(r)- Selective Amination

Abstract

The present invention relates to a method for the enzymatic synthesis of enantiomerically enriched (R)-amines of general formula [1][c], from the corresponding ketones of general formula [1][a], by using novel transaminases. These novel transaminases are selected from two different groups: either from a group of some 20 proteins with sequences as specified herein, or from a group of proteins having transaminase activity and isolated from a microorganism selected from the group of organisms consisting of Rahnella aquatilis, Ochrobactrum anthropi, Ochrobactrum tritici, Sinorhizobium morelense, Curtobacterium pusilllum, Paecilomyces lilacinus, Microbacterium ginsengisoli, Microbacterium trichothecenolyticum, Pseudomonas citronellolis, Yersinia kristensenii, Achromobacter spanius, Achromobacter insolitus, Mycobacterium fortuitum, Mycobacterium frederiksbergense, Mycobacterium sacrum, Mycobacterium fluoranthenivorans, Burkholderia sp., Burkholderia tropica, Cosmospora episphaeria, and Fusarium oxysporum. ##STR00001##

Description

[0001] The present invention relates to a method for the (R)-selective amination of ketones and to enzymes for use in this method.

[0002] A considerable number of pharmaceutically active compounds (existing and under development) contain a chiral amine functionality which can not be directly derived synthetically from natural amino acids. According to pharmaceutical manufacturers, asymmetric synthesis of amines from ketones is one of the most desirable reactions for the future (Constable et al., Green Chem. 2007, 9, 411). Furthermore, it is a goal of the pharmaceutical manufacturers to rely as much as possible on "green" routes in the synthesis of their pharmaceutically active compounds. Biocatalytic conversions are therefore preferred.

[0003] An attractive route for biocatalytic production of chiral amines is stereoselective transamination starting from a ketone and an amine donor, according to reaction [1]:

##STR00002##

[0004] The required stereoselective enzymes (interchangeably called transaminases, aminotransferases or aminopherases) have been reported for instance the (S)-selective w-transaminase from Vibrio fluvialis (Shin et al., Appl. Microbiol. Biotechnol. 2003, 61, 463), although (S)-selective enzymes are by far more abundant than (R)-selective enzymes.

[0005] Stereoselective transaminases have for example been described in the European patent publications EP1038953 and EP987332.

[0006] EP1038953 relates to a DNA encoding a transaminase capable of synthesizing optically active (R)-.alpha.-methylbenzylamine in the presence of sec-butylamine.

[0007] EP987332 relates in particular to a transaminase from a Mycobacterium aurum species which can catalyze the stereoselective transamination of acetophenone and sec-butylamine to an enantiomerically enriched (R)-.alpha.-methylbenzylamine and 2-butanone. The same transaminase was also used for the racemic resolution or (RS)-.alpha.-methylbenzylamine to obtain enantiomerically enriched (S)-.alpha.-methylbenzylamine as well as the synthesis of enantiomerically enriched D-alanine and D-serine.

[0008] U.S. Pat. No. 7,169,592 relates to a DNA from an Arthrobacter species encoding a recombinant transaminase, which can catalyze the (R)-stereoselective transamination of several ketones in the presence of an amino donor to produce enantiomerically enriched (R)-amines.

[0009] There is a need for further stereoselective transaminases in order to be able to apply stereoselective transamination reactions to a wider spectrum of compounds.

[0010] The present invention relates to a method for the enzymatic synthesis of enantiomerically enriched (R)-amines of general formula [1][c] from the corresponding ketones of the general formula [1][a] by using novel transaminases.

[0011] These novel transaminases are selected from the group consisting of [0012] a) a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 1; [0013] b) a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 3; [0014] c) a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 5; [0015] d) a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 7; [0016] e) a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 9; [0017] f) a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 11; [0018] g) a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 13; [0019] h) a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 15; [0020] i) a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 17; [0021] j) a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 19; [0022] k) a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 21; [0023] l) a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 23; [0024] m) a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 25; [0025] n) a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 30; [0026] o) a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 33; [0027] p) a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 35; [0028] q) a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 38; [0029] r) a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 40; [0030] s) a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 43; [0031] t) a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 46 and [0032] u) a protein having transaminase activity and isolated from a microorganism selected from the group of organisms consisting of Rahnella aquatilis, Ochrobactrum anthropi, Ochrobactrum tritici, Sinorhizobium morelense, Curtobacterium pusilllum, Paecilomyces lilacinus, Microbacterium ginsengisoli, Microbacterium trichothecenolyticum, Pseudomonas citronellolis, Yersinia kristensenii, Achromobacter spanius, Achromobacter insolitus, Mycobacterium fortuitum, Mycobacterium frederiksbergense, Mycobacterium sacrum, Mycobacterium fluoranthenivorans, Burkholderia sp., Burkholderia tropica, Cosmospora episphaeria, and Fusarium oxysporum.

[0033] According to the present invention it has been found, in sequence alignment studies using ClustalW2 multiple sequence alignment at default settings (http://www.ebi.ac.uk/Tools/clustalw2, Larkin et al., Bioinformatics 2007, 23, 2947), that the transaminases of SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25 according to this invention vary over a broad range of identity percentage with the wild-type amino acid sequence of SEQ ID No. 1 of the transaminase. Even at an identity percentage of about 30% to SEQ ID No. 1 still very suitable transaminases are being found according to the present invention.

[0034] The inventors have found, however, that the transaminases as can be used in the present invention (and the mutants derived there from) all have in common, that they have at least 37 conserved amino acids, namely A32, A44, D50, G52, D57, Y60, V65, G68, F71, L73, R79, V106, V116, R122, G123, P145, P177, K180, N181, W183, D185, E213, G216, N218, P230, L235, G237, R240, V243, E269, A276, G277, G278, P281, G296, W307, and Y321, when being compared to the wild-type amino acid sequence of SEQ ID No. 1 at the positions corresponding to the above positions in the amino acid sequence of SEQ ID No. 1.

[0035] Amino acid residues of wild-type or mutated protein sequences corresponding to positions of the amino acid residues in the wild-type amino acid sequence of the transaminase of SEQ ID No. 1 can be identified by performing ClustalW2 multiple sequence alignments at default settings. Amino acid residues, which are placed in the same column as an amino acid residue of the transaminase sequence as given in SEQ ID No. 1 in such alignments, are defined to be positions corresponding to this respective amino acid residue of the transaminase sequence of SEQ ID No. 1.

[0036] Samples of each of the microorganisms Rahnella aquatilis, Ochrobactrum anthropi, Ochrobactrum tritici, Sinorhizobium morelense, Curtobacterium pusilllum, Paecilomyces lilacinus, Microbacterium ginsengisoli, Microbacterium trichothecenolyticum, Pseudomonas citronellolis, Yersinia kristensenii, Achromobacter spanius, Achromobacter insolitus, Mycobacterium fortuitum, Mycobacterium frederiksbergense, Mycobacterium sacrum, Mycobacterium fluoranthenivorans, Burkholderia sp., Burkholderia tropica, Cosmospora episphaeria, and Fusarium oxysporum were deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) at Braunschweig, Germany on Jul. 13, 2010.

[0037] In the above chemical structures [1][a] and [1][c] R.sub.1 and R.sub.2 are different and can be independently linear or branched aliphatic, hetero-aliphatic, aromatic, hetero-aromatic or form a cyclic structure.

[0038] More in particular, R.sub.1 and R.sub.2 are different and R.sub.1 and R.sub.2 independently contain 1 to 30 carbon atoms and R.sub.1 and R.sub.2 are independently substituted or unsubstituted aliphatic; substituted or unsubstituted branched aliphatic; substituted or unsubstituted cyclic aliphatic; substituted or unsubstituted heterocyclic aliphatic, containing at least one oxygen, sulfur or nitrogen atom; substituted or unsubstituted aromatic; substituted or unsubstituted hetero-aromatic containing at least one sulfur, oxygen or nitrogen atom; or together form a substituted or unsubstituted cyclic structure or heterocyclic structure, containing at least one oxygen, sulfur or nitrogen atom; wherein the substituents are selected from, but not limited to, the group consisting of a halogen atom, an alkyl group having 1 to 6 carbon atoms, hydroxyl group, methoxy group, monofluoromethyl, difluoromethyl and trifluoromethyl group.

[0039] Preferably the final concentration of the enantiomerically enriched (R)-amine product lies between 1 and 50 weight % of the reaction mixture. Most preferably final concentration of the enantiomerically enriched (R)-amine product lies between 5 and 35 weight % of the reaction mixture.

[0040] A process according to this invention can be carried out in a aqueous reaction mixture with all reactions dissolved or in slurries with some of the reactants at least partially dissolved and some of the reactions at least partially as solid material.

[0041] The use of a non water-miscible solvent such as an organic solvent forming a second liquid phase next to the buffered aqueous phase containing a transaminase can be advantageous in transaminase reactions over purely aqueous or aqueous slurry reactions, because the organic solvent can act as a reservoir for poorly water-soluble ketones and amino donors. It can increase the mass transfer rate for the dissolution of poorly water-soluble ketones or amino donors into the transaminase containing aqueous phase compared to slurry reactions (solid-aqueous). Further it can reduce potential substrate or product inhibition by extractive removal of these potential inhibitors. Furthermore such extractive removal of at least one of the transaminase reaction products can pull the equilibrium of the transaminase reaction in the aqueous phase to the products side, thereby improving the yield and/or the efficiency of the transaminase catalysed reaction.

[0042] A process according to this invention can be carried out in a reaction mixture comprising an aqueous phase and second organic phase. In case a reaction mixture comprising an aqueous phase and second organic phase is used, the reaction mixture preferably comprises an aqueous phase and second organic phase and the volumetric ratio of water:organic phase is between 100 and 0.01. More preferably the reaction mixture comprises an aqueous phase and second organic phase and the volumetric ratio of water:organic phase is between 20 and 0.1. Most preferably the reaction mixture comprises an aqueous phase and second organic phase and the volumetric ratio of water:organic phase is between 20 and 1.

[0043] Suitable organic solvents can for instance be selected from the group of, but are not restricted to cyclohexanone, dichloromethane, pentane, heptane, MTBE (methyl-tert-butylether), toluene, 2-methyl-tetrahydrofurane, butylacetate and ethylacetate.

[0044] The transaminase a) through m) above according to the present invention have been identified in database searches in public nucleotide and polypeptide databases like EMBL/GenBank/DDBJ, Swiss-Prot/UniProtKB (released on Jun. 15, 2010), RefSeq or Non-redundant via the EMBL-EBI (http://www.ebi.ac.uk) or NCBI (http://www.ncbi.nlm.nih.gov) servers annotated as "hypothetical protein" with highest similarity to branched chain L-amino acid aminotransferases or 4-amino-4-deoxychorismate lyases (EC4.1.3.38) belonging to Pyridoxal 5'-Phosphate Dependent Enzymes class IV (PLPDE_IV) superfamily. Functional expression and activity have so far not been reported to support the annotation.

[0045] Surprisingly, it has been found that transaminase a) through m) are in fact (R)-selective .omega.-transaminases instead of (S)-selective amino acid aminotransferases as will be described below. Further the transaminases a) through m) differ in substrate spectrum than the (R)-transaminases described in EP1038953, EP987332 and U.S. Pat. No. 7,169,592.

[0046] The transaminases a) through t) above according to the present invention have been characterized by their activity and enantioselectivity in one or more of the following .omega.-transaminase conversions:

(i) Racemic Resolution of (RS)-.alpha.-Methylbenzylamine.

##STR00003##

[0048] The enantioselectivity of enzymes in general and transaminases specifically can be determined in a racemic resolution of a racemic mixture of a substrate comprising a chiral center. According to this invention an (R)-selective transaminase is an enzyme which preferentially transaminates (R)-.alpha.-methylbenzylamine (MBA) from racemic MBA in the presence of a ketone substrate such as pyruvic acid. An (R)-transaminase according to this invention is an enzyme which preferentially converts the (R)- over (S)-enantiomer of .alpha.-methylbenzylamine in the presence of pyruvic acid resulting in the enrichment of the remaining (S)-enantiomer with an enantiomeric excess (e.e.) of at least 10%. Preferably the resulting e.e. of (S)-MBA is at least 50%. More preferably the e.e. is at least 60%. Even more preferably the e.e. is at least 70%. Even more preferably the e.e. is at least 80%. Even more preferably the e.e. is at least 90%. Even more preferably the e.e. is at least 95%. Even more preferably the e.e. is at least 96%. Even more preferably the e.e. is at least 97%. Even more preferably the e.e. is at least 98%. Most preferably the e.e. is at least 99%.

[0049] Typically 80 mM of racemic .alpha.-methylbenzylamine (MBA) are reacted with 40 mM sodium pyruvate in 100 mM potassium phosphate (KP.sub.i) buffer pH 7.0 containing 0.1 mM pyridoxal 5'-phosphate (PLP) at 28.degree. C. for 20 h in the presence of a transaminase. The concentrations and enantiomeric excesses of (R)- and/or (S)-MBA can for instance be determined by high performance liquid chromatography (HPLC) or gas chromatography (GC) with suitable chiral column materials. The formulation of the (R)-transaminase is not critical; it can be added as (partially) purified enzyme, cell-free extract (CFE) or crude cell extract; liquid, powder or immobilized form; permeabilised cells, whole cells or culture broth containing cells comprising the transaminase or in any other form.

(ii) Activity and Selectivity on the Pure Enantiomers of .alpha.-Methylbenzylamine.

[0050] The enantioselectivity of an enzyme is also characterized by its specific activities towards the individual enantiomers of a specific substrate. These can be determined in separate activity assays with the individual enantiopure forms of the substrate. According to this invention the specific transaminase activities towards the two enantiomers of .alpha.-methylbenzylamine (MBA) are a measure for the (R)-selectivity of a transaminase. In the context of this invention the ratio of the specific activities on (R)-MBA over the specific activities on (S)-MBA is defined as the Transaminase Enantioselectivity Value (TEV). An (R)-selective transaminase is defined as a transaminase with a TEV value of >1. A good (R)-selectivity of a transaminase is defined as a specific activity ratio on (R)- over (S)-MBA of TEV 10. A high (R)-selectivity of a transaminase is defined as a specific activity ratio on (R)- over (S)-MBA of TEV .gtoreq.100.

##STR00004##

[0051] The specific transaminase activities on (R)- and (S)-MBA are separately determined using a spectrophotometric assay. In a final reaction volume of 1 ml 50 .mu.l of a suitable dilution of a transaminase in liquid form is mixed in a cuvette with 12.5 mM (R)- or (S)-MBA and 5 mM sodium pyruvate in the presence of 50 mM KP.sub.i buffer pH 7.5 containing 0.1 mM PLP. The reactions are started by addition of 10 .mu.l of 0.5 M sodium pyruvate (in 50 mM KP.sub.i buffer pH 7.5, 0.1 mM PLP) to the other assay components, after pre-incubation at 30.degree. C. for 5 min. After addition of sodium pyruvate the absorption at 300 nm is recorded and the transaminase activity in the samples is calculated according to the law of Lambert-Beer using the molar extinction coefficient for acetophenone at 300 nm of .epsilon.=0.28 cm.sup.2/.mu.mol. One unit (U) of transaminase activity is defined as 1 .mu.mol of acetophenone formed from 12.5 mM (R)-MBA or (S)-MBA and 5 mM sodium pyruvate at 30.degree. C. in 50 mM KP.sub.i buffer pH 7.5 containing 0.1 mM PLP per minute. The specific transaminase activities of the CFEs (U/mg total CFE protein) are calculated by dividing the volumetric activity values (U/ml CFE) by the total protein concentration in the liquid transaminase sample. The Transaminase Enantioselectivity Value (TEV) is calculated by dividing the volumetric or specific activity on (R)-MBA by the volumetric or specific activity, respectively, on (S)-MBA.

[0052] Preferably the (R)-transaminase has a TEV of at least 1, more preferably the (R)-transaminase has a TEV of at least 10, even more preferably the (R)-transaminase has a TEV of at least 100.

(iii) Synthesis of Enantiomerically Enriched (R)-Amines from the Corresponding Ketones and Suitable Amino Donors Such as Benzylamine or .alpha.-Methylbenzylamine.

[0053] The typical desired outcome of a reaction catalysed by an (R)-selective transaminase is the formation of an enantiomerically enriched (R)-amine according to formula 1 [c] from an amino donor and a ketone substrate. Suitable amino donors for (R)-selective transaminase reactions comprise for instance racemic or (R)-MBA, racemic or (R)-1-aminoindan, racemic or (R)-1-aminotetralin, racemic or D-alanine, isopropylamine, benzylamine, racemic or (R)-sec-butylamine (2-aminobutane), .beta.-alanine or racemic or D-3-aminobutyric acid.

[0054] Generally, with a particular transaminase, certain amino donors are preferred. Surprisingly, we have found that with the transaminase of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 21, SEQ NO ID 23, and SEQ ID No. 43 the preferred amino donors are racemic or (R)-MBA or racemic or (R)-sec-butylamine.

[0055] Typically 70 mM of amino donor is reacted with 70 mM of ketone substrate in the presence of an (R)-transaminase in an aqueous reaction mixture buffered with 100 mM potassium phosphate (KP.sub.i) at pH 7.5 at 28.degree. C. Optionally water miscible or immiscible solvents can be used to solubilise amino donor or ketone substrate. An (R)-selective transaminase according to this invention will exhibit at least low conversion of at least 0.5% to the desired enantiomerically enriched (R)-amine starting from equimolar amounts of amino donor and ketone substrate with the enantiomerically enriched (R)-amine having an enantiomeric excess of at least 50% after over night reaction at 28.degree. C. The form of the (R)-transaminase is not critical; it can be added as (partially) purified enzyme, cell-free extract or crude cell extract; liquid, powder or immobilized form; permeabilised cells, whole cells or culture broth containing cells comprising the transaminase or in any other form.

[0056] It is known to the person skilled in the art that transaminase reactions are reversible reactions and the degree of conversion is influenced by the equilibrium of the specific substrates and products. Further it is known to the person skilled in the art that the degree of substrate conversion at equimolar amino donor and ketone substrate concentrations can be increased by for instance in situ product removal by evaporation of one of the reaction products or using resins, or addition of enzymes which further convert one of the reaction products such as pyruvate decarboxylase or lactate dehydrogenase. Further it is known that the degree of conversion of ketone substrate 1[a] to amine product 1 [c] can be increased by an excess of amino donor 1[b].

[0057] Preferably the produced enantiomerically enriched (R)-amine has an e.e. of at least 50%. More preferably the e.e. is at least 60%. Even more preferably the e.e. is at least 70%. Even more preferably the e.e. is at least 80%. Even more preferably the e.e. is at least 90%. Even more preferably the e.e. is at least 95%. Even more preferably the e.e. is at least 96%. Even more preferably the e.e. is at least 97%. Even more preferably the e.e. is at least 98%. Even more preferably the e.e. is at least 99%. Most preferably the produced enantiomerically enriched (R)-amine has an e.e. of larger than 99%.

[0058] In the present application "a protein having at least 90% sequence identity to the amino acid sequence of (a reference sequence)" means that such protein is a homologue of the respective reference sequence having an amino acid sequence, which is for at least 90% identical to the amino acid sequence of the reference sequence as determined in sequence alignments performed with sequence alignment tools such as BLASTP (http://blast.ncbi.nlm.nih.gov/Blast), ClustalW (http://www.ebi.ac.uk/Tools/clustalw2) or Align Plus 5 (Scientific & Educational Software, Cary, N.C., USA).

[0059] The term "homologue" is used herein in particular for polynucleotides or polypeptides having a sequence identity of at least 60%, preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, in particular at least 85%, more in particular at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. The term homologue is also meant to include nucleic acid sequences (polynucleotide sequences) which differ from another nucleic acid sequence due to the degeneracy of the genetic code and encode the same polypeptide sequence.

[0060] 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., USA). 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).

[0061] The transaminase according to this invention may be used in any form. For example, the transaminase may be used--for example in the form of a dispersion, a solution or in immobilized form--as crude enzyme, as a commercially available enzyme, as an enzyme further purified from a commercially available preparation, as an enzyme obtained from its source by a combination of known purification methods, in whole (optionally permeabilized and/or immobilized) cells that naturally or through genetic modification possess the required tranasminase activity, or in a lysate of cells with such activity.

[0062] A cell comprising a transaminase in a method of the invention can be constructed using molecular biological techniques, which are known in the art per se. For instance, if a transaminase is to be produced in a heterologous system, such techniques can be used to provide a vector which comprises a gene encoding a transaminase.

[0063] A gene encoding a polypeptide with transaminase activity can be adapted to the preferred codon usage of the host cell used for the production of the polypeptide to improve expression level of the polypeptide. A suitable method to achieve such an adaptation is for instance Codon-Pair-Optimization as described in WO08000632.

[0064] A vector comprising such a gene can comprise one or more regulatory elements, e.g. one or more promoters, which may be operably linked to a gene encoding a transaminase. Examples of such vectors comprise plasmids like pBAD/Myc-His C, pBAD-DEST49, pET-DEST42 (all Invitrogen, Carlsbad, Calif., USA), plasmids of the pET series for instance pET-26b(+) (Novagen, Nottingham, UK) or pMS47008 (Balzer et al., Nucleic Acids Research, 1992, 20 (8): 1851-1858).

[0065] 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.

[0066] 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.

[0067] The promoter that could be used to achieve the expression of the nucleotide sequences coding for an enzyme for use in a method of the invention, in particular a transaminase, such as described herein above may be native to the nucleotide sequence coding for the enzyme to be expressed, or may be heterologous to the nucleotide sequence (coding sequence) to which it is operably linked. Preferably, the promoter is homologous, i.e. endogenous to the host cell.

[0068] If a heterologous promoter (to the nucleotide 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.

[0069] A "strong constitutive promoter" is one which causes mRNAs to be initiated at high frequency compared to a native host cell.

[0070] Examples of such strong constitutive promoters in Gram-positive micro-organisms include SP01-26, SP01-15, veg, pyc (pyruvate carboxylase promoter), and amyE.

[0071] Examples of inducible promoters in Gram-positive micro-organisms include, the IPTG inducible Pspac promoter, the xylose inducible PxylA promoter.

[0072] Examples of constitutive and inducible promoters in Gram-negative microorganisms include, but are not limited to tac, tet, trp-tet, lpp, lac, lpp-lac, laclq, T7, T5, T3, gal, trc, ara (P.sub.BAD), SP6, .lamda.-P.sub.R, and .lamda.-P.sub.L.

[0073] Examples of constitutive and inducible promoters in eukaryotic microorganisms such as yeasts and fungi include, but are not limited to, the AOX-, GAP-, and TEF-promoter.

[0074] 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.

[0075] The host cell or micro-organism may in particular be selected from the group of genera consisting of Aspergillus, Bacillus, Corynebacterium, Escherichia, Saccharomyces, Pseudomonas, Gluconobacter, Penicillium, and Pichia. In particular, the host strain and, thus, host cell suitable for the production of a transminase may be selected from the group of Escherichia coli, Bacillus subtilis, Bacillus amyloliquefaciens, Corynebacterium glutamicum, Aspergillus niger, Penicillium chrysogenum, and Pichia pastoris host cells.

[0076] The proteins having transaminase activity mentioned under n) above are produced by microorganisms present in soil samples collected in the Netherlands and Germany. These microorganisms were enriched from these soil samples in a culture medium using an (R)-amine as the sole nitrogen source for the microorganism for growth. To this end the following six structurally diverse (R)-amines were used as only nitrogen source: (R)-2-aminobutane, (R)-3,3-dimethyl-2-aminobutane, (R)-.alpha.-methylbenzylamine, (R)-.alpha.-ethylbenzylamine, (R)-1-aminoindan and (R)-1-aminotetralin.

[0077] Subsequent to enrichment in liquid medium, the microorganisms were isolated on agar plates containing the respective (R)-amine as sole nitrogen source, pure cultures were grown in the respective liquid enrichment medium again and used for transaminase reactions in whole cell systems. The resulting conversions and enantiomeric excesses of the produced (R)-amines were monitored by HPLC or GC analysis.

[0078] The typical desired outcome of a reaction catalysed by an (R)-selective transaminase is the formation of an enantiomerically enriched (R)-amine according to formula 1 [c] from an amino donor and a ketone substrate. Suitable amino donors for (R)-selective transaminase reactions comprise for instance racemic or (R)-MBA, racemic or (R)-1-aminoindan, racemic or (R)-1-aminotetralin, racemic or D-alanine, isopropylamine, benzylamine, racemic or (R)-sec-butylamine (2-aminobutane), .beta.-alanine or racemic or D-3-aminobutyric acid.

[0079] Typically 80 mM of amino donor is reacted with 40 mM of ketone substrate in the presence of an (R)-transaminase in an aqueous reaction mixture buffered with 100 mM potassium phosphate (KP.sub.i) at pH 7.5 at 28.degree. C. Preferably at least 100 mM of ketone substrate are used in the presence of an (R)-transaminase in an aqueous reaction mixture buffered with 100 mM potassium phosphate (KP.sub.i) at pH 7.5 at 28.degree. C. Optionally water miscible or immiscible solvents can be used to solubilise amino donor or ketone substrate. An (R)-selective transaminase according to this invention will exhibit at least low conversion of 0.5% to the desired enantiomerically enriched (R)-amine starting from equimolar amounts of amino donor and ketone substrate with the enantiomerically enriched (R)-amine having an enantiomeric excess of at least 50% after over night reaction at 28.degree. C.

[0080] The form of the (R)-transaminase is not critical; it can be added as (partially) purified enzyme, cell-free extract or crude cell extract; liquid, powder or immobilized form; permeabilised cells, whole cells or culture broth containing cells comprising the transaminase or in any other form.

[0081] It is known to the person skilled in the art that transaminase reactions are reversible reactions and the degree of conversion is influenced by the equilibrium of the specific substrates and products. Further it is known to the person skilled in the art that the degree of substrate conversion at equimolar amino donor and ketone substrate concentrations can be increased by for instance in situ product removal by evaporation of one of the reaction products or using resins, or addition of enzymes which further convert one of the reaction products such as pyruvate decarboxylase or lactate dehydrogenase.

[0082] Preferably the produced enantiomerically enriched (R)-amine has an e.e. of at least 50%. More preferably the e.e. is at least 60%. Even more preferably the e.e. is at least 70%. Even more preferably the e.e. is at least 80%. Even more preferably the e.e. is at least 90%. Even more preferably the e.e. is at least 95%. Even more preferably the e.e. is at least 96%. Even more preferably the e.e. is at least 97%. Most preferably the produced enantiomerically enriched (R)-amine has an e.e. of at least 99%.

[0083] The final result was a collection of 31 microorganisms, which were characterized as shown in table 1.

TABLE-US-00001 TABLE 1 The deposits at German Collection of Microorganisms and Cell Cultures (DSMZ) at Braunschweig, Germany were made on Jul. 13, 2010 Code Microorganism (scientific name) DSMZ deposition number 3Na Rahnella aquatilis DSM 23797 3Kb Rahnella aquatilis not applicable 3Ba Ochrobactrum anthropi not applicable 3Db Ochrobactrum anthropi DSM 23793 3H1 Sinorhizobium morelense DSM 23794 5BaB Curtobacterium pusillum DSM 23787 5BaS Paecilomyces lilacinus DSM 23771 2A2 Microbacterium ginsengisoli not applicable 2Ca Microbacterium ginsengisoli not applicable 2Cb Ochrobactrum anthropi not applicable 2M1 Pseudomonas citronellolis DSM 23795 2Da Yersinia kristensenii DSM 23792 2K1 Ochrobactrum anthropi not applicable 6Ab Ochrobactrum tritici DSM 23786 6Bb Mycobacterium fortuitum DSM 23789 6I Achromobacter spanius DSM 23791 6F Achromobacter spanius not applicable 1Ea Achromobacter insolitus DSM 23790 1Ia Mycobacterium frederiksbergense DSM 23798 1Ib Mycobacterium sacrum DSM 23785 1Eb Mycobacterium fluoranthenivorans not applicable 1A2 Mycobacterium fluoranthenivorans not applicable 1Nb Mycobacterium fluoranthenivorans DSM 23796 1Ja Mycobacterium fluoranthenivorans not applicable 1A1 Microbacterium ginsengisoli DSM 23784 4D1 Burkholderia sp. not applicable 4F1 Burkholderia tropica DSM 23799 4Bd Cosmospora episphaeria DSM 23772 4I Rahnella aquatilis not applicable 4Ba Fusarium oxysporum DSM 23770 4Bc Microbacterium trichothecenolyticum DSM 23788

[0084] For the synthesis of enantiomerically enriched (R)-aminotetralin from tetralon preferably the microorganism Sinorhizobium morelense, Rahnella aquatilis or Ochrobactrum anthropi or a transaminase obtainable from any of these species is used. More preferably the microorganism Rahnella aquatilis or Ochrobactrum anthropi or a transaminase obtainable from any of these species. Most preferably the microorganism Ochrobactrum anthropi or a transaminase obtainable from this species is used.

[0085] For the synthesis of enantiomerically enriched (R)-aminoindan from indanon preferably the microorganism Paecilomyces lilacinus or Curtobacterium pusillum or a transaminase obtainable from any of these species is used. Most preferably the microorganism Curtobacterium pusillum or a transaminase obtainable from this species is used.

[0086] For the synthesis of enantiomerically enriched (R)-.alpha.-ethylbenzylamine from propiophenone preferably the microorganism Microbacterium ginsengisoli, Yersinia kristensenii, Pseudomonas citronellolis or Ochrobactrum anthropi or a transaminase obtainable from any of these species is used. More preferably the microorganism Yersinia kristensenii, Pseudomonas citronellolis or Ochrobactrum anthropi or a transaminase obtainable from any of these species is used. Even more preferably the microorganism Pseudomonas citronellolis or Ochrobactrum anthropi or a transaminase obtainable from any of these species is used. Most preferably the microorganism Ochrobactrum anthropi or a transaminase obtainable from this species is used.

[0087] For the synthesis of enantiomerically enriched (R)-.alpha.-methylbenzylamine from acetophenone preferably the microorganism Mycobacterium fortuitum, Ochrobactrum tritici or Achromobacter spanius or a transaminase obtainable from any of these species is used. More preferably the microorganism Ochrobactrum tritici or Achromobacter spanius or a transaminase obtainable from any of these species is used. Most preferably the microorganism Achromobacter spanius or a transaminase obtainable from this species is used.

[0088] For the synthesis of enantiomerically enriched (R)-3,3-dimethyl-2-aminobutane from 3,3-dimethyl-2-butanone preferably a microorganism of the genus Mycobacterium or a transaminase obtainable from this genus is used. More preferably a microorganism from the group of Mycobacterium frederiksbergense, Mycobacterium sacrum or Mycobacterium fluoranthenivorans, or a transaminase obtainable from one of these species, respectively, is used. Even more preferably the microorganism Microbacterium ginsengisoli or Achromobacter insolitus or a transaminase obtainable from any of these species is used. Most preferably the microorganism Achromobacter insolitus or a transaminase obtainable from this species is used.

[0089] For the synthesis of enantiomerically enriched (R)-2-aminobutane from 2-butanone preferably a microorganism of the genus Burkholderia or a transaminase obtainable from this genus is used. More preferably a microorganism from the group of Cosmospora episphaeria or Fusarium oxysporum or a transaminase obtainable from any of these species, respectively, is used. Even more preferably the microorganism Microbacterium trichothecenolyticum or a transaminase obtainable from this species is used. Most preferably a microorganism from the group of Burkholderia sp., Burkholderia tropica or Rahnella aquatilis, or a transaminase obtainable from any of these species, respectively, is used.

[0090] The re-isolation of the individual microorganisms from the pools deposited can be achieved by plating of suitable dilutions of the pooled microorganisms and re-streaking on the selective enrichment medium containing the respective (R)-amine, on which they have been enriched originally. Repeated re-streaking on the respective (R)-amine containing selective enrichment medium is carried out until only colonies of uniform morphology are obtained. Subsequently 16S rRNA or D2-LSU rRNA sequencing is performed, e.g. by using the validated MicroSEQ.RTM. (Applied Biosystems, Carlsbad, Calif., USA) system, to re-identify the individual microorganisms.

[0091] Transaminases obtainable from the strains in table 1 include enzymes derived from transaminase gene sequences of the strains in table 1. These gene sequences can be identified and isolated by various methods known to the person skilled in the art such as genome sequencing and sequence comparison with known transaminase sequences, DNA isolation and using probes with DNA sequences having a high degree of identity (at least 80%) to known transaminase genes, enzyme purification and enzyme sequencing, transformation of DNA derived from these strains in other host organisms and selection for growth on (R)-amines followed by DNA sequencing, or combinations of these approaches (Ausubel et al., eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York 1987).

[0092] Accordingly, the present invention also relates to a microorganism comprising an enzyme having (R)-transaminase activity from the group of organisms consisting of Rahnella aquatilis (deposited as DSM 23797), Ochrobactrum anthropi (deposited as DSM 23793), Ochrobactrum tritici (deposited as DSM 23786), Sinorhizobium morelense (deposited as DSM 23794), Curtobacterium pusilllum (deposited as DSM 23787), Paecilomyces lilacinus (deposited as DSM 23771), Microbacterium ginsengisoli, Microbacterium trichothecenolyticum (deposited as DSM 23788), Pseudomonas citronellolis (deposited as DSM 23795), Yersinia kristensenii (deposited as DSM 23792), Achromobacter spanius (deposited as DSM 23791), Achromobacter insolitus (deposited as DSM 23790), Mycobacterium fortuitum (deposited as DSM 23789), Mycobacterium frederiksbergense (deposited as DSM 23798), Mycobacterium sacrum (deposited as DSM 23785), Mycobacterium fluoranthenivorans (deposited as DSM 23796), Burkholderia sp., Burkholderia tropica (deposited as DSM 23799), Cosmospora episphaeria (deposited as DSM 23772), and Fusarium oxysporum (deposited as DSM 23770).

[0093] According to a further embodiment the present invention relates to a microorganism comprising an enzyme having (R)-transaminase activity selected from the group consisting of Cpu-TA1 [SEQ ID No. 30], Cpu-TA2 [SEQ ID No. 33], Cpu-TA3 [SEQ ID No. 35], Raq-TA2 [SEQ ID No. 38], Raq-TA3 [SEQ ID No. 40], Asp-TA1 [SEQ ID No. 43] and Mgi-TA1 [SEQ ID No. 46] or a protein having at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% identity to any of these amino acid sequences.

[0094] According to a further embodiment the present invention relates to a polypeptide having (R)-transaminase activity and obtainable from an organism selected from the group of organisms consisting of Rahnella aquatilis (deposited as DSM 23797), Ochrobactrum anthropi (deposited as DSM 23793), Ochrobactrum tritici (deposited as DSM 23786), Sinorhizobium morelense (deposited as DSM 23794), Curtobacterium pusilllum (deposited as DSM 23787), Paecilomyces lilacinus (deposited as DSM 23771), Microbacterium ginsengisoli, Microbacterium trichothecenolyticum (deposited as DSM 23788), Pseudomonas citronellolis (deposited as DSM 23795), Yersinia kristensenii (deposited as DSM 23792), Achromobacter spanius (deposited as DSM 23791), Achromobacter insolitus (deposited as DSM 23790), Mycobacterium fortuitum (deposited as DSM 23789), Mycobacterium frederiksbergense (deposited as DSM 23798), Mycobacterium sacrum (deposited as DSM 23785), Mycobacterium fluoranthenivorans (deposited as DSM 23796), Burkholderia sp., Burkholderia tropica (deposited as DSM 23799), Cosmospora episphaeria (deposited as DSM 23772), and Fusarium oxysporum (deposited as DSM 23770).

[0095] According to a further embodiment the invention relates to a polypeptide having (R)-transaminase activity and at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to a polypeptide having (R)-transaminase activity and obtainable from an organism selected from the group of organisms consisting of Rahnella aquatilis (deposited as DSM 23797), Ochrobactrum anthropi (deposited as DSM 23793), Ochrobactrum tritici (deposited as DSM 23786), Sinorhizobium morelense (deposited as DSM 23794), Curtobacterium pusilllum (deposited as DSM 23787), Paecilomyces lilacinus (deposited as DSM 23771), Microbacterium ginsengisoli, Microbacterium trichothecenolyticum (deposited as DSM 23788), Pseudomonas citronellolis (deposited as DSM 23795), Yersinia kristensenii (deposited as DSM 23792), Achromobacter spanius (deposited as DSM 23791), Achromobacter insolitus (deposited as DSM 23790), Mycobacterium fortuitum (deposited as DSM 23789), Mycobacterium frederiksbergense (deposited as DSM 23798), Mycobacterium sacrum (deposited as DSM 23785), Mycobacterium fluoranthenivorans (deposited as DSM 23796), Burkholderia sp., Burkholderia tropica (deposited as DSM 23799), Cosmospora episphaeria (deposited as DSM 23772), and Fusarium oxysporum (deposited as DSM 23770).

[0096] According to a further embodiment the invention relates to a polypeptide having (R)-transaminase activity selected from the group consisting of Cpu-TA1 [SEQ ID No. 30], Cpu-TA2 [SEQ ID No. 33], Cpu-TA3 [SEQ ID No. 35], Raq-TA2 [SEQ ID No. 38], Raq-TA3 [SEQ ID No. 40], Asp-TA1 [SEQ ID No. 43] and Mgi-TA1 [SEQ ID No. 46] or a protein having at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% identity to any of these polypepetide sequences.

[0097] According to a further embodiment the present invention relates to a nucleic acid comprising a sequence encoding a polypeptide having (R)-transaminase activity and at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to a polypeptide having (R)-transaminase activity and obtainable from an organism selected from the group of organisms consisting of Rahnella aquatilis (deposited as DSM 23797), Ochrobactrum anthropi (deposited as DSM 23793), Ochrobactrum tritici (deposited as DSM 23786), Sinorhizobium morelense (deposited as DSM 23794), Curtobacterium pusilllum (deposited as DSM 23787), Paecilomyces lilacinus (deposited as DSM 23771), Microbacterium ginsengisoli, Microbacterium trichothecenolyticum (deposited as DSM 23788), Pseudomonas citronellolis (deposited as DSM 23795), Yersinia kristensenii (deposited as DSM 23792), Achromobacter spanius (deposited as DSM 23791), Achromobacter insolitus (deposited as DSM 23790), Mycobacterium fortuitum (deposited as DSM 23789), Mycobacterium frederiksbergense (deposited as DSM 23798), Mycobacterium sacrum (deposited as DSM 23785), Mycobacterium fluoranthenivorans (deposited as DSM 23796), Burkholderia sp., Burkholderia tropica (deposited as DSM 23799), Cosmospora episphaeria (deposited as DSM 23772), and Fusarium oxysporum (deposited as DSM 23770).

[0098] According to a further embodiment the present invention relates to a nucleic acid comprising a sequence encoding a polypeptide having (R)-transaminase activity selected from the group consisting of Cpu-TA1 [SEQ ID No. 30], Cpu-TA2 [SEQ ID No. 33], Cpu-TA3 [SEQ ID No. 35], Raq-TA2 [SEQ ID No. 38], Raq-TA3 [SEQ ID No. 40], Asp-TA1 [SEQ ID No. 43] and Mgi-TA1 [SEQ ID No. 46] or a protein having at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% identity to any of these polypepetide sequences.

[0099] Enzymes derived from gene sequences of the strains in table 1 also include enzymes with sequence identities of at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% which are obtained by mutagenesis of genes derived from the strains in table 1. Mutagenesis methods are known to the person skilled in the art and include gene synthesis as well as mutagenesis methods using mutagenic primers (Bloom & Arnold, Proc Natl Acad Sci USA 2009, 106, 9995).

EXAMPLES

General

Microbial Growth Media

LB (Luria Bertani) Medium

10 g/l Bacto Tryptone (BD, Le Pont de Claix, France)

5 g/l Bacto Yeast Extract (BD, Le Pont de Claix, France)

5 g/l NaCl (Sigma-Aldrich, Steinheim, Germany)

[0100] 15 g/l Bacto Agar (for solid media, BD, Le Pont de Claix, France)

[0101] The components were dissolved in demineralised water and if necessary the pH was adjusted to 7.0. The media were sterilized by autoclaving for 20 min at 121.degree. C. For solid media agar was added before autoclaving. Antibiotics were added after the autoclaved medium had cooled down to 60.degree. C.

Selective Enrichment Media (SEM)

10 g/l Difco Yeast Carbon Base (YCB, BD, Sparks, Md., USA)

[0102] 55 mM glycerol (Merck, Darmstadt, Germany) 10 mM pyruvic acid (Sigma-Aldrich, Steinheim, Germany) 5 mM (R)-amine substrate

15 g/l Difco Agar Noble (BD, Le Pont de Claix, France)

[0103] A stock solution of YCB medium (100 g/l), 550 mM glycerol and 100 mM pyruvic acid was prepared in MilliQ water (Millipore, Billerica, Mass., USA) and sterilized by filtration through 0.22 .mu.m sterile filters. Liquid enrichment media were prepared by adding an (R)-amine substrate selected from the group (R)-2-aminobutane, (R)-3,3-dimethyl-2-aminobutane, (R)-.alpha.-methylbenzylamine, (R)-.alpha.-ethylbenzylamine, (R)-1-aminoindan and (R)-1-aminotetralin to a 1:10 with sterile MilliQ water diluted stock solution of the enrichment medium. Solid enrichment media were prepared accordingly with autoclaved MilliQ water containing Agar Noble to obtain a final concentration of 15 g/l.

Antibiotics

[0104] LB medium containing carbenicillin or neomycine in final concentrations of 100 .mu.g/ml was used to select and cultivate recombinant Escherichia coli strains containing expression vectors comprising [SEQ IDs No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26] to maintain the plasmids. Carbenicllin and neomycine stock solutions (50 mg/ml) were sterilized by filtration through 0.22 .mu.m sterile filters.

Molecular and Genetic Techniques

[0105] 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

[0106] E. coli strains TOP10 (Invitrogen, Carlsbad, Calif., USA) was used for all cloning procedures. E. coli was also used for protein expression. For induction of gene expression L-arabinose was used at a final concentration of 0.02% (w/v).

Cloning of Target Genes

[0107] The target genes according to [SEQ IDs No. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26] were codon pair optimised according to a procedure described in WO08000632. attB sites were added to all genes upstream of the ribosomal binding site and start codon and downstream of the stop codon to facilitate cloning using the Gateway technology (Invitrogen, Carlsbad, Calif., USA). Synthetic genes were obtained from Geneart (Regensburg, Germany). The gene constructs were cloned into a pBAD/Myc-His C (Invitrogen, Carlsbad, Calif., USA) derived expression vector 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 pBAD expression vectors were obtained, respectively. The corresponding expression strains were obtained by transformation of chemically competent E. coli cells with the respective pBAD-expression vectors.

Identification of Plasmids

[0108] 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.

Determination Protein Concentrations in Solution

[0109] The concentrations of proteins in solutions such as cell-free extracts (CFEs) were determined using a modified protein-dye binding method as described by Bradford in Anal. Biochem. 72: 248-254 (1976). Of each sample 50 .mu.l in an appropriate dilution was incubated with 950 .mu.l reagent (100 mg Brilliant Blue G250 dissolved in 46 ml ethanol and 100 ml 85% ortho-phosphoric acid, filled up to 1,000 ml with milli-Q water) for at least five minutes at room temperature. The absorption of each sample at a wavelength of 595 nm was measured in a Perkin Elmer Lambda20 or a Lambda35 UV/VIS spectrophotometer. Using a calibration line determined with solutions containing known concentrations of bovine serum albumin (BSA, ranging from 0.0125 mg/ml to 0.20 mg/ml) the protein concentration in the samples was calculated.

Analysis

High-Pressure Liquid Chromatography (HPLC) Analysis

[0110] Ketones and amines containing aromatic groups were analysed on a Prevail C18 15 cm column. For the separation and quantification of the enantiomers of .alpha.-methylbenzylamine, .alpha.-ethylbenzylamine, 2-aminotetralin a Crownpak Cr (+) column (Daicel) with post-column dervatisation of the amines using o-phtalaldehyde plus mercaptoethanol and fluorescence detection was used. For the separation and quantification of the enantiomers of 1-aminotetralin and 1-aminoindan a Prevail C18 15 cm plus Crownpak Cr (+) columns were used with post-column dervatisation of the amines using o-phtalaldehyde plus mercaptoethanol and fluorescence detection was used.

Gas Chromatography

[0111] Alkyl ketones and alkyl amines such as 2-butanone, 2-aminobutane, 3,3-dimethyl-butanone, and 3,3-dimethyl-2-aminobutane were analysed on a CP-Sil 8 CB column (Varian) for amines column using commercially available reference materials (Sigma-Aldrich). For the separation and quantification of the (R)- and (S)-alkyl amines a Chiralsil-CB column (Agilent) was used.

Example 1

Enrichment of Microorganisms on (R)-Amines as Only Nitrogen Source

[0112] Soil samples from various places in the Netherlands and Germany were suspended in 100 mM potassium phosphate (KR) buffer pH 7.0 and incubated with shaking at 180 rotations per minute (rpm) and 28.degree. C. for 1 hour. The suspensions were filtered through Whatman filter paper. 100 .mu.l of filtrate, each, was used to inoculate 100 ml Erlenmeyer flasks containing 10 ml SEM containing one of the six (R)-amines selected from the group (R)-2-aminobutane, (R)-3,3-dimethyl-2-aminobutane, (R)-.alpha.-methylbenzylamine, (R)-.alpha.-ethylbenzylamine, (R)-1-aminoindan and (R)-1-aminotetralin. The flasks were incubated with shaking on an orbitary shaker at 180 rpm and 28.degree. C. When microbial growth was obtained in form of significant turibidity of the culture medium or "cell aggregates", 100 .mu.l of this culture medium was used to inoculate SEM containing 5 mM of the same (R)-amine. After two such passages 100 .mu.l of a 1:100 dilution of the last culture medium was plated on SEM agar plates containing 5 mM of the corresponding (R)-amine. Additionally approximately 10 .mu.l of the undiluted culture was streaked out on SEM agar plates containing 5 mM of the corresponding (R)-amine. The inoculated agar plates were incubated at 28.degree. C. until growth was observed. Colonies with different morphology were restreaked on fresh SEM plates to separate different microbial species. The re-streaking was continued until pure cultures with uniform morphologies were obtained after incubation at 28.degree. C. and storage at 4.degree. C.

[0113] Selected micro-organisms were sent for MicroSEQ.RTM. (Applied Biosystems, Carlsbad, Calif., USA) Identification to BaseClear (Leiden, The Netherlands). The bacterial or fungal 16S or D2-LSU rRNA sequences obtained were compared to the validated MicroSEQ.RTM. sequence database (at BaseClear, Leiden, The Netherlands) and in case no identity above 99% was obtained compared to the non-redundant (nr) nucleotide database using the BlastN algorithm on the NCBI Blast homepage (http://www.ncbi.nlm.nih.gov/BLAST/). An overview of the results of these sequence analyses is given in table 2.

TABLE-US-00002 TABLE 2 Identification of enrichment isolates by MicroSeq Identification % (R)-amine Isolate Genus species match 1-aminotetralin 3Na Rahnella aquatilis 99.8 1-aminotetralin 3Kb Rahnella aquatilis 99.8 1-aminotetralin 3Ba Ochrobactrum anthropi 100 1-aminotetralin 3Db Ochrobactrum anthropi 100 1-aminotetralin 3H1 Sinorhizobium morelense 100 1-aminoindan 5BaB Curtobacterium pusillum 100 1-aminoindan 5BaS Paecilomyces lilacinus 100 .alpha.- 2A2 Microbacterium ginsengisoli 99.8 ethylbenzylamine .alpha.- 2Ca Microbacterium ginsengisoli 99.8 ethylbenzylamine .alpha.- 2Cb Ochrobactrum anthropi 100 ethylbenzylamine .alpha.- 2M1 Pseudomonas citronellolis 99.0 ethylbenzylamine .alpha.- 2Da Yersinia kristensenii 99.1 ethylbenzylamine .alpha.- 2K1 Ochrobactrum anthropi 100 ethylbenzylamine .alpha.-methylbenzyl- 6Ab Ochrobactrum tritici 100 amine .alpha.-methylbenzyl- 6Bb Mycobacterium fortuitum 99.3 amine .alpha.-methylbenzyl- 6I Achromobacter spanius 100 amine .alpha.-methylbenzyl- 6F Achromobacter spanius 100 amine 3,3-dimethyl-2- 1Ea Achromobacter insolitus 99.6 aminobutane 3,3-dimethyl-2- 1Ia Mycobacterium frederiksbergense 99.4 aminobutane 3,3-dimethyl-2- 1Ib Mycobacterium sacrum 99.4 aminobutane 3,3-dimethyl-2- 1Eb Mycobacterium fluoranthenivorans 100 aminobutane 3,3-dimethyl-2- 1A2 Mycobacterium fluoranthenivorans 99.8 aminobutane 3,3-dimethyl-2- 1Nb Mycobacterium fluoranthenivorans 100 aminobutane 3,3-dimethyl-2- 1Ja Mycobacterium fluoranthenivorans 100 aminobutane 3,3-dimethyl-2- 1A1 Microbacterium ginsengisoli 99.8 aminobutane 2-aminobutane 4D1 Burkholderia sp. 100 2-aminobutane 4F1 Burkholderia tropica 100 2-aminobutane 4Bd Cosmospora episphaeria 99.0 2-aminobutane 4I Rahnella aquatilis 99.8 2-aminobutane 4Ba Fusarium oxysporum 100 2-aminobutane 4Bc Microbacterium trichothecenolyticum 99.8

Example-2

Detection of (R)-Transaminase Activity in Enrichment Isolates

[0114] From the pure cultures of the enrichment isolates on SEM agar plates 25 ml liquid SEM cultures containing 5 mM of the respective (R)-amine, on which the microorganisms had been enriched on, were inoculated and cultivated on an orbitary shaker at 180 rotations per minute (rpm) and at 28.degree. C. until the cultures turned turbid. Subsequently the cultures were transferred to 50 ml centrifuge tubes and centrifuged for 10 min at 50,000.times.g in JA-25.50 rotor in Beckman Avanti J-20.times.PI centrifuge (Beckman-Coulter, Woerden, The Netherlands). The cell pellets were resuspended in 2 ml 100 mM potassium phosphate (KP.sub.i) buffer pH 7.0 containing 0.1 mM pyridoxal 5'-phosphate (PLP). The cell suspensions were used to determine (R)-transaminase activity using 40 mM of amino donor and ketone substrate. Cell suspensions of enrichment isolates 3 Kb, 3Na, 3Ba, 3 Db, and 3H1 were tested with 40 mM (R)-.alpha.-methylbenzylamine as amine donor and 1-tetralone as ketone substrate yielding acetophenone and 1-aminotetraline as products. Cell suspensions of enrichment isolates 5BaB and 5BaS were tested with 40 mM (R)-.alpha.-methylbenzylamine as amine donor and 1-aminoindane as ketone substrate yielding acetophenone and 1-aminoindane as products. Cell suspensions of enrichment isolates 2A2, 2Ca, 2Cb, 2M1, 2Da, and 2K1 were tested with 40 mM (R)-.alpha.-methylbenzylamine as amine donor and propiophenone as ketone substrate yielding acetophenone and .alpha.-ethylbenzylamine as products. Cell suspensions of enrichment isolates 5BaB and 5BaS were tested with 40 mM (R)-.alpha.-methylbenzylamine as amine donor and 1-aminoindane as ketone substrate yielding acetophenone and 1-aminoindane as products. Cell suspensions of enrichment isolates 6Ab, 6Bb, 6I, and 6F were tested with 40 mM benzylamine as amine donor and acetophenone as ketone substrate yielding benzaldehyde and .alpha.-methylbenzylamine as products. The concentrations and the enantiomeric excess (e.e.) of the formed amine products were determined by HPLC as described in the general part. The results of the HPLC analyses are summarised in table 3.

TABLE-US-00003 TABLE 3 Concentrations and enantiomeric excesses of (R)-amines produced by the enriched microorganisms from amino donors and ketone substrates. (R)-amine produced Isolate Amine product [g/l] e.e. (R) 1-aminotetralin 3Na 0.31 99 1-aminotetralin 3Kb 0.24 99.6 1-aminotetralin 3Ba 0.40 98 1-aminotetralin 3Db 0.41 99 1-aminotetralin 3H1 0.12 88 1-aminoindan 5BaB 0.17 99.8 1-aminoindan 5BaS 0.17 99.8 .alpha.-ethylbenzylamine 2A2 0.32 95 .alpha.-ethylbenzylamine 2Ca 0.29 98 .alpha.-ethylbenzylamine 2Cb 0.25 98 .alpha.-ethylbenzylamine 2M1 0.33 98 .alpha.-ethylbenzylamine 2Da 0.34 96 .alpha.-ethylbenzylamine 2K1 0.30 98 .alpha.-methylbenzylamine 6Ab 0.08 94 .alpha.-methylbenzylamine 6Bb 0.17 64 .alpha.-methylbenzylamine 6I 0.09 94 .alpha.-methylbenzylamine 6F 0.10 70

[0115] These results demonstrate the presence of transaminases with good to excellent (R)-selectivity in the enriched microorganisms.

Example 3

Expression of (R)-Transaminases from Public Databases

[0116] The codon pair optimised genes encoding the polypeptides of the putative transaminases as summarized in table 4 were cloned into a pBAD/Myc-His C derived expression vector (as described in EP1513946) using the Gateway technology (Invitrogen) according to the manufacturer's protocols (www.invitrogen.com) as described in the general part.

TABLE-US-00004 TABLE 4 Overview of the polypeptide, nucleotide sequnces, and accession numbers Polypeptide Nucleotide Protein (amino acid (codon-pair- accession sequence) optimised gene) number Plasmid name SEQ ID No. 1 SEQ ID No. 2 XP_001209325 pBAD-XP_001209325 SEQ ID No. 3 SEQ ID No. 4 XP_002564064 pBAD-XP_002564064 SEQ ID No. 5 SEQ ID No. 6 EEU44019 pBAD-EEU44019 SEQ ID No. 7 SEQ ID No. 8 XP_001402221 pBAD-XP_001402221 SEQ ID No. 9 SEQ ID No. 10 YP_761201 pBAD-YP_761201 SEQ ID No. 11 SEQ ID No. 12 YP_838940 pBAD-YP_838940 SEQ ID No. 13 SEQ ID No. 14 YP_955297 pBAD-YP_955297 SEQ ID No. 15 SEQ ID No. 16 EDI73966 pBAD-EDI73966 SEQ ID No. 17 SEQ ID No. 18 NP_085750 pBAD-NP_085750 SEQ ID No. 19 SEQ ID No. 20 EDH25885 pBAD-EDH25885 SEQ ID No. 21 SEQ ID No. 22 EBP64591 pBAD-EBP64591 SEQ ID No. 23 SEQ ID No. 24 ECU93014 pBAD-ECU93014 SEQ ID No. 25 SEQ ID No. 26 YP_366475 pBAD-YP_366475

[0117] Additionally codon-pair optimised synthetic genes encoding for the polypeptides L-threonine aldolase (LTA_SAV, accession number Q82N15) from Streptomyces avelmitilis (as negative control), and (R)-transaminases ABN35871 (Sequence 2 from U.S. Pat. No. 7,169,592) and AAN21261 (Sequence 1 from U.S. Pat. No. 6,413,752) were ordered and cloned as described above. After transformation of competent E. coli TOP10 cells and plating on selective LB agar plates containing 100 .mu.g/ml antibiotic, the respective recombinant E. coli pBAD strains as given in table 4 were obtained. 5 ml LB precultures plus 50 .mu.g/ml antibiotic preculturures were inocululated with from the respective agar plates and cultivated over night at 28.degree. C. and 180 rpm on an orbitary shaker. From such precultures expression cultures were inoculated in Erlenmeyer flasks containing 50-100 ml LB plus 50 .mu.g/ml antibiotic to a start cell density of OD.sub.620=0.05. These cultures were incubated at 28.degree. C. and 180 rpm on an orbitary shaker. In the middle of the exponential growth phase (OD.sub.620 of about 0.6) the expression of the target genes was induced by the addition of 0.02% (w/v) L-arabinose to the culture flasks. After induction the cultivation was continued at 28.degree. C. and 180 rpm on an orbitary shaker over night (about 20 h). Subsequently the cells were harvested by centrifugation at 5,000.times.g for 10 min at 4.degree. C. The supernatant was discarded and the cells were resuspended and weighed. The cell pellets were resuspended in twice the volume of wet weight of ice-cold 50 mM KP.sub.i buffer pH 7.5 containing 0.1 mM PLP. Cell-free extracts (CFEs) were obtained by sonification of the cell suspensions using a Sonics (Meyrin/Satigny, Switzerland) Vibra-Cell VCX130 sonifier (output 100%, 10 s on/10 s off, for 10 min) with cooling in an ice/acetone bath and centrifugation in an Eppendorf (Hamburg, Germany) 5415R centrifuge at 13,000.times.g and 4.degree. C. for 30 min. The supernantants (=CFEs) were transferred to fresh tubes and stored on ice for immediate use or stored at -20.degree. C. Protein concentrations in the CFEs were determined using a modified method according to Bradford as described in the general part

Example 4

Racemic Resolution of (RS)-.alpha.-Methylbenzylamine

[0118] Cell-free extracts (CFEs) with heterologously in E. coli expressed transaminases XP.sub.--001209325 and EDH25885 were tested for (R)-transaminase activity and compared with CFEs containing heterologously in E. coli expressed L-threonine aldolase (LTA_SAV, accession number Q82N15) from Streptomyces avelmitilis (as negative control), and (R)-transaminases ABN35871 (Sequence 2 from U.S. Pat. No. 7,169,592) and AAN21261 (Sequence 1 from EP987332) in the racemic resolution of (RS)-.alpha.-methylbenzylamine. In a total reaction volume of 0.25 ml 0.1 ml of the CFEs were mixed with 80 mM (RS)-.alpha.-methylbenzylamine (MBA), 40 mM sodium pyruvate in 100 mM KP.sub.i buffer containing 0.1 mM PLP and incubated at 28.degree. C. for 20 h. The reactions were stopped by addition of 0.9 ml stopping reagent (50% (v/v) acetonitrile in H.sub.2O containing 0.1% (v/v) formic acid) to 0.1 ml reaction volume and analysed by HPLC on a chiral phase as described in the general part. The results of the HPLC analysis are given in table 5.

TABLE-US-00005 TABLE 5 concentration and e.e. of .alpha.-MBA in transaminase reactions % e.e. Enzyme (S)-.alpha.-MBA (S)-.alpha.-MBA [g/l] (R)-.alpha.-MBA [g/l] ABN35871 63 0.5 0.1 AAN21261 22 0.5 0.3 EDH25885 10 0.5 0.4 XP_001209325 >99 0.5 <0.01 LTA_SAV <2 0.5 0.5

[0119] These results show that XP.sub.--001209325 is a very efficient and selective (R)-transaminase, because it selectively converted all (R)-.alpha.-MBA but not (S)-.alpha.-MBA. Other (R)-transaminases like ABN35871 or AAN21261 were also selective, but at clearly lower productivities resulting in lower e.e. s in the racemic resolution of (RS)-.alpha.-MBA. EDH25885 also exhibited low (R)-selective transminase activity on (RS)-.alpha.-MBA.

Example 5

Synthesis of Enantiomerically Enriched (R)-Amines

[0120] In a final volume of 0.25 .mu.l buffered with 100 mM potassium phosphate (KP.sub.i) at pH 7.5 equimolar amounts of 70 mM of amino donor and 70 mM of ketone substrate were reacted in the presence of 0.1 ml CFE of a transaminase at 28.degree. C. for 24 h. CFE comprising transaminases XP.sub.--001209325, AAN21261 and ABN35871, respectively, was incubated with benzylacetone and .alpha.-methylbenzylamine yielding 4-phenyl-2-butylamine and acetophenone; propiophenone and .alpha.-methylbenzylamine yielding .alpha.-ethylbenzylamine and acetophenone; 1-indanone and .alpha.-methylbenzylamine yielding 1-aminoindan and acetophenone; 1-tetralone and .alpha.-methylbenzylamine yielding 1-aminotetralin and acetophenone; 2-tetralone and .alpha.-methylbenzylamine yielding 2-aminotetralin and acetophenone; butanone and .alpha.-methylbenzylamine yielding 2-aminobutane and acetophenone; and 3,3-dimethyl-2-butanone and .alpha.-methylbenzylamine yielding 3,3-dimethyl-2-aminobutane and acetophenone, respectively. The reactions were stopped by addition of 0.75 ml stopping reagent (50% (v/v) acetonitrile in H.sub.2O containing 0.1% (v/v) formic acid) to 0.25 ml reaction volume. The product concentrations and enantiomeric excesses were analysed by HPLC as described in the general part. The results of the HPLC analysis are given in table 6.

TABLE-US-00006 TABLE 6 Amine product concentrations and e.e.s with transaminase XP_001209325 compared to transaminases AAN21261 and ABN35871. XP_001209325 AAN21261 ABN35871 amine e.e. amine e.e. amine e.e. (R)-amine product [mM] [%] [mM] [%] [mM] [%] 4-phenyl-2-butylamine 16.0 n.d. <0.1 n.d. <0.1 n.d. 2-aminobutane 6.2 69 0.9 n.d. 6.9 -27 3,3-dimethyl-butylamine 0.7 50 0.3 n.d. 6.4 97 1-aminoindan <0.1 n.d. <0.1 n.d. <0.1 n.d. .alpha.-ethylbenzylamine 1.7 99 7.9 99 2.3 99 1-aminotetralin <0.1 n.d. <0.1 n.d. <0.1 n.d. 2-aminotetralin 0.4 96 0.7 61 0.3 97 n.d.: not determined

[0121] Further CFE comprising transaminase YP.sub.--955297 was incubated with propiophenone and .alpha.-methylbenzylamine yielding .alpha.-ethylbenzylamine and acetophenone. The reaction was stopped by addition of 0.75 ml stopping reagent (50% (v/v) acetonitrile in H.sub.2O containing 0.1% (v/v) formic acid) to 0.25 ml reaction volume. The product concentration and enantiomeric excess was analysed by HPLC as described in the general part. After 24 h incubation at 28.degree. C. 0.87 mmol/l (R)-.alpha.-ethylbenzylamine was obtained with an e.e. of 99%.

[0122] The above results show transaminases XP.sub.--001209325 and YP.sub.--955297 are highly selective (R)-transaminases. Further it becomes clear that XP.sub.--001209325 has a different substrate spectrum than the transaminases AAN21261 and ABN35871: 4-phenyl-2-butylamine was formed in significant concentrations by XP.sub.--001209325 but not by transaminases AAN21261 and ABN35871 (table 6). Additionally XP.sub.--001209325 produced enantiomerically enriched (R)-2-aminobutane from 2-butanone, while ABN35871 delivered enantiomerically enriched (S)-2-aminobutane (table 6).

Example 6

Selectivity of (R)-Transaminases on Chiral .alpha.-Methylbenzylamines

[0123] To examine the enantioselectivity of (R)-transaminases they were tested in the conversion of the pure enantiomers forms of .alpha.-methylbenzylamine (MBA). Cell-free extracts containing transaminses as prepared in EXAMPLE 3 were tested separately in their activity on (R)-MBA and (S)-MBA, respectively, with pyruvate as ketone substrate in a spectrophotometric assay in a Perkin Elmer Lambda35 UV/VIS spectrophotometer thermostated at 30.degree. C. at a wavelength of 300 nm.

[0124] In a final reaction volume of 1 ml 50 .mu.l of a suitable dilution of a CFE containing transaminase were mixed in disposable plastic UV or quartz cuvettes with 12.5 mM (R)- or (S)-MBA and 5 mM sodium pyruvate in the presence of 50 mM KP.sub.i buffer pH 7.5 containing 0.1 mM PLP. The reactions were started by addition of 10 .mu.l of 0.5 M sodium pyruvate (in 50 mM KP.sub.i buffer pH 7.5, 0.1 mM PLP) to the other assay components, which had been pre-incubated in the photometer at 30.degree. C. for 5 min. After addition of sodium pyruvate the absorption at 300 nm was recorded and the transaminase activity in the samples (CFEs) was calculated according to the law of Lambert-Beer with an molar extinction coefficient for acetophenone of .epsilon.=0.28 cm.sup.2/.mu.mol. One unit (U) of transaminase activity is defined as 1 .mu.mol of acetophenone formed from 12.5 mM (R)-MBA or (S)-MBA and 5 mM sodium pyruvate at 30.degree. C. in 50 mM KP.sub.i buffer pH 7.5 containing 0.1 mM PLP per minute. The specific transaminase activities of the CFEs (U/mg total CFE protein) were calculated by dividing the volumetric activity values (U/ml CFE) by the total protein concentration as determined according to the general procedures. The ratio of specific transaminase activities on (R)- over (S)-MBA are defined as the Transaminase Enantioselectivity Value (TEV). An (R)-selective transaminase is defined as a transaminase with a TEV value of >1. A good (R)-selectivity is defined as a specific activity ratio on (R)- over (S)-MBA of TEV .gtoreq.5. A high (R)-selectivity is defined as a specific activity ratio on (R)- over (S)-MBA of TEV .gtoreq.10. The specific activities of the transaminases in the CFEs on (R)- and (S)-MBA and the TEVs as determined in these experiments are given in table 7.

TABLE-US-00007 TABLE 7 specific transaminase activities on (R)- and (S)-MBA and TEVs Activity (R)-MBA Activity (S)-MBA Transaminase [mU/mg] [mU/mg] TEV XP_001209325 559 38 15 XP_002564064 12 0.9 13 YP_761201 32 7.2 4 YP_955297 210 26 8 NP_085750 4.6 0.6 8 EBP64591 18 13 1.3 EDI73966 15 14 1.1 ABN35871 1111 20 56 AAN21261 578 39 15

[0125] As becomes clear from this experiment the transaminases XP.sub.--001209325, XP.sub.--002564064, YP.sub.--761201, YP.sub.--955297, NP.sub.--085750, EBP64591, and ED173966 are (R)-selective transaminases as are transaminases ABN35871 and AAN21261. Transaminases YP.sub.--955297 and NP.sub.--085750 exhibited good (R)-selectivity with TEVs of above 5, while XP.sub.--001209325 and XP.sub.--002564064 and even showed high (R)-selectivity with TEVs of above 10.

Example 7

Re-Isolation of Microorganisms from Pooled Deposits

[0126] The re-isolation of the individual microorganisms from the pooled deposit is be achieved by plating of 1:1000, 1:10,000 and further dilutions of the pooled deposit on selective enrichment medium (SEM) agar plates containing one of the six (R)-amines as sole nitrogen source as described in the general part and EXAMPLE 1. By repeated re-streaking on the SEM agar plates containing the respective (R)-amine, on which they have been enriched originally, pure cultures are obtained. Repeated re-streaking on the respective (R)-amine containing selective enrichment medium is carried out until only colonies of uniform morphology are obtained. Subsequently 16S rRNA or D2-LSU rRNA sequencing is performed using the validated MicroSEQ.RTM. (Applied Biosystems, Carlsbad, Calif., USA) system, to re-identify the individual microorganisms.

Example 8

Enzymatic Transamination of Phenoxyacetone

[0127] Cell-free extract with heterologously in E. coli expressed (R)-transaminase XP.sub.--001209325 [SEQ ID No. 1] (2 U/ml) as produced and assayed as in EXAMPLE 3 and EXAMPLE 6 was incubated with 0.1 M of ketone substrate phenoxyacetone and 0.5 M of the amino donor isopropylamine, (RS)-2-butylamine (effectively 0.25 M (R)-2-butylamine) and (RS)-.alpha.-methylbenzylamine (effectively 0.25 M (R)-MBA), respectively, in 50 mM KP.sub.i buffer pH 7.5 containing 0.1 mM PLP at 30.degree. C. for 24 h. The amount of 1-phenoxy-2-propylamine formed was measured by HPLC analysis. The HPLC conditions were as follows:

[0128] 50 .mu.l of reaction mixture was added to 950 .mu.l of a 50:50 mixture of acetonitrile/water with 0.01% (v/v) formic acid and centrifuged for 5 min at 13,000 rpm.

HPLC Conditions:

Column: Purospher Star RP C18 (250.times.4.0, 5 .mu.m)

[0129] Eluent A: 0.01% formic acid in water

Eluent B: Acetonitrile

[0130] Flow: 0.8 ml/min Column temperature: 30.degree. C. Injection volume: 4 .mu.l

Detection: UV 210 nm (or 254 nm)

[0131] The results of these experiments are given in table 8.

TABLE-US-00008 TABLE 8 Conversion of phenoxyacetone to 1-phenoxy-2-propylamine catalysed by the (R)-transaminase XP_001209325 [SEQ ID No. 1] Conversion ketone 1-Phenoxy-2-propylamine Donor [%] [mol/l] [wt %] 2-butylamine 64 0.064 1.0 MBA 32 0.032 0.5 isopropylamine 31 0.031 0.5

[0132] 2-butylamine and MBA are effectively better amino donors than isopropylamine for the (R)-selective transaminase XP.sub.--001209325 [SEQ ID No. 1] as the actual concentration of (R)-2-butylamine and (R)-MBA are 0.25 M compared to 0.5 M of the achiral amino donor isopropylamine. Thus comparable or even better conversions are obtained with the amino donors 2-butylamine and MBA, respectively, at relatively lower amino donor concentrations compared to the amino donor isopropylamine. Even high product concentrations of 1.0 wt % were obtained (with 2-butylamine as amino donor).

Example 9

Transamination of Benzylacetone in the Presence of a Second Organic Solvent Phase

[0133] Cell-free extract with heterologously in E. coli expressed (R)-transaminase XP.sub.--001209325 [SEQ ID No. 1] (2 U/ml) as produced and assayed as in EXAMPLE 3 and EXAMPLE 6 was incubated with 0.1 M of ketone substrate benzylacetone and 0.25 M of the amino donor (RS)-.alpha.-methylbenzylamine in 50 mM KP.sub.i buffer pH 7.5 containing 0.5 mM PLP at 30.degree. C. for 24 h. The reactions were carried out in the presence and absence of 15% (v/v) of the non water-miscible organic solvent cyclohexane. The amount of 4-phenyl-2-propylamine formed and the enantiomeric excess (e.e.) of the reactions were measured by HPLC analysis as described in EXAMPLE 8. The determination of enantiomeric excesses was performed by HPLC analysis on a chiral stationary phase as follows:

[0134] 20 .mu.l of the reaction mixture was mixed with 50 .mu.l of Marfey's reagent solution (1% w/v N-.alpha.-[2,4-dinitrophenyl-5-fluorophenyl]-L-alanine amide in acetone) and 10 .mu.l saturated solution of NaHCO.sub.3. After incubation of the mixture at 40.degree. C. for 1 hour 10 .mu.l 2 N HCl and 920 .mu.l acetonitrile were added and the sample was centrifuged for 5 min prior to injection.

HPLC Conditions:

Column: Purospher Star RP C18 (250.times.4.0, 5 .mu.m)

[0135] Eluent A: 0.01% formic acid in water

Eluent B: Acetonitrile

[0136] Flow: 1 ml/min Column temperature: 30.degree. C. Injection volume: 2 .mu.l

Detection: UV 338.1 nm

[0137] Isocratic 50/50 eluent A/eluent B

[0138] The results of these experiments are given in table 9.

TABLE-US-00009 TABLE 9 Conversion and e.e. of (R)-4-phenyl-2-propylamine formed by XP_001209325 [SEQ ID No. 1] from benzylacetone in the presence and absence of the non water-miscible organic solvent cyclohexane Conversion to 4-phenyl- e.e. (R)-4-phenyl-2- Condition 2-propylamine [%] propylamine [%] no organic solvent 32 >99.9 15% (v/v) cyclohexane 22 >99.9

[0139] These results show that the (R)-selective transaminase XP.sub.--001209325 [SEQ ID No. 1] well tolerates the presence of the non water-miscible organic solvent cyclohexane and that its presence does not affect the enantioselectivity of the enzyme reaction.

Example 10

Recombinant (R)-Transaminases from Genomes of the Enriched Microorganisms

[0140] From the six strains, which were enriched on selective media for their (R)-selective transaminase activity (EXAMPLE 1 and 2), Rahnella aquatilis 3 Kb, Microbacterium ginsengisoli 1A1 DSM 23784, Sinorhizobium morelense 3H1 DSM 23794, Curtobacterium pusillum 5BaB DSM 23787, Mycobacterium frederiksbergense 1Ia DSM 23798, and Achromobacter spanius 6I DSM 23791 genomic DNA was isolated with the Easy-DNA kit (Invitrogen) according to the manufacturer's manual and finally eluted with TE buffer. The quality of the DNA was checked photometrically as well as by digest with Bsp143I or BamHI (Fermentas, St. Leon-Rot) followed by agarose gel electrophoresis. The thus isolated genomic DNA samples from Rahnella aquatilis 3 Kb, Microbacterium ginsengisoli 1A1 DSM 23784, Curtobacterium pusillum 5BaB DSM 23787, Achromobacter spanius 61 DSM 23791, and Microbacterium ginsengisoli 1A1 DSM 23784 were use for genome sequencing.

[0141] These genomic sequences were uploaded to a server and BLAST searches were conducted against the known (R)-transaminase sequences SEQ ID No. 1 and against hits found in one of the genomes. Several hits with different degree of similarity were identified and chosen for subsequent NdeI/HindIII cloning into pMS470.DELTA.8 (Balzer et al., Nucleic Acids Research, 1992, 20 (8): 1851-1858) and expression in E. coli. Three of the genes could not be amplified by PCR and one contained NdeI and HindIII in the native sequence and thus Asp-TA1, Cpu-TA1, Cpu-TA3 Raq-TA2 and Mgi-TA1 were ordered codon optimised for the expression in E. coli from Geneart/life technologies (Regensburg, Germany).

TABLE-US-00010 TABLE 10 BLAST search identity to the (R)-transaminase of SEQ ID No. 1 Sequence identity SEQ ID to SEQ protein Source organism No. ID No. 1 Cpu-TA1 Curtobacterium pusillum 5BaB DSM 23787 30 26% Cpu-TA2 Curtobacterium pusillum 5BaB DSM 23787 33 32% Cpu-TA3 Curtobacterium pusillum 5BaB DSM 23787 35 27% Raq-TA2 Rahnella aquatilis 3Kb 38 27% Raq-TA3 Rahnella aquatilis 3Kb 40 22% Asp-TA1 Achromobacter spanius 61 DSM 23791 43 27% Mgi-TA1 Microbacterium ginsengisoli 1A1 DSM 46 24% 23784

[0142] The 6 target genes were expressed in E. coli TOP10F' in LB medium supplemented with Ampicillin (100 .mu.g/ml) after induction with 0.5 mM IPTG at 25.degree. C. o/n. As a control served an E. coli TOP10F' culture containing a pMS470 vector without an inserted transaminase gene. The cells were harvested and lysed by sonication in 50 mM KP.sub.i buffer pH 7.5 containing 0.1 mM PLP and centrifuged. The lysates were concentrated using VivaSpin concentrators (Sartorius, Vienna, Austria). The protein concentrations of the lysates were determined by Bradford protein assay.

[0143] All lysates were tested in transamination reactions using the corresponding bulky amines on which the donor microorganism had been enriched on as a donor and pyruvate as an acceptor. Five times excess of a racemic or enantiopure amine (50 mM) was used over pyruvic acid (10 mM) in 50 mM KP.sub.i buffer pH 7.5 containing 0.1 mM PLP at 30.degree. C. for 24 h. In the reactions with Mgi-TA1 10 mM of pyruvic acid and 10 mM of amino donor MBA or 1-aminotetralin, respectively, were applied in 50 mM KP.sub.i buffer pH 7.5 containing 0.1 mM PLP at 30.degree. C. for 19 h. The formation of corresponding ketone was detected on HPLC as described in EXAMPLES 8 and 9. The selectivity of new transaminases was determined by formation of L- or D-alanine and the reactivity of different enantiomers of the respective amines. The new transaminases Cpu-TA1 [SEQ ID No. 30], Cpu-TA2 [SEQ ID No. 33], Cpu-TA3 [SEQ ID No. 35], Raq-TA2 [SEQ ID No. 38], Raq-TA3 [SEQ ID No. 40], Asp-TA1 [SEQ ID No. 43], and Mgi-TA1 [SEQ ID No. 46] showed (R)-selectivity (table 11). Cpu-TA2 was also tested with MBA and alanine as amino donors and phenoxyacetone and acetophenone as acceptor ketones, respectively. The amine products of these two reactions were enantiomerically enriched (R)-1-phenoxy-2-propylamine and (R)-MBA, respectively (table 11).

[0144] These results show that the new transaminases Cpu-TA1 [SEQ ID No. 30], Cpu-TA2 [SEQ ID No. 33], Cpu-TA3 [SEQ ID No. 35], Raq-TA2 [SEQ ID No. 38], Raq-TA3 [SEQ ID No. 40], Asp-TA1 [SEQ ID No. 43] and Mgi-TA1 [SEQ ID No. 46] are indeed (R)-selective transaminases.

TABLE-US-00011 TABLE 11 Transamination reactions catalyzed by new transaminases and their selectivities. Acceptor Conversion Selectivity Enzyme Amino donor ketone ketone [%] Product product Asp-TA1 MBA Pyruvate 1.5 alanine (R) (=D) Cpu-TA1 1-aminoindan Pyruvate 89.0 alanine (R) (=D) (R)-1-aminoindan Pyruvate 96.7 alanine (R) (=D) Cpu-TA2 1-aminoindan Pyruvate 15.4 alanine (R) (=D) MBA Phenoxyacetone 1.1 1-phenoxy-2-propylamine (R) alanine Acetophenone 1.3 MBA (R) Cpu-TA3 1-aminoindan Pyruvate 26.6 alanine (R) (=D) (R)-1-aminoindan Pyruvate 17.4 alanine (R) (=D) Raq-TA2 1-aminotetralin Pyruvate 19.2 alanine (R) (=D) (R)-1-aminotetralin Pyruvate 17.9 alanine (R) (=D) Raq-TA3 1-aminotetralin Pyruvate 17.0 alanine (R) (=D) (R)-1-aminotetralin Pyruvate 62.5 alanine (R) (=D) (S)-1-aminotetralin Pyruvate 6.5 alanine (R) (=D) Mgi-TA1 MBA Pyruvate 21.0 alanine (R) (=D) 1-aminotetralin Pyruvate 34.0 alanine (R) (=D) pMS vector w/o MBA Pyruvate 0.6 transaminase insert (-control)

Example 11

Synthesis of 4-Phenyl-2-Propylamine from Benzylacetone and (R)-.alpha.-Methylbenzylamine in the Presence of Non Water-Miscible Organic Solvent

[0145] Cell-free extract with heterologously in E. coli expressed (R)-transaminase XP.sub.--001209325 [SEQ ID No. 1] as produced and assayed as in EXAMPLE 3 and EXAMPLE 6 was incubated with 0.08 M of the amino donor (R)-.alpha.-methylbenzylamine (MBA) and a 1.5 fold excess of the ketone substrate benzylacetone (0.12 M) in 50 mM KP.sub.i buffer pH 7.5 containing 0.1 mM PLP at 28.degree. C. for 20 h. The reactions were carried out in the presence and absence of 10% (v/v) of the non water-miscible organic solvent cyclohexane. The amount of 4-phenyl-2-propylamine formed was measured by HPLC analysis as described in the general part.

[0146] Without addition of cyclohexanone 42.8 mM 4-phenyl-2-propylamine was formed, while with 10% (v/v) cyclohexanone an even higher concentration of 44.3 mM 4-phenyl-2-propylamine was formed from benzylacetone with (R)-MBA as amino donor.

Example 12

Enzymatic Synthesis of (R)-Sec-Butylamine from (R)-.alpha.-Methylbenzylamine

[0147] The transaminases XP.sub.--001209325 [SEQ ID No. 1], XP.sub.--002564064 [SEQ ID No. 3], EEU44019 [SEQ ID No. 5], XP.sub.--001402221 [SEQ ID No. 7], YP.sub.--761201 [SEQ ID No. 9], YP.sub.--838940 [SEQ ID No. 11], YP.sub.--955297 [SEQ ID No. 13], ED173966 [SEQ ID No. 15], NP.sub.--085750 [SEQ ID No. 17], EDH25885 [SEQ ID No. 19], EBP64591 [SEQ ID No. 21], and ECU93014 [SEQ ID No. 23] as well as ABN35871 and AAN21261 were produced as described in EXAMPLE 3. Cell-free extracts containing the heterologously expressed transaminases were reacted with 25 mM butanone and 50 mM (R)-.alpha.-methylbenzylamine at 28.degree. C. with shaking at 400 rpm for 24 h in 100 mM KP.sub.i buffer pH 7.5 containing 0.1 mM PLP in a total volume of 1 ml. After 20 h the reactions were quenched by addition of a 100 .mu.l sample of each reaction to 50 .mu.l of 2-hexanone solution and 850 .mu.l acetonitrile and centrifugation for 10 min at 3000.times.g. The samples were analysed by GC-FID detection on a CpSil 8 for amines column (30 m.times.0.25.times.0.5) using commercial MBA, acetophenone, butanone and sec-butylamine as references (Sigma-Aldrich). The results are summarized in table 12.

TABLE-US-00012 TABLE 12 Concentration of sec-butylamine in mM formed from (R)-MBA and butanone after 24 h using the respective transaminases Transaminase sec-Butylamine [mM] ABN35871 0.99 AAN21261 7.96 YP_955297 1.52 YP_838940 0.45 XP_001209325 15.74 EBP64591 0.06 EDI73966 0.42 XP_002564064 b 0.63 YP_761201 0.42 EEU44019 b 0.23 XP_001402221 0.71 NP_085750 b 0.08

Example 13

Enzymatic Synthesis of (R)-3,3-Dimethyl-2-Butylamine from (R)-MBA and 3,3-Dimethyl-Butanone

[0148] Cell-free extracts containing the heterologously expressed transaminases XP.sub.--001209325 [SEQ ID No. 1], YP.sub.--955297 [SEQ ID No. 13], ABN35871 and AAN21261 produced as described in EXAMPLE 3 were reacted with 25 mM 3,3-dimethyl-butanone and 50 mM (R)-.alpha.-methylbenzylamine at 28.degree. C. with shaking at 400 rpm for 24 h in 100 mM KP.sub.i buffer pH 7.5 containing 0.1 mM PLP in a total volume of 1 ml. After 20 h the reactions were quenched by addition of a 100 .mu.l sample of each reaction to 50 .mu.l of 2-hexanone solution and 850 .mu.l acetonitrile and centrifugation for 10 min at 3000.times.g. The samples were analysed by GC-FID detection on a CpSil 8 for amines column (30 m.times.0.25.times.0.5) using commercial MBA, acetophenone, butanone and sec-butylamine as references (Sigma-Aldrich). The results are summarized in table 13.

TABLE-US-00013 TABLE 13 Concentration of sec-butylamine in mM formed from (R)-MBA and 3,3- dimethyl-butanone after 24 h using the respective transaminases Transaminase 3,3-dimethyl-2-butylamine [mM] ABN35871 0.00 AAN21261 3.28 YP_955297 0.34 XP_001209325 1.49

Sequence CWU 1

1

461325PRTAspergillus terreus 1Met Ala Ser Met Asp Lys Val Phe Ala Gly Tyr Ala Ala Arg Gln Ala 1 5 10 15 Ile Leu Glu Ser Thr Glu Thr Thr Asn Pro Phe Ala Lys Gly Ile Ala 20 25 30 Trp Val Glu Gly Glu Leu Val Pro Leu Ala Glu Ala Arg Ile Pro Leu 35 40 45 Leu Asp Gln Gly Phe Met His Ser Asp Leu Thr Tyr Asp Val Pro Ser 50 55 60 Val Trp Asp Gly Arg Phe Phe Arg Leu Asp Asp His Ile Thr Arg Leu 65 70 75 80 Glu Ala Ser Cys Thr Lys Leu Arg Leu Arg Leu Pro Leu Pro Arg Asp 85 90 95 Gln Val Lys Gln Ile Leu Val Glu Met Val Ala Lys Ser Gly Ile Arg 100 105 110 Asp Ala Phe Val Glu Leu Ile Val Thr Arg Gly Leu Lys Gly Val Arg 115 120 125 Gly Thr Arg Pro Glu Asp Ile Val Asn Asn Leu Tyr Met Phe Val Gln 130 135 140 Pro Tyr Val Trp Val Met Glu Pro Asp Met Gln Arg Val Gly Gly Ser 145 150 155 160 Ala Val Val Ala Arg Thr Val Arg Arg Val Pro Pro Gly Ala Ile Asp 165 170 175 Pro Thr Val Lys Asn Leu Gln Trp Gly Asp Leu Val Arg Gly Met Phe 180 185 190 Glu Ala Ala Asp Arg Gly Ala Thr Tyr Pro Phe Leu Thr Asp Gly Asp 195 200 205 Ala His Leu Thr Glu Gly Ser Gly Phe Asn Ile Val Leu Val Lys Asp 210 215 220 Gly Val Leu Tyr Thr Pro Asp Arg Gly Val Leu Gln Gly Val Thr Arg 225 230 235 240 Lys Ser Val Ile Asn Ala Ala Glu Ala Phe Gly Ile Glu Val Arg Val 245 250 255 Glu Phe Val Pro Val Glu Leu Ala Tyr Arg Cys Asp Glu Ile Phe Met 260 265 270 Cys Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Thr Leu Asp Gly Met 275 280 285 Pro Val Asn Gly Gly Gln Ile Gly Pro Ile Thr Lys Lys Ile Trp Asp 290 295 300 Gly Tyr Trp Ala Met His Tyr Asp Ala Ala Tyr Ser Phe Glu Ile Asp 305 310 315 320 Tyr Asn Glu Arg Asn 325 2978DNAartificial sequenceCodon optimized sequence coding for SEQ ID 1 2atggcgtcta tggacaaagt attcgctggt tacgctgcac gtcaggctat cctggaatcc 60actgaaacta ccaacccgtt cgctaaaggt atcgcatggg ttgaaggtga actggtaccg 120ctggctgaag cgcgtatccc gctgctggat cagggcttca tgcactctga cctgacttac 180gacgttccgt ctgtatggga cggtcgtttc ttccgtctgg atgaccacat cactcgtctg 240gaagcgtcct gcaccaaact gcgtctgcgt ctgccgctgc cgcgcgacca ggttaagcag 300atcctggttg aaatggttgc taaatctggt atccgtgacg cattcgttga gctgatcgtt 360actcgcggtc tgaaaggcgt tcgtggtact cgtccggaag atatcgttaa caacctgtac 420atgttcgttc agccgtacgt atgggtaatg gaaccggata tgcagcgcgt tggtggttct 480gctgttgttg cgcgtaccgt tcgtcgcgtt ccgccaggtg caatcgaccc gaccgttaaa 540aacctgcagt ggggcgacct ggttcgtggt atgttcgaag cagctgaccg cggtgcaact 600tacccgttcc tgactgacgg tgacgcacac ctgactgaag gttctggctt caacatcgtt 660ctggttaaag acggcgtact gtacactccg gaccgcggtg ttctgcaggg cgtaactcgt 720aagtctgtta tcaacgctgc tgaagcgttc ggtatcgaag ttcgcgttga gttcgttccg 780gttgaactgg cttaccgctg cgacgaaatc ttcatgtgta ctactgcagg tggtatcatg 840ccaatcacca ctctggacgg tatgccggtt aacggtggtc agatcggtcc gatcaccaag 900aaaatctggg acggttactg ggcgatgcac tacgacgctg cttactcctt cgaaatcgac 960tacaacgaac gtaattaa 9783319PRTPenicillium chrysogenum 3Met Ala Thr Met Glu Lys Ile Phe Ala Ala Tyr His Glu Arg Gln Lys 1 5 10 15 Leu Leu Ala Ala Asn Thr His Pro Phe Ala Lys Gly Val Ala Trp Val 20 25 30 Glu Gly Glu Leu Thr Pro Leu His Glu Ala Arg Ile Pro Ile Leu Asp 35 40 45 Gln Gly Phe Met His Ser Asp Leu Thr Tyr Asp Val Pro Ser Val Trp 50 55 60 Asp Gly Arg Phe Phe Arg Leu Asp Asp His Ile Thr Arg Leu Glu Ala 65 70 75 80 Ser Cys Thr Lys Leu Arg Met Lys Leu Pro Leu Pro Arg Asp Glu Val 85 90 95 Lys Gln Ile Leu Val Asp Met Val Ala Lys Ser Gly Ile Arg Asp Ala 100 105 110 Phe Val Glu Ile Ile Val Thr Arg Gly Leu Lys Gly Val Arg Gly Ser 115 120 125 Arg Pro Glu Asp Ile Val Asn Arg Ile Tyr Met Phe Ile Gln Pro Tyr 130 135 140 Val Trp Cys Met Glu Pro Glu Val Gln Pro Val Gly Gly Ser Ala Ile 145 150 155 160 Ile Ala Arg Thr Val Arg Arg Val Pro Pro Gly Cys Ile Asp Pro Thr 165 170 175 Val Lys Asn Leu Gln Trp Gly Asp Leu Val Arg Gly Leu Phe Glu Ala 180 185 190 Ser Asp Arg Gly Ala Glu Tyr Pro Phe Leu Thr Asp Gly Asp Thr Asn 195 200 205 Leu Thr Glu Gly Ser Gly Phe Asn Ile Val Leu Val Lys Asp Asn Ile 210 215 220 Leu Tyr Thr Pro Ala Arg Gly Val Leu Glu Gly Val Thr Arg Lys Ser 225 230 235 240 Val Ile Asp Val Ala Arg Ala Ser Gly Phe Asp Ile Lys Val Glu Leu 245 250 255 Val Pro Val Gln Met Ala Tyr Asp Ala Asp Glu Ile Phe Met Cys Thr 260 265 270 Thr Ala Gly Gly Ile Met Pro Ile Thr Ser Leu Asp Gly Lys Pro Val 275 280 285 Asn Asp Gly Lys Val Gly Ser Val Thr Lys Lys Ile Trp Asp Gly Tyr 290 295 300 Trp Ala Ile His Tyr Asp Pro Ala Tyr Ser Phe Glu Ile Ala Tyr 305 310 315 4960DNAartificial sequencecodon optimized sequence coding for SEQ ID 3 4atggcgacca tggaaaaaat cttcgctgct taccacgagc gtcagaaact gctggcagct 60aacactcacc cgttcgctaa aggcgtagca tgggttgaag gtgaactgac tccgctgcac 120gaagcgcgta tcccgattct ggatcagggc ttcatgcact ctgacctgac ttacgacgtt 180ccgtctgtat gggacggtcg tttcttccgt ctggatgacc acatcactcg tctggaagcg 240tcctgcacca aactgcgtat gaagctgccg ctgccgcgcg acgaagttaa gcagatcctg 300gttgatatgg ttgctaaatc tggtatccgt gacgcattcg ttgaaatcat cgttactcgc 360ggtctgaaag gcgttcgtgg ttcccgtccg gaagatatcg ttaaccgtat ctacatgttc 420atccagccgt acgtatggtg catggaaccg gaagttcagc cggtaggtgg ttctgctatc 480atcgcgcgta ccgttcgtcg cgttccgcca ggttgcatcg acccgaccgt taaaaacctg 540cagtggggcg acctggttcg tggtctgttc gaagcgtctg accgcggtgc agaatacccg 600ttcctgactg acggtgacac caacctgact gaaggttctg gcttcaacat cgttctggtt 660aaagacaaca tcctgtacac tccggcacgt ggtgttctgg aaggcgtaac tcgtaagtct 720gttatcgacg ttgctcgcgc ttctggcttc gacatcaaag ttgagctggt tccggtacag 780atggcttacg acgctgacga aatcttcatg tgtactactg caggtggtat catgccaatc 840acttctctgg acggtaagcc ggttaacgac ggtaaagttg gttctgttac caagaaaatc 900tgggacggtt actgggcaat ccactacgac ccggcttact ccttcgaaat cgcttattaa 9605320PRTNectria haematococca 5Met Ala Thr Met Asp Lys Val Phe Ala Gly Tyr Ala Glu Arg Gln Ala 1 5 10 15 Val Leu Glu Ala Ser Lys Asn Pro Leu Ala Lys Gly Val Ala Trp Ile 20 25 30 Gln Gly Glu Leu Val Pro Leu His Glu Ala Arg Ile Pro Leu Leu Asp 35 40 45 Gln Gly Phe Met His Ser Asp Leu Thr Tyr Asp Val Pro Ser Val Trp 50 55 60 Asp Gly Arg Phe Phe Arg Leu Glu Asp His Leu Asn Arg Leu Glu Ala 65 70 75 80 Ser Cys Lys Lys Met Arg Leu Arg Met Pro Leu Pro Arg Glu Glu Val 85 90 95 Ile Lys Thr Leu Val Asp Met Val Ala Lys Ser Gly Ile Arg Asp Ala 100 105 110 Phe Val Glu Leu Ile Val Thr Arg Gly Leu Thr Gly Val Arg Gly Ala 115 120 125 Lys Pro Glu Glu Leu Leu Asn Asn Asn Leu Tyr Met Phe Ile Gln Pro 130 135 140 Tyr Val Trp Val Met Asp Pro Asp Val Gln Tyr Thr Gly Gly Arg Ala 145 150 155 160 Ile Val Ala Arg Thr Val Arg Arg Val Pro Pro Gly Ser Ile Asp Pro 165 170 175 Thr Ile Lys Asn Leu Gln Trp Gly Asp Leu Val Arg Gly Leu Phe Glu 180 185 190 Ala Asn Asp Arg Gly Ala Thr Tyr Pro Phe Leu Thr Asp Gly Asp Ala 195 200 205 Asn Leu Thr Glu Gly Ser Gly Phe Asn Val Val Leu Ile Lys Asp Gly 210 215 220 Val Leu Tyr Thr Pro Asp Arg Gly Val Leu Gln Gly Ile Thr Arg Lys 225 230 235 240 Ser Val Ile Asp Ala Ala Arg Ser Cys Gly Tyr Glu Ile Arg Val Glu 245 250 255 His Val Pro Ile Glu Ala Thr Tyr Gln Ala Asp Glu Ile Leu Met Cys 260 265 270 Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Thr Leu Asp Asp Lys Pro 275 280 285 Val Lys Asp Gly Lys Val Gly Pro Ile Thr Lys Ala Ile Trp Asp Arg 290 295 300 Tyr Trp Ala Met His Trp Glu Asp Glu Phe Ser Phe Lys Ile Asn Tyr 305 310 315 320 6963DNAartificial sequenceCodon optimized sequence coding for SEQ ID 5 6atggcaacca tggacaaagt attcgctggt tacgctgaac gtcaggctgt tctggaagcg 60tctaaaaacc cgctggcgaa aggcgttgca tggattcagg gcgaactggt tccgctgcac 120gaagcgcgta tcccgctgct ggatcagggc ttcatgcact ctgacctgac ttacgacgtt 180ccgtctgtat gggacggtcg tttcttccgt ctggaagatc acctgaaccg tctggaagcg 240tcctgcaaga aaatgcgtct gcgtatgccg ctgccgcgtg aagaagttat caaaactctg 300gttgatatgg ttgctaaatc tggtatccgt gacgcattcg ttgaactgat cgttactcgc 360ggtctgactg gcgttcgtgg tgcgaagccg gaagagctgc tgaacaacaa cctgtacatg 420ttcatccagc cgtacgtatg ggtaatggac ccggacgttc agtacaccgg tggtcgtgct 480atcgttgctc gtaccgttcg tcgcgtaccg ccaggttcta tcgacccgac tatcaaaaac 540ctgcagtggg gcgacctggt tcgtggtctg ttcgaagcta acgaccgcgg tgcaacttac 600ccgttcctga ctgacggtga cgctaacctg actgaaggtt ctggcttcaa cgttgttctg 660atcaaagacg gcgtactgta cactccggac cgcggtgttc tgcagggtat cactcgtaag 720tctgttatcg acgctgcacg ttcctgcggt tacgaaatcc gcgttgaaca cgttccgatc 780gaagcaactt accaggctga cgaaatcctg atgtgtacta ctgctggcgg tatcatgcca 840atcaccactc tggatgacaa gccggttaaa gacggtaaag ttggtccgat caccaaagct 900atctgggacc gttactgggc aatgcactgg gaagacgagt tctctttcaa gatcaactac 960taa 9637327PRTAspergillus niger 7Met Ala Ser Met Asn Gln Val Leu Thr Glu Tyr Ala Thr Arg Arg Ala 1 5 10 15 Thr Leu Glu Ala Ser Lys Asn Pro Tyr Ala Lys Gly Ile Ala Trp Val 20 25 30 Glu Gly Gln Leu Val Pro Leu Arg Glu Ala Arg Ile Pro Leu Ile Asp 35 40 45 Gln Gly Phe Leu Arg Ser Asp Leu Thr Tyr Asp Val Ile Ser Val Trp 50 55 60 Asp Gly Trp Phe Phe Arg Leu Asp Asp His Leu Ser Arg Leu Glu Leu 65 70 75 80 Ala Cys Ala Lys Ser Arg Leu Lys Leu Pro Ile Ser Arg Asp Glu Val 85 90 95 Lys Gln Ser Leu Val Arg Met Val Ala Gln Ser Gly Ile Arg Asp Ala 100 105 110 Tyr Val Ala Leu Ile Val Thr Arg Gly Leu Gln Ser Val Arg Gly Ala 115 120 125 Lys Pro Glu Asp Leu Val Asn Asn Leu Tyr Met Phe Val Gln Pro Tyr 130 135 140 Val Trp Val Met Glu Pro Glu Val Gln Arg Val Gly Gly Ser Ala Val 145 150 155 160 Val Thr Arg Thr Val Arg Arg Val Pro Pro Gly Ala Ile Tyr Pro Thr 165 170 175 Val Lys Asn Leu Gln Trp Gly Asp Leu Thr Arg Gly Met Leu Glu Ala 180 185 190 Ala Asp Arg Gly Ser Met Tyr Pro Phe Leu Thr Asp Gly Asp Gly His 195 200 205 Leu Thr Glu Gly Ser Gly Tyr Asn Ile Val Leu Ile Lys Ala Gly Ala 210 215 220 Ile Tyr Thr Pro Asp Arg Gly Val Leu His Gly Val Thr Arg Thr Ser 225 230 235 240 Val Ile Asp Val Ala Arg Ala Cys Gly Ile Gln Val His Leu Glu Ala 245 250 255 Val Pro Val Glu Leu Val Tyr Gln Cys Asp Glu Ile Phe Met Cys Thr 260 265 270 Thr Ala Gly Gly Ile Met Pro Ile Thr Glu Leu Asp Gly Lys Pro Val 275 280 285 Asn Gly Gly Arg Ile Gly Pro Ile Thr Lys Lys Ile Trp Asp Gly Tyr 290 295 300 Trp Gly Met His Tyr Asp Pro Ala Tyr Ser Phe Ala Val Ser Tyr Asp 305 310 315 320 Asp Gly Ser Lys Ala Lys Leu 325 8984DNAArtificial sequenceCodon optimized sequence coding for SEQ ID 7 8atggcttcta tgaaccaggt tctgactgaa tacgcaactc gtcgtgcaac tctggaagcg 60tctaaaaacc cgtacgctaa aggtatcgca tgggttgaag gtcagctggt accgctgcgt 120gaagcgcgta tcccgctgat cgaccagggc ttcctgcgtt ctgacctgac ttacgacgtt 180atctccgtat gggacggctg gttcttccgt ctggatgacc acctgtctcg cctggaactg 240gcatgtgcga aatctcgcct gaaactgccg atctcccgtg acgaagttaa gcagtctctg 300gtacgtatgg ttgctcagtc tggtatccgt gacgcttacg ttgctctgat cgttactcgc 360ggtctgcagt ctgtacgtgg tgcgaagccg gaagatctgg ttaacaacct gtacatgttc 420gttcagccgt acgtatgggt aatggaaccg gaagttcagc gcgttggtgg ttctgctgtt 480gttactcgta ctgttcgtcg cgttccgcca ggtgctatct acccgaccgt taaaaacctg 540cagtggggcg acctgactcg cggtatgctg gaagcagctg accgcggttc tatgtacccg 600ttcctgactg acggtgacgg tcacctgact gaaggttctg gttacaacat cgttctgatc 660aaagctggcg caatctacac tccggaccgt ggtgttctgc acggtgttac tcgtacttct 720gttatcgacg ttgctcgcgc ttgcggtatt caggttcacc tggaagcggt accggttgag 780ctggtttacc agtgcgacga aatcttcatg tgtactactg caggtggtat catgccaatc 840actgaactgg acggtaaacc ggttaacggt ggtcgtatcg gtccgatcac caagaaaatc 900tgggacggtt actggggtat gcactacgac ccggcttact ccttcgctgt ttcttacgac 960gacggttcta aagcgaagtt ataa 9849321PRTHyphomonas neptunium 9Met Leu Thr Phe Gln Lys Val Leu Thr Gly Phe Gln Thr Arg Ala Asp 1 5 10 15 Ala Arg Ala Glu Arg Thr Asp Ala Phe Ala Asp Gly Ile Ala Trp Ile 20 25 30 Glu Asn Glu Phe Val Pro Ile Gly Lys Ala Arg Ile Pro Ile Leu Asp 35 40 45 Gln Gly Phe Leu His Ser Asp Leu Thr Tyr Asp Val Pro Ala Val Trp 50 55 60 Asn Gly Arg Ile Phe Arg Leu Asp Asp His Leu Asp Arg Leu Glu Val 65 70 75 80 Ser Cys Ala Lys Met Arg Leu Pro Leu Pro Ile Ala Arg Pro Glu Leu 85 90 95 Arg Arg Leu Val Met Glu Leu Val Ser Arg Ser Gly Leu Arg Asp Ala 100 105 110 Tyr Val Glu Ile Ile Val Thr Arg Gly Leu Lys Phe Leu Arg Gly Ala 115 120 125 Gln Ala Glu Asp Ile Ile Pro Asn Leu Tyr Leu Met Ala Val Pro Tyr 130 135 140 Val Trp Ile Leu Pro Leu Glu Tyr Gln Asn His Gly Ala Pro Ala Val 145 150 155 160 Val Thr Arg Thr Val Arg Arg Thr Pro Pro Gly Ala Leu Asp Pro Thr 165 170 175 Ile Lys Asn Leu Gln Trp Gly Asp Leu Val Arg Gly Leu Met Glu Ala 180 185 190 Gly Asp Arg Asp Ser Phe Phe Pro Ile Leu Pro Asp Gly Asp Gly Asn 195 200 205 Ala Thr Glu Gly Ala Gly Tyr Asn Ile Val Leu Val Arg Asn Gly Glu 210 215 220 Leu His Thr Pro Arg Arg Gly Val Leu Glu Gly Ile Thr Arg Arg Thr 225 230 235 240 Val Leu Glu Ile Ala Ala Ala Arg Gly Leu Lys Thr His Val Thr Glu 245 250 255 Ile Pro Ile Gln Ala Leu Tyr Glu Cys Asp Glu Leu Phe Met Cys Ser 260 265 270 Thr Ala Gly Gly Ile Met Pro Leu Val Leu Leu Asp Gly Asn Ile Val 275 280 285 Gly Asp Gly Thr Val Gly Pro Val Thr Arg Met Ile Trp Glu Ala Tyr 290 295 300 Trp Asp Leu His Asp Asp Pro Gln Leu Ser Glu Pro Val Thr Tyr Ala 305 310 315 320 Pro 10966DNAArtificial sequenceCodon optimized sequence coding for SEQ ID 9 10atgctgactt tccagaaagt tctgactggc ttccagactc gcgctgacgc tcgcgctgaa

60cgtactgacg cattcgctga cggtatcgcc tggatcgaaa acgagttcgt tccgatcggt 120aaagcgcgta tcccgatcct ggatcagggc ttcctgcact ctgacctgac ttacgacgtt 180ccggcagtat ggaacggtcg tatcttccgt ctggatgacc acctggaccg tctggaagtt 240tcctgtgcga agatgcgtct gccgctgcca atcgcgcgtc cggaactgcg tcgtctggta 300atggaactgg tttcccgttc tggtctgcgt gacgcttacg ttgaaatcat cgttactcgc 360ggtctgaaat tcctgcgcgg tgctcaggct gaagatatca tcccgaacct gtacctgatg 420gctgttccgt acgtatggat tctgccgctg gaataccaga accacggtgc accggctgtt 480gttactcgta ccgttcgtcg tactccgcca ggtgcgctgg acccgactat caaaaacctg 540cagtggggcg acctggttcg tggtctgatg gaagctggcg accgtgactc cttcttcccg 600atcctgccgg acggtgacgg taacgcaact gaaggtgcag gttacaacat cgttctggtt 660cgtaacggtg aactgcacac tccgcgtcgc ggtgttctgg aaggtatcac tcgtcgtacc 720gttctggaaa tcgctgctgc tcgcggtctg aaaactcacg ttactgaaat cccgattcag 780gcgctgtacg agtgcgacga actgttcatg tgctccactg caggtggtat catgccgctg 840gttctgctgg acggtaacat cgttggtgac ggtactgttg gtccggtaac tcgtatgatc 900tgggaagcat actgggatct gcacgacgac ccgcagctgt ctgaaccggt aacttacgca 960ccgtaa 96611317PRTBurkholderia cenocepacia 11Met Pro Arg Glu Thr Arg Ser Ala His His Gly Ile Pro Ser Val Glu 1 5 10 15 Ala Pro Ala Phe Pro Gln Gly Ala Ala Tyr Met Asn Gly Arg Phe Ile 20 25 30 Pro Ile Ala Asp Ala Arg Val Ser Val Leu Asp Trp Gly Phe Leu His 35 40 45 Ser Asp Val Thr Tyr Asp Thr Val His Val Trp Asn Gly Arg Phe Phe 50 55 60 Arg Leu Asp Lys His Ile Glu Arg Phe Arg Arg Ser Leu Ala Arg Leu 65 70 75 80 Arg Leu Asn Val Pro Leu Thr Asp Asp Ala Leu Arg Asp Ile Leu Val 85 90 95 Glu Cys Val Arg Arg Ser Gly Leu Arg His Ala Tyr Val Glu Met Leu 100 105 110 Cys Thr Arg Gly Val Ser Pro Thr Phe Ser Arg Asp Pro Arg Asp Ala 115 120 125 Val Asn Gln Phe Ile Ala Phe Ala Val Pro Tyr Gly Ser Val Ala Asn 130 135 140 Glu Arg Gln Leu Arg Glu Gly Leu His Leu His Val Ile Asp Asp Val 145 150 155 160 Arg Arg Ile Pro Pro Glu Ser Val Asp Pro Gln Ile Lys Asn Tyr His 165 170 175 Trp Leu Asp Leu Val Ala Gly Leu Leu Lys Gly Tyr Asp Ala Gly Ala 180 185 190 Glu Ser Val Leu Leu Lys Cys Thr Asp Gly Ser Ile Ala Glu Gly Pro 195 200 205 Gly Phe Asn Val Phe Val Val Arg Asp Gly Arg Leu Arg Thr Pro Glu 210 215 220 Arg Gly Val Leu His Gly Ile Thr Arg Gln Thr Val Phe Glu Leu Ala 225 230 235 240 Thr Ala Met Gly Ile Asp Ala Gln Ala Ala Arg Ile Asp Asp Ala Gln 245 250 255 Leu Arg Asp Ala Asp Glu Val Phe Ile Thr Ser Thr Ala Gly Gly Ile 260 265 270 Met Pro Val Thr Arg Leu Asn Asp Ala Thr Ile Gly Asp Gly Arg Pro 275 280 285 Gly Pro Met Thr Arg Arg Leu Phe Asp Ala Tyr Trp Ala Lys His Gly 290 295 300 Asp Pro Ala Trp Ser Leu Ala Val Asp Tyr Ala Asp Gly 305 310 315 12954DNAArtificial sequenceCodon optimized sequence coding for SEQ ID 11 12atgccgcgtg aaactcgttc tgcacaccac ggtatcccgt ctgttgaagc gccggcattc 60ccgcagggcg cagcttacat gaacggtcgt ttcatcccaa tcgctgacgc tcgcgtttct 120gtactggact ggggcttcct gcactctgac gttacttacg acaccgttca cgtatggaac 180ggtcgtttct tccgtctgga caagcacatc gaacgtttcc gtcgttctct ggcgcgtctg 240cgtctgaacg ttccgctgac tgacgacgcg ctgcgtgaca tcctggttga gtgcgtacgt 300cgttctggtc tgcgtcacgc ttacgttgaa atgctgtgca ctcgcggtgt ttctccgact 360ttctctcgcg atccgcgcga cgctgttaac cagttcatcg cgttcgctgt tccgtacggt 420tctgttgcta acgaacgtca gctgcgtgaa ggtctgcacc tgcacgttat cgacgacgtt 480cgtcgtatcc cgccagaatc cgttgatccg cagatcaaaa actaccactg gctggatctg 540gttgctggtc tgctgaaagg ttacgacgct ggcgcagaat ccgttctgct gaaatgcact 600gacggttcta tcgctgaagg tccgggcttc aacgtattcg ttgttcgtga cggtcgtctg 660cgtactccgg aacgtggtgt tctgcacggt atcactcgtc agactgtatt cgaactggca 720actgcgatgg gtatcgacgc tcaggctgca cgtatcgacg acgctcagct gcgtgacgct 780gacgaagttt tcatcacttc tactgcaggt ggtatcatgc cggtaactcg tctgaacgac 840gcaactatcg gtgacggtcg tccgggtccg atgactcgtc gtctgttcga cgcttactgg 900gcgaagcacg gtgacccggc atggtctctg gcggttgact acgctgacgg ataa 95413337PRTMycobacterium vanbaalenii 13Met Gly Ile Asp Thr Gly Thr Ser Asn Leu Val Ala Val Glu Pro Gly 1 5 10 15 Ala Ile Arg Glu Asp Thr Pro Ala Gly Ser Val Ile Gln Tyr Ser Asp 20 25 30 Tyr Glu Ile Asp Tyr Ser Ser Pro Phe Ala Gly Gly Val Ala Trp Ile 35 40 45 Glu Gly Glu Tyr Leu Pro Ala Glu Asp Ala Lys Ile Ser Ile Phe Asp 50 55 60 Thr Gly Phe Gly His Ser Asp Leu Thr Tyr Thr Val Ala His Val Trp 65 70 75 80 His Gly Asn Ile Phe Arg Leu Gly Asp His Leu Asp Arg Leu Leu Asp 85 90 95 Gly Ala Arg Lys Leu Arg Leu Asp Ser Gly Tyr Thr Lys Asp Glu Leu 100 105 110 Ala Asp Ile Thr Lys Lys Cys Val Ser Leu Ser Gln Leu Arg Glu Ser 115 120 125 Phe Val Asn Leu Thr Ile Thr Arg Gly Tyr Gly Lys Arg Lys Gly Glu 130 135 140 Lys Asp Leu Ser Lys Leu Thr His Gln Val Tyr Ile Tyr Ala Ile Pro 145 150 155 160 Tyr Leu Trp Ala Phe Pro Pro Ala Glu Gln Ile Phe Gly Thr Thr Ala 165 170 175 Val Val Pro Arg His Val Arg Arg Ala Gly Arg Asn Thr Val Asp Pro 180 185 190 Thr Ile Lys Asn Tyr Gln Trp Gly Asp Leu Thr Ala Ala Ser Phe Glu 195 200 205 Ala Lys Asp Arg Gly Ala Arg Thr Ala Ile Leu Met Asp Ala Asp Asn 210 215 220 Cys Val Ala Glu Gly Pro Gly Phe Asn Val Cys Ile Val Lys Asp Gly 225 230 235 240 Lys Leu Ala Ser Pro Ser Arg Asn Ala Leu Pro Gly Ile Thr Arg Lys 245 250 255 Thr Val Phe Glu Ile Ala Gly Ala Met Gly Ile Glu Ala Ala Leu Arg 260 265 270 Asp Val Thr Ser His Glu Leu Tyr Asp Ala Asp Glu Ile Met Ala Val 275 280 285 Thr Thr Ala Gly Gly Val Thr Pro Ile Asn Thr Leu Asp Gly Val Pro 290 295 300 Ile Gly Asp Gly Glu Pro Gly Pro Val Thr Val Ala Ile Arg Asp Arg 305 310 315 320 Phe Trp Ala Leu Met Asp Glu Pro Gly Pro Leu Ile Glu Ala Ile Gln 325 330 335 Tyr 141014DNAArtificial sequenceCodon optimized sequence coding for SEQ ID NO 13 14atgggtatcg acaccggtac ttctaacctg gttgctgttg agccgggtgc tatccgtgaa 60gatactccgg caggttctgt tatccagtac tctgactacg aaatcgacta ctcttctccg 120ttcgctggcg gcgttgcatg gatcgaaggt gaatacctgc cggcagaaga tgcgaagatc 180tccatcttcg acactggctt cggtcactct gacctgactt acaccgttgc tcacgtatgg 240cacggtaaca tcttccgtct gggtgaccac ctggaccgtc tgctggacgg tgcgcgtaag 300ctgcgtctgg attccggtta caccaaagac gaactggctg acatcaccaa gaaatgcgta 360tctctgtctc agctgcgtga atccttcgtt aacctgacta tcactcgcgg ttacggtaag 420cgtaaaggtg aaaaagacct gtctaaactg actcaccagg tttacatcta cgcaatcccg 480tacctgtggg cattcccgcc agcagagcag atcttcggta ctactgctgt tgttccgcgt 540cacgttcgtc gcgctggtcg taacaccgtt gatccgacta tcaaaaacta ccagtggggc 600gacctgactg cagcttcttt cgaagcgaaa gaccgcggtg cacgtactgc gattctgatg 660gacgctgaca actgcgttgc tgaaggtccg ggcttcaacg tttgcatcgt taaagacggt 720aaactggctt ctccgtcccg taacgcgctg ccaggtatca ctcgtaaaac cgtattcgaa 780atcgctggcg caatgggtat cgaagctgca ctgcgtgacg taacttctca cgaactgtac 840gacgctgacg aaatcatggc ggtaactact gctggcggcg ttactccgat caacaccctg 900gatggcgttc cgatcggtga cggtgaaccg ggtccggtta ccgttgctat ccgtgaccgc 960ttctgggcgc tgatggacga accgggtccg ctgatcgaag cgattcagta ctaa 101415305PRTUnknownOrganism isolated from soil 15Met Val Ser Asp Ile Phe Pro Lys Gly Ser Ala Trp Met Asn Gly Glu 1 5 10 15 Phe Ile Gln Leu Ser Glu Ala Lys Ile Pro Ile Leu Asp Trp Gly Phe 20 25 30 Leu Arg Ser Asp Ala Thr Tyr Asp Val Val His Val Trp Lys Gly Ser 35 40 45 Phe Phe Gln Leu Asp Lys His Ile Asp Arg Phe Phe Lys Ser Thr Glu 50 55 60 Lys Leu Arg Met Pro Cys Arg Leu Ser Arg Glu Glu Ile Lys Arg Ile 65 70 75 80 Leu Ala Gly Cys Val Lys Lys Ala Asp Leu Glu Asp Ser Tyr Val Glu 85 90 95 Met Ile Gln Thr Arg Gly Met Ser Pro Asn Phe Val Arg Asp Pro Arg 100 105 110 Lys Ala Thr Pro Arg Phe Met Ala Phe Ala Val Pro Phe Gly Trp Ile 115 120 125 Leu Arg Pro Glu Asp Phe Glu Lys Gly Leu Asp Val Tyr Leu Thr Asp 130 135 140 Ile Thr Arg Ile Pro Pro Ser Ser Val Asp Pro Thr Ile Lys Asn Tyr 145 150 155 160 His Trp Met Asp Leu Val Thr Gly Met Leu Asp Ala Tyr Asp Arg Gly 165 170 175 His His Thr Ala Ile Leu Val Asp Glu Asp Asn Asn Val Ser Glu Gly 180 185 190 Pro Gly Phe Asn Ile Phe Ser Val Asp Glu Asn Glu Ile Asn Thr Pro 195 200 205 Asp His Gly Val Leu Glu Gly Ile Thr Arg Gln Thr Val Ile Asp Leu 210 215 220 Ala Lys Glu Leu Asn Ile Lys Val Asn Lys Lys Pro Ile Thr Ile Lys 225 230 235 240 Met Leu Lys Ser Ser Glu Glu Leu Phe Ala Thr Ser Thr Ala Gly Gly 245 250 255 Val Met Pro Ile Thr Lys Ile Ser Gly Lys Asn Ile Asn Lys Gly Thr 260 265 270 Val Gly Asp Ile Thr Arg Lys Ile His Lys Leu Tyr Trp Asp Lys His 275 280 285 Ser Asp Pro Asp Trp Ser Thr Ser Ile Asn Asp Ile Leu Leu Tyr Lys 290 295 300 Val 305 16915DNAArtificial seqienceCodon optimized sequence coding for SEQ ID NO 15 16atggtttctg acatcttccc gaaaggttct gcatggatga acggtgaatt catccagctg 60tctgaagcga aaatcccgat cctcgactgg ggcttcctgc gttctgacgc aacttacgac 120gttgttcacg tatggaaagg ttctttcttc cagctggaca agcacatcga ccgcttcttc 180aaatctactg aaaaactgcg tatgccatgc cgtctgtctc gtgaagaaat caagcgtatc 240ctggctggct gcgttaagaa agctgacctg gaagactcct acgttgaaat gatccagact 300cgcggtatgt ctccgaactt cgttcgtgac ccgcgtaaag caactccgcg cttcatggcg 360ttcgctgttc cgttcggctg gattctgcgt ccggaagact tcgaaaaagg tctggacgtt 420tacctgactg acatcactcg tatcccgccg tcttccgttg atccgactat caaaaactac 480cactggatgg acctggttac cggtatgctg gatgcttacg accgcggtca ccacactgcg 540attctggttg acgaagataa caacgtttct gaaggtccgg gcttcaacat cttctctgtt 600gacgaaaacg aaatcaacac tccggatcac ggcgtactgg aaggtatcac tcgtcagact 660gttatcgacc tggctaaaga gctgaacatc aaagttaaca agaaaccaat cactatcaaa 720atgctgaaat cttctgaaga gctgttcgca acttctactg ctggcggcgt aatgccgatc 780accaagatct ccggtaaaaa catcaacaaa ggcaccgttg gtgacatcac tcgtaagatc 840cacaaactgt actgggacaa gcactctgat ccggactggt ctacttccat caacgacatc 900ctgctgtaca aggta 91517291PRTMesorhizobium loti 17Met Ala Phe Met Asp Gly Gln Tyr Leu Pro Met Ser Glu Ala Lys Val 1 5 10 15 Ser Val Leu Asp Trp Gly Phe Leu His Ser Asp Ala Thr Tyr Asp Thr 20 25 30 Val His Val Trp Asp Gly Arg Phe Phe Arg Leu Asn Leu His Val Asp 35 40 45 Arg Phe Phe Arg Gly Met Glu Lys Leu Arg Met Lys Leu Pro Tyr Asn 50 55 60 Arg Ser Glu Ile Glu Lys Ile Leu Ser Thr Cys Val Ala Leu Ser Gly 65 70 75 80 His Lys Ser Ala Tyr Val Glu Met Ile Cys Thr Arg Gly Gly Ser Pro 85 90 95 Thr Phe Ser Arg Asp Pro Arg Gln Ser Glu Asn Arg Phe Ile Ala Phe 100 105 110 Ala Val Pro Phe Gly Ser Val Ala Asn Lys Glu Gln Leu Glu Arg Gly 115 120 125 Leu His Val Ala Ile Ser Asn Thr Val Arg Ile Pro Pro Lys Ser Ile 130 135 140 Asp Pro Thr Ile Lys Asn Tyr His Trp Leu Asp Leu Val Lys Gly Leu 145 150 155 160 Phe Asp Ala Tyr Asp Tyr Gly Ala Glu Thr Ala Leu Ile Val Asp Ile 165 170 175 Asn Asp Asn Ile Ala Glu Gly Pro Gly Phe Asn Val Phe Thr Val Lys 180 185 190 Asp Gly Arg Leu Lys Thr Pro Ala Tyr Gly Val Leu Ala Gly Ile Thr 195 200 205 Arg Gln Thr Val Phe Asp Leu Cys Asp Glu Leu Gly Leu Ser Val Ser 210 215 220 Ala Gly Asp Ile Asp Arg Asn Glu Leu Lys Gly Ala Asp Glu Val Phe 225 230 235 240 Ile Thr Ser Thr Ala Gly Gly Ile Met Pro Val Ser Lys Ile Asp Glu 245 250 255 Thr Val Val Gly Asp Gly Lys Val Gly Ala Leu Thr Arg Gln Leu Ala 260 265 270 Asp Leu Tyr Trp Glu Lys His Ala Asp Pro Ala Trp Ser Thr Ala Val 275 280 285 Asn Tyr Ala 290 18876DNAArtificial sequenceCodon optimized sequence coding for SEQ ID NO 17 18atggcgttca tggacggtca gtacctgccg atgtctgaag cgaaagtttc tgtactggac 60tggggcttcc tgcactctga cgcaacttac gacaccgttc acgtatggga cggtcgtttc 120ttccgtctga acctgcacgt tgaccgcttc ttccgcggta tggaaaaact gcgtatgaag 180ctgccgtaca accgttctga aatcgaaaaa atcctgtcta cttgcgtagc gctgtctggt 240cacaagtctg cttacgttga aatgatctgt actcgcggtg gttctccgac tttctctcgc 300gatccgcgtc agtctgaaaa ccgcttcatc gcgttcgctg ttccgttcgg ttctgttgct 360aacaaagagc agctggaacg tggtctgcac gttgctatct ccaacaccgt tcgtatcccg 420ccaaagtcta tcgacccgac tatcaaaaac taccactggc tggatctggt taaaggtctg 480ttcgacgctt acgactacgg cgcagaaact gcactgatcg ttgatatcaa cgacaacatc 540gctgaaggtc cgggcttcaa cgtattcacc gttaaagacg gtcgtctgaa aactccggct 600tacggcgttc tggcaggtat cactcgtcag actgtattcg acctgtgcga cgaactgggt 660ctgtctgttt ctgctggcga catcgaccgt aacgaactga aaggtgctga cgaagttttc 720atcacttcta ctgcaggtgg tatcatgccg gtttccaaga tcgacgaaac cgttgttggt 780gacggtaaag ttggtgcgct gactcgtcag ctggctgacc tgtactggga aaaacacgct 840gatccggcat ggtctactgc tgttaactac gcataa 87619299PRTUnknownOrganism isolated from soil 19Met Thr Tyr Ser Ala Gly Ala Ala Trp Met Asp Gly Lys Val Ile Pro 1 5 10 15 Val Ser Glu Ala Lys Ile Ser Val Phe Asp Trp Gly Leu Thr Arg Ser 20 25 30 Asp Ile Thr Tyr Asp Val Val His Val Trp Glu Gly Ala Phe Phe Arg 35 40 45 Leu Glu Asp Tyr Leu Asp Arg Phe Met Val Ser Met Asp Lys Leu Arg 50 55 60 Leu Asp Val Gly Met Thr Arg Ala Glu Ile Lys Ala Ala Leu Val Glu 65 70 75 80 Leu Val Ala Thr Ser Gly Leu Lys Ser Ala Tyr Val Ser Met Val Ala 85 90 95 Ser Arg Gly Thr Pro Gln Val Pro Gly Thr Arg Asp Pro Arg Ala Cys 100 105 110 Thr Asn His Phe Tyr Ala Trp Ala Val Pro Phe Ile Trp Val Ile Pro 115 120 125 Gln Glu Val Ala Gln Arg Gly Ala His Ile Ser Val Glu Glu Asn Leu 130 135 140 Arg Arg Ile Pro Pro His Ser Val Asp Pro Thr Val Lys Asn Tyr His 145 150 155 160 Trp Gly Asp Met Thr Ala Ala Leu Phe Asn Ala Leu Asp Ala Gly Tyr 165 170 175 Asp Thr Thr Val Leu Leu Asp Thr Asp Gly Tyr Val Thr Glu Gly Pro 180 185 190 Gly Phe Asn Ile Phe Ala Val Ile Asp Gly Lys Val Leu Thr Pro Arg 195 200 205 Ser Gly Met Leu Glu Gly Ile Ser Arg Lys Thr Val Leu Glu Ile Cys 210 215 220 Ala Asp Leu Gly Ile Pro Cys Ala Gln Thr Asp Ile Ser Leu Asp Glu 225 230 235 240 Phe Leu Ser Ala Asp Glu Val Phe Thr Ala Thr Thr Ala Gly Gly Pro 245 250

255 Val Pro Val Thr Arg Val Asn Lys Thr Ile Leu Gly Asn Asp Ala Val 260 265 270 Gly Pro Ile Thr Ala Arg Leu Leu Lys Thr Tyr Trp Asp Trp His Asn 275 280 285 Arg Asp Asp Leu Thr Glu Lys Ile Thr Tyr Val 290 295 20900DNAArtificial sequenceCodon optimized sequence coding for SEQ ID NO 19 20atgacttact ccgcaggtgc tgcatggatg gacggtaaag ttatcccggt ttctgaagcg 60aagatctccg tattcgactg gggtctgact cgttctgaca tcacttacga cgttgttcac 120gtatgggaag gtgcattctt ccgtctggaa gactacctgg atcgcttcat ggtttccatg 180gacaagctgc gtctggacgt tggtatgact cgcgctgaaa tcaaagctgc gctggttgaa 240ctggttgcaa cttctggtct gaaatctgct tacgtttcca tggttgcttc tcgcggtact 300ccgcaggttc cgggtactcg tgacccgcgc gcttgcacca accacttcta cgcatgggcg 360gtaccgttca tctgggttat cccgcaggaa gttgctcagc gtggtgctca catctccgtt 420gaagaaaacc tgcgtcgtat cccgccgcac tctgttgatc cgaccgttaa aaactaccac 480tggggcgaca tgactgctgc gctgttcaac gcgctggatg ctggttacga cactaccgtt 540ctgctggata ctgacggtta cgttactgaa ggtccgggct tcaacatctt cgctgttatc 600gacggtaaag ttctgactcc gcgttctggt atgctggaag gtatctcccg taaaaccgtt 660ctggaaatct gcgcagacct gggtatccca tgcgcacaga ctgacatctc tctggacgag 720ttcctgtctg ctgacgaagt attcactgca accactgctg gcggtccggt accggtaact 780cgcgttaaca aaactattct gggtaacgac gctgttggtc cgatcactgc gcgtctgctg 840aaaacttact gggactggca caaccgtgac gacctgaccg agaagatcac ttacgtctaa 90021299PRTUnknownOrganism isolated from soil 21Met Thr Tyr Ala Thr Gly Ala Ala Trp Met Asp Gly Gln Val Ile Pro 1 5 10 15 Val Ser Glu Ala Lys Ile Ser Val Phe Asp Trp Gly Leu Thr Arg Ser 20 25 30 Asp Ile Thr Tyr Asp Val Val His Val Trp Glu Gly Ala Phe Phe Arg 35 40 45 Leu Glu Asp Tyr Leu Asp Arg Phe Thr Val Ser Met Asp Lys Leu Arg 50 55 60 Leu Asp Val Gly Met Asn Arg Ala Asp Ile Lys Ala Ala Leu Val Glu 65 70 75 80 Leu Val Ala Thr Ser Gly Leu Gln Ser Ala Tyr Val Ser Met Val Ala 85 90 95 Ser Arg Gly Thr Pro Leu Val Pro Gly Thr Arg Asp Pro Arg Ala Cys 100 105 110 Thr Asn His Phe Tyr Ala Trp Ala Val Pro Phe Ile Trp Val Ile Pro 115 120 125 Gln Glu Val Ala Gln Arg Gly Ala His Ile Ser Val Glu Glu Asn Leu 130 135 140 Arg Arg Ile Pro Pro His Ser Val Asp Pro Thr Val Lys Asn Tyr His 145 150 155 160 Trp Gly Asp Met Thr Ala Ala Leu Phe Asn Ala Leu Asp Ala Gly Tyr 165 170 175 Asp Thr Thr Val Leu Leu Asp Thr Asn Gly Tyr Ile Thr Glu Gly Pro 180 185 190 Gly Phe Asn Ile Phe Ala Val Ile Asp Gly Lys Val Leu Thr Pro Arg 195 200 205 Leu Gly Met Leu Glu Gly Ile Ser Arg Lys Thr Val Leu Glu Ile Cys 210 215 220 Ala Asp Leu Gly Ile Pro Cys Ala Glu Ala Asp Ile Ser Leu Ala Glu 225 230 235 240 Phe Leu Ser Ala Asp Glu Leu Phe Thr Ala Thr Thr Ala Gly Gly Pro 245 250 255 Val Pro Val Thr Arg Val Asn Lys Thr Ile Leu Gly Asn Asp Ala Ile 260 265 270 Gly Pro Ile Thr Ala Gln Val Leu Lys Thr Tyr Trp Asp Trp His His 275 280 285 Arg Asp Asp Leu Thr Glu Lys Ile Ala Tyr Ile 290 295 22900DNAArtificial sequenceCodon optimized sequence coding for SEQ ID NO 21 22atgacttacg caactggcgc tgcatggatg gacggtcagg ttatcccggt ttctgaagcg 60aagatctccg tattcgactg gggtctgact cgttctgaca tcacttacga cgttgttcac 120gtatgggaag gtgcgttctt ccgtctggaa gactacctgg atcgcttcac cgtttccatg 180gacaagctgc gtctggacgt tggtatgaac cgcgctgaca tcaaagcggc actggttgaa 240ctggttgcaa cttctggtct gcagtctgct tacgtttcca tggttgcttc tcgcggtact 300ccgctggtac cgggtactcg tgacccgcgc gcttgcacca accacttcta cgcatgggcg 360gtaccgttca tctgggttat cccgcaggaa gttgctcagc gtggtgcgca catctccgtt 420gaagaaaacc tgcgtcgtat cccgccgcac tctgttgatc cgaccgttaa gaactaccac 480tggggcgaca tgactgctgc gctgttcaac gcgctggatg ctggttacga cactaccgtt 540ctgctggata ccaacggtta catcactgaa ggtccgggct tcaacatctt cgctgttatc 600gacggtaaag ttctgactcc gcgtctgggt atgctggaag gtatctcccg taaaaccgtt 660ctggaaatct gcgcagacct gggtatccca tgcgcagaag ctgacatctc tctggctgag 720ttcctgtctg ctgacgaact gttcactgca accactgcag gtggtccggt accggtaact 780cgcgttaaca aaactattct gggtaacgac gctatcggtc cgatcactgc tcaggttctg 840aaaacttact gggactggca ccaccgtgac gacctgactg aaaaaatcgc ttacatttaa 90023326PRTUnknownOrganism isolated from soil 23Met Ala Ile Ile Gln Val Gln Gln Ile Met His Glu Asn Pro Leu His 1 5 10 15 Ala Arg Ala Pro His Glu Pro Arg Tyr Glu Asp Gly Ser Ala Phe Cys 20 25 30 Asp Gly Lys Tyr Val Pro Ile Ser Glu Ala Thr Val Pro Leu Val Asp 35 40 45 Ala Gly Phe Leu His Ala Asp Ala Ala Tyr Asp Val Val Thr Val Ser 50 55 60 Arg Gly Asn Phe Phe Arg Leu Asp Asp His Leu Ala Arg Met Glu Glu 65 70 75 80 Ser Ser Ala Lys Phe Phe Leu Glu Asn Pro Phe Asn Arg Asp Gln Val 85 90 95 Arg Glu Ile Leu His Asn Leu Val Arg Asn Ala Gly Leu Lys Asp Ala 100 105 110 Tyr Val Trp Trp Cys Val Thr Arg Gly Pro Leu Ser Val Asp Arg Arg 115 120 125 Asp Arg Ser Ala Met Lys Asn Ala Met Phe Ala Phe Ala Val Pro Phe 130 135 140 Phe Phe Gln Ala Asp Asp Glu Val Arg Thr Arg Gly Ser Asn Leu Leu 145 150 155 160 Ile Ser Lys Arg Tyr Asn Arg Ile Ser Ala Lys Ala Val Asp Pro Thr 165 170 175 Ala Lys Asn Phe His Trp Met Asp Met Lys Leu Ala Leu Phe Glu Ala 180 185 190 Met Thr Gln Glu Lys Asp Trp Ala Val Leu Val Asp Glu His Asp Asn 195 200 205 Leu Thr Glu Ala Ala Gly Ala Asn Val Phe Phe Val Lys Asn Gly Glu 210 215 220 Leu Tyr Thr Pro Ala Glu Gly Cys Leu Leu Gly Ile Thr Arg Gln Ser 225 230 235 240 Val Phe Asp Ile Ala Ala Glu Leu Gly Ile Lys Val Asn Ile Gly Lys 245 250 255 Tyr Thr Ala Thr Gln Leu Arg Glu Ala Asp Glu Ala Phe Thr Ser Ser 260 265 270 Ser Ala Gly Gly Ile Met Pro Val Ser Ala Val Asp Asp Gln Pro Leu 275 280 285 Gly Asn His Asn Gly Pro Gly Pro Ile Ser Glu Lys Ile His Asn Leu 290 295 300 Tyr Trp Glu Lys Arg Trp Ala Gly Trp His Ala Gln Pro Ala Glu Tyr 305 310 315 320 Phe Ser Ser Ile Pro Ala 325 24981DNAArtificial sequenceCodon optimized sequence coding for SEQ ID NO 23 24atggctatca tccaggttca gcagatcatg cacgaaaacc cgctgcacgc gcgtgctccg 60cacgaaccgc gctacgaaga cggttctgca ttctgcgacg gtaaatacgt tccgatttct 120gaagcaaccg ttccgctggt tgatgctggc ttcctgcacg ctgacgctgc ttacgacgtt 180gttaccgttt ctcgcggtaa cttcttccgt ctggatgacc acctggcgcg tatggaagag 240tcttctgcga aattcttcct ggaaaacccg ttcaaccgtg accaggttcg tgaaatcctg 300cacaacctgg ttcgtaacgc aggtctgaaa gacgcttacg tatggtggtg cgtaactcgc 360ggtccgctgt ctgttgaccg tcgtgaccgt tctgcgatga agaacgcaat gttcgctttc 420gctgttccgt tcttcttcca ggctgacgac gaagttcgta ctcgcggttc taacctgctg 480atctccaagc gctacaaccg tatctctgcg aaagctgttg atccgactgc gaaaaacttc 540cactggatgg acatgaagct ggcgctgttc gaagcgatga ctcaggaaaa agactgggcg 600gtactggttg acgaacacga caacctgact gaagctgcag gtgctaacgt attcttcgtt 660aaaaacggtg aactgtacac tccggcagaa ggttgcctgc tgggtatcac tcgtcagtct 720gtattcgaca tcgctgctga actgggtatc aaagttaaca tcggtaaata cactgcaact 780cagctgcgtg aagctgacga agcattcact tcttcttccg caggtggtat catgccggtt 840tctgctgttg atgaccagcc gctgggtaac cacaacggtc cgggtccaat ctctgagaag 900atccacaacc tgtactggga aaaacgctgg gctggctggc acgctcagcc ggcagaatac 960ttctcttcca tcccggcata a 98125326PRTBurkholderia sp. 383 25Met Ala Ile Ile Gln Val Gln Gln Ile Met His Glu Asn Pro Leu His 1 5 10 15 Ala Arg Ala Pro His Glu Pro Arg Tyr Glu Asp Gly Ser Ala Phe Cys 20 25 30 Asp Gly Asn Tyr Val Pro Ile Thr Glu Ala Thr Val Pro Leu Val Asp 35 40 45 Ala Gly Phe Leu His Ala Asp Ala Ala Tyr Asp Val Val Thr Val Ser 50 55 60 Arg Gly Asn Phe Phe Arg Leu Asp Asp His Leu Thr Arg Met Glu Glu 65 70 75 80 Ser Ser Ala Lys Phe Phe Leu Glu Asn Pro Phe Asn Arg Asp Gln Val 85 90 95 Lys Glu Ile Leu His Asn Leu Val Arg Asn Ala Gly Leu Lys Asp Ala 100 105 110 Tyr Val Trp Trp Cys Val Thr Arg Gly Pro Leu Ser Val Asp Arg Arg 115 120 125 Asp Arg Gly Ala Met Lys Asn Ala Met Phe Ala Phe Ala Val Pro Phe 130 135 140 Phe Phe Gln Ala Asp Asp Glu Val Arg Thr Arg Gly Ser Asn Leu Leu 145 150 155 160 Ile Ser Lys Leu Tyr Asn Arg Ile Ser Ala Lys Ala Val Asp Pro Thr 165 170 175 Ala Lys Asn Phe His Trp Met Asp Met Lys Leu Ala Leu Phe Glu Ala 180 185 190 Met Thr Gln Glu Lys Asp Trp Ala Val Leu Val Asp Glu Ser Asp Asn 195 200 205 Leu Thr Glu Ala Ala Gly Ala Asn Val Phe Phe Ala Lys Asn Gly Glu 210 215 220 Leu Tyr Thr Pro Ala Glu Gly Cys Leu Leu Gly Ile Thr Arg Gln Ser 225 230 235 240 Val Phe Asp Ile Ala Ala Glu Leu Gly Ile Lys Val Asn Ile Gly Lys 245 250 255 Tyr Thr Ala Thr Gln Leu Arg Glu Ala Asp Glu Ala Phe Thr Ser Ser 260 265 270 Ser Ala Gly Gly Ile Met Pro Val Ser Ala Ile Asp Asp Gln Pro Leu 275 280 285 Gly Asn Arg Asn Gly Pro Gly Pro Ile Ser Glu Lys Ile His Asn Leu 290 295 300 Tyr Trp Glu Lys Arg Trp Ala Gly Trp His Ala Gln Pro Ala Glu Tyr 305 310 315 320 Phe Ser Ser Val Pro Ala 325 26981DNAArtificial sequenceCodon optimized sequence coding for SEQ ID NO 25 26atggctatca tccaggttca gcagatcatg cacgaaaacc cgctgcacgc gcgtgctccg 60cacgaaccgc gctacgaaga cggttctgca ttctgcgacg gtaactacgt tccgatcact 120gaagcaaccg ttccgctggt tgatgctggc ttcctgcacg ctgacgctgc ttacgacgtt 180gttaccgttt ctcgcggtaa cttcttccgt ctggatgacc acctgactcg tatggaagag 240tcttctgcga aattcttcct ggaaaacccg ttcaaccgtg accaggttaa agaaatcctg 300cacaacctgg ttcgtaacgc aggtctgaaa gacgcttacg tatggtggtg cgtaactcgc 360ggtccgctgt ctgttgaccg tcgtgaccgc ggtgcgatga agaacgcaat gttcgctttc 420gctgttccgt tcttcttcca ggctgatgac gaagttcgta ctcgcggttc taacctgctg 480atctccaagc tgtacaaccg tatctctgcg aaagcggttg atccgactgc gaaaaacttc 540cactggatgg acatgaagct ggcgctgttc gaagcgatga ctcaggaaaa agactgggcg 600gtactggttg acgaatctga caacctgact gaagctgcag gtgctaacgt attcttcgct 660aaaaacggtg aactgtacac tccggcagaa ggttgcctgc tgggtatcac tcgtcagtct 720gtattcgaca tcgctgctga actgggtatc aaagttaaca tcggtaaata cactgcaact 780cagctgcgtg aagctgacga agcattcact tcttcttccg caggtggtat catgccggtt 840tctgctatcg acgaccagcc gctgggtaac cgtaacggtc cgggtccgat ttctgagaag 900atccacaacc tgtactggga aaaacgctgg gctggctggc acgctcagcc ggcagaatac 960ttctcttccg ttcccgcata a 98127283PRTBacillus sp. YM-1 27Met Gly Tyr Thr Leu Trp Asn Asp Gln Ile Val Lys Asp Glu Glu Val 1 5 10 15 Lys Ile Asp Lys Glu Asp Arg Gly Tyr Gln Phe Gly Asp Gly Val Tyr 20 25 30 Glu Val Val Lys Val Tyr Asn Gly Glu Met Phe Thr Val Asn Glu His 35 40 45 Ile Asp Arg Leu Tyr Ala Ser Ala Glu Lys Ile Arg Ile Thr Ile Pro 50 55 60 Tyr Thr Lys Asp Lys Phe His Gln Leu Leu His Glu Leu Val Glu Lys 65 70 75 80 Asn Glu Leu Asn Thr Gly His Ile Tyr Phe Gln Val Thr Arg Gly Thr 85 90 95 Ser Pro Arg Ala His Gln Phe Pro Glu Asn Thr Val Lys Pro Val Ile 100 105 110 Ile Gly Tyr Thr Lys Glu Asn Pro Arg Pro Leu Glu Asn Leu Glu Lys 115 120 125 Gly Val Lys Ala Thr Phe Val Glu Asp Ile Arg Trp Leu Arg Cys Asp 130 135 140 Ile Lys Ser Leu Asn Leu Leu Gly Ala Val Leu Ala Lys Gln Glu Ala 145 150 155 160 His Glu Lys Gly Cys Tyr Glu Ala Ile Leu His Arg Asn Asn Thr Val 165 170 175 Thr Glu Gly Ser Ser Ser Asn Val Phe Gly Ile Lys Asp Gly Ile Leu 180 185 190 Tyr Thr His Pro Ala Asn Asn Met Ile Leu Lys Gly Ile Thr Arg Asp 195 200 205 Val Val Ile Ala Cys Ala Asn Glu Ile Asn Met Pro Val Lys Glu Ile 210 215 220 Pro Phe Thr Thr His Glu Ala Leu Lys Met Asp Glu Leu Phe Val Thr 225 230 235 240 Ser Thr Thr Ser Glu Ile Thr Pro Val Ile Glu Ile Asp Gly Lys Leu 245 250 255 Ile Arg Asp Gly Lys Val Gly Glu Trp Thr Arg Lys Leu Gln Lys Gln 260 265 270 Phe Glu Thr Lys Ile Pro Lys Pro Leu His Ile 275 280 28852DNAArtificial sequenceCodon optimized sequence coding for SEQ ID NO 27 28atgggttaca ctctgtggaa cgaccagatc gttaaagacg aagaagttaa gatcgacaaa 60gaagaccgtg gttaccagtt cggtgacggt gtttacgaag ttgttaaagt ttacaacggt 120gaaatgttca ccgttaacga acacatcgac cgtctgtacg cttctgctga aaaaatccgt 180atcactatcc cgtacaccaa agacaaattc caccagctgc tgcacgaact ggttgaaaaa 240aacgaactga acaccggtca catctacttc caggtaactc gcggtacttc tccgcgcgct 300caccagttcc cggaaaacac cgttaagccg gttatcatcg gttacaccaa agagaacccg 360cgtccgctgg aaaacctgga aaaaggcgtt aaagcaactt tcgttgaaga tatccgctgg 420ctgcgctgcg acatcaagtc tctgaacctg ctgggtgcgg tactggcgaa gcaggaagca 480cacgaaaaag gctgctacga agctatcctg caccgtaaca acaccgtaac tgaaggttct 540tcttctaacg tattcggtat caaagacggt attctgtaca ctcacccggc taacaacatg 600atcctgaaag gtatcactcg tgacgttgtt atcgcttgcg caaacgaaat caacatgccg 660gttaaagaga tcccgttcac cactcacgaa gcgctgaaaa tggacgaact gttcgtaact 720tctaccactt ctgaaatcac tccggttatc gaaatcgacg gtaaactgat ccgtgacggt 780aaagttggcg agtggactcg taagctgcag aagcagttcg aaaccaagat cccgaagccg 840ctgcacatat aa 85229918DNACurtobacterium pusillumCDS(1)..(918) 29atg acc cgt gcg acc ctc ctg acc gtg acc gcg ccg acc cgt ccc gcc 48Met Thr Arg Ala Thr Leu Leu Thr Val Thr Ala Pro Thr Arg Pro Ala 1 5 10 15 tcg gcg gac gag cac ggg gcg gac cgt gcc gcc ggc gac gcc ggc ttc 96Ser Ala Asp Glu His Gly Ala Asp Arg Ala Ala Gly Asp Ala Gly Phe 20 25 30 gtc ctc gcc gac ttc ggc gca ccg cag gtc cgc atc acc gac ctc ggc 144Val Leu Ala Asp Phe Gly Ala Pro Gln Val Arg Ile Thr Asp Leu Gly 35 40 45 atc acg cgc ggc gac ggg gtg ttc gag acc atc gcc gtg atc gac ggg 192Ile Thr Arg Gly Asp Gly Val Phe Glu Thr Ile Ala Val Ile Asp Gly 50 55 60 cac ccg cag gcg ctg gaa ctg cac ctc gga cgg ctg gcc cac tcg gcg 240His Pro Gln Ala Leu Glu Leu His Leu Gly Arg Leu Ala His Ser Ala 65 70 75 80 gcg ctc ctc gac ctc ccc gaa ccg gat gcg gcg gtg tgg cgg gag gcc 288Ala Leu Leu Asp Leu Pro Glu Pro Asp Ala Ala Val Trp Arg Glu Ala 85 90 95 gtc ctc gcg ggc gtg gcg gac tac cgg tcc cgc aac ggc gac ggc ggc 336Val Leu Ala Gly Val Ala Asp Tyr Arg Ser Arg Asn Gly Asp Gly Gly 100 105 110 gaa ctg ttc gcc aag ctc atc ctg acc cgc ggc atc gag ggc gag ggc 384Glu Leu Phe Ala Lys Leu Ile Leu Thr Arg Gly Ile Glu Gly Glu Gly 115 120 125

cgg ccg agc ggg tgg gtg ttc gtg gac gag ggc gag gac ttc tcg cag 432Arg Pro Ser Gly Trp Val Phe Val Asp Glu Gly Glu Asp Phe Ser Gln 130 135 140 cag cgc ctc ggg atc cgc gtc gtc acg ctc gac cgc ggc tac cgt cac 480Gln Arg Leu Gly Ile Arg Val Val Thr Leu Asp Arg Gly Tyr Arg His 145 150 155 160 gac gtg gcg gag acg tcc ccg tgg ctg ctg gcc gga gcg aag tcc ctg 528Asp Val Ala Glu Thr Ser Pro Trp Leu Leu Ala Gly Ala Lys Ser Leu 165 170 175 tcg tac gcg acc aat cgc gcc gcc ggc cgg gag gcg gcc cgc cgg ggc 576Ser Tyr Ala Thr Asn Arg Ala Ala Gly Arg Glu Ala Ala Arg Arg Gly 180 185 190 gcc gac gac gtg atc ttc gtc agc tcc gac gga tac gca ctg gag ggg 624Ala Asp Asp Val Ile Phe Val Ser Ser Asp Gly Tyr Ala Leu Glu Gly 195 200 205 ccg acc tcg aac gtc atc gtg ctt gcg gac ggc gtc gtg cgc acc ccg 672Pro Thr Ser Asn Val Ile Val Leu Ala Asp Gly Val Val Arg Thr Pro 210 215 220 cag acg gac cag ggc atc ctg gcc ggc acc acc cag gcg gcc gtg ttc 720Gln Thr Asp Gln Gly Ile Leu Ala Gly Thr Thr Gln Ala Ala Val Phe 225 230 235 240 gac ttc ttc gag gag cgc ggc tac ccc acc gag tac cgc cgc atc tcc 768Asp Phe Phe Glu Glu Arg Gly Tyr Pro Thr Glu Tyr Arg Arg Ile Ser 245 250 255 gcg gac gag ctg cgc gac gcg gag gcg ctg tgg ctc gtc tcc agc gtg 816Ala Asp Glu Leu Arg Asp Ala Glu Ala Leu Trp Leu Val Ser Ser Val 260 265 270 cgc cag gcc gca ccg atc acg gcg ctc gac gac cgc gag tac ccg gtc 864Arg Gln Ala Ala Pro Ile Thr Ala Leu Asp Asp Arg Glu Tyr Pro Val 275 280 285 gac gcg gcg ctc acg gcc gac ctg aac gcg tac ctg ctc gcc cgc acc 912Asp Ala Ala Leu Thr Ala Asp Leu Asn Ala Tyr Leu Leu Ala Arg Thr 290 295 300 gac tga 918Asp 305 30305PRTCurtobacterium pusillum 30Met Thr Arg Ala Thr Leu Leu Thr Val Thr Ala Pro Thr Arg Pro Ala 1 5 10 15 Ser Ala Asp Glu His Gly Ala Asp Arg Ala Ala Gly Asp Ala Gly Phe 20 25 30 Val Leu Ala Asp Phe Gly Ala Pro Gln Val Arg Ile Thr Asp Leu Gly 35 40 45 Ile Thr Arg Gly Asp Gly Val Phe Glu Thr Ile Ala Val Ile Asp Gly 50 55 60 His Pro Gln Ala Leu Glu Leu His Leu Gly Arg Leu Ala His Ser Ala 65 70 75 80 Ala Leu Leu Asp Leu Pro Glu Pro Asp Ala Ala Val Trp Arg Glu Ala 85 90 95 Val Leu Ala Gly Val Ala Asp Tyr Arg Ser Arg Asn Gly Asp Gly Gly 100 105 110 Glu Leu Phe Ala Lys Leu Ile Leu Thr Arg Gly Ile Glu Gly Glu Gly 115 120 125 Arg Pro Ser Gly Trp Val Phe Val Asp Glu Gly Glu Asp Phe Ser Gln 130 135 140 Gln Arg Leu Gly Ile Arg Val Val Thr Leu Asp Arg Gly Tyr Arg His 145 150 155 160 Asp Val Ala Glu Thr Ser Pro Trp Leu Leu Ala Gly Ala Lys Ser Leu 165 170 175 Ser Tyr Ala Thr Asn Arg Ala Ala Gly Arg Glu Ala Ala Arg Arg Gly 180 185 190 Ala Asp Asp Val Ile Phe Val Ser Ser Asp Gly Tyr Ala Leu Glu Gly 195 200 205 Pro Thr Ser Asn Val Ile Val Leu Ala Asp Gly Val Val Arg Thr Pro 210 215 220 Gln Thr Asp Gln Gly Ile Leu Ala Gly Thr Thr Gln Ala Ala Val Phe 225 230 235 240 Asp Phe Phe Glu Glu Arg Gly Tyr Pro Thr Glu Tyr Arg Arg Ile Ser 245 250 255 Ala Asp Glu Leu Arg Asp Ala Glu Ala Leu Trp Leu Val Ser Ser Val 260 265 270 Arg Gln Ala Ala Pro Ile Thr Ala Leu Asp Asp Arg Glu Tyr Pro Val 275 280 285 Asp Ala Ala Leu Thr Ala Asp Leu Asn Ala Tyr Leu Leu Ala Arg Thr 290 295 300 Asp 305 31918DNAArtificialCodon-optimized sequence encoding SEQ ID NO 30 31atgacccgtg caaccctgct gaccgttacc gcaccgaccc gtccggcaag cgcagatgaa 60catggtgcag atcgtgcagc cggtgatgca ggttttgttc tggcagattt tggtgcaccg 120caggttcgta ttaccgatct gggtattacc cgtggtgatg gtgtttttga aaccattgca 180gttattgatg gtcatccgca ggcactggaa ctgcatctgg gtcgtctggc acatagcgca 240gcactgctgg atctgccgga accggatgca gcagtttggc gtgaagccgt gctggcaggc 300gttgcagatt atcgtagccg taatggtgat ggcggtgaac tgtttgcaaa actgattctg 360acccgtggta ttgaaggtga aggtcgtccg agcggttggg tttttgttga tgaaggcgaa 420gattttagcc agcagcgcct gggtattcgt gttgttaccc tggatcgtgg ttatcgtcat 480gatgttgcag aaaccagccc gtggctgctg gctggtgcaa aaagcctgag ctatgcaacc 540aatcgtgccg caggtcgtga agcagcacgt cgtggtgccg atgatgttat ttttgttagc 600agtgatggtt atgcactgga aggtccgacc agcaatgtta ttgtgctggc cgatggtgtt 660gttcgtacac cgcagaccga tcagggcatt ctggcaggca ccacccaggc agccgttttt 720gatttttttg aagaacgcgg ttatccgacc gaatatcgtc gtatttcagc cgatgaactg 780cgtgatgccg aagcactgtg gctggttagc agcgttcgtc aggcagcacc gattacagca 840ctggatgatc gtgaatatcc ggttgatgca gcactgaccg cagatctgaa tgcatatctg 900ctggcacgta ccgattaa 918321131DNACurtobacterium pusillumCDS(1)..(1131) 32atg act tct acc agc atc tcc ctt acc agc ggc ccc acc gag acg agc 48Met Thr Ser Thr Ser Ile Ser Leu Thr Ser Gly Pro Thr Glu Thr Ser 1 5 10 15 ggc ctg ctc tgg cag gtc acc cgc aac gag gcg gcg cgc gac gcc gcc 96Gly Leu Leu Trp Gln Val Thr Arg Asn Glu Ala Ala Arg Asp Ala Ala 20 25 30 gag cgc gag gag atc ctc gcc gat ccc ggt ttc ggc aac cac ttc acc 144Glu Arg Glu Glu Ile Leu Ala Asp Pro Gly Phe Gly Asn His Phe Thr 35 40 45 gac cac atg gtc gac atc tgc tgg tcg gcg aag gga ggc tgg cac cgg 192Asp His Met Val Asp Ile Cys Trp Ser Ala Lys Gly Gly Trp His Arg 50 55 60 ccg cgc gtc tcg ccg tac ggc ccg atc cag ctc gac ccg tcg gcc gcc 240Pro Arg Val Ser Pro Tyr Gly Pro Ile Gln Leu Asp Pro Ser Ala Ala 65 70 75 80 gtg ctg cac tac gcg cag gag atc ttc gag ggg ctg aag gcg tac cgc 288Val Leu His Tyr Ala Gln Glu Ile Phe Glu Gly Leu Lys Ala Tyr Arg 85 90 95 cac gag gac ggc tcg atc tgg aca ttc cgc ccg gag gcc aac gcc gcc 336His Glu Asp Gly Ser Ile Trp Thr Phe Arg Pro Glu Ala Asn Ala Ala 100 105 110 cgc atg cag cgc tcg gcc tat cgc ctg gcg ctg ccc gag ctc ccg gtc 384Arg Met Gln Arg Ser Ala Tyr Arg Leu Ala Leu Pro Glu Leu Pro Val 115 120 125 gag cac ttc ctc gac tcg ttg aag cag ctc gtc gcg gtg gac ggc gat 432Glu His Phe Leu Asp Ser Leu Lys Gln Leu Val Ala Val Asp Gly Asp 130 135 140 tgg gtg ccg acc gcg ccg gag acc agc ctc tac ctg cgt ccg ttc atg 480Trp Val Pro Thr Ala Pro Glu Thr Ser Leu Tyr Leu Arg Pro Phe Met 145 150 155 160 ttc gcc aag gag gcg ttc ctg ggc gtg cgc ccg gcg aac aag gtc gcg 528Phe Ala Lys Glu Ala Phe Leu Gly Val Arg Pro Ala Asn Lys Val Ala 165 170 175 tac tac ctg atc gcg agc ccg gcg ggc gcc tat ttc tcg ggc ggc gtc 576Tyr Tyr Leu Ile Ala Ser Pro Ala Gly Ala Tyr Phe Ser Gly Gly Val 180 185 190 gca ccc gtc tcg atc tgg ctg tcc gac cgc tgg tcg cgc gcc ggc cac 624Ala Pro Val Ser Ile Trp Leu Ser Asp Arg Trp Ser Arg Ala Gly His 195 200 205 ggt ggc acc ggg gcg gcg aag acc ggc ggc aac tac gcc tcc agc ctc 672Gly Gly Thr Gly Ala Ala Lys Thr Gly Gly Asn Tyr Ala Ser Ser Leu 210 215 220 ctg cct cag gcc gag gcg gcc gag cac ggc tgc gca cag gtg ctg ttc 720Leu Pro Gln Ala Glu Ala Ala Glu His Gly Cys Ala Gln Val Leu Phe 225 230 235 240 ctc gac tcg gtc gaa ggc cga tac ctc gag gag ctc ggc ggg atg aac 768Leu Asp Ser Val Glu Gly Arg Tyr Leu Glu Glu Leu Gly Gly Met Asn 245 250 255 gtg gtg ctc gtc tac aag gac ggc acg gtg gtg acc ccg gag tcc gac 816Val Val Leu Val Tyr Lys Asp Gly Thr Val Val Thr Pro Glu Ser Asp 260 265 270 agc atc ctg gag ggc atc acg ctg gac tcg atc ctg cag ctc gcg cgt 864Ser Ile Leu Glu Gly Ile Thr Leu Asp Ser Ile Leu Gln Leu Ala Arg 275 280 285 gat cgc ggc cac cgg gtc gag cgc cgc cgg gtg acg atc gac gag tgg 912Asp Arg Gly His Arg Val Glu Arg Arg Arg Val Thr Ile Asp Glu Trp 290 295 300 cgc gac ggc gtc gag agc ggc gac atc gtc gag gtg ttc gcc tgc ggc 960Arg Asp Gly Val Glu Ser Gly Asp Ile Val Glu Val Phe Ala Cys Gly 305 310 315 320 acg gcc gcg gtg atc acc ccg atc ggc gag ctc aag tcg gac acc ttc 1008Thr Ala Ala Val Ile Thr Pro Ile Gly Glu Leu Lys Ser Asp Thr Phe 325 330 335 acc gtc ggc gac atc acc gcg cct ccc ggt gag ctg acg atg gcg ctg 1056Thr Val Gly Asp Ile Thr Ala Pro Pro Gly Glu Leu Thr Met Ala Leu 340 345 350 cgc cag gag ctc acc gac atc cag tac ggc cgc gtc cac gac cgg cac 1104Arg Gln Glu Leu Thr Asp Ile Gln Tyr Gly Arg Val His Asp Arg His 355 360 365 aac tgg atg acg cgc ctc gac gcg tag 1131Asn Trp Met Thr Arg Leu Asp Ala 370 375 33376PRTCurtobacterium pusillum 33Met Thr Ser Thr Ser Ile Ser Leu Thr Ser Gly Pro Thr Glu Thr Ser 1 5 10 15 Gly Leu Leu Trp Gln Val Thr Arg Asn Glu Ala Ala Arg Asp Ala Ala 20 25 30 Glu Arg Glu Glu Ile Leu Ala Asp Pro Gly Phe Gly Asn His Phe Thr 35 40 45 Asp His Met Val Asp Ile Cys Trp Ser Ala Lys Gly Gly Trp His Arg 50 55 60 Pro Arg Val Ser Pro Tyr Gly Pro Ile Gln Leu Asp Pro Ser Ala Ala 65 70 75 80 Val Leu His Tyr Ala Gln Glu Ile Phe Glu Gly Leu Lys Ala Tyr Arg 85 90 95 His Glu Asp Gly Ser Ile Trp Thr Phe Arg Pro Glu Ala Asn Ala Ala 100 105 110 Arg Met Gln Arg Ser Ala Tyr Arg Leu Ala Leu Pro Glu Leu Pro Val 115 120 125 Glu His Phe Leu Asp Ser Leu Lys Gln Leu Val Ala Val Asp Gly Asp 130 135 140 Trp Val Pro Thr Ala Pro Glu Thr Ser Leu Tyr Leu Arg Pro Phe Met 145 150 155 160 Phe Ala Lys Glu Ala Phe Leu Gly Val Arg Pro Ala Asn Lys Val Ala 165 170 175 Tyr Tyr Leu Ile Ala Ser Pro Ala Gly Ala Tyr Phe Ser Gly Gly Val 180 185 190 Ala Pro Val Ser Ile Trp Leu Ser Asp Arg Trp Ser Arg Ala Gly His 195 200 205 Gly Gly Thr Gly Ala Ala Lys Thr Gly Gly Asn Tyr Ala Ser Ser Leu 210 215 220 Leu Pro Gln Ala Glu Ala Ala Glu His Gly Cys Ala Gln Val Leu Phe 225 230 235 240 Leu Asp Ser Val Glu Gly Arg Tyr Leu Glu Glu Leu Gly Gly Met Asn 245 250 255 Val Val Leu Val Tyr Lys Asp Gly Thr Val Val Thr Pro Glu Ser Asp 260 265 270 Ser Ile Leu Glu Gly Ile Thr Leu Asp Ser Ile Leu Gln Leu Ala Arg 275 280 285 Asp Arg Gly His Arg Val Glu Arg Arg Arg Val Thr Ile Asp Glu Trp 290 295 300 Arg Asp Gly Val Glu Ser Gly Asp Ile Val Glu Val Phe Ala Cys Gly 305 310 315 320 Thr Ala Ala Val Ile Thr Pro Ile Gly Glu Leu Lys Ser Asp Thr Phe 325 330 335 Thr Val Gly Asp Ile Thr Ala Pro Pro Gly Glu Leu Thr Met Ala Leu 340 345 350 Arg Gln Glu Leu Thr Asp Ile Gln Tyr Gly Arg Val His Asp Arg His 355 360 365 Asn Trp Met Thr Arg Leu Asp Ala 370 375 34795DNACurtobacterium pusillumCDS(1)..(795) 34atg gac gcg agc agc acc ctc ttc gac tgg gcg ggc ggc gag ctc gtc 48Met Asp Ala Ser Ser Thr Leu Phe Asp Trp Ala Gly Gly Glu Leu Val 1 5 10 15 gcc cgg gac tcc tgc gag gtc gcc gag acc gcg ctg ctc gtc gcg gac 96Ala Arg Asp Ser Cys Glu Val Ala Glu Thr Ala Leu Leu Val Ala Asp 20 25 30 tcc ttc ctc gtc gcc gac ggc acc gct ctc gcc ctc ggt ctg cac ggc 144Ser Phe Leu Val Ala Asp Gly Thr Ala Leu Ala Leu Gly Leu His Gly 35 40 45 tcc cgg ttc cag gac tcc gcg cgt ctg cag ggg cac ccc gac cga gcg 192Ser Arg Phe Gln Asp Ser Ala Arg Leu Gln Gly His Pro Asp Arg Ala 50 55 60 gag ctg cag cgg ttc tgg gag gcg ggc gtc gcc gcg ctg ccg cgc acc 240Glu Leu Gln Arg Phe Trp Glu Ala Gly Val Ala Ala Leu Pro Arg Thr 65 70 75 80 ggc gcc tgg ttc ccg cgg ttc gag ctg gtg cgc acc cgc gac gcg ctg 288Gly Ala Trp Phe Pro Arg Phe Glu Leu Val Arg Thr Arg Asp Ala Leu 85 90 95 cgg ttg cga ttc cgg ttg cgc acc gcg ccg gcg ttg acg agc gag ctg 336Arg Leu Arg Phe Arg Leu Arg Thr Ala Pro Ala Leu Thr Ser Glu Leu 100 105 110 gtc gtc gcc acg gcg gcc tcc gac ccg cga cgg gct ccc gac atc aaa 384Val Val Ala Thr Ala Ala Ser Asp Pro Arg Arg Ala Pro Asp Ile Lys 115 120 125 ggg ccc gac atc gac cgg ctg tcg gtg ctg cgc cag cgc gca cag gcg 432Gly Pro Asp Ile Asp Arg Leu Ser Val Leu Arg Gln Arg Ala Gln Ala 130 135 140 gcc ggc gcc cag gag gcg atc ctg ctc gac gag gga ttc gtg gcc gac 480Ala Gly Ala Gln Glu Ala Ile Leu Leu Asp Glu Gly Phe Val Ala Asp 145 150 155 160 ggt gcg acc acc gcc ctg ctc tgg tgg cgc ggc gac acg ctc tac acg 528Gly Ala Thr Thr Ala Leu Leu Trp Trp Arg Gly Asp Thr Leu Tyr Thr 165 170 175 ccc ccg ctg tcc ctg ccc cgg gtg gac agc gtc gcc gcg cgc acc gtc 576Pro Pro Leu Ser Leu Pro Arg Val Asp Ser Val Ala Ala Arg Thr Val 180 185 190 cgc ggc atc gcc gcc gcc ctg cgg gtg ccg gtc gac gag gag gag gcg 624Arg Gly Ile Ala Ala Ala Leu Arg Val Pro Val Asp Glu Glu Glu Ala 195 200 205 cgc ccc gcg cag ctg gac ggc gtg acg ctc tgg gcc gtc aac gcc ctg 672Arg Pro Ala Gln Leu Asp Gly Val Thr Leu Trp Ala Val Asn Ala Leu 210 215 220 cac ggc atc cgg gcc gtc acg gcc tgg gtg gac ggc ccc gga ctg tcg 720His Gly Ile Arg Ala Val Thr Ala Trp Val Asp Gly Pro Gly Leu Ser 225 230 235 240 cag gat ccg gca cgc acg gac gcg tgg cgc gcc cgc ttc gcg atg ctg 768Gln Asp Pro Ala Arg Thr Asp Ala Trp Arg Ala Arg Phe Ala Met Leu 245 250 255 tcg cgt ccg ctg ccg ctc gca gcc tga 795Ser Arg Pro Leu Pro Leu Ala Ala

260 35264PRTCurtobacterium pusillum 35Met Asp Ala Ser Ser Thr Leu Phe Asp Trp Ala Gly Gly Glu Leu Val 1 5 10 15 Ala Arg Asp Ser Cys Glu Val Ala Glu Thr Ala Leu Leu Val Ala Asp 20 25 30 Ser Phe Leu Val Ala Asp Gly Thr Ala Leu Ala Leu Gly Leu His Gly 35 40 45 Ser Arg Phe Gln Asp Ser Ala Arg Leu Gln Gly His Pro Asp Arg Ala 50 55 60 Glu Leu Gln Arg Phe Trp Glu Ala Gly Val Ala Ala Leu Pro Arg Thr 65 70 75 80 Gly Ala Trp Phe Pro Arg Phe Glu Leu Val Arg Thr Arg Asp Ala Leu 85 90 95 Arg Leu Arg Phe Arg Leu Arg Thr Ala Pro Ala Leu Thr Ser Glu Leu 100 105 110 Val Val Ala Thr Ala Ala Ser Asp Pro Arg Arg Ala Pro Asp Ile Lys 115 120 125 Gly Pro Asp Ile Asp Arg Leu Ser Val Leu Arg Gln Arg Ala Gln Ala 130 135 140 Ala Gly Ala Gln Glu Ala Ile Leu Leu Asp Glu Gly Phe Val Ala Asp 145 150 155 160 Gly Ala Thr Thr Ala Leu Leu Trp Trp Arg Gly Asp Thr Leu Tyr Thr 165 170 175 Pro Pro Leu Ser Leu Pro Arg Val Asp Ser Val Ala Ala Arg Thr Val 180 185 190 Arg Gly Ile Ala Ala Ala Leu Arg Val Pro Val Asp Glu Glu Glu Ala 195 200 205 Arg Pro Ala Gln Leu Asp Gly Val Thr Leu Trp Ala Val Asn Ala Leu 210 215 220 His Gly Ile Arg Ala Val Thr Ala Trp Val Asp Gly Pro Gly Leu Ser 225 230 235 240 Gln Asp Pro Ala Arg Thr Asp Ala Trp Arg Ala Arg Phe Ala Met Leu 245 250 255 Ser Arg Pro Leu Pro Leu Ala Ala 260 36795DNAArtificialCodon optimized sequence of SEQ ID NO 35 36atggatgcaa gcagcaccct gtttgattgg gcaggcggtg aactggttgc acgtgatagc 60tgtgaagttg cagaaaccgc actgctggtt gcagatagct ttctggttgc cgatggcacc 120gcactggcac tgggtctgca tggtagccgt tttcaggata gcgcacgtct gcagggtcat 180ccggatcgtg cagaactgca gcgtttttgg gaagccggtg ttgcagcact gcctcgtacc 240ggtgcatggt ttccgcgttt tgaactggtg cgtacccgtg atgcactgcg tctgcgtttt 300cgtctgcgta cagcaccggc actgaccagc gaactggtgg ttgccaccgc agcaagcgat 360ccgcgtcgtg caccggatat taaaggtccg gatattgatc gtctgagcgt tctgcgtcag 420cgtgcacagg cagccggtgc acaagaggca attctgctgg atgaaggttt tgttgcagat 480ggtgcaacca cagccctgct gtggtggcgt ggtgataccc tgtatacccc tccgctgagc 540ctgcctcgtg ttgatagcgt tgcagcccgt accgttcgtg gtattgcagc agccctgcgt 600gttccggttg atgaagaaga agcacgtccg gcacagctgg atggtgtgac cctgtgggca 660gttaatgcac tgcatggcat tcgtgcagtt accgcatggg ttgatggtcc gggtctgagc 720caggatccgg cacgtaccga tgcctggcgt gcacgttttg caatgctgag ccgtccgctg 780ccgctggcag cataa 79537930DNARahnella aquatilisCDS(1)..(930) 37atg acg aag aaa gct gat tac att tgg ttc aac ggc gag atg gtt cca 48Met Thr Lys Lys Ala Asp Tyr Ile Trp Phe Asn Gly Glu Met Val Pro 1 5 10 15 tgg gca gaa gct aaa gtc cat gtc atg tca cac gca ctg cat tac ggc 96Trp Ala Glu Ala Lys Val His Val Met Ser His Ala Leu His Tyr Gly 20 25 30 act tcc gtt ttc gaa ggc gtg cgt tgc tac gac tcc cat aaa ggc cca 144Thr Ser Val Phe Glu Gly Val Arg Cys Tyr Asp Ser His Lys Gly Pro 35 40 45 gtc gta ttc cgt cac cgt gaa cat atg cag cgt ctg cgc gat tcc gca 192Val Val Phe Arg His Arg Glu His Met Gln Arg Leu Arg Asp Ser Ala 50 55 60 aaa att tac cgt atg cct gtt tcc cag agt gtg gat gag ctg atg gaa 240Lys Ile Tyr Arg Met Pro Val Ser Gln Ser Val Asp Glu Leu Met Glu 65 70 75 80 gct tgc cgc gaa acc ctg cgt aaa aac aat ctg gtc agc gcg tat atc 288Ala Cys Arg Glu Thr Leu Arg Lys Asn Asn Leu Val Ser Ala Tyr Ile 85 90 95 cgt ccg ctg gtg ttt gtc ggc gat gtg ggt atg ggc gtt aat ccg ccg 336Arg Pro Leu Val Phe Val Gly Asp Val Gly Met Gly Val Asn Pro Pro 100 105 110 gac ggc tac aaa act gat gtg atc atc gcc gcc ttc ccg tgg ggc gcg 384Asp Gly Tyr Lys Thr Asp Val Ile Ile Ala Ala Phe Pro Trp Gly Ala 115 120 125 tat ctg ggt gaa gaa gcg ctg gag cag ggt atc gac gcg atg gtg tct 432Tyr Leu Gly Glu Glu Ala Leu Glu Gln Gly Ile Asp Ala Met Val Ser 130 135 140 tca tgg aac cgc gtt gct gca aac acc att cca acc gct gcg aaa gcc 480Ser Trp Asn Arg Val Ala Ala Asn Thr Ile Pro Thr Ala Ala Lys Ala 145 150 155 160 ggt ggt aac tac ctg tcc tcc ctg ctg gtc ggc agc gaa gca cgt cgt 528Gly Gly Asn Tyr Leu Ser Ser Leu Leu Val Gly Ser Glu Ala Arg Arg 165 170 175 cac ggt tat cag gaa ggt atc gcg ctg gac att cac ggc tat gtg tct 576His Gly Tyr Gln Glu Gly Ile Ala Leu Asp Ile His Gly Tyr Val Ser 180 185 190 gaa ggc gct ggc gaa aac ctg ttt gaa gtg aaa gaa ggc att ctg ttc 624Glu Gly Ala Gly Glu Asn Leu Phe Glu Val Lys Glu Gly Ile Leu Phe 195 200 205 aca ccg cca ttt acc tct tct gcc ctg cca ggt atc acc cgt gac gct 672Thr Pro Pro Phe Thr Ser Ser Ala Leu Pro Gly Ile Thr Arg Asp Ala 210 215 220 att att aaa ctg gca aaa gac ctg ggt ctt gaa gtg cgt gag caa gtt 720Ile Ile Lys Leu Ala Lys Asp Leu Gly Leu Glu Val Arg Glu Gln Val 225 230 235 240 ctg tcc cgt gaa tcc ctg tat ctg gca gac gaa gtc ttc atg tcc ggt 768Leu Ser Arg Glu Ser Leu Tyr Leu Ala Asp Glu Val Phe Met Ser Gly 245 250 255 acc gct gca gaa atc acc ccg gtg cgc agc gtt gac ggc att cag gtc 816Thr Ala Ala Glu Ile Thr Pro Val Arg Ser Val Asp Gly Ile Gln Val 260 265 270 ggt atc ggt aaa cgt ggt ccg gtg acc aaa caa att cag gat gca ttc 864Gly Ile Gly Lys Arg Gly Pro Val Thr Lys Gln Ile Gln Asp Ala Phe 275 280 285 ttc ggc ctg ttc acc ggc aaa acc gaa gat aaa tgg ggt tgg ctg gat 912Phe Gly Leu Phe Thr Gly Lys Thr Glu Asp Lys Trp Gly Trp Leu Asp 290 295 300 cca atc aac cca caa taa 930Pro Ile Asn Pro Gln 305 38309PRTRahnella aquatilis 38Met Thr Lys Lys Ala Asp Tyr Ile Trp Phe Asn Gly Glu Met Val Pro 1 5 10 15 Trp Ala Glu Ala Lys Val His Val Met Ser His Ala Leu His Tyr Gly 20 25 30 Thr Ser Val Phe Glu Gly Val Arg Cys Tyr Asp Ser His Lys Gly Pro 35 40 45 Val Val Phe Arg His Arg Glu His Met Gln Arg Leu Arg Asp Ser Ala 50 55 60 Lys Ile Tyr Arg Met Pro Val Ser Gln Ser Val Asp Glu Leu Met Glu 65 70 75 80 Ala Cys Arg Glu Thr Leu Arg Lys Asn Asn Leu Val Ser Ala Tyr Ile 85 90 95 Arg Pro Leu Val Phe Val Gly Asp Val Gly Met Gly Val Asn Pro Pro 100 105 110 Asp Gly Tyr Lys Thr Asp Val Ile Ile Ala Ala Phe Pro Trp Gly Ala 115 120 125 Tyr Leu Gly Glu Glu Ala Leu Glu Gln Gly Ile Asp Ala Met Val Ser 130 135 140 Ser Trp Asn Arg Val Ala Ala Asn Thr Ile Pro Thr Ala Ala Lys Ala 145 150 155 160 Gly Gly Asn Tyr Leu Ser Ser Leu Leu Val Gly Ser Glu Ala Arg Arg 165 170 175 His Gly Tyr Gln Glu Gly Ile Ala Leu Asp Ile His Gly Tyr Val Ser 180 185 190 Glu Gly Ala Gly Glu Asn Leu Phe Glu Val Lys Glu Gly Ile Leu Phe 195 200 205 Thr Pro Pro Phe Thr Ser Ser Ala Leu Pro Gly Ile Thr Arg Asp Ala 210 215 220 Ile Ile Lys Leu Ala Lys Asp Leu Gly Leu Glu Val Arg Glu Gln Val 225 230 235 240 Leu Ser Arg Glu Ser Leu Tyr Leu Ala Asp Glu Val Phe Met Ser Gly 245 250 255 Thr Ala Ala Glu Ile Thr Pro Val Arg Ser Val Asp Gly Ile Gln Val 260 265 270 Gly Ile Gly Lys Arg Gly Pro Val Thr Lys Gln Ile Gln Asp Ala Phe 275 280 285 Phe Gly Leu Phe Thr Gly Lys Thr Glu Asp Lys Trp Gly Trp Leu Asp 290 295 300 Pro Ile Asn Pro Gln 305 39930DNAArtificialCodon-optimized sequence coding for SEQ ID NO 38 39atgaccaaaa aagccgacta catttggttt aatggtgaaa tggttccgtg ggctgaagca 60aaagttcatg ttatgagcca tgcactgcat tatggcacca gcgtttttga aggtgttcgt 120tgttatgata gccataaagg tccggttgtt tttcgtcatc gtgaacacat gcagcgtctg 180cgtgatagcg caaaaatcta tcgtatgccg gttagccaga gcgttgatga actgatggaa 240gcatgtcgtg aaaccctgcg taaaaacaat ctggttagcg catatattcg tccgctggtt 300tttgttggtg atgttggtat gggtgttaat ccgcctgatg gttataaaac cgatgttatt 360attgcagcat ttccgtgggg tgcatatctg ggtgaagaag cactggaaca gggtattgat 420gcaatggtta gcagctggaa tcgtgttgca gcaaatacca ttccgaccgc agcaaaagcc 480ggtggtaatt atctgagcag cctgctggtt ggtagcgaag cacgtcgtca tggttatcaa 540gaaggtattg cactggatat tcatggctat gttagcgaag gtgccggtga aaacctgttt 600gaagttaaag aaggcattct gtttacccca ccgtttacca gcagcgcact gcctggtatt 660acccgtgatg caattatcaa actggcaaaa gatctgggtc tggaagttcg tgaacaggtt 720ctgagccgtg aaagcctgta tctggcagat gaagttttta tgagcggcac cgcagcagaa 780attacaccgg ttcgtagcgt ggatggtatt caggttggta ttggtaaacg tggtccggtt 840accaaacaaa ttcaggatgc attttttggc ctgtttaccg gcaaaaccga agataaatgg 900ggttggctgg atccgattaa tccgcagtaa 93040798DNARahnella aquatilisCDS(1)..(798) 40atg tgg att aat ggt gtg gcg gcc gcg atg ttg tcg gca agt gac cgt 48Met Trp Ile Asn Gly Val Ala Ala Ala Met Leu Ser Ala Ser Asp Arg 1 5 10 15 tca gtg cag ttt ggc gac ggt tgc ttt acc aca gcc agg gtg tca gac 96Ser Val Gln Phe Gly Asp Gly Cys Phe Thr Thr Ala Arg Val Ser Asp 20 25 30 ggt gtg att gtg ttt ctg gcc ggg cat att cag cgt ctg caa cgt gct 144Gly Val Ile Val Phe Leu Ala Gly His Ile Gln Arg Leu Gln Arg Ala 35 40 45 gcg tcg gta ctg cgg att gaa ggt gtg gac tgg acg gct ctg gaa cag 192Ala Ser Val Leu Arg Ile Glu Gly Val Asp Trp Thr Ala Leu Glu Gln 50 55 60 gaa atg gtt ctg gct gcc gga cag cag aaa gag gca gtg gtt aaa gcc 240Glu Met Val Leu Ala Ala Gly Gln Gln Lys Glu Ala Val Val Lys Ala 65 70 75 80 gtc gtg acg cgc ggg cag ggc ggc cga ggt tac agc gcc gca ggt tgc 288Val Val Thr Arg Gly Gln Gly Gly Arg Gly Tyr Ser Ala Ala Gly Cys 85 90 95 tct gcg ccg acg cgt att gtt tct gcg tct gat tat ccg gtg cat tat 336Ser Ala Pro Thr Arg Ile Val Ser Ala Ser Asp Tyr Pro Val His Tyr 100 105 110 cac gcg tgg cgg caa cag ggc gtg aaa ctc gcg ctg agc ccg gtc aca 384His Ala Trp Arg Gln Gln Gly Val Lys Leu Ala Leu Ser Pro Val Thr 115 120 125 ctg agt aag aac ccg ttg ctg gcc gga ata aaa cat ctt aac cgg ctg 432Leu Ser Lys Asn Pro Leu Leu Ala Gly Ile Lys His Leu Asn Arg Leu 130 135 140 gaa cag gta atg atc cgc atg cat ctt gac cag aca gat gcc aat gaa 480Glu Gln Val Met Ile Arg Met His Leu Asp Gln Thr Asp Ala Asn Glu 145 150 155 160 gcg ctg gtg gtt gac acc tcg ggc tgc ctg gtg gaa tgc tgt gcg gca 528Ala Leu Val Val Asp Thr Ser Gly Cys Leu Val Glu Cys Cys Ala Ala 165 170 175 aat tta ttc tgg cgt aag gga aat cag gtg ttt act ccg gat tta tcg 576Asn Leu Phe Trp Arg Lys Gly Asn Gln Val Phe Thr Pro Asp Leu Ser 180 185 190 cag tcc ggc gtt gat ggt ctt atg cgt cag cac gtc atc cgc gta ctt 624Gln Ser Gly Val Asp Gly Leu Met Arg Gln His Val Ile Arg Val Leu 195 200 205 gaa gcg aca tcc ccc tgg gtt gtg aac atc gtc agt gaa tct gcg gaa 672Glu Ala Thr Ser Pro Trp Val Val Asn Ile Val Ser Glu Ser Ala Glu 210 215 220 aca tta tca gat gct gac gaa atc ctg att tgt aac gcc ctg atg ccc 720Thr Leu Ser Asp Ala Asp Glu Ile Leu Ile Cys Asn Ala Leu Met Pro 225 230 235 240 gtt ctg ccg gtg aat cag gtc gat gac aaa tat tac att tca cgg cgt 768Val Leu Pro Val Asn Gln Val Asp Asp Lys Tyr Tyr Ile Ser Arg Arg 245 250 255 ttg tgc gat ttc ctg ctc cag agc tgt taa 798Leu Cys Asp Phe Leu Leu Gln Ser Cys 260 265 41265PRTRahnella aquatilis 41Met Trp Ile Asn Gly Val Ala Ala Ala Met Leu Ser Ala Ser Asp Arg 1 5 10 15 Ser Val Gln Phe Gly Asp Gly Cys Phe Thr Thr Ala Arg Val Ser Asp 20 25 30 Gly Val Ile Val Phe Leu Ala Gly His Ile Gln Arg Leu Gln Arg Ala 35 40 45 Ala Ser Val Leu Arg Ile Glu Gly Val Asp Trp Thr Ala Leu Glu Gln 50 55 60 Glu Met Val Leu Ala Ala Gly Gln Gln Lys Glu Ala Val Val Lys Ala 65 70 75 80 Val Val Thr Arg Gly Gln Gly Gly Arg Gly Tyr Ser Ala Ala Gly Cys 85 90 95 Ser Ala Pro Thr Arg Ile Val Ser Ala Ser Asp Tyr Pro Val His Tyr 100 105 110 His Ala Trp Arg Gln Gln Gly Val Lys Leu Ala Leu Ser Pro Val Thr 115 120 125 Leu Ser Lys Asn Pro Leu Leu Ala Gly Ile Lys His Leu Asn Arg Leu 130 135 140 Glu Gln Val Met Ile Arg Met His Leu Asp Gln Thr Asp Ala Asn Glu 145 150 155 160 Ala Leu Val Val Asp Thr Ser Gly Cys Leu Val Glu Cys Cys Ala Ala 165 170 175 Asn Leu Phe Trp Arg Lys Gly Asn Gln Val Phe Thr Pro Asp Leu Ser 180 185 190 Gln Ser Gly Val Asp Gly Leu Met Arg Gln His Val Ile Arg Val Leu 195 200 205 Glu Ala Thr Ser Pro Trp Val Val Asn Ile Val Ser Glu Ser Ala Glu 210 215 220 Thr Leu Ser Asp Ala Asp Glu Ile Leu Ile Cys Asn Ala Leu Met Pro 225 230 235 240 Val Leu Pro Val Asn Gln Val Asp Asp Lys Tyr Tyr Ile Ser Arg Arg 245 250 255 Leu Cys Asp Phe Leu Leu Gln Ser Cys 260 265 42867DNAAchromobacter spaniusCDS(1)..(867) 42atg att ccg ggc gtg ccg ggc gaa agc cag gtg tat ctc aac ggc gag 48Met Ile Pro Gly Val Pro Gly Glu Ser Gln Val Tyr Leu Asn Gly Glu 1 5 10 15 ttc ctg cgt gtc gac gag gcg aaa gtc tcc gtc ctg gac cgc ggt ttt 96Phe Leu Arg Val Asp Glu Ala Lys Val Ser Val Leu Asp Arg Gly Phe 20 25 30 att ttt ggc gac ggc atc tac gaa gtc gtt ccc gtg tac cag ggc aag 144Ile Phe Gly Asp Gly Ile Tyr Glu Val Val Pro Val Tyr Gln Gly Lys 35 40 45 gca ttc cgc atg gcg gag cac ctt aac cgc ctg gac cgc agc ctg gcc 192Ala Phe Arg Met Ala Glu His Leu Asn Arg Leu Asp Arg Ser Leu Ala 50 55 60 gcc ttg cgc atc acg ccg ccg atg gat cgc gcg ggc tgg gtc gac ctg 240Ala Leu Arg Ile Thr Pro Pro Met Asp Arg Ala Gly Trp Val Asp Leu 65 70

75 80 atc gaa cag ttg ctg gcg cgc acc acg ctg gac acc tgc atc gtg tac 288Ile Glu Gln Leu Leu Ala Arg Thr Thr Leu Asp Thr Cys Ile Val Tyr 85 90 95 ttg cag gtc acg cgc ggc gtt gcc aag cgc gac cac cag ttc ccg gcc 336Leu Gln Val Thr Arg Gly Val Ala Lys Arg Asp His Gln Phe Pro Ala 100 105 110 acg ccg gtt acg ccc acc gtg ttc ggc atg att tcg gcg tgg tcc cct 384Thr Pro Val Thr Pro Thr Val Phe Gly Met Ile Ser Ala Trp Ser Pro 115 120 125 ccg ccc gcc gcg caa cgc acg cag ggc ttg acc gcc atc agc att ccc 432Pro Pro Ala Ala Gln Arg Thr Gln Gly Leu Thr Ala Ile Ser Ile Pro 130 135 140 gac gaa cgc tgg ctg cat tgc gag atc aag tcg gtg tcg ctg ttg ggt 480Asp Glu Arg Trp Leu His Cys Glu Ile Lys Ser Val Ser Leu Leu Gly 145 150 155 160 aac gtg ctg gcc aag cag cag gcg gtg gat gcg aac gcc gac gaa gtc 528Asn Val Leu Ala Lys Gln Gln Ala Val Asp Ala Asn Ala Asp Glu Val 165 170 175 gtg cag ttt cgc gat ggc tat ctg acc gaa ggc tcg tcc acc aac atc 576Val Gln Phe Arg Asp Gly Tyr Leu Thr Glu Gly Ser Ser Thr Asn Ile 180 185 190 tgg gtg gtg tct ggc ggc aag ttg ttg gcg ccg ccc aag aac aac ctg 624Trp Val Val Ser Gly Gly Lys Leu Leu Ala Pro Pro Lys Asn Asn Leu 195 200 205 atc ctg gaa ggc atc cgc tac ggt ctg atg ggc gag ctg gcc gaa gca 672Ile Leu Glu Gly Ile Arg Tyr Gly Leu Met Gly Glu Leu Ala Glu Ala 210 215 220 gcg ggc atc cca ttc gag tcg cgc cgc atc acc cag caa gag gtg gaa 720Ala Gly Ile Pro Phe Glu Ser Arg Arg Ile Thr Gln Gln Glu Val Glu 225 230 235 240 tcc gcc gac gaa ttg atg ctg tct tcc gcc acc aag gaa gtg ctg gcg 768Ser Ala Asp Glu Leu Met Leu Ser Ser Ala Thr Lys Glu Val Leu Ala 245 250 255 att gtt tcc ttg gac gga aag ccg gtg ggt tcg ggc aag ccc ggc cct 816Ile Val Ser Leu Asp Gly Lys Pro Val Gly Ser Gly Lys Pro Gly Pro 260 265 270 gtt ttt gag cag ttg cga gcg ggt tat gat gcc cgc atc gcc gcg ctg 864Val Phe Glu Gln Leu Arg Ala Gly Tyr Asp Ala Arg Ile Ala Ala Leu 275 280 285 taa 86743288PRTAchromobacter spanius 43Met Ile Pro Gly Val Pro Gly Glu Ser Gln Val Tyr Leu Asn Gly Glu 1 5 10 15 Phe Leu Arg Val Asp Glu Ala Lys Val Ser Val Leu Asp Arg Gly Phe 20 25 30 Ile Phe Gly Asp Gly Ile Tyr Glu Val Val Pro Val Tyr Gln Gly Lys 35 40 45 Ala Phe Arg Met Ala Glu His Leu Asn Arg Leu Asp Arg Ser Leu Ala 50 55 60 Ala Leu Arg Ile Thr Pro Pro Met Asp Arg Ala Gly Trp Val Asp Leu 65 70 75 80 Ile Glu Gln Leu Leu Ala Arg Thr Thr Leu Asp Thr Cys Ile Val Tyr 85 90 95 Leu Gln Val Thr Arg Gly Val Ala Lys Arg Asp His Gln Phe Pro Ala 100 105 110 Thr Pro Val Thr Pro Thr Val Phe Gly Met Ile Ser Ala Trp Ser Pro 115 120 125 Pro Pro Ala Ala Gln Arg Thr Gln Gly Leu Thr Ala Ile Ser Ile Pro 130 135 140 Asp Glu Arg Trp Leu His Cys Glu Ile Lys Ser Val Ser Leu Leu Gly 145 150 155 160 Asn Val Leu Ala Lys Gln Gln Ala Val Asp Ala Asn Ala Asp Glu Val 165 170 175 Val Gln Phe Arg Asp Gly Tyr Leu Thr Glu Gly Ser Ser Thr Asn Ile 180 185 190 Trp Val Val Ser Gly Gly Lys Leu Leu Ala Pro Pro Lys Asn Asn Leu 195 200 205 Ile Leu Glu Gly Ile Arg Tyr Gly Leu Met Gly Glu Leu Ala Glu Ala 210 215 220 Ala Gly Ile Pro Phe Glu Ser Arg Arg Ile Thr Gln Gln Glu Val Glu 225 230 235 240 Ser Ala Asp Glu Leu Met Leu Ser Ser Ala Thr Lys Glu Val Leu Ala 245 250 255 Ile Val Ser Leu Asp Gly Lys Pro Val Gly Ser Gly Lys Pro Gly Pro 260 265 270 Val Phe Glu Gln Leu Arg Ala Gly Tyr Asp Ala Arg Ile Ala Ala Leu 275 280 285 44867DNAArtificialCodon-optimized sequence coding for SEQ ID NO 43 44atgattccgg gtgttccagg tgaaagccag gtttatctga atggtgaatt tctgcgtgtt 60gatgaagcaa aagttagcgt tctggatcgc ggttttatct ttggtgatgg tatttatgaa 120gtggtgccgg tttatcaggg taaagcattt cgtatggcag aacatctgaa tcgtctggat 180cgtagcctgg cagcactgcg tattacccct ccgatggatc gtgcaggttg ggttgatctg 240attgaacagc tgctggcacg taccaccctg gatacctgta ttgtttatct gcaggttacc 300cgtggtgttg caaaacgtga tcatcagttt ccggcaacac cggttacccc gaccgttttt 360ggcatgatta gcgcatggtc accgcctccg gcagcccagc gtacccaggg tctgaccgca 420attagcattc cggatgaacg ttggctgcat tgtgaaatca aaagcgttag cctgctgggt 480aatgttctgg caaaacagca ggcagttgat gcaaatgcag atgaagttgt tcagtttcgt 540gatggttatc tgaccgaagg tagcagcacc aatatttggg ttgttagcgg tggtaaactg 600ctggctccgc ctaaaaacaa tctgattctg gaaggtattc gctatggtct gatgggtgaa 660ctggcagaag cagcaggtat tccgtttgaa agccgtcgta ttacacagca agaggttgaa 720agcgcagatg aactgatgct gagcagcgca accaaagaag ttctggccat tgttagcctg 780gatggtaaac cggttggtag cggcaaaccg ggtccggttt ttgagcagct gcgtgccggt 840tatgatgcac gtattgcagc actgtaa 86745885DNAMicrobacterium ginsengisoliCDS(1)..(885) 45atg acc tgg cgt ttc gcg ctc atc atc gag ccc gtg gca tcc gat gac 48Met Thr Trp Arg Phe Ala Leu Ile Ile Glu Pro Val Ala Ser Asp Asp 1 5 10 15 ccc cgc acc gac ttc gac acg acc ttc gcg ccc gtc gac gcc tcg gcc 96Pro Arg Thr Asp Phe Asp Thr Thr Phe Ala Pro Val Asp Ala Ser Ala 20 25 30 ccc gcc ctc agc atc ggc gag ctc agc acc cag cgc gga gac gga atc 144Pro Ala Leu Ser Ile Gly Glu Leu Ser Thr Gln Arg Gly Asp Gly Ile 35 40 45 ttc gag tcg atc ggt gtc gtc gac agg cac ccg cag gag gtc gag gcg 192Phe Glu Ser Ile Gly Val Val Asp Arg His Pro Gln Glu Val Glu Ala 50 55 60 cac ctg gcg cgg ctc gcg cac tcc gcc gag atc tgc gac ctt ccg gtg 240His Leu Ala Arg Leu Ala His Ser Ala Glu Ile Cys Asp Leu Pro Val 65 70 75 80 ccg aac ctc gcc cag tgg cgc gcc gcc gtc gcc cgc gcg gcg gcg cag 288Pro Asn Leu Ala Gln Trp Arg Ala Ala Val Ala Arg Ala Ala Ala Gln 85 90 95 tgc ccg gag ggc gag gcg gtc atc aag ctc atc ctg agt cgc ggc atc 336Cys Pro Glu Gly Glu Ala Val Ile Lys Leu Ile Leu Ser Arg Gly Ile 100 105 110 gag cac ggt ccg acc ccg acc gcg tgg gtg acc gcg tcg gcc gcg ccc 384Glu His Gly Pro Thr Pro Thr Ala Trp Val Thr Ala Ser Ala Ala Pro 115 120 125 aac tat gcc cgc ccg cgc gcc gaa ggc atc tcc gtc gtc gtg ctc gat 432Asn Tyr Ala Arg Pro Arg Ala Glu Gly Ile Ser Val Val Val Leu Asp 130 135 140 cgc ggg ctc gac ctc gcc gca ccc gcc cgc gcc ccc tgg ctg ctg ctc 480Arg Gly Leu Asp Leu Ala Ala Pro Ala Arg Ala Pro Trp Leu Leu Leu 145 150 155 160 ggt gcg aag acg ttg tcg tat gcg acc aac atg gcg gcg ctg cgc gag 528Gly Ala Lys Thr Leu Ser Tyr Ala Thr Asn Met Ala Ala Leu Arg Glu 165 170 175 gcg cac cga cgc ggc gcg gat gac gcc gtc ttc gcc acg tcc gat gga 576Ala His Arg Arg Gly Ala Asp Asp Ala Val Phe Ala Thr Ser Asp Gly 180 185 190 ttc ctg ctc gag gcg ccg acc gcg tcg ctc gtg ctg cgc cgc ggc gat 624Phe Leu Leu Glu Ala Pro Thr Ala Ser Leu Val Leu Arg Arg Gly Asp 195 200 205 gtg ttc gtg acc ccc gag ccc gcc gcc ggc atc ctg cac ggc acc act 672Val Phe Val Thr Pro Glu Pro Ala Ala Gly Ile Leu His Gly Thr Thr 210 215 220 cag ctg agc ctg ttc gcc cac ctc gcc gag cgg ggg ttc acg acc gcc 720Gln Leu Ser Leu Phe Ala His Leu Ala Glu Arg Gly Phe Thr Thr Ala 225 230 235 240 tac gag acc ctt ccg acg gcg gcc ctc gcc gac gcg gat gcc gcg tgg 768Tyr Glu Thr Leu Pro Thr Ala Ala Leu Ala Asp Ala Asp Ala Ala Trp 245 250 255 ctc gtc tcg agc gtc cgc ctc gcg gcc ccg atc acg gcc gtc gac ggc 816Leu Val Ser Ser Val Arg Leu Ala Ala Pro Ile Thr Ala Val Asp Gly 260 265 270 cgg gct ctc ccg cat gat gcg gcc ttc acg gcc gag ctg aac gcc tac 864Arg Ala Leu Pro His Asp Ala Ala Phe Thr Ala Glu Leu Asn Ala Tyr 275 280 285 ctg ctc tcg ccg cgc gac tga 885Leu Leu Ser Pro Arg Asp 290 46294PRTMicrobacterium ginsengisoli 46Met Thr Trp Arg Phe Ala Leu Ile Ile Glu Pro Val Ala Ser Asp Asp 1 5 10 15 Pro Arg Thr Asp Phe Asp Thr Thr Phe Ala Pro Val Asp Ala Ser Ala 20 25 30 Pro Ala Leu Ser Ile Gly Glu Leu Ser Thr Gln Arg Gly Asp Gly Ile 35 40 45 Phe Glu Ser Ile Gly Val Val Asp Arg His Pro Gln Glu Val Glu Ala 50 55 60 His Leu Ala Arg Leu Ala His Ser Ala Glu Ile Cys Asp Leu Pro Val 65 70 75 80 Pro Asn Leu Ala Gln Trp Arg Ala Ala Val Ala Arg Ala Ala Ala Gln 85 90 95 Cys Pro Glu Gly Glu Ala Val Ile Lys Leu Ile Leu Ser Arg Gly Ile 100 105 110 Glu His Gly Pro Thr Pro Thr Ala Trp Val Thr Ala Ser Ala Ala Pro 115 120 125 Asn Tyr Ala Arg Pro Arg Ala Glu Gly Ile Ser Val Val Val Leu Asp 130 135 140 Arg Gly Leu Asp Leu Ala Ala Pro Ala Arg Ala Pro Trp Leu Leu Leu 145 150 155 160 Gly Ala Lys Thr Leu Ser Tyr Ala Thr Asn Met Ala Ala Leu Arg Glu 165 170 175 Ala His Arg Arg Gly Ala Asp Asp Ala Val Phe Ala Thr Ser Asp Gly 180 185 190 Phe Leu Leu Glu Ala Pro Thr Ala Ser Leu Val Leu Arg Arg Gly Asp 195 200 205 Val Phe Val Thr Pro Glu Pro Ala Ala Gly Ile Leu His Gly Thr Thr 210 215 220 Gln Leu Ser Leu Phe Ala His Leu Ala Glu Arg Gly Phe Thr Thr Ala 225 230 235 240 Tyr Glu Thr Leu Pro Thr Ala Ala Leu Ala Asp Ala Asp Ala Ala Trp 245 250 255 Leu Val Ser Ser Val Arg Leu Ala Ala Pro Ile Thr Ala Val Asp Gly 260 265 270 Arg Ala Leu Pro His Asp Ala Ala Phe Thr Ala Glu Leu Asn Ala Tyr 275 280 285 Leu Leu Ser Pro Arg Asp 290

Claims

1. A method for the enzymatic synthesis of an enantiomerically enriched (R)-amine of general formula [1][c] ##STR00005## from the corresponding ketone of the general formula ##STR00006## and a suitable amino donor, wherein R.sub.1 and R.sub.2 are different and are independently linear or branched aliphatic, aromatic, hetero-aromatic or form a cyclic structure, by using a transaminase selected from the group consisting of a. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 1; b. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 3; c. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 5; d. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 7; e. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 9; f. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 11; g. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 13; h. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 15; i. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 17; j. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 19; k. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 21; l. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 23; m. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 25; n. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 30; o. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 33; p. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 35; q. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 38; r. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 40; s. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 43; t. a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 46 and u. a protein having transaminase activity and obtainable from an organism selected from the group of organisms consisting of Rahnella aquatilis, Ochrobactrum anthropi, Ochrobactrum tritici, Sinorhizobium morelense, Curtobacterium pusilllum, Paecilomyces lilacinus, Microbacterium ginsengisoli, Microbacterium trichothecenolyticum, Pseudomonas citronellolis, Yersinia kristensenii, Achromobacter spanius, Achromobacter insolitus, Mycobacterium fortuitum, Mycobacterium frederiksbergense, Mycobacterium sacrum, Mycobacterium fluoranthenivorans, Burkholderia sp., Burkholderia tropica, Cosmospora episphaeria, and Fusarium oxysporum.

2. A method according to claim 1, wherein R.sub.1 and R.sub.2 are different and R.sub.1 and R.sub.2 independently contain 1 to 30 carbon atoms and R.sub.1 and R.sub.2 are independently substituted or unsubstituted aliphatic; substituted or unsubstituted branched aliphatic; substituted or unsubstituted cyclic aliphatic; substituted or unsubstituted heterocyclic aliphatic, containing at least one oxygen, sulfur or nitrogen atom; substituted or unsubstituted aromatic; substituted or unsubstituted hetero-aromatic containing at least one sulfur or nitrogen atom; or together form a substituted or unsubstituted cyclic structure or heterocyclic structure, containing at least one oxygen, sulfur or nitrogen atom; wherein the substituents are selected from but not limited to the group consisting of a halogen atom, an alkyl group having 1 to 6 carbon atoms, hydroxyl group, methoxy group, monofluoromethyl, difluoromethyl and trifluoromethyl group.

3. A process according to claim 1, characterized in that the final concentration of the enantiomerically enriched (R)-amine product lies between 1 and 50 weight % of the reaction mixture.

4. A process according to claim 3, wherein the transaminase is a protein having at least 90% identity to the amino acid sequence of SEQ ID No. 1.

5. A process according to claim 4, wherein the amino donor is .alpha.-methylbenzylamine.

6. A process according to claim 4, wherein the amino donor is sec-butylamine.

7. A process according to claim 1, characterized in that the final concentration of the enantiomerically enriched (R)-amine product lies between 5 and 35 weight % of the reaction mixture.

8. A process according to claim 1, wherein the reaction mixture comprises an aqueous phase and second organic phase.

9. A process according to claim 1, wherein the reaction mixture comprises an aqueous phase and second organic phase and the volumetric ratio of water:organic phase is between 100 and 0.01.

10. A process according to claim 1, wherein the reaction mixture comprises an aqueous phase and second organic phase and the volumetric ratio of water:organic phase is between 20 and 0.1.

11. A process according to claim 1, wherein the reaction mixture comprises an aqueous phase and second organic phase and the volumetric ratio of water:organic phase is between 20 and 1.

12. A microorganism comprising an enzyme having (R)-transaminase activity from the group of organisms consisting of Rahnella aquatilis (deposited as DSM 23797), Ochrobactrum anthropi (deposited as DSM 23793), Ochrobactrum tritici (deposited as DSM 23786), Sinorhizobium morelense (deposited as DSM 23794), Curtobacterium pusilllum (deposited as DSM 23787), Paecilomyces lilacinus (deposited as DSM 23771), Microbacterium ginsengisoli, Microbacterium trichothecenolyticum (deposited as DSM 23788), Pseudomonas citronellolis (deposited as DSM 23795), Yersinia kristensenii (deposited as DSM 23792), Achromobacter spanius (deposited as DSM 23791), Achromobacter insolitus (deposited as DSM 23790), Mycobacterium fortuitum (deposited as DSM 23789), Mycobacterium frederiksbergense (deposited as DSM 23798), Mycobacterium sacrum (deposited as DSM 23785), Mycobacterium fluoranthenivorans (deposited as DSM 23796), Burkholderia sp., Burkholderia tropica (deposited as DSM 23799), Cosmospora episphaeria (deposited as DSM 23772), and Fusarium oxysporum (deposited as DSM 23770).

13. A microorganism according to claim 12, wherein the enzyme having (R)-transaminase activity is selected from the group consisting of Cpu-TA1 [SEQ ID No. 30], Cpu-TA2 [SEQ ID No. 33], Cpu-TA3 [SEQ ID No. 35], Raq-TA2 [SEQ ID No. 38], Raq-TA3 [SEQ ID No. 40], Asp-TA1 [SEQ ID No. 43] and Mgi-TA1 [SEQ ID No. 46] or a protein having at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% identity to any of these amino acid sequences.

14. A polypeptide having (R)-transaminase activity and obtainable from an organism selected from the group of organisms consisting of Rahnella aquatilis (deposited as DSM 23797), Ochrobactrum anthropi (deposited as DSM 23793), Ochrobactrum tritici (deposited as DSM 23786), Sinorhizobium morelense (deposited as DSM 23794), Curtobacterium pusilllum (deposited as DSM 23787), Paecilomyces lilacinus (deposited as DSM 23771), Microbacterium ginsengisoli, Microbacterium trichothecenolyticum (deposited as DSM 23788), Pseudomonas citronellolis (deposited as DSM 23795), Yersinia kristensenii (deposited as DSM 23792), Achromobacter spanius (deposited as DSM 23791), Achromobacter insolitus (deposited as DSM 23790), Mycobacterium fortuitum (deposited as DSM 23789), Mycobacterium frederiksbergense (deposited as DSM 23798), Mycobacterium sacrum (deposited as DSM 23785), Mycobacterium fluoranthenivorans (deposited as DSM 23796), Burkholderia sp., Burkholderia tropica (deposited as DSM 23799), Cosmospora episphaeria (deposited as DSM 23772), and Fusarium oxysporum (deposited as DSM 23770).

15. A polypeptide having (R)-transaminase activity and at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to a polypeptide having (R)-transaminase activity and obtainable from an organism selected from the group of organisms consisting of Rahnella aquatilis (deposited as DSM 23797), Ochrobactrum anthropi (deposited as DSM 23793), Ochrobactrum tritici (deposited as DSM 23786), Sinorhizobium morelense (deposited as DSM 23794), Curtobacterium pusilllum (deposited as DSM 23787), Paecilomyces lilacinus (deposited as DSM 23771), Microbacterium ginsengisoli, Microbacterium trichothecenolyticum (deposited as DSM 23788), Pseudomonas citronellolis (deposited as DSM 23795), Yersinia kristensenii (deposited as DSM 23792), Achromobacter spanius (deposited as DSM 23791), Achromobacter insolitus (deposited as DSM 23790), Mycobacterium fortuitum (deposited as DSM 23789), Mycobacterium frederiksbergense (deposited as DSM 23798), Mycobacterium sacrum (deposited as DSM 23785), Mycobacterium fluoranthenivorans (deposited as DSM 23796), Burkholderia sp., Burkholderia tropica (deposited as DSM 23799), Cosmospora episphaeria (deposited as DSM 23772), and Fusarium oxysporum (deposited as DSM 23770).

16. A polypeptide according to claim 14 having (R)-transaminase activity selected from the group consisting of Cpu-TA1 [SEQ ID No. 30], Cpu-TA2 [SEQ ID No. 33], Cpu-TA3 [SEQ ID No. 35], Raq-TA2 [SEQ ID No. 38], Raq-TA3 [SEQ ID No. 40], Asp-TA1 [SEQ ID No. 43] and Mgi-TA1 [SEQ ID No. 46] or a protein having at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% identity to any of these amino acid sequences.

17. A nucleic acid encoding for a polypeptide according to claim 14.