TW201033369A - Preparation of 6-aminocaproic acid from α-ketopimelic acid - Google Patents

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 ε -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

201033369 • _ » VI. Description of the invention: [Technical field of invention] The present invention relates to a method for preparing 6-aminohexanoic acid (hereinafter also referred to as "6-ACA"). The present invention further relates to a method of preparing _caprolactam (hereinafter referred to as "caprolactam") from 6_ACA. The invention further relates to host cells useful for the preparation of 6_ACA or caprolactam. The caprolactam is an internal guanamine which can be used in the manufacture of polyamines such as nylon-6 or nylon-6,12 (copolymer of caprolactam and laurylamine). The art has known a variety of ways to prepare caprolactam from bulk chemicals, including the preparation of caprolactam from cyclohexanone, methyl phenol, phenol, cyclohexanol, benzene or cyclohexane. These intermediate compounds are usually derived from mineral oil. In view of the growing demand for materials prepared using greener resiliency techniques, it is desirable to provide a process in which caprolactam is prepared from an intermediate compound 15 derived from a biorenewable source, or at least decylamine is obtained. An intermediate compound which has been converted to caprolactam by biochemical methods. It is further desirable to provide a method of comparing the chemical methods of utilizing bulk chemicals derived from petrochemical sources that require less energy. It is known to prepare caprolactam from 6-ACA, for example as described in US-A 6,194,572 K20. As described in WO 2005/068643, 6-ACA can be prepared biochemically via the conversion of 6-amine-2-diacid (6-oxime) via the presence of an enzyme having alpha, beta-enoate reductase activity. . 6-ΑΗΕΑ can be prepared, for example, from an amide acid by biochemical means or by a purification synthesis. Although it is feasible to prepare 6_ACa 3 201033369 by reduction of 6_AHEA by the method disclosed in WO 2005/068643, the inventors have found that under the reducing reaction conditions, 6 ahea may spontaneously and substantially irreversibly cyclize to form an undesired pair. The product, notable, is beta-homoamine. This cyclization can be a bottleneck in the manufacture of 6_aca and can result in significant loss in yield. 5 [Bright] The object of the present invention is to provide a novel process for the preparation of 6_ACA or caprolactam, which can be used for the preparation of intermediates in the preparation of polyamide or 6_ACA or caprolactam. As an alternative to known methods. A further object is to provide a new method that overcomes the aforementioned - or more than four points. The item or a plurality of other objects that can be solved according to the present invention are obtained by the following description. It is known to prepare 6_ACA from a specific starting material, in other words, it has been found that 6-aminocaproic acid (6-ACA) can be prepared, wherein 6-amine hexanoic acid is also referred to as 2_steel pimelic acid and 15 as α-ketone gly. Diacid (AKP) preparation. In particular, the method can be carried out in two or more reaction steps. For example, a method is provided in which AKp is first converted to 5-methylvalerate (5-valeric acid, 5 FVA), which is converted to 6-ACA. Further provided is a method wherein AKp is first converted to indoleamine pimelic acid (AAP) followed by AAP to 6_aca. 20 The inventors understand that in principle, 6_ACA can be prepared from AKP by means of full chemical (in other words, no biocatalyst). Individual reaction steps are carried out. Examples of suitable chemical means are listed below. However, the inventors also understand that 6-ACA can be prepared biochemically by AKp. Thus, the present invention relates in particular to a method for preparing 6_ACA 4 201033369, wherein 6-ACA uses at least A biocatalyst prepared from Ακρ. The invention further relates to a process wherein 6aca is prepared from 5-valeric acid ester (5-valeric acid, 5-FVA) using a biocatalyst. As indicated above, '5-FVA Available in AKP. 5 In one embodiment, 6_ACA prepared in the process of the present invention can be used to prepare caprolactam. This method comprises cyclizing 6-amine caproic acid in the presence of a biocatalyst, as desired. • [Embodiment 3] Resin or tare acid vinegar such as 6-AC A, 2-amine pimelic acid • 10 (α-amine pimelic acid, hereinafter also abbreviated as “AAP”), In the case of amino acids, 5_FVa, -- or AKP, these terms are meant to include protonated carboxylic acid groups (ie, neutral, base), their corresponding carboxylates (its conjugate bases), and salts thereof. When reference is made herein to amino acids such as 6-ACA, the term is meant to include amino acids in their zwitterionic form (wherein the amine group is in protonated form and the carboxylic acid group is in deprotonated form), wherein The amine group which is protonated and the carboxylic acid is in its neutral form, the acid, and the amino group in which the amine group is in its neutral form and the carboxylic acid group is in a deprotonated form, and salts thereof. Undesirable cyclization of the intermediate product leading to yield loss was not observed when 6-ACA was formed and, if desired, it was formed. The process of the invention is expected to allow comparison of the process described in WO 2005/68643 to obtain phase A preferred or even better yield. It is expected that the method of the invention is particularly advantageous when utilizing an organism's particular consideration of the growth and maintenance of the organism. It is further contemplated that in an embodiment of the invention, the method 5 of the invention may be modified in 201033369 6-ACA productivity ( The word "or" is used herein unless otherwise specified. It is defined as "and / or". As used herein, the word "一" is defined as "less than one" unless otherwise specified. When a noun (for example, a compound, an additive, etc.) is used in the singular, it is meant to include a plural. When referring to a compound in which a stereoisomer is present, the compound may be any of such stereoisomers. Or a composition thereof. Thus, when the amino acid of the enantiomer is present in Example 10, the amino acid may be the 1-enantiomer, the D-enantiomer or combination. In the case where a natural stereoisomer is present, the compound is preferably a natural stereoisomer. When an enzyme is mentioned and the enzyme class (EC) is included, the enzyme class is based on the enzyme system or may be based on the "National Association of Biochemistry and Molecular Biology Committee 15" (NC-IUBMB). The name of the enzyme is classified as a category, which can be found at http://www.chem.qmul.ac.uk/iubmb/enzyme/. Intents include other suitable enzymes that have not (not yet) been classified in a particular category but can be classified in this manner. The term "homolog" as used herein particularly means having at least 3%, more preferably at least 40%, more preferably at least 60%, more preferably at least 3%, more preferably at least 7%, and even more preferably at least 75%. 'Better at least 8〇%' is better than 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% sequence homology of polynucleic acid or polypeptide. The "homolog"-word also means a nucleotide sequence (polynucleotide sequence) which differs from another nucleotide sequence but encodes the same polynucleotide sequence due to the simplicity of the code 201033369. Sequence identity or similarity is defined herein as determining the relationship between two or more multi-peptide sequences or two or more nucleotide sequences via a comparison of two sequences. Usually the sequence identity or similarity compares the full length of the sequence 'but it is also possible to compare only the portion of the sequence aligned to each other. In the industry, "identity" or "similarity" also indicates the degree of sequence correlation between a multi-peptide sequence or a nucleotide sequence (as the case may be) determined by the matching between such sequences. Preferably, the assay for similarity or similarity is designed to achieve a maximum of 10 matches between the sequences tested. In the context of the present invention, preferred computer program methods for the degree of similarity and similarity between specific sequences include BLASTP and BLASTN (Altschul, SF et al, J. Mol. Biol. 1990, 215, 403-410, published From NCBI and other sources (BLAST Handbook, Altschul, S. et al., NCBI NLM NIH, Beira, Maryland, 20894)). Preferably, BLASTP is used to perform multi-peptide comparison. The parameters of the sequence comparison are gap opening 1〇.〇, gap extension 0.5, Blosum 62 matrix. Preferred parameters for nucleic acid sequence comparison using BLASTN are gap opening 10.0, gap extension 〇·5, DNA full matrix (DNA identity matrix). According to the invention, a biocatalyst is used, in other words a reaction step of the method is catalyzed by a biological material or biological moiety derived from a biological source such as an organism or a biological knife derived from an organism. The biocatalyst specifically comprises one or more enzymes. The biocatalyst can be used in any form. In one embodiment, for example, in the form of a solution, an emulsion, a dispersion, a bundle of stem cells (a suspension), a lysate, or a brake on a body, and isolated from the natural environment (separating itself to create the organism) - One or more of the 7 201033369 enzymes. In one embodiment, the one or more enzymes form part of a living organism (eg, a living whole cell). The enzyme can perform a catalytic function inside the cell. It is also possible that the enzyme can be secreted into the cell in which it is present. In the medium. 5 Living cells can be growing cells, resting quiescent cells or dormant cells (such as spores or spores) or cells in stable phase. Permeabilized cells can also be used (in other words, become permeable enzymes). An enzyme forming moiety of a matrix or an enzyme matrix precursor. The biocatalyst used in the method of the invention is primarily any organism 10 or may be derived or derived from any organism. The organic hydrazine may be a eukaryotic or prokaryotic organism. May be selected from animals (including humans), plants, bacteria, archaea, yeast, and fungi. In one embodiment, the biocatalyst system From animals, in particular from one part of an animal such as liver, pancreas, brain, kidney, heart or other organs. 15 Animals may in particular be selected from the group consisting of mammals, more particularly from the family of rabbits a group consisting of (Leporidae), Muridae, Suidae, and Bovidae. Suitable plants include, inter alia, from Asplenium; Cucurbitaceae, especially Cucurbita such as Cucurbita 20 moschata (pumpkin), or Cucumis; Mercurialis such as Mercurialis perennis; Hydnocarpus; and Ceratonia The plant of the group consisting of the appropriate bacteria is particularly selected from the group consisting of Vibrio, Pseudomonas 8 201033369 5 (Pseudomonas) 'Bacnius, Corynebacterium, short Brevibacterium, Enterococcus, Strept〇c〇ccus, Klebsiella 'Lact〇coccus, Lactobacillus, Clostridium Ridium), Escherichia, Thermus, Mycobacterium, Zymomonas, Proteus, Agrobacterium ), Geobacillus, Acinetobacter, Rastaea 10 « w (Ralstonia), Rhodobacter, Paracoccus, Novosphingobium , Nitrosomonas, Legionella, Neisseria, Rhodopseudomonas, Staphylococcus, Dinosum A group consisting of Deinococcus and Salmonella. Suitable archaea may in particular be selected from the group consisting of Archaeoglobus, Aeropyrum, Halobacterium, Methanosarcina, Methanococcus. , Thermoplasma, Pyrobaculum, Methanocaldococcus, Methanobacterium, Methanosphaera, Methanopyrus And a group consisting of Methanobrevibacter. Suitable fungi are particularly selected from the group consisting of Rhizopus, Rhizopus 9 201033369 (NeurosP〇ra), Penicillium and Aspergillus. Suitable yeasts are particularly selected from the group consisting of Candida, Hansenula, Kiuyveromyces, and Sacchar〇myces. It will be apparent to those skilled in the art that a mutant having a suitably active natural biocatalyst (wild type) or a natural biocatalyst can be utilized in the method according to the present invention. The nature of the natural biocatalyst can be improved by biotechnological techniques known to those skilled in the art, such as molecular evolution or theoretical design. The preparation method of the mutant strain of the wild type biocatalyst can be modified, for example, by using a mutation generating technique (random mutation occurrence, position-directed mutation occurrence, directed evolution, genetic recombination, etc.) known to those skilled in the art as a biocatalyst or a biocatalytic moiety. Prepared by encoding the DNA of an organism such as an enzyme. Specifically, DNA can be modified to allow the DNA to encode an enzyme that differs from the wild-type enzyme by at least one acid 10 15 'so can encode a wild-type enzyme that contains one or more amino acid substitutions, deletions, and/or insertions. , or a combination of mutants or a plurality of parental enzyme sequences, or by affecting the appropriate (host) performance of the modified Dna. The latter can be achieved by methods known to those skilled in the art, such as two:

Optimization method or codon pair optimization method, such as the method described in ', 2008/000632. Soil-to-WO mutant biocatalysts have improved properties in one or more of the following phases: selectivity to enzyme matrix, activity, climaticity, solvent odor resistance, data, temperature profile data, enzyme matrix profile data, Susceptibility to inhibition, cofactor utilization, and enzyme matrix affinity. p 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 A recombinant biocatalyst derived from a second organism, which is derived from a first organism but is actually (genically modified), in particular an enzyme comprising a biocatalyst derived from the first organism, in particular an enzyme. In a preferred method of the invention, the preparation of a biocatalyst comprising a decarboxylation reaction which catalyzes a CC-keto acid or an amino acid (ie, a compound comprising at least one carboxylic acid group and at least one amine group) is prepared. Biocatalytic (usually 1 〇 enzyme catalyzed) reaction. The enzymes having such catalytic activity are thus referred to as α•net decarboxylase or amino acid decarboxylase, respectively. The acid is preferably a dibasic acid in which the biocatalyst is selective for an acid group adjacent to a keto group or an amine group. Roughly 'appropriate decarboxylase has (X-ketopimelate decarboxylase activity, can catalyze the conversion of ruthenium to 5-FVA; or has alpha-amine pimelic acid decarburase activity, can catalyze the conversion of ruthenium to 6- ACA. The enzyme which can decarboxylate ex-keto acid or amino acid is especially selected from the decarboxylase group (EC 4.1.1), preferably selected from the grass detoxification enzyme (EC 4.1). .1.3), diamine pimelic acid decarboxylase (EC 4.1.1.20), branched chain α-keto acid de-20 carboxylase (EC 4.1.1.72), α-ketoisovalerate decarboxylase, α-keto a group consisting of diacid decarboxylase (EC 4.1.1.71) and pyruvate decarboxylase (EC 4.1.1.1). One or more other suitable decarboxylases may be selected from oxalic acid decarboxylase (EC) 4.1.1.2), acetamidine decarboxylase (EC 4.1.1.4), proline decarboxylase / leucine decarboxylase (EC 4.1.1.14), glutamic acid decarboxylase (EC 4.1.1.15) , 11 201033369 Aspartic acid 1-decarboxylase (EC 4.L1.11), 3-hydroxy faceamine decarboxylase (EC 4.1.1.16), ornithine decarboxylase (EC 4.1.1.17), from Amino acid decarboxylase (EC 4.1.1.18), arginine detoxification enzyme (EC 4.1.1.19), 2-ketoglutarate decarboxylase (EC 4.1.1.71), and diamine butyrate decarboxylase ( E C 4.1.1.86) Group 5 consisting of. The decarboxylase may especially be an organism decarboxylase selected from the group consisting of pumpkin, courgette; yeast; fungi such as Saccharomyces cerevisiae, trumpet-like silk Candida flareri, Hansenula sp., Kluyveromyces 10 marxianus, Rhizopus javanicus, and Neurospora crassa; Mammalian special line From mammalian brain and fine pain such as Escherichia coli, Lactococcus lactis, Mycobacterium tuberculosis, Pseudomonas sp., and active fermenting cells A group consisting of Zymomonas 15 mobilis. Pyruvate decarboxylase may be derived from Saccharomyces cerevisiae or Z. mobilis. In particular, pyruvate decarboxylase mutant I472A from Z. mobilis can be used. A glutamate decarboxylase, a diamine heptanedioate decarboxylase or an aspartate decarboxylase derived from Escherichia coli (E. c〇li) can be used. Use of glutamate decarboxylase from the red scutellaria, Mycobacterium leprae, Clostridium perfringens, Lactobacillus brevis, Mycobacterium tuberculosis, Streptococcus or Lactococcus . Examples of Lactococcus species that can obtain glutamate decarboxylase 201033369 specifically include Lactococcus lactis such as Lactococcus lactis b1157, Lactococcus lactis IFPL730, and more particularly Lactococcus lactis variant maltigenes (month il A strain of Strept〇c〇ccus iactis var. maltigenes) 〇5 In particular, a grasshopper acetic acid decarboxylase derived from Pseudomonas can be used. A branched chain α-keto acid decarboxylase derived from Lactococcus lactis can be used. More specifically, α-ketoisovalerate decarboxylase derived from Lactococcus lactis can be used. In particular, alpha-ketoglutarate decarboxylase from Mycobacterium tuberculosis can be used. In the preferred method of the invention, the preparation of 6-ACA comprises enzymatically catalyzed the reaction in the presence of an amine-based donor 10 which catalyzes the transamination reaction and is selected from the group of enzymes of the transaminase (EC 261). In general, the appropriate transaminase has 6-aminocaproic acid 6 transaminase activity, which can catalyze the conversion of 5-FVA to 6-ACA; or has α-amine pimelic acid 2_transaminase activity, which can catalyze the transfer. Cheng Yu. 15 transaminase is especially selected from β-amine isobutyric acid: α-ketoglutarate transaminase, β-alanine transaminase, aspartate transaminase, 4 amine butyrate transaminase 2.6 .1.19), L-lysine 6-transaminase (Ec 2·6136), 2-aminoadipate transaminase (EC 2.6.1.39), 5-aminopentanoate transaminase (EC 2 6丨) 48), 2 aminohexanoate transaminase (EC 2.6.1.67) and a group consisting of lysine: pyruvate 6_transaminase (EC 20 2.6.1.71). In one embodiment, the transaminase may be selected from the group consisting of alanine transaminase (EC 2.6.1.2), leucine transaminase (EC 2.6.1.6), alanine-ketoacid transaminase (EC 2.6) .1.12), 3-alanine-pyruvate transaminase (:26118), 〇3_amine_2_

A group consisting of methylpropionic acid transaminase (EC 2.6.1.22), l,L-diaminepimelate transaminase (EC 13 201033369 2.6.1.83). The transaminase is particularly selected from the group consisting of mammals; the genus Hawthorn is especially a perennial hawthorn, more particularly the perennial hawthorn; the genus Pteridium is more particularly the unilateral hornbeam fern (Asplenium unilaterale) or the north Asplenium Septentrionale · 'Celebria genus is more particularly Cemtonia siliqUa; Rhodobacter genus is especially RhRdd〇 sphaeroides; (4) Coccidia is especially Staphylococcus au (Staphylococcus au); Vibrio genus especially for Vibrio fluvialis (5) plus • "Eight τ 闽 闽 付 付 付 付 ( ( ( ( ( ( ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; Bacillus genus (BaciUus weihenstephanensis) and Bacillus subtilis 1 (4) also belongs to the genus Legionella; subsp. genus; Neisseria or yeast, especially the transaminase of Saccharomyces cerevisiae. In the case of enzymes, 5 kidneys, mammals, and t-systems are derived from mammals: from: heart or mammalian brain. For example, it is especially suitable for β-amine isobutyric acid derived from Xushen: glutaric acid : milk animal liver β-alanine transaminase, special 020 is the bis

Transaminase, derived from the mammalian heart aspartate transaminase, special: amine: especially 4-potassium butyrate transaminase from pig liver; obtained from m-amine, amine butyrate transaminase 'Specially derived from human, :: animal brain 4-enzyme; derived from the genus of the genus Red genus 4 M. amine butyrate transaminating _ adipic acid: diacid = especially from crude amine , 4-Amine 14 from Escherichia coli 201033369 Butyric acid transaminase, or the amine adipic acid transaminase from the genus Thermus, especially from Thermus thermophilus (X) - Amine adipate transaminase, and 5-amine valerate transaminase from Clostridium, especially 5-10-aminovalerate transaminase from Clostridium aminovaledcum 15 20 Groups. Suitable 2-amine adipic acid transaminase may, for example, also be provided by Pyrobaculum islandicum. In particular, the amine donor may be selected from the group consisting of ammonia, ammonium ions, amines and amino acids. a group consisting of a suitable amine as a first amine and a second amine. The amino acid may have a D-configuration or an L-configuration. Examples of amine-based donors are alanine, a face acid, an isopropylamine, 2-amine butyl, 2-amine Burning, benzylamine, ι_phenyl acetamide, face amine, citrate, phenylalanine, aspartic acid, β-amine isobutyric acid, 戸-alanine, 4-amine butyric acid, And (X-amine adipic acid. In a preferred embodiment, the method for preparing 6-ACA comprises an enzyme capable of catalytically reductive amination in the presence of an ammonia source selected from CH-NH2 acting on the donor. a group of oxidoreductases (EC 1.4), in particular a biocatalytic reaction in the presence of an enzyme selected from the group consisting of amino acid dehydrogenases (EC 1.4.1). Roughly 'appropriate amino acids Hydrogenase has 6-aminohexanoic acid 6-dehydrogenase activity 'can catalyze the conversion of 5-FVA to 6-ACA; or has a-amine pimelic acid 2-dehydrogenase activity' to catalyze the conversion to oxime. The appropriate amino acid dehydrogenase may be selected from the group consisting of diamine pimelate dehydrogenase (EC 1.4.1.16), lysine 6-dehydrogenase (EC 1.4.1.18), dehydrogenating glutamic acid. A group consisting of enzyme (EC 1.4.1.3; EC 1.4.1.4) and leucine dehydrogenase (EC 1.4· 1.9).

In one embodiment, the amino acid dehydrogenase may be selected from the group consisting of glutamate dehydrogenase (EC 15 201033369 1 ·4.1 ·3), which is classified as a receptor with NAD or NADP, with NADP as The glutamate dehydrogenase (Ec 1.4.1 ·4), leucine dehydrogenase (EC 1.4· 1.9), diamine pimelate dehydrogenase (EC 1.4.1.16), and Amino acid dehydrogenase of amine 6-dehydrogenase (EC 1.4.1.18). The amino acid dehydrogenase is particularly derived from an organism selected from the group consisting of 5 in the group consisting of: Corynebacterium glutamicum, in particular, of the genus Proteus; Proteus vulgaris; Agrobacterium is particularly Agrobacterium tumefaciens; Geobacterium genus is Geobacillus stearothermophilus; Acinetobacter is a genus of ADP1; The genus is particularly Ralstonia solanacearum; the Salmonella is particularly Salmonella typhimurium; the genus Saccharomyces is particularly Saccharomyces cerevisiae; the Brevibacterium genus is Brevibacterium flavum; and Bacillus The genus is particularly Bacillus sphaericus, Bacillus cereus and Bacillus subtilis. For example, a suitable amino acid dehydrogenase may be selected from the group consisting of diamine pimelic acid dehydrogenase derived from Bacillus, particularly Bacillus licheniformis; diamine pimelic acid dechlorinase obtained from Brevibacterium; a diamine pimelic acid dehydrogenase from the genus Corynebacterium, in particular a diamine pimelic acid dehydrogenase obtained from Corynebacterium faecalis; a diamine 20 pimelic acid dehydrogenase derived from the genus Proteus, in particular a diamine pimelic acid deaerator obtained from the common Proteus; an lysine 6-deaerator derived from Agrobacterium, particularly Agrobacterium tumefaciens; a genus of the genus Bacillus Amino acid 6-dehydrogenase; glutamate dehydrogenase (EC 1.4.1.3) derived from Acinetobacter with NADH or NADPH as a cofactor, especially from immobilized rod 201033369 5 10 15 20 A face acid dehydrogenase of the genus ADP1; a face acid dehydrogenase (EC 1.4.1.3) from the genus Rasta, especially for amygdamine from the genus R. serrata Hydrogenase; glutamate dehydrogenase (EC 1.4.1.4) derived from Salmonella and acting as a cofactor with NADPH, especially for tyrosine from typhoid bacillus Dehydrogenase; glutamate dehydrogenase (EC丨4丨4) from the genus Saccharomyces, especially the acid-dehydrogenase derived from the fermented mother; the amylin dehydrogenase from the short (four) genus ( EC 1.4.1.4) 'Specially for the sulphate deaminase from Brevibacterium flavum; and leucine dehydrogenase from the genus Spore, especially for leucine from Bacillus cereus or Bacillus subtilis Hydrogenase. In a particular embodiment, AKP is biocatalyzed to 5-potted valeric acid (5_FVA) in the presence of de- or other biocatalysts that catalyze such conversion. According to the invention (4), it is difficult to select from the group consisting of: lactate lactate g, lactate milk (four) variant malt gene or Lactobacillus lactis subspecies stone (Lact〇c〇ccus (8) 叱cremoris a cc-keto acid decarboxylase; a branch of the Lactobacillus lactis strain Bu57 or Lactobacillus lactis IFPL730, a keto acid decarboxylase; obtained from Saccharomyces cerevisiae, Candida faecalis, and Z. mobilis , Hansenula species, Rhizopus oryzae, Mycorrhizal, or Kluyveromyces cerevisiae; A-ketoglutarate decarboxylase from Mycobacterium tuberculosis; obtained from Escherichia coli, A small acid lactobacillus, leprosy, Rhizopus oryzae, and a face acid decarboxylase; and an aspartic acid decarboxylase obtained from Escherichia coli. In particular, it has been found that it is suitable for the catalysis of cockroaches from the large intestine (4), the active fermentation singular, the Saccharomyces cerevisiae, the tuberculosis branch scorpion, the pseudo-singular genus or the lactic acid milk (4). More specifically, it can be decarboxylase having the amino acid sequence identified by the sequence of the 2010 2010, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 4, 4, 4, 4, or 4, Bio-catalyst. It is also contemplated that such decarboxylase can be used to prepare 6-ACA from AAP. The 5-FVA is then converted to 6-ACA. It can be carried out in a chemical manner: by using a hydrogenation catalyst such as nickel in a SiO 2 /Al 2 2 support, a reductive amination reaction of 5-FVA with ammonia can be used to prepare 6-ACA in high yield, such as EP-A 628 535 or DE 4 322 065 to 9 - Aminic acid (9-amine geranyl acid) and 12-amine dodecanoic acid (12-amine lauric acid). In addition, 6-ACA can be prepared by using 6-decanoic acid prepared by reacting 5-FVA with hydroxylamine, and hydrogenating 6-mercaptoic acid using Pt〇2 (for example, F. Ayorinde, EY Nana, PD Nicely, AS Woods, EO Price, CP Nwaonicha J. Am. Oil Chem. Soc. 1997, 74, 531-538 for the synthesis of the homologous 12-amine dodecanoic acid. In one embodiment '5-FVA is converted to 6-ACA The transformation is carried out in a biocatalytical manner in the presence of (1) an amine donor and (ii) a transaminase, an amino acid dehydrogenase or other 15 biocatalysts which catalyze such conversion. In particular, in such an implementation In one embodiment, the transaminase may be selected from the group consisting of: transaminase from Vibrio fluvialis, Pseudomonas aeruginosa, Bacillus subtilis, Bacillus vegetative or Escherichia coli; β-derived from porcine kidney Aminoisobutyric acid: 〇c-ketoglutarate transaminase; β-alanine transaminase obtained from rabbit liver; transaminase 20 obtained from perennial hawthorn shoots; obtained from pig liver or from human 4-amine butyrate transaminase in rabbit or pig brain; β-alanine transaminase obtained from rabbit liver; and L-lysine: a-ketoglutarate-ε-transaminase. In the case of an amino acid dehydrogenase, such an amino acid dehydrogenase is particularly selected from the group consisting of lysine 6-dehydrogenase derived from Agrobacterium tumefaciens or B. thermophilus. Other suitable amino acid dehydrogenases may be selected from the group consisting of Bacillus bacillus, Brevibacterium species, Corynebacterium glutamicum, or 曰l variants; diamine pimelic acid dehydrogenase a group consisting of a group consisting of amylin dehydrogenase (ec 1.4.1.3) 5 derived from ADP1 or A. platensis with NADH or NADPH as cofactors a group consisting of a face acid dehydrogenase (EC Shan. 4) derived from the typhoid bacillus as a cofactor; a face acid dehydrogenase derived from Saccharomyces cerevisiae or Brevibacterium flavum (EC 141) 4) a group consisting of, or a group consisting of leucine degassing enzymes derived from Bacillus cereus or Bacillus subtilis. 1 In a specific embodiment, by serial ID 2, sequence ID 5 , a sequence ID 8, a sequence ID illusion, a sequence ID π, a sequence of or a homologue of any such sequence, the amino acid sequence-like biocatalyst Conversion of 5-FVA to 6-ACA. In a specific implementation, ΑΚΡ, χ 方 转 转 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由 经由The formation of 2__acids can be effectively chemically de-neutralized into a corresponding method, for example, based on the method described in the tetrahedral letter 1982, 23(4) 459-462. The intermediate terminal dilute amine is subsequently hydrolyzed. Become the corresponding road. The enazylation reaction is carried out in the presence of a transaminase, or by enzymatic reductive amination via a 20-amino acid deaminase or other biocatalyst that catalyzes such conversion, 5-cis followed by biocatalytic conversion to 6aca . Such a transaminase or amino acid dehydrogenase may be particularly selected from the above-described biocatalysts described when converting 5_fva to 6-ACA. Alternatively, the 5-hidden conversion to 6-ACA can be carried out by chemical methods such as the previously described method of 19 201033369. In a particular embodiment, the process is biocatalyzed to AAP in the presence of (1) a transaminase 'amino acid dehydrogenase, or another biocatalyst that catalyzes such conversion, and (9) an amine-based donor. Such a transaminase which converts Ακρ 5 into oxime according to the present invention (4) is particularly selected from the group consisting of the transaminase described above, and is more particularly selected from the group consisting of aspartic acid from pig heart. Transaminase; ketoadipate: glutamate transaminase from rhesus red mold or yeast; transaminase from perennial mountain shoots; 4 amine butyrate transaminase from E. coli ; α-amine adipic acid transaminase derived from the thermophilic thermophilic bacteria; transaminase obtained from the North # 〇10 hornhorn fern or the unilateral iron horn fern; and the transamine from the long pod Jiale tree Enzyme. In a preferred embodiment, the transaminase system for converting AKP to AAP is selected from the group consisting of Vibrio, Pseudomonas, Bacillus, Legionella, nitrite, and nitrite. A group consisting of a transaminase of the genus, Neisseria, Rhodobacter, 15 genus Escherichia, and Rhodopseudomonas. It is particularly found that a transaminase derived from an organism selected from the group consisting of the following is suitable for catalyzing the conversion of AKP to AAP: Bacillus subtilis, Rhodobacter sphaeroides, Legionella vulgaris, European nitrite bacteria , Neisseria gonorrhoeae, Pseudomonas syringae, RhodoPseudomonas palustris, Vibrio fluvialis, Escherichia coli and Pseudomonas aeruginosa. ^ In a specific embodiment, 'in order to convert AKP to AAP, use the root sequence data ID 2, sequence ID 8, sequence ID 12, sequence ID 15, sequence ID 17, sequence ID 19, sequence ID 2 sequence ID 23 , Sequence ID 25, Sequence ID 27, 20 201033369 Sequence ID 2 9 or a transaminase of the amino acid sequence of a homologue of any of these sequences. In yet another embodiment, the method for preparing AAP comprises an enzyme capable of catalytically reductive amination in the presence of Dunyuan selected from an oxidoreductase (EC M) acting on a 5 CH-NH 2 group of a donor. The group is specifically subjected to a biocatalytic reaction in the presence of an enzyme selected from the group consisting of amino acid dehydrogenases (EC 1.4.1). In general, an appropriate amino acid dehydrogenase has alpha-amine pimelic acid 2 dehydrogenase activity which catalyzes the conversion of hydrazine to hydrazine. Particularly suitable amino acid dehydrogenases may be selected from the group consisting of diamine pimelic acid dechlorinase 10 (EC 1.4.1.16), facial acid dehydrogenase (EC 1.4.1 3; EC J 4] 4) and white A group consisting of amino acid dehydrogenase (EC 1.4.1.9). Dehydrogenation of amino acid is selected from the group consisting of a faceted acid dehydrogenase (ec 1.4.1.3) that functions as a receptor with NAD and NADP, and a bran that acts as a receptor with NADP. Amino acid dehydrogenase (跎15 Μ丄4), leucine dehydrogenase (EC1A1.9), and diamine pimelate deaerator (EC 1.4.1.16) amino acid dehydrogenase. The amino acid dehydrogenase is particularly derived from an organism selected from the group consisting of: Corynebacterium, particularly Corynebacterium koegii; Proteus, particularly Proteus; Agrobacterium, especially nodule Agrobacterium; 2 genus of Bacillus licheniformis; especially Acinetobacter spp. ADP1; A. genus, especially of the genus The genus Saccharomyces is particularly Saccharomyces cerevisiae; the genus Brevibacterium is in particular B. brevis; and the genus Bacillus is in particular Bacillus licheniformis, Bacillus cereus and Bacillus licheniformis. 21 201033369 For example, a suitable amino acid dehydrogenase may be selected from diamine pimelic acid dehydrogenase derived from Bacillus, particularly Bacillus licheniformis; diamine pimelic acid, obtained from Brevibacterium; Obtained from the rod (four) two-filament read dechlorination, especially the diamine pimelic acid deaerator obtained from Corynebacterium faecalis; obtained from the diamine pimelate dehydrogenase of the genus 5 bacillus, especially A diamine pimelate dehydrogenase from the common Proteus; a face acid dehydrogenase (Ec 141 3) derived from Acinetobacter with NADH or NADpH as a cofactor, especially from Acinetobacter species ATP1 glutamate dehydrogenase; a sulphate dehydrogenase from the genus Rasta (EC 1.4.1.3), especially dehydrogenated by glutamic acid from the genus 1 Enzyme; glutamate dehydrogenase derived from Salmonella with NADPH as a cofactor (EC ^ haiku, especially glutamate dehydrogenase from Salmonella typhi; dehydrogenation of amygdalin from Saccharomyces) Enzyme (EC 1.4.1.4), in particular glutamate dehydrogenase from Saccharomyces cerevisiae; glutamate dehydrogenase (EC 1.4.1.4) from Brevibacterium 15 glutamate dehydrogenase from Brevibacterium flavum; and leucine dehydrogenase from Bacillus, especially leucine dehydrogenase from Bacillus cereus or Bacillus subtilis. The base acid dehydrogenase may be selected from the group consisting of a lysine 6-dehydrogenase derived from Agrobacterium tumefaciens or B. thermophilus; or an amine obtained from Bacillus cereus or Bacillus 20 groups consisting of acid dehydrogenase. The AAP prepared by the method of the invention can be further used for the preparation of 6-ACA. The inventors have realized that AAP made of AKP can be converted into 6- by carboxylation reaction. ACA. This reaction can be carried out chemically, for example by heating in a high boiling solvent in the presence of a ketone or a catalyst. For example, amino acid 22 201033369 is used in good yield in cyclohexanol at 150-16 (TC). 1-2 ν/ν% cyclohexanone decarboxylation, as described by M. Hashimoto, Y. Eda, Y. Osanai, T. Iwai and S. Aoki, Chem. Lett. 1986, 893-896. Described in Eiso. Pat. Appl. 1586553 by Daiso, and SD Brandt, D. Mansell, S. 5 Freeman, IA Fleet, JF Alder J. Pharm. Biomed. Anal. 2006, 41, 872-882. In addition, AAP de-synthesis into 6-ACA can be carried out in a biocatalytic manner in the presence of de-enzymes or other biocatalysts that catalyze such decarboxylation. The decarbase can be selected from a detoxification enzyme that catalyzes the de-catalyzed oc-amino acid. 10 catalyzed (X-amino acid enzymes may be selected in particular from the group consisting of de-reducing enzymes (ec 4.1.1), preferably selected from pyruvate decarboxylase (EC 4.1.1.1), Amine pimelic acid decarboxylase (EC 4.1.1.20), diamine pimelic acid decarboxylase (EC 4.1.1.20), branched chain alpha-keto acid decarboxylase (EC 4.1.1.72) which includes ex-ketoisoform Group 15 consisting of valerate decarboxylase and a-ketoglutarate decarboxylase (EC 4.1.1.71). One or more other suitable decarboxylases may be selected in particular from the group consisting of oxalic acid Decarboxylase (EC 4.1.1.2), oxalic acid decarboxylase (EC 4.1.1.3), acetamidine decarboxylase (EC 4.1.1.4), aspartate 1-decarboxylase (EC 4.1.1.11) , valine decarboxylase / leucine decarboxylase (EC 4.1.1.14), indoleamine 2 decanoate decarboxylase (EC 4.1.1.15), 3-hydroxyglutamate decarboxylase (EC 4.1.1.16 ), amphoteric acid decarboxylase (EC 4.1.1.17), lysine decarboxylase (EC 4.1.1.18), arginine decarboxylase (EC 4.1.1.19), 2-ketoglutarate decarboxylase (EC 4.1.1.71), and diamine butyrate decarboxylase (EC 4.1.1.86). Decarboxylase is especially selected from the following groups of organisms 23 201033369 _.Pumpkin' For example, pumpkin; courgette; yeast; fungi such as Saccharomyces cerevisiae (four) Candida, Hansenula species, Kluyveromyces marxianus, C. serrata, and Rhodopseudomonas fuliginea; mammals are particularly derived from mammalian brain and bacteria a group consisting of Escherichia coli, Lactococcus lactis, Mycobacterium tuberculosis, 5 strains, and Zymomonas active. Pyruvate de-spray is derived from yeast or Z. mobilis. Pyruvate de-mutation I47jA from Zymomonas mobilis. In particular, it can be obtained from Pseudomonas vaginal acetic acid. A face acid ribozyme, diamine derived from E. coli (E eQli) can be used. Gimelic acid money φ 1 〇 enzyme or aspartic acid to _, or from the red sugar of red sugar, M. leprae, Clostridium perfringens, Lactobacillus brevis, Mycobacterium tuberculosis, Streptococcus or lactobacillus Examples of the genus Lactococcus species that obtain a face acid de-sustaining enzyme from a sputum · · · · · · 诸如 诸如 BU BU BU 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 57 IF IF IF IF IF Formerly known as lactic acid chain 15 cocci variant malt gene) The diamine pimelic acid dewaxing enzyme can be obtained, for example, from an amino acid which can be synthesized from diamine pimelic acid. The organic system is derived from bacteria, archaea and plants. Special diamine pimelic acid decarboxylase can be obtained. From Gram-negative bacteria such as Escherichia coli. The branching bond derived from Lactobacillus lactis can be used to deactivate the enzyme with keto acid. More specifically, the branched chain derived from Lactobacillus lactis can be used as the steel 20 acid decarboxylase and Ketoisovalerate decarboxylase. In particular, alpha-ketoglutarate decarboxylase from Mycobacterium tuberculosis can be used. The inventors have found that alpha ketoglutarate decarboxylase (Kgd) from Mycobacterium tuberculosis can be used to convert AAP to 6-ACA. In particular, the inventors have found that such a decarboxylase comprising a sequence other than sequence ID 46 or a functional analog thereof catalyzes the formation of 6-ACA from aap 24 201033369. The face amine decarboxylation enzyme is particularly selected from the group consisting of pumpkin, courgette, yeast, or calf brain; and diamine pimelic acid decarboxylase (EC 4.1.1.20). The monoamine pimelic acid destabilizing enzyme can be obtained, for example, from an organic acid which can be synthesized from diamine pimelic acid. Such organisms are particularly derived from bacteria, archaea and plants. Particularly diamine pimelic acid decarboxylase can be obtained from Gram-negative bacteria such as Escherichia coli. In a particular embodiment, the AKP is chemically converted to AAP. AAP can be prepared from 2-keto pimelic acid by catalyzing the Leuckart-Wallach reaction as described for the analogous compound. This reaction was carried out using citric acid in methanol and [RhCp*Cl2]2 as a homogeneous catalyst (M Kitamura, D. Lee, S. Hayashi, S. Tanaka, M. Yoshimura J. Org. Chem. 2002, 67, 8685). -8687). In addition, the Rukat-Wara reaction can be used as aqueous catalyst for aqueous ammonium formate [Ir Cp*(bpy)H2〇]S〇4 as a catalyst, such as s. 〇go, κ· Uehara and 15 S. Fukuzumi at J. Am. Chem. Soc. 2004, 126, 3020-3021

get on. Conversion of an oc-keto acid to an (enantiomerically enriched) amino acid by reaction with a (preferable) benzylamine followed by hydrogenation of the intermediate imine with Pd/C or pd(〇H) 2/C It is also possible to achieve. See for example R.G. Hiskey R C N〇rthr〇pJ

Am. Chem. Soc. 1961, 83, 4798. 20 AAP is then biocatalytically converted to 6 ACA in the presence of decarboxylase or other biocatalyst that can undergo such decarboxylation. Such a decarboxylase may be particularly selected from the above-described biocatalysts when describing a biocatalyst for converting AAp to 6_ACA. Alternatively, the conversion of AAP to 6-ACA can be carried out by chemical methods such as the aforementioned 25 201033369 method. In a particular embodiment, AKP is converted to 5_FVA in a biocatalytical manner in the presence of a decarboxylase or other biocatalyst that catalyzes such conversion; and subsequently catalyzed by a transaminase, an amino acid dehydrogenase, or other catalyzed In the presence of such a converted raw material catalyst, 5_FVA was converted to 6_ACA. The decarboxylase suitable for use in such a reaction is particularly selected from the group consisting of the decarboxylase described above for the AKP biocatalysis to 5 FVA. Suitable transaminase or amino acid dehydrogenase for the conversion of 5_FVA can be selected, inter alia, from the biocatalyst described above for the biocatalytic conversion of 5 Å to 8 ACA. · 10 In certain embodiments, in the presence of a transaminase, an amino acid dehydrogenase, or other biocatalyst that catalyzes such conversion, AKP is biocatalyzed to AAP; and subsequently to decarboxylase or other In the presence of a biocatalyst that catalyzes such conversion, AAP is converted to 6-ACA. Enzymes suitable for use in such reactions are selected, inter alia, from the transaminase, amino acid dehydrogenase described above when describing the biotransformation of AKp 15 to AAP and the biotransformation of AAP to 6-ACA. A group consisting of carboxylases. The AKP used to prepare 6-ACA can in principle be obtained in any manner. ® AKP can be obtained, for example, according to the method described by H. Jager et al., Chem. Ber. 1959, 92, 2492-2499. AKP can be prepared by alkylating cyclopentanone with diethyl oxalate as a base, diethyl oxalate, refluxing the resulting product in a strong acid (2M hydrochloric acid) and recovering the product, for example, by crystallization of toluene, to prepare AKP. . It can also be derived from natural sources such as methane-producing archaea, from the northern iron horn, or from the AKP of Hydnocarpusant helminthica. The AKP can, for example, be extracted from such organisms or parts thereof such as the deworming windy species 26 201033369. Suitable extraction methods are based, for example, on VIII.1.\^like 11611 and eight1.66

The method described in Acta Chemica Scandinavica 1954, 6, 1085-1086, which describes the extraction of amino acids and AKP from the fern, using 60% ethanol. In a specific embodiment, the AKP is prepared by a method comprising converting alpha-5 ketoglutarate (AKG) to a-ketoadipate (AKA) and converting a-ketoadipate to ct-ketone acid. This reaction can be catalyzed by a biocatalyst. Akg can be prepared, for example, in a biocatalytic manner from a carbon source such as a carbohydrate, in a manner known in the art. Suitable catalysts for the preparation of AKP from AKG may in particular be selected from the group consisting of catalyzing the elongation of 10 α-ketoglutaric acid to α-ketoadipate and/or the extension of α-keto adipic acid to α-keto Biocatalyst of pimelic acid. In a particular embodiment, the preparation of hydrazine is catalyzed by a biocatalyst comprising a. AksA enzyme or a homolog thereof;

15b. at least one enzyme selected from the group consisting of AksD enzyme, AksE enzyme, AksD enzyme homologue, and AksE enzyme homolog; and c. AksF enzyme or a homolog thereof.

One or more of AksA' AksD, AksE' AksF enzyme or a homolog thereof may be obtained from an organism selected from the group consisting of methane-producing archaea, and 20 is preferably selected from tropococcus, methane. A group consisting of Warmococcus, Octane bacillus, Methanothermobacter, Artemisia, A. sinensis, and Brevibacterium brevis. In a particular embodiment, the biocatalyst for catalyzing the preparation of AKP from 〇c-ketoglutarate (AKG) comprises a catalyzed (transformation of X-ketoglutarate to ketone adipic acid conversion 27 201033369, wherein the enzyme system Forming a portion of the alpha-amine adipic acid pathway for the biosynthesis of amino acids. The term "enzyme" is used herein specifically to mean a single enzyme or group of enzymes that catalyze a particular transformation. Preparation of ΑΚΡ from AKG Including one or more than 5 biocatalytic reactions using known or unknown intermediates, such as AKG, which are converted to hydrazine or hydrazine into hydrazine. These systems may be present inside or separated from the cells. The enzyme system is particularly selected from the group consisting of yeast a group consisting of fungi, archaea and bacteria, in particular from a group consisting of Penicillium, Cephalosporium, Paelicomyces, and Bacillus 10 (Trichophytum), Genus, Phanerochaete, Emericella, Ustilago, Schizosaccharomyces, Saccharomyces, Candida, Jarrow Syzygium (Yarrowi a), Pichia, Kluyveromyces, Thermus, Dinoflagellates, Pyrococcus, Sulfolobus, Thermococcus, A genus of genus, a genus of genus, a bacterium of the genus, a bacterium of the genus, a bacterium, a bacterium, a bacterium, a bacterium, a bacterium, a bacterium, a bacterium, a bacterium, a bacterium, a bacterium, a bacterium, a bacterium, a bacterium, a bacterium, a bacterium, a bacterium, a genus The biocatalyst for preparing AKP from glutaric acid comprises an enzyme 20 system for catalyzing the conversion of α-ketoglutaric acid to α-ketoadipate, wherein at least one of the enzymes in the enzyme system is derived from A nitrogen-fixing bacterium of the group consisting of cyanobacteria, rhizobium, γ-proteobacteria and actinobacillus, in particular selected from the group consisting of Anabaena, Microcystis, Synechocystis, Rhizobium, Bradyrhizobium, Pseudomonas, 201033369 Azotobacter, Klebsiella, and Frankia. Examples of homologs of Aks enzymes and genes encoding such enzymes are listed below Table 1A and Table 1B of the pages.

29 201033369

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Protein ΝΡ_247479 NP_276742 ΝΡ_987273 YP_001098033 YP_001330370 YP_447259 NP_614492 YP_001273295 YP_001323668 YP—001325184 NP_247997 NP-276502 ΝΡ_988600 Ο cn Os Ο ο ο 1 CU YP_001329942 YP448499 NP_614723 〇\ CN 〇〇1 0. YP_001323307 Art τ—Η Ο 1 cu Gene MJ0503 MTH1630 ΜΜΡ0153 MmarC5_1522 MmarC7 - 1153 Msp_0199 MK1209 Msm - 0722 Mevan - 1158 Maeo_0994 MJ1003 MTH1386 Mmpl480 MmarC5_0098 MmarC7_0724 Msp_1486 MK1440 Msm_0723 Mevan_0789 Maeo_0311 Organism 曱 暖 暖 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷 甲烷AV19 Brevibacterium brevis ATCC35061 Cyclohexane SB Cyclohexane 3 Methane thermococci temperate ΔΗ Methanococcus S2 Cyclohexane C5 Methanococcus C7 Methanococcus DSM 3091 Methane archaea AV19 Brevibacterium bacillus ATCC35061 Sterolococcus SB Methanococcus 3 Invited W AksA AksD (Rose 3 > 01 inch ^ 8003) VAO&a.qcl.scl.iqou.MMM Life Girl.t 铋Φ 咖女回硪30 201033369

Hi Protein NP—248267 NP_276503 NP_987501 〇\ 〇\ 〇〇〇1 Dh ON ο cn CO Ο ο 1 CL, ΗYP448498 NP_614065 YP_001273420 YP—001323877 YP_001324848 NP—248605 NP—275327 ΝΡ988000 ΥΡ001097214 ON inch m On CS cn ο ο 1 >Η γρ_447715 NP_614066 YP001272946 YP.001322567 YP-001325672 Gene MJ1271 MTH1387 MMP0381 MmarC5_1257 MmarC7_1379 Msp_1485 MK0781 Msm_0847 Mevan-1368 Maeo_0652 MJ1596 MTH184 ΜΜΡ0880 MmarC5_0688 MmarC7_0128 Msp_0674 MK0782 Msm A0373 Mevan_0040 Maeo_1484 Organism Cyclohexane thermococcal MH Methanococcus S2 Methane Coccus C5 Cyclohexane C7 Cyclohexane bacterium DSM 3091 Methane archaea AV19 Brevibacterium brevis ATCC35061 Methanococcus SB Cyclohexane 3 Methane thermococci Thermotoxin ΔΗ Methanococcus S2 Methanococcus C5 Methanococcus C7 Methanococcus DSM 3091 AV19 Brevibacterium brevis ATCC35061 Cyclohexane SB Cyclohexane 3 Training spectrum AksE AksF (attack Β ΠΠΙ: inch Zhu 00 ΟΟΖ;) VAOba.xlfi-UItlnqoU.MMM%^b 铋-ffi coffee βθ 砩31 201033369 The 6_ACA obtained according to the present invention may be cyclized to form caprolactam as described in still-A-6,194,572. The reaction conditions for any of the biocatalytic steps in the context of the present invention can be selected based on the biocatalyst, particularly the known conditions of the enzyme, the information disclosed herein, and optionally a number of routine experiments. In principle, what is the reaction medium used? 11 can be selected from a wide range, as long as the biocatalyst is active under pH conditions. Depending on the biocatalyst and other factors, the test conditions, neutral conditions and acidic conditions can be used. In the case where the method comprises a microorganism, for example, for expressing an enzyme which catalyzes the method of the present invention, 1) the selection of the line 11 allows the microorganism to perform its desired function. In the case where 25 C is mainly an aqueous system, {11) may be selected, in particular, from a range of four pH units below neutral pH to two pH units above neutral pH, that is, selected from pH 3 to pH 9. . If water is the sole solvent or main solvent (> 5 〇 wt %, especially > 90 wt. %, based on total liquid), the system is considered to be aqueous, 5 of which is a small amount of alcohol or other solvent (<5〇) The wt.%, particularly <i〇wt.%, based on the total liquid, may be dissolved (e.g., as a carbon source) at a concentration that maintains the presence of microbial activity. Especially in the case of using yeast and/or fungi, based on 25. The aqueous system is preferably an acidic condition, particularly pH ranging from pH 3 to pH 8. If desired, the pH can be adjusted using an acid and/or base or using an appropriate combination of acid and base. In principle, the culture conditions can be selected in a wide range as long as the biocatalyst exhibits sufficient activity and/or growth. This includes aerobic, microaerobic, oxygen-limited, and anaerobic conditions. The anaerobic conditions are defined herein as not containing any oxygen or substantially without any 32 201033369 Oxygen is biologically (four) secret H _ is a microorganism, usually the phase is depleted in oxygen consumption, low m mole / liter · hour, Low consumption: 2.5 millimoles per liter. hours or less than 丨 millimoles per liter hour. 5 10 15 20 Aerobic conditions in which the high oxygen concentration for unrestricted growth dissolves in the medium and supports at least 10 millimoles per liter. hour, more preferably greater than 2 millimoles per liter hour and more preferably greater than 50 millimoles per liter. hour, and optimally greater than the oxygen consumption rate of just millimoles per liter per hour. Oxygen limiting conditions are defined as conditions under which oxygen consumption is limited by the conversion of oxygen from gas to liquid. The lower limit of the oxygen limiting condition is determined by the upper limit of the anaerobic condition, in other words, usually at least i millimoles per liter hour, and particularly at least 2 ng m/l hr or at least 5 mM hr. The upper limit of the oxygen limiting condition is determined by the lower limit of the aerobic condition, that is, less than 1 〇〇 millimol/liter hour, less than 5 〇 millimol/liter. hour, and less than 2 〇 millimol/L. , or less than (1) millimoles per liter hour. The conditions are aerobic, anaerobic or oxygen-free, depending on the conditions under which the process is carried out, in particular by the amount and composition of the influent gas stream, the actual mixing/mass transfer properties of the equipment used, and the microbial and microbial density of the money Decide. The temperature used in principle is not particularly limited as long as the biocatalyst exhibits substantial activity especially for the enzyme. The temperature is substantially at least, in particular at least 15 ° C, more particularly at least 2 (TC ^ the desired maximum temperature is determined according to the biocatalyst. Usually such maximum temperatures are known to the art, for example commercially available biocatalysts The case is indicated on the product information sheet or can be routinely determined based on common knowledge and information disclosed herein. Temperature 33 201033369 is usually 90 ° C or below, preferably 7 ° C or below, especially 5 (rc or below 'more specifically 40. (: or below. Especially if the biocatalytic reaction is carried out outside the host organism, a high concentration can be used when using an enzyme that retains sufficient activity in such a medium (Example 5) A reaction medium comprising an organic solvent, such as greater than 50 wt.% or greater than 90 wt.%. In a preferred method, whole cell biotransformation using a 6-ACA enzyme matrix or intermediate for forming 6-ACA (AKP) , AAP or 5-FVA) Preparation of 6-ACA 'comprising a microorganism in which one or more biocatalyst 10 catalysts (typically one or more enzymes) capable of catalyzing bioconversion are produced, such as selected from the group consisting of One or more Biocatalyst: a biocatalyst that can catalyze the conversion of Ακρ to AAP, a biocatalyst that catalyzes the conversion of aaP to 6-ACA, a biocatalyst that can catalyze the conversion of AKP to 5-FVA, and a biocatalyst that can catalyze the conversion of 5_fva to 6-ACA. In a preferred embodiment, the microorganism can produce 15 dehydronase and/or at least one enzyme selected from the group consisting of amino acid dehydrogenase and transaminase; catalyzing the aforementioned reaction step, and carbon source for microorganisms The carbon source particularly contains at least one selected from the group consisting of a monohydric alcohol, a polyhydric alcohol, a decanoic acid, a dioxetane carbon, a fatty acid, a glyceride, and a mixture comprising any of the compounds. Suitable monohydric alcohols include methanol and ethanol. Suitable polyols include glycerol and carbohydrates. Suitable fatty acids or glycerides are especially provided in the form of edible oils, preferably in plant form. · In particular, water-breaking compounds can be used because of the usual carbon water The compound is obtained in a large amount from a biorenewable source, such as an agricultural product, preferably agricultural waste 34 201033369. Preferably, it is selected from the group consisting of glucose, fructose, and Yan. , lactose, and vegetable sugar; knowledge: carbohydrates of a group consisting of cellulose and hemicellulose. Particularly preferred are glucose, glucose-containing oligosaccharides, and polysaccharides containing glucose. Containing species or a plurality of methods for use in the present invention Catalytic reaction step t biological 5 catalyst (usually one or more enzymes) of cells, particularly recombinant cells, < using molecular biotechnology known in the art. For example, if a recombinant cell (which may be a non-homogeneous system) is produced Alternatively to a plurality of biocatalysts, such a technique 10 can be used to provide a vector (such as a recombinant vector) comprising one or more genes encoding one or more biocatalysts. One or more vectors each comprising one or more genes can be used. Such vectors comprise one or more regulatory elements, such as one or more promoter genes, that are linked to the gene encoding the biocatalyst in a working manner. The term "working bond" as used herein refers to a multi-nucleotide element (or coding sequence or nucleic acid sequence) that is functionally linked. A nucleic acid sequence is a "workable link" when placed in a functional relationship with another 15 nucleic acid sequences. For example, if the promoter gene or the booster gene affects the transcription of the coding sequence, a working bond is ligated to the coding sequence. As used herein, the term "initiating gene" refers to a nucleic acid fragment that functions to control the transcription of one or more genes, upstream of the transcriptional direction relative to the start of transcription of the gene, and is employed on the structure. Recognition of the binding site of the polymerase-like polymerase, the location of transcription, and the presence of any other dna sequence, including but not limited to transcription factor binding sites, repressor genes, and activation gene protein binding sites, and skilled artisans It is known to act directly or indirectly on any other nuclear (four) sequence that modulates the amount of transcription from the promoter gene 35 201033369. A "constitutive" promoter gene is a promoter that is active under most environmental conditions and developmental conditions. An "inducible" promoter is a promoter that is active under environmental regulation or developmental regulation. "homologous" - the word (d) is used to indicate that a given (recombinant) nucleic acid or a multi-peptide molecule is associated with a given host organism or host cell, and that the nucleic acid or multi-peptide peptide is expressed in nature. Made from host cells or host organisms of the same species and preferably of the same variety or of the same germline. A promoter gene that can be used to achieve the expression of a nucleic acid sequence encoding an enzyme for use in the methods of the invention, particularly a transaminase, amino acid dehydrogenase or holase, as described above, may be encoded for expression The non-homologous (four) factor of the nucleic acid sequence of the enzyme, or the nucleic acid sequence (coding sequence) of its working linkage. Preferably, the promoter gene is the homology of the host cell, that is, the host cell: · endogenous. If a non-homologous promoter gene is used (relative to the nucleotide sequence of the enzyme of interest, the 15-acid sequence is non-homologous), then the non-homologous promoter gene preferably compares the original promoter gene of the editing sequence to produce more A highly stable concentration comprises a coding sequence t transcript (or more transcript molecules per unit time, i.e., mRNA_molecules). Such promoter genes in this context include constitutive natural promoter genes and inducible natural promoter genes as well as genetically engineered promoter genes, which are well known to those skilled in the art. ... "Strong constitutive promoter" is a promoter that causes mRNA to start at a higher frequency than the native host cell. Examples of such potent constitutive promoter genes in Gram-positive microorganisms include sp〇126, sp〇i7, veg, pyc (pyruvate carboxylase promoter gene), &amyE. 36 201033369 Examples of inducible promoter genes in Gram-positive microorganisms include the IPTG-inducible Pspac promoter gene, the xylose-inducible PxylA promoter gene. Examples of constitutive promoter genes and inducible promoter genes in Gram-negative microorganisms include, but are not limited to, tac, tet, trp-tet, lpp, lac, lpp-lac, 5 laclq, T7, T5, T3, gal, trc , ara (PBAd), SP6, λ-PR, and λ-PL. The promoter gene of (filamentous) fungal cells is known in the art and may be, for example, a glucose-6-phosphate dehydrogenase gpdA promoter gene, a protease promoter gene such as pepA, pepB, pepC, a glucose phosphatase glaA promoter gene, starch. Enzyme 10 amyA, amyB promoter gene, catalytic enzyme catR or catA promoter gene, glucose oxidase goxC promoter gene, β-galactosidase lacA promoter gene, α-glucosidase aglA promoter gene, translation elongation factor tefA promoter gene, wood Glycanase promoter genes such as xlnA, xlnB, xlnC, xlnD, cellulase promoter genes such as eglA, eglB, cbhA, promoters of transcriptional regulatory genes such as areA, creA, xlnR, pacC, prtT, or other promoter genes, For reference to the NCBI website (httD://www.ncbi.nlm.nih.gov/emre7A), when used in relation to nucleic acids (DNA or RNA) or proteins, the term "homologous" refers to the absence of naturally occurring organisms, cells, genes. A nucleic acid or protein that is part of a DNA or RNA sequence present; or a cell or genome present in a cell or 20 that appears in a different location or DNA or RNA sequence than the natural one Nucleic acid or protein. A non-homologous nucleic acid or protein is not endogenous to the cell into which the nucleic acid or protein is introduced, but is derived from other cells or produced synthetically or recombinantly. Generally, but not necessarily, such nucleic acid is encoded by A protein that is not naturally produced by cells transcribed or expressed by DNA. Similar to 37 201033369 Extracellular RΝΑ encodes a protein in which the exogenous RNA is not naturally expressed. Non-homologous nucleic acids and proteins are also referred to as foreign nucleic acids or Proteins. Those skilled in the art understand that any nucleic acid or protein that is recognized as non-homologous or foreign to a expressing cell is encompassed herein by a term that is not a homologous nucleic acid 5 or protein. The method according to the invention can be carried out in a host organism. The host organism can be novel.

Thus, the invention also relates to a host cell comprising one or more biocatalysts which catalyzes at least one of the 10 reaction steps of the method of the invention 'particularly catalyzing the conversion of AKP, AAP or 5-FVA to 6-ACA At least one of the reaction steps. The invention also relates to novel vectors comprising one or more genes encoding one or more enzymes which catalyze at least one of the reaction steps of the method of the invention 'particularly catalyzing the conversion of Ακρ to 6-ACA at least- Reaction step; and the present invention relates to a novel host cell comprising a gene encoding one or more genes of one or more enzymes, the enzyme catalyzing at least one reaction step in the method of the invention, particularly for converting hydrazine into At least one reaction step in the transformation of 6-ACA (the one of the plurality of genes may constitute part of one or more vectors). In a particular embodiment, the host cell according to the invention is a recombinant cell comprising a nucleic acid sequence encoding a 20-symbol biocatalyst which catalyzes a transamination or reductive amination reaction to form a cardinalpimelate. Amine pimelic acid. Such a sequence may be part of a vector, or may be inserted: dyed:: DNA. In particular, the host cell or vector according to the invention comprises at least one nucleic acid sequence selected from the group consisting of the following 38 201033369 column, in particular at least two nucleic acid sequences encoding a ketone pimelic acid decarboxylase activity a nucleic acid sequence of the enzyme, a nucleic acid sequence encoding an enzyme having 5-methylvalerate transaminase activity, a nucleic acid sequence encoding an enzyme having 〇t--pimelate transaminase activity, and encoding having 5 α-keto a nucleic acid sequence of an enzyme having diacid dehydrogenase activity, and a nucleic acid sequence encoding an enzyme having (X-amine pimelic acid decarboxylase activity. Typically, one or more of such sequences are particularly two or more Is a recombinant sequence. In a preferred embodiment, the host cell according to the invention is typically a recombinant host cell or vector comprising a nucleic acid sequence encoding a biocatalyst having less than 10 X-ketopimelate dehydroampase activity, and Or alternatively selected from at least one nucleic acid sequence encoding a biocatalyst sequence having 5-mercaptopurine transaminase activity. 15 20 In such an embodiment, the 'encoded with OC-ketopimelate dehydrogenase activity Nucleic acid sequence of enzyme Specifically comprising an amino acid sequence according to sequence ID 31, sequence ID 34, sequence m 37, sequence id 40, sequence ID 43 or sequence ID 46 or a homologue of any such sequence; and/or having a 5-methylidene The nucleic acid sequence of the acid transaminase activity enzyme comprises, in particular, according to the sequence 10 2, the sequence 1〇5 sequence ID 8, the sequence ID 65, the sequence ID 67, the sequence id 69 or its homolog acid sequence. One or more nucleic acid sequences In a further preferred embodiment, the vector or host cell comprises a nucleic acid sequence encoding a pimelic acid transaminase activity and/or a coding having = 0 acid decarboxylase An active nucleic acid sequence encoding a nucleic acid sequence having alpha steel, Gem II, and transamination, according to sequence ID 2, sequence m

8 'Sequence ID 39 201033369 12, Sequence ID 15, Sequence ID 17, Sequence π) 19. Sequence 1D 21, Sequence ID 23, Sequence ID 25, Sequence ID 27, Sequence ϊ ρ 29 is the amino acid sequence of its homologue. Wherein the nucleic acid sequence or nucleic acid sequences are part of one or more of the recombinant vectors. 5 10 15 20 In a particularly preferred embodiment, the host cell according to the invention comprises an amino acid sequence encoding a-amine pimelic acid 2-dehydrogenase activity and encoding an amine pimelic acid decarboxylase activity. Amine base sequence. In a particularly preferred embodiment, the host cell according to the present invention comprises a nucleic acid sequence encoding an enzyme having 6-amine adipic acid 6-dehydrogenase activity and an enzyme encoding an enzyme having 〇[-epimedenate decarboxylase activity Nucleic acid sequence. One or more suitable genes of the host cell or vector according to the invention are in particular selected from the genes encoding the enzymes described above. In a particular embodiment, the host cell is comprised of a group selected from the group consisting of

Recombinant cells of at least one nucleic acid sequence in the group: sequence ID 1 sequence ID 3, sequence ID 4, sequence ID 6, sequence ID 7, sequence still u, sequence 歹 JID 13, sequence 14, sequence i6, Sequence 18, sequence addition, 歹jID 22, sequence iD 24, sequence 1〇%, sequence 28, sequence 〇, sequence 1〇32, sequence 11^1 wide D 33, sequence ID 35, sequence ID 36, sequence ID 38 , jin column 1〇39, sequence 11) 41 wide, chuanchuan 41, sequence ID 42, sequence ID 44, sequence ID 45, jin column ID 47, sequence τη " + like ^ I, sequence touch 66, preface a sequence recognized by any one of _68 and its functional classes. Encoding a nucleic acid sequence having a 5-FVA nucleus from an enzyme known as the activity of the sequence ID i, 3, 4, particularly selected functional analogues A sequence of 201033369 of groups consisting of #6, 7, 64, 66, 68 and any sequence represented by such a sequencer. As used herein, the term "functional analogue" includes at least the code having the same amine. Other sequences of the enzymes of the base acid sequence and other sequences encoding homologs of such enzymes. 5 a nucleic acid sequence encoding an enzyme having AKP decarboxylase activity, particularly selected from the group consisting of sequence IDs 30, 32, 33, 35, 36, 38, 39, 41, 42, 44, 45, 47 and any such sequences A sequence of a group consisting of sequences represented by any of the functional analogs. In a preferred embodiment, the host cell comprises a nucleic acid sequence encoding an enzyme that catalyzes the conversion of AAp to 10 AKP, which is based on sequence IDs 1, 3, 7, η, 13, 14, 16, 18, 20, 22, 24, 26, 28, or a functional analog thereof and may be a wild-type sequence or a non-wild-type sequence. In a particular embodiment, the host cell comprises at least one nucleic acid sequence encoding a biocatalyst having a sense amine pimelic acid decarboxylase activity, which sequence may be homologous or non-homologous to the injured host cell. In particular, such a biocatalyst may be selected from the group consisting of de-septase (EC 4.1.1), more particularly from the group consisting of glutamate decarboxylase (EC 4.1.1.15). ), diamine pimelic acid decarboxylase (EC 4.1.1.20), aspartate 1-decarboxylase (EC 4.1.1.Π), branched chain (X-keto acid decarboxylase, ketoisovalerate) Carboxylase, alpha-ketoglutarate de-20 carboxylase, pyruvate decarboxylase (EC 4.1.1.1) and oxaloacetate de-negotase (EC 4.1.1.3). In a specific embodiment, the host cell comprises a catalytic AKG (also referred to above) forms one or more enzymes of AKP. An enzyme system that forms part of the X-amine adipic acid pathway for the biosynthesis of lysine may be used. The term "enzyme system" is used for 41 201033369 In particular, it refers to a single enzyme or a group of enzymes that catalyze a particular transformation reaction. Such transformations involve one or more chemical reactions with known intermediates or unknown intermediates, such as conversion of AKG to AKA or AKA to AKP. The system may be present inside or isolated from the cell. It is known that transaminase often has a broad enzyme 5 matrix range. If an enzyme matrix is present, it is desirable to reduce one or The activity of such enzymes in host cells reduces the conversion activity of AKA to alpha-amine adipic acid (AAA) while maintaining the catalytic function associated with the biosynthesis of other amino acids or cellular components. Host cells that do not contain any other catalytic activity will result in the conversion of AKA to an undesired by-product. ® Suitable for use in a preferred host cell for the preparation of AAp using whole-cell biotransformation, encoding a catalyzed by 1 ketoglutarate Preparing one or more biocatalysts of at least one of the alpha ketopimelic acid. Suitable biocatalysts are for example discussed in the preparation of hydrazine. The host cell may be selected from bacteria, yeast or fungi. In particular, the host 5 cells may be selected from the group consisting of genus Fusarium, Penicillium, Saccharomyces, Kluyveromyces, Pichia, Candida, Hansenula, Bacillus, Corynebacterium, Fake a group consisting of the genus of the genus such as the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus of the genus group Here, it is generally selected and expressed one or more encoding nucleic acid sequences as described above. In particular, host cells and host cells so suitable for biosynthesis of 6 ACA may be selected from the group consisting of the following host cells. Group: Escherichia coli, Bacillus subtilis, BaciUus amyloliquefaciens, Corynebacterium glutamicum (C〇rynebacterium 42 201033369 5 10 15 20 glutamicum), Aspergillus niger, Penicillium chrysogenum , Saccharomyces cerevisiae, Hansenula polymorpha, Candida albicans, Kluyveromyces lactis, Pichia stipitis, Pichia Pastoris), Methanobacterium thermoautothrophicum AH, Methanococcus maripaludis, Methanococcus voltae, Methanosarcina acetivorans, Pap. Methanosarcina barker and Methalonia serrata (Metha Nosarcina mazei) and other host cells. In a preferred embodiment, the host cell can produce an exogenous acid (in the case of a precursor host cell, in principle, a naturally occurring organism or can be a genetically engineered organism. Such organisms can use mutation screening strategies or metabolism known in the art. The genetic engineering strategy is genetically engineered. In a particular embodiment, the host cell naturally comprises (or can be made) one or more enzymes suitable for use in catalyzing the reaction step in the methods of the invention, such as selected from the methods of catalyzing the invention One or more activities in the group consisting of decarboxylase, transaminase, and amino acid dehydrogenase. For example, E. coli can be naturally produced to catalyze the transamination reaction in the method of the present invention. Enzymes. It is also possible to provide a recombinant host cell carrying two recombinant genes, a recombinant gene encoding a transaminase or amino acid dehydrogenase which catalyzes a reaction step of the method of the invention, and a recombinant gene encoding A decarboxylase gene catalyzing the reaction step of the method of the invention. For example, the host cell may be selected from the group consisting of Corynebacterium, particularly glutamine rods 43 2 01033369 Bacteria and enteric bacteria are especially Escherichia coli, Bacillus, especially Bacillus subtilis and B. methan〇licus, and Saccharomyces cerevisiae. Especially suitable for industrial production. Amino acids of Corynebacterium glutamicum or Bacillus licheniformis. 5 The present invention relates to a microorganism which may be a wild-type microorganism or a recombinant microorganism isolated from the natural environment, comprising, for example, selected from the sequence ID. No. 3, sequence id No. 6, sequence ID No. 13, sequence ID No. 32, sequence ID No. 35, sequence ID No. 41, sequence ID No. 44, sequence ID No. 47, and functional analogs thereof The DNA of the nucleic acid sequence recognized by any one of the sequence ❹ 10 Π). A functional analog of a nucleotide sequence as described herein is particularly such a nucleotide sequence which encodes the same amino acid sequence as the nucleotide sequence or a homologue encoding the nucleotide sequence. In particular, a preferred functional analog is a nucleotide sequence having a similar, identical or better degree of expression than a nucleotide sequence referred to as a functional analog thereof in a host 15 cell of interest. The invention further relates to a polynucleotide or vector comprising: selected from the group consisting of: Sequence ID No. 3, Sequence ID No. 6, Sequence ID No. 13, Sequence ID No. 32, Sequence ID No. 35, Sequence Nucleic acid sequence recognized by any one of 20 of the group consisting of ID No. 41, Sequence ID No. 44, Sequence ID No. 47 and its non-wild type functional analog. Such a polynucleotide or vector is compared. The corresponding wild-type gene can provide host cells more excellently, particularly for E. coli host cells, or at least other transformation processes that can catalyze the conversion of AKP to 6-ACA in high yield. Another host cell for a transformation step. Optionally, the polynucleotide or vector comprises one or more nucleic acid sequences thereof. 44 201033369 encodes one or more other biocatalysts suitable for catalyzing the reaction step in the process according to the invention, particularly in the catalysts described above. One or more. 5 10 15 20 The invention further relates to a process for the preparation of alpha amine pimelic acid (AAp) comprising converting AKP to AAp which is catalyzed by a biocatalyst. For use in such methods, in particular, a biocatalyst having transaminase activity or reductive amination activity as described above can be used. As previously indicated, AAP can then be used for the preparation of 6-ACA. Alternatively, AAP can be used as such, for example, as a chemical for biochemical research or as a pH buffering compound, e.g., for preparative separation techniques or analytical separation techniques such as liquid chromatography or capillary electrophoresis. Further, the AAP prepared by the method of the present invention can be further used for the preparation of other compounds, for example, AAP can be converted to caprolactam. As explained above, based on the exemplification in the following examples, AAP can be converted to caprolactam by chemical means such as exposure to elevated temperatures. Without wishing to be bound by theory, it is contemplated that 6-ACA may form a short-lived intermediate in this reaction. Next, the present invention will be exemplified by the following examples. Example summary method Early and far-reaching technical standards Genetic and molecular biology techniques are generally known to the art world and are described above (Maniatis et al., 1982, "Molecular Selection: Laboratory Manual", Cold Spring Harbor Experiment to 'Cold Springs Port, New York; Miller, 1972, "Molecular Genetics 45 201033369 Experiment", Cold Spring Harbor Laboratory, Cold Spring Harbor; Sambrook and Russell 2001 "Molecular Selection: Laboratory Manual" (3rd Edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; edited by F. Ausubel et al., "Popular Programs in Molecular Biology", Green Press and Willy Technology, New York, 1987). 5 Physique and lineage The pBAD/Myc-His C line was obtained from Invitrogen (Casper, CA). The plastid pBAD/Myc-His-DEST consisting of the composition described in WO2005/068643 is used for protein expression. E. coli TOP10 (Invitrogen, Inc., Casper, CA) was used for all selection procedures and for the performance of target 10 genes. Medium LB medium (10 g/L trypsin gland, 5 g/L yeast extract, 5 g/L NaCl) was used for the growth of E. coli. Supplement the antibiotic (5 〇 microgram / ml card carbenicillin) to maintain the plastid. To induce gene expression in the plastids derived from 15 pBAD/Myc-His-DEST under the control of the pBAD promoter, L-arabinose was added to a final concentration of 0.2% (w/v). Identification of plastids The system of genes carrying different genes is identified by genetics, biochemistry, and/or phenotypic means generally known in the art world, such as resistance of antibiotics to transgenic plants, PCR analysis of transformed plants. Or purification of plastid DNA, restriction analysis of purified plastid DNA or DNA sequence analysis. The HPLC-MS bifurcation method for the determination of 5-FVA was carried out by selective reaction monitoring (SRM)-MS 'measurement change 129 -> 83 and 201033369 to detect 5-FVA. The 5-FVA concentration was calculated by measuring the peak area of the 5-FVA peak eluted in about 6 minutes. Calibrate using an external standard program. All LC-MS experiments were performed on an Agilent 1200 LC system containing a fourth stage pump, autosampler and column oven coupling Aegean 5 6410 QQQ triple quaternary MS. LC conditions: Column: 50 X 4.6 mm Nucleosil C18, 5 micron (Machery & Nagel) pre-column coupled to 250 x 4.6 mm inner diameter Pavel ( Prevail) C18, 5 micron (Alltech). Column temperature: room temperature Eluent: A: Water containing 0.1% formic acid

B: Acetonitrile with 0.1% formic acid Gradient: Time (minutes) % Eluent B 15 20 0 10 6 50 6.1 10 11 10 Flow rate: 1.2 ml/min, the flow was split 1:3 before entering MS. Injection volume: 2 μl MS condition: Ionization: Negative ion electric spraying

Source conditions: ion spray voltage: 5kV Temperature: 350 °C Segmenter voltage and collision energy optimization Scan mode: Select reaction mode: change m/z 129-83 47 201033369 HPLC-MS for determination of AAP AAP was detected by measuring the protonated molecules of AAP with m/z 176 by selected ion monitoring (SIM)-MS. The aap concentration was calculated by measuring the peak area of the AAP peak eluted in the sample at a retention time of 27 minutes. Calibrate via 5 using an external standard procedure. All LC-MS experiments are performed on the Aijilan 1100 LC system coupled with a fourth stage pump, deaerator, autosampler and column oven. API 2000 Triple Quaternary MS (Applied Biosystems (Applied) Biosystems)).

The LC conditions are as follows: 0 Column: 50*4 Newquay Sil C18, 5 micron (McKelly-Nagy) +250x4.6 Puwei C18, 5 micron (Aer Technology) are all at room temperature (RT). Eluent: Α=0·1% (ν/ν) formic acid in ultrapure water Β=〇. 1 % (ν/ν) citric acid in acetonitrile (pa, Merck) 15 Flow rate: 1_2 ml /min, the traffic splits 1:3 before entering MS.

Gradient: The gradient was changed to 50% (v/v) A within 6 minutes starting at 90% (v/v) A at t = 0 minutes. The gradient was changed to the original condition at 6.1 minutes. Injection volume: 2 μL • ° MS conditions: Positive ion electrospray for ionization. Detection: SIM mode is at m/z 176, and the residence time is 1 〇〇 millisecond. ^^1^-ACA HPLr-TU.S Analysis Calibration: Calibration is performed by an external calibration line of 6-ACA (m/z 132-m/z 114, room temperature 7.5 minutes 48 201033369 clocks). All LC-MS experiments were performed on the Aijilan 1100, which was equipped with a fourth-stage pump, deaerator, autosampler, column oven, and single quaternary MS (Aijilan, Germany) Bonn hLC-MS conditions are: Column: 50*4 Newquay (McKelly-Nagy) + 250 X 4.6 Pauville C18 (Aer Technology), all at room temperature (RT). Extractant: Α=0·1% (ν/ν) formic acid in ultrapure water Β = acetonitrile (pa, Merck)

W Flow rate: 1.0 ml/min. The flow rate was split 1:3 before entering MS. 10 Gradient: The gradient starts at 100% (v/v) A at t = 0 minutes and is maintained at 80% (v/v) B (t = 30 minutes) in 15 minutes. ' From 30 to 31 minutes, the gradient is maintained constant at 80% (v/v) B. Injection volume: 5 microliters

MS detection: ESI〇)-MS 15 Electrospray ionization (ESI) is performed in the positive scan mode with the following conditions: m/z 50-500, 50V segmenter, 0.1 m/z step size, 35 (TC) Dry gas temperature, 10 liters of nitrogen/min dry gas, 50 psig nebulizer pressure, and 2.5 capillary voltage. 20 Targeting gene selection of the composition of the composition of the meta-tatB position added to the ribosome binding site and the start codon All genes from the upstream and the male and the lower reaches of the horse are used to assist in the selection of the Gateway technology (Invitrogen, Inc., Casper, California, USA). 49 201033369 Gene synthesis of plaques

Synthetic genes are obtained from DNA2.0 and codons that are optimized for expression in E. coli according to the standard procedure of DNA 2.0. The amino acid sequence encoding the Vibrio fluvialis JS17 ω-transaminase [SEQ ID Νο. 2] and the amino acid sequence of B. vaginalis ΚΒΑΒ4 transaminase (ΖΡ_01186960) [SEQ ID Νο·5] Sequences obtained by codon optimisation of the transaminase gene of Vibrio fluvialis JS17 [SEQ ID No. 1] and B. faecalis KBAB4 [SEQ ID No. 4] [SEQ ID No. 3] and [SEQ ID No. 6 ] obtained by DNA synthesis.

10 Amino acid sequence encoding the Vibrio fluvialis J S17 ω-transaminase [SEQ ID Νο. 3], B. vaginalis ΚΒΑΒ4 transaminase (ΖΡ_01186960) [SEQ ID Νο. 6], Escherichia coli diamine Gem Diacid decarboxylase LysA [SEQ ID Νο. 31], Alcoholic yeast pyruvate dehydrogenase Pdc [SEQ ID No. 34], Z. mobilis pyruvate decarboxylase Pdcl472A [SEQ ID No. 37], lactic acid 15 Lactobacillus branched chain α-keto acid decarboxylase KdcA [SEQ ID No. 40] and oc-ketoisovalerate de-sustaining enzyme KivD [SEQ ID Νο·43], and Mycobacterium tuberculosis 〇t-ketopentane The amino acid sequence of acid decarboxylase Kgd [SEQ ID No. 46] was obtained from Escherichia coli [SEQ ID No. 30], Saccharomyces cerevisiae [SEQ ID No. 33], Z. mobilis [SEQ ID No. 36], lactic acid. The decarboxylase genes of Lactobacillus [SEQ ID No. 39], [SEQ 20 ID Ν ο 42], and Mycobacterium tuberculosis [SEQ ID No. 45] were also codon-optimized, and the resulting sequence [SEQ ID No] .32], [SEQ ID Νο.35], [SEQ ID Νο. 38], [SEQ ID No. 41], [SEQ ID No. 44], and [SEQ ID No. 47] were obtained by DNA synthesis, respectively. . The gene composition, such as the manufacturer's protocol (www.invitrogen.com, stated, 201033369) was imported into the vector through the introduced attB position and pd〇NR201 (Invitrogen), and was selected using Jiawei Technology (Invitrogen). The pBAD/Myc-His-DEST expression vector was obtained, thereby obtaining the expression vectors pBAD-Vfl_AT and pBAD-Bwe_AT, respectively. The phase was obtained by using individual pBAD table 5 to transform the chemical into E. coli TOP10 (Invitrogen). Corresponding performance of the germline. Using PCR to write a variety of genes encoding biocatalysts according to the manufacturer's specifications, using the primers listed in the table below, using PCR 10 SuPermix High Fidelity (Invitrogen Gene Inc. ), amplified by genomic DNA by pCR.

❿ 51 201033369 PCR reaction by agarose gel electrophoresis analysis 'The PCR product with the correct size was eluted by gel using QIAquick PCR purification kit group (Qiagen, Hilden, Germany) . As described in the manufacturer's protocol, the purified PCR product was cloned into pBAD/Myc using the introduced attB position and PDONR-zeo (Invitrogen 5) as the entry vector, using Jiawei Technology (Invitrogen) -His-DEST expression vector. The genetic sequence cloned by PCR was confirmed by DNA sequencing. In this way, the performance carrier is obtained? 8八0436_2丨9946143_八丁, pBAD-Bsu_gil6078032—AT, pBAD-Bsu_gil6080075_AT, 10 pB AD-B su_gi 16077991_AT , pBAD-Rsp_AT , pBAD-Lpn-AT, pBAD-Neu-AT, pBAD-Ngo_AT, pBAD-Pae_gi9951299_AT, pB AD-Pae_gi9951072_AT, pBAD-Pae_gi9951630_AT and pBAD-Rpa_AT. Corresponding performance The germline was obtained by transforming the chemically competent E. coli 15 TOP10 (Invitrogen) with the pB AD composition. Growth of E. coli for protein expression Small scale growth was performed on 96 deep well plates using 940 microliters of medium containing 0.02% (w/v) L-arabine. The cells obtained from the frozen spare culture were transferred to a fine cell using a 96-well punch (Ktihner, Bofeden, Switzerland). The plates were incubated at 25 ° C for 48 hours on an orbital shaker (300 rpm, 5 cm amplitude). Typically 〇D62() nm is 2-4. Preparation of Cell Lysate Dissolution Buffer Hehe # Dissolution buffer contains the following ingredients: 201033369 Table 3 1M MOPS pH 7.5 5 ml DNAse I grade II (Roche (R0Che)) 1 mg lysozyme 200 mg MgS04-7H20 123.2 mg Dithiothreitol (DTT) 154.2 mg H20 (MilliQ) Balanced to 100 ml solution is prepared just before use. Preparation of cell-free extract by dissolving is obtained by small-scale growth by centrifugation. (Refer to the previous paragraph) of the cells, discard the supernatant 5. The pellets formed during centrifugation are at -2 0. (: at least 16 hours of freezing and then thawed on ice. 500 μl of freshly prepared lysis buffer is added to The wells were resuspended by vigorous vortexing for 2-5 minutes. To achieve cell lysis, the plates were incubated for 30 minutes at room temperature. To remove cell debris, the plates were centrifuged at 4 ° C and 6000 g for 20 minutes. The supernatant is transferred to a new well plate and maintained on ice until further use. 畺_音振虚理_The cell-free extract is obtained from the medium-sized cells (refer to the previous paragraph) by centrifugation and abandonment Supernatant 1 ml of potassium phosphate buffer pH 7 was added to 克. 5 g of wet cell pellets, and the cells were resuspended by vigorous vortexing. To achieve cell lysis, cells 15 were sonicated for 20 minutes. To remove cell debris, lysate After centrifugation at 4:(: and 6000g for 20 minutes, the supernatant was transferred to a new tube and cooled at -20 °C until further use. Preparation of 5-methyl hydrazine by chemical hydrolysis of methyl S-methylvalerate The acid is hydrolyzed by methyl 5-methylvalerate to prepare a transaminase reaction. The 20 enzyme substrate, 5-methylvaleric acid, is as follows: (w/v) methyl 5-methylvalerate at 53 201033369 It was fixed to pH 14.1 with sodium hydroxide. After incubation at 20 ° C for 24 hours, the pH was fixed to 7.1 with hydrochloric acid. 5. Enzymatic reaction of conversion of formazanic acid to 6-ACA Unless otherwise specified, the preparation reaction mixture contained 10 mM 5-methylpenta-5 acid '20 mM racemic ex-methylbenzylamine, and 200 μΜβ than furfural 5'-decanoic acid in 50 mM potassium phosphate buffer, pH 7.0. 100 μl reaction mixture delivery To each well of the well plate. To initiate the reaction, 20 μl of cell-free extract was added to each well. The cells were incubated on a shaker at 37 ° C for 24 hours. In addition, the chemical blank group (without cell-free extract) and the biological blank group 10 (with PBAD/Myc-His C-based E. coli TOP10) under the same conditions Culture. Samples were analyzed by HPLC-MS. The results are summarized in the table below.

Table 4: Formation of 6-ACA biocatalyst from 5-FVA in the presence of transaminase

E. coli TOP10/pBAD-Vfl_AT

E. coli TOP10/pBAD-Pae_AT

E. coli TOPlO/pB AD-Pae_AT

E. coli TOP10/pBAD-Bwe_AT

Escherichia coli TOP10/pBAD-Pae_gi9951072_AT E. coli TOP10/pBAD-Pae_gi9951630_AT 6-ACA concentration I mg/kg] 43* 930 25* 大肠杆菌 E. coli TOPIO/ pBAD-Bsu_gil6077991_AT 24* 288

Nd: Undetectable * The difference between this method is that 10 μl of cell-free extract is used instead of 2 μl of microliter, furfural 5, phosphoric acid concentration of 15 degrees is 50^ instead of 200_ and the volume of the reaction mixture in the well is 190 μm. Rise instead of 100 microliters. ‘

6-ACA formed by 5-FVA in the presence of transaminase 54 201033369 AKP converted to s-mevalonic acid enzyme catalytic reaction preparation reaction mixture containing 50 mM ΑΚΡ, 5 mM magnesium hydride, 1 〇〇μΜ ° 5'-phosphoric acid (for LysA) and 1 mM guanamine dichromate (for all other enzymes) were dispensed into 100 mM acid buffer buffer, pH 6.5. 4 ml reaction mixture 5 was dispensed into the reaction vessel. To initiate the reaction, 1 ml of the cell-free extract obtained by sonication was added to each well. 50 units were used in the case of commercial grass 醯 acetic acid decarboxylase (Sigma-Aldrich product number 04878). The reaction mixture was used at 37 using a magnetic stirrer. (: culture for 48 hours. In addition, the chemical blank group mixture (excluding cell-free extract) and the bio-empty white group (pBAD/Myc-His C-containing E. coli TOP10) were cultured under the same conditions. Samples at time points were analyzed by HPLC-MS. The results are summarized in the table below.

Table 5: Formation of 5-FVA biocatalyst from AKP in the presence of decarboxylase 5-FVA concentration 1 mg/kg 1 3 hours 18 hours 48 hours E. coli TOPlO/pBAD-LysA 150 590 720 E. coli TOPlO/pBAD-Pdc 1600 1700 1300 E. coli TOP10/pBAD-Pdcl472A 2000 2000 1600 E. coli TOPlO/pBAD-KdcA 3300 2300 2200 E. coli TOPlO/pBAD-KivD 820 1400 1500 oxalic acid decarboxylase — nd 6 10 with pBAD/Myc-His C Escherichia coli TOP10 (biological blank group) ndnd rud·n (chemical blank group) ndnd·nd nd·: Undetectable shows that 5-FVA is formed from AKP in the presence of decarboxylase. 55 201033369 Enzymatic reaction to convert AKP to 6-ACA in the presence of recombinant decarboxylase to prepare a reaction mixture containing 50 mM AKP, 5 mM chlorinated town, 1 〇〇μΜ° 哆 5 5'- citric acid (for LysA) and 1 mM of amine diphosphate (for all other tested biocatalysts) in 100 mM potassium phosphate buffer, pH 6.5. 5 4 ml of the reaction mixture was dispensed into the reaction vessel. To initiate the reaction, the resulting cell-free extract was treated with i ml of sonication to each well. The reaction mixture was incubated at 37 ° C for 48 hours using a magnetic stirrer. Further, a chemical blank mixture (without cell-free extract) and a biological blank group (E. coli TOP10 containing pBAD/Myc-His C) were cultured under the same conditions. During the reaction, samples from the same time point were analyzed by HPLC-MS. The results are summarized in the table below.

Table 6: Formation of 6-ACA biocatalyst 6-ACA concentration in the presence of decarboxylase [mg/kg] 3 hours 18 hours 48 hours E. coli TOPlO/pBAD-LysA na 0.01 0 E. coli TOPlO/pBAD-Pdc 0.1 0.3 na 2 Enterobacter TOP10/pBAD-Pdcl472A 0.03 0.1 0.2 E. coli TOPlO/pBAD-KdcA 0.04 0.1 0.3 E. coli TOPlO/pBAD-KivD na 0.3 0.6 PBAD/Myc-His C E. coli TOP10 (bio blank group) ndndnd M 4b learning blank group) ndndnd Unanalyzed Undetectable shows that 6-ACA is formed by AKP in the presence of de-enzyme. E. coli is expected to contain natural 5-FVA transaminase activity. 56 201033369 Preparation of a reaction mixture comprising 405 AKP, 5 mM magnesium chloride, ΙΟΟμΜpyraldehyde 5,-phosphate, 1 mM thiamine Phosphoric acid and 50 mM of racemic CC-5 methylbenzylamine in 100 mM acid clock buffer, pH 6.5. 1.6 ml of the reaction mixture was dispensed into the reaction vessel. To initiate the reaction, 0.2 ml of decarboxylase containing cell-free extract and 0.2 ml of transaminase containing cell-free extract were added to each reaction vessel. The reaction mixture was incubated at 37 ° C for 48 hours with a magnetic stirrer. In addition, a chemical blank mixture (without cell-free extract) and a biological 10 blank group (E. coli TOP10 containing pBAD/Myc-His C) were cultured under the same conditions. Samples from different time points during the reaction were analyzed by HPLC-MS. The results are summarized in the table below.

Table 7: 6-ACA formation by akP in the presence of recombinant decarboxylase and recombinant transaminase 6-ACA concentration after your hour [mg/kg] AT DC E. coli TOP10/ pBAD-Vfl_AT E. coli TOP10/ pBAD-Bwe_AT Escherichia coli TOP 10/ pBAD- PAE_gi9946143_AT Big bacillus TOP lO/pBAD-Pdc 183.4 248.9 117.9 E. coli TOPI 0/pB AD-Pdc 1472A 458.5 471.6 170.3 Large intestine culvert TOP lO/pBAD-KdcA 497.8 497.8 275.1 E. coli TOPlO/pBAD-KivD 510.9 510.9 314.4 ΑΤ=Transaminase 15 DC=Decarboxylase No 6ACA was detected in the chemical blank group and the biological blank group. Furthermore, the results show an improvement in the conversion rate of the example 6-ACA in which the host cell contains only the recombinant decarboxylase (and 57 201033369 does not contain the recombinant transaminase). Composition of Saccharomyces cerevisiae for the expression of transaminase and decarboxylase according to the manufacturer's specifications and using specific primers [SEQ ID No. 76 and 77] using Phusion DNA polymerase (pinnZymes )), by 5 PCR, PBAD_vfl-AT [SEQ Νο·3] amplifies the amino acid sequence of the Vibrio fluvialis JS17 ω-transaminase obtained from Vibrio fluvialis JS17 [SEq ID ν〇.2] Transaminase gene. P. aeruginosa transaminase derived from Pseudomonas aeruginosa amplified by pBAD-Pae_AT 10 according to manufacturer's specifications and using specific primers [SEQ ID Ν ο. 78 and 79] using Fussen DNA polymerase (Finzan) The transaminase gene of the enzyme [SEQ ID No. 8] [5 £ (3 «) 1 ^ 〇. 7]. The resulting PCR product was transfected into the vector pAKP-41 using Spel and BamHI restriction enzymes to obtain pAKP-79 and pAKP-80, respectively. The transaminase 15 gene contained in the Saccharomyces cerevisiae gateO promoter gene and the Saccharomyces cerevisiae adh2 terminator gene today. . Saccharomyces cerevisiae pyruvate decarboxylase from Saccharomyces cerevisiae amplified by pBAD-Pdc by PCR using Füssen DNA polymerase (Finzan) according to manufacturer's specifications and using specific primers [SEQ ID No. 80 and 81] Decarboxylase gene of Pdc [SEQ ID No. 34] [SEQ ID No. 33]. 20 According to the manufacturer's specifications and using specific primers [SEQ ID No. 82 and 83], Fusin DNA polymerase (Finzan) was used to amplify the Lactobacillus lactis branched from Lactobacillus by PCR using pBAD-KdcA. Decarboxylase gene of the α-keto acid decarboxylase KdcA [SEQ ID Νο. 40] [SEQ ID Ν ο. 39]. The obtained PCR product was transfected into vector 201033369 ρΑΚΡ-44 using AscI and BamHI restriction enzymes to obtain ρΑΚΡ-81 and ρΑΚΡ-82, respectively. The transaminase gene contained in the Saccharomyces cerevisiae gal2 promoter gene and Saccharomyces cerevisiae pmal terminator gene was obtained. . The plastids pAKP-79 and pAKP-80 were digested with SacI and Xbal by restriction enzymes; the plastids ρΑΚΡ-81 and ρΑΚΡ-82 were digested with Sail and Xbal. The Sacl/Xbal transaminase fragment and the fragment combined with the Sall/Xbal decarboxylase fragment became the S. cerevisiae low-replication free gene vector pRS414, which was digested with Sail and SacI. The resulting plastid is:

Transformation and growth of Saccharomyces cerevisiae Saccharomyces cerevisiae line CEN.PK113-3C according to the method described by Gietz and Woods (Gietz, R.D. and Woods, R.A. (2002)). Yeast transformation is performed by Liac/SS vector DNA/PEG method. Enzymology Method 350: 87-96), transformed with 1 microgram of 15 plastid DNA. The cells were seeded on agar plates containing lx yeast nitrogen bases without amino acids and 2% glucose. The resulting strain was aerobically grown for 48 hours at 30 ° C in Verduyn minimal medium containing 0.05% glucose and 4% galactose. Preparation of cell-free extract 2 〇 1 ml of potassium phosphate buffer (pH 7) was added to 克. 5 g of cell pellets. This mixture was added to a 2 ml coat of cotton with a diameter of 0.4-0.5 mm containing 0.5 g of glass beads 59 201033369 in an eppendorf test tube. The sample was shaken vigorously for 20 seconds with the Ibn VIBRAX-VXR. The resulting cell-free extract was centrifuged at kmooo rpm for 5 minutes at 4 °C. The supernatant was used for enzyme activity assay analysis. Preparation of a reaction mixture by the enzyme-catalyzed reaction of S. cerevisiae to a 6-ACA in the presence of a common decarboxylase and transaminase. 'Contains 50 mM AKP, 5 mM gasification, 100 μM pyridoxal 5' - Phosphoric acid, 1 mM thiamine diphosphate and 5 mM of racemic ?-methylbenzylamine in 100 mM potassium phosphate buffer, ρ 6.5. 1.6 ml of the reaction mixture was dispensed into a reaction vessel. To initiate the reaction, 4 ml of cell-free extract from Saccharomyces cerevisiae containing decarboxylase and 10 transaminase was added to each reaction vessel. The reaction mixture was incubated at 37 ° C with a magnetic stirrer. In addition, the chemical blank mixture (without cell-free extract) and the biological blank group (wheat yeast) were cultured under the same conditions. The samples obtained after 19 hours of incubation were analyzed by HPLC-MS. The results are summarized in the table below.

15 Table 8: Formation of 6-ACA biocatalyst concentration by AKP using biomicrocatalyst as biocatalyst 1 mg/kg] Saccharomyces cerevisiae pAKP-85 63 Saccharomyces cerevisiae pAKP-86 226 Saccharomyces cerevisiae pAKP-87 1072 Alcohol yeast pAKP-88 4783 Saccharomyces cerevisiae ( Biological blank group) 3.9 None (chemical blank group) 1.3 The reaction mixture of α-ketopimelate converted to α-amine pimelic acid was prepared by catalytic reaction, containing 10 mM ex-ketopimelic acid, 20 mM L- 60 201033369 Alanine, and 50 μΜ° awake · 5'_acid in 50 mM potassium carbonate buffer 'PH 7.0. 800 μl of the reaction mixture was dispensed into the wells of the well plate. To initiate the reaction, 200 microliters of cell lysate was added to each well. The reaction mixture was incubated at 37 ° C for 24 hours on a shaker. In addition, a chemical blank group mixture (excluding extracts without 5 cells) and a biological blank group (E. coli TOP10 containing pBAD/Myc-His C) were cultured under the same conditions. The samples were analyzed by HPLC_MS. The results are summarized in the table below.

Table 9: AAP biocatalyst concentration from AKP in the presence of transaminase [mg/kg] [14 hours later, E. coli TOP10/pBAD-Vfl_AT 3.7 E. coli TOP10/pBAD-Psy_AT 15.8 E. coli TOP10/pBAD-Bsu_gil6078032_AT — 11.2 E. coli TOP10/pBAD-Rsp_AT 9.8 E. coli TOP10/pBAD-Bsu_gil6080075_AT 4.6 E. coli TOP10/pBAD-Lpn_AT E. coli TOP10/pBAD-Neu_AT ---- 7.7 E. coli TOP10/pBAD-Ngo_AT ----- 5.1 Large intestine Bacillus TOP 10/pBAD-Pae_gi9951299_AT 5.6 ' E. coli TOP10/pBAD-Rpa_AT 5.4 E. coli TOP10 containing PBAD/Myc-His C (bio blank group) 1.4 None (chemical blank group) 0

It is shown that AAP is formed by AKP and catalyzed by a biocatalyst. Chemical conversion of AAP to caprolactam 0.5 ml of cyclohexenone was added to a suspension of 1.5 g of D,L-2-amine pimelic acid in 21 ml of cyclohexanone. The mixture was heated for about 20 hours (about 61 201033369 160 ° C). After cooling to room temperature, the reaction mixture was decanted and evaporated under reduced pressure to remove a clear solution. The remaining 2 g of brown oil was analyzed by 1 H-NMR and HPLC to contain a cyclic oligomer of 0.8 wt% of caprolactam and 6 wt% of caprolactam. Simple description of C pattern 3 5 (none) [Explanation of main component symbols] (none)

❿ 62 201033369

Sequence Listing <110> DSM IP Assets B.V.

Raemakers-Franken, Petronella, Catharina Schurmann, Martin Trefzer, Axel Christoph De Wildeman, Stefaan Marie Andre <120> Technique for the preparation of 6-aminocaproic acid from a-ketopimelic acid <130> P84198PC00 <150> EP 08152584.2 <151> 2008-03-11 <160> 83 <170> Patentln version 3.3 <210> 1 <211> 1362 <212> DNA <213> Vibrio fluvialis<220>

- <221> CDS <222> (1)..(1362) <400> 1 atg aac aaa ccg caa age tgg gaa gcc egg gcc gag acc tat teg etc

Met Asn Lys Pro Gin Ser Trp Glu Ala Arg Ala Glu Thr Tyr Ser Leu 15 10 15 tat ggt ttc acc gac atg cct teg ctg cat cag ege ggc aeg gtc gtc

TGg acc cat ggc gag gga ccc tat ate gtc gat gg

Val Thr His Gly Glu Gly Pro Tyr lie Val Asp Val Asn Gly Arg Arg 35 40 45 tat ctg gac gcc aac teg ggc ctg tgg aac atg gtc geg ggc ttt gac

Tyr Leu Asp Ala Asn Ser Gly Leu Trp Asn Met Val Ala Gly Phe Asp 50 55 60 • cac aag ggg ctg ate gac gcc gcc aag gcc caa tac gag cgt ttt ccc

His Lys Gly Leu lie Asp Ala Ala Lys Ala Gin Tyr Glu Arg Phe Pro 65 70 75 80 ggt tat cac gcc ttt ttc ggc ege atg tcc gat cag aeg gta atg ctg

Gly Tyr His Ala Phe Phe Gly Arg Met Ser Asp Gin Thr Val Met Leu 85 90 95 teg gaa aag ctg gtc gag gtg teg ccc ttt gat teg ggc egg gtg ttc

Ser Glu Lys Leu Val Glu Val Ser Pro Phe Asp Ser Gly Arg Val Phe 100 105 110 tat aca aac teg ggg tcc gag geg aat gac acc atg gtc aag atg eta 48 96 144 192 240 288 336 384 1 201033369

Tyr 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 ate ctg 432

Trp Phe Leu His Ala Ala Glu Gly Lys Pro Gin Lys Arg Lys lie Leu 130 135 140 acc cgc tgg aac gcc tat cac ggc gtg acc gcc gtt teg gcc age atg 480

Thr 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 teg gtc ttt ggc ctg ccg ctg ccg ggc ttt 528

Th G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G

Val 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 etc gcc cgc gag ctg gag gaa aeg 624

Thr Glu Glu Gin Phe Val Ala Arg Leu Ala Arg Glu Leu Glu Glu Thr 195 200 205 ate cag cgc gag ggc gcc gac acc ate gcc ggt ttc ttt gcc gaa ccg 672 lie Gin Arg Glu Gly Ala Asp Thr lie Ala Gly Phe Phe Ala Glu Pro 210 215 220 gtg atg ggc geg ggc ggc gtg att ccc ccg gcc aag ggc tat ttc cag 720

Val Met Gly Ala Gly Gly Val lie Pro Pro Ala Lys Gly Tyr Phe Gin 225 230 235 240 geg ate ctg cca ate ctg cgc aaa tat gac ate ccg gtc ate teg gac 768

Ala lie Leu Pro lie Leu Arg Lys Tyr Asp lie Pro Val lie Ser Asp 245 250 255 gag gtg ate tgc ggt ttc gga cgc acc ggt aac acc tgg ggc tgc gtg 816

Glu Val lie Cys Gly Phe Gly Arg Thr Gly Asn Thr Trp Gly Cys Val

260 265 270 acc tat gac ttt aca ccc gat gca ate ate teg tee aag aat ctt aca 864

Thr Tyr Asp Phe Thr Pro Asp Ala He lie Ser Ser Lys Asn Leu Thr 275 280 285 geg ggc ttt ttc ccc atg ggg geg gtg ate ett ggc ccg gaa ett tee 912

Ala Gly Phe Phe Pro Met Gly Ala Val lie Leu Gly Pro Glu Leu Ser 290 295 300 aaa egg ctg gaa acc gca ate gag geg ate gag gaa ttc ccc cat ggc 960

Lys Arg Leu Glu Thr Ala He Glu Ala He Glu Glu Phe Pro His Gly 305 310 315 320 ttt acc gcc teg ggc cat ccg gtc ggc tgt get att geg ctg aaa gca 1008

Phe Thr Ala Ser Gly His Pro Val Gly Cys Ala He Ala Leu Lys Ala 325 330 335 ate gac gtg gtg atg aat gaa ggg ctg get gag aac gtc cgc cgc ett 1056 lie Asp Val Val Met Asn Glu Gly Leu Ala Glu Asn Val Arg Arg Leu 340 345 350 2 1104 1104 ❹ 201033369 gcc ccc cgt ttc gag gaa agg ctg aaa cat ate gee gag ege ccg aac

Ala Pro Arg Phe Glu Glu Arg Leu Lys His lie Ala Glu Arg Pro Asn 355 360 365 ate ggt gaa tat ege ggc ate ggc ttc atg tgg geg ctg gag get gtc lie Gly Glu Tyr Arg Gly lie Gly Phe Met Trp Ala Leu Glu Ala Val 370 375 380 aag gac aag gca age aag aeg ccg ttc gac ggc aac ctg teg gtc age

Lys Asp Lys Ala Ser Lys Thr Pro Phe Asp Gly Asn Leu Ser Val Ser 385 390 395 400 gag cgt ate gcc aat acc tgc acc gat ctg ggg ctg att tgc egg ccg

Glu Arg He Ala Asn Thr Cys Thr Asp Leu Gly Leu lie Cys Arg Pro 405 410 415 ett ggt cag tee gtc gtc ett tgt ccg ccc ttt ate ctg acc gag geg

Leu Gly Gin Ser Val Val Leu Cys Pro Pro Phe lie Leu Thr Glu Ala 420 425 430 cag atg gat gag atg ttc gat aaa etc gaa aaa gcc ett gat aag gtc

Gin Met Asp Glu Met Phe Asp Lys Leu Glu Lys Ala Leu Asp Lys Val 435 440 445 ttt gcc gag gtt gcc tga Phe Ala Glu Val Ala 450 <210〉 2 <211> 453 <212> PRT <213> River Vibrio <400> 2

Met Asn Lys Pro Gin Ser Trp Glu Ala Arg Ala Glu Thr Tyr Ser Leu 1152 1200 1248 1296 1344 1362

Tyr Gly Phe Thr Asp Met Pro Ser Leu His Gin Arg Gly Thr Val Val 20 25 30

Val Thr His Gly Glu Gly Pro Tyr lie 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 He Asp Ala Ala Lys Ala Gin Tyr Glu Arg Phe Pro 65 70 75 80

Gly Tyr His Ala Phe Phe Gly Arg Met Ser Asp Gin Thr Val Met Leu 85 90 95 3 201033369

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 Gin Lys Arg Lys He 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 Gin Phe Val Ala Arg Leu Ala Arg Glu Leu Glu Glu Thr 195 200 205 lie Gin Arg Glu Gly Ala Asp Thr He Ala Gly Phe Phe Ala Glu Pro 210 215 220

Val Met Gly Ala Gly Gly Val lie Pro Pro Ala Lys Gly Tyr Phe Gin 225 230 235 240

Ala He Leu Pro lie Leu Arg Lys Tyr Asp He Pro Val lie Ser Asp 245 250 255

Glu Val lie Cys Gly Phe Gly Arg Thr Gly Asn Thr Trp Gly Cys Val 260 265 270

Thr Tyr Asp Phe Thr Pro Asp Ala He He Ser Ser Lys Asn Leu Thr 275 280 285

Ala Gly Phe Phe Pro Met Gly Ala Val He Leu Gly Pro Glu Leu Ser 290 295 300

Lys Arg Leu Glu Thr Ala lie Glu Ala lie Glu Glu Phe Pro His Gly 305 310 315 320

Phe Thr Ala Ser Gly His Pro Val Gly Cys Ala He Ala Leu Lys Ala 325 330 335 201033369 lie 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 lie Ala Glu Arg Pro Asn 355 360 365 lie Gly Glu Tyr Arg Gly lie 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 lie Ala Asn Thr Cys Thr Asp Leu Gly Leu He Cys Arg Pro 405 410 415

Leu Gly Gin Ser Val Val Leu Cys Pro Pro Phe lie Leu Thr Glu Ala 420 425 430

Gin Met Asp Glu Met Phe Asp Lys Leu Glu Lys Ala Leu Asp Lys Val 435 440 445

Phe Ala Glu Val Ala 450 <210> 3 <211> 1362 <212> DNA <213>manmade <220><223> Vibrio fluvialis JS17 ω-transaminator codon optimization gene <; 400> 3 60 120 180 240 300 360 420 atgaataaac cacagtcttg ggaagctcgt gctgaaacct atagcctgta cggctttacc gatatgccgt ctctgcacca gcgtggtact gtagtggtaa cgcacggtga gggcccgtac atcgtggacg ttaatggccg ccgttacctg gatgcaaaca gcggcctgtg gaacatggtt gcgggcttcg accacaaagg cctgatcgat gccgcaaaag cgcagtacga acgcttcccg ggttatcacg cgttctttgg ccgtatgagc gaccagactg tgatgctgag cgaaaaactg gttgaagtgt ccccgttcga tagcggtcgt gtcttttaca ctaactctgg cagcgaggct aacgatacca tggttaagat gctgtggttc ctgcacgcag cggaaggcaa acctcagaaa cgtaaaattc tgacccgttg gaacgcttat cacggtgtga ctgctgtttc cgcatctatg 480 5 201033369 accggtaaac cgtataacag cgtgttcggt ctgccgctgc ctggcttcgt gcatctgacc 540 tgcccgcact actggcgtta tggtgaggaa ggcgaaactg aggaacagtt cgtggcgcgt 600 ctggctcgtg aactggaaga aaccattcaa cgcgaaggtg cagatactat cgcgggcttc 660 tttgcggagc ctgttatggg tgccggcggt gtgattcc gc cggcgaaggg ctatttccag 720 gcaatcctgc cgatcctgcg caagtacgac attccggtta tttctgacga agtgatctgc 780 ggcttcggcc gcaccggtaa cacctggggc tgcgtgacgt atgacttcac tccggacgca 840 atcattagct ctaaaaacct gactgcgggt ttcttcccta tgggcgccgt aatcctgggc 900 ccagaactgt ctaagcgcct ggaaaccgcc atcgaggcaa tcgaagagtt cccgcacggt 960 ttcactgcta gcggccatcc ggtaggctgc gcaatcgcgc tgaaggcgat cgatgttgtc 1020 atgaacgagg gcctggcgga aaacgtgcgc cgcctggcgc cgcgttttga agaacgtctg 1080 aaacacattg ctgagcgccc gaacattggc gaatatcgcg gcatcggttt 1140 ctggaagcag ttaaagataa agctagcaag accccgttcg acggcaacct gtccgtgagc 1200 gaacgtatcg ctaatacctg tacggacctg ggtctgatct gccgtccgct gggtcagtcc 1260 gtagttctgt gcccaccatt tatcctgacc gaagcgcaga tggatgaaat gttcgataaa 1320 ctggagaaag ctctggataa agtgttcgct gaagtcgcgt aa 1362 & lt catgtgggcc; 210 > 4 < 211 > 1350 < 212 > DNA < 213 > Wei Bacillus sputum <220><221> CDS <222> (1)..(1350) <400> 4 gtg caa gcg acg gag caa aca caa agt t tg aaa aaa aca gat gaa aag 4 8

Val Gin Ala Thr Glu Gin Thr Gin Ser Leu Lys Lys Thr Asp Glu Lys 15 10 15 tac ctt tgg cat gcg atg aga gga gca gcc cct agt cca acg aat tta 96

Tyr Leu Trp His Ala Met Arg Gly Ala Ala Pro Ser Pro Thr Asn Leu 20 25 30 att ate aca aaa gca gaa ggg gca tgg gtg acg gat att gat gga aac 144 lie He Thr Lys Ala Glu Gly Ala Trp Val Thr Asp He Asp Gly Asn 35 40 45 cgt tat tta gac ggt atg tcc ggt ctt tgg tgc gtg aat gtt ggg tat 192

Arg Tyr Leu Asp Gly Met Ser Gly Leu Trp Cys Val Asn Val Gly Tyr 50 55 60 ggt ega aaa gaa ctt gca aga gcg gcg ttt gaa cag ctt gaa gaa atg 240 6 288 288

201033369

Gly Arg Lys Glu Leu Ala Arg Ala Ala Phe Glu Gin Leu Glu Glu Met 65 70 75 80 ccg tat ttc cct ctg act caa agt cat gtt cct get att aaa tta gca

Pro Tyr Phe Pro Leu Thr Gin Ser His Val Pro Ala lie Lys Leu Ala 85 90 95 gaa aaa ttg aat gaa tgg ett gat gat gaa tac gtc att ttc ttt tet

Glu Lys Leu Asn Glu Trp Leu Asp Asp Glu Tyr Val lie Phe Phe Ser 100 105 110 aac agt gga teg gaa geg aat gaa aca gca ttt aaa att get cgt caa

Asn Ser Gly Ser Glu Ala Asn Glu Thr Ala Phe Lys He Ala Arg Gin 115 120 125 tat cat caa caa aaa ggt gat cat gga ege tat aag ttt att tcc ege

Tyr His Gin Gin Lys Gly Asp His Gly Arg Tyr Lys Phe He Ser Arg 130 135 140 tac ege get tat cac ggt aac tea atg gga get ett gca gca aca ggt

Tyr Arg Ala Tyr His Gly Asn Ser Met Gly Ala Leu Ala Ala Thr Gly 145 150 155 160 - caa gca cag ega aag tat aaa tat gaa cca etc ggg caa gga ttc ctg

Gin Ala Gin Arg Lys Tyr Lys Tyr Glu Pro Leu Gly Gin Gly Phe Leu -. 165 170 175 cat gta gca ccg cct gat aeg tat ega aat cca gag gat gtt cat aca

His Val Ala Pro Pro Asp Thr Tyr Arg Asn Pro Glu Asp Val His Thr 180 185 190 ctg gca agt get gag gaa ate gat cgt gtc atg aca tgg gag tta age

Leu Ala Ser Ala Glu Glu He Asp Arg Val Met Thr Trp Glu Leu Ser 195 200 205 - caa aca gta gee ggt gtg att atg gag cca ate att act ggg ggc gga Gin Thr Val Ala Gly Val lie Met Glu Pro lie lie Thr Gly Gly Gly 210 215 220 att tta atg cct cct gat gga tat atg gga aaa gta aaa gaa att tgc

He Leu Met Pro Pro Asp Gly Tyr Met Gly Lys Val Lys Glu lie Cys 225 230 235 240 gag aag cac ggt geg ttg etc att tgt gat gaa gtt ata tgt gga ttt

Glu Lys His Gly Ala Leu Leu He Cys Asp Glu Val He Cys Gly Phe 245 250 255 ggc egg aca ggg aag cca ttt gga ttt atg aat tat ggc gtc aaa cca

Gly Arg Thr Gly Lys Pro Phe Gly Phe Met Asn Tyr Gly Val Lys Pro 260 265 270 gat ate att aca atg gca aaa ggt att aca agt geg tat ett cct ttg

Asp lie He Thr Met Ala Lys Gly lie Thr Ser Ala Tyr Leu Pro Leu 275 280 285 tea gca aca gca gtt aga ega gag gtt tat gag gca ttc gta ggt agt

Ser Ala Thr Ala Val Arg Arg Glu Val Tyr Glu Ala Phe Val Gly Ser 290 295 300 336 384 432 480 528 576 624 672 720 768 816 864 7 912 960 201033369 gat gat tat gat cgc ttc cgc cat gta aat acg ttc gga ggg aat Cct

Asp Asp Tyr Asp Arg Phe Arg His Val Asn Thr Phe Gly Gly Asn Pro 305 310 315 320 get get tgc get tta get ttg aag aat tta gaa att atg gag aat gag

Ala Ala Cys Ala Leu Ala Leu Lys Asn Leu Glu lie Met Glu Asn Glu 325 330 335 aaa etc att gaa cgt tee aaa gaa ttg ggt gaa ega ctg tta tat gag

Lys Leu He Glu Arg Ser Lys Glu Leu Gly Glu Arg Leu Leu Tyr Glu 340 345 350 eta gag gat gta aaa gag cat cca aac gta ggg gat gtt cgc gga aag

Leu Glu Asp Val Lys Glu His Pro Asn Val Gly Asp Val Arg Gly Lys 355 360 365 ggc ett ett tta ggc att gaa eta gtg gaa gat aag caa aca aaa gaa

Gly Leu Leu Leu Gly He Glu Leu Val Glu Asp Lys Gin Thr Lys Glu 370 375 380 ccg get tee att gaa aag atg aac aaa gtc ate aat get tgt aaa gaa

Pro Ala Ser lie Glu Lys Met Asn Lys Val lie Asn Ala Cys Lys Glu 385 390 395 400 aaa ggt eta att att ggt aaa aat ggt gac act gtc gca ggt tac aat

Lys Gly Leu lie He Gly Lys Asn Gly Asp Thr Val Ala Gly Tyr Asn 405 410 415 aat att ttg cag ett gca cct cca tta age ate aca gag gaa gac ttt

Asn He Leu Gin Leu Ala Pro Pro Leu Ser He Thr Glu Glu Asp Phe 420 425 430 act ttt ate gtt aaa aca atg aaa gaa tgt tta tee cgc att aac ggg

Thr Phe He Val Lys Thr Met Lys Glu Cys Leu Ser Arg lie Asn Gly 435 440 445 cag taa Gin <210> 5 <211> 449 <212> PRT <213> Bacillus vegetative <400>

Val Gin Ala Thr Glu Gin Thr Gin 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 lie He Thr Lys Ala Glu Gly Ala Trp Val Thr Asp lie Asp Gly Asn 35 40 45 1008 1056 1104 1152 1200 1248 1296 1344 1350 201033369

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 Gin Leu Glu Glu Met 65 70 75 80

Pro Tyr Phe Pro Leu Thr Gin Ser His Val Pro Ala lie Lys Leu Ala 85 90 95

Glu Lys Leu Asn Glu Trp Leu Asp Asp Glu Tyr Val lie Phe Phe Ser 100 105 110

Asn Ser Gly Ser Glu Ala Asn Glu Thr Ala Phe Lys lie Ala Arg Gin 115 120 125

Tyr His Gin Gin Lys Gly Asp His Gly Arg Tyr Lys Phe lie 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

Gin Ala Gin Arg Lys Tyr Lys Tyr Glu Pro Leu Gly Gin 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 lie Asp Arg Val Met Thr Trp Glu Leu Ser 195 200 205

Gin Thr Val Ala Gly Val lie Met Glu Pro lie lie Thr Gly Gly Gly 210 215 220

He Leu Met Pro Pro Asp Gly Tyr Met Gly Lys Val Lys Glu He Cys 225 230 235 240

Glu Lys His Gly Ala Leu Leu lie Cys Asp Glu Val lie 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 He lie Thr Met Ala Lys Gly lie Thr Ser Ala Tyr Leu Pro Leu 275 280 285 9 201033369

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 He Met Glu Asn Glu 325 330 335

Lys Leu lie 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 He Glu Leu Val Glu Asp Lys Gin Thr Lys Glu 370 375 380

Pro Ala Ser lie Glu Lys Met Asn Lys Val He Asn Ala Cys Lys Glu 385 390 395 400

Lys Gly Leu lie He Gly Lys Asn Gly Asp Thr Val Ala Gly Tyr Asn 405 410 415

Asn lie Leu Gin Leu Ala Pro Pro Leu Ser He Thr Glu Glu Asp Phe 420 425 430

Thr Phe lie Val Lys Thr Met Lys Glu Cys Leu Ser Arg He Asn Gly Xin 435 440 445

Gin <210> 6 <211> 1350 <212> DNA <213>artificial<220><223> B. vesenida KBAB4 transaminator codon optimization gene <400> 6 atgcaggcta ccgaacaaac Ccaatctctg aaaaagactg acgaaaaata tctgtggcac 60 gcgatgcgcg gtgcagctcc gtctccgacc aacctgatta ttaccaaagc tgaaggcgcg 120 10 201033369

tgggtgaccg aatgtcggtt ccgtacttcc gaatggctgg accgcattca ttcatcagcc caggctcagc ccggatacct cgtgttatga accggtggtg gaaaaacacg aaaccatttg attacttccg tttgttggtt gcggcatgtg cgtagcaaag aacgtgggcg cagaccaagg aaaggcctga ctggcgccgc gagtgcctga acattgacgg atggccgtaa cgctgactca acgacgaata aaatcgcccg gttatcgtgc gcaaatacaa accgtaaccc cctgggagct gcattctgat gcgcgctgct gcttcatgaa cttatctgcc ctgatgatta cgctggcgct aactgggtga atgttcgcgg aaccggcttc tcattggtaa ctctgagcat gccgcatcaa taaccgttat ggagctggcg aagccatgtg cgtgattttc tcaatatcac ataccatggt gtacgaaccg ggaagacgtc gtcccagact gccgccggac gatctgcgat ttatggcgta gctgagcgcg cgaccgtttc gaaaaacctg acgtctgctg taaaggcctg cattgaaaag gaacggtgat cactgaagaa tggtcagtaa ctggatggca cgcgcggcat ccggctatca ttctctaatt cagcagaaag aattctatgg ctgggtcagg cacaccctgg gttgcgggtg ggttatatgg gaagttatct aaacctgaca accgcagttc cgtcatgtaa gaaatcatgg tacgaactgg ctgctgggta atgaacaaag accgtggcag gatttcacct tgagcggcct ttgaacaact aactggcgga ctggctccga gtgaccacgg gtgcgctggc gttttctgca cttctgccga ttatcatgga gtaaagtcaa gtggcttcgg ttattaccat gccgcgaagt acacgtttgg aaaacgaaaa aagatgtcaa ttgaactggt tgattaacgc gttataacaa tcatcgtcaa gtggtgtgtt ggaagaaatg aaaactgaac agcaaacgaa ccgctataaa tgctaccggt cgttgcacca agaaatcgat acctattatt ggaaatctgc tcgcaccggc ggctaaaggc ttatgaagcg cggtaaccca gctgatcgaa agaacacccg tgaagacaaa gtgcaaagag cattctgcag aactatgaag 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1350

<210> 7 <211> 1371 <212> DNA <213> Pseudomonas aeruginosa (gi 9946143) <220> <221> CDS <222> (1)..(1371) <400 〉 7 atg aac age caa ate acc aac gee aag acc cgt gag tgg cag geg ttg Met Asn Ser Gin lie Thr Asn Ala Lys Thr Arg Glu Trp Gin Ala Leu 15 10 15 96 age ege gac cac cat ctg ccg ccg ttc acc gac tac Aag cag ttg aac 11 201033369

Ser Arg Asp His His Leu Pro Pro Phe Thr Asp Tyr Lys Gin Leu Asn 20 25 30 gag aag ggc gcg egg ate ate acc aag gee gaa ggc gtc tat ate tgg 144

Glu Lys Gly Ala Arg lie lie Thr Lys Ala Glu Gly Val Tyr He Trp 35 40 45 gac age gag ggc aac aag ate etc gat gcg atg gee ggc etc tgg tgc 192

Asp Ser Glu Gly Asn Lys lie Leu Asp Ala Met Ala Gly Leu Trp Cys 50 55 60 gtc aac gtc ggc tac ggc ege gag gag ctg gtc cag gee gee acc egg 240

Val Asn Val Gly Tyr Gly Arg Glu Glu Leu Val Gin Ala Ala Thr Arg 65 70 75 80 cag atg ege gag ttg ccg tie tac aac ctg ttc ttc cag acc gee cac 288

Gin Met Arg Glu Leu Pro Phe Tyr Asn Leu Phe Phe Gin Thr Ala His 85 90 95

Ccg ccg gtg gtc gag ctg gee aag gcg ate gee gac gtc get ccg gaa 336

Pro Pro Val Val Glu Leu Ala Lys Ala He Ala Asp Val Ala Pro Glu 100 105 110 ggc atg aac cac gtg ttc ttc acc ggc tee ggc tee gag gee aac gac 384

Gly Met Asn His Val Phe Phe Thr Gly Ser Gly Ser Glu Ala Asn Asp 115 120 125 acc gtg ctg cgt atg gtc ege cac tat tgg gcg acc aag ggc cag ccg 432

Thr Val Leu Arg Met Val Arg His Tyr Trp Ala Thr Lys Gly Gin Pro 130 135 140 cag aag aaa gtg gt ate ggc ege tgg aac ggc tac cac ggc tee acc 480

Gin Lys Lys Val Val lie Gly Arg Trp Asn Gly Tyr His Gly Ser Thr 145 150 155 160 gtc gee ggc gtc age ctg ggc ggc atg aag gcg ttg cat gag cag ggt 528

Val Ala Gly Val Ser Leu Gly Gly Met Lys Ala Leu His Glu Gin Gly 165 170 175 gat ttc ccc ate ccg ggc ate gtc cac ate gee cag ccc tac tgg tac 576

Asp Phe Pro lie Pro Gly lie Val His He Ala Gin Pro Tyr Trp Tyr 180 185 190 ggc gag ggc ggc gac atg teg ccg gac gag ttc ggc gtc tgg gee gee 624

Gly 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 etc gaa gtg ggc gag gaa aac gtc gee 672

Glu Gin Leu Glu Lys Lys lie Leu Glu Val Gly Glu Glu Asn Val Ala 210 215 220 gee ttc ate gee gag ccg ate cag ggc gee ggc ggc gtg ate gtc ccg 720

Ala Phe lie Ala Glu Pro lie Gin Gly Ala Gly Gly Val lie Val Pro 225 230 235 240 ccg gac acc tac tgg ccg aag ate ege gag ate etc gee aag tac gac 768

Pro Asp Thr Tyr Trp Pro Lys lie Arg Glu lie Leu Ala Lys Tyr Asp 245 250 255 12 816 816

201033369 ate ctg ttc ate gee gac gaa gtg ate tgc ggc ttc ggc cgt acc ggc lie Leu Phe lie Ala Asp Glu Val He Cys Gly Phe Gly Arg Thr Gly 260 265 270 gag tgg ttc ggc age cag tac tac ggc aac gee ccg gac ctg Atg ccg

Glu Trp Phe Gly Ser Gin Tyr Tyr Gly Asn Ala Pro Asp Leu Met Pro 275 280 285 ate gee aag ggc etc acc tee ggc tac ate ccc atg ggc ggg gtg gtg lie Ala Lys Gly Leu Thr Ser Gly Tyr He Pro Met Gly Gly Val Val 290 295 300 gtg ege gac gag ate gtc gaa gtg etc aac cag ggc ggc gag ttc tac

Val Arg Asp Glu lie Val Glu Val Leu Asn Gin Gly Gly Glu Phe Tyr 305 310 315 320 cac ggc ttc acc tat tee ggt cac ccg gtg geg gee gee gtg gee ctg

His Gly Phe Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu 325 330 335 gag aac ate ege ate ctg ege gaa gag aag ate ate gag aag gtg aag

Glu Asn lie Arg He Leu Arg Glu Glu Lys He He Glu Lys Val Lys 340 345 350 geg gaa aeg gca ccg tat ttg cag aaa ege tgg cag gag ctg gee gac

Ala Glu Thr Ala Pro Tyr Leu Gin Lys Arg Trp Gin Glu Leu Ala Asd 355 360 365 cac ccg ttg gtg ggc gaa geg ege ggg gtc ggc atg gtc gee gee ctg

His 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 ege gag cgt ttc acc gac aag ggc

Glu Leu Val Lys Asn Lys Lys Thr Arg Glu Arg Phe Thr Asp Lvs Glv 385 390 395 4〇〇 gtc ggg atg ctg tgc egg gaa cat tgt ttc ege aac ggt ttg ate atg

Val Gly Met Leu Cys Arg Glu His Cys Phe Arg Asn Gly Leu lie Met 405 410 415 ege geg gtg ggc gac act atg att ate teg ccg ccg ctg gtg ate gat

Arg Ala Val Gly Asp Thr Met lie lie Ser Pro Pro Leu Val lie Asd 420 425 430 ccg teg cag ate gat gag ttg ate acc ctg geg ege aag tgc etc gat

Pro Ser Gin lie Asp Glu Leu lie Thr Leu Ala Arg Lys Cys Leu Asd 435 440 445 cag acc gee gee gee gtc ctg get tga

Gin Thr Ala Ala Ala Val Leu Ala 450 455

<210> 8 <211> 456 <212> PRT <213> Pseudomonas aeruginosa (gi 9946143) <400> 8 864 912 960 1008 1056 1104 1152 1200 1248 1296 1344 13 1371 201033369

Met Asn Ser Gin He Thr Asn Ala Lys Thr Arg Glu Trp Gin Ala Leu 15 10 15

Ser Arg Asp His His Leu Pro Pro Phe Thr Asp Tyr Lys Gin Leu Asn 20 25 30

Glu Lys Gly Ala Arg lie He Thr Lys Ala Glu Gly Val Tyr lie Trp 35 40 45

Asp Ser Glu Gly Asn Lys He Leu Asp Ala Met Ala Gly Leu Trp Cys 50 55 60

Val Asn Val Gly Tyr Gly Arg Glu Glu Leu Val Gin Ala Ala Thr Arg 65 70 75 80

Gin Met Arg Glu Leu Pro Phe Tyr Asn Leu Phe Phe Gin Thr Ala His 85 90 95

Pro Pro Val Val Glu Leu Ala Lys Ala lie 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 Gin Pro 130 135 140 #

Gin Lys Lys Val Val He 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 Gin Gly 165 170 175

Asp Phe Pro lie Pro Gly lie Val His lie Ala Gin 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 Gin Leu Glu Lys Lys He Leu Glu Val Gly Glu Glu Asn Val Ala 210 215 220

Ala Phe lie Ala Glu Pro He Gin Gly Ala Gly Gly Val He Val Pro 225 230 235 240 14 201033369

Pro Asp Thr Tyr Trp Pro Lys lie Arg Glu He Leu Ala Lys Tyr Asp 245 250 255 lie Leu Phe He Ala Asp Glu Val He Cys Gly Phe Gly Arg Thr Gly 260 265 270

Glu Trp Phe Gly Ser Gin Tyr Tyr Gly Asn Ala Pro Asp Leu Met Pro 275 280 285 lie Ala Lys Gly Leu Thr Ser Gly Tyr lie Pro Met Gly Gly Val Val 290 295 300 _ Val Arg Asp Glu He Val Glu Val Leu Asn Gin Gly Gly Glu Phe Tyr φ 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 He Arg lie Leu Arg Glu Glu Lys lie He Glu Lys Val Lys 340 345 350

I

Ala Glu Thr Ala Pro Tyr Leu Gin Lys Arg Trp Gin 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 He Met 405 410 415

Arg Ala Val Gly Asp Thr Met lie He Ser Pro Pro Leu Val He Asp ' 420 425 430 ' Pro Ser Gin lie Asp Glu Leu He Thr Leu Ala Arg Lys Cys Leu Asp 435 440 445

Gin Thr Ala Ala Ala Val Leu Ala 450 455 <210> 9 15 201033369 <211> 70 <212> DNA <213>manmade<220><223>Introduction<400> 9 ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta Accatgaaca gccaaatcac 60 caacgccaag 70 <210> 10 <211> 49 <212> DNA <213> Manmade <220><223>Introduction φ <400> 10 ggggaccact ttgtacaaga aagctgggtt caagccagga cggcggcgg 49 <210> 11 <211> 1365 <212> DNA <213> Pseudomonas aeruginosa <220><221> CDS <222> (1)..(1365) <400> 11 atg agt gcc Aac aac ccg caa acc etc gaa tgg cag gee ctg age age 48

Met Ser Ala Asn Asn Pro Gin Thr Leu Glu Trp Gin Ala Leu Ser Ser ^ 15 10 15 0 gag cat cac ctg gca ccg ttc age gac tac aaa caa ctg aaa gag aaa 96

Glu His His Leu Ala Pro Phe Ser Asp Tyr Lys Gin Leu Lys Glu Lys 20 25 30 ggc ccg ege ate ate acc cgt gcc gag ggc gtt tat ctg tgg gac age 144

Gly Pro Arg lie He Thr Arg Ala Glu Gly Val Tyr Leu Trp Asp Ser 35 40 45 gag ggc aac aag ate etc gat ggc atg tee ggc ctg tgg tgc gtg gcc 192

Glu Gly Asn Lys lie Leu Asp Gly Met Ser Gly Leu Trp Cys Val Ala 50 55 60 · ate ggt tat ggc ege gaa gaa ctg gcc gac gca gcc age aaa cag atg 240 lie Gly Tyr Gly Arg Glu Glu Leu Ala Asp Ala Ala Ser Lys Gin Met 65 70 75 80 ege gag ctg ccg tac tac aac ctg ttc ttc cag acc gcc cac ccg ccg 288

Arg Glu Leu Pro Tyr Tyr Asn Leu Phe Phe Gin Thr Ala His Pro Pro 85 90 95 16 336 336

201033369 gtg ctg gaa ctg gcc aag gcc ate tee gac ate get ccc gag ggc atg

Val Leu Glu Leu Ala Lys Ala lie Ser Asp lie Ala Pro Glu Gly Met 100 105 110 aac cat gtg ttc ttc acc ggt tea ggc tet gaa ggc aat gac aeg atg

Asn His Val Phe Phe Thr Gly Ser Gly Ser Glu Gly Asn Asp Thr Met 115 120 125 ctg ege atg gtt cgt cat tac tgg geg ctg aaa ggc cag ccg aac aag

Leu Arg Met Val Arg His Tyr Trp Ala Leu Lys Gly Gin Pro Asn Lys 130 135 140 aaa acc ate ate age ege gtc aat ggc tac cac ggc tee acc gtc gcc

Lys Thr lie lie Ser Arg Val Asn Gly Tyr His Gly Ser Thr Val Ala 145 150 155 160 ggt gcc age ctg ggt ggc atg acc tac atg cac gaa cag ggc gac ctg

Gly Ala Ser Leu Gly Gly Met Thr Tyr Met His Glu Gin Gly Asp Leu 165 170 175 ccg ate ccg ggg gtg gtg cac att cca cag cct tac tgg ttc ggc gaa

Pro lie Pro Gly Val Val His lie Pro Gin Pro Tyr Trp Phe Gly Glu 180 185 190 ggc ggc gac atg aeg ccg gac gag ttc ggc ate tgg geg gcc gag caa

Gly Gly Asp Met Thr Pro Asp Glu Phe Gly lie Trp Ala Ala Glu Gin 195 200 205 ctg gaa aag aaa att etc gag ctg ggc gtc gag aac gtc ggt geg ttc

Leu Glu Lys Lys He Leu Glu Leu Gly Val Glu Asn Val Gly Ala Phe 210 215 220 att gcc gag cca ate cag ggc geg ggc ggt gtg att gtc ccg cct gat lie Ala Glu Pro lie Gin Gly Ala Gly Gly Val lie Val Pro Pro Asp 225 230 235 240 tee tac tgg ccg aag ate aag ga ate ett tee ege tac gac ate ctg

Ser Tyr Trp Pro Lys He Lys Glu He Leu Ser Arg Tyr Asp lie Leu 245 250 255 ttc gcc gcc gat gag gtg att tgt ggc ttc ggg cgt acc agt gag tgg

Phe Ala Ala Asp Glu Val He Cys Gly Phe Gly Arg Thr Ser Glu Trp 260 265 270 ttc ggt age gat ttc tat ggc etc agg ccg gac atg atg acc ate gcc

Phe Gly Ser Asp Phe Tyr Gly Leu Arg Pro Asp Met Met Thr lie Ala 275 280 285 aaa ggc ctg acc tee ggt tac gta ccg atg ggc ggc ctg ate gtg ege

Lys Gly Leu Thr Ser Gly Tyr Val Pro Met Gly Gly Leu He Val Arg 290 295 300 gat gaa ate gtt geg gtg etc aat gag ggt ggc gat ttc aat cac ggc

Asp Glu lie Val Ala Val Leu Asn Glu Gly Gly Asp Phe Asn His Gly 305 310 315 320 ttt acc tac tee ggg cac ccg gtg geg gcc geg gtt geg ctg gag aac

Phe Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu Glu Asn 384 432 480 528 576 624 672 720 768 816 864 912 960 960 17 1008 201033369 325 330 335 ate cgt ate ctg ege gaa gaa aag ate gtc gaa egg gtc agg teg Gaa 1056 lie Arg lie Leu Arg Glu Glu Lys He Val Glu Arg Val Arg Ser Glu 340 345 350 aeg gca ccg tat ttg caa aag cgt ttg cgt gag ttg age gat cat ccg 1104

Thr Ala Pro Tyr Leu Gin Lys Arg Leu Arg Glu Leu Ser Asp His Pro 355 360 365 ctg gtg ggc gaa gtc egg ggt gtc ggg ctg etc ggg gee att gag ctg 1152

Leu Val Gly Glu Val Arg Gly Val Gly Leu Leu Gly Ala lie Glu Leu 370 375 380 gtg aag gac aag acc acc ege gag ege tat acc gac aag ggc geg gga 1200

Val Lys Asp Lys Thr Thr Arg Glu Arg Tyr Thr Asp Lys Gly Ala Gly 385 390 395 400

Atg ate tgt ega acc ttc tgc ttc gac aat ggc ctg ate atg egg get 1248

Met lie Cys Arg Thr Phe Cys Phe Asp Asn Gly Leu lie Met Arg Ala 405 410 415 gtg ggc gat acc atg ate att geg ccg cca ctg gtg ate agt ttt geg 1296

Val Gly Asp Thr Met lie He Ala Pro Pro Leu Val lie Ser Phe Ala 420 425 430 caa ate gat gag ctg gta gag aag geg ege aeg tgt ctg gat ctg aeg 1344

Gin lie Asp Glu Leu Val Glu Lys Ala Arg Thr Cys Leu Asp Leu Thr 435 440 445 ctg geg gtg ttg cag ggc tga 1365

Leu Ala Val Leu Gin Gly 450 <210> 12 <211> 454

<212> PRT <213> Pseudomonas aeruginosa <400> 12

Met Ser Ala Asn Asn Pro Gin Thr Leu Glu Trp Gin Ala Leu Ser Ser 15 10 15

Glu His His Leu Ala Pro Phe Ser Asp Tyr Lys Gin Leu Lys Glu Lys 20 25 30

Gly Pro Arg He lie Thr Arg Ala Glu Gly Val Tyr Leu Trp Asp Ser 35 40 45

Glu Gly Asn Lys He Leu Asp Gly Met Ser Gly Leu Trp Cys Val Ala 50 55 60 lie Gly Tyr Gly Arg Glu Glu Leu Ala Asp Ala Ala Ser Lys Gin Met 18 201033369 65 70 75 80

Arg Glu Leu Pro Tyr Tyr Asn Leu Phe Phe Gin Thr Ala His Pro Pro 85 90 95

Val Leu Glu Leu Ala Lys Ala lie Ser Asp lie 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 Gin Pro Asn Lys 130 135 140 _ Lys Thr He He 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 Gin Gly Asp Leu 165 170 175

Pro lie Pro Gly Val Val His He Pro Gin Pro Tyr Trp Phe Gly Glu 180 185 190

Gly Gly Asp Met Thr Pro Asp Glu Phe Gly He Trp Ala Ala Glu Gin 195 200 205 - Leu Glu Lys Lys He Leu Glu Leu Gly Val Glu Asn Val Gly Ala Phe 210 215 220 lie Ala Glu Pro lie Gin Gly Ala Gly Gly Val Lie Val Pro Pro Asp 225 230 235 240

Ser Tyr Trp Pro Lys lie Lys Glu He Leu Ser Arg Tyr Asp lie Leu 245 250 255

Phe Ala Ala Asp Glu Val lie 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 lie Ala 275 280 285

Lys Gly Leu Thr Ser Gly Tyr Val Pro Met Gly Gly Leu He Val Arg 290 295 300 19 201033369

Asp Glu He 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

He Arg lie Leu Arg Glu Glu Lys lie Val Glu Arg Val Arg Ser Glu 340 345 350

Thr Ala Pro Tyr Leu Gin 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 lie 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 lie Cys Arg Thr Phe Cys Phe Asp Asn Gly Leu lie Met Arg Ala 405 410 415

Val Gly Asp Thr Met He He Ala Pro Pro Leu Val lie Ser Phe Ala 420 425 430

Gin lie Asp Glu Leu Val Glu Lys Ala Arg Thr Cys Leu Asp Leu Thr 435 440 445

Leu Ala Val Leu Gin Gly ❺ <210> 13 <211> 1365 <212> DNA <213>artificial<220><223> Pseudomonas aeruginosa codon-optimized transaminase gene < 400> 13 atgtctgcta acaatccaca aactctggaa tggcaggcac tgagctccga acatcacctg 60 gctccgttct ccgactacaa acaactgaaa gagaaaggcc cgcgtatcat tacccgcgct 120 gaaggtgtgt acctgtggga ttctgaaggc aacaaaattc tggacggtat gagcggcctg 180 tggtgcgtag caatcggtta tggccgtgaa gaactggctg acgcggcgag caaacagatg 240 cgtgaactgc cgtattataa cctgttcttc caaaccgcac acccgccggt tctggaactg 300 20 201033369

gctaaagcta ggtagcgaag cagccgaaca ggtgcgagcc gtagtgcaca ttcggcatct gtcggcgcgt agctactggc gaagtgatct cgtccggaca ctgatcgtgc ttcacctatt cgtgaagaaa ctgcgcgaac gcgatcgagc atgatctgcc atgatcattg gcgcgcactt tcagcgatat gcaacgacac agaaaacgat tgggcggtat ttccgcagcc gggcggcaga tcatcgcgga caaaaatcaa gcggttttgg tgatgaccat gcgacgaaat ccggtcaccc agatcgtaga tgagcgacca tggtgaaaga gtaccttttg ctccgcctct gtctggatct cgcaccggag gatgctgcgc tatcagccgt gacctacatg gtattggttc gcagctggaa accgattcag agagatcctg ccgcacctct cgccaaaggc tgttgcggtt agttgctgct acgcgtacgt ccctctggta caaaactacc cttcgataac ggttatttct gactctggct ggcatgaatc atggtacgtc gtaaacggtt cacgaacagg ggtgaaggcg aagaaaatcc ggcgcgggcg tctcgttacg gaatggttcg ctgacctccg ctgaacgaag gctgtagcac tccgaaaccg ggtgaagttc cgtgaacgtt ggtctgatca tttgcccaga gttctgcagg acgtcttctt actattgggc atcacggcag gtgacctgcc gtgacatgac tggaactggg gcgtaattgt acatcctgtt gctccgactt gttatgttcc gcggcgattt tggaaaacat caccttacct gcggcgtggg acaccgacaa tgcgcgcagt ttgatgagct gttaa cactggttcc gctg Aagggc caccgttgcg gatcccgggt gccggacgaa cgtggaaaac tccgccggac cgccgcagac ctacggtctg tatgggtggc caaccacggc ccgcatcctg gcagaagcgc cctgctgggc aggcgcaggc cggtgatacc ggtcgaaaaa 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1365

<210> 14 <211> 849 <212> DNA <213> Bacillus subtilis (gi 16078032) <220> <221> CDS <222> (1)..(849) <400> 14 48 96 atg aag gtt tta gtc aat ggc egg ctg att ggg ege agt gaa gca tea

Met Lys Val Leu Val Asn Gly Arg Leu He Gly Arg Ser Glu Ala Ser 15 10 15 ate gat ttg gaa gat ege ggt tat cag ttt ggt gac ggc ate tat gaa lie Asp Leu Glu Asp Arg Gly Tyr Gin Phe Gly Asp Gly lie Tyr Glu 20 25 30 gtg ate agg gtg tac aaa gga gta ttg ttc ggc tta cgt gag cat gca

Val He Arg Val Tyr Lys Gly Val Leu Phe Gly Leu Arg Glu His Ala 35 40 45 21 144 201033369 gag cgt ttt ttc aga agt get get gaa ate gga att tea ctg cca ttc 192

Glu Arg Phe Phe Arg Ser Ala Ala Glu lie Gly lie Ser Leu Pro Phe 50 55 60 agt ata gaa gat etc gag tgg gac ctg caa aag ett gta cag gaa aat 240

Ser lie Glu Asp Leu Glu Trp Asp Leu Gin Lys Leu Val Gin Glu Asn 65 70 75 80 geg gtc agt gag gga geg gta tac att cag aca aca aga ggt gtg gee 288

Ala Val Ser Glu Gly Ala Val Tyr lie Gin Thr Thr Arg Gly Val Ala 85 90 95 ccg ega aaa cac cag tat gaa gee ggc etc gag ccg cag act act gee 336

Pro Arg Lys His Gin Tyr Glu Ala Gly Leu Glu Pro Gin Thr Thr Ala 100 105 110 tat aeg ttt aeg gtg aaa aaa ccg gag caa gag cag gca tac gga gtg 384

Tyr Thr Phe Thr Val Lys Lys Pro Glu Gin Glu Gin Ala Tyr Gly Val 115 120 125 geg gee att aca gat gag gat ett ege tgg tta aga tgt gat ate aaa 432

Ala Ala lie Thr Asp Glu Asp Leu Arg Trp Leu Arg Cys Asp lie Lys 130 135 140 agt ctg aat tta ctg tat aat gtc atg aeg aag caa agg gee tat gaa 480

Ser Leu Asn Leu Leu Tyr Asn Val Met Thr Lys Gin Arg Ala Tyr Glu 145 150 155 160 gee gga gca ttt gaa gee att tta ett agg gac ggc gtt gtt aeg gag 528

Ala Gly Ala Phe Glu Ala lie Leu Leu Arg Asp Gly Val Val Thr Glu 165 170 175 ggt aca tee tet aac gtt tat gee gtt ate aac ggc aca gtg ega aca 576

Gly Thr Ser Ser Asn Val Tyr Ala Val He Asn Gly Thr Val Arg Thr 180 185 190 s cat ccg get aat egg etc att etc aat gga att aca egg atg aat att 624

His Pro Ala Asn Arg Leu lie Leu Asn Gly lie Thr Arg Met Asn lie 195 200 205 tta gga ctg att gag aag aat ggg ate aaa ctg gat gag act cct gtc 672

Leu Gly Leu He Glu Lys Asn Gly lie Lys Leu Asp Glu Thr Pro Val 210 215 220 agt gaa gaa gag ttg aaa cag geg gaa gag ate ttt att teg tea aeg 720

Ser Glu Glu Glu Leu Lys Gin Ala Glu Glu lie Phe He Ser Ser Thr 225 230 235 240 aeg gca gaa att att ccg gtc gtg aeg etc gat gga caa teg ate gga 768

Thr Ala Glu lie lie Pro Val Val Thr Leu Asp Gly Gin Ser lie Gly 245 250 255 age ggg aaa ccc gga ccg gtg acc aaa cag ett cag get get ttt caa 816

Ser Gly Lys Pro Gly Pro Val Thr Lys Gin Leu Gin Ala Ala Phe Gin 260 265 270 gaa age att caa cag get get age att tea taa 849

Glu Ser He Gin Gin Ala Ala Ser He Ser 22 201033369 275 280

<210> 15 <211> 282 <212> PRT <213> Bacillus subtilis (gi 16078032) <400> 15

Met Lys Val Leu Val Asn Gly Arg Leu He Gly Arg Ser Glu Ala Ser 15 10 15 lie Asp Leu Glu Asp Arg Gly Tyr Gin Phe Gly Asp Gly He Tyr Glu 20 25 30 _ Val lie Arg Val Tyr Lys Gly Val Leu Phe Gly Leu Arg Glu His Ala ❿ 35 40 45

Glu Arg Phe Phe Arg Ser Ala Ala Glu lie Gly lie Ser Leu Pro Phe 50 55 60

Ser lie Glu Asp Leu Glu Trp Asp Leu Gin Lys Leu Val Gin Glu Asn 65 70 75 80

Ala Val Ser Glu Gly Ala Val Tyr lie Gin Thr Thr Arg Gly Val Ala 85 90 95

Pro Arg Lys His Gin Tyr Glu Ala Gly Leu Glu Pro Gin Thr Thr Ala 100 105 110 _ Tyr Thr Phe Thr Val Lys Lys Pro Glu Gin Glu Gin Ala Tyr Gly Val 115 120 125

Ala Ala lie Thr Asp Glu Asp Leu Arg Trp Leu Arg Cys Asp lie Lys 130 135 140

Ser Leu Asn Leu Leu Tyr Asn Val Met Thr Lys Gin Arg Ala Tyr Glu 145 150 155 160

Ala Gly Ala Phe Glu Ala lie Leu Leu Arg Asp Gly Val Val Thr Glu 165 170 175

Gly Thr Ser Ser Asn Val Tyr Ala Val He Asn Gly Thr Val Arg Thr 180 185 190

His Pro Ala Asn Arg Leu lie Leu Asn Gly He Thr Arg Met Asn lie 23 201033369 195 200 205

Leu Gly Leu lie Glu Lys Asn Gly lie Lys Leu Asp Glu Thr Pro Val 210 215 220

Ser Glu Glu Glu Leu Lys Gin Ala Glu Glu lie Phe lie Ser Ser Thr 225 230 235 240

Thr Ala Glu lie lie Pro Val Val Thr Leu Asp Gly Gin Ser lie Gly 245 250 255

Ser Gly Lys Pro Gly Pro Val Thr Lys Gin Leu Gin Ala Ala Phe Gin 260 265 270

Glu Ser lie Gin Gin Ala Ala Ser He Ser 275 280

<210> 16 <211> 1347 <212> DNA <213> Bacillus subtilis (gi 16080075) <220> <221> CDS <222> (1)..(1347) <400> 16 atg act cat gat ttg ata gaa aaa agt aaa aag cac etc tgg ctg cca 48

Met Thr His Asp Leu lie Glu Lys Ser Lys Lys His Leu Trp Leu Pro 15 10 15 ❺ ttt acc caa atg aaa gat tat gat gaa aac ccc tta ate ate gaa age 96

Phe Thr Gin Met Lys Asp Tyr Asp Glu Asn Pro Leu lie He Glu Ser 20 25 30 ggg act gga ate aaa gtc aaa gac ata aac ggc aag gaa tac tat gac 144

Gly Thr Gly lie Lys Val Lys Asp lie Asn Gly Lys Glu Tyr Tyr Asp 35 40 45 ggt ttt tea teg gtt tgg ett aat gtc cac gga cac ege aaa aaa gaa 192

Gly Phe Ser Ser Val Trp Leu Asn Val His Gly His Arg Lys Lys Glu 50 55 60 eta gat gac gcc ata aaa aaa cag etc gga aaa att geg cac tcc aeg 240

Leu Asp Asp Ala He Lys Lys Gin Leu Gly Lys lie Ala His Ser Thr 65 70 75 80 tta ttg ggc atg acc aat gtt cca gca acc cag ett gcc gaa aca tta 288

Leu Leu Gly Met Thr Asn Val Pro Ala Thr Gin Leu Ala Glu Thr Leu 85 90 95 ate gac ate age cca aaa aag etc aeg egg gtc ttt tat tea gac age 336 24 384 384

201033369 lie Asp He Ser Pro Lys Lys Leu Thr Arg Val Phe Tyr Ser Asp Ser 100 105 110 ggc gca gag gcg atg gaa ata gcc eta aaa atg geg ttt cag tat tgg

Gly Ala Glu Ala Met Glu lie Ala Leu Lys Met Ala Phe Gin Tyr Trp 115 120 125 aag aac ate ggg aag ccc gag aaa caa aaa ttc ate gca atg aaa aac

Lys Asn lie Gly Lys Pro Glu Lys Gin Lys Phe lie Ala Met Lys Asn 130 135 140 ggg tat cac ggt gat aeg att ggc gcc gtc agt gtc ggt tea att gag

Gly Tyr His Gly Asp Thr lie Gly Ala Val Ser Val Gly Ser He Glu 145 150 155 160 ett ttt cac cac gta tac ggc ccg ttg atg ttc gag agt tac aag gcc

Leu 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 tet gaa age ggt gat cct gat gag tgc

Pro He Pro Tyr Val Tyr Arg Ser Glu Ser Gly Asp Pro Asp Glu Cys 180 185 190 • cgt gat cag tgc etc ega gag ett gca cag ctg ett gag gaa cat cat

Arg Asp Gin Cys Leu Arg Glu Leu Ala Gin Leu Leu Glu Glu His His . 195 200 205 gag gaa att gcc gcg ett tcc att gaa tea atg gta caa ggc gcg tcc - Glu Glu He Ala Ala Leu Ser lie Glu Ser Met Val Gin Gly Ala Ser 210 215 220 ggt atg ate gtg atg ccg gaa gga tat ttg gca ggc gtg ege gag eta

Gly Met lie 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 ate gtt gat gaa gtc get aca ggc

Cys Thr Thr Tyr Asp Val Leu Met lie 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

Phe Gly Arg Thr Gly Lys Met Phe Ala Cys Glu His Glu Asn Val Gin 260 265 270 cct gat ctg atg get gcc ggt aaa ggc att aca gga ggc tat ttg cca

Pro Asp Leu Met Ala Ala Gly Lys Gly He Thr Gly Gly Tyr Leu Pro 275 280 285 att gcc gtt aeg ttt gcc act gaa gac ate tat aag gca ttc tat gat lie Ala Val Thr Phe Ala Thr Glu Asp He Tyr Lys Ala Phe Tyr Asp 290 295 300 gat tat gaa aac eta aaa acc ttt ttc cat ggc cat tcc tat aca ggc

Asp Tyr Glu Asn Leu Lys Thr Phe Phe His Gly His Ser Tyr Thr Gly 305 310 315 320 aat cag ett ggc tgt gcg gtt gcg ett gaa aat ctg gca tta ttt gaa

Asn Gin Leu Gly Cys Ala Val Ala Leu Glu Asn Leu Ala Leu Phe Glu 325 330 335 432 480 528 576 624 672 720 768 816 864 912 912 960 25 1008 201033369 tct gaa aac att gtg gaa caa gta gcg gaa aaa agt aaa aag etc cat 1056

Ser Glu Asn lie Val Glu Gin Val Ala Glu Lys Ser Lys Lys Leu His 340 345 350 ttt ett ett caa gat ctg cac get ett cct cat gtt ggg gat att egg 1104

Phe Leu Leu Gin Asp Leu His Ala Leu Pro His Val Gly Asp He Arg 355 360 365 cag ett ggc ttt atg tgc ggt gca gag ett gta ega tea aag gaa act 1152

Gin Leu Gly Phe Met Cys Gly Ala Glu Leu Val Arg Ser Lys Glu Thr 370 375 380 aaa gaa cct tac ccg get gat egg egg att gga tac aaa gtt tee tta 1200

Lys Glu Pro Tyr Pro Ala Asp Arg Arg lie Gly Tyr Lys Val Ser Leu 385 390 395 400 aaa atg aga gag tta gga atg ctg aca aga ccg ett ggg gac gtg att 1248

Lys Met Arg Glu Leu Gly Met Leu Thr Arg Pro Leu Gly Asp Val lie ❹ 405 410 415 gca ttt ett cct cct ett gee age aca get gaa gag etc teg gaa atg 1296

Ala Phe Leu Pro Pro Leu Ala Ser Thr Ala Glu Glu Leu Ser Glu Met 420 425 430 gtt gee att atg aaa caa gcg ate cac gag gtt aeg age ett gaa gat 1344

Val Ala lie Met Lys Gin Ala lie His Glu Val Thr Ser Leu Glu Asp 435 440 445 tga 1347

<210> 17 <211> 448 <212> PRT < 213 > Bacillus subtilis (gi 16080075) <400>

Met Thr His Asp Leu He Glu Lys Ser Lys Lys His Leu Trp Leu Pro 15 10 15

Phe Thr Gin Met Lys Asp Tyr Asp Glu Asn Pro Leu He lie Glu Ser 20 25 30

Gly Thr Gly He Lys Val Lys Asp lie 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 lie Lys Lys Gin Leu Gly Lys lie Ala His Ser Thr 65 70 75 80

Leu Leu Gly Met Thr Asn Val Pro Ala Thr Gin Leu Ala Glu Thr Leu 26 201033369 85 90 95 lie Asp lie Ser Pro Lys Lys Leu Thr Arg Val Phe Tyr Ser Asp Ser 100 105 110

Gly Ala Glu Ala Met Glu lie Ala Leu Lys Met Ala Phe Gin Tyr Trp 115 120 125

Lys Asn lie Gly Lys Pro Glu Lys Gin Lys Phe lie Ala Met Lys Asn 130 135 140

Gly Tyr His Gly Asp Thr He Gly Ala Val Ser Val Gly Ser lie Glu 145 150 155 160 Φ

Leu Phe His His Val Tyr Gly Pro Leu Met Phe Glu Ser Tyr Lys Ala 165 170 175

Pro lie Pro Tyr Val Tyr Arg Ser Glu Ser Gly Asp Pro Asp Glu Cys 180 185 190

Arg Asp Gin Cys Leu Arg Glu Leu Ala Gin Leu Leu Glu Glu His His 195 200 205

Glu Glu lie Ala Ala Leu Ser He Glu Ser Met Val Gin Gly Ala Ser 210 215 220

Gly Met He 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 lie 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 Gin 260 265 270

Pro Asp Leu Met Ala Ala Gly Lys Gly lie Thr Gly Gly Tyr Leu Pro 275 280 285

He Ala Val Thr Phe Ala Thr Glu Asp He 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 27 201033369

Asn Gin Leu Gly Cys Ala Val Ala Leu Glu Asn Leu Ala Leu Phe Glu 325 330 335

Ser Glu Asn lie Val Glu Gin Val Ala Glu Lys Ser Lys Lys Leu His 340 345 350

Phe Leu Leu Gin Asp Leu His Ala Leu Pro His Val Gly Asp lie Arg 355 360 365

Gin 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 He 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 lie 405 410 415

Ala Phe Leu Pro Pro Leu Ala Ser Thr Ala Glu Glu Leu Ser Glu Met 420 425 430

Val Ala He Met Lys Gin Ala He His Glu Val Thr Ser Leu Glu Asp 435 440 445 <210> 18 <211> 1467 <212> DNA <213>

<220〉 <221> CDS <222> (1)..(1467) <400> 18 atg ccc ggt tgc ggg ggc ttg ccc ggg aat gaa ccg aaa tgc gga cga 48

Met Pro Gly Cys Gly Gly Leu Pro Gly Asn Glu Pro Lys Cys Gly Arg 15 10 15 gag ggg agg teg geg atg aeg egg aat gac geg aeg aat get gcc gga 96

Glu Gly Arg Ser Ala Met Thr Arg Asn Asp Ala Thr Asn Ala Ala Gly 20 25 30 geg gtg ggc geg geg atg egg gat cac ate etc ttg cct gca cag gaa 144

Ala Val Gly Ala Ala Met Arg Asp His lie Leu Leu Pro Ala Gin Glu 35 40 45 atg geg aag etc ggc aag tcc geg cag ccg gtg ctg act cat gcc gag 192

Met Ala Lys Leu Gly Lys Ser Ala Gin Pro Val Leu Thr His Ala Glu 50 55 60 28 240 240

201033369 ggc ate tat gtc cat acc gag gac ggc ege ege ctg ate gac ggg ccg

Gly lie Tyr Val His Thr Glu Asp Gly Arg Arg Leu lie Asp Gly Pro 65 70 75 80 geg ggc atg tgg tgc geg cag gtg ggc tac ggc ege ege gag ate gtc

Ala Gly Met Trp Cys Ala Gin Val Gly Tyr Gly Arg Arg Glu lie Val 85 90 95 gat gee atg geg cat cag geg atg gtg ctg ccc tat gee teg ccc tgg

Asp Ala Met Ala His Gin Ala Met Val Leu Pro Tyr Ala Ser Pro Trp 100 105 110 tat atg gee aeg age ccc geg geg egg ctg geg gag aag ate gee aeg

Tyr Met Ala Thr Ser Pro Ala Ala Arg Leu Ala Glu Lys lie Ala Thr 115 120 125 ctg aeg ccg ggc gat etc aac egg ate ttt ttc acc aeg ggc ggg teg

Leu Thr Pro Gly Asp Leu Asn Arg He Phe Phe Thr Thr Gly Gly Ser 130 135 140 acc geg gtg gac age geg ctg ege ttc teg gaa ttc tac aac aac gtg

Thr Ala Val Asp Ser Ala Leu Arg Phe Ser Glu Phe Tyr Asn Asn Val 145 150 155 160 ctg ggc egg ccg cag aag aag ege ate ate gtg ege tac gac ggc tat

Leu Gly Arg Pro Gin Lys Lys Arg He He Val Arg Tyr Asp Gly Tyr 165 170 175 cac ggc teg aeg geg etc acc gee gee tgc acc ggc ege acc ggc aac

His Gly Ser Thr Ala Leu Thr Ala Ala Cys Thr Gly Arg Thr Gly Asn 180 185 190 tgg ccg aac ttc gac ate geg cag gac egg ate teg ttc etc teg age

Trp Pro Asn Phe Asp lie Ala Gin Asp Arg lie Ser Phe Leu Ser Ser 195 200 205 ccc aat ccg ege cac gee ggc aac ege age cag gag geg ttc etc gac

Pro Asn Pro Arg His Ala Gly Asn Arg Ser Gin Glu Ala Phe Leu Asp 210 215 220 gat ctg gtg cag gaa ttc gag gac egg ate gag age etc ggc ccc gac

Asp Leu Val Gin Glu Phe Glu Asp Arg lie Glu Ser Leu Gly Pro Asp 225 230 235 240 aeg ate geg gee ttc ctg gee gag ccg ate etc gee teg ggc ggc gtc

Thr lie Ala Ala Phe Leu Ala Glu Pro lie Leu Ala Ser Gly Gly Val 245 250 255 att att ccg ccc gca ggc tat cat geg ege ttc aag geg ate tgc gag lie He Pro Pro Ala Gly Tyr His Ala Arg Phe Lys Ala He Cys Glu 260 265 270 aag cac gac ate etc tat ate teg gac gag gtg gtg aeg ggc ttc ggc

Lys His Asp He Leu Tyr lie Ser Asp Glu Val Val Thr Gly Phe Gly 275 280 285 cgt tgc ggc gag tgg ttc gee teg gag aag gtg ttc ggg gtg gtg ccg

Arg Cys Gly Glu Trp Phe Ala Ser Glu Lys Val Phe Gly Val Val Pro 290 295 300 288 336 384 432 480 528 576 624 672 720 768 816 864 29 912 201033369 gac ate ate acc ttc gee aag ggc gtg acc teg ggc tat gtg ccg Etc 960

Asp He lie Thr Phe Ala Lys Gly Val Thr Ser Gly Tyr Val Pro Leu 305 310 315 320 ggc ggc ett geg ate tee gag geg gtg ctg geg egg ate teg ggc gag 1008

Gly Gly Leu Ala lie Ser Glu Ala Val Leu Ala Arg lie Ser Gly Glu 325 330 335 aat gee aag gga age tgg ttc acc aac ggc tat acc tac age aat cag 1056

Asn Ala Lys Gly Ser Trp Phe Thr Asn Gly Tyr Thr Tyr Ser Asn Gin 340 345 350 ccg gtg gee tgc gee geg geg ett gee aac ate gag ctg atg gag ege 1104

Pro Val Ala Cys Ala Ala Ala Leu Ala Asn lie Glu Leu Met Glu Arg 355 360 365 gag ggc ate gtc gat cag geg ege gag atg geg gac tat ttc gee geg 1152 ❹

Glu Gly lie Val Asp Gin Ala Arg Glu Met Ala Asp Tyr Phe Ala Ala 370 375 380 geg ctg get teg ctg ege gat ctg ccg ggc gtg geg gaa acc egg teg 1200

Ala Leu Ala Ser Leu Arg Asp Leu Pro Gly Val Ala Glu Thr Arg Ser 385 390 395 400 gtg ggc etc gtg ggt tgc gtg caa tgc ctg etc gac ccg acc egg geg 1248

Val Gly Leu Val Gly Cys Val Gin Cys Leu Leu Asp Pro Thr Arg Ala 405 410 415 gac ggc aeg gee gag gac aag gee ttc acc ctg aag ate gac gag ege 1296

Asp Gly Thr Ala Glu Asp Lys Ala Phe Thr Leu Lys lie Asp Glu Arg 420 425 430 tgc ttc gag etc ggg ctg ate gtg ege ccg ctg ggc gat etc tgc gtg 1344

Cys Phe Glu Leu Gly Leu lie Val Arg Pro Leu Gly Asp Leu Cys Val 435 440 445 ❹ ate teg ccg ccg ate ate teg ege geg cag ate gac gag atg gtc 1392 lie Ser Pro Pro Leu lie He Ser Arg Ala Gin lie Asp Glu Met Val 450 455 460 geg ate atg egg cag gee ate acc gaa gtg age gee gee cac ggt ctg 1440

Ala lie Met Arg Gin Ala He Thr Glu Val Ser Ala Ala His Gly Leu 465 470 475 480 acc geg aaa gaa ccg gee gee gtc tga 1467

Thr Ala Lys Glu Pro Ala Ala Val 485 <210> 19 <211> 488 <212> PRT <213> Rhodobacter sphaeroides <400> 19

Met Pro Gly Cys Gly Gly Leu Pro Gly Asn Glu Pro Lys Cys Gly Arg 1 5 10 15 30 201033369

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 lie Leu Leu Pro Ala Gin Glu 35 40 45

Met Ala Lys Leu Gly Lys Ser Ala Gin Pro Val Leu Thr His Ala Glu 50 55 60

Gly He Tyr Val His Thr Glu Asp Gly Arg Arg Leu lie Asp Gly Pro 65 70 75 80

Ala Gly Met Trp Cys Ala Gin Val Gly Tyr Gly Arg Arg Glu lie Val 85 90 95

Asp Ala Met Ala His Gin 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 He Ala Thr 115 120 125

Leu Thr Pro Gly Asp Leu Asn Arg lie 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 Gin Lys Lys Arg He He 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 He Ala Gin Asp Arg lie Ser Phe Leu Ser Ser 195 200 205

Pro Asn Pro Arg His Ala Gly Asn Arg Ser Gin Glu Ala Phe Leu Asp 210 215 220

Asp Leu Val Gin Glu Phe Glu Asp Arg He Glu Ser Leu Gly Pro Asp 225 230 235 240

Thr lie Ala Ala Phe Leu Ala Glu Pro He Leu Ala Ser Gly Gly Val 31 201033369 245 250 255 lie He Pro Pro Ala Gly Tyr His Ala Arg Phe Lys Ala He Cys Glu 260 265 270

Lys His Asp lie Leu Tyr lie 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 He lie Thr Phe Ala Lys Gly Val Thr Ser Gly Tyr Val Pro Leu 305 310 315 320

Gly Gly Leu Ala lie Ser Glu Ala Val Leu Ala Arg lie Ser Gly Glu 325 330 335

Asn Ala Lys Gly Ser Trp Phe Thr Asn Gly Tyr Thr Tyr Ser Asn Gin 340 345 350

Pro Val Ala Cys Ala Ala Ala Leu Ala Asn He Glu Leu Met Glu Arg 355 360 365

Glu Gly lie Val Asp Gin 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 Gin Cys Leu Leu Asp Pro Thr Arg Ala 405 410 415

Asp Gly Thr Ala Glu Asp Lys Ala Phe Thr Leu Lys He Asp Glu Arg 420 425 430

Cys Phe Glu Leu Gly Leu lie Val Arg Pro Leu Gly Asp Leu Cys Val 435 440 445 lie Ser Pro Pro Leu lie lie Ser Arg Ala Gin lie Asp Glu Met Val 450 455 460

Ala lie Met Arg Gin Ala lie Thr Glu Val Ser Ala Ala His Gly Leu 465 470 475 480 32

201033369

Thr Ala Lys Glu Pro Ala Ala Val 485

<210> 20 <211> 837 <212> DNA <213>##性退军人病<220><221> CDS <222> (1)..(837) <400&gt 20 atg agt ate gca ttt gtt aac ggc aag tat tgt tgt caa tet gaa gca

Met Ser He Ala Phe Val Asn Gly Lys Tyr Cys Cys Gin Ser Glu Ala 1 5 10 15 Lu aaa att tea ata ttt gat ega ggg ttt ett ttt ggt gac teg gtt tat

Lys lie Ser lie Phe Asp Arg Gly Phe Leu Phe Gly Asp Ser Val Tyr 2〇 25 30 - gaa gtg ctg cct gtt tac cat ggg cag cct tac ttt gta gac caa cat

Glu Val Leu Pro Val Tyr His Gly Gin Pro Tyr Phe Val Asp Gin His . 35 40 45 ett gac ega tta ttc tea aat atg aaa aaa att ag atg att ata cca • Leu Asp Arg Leu Phe Ser Asn Met Lys Lys lie Lys Met Lie lie Pro 5〇55 60 aat tat gat tgg cat ggt tta att cat aga eta ata tea gaa aat aat

Asn Tyr Asp Trp His Gly Leu lie His Arg Leu lie Ser Glu Asn Asn 65 70 75 80 ,- ggc ggt aat tta caa gta tat ate caa gtc aca ega ggg aat caa ggg

Gly Gly Asn Leu Gin Val Tyr He Gin Val Thr Arg Gly Asn Gin Gly gtg ege aag cat gat ate cct act tee ate aca cct tet gtt ate gca

Val Arg Lys His Asp He Pro Thr Ser He Thr Pro Ser Val lie Ala 100 105 110 ttc act atg cat aat cca ttt ccc acc etc gaa gat aag gaa cag gga

Phe Thr Met His Asn Pro Phe Pro Thr Leu Glu Asp Lys Glu Gin Gly 115 120 125 atg tea gca aaa ctg gtt gaa gat ttt egg tgg atg aga tgt gat ata

Met Ser Ala Lys Leu Val Glu Asp Phe Arg Trp Met Arg Cys Asp lie 130 135 140 aaa act act tet tta att gee aat ata tta ctg aat gat gag get gta

Lys Thr Thr Ser Leu lie Ala Asn lie Leu Leu Asn Asp Glu Ala Val 145 150 155 160 tet gca gga ttc cac act gca att ett gee egg aac ggt eta att aca

Ser Ala Gly Phe His Thr Ala He Leu Ala Arg Asn Gly Leu lie Thr 165 170 175 48 96 144 192 240 288 336 384 432 480 33 528 201033369 gag gga agt agt acc aac gta ttt att gtc gca cag gat ggt gtt att 576

Glu Gly Ser Ser Thr Asn Val Phe lie Val Ala Gin Asp Gly Val lie 180 185 190 aag aca cca ccc atg aat aat ttc tgt tta cca gga att act egg caa 624

Lys Thr Pro Pro Met Asn Asn Phe Cys Leu Pro Gly lie Thr Arg Gin 195 200 205 gtt gtt att gaa ata att aaa aaa tta gat tta aag ttc aga gaa ata 672

Val Val lie Glu lie lie Lys Lys Leu Asp Leu Lys Phe Arg Glu lie 210 215 220 gaa att age att tea gag ett ttt tet get cag gaa gtt tgg ata aca 720

Glu lie Ser lie Ser Glu Leu Phe Ser Ala Gin Glu Val Trp lie Thr 225 230 235 240 agt aeg aca aaa gaa gta ttc cct att aca aag att aat gac tet ttg 768

Ser Thr Thr Lys Glu Val Phe Pro He Thr Lys He Asn Asp Ser Leu 245 250 255

Att aat ggc gga aaa gtt ggc gaa tat tgg egg ata att aat gat tcc 816 lie Asn Gly Gly Lys Val Gly Glu Tyr Trp Arg lie lie Asn Asp Ser 260 265 270 tac caa caa eta gta aac taa 837

Tyr Gin Gin Leu Val Asn 275

<210> 21 <211> 278 <212> PRT <213> Lungophilic Legionnaires' Disease <400> 21

Met Ser lie Ala Phe Val Asn Gly Lys Tyr Cys Cys Gin Ser Glu Ala 15 10 15

Lys lie Ser lie Phe Asp Arg Gly Phe Leu Phe Gly Asp Ser Val Tyr 20 25 30

Glu Val Leu Pro Val Tyr His Gly Gin Pro Tyr Phe Val Asp Gin His 35 40 45

Leu Asp Arg Leu Phe Ser Asn Met Lys Lys lie Lys Met He lie Pro 50 55 60

Asn Tyr Asp Trp His Gly Leu lie His Arg Leu lie Ser Glu Asn Asn 65 70 75 80

Gly Gly Asn Leu Gin Val Tyr lie Gin Val Thr Arg Gly Asn Gin Gly 85 90 95 34 201033369

Val Arg Lys His Asp lie Pro Thr Ser lie Thr Pro Ser Val lie Ala 100 105 110

Phe Thr Met His As Pro Pro Phe Pro Thr Leu Glu Asp Lys Glu Gin Gly 115 120 125

Met Ser Ala Lys Leu Val Glu Asp Phe Arg Trp Met Arg Cys Asp lie 130 135 140

Lys Thr Thr Ser Leu lie Ala Asn He Leu Leu Asn Asp Glu Ala Val 145 150 155 160

Ser Ala Gly Phe His Thr Ala lie Leu Ala Arg Asn Gly Leu lie Thr _ 165 170 175

Glu Gly Ser Ser Thr Asn Val Phe He Val Ala Gin Asp Gly Val He 180 185 190

Lys Thr Pro Pro Met Asn Asn Phe Cys Leu Pro Gly He Thr Arg Gin 195 200 205

Val Val lie Glu He lie Lys Lys Leu Asp Leu Lys Phe Arg Glu lie 210 215 220

Glu He Ser lie Ser Glu Leu Phe Ser Ala Gin Glu Val Trp He Thr 225 230 235 240

Ser Thr Thr Lys Glu Val Phe Pro lie Thr Lys lie Asn Asp Ser Leu φ 245 250 255 lie Asn Gly Gly Lys Val Gly Glu Tyr Trp Arg lie lie Asn Asp Ser 260 265 270

Tyr Gin Gin Leu Val Asn 275 <210> 22 <211> 861 <212> DNA <213> European nitrite bacteria <220>

<221> CDS <222> (1)..(861) 35 201033369 <400> 22 atg att tac etc aat ggc aaa ttt ctg ccg atg gaa cag get acc gtt 48

Met He Tyr Leu Asn Gly Lys Phe Leu Pro Met Glu Gin Ala Thr Val 15 10 15 cca gtg ctg gat aga ggc ttc ate ttc ggt gat ggt gtc tat gaa gtc 96

Pro Val Leu Asp Arg Gly Phe lie Phe Gly Asp Gly Val Tyr Glu Val 20 25 30 ata ccg gtt tat tea cgt aaa ccg ttc egg ctg ggc gaa cat ett tee 144 lie Pro Val Tyr Ser Arg Lys Pro Phe Arg Leu Gly Glu His Leu Ser 35 40 45 egg ctg cag cac agt ctg gat ggc ata cgt etc cag aat ccg cac act 192

Arg Leu Gin His Ser Leu Asp Gly lie Arg Leu Gin Asn Pro His Thr 50 55 60 gaa gaa caa tgg get ggt ctg ate gaa ege ate ate gag ctg aat gaa 240

Glu Glu Gin Trp Ala Gly Leu lie Glu Arg He lie Glu Leu Asn Glu 65 70 75 80 ggt gat gat cag tac ett tac ctg cac att aca ege ggg gtg gca aaa 288

Gly Asp Asp Gin Tyr Leu Tyr Leu His He Thr Arg Gly Val Ala Lys 85 90 95 cgt gac cat gee ttt cct ege gaa gta aeg ccc act gtc ttc ate atg 336

Arg Asp His Ala Phe Pro Arg Glu Val Thr Pro Thr Val Phe lie Met 100 105 110 age aac ccg ett ccg get cca cct gca aaa ttg etc gtt tee gga gtt 384

Ser Asn Pro Leu Pro Ala Pro Pro Ala Lys Leu Leu Val Ser Gly Val 115 120 125 tea geg att acc gee agg gat aat ege tgg ggg ege tgt gat ate aaa 432

Ser Ala lie Thr Ala Arg Asp Asn Arg Trp Gly Arg Cys Asp He Lys 130 135 140

Gee att tea ctg ttg cca aat ate tta ttg ege cag ett gee gtg gac 480

Ala lie Ser Leu Leu Pro Asn He Leu Leu Arg Gin Leu Ala Val Asp 145 150 155 160 gca caa gee atg gaa aeg ate ctg tta ege gat ggt ctg ttg acc gaa 528

Ala Gin Ala Met Glu Thr He Leu Leu Arg Asp Gly Leu Leu Thr Glu 165 170 175 ggg gee gee age aat att ttc ate gta aaa gac gac ctg ctg ctg acc 576

Gly Ala Ala Ser Asn lie Phe lie 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 624

Pro Pro Lys Asp His Arg lie Leu Pro Gly lie Thr Tyr Asp Val Val 195 200 205 ctg gaa ctg get gaa aca cat ggt gtt cca cat geg aca aga gaa ata 672

Leu Glu Leu Ala Glu Thr His Gly Val Pro His Ala Thr Arg Glu lie 210 215 220 tea gag ett gag tta cgt act gca egg gaa ate atg ctg act tet tee 720

Ser Glu Leu Glu Leu Arg Thr Ala Arg Glu He Met Leu Thr Ser Ser 36 768 201033369 225 230 235 240 acc aaa gaa att etc ccg ate aca cag ctg gat gga caa ccg ate ggt

Thr Lys Glu He Leu Pro lie Thr Gin Leu Asp Gly Gin Pro He Gly 245 250 255 aat ggc acc cca ggg cca gta ttt cag caa ctg gat egg etc tat cag

Asn Gly Thr Pro Gly Pro Val Phe Gin Gin Leu Asp Arg Leu Tyr Gin 260 265 270 gca tat aag ctg gaa gtc atg ege ggg cat get cca ege cag taa

Ala Tyr Lys Leu Glu Val Met Arg Gly His Ala Pro Arg Gin 275 280 285

816 861 <210> 23 <211> 286 <212> PRT <213> European nitrite bacteria <400> 23

Met lie Tyr Leu Asn Gly Lys Phe Leu Pro Met Glu Gin Ala Thr Val 15 10 15

Pro Val Leu Asp Arg Gly Phe He Phe Gly Asp Gly Val Tyr Glu Val 20 25 30 lie Pro Val Tyr Ser Arg Lys Pro Phe Arg Leu Gly Glu His Leu Ser 35 40 45

Arg Leu Gin His Ser Leu Asp Gly lie Arg Leu Gin Asn Pro His Thr 50 55 60 A Glu Glu Gin Trp Ala Gly Leu lie Glu Arg lie lie Glu Leu Asn Glu 65 70 75 80

Gly Asp Asp Gin Tyr Leu Tyr Leu His He Thr Arg Gly Val Ala Lys 85 90 95

Arg Asp His Ala Phe Pro Arg Glu Val Thr Pro Thr Val Phe lie 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 He Thr Ala Arg Asp Asn Arg Trp Gly Arg Cys Asp lie Lys 130 135 140

Ala lie Ser Leu Leu Pro Asn lie Leu Leu Arg Gin Leu Ala Val Asp 37 201033369 160 145 150 155

Ala Gin Ala Met Glu Thr lie Leu Leu Arg Asp Gly Leu Leu Thr Glu 165 170 175

Gly Ala Ala Ser Asn lie Phe lie Val Lys Asp Asp Leu Leu Leu Thr 180 185 190

Pro Pro Lys Asp His Arg lie Leu Pro Gly lie Thr Tyr Asp Val Val 195 200 205

Leu Glu Leu Ala Glu Thr His Gly Val Pro His Ala Thr Arg Glu lie 210 215 220

Ser Glu Leu Glu Leu Arg Thr Ala Arg Glu lie Met Leu Thr Ser Ser 225 230 235 240

Thr Lys Glu lie Leu Pro lie Thr Gin Leu Asp Gly Gin Pro He Gly 245 250 255

Asn Gly Thr Pro Gly Pro Val Phe Gin Gin Leu Asp Arg Leu Tyr Gin 260 265 270

Ala Tyr Lys Leu Glu Val Met Arg Gly His Ala Pro Arg Gin 275 280 285

<210> 24 <211> 1293 <212> DNA <213> Neisseria gonorrhoeae<220><221> CDS <222> (1)..(1293) <400> 24 Atg agg ata aat atg aac cgt aac gaa att tta ttc gac cgc gcc aag 48

Met Arg lie Asn Met Asn Arg Asn Glu lie Leu Phe Asp Arg Ala Lys 15 10 15 gcc ate ate ccc ggc ggc gtg aat teg ccc gtg cgc gca ttc ggc age 96

Ala lie lie Pro Gly Gly Val Asn Ser Pro Val Arg Ala Phe Gly Ser 20 25 30 gtc ggc ggc gtg ccg cgc ttc ate aaa aaa gcc gaa ggc geg tat gtt 144

Val Gly Gly Val Pro Arg Phe lie Lys Lys Ala Glu Gly Ala Tyr Val 35 40 45 192 tgg gac gaa aac ggc aeg cgc tac acc gat tat gtc ggc tet tgg ggg 38 240 240

201033369

Trp 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

Pro Ala lie Val Gly His Ala His Pro Glu Val Val Glu Ala Val Arg 65 70 75 80 gaa get gcg ttg ggc ggt ttg teg ttc ggc gcg ccc acc gaa ggc gaa

Glu Ala Ala Leu Gly Gly Leu Ser Phe Gly Ala Pro Thr Glu Gly Glu 85 90 95 ate gcc att gcc gaa caa att gcc gaa att ccg tet gtc gaa egg lie Ala He Ala Glu Gin lie Ala Glu He Met Pro Ser Val Glu Arg 100 105 110 ctg cgc etc gtc age tee ggc aeg gaa gcg aeg atg act gcc ate cgt

Gu gu gu gu gu gu gu gu gu gu gu gu gu gu

Leu Ala Arg Gly Phe Thr Gly Arg Asp Lys lie lie Lys Phe Glu Gly 130 135 140 tgc tac cac ggc cat tee gac age ctg ttg gtg aaa gca ggc age ggt

Cys Tyr His Gly His Ser Asp Ser Leu Leu Val Lys Ala Gly Ser Gly . 145 150 155 160 ctg ett acc ttc ggc aat cct tet tee gcc ggt gtg cct gcc gac ttt . Leu Leu Thr Phe Gly Asn Pro Ser Ser Ala Gly Val Pro Ala Asp Phe 165 170 175 acc aaa cat act ttg gta etc gaa tac aac aac ate gcc caa etc gaa

Thr Lys His Thr Leu Val Leu Glu Tyr Asn Asn lie Ala Gin Leu Glu 180 185 190 • gaa gcc ttt gcc caa age ggc gac gaa ate gcc tgc gtg att gtc gaa

Glu Ala Phe Ala Gin Ser Gly Asp Glu lie Ala Cys Val lie Val Glu 195 200 205 ccc ttc gtc ggc aat atg aac etc gtc cgc ccg acc gaa gcc ttt gtc

Pro 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

Lys Ala Leu Arg Gly Leu Thr Glu Lys His Gly Ala Val Leu lie Tyr 225 230 235 240 gac gaa gtg atg acc ggt ttc cgc gtc gcg etc ggc ggc gcg cag teg

Asp Glu Val Met Thr Gly Phe Arg Val Ala Leu Gly Gly Ala Gin Ser 245 250 255 ctg cac ggc ate aeg ccc gac ctg acc aeg atg ggc aaa gtc ate ggc

Leu His Gly lie Thr Pro Asp Leu Thr Thr Met Gly Lys Val He Gly 260 265 270 ggc ggt atg ccg ett gcc gcg ttc ggc gga cgc aaa gac ate atg gaa

Gly Gly Met Pro Leu Ala Ala Phe Gly Gly Arg Lys Asp lie Met Glu 275 280 285 288 336 384 432 480 528 576 624 672 720 768 816 39 864 912 201033369 tgt att tcc ccg ttg ggc ggc gtg tat cag gca ggt aca tta tea Ggc

Cys lie Ser Pro Leu Gly Gly Val Tyr Gin Ala Gly Thr Leu Ser Gly 290 295 300 aac ccg att gcc gtc gcc gcc ggc ttg aaa aeg ctg gaa ate ate cag

Asn Pro lie Ala Val Ala Ala Gly Leu Lys Thr Leu Glu lie He Gin 305 310 315 320 ege gaa ggc ttc tat gaa aac ctg acc gcc ttg aca caa ege ett gcc

Arg Glu Gly Phe Tyr Glu Asn Leu Thr Ala Leu Thr Gin Arg Leu Ala 325 330 335 aac ggt att gcc gcc gcc aaa geg cac ggt ate gag ttt gcc gcc gac

Asn Gly He Ala Ala Ala Lys Ala His Gly lie Glu Phe Ala Ala Asp 340 345 350 age gtg ggc ggt atg ttc ggt ctg tat ttc gcc gca cac gtg ccg ega

Ser Val Gly Gly Met Phe Gly Leu Tyr Phe Ala Ala His Val Pro Arg 355 360 365 aac tat gcc gat atg geg ege tec aat ate gac get ttc aaa ege ttc

Asn Tyr Ala Asp Met Ala Arg Ser Asn lie Asp Ala Phe Lys Arg Phe 370 375 380 ttc cac ggc atg etc gac ege ggc att gcc ttc ggc ccg tec get tat

Phe His Gly Met Leu Asp Arg Gly lie Ala Phe Gly Pro Ser Ala Tyr 385 390 395 400 gaa geg ggt ttc gtt tec gcc geg cat aeg ccc gag ctg att gac gaa Glu Ala Gly Phe Val Ser Ala Ala His Thr Pro Glu Leu lie Asp Glu 405 410 415 aeg gtt geg gtt geg gtt gaa gtg ttc aag geg atg get gca tga Thr Val Ala Val Ala Val Glu Val Phe Lys Ala Met Ala Ala 420 425 430 <210> 25 <211> 430 <212 〉 PRT <213> Neisseria gonorrhoeae <400〉 25

Met Arg lie Asn Met Asn Arg Asn Glu He Leu Phe Asp Arg Ala Lys 1 5 10 15

Ala lie lie Pro Gly Gly Val Asn Ser Pro Val Arg Ala Phe Gly Ser 20 25 30

Val Gly Gly Val Pro Arg Phe He 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 60 960 1008 1056 1104 1152 1200 1248 1293

40 201033369

Pro Ala lie 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 lie Ala lie Ala Glu Gin lie Ala Glu He Met Pro Ser Val Glu Arg 100 105 110

Leu Arg Leu Val Ser Ser Gly Thr Glu Ala Thr Met Thr Ala He Arg 115 120 125

Leu Ala Arg Gly Phe Thr Gly Arg Asp Lys lie lie 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 lie Ala Gin Leu Glu 180 185 190

Glu Ala Phe Ala Gin Ser Gly Asp Glu He Ala Cys Val He Val Glu 195 200 205 ^ Pro Phe Val Gly Asn Met Asn Leu Val Arg Pro Thr Glu Ala Phe Val φ 210 215 220

Lys Ala Leu Arg Gly Leu Thr Glu Lys His Gly Ala Val Leu lie Tyr 225 230 235 240

Asp Glu Val Met Thr Gly Phe Arg Val Ala Leu Gly Gly Ala Gin Ser 245 250 255

Leu His Gly He Thr Pro Asp Leu Thr Thr Met Gly Lys Val lie Gly 260 265 270

Gly Gly Met Pro Leu Ala Ala Phe Gly Gly Arg Lys Asp lie Met Glu 275 280 285

Cys He Ser Pro Leu Gly Gly Val Tyr Gin Ala Gly Thr Leu Ser Gly 290 295 300 41 201033369

Asn Pro lie Ala Val Ala Ala Gly Leu Lys Thr Leu Glu He lie Gin 305 310 315 320

Arg Glu Gly Phe Tyr Glu Asn Leu Thr Ala Leu Thr Gin Arg Leu Ala 325 330 335

Asn Gly lie Ala Ala Ala Lys Ala His Gly lie 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 He Asp Ala Phe Lys Arg Phe 370 375 380 φ

Phe His Gly Met Leu Asp Arg Gly He Ala Phe Gly Pro Ser Ala Tyr 385 390 395 400

Glu Ala Gly Phe Val Ser Ala Ala His Thr Pro Glu Leu He Asp Glu 405 410 415

Thr Val Ala Val Ala Val Glu Val Phe Lys Ala Met Ala Ala 420 425 430

<210> 26 <211> 924 <212> DNA <213> Pseudomonas aeruginosa (gi 9951299) 参<220> <221> CDS <222> (1).. (924) < 400〉 26 atg teg atg gcc gat cgt gat ggc gtg ate tgg tat gac ggt gaa ctg 48

Met Ser Met Ala Asp Arg Asp Gly Val lie Trp Tyr Asp Gly Glu Leu 15 10 15 · gtg cag tgg ege gac geg acc aeg cac gtg ctg acc cat acc ctg cac 96

Val Gin 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 ege gcc tac gac acc ccg cag 144

Tyr Gly Met Gly Val Phe Glu Gly Val Arg Ala Tyr Asp Thr Pro Gin 35 40 45 ggc aeg geg ate ttc ege ctg cag geg cat acc gac egg ctg ttc gac 192

Gly Thr Ala lie Phe Arg Leu Gin Ala His Thr Asp Arg Leu Phe Asp 42 240 240

201033369 50 55 60 tcc gcg cac ate atg aac atg cag ate ccg tac age ege gac gag ate

Ser Ala His lie Met Asn Met Gin He Pro Tyr Ser Arg Asp Glu lie 65 70 75 80 aac gag gcg acc ege gee gee gtg ege gag aac aac ctg gaa age gee

Asn Glu Ala Thr Arg Ala Ala Val Arg Glu Asn Asn Leu Glu Ser Ala 85 90 95 tat ate ege ccg atg gtg ttc tac gga age gaa ggc atg ggc ctg ege

Tyr He Arg Pro Met Val Phe Tyr Gly Ser Glu Gly Met Gly Leu Arg 100 105 110 gee age ggc ctg aag gtc cat gtg ate ate gee gee tgg age tgg ggc

Ala Ser Gly Leu Lys Val His Val lie lie Ala Ala Trp Ser Trp Gly 115 120 125 gee tac atg ggc gag gaa gee ctg cag caa ggc ate aag gtg ege acc

Ala Tyr Met Gly Glu Glu Ala Leu Gin Gin Gly ly Lys Val Arg Thr 130 135 140 agt tec ttc acc ege cac cac gtc aac ate teg atg acc ege gee aag

Ser Ser Phe Thr Arg His His Val Asn lie Ser Met Thr Arg Ala Lys 145 150 155 160 tec aac ggc gee tac ate aac teg atg ctg gee etc cag gaa gcg ate

Ser Asn Gly Ala Tyr lie Asn Ser Met Leu Ala Leu Gin Glu Ala lie 165 170 175 tec ggc ggc gee gac gag gee atg atg etc gat ccg gaa ggc tac gtg

Ser Gly Gly Ala Asp Glu Ala Met Met Leu Asp Pro Glu Gly Tyr Val 180 185 190 gee gaa ggc tec ggc gag aac ate ttc ate ate aag gat ggc gtg ate

Ala Glu Gly Ser Gly Glu Asn He Phe lie lie Lys Asp Gly Val lie 195 200 205 tac acc ccg gaa gtc acc gee tgc ctg aac ggc ate act cgt aac act

Tyr Thr Pro Glu Val Thr Ala Cys Leu Asn Gly lie Thr Arg Asn Thr 210 215 220 ate ctg acc ctg gee gee gaa cac ggt ttt aaa ctg gtc gag aag ege lie Leu Thr Leu Ala Ala Glu His Gly Phe Lys Leu Val Glu Lys Arg 225 230 235 240 ate acc ege gac gag gtg tac ate gee gac gag gee ttc ttc act ggc lie Thr Arg Asp Glu Val Tyr lie Ala Asp Glu Ala Phe Phe Thr Gly 245 250 255 act gee gcg gaa gtc aeg ccg ate ege gaa Gtg gac ggt ege aag ate

Thr Ala Ala Glu Val Thr Pro lie Arg Glu Val Asp Gly Arg Lys lie 260 265 270 ggc gee ggc ege cgt ggc ccg gtc acc gaa aag ctg cag aaa gee tat

Gly Ala Gly Arg Arg Gly Pro Val Thr Glu Lys Leu Gin Lys Ala Tyr 275 280 285 ttc gac ctg gtc age ggc aag acc gag gee cac gee gag tgg cgt acc 288 336 384 432 480 528 576 624 672 720 768 816 864 912 43 201033369

Phe Asp Leu Val Ser Gly Lys Thr Glu Ala His Ala Glu Trp Arg Thr 290 295 300 ctg gtc aag taa 924 Leu Val Lys 305

<210> 27 <211> 307 <212> PRT <213> Bacillus licheniformis Ui 9951299) <400>

Met Ser Met Ala Asp Arg Asp Gly Val lie Trp Tyr Asp Gly Glu Leu 15 10 15

Val Gin 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 Gin 35 40 45

Gly Thr Ala He Phe Arg Leu Gin Ala His Thr Asp Arg Leu Phe Asp 50 55 60

Ser Ala His lie Met Asn Met Gin lie Pro Tyr Ser Arg Asp Glu lie 65 70 75 80

Asn Glu Ala Thr Arg Ala Ala Val Arg Glu Asn Asn Leu Glu Ser Ala 85 90 95

Tyr lie 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 lie He Ala Ala Trp Ser Trp Gly 115 120 125

Ala Tyr Met Gly Glu Glu Ala Leu Gin Gin Gly lie Lys Val Arg Thr 130 135 140

Ser Ser Phe Thr Arg His His Val Asn lie Ser Met Thr Arg Ala Lys 145 150 155 160

Ser Asn Gly Ala Tyr lie Asn Ser Met Leu Ala Leu Gin Glu Ala lie 165 170 175 44 201033369

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 lie Phe lie lie Lys Asp Gly Val He 195 200 205

Tyr Thr Pro Glu Val Thr Ala Cys Leu Asn Gly lie Thr Arg Asn Thr 210 215 220 lie Leu Thr Leu Ala Ala Glu His Gly Phe Lys Leu Val Glu Lys Arg 225 230 235 240 lie Thr Arg Asp Glu Val Tyr He Ala Asp Glu Ala Phe Phe Thr Gly 245 250 255 ❿

Thr Ala Ala Glu Val Thr Pro lie Arg Glu Val Asp Gly Arg Lys lie 260 265 270

Gly Ala Gly Arg Arg Gly Pro Val Thr Glu Lys Leu Gin 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

<210> 28 <211> 1407 <212> DNA <213> Rhodopseudomonas palustris <220><221> CDS <222> (1).. (1407) <400> 28 48 96 atg aag ctg ata ccg tgc cgc gcc ttt cac ccc ccg gcc gcg cag tgc

Met Lys Leu lie Pro Cys Arg Ala Phe His Pro Pro Ala Ala Gin Cys 15 10 15 atg agg age gcc atg tta gac aag ate aag ccc aeg tcc gcc gtc aac

Met Arg Ser Ala Met Leu Asp Lys lie Lys Pro Thr Ser Ala Val Asn 20 25 30 gcg ccg aac gat etc aac gcg ttc tgg atg ccg ttc acc gcg aac egg

Ala Pro Asn Asp Leu Asn Ala Phe Trp Met Pro Phe Thr Ala Asn Arg 35 40 45 45 144 201033369 gcc ttc aag cgc gcg ccg aag atg gtc gtg ggt gcc gaa ggc atg cac 192

Ala Phe Lys Arg Ala Pro Lys Met Val Val Gly Ala Glu Gly Met His 50 55 60 tac ate acc gcc gat ggt cgc aag ate ate gac gcc gcc teg ggc atg 240

Tyr lie Thr Ala Asp Gly Arg Lys He He Asp Ala Ala Ser Gly Met 65 70 75 80 tgg tgc acc aat gcg ggc cat ggc cgc aag gaa ate gcc gag gcg ate 288

Trp Cys Thr Asn Ala Gly His Gly Arg Lys Glu lie Ala Glu Ala lie 85 90 95 aag gcg cag gcc gat gaa etc gac ttc teg ccg ccg ttc cag ttc ggc 336

Lys Ala Gin Ala Asp Glu Leu Asp Phe Ser Pro Pro Phe Gin Phe Gly 100 105 110 cag ccg aag gcg ttc gaa etc gcc age egg ate gcc gat ctg gcg ccg 384

Gin Pro Lys Ala Phe Glu Leu Ala Ser Arg He Ala Asp Leu Ala Pro

115 120 125 gaa ggc etc gat cac gtg ttc ttc tgc aat teg ggc teg gaa gcc ggc 432

Glu Gly Leu Asp His Val Phe Phe Cys Asn Ser Gly Ser Glu Ala Gly 130 135 140 gac acc gcg ctg aag ate gcg gtc gcc tat cag cag ate aag ggc cag 480

Asp Thr Ala Leu Lys lie Ala Val Ala Tyr Gin Gin He Lys Gly Gin 145 150 155 160 ggc tea cgc acc cgc ctg ate ggc cgc gag cgc ggc tat cac ggc gtc 528

Gly Ser Arg Thr Arg Leu lie Gly Arg Glu Arg Gly Tyr His Gly Val 165 170 175 ggc ttc ggc ggc acc gcg gtc ggc ggc ate ggc aac aac cgc aag atg 576

Gly Phe Gly Gly Thr Ala Val Gly Gly lie Gly Asn Asn Arg Lys Met 180 185 190 ttc ggt ccg ctg etc aac ggc gtc gat cat ctg cct gcg act tat gat 624

Phe Gly Pro Leu Leu Asn Gly Val Asp His Leu Pro Ala Thr Tyr Asp 195 200 205 cgc gac aag cag get ttc acc ate ggc gag ccg gaa tac ggc gcg cac 672

Arg Asp Lys Gin Ala Phe Thr lie Gly Glu Pro Glu Tyr Gly Ala His 210 215 220 ttc gcc gaa gcg ett gaa ggc etc gtc aat ctg cac ggc gcc aac acc 720

Phe Ala Glu Ala Leu Glu Gly Leu Val Asn Leu His Gly Ala Asn Thr 225 230 235 240 ate gcg gcg gtg ate gtc gag ccg atg gcc ggc tee acc ggc gtg ctg 768 lie Ala Ala Val lie Val Glu Pro Met Ala Gly Ser Thr Gly Val Leu 245 250 255 ccg gcg ccg aag ggc tat etc aag aag ctg cgc gag ate acc aag aag 816

Pro Ala Pro Lys Gly Tyr Leu Lys Lys Leu Arg Glu lie Thr Lys Lys 260 265 270 cac ggc ate ctg ctg ate ttc gac gag gtc ate acc ggc tac ggc cgt 864

His Gly He Leu Leu lie Phe Asp Glu Val lie Thr Gly Tyr Gly Arg 275 280 285 46 912 912

201033369 etc ggc tat gee ttc geg tee gaa cgt tac ggc gtc acc ccg gac atg

Leu Gly Tyr Ala Phe Ala Ser Glu Arg Tyr Gly Val Thr Pro Asp Met 290 295 300 ate acc ttc gee aag ggc gtc acc aat ggt geg gtg ccg atg ggc ggc lie Thr Phe Ala Lys Gly Val Thr Asn Gly Ala Val Pro Met Gly Gly 305 310 315 320 gtg ate acc teg geg gate ate cac gat geg ttc atg acc ggc ccc gag

Val lie Thr Ser Ala Glu lie His Asp Ala Phe Met Thr Gly Pro Glu 325 330 335 cac geg gtc gag ctg geg cac ggc tac acc tat teg geg cat ccg etc

His Ala Val Glu Leu Ala His Gly Tyr Thr Tyr Ser Ala His Pro Leu 340 345 350 gee tgc geg gee ggc ate gee acc etc gac ate tac ege gac gag aag

Ala Cys Ala Ala Gly lie Ala Thr Leu Asp He Tyr Arg Asp Glu Lys 355 360 365 ctg ttc gag ege gee aag geg ctg gag ccg aag ttt gee gag geg gtg

Leu Phe Glu Arg Ala Lys Ala Leu Glu Pro Lys Phe Ala Glu Ala Val 370 375 380 atg teg ctg aag teg gee ccg aac gtg gtc gac ate ege acc gtc ggc

Met Ser Leu Lys Ser Ala Pro Asn Val Val Asp He Arg Thr Val Gly 385 390 395 400 ctg aeg geg ggt ate gac etc get teg ate gee gat geg gtc ggc aag

Leu Thr Ala Gly He Asp Leu Ala Ser lie Ala Asp Ala Val Gly Lys 405 410 415 cgt ggc ttc gaa geg atg aat gee ggc ttc cac gac cac gag ctg atg

Arg Gly Phe Glu Ala Met Asn Ala Gly Phe His Asp His Glu Leu Met 420 425 430 ctg egg ate gee ggc gac acc ctg geg ctg acc ccg ccg ctg ate etc

Leu Arg He Ala Gly Asp Thr Leu Ala Leu Thr Pro Pro Leu lie Leu 435 440 445 age gag gac cac ate ggt gag ate gtc gac aag gtc ggc aag gtg ate

Ser Glu Asp His lie Gly Glu lie Val Asp Lys Val Gly Lys Val lie 450 455 460 ege geg gtc gee tga Arg Ala Val Ala 465

<210> 29 <211> 468 <212> PRT <213> Rhodopseudomonas palustris <400> 29

Met Lys Leu lie Pro Cys Arg Ala Phe His Pro Pro Ala Ala Gin Cys 1 5 10 15 960 1008 1056 1104 1152 1200 1248 1296 1344 1392 1407 47 201033369

Met Arg Ser Ala Met Leu Asp Lys He 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 lie Thr Ala Asp Gly Arg Lys lie lie Asp Ala Ala Ser Gly Met 65 70 75 80

Trp Cys Thr Asn Ala Gly His Gly Arg Lys Glu He Ala Glu Ala lie 85 90 95

Lys Ala Gin Ala Asp Glu Leu Asp Phe Ser Pro Pro Phe Gin Phe Gly 100 105 110

Gin Pro Lys Ala Phe Glu Leu Ala Ser Arg lie 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 lie Ala Val Ala Tyr Gin Gin lie Lys Gly Gin 145 150 155 160

Gly Ser Arg Thr Arg Leu lie Gly Arg Glu Arg Gly Tyr His Gly Val 165 170 175

Gly Phe Gly Gly Thr Ala Val Gly Gly lie 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 Gin Ala Phe Thr He 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 lie Ala Ala Val He Val Glu Pro Met Ala Gly Ser Thr Gly Val Leu 48 201033369 245 250 255

Pro Ala Pro Lys Gly Tyr Leu Lys Lys Leu Arg Glu He Thr Lys Lys 260 265 270

His Gly He Leu Leu lie Phe Asp Glu Val lie 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 lie Thr Phe Ala Lys Gly Val Thr Asn Gly Ala Val Pro Met Gly Gly 305 310 315 320

Val lie Thr Ser Ala Glu lie 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 lie Ala Thr Leu Asp lie 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 lie Arg Thr Val Gly 385 390 395 400

Leu Thr Ala Gly lie Asp Leu Ala Ser lie 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 lie Ala Gly Asp Thr Leu Ala Leu Thr Pro Pro Leu He Leu 435 440 445

Ser Glu Asp His He Gly Glu He Val Asp Lys Val Gly Lys Val lie 450 455 460

Arg Ala Val Ala 465 49 201033369 <210> 30 <211> 1263 <212> DNA <213> E. <220><221> CDS <222> (1).. (1263) <;400〉30 atg cca cat tea ctg ttc age acc gat acc gat etc acc gee gaa aat 48

Met Pro His Ser Leu Phe Ser Thr Asp Thr Asp Leu Thr Ala Glu Asn 15 10 15 ctg ctg cgt ttg ccc get gaa ttt ggc tgc ccg gtg tgg gtc tac gat 96

Leu Leu Arg Leu Pro Ala Glu Phe Gly Cys Pro Val Trp Val Tyr Asp 20 25 30

Geg caa att att cgt egg cag att gca geg ctg aaa cag ttt gat gtg 144

Ala Gin He lie Arg Arg Gin He Ala Ala Leu Lys Gin Phe Asp Val 35 40 45 gtg ege ttt gca cag aaa gee tgt tee aat att cat att ttg ege tta 192

Val Arg Phe Ala Gin Lys Ala Cys Ser Asn He His lie Leu Arg Leu 50 55 60 atg cgt gag cag ggc gtg aaa gtg gat tee gtc teg tta ggc gaa ata 240

Met Arg Glu Gin Gly Val Lys Val Asp Ser Val Ser Leu Gly Glu He 65 70 75 80 gag cgt geg ttg geg geg ggt tac aat ccg caa aeg cac ccc gat gat 288

Glu Arg Ala Leu Ala Ala Gly Tyr Asn Pro Gin Thr His Pro Asp Asp 85 90 95 att gtt ttt aeg gca gat gtt ate gat cag geg aeg ett gaa ege gtc 336 lie Val Phe Thr Ala Asp Val He Asp Gin Ala Thr Leu Glu Arg Val 100 105 110 agt gaa ttg caa att ccg gtg aat geg ggt tet gtt gat atg etc gac 384

Ser Glu Leu Gin lie Pro Val Asn Ala Gly Ser Val Asp Met Leu Asp 115 120 125 caa ctg ggc cag gtt teg cca ggg cat egg gta tgg ctg ege gtt aat 432

Gin Leu Gly Gin Val Ser Pro Gly His Arg Val Trp Leu Arg Val Asn 130 135 140 ccg ggg ttt ggt cac gga cat age caa aaa acc aat acc ggt ggc gaa 480

Pro Gly Phe Gly His Gly His Ser Gin Lys Thr Asn Thr Gly Gly Glu 145 150 155 160 aac age aag cac ggt ate tgg tac acc gat ctg ccc gee gca ctg gac 528

Asn Ser Lys His Gly lie 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 576

Val lie Gin Arg His His Leu Gin Leu Val Gly lie His Met His He 180 185 190 50 201033369 ggt tct ggc gtt gat tat gcc cat ctg gaa cag gtg tgt ggt get atg 624

Gly Ser Gly Val Asp Tyr Ala His Leu Glu Gin Val Cys Gly Ala Met 195 200 205 gtg cgt cag gtc ate gaa ttc ggt cag gat tta cag get att tct geg 672

Val Arg Gin Val lie Glu Phe Gly Gin Asp Leu Gin Ala lie Ser Ala 210 215 220 ggc ggt ggg ett tct gtt cct tat caa cag ggt gaa gag geg gtt gat 720

Gly Gly Gly Leu Ser Val Pro Tyr Gin Gin Gly Glu Glu Ala Val Asp 225 230 235 240 acc gaa cat tat tat ggt ctg tgg aat gcc geg cgt gag caa ate gcc 768

Th G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G

Arg His Leu Gly His Pro Val Lys Leu Glu lie Glu Pro Gly Arg Phe • 260 265 270 ctg gta geg cag tct ggc gta tta att act cag gtg egg age gtc aaa 864

Leu Val Ala Gin Ser Gly Val Leu lie Thr Gin Val Arg Ser Val Lys 275 280 285 caa atg ggg age ege cac ttt gtg ctg gtt gat gcc ggg ttc aac gat 912 . Gin Met Gly Ser Arg His Phe Val Leu Val Asp Ala Gly Phe Asn Asp . 290 295 300 . ctg atg ege ccg gca atg tac ggt agt tac cac cat ate agt gcc ctg 960

Leu Met Arg Pro Ala Met Tyr Gly Ser Tyr His His lie Ser Ala Leu 305 310 315 320 gca get gat ggt cgt tct ctg gaa cac geg cca aeg gtg gaa acc gtc 1008

Ala 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 teg ggc gat gtc ttt acc cag cag gaa 1056 _ Val Ala Gly Pro Leu Cys Glu Ser Gly Asp Val Phe Thr Gin Gin Glu φ 340 345 350 ggg gga aat gtt gaa acc ege gcc ttg ccg gaa gtg aag gca ggt gat 1104

Gly 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 tea atg tea tee 1152

Tyr Leu Val Leu His Asp Thr Gly Ala Tyr Gly Ala Ser Met Ser Ser 370 375 380 aac tac aat age cgt ccg ctg tta cca gaa gtt ctg ttt gat aat ggt 1200

Asn Tyr Asn Ser Arg Pro Leu Leu Pro Glu Val Leu Phe Asp Asn Gly 385 390 395 400 cag geg egg ttg att ege cgt ege cag acc ate gaa ga tta ctg geg 1248

Gin Ala Arg Leu He Arg Arg Arg Gin Thr lie Glu Glu Leu Leu Ala 405 410 415 ctg gaa ttg ett taa 1263

Leu Glu Leu Leu 420 51 201033369 <210> 31 <211> 420 <212> PRT <213> E. coli <400> 31

Met Pro His Ser Leu Phe Ser Thr Asp Thr Asp Leu Thr Ala Glu Asn 15 10 15

Leu Leu Arg Leu Pro Ala Glu Phe Gly Cys Pro Val Trp Val Tyr Asp 20 25 30

Ala Gin He lie Arg Arg Gin lie Ala Ala Leu Lys Gin Phe Asp Val 35 40 45

Val Arg Phe Ala Gin Lys Ala Cys Ser Asn He His lie Leu Arg Leu 50 55 60

Met Arg Glu Gin Gly Val Lys Val Asp Ser Val Ser Leu Gly Glu lie 65 70 75 80

Glu Arg Ala Leu Ala Ala Gly Tyr Asn Pro Gin Thr His Pro Asp Asp 85 90 95

He Val Phe Thr Ala Asp Val lie Asp Gin Ala Thr Leu Glu Arg Val 100 105 110

Ser Glu Leu Gin He Pro Val Asn Ala Gly Ser Val Asp Met Leu Asp 115 120 125 ❿

Gin Leu Gly Gin Val Ser Pro Gly His Arg Val Trp Leu Arg Val Asn 130 135 140

Pro Gly Phe Gly His Gly His Ser Gin Lys Thr Asn Thr Gly Gly Glu 145 150 155 160

Asn Ser Lys His Gly He Trp Tyr Thr Asp Leu Pro Ala Ala Leu Asp 165 170 175

Val He Gin Arg His His Leu Gin Leu Val Gly lie His Met His lie 180 185 190

Gly Ser Gly Val Asp Tyr Ala His Leu Glu Gin Val Cys Gly Ala Met 195 200 205 52 201033369

Val Arg Gin Val He Glu Phe Gly Gin Asp Leu Gin Ala He Ser Ala 210 215 220

Gly Gly Gly Leu Ser Val Pro Tyr Gin Gin Gly Glu Glu Ala Val Asp 225 230 235 240

Thr Glu His Tyr Tyr Gly Leu Trp Asn Ala Ala Arg Glu Gin He Ala 245 250 255

Arg His Leu Gly His Pro Val Lys Leu Glu He Glu Pro Gly Arg Phe 260 265 270

Leu Val Ala Gin Ser Gly Val Leu He Thr Gin Val Arg Ser Val Lys 275 280 285

Gin 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 lie 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 Gin Gin 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

Gin Ala Arg Leu He Arg Arg Arg Gin Thr lie Glu Glu Leu Leu Ala 405 410 415

Leu Glu Leu Leu 420 <210> 32 53 201033369 <211> 1265 <212> DNA <213>manmade <220><223> 223 E. coli diamine pimelate decarboxylated glycine LysA codon is best of the gene < 400> 32 atatgccaca ctctctgttt tctactgata ctgatctgac tgcggaaaac ctgctgcgtc 60 tgccggctga attcggttgt ccggtatggg tgtacgacgc tcagattatt cgtcgccaga 120 tcgcagcact gaagcagttc gatgtagtgc gttttgcaca gaaggcgtgc tccaacatcc 180 atatcctgcg cctgatgcgt gagcagggcg ttaaagttga ctccgtctct ctgggtgaga 240 ttgagcgcgc cctggcagcc ggctataacc cacagaccca tcctgacgac attgtattta 300 ❹ ctgccgacgt gatcgaccag gctactctgg aacgcgtttc tgaactgcag atcccggtta 360 atgctggttc tgtggacatg ctggaccagc tgggccaggt atccccaggt catcgtgtgt 420 ggctgcgtgt caacccaggt ttcggccacg gccactctca gaaaactaac actggtggtg 480 agaactccaa gcatggcatt tggtataccg atctgccggc tgcactggac gtaatccagc 540 gtcaccacct gcagctggtg ggcatccaca tgcacattgg ctccggcgta gactacgccc 600 acctggagca agtctgcggt gctatggtac gtcaggtaat cgagttcggc caagatctgc 660 aggcaatcag cgctggtggc ggcctg Tctg taccttatca gcagggcgag gaggcggttg 720 acactgagca ctactacggt ctgtggaacg ccgctcgtga gcaaattgca cgtcacctgg 780 gccacccggt gaaactggag atcgagccgg gccgcttcct ggtagcacag tccggcgtac 840 tgattaccca ggtacgctct gttaaacaga tgggctcccg tcactttgtg ctggtagacg 900

caggcttcaa cgacctgatg cgtccggcta tgtatggttc ctatcatcac atctctgcgc 960 tggccgccga cggccgctct ctggaacacg cgccgacggt tgaaacggtg gtggctggtc 1020 cgctgtgcga gtccggcgac gttttcactc agcaggaggg cggcaatgta gagacgcgtg 1080 cgctgccgga agtgaaagcc ggtgattatc tggtgctgca tgataccggc gcctatggtg 1140 cgagcatgag cagcaactac aactctcgcc cgctgctgcc ggaggtcctg ttcgataacg 1200 gccaagcccg cctgatccgt cgtcgtcaga ccatcgagga actgctggca ctggagctgc 1260 tgtaa 1265 < 210 > 33 <211> 1692 <212> DNA <213> Saccharomyces Cerevisiae 54 48 48

201033369 <220〉 <221> CDS <222> (1)..(1692) <400> 33 atg tct gaa att act ttg ggt aaa tat ttg ttc gaa aga tta aag caa

Met Ser Glu lie Thr Leu Gly Lys Tyr Leu Phe Glu Arg Leu Lys Gin 15 10 15 gtc aac gtt aac acc gtt ttc ggt ttg cca ggt gac ttc aac ttg tcc

Val Asn Val Asn Thr Val Phe Gly Leu Pro Gly Asp Phe Asn Leu Ser 20 25 30 ttg ttg gac aag ate tac gaa gtt gaa ggt atg aga tgg get ggt aac

Leu Leu Asp Lys lie Tyr Glu Val Glu Gly Met Arg Trp Ala Gly Asn 35 40 45 gcc aac gaa ttg aac get get tac gcc get gat ggt tac get cgt ate

Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg lie 50 55 60 aag ggt atg tct tgt ate ate acc acc ttc ggt gtc ggt gaa ttg tct

Lys Gly Met Ser Cys lie He Thr Thr Phe Gly Val Gly Glu Leu Ser 65 70 75 80 get ttg aac ggt att gcc ggt tct tac get gaa cac gtc ggt gtt ttg

Ala Leu Asn Gly lie Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu 85 90 95 cac gtt gtt ggt gtc cca tcc ate tct get caa get aag caa ttg ttg

His Val Val Gly Val Pro Ser He Ser Ala Gin Ala Lys Gin Leu Leu 100 105 110 ttg cac cac acc ttg ggt aac ggt gac ttc act gtt ttc cac aga atg

Leu 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 get atg ate act gac att get acc

Ser Ala Asn lie Ser Glu Thr Thr Ala Met lie Thr Asp He Ala Thr 130 135 140 gcc cca get gaa att gac aga tgt ate aga acc act tac gtc acc caa

Ala Pro Ala Glu lie Asp Arg Cys lie Arg Thr Thr Tyr Val Thr Gin 145 150 155 160 aga cca gtc tac tta ggt ttg cca get aac ttg gtc gac ttg aac gtc

Arg Pro Val Tyr Leu Gly Leu Pro Ala Asn Leu Val Asp Leu Asn Val 165 170 175 cca get aag ttg ttg caa act cca att gac atg tct ttg aag cca aac

Pro Ala Lys Leu Leu Gin Thr Pro He Asp Met Ser Leu Lys Pro Asn 180 185 190 gat get gaa tcc gaa aag gaa gtc att gac acc ate ttg get ttg gtc

Asp Ala Glu Ser Glu Lys Glu Val He Asp Thr lie Leu Ala Leu Val 195 200 205 aag gat get aag aac cca gtt ate ttg get gat get tgt tgt tcc aga

Lys Asp Ala Lys Asn Pro Val lie Leu Ala Asp Ala Cys Cys Ser Arg 96 144 192 240 288 336 384 432 480 528 576 624 55 672 201033369 210 215 220 cac gac gtc aag get gaa act aag aag ttg att gac ttg act caa ttc 720

His Asp Val Lys Ala Glu Thr Lys Lys Leu lie Asp Leu Thr Gin Phe 225 230 235 240 cca get ttc gtc acc cca atg ggt aag ggt tcc att gac gaa caa cac 768

Pro Ala Phe Val Thr Pro Met Gly Lys Gly Ser lie Asp Glu Gin His 245 250 255 cca aga tac ggt ggt gtt tac gtc ggt acc ttg tcc aag cca gaa gtt 816

Pro Arg Tyr Gly Gly Val Tyr Val Gly Thr Leu Ser Lys Pro Glu Val 260 265 270 aag gaa gcc gtt gaa tet get gac ttg att ttg tet gtc ggt get ttg 864

Lys Glu Ala Val Glu Ser Ala Asp Leu lie Leu Ser Val Gly Ala Leu 275 280 285 ttg tet gat ttc aac acc ggt tet ttc tet tac tet tac aag acc aag 912

Leu 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 ate aga aac gcc act 960

Asn lie Val Glu Phe His Ser Asp His Met Lys lie Arg Asn Ala Thr 305 310 315 320 ttc cca ggt gtc caa atg aaa ttc gtt ttg caa aag ttg ttg acc act 1008

Phe Pro Gly Val Gin Met Lys Phe Val Leu Gin Lys Leu Leu Thr Thr 325 330 335 att get gac gcc get aag ggt tac aag cca gtt get gtc cca get aga 1056 lie Ala Asp Ala Ala Lys Gly Tyr Lys Pro Val Ala Val Pro Ala Arg 340 345 350 act cca get aac get get gtc cca get tet acc cca ttg aag caa gaa 1104

Thr Pro Ala Asn Ala Ala Val Pro Ala Ser Thr Pro Leu Lys Gin Glu 355 360 365

Tgg atg tgg aac caa ttg ggt aac ttc ttg caa gaa ggt gat gtt gtc 1152

Trp Met Trp Asn Gin Leu Gly Asn Phe Leu Gin Glu Gly Asp Val Val 370 375 380 att get gaa acc ggt acc tcc get ttc ggt ate aac caa acc act ttc 1200 lie Ala Glu Thr Gly Thr Ser Ala Phe Gly lie Asn Gin Thr Thr Phe 385 390 395 400 cca aac aac acc tac ggt ate tet caa gtc tta tgg ggt tcc att ggt 1248

Pro Asn Asn Thr Tyr Gly lie Ser Gin Val Leu Trp Gly Ser lie Gly 405 410 415 ttc acc act ggt get acc ttg ggt get get ttc get get gaa gaa att 1296

Phe Thr Thr Gly Ala Thr Leu Gly Ala Ala Phe Ala Ala Glu Glu lie 420 425 430 gat cca aag aag aga gtt ate tta ttc att ggt gac ggt tet ttg caa 1344

Asp Pro Lys Lys Arg Val He Leu Phe lie Gly Asp Gly Ser Leu Gin 435 440 445 ttg act gtt caa gaa ate tcc acc atg ate aga tgg ggc ttg aag cca 1392 56 1440 1440

201033369

Leu Thr Val Gin Glu lie Ser Thr Met He 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

Tyr Leu Phe Val Leu Asn Asn Asp Gly Tyr Thr lie Glu Lys Leu lie 465 470 475 480 cac ggt cca aag get caa tac aac gaa att caa ggt tgg gac cac eta

His Gly Pro Lys Ala Gin Tyr Asn Glu lie Gin Gly Trp Asp His Leu 485 490 495 tcc ttg ttg cca act ttc ggt get aag gac tat gaa acc cac aga gtc

Ser Leu Leu Pro Thr Phe Gly Ala Lys Asp Tyr Glu Thr His Arg Val 500 505 510 get acc acc ggt gaa tgg gac aag ttg acc caa gac aag tet ttc aac

Ala Thr Thr Gly Glu Trp Asp Lys Leu Thr Gin Asp Lys Ser Phe Asn 515 520 525 gac aac tet aag ate aga atg att gaa ate atg ttg cca gtc ttc gat

Asp Asn Ser Lys lie Arg Met He Glu He Met Leu Pro Val Phe Asp 530 535 540 get cca caa aac ttg gtt gaa caa get aag ttg act get get acc aac

Ala Pro Gin Asn Leu Val Glu Gin Ala Lys Leu Thr Ala Ala Thr Asn 545 550 555 560 get aag caa taa Ala Lys Gin <210> 34 <211> 563 <212> PRT <213> Saccharomyces cerevisiae<400> 34

Met Ser Glu He Thr Leu Gly Lys Tyr Leu Phe Glu Arg Leu Lys Gin 15 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 lie 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 He 50 55 60

Lys Gly Met Ser Cys lie He Thr Thr Phe Gly Val Gly Glu Leu Ser 65 70 75 80 1488 1536 1584 1632 1680 1692 57 201033369

Ala Leu Asn Gly lie Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu 85 90 95

His Val Val Gly Val Pro Ser lie Ser Ala Gin Ala Lys Gin 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 He Ser Glu Thr Thr Ala Met lie Thr Asp He Ala Thr 130 135 140

Ala Pro Ala Glu lie Asp Arg Cys lie Arg Thr Thr Tyr Val Thr Gin 145 150 155 160 ❹

Arg Pro Val Tyr Leu Gly Leu Pro Ala Asn Leu Val Asp Leu Asn Val 165 170 175

Pro Ala Lys Leu Leu Gin Thr Pro He Asp Met Ser Leu Lys Pro Asn 180 185 190

Asp Ala Glu Ser Glu Lys Glu Val lie Asp Thr lie Leu Ala Leu Val 195 200 205

Lys Asp Ala Lys Asn Pro Val lie Leu Ala Asp Ala Cys Cys Ser Arg 210 215 220

His Asp Val Lys Ala Glu Thr Lys Lys Leu lie Asp Leu Thr Gin Phe 225 230 235 240

Pro Ala Phe Val Thr Pro Met Gly Lys Gly Ser lie Asp Glu Gin 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 lie 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 lie Val Glu Phe His Ser Asp His Met Lys lie Arg Asn Ala Thr 305 310 315 320 58 201033369

Phe Pro Gly Val Gin Met Lys Phe Val Leu Gin Lys Leu Leu Thr Thr 325 330 335 lie 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 Gin Glu 355 360 365

Trp Met Trp Asn Gin Leu Gly Asn Phe Leu Gin Glu Gly Asp Val Val 370 375 380 ❿ lie Ala Glu Thr Gly Thr Ser Ala Phe Gly lie Asn Gin Thr Thr Phe 385 390 395 400

Pro Asn Asn Thr Tyr Gly He Ser Gin Val Leu Trp Gly Ser lie Gly 405 410 415

Phe Thr Thr Gly Ala Thr Leu Gly Ala Ala Phe Ala Ala Glu Glu lie 420 425 430

Asp Pro Lys Lys Arg Val lie Leu Phe lie Gly Asp Gly Ser Leu Gin 435 440 445

Leu Thr Val Gin Glu lie Ser Thr Met lie Arg Trp Gly Leu Lys Pro 450 455 460

Tyr Leu Phe Val Leu Asn Asn Asp Gly Tyr Thr lie Glu Lys Leu lie 465 470 475 480

His Gly Pro Lys Ala Gin Tyr Asn Glu lie Gin 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 Gin Asp Lys Ser Phe Asn 515 520 525

Asp Asn Ser Lys He Arg Met He Glu He Met Leu Pro Val Phe Asp 530 535 540

Ala Pro Gin Asn Leu Val Glu Gin Ala Lys Leu Thr Ala Ala Thr Asn 545 550 555 560 59 201033369

Ala Lys Gin <210> 35 <211> 1692 <212> DNA <213>manmade <220><223> Saccharomyces cerevisiae pyruvate decarboxylation Pdc codon optimization gene <400> atgtccgaga tcactctggg caaatacctg tttgaacgtc tgaaacaggt gaacgttaat 60 accgtattcg gcctgccggg tgatttcaac ctgtccctgc tggacaaaat ctatgaagtt 120 ❺ gaaggtatgc gttgggctgg caacgctaac gagctgaacg cagcgtacgc ggcagatggt 180 tacgctcgta tcaaaggtat gtcttgtatc atcaccacct tcggtgttgg tgagctgagc 240 gccctgaacg gcatcgccgg ctcctatgca gagcacgtgg gcgtgctgca cgttgtgggt 300 gtaccgtcca tcagcgccca ggcaaaacag ctgctgctgc accacaccct gggtaacggc 360 gactttaccg ttttccatcg tatgtctgcg aacatcagcg aaactactgc aatgattact 420 gacatcgcta cggcaccggc agaaatcgac cgttgcattc gtaccacgta cgttactcag 480 cgcccggttt atctgggcct gccagccaac ctggtggatc tgaacgtccc ggctaaactg 540 ctgcagactc cgatcgatat gtctctgaaa cctaacgacg cagaatctga gaaagaagtt 600 atcgatacta ttctggctct ggtgaaagat gcaaagaacc cagttatcct ggctgacgca 660

tgttgctctc gtcatgatgt aaaggcagaa accaaaaagc tgatcgacct gacgcagttc 720 ccggcgttcg ttaccccgat gggcaagggt tccatcgatg agcagcaccc gcgttatggt 780 ggtgtatacg ttggcacgct gtccaaaccg gaggtaaaag aagcggttga aagcgcagat 840 ctgatcctgt ctgttggtgc actgctgagc gacttcaaca ccggttcttt ctcctatagc 900 tacaagacca aaaacattgt ggagtttcac tccgatcaca tgaaaatccg caacgcgacc 960 tttcctggtg tgcagatgaa attcgtactg cagaaactgc tgaccaccat cgccgacgct 1020 gcgaaaggtt ataaaccggt agctgtgccg gcacgtaccc cggcgaacgc cgcggttcct 1080 gcatccactc cactgaagca ggaatggatg tggaatcagc tgggtaattt cctgcaagaa 1140 ggcgacgttg taatcgcaga aaccggcact agcgcgtttg gcattaacca gacgaccttc 1200 ccaaacaaca cctacggtat cagccaagtc ctgtggggct ctatcggctt caccaccggt 1260 gcaaccctgg gtgcggcttt cgctgctgag gagatcgacc cgaagaaacg tgttatcctg 1320 60 201033369 ttcatcggtg acggctccct gcagctgacc gtccaggaga tttctaccat gatccgctgg 1380 ggcctgaaac cgtacctgtt tgtgctgaac aacgacggct acactattga gaaactgatc 1440 cacggtccga aagcacagta taatgagatc cagggttggg atcatctgtc tctgctgccg 150 0 acctttggcg ctaaagacta cgagacccac cgcgtggcta ccaccggcga gtgggataaa 1560 ctgacgcagg ataaatcctt caatgacaat agcaagattc gtatgatcga aatcatgctg 1620 ccggtctttg atgctccgca gaacctggta gagcaagcaa aactgaccgc ggcaactaac 1680 gctaaacagt aa 1692

<210> 36 <211> 1707 <212> DNA <213>activator Zymomonas

<220> <221> CDS <222> (1)..(1707) <400> 36 48 96 144 192 240 288 336 384 432 atg agt tat act gtc ggt acc tat tta gcg gag egg ett gtc cag Att

Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gin He 15 10 15 ggt etc aag cat cac ttc gca gtc gcg ggc gac tac aac etc gtc ett

Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu 20 25 30 ett gac aac ctg ett ttg aac aaa aac atg gag cag gtt tat tgc tgt

Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gin Val Tyr Cys Cys 35 40 45 aac gaa ctg aac tgc ggt ttc agt gca gaa ggt tat get cgt gee aaa

Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys 50 55 60 ggc gca gca gca gee gtc gtt acc tac age gtc ggt gcg ett tee gca

Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser Ala 65 70 75 80 ttt gat get ate ggt ggc gee tat gca gaa aac ett ccg gtt ate ctg

Phe Asp Ala lie Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val lie Leu 85 90 95 ate tee ggt get ccg aac aac aat gat cac get get ggt cac gtg ttg lie Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu 100 105 110 cat cac get ett ggc aaa acc gac tat cac tat cag ttg gaa atg gee

His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gin Leu Glu Met Ala 115 120 125 aag aac ate aeg gee gee get gaa gcg att tac acc ccg gaa gaa get 61 201033369

Lys Asn lie Thr Ala Ala Ala Glu Ala He Tyr Thr Pro Glu Glu Ala 130 135 140 ccg get aaa ate gat cac gtg att aaa act get ett cgt gag aag aag 480

Pro Ala Lys lie Asp His Val lie Lys Thr Ala Leu Arg Glu Lys Lys 145 150 155 160 ccg gtt tat etc gaa ate get tgc aac att get tee atg ccc tgc gee 528

Pro Val Tyr Leu Glu He Ala Cys Asn lie Ala Ser Met Pro Cys Ala 165 170 175 get cct gga ccg gca age gca ttg ttc aat gac gaa gee age gac gaa 576

Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu 180 185 190 get tet ttg aat gca geg gtt gaa gaa acc ctg aaa ttc ate gee aac 624

Ala Ser Leu Asn Ala Ala Val Glu Glu Thr Leu Lys Phe He Ala Asn 195 200 205

Ege gac aaa gtt gee gtc etc gtc ggc age aag ctg ege gca get ggt 672

Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala Gly 210 215 220 get gaa gaa get get gtc aaa ttt get gat get etc ggt ggc gca gtt 720

Ala Glu Glu Ala Ala Val Lys Phe Ala Asp Ala Leu Gly Gly Ala Val 225 230 235 240 get acc atg get get gca aaa age ttc ttc cca gaa gaa aac ccg cat 768

Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Pro His 245 250 255 tac ate ggc acc tea tgg ggt gaa gtc age tat ccg ggc gtt gaa aag 816

Tyr lie Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu Lys 260 265 270 aeg atg aaa gaa gee gat geg gtt ate get ctg get cct gtc ttc aac 864

Thr Met Lys Glu Ala Asp Ala Val lie Ala Leu Ala Pro Val Phe Asn 275 280 285 gac tac tee acc act ggt tgg aeg gat att cct gat cct aag aaa ctg 912

Asp Tyr Ser Thr Thr Gly Trp Thr Asp He Pro Asp Pro Lys Lys Leu 290 295 300 gtt etc get gaa ccg cgt tet gtc gtc gtt aac ggc att ege ttc ccc 960

Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly lie Arg Phe Pro 305 310 315 320 age gtc cat ctg aaa gac tat ctg acc cgt ttg get cag aaa gtt tee 1008

Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gin Lys Val Ser 325 330 335 aag aaa acc ggt gca ttg gac ttc ttc aaa tee etc aat gca ggt gaa 1056

Lys Lys Thr Gly Ala Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly Glu 340 345 350 ctg aag aaa gee get ccg get gat ccg agt get ccg ttg gtc aac gca 1104

Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn Ala 355 360 365 62 1152 1152

201033369 gag〒c 〒gt gg gtc gaa get ctt ctg acc ccg aac acg acg gtt

Glu Ala Arg Gin Val δΐιι Xia Leu Leu Thr Pro Asn Thr Thr Vai 370 375 380 τι1 f?1· η?1 ?ac tct ttc aat get cag ege atg aag etc lie Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gin Arg Met Lys Leu 385 390 395 400 ccg aac ggt get ege gtt gaa tat gaa atg cag tgg ggt cac att ggt

Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gin Trp Gly His lie Gly 4〇5 410 415 tgg tee gtt cct gee gee ttc ggt tat gee gtc ggt get ccg gaa cgt

Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg 42〇425 430 ege aac ate etc atg gtt ggt gat ggt tee ttc cag ctg aeg get cag Arg Asn lie Leu Met Val Gly Asp Gly Ser Phe Gin Leu Thr Ala Gin 435 440 445 a gt〒 g〒t cag atg gtt ege ctg aaa ctg ccg gtt ate ate ttc ttg Glu Val Ala Gin Met Val Arg Leu Lys Leu Pro Val lie He Phe Leu 450 455 460 ate aat aac tat ggt tac Acc gee gaa gtt atg ate cat gat ggt ccg He Asn Asn Tyr Gly Tyr Thr Ala Glu Val Met lie His Asp Gly Pro 465 470 475 480 tac aac aac ate aag aac tgg gat tat gee ggt ctg atg gaa gtg ttc Tyr Asn Asn lie Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val Phe 485 490 495 aac ggt aac ggt ggt tat gac age ggt get ggt aaa ggc ctg aag get Asn 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 get ate aag gtt get ctg gca aac Lys Thr Gly Gly Glu Leu Ala Glu Ala lie Lys Val Ala Leu Ala Asn 515 520 525 acc gac ggc cca acc ctg ate gaa tgc ttc ate ggt cgt gaa gac Tgc Thr Asp Gly Pro Thr Leu lie Glu Cys Phe He Gly Arg Glu Asp Cys 530 535 540 act gaa gaa ttg gtc aaa tgg ggt aag ege gtt get gee gee aac age

Thr 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 etc etc tag

Arg Lys Pro Val Asn Lys Leu Leu 565

<210> 37 <211> 568 <212> PRT <213> Active Zymomonas <400> 37 1200 1248 1296 1344 1392 1440 1488 1536 1584 1632 1680 1707 63 201033369

Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gin lie 15 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 Gin 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 lie Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val lie Leu 85 90 95

He 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 Gin Leu Glu Met Ala 115 120 125

Lys Asn lie Thr Ala Ala Ala Glu Ala lie Tyr Thr Pro Glu Glu Ala 130 135 140

Pro Ala Lys He Asp His Val He Lys Thr Ala Leu Arg Glu Lys Lys 145 150 155 160

Pro Val Tyr Leu Glu lie Ala Cys Asn lie 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 lie 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 64 201033369

Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Pro His 245 250 255

Tyr lie 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 lie Ala Leu Ala Pro Val Phe Asn 275 280 285

Asp Tyr Ser Thr Thr Gly Trp Thr Asp lie Pro Asp Pro Lys Lys Leu 290 295 300

Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly lie Arg Phe Pro 305 310 315 320

Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gin 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 lie Ala Arg Gin Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val 370 375 380 lie Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gin Arg Met Lys Leu 385 390 395 400

Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gin Trp Gly His He 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 He Leu Met Val Gly Asp Gly Ser Phe Gin Leu Thr Ala Gin 435 440 445

Glu Val Ala Gin Met Val Arg Leu Lys Leu Pro Val He lie Phe Leu 450 455 460

He Asn Asn Tyr Gly Tyr Thr Ala Glu Val Met lie His Asp Gly Pro 65 201033369 465 470 475 480

Tyr Asn Asn lie 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 lie Lys Val Ala Leu Ala Asn 515 520 525

Thr Asp Gly Pro Thr Leu lie Glu Cys Phe lie 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 <210> 38 <211> 1707 <212> DNA <213>artificial<220><223> Activity Zymomonas pyruvate decarboxylation PdcI472A codon Optimized gene <400> 38 atgtcttata ctgttggtac ttatctggct gagcgtctgg tgcaaatcgg cctgaaacac 60

cactttgcag ttgctggcga ctacaacctg gttctgctgg ataacctgct gctgaacaaa 120 aacatggagc aagtttattg ctgtaacgag ctgaactgcg gcttctctgc ggagggttat 180 gcgcgtgcga aaggtgccgc tgcagcagtc gtaacctact ctgtgggcgc tctgtccgcg 240 ttcgacgcaa tcggtggcgc ttacgctgaa aacctgccgg tgatcctgat tagcggtgcg 300 ccgaataata acgaccatgc tgctggccac gttctgcacc acgccctggg taaaactgat 360 taccattacc agctggagat ggctaaaaac atcactgcag cagcagaagc gatctacacc 420 ccggaagagg ctccggcaaa aatcgaccac gtgattaaaa ccgctctgcg tgagaaaaag 480 ccggtatacc tggaaatcgc gtgcaacatc gcgtctatgc cgtgcgccgc accgggtccg 540 gcttctgccc tgttcaacga tgaggcgagc gatgaggcat ctctgaacgc agcagtagaa 600 gaaaccctga aatttatcgc aaaccgtgac aaagtagcag tcctggtagg ttctaaactg 660 cgtgcggctg gtgcggaaga ggctgcggta aagttcgcgg atgctctggg cggtgcagtg 720 66 780 780

201033369 gcgaccatgg cagcggctaa atccttcttc ccagaggaga acccgcatta cattggtacc tcctggggcg aagtttccta ccctggtgtg gagaaaacca tgaaagaagc cgatgctgtg attgccctgg cgcctgtatt caacgattat tccaccaccg gttggaccga tatcccggac ccgaagaaac tggtcctggc tgaaccgcgc tccgtagtag tgaatggcat tcgtttcccg tccgtacacc tgaaggatta cctgacgcgt ctggcacaga aagtatccaa gaaaactggc gcgctggact tctttaaatc cctgaacgct ggtgagctga aaaaggcggc tccggccgat ccgtccgcac cgctggtgaa cgcagagatt gcacgtcagg ttgaggcact gctgacgccg aacaccaccg taatcgcgga aacgggcgac tcttggttca acgcacagcg catgaaactg ccgaacggtg cccgcgttga atatgaaatg cagtggggtc acatcggctg gtctgtccca reference gcagcgtttg gttacgcggt tggtgcaccg gagcgtcgca acatcctgat ggtgggtgac ggctccttcc agctgactgc tcaggaggtg gcgcagatgg tgcgcctgaa gctgccggtt. atcattttcc tgatcaacaa ctacggctac accgccgagg taatgatcca cgatggtccg, tacaacaaca tcaaaaactg ggactacgcc ggtctgatgg aggtttttaa cggtaacggc ggttacgaca gcggtgctgg taagggtctg aaagccaaaa ccggtggcga actggcagag gcgattaaag ttgcgctggc aaacaccgat ggcccgaccc tgatcgagtg ctt Catcggc cgtgaggact gcaccgagga gctggtcaaa tggggcaaac gtgtggcggc tgctaactct cgcaagccgg taaacaaact gctgtaa <210> 39 <211> 1644 <212> DNA <213> Lactococcus lactis <220>221> CDS <222> (1644) <400> 39 atg tat aca gta gga gat tac ctg tta gac cga tta cac gag ttg gga

Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 15 10 15 att gaa gaa att ttt gga gtt cct ggt gac tat aac tta caa ttt tta

He Glu Glu He Phe Gly Val Pro Gly Asp Tyr Asn Leu Gin Phe Leu 20 25 30 gat caa att att tea ege gaa gat atg aaa tgg att gga aat get aat

Asp Gin lie lie Ser Arg Glu Asp Met Lys Trp lie Gly Asn Ala Asn 35 40 45 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1707 48 96 67 144 201033369 gaa tta aat get tet tat atg get gat ggt Tat get cgt act aaa aaa 192

Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys 50 55 60 get gee gca ttt etc acc aca ttt gga gtc ggc gaa ttg agt geg ate 240

Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala He 65 70 75 80 aat gga ctg gca gga agt tat gee gaa aat tta cca gta gta gaa att 288

Asn Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu He 85 90 95 gtt ggt tea cca act tea aaa gta caa aat gac gga aaa ttt gtc cat 336

Val Gly Ser Pro Thr Ser Lys Val Gin Asn Asp Gly Lys Phe Val His 100 105 110 cat aca eta gca gat ggt gat ttt aaa cac ttt atg aag atg cat gaa 384

His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu 115 120 125

Cct gtt aca gca geg egg act tta ctg aca gca gaa aat gee aca tat 432

Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr Tyr 130 135 140 gaa att gac ega gta ett tet caa tta eta aaa gaa aga aaa cca gtc 480

Glu lie Asp Arg Val Leu Ser Gin Leu Leu Lys Glu Arg Lys Pro Val 145 150 155 160 tat att aac tta cca gtc gat gtt get gca gca aaa gca gag aag cct 528

Tyr He Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro 165 170 175 gca tta tet tta gaa aaa gaa age tet aca aca aat aca act gaa caa 576

Ala Leu Ser Leu Glu Lys Glu Ser Ser Thr Thr Asn Thr Thr Glu Gin 180 185 190 gtg att ttg agt ag att gaa gaa agt ttg aaa aat gee caa aaa cca 624

Val lie Leu Ser Lys lie Glu Glu Ser Leu Lys Asn Ala Gin Lys Pro 195 200 205 gta gtg att gca gga cac gaa gta att agt ttt ggt tta gaa aaa aeg 672

Val Val lie Ala Gly His Glu Val lie Ser Phe Gly Leu Glu Lys Thr 210 215 220 gta act cag ttt gtt tea gaa aca aaa eta ccg att aeg aca eta aat 720

Val Thr Gin Phe Val Ser Glu Thr Lys Leu Pro lie Thr Thr Leu Asn 225 230 235 240 ttt ggt aaa agt get gtt gat gaa tet ttg ccc tea ttt tta gga ata 768

Phe Gly Lys Ser Ala Val Asp Glu Ser Leu Pro Ser Phe Leu Gly lie 245 250 255 tat aac ggg aaa ett tea gaa ate agt ett aaa aat ttt gtg gag tee 816

Tyr Asn Gly Lys Leu Ser Glu He Ser Leu Lys Asn Phe Val Glu Ser 260 265 270 gca gac ttt ate eta atg ett gga gtg aag ett aeg gac tee tea aca 864

Ala Asp Phe lie Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr 275 280 285 68 912 912

201033369 ggt gca ttc aca cat cat tta gat gaa aat aaa atg att tea eta aac

Gly Ala Phe Thr His His Leu Asp Glu Asn Lys Met lie Ser Leu Asn 290 295 300 ata gat gaa gga ata att ttc aat aaa gtg gta gaa gat ttt gat ttt lie Asp Glu Gly lie He Phe Asn Lys Val Val Glu Asp Phe Asp Phe 305 310 315 320 aga gca gtg gtt tet tet tta tea gaa tta aaa gga ata gaa tat gaa

Arg Ala Val Val Ser Ser Leu Ser Glu Leu Lys Gly lie Glu Tyr Glu 325 330 335 gga caa tat att gat aag caa tat gaa gaa ttt att cca tea agt get

Gly Gin Tyr He Asp Lys Gin Tyr Glu Glu Phe He Pro Ser Ser Ala 340 345 350 ccc tta tea caa gac cgt eta tgg cag gca gtt gaa agt ttg act caa

Pro Leu Ser Gin Asp Arg Leu Trp Gin Ala Val Glu Ser Leu Thr Gin 355 360 365 age aat gaa aca ate gtt get gaa caa gga acc tea ttt ttt gga get

Ser Asn Glu Thr lie Val Ala Glu Gin Gly Thr Ser Phe Phe Gly Ala 370 375 380 tea aca att ttc tta aaa tea aat agt cgt ttt att gga caa cct tta

Ser Thr He He He He He He He He He He He He He He He He He He He He He He He He He He He

Trp Gly Ser lie Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gin He 405 410 415 geg gat aaa gag age aga cac ett tta ttt att ggt gat ggt tea ett

Ala Asp Lys Glu Ser Arg His Leu Leu Phe He Gly Asp Gly Ser Leu 420 425 430 caa ett acc gta caa gaa tta gga eta tea ate aga gaa aaa etc aat

Gin Leu Thr Val Gin Glu Leu Gly Leu Ser He Arg Glu Lys Leu Asn 435 440 445 cca att tgt ttt ate ata aat aat gat ggt tat aca gtt gaa aga gaa

Pro lie Cys Phe He He Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu 450 455 460 ate cac gga cct act caa agt tat aac gac att cca atg tgg aat tac lie His Gly Pro Thr Gin Ser Tyr Asn Asp He Pro Met Trp Asn Tyr 465 470 475 480 teg aaa tta cca gaa aca ttt gga gca aca gaa gat cgt gta gta tea

Ser 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 tet gtc atg aaa gaa gcc

Lys He 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 eta gtt ttg gaa aaa

Gin Ala Asp Val Asn Arg Met Tyr Trp lie Glu Leu Val Leu Glu Lys 960 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 1488 1536 1584 69 201033369 515 520 525 gaa gat gcg cca aaa tta ctg aaa aaa atg ggt aaa tta ttt get Gag 1632

Glu Asp Ala Pro Lys Leu Leu Lys Lys Met Gly Lys Leu Phe Ala Glu 530 535 540 caa aat aaa tag 1644

Gin Asn Lys 545 <210> 40 <211> 547 <212> PRT <213> Lactococcus lactis <400> 40 φ

Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 15 10 15 lie Glu Glu He Phe Gly Val Pro Gly Asp Tyr Asn Leu Gin Phe Leu 20 25 30

Asp Gin lie lie Ser Arg Glu Asp Met Lys Trp lie 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 He 65 70 75 80

Asn Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu lie 85 90 95

Val Gly Ser Pro Thr Ser Lys Val Gin 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 He Asp Arg Val Leu Ser Gin Leu Leu Lys Glu Arg Lys Pro Val 145 150 155 160

Tyr He Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro 70 201033369 165 170 175

Ala Leu Ser Leu Glu Lys Glu Ser Ser Thr Thr Asn Thr Thr Glu Gin 180 185 190

Val He Leu Ser Lys lie Glu Glu Ser Leu Lys Asn Ala Gin Lys Pro 195 200 205

Val Val lie Ala Gly His Glu Val He Ser Phe Gly Leu Glu Lys Thr 210 215 220

Val Thr Gin Phe Val Ser Glu Thr Lys Leu Pro lie Thr Thr Leu Asn 225 230 235 240

Phe Gly Lys Ser Ala Val Asp Glu Ser Leu Pro Ser Phe Leu Gly lie 245 250 255

Tyr Asn Gly Lys Leu Ser Glu lie Ser Leu Lys Asn Phe Val Glu Ser 260 265 270

Ala Asp Phe He 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 He Ser Leu Asn 290 295 300 lie Asp Glu Gly lie lie 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 lie Glu Tyr Glu 325 330 335

Gly Gin Tyr lie Asp Lys Gin Tyr Glu Glu Phe lie Pro Ser Ser Ala 340 345 350

Pro Leu Ser Gin Asp Arg Leu Trp Gin Ala Val Glu Ser Leu Thr Gin 355 360 365

Ser Asn Glu Thr He Val Ala Glu Gin Gly Thr Ser Phe Phe Gly Ala 370 375 380

Ser Thr lie Phe Leu Lys Ser Asn Ser Arg Phe lie Gly Gin Pro Leu 385 390 395 400 71 201033369

Trp Gly Ser lie Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gin He 405 410 415

Ala Asp Lys Glu Ser Arg His Leu Leu Phe lie Gly Asp Gly Ser Leu 420 425 430

Gin Leu Thr Val Gin Glu Leu Gly Leu Ser He Arg Glu Lys Leu Asn 435 440 445

Pro lie Cys Phe lie lie Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu 450 455 460 lie His Gly Pro Thr Gin Ser Tyr Asn Asp lie 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 He Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala 500 505 510

Gin Ala Asp Val Asn Arg Met Tyr Trp He 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

Gin Asn Lys 545

<210> 41 <211> 1644 <212> DNA <213>artificial<220><223> Lactococcus lactis branched chain alpha ketoacid decarboxylation KdcA codon-optimized gene <400> 41 atgtatactg ttggtgatta tctgctggac cgtctgcatg aactgggcat tgaagaaatc 60 ttcggtgtcc caggcgacta caacctgcag ttcctggacc agatcatctc ccgcgaagat 120 atgaaatgga tcggtaacgc aaacgagctg aacgcgtctt atatggctga tggttatgct 180 cgcaccaaaa aggctgcggc ctttctgacc acctttggtg tgggcgagct gagcgcgatc 240 aacggcctgg caggttccta cgctgagaac ctgccggtag tagaaatcgt tggttccccg 300 72 360 360

201033369 acctctaagg ttcagaacga cggcaaattc gtacatcaca ccctggcgga cggcgatttt aagcacttta tgaaaatgca cgaaccggtc accgccgctc gcactctgct gaccgcggaa aacgcaacgt acgagatcga tcgtgtactg tcccagctgc tgaaagaacg taaaccggtg tatatcaatc tgccggttga tgtcgctgcg gccaaagcag agaaaccggc actgtccctg gagaaggaga gctccactac taacaccacc gaacaggtta tcctgtccaa aattgaagaa tctctgaaaa acgcacagaa accggtggtt atcgcaggtc acgaggttat ctccttcggc ctggagaaaa ctgttactca attcgtctct gaaacgaaac tgccgatcac gaccctgaac tttggcaagt ccgcagttga cgaatctctg ccttctttcc tgggcattta caacggcaaa ctgtccgaga tctccctgaa gaacttcgta gaatccgctg actttatcct gatgctgggt gtgaaactga ccgactcctc taccggtgcg ttcacgcacc atctggatga aaacaaaatg atcagcctga acatcgacga gggtatcatc ttcaacaagg tagttgaaga tttcgacttc cgtgctgttg tcagcagcct gtccgagctg aaaggcattg agtacgaggg tcaatacatc gataaacagt acgaagagtt tattccgtct tctgcaccgc tgagccagga ccgcctgtgg caggcagttg agtccctgac gcagtccaac gaaactatcg tagcggaaca aggtacctct ttcttcggtg cttctaccat ctttctgaag tccaactctc gctttatcgg tcagccgctg tggggttcta tcggttacac gttcccggct gcgctgggta gccagatcgc tgataaagag tctcgtcatc tgctgttcat cggtgatggt tccctgcagc tgactgtaca ggaactgggt ctgtctatcc gtgaaaaact gaacccgatt tgttttatca tcaataacga tggctacact gttgagcgtg aaattcatgg tccgactcag tcttacaacg atattccgat gtggaactac tctaaactgc cggaaacctt cggtgcaact gaggatcgcg tcgtgagcaa gattgtgcgt actgagaacg agttcgtatc tgttatgaaa gaggcgcagg cagatgtgaa ccgcatgtac tggatcgaac tggttctgga aaaagaggat gcaccgaaac tgctgaagaa aatgggtaaa ctgtttgcgg agcagaacaa gtaa < 210> 42 <211> 1647 <212> DNA <213> Lactococcus lactis <220>221>221> CDS <222> (1)..(1647) <400> 42 atg tat aca gta gga gat tac Eta tta gac ega tta cac gag tta gga 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1644 48 73 201033369

Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 15 10 15 att gaa gaa att ttt gga gtc cct gga gac tat aac tta caa ttt tta 96 lie Glu Glu lie Phe Gly Val Pro Gly Asp Tyr Asn Leu Gin Phe Leu 20 25 30 gat caa att att tcc cac aag gat atg aaa tgg gtc gga aat get aat 144

Asp Gin He lie Ser His Lys Asp Met Lys Trp Val Gly Asn Ala Asn 35 40 45 gaa tta aat get tea tat atg get gat ggc tat get cgt act aaa aaa 192

Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys 50 55 60 get gcc gca ttt ett aca acc ttt gga gta ggt gaa ttg agt gca gtt 240

Ala 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 288

Asn Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu He 85 90 95 gtg gga tea cct aca tea aaa gtt caa aat gaa gga aaa ttt gtt cat 336

Val Gly Ser Pro Thr Ser Lys Val Gin Asn Glu Gly Lys Phe Val His 100 105 110 cat aeg ctg get gac ggt gat ttt aaa cac ttt atg aaa atg cac gaa 384

His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu 115 120 125 cct gtt aca gca get ega act tta ctg aca gca gaa aat gca acc gtt 432

Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr Val 130 135 140 gaa att gac ega gta ett tet gca eta tta aaa gaa aga aaa cct gtc 480

Glu lie Asp Arg Val Leu Ser Ala Leu Leu Lys Glu Arg Lys Pro Val 145 150 155 160 tat ate aac tta cca gtt gat gtt get get gca aaa gca gag aaa ccc 528

Tyr lie Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro 165 170 175 tea etc cct ttg aaa aag gaa aac tea act tea aat aca agt gac caa 576

Ser Leu Pro Leu Lys Lys Glu Asn Ser Thr Ser Asn Thr Ser Asp Gin 180 185 190 gaa att ttg aac aaa att caa gaa age ttg aaa aat gcc aaa aaa cca 624

Glu lie Leu Asn Lys lie Gin Glu Ser Leu Lys Asn Ala Lys Lys Pro 195 200 205 ate gtg att aca gga cat gaa ata att agt ttt ggc tta gaa aaa aca 672 lie Val lie Thr Gly His Glu lie lie Ser Phe Gly Leu Glu Lys Thr 210 215 220 gtc act caa ttt att tea aag aca aaa eta cct att aeg aca tta aac 720

Val Thr Gin Phe lie Ser Lys Thr Lys Leu Pro lie Thr Thr Leu Asn 225 230 235 240 74 768 768

201033369 ttt ggt aaa agt tea gtt gat gaa gee etc cct tea ttt tta gga ate

Phe Gly Lys Ser Ser Val Asp Glu Ala Leu Pro Ser Phe Leu Gly lie 245 250 255 tat aat ggt aca etc tea gag cct aat ett aaa gaa tie gtg gaa tea

Tyr Asn Gly Thr Leu Ser Glu Pro Asn Leu Lys Glu Phe Val Glu Ser 260 265 270 gee gac ttc ate ttg atg ett gga gtt aaa etc aca gac tet tea aca

Ala Asp Phe lie Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr 275 280 285 gga gee ttc act cat cat tta aat gaa aat aaa atg att tea ctg aat

Gly Ala Phe Thr His His Leu Asn Glu Asn Lys Met He Ser Leu Asn 290 295 300 ata gat gaa gga aaa ata ttt aac gaa aga ate caa aat ttt gat ttt

He Asp Glu Gly Lys lie Phe Asn Glu Arg lie Gin Asn Phe Asp Phe 305 310 315 320 gaa tee etc ate tee tet etc tta gac eta age gaa ata gaa tac aaa

Glu Ser Leu lie Ser Ser Leu Leu Asp Leu Ser Glu He Glu Tyr Lys 325 330 335 gga aaa tat ate gat aaa aag caa gaa gac ttt gtt cca tea aat geg - Gly Lys Tyr lie Asp Lys Lys Gin Glu Asp Phe Val Pro Ser Asn Ala . 340 345 350 . ett tta tea caa gac ege eta tgg caa gca gtt gaa aac eta act caa

Leu Leu Ser Gin Asp Arg Leu Trp Gin Ala Val Glu Asn Leu Thr Gin 355 360 365 age aat gaa aca ate gtt get gaa caa ggg aca tea ttc ttt ggc get

Ser Asn Glu Thr lie Val Ala Glu Gin Gly Thr Ser Phe Phe Gly Ala 370 375 380 •4 tea tea att ttc tta aaa tea aag agt cat ttt att ggt caa ccc tta

Ser Ser lie Phe Leu Lys Ser Lys Ser His Phe lie Gly Gin Pro Leu 0 385 390 395 400 tgg gga tea att gga tat aca ttc cca gca gca tta gga age caa att

Trp Gly Ser lie Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gin lie 405 410 415 gca gat aaa gaa age aga cac ett tta ttt att ggt gat ggt tea ett

Ala Asp Lys Glu Ser Arg His Leu Leu Phe lie Gly Asp Gly Ser Leu 420 425 430 caa ett aca gtg caa gaa tta gga tta gca ate aga ga gaa aaa att aat

Gin Leu Thr Val Gin Glu Leu Gly Leu Ala lie Arg Glu Lys lie Asn - 435 440 445 cca att tgc ttt att ate aat aat gat ggt tat aca gtc gaa aga gaa

Pro lie Cys Phe He lie Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu 450 455 460 att cat gga cca aat caa age tac aat gat att cca atg tgg aat tac lie His Gly Pro Asn Gin Ser Tyr Asn Asp lie Pro Met Trp Asn Tyr 465 470 475 480 480 816 864 912 960 1008 1056 1104 1152 1200 1248 1296 1344 1392 75 1440 201033369 tea aaa tta cca gaa teg ttt gga gca aca gaa gat ega gta gtc tea 1488 Ser Lys Leu Pro Glu Ser Phe Gly Ala Thr Glu Asp Arg Val Val Ser 485 490 495 aaa ate gtt aga act gaa aat gaa ttt gtg tet gtc atg aaa gaa get 1536 Lys lie 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 at t ttg gca aaa 1584 Gin Ala Asp Pro Asn Arg Met Tyr Trp lie Glu Leu lie Leu Ala Lys 515 520 525 gaa ggt gca cca aaa gta ctg aaa aaa atg ggc aaa eta ttt get gaa 1632 Glu Gly Ala Pro Lys Val Leu Lys Lys Met Gly Lys Leu Phe Ala Glu 530 535 540 caa aat aaa tea taa 1647 Gin Asn Lys Ser 545

<210> 43 <211> 548 <212> PRT <213> Lactococcus lactis <400>

Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 15 10 15 lie Glu Glu lie Phe Gly Val Pro Gly Asp Tyr Asn Leu Gin Phe Leu 20 25 30

Asp Gin lie lie 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 lie 85 90 95

Val Gly Ser Pro Thr Ser Lys Val Gin 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 76 201033369

Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr Val 130 135 140

Glu lie Asp Arg Val Leu Ser Ala Leu Leu Lys Glu Arg Lys Pro Val 145 150 155 160

Tyr lie 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 Gin 180 185 190

Glu lie Leu Asn Lys He Gin Glu Ser Leu Lys Asn Ala Lys Lys Pro 195 200 205 lie Val lie Thr Gly His Glu lie He Ser Phe Gly Leu Glu Lys Thr 210 215 220

Val Thr Gin Phe lie Ser Lys Thr Lys Leu Pro He Thr Thr Leu Asn 225 230 235 240

Phe Gly Lys Ser Ser Val Asp Glu Ala Leu Pro Ser Phe Leu Gly lie 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 He 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 lie Ser Leu Asn 290 295 300

He Asp Glu Gly Lys lie Phe Asn Glu Arg lie Gin Asn Phe Asp Phe 305 310 315 320

Glu Ser Leu He Ser Ser Leu Leu Asp Leu Ser Glu lie Glu Tyr Lys 325 330 335

Gly Lys Tyr He Asp Lys Lys Gin Glu Asp Phe Val Pro Ser Asn Ala 340 345 350

Leu Leu Ser Gin Asp Arg Leu Trp Gin Ala Val Glu Asn Leu Thr Gin 77 201033369 355 360 365

Ser Asn Glu Thr lie Val Ala Glu Gin Gly Thr Ser Phe Phe Gly Ala 370 375 380

Ser Ser lie Phe Leu Lys Ser Lys Ser His Phe lie Gly Gin Pro Leu 385 390 395 400

Trp Gly Ser lie Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gin lie 405 410 415

Ala Asp Lys Glu Ser Arg His Leu Leu Phe lie Gly Asp Gly Ser Leu 420 425 430

Gin Leu Thr Val Gin Glu Leu Gly Leu Ala lie Arg Glu Lys lie Asn 435 440 445

Pro lie Cys Phe lie lie Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu 450 455 460

He His Gly Pro Asn Gin Ser Tyr Asn Asp He 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 lie Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala 500 505 510

Gin Ala Asp Pro Asn Arg Met Tyr Trp lie Glu Leu lie 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

Gin Asn Lys Ser 545 <210> 44 <211> 1647 <212>DNA <213>manmade <220><223> Lactococcus lactis alpha valerate decarboxylated KivD codon optimization Gene 78 60 60

201033369 < 400> 44 atgtatactg ttggtgatta cctgctggat cgtctgcatg aactgggcat cgaggaaatt ttcggcgtac ctggtgacta taacctgcag ttcctggatc agatcatttc ccacaaagat atgaaatggg ttggtaacgc gaacgagctg aatgcaagct acatggctga cggttatgca cgcaccaaga aagctgcggc gttcctgact acttttggcg tcggcgagct gtctgcggta aacggtctgg ccggctccta cgcggaaaac ctgccggtag tagaaatcgt cggttccccg acctctaaag ttcagaacga gggtaaattc gtgcaccata ctctggccga tggtgacttc aaacacttca tgaagatgca cgaaccggtc actgctgctc gtacgctgct gaccgcggaa aatgcgactg tcgagattga tcgtgtactg agcgcactgc tgaaagaacg caagcctgta tacatcaacc tgccggttga tgtcgcggcc gccaaagcgg aaaaaccatc tctgccgctg aaaaaggaga acagcacctc taacaccagc gaccaggaaa tcctgaacaa gatccaggag tctctgaaga acgctaaaaa gccgatcgta atcaccggcc atgagattat ctctttcggt ctggagaaaa ctgtcaccca gttcatcagc aaaaccaaac tgccgatcac caccctgaac ttcggtaaat cctccgttga cgaagcgctg ccgtcctttc tgggtattta caacggcact ctgtctgagc cgaacctgaa agagttcgtg gagtctgcgg attttatcct gatgctgggc gtgaaactga cggattcctc caccggtgca ttcacccacc acctgaat ga gaataaaatg atctctctga acattgatga gggcaaaatc ttcaacgagc gtattcagaa cttcgatttc gaatccctga tctcctccct gctggatctg tccgagattg aatataaagg caaatacatt gataagaagc aagaggactt cgtaccgtct aacgcgctgc tgagccagga ccgtctgtgg caagctgtgg aaaacctgac ccagtccaac gaaaccatcg tggcggaaca gggtacctcc ttcttcggtg ctagctctat cttcctgaaa tctaaaagcc acttcatcgg tcagccactg tggggctcta ttggctacac cttcccggca gcgctgggtt cccaaatcgc agacaaagaa tcccgccacc tgctgttcat tggtgacggc tctctgcaac tgaccgtaca ggagctgggt ctggcgattc gtgagaaaat caacccgatt tgtttcatca tcaacaacga tggctacact gttgagcgtg agatccacgg cccgaaccag tcctacaacg acattccgat gtggaactac tctaaactgc cggaatcctt cggtgcgact gaagaccgtg tcgtaagcaa gatcgtccgt accgaaaacg aattcgtgtc tgtcatgaaa gaagcacagg cggacccgaa ccgcatgtac tggatcgagc tgattctggc taaagagggc gcgccaaaag tactgaaaaa gatgggtaaa ctgttcgcag aacagaacaa atcctaa < 210> 45 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1647 79 201033369 <211> 3696 <212> DNA <213>Mycobacterium tuberculosis<220>221> CDS <222> (1)..(3696) <400> 45 gtg gcc aac ata agt tea cca tie ggg caa aac Gaa tgg ctg gtc gaa 48

Val Ala Asn lie Ser Ser Pro Phe Gly Gin Asn Glu Trp Leu Val Glu 15 10 15 gag atg tac ege aag ttc ege gac gac ccc tee teg gtc gat ccc age 96

Glu 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 age ccc gaa ccc acc tee caa cca 144

Trp His Glu Phe Leu Val Asp Tyr Ser Pro Glu Pro Thr Ser Gin Pro 35 40 45 get gcc gaa cca acc egg gtt acc teg cca etc gtt gcc gag egg gcc 192

Ala Ala Glu Pro Thr Arg Val Thr Ser Pro Leu Val Ala Glu Arg Ala 50 55 60 get geg gcc gcc ccg cag gca ccc ccc aag ccg gcc gac acc geg gcc 240

Ala Ala Ala Ala Pro Gin Ala Pro Pro Lys Pro Ala Asp Thr Ala Ala 65 70 75 80 geg ggc aac ggc gtg gtc gcc gca ctg gee gcc aaa act gcc gtt ccc 288

Ala 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 geg gtg ctg ege ggc gcc gcc geg 336

Pro Pro Ala Glu Gly Asp Glu Val Ala Val Leu Arg Gly Ala Ala Ala 100 105 110

Gcc gtc gtc aag aac atg tee geg teg ttg gag gtg ccg aeg geg acc 384

Ala Val Val Lys Asn Met Ser Ala Ser Leu Glu Val Pro Thr Ala Thr 115 120 125 age gtc egg geg gtc ccg gcc aag eta ctg ate gac aac egg ate gtc 432

Ser Val Arg Ala Val Pro Ala Lys Leu Leu lie Asp Asn Arg lie Val 130 135 140 ate aac aac cag ttg aag egg acc ege ggc ggc aag ate teg ttc aeg 480 lie Asn Asn Gin Leu Lys Arg Thr Arg Gly Gly Lys lie Ser Phe Thr 145 150 155 160 cat ttg ctg ggc tac gcc ctg gtg cag geg gtg aag aaa ttc ccg aac 528

His Leu Leu Gly Tyr Ala Leu Val Gin Ala Val Lys Lys Phe Pro Asn 165 170 175 atg aac egg cac tac acc gaa gtc gac ggc aag ccc acc geg gtc aeg 576

Met Asn Arg His Tyr Thr Glu Val Asp Gly Lys Pro Thr Ala Val Thr 180 185 190 ccg geg cac acc aat etc ggc ctg geg ate gac ctg caa ggc aag gac 624 80 672 672

201033369

Pro Ala His Thr Asn Leu Gly Leu Ala lie Asp Leu Gin Gly Lys Asp 195 200 205 ggg aag cgt tcc ctg gtg gtg gcc ggc ate aag egg tgc gag acc atg

Gly Lys Arg Ser Leu Val Val Ala Gly He Lys Arg Cys Glu Thr Met 210 215 220 ega ttc geg cag ttc gtc aeg gcc tac gaa gac ate gta ege egg gcc

Arg Phe Ala Gin Phe Val Thr Ala Tyr Glu Asp lie Val Arg Arg Ala 225 230 235 240 ege gac ggc aag ctg acc act gaa gac ttt gcc ggc gtg aeg att teg

Arg Asp Gly Lys Leu Thr Thr Glu Asp Phe Ala Gly Val Thr lie Ser 245 250 255 ctg acc aat ccc gga acc ate ggc acc gtg cat teg gtg cog egg ctg

Leu Thr Asn Pro Gly Thr lie Gly Thr Val His Ser Val Pro Arg Leu 260 265 270 atg ccc ggc cag ggc gcc ate ate ggc gtg ggc gcc atg gaa tac ccc

Met Pro Gly Gin Gly Ala lie lie Gly Val Gly Ala Met Glu Tyr Pro 275 280 285 gcc gag ttt caa ggc gcc age gag gaa ege ate gcc gag ctg ggc ate

Ala Glu Phe Gin Gly Ala Ser Glu Glu Arg lie Ala Glu Leu Gly lie 290 295 300 ggc aaa ttg ate act ttg acc tec acc tac gac cac ege ate ate cag

Gly Lys Leu He Thr Leu Thr Ser Thr Tyr Asp His Arg He lie Gin 305 310 315 320 ggc geg gaa teg ggc gac ttc ctg ege acc ate cac gag ttg ctg etc

Gly Ala Glu Ser Gly Asp Phe Leu Arg Thr He His Glu Leu Leu Leu 325 330 335 teg gat ggc ttc tgg gac gag gtc ttc ege gaa ctg age ate cca tat

Ser Asp Gly Phe Trp Asp Glu Val Phe Arg Glu Leu Ser lie Pro Tyr 340 345 350 ctg ccg gtg ege tgg age acc gac aac ccc gac teg ate gtc gac aag

Leu Pro Val Arg Trp Ser Thr Asp Asn Pro Asp Ser lie Val Asp Lys 355 360 365 aac get ege gtc atg aac ttg ate geg gcc tac ege aac ege ggc cat

Asn Ala Arg Val Met Asn Leu He Ala Ala Tyr Arg Asn Arg Gly His 370 375 380 ctg atg gcc gat acc gac ccg ctg egg ttg gac aaa get egg ttc ege

Leu Met Ala Asp Thr Asp Pro Leu Arg Leu Asp Lys Ala Arg Phe Arg 385 390 395 400 agt cac ccc gac etc gaa gtg ctg acc cac ggc ctg aeg ctg tgg gat

Ser His Pro Asp Leu Glu Val Leu Thr His Gly Leu Thr Leu Trp Asp 405 410 415 etc gat egg gtg ttc aag gtc gac ggc ttt gcc ggt geg cag tac aag

Leu Asp Arg Val Phe Lys Val Asp Gly Phe Ala Gly Ala Gin Tyr Lys 420 425 430 720 768 816 864 912 960 1008 1056 1104 1152 1200 1248 81 1296 201033369 aaa ctg cgc gac gtg ctg ggc ttg ctg cgc gat gcc tac tgc cgc cac 1344

Lys Leu Arg Asp Val Leu Gly Leu Leu Arg Asp Ala Tyr Cys Arg His 435 440 445 ate ggc gtg gag tac gcc cat ate etc gac ccc gaa caa aag gag tgg 1392

He Gly Val Glu Tyr Ala His lie Leu Asp Pro Glu Gin Lys Glu Trp 450 455 460 etc gaa caa egg gtc gag acc aag cac gtc aaa ccc act gtg gcc caa 1440

Leu Glu Gin Arg Val Glu Thr Lys His Val Lys Pro Thr Val Ala Gin 465 470 475 480 cag aaa tac ate etc age aag etc aac gcc gcc gag gcc ttt gaa aeg 1488

Gin Lys Tyr lie Leu Ser Lys Leu Asn Ala Ala Glu Ala Phe Glu Thr 485 490 495 ttc eta cag acc aag tac gtc ggc cag aag egg ttc teg ctg gaa ggc 1536

Phe Leu Gin Thr Lys Tyr Val Gly Gin Lys Arg Phe Ser Leu Glu Gly 500 505 510

Gcc gaa age gtg ate ccg atg atg gac geg geg ate gac cag tgc get 1584

Ala Glu Ser Val lie Pro Met Met Asp Ala Ala lie Asp Gin Cys Ala 515 520 525 gag cac ggc etc gac gag gtg gtc ate ggg atg ccg cac egg ggc egg 1632

Glu His Gly Leu Asp Glu Val Val lie Gly Met Pro His Arg Gly Arg 530 535 540 etc aac gtg ctg gcc aac ate gtc ggc aag ccg tac teg cag ate ttc 1680

Leu Asn Val Leu Ala Asn lie Val Gly Lys Pro Tyr Ser Gin lie Phe 545 550 555 560 acc gag ttc gag ggc aac ctg aat ccg teg cag geg cac ggc tee ggt 1728

Thr Glu Phe Glu Gly Asn Leu Asn Pro Ser Gin Ala His Gly Ser Gly 565 570 575 gac gtc aag tac cac ctg ggc gcc acc ggg ctg tac ctg cag atg ttc 1776

Asp Val Lys Tyr His Leu Gly Ala Thr Gly Leu Tyr Leu Gin Met Phe

580 585 590 ggc gac aac gac att cag gtg teg ctg acc gcc aac ccg teg cat ctg 1824

Gly Asp Asn Asp lie Gin Val Ser Leu Thr Ala Asn Pro Ser His Leu 595 600 605 gag gcc gtc gac ccg gtg ctg gag gga ttg gtg egg gcc aag cag gat 1872

Glu Ala Val Asp Pro Val Leu Glu Gly Leu Val Arg Ala Lys Gin Asp 610 615 620 ctg etc gac cac gga age ate gac age gac ggc caa egg geg ttc teg 1920

Leu Leu Asp His Gly Ser He Asp Ser Asp Gly Gin Arg Ala Phe Ser 625 630 635 640 gtg gtg ccg ctg atg ttg cat ggc gat gcc geg ttc gcc ggt cag ggt 1968

Val Val Pro Leu Met Leu His Gly Asp Ala Ala Phe Ala Gly Gin Gly 645 650 655 gtg gtc gcc gag aeg ctg aac ctg geg aat ctg ccg ggc tac cgc gtc 2016

Val Val Ala Glu Thr Leu Asn Leu Ala Asn Leu Pro Gly Tyr Arg Val 660 665 670 82

201033369 ggc ggc acc ate cac ate ate gtc aac aac cag ate ggc ttc acc acc

Gly Gly Thr lie His lie lie Val Asn Asn Gin lie Gly Phe Thr Thr 675 680 685 geg ccc gag tat tee agg tee age gag tac tgc acc gac gtc gca aag

Ala Pro Glu Tyr Ser Arg Ser Ser Glu Tyr Cys Thr Asp Val Ala Lys 690 695 700 atg ate ggg gca ccg ate ttt cac gtc aac ggc gac gac ccg gag geg

Met lie Gly Ala Pro lie Phe His Val Asn Gly Asp Asp Pro Glu Ala 705 710 715 720 tgt gtc tgg gtg geg egg ttg geg gtg gac ttc ega caa egg ttc aag

Cys Val Trp Val Ala Arg Leu Ala Val Asp Phe Arg Gin Arg Phe Lys 725 730 735 aag gac gtc gtc ate gac atg ctg tgc tac ege ege ege ggg cac aac

Lys Asp Val Val lie Asp Met Leu Cys Tyr Arg Arg Arg Gly His Asn 740 745 750 gag ggt gac gac ccg teg atg acc aac ccc tac gtg tac gac gtc gtc

Glu Gly Asp Asp Pro Ser Met Thr Asn Pro Tyr Val Tyr Asp Val Val 755 760 765 gac acc aag ege ggg gee ege aaa age tac acc gaa gee ctg ate gga

Asp Thr Lys Arg Gly Ala Arg Lys Ser Tyr Thr Glu Ala Leu lie Gly 770 775 780 cgt ggc gac ate teg atg aag gag gee gag gac geg ctg ege gac tac

Arg Gly Asp lie Ser Met Lys Glu Ala Glu Asp Ala Leu Arg Asp Tyr 785 790 795 800 cag ggc cag ctg gaa egg gtg ttc aac gaa gtg ege gag ctg gag aag

Gin Gly Gin Leu Glu Arg Val Phe Asn Glu Val Arg Glu Leu Glu Lys 805 810 815 cac ggt gtg cag ccg age gag teg gtc gag tee gac cag atg att ccc

His Gly Val Gin Pro Ser Glu Ser Val Glu Ser Asp Gin Met lie Pro 820 825 830 geg ggg ctg gee act geg gtg gac aag teg ctg ctg gee egg ate ggc

Ala Gly Leu Ala Thr Ala Val Asp Lys Ser Leu Leu Ala Arg lie Gly 835 840 845 gat geg ttc etc gee ttg ccg aac ggc ttc acc geg cac ccg ega gtc

Asp Ala Phe Leu Ala Leu Pro Asn Gly Phe Thr Ala His Pro Arg Val 850 855 860 caa ccg gtg ctg gag aag ege egg gag atg gee tat gaa ggc aag ate

Gin Pro Val Leu Glu Lys Arg Arg Glu Met Ala Tyr Glu Gly Lys lie 865 870 875 880 gac tgg gee ttt ggc gag ctg ctg geg ctg ggc teg ctg gtg gee gaa

Asp Trp Ala Phe Gly Glu Leu Leu Ala Leu Gly Ser Leu Val Ala Glu 885 890 895 ggc aag ctg gtg ege ttg teg ggg cag gac age ege ege ggc acc ttc

Gly Lys Leu Val Arg Leu Ser Gly Gin Asp Ser Arg Arg Gly Thr Phe 2064 2112 2160 2208 2256 2304 2352 2400 2448 2496 2544 2592 2640 2688 2736 83 201033369 900 905 910 tcc cag egg cat teg gtt etc ate gac ege cac act ggc gag Gag ttc 2784

Ser Gin Arg His Ser Val Leu lie Asp Arg His Thr Gly Glu Glu Phe 915 920 925 aca cca ctg cag ctg ctg geg acc aac tec gac ggc age ccg acc ggc 2832

Thr Pro Leu Gin Leu Leu Ala Thr Asn Ser Asp Gly Ser Pro Thr Gly 930 935 940 gga aag ttc ctg gtc tac gac teg cca ctg teg gag tac gee gee gtc 2880

Gly 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 gee gtg gtg etc 2928

Gly Phe Glu Tyr Gly Tyr Thr Val Gly Asn Pro Asp Ala Val Val Leu 965 970 975 Φ tgg gag geg cag ttc ggc gac ttc gtc aac ggc geg cag teg ate ate 2976

Trp Glu Ala Gin Phe Gly Asp Phe Val Asn Gly Ala Gin Ser lie 980 985 990 gac gag ttc ate age tec ggt gag gee aag tgg ggc caa ttg tec aac 3024

Asp Glu Phe lie Ser Ser Gly Glu Ala Lys Trp Gly Gin Leu Ser Asn 995 1000 1005 gtc gtg ctg ctg tta ccg cac ggg cac gag ggg cag gga ccc gac 3069

Val Val Leu Leu Leu Pro His Gly His Glu Gly Gin Gly Pro Asp 1010 1015 1020 cac act tet gee egg ate gaa ege ttc ttg cag ttg tgg geg gaa 3114

His Thr Ser Ala Arg lie Glu Arg Phe Leu Gin Leu Trp Ala Glu 1025 1030 1035 ggt teg atg acc ate geg atg ccg teg act ccg teg aac tac ttc 3159

Gly Ser Met Thr He Ala Met Pro Ser Thr Pro Ser Asn Tyr Phe 1040 1045 1050

Cac ctg eta ege egg cat gee ctg gac ggc ate caa ege ccg ctg 3204

His Leu Leu Arg Arg His Ala Leu Asp Gly He Gin Arg Pro Leu 1055 1060 1065 ate gtg ttc aeg ccc aag teg atg ttg cgt cac aag gee gee gtc 3249 lie Val Phe Thr Pro Lys Ser Met Leu Arg His Lys Ala Ala Val 1070 1075 1080 age gaa ate aag gac ttc acc gag ate aag ttc ege tea gtg ctg 3294

Ser Glu He Lys Asp Phe Thr Glu lie Lys Phe Arg Ser Val Leu 1085 1090 1095 gag gaa ccc acc tat gag gac ggc ate gga gac ege aac aag gtc 3339

Glu Glu Pro Thr Tyr Glu Asp Gly lie Gly Asp Arg Asn Lys Val 1100 1105 1110 age egg ate ctg ctg acc agt ggc aag ctg tat tac gag ctg gee 3384

Ser Arg lie Leu Leu Thr Ser Gly Lys Leu Tyr Tyr Glu Leu Ala 1115 1120 1125 gee ege aag gee aag gac aac ege aat gac etc geg ate gtg egg 3429 84

201033369

Ala Arg Lys Ala Lys Asp Asn Arg Asn Asp Leu Ala lie Val Arg 1130 1135 1140 ctt gaa cag etc gcc ccg ctg ccc agg cgt ega ctg cgt gaa aeg

Leu Glu Gin Leu Ala Pro Leu Pro Arg Arg Arg Leu Arg Glu Thr 1145 1150 1155 ctg gac ege tac gag aac gtc aag gag ttc ttc tgg gtc caa gag

Leu Asp Arg Tyr Glu Asn Val Lys Glu Phe Phe Trp Val Gin Glu 1160 1165 1170 gaa ccg gcc aac cag ggt geg tgg ccg ega ttc ggg etc gaa eta

Glu Pro Ala Asn Gin Gly Ala Trp Pro Arg Phe Gly Leu Glu Leu 1175 1180 1185 ccc gag ctg ctg cct gac aag ttg gcc ggg ate aag ega ate teg

Pro Glu Leu Leu Pro Asp Lys Leu Ala Gly lie Lys Arg lie Ser 1190 1195 1200 ege egg geg atg tea gcc ccg teg tea ggc teg teg aag gtg cac

Arg Arg Ala Met Ser Ala Pro Ser Ser Gly Ser Ser Lys Val His 1205 1210 1215 gcc gtc gaa cag cag gag ate etc gac gag geg ttc ggc tga

Ala Val Glu Gin Gin Glu lie Leu Asp Glu Ala Phe Gly 1220 1225 1230 . <210> 46 <211> 1231 <212> PRT <213> Mycobacterium tuberculosis <400> 46

Val Ala Asn lie Ser Ser Pro Phe Gly Gin Asn Glu Trp Leu Val Glu 15 10 15 ❹

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 Gin 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 Gin 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 3474 3519 3564 3609 3654 3696 85 201033369

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 lie Asp Asn Arg He Val 130 135 140 lie Asn Asn Gin Leu Lys Arg Thr Arg Gly Gly Lys lie Ser Phe Thr 145 150 155 160

His Leu Leu Gly Tyr Ala Leu Val Gin 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 He Asp Leu Gin Gly Lys Asp 195 200 205

Gly Lys Arg Ser Leu Val Val Ala Gly He Lys Arg Cys Glu Thr Met 210 215 220

Arg Phe Ala Gin Phe Val Thr Ala Tyr Glu Asp lie Val Arg Arg Ala 225 230 235 240

Arg Asp Gly Lys Leu Thr Thr Glu Asp Phe Ala Gly Val Thr lie Ser 245 250 255

Leu Thr Asn Pro Gly Thr He Gly Thr Val His Ser Val Pro Arg Leu 260 265 270

Met Pro Gly Gin Gly Ala lie He Gly Val Gly Ala Met Glu Tyr Pro 275 280 285

Ala Glu Phe Gin Gly Ala Ser Glu Glu Arg lie Ala Glu Leu Gly lie 290 295 300

Gly Lys Leu He Thr Leu Thr Ser Thr Tyr Asp His Arg lie He Gin 305 310 315 320

Gly Ala Glu Ser Gly Asp Phe Leu Arg Thr He His Glu Leu Leu Leu 325 330 335 86 201033369

Ser Asp Gly Phe Trp Asp Glu Val Phe Arg Glu Leu Ser lie Pro Tyr 340 345 350

Leu Pro Val Arg Trp Ser Thr Asp Asn Pro Asp Ser lie Val Asp Lys 355 360 365

Asn Ala Arg Val Met Asn Leu lie 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 Gin Tyr Lys 420 425 430

Lys Leu Arg Asp Val Leu Gly Leu Leu Arg Asp Ala Tyr Cys Arg His 435 440 445

He Gly Val Glu Tyr Ala His lie Leu Asp Pro Glu Gin Lys Glu Trp 450 455 460

Leu Glu Gin Arg Val Glu Thr Lys His Val Lys Pro Thr Val Ala Gin 465 470 475 480

Gin Lys Tyr lie Leu Ser Lys Leu Asn Ala Ala Glu Ala Phe Glu Thr 485 490 495

Phe Leu Gin Thr Lys Tyr Val Gly Gin Lys Arg Phe Ser Leu Glu Gly 500 505 510

Ala Glu Ser Val He Pro Met Met Asp Ala Ala lie Asp Gin Cys Ala 515 520 525

Glu His Gly Leu Asp Glu Val Val He Gly Met Pro His Arg Gly Arg 530 535 540

Leu Asn Val Leu Ala Asn He Val Gly Lys Pro Tyr Ser Gin lie Phe 545 550 555 560

Thr Glu Phe Glu Gly Asn Leu Asn Pro Ser Gin Ala His Gly Ser Gly 565 570 575 87 201033369

Asp Val Lys Tyr His Leu Gly Ala Thr Gly Leu Tyr Leu Gin Met Phe 580 585 590

Gly Asp Asn Asp He Gin 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 Gin Asp 610 615 620

Leu Leu Asp His Gly Ser lie Asp Ser Asp Gly Gin Arg Ala Phe Ser 625 630 635 640

Val Val Pro Leu Met Leu His Gly Asp Ala Ala Phe Ala Gly Gin 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 He His He He Val Asn Asn Gin He 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 lie Gly Ala Pro lie 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 Gin Arg Phe Lys 725 730 735

Lys Asp Val Val He 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 He Gly 770 775 780

Arg Gly Asp He Ser Met Lys Glu Ala Glu Asp Ala Leu Arg Asp Tyr 785 790 795 800

Gin Gly Gin Leu Glu Arg Val Phe Asn Glu Val Arg Glu Leu Glu Lys 88 201033369 805 810 815

His Gly Val Gin Pro Ser Glu Ser Val Glu Ser Asp Gin Met He Pro 820 825 830

Ala Gly Leu Ala Thr Ala Val Asp Lys Ser Leu Leu Ala Arg lie Gly 835 840 845

Asp Ala Phe Leu Ala Leu Pro Asn Gly Phe Thr Ala His Pro Arg Val 850 855 860

Gin Pro Val Leu Glu Lys Arg Arg Glu Met Ala Tyr Glu Gly Lys lie 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 Gin Asp Ser Arg Arg Gly Thr Phe 900 905 910

Ser Gin Arg His Ser Val Leu He Asp Arg His Thr Gly Glu Glu Phe 915 920 925

Thr Pro Leu Gin 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 Gin Phe Gly Asp Phe Val Asn Gly Ala Gin Ser lie He 980 985 990

Asp Glu Phe lie Ser Ser Gly Glu Ala Lys Trp Gly Gin Leu Ser Asn 995 1000 1005

Val Val Leu Leu Leu Pro His Gly His Glu Gly Gin Gly Gly Pro Asp 1010 1015 1020

His Thr Ser Ala Arg He Glu Arg Phe Leu Gin Leu Trp Ala Glu 1025 1030 1035 89 201033369

Gly Ser Met Thr lie Ala Met Pro Ser Thr Pro Ser Asn Tyr Phe 1040 1045 1050

His Leu Leu Arg Arg His Ala Leu Asp Gly lie Gin Arg Pro Leu 1055 1060 1065 lie Val Phe Thr Pro Lys Ser Met Leu Arg His Lys Ala Ala Val 1070 1075 1080

Ser Glu lie Lys Asp Phe Thr Glu lie Lys Phe Arg Ser Val Leu 1085 1090 1095

Glu Glu Pro Thr Tyr Glu Asp Gly He Gly Asp Arg Asn Lys Val 1100 1105 1110

Ser Arg He 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 lie Val Arg 1130 1135 1140

Leu Glu Gin 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 Gin Glu 1160 1165 1170

Glu Pro Ala Asn Gin Gly Ala Trp Pro Arg Phe Gly Leu Glu Leu 1175 1180 1185

Pro Glu Leu Leu Pro Asp Lys Leu Ala Gly He Lys Arg He 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 Gin Gin Glu He Leu Asp Glu Ala Phe Gly 1220 1225 1230 <210> 47 <211> 3696 <212> DNA <213>artificial<220><223> Glutaric acid decarboxylation KgD codon optimization gene 90 60 60

201033369 < 400 > 47 atggctaata tctcctctcc gtttggtcag aatgaatggc tggtagaaga aatgtaccgt aaattccgcg atgacccgtc ctctgtggac ccgtcctggc atgaattcct ggtagactac agcccggagc cgaccagcca accggcagcg gaaccaaccc gcgttacttc tccgctggta gcggaacgtg cagctgctgc cgcgcctcag gcgccgccta aaccggcgga tactgccgca gccggtaacg gtgtggtggc cgcactggct gctaagactg cggttccgcc gccagcagaa ggcgatgaag ttgcagtcct gcgcggtgcg gcggctgcag tggtgaaaaa catgagcgcg tccctggagg taccgaccgc cacgagcgtg cgcgcggtcc ctgctaaact gctgattgat aaccgtattg tgatcaacaa ccagctgaaa cgtacccgtg gtggcaagat ctccttcact catctgctgg gttatgcact ggtacaagcg gttaagaaat tccctaacat gaaccgtcat tacactgagg tcgacggtaa accgacggct gttactccgg cacacacgaa cctgggcctg gcgatcgacc tgcaaggtaa agatggtaag cgctccctgg tagttgcggg tattaaacgt tgcgaaacca tgcgtttcgc acaattcgta accgcctacg aggacattgt ccgccgtgct cgtgatggca aactgaccac cgaagatttt gcgggcgtta ctattagcct gaccaaccca ggcaccatcg gcaccgtgca cagcgtacct cgtctgatgc cgggccaagg tgcgattatc ggtgtgggtg ccatggagta cccggcagaa tttcagggtg cttctga aga gcgcatcgcc gagctgggta ttggtaaact gatcaccctg acttctacct atgaccaccg catcattcag ggcgcagaat ccggtgactt cctgcgcact attcacgaac tgctgctgtc cgacggtttc tgggatgaag tttttcgtga actgagcatc ccatatctgc cagttcgctg gtccaccgac aatccggact ctatcgttga caaaaacgct cgcgtaatga acctgatcgc tgcttatcgt aatcgtggtc acctgatggc tgatacggat ccgctgcgcc tggataaagc tcgtttccgt tcccacccgg acctggaagt gctgacccat ggtctgactc tgtgggatct ggaccgcgtg ttcaaagtag atggtttcgc gggtgctcag tacaagaagc tgcgtgacgt gctgggtctg ctgcgtgatg cgtactgtcg tcacattggt gtggagtacg cccacattct ggatccggaa cagaaagaat ggctggagca gcgtgtcgag accaaacacg taaaaccgac cgtagcgcag cagaaatata tcctgtccaa actgaacgcc gccgaggctt tcgaaacttt cctgcagacc aagtacgtgg gccagaaacg cttcagcctg gagggtgcgg aaagcgttat tccgatgatg gatgcagcta tcgatcagtg cgcggaacat ggtctggatg aagtcgttat cggtatgccg caccgtggtc gcctgaacgt actggcaaac atcgtcggta aaccatattc tcagatcttc acggaatteg agggcaacct gaacccgtcc caagcccacg gctccggcga cgtaaaatat 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1 020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 91 201033369 catctgggtg ctaccggcct gtatctgcag atgttcggtg ataacgacat ccaggtatct 1800 ctgactgcta acccgagcca cctggaggcg gttgatcctg ttctggaagg tctggttcgc 1860 gccaaacagg atctgctgga ccacggctct atcgacagcg atggccagcg tgcattcagc 1920 gttgtaccgc tgatgctgca tggcgacgcg gcgttcgccg gtcagggtgt cgtagcagaa 1980 actctgaacc tggcgaacct gcctggctat cgcgtgggtg gcaccattca catcatcgtt 2040 aacaaccaaa tcggtttcac cacggcaccg gagtatagcc gttctagcga atattgcacc 2100 gacgtagcca aaatgatcgg tgcgccgatc ttccatgtaa acggtgacga tccagaggcc 2160 tgcgtgtggg tggctcgtct ggccgtagac ttccgccagc gttttaagaa agatgtggtt 2220 atcgacatgc tgtgctaccg ccgtcgtggt cacaacgaag gtgatgatcc gtctatgact 2280 aacccgtatg tctatgacgt ggtggacacc aagcgtggtg cacgcaaatc ttacacggag 2340 gccctgatcg gtcgtggcga catctctatg aaagaagcgg aagacgctct gcgtgattac 2400 cagggtcagc tggaacgtgt gttcaatgag gtgcgtgagc tggaaaagca cggcgtacaa 2460 ccgtccgaat Ccgtagagtc cgatcagatg atccctgctg gtctggcaac tgct gttgat 2520 aaaagcctgc tggcgcgtat cggcgacgca ttcctggcgc tgccgaatgg ctttaccgcg 2580 cacccgcgcg tacagccggt actggaaaaa cgtcgtgaaa tggcctacga aggtaaaatc 2640 gattgggcct tcggtgagct gctggccctg ggctctctgg tggctgaggg caagctggta 2700 cgcctgagcg gccaggactc ccgtcgcggc actttttctc agcgtcacag cgtcctgatc 2760 gatcgtcaca ccggcgaaga attcacgccg ctgcaactgc tggctactaa ctccgatggt 2820 agcccgaccg gtggtaagtt cctggtgtac gattccccgc tgtccgaata tgctgcagtt 2880 ggtttcgagt atggttacac cgt tggcaac ccggacgcag tggttctgtg ggaagcgcag 2940 ttcggcgatt tcgttaacgg tgcccagtcc attatcgatg agtttattag cagcggcgag 3000 gccaaatggg gccagctgtc taacgttgtg ctgctgctgc ctcacggcca cgagggtcaa 3060 ggcccggacc acacctccgc ccgtatcgaa cgcttcctgc agctgtgggc tgaaggctct 3120 atgaccatcg cgatgccgtc taccccaagc aactacttcc acctgctgcg tcgccacgca 3180 ctggacggca ttcagcgccc gctgatcgtt ttcaccccaa aatccatgct gcgccacaaa 3240 gcagctgttt ctgaaatcaa agattttacg gaaattaaat tccgttctgt gctggaagaa 3300 ccaacctacg aagacggtat tggcgaccgc aacaaggtaa gccgta Tcct gctgacctcc 3360 ggcaaactgt actacgagct ggcagcacgt aaggcaaaag ataaccgcaa cgacctggcc 3420 atcgtccgcc tggaacagct ggcgccactg ccacgccgtc gcctgcgtga aaccctggat 3480

92 201033369 cgctacgaaa tggccgcgct cgcatcagcc gaacagcaag acgtaaaaga ttggtctgga gtcgcgctat agatcctgga attcttctgg actgccggaa gagcgccccg tgaggccttc gtgcaggaag ctgctgccgg tcttctggta ggctaa aaccggcaaa ataaactggc gctctaaagt ccagggtgcg aggtatcaag acacgctgta 3540 3600 3660 3696

<210> 48 <211> 74 <212> DNA <213> Artificial sequence <220><223> transcript for amplification of bacillus transgenic aminotransferase x <400> Ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgaagg ttttagtcaa tggccggctg attg 60 74 <210> 49 <211> 62 <212> DNA <213> artificial sequence <220><223> for expansion of bacillus Add anti-recording primer <400> 49 ggggaccact ttgtacaaga aagctgggtt tatgaaatgc tagcagcctg ttgaatgctt tc 60 62

<210> 50 <211>82 <212> DNA <213> artificial sequence<220><223> for the amplification of Bacillus subtilis transaminase <400>400 ggggacaagt Ttgtacaaaa aagcaggcta ggaggaatta accatgactc atgatttgat 60 agaaaaaagt aaaaagcacc tc <210> 51 <211> 57 <212> DNA <213> artificial sequence <220><223> for expansion of Bacillus subtilis transaminase y Reverse recording 93 82 57201033369 <400> 51 ggggaccact ttgtacaaga aagctgggtt caatcttcaa ggctcgtaac ctcgtgg <210> 52 <211> 64 <212> DNA <213> artificial sequence <220><223> for spherical The amplification of the transaminant of the erythromycin transaminase <400> 52 gggg g ttgtacaaaa aagcaggcta ggaggaatta accatgcccg gttgcggggg cttg 60 64 <210> 53 <211> 51 <212> DNA <213>

<220><223>Recounting primer for amplification of Rhodobacter sphaeroides to the amine <400> 53 ggggaccact ttgtacaaga aagctgggtt cagacggcgg ccggttcttt c 51 <210> 54 <211> 78 <212> DNA <213>Artificial Sequence<220><223> xiao>Augmentation of the augmentation of the pulmonary tuberculosis of the Legionnaires disease <400> 54 ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgagta tcgcatttgt taacggcaag tattgttg <210> 55 <211> 67 <212> DNA <213> artificial sequence <220><223> anti-recording primer for augmentation of the lung disease of Legionnella vulgaris <400> 55 ggggaccact ttgtacaaga Aagctgggtt tagtttacta gttgttggta ggaatcatta attatcc 60 78 60

<210> 56 94 67 201033369 <211> 76 <212> DNA <213> Human ▲ sequence <220><223> transcript for the amplification of nitrite bacteria in Europe ;400> 56 ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgattt acctcaatgg 60 caaatttctg ccgatg 76 <210> 57 <211> 50 <212> DNA <213> artificial sequence <220>

<223> Reverse transcription primer for amplification of nitrous acid transaminase in Europe <400> 57 50 ggggaccact ttgtacaaga aagctgggtt tactggcgtg gagcatgccc <210> 58 <211> 79 <212> DNA <213> Artificial sequence <220> <223> transcript for amplification of Neisseria gonorrhoeae transaminase <400> 58 ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgagga taaatatgaa 60 ccgtaacgaa attttattc 79 <210> 59 <211 〉 56 <212> DNA <213> artificial sequence <220><223><223>>400 for the amplification of Neisseria gonorrhoeae transaminase <400> 59 ggggaccact ttgtacaaga aagctgggtt catgcagcca tcgccttgaa cacttc 56 <210> 60 <211> 66 <212>DNA <213> Artificial sequence <220><223> transcript for the amplification of Pseudomonas aeruginosa transaminase 95 201033369 <400> 60 ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgtcga tggccgatcg 60 tgatgg 66 <210> 61 <211> 53 <212> DNA <213> artificial sequence <220><223> for amplification of Pseudomonas aeruginosa transamin It Reverse Recording <400> 61 53 ggggaccact ttgtacaaga aagctgggtt tacttgacca gggtacgcca etc

<210> 62 <211> 67 <212> DNA <213>Artificial sequence <220><223> transcript for amplification of Rhodopseudomonas pallidum transaminase <400> 62 ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgaagc tgataccgtg 60 ccgcgcc 57 <210> 63 <211> 51 <212>DNA <213>artificial sequence<220>

<223>Reversal primer for amplification of Rhodopseudomonas pallidum transaminase aminotransferase <400> 63 ggggaccact ttgtacaaga aagctgggtt caggcgaccg cgcggatcac c 51

<210> 64 <211> 1353 <212> DNA <213> Bacillus subtilis transaminase gene (gii6〇77991) <400> 64 atggagatga tggggatgga aaacattcag caaaatcagg gattaaagea aaaagatgag 60 caatttgtgt ggcatgccat gaagggagcg catcaagcgg acagcctgat agcccagaag 120 gccgaagggg Cctgggtaac cgacacagac ggacgccgct atttggatgc gatgtccggt 180 ttgtggtgcg tcaacattgg ttaeggeaga aaggagcttg cggaggctgc ctatgagcaa 240 96 300 201033369

<213> Amino acid sequence of Bacillus subtilis transamin (gi 16077991) <400> 65 Met Glu Met Met Gly Met Glu Asn lie Gin Gin Asn Gin Gly Leu Lys 15 10 15 ctaaaggagc tgccttacta gaaaagctga atgaatggct gaagcaaacg aaactgcttt agccgttata aattcatctc tcagctaccg gacaggcgca catgcagctc cgccagatat aatgaaatcg accgcatcat gagcccatca ttacaggcgg gaggacattt gccggcgcca ggacggacag gtgagccgtt atggcaaagg gaatcacaag attttcgaag cgtatcaggg ggcggaagcc cggctgcctg cagctgattc agcgatcccg agagaacacc cggcagtcgg gtcaaagaca aattgactaa gcgtgcaaag aaaaagggct aatgtcatcc acgttgcgcc aaaacggtga aagaaagctt

≪ 210 > 65 < 211> 450 < 212 > PRT cccgttaacg caaagtcacg tggcggcgat tatgttattt taaaattgcc cgccagtacc aagatatcgg gcataccacg gcggaaatat aaatacgagc ataccggaat cctgatgatg gacatgggaa ttaagcgaaa aggcatccta atgccgccgg cggagccctt ttgatttgcg cgggtttatg cactacggtg cgcgtatctg ccattgtcag ggaagctcct tatgaccgtt tgctttggcg ttgaaaaacc tgatcttgga gcaaagcttt ggatgttaga ggaaaagggc agagccggct gatgccgcca gatcatcggc aaaaacggcg gccattttgc ctgacagaag tcaaacgata taa cacccgcaat tcaactggcg ttttttccaa cagcggatcg atctgcaaaa cggcgaccac gcaatacatt gggagcgctc ctttgagcca agggttcctg cagacacgct tgaaagcgca cgattgccgg ggtcattatg acggatatat gaagaaggtg atgaagtgat ctgcgggttt tgaagcctga tatcattacg cgactgctgt gaaacgggac tccgccacgt gaacacgttc tgcaaattat ggaggacgaa taggtgagct tcaagctctg tgctgatcgg aatcgaactc aagtaaacca agtggttgcg atacagtcgc cggctacaac aggacctttc ctttatcgtg 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1353

Gin Lys Asp Glu Gin Phe Val Trp His Ala Met Lys Gly Ala His Gin 20 25 30

Ala Asp Ser Leu lie Ala Gin 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 97 201033369 50 55 60

Asn lie Gly Tyr Gly Arg Lys Glu Leu Ala Glu Ala Ala Tyr Glu Gin 65 70 75 80

Leu Lys Glu Leu Pro Tyr Tyr Pro Leu Thr Gin Ser His Ala Pro Ala 85 90 95

He Gin Leu Ala Glu Lys Leu Asn Glu Trp Leu Gly Gly Asp Tyr Val 100 105 110 lie Phe Phe Ser Asn Ser Gly Ser Glu Ala Asn Glu Thr Ala Phe Lys 115 120 125 ❿ lie Ala Arg Gin Tyr His Leu Gin Asn Gly Asp His Ser Arg Tyr Lys 130 135 140

Phe lie Ser Arg Tyr Arg Ala Tyr His Gly Asn Thr Leu Gly Ala Leu 145 150 155 160

Ser Ala Thr Gly Gin Ala Gin Arg Lys Tyr Lys Tyr Glu Pro Leu Ser 165 170 175

Gin Gly Phe Leu His Ala Ala Pro Pro Asp He Tyr Arg Asn Pro Asp 180 185 190

Asp Ala Asp Thr Leu Glu Ser Ala Asn Glu lie Asp Arg He Met Thr 195 200 205

Trp Glu Leu Ser Glu Thr lie Ala Gly Val lie Met Glu Pro lie lie 210 215 220

Thr Gly Gly Gly lie Leu Met Pro Pro Asp Gly Tyr Met Lys Lys Val 225 230 235 240

Glu Asp He Cys Arg Arg His Gly Ala Leu Leu lie Cys Asp Glu Val 245 250 255 lie Cys Gly Phe Gly Arg Thr Gly Glu Pro Phe Gly Phe Met His Tyr 260 265 270

Gly Val Lys Pro Asp He lie Thr Met Ala Lys Gly He Thr Ser Ala 275 280 285 98 201033369

Tyr Leu Pro Leu Ser Ala Thr Ala Val Lys Arg Asp lie Phe Glu Ala 290 295 300

Tyr Gin 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 Gin He 325 330 335

Met Glu Asp Glu Gin Leu lie Gin Arg Ser Arg Asp Leu Gly Ala Lys 340 345 350

Leu Leu Gly Glu Leu Gin Ala Leu Arg Glu His Pro Ala Val Gly Asp 355 360 365 Lu

Val Arg Gly Lys Gly Leu Leu He Gly lie Glu Leu Val Lys Asp Lys 370 375 380

Leu Thr Lys Glu Pro Ala Asp Ala Ala Lys Val Asn Gin Val Val Ala 385 390 395 400

Ala Cys Lys Glu Lys Gly Leu lie He Gly Lys Asn Gly Asp Thr Val 405 410 415

Ala Gly Tyr Asn Asn Val lie His Val Ala Pro Pro Phe Cys Leu Thr 420 425 430

Glu Glu Asp Leu Ser Phe lie Val Lys Thr Val Lys Glu Ser Phe Gin 435 440 445

Thr lie 450

<210> 66 <211> 1407 <212> DNA <213> Pseudomonas aeruginosa transaminase gene (seq iDgi9951072) <400> 66 60 120 180 240 atgaacgcaa gactgcacgc cacgtccccc ctcggcgacg ccgacctggt ccgtgccgac caggcccact acatgcacgg ctaccacgtg ttcgacgacc accgcgtcaa cggctcgctg aacatcgccg ccggcgacgg cgcctatatc tacgacaccg ccggcaaccg ctacctcgac gcggtgggcg gcatgtggtg caccaacatc ggcctggggc gcgaggaaat ggctcgcacc gtggccgagc agacccgcct gctggcctat tccaatccct tctgcgacat ggccaacccg 99 201033369 cgcgccatcg aactctgccg caagctcgcc gagctggccc ccggcgacct cgaccacgtg 360 ttcctcacca ccggcggttc 300 caccgccgtg gacaccgcga tccgcctcat gcactactac 420 cagaactgcc gcggcaagcg cgccaagaag cacgtcatca cgcggatcaa cgcctaccac 480 ggctcgacct tcctcggcat gtcgctgggc ggcaagagcg ccgaccggcc ggccgagttc 540 Gactccctcg acgagcgcat ccaccacctc gcctgtccct attactaccg cgctccggaa 600 gggctgggcg aagccgagtt cctcgatggc ctggtggacg agttcgaacg caagatcctc 660 gaactgggcg ccgaccgggt gggggcgttc atctccgagc cggtgttcgg ctccggcggc 720 gtgatcgtcc cgcccgcggg ctacca Cagg cggatgtggg agctgtgcca gcgctacgac 780 gtgctgtaca tctccgacga agtggtgacc tccttcggcc gcctcggcca cttcttcgcc 840

agccaggcgg tgttcggcgt acagccggac atcatcctca ccgccaaggg cctcacctcc 900 ggctaccagc cgctgggcgc gtgcatcttc tcccggcgca tctgggaggt gatcgccgag 960 ccggacaagg gccgctgctt cagccatggt ttcacctact ccggccaccc ggtggcctgc 1020 gcggcggcgc tgaagaacat cgagatcatc gagcgcgagg gcttgctcgc ccacgccgac 1080 gaggtcggcc gctacttcga ggagcgcctg caaagcctcc gcgacctgcc catcgtcggc 1140 gacgtgcgcg ggatgcgctt catggcctgt gtcgagttcg tcgccgacaa ggcgagcaag 1200 gcgctgtttc cggaaagcct gaacatcggc gagtgggtcc acctgcgggc gcagaagcgc 1260 ggcctgctgg Ttcgtccgat cgtccacctg aacgtgatgt cgccgccgct gatcctcacc 1320 cgcgaacagg tcgataccgt ggtccgggtg ctgcgcgaga gcatcgagga aaccgtggag 1380

Gatcttgtcc gcgccggtca ccggtaa 1407

<210> 67 <211> 468 <212> PRT <213> Amino acid sequence of Pseudomonas aeruginosa transaminase (seq ID gi9951072) <400>

Met Asn Ala Arg Leu His Ala Thr Ser Pro Leu Gly Asp Ala Asp Leu 15 10 15

Val Arg Ala Asp Gin Ala His Tyr Met His Gly Tyr His Val Phe Asp 20 25 30

Asp His Arg Val Asn Gly Ser Leu Asn He Ala Ala Gly Asp Gly Ala 35 40 45 100 201033369

Tyr He Tyr Asp Thr Ala Gly Asn Arg Tyr Leu Asp Ala Val Gly Gly 50 55 60

Met Trp Cys Thr Asn lie Gly Leu Gly Arg Glu Glu Met Ala Arg Thr 65 70 75 80

Val Ala Glu Gin Thr Arg Leu Leu Ala Tyr Ser Asn Pro Phe Cys Asp 85 90 95

Met Ala Asn Pro Arg Ala lie 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 lie Arg Leu Met His Tyr Tyr Gin Asn Cys Arg 130 135 140

Gly Lys Arg Ala Lys Lys His Val lie Thr Arg He 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 lie 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 He Leu Glu Leu Gly Ala 210 215 220

Asp Arg Val Gly Ala Phe He Ser Glu Pro Val Phe Gly Ser Gly Gly

Val lie Val Pro Pro Ala Gly Tyr His Arg Arg Met Trp Glu Leu Cys 245 250 255

Gin Arg Tyr Asp Val Leu Tyr lie Ser Asp Glu Val Val Thr Ser Phe 260 265 270

Gly Arg Leu Gly His Phe Phe Ala Ser Gin Ala Val Phe Gly Val Gin 275 280 285 101 201033369

Pro Asp lie lie Leu Thr Ala Lys Gly Leu Thr Ser Gly Tyr Gin Pro 290 295 300

Leu Gly Ala Cys lie Phe Ser Arg Arg lie Trp Glu Val lie 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 He Glu lie lie 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 Gin Ser Leu Arg Asp Leu Pro lie 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 He Gly Glu Trp Val His Leu Arg 405 410 415

Ala Gin Lys Arg Gly Leu Leu Val Arg Pro lie Val His Leu Asn Val 420 425 430

Met Ser Pro Pro Leu lie Leu Thr Arg Glu Gin Val Asp Thr Val Val 435 440 445

Are Val Leu Arg Glu Ser lie Glu Glu Thr Val Glu Asp Leu Val Arg

Ala Gly His Arg 465 <210> 68 <211> 1335 <212> DNA <213> Pseudomonas aeruginosa transaminase gene (seq iDgi995i630) <400> 68 atgacaatga atgacgagcc gcagtcgagc agcctcgaca acttctggat gcccttcacc gccaaccgcc agttcaaggc gcggccgcgc ctgctggaaa Gcgccgaagg catccactat 201033369

atcgcccagg ggccacggcc gccccgccgt atcgccccgc accgcgctga ctgatcggcc atggtcaaca cacaccctgg aaggccgagg atcgtcgagc cggctgcgcg ggtttcggcc ctgacctgcg gagaagatcc tatacctatt cgtcgcgacg ttcagcctgc gtgcaactgg tgcttctggg ccgctgatca cgcgccagcg gcgggcgccg ggcgcgagat tccagatggg cgagcctgaa agatcgccct gcgaactggg accgcaaggc acgtcgcccg agctggaacg cgatgtccgg agataacccg gcgtcggcga ccaaggggct atgctgcctt ccggccatcc acctgttcca gcgacctgcc cgccgcacgc agcacgacct tcgacaagcc tctga catcctcgac cagcgaagcg tcacccgctg caaagtattc tgcctaccag ctaccacggg cttctccgcc caacgccttc cctggtgacc ctcggccggc caagcatggc agccttcgcc gaccaacggc catgcaaggc ggtagcctgc gcgggccgtc caacgtggtc ggacggcccc gatggtccgg ccacatcgac ggcaccgccg gtggcccggc ccgttcgaac ttcaccaact cgcgccatcg gtcggcttcg aacctgctgc accgtcggcc ctgcacggcg gtggtgctgc atcctgctga gcgcagcgct agcatcccga ccgcagggcg gccgccgccc gaactggaag gacatccgcg ggcaagcgcg gtgaccggcg cagatcgtcg gcctctggtg agatcgccac tcgccgcgcg ccggctcgga gccagggcac gcggcctgtc cgggggtcga tgcccgagca ccgagaatat cgcc caaggg tcttcgacga ggggcgtcgt tgggcgccgt ccatcgagtt tggcgaccct gctactggca ccgtaggcct gctacgacgt acatcatcgc agcgcctggc ctgcaatgcc cctcgactac gctgacggaa atcggcggac ccgcacccgc ggtaggcggt ccacctgccg tggcgtggaa cgccgcggtg ctaccttcag agtgatcacc cccggacctg attcgtcgac cttccacggc ggacatctac ggacgcgctg ggtcggcggc cttcgagcgc catgtcgccg ccaggccatc 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1335

<210> 69 <211> 444 <212> PRT <213> Amino acid sequence of Pseudomonas aeruginosa transaminase (seq ID gi9951630) <400> 69

Met Thr Met Asn

Asp Glu Pro Gin Ser Ser Ser Leu Asp Asn Phe Trp 5 10 15

Met Pro Phe Thr Ala Asn Arg Gin Phe Lys Ala Arg Pro Arg Leu Leu 20 25 30

Glu Ser Ala Glu Gly lie His Tyr lie Ala Gin Gly Gly Arg Arg lie 103 201033369 35 40 45

Leu Asp Gly Thr Ala Gly Leu Trp Cys Cys Asn Ala Gly His Gly Arg 50 55 60

Arg Glu lie Ser Glu Ala Val Ala Arg Gin lie Ala Thr Leu Asp Tyr 65 70 75 80

Ala Pro Pro Phe Gin Met Gly His Pro Leu Pro Phe Glu Leu Ala Ala 85 90 95

Arg Leu Thr Glu lie 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 lie Ala Leu Ala 115 120 125

Tyr Gin Arg Ala He Gly Gin Gly Thr Arg Thr Arg Leu lie 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 lie Ala Ala Val lie Val Glu Pro 210 215 220

Met Ser Gly Ser Ala Gly Val Val Leu Pro Pro Lys Gly Tyr Leu Gin 225 230 235 240

Arg Leu Arg Glu lie Thr Arg Lys His Gly lie Leu Leu lie Phe Asp 245 250 255

Glu Val He Thr Gly Phe Gly Arg Val Gly Glu Ala Phe Ala Ala Gin 260 265 270 104 201033369

Arg Trp Gly Val Val Pro Asp Leu Leu Thr Cys Ala Lvs Glv Leu Thr 275 280 285

Asn Gly Ser lie Pro Met Gly Ala Val Phe Val Asp Glu Lvs lie His 290 295 300

Ala Ala Phe Met Gin Gly Pro Gin Gly Ala lie Glu Phe Phe His Glv 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 lie Tyr Arg Arg Asp Asp Leu Phe Gin Arg Ala Val Glu Leu 340 345 350

Glu Gly Tyr Trp Gin Asp Ala Leu Phe Ser Leu Arg Asd Leu Pro Asn 355 360 365

Val Val Asp lie Arg Ala Val Gly Leu Val Gly Gly Val Gin Leu Ala 370 375 380

Pro His Ala Asp Gly Pro Gly Lys Arg Gly Tyr Asn Val Phe Glu Are 385 390 395 4〇〇

Cys Phe Trp Glu His Asp Leu Met Val Arg Val Thr Gly Asd He He 405 410 4i5

Ala Met Ser Pro Pro Leu He He Asp Lys Pro His He Asd Gin lie 420 425 430

Val Glu Arg Leu Ala Gin Ala He Arg Ala Ser Val 435 440

<210> 70 <211> 71 <212> DNA <213> transcript for the amplification of Bacillus subtilis transaminating (gil6077991) <400> 70 60 ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatggaga tgatggggat ggaaaacatt c

<210> 71 <211> 65 <212> DNA <213> Reverse transcription primer for amplification of Bacillus subtilis transaminide (gi 16077991) 105 71 60 201033369 <400> 71 ggggaccact ttcac <210> 72 <211> 66 <212> DNA <213> For <400> 72 65 For the amplification of Pseudomonas aeruginosa transamination (gi9951072) GGggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgaacg caagactgca 60 Cgccac 66 <210> 73 <211> 48 <212> DNA <213>Reversal primer for amplification of Pseudomonas aeruginosa transgenic moss (gi995H) 72) <400> 73 ggggaccact ttgtacaaga aagctgggtt taccggtgac Cggcgcgg ❹ 48 · <210> 74 <211> 69 <212> DNA <213> transcript for amplification of Pseudomonas aeruginosa transaminase (gi9951630) <400> 74 ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta Accatgacaa tgaatgacga 60 gccgcagtc 69 <210> 75 <211> 49 <212> DNA <213>Reversal primer for amplification of Pseudomonas aeruginosa (gi 995! mo) <400> 75 Ggggaccact ttgtacaaga aagctgggtt caga Cgctgg cgcggatgg 49 <210> 76 <211> 57 <212> DNA <213> Artificial sequence <220><223> Positive recording <400> 76 aaatttacta gtaagaattt ttgaggaggc aatataaatg aataaaccac agtcttg 57 106 201033369

<210> 77 <211> 32 <212> DNA < 213 > artificial sequence <220 < 223 > reverse recording primer <400> 77 aaatttggat cctacaagaa agctgggttt ac 32 <210> 78 <211 〉 58 <212> DNA <213> artificial sequence <220> <223> positive recording introduction <400> 78 aaatttacta gtaagaattt ttgaggaggc aatataaatg aacagccaaa tcaccaac 58 <210> 79 <211> 37 <212〉 DNA <213> Artificial Sequence <220〉 <223> Reverse Recording <400> 79 aaatttggat ccactttgta caagaaagct gggttca 37

<210〉 80 <211> 57 <212> DNA <213> Artificial sequence <220><223> Positive recording <400> 80 aaatttggat ccgttgagga ggcctcaaaa atgtccgaga tcactctggg caaatac 57 <210> 81 &lt ;211> 35 <212> DNA <213> Artificial sequence <220>223> Reverse recording primer <400> 81 aaatttggcg cgccattact gtttagcgtt agttg 35 107 201033369 <210> 82 <211> 52 < 212> DNA <213> Artificial sequence <220> <223> Positive recording <400> 82 aaatttggat ccgttgagga ggcctcaaaa atgtatactg ttggtgatta tc <210> 83 <211> 37 <212> DNA <213> Artificial sequence <220> <223>reverse entry <400> 83 aaatttggcg cgccattact tgttctgctc cgcaaac 3 105 105 52

37

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Claims (1)

201033369 VII. Patent Application Range: 1. A method for preparing 6-amine hexanoic acid, wherein the 6-aminocaproic acid is prepared from α--pimelic acid using at least one biocatalyst. 2. A process for the preparation of 6-aminocaproic acid, wherein the 6-aminocaproic acid is prepared from 5-methylvalerate using at least one biocatalyst. 3. The method of claim 1 or 2, wherein the biocatalyst comprises an enzyme that catalyzes a transamination reaction and/or a reductive amination reaction. 4) The method of claim 3, wherein the enzyme that catalyzes the transamination reaction and/or the reductive amination reaction is selected from the group consisting of a transaminase (EC 2.6.1) and an amino acid dehydrogenase. Group of (EC 1.4.1). 5. The method of claim 4, wherein the transaminase or amino acid dehydrogenase is selected from the group consisting of β-amine isobutyric acid: α-ketoglutarate transaminase, β-alanine Aminease, aspartate transaminase, 4-amine butyrate transaminase (EC 2.6.1.19), L-lysine 6-transaminase (EC 2.6.1.36), 2-amine adipate transamin Enzyme (EC 2.6.1.39), 5-aminopentanoate transaminase (EC 2.6.1.48), 2-aminohexanoate transaminase (EC 2.6.1.67), lysine: pyruvate 6-transaminase ( EC 2.6.1.71) and a group consisting of lysine-6-dehydrogenase (EC 1.4.1.18). 6. The method of claim 3, 4 or 5, wherein the enzyme is selected from the group consisting of an enzyme capable of catalyzing a transamination reaction and/or a reductive amination reaction in an organism of the group consisting of: Vibrio; Pseudomonas; Bacillus; Mercurialis; Asplenium; Ceratonia; mammal; Neurospora; Escherichia 201033369 (Escherichia); Thermus; Saccharomyces; Brevibacterium; Corynebacterium; Proteus ; Agrobacterium; Geobacillus; Acinetobacter; Ralstonia; Salmonella (Salmonella); Rhodobacter and Staphylococcus, especially derived from Bacillus subtilis, Bacillus weihenstephanensis, Rhodobacter 10 sphaeroides , Staphylococcus aureus, Legionella pneumophilia, Nitrosomas europaea, Neisseria gonorrhoeae, Pseudomonas syringae An organism in a group consisting of Rhodopseudomonas palustris, Vibrio fluvialis, and Pseudomonas aeruginosa. 7. The method according to any one of claims 4-6, wherein the transaminase used comprises according to sequence ID 2, sequence ID 5, sequence ID 8, sequence ID 12, sequence ID 15, sequence ID 17. Sequence ID 19, Sequence ID 21, 20 Sequence 1D 23, Sequence ID 25, Sequence ID 27, Sequence ID 29, Sequence ID 65, Sequence ID 67, Sequence ID 69 or any amino acid of a homologue of such sequences sequence. 8. The method of any of the preceding claims, wherein the biocatalyst comprises an enzyme that catalyzes the decarboxylation of an OC-keto acid or an amino acid. 2 201033369 9. The method of claim 8, wherein the enzyme that catalyzes decarboxylation is a decarboxylase (E.C. 4丄1). 10. The method of claim 9, wherein the decarboxylase is selected from the group consisting of a face acid decarboxylase (EC 4.1.1.15) and a diamine pimelic acid decarboxylase (EC 5 4.1.1.20). , Aspartic acid 1-decarboxylase (EC 4.1.1.11), branched chain α-keto acid decarboxylase, oc-ketoisovalerate decarboxylase, ex-ketoglutarate decarboxylase, pyruvate decarboxylation A group consisting of an enzyme (EC 4.1.1.1) and a grasshopper acetic acid decarboxylase (EC 4.1.1.3). ❹ 11. The method of claim 8, wherein the catalyzed decarboxylation enzyme is selected from the group consisting of Cucurbitaceae; Saccharomyces; Candida. Candida; Hansenula. (Hansenula); Kluyveromyces; Rhizopus; Neurospora; Zymomonas; Escherichia; Mycobacterium, 15 (Mycobacterium); Clostridium; Lactobacillus; Streptococcus; organisms of the group consisting of Pseudomonas and Lactococcus or Part of the enzyme. 12. The method of any one of claims 8-11, wherein the enzyme capable of decarboxylation comprises according to sequence ID 31, sequence ID 34, sequence ID 37, sequence ID 40, sequence id 43 or sequence ID 46 or an amino acid sequence of a homologue of any such sequence. 13. The method according to any one of the preceding claims, wherein the α-pyridimedioic acid is converted to 5- in the presence of a biocatalyst which catalyzes the decarboxylation of the α-keto acid 3 201033369 by biocatalysis Mevalonate, and 5_valerate are derived from at least one amine donor and at least one biocatalyst that catalyzes the transamination and/or reductive amination of 5-methylpentanoate Biocatalytic conversion to 6-aminocaproic acid. The method of any one of the preceding claims, wherein the α-ketoheptanoic acid is at least one amine donor and at least one transamination and/or reductive amination of the catalytic ketone pimelic acid In the presence of a biocatalyst for the formation of α-amine pimelic acid, it is converted into ... amine pimelic acid by biocatalysis, and α-amine pimelic acid is used in the biocatalyst for decarboxylation of a catalytic amino acid. It is converted to 6-aminocaproic acid by biocatalysis. The method of any of the preceding claims, wherein the ketone pimelic acid has been obtained from a natural source. 16. A process for the preparation of caprolactam comprising the cyclization of 6-amine caproic acid prepared by the process of any of the preceding claims, whereby 15 caprolactam is formed. 17. A recombinant host cell comprising one of a nucleic acid sequence encoding an alpha-ketopimelate decarboxylase, a tongue licking enzyme, and/or one of the enzymes encoding 5-methylvalerate transaminase, a tongue Nucleic acid sequence. 18. The recombinant host cell according to claim 17, wherein the nucleic acid sequence comprising one of the enzymes having the activity of 5 mevalonate transaminase comprises according to the sequence IDI ID 2, sequence ID 5, sequence ID 8, sequence ID 65' sequence m 67, sequence ID 69 or one of its homologs amino acid sequence. The recombinant host cell of claim 17 or 18, wherein the nucleic acid sequence comprising cyclase alpha-ketopimelate decarboxylase activity comprises according to 19 20 201033369 5 sequence ID 31, sequence ID 34, sequence id 37. Sequence ID 40, Sequence 1D 43 or Sequence ID 46 or an amino acid sequence of one of the homologs of any such sequence. 20. A recombinant host cell comprising a nucleic acid sequence encoding an alpha-ketopimelate transaminase activity or an enzyme of X-ketopimelate dehydrogenase activity and/or encoding alpha-amine pimelic acid A nucleic acid sequence which is one of the enzymes of the enzyme activity of claim 2. The recombinant host cell of claim 2, wherein the biological stimulating agent comprises a nucleic acid sequence encoding a transaminase, and the transaminase comprises Sequence ID 2, Sequence ID 8, Sequence ID 12, Sequence ID 15, Sequence ID 10 Λ 17, Sequence ID 19, Sequence ID 21, Sequence ID 23, Sequence ID 25, Sequence ID 27, Sequence ID 29 or its homologs A recombinant amino acid cell according to any one of claims 17 to 21, which comprises one or more nucleic acid sequences encoding one or more biocatalysts which can be catalyzed by (X) - ketoglutaric acid preparation (at least one of the reaction steps of X-keto-glycol, .15 diacid). The recombinant host cell according to any one of claims 17-22, wherein the host cell line is selected From Aspergillus, Penicillium, Yeast Genus (Saccharomyces), Kluyveromyces, Pichia, Candida, Hansenula, Bacillus, Corynebacterium (Corynebacterium), and a group consisting of Escherichia. The microorganism of any one of claims 17-23, comprising a group selected from the group consisting of: The group in the group—the sequence represents the DNA of one of the sequence groups of the 201033369 nucleic acid sequence: sequence ID, 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, 5 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 analogs thereof. 25. Polynucleotide, including Selected from sequence ID 3, sequence ID 6, 10 sequence ID 13, sequence ID 32. A nucleic acid sequence of a group consisting of a sequence ID 35, a sequence ID 38, a sequence ID 41, a sequence ID 44, a sequence ID 47, and a sequence recognized by the functional analog thereof. 201033369 IV. Designated representative map: (1) The representative representative of the case is: ( ). (None) (2) A brief description of the symbol of the representative figure: 5. If there is a chemical formula in this case, please disclose the chemical formula that best shows the characteristics of the invention: