperbutylacetonicum (Identities = 123/409 (31%), Positives = 205/409 (51%), e-value = 1e-48) Seq. ID 99-100 Butyraldehyde dehydrogenase bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. beijerinckii NCIMB 8052 (Identities = 188/477 (40%), Positives = 270/477 (57%), e-value = 9e-84) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (Identities = 164/428 (39%), Positives = 256/428 (60%), e-value = 1e-79) Seq. ID 101-102 Butanol dehydrogenase NADPH-dependet butanol dehydrogenase from C. saccharobutylicum (Identities = 285/388 (74%), Positives = 335/388 (87%), e-value = 9e-177) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (Identities = 164/396 (42%), Positives = 238/396 (61%), e-value = 1e-80) Seq. ID 103-104 Butanol dehydrogenase NADPH-dependet butanol dehydrogenase from C. saccharobutylicum (Identities = 281/388 (73%), Positives = 327/388 (85%), e-value = 2e-173) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (Identities = 169/403 (42%), Positives = 240/403 (60%), e-value = 3e-83) Seq. ID 105-106 Butanol dehydrogenase NADPH-dependet butanol dehydrogenase from C. saccharobutylicum (Identities = 264/388 (69%), Positives = 326/388 (85%), e-value = 5e-163) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. beijerinckii NCIMB8052 (Identities = 169/410 (42%), Positives = 246/410 (60%), e-value = 4e-82) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (Identities = 162/402 (41%), Positives = 240/402 (60%), e-value = 2e-78) Seq. ID 107-108 Butanol dehydrogenase NADH-dependent butanol dehydrogenase BdhA from C. acetobutylicum ATCC824 (Identities = 162/388 (42%), Positives = 243/388 (63%), e-value = 3e-92) NADH-dependent butanol dehydrogenase BdhB from C. acetobutylicum ATCC824 (Identities = 155/389 (40%), Positives = 242/389 (63%), e-value = 6e-85) Seq. ID 109-110 Butanol dehydrogenase NADPH-dependet butanol dehydrogenase from C. saccharobutylicum (Identities = 147/389 (38%), Positives = 227/389 (59%), e-value = 3e-71) bifunctional aldehyde/alcohol dehydrogenase AdhE2 from C. acetobutylicum ATCC824 (Identities = 155/412 (38%), Positives = 233/412 (57%), e-value = 2e-70) Seq. ID 111-112 Phosphate acetyl/butyryl phosphate butyryltransferase from C. acetobutylicum ATCC 824 87/325 (27%), transferase Positives = 148/325 (46%), e-value = 2e-16) Seq ID 113-114 Acetate/butyrate kinase butyrate kinase from C. acetobutylicum ATCC 824 (Identities = 49/162 (31%), Positives = 77/162 (48%), e-value 4e-11) Seq ID 115-116 Aldehyde: ferredoxin aldehyde: ferredoxin oxidoreductase from C. acetobutylicum ATCC 824 oxidoreductase (Identities = 187/633 (30%), Positives = 319/633 (51%), e-value = 3e-74) Seq ID 117-118 Aldehyde: ferredoxin aldehyde: ferredoxin oxidoreductase from C. acetobutylicum ATCC 824 oxidoreductase (Identities = 187/633 (30%), Positives = 302/633 (48%), e-value = 1e-69)
Gene Expression Studies
[0311] Successful expression of introduced Thiolase, 3-hydroxybutyryl-CoA dehydrogenase, Crotonase, Butyryl-CoA dehydrogenase, Electron Transfer Flavoprotein A and Electron Transfer Flavoprotein B were confirmed by RT-PCR studies.
[0312] A 50-ml culture of C. autoethanogenum harboring butanol plasmid pMTL85245-thlA-crt-hbd grown in a serum bottle with 30 psi steel mill gas as substrate was harvested by centrifugation (6,000×g, 5 min, 4° C.). RNA was isolated by suspending the cell pellet in 100 μL of lysozyme solution (50,000 U lysozyme, 0.5 μL 10% SDS, 10 mM Tris-HCl, 0.1 mM EDTA; pH 8). After 5 min, 350 μL of lysis buffer (containing 10 μL of 2-mercaptoethanol) was added. The cell suspension was mechanistically disrupted by passing five times through an 18-21 gauge needle. RNA was then isolated using PureLink® RNA Mini Kit (Invitrogen) and eluted in 100 μL of RNase-free water. The RNA was checked via PCR and gel electrophoresis and quantified spectrophotometrically, and treated with DNase I (Roche) if necessary. Quality and integrity of RNA was checked using a BioAnalyzer (Agilent Technologies). The reverse transcription step was carried out using SuperScript III Reverse Transcriptase Kit (Invitrogen). RT-PCR reactions were performed in MyiQ Single Colour Real-Time PCR Detection System (Bio-Rad Labratories) in a reaction volume of 15 μL with 25 ng of cDNA template, 67 nM of each primer (Tab. 11), and 1× iQ SYBR Green Supermix (Bio-Rad Labratories, Hercules, Calif. 94547, USA). Guanylate kinase and formate tetrahydrofolate ligase were used as housekeeping gene and non-template controls were included. 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. A melting-curve analysis was performed immediately after completion of the RT PCR (38 cycles of 58° C. to 95° C. at 1° C./s), for detection of primer dimerisation or other artifacts of amplification. mRNA from housekeeping and all target genes were successfully detected.
TABLE-US-00010 TABLE 11 Oligonucleotides for RT-PCR Oligonu- cleotide SEQ_ID Target Name DNA Sequence (5' to 3') NO. Guanylate kinase GnK-F TCAGGACCTTCTGGAACTGG 131 GnK-R ACCTCCCCTTTTCTTGGAGA 132 Formate FoT4L-F CAGGTTTCGGTGCTGACCTA 133 tetrahydrofolate FoT4L-R AACTCCGCCGTTGTATTTCA 134 ligase Thiolase thlA-RT-F TTGATGAAATGATCACTGACGGATT 123 thlA-RT-R GAAATGTTCCATCTCTCAGCTATGT 124 3-hydroxybutyryl- hdb-RT-F CATCACTTTCAATAACAGAAGTGGC 125 CoA dehydrogenase hbd-RT-R TACCTCTACAAGCTTCATAACAGGA 126 Butyryl-CoA bcd-RT-F AAAATGGGTCAGTATGGTATGATGG 127 dehydrogenase bcd-RT-R TGTAGTACCGCAAACCTTTGATAAT 128 Electron Transfer etfA-RT-F CAAGTTTACTTGGTGGAACAATAGC 129 Flavoprotein A etfA-RT-R GAGTTGGTCTTACAGTTTTACCAGT 130
[0313] 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.
[0314] 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.
[0315] 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
13511179DNAC. acetobutylicum 1atgaaagaag ttgtaatagc tagtgcagta agaacagcga
ttggatctta tggaaagtct 60cttaaggatg taccagcagt agatttagga gctacagcta
taaaggaagc agttaaaaaa 120gcaggaataa aaccagagga tgttaatgaa gtcattttag
gaaatgttct tcaagcaggt 180ttaggacaga atccagcaag acaggcatct tttaaagcag
gattaccagt tgaaattcca 240gctatgacta ttaataaggt ttgtggttca ggacttagaa
cagttagctt agcagcacaa 300attataaaag caggagatgc tgacgtaata atagcaggtg
gtatggaaaa tatgtctaga 360gctccttact tagcgaataa cgctagatgg ggatatagaa
tgggaaacgc taaatttgtt 420gatgaaatga tcactgacgg attgtgggat gcatttaatg
attaccacat gggaataaca 480gcagaaaaca tagctgagag atggaacatt tcaagagaag
aacaagatga gtttgctctt 540gcatcacaaa aaaaagctga agaagctata aaatcaggtc
aatttaaaga tgaaatagtt 600cctgtagtaa ttaaaggcag aaagggagaa actgtagttg
atacagatga gcaccctaga 660tttggatcaa ctatagaagg acttgcaaaa ttaaaacctg
ccttcaaaaa agatggaaca 720gttacagctg gtaatgcatc aggattaaat gactgtgcag
cagtacttgt aatcatgagt 780gcagaaaaag ctaaagagct tggagtaaaa ccacttgcta
agatagtttc ttatggttca 840gcaggagttg acccagcaat aatgggatat ggacctttct
atgcaacaaa agcagctatt 900gaaaaagcag gttggacagt tgatgaatta gatttaatag
aatcaaatga agcttttgca 960gctcaaagtt tagcagtagc aaaagattta aaatttgata
tgaataaagt aaatgtaaat 1020ggaggagcta ttgcccttgg tcatccaatt ggagcatcag
gtgcaagaat actcgttact 1080cttgtacacg caatgcaaaa aagagatgca aaaaaaggct
tagcaacttt atgtataggt 1140ggcggacaag gaacagcaat attgctagaa aagtgctag
11792849DNAC. acetobutylicum 2atgaaaaagg tatgtgttat
aggtgcaggt actatgggtt caggaattgc tcaggcattt 60gcagctaaag gatttgaagt
agtattaaga gatattaaag atgaatttgt tgatagagga 120ttagatttta tcaataaaaa
tctttctaaa ttagttaaaa aaggaaagat agaagaagct 180actaaagttg aaatcttaac
tagaatttcc ggaacagttg accttaatat ggcagctgat 240tgcgatttag ttatagaagc
agctgttgaa agaatggata ttaaaaagca gatttttgct 300gacttagaca atatatgcaa
gccagaaaca attcttgcat caaatacatc atcactttca 360ataacagaag tggcatcagc
aactaaaaga cctgataagg ttataggtat gcatttcttt 420aatccagctc ctgttatgaa
gcttgtagag gtaataagag gaatagctac atcacaagaa 480acttttgatg cagttaaaga
gacatctata gcaataggaa aagatcctgt agaagtagca 540gaagcaccag gatttgttgt
aaatagaata ttaataccaa tgattaatga agcagttggt 600atattagcag aaggaatagc
ttcagtagaa gacatagata aagctatgaa acttggagct 660aatcacccaa tgggaccatt
agaattaggt gattttatag gtcttgatat atgtcttgct 720ataatggatg ttttatactc
agaaactgga gattctaagt atagaccaca tacattactt 780aagaagtatg taagagcagg
atggcttgga agaaaatcag gaaaaggttt ctacgattat 840tcaaaataa
8493786DNAC. acetobutylicum
3atggaactaa acaatgtcat ccttgaaaag gaaggtaaag ttgctgtagt taccattaac
60agacctaaag cattaaatgc gttaaatagt gatacactaa aagaaatgga ttatgttata
120ggtgaaattg aaaatgatag cgaagtactt gcagtaattt taactggagc aggagaaaaa
180tcatttgtag caggagcaga tatttctgag atgaaggaaa tgaataccat tgaaggtaga
240aaattcggga tacttggaaa taaagtgttt agaagattag aacttcttga aaagcctgta
300atagcagctg ttaatggttt tgctttagga ggcggatgcg aaatagctat gtcttgtgat
360ataagaatag cttcaagcaa cgcaagattt ggtcaaccag aagtaggtct cggaataaca
420cctggttttg gtggtacaca aagactttca agattagttg gaatgggcat ggcaaagcag
480cttatattta ctgcacaaaa tataaaggca gatgaagcat taagaatcgg acttgtaaat
540aaggtagtag aacctagtga attaatgaat acagcaaaag aaattgcaaa caaaattgtg
600agcaatgctc cagtagctgt taagttaagc aaacaggcta ttaatagagg aatgcagtgt
660gatattgata ctgctttagc atttgaatca gaagcatttg gagaatgctt ttcaacagag
720gatcaaaagg atgcaatgac agctttcata gagaaaagaa aaattgaagg cttcaaaaat
780agatag
78641140DNAC. acetobutylicum 4atggatttta atttaacaag agaacaagaa ttagtaagac
agatggttag agaatttgct 60gaaaatgaag ttaaacctat agcagcagaa attgatgaaa
cagaaagatt tccaatggaa 120aatgtaaaga aaatgggtca gtatggtatg atgggaattc
cattttcaaa agagtatggt 180ggcgcaggtg gagatgtatt atcttatata atcgccgttg
aggaattatc aaaggtttgc 240ggtactacag gagttattct ttcagcacat acatcacttt
gtgcttcatt aataaatgaa 300catggtacag aagaacaaaa acaaaaatat ttagtacctt
tagctaaagg tgaaaaaata 360ggtgcttatg gattgactga gccaaatgca ggaacagatt
ctggagcaca acaaacagta 420gctgtacttg aaggagatca ttatgtaatt aatggttcaa
aaatattcat aactaatgga 480ggagttgcag atacttttgt tatatttgca atgactgaca
gaactaaagg aacaaaaggt 540atatcagcat ttataataga aaaaggcttc aaaggtttct
ctattggtaa agttgaacaa 600aagcttggaa taagagcttc atcaacaact gaacttgtat
ttgaagatat gatagtacca 660gtagaaaaca tgattggtaa agaaggaaaa ggcttcccta
tagcaatgaa aactcttgat 720ggaggaagaa ttggtatagc agctcaagct ttaggtatag
ctgaaggtgc tttcaacgaa 780gcaagagctt acatgaagga gagaaaacaa tttggaagaa
gccttgacaa attccaaggt 840cttgcatgga tgatggcaga tatggatgta gctatagaat
cagctagata tttagtatat 900aaagcagcat atcttaaaca agcaggactt ccatacacag
ttgatgctgc aagagctaag 960cttcatgctg caaatgtagc aatggatgta acaactaagg
cagtacaatt atttggtgga 1020tacggatata caaaagatta tccagttgaa agaatgatga
gagatgctaa gataactgaa 1080atatatgaag gaacttcaga agttcagaaa ttagttattt
caggaaaaat ttttagataa 114051011DNAC. acetobutylicum 5atgaataaag
cagattacaa gggcgtatgg gtgtttgctg aacaaagaga cggagaatta 60caaaaggtat
cattggaatt attaggtaaa ggtaaggaaa tggctgagaa attaggcgtt 120gaattaacag
ctgttttact tggacataat actgaaaaaa tgtcaaagga tttattatct 180catggagcag
ataaggtttt agcagcagat aatgaacttt tagcacattt ttcaacagat 240ggatatgcta
aagttatatg tgatttagtt aatgaaagaa agccagaaat attattcata 300ggagctactt
tcataggaag agatttagga ccaagaatag cagcaagact ttctactggt 360ttaactgctg
attgtacatc acttgacata gatgtagaaa atagagattt attggctaca 420agaccagcgt
ttggtggaaa tttgatagct acaatagttt gttcagacca cagaccacaa 480atggctacag
taagacctgg tgtgtttgaa aaattacctg ttaatgatgc aaatgtttct 540gatgataaaa
tagaaaaagt tgcaattaaa ttaacagcat cagacataag aacaaaagtt 600tcaaaagttg
ttaagcttgc taaagatatt gcagatatcg gagaagctaa ggtattagtt 660gctggtggta
gaggagttgg aagcaaagaa aactttgaaa aacttgaaga gttagcaagt 720ttacttggtg
gaacaatagc cgcttcaaga gcagcaatag aaaaagaatg ggttgataag 780gaccttcaag
taggtcaaac tggtaaaact gtaagaccaa ctctttatat tgcatgtggt 840atatcaggag
ctatccagca tttagcaggt atgcaagatt cagattacat aattgctata 900aataaagatg
tagaagcccc aataatgaag gtagcagatt tggctatagt tggtgatgta 960aataaagttg
taccagaatt aatagctcaa gttaaagctg ctaataatta a 10116780DNAC.
acetobutylicum 6atgaatatag ttgtttgttt aaaacaagtt ccagatacag cggaagttag
aatagatcca 60gttaagggaa cacttataag agaaggagtt ccatcaataa taaatccaga
tgataaaaac 120gcacttgagg aagctttagt attaaaagat aattatggtg cacatgtaac
agttataagt 180atgggacctc cacaagctaa aaatgcttta gtagaagctt tggctatggg
tgctgatgaa 240gctgtacttt taacagatag agcatttgga ggagcagata cacttgcgac
ttcacataca 300attgcagcag gaattaagaa gctaaaatat gatatagttt ttgctggaag
gcaggctata 360gatggagata cagctcaggt tggaccagaa atagctgagc atcttggaat
acctcaagta 420acttatgttg agaaagttga agttgatgga gatactttaa agattagaaa
agcttgggaa 480gatggatatg aagttgttga agttaagaca ccagttcttt taacagcaat
taaagaatta 540aatgttccaa gatatatgag tgtagaaaaa atattcggag catttgataa
agaagtaaaa 600atgtggactg ccgatgatat agatgtagat aaggctaatt taggtcttaa
aggttcacca 660actaaagtta agaagtcatc aactaaagaa gttaaaggac agggagaagt
tattgataag 720cctgttaagg aagcagctgc atatgttgtc tcaaaattaa aagaagaaca
ctatatttaa 7807498DNAC. autoethanogenum 7gagcggccgc aatatgatat
ttatgtccat tgtgaaaggg attatattca actattattc 60cagttacgtt catagaaatt
ttcctttcta aaatatttta ttccatgtca agaactctgt 120ttatttcatt aaagaactat
aagtacaaag tataaggcat ttgaaaaaat aggctagtat 180attgattgat tatttatttt
aaaatgccta agtgaaatat atacatatta taacaataaa 240ataagtatta gtgtaggatt
tttaaataga gtatctattt tcagattaaa tttttgatta 300tttgatttac attatataat
attgagtaaa gtattgacta gcaaaatttt ttgatacttt 360aatttgtgaa atttcttatc
aaaagttata tttttgaata atttttattg aaaaatacaa 420ctaaaaagga ttatagtata
agtgtgtgta attttgtgtt aaatttaaag ggaggaaatg 480aacatgaaac atatggaa
498828DNAArtificial
sequencesynthetic primer 8gagcggccgc aatatgatat ttatgtcc
28928DNAArtificial sequencesynthetic primer
9ttccatatgt ttcatgttca tttcctcc
281026DNAArtificial sequencesynthetic primer 10gttcatatga aagaagttgt
aatagc 261125DNAArtificial
sequencesynthetic primer 11caagaattcc tagcactttt ctagc
251227DNAArtificial sequencesynthetic primer
12aaggtacctt aggaggatta gtcatgg
271330DNAArtificial sequencesynthetic primer 13gaggatccgg attcttgtaa
acttattttg 30142963DNAE. coli
14cctgcaggat aaaaaaattg tagataaatt ttataaaata gttttatcta caattttttt
60atcaggaaac agctatgacc gcggccgctg tatccatatg accatgatta cgaattcgag
120ctcggtaccc ggggatcctc tagagtcgac gtcacgcgtc catggagatc tcgaggcctg
180cagacatgca agcttggcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
240cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
300agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcta
360gcataaaaat aagaagcctg catttgcagg cttcttattt ttatggcgcg ccgcattcac
420ttcttttcta tataaatatg agcgaagcga ataagcgtcg gaaaagcagc aaaaagtttc
480ctttttgctg ttggagcatg ggggttcagg gggtgcagta tctgacgtca atgccgagcg
540aaagcgagcc gaagggtagc atttacgtta gataaccccc tgatatgctc cgacgcttta
600tatagaaaag aagattcaac taggtaaaat cttaatatag gttgagatga taaggtttat
660aaggaatttg tttgttctaa tttttcactc attttgttct aatttctttt aacaaatgtt
720cttttttttt tagaacagtt atgatatagt tagaatagtt taaaataagg agtgagaaaa
780agatgaaaga aagatatgga acagtctata aaggctctca gaggctcata gacgaagaaa
840gtggagaagt catagaggta gacaagttat accgtaaaca aacgtctggt aacttcgtaa
900aggcatatat agtgcaatta ataagtatgt tagatatgat tggcggaaaa aaacttaaaa
960tcgttaacta tatcctagat aatgtccact taagtaacaa tacaatgata gctacaacaa
1020gagaaatagc aaaagctaca ggaacaagtc tacaaacagt aataacaaca cttaaaatct
1080tagaagaagg aaatattata aaaagaaaaa ctggagtatt aatgttaaac cctgaactac
1140taatgagagg cgacgaccaa aaacaaaaat acctcttact cgaatttggg aactttgagc
1200aagaggcaaa tgaaatagat tgacctccca ataacaccac gtagttattg ggaggtcaat
1260ctatgaaatg cgattaaggg ccggccagtg ggcaagttga aaaattcaca aaaatgtggt
1320ataatatctt tgttcattag agcgataaac ttgaatttga gagggaactt agatggtatt
1380tgaaaaaatt gataaaaata gttggaacag aaaagagtat tttgaccact actttgcaag
1440tgtaccttgt acctacagca tgaccgttaa agtggatatc acacaaataa aggaaaaggg
1500aatgaaacta tatcctgcaa tgctttatta tattgcaatg attgtaaacc gccattcaga
1560gtttaggacg gcaatcaatc aagatggtga attggggata tatgatgaga tgataccaag
1620ctatacaata tttcacaatg atactgaaac attttccagc ctttggactg agtgtaagtc
1680tgactttaaa tcatttttag cagattatga aagtgatacg caacggtatg gaaacaatca
1740tagaatggaa ggaaagccaa atgctccgga aaacattttt aatgtatcta tgataccgtg
1800gtcaaccttc gatggcttta atctgaattt gcagaaagga tatgattatt tgattcctat
1860ttttactatg gggaaatatt ataaagaaga taacaaaatt atacttcctt tggcaattca
1920agttcatcac gcagtatgtg acggatttca catttgccgt tttgtaaacg aattgcagga
1980attgataaat agttaacttc aggtttgtct gtaactaaaa acaagtattt aagcaaaaac
2040atcgtagaaa tacggtgttt tttgttaccc taagtttaaa ctcctttttg ataatctcat
2100gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
2160caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
2220accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
2280ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
2340aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
2400accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
2460gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
2520ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
2580gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
2640gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
2700ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
2760aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
2820gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
2880tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
2940agagcgccca atacgcaggg ccc
2963155935DNAE. coli 15cctgcaggat aaaaaaattg tagataaatt ttataaaata
gttttatcta caattttttt 60atcaggaaac agctatgacc gcggccgctg tatccatatg
gtatttgaaa aaattgataa 120aaatagttgg aacagaaaag agtattttga ccactacttt
gcaagtgtac cttgtaccta 180cagcatgacc gttaaagtgg atatcacaca aataaaggaa
aagggaatga aactatatcc 240tgcaatgctt tattatattg caatgattgt aaaccgccat
tcagagttta ggacggcaat 300caatcaagat ggtgaattgg ggatatatga tgagatgata
ccaagctata caatatttca 360caatgatact gaaacatttt ccagcctttg gactgagtgt
aagtctgact ttaaatcatt 420tttagcagat tatgaaagtg atacgcaacg gtatggaaac
aatcatagaa tggaaggaaa 480gccaaatgct ccggaaaaca tttttaatgt atctatgata
ccgtggtcaa ccttcgatgg 540ctttaatctg aatttgcaga aaggatatga ttatttgatt
cctattttta ctatggggaa 600atattataaa gaagataaca aaattatact tcctttggca
attcaagttc atcacgcagt 660atgtgacgga tttcacattt gccgttttgt aaacgaattg
caggaattga taaatagtta 720aacgcgtcca tggagatctc gaggcctgca gacatgcaag
cttggcactg gccgtcgttt 780tacaacgtcg tgactgggaa aaccctggcg ttacccaact
taatcgcctt gcagcacatc 840cccctttcgc cagctggcgt aatagcgaag aggcccgcac
cgatcgccct tcccaacagt 900tgcgcagcct gaatggcgaa tggcgctagc ataaaaataa
gaagcctgca tttgcaggct 960tcttattttt atggcgcgcc gttctgaatc cttagctaat
ggttcaacag gtaactatga 1020cgaagatagc accctggata agtctgtaat ggattctaag
gcatttaatg aagacgtgta 1080tataaaatgt gctaatgaaa aagaaaatgc gttaaaagag
cctaaaatga gttcaaatgg 1140ttttgaaatt gattggtagt ttaatttaat atattttttc
tattggctat ctcgatacct 1200atagaatctt ctgttcactt ttgtttttga aatataaaaa
ggggcttttt agcccctttt 1260ttttaaaact ccggaggagt ttcttcattc ttgatactat
acgtaactat tttcgatttg 1320acttcattgt caattaagct agtaaaatca atggttaaaa
aacaaaaaac ttgcattttt 1380ctacctagta atttataatt ttaagtgtcg agtttaaaag
tataatttac caggaaagga 1440gcaagttttt taataaggaa aaatttttcc ttttaaaatt
ctatttcgtt atatgactaa 1500ttataatcaa aaaaatgaaa ataaacaaga ggtaaaaact
gctttagaga aatgtactga 1560taaaaaaaga aaaaatccta gatttacgtc atacatagca
cctttaacta ctaagaaaaa 1620tattgaaagg acttccactt gtggagatta tttgtttatg
ttgagtgatg cagacttaga 1680acattttaaa ttacataaag gtaatttttg cggtaataga
ttttgtccaa tgtgtagttg 1740gcgacttgct tgtaaggata gtttagaaat atctattctt
atggagcatt taagaaaaga 1800agaaaataaa gagtttatat ttttaactct tacaactcca
aatgtaaaaa gttatgatct 1860taattattct attaaacaat ataataaatc ttttaaaaaa
ttaatggagc gtaaggaagt 1920taaggatata actaaaggtt atataagaaa attagaagta
acttaccaaa aggaaaaata 1980cataacaaag gatttatgga aaataaaaaa agattattat
caaaaaaaag gacttgaaat 2040tggtgattta gaacctaatt ttgatactta taatcctcat
tttcatgtag ttattgcagt 2100taataaaagt tattttacag ataaaaatta ttatataaat
cgagaaagat ggttggaatt 2160atggaagttt gctactaagg atgattctat aactcaagtt
gatgttagaa aagcaaaaat 2220taatgattat aaagaggttt acgaacttgc gaaatattca
gctaaagaca ctgattattt 2280aatatcgagg ccagtatttg aaatttttta taaagcatta
aaaggcaagc aggtattagt 2340ttttagtgga ttttttaaag atgcacacaa attgtacaag
caaggaaaac ttgatgttta 2400taaaaagaaa gatgaaatta aatatgtcta tatagtttat
tataattggt gcaaaaaaca 2460atatgaaaaa actagaataa gggaacttac ggaagatgaa
aaagaagaat taaatcaaga 2520tttaatagat gaaatagaaa tagattaaag tgtaactata
ctttatatat atatgattaa 2580aaaaataaaa aacaacagcc tattaggttg ttgtttttta
ttttctttat taattttttt 2640aatttttagt ttttagttct tttttaaaat aagtttcagc
ctctttttca atatttttta 2700aagaaggagt atttgcatga attgcctttt ttctaacaga
cttaggaaat attttaacag 2760tatcttcttg cgccggtgat tttggaactt cataacttac
taatttataa ttattatttt 2820cttttttaat tgtaacagtt gcaaaagaag ctgaacctgt
tccttcaact agtttatcat 2880cttcaatata atattcttga cctatatagt ataaatatat
ttttattata tttttacttt 2940tttctgaatc tattatttta taatcataaa aagttttacc
accaaaagaa ggttgtactc 3000cttctggtcc aacatatttt tttactatat tatctaaata
atttttggga actggtgttg 3060taatttgatt aatcgaacaa ccagttatac ttaaaggaat
tataactata aaaatatata 3120ggattatctt tttaaatttc attattggcc tcctttttat
taaatttatg ttaccataaa 3180aaggacataa cgggaatatg tagaatattt ttaatgtaga
caaaatttta cataaatata 3240aagaaaggaa gtgtttgttt aaattttata gcaaactatc
aaaaattagg gggataaaaa 3300tttatgaaaa aaaggttttc gatgttattt ttatgtttaa
ctttaatagt ttgtggttta 3360tttacaaatt cggccggccg aagcaaactt aagagtgtgt
tgatagtgca gtatcttaaa 3420attttgtata ataggaattg aagttaaatt agatgctaaa
aatttgtaat taagaaggag 3480tgattacatg aacaaaaata taaaatattc tcaaaacttt
ttaacgagtg aaaaagtact 3540caaccaaata ataaaacaat tgaatttaaa agaaaccgat
accgtttacg aaattggaac 3600aggtaaaggg catttaacga cgaaactggc taaaataagt
aaacaggtaa cgtctattga 3660attagacagt catctattca acttatcgtc agaaaaatta
aaactgaata ctcgtgtcac 3720tttaattcac caagatattc tacagtttca attccctaac
aaacagaggt ataaaattgt 3780tgggagtatt ccttaccatt taagcacaca aattattaaa
aaagtggttt ttgaaagcca 3840tgcgtctgac atctatctga ttgttgaaga aggattctac
aagcgtacct tggatattca 3900ccgaacacta gggttgctct tgcacactca agtctcgatt
cagcaattgc ttaagctgcc 3960agcggaatgc tttcatccta aaccaaaagt aaacagtgtc
ttaataaaac ttacccgcca 4020taccacagat gttccagata aatattggaa gctatatacg
tactttgttt caaaatgggt 4080caatcgagaa tatcgtcaac tgtttactaa aaatcagttt
catcaagcaa tgaaacacgc 4140caaagtaaac aatttaagta ccgttactta tgagcaagta
ttgtctattt ttaatagtta 4200tctattattt aacgggagga aataattcta tgagtcgctt
ttgtaaattt ggaaagttac 4260acgttactaa agggaatgtg tttaaactcc tttttgataa
tctcatgacc aaaatccctt 4320aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
aaagatcaaa ggatcttctt 4380gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
aaaaaaacca ccgctaccag 4440cggtggtttg tttgccggat caagagctac caactctttt
tccgaaggta actggcttca 4500gcagagcgca gataccaaat actgttcttc tagtgtagcc
gtagttaggc caccacttca 4560agaactctgt agcaccgcct acatacctcg ctctgctaat
cctgttacca gtggctgctg 4620ccagtggcga taagtcgtgt cttaccgggt tggactcaag
acgatagtta ccggataagg 4680cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
cagcttggag cgaacgacct 4740acaccgaact gagataccta cagcgtgagc tatgagaaag
cgccacgctt cccgaaggga 4800gaaaggcgga caggtatccg gtaagcggca gggtcggaac
aggagagcgc acgagggagc 4860ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
gtttcgccac ctctgacttg 4920agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
atggaaaaac gccagcaacg 4980cggccttttt acggttcctg gccttttgct ggccttttgc
tcacatgttc tttcctgcgt 5040tatcccctga ttctgtggat aaccgtatta ccgcctttga
gtgagctgat accgctcgcc 5100gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
agcggaagag cgcccaatac 5160gcagggcccc ctgcttcggg gtcattatag cgattttttc
ggtatatcca tcctttttcg 5220cacgatatac aggattttgc caaagggttc gtgtagactt
tccttggtgt atccaacggc 5280gtcagccggg caggataggt gaagtaggcc cacccgcgag
cgggtgttcc ttcttcactg 5340tcccttattc gcacctggcg gtgctcaacg ggaatcctgc
tctgcgaggc tggccggcta 5400ccgccggcgt aacagatgag ggcaagcgga tggctgatga
aaccaagcca accaggaagg 5460gcagcccacc tatcaaggtg tactgccttc cagacgaacg
aagagcgatt gaggaaaagg 5520cggcggcggc cggcatgagc ctgtcggcct acctgctggc
cgtcggccag ggctacaaaa 5580tcacgggcgt cgtggactat gagcacgtcc gcgagctggc
ccgcatcaat ggcgacctgg 5640gccgcctggg cggcctgctg aaactctggc tcaccgacga
cccgcgcacg gcgcggttcg 5700gtgatgccac gatcctcgcc ctgctggcga agatcgaaga
gaagcaggac gagcttggca 5760aggtcatgat gggcgtggtc cgcccgaggg cagagccatg
acttttttag ccgctaaaac 5820ggccgggggg tgcgcgtgat tgccaagcac gtccccatgc
gctccatcaa gaagagcgac 5880ttcgcggagc tggtgaagta catcaccgac gagcaaggca
agaccgatcg ggccc 59351620DNAArtificial sequencesynthetic primer
16cagaggatgt taatgaagtc
201720DNAArtificial sequencesynthetic primer 17gcatcaggat taaatgactg
201815DNAArtificial
sequencesynthetic primer 18atagcgaagt acttg
151918DNAArtificial sequencesynthetic primer
19gatgcaatga cagctttc
182020DNAArtificial sequencesynthetic primer 20ggaacaaaag gtatatcagc
202119DNAArtificial
sequencesynthetic primer 21cggagcattt gataaagaa
192220DNAArtificial sequencesynthetic primer
22gctgattgta catcacttga
202320DNAArtificial sequencesynthetic primerr 23ccagaattaa tagctcaagt
20241764DNAC. Ljungdahlii
24atgaacagtt ttattgaaga tgttgaacaa atttacaatt ttattaaaaa aaatatagat
60gtagaagaga agatgcattt tatagaaact tataagcaaa aatctaatat gaagaaagaa
120attagctttt cagaagaata ctataaacag aaaattatga atggaaaaaa tggagtagtg
180tatactcctc cggaaatggc agcatttatg gttaaaaact tgataaatgt caatgatgta
240attggaaatc catttataaa aataatagat ccttcctgtg gatctgggaa tttaatttgt
300aagtgctttc tatatttaaa tcgaatattt attaagaata ttgaagttat aaatagtaaa
360aacaatttaa atttgaaact agaagatata agttaccata tagtacgtaa caatctattt
420ggatttgata tagatgaaac tgcaataaaa gttttaaaaa tagacttatt tttgattagc
480aatcagttta gtgaaaaaaa ttttcaagta aaggattttc tagtggaaaa tatagataga
540aaatatgatg tgtttatagg aaatcctccg tatataggac ataaatctgt agattctagt
600tattcatatg ttttaagaaa aatatatgga agtatatata gagacaaagg agacatatcc
660tactgttttt ttcaaaaatc attaaagtgt ttaaaggagg gaggaaaact ggtttttgtt
720acttctaggt atttttgtga atcttgcagc ggaaaagaac ttagaaagtt tttaattgaa
780aatacctcta tttataaaat tatagatttt tatggtataa gaccttttaa aagagtaggt
840atagacccaa tgataatatt tttagtaaga acaaaaaatt ggaacaataa tatagaaatc
900ataagaccca ataaaattga aaaaaatgaa aaaaataaat ttcttgattc cttgttttta
960gataaatctg aaaaatgcaa aaagttttct atttctcaaa agtctataaa taatgatgga
1020tgggtatttg ttgacgaagt tgagaaaaat ataatagata aaataaaaga aaaaagtaaa
1080tttattttaa aggatatatg ccatagttgt cagggtataa taacgggatg tgatagggct
1140tttatagttg atagagacat aataaatagt agaaaaattg aattaaggtt aataaaaccc
1200tggataaaaa gtagccatat acgaaaaaac gaagtaatta aaggtgaaaa atttattata
1260tactcaaatt taatagaaaa tgaaacagaa tgtcctaatg ctataaagta tatagagcag
1320tacaaaaaaa ggcttatgga aagaagagaa tgtaaaaaag gaacaagaaa gtggtatgaa
1380cttcaatggg ggagaaaacc ggaaattttt gaagaaaaga aaattgtgtt cccatacaag
1440tcctgtgaca atagatttgc tcttgacaag ggaagctatt ttagtgcaga tatatattcc
1500ttagtattaa aaaaaaatgt accttttacc tatgaaatac ttttaaatat attaaacagt
1560cctttgtatg aattttactt taaaactttc gcaaaaaaat taggagaaaa tctatatgag
1620tattacccta ataatctaat gaaattgtgt attccttcta ttgattttgg aggagaaaat
1680aatatagaaa aaaagctgta tgattttttt ggactgacag ataaggaaat tgagattgta
1740gaaaagataa aagataattg ctga
1764251693DNAC. autoethanogenum 25atgcatttta tagaaactta taagcaaaaa
tctaatatga agaaagaaat tagcttttca 60gaagaatact ataaacagaa aattatgaat
ggaaaaaatg gagtagtgta tactcctccg 120gaaatggcag catttatggt taaaaacttg
ataaatgtca atgatgtaat tggaaatcca 180tttataaaaa taatagatcc ttcctgtgga
tctgggaatt taatttgtaa gtgctttcta 240tatttaaatc gaatatttat taagaatatt
gaagttataa atagtaaaaa caatttaaat 300ttgaaactag aagatataag ttaccatata
gtacgtaaca atctatttgg atttgatata 360gatgaaactg caataaaagt tttaaaaata
gacttatttt tgattagcaa tcagtttagt 420gaaaaaaatt ttcaagtaaa ggattttcta
gtggaaaata tagatagaaa atatgatgtg 480tttataggaa atcctccgta tataggacat
aaatctgtag attctagtta ttcatatgtt 540ttaagaaaaa tatatggaag tatatataga
gacaaaggag acatatccta ctgttttttt 600caaaaatcat taaagtgttt aaaggaggga
ggaaaactgg tttttgttac ttctaggtat 660ttttgtgaat cttgcagcgg aaaagaactt
agaaagtttt taattgaaaa tacctctatt 720tataaaatta tagattttta tggtataaga
ccttttaaaa gagtaggtat agacccaatg 780ataatatttt tagtaagaac aaaaaattgg
aacaataata tagaaatcat aagacccaat 840aaaattgaaa aaaatgaaaa aaataaattt
cttgattcct tgtttttaga taaatctgaa 900aaatgcaaaa agttttctat ttctcaaaag
tctataaata atgatggatg ggtatttgtt 960gacgaagttg agaaaaatat aatagataaa
ataaaagaaa aaagtaaatt tattttaaag 1020gatatatgcc atagttgtca gggtataata
acgggatgtg atagggcttt tatagttgat 1080agagacataa taaatagtag aaaaattgaa
ttaaggttaa taaaaccctg gataaaaagt 1140agccatatac gaaaaaacga agtaattaaa
ggtgaaaaat ttattatata ctcaaattta 1200atagaaaatg aaacagaatg tcctaatgct
ataaagtata tagagcagta caaaaaaaag 1260gcttatggaa agaagagaat gtaaaaaagg
aacaagaaag tggtatgaac ttcaatgggg 1320gagaaaaccg gaaatttttg aagaaaagaa
aattgtgttc ccatacaagt cctgtgacaa 1380tagatttgct cttgacaagg gaagctattt
tagtgcagat atatattcct tagtattaaa 1440aaaaaatgta ccttttacct atgaaatact
tttaaatata ttaaacagtc ctttgtatga 1500attttacttt aaaactttcg caaaaaaatt
aggagaaaat ctatatgagt attaccctaa 1560taatctaatg aaattgtgta ttccttctat
tgattttgga ggagaaaata atatagaaaa 1620aaagctgtat gatttttttg gactgacaga
taaggaaatt gagattgtag aaaagataaa 1680agataattgc tga
1693261805DNAC. ragsdalei 26atgtttccct
gtaatgcata tattcagcac ggagatagga atatgaataa ttttattgaa 60gatattgaag
aaatttataa ttttattaaa aaaaatacag atgtagaaga gaatattcat 120tttatagaaa
cttataggca aagacttaat atgaagaaag aaattagctt ttcagaagaa 180tactataaac
agaaaattat gaatggaaaa aacggagtag tgtatactcc tccggaaatg 240gcagcattta
tggttaaaaa cttgataaat gtcaatgatg taattgaaaa tccatttata 300aaagtagtag
atccttcctg tggatctgga aatttaattt gtaagtgctt tctatactta 360aatcaaatat
tcattaaaaa tattgaagtt ataaatagta aaaataattt aaatttgaaa 420ctaaaagata
taagttacca tatagtacat aacaatctat ttggatttga tgtagatgaa 480actgcaataa
aagttttaaa atagacttat ttttgattag caatcagttt agtgaaaaaa 540attttcaagt
aaaggatttt ctagtggaaa atatagatag aaaatttgat gtgtttatag 600gaaatccccc
atatatagga cataaatctg tagattccag ttattcatat attttaagga 660aaatatatgg
aagtatatat agagataaag gagacatatc ttactgtttt tttcaaaaat 720cattaaagtg
cttaaaagag ggaggaaaat tactttttgt tacctccaga tatttttgcg 780aatcttgcag
cggaaaagaa cttagaaagt ttttaattga aaatacctct atttataaaa 840ttatagattt
ttatggtata agacctttta aaagagtagg tatagatcca atgataatat 900ttttagtaag
aacaaaaaat tgggacaata atatagaaat cataagaccc aataaaagtg 960gaaaagatga
aaaaaataaa ttccttgatt ctttgctttt agataaatct gaaaaataca 1020aaaaattttc
tattcctcaa aagtctataa atagtgatgg atgggtattt gttaatgaag 1080ttgagaaaaa
tataatggat aaaatagaag caaaaagtga atttatttta aaggatatat 1140gccatagtta
tcagggtata ataacgggat gtgatagggc ttttatagtt gatagagaca 1200caataaatag
tagaaaaatt gaattaaggt taataaaacc ctgggtgaaa agcagccata 1260tacgaaaaaa
cgaagtaatt aaaggtgaaa aatttattat atactcaaat ttaatagaaa 1320atgagataga
atgtcctaat gctataaagt atatagagca gtacaaaaaa aagcttatgg 1380aaagaagaga
atgtaaaaaa ggaacgagaa agtggtatga gcttcaatgg gggagaaaac 1440cggaaatttt
cgaagaaaag aaaattgtat tcccatacaa atcgtgtgat aatagatttg 1500ctcttgataa
gggaagctat tttagtgcag atatatattc tttagtatta aaaaaaaatg 1560taccttttac
ctatgaaatg cttttaaata tattaaatag ttctttgtat gaattttact 1620ttaaaacttt
cgggaaaaaa ttaggagaaa atctatatga gtattatcct aataatctga 1680tgaaattgtg
tattccttct attggttttc gagaagaaaa taatgtagaa aaaaggttgt 1740atgatttttt
tgggctgaca gataaggaaa ttcagattgt agaaaaaata aaagataatt 1800gctga
1805271940DNAArtificial sequencesynthetic oligonucleotide 27gcggccgcgc
aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca 60ctttatgctt
ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg 120aaacacatat
gtttccgtgc aatgcctata tcgaatatgg tgataaaaat atgaacagct 180ttatcgaaga
tgtggaacag atctacaact tcattaaaaa gaacattgat gtggaagaaa 240agatgcattt
cattgaaacc tataaacaga aaagcaacat gaagaaagag attagcttta 300gcgaagaata
ctataaacag aagattatga acggcaaaaa tggcgttgtg tacaccccgc 360cggaaatggc
ggcctttatg gttaaaaatc tgatcaacgt taacgatgtt attggcaatc 420cgtttattaa
aatcattgac ccgagctgcg gtagcggcaa tctgatttgc aaatgttttc 480tgtatctgaa
tcgcatcttt attaagaaca ttgaggtgat taacagcaaa aataacctga 540atctgaaact
ggaagacatc agctaccaca tcgttcgcaa caatctgttt ggcttcgata 600ttgacgaaac
cgcgatcaaa gtgctgaaaa ttgatctgtt tctgatcagc aaccaattta 660gcgagaaaaa
tttccaggtt aaagactttc tggtggaaaa tattgatcgc aaatatgacg 720tgttcattgg
taatccgccg tatatcggtc acaaaagcgt ggacagcagc tacagctacg 780tgctgcgcaa
aatctacggc agcatctacc gcgacaaagg cgatatcagc tattgtttct 840ttcagaagag
cctgaaatgt ctgaaggaag gtggcaaact ggtgtttgtg accagccgct 900acttctgcga
gagctgcagc ggtaaagaac tgcgtaaatt cctgatcgaa aacacgagca 960tttacaagat
cattgatttt tacggcatcc gcccgttcaa acgcgtgggt atcgatccga 1020tgattatttt
tctggttcgt acgaagaact ggaacaataa cattgaaatt attcgcccga 1080acaagattga
aaagaacgaa aagaacaaat tcctggatag cctgttcctg gacaaaagcg 1140aaaagtgtaa
aaagtttagc attagccaga aaagcattaa taacgatggc tgggttttcg 1200tggacgaagt
ggagaaaaac attatcgaca aaatcaaaga gaaaagcaag ttcattctga 1260aagatatttg
ccatagctgt caaggcatta tcaccggttg tgatcgcgcc tttattgtgg 1320accgtgatat
catcaatagc cgtaagatcg aactgcgtct gattaaaccg tggattaaaa 1380gcagccatat
ccgtaagaat gaagttatta agggcgaaaa attcatcatc tatagcaacc 1440tgattgagaa
tgaaaccgag tgtccgaatg cgattaaata tatcgaacag tacaagaaac 1500gtctgatgga
gcgccgcgaa tgcaaaaagg gcacgcgtaa gtggtatgaa ctgcaatggg 1560gccgtaaacc
ggaaatcttc gaagaaaaga aaattgtttt cccgtataaa agctgtgaca 1620atcgttttgc
actggataag ggtagctatt ttagcgcaga catttatagc ctggttctga 1680agaaaaatgt
gccgttcacc tatgagatcc tgctgaatat cctgaatagc ccgctgtacg 1740agttttactt
taagaccttc gcgaaaaagc tgggcgagaa tctgtacgag tactatccga 1800acaacctgat
gaagctgtgc atcccgagca tcgatttcgg cggtgagaac aatattgaga 1860aaaagctgta
tgatttcttt ggtctgacgg ataaagaaat tgagattgtg gagaagatca 1920aagataactg
ctaagaattc
194028601PRTArtificial sequencesynthetic protein 28Met Phe Pro Cys Asn
Ala Tyr Ile Glu Tyr Gly Asp Lys Asn Met Asn1 5
10 15Ser Phe Ile Glu Asp Val Glu Gln Ile Tyr Asn
Phe Ile Lys Lys Asn 20 25
30Ile Asp Val Glu Glu Lys Met His Phe Ile Glu Thr Tyr Lys Gln Lys
35 40 45Ser Asn Met Lys Lys Glu Ile Ser
Phe Ser Glu Glu Tyr Tyr Lys Gln 50 55
60Lys Ile Met Asn Gly Lys Asn Gly Val Val Tyr Thr Pro Pro Glu Met65
70 75 80Ala Ala Phe Met Val
Lys Asn Leu Ile Asn Val Asn Asp Val Ile Gly 85
90 95Asn Pro Phe Ile Lys Ile Ile Asp Pro Ser Cys
Gly Ser Gly Asn Leu 100 105
110Ile Cys Lys Cys Phe Leu Tyr Leu Asn Arg Ile Phe Ile Lys Asn Ile
115 120 125Glu Val Ile Asn Ser Lys Asn
Asn Leu Asn Leu Lys Leu Glu Asp Ile 130 135
140Ser Tyr His Ile Val Arg Asn Asn Leu Phe Gly Phe Asp Ile Asp
Glu145 150 155 160Thr Ala
Ile Lys Val Leu Lys Ile Asp Leu Phe Leu Ile Ser Asn Gln
165 170 175Phe Ser Glu Lys Asn Phe Gln
Val Lys Asp Phe Leu Val Glu Asn Ile 180 185
190Asp Arg Lys Tyr Asp Val Phe Ile Gly Asn Pro Pro Tyr Ile
Gly His 195 200 205Lys Ser Val Asp
Ser Ser Tyr Ser Tyr Val Leu Arg Lys Ile Tyr Gly 210
215 220Ser Ile Tyr Arg Asp Lys Gly Asp Ile Ser Tyr Cys
Phe Phe Gln Lys225 230 235
240Ser Leu Lys Cys Leu Lys Glu Gly Gly Lys Leu Val Phe Val Thr Ser
245 250 255Arg Tyr Phe Cys Glu
Ser Cys Ser Gly Lys Glu Leu Arg Lys Phe Leu 260
265 270Ile Glu Asn Thr Ser Ile Tyr Lys Ile Ile Asp Phe
Tyr Gly Ile Arg 275 280 285Pro Phe
Lys Arg Val Gly Ile Asp Pro Met Ile Ile Phe Leu Val Arg 290
295 300Thr Lys Asn Trp Asn Asn Asn Ile Glu Ile Ile
Arg Pro Asn Lys Ile305 310 315
320Glu Lys Asn Glu Lys Asn Lys Phe Leu Asp Ser Leu Phe Leu Asp Lys
325 330 335Ser Glu Lys Cys
Lys Lys Phe Ser Ile Ser Gln Lys Ser Ile Asn Asn 340
345 350Asp Gly Trp Val Phe Val Asp Glu Val Glu Lys
Asn Ile Ile Asp Lys 355 360 365Ile
Lys Glu Lys Ser Lys Phe Ile Leu Lys Asp Ile Cys His Ser Cys 370
375 380Gln Gly Ile Ile Thr Gly Cys Asp Arg Ala
Phe Ile Val Asp Arg Asp385 390 395
400Ile Ile Asn Ser Arg Lys Ile Glu Leu Arg Leu Ile Lys Pro Trp
Ile 405 410 415Lys Ser Ser
His Ile Arg Lys Asn Glu Val Ile Lys Gly Glu Lys Phe 420
425 430Ile Ile Tyr Ser Asn Leu Ile Glu Asn Glu
Thr Glu Cys Pro Asn Ala 435 440
445Ile Lys Tyr Ile Glu Gln Tyr Lys Lys Arg Leu Met Glu Arg Arg Glu 450
455 460Cys Lys Lys Gly Thr Arg Lys Trp
Tyr Glu Leu Gln Trp Gly Arg Lys465 470
475 480Pro Glu Ile Phe Glu Glu Lys Lys Ile Val Phe Pro
Tyr Lys Ser Cys 485 490
495Asp Asn Arg Phe Ala Leu Asp Lys Gly Ser Tyr Phe Ser Ala Asp Ile
500 505 510Tyr Ser Leu Val Leu Lys
Lys Asn Val Pro Phe Thr Tyr Glu Ile Leu 515 520
525Leu Asn Ile Leu Asn Ser Pro Leu Tyr Glu Phe Tyr Phe Lys
Thr Phe 530 535 540Ala Lys Lys Leu Gly
Glu Asn Leu Tyr Glu Tyr Tyr Pro Asn Asn Leu545 550
555 560Met Lys Leu Cys Ile Pro Ser Ile Asp Phe
Gly Gly Glu Asn Asn Ile 565 570
575Glu Lys Lys Leu Tyr Asp Phe Phe Gly Leu Thr Asp Lys Glu Ile Glu
580 585 590Ile Val Glu Lys Ile
Lys Asp Asn Cys 595 600292781DNAArtificial
sequenceSynthetic plasmid DNA 29tttgccacct gacgtctaag aaaaggaata
ttcagcaatt tgcccgtgcc gaagaaaggc 60ccacccgtga aggtgagcca gtgagttgat
tgctacgtaa ttagttagtt agcccttagt 120gactcgtaat acgactcact atagggctcg
agtctagaga attcgatatc acccgggaac 180tagtctgcag ccctttagtg agggttaatt
ggagtcacta agggttagtt agttagatta 240gcagaaagtc aaaagcctcc gaccggaggc
ttttgactaa aacttccctt ggggttatca 300ttggggctca ctcaaaggcg gtaatcagat
aaaaaaaatc cttagctttc gctaaggatg 360atttctgcta gagatggaat agactggatg
gaggcggata aagttgcagg accacttctg 420cgctcggccc ttccggctgg ctggtttatt
gctgataaat ctggagccgg tgagcgtggg 480tctcgcggta tcattgcagc actggggcca
gatggtaagc cctcccgtat cgtagttatc 540tacacgacgg ggagtcaggc aactatggat
gaacgaaata gacagatcgc tgagataggt 600gcctcactga ttaagcattg gtaactgtca
gaccaagttt actcatatat actttagatt 660gatttaaaac ttcattttta atttaaaagg
atctaggtga agatcctttt tgataatctc 720atgaccaaaa tcccttaacg tgagttttcg
ttccactgag cgtcagaccc cttaataaga 780tgatcttctt gagatcgttt tggtctgcgc
gtaatctctt gctctgaaaa cgaaaaaacc 840gccttgcagg gcggtttttc gaaggttctc
tgagctacca actctttgaa ccgaggtaac 900tggcttggag gagcgcagtc accaaaactt
gtcctttcag tttagcctta accggcgcat 960gacttcaaga ctaactcctc taaatcaatt
accagtggct gctgccagtg gtgcttttgc 1020atgtctttcc gggttggact caagacgata
gttaccggat aaggcgcagc ggtcggactg 1080aacggggggt tcgtgcatac agtccagctt
ggagcgaact gcctacccgg aactgagtgt 1140caggcgtgga atgagacaaa cgcggccata
acagcggaat gacaccggta aaccgaaagg 1200caggaacagg agagcgcacg agggagccgc
caggggaaac gcctggtatc tttatagtcc 1260tgtcgggttt cgccaccact gatttgagcg
tcagatttcg tgatgcttgt caggggggcg 1320gagcctatgg aaaaacggct ttgccgcggc
cctctcactt ccctgttaag tatcttcctg 1380gcatcttcca ggaaatctcc gccccgttcg
taagccattt ccgctcgccg cagtcgaacg 1440accgagcgta gcgagtcagt gagcgaggaa
gcggaatata tcctgtatca catattctgc 1500tgacgcaccg gtgcagcctt ttttctcctg
ccacatgaag cacttcactg acaccctcat 1560cagtgccaac atagtaagcc agtatacact
ccgctagcgc tgaggtctgc ctcgtgaaga 1620aggtgttgct gactcatacc aggcctgaat
cgccccatca tccagccaga aagtgaggga 1680gccacggttg atgagagctt tgttgtaggt
ggaccagttg gtgattttga acttttgctt 1740tgccacggaa cggtctgcgt tgtcgggaag
atgcgtgatc tgatccttca actcagcaaa 1800agttcgattt attcaacaaa gccacgttgt
gtctcaaaat ctctgatgtt acattgcaca 1860agataaaaat atatcatcat gaacaataaa
actgtctgct tacataaaca gtaatacaag 1920gggtgtttac tagaggttga tcgggcacgt
aagaggttcc aactttcacc ataatgaaat 1980aagatcacta ccgggcgtat tttttgagtt
atcgagattt tcaggagcta aggaagctaa 2040aatggagaaa aaaatcacgg gatataccac
cgttgatata tcccaatggc atcgtaaaga 2100acattttgag gcatttcagt cagttgctca
atgtacctat aaccagaccg ttcagctgga 2160tattacggcc tttttaaaga ccgtaaagaa
aaataagcac aagttttatc cggcctttat 2220tcacattctt gcccgcctga tgaacgctca
cccggagttt cgtatggcca tgaaagacgg 2280tgagctggtg atctgggata gtgttcaccc
ttgttacacc gttttccatg agcaaactga 2340aacgttttcg tccctctgga gtgaatacca
cgacgatttc cggcagtttc tccacatata 2400ttcgcaagat gtggcgtgtt acggtgaaaa
cctggcctat ttccctaaag ggtttattga 2460gaatatgttt tttgtctcag ccaatccctg
ggtgagtttc accagttttg atttaaacgt 2520ggccaatatg gacaacttct tcgcccccgt
tttcacgatg ggcaaatatt atacgcaagg 2580cgacaaggtg ctgatgccgc tggcgatcca
ggttcatcat gccgtttgtg atggcttcca 2640tgtcggccgc atgcttaatg aattacaaca
gtactgtgat gagtggcagg gcggggcgta 2700ataatactag ctccggcaaa aaaacgggca
aggtgtcacc accctgccct ttttctttaa 2760aaccgaaaag attacttcgc g
2781301460DNAC. autoethanogenum
30ggctcaggac gaacgctggc ggcgtgctta acacatgcaa gtcgagcgat gaagctcctt
60cgggagtgga ttagcggcgg acgggtgagt aacacgtggg taacctacct caaagagggg
120gatagcctcc cgaaagggag attaataccg cataataatc agttttcaca tggagactga
180tttaaaggag taatccgctt tgagatggac ccgcggcgca ttagctagtt ggtagggtaa
240cggcctacca aggcgacgat gcgtagccga cctgagaggg tgatcggcca cattggaact
300gagagacggt ccagactcct acgggaggca gcagtgggga atattgcaca atgggcgaaa
360gcctgatgca gcaacgccgc gtgagtgaag aaggttttcg gattgtaaag ctctgtcttt
420ggggacgata atgacggtac ccaaggagga agccacggct aactacgtgc cagcagccgc
480ggtaatacgt aggtggcgag cgttgtccgg aattactggg cgtaaagagt gcgtaggcgg
540atatttaagt gagatgtgaa atacccgggc ttaacccggg cactgcattt caaactggat
600atctagagtg cgggagagga gaatggaatt cctagtgtag cggtgaaatg cgtagagatt
660aggaagaaca ccagtggcga aggcgattct ctggaccgta actgacgctg aggcacgaaa
720gcgtgggtag caaacaggat tagataccct ggtagtccac gccgtaaacg atgagtacta
780ggtgtaggag gtatcgaccc cttctgtgcc gcagtaaaca caataagtac tccgcctggg
840aagtacgatc gcaagattaa aactcaaagg aattgacggg ggcccgcaca agcagcggag
900catgtggttt aattcgaagc aacgcgaaga accttacctg gacttgacat accctgaata
960tcttagagat aagagaagcc cttcggggca gggatacagg tggtgcatgg ttgtcgtcag
1020ctcgtgtcgt gagatgttag gttaagtcct gcaacgagcg caacccctgt tgttagttgc
1080taacatttag ttgagcactc tagcaagact gccgcggtta acgcggagga aggtggggat
1140gacgtcaaat catcatgccc cttatgtcca gggcaacaca cgtgctacaa tgggcagtac
1200agagagaagc aagaccgcaa ggtggagcaa acctcaaaaa ctgcccccag ttcggattgc
1260aggctgaaac tcgcctacat gaagttggag ttgctagtaa tcgcgaatca gaatgtcgcg
1320gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccatgagagc tggcaacacc
1380cgaagtccgt agtctaactt aggaggacgc ggccgaaggt ggggttagta attggggtga
1440agtcgtaaca aggtagccgt
1460319459DNAArtificial sequencesynthetic plasmid DNA 31ataaaaaaat
tgtagataaa ttttataaaa tagttttatc tacaattttt ttatcaggaa 60acagctatga
ccgcggccgc aatatgatat ttatgtccat tgtgaaaggg attatattca 120actattattc
cagttacgtt catagaaatt ttcctttcta aaatatttta ttccatgtca 180agaactctgt
ttatttcatt aaagaactat aagtacaaag tataaggcat ttgaaaaaat 240aggctagtat
attgattgat tatttatttt aaaatgccta agtgaaatat atacatatta 300taacaataaa
ataagtatta gtgtaggatt tttaaataga gtatctattt tcagattaaa 360tttttgatta
tttgatttac attatataat attgagtaaa gtattgacta gcaaaatttt 420ttgatacttt
aatttgtgaa atttcttatc aaaagttata tttttgaata atttttattg 480aaaaatacaa
ctaaaaagga ttatagtata agtgtgtgta attttgtgtt aaatttaaag 540ggaggaaatg
aacatgaaac atatgaaaga agttgtaata gctagtgcag taagaacagc 600gattggatct
tatggaaagt ctcttaagga tgtaccagca gtagatttag gagctacagc 660tataaaggaa
gcagttaaaa aagcaggaat aaaaccagag gatgttaatg aagtcatttt 720aggaaatgtt
cttcaagcag gtttaggaca gaatccagca agacaggcat cttttaaagc 780aggattacca
gttgaaattc cagctatgac tattaataag gtttgtggtt caggacttag 840aacagttagc
ttagcagcac aaattataaa agcaggagat gctgacgtaa taatagcagg 900tggtatggaa
aatatgtcta gagctcctta cttagcgaat aacgctagat ggggatatag 960aatgggaaac
gctaaatttg ttgatgaaat gatcactgac ggattgtggg atgcatttaa 1020tgattaccac
atgggaataa cagcagaaaa catagctgag agatggaaca tttcaagaga 1080agaacaagat
gagtttgctc ttgcatcaca aaaaaaagct gaagaagcta taaaatcagg 1140tcaatttaaa
gatgaaatag ttcctgtagt aattaaaggc agaaagggag aaactgtagt 1200tgatacagat
gagcacccta gatttggatc aactatagaa ggacttgcaa aattaaaacc 1260tgccttcaaa
aaagatggaa cagttacagc tggtaatgca tcaggattaa atgactgtgc 1320agcagtactt
gtaatcatga gtgcagaaaa agctaaagag cttggagtaa aaccacttgc 1380taagatagtt
tcttatggtt cagcaggagt tgacccagca ataatgggat atggaccttt 1440ctatgcaaca
aaagcagcta ttgaaaaagc aggttggaca gttgatgaat tagatttaat 1500agaatcaaat
gaagcttttg cagctcaaag tttagcagta gcaaaagatt taaaatttga 1560tatgaataaa
gtaaatgtaa atggaggagc tattgccctt ggtcatccaa ttggagcatc 1620aggtgcaaga
atactcgtta ctcttgtaca cgcaatgcaa aaaagagatg caaaaaaagg 1680cttagcaact
ttatgtatag gtggcggaca aggaacagca atattgctag aaaagtgcta 1740ggaattcgag
ctcggtacct taggaggatt agtcatggaa ctaaacaatg tcatccttga 1800aaaggaaggt
aaagttgctg tagttaccat taacagacct aaagcattaa atgcgttaaa 1860tagtgataca
ctaaaagaaa tggattatgt tataggtgaa attgaaaatg atagcgaagt 1920acttgcagta
attttaactg gagcaggaga aaaatcattt gtagcaggag cagatatttc 1980tgagatgaag
gaaatgaata ccattgaagg tagaaaattc gggatacttg gaaataaagt 2040gtttagaaga
ttagaacttc ttgaaaagcc tgtaatagca gctgttaatg gttttgcttt 2100aggaggcgga
tgcgaaatag ctatgtcttg tgatataaga atagcttcaa gcaacgcaag 2160atttggtcaa
ccagaagtag gtctcggaat aacacctggt tttggtggta cacaaagact 2220ttcaagatta
gttggaatgg gcatggcaaa gcagcttata tttactgcac aaaatataaa 2280ggcagatgaa
gcattaagaa tcggacttgt aaataaggta gtagaaccta gtgaattaat 2340gaatacagca
aaagaaattg caaacaaaat tgtgagcaat gctccagtag ctgttaagtt 2400aagcaaacag
gctattaata gaggaatgca gtgtgatatt gatactgctt tagcatttga 2460atcagaagca
tttggagaat gcttttcaac agaggatcaa aaggatgcaa tgacagcttt 2520catagagaaa
agaaaaattg aaggcttcaa aaatagatag gaggtaagtt tatatggatt 2580ttaatttaac
aagagaacaa gaattagtaa gacagatggt tagagaattt gctgaaaatg 2640aagttaaacc
tatagcagca gaaattgatg aaacagaaag atttccaatg gaaaatgtaa 2700agaaaatggg
tcagtatggt atgatgggaa ttccattttc aaaagagtat ggtggcgcag 2760gtggagatgt
attatcttat ataatcgccg ttgaggaatt atcaaaggtt tgcggtacta 2820caggagttat
tctttcagca catacatcac tttgtgcttc attaataaat gaacatggta 2880cagaagaaca
aaaacaaaaa tatttagtac ctttagctaa aggtgaaaaa ataggtgctt 2940atggattgac
tgagccaaat gcaggaacag attctggagc acaacaaaca gtagctgtac 3000ttgaaggaga
tcattatgta attaatggtt caaaaatatt cataactaat ggaggagttg 3060cagatacttt
tgttatattt gcaatgactg acagaactaa aggaacaaaa ggtatatcag 3120catttataat
agaaaaaggc ttcaaaggtt tctctattgg taaagttgaa caaaagcttg 3180gaataagagc
ttcatcaaca actgaacttg tatttgaaga tatgatagta ccagtagaaa 3240acatgattgg
taaagaagga aaaggcttcc ctatagcaat gaaaactctt gatggaggaa 3300gaattggtat
agcagctcaa gctttaggta tagctgaagg tgctttcaac gaagcaagag 3360cttacatgaa
ggagagaaaa caatttggaa gaagccttga caaattccaa ggtcttgcat 3420ggatgatggc
agatatggat gtagctatag aatcagctag atatttagta tataaagcag 3480catatcttaa
acaagcagga cttccataca cagttgatgc tgcaagagct aagcttcatg 3540ctgcaaatgt
agcaatggat gtaacaacta aggcagtaca attatttggt ggatacggat 3600atacaaaaga
ttatccagtt gaaagaatga tgagagatgc taagataact gaaatatatg 3660aaggaacttc
agaagttcag aaattagtta tttcaggaaa aatttttaga taatttaagg 3720aggttaagag
gatgaatata gttgtttgtt taaaacaagt tccagataca gcggaagtta 3780gaatagatcc
agttaaggga acacttataa gagaaggagt tccatcaata ataaatccag 3840atgataaaaa
cgcacttgag gaagctttag tattaaaaga taattatggt gcacatgtaa 3900cagttataag
tatgggacct ccacaagcta aaaatgcttt agtagaagct ttggctatgg 3960gtgctgatga
agctgtactt ttaacagata gagcatttgg aggagcagat acacttgcga 4020cttcacatac
aattgcagca ggaattaaga agctaaaata tgatatagtt tttgctggaa 4080ggcaggctat
agatggagat acagctcagg ttggaccaga aatagctgag catcttggaa 4140tacctcaagt
aacttatgtt gagaaagttg aagttgatgg agatacttta aagattagaa 4200aagcttggga
agatggatat gaagttgttg aagttaagac accagttctt ttaacagcaa 4260ttaaagaatt
aaatgttcca agatatatga gtgtagaaaa aatattcgga gcatttgata 4320aagaagtaaa
aatgtggact gccgatgata tagatgtaga taaggctaat ttaggtctta 4380aaggttcacc
aactaaagtt aagaagtcat caactaaaga agttaaagga cagggagaag 4440ttattgataa
gcctgttaag gaagcagctg catatgttgt ctcaaaatta aaagaagaac 4500actatattta
agttaggagg gatttttcaa tgaataaagc agattacaag ggcgtatggg 4560tgtttgctga
acaaagagac ggagaattac aaaaggtatc attggaatta ttaggtaaag 4620gtaaggaaat
ggctgagaaa ttaggcgttg aattaacagc tgttttactt ggacataata 4680ctgaaaaaat
gtcaaaggat ttattatctc atggagcaga taaggtttta gcagcagata 4740atgaactttt
agcacatttt tcaacagatg gatatgctaa agttatatgt gatttagtta 4800atgaaagaaa
gccagaaata ttattcatag gagctacttt cataggaaga gatttaggac 4860caagaatagc
agcaagactt tctactggtt taactgctga ttgtacatca cttgacatag 4920atgtagaaaa
tagagattta ttggctacaa gaccagcgtt tggtggaaat ttgatagcta 4980caatagtttg
ttcagaccac agaccacaaa tggctacagt aagacctggt gtgtttgaaa 5040aattacctgt
taatgatgca aatgtttctg atgataaaat agaaaaagtt gcaattaaat 5100taacagcatc
agacataaga acaaaagttt caaaagttgt taagcttgct aaagatattg 5160cagatatcgg
agaagctaag gtattagttg ctggtggtag aggagttgga agcaaagaaa 5220actttgaaaa
acttgaagag ttagcaagtt tacttggtgg aacaatagcc gcttcaagag 5280cagcaataga
aaaagaatgg gttgataagg accttcaagt aggtcaaact ggtaaaactg 5340taagaccaac
tctttatatt gcatgtggta tatcaggagc tatccagcat ttagcaggta 5400tgcaagattc
agattacata attgctataa ataaagatgt agaagcccca ataatgaagg 5460tagcagattt
ggctatagtt ggtgatgtaa ataaagttgt accagaatta atagctcaag 5520ttaaagctgc
taataattaa gataaataaa aagaattatt taaagcttat tatgccaaaa 5580tacttatata
gtattttggt gtaaatgcat tgatagtttc tttaaattta gggaggtctg 5640tttaatgaaa
aaggtatgtg ttataggtgc aggtactatg ggttcaggaa ttgctcaggc 5700atttgcagct
aaaggatttg aagtagtatt aagagatatt aaagatgaat ttgttgatag 5760aggattagat
tttatcaata aaaatctttc taaattagtt aaaaaaggaa agatagaaga 5820agctactaaa
gttgaaatct taactagaat ttccggaaca gttgacctta atatggcagc 5880tgattgcgat
ttagttatag aagcagctgt tgaaagaatg gatattaaaa agcagatttt 5940tgctgactta
gacaatatat gcaagccaga aacaattctt gcatcaaata catcatcact 6000ttcaataaca
gaagtggcat cagcaactaa aagacctgat aaggttatag gtatgcattt 6060ctttaatcca
gctcctgtta tgaagcttgt agaggtaata agaggaatag ctacatcaca 6120agaaactttt
gatgcagtta aagagacatc tatagcaata ggaaaagatc ctgtagaagt 6180agcagaagca
ccaggatttg ttgtaaatag aatattaata ccaatgatta atgaagcagt 6240tggtatatta
gcagaaggaa tagcttcagt agaagacata gataaagcta tgaaacttgg 6300agctaatcac
ccaatgggac cattagaatt aggtgatttt ataggtcttg atatatgtct 6360tgctataatg
gatgttttat actcagaaac tggagattct aagtatagac cacatacatt 6420acttaagaag
tatgtaagag caggatggct tggaagaaaa tcaggaaaag gtttctacga 6480ttattcaaaa
taagtttaca agaatccgga tcctctagag tcgacgtcac gcgtccatgg 6540agatctcgag
gcctgcagac atgcaagctt ggcactggcc gtcgttttac aacgtcgtga 6600ctgggaaaac
cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag 6660ctggcgtaat
agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa 6720tggcgaatgg
cgctagcata aaaataagaa gcctgcattt gcaggcttct tatttttatg 6780gcgcgccgca
ttcacttctt ttctatataa atatgagcga agcgaataag cgtcggaaaa 6840gcagcaaaaa
gtttcctttt tgctgttgga gcatgggggt tcagggggtg cagtatctga 6900cgtcaatgcc
gagcgaaagc gagccgaagg gtagcattta cgttagataa ccccctgata 6960tgctccgacg
ctttatatag aaaagaagat tcaactaggt aaaatcttaa tataggttga 7020gatgataagg
tttataagga atttgtttgt tctaattttt cactcatttt gttctaattt 7080cttttaacaa
atgttctttt ttttttagaa cagttatgat atagttagaa tagtttaaaa 7140taaggagtga
gaaaaagatg aaagaaagat atggaacagt ctataaaggc tctcagaggc 7200tcatagacga
agaaagtgga gaagtcatag aggtagacaa gttataccgt aaacaaacgt 7260ctggtaactt
cgtaaaggca tatatagtgc aattaataag tatgttagat atgattggcg 7320gaaaaaaact
taaaatcgtt aactatatcc tagataatgt ccacttaagt aacaatacaa 7380tgatagctac
aacaagagaa atagcaaaag ctacaggaac aagtctacaa acagtaataa 7440caacacttaa
aatcttagaa gaaggaaata ttataaaaag aaaaactgga gtattaatgt 7500taaaccctga
actactaatg agaggcgacg accaaaaaca aaaatacctc ttactcgaat 7560ttgggaactt
tgagcaagag gcaaatgaaa tagattgacc tcccaataac accacgtagt 7620tattgggagg
tcaatctatg aaatgcgatt aagggccggc cgaagcaaac ttaagagtgt 7680gttgatagtg
cagtatctta aaattttgta taataggaat tgaagttaaa ttagatgcta 7740aaaatttgta
attaagaagg agtgattaca tgaacaaaaa tataaaatat tctcaaaact 7800ttttaacgag
tgaaaaagta ctcaaccaaa taataaaaca attgaattta aaagaaaccg 7860ataccgttta
cgaaattgga acaggtaaag ggcatttaac gacgaaactg gctaaaataa 7920gtaaacaggt
aacgtctatt gaattagaca gtcatctatt caacttatcg tcagaaaaat 7980taaaactgaa
tactcgtgtc actttaattc accaagatat tctacagttt caattcccta 8040acaaacagag
gtataaaatt gttgggagta ttccttacca tttaagcaca caaattatta 8100aaaaagtggt
ttttgaaagc catgcgtctg acatctatct gattgttgaa gaaggattct 8160acaagcgtac
cttggatatt caccgaacac tagggttgct cttgcacact caagtctcga 8220ttcagcaatt
gcttaagctg ccagcggaat gctttcatcc taaaccaaaa gtaaacagtg 8280tcttaataaa
acttacccgc cataccacag atgttccaga taaatattgg aagctatata 8340cgtactttgt
ttcaaaatgg gtcaatcgag aatatcgtca actgtttact aaaaatcagt 8400ttcatcaagc
aatgaaacac gccaaagtaa acaatttaag taccgttact tatgagcaag 8460tattgtctat
ttttaatagt tatctattat ttaacgggag gaaataattc tatgagtcgc 8520ttttgtaaat
ttggaaagtt acacgttact aaagggaatg tgtttaaact cctttttgat 8580aatctcatga
ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 8640gaaaagatca
aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 8700acaaaaaaac
caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 8760tttccgaagg
taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag 8820ccgtagttag
gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 8880atcctgttac
cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 8940agacgatagt
taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 9000cccagcttgg
agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 9060agcgccacgc
ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 9120acaggagagc
gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 9180gggtttcgcc
acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 9240ctatggaaaa
acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 9300gctcacatgt
tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt 9360gagtgagctg
ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag 9420gaagcggaag
agcgcccaat acgcagggcc ccctgcagg
94593237DNAArtificial sequencesynthetic primer 32cccgggatcc aagcttacgg
ctaccttgtt acgactt 373318DNAArtificial
sequencesynthetic primer 33tttgtaatta agaaggag
183418DNAArtificial sequencesynthetic primer
34gtagaatcct tcttcaac
183514DNAArtificial sequencesynthetic primer 35gctggagcag atat
143614DNAArtificial
sequencesynthetic primer 36gctgtcattc cttc
143718DNAArtificial sequencesynthetic primer
37cgtcagaccc cgtagaaa
183818DNAArtificial sequencesynthetic primer 38ctctcctgtt ccgaccct
18392688DNAC. autoethanogenum
39atgagaaatt tgtttatatt taacagcata aaaaataaga aagaggtgtc attaatgaag
60gtaactaagg taactaacgt tgaagaatta atgaaaaagt tagatgaagt aacggctgct
120caaaagaaat tttctagcta tactcaagaa caagtggatg aaattttcag gcaggcagct
180atggcagcca atagtgctag aatagactta gctaaaatgg cagtggaaga aagcggaatg
240ggaattgtag aagacaaggt cattaaaaat cattttgttg cagaatatat atataacaaa
300tataagggtg aaaagacctg cggagttctg gaacaagatg aaggctttgg tatggttaga
360attgcagaac ctgtaggagt tattgcagca gtagttccaa caactaatcc aacatctaca
420gcaatattta aatcactaat agctttaaaa actagaaatg gtatagtttt ttcaccacat
480ccaagggcaa aaaaatcaac tattgcagca gctaagatag tacttgatgc agcagttaaa
540gctggtgccc ctgaaggaat tataggctgg atagatgaac cttctattga actttcacag
600gtggtaatga aagaagcaga tctaattctt gcaactggtg gaccaggtat ggttaaggct
660gcctattctt caggaaagcc tgctatagga gttggtccag gtaatacacc tgctgtaatt
720gatgaaagtg ccgacattaa aatggcagta aattcaatac tactttcaaa aacttttgat
780aatggtatga tttgtgcttc agagcagtca gtaatagttg caagctcaat atacgatgaa
840gtcaagaaag agtttgcaga tagaggagca tatatattaa gtaaggatga aacagataag
900gttggaaaaa caatcatgat taatggagct ttaaatgctg gaattgtagg gcaaagtgcc
960tttaaaatag ctcagatggc gggagtcagt gtaccggaag atgctaaaat acttatagga
1020gaagttaaat cggtagaacc tgaagaagag ccctttgctc atgaaaagct gtctccagtt
1080ctagccatgt acaaagcaaa agattttgat gaagcacttc taaaggctgg aagattagtt
1140gaacgaggtg gaatagggca tacatctgta ttgtatgtaa attcgatgac ggaaaaagta
1200aaagtagaaa agttcagaga aactatgaag accggtagaa cattgataaa tatgccttca
1260gcgcaaggcg ctataggaga tatatataac tttaaactag ctccttcttt gacattaggc
1320tgtggttcct ggggaggaaa ctctgtatca gaaaatgttg gacctaaaca tttgttaaac
1380ataaagagtg ttgctgagag gagagaaaat atgctttggt ttagagtacc tgaaaaggtt
1440tatttcaaat atggcagcct tggagttgca ctaaaagaac tgagaattat ggagaagaaa
1500aaggcgttta tagtaacgga taaagttctt tatcaattag gttatgtaga taaaattaca
1560aagaacctcg atgaattaag agtttcatat aaaatattta cagatgtaga accagatcca
1620acccttgcta cagctaaaaa aggtgcagca gaactgcttt cctatgaacc agatacaatt
1680atagcagttg gtggtggttc ggcaatggat gctgccaaga tcatgtgggt aatgtatgag
1740catccagaag taagatttga agatttggcc atgagattta tggatataag aaagagagta
1800tatgtttttc ctaagatggg agaaaaggca atgatgattt cagtagcaac atccgcagga
1860acagggtcag aagttactcc atttgcagta attacggacg aaagaacagg agctaaatat
1920cctctggctg attatgaatt aactccaaac atggctatag ttgatgcaga acttatgatg
1980ggaatgccaa aggggctaac agcagcttca ggtatagatg cgttgactca tgcactggag
2040gcctatgtgt caataatggc ttcagaatat accaacggat tggctcttga agcaacaaga
2100ttagtattca aatatttgcc aatagcttat acagaaggta caattaatgt aaaggcaaga
2160gaaaaaatgg ctcatgcttc atgtattgca ggtatggcct ttgccaatgc atttttaggg
2220gtatgccact ctatggcaca taaattggga gcacagcacc acataccaca tggaattgcc
2280aatgcactta tgatagatga agttataaaa ttcaatgctg tagaggctcc aaggaaacaa
2340gcggcatttc cacaatataa atatccaaat gttaaaagaa gatatgctag aatagctgat
2400tacctaaatt taggtggaag tacagatgat gaaaaagtac aattgctaat aaatgctata
2460gatgacttaa aaactaagtt aaatattcca aagactatta aagaagcagg agtttcagaa
2520gataaattct atgctacttt agatacaatg tcagaactgg cttttgatga tcaatgtaca
2580ggagctaatc cacgatatcc actaatagga gaaataaaac aaatgtatat aaatgcattt
2640gatacaccaa aggcaactgt ggagaagaaa acaagaaaga aaaagtaa
268840895PRTC. autoethanogenum 40Met Arg Asn Leu Phe Ile Phe Asn Ser Ile
Lys Asn Lys Lys Glu Val1 5 10
15Ser Leu Met Lys Val Thr Lys Val Thr Asn Val Glu Glu Leu Met Lys
20 25 30Lys Leu Asp Glu Val Thr
Ala Ala Gln Lys Lys Phe Ser Ser Tyr Thr 35 40
45Gln Glu Gln Val Asp Glu Ile Phe Arg Gln Ala Ala Met Ala
Ala Asn 50 55 60Ser Ala Arg Ile Asp
Leu Ala Lys Met Ala Val Glu Glu Ser Gly Met65 70
75 80Gly Ile Val Glu Asp Lys Val Ile Lys Asn
His Phe Val Ala Glu Tyr 85 90
95Ile Tyr Asn Lys Tyr Lys Gly Glu Lys Thr Cys Gly Val Leu Glu Gln
100 105 110Asp Glu Gly Phe Gly
Met Val Arg Ile Ala Glu Pro Val Gly Val Ile 115
120 125Ala Ala Val Val Pro Thr Thr Asn Pro Thr Ser Thr
Ala Ile Phe Lys 130 135 140Ser Leu Ile
Ala Leu Lys Thr Arg Asn Gly Ile Val Phe Ser Pro His145
150 155 160Pro Arg Ala Lys Lys Ser Thr
Ile Ala Ala Ala Lys Ile Val Leu Asp 165
170 175Ala Ala Val Lys Ala Gly Ala Pro Glu Gly Ile Ile
Gly Trp Ile Asp 180 185 190Glu
Pro Ser Ile Glu Leu Ser Gln Val Val Met Lys Glu Ala Asp Leu 195
200 205Ile Leu Ala Thr Gly Gly Pro Gly Met
Val Lys Ala Ala Tyr Ser Ser 210 215
220Gly Lys Pro Ala Ile Gly Val Gly Pro Gly Asn Thr Pro Ala Val Ile225
230 235 240Asp Glu Ser Ala
Asp Ile Lys Met Ala Val Asn Ser Ile Leu Leu Ser 245
250 255Lys Thr Phe Asp Asn Gly Met Ile Cys Ala
Ser Glu Gln Ser Val Ile 260 265
270Val Ala Ser Ser Ile Tyr Asp Glu Val Lys Lys Glu Phe Ala Asp Arg
275 280 285Gly Ala Tyr Ile Leu Ser Lys
Asp Glu Thr Asp Lys Val Gly Lys Thr 290 295
300Ile Met Ile Asn Gly Ala Leu Asn Ala Gly Ile Val Gly Gln Ser
Ala305 310 315 320Phe Lys
Ile Ala Gln Met Ala Gly Val Ser Val Pro Glu Asp Ala Lys
325 330 335Ile Leu Ile Gly Glu Val Lys
Ser Val Glu Pro Glu Glu Glu Pro Phe 340 345
350Ala His Glu Lys Leu Ser Pro Val Leu Ala Met Tyr Lys Ala
Lys Asp 355 360 365Phe Asp Glu Ala
Leu Leu Lys Ala Gly Arg Leu Val Glu Arg Gly Gly 370
375 380Ile Gly His Thr Ser Val Leu Tyr Val Asn Ser Met
Thr Glu Lys Val385 390 395
400Lys Val Glu Lys Phe Arg Glu Thr Met Lys Thr Gly Arg Thr Leu Ile
405 410 415Asn Met Pro Ser Ala
Gln Gly Ala Ile Gly Asp Ile Tyr Asn Phe Lys 420
425 430Leu Ala Pro Ser Leu Thr Leu Gly Cys Gly Ser Trp
Gly Gly Asn Ser 435 440 445Val Ser
Glu Asn Val Gly Pro Lys His Leu Leu Asn Ile Lys Ser Val 450
455 460Ala Glu Arg Arg Glu Asn Met Leu Trp Phe Arg
Val Pro Glu Lys Val465 470 475
480Tyr Phe Lys Tyr Gly Ser Leu Gly Val Ala Leu Lys Glu Leu Arg Ile
485 490 495Met Glu Lys Lys
Lys Ala Phe Ile Val Thr Asp Lys Val Leu Tyr Gln 500
505 510Leu Gly Tyr Val Asp Lys Ile Thr Lys Asn Leu
Asp Glu Leu Arg Val 515 520 525Ser
Tyr Lys Ile Phe Thr Asp Val Glu Pro Asp Pro Thr Leu Ala Thr 530
535 540Ala Lys Lys Gly Ala Ala Glu Leu Leu Ser
Tyr Glu Pro Asp Thr Ile545 550 555
560Ile Ala Val Gly Gly Gly Ser Ala Met Asp Ala Ala Lys Ile Met
Trp 565 570 575Val Met Tyr
Glu His Pro Glu Val Arg Phe Glu Asp Leu Ala Met Arg 580
585 590Phe Met Asp Ile Arg Lys Arg Val Tyr Val
Phe Pro Lys Met Gly Glu 595 600
605Lys Ala Met Met Ile Ser Val Ala Thr Ser Ala Gly Thr Gly Ser Glu 610
615 620Val Thr Pro Phe Ala Val Ile Thr
Asp Glu Arg Thr Gly Ala Lys Tyr625 630
635 640Pro Leu Ala Asp Tyr Glu Leu Thr Pro Asn Met Ala
Ile Val Asp Ala 645 650
655Glu Leu Met Met Gly Met Pro Lys Gly Leu Thr Ala Ala Ser Gly Ile
660 665 670Asp Ala Leu Thr His Ala
Leu Glu Ala Tyr Val Ser Ile Met Ala Ser 675 680
685Glu Tyr Thr Asn Gly Leu Ala Leu Glu Ala Thr Arg Leu Val
Phe Lys 690 695 700Tyr Leu Pro Ile Ala
Tyr Thr Glu Gly Thr Ile Asn Val Lys Ala Arg705 710
715 720Glu Lys Met Ala His Ala Ser Cys Ile Ala
Gly Met Ala Phe Ala Asn 725 730
735Ala Phe Leu Gly Val Cys His Ser Met Ala His Lys Leu Gly Ala Gln
740 745 750His His Ile Pro His
Gly Ile Ala Asn Ala Leu Met Ile Asp Glu Val 755
760 765Ile Lys Phe Asn Ala Val Glu Ala Pro Arg Lys Gln
Ala Ala Phe Pro 770 775 780Gln Tyr Lys
Tyr Pro Asn Val Lys Arg Arg Tyr Ala Arg Ile Ala Asp785
790 795 800Tyr Leu Asn Leu Gly Gly Ser
Thr Asp Asp Glu Lys Val Gln Leu Leu 805
810 815Ile Asn Ala Ile Asp Asp Leu Lys Thr Lys Leu Asn
Ile Pro Lys Thr 820 825 830Ile
Lys Glu Ala Gly Val Ser Glu Asp Lys Phe Tyr Ala Thr Leu Asp 835
840 845Thr Met Ser Glu Leu Ala Phe Asp Asp
Gln Cys Thr Gly Ala Asn Pro 850 855
860Arg Tyr Pro Leu Ile Gly Glu Ile Lys Gln Met Tyr Ile Asn Ala Phe865
870 875 880Asp Thr Pro Lys
Ala Thr Val Glu Lys Lys Thr Arg Lys Lys Lys 885
890 895412613DNAC. autoethanogenum 41atgaaagtta
caaacgtaga agaactaatg aaaagactag aagaaataaa ggatgctcaa 60aagaaatttg
ctacatatac tcaagaacaa gtggatgaaa tttttagaca agcagctatg 120gcagctaata
gtgctagaat agaactagct aaaatggcag tagaagaaag cggaatggga 180attgtagaag
acaaggttat taaaaatcac tttgcttcag aatatatata taacaaatat 240aaggatgaaa
aaacctgtgg agttttagag agagatgcag gctttggtat agttagaatt 300gcggaacctg
taggagttat tgcagcagta gttccaacaa ctaatccaac atctacagca 360atatttaaat
cactaatagc tttaaaaact agaaatggta taattttttc accccatcca 420agggcaaaga
aatcaactat tgcagcagct aaaatagtac ttgacgctgc agttaaagct 480ggtgctcctg
aaggaattat aggatggata gatgaacctt ccattgaact ttcacaggtg 540gtaatgggag
aagcaaattt aattcttgca actggtggtc cgggtatggt taaggctgcc 600tattcttcag
gcaaacctgc tgtgggagtt ggtccaggta acacacctgc tgtaattgat 660gaaagtgccg
acattaaaat ggcagtaaat tcaatattac tatcaaaaac ttttgataat 720ggtatgattt
gtgcctcaga gcagtcagta atagttttag actcaatata tgaggaagtt 780aaaaaagaat
ttgcttatag gggtgcttat atattaagta aggatgaaac agataaggtt 840ggaaaaataa
ttttaaaaaa tggagcctta aatgcaggta ttgtaggaca acctgctttt 900aaaatagcac
agctggcagg agtggatgta ccagaaaaag ctaaagtact tataggagag 960gtagaatcgg
tagaacttga agaaccattt tctcatgaaa agttatctcc agttttagct 1020atgtacaggg
caagaaattt tgaggatgcc attgcaaaaa ctgataaact ggttagggca 1080ggtggatttg
gacatacatc ttcattgtat ataaatccaa tgacagaaaa agcaaaagta 1140gaaaaattta
gtactatgat gaaaacatca agaactataa ttaacacacc ttcatcccaa 1200ggtggtatag
gtgatatata taactttaaa ctagctcctt ctttgacatt aggctgcggt 1260tcctgggggg
gaaattctgt atccgaaaat gttgggccta aacatttatt aaacataaaa 1320agtgttgctg
agaggagaga aaatatgctt tggtttagag tacctgaaaa ggtttatttc 1380aaatatggta
gtcttggagt tgcattaaaa gagttaaaag ttatgaataa gaagaaagta 1440tttatagtaa
cagataaagt tctttatcaa ttaggttatg tggacaaagt tacaaaagtt 1500cttgaggaac
taaaaatttc ctataaggta tttacagatg tagaaccaga tccaaccctt 1560gctacagcta
aaaaaggtgc agcagaactg ctttcctatg aaccggatac aattatatca 1620gttggtggtg
gttcagcaat ggatgcagct aagatcatgt gggtaatgta tgagcatcca 1680gaagtaaaat
ttgaagattt agctatgaga tttatggata taagaaagag agtatatgtt 1740ttccctaaga
tgggagaaaa ggcaatgatg atttcagtag caacatccgc aggaacaggg 1800tcggaagtta
ctccatttgc agtaatcact gatgaaaaaa caggagctaa atatccatta 1860gctgattatg
aactaactcc agacatggct atagttgatg cagaacttat gatgggaatg 1920ccaagaggac
ttacagcagc ttcgggtata gatgcattaa cccatgcact ggaggcgtat 1980gtgtcaataa
tggctacaga atttaccaat ggattagccc ttgaagcagt aaagttgata 2040tttgaatatt
taccaaaagc ttatacagaa ggtacaacta atgtaaaggc aagagaaaag 2100atggctcatg
cttcatgtat tgcaggtatg gcctttgcaa atgcattttt aggggtatgc 2160cactctatgg
cacataaatt gggagcacag catcacatac cacatggaat tgccaatgca 2220cttatgatag
atgaagttat aaaattcaat gctgtagatg atccaataaa acaagctgca 2280tttccccaat
acgagtatcc aaatgctagg tatagatatg ctcagatagc tgattgtctg 2340aacttgggag
gaaatacaga agaggaaaag gtacaactat taataaatgc tatagatgat 2400ttaaaagcta
agttaaatat tccagaaact ataaaagaag caggagtttc agaagataaa 2460ttctatgcta
ctttagataa aatgtcagaa ttagcttttg atgatcagtg tacaggagct 2520aatccaagat
atccactgat aagtgaaata aaacaaatgt atataaatgt ttttgataaa 2580accgaaccaa
ttgtagaaga tgaagaaaag taa 261342508PRTC.
autoethanogenum 42Met Lys Val Thr Asn Val Glu Glu Leu Met Lys Arg Leu Glu
Glu Ile1 5 10 15Lys Asp
Ala Gln Lys Lys Phe Ala Thr Tyr Thr Gln Glu Gln Val Asp 20
25 30Glu Ile Phe Arg Gln Ala Ala Met Ala
Ala Asn Ser Ala Arg Ile Glu 35 40
45Leu Ala Lys Met Ala Val Glu Glu Ser Gly Met Gly Ile Val Glu Asp 50
55 60Lys Val Ile Lys Asn His Phe Ala Ser
Glu Tyr Ile Tyr Asn Lys Tyr65 70 75
80Lys Asp Glu Lys Thr Cys Gly Val Leu Glu Arg Asp Ala Gly
Phe Gly 85 90 95Ile Val
Arg Ile Ala Glu Pro Val Gly Val Ile Ala Ala Val Val Pro 100
105 110Thr Thr Asn Pro Thr Ser Thr Ala Ile
Phe Lys Ser Leu Ile Ala Leu 115 120
125Lys Thr Arg Asn Gly Ile Ile Phe Ser Pro His Pro Arg Ala Lys Lys
130 135 140Ser Thr Ile Ala Ala Ala Lys
Ile Val Leu Asp Ala Ala Val Lys Ala145 150
155 160Gly Ala Pro Glu Gly Ile Ile Gly Trp Ile Asp Glu
Pro Ser Ile Glu 165 170
175Leu Ser Gln Val Val Met Gly Glu Ala Asn Leu Ile Leu Ala Thr Gly
180 185 190Gly Pro Gly Met Val Lys
Ala Ala Tyr Ser Ser Gly Lys Pro Ala Val 195 200
205Gly Val Gly Pro Gly Asn Thr Pro Ala Val Ile Asp Glu Ser
Ala Asp 210 215 220Ile Lys Met Ala Val
Asn Ser Ile Leu Leu Ser Lys Thr Phe Asp Asn225 230
235 240Gly Met Ile Cys Ala Ser Glu Gln Ser Val
Ile Val Leu Asp Ser Ile 245 250
255Tyr Glu Glu Val Lys Lys Glu Phe Ala Tyr Arg Gly Ala Tyr Ile Leu
260 265 270Ser Lys Asp Glu Thr
Asp Lys Val Gly Lys Ile Ile Leu Lys Asn Gly 275
280 285Ala Leu Asn Ala Gly Ile Val Gly Gln Pro Ala Phe
Lys Ile Ala Gln 290 295 300Leu Ala Gly
Val Asp Val Pro Glu Lys Ala Lys Val Leu Ile Gly Glu305
310 315 320Val Glu Ser Val Glu Leu Glu
Glu Pro Phe Ser His Glu Lys Leu Ser 325
330 335Pro Val Leu Ala Met Tyr Arg Ala Arg Asn Phe Glu
Asp Ala Ile Ala 340 345 350Lys
Thr Asp Lys Leu Val Arg Ala Gly Gly Phe Gly His Thr Ser Ser 355
360 365Leu Tyr Ile Asn Pro Met Thr Glu Lys
Ala Lys Val Glu Lys Phe Ser 370 375
380Thr Met Met Lys Thr Ser Arg Thr Ile Ile Asn Thr Pro Ser Ser Gln385
390 395 400Gly Gly Ile Gly
Asp Ile Tyr Asn Phe Lys Leu Ala Pro Ser Leu Thr 405
410 415Leu Gly Cys Gly Ser Trp Gly Gly Asn Ser
Val Ser Glu Asn Val Gly 420 425
430Pro Lys His Leu Leu Asn Ile Lys Ser Val Ala Glu Arg Arg Glu Asn
435 440 445Met Leu Trp Phe Arg Val Pro
Glu Lys Val Tyr Phe Lys Tyr Gly Ser 450 455
460Leu Gly Val Ala Leu Lys Glu Leu Lys Val Met Asn Lys Lys Lys
Val465 470 475 480Phe Ile
Val Thr Asp Lys Val Leu Tyr Gln Leu Gly Tyr Val Asp Lys
485 490 495Val Thr Lys Val Leu Glu Glu
Leu Lys Asn Phe Leu 500 505431554DNAC.
autoethanogenum 43ttggaaaatt ttgataaaga cttacgttct atacaagaag caagagatct
tgcacgttta 60ggaaaaattg cagcagacca aattgctgat tatactgaag aacaaattga
taaaatccta 120tgtaatatgg ttagggtagc agaagaaaat gcagtttgcc ttggtaaaat
ggctgcagaa 180gaaactggtt ttggaaaagc tgaagataag gcttataaga accatatggc
tgctactaca 240gtatataatt acatcaagga tatgaagact attggtgtta taaaagaaga
taaaagtgaa 300ggtgtaattg aatttgcaga accagttggt ttattaatgg gtattgtacc
atctacaaat 360ccaacatcta ctgttattta taaatcaatc attgcaatta aatcaagaaa
tgcaattgta 420ttctcaccac acccagctgc attaaaatgt tcaacaaaag caatagaact
tatgcgtgat 480gcagcagtag cagcaggagc tcctgcaaat gtaattggtg gtattgttac
accatctata 540caagctacaa atgaacttat gaaagctaaa gaagttgcta tgataattgc
aactggaggc 600cctggaatgg taaaggctgc atatagttca ggaacacctg caataggcgt
tggtgctggt 660aactctccat cctatattga aagaactgct gatgttcatc aatcagttaa
agatataata 720gctagtaaga gttttgacta tggtactatt tgtgcatccg agcagtctgt
aattgcagaa 780gaatgcaacc atgatgaaat agtagctgaa tttaagaaac aaggcggata
tttcatgaca 840gctgaagaaa ctgcaaaagt ttgcagcgta ctttttaaac ctggtacaca
cagcatgagc 900gctaagtttg taggaagagc tcctcaggtt atagcagaag ctgcaggttt
cacagttcca 960gaaggaacaa aagtattaat aggagaacaa ggcggagttg gtaatggtta
ccctctatct 1020tatgagaaac ttacaacagt acttgctttc tatacagtta aagattggca
tgaagcatgt 1080gagcttagta taagattact tcaaaatggt cttggacata caatgaacat
tcatacaaat 1140gatagagact tagtaatgaa gtttgctaaa aaaccagcat cccgtatctt
agttaatact 1200ggtggaagcc agggaggtac tggtgcaagc acaggattag cacctgcatt
tacattaggt 1260tgtggtacat ggggaggaag ctctgtttct gaaaatgtta ctccattaca
tttaatcaat 1320ataaagagag tagcatatgg tcttaaagat tgtactacat tagctgcaga
cgatacaact 1380ttcaatcatc ctgaactttg cggaagcaaa aatgacttag gattctgtgc
tacaagccct 1440gcagaatttg cagcaaagag caattgtgat agcactgctg cagatactac
tgataatgat 1500aaacttgcta gactcgtaag tgaattagta gctgcaatga agggagctaa
ctaa 155444517PRTC. autoethanogenum 44Met Glu Asn Phe Asp Lys Asp
Leu Arg Ser Ile Gln Glu Ala Arg Asp1 5 10
15Leu Ala Arg Leu Gly Lys Ile Ala Ala Asp Gln Ile Ala
Asp Tyr Thr 20 25 30Glu Glu
Gln Ile Asp Lys Ile Leu Cys Asn Met Val Arg Val Ala Glu 35
40 45Glu Asn Ala Val Cys Leu Gly Lys Met Ala
Ala Glu Glu Thr Gly Phe 50 55 60Gly
Lys Ala Glu Asp Lys Ala Tyr Lys Asn His Met Ala Ala Thr Thr65
70 75 80Val Tyr Asn Tyr Ile Lys
Asp Met Lys Thr Ile Gly Val Ile Lys Glu 85
90 95Asp Lys Ser Glu Gly Val Ile Glu Phe Ala Glu Pro
Val Gly Leu Leu 100 105 110Met
Gly Ile Val Pro Ser Thr Asn Pro Thr Ser Thr Val Ile Tyr Lys 115
120 125Ser Ile Ile Ala Ile Lys Ser Arg Asn
Ala Ile Val Phe Ser Pro His 130 135
140Pro Ala Ala Leu Lys Cys Ser Thr Lys Ala Ile Glu Leu Met Arg Asp145
150 155 160Ala Ala Val Ala
Ala Gly Ala Pro Ala Asn Val Ile Gly Gly Ile Val 165
170 175Thr Pro Ser Ile Gln Ala Thr Asn Glu Leu
Met Lys Ala Lys Glu Val 180 185
190Ala Met Ile Ile Ala Thr Gly Gly Pro Gly Met Val Lys Ala Ala Tyr
195 200 205Ser Ser Gly Thr Pro Ala Ile
Gly Val Gly Ala Gly Asn Ser Pro Ser 210 215
220Tyr Ile Glu Arg Thr Ala Asp Val His Gln Ser Val Lys Asp Ile
Ile225 230 235 240Ala Ser
Lys Ser Phe Asp Tyr Gly Thr Ile Cys Ala Ser Glu Gln Ser
245 250 255Val Ile Ala Glu Glu Cys Asn
His Asp Glu Ile Val Ala Glu Phe Lys 260 265
270Lys Gln Gly Gly Tyr Phe Met Thr Ala Glu Glu Thr Ala Lys
Val Cys 275 280 285Ser Val Leu Phe
Lys Pro Gly Thr His Ser Met Ser Ala Lys Phe Val 290
295 300Gly Arg Ala Pro Gln Val Ile Ala Glu Ala Ala Gly
Phe Thr Val Pro305 310 315
320Glu Gly Thr Lys Val Leu Ile Gly Glu Gln Gly Gly Val Gly Asn Gly
325 330 335Tyr Pro Leu Ser Tyr
Glu Lys Leu Thr Thr Val Leu Ala Phe Tyr Thr 340
345 350Val Lys Asp Trp His Glu Ala Cys Glu Leu Ser Ile
Arg Leu Leu Gln 355 360 365Asn Gly
Leu Gly His Thr Met Asn Ile His Thr Asn Asp Arg Asp Leu 370
375 380Val Met Lys Phe Ala Lys Lys Pro Ala Ser Arg
Ile Leu Val Asn Thr385 390 395
400Gly Gly Ser Gln Gly Gly Thr Gly Ala Ser Thr Gly Leu Ala Pro Ala
405 410 415Phe Thr Leu Gly
Cys Gly Thr Trp Gly Gly Ser Ser Val Ser Glu Asn 420
425 430Val Thr Pro Leu His Leu Ile Asn Ile Lys Arg
Val Ala Tyr Gly Leu 435 440 445Lys
Asp Cys Thr Thr Leu Ala Ala Asp Asp Thr Thr Phe Asn His Pro 450
455 460Glu Leu Cys Gly Ser Lys Asn Asp Leu Gly
Phe Cys Ala Thr Ser Pro465 470 475
480Ala Glu Phe Ala Ala Lys Ser Asn Cys Asp Ser Thr Ala Ala Asp
Thr 485 490 495Thr Asp Asn
Asp Lys Leu Ala Arg Leu Val Ser Glu Leu Val Ala Ala 500
505 510Met Lys Gly Ala Asn 515451446DNAC.
autoethanogenum 45gtggaaaatg ctgcacgagc acaaaaaatg ttagcaacct ttccacaaga
aaagctagat 60gagattgttg aacgtatggc ggaagaaatc ggaaaacata cccgagagct
tgctgtaatg 120tcacaggatg aaactggtta tggaaaatgg caggataaat gcatcaaaaa
ccgatttgcc 180tgtgagtatt tgccagctaa gcttagagga atgcgatgtg taggtattat
taatgaaaat 240ggtcaggata agaccatgga tgtaggtgta cctatgggtg taattattgc
attatgtcct 300gcaactagtc cggtttctac taccatatat aaggcattga ttgcaattaa
gtctggtaat 360gcaattatct tttctccaca tcctagagca aaggagacaa tttgtaaggc
gcttgacatc 420atgattcgtg cagctgaagg atatgggctt ccagaaggag ctcttgcata
cttacatact 480gtgacgccta gtggaacaat cgaattgatg aaccatattg cgacttcttt
gattatgaat 540acaggtgttc ccgggatgct taaagcagca tataattctg ggaaacctgt
tatatatgga 600ggaactggta atggaccagc atttattgaa cgtacagctg acatcaaaca
ggcggtaaaa 660gatattattg ctagtaagac ctttgataac ggaatagtac catcagctga
acaatctatt 720gttgtagata gctgtgttgc atctgatgtt aaacgtgagt tgcaaaataa
tggtgcatat 780ttcatgacag aggaggaagc acaaaaacta ggttctctct ttttccgttc
tgatggcagt 840atggattcag aaatggttgg caaatccgca caaagattgg ctaaaaaagc
aggtttcagc 900attcctgaaa gtagcacagt gctaatttca gagcagaaat atgtttctca
agataatcct 960tattccaagg agaaactttg tccggtacta gcttactaca ttgaagatga
ttggatgcat 1020gcatgtgaaa agtgtattga actgctgtta agtgagagac atggtcacac
tcttgttata 1080cattcaaaag acgaagatgt aattcgccag tttgcattaa aaaaacctgt
aggtaggata 1140cttgttaata cgcctgcttc ctttggtagt atgggtgcta caagtaattt
atttcctgct 1200ttaactttag gtagtggatc ggcaggtaaa ggtattacct ccgataatgt
ttcaccaatg 1260aatcttattt acgtccgcaa agtcggatat ggcgtacgga atgtagaaga
gattgtcaat 1320actaatggat tgtttacaga agaaaaaagt gatttgaatg gaatgacaaa
aaagtcagac 1380tataatccag aggatataca aatgttacag catattttaa aaaaagctat
ggaaaaaatt 1440aaatag
144646481PRTC. autoethanogenum 46Met Glu Asn Ala Ala Arg Ala
Gln Lys Met Leu Ala Thr Phe Pro Gln1 5 10
15Glu Lys Leu Asp Glu Ile Val Glu Arg Met Ala Glu Glu
Ile Gly Lys 20 25 30His Thr
Arg Glu Leu Ala Val Met Ser Gln Asp Glu Thr Gly Tyr Gly 35
40 45Lys Trp Gln Asp Lys Cys Ile Lys Asn Arg
Phe Ala Cys Glu Tyr Leu 50 55 60Pro
Ala Lys Leu Arg Gly Met Arg Cys Val Gly Ile Ile Asn Glu Asn65
70 75 80Gly Gln Asp Lys Thr Met
Asp Val Gly Val Pro Met Gly Val Ile Ile 85
90 95Ala Leu Cys Pro Ala Thr Ser Pro Val Ser Thr Thr
Ile Tyr Lys Ala 100 105 110Leu
Ile Ala Ile Lys Ser Gly Asn Ala Ile Ile Phe Ser Pro His Pro 115
120 125Arg Ala Lys Glu Thr Ile Cys Lys Ala
Leu Asp Ile Met Ile Arg Ala 130 135
140Ala Glu Gly Tyr Gly Leu Pro Glu Gly Ala Leu Ala Tyr Leu His Thr145
150 155 160Val Thr Pro Ser
Gly Thr Ile Glu Leu Met Asn His Ile Ala Thr Ser 165
170 175Leu Ile Met Asn Thr Gly Val Pro Gly Met
Leu Lys Ala Ala Tyr Asn 180 185
190Ser Gly Lys Pro Val Ile Tyr Gly Gly Thr Gly Asn Gly Pro Ala Phe
195 200 205Ile Glu Arg Thr Ala Asp Ile
Lys Gln Ala Val Lys Asp Ile Ile Ala 210 215
220Ser Lys Thr Phe Asp Asn Gly Ile Val Pro Ser Ala Glu Gln Ser
Ile225 230 235 240Val Val
Asp Ser Cys Val Ala Ser Asp Val Lys Arg Glu Leu Gln Asn
245 250 255Asn Gly Ala Tyr Phe Met Thr
Glu Glu Glu Ala Gln Lys Leu Gly Ser 260 265
270Leu Phe Phe Arg Ser Asp Gly Ser Met Asp Ser Glu Met Val
Gly Lys 275 280 285Ser Ala Gln Arg
Leu Ala Lys Lys Ala Gly Phe Ser Ile Pro Glu Ser 290
295 300Ser Thr Val Leu Ile Ser Glu Gln Lys Tyr Val Ser
Gln Asp Asn Pro305 310 315
320Tyr Ser Lys Glu Lys Leu Cys Pro Val Leu Ala Tyr Tyr Ile Glu Asp
325 330 335Asp Trp Met His Ala
Cys Glu Lys Cys Ile Glu Leu Leu Leu Ser Glu 340
345 350Arg His Gly His Thr Leu Val Ile His Ser Lys Asp
Glu Asp Val Ile 355 360 365Arg Gln
Phe Ala Leu Lys Lys Pro Val Gly Arg Ile Leu Val Asn Thr 370
375 380Pro Ala Ser Phe Gly Ser Met Gly Ala Thr Ser
Asn Leu Phe Pro Ala385 390 395
400Leu Thr Leu Gly Ser Gly Ser Ala Gly Lys Gly Ile Thr Ser Asp Asn
405 410 415Val Ser Pro Met
Asn Leu Ile Tyr Val Arg Lys Val Gly Tyr Gly Val 420
425 430Arg Asn Val Glu Glu Ile Val Asn Thr Asn Gly
Leu Phe Thr Glu Glu 435 440 445Lys
Ser Asp Leu Asn Gly Met Thr Lys Lys Ser Asp Tyr Asn Pro Glu 450
455 460Asp Ile Gln Met Leu Gln His Ile Leu Lys
Lys Ala Met Glu Lys Ile465 470 475
480Lys47490DNAC. autoethanogenum 47aagcggccgc aaaatagttg
ataataatgc agagttataa acaaaggtga aaagcattac 60ttgtattctt ttttatatat
tattataaat taaaatgaag ctgtattaga aaaaatacac 120acctgtaata taaaatttta
aattaatttt taattttttc aaaatgtatt ttacatgttt 180agaattttga tgtatattaa
aatagtagaa tacataagat acttaattta attaaagata 240gttaagtact tttcaatgtg
cttttttaga tgtttaatac aaatctttaa ttgtaaaaga 300aatgctgtac tatttactgt
actagtgacg ggattaaact gtattaatta taaataaaaa 360ataagtacag ttgtttaaaa
ttatattttg tattaaatct aatagtacga tgtaagttat 420tttatactat tgctagttta
ataaaaagat ttaattatat gcttgaaaag gagaggaatc 480catatgcgta
49048500DNAC.
autoethanogenum 48ataccataaa ttacttgaaa aatagttgat aataatgtag agttataaac
aaaggtgaaa 60agcattactt gtattctttt ttatatatta ttataaatta aaatgaagct
gtattagaaa 120aaatacacac ctgtaatata aaattttaaa ttaattttta attttttcaa
aatgtatttt 180acatgtttag aattttgatg tatattaaaa tagtagaata cataagatac
ttaatttaat 240taaagatagt taagtacttt tcaatgtgct tttttagatg tttaatacaa
atctttaatt 300gtaaaagaaa tgctgtacta tttactgtac tagtgacggg attaaactgt
attaattata 360aataaaaaat aagtacagtt gtttaaaatt atattttgta ttaaatctaa
tagtacgatg 420taagttattt tatactattg ctagtttaat aaaaagattt aattatatac
ttgaaaagga 480gaggaatttt tatgcgtaaa
50049200DNAC. autoethanogenum 49tagaaaaaca tgtatacaaa
attaaaaaac tattataaca catagtatca atattgaagg 60taatactgtt caatatcgat
acagataaaa aaaatatata atacagaaga aaaaattata 120aatttgtggt ataatataaa
gtatagtaat ttaagtttaa acctcgtgaa aacgctaaca 180aataatagga ggtgtattat
20050300DNAC.
autoethanogenum 50atctgtatat tttttcccat tttaattatt tgtactataa tattacactg
agtgtattgt 60atatttaaaa aatatttggt acaattagtt agttaaataa attctaaatt
gtaaattatc 120agaatcctta ttaaggaaat acatagattt aaggagaaat cataaaaagg
tgtaatataa 180actggctaaa attgagcaaa aattgagcaa ttaagacttt ttgattgtat
ctttttatat 240atttaaggta tataatctta tttatattgg gggaacttga tgaataaaca
tattctagac 300512613DNAC. autoethanogenum 51atgaaagtta caaacgtaga
agaactaatg aaaagactag aagaaataaa ggatgctcaa 60aagaaatttg ctacatatac
tcaagaacaa gtggatgaaa tttttagaca agcagctatg 120gcagctaata gtgctagaat
agaactagct aaaatggcag tagaagaaag cggaatggga 180attgtagaag acaaggttat
taaaaatcac tttgcttcag aatatatata taacaaatat 240aaggatgaaa aaacctgtgg
agttttagag agagatgcag gctttggtat agttagaatt 300gcggaacctg taggagttat
tgcagcagta gttccaacaa ctaatccaac atctacagca 360atatttaaat cactaatagc
tttaaaaact agaaatggta taattttttc accccatcca 420agggcaaaga aatcaactat
tgcagcagct aaaatagtac ttgacgctgc agttaaagct 480ggtgctcctg aaggaattat
aggatggata gatgaacctt ccattgaact ttcacaggtg 540gtaatgggag aagcaaattt
aattcttgca actggtggtc cgggtatggt taaggctgcc 600tattcttcag gcaaacctgc
tgtgggagtt ggtccaggta acacacctgc tgtaattgat 660gaaagtgccg acattaaaat
ggcagtaaat tcaatattac tatcaaaaac ttttgataat 720ggtatgattt gtgcctcaga
gcagtcagta atagttttag actcaatata tgaggaagtt 780aaaaaagaat ttgcttatag
gggtgcttat atattaagta aggatgaaac agataaggtt 840ggaaaaataa ttttaaaaaa
tggagcctta aatgcaggta ttgtaggaca acctgctttt 900aaaatagcac agctggcagg
agtggatgta ccagaaaaag ctaaagtact tataggagag 960gtagaatcgg tagaacttga
agaaccattt tctcatgaaa agttatctcc agttttagct 1020atgtacaggg caagaaattt
tgaggatgcc attgcaaaaa ctgataaact ggttagggca 1080ggtggatttg gacatacatc
ttcattgtat ataaatccaa tgacagaaaa agcaaaagta 1140gaaaaattta gtactatgat
gaaaacatca agaactataa ttaacacacc ttcatcccaa 1200ggtggtatag gtgatatata
taactttaaa ctagctcctt ctttgacatt aggctgcggt 1260tcctgggggg gaaattctgt
atccgaaaat gttgggccta aacatttatt aaacataaaa 1320agtgttgctg agaggagaga
aaatatgctt tggtttagag tacctgaaaa ggtttatttc 1380aaatatggta gtcttggagt
tgcattaaaa gagttaaaag ttatgaataa gaagaaagta 1440tttatagtaa cagataaagt
tctttatcaa ttaggttatg tggacaaagt tacaaaagtt 1500cttgaggaac taaaaatttc
ctataaggta tttacagatg tagaaccaga tccaaccctt 1560gctacagcta aaaaaggtgc
agcagaactg ctttcctatg aaccggatac aattatatca 1620gttggtggtg gttcagcaat
ggatgcagct aagatcatgt gggtaatgta tgagcatcca 1680gaagtaaaat ttgaagattt
agctatgaga tttatggata taagaaagag agtatatgtt 1740ttccctaaga tgggagaaaa
ggcaatgatg atttcagtag caacatccgc aggaacaggg 1800tcggaagtta ctccatttgc
agtaatcact gatgaaaaaa caggagctaa atatccatta 1860gctgattatg aactaactcc
agacatggct atagttgatg cagaacttat gatgggaatg 1920ccaagaggac ttacagcagc
ttcgggtata gatgcattaa cccatgcact ggaggcgtat 1980gtgtcaataa tggctacaga
atttaccaat ggattagccc ttgaagcagt aaagttgata 2040tttgaatatt taccaaaagc
ttatacagaa ggtacaacta atgtaaaggc aagagaaaag 2100atggctcatg cttcatgtat
tgcaggtatg gcctttgcaa atgcattttt aggggtatgc 2160cactctatgg cacataaatt
gggagcacag catcacatac cacatggaat tgccaatgca 2220cttatgatag atgaagttat
aaaattcaat gctgtagatg atccaataaa acaagctgca 2280tttccccaat acgagtatcc
aaatgctagg tatagatatg ctcagatagc tgattgtctg 2340aacttgggag gaaatacaga
agaggaaaag gtacaactat taataaatgc tatagatgat 2400ttaaaagcta agttaaatat
tccagaaact ataaaagaag caggagtttc agaagataaa 2460ttctatgcta ctttagataa
aatgtcagaa ttagcttttg atgatcagtg tacaggagct 2520aatccaagat atccactgat
aagtgaaata aaacaaatgt atataaatgt ttttgataaa 2580accgaaccaa ttgtagaaga
tgaagaaaag taa 261352324PRTC.
autoethanogenum 52Met Asp Ala Ala Lys Ile Met Trp Val Met Tyr Glu His Pro
Glu Val1 5 10 15Lys Phe
Glu Asp Leu Ala Met Arg Phe Met Asp Ile Arg Lys Arg Val 20
25 30Tyr Val Phe Pro Lys Met Gly Glu Lys
Ala Met Met Ile Ser Val Ala 35 40
45Thr Ser Ala Gly Thr Gly Ser Glu Val Thr Pro Phe Ala Val Ile Thr 50
55 60Asp Glu Lys Thr Gly Ala Lys Tyr Pro
Leu Ala Asp Tyr Glu Leu Thr65 70 75
80Pro Asp Met Ala Ile Val Asp Ala Glu Leu Met Met Gly Met
Pro Arg 85 90 95Gly Leu
Thr Ala Ala Ser Gly Ile Asp Ala Leu Thr His Ala Leu Glu 100
105 110Ala Tyr Val Ser Ile Met Ala Thr Glu
Phe Thr Asn Gly Leu Ala Leu 115 120
125Glu Ala Val Lys Leu Ile Phe Glu Tyr Leu Pro Lys Ala Tyr Thr Glu
130 135 140Gly Thr Thr Asn Val Lys Ala
Arg Glu Lys Met Ala His Ala Ser Cys145 150
155 160Ile Ala Gly Met Ala Phe Ala Asn Ala Phe Leu Gly
Val Cys His Ser 165 170
175Met Ala His Lys Leu Gly Ala Gln His His Ile Pro His Gly Ile Ala
180 185 190Asn Ala Leu Met Ile Asp
Glu Val Ile Lys Phe Asn Ala Val Asp Asp 195 200
205Pro Ile Lys Gln Ala Ala Phe Pro Gln Tyr Glu Tyr Pro Asn
Ala Arg 210 215 220Tyr Arg Tyr Ala Gln
Ile Ala Asp Cys Leu Asn Leu Gly Gly Asn Thr225 230
235 240Glu Glu Glu Lys Val Gln Leu Leu Ile Asn
Ala Ile Asp Asp Leu Lys 245 250
255Ala Lys Leu Asn Ile Pro Glu Thr Ile Lys Glu Ala Gly Val Ser Glu
260 265 270Asp Lys Phe Tyr Ala
Thr Leu Asp Lys Met Ser Glu Leu Ala Phe Asp 275
280 285Asp Gln Cys Thr Gly Ala Asn Pro Arg Tyr Pro Leu
Ile Ser Glu Ile 290 295 300Lys Gln Met
Tyr Ile Asn Val Phe Asp Lys Thr Glu Pro Ile Val Glu305
310 315 320Asp Glu Glu Lys531194DNAC.
autoethanogenum 53atggaaataa aattaggggg aataataatg gagagattta cgttgccaag
agacatttac 60tttggagaag atgctttggg tgctttgaaa acgttaaaag gtaagaaagc
tgtagtagtt 120gttggaggag gatccatgaa gagattcggt ttccttgaca aggtagaaga
atacttaaaa 180gaagcaaaca tagaagttaa actaatagaa ggtgttgaac cagatccgtc
tgtggaaacc 240gttatgaaag gtgccaaaat aatgacagaa tttgggccag attggatagt
tgctattgga 300ggaggttcac caatagatgc tgcaaaggct atgtggctat tttatgaata
tccagatttt 360acttttaaac aagcaattgt tccgtttgga ttaccagaat taagacaaaa
agctaaattt 420gtagctatag cttctactag tggaacagct actgaagtta cttcattttc
agtaataact 480gattataaag ctaaaataaa gtatccttta gctgacttca atttgacacc
ggatatagct 540atagttgatc cagcattagc ccagacaatg ccacctaaat taactgcaca
tactggtatg 600gatgcattaa ctcatgcact agaagcttat gtagcatcag ctagatcaga
tatttcagat 660ccacttgcaa tacattccat aattatgaca agggataact tacttaaatc
ctataagggt 720gataaagatg ctagaaataa gatgcatata tcacaatgtt tagcaggtat
ggcattttct 780aatgcacttc ttggtataac tcatagttta gcacataaaa caggagctgt
atggcacata 840ccacatggat gcgctaatgc aatatatctt ccatatgttt tagattttaa
taaaaaagct 900tgctcagata gatatgctaa tatagctaaa atattaggac ttaaaggaac
tactgaagat 960gaattggtag attctctagt taaaatggta caagatatgg ataaggaatt
gaatatacct 1020ttgaccttaa aagattatgg tataagcaaa gatgatttca attcaaatgt
tgattttata 1080gcaaagaatg cgctcttaga tgcatgtaca ggagctaatc caaggcctat
agattttgat 1140caaatgaaaa agatacttca atgtatatat gatggaaaaa aggtaacttt
ttaa 119454397PRTC. autoethanogenum 54Met Glu Ile Lys Leu Gly Gly
Ile Ile Met Glu Arg Phe Thr Leu Pro1 5 10
15Arg Asp Ile Tyr Phe Gly Glu Asp Ala Leu Gly Ala Leu
Lys Thr Leu 20 25 30Lys Gly
Lys Lys Ala Val Val Val Val Gly Gly Gly Ser Met Lys Arg 35
40 45Phe Gly Phe Leu Asp Lys Val Glu Glu Tyr
Leu Lys Glu Ala Asn Ile 50 55 60Glu
Val Lys Leu Ile Glu Gly Val Glu Pro Asp Pro Ser Val Glu Thr65
70 75 80Val Met Lys Gly Ala Lys
Ile Met Thr Glu Phe Gly Pro Asp Trp Ile 85
90 95Val Ala Ile Gly Gly Gly Ser Pro Ile Asp Ala Ala
Lys Ala Met Trp 100 105 110Leu
Phe Tyr Glu Tyr Pro Asp Phe Thr Phe Lys Gln Ala Ile Val Pro 115
120 125Phe Gly Leu Pro Glu Leu Arg Gln Lys
Ala Lys Phe Val Ala Ile Ala 130 135
140Ser Thr Ser Gly Thr Ala Thr Glu Val Thr Ser Phe Ser Val Ile Thr145
150 155 160Asp Tyr Lys Ala
Lys Ile Lys Tyr Pro Leu Ala Asp Phe Asn Leu Thr 165
170 175Pro Asp Ile Ala Ile Val Asp Pro Ala Leu
Ala Gln Thr Met Pro Pro 180 185
190Lys Leu Thr Ala His Thr Gly Met Asp Ala Leu Thr His Ala Leu Glu
195 200 205Ala Tyr Val Ala Ser Ala Arg
Ser Asp Ile Ser Asp Pro Leu Ala Ile 210 215
220His Ser Ile Ile Met Thr Arg Asp Asn Leu Leu Lys Ser Tyr Lys
Gly225 230 235 240Asp Lys
Asp Ala Arg Asn Lys Met His Ile Ser Gln Cys Leu Ala Gly
245 250 255Met Ala Phe Ser Asn Ala Leu
Leu Gly Ile Thr His Ser Leu Ala His 260 265
270Lys Thr Gly Ala Val Trp His Ile Pro His Gly Cys Ala Asn
Ala Ile 275 280 285Tyr Leu Pro Tyr
Val Leu Asp Phe Asn Lys Lys Ala Cys Ser Asp Arg 290
295 300Tyr Ala Asn Ile Ala Lys Ile Leu Gly Leu Lys Gly
Thr Thr Glu Asp305 310 315
320Glu Leu Val Asp Ser Leu Val Lys Met Val Gln Asp Met Asp Lys Glu
325 330 335Leu Asn Ile Pro Leu
Thr Leu Lys Asp Tyr Gly Ile Ser Lys Asp Asp 340
345 350Phe Asn Ser Asn Val Asp Phe Ile Ala Lys Asn Ala
Leu Leu Asp Ala 355 360 365Cys Thr
Gly Ala Asn Pro Arg Pro Ile Asp Phe Asp Gln Met Lys Lys 370
375 380Ile Leu Gln Cys Ile Tyr Asp Gly Lys Lys Val
Thr Phe385 390 395551191DNAC.
autoethanogenum 55gtgagggatg ttattatgga aaactttatt tttaaaaatg ctacagaaat
tatttttggt 60aaggataccg aaaatcttgt aggaagtaaa gtaaaggagt attcaaagtc
agataaaata 120ctcttttgct atgggggagg aagcataaaa agatctggtc tatatgatag
agttataaag 180tccttaaaag aaaatggaat tgaatttata gaacttccag gaattaaacc
taatccaaga 240ttaggacctg ttaaagaagg tataagacta tgtagagaaa ataatataaa
atttgtacta 300tctgtaggag gaggaagttc agcagatacg gctaaagcta ttgctgtagg
agtaccttat 360aaaggagacg tatgggattt ttatacgggc aaagctgaag tgaaagaggc
tcttcctgta 420ggagttgtaa taacattacc tgctacaggt acagaatcta gtaatagttc
tgttattatg 480aatgaagatg gttggtttaa aaaaggatta aatacagtac ttataagacc
tgctttttca 540attatgaatc ctgaacttac ttttacacta ccagagtatc aaactgcttg
tggtgcttgt 600gacattatgg cacatataat ggaaagatat tttacaaatg tgaaacatgt
agatataact 660gataggcttt gcgaagctgc acttagaaat gttataaata atgccccaat
agttttaaaa 720gatcccaaaa actatgatgc tagggcagaa attatgtgga ccggtactat
agctcataat 780gatgtgctta gtgcgggtag aataggtgat tgggcttctc acaaaattga
acatgaattg 840agtggggaaa cagacattgc ccatggagca ggacttgcaa ttgtatttcc
tgcatggatg 900aaatatgtat ataaacacga tatcaataga tttgtacaat ttgcagtaag
ggtatgggat 960gtagatttat cttatagttc ctgcgaagat attgtacttg aaggcataag
gagaatgaca 1020gcatttttca agagcatggg gttacctgta actttaaaag aaggaagtat
aggagaagat 1080aaaattgaag aaatggctaa taagtgcacg gataatggaa ctaaaactgt
aggacaattt 1140gtaaaattaa ataaagatga tattgtaaaa atattaaatt tagctaaata a
119156396PRTC. autoethanogenum 56Val Arg Asp Val Ile Met Glu
Asn Phe Ile Phe Lys Asn Ala Thr Glu1 5 10
15Ile Ile Phe Gly Lys Asp Thr Glu Asn Leu Val Gly Ser
Lys Val Lys 20 25 30Glu Tyr
Ser Lys Ser Asp Lys Ile Leu Phe Cys Tyr Gly Gly Gly Ser 35
40 45Ile Lys Arg Ser Gly Leu Tyr Asp Arg Val
Ile Lys Ser Leu Lys Glu 50 55 60Asn
Gly Ile Glu Phe Ile Glu Leu Pro Gly Ile Lys Pro Asn Pro Arg65
70 75 80Leu Gly Pro Val Lys Glu
Gly Ile Arg Leu Cys Arg Glu Asn Asn Ile 85
90 95Lys Phe Val Leu Ser Val Gly Gly Gly Ser Ser Ala
Asp Thr Ala Lys 100 105 110Ala
Ile Ala Val Gly Val Pro Tyr Lys Gly Asp Val Trp Asp Phe Tyr 115
120 125Thr Gly Lys Ala Glu Val Lys Glu Ala
Leu Pro Val Gly Val Val Ile 130 135
140Thr Leu Pro Ala Thr Gly Thr Glu Ser Ser Asn Ser Ser Val Ile Met145
150 155 160Asn Glu Asp Gly
Trp Phe Lys Lys Gly Leu Asn Thr Val Leu Ile Arg 165
170 175Pro Ala Phe Ser Ile Met Asn Pro Glu Leu
Thr Phe Thr Leu Pro Glu 180 185
190Tyr Gln Thr Ala Cys Gly Ala Cys Asp Ile Met Ala His Ile Met Glu
195 200 205Arg Tyr Phe Thr Asn Val Lys
His Val Asp Ile Thr Asp Arg Leu Cys 210 215
220Glu Ala Ala Leu Arg Asn Val Ile Asn Asn Ala Pro Ile Val Leu
Lys225 230 235 240Asp Pro
Lys Asn Tyr Asp Ala Arg Ala Glu Ile Met Trp Thr Gly Thr
245 250 255Ile Ala His Asn Asp Val Leu
Ser Ala Gly Arg Ile Gly Asp Trp Ala 260 265
270Ser His Lys Ile Glu His Glu Leu Ser Gly Glu Thr Asp Ile
Ala His 275 280 285Gly Ala Gly Leu
Ala Ile Val Phe Pro Ala Trp Met Lys Tyr Val Tyr 290
295 300Lys His Asp Ile Asn Arg Phe Val Gln Phe Ala Val
Arg Val Trp Asp305 310 315
320Val Asp Leu Ser Tyr Ser Ser Cys Glu Asp Ile Val Leu Glu Gly Ile
325 330 335Arg Arg Met Thr Ala
Phe Phe Lys Ser Met Gly Leu Pro Val Thr Leu 340
345 350Lys Glu Gly Ser Ile Gly Glu Asp Lys Ile Glu Glu
Met Ala Asn Lys 355 360 365Cys Thr
Asp Asn Gly Thr Lys Thr Val Gly Gln Phe Val Lys Leu Asn 370
375 380Lys Asp Asp Ile Val Lys Ile Leu Asn Leu Ala
Lys385 390 395571149DNAC. autoethanogenum
57atggaagaca agtttgaaaa ttttaatttg aaatccaaga tttattttaa tagggaatct
60attcaacttt tagagcaagt cactggttct cgagcattta ttgttgcaga tgctattatg
120ggaaaacttg gatatcttca aaaagtaata gattacctaa gcaaagctgg aataagttcc
180gttgttttta cgggggtaca ccctgatcca gacgtcaatg taattgcaga tgcaatgaaa
240ttgtacaaaa aaagcgacgc agatgttctc gtagcactag gtggaggatc cagtattgat
300accgctaagg gaataatgta ttttgcatgt aatttaggaa aagcaatggg ccaagaaatg
360aaaaaacctc tatttattgc aattccatca acaagtggta caggctctga agtaacaaac
420tttactgtta ttacttctca gaaagaaaag gtatgcatta tagatgattt tattgcacca
480gatgttgcaa tacttgactc aagttgtatt gatggtctgc ctcagcgtat tgtagcagat
540actggtatag atgttctagt tcattctatt gaagcctatg tttccaaaaa agcaactgac
600tttacagacg ctcttgctga aaaagcagtt aaattaattt ttgagaatct tccaaaaatt
660tataacgata gtaaggattc cgaagctcga gatcatgttc aaaacgcttc ctgtatagca
720ggaatagcat ttacaaatgc tggtcttgga attaatcaca gcttggctca tgctatgggt
780ggatctttcc acattcctca cggccgatcc aatgcacttc tacttaatgc agtaatggaa
840tacaacgcta gcttggttgg aaatgcaagc gaacatgcta tggaaaaata cgcaaaacta
900gcatcaattc tacaccttcc agctcgaaca actcgcgaag gcgctgtaag ttttattgaa
960gctgtagata aattaataaa atccctaggt gttgaagata atattcgatc tcttgggatt
1020aaagaagatg agtttcaaag tgctctaaat catatggcag aaacagcaat gcaagataga
1080tgcactccaa ctaatcctag aaaaccttct aaagaagaac ttatacatat ttatcaaaaa
1140tgttattaa
114958307PRTC. autoethanogenum 58Met Glu Asp Lys Phe Glu Asn Phe Asn Leu
Lys Ser Lys Ile Tyr Phe1 5 10
15Asn Arg Glu Ser Ile Gln Leu Leu Glu Gln Val Thr Gly Ser Arg Ala
20 25 30Phe Ile Val Ala Asp Ala
Ile Met Gly Lys Leu Gly Tyr Leu Gln Lys 35 40
45Val Ile Asp Tyr Leu Ser Lys Ala Gly Ile Ser Ser Val Val
Phe Thr 50 55 60Gly Val His Pro Asp
Pro Asp Val Asn Val Ile Ala Asp Ala Met Lys65 70
75 80Leu Tyr Lys Lys Ser Asp Ala Asp Val Leu
Val Ala Leu Gly Gly Gly 85 90
95Ser Ser Ile Asp Thr Ala Lys Gly Ile Met Tyr Phe Ala Cys Asn Leu
100 105 110Gly Lys Ala Met Gly
Gln Glu Met Lys Lys Pro Leu Phe Ile Ala Ile 115
120 125Pro Ser Thr Ser Gly Thr Gly Ser Glu Val Thr Asn
Phe Thr Val Ile 130 135 140Thr Ser Gln
Lys Glu Lys Val Cys Ile Ile Asp Asp Phe Ile Ala Pro145
150 155 160Asp Val Ala Ile Leu Asp Ser
Ser Cys Ile Asp Gly Leu Pro Gln Arg 165
170 175Ile Val Ala Asp Thr Gly Ile Asp Val Leu Val His
Ser Ile Glu Ala 180 185 190Tyr
Val Ser Lys Lys Ala Thr Asp Phe Thr Asp Ala Leu Ala Glu Lys 195
200 205Ala Val Lys Leu Ile Phe Glu Asn Leu
Pro Lys Ile Tyr Asn Asp Ser 210 215
220Lys Asp Ser Glu Ala Arg Asp His Val Gln Asn Ala Ser Cys Ile Ala225
230 235 240Gly Ile Ala Phe
Thr Asn Ala Gly Leu Gly Ile Asn His Ser Leu Ala 245
250 255His Ala Met Gly Gly Ser Phe His Ile Pro
His Gly Arg Ser Asn Ala 260 265
270Leu Leu Leu Asn Ala Val Met Glu Tyr Asn Ala Ser Leu Val Gly Asn
275 280 285Ala Ser Glu His Ala Met Glu
Lys Tyr Ala Lys Leu Ala Ser Ile Leu 290 295
300His Leu Pro30559993DNAC. autoethanogenum 59atggaaaaaa tttggagtaa
ggcaaaggaa gacaaaaaaa agattgtctt agctgaagga 60gaagaagaaa gaactcttca
agcttgtgaa aaaataatta aagagggtat tgcaaattta 120atccttgtag ggaatgaaaa
ggtaataaaa gaaaaagcgt caaaattagg tgtaagttta 180aatggagcag aaatagtaga
tccagagatt tcagataaac taaaggcata tgcagatgct 240ttttatgaat tgagaaagaa
gaagggaata acgccagaaa aagcggataa aatagtaaga 300gatccaatat actttgctac
aatgatggtt aaacttggag atgcagatgg attggtttca 360ggtgcggttc atactacagg
cgatcttttg agaccaggac ttcaaatagt aaagacagct 420ccaggtacat cagtagtttc
cagtacattt ataatggaag taccaaattg tgagtatggt 480gacaatggtg tacttctatt
tgctgattgt gctgtaaatc catgcccaga tagtgatcaa 540ttggcttcaa ttgcaataag
tacagcagaa actgcaaaga acttatgtgg aatggatcca 600aaagtagcaa tgctttcatt
ttctactaag ggaagtgcaa aacacgaatt agtagacaaa 660gttagaaatg ctgtagagat
tgcaaaaaaa gctaaaccag atttaagttt agacggagaa 720ttacaattag atgcctctat
cgtagaaaag gttgcaagtt taaaggctcc tggaagtgaa 780gtagcaggaa aagcaaatgt
acttgtattt ccagatctcc aagcaggaaa tataggctat 840aaactcgttc aaagatttgc
aaaagcagat gctataggac ctgtatgcca aggatttgca 900aaacctataa atgatttgtc
aagaggatgt aattctgatg atatagtaaa tgtagtagct 960gtaacagcag ttcaagcaca
agctcaaaag taa 99360330PRTC.
autoethanogenum 60Met Glu Lys Ile Trp Ser Lys Ala Lys Glu Asp Lys Lys Lys
Ile Val1 5 10 15Leu Ala
Glu Gly Glu Glu Glu Arg Thr Leu Gln Ala Cys Glu Lys Ile 20
25 30Ile Lys Glu Gly Ile Ala Asn Leu Ile
Leu Val Gly Asn Glu Lys Val 35 40
45Ile Lys Glu Lys Ala Ser Lys Leu Gly Val Ser Leu Asn Gly Ala Glu 50
55 60Ile Val Asp Pro Glu Ile Ser Asp Lys
Leu Lys Ala Tyr Ala Asp Ala65 70 75
80Phe Tyr Glu Leu Arg Lys Lys Lys Gly Ile Thr Pro Glu Lys
Ala Asp 85 90 95Lys Ile
Val Arg Asp Pro Ile Tyr Phe Ala Thr Met Met Val Lys Leu 100
105 110Gly Asp Ala Asp Gly Leu Val Ser Gly
Ala Val His Thr Thr Gly Asp 115 120
125Leu Leu Arg Pro Gly Leu Gln Ile Val Lys Thr Ala Pro Gly Thr Ser
130 135 140Val Val Ser Ser Thr Phe Ile
Met Glu Val Pro Asn Cys Glu Tyr Gly145 150
155 160Asp Asn Gly Val Leu Leu Phe Ala Asp Cys Ala Val
Asn Pro Cys Pro 165 170
175Asp Ser Asp Gln Leu Ala Ser Ile Ala Ile Ser Thr Ala Glu Thr Ala
180 185 190Lys Asn Leu Cys Gly Met
Asp Pro Lys Val Ala Met Leu Ser Phe Ser 195 200
205Thr Lys Gly Ser Ala Lys His Glu Leu Val Asp Lys Val Arg
Asn Ala 210 215 220Val Glu Ile Ala Lys
Lys Ala Lys Pro Asp Leu Ser Leu Asp Gly Glu225 230
235 240Leu Gln Leu Asp Ala Ser Ile Val Glu Lys
Val Ala Ser Leu Lys Ala 245 250
255Pro Gly Ser Glu Val Ala Gly Lys Ala Asn Val Leu Val Phe Pro Asp
260 265 270Leu Gln Ala Gly Asn
Ile Gly Tyr Lys Leu Val Gln Arg Phe Ala Lys 275
280 285Ala Asp Ala Ile Gly Pro Val Cys Gln Gly Phe Ala
Lys Pro Ile Asn 290 295 300Asp Leu Ser
Arg Gly Cys Asn Ser Asp Asp Ile Val Asn Val Val Ala305
310 315 320Val Thr Ala Val Gln Ala Gln
Ala Gln Lys 325 330611197DNAC.
autoethanogenum 61atgaaaatat tagtagtaaa ctgtggaagt tcatctttaa aatatcaact
tattgatatg 60caagatgaaa gtgttgtagc aaagggtctt gtagaaagaa taggaatgga
cggttcaatt 120ttaacacaca aagttaatgg agaaaagttt gttacagagc aaccaatgga
agaccacaaa 180gttgctatac aattagtatt aaatgctctt gtagataaaa aacatggtgt
aataaaagac 240atgtcagaaa tatccgctgt aggacataga gttttgcacg gtggaaagaa
atatgcagca 300tccattctta ttgacgaaaa tgtaatgaaa gcaatagaag aatgtatccc
actaggacca 360ctacataatc cagctaatat aatgggaata gatgcttgta aaaaattaat
gccaaatact 420ccaatggtag cagtatttga tacagcattt catcagacaa tgccagatta
tgcttatact 480tatgcaatac cttatgatat atctgaaaag tatgatatca gaaaatatgg
ttttcatgga 540acttctcata gattcgtttc aattgaagca gctaaattat taaagaaaga
tccaaaagat 600cttaagttaa taacttgtca tttaggaaat ggagctagca tatgtgcagt
aaaccaagga 660aaagcagtag atacaactat gggacttact cctcttgcag gacttgtaat
gggaactaga 720tgcggtgata tagatccagc tatagtacca tttgtaatga aaagaacagg
catgtctgta 780gatgaagtgg ataccttaat gaataaaaag tcaggaatac ttggagtatc
aggagtaagc 840agtgatttta gagatgtaga agaagctgca aattcaggaa atgatagagc
aaaacttgca 900ttaaatatgt attatcacaa agttaaatct ttcataggag cttatgttgc
agttttaaat 960ggagcagatg ctataatatt tacggcagga cttggagaaa attcagcaac
tagcagatct 1020gctatatgta atggattaag ctattttgga attaaaatag atgaagaaaa
gaataagaaa 1080aggggagagg cactagaaat aagcacacct gattcaaaga taaaagtatt
agtaattcct 1140acaaatgaag aacttatgat agctagggat acaaaagaaa tagttgaaaa
taaataa 119762398PRTC. autoethanogenum 62Met Lys Ile Leu Val Val Asn
Cys Gly Ser Ser Ser Leu Lys Tyr Gln1 5 10
15Leu Ile Asp Met Gln Asp Glu Ser Val Val Ala Lys Gly
Leu Val Glu 20 25 30Arg Ile
Gly Met Asp Gly Ser Ile Leu Thr His Lys Val Asn Gly Glu 35
40 45Lys Phe Val Thr Glu Gln Pro Met Glu Asp
His Lys Val Ala Ile Gln 50 55 60Leu
Val Leu Asn Ala Leu Val Asp Lys Lys His Gly Val Ile Lys Asp65
70 75 80Met Ser Glu Ile Ser Ala
Val Gly His Arg Val Leu His Gly Gly Lys 85
90 95Lys Tyr Ala Ala Ser Ile Leu Ile Asp Glu Asn Val
Met Lys Ala Ile 100 105 110Glu
Glu Cys Ile Pro Leu Gly Pro Leu His Asn Pro Ala Asn Ile Met 115
120 125Gly Ile Asp Ala Cys Lys Lys Leu Met
Pro Asn Thr Pro Met Val Ala 130 135
140Val Phe Asp Thr Ala Phe His Gln Thr Met Pro Asp Tyr Ala Tyr Thr145
150 155 160Tyr Ala Ile Pro
Tyr Asp Ile Ser Glu Lys Tyr Asp Ile Arg Lys Tyr 165
170 175Gly Phe His Gly Thr Ser His Arg Phe Val
Ser Ile Glu Ala Ala Lys 180 185
190Leu Leu Lys Lys Asp Pro Lys Asp Leu Lys Leu Ile Thr Cys His Leu
195 200 205Gly Asn Gly Ala Ser Ile Cys
Ala Val Asn Gln Gly Lys Ala Val Asp 210 215
220Thr Thr Met Gly Leu Thr Pro Leu Ala Gly Leu Val Met Gly Thr
Arg225 230 235 240Cys Gly
Asp Ile Asp Pro Ala Ile Val Pro Phe Val Met Lys Arg Thr
245 250 255Gly Met Ser Val Asp Glu Val
Asp Thr Leu Met Asn Lys Lys Ser Gly 260 265
270Ile Leu Gly Val Ser Gly Val Ser Ser Asp Phe Arg Asp Val
Glu Glu 275 280 285Ala Ala Asn Ser
Gly Asn Asp Arg Ala Lys Leu Ala Leu Asn Met Tyr 290
295 300Tyr His Lys Val Lys Ser Phe Ile Gly Ala Tyr Val
Ala Val Leu Asn305 310 315
320Gly Ala Asp Ala Ile Ile Phe Thr Ala Gly Leu Gly Glu Asn Ser Ala
325 330 335Thr Ser Arg Ser Ala
Ile Cys Asn Gly Leu Ser Tyr Phe Gly Ile Lys 340
345 350Ile Asp Glu Glu Lys Asn Lys Lys Arg Gly Glu Ala
Leu Glu Ile Ser 355 360 365Thr Pro
Asp Ser Lys Ile Lys Val Leu Val Ile Pro Thr Asn Glu Glu 370
375 380Leu Met Ile Ala Arg Asp Thr Lys Glu Ile Val
Glu Asn Lys385 390 395631767DNAC.
autoethanogenum 63gtggaagaat tgaaaattga caaagctaaa aaatttatag gtgcaagagg
gttaggcgta 60aaaaccttat ttgacgaagt agatccaaag gtagatccat tatcacctga
taacaaattt 120attatagcag cgggaccact tacaggtgca cctgttccaa caagcggaag
attcatggta 180gttactaaat cacctttaac aggaactatt gctattgcaa attcaggtgg
aaaatgggga 240gcagaattca aagcagctgg atacgatatg ataatcgttg aaggtaaatc
tgataaagaa 300gtttatgtaa atatagtaga tgataaagta gaatttaggg atgcttctca
tgtttgggga 360aaactaacag aagaaactac aaaaatgctt caacaggaaa cagattcgag
agctaaggtt 420ttatgcatag gaccagctgg ggaaaagtta tcacttatgg cagcagttat
gaatgatgtt 480gatagaacag caggacgtgg tggtgttgga gctgttatgg gttcaaagaa
cttaaaagct 540attgtagtta aaggaagcgg aaaagtaaaa ttatttgatg aacaaaaagt
gaaggaagta 600gcacttgaga aaacaaatat tttaagaaaa gatccagtag ctggtggagg
acttccaaca 660tacggaacag ctgtacttgt taatattata aatgaaaatg gtgtacatcc
agtaaagaat 720tttcaaaaat cttatacaga tcaagcagat aagatcagtg gagaaacttt
aactaaagat 780tgcttagtta gaaaaaatcc ttgctatagg tgtccaattg cctgtggaag
atgggtaaaa 840cttgatgatg gaactgaatg tggaggacca gaatatgaaa cattatggtc
atttggatct 900gattgtgatg tatacgatat aaatgctgta aatacagcaa atatgttgtg
taatgaatat 960ggactagata ccattacagc aggatgtact attgcagcag ctatggaact
ttatcaaaga 1020ggttatatta aggatgaaga aatagcagca gatggattgt cacttaattg
gggagatgct 1080aagtccatgg ttgaatgggt aaagaaaatg ggacttagag aaggatttgg
agacaagatg 1140gcagatggtt catacagact ttgtgactca tacggtgtac ctgagtattc
aatgactgta 1200aaaaaacagg aacttccagc atatgaccca agaggaatac agggacatgg
cattacttat 1260gctgttaaca ataggggagg atgtcacatt aagggatata tggtaagtcc
tgaaatactt 1320ggctatccag aaaaacttga tagacttgca gtggaaggaa aagcaggata
tgctagagta 1380ttccatgatt taacagctgt tatagattca cttggattat gtatttttac
aacatttggt 1440cttggtgcac aggattatgt tgatatgtat aatgcagtag ttggtggaga
attacatgat 1500gtaaattctt taatgttagc tggagataga atatggactt tagaaaaaat
atttaactta 1560aaagcaggca tagatagttc acaggatact cttccaaaga gattgcttga
agaacaaatt 1620ccagaaggac catcaaaagg agaagttcat aagttagatg tactactacc
tgaatattat 1680tcagtacgtg gatgggataa aaatggtatt cctacagagg aaacgttaaa
gaaattagga 1740ttagatgaat acgtaggtaa gctttag
176764588PRTC. autoethanogenum 64Met Glu Glu Leu Lys Ile Asp
Lys Ala Lys Lys Phe Ile Gly Ala Arg1 5 10
15Gly Leu Gly Val Lys Thr Leu Phe Asp Glu Val Asp Pro
Lys Val Asp 20 25 30Pro Leu
Ser Pro Asp Asn Lys Phe Ile Ile Ala Ala Gly Pro Leu Thr 35
40 45Gly Ala Pro Val Pro Thr Ser Gly Arg Phe
Met Val Val Thr Lys Ser 50 55 60Pro
Leu Thr Gly Thr Ile Ala Ile Ala Asn Ser Gly Gly Lys Trp Gly65
70 75 80Ala Glu Phe Lys Ala Ala
Gly Tyr Asp Met Ile Ile Val Glu Gly Lys 85
90 95Ser Asp Lys Glu Val Tyr Val Asn Ile Val Asp Asp
Lys Val Glu Phe 100 105 110Arg
Asp Ala Ser His Val Trp Gly Lys Leu Thr Glu Glu Thr Thr Lys 115
120 125Met Leu Gln Gln Glu Thr Asp Ser Arg
Ala Lys Val Leu Cys Ile Gly 130 135
140Pro Ala Gly Glu Lys Leu Ser Leu Met Ala Ala Val Met Asn Asp Val145
150 155 160Asp Arg Thr Ala
Gly Arg Gly Gly Val Gly Ala Val Met Gly Ser Lys 165
170 175Asn Leu Lys Ala Ile Val Val Lys Gly Ser
Gly Lys Val Lys Leu Phe 180 185
190Asp Glu Gln Lys Val Lys Glu Val Ala Leu Glu Lys Thr Asn Ile Leu
195 200 205Arg Lys Asp Pro Val Ala Gly
Gly Gly Leu Pro Thr Tyr Gly Thr Ala 210 215
220Val Leu Val Asn Ile Ile Asn Glu Asn Gly Val His Pro Val Lys
Asn225 230 235 240Phe Gln
Lys Ser Tyr Thr Asp Gln Ala Asp Lys Ile Ser Gly Glu Thr
245 250 255Leu Thr Lys Asp Cys Leu Val
Arg Lys Asn Pro Cys Tyr Arg Cys Pro 260 265
270Ile Ala Cys Gly Arg Trp Val Lys Leu Asp Asp Gly Thr Glu
Cys Gly 275 280 285Gly Pro Glu Tyr
Glu Thr Leu Trp Ser Phe Gly Ser Asp Cys Asp Val 290
295 300Tyr Asp Ile Asn Ala Val Asn Thr Ala Asn Met Leu
Cys Asn Glu Tyr305 310 315
320Gly Leu Asp Thr Ile Thr Ala Gly Cys Thr Ile Ala Ala Ala Met Glu
325 330 335Leu Tyr Gln Arg Gly
Tyr Ile Lys Asp Glu Glu Ile Ala Ala Asp Gly 340
345 350Leu Ser Leu Asn Trp Gly Asp Ala Lys Ser Met Val
Glu Trp Val Lys 355 360 365Lys Met
Gly Leu Arg Glu Gly Phe Gly Asp Lys Met Ala Asp Gly Ser 370
375 380Tyr Arg Leu Cys Asp Ser Tyr Gly Val Pro Glu
Tyr Ser Met Thr Val385 390 395
400Lys Lys Gln Glu Leu Pro Ala Tyr Asp Pro Arg Gly Ile Gln Gly His
405 410 415Gly Ile Thr Tyr
Ala Val Asn Asn Arg Gly Gly Cys His Ile Lys Gly 420
425 430Tyr Met Val Ser Pro Glu Ile Leu Gly Tyr Pro
Glu Lys Leu Asp Arg 435 440 445Leu
Ala Val Glu Gly Lys Ala Gly Tyr Ala Arg Val Phe His Asp Leu 450
455 460Thr Ala Val Ile Asp Ser Leu Gly Leu Cys
Ile Phe Thr Thr Phe Gly465 470 475
480Leu Gly Ala Gln Asp Tyr Val Asp Met Tyr Asn Ala Val Val Gly
Gly 485 490 495Glu Leu His
Asp Val Asn Ser Leu Met Leu Ala Gly Asp Arg Ile Trp 500
505 510Thr Leu Glu Lys Ile Phe Asn Leu Lys Ala
Gly Ile Asp Ser Ser Gln 515 520
525Asp Thr Leu Pro Lys Arg Leu Leu Glu Glu Gln Ile Pro Glu Gly Pro 530
535 540Ser Lys Gly Glu Val His Lys Leu
Asp Val Leu Leu Pro Glu Tyr Tyr545 550
555 560Ser Val Arg Gly Trp Asp Lys Asn Gly Ile Pro Thr
Glu Glu Thr Leu 565 570
575Lys Lys Leu Gly Leu Asp Glu Tyr Val Gly Lys Leu 580
585651824DNAC. autoethanogenum 65atgtatggtt atgatggtaa agtattaaga
attaatttaa aagaaagaac ttgcaaatca 60gaaaatttag atttagataa agctaaaaag
tttataggtt gtaggggact aggtgttaaa 120actttatttg atgaaataga tcctaaaata
gatgcattat caccagaaaa taaatttata 180attgtaacag gtcctttaac tggagctccg
gttccaacta gtggaaggtt tatggtagtt 240actaaagcac cgcttacagg aactatagga
atttcaaatt cgggtggaaa atggggagta 300gacttaaaaa aagctggttg ggatatgata
atagtagagg ataaggctga ttcaccagtt 360tacattgaaa tagtagatga taaggtagaa
attaaagacg cgtcacagct ttggggaaaa 420gttacatcag aaactacaaa agagttagaa
aagataactg agaataaatc aaaggtatta 480tgtataggac ctgctggtga acgattgtct
cttatggcag cagttatgaa tgatgtagat 540agaactgcag caagaggcgg cgttggtgca
gttatgggat ctaaaaactt aaaagctatt 600acagttaaag gaactggaaa aatagcttta
gctgataaag aaaaagtaaa aaaagtgtcc 660gtagaaaaaa ttacaacatt aaaaaatgat
ccagtagctg gtcagggaat gccaacttat 720ggtacagcta tactggttaa tataataaat
gaaaatggag ttcatcctgt aaagaatttt 780caagagtctt atacgaatca agcagataaa
ataagtggag agactcttac tgctaaccaa 840ctagtaagga aaaatccttg ttacagctgt
cctataggtt gtggaagatg ggttagacta 900aaagatggca cagagtgcgg aggaccagaa
tatgaaacac tgtggtgttt tggatctgac 960tgtggttcat atgatttaga tgctataaat
gaagctaata tgttatgtaa tgaatatggt 1020attgatacta ttacttgtgg tgcaacaatt
gctgcagcta tggaacttta tcaaagagga 1080tatataaaag acgaagaaat agctggagat
aacctatctc tcaagtgggg tgatacggaa 1140tctatgattg gctggataaa gagaatggta
tatagtgaag gctttggagc aaagatgaca 1200aatggttcat ataggctttg tgaaggttat
ggagcaccgg agtattctat gacagttaaa 1260aagcaggaaa ttccagcata tgatccaagg
ggaatacagg gacacggtat tacctatgca 1320gttaataata gaggaggctg tcatattaag
ggatacatga ttaaccctga aatattaggt 1380tatcctgaaa aacttgatag atttgcatta
gatggtaaag cagcttatgc caaattattt 1440catgatttaa ctgctgtaat tgattcttta
ggattgtgca tattcactac atttgggctt 1500ggaatacagg attatgtaga tatgtataat
gcagtagtag gagaatctac ttatgatgca 1560gattcactat tagaggcagg agatagaatc
tggactcttg agaaattatt taatcttgca 1620gctggaatag acagcagcca ggatactcta
ccaaagagat tgttagaaga acctattcca 1680gatggcccat caaagggaga agttcatagg
ctagatgttc ttctgccaga atattactca 1740gtacgaggat ggagtaaaga gggtatacct
acagaagaaa cattaaagaa attaggatta 1800gatgaatata taggtaagtt ctag
182466607PRTC. autoethanogenum 66Met Tyr
Gly Tyr Asp Gly Lys Val Leu Arg Ile Asn Leu Lys Glu Arg1 5
10 15Thr Cys Lys Ser Glu Asn Leu Asp
Leu Asp Lys Ala Lys Lys Phe Ile 20 25
30Gly Cys Arg Gly Leu Gly Val Lys Thr Leu Phe Asp Glu Ile Asp
Pro 35 40 45Lys Ile Asp Ala Leu
Ser Pro Glu Asn Lys Phe Ile Ile Val Thr Gly 50 55
60Pro Leu Thr Gly Ala Pro Val Pro Thr Ser Gly Arg Phe Met
Val Val65 70 75 80Thr
Lys Ala Pro Leu Thr Gly Thr Ile Gly Ile Ser Asn Ser Gly Gly
85 90 95Lys Trp Gly Val Asp Leu Lys
Lys Ala Gly Trp Asp Met Ile Ile Val 100 105
110Glu Asp Lys Ala Asp Ser Pro Val Tyr Ile Glu Ile Val Asp
Asp Lys 115 120 125Val Glu Ile Lys
Asp Ala Ser Gln Leu Trp Gly Lys Val Thr Ser Glu 130
135 140Thr Thr Lys Glu Leu Glu Lys Ile Thr Glu Asn Lys
Ser Lys Val Leu145 150 155
160Cys Ile Gly Pro Ala Gly Glu Arg Leu Ser Leu Met Ala Ala Val Met
165 170 175Asn Asp Val Asp Arg
Thr Ala Ala Arg Gly Gly Val Gly Ala Val Met 180
185 190Gly Ser Lys Asn Leu Lys Ala Ile Thr Val Lys Gly
Thr Gly Lys Ile 195 200 205Ala Leu
Ala Asp Lys Glu Lys Val Lys Lys Val Ser Val Glu Lys Ile 210
215 220Thr Thr Leu Lys Asn Asp Pro Val Ala Gly Gln
Gly Met Pro Thr Tyr225 230 235
240Gly Thr Ala Ile Leu Val Asn Ile Ile Asn Glu Asn Gly Val His Pro
245 250 255Val Lys Asn Phe
Gln Glu Ser Tyr Thr Asn Gln Ala Asp Lys Ile Ser 260
265 270Gly Glu Thr Leu Thr Ala Asn Gln Leu Val Arg
Lys Asn Pro Cys Tyr 275 280 285Ser
Cys Pro Ile Gly Cys Gly Arg Trp Val Arg Leu Lys Asp Gly Thr 290
295 300Glu Cys Gly Gly Pro Glu Tyr Glu Thr Leu
Trp Cys Phe Gly Ser Asp305 310 315
320Cys Gly Ser Tyr Asp Leu Asp Ala Ile Asn Glu Ala Asn Met Leu
Cys 325 330 335Asn Glu Tyr
Gly Ile Asp Thr Ile Thr Cys Gly Ala Thr Ile Ala Ala 340
345 350Ala Met Glu Leu Tyr Gln Arg Gly Tyr Ile
Lys Asp Glu Glu Ile Ala 355 360
365Gly Asp Asn Leu Ser Leu Lys Trp Gly Asp Thr Glu Ser Met Ile Gly 370
375 380Trp Ile Lys Arg Met Val Tyr Ser
Glu Gly Phe Gly Ala Lys Met Thr385 390
395 400Asn Gly Ser Tyr Arg Leu Cys Glu Gly Tyr Gly Ala
Pro Glu Tyr Ser 405 410
415Met Thr Val Lys Lys Gln Glu Ile Pro Ala Tyr Asp Pro Arg Gly Ile
420 425 430Gln Gly His Gly Ile Thr
Tyr Ala Val Asn Asn Arg Gly Gly Cys His 435 440
445Ile Lys Gly Tyr Met Ile Asn Pro Glu Ile Leu Gly Tyr Pro
Glu Lys 450 455 460Leu Asp Arg Phe Ala
Leu Asp Gly Lys Ala Ala Tyr Ala Lys Leu Phe465 470
475 480His Asp Leu Thr Ala Val Ile Asp Ser Leu
Gly Leu Cys Ile Phe Thr 485 490
495Thr Phe Gly Leu Gly Ile Gln Asp Tyr Val Asp Met Tyr Asn Ala Val
500 505 510Val Gly Glu Ser Thr
Tyr Asp Ala Asp Ser Leu Leu Glu Ala Gly Asp 515
520 525Arg Ile Trp Thr Leu Glu Lys Leu Phe Asn Leu Ala
Ala Gly Ile Asp 530 535 540Ser Ser Gln
Asp Thr Leu Pro Lys Arg Leu Leu Glu Glu Pro Ile Pro545
550 555 560Asp Gly Pro Ser Lys Gly Glu
Val His Arg Leu Asp Val Leu Leu Pro 565
570 575Glu Tyr Tyr Ser Val Arg Gly Trp Ser Lys Glu Gly
Ile Pro Thr Glu 580 585 590Glu
Thr Leu Lys Lys Leu Gly Leu Asp Glu Tyr Ile Gly Lys Phe 595
600 605672634DNAC. ljungdahlii 67atgaaggtaa
ctaaggtaac taacgttgaa gaattaatga aaaagttaga tgaagtaacg 60gctgctcaaa
agaaattttc tagctatact caagaacaag tggatgaaat tttcaggcag 120gcagctatgg
cagccaatag tgctagaata gacttagcta aaatggcagt ggaagaaagc 180ggaatgggaa
ttgtagaaga caaggtcatt aaaaatcatt ttgttgcaga gtatatatat 240aacaaatata
agggtgaaaa aacctgtgga gttctggaac aagatgaagg ctttggtatg 300gttagaattg
cagaacctgt aggagttatt gcagcagtag tcccaacaac taatccaaca 360tctacagcaa
tatttaaatc actaatagct ttaaaaacta gaaatggtat agttttttcg 420ccacatccaa
gggcaaaaaa atcaactatt gcagcagcta agatagtact tgatgctgca 480gttaaagctg
gtgctcctga aggaattata ggatggatag atgaaccttc tattgaactt 540tcacaggtgg
taatgaaaga agcagatcta attcttgcaa ctggtggacc aggtatggtt 600aaggctgcct
attcttcagg aaagcctgct ataggagttg gtccaggtaa cacgcctgct 660gtaattgatg
aaagtgctga cattaaaatg gcagtaaatt caatactatt atcaaaaact 720tttgataatg
gtatgatttg tgcttcagag cagtcagtag tagttgcaag ctcaatatac 780gatgaagtca
agaaagagtt tgcagataga ggagcatata tattaagtaa ggatgaaaca 840gagaaggttg
gaaaaacaat tataattaat ggagccttaa atgctggcat tgtagggcaa 900agtgctttta
aaatagcaca gatggcagga gtgagtgtac cagaagatgc taaagtactt 960ataggagaag
ttaaatcagt agaaccggaa gaagagccct ttgcgcatga aaagctatct 1020ccagttttag
ctatgtacaa agcaaaagat tttgacgaag cactcctaaa ggctggaaga 1080ttagttgaac
gaggtggaat tgggcataca tctgtattat atgtaaatgc aatgacggaa 1140aaagtaaagg
tagaaaagtt cagagaaact atgaagactg gtagaacatt gataaatatg 1200ccttcagcac
aaggtgctat aggagatata tataacttta agctagctcc ttctttgaca 1260ctaggttgtg
gttcctgggg aggaaactct gtatcagaaa atgttggtcc taaacattta 1320ttaaacataa
agagtgttgc tgagaggaga gaaaatatgc tttggtttag agtacctgaa 1380aaggtttatt
tcaaatatgg tagtcttgga gttgcactaa aagaactgag aattatggag 1440aagaaaaagg
catttatagt aacggataaa gttctttatc aattaggtta tgtagataaa 1500attacaaaaa
atctggatga attaagagtt tcatataaaa tatttacaga tgtagaacca 1560gatccaaccc
ttgctacagc taaaaaaggt gcagcagaac tgttagctta tgaaccagat 1620acaattatag
cagtcggtgg tggttcagca atggatgcag ccaagatcat gtgggtaatg 1680tatgagcatc
cagaagtaag atttgaagat ttagctatga gatttatgga tataagaaag 1740agagtgtatg
ttttccctaa aatgggagaa aaggcaatga tgatttcagt agcaacatcc 1800gcaggaacag
ggtcggaagt tacgccattt gcagtaatta cggatgaaag aacaggagct 1860aaatatcctc
tggctgatta tgaattgact ccaaacatgg ctatagttga tgcagaactt 1920atgatgggaa
tgccaaaggg actaacagca gcttcaggta tagatgcatt aacccatgcg 1980ctggaggcct
atgtatcaat aatggcttca gaatatacca atggattggc tcttgaagca 2040acaagattag
tatttaaata tttgccaata gcttatacag aaggtacaac taatgtaaag 2100gcaagagaaa
aaatggctca tgcttcatgt attgcaggta tggcctttgc caatgcattt 2160ttaggggtat
gccactccat ggcacataaa ttgggagcac agcaccacat accacatgga 2220attgccaatg
cacttatgat agatgaagtt ataaagttca atgctgtaga ggctccaagg 2280aaacaagcgg
catttccaca atataaatat ccaaatgtta aaagaagata tgctagaata 2340gctgattact
taaatttagg tggaagtaca gatgatgaaa aagtacaatt tttaataaat 2400gctatagatg
acttgaaaac caagttaaat attccaaaga ctattaaaga agcgggagtt 2460tcagaagata
aattctatgc tactttagat acaatgtcag aactggcttt tgatgatcaa 2520tgtacaggag
ctaatccaag atatccatta ataggagaaa taaaacaaat gtatataaat 2580gcatttgata
caccaaaggc aactgtggag aagaaaacaa gaaagaaaaa ataa 263468877PRTC.
ljungdahlii 68Met Lys Val Thr Lys Val Thr Asn Val Glu Glu Leu Met Lys Lys
Leu1 5 10 15Asp Glu Val
Thr Ala Ala Gln Lys Lys Phe Ser Ser Tyr Thr Gln Glu 20
25 30Gln Val Asp Glu Ile Phe Arg Gln Ala Ala
Met Ala Ala Asn Ser Ala 35 40
45Arg Ile Asp Leu Ala Lys Met Ala Val Glu Glu Ser Gly Met Gly Ile 50
55 60Val Glu Asp Lys Val Ile Lys Asn His
Phe Val Ala Glu Tyr Ile Tyr65 70 75
80Asn Lys Tyr Lys Gly Glu Lys Thr Cys Gly Val Leu Glu Gln
Asp Glu 85 90 95Gly Phe
Gly Met Val Arg Ile Ala Glu Pro Val Gly Val Ile Ala Ala 100
105 110Val Val Pro Thr Thr Asn Pro Thr Ser
Thr Ala Ile Phe Lys Ser Leu 115 120
125Ile Ala Leu Lys Thr Arg Asn Gly Ile Val Phe Ser Pro His Pro Arg
130 135 140Ala Lys Lys Ser Thr Ile Ala
Ala Ala Lys Ile Val Leu Asp Ala Ala145 150
155 160Val Lys Ala Gly Ala Pro Glu Gly Ile Ile Gly Trp
Ile Asp Glu Pro 165 170
175Ser Ile Glu Leu Ser Gln Val Val Met Lys Glu Ala Asp Leu Ile Leu
180 185 190Ala Thr Gly Gly Pro Gly
Met Val Lys Ala Ala Tyr Ser Ser Gly Lys 195 200
205Pro Ala Ile Gly Val Gly Pro Gly Asn Thr Pro Ala Val Ile
Asp Glu 210 215 220Ser Ala Asp Ile Lys
Met Ala Val Asn Ser Ile Leu Leu Ser Lys Thr225 230
235 240Phe Asp Asn Gly Met Ile Cys Ala Ser Glu
Gln Ser Val Val Val Ala 245 250
255Ser Ser Ile Tyr Asp Glu Val Lys Lys Glu Phe Ala Asp Arg Gly Ala
260 265 270Tyr Ile Leu Ser Lys
Asp Glu Thr Glu Lys Val Gly Lys Thr Ile Ile 275
280 285Ile Asn Gly Ala Leu Asn Ala Gly Ile Val Gly Gln
Ser Ala Phe Lys 290 295 300Ile Ala Gln
Met Ala Gly Val Ser Val Pro Glu Asp Ala Lys Val Leu305
310 315 320Ile Gly Glu Val Lys Ser Val
Glu Pro Glu Glu Glu Pro Phe Ala His 325
330 335Glu Lys Leu Ser Pro Val Leu Ala Met Tyr Lys Ala
Lys Asp Phe Asp 340 345 350Glu
Ala Leu Leu Lys Ala Gly Arg Leu Val Glu Arg Gly Gly Ile Gly 355
360 365His Thr Ser Val Leu Tyr Val Asn Ala
Met Thr Glu Lys Val Lys Val 370 375
380Glu Lys Phe Arg Glu Thr Met Lys Thr Gly Arg Thr Leu Ile Asn Met385
390 395 400Pro Ser Ala Gln
Gly Ala Ile Gly Asp Ile Tyr Asn Phe Lys Leu Ala 405
410 415Pro Ser Leu Thr Leu Gly Cys Gly Ser Trp
Gly Gly Asn Ser Val Ser 420 425
430Glu Asn Val Gly Pro Lys His Leu Leu Asn Ile Lys Ser Val Ala Glu
435 440 445Arg Arg Glu Asn Met Leu Trp
Phe Arg Val Pro Glu Lys Val Tyr Phe 450 455
460Lys Tyr Gly Ser Leu Gly Val Ala Leu Lys Glu Leu Arg Ile Met
Glu465 470 475 480Lys Lys
Lys Ala Phe Ile Val Thr Asp Lys Val Leu Tyr Gln Leu Gly
485 490 495Tyr Val Asp Lys Ile Thr Lys
Asn Leu Asp Glu Leu Arg Val Ser Tyr 500 505
510Lys Ile Phe Thr Asp Val Glu Pro Asp Pro Thr Leu Ala Thr
Ala Lys 515 520 525Lys Gly Ala Ala
Glu Leu Leu Ala Tyr Glu Pro Asp Thr Ile Ile Ala 530
535 540Val Gly Gly Gly Ser Ala Met Asp Ala Ala Lys Ile
Met Trp Val Met545 550 555
560Tyr Glu His Pro Glu Val Arg Phe Glu Asp Leu Ala Met Arg Phe Met
565 570 575Asp Ile Arg Lys Arg
Val Tyr Val Phe Pro Lys Met Gly Glu Lys Ala 580
585 590Met Met Ile Ser Val Ala Thr Ser Ala Gly Thr Gly
Ser Glu Val Thr 595 600 605Pro Phe
Ala Val Ile Thr Asp Glu Arg Thr Gly Ala Lys Tyr Pro Leu 610
615 620Ala Asp Tyr Glu Leu Thr Pro Asn Met Ala Ile
Val Asp Ala Glu Leu625 630 635
640Met Met Gly Met Pro Lys Gly Leu Thr Ala Ala Ser Gly Ile Asp Ala
645 650 655Leu Thr His Ala
Leu Glu Ala Tyr Val Ser Ile Met Ala Ser Glu Tyr 660
665 670Thr Asn Gly Leu Ala Leu Glu Ala Thr Arg Leu
Val Phe Lys Tyr Leu 675 680 685Pro
Ile Ala Tyr Thr Glu Gly Thr Thr Asn Val Lys Ala Arg Glu Lys 690
695 700Met Ala His Ala Ser Cys Ile Ala Gly Met
Ala Phe Ala Asn Ala Phe705 710 715
720Leu Gly Val Cys His Ser Met Ala His Lys Leu Gly Ala Gln His
His 725 730 735Ile Pro His
Gly Ile Ala Asn Ala Leu Met Ile Asp Glu Val Ile Lys 740
745 750Phe Asn Ala Val Glu Ala Pro Arg Lys Gln
Ala Ala Phe Pro Gln Tyr 755 760
765Lys Tyr Pro Asn Val Lys Arg Arg Tyr Ala Arg Ile Ala Asp Tyr Leu 770
775 780Asn Leu Gly Gly Ser Thr Asp Asp
Glu Lys Val Gln Phe Leu Ile Asn785 790
795 800Ala Ile Asp Asp Leu Lys Thr Lys Leu Asn Ile Pro
Lys Thr Ile Lys 805 810
815Glu Ala Gly Val Ser Glu Asp Lys Phe Tyr Ala Thr Leu Asp Thr Met
820 825 830Ser Glu Leu Ala Phe Asp
Asp Gln Cys Thr Gly Ala Asn Pro Arg Tyr 835 840
845Pro Leu Ile Gly Glu Ile Lys Gln Met Tyr Ile Asn Ala Phe
Asp Thr 850 855 860Pro Lys Ala Thr Val
Glu Lys Lys Thr Arg Lys Lys Lys865 870
875692613DNAC. ljungdahlii 69atgaaagtta caaacgtaga agaactaatg aaaagactag
aagaaataaa ggatgctcaa 60aagaaatttg ctacatatac tcaagaacaa gtggatgaaa
tttttagaca agcagctatg 120gcagctaata gtgctagaat agaactagct aaaatggcag
tagaagaaag cggaatggga 180attgtagaag acaaggtcat taaaaatcac tttgcctcag
aatatatata taacaaatat 240aaggatgaaa aaacctgtgg agttttagag agagatgcag
gatttggtat agttagaatt 300gcggaacctg taggagttat cgcagcagta gttccaacaa
ctaatccaac atctacagca 360atatttaaat cactaatagc tttaaaaact agaaatggta
taattttttc accccatcca 420agggcaaaga aatcaactat tgcagcagct aaaatagtac
ttgacgctgc agttaaagct 480ggtgctcctg aaggaattat aggatggata gatgaacctt
ccattgaact ttcacaggtg 540gtaatgggag aagcaaattt aattcttgca actggtggcc
cgggtatggt taaggctgcc 600tattcttcag gcaaacctgc tgtgggagtt ggtccaggta
acacacctgc tgtaattgat 660gaaagtgccg acattaaaat ggcagtaaat tcaatattac
tatcaaagac ttttgataat 720ggtatgattt gtgcctcaga gcagtcagta atagttttag
actcaatata tgaggaagtt 780aaaaaagaat ttgcttatag gggtgcttat atattaagta
aggatgaaac agataaggtt 840ggaaaaataa ttttaaaaaa tggagcctta aatgcaggta
ttgtaggaca acctgctttt 900aaaatagcac agctggcagg agtggatgta ccagaaaaag
ctaaagtact tataggagag 960gtagaatcgg tagaacttga agaaccattt tctcatgaaa
agttatctcc agttttagct 1020atgtacaggg caagaaattt tgaggatgcc attgcaaaaa
ctgataaact ggttaggtca 1080ggtggatttg gacatacatc ttcattatat gtaaatccaa
tgacagagaa agcaaaagta 1140gaaaaattta gtactatgat gaaaacatca agaactataa
ttaacacacc ttcatcccaa 1200ggtggtatag gtgatatata taactttaaa ctagctcctt
ctttgacatt aggctgcggt 1260tcctggggag gaaattctgt atccgaaaat gttgggccta
aacatttatt aaacataaaa 1320agtgttgctg agaggagaga aaatatgctt tggtttagag
tacctgaaaa ggtttatttc 1380aaatatggta gtcttggagt tgcattaaaa gaattaaaag
ttatgaataa gaagaaagta 1440tttatagtaa cagataaagt tctttatcaa ttaggttatg
tggacaaagt tacaaaagtt 1500cttgaggaac taaaaatttc ctataaggta tttacagatg
tagaaccaga tccaaccctt 1560gctacagcta aaaaaggtgc agcagaactg ctttcctatg
aaccggatac aattatatca 1620gttggtggtg gctcagcaat ggatgcagct aagatcatgt
gggtaatgta tgagcatcca 1680gaagtaaaat ttgaagattt agctatgaga tttatggata
taagaaagag agtatatgtt 1740ttccctaaga tgggagaaaa ggcaatgatg atttcagtag
caacatccgc aggaacaggg 1800tcggaagtta ctccatttgc agtaatcact gatgaaaaaa
caggagctaa atatccatta 1860gctgattatg aactaactcc agacatggct atagttgatg
cagaacttat gatgggaatg 1920ccaagaggac ttacagcagc ttcgggtata gatgcattaa
cccatgcact ggaggcatat 1980gtgtcaataa tggctacaga atttaccaat ggattagccc
ttgaagcagt aaagttgata 2040tttgaatatt taccaaaagc ttatacagaa ggtacaacta
atgtaaaggc aagagaaaag 2100atggttcatg cttcatgtat tgcaggtatg gcctttgcaa
atgcattttt aggggtatgc 2160cactctatgg cacataaatt gggagcacag catcacatac
cacatggaat tgccaatgca 2220cttatgatag atgaagttat aaaattcaat gctgtagatg
atccaataaa acaagctgca 2280tttccccaat acgagtatcc aaatgctagg tatagatatg
ctcagatagc tgattgtctg 2340aacttgggag gaaatacaga agaggaaaag gtacaactat
taataaatgc tatagatgat 2400ttaaaagcta agttaaatat tccagaaact ataaaagaag
caggagtttc agaagataaa 2460ttctatgcta ctttagataa aatgtcagaa ttagcttttg
atgatcagtg tacaggagct 2520aatccaagat atccactgat aagtgaaata aaacaaatgt
atataaatgt ttttgataaa 2580accgaaccaa ttgtagaaga tgaagaaaag taa
261370870PRTC. ljungdahlii 70Met Lys Val Thr Asn
Val Glu Glu Leu Met Lys Arg Leu Glu Glu Ile1 5
10 15Lys Asp Ala Gln Lys Lys Phe Ala Thr Tyr Thr
Gln Glu Gln Val Asp 20 25
30Glu Ile Phe Arg Gln Ala Ala Met Ala Ala Asn Ser Ala Arg Ile Glu
35 40 45Leu Ala Lys Met Ala Val Glu Glu
Ser Gly Met Gly Ile Val Glu Asp 50 55
60Lys Val Ile Lys Asn His Phe Ala Ser Glu Tyr Ile Tyr Asn Lys Tyr65
70 75 80Lys Asp Glu Lys Thr
Cys Gly Val Leu Glu Arg Asp Ala Gly Phe Gly 85
90 95Ile Val Arg Ile Ala Glu Pro Val Gly Val Ile
Ala Ala Val Val Pro 100 105
110Thr Thr Asn Pro Thr Ser Thr Ala Ile Phe Lys Ser Leu Ile Ala Leu
115 120 125Lys Thr Arg Asn Gly Ile Ile
Phe Ser Pro His Pro Arg Ala Lys Lys 130 135
140Ser Thr Ile Ala Ala Ala Lys Ile Val Leu Asp Ala Ala Val Lys
Ala145 150 155 160Gly Ala
Pro Glu Gly Ile Ile Gly Trp Ile Asp Glu Pro Ser Ile Glu
165 170 175Leu Ser Gln Val Val Met Gly
Glu Ala Asn Leu Ile Leu Ala Thr Gly 180 185
190Gly Pro Gly Met Val Lys Ala Ala Tyr Ser Ser Gly Lys Pro
Ala Val 195 200 205Gly Val Gly Pro
Gly Asn Thr Pro Ala Val Ile Asp Glu Ser Ala Asp 210
215 220Ile Lys Met Ala Val Asn Ser Ile Leu Leu Ser Lys
Thr Phe Asp Asn225 230 235
240Gly Met Ile Cys Ala Ser Glu Gln Ser Val Ile Val Leu Asp Ser Ile
245 250 255Tyr Glu Glu Val Lys
Lys Glu Phe Ala Tyr Arg Gly Ala Tyr Ile Leu 260
265 270Ser Lys Asp Glu Thr Asp Lys Val Gly Lys Ile Ile
Leu Lys Asn Gly 275 280 285Ala Leu
Asn Ala Gly Ile Val Gly Gln Pro Ala Phe Lys Ile Ala Gln 290
295 300Leu Ala Gly Val Asp Val Pro Glu Lys Ala Lys
Val Leu Ile Gly Glu305 310 315
320Val Glu Ser Val Glu Leu Glu Glu Pro Phe Ser His Glu Lys Leu Ser
325 330 335Pro Val Leu Ala
Met Tyr Arg Ala Arg Asn Phe Glu Asp Ala Ile Ala 340
345 350Lys Thr Asp Lys Leu Val Arg Ser Gly Gly Phe
Gly His Thr Ser Ser 355 360 365Leu
Tyr Val Asn Pro Met Thr Glu Lys Ala Lys Val Glu Lys Phe Ser 370
375 380Thr Met Met Lys Thr Ser Arg Thr Ile Ile
Asn Thr Pro Ser Ser Gln385 390 395
400Gly Gly Ile Gly Asp Ile Tyr Asn Phe Lys Leu Ala Pro Ser Leu
Thr 405 410 415Leu Gly Cys
Gly Ser Trp Gly Gly Asn Ser Val Ser Glu Asn Val Gly 420
425 430Pro Lys His Leu Leu Asn Ile Lys Ser Val
Ala Glu Arg Arg Glu Asn 435 440
445Met Leu Trp Phe Arg Val Pro Glu Lys Val Tyr Phe Lys Tyr Gly Ser 450
455 460Leu Gly Val Ala Leu Lys Glu Leu
Lys Val Met Asn Lys Lys Lys Val465 470
475 480Phe Ile Val Thr Asp Lys Val Leu Tyr Gln Leu Gly
Tyr Val Asp Lys 485 490
495Val Thr Lys Val Leu Glu Glu Leu Lys Ile Ser Tyr Lys Val Phe Thr
500 505 510Asp Val Glu Pro Asp Pro
Thr Leu Ala Thr Ala Lys Lys Gly Ala Ala 515 520
525Glu Leu Leu Ser Tyr Glu Pro Asp Thr Ile Ile Ser Val Gly
Gly Gly 530 535 540Ser Ala Met Asp Ala
Ala Lys Ile Met Trp Val Met Tyr Glu His Pro545 550
555 560Glu Val Lys Phe Glu Asp Leu Ala Met Arg
Phe Met Asp Ile Arg Lys 565 570
575Arg Val Tyr Val Phe Pro Lys Met Gly Glu Lys Ala Met Met Ile Ser
580 585 590Val Ala Thr Ser Ala
Gly Thr Gly Ser Glu Val Thr Pro Phe Ala Val 595
600 605Ile Thr Asp Glu Lys Thr Gly Ala Lys Tyr Pro Leu
Ala Asp Tyr Glu 610 615 620Leu Thr Pro
Asp Met Ala Ile Val Asp Ala Glu Leu Met Met Gly Met625
630 635 640Pro Arg Gly Leu Thr Ala Ala
Ser Gly Ile Asp Ala Leu Thr His Ala 645
650 655Leu Glu Ala Tyr Val Ser Ile Met Ala Thr Glu Phe
Thr Asn Gly Leu 660 665 670Ala
Leu Glu Ala Val Lys Leu Ile Phe Glu Tyr Leu Pro Lys Ala Tyr 675
680 685Thr Glu Gly Thr Thr Asn Val Lys Ala
Arg Glu Lys Met Val His Ala 690 695
700Ser Cys Ile Ala Gly Met Ala Phe Ala Asn Ala Phe Leu Gly Val Cys705
710 715 720His Ser Met Ala
His Lys Leu Gly Ala Gln His His Ile Pro His Gly 725
730 735Ile Ala Asn Ala Leu Met Ile Asp Glu Val
Ile Lys Phe Asn Ala Val 740 745
750Asp Asp Pro Ile Lys Gln Ala Ala Phe Pro Gln Tyr Glu Tyr Pro Asn
755 760 765Ala Arg Tyr Arg Tyr Ala Gln
Ile Ala Asp Cys Leu Asn Leu Gly Gly 770 775
780Asn Thr Glu Glu Glu Lys Val Gln Leu Leu Ile Asn Ala Ile Asp
Asp785 790 795 800Leu Lys
Ala Lys Leu Asn Ile Pro Glu Thr Ile Lys Glu Ala Gly Val
805 810 815Ser Glu Asp Lys Phe Tyr Ala
Thr Leu Asp Lys Met Ser Glu Leu Ala 820 825
830Phe Asp Asp Gln Cys Thr Gly Ala Asn Pro Arg Tyr Pro Leu
Ile Ser 835 840 845Glu Ile Lys Gln
Met Tyr Ile Asn Val Phe Asp Lys Thr Glu Pro Ile 850
855 860Val Glu Asp Glu Glu Lys865
870711554DNAC. ljundahlii 71ttggaaaatt ttgataaaga cttacgttct atacaagaag
caagagatct tgcacgttta 60ggaaaaattg cagcagacca aattgctgat tatactgaag
aacaaattga taaaatccta 120tgtaatatgg ttagggtagc agaagaaaat gcagtttgcc
ttggtaaaat ggctgcagaa 180gaaactggtt ttggaaaagc tgaagataag gcttataaga
accatatggc tgctactaca 240gtatataatt acatcaagga tatgaagact attggtgtta
taaaagaaga taaaagtgaa 300ggtgtaattg aatttgcaga accagttggt ttattaatgg
gtattgtacc atctacaaat 360ccaacatcta ctgttattta taaatcaatc attgcaatta
aatcaagaaa tgcaattgta 420ttctcaccac acccagctgc attaaaatgt tcaacaaaag
caatagaact tatgcgtgat 480gcagcagtag cagcaggagc tcctgcaaat gtaattggtg
gtattgttac accatctata 540caagctacaa atgaacttat gaaagctaaa gaagttgcta
tgataattgc aactggaggc 600cctggaatgg taaaggctgc atatagttca ggaacacctg
caataggcgt tggtgctggt 660aactctccat cctatattga aagaactgct gatgttcatc
aatcagttaa agatataata 720gctagtaaga gttttgacta tggtactatt tgtgcatccg
agcagtctgt aattgcagaa 780gaatgcaacc atgatgaaat agtagctgaa tttaagaaac
aaggcggata tttcatgaca 840gctgaagaaa ctgcaaaagt ttgcagcgta ctttttaaac
ctggtacaca cagcatgagc 900gctaagtttg taggaagagc tcctcaggtt atagcagaag
ctgcaggttt cacagttcca 960gaaggaacaa aagtattaat aggagaacaa ggcggagttg
gtaatggtta ccctctatct 1020tatgagaaac ttacaacagt acttgctttc tatacagtta
aagattggca tgaagcatgt 1080gagcttagta taagattact tcaaaatggt cttggacata
caatgaacat tcatacaaat 1140gatagagact tagtaatgaa gtttgctaaa aaaccagcat
cccgtatctt agttaatact 1200ggtggaagcc agggaggtac tggtgcaagc acaggattag
cacctgcatt tacattaggt 1260tgtggtacat ggggaggaag ctctgtttct gaaaatgtta
ctccattaca tttaatcaat 1320ataaagagag tagcatatgg tcttaaagat tgtactacat
tagctgcaga cgatacaact 1380ttcaatcatc ctgaactttg cggaagcaaa aatgacttag
gattctgtgc tacaagccct 1440gcagaatttg cagcaaagag caattgtgat agcactgctg
cagatactac tgataatgat 1500aaacttgcta gactcgtaag tgaattagta gctgcaatga
agggagctaa ctaa 155472517PRTC. ljungdahlii 72Met Glu Asn Phe Asp
Lys Asp Leu Arg Ser Ile Gln Glu Ala Arg Asp1 5
10 15Leu Ala Arg Leu Gly Lys Ile Ala Ala Asp Gln
Ile Ala Asp Tyr Thr 20 25
30Glu Glu Gln Ile Asp Lys Ile Leu Cys Asn Met Val Arg Val Ala Glu
35 40 45Glu Asn Ala Val Cys Leu Gly Lys
Met Ala Ala Glu Glu Thr Gly Phe 50 55
60Gly Lys Ala Glu Asp Lys Ala Tyr Lys Asn His Met Ala Ala Thr Thr65
70 75 80Val Tyr Asn Tyr Ile
Lys Asp Met Lys Thr Ile Gly Val Ile Lys Glu 85
90 95Asp Lys Ser Glu Gly Val Ile Glu Phe Ala Glu
Pro Val Gly Leu Leu 100 105
110Met Gly Ile Val Pro Ser Thr Asn Pro Thr Ser Thr Val Ile Tyr Lys
115 120 125Ser Ile Ile Ala Ile Lys Ser
Arg Asn Ala Ile Val Phe Ser Pro His 130 135
140Pro Ala Ala Leu Lys Cys Ser Thr Lys Ala Ile Glu Leu Met Arg
Asp145 150 155 160Ala Ala
Val Ala Ala Gly Ala Pro Ala Asn Val Ile Gly Gly Ile Val
165 170 175Thr Pro Ser Ile Gln Ala Thr
Asn Glu Leu Met Lys Ala Lys Glu Val 180 185
190Ala Met Ile Ile Ala Thr Gly Gly Pro Gly Met Val Lys Ala
Ala Tyr 195 200 205Ser Ser Gly Thr
Pro Ala Ile Gly Val Gly Ala Gly Asn Ser Pro Ser 210
215 220Tyr Ile Glu Arg Thr Ala Asp Val His Gln Ser Val
Lys Asp Ile Ile225 230 235
240Ala Ser Lys Ser Phe Asp Tyr Gly Thr Ile Cys Ala Ser Glu Gln Ser
245 250 255Val Ile Ala Glu Glu
Cys Asn His Asp Glu Ile Val Ala Glu Phe Lys 260
265 270Lys Gln Gly Gly Tyr Phe Met Thr Ala Glu Glu Thr
Ala Lys Val Cys 275 280 285Ser Val
Leu Phe Lys Pro Gly Thr His Ser Met Ser Ala Lys Phe Val 290
295 300Gly Arg Ala Pro Gln Val Ile Ala Glu Ala Ala
Gly Phe Thr Val Pro305 310 315
320Glu Gly Thr Lys Val Leu Ile Gly Glu Gln Gly Gly Val Gly Asn Gly
325 330 335Tyr Pro Leu Ser
Tyr Glu Lys Leu Thr Thr Val Leu Ala Phe Tyr Thr 340
345 350Val Lys Asp Trp His Glu Ala Cys Glu Leu Ser
Ile Arg Leu Leu Gln 355 360 365Asn
Gly Leu Gly His Thr Met Asn Ile His Thr Asn Asp Arg Asp Leu 370
375 380Val Met Lys Phe Ala Lys Lys Pro Ala Ser
Arg Ile Leu Val Asn Thr385 390 395
400Gly Gly Ser Gln Gly Gly Thr Gly Ala Ser Thr Gly Leu Ala Pro
Ala 405 410 415Phe Thr Leu
Gly Cys Gly Thr Trp Gly Gly Ser Ser Val Ser Glu Asn 420
425 430Val Thr Pro Leu His Leu Ile Asn Ile Lys
Arg Val Ala Tyr Gly Leu 435 440
445Lys Asp Cys Thr Thr Leu Ala Ala Asp Asp Thr Thr Phe Asn His Pro 450
455 460Glu Leu Cys Gly Ser Lys Asn Asp
Leu Gly Phe Cys Ala Thr Ser Pro465 470
475 480Ala Glu Phe Ala Ala Lys Ser Asn Cys Asp Ser Thr
Ala Ala Asp Thr 485 490
495Thr Asp Asn Asp Lys Leu Ala Arg Leu Val Ser Glu Leu Val Ala Ala
500 505 510Met Lys Gly Ala Asn
515731497DNAC. ljungdahlii 73atgaatatta ttgataatga tttgctctcc atccaagaat
cccgaatcct tgtggaaaat 60gctgcacgag cacaaaaaat gttagcaacc tttccacaag
aaaagctaga tgagattgtt 120gaacgtatgg cggaagaaat cggaaaacat acccgagagc
ttgctgtaat gtcacaggat 180gaaactggtt atggaaaatg gcaggataaa tgcatcaaaa
accgatttgc ctgtgagtat 240ttgccagcta agcttagagg aatgcgatgt gtaggtatta
ttaatgaaaa tggtcaggat 300aagaccatgg atgtaggtgt acctatgggt gtaattattg
cattatgtcc tgcaactagt 360ccggtttcta ctaccatata taaggcattg attgcaatta
agtctggtaa tgcaattatc 420ttttctccac atcctagagc aaaggagaca atttgtaagg
cgcttgacat catgattcgt 480gcagctgaag gatatgggct tccagaagga gctcttgcat
acttacatac tgtgacgcct 540agtggaacaa tcgaattgat gaaccatatt gcgacttctt
tgattatgaa tacaggtgtt 600cccgggatgc ttaaagcagc atataattct gggaaacctg
ttatatatgg aggaactggt 660aatggaccag catttattga acgtacagct gacatcaaac
aggcggtaaa agatattatt 720gctagtaaga cctttgataa cggaatagta ccatcagctg
aacaatctat tgttgtagat 780agctgtgttg catctgatgt taaacgtgag ttgcaaaata
atggtgcata tttcatgaca 840gaggaggaag cacaaaaact aggttctctc tttttccgtt
ctgatggcag tatggattca 900gaaatggttg gcaaatccgc acaaagattg gctaaaaaag
caggtttcag cattcctgaa 960agtagcacag tgctaatttc agagcagaaa tatgtttctc
aagataatcc ttattccaag 1020gagaaacttt gtccggtact agcttactac attgaagatg
attggatgca tgcatgtgaa 1080aagtgtattg aactgctgtt aagtgagaga catggtcaca
ctcttgttat acattcaaaa 1140gacgaagatg taattcgcca gtttgcatta aaaaaacctg
taggtaggat acttgttaat 1200acgcctgctt cctttggtag tatgggtgct acaagtaatt
tatttcctgc tttaacttta 1260ggtagtggat cggcaggtaa aggtattacc tccgataatg
tttcaccaat gaatcttatt 1320tacgtccgca aagtcggata tggcgtacgg aatgtagaag
agattgtcaa tactaatgga 1380ttgtttacag aagaaaaaag tgatttgaat ggaatgacaa
aaaagtcaga ctataatcca 1440gaggatatac aaatgttaca gcatatttta aaaaaagcta
tggaaaaaat taaatag 149774498PRTC. ljungdahlii 74Met Asn Ile Ile Asp
Asn Asp Leu Leu Ser Ile Gln Glu Ser Arg Ile1 5
10 15Leu Val Glu Asn Ala Ala Arg Ala Gln Lys Met
Leu Ala Thr Phe Pro 20 25
30Gln Glu Lys Leu Asp Glu Ile Val Glu Arg Met Ala Glu Glu Ile Gly
35 40 45Lys His Thr Arg Glu Leu Ala Val
Met Ser Gln Asp Glu Thr Gly Tyr 50 55
60Gly Lys Trp Gln Asp Lys Cys Ile Lys Asn Arg Phe Ala Cys Glu Tyr65
70 75 80Leu Pro Ala Lys Leu
Arg Gly Met Arg Cys Val Gly Ile Ile Asn Glu 85
90 95Asn Gly Gln Asp Lys Thr Met Asp Val Gly Val
Pro Met Gly Val Ile 100 105
110Ile Ala Leu Cys Pro Ala Thr Ser Pro Val Ser Thr Thr Ile Tyr Lys
115 120 125Ala Leu Ile Ala Ile Lys Ser
Gly Asn Ala Ile Ile Phe Ser Pro His 130 135
140Pro Arg Ala Lys Glu Thr Ile Cys Lys Ala Leu Asp Ile Met Ile
Arg145 150 155 160Ala Ala
Glu Gly Tyr Gly Leu Pro Glu Gly Ala Leu Ala Tyr Leu His
165 170 175Thr Val Thr Pro Ser Gly Thr
Ile Glu Leu Met Asn His Ile Ala Thr 180 185
190Ser Leu Ile Met Asn Thr Gly Val Pro Gly Met Leu Lys Ala
Ala Tyr 195 200 205Asn Ser Gly Lys
Pro Val Ile Tyr Gly Gly Thr Gly Asn Gly Pro Ala 210
215 220Phe Ile Glu Arg Thr Ala Asp Ile Lys Gln Ala Val
Lys Asp Ile Ile225 230 235
240Ala Ser Lys Thr Phe Asp Asn Gly Ile Val Pro Ser Ala Glu Gln Ser
245 250 255Ile Val Val Asp Ser
Cys Val Ala Ser Asp Val Lys Arg Glu Leu Gln 260
265 270Asn Asn Gly Ala Tyr Phe Met Thr Glu Glu Glu Ala
Gln Lys Leu Gly 275 280 285Ser Leu
Phe Phe Arg Ser Asp Gly Ser Met Asp Ser Glu Met Val Gly 290
295 300Lys Ser Ala Gln Arg Leu Ala Lys Lys Ala Gly
Phe Ser Ile Pro Glu305 310 315
320Ser Ser Thr Val Leu Ile Ser Glu Gln Lys Tyr Val Ser Gln Asp Asn
325 330 335Pro Tyr Ser Lys
Glu Lys Leu Cys Pro Val Leu Ala Tyr Tyr Ile Glu 340
345 350Asp Asp Trp Met His Ala Cys Glu Lys Cys Ile
Glu Leu Leu Leu Ser 355 360 365Glu
Arg His Gly His Thr Leu Val Ile His Ser Lys Asp Glu Asp Val 370
375 380Ile Arg Gln Phe Ala Leu Lys Lys Pro Val
Gly Arg Ile Leu Val Asn385 390 395
400Thr Pro Ala Ser Phe Gly Ser Met Gly Ala Thr Ser Asn Leu Phe
Pro 405 410 415Ala Leu Thr
Leu Gly Ser Gly Ser Ala Gly Lys Gly Ile Thr Ser Asp 420
425 430Asn Val Ser Pro Met Asn Leu Ile Tyr Val
Arg Lys Val Gly Tyr Gly 435 440
445Val Arg Asn Val Glu Glu Ile Val Asn Thr Asn Gly Leu Phe Thr Glu 450
455 460Glu Lys Ser Asp Leu Asn Gly Met
Thr Lys Lys Ser Asp Tyr Asn Pro465 470
475 480Glu Asp Ile Gln Met Leu Gln His Ile Leu Lys Lys
Ala Met Glu Lys 485 490
495Ile Lys751167DNAC. ljungdahlii 75atggcaagat ttactttacc aagagacatt
tattttggag aaaattcatt agaaaccttg 60aaagacctag atggaaaaaa agctgttatt
gtcgtaggtg gtggatccat gaaacgattt 120ggattccttg ataaggtagt aaactactta
aaagaagcag gtattgaatc aaaattaata 180gaaggagttg aaccagatcc atctgtagaa
actgttatga atggcgctaa actaatgaga 240gaatatgaac cagatttaat agtatcaata
ggtggaggtt caccaattga cgcagcaaaa 300gctatgtgga tattctatga ataccctgag
tttactttta aagaggctgt ggttcctttt 360ggtcttccta aattaagaca aaaagcaaca
tttatagcta taccttctac aagtggtact 420gcaacagaag taacggcatt ttctgtaata
acagactata aagctaaaat taaatatcct 480ttagctgact tcaatttaac accagatata
gctataattg atccagcatt agctcaaaca 540atgccaccta aattaactgc acatactgga
atggatgcac ttacccatgc tattgaagca 600tatgttgcag gacttcattc agttttctca
gatcctcttg ctattcaagc tatagttatg 660gtaaatcagt atttaattaa atcttacaat
gaagataaag aagctagaaa ccaaatgcat 720ttagctcaat gtttagctgg aatggcattt
tcaaatgcac ttcttggaat aactcacagt 780ttagcacata aaacaggtgc agtattccat
atccctcatg gatgtgccaa tgcaatatat 840cttccttatg ttatagattt caataaaaaa
gcttgtgcac caagatatgc tgaaatagct 900aggagtctta aacttccagg aaatactgat
gatgaattag tagattcatt aaccaacatg 960attaaagata tgaataagag tatggatatt
cctttaacat taaaagatta cggagtagat 1020gaaaaagaat ttaaagatag tgaagatttt
atagctcaca atgccgtatt agatgcctgc 1080actggatcaa atcctagaag tataaatgat
actgaaatga aaaagttatt agaatacatc 1140tattatggta aaaaggttga tttttaa
116776388PRTC. ljundahlii 76Met Ala Arg
Phe Thr Leu Pro Arg Asp Ile Tyr Phe Gly Glu Asn Ser1 5
10 15Leu Glu Thr Leu Lys Asp Leu Asp Gly
Lys Lys Ala Val Ile Val Val 20 25
30Gly Gly Gly Ser Met Lys Arg Phe Gly Phe Leu Asp Lys Val Val Asn
35 40 45Tyr Leu Lys Glu Ala Gly Ile
Glu Ser Lys Leu Ile Glu Gly Val Glu 50 55
60Pro Asp Pro Ser Val Glu Thr Val Met Asn Gly Ala Lys Leu Met Arg65
70 75 80Glu Tyr Glu Pro
Asp Leu Ile Val Ser Ile Gly Gly Gly Ser Pro Ile 85
90 95Asp Ala Ala Lys Ala Met Trp Ile Phe Tyr
Glu Tyr Pro Glu Phe Thr 100 105
110Phe Lys Glu Ala Val Val Pro Phe Gly Leu Pro Lys Leu Arg Gln Lys
115 120 125Ala Thr Phe Ile Ala Ile Pro
Ser Thr Ser Gly Thr Ala Thr Glu Val 130 135
140Thr Ala Phe Ser Val Ile Thr Asp Tyr Lys Ala Lys Ile Lys Tyr
Pro145 150 155 160Leu Ala
Asp Phe Asn Leu Thr Pro Asp Ile Ala Ile Ile Asp Pro Ala
165 170 175Leu Ala Gln Thr Met Pro Pro
Lys Leu Thr Ala His Thr Gly Met Asp 180 185
190Ala Leu Thr His Ala Ile Glu Ala Tyr Val Ala Gly Leu His
Ser Val 195 200 205Phe Ser Asp Pro
Leu Ala Ile Gln Ala Ile Val Met Val Asn Gln Tyr 210
215 220Leu Ile Lys Ser Tyr Asn Glu Asp Lys Glu Ala Arg
Asn Gln Met His225 230 235
240Leu Ala Gln Cys Leu Ala Gly Met Ala Phe Ser Asn Ala Leu Leu Gly
245 250 255Ile Thr His Ser Leu
Ala His Lys Thr Gly Ala Val Phe His Ile Pro 260
265 270His Gly Cys Ala Asn Ala Ile Tyr Leu Pro Tyr Val
Ile Asp Phe Asn 275 280 285Lys Lys
Ala Cys Ala Pro Arg Tyr Ala Glu Ile Ala Arg Ser Leu Lys 290
295 300Leu Pro Gly Asn Thr Asp Asp Glu Leu Val Asp
Ser Leu Thr Asn Met305 310 315
320Ile Lys Asp Met Asn Lys Ser Met Asp Ile Pro Leu Thr Leu Lys Asp
325 330 335Tyr Gly Val Asp
Glu Lys Glu Phe Lys Asp Ser Glu Asp Phe Ile Ala 340
345 350His Asn Ala Val Leu Asp Ala Cys Thr Gly Ser
Asn Pro Arg Ser Ile 355 360 365Asn
Asp Thr Glu Met Lys Lys Leu Leu Glu Tyr Ile Tyr Tyr Gly Lys 370
375 380Lys Val Asp Phe385771167DNAC. ljundahlii
77atgggaagat ttactttgcc tagggatatt tactttggtg aaaatgcctt agaaaattta
60aaaaatttag atggaaataa agcagtagtt gttgtaggtg ggggatctat gaagagattt
120ggattcttag ccaaagttga aaaatactta aaagaaactg gtatggaagt taaattaata
180gaaggtgttg agcctgatcc gtctgttgat actgttatga atggcgctaa aataatgaga
240gactttaacc cagattggat agtatcaata ggtggaggat ctcccataga tgctgctaaa
300gcaatgtgga tattttatga ataccccgac tttacatttg aaaaagcggt agtccctttt
360ggaattccta aattaaggca gaaggcacaa tttgttgcta taccttctac aagtggaaca
420gcaactgaag taacatcatt ttctgtaata acagactata aagctaaaat aaaatatcct
480cttgcagatt ttaaccttac ccctgatata gctataatag atccgtctct tgcagaaaca
540atgcccaaaa agcttacagc acacactgga atggatgcac ttactcacgc aatagaagca
600tatgtagcaa gtttacattc agatttctca gatccacttg ctatgcatgc tataaccatg
660attcataaat atttattgaa atcctatgaa gaagataaag aagctagagg acatatgcat
720atagcccaat gtctagctgg gatggcattt tcaaatgctc tccttggaat aactcatagt
780atagcacata aaactggtgc agtatttcac atacctcatg ggtgtgctaa tgccatatac
840ttaccttatg ttatagattt taacaagaaa gcttgttcag aaagatatgc taaaatagcc
900aaaaagctgc atctatcagg aaatagtgaa gatgagctaa tagattcatt aactgaaatg
960attcgtacta tgaacaaaaa gatggatatt cctctcacca taaaagatta tggtataagc
1020gaaaacgatt ttaatgaaaa cctagatttt atagctcaca atgccatgat ggatgcctgc
1080actggatcca atcctagagc aataactgag gaagaaatga aaaagctctt gcagtatatg
1140tataatgggc aaaaggttaa tttctag
116778388PRTC. ljundahlii 78Met Gly Arg Phe Thr Leu Pro Arg Asp Ile Tyr
Phe Gly Glu Asn Ala1 5 10
15Leu Glu Asn Leu Lys Asn Leu Asp Gly Asn Lys Ala Val Val Val Val
20 25 30Gly Gly Gly Ser Met Lys Arg
Phe Gly Phe Leu Ala Lys Val Glu Lys 35 40
45Tyr Leu Lys Glu Thr Gly Met Glu Val Lys Leu Ile Glu Gly Val
Glu 50 55 60Pro Asp Pro Ser Val Asp
Thr Val Met Asn Gly Ala Lys Ile Met Arg65 70
75 80Asp Phe Asn Pro Asp Trp Ile Val Ser Ile Gly
Gly Gly Ser Pro Ile 85 90
95Asp Ala Ala Lys Ala Met Trp Ile Phe Tyr Glu Tyr Pro Asp Phe Thr
100 105 110Phe Glu Lys Ala Val Val
Pro Phe Gly Ile Pro Lys Leu Arg Gln Lys 115 120
125Ala Gln Phe Val Ala Ile Pro Ser Thr Ser Gly Thr Ala Thr
Glu Val 130 135 140Thr Ser Phe Ser Val
Ile Thr Asp Tyr Lys Ala Lys Ile Lys Tyr Pro145 150
155 160Leu Ala Asp Phe Asn Leu Thr Pro Asp Ile
Ala Ile Ile Asp Pro Ser 165 170
175Leu Ala Glu Thr Met Pro Lys Lys Leu Thr Ala His Thr Gly Met Asp
180 185 190Ala Leu Thr His Ala
Ile Glu Ala Tyr Val Ala Ser Leu His Ser Asp 195
200 205Phe Ser Asp Pro Leu Ala Met His Ala Ile Thr Met
Ile His Lys Tyr 210 215 220Leu Leu Lys
Ser Tyr Glu Glu Asp Lys Glu Ala Arg Gly His Met His225
230 235 240Ile Ala Gln Cys Leu Ala Gly
Met Ala Phe Ser Asn Ala Leu Leu Gly 245
250 255Ile Thr His Ser Ile Ala His Lys Thr Gly Ala Val
Phe His Ile Pro 260 265 270His
Gly Cys Ala Asn Ala Ile Tyr Leu Pro Tyr Val Ile Asp Phe Asn 275
280 285Lys Lys Ala Cys Ser Glu Arg Tyr Ala
Lys Ile Ala Lys Lys Leu His 290 295
300Leu Ser Gly Asn Ser Glu Asp Glu Leu Ile Asp Ser Leu Thr Glu Met305
310 315 320Ile Arg Thr Met
Asn Lys Lys Met Asp Ile Pro Leu Thr Ile Lys Asp 325
330 335Tyr Gly Ile Ser Glu Asn Asp Phe Asn Glu
Asn Leu Asp Phe Ile Ala 340 345
350His Asn Ala Met Met Asp Ala Cys Thr Gly Ser Asn Pro Arg Ala Ile
355 360 365Thr Glu Glu Glu Met Lys Lys
Leu Leu Gln Tyr Met Tyr Asn Gly Gln 370 375
380Lys Val Asn Phe385791167DNAC. ljundahlii 79atggagagat ttacgttgcc
aagagacatt tactttggag aagatgcttt gggtgctttg 60aaaacgttaa aaggtaagaa
agctgtagta gttgttggag gaggatccat gaagagattc 120ggtttccttg acaaggtaga
agaatactta aaagaagcaa acatagaagt taaactaata 180gaaggtgttg aaccagatcc
gtctgtggaa accgttatga aaggtgccaa aataatgaca 240gaatttgggc cagattggat
agttgctatt ggaggaggtt caccaataga tgctgcaaag 300gctatgtggc tattttatga
atatccagat tttactttta aacaagcaat tgttccgttt 360ggattaccag aattaagaca
aaaagctaaa tttgtagcta tagcttctac tagtggaaca 420gctactgaag ttacttcatt
ttcagtaata actgattata aagctaaaat aaagtatcct 480ttagctgact tcaatttgac
accggatata gctatagttg atccagcatt agcccagaca 540atgccaccta aattaactgc
acatactggt atggatgcat taactcatgc actagaagct 600tatgtagcat cagctagatc
agatatttca gatccacttg caatacattc cataattatg 660acaagggata acttacttaa
atcctataag ggtgataaag atgctagaaa taagatgcat 720atatcacaat gtttagcagg
tatggcattt tctaatgcac ttcttggtat aactcatagt 780ttagcacata aaacaggagc
tgtatggcac ataccacatg gatgcgctaa tgcaatatat 840cttccatatg ttttagattt
taataaaaaa gcttgctcag atagatatgc taatatagct 900aaaatattag gacttaaagg
aactactgaa gatgaattgg tagattctct agttaaaatg 960gtacaagata tggataagga
attgaatata cctttgacct taaaagatta tggtataagc 1020aaagatgatt tcaattcaaa
tgttgatttt atagcaaaga atgcgctctt agatgcatgt 1080acaggagcta atccaaggcc
tatagatttt gatcaaatga aaaagatact tcaatgtata 1140tatgatggaa aaaaggtaac
tttttaa 116780388PRTC. ljungdahlii
80Met Glu Arg Phe Thr Leu Pro Arg Asp Ile Tyr Phe Gly Glu Asp Ala1
5 10 15Leu Gly Ala Leu Lys Thr
Leu Lys Gly Lys Lys Ala Val Val Val Val 20 25
30Gly Gly Gly Ser Met Lys Arg Phe Gly Phe Leu Asp Lys
Val Glu Glu 35 40 45Tyr Leu Lys
Glu Ala Asn Ile Glu Val Lys Leu Ile Glu Gly Val Glu 50
55 60Pro Asp Pro Ser Val Glu Thr Val Met Lys Gly Ala
Lys Ile Met Thr65 70 75
80Glu Phe Gly Pro Asp Trp Ile Val Ala Ile Gly Gly Gly Ser Pro Ile
85 90 95Asp Ala Ala Lys Ala Met
Trp Leu Phe Tyr Glu Tyr Pro Asp Phe Thr 100
105 110Phe Lys Gln Ala Ile Val Pro Phe Gly Leu Pro Glu
Leu Arg Gln Lys 115 120 125Ala Lys
Phe Val Ala Ile Ala Ser Thr Ser Gly Thr Ala Thr Glu Val 130
135 140Thr Ser Phe Ser Val Ile Thr Asp Tyr Lys Ala
Lys Ile Lys Tyr Pro145 150 155
160Leu Ala Asp Phe Asn Leu Thr Pro Asp Ile Ala Ile Val Asp Pro Ala
165 170 175Leu Ala Gln Thr
Met Pro Pro Lys Leu Thr Ala His Thr Gly Met Asp 180
185 190Ala Leu Thr His Ala Leu Glu Ala Tyr Val Ala
Ser Ala Arg Ser Asp 195 200 205Ile
Ser Asp Pro Leu Ala Ile His Ser Ile Ile Met Thr Arg Asp Asn 210
215 220Leu Leu Lys Ser Tyr Lys Gly Asp Lys Asp
Ala Arg Asn Lys Met His225 230 235
240Ile Ser Gln Cys Leu Ala Gly Met Ala Phe Ser Asn Ala Leu Leu
Gly 245 250 255Ile Thr His
Ser Leu Ala His Lys Thr Gly Ala Val Trp His Ile Pro 260
265 270His Gly Cys Ala Asn Ala Ile Tyr Leu Pro
Tyr Val Leu Asp Phe Asn 275 280
285Lys Lys Ala Cys Ser Asp Arg Tyr Ala Asn Ile Ala Lys Ile Leu Gly 290
295 300Leu Lys Gly Thr Thr Glu Asp Glu
Leu Val Asp Ser Leu Val Lys Met305 310
315 320Val Gln Asp Met Asp Lys Glu Leu Asn Ile Pro Leu
Thr Leu Lys Asp 325 330
335Tyr Gly Ile Ser Lys Asp Asp Phe Asn Ser Asn Val Asp Phe Ile Ala
340 345 350Lys Asn Ala Leu Leu Asp
Ala Cys Thr Gly Ala Asn Pro Arg Pro Ile 355 360
365Asp Phe Asp Gln Met Lys Lys Ile Leu Gln Cys Ile Tyr Asp
Gly Lys 370 375 380Lys Val Thr
Phe385811176DNAC. ljundahlii 81atggaaaact ttatttttaa aaatgctaca
gaaattattt ttggtaagga taccgaaaat 60cttgtaggaa gtaaagtaaa ggagtattca
aagtcagata aaatactctt ttgctatggg 120ggaggaagca taaaaagatc tggtctatat
gatagagtta taaagtcctt aaaagaaaat 180ggaattgaat ttatagaact tccaggaatt
aaacctaatc caagattagg acctgttaaa 240gaaggtataa gactatgtag agaaaataat
ataaaatttg tactatctgt aggaggagga 300agttcagcag atacggctaa agctattgct
gtaggagtac cttataaagg agacgtatgg 360gatttttata cgggcaaagc tgaagtgaaa
gaggctcttc ctgtaggagt tgtaataaca 420ttacctgcta caggtacaga atctagtaat
agttctgtta ttatgaatga agatggttgg 480tttaaaaaag gattaaatac agtacttata
agacctgctt tttcaattat gaatcctgaa 540cttactttta cactaccaga gtatcaaact
gcttgtggtg cttgtgacat tatggcacat 600ataatggaaa gatattttac aaatgtgaaa
catgtagata taactgatag gctttgcgaa 660gctgcactta gaaatgttat aaataatgcc
ccaatagttt taaaagatcc caaaaactat 720gatgctaggg cagaaattat gtggaccggt
actatagctc ataatgatgt gcttagtgcg 780ggtagaatag gtgattgggc ttctcacaaa
attgaacatg aattgagtgg ggaaacagac 840attgcccatg gagcaggact tgcaattgta
tttcctgcat ggatgaaata tgtatataaa 900cacgatatca atagatttgt acaatttgca
gtaagggtat gggatgtaga tttatcttat 960agttcctgcg aagatattgt acttgaaggc
ataaggagaa tgacagcatt tttcaagagc 1020atggggttac ctgtaacttt aaaagaagga
agtataggag aagataaaat tgaagaaatg 1080gctaataagt gcacggataa tggaactaaa
actgtaggac aatttgtaaa attaaataaa 1140gatgatattg taaaaatatt aaatttagct
aaataa 117682391PRTC. ljundahlii 82Met Glu
Asn Phe Ile Phe Lys Asn Ala Thr Glu Ile Ile Phe Gly Lys1 5
10 15Asp Thr Glu Asn Leu Val Gly Ser
Lys Val Lys Glu Tyr Ser Lys Ser 20 25
30Asp Lys Ile Leu Phe Cys Tyr Gly Gly Gly Ser Ile Lys Arg Ser
Gly 35 40 45Leu Tyr Asp Arg Val
Ile Lys Ser Leu Lys Glu Asn Gly Ile Glu Phe 50 55
60Ile Glu Leu Pro Gly Ile Lys Pro Asn Pro Arg Leu Gly Pro
Val Lys65 70 75 80Glu
Gly Ile Arg Leu Cys Arg Glu Asn Asn Ile Lys Phe Val Leu Ser
85 90 95Val Gly Gly Gly Ser Ser Ala
Asp Thr Ala Lys Ala Ile Ala Val Gly 100 105
110Val Pro Tyr Lys Gly Asp Val Trp Asp Phe Tyr Thr Gly Lys
Ala Glu 115 120 125Val Lys Glu Ala
Leu Pro Val Gly Val Val Ile Thr Leu Pro Ala Thr 130
135 140Gly Thr Glu Ser Ser Asn Ser Ser Val Ile Met Asn
Glu Asp Gly Trp145 150 155
160Phe Lys Lys Gly Leu Asn Thr Val Leu Ile Arg Pro Ala Phe Ser Ile
165 170 175Met Asn Pro Glu Leu
Thr Phe Thr Leu Pro Glu Tyr Gln Thr Ala Cys 180
185 190Gly Ala Cys Asp Ile Met Ala His Ile Met Glu Arg
Tyr Phe Thr Asn 195 200 205Val Lys
His Val Asp Ile Thr Asp Arg Leu Cys Glu Ala Ala Leu Arg 210
215 220Asn Val Ile Asn Asn Ala Pro Ile Val Leu Lys
Asp Pro Lys Asn Tyr225 230 235
240Asp Ala Arg Ala Glu Ile Met Trp Thr Gly Thr Ile Ala His Asn Asp
245 250 255Val Leu Ser Ala
Gly Arg Ile Gly Asp Trp Ala Ser His Lys Ile Glu 260
265 270His Glu Leu Ser Gly Glu Thr Asp Ile Ala His
Gly Ala Gly Leu Ala 275 280 285Ile
Val Phe Pro Ala Trp Met Lys Tyr Val Tyr Lys His Asp Ile Asn 290
295 300Arg Phe Val Gln Phe Ala Val Arg Val Trp
Asp Val Asp Leu Ser Tyr305 310 315
320Ser Ser Cys Glu Asp Ile Val Leu Glu Gly Ile Arg Arg Met Thr
Ala 325 330 335Phe Phe Lys
Ser Met Gly Leu Pro Val Thr Leu Lys Glu Gly Ser Ile 340
345 350Gly Glu Asp Lys Ile Glu Glu Met Ala Asn
Lys Cys Thr Asp Asn Gly 355 360
365Thr Lys Thr Val Gly Gln Phe Val Lys Leu Asn Lys Asp Asp Ile Val 370
375 380Lys Ile Leu Asn Leu Ala Lys385
390831149DNAC. ljungdahlii 83atggaagaca agtttgaaaa ttttaatttg
aaatccaaga tttattttaa tagggaatct 60attcaacttt tagagcaagt cactggttct
cgagcattta ttgttgcaga tgctattatg 120ggaaaacttg gatatcttca aaaagtaata
gattacctaa gcaaagctgg aataagttcc 180gttgttttta cgggggtaca ccctgatcca
gacgtcaatg taattgcaga tgcaatgaaa 240ttgtacaaaa aaagcgacgc agatgttctc
gtagcactag gtggaggatc cagtattgat 300accgctaagg gaataatgta ttttgcatgt
aatttaggaa aagcaatggg ccaagaaatg 360aaaaaacctc tatttattgc aattccatca
acaagtggta caggctctga agtaacaaac 420tttactgtta ttacttctca gaaagaaaag
gtatgcatta tagatgattt tattgcacca 480gatgttgcaa tacttgactc aagttgtatt
gatggtctgc ctcagcgtat tgtagcagat 540actggtatag atgttctagt tcattctatt
gaagcctatg tttccaaaaa agcaactgac 600tttacagacg ctcttgctga aaaagcagtt
aaattaattt ttgagaatct tccaaaaatt 660tataacgata gtaaggattc cgaagctcga
gatcatgttc aaaacgcttc ctgtatagca 720ggaatagcat ttacaaatgc tggtcttgga
attaatcaca gcttggctca tgctatgggt 780ggatctttcc acattcctca cggccgatcc
aatgcacttc tacttaatgc agtaatggaa 840tacaacgcta gcttggttgg aaatgcaagc
gaacatgcta tggaaaaata cgcaaaacta 900gcatcaattc tacaccttcc agctcgaaca
actcgcgaag gcgctgtaag ttttattgaa 960gctgtagata aattaataaa atccctaggt
gttgaagata atattcgatc tcttgggatt 1020aaagaagatg agtttcaaag tgctctaaat
catatggcag aaacagcaat gcaagataga 1080tgcactccaa ctaatcctag aaaaccttct
aaagaagaac ttatacatat ttatcaaaaa 1140tgttattaa
114984382PRTC. lungdahlii 84Met Glu Asp
Lys Phe Glu Asn Phe Asn Leu Lys Ser Lys Ile Tyr Phe1 5
10 15Asn Arg Glu Ser Ile Gln Leu Leu Glu
Gln Val Thr Gly Ser Arg Ala 20 25
30Phe Ile Val Ala Asp Ala Ile Met Gly Lys Leu Gly Tyr Leu Gln Lys
35 40 45Val Ile Asp Tyr Leu Ser Lys
Ala Gly Ile Ser Ser Val Val Phe Thr 50 55
60Gly Val His Pro Asp Pro Asp Val Asn Val Ile Ala Asp Ala Met Lys65
70 75 80Leu Tyr Lys Lys
Ser Asp Ala Asp Val Leu Val Ala Leu Gly Gly Gly 85
90 95Ser Ser Ile Asp Thr Ala Lys Gly Ile Met
Tyr Phe Ala Cys Asn Leu 100 105
110Gly Lys Ala Met Gly Gln Glu Met Lys Lys Pro Leu Phe Ile Ala Ile
115 120 125Pro Ser Thr Ser Gly Thr Gly
Ser Glu Val Thr Asn Phe Thr Val Ile 130 135
140Thr Ser Gln Lys Glu Lys Val Cys Ile Ile Asp Asp Phe Ile Ala
Pro145 150 155 160Asp Val
Ala Ile Leu Asp Ser Ser Cys Ile Asp Gly Leu Pro Gln Arg
165 170 175Ile Val Ala Asp Thr Gly Ile
Asp Val Leu Val His Ser Ile Glu Ala 180 185
190Tyr Val Ser Lys Lys Ala Thr Asp Phe Thr Asp Ala Leu Ala
Glu Lys 195 200 205Ala Val Lys Leu
Ile Phe Glu Asn Leu Pro Lys Ile Tyr Asn Asp Ser 210
215 220Lys Asp Ser Glu Ala Arg Asp His Val Gln Asn Ala
Ser Cys Ile Ala225 230 235
240Gly Ile Ala Phe Thr Asn Ala Gly Leu Gly Ile Asn His Ser Leu Ala
245 250 255His Ala Met Gly Gly
Ser Phe His Ile Pro His Gly Arg Ser Asn Ala 260
265 270Leu Leu Leu Asn Ala Val Met Glu Tyr Asn Ala Ser
Leu Val Gly Asn 275 280 285Ala Ser
Glu His Ala Met Glu Lys Tyr Ala Lys Leu Ala Ser Ile Leu 290
295 300His Leu Pro Ala Arg Thr Thr Arg Glu Gly Ala
Val Ser Phe Ile Glu305 310 315
320Ala Val Asp Lys Leu Ile Lys Ser Leu Gly Val Glu Asp Asn Ile Arg
325 330 335Ser Leu Gly Ile
Lys Glu Asp Glu Phe Gln Ser Ala Leu Asn His Met 340
345 350Ala Glu Thr Ala Met Gln Asp Arg Cys Thr Pro
Thr Asn Pro Arg Lys 355 360 365Pro
Ser Lys Glu Glu Leu Ile His Ile Tyr Gln Lys Cys Tyr 370
375 380851002DNAC. ljungdahlii 85atgaaattga tggaaaaaat
ttggagtaag gcaaaggaag acaaaaaaaa gattgtctta 60gctgaaggag aagaagaaag
aactcttcaa gcttgtgaaa aaataattaa agagggtatt 120gcaaatttaa tccttgtagg
gaatgaaaag gtaataaaag aaaaagcgtc aaaattaggt 180gtaagtttaa atggagcaga
aatagtagat ccagagactt cagataaact aaaggcatat 240gcagatgctt tttatgaatt
gagaaagaag aagggaataa cgccagaaaa agcggataaa 300atagtaagag atccaatata
ctttgctaca atgatggtta aacttggaga tgcagatgga 360ttggtttcag gtgcggttca
tactacaggt gatcttttga gaccaggact tcaaatagta 420aagacagctc caggtacatc
agtagtttcc agtacattta taatggaagt accaaattgt 480gagtatggtg acaatggtgt
acttctattt gctgattgtg ctgtaaatcc atgcccagat 540agtgatcaat tggcttcaat
tgcaataagt acagcagaaa ctgcaaagaa cttatgtgga 600atggatccaa aagtagcaat
gctttcattt tctactaagg gaagtgcaaa acacgaatta 660gtagacaaag ttagaaatgc
tgtagagatt gcaaaaaaag ctaaaccaga tttaagttta 720gacggagaat tacaattaga
tgcctctatc gtagaaaagg ttgcaagttt aaaggctcct 780ggaagtgaag tagcaggaaa
agcaaatgta cttgtatttc cagatctcca agcaggaaat 840ataggctata aactcgttca
aagatttgca aaagcagatg ctataggacc tgtatgccaa 900ggatttgcaa aacctataaa
tgatttgtca agaggatgta attctgatga tatagtaaat 960gtagtagctg taacagcagt
tcaagcacaa gctcaaaagt aa 100286333PRTC. ljungdahlii
86Met Lys Leu Met Glu Lys Ile Trp Ser Lys Ala Lys Glu Asp Lys Lys1
5 10 15Lys Ile Val Leu Ala Glu
Gly Glu Glu Glu Arg Thr Leu Gln Ala Cys 20 25
30Glu Lys Ile Ile Lys Glu Gly Ile Ala Asn Leu Ile Leu
Val Gly Asn 35 40 45Glu Lys Val
Ile Lys Glu Lys Ala Ser Lys Leu Gly Val Ser Leu Asn 50
55 60Gly Ala Glu Ile Val Asp Pro Glu Thr Ser Asp Lys
Leu Lys Ala Tyr65 70 75
80Ala Asp Ala Phe Tyr Glu Leu Arg Lys Lys Lys Gly Ile Thr Pro Glu
85 90 95Lys Ala Asp Lys Ile Val
Arg Asp Pro Ile Tyr Phe Ala Thr Met Met 100
105 110Val Lys Leu Gly Asp Ala Asp Gly Leu Val Ser Gly
Ala Val His Thr 115 120 125Thr Gly
Asp Leu Leu Arg Pro Gly Leu Gln Ile Val Lys Thr Ala Pro 130
135 140Gly Thr Ser Val Val Ser Ser Thr Phe Ile Met
Glu Val Pro Asn Cys145 150 155
160Glu Tyr Gly Asp Asn Gly Val Leu Leu Phe Ala Asp Cys Ala Val Asn
165 170 175Pro Cys Pro Asp
Ser Asp Gln Leu Ala Ser Ile Ala Ile Ser Thr Ala 180
185 190Glu Thr Ala Lys Asn Leu Cys Gly Met Asp Pro
Lys Val Ala Met Leu 195 200 205Ser
Phe Ser Thr Lys Gly Ser Ala Lys His Glu Leu Val Asp Lys Val 210
215 220Arg Asn Ala Val Glu Ile Ala Lys Lys Ala
Lys Pro Asp Leu Ser Leu225 230 235
240Asp Gly Glu Leu Gln Leu Asp Ala Ser Ile Val Glu Lys Val Ala
Ser 245 250 255Leu Lys Ala
Pro Gly Ser Glu Val Ala Gly Lys Ala Asn Val Leu Val 260
265 270Phe Pro Asp Leu Gln Ala Gly Asn Ile Gly
Tyr Lys Leu Val Gln Arg 275 280
285Phe Ala Lys Ala Asp Ala Ile Gly Pro Val Cys Gln Gly Phe Ala Lys 290
295 300Pro Ile Asn Asp Leu Ser Arg Gly
Cys Asn Ser Asp Asp Ile Val Asn305 310
315 320Val Val Ala Val Thr Ala Val Gln Ala Gln Ala Gln
Lys 325 330871197DNAC. ljungdahlii
87atgaaaatat tagtagtaaa ctgtggaagt tcatctttaa aatatcaact tattgatatg
60caagatgaaa gtgttgtagc aaagggtctt gtagaaagaa taggaatgga cggttcaatt
120ttaacacaca aagttaatgg agaaaagttt gttacagagc aaacaatgga agaccacaaa
180gttgctatac aattagtatt aaatgctctt gtagataaaa aacatggtgt aataaaagac
240atgtcagaaa tatccgctgt aggacataga gtcttgcacg gtggaaagaa atatgcagca
300tccattctta ttgacgaaaa tgtaatgaaa gcaatagaag aatgtatccc actaggacca
360ctacataatc cagctaatat aatgggaata gatgcttgta aaaaattaat gccaaatact
420ccaatggtag cagtatttga tacagcattt catcagacaa tgccagatta tgcttatact
480tatgcaatac cttatgatat atctgaaaag tatgatatca gaaaatatgg ttttcatgga
540acttctcata gattcgtttc aattgaagca gctaaattat taaagaaaga tccaaaagat
600cttaagttaa taacttgtca tttaggaaat ggagctagca tatgtgcagt aaaccaagga
660aaagcagtag atacaacgat gggacttact cctcttgcag gacttgtaat gggaactaga
720tgcggtgata tagatccagc tatagtacca tttgtaatga aaagaacagg catgtctgta
780gatgaagtgg ataccttaat gaataaaaag tcaggaatac ttggagtatc aggagtaagc
840agtgatttta gagatgtaga agaagctgca aattcaggaa atgatagagc aaaacttgca
900ttaaatatgt attatcacaa agttaaatct ttcataggag cttatgttgc agttttaaat
960ggagcagatg ctataatatt tacagcagga cttggagaaa attcagcaac tagcagatct
1020gctatatgta atggattaag ctattttgga attaaaatag atgaagaaaa gaataagaaa
1080aggggagagg cactagaaat aagcacacct gattcaaaga taaaagtatt agtaattcct
1140acaaatgaag aacttatgat agctagggat acaaaagaaa tagttgaaaa taaataa
119788398PRTC. ljungdahlii 88Met Lys Ile Leu Val Val Asn Cys Gly Ser Ser
Ser Leu Lys Tyr Gln1 5 10
15Leu Ile Asp Met Gln Asp Glu Ser Val Val Ala Lys Gly Leu Val Glu
20 25 30Arg Ile Gly Met Asp Gly Ser
Ile Leu Thr His Lys Val Asn Gly Glu 35 40
45Lys Phe Val Thr Glu Gln Thr Met Glu Asp His Lys Val Ala Ile
Gln 50 55 60Leu Val Leu Asn Ala Leu
Val Asp Lys Lys His Gly Val Ile Lys Asp65 70
75 80Met Ser Glu Ile Ser Ala Val Gly His Arg Val
Leu His Gly Gly Lys 85 90
95Lys Tyr Ala Ala Ser Ile Leu Ile Asp Glu Asn Val Met Lys Ala Ile
100 105 110Glu Glu Cys Ile Pro Leu
Gly Pro Leu His Asn Pro Ala Asn Ile Met 115 120
125Gly Ile Asp Ala Cys Lys Lys Leu Met Pro Asn Thr Pro Met
Val Ala 130 135 140Val Phe Asp Thr Ala
Phe His Gln Thr Met Pro Asp Tyr Ala Tyr Thr145 150
155 160Tyr Ala Ile Pro Tyr Asp Ile Ser Glu Lys
Tyr Asp Ile Arg Lys Tyr 165 170
175Gly Phe His Gly Thr Ser His Arg Phe Val Ser Ile Glu Ala Ala Lys
180 185 190Leu Leu Lys Lys Asp
Pro Lys Asp Leu Lys Leu Ile Thr Cys His Leu 195
200 205Gly Asn Gly Ala Ser Ile Cys Ala Val Asn Gln Gly
Lys Ala Val Asp 210 215 220Thr Thr Met
Gly Leu Thr Pro Leu Ala Gly Leu Val Met Gly Thr Arg225
230 235 240Cys Gly Asp Ile Asp Pro Ala
Ile Val Pro Phe Val Met Lys Arg Thr 245
250 255Gly Met Ser Val Asp Glu Val Asp Thr Leu Met Asn
Lys Lys Ser Gly 260 265 270Ile
Leu Gly Val Ser Gly Val Ser Ser Asp Phe Arg Asp Val Glu Glu 275
280 285Ala Ala Asn Ser Gly Asn Asp Arg Ala
Lys Leu Ala Leu Asn Met Tyr 290 295
300Tyr His Lys Val Lys Ser Phe Ile Gly Ala Tyr Val Ala Val Leu Asn305
310 315 320Gly Ala Asp Ala
Ile Ile Phe Thr Ala Gly Leu Gly Glu Asn Ser Ala 325
330 335Thr Ser Arg Ser Ala Ile Cys Asn Gly Leu
Ser Tyr Phe Gly Ile Lys 340 345
350Ile Asp Glu Glu Lys Asn Lys Lys Arg Gly Glu Ala Leu Glu Ile Ser
355 360 365Thr Pro Asp Ser Lys Ile Lys
Val Leu Val Ile Pro Thr Asn Glu Glu 370 375
380Leu Met Ile Ala Arg Asp Thr Lys Glu Ile Val Glu Asn Lys385
390 395891824DNAC. ljungdahlii 89atgtacggat
ataagggtaa ggtattaaga attaatctaa gtagtaaaac ttatatagtg 60gaagaattga
aaattgacaa agctaaaaaa tttataggtg caagagggtt aggcgtaaaa 120accttatttg
acgaagtaga tccaaaggta gatccattat cacctgataa caaatttatt 180atagcagcgg
gaccacttac aggtgcacct gttccaacaa gcggaagatt catggtagtt 240actaaatcac
ctttaacagg aactattgct attgcaaatt caggtggaaa atggggagca 300gaattcaaag
cagctggata cgatatgata atcgttgaag gtaaatctga taaagaagtt 360tatgtaaata
tagtagatga taaagtagaa tttagggatg cttctcatgt ttggggaaaa 420ctaacagaag
aaactacaaa aatgcttcaa caggaaacag attcgagagc taaggtttta 480tgcataggac
cagctgggga aaagttatca cttatggcag cagttatgaa tgatgttgat 540agaacagcag
gacgtggtgg tgttggagct gttatgggtt caaagaactt aaaagctatt 600gtagttaaag
gaagcggaaa agtaaaatta tttgatgaac aaaaagtgaa ggaagtagca 660cttgagaaaa
caaatatttt aagaaaagat ccagtagctg gtggaggact tccaacatac 720ggaacagctg
tacttgttaa tattataaat gaaaatggtg tacatccagt aaagaatttt 780caaaaatctt
atacagatca agcagataag atcagtggag aaactttaac taaagattgc 840ttagttagaa
aaaatccttg ctataggtgt ccaattgcct gtggaagatg ggtaaaactt 900gatgatggaa
ctgaatgtgg aggaccagaa tatgaaacat tatggtcatt tggatctgat 960tgtgatgtat
acgatataaa tgctgtaaat acagcaaata tgttgtgtaa tgaatatgga 1020ttagatacca
ttacagcagg atgtactatt gcagcagcta tggaacttta tcaaagaggt 1080tatattaagg
atgaagaaat agcagcagat ggattgtcac ttaattgggg agatgctaag 1140tccatggttg
aatgggtaaa gaaaatggga cttagagaag gatttggaga caagatggca 1200gatggttcat
acagactttg tgactcatac ggtgtacctg agtattcaat gactgtaaaa 1260aaacaggaac
ttccagcata tgacccaaga ggaatacagg gacatggtat tacttatgct 1320gttaacaata
ggggaggatg tcacattaag ggatatatgg taagtcctga aatacttggc 1380tatccagaaa
aacttgatag acttgcagtg gaaggaaaag caggatatgc tagagtattc 1440catgatttaa
cagctgttat agattcactt ggattatgta tttttacaac atttggtctt 1500ggtgcacagg
attatgttga tatgtataat gcagtagttg gtggagaatt acatgatgta 1560aattctttaa
tgttagctgg agatagaata tggactttag aaaaaatatt taacttaaag 1620gcaggcatag
atagttcaca ggatactctt ccaaagagat tgcttgaaga acaaattcca 1680gaaggaccat
caaaaggaga agttcataag ttagatgtac tactacctga atattattca 1740gtacgtggat
gggataaaaa tggtattcct acagaggaaa cgttaaagaa attaggatta 1800gatgaatacg
taggtaagct ttag 182490607PRTC.
ljungdahlii 90Met Tyr Gly Tyr Lys Gly Lys Val Leu Arg Ile Asn Leu Ser Ser
Lys1 5 10 15Thr Tyr Ile
Val Glu Glu Leu Lys Ile Asp Lys Ala Lys Lys Phe Ile 20
25 30Gly Ala Arg Gly Leu Gly Val Lys Thr Leu
Phe Asp Glu Val Asp Pro 35 40
45Lys Val Asp Pro Leu Ser Pro Asp Asn Lys Phe Ile Ile Ala Ala Gly 50
55 60Pro Leu Thr Gly Ala Pro Val Pro Thr
Ser Gly Arg Phe Met Val Val65 70 75
80Thr Lys Ser Pro Leu Thr Gly Thr Ile Ala Ile Ala Asn Ser
Gly Gly 85 90 95Lys Trp
Gly Ala Glu Phe Lys Ala Ala Gly Tyr Asp Met Ile Ile Val 100
105 110Glu Gly Lys Ser Asp Lys Glu Val Tyr
Val Asn Ile Val Asp Asp Lys 115 120
125Val Glu Phe Arg Asp Ala Ser His Val Trp Gly Lys Leu Thr Glu Glu
130 135 140Thr Thr Lys Met Leu Gln Gln
Glu Thr Asp Ser Arg Ala Lys Val Leu145 150
155 160Cys Ile Gly Pro Ala Gly Glu Lys Leu Ser Leu Met
Ala Ala Val Met 165 170
175Asn Asp Val Asp Arg Thr Ala Gly Arg Gly Gly Val Gly Ala Val Met
180 185 190Gly Ser Lys Asn Leu Lys
Ala Ile Val Val Lys Gly Ser Gly Lys Val 195 200
205Lys Leu Phe Asp Glu Gln Lys Val Lys Glu Val Ala Leu Glu
Lys Thr 210 215 220Asn Ile Leu Arg Lys
Asp Pro Val Ala Gly Gly Gly Leu Pro Thr Tyr225 230
235 240Gly Thr Ala Val Leu Val Asn Ile Ile Asn
Glu Asn Gly Val His Pro 245 250
255Val Lys Asn Phe Gln Lys Ser Tyr Thr Asp Gln Ala Asp Lys Ile Ser
260 265 270Gly Glu Thr Leu Thr
Lys Asp Cys Leu Val Arg Lys Asn Pro Cys Tyr 275
280 285Arg Cys Pro Ile Ala Cys Gly Arg Trp Val Lys Leu
Asp Asp Gly Thr 290 295 300Glu Cys Gly
Gly Pro Glu Tyr Glu Thr Leu Trp Ser Phe Gly Ser Asp305
310 315 320Cys Asp Val Tyr Asp Ile Asn
Ala Val Asn Thr Ala Asn Met Leu Cys 325
330 335Asn Glu Tyr Gly Leu Asp Thr Ile Thr Ala Gly Cys
Thr Ile Ala Ala 340 345 350Ala
Met Glu Leu Tyr Gln Arg Gly Tyr Ile Lys Asp Glu Glu Ile Ala 355
360 365Ala Asp Gly Leu Ser Leu Asn Trp Gly
Asp Ala Lys Ser Met Val Glu 370 375
380Trp Val Lys Lys Met Gly Leu Arg Glu Gly Phe Gly Asp Lys Met Ala385
390 395 400Asp Gly Ser Tyr
Arg Leu Cys Asp Ser Tyr Gly Val Pro Glu Tyr Ser 405
410 415Met Thr Val Lys Lys Gln Glu Leu Pro Ala
Tyr Asp Pro Arg Gly Ile 420 425
430Gln Gly His Gly Ile Thr Tyr Ala Val Asn Asn Arg Gly Gly Cys His
435 440 445Ile Lys Gly Tyr Met Val Ser
Pro Glu Ile Leu Gly Tyr Pro Glu Lys 450 455
460Leu Asp Arg Leu Ala Val Glu Gly Lys Ala Gly Tyr Ala Arg Val
Phe465 470 475 480His Asp
Leu Thr Ala Val Ile Asp Ser Leu Gly Leu Cys Ile Phe Thr
485 490 495Thr Phe Gly Leu Gly Ala Gln
Asp Tyr Val Asp Met Tyr Asn Ala Val 500 505
510Val Gly Gly Glu Leu His Asp Val Asn Ser Leu Met Leu Ala
Gly Asp 515 520 525Arg Ile Trp Thr
Leu Glu Lys Ile Phe Asn Leu Lys Ala Gly Ile Asp 530
535 540Ser Ser Gln Asp Thr Leu Pro Lys Arg Leu Leu Glu
Glu Gln Ile Pro545 550 555
560Glu Gly Pro Ser Lys Gly Glu Val His Lys Leu Asp Val Leu Leu Pro
565 570 575Glu Tyr Tyr Ser Val
Arg Gly Trp Asp Lys Asn Gly Ile Pro Thr Glu 580
585 590Glu Thr Leu Lys Lys Leu Gly Leu Asp Glu Tyr Val
Gly Lys Leu 595 600 605911824DNAC.
ljungdahlii 91atgtatggtt atgatggtaa agtattaaga attaatttaa aagaaagaac
ttgcaaatca 60gaaaatttag atttagataa agctaaaaag tttataggtt gtaggggact
aggtgttaaa 120actttatttg atgaaataga tcctaaaata gatgcattat caccagaaaa
taaatttata 180attgtaacag gtcctttaac tggagctccg gttccaacta gtggaaggtt
tatggtagtt 240actaaagcac cgcttacagg aactatagga atttcaaatt cgggtggaaa
atggggagta 300gacttaaaaa aagctggttg ggatatgata atagtagagg ataaggctga
ttcaccagtt 360tacattgaaa tagtagatga taaggtagaa attaaagacg cgtcacagct
ttggggaaaa 420gttacatcag aaactacaaa agagttagaa aagataactg agaataaatc
aaaggtatta 480tgtataggac ctgctggtga acgattgtct cttatggcag cagttatgaa
tgatgtagat 540agaactgcag caagaggcgg cgttggtgca gttatgggat ctaaaaactt
aaaagctatt 600acagttaaag gaactggaaa aatagcttta gctgataaag aaaaagtaaa
aaaagtgtcc 660gtagaaaaaa ttacaacatt aaaaaatgat ccagtagctg gtcagggaat
gccaacttat 720ggtacagcta tactggttaa tataataaat gaaaatggag ttcatcctgt
aaagaatttt 780caagagtctt atacgaatca agcagataaa ataagtggag agactcttac
tgctaaccaa 840ctagtaagga aaaatccttg ttacagctgt cctataggtt gtggaagatg
ggttagacta 900aaagatggca cagagtgcgg aggaccagaa tatgaaacac tgtggtgttt
tggatctgac 960tgtggttcat atgatttaga tgctataaat gaagctaata tgttatgtaa
tgaatatggt 1020attgatacta ttacttgtgg tgcaacaatt gctgcagcta tggaacttta
tcaaagagga 1080tatataaaag acgaagaaat agctggagat aacctatctc tcaagtgggg
tgatacggaa 1140tctatgattg gctggataaa gagaatggta tatagtgaag gctttggagc
aaagatgaca 1200aatggttcat ataggctttg tgaaggttat ggagcaccgg agtattctat
gacagttaaa 1260aagcaggaaa ttccagcata tgatccaagg ggaatacagg gacacggtat
tacctatgca 1320gttaataata gaggaggctg tcatattaag ggatatatga ttaaccctga
aatattaggt 1380tatcctgaaa aacttgatag atttgcatta gatggtaaag cagcttatgc
caaattattt 1440catgatttaa ctgctgtaat tgattcttta ggattgtgca tattcactac
atttgggctt 1500ggaatacagg attatgtaga tatgtataat gcagtagtag gagaatctac
ttatgatgca 1560gattcactat tagaggcagg agatagaatc tggactcttg agaaattatt
taatcttgca 1620gctggaatag acagcagcca ggatactcta ccaaagagat tgttagaaga
acctattcca 1680gatggcccat caaagggaga agttcatagg ctagatgttc ttctgccaga
atattactca 1740gtacgaggat ggagtaaaga gggtatacct acagaagaaa cattaaagaa
attaggatta 1800gatgaatata taggtaagtt ctag
182492607PRTC. lungdahlii 92Met Tyr Gly Tyr Asp Gly Lys Val
Leu Arg Ile Asn Leu Lys Glu Arg1 5 10
15Thr Cys Lys Ser Glu Asn Leu Asp Leu Asp Lys Ala Lys Lys
Phe Ile 20 25 30Gly Cys Arg
Gly Leu Gly Val Lys Thr Leu Phe Asp Glu Ile Asp Pro 35
40 45Lys Ile Asp Ala Leu Ser Pro Glu Asn Lys Phe
Ile Ile Val Thr Gly 50 55 60Pro Leu
Thr Gly Ala Pro Val Pro Thr Ser Gly Arg Phe Met Val Val65
70 75 80Thr Lys Ala Pro Leu Thr Gly
Thr Ile Gly Ile Ser Asn Ser Gly Gly 85 90
95Lys Trp Gly Val Asp Leu Lys Lys Ala Gly Trp Asp Met
Ile Ile Val 100 105 110Glu Asp
Lys Ala Asp Ser Pro Val Tyr Ile Glu Ile Val Asp Asp Lys 115
120 125Val Glu Ile Lys Asp Ala Ser Gln Leu Trp
Gly Lys Val Thr Ser Glu 130 135 140Thr
Thr Lys Glu Leu Glu Lys Ile Thr Glu Asn Lys Ser Lys Val Leu145
150 155 160Cys Ile Gly Pro Ala Gly
Glu Arg Leu Ser Leu Met Ala Ala Val Met 165
170 175Asn Asp Val Asp Arg Thr Ala Ala Arg Gly Gly Val
Gly Ala Val Met 180 185 190Gly
Ser Lys Asn Leu Lys Ala Ile Thr Val Lys Gly Thr Gly Lys Ile 195
200 205Ala Leu Ala Asp Lys Glu Lys Val Lys
Lys Val Ser Val Glu Lys Ile 210 215
220Thr Thr Leu Lys Asn Asp Pro Val Ala Gly Gln Gly Met Pro Thr Tyr225
230 235 240Gly Thr Ala Ile
Leu Val Asn Ile Ile Asn Glu Asn Gly Val His Pro 245
250 255Val Lys Asn Phe Gln Glu Ser Tyr Thr Asn
Gln Ala Asp Lys Ile Ser 260 265
270Gly Glu Thr Leu Thr Ala Asn Gln Leu Val Arg Lys Asn Pro Cys Tyr
275 280 285Ser Cys Pro Ile Gly Cys Gly
Arg Trp Val Arg Leu Lys Asp Gly Thr 290 295
300Glu Cys Gly Gly Pro Glu Tyr Glu Thr Leu Trp Cys Phe Gly Ser
Asp305 310 315 320Cys Gly
Ser Tyr Asp Leu Asp Ala Ile Asn Glu Ala Asn Met Leu Cys
325 330 335Asn Glu Tyr Gly Ile Asp Thr
Ile Thr Cys Gly Ala Thr Ile Ala Ala 340 345
350Ala Met Glu Leu Tyr Gln Arg Gly Tyr Ile Lys Asp Glu Glu
Ile Ala 355 360 365Gly Asp Asn Leu
Ser Leu Lys Trp Gly Asp Thr Glu Ser Met Ile Gly 370
375 380Trp Ile Lys Arg Met Val Tyr Ser Glu Gly Phe Gly
Ala Lys Met Thr385 390 395
400Asn Gly Ser Tyr Arg Leu Cys Glu Gly Tyr Gly Ala Pro Glu Tyr Ser
405 410 415Met Thr Val Lys Lys
Gln Glu Ile Pro Ala Tyr Asp Pro Arg Gly Ile 420
425 430Gln Gly His Gly Ile Thr Tyr Ala Val Asn Asn Arg
Gly Gly Cys His 435 440 445Ile Lys
Gly Tyr Met Ile Asn Pro Glu Ile Leu Gly Tyr Pro Glu Lys 450
455 460Leu Asp Arg Phe Ala Leu Asp Gly Lys Ala Ala
Tyr Ala Lys Leu Phe465 470 475
480His Asp Leu Thr Ala Val Ile Asp Ser Leu Gly Leu Cys Ile Phe Thr
485 490 495Thr Phe Gly Leu
Gly Ile Gln Asp Tyr Val Asp Met Tyr Asn Ala Val 500
505 510Val Gly Glu Ser Thr Tyr Asp Ala Asp Ser Leu
Leu Glu Ala Gly Asp 515 520 525Arg
Ile Trp Thr Leu Glu Lys Leu Phe Asn Leu Ala Ala Gly Ile Asp 530
535 540Ser Ser Gln Asp Thr Leu Pro Lys Arg Leu
Leu Glu Glu Pro Ile Pro545 550 555
560Asp Gly Pro Ser Lys Gly Glu Val His Arg Leu Asp Val Leu Leu
Pro 565 570 575Glu Tyr Tyr
Ser Val Arg Gly Trp Ser Lys Glu Gly Ile Pro Thr Glu 580
585 590Glu Thr Leu Lys Lys Leu Gly Leu Asp Glu
Tyr Ile Gly Lys Phe 595 600
605932697DNAC. ragsdalei 93atgccaagaa atctgtttat atttaacagc atgaaaaata
agaaagaggt gtcattaatg 60aaggtaacta aggtaactaa cgttgaagaa ttaatgaaaa
agttagatga agtaacggct 120gctcaaaaaa aattctctag ttatagtcag gaacaagtgg
atgagatctt taggcaggca 180gctatggcag ccaatagtgc tagaatagat ctagctaaaa
tggcagtgga agaaagcgga 240atgggaattg tagaagacaa ggttattaaa aatcattttg
tttcagaata tatatataac 300aaatataagg atgaaaagac ctgtggagtt ttagaagaag
accaaggttt tggtatggtt 360agaattgcgg aacctgtagg ggttatagca gcagtagttc
caacaactaa tccaacatcc 420acagcaatct ttaaatcttt aatagctttg aaaactagaa
atggtatagt tttttcacca 480catccaagag caaaaaaatc aactattgca gcagctaaga
tagtacttga tgcagcagtt 540aaagctggtg ctcctgaagg aattatagga tggatagatg
aaccttccat tgaactctca 600caggtggtaa tgaaagaagc agatttaatt cttgcaactg
gtggcccggg tatggttaag 660gctgcctatt cttcaggaaa gcctgctata ggagttggcc
caggtaacac acctgctgta 720attgatgaaa gtgctgatat taaaatggca gtaaattcaa
tactcctttc aaaaactttt 780gataatggta tgatttgtgc ttcagagcag tcagtagtag
ttgtaagctc aatatacgat 840gaagtcaaga aagaatttgc agatagagga gcgtatatat
taagtaagga tgaaacagat 900aaggttggaa aaacaattat gattaatggc gctctaaatg
ctggcattgt agggcaaagt 960gcttttaaaa tagcacagat ggcaggagtg agtgtaccag
aggatgctaa agtacttata 1020ggagaagtta aatcagtaga acctgaagaa gagccctttg
ctcatgaaaa gctgtctcca 1080gttttagcta tgtacaaagc aaaagatttt gatgaagcac
ttctaaaggc tggaagatta 1140gttgaacgag gtggaattgg gcatacatct gtattatatg
taaattcaat gacggaaaaa 1200gtaaaagtag aaaagttcag agaaactatg aagactggta
gaacattgat aaatatgcct 1260tcagcacaag gtgctatagg agatatatat aactttaaac
tagctccttc tttgacgcta 1320ggatgtggtt cctggggagg aaactctgta tcagaaaatg
ttggacctaa acatttatta 1380aacataaaaa gtgttgctga gaggagagaa aatatgcttt
ggtttagagt acctgaaaaa 1440gtttatttca aatatggtag tcttggagtt gcattaaagg
aattgagaac tttggagaag 1500aaaaaggcat ttatagtaac ggataaggtt ctttatcaat
taggttatgt agataaaatt 1560acaaaaaatc tcgatgaatt aagagtttca tataaaatat
ttacagatgt agaaccagat 1620ccaacccttg ctacagctaa aaaaggtgca tcagaactgc
tttcctatga accagataca 1680attatagcag ttggtggtgg ttcggcaatg gatgcagcca
agatcatgtg ggtaatgtat 1740gagcatccag aagtaagatt tgaagatttg gctatgagat
ttatggatat aagaaagaga 1800gtatatgttt ttcctaagat gggtgaaaaa gcaatgatga
tttcagtagc aacatccgca 1860ggaacaggat ctgaagttac tccatttgca gtaattacgg
atgaaagaac aggagctaaa 1920tatccactgg ctgattatga attgactcca aacatggcta
taattgatgc agaacttatg 1980atgggaatgc caaaagggct tacagcagct tcgggtatag
atgcattaac ccatgcactg 2040gaggcgtatg tatcaataat ggcttcagaa tataccaatg
gattggctct tgaagcaaca 2100agattagtat ttaaatattt gccaatagct tatacagaag
gtacaactaa tgtaaaggca 2160agagaaaaaa tggctcatgc ttcaactata gcaggtatgg
cttttgccaa tgcattctta 2220ggggtatgtc actctatggc acataaattg ggagcacagc
accatatacc acatggaatt 2280gccaatgcgc ttatgataga tgaagttata aaattcaatg
ctgtagaggc tccaaggaaa 2340caagcggcat ttccacaata taagtaccca aatgttaaaa
gaagatatgc tagaatagct 2400gattacttaa atttaggagg aagcacagat gatgaaaaag
tacaattgct aataaatgct 2460atagatgact taaaaactaa gttaaatatt ccaaagacta
ttaaagaggc aggagtttca 2520gaagataaat tctatgctac tttagacaca atgtcagaac
tggcttttga tgatcaatgt 2580acaggagcta atccaagata tccactaata ggagaaataa
aacaaatgta tataaatgca 2640tttgatacac caaaggcaac tgtggagaag aaaacaaaaa
gaaaaataaa catataa 269794898PRTC. ragsdalei 94Met Pro Arg Asn Leu
Phe Ile Phe Asn Ser Met Lys Asn Lys Lys Glu1 5
10 15Val Ser Leu Met Lys Val Thr Lys Val Thr Asn
Val Glu Glu Leu Met 20 25
30Lys Lys Leu Asp Glu Val Thr Ala Ala Gln Lys Lys Phe Ser Ser Tyr
35 40 45Ser Gln Glu Gln Val Asp Glu Ile
Phe Arg Gln Ala Ala Met Ala Ala 50 55
60Asn Ser Ala Arg Ile Asp Leu Ala Lys Met Ala Val Glu Glu Ser Gly65
70 75 80Met Gly Ile Val Glu
Asp Lys Val Ile Lys Asn His Phe Val Ser Glu 85
90 95Tyr Ile Tyr Asn Lys Tyr Lys Asp Glu Lys Thr
Cys Gly Val Leu Glu 100 105
110Glu Asp Gln Gly Phe Gly Met Val Arg Ile Ala Glu Pro Val Gly Val
115 120 125Ile Ala Ala Val Val Pro Thr
Thr Asn Pro Thr Ser Thr Ala Ile Phe 130 135
140Lys Ser Leu Ile Ala Leu Lys Thr Arg Asn Gly Ile Val Phe Ser
Pro145 150 155 160His Pro
Arg Ala Lys Lys Ser Thr Ile Ala Ala Ala Lys Ile Val Leu
165 170 175Asp Ala Ala Val Lys Ala Gly
Ala Pro Glu Gly Ile Ile Gly Trp Ile 180 185
190Asp Glu Pro Ser Ile Glu Leu Ser Gln Val Val Met Lys Glu
Ala Asp 195 200 205Leu Ile Leu Ala
Thr Gly Gly Pro Gly Met Val Lys Ala Ala Tyr Ser 210
215 220Ser Gly Lys Pro Ala Ile Gly Val Gly Pro Gly Asn
Thr Pro Ala Val225 230 235
240Ile Asp Glu Ser Ala Asp Ile Lys Met Ala Val Asn Ser Ile Leu Leu
245 250 255Ser Lys Thr Phe Asp
Asn Gly Met Ile Cys Ala Ser Glu Gln Ser Val 260
265 270Val Val Val Ser Ser Ile Tyr Asp Glu Val Lys Lys
Glu Phe Ala Asp 275 280 285Arg Gly
Ala Tyr Ile Leu Ser Lys Asp Glu Thr Asp Lys Val Gly Lys 290
295 300Thr Ile Met Ile Asn Gly Ala Leu Asn Ala Gly
Ile Val Gly Gln Ser305 310 315
320Ala Phe Lys Ile Ala Gln Met Ala Gly Val Ser Val Pro Glu Asp Ala
325 330 335Lys Val Leu Ile
Gly Glu Val Lys Ser Val Glu Pro Glu Glu Glu Pro 340
345 350Phe Ala His Glu Lys Leu Ser Pro Val Leu Ala
Met Tyr Lys Ala Lys 355 360 365Asp
Phe Asp Glu Ala Leu Leu Lys Ala Gly Arg Leu Val Glu Arg Gly 370
375 380Gly Ile Gly His Thr Ser Val Leu Tyr Val
Asn Ser Met Thr Glu Lys385 390 395
400Val Lys Val Glu Lys Phe Arg Glu Thr Met Lys Thr Gly Arg Thr
Leu 405 410 415Ile Asn Met
Pro Ser Ala Gln Gly Ala Ile Gly Asp Ile Tyr Asn Phe 420
425 430Lys Leu Ala Pro Ser Leu Thr Leu Gly Cys
Gly Ser Trp Gly Gly Asn 435 440
445Ser Val Ser Glu Asn Val Gly Pro Lys His Leu Leu Asn Ile Lys Ser 450
455 460Val Ala Glu Arg Arg Glu Asn Met
Leu Trp Phe Arg Val Pro Glu Lys465 470
475 480Val Tyr Phe Lys Tyr Gly Ser Leu Gly Val Ala Leu
Lys Glu Leu Arg 485 490
495Thr Leu Glu Lys Lys Lys Ala Phe Ile Val Thr Asp Lys Val Leu Tyr
500 505 510Gln Leu Gly Tyr Val Asp
Lys Ile Thr Lys Asn Leu Asp Glu Leu Arg 515 520
525Val Ser Tyr Lys Ile Phe Thr Asp Val Glu Pro Asp Pro Thr
Leu Ala 530 535 540Thr Ala Lys Lys Gly
Ala Ser Glu Leu Leu Ser Tyr Glu Pro Asp Thr545 550
555 560Ile Ile Ala Val Gly Gly Gly Ser Ala Met
Asp Ala Ala Lys Ile Met 565 570
575Trp Val Met Tyr Glu His Pro Glu Val Arg Phe Glu Asp Leu Ala Met
580 585 590Arg Phe Met Asp Ile
Arg Lys Arg Val Tyr Val Phe Pro Lys Met Gly 595
600 605Glu Lys Ala Met Met Ile Ser Val Ala Thr Ser Ala
Gly Thr Gly Ser 610 615 620Glu Val Thr
Pro Phe Ala Val Ile Thr Asp Glu Arg Thr Gly Ala Lys625
630 635 640Tyr Pro Leu Ala Asp Tyr Glu
Leu Thr Pro Asn Met Ala Ile Ile Asp 645
650 655Ala Glu Leu Met Met Gly Met Pro Lys Gly Leu Thr
Ala Ala Ser Gly 660 665 670Ile
Asp Ala Leu Thr His Ala Leu Glu Ala Tyr Val Ser Ile Met Ala 675
680 685Ser Glu Tyr Thr Asn Gly Leu Ala Leu
Glu Ala Thr Arg Leu Val Phe 690 695
700Lys Tyr Leu Pro Ile Ala Tyr Thr Glu Gly Thr Thr Asn Val Lys Ala705
710 715 720Arg Glu Lys Met
Ala His Ala Ser Thr Ile Ala Gly Met Ala Phe Ala 725
730 735Asn Ala Phe Leu Gly Val Cys His Ser Met
Ala His Lys Leu Gly Ala 740 745
750Gln His His Ile Pro His Gly Ile Ala Asn Ala Leu Met Ile Asp Glu
755 760 765Val Ile Lys Phe Asn Ala Val
Glu Ala Pro Arg Lys Gln Ala Ala Phe 770 775
780Pro Gln Tyr Lys Tyr Pro Asn Val Lys Arg Arg Tyr Ala Arg Ile
Ala785 790 795 800Asp Tyr
Leu Asn Leu Gly Gly Ser Thr Asp Asp Glu Lys Val Gln Leu
805 810 815Leu Ile Asn Ala Ile Asp Asp
Leu Lys Thr Lys Leu Asn Ile Pro Lys 820 825
830Thr Ile Lys Glu Ala Gly Val Ser Glu Asp Lys Phe Tyr Ala
Thr Leu 835 840 845Asp Thr Met Ser
Glu Leu Ala Phe Asp Asp Gln Cys Thr Gly Ala Asn 850
855 860Pro Arg Tyr Pro Leu Ile Gly Glu Ile Lys Gln Met
Tyr Ile Asn Ala865 870 875
880Phe Asp Thr Pro Lys Ala Thr Val Glu Lys Lys Thr Lys Arg Lys Ile
885 890 895Asn Ile952613DNAC.
ragsdalei 95atgaaagtta caaacgtgga agaattaatg aaaagactag aagagataaa
ggatgctcaa 60aagaaatttg ctacatatac tcaagaacaa gtggatgaaa tttttagaca
agcagctatg 120gcagccaata gtgctagaat agaactagct aaaatggcag tggaagaaag
cggaatggga 180attgtagaag acaaggttat taaaaatcac tttgcctcag aatatatata
taacaaatat 240aaggatgaaa agacctgtgg agttttagaa agagatgcag gctttggtat
agttagaatt 300gcggaacctg taggggttat tgcagcagta gttccaacaa ctaatccaac
atctacagca 360atctttaaat cactaatagc tttaaaaact agaaatggta taattttttc
accgcatcca 420agggcaaaga aatcaactat tgcagcagct aaaatagtac ttgatgctgc
agttaaagct 480ggtgctcccg aaggaattat aggatggata gatgaacctt ccattgaact
ttcacaggtg 540gtaatgggag aagcaaattt aattcttgca actggtggcc cgggtatggt
taaggctgcc 600tattcttcag gaaaacctgc tgtaggagtt ggcccaggta atacacctgc
tataattgat 660gaaagtgccg atattaaaat ggcagtaaat tcaatattac tctcaaaaac
ttttgataat 720ggtatgattt gtgcctcaga gcagtcagta atagttttag actcaatata
tgaggaagtt 780aaaaaagaat ttgcttatag gggagcttat atattgagtg aggatgaaac
agataaggtt 840ggaaaaataa ttttaaaaaa tggagcctta aatgctggta ttgtaggaca
aagtgctttt 900aaaatagcac agctggcagg agtgaacgta ccagaaaaag ctaaagtact
tataggagag 960gtagaatcag tagaacttga agaaccattt tctcatgaaa agttatctcc
agttttagct 1020atgtacaggg caagagattt tgaggatgcc attgcaaaaa ctgataaact
ggttagggca 1080ggtggatttg gacatacatc ttcattatat gtaaatccaa tgacagaaaa
agcaaaagta 1140gaaaaattta gtactatgat gaaaacatca agaactataa ttaacacacc
ttcatctcaa 1200ggtggtatag gtgacatata taactttaag ctagctcctt cgctgacgct
aggctgcgga 1260tcttggggag gaaactctgt atccgaaaat gttgggccta aacatttatt
aaacataaaa 1320agtgttgctg agaggagaga aaatatgctt tggtttagag tgcctgaaaa
ggtttatttc 1380aaatacggta gtcttggagt tgcattaaaa gaattaaaag ttatgaataa
gaagaaagta 1440tttatagtaa cagataaagt cctttatcaa ttaggttatg tggacaaagt
tacaaaagtt 1500cttgaggaac taaaaatttc ctataaagta tttacagatg tagaaccaga
tccaaccctt 1560gctacagcta aaaaaggtgc agcagaattg ctgtcatatg aaccggatac
aattatatca 1620gttggtggtg gttcagcaat ggatgcagcc aagattatgt gggtaatgta
tgagcatcca 1680gaagtaaaat ttgaagattt agctatgaga tttatggata taagaaagag
agtatatgtt 1740ttccctaaga tgggagaaaa agcaatgatg atttcagtag caacatccgc
aggtacagga 1800tcagaagtta ctccatttgc agtaattaca gatgaaaaaa caggagctaa
atatccatta 1860gctgattatg agttaactcc aaacatggct atagttgatg cagaacttat
gatgggaatg 1920ccaagaggac ttacggcagc gtcaggtata gatgcattaa ctcatgcact
ggaagcttat 1980gtatcaataa tggctacaga atttaccaat ggattagccc ttgaagcagt
aaagttgata 2040tttgaatatt taccaaaagc ttatacagaa ggtacaacta atgtaaaggc
aagagaaaaa 2100atggctcatg cttcatgtat tgctggtatg gcttttgcaa atgcattctt
aggggtatgc 2160cactctatgg cacataaatt aggagcacag caccacatac cacatggaat
tgctaatgca 2220cttatgatag atgaagttat aaaattcaat gctgtagatg atccaataaa
acaagctgca 2280tttcctcaat acgagtatcc aaatgccaag tatagatatg ctcagatagc
tgattgtctc 2340aacttaggag gaaatacaga agatgaaaag gtgcaattat taataaatgc
tatagatgat 2400ctaaaagcta agttaaatat tccagaaacg attaaagaag caggagtttc
agaagaaaaa 2460ttctatacta ctttagataa aatgtcagaa ttagcttttg atgatcaatg
tacaggagct 2520aacccaaggt atccactaat aagtgaaata aaacaaatgt atataaatgt
ttttgataaa 2580actgaaccaa ttgtagaaga tgaagaaaag taa
261396870PRTC. ragsdalei 96Met Lys Val Thr Asn Val Glu Glu Leu
Met Lys Arg Leu Glu Glu Ile1 5 10
15Lys Asp Ala Gln Lys Lys Phe Ala Thr Tyr Thr Gln Glu Gln Val
Asp 20 25 30Glu Ile Phe Arg
Gln Ala Ala Met Ala Ala Asn Ser Ala Arg Ile Glu 35
40 45Leu Ala Lys Met Ala Val Glu Glu Ser Gly Met Gly
Ile Val Glu Asp 50 55 60Lys Val Ile
Lys Asn His Phe Ala Ser Glu Tyr Ile Tyr Asn Lys Tyr65 70
75 80Lys Asp Glu Lys Thr Cys Gly Val
Leu Glu Arg Asp Ala Gly Phe Gly 85 90
95Ile Val Arg Ile Ala Glu Pro Val Gly Val Ile Ala Ala Val
Val Pro 100 105 110Thr Thr Asn
Pro Thr Ser Thr Ala Ile Phe Lys Ser Leu Ile Ala Leu 115
120 125Lys Thr Arg Asn Gly Ile Ile Phe Ser Pro His
Pro Arg Ala Lys Lys 130 135 140Ser Thr
Ile Ala Ala Ala Lys Ile Val Leu Asp Ala Ala Val Lys Ala145
150 155 160Gly Ala Pro Glu Gly Ile Ile
Gly Trp Ile Asp Glu Pro Ser Ile Glu 165
170 175Leu Ser Gln Val Val Met Gly Glu Ala Asn Leu Ile
Leu Ala Thr Gly 180 185 190Gly
Pro Gly Met Val Lys Ala Ala Tyr Ser Ser Gly Lys Pro Ala Val 195
200 205Gly Val Gly Pro Gly Asn Thr Pro Ala
Ile Ile Asp Glu Ser Ala Asp 210 215
220Ile Lys Met Ala Val Asn Ser Ile Leu Leu Ser Lys Thr Phe Asp Asn225
230 235 240Gly Met Ile Cys
Ala Ser Glu Gln Ser Val Ile Val Leu Asp Ser Ile 245
250 255Tyr Glu Glu Val Lys Lys Glu Phe Ala Tyr
Arg Gly Ala Tyr Ile Leu 260 265
270Ser Glu Asp Glu Thr Asp Lys Val Gly Lys Ile Ile Leu Lys Asn Gly
275 280 285Ala Leu Asn Ala Gly Ile Val
Gly Gln Ser Ala Phe Lys Ile Ala Gln 290 295
300Leu Ala Gly Val Asn Val Pro Glu Lys Ala Lys Val Leu Ile Gly
Glu305 310 315 320Val Glu
Ser Val Glu Leu Glu Glu Pro Phe Ser His Glu Lys Leu Ser
325 330 335Pro Val Leu Ala Met Tyr Arg
Ala Arg Asp Phe Glu Asp Ala Ile Ala 340 345
350Lys Thr Asp Lys Leu Val Arg Ala Gly Gly Phe Gly His Thr
Ser Ser 355 360 365Leu Tyr Val Asn
Pro Met Thr Glu Lys Ala Lys Val Glu Lys Phe Ser 370
375 380Thr Met Met Lys Thr Ser Arg Thr Ile Ile Asn Thr
Pro Ser Ser Gln385 390 395
400Gly Gly Ile Gly Asp Ile Tyr Asn Phe Lys Leu Ala Pro Ser Leu Thr
405 410 415Leu Gly Cys Gly Ser
Trp Gly Gly Asn Ser Val Ser Glu Asn Val Gly 420
425 430Pro Lys His Leu Leu Asn Ile Lys Ser Val Ala Glu
Arg Arg Glu Asn 435 440 445Met Leu
Trp Phe Arg Val Pro Glu Lys Val Tyr Phe Lys Tyr Gly Ser 450
455 460Leu Gly Val Ala Leu Lys Glu Leu Lys Val Met
Asn Lys Lys Lys Val465 470 475
480Phe Ile Val Thr Asp Lys Val Leu Tyr Gln Leu Gly Tyr Val Asp Lys
485 490 495Val Thr Lys Val
Leu Glu Glu Leu Lys Ile Ser Tyr Lys Val Phe Thr 500
505 510Asp Val Glu Pro Asp Pro Thr Leu Ala Thr Ala
Lys Lys Gly Ala Ala 515 520 525Glu
Leu Leu Ser Tyr Glu Pro Asp Thr Ile Ile Ser Val Gly Gly Gly 530
535 540Ser Ala Met Asp Ala Ala Lys Ile Met Trp
Val Met Tyr Glu His Pro545 550 555
560Glu Val Lys Phe Glu Asp Leu Ala Met Arg Phe Met Asp Ile Arg
Lys 565 570 575Arg Val Tyr
Val Phe Pro Lys Met Gly Glu Lys Ala Met Met Ile Ser 580
585 590Val Ala Thr Ser Ala Gly Thr Gly Ser Glu
Val Thr Pro Phe Ala Val 595 600
605Ile Thr Asp Glu Lys Thr Gly Ala Lys Tyr Pro Leu Ala Asp Tyr Glu 610
615 620Leu Thr Pro Asn Met Ala Ile Val
Asp Ala Glu Leu Met Met Gly Met625 630
635 640Pro Arg Gly Leu Thr Ala Ala Ser Gly Ile Asp Ala
Leu Thr His Ala 645 650
655Leu Glu Ala Tyr Val Ser Ile Met Ala Thr Glu Phe Thr Asn Gly Leu
660 665 670Ala Leu Glu Ala Val Lys
Leu Ile Phe Glu Tyr Leu Pro Lys Ala Tyr 675 680
685Thr Glu Gly Thr Thr Asn Val Lys Ala Arg Glu Lys Met Ala
His Ala 690 695 700Ser Cys Ile Ala Gly
Met Ala Phe Ala Asn Ala Phe Leu Gly Val Cys705 710
715 720His Ser Met Ala His Lys Leu Gly Ala Gln
His His Ile Pro His Gly 725 730
735Ile Ala Asn Ala Leu Met Ile Asp Glu Val Ile Lys Phe Asn Ala Val
740 745 750Asp Asp Pro Ile Lys
Gln Ala Ala Phe Pro Gln Tyr Glu Tyr Pro Asn 755
760 765Ala Lys Tyr Arg Tyr Ala Gln Ile Ala Asp Cys Leu
Asn Leu Gly Gly 770 775 780Asn Thr Glu
Asp Glu Lys Val Gln Leu Leu Ile Asn Ala Ile Asp Asp785
790 795 800Leu Lys Ala Lys Leu Asn Ile
Pro Glu Thr Ile Lys Glu Ala Gly Val 805
810 815Ser Glu Glu Lys Phe Tyr Thr Thr Leu Asp Lys Met
Ser Glu Leu Ala 820 825 830Phe
Asp Asp Gln Cys Thr Gly Ala Asn Pro Arg Tyr Pro Leu Ile Ser 835
840 845Glu Ile Lys Gln Met Tyr Ile Asn Val
Phe Asp Lys Thr Glu Pro Ile 850 855
860Val Glu Asp Glu Glu Lys865 870971569DNAC. ragsdalei
97atggagggaa cacaattgga aaattttgat aaagacttac gctctataca agaagcaaga
60gatcttgcac gtttaggaaa aattgcagca tgtgaaattg ctgattatac tgaagaacaa
120attgataaaa tcctatgtaa tatggttagg gtagcagagg aaaatgcagt ttgccttggt
180aaaatggctg cagaagaaac tggttttgga aaagctgaag ataaggctta taagaaccat
240atggctgcta ctacagtata taattatatc aaggatatga agactattgg tgttataaaa
300gaagataaaa gtcaaggtgt aattgaattt gctgaaccag ttggtttatt aatgggtatt
360gtaccatcta caaatccaac atctactgtt atctataaat caatcattgc aattaaatca
420agaaatgcaa ttgtattctc accacaccca gctgcattaa aatgttcaac aaaagcaata
480gaacttatgc gtgatgcagc agtagcagca ggagctcctg caaatgtaat tggcggtatt
540gttacaccat ctatacaagc tacaaatgaa cttatgaaag ctaaagaagt tgctatgata
600attgccactg gaggccctgg aatggtaaag gctgcttata gttcaggaac acctgcaata
660ggcgttggtg ctggtaactc tccatcttat atagaaagaa ctgctgatgt tcatcaatca
720gttaaagata taattgctag taagagtttt gactatggta ctatttgtgc atctgagcaa
780tcaataattg ttgaagaatg caaccatgat gaagtaatag ctgagttgaa gaaacaaggc
840ggatatttca tgacagctga agaaactgca aaagtttgca gtatactttt taagcctggt
900acacacagta tgagtgctaa gtttgtagga agagctcctc aggttatagc agcagctgca
960ggtttctcag ttccagaagg aacaaaagtt ttagtaggag aacaaggcgg agttggtaat
1020ggttaccctc tatcttatga gaaacttaca acagtacttg ctttctatac agttaaagat
1080tggcatgaag catgtgatct tagtataaga ttacttcaaa atggtcttgg acatactatg
1140aacattcata caaatgacag agacttagta atgaagtttg ctaaaaaacc agcatcccgt
1200atattagtta atactggtgg aagccaagga ggtactggtg caagcacagg attagcacct
1260gcatttacat taggttgtgg tacatgggga ggaagctctg tttccgaaaa tgttactcca
1320ttacatttaa tcaatataaa gagagttgca tatggtctta aagattgttc tacattagct
1380gcagatgata caactttcaa tcatcctgaa ctttgtggaa gcaaaaatga cttaggatgc
1440tgtgctacaa gccctgcaga atttgcagca aatagcaatt gtgctagcac tgctgcggat
1500actactgata atgataaact tgctagactc gtaagtgaat tagtagctgc aatgaaggga
1560gctaactaa
156998522PRTC. ragsdalei 98Met Glu Gly Thr Gln Leu Glu Asn Phe Asp Lys
Asp Leu Arg Ser Ile1 5 10
15Gln Glu Ala Arg Asp Leu Ala Arg Leu Gly Lys Ile Ala Ala Cys Glu
20 25 30Ile Ala Asp Tyr Thr Glu Glu
Gln Ile Asp Lys Ile Leu Cys Asn Met 35 40
45Val Arg Val Ala Glu Glu Asn Ala Val Cys Leu Gly Lys Met Ala
Ala 50 55 60Glu Glu Thr Gly Phe Gly
Lys Ala Glu Asp Lys Ala Tyr Lys Asn His65 70
75 80Met Ala Ala Thr Thr Val Tyr Asn Tyr Ile Lys
Asp Met Lys Thr Ile 85 90
95Gly Val Ile Lys Glu Asp Lys Ser Gln Gly Val Ile Glu Phe Ala Glu
100 105 110Pro Val Gly Leu Leu Met
Gly Ile Val Pro Ser Thr Asn Pro Thr Ser 115 120
125Thr Val Ile Tyr Lys Ser Ile Ile Ala Ile Lys Ser Arg Asn
Ala Ile 130 135 140Val Phe Ser Pro His
Pro Ala Ala Leu Lys Cys Ser Thr Lys Ala Ile145 150
155 160Glu Leu Met Arg Asp Ala Ala Val Ala Ala
Gly Ala Pro Ala Asn Val 165 170
175Ile Gly Gly Ile Val Thr Pro Ser Ile Gln Ala Thr Asn Glu Leu Met
180 185 190Lys Ala Lys Glu Val
Ala Met Ile Ile Ala Thr Gly Gly Pro Gly Met 195
200 205Val Lys Ala Ala Tyr Ser Ser Gly Thr Pro Ala Ile
Gly Val Gly Ala 210 215 220Gly Asn Ser
Pro Ser Tyr Ile Glu Arg Thr Ala Asp Val His Gln Ser225
230 235 240Val Lys Asp Ile Ile Ala Ser
Lys Ser Phe Asp Tyr Gly Thr Ile Cys 245
250 255Ala Ser Glu Gln Ser Ile Ile Val Glu Glu Cys Asn
His Asp Glu Val 260 265 270Ile
Ala Glu Leu Lys Lys Gln Gly Gly Tyr Phe Met Thr Ala Glu Glu 275
280 285Thr Ala Lys Val Cys Ser Ile Leu Phe
Lys Pro Gly Thr His Ser Met 290 295
300Ser Ala Lys Phe Val Gly Arg Ala Pro Gln Val Ile Ala Ala Ala Ala305
310 315 320Gly Phe Ser Val
Pro Glu Gly Thr Lys Val Leu Val Gly Glu Gln Gly 325
330 335Gly Val Gly Asn Gly Tyr Pro Leu Ser Tyr
Glu Lys Leu Thr Thr Val 340 345
350Leu Ala Phe Tyr Thr Val Lys Asp Trp His Glu Ala Cys Asp Leu Ser
355 360 365Ile Arg Leu Leu Gln Asn Gly
Leu Gly His Thr Met Asn Ile His Thr 370 375
380Asn Asp Arg Asp Leu Val Met Lys Phe Ala Lys Lys Pro Ala Ser
Arg385 390 395 400Ile Leu
Val Asn Thr Gly Gly Ser Gln Gly Gly Thr Gly Ala Ser Thr
405 410 415Gly Leu Ala Pro Ala Phe Thr
Leu Gly Cys Gly Thr Trp Gly Gly Ser 420 425
430Ser Val Ser Glu Asn Val Thr Pro Leu His Leu Ile Asn Ile
Lys Arg 435 440 445Val Ala Tyr Gly
Leu Lys Asp Cys Ser Thr Leu Ala Ala Asp Asp Thr 450
455 460Thr Phe Asn His Pro Glu Leu Cys Gly Ser Lys Asn
Asp Leu Gly Cys465 470 475
480Cys Ala Thr Ser Pro Ala Glu Phe Ala Ala Asn Ser Asn Cys Ala Ser
485 490 495Thr Ala Ala Asp Thr
Thr Asp Asn Asp Lys Leu Ala Arg Leu Val Ser 500
505 510Glu Leu Val Ala Ala Met Lys Gly Ala Asn
515 520991446DNAC. ragsdalei 99gtggaaaatg ctgcacgagc
acaaaaaatg ttagcaactt ttccgcaaga aaagttagat 60gagattgttg aacgtatggc
tgaagaaatc ggaaaacata cccgagagct tgctgtaatg 120tcacaggatg aaactggtta
tggaaaatgg caggataaat gcatcaaaaa ccgatttgcc 180tgtgaatatt tgccagctaa
gcttagagga atgcgatgtg taggtattat taacgaaaat 240ggtcaggata agaccatgga
tgtaggtgta cctatgggtg taattattgc attatgtcct 300gcaactagtc cggtttctac
taccatatat aaggcattaa ttgcaattaa gtctggtaat 360gcaattatct tttctccaca
tcctagagca aaggagacaa tttgtaaggc gcttgacatc 420atgattcgtg cagctgaagg
atatgggctg ccagaaggag ctcttgcata cttacatact 480gtgacgccta gtggaacaat
cgaattgatg aaccatgagg cgacttcttt gattatgaat 540acaggcgttc ccgggatgct
taaagcgtca tatagatctg gaaaacctgt gatctatgga 600ggaactggta atggaccagc
atttattgaa cgtacagctg acatcaagca ggcggtaaga 660gatattattg ctagtaagac
ctttgataac ggaatagtac catcatctga acaatctatt 720gttgtagata gctgtgttgc
atctgatgtt aaacgtgagt tgcaaaatag tggtgcatat 780ttcatgacag aggaggaagc
acaaaaactg ggttctctct ttttccgttc tgatggtagt 840atggattcag aaatggttgg
caaatccgca cagagattgg ctaagaaagc aggtttcagt 900attcctgaaa gtagcacagt
gctaatttca gagcagaaat atgtttccca agataatcct 960tattccaagg agaaactttg
tccggtacta gcttactaca ttgaagatga ttggatgcat 1020gcatgtgaaa agtgtattga
gctgctatta agtgagagac atggtcacac tcttgttata 1080cattcaaaag acgaagatgt
aattcgccag tttgcattaa aaaaacctgt aggcaggata 1140cttgttaata cgcctgcttc
ctttggtagt atgggtgcta caagtaattt atttcctgct 1200ttaactttag gtagtggatc
ggcaggtaaa ggtattacct ccgataatgt ttcaccaatg 1260aatcttattt acgtccgtaa
agtcggatat ggcgtacgga atgtagaaga gattattaat 1320actaatggat tgtttacaga
agaaaaaagt gatttgagtg gtatgacaaa gcagtcagac 1380tataatccag aggatataca
aatgttgcag catattttga aaaaagctat ggaaaaaatt 1440aaatag
1446100481PRTC. ragsdalei
100Val Glu Asn Ala Ala Arg Ala Gln Lys Met Leu Ala Thr Phe Pro Gln1
5 10 15Glu Lys Leu Asp Glu Ile
Val Glu Arg Met Ala Glu Glu Ile Gly Lys 20 25
30His Thr Arg Glu Leu Ala Val Met Ser Gln Asp Glu Thr
Gly Tyr Gly 35 40 45Lys Trp Gln
Asp Lys Cys Ile Lys Asn Arg Phe Ala Cys Glu Tyr Leu 50
55 60Pro Ala Lys Leu Arg Gly Met Arg Cys Val Gly Ile
Ile Asn Glu Asn65 70 75
80Gly Gln Asp Lys Thr Met Asp Val Gly Val Pro Met Gly Val Ile Ile
85 90 95Ala Leu Cys Pro Ala Thr
Ser Pro Val Ser Thr Thr Ile Tyr Lys Ala 100
105 110Leu Ile Ala Ile Lys Ser Gly Asn Ala Ile Ile Phe
Ser Pro His Pro 115 120 125Arg Ala
Lys Glu Thr Ile Cys Lys Ala Leu Asp Ile Met Ile Arg Ala 130
135 140Ala Glu Gly Tyr Gly Leu Pro Glu Gly Ala Leu
Ala Tyr Leu His Thr145 150 155
160Val Thr Pro Ser Gly Thr Ile Glu Leu Met Asn His Glu Ala Thr Ser
165 170 175Leu Ile Met Asn
Thr Gly Val Pro Gly Met Leu Lys Ala Ser Tyr Arg 180
185 190Ser Gly Lys Pro Val Ile Tyr Gly Gly Thr Gly
Asn Gly Pro Ala Phe 195 200 205Ile
Glu Arg Thr Ala Asp Ile Lys Gln Ala Val Arg Asp Ile Ile Ala 210
215 220Ser Lys Thr Phe Asp Asn Gly Ile Val Pro
Ser Ser Glu Gln Ser Ile225 230 235
240Val Val Asp Ser Cys Val Ala Ser Asp Val Lys Arg Glu Leu Gln
Asn 245 250 255Ser Gly Ala
Tyr Phe Met Thr Glu Glu Glu Ala Gln Lys Leu Gly Ser 260
265 270Leu Phe Phe Arg Ser Asp Gly Ser Met Asp
Ser Glu Met Val Gly Lys 275 280
285Ser Ala Gln Arg Leu Ala Lys Lys Ala Gly Phe Ser Ile Pro Glu Ser 290
295 300Ser Thr Val Leu Ile Ser Glu Gln
Lys Tyr Val Ser Gln Asp Asn Pro305 310
315 320Tyr Ser Lys Glu Lys Leu Cys Pro Val Leu Ala Tyr
Tyr Ile Glu Asp 325 330
335Asp Trp Met His Ala Cys Glu Lys Cys Ile Glu Leu Leu Leu Ser Glu
340 345 350Arg His Gly His Thr Leu
Val Ile His Ser Lys Asp Glu Asp Val Ile 355 360
365Arg Gln Phe Ala Leu Lys Lys Pro Val Gly Arg Ile Leu Val
Asn Thr 370 375 380Pro Ala Ser Phe Gly
Ser Met Gly Ala Thr Ser Asn Leu Phe Pro Ala385 390
395 400Leu Thr Leu Gly Ser Gly Ser Ala Gly Lys
Gly Ile Thr Ser Asp Asn 405 410
415Val Ser Pro Met Asn Leu Ile Tyr Val Arg Lys Val Gly Tyr Gly Val
420 425 430Arg Asn Val Glu Glu
Ile Ile Asn Thr Asn Gly Leu Phe Thr Glu Glu 435
440 445Lys Ser Asp Leu Ser Gly Met Thr Lys Gln Ser Asp
Tyr Asn Pro Glu 450 455 460Asp Ile Gln
Met Leu Gln His Ile Leu Lys Lys Ala Met Glu Lys Ile465
470 475 480Lys1011167DNAC. ragsdalei
101atggcaagat ttactttacc aagagacatt tattttggag aaaattcatt agagaccttg
60aaaaacctag atggaaaaaa agctgtcatt gtcgtaggtg gaggatccat gaaaagattt
120ggattccttg ataaggtagt agactactta aaagaagcag gtattgaatc aaaattaata
180gaaggcgttg agccagatcc atccgtagaa actgttatga atggtgctaa actaatgagg
240gaatatgggc cagatttaat aatatcaata ggtggaggtt caccaattga tgcagcaaaa
300gctatgtgga tattctatga ataccctgag tttactttta aagaagctgt agttcctttt
360ggtcttccta aattaagaca aaaagcaaca tttatagcta tcccttctac aagtggtact
420gcaacggaag taactgcatt ttctgtaata acagactata aagctaaaat taaatatcct
480ttggctgact tcaatttaac accagatata gctataattg atccagtatt agctcaaaca
540atgccgccta aattaactgc acatactgga atggatgcac ttactcacgc tattgaagca
600tatgttgcag gacttcattc agttttctcg gacccacttg ctattcaagc tatagtcatg
660gtaaatcaat atttaattaa atcttacaat gaagataaag aagctaggga tcaaatgcat
720ttagctcaat gtttagctgg aatggcattt tcaaatgcac ttcttggaat aactcacagt
780ttagcacata aaacaggtgc agtattccat atccctcatg gatgtgctaa tgcaatatat
840cttccttatg ttatagattt caataaaaaa gcttgtgcac caagatatgc tgatatagct
900aggagtctta aacttccagg aaatactgat gatgaattag tagattcatt aactaatatg
960attaaagata tgaacaagag tatggatatt cctttgacat taaaagatta tggagtagat
1020gaaaaagaat ttaaagatag tgaagatttt atagctcata atgccgtatt agatgcctgt
1080actggatcaa atcctagaag cataaatgat gctgaaatga aaaagttgtt agaatacatc
1140tattatggta aaaaggttga tttttaa
1167102388PRTC. ragsdalei 102Met Ala Arg Phe Thr Leu Pro Arg Asp Ile Tyr
Phe Gly Glu Asn Ser1 5 10
15Leu Glu Thr Leu Lys Asn Leu Asp Gly Lys Lys Ala Val Ile Val Val
20 25 30Gly Gly Gly Ser Met Lys Arg
Phe Gly Phe Leu Asp Lys Val Val Asp 35 40
45Tyr Leu Lys Glu Ala Gly Ile Glu Ser Lys Leu Ile Glu Gly Val
Glu 50 55 60Pro Asp Pro Ser Val Glu
Thr Val Met Asn Gly Ala Lys Leu Met Arg65 70
75 80Glu Tyr Gly Pro Asp Leu Ile Ile Ser Ile Gly
Gly Gly Ser Pro Ile 85 90
95Asp Ala Ala Lys Ala Met Trp Ile Phe Tyr Glu Tyr Pro Glu Phe Thr
100 105 110Phe Lys Glu Ala Val Val
Pro Phe Gly Leu Pro Lys Leu Arg Gln Lys 115 120
125Ala Thr Phe Ile Ala Ile Pro Ser Thr Ser Gly Thr Ala Thr
Glu Val 130 135 140Thr Ala Phe Ser Val
Ile Thr Asp Tyr Lys Ala Lys Ile Lys Tyr Pro145 150
155 160Leu Ala Asp Phe Asn Leu Thr Pro Asp Ile
Ala Ile Ile Asp Pro Val 165 170
175Leu Ala Gln Thr Met Pro Pro Lys Leu Thr Ala His Thr Gly Met Asp
180 185 190Ala Leu Thr His Ala
Ile Glu Ala Tyr Val Ala Gly Leu His Ser Val 195
200 205Phe Ser Asp Pro Leu Ala Ile Gln Ala Ile Val Met
Val Asn Gln Tyr 210 215 220Leu Ile Lys
Ser Tyr Asn Glu Asp Lys Glu Ala Arg Asp Gln Met His225
230 235 240Leu Ala Gln Cys Leu Ala Gly
Met Ala Phe Ser Asn Ala Leu Leu Gly 245
250 255Ile Thr His Ser Leu Ala His Lys Thr Gly Ala Val
Phe His Ile Pro 260 265 270His
Gly Cys Ala Asn Ala Ile Tyr Leu Pro Tyr Val Ile Asp Phe Asn 275
280 285Lys Lys Ala Cys Ala Pro Arg Tyr Ala
Asp Ile Ala Arg Ser Leu Lys 290 295
300Leu Pro Gly Asn Thr Asp Asp Glu Leu Val Asp Ser Leu Thr Asn Met305
310 315 320Ile Lys Asp Met
Asn Lys Ser Met Asp Ile Pro Leu Thr Leu Lys Asp 325
330 335Tyr Gly Val Asp Glu Lys Glu Phe Lys Asp
Ser Glu Asp Phe Ile Ala 340 345
350His Asn Ala Val Leu Asp Ala Cys Thr Gly Ser Asn Pro Arg Ser Ile
355 360 365Asn Asp Ala Glu Met Lys Lys
Leu Leu Glu Tyr Ile Tyr Tyr Gly Lys 370 375
380Lys Val Asp Phe3851031167DNAC. ragsdalei 103atgggaagat ttactttgcc
tagggatatt tactttggtg aaaatgcctt agaaaattta 60aaaaatttag atggaaataa
agcagtagtt gttgtaggtg gaggatctat gaagagattt 120gggttcttag ccaaagttga
agaatactta aaagaagcag gtatggaagt taaattaata 180gaaggtgttg agcctgatcc
atctgttgat actgttatga atggtgctaa aataatgaga 240gactttaatc cagactggat
agtatcaata ggtggaggat ctcccatcga tgctgccaaa 300gcaatgtgga tattttatga
ataccctgac tttacatttg aaaaagcggt agtccctttt 360gggattccta aattaaggca
aaaggcacaa tttgttgcta taccttctac aagtggaaca 420gcaactgaag taacatcatt
ttctgtaata acagactata aagctaaaat aaaatatcct 480cttgcagatt ttaaccttac
ccctgatata gctataatag atccgtctct tgcagaaaca 540atgcctaaaa agcttacagc
acacactgga atggatgcac ttactcacgc aatagaagca 600tatgtggcaa gtttacattc
agatttctca gatccacttg ctatgcatgc tataaccatg 660attcataaat atttattgaa
atcctatgaa gaagataaag aagctagggg ccatatgcac 720atagcccaat gtctagctgg
aatggcattt tcaaatgcac tccttggaat aactcatagt 780atagcacata aaactggcgc
agtattccac atacctcatg ggtgtgctaa tgccatatac 840ttaccttatg ttatagattt
taacaagaaa gcttgttcag aaagatatgc taaaatagct 900aaaaagcttc atctatcagg
gaatagtgaa gatgaattaa tagattcatt aacagaaatg 960atttgtacta tgaataaaaa
gatggatatt cctcttacta taaaagatta tggtataagc 1020gaaaacgatt ttaatgaaaa
cctagatttt atagctcaca atgctatgat ggatgcttgc 1080actggatcta atcctagagc
aataactgag gaagaaatga aaaagctctt gcagtatatg 1140tataatgggc aaaaggttaa
tttctag 1167104388PRTC. ragsdalei
104Met Gly Arg Phe Thr Leu Pro Arg Asp Ile Tyr Phe Gly Glu Asn Ala1
5 10 15Leu Glu Asn Leu Lys Asn
Leu Asp Gly Asn Lys Ala Val Val Val Val 20 25
30Gly Gly Gly Ser Met Lys Arg Phe Gly Phe Leu Ala Lys
Val Glu Glu 35 40 45Tyr Leu Lys
Glu Ala Gly Met Glu Val Lys Leu Ile Glu Gly Val Glu 50
55 60Pro Asp Pro Ser Val Asp Thr Val Met Asn Gly Ala
Lys Ile Met Arg65 70 75
80Asp Phe Asn Pro Asp Trp Ile Val Ser Ile Gly Gly Gly Ser Pro Ile
85 90 95Asp Ala Ala Lys Ala Met
Trp Ile Phe Tyr Glu Tyr Pro Asp Phe Thr 100
105 110Phe Glu Lys Ala Val Val Pro Phe Gly Ile Pro Lys
Leu Arg Gln Lys 115 120 125Ala Gln
Phe Val Ala Ile Pro Ser Thr Ser Gly Thr Ala Thr Glu Val 130
135 140Thr Ser Phe Ser Val Ile Thr Asp Tyr Lys Ala
Lys Ile Lys Tyr Pro145 150 155
160Leu Ala Asp Phe Asn Leu Thr Pro Asp Ile Ala Ile Ile Asp Pro Ser
165 170 175Leu Ala Glu Thr
Met Pro Lys Lys Leu Thr Ala His Thr Gly Met Asp 180
185 190Ala Leu Thr His Ala Ile Glu Ala Tyr Val Ala
Ser Leu His Ser Asp 195 200 205Phe
Ser Asp Pro Leu Ala Met His Ala Ile Thr Met Ile His Lys Tyr 210
215 220Leu Leu Lys Ser Tyr Glu Glu Asp Lys Glu
Ala Arg Gly His Met His225 230 235
240Ile Ala Gln Cys Leu Ala Gly Met Ala Phe Ser Asn Ala Leu Leu
Gly 245 250 255Ile Thr His
Ser Ile Ala His Lys Thr Gly Ala Val Phe His Ile Pro 260
265 270His Gly Cys Ala Asn Ala Ile Tyr Leu Pro
Tyr Val Ile Asp Phe Asn 275 280
285Lys Lys Ala Cys Ser Glu Arg Tyr Ala Lys Ile Ala Lys Lys Leu His 290
295 300Leu Ser Gly Asn Ser Glu Asp Glu
Leu Ile Asp Ser Leu Thr Glu Met305 310
315 320Ile Cys Thr Met Asn Lys Lys Met Asp Ile Pro Leu
Thr Ile Lys Asp 325 330
335Tyr Gly Ile Ser Glu Asn Asp Phe Asn Glu Asn Leu Asp Phe Ile Ala
340 345 350His Asn Ala Met Met Asp
Ala Cys Thr Gly Ser Asn Pro Arg Ala Ile 355 360
365Thr Glu Glu Glu Met Lys Lys Leu Leu Gln Tyr Met Tyr Asn
Gly Gln 370 375 380Lys Val Asn
Phe3851051359DNAC. ragsdalei 105atgattttaa aaactaaact ttttgggcaa
acttatgaat ttaaaaatat gaaggaagta 60ttggcaaaag ctaatgaaga aaaatcggga
gatgctttag ctggaatcat agcaaaaagt 120acagcggaga gagttgcagc aaaggttgtt
ttgtctgaaa taactcttga ggaattaagg 180aataatcctg tagttcctta tgaggaggat
gaagtaacaa gagtaataca agatatgatt 240gataaagaag cctataataa aatcaaagct
atgacagttg gcgaatttag agaatttata 300ttaaaatcag aagaagccga tataaaagaa
ataagagatg gattaacttc tgagatgata 360gcaggtgtaa ctaagcttat gagtaatatg
gacttagtat atgcttctaa aaaaataaga 420aatattgcta cttgcaatac tactattggt
gaaaagggaa cagtctcttc aagacttcag 480cctaatcatg cagcagatag tatagatgga
attatggctt ctgtaatgga agggataagc 540tatggtatag gtgatgctgt aataggttta
aaccctgtag tagataccat agataatata 600tcagagattt tgaaaaattt taagcagttc
atgataaaat gggatatacc tacacaaaat 660tgtgtacttg ctcatataac aacgcaaatg
gaggctttaa agaaaggagt tcctatggat 720ctgatgttcc agagtatagc tggttcacaa
aaatccaata aaggctttgg aataagtgtg 780aagcttatgg atgaagctta tgaacttatg
aaggaaaaaa agagctccaa aggtcctaat 840tttatgtatt ttgaaacagg ccagggttct
gagctttctt cagaaggcca taatggagca 900gatcagctta caatggaagc aagatgttat
ggtcttgcaa aaaaatataa tccattcctt 960gtaaactctg tggtgggatt cataggacca
gaatatctat atgatggaaa acaaattata 1020agagcaggct tagaagatca ttttatgggt
aagttaacag gacttcctat gggtgttgat 1080gtatgttata caaaccatat gaaagcagat
caaaatgatt tggaaaattt agcattactc 1140cttgcagcag ctgactgtac ttattttatg
ggtatacctg gaggagatga cgtaatgctt 1200atgtatcaaa ctaccagcta tcatgatgta
gcttctatca gggacattat gcgtaaaaat 1260cctataaaag aatttgaaga aagaatggaa
gctctaggaa taatgaaaaa tggaaggctc 1320acagaaatag ctggtgatcc atctatattt
atgatttag 1359106452PRTC. ragsdalei 106Met Ile
Leu Lys Thr Lys Leu Phe Gly Gln Thr Tyr Glu Phe Lys Asn1 5
10 15Met Lys Glu Val Leu Ala Lys Ala
Asn Glu Glu Lys Ser Gly Asp Ala 20 25
30Leu Ala Gly Ile Ile Ala Lys Ser Thr Ala Glu Arg Val Ala Ala
Lys 35 40 45Val Val Leu Ser Glu
Ile Thr Leu Glu Glu Leu Arg Asn Asn Pro Val 50 55
60Val Pro Tyr Glu Glu Asp Glu Val Thr Arg Val Ile Gln Asp
Met Ile65 70 75 80Asp
Lys Glu Ala Tyr Asn Lys Ile Lys Ala Met Thr Val Gly Glu Phe
85 90 95Arg Glu Phe Ile Leu Lys Ser
Glu Glu Ala Asp Ile Lys Glu Ile Arg 100 105
110Asp Gly Leu Thr Ser Glu Met Ile Ala Gly Val Thr Lys Leu
Met Ser 115 120 125Asn Met Asp Leu
Val Tyr Ala Ser Lys Lys Ile Arg Asn Ile Ala Thr 130
135 140Cys Asn Thr Thr Ile Gly Glu Lys Gly Thr Val Ser
Ser Arg Leu Gln145 150 155
160Pro Asn His Ala Ala Asp Ser Ile Asp Gly Ile Met Ala Ser Val Met
165 170 175Glu Gly Ile Ser Tyr
Gly Ile Gly Asp Ala Val Ile Gly Leu Asn Pro 180
185 190Val Val Asp Thr Ile Asp Asn Ile Ser Glu Ile Leu
Lys Asn Phe Lys 195 200 205Gln Phe
Met Ile Lys Trp Asp Ile Pro Thr Gln Asn Cys Val Leu Ala 210
215 220His Ile Thr Thr Gln Met Glu Ala Leu Lys Lys
Gly Val Pro Met Asp225 230 235
240Leu Met Phe Gln Ser Ile Ala Gly Ser Gln Lys Ser Asn Lys Gly Phe
245 250 255Gly Ile Ser Val
Lys Leu Met Asp Glu Ala Tyr Glu Leu Met Lys Glu 260
265 270Lys Lys Ser Ser Lys Gly Pro Asn Phe Met Tyr
Phe Glu Thr Gly Gln 275 280 285Gly
Ser Glu Leu Ser Ser Glu Gly His Asn Gly Ala Asp Gln Leu Thr 290
295 300Met Glu Ala Arg Cys Tyr Gly Leu Ala Lys
Lys Tyr Asn Pro Phe Leu305 310 315
320Val Asn Ser Val Val Gly Phe Ile Gly Pro Glu Tyr Leu Tyr Asp
Gly 325 330 335Lys Gln Ile
Ile Arg Ala Gly Leu Glu Asp His Phe Met Gly Lys Leu 340
345 350Thr Gly Leu Pro Met Gly Val Asp Val Cys
Tyr Thr Asn His Met Lys 355 360
365Ala Asp Gln Asn Asp Leu Glu Asn Leu Ala Leu Leu Leu Ala Ala Ala 370
375 380Asp Cys Thr Tyr Phe Met Gly Ile
Pro Gly Gly Asp Asp Val Met Leu385 390
395 400Met Tyr Gln Thr Thr Ser Tyr His Asp Val Ala Ser
Ile Arg Asp Ile 405 410
415Met Arg Lys Asn Pro Ile Lys Glu Phe Glu Glu Arg Met Glu Ala Leu
420 425 430Gly Ile Met Lys Asn Gly
Arg Leu Thr Glu Ile Ala Gly Asp Pro Ser 435 440
445Ile Phe Met Ile 4501071176DNAC. ragsdalei
107atggaaaact ttatttttaa aaatgctaca gaaattattt ttggtaagga taccgaagat
60cttgtaggaa gtaaagtaaa ggagtattca aagtcagata aaatactctt ttgctatggg
120ggaggaagta taaagagatc gggcctctat gatagagtta taaagtcctt aaaagaaaat
180ggaattgaat ttatagaact tccaggaatt aaacctaatc caagattagg acctgttaaa
240gaaggtataa gactatgtag agaaaataat ataaaatttg tactatctgt aggaggagga
300agttcagcag atacagctaa agctattgct gtaggagtac cttataaagg agatgtatgg
360gatttttata cgggcaaagc tgaagtaaaa gaggctcttc ctgtaggagt tgtaataaca
420ttacctgcta caggtacaga atctagtaat agttctgtta ttatgaatga agatggttgg
480tttaaaaaag gattaaatac ggtacttata agacctgctt tttcaattat gaatcctgaa
540cttactttta cactaccaga atatcaaact gcttgtggtg cttgtgacat tatggcacat
600ataatggaaa gatattttac aaatgtgaaa catgtagatt taactgatag gctttgcgaa
660gctgcactta gaaatgttat aaataatgcc ccaatagttt taaaagatcc taaaaattat
720gatgctaggg cagaaattat gtggactggt actatagctc ataatgatgt gcttagtaca
780ggtagaatag gtgattgggc ttctcacaaa attgaacatg aattaagtgg ggaaacagat
840attgcccatg gagcaggact tgcaattgta tttcctgcat ggatgaaata tgtatataaa
900catgatatca atagatttgt acaatttgca gtaagggtat gggatgtaga tttatcttat
960agttcctgtg aagatattgt acttgaaggc ataaggagaa tgacagcatt tttcaagagc
1020atggggttac ctataacttt aaaagaagga agtataggag aagataaaat tgaagaaatg
1080gctaataagt gcacggataa tggaaccaaa actgtaggac aatttgtaaa actaaataaa
1140gatgatattg taaaaatatt aaatttagct agataa
1176108391PRTC. ragsdalei 108Met Glu Asn Phe Ile Phe Lys Asn Ala Thr Glu
Ile Ile Phe Gly Lys1 5 10
15Asp Thr Glu Asp Leu Val Gly Ser Lys Val Lys Glu Tyr Ser Lys Ser
20 25 30Asp Lys Ile Leu Phe Cys Tyr
Gly Gly Gly Ser Ile Lys Arg Ser Gly 35 40
45Leu Tyr Asp Arg Val Ile Lys Ser Leu Lys Glu Asn Gly Ile Glu
Phe 50 55 60Ile Glu Leu Pro Gly Ile
Lys Pro Asn Pro Arg Leu Gly Pro Val Lys65 70
75 80Glu Gly Ile Arg Leu Cys Arg Glu Asn Asn Ile
Lys Phe Val Leu Ser 85 90
95Val Gly Gly Gly Ser Ser Ala Asp Thr Ala Lys Ala Ile Ala Val Gly
100 105 110Val Pro Tyr Lys Gly Asp
Val Trp Asp Phe Tyr Thr Gly Lys Ala Glu 115 120
125Val Lys Glu Ala Leu Pro Val Gly Val Val Ile Thr Leu Pro
Ala Thr 130 135 140Gly Thr Glu Ser Ser
Asn Ser Ser Val Ile Met Asn Glu Asp Gly Trp145 150
155 160Phe Lys Lys Gly Leu Asn Thr Val Leu Ile
Arg Pro Ala Phe Ser Ile 165 170
175Met Asn Pro Glu Leu Thr Phe Thr Leu Pro Glu Tyr Gln Thr Ala Cys
180 185 190Gly Ala Cys Asp Ile
Met Ala His Ile Met Glu Arg Tyr Phe Thr Asn 195
200 205Val Lys His Val Asp Leu Thr Asp Arg Leu Cys Glu
Ala Ala Leu Arg 210 215 220Asn Val Ile
Asn Asn Ala Pro Ile Val Leu Lys Asp Pro Lys Asn Tyr225
230 235 240Asp Ala Arg Ala Glu Ile Met
Trp Thr Gly Thr Ile Ala His Asn Asp 245
250 255Val Leu Ser Thr Gly Arg Ile Gly Asp Trp Ala Ser
His Lys Ile Glu 260 265 270His
Glu Leu Ser Gly Glu Thr Asp Ile Ala His Gly Ala Gly Leu Ala 275
280 285Ile Val Phe Pro Ala Trp Met Lys Tyr
Val Tyr Lys His Asp Ile Asn 290 295
300Arg Phe Val Gln Phe Ala Val Arg Val Trp Asp Val Asp Leu Ser Tyr305
310 315 320Ser Ser Cys Glu
Asp Ile Val Leu Glu Gly Ile Arg Arg Met Thr Ala 325
330 335Phe Phe Lys Ser Met Gly Leu Pro Ile Thr
Leu Lys Glu Gly Ser Ile 340 345
350Gly Glu Asp Lys Ile Glu Glu Met Ala Asn Lys Cys Thr Asp Asn Gly
355 360 365Thr Lys Thr Val Gly Gln Phe
Val Lys Leu Asn Lys Asp Asp Ile Val 370 375
380Lys Ile Leu Asn Leu Ala Arg385 3901091149DNAC.
ragsdalei 109atggaagaca agtttgaaaa ttttaatttg aaatccaaga tttattttaa
tagggaatcc 60atacaacttt tagagcaggt tactggctct cgagcattta ttgttgcaga
tgccattatg 120ggaaaacttg gatatcttca aaaagtaata gattccctaa gtaaagccgg
aataagttcc 180gttgttttta cgggagtaca ccctgatcca gatgtcaatg taattgcaga
tgcaatgaaa 240ttgtacaaca aaagcgatgc agatgttctc gttgcactag gtggaggctc
cagcattgat 300accgccaaag gaataatgta ttttgcatgt aatttaggaa aagcaatggg
ccaggaaatg 360aaaaagcccc tgtttattgc aattccatca acaagtggaa caggctctga
agtaacaaac 420tttactgtta ttacttctca gaaagaaaag gtatgcattg tagatgattt
tattgcacca 480gacgttgcaa tacttgactc tagttgtatt gatggtctgc ctcaacgtat
tgtagcagat 540actggtatag atgttctagt tcattctatt gaagcctatg tttccaaaaa
agcaactgac 600tttacagacg ctcttgctga aaaagcagtt aaattgattt ttgagaatct
tccaaaaatt 660tataacgata gtaaagattc tgaagctcga gatcatgttc aaaacgcttc
ttgtatagca 720ggaatagcat ttacaaatgc tggtcttgga attaatcaca gcttggctca
tgctatgggt 780ggatcttttc acattcctca cggccgatcc aatgcacttt tacttaatgc
agtaatggaa 840tacaatgcta gcttagtggg aaatgcaaac gatcatgcta tggaaaaata
cgcaaaacta 900gcatcagttc tacaccttcc agctcgaaca actcgtgaag gcgctgtaag
ttttatcgaa 960gctgtaaata aattaataaa atccctaggt gttgaagata atattcgagc
tcttggaatt 1020aaagaagacg attttcaagg tgctctaaat catatggcag aaacagcaat
gcaagataga 1080tgcactccaa ctaatcctag aaaaccttct aaagaagaac tgatacatat
ttatcaaaaa 1140tgctattaa
1149110382PRTC. ragsdalei 110Met Glu Asp Lys Phe Glu Asn Phe
Asn Leu Lys Ser Lys Ile Tyr Phe1 5 10
15Asn Arg Glu Ser Ile Gln Leu Leu Glu Gln Val Thr Gly Ser
Arg Ala 20 25 30Phe Ile Val
Ala Asp Ala Ile Met Gly Lys Leu Gly Tyr Leu Gln Lys 35
40 45Val Ile Asp Ser Leu Ser Lys Ala Gly Ile Ser
Ser Val Val Phe Thr 50 55 60Gly Val
His Pro Asp Pro Asp Val Asn Val Ile Ala Asp Ala Met Lys65
70 75 80Leu Tyr Asn Lys Ser Asp Ala
Asp Val Leu Val Ala Leu Gly Gly Gly 85 90
95Ser Ser Ile Asp Thr Ala Lys Gly Ile Met Tyr Phe Ala
Cys Asn Leu 100 105 110Gly Lys
Ala Met Gly Gln Glu Met Lys Lys Pro Leu Phe Ile Ala Ile 115
120 125Pro Ser Thr Ser Gly Thr Gly Ser Glu Val
Thr Asn Phe Thr Val Ile 130 135 140Thr
Ser Gln Lys Glu Lys Val Cys Ile Val Asp Asp Phe Ile Ala Pro145
150 155 160Asp Val Ala Ile Leu Asp
Ser Ser Cys Ile Asp Gly Leu Pro Gln Arg 165
170 175Ile Val Ala Asp Thr Gly Ile Asp Val Leu Val His
Ser Ile Glu Ala 180 185 190Tyr
Val Ser Lys Lys Ala Thr Asp Phe Thr Asp Ala Leu Ala Glu Lys 195
200 205Ala Val Lys Leu Ile Phe Glu Asn Leu
Pro Lys Ile Tyr Asn Asp Ser 210 215
220Lys Asp Ser Glu Ala Arg Asp His Val Gln Asn Ala Ser Cys Ile Ala225
230 235 240Gly Ile Ala Phe
Thr Asn Ala Gly Leu Gly Ile Asn His Ser Leu Ala 245
250 255His Ala Met Gly Gly Ser Phe His Ile Pro
His Gly Arg Ser Asn Ala 260 265
270Leu Leu Leu Asn Ala Val Met Glu Tyr Asn Ala Ser Leu Val Gly Asn
275 280 285Ala Asn Asp His Ala Met Glu
Lys Tyr Ala Lys Leu Ala Ser Val Leu 290 295
300His Leu Pro Ala Arg Thr Thr Arg Glu Gly Ala Val Ser Phe Ile
Glu305 310 315 320Ala Val
Asn Lys Leu Ile Lys Ser Leu Gly Val Glu Asp Asn Ile Arg
325 330 335Ala Leu Gly Ile Lys Glu Asp
Asp Phe Gln Gly Ala Leu Asn His Met 340 345
350Ala Glu Thr Ala Met Gln Asp Arg Cys Thr Pro Thr Asn Pro
Arg Lys 355 360 365Pro Ser Lys Glu
Glu Leu Ile His Ile Tyr Gln Lys Cys Tyr 370 375
380111993DNAC. ragsdalei 111atggaaaaaa tttggaataa ggcaaaggaa
gacaaaaaaa agattgtctt agctgaagga 60gaagaagaaa gaactcttca agcttgtgaa
aaaataatta aagaaggtat tgcaaattta 120atccttgtag ggaatgaaaa ggtaatagag
gagaaggcat caaaattagg cgtaagttta 180aatggagcag aaatagtaga tccagaaacc
tcggataaac taaaaaaata tgcagatgct 240ttttatgaat tgagaaagaa gaagggaata
acaccagaaa aagcggataa aatagtaaga 300gatccaatat attttgctac gatgatggtt
aagcttggag atgcagatgg attggtttca 360ggtgcagtgc atactacagg tgatcttttg
agaccaggac ttcaaatagt aaagacagct 420ccaggtacat cagtagtttc cagcacattt
ataatggaag taccaaattg tgaatatggt 480gacaatggtg tacttctatt tgctgattgt
gctgtaaatc catgcccaga tagtgatcaa 540ttggcttcaa ttgcaataag tacagcagaa
actgcaaaga acttatgtgg aatggatcca 600aaagtagcaa tgctttcatt ttctactaag
ggaagtgcaa aacacgaatt agtagataaa 660gttagaaatg ctgtagaaat tgccaaaaaa
gctaaaccag atttaagttt ggacggagaa 720ttacaattag atgcctctat cgtagaaaag
gttgcaagtt taaaggctcc tgaaagtgaa 780gtagcaggaa aagcaaatgt acttgtattt
ccagatctcc aagcaggaaa tataggttat 840aaacttgttc aaagatttgc aaaagctgat
gctataggac ctgtatgcca gggatttgca 900aaacctataa atgatttgtc aagaggatgt
aactccgatg atatagtaaa tgtagtagct 960gtaacagcag ttcaggcaca agctcaaaag
taa 993112330PRTC. ragsdalei 112Met Glu
Lys Ile Trp Asn Lys Ala Lys Glu Asp Lys Lys Lys Ile Val1 5
10 15Leu Ala Glu Gly Glu Glu Glu Arg
Thr Leu Gln Ala Cys Glu Lys Ile 20 25
30Ile Lys Glu Gly Ile Ala Asn Leu Ile Leu Val Gly Asn Glu Lys
Val 35 40 45Ile Glu Glu Lys Ala
Ser Lys Leu Gly Val Ser Leu Asn Gly Ala Glu 50 55
60Ile Val Asp Pro Glu Thr Ser Asp Lys Leu Lys Lys Tyr Ala
Asp Ala65 70 75 80Phe
Tyr Glu Leu Arg Lys Lys Lys Gly Ile Thr Pro Glu Lys Ala Asp
85 90 95Lys Ile Val Arg Asp Pro Ile
Tyr Phe Ala Thr Met Met Val Lys Leu 100 105
110Gly Asp Ala Asp Gly Leu Val Ser Gly Ala Val His Thr Thr
Gly Asp 115 120 125Leu Leu Arg Pro
Gly Leu Gln Ile Val Lys Thr Ala Pro Gly Thr Ser 130
135 140Val Val Ser Ser Thr Phe Ile Met Glu Val Pro Asn
Cys Glu Tyr Gly145 150 155
160Asp Asn Gly Val Leu Leu Phe Ala Asp Cys Ala Val Asn Pro Cys Pro
165 170 175Asp Ser Asp Gln Leu
Ala Ser Ile Ala Ile Ser Thr Ala Glu Thr Ala 180
185 190Lys Asn Leu Cys Gly Met Asp Pro Lys Val Ala Met
Leu Ser Phe Ser 195 200 205Thr Lys
Gly Ser Ala Lys His Glu Leu Val Asp Lys Val Arg Asn Ala 210
215 220Val Glu Ile Ala Lys Lys Ala Lys Pro Asp Leu
Ser Leu Asp Gly Glu225 230 235
240Leu Gln Leu Asp Ala Ser Ile Val Glu Lys Val Ala Ser Leu Lys Ala
245 250 255Pro Glu Ser Glu
Val Ala Gly Lys Ala Asn Val Leu Val Phe Pro Asp 260
265 270Leu Gln Ala Gly Asn Ile Gly Tyr Lys Leu Val
Gln Arg Phe Ala Lys 275 280 285Ala
Asp Ala Ile Gly Pro Val Cys Gln Gly Phe Ala Lys Pro Ile Asn 290
295 300Asp Leu Ser Arg Gly Cys Asn Ser Asp Asp
Ile Val Asn Val Val Ala305 310 315
320Val Thr Ala Val Gln Ala Gln Ala Gln Lys 325
3301131197DNAC. ragsdalei 113atgaaaatat tagtagtaaa
ctgtggaagt tcatctttaa aatatcaact tattgatatg 60aaagatgaaa gcgttgtggc
aaaaggactt gtagaaagaa taggagcaga aggttcagtt 120ttaacacata aagttaacgg
agaaaagttt gttacagagc agccaatgga agatcataaa 180gttgctatac aattagtatt
aaatgctctt gtagataaaa aacatggtgt aataaaagat 240atgtcagaaa tatctgctgt
agggcataga gttttgcatg gtggaaaaaa atatgcggca 300tccattctta ttgatgacaa
tgtaatgaaa gcaatagaag aatgtattcc attaggacca 360ttacataatc cagctaatat
aatgggaata gatgcttgta aaaaactaat gccaaatact 420ccaatggtag cagtatttga
tacagcattt catcagacaa tgccagatta tgcttatact 480tatgcaatac cttatgatat
atctgaaaag tatgatatca gaaaatatgg ttttcatgga 540acttctcata gattcgtttc
aattgaagca gccaagttgt taaagaaaga tccaaaagat 600cttaagctaa taacttgtca
tttaggaaat ggagctagta tatgtgcagt aaaccaggga 660aaagcagtag atacaactat
gggacttact ccccttgcag gacttgtaat gggaactaga 720tgtggtgata tagatccagc
tataatacca tttgtaatga aaagaacagg tatgtctgta 780gatgaaatgg atactttaat
gaacaaaaag tcaggaatac ttggagtatc aggagtaagc 840agcgatttta gagatgtaga
agaagctgca aattcaggaa atgatagagc aaaacttgca 900ttaaatatgt attatcacaa
agttaaatct ttcataggag cttatgttgc agttttaaat 960ggagcagatg ctataatatt
tacagcagga cttggagaaa attcagctac tagcagatct 1020gctatatgta agggattaag
ctattttgga attaaaatag atgaagaaaa gaataagaaa 1080aggggagaag cactagaaat
aagcacacct gattcaaaga taaaagtatt agtaattcct 1140acaaatgaag aacttatgat
agctagggat acaaaagaaa tagttgaaaa taaataa 1197114398PRTC. ragsdalei
114Met Lys Ile Leu Val Val Asn Cys Gly Ser Ser Ser Leu Lys Tyr Gln1
5 10 15Leu Ile Asp Met Lys Asp
Glu Ser Val Val Ala Lys Gly Leu Val Glu 20 25
30Arg Ile Gly Ala Glu Gly Ser Val Leu Thr His Lys Val
Asn Gly Glu 35 40 45Lys Phe Val
Thr Glu Gln Pro Met Glu Asp His Lys Val Ala Ile Gln 50
55 60Leu Val Leu Asn Ala Leu Val Asp Lys Lys His Gly
Val Ile Lys Asp65 70 75
80Met Ser Glu Ile Ser Ala Val Gly His Arg Val Leu His Gly Gly Lys
85 90 95Lys Tyr Ala Ala Ser Ile
Leu Ile Asp Asp Asn Val Met Lys Ala Ile 100
105 110Glu Glu Cys Ile Pro Leu Gly Pro Leu His Asn Pro
Ala Asn Ile Met 115 120 125Gly Ile
Asp Ala Cys Lys Lys Leu Met Pro Asn Thr Pro Met Val Ala 130
135 140Val Phe Asp Thr Ala Phe His Gln Thr Met Pro
Asp Tyr Ala Tyr Thr145 150 155
160Tyr Ala Ile Pro Tyr Asp Ile Ser Glu Lys Tyr Asp Ile Arg Lys Tyr
165 170 175Gly Phe His Gly
Thr Ser His Arg Phe Val Ser Ile Glu Ala Ala Lys 180
185 190Leu Leu Lys Lys Asp Pro Lys Asp Leu Lys Leu
Ile Thr Cys His Leu 195 200 205Gly
Asn Gly Ala Ser Ile Cys Ala Val Asn Gln Gly Lys Ala Val Asp 210
215 220Thr Thr Met Gly Leu Thr Pro Leu Ala Gly
Leu Val Met Gly Thr Arg225 230 235
240Cys Gly Asp Ile Asp Pro Ala Ile Ile Pro Phe Val Met Lys Arg
Thr 245 250 255Gly Met Ser
Val Asp Glu Met Asp Thr Leu Met Asn Lys Lys Ser Gly 260
265 270Ile Leu Gly Val Ser Gly Val Ser Ser Asp
Phe Arg Asp Val Glu Glu 275 280
285Ala Ala Asn Ser Gly Asn Asp Arg Ala Lys Leu Ala Leu Asn Met Tyr 290
295 300Tyr His Lys Val Lys Ser Phe Ile
Gly Ala Tyr Val Ala Val Leu Asn305 310
315 320Gly Ala Asp Ala Ile Ile Phe Thr Ala Gly Leu Gly
Glu Asn Ser Ala 325 330
335Thr Ser Arg Ser Ala Ile Cys Lys Gly Leu Ser Tyr Phe Gly Ile Lys
340 345 350Ile Asp Glu Glu Lys Asn
Lys Lys Arg Gly Glu Ala Leu Glu Ile Ser 355 360
365Thr Pro Asp Ser Lys Ile Lys Val Leu Val Ile Pro Thr Asn
Glu Glu 370 375 380Leu Met Ile Ala Arg
Asp Thr Lys Glu Ile Val Glu Asn Lys385 390
3951151824DNAC. ragsdalei 115atgtacggat ataatggtaa ggtattaaga attaatctaa
gtagtaaaac ttatatagtg 60gaagaattga aaattgacaa agctaaaaaa tttataggtg
caagaggttt aggcgtaaaa 120accttatttg acgaagtaga tccaaaggta gatccattat
cacctgataa caaatttatt 180atagcagcgg gaccacttac aggtgcgcct gttccaacaa
gcggaagatt catggtagtt 240actaaatcac ctttaacagg aactattgct attgcaaatt
caggtggaaa atggggagca 300gaattcaaag cagctggata cgatatgata atcgttgaag
gtaaatctga taaagaagtt 360tatgtaaata tagtagatga taaagtagaa tttagggatg
cttctcatgt ttggggaaaa 420ctaacagaag aaactacaaa aatgcttcaa caggaaacag
attcgagagc taaggtttta 480tgcataggac cagctgggga aaaattatca cttatggcag
cagttatgaa tgatgttgat 540agaacagcag gacgtggtgg tgttggagct gttatgggct
caaagaactt aaaagctatt 600gtagttaaag gaagcggaaa agtaaaatta tttgatgagc
aaaaagtgaa agaagtagca 660cttgagaaaa caaatatttt aagaaaagat ccagtagctg
gtggaggact tccaacatac 720ggaacagctg tacttgttaa tattataaat gaaaatggcg
tacatccagt aaaaaatttc 780caaaaatctt atacagatca ggcagataag atcagtggag
aaactttaac taaagattgc 840ttagttagaa aaaatccttg ctataggtgt ccaattgcct
gtggaagatg ggtaaaactt 900gatgatggaa ctgaatgtgg aggaccagaa tatgaaacat
tatggtcatt tggatctgat 960tgtgatgtat acgatataaa tgctgtaaat acagcaaata
tgttgtgtaa tgaatatgga 1020ttagatacca ttacagcagg atgtactatt gcagcagcta
tggaacttta tcaaagaggt 1080tatattaagg atgaagaaat agcagcagat ggattgtcac
ttaattgggg agatgctaag 1140tccatggttg aatgggtaaa gaaaatggga cttagagaag
gatttggaga caagatggca 1200gatggttcat acagactttg tgactcatac ggtgtacctg
agtattcaat gactgtaaaa 1260aaacaagaaa tcccagcata tgacccaaga ggaatacagg
gacatggtat aacttatgct 1320gttaacaata ggggagggtg tcatattaag ggatatatgg
taagccctga aatacttggt 1380tatccagaaa aacttgatag acttgcagtg gaaggaaaag
caggatatgc tagagtattc 1440catgatttaa cagctgttat agattcactt ggattatgta
tttttacaac atttggtctt 1500ggtgcacagg attatgttga tttgtataat gcagtagttg
gtggagaatt acatgatgta 1560gactctttaa tgttagctgg agatagaata tggactttag
aaaaaatatt taacttaaag 1620gcaggcatag atagttcaca ggatactctt ccaaagagat
tgcttgagga accagttcca 1680gaaggaccat caaaaggaga gattcataga ttagatgtac
ttcttcctga atattattca 1740gtacgtggat gggataaaaa tggtatacct acagaggaaa
cgttaaagaa attaggatta 1800gatgaatatg taggtaagtt ttaa
1824116607PRTC. ragsdalei 116Met Tyr Gly Tyr Asn
Gly Lys Val Leu Arg Ile Asn Leu Ser Ser Lys1 5
10 15Thr Tyr Ile Val Glu Glu Leu Lys Ile Asp Lys
Ala Lys Lys Phe Ile 20 25
30Gly Ala Arg Gly Leu Gly Val Lys Thr Leu Phe Asp Glu Val Asp Pro
35 40 45Lys Val Asp Pro Leu Ser Pro Asp
Asn Lys Phe Ile Ile Ala Ala Gly 50 55
60Pro Leu Thr Gly Ala Pro Val Pro Thr Ser Gly Arg Phe Met Val Val65
70 75 80Thr Lys Ser Pro Leu
Thr Gly Thr Ile Ala Ile Ala Asn Ser Gly Gly 85
90 95Lys Trp Gly Ala Glu Phe Lys Ala Ala Gly Tyr
Asp Met Ile Ile Val 100 105
110Glu Gly Lys Ser Asp Lys Glu Val Tyr Val Asn Ile Val Asp Asp Lys
115 120 125Val Glu Phe Arg Asp Ala Ser
His Val Trp Gly Lys Leu Thr Glu Glu 130 135
140Thr Thr Lys Met Leu Gln Gln Glu Thr Asp Ser Arg Ala Lys Val
Leu145 150 155 160Cys Ile
Gly Pro Ala Gly Glu Lys Leu Ser Leu Met Ala Ala Val Met
165 170 175Asn Asp Val Asp Arg Thr Ala
Gly Arg Gly Gly Val Gly Ala Val Met 180 185
190Gly Ser Lys Asn Leu Lys Ala Ile Val Val Lys Gly Ser Gly
Lys Val 195 200 205Lys Leu Phe Asp
Glu Gln Lys Val Lys Glu Val Ala Leu Glu Lys Thr 210
215 220Asn Ile Leu Arg Lys Asp Pro Val Ala Gly Gly Gly
Leu Pro Thr Tyr225 230 235
240Gly Thr Ala Val Leu Val Asn Ile Ile Asn Glu Asn Gly Val His Pro
245 250 255Val Lys Asn Phe Gln
Lys Ser Tyr Thr Asp Gln Ala Asp Lys Ile Ser 260
265 270Gly Glu Thr Leu Thr Lys Asp Cys Leu Val Arg Lys
Asn Pro Cys Tyr 275 280 285Arg Cys
Pro Ile Ala Cys Gly Arg Trp Val Lys Leu Asp Asp Gly Thr 290
295 300Glu Cys Gly Gly Pro Glu Tyr Glu Thr Leu Trp
Ser Phe Gly Ser Asp305 310 315
320Cys Asp Val Tyr Asp Ile Asn Ala Val Asn Thr Ala Asn Met Leu Cys
325 330 335Asn Glu Tyr Gly
Leu Asp Thr Ile Thr Ala Gly Cys Thr Ile Ala Ala 340
345 350Ala Met Glu Leu Tyr Gln Arg Gly Tyr Ile Lys
Asp Glu Glu Ile Ala 355 360 365Ala
Asp Gly Leu Ser Leu Asn Trp Gly Asp Ala Lys Ser Met Val Glu 370
375 380Trp Val Lys Lys Met Gly Leu Arg Glu Gly
Phe Gly Asp Lys Met Ala385 390 395
400Asp Gly Ser Tyr Arg Leu Cys Asp Ser Tyr Gly Val Pro Glu Tyr
Ser 405 410 415Met Thr Val
Lys Lys Gln Glu Ile Pro Ala Tyr Asp Pro Arg Gly Ile 420
425 430Gln Gly His Gly Ile Thr Tyr Ala Val Asn
Asn Arg Gly Gly Cys His 435 440
445Ile Lys Gly Tyr Met Val Ser Pro Glu Ile Leu Gly Tyr Pro Glu Lys 450
455 460Leu Asp Arg Leu Ala Val Glu Gly
Lys Ala Gly Tyr Ala Arg Val Phe465 470
475 480His Asp Leu Thr Ala Val Ile Asp Ser Leu Gly Leu
Cys Ile Phe Thr 485 490
495Thr Phe Gly Leu Gly Ala Gln Asp Tyr Val Asp Leu Tyr Asn Ala Val
500 505 510Val Gly Gly Glu Leu His
Asp Val Asp Ser Leu Met Leu Ala Gly Asp 515 520
525Arg Ile Trp Thr Leu Glu Lys Ile Phe Asn Leu Lys Ala Gly
Ile Asp 530 535 540Ser Ser Gln Asp Thr
Leu Pro Lys Arg Leu Leu Glu Glu Pro Val Pro545 550
555 560Glu Gly Pro Ser Lys Gly Glu Ile His Arg
Leu Asp Val Leu Leu Pro 565 570
575Glu Tyr Tyr Ser Val Arg Gly Trp Asp Lys Asn Gly Ile Pro Thr Glu
580 585 590Glu Thr Leu Lys Lys
Leu Gly Leu Asp Glu Tyr Val Gly Lys Phe 595 600
6051171824DNAC. ragsdalei 117atgtatggtt ataatggtaa
agtattaaga attaatttaa aagaaagaac ttgcaaatca 60gaaaatttag atttagataa
agctaaaaag tttataggct gtaggggact aggtgttaaa 120actttatttg atgaaataga
tcctaaaata gatgcattat caccagaaaa taaatttata 180attgtaacag gtccgttaac
tggagctcca gttccaacta gtggaaggtt tatggtagtt 240actaaagcac cgcttacagg
aactatagga atttcaaatt cgggtggaaa atggggagta 300gacttgaaaa aagctggctg
ggatatgata atagtagagg ataaggctga ttcaccagtt 360tacattgaaa tagtagatga
taaagtagaa attaaagatg cgtcacagct ttggggaaaa 420gttacatcag aaactacaaa
agagttagaa aagataactg agaatagatc aaaggtatta 480tgtataggac ctgctggtga
aagattgtcc cttatggcag cagttatgaa tgatgtagat 540agaactgcag caagaggcgg
cgttggtgca gttatgggat ctaaaaactt aaaagctatt 600acagttaaag gaactggaaa
aatagcttta gctgataaag aaaaagtaaa aaaagtgtcc 660gtagaaaaaa ttacaacatt
aaaaaatgat ccagtagctg gtcagggaat gccaacttat 720ggtacagcta tactggttaa
tataataaat gaaaatggag ttcatcctgt aaataatttt 780caagaatctt atacggatca
agcagataaa ataagtggag agactcttac tgctaaccaa 840ctagtaagga aaaatccttg
ttacagctgt cctataggtt gtggaagatg ggttagacta 900aaagatggta cagagtgcgg
aggaccggag tatgaaacac tgtggtgttt tggctctgac 960tgtggttcat atgatttaga
tgctataaat gaagctaata tgttatgtaa tgaatatggt 1020attgatacta ttacctgtgg
tgcaacaatt gctgcagcta tggaacttta tcaaagagga 1080tatgtaaaag atgaagaaat
agccggagat aacctatctc tcaagtgggg agatacggag 1140tctatgattg gctggataaa
gaaaatggta tatagtgaag gctttggagc aaagatgaca 1200aatggttcat ataggctttg
tgaaggttat ggagtacctg agtattctat gacagttaaa 1260aagcaagaaa ttccagcata
tgatccaagg ggaatacagg gacatggtat tacctatgca 1320gttaataata gaggaggatg
tcatattaag ggatatatga ttaatcctga aatattaggt 1380tatccggaaa aacttgatag
atttgcatta gatggtaaag cagcctatgc caaaatgatg 1440catgatttaa ctgctgtaat
tgattcttta ggattgtgca tattcactac atttgggctt 1500ggaatacagg attatgtaga
tatgtataat gcagtagtag gagaatctac ttgtgattca 1560gattcactat tagaggcagg
agatagagta tggactcttg aaaaattatt taatcttgca 1620gctggaatag acagcagcca
ggatactcta ccaaagagat tgttagaaga acctattcca 1680gatggtccat caaagggaca
cgttcatagg ctagatgttc ttctgccaga atattactca 1740gtacgaggat ggagtaaaga
gggtatacct acagaagaaa cattaaagaa attaggatta 1800gatgaatata taggtaagtt
ctag 1824118607PRTC. ragsdalei
118Met Tyr Gly Tyr Asn Gly Lys Val Leu Arg Ile Asn Leu Lys Glu Arg1
5 10 15Thr Cys Lys Ser Glu Asn
Leu Asp Leu Asp Lys Ala Lys Lys Phe Ile 20 25
30Gly Cys Arg Gly Leu Gly Val Lys Thr Leu Phe Asp Glu
Ile Asp Pro 35 40 45Lys Ile Asp
Ala Leu Ser Pro Glu Asn Lys Phe Ile Ile Val Thr Gly 50
55 60Pro Leu Thr Gly Ala Pro Val Pro Thr Ser Gly Arg
Phe Met Val Val65 70 75
80Thr Lys Ala Pro Leu Thr Gly Thr Ile Gly Ile Ser Asn Ser Gly Gly
85 90 95Lys Trp Gly Val Asp Leu
Lys Lys Ala Gly Trp Asp Met Ile Ile Val 100
105 110Glu Asp Lys Ala Asp Ser Pro Val Tyr Ile Glu Ile
Val Asp Asp Lys 115 120 125Val Glu
Ile Lys Asp Ala Ser Gln Leu Trp Gly Lys Val Thr Ser Glu 130
135 140Thr Thr Lys Glu Leu Glu Lys Ile Thr Glu Asn
Arg Ser Lys Val Leu145 150 155
160Cys Ile Gly Pro Ala Gly Glu Arg Leu Ser Leu Met Ala Ala Val Met
165 170 175Asn Asp Val Asp
Arg Thr Ala Ala Arg Gly Gly Val Gly Ala Val Met 180
185 190Gly Ser Lys Asn Leu Lys Ala Ile Thr Val Lys
Gly Thr Gly Lys Ile 195 200 205Ala
Leu Ala Asp Lys Glu Lys Val Lys Lys Val Ser Val Glu Lys Ile 210
215 220Thr Thr Leu Lys Asn Asp Pro Val Ala Gly
Gln Gly Met Pro Thr Tyr225 230 235
240Gly Thr Ala Ile Leu Val Asn Ile Ile Asn Glu Asn Gly Val His
Pro 245 250 255Val Asn Asn
Phe Gln Glu Ser Tyr Thr Asp Gln Ala Asp Lys Ile Ser 260
265 270Gly Glu Thr Leu Thr Ala Asn Gln Leu Val
Arg Lys Asn Pro Cys Tyr 275 280
285Ser Cys Pro Ile Gly Cys Gly Arg Trp Val Arg Leu Lys Asp Gly Thr 290
295 300Glu Cys Gly Gly Pro Glu Tyr Glu
Thr Leu Trp Cys Phe Gly Ser Asp305 310
315 320Cys Gly Ser Tyr Asp Leu Asp Ala Ile Asn Glu Ala
Asn Met Leu Cys 325 330
335Asn Glu Tyr Gly Ile Asp Thr Ile Thr Cys Gly Ala Thr Ile Ala Ala
340 345 350Ala Met Glu Leu Tyr Gln
Arg Gly Tyr Val Lys Asp Glu Glu Ile Ala 355 360
365Gly Asp Asn Leu Ser Leu Lys Trp Gly Asp Thr Glu Ser Met
Ile Gly 370 375 380Trp Ile Lys Lys Met
Val Tyr Ser Glu Gly Phe Gly Ala Lys Met Thr385 390
395 400Asn Gly Ser Tyr Arg Leu Cys Glu Gly Tyr
Gly Val Pro Glu Tyr Ser 405 410
415Met Thr Val Lys Lys Gln Glu Ile Pro Ala Tyr Asp Pro Arg Gly Ile
420 425 430Gln Gly His Gly Ile
Thr Tyr Ala Val Asn Asn Arg Gly Gly Cys His 435
440 445Ile Lys Gly Tyr Met Ile Asn Pro Glu Ile Leu Gly
Tyr Pro Glu Lys 450 455 460Leu Asp Arg
Phe Ala Leu Asp Gly Lys Ala Ala Tyr Ala Lys Met Met465
470 475 480His Asp Leu Thr Ala Val Ile
Asp Ser Leu Gly Leu Cys Ile Phe Thr 485
490 495Thr Phe Gly Leu Gly Ile Gln Asp Tyr Val Asp Met
Tyr Asn Ala Val 500 505 510Val
Gly Glu Ser Thr Cys Asp Ser Asp Ser Leu Leu Glu Ala Gly Asp 515
520 525Arg Val Trp Thr Leu Glu Lys Leu Phe
Asn Leu Ala Ala Gly Ile Asp 530 535
540Ser Ser Gln Asp Thr Leu Pro Lys Arg Leu Leu Glu Glu Pro Ile Pro545
550 555 560Asp Gly Pro Ser
Lys Gly His Val His Arg Leu Asp Val Leu Leu Pro 565
570 575Glu Tyr Tyr Ser Val Arg Gly Trp Ser Lys
Glu Gly Ile Pro Thr Glu 580 585
590Glu Thr Leu Lys Lys Leu Gly Leu Asp Glu Tyr Ile Gly Lys Phe
595 600 6051191167DNAC. autoethanogenum
119atggcaagat ttactttacc aagagacatt tattttggag aaaattcatt agaaaccttg
60aaagacctag atggaaaaaa agctgttatt gtcgtaggtg gtggatccat gaaacgattt
120ggattccttg ataaggtagt aaactactta aaagaagcag gtattgaatc aaaattaata
180gaaggagttg aaccagatcc atctgtagaa actgttatga atggcgctaa actaatgaga
240gaatatgaac cagatttaat agtatcaata ggtggaggtt caccaattga cgcagcaaaa
300gctatgtgga tattctatga ataccctgag tttactttta aagaggctgt ggttcctttt
360ggtcttccta aattaagaca aaaagcaaca tttatagcta taccttctac aagtggtact
420gcaacagaag taacggcatt ttctgtaata acagactata aagctaaaat taaatatcct
480ttagctgact tcaatttaac accagatata gctataattg atccagcatt agctcaaaca
540atgccaccta aattaactgc acatactgga atggatgcac ttacccatgc tattgaagca
600tatgttgcag gacttcattc agttttctca gatcctcttg ctattcaagc tatagttatg
660gtaaatcagt atttaattaa atcttacaat gaagataaag aagctagaaa ccaaatgcat
720ttagctcaat gtttagctgg aatggcattt tcaaatgcac ttcttggaat aactcacagt
780ttagcacata aaacaggtgc agtattccat attcctcatg gatgtgccaa tgcaatatat
840cttccctatg ttatagattt caataaaaaa gcttgtacac caagatatgc tgatatagct
900aggagtctta aacttccagg aaatactgat gatgaattag tagattcatt aactaacatg
960attaaagata tgaacaagag tatggatatt cctttgacat taaaagatta cggagtagat
1020gaaaaagaat ttaaagataa tgaagatttt atagctcata atgccgtatt agatgcctgc
1080actggatcaa atcctagaag tataaatgat gctgaaatga aaaaattgtt agaatacatc
1140tattatggta aaaaggttga tttttaa
1167120388PRTC. autoethanogenum 120Met Ala Arg Phe Thr Leu Pro Arg Asp
Ile Tyr Phe Gly Glu Asn Ser1 5 10
15Leu Glu Thr Leu Lys Asp Leu Asp Gly Lys Lys Ala Val Ile Val
Val 20 25 30Gly Gly Gly Ser
Met Lys Arg Phe Gly Phe Leu Asp Lys Val Val Asn 35
40 45Tyr Leu Lys Glu Ala Gly Ile Glu Ser Lys Leu Ile
Glu Gly Val Glu 50 55 60Pro Asp Pro
Ser Val Glu Thr Val Met Asn Gly Ala Lys Leu Met Arg65 70
75 80Glu Tyr Glu Pro Asp Leu Ile Val
Ser Ile Gly Gly Gly Ser Pro Ile 85 90
95Asp Ala Ala Lys Ala Met Trp Ile Phe Tyr Glu Tyr Pro Glu
Phe Thr 100 105 110Phe Lys Glu
Ala Val Val Pro Phe Gly Leu Pro Lys Leu Arg Gln Lys 115
120 125Ala Thr Phe Ile Ala Ile Pro Ser Thr Ser Gly
Thr Ala Thr Glu Val 130 135 140Thr Ala
Phe Ser Val Ile Thr Asp Tyr Lys Ala Lys Ile Lys Tyr Pro145
150 155 160Leu Ala Asp Phe Asn Leu Thr
Pro Asp Ile Ala Ile Ile Asp Pro Ala 165
170 175Leu Ala Gln Thr Met Pro Pro Lys Leu Thr Ala His
Thr Gly Met Asp 180 185 190Ala
Leu Thr His Ala Ile Glu Ala Tyr Val Ala Gly Leu His Ser Val 195
200 205Phe Ser Asp Pro Leu Ala Ile Gln Ala
Ile Val Met Val Asn Gln Tyr 210 215
220Leu Ile Lys Ser Tyr Asn Glu Asp Lys Glu Ala Arg Asn Gln Met His225
230 235 240Leu Ala Gln Cys
Leu Ala Gly Met Ala Phe Ser Asn Ala Leu Leu Gly 245
250 255Ile Thr His Ser Leu Ala His Lys Thr Gly
Ala Val Phe His Ile Pro 260 265
270His Gly Cys Ala Asn Ala Ile Tyr Leu Pro Tyr Val Ile Asp Phe Asn
275 280 285Lys Lys Ala Cys Thr Pro Arg
Tyr Ala Asp Ile Ala Arg Ser Leu Lys 290 295
300Leu Pro Gly Asn Thr Asp Asp Glu Leu Val Asp Ser Leu Thr Asn
Met305 310 315 320Ile Lys
Asp Met Asn Lys Ser Met Asp Ile Pro Leu Thr Leu Lys Asp
325 330 335Tyr Gly Val Asp Glu Lys Glu
Phe Lys Asp Asn Glu Asp Phe Ile Ala 340 345
350His Asn Ala Val Leu Asp Ala Cys Thr Gly Ser Asn Pro Arg
Ser Ile 355 360 365Asn Asp Ala Glu
Met Lys Lys Leu Leu Glu Tyr Ile Tyr Tyr Gly Lys 370
375 380Lys Val Asp Phe3851211167DNAC. autoethanogenum
121atgggaagat ttactttgcc tagggatatt tactttggtg aaaatgcctt agaaaattta
60aaaaatttag atggaaataa agcagtagtt gttgtaggtg ggggatctat gaagagattt
120ggattcttag ccaaagttga aaaatactta aaagaaactg gtatggaagt taaattaata
180gaaggtgttg agcctgatcc gtctgttgat actgttatga atggcgctaa aataatgaga
240gactttaacc cagattggat agtatcaata ggtggaggat ctcccataga tgctgctaaa
300gcaatgtgga tattttatga ataccccgac tttacatttg aaaaagcggt agtccctttt
360ggaattccta aattaaggca gaaggcacaa tttgttgcta taccttctac aagtggaaca
420gcaactgaag taacatcatt ttctgtaata acagactata aagctaaaat aaaatatcct
480cttgcagatt ttaaccttac ccctgatata gctataatag atccgtctct tgcagaaaca
540atgcccaaaa agcttacagc acacactgga atggatgcac ttactcacgc aatagaagca
600tatgtagcaa gtttacattc agatttctca gatccacttg ctatgcatgc tataaccatg
660attcataaat atttattgaa atcctatgaa gaagataaag aagctagagg acatatgcat
720atagcccaat gtctagctgg gatggcattt tcaaatgctc tccttggaat aactcatagt
780atagcacata aaactggtgc agtatttcac atacctcatg ggtgtgctaa tgccatatac
840ttaccttatg ttatagattt taacaagaaa gcttgttcag aaagatatgc taaaatagcc
900aaaaagctgc atctatcagg aaatagtgaa gatgagctaa tagattcatt aactgaaatg
960attcgtacta tgaacaaaaa gatggatatt cctctcacca taaaagatta tggtataagc
1020gaaaacgatt ttaatgaaaa cctagatttt atagctcaca atgccatgat ggatgcctgc
1080actggatcca atcctagagc aataactgag gaagaaatga aaaagctctt gcagtatatg
1140tataatgggc aaaaggttaa tttctag
1167122388PRTC. autoethanogenum 122Met Gly Arg Phe Thr Leu Pro Arg Asp
Ile Tyr Phe Gly Glu Asn Ala1 5 10
15Leu Glu Asn Leu Lys Asn Leu Asp Gly Asn Lys Ala Val Val Val
Val 20 25 30Gly Gly Gly Ser
Met Lys Arg Phe Gly Phe Leu Ala Lys Val Glu Lys 35
40 45Tyr Leu Lys Glu Thr Gly Met Glu Val Lys Leu Ile
Glu Gly Val Glu 50 55 60Pro Asp Pro
Ser Val Asp Thr Val Met Asn Gly Ala Lys Ile Met Arg65 70
75 80Asp Phe Asn Pro Asp Trp Ile Val
Ser Ile Gly Gly Gly Ser Pro Ile 85 90
95Asp Ala Ala Lys Ala Met Trp Ile Phe Tyr Glu Tyr Pro Asp
Phe Thr 100 105 110Phe Glu Lys
Ala Val Val Pro Phe Gly Ile Pro Lys Leu Arg Gln Lys 115
120 125Ala Gln Phe Val Ala Ile Pro Ser Thr Ser Gly
Thr Ala Thr Glu Val 130 135 140Thr Ser
Phe Ser Val Ile Thr Asp Tyr Lys Ala Lys Ile Lys Tyr Pro145
150 155 160Leu Ala Asp Phe Asn Leu Thr
Pro Asp Ile Ala Ile Ile Asp Pro Ser 165
170 175Leu Ala Glu Thr Met Pro Lys Lys Leu Thr Ala His
Thr Gly Met Asp 180 185 190Ala
Leu Thr His Ala Ile Glu Ala Tyr Val Ala Ser Leu His Ser Asp 195
200 205Phe Ser Asp Pro Leu Ala Met His Ala
Ile Thr Met Ile His Lys Tyr 210 215
220Leu Leu Lys Ser Tyr Glu Glu Asp Lys Glu Ala Arg Gly His Met His225
230 235 240Ile Ala Gln Cys
Leu Ala Gly Met Ala Phe Ser Asn Ala Leu Leu Gly 245
250 255Ile Thr His Ser Ile Ala His Lys Thr Gly
Ala Val Phe His Ile Pro 260 265
270His Gly Cys Ala Asn Ala Ile Tyr Leu Pro Tyr Val Ile Asp Phe Asn
275 280 285Lys Lys Ala Cys Ser Glu Arg
Tyr Ala Lys Ile Ala Lys Lys Leu His 290 295
300Leu Ser Gly Asn Ser Glu Asp Glu Leu Ile Asp Ser Leu Thr Glu
Met305 310 315 320Ile Arg
Thr Met Asn Lys Lys Met Asp Ile Pro Leu Thr Ile Lys Asp
325 330 335Tyr Gly Ile Ser Glu Asn Asp
Phe Asn Glu Asn Leu Asp Phe Ile Ala 340 345
350His Asn Ala Met Met Asp Ala Cys Thr Gly Ser Asn Pro Arg
Ala Ile 355 360 365Thr Glu Glu Glu
Met Lys Lys Leu Leu Gln Tyr Met Tyr Asn Gly Gln 370
375 380Lys Val Asn Phe38512325DNAArtificial
sequencesynthetic primer 123ttgatgaaat gatcactgac ggatt
2512425DNAArtificial sequencesynthetic primer
124gaaatgttcc atctctcagc tatgt
2512525DNAArtificial sequencesynthetic primer 125catcactttc aataacagaa
gtggc 2512625DNAArtificial
sequencesynthetic primer 126tacctctaca agcttcataa cagga
2512725DNAArtificial sequencesynthetic primer
127aaaatgggtc agtatggtat gatgg
2512825DNAArtificial sequencesynthetic primer 128tgtagtaccg caaacctttg
ataat 2512925DNAArtificial
sequencesynthetic primer 129caagtttact tggtggaaca atagc
2513025DNAArtificial sequencesynthetic primerr
130gagttggtct tacagtttta ccagt
2513120DNAArtificial sequencesynthetic primer 131tcaggacctt ctggaactgg
2013220DNAArtificial
sequencesynthetic primer 132acctcccctt ttcttggaga
2013320DNAArtificial sequencesynthetic primer
133caggtttcgg tgctgaccta
2013420DNAArtificial sequencesynthetic primer 134aactccgccg ttgtatttca
2013537DNAArtificial
sequencesynthetic primerr 135ccgaattcgt cgacaacaga gtttgatcct ggctcag
37
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