WO2011031147A1

WO2011031147A1 - Preparation of a compound comprising an amine group from an alpha-keto acid

Info

Publication number: WO2011031147A1
Application number: PCT/NL2010/050574
Authority: WO
Inventors: Petronella Catharina Raemakers-Franken; Martin SCHÜRMANN; Monika MÜLLER; Stefaan Marie André DE WILDEMAN; Stefanus Cornelis Hendikus Jozef Turk; Axel Christoph Trefzer
Original assignee: Dsm Ip Assets B.V.
Priority date: 2009-09-11
Filing date: 2010-09-10
Publication date: 2011-03-17
Also published as: TW201127961A

Abstract

The invention relates to a method for preparing a compound comprising an amine group from an alpha-keto acid, wherein the preparation comprises using at least one reaction step catalysed by a biocatalyst. The invention further relates to a method, wherein an amine is prepared from an aldehyde or an alpha-amino acid. The invention further relates to a method for cyclising or polymerizing the amine.

Description

Preparation of a compound comprising an amine group from an alpha-keto acid

The invention relates to a method for preparing a compound comprising an amine group (hereinafter 'amine') from an alpha-keto acid. The invention further relates to a method for cyclising the amine prepared from an alpha-keto acid.

Diamines and amino acids are currently mostly prepared using chemical synthesis methods starting from feedstocks derived from mineral oil. Such compounds may be used as intermediates for polymers and/or cyclic compounds.

In view of a growing desire to prepare materials using more sustainable technology, it would be desirable to provide a method wherein an amine, for example an amino acid or diamine, is prepared using a biochemical process.

Further, it would be desirable to provide a method that requires less energy than conventional chemical processes.

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

It is known to prepare caprolactam from the amino acid

6-aminocaproic acid (6-ACA), e.g. as described in US-A 6,194,572. As disclosed in WO 2005/068643, 6-ACA may be prepared biochemically by converting 6-aminohex-2- enoic acid (6-AHEA) in the presence of an enzyme having alpha, beta-enoate reductase activity. The 6-AHEA may be prepared from lysine, e.g. biochemically or by pure chemical synthesis. Although the preparation of 6-ACA via the reduction of 6- AHEA is feasible by the methods disclosed in WO 2005/068643, the inventors have found that - under the reduction reaction conditions - 6-AHEA may spontaneously and substantially irreversibly cyclise to form an undesired side-product, notably beta- homoproline. This cyclisation may be a bottleneck in the production of 6-ACA, and may lead to a considerable loss in yield.

It is an object of the invention to provide a novel method for preparing an amine, for example an amino acid such as 6-ACA, or a cyclised amine, such as caprolactam- which may, inter alia, be used for the preparation of polyamides -, or an intermediate compound for the preparation of an amine or a cyclised amine, that can serve as an alternative for known methods.

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

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

It has now been found possible to prepare an amine, in particular an amino acid, from an alpha-keto acid, namely it has been found possible to prepare 6- aminocaproic acid (6-ACA), wherein the 6-aminocaproic acid is prepared from 2-oxo- heptanedioic acid, also known as alpha-ketopimelic acid (AKP), in a process comprising at least one biochemical step. The inventors have contemplated that this specific preparation of 6-ACA has opened up the possibility to the more general conversion of an alpha-keto acid into an amine.

In particular, the preparation of the amine may be carried out in two or more reaction steps. For instance, a method is provided wherein an alpha-keto acid is first converted into an aldehyde, which aldehyde is converted into the amine. Further a method is provided wherein the alpha-keto acid is first converted into an alpha-amino acid and is thereafter converted into the amine. For example, in case of preparation of 6-ACA, AKP may first be converted into 5-formylpentanoate (5-formylvaleric acid, 5- FVA), which 5-FVA is then converted into 6-ACA. Alternatively, AKP is first converted into alpha-aminopimelic acid (AAP) and is then converted into 6-ACA.

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

Accordingly, the present invention in particular relates to a method for preparing a compound comprising an amine group represented by the formula

H₂N CH₂ A R from an alpha-keto acid represented by the formula

0

HO— C C A R

⁰ (2) ; wherein

A represents a hydrocarbon group, which hydrocarbon group may comprise one or more substituents and/or contain one or more heteroatoms, in particular a hydrocarbon group comprising 2-10 carbon atoms;

R represents a functional group, in particular a functional group selected from the group of CN, COOH and NH₂; and

wherein the preparation comprises using at least one reaction step catalysed by a biocatalyst.

R may further in particular be selected from functional groups that can be converted in a manner known per se (e.g. using a biocatalyst) into a functional group selected from the group of CN, COOH and NH₂

The invention further relates to a method, wherein a compound comprising an amine according to formula (1 ) is prepared from an aldehyde represented by the formula

H C A R

⁰ (3) ; wherein A and R are as defined for formulas (1 ) and (2), using a biocatalyst. As indicated above, the aldehyde according to formula (3) may be obtained from the alpha-keto acid according to formula (2).

The invention further relates to a method, wherein an amine according to formula (1 ) is prepared from an alpha-amino acid represented by the formula

0

HO C CH A R

NH₂

(4) ; wherein A and R are as defined for formulas (1 ) and (2), using a biocatalyst. As indicated above, the alpha-amino acid according to formula (4) may be obtained from the alpha-keto acid according to formula (2).

In an embodiment, the amine according to formula (1 ) prepared in a method of the invention may be used for preparing a cyclic product. Such a cyclic product may for example be a lactam in case the amine is an amino acid. Such a method comprises cyclising the amine, e.g. an amino acid, optionally in the presence of a biocatalyst. For example, 6-ACA prepared in a method of the invention may be used for preparing caprolactam.

The term "amine" as used herein refers to a compound comprising an amine group. In particular, the term "amine" refers to a compound comprising an amine group according to formula (1 ). In case R is a COOH group, the amine according to formula (1 ) may also be referred to as an "amino acid". In case R is a NH₂ group, the amine according to formula (1 ) may also be referred to as a "diamine". However, in case group A of formula (1 ) also comprises one or more amine groups, such a

"diamine" will comprise more than one amino group and may also be referred to as a triamine (when A comprises 1 amine group), tetra-amine (when A comprises 2 amine groups), etc.

The term "alpha-amino acid" as used herein refers to an amino acid, wherein an amino group and a carboxylic acid group are attached to the same carbon atom. In particular, the term refers to a compound according to formula (4).The amine according to formula (1 ) that is prepared by the method of the invention may be 6- aminocaproic acid (6-ACA). In this case, the alpha-keto acid from which the amine is prepared is alpha-ketopimelic acid.

The amine according to formula (1 ) that is prepared by the method of the invention may also be a compound other than 6-ACA. In this case, group A in formulas (1 )-(4) preferably represents a (CH₂)_X, wherein x is an integer selected from the group of 2, 3, 5, 6, 7, 8 and 9.

For example, the amine according to formula (1 ) may be selected from the group of 1 ,5-diaminopentane, 1 ,6-diaminohexane, 1 ,7-diaminoheptane, omega-amino-butanoic acid, omega-amino-pentanoic acid (5-AVA, also known as 5- amino valeric acid), omega-amino-heptanoic acid and omega-amino-octanoic acid. In particular, the amine may be selected from the group of omega-amino-pentanoic acid, omega-amino-heptanoic acid (7-amino-heptanoic acid), 1 ,6-diaminohexane and 1 ,7-diaminoheptane.

In case 'R' is a COOH group, the alpha-keto acid from which the amine is made is preferably alpha-keto glutaric acid (AKG), alpha-ketoadipic acid (AKA) or alpha-keto suberic acid (AKS). When referring herein to carboxylic acids or carboxylates, e.g.

alpha-keto acid, amino acid, 6-ACA, 2-aminoheptanedioic acid (alpha-aminopimelic acid, herein after abbreviated as ΆΑΡ'), 5-FVA or AKP, these terms are meant to include the protonated carboxylic acid group (i.e. the neutral group), their

corresponding carboxylate (their conjugated bases) as well as salts thereof. When referring herein to amino acids, e.g. alpha-amino acids or 6-ACA, this term is meant to include amino acids in their zwitterionic form (in which the amino group is in the protonated and the carboxylate group is in the deprotonated form), the amino acid in which the amino group is protonated and the carboxylic group is in its neutral form, and the amino acid in which the amino group is in its neutral form and the carboxylate group is in the deprotonated form, as well as salts thereof.

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

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

It is further envisaged that in an embodiment of the invention the productivity of an amine according to formula (1 ) (g/l.h formed) in a method of the invention may be improved.

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

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

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

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

When an enzyme is mentioned with reference to an enzyme class (EC) between brackets, the enzyme class is a class wherein the enzyme is classified or may be classified, on the basis of the Enzyme Nomenclature provided by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-I UBMB), which nomenclature may be found at

http://www.chem.gmul.ac.uk/iubmb/enzyme/. Other suitable enzymes that have not (yet) been classified in a specified class but may be classified as such, are meant to be included.

The term "homologue" is used herein in particular for polynucleotides or polypeptides having a sequence identity of at least 30 %, preferably at least 40 %, more preferably at least 60%, more preferably at least 65%, more preferably at least 70 %, more preferably at least 75%, more preferably at least 80%, in particular at least 85 %, more in particular at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 %.

Further, homologues usually have a significant sequence similarity, usually of more than 30 %, in particular a sequence similarity of at least 35 %, preferably at least 40 %, more preferably at least 60%, more preferably at least 65%, more preferably at least 70 %, more preferably at least 75%, more preferably at least 80%, in particular at least 85 %, more in particular at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 %.

Homologues generally have an intended function in common with the polynucleotide respectively polypeptide of which it is a homologue, such as encoding the same peptide respectively being capable of catalysing the same reaction (typically the conversion of the same substrate into the same compound) or a similar reaction. A 'similar reaction' typically is a reaction of the same type, e.g. a decarboxylation, an aminotransfer, a C1 -elongation. Accordingly, as a rule of thumb, homologous enzymes can be classified in an EC class sharing the first three numerals of the EC class (x.y.z), for example EC 4.1 .1 for carboxy lyases. Typically, in the similar reaction, a substrate of the same class (e.g. an amine, a carboxylic acid, an amino acid) as the substrate for the reaction to which the similar reaction is similar is converted into a product of the same class as the product of the reaction to which the similar reaction is similar. Similar reactions in particular include reactions that are defined by the same chemical conversion as defined by the same KEGG RDM patterns, wherein the R-atoms and D- atoms describe the chemical conversion (KEGG RDM patterns: Oh, M. et al. (2007) Systematic analysis of enzyme-catalyzed reaction patterns and prediction of microbial biodegradation pathways. J. Chem. Inf. Model., 47, 1702-1712).

The term homologue is also meant to include nucleic acid sequences

(polynucleotide sequences) which differ from another nucleic acid sequence due to the degeneracy or experimental adaptation of the genetic code and encode the same polypeptide sequence.

The term "functional analogue" is used herein for nucleic acid sequences that differ from a given sequence of which said analogue is an analogue, yet that encode a peptide (protein, enzyme) having the same amino acid sequence or that encode a homologue of such peptide. In particular, preferred functional analogues are nucleotide sequences having a similar, the same or a better level of expression in a host cell of interest as the nucleotide sequence of which it is referred to as being a functional analogue of. In this respect it is observed that, as the skilled person understands, a better level of expression usually is a higher level of expression if the expression of the peptide (protein, enzyme) is desired. However, in specific

embodiment a better level of expression may be a lower expression level since this might be desirable in context of a metabolic pathway in said host cell. The functional analogue can be a naturally occurring sequence, i.e. a wild-type functional analogue, or a genetically modified sequence, i.e. a non-wild type functional analogue. Codon optimised sequences encoding a specific peptide, are generally non-wild type functional analogues of a wild-type sequence, designed to achieve a desired expression level.

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

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

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

Living cells may be growing cells, resting or dormant cells (e.g.

spores) or cells in a stationary phase. It is also possible to use an enzyme forming part of a permeabilised cell (i.e. made permeable to a substrate for the enzyme or a precursor for a substrate for the enzyme or enzymes).

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

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

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

Suitable bacteria may in particular be selected amongst the group of Vibrio, Pseudomonas, Bacillus, Corynebacterium, Brevibacterium, Enterococcus, Streptococcus, Actinomycetales, Klebsiella, Lactococcus, Lactobacillus, Clostridium, Escherichia, Anabaena, Microcystis, Synechocystis, Rhizobium, Bradyrhizobium, Therm us, Azotobacter, Aerococcus, Therm us, Mycobacterium, Zymomonas, Proteus, Agrobacterium, Geobacillus, Acinetobacter, Ralstonia, Rhodobacter, Paracoccus, Novosphingobium, Nitrosomonas, Legionella, Neisseria, Rhodopseudomonas, Staphylococcus, Deinococcus and Salmonella.

Suitable archaea may in particular be selected amongst the group of Archaeoglobus, Aeropyrum, Halobacterium, Methanosarcina, Methanococcus, Thermoplasma, Pyrobaculum, Methanocaldococcus, Methanobacterium,

Methanosphaera, Methanopyrus and Methanobrevibacter.

Suitable fungi may in particular be selected amongst the group of Phanerochaete, Emericella, Ustilago, Cephalospo um, Paecilomyces,

Trichophytum,.Rhizopus, Neurospora, Penicillium and Aspergillus.

A suitable yeast may in particular be selected amongst the group of Schizosaccharomyces, Pichia, Candida, Hansenula, Kluyveromyces, Yarrowia and Saccharomyces.

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

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

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

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

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

In case the R in the alpha-keto acid according to formula (2) is a COOH, a suitable decarboxylase has alpha-ketodicarboxylate decarboxylase activity, capable of catalysing the conversion of the alpha-keto acid into the aldehyde according to formula (3) or alpha-aminodicarboxylate decarboxylase activity, capable of catalysing the conversion of the alpha-keto acid to the amine according to formula (1 ).

An enzyme capable of decarboxylating an alpha-keto acid or an amino acid is preferably selected from the group of decarboxylases (E.C. 4.1 .1 ), and is more preferably selected from the group of glutamate decarboxylases (EC 4.1 .1.15), oxaloacetate decarboxylases (EC 4.1.1 .3), diaminopimelate decarboxylases (EC 4.1 .1.20), aspartate 1 -decarboxylases (EC 4.1 .1 .1 1 ), branched chain alpha-keto acid decarboxylases (EC 4.1 .1 .72), alpha-ketoisovalerate decarboxylases (EC 1 .2.4.4), alpha-ketoglutarate decarboxylases (EC 4.1 .1 .71 ), and pyruvate decarboxylases (EC 4.1 .1.1 ).

One or more other suitable decarboxylases may be selected amongst the group of oxalate decarboxylases (EC 4.1 .1 .2), acetoacetate decarboxylases (EC 4.1 .1.4), valine decarboxylases/leucine decarboxylases (EC 4.1 .1.14),

3-hydroxyglutamate decarboxylases (EC 4.1 .1.16), ornithine decarboxylases (EC 4.1 .1.17), lysine decarboxylases (EC 4.1.1 .18), arginine decarboxylases (EC 4.1 .1 .19), 2-oxoglutarate decarboxylases (EC 4.1 .1 .71 ), and diaminobutyrate decarboxylases (EC 4.1 .1 .86).

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

Pseudomonas sp.) and Zymomonas mobilis.

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

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

Glutamate decarboxylase from Neurospora crassa, Mycobacterium leprae, Clostridium perfringens, Lactobacillus brevis, Mycobacterium tuberculosis, Streptococcus or Lactococcus may be used. Examples of Lactococcus species from which the glutamate decarboxylase may originate in particular include Lactococcus lactis, such as Lactococcus lactis strain B1 157, Lactococcus lactis IFPL730, more in particular Lactococcus lactis var. maltigenes formerly named Streptococcus lactis var. maltigenes).

An oxaloacetate decarboxylase from Pseudomonas may in particular be used.

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

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

In a preferred method of the invention, the preparation of the amine according to formula (1 ) comprises an enzymatic reaction in the presence of a biocatalyst , which biocatalyst comprises an enzyme having catalytic activity with respect to catalysing transamination and/or reductive amination, preferably an enzyme having at least one of said catalytic activities selected from the group of

aminotransferases (E.C. 2.6.1 ) and amino acid dehydrogenases (E.C.1 .4.1 ). In addition, the enzymatic reaction may occur in the presence of an amino donor.

In case of the amine being 6-ACA, a suitable aminotransferase has 6- aminocaproic acid 6-aminotransferase activity, capable of catalysing the conversion of 5-FVA into 6-ACA or alpha-aminopimelate 2-aminotransf erase activity, capable of catalysing the conversion of AKP into AAP.

The aminotransferase may in particular be selected from the group of beta-aminoisobutyrate:alpha-ketoglutarate aminotransferases, beta-alanine aminotransferases, aspartate aminotransferases, 4-amino-butyrate aminotransferases (EC 2.6.1 .19), L-lysine 6-aminotransferase (EC 2.6.1 .36), 2-aminoadipate

aminotransferases (EC 2.6.1 .39), 5-aminovalerate aminotransferases (EC 2.6.1.48), 2- aminohexanoate aminotransferases (EC 2.6.1 .67), lysine:pyruvate 6- aminotransferases (EC 2.6.1 .71 ) and aromatic amino acid aminotransferase (EC 2.6.1.57).

In an embodiment an aminotransferase may be selected amongst the group of alanine aminotransferases (EC 2.6.1 .2), leucine aminotransferases (EC 2.6.1.6), alanine-oxo-acid aminotransferases (EC 2.6.1 .12), beta-alanine-pyruvate aminotransferases (EC 2.6.1 .18), (S)-3-amino-2-methylpropionate aminotransferases (EC 2.6.1 .22), L,L-diaminopimelate aminotransferase (EC 2.6.1 .83).

The enzyme having catalytic activity with respect to catalysing transamination and/or reductive amination, e.g. aminotransferase or amino acid dehydrogenase, may be an enzyme from an organism selected from the group of Vibrio; Pseudomonas; Bacillus; Mercurialis; Asplenium; Ceratonia; mammals;

Neurospora; Escherichia; Thermus; Saccharomyces; Brevibacterium;

Corynebacterium; Proteus; Agrobacterium; Geobacillus; Acinetobacter; Ralstonia; Salmonella; Rhodobacter and Staphylococcus, in particular from an organism selected from the group of Bacillus subtilis, Bacillus weihenstephanensis, Rhodobacter sphaeroides, Staphylococcus aureus, Legionella pneumophila, Nitrosomonas europaea, Neisseria gonorrhoeae, Pseudomonas syringae, Rhodopseudomonas palustris, Vibrio fluvialis and Pseudomonas aeruginosa.

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

In case the enzyme is of a mammal, it may in particular originate from mammalian kidney, from mammalian liver, from mammalian heart or from mammalian brain. For instance a suitable enzyme may be selected amongst the group of beta- aminoisobutyrate:alpha-ketoglutarate aminotransferase from mammalian kidney, in particular beta-aminoisobutyrate:alpha-ketoglutarate aminotransferase from hog kidney; beta-alanine aminotransferase from mammalian liver, in particular beta-alanine aminotransferase from rabbit liver; aspartate aminotransferase from mammalian heart; in particular aspartate aminotransferase from pig heart; 4-amino-butyrate

aminotransferase from mammalian liver, in particular 4-amino-butyrate

aminotransferase from pig liver; 4-amino-butyrate aminotransferase from mammalian brain, in particular 4-aminobutyrate aminotransferase from human, pig, or rat brain.

In an embodiment, the aminotransferase is selected from the group of alpha-ketoadipate-glutamate aminotransferase from Neurospora, in particular alpha- ketoadipate:glutamate aminotransferase from Neurospora crassa; 4-amino-butyrate aminotransferase from E. coli, or alpha-aminoadipate aminotransferase from Thermus, in particular alpha-aminoadipate aminotransferase from Thermus thermophilus, and 5- aminovalerate aminotransferase from Clostridium in particular from Clostridium aminovalericum. A suitable 2-aminoadipate aminotransferase may e.g. be provided by Pyrobaculum islandicum.

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

In a further preferred embodiment, the method for preparing the amine according to formula (1 ) comprises a biocatalytic reaction in the presence of an enzyme capable of catalysing a reductive amination reaction in the presence of an ammonia source, selected from the group of oxidoreductases acting on the CH-NH₂ group of donors (EC 1.4), in particular from the group of amino acid dehydrogenases (E.C. 1 .4.1 ). In case 6-ACA is prepared, a suitable amino acid dehydrogenase has 6- aminocaproic acid 6-dehydrogenase activity, catalysing the conversion of 5-FVA into 6- ACA or has alpha-aminopimelate 2-dehydrogenase activity, catalysing the conversion of AKP into AAP. In particular a suitable amino acid dehydrogenase be selected amongst the group of diaminopimelate dehydrogenases (EC 1 .4.1 .16), lysine 6- dehydrogenases (EC 1.4.1 .18), glutamate dehydrogenases (EC 1 .4.1 .3; EC 1 .4.1 .4), and leucine dehydrogenases (EC 1 .4.1 .9). Preferably, the amino acid dehydrogenase is a lysine 6-dehydrogenase (EC 1.4.1.18). In an embodiment, an amino acid dehydrogenase may be selected amongst an amino acid dehydrogenases classified as glutamate dehydrogenases acting with NAD or NADP as acceptor (EC 1 .4.1 .3), glutamate dehydrogenases acting with NADP as acceptor (EC 1 .4.1 .4), leucine dehydrogenases (EC 1.4.1.9), diaminopimelate dehydrogenases (EC 1 .4.1 .16), and lysine 6-dehydrogenases (EC 1.4.1.18).

An amino acid dehydrogenase may in particular originate from an organism selected from the group of Corynebacterium, in particular Corynebacterium glutamicum; Proteus, in particular Proteus vulgaris; Agrobacterium, in particular Agrobacterium tumefaciens; Geobacillus, in particular Geobacillus stearothermophilus; Acinetobacter, in particular Acinetobacter sp. ADP1 ; Ralstonia, in particular Ralstonia solanacearum; Salmonella, in particular Salmonella typhimurium; Saccharomyces, in particular Saccharomyces cerevisiae; Brevibacterium, in particular Brevibacterium flavum; and Bacillus, in particular Bacillus sphaericus, Bacillus cereus or Bacillus subtilis. For instance a suitable amino acid dehydrogenase may be selected amongst diaminopimelate dehydrogenases from Bacillus, in particular Bacillus sphaericus;

diaminopimelate dehydrogenases from Brevibacterium sp.; diaminopimelate dehydrogenases from Corynebacterium, in particular diaminopimelate dehydrogenases from Corynebacterium glutamicum; diaminopimelate dehydrogenases from Proteus, in particular diaminopimelate dehydrogenase from Proteus vulgaris; lysine 6- dehydrogenases from Agrobacterium, in particular Agrobacterium tumefaciens, lysine 6-dehydrogenases from Geobacillus, in particular from Geobacillus stearothermophilus; glutamate dehydrogenases acting with NADH or NADPH as cofactor (EC 1 .4.1 .3) from Acinetobacter, in particular glutamate dehydrogenases from Acinetobacter sp. ADP1 ; glutamate dehydrogenases (EC 1.4.1.3) from Ralstonia, in particular glutamate dehydrogenases from Ralstonia solanacearum; glutamate dehydrogenases acting with NADPH as cofactor (EC 1 .4.1.4) from Salmonella, in particular glutamate

dehydrogenases from Salmonella typhimurium; glutamate dehydrogenases (EC 1 .4.1.4) from Saccharomyces, in particular glutamate dehydrogenases from

Saccharomyces cerevisiae; glutamate dehydrogenases (EC 1 .4.1 .4) from

Brevibacterium, in particular glutamate dehydrogenases from Brevibacterium flavum; and leucine dehydrogenases from Bacillus, in particular leucine dehydrogenases from Bacillus cereus or Bacillus subtilis.

In a specific embodiment, the alpha-keto acid according to formula (2) is biocatalytically converted into an aldehyde according to formula (3) in the presence of a decarboxylase or other biocatalyst catalysing such conversion. For example, AKP may be biocatalytically converted into 5-FVA using such a method. A decarboxylase used in accordance with the invention may in particular be selected from the group of alpha-keto acid decarboxylases from Lactococcus lactis, Lactococcus lactis var.

maltigenes or Lactococcus lactis subsp. cremoris; branched chain alpha-Weto acid decarboxylases from Lactococcus lactis strain B1157 or Lactococcus lactis IFPL730; pyruvate decarboxylases from Saccharomyces cerevisiae, Candida flareri, Zymomonas mobilis, Hansenula sp., Rhizopus javanicus, Neurospora crassa, or Kluyveromyces marxianus; a/pfra-ketoglutarate decarboxylases from Mycobacterium tuberculosis; glutamate decarboxylases from E. coli, Lactobacillus brevis, Mycobacterium leprae, Neurospora crassa or Clostridium perfringens; and aspartate decarboxylases from E. coli.

In particular, a decarboxylase from Escherichia coli, Zymomonas mobilis, Saccharomyces cerevisiae, Mycobacterium tuberculosis, Pseudomonas species, or Lactococcus lactis has been found suitable to catalyse the conversion of the alpha-keto acid of formula (1 ) into the aldehyde of formula (3), for example for the conversion of AKP into 5-FVA. More in particular, a biocatalyst comprising a decarboxylase having an amino acid sequence as identified by Sequence ID 31 , Sequence ID 34, Sequence ID 37, Sequence ID 40, Sequence ID 43, Sequence ID 46, Sequence ID 143, Sequence ID 146, Sequence ID 149, Sequence 152 or a homologue of any of these sequences may be used. It is also envisaged that such decarboxylase may be used to prepare the amine according to formula (1 ) from the alpha-amino acid according to formula (4).

Thereafter the aldehyde is converted into the amine according to formula (1 ), for example 5-FVA into 6-ACA. This can be done chemically: the amine can be prepared in high yield by reductive amination of the aldehyde with ammonia over a hydrogenation catalyst, for example Ni on Si0₂/Al₂0₃ support, as described for 9-aminononanoic acid (9-aminopelargonic acid) and 12-aminododecanoic acid (12- aminolauric acid) in EP-A 628 535 or DE 4 322 065.

In case the amine according to formula (1 ) is 6-ACA, 6-ACA can alternatively be obtained by hydrogenation over Pt0₂ of 6-oximocaproic acid, prepared by reaction of 5-FVA and hydroxylamine. (see e.g. F.O. Ayorinde, E.Y. Nana, P.D. Nicely, A.S. Woods, E.O. Price, CP. Nwaonicha J. Am. Oil Chem. Soc. 1997, 74, 531 - 538 for synthesis of the homologous 12-aminododecanoic acid).

In an embodiment, the conversion of the aldehyde according to formula (3) into the amine according to formula (1 ) is performed biocatalytically in the presence of (i) an amino donor and (ii) an aminotransferase, an amino acid

dehydrogenase or another biocatalyst capable of catalysing such conversion. In particular in such an embodiment the aminotransferase may be selected from the group of aminotransferases from Vibrio fluvialis, Pseudomonas aeruginosa, Bacillus subtilis, Bacillus weihenstephanensis or Escherichia coli; 0efa-aminoisobutyrate:alpha- ketoglutarate aminotransferase from hog kidney; beta-alanine aminotransferase from rabbit liver; aminotransferase from shoots from Mercurialis perennis; 4-aminobutyrate aminotransferase from pig liver or from human, rat, or pig brain; beta-alanine aminotransferase from rabbit liver; and L-lysine:alpha-ketoglutarate-£- aminotransferase. In case an amino acid dehydrogenase is used, such amino acid dehydrogenase may in particular be selected from the group of lysine 6- dehydrogenases from Agrobacterium tumefaciens or Geobacillus stearothermophilus. Another suitable amino acid dehydrogenase may be selected from the group of diaminopimelate dehydrogenases from Bacillus sphaericus, Brevibacterium sp., Corynebacterium glutamicum, or Proteus vulgaris; from the group of glutamate dehydrogenases acting with NADH or NADPH as cofactor (EC 1 .4.1 .3) from

Acinetobacter sp. ADP1 or Ralstonia solanacearum; from the group of glutamate dehydrogenases acting with NADPH as cofactor (EC 1 .4.1 .4) from Salmonella typhimurium; from the group of glutamate dehydrogenases (EC 1 .4.1 .4) from

Saccharomyces cerevisiae or Brevibacterium flavum; or from the group of leucine dehydrogenases from Bacillus cereus or Bacillus subtilis.

In a specific embodiment, the conversion of the aldehyde according to formula (3) to the amine according to formula (1 ) is catalysed by a biocatalyst comprising an aminotransferase comprising an amino acid sequence according to Sequence ID 2, Sequence ID 5, Sequence ID 8, Sequence ID 65, Sequence ID 67, Sequence ID 69 or a homologue of any of these sequences.

In a specific embodiment, the alpha-keto acid according to formula (2) is chemically converted into the aldehyde according to formula (3). Efficient chemical decarboxylation of a 2-keto acid into the corresponding aldehyde can be performed by intermediate enamine formation using a secondary amine, for instance morpholine, under azeotropic water removal and simultaneous loss of C0₂, e.g. based on a method as described in Tetrahedron Lett. 1982, 23(4), 459-462. The intermediate terminal enamide is subsequently hydrolysed to the corresponding aldehyde. The aldehyde according to formula (3) may thereafter be biocatalytically converted into the amine according to formula (1 ) by transamination in the presence of an aminotransferase or by enzymatic reductive amination by an amino acid dehydrogenase or another biocatalyst able of catalysing such conversion. Such aminotransferase or amino acid dehydrogenase may in particular be selected from the biocatalysts mentioned above when describing the conversion of the aldehyde according to formula (3) to the amine according to formula (1 ).

Alternatively, the conversion of the aldehyde according to formula (3) to the amine according to formula (1 ) may be performed by a chemical method, e.g. as mentioned above.

In a specific embodiment, the alpha-keto acid according to formula (2) is biocatalytically converted into the alpha-amino acid according to formula (4) in the presence of (i) an aminotransferase, an amino acid dehydrogenase, or another biocatalyst capable of catalysing such conversion and (ii) an amino donor. Such aminotransferase used in accordance with the invention for the conversion of the alpha-keto acid to the alpha-amino acid may in particular be selected from

aminotransferases mentioned above, more in particular from the group of aspartate aminotransferases from pig heart; alpha-ketoadipate:glutamate aminotransferases from Neurospora crassa or yeast; aminotransferases from shoots from Mercu alis perennis; 4-aminobutyrate aminotransferases from E. coli; alpha-aminoadipate

aminotransferases from Thermus thermophilus; aminotransferases from Asplenium septentrionale or Asplenium unilateral; and aminotransferases from Ceratonia siliqua.

In a preferred embodiment, the aminotransferase for the conversion of the alpha-keto acid according to formula (2) to the alpha-amino acid according to formula (4)is selected from the group of aminotransferases from Vibrio, Pseudomonas, Bacillus, Legionella, Nitrosomonas, Neisseria, Rhodobacter, Escherichia and

Rhodopseudomonas.

In particular, aminotransferases from an organism selected from the group of Bacillus subtilis, Rhodobacter sphaeroides, Legionella pneumophila,

Nitrosomonas europaea, Neisseria gonorrhoeae, Pseudomonas syringae,

Rhodopseudomonas palustris, Vibrio fluvialis, Escherichia coli and Pseudomonas aeruginosa, have been found suitable to catalyse the conversion of the alpha-keto acid according to formula (2) to the alpha-amino acid according to formula (4), especially the conversion of AKP to AAP.

In a specific embodiment, for the conversion of the alpha-keto acid according to formula (2) to the alpha-amino acid according to formula (4) an aminotransferase is used comprising an amino acid sequence according to Sequence ID 2, Sequence ID 8, Sequence ID 12, Sequence ID 15, Sequence ID 17, Sequence ID 19, Sequence ID 21 , Sequence ID 23, Sequence ID 25, Sequence ID 27, Sequence ID 29 or a homologue of any of these sequences.

In a further embodiment, the method for preparing alpha-amino acid according to formula (4) comprises a biocatalytic reaction in the presence of an enzyme capable of catalysing a reductive amination reaction in the presence of an ammonia source, selected from the group of oxidoreductases acting on the CH-NH₂ group of donors (EC 1.4), in particular from the group of amino acid dehydrogenases (E.C. 1 .4.1 ). In case the amine according to formula (1 ) is 6-ACA, a suitable amino acid dehydrogenase has alpha-aminopimelate 2-dehydrogenase activity, catalysing the conversion of AKP into AAP.

In particular a suitable amino acid dehydrogenase may be selected from the group of diaminopimelate dehydrogenases (EC 1.4.1.16), glutamate dehydrogenases (EC 1.4.1 .3; EC 1 .4.1 .4), and leucine dehydrogenases (EC 1 .4.1 .9).

In an embodiment, an amino acid dehydrogenase is selected amongst amino acid dehydrogenases classified as glutamate dehydrogenases acting with NAD or NADP as acceptor (EC 1 .4.1.3), glutamate dehydrogenases acting with NADP as acceptor (EC 1 .4.1 .4), leucine dehydrogenases (EC 1 .4.1.9), and diaminopimelate dehydrogenases (EC 1.4.1 .16).

An amino acid dehydrogenase may in particular originate from an organism selected from the group of Corynebacterium, in particular Corynebacterium glutamicum; Proteus, in particular Proteus vulgaris; Agrobacterium, in particular

Agrobacterium tumefaciens; Geobacillus, in particular Geobacillus stearothermophilus; Acinetobacter, in particular Acinetobacter sp. ADP1 ; Ralstonia, in particular Ralstonia solanacearum; Salmonella, in particular Salmonella typhimurium; Saccharomyces, in particular Saccharomyces cerevisiae; Brevibacterium, in particular Brevibacterium flavum; and Bacillus, in particular Bacillus sphaericus, Bacillus cereus or Bacillus subtilis.

For instance a suitable amino acid dehydrogenase may be selected amongst diaminopimelate dehydrogenases from Bacillus, in particular Bacillus sphaericus; diaminopimelate dehydrogenases from Brevibacterium sp. ;

diaminopimelate dehydrogenases from Corynebacterium, in particular diaminopimelate dehydrogenases from Corynebacterium glutamicum; diaminopimelate dehydrogenases from Proteus, in particular diaminopimelate dehydrogenase from Proteus vulgaris; glutamate dehydrogenases acting with NADH or NADPH as cofactor (EC 1 .4.1 .3) from Acinetobacter, in particular glutamate dehydrogenases from Acinetobacter sp. ADP1 ; glutamate dehydrogenases (EC 1.4.1.3) from Ralstonia, in particular glutamate dehydrogenases from Ralstonia solanacearum; glutamate dehydrogenases acting with NADPH as cofactor (EC 1 .4.1.4) from Salmonella, in particular glutamate

dehydrogenases from Salmonella typhimurium; glutamate dehydrogenases (EC 1.4.1.4) from Saccharomyces, in particular glutamate dehydrogenases from

Saccharomyces cerevisiae; glutamate dehydrogenases (EC 1 .4.1 .4) from

Brevibacte um, in particular glutamate dehydrogenases from Brevibacte um flavum; and leucine dehydrogenases from Bacillus, in particular leucine dehydrogenases from Bacillus cereus or Bacillus subtilis.

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

The alpha-amino acid according to formula (4) prepared in a method of the invention may further be used for the preparation of the amine according to formula (1 ). The inventors have realised that the alpha-amino acid according to formula (4), prepared from the alpha-keto acid according to formula (2), can be converted into the amine according to formula (1 ) by a decarboxylation reaction. This can be performed chemically, for instance by heating in a high boiling solvent in the presence of a ketone or aldehyde catalyst. For example, amino acids are decarboxylated in good yields in cyclohexanol at 150-160°C with 1 -2 v/v% of cyclohexenone as described by M. Hashimoto, Y. Eda, Y. Osanai, T. Iwai and S. Aoki in Chem. Lett. 1986, 893-896. Similar methods are described in Eur. Pat. Appl. 1586553, 2005 by Daiso, and by S.D. Brandt, D. Mansell, S. Freeman, I.A. Fleet, J.F. Alder J. Pharm. Biomed. Anal. 2006, 41, 872-882.

Alternatively, the decarboxylation of the alpha-amino acid according to formula (4) to the amine according to formula (1 ) may be performed biocatalytically in the presence of a decarboxylase or other biocatalyst catalysing such

decarboxylation.

The decarboxylase may be selected amongst decarboxylases capable of catalysing the decarboxylation of an alpha-amino acid. An enzyme capable of decarboxylating an alpha-amino acid may in particular be selected from the group of decarboxylases (E.C. 4.1 .1 ), preferably from the group of pyruvate decarboxylases (EC 4.1 .1.1 ), diaminopimelate decarboxylases (EC 4.1 .1 .20), diaminopimelate

decarboxylases (EC 4.1 .1 .20), branched chain alpha-keto acid decarboxylases (EC 4.1 .1.72), which include alpha-ketoisovalerate decarboxylases, and alpha-ketoglutarate decarboxylases (EC 4.1 .1 .71 ).

One or more other suitable decarboxylases may in particular be selected amongst the group of oxalate decarboxylases (EC 4.1 .1 .2), oxaloacetate decarboxylases (EC 4.1 .1 .3), acetoacetate decarboxylases (EC 4.1.1 .4), aspartate 1 - decarboxylases (EC 4.1 .1 .1 1 ), valine decarboxylases/leucine decarboxylases (EC 4.1 .1.14), glutamate decarboxylases (EC 4.1 .1.15), 3-hydroxyglutamate

decarboxylases (EC 4.1 .1 .16), ornithine decarboxylases (EC 4.1 .1 .17), lysine decarboxylases (EC 4.1 .1 .18), arginine decarboxylases (EC 4.1 .1.19), 2-oxoglutarate decarboxylases (EC 4.1 .1 .71 ), and diaminobutyrate decarboxylases (EC 4.1 .1.86).

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

Mycobacterium tuberculosis, Pseudomonas sp. and Zymomonas mobilis.

The pyruvate decarboxylase may originate from Saccharomyces cerevisiae or Zymomonas mobilis. In particular, pyruvate decarboxylase mutant I472A from Zymomonas mobilis may be used. An oxaloacetate decarboxylase from

Pseudomonas may in particular be used. Glutamate decarboxylase or aspartate decarboxylase from Escherichia coli (E. coli) may be used, or glutamate decarboxylase from Neurospora crassa, Mycobacterium leprae, Clostridium perfringens, Lactobacillus brevis, Mycobacterium tuberculosis, Streptococcus or Lactococcus may be used. Examples of Lactococcus species from which the glutamate decarboxylase may originate in particular include Lactococcus lactis, such as Lactococcus lactis strain B1 157, Lactococcus lactis IFPL730, more in particular Lactococcus lactis var.

maltigenes formerly named Streptococcus lactis var. maltigenes). A diaminopimelate decarboxylase may, e.g., be from an organism capable of synthesising lysine from diaminopimelate. Such organism may in particular be found amongst bacteria, archaea and plants. In particular, the diaminopimelate decarboxylase may be from a gram negative bacterium, for instance E. coli. Branched-chain alpha-keto acid

decarboxylases from Lactococcus lactis may be used. More in particular, branched chain alpha-keto acid decarboxylases and alpha-ketoisovalerate decarboxylases from Lactococcus lactis may be used.

An alpha-ketoglutarate decarboxylase from Mycobacterium tuberculosis may in particular be used. The inventors have found that

alpha-ketoglutarate decarboxylase (Kgd) from Mycobacterium tuberculosis may be used for converting an alpha-amino acid according to formula (4) to the amine according to formula (1 ), in particular for converting AAP into 6-ACA. In particular, the inventors have found that such decarboxylase comprising a sequence as shown in SEQUENCE ID No. 46 or a functional analogue thereof may be capable of catalysing the formation of the amine according to formula (1 ) from the alpha-amino acid according to formula (4), more in particular the formation of 6-ACA from AAP.

A glutamate decarboxylase may in particular be selected from

Curcurbita moschata, cucumber, yeast, or calf brain; and diaminopimelate

decarboxylases (EC 4.1 .1 .20).

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

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

In a specific embodiment, the alpha-keto acid according to formula (2) is chemically converted into the alpha-amino acid according to formula (4). The alpha-amino acid can be prepared from the alpha-keto acid by catalytic Leuckart- Wallach reaction as described for similar compounds. This reaction is performed with ammonium formate in methanol and [RhCp^*CI₂]₂ as homogeneous catalyst (M.

Kitamura, D. Lee, S. Hayashi, S. Tanaka, M. Yoshimura J. Org. Chem. 2002, 67, 8685- 8687). Alternatively, the Leuckart-Wallach reaction can be performed with aqueous ammonium formate using [lr^l"Cp^*(bpy)H₂0]S0₄ as catalyst as described by S. Ogo, K. Uehara and S. Fukuzumi in J. Am. Chem. Soc. 2004, 126, 3020-3021 . Transformation of alpha-keto acids into (enantiomerically enriched) amino acids is also possible by reaction with (chiral) benzylamines and subsequent hydrogenation of the intermediate imine over Pd/C or Pd(OH)₂/C. See for example, R.G. Hiskey, R.C. Northrop J. Am. Chem. Soc. 1961 , 83, 4798.

Thereafter the alpha-amino acid according to formula (4) is biocatalytically converted into the amine according to formula (1 ), in the presence of a decarboxylase or another biocatalyst capable of performing such decarboxylation. Such decarboxylase may in particular be selected amongst the biocatalysts referred to above, when describing biocatalysts for the conversion of AAP to 6-ACA.

Alternatively, the conversion of the alpha-amino acid according to formula (4) to the amine according to formula (1 ) may be performed by a chemical method, e.g. as mentioned above.

In a specific embodiment, the alpha-keto acid according to formula (2) is biocatalytically converted into the aldehyde according to formula (3) in the presence of a decarboxylase or other biocatalyst capable of catalysing such conversion and the aldehyde is thereafter converted into the amine according to formula (1 ) in the presence of an amino donor and an aminotransferase, amino acid dehydrogenase, or other biocatalyst capable of catalysing such conversion. Decarboxylases suitable for these reactions may in particular be selected from the group of decarboxylases mentioned above, when describing the biocatalytic conversion of the alpha-keto acid according to formula (2) into the aldehyde according to formula (3). A suitable aminotransferase or amino acid dehydrogenase for the conversion of the aldehyde according to formula (3) may in particular be selected from those mentioned above, when describing the biocatalytic conversion of the aldehyde according to formula (3) to the amine according to formula (1 ).

In a specific embodiment, the alpha-keto acid according to formula (2) is biocatalytically converted into the alpha-amino acid according to formula (4) in the presence of an amino donor and an aminotransferase, amino acid dehydrogenase, or other biocatalyst capable of catalysing such conversion and the alpha-amino acid is thereafter converted into the amine according to formula (1 ) in the presence of a decarboxylase or other biocatalyst capable of catalysing such conversion.

Enzymes suitable for these reactions may in particular be selected from the group of aminotransferases, amino acid dehydrogenases, and decarboxylases which have been described above when describing the biocatalytic conversion of the alpha-keto acid into the alpha-amino acid and the biocatalytic conversion of the alpha- amino acid into the amine respectively.

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

It is also possible to obtain the alpha-keto acid according to formula (2) from a natural source, e.g. from methanogenic Archaea, from Asplenium

septentrionale, or from Hydnocarpus anthelminthica. The alpha-keto acid may for instance be extracted from such organism, or a part thereof, e.g. from Hydnocarpus anthelminthica seeds. A suitable extraction method may e.g. be based on the method described in A.I. Virtanen and A.M. Berg in Acta Chemica Scandinavica 1954, 6,1085- 1086, wherein the extraction of amino acids and AKP from Asplenium, using 70% ethanol, is described. In a specific embodiment, the alpha-keto acid according to formula (2) is prepared in a method comprising carbon chain elongation of a smaller alpha-keto acid. Such carbon chain elongation may be a C_x elongation, wherein x is an integer of 1 or more. The elongation is preferably a d elongation. The elongation reaction may be catalysed by a biocatalyst. For example, Alpha-ketoadipic acid (AKA) may be prepared in a method comprising converting alpha-ketoglutaric acid (AKG) into alpha- ketoadipic acid (AKA) by d elongation. AKP may be prepared by converting alpha- ketoadipic acid into alpha-ketopimelic acid, etc. AKG may, e.g., be prepared biocatalytically from a carbon source, such as a carbohydrate, in a manner known in the art per se.

A suitable biocatalyst for preparing the alpha-keto acid from AKG may in particular be selected amongst biocatalysts catalysing d -elongation of alpha- ketoglutaric acid into alpha-ketoadipic acid and/or d -elongation of alpha-ketoadipic acid into alpha-ketopimelic acid and/or d -elongation of alpha-ketopimelic acid into alpha-ketosuberic acid.

In a specific embodiment, the preparation of the alpha-keto acid is catalysed by a biocatalyst comprising

a. an AksA enzyme or an homologue thereof having homo_(n)Citrate synthase activity; and

b. at least one enzyme selected from the group of AksD enzymes,

AksE enzymes, homologues of AksD enzymes and homologues of AksE enzymes having homo^aconitase activity; and

c. an AksF enzyme or a homologue thereof having homO₍n₎-isocitrate dehydrogenase activity;

wherein n is preferably an integer of 1 -5, more preferably an integer of 1 -3.

Preferably, the catalyst comprises both an enzyme selected from the group of AksD enzymes and homologues thereof and an enzyme selected from the group of AksE enzymes and homologues thereof. Said AksD enzyme or its homologue and said AksE enzyme typically form a heterodimer.

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

Methanobrevibacter.

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

The preparation of the alpha-keto acid from AKG may comprise one or more biocatalytic reactions with known or unknown intermediates e.g. the conversion of AKG into AKA or the conversion of AKA into AKP. Such system may be present inside a cell or isolated from a cell. The enzyme system may in particular be from an organism selected from the group of yeasts, fungi, archaea and bacteria, in particular from the group of Penicillium, Cephalosporium, Paelicomyces, Trichophytum,

Aspergillus, Phanerochaete, Eme cella, Ustilago, Schizosaccharomyces,

Saccharomyces, Candida, Yarrowia, Pichia, Kluyveromyces, Thermus, Deinococcus, Pyrococcus, Sulfolobus, Thermococcus, Methanococcus, Methanocaldococcus, Methanosphaera, Methanopyrus, Methanobrevibacter, Methanosarcina and

Methanothermobacter.

In a specific embodiment, the biocatalyst catalysing the preparation of the alpha-keto acid according to formula (2) from alpha-ketoglutaric acid comprises an enzyme system catalysing the conversion of alpha-ketoglutaric acid into alpha- ketoadipic acid, wherein at least one of the enzymes of the enzyme system originates from nitrogen fixing bacteria selected from the group of cyanobacteria, rhizobiales, γ- proteobacteria and actinobacteria, in particular from the group of Anabaena,

Microcystis, Synechocystis, Rhizobium, Brady rhizobium, Pseudomonas, Azotobacter, Klebsiella and Frankia.

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

Step Enzyme Organism gene Protein name

1 AksA Methanocaldococcus MJ0503

jannashii NP_247479

Methanothermobacter MTH1630

thermoautotropicum□ H NP 276742 Methanococcus ma paludis MMP0153 NP_987273 S2

Methanococcus ma paludis MmarC5_1522 YP_001098033 C5

Methanococcus mahpaludis MmarC7_1 153 C7 YP 001330370

Methanospaera stadtmanae Msp_0199 YP_447259 DSM 3091

Methanopyrus kandleri AV19 MK1209 NP_614492 Methanobrevibacter smithii Msm_0722 YP_001273295 ATCC35061

Methanococcus vannielii SB Mevan l 158 YP_001323668 Klebsiella pneumoniae nifV P05345 Azotobacter vinelandii nifV P05342 Pseudomonas stutzehi nifV ABP79047 Methanococcus aeolicus Maeo 0994 YP 001325184 Nankai 3

2, 3 AksD Methanocaldococcus MJ1003 NP_ _247997 jannashii

Methanothermobacter MTH1386 NP_ _276502 thermoautotropicum□ H

Methanococcus mahpaludis Mmp1480 NP_ _988600 S2

Methanococcus mahpaludis MmarC5_0098 YP_ _,001096630

C5

Methanococcus mahpaludis MmarC7_0724 YP_ _001329942 C7

Methanospaera stadtmanae Msp_1486 YP_ _448499

DSM 3091

Methanopyrus kandleri AV19 MK1440 NP_ _614723

Methanobrevibacter smithii Msm_0723 YP_ _001273296

ATCC35061

Methanococcus vannielii SB Mevan_0789 YP_ _,001323307

Methanococcus aeolicus Maeo_031 1 YP_ _00132451 1

Nankai 3

Methanosarcina acetivorans MA3085* NP_ _617978*

Methanospirillum hungatei Mhun_1800* YP_ _503240*

JF-1

Methanosaeta thermophila Mthe_0788* YP_ _843217*

PT

Methanosphaera stadtmanae Msp_1 100* YP_ _448126*

DSM 3091

References to gene and protein can be found via www.ncbi.nlm.nih.gov/ (for listed gene/protein marked with an *: as available on 2 March 2010, for the others: as available on 15 April 2008). Step Enzyme Orgamism gene Protein name

2, 3 AksE Methanocaldococcus jannashii MJ1271 NP_ _248267

Methanothermobacter MTH1387 NP_ _276503 thermoautotropicum□ H

Methanococcus ma paludis S2 MMP0381 NP_ _987501

Methanococcus ma paludis C5 MmarC5_1257 YP_ _,001097769

Methanococcus mahpaludis C7 MmarC7_1379 YP_ _,001330593

Methanospaera stadtmanae Msp_1485 YP_ _448498

DSM 3091

Methanopyrus kandleri AV19 MK0781 NP_ _614065

Methanobrevibacter smithii Msm_0847 YP_ _001273420

ATCC35061

Methanococcus vannielii SB Mevan_1368 YP_ _001323877

Methanococcus aeolicus Nankai Maeo_0652 YP_ _001324848 o

Methanosarcina acetivorans MA3751 ^* NP_ _618624^*

Methanospirillum hungatei JF-1 Mhun_1799^* YP_ _503239^*

Methanosphaera stadtmanae Msp_0374^* YP_ 447420^*

DSM 3091

Methanosaeta thermophila PT Mthe_0853^* YP_ _843282^*

4 AksF Methanocaldococcus jannashii MJ1596 NP_ _248605

Methanothermobacter MTH184 NP_ _275327 thermoautotropicum□ H

Methanococcus mahpaludis S2 MMP0880 NP988000

Methanococcus mahpaludis C5 MmarC5_0688 YP001097214

Methanococcus mahpaludis C7 MmarC7_0128 YP_ _,001329349

Methanospaera stadtmanae Msp_0674 YP_ _447715

DSM 3091

Methanopyrus kandleri AV19 MK0782 NP_ _614066

Methanobrevibacter smithii Msm_0373 YP001272946

ATCC35061

Methanococcus vannielii SB Mevan_0040 YP_ _001322567

Methanococcus aeolicus Nankai

3 Maeo_1484 YP 001325672

Methanosarcina acetivorans MA3748^* NP_ _618621 ^*

Methanospirillum hungatei JF ^:-1 Mhun_1797^* YP_ _503237^*

Methanosphaera stadtmanae YP_ _447715^*

DSM 3091 Msp_0674^*

Methanosaeta thermophila PT Mthe_0855^* YP_ _843284^*

Methanobrevibacter smithii

ATCC 35061 Msm 1298^* YP 001273871 ^*

References to gene and protein can be found via www.ncbi.nlm.nih.gov/ ((for listed gene/protein marked with an ^*: as available on 2 March 2010, for the others:as available on 15 April 2008). In particular, good results have been achieved in a method wherein one or more enzymes have been used represented by SEQ ID NOs 1 1 1 , 114, 117, 120, 123, 126, 129, 132, 135, 138 or 141 . Accordingly, in a preferred embodiment, the invention relates to a method for preparing a compound comprising an amine group wherein one or more of these enzymes or their homologues are used.

The invention also provides novel genes that are codon pair optimised, in particular for Escherichia, more in particular for E. coli. Accordingly, the invention further relates to a polynucleotide comprising a sequence according to any of the: 1 12, 1 15, 1 18, 121 , 124, 127, 130, 133, 136, 139 and functional analogues thereof having a similar, the same or a better level of expression in a Escherichia host cell. To the best of the inventors knowledge these polynucleotides do not occur in nature. In particular in as far as they would occur in nature, any of these polynucleotides is claimed isolated from any organism in which it naturally occurs.

An alpha-keto acid according to formula (2) may be prepared by converting a fatty (di-)acid into an alpha-keto acid, which conversion may be catalysed using a biocatalyst, in particular a heterologous biocatalyst. The biocatalyst may be a heterologous cell, comprising one or more nucleic acid sequence encoding on or more enzymes having catalytic activity in the conversion of a fatty acid into alpha-keto acid.

For preparing an alpha-keto acid from a fatty di-acid, one of the carboxylic acid groups of the acids is converted into an aldehyde group to yield the alpha-keto acid. For example, 2-hydroxyalkanedioic acid may be converted into an alpha-keto acid in such a way. For example, AKP may be prepared from 2- hydroxyheptanedioic acid in such a way.

The conversion may comprise the step of hydroxylation of a dicarboxylic acid into a 2-hydroxy alkanedioic acid (or alpha-hydroxy dicarboxylic acid), which conversion may be catalyzed by a hydroxylase. The conversion may further comprise the step of oxidation of the 2-hydroxy fatty acid into an alpha-keto acid. Said hydroxylation may be catalysed by a biocatalyst comprising an enzyme selected from the group of Oxidoreductases acting on paired donors (with 0₂ as oxidant) and incorporation or reduction of oxygen (EC 1 .14), Oxidoreductases acting on CH or CH2 groups (EC1 .17), hydrolases (EC 3) with pimelate hydrolase activity and hydrolases (EC 3) with pimelate-2-monooxygenase activity.

Further, for the catalysis of the hydroxalation of the dicarboxylic acid, an enzyme may be used selected from the group of enzymes comprising an amino acid sequence as shown in any of the Seq ID No's: 90-109 or a homologue of any of these sequences. The oxidation may be catalysed by a biocatalyst comprising an enzyme is selected from the group of

- oxidoreductases with oxygen as acceptor (EC 1.1 .3), such as a lactate oxidase or another hydroxy acid oxidase

- L-lactate dehydrogenases (EC 1 .1 .1 .27);

- hydroxypyruvate reductases, beta-hydroxypyruvate reductases;

NADH:hydropyruvatereductases and D-glycerate dehydrogenases (EC1 .1.1 .81 ) ;

- malate dehydrogenases [NADP+], NADP+-malic enzymes, NADP+- malicdehydrogenases (nicotinamide adenine dicnucleotide phosphate); malate NADP dehydrogenases; NADP+ malate dehydrogenases; NADP+-linked malate

dehydrogenase and malate dehydrogenases (NADP+) (EC 1.1 .1.82);

- 3-isopropylmalate dehydrogenases, beta-isopropylmalic enzymes; beta- isopropylmalatedehydrogenases; threo-Ds-3-isopropylmalate dehydrogenases, 3- carboxy-2-hydroxy-4-methylpentanoate:NAD+ oxidoreductases (EC 1 .1.1 .85);

- tartrate dehydrogenases, mesotartrate dehydrogenases (EC 1 .1.1.93);

- (R)-2-hydroxy-fatty-acid dehydrogenases (EC1 .1 .1 .98);

- (S)-2-hydroxy-fatty-acid dehydrogenases (EC 1 .1 .1 .99);

- 1 .1 .1 .172 2-oxoadipate reductases, 2-ketoadipate reductses, alpha-ketoadipate reductases2-ketoadipate reductases

- 2-hydroxyglutarate dehydrogenase (EC 1 .1 .99.2); and

- D-2-hydroxy-acid dehydrogenase (EC 1 .1.99.6).

Such enzyme may in particular originate from an organism selected from the group of Hominidae and Aerococcus, more in particular from the group of Homininae, such as from Homo sapiens, and Aerococcus viridans. For instance, Homo sapiens hydroxyacid oxidase (glycolate oxidase) or Lactate oxidase - Aerococcus viridans may be used.

The oxidation of the 2-hydroxy fatty acid into an alpha-keto acid may in particular be catalysed by an enzyme comprising an amino acid sequence represented by SEQ ID NO: 85 or 88 (for which encoding nucleic acid sequences are shown in SEQ ID NO: 84 and 86, respectively in SEQ ID NO: 87 and 89), or a homologue of any of these sequences.

Preferably, a biocatalyst used for the preparation of the alpha-keto acid comprises an enzyme system for preparing an alpha-keto acid from a suitable carbon source that can be converted into an alpha-keto acid, for instance by fermentation of the carbon source.

In an advantageous method an alpha-keto acid is prepared making use of a whole cell biotransformation of the carbon source to form an alpha-keto acid. It is known that dicarboxylic acid may be formed from long chain fatty acids via oxidative cleavage. Such fatty acids may therefore be provided as a carbon source, e.g. by supplying plant oils, fatty acid esters (bio-diesel) or the like to a biocatalyst (in particular in case it is a host cell) in a method of the invention. For instance a host cell may be selected naturally comprising such system - such as E. coli or B. sphaericus - or the host cell may be obtained by genetic modification.

2-Hydroxy fatty acids may also be naturally occurring compounds and make a very suitable carbon source (see reference Chembiochem. 2009 Aug

17;10(12):2003-10). Biosynthesis of 2-hydroxy and iso-even fatty acids is connected to sphingolipid formation in myxobacteria.Ring MW, Schwar G, Bode HB.). When using 2- hydroxy fatty acids as a starting material, the hydroxylation step may not be needed to obtain the alpha-keto acid.

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

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

If desired, the amine according to formula (1 ) obtained in accordance with the invention can be cyclised to form a lactam. This may be accomplished based on methodology known per se. E.g. 6-ACA may be converted to form caprolactam, e.g. as described in US-A 6,194,572.

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

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

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

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

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

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

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

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

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

In an advantageous method the amine according to formula (1 ) is prepared making use of a whole cell biotransformation of the substrate for the amine or an intermediate for forming the amine (the alpha-keto acid, aldehyde or alpha-amino acid according to formula (2), (3) and (4) respectively), comprising a micro-organism wherein one or more biocatalysts (usually one or more enzymes) catalysing the biotransformation are produced, such as one or more biocatalysts selected from the group of biocatalysts capable of catalysing the conversion of the alpha-keto acid to the alpha-amino acid, biocatalysts capable of catalysing the conversion of the alpha-amino acid to the amine, biocatalysts capable of catalysing the conversion of the alpha-keto acid to the aldehyde and biocatalysts capable of catalysing the conversion of the aldehyde to the amine. In a preferred embodiment the micro-organism is capable of producing a decarboxylase and/or at least one enzyme selected from amino acid dehydrogenases and aminotransferases are produced.

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

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

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

As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skilled in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "homologous" when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain. The promoter that could be used to achieve the expression of the nucleic acid sequences coding for an enzyme for use in a method of the invention, in particular an aminotransferase, an amino acid dehydrogenase or a decarboxylase, such as described herein above may be native to the nucleic acid sequence coding for the enzyme to be expressed, or may be heterologous to the nucleic acid sequence (coding sequence) to which it is operably linked. Preferably, the promoter is homologous, i.e. endogenous to the host cell.

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

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

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

Examples of constitutive and inducible promoters in Gram-negative microorganisms include, but are not limited to, tac, tet, trp-tet, Ipp, lac, Ipp-lac, laclq, T7, T5, 73, gal, trc, ara (P_BAD), SP6, A-P_R> and A-P_L.

Promoters for (filamentous) fungal cells are known in the art and can be, for example, the glucose-6-phosphate dehydrogenase gpdk promoters, protease promoters such as pepk, pep , pepC, the glucoamylase glak promoters, amylase amyk, amyB promoters, the catalase catR or catk promoters, glucose oxidase goxC promoter, beta-galactosidase lack promoter, alpha-glucosidase aglk promoter, translation elongation factor tefk promoter, xylanase promoters such as xlnk, xlnB, xlnC, xlnD, cellulase promoters such as eglk, eglB, cbhk, promoters of transcriptional regulators such as arek, crek, xlnR, pacC, prfT, or another promotor, and can be found among others at the NCBI website (http://www.ncbi.nlm.nih.gov/entrez/).

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

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

Accordingly, the invention also relates to a host cell comprising one or more biocatalysts capable of catalysing at least one reaction step in a method of the invention, in particular capable of catalysing at least one reaction step in the conversion of the alpha-keto acid, aldehyde or alpha-amino acid according to formula (2), (3) and (4) respectively to the amine according to formula (1 ). The invention also relates to a novel vector comprising one or more genes encoding for one or more enzymes capable of catalysing at least one reaction step in a method of the invention, in particular capable of catalysing at least one reaction step in the conversion of the alpha-keto acid to the amine and to a novel host cell comprising one or more genes encoding for one or more enzymes capable of catalysing at least one reaction step in a method of the invention, in particular capable of catalysing at least one reaction step in the conversion of the alpha-keto acid to the amine (which one or more genes may form part of one or more vectors), more in particular in the conversion of AKP to 6-ACA.

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

In particular, a host cell or vector according to the invention may comprise at least one nucleic acid sequence, in particular at least two nucleic acid sequences, selected from the group of nucleic acid sequences encoding an enzyme with alpha-keto acid decarboxylase activity, nucleic acid sequences encoding an enzyme with aldehyde ( e.g. 5-formylpentanoate) aminotransferase activity, nucleic acid sequences encoding an enzyme with alpha-keto acid aminotransferase activity, nucleic acid sequences encoding an enzyme with alpha-keto acid dehydrogenase activity and nucleic acid sequences encoding an enzyme with alpha-amino acid decarboxylase activity. Of these sequences, typically one or more, in particular two or more, are recombinant sequences.

In preferred embodiment the host cell, typically a recombinant host cell, or the vector according to the invention comprises a nucleic acid sequence encoding at least one biocatalyst having alpha-keto acid decarboxylase activity, and/or at least one nucleic acid sequence selected from sequences encoding a biocatalyst with aldehyde (e.g. 5-formylpentanoate) aminotransferase activity.

In such an embodiment, the nucleic acid sequence encoding an enzyme with alpha-keto acid decarboxylase activity may in particular comprise a nucleic acid sequence encoding an amino acid sequence according to Sequence ID 31 , Sequence ID 34, Sequence I D 37, Sequence ID 40, Sequence ID 43, Sequence ID 46, Sequence ID 143, Sequence I D 146, Sequence ID 149, Sequence 152 or a homologue of any of these sequences and/or the nucleic acid sequence encoding an enzyme with aldehyde aminotransferase may in particular comprise an amino acid sequence according to Sequence I D 2, Sequence ID 5, Sequence ID 8, Sequence ID 65 Sequence I D 67, Sequence ID 69 or a homologue thereof. One or more of said nucleic acid sequences may form part of one or more recombinant vectors.

In a further preferred embodiment, the vector or host cell comprises a nucleic acid sequence encoding an enzyme with alpha-keto acid aminotransferase activity and/or a nucleic acid sequence encoding an enzyme with alpha-amino acid decarboxylase activity. The nucleic acid sequence encoding an enzyme with alpha- keto acid aminotransferase activity may in particular comprise an amino acid sequence according to Sequence ID 2, Sequence I D 8, Sequence ID 12, Sequence ID 15,

Sequence ID 17, Sequence ID 19, Sequence I D 21 , Sequence ID 23, Sequence I D 25, Sequence ID 27, Sequence ID 29, or a homologue thereof. One or more of said nucleic acid sequences may form part of one or more recombinant vectors.

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

In a specific preferred embodiment, a host cell according to the invention comprises a nucleic acid sequence encoding an enzyme with 6-aminocaproic acid 6- dehydrogenase activity and a nucleic acid sequence encoding an enzyme with alpha-ketopimelic acid decarboxylase activity. One or more suitable genes of a host cell or vectors according to the invention may in particular be selected amongst genes encoding an enzyme as mentioned herein above.

In a specific embodiment, the host cell is a recombinant cell comprising at least one nucleic acid sequence selected from the group of sequences as identified in any of Sequence ID 1 , Sequence ID 3, Sequence ID 4, Sequence ID 6, Sequence ID 7, Sequence ID 1 1 , Sequence I D 13, Sequence ID 14, Sequence I D 16, Sequence ID 18, Sequence ID 20, Sequence I D 22, Sequence ID 24, Sequence I D 26, Sequence ID 28, Sequence ID 30, Sequence I D 32, Sequence ID 33, Sequence I D 35, Sequence ID 36, Sequence ID 38, Sequence I D 39, Sequence ID 41 , Sequence I D 42, Sequence ID 44, Sequence ID 45, Sequence I D 47, Sequence ID 64, Sequence I D 66, Sequence ID 68, Sequence ID 142, Sequence ID 144, Sequence ID 145, Sequence ID 147, Sequence ID 151 and functional analogues thereof.

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

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

In a preferred embodiment, the host cell comprises a nucleic acid sequence encoding an enzyme, capable of catalysing the conversion of alpha-amino acid according to formula (4) to alpha-keto acid according to formula (2), according to Sequence ID No. : 1 , 3, 7, 1 1 , 13, 14, 16, 18, 20, 22, 24, 26, 28, or a functional analogue thereof, which may be a wild type or non-wild type sequence

In a specific embodiment, the host cell comprises at least one nucleic acid sequence encoding a biocatalyst having alpha-aminopimelic acid decarboxylase activity, which may be homologous or heterologous to the host cell. In particular such biocatalyst may be selected from the group of decarboxylases (E.C. 4.1 .1 ), more in particular from the group of glutamate decarboxylases (EC 4.1 .1 .15), diaminopimelate decarboxylases (EC 4.1 .1 .20) aspartate 1 -decarboxylases (EC 4.1 .1 .1 1 ), branched chain alpha-keto acid decarboxylases, alpha-ketoisovalerate decarboxylases, alpha- ketoglutarate decarboxylases, pyruvate decarboxylases (EC 4.1 .1 .1 ) and oxaloacetate decarboxylases (E.C. 4.1 .1 .3).

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

components, such as the specific compound of interest. Also a host cell devoid of any other enzymatic activity resulting in the conversion of AKA to an undesired side product is preferred.

Preferably, the organism or host cell is capable of converting a fatty acid to an alpha-keto acid in a method as described above.

In a specific embodiment, the host cell comprises one or more enzymes catalysing the formation of the alpha-keto acid according to formula 2. Use may be made of an enzyme system forming part of the amino acid degradation pathways as described in Atsumi S, Hanai T, Liao JC. Nature. 2008 Jan

3;451 (7174):86-9. or an enzyme system capable of modifying fatty acids in a suitable way, e.g. by hydroxylation and/ or oxidation. The enzyme system may be naturally occurring in the host cell or heterologously engineered. For example, the system may be isolated from a cell other than the host cell. The term ^'enzyme system^' is in particular used herein for a single enzyme or a group of enzymes whereby a specific conversion can be catalysed. Said conversion may comprise one or more chemical reactions with known or unknown intermediates.

In a preferred host cell, suitable for preparing the alpha-amino acid according to formula (4) making use of a whole cell biotransformation process, one or more biocatalysts capable of catalysing at least one reaction step in the preparation of alpha-ketopimelic acid from alpha-ketoglutaric acid are encoded for. Suitable biocatalysts are, e.g., as described above when discussing the preparation of alpha- keto acid.

The host cell may for instance be selected from bacteria, yeasts or fungi. In particular the host cell may be selected from the genera selected from the group of Aspergillus, Penicillium, Saccharomyces, Kluyveromyces, Pichia, Candida, Hansenula, Bacillus, Corynebacterium, Pseudomonas, Gluconobacter,

Methanococcus, Methanobacterium, Methanocaldococcus and Methanosarcina and Escherichia. Herein, usually one or more encoding nucleic acid sequences as mentioned above have been cloned and expressed.

In particular, the host strain and, thus, a host cell suitable for the biochemical synthesis of the amine according to formula (1 ) may be selected from the group of Escherichia coli, Bacillus subtilis, Bacillus amyloliquefaciens, Corynebacterium glutamicum, Aspergillus niger, Penicillium chrysogenum, Saccharomyces cerevisiae, Hansenula polymorpha, Candida albicans, Kluyveromyces lactis, Pichia stipitis, Pichia pastoris, Methanobacterium thermoautothrophicum ΔΗ, Methanococcus maripaludis, Methanococcus voltae, Methanosarcina acetivorans, Methanosarcina barker! and Methanosarcina mazei host cells.

The host cell may be in principle a naturally occurring organism or may be an engineered organism. Such an organism can be engineered using a mutation screening or metabolic engineering strategies known in the art. In a specific embodiment, the host cell naturally comprises (or is capable of producing) one or more of the enzymes suitable for catalysing a reaction step in a method of the invention, such as one or more activities selected from the group of decarboxylases,

aminotransferases and amino acid dehydrogenases capable of catalysing a reaction step in a method of the invention. For instance E. coli may naturally be capable of producing an enzyme catalysing a transamination in a method of the invention. It is also possible to provide a recombinant host cell with both a recombinant gene encoding an aminotransferase or amino acid dehydrogenase capable of catalysing a reaction step in a method of the invention and a recombinant gene encoding a decarboxylase gene capable of catalysing a reaction step in a method of the invention.

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

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

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

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

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

The invention further relates to a method for preparing alpha-amino acid, comprising converting alpha-keto acid into alpha-amino acid, which conversion is catalysed by a biocatalyst.

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

As indicated above, the alpha-amino acid may thereafter be used for the preparation of the amine according to formula (1 ). Alternatively, the alpha-amino acid may be used as such, e.g. as a chemical for biochemical research or as a pH- buffer compound, e.g. for use in a preparative or analytical separation technique such as liquid chromatography or capillary electrophoresis.

Further, the alpha-amino acid according to formula (4) prepared in a method of the invention may further be used in the preparation of another compound, for instance, the alpha-amino acid according to formula (1 ) may be converted into a cyclic compound, such as a lactam. For example, the lactam obtained by cyclisation may be a lactam represented by the formula O

N

H-C A

H (5), wherein A is defined is as defined for formula (1 ) and (2). As described above, and illustrated in an example, below, AAP can be chemically converted in caprolactam, e.g. by exposure to a high temperature. Without being bound by theory, it is contemplated that also in this reaction the amine 6-ACA may be formed as a short-lived intermediate. Alternatively, the amine according to formula (1 ) with R=COOH may also be used in the preparation of a cyclic compound.

Further, the amine according to formula (1 ) may be used in a method for preparing a polymer. Such a method comprises subjecting the amine or a mixture of compounds containing at least one amine according to formula (1 ) to a polymerisation reaction, optionally in the presence of one or more further monomers. Preferably, amines according to formula (1 ) may be polymerized into polyamides. For example, amines according to formula (1 ) with R=NH₂ (diamines) may be polymerized into polyamides, for example by copolymerization with diacids. Amines according to formula (1 ) with R=COOH (amino acids) may be polymerized into polyamides, optionally with one or more further monomers, such as other amino acids.

Polymerization reactions may be conducted in host cells, for example in host cells such as described hereinabove.

Next, the invention will be illustrated by the following examples.

EXAMPLES

/. Examples relating to the preparation of a compound comprising an amino group

1.1 General Methods

Molecular and genetic techniques

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

Publishing and Wiley Interscience, New York 1987).

Plasmids and Strains

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

Media

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

Identification of plasmids

Plasmids carrying the different genes were identified by genetic, biochemical, and/or phenotypic means generally known in the art, such as resistance of transformants to antibiotics, PCR diagnostic analysis of transformant or purification of plasmid DNA, restriction analysis of the purified plasmid DNA or DNA sequence analysis. HPLC-MS analysis method for the determination of 5-FVA

5-FVA was detected by selective reaction monitoring (SRM)-MS, measuring the transition m/z 129-> 83. Concentrations for 5-FVA were calculated by measuring the peak area of the 5-FVA peak eluting at approximately 6 min. Calibration was performed by using an external standard procedure. All the LC-MS experiments were performed on an Agilent 1200 LC system, consisting of a quaternary pump, autosampler and column oven, coupled with an Agilent 6410 QQQ triple quadrupole MS.

LC conditions:

Column: 50 x 4.6 mm Nucleosil C18, 5 μιη (Machery & Nagel) pre column coupled to a 250 x 4.6 mm id. Prevail C18, 5 μιη (Alltech)

Column temperature : room temperature

Eluent: A: water containing 0.1 % formic acid

B: acetonitrile containing 0.1 % formic acid

Gradient: time (min) % eluent B

0 10

6 50

6.1 10

11 10

Flow: 1 .2 ml/min, before entering the MS the flow is split 1 :3

Injection volume: 2 ul MS conditions:

lonisation: negative ion electrospray

source conditions: ionspray voltage : 5kV

temperature : 350^eC

fragmentor voltage and collision energy optimized Scan mode: selective reaction mode : transition m/z 129 -> 83

HPLC-MS analysis for the determination of AAP

AAP was detected by selected ion monitoring (SIM)-MS, measuring the protonated molecule for AAP with m/z 176. Concentrations for AAP were calculated by measuring the peak area of the AAP peak eluting at a retention time of 2.7 minutes in the samples. Calibration was performed by using an external standard procedure. All the LC-MS experiments were performed on an Agilent 1 100 LC system consisting of a quaternary pump, degasser, autosampler and column oven, coupled with an API 2000 triple quadrupole MS (Applied Biosystems).

LC conditions were as follows:

Column: 50^*4 Nucleosil C18, 5 μιη (Macherey-Nagel) + 250 x 4.6 Prevail C18, 5 μιη (Alltech), both at room temperature (RT)

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

B = 0.1 % (v/v) formic acid in acetonitrile (pa, Merck) Flow: 1 .2 ml/min, before entering the MS the flow was split 1 :3

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

Injection volume: 2 μΙ

MS conditions: Positive ion electrospray was used for ionization

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

HPLC-MS analysis for the determination of 6-ACA

Calibration:

The calibration was performed by an external calibration line of 6- ACA (m/z 132 -> m/z 1 14, Rt 7.5 min). All the LC-MS experiments were performed on an Agilent 1 100, equipped with a quaternary pump, degasser, autosampler, column oven, and a single-quadrupole MS (Agilent, Waldbronn, Germany). The LC-MS conditions were:

Column: 50^*4 Nucleosil (Mancherey-Nagel) + 250 x 4.6 Prevail C18 (Alltech), both at room temperature (RT)

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

B = Acetonitrile (pa, Merck)

Flow: 1 .0 ml/min, before entering the MS the flow was split 1 :3

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

80% (v/v) B.

Injection volume: 5 μΙ

MS detection: ESI(+)-MS

The electrospray ionization (ESI) was run in the positive scan mode with the following conditions; m/z 50-500, 50 V fragmentor, 0.1 m/z step size, 350 °C drying gas temperature, 10 L N₂/min drying gas, 50 psig nebuliser pressure and 2.5 kV capillary voltage.

1.2 Cloning of target genes

Design of expression constructs

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

Gene synthesis and construction of plasm ids

Synthetic genes were obtained from DNA2.0 and codon optimised for expression in E. coli according to standard procedures of DNA2.0. The

aminotransferase genes from Vibrio fluvialis JS17 [SEQ ID No. 1 ] and Bacillus weihenstephanensis KBAB4 [SEQ ID No. 4] encoding the amino acid sequences of the V. fluvialis JS17 ω-aminotransf erase [SEQ ID No. 2] and the B. weihenstephanensis KBAB4 aminotransferase (ZP_01 186960) [SEQ ID No. 5], respectively, were codon optimised and the resulting sequences [SEQ ID No. 3] and [SEQ ID No. 6] were obtained by DNA synthesis.

The decarboxylase genes from Escherichia coli [SEQ ID No. 30],

Saccharomyces cerevisiae [SEQ ID No. 33], Zymomonas mobilis [SEQ ID No. 36], Lactococcus lactis [SEQ ID No. 39], [SEQ ID No. 42], and Mycobacterium tuberculosis [SEQ ID No. 45] encoding the amino acid sequences of the Escherichia coli diaminopimelate decarboxylase LysA [SEQ ID No. 31], the Saccharomyces cerevisiae pyruvate decarboxylase Pdc [SEQ ID No. 34], the Zymomonas mobilis pyruvate decarboxylase Pdcl472A [SEQ ID No. 37], the Lactococcus lactis branched chain alpha-keto acid decarboxylase KdcA [SEQ ID No. 40] and alpha-ketoisovalerate decarboxylase KivD [SEQ ID No. 43], and the Mycobacterium tuberculosis

alpha-ketoglutarate decarboxylase Kgd [SEQ ID No. 46], respectively, were also codon optimised and the resulting sequences [SEQ ID No. 32], [SEQ ID No. 35], [SEQ ID No. 38], [SEQ ID No. 41 ], [SEQ ID No. 44], and [SEQ ID No. 47] were obtained by DNA synthesis, respectively.

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

Cloning by PCR

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

Table 2

origin of gene gene Sequence ID enzyme primer

Sequence ID Sequence ID's

Pseudomonas 7 8 9&10 aeruginosa

Pseudomonas 26 27 60&61 aeruginosa

Pseudomonas 66 67 72&73 aeruginosa

Pseudomonas 68 69 74&75 aeruginosa

Bacillus subtilis 14 15 48&49

Bacillus subtilis 16 17 50&51

Bacillus subtilis 64 65 70&71

Rhodobacter 18 19 52&53 sphaeroides

Legionella 20 21 54&55 pneumophila

Nitrosomas europaea 22 23 56&57

Neisseria 24 25 58&59 gonorrhoeae

Rhodopseudomonas 28 29 62&63 palustris PCR reactions were analysed by agarose gel electrophoresis and PCR products of the correct size were eluted from the gel using the QIAquick PCR purification kit (Qiagen, Hilden, Germany). Purified PCR products were cloned into pBAD/Myc-His-DEST expression vectors using the Gateway technology (Invitrogen) via the introduced attB sites and pDONR-zeo (Invitrogen) as entry vector as described in the manufacturer's protocols. The sequence of genes cloned by PCR was verified by DNA sequencing. This way the expression vectors pBAD-Pae-_gi9946143_AT, pBAD- Bsu_gi16078032_AT, pBAD- Bsu_gi16080075_AT, pBAD-6sL_gi16077991_AT, pBAD- /¾p_AT, pBAD-/.pn_AT, pBAD-Neu_AT, pBAD-Ngo AT, pBAD-Pae_gi9951299_AT, pBAD-Pae_gi9951072_AT, pBAD-Pae_gi9951630_AT and pBAD-Ppa_AT were obtained. The corresponding expression strains were obtained by transformation of chemically competent E. coli TOP10 (Invitrogen) with the pBAD constructs.

1.3 Growth of E. coli for protein expression

Small scale growth was carried out in 96-deep-well plates with 940 μΙ media containing 0.02% (w/v) L-arabinose. Inoculation was performed by transferring cells from frozen stock cultures with a 96-well stamp (Kuhner, Birsfelden, Switzerland). Plates were incubated on an orbital shaker (300 rpm, 5 cm amplitude) at 25 °C for 48 h. Typically an OD_620nm of 2 - 4 was reached.

1.4 Preparation of cell lysates

Preparation of lysis buffer

The lysis buffer contained the following ingredients:

Table 3

The solution was freshly prepared directly before use. Preparation of cell free extract by lysis

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

Preparation of cell free extract by sonification

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

1.5 Preparation of 5-formylpentanoic acid by chemical hydrolysis of methyl 5-formylpentanoate

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

1.6 Enzymatic reactions for conversion of 5-formylpentanoic acid to 6- AC A Unless specified otherwise, a reaction mixture was prepared comprising 10 mM 5-formylpentanoic acid, 20 mM racemic omethylbenzylamine, and 200 μΜ pyridoxal 5'-phosphate in 50 mM potassium phosphate buffer, pH 7.0. 100 μΙ of the reaction mixture were dispensed into each well of the well plates. To start the reaction, 20 μΙ of the cell free extracts were added, to each of the wells. Reaction mixtures were incubated on a shaker at 37 °C for 24 h. Furthermore, a chemical blank mixture (without cell free extract) and a biological blank (E. coli TOP10 with pBAD/Myc- His C) were incubated under the same conditions. Samples were analysed by HPLC- MS. The results are summarised in the following table.

Table 4: 6-ACA formation from 5-FVA in the presence of aminotransferases

n.d.: not detectable

^* method differed in that 10 μΙ cell free extract was used instead of 20 μΙ, the pyridoxal- 5'-phosphate concentration was 50 μΜ instead of 200 μΜ and the reaction mixture volume in the wells was 190 μΙ instead of 100 μΙ. It is shown that 6-ACA is formed from 5-FVA in the presence of an aminotransferase.

/.7 Enzymatic reactions for conversion of AKP to 5- formylpentanoic acid

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

Table 5: 5-FVA formation from AKP in the presence of decarboxylases

n.d. : not detectable

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

1.8 Enzymatic reactions for conversion of AKP to 6-ACA in presence of recombinant decarboxylase

A reaction mixture was prepared comprising 50 mM AKP, 5 mM magnesium chloride, 100 μΜ pyridoxal 5'-phosphate (for LysA) or 1 mM thiamine diphosphate (for all other tested biocatalysts) in 100 mM potassium phosphate buffer, pH 6.5. 4 ml of the reaction mixture were dispensed into a reaction vessel. To start the reaction, 1 ml of the cell free extracts were added, to each of the wells. Reaction mixtures were incubated with a magnetic stirrer at 37 °C for 48 h. Furthermore, a chemical blank mixture (without cell free extract) and a biological blank (E. coli TOP10 with pBAD/Myc-His C) were incubated under the same conditions. Samples from different time points during the reaction were analysed by HPLC-MS. The results are summarised in the following table. Table 6: 6- AC A formation from AKP in the presence of decarboxylases

n.a. = not analysed

n.d. = not detectable It is shown that 6-ACA is formed from AKP in the presence of a decarboxylase. It is contemplated that the E. coli contained natural 5-FVA

aminotransferase activity.

1.9 Enzymatic reactions for conversion of AKP to 6-ACA in presence of recombinant decarboxylase and recombinant aminotransferase

A reaction mixture was prepared comprising 50 mM AKP, 5 mM magnesium chloride, 100 μΜ pyridoxal 5'-phosphate, 1 mM thiamine diphosphate and 50 mM racemic a-methylbenzylamine in 100 mM potassium phosphate buffer, pH 6.5. 1 .6 ml of the reaction mixture were dispensed into a reaction vessel. To start the reaction, 0.2 ml of the decarboxylase containing cell free extract and 0.2 ml of the aminotransferase containing cell free extract were added, to each of the reaction vessels. Reaction mixtures were incubated with a magnetic stirrer at 37 °C for 48 h. Furthermore, a chemical blank mixture (without cell free extract) and a biological blank (E. coli TOP10 with pBAD/Myc-His C) were incubated under the same conditions. Samples from different time points during the reaction were analysed by HPLC-MS. The results are summarised in the following table. Table 7: 6- AC A formation from AKP in the presence of a recombinant decarboxylase and a recombinant aminotransferase

DC=decarboxylase

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

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

1.10 Construction of plasm ids for expression of aminotransferases and decarboxylases in S. cerevisiae

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

The aminotransferase gene from Pseudomonas aeruginosa [SEQ ID No. 7] coding for P. aeruginosa aminotransferase [SEQ ID No. 8] was amplified from pBAD-Pae_AT by PCR using Phusion DNA polymerase (Finnzymes) according to the manufacturers specifications and using specific primers [SEQ ID No. 78 & 79]. The resulting PCR products were cloned into vector pAKP-41 using Spe\ and BamH\ restriction enzymes resulting in vectors pAKP-79 and pAKP-80 respectively, which now contain the aminotransferase gene under the S. cerevisiae gaUO promoter and the S. cerevisiae adh2 terminator.

The decarboxylase gene from Saccharomyces cerevisiae [SEQ ID

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

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

The resulting PCR products were cloned into vector pAKP-44 using

Asc\ and BamYW restriction enzymes resulting in vectors pAKP-81 and pAKP-82 respectively, which now contain the decarboxylase gene under the S. cerevisiae gal2 promoter and the S. cerevisiae pma1 terminator.

Plasmids pAKP-79 and pAKP-80 were restriction enzyme digested with Sad and Xba\ and plasmids pAKP-81 and pAKP-82 were restriction enzyme digested with Sa/I and Xba\. A Sac\/Xba\ aminotransferase fragment was combined with a Sal\/Xba\ decarboxylase fragment into the S. cerevisiae low copy episomal vector pRS414, which was restriction enzyme digested with Sal\ and Sad.

The resulting plasmids were obtained:

pAKP-85: Pgal10-Pae_AT-Tadh2 Pgal2-Pdc_DC-Tpma1

pAKP-86: Pgal10-Pae_AT-Tadh2 Pgal2-KdcA_DC-Tpma1

pAKP-87: Pgal10-Vfl_AT-Tadh2 Pgal2-Pdc_DC-Tpma1

pAKP-88: Pgal10-Vfl_AT-Tadh2 Pgal2-KdcA_DC-Tpma1 1.11 Transformation and growth of S. cerevisiae

S. cerevisiae strain CEN.PK1 13-3C was transformed with 1 μς of plasmid DNA according to the method as described by Gietz and Woods (Gietz, R.D. and Woods, R.A. (2002). Transformation of yeast by the Liac/SS carrier DNA/PEG method. Methods in Enzymology 350: 87-96). Cells were plated on agar plates with 1 x Yeast Nitrogen Base without amino acids and 2% glucose.

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

/.12 Preparation of cell free extract

1 ml of potassium phosphate buffer (pH 7) was added to 0.5 g of the cell pellet. This mixture was added to a 2 ml eppendorf tube which contained 0.5 g of glassbeads with a diameter of 0.4-0.5 mM. Samples were vigorously shaken with an eppendorf shaker (IKA VIBRAX-VXR) for 20 s. The resulting cell free extract was centrifuged for 5 minutes at 14000 rpm and 4°C. The supernatant was used for enzyme activity assays. /.13 Enzymatic reactions for conversion of AKP to 6-ACA in presence of decarboxylase and aminotransferase co-expressed in S. cerevisiae

A reaction mixture was prepared comprising 50 mM AKP, 5 mM magnesium chloride, 100 μΜ pyridoxal 5'-phosphate, 1 mM thiamine diphosphate and 50 mM racemic omethylbenzylamine in 100 mM potassium phosphate buffer, pH 6.5. 1.6 ml of the reaction mixture were dispensed into a reaction vessel. To start the reaction, 0.4 ml of the cell free extract from S. cerevisiae containing decarboxylase and aminotransferase were added, to each of the reaction vessels. Reaction mixtures were incubated with a magnetic stirrer at 37 °C. Furthermore, a chemical blank mixture (without cell free extract) and a biological blank (S. cerevisiae) were incubated under the same conditions. Samples, taken after 19 hours of incubation, were analysed by HPLC-MS. The results are summarised in the following table. Table 8: 6- AC A formation from AKP using a micro-organism as a biocatalyst

/.14 Enzymatic reactions for conversion of alpha-ketopimelic acid to alpha-aminopimelic acid

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

Table 9: AAP formation from AKP in the presence of aminotransferases

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

//. Examples relating to the preparation of an alpha-keto acid

II.1 General methods Molecular and genetic techniques

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

Publishing and Wiley Interscience, New York 1987). Plasmids and Strains

pMS470 (Balzer, D.; Ziegelin, G.; Pansegrau, W.; Kruft, V.; Lanka, E. Nucleic Acids Research 1992, 20(8), 1851 -1858.) and pBBRI MCS (Kovach ME, Phillips RW, Elzer PH, Roop RM 2nd, Peterson KM. Biotechniques. 1994

May;16(5):800-2. pBBRI MCS: a broad-host-range cloning vector) have been described previously. E. coli strains TOP10 and DH10B (Invitrogen, Carlsbad, CA, USA) were used for all cloning procedures. E. coli strains BL21 A1 (Invitrogen, Carlsbad, CA, USA) and BL21 (Novagen (EMD/Merck), Nottingham, UK) were used for protein expression.

Media

2xTY medium (16 g/l tryptopeptone, 10 g/l yeast extract, 5 g/l NaCI) was used for growth of E. coli. Antibiotics (100 \g/m\ ampicillin, 50-100 \g/m\ neomycin) were supplemented to maintain plasmids in E. coli. For induction of gene expression in E. coli arabinose (for BL21 -AI derivatives) and IPTG (for pMS470, pBBRI MCS derivatives) were used at 0.02% (L-arabinose) and 0.2mM (IPTG) final concentrations. AKP production by E. coli was done in M9 minimal medium (12.8 g/L Na₂HP0₄.7H₂0, 3 g/L KH₂P0_4, 0.5 g/L NaCI, 1 g/L NH₄CI, 2 mM MgS0₄, 0.1 mM CaCI₂) with 1 % glucose.

Identification of plasmids

Plasmids carrying the different genes were identified by genetic, biochemical, and/or phenotypic means generally known in the art, such as resistance of transformants to antibiotics, PCR diagnostic analysis of transformant or purification of plasmid DNA, restriction analysis of the purified plasmid DNA or DNA sequence analysis. Integrity of all new constructs described was confirmed by restriction digest and, if PCR steps were involved, additionally by sequencing

UPLC-MS/MS analysis method for the determination of a-keto acids A Waters HSS T3 column 1 .8 Mm, 100 mm^*2.1 mm was used for the separation of a-keto acids with gradient elution as depicted in Table 10. Eluent A consists of LC/MS grade water, containing 0.1 % formic acid, and eluent B consists of acetonitrile, containing 0.1 % formic acid. The flow-rate was 0.25 ml/min and the column was thermostated at a temperature of 40 °C. Table 10: gradient elution program used for the separation of o-keto acids, 6- AC A, 5- FVA and homo_(n)Citrate

A Waters micromass Quattro micro API was used in electrospray either positive or negative ionization mode, depending on the compounds to be analyzed, using multiple reaction monitoring (MRM). The ion source temperature was kept at 130 °C, whereas the desolvation temperature is 350 °C, at a flow-rate of 500 L/hr.

For AKP the deprotonated molecule was fragmented with 10-14 eV, resulting in specific fragments from losses of e.g. H₂0, CO and C0₂.

To determine concentrations a standard curve of synthetically prepared compounds was run to calculate a response factor for the respective ions. This was used to calculate the concentrations in unknown samples. 11.2 Synthesis of 2-hydroxyheptanedioic acid (method serving to illustrate how AKP was made to be used for testing purposes)

2-Hydroxyheptanedioic acid for use as a substrate for the biocatalytic production of AKP was synthesised by hydrogenation of AKP (provided by Syncom). AKP (2.2 g, 12.6 mmol) was dissolved in methanol (50 ml_) to this 30 mg of Pd on charcoal was added (Pd/C, 5 %) and placed in an autoclave under a hydrogen pressure of 30 bar at 50 °C for 48 hours. The reaction mixture was allowed reach room temperature and subsequently filtered over Celite® and concentrated in vacuo to yield the title compound as oil (2.2 g, 99 %).

The product was characterised by ¹ H-NMR and ¹³C-NMR

¹ H-NMR (300 MHz, DMSO): δ 4.02-3.98 and 3.92-3.89 (dd, ³J= 7.6 Hz, ³J= 4.8 Hz, 1 H), 2.28 and 2.18 (f, ³J= 7.2 Hz, 2H), 1 .66-1 .28 (m, 6 H)

1³C-NMR (75 MHz, DMSO) : δ 174.9, 173.6, 70.0, 51 .6, 34.0, 33.6, 24.6 11.3 preparation of pBAD-DEST Top10 cell with heterologous hydroxyacid oxidase

HAOX5B and LA0X8C were obtained by DNA synthesis._affS sites were added to all genes upstream of the ribosomal binding site and start codon and downstream of the stop codon to facilitate cloning using the Gateway technology (Invitrogen, Carlsbad, CA, USA). The gene constructs were cloned into pBAD/Myc-His- DEST expression vectors using the Gateway technology (Invitrogen) via the introduced attB sites and pDONR201 (Invitrogen) as entry vector as described in the

manufacturer's protocols (www.invitrogen.com). This way the expression vectors pBAD-HAOX5B and pBAD- LAOX8C were obtained, respectively. The corresponding expression strains were obtained by transformation of chemically competent E. coli TOP10 (Invitrogen) with the respective pBAD-expression vectors. 11.4 Growth of E. coli for protein expression

Small scale growth of the cells prepared in Example 11.3 was carried out in 96-deep-well plates with 940 μΙ media containing 0.02% (w/v) L-arabinose. Inoculation was performed by transferring cells from frozen stock cultures with a 96- well stamp (Kuhner, Birsfelden, Switzerland). Plates were incubated on an orbital shaker (300 rpm, 5 cm amplitude) at 25 °C for 48 h. Typically an OD₆₂₀nm of 2 - 4 was reached.

11.5 Preparation of cell ly sates

The lysis buffer contained the following ingredients:

Table 11 lysis buffer contents

1 M MOPS pH 7.5 5 ml

DNAse I grade II (Roche) 10 mg

Lysozyme 200 mg

MgS0₄.7H₂0 123.2 mg

dithiothreitol (DTT) 154.2 mg

H₂0 (MilliQ) Balance to 100 ml The solution was freshly prepared directly before use.

Cells from small scales growth (see Example 2) were harvested by centrifugation and the supernatant was discarded. The cell pellets formed during centrifugation were frozen at -20 °C for at least 16 h and then thawed on ice. 500 μΙ of freshly prepared lysis buffer were added to each well and cells were resuspended by vigorously vortexing the plate for 2-5 min. To achieve lysis, the plate was incubated at room temperature for 30 min. To remove cell debris, the plate was centrifuged at 4 ^<€ and 6000 g for 20 min. The supernatant (containing hydroxyacid oxidase, either HAOX 5B or LAOX 8C) was transferred to a fresh plate and kept on ice until further use.

II.6 enzymatic preparation of AKP

2-Hydroxyheptanedioic acid (final concentration 50m M, >95 % purity, obtained as described above) was contacted with hydroxyacid oxidase (either HAOX 5B or LAOX 8C), obtained as described above 3 in a buffer solution containing the following.

- 4-aminoantipyrine (1 mM)

- 3,5-dichloro-2-hydroxybenzenesulfonic acid (DCHBS) (10 mM)

- 50 mM potassium phosphate buffer, pH 7.5

- Horseradish peroxidase (200 U/ml)

Reactions were incubated for 20h at 37C. Samples were frozen and prior to analysis heated to 95 °C for 2min to precipitate protein. After centrifugation the supernatant was analyzed by UPLC-MS.AKP concentration in the sample from the test with HAOX 5B was 59 mg/l, and APK concentration in the sample from the test with LAOX 8C was 58 mg/l.

Example III production of 5-A VA in E. coli

III.1 Cloning of the genes

Protein sequences for the Methanococcus jannaschii proteins homoaconitase small subunit (AksE, MJ1271 , [SEQ ID No 1 17]), homoaconitase large subunit (AksD, MJ1003, [SEQ ID No 1 14]) and homoisocitrate dehydrogenase (AksF, MJ1596, [SEQ ID No 120]), homologues thereof from Methanococcus vannielii SB homoisocitrate dehydrogenase (AksF, Mevan_0040, [SEQ ID No 123), homologues thereof from Methanococcus aeolicus Nankai 3 homoaconitase small subunit (AksE, Maeo_0652 [SEQ ID No 135]), homoaconitase large subunit (AksD, Maeo_031 1 , [SEQ ID No 138], homologues thereof from Methanococcus maripaludis S2 homoaconitase small subunit (AksE, MMP0381 , [SEQ ID No 129]), homoaconitase large subunit (AksD, MMP1480, [SEQ ID No 132]), homoisocitrate dehydrogenase (AksF, MMP0880 [SEQ ID No 126), ]) the A. vinelandii homocitrate synthase NifV, [SEQ ID 141 ]), the aminotransferase protein from Vibrio fluvialis JS17 [SEQ ID No. 2] and the Lactococcus lactis branched chain alpha-keto acid decarboxylase KdcA [SEQ ID No. 40] were retrieved from databases.

All genes, except for the A. vinelandii homocitrate synthase nifV (Sequence ID No 140), were codon pair optimized for E. coli using methodology described in WO 2008/000632. (Table 13) and the constructs were made synthetically (Geneart, Regensburg, Germany). In the optimization procedure internal restriction sites were avoided and common restriction sites were introduced at the start and stop to allow subcloning in expression vectors. The codon optimised aminotransferase gene from Vibrio fluvialis JS17 (Seq ID NO: 3) was PCR amplified using Phusion DNA polymerase according to the manufacturers specifications using primer pairs AT- Vfl_for_Ec (AAATTT GGTACC GCTAGGAGGAATTAACCATG) + AT-Vfl_rev_Ec (AAATTT ACTAGT AAGCTGGGTTTACGCGACTTC). The codon optimised decarboxylase gene from Lactococcus lactis coding for Lactococcus lactis branched chain alpha-keto acid decarboxylase KdcA (Seq ID NO: 41 ) was amplified using Phusion DNA polymerase according to the manufacturers specifications and using primers Kdc_for_Ec (AAATTT ACTAGT GGCTAGGAGGAATTACATATG) and Kdc_rev_Ec (AAATTT AAGCTT ATTACTTGTTCTGCTCCGCAAAC). The

aminotransferase fragments were digested with Kpnl/Spel and the decarboxylase fragment was digested with Spel/Hindlll. Both fragments were ligated to Kpnl/Hindlll digested pBBR-lac to obtain pAKP-96.

Genes encoding the homoaconitase small subunit (AksE),, homoaconitase large subunit (AksD) and homoisocitrate dehydrogenase (AksF) from either M. jannaschii, M. vannielii, M. aeolicus or M. maripaludis were codon pair optimized for E. coli (using methodology described in WO08000632; Table 13).

Sequences of codon pair optimized genes are shown in Seq ID Nos: 115, 1 18, 121 , 124, 127, 130, 133, 136, and 139. Constructs were made synthetically (Geneart, Regensburg, Germany) containing the optimized genes together with the wild-type nifV gene (SEQ ID No 140), see also Table 12 for the genes in each of the constructs . In the optimization procedure internal restriction sites were avoided and common restriction sites were introduced at the start and stop to allow subcloning in expression vectors. Also, upstream of AksD the sequence of the tac promoter from pMS470 was added. Each ORF was preceded by a consensus ribosomal binding site and leader sequence to drive transcription and translation in E. coli. A synthetic AksA /AksF cassette was cut with Ndel/Xbal and a synthetic AksD/AksE cassette was cut with Xbal/Hindlll. Fragments containing Aks genes were inserted in the Ndel/Hindlll sites of pMS470 to obtain the various vectors. These plasmids were co-transformed with plasmid pAKP96, a vector containing the aminotransferase gene (AT) from V. fluvialis and the decarboxylase gene (DC) from Lactococcus lactis to BL21 to obtain the strains listed in Table 12. Table 12 Sequences of the various genes present in eAKP236, eAKP-489 and eAKP^■ 491

(AT= aminotransferase, DC=decarboxy ase

111.2 Protein expression and metabolite production in E. coli

Cultures eAKP236, eAKP-489 and eAKP -491 (shown in Table 12 ) were grown overnight in tubes with 10 ml 2^*TY medium. 200 μΙ culture was transferred to shake flasks with 20 ml 2xTY medium. Flasks were incubated in an orbital shaker at 30 °C and 280 rpm. After 4h IPTG was added at a final concentration of 0.1 mM and flasks were incubated for 16h at 30 °C and 120 rpm. Cells from 20 ml culture were collected by centrifugation, resuspended in 4 ml M9 medium with 1 % glycerol and incubated at 30 °C and 210 rpm in a 24 wells plate. After 24 hours the supernatant was collected by centrifugation and stored at -20C for analysis. I I 1.3 Detection of 5- AVA in culture samples

HPLC-MS analysis for the determination of 5-AVA

The calibration was performed by an external calibration line of 6- AVA (m/z 1 16, retention time 4.1 min). All the LC-MS experiments were performed on an Agilent 1200 LC-, system equipped with a quaternary pump, degasser, autosampler, column oven, and a triple-quadrupole 6410 LC-MS (Agilent, Waldbronn, Germany). The LC-MS conditions were:

Column: 50^*4 mm Prevail C18 (Alltech) + 250 ^* 4.6 mm Prevail C18 (Alltech), both at room temperature (RT)

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

B = 0.1 (v/v) formic acid in acetonitrile (Lichrosolv, Merck)

Flow: 1 .2 ml/min, before entering the MS the flow was split 1 :3

Gradient: The gradient was started at t=0 minutes with 100% (v/v) A, changed within 8 minutes to 15% (v/v) B and from 8-9 min to 50% (v/v) B. From 9 to 12 minutes the gradient was kept constant at 50% (v/v) B. Injection volume: 5 μΙ

MS detection: ESI(+)-MS

The electrospray ionization (ESI) was run in the negative ion SIM mode with the following conditions; MS2 SIM on m/z 116, dwell time 100 msec, 70 V fragmentor, 350 °C drying gas temperature, 12 L N₂/min drying gas, 50 psig nebuliser pressure and 3 kV capillary voltage.

Table 13 results

Results clearly show the presence of 5-AVA in recombinant strains while this can not be detected in the non-transformed BL-21 strain.

Example IV Activity of 5-FVA aminotransferases on shorter substrate analogues

IV.1 Preparation of 4-formylbutanoic acid

4-formulbutanoic acid (4-FBA) was prepared by chemical hydrolysis of methyl 4-formylbutanoate: (Syncom, Groningen, The Netherlands). This was done as follows. A 10% (w/v) solution of methyl 4-formylbutanoate in water was set at pH 14.1 with NaOH. After 24 h of incubation at 20 °C the pH was set to 7.1 with HCI. The thus obtained 4-FBA solution was diluted appropriately and used as such.

IV.2 Activity of 5-FVA-ATs on shorter substrate analogues

Cell free extracts (CFE) containing aminotransferases as prepared above were tested separately for their activity on 4-FBA and 3-formylpropanoic acid (3- FPA), respectively, in a spectro-photometric assay. 4-FBA was obtained as described above and 3-FPA was obtained from Sigma-Aldrich (Schnelldorf, Germany).

The activity was assayed with omethylbenzylamine (MBA) as amino donor and detection of the by-product acetophenone in a Perkin Elmer Lambda35 UV/VIS spectrophotometer thermostated at 30 ¾ at a wavelength of 300 nm. In a final reaction volume of 1 ml 50 μΙ of a suitable dilution of a CFE containing an

aminotransferase were mixed in disposable plastic UV cuvettes with 25 mM MBA and 10 mM 5-FVA, 4-FBA, or 3-FPA, respectively, in the presence of 50 mM potassium phosphate buffer pH 7.5 containing 0.1 mM pyridoxal 5'-phosphate (PLP). The reactions were started by addition of 10 μΙ of the respective formyl acid substrate to the other assay components, which had been pre-incubated in the photometer at 30 for 5 min. After addition of the respective formyl acid, the absorption at 300 nm was recorded and the aminotransferase activity in the CFE samples was calculated from triplicate measurements according to the law of Lambert-Beer with a molar extinction coefficient for acetophenone of ε = 0.28 αη²/μιηοΙ. One unit (U) of aminotransferase activity is defined as 1 μιηοΙ of acetophenone formed from 25 mM MBA and 10 mM of the respective formyl acid at 30 °C in 50 mM potassium phosphate buffer pH 7.5 containing 0.1 mM PLP per minute.

The volumetric activities of the aminotransferases Vfl / J (Sequence ID NO 2), Pae_AT (Sequence ID NO 8), Bwe_AT (Sequence ID NO 5), and

Pae_AT_g Ϊ9951072 (Sequence ID NO 67) in U per ml CFE are given in Table 14.

Table 14: Volumetric activities of CFEs containing the 5- FVA aminotransferases Vfl_A T, Pae_A T, Bwe_A T, and Pae_A T_gi9951072 with 10 mM 5-formylvaleric acid (5-

FVA), 4-formylbutanoic acid (4-FBA), or 3-formylpropanoic acid (3-FPA), respectively.

These results show that aminotransferases W/_AT (Sequence ID NO 2), Pae_AT (Sequence ID NO 8), Bwe_W (Sequence ID NO 5), and

Pae_AT_gi9951072 (Sequence ID NO 67) are also suitable 4-formylbutanoic acid (4- FBA) aminotransferases and 3-formylpropanoic acid (3-FPA) aminotransferases.

EXAMPLE V Activity of AKP-DCs on shorter substrates Cell free extracts containing AKP-decarboxylases KdcA (Sequence

ID NO 40), KivD (Sequence ID NO 43) and PDC I472A (Sequence ID NO 37) were freshly prepared as described above and separately tested in the decarboxylation of AKP and a-ketoadipic acid (AKA), respectively. In total reaction volumes of 2.5 ml 0.5ml of CFEs containing overexpressed PDC variant I472 (undiluted), KivD

(undiluted), and KdcA (1 :10 diluted), respectively, were reacted with 25 mM AKP or AKA, respectively in 100 mM potassium phosphate buffer pH 6.5 containing 1 mM thiamine diphosphate and 5 mM MgCI₂ at 37°C and 400 rpm. After 5 min reaction 0.25 ml samples were taken and stopped by addition of 0.75 ml of a 1 :1 acetonitrile/water mixture and centrifugation (20 min at 5000x g).

The samples were analysed by HPLC-MS as described in the general methods for the decarboxylation of AKP to 5-formylvaleric acid (5-FVA) and AKA to 4- formylbutanoic acid (4-FBA), respectively. It was found that some of the 5-FVA or 4- FVA, respectively, had been further converted by enzymes in the cell free extracts to adipic acid or glutaric acid, respectively. Therefore, the AKP decarboxylase and the AKA decarboxylase activity, respectively, was calculated from the sum of the concentrations of the formed 5-FVA plus adipic acid and 4-FBA plus glutaric acid. One unit of AKP decarboxylase activity is herefore defined as the sum of 5-FVA and adipic acid in μιηοΙ formed from 25 mM AKP per min at 37 °C in the presence of a 100 mM potassium phosphate buffer pH 6.5 containing 1 mM thiamine diphosphate and 5 mM MgCI₂. One unit of AKA decarboxylase activity is here fore defined as the sum of 4- FBA and glutaric acid in μιηοΙ formed from 25 mM AKA per min at 37 °C in the presence of a 100 mM potassium phosphate buffer pH 6.5 containing 1 mM thiamine diphosphate and 5 mM MgCI₂.

The results are given in Table 15.

Table 15: Activity of AKP decarboxylases PDC I472A, KdcA, and KivD on a-ketopimelic acid (AKP) and a-ketoadipic acid (AKA) at 50 mM substrate concentration.

These results show that decarboxylases KdcA (Sequence ID NO 40),

KivD (Sequence ID NO 43) and PDC I472A (Sequence ID NO 37) are also suitable a- ketoadipic acid decarboxylases.

SEQUENCES

<210> 1

<211> 1362

<212> DNA

<213> Vibrio fluvial

<220>

<221> CDS

<222> (1) .. (1362)

<400> 1

atg aac aaa ccg caa age tgg gaa gee egg gee gag ace tat teg etc 48

Met Asn Lys Pro Gin Ser Trp Glu Ala Arg Ala Glu Thr Tyr Ser Leu 1 5 10 15 tat ggt ttc acc gac atg cct teg ctg cat cag cgc ggc acg gtc gtc 96

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

20 25 30 gtg acc cat ggc gag gga ccc tat ate gtc gat gtg aat ggc egg cgt 144

Val Thr His Gly Glu Gly Pro Tyr lie Val Asp Val Asn Gly Arg Arg

35 40 45

tat ctg gac gec aac teg ggc ctg tgg aac atg gtc gcg ggc ttt gac 192

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 gec gec aag gec caa tac gag cgt ttt ccc 240

His Lys Gly Leu lie Asp Ala Ala Lys Ala Gin Tyr Glu Arg Phe Pro 65 70 75 80 ggt tat cac gec ttt ttc ggc cgc atg tec gat cag acg gta atg ctg 288

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 336

Ser Glu Lys Leu Val Glu Val Ser Pro Phe Asp Ser Gly Arg Val Phe

100 105 110 tat aca aac teg ggg tec gag gcg aat gac acc atg gtc aag atg eta 384

Tyr Thr Asn Ser Gly Ser Glu Ala Asn Asp Thr Met Val Lys Met Leu

115 120 125

tgg ttc ctg cat gca gec 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

Thr Gly Lys Pro Tyr Asn Ser Val Phe Gly Leu Pro Leu Pro Gly Phe

165 170 175 gtg cat ctg acc tgc ccg cat tac tgg cgc tat ggc gaa gag ggc gaa 576

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 acg 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

He Gin Arg Glu Gly Ala Asp Thr He Ala Gly Phe Phe Ala Glu Pro 210 215 220

gtg atg ggc gcg ggc ggc gtg att ccc ccg gcc aag ggc tat ttc cag 720

Val Met Gly Ala Gly Gly Val He Pro Pro Ala Lys Gly Tyr Phe Gin

225 230 235 240 gcg ate ctg cca ate ctg cgc aaa tat gac ate ccg gtc ate teg gac 768

Ala He Leu Pro He Leu Arg Lys Tyr Asp He Pro Val He Ser Asp

245 250 255 gag gtg ate tgc ggt ttc gga cgc acc ggt aac acc tgg ggc tgc gtg 816

Glu Val He 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 tec aag aat ctt aca 864

Thr Tyr Asp Phe Thr Pro Asp Ala He He Ser Ser Lys Asn Leu Thr

275 280 285

gcg ggc ttt ttc ccc atg ggg gcg gtg ate ctt ggc ccg gaa ctt tec 912

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

aaa egg ctg gaa acc gca ate gag gcg 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 gcg ctg aaa gca 1008

Phe Thr Ala Ser Gly His Pro Val Gly Cys Ala He Ala Leu Lys Ala

325 330 335 ate gae gtg gtg atg aat gaa ggg ctg get gag aac gtc cgc cgc ctt 1056

lie Asp Val Val Met Asn Glu Gly Leu Ala Glu Asn Val Arg Arg Leu

340 345 350 gee ccc cgt ttc gag gaa agg ctg aaa cat ate gee gag cgc ccg aac 1104

Ala Pro Arg Phe Glu Glu Arg Leu Lys His lie Ala Glu Arg Pro Asn

355 360 365

ate ggt gaa tat cgc ggc ate ggc ttc atg tgg gcg ctg gag get gtc 1152

lie Gly Glu Tyr Arg Gly lie Gly Phe Met Trp Ala Leu Glu Ala Val 370 375 380

aag gae aag gca age aag acg ccg ttc gae ggc aac ctg teg gtc age 1200

Lys Asp Lys Ala Ser Lys Thr Pro Phe Asp Gly Asn Leu Ser Val Ser 385 390 395 400 gag cgt ate gee aat ace tgc ace gat ctg ggg ctg att tgc egg ccg 1248

Glu Arg lie Ala Asn Thr Cys Thr Asp Leu Gly Leu lie Cys Arg Pro

405 410 415 ctt ggt cag tec gtc gtc ctt tgt ccg ccc ttt ate ctg acc gag gcg 1296

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 gec ctt gat aag gtc 1344

Gin Met Asp Glu Met Phe Asp Lys Leu Glu Lys Ala Leu Asp Lys Val

435 440 445

ttt gec gag gtt gec tga

1362

Phe Ala Glu Val Ala

450

<210> 2

<211> 453

<212> PRT

<213> Vibrio fluvialis

<400> 2

Met Asn Lys Pro Gin Ser Trp Glu Ala Arg Ala Glu Thr Tyr Ser Leu 1 5 10 15

Tyr Gly Phe Thr Asp Met Pro Ser Leu His 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 lie 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

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 lie 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

He 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 He Pro Pro Ala Lys Gly Tyr Phe Gin 225 230 235 240

Ala He Leu Pro He Leu Arg Lys Tyr Asp He Pro Val He Ser Asp

245 250 255

Glu Val He 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 He Glu Ala He 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

He 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 He Ala Glu Arg Pro Asn

355 360 365

He Gly Glu Tyr Arg Gly He 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 He 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 He 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> Artificial

<220>

<223> Vibrio fluvialis JS17 omega-aminotransferase codon optimised gene

<400> 3

atgaataaac cacagtcttg ggaagctcgt gctgaaacct atagcctgta cggctttacc 60 gatatgccgt ctctgcacca gcgtggtact gtagtggtaa cgcacggtga gggcccgtac 120

atcgtggacg ttaatggccg ccgttacctg gatgcaaaca gcggcctgtg gaacatggtt 180

gcgggcttcg accacaaagg cctgatcgat gccgcaaaag cgcagtacga acgcttcccg 240

ggttatcacg cgttctttgg ccgtatgagc gaccagactg tgatgctgag cgaaaaactg 300

gttgaagtgt ccccgttcga tagcggtcgt gtcttttaca ctaactctgg cagcgaggct 360

aacgatacca tggttaagat gctgtggttc ctgcacgcag cggaaggcaa acctcagaaa 420

cgtaaaattc tgacccgttg gaacgcttat cacggtgtga ctgctgtttc cgcatctatg 480

accggtaaac cgtataacag cgtgttcggt ctgccgctgc ctggcttcgt gcatctgacc 540

tgcccgcact actggcgtta tggtgaggaa ggcgaaactg aggaacagtt cgtggcgcgt 600

ctggctcgtg aactggaaga aaccattcaa cgcgaaggtg cagatactat cgcgggcttc 660

tttgcggagc ctgttatggg tgccggcggt gtgattccgc 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 catgtgggcc 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

<210> 4

<211> 1350

<212> DNA

<213> Bacillus weihenstephanensis

<220>

<221> CDS

<222> (1) .. (1350)

<400> 4

gtg caa gcg acg gag caa aca caa agt ttg aaa aaa aca gat gaa aag 48

Val Gin Ala Thr Glu Gin Thr Gin Ser Leu Lys Lys Thr Asp Glu Lys 1 5 10 15 tac ctt tgg cat gcg atg aga gga gca gcc cct agt cca acg aat tta 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 lie Thr Lys Ala Glu Gly Ala Trp Val Thr Asp lie Asp Gly Asn

35 40 45

cgt tat tta gac ggt atg tec 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 cga aaa gaa ctt gca aga gcg gcg ttt gaa cag ctt gaa gaa atg 240

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 288

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 ctt gat gat gaa tac gtc att ttc ttt tct 336

Glu Lys Leu Asn Glu Trp Leu Asp Asp Glu Tyr Val lie Phe Phe Ser

100 105 110 aac agt gga teg gaa gcg aat gaa aca gca ttt aaa att get cgt caa 384

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

tat cat caa caa aaa ggt gat cat gga cgc tat aag ttt att tec cgc 432

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

130 135 140

tac cgc get tat cac ggt aac tea atg gga get ctt gca gca aca ggt 480

Tyr Arg Ala Tyr His Gly Asn Ser Met Gly Ala Leu Ala Ala Thr Gly

145 150 155 160 caa gca cag cga aag tat aaa tat gaa cca etc ggg caa gga ttc ctg 528

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 acg tat cga aat cca gag gat gtt cat aca 576

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 624

Leu Ala Ser Ala Glu Glu lie Asp Arg Val Met Thr Trp Glu Leu Ser

195 200 205

caa aca gta gec ggt gtg att atg gag cca ate att act ggg ggc gga 672

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 720

lie Leu Met Pro Pro Asp Gly Tyr Met Gly Lys Val Lys Glu lie Cys

225 230 235 240 gag aag cac ggt gcg ttg etc att tgt gat gaa gtt ata tgt gga ttt 768

Glu Lys His Gly Ala Leu Leu lie Cys Asp Glu Val lie Cys Gly Phe

245 250 255 ggc egg aca ggg aag cca ttt gga ttt atg aat tat ggc gtc aaa cca 816

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 gcg tat ctt cct ttg 864

Asp lie lie Thr Met Ala Lys Gly lie Thr Ser Ala Tyr Leu Pro Leu

275 280 285

tea gca aca gca gtt aga cga gag gtt tat gag gca ttc gta ggt agt 912

Ser Ala Thr Ala Val Arg Arg Glu Val Tyr Glu Ala Phe Val Gly Ser 290 295 300

gat gat tat gat cgc ttc cgc cat gta aat acg ttc gga ggg aat cct 960 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 1008

Ala Ala Cys Ala Leu Ala Leu Lys Asn Leu Glu He Met Glu Asn Glu

325 330 335 aaa etc att gaa cgt tec aaa gaa ttg ggt gaa cga ctg tta tat gag 1056

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 1104

Leu Glu Asp Val Lys Glu His Pro Asn Val Gly Asp Val Arg Gly Lys

355 360 365

ggc ctt ctt tta ggc att gaa eta gtg gaa gat aag caa aca aaa gaa 1152

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

ccg get tec att gaa aag atg aac aaa gtc ate aat get tgt aaa gaa 1200

Pro Ala Ser He Glu Lys Met Asn Lys Val He 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 1248

Lys Gly Leu He He Gly Lys Asn Gly Asp Thr Val Ala Gly Tyr Asn

405 410 415 aat att ttg cag ctt gca cct cca tta age ate aca gag gaa gac ttt 1296

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 tec cgc att aac ggg 1344

Thr Phe He Val Lys Thr Met Lys Glu Cys Leu Ser Arg He Asn Gly

435 440 445

cag taa

1350

Gin

<210> 5

<211> 449

<212> PRT

<213> Bacillus weihenstephanensis

<400> 5

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 lie Thr Lys Ala Glu Gly Ala Trp Val Thr Asp lie Asp Gly Asn

35 40 45 Arg Tyr Leu Asp Gly Met Ser Gly Leu Trp Cys Val Asn Val Gly Tyr 50 55 60

Gly Arg Lys Glu Leu Ala Arg Ala Ala Phe Glu 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 lie Leu Met Pro Pro Asp Gly Tyr Met Gly Lys Val Lys Glu lie 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 He Thr Met Ala Lys Gly He Thr Ser Ala Tyr Leu Pro Leu

275 280 285

Ser Ala Thr Ala Val Arg Arg Glu Val Tyr Glu Ala Phe Val Gly Ser 290 295 300 Asp Asp Tyr Asp Arg Phe Arg His Val Asn Thr Phe Gly Gly Asn Pro 305 310 315 320

Ala Ala Cys Ala Leu Ala Leu Lys Asn Leu Glu He Met Glu Asn Glu

325 330 335

Lys Leu He 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 He Glu Lys Met Asn Lys Val He Asn Ala Cys Lys Glu 385 390 395 400

Lys Gly Leu He He Gly Lys Asn Gly Asp Thr Val Ala Gly Tyr Asn

405 410 415

Asn He Leu Gin Leu Ala Pro Pro Leu Ser He Thr Glu Glu Asp Phe

420 425 430

Thr Phe He Val Lys Thr Met Lys Glu Cys Leu Ser Arg He Asn Gly

435 440 445

Gin <210> 6

<211> 1350

<212> DNA

<213> Artificial <220>

<223> B. weihenstephanensis KBAB4 aminotransferase codon-optimised gene

<400> 6

atgcaggcta ccgaacaaac ccaatctctg aaaaagactg acgaaaaata tctgtggcac 60

gcgatgcgcg gtgcagctcc gtctccgacc aacctgatta ttaccaaagc tgaaggcgcg 120

tgggtgaccg acattgacgg taaccgttat ctggatggca tgagcggcct gtggtgtgtt 180

aatgtcggtt atggccgtaa ggagctggcg cgcgcggcat ttgaacaact ggaagaaatg 240

ccgtacttcc cgctgactca aagccatgtg ccggctatca aactggcgga aaaactgaac 300

gaatggctgg acgacgaata cgtgattttc ttctctaatt ctggctccga agcaaacgaa 360

accgcattca aaatcgcccg tcaatatcac cagcagaaag gtgaccacgg ccgctataaa 420

ttcatcagcc gttatcgtgc ataccatggt aattctatgg gtgcgctggc tgctaccggt 480

caggctcagc gcaaatacaa gtacgaaccg ctgggtcagg gttttctgca cgttgcacca 540

ccggatacct accgtaaccc ggaagacgtc cacaccctgg cttctgccga agaaatcgat 600

cgtgttatga cctgggagct gtcccagact gttgcgggtg ttatcatgga acctattatt 660

accggtggtg gcattctgat gccgccggac ggttatatgg gtaaagtcaa ggaaatctgc 720

gaaaaacacg gcgcgctgct gatctgcgat gaagttatct gtggcttcgg tcgcaccggc 780

aaaccatttg gcttcatgaa ttatggcgta aaacctgaca ttattaccat ggctaaaggc 840

attacttccg cttatctgcc gctgagcgcg accgcagttc gccgcgaagt ttatgaagcg 900

tttgttggtt ctgatgatta cgaccgtttc cgtcatgtaa acacgtttgg cggt

960

gcggcatgtg cgctggcgct gaaaaacctg gaaatcatgg aaaacgaaaa gctgatcgaa 1020

cgtagcaaag aactgggtga acgtctgctg tacgaactgg aagatgtcaa agaacacccg 1080 aacgtgggcg atgttcgcgg taaaggcctg ctgctgggta ttgaactggt tgaagacaaa 1140

cagaccaagg aaccggcttc cattgaaaag atgaacaaag tgattaacgc gtgcaaagag 1200

aaaggcctga tcattggtaa gaacggtgat accgtggcag gttataacaa cattctgcag 1260

ctggcgccgc ctctgagcat cactgaagaa gatttcacct tcatcgtcaa aactatgaag 1320

gagtgcctga gccgcatcaa tggt

1350

<210> 7

<211> 1371

<212> DNA

<213> Pseudomonas aeruginosa

<220>

<221> CDS

<222> (1) . (1371)

<400> 7

atg aac age caa ate acc aac gcc aag acc cgt gag tgg cag gcg ttg 48

Met Asn Ser Gin lie Thr Asn Ala Lys Thr Arg Glu Trp Gin Ala Leu 1 5 10 15 age cgc gac cac cat ctg ccg ccg ttc acc gac tac aag cag ttg aac 96

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 gcc gaa ggc gtc tat ate tgg 144

Glu Lys Gly Ala Arg lie lie Thr Lys Ala Glu Gly Val Tyr lie Trp

35 40 45

gac age gag ggc aac aag ate etc gat gcg atg gcc 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 cgc gag gag ctg gtc cag gcc gcc 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 cgc gag ttg ccg ttc tac aac ctg ttc ttc cag acc gcc 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 gcc aag gcg ate gcc gac gtc get ccg gaa 336 Pro Pro Val Val Glu Leu Ala Lys Ala lie Ala Asp Val Ala Pro Glu 100 105 110 ggc atg aac cac gtg ttc ttc acc ggc tec ggc tec gag gcc 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 cgc 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 gtg ate ggc cgc tgg aac ggc tac cac ggc tec acc 480

Gin Lys Lys Val Val lie Gly Arg Trp Asn Gly Tyr His Gly Ser Thr

145 150 155 160 gtc gcc 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 gcc cag ccc tac tgg tac 576

Asp Phe Pro lie Pro Gly lie Val His lie Ala Gin Pro Tyr Trp Tyr

180 185 190 ggc gag ggc ggc gac atg teg ccg gac gag ttc ggc gtc tgg gcc gcc 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 gcc 672

Glu Gin Leu Glu Lys Lys lie Leu Glu Val Gly Glu Glu Asn Val Ala 210 215 220

gcc ttc ate gcc gag ccg ate cag ggc gcc 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 cgc gag ate etc gcc aag tac gac 768

Pro Asp Thr Tyr Trp Pro Lys lie Arg Glu lie Leu Ala Lys Tyr Asp

245 250 255 ate ctg ttc ate gcc gac gaa gtg ate tgc ggc ttc ggc cgt acc ggc 816

lie Leu Phe lie Ala Asp Glu Val lie Cys Gly Phe Gly Arg Thr Gly

260 265 270 gag tgg ttc ggc age cag tac tac ggc aac gcc ccg gac ctg atg ccg 864

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 tec ggc tac ate ccc atg ggc ggg gtg gtg 912

He Ala Lys Gly Leu Thr Ser Gly Tyr He Pro Met Gly Gly Val Val 290 295 300

gtg cgc gac gag ate gtc gaa gtg etc aac cag ggc ggc gag ttc tac 960

Val Arg Asp Glu He Val Glu Val Leu Asn Gin Gly Gly Glu Phe Tyr

305 310 315 320 cac ggc ttc acc tat tec ggt cac ccg gtg gcg gec gec gtg gec ctg 1008

His Gly Phe Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu

325 330 335 gag aac ate cgc ate ctg cgc gaa gag aag ate ate gag aag gtg aag 1056

Glu Asn He Arg He Leu Arg Glu Glu Lys He He Glu Lys Val Lys

340 345 350 gcg gaa acg gca ccg tat ttg cag aaa cgc tgg cag gag ctg gec gac 1104

Ala Glu Thr Ala Pro Tyr Leu Gin Lys Arg Trp Gin Glu Leu Ala Asp

355 360 365

cac ccg ttg gtg ggc gaa gcg cgc ggg gtc ggc atg gtc gec gec ctg 1152

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 cgc gag cgt ttc acc gac aag ggc 1200

Glu Leu Val Lys Asn Lys Lys Thr Arg Glu Arg Phe Thr Asp Lys Gly

385 390 395 400 gtc ggg atg ctg tgc egg gaa cat tgt ttc cgc aac ggt ttg ate atg 1248

Val Gly Met Leu Cys Arg Glu His Cys Phe Arg Asn Gly Leu He Met

405 410 415 cgc gcg gtg ggc gac act atg att ate teg ccg ccg ctg gtg ate gat 1296

Arg Ala Val Gly Asp Thr Met He He Ser Pro Pro Leu Val He Asp

420 425 430 ccg teg cag ate gat gag ttg ate acc ctg gcg cgc aag tgc etc gat 1344

Pro Ser Gin He Asp Glu Leu He Thr Leu Ala Arg Lys Cys Leu Asp

435 440 445

cag acc gec gec gee gtc ctg get tga

1371

Gin Thr Ala Ala Ala Val Leu Ala

450 455

<210>

<211>

<212> <213> Pseudomonas aeruginosa

<400> 8

Met Asn Ser Gin He Thr Asn Ala Lys Thr Arg Glu Trp Gin Ala Leu 1 5 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 He He Thr Lys Ala Glu Gly Val Tyr He 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 He 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 He Pro Gly He Val His He 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 He Ala Glu Pro He Gin Gly Ala Gly Gly Val He Val Pro 225 230 235 240

Pro Asp Thr Tyr Trp Pro Lys He Arg Glu He Leu Ala Lys Tyr Asp

245 250 255

He 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

He Ala Lys Gly Leu Thr Ser Gly Tyr He 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 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 He Leu Arg Glu Glu Lys He He Glu Lys Val Lys

340 345 350

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 He He Ser Pro Pro Leu Val He Asp

420 425 430

Pro Ser Gin He 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

<211> 70

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 9

ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgaaca gccaaatcac 60

caacgccaag

70

<210> 10

<211> 49

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 10

ggggaeeaet ttgtaeaaga aagetgggtt caagecagga eggeggegg

49

<210> 11

<211> 1365

<212> DNA

<213> Pseudomonas syringae

<220>

<221> CDS

<222> (1) .. (1365)

<400> 11

atg agt gec aac aac ccg caa acc etc gaa tgg cag gee etg age age 48

Met Ser Ala Asn Asn Pro Gin Thr Leu Glu Trp Gin Ala Leu Ser Ser 1 5 10 15 gag cat cac etg gca ccg ttc age gac tac aaa caa etg 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 cgc ate ate acc cgt gec gag ggc gtt tat etg tgg gac age 144

Gly Pro Arg He He Thr Arg Ala Glu Gly Val Tyr Leu Trp Asp Ser

35 40 45

gag ggc aac aag ate etc gat ggc atg tec ggc etg tgg tgc gtg gec 192 Glu Gly Asn Lys He Leu Asp Gly Met Ser Gly Leu Trp Cys Val Ala 50 55 60

ate ggt tat ggc cgc gaa gaa ctg gcc gac gca gcc age aaa cag atg 240

He Gly Tyr Gly Arg Glu Glu Leu Ala Asp Ala Ala Ser Lys Gin Met 65 70 75 80 cgc 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 gtg ctg gaa ctg gcc aag gcc ate tec gac ate get ccc gag ggc atg 336

Val Leu Glu Leu Ala Lys Ala He Ser Asp He Ala Pro Glu Gly Met

100 105 110 aac cat gtg ttc ttc acc ggt tea ggc tct gaa ggc aat gac acg atg 384

Asn His Val Phe Phe Thr Gly Ser Gly Ser Glu Gly Asn Asp Thr Met

115 120 125

ctg cgc atg gtt cgt cat tac tgg gcg ctg aaa ggc cag ccg aac aag 432

Leu Arg Met Val Arg His Tyr Trp Ala Leu Lys Gly Gin Pro Asn Lys 130 135 140

aaa acc ate ate age cgc gtc aat ggc tac cac ggc tec acc gtc gcc 480

Lys Thr He He 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 528

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 576

Pro He Pro Gly Val Val His He Pro Gin Pro Tyr Trp Phe Gly Glu

180 185 190 ggc ggc gac atg acg ccg gac gag ttc ggc ate tgg gcg gcc gag caa 624

Gly Gly Asp Met Thr Pro Asp Glu Phe Gly He Trp Ala Ala Glu Gin

195 200 205

ctg gaa aag aaa att etc gag ctg ggc gtc gag aac gtc ggt gcg ttc 672

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 gcg ggc ggt gtg att gtc ccg cct gat 720

He Ala Glu Pro He Gin Gly Ala Gly Gly Val He Val Pro Pro Asp

225 230 235 240 tec tac tgg ccg aag ate aag gaa ate ctt tec cgc tac gac ate etg 768

Ser Tyr Trp Pro Lys He Lys Glu He Leu Ser Arg Tyr Asp He Leu

245 250 255 ttc gee gee gat gag gtg att tgt gge ttc ggg cgt ace agt gag tgg 816

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 gge etc agg ccg gac atg atg acc ate gee 864

Phe Gly Ser Asp Phe Tyr Gly Leu Arg Pro Asp Met Met Thr He Ala

275 280 285

aaa gge etg acc tec ggt tac gta ccg atg gge gge etg ate gtg cgc 912

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

gat gaa ate gtt gcg gtg etc aat gag ggt gge gat ttc aat cac gge 960

Asp Glu He Val Ala Val Leu Asn Glu Gly Gly Asp Phe Asn His Gly

305 310 315 320 ttt acc tac tec ggg cac ccg gtg gcg gec gcg gtt gcg etg gag aac 1008

Phe Thr Tyr Ser Gly His Pro Val Ala Ala Ala Val Ala Leu Glu Asn

325 330 335 ate cgt ate etg cgc gaa gaa aag ate gte gaa egg gte agg teg gaa 1056

He Arg He Leu Arg Glu Glu Lys He Val Glu Arg Val Arg Ser Glu

340 345 350 acg 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

etg gtg gge gaa gte egg ggt gte ggg etg etc ggg gee att gag etg 1152

Leu Val Gly Glu Val Arg Gly Val Gly Leu Leu Gly Ala He Glu Leu

370 375 380

gtg aag gac aag acc acc cgc gag cgc tat acc gac aag gge gcg 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 cga acc ttc tgc ttc gac aat gge etg ate atg egg get 1248

Met He Cys Arg Thr Phe Cys Phe Asp Asn Gly Leu He Met Arg Ala

405 410 415 gtg gge gat acc atg ate att gcg ccg cca etg gtg ate agt ttt gcg 1296

Val Gly Asp Thr Met He He Ala Pro Pro Leu Val He Ser Phe Ala

420 425 430 caa ate gat gag ctg gta gag aag gcg cgc acg tgt ctg gat ctg acg 1344

Gin He Asp Glu Leu Val Glu Lys Ala Arg Thr Cys Leu Asp Leu Thr

435 440 445

ctg gcg gtg ttg cag ggc tga

1365

Leu Ala Val Leu Gin Gly

450

<210> 12

<211> 454

<212> PRT

<213> Pseudomonas syringae

<400> 12

Met Ser Ala Asn Asn Pro Gin Thr Leu Glu Trp Gin Ala Leu Ser 1 5 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 He 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

He Gly Tyr Gly Arg Glu Glu Leu Ala Asp Ala Ala Ser Lys Gin Met 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 He Ser Asp He 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 He 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

He Ala Glu Pro He Gin Gly Ala Gly Gly Val He Val Pro Pro Asp 225 230 235 240

Ser Tyr Trp Pro Lys He Lys Glu He Leu Ser Arg Tyr Asp He Leu

245 250 255

Phe Ala Ala Asp Glu Val He 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 He Ala

275 280 285

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

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 He Leu Arg Glu Glu Lys He 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 He 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 lie lie 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

450

<210> 13

<211> 1365

<212> DNA

<213> Artificial

<220>

<223> Pseudomonas syringae codon optimised aminotransferase 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

gctaaagcta tcagcgatat cgcaccggag ggcatgaatc acgtcttctt cactggttcc 360

ggtagcgaag gcaacgacac gatgctgcgc atggtacgtc actattgggc gctgaagggc 420

cagccgaaca agaaaacgat tatcagccgt gtaaacggtt atcacggcag caccgttgcg 480

ggtgcgagcc tgggcggtat gacctacatg cacgaacagg gtgacctgcc gatcccgggt 540

gtagtgcaca ttccgcagcc gtattggttc ggtgaaggcg gtgacatgac gccggacgaa 600

ttcggcatct gggcggcaga gcagctggaa aagaaaatcc tggaactggg cgtggaaaac 660 gtcggcgcgt tcatcgcgga accgattcag ggcgcgggcg gcgtaattgt tccgccggac 720

agctactggc caaaaatcaa agagatcctg tctcgttacg acatcctgtt cgccgcagac 780

gaagtgatct gcggttttgg ccgcacctct gaatggttcg gctccgactt ctacggtctg 840

cgtccggaca tgatgaccat cgccaaaggc ctgacctccg gttatgttcc tatgggtggc 900

ctgatcgtgc gcgacgaaat tgttgcggtt ctgaacgaag gcggcgattt caaccacggc 960

ttcacctatt ccggtcaccc agttgctgct gctgtagcac tggaaaacat ccgcatcctg 1020

cgtgaagaaa agatcgtaga acgcgtacgt tccgaaaccg caccttacct gcagaagcgc 1080

ctgcgcgaac tgagcgacca ccctctggta ggtgaagttc gcggcgtggg cctgctgggc 1140

gcgatcgagc tggtgaaaga caaaactacc cgtgaacgtt acaccgacaa aggcgcaggc 1200

atgatctgcc gtaccttttg cttcgataac ggtctgatca tgcgcgcagt cggtgatacc 1260

atgatcattg ctccgcctct ggttatttct tttgcccaga ttgatgagct ggtcgaaaaa 1320

gcgcgcactt gtctggatct gactctggct gttctgcagg gtt

1365

<210> 14

<211> 849

<212> DNA

<213> Bacillus subtilis

<220>

<221> CDS

<222> (1) .. (849)

<400> 14

atg aag gtt tta gtc aat ggc egg ctg att ggg cgc agt gaa gca tea 48

Met Lys Val Leu Val Asn Gly Arg Leu lie Gly Arg Ser Glu Ala Ser 1 5 10 15 ate gat ttg gaa gat cgc ggt tat cag ttt ggt gac ggc ate tat gaa 96

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 144

Val lie Arg Val Tyr Lys Gly Val Leu Phe Gly Leu Arg Glu His Ala

35 40 45

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 ctt 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 gcg gtc agt gag gga gcg gta tac att cag aca aca aga ggt gtg gec 288

Ala Val Ser Glu Gly Ala Val Tyr lie Gin Thr Thr Arg Gly Val Ala

85 90 95 ccg cga aaa cac cag tat gaa gec ggc etc gag ccg cag act act gec 336

Pro Arg Lys His Gin Tyr Glu Ala Gly Leu Glu Pro Gin Thr Thr Ala

100 105 110 tat acg ttt acg 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

gcg gec att aca gat gag gat ctt cgc 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 acg aag caa agg gec 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 gec att tta ctt agg gac ggc gtt gtt acg gag 528

Ala Gly Ala Phe Glu Ala lie Leu Leu Arg Asp Gly Val Val Thr Glu

165 170 175 ggt aca tec tct aac gtt tat gec gtt ate aac ggc aca gtg cga aca 576

Gly Thr Ser Ser Asn Val Tyr Ala Val lie Asn Gly Thr Val Arg Thr

180 185 190 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 lie Glu Lys Asn Gly lie Lys Leu Asp Glu Thr Pro Val 210 215 220 agt gaa gaa gag ttg aaa cag gcg gaa gag ate ttt att teg tea acg 720

Ser Glu Glu Glu Leu Lys Gin Ala Glu Glu He Phe He Ser Ser Thr

225 230 235 240 acg gca gaa att att ccg gtc gtg acg etc gat gga caa teg ate gga 768

Thr Ala Glu He He Pro Val Val Thr Leu Asp Gly Gin Ser He Gly

245 250 255 age ggg aaa ccc gga ccg gtg acc aaa cag ctt cag get get ttt caa 816

Ser Gly Lys Pro Gly Pro Val Thr Lys Gin Leu Gin Ala Ala Phe Gin

260 265 2 70 gaa age att caa cag get get age att tea taa

849

Glu Ser He Gin Gin Ala Ala Ser He Ser

2 75 280

<210> 15

<21 1> 282

<212> PRT

<213> Baci llus subti li s

<400> 15

Met Lys Val Leu Val Asn Gly Arg Leu He Gly Arg Ser Glu Ala Ser 1 5 10 15

He Asp Leu Glu Asp Arg Gly Tyr Gin Phe Gly Asp Gly He Tyr Glu

20 25 30

Val He Arg Val Tyr Lys Gly Val Leu Phe Gly Leu Arg Glu His Ala

35 40 45

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

Ser He 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 He 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 1 10

Tyr Thr Phe Thr Val Lys Lys Pro Glu Gin Glu Gin Ala Tyr Gly Val

1 15 120 125 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 lie Asn Gly Thr Val Arg Thr

180 185 190

His Pro Ala Asn Arg Leu lie Leu Asn Gly lie Thr Arg Met Asn lie

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 lie Ser

275 280

<210> 16

<211> 1347

<212> DNA

<213> Bacillus subtilis

<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 1 5 10 15 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 lie 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 ctt aat gtc cac gga cac cgc 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 gec ata aaa aaa cag etc gga aaa att gcg cac tec acg 240

Leu Asp Asp Ala lie 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 ctt gec 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 acg egg gtc ttt tat tea gac age 336

lie Asp lie Ser Pro Lys Lys Leu Thr Arg Val Phe Tyr Ser Asp Ser

100 105 110 ggc gca gag gcg atg gaa ata gec eta aaa atg gcg ttt cag tat tgg 384

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 432

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

ggg tat cac ggt gat acg att ggc gec gtc agt gtc ggt tea att gag 480

Gly Tyr His Gly Asp Thr lie Gly Ala Val Ser Val Gly Ser lie Glu

145 150 155 160 ctt ttt cac cac gta tac ggc ccg ttg atg ttc gag agt tac aag gec 528

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 tct gaa age ggt gat cct gat gag tgc 576

Pro lie Pro Tyr Val Tyr Arg Ser Glu Ser Gly Asp Pro Asp Glu Cys

180 185 190 cgt gat cag tgc etc cga gag ctt gca cag ctg ctt gag gaa cat cat 624

Arg Asp Gin Cys Leu Arg Glu Leu Ala Gin Leu Leu Glu Glu His His

195 200 205 gag gaa att gcc gcg ctt tec att gaa tea atg gta caa ggc gcg tec 672

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

ggt atg ate gtg atg ccg gaa gga tat ttg gca ggc gtg cgc gag eta 720

Gly Met He Val Met Pro Glu Gly Tyr Leu Ala Gly Val Arg Glu Leu

225 230 235 240 tgt aca aca tac gat gte tta atg ate gtt gat gaa gte get aca ggc 768

Cys Thr Thr Tyr Asp Val Leu Met He Val Asp Glu Val Ala Thr Gly

245 250 255 ttt ggc cgt aca gga aaa atg ttt gcg tgc gag cac gag aat gte cag 816

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 864

Pro Asp Leu Met Ala Ala Gly Lys Gly He Thr Gly Gly Tyr Leu Pro

275 280 285

att gcc gtt acg ttt gcc act gaa gac ate tat aag gca ttc tat gat 912

He Ala Val Thr Phe Ala Thr Glu Asp He Tyr Lys Ala Phe Tyr Asp

290 295 300

gat tat gaa aac eta aaa ace ttt ttc cat ggc cat tec tat aca ggc 960

Asp Tyr Glu Asn Leu Lys Thr Phe Phe His Gly His Ser Tyr Thr Gly

305 310 315 320 aat cag ctt ggc tgt gcg gtt gcg ctt gaa aat ctg gca tta ttt gaa 1008

Asn Gin Leu Gly Cys Ala Val Ala Leu Glu Asn Leu Ala Leu Phe Glu

325 330 335 tct gaa aac att gtg gaa caa gta gcg gaa aaa agt aaa aag etc cat 1056

Ser Glu Asn He Val Glu Gin Val Ala Glu Lys Ser Lys Lys Leu His

340 345 350 ttt ctt ctt caa gat ctg cac get ctt 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 ctt ggc ttt atg tgc ggt gca gag ctt gta cga 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 tec tta 1200

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

Lys Met Arg Glu Leu Gly Met Leu Thr Arg Pro Leu Gly Asp Val lie

405 410 415 gca ttt ctt cct cct ctt gcc 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 gcc att atg aaa caa gcg ate cac gag gtt acg age ctt 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

<400> 17

Met Thr His Asp Leu lie Glu Lys Ser Lys Lys His Leu Trp Leu Pro 1 5 10 15

Phe Thr Gin Met Lys Asp Tyr Asp Glu Asn Pro Leu lie lie Glu Ser

20 25 30

Gly Thr Gly lie 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

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 lie Gly Ala Val Ser Val Gly Ser lie Glu 145 150 155 160

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

165 170 175

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

Gly Met lie 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

Asn Gin Leu Gly Cys Ala Val Ala Leu Glu Asn Leu Ala Leu Phe Glu

325 330 335

Ser Glu Asn He 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 He 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 lie 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 lie Met Lys Gin Ala lie His Glu Val Thr Ser Leu Glu Asp

435 440 445

<210> 18

<211> 1467

<212> DNA

<213> Rhodobacter sphaeroides

<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 1 5 10 15 gag ggg agg teg gcg atg acg egg aat gac gcg acg aat get gcc gga 96

Glu Gly Arg Ser Ala Met Thr Arg Asn Asp Ala Thr Asn Ala Ala Gly

20 25 30 gcg gtg ggc gcg gcg 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 gcg aag etc ggc aag tec gcg 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

ggc ate tat gtc cat acc gag gac ggc cgc cgc ctg ate gac ggg ccg 240

Gly lie Tyr Val His Thr Glu Asp Gly Arg Arg Leu lie Asp Gly Pro 65 70 75 80 gcg ggc atg tgg tgc gcg cag gtg ggc tac ggc cgc cgc gag ate gtc 288

Ala Gly Met Trp Cys Ala Gin Val Gly Tyr Gly Arg Arg Glu He Val

85 90 95 gat gcc atg gcg cat cag gcg atg gtg ctg ccc tat gcc teg ccc tgg 336

Asp Ala Met Ala His Gin Ala Met Val Leu Pro Tyr Ala Ser Pro Trp

100 105 110 tat atg gcc acg age ccc gcg gcg egg ctg gcg gag aag ate gcc acg 384

Tyr Met Ala Thr Ser Pro Ala Ala Arg Leu Ala Glu Lys He Ala Thr

115 120 125

ctg acg ccg ggc gat etc aac egg ate ttt ttc acc acg ggc ggg teg 432

Leu Thr Pro Gly Asp Leu Asn Arg He Phe Phe Thr Thr Gly Gly Ser 130 135 140

acc gcg gtg gac age gcg ctg cgc ttc teg gaa ttc tac aac aac gtg 480

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 cgc ate ate gtg cgc tac gac ggc tat 528

Leu Gly Arg Pro Gin Lys Lys Arg He He Val Arg Tyr Asp Gly Tyr

165 170 175 cac ggc teg acg gcg etc acc gcc gcc tgc acc ggc cgc acc ggc aac 576

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 gcg cag gac egg ate teg ttc etc teg age 624

Trp Pro Asn Phe Asp He Ala Gin Asp Arg He Ser Phe Leu Ser Ser

195 200 205

ccc aat ccg cgc cac gcc ggc aac cgc age cag gag gcg ttc etc gac

672

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 720

Asp Leu Val Gin Glu Phe Glu Asp Arg He Glu Ser Leu Gly Pro Asp

225 230 235 240 acg ate gcg gcc ttc ctg gcc gag ccg ate etc gcc teg ggc ggc gtc 768

Thr He Ala Ala Phe Leu Ala Glu Pro He Leu Ala Ser Gly Gly Val

245 250 255 att att ccg ccc gca ggc tat cat gcg cgc ttc aag gcg ate tgc gag 816

He 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 gae gag gtg gtg acg ggc ttc ggc 864

Lys His Asp He Leu Tyr He Ser Asp Glu Val Val Thr Gly Phe Gly

275 280 285

cgt tgc ggc gag tgg ttc gcc teg gag aag gtg ttc ggg gtg gtg ccg 912

Arg Cys Gly Glu Trp Phe Ala Ser Glu Lys Val Phe Gly Val Val Pro 290 295 300

gac ate ate ace ttc gcc aag ggc gtg acc teg ggc tat gtg ccg etc 960

Asp He He Thr Phe Ala Lys Gly Val Thr Ser Gly Tyr Val Pro Leu

305 310 315 320 ggc ggc ctt gcg ate tec gag gcg gtg ctg gcg egg ate teg ggc gag 1008

Gly Gly Leu Ala He Ser Glu Ala Val Leu Ala Arg He Ser Gly Glu

325 330 335 aat gcc 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 gcc tgc gcc gcg gcg ctt gcc aac ate gag ctg atg gag cgc 1104

Pro Val Ala Cys Ala Ala Ala Leu Ala Asn He Glu Leu Met Glu Arg

355 360 365

gag ggc ate gtc gat cag gcg cgc gag atg gcg gac tat ttc gcc gcg 1152

Glu Gly He Val Asp Gin Ala Arg Glu Met Ala Asp Tyr Phe Ala Ala 370 375 380

gcg ctg get teg ctg cgc gat ctg ccg ggc gtg gcg 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 gcg 1248

Val Gly Leu Val Gly Cys Val Gin Cys Leu Leu Asp Pro Thr Arg Ala

405 410 415 gac ggc acg gcc gag gac aag gcc ttc acc ctg aag ate gac gag cgc 1296

Asp Gly Thr Ala Glu Asp Lys Ala Phe Thr Leu Lys He Asp Glu Arg

420 425 430 tgc ttc gag etc ggg ctg ate gtg cgc ccg ctg ggc gat etc tgc gtg 1344

Cys Phe Glu Leu Gly Leu He Val Arg Pro Leu Gly Asp Leu Cys Val

435 440 445

ate teg ccg ccg etc ate ate teg cgc gcg cag ate gac gag atg gtc 1392

He Ser Pro Pro Leu He He Ser Arg Ala Gin He Asp Glu Met Val 450 455 460

gcg ate atg egg cag gec ate acc gaa gtg age gec gec cac ggt ctg 1440

Ala He Met Arg Gin Ala He Thr Glu Val Ser Ala Ala His Gly Leu

465 470 475 480 acc gcg aaa gaa ccg gec gec 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

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 He 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 He Asp Gly Pro 65 70 75 80

Ala Gly Met Trp Cys Ala Gin Val Gly Tyr Gly Arg Arg Glu He 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 He 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 He 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 He Ala Ala Phe Leu Ala Glu Pro He Leu Ala Ser Gly Gly Val

245 250 255

He He Pro Pro Ala Gly Tyr His Ala Arg Phe Lys Ala He Cys Glu

260 265 270 Lys His Asp He Leu Tyr He 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 He Thr Phe Ala Lys Gly Val Thr Ser Gly Tyr Val Pro Leu 305 310 315 320

Gly Gly Leu Ala He Ser Glu Ala Val Leu Ala Arg He 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 He 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 4 10 4 15

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 He Val Arg Pro Leu Gly Asp Leu Cys Val

435 440 445

He Ser Pro Pro Leu He He Ser Arg Ala Gin He Asp Glu Met Val 450 455 460

Ala He Met Arg Gin Ala He Thr Glu Val Ser Ala Ala His Gly Leu 465 4 70 4 75 480

Thr Ala Lys Glu Pro Ala Ala Val

485

<210> 20

<21 1> 83 7

<212> DNA

<213> Legionel la pneumophi

<220>

<22 1> CDS

<222> ( 1 ) . . ( 83 7 )

<400> 20

atg agt ate gca ttt gtt aac ggc aag tat tgt tgt caa tct gaa gca 48

Met Ser He Ala Phe Val Asn Gly Lys Tyr Cys Cys Gin Ser Glu Ala 1 5 10 15 aaa att tea ata ttt gat cga ggg ttt ctt ttt ggt gac teg gtt tat 96

Lys He Ser He Phe Asp Arg Gly Phe Leu Phe Gly Asp Ser Val Tyr

20 25 30 gaa gtg ctg cct gtt tac cat ggg cag cct tac ttt gta gac caa cat 144

Glu Val Leu Pro Val Tyr His Gly Gin Pro Tyr Phe Val Asp Gin His

35 40 45

ctt gac cga tta ttc tea aat atg aaa aaa att aag atg att ata cca 192

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

aat tat gat tgg cat ggt tta att cat aga eta ata tea gaa aat aat 240 Asn Tyr Asp Trp His Gly Leu He His Arg Leu He Ser Glu Asn Asn 65 70 75 80 ggc ggt aat tta caa gta tat ate caa gtc aca cga ggg aat caa ggg 288

Gly Gly Asn Leu Gin Val Tyr He Gin Val Thr Arg Gly Asn Gin Gly

85 90 95 gtg cgc aag cat gat ate cct act tec ate aca cct tct gtt ate gca 336

Val Arg Lys His Asp He Pro Thr Ser He Thr Pro Ser Val He Ala

100 105 110 ttc act atg cat aat cca ttt ccc acc etc gaa gat aag gaa cag gga 384

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 432

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

aaa act act tct tta att gec aat ata tta ctg aat gat gag get gta 480

Lys Thr Thr Ser Leu He Ala Asn He Leu Leu Asn Asp Glu Ala Val

145 150 155 160 tct gca gga ttc cac act gca att ctt gec egg aac ggt eta att aca 528

Ser Ala Gly Phe His Thr Ala He Leu Ala Arg Asn Gly Leu He Thr

165 170 175 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 He Val Ala Gin Asp Gly Val He

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

gaa att age att tea gag ctt ttt tct get cag gaa gtt tgg ata aca 720

Glu He Ser He Ser Glu Leu Phe Ser Ala Gin Glu Val Trp He Thr

225 230 235 240 agt acg aca aaa gaa gta ttc cct att aca aag att aat gac tct 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 tec 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> Legionella pneumophi

<400> 21

Met Ser lie Ala Phe Val Asn Gly Lys Tyr Cys Cys Gin Ser Glu Ala 1 5 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 lie 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

Val Arg Lys His Asp lie Pro Thr Ser lie Thr Pro Ser Val lie Ala

100 105 110

Phe Thr Met His Asn 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 lie 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 lie Val Ala Gin Asp Gly Val lie

180 185 190

Lys Thr Pro Pro Met Asn Asn Phe Cys Leu Pro Gly lie Thr Arg Gin

195 200 205

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

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

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> Nitrosomonas europaea

<220>

<221> CDS

<222> (1) .. (861)

<400> 22

atg att tac etc aat ggc aaa ttt ctg ccg atg gaa cag get ace gtt 48

Met lie Tyr Leu Asn Gly Lys Phe Leu Pro Met Glu Gin Ala Thr Val 1 5 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 ctt tec 144

He 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 He Arg Leu Gin Asn Pro His Thr 50 55 60

gaa gaa caa tgg get ggt ctg ate gaa cgc ate ate gag ctg aat gaa 240

Glu Glu Gin Trp Ala Gly Leu He Glu Arg He He Glu Leu Asn Glu 65 70 75 80 ggt gat gat cag tac ctt tac ctg cac att aca cgc 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 gec ttt cct cgc gaa gta acg ccc act gtc ttc ate atg 336

Arg Asp His Ala Phe Pro Arg Glu Val Thr Pro Thr Val Phe He Met

100 105 110 age aac ccg ctt ccg get cca cct gca aaa ttg etc gtt tec gga gtt 384

Ser Asn Pro Leu Pro Ala Pro Pro Ala Lys Leu Leu Val Ser Gly Val

115 120 125

tea gcg att acc gec agg gat aat cgc tgg ggg cgc tgt gat ate aaa 432

Ser Ala He 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 cgc cag ctt gec gtg gac 480

Ala He Ser Leu Leu Pro Asn He Leu Leu Arg Gin Leu Ala Val Asp

145 150 155 160 gca caa gec atg gaa acg ate ctg tta cgc 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 gec gec age aat att ttc ate gta aaa gac gac ctg ctg ctg acc 576

Gly Ala Ala Ser Asn He Phe He 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 He Leu Pro Gly He Thr Tyr Asp Val Val

195 200 205

ctg gaa ctg get gaa aca cat ggt gtt cca cat gcg aca aga gaa ata 672

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

tea gag ctt gag tta cgt act gca egg gaa ate atg ctg act tct tec 720

Ser Glu Leu Glu Leu Arg Thr Ala Arg Glu He Met Leu Thr Ser Ser

225 230 235 240 acc aaa gaa att etc ccg ate aca cag ctg gat gga caa ccg ate ggt 768

Thr Lys Glu He Leu Pro He 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 816

Asn Gly Thr Pro Gly Pro Val Phe Gin Gin Leu Asp Arg Leu Tyr Gin

260 265 2 70 gca tat aag ctg gaa gtc atg cgc ggg cat get cca cgc cag taa 861

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

2 75 280 285

<210> 23

<21 1> 286

<212> PRT

<213> Nitros oraonas europaea

<400> 23

Met He Tyr Leu Asn Gly Lys Phe Leu Pro Met Glu Gin Ala Thr Val 1 5 10 15

Pro Val Leu Asp Arg Gly Phe He Phe Gly Asp Gly Val Tyr Glu Val

20 25 30

He 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 He Arg Leu Gin Asn Pro His Thr 50 55 60

Glu Glu Gin Trp Ala Gly Leu He Glu Arg He He 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 He Met

100 105 1 10

Ser Asn Pro Leu Pro Ala Pro Pro Ala Lys Leu Leu Val Ser Gly Val

1 15 120 125

Ser Ala He Thr Ala Arg Asp Asn Arg Trp Gly Arg Cys Asp He Lys 130 135 140 Ala He Ser Leu Leu Pro Asn He Leu Leu Arg Gin Leu Ala Val Asp 145 150 155 160

Ala Gin Ala Met Glu Thr He Leu Leu Arg Asp Gly Leu Leu Thr Glu

165 1 70 1 75

Gly Ala Ala Ser Asn He Phe He Val Lys Asp Asp Leu Leu Leu Thr

180 185 190

Pro Pro Lys Asp His Arg He Leu Pro Gly He Thr Tyr Asp Val Val

195 200 205

Leu Glu Leu Ala Glu Thr His Gly Val Pro His Ala Thr Arg Glu He 2 10 2 15 220

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

Thr Lys Glu He Leu Pro He 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 2 70

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

2 75 280 285

<210> 24

<21 1> 1293

<212> DNA

<213> Ne is seria gonorrhoeae

<220>

<22 1> 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 He Asn Met Asn Arg Asn Glu He Leu Phe Asp Arg Ala Lys 1 5 10 15 gcc ate ate ccc ggc ggc gtg aat teg ccc gtg cgc gca ttc ggc age 96

Ala He He 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 gcg tat gtt 144 Val Gly Gly Val Pro Arg Phe He Lys Lys Ala Glu Gly Ala Tyr Val 35 40 45

tgg gac gaa aac ggc aeg cgc tac acc gat tat gtc ggc tct tgg ggg 192

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 gec gtg cgc 240

Pro Ala He 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 288

Glu Ala Ala Leu Gly Gly Leu Ser Phe Gly Ala Pro Thr Glu Gly Glu

85 90 95 ate gec att gec gaa caa att gec gaa att atg ccg tct gtc gaa egg 336

He Ala He Ala Glu Gin He Ala Glu He Met Pro Ser Val Glu Arg

100 105 110 ctg cgc etc gtc age tec ggc aeg gaa gcg aeg atg act gec ate egt 384

Leu Arg Leu Val Ser Ser Gly Thr Glu Ala Thr Met Thr Ala He Arg

115 120 125

ctg gca cgc ggt ttt acc ggc cgc gac aaa ate ate aaa ttt gaa ggc 432

Leu Ala Arg Gly Phe Thr Gly Arg Asp Lys He He Lys Phe Glu Gly 130 135 140

tgc tac cac ggc cat tec gac age ctg ttg gtg aaa gca ggc age ggt 480

Cys Tyr His Gly His Ser Asp Ser Leu Leu Val Lys Ala Gly Ser Gly 145 150 155 160 ctg ctt acc ttc ggc aat cct tct tec gec ggt gtg cct gec gac ttt 528

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 gee caa etc gaa 576

Thr Lys His Thr Leu Val Leu Glu Tyr Asn Asn He Ala Gin Leu Glu

180 185 190 gaa gec ttt gee caa age ggc gac gaa ate gec tgc gtg att gtc gaa 624

Glu Ala Phe Ala Gin Ser Gly Asp Glu He Ala Cys Val He Val Glu

195 200 205

ccc ttc gtc ggc aat atg aac etc gtc cgc ccg acc gaa gec ttt gtc

672

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 720

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 768

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

245 250 255 ctg cac ggc ate acg ccc gac ctg acc acg atg ggc aaa gtc ate ggc 816

Leu His Gly lie Thr Pro Asp Leu Thr Thr Met Gly Lys Val lie Gly

260 265 270 ggc ggt atg ccg ctt gcc gcg ttc ggc gga cgc aaa gac ate atg gaa 864

Gly Gly Met Pro Leu Ala Ala Phe Gly Gly Arg Lys Asp lie Met Glu

275 280 285

tgt att tec ccg ttg ggc ggc gtg tat cag gca ggt aca tta tea ggc 912

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 acg ctg gaa ate ate cag 960

Asn Pro lie Ala Val Ala Ala Gly Leu Lys Thr Leu Glu lie lie Gin

305 310 315 320 cgc gaa ggc ttc tat gaa aac ctg acc gcc ttg aca caa cgc ctt gcc 1008

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 gcg cac ggt ate gag ttt gcc gcc gac 1056

Asn Gly lie 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 cga 1104

Ser Val Gly Gly Met Phe Gly Leu Tyr Phe Ala Ala His Val Pro Arg

355 360 365

aac tat gcc gat atg gcg cgc tec aat ate gac get ttc aaa cgc ttc 1152

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 cgc ggc att gcc ttc ggc ccg tec get tat 1200

Phe His Gly Met Leu Asp Arg Gly lie Ala Phe Gly Pro Ser Ala Tyr

385 390 395 400 gaa gcg ggt ttc gtt tec gcc gcg cat acg ccc gag ctg att gac gaa 1248

Glu Ala Gly Phe Val Ser Ala Ala His Thr Pro Glu Leu lie Asp Glu

405 410 415 acg gtt gcg gtt gcg gtt gaa gtg ttc aag gcg atg get gca tga 1293

Thr Val Ala Val Ala Val Glu Val Phe Lys Ala Met Ala Ala

420 425 430

<210> 25

<21 1> 430

<212> PRT

<213> Ne is seria gonorrhoeae

<400> 25

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

Ala He He 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

Pro Ala He 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

He Ala He Ala Glu Gin He Ala Glu He Met Pro Ser Val Glu Arg

100 105 1 10

Leu Arg Leu Val Ser Ser Gly Thr Glu Ala Thr Met Thr Ala He Arg

1 15 120 125

Leu Ala Arg Gly Phe Thr Gly Arg Asp Lys He He 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 1 70 1 75

Thr Lys His Thr Leu Val Leu Glu Tyr Asn Asn He Ala Gin Leu Glu 180 185 190

Glu Ala Phe Ala Gin Ser Gly Asp Glu lie Ala Cys Val lie Val Glu

195 200 205

Pro Phe Val Gly Asn Met Asn Leu Val Arg Pro Thr Glu Ala Phe Val 210 215 220

Lys Ala Leu Arg Gly Leu Thr Glu Lys His Gly Ala Val Leu 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 lie 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 lie Ser Pro Leu Gly Gly Val Tyr Gin Ala Gly Thr Leu Ser Gly 290 295 300

Asn Pro lie Ala Val Ala Ala Gly Leu Lys Thr Leu Glu lie 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 lie Asp Ala Phe Lys Arg Phe 370 375 380

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

Glu Ala Gly Phe Val Ser Ala Ala His Thr Pro Glu Leu lie 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

<220>

<221> CDS

<222> (1) .. (924)

<400> 26

atg teg atg gec gat cgt gat ggc gtg ate tgg tat gac ggt gaa ctg 48

Met Ser Met Ala Asp Arg Asp Gly Val He Trp Tyr Asp Gly Glu Leu 1 5 10 15 gtg cag tgg cgc gac gcg acc acg cac gtg ctg acc cat acc ctg cac 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 cgc gec 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 acg gcg ate ttc cgc ctg cag gcg cat acc gac egg ctg ttc gac 192

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

tec gcg cac ate atg aac atg cag ate ccg tac age cgc gac gag ate 240

Ser Ala His He Met Asn Met Gin He Pro Tyr Ser Arg Asp Glu He 65 70 75 80 aac gag gcg acc cgc gec gee gtg cgc gag aac aac ctg gaa age gec 288

Asn Glu Ala Thr Arg Ala Ala Val Arg Glu Asn Asn Leu Glu Ser Ala

85 90 95 tat ate cgc ccg atg gtg ttc tac gga age gaa ggc atg ggc ctg cgc 336

Tyr He Arg Pro Met Val Phe Tyr Gly Ser Glu Gly Met Gly Leu Arg

100 105 110 gec age ggc ctg aag gtc cat gtg ate ate gee gee tgg age tgg ggc 384

Ala Ser Gly Leu Lys Val His Val He He Ala Ala Trp Ser Trp Gly

115 120 125

gec tac atg ggc gag gaa gec ctg cag caa ggc ate aag gtg cgc acc 432

Ala Tyr Met Gly Glu Glu Ala Leu Gin Gin Gly He Lys Val Arg Thr 130 135 140 agt tec ttc acc cgc cac cac gtc aac ate teg atg acc cgc gcc aag 480

Ser Ser Phe Thr Arg His His Val Asn He Ser Met Thr Arg Ala Lys

145 150 155 160 tec aac ggc gcc tac ate aac teg atg ctg gcc etc cag gaa gcg ate 528

Ser Asn Gly Ala Tyr He Asn Ser Met Leu Ala Leu Gin Glu Ala He

165 170 175 tec ggc ggc gcc gac gag gcc atg atg etc gat ccg gaa ggc tac gtg 576

Ser Gly Gly Ala Asp Glu Ala Met Met Leu Asp Pro Glu Gly Tyr Val

180 185 190 gcc gaa ggc tec ggc gag aac ate ttc ate ate aag gat ggc gtg ate 624

Ala Glu Gly Ser Gly Glu Asn He Phe He He Lys Asp Gly Val He

195 200 205

tac acc ccg gaa gtc acc gcc tgc ctg aac ggc ate act cgt aac act 672

Tyr Thr Pro Glu Val Thr Ala Cys Leu Asn Gly He Thr Arg Asn Thr 210 215 220

ate ctg acc ctg gcc gcc gaa cac ggt ttt aaa ctg gtc gag aag cgc 720

He Leu Thr Leu Ala Ala Glu His Gly Phe Lys Leu Val Glu Lys Arg

225 230 235 240 ate acc cgc gac gag gtg tac ate gcc gac gag gcc ttc ttc act ggc 768

He Thr Arg Asp Glu Val Tyr He Ala Asp Glu Ala Phe Phe Thr Gly

245 250 255 act gcc gcg gaa gtc acg ccg ate cgc gaa gtg gac ggt cgc aag ate 816

Thr Ala Ala Glu Val Thr Pro He Arg Glu Val Asp Gly Arg Lys He

260 265 270 ggc gcc ggc cgc cgt ggc ccg gtc acc gaa aag ctg cag aaa gcc tat 864

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 gcc cac gcc gag tgg cgt acc 912

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> Pseudomonas aeruginosa

<400> 27

Met Ser Met Ala Asp Arg Asp Gly Val He Trp Tyr Asp Gly Glu Leu 1 5 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 He Met Asn Met Gin He Pro Tyr Ser Arg Asp Glu He 65 70 75 80

Asn Glu Ala Thr Arg Ala Ala Val Arg Glu Asn Asn Leu Glu Ser Ala

85 90 95 Tyr He Arg Pro Met Val Phe Tyr Gly Ser Glu Gly Met Gly Leu Arg

100 105 1 10

Ala Ser Gly Leu Lys Val His Val He He Ala Ala Trp Ser Trp Gly

1 15 120 125

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

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

Ser Asn Gly Ala Tyr He Asn Ser Met Leu Ala Leu Gin Glu Ala He

165 1 70 1 75 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 He Phe He He Lys Asp Gly Val He

195 200 205

Tyr Thr Pro Glu Val Thr Ala Cys Leu Asn Gly He Thr Arg Asn Thr 2 10 2 15 220 He Leu Thr Leu Ala Ala Glu His Gly Phe Lys Leu Val Glu Lys Arg 225 230 235 240

He Thr Arg Asp Glu Val Tyr He Ala Asp Glu Ala Phe Phe Thr Gly

245 250 255

Thr Ala Ala Glu Val Thr Pro He Arg Glu Val Asp Gly Arg Lys He

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

305

<210> 28

<211> 1407

<212> DNA

<213> Rhodopseudomonas palust

<220>

<221> CDS

<222> (1) .. (1407)

<400> 28

atg aag ctg ata ccg tgc cgc gcc ttt cac ccc ccg gcc gcg cag tgc 48

Met Lys Leu He Pro Cys Arg Ala Phe His Pro Pro Ala Ala Gin Cys 1 5 10 15 atg agg age gcc atg tta gac aag ate aag ccc acg tec gcc gtc aac 96

Met Arg Ser Ala Met Leu Asp Lys He 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 144

Ala Pro Asn Asp Leu Asn Ala Phe Trp Met Pro Phe Thr Ala Asn Arg

35 40 45

gcc ttc aag cgc gcg ccg aag atg gtc gtg ggt gcc gaa ggc atg cac 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 He 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 He Ala Glu Ala He

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 He 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 He 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 He 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 He Gly Glu Pro Glu Tyr Gly Ala His 210 215 220

ttc gcc gaa gcg ctt 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 tec acc ggc gtg ctg 768

He Ala Ala Val He 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 ace 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 ace ggc tac ggc cgt 864

His Gly lie Leu Leu lie Phe Asp Glu Val lie Thr Gly Tyr Gly Arg

275 280 285

etc ggc tat gcc ttc gcg tec gaa cgt tac ggc gtc acc ccg gac atg 912

Leu Gly Tyr Ala Phe Ala Ser Glu Arg Tyr Gly Val Thr Pro Asp Met 290 295 300

ate acc ttc gcc aag ggc gtc acc aat ggt gcg gtg ccg atg ggc ggc 960

lie Thr Phe Ala Lys Gly Val Thr Asn Gly Ala Val Pro Met Gly Gly

305 310 315 320 gtg ate acc teg gcg gag ate cac gat gcg ttc atg acc ggc ccc gag 1008

Val lie Thr Ser Ala Glu lie His Asp Ala Phe Met Thr Gly Pro Glu

325 330 335 cac gcg gtc gag ctg gcg cac ggc tac acc tat teg gcg cat ccg etc 1056

His Ala Val Glu Leu Ala His Gly Tyr Thr Tyr Ser Ala His Pro Leu

340 345 350 gcc tgc gcg gcc ggc ate gcc acc etc gac ate tac cgc gac gag aag 1104

Ala Cys Ala Ala Gly lie Ala Thr Leu Asp lie Tyr Arg Asp Glu Lys

355 360 365

ctg ttc gag cgc gcc aag gcg ctg gag ccg aag ttt gcc gag gcg gtg 1152

Leu Phe Glu Arg Ala Lys Ala Leu Glu Pro Lys Phe Ala Glu Ala Val 370 375 380

atg teg ctg aag teg gcc ccg aac gtg gtc gac ate cgc acc gtc ggc 1200

Met Ser Leu Lys Ser Ala Pro Asn Val Val Asp lie Arg Thr Val Gly

385 390 395 400 ctg acg gcg ggt ate gac etc get teg ate gcc gat gcg gtc ggc aag 1248

Leu Thr Ala Gly lie Asp Leu Ala Ser lie Ala Asp Ala Val Gly Lys

405 410 415 cgt ggc ttc gaa gcg atg aat gcc ggc ttc cac gac cac gag ctg atg 1296

Arg Gly Phe Glu Ala Met Asn Ala Gly Phe His Asp His Glu Leu Met

420 425 430 ctg egg ate gcc ggc gac acc ctg gcg ctg acc ccg ccg ctg ate etc 1344

Leu Arg lie Ala Gly Asp Thr Leu Ala Leu Thr Pro Pro Leu lie Leu

435 440 445 age gag gae cac ate ggt gag ate gte gae aag gte gge aag gtg ate 1392

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

cgc gcg gte gec tga

1407

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

Met Arg Ser Ala Met Leu Asp Lys lie 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 lie 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 He Gly Arg Glu Arg Gly Tyr His Gly Val 165 170 175

Gly Phe Gly Gly Thr Ala Val Gly Gly He 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

He Ala Ala Val He Val Glu Pro Met Ala Gly Ser Thr Gly Val Leu

245 250 255 Pro Ala Pro Lys Gly Tyr Leu Lys Lys Leu Arg Glu He Thr Lys Lys

260 265 270

His Gly He Leu Leu He Phe Asp Glu Val He 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

He Thr Phe Ala Lys Gly Val Thr Asn Gly Ala Val Pro Met Gly Gly 305 310 315 320

Val He Thr Ser Ala Glu He 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 He Ala Thr Leu Asp He 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 He 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 lie Leu

435 440 445

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

Arg Ala Val Ala

465

<210> 30

<211> 1263

<212> DNA

<213> Escherichia

<220>

<221> CDS

<222> (1) . (1263)

<400> 30

atg cca cat tea ctg ttc age ace gat acc gat etc acc gec gaa aat 48

Met Pro His Ser Leu Phe Ser Thr Asp Thr Asp Leu Thr Ala Glu Asn 1 5 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 gcg caa att att cgt egg cag att gca gcg ctg aaa cag ttt gat gtg 144

Ala Gin lie lie Arg Arg Gin lie Ala Ala Leu Lys Gin Phe Asp Val

35 40 45

gtg cgc ttt gca cag aaa gec tgt tec aat att cat att ttg cgc tta 192

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

atg cgt gag cag ggc gtg aaa gtg gat tec gtc teg tta ggc gaa ata 240

Met Arg Glu Gin Gly Val Lys Val Asp Ser Val Ser Leu Gly Glu lie 65 70 75 80 gag cgt gcg ttg gcg gcg ggt tac aat ccg caa acg 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 acg gca gat gtt ate gat cag gcg acg ctt gaa cgc gtc 336

He 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 gcg ggt tct gtt gat atg etc gac 384

Ser Glu Leu Gin He 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 cgc 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 gec gca ctg gac 528

Asn Ser Lys His Gly He 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 He Gin Arg His His Leu Gin Leu Val Gly He His Met His He

180 185 190 ggt tct ggc gtt gat tat gec 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 gcg

672

Val Arg Gin Val He Glu Phe Gly Gin Asp Leu Gin Ala He Ser Ala

210 215 220

ggc ggt ggg ctt tct gtt cct tat caa cag ggt gaa gag gcg 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 gec gcg cgt gag caa ate gee 768

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

245 250 255 cgc cat ttg ggc cac cct gtg aaa ctg gaa att gaa ccg ggt cgc ttc 816

Arg His Leu Gly His Pro Val Lys Leu Glu He Glu Pro Gly Arg Phe

260 265 270 ctg gta gcg 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 cgc 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 cgc 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 gcg cca acg 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

340 345 350 ggg gg^a aat gtt gaa acc cgc 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 tec 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 gcg egg ttg att cgc cgt cgc cag acc ate gaa gaa tta ctg gcg 1248

Gin Ala Arg Leu lie Arg Arg Arg Gin Thr lie Glu Glu Leu Leu Ala

405 410 415 ctg gaa ttg ctt taa

1263

Leu Glu Leu Leu

420

<210> 31

<211> 420

<212> PRT

<213> Escherichia

<400> 31

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

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

20 25 30

Ala Gin He He Arg Arg Gin He Ala Ala Leu Lys Gin Phe Asp Val

35 40 45

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

Met Arg Glu Gin Gly Val Lys Val Asp Ser Val Ser Leu Gly Glu He 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 He 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

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 He His Met His He

180 185 190

Gly Ser Gly Val Asp Tyr Ala His Leu Glu Gin Val Cys Gly Ala Met

195 200 205

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 lie Ala 245 250 255

Arg His Leu Gly His Pro Val Lys Leu Glu lie Glu Pro Gly Arg Phe

260 265 270

Leu Val Ala Gin Ser Gly Val Leu lie 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 lie Arg Arg Arg Gin Thr lie Glu Glu Leu Leu Ala

405 410 415 Leu Glu Leu Leu

420

<210> 32

<211> 1265

<212> DNA

<213> Artificial

<220>

<223> Escherichia . coli diaminopimelate decarboxylase LysA codon optimised 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

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 ggcctgtctg 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

<220>

<221> CDS

<222> (1) .. (1692)

<400> 33

atg tct gaa att act ttg ggt aaa tat ttg ttc gaa aga tta aag caa 48

Met Ser Glu lie Thr Leu Gly Lys Tyr Leu Phe Glu Arg Leu Lys Gin 1 5 10 15 gtc aac gtt aac acc gtt ttc ggt ttg cca ggt gac ttc aac ttg tec 96

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 144

Leu Leu Asp Lys lie Tyr Glu Val Glu Gly Met Arg Trp Ala Gly Asn

35 40 45

gee aac gaa ttg aac get get tac gee get gat ggt tac get cgt ate 192

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 240

Lys Gly Met Ser Cys lie lie Thr Thr Phe Gly Val Gly Glu Leu Ser

65 70 75 80 get ttg aac ggt att gee ggt tct tac get gaa cac gtc ggt gtt ttg 288

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 tec ate tct get caa get aag caa ttg ttg 336

His Val Val Gly Val Pro Ser lie 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 384

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 432

Ser Ala Asn lie Ser Glu Thr Thr Ala Met lie Thr Asp lie Ala Thr

130 135 140

gcc cca get gaa att gac aga tgt ate aga acc act tac gtc acc caa 480

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 528

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 576

Pro Ala Lys Leu Leu Gin Thr Pro lie Asp Met Ser Leu Lys Pro Asn

180 185 190 gat get gaa tec gaa aag gaa gtc att gac acc ate ttg get ttg gtc 624

Asp Ala Glu Ser Glu Lys Glu Val lie Asp Thr lie Leu Ala Leu Val

195 200 205

aag gat get aag aac cca gtt ate ttg get gat get tgt tgt tec aga 672

Lys Asp Ala Lys Asn Pro Val lie Leu Ala Asp Ala Cys Cys Ser Arg

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 tec 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 tec 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 tct get gac ttg att ttg tct 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 tct gat ttc aac acc ggt tct ttc tct tac tct 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 tec 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 gec 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 tct 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 tec 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 tct caa gtc tta tgg ggt tec 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 tct ttg caa 1344

Asp Pro Lys Lys Arg Val lie Leu Phe lie Gly Asp Gly Ser Leu Gin

435 440 445

ttg act gtt caa gaa ate tec acc atg ate aga tgg ggc ttg aag cca 1392

Leu Thr Val Gin Glu lie Ser Thr Met lie 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 1440

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 1488

His Gly Pro Lys Ala Gin Tyr Asn Glu lie Gin Gly Trp Asp His Leu

485 490 495 tec ttg ttg cca act ttc ggt get aag gac tat gaa acc cac aga gtc 1536

Ser Leu Leu Pro Thr Phe Gly Ala Lys Asp Tyr Glu Thr His Arg Val 500 505 510 get ace acc ggt gaa tgg gac aag ttg acc caa gac aag tct ttc aac 1584

Ala Thr Thr Gly Glu Trp Asp Lys Leu Thr Gin Asp Lys Ser Phe Asn

515 520 525

gac aac tct aag ate aga atg att gaa ate atg ttg cca gtc ttc gat 1632

Asp Asn Ser Lys lie Arg Met lie Glu lie 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 1680

Ala Pro Gin Asn Leu Val Glu Gin Ala Lys Leu Thr Ala Ala Thr Asn

545 550 555 560 get aag caa taa

1692

Ala Lys Gin

<210> 34

<211> 563

<212> PRT

<213> Saccharomyces cerevisiae

<400> 34

Met Ser Glu lie Thr Leu Gly Lys Tyr Leu Phe Glu Arg Leu Lys Gin 1 5 10 15

Val Asn Val Asn Thr Val Phe Gly Leu Pro Gly Asp Phe Asn Leu Ser

20 25 30

Leu Leu Asp Lys 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 lie 50 55 60

Lys Gly Met Ser Cys lie lie Thr Thr Phe Gly Val Gly Glu Leu Ser 65 70 75 80

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 lie Ser Glu Thr Thr Ala Met lie Thr Asp lie 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

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 lie 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

Phe Pro Gly Val Gin Met Lys Phe Val Leu Gin Lys Leu Leu Thr Thr

325 330 335

He 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 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 lie 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 lie Arg Met lie Glu lie 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

Ala Lys Gin

<210> 35

<211> 1692

<212> DNA <213> Artificial

<220>

<223> Saccharomyces cerevisiae pyruvate decarboxylase Pdc codon optimised gene

<400> 35

atgtccgaga tcactctggg caaatacctg tttgaacgtc tgaaacaggt gaacgttaat 60

accgtattcg gcctgccggg tgatttcaac ctgtccctgc tggacaaaat ctatgaagtt 120

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

ttcatcggtg acggctccct gcagctgacc gtccaggaga tttctaccat gatccgctgg 1380

ggcctgaaac cgtacctgtt tgtgctgaac aacgacggct acactattga gaaactgatc 1440

cacggtccga aagcacagta taatgagatc cagggttggg atcatctgtc tctgctgccg 1500

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> Zymomonas mobilis

<220>

<221> CDS

<222> (1) .. (1707)

<400> 36

atg agt tat act gtc ggt acc tat tta gcg gag egg ctt gtc cag att 48

Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gin lie 1 5 10 15 ggt etc aag cat cac ttc gca gtc gcg ggc gac tac aac etc gtc ctt 96

Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu

20 25 30 ctt gac aac ctg ctt ttg aac aaa aac atg gag cag gtt tat tgc tgt 144

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 gea gaa ggt tat get cgt gcc aaa 192

Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Lys 50 55 60

ggc gea gea gea gcc gtc gtt acc tac age gtc ggt gcg ctt tec gea 240

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 gcc tat gea gaa aac ctt ccg gtt ate ctg 288

Phe Asp Ala lie Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val lie Leu

85 90 95 ate tec ggt get ccg aac aac aat gat cac get get ggt cac gtg ttg 336

lie Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val Leu

100 105 110 cat cac get ctt ggc aaa acc gac tat cac tat cag ttg gaa atg gcc 384

His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gin Leu Glu Met Ala

115 120 125

aag aac ate acg gcc gcc get gaa gcg att tac acc ccg gaa gaa get 432

Lys Asn lie Thr Ala Ala Ala Glu Ala lie Tyr Thr Pro Glu Glu Ala

130 135 140

ccg get aaa ate gat cac gtg att aaa act get ctt 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 tec atg ccc tgc gcc 528

Pro Val Tyr Leu Glu lie Ala Cys Asn lie Ala Ser Met Pro Cys Ala

165 170 175 get cct gga ccg gea age gea ttg ttc aat gac gaa gcc age gac gaa 576

Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp Glu

180 185 190 get tct ttg aat gea gcg gtt gaa gaa acc ctg aaa ttc ate gcc aac 624

Ala Ser Leu Asn Ala Ala Val Glu Glu Thr Leu Lys Phe lie Ala Asn

195 200 205

cgc gac aaa gtt gcc gtc etc gtc ggc age aag ctg cgc gea 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 ace 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 acg atg aaa gaa gec gat gcg 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 tec acc act ggt tgg acg gat att cct gat cct aag aaa ctg 912

Asp Tyr Ser Thr Thr Gly Trp Thr Asp lie Pro Asp Pro Lys Lys Leu

290 295 300

gtt etc get gaa ccg cgt tct gtc gtc gtt aac ggc att cgc 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 tec 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 tec 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 gec 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

gaa ate gee cgt cag gtc gaa get ctt ctg acc ccg aac acg acg gtt 1152

Glu lie Ala Arg Gin Val Glu Ala Leu Leu Thr Pro Asn Thr Thr Val 370 375 380

att get gaa acc ggt gac tct tgg ttc aat get cag cgc atg aag etc 1200

lie Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gin Arg Met Lys Leu

385 390 395 400 ccg aac ggt get cgc gtt gaa tat gaa atg cag tgg ggt cac att ggt 1248

Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gin Trp Gly His lie Gly 405 410 415 tgg tec gtt cct gcc gcc ttc ggt tat gcc gtc ggt get ccg gaa cgt 1296

Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu Arg

420 425 430 cgc aac ate etc atg gtt ggt gat ggt tec ttc eag ctg aeg get eag 1344

Arg Asn He Leu Met Val Gly Asp Gly Ser Phe Gin Leu Thr Ala Gin

435 440 445

gaa gtc get cag atg gtt cgc ctg aaa ctg ccg gtt ate ate ttc ttg 1392

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

ate aat aac tat ggt tac acc gcc gaa gtt atg ate cat gat ggt ccg 1440

He Asn Asn Tyr Gly Tyr Thr Ala Glu Val Met He His Asp Gly Pro

465 470 475 480 tac aac aac ate aag aac tgg gat tat gcc ggt ctg atg gaa gtg ttc 1488

Tyr Asn Asn He 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 1536

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 1584

Lys Thr Gly Gly Glu Leu Ala Glu Ala He 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 1632

Thr Asp Gly Pro Thr Leu He Glu Cys Phe He Gly Arg Glu Asp Cys 530 535 540

act gaa gaa ttg gtc aaa tgg ggt aag cgc gtt get gcc gcc aac age 1680

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

1707

Arg Lys Pro Val Asn Lys Leu Leu

565

<210> 37

<211> 568

<212> PRT

<213> Zymomonas mobil^J

<400> 37 Met Ser Tyr Thr Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gin lie 1 5 10 15

Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu

20 25 30

Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu 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 lie 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 lie Asp His Val lie 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 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 lie 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 lie 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 lie lie Phe Leu 450 455 460 lie Asn Asn Tyr Gly Tyr Thr Ala Glu Val Met lie His Asp Gly Pro 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> Zymomonas mobilis pyruvate decarboxylase PdcI472A codon optimised

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

gcgaccatgg cagcggctaa atccttcttc ccagaggaga acccgcatta cattggtacc 780

tcctggggcg aagtttccta ccctggtgtg gagaaaacca tgaaagaagc cgatgctgtg 840

attgccctgg cgcctgtatt caacgattat tccaccaccg gttggaccga tatcccggac 900

ccgaagaaac tggtcctggc tgaaccgcgc tccgtagtag tgaatggcat tcgtttcccg 960

tccgtacacc tgaaggatta cctgacgcgt ctggcacaga aagtatccaa gaaaactggc 1020

gcgctggact tctttaaatc cctgaacgct ggtgagctga aaaaggcggc tccggi 1080

ccgtccgcac cgctggtgaa cgcagagatt gcacgtcagg ttgaggcact gctgacgccg 1140

aacaccaccg taatcgcgga aacgggcgac tcttggttca acgcacagcg catgaaactg 1200

ccgaacggtg cccgcgttga atatgaaatg cagtggggtc acatcggctg gtctgtccca 1260

gcagcgtttg gttacgcggt tggtgcaccg gagcgtcgca acatcctgat ggtgggtgac 1320

ggctccttcc agctgactgc tcaggaggtg gcgcagatgg tgcgcctgaa gctgccggtt 1380

atcattttcc tgatcaacaa ctacggctac accgccgagg taatgatcca cgatggtccg 1440

tacaacaaca tcaaaaactg ggactacgcc ggtctgatgg aggtttttaa cggtaacggc 1500

ggttacgaca gcggtgctgg taagggtctg aaagccaaaa ccggtggcga actggcagag 1560

gcgattaaag ttgcgctggc aaacaccgat ggcccgaccc tgatcgagtg cttcatcggc 1620 cgtgaggact gcaccgagga gctggtcaaa tggggcaaac gtgtggcggc tgctaactct 1680

cgcaagccgg taaacaaact gctgtaa

1707

<210> 39

<211> 1644

<212> DNA

<213> Lactococcus lact

<220>

<221> CDS

<222> (1) .. (1644)

<400> 39

atg tat aca gta gga gat tac ctg tta gac cga tta cac gag ttg gga 48

Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 1 5 10 15 att gaa gaa att ttt gga gtt cct ggt gac tat aac tta caa ttt tta 96

He Glu Glu He Phe Gly Val Pro Gly Asp Tyr Asn Leu Gin Phe Leu

20 25 30 gat caa att att tea cgc gaa gat atg aaa tgg att gga aat get aat 144

Asp Gin He He Ser Arg Glu Asp Met Lys Trp He Gly Asn Ala Asn

35 40 45

gaa tta aat get tct 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 gcc gca ttt etc acc aca ttt gga gtc ggc gaa ttg agt gcg 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 gcc 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 gcg egg act tta ctg aca gca gaa aat gec 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 cga gta ctt tct 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 lie Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro

165 170 175 gca tta tct tta gaa aaa gaa age tct 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 aag att gaa gaa agt ttg aaa aat gec 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 acg 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 acg 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 tct 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 ctt tea gaa ate agt ctt aaa aat ttt gtg gag tec 816

Tyr Asn Gly Lys Leu Ser Glu lie Ser Leu Lys Asn Phe Val Glu Ser

260 265 270 gca gac ttt ate eta atg ctt gga gtg aag ctt acg gac tec tea aca 864

Ala Asp Phe lie Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr

275 280 285

ggt gca ttc aca cat cat tta gat gaa aat aaa atg att tea eta aac 912

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 960

lie Asp Glu Gly lie lie Phe Asn Lys Val Val Glu Asp Phe Asp Phe

305 310 315 320 aga gca gtg gtt tct tct tta tea gaa tta aaa gga ata gaa tat gaa 1008

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 1056

Gly Gin Tyr lie Asp Lys Gin Tyr Glu Glu Phe lie Pro Ser Ser Ala

340 345 350 ccc tta tea caa gac cgt eta tgg cag gca gtt gaa agt ttg act caa 1104

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 1152

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 1200

Ser Thr lie Phe Leu Lys Ser Asn Ser Arg Phe lie Gly Gin Pro Leu

385 390 395 400 tgg ggt tct att gga tat act ttt cca gcg get tta gga age caa att 1248

Trp Gly Ser lie Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gin lie

405 410 415 gcg gat aaa gag age aga cac ctt tta ttt att ggt gat ggt tea ctt 1296

Ala Asp Lys Glu Ser Arg His Leu Leu Phe lie Gly Asp Gly Ser Leu

420 425 430 caa ctt acc gta caa gaa tta gga eta tea ate aga gaa aaa etc aat 1344

Gin Leu Thr Val Gin Glu Leu Gly Leu Ser lie Arg Glu Lys Leu Asn

435 440 445

cca att tgt ttt ate ata aat aat gat ggt tat aca gtt gaa aga gaa 1392

Pro lie Cys Phe lie lie 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 1440

lie His Gly Pro Thr Gin Ser Tyr Asn Asp lie 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 1488

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 tct gtc atg aaa gaa gec 1536

Lys lie 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 1584

Gin Ala Asp Val Asn Arg Met Tyr Trp lie Glu Leu Val Leu Glu Lys

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 lact

<400> 40

Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 1 5 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 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 lie 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 lie Asp Arg Val Leu Ser Gin 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

Ala Leu Ser Leu Glu Lys Glu Ser Ser Thr Thr Asn Thr Thr Glu Gin

180 185 190

Val lie 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 lie 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 lie 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 lie Ser Leu Asn 290 295 300

He Asp Glu Gly He He 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 He Glu Tyr Glu

325 330 335

Gly Gin Tyr He Asp Lys Gin Tyr Glu Glu Phe He 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 He Phe Leu Lys Ser Asn Ser Arg Phe He Gly Gin Pro Leu 385 390 395 400

Trp Gly Ser He 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 He 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 He Cys Phe He He Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu

450 455 460

He His Gly Pro Thr Gin Ser Tyr Asn Asp He 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 decarboxylase KdcA codon optimised 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

acctctaagg ttcagaacga cggcaaattc gtacatcaca ccctggcgga cggcgatttt 360

aagcacttta tgaaaatgca cgaaccggtc accgccgctc gcactctgct gaccgcggaa 420

aacgcaacgt acgagatcga tcgtgtactg tcccagctgc tgaaagaacg taaaccggtg 480

tatatcaatc tgccggttga tgtcgctgcg gccaaagcag agaaaccggc actgtccctg 540

gagaaggaga gctccactac taacaccacc gaacaggtta tcctgtccaa aattgaagaa 600

tctctgaaaa acgcacagaa accggtggtt atcgcaggtc acgaggttat ctccttcggc 660

ctggagaaaa ctgttactca attcgtctct gaaacgaaac tgccgatcac gaccctgaac 720

tttggcaagt ccgcagttga cgaatctctg ccttctttcc tgggcattta caacggcaaa 780

ctgtccgaga tctccctgaa gaacttcgta gaatccgctg actttatcct gatgctgggt 840

gtgaaactga ccgactcctc taccggtgcg ttcacgcacc atctggatga aaacaaaatg 900

atcagcctga acatcgacga gggtatcatc ttcaacaagg tagttgaaga tttcgacttc 960

cgtgctgttg tcagcagcct gtccgagctg aaaggcattg agtacgaggg tcaatacatc 1020

gataaacagt acgaagagtt tattccgtct tctgcaccgc tgagccagga ccgcctgtgg 1080

caggcagttg agtccctgac gcagtccaac gaaactatcg tagcggaaca aggtacctct 1140

ttcttcggtg cttctaccat ctttctgaag tccaactctc gctttatcgg tcagccgctg 1200 tggggttcta tcggttacac gttcccggct gcgctgggta gccagatcgc tgataaagag 1260

tctcgtcatc tgctgttcat cggtgatggt tccctgcagc tgactgtaca ggaactgggt 1320

ctgtctatcc gtgaaaaact gaacccgatt tgttttatca tcaataacga tggctacact 1380

gttgagcgtg aaattcatgg tccgactcag tcttacaacg atattccgat gtggaactac 1440

tctaaactgc cggaaacctt cggtgcaact gaggatcgcg tcgtgagcaa gattgtgcgt 1500

actgagaacg agttcgtatc tgttatgaaa gaggcgcagg cagatgtgaa ccgcatgtac 1560

tggatcgaac tggttctgga aaaagaggat gcaccgaaac tgctgaagaa aatgggt 1620

ctgtttgcgg agcagaacaa gtaa

1644

<210> 42

<211> 1647

<212> DNA

<213> Lactococcus lact

<220>

<221> CDS

<222> (1) .. (1647)

<400> 42

atg tat aca gta gga gat tac eta tta gac cga tta cac gag tta gga

48

Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 1 5 10 15 att gaa gaa att ttt gga gtc cct gga gac tat aac tta caa ttt tta 96

lie Glu Glu lie Phe Gly Val Pro Gly Asp Tyr Asn Leu Gin Phe Leu

20 25 30 gat caa att att tec cac aag gat atg aaa tgg gtc gga aat get aat 144

Asp Gin lie 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 gec gca ttt ctt aca ace 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 lie

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 acg 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 cga 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 cga gta ctt tct 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 acg aca tta aac 720

Val Thr Gin Phe lie Ser Lys Thr Lys Leu Pro lie Thr Thr Leu Asn

225 230 235 240 ttt ggt aaa agt tea gtt gat gaa gcc etc cct tea ttt tta gga ate 768

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 ctt aaa gaa ttc gtg gaa tea 816 Tyr Asn Gly Thr Leu Ser Glu Pro Asn Leu Lys Glu Phe Val Glu Ser 260 265 270 gcc gac ttc ate ttg atg ctt gga gtt aaa etc aca gac tct tea aca 864

Ala Asp Phe lie Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr

275 280 285

gga gcc ttc act cat cat tta aat gaa aat aaa atg att tea ctg aat 912

Gly Ala Phe Thr His His Leu Asn Glu Asn Lys Met lie Ser Leu Asn 290 295 300

ata gat gaa gga aaa ata ttt aac gaa aga ate caa aat ttt gat ttt 960

lie Asp Glu Gly Lys lie Phe Asn Glu Arg lie Gin Asn Phe Asp Phe

305 310 315 320 gaa tec etc ate tec tct etc tta gac eta age gaa ata gaa tac aaa 1008

Glu Ser Leu lie Ser Ser Leu Leu Asp Leu Ser Glu lie Glu Tyr Lys

325 330 335 gga aaa tat ate gat aaa aag caa gaa gac ttt gtt cca tea aat gcg 1056

Gly Lys Tyr lie Asp Lys Lys Gin Glu Asp Phe Val Pro Ser Asn Ala

340 345 350 ctt tta tea caa gac cgc eta tgg caa gca gtt gaa aac eta act caa 1104

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 1152

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

tea tea att ttc tta aaa tea aag agt cat ttt att ggt caa ccc tta 1200

Ser Ser lie Phe Leu Lys Ser Lys Ser His Phe lie Gly Gin Pro Leu

385 390 395 400 tgg gga tea att gga tat aca ttc cca gca gca tta gga age caa att 1248

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 ctt tta ttt att ggt gat ggt tea ctt 1296

Ala Asp Lys Glu Ser Arg His Leu Leu Phe lie Gly Asp Gly Ser Leu

420 425 430 caa ctt aca gtg caa gaa tta gga tta gca ate aga gaa aaa att aat 1344

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 1392

Pro He Cys Phe He He 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 1440

He His Gly Pro Asn Gin Ser Tyr Asn Asp He Pro Met Trp Asn Tyr

465 470 475 480 tea aaa tta cca gaa teg ttt gga gca aca gaa gat cga 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 tct gtc atg aaa gaa get 1536

Lys He Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala

500 505 510 caa gca gat cca aat aga atg tac tgg att gag tta att ttg gca aaa 1584

Gin Ala Asp Pro Asn Arg Met Tyr Trp He Glu Leu He 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> 43

Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 1 5 10 15

He Glu Glu He Phe Gly Val Pro Gly Asp Tyr Asn Leu Gin Phe Leu

20 25 30

Asp Gin He He 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

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 lie Gin Glu Ser Leu Lys Asn Ala Lys Lys Pro

195 200 205

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

Val Thr Gin Phe He 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 He

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 He Ser Leu Asn 290 295 300 He Asp Glu Gly Lys He Phe Asn Glu Arg He Gin Asn Phe Asp Phe 305 310 315 320

Glu Ser Leu He Ser Ser Leu Leu Asp Leu Ser Glu He 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

355 360 365

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

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

Trp Gly Ser He 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 He Gly Asp Gly Ser Leu

420 425 430

Gin Leu Thr Val Gin Glu Leu Gly Leu Ala He Arg Glu Lys He Asn

435 440 445

Pro He Cys Phe He He 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 He 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 He Glu Leu He 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> Artificial

<220>

<223> Lactococcus lactis -ketoisovalerate decarboxylase KivD codon optimised gene

<400> 44

atgtatactg ttggtgatta cctgctggat cgtctgcatg aactgggcat cgaggaaatt 60

ttcggcgtac ctggtgacta taacctgcag ttcctggatc agatcatttc ccacaaagat 120

atgaaatggg ttggtaacgc gaacgagctg aatgcaagct acatggctga cggttatgca 180

cgcaccaaga aagctgcggc gttcctgact acttttggcg tcggcgagct gtctgcggta 240

aacggtctgg ccggctccta cgcggaaaac ctgccggtag tagaaatcgt cggttccccg 300

acctctaaag ttcagaacga gggtaaattc gtgcaccata ctctggccga tggtgacttc 360

aaacacttca tgaagatgca cgaaccggtc actgctgctc gtacgctgct gaccgcggaa 420

aatgcgactg tcgagattga tcgtgtactg agcgcactgc tgaaagaacg caagcctgta 480

tacatcaacc tgccggttga tgtcgcggcc gccaaagcgg aaaaaccatc tctgccgctg 540

aaaaaggaga acagcacctc taacaccagc gaccaggaaa tcctgaacaa gatccaggag 600

tctctgaaga acgctaaaaa gccgatcgta atcaccggcc atgagattat ctctttcggt 660

ctggagaaaa ctgtcaccca gttcatcagc aaaaccaaac tgccgatcac caccctgaac 720

ttcggtaaat cctccgttga cgaagcgctg ccgtcctttc tgggtattta caacggcact 780

ctgtctgagc cgaacctgaa agagttcgtg gagtctgcgg attttatcct gatgctgggc 840 gtgaaactga cggattcctc caccggtgca ttcacccacc acctgaatga gaataaaatg 900

atctctctga acattgatga gggcaaaatc ttcaacgagc gtattcagaa cttcgatttc 960

gaatccctga tctcctccct gctggatctg tccgagattg aatataaagg caaatacatt 1020

gataagaagc aagaggactt cgtaccgtct aacgcgctgc tgagccagga ccgtctgtgg 1080

caagctgtgg aaaacctgac ccagtccaac gaaaccatcg tggcggaaca gggtacctcc 1140

ttcttcggtg ctagctctat cttcctgaaa tctaaaagcc acttcatcgg tcagccactg 1200

tggggctcta ttggctacac cttcccggca gcgctgggtt cccaaatcgc agacaaagaa 1260

tcccgccacc tgctgttcat tggtgacggc tctctgcaac tgaccgtaca ggagctgggt 1320

ctggcgattc gtgagaaaat caacccgatt tgtttcatca tcaacaacga tggctacact 1380

gttgagcgtg agatccacgg cccgaaccag tcctacaacg acattccgat gtggaactac 1440

tctaaactgc cggaatcctt cggtgcgact gaagaccgtg tcgtaagcaa gatcgtccgt 1500

accgaaaacg aattcgtgtc tgtcatgaaa gaagcacagg cggacccgaa ccgcatgtac 1560

tggatcgagc tgattctggc taaagagggc gcgccaaaag tactgaaaaa gatgggtaaa 1620

ctgttcgcag aacagaacaa atcctaa

1647

<210> 45

<211> 3696

<212> DNA

<213> Mycobacterium tuberculosis <220>

<221> CDS

<222> (1) .. (3696)

<400> 45

gtg gcc aac ata agt tea cca ttc 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 1 5 10 15 gag atg tac cgc aag ttc cgc gac gac ccc tec 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 tec 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 gcg gcc gcc ccg cag gca ccc ccc aag ccg gcc gac acc gcg gcc 240

Ala Ala Ala Ala Pro Gin Ala Pro Pro Lys Pro Ala Asp Thr Ala Ala 65 70 75 80 gcg ggc aac ggc gtg gtc gcc gca ctg gcc gcc aaa act gcc gtt ccc 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 gcg gtg ctg cgc ggc gcc gcc gcg 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 tec gcg teg ttg gag gtg ccg acg gcg acc 384

Ala Val Val Lys Asn Met Ser Ala Ser Leu Glu Val Pro Thr Ala Thr

115 120 125

age gtc egg gcg 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 cgc ggc ggc aag ate teg ttc acg 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 gcg 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 gcg gtc acg 576

Met Asn Arg His Tyr Thr Glu Val Asp Gly Lys Pro Thr Ala Val Thr

180 185 190 ccg gcg cac acc aat etc ggc ctg gcg ate gac ctg caa ggc aag gac 624

Pro Ala His Thr Asn Leu Gly Leu Ala lie Asp Leu Gin Gly Lys Asp

195 200 205 ggg aag cgt tec ctg gtg gtg gcc ggc ate aag egg tgc gag acc atg

672

Gly Lys Arg Ser Leu Val Val Ala Gly lie Lys Arg Cys Glu Thr Met 210 215 220

cga tte gcg cag tte gtc aeg gcc tac gaa gac ate gta cgc egg gcc 720

Arg Phe Ala Gin Phe Val Thr Ala Tyr Glu Asp lie Val Arg Arg Ala

225 230 235 240 cgc gac ggc aag ctg acc act gaa gac ttt gcc ggc gtg aeg att teg 768

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 ccg egg ctg 816

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 864

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 cgc ate gcc gag ctg ggc ate 912

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 cgc ate ate cag 960

Gly Lys Leu lie Thr Leu Thr Ser Thr Tyr Asp His Arg lie lie Gin

305 310 315 320 ggc gcg gaa teg ggc gac tte ctg cgc acc ate cac gag ttg ctg etc 1008

Gly Ala Glu Ser Gly Asp Phe Leu Arg Thr lie His Glu Leu Leu Leu

325 330 335 teg gat ggc tte tgg gac gag gtc tte cgc gaa ctg age ate cca tat 1056

Ser Asp Gly Phe Trp Asp Glu Val Phe Arg Glu Leu Ser lie Pro Tyr

340 345 350 ctg ccg gtg cgc tgg age acc gac aac ccc gac teg ate gtc gac aag 1104

Leu Pro Val Arg Trp Ser Thr Asp Asn Pro Asp Ser lie Val Asp Lys

355 360 365

aac get cgc gtc atg aac ttg ate gcg gcc tac cgc aac cgc ggc cat 1152

Asn Ala Arg Val Met Asn Leu lie 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 tte cgc 1200

Leu Met Ala Asp Thr Asp Pro Leu Arg Leu Asp Lys Ala Arg Phe Arg 385 390 395 400 agt cac ccc gae etc gaa gtg ctg acc cac gge ctg acg ctg tgg gat 1248

Ser His Pro Asp Leu Glu Val Leu Thr His Gly Leu Thr Leu Trp Asp

405 410 415 etc gat egg gtg tte aag gte gae gge ttt gcc ggt geg cag tac aag 1296

Leu Asp Arg Val Phe Lys Val Asp Gly Phe Ala Gly Ala Gin Tyr Lys

420 425 430 aaa ctg cgc gae gtg ctg gge 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 gge gtg gag tac gcc cat ate etc gae ccc gaa caa aag gag tgg 1392

lie Gly Val Glu Tyr Ala His lie Leu Asp Pro Glu Gin Lys Glu Trp

450 455 460

etc gaa caa egg gte gag acc aag cac gte 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 acg 1488

Gin Lys Tyr lie Leu Ser Lys Leu Asn Ala Ala Glu Ala Phe Glu Thr

485 490 495 tte eta cag acc aag tac gte gge cag aag egg tte teg ctg gaa gge 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 gae geg geg ate gae 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 gge etc gae gag gtg gte ate ggg atg ccg cac egg gge 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 gte gge aag ccg tac teg cag ate tte 1680

Leu Asn Val Leu Ala Asn lie Val Gly Lys Pro Tyr Ser Gin lie Phe

545 550 555 560 acc gag tte gag gge aac ctg aat ccg teg cag geg cac gge tec ggt 1728

Thr Glu Phe Glu Gly Asn Leu Asn Pro Ser Gin Ala His Gly Ser Gly

565 570 575 gae gte aag tac cac ctg gge gcc acc ggg ctg tac ctg cag atg tte 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 He 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 gcg 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 gcg 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 acg ctg aac ctg gcg 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 ggc ggc acc ate cac ate ate gtc aac aac cag ate ggc ttc acc acc 2064

Gly Gly Thr He His He He Val Asn Asn Gin He Gly Phe Thr Thr

675 680 685

gcg ccc gag tat tec agg tec age gag tac tgc acc gac gtc gca aag 2112

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 gcg 2160

Met He Gly Ala Pro He Phe His Val Asn Gly Asp Asp Pro Glu Ala

705 710 715 720 tgt gtc tgg gtg gcg egg ttg gcg gtg gac ttc cga caa egg ttc aag 2208

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 cgc cgc cgc ggg cac aac 2256

Lys Asp Val Val He 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 2304

Glu Gly Asp Asp Pro Ser Met Thr Asn Pro Tyr Val Tyr Asp Val Val

755 760 765 gac acc aag cgc ggg gcc cgc aaa age tac acc gaa gcc ctg ate gga 2352

Asp Thr Lys Arg Gly Ala Arg Lys Ser Tyr Thr Glu Ala Leu He Gly 770 775 780

cgt ggc gac ate teg atg aag gag gcc gag gac gcg ctg cgc gac tac 2400

Arg Gly Asp He 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 cgc gag ctg gag aag 2448

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 tec gac cag atg att ccc 2496

His Gly Val Gin Pro Ser Glu Ser Val Glu Ser Asp Gin Met He Pro

820 825 830 gcg ggg ctg gcc act gcg gtg gac aag teg ctg ctg gcc egg ate ggc 2544

Ala Gly Leu Ala Thr Ala Val Asp Lys Ser Leu Leu Ala Arg He Gly

835 840 845

gat gcg ttc etc gcc ttg ccg aac ggc ttc acc gcg cac ccg cga gtc 2592

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 cgc egg gag atg gcc tat gaa ggc aag ate 2640

Gin Pro Val Leu Glu Lys Arg Arg Glu Met Ala Tyr Glu Gly Lys He 865 870 875

gac tgg gcc ttt ggc gag ctg ctg gcg ctg ggc teg ctg gtg gcc gaa 2688

Asp Trp Ala Phe Gly Glu Leu Leu Ala Leu Gly Ser Leu Val Ala Glu

885 890 895 ggc aag ctg gtg cgc ttg teg ggg cag gac age cgc cgc ggc acc ttc 2736

Gly Lys Leu Val Arg Leu Ser Gly Gin Asp Ser Arg Arg Gly Thr Phe

900 905 910 tec cag egg cat teg gtt etc ate gac cgc cac act ggc gag gag ttc 2784

Ser Gin Arg His Ser Val Leu He Asp Arg His Thr Gly Glu Glu Phe

915 920 925

aca cca ctg cag ctg ctg gcg 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 gcc gcc 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 gcc gtg gtg etc 2928

Gly Phe Glu Tyr Gly Tyr Thr Val Gly Asn Pro Asp Ala Val Val Leu

965 970 975 tgg gag gcg cag ttc ggc gac ttc gtc aac ggc gcg cag teg ate ate 2976

Trp Glu Ala Gin Phe Gly Asp Phe Val Asn Gly Ala Gin Ser lie lie

980 985 990 gac gag ttc ate age tec ggt gag gcc 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 tct gcc egg ate gaa cgc ttc ttg cag ttg tgg gcg gaa 3114

His Thr Ser Ala Arg lie Glu Arg Phe Leu Gin Leu Trp Ala Glu 1025 1030 1035

ggt teg atg ace ate gcg atg ccg teg act ccg teg aac tac ttc 3159

Gly Ser Met Thr lie Ala Met Pro Ser Thr Pro Ser Asn Tyr Phe 1040 1045 1050

cac ctg eta cgc egg cat gcc ctg gac ggc ate caa cgc ccg ctg 3204

His Leu Leu Arg Arg His Ala Leu Asp Gly lie Gin Arg Pro Leu 1055 1060 1065

ate gtg ttc acg ccc aag teg atg ttg cgt cac aag gcc gcc 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 cgc tea gtg ctg 3294

Ser Glu lie 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 cgc 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 gcc 3384

Ser Arg lie Leu Leu Thr Ser Gly Lys Leu Tyr Tyr Glu Leu Ala 1115 1120 1125

gcc cgc aag gcc aag gac aac cgc aat gac etc gcg ate gtg egg 3429

Ala Arg Lys Ala Lys Asp Asn Arg Asn Asp Leu Ala lie Val Arg 1130 1135 1140

ctt gaa cag etc gec ccg ctg ccc agg cgt cga ctg cgt gaa acg 3474

Leu Glu Gin Leu Ala Pro Leu Pro Arg Arg Arg Leu Arg Glu Thr

1145 1150 1155

ctg gac cgc tac gag aac gtc aag gag ttc ttc tgg gtc caa gag 3519

Leu Asp Arg Tyr Glu Asn Val Lys Glu Phe Phe Trp Val Gin Glu

1160 1165 1170

gaa ccg gec aac cag ggt gcg tgg ccg cga ttc ggg etc gaa eta 3564

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 gec ggg ate aag cga ate teg 3609

Pro Glu Leu Leu Pro Asp Lys Leu Ala Gly lie Lys Arg lie Ser

1190 1195 1200

cgc egg gcg atg tea gee ccg teg tea ggc teg teg aag gtg cac 3654

Arg Arg Ala Met Ser Ala Pro Ser Ser Gly Ser Ser Lys Val His

1205 1210 1215

gec gtc gaa cag cag gag ate etc gac gag gcg ttc ggc tga 3696

Ala Val Glu Gin Gin Glu lie Leu Asp Glu Ala Phe Gly

1220 1225 1230

<210> 46

<211> 1231

<212> PRT

<213> Mycobacterium tubercul

<400> 46

Val Ala Asn lie Ser Ser Pro Phe Gly Gin Asn Glu Trp Leu Val Glu 1 5 10 15 Glu Met Tyr Arg Lys Phe Arg Asp Asp Pro Ser Ser Val Asp Pro Ser

20 25 30

Trp His Glu Phe Leu Val Asp Tyr Ser Pro Glu Pro Thr Ser 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

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 He Asp Asn Arg He Val 130 135 140

He Asn Asn Gin Leu Lys Arg Thr Arg Gly Gly Lys He 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 He Val Arg Arg Ala 225 230 235 240

Arg Asp Gly Lys Leu Thr Thr Glu Asp Phe Ala Gly Val Thr He 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 He He Gly Val Gly Ala Met Glu Tyr Pro

275 280 285

Ala Glu Phe Gin Gly Ala Ser Glu Glu Arg He Ala Glu Leu Gly He 290 295 300

Gly Lys Leu He Thr Leu Thr Ser Thr Tyr Asp His Arg He He Gin 305 310 315 320 Gly Ala Glu Ser Gly Asp Phe Leu Arg Thr lie His Glu Leu Leu Leu 325 330 335

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 lie 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 lie Pro Met Met Asp Ala Ala lie Asp Gin Cys Ala

515 520 525

Glu His Gly Leu Asp Glu Val Val lie Gly Met Pro His Arg Gly Arg 530 535 540

Leu Asn Val Leu Ala Asn lie 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

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 He 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 He Gly Ala Pro He 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

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 He 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 Glu Gly Lys He 865 870 875

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 He He

980 985 990

Asp Glu Phe He 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 Pro Asp 1010 1015 1020 His Thr Ser Ala Arg He Glu Arg Phe Leu Gin Leu Trp Ala Glu 1025 1030 1035

Gly Ser Met Thr He Ala Met Pro Ser Thr Pro Ser Asn Tyr Phe 1040 1045 1050

His Leu Leu Arg Arg His Ala Leu Asp Gly He Gin Arg Pro Leu 1055 1060 1065

He Val Phe Thr Pro Lys Ser Met Leu Arg His Lys Ala Ala Val 1070 1075 1080

Ser Glu He Lys Asp Phe Thr Glu He 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 He 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> Mycobacterium tuberculosis -ketoglutarate decarboxylase Kgd codon optimised gene

<400> 47

atggctaata tctcctctcc gtttggtcag aatgaatggc tggtagaaga aatgtaccgt 60

aaattccgcg atgacccgtc ctctgtggac ccgtcctggc atgaattcct ggtagactac 120

agcccggagc cgaccagcca accggcagcg gaaccaaccc gcgttacttc tccgctggta 180

gcggaacgtg cagctgctgc cgcgcctcag gcgccgccta aaccggcgga tactgccgca 240

gccggtaacg gtgtggtggc cgcactggct gctaagactg cggttccgcc gccagcagaa 300

ggcgatgaag ttgcagtcct gcgcggtgcg gcggctgcag tggtgaaaaa catgagcgcg 360

tccctggagg taccgaccgc cacgagcgtg cgcgcggtcc ctgctaaact gctgattgat 420

aaccgtattg tgatcaacaa ccagctgaaa cgtacccgtg gtggcaagat ctccttcact 480

catctgctgg gttatgcact ggtacaagcg gttaagaaat tccctaacat gaaccgtcat 540

tacactgagg tcgacggtaa accgacggct gttactccgg cacacacgaa cctgggcctg 600

gcgatcgacc tgcaaggtaa agatggtaag cgctccctgg tagttgcggg tattaaacgt 660

tgcgaaacca tgcgtttcgc acaattcgta accgcctacg aggacattgt ccgccgtgct 720

cgtgatggca aactgaccac cgaagatttt gcgggcgtta ctattagcct gaccaaccca 780

ggcaccatcg gcaccgtgca cagcgtacct cgtctgatgc cgggccaagg tgcgattatc 840

ggtgtgggtg ccatggagta cccggcagaa tttcagggtg cttctgaaga gcgcatcgcc 900

gagctgggta ttggtaaact gatcaccctg acttctacct atgaccaccg catcattcag 960

ggcgcagaat ccggtgactt cctgcgcact attcacgaac tgctgctgtc cgacggtttc 1020 tgggatgaag tttttcgtga actgagcatc ccatatctgc cagttcgctg gtccaccgac 1080

aatccggact ctatcgttga caaaaacgct cgcgtaatga acctgatcgc tgcttatcgt 1140

aatcgtggtc acctgatggc tgatacggat ccgctgcgcc tggataaagc tcgtttccgt 1200

tcccacccgg acctggaagt gctgacccat ggtctgactc tgtgggatct ggaccgcgtg 1260

ttcaaagtag atggtttcgc gggtgctcag tacaagaagc tgcgtgacgt gctgggtctg 1320

ctgcgtgatg cgtactgtcg tcacattggt gtggagtacg cccacattct ggatccggaa 1380

cagaaagaat ggctggagca gcgtgtcgag accaaacacg taaaaccgac cgtagcgcag 1440

cagaaatata tcctgtccaa actgaacgcc gccgaggctt tcgaaacttt cctgcagacc 1500

aagtacgtgg gccagaaacg cttcagcctg gagggtgcgg aaagcgttat tccgatgatg 1560

gatgcagcta tcgatcagtg cgcggaacat ggtctggatg aagtcgttat cggtatgccg 1620

caccgtggtc gcctgaacgt actggcaaac atcgtcggta aaccatattc tcagatcttc 1680

acggaattcg agggcaacct gaacccgtcc caagcccacg gctccggcga cgtaaaatat 1740

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 tgctgttgat 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 cgttggcaac 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 gccgtatcct gctgacctcc 3360 ggcaaactgt actacgagct ggcagcacgt aaggcaaaag ataaccgcaa cgacctggcc 3420

atcgtccgcc tggaacagct ggcgccactg ccacgccgtc gcctgcgtga aaccctggat 3480

cgctacgaaa acgtaaaaga attcttctgg gtgcaggaag aaccggcaaa ccagggtgcg 3540

tggccgcgct ttggtctgga actgccggaa ctgctgccgg ataaactggc aggtatcaag 3600

cgcatcagcc gtcgcgctat gagcgccccg tcttctggta gctctaaagt acacgctgta 3660

gaacagcaag agatcctgga tgaggccttc ggctaa

3696

<210> 48

<211> 74

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer for amplification of Bacillus subtilis aminotransferase x

<400> 48

ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgaagg ttttagt 60

tggccggctg attg

74

<210> 49

<211> 62

<212> DNA

<213> Artifici sequence

<220>

<223> Reverse primer for amplification of Bacillus subtilis aminotransferase x

<400> 49

ggggaccact ttgtacaaga aagctgggtt tatgaaatgc tagcagcctg ttgaatgctt 60

tc

62

<210> 50

<211> 82

<212> DNA

<213> Artificial sequence

<220> <223> Forward primer for amplification of Bacillus subtilis aminotransferase y

<400> 50

ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgactc atgatttgat 60

agaaaaaagt aaaaagcacc

82

<210> 51

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer for amplification of Bacillus subtilis

aminotransferase y

<400> 51

ggggaccact ttgtacaaga aagctgggtt caatcttcaa ggctcgtaac ctcgtgg 57

<210> 52

<211> 64

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer for amplification of Rhodobacter sphaeroides aminotransferase

<400> 52

ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgcccg gttgcggggg 60

cttg

64

<210> 53

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer for amplification of Rhodobacter sphaeroides aminotransferase

<400> 53

ggggaccact ttgtacaaga aagctgggtt cagacggcgg ccggttcttt c

51

<210> 54

<211> 78

<212> DNA <213> Artificial sequence

<220>

<223> Forward primer for amplification of Legionella pneumophila aminotransferase

<400> 54

ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgagta tcgcatttgt 60

taacggcaag tattgttg

78

<210> 55

<211> 67

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer for amplification of Legionella pneumophila amino r nsfer se

<400> 55

ggggaccact ttgtacaaga aagctgggtt tagtttacta gttgttggta ggaatcatta 60

attatcc

67

<210> 56

<211> 76

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer for amplification of Nitrosomonas europaea aminotransferase

<400> 56

ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgattt acctcaatgg 60

caaatttctg ccgatg

76

<210> 57

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer for amplification of Nitrosomonas europaea aminotransferase

<400> 57 ggggaccact ttgtacaaga aagctgggtt tactggcgtg gagcatgccc

50

<210> 58

<211> 79

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer for amplification of Neisseria gonorrhoeae aminotransferase

<400> 58

ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgagga taaatatgaa 60

ccgtaacgaa attttattc

79

<210> 59

<211> 56

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer for amplification of Neisseria gonorrhoeae aminotransferase

<400> 59

ggggaccact ttgtacaaga aagctgggtt catgcagcca tcgccttgaa cacttc 56

<210> 60

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer for amplification of Pseudomonas aeruginosa aminotransferase

<400> 60

ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgtcga tggccgatcg 60

tgatgg

66

<210> 61

<211> 53

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer for amplification of Pseudomonas aeruginosa aminotr nsfer se

<400> 61

ggggaccact ttgtacaaga aagctgggtt tacttgacca gggtacgcca etc

53

<210> 62

<211> 67

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer for amplification of Rhodopseudomonas palust aminotransferase

<400> 62

ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgaagc tgataccgtg 60

ccgcgcc

67

<210> 63

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer for amplification of Rhodopseudomonas palust aminotransferase

<400> 63

ggggaccact ttgtacaaga aagctgggtt caggcgaccg cgcggatcac c

51

<210> 64

<211> 1353

<212> DNA

<213> Bacillus subtilis

<400> 64

atggagatga tggggatgga aaacattcag caaaatcagg gattaaagca aaaagatgag 60

caatttgtgt ggcatgecat gaagggagcg catcaagegg acagectgat ageccagaag 120

gecgaagggg cctgggtaac cgacacagac ggacgccgct atttggatgc gatgtccggt 180

ttgtggtgcg tcaacattgg ttacggcaga aaggagcttg cggaggctgc ctatgagcaa 240

ctaaaggagc tgccttacta cccgttaacg caaagtcacg cacccgcaat tcaactggcg 300 gaaaagctga atgaatggct tggcggcgat tatgttattt ttttttccaa cagcggatcg 360

gaagcaaacg aaactgcttt taaaattgcc cgccagtacc atctgcaaaa cggcgaccac 420

agccgttata aattcatctc aagatatcgg gcataccacg gcaatacatt gggagcgctc 480

tcagctaccg gacaggcgca gcggaaatat aaatacgagc ctttgagcca agggttcctg 540

catgcagctc cgccagatat ataccggaat cctgatgatg cagacacgct tgaaagcgca 600

aatgaaatcg accgcatcat gacatgggaa ttaagcgaaa cgattgccgg ggtcattatg 660

gagcccatca ttacaggcgg aggcatccta atgccgccgg acggatatat gaagaaggtg 720

gaggacattt gccggcgcca cggagccctt ttgatttgcg atgaagtgat ctgcgggttt 780

ggacggacag gtgagccgtt cgggtttatg cactacggtg tgaagcctga tatcattacg 840

atggcaaagg gaatcacaag cgcgtatctg ccattgtcag cgactgctgt gaaacgggac 900

attttcgaag cgtatcaggg ggaagctcct tatgaccgtt tccgccacgt gaacacgttc 960

ggcggaagcc cggctgcctg tgctttggcg ttgaaaaacc tgcaaattat ggaggacgaa 1020

cagctgattc agcgatcccg tgatcttgga gcaaagcttt taggtgagct tcaagctctg 1080

agagaacacc cggcagtcgg ggatgttaga ggaaaagggc tgctgatcgg aatcgaactc 1140

gtcaaagaca aattgactaa agagccggct gatgccgcca aagtaaacca agtggttgcg 1200

gcgtgcaaag aaaaagggct gatcatcggc aaaaacggcg atacagtcgc cggctacaac 1260

aatgtcatcc acgttgcgcc gccattttgc ctgacagaag aggacctttc ctttatcgtg 1320

aaaacggtga aagaaagctt tcaaacgata taa

1353

<210> 65

<211> 450

<212> PRT

<213> Bacillus subtilis <400> 65

Met Glu Met Met Gly Met Glu Asn lie Gin Gin Asn Gin Gly Leu Lys 1 5 10 15

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 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 lie Gin Leu Ala Glu Lys Leu Asn Glu Trp Leu Gly Gly Asp Tyr Val

100 105 110

He Phe Phe Ser Asn Ser Gly Ser Glu Ala Asn Glu Thr Ala Phe Lys

115 120 125

He Ala Arg Gin Tyr His Leu Gin Asn Gly Asp His Ser Arg Tyr Lys 130 135 140 Phe He 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 He Asp Arg He Met Thr

195 200 205

Trp Glu Leu Ser Glu Thr He Ala Gly Val He Met Glu Pro He He 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 lie 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 lie lie Thr Met Ala Lys Gly lie Thr Ser Ala

275 280 285

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 lie

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

Val Arg Gly Lys Gly Leu Leu lie 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 lie 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

<400> 66

atgaacgcaa gactgcacgc cacgtccccc ctcggcgacg ccgacctggt ccgtgccgac 60

caggcccact acatgcacgg ctaccacgtg ttcgacgacc accgcgtcaa cggctcgctg 120

aacatcgccg ccggcgacgg cgcctatatc tacgacaccg ccggcaaccg ctacctcgac 180

gcggtgggcg gcatgtggtg caccaacatc ggcctggggc gcgaggaaat ggctcgcacc 240

gtggccgagc agacccgcct gctggcctat tccaatccct tctgcgacat ggccaacccg 300

cgcgccatcg aactctgccg caagctcgcc gagctggccc ccggcgacct cgaccacgtg 360

ttcctcacca ccggcggttc caccgccgtg gacaccgcga tccgcctcat gcactactac 420

cagaactgcc gcggcaagcg cgccaagaag cacgtcatca cgcggatcaa cgcctaccac 480

ggctcgacct tcctcggcat gtcgctgggc ggcaagagcg ccgaccggcc ggccgagttc 540

gacttcctcg acgagcgcat ccaccacctc gcctgtccct attactaccg cgctccggaa 600

gggctgggcg aagccgagtt cctcgatggc ctggtggacg agttcgaacg caagatcctc 660

gaactgggcg ccgaccgggt gggggcgttc atctccgagc cggtgttcgg ctccggcggc 720

gtgatcgtcc cgcccgcggg ctaccacagg 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 ccggt

1407

<210> 67

<211> 468

<212> PRT

<213> Pseudomonas aeruginosa

<400> 67

Met Asn Ala Arg Leu His Ala Thr Ser Pro Leu Gly Asp Ala Asp Leu 1 5 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 lie Ala Ala Gly Asp Gly Ala

35 40 45

Tyr lie 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 lie 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 lie Leu Glu Leu Gly Ala 210 215 220

Asp Arg Val Gly Ala Phe lie Ser Glu Pro Val Phe Gly Ser Gly Gly 225 230 235 240

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

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 lie 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 lie 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

Arg Val Leu Arg Glu Ser lie Glu Glu Thr Val Glu Asp Leu Val Arg 450 455 460

Ala Gly His Arg

465

<210> 68

<211> 1335

<212> DNA

<213> Pseudomonas aeruginosa

<400> 68

atgacaatga atgacgagcc gcagtcgagc agcctcgaca acttctggat gcccttcacc 60

gccaaccgcc agttcaaggc gcggccgcgc ctgctggaaa gcgccgaagg catccactat 120

atcgcccagg gcgggcgccg catcctcgac ggcaccgccg gcctctggtg ctgcaatgcc 180

ggccacggcc ggcgcgagat cagcgaagcg gtggcccggc agatcgccac cctcgactac 240

gccccgccgt tccagatggg tcacccgctg ccgttcgaac tcgccgcgcg gctgacggaa 300

atcgccccgc cgagcctgaa caaagtattc ttcaccaact ccggctcgga atcggcggac 360

accgcgctga agatcgccct tgcctaccag cgcgccatcg gccagggcac ccgcacccgc 420 ctgatcggcc gcgaactggg ctaccacggg gtcggcttcg gcggcctgtc ggtaggcggt 480

atggtcaaca accgcaaggc cttctccgcc aacctgctgc cgggggtcga ccacctgccg 540

cacaccctgg acgtcgcccg caacgccttc accgtcggcc tgcccgagca tggcgtggaa 600

aaggccgagg agctggaacg cctggtgacc ctgcacggcg ccgagaatat cgccgcggtg 660

atcgtcgagc cgatgtccgg ctcggccggc gtggtgctgc cgcccaaggg ctaccttcag 720

cggctgcgcg agataacccg caagcatggc atcctgctga tcttcgacga agtgatcacc 780

ggtttcggcc gcgtcggcga agccttcgcc gcgcagcgct ggggcgtcgt cccggacctg 840

ctgacctgcg ccaaggggct gaccaacggc agcatcccga tgggcgccgt attcgtcgac 900

gagaagatcc atgctgcctt catgcaaggc ccgcagggcg ccatcgagtt cttccacggc 960

tatacctatt ccggccatcc ggtagcctgc gccgccgccc tggcgaccct ggacatctac 1020

cgtcgcgacg acctgttcca gcgggccgtc gaactggaag gctactggca ggacgcgctg 1080

ttcagcctgc gcgacctgcc caacgtggtc gacatccgcg ccgtaggcct ggtcggcggc 1140

gtgcaactgg cgccgcacgc ggacggcccc ggcaagcgcg gctacgacgt cttcgagcgc 1200

tgcttctggg agcacgacct gatggtccgg gtgaccggcg acatcatcgc catgtcgccg 1260

ccgctgatca tcgacaagcc ccacatcgac cagatcgtcg agcgcctggc ccaggccatc 1320

cgcgccagcg tctga

1335 <210> 69

<211> 444

<212> PRT

<213> Pseudomonas aeruginosa

<400> 69

Met Thr Met Asn Asp Glu Pro Gin Ser Ser Ser Leu Asp Asn Phe Trp 1 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

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 lie 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

Arg Trp Gly Val Val Pro Asp Leu Leu Thr Cys Ala Lys Gly Leu Thr

275 280 285

Asn Gly Ser He Pro Met Gly Ala Val Phe Val Asp Glu Lys He His 290 295 300 Ala Ala Phe Met Gin Gly Pro Gin Gly Ala He Glu Phe Phe His Gly 305 310 315 320

Tyr Thr Tyr Ser Gly His Pro Val Ala Cys Ala Ala Ala Leu Ala Thr

325 330 335

Leu Asp He 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 Asp Leu Pro Asn

355 360 365

Val Val Asp He 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 Asp Val Phe Glu Arg 385 390 395 400

Cys Phe Trp Glu His Asp Leu Met Val Arg Val Thr Gly Asp He He

405 410 415

Ala Met Ser Pro Pro Leu He He Asp Lys Pro His He Asp Gin He

420 425 430

Val Glu Arg Leu Ala Gin Ala He Arg Ala Ser Val

435 440

<210> 70

<211> 71

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer for amplification of Bacillus subtilis aminotransferase (gil6077991) <400> 70

ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatggaga tgatggggat 60

ggaaaacatt c

71

<210> 71

<211> 65

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer for amplification of Bacillus subtilis aminotransferase (gil6077991)

<400> 71

ggggaccact ttgtacaaga aagctgggtt tatatcgttt gaaagctttc tttcaccgtt 60

ttcac

65

<210> 72

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer for amplification of Pseudomonas aeruginosa aminotransferase (gi9951072)

<400> 72

ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgaacg caagactgca 60

cgccac

66

<210> 73

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer for amplification of Pseudomonas aeruginosa aminotransferase (gi9951072)

<400> 73

ggggaccact ttgtacaaga aagctgggtt taccggtgac cggcgcgg

48

<210> 74

<211> 69

<212> DNA <213> Artificial sequence

<220>

<223> Forward primer for amplification of Pseudomonas aeruginosa aminotransferase (gi9951630)

<400> 74

ggggacaagt ttgtacaaaa aagcaggcta ggaggaatta accatgacaa tgaatgacga 60

gccgcagtc

69

<210> 75

<211> 49

<212> DNA

<213> Artificial sequence

Reverse primer for amplification of Pseudomona

aminotransferase (gi9951630)

<400> 75

ggggaccact ttgtacaaga aagctgggtt cagacgctgg cgcggatgg

<210> 76

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400> 76

aaatttacta gtaagaattt ttgaggaggc aatataaatg aataaaccac agtcttg

<210> 77

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 77

aaatttggat cctacaagaa agctgggttt ac

32 <210> 78

<211> 58

<212> DNA

<213> Artificial sequence <220>

<223> Forward primer

<400> 78

aaatttacta gtaagaattt ttgaggaggc aatataaatg aacagccaaa tcaccaac

<210> 79

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 79

aaatttggat ccactttgta caagaaagct gggtt

37

<210> 80

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400> 80

aaatttggat ccgttgagga ggcctcaaaa atgtccgaga tcactctggg caaatac 57

<210> 81

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 81

aaatttggcg cgccattact gtttagcgtt agttg

35

<210> 82

<211> 52

<212> DNA

<213> Artificial sequence

<220>

<223> Forward primer

<400> 82

aaatttggat ccgttgagga ggcctcaaaa atgtatactg ttggtgatta

52 <210> 83

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> Reverse primer

<400> 83

aaatttggcg cgccattact tgttctgctc cgcaaac

37 <210> 84

<211> 1113

<212> DNA

<213> Artificial Sequence

<220>

<223> Homo sapiens hydroxyacid oxidase (glycolate oxidase) (HAOX-5B) <400> 84

atgctccccc ggctaatttg tatcaatgat tatgaacaac atgctaaatc agtacttcca 60

aagtctatat atgactatta caggtctggg gcaaatgatg aagaaacttt ggctgataat 120

attgcagcat tttccagatg gaagctgtat ccaaggatgc tccggaatgt tgctgaaaca 180

gatctgtcga cttctgtttt aggacagagg gtcagcatgc caatatgtgt gggggctacg 240

gccatgcagc gcatggctca tgtggacggc gagcttgcca ctgtgagagc ctgtcagtcc 300

ctgggaacgg gcatgatgtt gagttcctgg gccacctcct caattgaaga agtggcggaa 360

gctggtcctg aggcacttcg ttggctgcaa ctgtatatct acaaggaccg agaagtcacc 420

aagaagctag tgcggcaggc agagaagatg ggctacaagg ccatatttgt gacagtggac 480

acaccttacc tgggcaaccg tctggatgat gtgcgtaaca gattcaaact gccgccacaa 540

ctcaggatga aaaattttga aaccagtact ttatcatttt ctcctgagga aaattttgga 600

gacgacagtg gacttgctgc atatgtggct aaagcaatag acccatctat cagctgggaa 660

gatatcaaat ggctgagaag actgacatca ttgccaattg ttgcaaaggg cattttgaga 720 ggtgatgatg ccagggaggc tgttaaacat ggcttgaatg ggatcttggt gtcgaatcat 780

ggggctcgac aactcgatgg ggtgccagcc actattgatg ttctgccaga aattgtggag 840

gctgtggaag ggaaggtgga agtcttcctg gacgggggtg tgcggaaagg cactgatgtt 900

ctgaaagctc tggctcttgg cgccaaggct gtgtttgtgg ggagaccaat cgtttggggc 960

ttagctttcc agggggagaa aggtgttcaa gatgtcctcg agatactaaa ggaagaattc 1020

cggttggcca tggctctgag tgggtgccag aatgtgaaag tcatcgacaa gacattggtg 1080

aggaaaaatc ctttggccgt ttccaagatc tga

1113

<210> 85

<211> 370

<212> PRT

<213> HAOX1_HUMAN Hydroxyacid oxidase 1 - Homo sapiens (Human

<400> 85

Met Leu Pro Arg Leu lie Cys lie Asn Asp Tyr Glu Gin His Ala 1 5 10 15

Ser Val Leu Pro Lys Ser lie Tyr Asp Tyr Tyr Arg Ser Gly Ala Asn

20 25 30

Asp Glu Glu Thr Leu Ala Asp Asn lie Ala Ala Phe Ser Arg Trp Lys

35 40 45

Leu Tyr Pro Arg Met Leu Arg Asn Val Ala Glu Thr Asp Leu Ser Thr 50 55 60

Ser Val Leu Gly Gin Arg Val Ser Met Pro lie Cys Val Gly Ala Thr 65 70 75 80

Ala Met Gin Arg Met Ala His Val Asp Gly Glu Leu Ala Thr Val Arg

85 90 95

Ala Cys Gin Ser Leu Gly Thr Gly Met Met Leu Ser Ser Trp Ala Thr

100 105 110

Ser Ser lie Glu Glu Val Ala Glu Ala Gly Pro Glu Ala Leu Arg Trp

115 120 125 Leu Gin Leu Tyr lie Tyr Lys Asp Arg Glu Val Thr Lys Lys Leu Val 130 135 140

Arg Gin Ala Glu Lys Met Gly Tyr Lys Ala lie Phe Val Thr Val Asp 145 150 155 160

Thr Pro Tyr Leu Gly Asn Arg Leu Asp Asp Val Arg Asn Arg Phe Lys

165 170 175

Leu Pro Pro Gin Leu Arg Met Lys Asn Phe Glu Thr Ser Thr Leu Ser

180 185 190

Phe Ser Pro Glu Glu Asn Phe Gly Asp Asp Ser Gly Leu Ala Ala Tyr

195 200 205

Val Ala Lys Ala lie Asp Pro Ser lie Ser Trp Glu Asp lie Lys Trp 210 215 220

Leu Arg Arg Leu Thr Ser Leu Pro lie Val Ala Lys Gly lie Leu Arg 225 230 235 240

Gly Asp Asp Ala Arg Glu Ala Val Lys His Gly Leu Asn Gly lie Leu

245 250 255

Val Ser Asn His Gly Ala Arg Gin Leu Asp Gly Val Pro Ala Thr lie

260 265 270

Asp Val Leu Pro Glu lie Val Glu Ala Val Glu Gly Lys Val Glu Val

275 280 285

Phe Leu Asp Gly Gly Val Arg Lys Gly Thr Asp Val Leu Lys Ala Leu 290 295 300

Ala Leu Gly Ala Lys Ala Val Phe Val Gly Arg Pro lie Val Trp Gly 305 310 315 320

Leu Ala Phe Gin Gly Glu Lys Gly Val Gin Asp Val Leu Glu lie Leu

325 330 335 Lys Glu Glu Phe Arg Leu Ala Met Ala Leu Ser Gly Cys Gin Asn Val

340 345 350

Lys Val lie Asp Lys Thr Leu Val Arg Lys Asn Pro Leu Ala Val Ser 355 360 365

Lys lie

370

<210> 86

<211> 1113

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic DNA (Homo sapiens hydroxyacid oxidase (glycolate oxidase) (HAOX-5B), codon pair optimised)

<400> 86

atgctgccac gtctgatttg tattaacgat tacgaacaac acgcgaagag cgtactgccg 60

aaatccattt acgattatta ccgttctggt gcaaacgatg aagaaacgct ggctgataac 120

atcgccgctt tttcccgttg gaaactgtac ccacgtatgc tgcgtaacgt tgccgaaacc 180

gacctgtcca ccagcgtcct gggtcagcgt gtgtccatgc caatctgcgt gggtgcaacc 240

gcaatgcagc gtatggcaca cgttgacggc gaactggcaa ccgtccgtgc gtgccagagc 300

ctgggtaccg gtatgatgct gagcagctgg gctacctcta gcatcgagga agtggcagaa 360

gctggtccgg aagcactgcg ctggctgcag ctgtacatct acaaagatcg cgaagtcact 420

aagaaactgg tgcgccaggc ggaaaagatg ggttacaagg caatctttgt gactgttgac 480

accccgtacc tgggtaaccg cctggatgac gttcgtaacc gcttcaagct gccgccgcag 540

ctgcgtatga agaactttga aaccagcacc ctgtcctttt ccccagaaga aaatttcggt 600

gatgactctg gtctggccgc gtacgtcgcg aaagctatcg atccgtccat ctcctgggaa 660

gatatcaaat ggctgcgtcg tctgacttcc ctgccgatcg ttgctaaggg tattctgcgt 720

ggtgacgacg cgcgtgaagc tgttaaacat ggtctgaacg gcattctggt aagcaaccat 780

ggcgcacgcc agctggatgg tgtacctgct actattgatg tgctgccgga aatcgtggaa 840 gcggttgaag gtaaagttga agtgttcctg gacggtggtg tgcgcaaagg caccgatgta 900

ctgaaagcac tggcgctggg tgcgaaagcc gtctttgttg gccgtcctat tgtttggggt 960

ctggcattcc agggtgagaa aggtgtacag gacgttctgg agatcctgaa agaggagttc 1020

cgcctggcta tggcgctgtc tggttgtcaa aacgtgaaag taatcgataa aaccctggta 1080

cgtaaaaacc ctctggcagt aagcaagatc taa

1113

<210> 87

<211> 1125

<212> DNA

<213> Artificial Sequence

<220>

<223> Lactate oxidase - Aerococcus viridans (wt) (LAOX-8C) <400> 87

atgaataaca atgacattga atataatgca cctagtgaaa tcaagtacat tgatgttgtc 60

aatacttacg acttagaaga agaagcaagt aaagtggtac cacatggtgg ttttaactat 120

attgccggtg catctggtga tgagtggact aaacgcgcta atgaccgtgc ttggaaacat 180

aaattactat acccacgtct agcgcaagat gttgaagcgc ccgatacaag tactgaaatt 240

ttaggtcata aaattaaagc cccattcatc atggcaccaa ttgctgcaca tggtttagcc 300

cacactacta aagaagctgg tactgcacgt gcagtttcag aatttggtac aattatgtcc 360

atctcagctt attctggtgc aacatttgaa gaaatttctg aaggcttaaa tggcggaccc 420

cgttggttcc aaatctatat ggctaaagat gaccaacaaa accgtgatat cttagacgaa 480

gctaaatctg atggtgcaac tgctatcatc cttacagctg actcaactgt ttctggaaac 540

cgtgaccgtg atgtgaagaa taaattcgtt tacccatttg gtatgccaat tgttcaacgt 600

tacttacgtg gtacagcaga aggtatgtca ttaaacaata tctacggtgc ttcaaaacaa 660 aaaatctcac caagagatat tgaggaaatc gccggtcatt ctggattacc agtattcgtt 720

aaaggtattc aacacccaga agatgcagat atggcaatca aacgtggtgc atcaggtatc 780

tgggtatcta accacggtgc tcgtcaacta tatgaagctc caggttcatt tgacaccctt 840

ccagctattg ctgaacgtgt aaacaaacgt gtaccaatcg tctttgattc aggtgtacgt 900

cgtggtgaac acgttgccaa agcgctagct tcaggggcag acgttgttgc tttaggacgc 960

ccagtcttat ttggtttagc tttaggtggc tggcaaggtg cttactcagt acttgactac 1020

ttccaaaaag acttaacacg cgtaatgcaa ttaacaggtt cacaaaatgt ggaagacttg 1080

aagggtctag atttattcga taacccatac ggttatgaat actag

1125

<210> 88

<211> 374

<212> PRT

<213> LAOX-8C Lactate oxidase - Aerococcus viridans

<400> 88

Met Asn Asn Asn Asp lie Glu Tyr Asn Ala Pro Ser Glu lie Lys Tyr 1 5 10 15 lie Asp Val Val Asn Thr Tyr Asp Leu Glu Glu Glu Ala Ser Lys Val

20 25 30

Val Pro His Gly Gly Phe Asn Tyr lie Ala Gly Ala Ser Gly Asp Glu

35 40 45 Trp Thr Lys Arg Ala Asn Asp Arg Ala Trp Lys His Lys Leu Leu Tyr 50 55 60

Pro Arg Leu Ala Gin Asp Val Glu Ala Pro Asp Thr Ser Thr Glu lie 65 70 75 80

Leu Gly His Lys lie Lys Ala Pro Phe lie Met Ala Pro lie Ala Ala

85 90 95

His Gly Leu Ala His Thr Thr Lys Glu Ala Gly Thr Ala Arg Ala Val

100 105 110 Ser Glu Phe Gly Thr He Met Ser He Ser Ala Tyr Ser Gly Ala Thr 115 120 125

Phe Glu Glu He Ser Glu Gly Leu Asn Gly Gly Pro Arg Trp Phe Gin 130 135 140 He Tyr Met Ala Lys Asp Asp Gin Gin Asn Arg Asp He Leu Asp Glu 145 150 155 160

Ala Lys Ser Asp Gly Ala Thr Ala He He Leu Thr Ala Asp Ser Thr

165 170 175

Val Ser Gly Asn Arg Asp Arg Asp Val Lys Asn Lys Phe Val Tyr Pro

180 185 190

Phe Gly Met Pro He Val Gin Arg Tyr Leu Arg Gly Thr Ala Glu Gly

195 200 205

Met Ser Leu Asn Asn He Tyr Gly Ala Ser Lys Gin Lys He Ser Pro 210 215 220

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

Lys Gly He Gin His Pro Glu Asp Ala Asp Met Ala He Lys Arg Gly

245 250 255

Ala Ser Gly He Trp Val Ser Asn His Gly Ala Arg Gin Leu Tyr Glu

260 265 270

Ala Pro Gly Ser Phe Asp Thr Leu Pro Ala He Ala Glu Arg Val Asn

275 280 285

Lys Arg Val Pro He Val Phe Asp Ser Gly Val Arg Arg Gly Glu His 290 295 300 Val Ala Lys Ala Leu Ala Ser Gly Ala Asp Val Val Ala Leu Gly Arg 305 310 315 320

Pro Val Leu Phe Gly Leu Ala Leu Gly Gly Trp Gin Gly Ala Tyr Ser

325 330 335

Val Leu Asp Tyr Phe Gin Lys Asp Leu Thr Arg Val Met Gin Leu Thr

340 345 350 Gly Ser Gin Asn Val Glu Asp Leu Lys Gly Leu Asp Leu Phe Asp Asn 355 360 365

Pro Tyr Gly Tyr Glu

370

<210> 89

<211> 1125

<212> DNA

<213> Artificial Sequence

<220>

<223> gene for Lactate oxidase (codon pair optimised)

<400> 89

atgaacaaca acgacatcga atataacgct ccttctgaaa tcaaatatat cgacgtggtt 60

aacacctatg acctggagga agaagcgtct aaggtcgtac cgcacggtgg tttcaattac 120

attgcaggtg cctctggtga tgaatggacc aaacgcgcaa acgatcgtgc atggaaacac 180

aaactgctgt atccgcgcct ggcccaggat gtggaagcac cggatacttc cactgaaatc 240

ctgggtcaca aaatcaaggc accgtttatt atggctccga tcgcagcgca cggcctggca 300

cacaccacca aagaagctgg caccgctcgt gcggtttctg agttcggcac cattatgtct 360

atctctgcgt atagcggtgc cactttcgag gaaatttccg agggcctgaa cggtggcccg 420

cgttggtttc agatttacat ggcgaaagat gaccagcaga accgcgatat cctggatgaa 480

gccaaatctg acggcgcgac tgctatcatc ctgaccgcgg actctaccgt atccggtaac 540

cgtgaccgtg atgtgaagaa caagttcgtc tatcctttcg gtatgccgat tgttcagcgc 600

tatctgcgcg gtaccgctga gggtatgagc ctgaacaaca tctatggtgc gtccaaacag 660

aaaatcagcc cacgtgacat cgaagaaatt gctggtcata gcggtctgcc ggtgtttgtg 720

aaaggtatcc agcatccaga agatgcggac atggcaatca aacgtggtgc gtctggcatc 780 tgggttagca accacggtgc gcgtcagctg tacgaagctc cgggtagctt cgataccctg 840

ccggccatcg cggaacgtgt gaataaacgc gtgccgatcg ttttcgattc cggtgtgcgt 900

cgtggtgaac atgtggcaaa agcactggcg tctggcgctg atgtcgtagc actgggccgt 960

ccagtgctgt tcggtctggc tctgggtggc tggcagggcg cttactccgt cctggattac 1020

tttcagaaag acctgacccg tgttatgcag ctgaccggtt cccagaacgt agaggacctg 1080

aaaggcctgg acctgttcga caacccttac ggttacgaat actaa

1125

<210> 90

<211> 314

<212> PRT

<213> EC 1.1.1.27 - L-lactate dehydrogenases >Q8NLN0_Corynebacter ^' glutamicum

<400> 90

Met Lys Glu Thr Val Gly Asn Lys He Val Leu He Gly Ala Gly Asp 1 5 10 15

Val Gly Val Ala Tyr Ala Tyr Ala Leu He Asn Gin Gly Met Ala Asp

20 25 30

His Leu Ala He He Asp He Asp Glu Lys Lys Leu Glu Gly Asn Val

35 40 45

Met Asp Leu Asn His Gly Val Val Trp Ala Asp Ser Arg Thr Arg Val 50 55 60

Thr Lys Gly Thr Tyr Ala Asp Cys Glu Asp Ala Ala Met Val Val He 65 70 75 80

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

85 90 95

Asp Lys Asn Val Lys He Met Lys Ser He Val Gly Asp Val Met Asp

100 105 110

Ser Gly Phe Asp Gly He Phe Leu Val Ala Ser Asn Pro Val Asp He

115 120 125 Leu Thr Tyr Ala Val Trp Lys Phe Ser Gly Leu Glu Trp Asn Arg Val 130 135 140

He Gly Ser Gly Thr Val Leu Asp Ser Ala Arg Phe Arg Tyr Met Leu 145 150 155 160

Gly Glu Leu Tyr Glu Val Ala Pro Ser Ser Val His Ala Tyr He He

165 1 70 1 75

Gly Glu His Gly Asp Thr Glu Leu Pro Val Leu Ser Ser Ala Thr He

180 185 190

Ala Gly Val Ser Leu Ser Arg Met Leu Asp Lys Asp Pro Glu Leu Glu

195 200 205

Gly Arg Leu Glu Lys He Phe Glu Asp Thr Arg Asp Ala Ala Tyr His

2 10 2 15 220

He He Asp Ala Lys Gly Ser Thr Ser Tyr Gly He Gly Met Gly Leu 225 230 235 240

Ala Arg He Thr Arg Ala He Leu Gin Asn Gin Asp Val Ala Val Pro

245 250 255

Val Ser Ala Leu Leu His Gly Glu Tyr Gly Glu Glu Asp He Tyr He

260 265 2 70

Gly Thr Pro Ala Val Val Asn Arg Arg Gly He Arg Arg Val Val Glu

2 75 280 285

Leu Glu He Thr Asp His Glu Met Glu Arg Phe Lys His Ser Ala Asn

290 295 300 Thr Leu Arg Glu He Gin Lys Gin Phe Phe

305 3 10

<210> 91

<21 1> 329

<212> PRT

<213> EC 1 . 1 . 1 . 28 - D- lactate dehydrogenases >P52643_E scherichia col i <400> 91

Met Lys Leu Ala Val Tyr Ser Thr Lys Gin Tyr Asp Lys Lys Tyr Leu 1 5 10 15 Gin Gin Val Asn Glu Ser Phe Gly Phe Glu Leu Glu Phe Phe Asp Phe 20 25 30

Leu Leu Thr Glu Lys Thr Ala Lys Thr Ala Asn Gly Cys Glu Ala Val

35 40 45

Cys lie Phe Val Asn Asp Asp Gly Ser Arg Pro Val Leu Glu Glu Leu 50 55 60

Lys Lys His Gly Val Lys Tyr lie Ala Leu Arg Cys Ala Gly Phe Asn 65 70 75 80

Asn Val Asp Leu Asp Ala Ala Lys Glu Leu Gly Leu Lys Val Val Arg

85 90 95

Val Pro Ala Tyr Asp Pro Glu Ala Val Ala Glu His Ala lie Gly Met

100 105 110

Met Met Thr Leu Asn Arg Arg lie His Arg Ala Tyr Gin Arg Thr Arg

115 120 125

Asp Ala Asn Phe Ser Leu Glu Gly Leu Thr Gly Phe Thr Met Tyr Gly 130 135 140

Lys Thr Ala Gly Val lie Gly Thr Gly Lys lie Gly Val Ala Met Leu 145 150 155 160

Arg lie Leu Lys Gly Phe Gly Met Arg Leu Leu Ala Phe Asp Pro Tyr

165 170 175

Pro Ser Ala Ala Ala Leu Glu Leu Gly Val Glu Tyr Val Asp Leu Pro

180 185 190

Thr Leu Phe Ser Glu Ser Asp Val lie Ser Leu His Cys Pro Leu Thr

195 200 205

Pro Glu Asn Tyr His Leu Leu Asn Glu Ala Ala Phe Glu Gin Met Lys 210 215 220

Asn Gly Val Met lie Val Asn Thr Ser Arg Gly Ala Leu lie Asp Ser 225 230 235 240

Gin Ala Ala lie Glu Ala Leu Lys Asn Gin Lys lie Gly Ser Leu Gly

245 250 255 Met Asp Val Tyr Glu Asn Glu Arg Asp Leu Phe Phe Glu Asp Lys Ser 260 265 270

Asn Asp Val lie Gin Asp Asp Val Phe Arg Arg Leu Ser Ala Cys His

275 280 285

Asn Val Leu Phe Thr Gly His Gin Ala Phe Leu Thr Ala Glu Ala Leu 290 295 300

Thr Ser lie Ser Gin Thr Thr Leu Gin Asn Leu Ser Asn Leu Glu Lys 305 310 315 320

Gly Glu Thr Cys Pro Asn Glu Leu Val

325

<210> 92

<211> 312

<212> PRT

<213> EC 1.1.1.37 - malate dehydrogenases >P61889_Escherichia coli

<400> 92

Met Lys Val Ala Val Leu Gly Ala Ala Gly Gly lie Gly Gin Ala Leu 1 5 10 15

Ala Leu Leu Leu Lys Thr Gin Leu Pro Ser Gly Ser Glu Leu Ser Leu

20 25 30

Tyr Asp lie Ala Pro Val Thr Pro Gly Val Ala Val Asp Leu Ser His

35 40 45

He Pro Thr Ala Val Lys He Lys Gly Phe Ser Gly Glu Asp Ala Thr 50 55 60 Pro Ala Leu Glu Gly Ala Asp Val Val Leu He Ser Ala Gly Val Ala 65 70 75 80

Arg Lys Pro Gly Met Asp Arg Ser Asp Leu Phe Asn Val Asn Ala Gly

85 90 95

He Val Lys Asn Leu Val Gin Gin Val Ala Lys Thr Cys Pro Lys Ala

100 105 110

Cys He Gly He He Thr Asn Pro Val Asn Thr Thr Val Ala He Ala

115 120 125 Ala Glu Val Leu Lys Lys Ala Gly Val Tyr Asp Lys Asn Lys Leu Phe 130 135 140

Gly Val Thr Thr Leu Asp He He Arg Ser Asn Thr Phe Val Ala Glu 145 150 155 160

Leu Lys Gly Lys Gin Pro Gly Glu Val Glu Val Pro Val He Gly Gly

165 170 175

His Ser Gly Val Thr He Leu Pro Leu Leu Ser Gin Val Pro Gly Val

180 185 190

Ser Phe Thr Glu Gin Glu Val Ala Asp Leu Thr Lys Arg He Gin Asn

195 200 205

Ala Gly Thr Glu Val Val Glu Ala Lys Ala Gly Gly Gly Ser Ala Thr 210 215 220

Leu Ser Met Gly Gin Ala Ala Ala Arg Phe Gly Leu Ser Leu Val Arg 225 230 235 240

Ala Leu Gin Gly Glu Gin Gly Val Val Glu Cys Ala Tyr Val Glu Gly

245 250 255

Asp Gly Gin Tyr Ala Arg Phe Phe Ser Gin Pro Leu Leu Leu Gly Lys

260 265 270

Asn Gly Val Glu Glu Arg Lys Ser He Gly Thr Leu Ser Ala Phe Glu

275 280 285

Gin Asn Ala Leu Glu Gly Met Leu Asp Thr Leu Lys Lys Asp He Ala 290 295 300

Leu Gly Glu Glu Phe Val Asn Lys

305 310 <210> 93

<211> 312

<212> PRT

<213> >P49814_Bacillus subtilis

<400> 93

Met Gly Asn Thr Arg Lys Lys Val Ser Val He Gly Ala Gly Phe Thr 1 5 10 15 Gly Ala Thr Thr Ala Phe Leu He Ala Gin Lys Glu Leu Ala Asp Val 20 25 30

Val Leu Val Asp He Pro Gin Leu Glu Asn Pro Thr Lys Gly Lys Ala

35 40 45

Leu Asp Met Leu Glu Ala Ser Pro Val Gin Gly Phe Asp Ala Lys He 50 55 60

Thr Gly Thr Ser Asn Tyr Glu Asp Thr Ala Gly Ser Asp He Val Val 65 70 75 80

He Thr Ala Gly He Ala Arg Lys Pro Gly Met Ser Arg Asp Asp Leu

85 90 95

Val Ser Thr Asn Glu Lys He Met Arg Ser Val Thr Gin Glu He Val

100 105 110

Lys Tyr Ser Pro Asp Ser He He Val Val Leu Thr Asn Pro Val Asp

115 120 125 Ala Met Thr Tyr Ala Val Tyr Lys Glu Ser Gly Phe Pro Lys Glu Arg 130 135 140

Val He Gly Gin Ser Gly Val Leu Asp Thr Ala Arg Phe Arg Thr Phe 145 150 155 160

Val Ala Glu Glu Leu Asn Leu Ser Val Lys Asp Val Thr Gly Phe Val

165 170 175

Leu Gly Gly His Gly Asp Asp Met Val Pro Leu Val Arg Tyr Ser Tyr

180 185 190

Ala Gly Gly He Pro Leu Glu Thr Leu He Pro Lys Glu Arg He Asp

195 200 205

Ala He Val Glu Arg Thr Arg Lys Gly Gly Gly Glu He Val Asn Leu 210 215 220

Leu Gly Asn Gly Ser Ala Tyr Tyr Ala Pro Ala Ala Ser Leu Thr Glu 225 230 235 240

Met Val Glu Ala He Leu Lys Asp Gin Arg Arg Val Leu Pro Thr He

245 250 255 Ala Tyr Leu Glu Gly Glu Tyr Gly Tyr Glu Gly He Tyr Leu Gly Val 260 265 2 70

Pro Thr He Val Gly Gly Asn Gly Leu Glu Gin He He Glu Leu Glu

2 75 280 285

Leu Thr Asp Tyr Glu Arg Ala Gin Leu Asn Lys Ser Val Glu Ser Val

290 295 300

Lys Asn Val Met Lys Val Leu Ser

305 3 10

<210> 94

<21 1> 365

<212> PRT

<213> EC 1 . 1 . 1 . 8 1 - hydr oxypyruvate reductase >A3LRN9_Pichia stipiti

<400> 94

Met Thr Leu Lys Gin Gin Val Leu Phe Val Gly Lys Pro Asn Thr Asn 1 5 10 15 Thr Glu Ala Tyr Lys Lys Phe Ser Ala Asn Phe Glu Val He Asn Tyr

20 25 30

Lys He Thr Ser Lys Ser Gin Leu He Glu Asp Phe Glu Gly Arg Leu

35 40 45

Arg Tyr He Glu Ala He Tyr Ala Gly Trp Gly Gly Phe Asp Gly Val

50 55 60

Gly Gly Phe Gin Gly Glu Val Leu Arg His Cys Pro Pro Asn Val Lys 65 70 75 80

Val Val Ala He Cys Ser He Gly His Asp Gly Tyr Asp Thr Glu Gly

85 90 95 Met Ser Lys Arg Gly He Thr Leu Thr Asn Val Pro Ser Val He Ala

100 105 1 10

Ser Glu Ala Val Ala Asp Leu Val Leu Tyr Asn Thr Leu Ser Ser Phe

1 15 120 125

Arg Asn Phe Lys Met Phe Glu Lys Asn Leu Gly Gly Lys Leu Thr Asn

130 135 140 Thr Gly Ala Leu Arg Thr Ala Leu Val Arg Gly Glu Phe Asp Gin Phe 145 150 155 160

Asn Gly Val Pro Val lie Lys Pro Thr Val Gly Gly Ala Phe Ala Ser

165 170 175

Ser Cys Cys Gly Arg Asp lie Leu Ser Pro Arg Gly His Asn Val Val

180 185 190 lie Val Gly Phe Gly Ser lie Gly Lys Leu lie Gly Glu Arg Leu Ala

195 200 205

Cys lie Gly Met Asn lie His Tyr Val Lys Arg Ser Lys Leu Ser Glu 210 215 220

Gin Glu Glu Ala Ser Leu Gly Tyr Lys Val Thr Tyr His Ala Thr Leu 225 230 235 240

Lys Asp Thr Lys Asn lie Ala Asp Leu Val Val lie Ala Cys Pro Gly

245 250 255

Thr Ala His Thr Arg His Met Val Asn Glu Glu Met lie Asn Asp Phe

260 265 270

Ala Lys Pro Phe Arg Leu lie Asn lie Gly Arg Gly Tyr Val Val Asp

275 280 285

Glu Lys Ala Leu Val Asn Gly Leu Gin Ser Gly Lys lie Leu Phe Ala 290 295 300

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

Asn Arg Gin Asp Val Val Leu Thr Pro His lie Gly Ser Ser Thr Thr

325 330 335

Glu Asn Phe Asn Tyr Thr Ala Ala Ala Ala Met Phe Asn lie Glu Thr

340 345 350

Val Leu Tyr Asp Arg Glu Asp Thr lie Thr Arg Val Asn

355 360 365

<210> 95 <211> 424

<212> PRT

<213> >Q88F00_Pseudomonas putida

<400> 95

Met Ser Val Asp Pro Gin Lys Leu Leu Arg Glu Leu Phe Asp Thr Ala 1 5 10 15 lie Ala Ala Ala His Pro Arg Gin Val Leu Glu Pro Tyr Leu Pro Ala

20 25 30

Asp Arg Ser Gly Arg Val lie Val lie Gly Ala Gly Lys Ala Ala Ala

35 40 45

Ala Met Ala Glu Val Val Glu Lys Ser Trp Gin Gly Glu Val Ser Gly 50 55 60

Leu Val Val Thr Arg Tyr Gly His Gly Ala Asn Cys Gin Lys lie Glu 65 70 75 80

Val Val Glu Ala Ala His Pro Val Pro Asp Ala Ala Gly Leu Ala Val

85 90 95

Ala Lys Arg Val Leu Glu Leu Val Ser Asn Leu Asn Glu Glu Asp Arg

100 105 110

Val lie Phe Leu Leu Ser Gly Gly Gly Ser Ala Leu Leu Ala Leu Pro

115 120 125

Ala Glu Gly Leu Thr Leu Ala Asp Lys Gin Gin lie Asn Lys Ala Leu 130 135 140

Leu Lys Ser Gly Ala Thr lie Gly Glu Met Asn Cys Val Arg Lys His 145 150 155 160

Leu Ser Ala lie Lys Gly Gly Arg Leu Ala Lys Ala Cys Trp Pro Ala

165 170 175

Thr Val Tyr Thr Tyr Ala lie Ser Asp Val Pro Gly Asp Leu Ala Thr

180 185 190 Val lie Ala Ser Gly Pro Thr Val Ala Asp Pro Ser Thr Ser Ala Asp

195 200 205

Ala Leu Ala lie Leu Lys Arg Tyr Asn lie Glu Ala Pro Lys Ala Val 210 215 220

He Asp Trp Leu Asn Asn Pro Ala Ser Glu Thr Val Lys Ala Asp Asp 225 230 235 240

Pro Ala Leu Ala Arg Ser His Phe Gin Leu He Ala Lys Pro Gin Gin

245 250 255

Ser Leu Glu Ala Ala Ala Val Lys Ala Arg Gin Ala Gly Phe Ser Pro

260 265 270

Leu He Leu Gly Asp Leu Glu Gly Glu Ser Arg Glu Val Ala Lys Val

275 280 285 His Ala Gly He Ala Arg Gin He Val Gin His Gly Gin Pro Leu Lys 290 295 300

Ala Pro Cys Val He Leu Ser Gly Gly Glu Thr Thr Val Thr Val Arg 305 310 315 320

Gly Asn Gly Arg Gly Gly Arg Asn Ala Glu Phe Leu Leu Ser Leu Thr

325 330 335

Glu Ser Leu Lys Gly Leu Pro Gly Val Tyr Ala Leu Ala Gly Asp Thr

340 345 350

Asp Gly He Asp Gly Ser Glu Glu Asn Ala Gly Ala Phe Met Thr Pro

355 360 365 Ala Ser Tyr Ala Ser Ala Glu Ala Leu Gly Leu Ser Ala Ser Asp Glu 370 375 380

Leu Asp Asn Asn Asn Gly Tyr Gly Tyr Phe Ala Ala Leu Asp Ala Leu 385 390 395 400

He Val Thr Glu Pro Thr Arg Thr Asn Val Asn Asp Phe Arg Ala He

405 410 415

Leu He Leu Glu Thr Ala Gin Ser

420

<210> 96

<211> 347

<212> PRT <213> EC 1.1.1.82 - raalate dehydrogenases [NADP+]

>Q8NSK9_Corynebacteriura glutamicum

<400> 96

Met Pro Glu Val Thr Val Asn Ala Gin Gin Leu Thr Val Leu Cys Thr 1 5 10 15

Asp He Leu Thr Lys Thr Gly Val Pro Ala Ala Asp Ala His Leu Val

20 25 30

Gly Asp Ser Leu Val Gin Ala Asp Leu Trp Gly His Pro Ser His Gly

35 40 45

Val Leu Arg Leu Pro Trp Tyr Val Arg Arg Leu His Ser Gly Ala Met 50 55 60

Thr Thr His Ala His Val Glu Val Leu Asn Asp Leu Gly Ala Val Leu 65 70 75 80

Ala Leu Asp Gly His Asn Gly He Gly Gin Val Leu Ala Asp His Ala

85 90 95

Arg Lys Glu Ala Val Thr Arg Ala Met Met Phe Gly He Gly Ala Val

100 105 110

Ser Val Arg Asn Ser Asn His Phe Gly Thr Ala Met Tyr Tyr Thr Arg

115 120 125

Lys Ala Ala Ala Gin Gly Cys Val Ser He Leu Thr Thr Asn Ala Ser 130 135 140

Pro Ala Met Ala Pro Trp Gly Gly Arg Glu Lys Arg He Gly Thr Asn 145 150 155 160

Pro Trp Ser He Ala Ala Pro Phe Gly Glu Thr Ala Thr Val Val Asp

165 170 175 He Ala Asn Thr Ala Val Ala Arg Gly Lys He Tyr His Ala Arg Gin

180 185 190

Thr Asn Met Pro He Pro Glu Thr Trp Ala He Thr Ser Glu Gly Ala

195 200 205

Pro Thr Thr Asp Pro Ala Glu Ala He Asn Gly Val Val Leu Pro Met 210 215 220 Ala Gly His Lys Gly Tyr Ala lie Ser Phe Met Met Asp Val Leu Ser 225 230 235 240

Gly Val Leu Thr Gly Ser Gin His Ser Thr Lys Val His Gly Pro Tyr

245 250 255

Asp Pro Thr Pro Pro Gly Gly Ala Gly His Leu Phe lie Ala Leu Asp

260 265 270

Val Ala Ala Phe Arg Asp Pro Gin Asp Phe Asp Asp Ala Leu Ser Asp

275 280 285

Leu Val Gly Glu Val Lys Ser Thr Pro Lys Ala Gin Asn Thr Glu Glu 290 295 300 lie Phe Tyr Pro Gly Glu Ser Glu Asp Arg Ala His Arg Lys Asn Ser 305 310 315 320

Ala His Gly lie Ser Leu Pro Glu Lys Thr Trp Met Glu Leu Gin Glu

325 330 335

Leu Ala lie Glu Asn His Val Val Thr His Arg

340 345 <210> 97

<211> 315

<212> PRT

<213> >Q5E5E9_Vibrio fischeri

<400> 97

Met Lys Val Ser Tyr Tyr Glu Val Lys Glu Arg Leu lie Arg Lys Phe 1 5 10 15

He Ala Ser Gly Leu Ala Trp Asp Asp Ala Asn Trp Val Thr Asp Val

20 25 30 Leu He Ser Ser Glu Gin Arg Gly Asp Lys Ser His Gly He Lys His

35 40 45

Ala Lys Asn He Phe Asp Val He Asn Ser Glu Cys Tyr He Ala Gin 50 55 60

Ala Pro He He His Asp Glu Arg Ser He Thr He Leu Asp Gly Gin 65 70 75 80 Asn Ser lie Gly Pro lie Val Ala Lys Gin Ala lie Asp lie Ala lie 85 90 95

Lys Lys Ala Lys Lys Tyr Gly Thr Ala Ala lie Ser Leu Arg Ser Ser

100 105 110

Asn His Leu Phe Ser Leu Ser His Tyr Val Arg Tyr lie Ala Asn Asn

115 120 125

Asn Met lie Gly Phe lie Cys Ser Ser Ser Ser Pro Ala Met Ala Ala 130 135 140

Pro Asn Ser Leu Asn Ala Thr lie Gly Thr Asn Pro Phe Ala Phe Gly 145 150 155 160

Ala Pro Ser Ser Lys Asp Pro lie Val lie Asp Met Ser Ser Thr Asn

165 170 175

Val Ala Arg Gly Lys lie Lys Glu Tyr Lys Asp Ala Glu Leu Asp lie

180 185 190

Pro Val Ser Trp Ala Leu Asp Glu Tyr Gly Asn Pro Thr Thr Cys Ala

195 200 205 lie Glu Ala Leu Lys Gly Thr Leu Ser Pro Leu Gly Gly Tyr Lys Gly 210 215 220

Phe Ala Leu Gly Cys Met lie Asp lie Phe Ser Ser Val Leu Ser Gly 225 230 235 240

Ser Ala Phe Ser Thr Gin lie Thr Gly Thr Ser Leu His Met Glu Glu

245 250 255

Ala Asp Val Asn Lys Lys Gly Asp Phe Leu Phe Val Leu Asp lie Ser

260 265 270

Lys Phe lie Gin Leu Ser Glu Phe Lys lie Arg Met Asp Glu Phe lie

275 280 285

His lie lie Glu Ser Asn Gly Gly Tyr lie Pro Gly Thr Asn Tyr lie 290 295 300

Asn Asn Gin Phe Ala Asp lie Glu lie Leu Asn 305 310 315

<210> 98

<211> 354

<212> PRT

<213> EC 1.1.1.85 - 3-isopropylmalate dehydrogenases >A9VLG8_Bacillus weihenstephanensis

<400> 98

Met Glu Lys Arg lie Val Cys Leu Ala Gly Asp Gly Val Gly Pro Glu 1 5 10 15 lie Met Glu Ser Ala Lys Glu Val Leu His Met Val Glu Arg Leu Tyr

20 25 30 Gly His His Phe His Leu Gin Asp Glu Tyr Phe Gly Gly Ala Ala lie

35 40 45

Asp Leu Asn Gly Gin Pro Leu Pro Gin Arg Thr Leu Ala Ala Cys Leu

50 55 60

Ala Ser Asp Ala Val Leu Leu Gly Ala Val Gly Gly Pro Arg Trp Asp 65 70 75 80

Asp Ala Lys Glu Arg Pro Glu Lys Gly Leu Leu Ala Leu Arg Lys Gly

85 90 95

Leu Gly Val Phe Ala Asn Val Arg Pro Val Thr Val Glu Ser Ala Thr

100 105 110 Ala His Leu Ser Pro Leu Lys Asn Ala Asp Glu lie Asp Phe Val Val

115 120 125

Val Arg Glu Leu Thr Gly Gly lie Tyr Phe Ser Tyr Pro Lys Glu Arg

130 135 140

Thr Glu Glu Ser Ala Thr Asp Thr Leu Thr Tyr His Arg His Glu lie 145 150 155 160

Glu Arg lie Val Ser Tyr Ala Phe Gin Leu Ala Ser Lys Arg Glu Lys

165 170 175

Lys Val Thr Ser lie Asp Lys Ala Asn Val Leu Glu Ser Ser Lys Leu

180 185 190 Trp Arg Ala Val Thr Glu Glu Val Ala Leu Arg Tyr Pro Asn Val Glu 195 200 205

Leu Glu His He Leu Val Asp Ala Ala Ala Met Glu Leu He Arg Asn 2 10 2 15 220

Pro Arg Arg Phe Asp Val He Val Thr Glu Asn Leu Phe Gly Asp He 225 230 235 240

Leu Ser Asp Glu Ala Ser Val Leu Ala Gly Ser Leu Gly Met Leu Pro

245 250 255

Ser Ala Ser His Ala Glu Asn Gly Pro Ser Leu Tyr Glu Pro He His

260 265 2 70

Gly Ser Ala Pro Asp He Ala Gly Lys Asn Lys Ala Asn Pro He Ala

2 75 280 285

Met Met Arg Ser Val Ala Met Met Leu Gly Gin Ser Phe Gly Leu Thr 290 295 300

Arg Glu Gly Tyr Ala He Glu Glu Ala He Ser Ala Val Leu Gin Ser 305 3 10 3 15 320

Gly Lys Cys Thr Ala Asp He Gly Gly Asn Glu Thr Thr Thr Ser Phe

325 330 335

Thr Arg Ala Val He Gin Glu Met Glu Glu Gin Ala Leu Val Gly Arg

340 345 350

Gly Arg <210> 99

<21 1> 349

<212> PRT

<213> >Q5NPQ9_Zymomonas mobi li s

<400> 99

Met Arg He Ala Leu Leu Ala Gly Asp Gly He Gly Pro Glu He Thr 1 5 10 15

Ala Glu Ala Val Lys He Leu Lys Ala Val Val Gly Gin Glu He Glu

20 25 30 Phe Asp Glu Ala Leu lie Gly Gly Ala Ala Trp Lys Val Thr Gly Ser 35 40 45

Pro Leu Pro Glu Glu Thr Leu Lys Leu Cys Lys Asn Ser Asp Ala lie 50 55 60

Leu Phe Gly Ser Val Gly Asp Pro Glu Cys Asp His Leu Glu Arg Ala 65 70 75 80

Leu Arg Pro Glu Gin Ala lie Leu Gly Leu Arg Lys Glu Leu Asp Leu

85 90 95

Phe Ala Asn Leu Arg Pro Ala Arg Leu Phe Pro Glu Leu Gin Ala Glu

100 105 110

Ser Pro Leu Lys Glu Asn lie Val Thr Gly Thr Asp Leu Met lie Val

115 120 125

Arg Glu Leu Thr Gly Asp Val Tyr Phe Gly Thr Pro Arg Gly Gin Arg 130 135 140

Lys Asp Asp Gin Asn Arg Arg Glu Gly Phe Asp Thr Met Arg Tyr Asn 145 150 155 160

Glu Asp Glu Val Lys Arg lie Ala Arg lie Gly Phe Glu Thr Ala Arg

165 170 175

Ser Arg Ser Gly Asn Leu Cys Ser lie Asp Lys Ser Asn Val Leu Glu

180 185 190

Thr Ser Gin Leu Trp Arg Thr Val Val Leu Glu lie Ala Gin Glu Tyr

195 200 205

Pro Asp Val Glu Leu Ser His Met Tyr Val Asp Asn Ala Ala Met Gin 210 215 220

Leu Val Arg Ala Pro Asp Gin Phe Asp Val lie Val Thr Gly Asn Leu 225 230 235 240

Phe Gly Asp lie Leu Ser Asp Leu Ala Ser Ala Cys Val Gly Ser lie

245 250 255

Gly Leu Leu Pro Ser Ala Ser Leu Asn Ser Glu Gly Lys Gly Leu Tyr

260 265 270 Glu Pro lie His Gly Ser Ala Pro Asp lie Ala Gly Leu Gly Lys Ala 275 280 285

Asn Pro Leu Ala Thr lie Leu Ser Gly Ala Met Met Leu Arg Tyr Ser

290 295 300

Leu Lys Arg Glu Ala Asp Ala Asp Arg lie Glu Lys Ala Val Ser Thr 305 310 315 320

Ala Leu Glu Lys Gly Ala Arg Thr Ala Asp Leu Gly Gly Lys Met Thr

325 330 335

Thr Ser Glu Met Gly Asn Ala Val Leu Ala Ala Leu Asn

340 345

<210> 100

<211> 361

<212> PRT

<213> EC 1.1.1.93 - tartrate dehydrogenases >P7625 l_Escherichia coli

<400> 100

Met Met Lys Thr Met Arg lie Ala Ala lie Pro Gly Asp Gly lie Gly 1 5 10 15

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

20 25 30

Arg Trp Gly Phe Ala Leu Ser Phe Glu Gin Met Glu Trp Ala Ser Cys

35 40 45

Glu Tyr Tyr Ser His His Gly Lys Met Met Pro Asp Asp Trp His Glu

50 55 60 Gin Leu Ser Arg Phe Asp Ala lie Tyr Phe Gly Ala Val Gly Trp Pro 65 70 75 80

Asp Thr Val Pro Asp His lie Ser Leu Trp Gly Ser Leu Leu Lys Phe

85 90 95

Arg Arg Glu Phe Asp Gin Tyr Val Asn Leu Arg Pro Val Arg Leu Phe

100 105 110

Pro Gly Val Pro Cys Pro Leu Ala Gly Lys Gin Pro Gly Asp lie Asp

115 120 125 Phe Tyr Val Val Arg Glu Asn Thr Glu Gly Glu Tyr Ser Ser Leu Gly 130 135 140

Gly Arg Val Asn Glu Gly Thr Glu His Glu Val Val lie Gin Glu Ser 145 150 155 160

Val Phe Thr Arg Arg Gly Val Asp Arg lie Leu Arg Tyr Ala Phe Glu

165 170 175

Leu Ala Gin Ser Arg Pro Arg Lys Thr Leu Thr Ser Ala Thr Lys Ser

180 185 190

Asn Gly Leu Ala lie Ser Met Pro Tyr Trp Asp Glu Arg Val Glu Ala

195 200 205

Met Ala Glu Asn Tyr Pro Glu lie Arg Trp Asp Lys Gin His lie Asp 210 215 220 lie Leu Cys Ala Arg Phe Val Met Gin Pro Glu Arg Phe Asp Val Val 225 230 235 240

Val Ala Ser Asn Leu Phe Gly Asp lie Leu Ser Asp Leu Gly Pro Ala

245 250 255

Cys Thr Gly Thr lie Gly lie Ala Pro Ser Ala Asn Leu Asn Pro Glu

260 265 270

Arg Thr Phe Pro Ser Leu Phe Glu Pro Val His Gly Ser Ala Pro Asp

275 280 285

He Tyr Gly Lys Asn He Ala Asn Pro He Ala Thr He Trp Ala Gly 290 295 300

Ala Met Met Leu Asp Phe Leu Gly Asn Gly Asp Glu Arg Phe Gin Gin 305 310 315 320

Ala His Asn Gly He Leu Ala Ala He Glu Glu Val He Ala His Gly

325 330 335

Pro Lys Thr Pro Asp Met Lys Gly Asn Ala Thr Thr Pro Gin Val Ala

340 345 350

Asp Ala He Cys Lys He He Leu Arg

355 360 <210> 101

<211> 362

<212> PRT

<213> >A2Q846_Aspergillus niger

<400> 101

Met Thr Thr Glu Thr Thr Thr Tyr Arg He Ala Ser He Pro Gly Asp 1 5 10 15

Gly He Gly Glu Glu Val Val Arg Ala Thr He Glu Val He Asn Lys

20 25 30

Leu Ala Gin Thr Leu Asn Thr Phe Asn He Glu Phe Thr His Leu Pro

35 40 45

Trp Gly Thr Glu Tyr Tyr Lys Gin His Gly Arg Tyr Val Ser Glu Gly 50 55 60

Tyr Leu Asp Thr Leu Arg Gin Phe Asp Ala Gly Leu Phe Gly Ser Val 65 70 75 80

Gly His Pro Asp Val Pro Asp His Val Ser Leu Trp Gly Leu Leu Leu

85 90 95

Ala Leu Arg Ser Pro Leu Gin Leu Tyr Ala Asn Val Arg Pro Val Arg

100 105 110

Thr Phe Pro Gly Thr Lys Ser Pro Leu Thr Thr Ala Val Asn Gly He

115 120 125

Asp Trp Val Leu Val Arg Glu Asn Ser Glu Gly Glu Tyr Cys Gly Gin 130 135 140

Gly Gly Arg Ser His Thr Gly Gin Pro Trp Glu Ala Ala Thr Glu Val 145 150 155 160

Ala He Phe Thr Arg Val Gly Val Glu Arg He Met Arg Phe Ala Phe

165 170 175

Glu Thr Ala Arg Ser Arg Pro Arg Arg His Leu Thr Val Val Thr Lys

180 185 190

Ser Asn Ala Met Arg His Gly Met Val Leu Trp Asp Glu Val Ala Glu

195 200 205 Glu Val Ala Lys Asp Phe Pro Asp Val Thr Trp Asp Lys Met Leu Val 210 215 220

Asp Ala Met Thr Leu Arg Met He Ser Lys Pro Glu Ser Leu Asp Thr 225 230 235 240

He Val Gly Thr Asn Leu His Met Asp He Leu Ser Asp Leu Ala Ala

245 250 255

Gly Leu Ala Gly Ser He Gly Val Ala Pro Ser Ser Asn Leu Asp Pro

260 265 270

Thr Arg Lys Asn Pro Ser Leu Phe Glu Pro Val His Gly Ser Ala Phe

275 280 285

Asp He Met Gly Lys Gly Val Ala Asn Pro Val Ala Thr Phe Trp Ser 290 295 300

Ala Ala Glu Met Leu Ala Trp Leu Gly Glu Lys Asp Ala Ala Lys Lys 305 310 315 320

Leu Met Asp Cys Val Glu Lys Val Cys Ala Ala Gly He Leu Thr Pro

325 330 335 Asp Leu Gly Gly Ser Ala Asn Thr Gin Gly Val Val Asp Ala Val Cys

340 345 350

Lys Glu He Glu Gin Gin Leu Ala Ser Ser

355 360

<210> 102

<211> 591

<212> PRT

<213> EC 1.1.2.3 - L-lactate dehydrogenase (cytochrome) >P00175_Saccharomyces cerevisiae

<400> 102

Met Leu Lys Tyr Lys Pro Leu Leu Lys He Ser Lys Asn Cys Glu Ala 1 5 10 15 Ala He Leu Arg Ala Ser Lys Thr Arg Leu Asn Thr He Arg Ala Tyr

20 25 30

Gly Ser Thr Val Pro Lys Ser Lys Ser Phe Glu Gin Asp Ser Arg Lys 35 40 45

Arg Thr Gin Ser Trp Thr Ala Leu Arg Val Gly Ala lie Leu Ala Ala 50 55 60

Thr Ser Ser Val Ala Tyr Leu Asn Trp His Asn Gly Gin lie Asp Asn 65 70 75 80

Glu Pro Lys Leu Asp Met Asn Lys Gin Lys lie Ser Pro Ala Glu Val

85 90 95

Ala Lys His Asn Lys Pro Asp Asp Cys Trp Val Val lie Asn Gly Tyr

100 105 110

Val Tyr Asp Leu Thr Arg Phe Leu Pro Asn His Pro Gly Gly Gin Asp

115 120 125

Val lie Lys Phe Asn Ala Gly Lys Asp Val Thr Ala lie Phe Glu Pro 130 135 140

Leu His Ala Pro Asn Val lie Asp Lys Tyr lie Ala Pro Glu Lys Lys 145 150 155 160

Leu Gly Pro Leu Gin Gly Ser Met Pro Pro Glu Leu Val Cys Pro Pro

165 170 175

Tyr Ala Pro Gly Glu Thr Lys Glu Asp lie Ala Arg Lys Glu Gin Leu

180 185 190 Lys Ser Leu Leu Pro Pro Leu Asp Asn lie lie Asn Leu Tyr Asp Phe

195 200 205

Glu Tyr Leu Ala Ser Gin Thr Leu Thr Lys Gin Ala Trp Ala Tyr Tyr 210 215 220

Ser Ser Gly Ala Asn Asp Glu Val Thr His Arg Glu Asn His Asn Ala 225 230 235 240

Tyr His Arg lie Phe Phe Lys Pro Lys lie Leu Val Asp Val Arg Lys

245 250 255

Val Asp lie Ser Thr Asp Met Leu Gly Ser His Val Asp Val Pro Phe

260 265 270 Tyr Val Ser Ala Thr Ala Leu Cys Lys Leu Gly Asn Pro Leu Glu Gly 275 280 285

Glu Lys Asp Val Ala Arg Gly Cys Gly Gin Gly Val Thr Lys Val Pro 290 295 300

Gin Met lie Ser Thr Leu Ala Ser Cys Ser Pro Glu Glu lie lie Glu 305 310 315 320

Ala Ala Pro Ser Asp Lys Gin lie Gin Trp Tyr Gin Leu Tyr Val Asn

325 330 335

Ser Asp Arg Lys lie Thr Asp Asp Leu Val Lys Asn Val Glu Lys Leu

340 345 350

Gly Val Lys Ala Leu Phe Val Thr Val Asp Ala Pro Ser Leu Gly Gin

355 360 365 Arg Glu Lys Asp Met Lys Leu Lys Phe Ser Asn Thr Lys Ala Gly Pro 370 375 380

Lys Ala Met Lys Lys Thr Asn Val Glu Glu Ser Gin Gly Ala Ser Arg 385 390 395 400

Ala Leu Ser Lys Phe lie Asp Pro Ser Leu Thr Trp Lys Asp lie Glu

405 410 415

Glu Leu Lys Lys Lys Thr Lys Leu Pro lie Val lie Lys Gly Val Gin

420 425 430

Arg Thr Glu Asp Val lie Lys Ala Ala Glu lie Gly Val Ser Gly Val

435 440 445

Val Leu Ser Asn His Gly Gly Arg Gin Leu Asp Phe Ser Arg Ala Pro 450 455 460 lie Glu Val Leu Ala Glu Thr Met Pro lie Leu Glu Gin Arg Asn Leu 465 470 475 480

Lys Asp Lys Leu Glu Val Phe Val Asp Gly Gly Val Arg Arg Gly Thr

485 490 495

Asp Val Leu Lys Ala Leu Cys Leu Gly Ala Lys Gly Val Gly Leu Gly

500 505 510 Arg Pro Phe Leu Tyr Ala Asn Ser Cys Tyr Gly Arg Asn Gly Val Glu 515 520 525

Lys Ala lie Glu lie Leu Arg Asp Glu lie Glu Met Ser Met Arg Leu 530 535 540 Leu Gly Val Thr Ser lie Ala Glu Leu Lys Pro Asp Leu Leu Asp Leu 545 550 555 560

Ser Thr Leu Lys Ala Arg Thr Val Gly Val Pro Asn Asp Val Leu Tyr

565 570 575

Asn Glu Val Tyr Glu Gly Pro Thr Leu Thr Glu Phe Glu Asp Ala

580 585 590

<210> 103

<211> 396

<212> PRT

<213> >P33232_Escherichia

<400> 103

Met lie lie Ser Ala Ala Ser Asp Tyr Arg Ala Ala Ala Gin Arg lie 1 5 10 15

Leu Pro Pro Phe Leu Phe His Tyr Met Asp Gly Gly Ala Tyr Ser Glu

20 25 30

Tyr Thr Leu Arg Arg Asn Val Glu Asp Leu Ser Glu Val Ala Leu Arg

35 40 45

Gin Arg lie Leu Lys Asn Met Ser Asp Leu Ser Leu Glu Thr Thr Leu 50 55 60 Phe Asn Glu Lys Leu Ser Met Pro Val Ala Leu Ala Pro Val Gly Leu 65 70 75 80

Cys Gly Met Tyr Ala Arg Arg Gly Glu Val Gin Ala Ala Lys Ala Ala

85 90 95

Asp Ala His Gly lie Pro Phe Thr Leu Ser Thr Val Ser Val Cys Pro

100 105 110

He Glu Glu Val Ala Pro Ala He Lys Arg Pro Met Trp Phe Gin Leu

115 120 125 Tyr Val Leu Arg Asp Arg Gly Phe Met Arg Asn Ala Leu Glu Arg Ala 130 135 140

Lys Ala Ala Gly Cys Ser Thr Leu Val Phe Thr Val Asp Met Pro Thr 145 150 155 160

Pro Gly Ala Arg Tyr Arg Asp Ala His Ser Gly Met Ser Gly Pro Asn

165 170 175

Ala Ala Met Arg Arg Tyr Leu Gin Ala Val Thr His Pro Gin Trp Ala

180 185 190

Trp Asp Val Gly Leu Asn Gly Arg Pro His Asp Leu Gly Asn lie Ser

195 200 205

Ala Tyr Leu Gly Lys Pro Thr Gly Leu Glu Asp Tyr lie Gly Trp Leu 210 215 220

Gly Asn Asn Phe Asp Pro Ser lie Ser Trp Lys Asp Leu Glu Trp lie 225 230 235 240

Arg Asp Phe Trp Asp Gly Pro Met Val lie Lys Gly lie Leu Asp Pro

245 250 255

Glu Asp Ala Arg Asp Ala Val Arg Phe Gly Ala Asp Gly lie Val Val

260 265 270

Ser Asn His Gly Gly Arg Gin Leu Asp Gly Val Leu Ser Ser Ala Arg

275 280 285

Ala Leu Pro Ala lie Ala Asp Ala Val Lys Gly Asp lie Ala lie Leu 290 295 300

Ala Asp Ser Gly lie Arg Asn Gly Leu Asp Val Val Arg Met lie Ala 305 310 315 320

Leu Gly Ala Asp Thr Val Leu Leu Gly Arg Ala Phe Leu Tyr Ala Leu

325 330 335

Ala Thr Ala Gly Gin Ala Gly Val Ala Asn Leu Leu Asn Leu lie Glu

340 345 350

Lys Glu Met Lys Val Ala Met Thr Leu Thr Gly Ala Lys Ser lie Ser

355 360 365 Glu He Thr Gin Asp Ser Leu Val Gin Gly Leu Gly Lys Glu Leu Pro 370 375 380

Ala Ala Leu Ala Pro Met Ala Lys Gly Asn Ala Ala

385 390 395

<210> 104

<211> 587

<212> PRT

<213> EC 1.1.2.4 - D-lactate dehydrogenase (cytochrome) >P32891_Saccharomyces cerevisiae

<400> 104

Met Leu Trp Lys Arg Thr Cys Thr Arg Leu He Lys Pro He Ala Gin 1 5 10 15

Pro Arg Gly Arg Leu Val Arg Arg Ser Cys Tyr Arg Tyr Ala Ser Thr

20 25 30

Gly Thr Gly Ser Thr Asp Ser Ser Ser Gin Trp Leu Lys Tyr Ser Val

35 40 45

He Ala Ser Ser Ala Thr Leu Phe Gly Tyr Leu Phe Ala Lys Asn Leu 50 55 60 Tyr Ser Arg Glu Thr Lys Glu Asp Leu He Glu Lys Leu Glu Met Val 65 70 75 80

Lys Lys He Asp Pro Val Asn Ser Thr Leu Lys Leu Ser Ser Leu Asp

85 90 95

Ser Pro Asp Tyr Leu His Asp Pro Val Lys He Asp Lys Val Val Glu

100 105 110

Asp Leu Lys Gin Val Leu Gly Asn Lys Pro Glu Asn Tyr Ser Asp Ala

115 120 125

Lys Ser Asp Leu Asp Ala His Ser Asp Thr Tyr Phe Asn Thr His His 130 135 140 Pro Ser Pro Glu Gin Arg Pro Arg He He Leu Phe Pro His Thr Thr 145 150 155 160

Glu Glu Val Ser Lys He Leu Lys He Cys His Asp Asn Asn Met Pro 165 170 175

Val Val Pro Phe Ser Gly Gly Thr Ser Leu Glu Gly His Phe Leu Pro

180 185 190

Thr Arg He Gly Asp Thr He Thr Val Asp Leu Ser Lys Phe Met Asn

195 200 205

Asn Val Val Lys Phe Asp Lys Leu Asp Leu Asp He Thr Val Gin Ala 210 215 220

Gly Leu Pro Trp Glu Asp Leu Asn Asp Tyr Leu Ser Asp His Gly Leu 225 230 235 240

Met Phe Gly Cys Asp Pro Gly Pro Gly Ala Gin He Gly Gly Cys He

245 250 255

Ala Asn Ser Cys Ser Gly Thr Asn Ala Tyr Arg Tyr Gly Thr Met Lys

260 265 270

Glu Asn He He Asn Met Thr He Val Leu Pro Asp Gly Thr He Val

275 280 285

Lys Thr Lys Lys Arg Pro Arg Lys Ser Ser Ala Gly Tyr Asn Leu Asn 290 295 300

Gly Leu Phe Val Gly Ser Glu Gly Thr Leu Gly He Val Thr Glu Ala 305 310 315 320 Thr Val Lys Cys His Val Lys Pro Lys Ala Glu Thr Val Ala Val Val

325 330 335

Ser Phe Asp Thr He Lys Asp Ala Ala Ala Cys Ala Ser Asn Leu Thr

340 345 350

Gin Ser Gly He His Leu Asn Ala Met Glu Leu Leu Asp Glu Asn Met

355 360 365

Met Lys Leu He Asn Ala Ser Glu Ser Thr Asp Arg Cys Asp Trp Val 370 375 380

Glu Lys Pro Thr Met Phe Phe Lys He Gly Gly Arg Ser Pro Asn He 385 390 395 400 Val Asn Ala Leu Val Asp Glu Val Lys Ala Val Ala Gin Leu Asn His 405 410 415 Cys Asn Ser Phe Gin Phe Ala Lys Asp Asp Asp Glu Lys Leu Glu Leu

420 425 430

Trp Glu Ala Arg Lys Val Ala Leu Trp Ser Val Leu Asp Ala Asp Lys

435 440 445

Ser Lys Asp Lys Ser Ala Lys He Trp Thr Thr Asp Val Ala Val Pro 450 455 460

Val Ser Gin Phe Asp Lys Val He His Glu Thr Lys Lys Asp Met Gin 465 470 475 480

Ala Ser Lys Leu He Asn Ala He Val Gly His Ala Gly Asp Gly Asn

485 490 495 Phe His Ala Phe He Val Tyr Arg Thr Pro Glu Glu His Glu Thr Cys

500 505 510

Ser Gin Leu Val Asp Arg Met Val Lys Arg Ala Leu Asn Ala Glu Gly

515 520 525

Thr Cys Thr Gly Glu His Gly Val Gly He Gly Lys Arg Glu Tyr Leu 530 535 540

Leu Glu Glu Leu Gly Glu Ala Pro Val Asp Leu Met Arg Lys He Lys 545 550 555 560

Leu Ala He Asp Pro Lys Arg He Met Asn Pro Asp Lys He Phe Lys

565 570 575 Thr Asp Pro Asn Glu Pro Ala Asn Asp Tyr Arg

580 585

<210> 105

<211> 477

<212> PRT

<213> >Q5FP89_Gluconobacter oxydans

<400> 105

Met Pro Glu Pro Val Met Thr Ala Ser Ser Ala Ser Ala Pro Asp Arg 1 5 10 15 Leu Gin Ala Val Leu Lys Ala Leu Gin Pro Val Met Gly Glu Arg lie 20 25 30

Ser Thr Ala Pro Ser Val Arg Glu Glu His Ser His Gly Glu Ala Met

35 40 45

Asn Ala Ser Asn Leu Pro Glu Ala Val Val Phe Ala Glu Ser Thr Gin 50 55 60

Asp Val Ala Thr Val Leu Arg His Cys His Glu Trp Arg Val Pro Val 65 70 75 80

Val Ala Phe Gly Ala Gly Thr Ser Val Glu Gly His Val Val Pro Pro

85 90 95

Glu Gin Ala lie Ser Leu Asp Leu Ser Arg Met Thr Gly lie Val Asp

100 105 110

Leu Asn Ala Glu Asp Leu Asp Cys Arg Val Gin Ala Gly lie Thr Arg

115 120 125

Gin Thr Leu Asn Val Glu lie Arg Asp Thr Gly Leu Phe Phe Pro Val 130 135 140

Asp Pro Gly Gly Glu Ala Thr lie Gly Gly Met Cys Ala Thr Arg Ala 145 150 155 160

Ser Gly Thr Ala Ala Val Arg Tyr Gly Thr Met Lys Glu Asn Val Leu

165 170 175

Gly Leu Thr Val Val Leu Ala Thr Gly Glu lie lie Arg Thr Gly Gly

180 185 190 Arg Val Arg Lys Ser Ser Thr Gly Tyr Asp Leu Thr Ser Leu Phe Val

195 200 205

Gly Ser Glu Gly Thr Leu Gly lie lie Thr Glu Val Gin Leu Arg Leu 210 215 220

His Gly Arg Pro Asp Ser Val Ser Ala Ala lie Cys Gin Phe Glu Ser 225 230 235 240

Leu His Asp Ala lie Gin Thr Ala Met Glu lie lie Gin Cys Gly lie

245 250 255 Pro He Thr Arg Val Glu Leu Met Asp Ser Val Gin Met Ala Ala Ser 260 265 270

He Gin Tyr Ser Gly Leu Asn Glu Tyr Gin Pro Leu Thr Thr Leu Phe

275 280 285

Phe Glu Phe Thr Gly Ser Pro Ala Ala Val Arg Glu Gin Val Glu Thr 290 295 300

Thr Glu Ala He Ala Ser Gly Asn Asn Gly Leu Gly Phe Ala Trp Ala 305 310 315 320

Glu Ser Pro Glu Asp Arg Thr Arg Leu Trp Lys Ala Arg His Asp Ala

325 330 335

Tyr Trp Ala Ala Lys Ala He Val Pro Asp Ala Arg Val He Ser Thr

340 345 350

Asp Cys He Val Pro He Ser Arg Leu Gly Glu Leu He Glu Gly Val

355 360 365

His Arg Asp He Glu Ala Ser Gly Leu Arg Ala Pro Leu Leu Gly His 370 375 380

Val Gly Asp Gly Asn Phe His Thr Leu He He Thr Asp Asp Thr Pro 385 390 395 400

Glu Gly His Gin Gin Ala Leu Asp Leu Asp Arg Lys He Val Ala Arg

405 410 415

Ala Leu Ser Leu Asn Gly Ser Cys Ser Gly Glu His Gly Val Gly Met

420 425 430

Gly Lys Leu Glu Phe Leu Glu Thr Glu His Gly Pro Gly Ser Leu Ser

435 440 445

Val Met Arg Ala Leu Lys Asn Thr Met Asp Pro His His He Leu Asn 450 455 460

Pro Gly Lys Leu Leu Pro Pro Gly Ala Val Tyr Thr Gly

465 470 475

<210>

<211> <212> PRT

<213> EC 1 . 1 . 99 . 2 2-hydr oxyglutarate dehydrogenase

>Q9N4 Z0_Caenorhabditi s elegans

<400> 106

Met Leu Asn Arg Gly Thr Phe Gin Val Phe Arg Gly He Ser Gly Pro 1 5 10 15

Pro Lys Lys Ser Val Asp Leu Pro Lys Tyr Asp Leu Val He Val Gly

20 25 30 Gly Gly He Val Gly Cys Ala Thr Ala Arg Gin Leu Leu He Glu Lys

35 40 45

Pro Gin Leu Lys Val Ala Leu He Glu Lys Glu Lys Glu Leu Ala Val 50 55 60

His Gin Ser Gly His Asn Ser Gly Val He His Ala Gly He Tyr Tyr 65 70 75 80

Thr Pro Gly Ser Leu Lys Ala Lys Leu Cys Val Glu Gly Leu Asp Leu

85 90 95

Ser Tyr Glu Phe Phe Asp Lys Glu Lys Val Pro Tyr Lys Lys Thr Gly

100 105 1 10 Lys Leu He Val Ala Val Glu Pro Glu Glu Val Pro Arg Leu Asp Ala

1 15 120 125

Leu Phe Ser Arg Ala Gin Thr Asn Gly Cys Arg Asp He Glu Met He 130 135 140

Asp Ser Ser Lys He Thr Glu Leu Glu Pro His Cys Arg Gly Leu Lys 145 150 155 160

Ala Leu Trp Ser Pro His Thr Gly He Val Asp Trp Gly Tyr Val Thr

165 1 70 1 75

Lys Arg Phe Gly Glu Asp Phe Glu Lys Arg Gly Gly Lys He Tyr Thr

180 185 190 Ser Tyr Pro Leu Glu Lys He Ser Asp Asn His Asp Pro Gly Tyr Pro

195 200 205

He Arg Val Ser Ser Gly Pro Ala Leu Ala Glu Phe Glu Thr Lys Asn 210 215 220

Leu lie Thr Cys Ala Gly Leu Gin Ser Asp Arg Val Ala Ala Leu Ser 225 230 235 240

Gly Cys Ser Thr Asp Pro Lys lie Val Pro Phe Arg Gly Glu Tyr Leu

245 250 255

Leu Leu Lys Pro Glu Lys Arg His Leu Val Lys Thr Asn lie Tyr Pro

260 265 270

Val Pro Asp Pro Arg Phe Pro Phe Leu Gly Val His Phe Thr Pro Arg

275 280 285 Met Asn Gly Asp lie Trp Leu Gly Pro Asn Ala Val Leu Ala Tyr Lys 290 295 300

Arg Glu Gly Tyr Ser Tyr Phe Ser lie Ser Pro Ser Asp Leu Leu Glu 305 310 315 320

Ser Leu Ser Tyr Ser Gly Met Gin Lys Leu Val Lys Lys His Phe Thr

325 330 335

Phe Gly lie Lys Glu Leu Tyr Arg Gly Val Trp lie Ala Ala Gin Val

340 345 350

Lys Gin Leu Gin Arg Phe lie Pro Glu Leu Lys Leu Ser Asp Val Thr

355 360 365

Arg Gly Pro Ala Gly Val Arg Ala Gin Ala Met Asp Ser Ala Gly Asn 370 375 380

Leu Val Asp Asp Phe Val Phe Asp Ser Gly Thr Gly Lys Leu Ser Pro 385 390 395 400

Leu Leu Met His Val Arg Asn Ala Pro Ser Pro Ala Ala Thr Ser Ser

405 410 415

Leu Ala lie Ala Lys Met lie Thr Ser Glu Ala lie Asn Arg Phe Lys

420 425 430

Leu <210> 10 7

<21 1> 455

<212> PRT

<213> >Q9VJ28_Dros ophi la melanogaster

<400> 10 7

Met Ala Gin Val Arg Leu Leu Val Gin Gly Leu Arg Arg Ser Leu Leu 1 5 10 15

Asn Val Gly Val Ala Ala Pro Asn Glu Ser Thr Ala Thr His Lys Arg

20 25 30

Ser Gin His Ser Ser Ser Ser Cys Gly Asp Tyr Asp Leu Val Val Val

35 40 45

Gly Gly Gly He Val Gly Ala Ala Ser Ala Arg Glu He Val Leu Arg 50 55 60

His Pro Ser Leu Lys Val Ala Val Leu Glu Lys Glu Cys Lys Leu Ala 65 70 75 80

Lys His Gin Ser Gly His Asn Ser Gly Val He His Ala Gly He Tyr

85 90 95

Tyr Lys Pro Gly Thr Leu Lys Ala Arg Leu Cys Val Glu Gly Met His

100 105 1 10

Leu Ala Tyr Ala Tyr Leu Asp Glu Lys Lys He Pro Tyr Lys Lys Thr

1 15 120 125 Gly Lys Leu He Val Ala Thr Asp Glu Lys Glu Val Lys Leu Leu Lys 130 135 140

Asp Leu Glu Lys Arg Gly He Ala Asn Asn Val Pro Asp Leu Arg Met 145 150 155 160

He Glu Gly Ser Glu He Gin Glu He Glu Pro Tyr Cys Gin Gly Val

165 1 70 1 75

Met Ala Leu His Ser Pro His Thr Gly He Val Asp Trp Gly Leu Val

180 185 190

Thr Glu His Tyr Gly Gin Asp Phe Lys Gin Cys Gly Gly Asp He Tyr

195 200 205 Leu Asp Phe Asn Val Ser Lys Phe Thr Glu Thr Lys Glu Gly Thr Asp 210 215 220

Tyr Pro Val Thr lie His Gly Ala Lys Pro Gly Gin Thr Val Arg Thr 225 230 235 240

Lys Asn Val Leu Thr Cys Gly Gly Leu Gin Ser Asp Leu Leu Ala Glu

245 250 255

Lys Thr Gly Cys Pro Arg Asp Pro Arg lie Val Pro Phe Arg Gly Glu

260 265 270

Tyr Leu Leu Leu Thr Lys Glu Lys Gin His Met Val Lys Gly Asn lie

275 280 285

Tyr Pro Val Pro Asp Pro Arg Phe Pro Phe Leu Gly Val His Phe Thr 290 295 300 Pro Arg Met Asp Gly Ser lie Trp Leu Gly Pro Asn Ala Val Leu Ala 305 310 315 320

Leu Lys Arg Glu Gly Tyr Thr Trp Gly Asp lie Asn Leu Phe Glu Leu

325 330 335

Phe Asp Ala Leu Arg Tyr Pro Gly Phe Val Lys Met Ala Ser Lys Tyr

340 345 350

He Gly Phe Gly Leu Ser Glu Met Ser Lys Ser Trp Phe He Asn Leu

355 360 365

Gin He Lys Ala Leu Gin Lys Tyr He Pro Asp He Thr Glu Tyr Asp 370 375 380 He Gin Arg Gly Pro Ala Gly Val Arg Ala Gin Ala Met Asp Leu Asp 385 390 395 400

Gly Asn Leu Val Asp Asp Phe Val Phe Asp Arg Gly Gin Gly Ser Gly

405 410 415

Ala Leu Ala Lys Arg Val Leu His Cys Arg Asn Ala Pro Ser Pro Gly

420 425 430

Ala Thr Ser Ser Leu Ala He Ala Lys Met He Ala Asp Lys He Glu

435 440 445 Asn Glu Phe Ser lie Gly Lys

450 455

<210> 108

<211> 321

<212> PRT

<213> EC 1.1.1.27 - L-lactate dehydrogenases >P13714_Bacillus subtilis

<400> 108

Met Met Asn Lys His Val Asn Lys Val Ala Leu lie Gly Ala Gly Phe 1 5 10 15

Val Gly Ser Ser Tyr Ala Phe Ala Leu lie Asn Gin Gly lie Thr Asp

20 25 30

Glu Leu Val Val lie Asp Val Asn Lys Glu Lys Ala Met Gly Asp Val

35 40 45

Met Asp Leu Pro His Gly Lys Ala Phe Gly Leu Gin Pro Val Lys Thr 50 55 60 Ser Tyr Gly Thr Tyr Glu Asp Cys Lys Asp Ala Asp lie Val Cys lie 65 70 75 80

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

85 90 95

Glu Lys Asn Leu Lys lie Phe Lys Gly lie Val Ser Glu Val Met Ala

100 105 110

Ser Gly Phe Asp Gly lie Phe Leu Val Ala Thr Asn Pro Val Asp lie

115 120 125

Leu Thr Tyr Ala Thr Trp Lys Phe Ser Gly Leu Pro Lys Glu Arg Val 130 135 140 lie Gly Ser Gly Thr Thr Leu Asp Ser Ala Arg Phe Arg Phe Met Leu 145 150 155 160

Ser Glu Tyr Phe Gly Ala Ala Pro Gin Asn Val His Ala His lie lie

165 170 175

Gly Glu His Gly Asp Thr Glu Leu Pro Val Trp Ser His Ala Asn Val

180 185 190 Gly Gly Val Pro Val Ser Glu Leu Val Glu Lys Asn Asp Ala Tyr Lys 195 200 205

Gin Glu Glu Leu Asp Gin lie Val Asp Asp Val Lys Asn Ala Ala Tyr 210 215 220

His lie lie Glu Lys Lys Gly Ala Thr Tyr Tyr Gly Val Ala Met Ser 225 230 235 240

Leu Ala Arg lie Thr Lys Ala lie Leu His Asn Glu Asn Ser lie Leu

245 250 255

Thr Val Ser Thr Tyr Leu Asp Gly Gin Tyr Gly Ala Asp Asp Val Tyr

260 265 270 lie Gly Val Pro Ala Val Val Asn Arg Gly Gly lie Ala Gly lie Thr

275 280 285

Glu Leu Asn Leu Asn Glu Lys Glu Lys Glu Gin Phe Leu His Ser Ala 290 295 300

Gly Val Leu Lys Asn lie Leu Lys Pro His Phe Ala Glu Gin Lys Val 305 310 315 320 Asn

<210> 109

<211> 342

<212> PRT

<213> EC 1.1.1.28 - D-lactate dehydrogenases >Q88MC4_Pseudomonas putida

<400> 109

Met Thr His Pro Arg His Ala Leu Gin Arg Ser Ser Thr Met Arg Ala 1 5 10 15

Leu Leu Phe Ser Ser Gin His Tyr Asp Gin Glu Ser Phe Thr Lys Ala

20 25 30 Ala Gly Gly Thr Ala Leu Glu Leu His Phe Gin Pro Ala Arg Leu Thr

35 40 45

Leu Asp Thr Ala Ala Leu Ala Asp Gly Phe Glu Val Val Cys Ala Phe 50 55 60

He Asn Asp Glu Leu Asp Ala Pro Val Leu Gin Arg Leu Ala Ala Ala 65 70 75 80

Gly Thr Arg Leu He Ala Leu Arg Ser Ala Gly Tyr Asn His Val Asp

85 90 95

Leu Ala Ala Ala Gin Arg Leu Gly Leu Ala Val Val Arg Val Pro Ala

100 105 110

Tyr Ser Pro His Ala Val Ala Glu His Ala Val Ala Leu He Leu Ala

115 120 125

Leu Asn Arg Arg Leu His Arg Ala Tyr Asn Arg Thr Arg Glu Gly Asp 130 135 140

Phe Thr Leu His Gly Leu Thr Gly Phe Asp Leu His Gly Lys Thr Val 145 150 155 160

Gly Val Val Gly Thr Gly Gin He Gly Val Ala Phe Ala Arg He Met

165 170 175

Ala Gly Phe Gly Cys Gin Leu Leu Ala Tyr Asp Pro Tyr Pro Asn Pro

180 185 190

Glu Leu Leu Ala Leu Gly Ala Arg Tyr Leu Pro Leu Pro Glu Leu Leu

195 200 205

Arg Glu Ala Arg He He Ser Leu His Cys Pro Leu Thr Glu His Thr 210 215 220

Arg His Leu He Asn Ala Gin Ser Leu Ala Gin Leu Gin Pro Gly Ala 225 230 235 240

Met Leu He Asn Thr Gly Arg Gly Ala Leu Val Asp Thr Pro Ala Leu

245 250 255

He Asp Ala Leu Lys Ser Gly Gin Leu Gly Tyr Leu Gly Leu Asp Val

260 265 270

Tyr Glu Glu Glu Ala Gin Leu Phe Phe Glu Asp Arg Ser Asp Leu Pro

275 280 285 Leu Gin Asp Asp Val Leu Ala Arg Leu Leu Thr Phe Pro Asn Val lie 290 295 300

He Thr Ala His Gin Ala Phe Leu Thr Arg Glu Ala Leu Asp Ala He 305 310 315 320

Ala Ala Thr Thr Leu Asp Asn He Asn Arg Trp Ala Ala Gly Asn Pro

325 330 335

Gin Asn Leu Val Met Gly

340

[Seq ID NO 110]

Methanocaldococcus ja.nna.sch.ii (motif) aksA wild-type gene

ATGACAAAAGTGCTGGTGATGTTTATGGATTTCTTATTTGAGAACAGCTGGAAAGCAGTTTGTCCCTACA ATCCAAAGTTGGATTTAAAGGACATTTATATTTATGACACAACCCTAAGAGATGGAGAGCAAACCCCAGG AGTTTGCTTTACCAAAGAACAAAAATTGGAGATTGCAAGGAAGTTGGATGAACTTGGATTAAAGCAGATT GAAGCTGGCTTCCCAATAGTATCTGAAAGAGAAGCAGATATAGTTAAAACAATTGCTAATGAAGGGCTAA ATGCTGATATCTTAGCTTTATGCAGGGCTTTAAAGAAAGATATAGATAAAGCAATAGAGTGCGATGTAGA TGGGATTATTACCTTCATAGCAACATCTCCTCTCCACTTAAAATATAAATTCAACAACAAAAGCTTAGAT GAAATATTAGAGATGGGAGTTGAGGCAGTTGAGTATGCAAAGGAACATGGCTTATTTGTTGCTTTCTCTG CAGAGGATGCGACAAGAACACCAATAGAGGACTTGATTAAAGTGCATAAAGCCGCTGAAGAGGCTGGAGC AGATAGGGTTCATATAGCAGACACAACTGGCTGTGCTACCCCCCAAAGTATGGAGTTTATATGTAAAACA TTGAAGGAGAACTTAAAAAAGGCACATATTGGAGTGCATTGTCACAACGACTTTGGATTTGCAGTTATAA ATTCAATATATGGTTTAATTGGAGGAGCTAAGGCAGTTTCAACAACAGTTAATGGTATTGGAGAGAGGGC AGGGAATGCAGCTTTAGAAGAGCTAATTATGGCTTTAACTGTCTTGTATGATGTTGATTTGGGATTAAAC TTGGAGGTTCTTCCAGAGTTATGCAGAATGGTTGAGGAATACTCTGGAATAAAGATGCCAAAGAACAAAC CAATAGTTGGAGAGCTTGTATTTGCTCATGAAAGTGGAATTCACGTTGATGCTGTCATAGAGAATCCATT AACCTATGAACCCTTCCTTCCAGAGAAAATAGGGCTTAAGAGAAATATTTTGTTAGGGAAGCATTCTGGA TGCAGAGCCGTTGCCTATAAGCTAAAACTTATGGGAATTGATTACGATAGAGAGATGTTGTGCGAGATTG TTAAAAAGGTTAAAGAGATTAGAGAGGAAGGTAAATTTATAACTGATGAAGTCTTTAAGGAGATTGTTGA AGAAGTTTTAAGGAAGAGAAATAAAAATTAA

[Seq ID NO 1 1 1 ]

Methanococcus jannaschii AksA, MJ0503

MTKVLVMFMDFLFENSWKAVCPYNPKLDLKDIYIYDTTLRDGEQTPGVCFTKEQKLE IARKLDELGLKQI EAGFPIVSEREADIVKTIANEGLNADILALCRALKKDIDKAIECDVDGI ITFIATSPLHLKYKFNNKSLD EILEMGVEAVEYAKEHGLFVAFSAEDATRTPIEDLIKVHKAAEEAGADRVHIADTTGCATPQSMEFICKT LKENLKKAHIGVHCHNDFGFAVINS IYGLIGGAKAVSTTVNGIGERAGNAALEELIMALTVLYDVDLGLN LEVLPELCRMVEEYSGIKMPKNKPIVGELVFAHESGIHVDAVIENPLTYEPFLPEKIGLKRNILLGKHSG CRAVAYKLKLMGIDYDREMLCEIVKKVKE IREEGKFITDEVFKEIVEEVLRKRNKN [Seq ID NO 112]

Methanococcus ja.nna.sch.ii AksA, MJ0503 Codon Pair optimized gene,

ATGACCAAAGTTCTGGTAATGTTCATGGACTTCCTGTTCGAAAACTCCTGGAAAGCGGTTTGCCCGTACA ACCCGAAACTGGATCTGAAAGACATCTACATCTACGACACCACTCTGCGTGACGGTGAACAGACTCCGGG CGTTTGCTTCACCAAAGAGCAGAAGCTGGAAATCGCTCGTAAGCTGGACGAACTGGGTCTGAAGCAGATC GAAGCTGGCTTCCCGATCGTTTCTGAACGTGAAGCTGACATCGTTAAAACTATCGCTAACGAAGGTCTGA ACGCTGACATCCTGGCACTGTGCCGTGCGCTGAAGAAAGACATCGACAAAGCAATCGAATGCGACGTTGA CGGTATCATCACTTTCATCGCAACTTCTCCGCTGCACCTGAAATACAAATTCAACAACAAATCTCTGGAT GAAATCCTGGAAATGGGCGTTGAAGCGGTAGAATACGCTAAAGAGCACGGTCTGTTCGTTGCATTCTCTG CAGAAGATGCAACTCGTACTCCGATCGAAGATCTGATCAAAGTTCACAAAGCAGCTGAAGAAGCGGGTGC TGACCGCGTTCACATCGCTGACACCACTGGCTGCGCAACTCCGCAGTCTATGGAATTCATCTGCAAAACT CTGAAAGAAAACCTGAAGAAAGCACACATCGGCGTACACTGCCACAACGACTTCGGTTTCGCTGTTATCA ACTCCATCTACGGTCTGATCGGTGGTGCGAAAGCGGTATCTACTACCGTTAACGGTATCGGTGAACGTGC TGGTAACGCTGCACTGGAAGAGCTGATCATGGCGCTGACCGTACTGTACGACGTTGACCTGGGTCTGAAC CTGGAAGTTCTGCCGGAACTGTGCCGTATGGTTGAAGAATACTCCGGTATCAAGATGCCGAAAAACAAGC CAATCGTTGGTGAACTGGTATTCGCTCACGAATCCGGTATCCACGTTGACGCTGTTATCGAAAACCCGCT GACTTACGAACCGTTCCTGCCGGAAAAAATCGGTCTGAAACGTAACATCCTGCTGGGTAAGCACTCTGGT TGCCGTGCTGTTGCTTACAAGCTGAAACTGATGGGTATCGACTACGACCGTGAAATGCTGTGCGAAATCG TTAAGAAAGTTAAAGAAATCCGTGAAGAAGGTAAATTCATCACTGACGAAGTTTTCAAAGAGATCGTTGA AGAAGTTCTGCGTAAGCGTAACAAAAAC

[Seq ID NO 113]

Methanocaldococcus jannaschii DSM 2661 (motif) wild type gene

TTGACATTGGTAGAGAAGATACTATCAAAAAAAGTTGGTTATGAAGTTTGTGCAGGAGATAGCATAGAGG TTGAAGTTGATTTGGCAATGACACACGATGGAACAACACCTTTAGCATACAAAGCTTTAAAGGAAATGAG TGATAGTGTTTGGAATCCAGATAAAATAGTCGTTGCCTTTGACCACAATGTTCCACCAAACACAGTTAAA GCTGCTGAAATGCAAAAATTAGCTTTGGAGTTTGTTAAAAGATTTGGCATTAAAAATTTCCATAAAGGTG GAGAAGGCATCTGTCATCAAATCTTAGCTGAAAATTATGTTTTGCCAAACATGTTTGTAGCTGGTGGAGA CAGCCATACATGCACACATGGAGCTTTTGGAGCTTTTGCTACTGGCTTTGGAGCTACTGATATGGCTTAC ATCTATGCAACAGGAGAAACATGGATTAAAGTGCCAAAAACAATTAGGGTAGATATAGTTGGAAAAAATG AAAATGTTTCTGCCAAAGATATTGTTTTAAGGGTTTGTAAGGAAATTGGGAGAAGAGGAGCAACATACAT GGCTATTGAGTATGGTGGAGAGGTTGTTAAAAACATGGACATGGATGGAAGGCTAACTTTATGCAACATG GCAATAGAGATGGGAGGAAAAACAGGAGTGATAGAGGCTGATGAAATTACTTATGATTATTTAAAGAAAG AGAGAGGACTTTCTGATGAGGATATAGCTAAATTAAAAAAAGAGAGAATAACAGTAAATAGAGATGAAGC AAACTACTATAAGGAGATAGAAATTGACATAACAGATATGGAAGAACAAGTTGCTGTTCCACACCACCCA GATAACGTAAAGCCAATTAGTGATGTTGAAGGGACTGAGATAAATCAAGTTTTTATTGGGAGTTGCACAA ATGGAAGGTTGAGTGATTTAAGAGAAGCAGCTAAATATTTAAAAGGTAGGGAGGTTCATAAAGATGTTAA GCTAATTGTTATCCCGGCATCAAAAAAGGTATTTTTGCAAGCGTTAAAAGAGGGTATTATAGATATCTTT GTTAAAGCTGGGGCGATGATTTGCACTCCGGGATGCGGACCTTGCTTAGGAGCTCATCAAGGGGTTTTGG CTGAGGGAGAAATTTGTTTATCAACAACAAACAGAAACTTTAAAGGAAGGATGGGGCATATAAATAGCTA TATTTACTTGGCATCTCCAAAGATTGCCGCAATAAGTGCAGTTAAGGGATATATAACCAACAAATTGGAT TAA

[Seq ID NO 114]

Methanococcus ja.nna.sch.ii AksD, MJ1003

MTLVEKILSKKVGYEVCAGDS IEVEVDLAMTHDGTTPLAYKALKEMSDSVWNPDKIVVAFDHNVPPNTVK AAEMQKLALEFVKRFGIKNFHKGGEGICHQILAENYVLPNMFVAGGDSHTCTHGAFGAFATGFGATDMAY IYATGETWIKVPKTIRVDIVGKNENVSAKDIVLRVCKEIGRRGATYMAIEYGGEVVKNMDMDGRLTLCNM AIEMGGKTGVIEADEITYDYLKKERGLSDEDIAKLKKERITVNRDEANYYKEIEIDITDMEEQVAVPHHP DNVKPISDVEGTE INQVF IGSCTNGRLSDLREAAKYLKGREVHKDVKLIVIPASKKVFLQALKEGI I DIF VKAGAMICTPGCGPCLGAHQGVLAEGE ICLSTTNRNFKGRMGHINSYIYLASPKIAAISAVKGYITNKLD

[Seq ID NO 115]

Methanococcus jannaschii AksD, MJ1003 Codon Pair optimized gene

ATGACTCTGGTTGAGAAGATCCTCTCCAAGAAAGTTGGTTACGAAGTTTGCGCAGGCGACTCCATCGAAG TTGAAGTTGACCTGGCGATGACTCACGACGGTACTACTCCGCTGGCTTACAAAGCGCTGAAAGAGATGTC TGACTCCGTATGGAACCCGGACAAGATCGTTGTTGCATTCGACCACAACGTACCGCCGAACACCGTTAAA GCAGCTGAAATGCAGAAGCTGGCGCTGGAATTCGTTAAGCGCTTCGGTATCAAAAACTTCCACAAAGGTG GTGAAGGTATCTGCCACCAGATCCTGGCTGAAAACTACGTTCTGCCGAACATGTTCGTTGCTGGCGGCGA CTCTCACACCTGTACTCACGGTGCATTCGGTGCATTCGCAACTGGCTTCGGTGCAACTGACATGGCTTAC ATCTACGCAACTGGCGAAACCTGGATCAAAGTTCCGAAAACTATCCGCGTTGATATCGTTGGTAAAAACG AAAACGTATCTGCGAAAGACATCGTTCTGCGCGTTTGCAAAGAAATCGGTCGTCGCGGTGCAACTTACAT GGCTATCGAATACGGTGGTGAAGTTGTTAAAAACATGGACATGGACGGTCGTCTGACTCTGTGCAACATG GCTATCGAAATGGGTGGTAAAACTGGCGTTATCGAAGCTGACGAAATCACTTACGACTACCTGAAGAAAG AGCGTGGTCTGTCTGACGAAGATATCGCTAAACTGAAGAAAGAGCGTATCACCGTTAACCGTGACGAAGC TAACTACTACAAAGAAATCGAAATCGACATCACTGACATGGAAGAACAGGTTGCTGTACCGCACCACCCG GATAACGTTAAGCCAATCTCTGACGTTGAAGGTACTGAAATCAACCAGGTATTCATCGGTTCCTGCACCA ACGGTCGTCTGTCTGATCTGCGTGAAGCTGCGAAATACCTGAAAGGTCGTGAAGTTCACAAAGACGTTAA GCTGATCGTTATCCCGGCTTCCAAGAAAGTATTCCTGCAGGCGCTGAAAGAAGGTATCATCGACATCTTC GTTAAAGCGGGTGCGATGATCTGTACTCCGGGTTGCGGTCCGTGCCTGGGTGCACACCAGGGCGTACTGG CAGAAGGTGAAATCTGCCTGTCTACTACCAACCGTAACTTCAAAGGTCGTATGGGTCACATCAACTCTTA CATCTACCTGGCTTCTCCGAAAATCGCTGCTATCTCTGCTGTTAAAGGTTACATCACTAACAAGCTGGAT

[Seq ID NO 116] Methanocaldococcus jannaschii DSM 2661 (motif) wild type gene

ATGATTATTAAGGGAAGAGCTCACAAATTTGGGGATGATGTAGATACAGACGCAATAATTCCAGGACCTT ACTTAAGGACTACAGACCCTTACGAGTTAGCTTCACACTGCATGGCAGGGATAGATGAAAACTTCCCGAA AAAGGTTAAGGAGGGGGATGTGATAGTTGCTGGAGAGAATTTTGGTTGTGGTTCAAGTAGGGAGCAGGCT GTAATAGCAATAAAATACTGTGGTATTAAGGCTGTGATAGCAAAAAGCTTTGCAAGAATATTCTATAGAA ATGCAATAAACGTTGGATTAATACCAATAATAGCAAATACAGATGAAATTAAAGACGGAGACATAGTAGA GATTGATTTAGATAAAGAAGAGATTGTAATAACCAATAAAAACAAAACAATAAAGTGTGAAACACCAAAA GGTTTAGAAAGAGAAATATTGGCTGCTGGTGGCTTAGTCAATTATTTAAAAAAGAGAAAACTAATACAAT CAAAAAAAGGTGTAAAAACATGA

[Seq ID NO 117]

Methanococcus jannaschii AksE, MJ1271

MI IKGRAHKFGDDVDTDAI IPGPYLRTTDPYELASHCMAGIDENFPKKVKEGDVIVAGENFGCGSSREQA VIAIKYCGIKAVIAKSFARIFYRNAINVGLIPI IANTDE IKDGDIVE IDLDKEEIVITNKNKTIKCETPK GLERE ILAAGGLVNYLKKRKLIQSKKGVKT

[Seq ID NO 118]

Methanococcus jannaschii AksE, MJ1271 Codon Pair optimized gene

ATGATCATCAAAGGTCGTGCGCACAAGTTCGGTGACGACGTTGACACTGACGCTATCATCCCAGGTCCGT ACCTCCGTACTACTGACCCGTACGAACTGGCATCTCACTGCATGGCGGGTATCGACGAAAACTTCCCGAA GAAAGTTAAAGAAGGTGACGTTATCGTTGCTGGCGAAAACTTCGGTTGCGGTTCTTCCCGTGAGCAGGCT GTTATCGCTATCAAATACTGCGGTATCAAAGCGGTTATCGCTAAATCTTTCGCACGTATCTTCTACCGTA ACGCAATCAACGTAGGTCTGATCCCGATCATCGCTAACACCGACGAAATCAAAGACGGTGACATCGTTGA AATCGACCTGGATAAAGAAGAAATCGTTATCACTAACAAAAACAAAACTATCAAGTGCGAAACTCCGAAA GGTCTGGAACGTGAAATCCTGGCAGCTGGCGGTCTGGTTAACTACCTGAAGAAACGTAAGCTGATTCAGT CCAAGAAAGGCGTAAAAACT

[Seq ID NO 119]

Methanocaldococcus jannaschii DSM 2661 (motif) wild type gene

ATGATGAAGGTGTGTGTTATAGAAGGGGATGGAATAGGAAAAGAAGTGATTCCAGAGGCCATAAAAATAT TAAATGAGTTGGGAGAGTTTGAAATAATAAAAGGAGAGGCAGGATTAGAATGTTTAAAAAAATATGGTAA TGCACTTCCAGAGGATACAATAGAAAAAGCTAAAGAGGCAGATATTATTTTGTTTGGGGCTATAACCTCA CCAAAGCCAGGGGAAGTTCAAAATTATAAAAGCCCTATAATAACGTTGAGGAAGATGTTTCATTTATATG CAAATGTAAGACCAATAAACAACTTTGGAATTGGACAATTAATTGGGAAAATTGCAGATTATGAATTCTT AAATGCTAAGAATATTGATATAGTTATTATAAGAGAGAATACGGAAGATTTATATGTTGGTAGAGAGAGA TTAGAAAATGATACAGCAATAGCTGAGAGGGTTATAACAAGAAAGGGTAGCGAGAGAATAATAAGATTTG CATTTGAATATGCTATAAAAAATAATAGGAAAAAGGTATCTTGCATCCATAAAGCTAATGTTTTAAGAAT AACTGATGGTTTATTCTTAGAGGTTTTTAATGAAATAAAAAAACATTATAATATAGAGGCAGATGATTAT TTAGTTGATTCAACAGCTATGAACTTAATAAAACATCCTGAAAAATTTGATGTTATTGTTACAACAAACA TGTTTGGGGATATTTTATCAGATGAGGCATCTGCATTAATTGGAGGACTTGGTTTAGCTCCTTCAGCAAA TATAGGAGATGATAAAGCATTATTTGAGCCAGTTCATGGTTCAGCTCCAGATATAGCTGGGAAAGGTATA GCAAATCCAATGGCATCTATATTAAGTATTGCTATGCTTTTTGATTATATTGGAGAGAAAGAAAAGGGAG ATTTGATTAGAGAGGCAGTGAAATACTGCTTAATAAACAAAAAAGTTACTCCTGACTTGGGAGGGGATTT AAAGACAAAAGATGTTGGAGACGAAATTCTAAATTACATTAGAAAGAAGTTAAAGGGATATTGA

[Seq ID NO 120]

Methanococcus ja.nna.sch.ii AksF, MJ1596

MMKVCVIEGDGIGKEVIPEAIKILNELGEFE I IKGEAGLECLKKYGNALPEDT IEKAKEAD I I LFGAITS PKPGEVQNYKSPI ITLRKMFHLYANVRPINNFGIGQL IGKIADYEFLNAKNID IVI IRENTEDLYVGRER LENDTAIAERVITRKGSERI IRFAFEYAIKNNRKKVSCIHKANVLRITDGLFLEVFNEIKKHYNIEADDY LVDSTAMNLIKHPEKFDVIVTTNMFGDILSDEASALIGGLGLAPSANIGDDKALFEPVHGSAPDIAGKGI ANPMASILS IAMLFDYIGEKEKGDL IREAVKYCLINKKVTPDLGGDLKTKDVGDE ILNYIRKKLKGY

[Seq ID NO 121]

Methanococcus jannaschii AksF, MJ1596 Codon Pair optimized gene

ATGATGAAAGTTTGCGTTATCGAAGGTGACGGTATCGGTAAAGAAGTTATCCCGGAAGCTATCAAGATCC TGAACGAACTGGGTGAATTCGAAATCATCAAAGGTGAAGCGGGTCTGGAATGCCTGAAGAAATACGGTAA CGCACTGCCAGAAGATACCATCGAAAAAGCGAAAGAAGCTGACATCATCCTGTTCGGTGCAATCACTTCT CCGAAGCCGGGTGAAGTTCAGAACTACAAATCTCCGATCATCACTCTGCGTAAGATGTTCCACCTGTACG CTAACGTACGTCCGATCAACAACTTCGGTATCGGTCAGCTGATCGGTAAGATCGCTGACTACGAGTTCCT GAACGCTAAAAACATCGACATCGTTATCATCCGTGAAAACACTGAAGATCTGTACGTTGGTCGTGAACGT CTGGAAAACGACACTGCTATCGCTGAGCGCGTTATCACTCGTAAAGGTTCTGAACGTATCATCCGCTTCG CATTCGAATACGCAATCAAAAACAACCGTAAGAAAGTTTCCTGCATCCACAAAGCTAACGTACTGCGTAT CACTGACGGTCTGTTCCTGGAAGTATTCAACGAAATCAAGAAACACTACAACATCGAAGCTGACGACTAC CTGGTTGACTCCACTGCAATGAACCTGATCAAGCACCCGGAAAAATTCGACGTTATCGTTACCACTAACA TGTTCGGTGACATCCTGTCTGACGAAGCGTCTGCACTGATCGGTGGTCTGGGTCTGGCACCGTCTGCTAA CATCGGTGACGACAAAGCGCTGTTCGAACCGGTTCACGGTTCTGCACCGGATATCGCTGGTAAAGGTATC GCTAACCCGATGGCTTCTATCCTGTCTATCGCGATGCTGTTCGACTACATCGGTGAAAAAGAGAAAGGCG ACCTGATCCGTGAAGCGGTAAAATACTGCCTGATCAACAAGAAAGTTACTCCGGATCTGGGTGGTGACCT GAAAACCAAAGACGTTGGTGACGAAATCCTGAACTACATCCGTAAGAAACTGAAAGGTTAC

[Seq ID NO 122]

Methanococcus vannielii SB AksF, Mevan_0040 Wild type ATGGGCTATA TGCCAAAAAT CTGTGTCATA ACTGGTGATG GAATTGGAAA AGAAGTCGTG CCTGAAACAT TAAGAGTTTT AAACGAAGTT CACGACTTTG AATATATTGA AGCCCATGCT GGGTACGAGT GTTTTAAGAG ATGCGGAGAA TCAATACCTG AAAGCACGAT TCAAACAGCT AAAAATTCTG ATTCAATTCT TTTTGGTTCA GTAACTACTC CAAAACCAAC TGAATTAAAA AATAAGCCAT ATCGTAGTCC AATACTTACG TTAAGGCAGG AATTAGACCT TTATGCAAAC ATACGGCCCA CTTATAATTT TAAAGACTTG GATTTTGTCA TAATACGGGA AAATACTGAG TGTCTTTACG TAAAAAGGGA GTATTATGAC GAAATAAACG AAGTAGCAAT TGCCGAAAGA ATAATTTCAA AAAAGGGAAG CGAAAGAATA ATAAAATTTG CATTTGAATA TGCAAGGTTA AATAATCGAA AAAAAGTGTC TTGCATACAC AAAGCGAATG TATTAAGAGT AACTGACGGA TTATTTTTGG AAATTTTCGA AAAAATAGCT AAACTTTATG AAAACTTTGG TATATCGAGC AATGATTACT TAATAGATGC AACAGCAATG TACCTTATTA AAAATCCATA TATGTTTGAT GTAATGGTTA CAACAAACCT TTTTGGAGAT ATTTTATCTG ATGAGGCCGC AGGACTTATT GGGGGTCTTG GAATGTCGCC TTCTGCAAAT ATTGGGGATA ATTTAGGATT ATTTGAGCCT GTTCATGGTT CAGCCCCAGA TATTGCTGGA AAAGGAATAT CTAATCCGAT TGCGACAATT TTAAGTGCTT CAATGATGCT TGACCATTTA AAAATGAATA AAAAGGCGGA AATTATAAGA AATGCAGTTA AAAAAACGAT AAATAATGGT TATTTGACAC CCGATCTTGG TGGAAGCCTG AAAACTTCCG AAGTTGTAAA TAAAGTTATA GAATTTATTC GGGATGAAAT CTAA

[Seq ID NO 123]

Metha.nocoecus vannielii SB AksF, Mevan_0040

MGYMPKICVITGDGIGKEVVPETLRVLNEVHDFEYIEAHAGYECFKRCGES IPESTIQTAKNSDS ILFGS VTTPKPTELKNKPYRSPILTLRQELDLYANIRPTYNFKDLDFVI IRENTECLYVKREYYDE INEVAIAER I ISKKGSERI IKFAFEYARLNNRKKVSCIHKANVLRVTDGLFLEIFEKIAKLYENFGISSNDYLIDATAM YLIKNPYMFDVMVTTNLFGDILSDEAAGLIGGLGMSPSANIGDNLGLFEPVHGSAPDIAGKGISNPIATI LSASMMLDHLKMNKKAE I IRNAVKKTINNGYLTPDLGGSLKTSEVVNKVIEF IRDE I

[Seq ID NO 124]

Codon Pair optimized gene, Mevan_0040

ATGGGTTACATGCCGAAAATCTGCGTTATCACTGGCGACGGTATCGGTAAAGAAGTTGTTCCGGAAACTC TGCGCGTACTGAACGAAGTTCACGACTTCGAATACATCGAAGCACACGCGGGTTACGAGTGCTTCAAGCG CTGCGGTGAATCCATCCCGGAATCCACTATTCAGACTGCGAAAAACTCTGACTCCATCCTGTTCGGTTCT GTTACCACTCCGAAACCAACTGAACTGAAAAACAAGCCGTACCGCTCTCCGATTCTGACTCTGCGTCAGG AACTGGATCTGTACGCTAACATCCGTCCGACTTACAACTTCAAAGACCTGGACTTCGTTATCATCCGTGA AAACACTGAATGCCTGTACGTTAAGCGTGAATACTACGACGAAATCAACGAAGTTGCTATCGCTGAACGT ATCATCTCCAAGAAAGGTTCTGAACGTATCATCAAATTCGCTTTCGAATACGCACGTCTGAACAACCGTA AGAAAGTTTCCTGCATCCACAAAGCTAACGTACTGCGCGTAACTGACGGTCTGTTCCTGGAAATCTTCGA GAAGATCGCGAAACTGTACGAAAACTTCGGTATCTCTTCTAACGACTACCTGATCGACGCAACTGCAATG TACCTGATCAAAAACCCGTACATGTTCGACGTAATGGTTACCACTAACCTGTTCGGTGACATCCTGTCTG ACGAAGCTGCTGGTCTGATCGGTGGTCTGGGTATGTCTCCGTCTGCTAACATCGGTGACAACCTGGGTCT GTTCGAACCGGTTCACGGTTCTGCACCGGATATCGCTGGTAAAGGTATCTCCAACCCGATCGCGACTATC CTGTCTGCGTCTATGATGCTGGATCACCTGAAAATGAACAAGAAAGCAGAAATCATCCGTAACGCTGTTA AGAAAACTATCAACAACGGTTACCTGACTCCGGACCTGGGTGGTTCTCTGAAAACTTCTGAAGTTGTTAA CAAAGTTATCGAATTCATCCGCGACGAGATT [Seq ID NO 125]

Methanococcus maripaludis S2 §§§ § :ί:ί:ί:ΜΜΡ0880 Wild type gene

atgagaaacactcccaaaatttgtgtt tcaatggtgacggt ttggaaacgaagt gttcctgaaacgg tgcgagttttaaatgaacttggtgacttcgaattcattcatgcccatgcaggttacgaatgttttaaaag atgtggcgatgcgataccagaaaacacaattgaaattgcaaaagaatctgattgtattttatttggatca gttaccactccaaaaccgactgaattaaaaaataaatcatatagaagtccaatattaactttaagaaaag aacttgacctttatgcaaatattaggccaacttataactttgataatcttgattttgttataattcgaga aaatactgaaggactctatgtaaaaaaagaatattacgacgaaaaaaacgaagttgcaattgctgagcga ataatttcaaaatttggaagttctagaattgtaaaatttgcttttgattatgcggttcaaaataacagaa aaaaagtatcctgcatacataaagcaaacgtattacgggttactgacggattatttttagaagttttcga agaaatgtctaaacattacgaaaaattaggaataaagtctgatgactacctaattgacgcgacagcaatg tatttgattagaaacccgcaaatgtttgatgtattggttacaacaaatctttttggagatattttatctg atgaagctgcaggacttattggcggacttggaatgtctccttcagcaaacattggtgataaaaacggatt atttgagccagttcatggatctgcaccagacattgctggaaaaggaatttcaaacccgattgcaacaata ttgagtgctgcaatgatgcttgaccatttaaaaatgaataaagaagccgaatacattagaaaagcggtta aaaaaacggttgaatgtaaatatttaactcctgatcttgggggaaacttaaaaacttttgaagttacgga aaaaatcattgaatccataaggtctcagatgattcagtga

[Seq ID NO 126]

Methanococcus maripaludis S2 AksF, MMP0880

MRNTPKICVINGDGIGNEVVPETVRVLNELGDFEF IHAHAGYECFKRCGDAIPENTIEIAKESDCILFGS VTTPKPTELKNKSYRSPILTLRKELDLYANIRPTYNFDNLDFVI IRENTEGLYVKKEYYDEKNEVAIAER I ISKFGSSRIVKFAFDYAVQNNRKKVSCIHKANVLRVTDGLFLEVFEEMSKHYEKLGIKSDDYLIDATAM YLIRNPQMFDVLVTTNLFGDILSDEAAGLIGGLGMSPSANIGDKNGLFEPVHGSAPDIAGKGISNPIATI LSAAMMLDHLKMNKEAEYIRKAVKKTVECKYLTPDLGGNLKTFEVTEKI IESIRSQMIQ

[Seq ID NO 127]

Codon Pair optimized gene, MMP0880

ATGCGTAACACTCCGAAAATCTGCGTTATCAACGGTGACGGTATCGGTAACGAAGTTGTTCCGGAAACCG TTCGCGTACTGAACGAACTGGGTGACTTCGAATTCATCCACGCGCACGCTGGTTACGAATGCTTCAAGCG CTGCGGTGACGCTATCCCGGAAAACACCATCGAAATCGCTAAAGAGTCTGACTGCATCCTGTTCGGTTCT GTAACTACTCCGAAACCAACTGAACTGAAAAACAAGTCTTACCGCTCTCCGATTCTGACTCTGCGTAAAG AGCTGGATCTGTACGCTAACATCCGTCCGACTTACAACTTCGACAACCTGGATTTCGTTATCATCCGTGA AAACACTGAAGGTCTGTACGTTAAGAAAGAATACTACGACGAGAAAAACGAAGTTGCTATCGCTGAACGT ATCATCTCCAAGTTCGGTTCTTCTCGCATCGTTAAATTCGCATTCGACTACGCAGTACAGAACAACCGTA AGAAAGTTTCCTGCATCCACAAAGCGAACGTTCTGCGCGTAACTGACGGTCTGTTCCTGGAAGTTTTCGA AGAAATGTCCAAGCACTACGAAAAACTGGGTATCAAATCTGACGACTACCTGATCGACGCAACTGCGATG TACCTGATCCGTAACCCGCAGATGTTCGACGTTCTGGTTACTACCAACCTGTTCGGTGACATCCTGTCTG ACGAAGCAGCTGGTCTGATTGGTGGTCTGGGTATGTCTCCGTCTGCTAACATCGGTGACAAAAACGGTCT GTTCGAACCGGTTCACGGTTCTGCACCGGATATCGCTGGTAAAGGTATCTCCAACCCGATCGCGACTATC CTGTCTGCTGCAATGATGCTGGATCACCTGAAAATGAACAAAGAAGCTGAATACATCCGTAAAGCGGTTA AGAAAACCGTTGAATGCAAATACCTGACTCCGGACCTGGGTGGTAACCTGAAAACTTTCGAAGTTACTGA AAAGATCATCGAATCCATCCGTTCTCAGATGATTCAA

[Seq ID NO 128]

Methanococcus maripaludis S2 AksE, MMP0381 Wild type

ATGAAAATAA CTGGTAAGGT GCACTTATTT GGGGATGACA TCGATACTGA TGCGATAATT

CCCGGAGCTT ATTTAAAAAC GACTGATGAA TATGAGCTTG CATCGCACTG TATGGCAGGA

ATTGACGAAA ATTTTCCAGA AAGGGTCGAA GATGGTGACT TTTTAGTTGC AGGTGAAAAT

TTTGGATGCG GAAGTTCAAG GGAACAGGCC CCAATTGCCA TAAAATACTG CGGAATCAAG

GCAATAATTG TTGAGAGTTT TGCAAGGATA TTTTACAGAA ATTGCATAAA TTTAGGAGTA

TTTCCAATTG AATGCAAGGG AATATCAAAA CACGTCAAAG ATGGGGATGT AATAGAATTA

GATCTTGAAG AAAAAAAAGT TATCTTAAAA GACACGGTTC TTGACTGCAA TCTTCCGACA

GGGACTGCAA AAGATATAAT GGATGAAGGC GGGCTTATAA ATTACGCAAA GAAACAAAAA AATTAA

[Seq ID NO 129]

Methanococcus maripaludis S2 AksE, MMP0381

MKITGKVHLFGDDIDTDAI IPGAYLKTTDEYELASHCMAGIDENFPERVEDGDFLVAGENFGCGSSREQA PIAIKYCGIKAI IVESFARIFYRNCINLGVFPIECKGISKHVKDGDVIELDLEEKKVILKDTVLDCNLPT GTAKDIMDEGGLINYAKKQKN

[Seq ID NO 130]

Codon Pair optimized gene, MMP0381

ATGAAGATCACCGGTAAAGTTCACCTGTTCGGTGACGACATCGACACTGACGCTATCATTCCGGGTGCTT ACCTGAAAACCACTGACGAATACGAACTGGCTTCTCACTGCATGGCGGGTATCGACGAAAACTTCCCGGA ACGCGTTGAAGATGGCGACTTCCTGGTTGCTGGCGAAAACTTCGGTTGCGGTTCTTCCCGTGAACAGGCA CCGATTGCTATCAAATACTGCGGTATCAAAGCAATCATCGTTGAATCCTTCGCACGTATCTTCTACCGTA ACTGCATCAACCTGGGCGTATTCCCGATCGAATGCAAAGGTATCTCCAAGCACGTTAAAGACGGTGACGT TATCGAACTGGATCTGGAAGAGAAGAAAGTTATCCTGAAAGACACCGTACTGGACTGCAACCTCCCGACT GGTACTGCGAAAGATATCATGGACGAAGGTGGTCTGATCAACTACGCTAAGAAGCAGAAAAAC

[Seq ID NO 131]

Methanococcus maripaludis S2 AksD, MMP1480 Wild type

ATGACACTTG CTGAGAAAAT CATTTCAAAA AATGTTGGAA AAAATGTTTA CGCCAAAGAC

AGCGTCGAAA TAAGCGTAGA TATTGCAATG ACACATGACG GGACCACCCC GCTTACGGTA

AAAGCCTTTG AGCAGATTTC AGATAAAGTA TGGGATAATG AAAAGATAGT CATTATTTTT GACCACAATA TCCCTGCAAA CACGTCAAAA GCTGCAAATA TGCAGGTTAT AACGAGAGAA

TTCATAAAAA AGCAGGGGAT TAAAAATTAC TACCTCGATG GCGAAGGAAT ATGCCATCAA

GTTCTACCTG AAAAGGGCCA TGTAAAACCA AACATGATAA TTGCGGGAGC TGACAGCCAC

ACATGTACTC ATGGGGCCTT TGGAGCATTT GCTACAGGTT TTGGTGCTAC AGACATGGGT

TACGTCTATG CAACAGGAAA AACCTGGCTT AGAGTTCCTG AAACTATCCG CGTAAATGTA ACTGGAGAAA ATGAAAATAT TTCTGGAAAA GACATTATTT TAAAAACTTG TAAGGAAGTT

GGAAGACGTG GGGCTACGTA CATGTCTTTA GAATACGGTG GAAATGCAGT CCACAATCTT

TCAATGGATG AAAGAATGGT TCTGTCAAAC ATGGCTATTG AAATGGGCGG AAAAGCAGGA

ATTATCGAAG CTGATGATAC TACATATAGA TATCTTGAAA ATGCAGGAGT TTCGCGCGAA GAAATTCTTG AATTGAAAAA AAATAAAATA ACAGTTGATG AATCCGAAGA AGACTACTAC AAAACAATTG AATTTGACAT AACCGGTATG GAAGAACAGG TTGCATGCCC TCACCACCCT GATAACGTAA AAGGAGTTTC AGAAGTTGAA GGAACAGAAT TAAACCAGGT ATTCATCGGT TCATGCACAA ACGGAAGATT AAACGACTTA AGAATTGCTG CAAAATATTT GAAAGGAAAA AAAGTTAATG AAAACACAAG ATTAATTGTA ATCCCTGCAT CAAAGTCAAT ATTTAAAGAA GCCCTAAATG AAGGATTAAT TGATATCTTT GTAGATTCCG GAGCATTAAT ATGTACCCCT GGATGCGGAC CATGTCTTGG AGCCCATCAG GGGGTTTTAG GTGATGGAGA AGTATGCCTT GCTACAACTA ACCGGAACTT TAAAGGAAGA ATGGGAAACA CGAACGCACA AGTTTACCTC TCTTCTCCAA AAATAGCTGC AAAATCTGCG GTTAAAGGCT ACATTACAAA TGAATAA

[Seq ID NO 132]

Methanococcus maripaludis S2 AksD, MMP1480

MTLAEKI ISKNVGKNVYAKDSVE ISVDIAMTHDGTTPLTVKAFEQISDKVWDNEKIVI IFDHNIPANTSK AANMQVITREF IKKQGIKNYYLDGEGICHQVLPEKGHVKPNMI IAGADSHTCTHGAFGAFATGFGATDMG YVYATGKTWLRVPETIRVNVTGENENISGKDI ILKTCKEVGRRGATYMSLEYGGNAVHNLSMDERMVLSN MAIEMGGKAGI IEADDTTYRYLENAGVSREE ILELKKNKITVDESEEDYYKTIEFDI TGMEEQVACPHHP DNVKGVSEVEGTELNQVFIGSCTNGRLNDLRIAAKYLKGKKVNENTRLIVIPASKSIFKEALNEGLIDIF VDSGALICTPGCGPCLGAHQGVLGDGEVCLATTNRNFKGRMGNTNAQVYLSSPKIAAKSAVKGYITNE

[Seq ID NO 133]

Codon Pair optimized gene, MMP1480

ATGACTCTGGCTGAGAAGATCATCTCCAAAAACGTTGGTAAAAACGTTTACGCGAAAGACTCCGTTGAAA TCTCCGTTGACATCGCGATGACTCACGACGGTACTACTCCGCTGACCGTTAAAGCGTTCGAACAGATCTC TGACAAAGTATGGGATAACGAGAAGATCGTTATCATCTTCGACCACAACATCCCGGCTAACACCTCTAAA GCTGCTAACATGCAAGTTATCACTCGTGAATTCATCAAGAAGCAGGGTATCAAAAACTACTACCTGGATG GTGAAGGTATCTGCCACCAGGTACTGCCGGAAAAAGGTCACGTTAAGCCGAACATGATCATCGCTGGCGC AGACTCTCACACTTGCACTCACGGTGCATTCGGTGCATTCGCTACCGGTTTCGGTGCAACTGACATGGGT TACGTTTACGCAACTGGTAAAACCTGGCTGCGCGTACCGGAAACCATTCGCGTTAACGTAACTGGCGAAA ACGAAAACATCTCCGGTAAAGACATCATCCTGAAAACTTGCAAAGAAGTTGGTCGTCGCGGTGCAACTTA CATGTCTCTGGAATACGGTGGTAACGCTGTTCACAACCTGTCTATGGACGAACGTATGGTTCTGTCTAAC ATGGCTATCGAAATGGGTGGTAAAGCTGGTATCATCGAAGCTGACGACACCACTTACCGCTACCTGGAAA ACGCTGGCGTTTCCCGTGAAGAAATCCTGGAACTGAAGAAAAACAAGATCACCGTTGACGAATCTGAAGA AGATTACTACAAAACTATTGAATTCGACATCACCGGTATGGAAGAACAGGTTGCTTGCCCACACCACCCG GACAACGTTAAAGGCGTTTCTGAAGTTGAAGGTACTGAACTGAACCAGGTATTCATCGGTTCCTGCACCA ACGGTCGTCTGAACGATCTGCGTATTGCTGCGAAATACCTGAAAGGTAAGAAAGTTAACGAAAACACCCG TCTGATCGTTATCCCGGCATCTAAATCTATCTTCAAAGAAGCGCTGAACGAAGGTCTGATCGACATCTTC GTTGACTCCGGTGCACTGATCTGCACTCCGGGTTGCGGTCCGTGCCTGGGTGCACACCAGGGCGTTCTGG GTGACGGTGAAGTTTGCCTGGCAACCACTAACCGTAACTTCAAAGGTCGTATGGGTAACACCAACGCTCA GGTTTACCTGTCCTCTCCGAAGATCGCTGCGAAGTCTGCGGTAAAAGGTTACATCACTAATGAG

[Seq ID NO 134] Methanococcus aeolicus Nankai 3, aksE Maeo_0652, wild-type

atgataataaaaggaaatattcatttatttggtgatgatattgataccgatgccataattcccggggcct accttaaaacaacagacccaaaggagttggcatctcattgcatggctggaattgatgaaaaattttcaac aaaggtaaaagacggcgatataattgttgcaggtgaaaattttggctgtggaagtagtagggaacaggca ccaatatccataaaacacaccggaataaaggcagtagttgctgaaagttttgcacggatattttatagaa attgtattaatataggattaatacctataacttgcgaaggaataaatgaacaaatccaaaacctaaaaga tggcgacacaatagaaattgatttgcaaaatgaaacaataaaaataaattctatgatgttaaattgtgga gctcccaaagggatagaaaaagaaattttagatgctggtggattagtacaatatacaaaaaataagttaa aaaaataa

[Seq ID NO 135]

Methanococcus aeolicus Nankai 3, aksE, Maeo_0652

MIIKGNIHLFGDDIDTDAIIPGAYLKTTDPKELASHCMAGIDEKFSTKVKDGDIIVAGENF

GCGSSREQAPISIKHTGIKAVVAESFARIFYRNCINIGLIPITCEGINEQIQNLKDGDTIEID

LQNETIKINSMMLNCGAPKGIEKEILDAGGLVQYTKNKLKK

[Seq ID NO 136]

Codon Pair optimized gene, MaeO_0652

ATGATCATCAAAGGTAACATCCACCTGTTCGGTGACGACATCGACACTGACGCTATCATCCCAGGTGCTT ACCTGAAAACCACTGACCCGAAAGAGCTGGCATCTCACTGCATGGCGGGTATCGACGAAAAATTCTCTAC CAAAGTTAAAGACGGTGACATCATCGTTGCTGGCGAAAACTTCGGTTGCGGTTCTTCCCGTGAACAGGCA CCGATCTCCATCAAGCACACCGGTATCAAAGCGGTTGTTGCTGAATCCTTCGCTCGCATTTTCTACCGTA ACTGCATCAACATCGGTCTGATCCCGATCACCTGTGAAGGTATCAACGAACAGATTCAGAACCTGAAAGA CGGTGACACCATCGAAATCGATCTGCAGAACGAAACCATCAAGATCAACTCCATGATGCTGAACTGCGGT GCACCGAAAGGTATCGAAAAAGAAATCCTGGATGCTGGCGGTCTGGTACAGTACACCAAGAACAAGCTGA AGAAA

[Seq ID NO 137]

Methanococcus aeolicus Nankai 3, AksD Maeo_0311, wild-type

atgacattggcagaggaaatattatcaaaaaaagtaggaaaaaaagtaaaagcaggagatgttgtagaaa tagatatagatttagcaatgactcatgatggaacaacaccattatctgcaaaggcatttaaacagataac cgataaggtatgggacaataaaaaaatagtcatagtatttgaccataatgtcccagcaaatacattaaaa gccgcaaatatgcaaaaaattacacgagaatttataaaagaacaaaatataataaatcattatttggatg gtgaaggcgtatgccatcaagtgctacctgaaaacggacatatacaaccaaacatggttatagctggtgg agatagtcacacatgcacctatggggcatttggggcatttgcaacaggatttggggctaccgacatgggg aatatatatgcaacaggaaaaacttggttaaaagttccaaaaaccataagaataaatgttaatggagaaa atgataaaattactggaaaagatattattttaaaaatttgtaaagaagttggacgaagtggagctactta catggcacttgaatacggcggggaagcaataaaaaaattaagtatggacgaaagaatggttttaagcaat atggctatcgaaatgggcggaaaagttgggcttatcgaagccgatgaaaccacatataattaccttagaa atgtaggaattagtgaagaaaaaatattagaattgaaaaaaaatcaaataaccattgatgagaacaatat agataatgataattattataaaattataaatatagatattacagacatggaggagcaagtggcatgccct caccatcccgataatgttaaaaatatttcggaagtaaaaggagctcccataaatcaggtgttcataggtt catgcacaaatggtaggttgaacgatttaagaatagcctcaaaatatttaaaaggaaaaaaggttcataa tgatgttagattaatagtaatacctgcttcaaaatcaatatttaaacaggcattaaaagaaggattaatt gatatttttgtagatgctggagctttaatttgcacccccggatgcggtccttgtttgggggcccaccaag gagtttt ggagatggtgaagtttgttt gccaccacaaat gaaatttcaaaggaagaatgggaaat c gacagcggaaatatatttatcctcccccgctattgccgcaaaaagtgcaattaaaggatatatcacaaat gaataa

[Seq ID NO 138]

Methanococcus aeolicus Nankai 3, AksD, Maeo_031 1

MTLAEEILSKKVGKKVKAGDVVE IDIDLAMTHDGTTPLSAKAFKQITDKVWDNKKIVIVFDHNVPANTLK AANMQKITREF IKEQNI INHYLDGEGVCHQVLPENGHIQPNMVIAGGDSHTCTYGAFGAFATGFGATDMG NIYATGKTWLKVPKTIRINVNGENDKITGKDI ILKICKEVGRSGATYMALEYGGEAIKKLSMDERMVLSN MAIEMGGKVGLIEADETTYNYLRNVGISEEKILELKKNQITIDENNIDNDNYYKI INIDITDMEEQVACP HHPDNVKNISEVKGAPINQVF IGSCTNGRLNDLRIASKYLKGKKVHNDVRLIVIPASKS IFKQALKEGLI DIFVDAGALICTPGCGPCLGAHQGVLGDGEVCLATTNRNFKGRMGNTTAEIYLSSPAIAAKSAIKGYITN

E

[Seq ID NO 139]

Codon Pair optimized gene, MaeO_031 1

ATGACTCTGGCTGAAGAAATCCTGTCCAAGAAAGTTGGTAAGAAAGTTAAAGCGGGTGACGTTGTTGAAA TCGATATCGACCTGGCGATGACTCACGACGGTACTACTCCGCTGTCTGCGAAAGCATTCAAGCAGATCAC TGACAAAGTATGGGATAACAAGAAAATCGTTATCGTTTTCGACCACAACGTTCCGGCTAACACCCTGAAA GCTGCTAACATGCAGAAGATCACTCGCGAATTCATCAAAGAGCAGAACATCATCAACCACTACCTGGACG GTGAAGGTGTTTGCCACCAGGTACTGCCGGAAAACGGTCACATTCAGCCGAACATGGTTATCGCTGGCGG CGATTCTCACACCTGTACTTACGGCGCATTCGGTGCGTTCGCTACTGGCTTCGGTGCAACTGACATGGGT AACATCTACGCAACTGGTAAAACCTGGCTGAAAGTTCCGAAAACTATTCGTATCAACGTTAACGGTGAAA ACGACAAGATCACCGGTAAAGACATCATCCTGAAAATCTGCAAAGAAGTTGGTCGTTCTGGTGCAACTTA CATGGCGCTGGAATACGGTGGTGAAGCAATCAAGAAACTGTCTATGGACGAACGTATGGTTCTGTCTAAC ATGGCTATCGAAATGGGTGGTAAAGTTGGTCTGATCGAAGCTGACGAAACCACTTACAACTATCTGCGTA ACGTTGGTATTTCTGAAGAGAAGATCCTGGAACTGAAGAAAAACCAGATCACTATCGACGAAAACAACAT CGACAACGACAACTACTACAAAATCATCAACATCGACATCACTGACATGGAAGAACAGGTTGCTTGCCCG CACCACCCGGATAACGTTAAAAACATCTCTGAAGTTAAAGGCGCACCAATCAACCAGGTATTCATCGGTT CCTGCACCAACGGTCGCCTGAACGATCTGCGCATTGCTTCTAAATACCTGAAAGGTAAGAAAGTTCACAA CGACGTACGTCTGATCGTTATCCCGGCTTCCAAGTCTATCTTCAAGCAGGCGCTGAAAGAAGGTCTGATC GACATCTTCGTTGACGCTGGCGCGCTGATCTGCACTCCGGGTTGCGGTCCGTGCCTGGGTGCACACCAGG GCGTACTGGGTGACGGTGAAGTTTGCCTGGCAACTACCAACCGTAACTTCAAAGGTCGTATGGGTAACAC CACTGCTGAAATCTACCTGTCCTCTCCGGCAATCGCTGCTAAATCTGCTATCAAAGGTTACATCACTAAC GAG

[Seq ID NO 140]

Wild-type gene, nifV ATGGCTAGCGTGATCATCGACGACACTACCCTGCGTGACGGTGAACAGAGTGCCGGGGTCGCCTTCAATG CCGACGAGAAGATCGCTATCGCCCGCGCGCTCGCCGAACTGGGCGTGCCGGAGTTGGAGATCGGCATTCC CAGCATGGGCGAGGAAGAGCGCGAGGTGATGCACGCCATCGCCGGTCTCGGCCTGTCGTCTCGCCTGCTG GCCTGGTGCCGGCTATGCGACGTCGATCTCGCGGCGGCGCGCTCCACCGGGGTGACCATGGTCGACCTTT CGCTGCCGGTCTCCGACCTGATGCTGCACCACAAGCTCAATCGCGATCGCGACTGGGCCTTGCGCGAAGT GGCCAGGCTGGTCGGCGAAGCGCGCATGGCCGGGCTCGAGGTGTGCCTGGGCTGCGAGGACGCCTCGCGG GCGGATCTGGAGTTCGTCGTGCAGGTGGGCGAAGTGGCGCAGGCCGCCGGCGCCCGTCGGCTGCGCTTCG CCGACACCGTCGGGGTCATGGAGCCCTTCGGCATGCTCGACCGCTTCCGTTTCCTCAGCCGGCGCCTGGA CATGGAGCTGGAAGTGCACGCCCACGATGATTTCGGGCTGGCCACGGCCAACACCCTGGCCGCGGTGATG GGCGGGGCGACTCATATCAACACCACGGTCAACGGGCTCGGCGAGCGTGCCGGCAACGCCGCGCTGGAAG AGTGCGTGCTGGCGCTCAAGAACCTCCACGGTATCGACACCGGTATCGATACCCGCGGCATCCCGGCCAT CTCCGCGCTGGTCGAGCGGGCCTCGGGGCGCCAGGTGGCCTGGCAGAAGAGCGTGGTCGGCGCCGGGGTG TTCACTCACGAGGCCGGTATCCACGTCGACGGACTGCTCAAGCATCGGCGCAACTACGAGGGGCTGAATC CCGACGAACTCGGTCGCAGCCACAGTCTGGTGCTGGGCAAGCATTCCGGGGCGCACATGGTGCGCAACAC GTACCGCGATCTGGGTATCGAGCTGGCGGACTGGCAGAGCCAAGCGCTGCTCGGCCGCATCCGTGCCTTC TCCACCAGGACCAAGCGCAGCCCGCAGCCTGCCGAGCTGCAGGATTTCTATCGGCAGTTGTGCGAGCAAG GCAATCCCGAACTGGCCGCAGGAGGAATGGCATGA

[Seq ID NO 141]

A.vinelandii nifV

MASVI IDDTTLRDGEQSAGVAFNADEKIAIARALAELGVPELE IGIPSMG EEEREVMHAIAGLGLSSRLLAWCRLCDVDLAAARSTGVTMVDLSLPVSDL MLHHKLNRDRDWALREVARLVGEARMAGLEVCLGCEDASRADLEFWQVG EVAQAAGARRLRFADTVGVMEPFGMLDRFRFLSRRLDMELEVHAHDDFGL ATANTLAAVMGGATHINTTVNGLGERAGNAALEECVLALKNLHGIDTGID TRGIPAISALVERASGRQVAWQKSVVGAGVFTHEAGIHVDGLLKHRRNYE GLNPDELGRSHSLVLGKHSGAHMVRNTYRDLGIELADWQSQALLGRIRAF STRTKRSPQPAELQDFYRQLCEQGNPELAAGGMA

[SEQ I D No. 142] wild-type T. maritima diaminopimelate decarboxylase gene

ATGGACATCCTGAGGAAAGTGGCAGAGATTCATGGGACACCCACCTACGTATACTTCGAG

GAAACACTGCGAAAAAGGTCACGTCTTGTAAAAGAGGTCTTCGAGGGAGTGAATCTCCTTC

CAACGTTTGCCGTGAAAGCGAACAACAATCCTGTTCTTTTGAAGATTCTAAGAGAAGAGGG

TTTCGGCATGGACGTGGTGACAAAGGGGGAACTCCTCGCGGCTAAACTGGCGGGAGTTC

CTTCCCATACCGTTGTATGGAACGGCAACGGAAAGAGCAGGGATCAAATGGAACACTTTTT

GAGAGAAGATGTGAGAATCGTCAACGTGGATTCGTTCGAGGAGATGGAGATCTGGAGAGA

ATTGAACCCGGAAGGCGTGGAGTATTTCATCAGGGTGAATCCGGAGGTCGATGCGAAGAC

ACACCCTCACATCTCCACCGGCTTGAAAAAGCACAAGTTCGGAATACCACTGGAAGATCTG

GATTCGTTCATGGAAAGATTCAGATCAATGAACATAAGAGGTCTCCATGTTCACATAGGATC

GCAGATAACCCGGGTTGAACCCTTTGTGGAAGCCTTCAGTAAAGTTGTTCGGGCTTCTGAA AGGTATGGATTCGAAGAGATCAACATCGGCGGCGGCTGGGGAATAAACTACAGCGGAGAG GAACTCGACCTGTCCAGTTACAGAGAAAAGGTTGTTCCTGATTTGAAGAGATTCAAAAGAG TCATCGTCGAAATAGGAAGGTACATCGTAGCACCTTCTGGGTATCTGCTCCTCAGAGTGGT GCTCGTCAAAAGAAGACATAACAAGGCGTTCGTTGTAGTCGATGGTGGGATGAATGTCCTC ATAAGACCGGCACTTTATTCCGCATATCACAGGATCTTTGTGCTCGGAAAACAGGGTAAAG AGATGAGGGCAGATGTGGTTGGTCCACTGTGCGAAAGCGGTGACGTGATCGCGTACGAC CGGGAACTTCCAGAGGTCGAACCGGGTGACATCATCGCTGTGGAAAACGCGGGAGCTTAC GGTTACACTATGTCGAACAACTACAACTCGACCACACGTCCAGCTGAAGTGCTCGTCAGAG AAAACGGAAGAATTTCTCTGATAAGAAGAAGGGAAACGGAGATGGATATTTTCAAAGACGT GGTGATGTGA

[SEQ I D No. 143] amino acid sequence T. maritima diaminopimelate decarboxylase Q9X1 K5 MDILRKVAEIHGTPTYVYFEETLRKRSRLVKEVFEGVNLLPTFAVKANNN PVLLKILREEGFGM D VVTKGELLAAKLAGVPSHTVVWNGNGKSRDQM EH FLREDVRIVNVDSFEEM EIWRELNPEGV EYFI RVN PEVDAKTH PHISTGLKKHKFGI PLEDLDSFMERFRSMN IRGLHVHIGSQITRVEPFVEA FSKVVRASERYGFEEINIGGGWGINYSGEELDLSSYREKVVPDLKRFKRVIVEIGRYIVAPSGYL LLRVVLVKRRHNKAFVVVDGGMNVLI RPALYSAYHRIFVLGKQGKEMRADVVGPLCESGDVIAY DRELPEVEPGDI IAVENAGAYGYTMSNNYNSTTRPAEVLVRENGRISLI RRRETEMDI FKDVVM

[SEQ I D No. 144] codon optimized T. maritima diaminopimelate decarboxylase gene

ATGGACATCCTGAGAAAGGTCGCGGAGATTCACGGTACTCCGACGTACGTCTACTTCGAA GAGACTTTGCGTAAACGCAGCCGCTTGGTGAAAGAGGTCTTTGAGGGCGTTAATCTGCTG CCGACGTTCGCGGTGAAGGCGAATAACAATCCGGTCCTGCTGAAGATCCTGCGCGAGGA GGGTTTTGGTATGGACGTGGTCACCAAGGGCGAGCTGCTGGCGGCAAAACTGGCGGGTG TCCCGAGCCATACCGTCGTTTGGAATGGTAATGGCAAATCGCGTGACCAGATGGAGCATTT TCTGCGTGAGGACGTTCGTATCGTTAATGTGGACTCTTTTGAAGAGATGGAAATCTGGCGT GAACTGAATCCGGAGGGTGTCGAGTATTTCATCCGTGTCAACCCAGAAGTGGACGCTAAA ACGCATCCGCACATCAGCACGGGCCTGAAGAAACACAAGTTCGGTATCCCGCTGGAAGAT CTGGACAGCTTCATGGAACGTTTCCGTAGCATGAACATTCGCGGCCTGCACGTTCACATCG GTTCCCAGATTACCCGCGTCGAACCGTTCGTTGAGGCTTTTAGCAAGGTGGTTCGTGCGA GCGAGCGTTATGGTTTCGAAGAAATCAACATCGGTGGTGGTTGGGGCATTAACTACTCCG GTGAAGAGCTGGATCTGAGCTCTTATCGTGAAAAGGTGGTCCCGGACCTGAAACGCTTCA AGCGTGTGATTGTTGAGATTGGCCGCTACATCGTGGCGCCGTCTGGTTACTTGCTGCTGC GTGTTGTGCTGGTGAAACGTCGCCATAACAAAGCCTTTGTTGTGGTGGATGGTGGCATGAA TGTGTTGATTCGTCCGGCACTGTACAGCGCCTACCACCGCATTTTCGTCTTGGGCAAGCAA GGCAAAGAGATGCGTGCGGACGTCGTCGGCCCTCTGTGCGAGAGCGGTGATGTTATTGCT TACGATCGTGAGCTGCCTGAAGTTGAACCGGGTGACATCATTGCCGTTGAGAACGCAGGC GCGTACGGTTATACCATGAGCAATAACTATAACAGCACCACCCGTCCAGCCGAGGTGCTG GTTCGCGAGAACGGTCGTATTAGCCTGATTCGTCGCCGTGAAACGGAAATGGATATCTTTA AGGATGTGGTTATGTAA

[SEQ I D No. 145] wild-type C. glutamicum diaminopimelate decarboxylase gene

ATGGCTACAGTTGAAAATTTCAATGAACTTCCCGCACACGTATGGCCACGCAATGCCGTGC

GCCAAGAAGACGGCGTTGTCACCGTCGCTGGTGTGCCTCTGCCTGACCTCGCTGAAGAAT ACGGAACCCCACTGTTCGTAGTCGACGAGGACGATTTCCGTTCCCGCTGTCGCGACATGG CTACCGCATTCGGTGGACCAGGCAATGTGCACTACGCATCTAAAGCGTTCCTGACCAAGA CCATTGCACGTTGGGTTGATGAAGAGGGGCTGGCACTGGACATTGCATCCATCAACGAAC TGGGCATTGCCCTGGCCGCTGGTTTCCCCGCCAGCCGTATCACCGCGCACGGCAACAAC AAAGGCGTAGAGTTCCTGCGCGCGTTGGTTCAAAACGGTGTGGGACACGTGGTGCTGGAC TCCGCACAGGAACTAGAACTGTTGGATTACGTTGCCGCTGGTGAAGGCAAGATTCAGGAC GTGTTGATCCGCGTAAAGCCAGGCATCGAAGCACACACCCACGAGTTCATCGCCACTAGC CACGAAGACCAGAAGTTCGGATTCTCCCTGGCATCCGGTTCCGCATTCGAAGCAGCAAAA GCCGCCAACAACGCAGAAAACCTGAACCTGGTTGGCCTGCACTGCCACGTTGGTTCCCAG GTGTTCGACGCCGAAGGCTTCAAGCTGGCAGCAGAACGCGTGTTGGGCCTGTACTCACAG ATCCACAGCGAACTGGGCGTTGCCCTTCCTGAACTGGATCTCGGTGGCGGATACGGCATT GCCTATACCGCAGCTGAAGAACCACTCAACGTCGCAGAAGTTGCCTCCGACCTGCTCACC GCAGTCGGAAAAATGGCAGCGGAACTAGGCATCGACGCACCAACCGTGCTTGTTGAGCCC GGCCGCGCTATCGCAGGCCCCTCCACCGTGACCATCTACGAAGTCGGCACCACCAAAGA CGTCCACGTAGACGACGACAAAACCCGCCGTTACATCGCCGTGGACGGAGGCATGTCCG ACAACATCCGCCCAGCACTCTACGGCTCCGAATACGACGCCCGCGTAGTATCCCGCTTCG CCGAAGGAGACCCAGTAAGCACCCGCATCGTGGGCTCCCACTGCGAATCCGGCGATATC CTGATCAACGATGAAATCTACCCATCTGACATCACCAGCGGCGACTTCCTTGCACTCGCAG CCACCGGCGCATACTGCTACGCCATGAGCTCCCGCTACAACGCCTTCACACGGCCCGCC GTCGTGTCCGTCCGCGCTGGCAGCTCCCGCCTCATGCTGCGCCGCGAAACGCTCGACGA CATCCTCTCACTAGAGGCATAA

[SEQ I D No. 146] amino acid sequence C. glutamicum diaminopimelate decarboxylase P09890 MATVENFN ELPAHVWPRNAVRQEDGVVTVAGVPLPDLAEEYGTPLFVVDEDDFRSRCRDMAT AFGGPGNVHYASKAFLTKTIARWVDEEGLALDIASINELGIALAAGFPASRITAHGNNKGVEFLR ALVQNGVGHVVLDSAQELELLDYVAAGEGKIQDVLI RVKPGI EAHTH EFIATSHEDQKFGFSLAS GSAFEAAKAANNAENLNLVGLHCHVGSQVFDAEGFKLAAERVLGLYSQI HSELGVALPELDLG GGYGIAYTAAEEPLNVAEVASDLLTAVGKMAAELGI DAPTVLVEPGRAIAGPSTVTIYEVGTTKD VHVDDDKTRRYIAVDGGMSDN I RPALYGSEYDARVVSRFAEGDPVSTRIVGSHCESGDILINDE

lYPSDITSGDFLALAATGAYCYAMSSRYNAFTRPAVVSVRAGSSRLMLRRETLDDILSLEA

[SEQ I D No. 147] codon optimized C. glutamicum diaminopimelate decarboxylase gene ATGGCCACGGTCGAAAATTTTAATGAGCTGCCGGCGCACGTCTGGCCTCGTAACGCGGTC CGCCAAGAGGACGGTGTGGTTACCGTCGCCGGTGTTCCGCTGCCGGACCTGGCAGAAGA ATATGGTACTCCGCTGTTCGTGGTTGACGAGGATGACTTTCGTAGCCGTTGTCGTGATATG GCGACCGCTTTTGGTGGCCCTGGTAACGTGCATTATGCCTCCAAGGCGTTTCTGACCAAAA CGATTGCACGTTGGGTCGATGAAGAGGGCCTGGCACTGGACATTGCATCGATTAACGAAC TGGGCATTGCTCTGGCAGCGGGTTTTCCGGCGAGCCGTATTACCGCCCATGGCAACAACA AAGGCGTGGAATTCTTGCGTGCGCTGGTGCAGAATGGTGTTGGCCACGTTGTTCTGGACA GCGCGCAGGAGCTGGAGCTGCTGGACTACGTCGCGGCAGGCGAAGGTAAGATCCAAGAC GTGCTGATCCGCGTCAAACCGGGTATCGAAGCGCATACGCACGAATTCATCGCGACCAGC CACGAGGATCAGAAATTCGGTTTCAGCCTGGCCTCGGGTAGCGCATTTGAGGCGGCGAAA GCGGCAAACAATGCGGAGAATCTGAATTTGGTTGGTCTGCATTGCCATGTCGGTAGCCAG GTTTTCGACGCCGAGGGCTTCAAACTGGCGGCAGAGCGTGTTCTGGGTTTGTACAGCCAA ATTCACTCTGAGCTGGGCGTGGCGTTGCCGGAACTGGATCTGGGTGGTGGCTATGGCATC GCATATACTGCGGCCGAAGAGCCGCTGAATGTTGCCGAGGTCGCAAGCGACCTGCTGAC GGCAGTGGGCAAGATGGCGGCTGAACTGGGTATTGATGCGCCGACCGTCCTGGTGGAAC CGGGTCGTGCCATCGCCGGTCCATCCACGGTTACCATCTATGAGGTGGGTACCACCAAGG ATGTTCACGTCGACGATGATAAGACTCGCCGCTACATTGCAGTGGATGGTGGCATGAGCG ACAACATCCGTCCAGCGCTGTATGGTAGCGAGTACGATGCGCGTGTTGTGAGCCGCTTTG CTGAAGGCGACCCGGTCAGCACGCGTATTGTCGGCAGCCACTGTGAGAGCGGCGACATC CTGATTAACGATGAGATTTACCCGTCCGACATCACGAGCGGTGATTTTCTGGCTCTGGCCG CCACTGGCGCGTACTGCTACGCGATGAGCAGCCGCTACAATGCGTTCACGCGTCCTGCTG TTGTGTCTGTCCGTGCGGGTAGCAGCCGCCTGATGCTGCGCCGTGAAACCTTGGATGACA TCTTGTCCCTGGAGGCATAA

[SEQ I D No. 148] wild-type B. subtilis diaminopimelate decarboxylase gene

ATGACATTGTTCTTACACGGCACAAGCAGACAAAATCAACATGGTCATTTAGAAATCGGAG GTGTGGATGCTCTCTATTTAGCGGAGAAATATGGTACACCTCTTTACGTATATGATGTGGCT TTAATACGTGAGCGTGCTAAAAGCTTTAAGCAGGCGTTTATTTCTGCAGGGCTGAAAGCAC AGGTGGCATATGCGAGCAAAGCATTCTCATCAGTCGCAATGATTCAGCTCGCTGAGGAAG AGGGACTTTCTTTAGATGTCGTATCCGGAGGAGAGCTATATACGGCTGTTGCAGCAGGCTT TCCGGCAGAACGCATCCACTTTCATGGAAACAATAAGAGCAGGGAAGAACTGCGGATGGC GCTTGAGCACCGCATCGGCTGCATTGTGGTGGATAATTTCTATGAAATCGCGCTTCTTGAA GACCTATGTAAAGAAACGGGTCACTCCATCGATGTTCTTCTTCGGATCACGCCCGGAGTAG AAGCGCATACGCATGACTACATTACAACGGGCCAGGAAGATTCAAAGTTTGGTTTCGATCT TCATAACGGACAAACTGAACGGGCCATTGAACAAGTATTACAATCGGAACACATTCAGCTG CTGGGTGTCCATTGCCATATCGGCTCGCAAATCTTTGATACGGCCGGTTTTGTGTTAGCAG CGGAAAAAATCTTCAAAAAACTAGACGAATGGAGAGATTCATATTCATTTGTATCCAAGGTG CTGAATCTTGGAGGAGGTTTCGGCATTCGTTATACGGAAGATGATGAACCGCTTCATGCCA CTGAATACGTTGAAAAAATTATCGAAGCTGTGAAAGAAAATGCTTCCCGTTACGGTTTTGAC ATTCCGGAAATTTGGATCGAACCGGGCCGTTCTCTCGTGGGAGACGCAGGCACAACTCTT TATACGGTTGGCTCTCAAAAAGAAGTGCCGGGTGTCCGCCAATATGTGGCTGTAGACGGA GGCATGAACGACAATATTCGTCCTGCGCTTTACCAAGCTAAATATGAAGCTGCGGCAGCCA ACAGGATCGGAGAAGCGCATGACAAAACGGTATCAATTGCCGGAAAGTGCTGTGAAAGCG GAGATATGCTGATTTGGGATATTGACCTGCCGGAAGTAAAAGAAGGCGATCTTCTTGCCGT TTTTTGTACAGGCGCTTATGGATACAGCATGGCCAACAATTATAACCGTATTCCGAGACCC GCCGTTGTATTTGTCGAAAACGGTGAGGCTCATTTAGTCGTGAAGCGAGAAACATACGAAG ATATTGTAAAACTTGATCTGCCATTTAAAACGGGTGTAAAGCAATAA

[SEQ I D No. 149] amino acid sequence B. subtilis diaminopimelate decarboxylase P23630 MTLFLHGTSRQNQHGHLEIGGVDALYLAEKYGTPLYVYDVALI RERAKSFKQAFISAGLKAQVA YASKAFSSVAM IQLAEEEGLSLDVVSGGELYTAVAAGFPAERI HFHGNNKSREELRMALEHRIG CIVVDNFYEIALLEDLCKETGHSIDVLLRITPGVEAHTHDYITTGQEDSKFGFDLHNGQTERAI EQ VLQSEHIQLLGVHCHIGSQIFDTAGFVLAAEKI FKKLDEWRDSYSFVSKVLNLGGGFGI RYTEDD EPLHATEYVEKI I EAVKENASRYGFDI PEIWI EPGRSLVGDAGTTLYTVGSQKEVPGVRQYVAVD GGMNDN IRPALYQAKYEAAAAN RIGEAH DKTVSIAGKCCESGDMLIWDIDLPEVKEGDLLAVFC TGAYGYSMANNYNRI PRPAVVFVENGEAHLVVKRETYEDIVKLDLPFKTGVKQ

[SEQ I D No. 150] codon optimized B. subtilis diaminopimelate decarboxylase gene

ATGACCTTATTCCTGCACGGTACCTCTCGCCAGAACCAACACGGCCACTTGGAAATCGGTG GTGTTGACGCACTGTATCTGGCGGAGAAGTACGGTACCCCGTTGTATGTCTACGACGTGG CCCTGATCCGTGAGCGCGCAAAGAGCTTCAAACAGGCTTTCATTAGCGCTGGTCTGAAGG CGCAAGTTGCGTATGCGAGCAAAGCGTTTAGCAGCGTTGCCATGATCCAACTGGCGGAAG AAGAGGGTCTGAGCCTGGACGTCGTGTCTGGCGGTGAGCTGTACACCGCGGTTGCTGCG GGCTTCCCTGCAGAACGCATTCACTTCCATGGCAACAATAAGAGCCGTGAAGAGCTGCGT ATGGCGCTGGAGCATCGTATTGGTTGCATCGTTGTGGATAACTTTTACGAGATTGCACTGC TGGAAGATCTGTGCAAAGAAACGGGTCACAGCATCGATGTGCTGCTGCGCATTACTCCGG GCGTCGAGGCCCACACCCACGACTACATTACGACGGGCCAGGAAGATAGCAAGTTCGGTT TCGACCTGCATAATGGTCAAACGGAGCGTGCCATCGAACAGGTGCTGCAATCGGAGCATA TTCAACTGTTGGGTGTGCACTGTCACATCGGCAGCCAGATTTTCGACACCGCAGGCTTTGT CCTGGCTGCAGAGAAGATTTTCAAGAAACTGGATGAATGGCGCGATTCCTACAGCTTTGTG TCCAAGGTGCTGAATCTGGGCGGTGGTTTTGGCATCCGCTATACCGAAGATGACGAACCG CTGCACGCAACGGAGTACGTTGAGAAAATCATTGAGGCGGTGAAAGAGAACGCGAGCCGC TATGGTTTCGATATTCCGGAGATTTGGATCGAGCCAGGCCGCAGCCTGGTGGGTGACGCC GGCACGACGCTGTATACTGTCGGTTCTCAGAAAGAAGTTCCAGGCGTCCGTCAGTATGTT GCTGTGGACGGTGGTATGAACGACAATATCCGTCCGGCGCTGTATCAGGCGAAATACGAG GCAGCTGCAGCGAACCGTATCGGCGAAGCCCACGACAAAACCGTCAGCATCGCGGGCAA ATGCTGTGAAAGCGGTGATATGCTGATTTGGGACATCGATTTGCCGGAGGTCAAAGAGGG CGACTTGCTGGCTGTTTTCTGTACCGGTGCGTATGGTTACAGCATGGCCAATAACTACAAT CGTATTCCGCGTCCGGCCGTTGTGTTTGTTGAGAATGGTGAAGCACATTTGGTTGTGAAGC GTGAAACCTACGAGGACATCGTCAAACTGGATCTGCCGTTTAAGACCGGTGTCAAGCAATA

A

[SEQ I D No. 151 ] wild-type diaminopimelate decarboxylase P. putida gene

ATGAACGCTTTCAACTACCGCGACGGCCAGCTGTTCGCGGAAGGGGTGGCCCTGTCGGC CGTCGCCGAACGTTTCGGCACCCCCACCTACGTGTATTCGCGCGCCCACATCGAGGCCCA GTACCGCAGCTACACCGACGCCCTGCAAGGCGCCGAGCACCTGGTGTGCTTCGCGGTCA AGGCCAACTCCAACCTCGGCGTGCTGAACGTGCTGGCACGCCTGGGCGCAGGCTTCGAC ATTGTCTCCGGCGGTGAGCTGGAGCGCGTGCTGGCTGCTGGCGGGCGCGCCGACCGCG TGGTGTTCTCCGGCGTCGGCAAAACCCGCGAAGACATGCGCCGCGCCCTGGAAGTGGGC GTGCACTGCTTCAACGTCGAATCCACCGACGAGCTGGAGCGCCTGCAGGTCGTGGCCGC CGAAATGGGCAAGGTCGCCCCGGTGTCGCTGCGGGTAAACCCGGATGTAGACGCCGGCA CCCACCCGTACATCTCCACGGGCCTTAAAGAAAACAAGTTCGGTATCGCCATCGCCGACG CCGAGGCCATCTACGTGCGTGCCGCGCAGCTTCCGAACCTGGAAGTGGTCGGCGTCGAC TGCCACATCGGCTCACAGCTGACCACCGTGGAGCCGTTCCTCGATGCCCTCGACCGCCTG CTGGACCTGGTCGATCGCCTCGCCGACTGCGGCATCCACCTGCGCCATCTGGACCTGGG TGGCGGCGTTGGCGTGCGCTACCGCGACGAGGAGCCACCGCTGGTGGCCGACTACATCA AGGCTATTCGCGAACGCGTAGGCAAGCGCGACCTGGCCCTGGTGTTCGAGCCGGGCCGC TACATCGTGGCCAACGCCGGCGTGTTGCTGACCCGCGTGGAATACCTCAAGCACACCGAA CACAAAGACTTCGCCATCATCGATGCGGCAATGAACGACCTGATCCGCCCGGCCCTTTAC CAGGCCTGGATGGGTGTCAGCGCGGTCATCCCACGCGAAGGCGAAGGGCGTGCCTACGA CCTGGTCGGCCCAATCTGCGAGACCGGCGACTTCCTCGGCAAGGACCGCGTGTTGAACC TGGCCGAAGGCGACCTGCTGGCCGTGCAGTCCGCGGGCGCCTATGGTTTTGTCATGAGTT CCAACTACAACACCCGTGGCCGTTGCGCTGAAATCCTGGTCGACGGCGACCAGGCGTTCG AAGTACGCCGCCGCGAGACCATCGCCGAACTGTACGCTGGCGAAAGCCTGCTGCCGGAG TAA

[SEQ I D No. 152] amino acid sequence P. putida diaminopimelate decarboxylase

MNAFNYRDGQLFAEGVALSAVAERFGTPTYVYSRAHI EAQYRSYTDALQGAEHLVCFAVKANS NLGVLNVLARLGAGFDIVSGGELERVLAAGGRADRVVFSGVGKTREDM RRALEVGVHCFNVE STDELERLQVVAAEMGKVAPVSLRVNPDVDAGTH PYISTGLKENKFGIAIADAEAIYVRAAQLPN LEVVGVDCH IGSQLTTVEPFLDALDRLLDLVDRLADCGI HLRHLDLGGGVGVRYRDEEPPLVAD YIKAI RERVGKRDLALVFEPGRYIVANAGVLLTRVEYLKHTEHKDFAI I DAAMN DLI RPALYQAWM GVSAVI PREGEGRAYDLVGPICETGDFLGKDRVLNLAEGDLLAVQSAGAYGFVMSSNYNTRGR CAEILVDGDQAFEVRRRETIAELYAGESLLPE

Claims

Method for preparing a compound comprising an amine group represented by the formula

H₂N CH₂ A R _{(1 )} from an alpha-keto acid represented by the formula

0

HO— C— C— A— R

⁰ (2); wherein

A represents a hydrocarbon group, which hydrocarbon group may comprise one or more substituents and/or contain one or more heteroatoms;

R represents a functional group; and

Method according to claim 1 , wherein the biocatalyst comprises an enzyme having catalytic activity with respect to catalysing transamination and/or reductive amination, preferably an enzyme having at least one of said catalytic activities selected from the group of aminotransferases (E.C. 2.6.1 ) and amino acid dehydrogenases (E.C.1 .4.1 ).

Method according to claim 2, wherein the aminotransferase or amino acid dehydrogenase is selected from the group of beta-aminoisobutyrate:alpha- ketoglutarate aminotransferases, beta-alanine aminotransferases, aspartate aminotransferases, 4-amino-butyrate aminotransferases (EC 2.6.1.19), L-lysine 6-aminotransferase (EC 2.6.1.36), 2-aminoadipate aminotransferases (EC 2.6.1 .39), 5-aminovalerate aminotransferases (EC 2.6.1 .48), 2-aminohexanoate aminotransferases (EC 2.6.1.67), lysine:pyruvate 6-aminotransferases (EC 2.6.1 .71 ), and lysine-6-dehydrogenases (EC 1.4.1 .18).

Method according to claim 2 or 3, wherein the enzyme is selected from the group of enzymes having catalytic activity with respect to catalysing

transamination and/or reductive amination from an organism selected from the group of Vibrio; Pseudomonas; Bacillus; Mercu alis; Asplenium; Ceratonia; mammals; Neurospora; Escherichia; Thermus; Saccharomyces; Brevibacterium; Corynebacterium; Proteus; Agrobacterium; Geobacillus;

Acinetobacter; Ralstonia; Salmonella; Rhodobacter and Staphylococcus, in particular from an organism selected from the group of Bacillus subtilis, Bacillus weihenstephanensis, Rhodobacter sphaeroides, Staphylococcus aureus, Legionella pneumophila, Nitrosomonas europaea, Neisseria gonorrhoeae, Pseudomonas syringae, Rhodopseudomonas palustris, Vibrio fluvialis and Pseudomonas aeruginosa.

Method according to any of the claims 2-4, wherein an aminotransferase is used comprising an amino acid sequence 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 , Sequence ID 23, Sequence ID 25, Sequence ID 27, Sequence ID 29, Sequence ID 65, Sequence ID 67, Sequence ID 69 or a homologue of any of these sequences.

Method according to any of the preceding claims, wherein the biocatalyst comprises an enzyme having catalytic activity with respect to catalysing the decarboxylation of an alpha-keto acid or an amino acid, preferably a decarboxylase (E.C. 4.1 .1 ), in particular a decarboxylase is selected from the group of glutamate decarboxylases (EC 4.1.1 .15), diaminopimelate

decarboxylases (EC 4.1 .1 .20) aspartate 1 -decarboxylases (EC 4.1 .1 .1 1 ), branched chain alpha-keto acid decarboxylases, alpha-ketoisovalerate decarboxylases, alpha-ketoglutarate decarboxylases, pyruvate decarboxylases (EC 4.1 .1 .1 ), and oxaloacetate decarboxylases (E.C. 4.1 .1 .3).

Method according to claim 6, wherein the enzyme having catalytic activity with respect to catalysing the decarboxylation is an enzyme from an organism selected from the group of Cucurbitaceae; Saccharomyces; Candida;

Hansenula; Kluyveromyces; Rhizopus; Neurospora; Zymomonas; Escherichia; Mycobacterium; Clostridium; Lactobacillus; Streptococcus; Pseudomonas and Lactococcus.

Method according to claims 6 or 7, wherein the enzyme having catalytic activity with respect to catalysing the decarboxylation comprises an amino acid sequence according to Sequence ID 31 , Sequence ID 34, Sequence ID 37, Sequence ID 40, Sequence ID 43, Sequence ID 46, 143, Sequence ID 146, Sequence ID 149, Sequence 152 or a homologue of any of these sequences. Method according to any of the preceding claims, wherein the alpha-keto acid is biocatalytically converted into a an aldehyde represented by the formula H C A R

⁰ (3) wherein A and R are as mentioned in claim 1 , in the presence of a biocatalyst having alpha-keto acid decarboxylation activity, whereafter said aldehyde is biocatalytically converted into the compound represented by formula (1 ) in the presence of at least one amino donor and at least one biocatalyst capable of catalysing a transamination and/or a reductive amination of said compound comprising an aldehyde group.

10. Method according to any of the preceding claims, wherein the alpha-keto acid is biocatalytically converted into an alpha-amino acid represented by the formula

0

HO C CH A R

NH₂

(4) wherein A and R are as mentioned in claim 1 , in the presence of at least one amino donor and at least one biocatalyst capable of catalysing a transamination and/or a reductive amination of the alpha-keto acid, whereafter said alpha- amino acid is biocatalytically converted into the compound represented by Formula (1 ) in the presence of a biocatalyst capable of catalysing the decarboxylation of an amino acid.

1 1 . Method according to any of the preceding claims, wherein R represents a

functional group selected from the group of -CN, -COOH and -NH₂.

12. Method according to any of the preceding claims, wherein A represents a

hydrocarbon group comprising 2-10 carbon atoms, which hydrocarbon group may comprise one or more substituents and/or contain one or more heteroatoms,

13. Method according to any of the preceding claims, wherein the compound

represented by Formula (1 ) that is prepared is a compound other than 6- aminocaproic acid.

14. Method according to claim 13, wherein A represents a (CH₂)_X group, x being an integer selected from the group of 2, 3, 5, 6, 7 and 8.

15. Method according to claim 14, wherein the compound represented by Formula (1 ) is selected from the group of diaminopentane, diaminohexane,

diaminoheptane, omega-amino-butanoic acid, omega-amino-pentanoic acid, omega-amino-heptanoic acid and omega-amino-octanoic acid, in particular from the group of omega-amino-heptanoic acid and diaminoheptane.

Method according to any of the claims 1 -12, wherein the compound represented by Formula (1 ) that is prepared is 6-aminocaproic acid and the alpha-keto acid from which 6-aminocaproic acid is prepared is alpha-ketopimelic acid.

Method for preparing a polymer, comprising subjecting the compound or a mixture of compounds containing at least one compound as represented by Formula (1 ) prepared in a method according to any of the preceding claims, optionally in the presence of one or more further monomers, to a polymerisation reaction.

Polynucleotide comprising a sequence according to any of the SEQUENCE ID NO's: 1 12, 1 15, 1 18, 121 , 124, 127, 130, 133, 136, 139 and functional analogues thereof having a similar, the same or a better level of expression in a Escherichia host cell.