PREPARATION OF 6-AMINOCAPROIC ACID FROM 5-FORMYL VALERIC ACID

Abstract

The invention relates to a method for preparing 6-aminocaproic acid (hereinafter also referred to as `6-ACA`) using a biocatalyst. The invention further relates to a method for preparing -caprolactam (hereafter referred to as `caprolactam`) by cyclising such 6-ACA. The invention further relates to a host cell, a micro-organism, or a polynucleotide which may be used in the preparation of 6-ACA or caprolactam.

Description

[0001] This application is a divisional of U.S. patent application Ser. No. 14,105,705, filed Dec. 13, 2013, which is a continuation of U.S. patent application Ser. No. 12/921,733, filed Dec. 21, 2010, now issued U.S. Pat. No. 8,673,599, issued Mar. 18, 2014, which is a U.S. National Stage Application under 35 U.S.C. .sctn. 371 of International Patent Application No. PCT/NL2009/050117, filed Mar. 11, 2009, which claims the benefit of European Patent Application No. 08152584.2, filed Mar. 11, 2008, the entire contents of which are each incorporated herein by reference.

[0002] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 17, 2017, is named Sequence_Listing_12956-416-999.txt and is 249,748 bytes in size.

[0003] The invention relates to a method for preparing 6-aminocaproic acid (hereinafter also referred to as `6-ACA`). The invention further relates to a method for preparing -caprolactam (hereafter referred to as `caprolactam`) from 6-ACA. The invention further relates to a host cell which may be used in the preparation of 6-ACA or caprolactam.

[0004] Caprolactam is a lactam which may be used for the production of polyamide, for instance nylon-6 or nylon-6,12 (a copolymer of caprolactam and laurolactam). Various manners of preparing caprolactam from bulk chemicals are known in the art and include the preparation of caprolactam from cyclohexanone, toluene, phenol, cyclohexanol, benzene or cyclohexane. These intermediate compounds are generally obtained from mineral oil. In view of a growing desire to prepare materials using more sustainable technology it would be desirable to provide a method wherein caprolactam is prepared from an intermediate compound that can be obtained from a biologically renewable source or at least from an intermediate compound that is converted into caprolactam using a biochemical method. Further, it would be desirable to provide a method that requires less energy than conventional chemical processes making use of bulk chemicals from petrochemical origin.

[0005] It is known to prepare caprolactam from 6-ACA, e.g. as described in U.S. Pat. No. 6,194,572. As disclosed in WO 2005/068643, 6-ACA may be prepared biochemically by converting 6-aminohex-2-enoic acid (6-AHEA) in the presence of an enzyme having .alpha.,.beta.-enoate reductase activity. The 6-AHEA may be prepared from lysine, e.g. biochemically or by pure chemical synthesis. Although the preparation of 6-ACA via the reduction of 6-AHEA is feasible by the methods disclosed in WO 2005/068643, the inventors have found that--under the reduction reaction conditions--6-AHEA may spontaneously and substantially irreversibly cyclise to form an undesired side-product, notably .beta.-homoproline. This cyclisation may be a bottleneck in the production of 6-ACA, and may lead to a considerable loss in yield.

[0006] It is an object of the invention to provide a novel method for preparing 6-ACA or caprolactam--which may, inter alia, be used for the preparation of polyamide--or an intermediate compound for the preparation of 6-ACA or caprolactam, that can serve as an alternative for known methods.

[0007] It is a further object to provide a novel method that would overcome one or more of the drawbacks mentioned above.

[0008] One or more further objects which may be solved in accordance with the invention, will follow from the description, below.

[0009] It has now been found possible to prepare 6-ACA from a specific starting compound, namely it has been found possible to prepare 6-aminocaproic acid (6-ACA), wherein the 6-aminocaproic acid is prepared from 2-oxo-heptanedioic acid also known as .alpha.-ketopimelic acid (AKP). In particular, the preparation may be carried out in two or more reaction steps. For instance, a method is provided wherein AKP is first converted into 5-formylpentanoate (5-formylvaleric acid, 5-FVA), which 5-FVA is converted into 6-ACA. Further a method is provided wherein AKP is first converted into alpha-aminopimelic acid (AAP). Thereafter, AAP is converted into 6-ACA.

[0010] The inventors realised that in principle, it is possible to prepare 6-ACA from AKP in an entirely chemical (i.e. without the use of a biocatalyst) manner. Examples of suitable chemical ways of carrying out individual reaction steps are given herein below. However, the inventors also realised that it is possible to prepare 6-ACA biochemically from AKP.

[0011] Accordingly, the present invention in particular relates to a method for preparing 6-ACA, wherein the 6-ACA is prepared from AKP, using at least one biocatalyst.

[0012] The invention further relates to a method, wherein 6-ACA is prepared from 5-formylpentanoate (5-formylvaleric acid, 5-FVA), using a biocatalyst. As indicated above, the 5-FVA may be obtained from AKP.

[0013] In an embodiment, 6-ACA prepared in a method of the invention is used for preparing caprolactam. Such method comprises cyclising the 6-amino-caproic acid, optionally in the presence of a biocatalyst.

[0014] When referring herein to carboxylic acids or carboxylates, e.g. 6-ACA, 2-aminoheptanedioic acid (.alpha.-aminopimelic acid, herein after abbreviated as `AAP`), another amino acid, 5-FVA or AKP, these terms are meant to include the protonated carboxylic acid group (i.e. the neutral group), their corresponding carboxylate (their conjugated bases) as well as salts thereof. When referring herein to amino acids, e.g. 6-ACA, this term is meant to include amino acids in their zwitterionic form (in which the amino group is in the protonated and the carboxylate group is in the deprotonated form), the amino acid in which the amino group is protonated and the carboxylic group is in its neutral form, and the amino acid in which the amino group is in its neutral form and the carboxylate group is in the deprotonated form, as well as salts thereof.

[0015] In accordance with the invention, no problems have been noticed with respect to an undesired cyclisation of an intermediate product, when forming 6-ACA and optionally caprolactam, resulting in a loss of yield.

[0016] It is envisaged that a method of the invention allows a comparable or even better yield than the method described in WO 2005/68643. It is envisaged that a method of the invention may in particular be favourable if a use is made of a living organism--in particular in a method wherein growth and maintenance of the organism is taken into account.

[0017] It is further envisaged that in an embodiment of the invention the productivity of 6-ACA (g/l.h formed) in a method of the invention may be improved.

[0018] The term "or" as used herein is defined as "and/or" unless specified otherwise.

[0019] The term "a" or "an" as used herein is defined as "at least one" unless specified otherwise.

[0020] When referring to a noun (e.g. a compound, an additive, etc.) in the singular, the plural is meant to be included.

[0021] When referring to a compound of which stereoisomers exist, the compound may be any of such stereoisomers or a combination thereof. Thus, when referred to, e.g., an amino acid of which enantiomers exist, the amino acid may be the L-enantiomer, the D-enantiomer or a combination thereof. In case a natural stereoisomer exists, the compound is preferably a natural stereoisomer.

[0022] When an enzyme is mentioned with reference to an enzyme class (EC) between brackets, the enzyme class is a class wherein the enzyme is classified or may be classified, on the basis of the Enzyme Nomenclature provided by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), which nomenclature may be found at http://www.chem.qmul.ac.uk/iubmb/enzyme/. Other suitable enzymes that have not (yet) been classified in a specified class but may be classified as such, are meant to be included.

[0023] The term "homologue" is used herein in particular for polynucleotides or polypeptides having a sequence identity of at least 30%, preferably at least 40%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, in particular at least 85%, more in particular at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. The term homologue is also meant to include nucleic acid sequences (polynucleotide sequences) which differ from another nucleic acid sequence due to the degeneracy of the genetic code and encode the same polypeptide sequence.

[0024] Sequence identity or similarity is herein defined as a relationship between two or more polypeptide sequences or two or more nucleic acid sequences, as determined by comparing the sequences. Usually, sequence identities or similarities are compared over the whole length of the sequences, but may however also be compared only for a part of the sequences aligning with each other. In the art, "identity" or "similarity" also means the degree of sequence relatedness between polypeptide sequences or nucleic acid sequences, as the case may be, as determined by the match between such sequences. Preferred methods to determine identity or similarity are designed to give the largest match between the sequences tested. In context of this invention a preferred computer program method to determine identity and similarity between two sequences includes BLASTP and BLASTN (Altschul, S. F. et al., J. Mol. Biol. 1990, 215, 403-410, publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894). Preferred parameters for polypeptide sequence comparison using BLASTP are gap open 10.0, gap extend 0.5, Blosum 62 matrix. Preferred parameters for nucleic acid sequence comparison using BLASTN are gap open 10.0, gap extend 0.5, DNA full matrix (DNA identity matrix).

[0025] In accordance with the invention, a biocatalyst is used, i.e. at least one reaction step in the method is catalysed by a biological material or moiety derived from a biological source, for instance an organism or a biomolecule derived there from. The biocatalyst may in particular comprise one or more enzymes. The biocatalyst may be used in any form. In an embodiment, one or more enzymes are used isolated from the natural environment (isolated from the organism it has been produced in), for instance as a solution, an emulsion, a dispersion, (a suspension of) freeze-dried cells, as a lysate, or immobilised on a support. In an embodiment, one or more enzymes form part of a living organism (such as living whole cells).

[0026] The enzymes may perform a catalytic function inside the cell. It is also possible that the enzyme may be secreted into a medium, wherein the cells are present.

[0027] Living cells may be growing cells, resting or dormant cells (e.g. spores) or cells in a stationary phase. It is also possible to use an enzyme forming part of a permeabilised cell (i.e. made permeable to a substrate for the enzyme or a precursor for a substrate for the enzyme or enzymes).

[0028] A biocatalyst used in a method of the invention may in principle be any organism, or be obtained or derived from any organism. The organism may be eukaryotic or prokaryotic. In particular the organism may be selected from animals (including humans), plants, bacteria, archaea, yeasts and fungi.

[0029] In an embodiment a biocatalyst originates from an animal, in particular from a part thereof--e.g. liver, pancreas, brain, kidney, heart or other organ. The animal may in particular be selected from the group of mammals, more in particular selected from the group of Leporidae, Muridae, Suidae and Bovidae.

[0030] Suitable plants in particular include plants selected from the group of Asplenium; Cucurbitaceae, in particular Curcurbita, e.g. Curcurbita moschata (squash), or Cucumis; Mercurialis, e.g. Mercurialis perennis; Hydnocarpus; and Ceratonia.

[0031] Suitable bacteria may in particular be selected amongst the group of Vibrio, Pseudomonas, Bacillus, Corynebacterium, Brevibacterium, Enterococcus, Streptococcus, Klebsiella, Lactococcus, Lactobacillus, Clostridium, Escherichia, Thermus, Mycobacterium, Zymomonas, Proteus, Agrobacterium, Geobacillus, Acinetobacter, Ralstonia, Rhodobacter, Paracoccus, Novosphingobium, Nitrosomonas, Legionella, Neisseria, Rhodopseudomonas, Staphylococcus, Deinococcus and Salmonella.

[0032] Suitable archaea may in particular be selected amongst the group of Archaeoglobus, Aeropyrum, Halobacterium, Methanosarcina, Methanococcus, Thermoplasma, Pyrobaculum, Methanocaldococcus, Methanobacterium, Methanosphaera, Methanopyrus and Methanobrevibacter.

[0033] Suitable fungi may in particular be selected amongst the group of Rhizopus, Neurospora, Penicillium and Aspergillus.

[0034] A suitable yeast may in particular be selected amongst the group of Candida, Hansenula, Kluyveromyces and Saccharomyces.

[0035] It will be clear to the person skilled in the art that use can be made of a naturally occurring biocatalyst (wild type) or a mutant of a naturally occurring biocatalyst with suitable activity in a method according to the invention. Properties of a naturally occurring biocatalyst may be improved by biological techniques known to the skilled person in the art, such as e.g. molecular evolution or rational design. Mutants of wild-type biocatalysts can for example be made by modifying the encoding DNA of an organism capable of acting as a biocatalyst or capable of producing a biocatalytic moiety (such as an enzyme) using mutagenesis techniques known to the person skilled in the art (random mutagenesis, site-directed mutagenesis, directed evolution, gene recombination, etc.). In particular the DNA may be modified such that it encodes an enzyme that differs by at least one amino acid from the wild-type enzyme, so that it encodes an enzyme that comprises one or more amino acid substitutions, deletions and/or insertions compared to the wild-type, or such that the mutants combine sequences of two or more parent enzymes or by effecting the expression of the thus modified DNA in a suitable (host) cell. The latter may be achieved by methods known to the skilled person in the art such as codon optimisation or codon pair optimisation, e.g. based on a method as described in WO 2008/000632.

[0036] A mutant biocatalyst may have improved properties, for instance with respect to one or more of the following aspects: selectivity towards the substrate, activity, stability, solvent tolerance, pH profile, temperature profile, substrate profile, susceptibility to inhibition, cofactor utilisation and substrate-affinity. Mutants with improved properties can be identified by applying e.g. suitable high through-put screening or selection methods based on such methods known to the skilled person in the art.

[0037] When referred to a biocatalyst, in particular an enzyme, from a particular source, recombinant biocatalysts, in particular enzymes, originating from a first organism, but actually produced in a (genetically modified) second organism, are specifically meant to be included as biocatalysts, in particular enzymes, from that first organism.

[0038] In a preferred method of the invention, the preparation comprises a biocatalytic (usually an enzymatic) reaction in the presence of a biocatalyst capable of catalysing the decarboxylation of an .alpha.-keto acid or an amino acid (i.e. a compound comprising at least one carboxylic acid group and at least one amino group). An enzyme having such catalytic activity may therefore be referred to as an .alpha.-keto acid decarboxylase respectively an amino acid decarboxylase.

[0039] Said acid preferably is a diacid, wherein the said biocatalyst is selective towards the acid group next to the keto- or amino-group.

[0040] In general, a suitable decarboxylase has .alpha.-ketopimelate decarboxylase activity, capable of catalysing the conversion of AKP into 5-FVA or .alpha.-aminopimelate decarboxylase activity, capable of catalysing the conversion of AAP to 6-ACA.

[0041] An enzyme capable of decarboxylating an .alpha.-keto acid or an amino acid may in particular be selected from the group of decarboxylases (E.C. 4.1.1), preferably from the group of oxaloacetate decarboxylases (EC 4.1.1.3), diaminopimelate decarboxylases (EC 4.1.1.20), branched chain .alpha.-keto acid decarboxylases (EC 4.1.1.72), .alpha.-ketoisovalerate decarboxylases, .alpha.-ketoglutarate decarboxylases (EC 4.1.1.71), and pyruvate decarboxylases (EC 4.1.1.1).

[0042] One or more other suitable decarboxylases may be selected amongst the group of oxalate decarboxylases (EC 4.1.1.2), acetoacetate decarboxylases (EC 4.1.1.4), valine decarboxylases/leucine decarboxylases (EC 4.1.1.14), glutamate decarboxylases (EC 4.1.1.15), aspartate 1-decarboxylases (EC 4.1.1.11), 3-hydroxyglutamate decarboxylases (EC 4.1.1.16), ornithine decarboxylases (EC 4.1.1.17), lysine decarboxylases (EC 4.1.1.18), arginine decarboxylases (EC 4.1.1.19), 2-oxoglutarate decarboxylases (EC 4.1.1.71), and diaminobutyrate decarboxylases (EC 4.1.1.86)

[0043] A decarboxylase may in particular be a decarboxylase of an organism selected from the group of squashes; cucumbers; yeasts; fungi, e.g. Saccharomyces cerevisiae, Candida flareri, Hansenula sp., Kluyveromyces marxianus, Rhizopus javanicus, and Neurospora crassa; mammals, in particular from mammalian brain; and bacteria, such as Escherichia coli, Lactococcus lactis, Mycobacterium tuberculosis, Pseudomonas sp. and Zymomonas mobilis.

[0044] The pyruvate decarboxylase may originate from Saccharomyces cerevisiae or Zymomonas mobilis. In particular, pyruvate decarboxylase mutant I472A from Zymomonas mobilis may be used.

[0045] Glutamate decarboxylase, diaminopimelate decarboxylase or aspartate decarboxylase from Escherichia coli (E. coli) may be used.

[0046] Glutamate decarboxylase from Neurospora crassa, Mycobacterium leprae, Clostridium perfringens, Lactobacillus brevis, Mycobacterium tuberculosis, Streptococcus or Lactococcus may be used. Examples of Lactococcus species from which the glutamate decarboxylase may originate in particular include Lactococcus lactis, such as Lactococcus lactis strain B1157, Lactococcus lactis IFPL730, more in particular Lactococcus lactis var. maltigenes (formerly named Streptococcus lactis var. maltigenes).

[0047] An oxaloacetate decarboxylase from Pseudomonas may in particular be used.

[0048] A branched-chain alpha-keto acid decarboxylase from Lactococcus lactis may be used. More in particular, an alpha-ketoisovalerate decarboxylase from Lactococcus lactis may be used.

[0049] An alpha-ketoglutarate decarboxylase from Mycobacterium tuberculosis may in particular be used.

[0050] In a preferred method of the invention, the preparation of 6-ACA comprises an enzymatic reaction in the presence of an enzyme capable of catalysing a transamination reaction in the presence of an amino donor, selected from the group of aminotransferases (E.C. 2.6.1).

[0051] In general, a suitable aminotransferase has 6-aminocaproic acid 6-aminotransferase activity, capable of catalysing the conversion of 5-FVA into 6-ACA or .alpha.-aminopimelate 2-aminotransferase activity, capable of catalysing the conversion of AKP into AAP.

[0052] The aminotransferase may in particular be selected amongst the group of .beta.-aminoisobutyrate:.alpha.-ketoglutarate aminotransferases, .beta.-alanine aminotransferases, aspartate aminotransferases, 4-amino-butyrate aminotransferases (EC 2.6.1.19), L-lysine 6-aminotransferase (EC 2.6.1.36), 2-aminoadipate aminotransferases (EC 2.6.1.39), 5-aminovalerate aminotransferases (EC 2.6.1.48), 2-aminohexanoate aminotransferases (EC 2.6.1.67) and lysine:pyruvate 6-aminotransferases (EC 2.6.1.71).

[0053] In an embodiment an aminotransferase may be selected amongst the group of alanine aminotransferases (EC 2.6.1.2), leucine aminotransferases (EC 2.6.1.6), alanine-oxo-acid aminotransferases (EC 2.6.1.12), .beta.-alanine-pyruvate aminotransferases (EC 2.6.1.18), (S)-3-amino-2-methylpropionate aminotransferases (EC 2.6.1.22), L,L-diaminopimelate aminotransferase (EC 2.6.1.83).

[0054] The aminotransferase may in particular be selected amongst aminotransferases from a mammal; Mercurialis, in particular Mercurialis perennis, more in particular shoots of Mercurialis perennis; Asplenium, more in particular Asplenium unilaterale or Asplenium septentrionale; Ceratonia, more in particular Ceratonia siliqua; Rhodobacter, in particular Rhodobacter sphaeroides, Staphylococcus, in particular Staphylococcus aureus; Vibrio, in particular Vibrio fluvialis; Pseudomonas, in particular Pseudomonas aeruginosa; Rhodopseusomonas; Bacillus, in particular Bacillus weihenstephanensis and Bacillus subtilis; Legionella; Nitrosomas; Neisseria; or yeast, in particular Saccharomyces cerevisiae.

[0055] In case the enzyme is of a mammal, it may in particular originate from mammalian kidney, from mammalian liver, from mammalian heart or from mammalian brain. For instance a suitable enzyme may be selected amongst the group of .beta.-aminoisobutyrate:.alpha.-ketoglutarate aminotransferase from mammalian kidney, in particular .beta.-aminoisobutyrate:.alpha.-ketoglutarate aminotransferase from hog kidney; .beta.-alanine aminotransferase from mammalian liver, in particular .beta.-alanine aminotransferase from rabbit liver; aspartate aminotransferase from mammalian heart; in particular aspartate aminotransferase from pig heart; 4-amino-butyrate aminotransferase from mammalian liver, in particular 4-amino-butyrate aminotransferase from pig liver; 4-amino-butyrate aminotransferase from mammalian brain, in particular 4-aminobutyrate aminotransferase from human, pig, or rat brain; .alpha.-ketoadipate-glutamate aminotransferase from Neurospora, in particular .alpha.-ketoadipate:glutamate aminotransferase from Neurospora crassa; 4-amino-butyrate aminotransferase from E. coli, or .alpha.-aminoadipate aminotransferase from Thermus, in particular .alpha.-aminoadipate aminotransferase from Thermus thermophilus, and 5-aminovalerate aminotransferase from Clostridium in particular from Clostridium aminovalericum. A suitable 2-aminoadipate aminotransferase may e.g. be provided by Pyrobaculum islandicum.

[0056] In particular, the amino donor can be selected from the group of ammonia, ammonium ions, amines and amino acids. Suitable amines are primary amines and secondary amines. The amino acid may have a D- or L-configuration. Examples of amino donors are alanine, glutamate, isopropylamine, 2-aminobutane, 2-aminoheptane, phenylmethanamine, 1-phenyl-1-aminoethane, glutamine, tyrosine, phenylalanine, aspartate, .beta.-aminoisobutyrate, 4-aminobutyrate, and .alpha.-aminoadipate.

[0057] In a further preferred embodiment, the method for preparing 6-ACA comprises a biocatalytic reaction in the presence of an enzyme capable of catalysing a reductive amination reaction in the presence of an ammonia source, selected from the group of oxidoreductases acting on the CH--NH.sub.2 group of donors (EC 1.4), in particular from the group of amino acid dehydrogenases (E.C. 1.4.1). In general, a suitable amino acid dehydrogenase has 6-aminocaproic acid 6-dehydrogenase activity, catalysing the conversion of 5-FVA into 6-ACA or has .alpha.-aminopimelate 2-dehydrogenase activity, catalysing the conversion of AKP into AAP. In particular a suitable amino acid dehydrogenase be selected amongst the group of diaminopimelate dehydrogenases (EC 1.4.1.16), lysine 6-dehydrogenases (EC 1.4.1.18), glutamate dehydrogenases (EC 1.4.1.3; EC 1.4.1.4), and leucine dehydrogenases (EC 1.4.1.9).

[0058] In an embodiment, an amino acid dehydrogenase may be selected amongst an amino acid dehydrogenases classified as glutamate dehydrogenases acting with NAD or NADP as acceptor (EC 1.4.1.3), glutamate dehydrogenases acting with NADP as acceptor (EC 1.4.1.4), leucine dehydrogenases (EC 1.4.1.9), diaminopimelate dehydrogenases (EC 1.4.1.16), and lysine 6-dehydrogenases (EC 1.4.1.18).

[0059] An amino acid dehydrogenase may in particular originate from an organism selected from the group of Corynebacterium, in particular Corynebacterium glutamicum; Proteus, in particular Proteus vulgaris; Agrobacterium, in particular Agrobacterium tumefaciens; Geobacillus, in particular Geobacillus stearothermophilus; Acinetobacter, in particular Acinetobacter sp. ADP1; Ralstonia, in particular Ralstonia solanacearum; Salmonella, in particular Salmonella typhimurium; Saccharomyces, in particular Saccharomyces cerevisiae; Brevibacterium, in particular Brevibacterium flavum; and Bacillus, in particular Bacillus sphaericus, Bacillus cereus or Bacillus subtilis. For instance a suitable amino acid dehydrogenase may be selected amongst diaminopimelate dehydrogenases from Bacillus, in particular Bacillus sphaericus; diaminopimelate dehydrogenases from Brevibacterium sp.; diaminopimelate dehydrogenases from Corynebacterium, in particular diaminopimelate dehydrogenases from Corynebacterium glutamicum; diaminopimelate dehydrogenases from Proteus, in particular diaminopimelate dehydrogenase from Proteus vulgaris; lysine 6-dehydrogenases from Agrobacterium, in particular Agrobacterium tumefaciens, lysine 6-dehydrogenases from Geobacillus, in particular from Geobacillus stearothermophilus; glutamate dehydrogenases acting with NADH or NADPH as cofactor (EC 1.4.1.3) from Acinetobacter, in particular glutamate dehydrogenases from Acinetobacter sp. ADP1; glutamate dehydrogenases (EC 1.4.1.3) from Ralstonia, in particular glutamate dehydrogenases from Ralstonia solanacearum; glutamate dehydrogenases acting with NADPH as cofactor (EC 1.4.1.4) from Salmonella, in particular glutamate dehydrogenases from Salmonella typhimurium; glutamate dehydrogenases (EC 1.4.1.4) from Saccharomyces, in particular glutamate dehydrogenases from Saccharomyces cerevisiae; glutamate dehydrogenases (EC 1.4.1.4) from Brevibacterium, in particular glutamate dehydrogenases from Brevibacterium flavum; and leucine dehydrogenases from Bacillus, in particular leucine dehydrogenases from Bacillus cereus or Bacillus subtilis.

[0060] In a specific embodiment, AKP is biocatalytically converted into 5-formylpentanoate (5-FVA) in the presence of a decarboxylase or other biocatalyst catalysing such conversion. A decarboxylase used in accordance with the invention may in particular be selected from the group of .alpha.-keto acid decarboxylases from Lactococcus lactis, Lactococcus lactis var. maltigenes or Lactococcus lactis subsp. cremoris; branched chain .alpha.-keto acid decarboxylases from Lactococcus lactis strain B1157 or Lactococcus lactis IFPL730; pyruvate decarboxylases from Saccharomyces cerevisiae, Candida flareri, Zymomonas mobilis, Hansenula sp., Rhizopus javanicus, Neurospora crassa, or Kluyveromyces marxianus; .alpha.-ketoglutarate decarboxylases from Mycobacterium tuberculosis; glutamate decarboxylases from E. coli, Lactobacillus brevis, Mycobacterium leprae, Neurospora crassa or Clostridium perfringens; and aspartate decarboxylases from E. coli.

[0061] In particular, a decarboxylase from Escherichia coli, Zymomonas mobilis, Saccharomyces cerevisiae, Mycobacterium tuberculosis, Pseudomonas species, or Lactococcus lactis has been found suitable to catalyse the conversion of AKP into 5-FVA. More in particular, a biocatalyst comprising a decarboxylase having a amino acid sequence as identified by Sequence ID 31, Sequence ID 34, Sequence ID 37, Sequence ID 40, Sequence ID 43, Sequence ID 46 or a homologue thereof may be used. It is also envisaged that such decarboxylase may be used to prepare 6-ACA from AAP.

[0062] Thereafter 5-FVA is converted into 6-ACA. This can be done chemically: 6-ACA can be prepared in high yield by reductive amination of 5-FVA with ammonia over a hydrogenation catalyst, for example Ni on SiO.sub.2/Al.sub.2O.sub.3 support, as described for 9-aminononanoic acid (9-aminopelargonic acid) and 12-aminododecanoic acid (12-aminolauric acid) in EP-A 628 535 or DE 4 322 065.

[0063] Alternatively, 6-ACA can be obtained by hydrogenation over PtO.sub.2 of 6-oximocaproic acid, prepared by reaction of 5-FVA and hydroxylamine. (see e.g. F. O. Ayorinde, E. Y. Nana, P. D. Nicely, A. S. Woods, E. O. Price, C. P. Nwaonicha J. Am. Oil Chem. Soc. 1997, 74, 531-538 for synthesis of the homologous 12-aminododecanoic acid).

[0064] In an embodiment, the conversion of 5-FVA to 6-ACA is performed biocatalytically in the presence of (i) an amino donor and (ii) an aminotransferase, an amino acid dehydrogenase or another biocatalyst capable of catalysing such conversion. In particular in such an embodiment the aminotransferase may be selected from the group of aminotransferases from Vibrio fluvialis, Pseudomonas aeruginosa, Bacillus subtilis, Bacillus weihenstephanensis or Escherichia coli; .beta.-aminoisobutyrate:.alpha.-ketoglutarate aminotransferase from hog kidney; .beta.-alanine aminotransferase from rabbit liver; aminotransferase from shoots from Mercurialis perennis; 4-aminobutyrate aminotransferase from pig liver or from human, rat, or pig brain; .beta.-alanine aminotransferase from rabbit liver; and L-lysine:.alpha.-ketoglutarate- -aminotransferase. In case an amino acid dehydrogenase is used, such amino acid dehydrogenase may in particular be selected from the group of lysine 6-dehydrogenases from Agrobacterium tumefaciens or Geobacillus stearothermophilus. Another suitable amino acid dehydrogenase may be selected from the group of diaminopimelate dehydrogenases from Bacillus sphaericus, Brevibacterium sp., Corynebacterium glutamicum, or Proteus vulgaris; from the group of glutamate dehydrogenases acting with NADH or NADPH as cofactor (EC 1.4.1.3) from Acinetobacter sp. ADP1 or Ralstonia solanacearum; from the group of glutamate dehydrogenases acting with NADPH as cofactor (EC 1.4.1.4) from Salmonella typhimurium; from the group of glutamate dehydrogenases (EC 1.4.1.4) from Saccharomyces cerevisiae or Brevibacterium flavum; or from the group of leucine dehydrogenases from Bacillus cereus or Bacillus subtilis.

[0065] In a specific embodiment, the conversion of 5-FVA to 6-ACA is catalysed by a biocatalyst comprising an aminotransferase comprising an amino acid sequence according to Sequence ID 2, Sequence ID 5, Sequence ID 8, Sequence ID 65, Sequence ID 67, Sequence ID 69 or a homologue of any of these sequences.

[0066] In a specific embodiment, AKP is chemically converted into 5-FVA. Efficient chemical decarboxylation of a 2-keto carboxylic acid into the corresponding aldehyde can be performed by intermediate enamine formation using a secondary amine, for instance morpholine, under azeotropic water removal and simultaneous loss of CO.sub.2, e.g. based on a method as described in Tetrahedron Lett. 1982, 23(4), 459-462. The intermediate terminal enamide is subsequently hydrolysed to the corresponding aldehyde. 5-FVA may thereafter be biocatalytically converted into 6-ACA by transamination in the presence of an aminotransferase or by enzymatic reductive amination by an amino acid dehydrogenase or another biocatalyst able of catalysing such conversion. Such aminotransferase or amino acid dehydrogenase may in particular be selected from the biocatalysts mentioned above when describing the conversion of 5-FVA to 6-ACA.

[0067] Alternatively, the conversion of 5-FVA to 6-ACA may be performed by a chemical method, e.g. as mentioned above.

[0068] In a specific embodiment, AKP is biocatalytically converted into AAP in the presence of (i) an aminotransferase, an amino acid dehydrogenase, or another biocatalyst capable of catalysing such conversion and (ii) an amino donor. Such aminotransferase used in accordance with the invention for the conversion of AKP to AAP may in particular be selected from aminotransferases mentioned above, more in particular from the group of aspartate aminotransferases from pig heart; .alpha.-ketoadipate:glutamate aminotransferases from Neurospora crassa or yeast; aminotransferases from shoots from Mercurialis perennis; 4-aminobutyrate aminotransferases from E. coli; .alpha.-aminoadipate aminotransferases from Thermus thermophilus; aminotransferases from Asplenium septentrionale or Asplenium unilaterale; and aminotransferases from Ceratonia siliqua.

[0069] In a preferred embodiment, the aminotransferase for the conversion of AKP to AAP is selected from the group of aminotransferases from Vibrio, Pseudomonas, Bacillus, Legionella, Nitrosomonas, Neisseria, Rhodobacter, Escherichia and Rhodopseudomonas.

[0070] In particular, aminotransferases from an organism selected from the group of Bacillus subtilis, Rhodobacter sphaeroides, Legionella pneumophila, Nitrosomonas europaea, Neisseria gonorrhoeae, Pseudomonas syringae, Rhodopseudomonas palustris, Vibrio fluvialis, Escherichia coli and Pseudomonas aeruginosa, have been found suitable to catalyse the conversion of AKP to AAP.

[0071] In a specific embodiment, for the conversion of AKP to AAP an aminotransferase is used comprising an amino acid sequence according to Sequence ID 2, Sequence ID 8, Sequence ID 12, Sequence ID 15, Sequence ID 17, Sequence ID 19, Sequence ID 21, Sequence ID 23, Sequence ID 25, Sequence ID 27, Sequence ID 29 or a homologue of any of these sequences.

[0072] In a further embodiment, the method for preparing AAP comprises a biocatalytic reaction in the presence of an enzyme capable of catalysing a reductive amination reaction in the presence of an ammonia source, selected from the group of oxidoreductases acting on the CH--NH.sub.2 group of donors (EC 1.4), in particular from the group of amino acid dehydrogenases (E.C. 1.4.1). In general, a suitable amino acid dehydrogenase has .alpha.-aminopimelate 2-dehydrogenase activity, catalysing the conversion of AKP into AAP.

[0073] In particular a suitable amino acid dehydrogenase may be selected from the group of diaminopimelate dehydrogenases (EC 1.4.1.16), glutamate dehydrogenases (EC 1.4.1.3; EC 1.4.1.4), and leucine dehydrogenases (EC 1.4.1.9).

[0074] In an embodiment, an amino acid dehydrogenase is selected amongst amino acid dehydrogenases classified as glutamate dehydrogenases acting with NAD or NADP as acceptor (EC 1.4.1.3), glutamate dehydrogenases acting with NADP as acceptor (EC 1.4.1.4), leucine dehydrogenases (EC 1.4.1.9), and diaminopimelate dehydrogenases (EC 1.4.1.16).

[0075] An amino acid dehydrogenase may in particular originate from an organism selected from the group of Corynebacterium, in particular Corynebacterium glutamicum; Proteus, in particular Proteus vulgaris; Agrobacterium, in particular Agrobacterium tumefaciens; Geobacillus, in particular Geobacillus stearothermophilus; Acinetobacter, in particular Acinetobacter sp. ADP1; Ralstonia, in particular Ralstonia solanacearum; Salmonella, in particular Salmonella typhimurium; Saccharomyces, in particular Saccharomyces cerevisiae; Brevibacterium, in particular Brevibacterium flavum; and Bacillus, in particular Bacillus sphaericus, Bacillus cereus or Bacillus subtilis.

[0076] For instance a suitable amino acid dehydrogenase may be selected amongst diaminopimelate dehydrogenases from Bacillus, in particular Bacillus sphaericus; diaminopimelate dehydrogenases from Brevibacterium sp.; diaminopimelate dehydrogenases from Corynebacterium, in particular diaminopimelate dehydrogenases from Corynebacterium glutamicum; diaminopimelate dehydrogenases from Proteus, in particular diaminopimelate dehydrogenase from Proteus vulgaris; glutamate dehydrogenases acting with NADH or NADPH as cofactor (EC 1.4.1.3) from Acinetobacter, in particular glutamate dehydrogenases from Acinetobacter sp. ADP1; glutamate dehydrogenases (EC 1.4.1.3) from Ralstonia, in particular glutamate dehydrogenases from Ralstonia solanacearum; glutamate dehydrogenases acting with NADPH as cofactor (EC 1.4.1.4) from Salmonella, in particular glutamate dehydrogenases from Salmonella typhimurium; glutamate dehydrogenases (EC 1.4.1.4) from Saccharomyces, in particular glutamate dehydrogenases from Saccharomyces cerevisiae; glutamate dehydrogenases (EC 1.4.1.4) from Brevibacterium, in particular glutamate dehydrogenases from Brevibacterium flavum; and leucine dehydrogenases from Bacillus, in particular leucine dehydrogenases from Bacillus cereus or Bacillus subtilis.

[0077] Another suitable amino acid dehydrogenase may be selected from the group of lysine 6-dehydrogenases from Agrobacterium tumefaciens or Geobacillus stearothermophilus; or from the group of leucine dehydrogenases from Bacillus cereus or Bacillus subtilis.

[0078] AAP prepared in a method of the invention may further be used for the preparation of 6-ACA. The inventors have realised that AAP, prepared from AKP, can be converted into 6-ACA by a decarboxylation reaction. This can be performed chemically, for instance by heating in a high boiling solvent in the presence of a ketone or aldehyde catalyst. For example, amino acids are decarboxylated in good yields in cyclohexanol at 150-160.degree. C. with 1-2 v/v % of cyclohexenone as described by M. Hashimoto, Y. Eda, Y. Osanai, T. Iwai and S. Aoki in Chem. Lett. 1986, 893-896. Similar methods are described in Eur. Pat. Appl. 1586553, 2005 by Daiso, and by S. D. Brandt, D. Mansell, S. Freeman, I. A. Fleet, J. F. Alder J. Pharm. Biomed. Anal. 2006, 41, 872-882.

[0079] Alternatively, the decarboxylation of AAP to 6-ACA may be performed biocatalytically in the presence of a decarboxylase or other biocatalyst catalysing such decarboxylation.

[0080] The decarboxylase may be selected amongst decarboxylases capable of catalysing the decarboxylation of an .alpha.-amino acid. An enzyme capable of decarboxylating an alpha-amino acid may in particular be selected from the group of decarboxylases (E.C. 4.1.1), preferably from the group of pyruvate decarboxylases (EC 4.1.1.1), diaminopimelate decarboxylases (EC 4.1.1.20), diaminopimelate decarboxylases (EC 4.1.1.20), branched chain alpha-keto acid decarboxylases (EC 4.1.1.72), which include alpha-ketoisovalerate decarboxylases, and alpha-ketoglutarate decarboxylases (EC 4.1.1.71).

[0081] One or more other suitable decarboxylases may in particular be selected amongst the group of oxalate decarboxylases (EC 4.1.1.2), oxaloacetate decarboxylases (EC 4.1.1.3), acetoacetate decarboxylases (EC 4.1.1.4), aspartate 1-decarboxylases (EC 4.1.1.11), valine decarboxylases/leucine decarboxylases (EC 4.1.1.14), glutamate decarboxylases (EC 4.1.1.15), 3-hydroxyglutamate decarboxylases (EC 4.1.1.16), ornithine decarboxylases (EC 4.1.1.17), lysine decarboxylases (EC 4.1.1.18), arginine decarboxylases (EC 4.1.1.19), 2-oxoglutarate decarboxylases (EC 4.1.1.71), and diaminobutyrate decarboxylases (EC 4.1.1.86).

[0082] A decarboxylase may in particular be a decarboxylase of an organism selected from the group of squashes, e.g. Curcurbita moschata; cucumbers; yeasts; fungi, e.g. Saccharomyces cerevisiae, Candida flareri, Hansenula sp., Kluyveromyces marxianus, Rhizopus javanicus, and Neurospora crassa; mammals, in particular from mammalian brain; and bacteria such as Escherichia coli, Lactococcus lactis, Mycobacterium tuberculosis, Pseudomonas sp. and Zymomonas mobilis.

[0083] The pyruvate decarboxylase may originate from Saccharomyces cerevisiae or Zymomonas mobilis. In particular, pyruvate decarboxylase mutant I472A from Zymomonas mobilis may be used. An oxaloacetate decarboxylase from Pseudomonas may in particular be used. Glutamate decarboxylase or aspartate decarboxylase from Escherichia coli (E. coli) may be used, or glutamate decarboxylase from Neurospora crassa, Mycobacterium leprae, Clostridium perfringens, Lactobacillus brevis, Mycobacterium tuberculosis, Streptococcus or Lactococcus may be used. Examples of Lactococcus species from which the glutamate decarboxylase may originate in particular include Lactococcus lactis, such as Lactococcus lactis strain B1157, Lactococcus lactis IFPL730, more in particular Lactococcus lactis var. maltigenes (formerly named Streptococcus lactis var. maltigenes). A diaminopimelate decarboxylase may, e.g., be from an organism capable of synthesising lysine from diaminopimelate. Such organism may in particular be found amongst bacteria, archaea and plants. In particular, the diaminopimelate decarboxylase may be from a gram negative bacterium, for instance E. coli. Branched-chain alpha-keto acid decarboxylases from Lactococcus lactis may be used. More in particular, branched chain alpha-keto acid decarboxylases and alpha-ketoisovalerate decarboxylases from Lactococcus lactis may be used.

[0084] An alpha-ketoglutarate decarboxylase from Mycobacterium tuberculosis may in particular be used. The inventors have found that alpha-ketoglutarate decarboxylase (Kgd) from Mycobacterium tuberculosis may be used for converting AAP into 6-ACA. In particular, the inventors have found that such decarboxylase comprising a sequence as shown in SEQUENCE ID No. 46 or a functional analogue thereof may be capable of catalysing the formation of 6-ACA from AAP.

[0085] A glutamate decarboxylase may in particular be selected from Curcurbita moschata, cucumber, yeast, or calf brain; and diaminopimelate decarboxylases (EC 4.1.1.20).

[0086] A diaminopimelate decarboxylase may, e.g., be from an organism capable of synthesising lysine from diaminopimelate. Such organism may in particular be found amongst bacteria, archaea and plants.

[0087] In particular, the diaminopimelate decarboxylase may be from a gram negative bacterium, for instance E. coli.

[0088] In a specific embodiment, AKP is chemically converted into AAP. AAP can be prepared from 2-oxopimelic acid by catalytic Leuckart-Wallach reaction as described for similar compounds. This reaction is performed with ammonium formate in methanol and [RhCp*Cl.sub.2].sub.2 as homogeneous catalyst (M. Kitamura, D. Lee, S. Hayashi, S. Tanaka, M. Yoshimura J. Org. Chem. 2002, 67, 8685-8687). Alternatively, the Leuckart-Wallach reaction can be performed with aqueous ammonium formate using [Ir.sup.IIICp*(bpy)H.sub.2O]SO.sub.4 as catalyst as described by S. Ogo, K. Uehara and S. Fukuzumi in J. Am. Chem. Soc. 2004, 126, 3020-3021. Transformation of .alpha.-keto acids into (enantiomerically enriched) amino acids is also possible by reaction with (chiral) benzylamines and subsequent hydrogenation of the intermediate imine over Pd/C or Pd(OH).sub.2/C. See for example, R. G. Hiskey, R. C. Northrop J. Am. Chem. Soc. 1961, 83, 4798.

[0089] Thereafter AAP is biocatalytically converted into 6-ACA, in the presence of a decarboxylase or another biocatalyst capable of performing such decarboxylation. Such decarboxylase may in particular be selected amongst the biocatalysts referred to above, when describing biocatalysts for the conversion of AAP to 6-ACA.

[0090] Alternatively, the conversion of AAP to 6-ACA may be performed by a chemical method, e.g. as mentioned above.

[0091] In a specific embodiment, AKP is biocatalytically converted into 5-FVA in the presence of a decarboxylase or other biocatalyst capable of catalysing such conversion and 5-FVA is thereafter converted into 6-ACA in the presence of an aminotransferase, amino acid dehydrogenase, or other biocatalyst capable of catalysing such conversion. Decarboxylases suitable for these reactions may in particular be selected from the group of decarboxylases mentioned above, when describing the biocatalytic conversion of AKP into 5-FVA. A suitable aminotransferase or amino acid dehydrogenase for the conversion of 5-FVA may in particular be selected from those mentioned above, when describing the biocatalytic conversion of 5-FVA to 6-ACA.

[0092] In a specific embodiment, AKP is biocatalytically converted into AAP in the presence of an aminotransferase, amino acid dehydrogenase, or other biocatalyst capable of catalysing such conversion and AAP is thereafter converted into 6-ACA in the presence of a decarboxylase or other biocatalyst capable of catalysing such conversion.

[0093] Enzymes suitable for these reactions may in particular be selected from the group of aminotransferases, amino acid dehydrogenases, and decarboxylases which have been described above when describing the biocatalytic conversion of AKP into AAP and the biocatalytic conversion of AAP into 6-ACA respectively.

[0094] AKP used to prepare 6-ACA may in principle be obtained in any way. For instance, AKP may be obtained based on a method as described by H. Jager et al. Chem. Ber. 1959, 92, 2492-2499. AKP can be prepared by alkylating cyclopentanone with diethyl oxalate using sodium ethoxide as a base, refluxing the resultant product in a strong acid (2 M HCl) and recovering the product, e.g. by crystallisation from toluene.

[0095] It is also possible to obtain AKP from a natural source, e.g. from methanogenic Archaea, from Asplenium septentrionale, or from Hydnocarpus anthelminthica. AKP may for instance be extracted from such organism, or a part thereof, e.g. from Hydnocarpus anthelminthica seeds. A suitable extraction method may e.g. be based on the method described in A. I. Virtanen and A. M. Berg in Acta Chemica Scandinavica 1954, 6,1085-1086, wherein the extraction of amino acids and AKP from Asplenium, using 70% ethanol, is described.

[0096] In a specific embodiment, AKP is prepared in a method comprising converting alpha-ketoglutaric acid (AKG) into alpha-ketoadipic acid (AKA) and converting alpha-ketoadipic acid into alpha-ketopimelic acid. This reaction may be catalysed by a biocatalyst. AKG may, e.g., be prepared biocatalytically from a carbon source, such as a carbohydrate, in a manner known in the art per se.

[0097] A suitable biocatalyst for preparing AKP from AKG may in particular be selected amongst biocatalysts catalysing C.sub.1-elongation of alpha-ketoglutaric acid into alpha-ketoadipic acid and/or C.sub.1-elongation of alpha-ketoadipic acid into alpha-ketopimelic acid.

[0098] In a specific embodiment, the preparation of AKP is catalysed by a biocatalyst comprising

[0099] a. an AksA enzyme or an homologue thereof;

[0100] b. at least one enzyme selected from the group of AksD enzymes, AksE enzymes, homologues of AksD enzymes and homologues of AksE enzymes; and

[0101] c. an AksF enzyme or a homologue thereof.

[0102] One or more of the AksA, AksD, AksE, AksF enzymes or homologues thereof may be found in an organism selected from the group of methanogenic archaea, preferably selected from the group of Methanococcus, Methanocaldococcus, Methanosarcina, Methanothermobacter, Methanosphaera, Methanopyrus and Methanobrevibacter.

[0103] In a specific embodiment, the biocatalyst catalysing the preparation of AKP from alpha-ketoglutaric acid (AKG) comprises an enzyme system catalysing the conversion of alpha-ketoglutaric acid into alpha-ketoadipic acid, wherein said enzyme system forms part of the alpha-amino adipate pathway for lysine biosynthesis. The term `enzyme system` is in particular used herein for a single enzyme or a group of enzymes whereby a specific conversion can be catalysed.

[0104] The preparation of AKP from AKG may comprise one or more biocatalytic reactions with known or unknown intermediates e.g. the conversion of AKG into AKA or the conversion of AKA into AKP. Such system may be present inside a cell or isolated from a cell. The enzyme system may in particular be from an organism selected from the group of yeasts, fungi, archaea and bacteria, in particular from the group of Penicillium, Cephalosporium, Paelicomyces, Trichophytum, Aspergillus, Phanerochaete, Emericella, Ustilago, Schizosaccharomyces, Saccharomyces, Candida, Yarrowia, Pichia, Kluyveromyces, Thermus, Deinococcus, Pyrococcus, Sulfolobus, Thermococcus, Methanococcus, Methanocaldococcus, Methanosphaera, Methanopyrus, Methanobrevibacter, Methanosarcina and Methanothermobacter.

[0105] In a specific embodiment, the biocatalyst catalysing the preparation of AKP from alpha-ketoglutaric acid comprises an enzyme system catalysing the conversion of alpha-ketoglutaric acid into alpha-ketoadipic acid, wherein at least one of the enzymes of the enzyme system originates from nitrogen fixing bacteria selected from the group of cyanobacteria, rhizobiales, .gamma.-proteobacteria and actinobacteria, in particular from the group of Anabaena, Microcystis, Synechocystis, Rhizobium, Bradyrhizobium, Pseudomonas, Azotobacter, Klebsiella and Frankia.

[0106] Examples of homologues for these Aks enzymes and the genes encoding these enzymes are given in the Tables 1A and 1B on the following pages.

TABLE-US-00001 TABLE 1A Enzyme name Organism gene Protein AksA Methanocaldococcus jannashii MJ0503 NP_247479 Methanothermobacter thermoautotropicum .DELTA.H MTH1630 NP_276742 Methanococcus maripaludis S2 MMP0153 NP_987273 Methanococcus maripaludis C5 MmarC5_1522 YP_001098033 Methanococcus maripaludis C7 MmarC7_1153 YP_001330370 Methanosphaera stadtmanae DSM 3091 Msp_0199 YP_447259 Methanopyrus kandleri AV19 MK1209 NP_614492 Methanobrevibacter smithii ATCC35061 Msm_0722 YP_001273295 Methanococcus vannielii SB Mevan_1158 YP_001323668 Methanococcus aeolicus Nankai 3 Maeo_0994 YP_001325184 AksD Methanocaldococcus jannashii MJ1003 NP_247997 Methanothermobacter thermoautotropicum .DELTA.H MTH1386 NP_276502 Methanococcus maripaludis S2 Mmp1480 NP_988600 Methanococcus maripaludis C5 MmarC5_0098 YP_001096630 Methanococcus maripaludis C7 MmarC7_0724 YP_001329942 Methanosphaera stadtmanae DSM 3091 Msp_1486 YP_448499 Methanopyrus kandleri AV19 MK1440 NP_614723 Methanobrevibacter smithii ATCC35061 Msm_0723 YP_001273296 Methanococcus vannielii SB Mevan_0789 YP_001323307 Methanococcus aeolicus Nankai 3 Maeo_0311 YP_001324511 References to gene and protein can be found via www.ncbi.nlm.nih.gov/, (as available on 15 Apr. 2008)

TABLE-US-00002 TABLE 1B Enzyme name Orgamism gene Protein AksE Methanocaldococcus jannashii MJ1271 NP_248267 Methanothermobacter thermoautotropicum .DELTA.H MTH1387 NP_276503 Methanococcus maripaludis S2 MMP0381 NP_987501 Methanococcus maripaludis C5 MmarC5_1257 YP_001097769 Methanococcus maripaludis C7 MmarC7_1379 YP_001330593 Methanosphaera stadtmanae DSM 3091 Msp_1485 YP_448498 Methanopyrus kandleri AV19 MK0781 NP_614065 Methanobrevibacter smithii ATCC35061 Msm_0847 YP_001273420 Methanococcus vannielii SB Mevan_1368 YP_001323877 Methanococcus aeolicus Nankai 3 Maeo_0652 YP_001324848 AksF Methanocaldococcus jannashii MJ1596 NP_248605 Methanothermobacter thermoautotropicum .DELTA.H MTH184 NP_275327 Methanococcus maripaludis S2 MMP0880 NP988000 Methanococcus maripaludis C5 MmarC5_0688 YP001097214 Methanococcus maripaludis C7 MmarC7_0128 YP_001329349 Methanosphaera stadtmanae DSM 3091 Msp_0674 YP_447715 Methanopyrus kandleri AV19 MK0782 NP_614066 Methanobrevibacter smithii ATCC35061 Msm_0373 YP001272946 Methanococcus vannielii SB Mevan_0040 YP_001322567 Methanococcus aeolicus Nankai 3 Maeo_1484 YP_001325672 References to gene and protein can be found via www.ncbi.nlm.nih.gov/, (as available on 15 Apr. 2008)

[0107] If desired, 6-ACA obtained in accordance with the invention can be cyclised to form caprolactam, e.g. as described in U.S. Pat. No. 6,194,572.

[0108] Reaction conditions for any biocatalytic step in the context of the present invention may be chosen depending upon known conditions for the biocatalyst, in particular the enzyme, the information disclosed herein and optionally some routine experimentation.

[0109] In principle, the pH of the reaction medium used may be chosen within wide limits, as long as the biocatalyst is active under the pH conditions. Alkaline, neutral or acidic conditions may be used, depending on the biocatalyst and other factors. In case the method includes the use of a micro-organism, e.g. for expressing an enzyme catalysing a method of the invention, the pH is selected such that the micro-organism is capable of performing its intended function or functions. The pH may in particular be chosen within the range of four pH units below neutral pH and two pH units above neutral pH, i.e. between pH 3 and pH 9 in case of an essentially aqueous system at 25.degree. C. A system is considered aqueous if water is the only solvent or the predominant solvent (>50 wt. %, in particular >90 wt. %, based on total liquids), wherein e.g. a minor amount of alcohol or another solvent (<50 wt. %, in particular <10 wt. %, based on total liquids) may be dissolved (e.g. as a carbon source) in such a concentration that micro-organisms which may be present remain active. In particular in case a yeast and/or a fungus is used, acidic conditions may be preferred, in particular the pH may be in the range of pH 3 to pH 8, based on an essentially aqueous system at 25.degree. C. If desired, the pH may be adjusted using an acid and/or a base or buffered with a suitable combination of an acid and a base.

[0110] In principle, the incubation conditions can be chosen within wide limits as long as the biocatalyst shows sufficient activity and/or growth. This includes aerobic, micro-aerobic, oxygen limited and anaerobic conditions.

[0111] Anaerobic conditions are herein defined as conditions without any oxygen or in which substantially no oxygen is consumed by the biocatalyst, in particular a micro-organism, and usually corresponds to an oxygen consumption of less than 5 mmol/l.h, in particular to an oxygen consumption of less than 2.5 mmol/l.h, or less than 1 mmol/l.h.

[0112] Aerobic conditions are conditions in which a sufficient level of oxygen for unrestricted growth is dissolved in the medium, able to support a rate of oxygen consumption of at least 10 mmol/l.h, more preferably more than 20 mmol/l.h, even more preferably more than 50 mmol/l.h, and most preferably more than 100 mmol/l.h.

[0113] Oxygen-limited conditions are defined as conditions in which the oxygen consumption is limited by the oxygen transfer from the gas to the liquid. The lower limit for oxygen-limited conditions is determined by the upper limit for anaerobic conditions, i.e. usually at least 1 mmol/l.h, and in particular at least 2.5 mmol/l.h, or at least 5 mmol/l.h. The upper limit for oxygen-limited conditions is determined by the lower limit for aerobic conditions, i.e. less than 100 mmol/l.h, less than 50 mmol/l.h, less than 20 mmol/l.h, or less than to 10 mmol/l.h.

[0114] Whether conditions are aerobic, anaerobic or oxygen limited is dependent on the conditions under which the method is carried out, in particular by the amount and composition of ingoing gas flow, the actual mixing/mass transfer properties of the equipment used, the type of micro-organism used and the micro-organism density.

[0115] In principle, the temperature used is not critical, as long as the biocatalyst, in particular the enzyme, shows substantial activity. Generally, the temperature may be at least 0.degree. C., in particular at least 15.degree. C., more in particular at least 20.degree. C. A desired maximum temperature depends upon the biocatalyst. In general such maximum temperature is known in the art, e.g. indicated in a product data sheet in case of a commercially available biocatalyst, or can be determined routinely based on common general knowledge and the information disclosed herein. The temperature is usually 90.degree. C. or less, preferably 70.degree. C. or less, in particular 50.degree. C. or less, more in particular or 40.degree. C. or less.

[0116] In particular if a biocatalytic reaction is performed outside a host organism, a reaction medium comprising an organic solvent may be used in a high concentration (e.g. more than 50%, or more than 90 wt. %), in case an enzyme is used that retains sufficient activity in such a medium.

[0117] In an advantageous method 6-ACA is prepared making use of a whole cell biotransformation of the substrate for 6-ACA or an intermediate for forming 6-ACA (AKP, AAP or 5-FVA), comprising a micro-organism wherein one or more biocatalysts (usually one or more enzymes) catalysing the biotransformation are produced, such as one or more biocatalysts selected from the group of biocatalysts capable of catalysing the conversion of AKP to AAP, biocatalysts capable of catalysing the conversion of AAP to 6-ACA, biocatalysts capable of catalysing the conversion of AKP to 5-FVA and biocatalysts capable of catalysing the conversion of 5-FVA to 6-ACA. In a preferred embodiment the micro-organism is capable of producing a decarboxylase and/or at least one enzyme selected from amino acid dehydrogenases and aminotransferases are produced. capable of catalysing a reaction step as described above, and a carbon source for the micro-organism.

[0118] The carbon source may in particular contain at least one compound selected from the group of monohydric alcohols, polyhydric alcohols, carboxylic acids, carbon dioxide, fatty acids, glycerides, including mixtures comprising any of said compounds. Suitable monohydric alcohols include methanol and ethanol, Suitable polyols include glycerol and carbohydrates. Suitable fatty acids or glycerides may in particular be provided in the form of an edible oil, preferably of plant origin.

[0119] In particular a carbohydrate may be used, because usually carbohydrates can be obtained in large amounts from a biologically renewable source, such as an agricultural product, preferably an agricultural waste-material. Preferably a carbohydrate is used selected from the group of glucose, fructose, sucrose, lactose, saccharose, starch, cellulose and hemi-cellulose. Particularly preferred are glucose, oligosaccharides comprising glucose and polysaccharides comprising glucose.

[0120] A cell, in particular a recombinant cell, comprising one or more biocatalysts (usually one or more enzymes) for catalysing a reaction step in a method of the invention can be constructed using molecular biological techniques, which are known in the art per se. For instance, if one or more biocatalysts are to be produced in a recombinant cell (which may be a heterologous system), such techniques can be used to provide a vector (such as a recombinant vector) which comprises one or more genes encoding one or more of said biocatalysts. One or more vectors may be used, each comprising one or more of such genes. Such vector can comprise one or more regulatory elements, e.g. one or more promoters, which may be operably linked to a gene encoding an biocatalyst.

[0121] As used herein, the term "operably linked" refers to a linkage of polynucleotide elements (or coding sequences or nucleic acid sequence) in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0122] As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skilled in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "homologous" when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain.

[0123] The promoter that could be used to achieve the expression of the nucleic acid sequences coding for an enzyme for use in a method of the invention, in particular an aminotransferase, an amino acid dehydrogenase or a decarboxylase, such as described herein above may be native to the nucleic acid sequence coding for the enzyme to be expressed, or may be heterologous to the nucleic acid sequence (coding sequence) to which it is operably linked. Preferably, the promoter is homologous, i.e. endogenous to the host cell.

[0124] If a heterologous promoter (to the nucleic acid sequence encoding for the enzyme of interest) is used, the heterologous promoter is preferably capable of producing a higher steady state level of the transcript comprising the coding sequence (or is capable of producing more transcript molecules, i.e. mRNA molecules, per unit of time) than is the promoter that is native to the coding sequence. Suitable promoters in this context include both constitutive and inducible natural promoters as well as engineered promoters, which are well known to the person skilled in the art.

[0125] A "strong constitutive promoter" is one which causes mRNAs to be initiated at high frequency compared to a native host cell. Examples of such strong constitutive promoters in Gram-positive micro-organisms include SP01-26, SP01-15, veg, pyc (pyruvate carboxylase promoter), and amyE.

[0126] Examples of inducible promoters in Gram-positive micro-organisms include, the IPTG inducible Pspac promoter, the xylose inducible PxylA promoter.

[0127] Examples of constitutive and inducible promoters in Gram-negative microorganisms include, but are not limited to, tac, tet, trp-tet, lpp, lac, lpp-lac, laclq, T7, T5, T3, gal, trc, ara (P.sub.BAD), SP6, .lamda.-P.sub.R, and .lamda.-P.sub.L.

[0128] Promoters for (filamentous) fungal cells are known in the art and can be, for example, the glucose-6-phosphate dehydrogenase gpdA promoters, protease promoters such as pepA, pepB, pepC, the glucoamylase glaA promoters, amylase amyA, amyB promoters, the catalase catR or catA promoters, glucose oxidase goxC promoter, beta-galactosidase lacA promoter, alpha-glucosidase aglA promoter, translation elongation factor tefA promoter, xylanase promoters such as xlnA, xlnB, xlnC, xlnD, cellulase promoters such as eglA, eglB, cbhA, promoters of transcriptional regulators such as areA, creA, xlnR, pacC, prtT, or another promotor, and can be found among others at the NCBI website (http://www.ncbi.nlm.nih.gov/entrez/).

[0129] The term "heterologous" when used with respect to a nucleic acid (DNA or RNA) or protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous nucleic acids or proteins are not endogenous to the cell into which it is introduced, but has been obtained from another cell or synthetically or recombinantly produced. Generally, though not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly exogenous RNA encodes for proteins not normally expressed in the cell in which the exogenous RNA is present. Heterologous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as heterologous or foreign to the cell in which it is expressed is herein encompassed by the term heterologous nucleic acid or protein.

[0130] A method according to the invention may be carried out in a host organism, which may be novel.

[0131] Accordingly, the invention also relates to a host cell comprising one or more biocatalysts capable of catalysing at least one reaction step in a method of the invention, in particular capable of catalysing at least one reaction step in the conversion of AKP, AAP or 5-FVA to 6-ACA. The invention also relates to a novel vector comprising one or more genes encoding for one or more enzymes capable of catalysing at least one reaction step in a method of the invention, in particular capable of catalysing at least one reaction step in the conversion of AKP to 6-ACA and to a novel host cell comprising one or more genes encoding for one or more enzymes capable of catalysing at least one reaction step in a method of the invention, in particular capable of catalysing at least one reaction step in the conversion of AKP to 6-ACA (which one or more genes may form part of one or more vectors).

[0132] In a specific embodiment, a host cell according to the invention is a recombinant cell comprising a nucleic acid sequence encoding a biocatalyst capable of catalysing a transamination reaction or a reductive amination reaction to form alpha-aminopimelic acid from alpha-ketopimelic acid. Said sequence may be part of a vector or may have been inserted into the chromosomal DNA.

[0133] In particular, a host cell or vector according to the invention may comprise at least one nucleic acid sequence, in particular at least two nucleic acid sequences, selected from the group of nucleic acid sequences encoding an enzyme with .alpha.-ketopimelic acid decarboxylase activity, nucleic acid sequences encoding an enzyme with 5-formylpentanoate aminotransferase activity, nucleic acid sequences encoding an enzyme with .alpha.-ketopimelic acid aminotransferase activity, nucleic acid sequences encoding an enzyme with .alpha.-ketopimelic acid dehydrogenase activity and nucleic acid sequences encoding an enzyme with .alpha.-aminopimelic acid decarboxylase activity. Of these sequences, typically one or more, in particular two or more, are recombinant sequences.

[0134] In preferred embodiment the host cell, typically a recombinant host cell, or the vector according to the invention comprises a nucleic acid sequence encoding at least one biocatalyst having .alpha.-ketopimelic acid decarboxylase activity, and/or at least one nucleic acid sequence selected from sequences encoding a biocatalyst with 5-formylpentanoate aminotransferase activity.

[0135] In such an embodiment, the nucleic acid sequence encoding an enzyme with .alpha.-ketopimelic acid decarboxylase activity may in particular comprise an amino acid sequence according to Sequence ID 31, Sequence ID 34, Sequence ID 37, Sequence ID 40, Sequence ID 43 or Sequence ID 46 or a homologue of any of these sequences and/or the nucleic acid sequence encoding an enzyme with 5-formylpentanoate aminotransferase may in particular comprise an amino acid sequence according to Sequence ID 2, Sequence ID 5, Sequence ID 8, Sequence ID 65 Sequence ID 67, Sequence ID 69 or a homologue thereof. One or more of said nucleic acid sequences may form part of one or more recombinant vectors.

[0136] In a further preferred embodiment, the vector or host cell comprises a nucleic acid sequence encoding an enzyme with .alpha.-ketopimelic acid aminotransferase activity and/or a nucleic acid sequence encoding an enzyme with .alpha.-aminopimelic acid decarboxylase activity. The nucleic acid sequence encoding an enzyme with .alpha.-ketopimelic acid aminotransferase activity may in particular comprise an amino acid sequence according to Sequence ID 2, Sequence ID 8, Sequence ID 12, Sequence ID 15, Sequence ID 17, Sequence ID 19, Sequence ID 21, Sequence ID 23, Sequence ID 25, Sequence ID 27, Sequence ID 29, or a homologue thereof. One or more of said nucleic acid sequences may form part of one or more recombinant vectors.

[0137] In a specific preferred embodiment, a host cell according to the invention comprises a nucleic acid sequence encoding an enzyme with .alpha.-aminopimelate 2-dehydrogenase activity and a nucleic acid sequence encoding an enzyme with .alpha.-aminopimelate decarboxylase activity.

[0138] In a specific preferred embodiment, a host cell according to the invention comprises a nucleic acid sequence encoding an enzyme with 6-aminocaproic acid 6-dehydrogenase activity and a nucleic acid sequence encoding an enzyme with .alpha.-ketopimelic acid decarboxylase activity.

[0139] One or more suitable genes of a host cell or vectors according to the invention may in particular be selected amongst genes encoding an enzyme as mentioned herein above.

[0140] In a specific embodiment, the host cell is a recombinant cell comprising at least one nucleic acid sequence selected from the group of sequences as identified in any of Sequence ID 1, Sequence ID 3, Sequence ID 4, Sequence ID 6, Sequence ID 7, Sequence ID 11, Sequence ID 13, Sequence ID 14, Sequence ID 16, Sequence ID 18, Sequence ID 20, Sequence ID 22, Sequence ID 24, Sequence ID 26, Sequence ID 28, Sequence ID 30, Sequence ID 32, Sequence ID 33, Sequence ID 35, Sequence ID 36, Sequence ID 38, Sequence ID 39, Sequence ID 41, Sequence ID 42, Sequence ID 44, Sequence ID 45, Sequence ID 47, Sequence ID 64, Sequence ID 66, Sequence ID 68 and functional analogues thereof.

[0141] A nucleic acid sequence encoding an enzyme with 5-FVA aminotransferase activity, may in particular be a sequence selected from the group of sequences represented by any of the Sequence ID's 1, 3, 4, 6, 7, 64, 66, 68, and functional analogues of any of these sequences.

[0142] As used herein, the term "functional analogues" at least includes other sequences encoding an enzyme having the same amino acid sequence and other sequences encoding a homologue of such enzyme.

[0143] A nucleic acid sequence encoding an enzyme with AKP decarboxylase activity may in particular be a sequence selected from the group of sequences represented by any of the Sequence ID's 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47 and functional analogues of any of these sequences.

[0144] In a preferred embodiment, the host cell comprises a nucleic acid sequence encoding an enzyme, capable of catalysing the conversion of AAP to AKP, according to Sequence ID No.: 1, 3, 7, 11, 13, 14, 16, 18, 20, 22, 24, 26, 28, or a functional analogue thereof, which may be a wild type or non-wild type sequence

[0145] In a specific embodiment, the host cell comprises at least one nucleic acid sequence encoding a biocatalyst having alpha-aminopimelic acid decarboxylase activity, which may be homologous or heterologous to the host cell. In particular such biocatalyst may be selected from the group of decarboxylases (E.C. 4.1.1), more in particular from the group of glutamate decarboxylases (EC 4.1.1.15), diaminopimelate decarboxylases (EC 4.1.1.20) aspartate 1-decarboxylases (EC 4.1.1.11), branched chain alpha-keto acid decarboxylases, alpha-ketoisovalerate decarboxylases, alpha-ketoglutarate decarboxylases, pyruvate decarboxylases (EC 4.1.1.1) and oxaloacetate decarboxylases (E.C. 4.1.1.3).

[0146] In a specific embodiment, the host cell comprises one or more enzymes catalysing the formation of AKP from AKG (see also above). Use may be made of an enzyme system forming part of the alpha-amino adipate pathway for lysine biosynthesis. The term `enzyme system` is in particular used herein for a single enzyme or a group of enzymes whereby a specific conversion can be catalysed. Said conversion may comprise one or more chemical reactions with known or unknown intermediates e.g. the conversion of AKG into AKA or the conversion of AKA into AKP. Such system may be present inside a cell or isolated from a cell. It is known that aminotransferases often have a wide substrate range. If present, it may be desired to decrease activity of one or more such enzymes in a host cell such that activity in the conversion of AKA to alpha-aminoadipate (AAA) is reduced, whilst maintaining relevant catalytic functions for biosynthesis of other amino acids or cellular components. Also a host cell devoid of any other enzymatic activity resulting in the conversion of AKA to an undesired side product is preferred.

[0147] In a preferred host cell, suitable for preparing AAP making use of a whole cell biotransformation process, one or more biocatalysts capable of catalysing at least one reaction step in the preparation of alpha-ketopimelic acid from alpha-ketoglutaric acid are encoded for. Suitable biocatalysts are, e.g., as described above when discussing the preparation of AKP.

[0148] The host cell may for instance be selected from bacteria, yeasts or fungi. In particular the host cell may be selected from the genera selected from the group of Aspergillus, Penicillium, Saccharomyces, Kluyveromyces, Pichia, Candida, Hansenula, Bacillus, Corynebacterium, Pseudomonas, Gluconobacter, Methanococcus, Methanobacterium, Methanocaldococcus and Methanosarcina and Escherichia. Herein, usually one or more encoding nucleic acid sequences as mentioned above have been cloned and expressed.

[0149] In particular, the host strain and, thus, a host cell suitable for the biochemical synthesis of 6-ACA may be selected from the group of Escherichia coli, Bacillus subtilis, Bacillus amyloliquefaciens, Corynebacterium glutamicum, Aspergillus niger, Penicillium chrysogenum, Saccharomyces cervisiae, Hansenula polymorpha, Candida albicans, Kluyveromyces lactis, Pichia stipitis, Pichia pastoris, Methanobacterium thermoautothrophicum .DELTA.H, Methanococcus maripaludis, Methanococcus voltae, Methanosarcina acetivorans, Methanosarcina barkeri and Methanosarcina mazei host cells. In a preferred embodiment, the host cell is capable of producing lysine (as a precursor).

[0150] The host cell may be in principle a naturally occurring organism or may be an engineered organism. Such an organism can be engineered using a mutation screening or metabolic engineering strategies known in the art. In a specific embodiment, the host cell naturally comprises (or is capable of producing) one or more of the enzymes suitable for catalysing a reaction step in a method of the invention, such as one or more activities selected from the group of decarboxylases, aminotransferases and amino acid dehydrogenases capable of catalysing a reaction step in a method of the invention. For instance E. coli may naturally be capable of producing an enzyme catalysing a transamination in a method of the invention. It is also possible to provide a recombinant host cell with both a recombinant gene encoding an aminotransferase or amino acid dehydrogenase capable of catalysing a reaction step in a method of the invention and a recombinant gene encoding a decarboxylase gene capable of catalysing a reaction step in a method of the invention.

[0151] For instance a host cell may be selected of the genus Corynebacterium, in particular C. glutamicum, enteric bacteria, in particular Escherichia coli, Bacillus, in particular B. subtilis and B. methanolicus, and Saccharomyces, in particular S. cerevisiae. Particularly suitable are C. glutamicum or B. methanolicus strains which have been developed for the industrial production of lysine.

[0152] The invention further relates to a micro-organism, which may be a wild-type micro-organism isolated from its natural environment or a recombinant micro-organism, comprising DNA containing a nucleic acid sequence as identified in any Sequence ID selected from the group of Sequence ID 3, Sequence ID 6, Sequence ID 13, Sequence ID No. 32, Sequence ID No. 35, Sequence ID No. 41, Sequence ID No. 44, Sequence ID No. 47, and functional analogues thereof.

[0153] Functional analogues of a nucleotides sequence, as referred to herein, are in particular nucleotide sequences encoding the same amino acid sequence as that nucleotide sequence or encoding a homologue of that nucleotide sequence. In particular, preferred functional analogues are nucleotide sequence having a similar, the same or a better level of expression in a host cell of interest as the nucleotide sequence of which it is referred to as being a functional analogue of.

[0154] The invention further relates to a polynucleotide or vector comprising a nucleic acid sequence as identified in any Sequence ID selected from the group of Sequence ID 3, Sequence ID 6, Sequence ID 13, Sequence ID No. 32, Sequence ID No. 35, Sequence ID No. 41, Sequence ID No. 44, Sequence ID No. 47 and non-wild-type functional analogues thereof. Such polynucleotide or vector is in particular advantageous for providing a host cell, especially an E. coli host cell, or another host cell which is capable of catalysing at least one reaction step in the conversion of AKP to 6-ACA with a high yield, compared to a corresponding wild-type gene.

[0155] Optionally, the polynucleotide or vector comprises one or more nucleic acid sequences encoding one or more other biocatalysts suitable for catalysing a reaction step in a method according to the invention, in particular such one or more of the biocatalyst referred to above.

[0156] The invention further relates to a method for preparing alpha-aminopimelic acid (AAP), comprising converting AKP into AAP, which conversion is catalysed by a biocatalyst.

[0157] For such method in particular a biocatalyst may be used having aminotransferase activity or reductive amination activity as described above.

[0158] As indicated above, the AAP may thereafter be used for the preparation of 6-ACA. Alternatively, AAP may be used as such, e.g. as a chemical for biochemical research or as a pH-buffer compound, e.g. for use in an preparative or analytical separation technique such as liquid chromatography or capillary electrophoresis.

[0159] Further, AAP prepared in a method of the invention may further be used in the preparation of another compound, for instance, AAP may be converted into caprolactam. As described above, and illustrated in an example, below. AAP can be chemically converted in caprolactam, e.g. by exposure to a high temperature. Without being bound by theory, it is contemplated that also in this reaction 6-ACA may be formed as a short-lived intermediate.

[0160] Next, the invention will be illustrated by the following examples.

EXAMPLES

[0161] General Methods

[0162] Molecular and Genetic Techniques

[0163] Standard genetic and molecular biology techniques are generally known in the art and have been previously described (Maniatis et al. 1982 "Molecular cloning: a laboratory manual". Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Miller 1972 "Experiments in molecular genetics", Cold Spring Harbor Laboratory, Cold Spring Harbor; Sambrook and Russell 2001 "Molecular cloning: a laboratory manual" (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; F. Ausubel et al, eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York 1987).

[0164] Plasmids and Strains

[0165] pBAD/Myc-His C was obtained from Invitrogen (Carlsbad, Calif., USA). Plasmid pBAD/Myc-His-DEST constructed as described in WO2005/068643, was used for protein expression. E. coli TOP10 (Invitrogen, Carlsbad, Calif., USA) was used for all cloning procedures and for expression of target genes.

[0166] Media

[0167] LB medium (10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl) was used for growth of E. coli. Antibiotics (50 .mu.g/ml carbenicillin) were supplemented to maintain plasmids. For induction of gene expression under control of the P.sub.BAD promoter in pBAD/Myc-His-DEST derived plasmids, L-arabinose was added to a final concentration of 0.2% (w/v).

[0168] Identification of Plasmids

[0169] Plasmids carrying the different genes were identified by genetic, biochemical, and/or phenotypic means generally known in the art, such as resistance of transformants to antibiotics, PCR diagnostic analysis of transformant or purification of plasmid DNA, restriction analysis of the purified plasmid DNA or DNA sequence analysis.

[0170] HPLC-MS Analysis Method for the Determination of 5-FVA

[0171] 5-FVA was detected by selective reaction monitoring (SRM)-MS, measuring the transition m/z 1294.fwdarw.83. Concentrations for 5-FVA were calculated by measuring the peak area of the 5-FVA peak eluting at approximately 6 min. Calibration was performed by using an external standard procedure. All the LC-MS experiments were performed on an Agilent 1200 LC system, consisting of a quaternary pump, autosampler and column oven, coupled with an Agilent 6410 QQQ triple quadrupole MS.

LC Conditions:



[0172] Column: 50.times.4.6 mm Nucleosil C18, 5 .mu.m (Machery & Nagel) pre column coupled to a 250.times.4.6 mm id. Prevail C18, 5 .mu.m (Alltech)

[0173] Column temperature: room temperature

[0174] Eluent: A: water containing 0.1% formic acid

[0175] B: acetonitrile containing 0.1% formic acid

TABLE-US-00003 time (min) % eluent B Gradient: 0 10 6 50 6.1 10 11 10



[0176] Flow: 1.2 ml/min, before entering the MS the flow is split 1:3 Injection volume: 2 .mu.l

MS Conditions:

[0176]

[0177] Ionisation: negative ion electrospray

[0178] source conditions: ionspray voltage: 5 kV

[0179] temperature: 350.degree. C.

[0180] fragmentor voltage and collision energy optimized

[0181] Scan mode: selective reaction mode: transition m/z 129.fwdarw.83

[0182] HPLC-MS Analysis for the Determination of AAP

[0183] AAP was detected by selected ion monitoring (SIM)-MS, measuring the protonated molecule for AAP with m/z 176. Concentrations for AAP were calculated by measuring the peak area of the AAP peak eluting at a retention time of 2.7 minutes in the samples. Calibration was performed by using an external standard procedure. All the LC-MS experiments were performed on an Agilent 1100 LC system consisting of a quaternary pump, degasser, autosampler and column oven, coupled with an API 2000 triple quadrupole MS (Applied Biosystems).

[0184] LC conditions were as follows:

[0185] Column: 50*4 Nucleosil C18, 5 .mu.m (Macherey-Nagel)+250.times.4.6 Prevail C18, 5 .mu.m (Alltech), both at room temperature (RT)

[0186] Eluent: A=0.1% (v/v) formic acid in ultrapure water

[0187] B=0.1% (v/v) formic acid in acetonitrile (pa, Merck)

[0188] Flow: 1.2 ml/min, before entering the MS the flow was split 1:3

[0189] Gradient: The gradient was started at t=0 minutes with 90% (v/v) A and changed within 6 minutes to 50% (v/v) A. At 6.1 minutes the gradient was changed to the original condition.

[0190] Injection volume: 2 .mu.l

[0191] MS conditions: Positive ion electrospray was used for ionization

[0192] Detection: in SIM mode on m/z 176, with a dwell time of 100 msec.

[0193] HPLC-MS Analysis for the Determination of 6-ACA

[0194] Calibration:

[0195] The calibration was performed by an external calibration line of 6-ACA (m/z 132.fwdarw.m/z 114, Rt 7.5 min). All the LC-MS experiments were performed on an Agilent 1100, equipped with a quaternary pump, degasser, autosampler, column oven, and a single-quadrupole MS (Agilent, Waldbronn, Germany). The LC-MS conditions were:

[0196] Column: 50*4 Nucleosil (Mancherey-Nagel)+250.times.4.6 Prevail C18 (Alltech), both at room temperature (RT)

[0197] Eluent: A=0.1(v/v) formic acid in ultrapure water

[0198] B=Acetonitrile (pa, Merck)

[0199] Flow: 1.0 ml/min, before entering the MS the flow was split 1:3

[0200] Gradient: The gradient was started at t=0 minutes with 100% (v/v) A, remaining for 15 minutes and changed within 15 minutes to 80% (v/v) B (t=30 minutes). From 30 to 31 minutes the gradient was kept at constant at 80% (v/v) B.

[0201] Injection volume: 5 .mu.l

[0202] MS detection: ESI(+)-MS

[0203] The electrospray ionization (ESI) was run in the positive scan mode with the following conditions; m/z 50-500, 50 V fragmentor, 0.1 m/z step size, 350.degree. C. drying gas temperature, 10 L N.sub.2/min drying gas, 50 psig nebuliser pressure and 2.5 kV capillary voltage.

[0204] Cloning of Target Genes

[0205] Design of Expression Constructs

[0206] attB sites were added to all genes upstream of the ribosomal binding site and start codon and downstream of the stop codon to facilitate cloning using the Gateway technology (Invitrogen, Carlsbad, Calif., USA).

[0207] Gene Synthesis and Construction of Plasmids

[0208] Synthetic genes were obtained from DNA2.0 and codon optimised for expression in E. coli according to standard procedures of DNA2.0. The aminotransferase genes from Vibrio fluvialis JS17 [SEQ ID No. 1] and Bacillus weihenstephanensis KBAB4 [SEQ ID No. 4] encoding the amino acid sequences of the V. fluvialis JS17 .omega.-aminotransferase [SEQ ID No. 2] and the B. weihenstephanensis KBAB4 aminotransferase (ZP_01186960) [SEQ ID No. 5], respectively, were codon optimised and the resulting sequences [SEQ ID No. 3] and [SEQ ID No. 6] were obtained by DNA synthesis.

[0209] The decarboxylase genes from Escherichia coli [SEQ ID No. 30], Saccharomyces cerevisiae [SEQ ID No. 33], Zymomonas mobilis [SEQ ID No. 36], Lactococcus lactis [SEQ ID No. 39], [SEQ ID No. 42], and Mycobacterium tuberculosis [SEQ ID No. 45] encoding the amino acid sequences of the V. fluvialis JS17 .omega.-aminotransferase [SEQ ID No. 3], the B. weihenstephanensis KBAB4 aminotransferase (ZP_01186960) [SEQ ID No. 6], the Escherichia coli diaminopimelate decarboxylase LysA [SEQ ID No. 31], the Saccharomyces cerevisiae pyruvate decarboxylase Pdc [SEQ ID No. 34], the Zymomonas mobilis pyruvate decarboxylase Pdc1472A [SEQ ID No. 37], the Lactococcus lactis branched chain alpha-keto acid decarboxylase KdcA [SEQ ID No. 40] and alpha-ketoisovalerate decarboxylase KivD [SEQ ID No. 43], and the Mycobacterium tuberculosis alpha-ketoglutarate decarboxylase Kgd [SEQ ID No. 46], respectively, were also codon optimised and the resulting sequences [SEQ ID No. 32], [SEQ ID No. 35], [SEQ ID No. 38], [SEQ ID No. 41], [SEQ ID No. 44], and [SEQ ID No. 47] were obtained by DNA synthesis, respectively.

[0210] The gene constructs were cloned into pBAD/Myc-His-DEST expression vectors using the Gateway technology (Invitrogen) via the introduced attB sites and pDONR201 (Invitrogen) as entry vector as described in the manufacturer's protocols (www.invitrogen.com). This way the expression vectors pBAD-Vfl_AT and pBAD-Bwe_AT were obtained, respectively. The corresponding expression strains were obtained by transformation of chemically competent E. coli TOP10 (Invitrogen) with the respective pBAD-expression vectors.

[0211] Cloning by PCR

[0212] Various genes encoding a biocatalyst were amplified from genomic DNA by PCR using PCR Supermix High Fidelity (Invitrogen) according to the manufacturer's specifications, using primers as listed in the following table.

TABLE-US-00004 TABLE 2 enzyme primer origin of gene gene Sequence ID Sequence ID Sequence ID's Pseudomonas 7 8 9&10 aeruginosa Pseudomonas 26 27 60&61 aeruginosa Pseudomonas 66 67 72&73 aeruginosa Pseudomonas 68 69 74&75 aeruginosa Bacillus subtilis 14 15 48&49 Bacillus subtilis 16 17 50&51 Bacillus subtilis 64 65 70&71 Rhodobacter 18 19 52&53 sphaeroides Legionella 20 21 54&55 pneumophilia Nitrosomas europaea 22 23 56&57 Neisseria 24 25 58&59 gonorrhoeae Rhodopseudomonas 28 29 62&63 palustris

[0213] PCR reactions were analysed by agarose gel electrophoresis and PCR products of the correct size were eluted from the gel using the QIAquick PCR purification kit (Qiagen, Hilden, Germany). Purified PCR products were cloned into pBAD/Myc-His-DEST expression vectors using the Gateway technology (Invitrogen) via the introduced attB sites and pDONR-zeo (Invitrogen) as entry vector as described in the manufacturer's protocols. The sequence of genes cloned by PCR was verified by DNA sequencing. This way the expression vectors pBAD-Pae-_gi9946143_AT, pBAD-Bsu_gi16078032_AT, pBAD-Bsu_gi16080075_AT, pBAD-Bsu_gi16077991_AT, pBAD-Rsp_AT , pBAD-Lpn_AT, pBAD-Neu_AT, pBAD-Ngo_AT, pBAD-Pae_gi9951299_AT, pBAD-Pae_gi9951072_AT, pBAD-Pae_gi9951630_AT and pBAD-Rpa_AT were obtained. The corresponding expression strains were obtained by transformation of chemically competent E. coli TOP10 (Invitrogen) with the pBAD constructs.

[0214] Growth of E. coli for Protein Expression

[0215] Small scale growth was carried out in 96-deep-well plates with 940 .mu.l media containing 0.02% (w/v) L-arabinose. Inoculation was performed by transferring cells from frozen stock cultures with a 96-well stamp (Kuhner, Birsfelden, Switzerland). Plates were incubated on an orbital shaker (300 rpm, 5 cm amplitude) at 25.degree. C. for 48 h. Typically an OD.sub.620nm of 2-4 was reached.

[0216] Preparation of Cell Lysates

[0217] Preparation of Lysis Buffer

[0218] The lysis buffer contained the following ingredients:

TABLE-US-00005 TABLE 3 1M MOPS pH 7.5 5 ml DNAse I grade II (Roche) 10 mg Lysozyme 200 mg MgSO.sub.4.cndot.7H.sub.2O 123.2 mg dithiothreitol (DTT) 154.2 mg H.sub.2O (MilliQ) Balance to 100 ml

[0219] The solution was freshly prepared directly before use.

[0220] Preparation of Cell Free Extract by Lysis

[0221] Cells from small scales growth (see previous paragraph) were harvested by centrifugation and the supernatant was discarded. The cell pellets formed during centrifugation were frozen at -20.degree. C. for at least 16 h and then thawed on ice. 500 .mu.l of freshly prepared lysis buffer were added to each well and cells were resuspended by vigorously vortexing the plate for 2-5 min. To achieve lysis, the plate was incubated at room temperature for 30 min. To remove cell debris, the plate was centrifuged at 4.degree. C. and 6000 g for 20 min. The supernatant was transferred to a fresh plate and kept on ice until further use.

[0222] Preparation of Cell Free Extract by Sonification

[0223] Cells from medium scales growth (see previous paragraph) were harvested by centrifugation and the supernatant was discarded. 1 ml of potassium phosphate buffer pH7 was added to 0.5 g of wet cell pellet and cells were resuspended by vigorously vortexing. To achieve lysis, the cells were sonicated for 20 min. To remove cell debris, the lysates were centrifuged at 4.degree. C. and 6000 g for 20 min. The supernatant was transferred to a fresh tube and frozen at -20.degree. C. until further use.

[0224] Preparation of 5-formylpentanoic Acid by Chemical Hydrolysis of Methyl 5-formylpentanoate

[0225] The substrate for the aminotransferase reaction i.e. 5-formylpentanoic acid was prepared by chemical hydrolysis of methyl 5-formylpentanoate as follows: a 10% (w/v) solution of methyl 5-formylpentanoate in water was set at pH 14.1 with NaOH. After 24 h of incubation at 20.degree. C. the pH was set to 7.1 with HCl.

[0226] Enzymatic Reactions for Conversion of 5-formylpentanoic Acid to 6-ACA

[0227] Unless specified otherwise, a reaction mixture was prepared comprising 10 mM 5-formylpentanoic acid, 20 mM racemic .alpha.-methylbenzylamine, and 200 .mu.M.quadrature. pyridoxal 5'-phosphate in 50 mM potassium phosphate buffer, pH 7.0. 100 .mu.l of the reaction mixture were dispensed into each well of the well plates. To start the reaction, 20 .mu.l of the cell free extracts were added, to each of the wells. Reaction mixtures were incubated on a shaker at 37.degree. C. for 24 h. Furthermore, a chemical blank mixture (without cell free extract) and a biological blank (E. coli TOP10 with pBAD/Myc-His C) were incubated under the same conditions. Samples were analysed by HPLC-MS. The results are summarised in the following table.

TABLE-US-00006 TABLE 4 6-ACA formation from 5-FVA in the presence of aminotransferases 6-ACA Biocatalyst concentration [mg/kg] E. coli TOP10/pBAD-Vfl_AT 43* E. coli TOP10/pBAD-Pae_AT 930 E. coli TOP10/pBAD-Pae_AT 25* E. coli TOP10/pBAD-Bwe_AT 24* E. coli TOP10/pBAD-Bsu_gi16077991_AT 288 E. coli TOP10/pBAD-Pae_gi9951072_AT 1087 E. coli TOP10/pBAD-Pae_gi9951630_AT 92 E. coli TOP10 with pBAD/Myc-His C (biological 0.6 blank) None (chemical blank) n.d. n.d.: not detectable *method differed in that 10 .mu.l cell free extract was used instead of 20 .mu.l, the pyridoxal-5'-phosphate concentration was 50 .mu.M instead of 200 .mu.M and the reaction mixture volume in the wells was 190 .mu.l instead of 100 .mu.l.

[0228] It is shown that 6-ACA is formed from 5-FVA in the presence of an aminotransferase.

[0229] Enzymatic Reactions for Conversion of AKP to 5-formylpentanoic Acid

[0230] A reaction mixture was prepared comprising 50 mM AKP, 5 mM magnesium chloride, 100 .mu.M.quadrature. pyridoxal 5'-phosphate (for LysA) or 1 mM thiamine diphosphate (for all other enzymes) in 100 mM potassium phosphate buffer, pH 6.5. 4 ml of the reaction mixture were dispensed into a reaction vessel. To start the reaction, 1 ml of the cell free extracts obtained by sonification were added, to each of the wells. In case of the commercial oxaloacetate decarboxylase (Sigma-Aldrich product number 04878), 50 U were used. Reaction mixtures were incubated with a magnetic stirrer at 37.degree. C. for 48 h. Furthermore, a chemical blank mixture (without cell free extract) and a biological blank (E. coli TOP10 with pBAD/Myc-His C) were incubated under the same conditions. Samples from different time points during the reaction were analysed by HPLC-MS. The results are summarised in the following table.

TABLE-US-00007 TABLE 5 5-FVA formation from AKP in the presence of decarboxylases 5-FVA concentration [mg/kg] Biocatalyst 3 h 18 h 48 h E. coli TOP10/pBAD-LysA 150 590 720 E. coli TOP10/pBAD-Pdc 1600 1700 1300 E. coli TOP10/pBAD-PdcI472A 2000 2000 1600 E. coli TOP10/pBAD-KdcA 3300 2300 2200 E. coli TOP10/pBAD-KivD 820 1400 1500 Oxaloacetate decarboxylase n.d. 6 10 E. coli TOP10 with pBAD/Myc- n.d. n.d. n.d. His C (biological blank) None (chemical blank) n.d. n.d. n.d. n.d.: not detectable

[0231] It is shown that 5-FVA is formed from AKP in the presence of a decarboxylase.

[0232] Enzymatic Reactions for Conversion of AKP to 6-ACA in Presence of Recombinant Decarboxylase

[0233] A reaction mixture was prepared comprising 50 mM AKP, 5 mM magnesium chloride, 100 .mu.M.quadrature. pyridoxal 5'-phosphate (for LysA) or 1 mM thiamine diphosphate (for all other tested biocatalysts) in 100 mM potassium phosphate buffer, pH 6.5. 4 ml of the reaction mixture were dispensed into a reaction vessel. To start the reaction, 1 ml of the cell free extracts were added, to each of the wells. Reaction mixtures were incubated with a magnetic stirrer at 37.degree. C. for 48 h. Furthermore, a chemical blank mixture (without cell free extract) and a biological blank (E. coli TOP10 with pBAD/Myc-His C) were incubated under the same conditions. Samples from different time points during the reaction were analysed by HPLC-MS. The results are summarised in the following table.

TABLE-US-00008 TABLE 6 6-ACA formation from AKP in the presence of decarboxylases 6-ACA concentration [mg/kg] Biocatalyst 3 h 18 h 48 h E. coli TOP10/pBAD-LysA n.a. 0.01 0 E. coli TOP10/pBAD-Pdc 0.1 0.3 n.a. E. coli TOP10/pBAD-PdcI472A 0.03 0.1 0.2 E. coli TOP10/pBAD-KdcA 0.04 0.1 0.3 E. coli TOP10/pBAD-KivD n.a. 0.3 0.6 E. coli TOP10 with pBAD/Myc- n.d. n.d. n.d. His C (biological blank) None (chemical blank) n.d. n.d. n.d. n.a. = not analysed n.d. = not detectable

[0234] It is shown that 6-ACA is formed from AKP in the presence of a decarboxylase. It is contemplated that the E. coli contained natural 5-FVA aminotransferase activity.

[0235] Enzymatic Reactions for Conversion of AKP to 6-ACA in Presence of Recombinant Decarboxylase and Recombinant Aminotransferase

[0236] A reaction mixture was prepared comprising 50 mM AKP, 5 mM magnesium chloride, 100 .mu.M.quadrature. pyridoxal 5'-phosphate, 1 mM thiamine diphosphate and 50 mM racemic .alpha.-methylbenzylamine in 100 mM potassium phosphate buffer, pH 6.5. 1.6 ml of the reaction mixture were dispensed into a reaction vessel. To start the reaction, 0.2 ml of the decarboxylase containing cell free extract and 0.2 ml of the aminotransferase containing cell free extract were added, to each of the reaction vessels. Reaction mixtures were incubated with a magnetic stirrer at 37.degree. C. for 48 h. Furthermore, a chemical blank mixture (without cell free extract) and a biological blank (E. coli TOP10 with pBAD/Myc-His C) were incubated under the same conditions. Samples from different time points during the reaction were analysed by HPLC-MS. The results are summarised in the following table.

TABLE-US-00009 TABLE 7 6-ACA formation from AKP in the presence of a recombinant decarboxylase and a recombinant aminotransferase 6-ACA concentration [mg/kg] after 48 hours AT E. coli TOP10/ E. coli TOP10/ E. coli TOP10/ pBAD- pBAD- DC pBAD-Vfl-AT Bwe-AT PAE_gi9946143_AT E. coli TOP10/ 183.4 248.9 117.9 pBAD-Pdc E. coli TOP10/ 458.5 471.6 170.3 pBAD-PdcI472A E. coli TOP10/ 497.8 497.8 275.1 pBAD-KdcA E. coli TOP10/ 510.9 510.9 314.4 pBAD-KivD AT = aminotransferase DC = decarboxylase

[0237] In the chemical blank and in the biological blank no 6-ACA was detectable.

[0238] Further, the results show that compared to the example wherein a host-cell with only recombinant decarboxylase (and no recombinant aminotransferase) the conversion to 6-ACA was improved.

[0239] Construction of Plasmids for Expression of Aminotransferases and Decarboxylases in S. cerevisiae

[0240] The aminotransferase gene from Vibrio fluvialis JS17 encoding the amino acid sequence of the V. fluvialis JS17 .omega.-aminotransferase [SEQ ID No. 2] was amplified by PCR from pBAD-Vfl_AT [SEQ ID No. 3] using Phusion DNA polymerase (Finnzymes) according to the manufacturers specifications and using specific primers [SEQ ID No. 76 & 77].

[0241] The aminotransferase gene from Pseudomonas aeruginosa [SEQ ID No. 7] coding for P. aeruginosa aminotransferase [SEQ ID No. 8] was amplified from pBAD-Pae_AT by PCR using Phusion DNA polymerase (Finnzymes) according to the manufacturers specifications and using specific primers [SEQ ID No. 78 & 79].

[0242] The resulting PCR products were cloned into vector pAKP-41 using SpeI and BamHI restriction enzymes resulting in vectors pAKP-79 and pAKP-80 respectively, which now contain the aminotransferase gene under the S. cerevisiae gal10 promoter and the S. cerevisiae adh2 terminator.

[0243] The decarboxylase gene from Saccharamyces cerevisiae [SEQ ID No. 33] coding for Saccharamyces cerevisiae pyruvate decarboxylase Pdc [SEQ ID No. 34] was amplified from pBAD-Pdc by PCR using Phusion DNA polymerase (Finnzymes) according to the manufacturers specifications and using specific primers [SEQ ID No 80 & 81].

[0244] The decarboxylase gene from Lactococcus lactis [SEQ ID No. 39] coding for Lactococcus lactis branched chain alpha-keto acid decarboxylase KdcA [SEQ ID No. 40] was amplified from pBAD-KdcA by PCR using Phusion DNA polymerase (Finnzymes) according to the manufacturers specifications and using specific primers [SEQ ID No 82 & 83].

[0245] The resulting PCR products were cloned into vector pAKP-44 using AscI and BamHI restriction enzymes resulting in vectors pAKP-81 and pAKP-82 respectively, which now contain the decarboxylase gene under the S. cerevisiae gal2 promoter and the S. cerevisiae pma1 terminator.

[0246] Plasmids pAKP-79 and pAKP-80 were restriction enzyme digested with SacI and XbaI and plasmids pAKP-81 and pAKP-82 were restriction enzyme digested with SalI and XbaI. A SacI/XbaI aminotransferase fragment was combined with a SalI/XbaI decarboxylase fragment into the S. cerevisiae low copy episomal vector pRS414, which was restriction enzyme digested with SalI and SacI.

[0247] The resulting plasmids were obtained:

[0248] pAKP-85: Pgal10-Pae_AT-Tadh2 Pgal2-Pdc_DC-Tpma1

[0249] pAKP-86: Pgal10-Pae_AT-Tadh2 Pgal2-KdcA_DC-Tpma1

[0250] pAKP-87: Pgal10-Vfl_AT-Tadh2 Pgal2-Pdc_DC-Tpma1

[0251] pAKP-88: Pgal10-Vfl_AT-Tadh2 Pgal2-KdcA_DC-Tpma1

[0252] Transformation and Growth of S. cerevisiae

[0253] S. cerevisiae strain CEN.PK113-3C was transformed with 1 .mu.g of plasmid DNA according to the method as described by Gietz and Woods (Gietz, R. D. and Woods, R. A. (2002). Transformation of yeast by the Liac/SS carrier DNA/PEG method. Methods in Enzymology 350: 87-96). Cells were plated on agar plates with 1.times. Yeast Nitrogen Base without amino acids and 2% glucose.

[0254] The resulting strains were grown aerobically at 30.degree. C. for 48 hour in Verduyn minimal medium containing 0.05% glucose and 4% galactose.

[0255] Preparation of Cell Free Extract

[0256] 1 ml of potassium phosphate buffer (pH 7) was added to 0.5 g of the cell pellet. This mixture was added to a 2 ml eppendorf tube which contained 0.5 g of glassbeads with a diameter of 0.4-0.5 mM. Samples were vigorously shaken with an eppendorf shaker (IKA VIBRAX-VXR) for 20 s. The resulting cell free extract was centrifuged for 5 minutes at 14000 rpm and 4.degree. C. The supernatant was used for enzyme activity assays.

[0257] Enzymatic Reactions for Conversion of AKP to 6-ACA in Presence of Decarboxylase and Aminotransferase Co-Expressed in S. cerevisiae

[0258] A reaction mixture was prepared comprising 50 mM AKP, 5 mM magnesium chloride, 100 .mu.M.quadrature. pyridoxal 5'-phosphate, 1 mM thiamine diphosphate and 50 mM racemic .alpha.-methylbenzylamine in 100 mM potassium phosphate buffer, pH 6.5. 1.6 ml of the reaction mixture were dispensed into a reaction vessel. To start the reaction, 0.4 ml of the cell free extract from S. cerevisiae containing decarboxylase and aminotransferase were added, to each of the reaction vessels. Reaction mixtures were incubated with a magnetic stirrer at 37.degree. C. Furthermore, a chemical blank mixture (without cell free extract) and a biological blank (S. cerevisiae) were incubated under the same conditions. Samples, taken after 19 hours of incubation, were analysed by HPLC-MS. The results are summarised in the following table.

TABLE-US-00010 TABLE 8 6-ACA formation from AKP using a micro-organism as a biocatalyst 6-ACA concentration Biocatalyst [mg/kg] S. cerevisiae pAKP-85 63 S. cerevisiae pAKP-86 226 S. cerevisiae pAKP-87 1072 S. cerevisiae pAKP-88 4783 S. cerevisiae 3.9 (biological blank) None (chemical blank) 1.3

[0259] Enzymatic Reactions for Conversion of Alpha-Ketopimelic Acid to Alpha-Aminopimelic Acid

[0260] A reaction mixture was prepared comprising 10 mM alpha-ketopimelic acid, 20 mM L-alanine, and 50 .mu.M.quadrature. pyridoxal 5'-phosphate in 50 mM potassium phosphate buffer, pH 7.0. 800 .mu.l of the reaction mixture were dispensed into each well of the well plates. To start the reaction, 200 .mu.l of the cell lysates were added, to each of the wells. Reaction mixtures were incubated on a shaker at 37.degree. C. for 24 h. Furthermore, a chemical blank mixture (without cell free extract) and a biological blank (E. coli TOP10 with pBAD/Myc-His C) were incubated under the same conditions. Samples were analysed by HPLC-MS. The results are summarised in the following table.

TABLE-US-00011 TABLE 9 AAP formation from AKP in the presence of aminotransferases AAP concentration [mg/kg] Biocatalyst (after 24 hrs) E. coli TOP10/pBAD-Vfl_AT 3.7 E. coli TOP10/pBAD-Psy_AT 15.8 E. coli TOP10/pBAD-Bsu_gi16078032_AT 11.2 E. coli TOP10/pBAD-Rsp_AT 9.8 E. coli TOP10/pBAD-Bsu_gi16080075_AT 4.6 E. coli TOP10/pBAD-Lpn_AT 5.4 E. coli TOP10/pBAD-Neu_AT 7.7 E. coli TOP10/pBAD-Ngo_AT 5.1 E. coli TOP10/pBAD-Pae_gi9951299_AT 5.6 E. coli TOP10/pBAD-Rpa_AT 5.4 E. coli TOP10 with pBAD/Myc-His C 1.4 (biological blank) None (chemical blank) 0

[0261] It is shown that the formation of AAP from AKP is catalysed by the biocatalyst.

[0262] Chemical Conversion of AAP to Caprolactam

[0263] To a suspension of 1.5 grams of D,L-2-aminopimelic acid in 21 ml cyclohexanone, 0.5 ml of cyclohexenone was added. The mixture was heated on an oil bath for 20 h at reflux (approximately 160.degree. C.). After cooling to room temperature the reaction mixture was decanted and the clear solution was evaporated under reduced pressure. The remaining 2 grams of brownish oil were analyzed by .sup.1H-NMR and HPLC and contained 0.8 wt % caprolactam and 6 wt % of cyclic oligomers of caprolactam.

Sequence CWU 1

1

8311362DNAVibrio fluvialisCDS(1)..(1362) 1atg aac aaa ccg caa agc tgg gaa gcc cgg gcc gag acc tat tcg ctc 48Met Asn Lys Pro Gln Ser Trp Glu Ala Arg Ala Glu Thr Tyr Ser Leu 1 5 10 15 tat ggt ttc acc gac atg cct tcg ctg cat cag cgc ggc acg gtc gtc 96Tyr Gly Phe Thr Asp Met Pro Ser Leu His Gln Arg Gly Thr Val Val 20 25 30 gtg acc cat ggc gag gga ccc tat atc gtc gat gtg aat ggc cgg cgt 144Val Thr His Gly Glu Gly Pro Tyr Ile Val Asp Val Asn Gly Arg Arg 35 40 45 tat ctg gac gcc aac tcg ggc ctg tgg aac atg gtc gcg ggc ttt gac 192Tyr Leu Asp Ala Asn Ser Gly Leu Trp Asn Met Val Ala Gly Phe Asp 50 55 60 cac aag ggg ctg atc gac gcc gcc aag gcc caa tac gag cgt ttt ccc 240His Lys Gly Leu Ile Asp Ala Ala Lys Ala Gln Tyr Glu Arg Phe Pro 65 70 75 80 ggt tat cac gcc ttt ttc ggc cgc atg tcc gat cag acg gta atg ctg 288Gly Tyr His Ala Phe Phe Gly Arg Met Ser Asp Gln Thr Val Met Leu 85 90 95 tcg gaa aag ctg gtc gag gtg tcg ccc ttt gat tcg ggc cgg gtg ttc 336Ser Glu Lys Leu Val Glu Val Ser Pro Phe Asp Ser Gly Arg Val Phe 100 105 110 tat aca aac tcg ggg tcc gag gcg aat gac acc atg gtc aag atg cta 384Tyr Thr Asn Ser Gly Ser Glu Ala Asn Asp Thr Met Val Lys Met Leu 115 120 125 tgg ttc ctg cat gca gcc gag ggc aaa ccg caa aag cgc aag atc ctg 432Trp Phe Leu His Ala Ala Glu Gly Lys Pro Gln Lys Arg Lys Ile Leu 130 135 140 acc cgc tgg aac gcc tat cac ggc gtg acc gcc gtt tcg gcc agc atg 480Thr Arg Trp Asn Ala Tyr His Gly Val Thr Ala Val Ser Ala Ser Met 145 150 155 160 acc ggc aag ccc tat aat tcg gtc ttt ggc ctg ccg ctg ccg ggc ttt 528Thr Gly Lys Pro Tyr Asn Ser Val Phe Gly Leu Pro Leu Pro Gly Phe 165 170 175 gtg cat ctg acc tgc ccg cat tac tgg cgc tat ggc gaa gag ggc gaa 576Val His Leu Thr Cys Pro His Tyr Trp Arg Tyr Gly Glu Glu Gly Glu 180 185 190 acc gaa gag cag ttc gtc gcc cgc ctc gcc cgc gag ctg gag gaa acg 624Thr Glu Glu Gln Phe Val Ala Arg Leu Ala Arg Glu Leu Glu Glu Thr 195 200 205 atc cag cgc gag ggc gcc gac acc atc gcc ggt ttc ttt gcc gaa ccg 672Ile Gln Arg Glu Gly Ala Asp Thr Ile Ala Gly Phe Phe Ala Glu Pro 210 215 220 gtg atg ggc gcg ggc ggc gtg att ccc ccg gcc aag ggc tat ttc cag 720Val Met Gly Ala Gly Gly Val Ile Pro Pro Ala Lys Gly Tyr Phe Gln 225 230 235 240 gcg atc ctg cca atc ctg cgc aaa tat gac atc ccg gtc atc tcg gac 768Ala Ile Leu Pro Ile Leu Arg Lys Tyr Asp Ile Pro Val Ile Ser Asp 245 250 255 gag gtg atc tgc ggt ttc gga cgc acc ggt aac acc tgg ggc tgc gtg 816Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Asn Thr Trp Gly Cys Val 260 265 270 acc tat gac ttt aca ccc gat gca atc atc tcg tcc aag aat ctt aca 864Thr Tyr Asp Phe Thr Pro Asp Ala Ile Ile Ser Ser Lys Asn Leu Thr 275 280 285 gcg ggc ttt ttc ccc atg ggg gcg gtg atc ctt ggc ccg gaa ctt tcc 912Ala Gly Phe Phe Pro Met Gly Ala Val Ile Leu Gly Pro Glu Leu Ser 290 295 300 aaa cgg ctg gaa acc gca atc gag gcg atc gag gaa ttc ccc cat ggc 960Lys Arg Leu Glu Thr Ala Ile Glu Ala Ile Glu Glu Phe Pro His Gly 305 310 315 320 ttt acc gcc tcg ggc cat ccg gtc ggc tgt gct att gcg ctg aaa gca 1008Phe Thr Ala Ser Gly His Pro Val Gly Cys Ala Ile Ala Leu Lys Ala 325 330 335 atc gac gtg gtg atg aat gaa ggg ctg gct gag aac gtc cgc cgc ctt 1056Ile Asp Val Val Met Asn Glu Gly Leu Ala Glu Asn Val Arg Arg Leu 340 345 350 gcc ccc cgt ttc gag gaa agg ctg aaa cat atc gcc gag cgc ccg aac 1104Ala Pro Arg Phe Glu Glu Arg Leu Lys His Ile Ala Glu Arg Pro Asn 355 360 365 atc ggt gaa tat cgc ggc atc ggc ttc atg tgg gcg ctg gag gct gtc 1152Ile Gly Glu Tyr Arg Gly Ile Gly Phe Met Trp Ala Leu Glu Ala Val 370 375 380 aag gac aag gca agc aag acg ccg ttc gac ggc aac ctg tcg gtc agc 1200Lys Asp Lys Ala Ser Lys Thr Pro Phe Asp Gly Asn Leu Ser Val Ser 385 390 395 400 gag cgt atc gcc aat acc tgc acc gat ctg ggg ctg att tgc cgg ccg 1248Glu Arg Ile Ala Asn Thr Cys Thr Asp Leu Gly Leu Ile Cys Arg Pro 405 410 415 ctt ggt cag tcc gtc gtc ctt tgt ccg ccc ttt atc ctg acc gag gcg 1296Leu Gly Gln Ser Val Val Leu Cys Pro Pro Phe Ile Leu Thr Glu Ala 420 425 430 cag atg gat gag atg ttc gat aaa ctc gaa aaa gcc ctt gat aag gtc 1344Gln Met Asp Glu Met Phe Asp Lys Leu Glu Lys Ala Leu Asp Lys Val 435 440 445 ttt gcc gag gtt gcc tga 1362Phe Ala Glu Val Ala 450 2453PRTVibrio fluvialis 2Met Asn Lys Pro Gln Ser Trp Glu Ala Arg Ala Glu Thr Tyr Ser Leu 1 5 10 15 Tyr Gly Phe Thr Asp Met Pro Ser Leu His Gln Arg Gly Thr Val Val 20 25 30 Val Thr His Gly Glu Gly Pro Tyr Ile Val Asp Val Asn Gly Arg Arg 35 40 45 Tyr Leu Asp Ala Asn Ser Gly Leu Trp Asn Met Val Ala Gly Phe Asp 50 55 60 His Lys Gly Leu Ile Asp Ala Ala Lys Ala Gln Tyr Glu Arg Phe Pro 65 70 75 80 Gly Tyr His Ala Phe Phe Gly Arg Met Ser Asp Gln Thr Val Met Leu 85 90 95 Ser Glu Lys Leu Val Glu Val Ser Pro Phe Asp Ser Gly Arg Val Phe 100 105 110 Tyr Thr Asn Ser Gly Ser Glu Ala Asn Asp Thr Met Val Lys Met Leu 115 120 125 Trp Phe Leu His Ala Ala Glu Gly Lys Pro Gln Lys Arg Lys Ile Leu 130 135 140 Thr Arg Trp Asn Ala Tyr His Gly Val Thr Ala Val Ser Ala Ser Met 145 150 155 160 Thr Gly Lys Pro Tyr Asn Ser Val Phe Gly Leu Pro Leu Pro Gly Phe 165 170 175 Val His Leu Thr Cys Pro His Tyr Trp Arg Tyr Gly Glu Glu Gly Glu 180 185 190 Thr Glu Glu Gln Phe Val Ala Arg Leu Ala Arg Glu Leu Glu Glu Thr 195 200 205 Ile Gln Arg Glu Gly Ala Asp Thr Ile Ala Gly Phe Phe Ala Glu Pro 210 215 220 Val Met Gly Ala Gly Gly Val Ile Pro Pro Ala Lys Gly Tyr Phe Gln 225 230 235 240 Ala Ile Leu Pro Ile Leu Arg Lys Tyr Asp Ile Pro Val Ile Ser Asp 245 250 255 Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Asn Thr Trp Gly Cys Val 260 265 270 Thr Tyr Asp Phe Thr Pro Asp Ala Ile Ile Ser Ser Lys Asn Leu Thr 275 280 285 Ala Gly Phe Phe Pro Met Gly Ala Val Ile Leu Gly Pro Glu Leu Ser 290 295 300 Lys Arg Leu Glu Thr Ala Ile Glu Ala Ile Glu Glu Phe Pro His Gly 305 310 315 320 Phe Thr Ala Ser Gly His Pro Val Gly Cys Ala Ile Ala Leu Lys Ala 325 330 335 Ile Asp Val Val Met Asn Glu Gly Leu Ala Glu Asn Val Arg Arg Leu 340 345 350 Ala Pro Arg Phe Glu Glu Arg Leu Lys His Ile Ala Glu Arg Pro Asn 355 360 365 Ile Gly Glu Tyr Arg Gly Ile Gly Phe Met Trp Ala Leu Glu Ala Val 370 375 380 Lys Asp Lys Ala Ser Lys Thr Pro Phe Asp Gly Asn Leu Ser Val Ser 385 390 395 400 Glu Arg Ile Ala Asn Thr Cys Thr Asp Leu Gly Leu Ile Cys Arg Pro 405 410 415 Leu Gly Gln Ser Val Val Leu Cys Pro Pro Phe Ile Leu Thr Glu Ala 420 425 430 Gln Met Asp Glu Met Phe Asp Lys Leu Glu Lys Ala Leu Asp Lys Val 435 440 445 Phe Ala Glu Val Ala 450 31362DNAArtificialVibrio fluvialis JS17 omega-aminotransferase codon optimised gene 3atgaataaac cacagtcttg ggaagctcgt gctgaaacct atagcctgta cggctttacc 60gatatgccgt ctctgcacca gcgtggtact gtagtggtaa cgcacggtga gggcccgtac 120atcgtggacg ttaatggccg ccgttacctg gatgcaaaca gcggcctgtg gaacatggtt 180gcgggcttcg accacaaagg cctgatcgat gccgcaaaag cgcagtacga acgcttcccg 240ggttatcacg cgttctttgg ccgtatgagc gaccagactg tgatgctgag cgaaaaactg 300gttgaagtgt ccccgttcga tagcggtcgt gtcttttaca ctaactctgg cagcgaggct 360aacgatacca tggttaagat gctgtggttc ctgcacgcag cggaaggcaa acctcagaaa 420cgtaaaattc tgacccgttg gaacgcttat cacggtgtga ctgctgtttc cgcatctatg 480accggtaaac cgtataacag cgtgttcggt ctgccgctgc ctggcttcgt gcatctgacc 540tgcccgcact actggcgtta tggtgaggaa ggcgaaactg aggaacagtt cgtggcgcgt 600ctggctcgtg aactggaaga aaccattcaa cgcgaaggtg cagatactat cgcgggcttc 660tttgcggagc ctgttatggg tgccggcggt gtgattccgc cggcgaaggg ctatttccag 720gcaatcctgc cgatcctgcg caagtacgac attccggtta tttctgacga agtgatctgc 780ggcttcggcc gcaccggtaa cacctggggc tgcgtgacgt atgacttcac tccggacgca 840atcattagct ctaaaaacct gactgcgggt ttcttcccta tgggcgccgt aatcctgggc 900ccagaactgt ctaagcgcct ggaaaccgcc atcgaggcaa tcgaagagtt cccgcacggt 960ttcactgcta gcggccatcc ggtaggctgc gcaatcgcgc tgaaggcgat cgatgttgtc 1020atgaacgagg gcctggcgga aaacgtgcgc cgcctggcgc cgcgttttga agaacgtctg 1080aaacacattg ctgagcgccc gaacattggc gaatatcgcg gcatcggttt catgtgggcc 1140ctggaagcag ttaaagataa agctagcaag accccgttcg acggcaacct gtccgtgagc 1200gaacgtatcg ctaatacctg tacggacctg ggtctgatct gccgtccgct gggtcagtcc 1260gtagttctgt gcccaccatt tatcctgacc gaagcgcaga tggatgaaat gttcgataaa 1320ctggagaaag ctctggataa agtgttcgct gaagtcgcgt aa 136241350DNABacillus weihenstephanensisCDS(1)..(1350) 4gtg caa gcg acg gag caa aca caa agt ttg aaa aaa aca gat gaa aag 48Val Gln Ala Thr Glu Gln Thr Gln Ser Leu Lys Lys Thr Asp Glu Lys 1 5 10 15 tac ctt tgg cat gcg atg aga gga gca gcc cct agt cca acg aat tta 96Tyr Leu Trp His Ala Met Arg Gly Ala Ala Pro Ser Pro Thr Asn Leu 20 25 30 att atc aca aaa gca gaa ggg gca tgg gtg acg gat att gat gga aac 144Ile Ile Thr Lys Ala Glu Gly Ala Trp Val Thr Asp Ile Asp Gly Asn 35 40 45 cgt tat tta gac ggt atg tcc ggt ctt tgg tgc gtg aat gtt ggg tat 192Arg Tyr Leu Asp Gly Met Ser Gly Leu Trp Cys Val Asn Val Gly Tyr 50 55 60 ggt cga aaa gaa ctt gca aga gcg gcg ttt gaa cag ctt gaa gaa atg 240Gly Arg Lys Glu Leu Ala Arg Ala Ala Phe Glu Gln Leu Glu Glu Met 65 70 75 80 ccg tat ttc cct ctg act caa agt cat gtt cct gct att aaa tta gca 288Pro Tyr Phe Pro Leu Thr Gln Ser His Val Pro Ala Ile Lys Leu Ala 85 90 95 gaa aaa ttg aat gaa tgg ctt gat gat gaa tac gtc att ttc ttt tct 336Glu Lys Leu Asn Glu Trp Leu Asp Asp Glu Tyr Val Ile Phe Phe Ser 100 105 110 aac agt gga tcg gaa gcg aat gaa aca gca ttt aaa att gct cgt caa 384Asn Ser Gly Ser Glu Ala Asn Glu Thr Ala Phe Lys Ile Ala Arg Gln 115 120 125 tat cat caa caa aaa ggt gat cat gga cgc tat aag ttt att tcc cgc 432Tyr His Gln Gln Lys Gly Asp His Gly Arg Tyr Lys Phe Ile Ser Arg 130 135 140 tac cgc gct tat cac ggt aac tca atg gga gct ctt gca gca aca ggt 480Tyr Arg Ala Tyr His Gly Asn Ser Met Gly Ala Leu Ala Ala Thr Gly 145 150 155 160 caa gca cag cga aag tat aaa tat gaa cca ctc ggg caa gga ttc ctg 528Gln Ala Gln Arg Lys Tyr Lys Tyr Glu Pro Leu Gly Gln Gly Phe Leu 165 170 175 cat gta gca ccg cct gat acg tat cga aat cca gag gat gtt cat aca 576His Val Ala Pro Pro Asp Thr Tyr Arg Asn Pro Glu Asp Val His Thr 180 185 190 ctg gca agt gct gag gaa atc gat cgt gtc atg aca tgg gag tta agc 624Leu Ala Ser Ala Glu Glu Ile Asp Arg Val Met Thr Trp Glu Leu Ser 195 200 205 caa aca gta gcc ggt gtg att atg gag cca atc att act ggg ggc gga 672Gln Thr Val Ala Gly Val Ile Met Glu Pro Ile Ile Thr Gly Gly Gly 210 215 220 att tta atg cct cct gat gga tat atg gga aaa gta aaa gaa att tgc 720Ile Leu Met Pro Pro Asp Gly Tyr Met Gly Lys Val Lys Glu Ile Cys 225 230 235 240 gag aag cac ggt gcg ttg ctc att tgt gat gaa gtt ata tgt gga ttt 768Glu Lys His Gly Ala Leu Leu Ile Cys Asp Glu Val Ile Cys Gly Phe 245 250 255 ggc cgg aca ggg aag cca ttt gga ttt atg aat tat ggc gtc aaa cca 816Gly Arg Thr Gly Lys Pro Phe Gly Phe Met Asn Tyr Gly Val Lys Pro 260 265 270 gat atc att aca atg gca aaa ggt att aca agt gcg tat ctt cct ttg 864Asp Ile Ile Thr Met Ala Lys Gly Ile Thr Ser Ala Tyr Leu Pro Leu 275 280 285 tca gca aca gca gtt aga cga gag gtt tat gag gca ttc gta ggt agt 912Ser Ala Thr Ala Val Arg Arg Glu Val Tyr Glu Ala Phe Val Gly Ser 290 295 300 gat gat tat gat cgc ttc cgc cat gta aat acg ttc gga ggg aat cct 960Asp Asp Tyr Asp Arg Phe Arg His Val Asn Thr Phe Gly Gly Asn Pro 305 310 315 320 gct gct tgc gct tta gct ttg aag aat tta gaa att atg gag aat gag 1008Ala Ala Cys Ala Leu Ala Leu Lys Asn Leu Glu Ile Met Glu Asn Glu 325 330 335 aaa ctc att gaa cgt tcc aaa gaa ttg ggt gaa cga ctg tta tat gag 1056Lys Leu Ile Glu Arg Ser Lys Glu Leu Gly Glu Arg Leu Leu Tyr Glu 340 345 350 cta gag gat gta aaa gag cat cca aac gta ggg gat gtt cgc gga aag 1104Leu Glu Asp Val Lys Glu His Pro Asn Val Gly Asp Val Arg Gly Lys 355 360 365 ggc ctt ctt tta ggc att gaa cta gtg gaa gat aag caa aca aaa gaa 1152Gly Leu Leu Leu Gly Ile Glu Leu Val Glu Asp Lys Gln Thr Lys Glu 370 375 380 ccg gct tcc att gaa aag atg aac aaa gtc atc aat gct tgt aaa gaa 1200Pro Ala Ser Ile Glu Lys Met Asn Lys Val Ile Asn Ala Cys Lys Glu 385 390 395 400 aaa ggt cta att att ggt aaa aat ggt gac act gtc gca ggt tac aat 1248Lys Gly Leu Ile Ile Gly Lys Asn Gly Asp Thr Val Ala Gly Tyr Asn 405 410 415 aat att ttg cag ctt gca cct cca tta agc atc aca gag gaa gac ttt 1296Asn Ile Leu Gln Leu Ala Pro Pro Leu Ser Ile Thr Glu Glu Asp Phe 420 425 430 act ttt atc gtt aaa aca atg aaa gaa tgt tta tcc cgc att aac ggg 1344Thr Phe Ile Val Lys Thr Met Lys Glu Cys Leu Ser Arg Ile Asn Gly 435 440 445 cag taa 1350Gln 5449PRTBacillus weihenstephanensis 5Val Gln Ala Thr Glu Gln Thr Gln Ser Leu Lys Lys Thr Asp Glu Lys 1 5 10 15 Tyr Leu Trp His Ala Met Arg Gly Ala Ala Pro Ser Pro Thr Asn Leu 20 25 30

Ile Ile Thr Lys Ala Glu Gly Ala Trp Val Thr Asp Ile Asp Gly Asn 35 40 45 Arg Tyr Leu Asp Gly Met Ser Gly Leu Trp Cys Val Asn Val Gly Tyr 50 55 60 Gly Arg Lys Glu Leu Ala Arg Ala Ala Phe Glu Gln Leu Glu Glu Met 65 70 75 80 Pro Tyr Phe Pro Leu Thr Gln Ser His Val Pro Ala Ile Lys Leu Ala 85 90 95 Glu Lys Leu Asn Glu Trp Leu Asp Asp Glu Tyr Val Ile Phe Phe Ser 100 105 110 Asn Ser Gly Ser Glu Ala Asn Glu Thr Ala Phe Lys Ile Ala Arg Gln 115 120 125 Tyr His Gln Gln Lys Gly Asp His Gly Arg Tyr Lys Phe Ile Ser Arg 130 135 140 Tyr Arg Ala Tyr His Gly Asn Ser Met Gly Ala Leu Ala Ala Thr Gly 145 150 155 160 Gln Ala Gln Arg Lys Tyr Lys Tyr Glu Pro Leu Gly Gln Gly Phe Leu 165 170 175 His Val Ala Pro Pro Asp Thr Tyr Arg Asn Pro Glu Asp Val His Thr 180 185 190 Leu Ala Ser Ala Glu Glu Ile Asp Arg Val Met Thr Trp Glu Leu Ser 195 200 205 Gln Thr Val Ala Gly Val Ile Met Glu Pro Ile Ile Thr Gly Gly Gly 210 215 220 Ile Leu Met Pro Pro Asp Gly Tyr Met Gly Lys Val Lys Glu Ile Cys 225 230 235 240 Glu Lys His Gly Ala Leu Leu Ile Cys Asp Glu Val Ile Cys Gly Phe 245 250 255 Gly Arg Thr Gly Lys Pro Phe Gly Phe Met Asn Tyr Gly Val Lys Pro 260 265 270 Asp Ile Ile Thr Met Ala Lys Gly Ile Thr Ser Ala Tyr Leu Pro Leu 275 280 285 Ser Ala Thr Ala Val Arg Arg Glu Val Tyr Glu Ala Phe Val Gly Ser 290 295 300 Asp Asp Tyr Asp Arg Phe Arg His Val Asn Thr Phe Gly Gly Asn Pro 305 310 315 320 Ala Ala Cys Ala Leu Ala Leu Lys Asn Leu Glu Ile Met Glu Asn Glu 325 330 335 Lys Leu Ile Glu Arg Ser Lys Glu Leu Gly Glu Arg Leu Leu Tyr Glu 340 345 350 Leu Glu Asp Val Lys Glu His Pro Asn Val Gly Asp Val Arg Gly Lys 355 360 365 Gly Leu Leu Leu Gly Ile Glu Leu Val Glu Asp Lys Gln Thr Lys Glu 370 375 380 Pro Ala Ser Ile Glu Lys Met Asn Lys Val Ile Asn Ala Cys Lys Glu 385 390 395 400 Lys Gly Leu Ile Ile Gly Lys Asn Gly Asp Thr Val Ala Gly Tyr Asn 405 410 415 Asn Ile Leu Gln Leu Ala Pro Pro Leu Ser Ile Thr Glu Glu Asp Phe 420 425 430 Thr Phe Ile Val Lys Thr Met Lys Glu Cys Leu Ser Arg Ile Asn Gly 435 440 445 Gln 61350DNAArtificialB. weihenstephanensis KBAB4 aminotransferase codon-optimised gene 6atgcaggcta ccgaacaaac ccaatctctg aaaaagactg acgaaaaata tctgtggcac 60gcgatgcgcg gtgcagctcc gtctccgacc aacctgatta ttaccaaagc tgaaggcgcg 120tgggtgaccg acattgacgg taaccgttat ctggatggca tgagcggcct gtggtgtgtt 180aatgtcggtt atggccgtaa ggagctggcg cgcgcggcat ttgaacaact ggaagaaatg 240ccgtacttcc cgctgactca aagccatgtg ccggctatca aactggcgga aaaactgaac 300gaatggctgg acgacgaata cgtgattttc ttctctaatt ctggctccga agcaaacgaa 360accgcattca aaatcgcccg tcaatatcac cagcagaaag gtgaccacgg ccgctataaa 420ttcatcagcc gttatcgtgc ataccatggt aattctatgg gtgcgctggc tgctaccggt 480caggctcagc gcaaatacaa gtacgaaccg ctgggtcagg gttttctgca cgttgcacca 540ccggatacct accgtaaccc ggaagacgtc cacaccctgg cttctgccga agaaatcgat 600cgtgttatga cctgggagct gtcccagact gttgcgggtg ttatcatgga acctattatt 660accggtggtg gcattctgat gccgccggac ggttatatgg gtaaagtcaa ggaaatctgc 720gaaaaacacg gcgcgctgct gatctgcgat gaagttatct gtggcttcgg tcgcaccggc 780aaaccatttg gcttcatgaa ttatggcgta aaacctgaca ttattaccat ggctaaaggc 840attacttccg cttatctgcc gctgagcgcg accgcagttc gccgcgaagt ttatgaagcg 900tttgttggtt ctgatgatta cgaccgtttc cgtcatgtaa acacgtttgg cggtaaccca 960gcggcatgtg cgctggcgct gaaaaacctg gaaatcatgg aaaacgaaaa gctgatcgaa 1020cgtagcaaag aactgggtga acgtctgctg tacgaactgg aagatgtcaa agaacacccg 1080aacgtgggcg atgttcgcgg taaaggcctg ctgctgggta ttgaactggt tgaagacaaa 1140cagaccaagg aaccggcttc cattgaaaag atgaacaaag tgattaacgc gtgcaaagag 1200aaaggcctga tcattggtaa gaacggtgat accgtggcag gttataacaa cattctgcag 1260ctggcgccgc ctctgagcat cactgaagaa gatttcacct tcatcgtcaa aactatgaag 1320gagtgcctga gccgcatcaa tggtcagtaa 135071371DNAPseudomonas aeruginosaCDS(1)..(1371) 7atg aac agc caa atc acc aac gcc aag acc cgt gag tgg cag gcg ttg 48Met Asn Ser Gln Ile Thr Asn Ala Lys Thr Arg Glu Trp Gln Ala Leu 1 5 10 15 agc cgc gac cac cat ctg ccg ccg ttc acc gac tac aag cag ttg aac 96Ser Arg Asp His His Leu Pro Pro Phe Thr Asp Tyr Lys Gln Leu Asn 20 25 30 gag aag ggc gcg cgg atc atc acc aag gcc gaa ggc gtc tat atc tgg 144Glu Lys Gly Ala Arg Ile Ile Thr Lys Ala Glu Gly Val Tyr Ile Trp 35 40 45 gac agc gag ggc aac aag atc ctc gat gcg atg gcc ggc ctc tgg tgc 192Asp Ser Glu Gly Asn Lys Ile Leu Asp Ala Met Ala Gly Leu Trp Cys 50 55 60 gtc aac gtc ggc tac ggc cgc gag gag ctg gtc cag gcc gcc acc cgg 240Val Asn Val Gly Tyr Gly Arg Glu Glu Leu Val Gln Ala Ala Thr Arg 65 70 75 80 cag atg cgc gag ttg ccg ttc tac aac ctg ttc ttc cag acc gcc cac 288Gln Met Arg Glu Leu Pro Phe Tyr Asn Leu Phe Phe Gln Thr Ala His 85 90 95 ccg ccg gtg gtc gag ctg gcc aag gcg atc gcc gac gtc gct ccg gaa 336Pro Pro Val Val Glu Leu Ala Lys Ala Ile Ala Asp Val Ala Pro Glu 100 105 110 ggc atg aac cac gtg ttc ttc acc ggc tcc ggc tcc gag gcc aac gac 384Gly Met Asn His Val Phe Phe Thr Gly Ser Gly Ser Glu Ala Asn Asp 115 120 125 acc gtg ctg cgt atg gtc cgc cac tat tgg gcg acc aag ggc cag ccg 432Thr Val Leu Arg Met Val Arg His Tyr Trp Ala Thr Lys Gly Gln Pro 130 135 140 cag aag aaa gtg gtg atc ggc cgc tgg aac ggc tac cac ggc tcc acc 480Gln Lys Lys Val Val Ile Gly Arg Trp Asn Gly Tyr His Gly Ser Thr 145 150 155 160 gtc gcc ggc gtc agc ctg ggc ggc atg aag gcg ttg cat gag cag ggt 528Val Ala Gly Val Ser Leu Gly Gly Met Lys Ala Leu His Glu Gln Gly 165 170 175 gat ttc ccc atc ccg ggc atc gtc cac atc gcc cag ccc tac tgg tac 576Asp Phe Pro Ile Pro Gly Ile Val His Ile Ala Gln Pro Tyr Trp Tyr 180 185 190 ggc gag ggc ggc gac atg tcg ccg gac gag ttc ggc gtc tgg gcc gcc 624Gly Glu Gly Gly Asp Met Ser Pro Asp Glu Phe Gly Val Trp Ala Ala 195 200 205 gag cag ttg gag aag aag att ctc gaa gtg ggc gag gaa aac gtc gcc 672Glu Gln Leu Glu Lys Lys Ile Leu Glu Val Gly Glu Glu Asn Val Ala 210 215 220 gcc ttc atc gcc gag ccg atc cag ggc gcc ggc ggc gtg atc gtc ccg 720Ala Phe Ile Ala Glu Pro Ile Gln Gly Ala Gly Gly Val Ile Val Pro 225 230 235 240 ccg gac acc tac tgg ccg aag atc cgc gag atc ctc gcc aag tac gac 768Pro Asp Thr Tyr Trp Pro Lys Ile Arg Glu Ile Leu Ala Lys Tyr Asp 245 250 255 atc ctg ttc atc gcc gac gaa gtg atc tgc ggc ttc ggc cgt acc ggc 816Ile Leu Phe Ile Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly 260 265 270 gag tgg ttc ggc agc cag tac tac ggc aac gcc ccg gac ctg atg ccg 864Glu Trp Phe Gly Ser Gln Tyr Tyr Gly Asn Ala Pro Asp Leu Met Pro 275 280 285 atc gcc aag ggc ctc acc tcc ggc tac atc ccc atg ggc ggg gtg gtg 912Ile Ala Lys Gly Leu Thr Ser Gly Tyr Ile Pro Met Gly Gly Val Val 290 295 300 gtg cgc gac gag atc gtc gaa gtg ctc aac cag ggc ggc gag ttc tac 960Val Arg Asp Glu Ile Val Glu Val Leu Asn Gln Gly Gly Glu Phe Tyr 305 310 315 320 cac ggc ttc acc tat tcc ggt cac ccg gtg gcg gcc gcc gtg gcc ctg 1008His Gly Phe Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu 325 330 335 gag aac atc cgc atc ctg cgc gaa gag aag atc atc gag aag gtg aag 1056Glu Asn Ile Arg Ile Leu Arg Glu Glu Lys Ile Ile Glu Lys Val Lys 340 345 350 gcg gaa acg gca ccg tat ttg cag aaa cgc tgg cag gag ctg gcc gac 1104Ala Glu Thr Ala Pro Tyr Leu Gln Lys Arg Trp Gln Glu Leu Ala Asp 355 360 365 cac ccg ttg gtg ggc gaa gcg cgc ggg gtc ggc atg gtc gcc gcc ctg 1152His Pro Leu Val Gly Glu Ala Arg Gly Val Gly Met Val Ala Ala Leu 370 375 380 gag ctg gtc aag aac aag aag acc cgc gag cgt ttc acc gac aag ggc 1200Glu Leu Val Lys Asn Lys Lys Thr Arg Glu Arg Phe Thr Asp Lys Gly 385 390 395 400 gtc ggg atg ctg tgc cgg gaa cat tgt ttc cgc aac ggt ttg atc atg 1248Val Gly Met Leu Cys Arg Glu His Cys Phe Arg Asn Gly Leu Ile Met 405 410 415 cgc gcg gtg ggc gac act atg att atc tcg ccg ccg ctg gtg atc gat 1296Arg Ala Val Gly Asp Thr Met Ile Ile Ser Pro Pro Leu Val Ile Asp 420 425 430 ccg tcg cag atc gat gag ttg atc acc ctg gcg cgc aag tgc ctc gat 1344Pro Ser Gln Ile Asp Glu Leu Ile Thr Leu Ala Arg Lys Cys Leu Asp 435 440 445 cag acc gcc gcc gcc gtc ctg gct tga 1371Gln Thr Ala Ala Ala Val Leu Ala 450 455 8456PRTPseudomonas aeruginosa 8Met Asn Ser Gln Ile Thr Asn Ala Lys Thr Arg Glu Trp Gln Ala Leu 1 5 10 15 Ser Arg Asp His His Leu Pro Pro Phe Thr Asp Tyr Lys Gln Leu Asn 20 25 30 Glu Lys Gly Ala Arg Ile Ile Thr Lys Ala Glu Gly Val Tyr Ile Trp 35 40 45 Asp Ser Glu Gly Asn Lys Ile Leu Asp Ala Met Ala Gly Leu Trp Cys 50 55 60 Val Asn Val Gly Tyr Gly Arg Glu Glu Leu Val Gln Ala Ala Thr Arg 65 70 75 80 Gln Met Arg Glu Leu Pro Phe Tyr Asn Leu Phe Phe Gln Thr Ala His 85 90 95 Pro Pro Val Val Glu Leu Ala Lys Ala Ile Ala Asp Val Ala Pro Glu 100 105 110 Gly Met Asn His Val Phe Phe Thr Gly Ser Gly Ser Glu Ala Asn Asp 115 120 125 Thr Val Leu Arg Met Val Arg His Tyr Trp Ala Thr Lys Gly Gln Pro 130 135 140 Gln Lys Lys Val Val Ile Gly Arg Trp Asn Gly Tyr His Gly Ser Thr 145 150 155 160 Val Ala Gly Val Ser Leu Gly Gly Met Lys Ala Leu His Glu Gln Gly 165 170 175 Asp Phe Pro Ile Pro Gly Ile Val His Ile Ala Gln Pro Tyr Trp Tyr 180 185 190 Gly Glu Gly Gly Asp Met Ser Pro Asp Glu Phe Gly Val Trp Ala Ala 195 200 205 Glu Gln Leu Glu Lys Lys Ile Leu Glu Val Gly Glu Glu Asn Val Ala 210 215 220 Ala Phe Ile Ala Glu Pro Ile Gln Gly Ala Gly Gly Val Ile Val Pro 225 230 235 240 Pro Asp Thr Tyr Trp Pro Lys Ile Arg Glu Ile Leu Ala Lys Tyr Asp 245 250 255 Ile Leu Phe Ile Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly 260 265 270 Glu Trp Phe Gly Ser Gln Tyr Tyr Gly Asn Ala Pro Asp Leu Met Pro 275 280 285 Ile Ala Lys Gly Leu Thr Ser Gly Tyr Ile Pro Met Gly Gly Val Val 290 295 300 Val Arg Asp Glu Ile Val Glu Val Leu Asn Gln Gly Gly Glu Phe Tyr 305 310 315 320 His Gly Phe Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu 325 330 335 Glu Asn Ile Arg Ile Leu Arg Glu Glu Lys Ile Ile Glu Lys Val Lys 340 345 350 Ala Glu Thr Ala Pro Tyr Leu Gln Lys Arg Trp Gln Glu Leu Ala Asp 355 360 365 His Pro Leu Val Gly Glu Ala Arg Gly Val Gly Met Val Ala Ala Leu 370 375 380 Glu Leu Val Lys Asn Lys Lys Thr Arg Glu Arg Phe Thr Asp Lys Gly 385 390 395 400 Val Gly Met Leu Cys Arg Glu His Cys Phe Arg Asn Gly Leu Ile Met 405 410 415 Arg Ala Val Gly Asp Thr Met Ile Ile Ser Pro Pro Leu Val Ile Asp 420 425 430 Pro Ser Gln Ile Asp Glu Leu Ile Thr Leu Ala Arg Lys Cys Leu Asp 435 440 445 Gln Thr Ala Ala Ala Val Leu Ala 450 455 970DNAArtificialprimer 9ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgaaca gccaaatcac 60caacgccaag 701049DNAArtificialprimer 10ggggaccact ttgtacaaga aagctgggtt caagccagga cggcggcgg 49111365DNAPseudomonas syringaeCDS(1)..(1365) 11atg agt gcc aac aac ccg caa acc ctc gaa tgg cag gcc ctg agc agc 48Met Ser Ala Asn Asn Pro Gln Thr Leu Glu Trp Gln Ala Leu Ser Ser 1 5 10 15 gag cat cac ctg gca ccg ttc agc gac tac aaa caa ctg aaa gag aaa 96Glu His His Leu Ala Pro Phe Ser Asp Tyr Lys Gln Leu Lys Glu Lys 20 25 30 ggc ccg cgc atc atc acc cgt gcc gag ggc gtt tat ctg tgg gac agc 144Gly Pro Arg Ile Ile Thr Arg Ala Glu Gly Val Tyr Leu Trp Asp Ser 35 40 45 gag ggc aac aag atc ctc gat ggc atg tcc ggc ctg tgg tgc gtg gcc 192Glu Gly Asn Lys Ile Leu Asp Gly Met Ser Gly Leu Trp Cys Val Ala 50 55 60 atc ggt tat ggc cgc gaa gaa ctg gcc gac gca gcc agc aaa cag atg 240Ile Gly Tyr Gly Arg Glu Glu Leu Ala Asp Ala Ala Ser Lys Gln Met 65 70 75 80 cgc gag ctg ccg tac tac aac ctg ttc ttc cag acc gcc cac ccg ccg 288Arg Glu Leu Pro Tyr Tyr Asn Leu Phe Phe Gln Thr Ala His Pro Pro 85 90 95 gtg ctg gaa ctg gcc aag gcc atc tcc gac atc gct ccc gag ggc atg 336Val Leu Glu Leu Ala Lys Ala Ile Ser Asp Ile Ala Pro Glu Gly Met 100 105 110 aac cat gtg ttc ttc acc ggt tca ggc tct gaa ggc aat gac acg atg 384Asn His Val Phe Phe Thr Gly Ser Gly Ser Glu Gly Asn Asp Thr Met 115 120 125 ctg cgc atg gtt cgt cat tac tgg gcg ctg aaa ggc cag ccg aac aag 432Leu Arg Met Val Arg His Tyr Trp Ala Leu Lys Gly Gln Pro Asn Lys 130 135 140 aaa acc atc atc agc cgc gtc aat ggc tac cac ggc tcc acc gtc gcc 480Lys Thr Ile Ile Ser Arg Val Asn Gly Tyr His Gly Ser Thr Val Ala 145 150 155 160 ggt gcc agc ctg ggt ggc atg acc tac atg cac gaa cag ggc gac ctg 528Gly Ala Ser Leu Gly Gly Met Thr Tyr Met His Glu Gln Gly Asp Leu 165 170 175 ccg atc ccg ggg gtg gtg cac att cca cag cct tac tgg ttc ggc gaa 576Pro Ile Pro Gly Val Val His Ile Pro Gln Pro Tyr Trp Phe Gly Glu 180 185 190 ggc ggc gac atg acg ccg gac gag ttc ggc atc tgg gcg gcc gag caa 624Gly Gly Asp Met Thr Pro Asp Glu Phe Gly Ile Trp Ala Ala Glu Gln 195 200 205 ctg gaa aag aaa att ctc gag ctg ggc gtc gag aac gtc ggt gcg ttc

672Leu Glu Lys Lys Ile Leu Glu Leu Gly Val Glu Asn Val Gly Ala Phe 210 215 220 att gcc gag cca atc cag ggc gcg ggc ggt gtg att gtc ccg cct gat 720Ile Ala Glu Pro Ile Gln Gly Ala Gly Gly Val Ile Val Pro Pro Asp 225 230 235 240 tcc tac tgg ccg aag atc aag gaa atc ctt tcc cgc tac gac atc ctg 768Ser Tyr Trp Pro Lys Ile Lys Glu Ile Leu Ser Arg Tyr Asp Ile Leu 245 250 255 ttc gcc gcc gat gag gtg att tgt ggc ttc ggg cgt acc agt gag tgg 816Phe Ala Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Ser Glu Trp 260 265 270 ttc ggt agc gat ttc tat ggc ctc agg ccg gac atg atg acc atc gcc 864Phe Gly Ser Asp Phe Tyr Gly Leu Arg Pro Asp Met Met Thr Ile Ala 275 280 285 aaa ggc ctg acc tcc ggt tac gta ccg atg ggc ggc ctg atc gtg cgc 912Lys Gly Leu Thr Ser Gly Tyr Val Pro Met Gly Gly Leu Ile Val Arg 290 295 300 gat gaa atc gtt gcg gtg ctc aat gag ggt ggc gat ttc aat cac ggc 960Asp Glu Ile Val Ala Val Leu Asn Glu Gly Gly Asp Phe Asn His Gly 305 310 315 320 ttt acc tac tcc ggg cac ccg gtg gcg gcc gcg gtt gcg ctg gag aac 1008Phe Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu Glu Asn 325 330 335 atc cgt atc ctg cgc gaa gaa aag atc gtc gaa cgg gtc agg tcg gaa 1056Ile Arg Ile Leu Arg Glu Glu Lys Ile Val Glu Arg Val Arg Ser Glu 340 345 350 acg gca ccg tat ttg caa aag cgt ttg cgt gag ttg agc gat cat ccg 1104Thr Ala Pro Tyr Leu Gln Lys Arg Leu Arg Glu Leu Ser Asp His Pro 355 360 365 ctg gtg ggc gaa gtc cgg ggt gtc ggg ctg ctc ggg gcc att gag ctg 1152Leu Val Gly Glu Val Arg Gly Val Gly Leu Leu Gly Ala Ile Glu Leu 370 375 380 gtg aag gac aag acc acc cgc gag cgc tat acc gac aag ggc gcg gga 1200Val Lys Asp Lys Thr Thr Arg Glu Arg Tyr Thr Asp Lys Gly Ala Gly 385 390 395 400 atg atc tgt cga acc ttc tgc ttc gac aat ggc ctg atc atg cgg gct 1248Met Ile Cys Arg Thr Phe Cys Phe Asp Asn Gly Leu Ile Met Arg Ala 405 410 415 gtg ggc gat acc atg atc att gcg ccg cca ctg gtg atc agt ttt gcg 1296Val Gly Asp Thr Met Ile Ile Ala Pro Pro Leu Val Ile Ser Phe Ala 420 425 430 caa atc gat gag ctg gta gag aag gcg cgc acg tgt ctg gat ctg acg 1344Gln Ile Asp Glu Leu Val Glu Lys Ala Arg Thr Cys Leu Asp Leu Thr 435 440 445 ctg gcg gtg ttg cag ggc tga 1365Leu Ala Val Leu Gln Gly 450 12454PRTPseudomonas syringae 12Met Ser Ala Asn Asn Pro Gln Thr Leu Glu Trp Gln Ala Leu Ser Ser 1 5 10 15 Glu His His Leu Ala Pro Phe Ser Asp Tyr Lys Gln Leu Lys Glu Lys 20 25 30 Gly Pro Arg Ile Ile Thr Arg Ala Glu Gly Val Tyr Leu Trp Asp Ser 35 40 45 Glu Gly Asn Lys Ile Leu Asp Gly Met Ser Gly Leu Trp Cys Val Ala 50 55 60 Ile Gly Tyr Gly Arg Glu Glu Leu Ala Asp Ala Ala Ser Lys Gln Met 65 70 75 80 Arg Glu Leu Pro Tyr Tyr Asn Leu Phe Phe Gln Thr Ala His Pro Pro 85 90 95 Val Leu Glu Leu Ala Lys Ala Ile Ser Asp Ile Ala Pro Glu Gly Met 100 105 110 Asn His Val Phe Phe Thr Gly Ser Gly Ser Glu Gly Asn Asp Thr Met 115 120 125 Leu Arg Met Val Arg His Tyr Trp Ala Leu Lys Gly Gln Pro Asn Lys 130 135 140 Lys Thr Ile Ile Ser Arg Val Asn Gly Tyr His Gly Ser Thr Val Ala 145 150 155 160 Gly Ala Ser Leu Gly Gly Met Thr Tyr Met His Glu Gln Gly Asp Leu 165 170 175 Pro Ile Pro Gly Val Val His Ile Pro Gln Pro Tyr Trp Phe Gly Glu 180 185 190 Gly Gly Asp Met Thr Pro Asp Glu Phe Gly Ile Trp Ala Ala Glu Gln 195 200 205 Leu Glu Lys Lys Ile Leu Glu Leu Gly Val Glu Asn Val Gly Ala Phe 210 215 220 Ile Ala Glu Pro Ile Gln Gly Ala Gly Gly Val Ile Val Pro Pro Asp 225 230 235 240 Ser Tyr Trp Pro Lys Ile Lys Glu Ile Leu Ser Arg Tyr Asp Ile Leu 245 250 255 Phe Ala Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Ser Glu Trp 260 265 270 Phe Gly Ser Asp Phe Tyr Gly Leu Arg Pro Asp Met Met Thr Ile Ala 275 280 285 Lys Gly Leu Thr Ser Gly Tyr Val Pro Met Gly Gly Leu Ile Val Arg 290 295 300 Asp Glu Ile Val Ala Val Leu Asn Glu Gly Gly Asp Phe Asn His Gly 305 310 315 320 Phe Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu Glu Asn 325 330 335 Ile Arg Ile Leu Arg Glu Glu Lys Ile Val Glu Arg Val Arg Ser Glu 340 345 350 Thr Ala Pro Tyr Leu Gln Lys Arg Leu Arg Glu Leu Ser Asp His Pro 355 360 365 Leu Val Gly Glu Val Arg Gly Val Gly Leu Leu Gly Ala Ile Glu Leu 370 375 380 Val Lys Asp Lys Thr Thr Arg Glu Arg Tyr Thr Asp Lys Gly Ala Gly 385 390 395 400 Met Ile Cys Arg Thr Phe Cys Phe Asp Asn Gly Leu Ile Met Arg Ala 405 410 415 Val Gly Asp Thr Met Ile Ile Ala Pro Pro Leu Val Ile Ser Phe Ala 420 425 430 Gln Ile Asp Glu Leu Val Glu Lys Ala Arg Thr Cys Leu Asp Leu Thr 435 440 445 Leu Ala Val Leu Gln Gly 450 131365DNAArtificialPseudomonas syringae codon optimised aminotransferase gene 13atgtctgcta acaatccaca aactctggaa tggcaggcac tgagctccga acatcacctg 60gctccgttct ccgactacaa acaactgaaa gagaaaggcc cgcgtatcat tacccgcgct 120gaaggtgtgt acctgtggga ttctgaaggc aacaaaattc tggacggtat gagcggcctg 180tggtgcgtag caatcggtta tggccgtgaa gaactggctg acgcggcgag caaacagatg 240cgtgaactgc cgtattataa cctgttcttc caaaccgcac acccgccggt tctggaactg 300gctaaagcta tcagcgatat cgcaccggag ggcatgaatc acgtcttctt cactggttcc 360ggtagcgaag gcaacgacac gatgctgcgc atggtacgtc actattgggc gctgaagggc 420cagccgaaca agaaaacgat tatcagccgt gtaaacggtt atcacggcag caccgttgcg 480ggtgcgagcc tgggcggtat gacctacatg cacgaacagg gtgacctgcc gatcccgggt 540gtagtgcaca ttccgcagcc gtattggttc ggtgaaggcg gtgacatgac gccggacgaa 600ttcggcatct gggcggcaga gcagctggaa aagaaaatcc tggaactggg cgtggaaaac 660gtcggcgcgt tcatcgcgga accgattcag ggcgcgggcg gcgtaattgt tccgccggac 720agctactggc caaaaatcaa agagatcctg tctcgttacg acatcctgtt cgccgcagac 780gaagtgatct gcggttttgg ccgcacctct gaatggttcg gctccgactt ctacggtctg 840cgtccggaca tgatgaccat cgccaaaggc ctgacctccg gttatgttcc tatgggtggc 900ctgatcgtgc gcgacgaaat tgttgcggtt ctgaacgaag gcggcgattt caaccacggc 960ttcacctatt ccggtcaccc agttgctgct gctgtagcac tggaaaacat ccgcatcctg 1020cgtgaagaaa agatcgtaga acgcgtacgt tccgaaaccg caccttacct gcagaagcgc 1080ctgcgcgaac tgagcgacca ccctctggta ggtgaagttc gcggcgtggg cctgctgggc 1140gcgatcgagc tggtgaaaga caaaactacc cgtgaacgtt acaccgacaa aggcgcaggc 1200atgatctgcc gtaccttttg cttcgataac ggtctgatca tgcgcgcagt cggtgatacc 1260atgatcattg ctccgcctct ggttatttct tttgcccaga ttgatgagct ggtcgaaaaa 1320gcgcgcactt gtctggatct gactctggct gttctgcagg gttaa 136514849DNABacillus subtilisCDS(1)..(849) 14atg aag gtt tta gtc aat ggc cgg ctg att ggg cgc agt gaa gca tca 48Met Lys Val Leu Val Asn Gly Arg Leu Ile Gly Arg Ser Glu Ala Ser 1 5 10 15 atc gat ttg gaa gat cgc ggt tat cag ttt ggt gac ggc atc tat gaa 96Ile Asp Leu Glu Asp Arg Gly Tyr Gln Phe Gly Asp Gly Ile Tyr Glu 20 25 30 gtg atc agg gtg tac aaa gga gta ttg ttc ggc tta cgt gag cat gca 144Val Ile Arg Val Tyr Lys Gly Val Leu Phe Gly Leu Arg Glu His Ala 35 40 45 gag cgt ttt ttc aga agt gct gct gaa atc gga att tca ctg cca ttc 192Glu Arg Phe Phe Arg Ser Ala Ala Glu Ile Gly Ile Ser Leu Pro Phe 50 55 60 agt ata gaa gat ctc gag tgg gac ctg caa aag ctt gta cag gaa aat 240Ser Ile Glu Asp Leu Glu Trp Asp Leu Gln Lys Leu Val Gln Glu Asn 65 70 75 80 gcg gtc agt gag gga gcg gta tac att cag aca aca aga ggt gtg gcc 288Ala Val Ser Glu Gly Ala Val Tyr Ile Gln Thr Thr Arg Gly Val Ala 85 90 95 ccg cga aaa cac cag tat gaa gcc ggc ctc gag ccg cag act act gcc 336Pro Arg Lys His Gln Tyr Glu Ala Gly Leu Glu Pro Gln Thr Thr Ala 100 105 110 tat acg ttt acg gtg aaa aaa ccg gag caa gag cag gca tac gga gtg 384Tyr Thr Phe Thr Val Lys Lys Pro Glu Gln Glu Gln Ala Tyr Gly Val 115 120 125 gcg gcc att aca gat gag gat ctt cgc tgg tta aga tgt gat atc aaa 432Ala Ala Ile Thr Asp Glu Asp Leu Arg Trp Leu Arg Cys Asp Ile Lys 130 135 140 agt ctg aat tta ctg tat aat gtc atg acg aag caa agg gcc tat gaa 480Ser Leu Asn Leu Leu Tyr Asn Val Met Thr Lys Gln Arg Ala Tyr Glu 145 150 155 160 gcc gga gca ttt gaa gcc att tta ctt agg gac ggc gtt gtt acg gag 528Ala Gly Ala Phe Glu Ala Ile Leu Leu Arg Asp Gly Val Val Thr Glu 165 170 175 ggt aca tcc tct aac gtt tat gcc gtt atc aac ggc aca gtg cga aca 576Gly Thr Ser Ser Asn Val Tyr Ala Val Ile Asn Gly Thr Val Arg Thr 180 185 190 cat ccg gct aat cgg ctc att ctc aat gga att aca cgg atg aat att 624His Pro Ala Asn Arg Leu Ile Leu Asn Gly Ile Thr Arg Met Asn Ile 195 200 205 tta gga ctg att gag aag aat ggg atc aaa ctg gat gag act cct gtc 672Leu Gly Leu Ile Glu Lys Asn Gly Ile Lys Leu Asp Glu Thr Pro Val 210 215 220 agt gaa gaa gag ttg aaa cag gcg gaa gag atc ttt att tcg tca acg 720Ser Glu Glu Glu Leu Lys Gln Ala Glu Glu Ile Phe Ile Ser Ser Thr 225 230 235 240 acg gca gaa att att ccg gtc gtg acg ctc gat gga caa tcg atc gga 768Thr Ala Glu Ile Ile Pro Val Val Thr Leu Asp Gly Gln Ser Ile Gly 245 250 255 agc ggg aaa ccc gga ccg gtg acc aaa cag ctt cag gct gct ttt caa 816Ser Gly Lys Pro Gly Pro Val Thr Lys Gln Leu Gln Ala Ala Phe Gln 260 265 270 gaa agc att caa cag gct gct agc att tca taa 849Glu Ser Ile Gln Gln Ala Ala Ser Ile Ser 275 280 15282PRTBacillus subtilis 15Met Lys Val Leu Val Asn Gly Arg Leu Ile Gly Arg Ser Glu Ala Ser 1 5 10 15 Ile Asp Leu Glu Asp Arg Gly Tyr Gln Phe Gly Asp Gly Ile Tyr Glu 20 25 30 Val Ile Arg Val Tyr Lys Gly Val Leu Phe Gly Leu Arg Glu His Ala 35 40 45 Glu Arg Phe Phe Arg Ser Ala Ala Glu Ile Gly Ile Ser Leu Pro Phe 50 55 60 Ser Ile Glu Asp Leu Glu Trp Asp Leu Gln Lys Leu Val Gln Glu Asn 65 70 75 80 Ala Val Ser Glu Gly Ala Val Tyr Ile Gln Thr Thr Arg Gly Val Ala 85 90 95 Pro Arg Lys His Gln Tyr Glu Ala Gly Leu Glu Pro Gln Thr Thr Ala 100 105 110 Tyr Thr Phe Thr Val Lys Lys Pro Glu Gln Glu Gln Ala Tyr Gly Val 115 120 125 Ala Ala Ile Thr Asp Glu Asp Leu Arg Trp Leu Arg Cys Asp Ile Lys 130 135 140 Ser Leu Asn Leu Leu Tyr Asn Val Met Thr Lys Gln Arg Ala Tyr Glu 145 150 155 160 Ala Gly Ala Phe Glu Ala Ile Leu Leu Arg Asp Gly Val Val Thr Glu 165 170 175 Gly Thr Ser Ser Asn Val Tyr Ala Val Ile Asn Gly Thr Val Arg Thr 180 185 190 His Pro Ala Asn Arg Leu Ile Leu Asn Gly Ile Thr Arg Met Asn Ile 195 200 205 Leu Gly Leu Ile Glu Lys Asn Gly Ile Lys Leu Asp Glu Thr Pro Val 210 215 220 Ser Glu Glu Glu Leu Lys Gln Ala Glu Glu Ile Phe Ile Ser Ser Thr 225 230 235 240 Thr Ala Glu Ile Ile Pro Val Val Thr Leu Asp Gly Gln Ser Ile Gly 245 250 255 Ser Gly Lys Pro Gly Pro Val Thr Lys Gln Leu Gln Ala Ala Phe Gln 260 265 270 Glu Ser Ile Gln Gln Ala Ala Ser Ile Ser 275 280 161347DNABacillus subtilisCDS(1)..(1347) 16atg act cat gat ttg ata gaa aaa agt aaa aag cac ctc tgg ctg cca 48Met Thr His Asp Leu Ile Glu Lys Ser Lys Lys His Leu Trp Leu Pro 1 5 10 15 ttt acc caa atg aaa gat tat gat gaa aac ccc tta atc atc gaa agc 96Phe Thr Gln Met Lys Asp Tyr Asp Glu Asn Pro Leu Ile Ile Glu Ser 20 25 30 ggg act gga atc aaa gtc aaa gac ata aac ggc aag gaa tac tat gac 144Gly Thr Gly Ile Lys Val Lys Asp Ile Asn Gly Lys Glu Tyr Tyr Asp 35 40 45 ggt ttt tca tcg gtt tgg ctt aat gtc cac gga cac cgc aaa aaa gaa 192Gly Phe Ser Ser Val Trp Leu Asn Val His Gly His Arg Lys Lys Glu 50 55 60 cta gat gac gcc ata aaa aaa cag ctc gga aaa att gcg cac tcc acg 240Leu Asp Asp Ala Ile Lys Lys Gln Leu Gly Lys Ile Ala His Ser Thr 65 70 75 80 tta ttg ggc atg acc aat gtt cca gca acc cag ctt gcc gaa aca tta 288Leu Leu Gly Met Thr Asn Val Pro Ala Thr Gln Leu Ala Glu Thr Leu 85 90 95 atc gac atc agc cca aaa aag ctc acg cgg gtc ttt tat tca gac agc 336Ile Asp Ile Ser Pro Lys Lys Leu Thr Arg Val Phe Tyr Ser Asp Ser 100 105 110 ggc gca gag gcg atg gaa ata gcc cta aaa atg gcg ttt cag tat tgg 384Gly Ala Glu Ala Met Glu Ile Ala Leu Lys Met Ala Phe Gln Tyr Trp 115 120 125 aag aac atc ggg aag ccc gag aaa caa aaa ttc atc gca atg aaa aac 432Lys Asn Ile Gly Lys Pro Glu Lys Gln Lys Phe Ile Ala Met Lys Asn 130 135 140 ggg tat cac ggt gat acg att ggc gcc gtc agt gtc ggt tca att gag 480Gly Tyr His Gly Asp Thr Ile Gly Ala Val Ser Val Gly Ser Ile Glu 145 150 155 160 ctt ttt cac cac gta tac ggc ccg ttg atg ttc gag agt tac aag gcc 528Leu Phe His His Val Tyr Gly Pro Leu Met Phe Glu Ser Tyr Lys Ala 165 170 175 ccg att cct tat gtg tat cgt tct gaa agc ggt gat cct gat gag tgc 576Pro Ile Pro Tyr Val Tyr Arg Ser Glu Ser Gly Asp Pro Asp Glu Cys 180 185 190 cgt gat cag tgc ctc cga gag ctt gca cag ctg ctt gag gaa cat cat 624Arg Asp Gln Cys Leu Arg Glu Leu Ala Gln Leu Leu Glu Glu His His 195 200 205 gag gaa att gcc gcg ctt tcc att gaa tca atg gta caa ggc gcg tcc 672Glu Glu Ile Ala Ala Leu Ser Ile Glu Ser Met Val Gln Gly Ala Ser 210 215 220 ggt atg atc gtg atg ccg gaa gga tat ttg gca ggc gtg cgc gag cta 720Gly Met Ile Val Met Pro Glu Gly Tyr Leu Ala Gly Val Arg Glu Leu 225 230 235

240 tgt aca aca tac gat gtc tta atg atc gtt gat gaa gtc gct aca ggc 768Cys Thr Thr Tyr Asp Val Leu Met Ile Val Asp Glu Val Ala Thr Gly 245 250 255 ttt ggc cgt aca gga aaa atg ttt gcg tgc gag cac gag aat gtc cag 816Phe Gly Arg Thr Gly Lys Met Phe Ala Cys Glu His Glu Asn Val Gln 260 265 270 cct gat ctg atg gct gcc ggt aaa ggc att aca gga ggc tat ttg cca 864Pro Asp Leu Met Ala Ala Gly Lys Gly Ile Thr Gly Gly Tyr Leu Pro 275 280 285 att gcc gtt acg ttt gcc act gaa gac atc tat aag gca ttc tat gat 912Ile Ala Val Thr Phe Ala Thr Glu Asp Ile Tyr Lys Ala Phe Tyr Asp 290 295 300 gat tat gaa aac cta aaa acc ttt ttc cat ggc cat tcc tat aca ggc 960Asp Tyr Glu Asn Leu Lys Thr Phe Phe His Gly His Ser Tyr Thr Gly 305 310 315 320 aat cag ctt ggc tgt gcg gtt gcg ctt gaa aat ctg gca tta ttt gaa 1008Asn Gln Leu Gly Cys Ala Val Ala Leu Glu Asn Leu Ala Leu Phe Glu 325 330 335 tct gaa aac att gtg gaa caa gta gcg gaa aaa agt aaa aag ctc cat 1056Ser Glu Asn Ile Val Glu Gln Val Ala Glu Lys Ser Lys Lys Leu His 340 345 350 ttt ctt ctt caa gat ctg cac gct ctt cct cat gtt ggg gat att cgg 1104Phe Leu Leu Gln Asp Leu His Ala Leu Pro His Val Gly Asp Ile Arg 355 360 365 cag ctt ggc ttt atg tgc ggt gca gag ctt gta cga tca aag gaa act 1152Gln Leu Gly Phe Met Cys Gly Ala Glu Leu Val Arg Ser Lys Glu Thr 370 375 380 aaa gaa cct tac ccg gct gat cgg cgg att gga tac aaa gtt tcc tta 1200Lys Glu Pro Tyr Pro Ala Asp Arg Arg Ile Gly Tyr Lys Val Ser Leu 385 390 395 400 aaa atg aga gag tta gga atg ctg aca aga ccg ctt ggg gac gtg att 1248Lys Met Arg Glu Leu Gly Met Leu Thr Arg Pro Leu Gly Asp Val Ile 405 410 415 gca ttt ctt cct cct ctt gcc agc aca gct gaa gag ctc tcg gaa atg 1296Ala Phe Leu Pro Pro Leu Ala Ser Thr Ala Glu Glu Leu Ser Glu Met 420 425 430 gtt gcc att atg aaa caa gcg atc cac gag gtt acg agc ctt gaa gat 1344Val Ala Ile Met Lys Gln Ala Ile His Glu Val Thr Ser Leu Glu Asp 435 440 445 tga 1347 17448PRTBacillus subtilis 17Met Thr His Asp Leu Ile Glu Lys Ser Lys Lys His Leu Trp Leu Pro 1 5 10 15 Phe Thr Gln Met Lys Asp Tyr Asp Glu Asn Pro Leu Ile Ile Glu Ser 20 25 30 Gly Thr Gly Ile Lys Val Lys Asp Ile Asn Gly Lys Glu Tyr Tyr Asp 35 40 45 Gly Phe Ser Ser Val Trp Leu Asn Val His Gly His Arg Lys Lys Glu 50 55 60 Leu Asp Asp Ala Ile Lys Lys Gln Leu Gly Lys Ile Ala His Ser Thr 65 70 75 80 Leu Leu Gly Met Thr Asn Val Pro Ala Thr Gln Leu Ala Glu Thr Leu 85 90 95 Ile Asp Ile Ser Pro Lys Lys Leu Thr Arg Val Phe Tyr Ser Asp Ser 100 105 110 Gly Ala Glu Ala Met Glu Ile Ala Leu Lys Met Ala Phe Gln Tyr Trp 115 120 125 Lys Asn Ile Gly Lys Pro Glu Lys Gln Lys Phe Ile Ala Met Lys Asn 130 135 140 Gly Tyr His Gly Asp Thr Ile Gly Ala Val Ser Val Gly Ser Ile Glu 145 150 155 160 Leu Phe His His Val Tyr Gly Pro Leu Met Phe Glu Ser Tyr Lys Ala 165 170 175 Pro Ile Pro Tyr Val Tyr Arg Ser Glu Ser Gly Asp Pro Asp Glu Cys 180 185 190 Arg Asp Gln Cys Leu Arg Glu Leu Ala Gln Leu Leu Glu Glu His His 195 200 205 Glu Glu Ile Ala Ala Leu Ser Ile Glu Ser Met Val Gln Gly Ala Ser 210 215 220 Gly Met Ile Val Met Pro Glu Gly Tyr Leu Ala Gly Val Arg Glu Leu 225 230 235 240 Cys Thr Thr Tyr Asp Val Leu Met Ile Val Asp Glu Val Ala Thr Gly 245 250 255 Phe Gly Arg Thr Gly Lys Met Phe Ala Cys Glu His Glu Asn Val Gln 260 265 270 Pro Asp Leu Met Ala Ala Gly Lys Gly Ile Thr Gly Gly Tyr Leu Pro 275 280 285 Ile Ala Val Thr Phe Ala Thr Glu Asp Ile Tyr Lys Ala Phe Tyr Asp 290 295 300 Asp Tyr Glu Asn Leu Lys Thr Phe Phe His Gly His Ser Tyr Thr Gly 305 310 315 320 Asn Gln Leu Gly Cys Ala Val Ala Leu Glu Asn Leu Ala Leu Phe Glu 325 330 335 Ser Glu Asn Ile Val Glu Gln Val Ala Glu Lys Ser Lys Lys Leu His 340 345 350 Phe Leu Leu Gln Asp Leu His Ala Leu Pro His Val Gly Asp Ile Arg 355 360 365 Gln Leu Gly Phe Met Cys Gly Ala Glu Leu Val Arg Ser Lys Glu Thr 370 375 380 Lys Glu Pro Tyr Pro Ala Asp Arg Arg Ile Gly Tyr Lys Val Ser Leu 385 390 395 400 Lys Met Arg Glu Leu Gly Met Leu Thr Arg Pro Leu Gly Asp Val Ile 405 410 415 Ala Phe Leu Pro Pro Leu Ala Ser Thr Ala Glu Glu Leu Ser Glu Met 420 425 430 Val Ala Ile Met Lys Gln Ala Ile His Glu Val Thr Ser Leu Glu Asp 435 440 445 181467DNARhodobacter sphaeroidesCDS(1)..(1467) 18atg ccc ggt tgc ggg ggc ttg ccc ggg aat gaa ccg aaa tgc gga cga 48Met Pro Gly Cys Gly Gly Leu Pro Gly Asn Glu Pro Lys Cys Gly Arg 1 5 10 15 gag ggg agg tcg gcg atg acg cgg aat gac gcg acg aat gct gcc gga 96Glu Gly Arg Ser Ala Met Thr Arg Asn Asp Ala Thr Asn Ala Ala Gly 20 25 30 gcg gtg ggc gcg gcg atg cgg gat cac atc ctc ttg cct gca cag gaa 144Ala Val Gly Ala Ala Met Arg Asp His Ile Leu Leu Pro Ala Gln Glu 35 40 45 atg gcg aag ctc ggc aag tcc gcg cag ccg gtg ctg act cat gcc gag 192Met Ala Lys Leu Gly Lys Ser Ala Gln Pro Val Leu Thr His Ala Glu 50 55 60 ggc atc tat gtc cat acc gag gac ggc cgc cgc ctg atc gac ggg ccg 240Gly Ile Tyr Val His Thr Glu Asp Gly Arg Arg Leu Ile Asp Gly Pro 65 70 75 80 gcg ggc atg tgg tgc gcg cag gtg ggc tac ggc cgc cgc gag atc gtc 288Ala Gly Met Trp Cys Ala Gln Val Gly Tyr Gly Arg Arg Glu Ile Val 85 90 95 gat gcc atg gcg cat cag gcg atg gtg ctg ccc tat gcc tcg ccc tgg 336Asp Ala Met Ala His Gln Ala Met Val Leu Pro Tyr Ala Ser Pro Trp 100 105 110 tat atg gcc acg agc ccc gcg gcg cgg ctg gcg gag aag atc gcc acg 384Tyr Met Ala Thr Ser Pro Ala Ala Arg Leu Ala Glu Lys Ile Ala Thr 115 120 125 ctg acg ccg ggc gat ctc aac cgg atc ttt ttc acc acg ggc ggg tcg 432Leu Thr Pro Gly Asp Leu Asn Arg Ile Phe Phe Thr Thr Gly Gly Ser 130 135 140 acc gcg gtg gac agc gcg ctg cgc ttc tcg gaa ttc tac aac aac gtg 480Thr Ala Val Asp Ser Ala Leu Arg Phe Ser Glu Phe Tyr Asn Asn Val 145 150 155 160 ctg ggc cgg ccg cag aag aag cgc atc atc gtg cgc tac gac ggc tat 528Leu Gly Arg Pro Gln Lys Lys Arg Ile Ile Val Arg Tyr Asp Gly Tyr 165 170 175 cac ggc tcg acg gcg ctc acc gcc gcc tgc acc ggc cgc acc ggc aac 576His Gly Ser Thr Ala Leu Thr Ala Ala Cys Thr Gly Arg Thr Gly Asn 180 185 190 tgg ccg aac ttc gac atc gcg cag gac cgg atc tcg ttc ctc tcg agc 624Trp Pro Asn Phe Asp Ile Ala Gln Asp Arg Ile Ser Phe Leu Ser Ser 195 200 205 ccc aat ccg cgc cac gcc ggc aac cgc agc cag gag gcg ttc ctc gac 672Pro Asn Pro Arg His Ala Gly Asn Arg Ser Gln Glu Ala Phe Leu Asp 210 215 220 gat ctg gtg cag gaa ttc gag gac cgg atc gag agc ctc ggc ccc gac 720Asp Leu Val Gln Glu Phe Glu Asp Arg Ile Glu Ser Leu Gly Pro Asp 225 230 235 240 acg atc gcg gcc ttc ctg gcc gag ccg atc ctc gcc tcg ggc ggc gtc 768Thr Ile Ala Ala Phe Leu Ala Glu Pro Ile Leu Ala Ser Gly Gly Val 245 250 255 att att ccg ccc gca ggc tat cat gcg cgc ttc aag gcg atc tgc gag 816Ile Ile Pro Pro Ala Gly Tyr His Ala Arg Phe Lys Ala Ile Cys Glu 260 265 270 aag cac gac atc ctc tat atc tcg gac gag gtg gtg acg ggc ttc ggc 864Lys His Asp Ile Leu Tyr Ile Ser Asp Glu Val Val Thr Gly Phe Gly 275 280 285 cgt tgc ggc gag tgg ttc gcc tcg gag aag gtg ttc ggg gtg gtg ccg 912Arg Cys Gly Glu Trp Phe Ala Ser Glu Lys Val Phe Gly Val Val Pro 290 295 300 gac atc atc acc ttc gcc aag ggc gtg acc tcg ggc tat gtg ccg ctc 960Asp Ile Ile Thr Phe Ala Lys Gly Val Thr Ser Gly Tyr Val Pro Leu 305 310 315 320 ggc ggc ctt gcg atc tcc gag gcg gtg ctg gcg cgg atc tcg ggc gag 1008Gly Gly Leu Ala Ile Ser Glu Ala Val Leu Ala Arg Ile Ser Gly Glu 325 330 335 aat gcc aag gga agc tgg ttc acc aac ggc tat acc tac agc aat cag 1056Asn Ala Lys Gly Ser Trp Phe Thr Asn Gly Tyr Thr Tyr Ser Asn Gln 340 345 350 ccg gtg gcc tgc gcc gcg gcg ctt gcc aac atc gag ctg atg gag cgc 1104Pro Val Ala Cys Ala Ala Ala Leu Ala Asn Ile Glu Leu Met Glu Arg 355 360 365 gag ggc atc gtc gat cag gcg cgc gag atg gcg gac tat ttc gcc gcg 1152Glu Gly Ile Val Asp Gln Ala Arg Glu Met Ala Asp Tyr Phe Ala Ala 370 375 380 gcg ctg gct tcg ctg cgc gat ctg ccg ggc gtg gcg gaa acc cgg tcg 1200Ala Leu Ala Ser Leu Arg Asp Leu Pro Gly Val Ala Glu Thr Arg Ser 385 390 395 400 gtg ggc ctc gtg ggt tgc gtg caa tgc ctg ctc gac ccg acc cgg gcg 1248Val Gly Leu Val Gly Cys Val Gln Cys Leu Leu Asp Pro Thr Arg Ala 405 410 415 gac ggc acg gcc gag gac aag gcc ttc acc ctg aag atc gac gag cgc 1296Asp Gly Thr Ala Glu Asp Lys Ala Phe Thr Leu Lys Ile Asp Glu Arg 420 425 430 tgc ttc gag ctc ggg ctg atc gtg cgc ccg ctg ggc gat ctc tgc gtg 1344Cys Phe Glu Leu Gly Leu Ile Val Arg Pro Leu Gly Asp Leu Cys Val 435 440 445 atc tcg ccg ccg ctc atc atc tcg cgc gcg cag atc gac gag atg gtc 1392Ile Ser Pro Pro Leu Ile Ile Ser Arg Ala Gln Ile Asp Glu Met Val 450 455 460 gcg atc atg cgg cag gcc atc acc gaa gtg agc gcc gcc cac ggt ctg 1440Ala Ile Met Arg Gln Ala Ile Thr Glu Val Ser Ala Ala His Gly Leu 465 470 475 480 acc gcg aaa gaa ccg gcc gcc gtc tga 1467Thr Ala Lys Glu Pro Ala Ala Val 485 19488PRTRhodobacter sphaeroides 19Met Pro Gly Cys Gly Gly Leu Pro Gly Asn Glu Pro Lys Cys Gly Arg 1 5 10 15 Glu Gly Arg Ser Ala Met Thr Arg Asn Asp Ala Thr Asn Ala Ala Gly 20 25 30 Ala Val Gly Ala Ala Met Arg Asp His Ile Leu Leu Pro Ala Gln Glu 35 40 45 Met Ala Lys Leu Gly Lys Ser Ala Gln Pro Val Leu Thr His Ala Glu 50 55 60 Gly Ile Tyr Val His Thr Glu Asp Gly Arg Arg Leu Ile Asp Gly Pro 65 70 75 80 Ala Gly Met Trp Cys Ala Gln Val Gly Tyr Gly Arg Arg Glu Ile Val 85 90 95 Asp Ala Met Ala His Gln Ala Met Val Leu Pro Tyr Ala Ser Pro Trp 100 105 110 Tyr Met Ala Thr Ser Pro Ala Ala Arg Leu Ala Glu Lys Ile Ala Thr 115 120 125 Leu Thr Pro Gly Asp Leu Asn Arg Ile Phe Phe Thr Thr Gly Gly Ser 130 135 140 Thr Ala Val Asp Ser Ala Leu Arg Phe Ser Glu Phe Tyr Asn Asn Val 145 150 155 160 Leu Gly Arg Pro Gln Lys Lys Arg Ile Ile Val Arg Tyr Asp Gly Tyr 165 170 175 His Gly Ser Thr Ala Leu Thr Ala Ala Cys Thr Gly Arg Thr Gly Asn 180 185 190 Trp Pro Asn Phe Asp Ile Ala Gln Asp Arg Ile Ser Phe Leu Ser Ser 195 200 205 Pro Asn Pro Arg His Ala Gly Asn Arg Ser Gln Glu Ala Phe Leu Asp 210 215 220 Asp Leu Val Gln Glu Phe Glu Asp Arg Ile Glu Ser Leu Gly Pro Asp 225 230 235 240 Thr Ile Ala Ala Phe Leu Ala Glu Pro Ile Leu Ala Ser Gly Gly Val 245 250 255 Ile Ile Pro Pro Ala Gly Tyr His Ala Arg Phe Lys Ala Ile Cys Glu 260 265 270 Lys His Asp Ile Leu Tyr Ile Ser Asp Glu Val Val Thr Gly Phe Gly 275 280 285 Arg Cys Gly Glu Trp Phe Ala Ser Glu Lys Val Phe Gly Val Val Pro 290 295 300 Asp Ile Ile Thr Phe Ala Lys Gly Val Thr Ser Gly Tyr Val Pro Leu 305 310 315 320 Gly Gly Leu Ala Ile Ser Glu Ala Val Leu Ala Arg Ile Ser Gly Glu 325 330 335 Asn Ala Lys Gly Ser Trp Phe Thr Asn Gly Tyr Thr Tyr Ser Asn Gln 340 345 350 Pro Val Ala Cys Ala Ala Ala Leu Ala Asn Ile Glu Leu Met Glu Arg 355 360 365 Glu Gly Ile Val Asp Gln Ala Arg Glu Met Ala Asp Tyr Phe Ala Ala 370 375 380 Ala Leu Ala Ser Leu Arg Asp Leu Pro Gly Val Ala Glu Thr Arg Ser 385 390 395 400 Val Gly Leu Val Gly Cys Val Gln Cys Leu Leu Asp Pro Thr Arg Ala 405 410 415 Asp Gly Thr Ala Glu Asp Lys Ala Phe Thr Leu Lys Ile Asp Glu Arg 420 425 430 Cys Phe Glu Leu Gly Leu Ile Val Arg Pro Leu Gly Asp Leu Cys Val 435 440 445 Ile Ser Pro Pro Leu Ile Ile Ser Arg Ala Gln Ile Asp Glu Met Val 450 455 460 Ala Ile Met Arg Gln Ala Ile Thr Glu Val Ser Ala Ala His Gly Leu 465 470 475 480 Thr Ala Lys Glu Pro Ala Ala Val 485 20837DNALegionella pneumophilaCDS(1)..(837) 20atg agt atc gca ttt gtt aac ggc aag tat tgt tgt caa tct gaa gca 48Met Ser Ile Ala Phe Val Asn Gly Lys Tyr Cys Cys Gln Ser Glu Ala 1 5 10 15 aaa att tca ata ttt gat cga ggg ttt ctt ttt ggt gac tcg gtt tat 96Lys Ile Ser Ile Phe Asp Arg Gly Phe Leu Phe Gly Asp Ser Val Tyr 20 25 30 gaa gtg ctg cct gtt tac cat ggg cag cct tac ttt gta gac caa cat 144Glu Val Leu Pro Val Tyr His Gly Gln Pro Tyr Phe Val Asp Gln His 35 40 45 ctt gac cga tta ttc tca aat atg aaa aaa att aag atg att ata cca 192Leu Asp Arg Leu Phe Ser Asn Met Lys Lys Ile Lys Met Ile Ile Pro 50 55 60 aat tat gat tgg cat ggt tta att cat aga cta ata tca gaa aat aat 240Asn Tyr Asp Trp His Gly Leu Ile His Arg Leu Ile Ser Glu Asn Asn 65 70 75

80 ggc ggt aat tta caa gta tat atc caa gtc aca cga ggg aat caa ggg 288Gly Gly Asn Leu Gln Val Tyr Ile Gln Val Thr Arg Gly Asn Gln Gly 85 90 95 gtg cgc aag cat gat atc cct act tcc atc aca cct tct gtt atc gca 336Val Arg Lys His Asp Ile Pro Thr Ser Ile Thr Pro Ser Val Ile Ala 100 105 110 ttc act atg cat aat cca ttt ccc acc ctc gaa gat aag gaa cag gga 384Phe Thr Met His Asn Pro Phe Pro Thr Leu Glu Asp Lys Glu Gln Gly 115 120 125 atg tca gca aaa ctg gtt gaa gat ttt cgg tgg atg aga tgt gat ata 432Met Ser Ala Lys Leu Val Glu Asp Phe Arg Trp Met Arg Cys Asp Ile 130 135 140 aaa act act tct tta att gcc aat ata tta ctg aat gat gag gct gta 480Lys Thr Thr Ser Leu Ile Ala Asn Ile Leu Leu Asn Asp Glu Ala Val 145 150 155 160 tct gca gga ttc cac act gca att ctt gcc cgg aac ggt cta att aca 528Ser Ala Gly Phe His Thr Ala Ile Leu Ala Arg Asn Gly Leu Ile Thr 165 170 175 gag gga agt agt acc aac gta ttt att gtc gca cag gat ggt gtt att 576Glu Gly Ser Ser Thr Asn Val Phe Ile Val Ala Gln Asp Gly Val Ile 180 185 190 aag aca cca ccc atg aat aat ttc tgt tta cca gga att act cgg caa 624Lys Thr Pro Pro Met Asn Asn Phe Cys Leu Pro Gly Ile Thr Arg Gln 195 200 205 gtt gtt att gaa ata att aaa aaa tta gat tta aag ttc aga gaa ata 672Val Val Ile Glu Ile Ile Lys Lys Leu Asp Leu Lys Phe Arg Glu Ile 210 215 220 gaa att agc att tca gag ctt ttt tct gct cag gaa gtt tgg ata aca 720Glu Ile Ser Ile Ser Glu Leu Phe Ser Ala Gln Glu Val Trp Ile Thr 225 230 235 240 agt acg aca aaa gaa gta ttc cct att aca aag att aat gac tct ttg 768Ser Thr Thr Lys Glu Val Phe Pro Ile Thr Lys Ile Asn Asp Ser Leu 245 250 255 att aat ggc gga aaa gtt ggc gaa tat tgg cgg ata att aat gat tcc 816Ile Asn Gly Gly Lys Val Gly Glu Tyr Trp Arg Ile Ile Asn Asp Ser 260 265 270 tac caa caa cta gta aac taa 837Tyr Gln Gln Leu Val Asn 275 21278PRTLegionella pneumophila 21Met Ser Ile Ala Phe Val Asn Gly Lys Tyr Cys Cys Gln Ser Glu Ala 1 5 10 15 Lys Ile Ser Ile Phe Asp Arg Gly Phe Leu Phe Gly Asp Ser Val Tyr 20 25 30 Glu Val Leu Pro Val Tyr His Gly Gln Pro Tyr Phe Val Asp Gln His 35 40 45 Leu Asp Arg Leu Phe Ser Asn Met Lys Lys Ile Lys Met Ile Ile Pro 50 55 60 Asn Tyr Asp Trp His Gly Leu Ile His Arg Leu Ile Ser Glu Asn Asn 65 70 75 80 Gly Gly Asn Leu Gln Val Tyr Ile Gln Val Thr Arg Gly Asn Gln Gly 85 90 95 Val Arg Lys His Asp Ile Pro Thr Ser Ile Thr Pro Ser Val Ile Ala 100 105 110 Phe Thr Met His Asn Pro Phe Pro Thr Leu Glu Asp Lys Glu Gln Gly 115 120 125 Met Ser Ala Lys Leu Val Glu Asp Phe Arg Trp Met Arg Cys Asp Ile 130 135 140 Lys Thr Thr Ser Leu Ile Ala Asn Ile Leu Leu Asn Asp Glu Ala Val 145 150 155 160 Ser Ala Gly Phe His Thr Ala Ile Leu Ala Arg Asn Gly Leu Ile Thr 165 170 175 Glu Gly Ser Ser Thr Asn Val Phe Ile Val Ala Gln Asp Gly Val Ile 180 185 190 Lys Thr Pro Pro Met Asn Asn Phe Cys Leu Pro Gly Ile Thr Arg Gln 195 200 205 Val Val Ile Glu Ile Ile Lys Lys Leu Asp Leu Lys Phe Arg Glu Ile 210 215 220 Glu Ile Ser Ile Ser Glu Leu Phe Ser Ala Gln Glu Val Trp Ile Thr 225 230 235 240 Ser Thr Thr Lys Glu Val Phe Pro Ile Thr Lys Ile Asn Asp Ser Leu 245 250 255 Ile Asn Gly Gly Lys Val Gly Glu Tyr Trp Arg Ile Ile Asn Asp Ser 260 265 270 Tyr Gln Gln Leu Val Asn 275 22861DNANitrosomonas europaeaCDS(1)..(861) 22atg att tac ctc aat ggc aaa ttt ctg ccg atg gaa cag gct acc gtt 48Met Ile Tyr Leu Asn Gly Lys Phe Leu Pro Met Glu Gln Ala Thr Val 1 5 10 15 cca gtg ctg gat aga ggc ttc atc ttc ggt gat ggt gtc tat gaa gtc 96Pro Val Leu Asp Arg Gly Phe Ile Phe Gly Asp Gly Val Tyr Glu Val 20 25 30 ata ccg gtt tat tca cgt aaa ccg ttc cgg ctg ggc gaa cat ctt tcc 144Ile Pro Val Tyr Ser Arg Lys Pro Phe Arg Leu Gly Glu His Leu Ser 35 40 45 cgg ctg cag cac agt ctg gat ggc ata cgt ctc cag aat ccg cac act 192Arg Leu Gln His Ser Leu Asp Gly Ile Arg Leu Gln Asn Pro His Thr 50 55 60 gaa gaa caa tgg gct ggt ctg atc gaa cgc atc atc gag ctg aat gaa 240Glu Glu Gln Trp Ala Gly Leu Ile Glu Arg Ile Ile Glu Leu Asn Glu 65 70 75 80 ggt gat gat cag tac ctt tac ctg cac att aca cgc ggg gtg gca aaa 288Gly Asp Asp Gln Tyr Leu Tyr Leu His Ile Thr Arg Gly Val Ala Lys 85 90 95 cgt gac cat gcc ttt cct cgc gaa gta acg ccc act gtc ttc atc atg 336Arg Asp His Ala Phe Pro Arg Glu Val Thr Pro Thr Val Phe Ile Met 100 105 110 agc aac ccg ctt ccg gct cca cct gca aaa ttg ctc gtt tcc gga gtt 384Ser Asn Pro Leu Pro Ala Pro Pro Ala Lys Leu Leu Val Ser Gly Val 115 120 125 tca gcg att acc gcc agg gat aat cgc tgg ggg cgc tgt gat atc aaa 432Ser Ala Ile Thr Ala Arg Asp Asn Arg Trp Gly Arg Cys Asp Ile Lys 130 135 140 gcc att tca ctg ttg cca aat atc tta ttg cgc cag ctt gcc gtg gac 480Ala Ile Ser Leu Leu Pro Asn Ile Leu Leu Arg Gln Leu Ala Val Asp 145 150 155 160 gca caa gcc atg gaa acg atc ctg tta cgc gat ggt ctg ttg acc gaa 528Ala Gln Ala Met Glu Thr Ile Leu Leu Arg Asp Gly Leu Leu Thr Glu 165 170 175 ggg gcc gcc agc aat att ttc atc gta aaa gac gac ctg ctg ctg acc 576Gly Ala Ala Ser Asn Ile Phe Ile Val Lys Asp Asp Leu Leu Leu Thr 180 185 190 ccc ccc aaa gat cac cgt ata ttg cct ggc att act tat gat gta gta 624Pro Pro Lys Asp His Arg Ile Leu Pro Gly Ile Thr Tyr Asp Val Val 195 200 205 ctg gaa ctg gct gaa aca cat ggt gtt cca cat gcg aca aga gaa ata 672Leu Glu Leu Ala Glu Thr His Gly Val Pro His Ala Thr Arg Glu Ile 210 215 220 tca gag ctt gag tta cgt act gca cgg gaa atc atg ctg act tct tcc 720Ser Glu Leu Glu Leu Arg Thr Ala Arg Glu Ile Met Leu Thr Ser Ser 225 230 235 240 acc aaa gaa att ctc ccg atc aca cag ctg gat gga caa ccg atc ggt 768Thr Lys Glu Ile Leu Pro Ile Thr Gln Leu Asp Gly Gln Pro Ile Gly 245 250 255 aat ggc acc cca ggg cca gta ttt cag caa ctg gat cgg ctc tat cag 816Asn Gly Thr Pro Gly Pro Val Phe Gln Gln Leu Asp Arg Leu Tyr Gln 260 265 270 gca tat aag ctg gaa gtc atg cgc ggg cat gct cca cgc cag taa 861Ala Tyr Lys Leu Glu Val Met Arg Gly His Ala Pro Arg Gln 275 280 285 23286PRTNitrosomonas europaea 23Met Ile Tyr Leu Asn Gly Lys Phe Leu Pro Met Glu Gln Ala Thr Val 1 5 10 15 Pro Val Leu Asp Arg Gly Phe Ile Phe Gly Asp Gly Val Tyr Glu Val 20 25 30 Ile Pro Val Tyr Ser Arg Lys Pro Phe Arg Leu Gly Glu His Leu Ser 35 40 45 Arg Leu Gln His Ser Leu Asp Gly Ile Arg Leu Gln Asn Pro His Thr 50 55 60 Glu Glu Gln Trp Ala Gly Leu Ile Glu Arg Ile Ile Glu Leu Asn Glu 65 70 75 80 Gly Asp Asp Gln Tyr Leu Tyr Leu His Ile Thr Arg Gly Val Ala Lys 85 90 95 Arg Asp His Ala Phe Pro Arg Glu Val Thr Pro Thr Val Phe Ile Met 100 105 110 Ser Asn Pro Leu Pro Ala Pro Pro Ala Lys Leu Leu Val Ser Gly Val 115 120 125 Ser Ala Ile Thr Ala Arg Asp Asn Arg Trp Gly Arg Cys Asp Ile Lys 130 135 140 Ala Ile Ser Leu Leu Pro Asn Ile Leu Leu Arg Gln Leu Ala Val Asp 145 150 155 160 Ala Gln Ala Met Glu Thr Ile Leu Leu Arg Asp Gly Leu Leu Thr Glu 165 170 175 Gly Ala Ala Ser Asn Ile Phe Ile Val Lys Asp Asp Leu Leu Leu Thr 180 185 190 Pro Pro Lys Asp His Arg Ile Leu Pro Gly Ile Thr Tyr Asp Val Val 195 200 205 Leu Glu Leu Ala Glu Thr His Gly Val Pro His Ala Thr Arg Glu Ile 210 215 220 Ser Glu Leu Glu Leu Arg Thr Ala Arg Glu Ile Met Leu Thr Ser Ser 225 230 235 240 Thr Lys Glu Ile Leu Pro Ile Thr Gln Leu Asp Gly Gln Pro Ile Gly 245 250 255 Asn Gly Thr Pro Gly Pro Val Phe Gln Gln Leu Asp Arg Leu Tyr Gln 260 265 270 Ala Tyr Lys Leu Glu Val Met Arg Gly His Ala Pro Arg Gln 275 280 285 241293DNANeisseria gonorrhoeaeCDS(1)..(1293) 24atg agg ata aat atg aac cgt aac gaa att tta ttc gac cgc gcc aag 48Met Arg Ile Asn Met Asn Arg Asn Glu Ile Leu Phe Asp Arg Ala Lys 1 5 10 15 gcc atc atc ccc ggc ggc gtg aat tcg ccc gtg cgc gca ttc ggc agc 96Ala Ile Ile Pro Gly Gly Val Asn Ser Pro Val Arg Ala Phe Gly Ser 20 25 30 gtc ggc ggc gtg ccg cgc ttc atc aaa aaa gcc gaa ggc gcg tat gtt 144Val Gly Gly Val Pro Arg Phe Ile Lys Lys Ala Glu Gly Ala Tyr Val 35 40 45 tgg gac gaa aac ggc acg cgc tac acc gat tat gtc ggc tct tgg ggg 192Trp Asp Glu Asn Gly Thr Arg Tyr Thr Asp Tyr Val Gly Ser Trp Gly 50 55 60 cct gcg att gtc gga cac gcg cat ccc gaa gtc gtc gaa gcc gtg cgc 240Pro Ala Ile Val Gly His Ala His Pro Glu Val Val Glu Ala Val Arg 65 70 75 80 gaa gct gcg ttg ggc ggt ttg tcg ttc ggc gcg ccc acc gaa ggc gaa 288Glu Ala Ala Leu Gly Gly Leu Ser Phe Gly Ala Pro Thr Glu Gly Glu 85 90 95 atc gcc att gcc gaa caa att gcc gaa att atg ccg tct gtc gaa cgg 336Ile Ala Ile Ala Glu Gln Ile Ala Glu Ile Met Pro Ser Val Glu Arg 100 105 110 ctg cgc ctc gtc agc tcc ggc acg gaa gcg acg atg act gcc atc cgt 384Leu Arg Leu Val Ser Ser Gly Thr Glu Ala Thr Met Thr Ala Ile Arg 115 120 125 ctg gca cgc ggt ttt acc ggc cgc gac aaa atc atc aaa ttt gaa ggc 432Leu Ala Arg Gly Phe Thr Gly Arg Asp Lys Ile Ile Lys Phe Glu Gly 130 135 140 tgc tac cac ggc cat tcc gac agc ctg ttg gtg aaa gca ggc agc ggt 480Cys Tyr His Gly His Ser Asp Ser Leu Leu Val Lys Ala Gly Ser Gly 145 150 155 160 ctg ctt acc ttc ggc aat cct tct tcc gcc ggt gtg cct gcc gac ttt 528Leu Leu Thr Phe Gly Asn Pro Ser Ser Ala Gly Val Pro Ala Asp Phe 165 170 175 acc aaa cat act ttg gta ctc gaa tac aac aac atc gcc caa ctc gaa 576Thr Lys His Thr Leu Val Leu Glu Tyr Asn Asn Ile Ala Gln Leu Glu 180 185 190 gaa gcc ttt gcc caa agc ggc gac gaa atc gcc tgc gtg att gtc gaa 624Glu Ala Phe Ala Gln Ser Gly Asp Glu Ile Ala Cys Val Ile Val Glu 195 200 205 ccc ttc gtc ggc aat atg aac ctc gtc cgc ccg acc gaa gcc ttt gtc 672Pro Phe Val Gly Asn Met Asn Leu Val Arg Pro Thr Glu Ala Phe Val 210 215 220 aaa gcc ttg cgc gga ttg acc gaa aaa cac ggc gcg gtg ttg att tac 720Lys Ala Leu Arg Gly Leu Thr Glu Lys His Gly Ala Val Leu Ile Tyr 225 230 235 240 gac gaa gtg atg acc ggt ttc cgc gtc gcg ctc ggc ggc gcg cag tcg 768Asp Glu Val Met Thr Gly Phe Arg Val Ala Leu Gly Gly Ala Gln Ser 245 250 255 ctg cac ggc atc acg ccc gac ctg acc acg atg ggc aaa gtc atc ggc 816Leu His Gly Ile Thr Pro Asp Leu Thr Thr Met Gly Lys Val Ile Gly 260 265 270 ggc ggt atg ccg ctt gcc gcg ttc ggc gga cgc aaa gac atc atg gaa 864Gly Gly Met Pro Leu Ala Ala Phe Gly Gly Arg Lys Asp Ile Met Glu 275 280 285 tgt att tcc ccg ttg ggc ggc gtg tat cag gca ggt aca tta tca ggc 912Cys Ile Ser Pro Leu Gly Gly Val Tyr Gln Ala Gly Thr Leu Ser Gly 290 295 300 aac ccg att gcc gtc gcc gcc ggc ttg aaa acg ctg gaa atc atc cag 960Asn Pro Ile Ala Val Ala Ala Gly Leu Lys Thr Leu Glu Ile Ile Gln 305 310 315 320 cgc gaa ggc ttc tat gaa aac ctg acc gcc ttg aca caa cgc ctt gcc 1008Arg Glu Gly Phe Tyr Glu Asn Leu Thr Ala Leu Thr Gln Arg Leu Ala 325 330 335 aac ggt att gcc gcc gcc aaa gcg cac ggt atc gag ttt gcc gcc gac 1056Asn Gly Ile Ala Ala Ala Lys Ala His Gly Ile Glu Phe Ala Ala Asp 340 345 350 agc gtg ggc ggt atg ttc ggt ctg tat ttc gcc gca cac gtg ccg cga 1104Ser Val Gly Gly Met Phe Gly Leu Tyr Phe Ala Ala His Val Pro Arg 355 360 365 aac tat gcc gat atg gcg cgc tcc aat atc gac gct ttc aaa cgc ttc 1152Asn Tyr Ala Asp Met Ala Arg Ser Asn Ile Asp Ala Phe Lys Arg Phe 370 375 380 ttc cac ggc atg ctc gac cgc ggc att gcc ttc ggc ccg tcc gct tat 1200Phe His Gly Met Leu Asp Arg Gly Ile Ala Phe Gly Pro Ser Ala Tyr 385 390 395 400 gaa gcg ggt ttc gtt tcc gcc gcg cat acg ccc gag ctg att gac gaa 1248Glu Ala Gly Phe Val Ser Ala Ala His Thr Pro Glu Leu Ile Asp Glu 405 410 415 acg gtt gcg gtt gcg gtt gaa gtg ttc aag gcg atg gct gca tga 1293Thr Val Ala Val Ala Val Glu Val Phe Lys Ala Met Ala Ala 420 425 430 25430PRTNeisseria gonorrhoeae 25Met Arg Ile Asn Met Asn Arg Asn Glu Ile Leu Phe Asp Arg Ala Lys 1 5 10 15 Ala Ile Ile Pro Gly Gly Val Asn Ser Pro Val Arg Ala Phe Gly Ser 20 25 30 Val Gly Gly Val Pro Arg Phe Ile Lys Lys Ala Glu Gly Ala Tyr Val 35 40 45 Trp Asp Glu Asn Gly Thr Arg Tyr Thr Asp Tyr Val Gly Ser Trp Gly 50 55 60 Pro Ala Ile Val Gly His Ala His Pro Glu Val Val Glu Ala Val Arg 65 70 75 80 Glu Ala Ala Leu Gly Gly Leu Ser Phe Gly Ala Pro Thr Glu Gly Glu 85 90 95 Ile Ala Ile Ala Glu Gln Ile Ala Glu Ile Met Pro Ser Val Glu Arg 100 105 110 Leu Arg Leu Val Ser Ser Gly Thr Glu Ala Thr Met Thr Ala Ile Arg 115 120

125 Leu Ala Arg Gly Phe Thr Gly Arg Asp Lys Ile Ile Lys Phe Glu Gly 130 135 140 Cys Tyr His Gly His Ser Asp Ser Leu Leu Val Lys Ala Gly Ser Gly 145 150 155 160 Leu Leu Thr Phe Gly Asn Pro Ser Ser Ala Gly Val Pro Ala Asp Phe 165 170 175 Thr Lys His Thr Leu Val Leu Glu Tyr Asn Asn Ile Ala Gln Leu Glu 180 185 190 Glu Ala Phe Ala Gln Ser Gly Asp Glu Ile Ala Cys Val Ile Val Glu 195 200 205 Pro Phe Val Gly Asn Met Asn Leu Val Arg Pro Thr Glu Ala Phe Val 210 215 220 Lys Ala Leu Arg Gly Leu Thr Glu Lys His Gly Ala Val Leu Ile Tyr 225 230 235 240 Asp Glu Val Met Thr Gly Phe Arg Val Ala Leu Gly Gly Ala Gln Ser 245 250 255 Leu His Gly Ile Thr Pro Asp Leu Thr Thr Met Gly Lys Val Ile Gly 260 265 270 Gly Gly Met Pro Leu Ala Ala Phe Gly Gly Arg Lys Asp Ile Met Glu 275 280 285 Cys Ile Ser Pro Leu Gly Gly Val Tyr Gln Ala Gly Thr Leu Ser Gly 290 295 300 Asn Pro Ile Ala Val Ala Ala Gly Leu Lys Thr Leu Glu Ile Ile Gln 305 310 315 320 Arg Glu Gly Phe Tyr Glu Asn Leu Thr Ala Leu Thr Gln Arg Leu Ala 325 330 335 Asn Gly Ile Ala Ala Ala Lys Ala His Gly Ile Glu Phe Ala Ala Asp 340 345 350 Ser Val Gly Gly Met Phe Gly Leu Tyr Phe Ala Ala His Val Pro Arg 355 360 365 Asn Tyr Ala Asp Met Ala Arg Ser Asn Ile Asp Ala Phe Lys Arg Phe 370 375 380 Phe His Gly Met Leu Asp Arg Gly Ile Ala Phe Gly Pro Ser Ala Tyr 385 390 395 400 Glu Ala Gly Phe Val Ser Ala Ala His Thr Pro Glu Leu Ile Asp Glu 405 410 415 Thr Val Ala Val Ala Val Glu Val Phe Lys Ala Met Ala Ala 420 425 430 26924DNAPseudomonas aeruginosaCDS(1)..(924) 26atg tcg atg gcc gat cgt gat ggc gtg atc tgg tat gac ggt gaa ctg 48Met Ser Met Ala Asp Arg Asp Gly Val Ile Trp Tyr Asp Gly Glu Leu 1 5 10 15 gtg cag tgg cgc gac gcg acc acg cac gtg ctg acc cat acc ctg cac 96Val Gln Trp Arg Asp Ala Thr Thr His Val Leu Thr His Thr Leu His 20 25 30 tat gga atg ggc gtg ttc gag ggc gtg cgc gcc tac gac acc ccg cag 144Tyr Gly Met Gly Val Phe Glu Gly Val Arg Ala Tyr Asp Thr Pro Gln 35 40 45 ggc acg gcg atc ttc cgc ctg cag gcg cat acc gac cgg ctg ttc gac 192Gly Thr Ala Ile Phe Arg Leu Gln Ala His Thr Asp Arg Leu Phe Asp 50 55 60 tcc gcg cac atc atg aac atg cag atc ccg tac agc cgc gac gag atc 240Ser Ala His Ile Met Asn Met Gln Ile Pro Tyr Ser Arg Asp Glu Ile 65 70 75 80 aac gag gcg acc cgc gcc gcc gtg cgc gag aac aac ctg gaa agc gcc 288Asn Glu Ala Thr Arg Ala Ala Val Arg Glu Asn Asn Leu Glu Ser Ala 85 90 95 tat atc cgc ccg atg gtg ttc tac gga agc gaa ggc atg ggc ctg cgc 336Tyr Ile Arg Pro Met Val Phe Tyr Gly Ser Glu Gly Met Gly Leu Arg 100 105 110 gcc agc ggc ctg aag gtc cat gtg atc atc gcc gcc tgg agc tgg ggc 384Ala Ser Gly Leu Lys Val His Val Ile Ile Ala Ala Trp Ser Trp Gly 115 120 125 gcc tac atg ggc gag gaa gcc ctg cag caa ggc atc aag gtg cgc acc 432Ala Tyr Met Gly Glu Glu Ala Leu Gln Gln Gly Ile Lys Val Arg Thr 130 135 140 agt tcc ttc acc cgc cac cac gtc aac atc tcg atg acc cgc gcc aag 480Ser Ser Phe Thr Arg His His Val Asn Ile Ser Met Thr Arg Ala Lys 145 150 155 160 tcc aac ggc gcc tac atc aac tcg atg ctg gcc ctc cag gaa gcg atc 528Ser Asn Gly Ala Tyr Ile Asn Ser Met Leu Ala Leu Gln Glu Ala Ile 165 170 175 tcc ggc ggc gcc gac gag gcc atg atg ctc gat ccg gaa ggc tac gtg 576Ser Gly Gly Ala Asp Glu Ala Met Met Leu Asp Pro Glu Gly Tyr Val 180 185 190 gcc gaa ggc tcc ggc gag aac atc ttc atc atc aag gat ggc gtg atc 624Ala Glu Gly Ser Gly Glu Asn Ile Phe Ile Ile Lys Asp Gly Val Ile 195 200 205 tac acc ccg gaa gtc acc gcc tgc ctg aac ggc atc act cgt aac act 672Tyr Thr Pro Glu Val Thr Ala Cys Leu Asn Gly Ile Thr Arg Asn Thr 210 215 220 atc ctg acc ctg gcc gcc gaa cac ggt ttt aaa ctg gtc gag aag cgc 720Ile Leu Thr Leu Ala Ala Glu His Gly Phe Lys Leu Val Glu Lys Arg 225 230 235 240 atc acc cgc gac gag gtg tac atc gcc gac gag gcc ttc ttc act ggc 768Ile Thr Arg Asp Glu Val Tyr Ile Ala Asp Glu Ala Phe Phe Thr Gly 245 250 255 act gcc gcg gaa gtc acg ccg atc cgc gaa gtg gac ggt cgc aag atc 816Thr Ala Ala Glu Val Thr Pro Ile Arg Glu Val Asp Gly Arg Lys Ile 260 265 270 ggc gcc ggc cgc cgt ggc ccg gtc acc gaa aag ctg cag aaa gcc tat 864Gly Ala Gly Arg Arg Gly Pro Val Thr Glu Lys Leu Gln Lys Ala Tyr 275 280 285 ttc gac ctg gtc agc ggc aag acc gag gcc cac gcc gag tgg cgt acc 912Phe Asp Leu Val Ser Gly Lys Thr Glu Ala His Ala Glu Trp Arg Thr 290 295 300 ctg gtc aag taa 924Leu Val Lys 305 27307PRTPseudomonas aeruginosa 27Met Ser Met Ala Asp Arg Asp Gly Val Ile Trp Tyr Asp Gly Glu Leu 1 5 10 15 Val Gln Trp Arg Asp Ala Thr Thr His Val Leu Thr His Thr Leu His 20 25 30 Tyr Gly Met Gly Val Phe Glu Gly Val Arg Ala Tyr Asp Thr Pro Gln 35 40 45 Gly Thr Ala Ile Phe Arg Leu Gln Ala His Thr Asp Arg Leu Phe Asp 50 55 60 Ser Ala His Ile Met Asn Met Gln Ile Pro Tyr Ser Arg Asp Glu Ile 65 70 75 80 Asn Glu Ala Thr Arg Ala Ala Val Arg Glu Asn Asn Leu Glu Ser Ala 85 90 95 Tyr Ile Arg Pro Met Val Phe Tyr Gly Ser Glu Gly Met Gly Leu Arg 100 105 110 Ala Ser Gly Leu Lys Val His Val Ile Ile Ala Ala Trp Ser Trp Gly 115 120 125 Ala Tyr Met Gly Glu Glu Ala Leu Gln Gln Gly Ile Lys Val Arg Thr 130 135 140 Ser Ser Phe Thr Arg His His Val Asn Ile Ser Met Thr Arg Ala Lys 145 150 155 160 Ser Asn Gly Ala Tyr Ile Asn Ser Met Leu Ala Leu Gln Glu Ala Ile 165 170 175 Ser Gly Gly Ala Asp Glu Ala Met Met Leu Asp Pro Glu Gly Tyr Val 180 185 190 Ala Glu Gly Ser Gly Glu Asn Ile Phe Ile Ile Lys Asp Gly Val Ile 195 200 205 Tyr Thr Pro Glu Val Thr Ala Cys Leu Asn Gly Ile Thr Arg Asn Thr 210 215 220 Ile Leu Thr Leu Ala Ala Glu His Gly Phe Lys Leu Val Glu Lys Arg 225 230 235 240 Ile Thr Arg Asp Glu Val Tyr Ile Ala Asp Glu Ala Phe Phe Thr Gly 245 250 255 Thr Ala Ala Glu Val Thr Pro Ile Arg Glu Val Asp Gly Arg Lys Ile 260 265 270 Gly Ala Gly Arg Arg Gly Pro Val Thr Glu Lys Leu Gln Lys Ala Tyr 275 280 285 Phe Asp Leu Val Ser Gly Lys Thr Glu Ala His Ala Glu Trp Arg Thr 290 295 300 Leu Val Lys 305 281407DNARhodopseudomonas palustrisCDS(1)..(1407) 28atg aag ctg ata ccg tgc cgc gcc ttt cac ccc ccg gcc gcg cag tgc 48Met Lys Leu Ile Pro Cys Arg Ala Phe His Pro Pro Ala Ala Gln Cys 1 5 10 15 atg agg agc gcc atg tta gac aag atc aag ccc acg tcc gcc gtc aac 96Met Arg Ser Ala Met Leu Asp Lys Ile Lys Pro Thr Ser Ala Val Asn 20 25 30 gcg ccg aac gat ctc aac gcg ttc tgg atg ccg ttc acc gcg aac cgg 144Ala Pro Asn Asp Leu Asn Ala Phe Trp Met Pro Phe Thr Ala Asn Arg 35 40 45 gcc ttc aag cgc gcg ccg aag atg gtc gtg ggt gcc gaa ggc atg cac 192Ala Phe Lys Arg Ala Pro Lys Met Val Val Gly Ala Glu Gly Met His 50 55 60 tac atc acc gcc gat ggt cgc aag atc atc gac gcc gcc tcg ggc atg 240Tyr Ile Thr Ala Asp Gly Arg Lys Ile Ile Asp Ala Ala Ser Gly Met 65 70 75 80 tgg tgc acc aat gcg ggc cat ggc cgc aag gaa atc gcc gag gcg atc 288Trp Cys Thr Asn Ala Gly His Gly Arg Lys Glu Ile Ala Glu Ala Ile 85 90 95 aag gcg cag gcc gat gaa ctc gac ttc tcg ccg ccg ttc cag ttc ggc 336Lys Ala Gln Ala Asp Glu Leu Asp Phe Ser Pro Pro Phe Gln Phe Gly 100 105 110 cag ccg aag gcg ttc gaa ctc gcc agc cgg atc gcc gat ctg gcg ccg 384Gln Pro Lys Ala Phe Glu Leu Ala Ser Arg Ile Ala Asp Leu Ala Pro 115 120 125 gaa ggc ctc gat cac gtg ttc ttc tgc aat tcg ggc tcg gaa gcc ggc 432Glu Gly Leu Asp His Val Phe Phe Cys Asn Ser Gly Ser Glu Ala Gly 130 135 140 gac acc gcg ctg aag atc gcg gtc gcc tat cag cag atc aag ggc cag 480Asp Thr Ala Leu Lys Ile Ala Val Ala Tyr Gln Gln Ile Lys Gly Gln 145 150 155 160 ggc tca cgc acc cgc ctg atc ggc cgc gag cgc ggc tat cac ggc gtc 528Gly Ser Arg Thr Arg Leu Ile Gly Arg Glu Arg Gly Tyr His Gly Val 165 170 175 ggc ttc ggc ggc acc gcg gtc ggc ggc atc ggc aac aac cgc aag atg 576Gly Phe Gly Gly Thr Ala Val Gly Gly Ile Gly Asn Asn Arg Lys Met 180 185 190 ttc ggt ccg ctg ctc aac ggc gtc gat cat ctg cct gcg act tat gat 624Phe Gly Pro Leu Leu Asn Gly Val Asp His Leu Pro Ala Thr Tyr Asp 195 200 205 cgc gac aag cag gct ttc acc atc ggc gag ccg gaa tac ggc gcg cac 672Arg Asp Lys Gln Ala Phe Thr Ile Gly Glu Pro Glu Tyr Gly Ala His 210 215 220 ttc gcc gaa gcg ctt gaa ggc ctc gtc aat ctg cac ggc gcc aac acc 720Phe Ala Glu Ala Leu Glu Gly Leu Val Asn Leu His Gly Ala Asn Thr 225 230 235 240 atc gcg gcg gtg atc gtc gag ccg atg gcc ggc tcc acc ggc gtg ctg 768Ile Ala Ala Val Ile Val Glu Pro Met Ala Gly Ser Thr Gly Val Leu 245 250 255 ccg gcg ccg aag ggc tat ctc aag aag ctg cgc gag atc acc aag aag 816Pro Ala Pro Lys Gly Tyr Leu Lys Lys Leu Arg Glu Ile Thr Lys Lys 260 265 270 cac ggc atc ctg ctg atc ttc gac gag gtc atc acc ggc tac ggc cgt 864His Gly Ile Leu Leu Ile Phe Asp Glu Val Ile Thr Gly Tyr Gly Arg 275 280 285 ctc ggc tat gcc ttc gcg tcc gaa cgt tac ggc gtc acc ccg gac atg 912Leu Gly Tyr Ala Phe Ala Ser Glu Arg Tyr Gly Val Thr Pro Asp Met 290 295 300 atc acc ttc gcc aag ggc gtc acc aat ggt gcg gtg ccg atg ggc ggc 960Ile Thr Phe Ala Lys Gly Val Thr Asn Gly Ala Val Pro Met Gly Gly 305 310 315 320 gtg atc acc tcg gcg gag atc cac gat gcg ttc atg acc ggc ccc gag 1008Val Ile Thr Ser Ala Glu Ile His Asp Ala Phe Met Thr Gly Pro Glu 325 330 335 cac gcg gtc gag ctg gcg cac ggc tac acc tat tcg gcg cat ccg ctc 1056His Ala Val Glu Leu Ala His Gly Tyr Thr Tyr Ser Ala His Pro Leu 340 345 350 gcc tgc gcg gcc ggc atc gcc acc ctc gac atc tac cgc gac gag aag 1104Ala Cys Ala Ala Gly Ile Ala Thr Leu Asp Ile Tyr Arg Asp Glu Lys 355 360 365 ctg ttc gag cgc gcc aag gcg ctg gag ccg aag ttt gcc gag gcg gtg 1152Leu Phe Glu Arg Ala Lys Ala Leu Glu Pro Lys Phe Ala Glu Ala Val 370 375 380 atg tcg ctg aag tcg gcc ccg aac gtg gtc gac atc cgc acc gtc ggc 1200Met Ser Leu Lys Ser Ala Pro Asn Val Val Asp Ile Arg Thr Val Gly 385 390 395 400 ctg acg gcg ggt atc gac ctc gct tcg atc gcc gat gcg gtc ggc aag 1248Leu Thr Ala Gly Ile Asp Leu Ala Ser Ile Ala Asp Ala Val Gly Lys 405 410 415 cgt ggc ttc gaa gcg atg aat gcc ggc ttc cac gac cac gag ctg atg 1296Arg Gly Phe Glu Ala Met Asn Ala Gly Phe His Asp His Glu Leu Met 420 425 430 ctg cgg atc gcc ggc gac acc ctg gcg ctg acc ccg ccg ctg atc ctc 1344Leu Arg Ile Ala Gly Asp Thr Leu Ala Leu Thr Pro Pro Leu Ile Leu 435 440 445 agc gag gac cac atc ggt gag atc gtc gac aag gtc ggc aag gtg atc 1392Ser Glu Asp His Ile Gly Glu Ile Val Asp Lys Val Gly Lys Val Ile 450 455 460 cgc gcg gtc gcc tga 1407Arg Ala Val Ala 465 29468PRTRhodopseudomonas palustris 29Met Lys Leu Ile Pro Cys Arg Ala Phe His Pro Pro Ala Ala Gln Cys 1 5 10 15 Met Arg Ser Ala Met Leu Asp Lys Ile Lys Pro Thr Ser Ala Val Asn 20 25 30 Ala Pro Asn Asp Leu Asn Ala Phe Trp Met Pro Phe Thr Ala Asn Arg 35 40 45 Ala Phe Lys Arg Ala Pro Lys Met Val Val Gly Ala Glu Gly Met His 50 55 60 Tyr Ile Thr Ala Asp Gly Arg Lys Ile Ile Asp Ala Ala Ser Gly Met 65 70 75 80 Trp Cys Thr Asn Ala Gly His Gly Arg Lys Glu Ile Ala Glu Ala Ile 85 90 95 Lys Ala Gln Ala Asp Glu Leu Asp Phe Ser Pro Pro Phe Gln Phe Gly 100 105 110 Gln Pro Lys Ala Phe Glu Leu Ala Ser Arg Ile Ala Asp Leu Ala Pro 115 120 125 Glu Gly Leu Asp His Val Phe Phe Cys Asn Ser Gly Ser Glu Ala Gly 130 135 140 Asp Thr Ala Leu Lys Ile Ala Val Ala Tyr Gln Gln Ile Lys Gly Gln 145 150 155 160 Gly Ser Arg Thr Arg Leu Ile Gly Arg Glu Arg Gly Tyr His Gly Val 165 170 175 Gly Phe Gly Gly Thr Ala Val Gly Gly Ile Gly Asn Asn Arg Lys Met 180 185 190 Phe Gly Pro Leu Leu Asn Gly Val Asp His Leu Pro Ala Thr Tyr Asp 195 200 205 Arg Asp Lys Gln Ala Phe Thr Ile Gly Glu Pro Glu Tyr Gly Ala His 210 215 220 Phe Ala Glu Ala Leu Glu Gly Leu Val Asn Leu His Gly Ala Asn Thr 225 230 235 240 Ile Ala Ala Val Ile Val Glu Pro Met Ala Gly Ser Thr Gly Val Leu 245 250 255 Pro Ala Pro Lys Gly Tyr Leu Lys Lys Leu Arg Glu Ile Thr Lys Lys 260 265 270 His Gly Ile Leu Leu Ile Phe Asp Glu Val Ile Thr Gly Tyr Gly Arg 275 280 285 Leu Gly Tyr Ala Phe Ala Ser Glu Arg Tyr Gly Val Thr Pro Asp Met 290 295 300 Ile Thr Phe Ala Lys Gly

Val Thr Asn Gly Ala Val Pro Met Gly Gly 305 310 315 320 Val Ile Thr Ser Ala Glu Ile His Asp Ala Phe Met Thr Gly Pro Glu 325 330 335 His Ala Val Glu Leu Ala His Gly Tyr Thr Tyr Ser Ala His Pro Leu 340 345 350 Ala Cys Ala Ala Gly Ile Ala Thr Leu Asp Ile Tyr Arg Asp Glu Lys 355 360 365 Leu Phe Glu Arg Ala Lys Ala Leu Glu Pro Lys Phe Ala Glu Ala Val 370 375 380 Met Ser Leu Lys Ser Ala Pro Asn Val Val Asp Ile Arg Thr Val Gly 385 390 395 400 Leu Thr Ala Gly Ile Asp Leu Ala Ser Ile Ala Asp Ala Val Gly Lys 405 410 415 Arg Gly Phe Glu Ala Met Asn Ala Gly Phe His Asp His Glu Leu Met 420 425 430 Leu Arg Ile Ala Gly Asp Thr Leu Ala Leu Thr Pro Pro Leu Ile Leu 435 440 445 Ser Glu Asp His Ile Gly Glu Ile Val Asp Lys Val Gly Lys Val Ile 450 455 460 Arg Ala Val Ala 465 301263DNAEscherichia coliCDS(1)..(1263) 30atg cca cat tca ctg ttc agc acc gat acc gat ctc acc gcc gaa aat 48Met Pro His Ser Leu Phe Ser Thr Asp Thr Asp Leu Thr Ala Glu Asn 1 5 10 15 ctg ctg cgt ttg ccc gct gaa ttt ggc tgc ccg gtg tgg gtc tac gat 96Leu Leu Arg Leu Pro Ala Glu Phe Gly Cys Pro Val Trp Val Tyr Asp 20 25 30 gcg caa att att cgt cgg cag att gca gcg ctg aaa cag ttt gat gtg 144Ala Gln Ile Ile Arg Arg Gln Ile Ala Ala Leu Lys Gln Phe Asp Val 35 40 45 gtg cgc ttt gca cag aaa gcc tgt tcc aat att cat att ttg cgc tta 192Val Arg Phe Ala Gln Lys Ala Cys Ser Asn Ile His Ile Leu Arg Leu 50 55 60 atg cgt gag cag ggc gtg aaa gtg gat tcc gtc tcg tta ggc gaa ata 240Met Arg Glu Gln Gly Val Lys Val Asp Ser Val Ser Leu Gly Glu Ile 65 70 75 80 gag cgt gcg ttg gcg gcg ggt tac aat ccg caa acg cac ccc gat gat 288Glu Arg Ala Leu Ala Ala Gly Tyr Asn Pro Gln Thr His Pro Asp Asp 85 90 95 att gtt ttt acg gca gat gtt atc gat cag gcg acg ctt gaa cgc gtc 336Ile Val Phe Thr Ala Asp Val Ile Asp Gln Ala Thr Leu Glu Arg Val 100 105 110 agt gaa ttg caa att ccg gtg aat gcg ggt tct gtt gat atg ctc gac 384Ser Glu Leu Gln Ile Pro Val Asn Ala Gly Ser Val Asp Met Leu Asp 115 120 125 caa ctg ggc cag gtt tcg cca ggg cat cgg gta tgg ctg cgc gtt aat 432Gln Leu Gly Gln Val Ser Pro Gly His Arg Val Trp Leu Arg Val Asn 130 135 140 ccg ggg ttt ggt cac gga cat agc caa aaa acc aat acc ggt ggc gaa 480Pro Gly Phe Gly His Gly His Ser Gln Lys Thr Asn Thr Gly Gly Glu 145 150 155 160 aac agc aag cac ggt atc tgg tac acc gat ctg ccc gcc gca ctg gac 528Asn Ser Lys His Gly Ile Trp Tyr Thr Asp Leu Pro Ala Ala Leu Asp 165 170 175 gtg ata caa cgt cat cat ctg cag ctg gtc ggc att cac atg cac att 576Val Ile Gln Arg His His Leu Gln Leu Val Gly Ile His Met His Ile 180 185 190 ggt tct ggc gtt gat tat gcc cat ctg gaa cag gtg tgt ggt gct atg 624Gly Ser Gly Val Asp Tyr Ala His Leu Glu Gln Val Cys Gly Ala Met 195 200 205 gtg cgt cag gtc atc gaa ttc ggt cag gat tta cag gct att tct gcg 672Val Arg Gln Val Ile Glu Phe Gly Gln Asp Leu Gln Ala Ile Ser Ala 210 215 220 ggc ggt ggg ctt tct gtt cct tat caa cag ggt gaa gag gcg gtt gat 720Gly Gly Gly Leu Ser Val Pro Tyr Gln Gln Gly Glu Glu Ala Val Asp 225 230 235 240 acc gaa cat tat tat ggt ctg tgg aat gcc gcg cgt gag caa atc gcc 768Thr Glu His Tyr Tyr Gly Leu Trp Asn Ala Ala Arg Glu Gln Ile Ala 245 250 255 cgc cat ttg ggc cac cct gtg aaa ctg gaa att gaa ccg ggt cgc ttc 816Arg His Leu Gly His Pro Val Lys Leu Glu Ile Glu Pro Gly Arg Phe 260 265 270 ctg gta gcg cag tct ggc gta tta att act cag gtg cgg agc gtc aaa 864Leu Val Ala Gln Ser Gly Val Leu Ile Thr Gln Val Arg Ser Val Lys 275 280 285 caa atg ggg agc cgc cac ttt gtg ctg gtt gat gcc ggg ttc aac gat 912Gln Met Gly Ser Arg His Phe Val Leu Val Asp Ala Gly Phe Asn Asp 290 295 300 ctg atg cgc ccg gca atg tac ggt agt tac cac cat atc agt gcc ctg 960Leu Met Arg Pro Ala Met Tyr Gly Ser Tyr His His Ile Ser Ala Leu 305 310 315 320 gca gct gat ggt cgt tct ctg gaa cac gcg cca acg gtg gaa acc gtc 1008Ala Ala Asp Gly Arg Ser Leu Glu His Ala Pro Thr Val Glu Thr Val 325 330 335 gtc gcc gga ccg tta tgt gaa tcg ggc gat gtc ttt acc cag cag gaa 1056Val Ala Gly Pro Leu Cys Glu Ser Gly Asp Val Phe Thr Gln Gln Glu 340 345 350 ggg gga aat gtt gaa acc cgc gcc ttg ccg gaa gtg aag gca ggt gat 1104Gly Gly Asn Val Glu Thr Arg Ala Leu Pro Glu Val Lys Ala Gly Asp 355 360 365 tat ctg gta ctg cat gat aca ggg gca tat ggc gca tca atg tca tcc 1152Tyr Leu Val Leu His Asp Thr Gly Ala Tyr Gly Ala Ser Met Ser Ser 370 375 380 aac tac aat agc cgt ccg ctg tta cca gaa gtt ctg ttt gat aat ggt 1200Asn Tyr Asn Ser Arg Pro Leu Leu Pro Glu Val Leu Phe Asp Asn Gly 385 390 395 400 cag gcg cgg ttg att cgc cgt cgc cag acc atc gaa gaa tta ctg gcg 1248Gln Ala Arg Leu Ile Arg Arg Arg Gln Thr Ile Glu Glu Leu Leu Ala 405 410 415 ctg gaa ttg ctt taa 1263Leu Glu Leu Leu 420 31420PRTEscherichia coli 31Met Pro His Ser Leu Phe Ser Thr Asp Thr Asp Leu Thr Ala Glu Asn 1 5 10 15 Leu Leu Arg Leu Pro Ala Glu Phe Gly Cys Pro Val Trp Val Tyr Asp 20 25 30 Ala Gln Ile Ile Arg Arg Gln Ile Ala Ala Leu Lys Gln Phe Asp Val 35 40 45 Val Arg Phe Ala Gln Lys Ala Cys Ser Asn Ile His Ile Leu Arg Leu 50 55 60 Met Arg Glu Gln Gly Val Lys Val Asp Ser Val Ser Leu Gly Glu Ile 65 70 75 80 Glu Arg Ala Leu Ala Ala Gly Tyr Asn Pro Gln Thr His Pro Asp Asp 85 90 95 Ile Val Phe Thr Ala Asp Val Ile Asp Gln Ala Thr Leu Glu Arg Val 100 105 110 Ser Glu Leu Gln Ile Pro Val Asn Ala Gly Ser Val Asp Met Leu Asp 115 120 125 Gln Leu Gly Gln Val Ser Pro Gly His Arg Val Trp Leu Arg Val Asn 130 135 140 Pro Gly Phe Gly His Gly His Ser Gln Lys Thr Asn Thr Gly Gly Glu 145 150 155 160 Asn Ser Lys His Gly Ile Trp Tyr Thr Asp Leu Pro Ala Ala Leu Asp 165 170 175 Val Ile Gln Arg His His Leu Gln Leu Val Gly Ile His Met His Ile 180 185 190 Gly Ser Gly Val Asp Tyr Ala His Leu Glu Gln Val Cys Gly Ala Met 195 200 205 Val Arg Gln Val Ile Glu Phe Gly Gln Asp Leu Gln Ala Ile Ser Ala 210 215 220 Gly Gly Gly Leu Ser Val Pro Tyr Gln Gln Gly Glu Glu Ala Val Asp 225 230 235 240 Thr Glu His Tyr Tyr Gly Leu Trp Asn Ala Ala Arg Glu Gln Ile Ala 245 250 255 Arg His Leu Gly His Pro Val Lys Leu Glu Ile Glu Pro Gly Arg Phe 260 265 270 Leu Val Ala Gln Ser Gly Val Leu Ile Thr Gln Val Arg Ser Val Lys 275 280 285 Gln Met Gly Ser Arg His Phe Val Leu Val Asp Ala Gly Phe Asn Asp 290 295 300 Leu Met Arg Pro Ala Met Tyr Gly Ser Tyr His His Ile Ser Ala Leu 305 310 315 320 Ala Ala Asp Gly Arg Ser Leu Glu His Ala Pro Thr Val Glu Thr Val 325 330 335 Val Ala Gly Pro Leu Cys Glu Ser Gly Asp Val Phe Thr Gln Gln Glu 340 345 350 Gly Gly Asn Val Glu Thr Arg Ala Leu Pro Glu Val Lys Ala Gly Asp 355 360 365 Tyr Leu Val Leu His Asp Thr Gly Ala Tyr Gly Ala Ser Met Ser Ser 370 375 380 Asn Tyr Asn Ser Arg Pro Leu Leu Pro Glu Val Leu Phe Asp Asn Gly 385 390 395 400 Gln Ala Arg Leu Ile Arg Arg Arg Gln Thr Ile Glu Glu Leu Leu Ala 405 410 415 Leu Glu Leu Leu 420 321265DNAArtificialEscherichia.coli diaminopimelate decarboxylase LysA codon optimised gene 32atatgccaca ctctctgttt tctactgata ctgatctgac tgcggaaaac ctgctgcgtc 60tgccggctga attcggttgt ccggtatggg tgtacgacgc tcagattatt cgtcgccaga 120tcgcagcact gaagcagttc gatgtagtgc gttttgcaca gaaggcgtgc tccaacatcc 180atatcctgcg cctgatgcgt gagcagggcg ttaaagttga ctccgtctct ctgggtgaga 240ttgagcgcgc cctggcagcc ggctataacc cacagaccca tcctgacgac attgtattta 300ctgccgacgt gatcgaccag gctactctgg aacgcgtttc tgaactgcag atcccggtta 360atgctggttc tgtggacatg ctggaccagc tgggccaggt atccccaggt catcgtgtgt 420ggctgcgtgt caacccaggt ttcggccacg gccactctca gaaaactaac actggtggtg 480agaactccaa gcatggcatt tggtataccg atctgccggc tgcactggac gtaatccagc 540gtcaccacct gcagctggtg ggcatccaca tgcacattgg ctccggcgta gactacgccc 600acctggagca agtctgcggt gctatggtac gtcaggtaat cgagttcggc caagatctgc 660aggcaatcag cgctggtggc ggcctgtctg taccttatca gcagggcgag gaggcggttg 720acactgagca ctactacggt ctgtggaacg ccgctcgtga gcaaattgca cgtcacctgg 780gccacccggt gaaactggag atcgagccgg gccgcttcct ggtagcacag tccggcgtac 840tgattaccca ggtacgctct gttaaacaga tgggctcccg tcactttgtg ctggtagacg 900caggcttcaa cgacctgatg cgtccggcta tgtatggttc ctatcatcac atctctgcgc 960tggccgccga cggccgctct ctggaacacg cgccgacggt tgaaacggtg gtggctggtc 1020cgctgtgcga gtccggcgac gttttcactc agcaggaggg cggcaatgta gagacgcgtg 1080cgctgccgga agtgaaagcc ggtgattatc tggtgctgca tgataccggc gcctatggtg 1140cgagcatgag cagcaactac aactctcgcc cgctgctgcc ggaggtcctg ttcgataacg 1200gccaagcccg cctgatccgt cgtcgtcaga ccatcgagga actgctggca ctggagctgc 1260tgtaa 1265331692DNASaccharomyces cerevisiaeCDS(1)..(1692) 33atg tct gaa att act ttg ggt aaa tat ttg ttc gaa aga tta aag caa 48Met Ser Glu Ile Thr Leu Gly Lys Tyr Leu Phe Glu Arg Leu Lys Gln 1 5 10 15 gtc aac gtt aac acc gtt ttc ggt ttg cca ggt gac ttc aac ttg tcc 96Val Asn Val Asn Thr Val Phe Gly Leu Pro Gly Asp Phe Asn Leu Ser 20 25 30 ttg ttg gac aag atc tac gaa gtt gaa ggt atg aga tgg gct ggt aac 144Leu Leu Asp Lys Ile Tyr Glu Val Glu Gly Met Arg Trp Ala Gly Asn 35 40 45 gcc aac gaa ttg aac gct gct tac gcc gct gat ggt tac gct cgt atc 192Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg Ile 50 55 60 aag ggt atg tct tgt atc atc acc acc ttc ggt gtc ggt gaa ttg tct 240Lys Gly Met Ser Cys Ile Ile Thr Thr Phe Gly Val Gly Glu Leu Ser 65 70 75 80 gct ttg aac ggt att gcc ggt tct tac gct gaa cac gtc ggt gtt ttg 288Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu 85 90 95 cac gtt gtt ggt gtc cca tcc atc tct gct caa gct aag caa ttg ttg 336His Val Val Gly Val Pro Ser Ile Ser Ala Gln Ala Lys Gln Leu Leu 100 105 110 ttg cac cac acc ttg ggt aac ggt gac ttc act gtt ttc cac aga atg 384Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His Arg Met 115 120 125 tct gcc aac att tct gaa acc act gct atg atc act gac att gct acc 432Ser Ala Asn Ile Ser Glu Thr Thr Ala Met Ile Thr Asp Ile Ala Thr 130 135 140 gcc cca gct gaa att gac aga tgt atc aga acc act tac gtc acc caa 480Ala Pro Ala Glu Ile Asp Arg Cys Ile Arg Thr Thr Tyr Val Thr Gln 145 150 155 160 aga cca gtc tac tta ggt ttg cca gct aac ttg gtc gac ttg aac gtc 528Arg Pro Val Tyr Leu Gly Leu Pro Ala Asn Leu Val Asp Leu Asn Val 165 170 175 cca gct aag ttg ttg caa act cca att gac atg tct ttg aag cca aac 576Pro Ala Lys Leu Leu Gln Thr Pro Ile Asp Met Ser Leu Lys Pro Asn 180 185 190 gat gct gaa tcc gaa aag gaa gtc att gac acc atc ttg gct ttg gtc 624Asp Ala Glu Ser Glu Lys Glu Val Ile Asp Thr Ile Leu Ala Leu Val 195 200 205 aag gat gct aag aac cca gtt atc ttg gct gat gct tgt tgt tcc aga 672Lys Asp Ala Lys Asn Pro Val Ile Leu Ala Asp Ala Cys Cys Ser Arg 210 215 220 cac gac gtc aag gct gaa act aag aag ttg att gac ttg act caa ttc 720His Asp Val Lys Ala Glu Thr Lys Lys Leu Ile Asp Leu Thr Gln Phe 225 230 235 240 cca gct ttc gtc acc cca atg ggt aag ggt tcc att gac gaa caa cac 768Pro Ala Phe Val Thr Pro Met Gly Lys Gly Ser Ile Asp Glu Gln His 245 250 255 cca aga tac ggt ggt gtt tac gtc ggt acc ttg tcc aag cca gaa gtt 816Pro Arg Tyr Gly Gly Val Tyr Val Gly Thr Leu Ser Lys Pro Glu Val 260 265 270 aag gaa gcc gtt gaa tct gct gac ttg att ttg tct gtc ggt gct ttg 864Lys Glu Ala Val Glu Ser Ala Asp Leu Ile Leu Ser Val Gly Ala Leu 275 280 285 ttg tct gat ttc aac acc ggt tct ttc tct tac tct tac aag acc aag 912Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr Ser Tyr Lys Thr Lys 290 295 300 aac att gtc gaa ttc cac tcc gac cac atg aag atc aga aac gcc act 960Asn Ile Val Glu Phe His Ser Asp His Met Lys Ile Arg Asn Ala Thr 305 310 315 320 ttc cca ggt gtc caa atg aaa ttc gtt ttg caa aag ttg ttg acc act 1008Phe Pro Gly Val Gln Met Lys Phe Val Leu Gln Lys Leu Leu Thr Thr 325 330 335 att gct gac gcc gct aag ggt tac aag cca gtt gct gtc cca gct aga 1056Ile Ala Asp Ala Ala Lys Gly Tyr Lys Pro Val Ala Val Pro Ala Arg 340 345 350 act cca gct aac gct gct gtc cca gct tct acc cca ttg aag caa gaa 1104Thr Pro Ala Asn Ala Ala Val Pro Ala Ser Thr Pro Leu Lys Gln Glu 355 360 365 tgg atg tgg aac caa ttg ggt aac ttc ttg caa gaa ggt gat gtt gtc 1152Trp Met Trp Asn Gln Leu Gly Asn Phe Leu Gln Glu Gly Asp Val Val 370 375 380 att gct gaa acc ggt acc tcc gct ttc ggt atc aac caa acc act ttc 1200Ile Ala Glu Thr Gly Thr Ser Ala Phe Gly Ile Asn Gln Thr Thr Phe 385 390 395 400 cca aac aac acc tac ggt atc tct caa gtc tta tgg ggt tcc att ggt 1248Pro Asn Asn Thr Tyr Gly Ile Ser Gln Val Leu Trp Gly Ser Ile Gly 405 410 415 ttc acc act ggt gct acc ttg ggt gct gct ttc gct gct gaa gaa att 1296Phe Thr Thr Gly Ala Thr Leu Gly Ala Ala Phe Ala Ala Glu Glu Ile 420 425 430 gat cca aag aag aga gtt atc tta ttc att ggt gac ggt tct ttg caa

1344Asp Pro Lys Lys Arg Val Ile Leu Phe Ile Gly Asp Gly Ser Leu Gln 435 440 445 ttg act gtt caa gaa atc tcc acc atg atc aga tgg ggc ttg aag cca 1392Leu Thr Val Gln Glu Ile Ser Thr Met Ile Arg Trp Gly Leu Lys Pro 450 455 460 tac ttg ttc gtc ttg aac aac gat ggt tac acc att gaa aag ttg att 1440Tyr Leu Phe Val Leu Asn Asn Asp Gly Tyr Thr Ile Glu Lys Leu Ile 465 470 475 480 cac ggt cca aag gct caa tac aac gaa att caa ggt tgg gac cac cta 1488His Gly Pro Lys Ala Gln Tyr Asn Glu Ile Gln Gly Trp Asp His Leu 485 490 495 tcc ttg ttg cca act ttc ggt gct aag gac tat gaa acc cac aga gtc 1536Ser Leu Leu Pro Thr Phe Gly Ala Lys Asp Tyr Glu Thr His Arg Val 500 505 510 gct acc acc ggt gaa tgg gac aag ttg acc caa gac aag tct ttc aac 1584Ala Thr Thr Gly Glu Trp Asp Lys Leu Thr Gln Asp Lys Ser Phe Asn 515 520 525 gac aac tct aag atc aga atg att gaa atc atg ttg cca gtc ttc gat 1632Asp Asn Ser Lys Ile Arg Met Ile Glu Ile Met Leu Pro Val Phe Asp 530 535 540 gct cca caa aac ttg gtt gaa caa gct aag ttg act gct gct acc aac 1680Ala Pro Gln Asn Leu Val Glu Gln Ala Lys Leu Thr Ala Ala Thr Asn 545 550 555 560 gct aag caa taa 1692Ala Lys Gln 34563PRTSaccharomyces cerevisiae 34Met Ser Glu Ile Thr Leu Gly Lys Tyr Leu Phe Glu Arg Leu Lys Gln 1 5 10 15 Val Asn Val Asn Thr Val Phe Gly Leu Pro Gly Asp Phe Asn Leu Ser 20 25 30 Leu Leu Asp Lys Ile Tyr Glu Val Glu Gly Met Arg Trp Ala Gly Asn 35 40 45 Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg Ile 50 55 60 Lys Gly Met Ser Cys Ile Ile Thr Thr Phe Gly Val Gly Glu Leu Ser 65 70 75 80 Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu 85 90 95 His Val Val Gly Val Pro Ser Ile Ser Ala Gln Ala Lys Gln Leu Leu 100 105 110 Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His Arg Met 115 120 125 Ser Ala Asn Ile Ser Glu Thr Thr Ala Met Ile Thr Asp Ile Ala Thr 130 135 140 Ala Pro Ala Glu Ile Asp Arg Cys Ile Arg Thr Thr Tyr Val Thr Gln 145 150 155 160 Arg Pro Val Tyr Leu Gly Leu Pro Ala Asn Leu Val Asp Leu Asn Val 165 170 175 Pro Ala Lys Leu Leu Gln Thr Pro Ile Asp Met Ser Leu Lys Pro Asn 180 185 190 Asp Ala Glu Ser Glu Lys Glu Val Ile Asp Thr Ile Leu Ala Leu Val 195 200 205 Lys Asp Ala Lys Asn Pro Val Ile Leu Ala Asp Ala Cys Cys Ser Arg 210 215 220 His Asp Val Lys Ala Glu Thr Lys Lys Leu Ile Asp Leu Thr Gln Phe 225 230 235 240 Pro Ala Phe Val Thr Pro Met Gly Lys Gly Ser Ile Asp Glu Gln His 245 250 255 Pro Arg Tyr Gly Gly Val Tyr Val Gly Thr Leu Ser Lys Pro Glu Val 260 265 270 Lys Glu Ala Val Glu Ser Ala Asp Leu Ile Leu Ser Val Gly Ala Leu 275 280 285 Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr Ser Tyr Lys Thr Lys 290 295 300 Asn Ile Val Glu Phe His Ser Asp His Met Lys Ile Arg Asn Ala Thr 305 310 315 320 Phe Pro Gly Val Gln Met Lys Phe Val Leu Gln Lys Leu Leu Thr Thr 325 330 335 Ile Ala Asp Ala Ala Lys Gly Tyr Lys Pro Val Ala Val Pro Ala Arg 340 345 350 Thr Pro Ala Asn Ala Ala Val Pro Ala Ser Thr Pro Leu Lys Gln Glu 355 360 365 Trp Met Trp Asn Gln Leu Gly Asn Phe Leu Gln Glu Gly Asp Val Val 370 375 380 Ile Ala Glu Thr Gly Thr Ser Ala Phe Gly Ile Asn Gln Thr Thr Phe 385 390 395 400 Pro Asn Asn Thr Tyr Gly Ile Ser Gln Val Leu Trp Gly Ser Ile Gly 405 410 415 Phe Thr Thr Gly Ala Thr Leu Gly Ala Ala Phe Ala Ala Glu Glu Ile 420 425 430 Asp Pro Lys Lys Arg Val Ile Leu Phe Ile Gly Asp Gly Ser Leu Gln 435 440 445 Leu Thr Val Gln Glu Ile Ser Thr Met Ile Arg Trp Gly Leu Lys Pro 450 455 460 Tyr Leu Phe Val Leu Asn Asn Asp Gly Tyr Thr Ile Glu Lys Leu Ile 465 470 475 480 His Gly Pro Lys Ala Gln Tyr Asn Glu Ile Gln Gly Trp Asp His Leu 485 490 495 Ser Leu Leu Pro Thr Phe Gly Ala Lys Asp Tyr Glu Thr His Arg Val 500 505 510 Ala Thr Thr Gly Glu Trp Asp Lys Leu Thr Gln Asp Lys Ser Phe Asn 515 520 525 Asp Asn Ser Lys Ile Arg Met Ile Glu Ile Met Leu Pro Val Phe Asp 530 535 540 Ala Pro Gln Asn Leu Val Glu Gln Ala Lys Leu Thr Ala Ala Thr Asn 545 550 555 560 Ala Lys Gln 351692DNAArtificialSaccharomyces cerevisiae pyruvate decarboxylase Pdc codon optimised gene 35atgtccgaga tcactctggg caaatacctg tttgaacgtc tgaaacaggt gaacgttaat 60accgtattcg gcctgccggg tgatttcaac ctgtccctgc tggacaaaat ctatgaagtt 120gaaggtatgc gttgggctgg caacgctaac gagctgaacg cagcgtacgc ggcagatggt 180tacgctcgta tcaaaggtat gtcttgtatc atcaccacct tcggtgttgg tgagctgagc 240gccctgaacg gcatcgccgg ctcctatgca gagcacgtgg gcgtgctgca cgttgtgggt 300gtaccgtcca tcagcgccca ggcaaaacag ctgctgctgc accacaccct gggtaacggc 360gactttaccg ttttccatcg tatgtctgcg aacatcagcg aaactactgc aatgattact 420gacatcgcta cggcaccggc agaaatcgac cgttgcattc gtaccacgta cgttactcag 480cgcccggttt atctgggcct gccagccaac ctggtggatc tgaacgtccc ggctaaactg 540ctgcagactc cgatcgatat gtctctgaaa cctaacgacg cagaatctga gaaagaagtt 600atcgatacta ttctggctct ggtgaaagat gcaaagaacc cagttatcct ggctgacgca 660tgttgctctc gtcatgatgt aaaggcagaa accaaaaagc tgatcgacct gacgcagttc 720ccggcgttcg ttaccccgat gggcaagggt tccatcgatg agcagcaccc gcgttatggt 780ggtgtatacg ttggcacgct gtccaaaccg gaggtaaaag aagcggttga aagcgcagat 840ctgatcctgt ctgttggtgc actgctgagc gacttcaaca ccggttcttt ctcctatagc 900tacaagacca aaaacattgt ggagtttcac tccgatcaca tgaaaatccg caacgcgacc 960tttcctggtg tgcagatgaa attcgtactg cagaaactgc tgaccaccat cgccgacgct 1020gcgaaaggtt ataaaccggt agctgtgccg gcacgtaccc cggcgaacgc cgcggttcct 1080gcatccactc cactgaagca ggaatggatg tggaatcagc tgggtaattt cctgcaagaa 1140ggcgacgttg taatcgcaga aaccggcact agcgcgtttg gcattaacca gacgaccttc 1200ccaaacaaca cctacggtat cagccaagtc ctgtggggct ctatcggctt caccaccggt 1260gcaaccctgg gtgcggcttt cgctgctgag gagatcgacc cgaagaaacg tgttatcctg 1320ttcatcggtg acggctccct gcagctgacc gtccaggaga tttctaccat gatccgctgg 1380ggcctgaaac cgtacctgtt tgtgctgaac aacgacggct acactattga gaaactgatc 1440cacggtccga aagcacagta taatgagatc cagggttggg atcatctgtc tctgctgccg 1500acctttggcg ctaaagacta cgagacccac cgcgtggcta ccaccggcga gtgggataaa 1560ctgacgcagg ataaatcctt caatgacaat agcaagattc gtatgatcga aatcatgctg 1620ccggtctttg atgctccgca gaacctggta gagcaagcaa aactgaccgc ggcaactaac 1680gctaaacagt aa 1692361707DNAZymomonas mobilisCDS(1)..(1707) 36atg agt tat act gtc ggt acc tat tta gcg gag cgg ctt gtc cag att 48Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile 1 5 10 15 ggt ctc aag cat cac ttc gca gtc gcg ggc gac tac aac ctc gtc ctt 96Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu 20 25 30 ctt gac aac ctg ctt ttg aac aaa aac atg gag cag gtt tat tgc tgt 144Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys 35 40 45 aac gaa ctg aac tgc ggt ttc agt gca gaa ggt tat gct cgt gcc aaa 192Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys 50 55 60 ggc gca gca gca gcc gtc gtt acc tac agc gtc ggt gcg ctt tcc gca 240Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala 65 70 75 80 ttt gat gct atc ggt ggc gcc tat gca gaa aac ctt ccg gtt atc ctg 288Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu 85 90 95 atc tcc ggt gct ccg aac aac aat gat cac gct gct ggt cac gtg ttg 336Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu 100 105 110 cat cac gct ctt ggc aaa acc gac tat cac tat cag ttg gaa atg gcc 384His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala 115 120 125 aag aac atc acg gcc gcc gct gaa gcg att tac acc ccg gaa gaa gct 432Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala 130 135 140 ccg gct aaa atc gat cac gtg att aaa act gct ctt cgt gag aag aag 480Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys 145 150 155 160 ccg gtt tat ctc gaa atc gct tgc aac att gct tcc atg ccc tgc gcc 528Pro Val Tyr Leu Glu Ile Ala Cys Asn Ile Ala Ser Met Pro Cys Ala 165 170 175 gct cct gga ccg gca agc gca ttg ttc aat gac gaa gcc agc gac gaa 576Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu 180 185 190 gct tct ttg aat gca gcg gtt gaa gaa acc ctg aaa ttc atc gcc aac 624Ala Ser Leu Asn Ala Ala Val Glu Glu Thr Leu Lys Phe Ile Ala Asn 195 200 205 cgc gac aaa gtt gcc gtc ctc gtc ggc agc aag ctg cgc gca gct ggt 672Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly 210 215 220 gct gaa gaa gct gct gtc aaa ttt gct gat gct ctc ggt ggc gca gtt 720Ala Glu Glu Ala Ala Val Lys Phe Ala Asp Ala Leu Gly Gly Ala Val 225 230 235 240 gct acc atg gct gct gca aaa agc ttc ttc cca gaa gaa aac ccg cat 768Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Pro His 245 250 255 tac atc ggc acc tca tgg ggt gaa gtc agc tat ccg ggc gtt gaa aag 816Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys 260 265 270 acg atg aaa gaa gcc gat gcg gtt atc gct ctg gct cct gtc ttc aac 864Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn 275 280 285 gac tac tcc acc act ggt tgg acg gat att cct gat cct aag aaa ctg 912Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu 290 295 300 gtt ctc gct gaa ccg cgt tct gtc gtc gtt aac ggc att cgc ttc ccc 960Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro 305 310 315 320 agc gtc cat ctg aaa gac tat ctg acc cgt ttg gct cag aaa gtt tcc 1008Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser 325 330 335 aag aaa acc ggt gca ttg gac ttc ttc aaa tcc ctc aat gca ggt gaa 1056Lys Lys Thr Gly Ala Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu 340 345 350 ctg aag aaa gcc gct ccg gct gat ccg agt gct ccg ttg gtc aac gca 1104Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala 355 360 365 gaa atc gcc cgt cag gtc gaa gct ctt ctg acc ccg aac acg acg gtt 1152Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val 370 375 380 att gct gaa acc ggt gac tct tgg ttc aat gct cag cgc atg aag ctc 1200Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu 385 390 395 400 ccg aac ggt gct cgc gtt gaa tat gaa atg cag tgg ggt cac att ggt 1248Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly 405 410 415 tgg tcc gtt cct gcc gcc ttc ggt tat gcc gtc ggt gct ccg gaa cgt 1296Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg 420 425 430 cgc aac atc ctc atg gtt ggt gat ggt tcc ttc cag ctg acg gct cag 1344Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln 435 440 445 gaa gtc gct cag atg gtt cgc ctg aaa ctg ccg gtt atc atc ttc ttg 1392Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu 450 455 460 atc aat aac tat ggt tac acc gcc gaa gtt atg atc cat gat ggt ccg 1440Ile Asn Asn Tyr Gly Tyr Thr Ala Glu Val Met Ile His Asp Gly Pro 465 470 475 480 tac aac aac atc aag aac tgg gat tat gcc ggt ctg atg gaa gtg ttc 1488Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe 485 490 495 aac ggt aac ggt ggt tat gac agc ggt gct ggt aaa ggc ctg aag gct 1536Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Gly Lys Gly Leu Lys Ala 500 505 510 aaa acc ggt ggc gaa ctg gca gaa gct atc aag gtt gct ctg gca aac 1584Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn 515 520 525 acc gac ggc cca acc ctg atc gaa tgc ttc atc ggt cgt gaa gac tgc 1632Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys 530 535 540 act gaa gaa ttg gtc aaa tgg ggt aag cgc gtt gct gcc gcc aac agc 1680Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser 545 550 555 560 cgt aag cct gtt aac aag ctc ctc tag 1707Arg Lys Pro Val Asn Lys Leu Leu 565 37568PRTZymomonas mobilis 37Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln Ile 1 5 10 15 Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu 20 25 30 Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys Cys 35 40 45 Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys 50 55 60 Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala 65 70 75 80 Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu 85 90 95 Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu 100 105 110 His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met Ala 115 120 125 Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu Ala 130 135 140 Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys Lys 145 150 155 160 Pro Val Tyr Leu Glu Ile Ala Cys Asn Ile Ala Ser Met Pro Cys Ala 165 170 175 Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu 180 185 190 Ala Ser Leu Asn Ala Ala Val Glu Glu Thr Leu Lys Phe Ile Ala Asn 195 200 205 Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly 210 215 220 Ala Glu Glu Ala Ala Val Lys Phe Ala Asp Ala Leu Gly Gly Ala Val 225 230

235 240 Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Pro His 245 250 255 Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys 260 265 270 Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe Asn 275 280 285 Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys Leu 290 295 300 Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Ile Arg Phe Pro 305 310 315 320 Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val Ser 325 330 335 Lys Lys Thr Gly Ala Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu 340 345 350 Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala 355 360 365 Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val 370 375 380 Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys Leu 385 390 395 400 Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile Gly 405 410 415 Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg 420 425 430 Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln 435 440 445 Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe Leu 450 455 460 Ile Asn Asn Tyr Gly Tyr Thr Ala Glu Val Met Ile His Asp Gly Pro 465 470 475 480 Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe 485 490 495 Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Gly Lys Gly Leu Lys Ala 500 505 510 Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala Asn 515 520 525 Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp Cys 530 535 540 Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn Ser 545 550 555 560 Arg Lys Pro Val Asn Lys Leu Leu 565 381707DNAArtificialZymomonas mobilis pyruvate decarboxylase PdcI472A codon optimised gene 38atgtcttata ctgttggtac ttatctggct gagcgtctgg tgcaaatcgg cctgaaacac 60cactttgcag ttgctggcga ctacaacctg gttctgctgg ataacctgct gctgaacaaa 120aacatggagc aagtttattg ctgtaacgag ctgaactgcg gcttctctgc ggagggttat 180gcgcgtgcga aaggtgccgc tgcagcagtc gtaacctact ctgtgggcgc tctgtccgcg 240ttcgacgcaa tcggtggcgc ttacgctgaa aacctgccgg tgatcctgat tagcggtgcg 300ccgaataata acgaccatgc tgctggccac gttctgcacc acgccctggg taaaactgat 360taccattacc agctggagat ggctaaaaac atcactgcag cagcagaagc gatctacacc 420ccggaagagg ctccggcaaa aatcgaccac gtgattaaaa ccgctctgcg tgagaaaaag 480ccggtatacc tggaaatcgc gtgcaacatc gcgtctatgc cgtgcgccgc accgggtccg 540gcttctgccc tgttcaacga tgaggcgagc gatgaggcat ctctgaacgc agcagtagaa 600gaaaccctga aatttatcgc aaaccgtgac aaagtagcag tcctggtagg ttctaaactg 660cgtgcggctg gtgcggaaga ggctgcggta aagttcgcgg atgctctggg cggtgcagtg 720gcgaccatgg cagcggctaa atccttcttc ccagaggaga acccgcatta cattggtacc 780tcctggggcg aagtttccta ccctggtgtg gagaaaacca tgaaagaagc cgatgctgtg 840attgccctgg cgcctgtatt caacgattat tccaccaccg gttggaccga tatcccggac 900ccgaagaaac tggtcctggc tgaaccgcgc tccgtagtag tgaatggcat tcgtttcccg 960tccgtacacc tgaaggatta cctgacgcgt ctggcacaga aagtatccaa gaaaactggc 1020gcgctggact tctttaaatc cctgaacgct ggtgagctga aaaaggcggc tccggccgat 1080ccgtccgcac cgctggtgaa cgcagagatt gcacgtcagg ttgaggcact gctgacgccg 1140aacaccaccg taatcgcgga aacgggcgac tcttggttca acgcacagcg catgaaactg 1200ccgaacggtg cccgcgttga atatgaaatg cagtggggtc acatcggctg gtctgtccca 1260gcagcgtttg gttacgcggt tggtgcaccg gagcgtcgca acatcctgat ggtgggtgac 1320ggctccttcc agctgactgc tcaggaggtg gcgcagatgg tgcgcctgaa gctgccggtt 1380atcattttcc tgatcaacaa ctacggctac accgccgagg taatgatcca cgatggtccg 1440tacaacaaca tcaaaaactg ggactacgcc ggtctgatgg aggtttttaa cggtaacggc 1500ggttacgaca gcggtgctgg taagggtctg aaagccaaaa ccggtggcga actggcagag 1560gcgattaaag ttgcgctggc aaacaccgat ggcccgaccc tgatcgagtg cttcatcggc 1620cgtgaggact gcaccgagga gctggtcaaa tggggcaaac gtgtggcggc tgctaactct 1680cgcaagccgg taaacaaact gctgtaa 1707391644DNALactococcus lactisCDS(1)..(1644) 39atg tat aca gta gga gat tac ctg tta gac cga tta cac gag ttg gga 48Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 1 5 10 15 att gaa gaa att ttt gga gtt cct ggt gac tat aac tta caa ttt tta 96Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu 20 25 30 gat caa att att tca cgc gaa gat atg aaa tgg att gga aat gct aat 144Asp Gln Ile Ile Ser Arg Glu Asp Met Lys Trp Ile Gly Asn Ala Asn 35 40 45 gaa tta aat gct tct tat atg gct gat ggt tat gct cgt act aaa aaa 192Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys 50 55 60 gct gcc gca ttt ctc acc aca ttt gga gtc ggc gaa ttg agt gcg atc 240Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Ile 65 70 75 80 aat gga ctg gca gga agt tat gcc gaa aat tta cca gta gta gaa att 288Asn Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu Ile 85 90 95 gtt ggt tca cca act tca aaa gta caa aat gac gga aaa ttt gtc cat 336Val Gly Ser Pro Thr Ser Lys Val Gln Asn Asp Gly Lys Phe Val His 100 105 110 cat aca cta gca gat ggt gat ttt aaa cac ttt atg aag atg cat gaa 384His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu 115 120 125 cct gtt aca gca gcg cgg act tta ctg aca gca gaa aat gcc aca tat 432Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr Tyr 130 135 140 gaa att gac cga gta ctt tct caa tta cta aaa gaa aga aaa cca gtc 480Glu Ile Asp Arg Val Leu Ser Gln Leu Leu Lys Glu Arg Lys Pro Val 145 150 155 160 tat att aac tta cca gtc gat gtt gct gca gca aaa gca gag aag cct 528Tyr Ile Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro 165 170 175 gca tta tct tta gaa aaa gaa agc tct aca aca aat aca act gaa caa 576Ala Leu Ser Leu Glu Lys Glu Ser Ser Thr Thr Asn Thr Thr Glu Gln 180 185 190 gtg att ttg agt aag att gaa gaa agt ttg aaa aat gcc caa aaa cca 624Val Ile Leu Ser Lys Ile Glu Glu Ser Leu Lys Asn Ala Gln Lys Pro 195 200 205 gta gtg att gca gga cac gaa gta att agt ttt ggt tta gaa aaa acg 672Val Val Ile Ala Gly His Glu Val Ile Ser Phe Gly Leu Glu Lys Thr 210 215 220 gta act cag ttt gtt tca gaa aca aaa cta ccg att acg aca cta aat 720Val Thr Gln Phe Val Ser Glu Thr Lys Leu Pro Ile Thr Thr Leu Asn 225 230 235 240 ttt ggt aaa agt gct gtt gat gaa tct ttg ccc tca ttt tta gga ata 768Phe Gly Lys Ser Ala Val Asp Glu Ser Leu Pro Ser Phe Leu Gly Ile 245 250 255 tat aac ggg aaa ctt tca gaa atc agt ctt aaa aat ttt gtg gag tcc 816Tyr Asn Gly Lys Leu Ser Glu Ile Ser Leu Lys Asn Phe Val Glu Ser 260 265 270 gca gac ttt atc cta atg ctt gga gtg aag ctt acg gac tcc tca aca 864Ala Asp Phe Ile Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr 275 280 285 ggt gca ttc aca cat cat tta gat gaa aat aaa atg att tca cta aac 912Gly Ala Phe Thr His His Leu Asp Glu Asn Lys Met Ile Ser Leu Asn 290 295 300 ata gat gaa gga ata att ttc aat aaa gtg gta gaa gat ttt gat ttt 960Ile Asp Glu Gly Ile Ile Phe Asn Lys Val Val Glu Asp Phe Asp Phe 305 310 315 320 aga gca gtg gtt tct tct tta tca gaa tta aaa gga ata gaa tat gaa 1008Arg Ala Val Val Ser Ser Leu Ser Glu Leu Lys Gly Ile Glu Tyr Glu 325 330 335 gga caa tat att gat aag caa tat gaa gaa ttt att cca tca agt gct 1056Gly Gln Tyr Ile Asp Lys Gln Tyr Glu Glu Phe Ile Pro Ser Ser Ala 340 345 350 ccc tta tca caa gac cgt cta tgg cag gca gtt gaa agt ttg act caa 1104Pro Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Ser Leu Thr Gln 355 360 365 agc aat gaa aca atc gtt gct gaa caa gga acc tca ttt ttt gga gct 1152Ser Asn Glu Thr Ile Val Ala Glu Gln Gly Thr Ser Phe Phe Gly Ala 370 375 380 tca aca att ttc tta aaa tca aat agt cgt ttt att gga caa cct tta 1200Ser Thr Ile Phe Leu Lys Ser Asn Ser Arg Phe Ile Gly Gln Pro Leu 385 390 395 400 tgg ggt tct att gga tat act ttt cca gcg gct tta gga agc caa att 1248Trp Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gln Ile 405 410 415 gcg gat aaa gag agc aga cac ctt tta ttt att ggt gat ggt tca ctt 1296Ala Asp Lys Glu Ser Arg His Leu Leu Phe Ile Gly Asp Gly Ser Leu 420 425 430 caa ctt acc gta caa gaa tta gga cta tca atc aga gaa aaa ctc aat 1344Gln Leu Thr Val Gln Glu Leu Gly Leu Ser Ile Arg Glu Lys Leu Asn 435 440 445 cca att tgt ttt atc ata aat aat gat ggt tat aca gtt gaa aga gaa 1392Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu 450 455 460 atc cac gga cct act caa agt tat aac gac att cca atg tgg aat tac 1440Ile His Gly Pro Thr Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr 465 470 475 480 tcg aaa tta cca gaa aca ttt gga gca aca gaa gat cgt gta gta tca 1488Ser Lys Leu Pro Glu Thr Phe Gly Ala Thr Glu Asp Arg Val Val Ser 485 490 495 aaa att gtt aga aca gag aat gaa ttt gtg tct gtc atg aaa gaa gcc 1536Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala 500 505 510 caa gca gat gtc aat aga atg tat tgg ata gaa cta gtt ttg gaa aaa 1584Gln Ala Asp Val Asn Arg Met Tyr Trp Ile Glu Leu Val Leu Glu Lys 515 520 525 gaa gat gcg cca aaa tta ctg aaa aaa atg ggt aaa tta ttt gct gag 1632Glu Asp Ala Pro Lys Leu Leu Lys Lys Met Gly Lys Leu Phe Ala Glu 530 535 540 caa aat aaa tag 1644Gln Asn Lys 545 40547PRTLactococcus lactis 40Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 1 5 10 15 Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu 20 25 30 Asp Gln Ile Ile Ser Arg Glu Asp Met Lys Trp Ile Gly Asn Ala Asn 35 40 45 Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys 50 55 60 Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Ile 65 70 75 80 Asn Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu Ile 85 90 95 Val Gly Ser Pro Thr Ser Lys Val Gln Asn Asp Gly Lys Phe Val His 100 105 110 His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu 115 120 125 Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr Tyr 130 135 140 Glu Ile Asp Arg Val Leu Ser Gln Leu Leu Lys Glu Arg Lys Pro Val 145 150 155 160 Tyr Ile Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro 165 170 175 Ala Leu Ser Leu Glu Lys Glu Ser Ser Thr Thr Asn Thr Thr Glu Gln 180 185 190 Val Ile Leu Ser Lys Ile Glu Glu Ser Leu Lys Asn Ala Gln Lys Pro 195 200 205 Val Val Ile Ala Gly His Glu Val Ile Ser Phe Gly Leu Glu Lys Thr 210 215 220 Val Thr Gln Phe Val Ser Glu Thr Lys Leu Pro Ile Thr Thr Leu Asn 225 230 235 240 Phe Gly Lys Ser Ala Val Asp Glu Ser Leu Pro Ser Phe Leu Gly Ile 245 250 255 Tyr Asn Gly Lys Leu Ser Glu Ile Ser Leu Lys Asn Phe Val Glu Ser 260 265 270 Ala Asp Phe Ile Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr 275 280 285 Gly Ala Phe Thr His His Leu Asp Glu Asn Lys Met Ile Ser Leu Asn 290 295 300 Ile Asp Glu Gly Ile Ile Phe Asn Lys Val Val Glu Asp Phe Asp Phe 305 310 315 320 Arg Ala Val Val Ser Ser Leu Ser Glu Leu Lys Gly Ile Glu Tyr Glu 325 330 335 Gly Gln Tyr Ile Asp Lys Gln Tyr Glu Glu Phe Ile Pro Ser Ser Ala 340 345 350 Pro Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Ser Leu Thr Gln 355 360 365 Ser Asn Glu Thr Ile Val Ala Glu Gln Gly Thr Ser Phe Phe Gly Ala 370 375 380 Ser Thr Ile Phe Leu Lys Ser Asn Ser Arg Phe Ile Gly Gln Pro Leu 385 390 395 400 Trp Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gln Ile 405 410 415 Ala Asp Lys Glu Ser Arg His Leu Leu Phe Ile Gly Asp Gly Ser Leu 420 425 430 Gln Leu Thr Val Gln Glu Leu Gly Leu Ser Ile Arg Glu Lys Leu Asn 435 440 445 Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu 450 455 460 Ile His Gly Pro Thr Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr 465 470 475 480 Ser Lys Leu Pro Glu Thr Phe Gly Ala Thr Glu Asp Arg Val Val Ser 485 490 495 Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala 500 505 510 Gln Ala Asp Val Asn Arg Met Tyr Trp Ile Glu Leu Val Leu Glu Lys 515 520 525 Glu Asp Ala Pro Lys Leu Leu Lys Lys Met Gly Lys Leu Phe Ala Glu 530 535 540 Gln Asn Lys 545 411644DNAArtificialLactococcus lactis branched chain alpha-ketoacid decarboxylase KdcA codon optimised gene 41atgtatactg ttggtgatta tctgctggac cgtctgcatg aactgggcat tgaagaaatc 60ttcggtgtcc caggcgacta caacctgcag ttcctggacc agatcatctc ccgcgaagat 120atgaaatgga tcggtaacgc aaacgagctg aacgcgtctt atatggctga tggttatgct 180cgcaccaaaa aggctgcggc ctttctgacc acctttggtg tgggcgagct gagcgcgatc 240aacggcctgg caggttccta cgctgagaac ctgccggtag tagaaatcgt tggttccccg 300acctctaagg ttcagaacga cggcaaattc gtacatcaca ccctggcgga cggcgatttt 360aagcacttta tgaaaatgca cgaaccggtc accgccgctc gcactctgct gaccgcggaa 420aacgcaacgt acgagatcga tcgtgtactg tcccagctgc tgaaagaacg taaaccggtg 480tatatcaatc tgccggttga tgtcgctgcg gccaaagcag agaaaccggc actgtccctg 540gagaaggaga gctccactac taacaccacc gaacaggtta tcctgtccaa aattgaagaa 600tctctgaaaa acgcacagaa accggtggtt atcgcaggtc acgaggttat ctccttcggc 660ctggagaaaa ctgttactca attcgtctct gaaacgaaac tgccgatcac gaccctgaac 720tttggcaagt ccgcagttga cgaatctctg ccttctttcc tgggcattta caacggcaaa 780ctgtccgaga tctccctgaa gaacttcgta gaatccgctg actttatcct gatgctgggt 840gtgaaactga ccgactcctc taccggtgcg ttcacgcacc atctggatga aaacaaaatg 900atcagcctga acatcgacga gggtatcatc ttcaacaagg tagttgaaga tttcgacttc 960cgtgctgttg tcagcagcct gtccgagctg aaaggcattg agtacgaggg tcaatacatc 1020gataaacagt acgaagagtt

tattccgtct tctgcaccgc tgagccagga ccgcctgtgg 1080caggcagttg agtccctgac gcagtccaac gaaactatcg tagcggaaca aggtacctct 1140ttcttcggtg cttctaccat ctttctgaag tccaactctc gctttatcgg tcagccgctg 1200tggggttcta tcggttacac gttcccggct gcgctgggta gccagatcgc tgataaagag 1260tctcgtcatc tgctgttcat cggtgatggt tccctgcagc tgactgtaca ggaactgggt 1320ctgtctatcc gtgaaaaact gaacccgatt tgttttatca tcaataacga tggctacact 1380gttgagcgtg aaattcatgg tccgactcag tcttacaacg atattccgat gtggaactac 1440tctaaactgc cggaaacctt cggtgcaact gaggatcgcg tcgtgagcaa gattgtgcgt 1500actgagaacg agttcgtatc tgttatgaaa gaggcgcagg cagatgtgaa ccgcatgtac 1560tggatcgaac tggttctgga aaaagaggat gcaccgaaac tgctgaagaa aatgggtaaa 1620ctgtttgcgg agcagaacaa gtaa 1644421647DNALactococcus lactisCDS(1)..(1647) 42atg tat aca gta gga gat tac cta tta gac cga tta cac gag tta gga 48Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 1 5 10 15 att gaa gaa att ttt gga gtc cct gga gac tat aac tta caa ttt tta 96Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu 20 25 30 gat caa att att tcc cac aag gat atg aaa tgg gtc gga aat gct aat 144Asp Gln Ile Ile Ser His Lys Asp Met Lys Trp Val Gly Asn Ala Asn 35 40 45 gaa tta aat gct tca tat atg gct gat ggc tat gct cgt act aaa aaa 192Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys 50 55 60 gct gcc gca ttt ctt aca acc ttt gga gta ggt gaa ttg agt gca gtt 240Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Val 65 70 75 80 aat gga tta gca gga agt tac gcc gaa aat tta cca gta gta gaa ata 288Asn Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu Ile 85 90 95 gtg gga tca cct aca tca aaa gtt caa aat gaa gga aaa ttt gtt cat 336Val Gly Ser Pro Thr Ser Lys Val Gln Asn Glu Gly Lys Phe Val His 100 105 110 cat acg ctg gct gac ggt gat ttt aaa cac ttt atg aaa atg cac gaa 384His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu 115 120 125 cct gtt aca gca gct cga act tta ctg aca gca gaa aat gca acc gtt 432Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr Val 130 135 140 gaa att gac cga gta ctt tct gca cta tta aaa gaa aga aaa cct gtc 480Glu Ile Asp Arg Val Leu Ser Ala Leu Leu Lys Glu Arg Lys Pro Val 145 150 155 160 tat atc aac tta cca gtt gat gtt gct gct gca aaa gca gag aaa ccc 528Tyr Ile Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro 165 170 175 tca ctc cct ttg aaa aag gaa aac tca act tca aat aca agt gac caa 576Ser Leu Pro Leu Lys Lys Glu Asn Ser Thr Ser Asn Thr Ser Asp Gln 180 185 190 gaa att ttg aac aaa att caa gaa agc ttg aaa aat gcc aaa aaa cca 624Glu Ile Leu Asn Lys Ile Gln Glu Ser Leu Lys Asn Ala Lys Lys Pro 195 200 205 atc gtg att aca gga cat gaa ata att agt ttt ggc tta gaa aaa aca 672Ile Val Ile Thr Gly His Glu Ile Ile Ser Phe Gly Leu Glu Lys Thr 210 215 220 gtc act caa ttt att tca aag aca aaa cta cct att acg aca tta aac 720Val Thr Gln Phe Ile Ser Lys Thr Lys Leu Pro Ile Thr Thr Leu Asn 225 230 235 240 ttt ggt aaa agt tca gtt gat gaa gcc ctc cct tca ttt tta gga atc 768Phe Gly Lys Ser Ser Val Asp Glu Ala Leu Pro Ser Phe Leu Gly Ile 245 250 255 tat aat ggt aca ctc tca gag cct aat ctt aaa gaa ttc gtg gaa tca 816Tyr Asn Gly Thr Leu Ser Glu Pro Asn Leu Lys Glu Phe Val Glu Ser 260 265 270 gcc gac ttc atc ttg atg ctt gga gtt aaa ctc aca gac tct tca aca 864Ala Asp Phe Ile Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr 275 280 285 gga gcc ttc act cat cat tta aat gaa aat aaa atg att tca ctg aat 912Gly Ala Phe Thr His His Leu Asn Glu Asn Lys Met Ile Ser Leu Asn 290 295 300 ata gat gaa gga aaa ata ttt aac gaa aga atc caa aat ttt gat ttt 960Ile Asp Glu Gly Lys Ile Phe Asn Glu Arg Ile Gln Asn Phe Asp Phe 305 310 315 320 gaa tcc ctc atc tcc tct ctc tta gac cta agc gaa ata gaa tac aaa 1008Glu Ser Leu Ile Ser Ser Leu Leu Asp Leu Ser Glu Ile Glu Tyr Lys 325 330 335 gga aaa tat atc gat aaa aag caa gaa gac ttt gtt cca tca aat gcg 1056Gly Lys Tyr Ile Asp Lys Lys Gln Glu Asp Phe Val Pro Ser Asn Ala 340 345 350 ctt tta tca caa gac cgc cta tgg caa gca gtt gaa aac cta act caa 1104Leu Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Asn Leu Thr Gln 355 360 365 agc aat gaa aca atc gtt gct gaa caa ggg aca tca ttc ttt ggc gct 1152Ser Asn Glu Thr Ile Val Ala Glu Gln Gly Thr Ser Phe Phe Gly Ala 370 375 380 tca tca att ttc tta aaa tca aag agt cat ttt att ggt caa ccc tta 1200Ser Ser Ile Phe Leu Lys Ser Lys Ser His Phe Ile Gly Gln Pro Leu 385 390 395 400 tgg gga tca att gga tat aca ttc cca gca gca tta gga agc caa att 1248Trp Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gln Ile 405 410 415 gca gat aaa gaa agc aga cac ctt tta ttt att ggt gat ggt tca ctt 1296Ala Asp Lys Glu Ser Arg His Leu Leu Phe Ile Gly Asp Gly Ser Leu 420 425 430 caa ctt aca gtg caa gaa tta gga tta gca atc aga gaa aaa att aat 1344Gln Leu Thr Val Gln Glu Leu Gly Leu Ala Ile Arg Glu Lys Ile Asn 435 440 445 cca att tgc ttt att atc aat aat gat ggt tat aca gtc gaa aga gaa 1392Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu 450 455 460 att cat gga cca aat caa agc tac aat gat att cca atg tgg aat tac 1440Ile His Gly Pro Asn Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr 465 470 475 480 tca aaa tta cca gaa tcg ttt gga gca aca gaa gat cga gta gtc tca 1488Ser Lys Leu Pro Glu Ser Phe Gly Ala Thr Glu Asp Arg Val Val Ser 485 490 495 aaa atc gtt aga act gaa aat gaa ttt gtg tct gtc atg aaa gaa gct 1536Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala 500 505 510 caa gca gat cca aat aga atg tac tgg att gag tta att ttg gca aaa 1584Gln Ala Asp Pro Asn Arg Met Tyr Trp Ile Glu Leu Ile Leu Ala Lys 515 520 525 gaa ggt gca cca aaa gta ctg aaa aaa atg ggc aaa cta ttt gct gaa 1632Glu Gly Ala Pro Lys Val Leu Lys Lys Met Gly Lys Leu Phe Ala Glu 530 535 540 caa aat aaa tca taa 1647Gln Asn Lys Ser 545 43548PRTLactococcus lactis 43Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 1 5 10 15 Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu 20 25 30 Asp Gln Ile Ile Ser His Lys Asp Met Lys Trp Val Gly Asn Ala Asn 35 40 45 Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys 50 55 60 Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Val 65 70 75 80 Asn Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu Ile 85 90 95 Val Gly Ser Pro Thr Ser Lys Val Gln Asn Glu Gly Lys Phe Val His 100 105 110 His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu 115 120 125 Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr Val 130 135 140 Glu Ile Asp Arg Val Leu Ser Ala Leu Leu Lys Glu Arg Lys Pro Val 145 150 155 160 Tyr Ile Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro 165 170 175 Ser Leu Pro Leu Lys Lys Glu Asn Ser Thr Ser Asn Thr Ser Asp Gln 180 185 190 Glu Ile Leu Asn Lys Ile Gln Glu Ser Leu Lys Asn Ala Lys Lys Pro 195 200 205 Ile Val Ile Thr Gly His Glu Ile Ile Ser Phe Gly Leu Glu Lys Thr 210 215 220 Val Thr Gln Phe Ile Ser Lys Thr Lys Leu Pro Ile Thr Thr Leu Asn 225 230 235 240 Phe Gly Lys Ser Ser Val Asp Glu Ala Leu Pro Ser Phe Leu Gly Ile 245 250 255 Tyr Asn Gly Thr Leu Ser Glu Pro Asn Leu Lys Glu Phe Val Glu Ser 260 265 270 Ala Asp Phe Ile Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr 275 280 285 Gly Ala Phe Thr His His Leu Asn Glu Asn Lys Met Ile Ser Leu Asn 290 295 300 Ile Asp Glu Gly Lys Ile Phe Asn Glu Arg Ile Gln Asn Phe Asp Phe 305 310 315 320 Glu Ser Leu Ile Ser Ser Leu Leu Asp Leu Ser Glu Ile Glu Tyr Lys 325 330 335 Gly Lys Tyr Ile Asp Lys Lys Gln Glu Asp Phe Val Pro Ser Asn Ala 340 345 350 Leu Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Asn Leu Thr Gln 355 360 365 Ser Asn Glu Thr Ile Val Ala Glu Gln Gly Thr Ser Phe Phe Gly Ala 370 375 380 Ser Ser Ile Phe Leu Lys Ser Lys Ser His Phe Ile Gly Gln Pro Leu 385 390 395 400 Trp Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gln Ile 405 410 415 Ala Asp Lys Glu Ser Arg His Leu Leu Phe Ile Gly Asp Gly Ser Leu 420 425 430 Gln Leu Thr Val Gln Glu Leu Gly Leu Ala Ile Arg Glu Lys Ile Asn 435 440 445 Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu 450 455 460 Ile His Gly Pro Asn Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr 465 470 475 480 Ser Lys Leu Pro Glu Ser Phe Gly Ala Thr Glu Asp Arg Val Val Ser 485 490 495 Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala 500 505 510 Gln Ala Asp Pro Asn Arg Met Tyr Trp Ile Glu Leu Ile Leu Ala Lys 515 520 525 Glu Gly Ala Pro Lys Val Leu Lys Lys Met Gly Lys Leu Phe Ala Glu 530 535 540 Gln Asn Lys Ser 545 441647DNAArtificialLactococcus lactis -ketoisovalerate decarboxylase KivD codon optimised gene 44atgtatactg ttggtgatta cctgctggat cgtctgcatg aactgggcat cgaggaaatt 60ttcggcgtac ctggtgacta taacctgcag ttcctggatc agatcatttc ccacaaagat 120atgaaatggg ttggtaacgc gaacgagctg aatgcaagct acatggctga cggttatgca 180cgcaccaaga aagctgcggc gttcctgact acttttggcg tcggcgagct gtctgcggta 240aacggtctgg ccggctccta cgcggaaaac ctgccggtag tagaaatcgt cggttccccg 300acctctaaag ttcagaacga gggtaaattc gtgcaccata ctctggccga tggtgacttc 360aaacacttca tgaagatgca cgaaccggtc actgctgctc gtacgctgct gaccgcggaa 420aatgcgactg tcgagattga tcgtgtactg agcgcactgc tgaaagaacg caagcctgta 480tacatcaacc tgccggttga tgtcgcggcc gccaaagcgg aaaaaccatc tctgccgctg 540aaaaaggaga acagcacctc taacaccagc gaccaggaaa tcctgaacaa gatccaggag 600tctctgaaga acgctaaaaa gccgatcgta atcaccggcc atgagattat ctctttcggt 660ctggagaaaa ctgtcaccca gttcatcagc aaaaccaaac tgccgatcac caccctgaac 720ttcggtaaat cctccgttga cgaagcgctg ccgtcctttc tgggtattta caacggcact 780ctgtctgagc cgaacctgaa agagttcgtg gagtctgcgg attttatcct gatgctgggc 840gtgaaactga cggattcctc caccggtgca ttcacccacc acctgaatga gaataaaatg 900atctctctga acattgatga gggcaaaatc ttcaacgagc gtattcagaa cttcgatttc 960gaatccctga tctcctccct gctggatctg tccgagattg aatataaagg caaatacatt 1020gataagaagc aagaggactt cgtaccgtct aacgcgctgc tgagccagga ccgtctgtgg 1080caagctgtgg aaaacctgac ccagtccaac gaaaccatcg tggcggaaca gggtacctcc 1140ttcttcggtg ctagctctat cttcctgaaa tctaaaagcc acttcatcgg tcagccactg 1200tggggctcta ttggctacac cttcccggca gcgctgggtt cccaaatcgc agacaaagaa 1260tcccgccacc tgctgttcat tggtgacggc tctctgcaac tgaccgtaca ggagctgggt 1320ctggcgattc gtgagaaaat caacccgatt tgtttcatca tcaacaacga tggctacact 1380gttgagcgtg agatccacgg cccgaaccag tcctacaacg acattccgat gtggaactac 1440tctaaactgc cggaatcctt cggtgcgact gaagaccgtg tcgtaagcaa gatcgtccgt 1500accgaaaacg aattcgtgtc tgtcatgaaa gaagcacagg cggacccgaa ccgcatgtac 1560tggatcgagc tgattctggc taaagagggc gcgccaaaag tactgaaaaa gatgggtaaa 1620ctgttcgcag aacagaacaa atcctaa 1647453696DNAMycobacterium tuberculosisCDS(1)..(3696) 45gtg gcc aac ata agt tca cca ttc ggg caa aac gaa tgg ctg gtc gaa 48Val Ala Asn Ile Ser Ser Pro Phe Gly Gln Asn Glu Trp Leu Val Glu 1 5 10 15 gag atg tac cgc aag ttc cgc gac gac ccc tcc tcg gtc gat ccc agc 96Glu Met Tyr Arg Lys Phe Arg Asp Asp Pro Ser Ser Val Asp Pro Ser 20 25 30 tgg cac gag ttc ctg gtt gac tac agc ccc gaa ccc acc tcc caa cca 144Trp His Glu Phe Leu Val Asp Tyr Ser Pro Glu Pro Thr Ser Gln Pro 35 40 45 gct gcc gaa cca acc cgg gtt acc tcg cca ctc gtt gcc gag cgg gcc 192Ala Ala Glu Pro Thr Arg Val Thr Ser Pro Leu Val Ala Glu Arg Ala 50 55 60 gct gcg gcc gcc ccg cag gca ccc ccc aag ccg gcc gac acc gcg gcc 240Ala Ala Ala Ala Pro Gln Ala Pro Pro Lys Pro Ala Asp Thr Ala Ala 65 70 75 80 gcg ggc aac ggc gtg gtc gcc gca ctg gcc gcc aaa act gcc gtt ccc 288Ala Gly Asn Gly Val Val Ala Ala Leu Ala Ala Lys Thr Ala Val Pro 85 90 95 ccg cca gcc gaa ggt gac gag gta gcg gtg ctg cgc ggc gcc gcc gcg 336Pro Pro Ala Glu Gly Asp Glu Val Ala Val Leu Arg Gly Ala Ala Ala 100 105 110 gcc gtc gtc aag aac atg tcc gcg tcg ttg gag gtg ccg acg gcg acc 384Ala Val Val Lys Asn Met Ser Ala Ser Leu Glu Val Pro Thr Ala Thr 115 120 125 agc gtc cgg gcg gtc ccg gcc aag cta ctg atc gac aac cgg atc gtc 432Ser Val Arg Ala Val Pro Ala Lys Leu Leu Ile Asp Asn Arg Ile Val 130 135 140 atc aac aac cag ttg aag cgg acc cgc ggc ggc aag atc tcg ttc acg 480Ile Asn Asn Gln Leu Lys Arg Thr Arg Gly Gly Lys Ile Ser Phe Thr 145 150 155 160 cat ttg ctg ggc tac gcc ctg gtg cag gcg gtg aag aaa ttc ccg aac 528His Leu Leu Gly Tyr Ala Leu Val Gln Ala Val Lys Lys Phe Pro Asn 165 170 175 atg aac cgg cac tac acc gaa gtc gac ggc aag ccc acc gcg gtc acg 576Met Asn Arg His Tyr Thr Glu Val Asp Gly Lys Pro Thr Ala Val Thr 180 185 190 ccg gcg cac acc aat ctc ggc ctg gcg atc gac ctg caa ggc aag gac 624Pro Ala His Thr Asn Leu Gly Leu Ala Ile Asp Leu Gln Gly Lys Asp 195 200 205 ggg aag cgt tcc ctg gtg gtg gcc ggc atc aag cgg tgc gag acc atg 672Gly Lys Arg Ser Leu Val Val Ala Gly Ile Lys Arg Cys Glu Thr Met 210 215 220 cga ttc gcg cag ttc gtc acg gcc tac gaa gac atc gta cgc cgg gcc 720Arg Phe Ala Gln Phe Val Thr Ala Tyr Glu Asp Ile Val Arg Arg Ala 225 230 235 240

cgc gac ggc aag ctg acc act gaa gac ttt gcc ggc gtg acg att tcg 768Arg Asp Gly Lys Leu Thr Thr Glu Asp Phe Ala Gly Val Thr Ile Ser 245 250 255 ctg acc aat ccc gga acc atc ggc acc gtg cat tcg gtg ccg cgg ctg 816Leu Thr Asn Pro Gly Thr Ile Gly Thr Val His Ser Val Pro Arg Leu 260 265 270 atg ccc ggc cag ggc gcc atc atc ggc gtg ggc gcc atg gaa tac ccc 864Met Pro Gly Gln Gly Ala Ile Ile Gly Val Gly Ala Met Glu Tyr Pro 275 280 285 gcc gag ttt caa ggc gcc agc gag gaa cgc atc gcc gag ctg ggc atc 912Ala Glu Phe Gln Gly Ala Ser Glu Glu Arg Ile Ala Glu Leu Gly Ile 290 295 300 ggc aaa ttg atc act ttg acc tcc acc tac gac cac cgc atc atc cag 960Gly Lys Leu Ile Thr Leu Thr Ser Thr Tyr Asp His Arg Ile Ile Gln 305 310 315 320 ggc gcg gaa tcg ggc gac ttc ctg cgc acc atc cac gag ttg ctg ctc 1008Gly Ala Glu Ser Gly Asp Phe Leu Arg Thr Ile His Glu Leu Leu Leu 325 330 335 tcg gat ggc ttc tgg gac gag gtc ttc cgc gaa ctg agc atc cca tat 1056Ser Asp Gly Phe Trp Asp Glu Val Phe Arg Glu Leu Ser Ile Pro Tyr 340 345 350 ctg ccg gtg cgc tgg agc acc gac aac ccc gac tcg atc gtc gac aag 1104Leu Pro Val Arg Trp Ser Thr Asp Asn Pro Asp Ser Ile Val Asp Lys 355 360 365 aac gct cgc gtc atg aac ttg atc gcg gcc tac cgc aac cgc ggc cat 1152Asn Ala Arg Val Met Asn Leu Ile Ala Ala Tyr Arg Asn Arg Gly His 370 375 380 ctg atg gcc gat acc gac ccg ctg cgg ttg gac aaa gct cgg ttc cgc 1200Leu Met Ala Asp Thr Asp Pro Leu Arg Leu Asp Lys Ala Arg Phe Arg 385 390 395 400 agt cac ccc gac ctc gaa gtg ctg acc cac ggc ctg acg ctg tgg gat 1248Ser His Pro Asp Leu Glu Val Leu Thr His Gly Leu Thr Leu Trp Asp 405 410 415 ctc gat cgg gtg ttc aag gtc gac ggc ttt gcc ggt gcg cag tac aag 1296Leu Asp Arg Val Phe Lys Val Asp Gly Phe Ala Gly Ala Gln Tyr Lys 420 425 430 aaa ctg cgc gac gtg ctg ggc ttg ctg cgc gat gcc tac tgc cgc cac 1344Lys Leu Arg Asp Val Leu Gly Leu Leu Arg Asp Ala Tyr Cys Arg His 435 440 445 atc ggc gtg gag tac gcc cat atc ctc gac ccc gaa caa aag gag tgg 1392Ile Gly Val Glu Tyr Ala His Ile Leu Asp Pro Glu Gln Lys Glu Trp 450 455 460 ctc gaa caa cgg gtc gag acc aag cac gtc aaa ccc act gtg gcc caa 1440Leu Glu Gln Arg Val Glu Thr Lys His Val Lys Pro Thr Val Ala Gln 465 470 475 480 cag aaa tac atc ctc agc aag ctc aac gcc gcc gag gcc ttt gaa acg 1488Gln Lys Tyr Ile Leu Ser Lys Leu Asn Ala Ala Glu Ala Phe Glu Thr 485 490 495 ttc cta cag acc aag tac gtc ggc cag aag cgg ttc tcg ctg gaa ggc 1536Phe Leu Gln Thr Lys Tyr Val Gly Gln Lys Arg Phe Ser Leu Glu Gly 500 505 510 gcc gaa agc gtg atc ccg atg atg gac gcg gcg atc gac cag tgc gct 1584Ala Glu Ser Val Ile Pro Met Met Asp Ala Ala Ile Asp Gln Cys Ala 515 520 525 gag cac ggc ctc gac gag gtg gtc atc ggg atg ccg cac cgg ggc cgg 1632Glu His Gly Leu Asp Glu Val Val Ile Gly Met Pro His Arg Gly Arg 530 535 540 ctc aac gtg ctg gcc aac atc gtc ggc aag ccg tac tcg cag atc ttc 1680Leu Asn Val Leu Ala Asn Ile Val Gly Lys Pro Tyr Ser Gln Ile Phe 545 550 555 560 acc gag ttc gag ggc aac ctg aat ccg tcg cag gcg cac ggc tcc ggt 1728Thr Glu Phe Glu Gly Asn Leu Asn Pro Ser Gln Ala His Gly Ser Gly 565 570 575 gac gtc aag tac cac ctg ggc gcc acc ggg ctg tac ctg cag atg ttc 1776Asp Val Lys Tyr His Leu Gly Ala Thr Gly Leu Tyr Leu Gln Met Phe 580 585 590 ggc gac aac gac att cag gtg tcg ctg acc gcc aac ccg tcg cat ctg 1824Gly Asp Asn Asp Ile Gln Val Ser Leu Thr Ala Asn Pro Ser His Leu 595 600 605 gag gcc gtc gac ccg gtg ctg gag gga ttg gtg cgg gcc aag cag gat 1872Glu Ala Val Asp Pro Val Leu Glu Gly Leu Val Arg Ala Lys Gln Asp 610 615 620 ctg ctc gac cac gga agc atc gac agc gac ggc caa cgg gcg ttc tcg 1920Leu Leu Asp His Gly Ser Ile Asp Ser Asp Gly Gln Arg Ala Phe Ser 625 630 635 640 gtg gtg ccg ctg atg ttg cat ggc gat gcc gcg ttc gcc ggt cag ggt 1968Val Val Pro Leu Met Leu His Gly Asp Ala Ala Phe Ala Gly Gln Gly 645 650 655 gtg gtc gcc gag acg ctg aac ctg gcg aat ctg ccg ggc tac cgc gtc 2016Val Val Ala Glu Thr Leu Asn Leu Ala Asn Leu Pro Gly Tyr Arg Val 660 665 670 ggc ggc acc atc cac atc atc gtc aac aac cag atc ggc ttc acc acc 2064Gly Gly Thr Ile His Ile Ile Val Asn Asn Gln Ile Gly Phe Thr Thr 675 680 685 gcg ccc gag tat tcc agg tcc agc gag tac tgc acc gac gtc gca aag 2112Ala Pro Glu Tyr Ser Arg Ser Ser Glu Tyr Cys Thr Asp Val Ala Lys 690 695 700 atg atc ggg gca ccg atc ttt cac gtc aac ggc gac gac ccg gag gcg 2160Met Ile Gly Ala Pro Ile Phe His Val Asn Gly Asp Asp Pro Glu Ala 705 710 715 720 tgt gtc tgg gtg gcg cgg ttg gcg gtg gac ttc cga caa cgg ttc aag 2208Cys Val Trp Val Ala Arg Leu Ala Val Asp Phe Arg Gln Arg Phe Lys 725 730 735 aag gac gtc gtc atc gac atg ctg tgc tac cgc cgc cgc ggg cac aac 2256Lys Asp Val Val Ile Asp Met Leu Cys Tyr Arg Arg Arg Gly His Asn 740 745 750 gag ggt gac gac ccg tcg atg acc aac ccc tac gtg tac gac gtc gtc 2304Glu Gly Asp Asp Pro Ser Met Thr Asn Pro Tyr Val Tyr Asp Val Val 755 760 765 gac acc aag cgc ggg gcc cgc aaa agc tac acc gaa gcc ctg atc gga 2352Asp Thr Lys Arg Gly Ala Arg Lys Ser Tyr Thr Glu Ala Leu Ile Gly 770 775 780 cgt ggc gac atc tcg atg aag gag gcc gag gac gcg ctg cgc gac tac 2400Arg Gly Asp Ile Ser Met Lys Glu Ala Glu Asp Ala Leu Arg Asp Tyr 785 790 795 800 cag ggc cag ctg gaa cgg gtg ttc aac gaa gtg cgc gag ctg gag aag 2448Gln Gly Gln Leu Glu Arg Val Phe Asn Glu Val Arg Glu Leu Glu Lys 805 810 815 cac ggt gtg cag ccg agc gag tcg gtc gag tcc gac cag atg att ccc 2496His Gly Val Gln Pro Ser Glu Ser Val Glu Ser Asp Gln Met Ile Pro 820 825 830 gcg ggg ctg gcc act gcg gtg gac aag tcg ctg ctg gcc cgg atc ggc 2544Ala Gly Leu Ala Thr Ala Val Asp Lys Ser Leu Leu Ala Arg Ile Gly 835 840 845 gat gcg ttc ctc gcc ttg ccg aac ggc ttc acc gcg cac ccg cga gtc 2592Asp Ala Phe Leu Ala Leu Pro Asn Gly Phe Thr Ala His Pro Arg Val 850 855 860 caa ccg gtg ctg gag aag cgc cgg gag atg gcc tat gaa ggc aag atc 2640Gln Pro Val Leu Glu Lys Arg Arg Glu Met Ala Tyr Glu Gly Lys Ile 865 870 875 880 gac tgg gcc ttt ggc gag ctg ctg gcg ctg ggc tcg ctg gtg gcc gaa 2688Asp Trp Ala Phe Gly Glu Leu Leu Ala Leu Gly Ser Leu Val Ala Glu 885 890 895 ggc aag ctg gtg cgc ttg tcg ggg cag gac agc cgc cgc ggc acc ttc 2736Gly Lys Leu Val Arg Leu Ser Gly Gln Asp Ser Arg Arg Gly Thr Phe 900 905 910 tcc cag cgg cat tcg gtt ctc atc gac cgc cac act ggc gag gag ttc 2784Ser Gln Arg His Ser Val Leu Ile Asp Arg His Thr Gly Glu Glu Phe 915 920 925 aca cca ctg cag ctg ctg gcg acc aac tcc gac ggc agc ccg acc ggc 2832Thr Pro Leu Gln Leu Leu Ala Thr Asn Ser Asp Gly Ser Pro Thr Gly 930 935 940 gga aag ttc ctg gtc tac gac tcg cca ctg tcg gag tac gcc gcc gtc 2880Gly Lys Phe Leu Val Tyr Asp Ser Pro Leu Ser Glu Tyr Ala Ala Val 945 950 955 960 ggc ttc gag tac ggc tac act gtg ggc aat ccg gac gcc gtg gtg ctc 2928Gly Phe Glu Tyr Gly Tyr Thr Val Gly Asn Pro Asp Ala Val Val Leu 965 970 975 tgg gag gcg cag ttc ggc gac ttc gtc aac ggc gcg cag tcg atc atc 2976Trp Glu Ala Gln Phe Gly Asp Phe Val Asn Gly Ala Gln Ser Ile Ile 980 985 990 gac gag ttc atc agc tcc ggt gag gcc aag tgg ggc caa ttg tcc aac 3024Asp Glu Phe Ile Ser Ser Gly Glu Ala Lys Trp Gly Gln Leu Ser Asn 995 1000 1005 gtc gtg ctg ctg tta ccg cac ggg cac gag ggg cag gga ccc gac 3069Val Val Leu Leu Leu Pro His Gly His Glu Gly Gln Gly Pro Asp 1010 1015 1020 cac act tct gcc cgg atc gaa cgc ttc ttg cag ttg tgg gcg gaa 3114His Thr Ser Ala Arg Ile Glu Arg Phe Leu Gln Leu Trp Ala Glu 1025 1030 1035 ggt tcg atg acc atc gcg atg ccg tcg act ccg tcg aac tac ttc 3159Gly Ser Met Thr Ile Ala Met Pro Ser Thr Pro Ser Asn Tyr Phe 1040 1045 1050 cac ctg cta cgc cgg cat gcc ctg gac ggc atc caa cgc ccg ctg 3204His Leu Leu Arg Arg His Ala Leu Asp Gly Ile Gln Arg Pro Leu 1055 1060 1065 atc gtg ttc acg ccc aag tcg atg ttg cgt cac aag gcc gcc gtc 3249Ile Val Phe Thr Pro Lys Ser Met Leu Arg His Lys Ala Ala Val 1070 1075 1080 agc gaa atc aag gac ttc acc gag atc aag ttc cgc tca gtg ctg 3294Ser Glu Ile Lys Asp Phe Thr Glu Ile Lys Phe Arg Ser Val Leu 1085 1090 1095 gag gaa ccc acc tat gag gac ggc atc gga gac cgc aac aag gtc 3339Glu Glu Pro Thr Tyr Glu Asp Gly Ile Gly Asp Arg Asn Lys Val 1100 1105 1110 agc cgg atc ctg ctg acc agt ggc aag ctg tat tac gag ctg gcc 3384Ser Arg Ile Leu Leu Thr Ser Gly Lys Leu Tyr Tyr Glu Leu Ala 1115 1120 1125 gcc cgc aag gcc aag gac aac cgc aat gac ctc gcg atc gtg cgg 3429Ala Arg Lys Ala Lys Asp Asn Arg Asn Asp Leu Ala Ile Val Arg 1130 1135 1140 ctt gaa cag ctc gcc ccg ctg ccc agg cgt cga ctg cgt gaa acg 3474Leu Glu Gln Leu Ala Pro Leu Pro Arg Arg Arg Leu Arg Glu Thr 1145 1150 1155 ctg gac cgc tac gag aac gtc aag gag ttc ttc tgg gtc caa gag 3519Leu Asp Arg Tyr Glu Asn Val Lys Glu Phe Phe Trp Val Gln Glu 1160 1165 1170 gaa ccg gcc aac cag ggt gcg tgg ccg cga ttc ggg ctc gaa cta 3564Glu Pro Ala Asn Gln Gly Ala Trp Pro Arg Phe Gly Leu Glu Leu 1175 1180 1185 ccc gag ctg ctg cct gac aag ttg gcc ggg atc aag cga atc tcg 3609Pro Glu Leu Leu Pro Asp Lys Leu Ala Gly Ile Lys Arg Ile Ser 1190 1195 1200 cgc cgg gcg atg tca gcc ccg tcg tca ggc tcg tcg aag gtg cac 3654Arg Arg Ala Met Ser Ala Pro Ser Ser Gly Ser Ser Lys Val His 1205 1210 1215 gcc gtc gaa cag cag gag atc ctc gac gag gcg ttc ggc tga 3696Ala Val Glu Gln Gln Glu Ile Leu Asp Glu Ala Phe Gly 1220 1225 1230 461231PRTMycobacterium tuberculosis 46Val Ala Asn Ile Ser Ser Pro Phe Gly Gln Asn Glu Trp Leu Val Glu 1 5 10 15 Glu Met Tyr Arg Lys Phe Arg Asp Asp Pro Ser Ser Val Asp Pro Ser 20 25 30 Trp His Glu Phe Leu Val Asp Tyr Ser Pro Glu Pro Thr Ser Gln Pro 35 40 45 Ala Ala Glu Pro Thr Arg Val Thr Ser Pro Leu Val Ala Glu Arg Ala 50 55 60 Ala Ala Ala Ala Pro Gln Ala Pro Pro Lys Pro Ala Asp Thr Ala Ala 65 70 75 80 Ala Gly Asn Gly Val Val Ala Ala Leu Ala Ala Lys Thr Ala Val Pro 85 90 95 Pro Pro Ala Glu Gly Asp Glu Val Ala Val Leu Arg Gly Ala Ala Ala 100 105 110 Ala Val Val Lys Asn Met Ser Ala Ser Leu Glu Val Pro Thr Ala Thr 115 120 125 Ser Val Arg Ala Val Pro Ala Lys Leu Leu Ile Asp Asn Arg Ile Val 130 135 140 Ile Asn Asn Gln Leu Lys Arg Thr Arg Gly Gly Lys Ile Ser Phe Thr 145 150 155 160 His Leu Leu Gly Tyr Ala Leu Val Gln Ala Val Lys Lys Phe Pro Asn 165 170 175 Met Asn Arg His Tyr Thr Glu Val Asp Gly Lys Pro Thr Ala Val Thr 180 185 190 Pro Ala His Thr Asn Leu Gly Leu Ala Ile Asp Leu Gln Gly Lys Asp 195 200 205 Gly Lys Arg Ser Leu Val Val Ala Gly Ile Lys Arg Cys Glu Thr Met 210 215 220 Arg Phe Ala Gln Phe Val Thr Ala Tyr Glu Asp Ile Val Arg Arg Ala 225 230 235 240 Arg Asp Gly Lys Leu Thr Thr Glu Asp Phe Ala Gly Val Thr Ile Ser 245 250 255 Leu Thr Asn Pro Gly Thr Ile Gly Thr Val His Ser Val Pro Arg Leu 260 265 270 Met Pro Gly Gln Gly Ala Ile Ile Gly Val Gly Ala Met Glu Tyr Pro 275 280 285 Ala Glu Phe Gln Gly Ala Ser Glu Glu Arg Ile Ala Glu Leu Gly Ile 290 295 300 Gly Lys Leu Ile Thr Leu Thr Ser Thr Tyr Asp His Arg Ile Ile Gln 305 310 315 320 Gly Ala Glu Ser Gly Asp Phe Leu Arg Thr Ile His Glu Leu Leu Leu 325 330 335 Ser Asp Gly Phe Trp Asp Glu Val Phe Arg Glu Leu Ser Ile Pro Tyr 340 345 350 Leu Pro Val Arg Trp Ser Thr Asp Asn Pro Asp Ser Ile Val Asp Lys 355 360 365 Asn Ala Arg Val Met Asn Leu Ile Ala Ala Tyr Arg Asn Arg Gly His 370 375 380 Leu Met Ala Asp Thr Asp Pro Leu Arg Leu Asp Lys Ala Arg Phe Arg 385 390 395 400 Ser His Pro Asp Leu Glu Val Leu Thr His Gly Leu Thr Leu Trp Asp 405 410 415 Leu Asp Arg Val Phe Lys Val Asp Gly Phe Ala Gly Ala Gln Tyr Lys 420 425 430 Lys Leu Arg Asp Val Leu Gly Leu Leu Arg Asp Ala Tyr Cys Arg His 435 440 445 Ile Gly Val Glu Tyr Ala His Ile Leu Asp Pro Glu Gln Lys Glu Trp 450 455 460 Leu Glu Gln Arg Val Glu Thr Lys His Val Lys Pro Thr Val Ala Gln 465 470 475 480 Gln Lys Tyr Ile Leu Ser Lys Leu Asn Ala Ala Glu Ala Phe Glu Thr 485 490 495 Phe Leu Gln Thr Lys Tyr Val Gly Gln Lys Arg Phe Ser Leu Glu Gly 500 505 510 Ala Glu Ser Val Ile Pro Met Met Asp Ala Ala Ile Asp Gln Cys Ala 515 520 525 Glu His Gly Leu Asp Glu Val Val Ile Gly Met Pro His Arg Gly Arg 530 535 540 Leu Asn Val Leu Ala Asn Ile Val Gly Lys Pro Tyr Ser Gln Ile Phe 545 550 555 560 Thr Glu Phe Glu Gly Asn Leu Asn Pro Ser

Gln Ala His Gly Ser Gly 565 570 575 Asp Val Lys Tyr His Leu Gly Ala Thr Gly Leu Tyr Leu Gln Met Phe 580 585 590 Gly Asp Asn Asp Ile Gln Val Ser Leu Thr Ala Asn Pro Ser His Leu 595 600 605 Glu Ala Val Asp Pro Val Leu Glu Gly Leu Val Arg Ala Lys Gln Asp 610 615 620 Leu Leu Asp His Gly Ser Ile Asp Ser Asp Gly Gln Arg Ala Phe Ser 625 630 635 640 Val Val Pro Leu Met Leu His Gly Asp Ala Ala Phe Ala Gly Gln Gly 645 650 655 Val Val Ala Glu Thr Leu Asn Leu Ala Asn Leu Pro Gly Tyr Arg Val 660 665 670 Gly Gly Thr Ile His Ile Ile Val Asn Asn Gln Ile Gly Phe Thr Thr 675 680 685 Ala Pro Glu Tyr Ser Arg Ser Ser Glu Tyr Cys Thr Asp Val Ala Lys 690 695 700 Met Ile Gly Ala Pro Ile Phe His Val Asn Gly Asp Asp Pro Glu Ala 705 710 715 720 Cys Val Trp Val Ala Arg Leu Ala Val Asp Phe Arg Gln Arg Phe Lys 725 730 735 Lys Asp Val Val Ile Asp Met Leu Cys Tyr Arg Arg Arg Gly His Asn 740 745 750 Glu Gly Asp Asp Pro Ser Met Thr Asn Pro Tyr Val Tyr Asp Val Val 755 760 765 Asp Thr Lys Arg Gly Ala Arg Lys Ser Tyr Thr Glu Ala Leu Ile Gly 770 775 780 Arg Gly Asp Ile Ser Met Lys Glu Ala Glu Asp Ala Leu Arg Asp Tyr 785 790 795 800 Gln Gly Gln Leu Glu Arg Val Phe Asn Glu Val Arg Glu Leu Glu Lys 805 810 815 His Gly Val Gln Pro Ser Glu Ser Val Glu Ser Asp Gln Met Ile Pro 820 825 830 Ala Gly Leu Ala Thr Ala Val Asp Lys Ser Leu Leu Ala Arg Ile Gly 835 840 845 Asp Ala Phe Leu Ala Leu Pro Asn Gly Phe Thr Ala His Pro Arg Val 850 855 860 Gln Pro Val Leu Glu Lys Arg Arg Glu Met Ala Tyr Glu Gly Lys Ile 865 870 875 880 Asp Trp Ala Phe Gly Glu Leu Leu Ala Leu Gly Ser Leu Val Ala Glu 885 890 895 Gly Lys Leu Val Arg Leu Ser Gly Gln Asp Ser Arg Arg Gly Thr Phe 900 905 910 Ser Gln Arg His Ser Val Leu Ile Asp Arg His Thr Gly Glu Glu Phe 915 920 925 Thr Pro Leu Gln Leu Leu Ala Thr Asn Ser Asp Gly Ser Pro Thr Gly 930 935 940 Gly Lys Phe Leu Val Tyr Asp Ser Pro Leu Ser Glu Tyr Ala Ala Val 945 950 955 960 Gly Phe Glu Tyr Gly Tyr Thr Val Gly Asn Pro Asp Ala Val Val Leu 965 970 975 Trp Glu Ala Gln Phe Gly Asp Phe Val Asn Gly Ala Gln Ser Ile Ile 980 985 990 Asp Glu Phe Ile Ser Ser Gly Glu Ala Lys Trp Gly Gln Leu Ser Asn 995 1000 1005 Val Val Leu Leu Leu Pro His Gly His Glu Gly Gln Gly Pro Asp 1010 1015 1020 His Thr Ser Ala Arg Ile Glu Arg Phe Leu Gln Leu Trp Ala Glu 1025 1030 1035 Gly Ser Met Thr Ile Ala Met Pro Ser Thr Pro Ser Asn Tyr Phe 1040 1045 1050 His Leu Leu Arg Arg His Ala Leu Asp Gly Ile Gln Arg Pro Leu 1055 1060 1065 Ile Val Phe Thr Pro Lys Ser Met Leu Arg His Lys Ala Ala Val 1070 1075 1080 Ser Glu Ile Lys Asp Phe Thr Glu Ile Lys Phe Arg Ser Val Leu 1085 1090 1095 Glu Glu Pro Thr Tyr Glu Asp Gly Ile Gly Asp Arg Asn Lys Val 1100 1105 1110 Ser Arg Ile Leu Leu Thr Ser Gly Lys Leu Tyr Tyr Glu Leu Ala 1115 1120 1125 Ala Arg Lys Ala Lys Asp Asn Arg Asn Asp Leu Ala Ile Val Arg 1130 1135 1140 Leu Glu Gln Leu Ala Pro Leu Pro Arg Arg Arg Leu Arg Glu Thr 1145 1150 1155 Leu Asp Arg Tyr Glu Asn Val Lys Glu Phe Phe Trp Val Gln Glu 1160 1165 1170 Glu Pro Ala Asn Gln Gly Ala Trp Pro Arg Phe Gly Leu Glu Leu 1175 1180 1185 Pro Glu Leu Leu Pro Asp Lys Leu Ala Gly Ile Lys Arg Ile Ser 1190 1195 1200 Arg Arg Ala Met Ser Ala Pro Ser Ser Gly Ser Ser Lys Val His 1205 1210 1215 Ala Val Glu Gln Gln Glu Ile Leu Asp Glu Ala Phe Gly 1220 1225 1230 473696DNAArtificialMycobacterium tuberculosis -ketoglutarate decarboxylase Kgd codon optimised gene 47atggctaata tctcctctcc gtttggtcag aatgaatggc tggtagaaga aatgtaccgt 60aaattccgcg atgacccgtc ctctgtggac ccgtcctggc atgaattcct ggtagactac 120agcccggagc cgaccagcca accggcagcg gaaccaaccc gcgttacttc tccgctggta 180gcggaacgtg cagctgctgc cgcgcctcag gcgccgccta aaccggcgga tactgccgca 240gccggtaacg gtgtggtggc cgcactggct gctaagactg cggttccgcc gccagcagaa 300ggcgatgaag ttgcagtcct gcgcggtgcg gcggctgcag tggtgaaaaa catgagcgcg 360tccctggagg taccgaccgc cacgagcgtg cgcgcggtcc ctgctaaact gctgattgat 420aaccgtattg tgatcaacaa ccagctgaaa cgtacccgtg gtggcaagat ctccttcact 480catctgctgg gttatgcact ggtacaagcg gttaagaaat tccctaacat gaaccgtcat 540tacactgagg tcgacggtaa accgacggct gttactccgg cacacacgaa cctgggcctg 600gcgatcgacc tgcaaggtaa agatggtaag cgctccctgg tagttgcggg tattaaacgt 660tgcgaaacca tgcgtttcgc acaattcgta accgcctacg aggacattgt ccgccgtgct 720cgtgatggca aactgaccac cgaagatttt gcgggcgtta ctattagcct gaccaaccca 780ggcaccatcg gcaccgtgca cagcgtacct cgtctgatgc cgggccaagg tgcgattatc 840ggtgtgggtg ccatggagta cccggcagaa tttcagggtg cttctgaaga gcgcatcgcc 900gagctgggta ttggtaaact gatcaccctg acttctacct atgaccaccg catcattcag 960ggcgcagaat ccggtgactt cctgcgcact attcacgaac tgctgctgtc cgacggtttc 1020tgggatgaag tttttcgtga actgagcatc ccatatctgc cagttcgctg gtccaccgac 1080aatccggact ctatcgttga caaaaacgct cgcgtaatga acctgatcgc tgcttatcgt 1140aatcgtggtc acctgatggc tgatacggat ccgctgcgcc tggataaagc tcgtttccgt 1200tcccacccgg acctggaagt gctgacccat ggtctgactc tgtgggatct ggaccgcgtg 1260ttcaaagtag atggtttcgc gggtgctcag tacaagaagc tgcgtgacgt gctgggtctg 1320ctgcgtgatg cgtactgtcg tcacattggt gtggagtacg cccacattct ggatccggaa 1380cagaaagaat ggctggagca gcgtgtcgag accaaacacg taaaaccgac cgtagcgcag 1440cagaaatata tcctgtccaa actgaacgcc gccgaggctt tcgaaacttt cctgcagacc 1500aagtacgtgg gccagaaacg cttcagcctg gagggtgcgg aaagcgttat tccgatgatg 1560gatgcagcta tcgatcagtg cgcggaacat ggtctggatg aagtcgttat cggtatgccg 1620caccgtggtc gcctgaacgt actggcaaac atcgtcggta aaccatattc tcagatcttc 1680acggaattcg agggcaacct gaacccgtcc caagcccacg gctccggcga cgtaaaatat 1740catctgggtg ctaccggcct gtatctgcag atgttcggtg ataacgacat ccaggtatct 1800ctgactgcta acccgagcca cctggaggcg gttgatcctg ttctggaagg tctggttcgc 1860gccaaacagg atctgctgga ccacggctct atcgacagcg atggccagcg tgcattcagc 1920gttgtaccgc tgatgctgca tggcgacgcg gcgttcgccg gtcagggtgt cgtagcagaa 1980actctgaacc tggcgaacct gcctggctat cgcgtgggtg gcaccattca catcatcgtt 2040aacaaccaaa tcggtttcac cacggcaccg gagtatagcc gttctagcga atattgcacc 2100gacgtagcca aaatgatcgg tgcgccgatc ttccatgtaa acggtgacga tccagaggcc 2160tgcgtgtggg tggctcgtct ggccgtagac ttccgccagc gttttaagaa agatgtggtt 2220atcgacatgc tgtgctaccg ccgtcgtggt cacaacgaag gtgatgatcc gtctatgact 2280aacccgtatg tctatgacgt ggtggacacc aagcgtggtg cacgcaaatc ttacacggag 2340gccctgatcg gtcgtggcga catctctatg aaagaagcgg aagacgctct gcgtgattac 2400cagggtcagc tggaacgtgt gttcaatgag gtgcgtgagc tggaaaagca cggcgtacaa 2460ccgtccgaat ccgtagagtc cgatcagatg atccctgctg gtctggcaac tgctgttgat 2520aaaagcctgc tggcgcgtat cggcgacgca ttcctggcgc tgccgaatgg ctttaccgcg 2580cacccgcgcg tacagccggt actggaaaaa cgtcgtgaaa tggcctacga aggtaaaatc 2640gattgggcct tcggtgagct gctggccctg ggctctctgg tggctgaggg caagctggta 2700cgcctgagcg gccaggactc ccgtcgcggc actttttctc agcgtcacag cgtcctgatc 2760gatcgtcaca ccggcgaaga attcacgccg ctgcaactgc tggctactaa ctccgatggt 2820agcccgaccg gtggtaagtt cctggtgtac gattccccgc tgtccgaata tgctgcagtt 2880ggtttcgagt atggttacac cgttggcaac ccggacgcag tggttctgtg ggaagcgcag 2940ttcggcgatt tcgttaacgg tgcccagtcc attatcgatg agtttattag cagcggcgag 3000gccaaatggg gccagctgtc taacgttgtg ctgctgctgc ctcacggcca cgagggtcaa 3060ggcccggacc acacctccgc ccgtatcgaa cgcttcctgc agctgtgggc tgaaggctct 3120atgaccatcg cgatgccgtc taccccaagc aactacttcc acctgctgcg tcgccacgca 3180ctggacggca ttcagcgccc gctgatcgtt ttcaccccaa aatccatgct gcgccacaaa 3240gcagctgttt ctgaaatcaa agattttacg gaaattaaat tccgttctgt gctggaagaa 3300ccaacctacg aagacggtat tggcgaccgc aacaaggtaa gccgtatcct gctgacctcc 3360ggcaaactgt actacgagct ggcagcacgt aaggcaaaag ataaccgcaa cgacctggcc 3420atcgtccgcc tggaacagct ggcgccactg ccacgccgtc gcctgcgtga aaccctggat 3480cgctacgaaa acgtaaaaga attcttctgg gtgcaggaag aaccggcaaa ccagggtgcg 3540tggccgcgct ttggtctgga actgccggaa ctgctgccgg ataaactggc aggtatcaag 3600cgcatcagcc gtcgcgctat gagcgccccg tcttctggta gctctaaagt acacgctgta 3660gaacagcaag agatcctgga tgaggccttc ggctaa 36964874DNAArtificial sequenceForward primer for amplification of Bacillus subtilis aminotransferase x 48ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgaagg ttttagtcaa 60tggccggctg attg 744962DNAArtificial sequenceReverse primer for amplification of Bacillus subtilis aminotransferase x 49ggggaccact ttgtacaaga aagctgggtt tatgaaatgc tagcagcctg ttgaatgctt 60tc 625082DNAArtificial sequenceForward primer for amplification of Bacillus subtilis aminotransferase y 50ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgactc atgatttgat 60agaaaaaagt aaaaagcacc tc 825157DNAArtificial sequenceReverse primer for amplification of Bacillus subtilis aminotransferase y 51ggggaccact ttgtacaaga aagctgggtt caatcttcaa ggctcgtaac ctcgtgg 575264DNAArtificial sequenceForward primer for amplification of Rhodobacter sphaeroides aminotransferase 52ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgcccg gttgcggggg 60cttg 645351DNAArtificial sequenceReverse primer for amplification of Rhodobacter sphaeroides aminotransferase 53ggggaccact ttgtacaaga aagctgggtt cagacggcgg ccggttcttt c 515478DNAArtificial sequenceForward primer for amplification of Legionella pneumophila aminotransferase 54ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgagta tcgcatttgt 60taacggcaag tattgttg 785567DNAArtificial sequenceReverse primer for amplification of Legionella pneumophila aminotransferase 55ggggaccact ttgtacaaga aagctgggtt tagtttacta gttgttggta ggaatcatta 60attatcc 675676DNAArtificial sequenceForward primer for amplification of Nitrosomonas europaea aminotransferase 56ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgattt acctcaatgg 60caaatttctg ccgatg 765750DNAArtificial sequenceReverse primer for amplification of Nitrosomonas europaea aminotransferase 57ggggaccact ttgtacaaga aagctgggtt tactggcgtg gagcatgccc 505879DNAArtificial sequenceForward primer for amplification of Neisseria gonorrhoeae aminotransferase 58ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgagga taaatatgaa 60ccgtaacgaa attttattc 795956DNAArtificial sequenceReverse primer for amplification of Neisseria gonorrhoeae aminotransferase 59ggggaccact ttgtacaaga aagctgggtt catgcagcca tcgccttgaa cacttc 566066DNAArtificial sequenceForward primer for amplification of Pseudomonas aeruginosa aminotransferase 60ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgtcga tggccgatcg 60tgatgg 666153DNAArtificial sequenceReverse primer for amplification of Pseudomonas aeruginosa aminotransferase 61ggggaccact ttgtacaaga aagctgggtt tacttgacca gggtacgcca ctc 536267DNAArtificial sequenceForward primer for amplification of Rhodopseudomonas palustris aminotransferase 62ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgaagc tgataccgtg 60ccgcgcc 676351DNAArtificial sequenceReverse primer for amplification of Rhodopseudomonas palustris aminotransferase 63ggggaccact ttgtacaaga aagctgggtt caggcgaccg cgcggatcac c 51641353DNABacillus subtilis 64atggagatga tggggatgga aaacattcag caaaatcagg gattaaagca aaaagatgag 60caatttgtgt ggcatgccat gaagggagcg catcaagcgg acagcctgat agcccagaag 120gccgaagggg cctgggtaac cgacacagac ggacgccgct atttggatgc gatgtccggt 180ttgtggtgcg tcaacattgg ttacggcaga aaggagcttg cggaggctgc ctatgagcaa 240ctaaaggagc tgccttacta cccgttaacg caaagtcacg cacccgcaat tcaactggcg 300gaaaagctga atgaatggct tggcggcgat tatgttattt ttttttccaa cagcggatcg 360gaagcaaacg aaactgcttt taaaattgcc cgccagtacc atctgcaaaa cggcgaccac 420agccgttata aattcatctc aagatatcgg gcataccacg gcaatacatt gggagcgctc 480tcagctaccg gacaggcgca gcggaaatat aaatacgagc ctttgagcca agggttcctg 540catgcagctc cgccagatat ataccggaat cctgatgatg cagacacgct tgaaagcgca 600aatgaaatcg accgcatcat gacatgggaa ttaagcgaaa cgattgccgg ggtcattatg 660gagcccatca ttacaggcgg aggcatccta atgccgccgg acggatatat gaagaaggtg 720gaggacattt gccggcgcca cggagccctt ttgatttgcg atgaagtgat ctgcgggttt 780ggacggacag gtgagccgtt cgggtttatg cactacggtg tgaagcctga tatcattacg 840atggcaaagg gaatcacaag cgcgtatctg ccattgtcag cgactgctgt gaaacgggac 900attttcgaag cgtatcaggg ggaagctcct tatgaccgtt tccgccacgt gaacacgttc 960ggcggaagcc cggctgcctg tgctttggcg ttgaaaaacc tgcaaattat ggaggacgaa 1020cagctgattc agcgatcccg tgatcttgga gcaaagcttt taggtgagct tcaagctctg 1080agagaacacc cggcagtcgg ggatgttaga ggaaaagggc tgctgatcgg aatcgaactc 1140gtcaaagaca aattgactaa agagccggct gatgccgcca aagtaaacca agtggttgcg 1200gcgtgcaaag aaaaagggct gatcatcggc aaaaacggcg atacagtcgc cggctacaac 1260aatgtcatcc acgttgcgcc gccattttgc ctgacagaag aggacctttc ctttatcgtg 1320aaaacggtga aagaaagctt tcaaacgata taa 135365450PRTBacillus subtilis 65Met Glu Met Met Gly Met Glu Asn Ile Gln Gln Asn Gln Gly Leu Lys 1 5 10 15 Gln Lys Asp Glu Gln Phe Val Trp His Ala Met Lys Gly Ala His Gln 20 25 30 Ala Asp Ser Leu Ile Ala Gln Lys Ala Glu Gly Ala Trp Val Thr Asp 35 40 45 Thr Asp Gly Arg Arg Tyr Leu Asp Ala Met Ser Gly Leu Trp Cys Val 50 55 60 Asn Ile Gly Tyr Gly Arg Lys Glu Leu Ala Glu Ala Ala Tyr Glu Gln 65 70 75 80 Leu Lys Glu Leu Pro Tyr Tyr Pro Leu Thr Gln Ser His Ala Pro Ala 85 90 95 Ile Gln Leu Ala Glu Lys Leu Asn Glu Trp Leu Gly Gly Asp Tyr Val 100 105 110 Ile Phe Phe Ser Asn Ser Gly Ser Glu Ala Asn Glu Thr Ala Phe Lys 115 120 125 Ile Ala Arg Gln Tyr His Leu Gln Asn Gly Asp His Ser Arg Tyr Lys 130 135 140 Phe Ile Ser Arg Tyr Arg Ala Tyr His Gly Asn Thr Leu Gly Ala Leu 145 150 155 160 Ser Ala Thr Gly Gln Ala Gln Arg Lys Tyr Lys Tyr Glu Pro Leu Ser 165 170 175 Gln Gly Phe Leu His Ala Ala Pro Pro Asp Ile Tyr Arg Asn Pro Asp 180 185 190 Asp Ala Asp Thr Leu Glu Ser Ala Asn Glu Ile Asp Arg Ile Met Thr 195 200 205 Trp Glu Leu Ser Glu Thr Ile Ala Gly Val Ile Met Glu Pro Ile Ile 210 215 220 Thr Gly Gly Gly Ile Leu Met Pro Pro Asp Gly Tyr Met Lys Lys Val 225 230 235 240 Glu Asp Ile Cys Arg Arg His Gly Ala Leu Leu Ile Cys Asp Glu Val 245 250 255 Ile Cys Gly Phe Gly Arg Thr Gly Glu Pro Phe Gly Phe Met His Tyr 260 265 270 Gly Val Lys Pro Asp Ile Ile Thr Met Ala Lys Gly Ile Thr Ser Ala 275 280 285 Tyr Leu Pro Leu Ser Ala Thr Ala Val Lys Arg Asp Ile Phe Glu Ala 290 295 300 Tyr Gln Gly Glu Ala Pro Tyr Asp Arg Phe Arg His Val Asn Thr Phe 305 310 315 320 Gly Gly Ser Pro Ala Ala Cys Ala Leu Ala Leu Lys Asn Leu Gln Ile 325 330 335 Met Glu Asp Glu Gln Leu Ile Gln Arg Ser Arg Asp Leu Gly Ala Lys 340 345 350 Leu Leu Gly Glu Leu Gln Ala Leu Arg Glu His Pro Ala Val

Gly Asp 355 360 365 Val Arg Gly Lys Gly Leu Leu Ile Gly Ile Glu Leu Val Lys Asp Lys 370 375 380 Leu Thr Lys Glu Pro Ala Asp Ala Ala Lys Val Asn Gln Val Val Ala 385 390 395 400 Ala Cys Lys Glu Lys Gly Leu Ile Ile Gly Lys Asn Gly Asp Thr Val 405 410 415 Ala Gly Tyr Asn Asn Val Ile His Val Ala Pro Pro Phe Cys Leu Thr 420 425 430 Glu Glu Asp Leu Ser Phe Ile Val Lys Thr Val Lys Glu Ser Phe Gln 435 440 445 Thr Ile 450 661407DNAPseudomonas aeruginosa 66atgaacgcaa gactgcacgc cacgtccccc ctcggcgacg ccgacctggt ccgtgccgac 60caggcccact acatgcacgg ctaccacgtg ttcgacgacc accgcgtcaa cggctcgctg 120aacatcgccg ccggcgacgg cgcctatatc tacgacaccg ccggcaaccg ctacctcgac 180gcggtgggcg gcatgtggtg caccaacatc ggcctggggc gcgaggaaat ggctcgcacc 240gtggccgagc agacccgcct gctggcctat tccaatccct tctgcgacat ggccaacccg 300cgcgccatcg aactctgccg caagctcgcc gagctggccc ccggcgacct cgaccacgtg 360ttcctcacca ccggcggttc caccgccgtg gacaccgcga tccgcctcat gcactactac 420cagaactgcc gcggcaagcg cgccaagaag cacgtcatca cgcggatcaa cgcctaccac 480ggctcgacct tcctcggcat gtcgctgggc ggcaagagcg ccgaccggcc ggccgagttc 540gacttcctcg acgagcgcat ccaccacctc gcctgtccct attactaccg cgctccggaa 600gggctgggcg aagccgagtt cctcgatggc ctggtggacg agttcgaacg caagatcctc 660gaactgggcg ccgaccgggt gggggcgttc atctccgagc cggtgttcgg ctccggcggc 720gtgatcgtcc cgcccgcggg ctaccacagg cggatgtggg agctgtgcca gcgctacgac 780gtgctgtaca tctccgacga agtggtgacc tccttcggcc gcctcggcca cttcttcgcc 840agccaggcgg tgttcggcgt acagccggac atcatcctca ccgccaaggg cctcacctcc 900ggctaccagc cgctgggcgc gtgcatcttc tcccggcgca tctgggaggt gatcgccgag 960ccggacaagg gccgctgctt cagccatggt ttcacctact ccggccaccc ggtggcctgc 1020gcggcggcgc tgaagaacat cgagatcatc gagcgcgagg gcttgctcgc ccacgccgac 1080gaggtcggcc gctacttcga ggagcgcctg caaagcctcc gcgacctgcc catcgtcggc 1140gacgtgcgcg ggatgcgctt catggcctgt gtcgagttcg tcgccgacaa ggcgagcaag 1200gcgctgtttc cggaaagcct gaacatcggc gagtgggtcc acctgcgggc gcagaagcgc 1260ggcctgctgg ttcgtccgat cgtccacctg aacgtgatgt cgccgccgct gatcctcacc 1320cgcgaacagg tcgataccgt ggtccgggtg ctgcgcgaga gcatcgagga aaccgtggag 1380gatcttgtcc gcgccggtca ccggtaa 140767468PRTPseudomonas aeruginosa 67Met Asn Ala Arg Leu His Ala Thr Ser Pro Leu Gly Asp Ala Asp Leu 1 5 10 15 Val Arg Ala Asp Gln Ala His Tyr Met His Gly Tyr His Val Phe Asp 20 25 30 Asp His Arg Val Asn Gly Ser Leu Asn Ile Ala Ala Gly Asp Gly Ala 35 40 45 Tyr Ile Tyr Asp Thr Ala Gly Asn Arg Tyr Leu Asp Ala Val Gly Gly 50 55 60 Met Trp Cys Thr Asn Ile Gly Leu Gly Arg Glu Glu Met Ala Arg Thr 65 70 75 80 Val Ala Glu Gln Thr Arg Leu Leu Ala Tyr Ser Asn Pro Phe Cys Asp 85 90 95 Met Ala Asn Pro Arg Ala Ile Glu Leu Cys Arg Lys Leu Ala Glu Leu 100 105 110 Ala Pro Gly Asp Leu Asp His Val Phe Leu Thr Thr Gly Gly Ser Thr 115 120 125 Ala Val Asp Thr Ala Ile Arg Leu Met His Tyr Tyr Gln Asn Cys Arg 130 135 140 Gly Lys Arg Ala Lys Lys His Val Ile Thr Arg Ile Asn Ala Tyr His 145 150 155 160 Gly Ser Thr Phe Leu Gly Met Ser Leu Gly Gly Lys Ser Ala Asp Arg 165 170 175 Pro Ala Glu Phe Asp Phe Leu Asp Glu Arg Ile His His Leu Ala Cys 180 185 190 Pro Tyr Tyr Tyr Arg Ala Pro Glu Gly Leu Gly Glu Ala Glu Phe Leu 195 200 205 Asp Gly Leu Val Asp Glu Phe Glu Arg Lys Ile Leu Glu Leu Gly Ala 210 215 220 Asp Arg Val Gly Ala Phe Ile Ser Glu Pro Val Phe Gly Ser Gly Gly 225 230 235 240 Val Ile Val Pro Pro Ala Gly Tyr His Arg Arg Met Trp Glu Leu Cys 245 250 255 Gln Arg Tyr Asp Val Leu Tyr Ile Ser Asp Glu Val Val Thr Ser Phe 260 265 270 Gly Arg Leu Gly His Phe Phe Ala Ser Gln Ala Val Phe Gly Val Gln 275 280 285 Pro Asp Ile Ile Leu Thr Ala Lys Gly Leu Thr Ser Gly Tyr Gln Pro 290 295 300 Leu Gly Ala Cys Ile Phe Ser Arg Arg Ile Trp Glu Val Ile Ala Glu 305 310 315 320 Pro Asp Lys Gly Arg Cys Phe Ser His Gly Phe Thr Tyr Ser Gly His 325 330 335 Pro Val Ala Cys Ala Ala Ala Leu Lys Asn Ile Glu Ile Ile Glu Arg 340 345 350 Glu Gly Leu Leu Ala His Ala Asp Glu Val Gly Arg Tyr Phe Glu Glu 355 360 365 Arg Leu Gln Ser Leu Arg Asp Leu Pro Ile Val Gly Asp Val Arg Gly 370 375 380 Met Arg Phe Met Ala Cys Val Glu Phe Val Ala Asp Lys Ala Ser Lys 385 390 395 400 Ala Leu Phe Pro Glu Ser Leu Asn Ile Gly Glu Trp Val His Leu Arg 405 410 415 Ala Gln Lys Arg Gly Leu Leu Val Arg Pro Ile Val His Leu Asn Val 420 425 430 Met Ser Pro Pro Leu Ile Leu Thr Arg Glu Gln Val Asp Thr Val Val 435 440 445 Arg Val Leu Arg Glu Ser Ile Glu Glu Thr Val Glu Asp Leu Val Arg 450 455 460 Ala Gly His Arg 465 681335DNAPseudomonas aeruginosa 68atgacaatga atgacgagcc gcagtcgagc agcctcgaca acttctggat gcccttcacc 60gccaaccgcc agttcaaggc gcggccgcgc ctgctggaaa gcgccgaagg catccactat 120atcgcccagg gcgggcgccg catcctcgac ggcaccgccg gcctctggtg ctgcaatgcc 180ggccacggcc ggcgcgagat cagcgaagcg gtggcccggc agatcgccac cctcgactac 240gccccgccgt tccagatggg tcacccgctg ccgttcgaac tcgccgcgcg gctgacggaa 300atcgccccgc cgagcctgaa caaagtattc ttcaccaact ccggctcgga atcggcggac 360accgcgctga agatcgccct tgcctaccag cgcgccatcg gccagggcac ccgcacccgc 420ctgatcggcc gcgaactggg ctaccacggg gtcggcttcg gcggcctgtc ggtaggcggt 480atggtcaaca accgcaaggc cttctccgcc aacctgctgc cgggggtcga ccacctgccg 540cacaccctgg acgtcgcccg caacgccttc accgtcggcc tgcccgagca tggcgtggaa 600aaggccgagg agctggaacg cctggtgacc ctgcacggcg ccgagaatat cgccgcggtg 660atcgtcgagc cgatgtccgg ctcggccggc gtggtgctgc cgcccaaggg ctaccttcag 720cggctgcgcg agataacccg caagcatggc atcctgctga tcttcgacga agtgatcacc 780ggtttcggcc gcgtcggcga agccttcgcc gcgcagcgct ggggcgtcgt cccggacctg 840ctgacctgcg ccaaggggct gaccaacggc agcatcccga tgggcgccgt attcgtcgac 900gagaagatcc atgctgcctt catgcaaggc ccgcagggcg ccatcgagtt cttccacggc 960tatacctatt ccggccatcc ggtagcctgc gccgccgccc tggcgaccct ggacatctac 1020cgtcgcgacg acctgttcca gcgggccgtc gaactggaag gctactggca ggacgcgctg 1080ttcagcctgc gcgacctgcc caacgtggtc gacatccgcg ccgtaggcct ggtcggcggc 1140gtgcaactgg cgccgcacgc ggacggcccc ggcaagcgcg gctacgacgt cttcgagcgc 1200tgcttctggg agcacgacct gatggtccgg gtgaccggcg acatcatcgc catgtcgccg 1260ccgctgatca tcgacaagcc ccacatcgac cagatcgtcg agcgcctggc ccaggccatc 1320cgcgccagcg tctga 133569444PRTPseudomonas aeruginosa 69Met Thr Met Asn Asp Glu Pro Gln Ser Ser Ser Leu Asp Asn Phe Trp 1 5 10 15 Met Pro Phe Thr Ala Asn Arg Gln Phe Lys Ala Arg Pro Arg Leu Leu 20 25 30 Glu Ser Ala Glu Gly Ile His Tyr Ile Ala Gln Gly Gly Arg Arg Ile 35 40 45 Leu Asp Gly Thr Ala Gly Leu Trp Cys Cys Asn Ala Gly His Gly Arg 50 55 60 Arg Glu Ile Ser Glu Ala Val Ala Arg Gln Ile Ala Thr Leu Asp Tyr 65 70 75 80 Ala Pro Pro Phe Gln Met Gly His Pro Leu Pro Phe Glu Leu Ala Ala 85 90 95 Arg Leu Thr Glu Ile Ala Pro Pro Ser Leu Asn Lys Val Phe Phe Thr 100 105 110 Asn Ser Gly Ser Glu Ser Ala Asp Thr Ala Leu Lys Ile Ala Leu Ala 115 120 125 Tyr Gln Arg Ala Ile Gly Gln Gly Thr Arg Thr Arg Leu Ile Gly Arg 130 135 140 Glu Leu Gly Tyr His Gly Val Gly Phe Gly Gly Leu Ser Val Gly Gly 145 150 155 160 Met Val Asn Asn Arg Lys Ala Phe Ser Ala Asn Leu Leu Pro Gly Val 165 170 175 Asp His Leu Pro His Thr Leu Asp Val Ala Arg Asn Ala Phe Thr Val 180 185 190 Gly Leu Pro Glu His Gly Val Glu Lys Ala Glu Glu Leu Glu Arg Leu 195 200 205 Val Thr Leu His Gly Ala Glu Asn Ile Ala Ala Val Ile Val Glu Pro 210 215 220 Met Ser Gly Ser Ala Gly Val Val Leu Pro Pro Lys Gly Tyr Leu Gln 225 230 235 240 Arg Leu Arg Glu Ile Thr Arg Lys His Gly Ile Leu Leu Ile Phe Asp 245 250 255 Glu Val Ile Thr Gly Phe Gly Arg Val Gly Glu Ala Phe Ala Ala Gln 260 265 270 Arg Trp Gly Val Val Pro Asp Leu Leu Thr Cys Ala Lys Gly Leu Thr 275 280 285 Asn Gly Ser Ile Pro Met Gly Ala Val Phe Val Asp Glu Lys Ile His 290 295 300 Ala Ala Phe Met Gln Gly Pro Gln Gly Ala Ile Glu Phe Phe His Gly 305 310 315 320 Tyr Thr Tyr Ser Gly His Pro Val Ala Cys Ala Ala Ala Leu Ala Thr 325 330 335 Leu Asp Ile Tyr Arg Arg Asp Asp Leu Phe Gln Arg Ala Val Glu Leu 340 345 350 Glu Gly Tyr Trp Gln Asp Ala Leu Phe Ser Leu Arg Asp Leu Pro Asn 355 360 365 Val Val Asp Ile Arg Ala Val Gly Leu Val Gly Gly Val Gln Leu Ala 370 375 380 Pro His Ala Asp Gly Pro Gly Lys Arg Gly Tyr Asp Val Phe Glu Arg 385 390 395 400 Cys Phe Trp Glu His Asp Leu Met Val Arg Val Thr Gly Asp Ile Ile 405 410 415 Ala Met Ser Pro Pro Leu Ile Ile Asp Lys Pro His Ile Asp Gln Ile 420 425 430 Val Glu Arg Leu Ala Gln Ala Ile Arg Ala Ser Val 435 440 7071DNAArtificial sequenceForward primer for amplification of Bacillus subtilis aminotransferase (gi16077991) 70ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatggaga tgatggggat 60ggaaaacatt c 717165DNAArtificial sequenceReverse primer for amplification of Bacillus subtilis aminotransferase (gi16077991) 71ggggaccact ttgtacaaga aagctgggtt tatatcgttt gaaagctttc tttcaccgtt 60ttcac 657266DNAArtificial sequenceForward primer for amplification of Pseudomonas aeruginosa aminotransferase (gi9951072) 72ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgaacg caagactgca 60cgccac 667348DNAArtificial sequenceReverse primer for amplification of Pseudomonas aeruginosa aminotransferase (gi9951072) 73ggggaccact ttgtacaaga aagctgggtt taccggtgac cggcgcgg 487469DNAArtificial sequenceForward primer for amplification of Pseudomonas aeruginosa aminotransferase (gi9951630) 74ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgacaa tgaatgacga 60gccgcagtc 697549DNAArtificial sequenceReverse primer for amplification of Pseudomonas aeruginosa aminotransferase (gi9951630) 75ggggaccact ttgtacaaga aagctgggtt cagacgctgg cgcggatgg 497657DNAArtificial sequenceForward primer 76aaatttacta gtaagaattt ttgaggaggc aatataaatg aataaaccac agtcttg 577732DNAArtificial sequenceReverse primer 77aaatttggat cctacaagaa agctgggttt ac 327858DNAArtificial sequenceForward primer 78aaatttacta gtaagaattt ttgaggaggc aatataaatg aacagccaaa tcaccaac 587937DNAArtificial sequenceReverse primer 79aaatttggat ccactttgta caagaaagct gggttca 378057DNAArtificial sequenceForward primer 80aaatttggat ccgttgagga ggcctcaaaa atgtccgaga tcactctggg caaatac 578135DNAArtificial sequenceReverse primer 81aaatttggcg cgccattact gtttagcgtt agttg 358252DNAArtificial sequenceForward primer 82aaatttggat ccgttgagga ggcctcaaaa atgtatactg ttggtgatta tc 528337DNAArtificial sequenceReverse primer 83aaatttggcg cgccattact tgttctgctc cgcaaac 37

Claims

1. (canceled)

2. Method for preparing 6-aminocaproic acid, wherein the 6-aminocaproic acid is prepared from 5-formylpentanoate, using at least one biocatalyst.

3-25. (canceled)