Nucleic acids encoding improved transaminase proteins

Abstract

The present invention relates to proteins having improved ω-transaminase (ω-TA) activity, nucleic acid molecules encoding the respective proteins having improved ω-TA activity, and methods for the stereoselective synthesis of chiral amines and amino acids or for increasing the chiral amine isomers in an enantiomeric mixture.

Description

Technical Field

[0001] The present invention relates to proteins having improved ω-transaminase (ω-TA) activity, nucleic acid molecules encoding each of the proteins having improved ω-TA activity, and methods for stereoselectively synthesizing chiral amines and amino acids or increasing chiral amine isomers in enantiomeric mixtures.

[0002] Biocatalysis can be based on enzymes available in nature. Generally, the need to produce a specific product gives rise to the need for a specific enzyme that is suitable for economically viable large-scale production of the desired product. Enzyme engineering is an option for optimizing enzymes for the economical production of a given product.

[0003] Amines and amino acids are ubiquitous in nature, not only as part of proteins and nucleic acids, but also as neurotransmitters (e.g., adrenaline and histamine), as coenzymes (e.g., the cysteamine of coenzyme A), or as precursors of complex lipids (e.g., the ethanolamine of phosphatidylethanolamine). In particular, higher substituted amines classified pharmaceutically as alkaloids exhibit a variety of structural forms, as well as biological effects found in various life forms. The biological activity of amines, such as antibiotic, analgesic, or neurotoxic activity, enhances their potential as drugs and thus makes them very promising candidates in the development of new drugs. The absolute configuration of the chiral amine stereocenter is crucial for the interaction with biomolecules and the type of action on biological systems. Generating the correct chirality is often a challenge for the production of the desired target molecule (Schaetzle, 2011, Inaugural Dissertation, Ernst-Moritz-Arndt-University of Greifswald, Germany, “Identification, characterization and application of novel (R)-selective amine transaminases”).

[0004] Many of the active compounds in the pharmaceutical company's R & D pipeline are chiral. Optically active amines belong to an important class of compounds used in the synthesis of many active pharmaceutical and agricultural products. For example, L-phenylalanine is an important additive in animal feed. There is no commercially viable method for the chemical synthesis of enantiomerically pure amino acids. However, the chemical synthesis of racemic amino acids is still important because in some cases the racemic mixture can be resolved into pure isomers by biocatalytic methods (Breuer et al., 2004, Angewandte Chemie International Edition 43, 788 - 824).

[0005] Amine transaminases or ω - transaminases (ω - TAs) are biocatalysts that are of great significance for the production of chiral primary amines. ω - TAs utilize pyridoxal - 5′ - phosphate (PLP) as a cofactor and catalyze the transfer of an amino group from an amino donor to a carbonyl moiety. Thus, the reaction mixture consists of two amines (the amino donor and the product) and two carbonyl compounds (the ketone substrate and the by - product). Both (S) - selective and (R) - selective transaminases have been found and have been well - described so far. These enzymes have a high degree of stereoselectivity and thus have great potential for direct asymmetric amination, in which chiral amines with high enantiomeric excess are produced directly from achiral ketones using inexpensive amino donors (Fesko et al., 2013, J. Molecular Catalysis B, Enzymatic 96, 103 - 110).

[0006] Transaminases have attracted attention in the biocatalytic synthesis of many chiral amines and amino acids. Transaminases can be used for the kinetic resolution of racemic amino acids (removing one isomer from the mixture) and also in asymmetric synthesis starting from the corresponding prochiral ketone substrates. The transaminase - catalyzed reaction can be considered a redox reaction in which the oxidative deamination of the donor is coupled with the reductive amination of the acceptor (Rudat et al., 2012, AMB Express 2:11).

[0007] Cann et al. (2012, Org. Process Res. Dev. 16, 1953 - 1966) disclosed the successful use of ω - transaminases for the stereoselective production of ω - transaminases, which are precursors for migraine drugs. The advantages and disadvantages of enzymatic synthesis and chemical synthesis were discussed.

[0008] US 4,950,606 describes a method for the production of optically active amines. In this method, ω - transaminases from Bacillus megaterium and Pseudomonas putida convert prochiral ketones or keto acids into amines by the enantioselective transfer of an amino group from an amino donor. The (R) - and (S) - configurations of the amines can be obtained.

[0009] Park et al. (2013, Organic & Biomolecular Chemistry 11, 6929 - 6933) disclosed the behavior of different transaminases in the enantioselective synthesis of non - natural amino acids from keto acids using isopropylamine and various other compounds as amine donors.

[0010] Park et al. (2013, ChemCatChem 5, 1734 - 1738) demonstrated the feasibility of using (R)- or (S)-selective ω-transaminases for the asymmetric amination of thermodynamically favorable prochiral alkyl ketones by using racemic arylalkylamines as amino donors in a one-pot reaction. This reaction does not require the addition of an excess of amino donor or the removal of coproducts.

[0011] ω-Transaminase-catalyzed reactions using 2-propylamine, 1-propylamine, and racemic-2-butylamine as amino donors have shown up to 3-fold higher conversions compared to reactions using alanine as the amino donor. The amino acids β-alanine and asparagine are poor amino donors. For some methyl ketones containing aromatic residues, high yields of optically active amino alcohols were obtained when using an excess of 2-butylamine or 1-phenylethylamine as the amino donor. No further steps were required to shift the equilibrium (Fesko et al., 2013, J. Molecular Catalysis B, Enzymatic 96, 103 - 110).

[0012] Shin & Kim (2001, Biosci. Biotechno. Biochem. 65(8), 1782 - 1788) disclosed the isolation of ω-transaminases using arylamines, including (S)-α-methylbenzylamine ((S)-α-MBA), 1-methyl-3-phenylpropyl-amine, 1-aminotetralin, or 1-aminoindane as amine donors. The ketoacids pyruvate and glyoxylate or the aldehydes propionaldehyde and butaraldehyde were found to be good amino acceptors.

[0013] US 6,133,018 discloses the production of (S)-1-methoxy-2-aminopropane by contacting methoxyacetone with the achiral amino donor 2-aminopropane and ω-transaminase.

[0014] A four-enzyme system for the production of D-amino acids by converting keto acids into respective D-amino acids using D-alanine as an amino donor, catalyzed by D-amino acid aminotransferase (transaminase), was described in Galkin et al. (1997, J. Fermentation and Bioengeneering 83(3), 299 - 300). To drive the reaction equilibrium in the D-amino acid direction, further reactions were coupled with D-amino acid aminotransferase. Pyruvate and ammonia were converted to L-alanine by alanine dehydrogenase while reducing NADH to NAD. L-alanine was converted to D-alanine by alanine racemase. NADH recycling from NAD was established by the formation of carbon dioxide from formic acid catalyzed by formic acid dehydrogenase. Pyruvate was recycled from alanine through the D-amino acid aminotransferase reaction. D-enantiomers of glutamic acid, leucine, norleucine, and methionine could be produced in high yields, while D-phenylalanine and D-tyrosine were synthesized in low yields, D-norvaline was produced only at nearly 30%, and aminobutyric acid was produced only as a racemic mixture.

[0015] WO 2010 / 089171 A2 discloses a method for aminating at least one keto group to an amino group in a polycyclic ring system containing at least one keto group by a reaction catalyzed by an enzyme having transaminase activity.

[0016] WO 2015 / 195707 A1 (US2015361468 A1) discloses the production of pentose polymer building blocks by transgenic bacteria. The bacterial biosynthetic pathway was manipulated by introducing multiple enzymes including ω-transaminase. It has been found that ω-transaminase catalyzes the reaction and the reverse reaction of glutaric semialdehyde to 5-aminovaleric acid, as well as the reactions of 5-aminopentanol to 5-oxopentanol, cadaverine to 5-aminovaleraldehyde, and N5-acetyl-1,5-diaminopentane to N5-acetyl-5-aminovaleraldehyde. L-glutamic acid / 2-oxoglutaric acid or L-alanine / pyruvic acid was used as an amino donor / acceptor, respectively.

[0017] KR 20030072067 discloses the isolation of a thermophyllic Bacillus sp. T30 strain (L-selective aromatic amino acid transferase (transaminase)), and the use of this strain as a biocatalyst for the production of aromatic L-amino acids at high reaction temperatures, thereby increasing the solubility of keto acid substrates.

[0018] Koszelewski et al. (2010, ChemCat Chem 2(1), 73 - 77, including “Supporting Information”) disclosed the use of whole - cell catalysts for the synthesis of enantiomerically pure amines from the corresponding prochiral amines and the resolution of racemic amines. Different ω - transaminases from Bacillus megaterium SC6394, Alcaligenes denitrificans Y2k - 2, Chromobacterium violaceum DSM30191, a W57G mutant of Vibrio fluvialis ω - transaminase, and a mutant from Arthrobacter sp. designated CNB05 - 01 were expressed in Escherichia coli cells. The freeze - dried E. coli cells were used for kinetic resolution and in vitro selective amination reactions.

[0019] The range of products obtainable by using transaminases is limited by the properties of most naturally occurring ω - transaminases, namely that they do not accept substrates larger than ethyl at the position adjacent to the ketone (Savile et al., 2010, Science 329, 305 - 309, including “Supporting Information”). Park et al. (2014, Adv. Synth. Catal. 356, 212 - 220) discovered an (S) - selective ω - transaminase from Paracoccus denitrificans that accepts acceptor substrates with up to a n - butyl substituent (i.e., n - hexyl 2 - oxohexanoate), but does not accept branched - chain α - ketoacids. A variant (V153A) of the (S) - selective ω - transaminase from Paracoccus denitrificans did show increased activity towards the linear ketoacid (S) - 1 - phenylbutylamine, but did not accept branched - chain ketoacids.

[0020] In the reaction for the preparation of substituted (S) - aminotetralins from substituted tetralinones in the presence of isopropylamine as the amine donor, a variant of the mesophilic Arthrobacter citreus ω - transaminase containing 17 amino acid substitutions showed increased thermal stability and significantly improved specific activity compared to the amino acid sequences of the respective wild - type sequences (Martin et al., 2007, Biochemical Engineering Journal 37, 246 - 255).

[0021] Savile et al. (2010, Science 329, 305-309, including “Supporting Information”) disclose the preparation of the complex antidiabetic drug sitagliptin by a biocatalytic method involving ω-transaminase. Various variants of Arthrobacter (R)-selective ω-transaminase (ATA-117) were produced. These enzymes showed a broad substrate range and increased tolerance to isopropylamine and organic solvents. Various amines and anilines substituted with trifluoromethyl can be produced by these enzymes. An optimized variant of Arthrobacter (R)-selective ω-transaminase (ATA-117) containing 27 amino acid substitutions was used to produce sitagliptin by amination of prostaglandin ketone in the presence of isopropylamine as an amine donor, compared to the wild-type enzyme.

[0022] WO 2006 / 06339 (US 7,247,460) discloses Arthrobacter citreus ω-transaminase variants that are thermostable in each case, have an increased reaction rate and tolerance to high amine donor concentrations, compared to each wild-type enzyme.

[0023] Although several improvements of transaminases have been obtained so far, limitations that occur during the asymmetric synthesis of amines or the resolution of racemic amines, such as unfavorable equilibria, substrate and product inhibition, poor thermal stability, insufficient substrate specificity and sometimes low enantioselectivity of the transaminase, still have to be overcome in order to efficiently produce a wide range of amines on an industrial scale.

[0024] Therefore, further improvements of ω-transaminases are needed. In particular, ω-transaminases that need further improvement are preferred in the production of the desired aminated, enantiomerically enriched or pure products, preferably under specific and / or economically viable production methods. SUMMARY OF THE INVENTION

[0025] The present invention provides ω-transaminase (ω-TA) variants comprising a modification in their amino acid sequence or further modified ω-transaminase (ω-TA) variants comprising an additional modification in their amino sequence, which variants and further modified variants comprising further amino acid modifications have improved reaction kinetics, improved substrate acceptance and improved specific activity compared to the corresponding wild-type ω-TA. Thus, the variants of the present invention and variants comprising further amino acid modifications can be used to develop an economically efficient production process for aminated products by using them in production methods for new aminated products or corresponding product precursors that cannot be achieved using the corresponding wild-type ω-TA.

[0026] Variants or further modified variants of ω-TA described herein have advantages over known wild-type and other known ω-TAs. In particular, the modified or variant ω-TAs described herein have the advantage that they can produce enantiomerically enriched or enantiomerically nearly pure or pure compounds, such as, for example, branched-chain or aromatic amino acids that cannot be produced by the corresponding wild-type ω-transaminase. The further modified ω-TA variants described herein have the advantage that they can produce enantiomerically enriched, nearly pure or pure compounds of phosphorylated-amino acids.

[0027] Positions 1 to 477 in SEQ ID NO 3 represent the amino acid sequence of the wild-type ω-transaminase (ω-TA) of Bacillus megaterium with the accession number No 5G09_A from GenPept (PDB).

[0028] Positions 1 to 479 in SEQ ID NO 6 represent the amino acid sequence of the wild-type ω-TA of Arthrobacter genus with the accession number No 5G2P_A from GenPept (PDB).

[0029] Positions 1 to 476 in SEQ ID NO 9 represent the amino acid sequence of the wild-type ω-TA of Bacillus genus (Soil 76801D1) with the accession number No KRF52528.1 from GenPept (PDB).

[0030] Positions 1 to 476 in SEQ ID NO 12 represent the amino acid sequence of the ω-TA variant of Arthrobacter genus of SEQ ID NO 16 from WO 2006 / 06336 A2.

[0031] Positions 1 to 476 in SEQ ID NO 15 represent the amino acid sequence of the wild-type ω-TA of Arthrobacter genus of SEQ ID NO 2 from WO 2006 / 06336 A2.

[0032] This text describes proteins with ω-TA activity, where the amino acid sequences of these proteins represent variants of known proteins with ω-TA activity. In particular, the amino acid sequences of the proteins with ω-TA activity described herein represent the amino acids represented by positions 1 to 477 in SEQ ID NO 3, and / or the amino acids represented by positions 1 to 479 in SEQ ID NO 6, and / or the amino acids represented by positions 1 to 476 in SEQ ID NO 9, and / or the amino acids represented by positions 1 to 476 in SEQ ID NO 12, and / or the amino acids represented by positions 1 to 476 in SEQ ID NO 15, where each amino acid sequence shown in SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 12, and SEQ ID NO 15 is different from the amino acids at the corresponding amino acid positions of each sequence shown in SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 12, and SEQ ID NO 15 at least at positions 25, 64, 88, 157, 165, 169, 174, 187, 197, 239, 327, 328, 384, 389, 391, 396, 410, and 414.

[0033] The abbreviation "ω-TA" is used and refers to "ω-transaminase" herein.

[0034] As used herein, the term "variant" refers to a subject that is different from a subject known in the art. With respect to nucleic acid molecules and proteins, a variant should be understood to respectively include a nucleic acid sequence or an amino acid sequence that deviates from the corresponding known sequence but encodes a protein with the same function or catalyzes the same reaction, for example, the function of encoding a protein with ω-TA activity. The deviation of the nucleic acid molecule sequence and the amino acid sequence from the known nucleic acid sequence and protein sequence means that compared with the corresponding known nucleic acid, the sequences respectively contain substitutions (replacements) and / or deletions and / or insertions of nucleotides or amino acids.

[0035] The first aspect of the present invention relates to proteins with ω-TA activity, where the proteins are selected from the following:

[0036] a) A protein comprising the amino acid sequence from position 1 to 477 as shown in SEQ ID NO 3, except that the amino acid at position 25 is not F, the amino acid at position 64 is not L, the amino acid at position 88 is not T, the amino acid at position 157 is not T, the amino acid at position 165 is not R, the amino acid at position 169 is not V, the amino acid at position 174 is not E, the amino acid at position 187 is not S, the amino acid at position 197 is not M, the amino acid at position 239 is not S, the amino acid at position 327 is not S, the amino acid at position 328 is not V, the amino acid at position 384 is not Y, the amino acid at position 389 is not I, the amino acid at position 391 is not D, the amino acid at position 396 is not K, the amino acid at position 410 is not H, and the amino acid at position 414 is not P;

[0037] b) A protein comprising the amino acid sequence from position 1 to 479 as shown in SEQ ID NO 6, except that the amino acid at position 25 is not F, the amino acid at position 64 is not L, the amino acid at position 88 is not T, the amino acid at position 157 is not T, the amino acid at position 165 is not R, the amino acid at position 169 is not V, the amino acid at position 174 is not E, the amino acid at position 187 is not S, the amino acid at position 197 is not T, the amino acid at position 239 is not S, the amino acid at position 327 is not S, the amino acid at position 328 is not V, the amino acid at position 384 is not Y, the amino acid at position 389 is not I, the amino acid at position 391 is not D, the amino acid at position 396 is not K, the amino acid at position 410 is not H, and the amino acid at position 414 is not P;

[0038] c) A protein comprising the amino acid sequence from position 1 to 476 as shown in SEQ ID NO 9, except that the amino acid at position 25 is not F, the amino acid at position 64 is not L, the amino acid at position 88 is not T, the amino acid at position 157 is not T, the amino acid at position 165 is not R, the amino acid at position 169 is not V, the amino acid at position 174 is not E, the amino acid at position 187 is not S, the amino acid at position 197 is not M, the amino acid at position 239 is not S, the amino acid at position 327 is not S, the amino acid at position 328 is not V, the amino acid at position 384 is not Y, the amino acid at position 389 is not I, the amino acid at position 391 is not D, the amino acid at position 396 is not K, the amino acid at position 410 is not H, and the amino acid at position 414 is not P;

[0039] d) A protein comprising the amino acid sequence from position 1 to 476 as set forth in SEQ ID NO: 12, except that the amino acid at position 25 is not F, the amino acid at position 64 is not L, the amino acid at position 88 is not T, the amino acid at position 157 is not T, the amino acid at position 165 is not R, the amino acid at position 169 is not V, the amino acids at positions 174 and 187 are not S and not E, the amino acid at position 197 is not T, the amino acid at position 239 is not S, the amino acid at position 327 is not S, the amino acid at position 328 is not V, the amino acid at position 384 is not Y, the amino acid at position 389 is not I, the amino acid at position 391 is not D, the amino acid at position 396 is not K, the amino acid at position 410 is not H, and the amino acid at position 414 is not P;

[0040] e) A protein comprising the amino acid sequence from position 1 to 476 as set forth in SEQ ID NO: 15, except that the amino acid at position 25 is not F, the amino acid at position 64 is not L, the amino acid at position 88 is not T, the amino acid at position 157 is not T, the amino acid at position 165 is not R, the amino acid at position 169 is not V, the amino acids at positions 174 and 187 are not S and not E, the amino acid at position 197 is not M, the amino acid at position 239 is not S, the amino acid at position 327 is not S, the amino acid at position 328 is not V, the amino acid at position 384 is not Y, the amino acid at position 389 is not I, the amino acid at position 391 is not D, the amino acid at position 396 is not K, the amino acid at position 410 is not H, and the amino acid at position 414 is not P;

[0041] f) A protein having an amino acid sequence with at least 60%, preferably 70%, more preferably 80%, still more preferably 90%, even more preferably 95%, even still more preferably 96%, particularly preferably 97%, most preferably 98% or particularly preferably 99% identity to any of the amino acid sequences shown in a), b), c), d), e) or f), provided that in each case the amino acid corresponding to position 25 is not F, the amino acid corresponding to position 64 is not L, the amino acid corresponding to position 88 is not T, the amino acid corresponding to position 157 is not T, the amino acid corresponding to position 165 is not R, the amino acid corresponding to position 169 is not V, the amino acid corresponding to position 174 is not E, the amino acid corresponding to position 187 is not S, the amino acid corresponding to position 197 is not T or M, the amino acid corresponding to position 239 is not S, the amino acid corresponding to position 327 is not S, the amino acid corresponding to position 328 is not V, the amino acid corresponding to position 384 is not Y, the amino acid corresponding to position 389 is not I, the amino acid corresponding to position 391 is not D, the amino acid corresponding to position 396 is not K, the amino acid corresponding to position 410 is not H, and the amino acid corresponding to position 414 is not P.

[0042] The meanings of the amino acid abbreviations A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y can be derived from Table 4 in the "Sequence Listing" paragraph of the subtitle below.

[0043] The "amino acid corresponding to position x" in the first amino acid sequence (e.g., position 64 in SEQ ID NO 3) herein refers to, under the pairwise sequence alignment of the first amino acid sequence and the second amino acid sequence, if the amino acid numbering of the second amino acid sequence is different from that of the first amino acid sequence, the amino acid of the second amino acid sequence that appears at position x of the first amino acid sequence when compared with the first amino acid sequence.

[0044] In the context of the present invention, with respect to sequence identity or a sequence identical thereto, the term "identity" should be understood to refer to the number of identical amino acids or nucleotides shared by the first nucleic acid or amino acid sequence, respectively, with another (second) nucleic acid or amino acid sequence over the entire sequence length, expressed as a percentage.

[0045] "Sequence identity" can be determined by aligning two amino acid or two nucleotide sequences using global or local alignment algorithms such as those included in known software such as GAP or BESTFIT or the Emboss program "Needle". These software use the Needleman and Wunsch global alignment algorithm to align over the entire length of the two sequences, maximizing the number of matches and minimizing the number of gaps. Under normal circumstances, using default parameters, the gap creation penalty = 10 and the gap extension penalty = 0.5 (both for nucleotide and protein alignments). For nucleotides, the default scoring matrix is DNAFULL, and for proteins, the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 10915 - 10919). The scores for sequence alignment and the percentage of sequence identity can be determined, for example, using software such as EMBOSS that is accessible at the World Wide Web site of EBI (ebi.ac.uk / Tools / emboss / ). Alternatively, sequence similarity or identity can be determined by searching databases (e.g., EMBL, GenBank) using well-known algorithms and output formats such as FASTA, BLAST, etc., but preferably, hits should be retrieved and pairwise alignments should be made to finally determine sequence identity.

[0046] Preferably, with the aid of a computer program, the identity of a protein having ω-TA activity is determined by comparing the amino acid sequence given in SEQ ID NO 18, and the identity of a nucleic acid molecule encoding a protein having ω-TA activity is determined by comparing the nucleic acid sequence given in SEQ ID NO 16 or 17 with other protein or nucleic acid molecules, respectively. If the sequences to be compared are of different lengths, the identity should be determined by determining the percentage of the number of amino acids or nucleotides shared by the shorter sequence with the longer sequence, respectively. Preferably, the identity is determined using the well-known and publicly available computer program ClustalW (Thompson et al., Nucleic Acids Research 22 (1994), 4673 - 4680). ClustalW is by Julie Thompson ( Thompson@EMBL-Heidelberg.DE) and Toby Gibson (Gibson@EMBL-Heidelberg.DE), European Molecular Biology Laboratory, Meyerhofstrasse 1, D69117 Heidelberg, Germany. ClustalW can also be downloaded from a number of different web pages, including the IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire, B.P. 163, 67404 Illkirch Cedex, France; ftp: / / ftp-igbmc.u-strasbg.fr / pub / ) and the EBI ( ftp: / / ftp.ebi.ac.uk / pub / software / ) and all mirror sites of the EBI (European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK).

[0047] Preferably, the ClustalW computer program, version 1.8, is used to determine the identity between the proteins described in the context of the present invention and other proteins. Here, the parameters must be set as follows: KTUPLE = 1, TOPDIAG = 5, WINDOW = 5, PAIRGAP = 3, GAPOPEN = 10, GAPEXTEND = 0.05, GAPDIST = 8, MAXDIV = 40, MATRIX = GONNET, ENDGAPS (OFF), NOPGAP, NOHGAP.

[0048] Preferably, the ClustalW computer program, version 1.8, is used to determine, for example, the identity between the nucleotide sequences of the nucleic acid molecules described in the context of the present invention and the nucleotide sequences of other nucleic acid molecules. Here, the parameters must be set as follows:

[0049] KTUPLE = 2, TOPDIAGS = 4, PAIRGAP = 5, DNAMATRIX: IUB, GAPOPEN = 10, GAPEXT = 5, MAXDIV = 40, TRANSITIONS: unweighted.

[0050] Identity also refers to the functional and / or structural equivalence between the nucleic acid molecules under discussion or the proteins encoded thereby. Functional equivalence means that the nucleic acid molecule sequence or amino acid sequence encodes a protein with ω-TA activity. Nucleic acid molecules that are homologous to the above-mentioned molecules and represent derivatives of these molecules are usually variants of these molecules, which represent modifications with the same biological function or catalyzing the same reaction, i.e., encoding a protein with ω-TA activity. It can be a naturally occurring variant, such as a sequence from another species, or a mutation, where these mutations can occur in a natural manner or be introduced by site-directed mutagenesis. In addition, the variant can be a synthetically generated sequence. Allelic variants can be naturally occurring variants or synthetically generated variants or variants produced by recombinant DNA technology. However, with respect to the present invention, what is decisive is that those variants encode a protein with ω-TA activity and contain amino acid substitutions (replacements), deletions or insertions of the proteins according to the present invention described herein.

[0051] A special type of derivative is, for example, a nucleic acid molecule that differs from the nucleic acid molecules described in the context of the present invention due to the degeneracy of the genetic code.

[0052] According to the regulations of the NC-IUBMB (Nomenclature Committee of the International Union of Biochemistry and Molecular Biology), transaminases (TA) belong to the class of transferases (EC 2). Transferases are enzymes that transfer a group (e.g., methyl or glycosyl) from one compound (usually regarded as the donor) to another compound (usually regarded as the acceptor). The group of transferases includes enzymes that transfer nitrogen-containing groups (EC 2.6). The reaction catalyzed by TA can formally be regarded as an oxidation-reduction reaction. According to the general equation (I), the oxidative deamination of the (amine) donor and the reductive amination of the carbonyl acceptor are exchanged by transferring the -NH2 group and -H to the carbonyl-containing compound in exchange for the =OR of this group 1 -CH(-NH2)-R 2 +R 3 -CO-R 4 →R 1 -CO-R 2 +R 3 -CH(-NH2)-R 4 。

[0053] The reverse reaction also catalyzed by TA can formally be described according to the general equation (Ia) R 1 -CO-R 2 +R 3 -CH(-NH2)-R 4 →R 1 -CH(-NH2)-R 2 +R 3 -CO-R 4 。

[0054] TA is a pyridoxal 5′-phosphate (PLP)-dependent enzyme. A unique feature of the reaction catalyzed by TA is the transfer of an amino group (by a well-established mechanism involving a covalent substrate–coenzyme intermediate), which justifies the assignment of these enzymes to a special subclass among transferases, called transaminases or aminotransferases (EC 2.6.1).

[0055] TA is generally further classified in the art as α-TA and ω-TA. This nomenclature is based on the relative position of the amino group of the amino acid transferred by the respective TA. For amines, the carboxylic acid α-TA catalyzes only the transfer of the amino group at the α-carbon, where ω-TA also acts on non-α-amines and transfers the distal amino group of the corresponding substrate (Shin et al., 2003, Appl Microbiol Biotechnol 61, 463–471). However, it is known in the art that some ω-TAs are capable of catalyzing the transfer of the amino group of (primary) amine compounds without a carboxyl group (Rudat et al., 2012, AMB Express 2(11); Shin et al., 2003, Appl Microbiol Biotechnol 61, 463–471).

[0056] If a protein has TA activity, ω-TA can in particular be detected by methods known and described in the art. Hwang & Kim (2004, Enzyme and Microbiol Technology 34(5), 429–436) developed a method for detecting the ω-TA activity of proteins based on the blue staining of α-amino acids with Cu-SO4 / MeOH. Truppo et al. (2009, Org. Biomol. Chem. 7, 395–398) described an assay for high-throughput screening of ω-TA based on a multi-enzyme cascade pH indicator assay, and also disclosed a conventional HPLC analytical assay.

[0057] Which method is used to detect whether the protein according to the invention has ω-TA activity is not decisive. Preferably, with respect to the present invention, the method described under item 4 of “General methods” is used to detect whether the protein according to the invention has ω-TA activity, in particular this method is used to detect whether the ω-TA variant according to the invention has ω-TA activity.

[0058] With respect to ω-TA variants comprising further amino acid modifications, preferably the method described under item 7 of “General methods” is used to detect whether the protein according to the invention has ω-TA activity.

[0059] In a preferred embodiment of the present invention, the protein according to the invention is an (S)-selective ω-TA.

[0060] In the present invention, the term "(S)-selective" refers to the (S)-enantiomer of the enantiomeric excess over the (R)-enantiomer produced by reductive amination of an (amine) receptor according to the general equation (I).

[0061] The reaction catalyzed by (S)-selective ω-TA can formally be described according to the general equation (II)

[0062] R 1 -CH(-NH2)-R 2 +R 3 -CO-R 4 →R 1 -CO-R 2 +R 3 -CH((S)-NH2)-R 4 。

[0063] Compared with the amino acid sequences described in connection with the amino acid sequences shown in SEQ ID No 3, 6, 9, 12 or 15 above, the ω-TA variant proteins according to the present invention may show additional amino acid modifications (amino acid substitutions, deletions or insertions)

[0064] In addition to the ω-TA variants described in items a) or c) above, the amino acid sequence shown in SEQ ID NO 3 from positions 1 to 477 or the amino acid sequence shown in SEQ ID NO 9 from positions 1 to 477 may each have additional amino acid substitutions at positions 2 and / or 48 and / or 164 and / or 242 and / or 245 and / or 311 and / or 353 and / or 424, and / or the amino acid sequence shown in SEQ ID NO 3 may have additional amino acid substitutions at positions 202 and / or 205 and / or 359 and / or 475 and / or 476, and / or may have an amino acid deletion at position 477, and / or the amino acid sequence shown in SEQ ID NO 9 may have additional amino acid substitutions at positions 69 and / or 90 and / or 268 and / or 318 and / or 322 and / or 452.

[0065] In addition to the ω-TA variants described in items b) or d) above, the amino acid sequence shown in SEQ ID NO 6 from positions 1 to 479 or the amino acid sequence shown in SEQ ID NO 12 from positions 1 to 476 may each have additional amino acid substitutions at positions 46 and / or 60 and / or 185 and / or 186 and / or 195 and / or 205 and / or 252 and / or 268 and / or 409 and / or 436, and / or the amino acids at positions 477 and / or 478 and / or 479 in the amino acid sequence shown in SEQ ID NO 6 may be deleted.

[0066] In addition to the ω-TA variants described in item e) above, the amino acid sequence of SEQ ID NO 15 from positions 1 to 476 may have additional amino acid substitutions at position 48 and / or 164 and / or 242 and / or 245 and / or 255 and / or 424.

[0067] Accordingly, another embodiment of the present invention relates to a protein according to the present invention, which comprises additional amino acid modifications, preferably those embodiments are proteins having ω-TA activity, wherein said protein is selected from the following

[0068] a) a protein comprising the amino acid sequence from positions 1 to 477 as shown in SEQ ID NO 3, except that the amino acid at position 25 is not F, the amino acid at position 64 is not L, the amino acid at position 88 is not T, the amino acid at position 157 is not T, the amino acid at position 165 is not R, the amino acid at position 169 is not V, the amino acid at position 174 is not E, the amino acid at position 187 is not S, the amino acid at position 197 is not M, the amino acid at position 239 is not S, the amino acid at position 327 is not S, the amino acid at position 328 is not V, the amino acid at position 384 is not Y, the amino acid at position 389 is not I, the amino acid at position 391 is not D, the amino acid at position 396 is not K, the amino acid at position 410 is not H, the amino acid at position 414 is not P, the amino acid at position 2 is not S, the amino acid at position 48 is not D, the amino acid at position 164 is not Y, the amino acid at position 202 is not D, the amino acid at position 205 is not L, the amino acid at position 242 is not A, the amino acid at position 245 is not A, the amino acid at position 311 is not L, the amino acid at position 353 is not F, the amino acid at position 359 is not D, the amino acid at position 424 is not K, the amino acid at position 475 is not A, the amino acid at position 476 is not L, and the amino acid at position 477 is deleted;

[0069] b) A protein comprising the amino acid sequence from position 1 to 479 as shown in SEQ ID NO: 6, except that the amino acid at position 25 is not F, the amino acid at position 64 is not L, the amino acid at position 88 is not T, the amino acid at position 157 is not T, the amino acid at position 165 is not R, the amino acid at position 169 is not V, the amino acid at position 174 is not E, the amino acid at position 187 is not S, the amino acid at position 197 is not T, the amino acid at position 239 is not S, the amino acid at position 327 is not S, the amino acid at position 328 is not V, the amino acid at position 384 is not Y, the amino acid at position 389 is not I, the amino acid at position 391 is not D, the amino acid at position 384 is not Y, the amino acid at position 389 is not I, the amino acid at position 391 is not D, the amino acid at position 396 is not K, the amino acid at position 410 is not H, the amino acid at position 414 is not P, the amino acid at position 46 is not T, the amino acid at position 60 is not C, the amino acid at position 185 is not C, the amino acid at position 186 is not S, the amino acid at position 195 is not S, the amino acid at position 205 is not Y, the amino acid at position 252 is not V, the amino acid at position 268 is not S, the amino acid at position 409 is not R, the amino acid at position 436 is not A, and the amino acids at positions 477, 478, and 479 are deleted;

[0070] c) A protein comprising the amino acid sequence from position 1 to 476 as set forth in SEQ ID NO: 9, except that the amino acid at position 25 is not F, the amino acid at position 64 is not L, the amino acid at position 88 is not T, the amino acid at position 157 is not T, the amino acid at position 165 is not R, the amino acid at position 169 is not V, the amino acid at position 174 is not E, the amino acid at position 187 is not S, the amino acid at position 197 is not M, the amino acid at position 239 is not S, the amino acid at position 327 is not S, the amino acid at position 328 is not V, the amino acid at position 384 is not Y, the amino acid at position 389 is not I, the amino acid at position 391 is not D, the amino acid at position 396 is not K, the amino acid at position 410 is not H, the amino acid at position 414 is not P, the amino acid at position 2 is not S, the amino acid at position 48 is not D, the amino acid at position 69 is not P, the amino acid at position 90 is not S, the amino acid at position 164 is not Y, the amino acid at position 242 is not A, the amino acid at position 245 is not A, the amino acid at position 268 is not T, the amino acid at position 311 is not L, the amino acid at position 318 is not E, the amino acid at position 322 is not R, the amino acid at position 353 is not S, the amino acid at position 424 is not K, and the amino acid at position 452 is not E;

[0071] d) A protein comprising the amino acid sequence from position 1 to 476 as set forth in SEQ ID NO: 12, except that the amino acid at position 25 is not F, the amino acid at position 64 is not L, the amino acid at position 88 is not T, the amino acid at position 157 is not T, the amino acid at position 165 is not R, the amino acid at position 169 is not V, the amino acid at position 174 is not E, the amino acid at position 187 is not S, the amino acid at position 197 is not T, the amino acid at position 239 is not S, the amino acid at position 327 is not S, the amino acid at position 328 is not V, the amino acid at position 384 is not Y, the amino acid at position 389 is not I, the amino acid at position 391 is not D, the amino acid at position 396 is not K, the amino acid at position 410 is not H, and the amino acid at position 414 is not P, the amino acid at position 46 is not T, the amino acid at position 60 is not C, the amino acid at position 185 is not C, the amino acid at position 186 is not C, the amino acid at position 195 is not S, the amino acid at position 205 is not Y, the amino acid at position 252 is not V, the amino acid at position 268 is not S, the amino acid at position 409 is not R, and the amino acid at position 436 is not A;

[0072] e) A protein comprising the amino acid sequence from position 1 to 476 as set forth in SEQ ID NO: 15, except that the amino acid at position 25 is not F, the amino acid at position 64 is not L, the amino acid at position 88 is not T, the amino acid at position 157 is not T, the amino acid at position 165 is not R, the amino acid at position 169 is not V, the amino acid at position 174 is not E, the amino acid at position 187 is not S, the amino acid at position 197 is not M, the amino acid at position 239 is not S, the amino acid at position 327 is not S, the amino acid at position 328 is not V, the amino acid at position 384 is not Y, the amino acid at position 389 is not I, the amino acid at position 391 is not D, the amino acid at position 396 is not K, the amino acid at position 410 is not H, the amino acid at position 414 is not P, the amino acid at position 48 is not D, the amino acid at position 164 is not Y, the amino acid at position 242 is not A, the amino acid at position 245 is not A, the amino acid at position 255 is not F, and the amino acid at position 424 is not K;

[0073] f) A protein having an amino acid sequence that is at least 60%, preferably 70%, more preferably 80%, still more preferably 90%, even more preferably 95%, even still more preferably 96%, particularly preferably 97%, most preferably 98% or particularly preferably 99% identical to any of the amino acid sequences defined in a) (the amino acid sequence from position 1 to 477 as shown in SEQ ID NO 3), provided that the amino acid corresponding to position 25 is not F, the amino acid corresponding to position 64 is not L, the amino acid corresponding to position 88 is not T, the amino acid corresponding to position 157 is not T, the amino acid corresponding to position 165 is not R, the amino acid corresponding to position 169 is not V, the amino acid corresponding to position 174 is not E, the amino acid corresponding to position 187 is not S, the amino acid corresponding to position 197 is not M, the amino acid corresponding to position 239 is not S, the amino acid corresponding to position 327 is not S, the amino acid corresponding to position 328 is not V, the amino acid corresponding to position 384 is not Y, the amino acid corresponding to position 389 is not I, the amino acid corresponding to position 391 is not D, the amino acid corresponding to position 396 is not K, the amino acid corresponding to position 410 is not H, the amino acid corresponding to position 414 is not P, the amino acid corresponding to position 2 is not S, the amino acid corresponding to position 48 is not D, the amino acid corresponding to position 164 is not Y, the amino acid corresponding to position 202 is not D, the amino acid corresponding to position 205 is not L, the amino acid corresponding to position 242 is not A, the amino acid corresponding to position 245 is not A, the amino acid corresponding to position 311 is not L, the amino acid corresponding to position 353 is not F, the amino acid corresponding to position 359 is not D, the amino acid corresponding to position 424 is not K, the amino acid corresponding to position 475 is not A, the amino acid corresponding to position 476 is not L, and the amino acid corresponding to position 477 is deleted;

[0074] g) A protein having an amino acid sequence that is at least 60%, preferably 70%, more preferably 80%, still more preferably 90%, even more preferably 95%, even still more preferably 96%, particularly preferably 97%, most preferably 98% or particularly preferably 99% identical to any of the amino acid sequences defined in b) (the amino acid sequence from position 1 to 476 as shown in SEQ ID NO 6), provided that the amino acid corresponding to position 25 is not F, the amino acid corresponding to position 64 is not L, the amino acid corresponding to position 88 is not T, the amino acid corresponding to position 157 is not T, the amino acid corresponding to position 165 is not R, the amino acid corresponding to position 169 is not V, the amino acid corresponding to position 174 is not E, the amino acid corresponding to position 187 is not S, the amino acid corresponding to position 197 is not T, the amino acid corresponding to position 239 is not S, the amino acid corresponding to position 327 is not S, the amino acid corresponding to position 328 is not V, the amino acid corresponding to position 384 is not Y, the amino acid corresponding to position 389 is not I, the amino acid corresponding to position 391 is not D, the amino acid corresponding to position 396 is not K, the amino acid corresponding to position 410 is not H, the amino acid corresponding to position 414 is not P, the amino acid corresponding to position 46 is not T, the amino acid corresponding to position 60 is not C, the amino acid corresponding to position 185 is not C, the amino acid corresponding to position 186 is not S, the amino acid corresponding to position 195 is not S, the amino acid corresponding to position 205 is not Y, the amino acid corresponding to position 252 is not V, the amino acid corresponding to position 268 is not S, the amino acid corresponding to position 409 is not R, the amino acid corresponding to position 436 is not A, and the amino acids corresponding to positions 477, 478 and 479 are deleted;

[0075] h) A protein having an amino acid sequence that is at least 60%, preferably 70%, more preferably 80%, still more preferably 90%, even more preferably 95%, even still more preferably 96%, particularly preferably 97%, most preferably 98% or particularly preferably 99% identical to any of the amino acid sequences defined in c) (the amino acid sequence from position 1 to 479 as shown in SEQ ID NO 9), provided that the amino acid corresponding to position 25 is not F, the amino acid corresponding to position 64 is not L, the amino acid corresponding to position 88 is not T, the amino acid corresponding to position 157 is not T, the amino acid corresponding to position 165 is not R, the amino acid corresponding to position 169 is not V, the amino acid corresponding to position 174 is not E, the amino acid corresponding to position 187 is not S, the amino acid corresponding to position 197 is not M, the amino acid corresponding to position 239 is not S, the amino acid corresponding to position 327 is not S, the amino acid corresponding to position 328 is not V, the amino acid corresponding to position 384 is not Y, the amino acid corresponding to position 389 is not I, the amino acid corresponding to position 391 is not D, the amino acid corresponding to position 396 is not K, the amino acid corresponding to position 410 is not H, the amino acid corresponding to position 414 is not P, the amino acid corresponding to position 2 is not S, the amino acid corresponding to position 48 is not D, the amino acid corresponding to position 69 is not P, the amino acid corresponding to position 90 is not S, the amino acid corresponding to position 164 is not Y, the amino acid corresponding to position 242 is not A, the amino acid corresponding to position 245 is not A, the amino acid corresponding to position 268 is not T, the amino acid corresponding to position 311 is not L, the amino acid corresponding to position 318 is not E, the amino acid corresponding to position 322 is not R, the amino acid corresponding to position 353 is not S, the amino acid corresponding to position 424 is not K, and the amino acid corresponding to position 452 is not E;

[0076] i) A protein having an amino acid sequence that is at least 60%, preferably 70%, more preferably 80%, still more preferably 90%, even more preferably 95%, even still more preferably 96%, particularly preferably 97%, most preferably 98% or particularly preferably 99% identical to any of the amino acid sequences defined in d) (the amino acid sequence from position 1 to 476 as shown in SEQ ID NO 12), provided that the amino acid corresponding to position 25 is not F, the amino acid corresponding to position 64 is not L, the amino acid corresponding to position 88 is not T, the amino acid corresponding to position 157 is not T, the amino acid corresponding to position 165 is not R, the amino acid corresponding to position 169 is not V, the amino acid corresponding to position 174 is not E, the amino acid corresponding to position 187 is not S, the amino acid corresponding to position 197 is not T, the amino acid corresponding to position 239 is not S, the amino acid corresponding to position 327 is not S, the amino acid corresponding to position 328 is not V, the amino acid corresponding to position 384 is not Y, the amino acid corresponding to position 389 is not I, the amino acid corresponding to position 391 is not D, the amino acid corresponding to position 396 is not K, the amino acid corresponding to position 410 is not H, the amino acid corresponding to position 414 is not P, the amino acid corresponding to position 46 is not T, the amino acid corresponding to position 60 is not C, the amino acid corresponding to position 185 is not C, the amino acid corresponding to position 186 is not C, the amino acid corresponding to position 195 is not S, the amino acid corresponding to position 205 is not Y, the amino acid corresponding to position 252 is not V, the amino acid corresponding to position 268 is not S, the amino acid corresponding to position 409 is not R, and the amino acid corresponding to position 436 is not A;

[0077] j) A protein having an amino acid sequence that is at least 60%, preferably 70%, more preferably 80%, still more preferably 90%, even more preferably 95%, even still more preferably 96%, particularly preferably 97%, most preferably 98% or particularly preferably 99% identical to any of the amino acid sequences defined in e) (the amino acid sequence from position 1 to 476 as shown in SEQ ID NO 15), provided that the amino acid corresponding to position 25 is not F, the amino acid corresponding to position 64 is not L, the amino acid corresponding to position 88 is not T, the amino acid corresponding to position 157 is not T, the amino acid corresponding to position 165 is not R, the amino acid corresponding to position 169 is not V, the amino acid corresponding to position 174 is not E, the amino acid corresponding to position 187 is not S, the amino acid corresponding to position 197 is not M, the amino acid corresponding to position 239 is not S, the amino acid corresponding to position 327 is not S, the amino acid corresponding to position 328 is not V, the amino acid corresponding to position 384 is not Y, the amino acid corresponding to position 389 is not I, the amino acid corresponding to position 391 is not D, the amino acid corresponding to position 396 is not K, the amino acid corresponding to position 410 is not H, the amino acid corresponding to position 414 is not P, the amino acid corresponding to position 48 is not D, the amino acid corresponding to position 164 is not Y, the amino acid corresponding to position 242 is not A, the amino acid corresponding to position 245 is not A, the amino acid corresponding to position 255 is not F, and the amino acid corresponding to position 424 is not K.

[0078] Positions 1 to 476 in SEQ ID NO 18 represent the amino acid sequence of the ω-TA variant protein, which contains all of the amino acid modifications described above compared to each of the amino acid sequences shown in SEQ ID NO 3 (from position 1 to 477), SEQ ID NO 6 (from position 1 to 479), SEQ ID NO 9 (from position 1 to 476), SEQ ID NO 12 (from position 1 to 476) and SEQ ID NO 15 (from position 1 to 476).

[0079] Table 1 summarizes the modifications present in the amino acid sequence of the ω-TA variant protein according to the invention (positions 1 to 476 in SEQ ID NO 18) compared to each amino acid sequence of wild-type ω-TA (positions 1 to 477 in SEQ ID NO 3, or positions 1 to 479 in SEQ ID NO 6, or positions 1 to 476 in SEQ ID NO 9, or positions 1 to 476 in SEQ ID NO 15) and compared to the modified ω-TA from Arthrobacter sp. (positions 1 to 476 in SEQ ID NO 12).

[0080]

[0081]

[0082] Table 1

[0083] "Terminal" in Table 1 refers to the position after the last amino acid present in the amino acid sequence of the separately known (wild-type) sequence.

[0084] Accordingly, a preferred embodiment of the present invention relates to a protein having ω-TA activity according to the present invention, selected from the following

[0085] a) a protein comprising the amino acid sequence of positions 1 to 476 as shown in SEQ ID NO 18;

[0086] b) a protein having an amino acid sequence having at least 60%, preferably 70%, more preferably 80%, still more preferably 90%, even more preferably 95%, even still more preferably 96%, particularly preferably 97%, most preferably 98% or particularly preferably 99% identity to the amino acid sequence of positions 1 to 476 as shown in SEQ ID NO 18, provided that the amino acids corresponding to positions 25, 64, 88, 157, 165, 169, 174, 187, 197, 239, 327, 328, 384, 389, 391, 396, 410 and 414 of SEQ ID NO 18 represent those shown at the respective positions in the amino acid sequence shown in SEQ ID NO 18;

[0087] c) a protein having an amino acid sequence having at least 60%, preferably 70%, more preferably 80%, still more preferably 90%, even more preferably 95%, even still more preferably 96%, particularly preferably 97%, most preferably 98% or particularly preferably 99% identity to the amino acid sequence of positions 1 to 476 as shown in SEQ ID NO 18, provided that the amino acids corresponding to positions 2, 25, 46, 48, 60, 64, 69, 88, 90, 157, 164, 165, 169, 174, 185, 186, 187, 195, 197, 202, 205, 239, 242, 245, 252, 255, 268, 311, 318, 322, 327, 328, 353, 359, 384, 389, 391, 396, 409, 410, 414, 424, 436, 452, 475 and 476 of SEQ ID NO 18 represent those shown at the respective positions in the amino acid sequence shown in SEQ ID NO 18.

[0088] In a most preferred embodiment, the protein encoding ω-TA according to the invention is a protein comprising the amino acid sequence from position 1 to 476 as shown in SEQ ID NO 18.

[0089] Up to now, the proteins described above herein are generally referred to herein as ω-TA variants or protein variants according to the invention.

[0090] It has been found that introducing further amino acid modifications into the protein variants according to the invention further improves the activity of the ω-TA variants, especially in terms of their substrate specificity, which means that compared with the ω-TA variants described above herein, these further modified ω-TA variants are more suitable for producing enantiomerically enriched or nearly pure products. As proteins according to the invention, ω-TAs comprising further modifications are further modified compared with the ω-TA variants described herein. ω-TA variants comprising further modifications are particularly suitable for producing enantiomerically enriched or enantiomerically nearly pure phosphoamino acids, and herein refer to ω-TA variants comprising further amino acid modifications or proteins according to the invention comprising further amino acid modifications.

[0091] Regarding ω-TA variants with further amino acid modifications, preferred methods for showing that a protein has ω-TA activity are described, for example, in WO 2017 / 151573, and a particularly preferred method for showing ω-TA variants with further amino acid modifications is described in item 7 of the "General Methods" herein.

[0092] "Enantiomerically enriched" herein means that one of the two enantiomers is present in a higher amount than the other enantiomer in a composition, preferably at least 60% of one enantiomer is present in the composition, more preferably 65% of one enantiomer is present in the composition, still more preferably at least 70% of one enantiomer is present in the composition, even more preferably at least 75% of one enantiomer is present in the composition, even more preferably at least 80% of one enantiomer is present in the composition, particularly preferably at least 85% of one enantiomer is present in the composition, most preferably at least 90% of one enantiomer is present in the composition or particularly preferably at least 94% of one enantiomer is present in the composition.

[0093] "Enantiomerically enriched" means that in the context herein, one of the two enantiomers is present in the composition in an amount of at least 95.0%, preferably one of the two enantiomers is present in the composition in an amount of at least 95.5%, more preferably one of the two enantiomers is present in the composition in an amount of at least 96.0%, still more preferably one of the two enantiomers is present in the composition in an amount of at least 96.5%, even more preferably one of the two enantiomers is present in the composition in an amount of at least 97.0%, even more preferably one of the two enantiomers is present in the composition in an amount of at least 98.0%, particularly preferably one of the two enantiomers is present in the composition in an amount of at least 98.5%, most preferably one of the two enantiomers is present in the composition in an amount of at least 99.0% or particularly preferably one of the two enantiomers is present in the composition in an amount of at least 99.5%.

[0094] Thus, another embodiment according to the invention relates to a protein variant according to the invention having the activity of an ω-TA variant, wherein the amino acid sequence according to the invention further comprises an amino acid modification as compared to the protein according to the invention.

[0095] Preferably, another embodiment of the invention relates to an amino acid sequence of a protein having an ω-TA activity (ω-TA variant) according to the invention comprising further amino acid modifications, and thus being a protein according to the invention having ω-TA activity, selected from the following

[0096] a) a protein according to the invention, except that the amino acid at position 166 is G and the amino acid at position 327 is Q;

[0097] b) a protein according to the invention, except that the amino acid at position 327 is Q and the amino acid at position 384 is S;

[0098] c) a protein according to the invention, except that the amino acid at position 326 is Q and the amino acid at position 327 is Q;

[0099] d) a protein according to the invention, except that the amino acid at position 327 is Q;

[0100] e) a protein according to the invention, except that the amino acid at position 326 is F and the amino acid at position 327 is Q;

[0101] f) a protein according to the invention, except that the amino acid at position 327 is C;

[0102] g) a protein according to the invention, except that the amino acid at position 327 is I;

[0103] h) a protein according to the invention, except that the amino acid at position 327 is M;

[0104] i) A protein according to the present invention, except that the amino acid at position 164 is Y;

[0105] j) A protein according to the present invention, except that the amino acid at position 164 is S;

[0106] k) A protein according to the present invention, except that the amino acid at position 327 is V;

[0107] l) A protein according to the present invention, except that the amino acid at position 409 is R;

[0108] m) A protein according to the present invention, except that the amino acid at position 327 is S;

[0109] n) A protein according to the present invention, except that the amino acid at position 271 is I;

[0110] o) A protein according to the present invention, except that the amino acid at position 329 is G;

[0111] p) A protein according to the present invention, except that the amino acid at position 409 is P;

[0112] q) A protein according to the present invention, except that the amino acid at position 414 is M;

[0113] r) A protein according to the present invention, except that the amino acid at position 165 is K;

[0114] s) A protein according to the present invention, except that the amino acid at position 414 is R;

[0115] t) A protein according to the present invention, except that the amino acid at position 414 is H;

[0116] u) A protein according to the present invention, except that the amino acid at position 165 is C;

[0117] v) A protein according to the present invention, except that the amino acid at position 327 is V;

[0118] w) A protein according to the present invention, except that the amino acid at position 164 is C;

[0119] x) A protein according to the present invention, except that the amino acid at position 409 is K.

[0120] A more preferred embodiment of the present invention relates to an amino acid sequence of a protein having ω-TA activity with further amino acid modifications, and relates to a protein having ω-TA activity, which is selected from the following

[0121] a) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO 18, except that the amino acid S at position 166 in SEQ ID NO 18 is replaced by G and the amino acid T at position 327 in SEQ ID NO 18 is replaced by Q;

[0122] b) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO 18, except that the amino acid T at position 327 in SEQ ID NO 18 is replaced by Q and the amino acid C at position 384 in SEQ ID NO 18 is replaced by S;

[0123] c) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO 18, except that the amino acid E at position 326 in SEQ ID NO 18 is replaced by Q and the amino acid T at position 327 in SEQ ID NO 18 is replaced by Q;

[0124] d) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO 18, except that the amino acid T at position 327 in SEQ ID NO 18 is replaced by Q;

[0125] e) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO 18, except that the amino acid E at position 326 in SEQ ID NO 18 is replaced by F and the amino acid T at position 327 in SEQ ID NO 18 is replaced by Q;

[0126] f) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO 18, except that the amino acid T at position 327 in SEQ ID NO 18 is replaced by C;

[0127] g) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO 18, except that the amino acid T at position 327 in SEQ ID NO 18 is replaced by I;

[0128] h) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO 18, except that the amino acid T at position 327 in SEQ ID NO 18 is replaced by M;

[0129] i) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO 18, except that the amino acid F at position 164 in SEQ ID NO 18 is replaced by Y;

[0130] j) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO 18, except that the amino acid F at position 164 in SEQ ID NO 18 is replaced by S;

[0131] k) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO 18, except that the amino acid T at position 327 in SEQ ID NO 18 is replaced by V;

[0132] l) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO 18, except that the amino acid T at position 409 in SEQ ID NO 18 is replaced by R;

[0133] m) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO 18, except that the amino acid T at position 327 in SEQ ID NO 18 is replaced by S;

[0134] n) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO 18, except that the amino acid V at position 271 in SEQ ID NO 18 is replaced by I;

[0135] o) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO 18, except that the amino acid S at position 329 in SEQ ID NO 18 is replaced by G;

[0136] p) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO 18, except that the amino acid T at position 409 in SEQ ID NO 18 is replaced by P;

[0137] q) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO 18, except that the amino acid L at position 414 in SEQ ID NO 18 is replaced by M;

[0138] r) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO 18, except that the amino acid Q at position 165 in SEQ ID NO 18 is replaced by K;

[0139] s) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO: 18, except that the amino acid L at position 414 in SEQ ID NO: 18 is replaced by R;

[0140] t) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO: 18, except that the amino acid L at position 414 in SEQ ID NO: 18 is replaced by H;

[0141] u) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO: 18, except that the amino acid Q at position 165 in SEQ ID NO: 18 is replaced by C;

[0142] v) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO: 18, except that the amino acid T at position 327 in SEQ ID NO: 18 is replaced by V;

[0143] w) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO: 18, except that the amino acid F at position 164 in SEQ ID NO: 18 is replaced by C;

[0144] x) A protein having the amino acid sequence from position 1 to 476 of the amino acid sequence shown in SEQ ID NO: 18, except that the amino acid T at position 409 in SEQ ID NO: 18 is replaced by K;

[0145] y) A protein having an amino acid sequence with at least 60%, preferably 70%, more preferably 80%, still more preferably 90%, even more preferably 95%, even still more preferably 96%, particularly preferably 97%, most preferably 98% or particularly preferably 99% identity to any of the amino acid sequences defined in a), b), c), d), e), f), g), h), i), j), k), l), m), n), o), p), q), r), s), t), u), v), w) or x), provided that at each amino acid position defined in a), b), c), d), e), f), g), h), i), j), k), l), m), n), o), p), q), r), s), t), u), v), w) or x) is also present at the corresponding amino acid position in an amino acid sequence of a protein having at least 60%, preferably 70%, more preferably 80%, still more preferably 90%, even more preferably 95%, even still more preferably 96%, particularly preferably 97%, most preferably 98% or particularly preferably 99% identity to any of the amino acid sequences defined in a), b), c), d), e), f), g), h), i), j), k), l), m), n), o), p), q), r), s), t), u), v), w) or x).

[0146] As an embodiment of the present invention, preferred proteins having ω-TA variant activity comprising further amino acid modifications are those proteins defined under a), b), c), d), e), f), g), h), i), j), k), l), m), n), o) and p) as defined above, more preferably those proteins defined under a), b), c), d), e), f), g) and h) as defined above and most preferably those proteins defined under a), b) and c) as defined above.

[0147] Table 2 summarizes additional amino acid modifications present in the amino acid sequence of ω-TA comprising further amino acid modifications as compared to the amino acid sequence shown in SEQ ID NO 18 (from position 1 to 476).

[0148]

[0149] Table 2

[0150] A further embodiment of the present invention relates to a nucleic acid molecule encoding a protein according to the present invention.

[0151] The nucleic acid molecule according to the invention can be any type of nucleic acid, as long as the nucleic acid encodes a protein according to the invention. The nucleic acid can be a ribonucleic acid molecule (e.g., RNA, mRNA) or a deoxyribonucleic acid molecule (DNA, including genomic DNA which may or may not contain introns and coding DNA).

[0152] Particularly of interest in the present invention are nucleic acid molecules encoding a protein having ω-TA activity, said ω-TA comprising the amino acid sequence as shown in positions 1 to 476 of SEQ ID NO 18.

[0153] Accordingly, the present invention also relates to nucleic acid molecules encoding a protein having the activity of ω-TA, selected from the following

[0154] a) a nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 17;

[0155] b) a nucleic acid molecule encoding a protein comprising the amino acid sequence from position 1 to 476 in the amino acid sequence shown in SEQ ID NO 18;

[0156] c) A nucleic acid molecule having at least 60%, preferably 70%, more preferably 80%, still more preferably 90%, even more preferably 95%, even still more preferably 96%, particularly preferably 97%, most preferably 98% or particularly preferably 99% identity to the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 17, provided that the codon corresponding to nucleotide positions 73 to 75 in SEQ ID NO 17 has the nucleotide sequence mgn, the codon corresponding to nucleotide positions 190 to 192 in SEQ ID NO 17 has the nucleotide sequence ath, the codon corresponding to nucleotide positions 262 to 264 in SEQ ID NO 17 has the nucleotide sequence gcn, the codon corresponding to nucleotide positions 469 to 471 in SEQ ID NO 17 has the nucleotide sequence gcn, the codon corresponding to nucleotide positions 493 to 495 in SEQ ID NO 17 has the nucleotide sequence mgn, the codon corresponding to nucleotide positions 505 to 507 in SEQ ID NO 17 has the nucleotide sequence gcn, the codon corresponding to nucleotide positions 520 to 522 in SEQ ID NO 17 has the nucleotide sequence ggn, the codon corresponding to nucleotide positions 589 to 591 in SEQ ID NO 17 has the nucleotide sequence gcn, the codon corresponding to nucleotide positions 559 to 561 in SEQ ID NO 17 has the nucleotide sequence aay, the codon corresponding to nucleotide positions 715 to 717 in SEQ ID NO 17 has the nucleotide sequence ccn, the codon corresponding to nucleotide positions 979 to 981 in SEQ ID NO 17 has the nucleotide sequence acn, the codon corresponding to nucleotide positions 982 to 984 in SEQ ID NO 17 has the nucleotide sequence ggn, the codon corresponding to nucleotide positions 1150 to 1152 in SEQ ID NO 17 has the nucleotide sequence tgy, the codon corresponding to nucleotide positions 1165 to 1167 in SEQ ID NO 17 has the nucleotide sequence ytn, the codon corresponding to nucleotide positions 1171 to 1173 in SEQ ID NO 17 has the nucleotide sequence gar, the codon corresponding to nucleotide positions 1186 to 1188 in SEQ ID NO 17 has the nucleotide sequence gar, the codon corresponding to nucleotide positions 1228 to 1230 in SEQ ID NO 17 has the nucleotide sequence mgn and the codon corresponding to nucleotide positions 1240 to 1242 in SEQ ID NO 17 has the nucleotide sequence ytn;

[0157] d) A nucleic acid molecule having at least 60%, preferably 70%, more preferably 80%, still more preferably 90%, even more preferably 95%, even still more preferably 96%, particularly preferably 97%, most preferably 98% or particularly preferably 99% identity to the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 17, provided that the codon corresponding to nucleotide positions 4 to 6 in SEQ ID NO 17 has the nucleotide sequence ggn, the codon corresponding to nucleotide positions 73 to 75 in SEQ ID NO 17 has the nucleotide sequence mgn, the codon corresponding to nucleotide positions 136 to 138 in SEQ ID NO 17 has the nucleotide sequence atg, the codon corresponding to nucleotide positions 142 to 144 in SEQ ID NO 17 has the nucleotide sequence ggn, the codon corresponding to nucleotide positions 178 to 180 in SEQ ID NO 17 has the nucleotide sequence tay, the codon corresponding to nucleotide positions 190 to 192 in SEQ ID NO 17 has the nucleotide sequence ath, the codon corresponding to nucleotide positions 205 to 207 in SEQ ID NO 17 has the nucleotide sequence car, the codon corresponding to nucleotide positions 262 to 264 in SEQ ID NO 17 has the nucleotide sequence gcn, the codon corresponding to nucleotide positions 268 to 270 in SEQ ID NO 17 has the nucleotide sequence gcn, the codon corresponding to nucleotide positions 469 to 471 in SEQ ID NO 17 has the nucleotide sequence gcn, the codon corresponding to nucleotide positions 490 to 492 in SEQ ID NO 17 has the nucleotide sequence tty, the codon corresponding to nucleotide positions 493 to 495 in SEQ ID NO 17 has the nucleotide sequence car, the codon corresponding to nucleotide positions 505 to 507 in SEQ ID NO 17 has the nucleotide sequence gcn, the codon corresponding to nucleotide positions 520 to 522 in SEQ ID NO 17 has the nucleotide sequence ggn, the codon corresponding to nucleotide positions 553 to 555 in SEQ ID NO 17 has the nucleotide sequence tay, the codon corresponding to nucleotide positions 556 to 558 in SEQ ID NO 17 has the nucleotide sequence aay, the codon corresponding to nucleotide positions 559 to 561 in SEQ ID NO 17 has the nucleotide sequence aay, the codon corresponding to nucleotide positions 583 to 585 in SEQ ID NO 17 has the nucleotide sequence ccn, the codon corresponding to nucleotide positions 589 to 591 in SEQ ID NO 17 has the nucleotide sequence gcn, the codon corresponding to nucleotide positions 604 to 606 in SEQ ID NO 17 has the nucleotide sequence aay,The codon corresponding to nucleotide positions 613 to 615 in SEQ ID NO 17 has the nucleotide sequence tgy, the codon corresponding to nucleotide positions 715 to 717 in SEQ ID NO 17 has the nucleotide sequence ccn, the codon corresponding to nucleotide positions 724 to 726 in SEQ ID NO 17 has the nucleotide sequence gtn, the codon corresponding to nucleotide positions 733 to 735 in SEQ ID NO 17 has the nucleotide sequence acn, the codon corresponding to nucleotide positions 754 to 756 in SEQ ID NO 17 has the nucleotide sequence ath, the codon corresponding to nucleotide positions 763 to 765 in SEQ ID NO 17 has the nucleotide sequence ath, the codon corresponding to nucleotide positions 802 to 804 in SEQ ID NO 17 has the nucleotide sequence aay, the codon corresponding to nucleotide positions 931 to 933 in SEQ ID NO 17 has the nucleotide sequence gtn, the codon corresponding to nucleotide positions 952 to 954 in SEQ ID NO 17 has the nucleotide sequence gcn, the codon corresponding to nucleotide positions 964 to 966 in SEQ ID NO 17 has the nucleotide sequence aar, the codon corresponding to nucleotide positions 979 to 981 in SEQ ID NO 17 has the nucleotide sequence acn, the codon corresponding to nucleotide positions 982 to 984 in SEQ ID NO 17 has the nucleotide sequence ggn, the codon corresponding to nucleotide positions 1057 to 1059 in SEQ ID NO 17 has the nucleotide sequence ytn, the codon corresponding to nucleotide positions 1075 to 1077 in SEQ ID NO 17 has the nucleotide sequence aay, the codon corresponding to nucleotide positions 1150 to 1152 in SEQ ID NO 17 has the nucleotide sequence tay, the codon corresponding to nucleotide positions 1165 to 1167 in SEQ ID NO 17 has the nucleotide sequence ytn, the codon corresponding to nucleotide positions 1171 to 1173 in SEQ ID NO 17 has the nucleotide sequence gar, the codon corresponding to nucleotide positions 1186 to 1188 in SEQ ID NO 17 has the nucleotide sequence gar, the codon corresponding to nucleotide positions 1225 to 1227 in SEQ ID NO 17 has the nucleotide sequence acn, the codon corresponding to nucleotide positions 1228 to 1230 in SEQ ID NO 17 has the nucleotide sequence mgn, the codon corresponding to nucleotide positions 1240 to 1242 in SEQ ID NO 17 has the nucleotide sequence ytn, the codon corresponding to nucleotide positions 1270 to 1272 in SEQ ID NO 17 has the nucleotide sequence garThe codon corresponding to nucleotide positions 1306 to 1308 in SEQ ID NO 17 has the nucleotide sequence gtn and the codon corresponding to nucleotide positions 1354 to 1356 in SEQ ID NO 17 has the nucleotide sequence ggn;

[0158] e) A nucleic acid molecule that hybridizes to the complementary strand of the nucleic acid molecule defined in a), b), c) or d), provided that the codon corresponding to nucleotide positions 73 to 75 in SEQ ID NO 17 has the nucleotide sequence mgn, the codon corresponding to nucleotide positions 190 to 192 in SEQ ID NO 17 has the nucleotide sequence ath, the codon corresponding to nucleotide positions 262 to 264 in SEQ ID NO 17 has the nucleotide sequence gcn, the codon corresponding to nucleotide positions 469 to 471 in SEQ ID NO 17 has the nucleotide sequence gcn, the codon corresponding to nucleotide positions 493 to 495 in SEQ ID NO 17 has the nucleotide sequence mgn, the codon corresponding to nucleotide positions 505 to 507 in SEQ ID NO 17 has the nucleotide sequence gcn, the codon corresponding to nucleotide positions 520 to 522 in SEQ ID NO 17 has the nucleotide sequence ggn, the codon corresponding to nucleotide positions 559 to 561 in SEQ ID NO 17 has the nucleotide sequence aay, the codon corresponding to nucleotide positions 715 to 717 in SEQ ID NO 17 has the nucleotide sequence ccn, the codon corresponding to nucleotide positions 979 to 981 in SEQ ID NO 17 has the nucleotide sequence acn, the codon corresponding to nucleotide positions 982 to 984 in SEQ ID NO 17 has the nucleotide sequence ggn, the codon corresponding to nucleotide positions 1150 to 1152 in SEQ ID NO 17 has the nucleotide sequence tgy, the codon corresponding to nucleotide positions 1165 to 1167 in SEQ ID NO 17 has the nucleotide sequence ytn, the codon corresponding to nucleotide positions 1171 to 1173 in SEQ ID NO 17 has the nucleotide sequence gar, the codon corresponding to nucleotide positions 1186 to 1188 in SEQ ID NO 17 has the nucleotide sequence gar, the codon corresponding to nucleotide positions 1228 to 1230 in SEQ ID NO 17 has the nucleotide sequence mgn and the codon corresponding to nucleotide positions 1240 to 1242 in SEQ ID NO 17 has the nucleotide sequence ytn;

[0159] f) A nucleic acid molecule that hybridizes to the complementary strand of the nucleic acid molecule defined in a), b), c), or d), provided that the codon corresponding to nucleotide positions 4 to 6 in SEQ ID NO: 17 has the nucleotide sequence ggn, the codon corresponding to nucleotide positions 73 to 75 in SEQ ID NO: 17 has the nucleotide sequence mgn, the codon corresponding to nucleotide positions 136 to 138 in SEQ ID NO: 17 has the nucleotide sequence atg, the codon corresponding to nucleotide positions 142 to 144 in SEQ ID NO: 17 has the nucleotide sequence ggn, the codon corresponding to nucleotide positions 178 to 180 in SEQ ID NO: 17 has the nucleotide sequence tay, the codon corresponding to nucleotide positions 190 to 192 in SEQ ID NO: 17 has the nucleotide sequence ath, the codon corresponding to nucleotide positions 205 to 207 in SEQ ID NO: 17 has the nucleotide sequence car, the codon corresponding to nucleotide positions 262 to 264 in SEQ ID NO: 17 has the nucleotide sequence gcn, the codon corresponding to nucleotide positions 268 to 270 in SEQ ID NO: 17 has the nucleotide sequence gcn, the codon corresponding to nucleotide positions 469 to 471 in SEQ ID NO: 17 has the nucleotide sequence gcn, the codon corresponding to nucleotide positions 490 to 492 in SEQ ID NO: 17 has the nucleotide sequence tty, the codon corresponding to nucleotide positions 493 to 495 in SEQ ID NO: 17 has the nucleotide sequence car, the codon corresponding to nucleotide positions 505 to 507 in SEQ ID NO: 17 has the nucleotide sequence gcn, the codon corresponding to nucleotide positions 520 to 522 in SEQ ID NO: 17 has the nucleotide sequence ggn, the codon corresponding to nucleotide positions 553 to 555 in SEQ ID NO: 17 has the nucleotide sequence tay, the codon corresponding to nucleotide positions 556 to 558 in SEQ ID NO: 17 has the nucleotide sequence aay, the codon corresponding to nucleotide positions 559 to 561 in SEQ ID NO: 17 has the nucleotide sequence aay, the codon corresponding to nucleotide positions 583 to 585 in SEQ ID NO: 17 has the nucleotide sequence ccn, the codon corresponding to nucleotide positions 589 to 591 in SEQ ID NO: 17 has the nucleotide sequence gcn, the codon corresponding to nucleotide positions 604 to 606 in SEQ ID NO: 17 has the nucleotide sequence aay, the codon corresponding to nucleotide positions 613 to 615 in SEQ ID NO: 17 has the nucleotide sequence tgy, the codon corresponding to nucleotide positions 715 to 717 in SEQ ID NO: 17 has the nucleotide sequence ccn,The codon corresponding to nucleotide positions 724 to 726 in SEQ ID NO 17 has the nucleotide sequence gtn, the codon corresponding to nucleotide positions 733 to 735 in SEQ ID NO 17 has the nucleotide sequence acn, the codon corresponding to nucleotide positions 754 to 756 in SEQ ID NO 17 has the nucleotide sequence ath, the codon corresponding to nucleotide positions 763 to 765 in SEQ ID NO 17 has the nucleotide sequence ath, the codon corresponding to nucleotide positions 802 to 804 in SEQ ID NO 17 has the nucleotide sequence aay, the codon corresponding to nucleotide positions 931 to 933 in SEQ ID NO 17 has the nucleotide sequence gtn, the codon corresponding to nucleotide positions 952 to 954 in SEQ ID NO 17 has the nucleotide sequence gcn, the codon corresponding to nucleotide positions 964 to 966 in SEQ ID NO 17 has the nucleotide sequence aar, the codon corresponding to nucleotide positions 979 to 981 in SEQ ID NO 17 has the nucleotide sequence acn, the codon corresponding to nucleotide positions 982 to 984 in SEQ ID NO 17 has the nucleotide sequence ggn, the codon corresponding to nucleotide positions 1057 to 1059 in SEQ ID NO 17 has the nucleotide sequence ytn, the codon corresponding to nucleotide positions 1075 to 1077 in SEQ ID NO 17 has the nucleotide sequence aay, the codon corresponding to nucleotide positions 1150 to 1152 in SEQ ID NO 17 has the nucleotide sequence tay, the codon corresponding to nucleotide positions 1165 to 1167 in SEQ ID NO 17 has the nucleotide sequence ytn, the codon corresponding to nucleotide positions 1171 to 1173 in SEQ ID NO 17 has the nucleotide sequence gar, the codon corresponding to nucleotide positions 1186 to 1188 in SEQ ID NO 17 has the nucleotide sequence gar, the codon corresponding to nucleotide positions 1225 to 1227 in SEQ ID NO 17 has the nucleotide sequence acn, the codon corresponding to nucleotide positions 1228 to 1230 in SEQ ID NO 17 has the nucleotide sequence mgn, the codon corresponding to nucleotide positions 1240 to 1242 in SEQ ID NO 17 has the nucleotide sequence ytn, the codon corresponding to nucleotide positions 1270 to 1272 in SEQ ID NO 17 has the nucleotide sequence gar, the codon corresponding to nucleotide positions 1306 to 1308 in SEQ ID NO 17 has the nucleotide sequence gtn and the codon corresponding to nucleotide positions 1354 to 1356 in SEQ ID NO 17 has the nucleotide sequence ggn;,

[0160] g) a nucleic acid molecule derived from the nucleic acid molecule defined in a), b), c), d), e) or f) due to codon degeneracy;

[0161] h) a nucleic acid molecule encoding a protein having at least 60%, preferably 70%, more preferably 80%, still more preferably 90%, even more preferably 95%, even still more preferably 96%, particularly preferably 97%, most preferably 98% or particularly preferably 99% identity to the amino acid sequence of positions 1 to 476 as shown in SEQ ID NO 18, provided that the amino acids corresponding to positions 25, 64, 88, 157, 165, 169, 174, 187, 197, 239, 327, 328, 384, 389, 391, 396, 410 and 414 of SEQ ID NO 18 represent those shown at the respective positions in the amino acid sequence shown in SEQ ID NO 18;

[0162] i) a nucleic acid molecule encoding a protein having at least 60%, preferably 70%, more preferably 80%, still more preferably 90%, even more preferably 95%, even still more preferably 96%, particularly preferably 97%, most preferably 98% or particularly preferably 99% identity to the amino acid sequence of positions 1 to 476 as shown in SEQ ID NO 18, provided that the amino acids corresponding to positions 2, 25, 46, 48, 60, 64, 69, 88, 90, 157, 164, 165, 169, 174, 185, 186, 187, 195, 197, 202, 205, 239, 242, 245, 252, 255, 268, 311, 318, 322, 327, 328, 353, 359, 384, 389, 391, 396, 409, 410, 414, 424, 436, 452, 475 and 476 of SEQ ID NO 18 represent those shown at the respective positions in the amino acid sequence shown in SEQ ID NO 18;

[0163] j) a nucleic acid molecule comprising the nucleic acid sequence of positions 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 16.

[0164] SEQ ID NO 16 shows the nucleotide sequence obtained by back - translation of the protein having the amino acid sequence shown in SEQ ID NO 18, which reflects the degeneracy of the genetic code.

[0165] SEQ ID NO 17 is a synthetic nucleic acid molecule obtained by specific nucleotide substitution due to the degeneracy of the flexible nucleotides of the genetic code in SEQ ID NO 16. Both SEQ ID NO 16 and SEQ ID NO 17 encode a protein with ω-TA activity having the amino acid sequence shown in SEQ ID NO 18.

[0166] In the context of the present invention, the term "hybridizes with" refers to hybridization under conventional hybridization conditions (preferably under stringent conditions), for example, as described in Sambrook et al. (Molecular Cloning, A Laboratory Manual, 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. ISBN: 0879695773) or Ausubel et al. (Short Protocols in Molecular Biology, John Wiley & Sons; 5th Edition (2002), ISBN: 0471250929). Particularly preferably, "hybridization" refers to hybridization under the following conditions:

[0167] Hybridization buffer:

[0168] 2xSSC; 10xDenhardt's solution (Fikoll 400 + PEG + BSA; ratio 1:1:1); 0.1% SDS; 5 mM EDTA; 50 mM Na2HPO4; 250 μg / ml herring sperm DNA; 50 μg / ml tRNA;

[0169] Or

[0170] 25 M sodium phosphate buffer pH 7.2; 1 mM EDTA; 7% SDS

[0171] Hybridization temperature: T = 65 to 68 °C

[0172] Wash buffer: 0.1xSSC; 0.1% SDS

[0173] Wash temperature: T = 65 to 68 °C.

[0174] A nucleic acid molecule that hybridizes with a nucleic acid molecule encoding a protein with ω-TA activity can be derived from any organism; thus, it can be derived from bacteria, fungi, animals, humans, plants, or viruses.

[0175] Nucleic acid molecules that hybridize to nucleic acid molecules encoding proteins with ω-TA activity are preferably derived from microorganisms, more preferably from fungi or bacteria, and most preferably from bacteria.

[0176] The nucleic acid molecules that hybridize to the indicated molecules can be isolated, for example, from genomic or cDNA libraries. Such nucleic acid molecules can be identified and isolated using the nucleic acid molecules described herein, or a portion of these molecules or the reverse complements of these molecules can be used for identification and isolation, for example, by hybridization according to standard methods (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. ISBN: 0879695773; Ausubel et al., Short Protocols in Molecular Biology, John Wiley & Sons; 5th Edition (2002), ISBN: 0471250929) or by using PCR amplification.

[0177] Nucleic acid molecules having, for example, exactly or substantially the nucleic acid sequence of positions 1 to 1431 as set forth in SEQ ID NO 2, or substantially the nucleic acid sequence of positions 1 to 1437 as set forth in SEQ ID NO 5, or substantially the nucleic acid sequence as set forth in SEQ ID NO 8, or substantially the nucleic acid sequence as set forth in SEQ ID NO 11, or substantially the nucleic acid sequence as set forth in SEQ ID NO 14, or substantially the nucleic acid sequence as set forth in SEQ ID NO 17, or fragments of these nucleic acid sequences can be used as hybridization samples for isolating nucleic acid sequences encoding proteins with ω-TA activity.

[0178] The fragments used as hybridization samples can also be synthetic fragments or oligonucleotides prepared using conventional synthetic techniques, the sequences of which are substantially the same as the nucleic acid molecules described in the context of the present invention. Once a gene that hybridizes to the nucleic acid sequences described in the context of the present invention is identified and isolated, the sequence should be determined and the properties of the protein encoded by the sequence should be analyzed to determine whether it is a protein with ω-TA activity. Methods for determining whether a certain protein has the protein activity of a protein with ω-TA activity are well known to those skilled in the art and have been mentioned above in this text.

[0179] Molecules that hybridize to the nucleic acid molecules described in the context of the present invention particularly include fragments, derivatives, and allelic variants of the nucleic acid molecules mentioned. In the context of the present invention, the term "derivative" means that the sequences of these molecules differ from the sequences of the nucleic acid molecules described above at one or more positions and are highly identical to these sequences. The differences from the above nucleic acid molecules may be caused, for example, by deletions, additions, substitutions, insertions, or recombinations.

[0180] Another embodiment of the present invention relates to a nucleic acid molecule encoding a protein having ω-TA activity with further amino acid modifications, which relates to a nucleic acid molecule according to the present invention encoding a protein having ω-TA activity, and is selected from the following

[0181] a) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17. In addition, the codon at nucleotide positions 496 to 498 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence ggn, and the codon at nucleotide positions 979 to 981 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence car;

[0182] b) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17. In addition, the codon at nucleotide positions 979 to 981 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence car, and the codon at nucleotide positions 1150 to 1152 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence wsn;

[0183] c) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17. In addition, the codon at nucleotide positions 976 to 978 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence car, and the codon at nucleotide positions 979 to 981 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence car;

[0184] d) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17. In addition, the codon at nucleotide positions 979 to 981 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence car;

[0185] e) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17, except that the codon at nucleotide positions 976 to 978 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence tty and the codon at nucleotide positions 979 to 981 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence car;

[0186] f) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17, except that the codon at nucleotide positions 979 to 981 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence car;

[0187] g) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17, except that the codon at nucleotide positions 979 to 981 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence ath;

[0188] h) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17, except that the codon at nucleotide positions 979 to 981 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence atg;

[0189] i) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17, except that the codon at nucleotide positions 490 to 492 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence tay;

[0190] j) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17, except that the codon at nucleotide positions 490 to 492 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence wsn;

[0191] k) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17, except that the codon at nucleotide positions 979 to 981 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence gtn;

[0192] l) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17, except that the codon at nucleotide positions 1225 to 1227 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence mgn;

[0193] m) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17, except that the codon at nucleotide positions 979 to 981 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence wsn;

[0194] n) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17, except that the codon at nucleotide positions 811 to 813 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence ath;

[0195] o) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17, except that the codon at nucleotide positions 985 to 987 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence ggn;

[0196] p) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17, except that the codon at nucleotide positions 1225 to 1227 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence ccn;

[0197] q) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17, except that the codon at nucleotide positions 1240 to 1242 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence atg;

[0198] r) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17, except that the codon at nucleotide positions 493 to 495 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence aar;

[0199] s) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17, except that the codon at nucleotide positions 1240 to 1242 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence mgn;

[0200] t) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17, except that the codon at nucleotide positions 1240 to 1242 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence cay;

[0201] u) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17, except that the codon at nucleotide positions 493 to 495 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence tgy;

[0202] v) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17, except that the codon at nucleotide positions 979 to 981 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence gtn;

[0203] w) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17, except that the codon at nucleotide positions 490 to 492 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence tgy;

[0204] x) A nucleic acid molecule comprising the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17, except that the codon at nucleotide positions 1225 to 1227 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence aar;

[0205] y) A nucleic acid molecule having a nucleic acid sequence with at least 60%, preferably 70%, more preferably 80%, still more preferably 90%, even more preferably 95%, even still more preferably 96%, particularly preferably 97%, most preferably 98% or particularly preferably 99% identity to any of the nucleic acid sequences defined in a), b), c), d), e), f), g), h), i), j), k), l), m), n), o), p), q), r), s), t), u), v), w) or x), provided that the nucleotide sequence of each codon as defined in a), b), c), d), e), f), g), h), i), j), k), l), m), n), o), p), q), r), s), t), u), v), w) or x) is also present at the nucleotide position of the corresponding codon in a nucleic acid sequence having at least 60%, preferably 70%, more preferably 80%, still more preferably 90%, even more preferably 95%, even still more preferably 96%, particularly preferably 97%, most preferably 98% or particularly preferably 99% identity to the nucleic acid sequence defined in each of a), b), c), d), e), f), g), h), i), j), k), l), m), n), o), p), q), r), s), t), u), v), w) or x).

[0206] Preferred nucleic acid molecules according to the invention are those nucleic acid molecules defined under a), b), c), d), e), f), g), h), i), j), k), l), m), n), o) and p) above, more preferably those nucleic acid molecules defined under a) to k) above, even more preferably those nucleic acid molecules defined under a), b), c), d), e), f), g), h) above and most preferably those nucleic acid molecules defined under a), b) and c) above.

[0207] The meanings of the nucleotide abbreviations a, c, g, t and the abbreviations of the degenerate nucleotides r, y, s, w, k, m, b, d, h, v, n can be deduced from Table 3 below the "Sequence Listing" subheading. Which amino acids are encoded by codons containing degenerate nucleotides can be deduced from Table 5 below the "Sequence Listing" subheading.

[0208] Furthermore, the invention relates to a recombinant nucleic acid molecule comprising a nucleic acid molecule according to the invention.

[0209] In connection with the present invention, the term "recombinant nucleic acid molecule" should be understood to refer to a nucleic acid molecule which, in addition to the nucleic acid molecule according to the present invention, contains other sequences which are not naturally occurring in the combination in which they are present in the recombinant nucleic acid according to the present invention. Herein, said other sequences can be any sequences, preferably they are functional or regulatory sequences (promoters, termination signals, enhancers, ribosome binding sites (rbs), leader sequences which enhance transcription, translation or RNA stability, subcellular targeting sequences, etc.), particularly preferably they are functional or regulatory sequences which are active in microorganisms, and especially particularly preferably they are regulatory sequences which are active in fungi (especially in yeast) or in bacteria. Methods for producing the recombinant nucleic acid molecules according to the present invention are known to the person skilled in the art and include genetic methods such as the bonding of nucleic acid molecules by ligation, genetic recombination or de novo synthesis of nucleic acid molecules. Those methods are described, for example, in Sambrok et al. (Molecular Cloning, A Laboratory Manual, 3rd Edition (2001) Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY. ISBN: 0879695773) or Ausubel et al. (Short Protocols in Molecular Biology, John Wiley & Sons; 5th Edition (2002), ISBN: 0471250929).

[0210] In a further embodiment, the recombinant nucleic acid molecule according to the present invention comprises a nucleic acid molecule according to the present invention linked to a regulatory sequence which initiates transcription in a prokaryotic or eukaryotic cell.

[0211] A regulatory sequence which "initiates transcription" in a cell is also referred to as a promoter.

[0212] Information on regulatory sequences and plasmids is known to the person skilled in the art and is described, for example, in the Registry of Standard Biological Parts supported by the International Genetically Engineered Machine (iGEM) Foundation (One Kendall Square, Suite B6104, Cambridge, MA 02139, USA) in the World Wide Web (http: / / parts.igem.org / Catalog).

[0213] Regulatory sequences for initiating transcription in prokaryotes (e.g., E. coli) and in eukaryotes are well described in the literature, and in particular, expression in yeast (e.g., Saccharomyces cerevisiae) is described. An overview of various systems for expressing proteins in various host organisms can be found, for example, in Methods in Enzymology 153 (1987), 383-516 and Bitter et al. (Methods in Enzymology 153 (1987), 516-544) or Gomes et al. (2016, Advances in Animal and Veterinary Sciences, 4(4), 346) and Baghban et al. (2018, Current Pharmaceutical Biotechnology, 19(6)). Common yeast promoters are pAOX1, pHIS4, pGAL, pScADH2 (Baghban et al., 2018, see above). Common bacterial promoters are T5, T7, rhamnose-inducible, arabinose-inducible, PhoA, the artificial trc (trp-lac) promoter, as described by Marschall et al. (2017, Appl Microbiol Biotechnol 101, 501–512) and Tegel et al. (2011, FEBS Journal 278, 729–739).

[0214] A further embodiment of the recombinant nucleic acid molecule of the present invention is a vector or plasmid comprising the nucleic acid molecule according to the present invention.

[0215] "Vector" is as generally understood in the field of molecular biology and herein represents a nucleic acid sequence or vehicle comprising a nucleic acid sequence for transferring genetic material (DNA or RNA) into a target cell. The vector can be a plasmid, such as the T-DNA or binary vector for generating transgenic plants, an expression vector for expressing a nucleic acid sequence in a host cell, a shuttle vector capable of replicating in different hosts, or the vector can be a viral particle or phage that has been modified to deliver foreign genetic material to a host.

[0216] "Plasmid" is as generally understood in the field of molecular biology and herein refers to an autonomously self-replicating, usually circular DNA molecule when it is present in a host cell separate from chromosomal DNA.

[0217] The nucleic acid molecule according to the present invention, the recombinant nucleic acid molecule according to the present invention, the vector or plasmid according to the present invention, for example, by expressing the nucleic acid molecule according to the present invention in a host cell, can be used to produce the protein according to the present invention.

[0218] Another embodiment of the present invention relates to a host or host cell comprising or expressing a nucleic acid molecule according to the present invention, or comprising a protein according to the present invention, or comprising a recombinant nucleic acid molecule according to the present invention, or comprising a vector according to the present invention, or comprising a plasmid according to the present invention.

[0219] The nucleic acid molecule according to the present invention encoding a protein having ω-TA activity can be expressed in a host cell for, for example, its proliferation or production of the protein of the present invention. For expression in a host cell, the nucleic acid molecule according to the present invention can be included in a vector or plasmid, or can be stably integrated into the genome of each host cell. The nucleic acid molecule according to the present invention can also be included in a vector that supports its introduction into the host cell.

[0220] A further embodiment of the present invention relates to a host or host cell according to the present invention comprising a nucleic acid molecule according to the present invention, or comprising a recombinant nucleic acid molecule according to the present invention, or comprising a vector according to the present invention, or comprising a plasmid according to the present invention, and in each case comprising a protein according to the present invention.

[0221] Another embodiment of the present invention relates to a host or host cell according to the present invention comprising a nucleic acid molecule according to the present invention, or comprising a recombinant nucleic acid molecule according to the present invention, or comprising a vector according to the present invention, or comprising a plasmid according to the present invention, and in each case expressing a protein according to the present invention.

[0222] Another embodiment of the present invention relates to a host or host cell according to the present invention comprising a nucleic acid molecule according to the present invention, or comprising a recombinant nucleic acid molecule according to the present invention, or comprising a vector according to the present invention, or comprising a plasmid according to the present invention, and in each case expressing a certain protein, wherein the protein has the activity of ω-transaminase.

[0223] "Expressing a nucleic acid molecule" should be understood herein to mean that in the case where the nucleic acid molecule is RNA or mRNA, the nucleic acid molecule is translated into a protein, preferably into a protein having ω-TA activity, or in the case where the nucleic acid molecule is DNA or cDNA, it is transcribed (and in the case where genomic DNA containing introns is processed) into mRNA, preferably into mRNA encoding a protein having ω-TA activity, and subsequently translated into a protein, preferably into a protein having ω-TA activity.

[0224] Transcription of a given nucleic acid molecule in a host can be demonstrated by methods well known to those skilled in the art, for example, by Northern blot analysis or RT-PCR to detect specific transcripts (mRNA) of the foreign nucleic acid molecule.

[0225] Whether a host or host cell contains a given protein or a protein derived from the expression of a nucleic acid molecule can be determined by methods well known to those skilled in the art, for example, by immunological methods such as Western blot analysis, ELISA (enzyme-linked immunosorbent assay) or RIA (radioimmunoassay). Those skilled in the art are familiar with methods for preparing antibodies that specifically react with a certain protein (i.e., that specifically bind to a certain protein) (see, for example, Lottspeich and Zorbas (eds.), 1998, Bioanalytik, Spektrum akad, Verlag, Heidelberg, Berlin, ISBN 3-8274-0041-4). Some companies (Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA USA 0245; GenScript, 60 Centennial Ave., Piscataway, NJ 08854, USA) offer ordering services for the preparation of such antibodies.

[0226] In addition, those skilled in the art can test whether a host or host cell contains a protein according to the invention by detecting the (additional) activity of a protein having ω-TA activity in the corresponding host cell. Preferably, the activity of a protein having additional ω-TA activity in the corresponding host cell is detected by comparing the ω-TA activity of a host cell according to the invention with the corresponding activity of a host cell that does not contain a protein according to the invention.

[0227] As described above, it is possible to detect whether a protein has the activity of ω-TA.

[0228] A host or host cell according to the invention can be produced by those skilled in the art by known methods of genetic modification or transformation of an organism.

[0229] Accordingly, another subject of the invention is a host or host cell according to the invention, in particular a prokaryotic or eukaryotic host or host cell, which is genetically modified (or transformed) by a nucleic acid molecule according to the invention or by a recombinant nucleic acid molecule according to the invention or by a vector according to the invention or by a plasmid according to the invention. Preferably, the genetically modified (transformed) host or host cell according to the invention expresses a protein having ω-transaminase activity, and more preferably, the genetically modified (transformed) host or host cell according to the invention expresses a protein according to the invention.

[0230] "Genetically modified by a nucleic acid molecule" or "transformed by a nucleic acid molecule" as used herein shall be understood to mean the introduction of a nucleic acid molecule into a host or host cell by a technical and / or non-naturally occurring method, preferably by a technical method in the field of molecular biology, biotechnology or genetic modification.

[0231] Descendants, offspring or progeny of the host or host cell according to the invention are also embodiments of the invention. Preferably, these descendants, offspring or progeny contain a nucleic acid molecule according to the invention, or a recombinant nucleic acid molecule according to the invention, or a vector according to the invention, or a plasmid according to the invention, or a protein according to the invention. More preferably, these descendants, offspring or progeny contain a nucleic acid molecule according to the invention, or a recombinant nucleic acid molecule according to the invention, or a vector according to the invention, or a plasmid according to the invention, and in each case express a certain protein, wherein the protein has the activity of ω-TA. Even more preferably, these descendants, offspring or progeny contain a nucleic acid molecule according to the invention, or a recombinant nucleic acid molecule according to the invention, or a vector according to the invention, or a plasmid according to the invention, and in each case express a certain protein, wherein the protein has the activity of ω-TA according to the invention.

[0232] The host or host cell according to the invention can be a host or host cell from any prokaryotic or eukaryotic organism. The host or host cell can be a bacterium or a bacterial cell (e.g., E. coli, Bacillus bacteria, especially Bacillus subtilis, Agrobacterium, especially Agrobacterium tumefaciens or Agrobacterium rhizogenes, Pseudomonas, especially Pseudomonas fluorescens, Streptomyces, Rhodococcus, especially Rhodococcus rhodochrous, Vibrio natrigens, Corynebacterium, especially Corynebacterium glutamicum), or a fungus or a fungal cell (e.g., Agaricus, especially Agaricus bisporus, Aspergillus, Trichoderma or yeast, especially S. cerevisiae, Pichia, such as P. pastori), and a plant or a plant cell, or it can be an animal or an animal cell.

[0233] Preferred host cells according to the invention are cells of microorganisms. Within the framework of this patent application, this is understood to include all bacteria and protists (e.g., fungi, especially yeasts and algae), as defined, for example, in Schlegel "General Microbiology" (Georg Thieme Publishing House (1985), 1-2).

[0234] With respect to microorganisms, the host or host cell according to the invention is preferably a bacterium / bacterial cell or a yeast / yeast cell, and most preferably it is a bacterium / cell cell. With respect to bacterium / bacterial cells, the host or host cell according to the invention is preferably a Bacillus / Bacillus cell or an Escherichia coli / Escherichia coli cell, and most preferably it is an Escherichia coli / Escherichia coli cell.

[0235] Alternatively, Pseudomonas, especially Pseudomonas fluorescens, Streptomyces, Rhodococcus, especially Rhodococcus rhodochrous, Vibrio, especially Vibrio natriegens, Corynebacterium, especially Corynebacterium glutamicum, or others can be the host or host cell according to the invention.

[0236] A preferred embodiment of the invention relates to a host or host cell according to the invention comprising a nucleic acid molecule according to the invention, wherein the nucleic acid molecule according to the invention is characterized in that the codons of the nucleic acid molecule are altered such that they are respectively adapted to the codon usage frequency of the host or host cell.

[0237] The host cell according to the invention can be used for the production of a protein according to the invention. The protein according to the invention can be used in a method for producing an enantiomerically enriched or nearly enantiomerically pure amine from a carbonyl (receptor) in the presence of an amine (donor).

[0238] In the method for producing an enantiomerically enriched or nearly enantiomerically pure amine by the protein according to the invention, the catalytic reaction can formally be as described by the above general equation (I) herein.

[0239] Therefore, another embodiment of the invention relates to a method for producing an amine, which comprises the following steps

[0240] a) providing an amine receptor molecule;

[0241] b) providing an amine donor molecule;

[0242] c) contacting the amine receptor molecule provided in step a) and the amine donor molecule provided in step b) with the protein according to the invention;

[0243] d) optionally, obtaining the amine.

[0244] A preferred embodiment of the method according to the invention for preparing amines is a method for producing aliphatic amines (including but not limited to linear, branched or cyclic alkylamines, alkenylamines, alkynylamines), or a method for producing aromatic amines, or a method for producing amino acids, more preferably a method for producing α-amino acids, even more preferably a method for producing branched-chain α-amino acids, aromatic α-amino acids or α-amino acids containing a substituted phenyl group, and most preferably a method for producing the amino acids norvaline, leucine, phenylalanine or tyrosine.

[0245] For ω-TA variants according to the invention comprising further amino acid modifications, the method according to the invention for producing amines is preferably a method for producing aliphatic amines containing phosphorus (including but not limited to linear, branched or cyclic alkylamines, alkenylamines, alkynylamines containing phosphorus), or a method for producing aromatic amines containing phosphorus, or a method for producing amino acids containing phosphorus, more preferably a method for producing α-amino acids containing phosphorus, even more preferably a method for producing branched-chain α-amino acids containing phosphorus, aromatic α-amino acids containing phosphorus or α-amino acids containing a substituted phenyl group and containing phosphorus, even more preferably a method for producing α-amino acids containing phosphorus, and even further more preferably a method for producing α-amino acids containing methyl-substituted phosphorus, and most preferably a method for producing glufosinate.

[0246] The amine acceptor molecule in step a) of the method according to the invention for producing amines is a molecule containing a carbonyl group which accepts an amino group from an amine donor molecule, whereby the carbonyl group of the acceptor molecule becomes an amine.

[0247] Preferably, the amine acceptor molecule in step a) of the method according to the invention for producing amines is an aliphatic ketone (including but not limited to linear, branched or cyclic alkanones, alkenones, alkynones) or an aryl ketone or a keto acid, more preferably it is a keto acid, even more preferably an α-keto acid, and most preferably the amine acceptor molecule is selected from the following: 2-oxovaleric acid, 4-methyl-2-oxovaleric acid, phenylpyruvic acid or 4-hydroxyphenylpyruvic acid.

[0248] For protein ω-TA variants according to the invention comprising further amino acid modifications, the amine acceptor molecule in step a) of the method according to the invention for producing amines is preferably an aliphatic ketone containing phosphorus (including but not limited to linear, branched or cyclic alkanones, alkenones, alkynones containing phosphorus), or an aryl ketone containing phosphorus or a keto acid containing phosphorus, more preferably the amine acceptor molecule is a keto acid containing phosphorus, even more preferably the amine acceptor molecule is an α-keto acid containing phosphorus, and even more preferably an α-keto acid containing methyl-substituted phosphorus, and most preferably the amine acceptor molecule in step a) is 4-[hydroxy(methyl)phosphinyl]-2-oxobutyric acid.

[0249] Preferably, the amine acceptor molecule in step a) of the process according to the invention for producing an amine is provided in an amount between 30 g / l (grams per liter) and 300 g / l, more preferably between 30 g / l and 250 g / l, even more preferably between 40 g / l and 250 g / l, and further more preferably between 50 g / l and 250 g / l.

[0250] The amine donor molecule in step b) of the process according to the invention for producing an amine is a molecule containing an amine group that donates the amine group to the amine acceptor molecule, whereby the amine group of the amine donor molecule becomes a carbonyl group.

[0251] The amine donor molecule in step b) of the process according to the invention for producing an amine is a chiral, prochiral or achiral amine. Preferably, the amine donor molecule is a chiral, prochiral or achiral alkyl or aryl or aryl-alkyl amine, more preferably the amine donor molecule is an amino acid or an alkyl amine.

[0252] For amino or aryl amines, the preferred amino donor molecules used in step b) of the process for producing an amine according to the invention are β-alanine, 1-propylamine, (racemic) 2-butylamine, 6-aminohexanoic acid, isopropylamine, benzylamine, methylbenzylamine, 1-aminoindane, 1-methyl-3-phenylpropylamine.

[0253] In the case where the amino donor is an achiral amino acid, glycine is the preferred amino donor molecule provided in step b) of the process for producing an amine according to the invention. In the case where the amino donor in step b) of the process for producing an amine according to the invention is a chiral amino acid, the amino acid preferably represents its (S)-enantiomer. The preferred amino acid donor molecules having the (S)-configuration provided in step b) of the process for producing an amine according to the invention are (S)-methylbenzylamine, (S)-1-aminoindane, (S)-1-methyl-3-phenylpropylamine, (S)-aspartic acid, (S)-asparagine, (S)-alanine, (S)-glutamine, (S)-glutamic acid, (S)-ornithine, (S)-phosphoserine, (S)-phenylalanine, (S)-leucine, (S)-tyrosine, (S)-norvaline.

[0254] The most preferred amino donor molecule provided in step b) of the process for producing an amine according to the invention is isopropylamine.

[0255] When isopropylamine is used as the amino donor molecule in the process according to the invention, it is converted to acetone by the action of ω-TA. Acetone is a volatile compound and thus has the advantage of evaporating at relatively low temperatures. This allows the acetone produced by ω-TA to be removed from the reaction mixture during the reaction, resulting in the beneficial effect that the equilibrium of the reaction shifts towards the amine produced by the method for producing amines according to the invention. The reverse reaction catalyzed by ω-TA is reduced due to the lack of one reaction partner, which enables the desired amine to be obtained in large quantities.

[0256] Preferably, the amine donor molecule in step b) of the process according to the invention for producing amines is provided in an amount between 10 g / l (grams per liter) and 250 g / l, more preferably between 15 g / l and 200 g / l, even more preferably between 17 g / l and 180 g / l.

[0257] In step c) of the process for producing amines according to the invention, the amine acceptor molecule provided in step a) and the amine donor molecule provided in step b) are preferably brought into contact with the protein according to the invention in solution. The solution can be an aqueous solution containing only water or a solution containing water and an organic solvent. When the protein according to the invention in step c) of the process for preparing amines according to the invention is brought into contact with the amine acceptor molecule provided in step a) and the amine donor molecule provided in step b) in an aqueous solution containing an organic solvent, the organic solvent is preferably selected from DMSO (dimethyl sulfoxide), DMAc (dimethylacetamide), DMF (dimethylformamide), acetonitrile, toluene, tert-butyl methyl ether, hexane, heptane. Most preferably, they are DMSO, DMAc and toluene.

[0258] Preferably, the aqueous solution containing an organic solvent contains the organic solvent in an amount of up to 10%, more preferably up to 20%, even more preferably up to 30%, even more preferably up to 40%, most preferably up to 50%.

[0259] The advantage of using an aqueous solution containing an organic solvent is that the solubility of the amine acceptor molecule provided in step a) and / or the amine donor molecule provided in step b) of the process for producing amines according to the invention can be improved in the case where they have low solubility, resulting in a larger amount of substrate available for ω-TA. This leads to a higher reaction rate, which means that the desired amine can be produced in a higher amount in a smaller volume and in a shorter time, thus increasing the space-time yield.

[0260] In step c) of the process for producing an amine according to the invention, in the case where the protein according to the invention is brought into contact with the amine acceptor molecule provided in step a) and the amine donor molecule provided in step b) in an aqueous solution, the solution preferably contains a buffer system for adjusting the pH. Preferred buffer systems are those containing TRIS-HCl, MOPS, HEPES, TRIS, Bicine.

[0261] Preferably, the pH of the aqueous solution in which the protein according to the invention is brought into contact with the amine acceptor molecule provided in step a) and the amine donor molecule provided in step b) in step c) of the process for producing an amine according to the invention is adjusted to a value between pH 4 and pH 11, more preferably between pH 5 and pH 10, even more preferably between pH 6 and pH 10, even more preferably between pH 7 and pH 10, even further more preferably between pH 8 and pH 10, and most preferably between pH 8.5 and pH 9.5.

[0262] Preferably, in step c) of the process for producing an amine according to the invention, the contact of the amine acceptor molecule provided in step a) and the amine donor molecule provided in step b) with the protein according to the invention takes place at a temperature between 10°C and 60°C, more preferably between 20°C and 60°C, even more preferably between 25°C and 55°C, even more preferably between 30°C and 50°C, even further more preferably between 30°C and 45°C, and most preferably between 34°C and 42°C.

[0263] In step c) of the process for producing an amine according to the invention, the amine acceptor molecule provided in step a) and the amine donor molecule provided in step b) are brought into contact with the protein according to the invention for a period of time sufficient to produce the amine.

[0264] Preferably, in step c) of the process for producing an amine according to the invention, the amine acceptor molecule provided in step a) and the amine donor molecule provided in step b) are brought into contact with the protein according to the invention for 5 hours to 48 hours to produce the amine, more preferably 5 hours to 36 hours, even more preferably 5 hours to 30 hours, even more preferably 5 hours to 24 hours, even further more preferably 5 hours to 18 hours, most preferably 5 hours to 14 hours and particularly preferably 5 hours to 13 hours.

[0265] For the contact of the protein according to the invention with the amine receptor molecule provided in step a) and the amine donor molecule provided in step b) in step c) of the process for the production of amines according to the invention, the protein can be in different forms in contact with the amine receptor molecule and the amine donor molecule. Preferably, the protein is in a partially purified form in contact with the amine receptor molecule and the amine donor molecule, or the protein is in a purified form in contact with the amine receptor molecule and the amine donor molecule, or the protein is present in a crude cell extract when in contact with the amine receptor molecule and the amine donor molecule, or the protein is in contact with the amine receptor molecule and the amine donor molecule when the protein is present as a component of a living or inanimate host cell.

[0266] If the protein is in contact with the amine receptor molecule and the amine donor molecule as components of a host cell in step c) of the process for the production of amines according to the invention, the host cell can be those that contain the culture medium for culturing the host cell, or the host cell can be free of the culture medium for culturing the host cell, or the host cell can be (further) processed. Preferably, the host cell is almost free of the culture medium for culturing the host cell, more preferably the host cell has been (further) processed, and even more preferably the host cell is almost free of the culture medium for culturing the host cell and the host cell has been (further) processed.

[0267] "Crude cell extract" as used herein refers to an extract obtained by disrupting living cells, which contains all or substantially all of the inorganic or organic substances present in the cells (including other proteins and / or nucleic acid molecules).

[0268] "Partially purified" as used herein refers to a composition containing a protein that contains (only) a part of all the inorganic or organic substances (including other proteins and / or nucleic acid molecules) present in the living cells expressing the protein.

[0269] The partially purified extract can be obtained by fractionating the inorganic or organic substances from the crude cell extract by, for example, commonly known methods such as centrifugation, filtration, any type of chromatographic separation, dialysis, etc. The fractionation of the crude cell extract can be repeated using the same or different fractionation methods and can include precipitation steps.

[0270] "Purified" as used herein refers to a protein whose specific activity (the protein activity present in the dry weight fraction divided by the total amount of the substance, especially other proteins in the dry weight fraction) cannot be increased by further fractionation or purification steps.

[0271] As is self - evident from the generally accepted definition of the term "purified" given above, "purified" can but in most cases does not mean that the protein is completely free of any other inorganic and / or organic compounds. Preferably, "purified" as used herein means that the protein according to the present invention accounts for at least 95% of the total dry - weight matter containing said protein, more preferably at least 96%, still more preferably at least 97%, even more preferably at least 98%, even still more preferably at least 99%, and most preferably at least 99.5%.

[0272] The term "living cell" as used herein refers to a cell capable of growing and / or reproducing.

[0273] The term "non - living cell" as used herein refers to a cell that cannot grow and / or reproduce.

[0274] Although non - living cells can no longer reproduce and / or grow, they still exhibit enzymatic activity with respect to the present application, particularly the activity of the protein according to the present invention having ω - TA activity.

[0275] As used herein, the term "free of medium" means that the medium used for culturing (host) cells has been removed, for example, by centrifugation and / or filtration.

[0276] As is self - evident from the generally accepted definition of the term "free of medium" given above, "free of medium" can but in most cases does not mean that the cells are completely free of any other inorganic and / or organic compounds present in the medium. Preferably, "purified" as used herein means that the cells according to the present invention account for at least 95% of the total dry - weight matter containing said cells but free of medium, more preferably at least 96%, still more preferably at least 97%, even more preferably at least 98%, even still more preferably at least 99%, and most preferably at least 99.5%.

[0277] The term "the host cell has been (further) processed" as used herein means that before contacting it with an amine - receptor molecule and an amine - donor molecule in step c) of the amine - production method according to the present invention, the host cell containing the protein according to the present invention has been processed using physical and / or chemical methods, has been processed using physical methods, more preferably has been dried, still more preferably has been freeze - dried or spray - dried, and most preferably has been spray - dried.

[0278] The drying process of cells (especially the freeze - drying and spray - drying processes) is well - known to those skilled in the art. Preferably, the host cell containing the protein according to the present invention has been freeze - dried or spray - dried, and most preferably, before contacting it in step c) of the amine - production method according to the present invention, it has been spray - dried by the method described under item 9 of "conventional methods" herein.

[0279] It is well known to those skilled in the art that proteins with ω-TA activity are pyridoxal phosphate (PLP)-dependent enzymes. In a preferred embodiment, in the presence of PLP, in step c) of the amine production method according to the present invention, the protein is contacted with the amine receptor molecule provided in step a) and the amine donor molecule provided in step b). More preferably, PLP is present in an amount between 0.05 g / l and 2.0 g / l, even more preferably in an amount between 0.05 g / l and 1.5 g / l, even more preferably in an amount between 0.05 g / l and 1.0 g / l, even further more preferably in an amount between 0.075 g / l and 0.75 g / l, and most preferably in an amount between 0.1 g / l and 0.5 g / l.

[0280] Obtaining the amine in the mandatory step d) in the method for producing an amine can mean that the amine is present in the composition of step d) without further purifying the produced amine, or it can refer to further purifying the produced amine. The purification of the amine can be carried out by methods well known to those skilled in the art. Such methods for purifying amines include, but are not limited to, methods involving precipitation, including chromatography, distillation, extraction, adsorption, or filtration.

[0281] A preferred embodiment of the method for producing an amine according to the present invention is a method for producing a composition comprising an (S)-amine in excess of its (respective) (R)-amine enantiomer, which comprises the following steps

[0282] a) providing an amine receptor molecule;

[0283] b) providing an amine donor molecule;

[0284] c) contacting the amine receptor molecule provided in step a) and the amine donor molecule provided in step b) with the protein according to the present invention;

[0285] d) optionally, obtaining a composition comprising an (S)-amine in excess of its (respective) (R)-amine enantiomer.

[0286] As used herein, the term "optical isomer" has the meaning commonly understood in the chemical field, and the molecule is one of two stereoisomers that are non-superimposable structural mirror images of each other. The term "enantiomer" is also commonly referred to as "optical isomer".

[0287] The term "enantiomeric excess" (commonly abbreviated as "ee") is commonly understood in the art of chemical technology and is used herein to refer to the excess of one enantiomer over the other in a composition, which is defined as the absolute difference between the mole fractions of each enantiomer. Enantiomeric excess is typically expressed in the art as a percentage of enantiomeric excess. For example, a composition containing 70% of the (S)-enantiomer and 30% of the (R)-enantiomer has an ee of 40% with respect to the (S)-enantiomer (40% pure (S)-enantiomer + 60% racemate (= 30% (S) + 30% (R))). Finally, a racemic enantiomer mixture has an ee of 0%, and a pure (S)- or (R)-enantiomer has an ee of 100%.

[0288] A preferred embodiment of the method according to the invention for producing a composition containing an enantiomeric excess of (S)-amine is a method for producing an aliphatic (S)-amine with an enantiomeric excess (including but not limited to linear, branched or cyclic alkylamines, alkenamines, alkynamines), or a method for producing an aryl (S)-amine with an enantiomeric excess, or a method for producing an (S)-amino acid with an enantiomeric excess, more preferably a method for producing an (S)-α-amino acid with an enantiomeric excess, even more preferably a method for producing a branched (S)-α-amino acid, an aromatic (S)-α-amino acid or an aromatic (S)-α-amino acid containing a substituted phenyl, and most preferably a method for producing an amino acid (S)-norvaline, (S)-leucine, (S)-phenylalanine or (S)-tyrosine with an enantiomeric excess.

[0289] For ω-TA variants according to the invention that contain further amino acid modifications, the process according to the invention for producing a composition comprising an enantiomerically enriched (S)-amine is preferably a process for producing an enantiomerically enriched aliphatic (S)-amine containing phosphorus (including but not limited to linear, branched or cyclic alkyl (S)-amines, alkenyl (S)-amines, alkynyl (S)-amines containing phosphorus), a process for producing an enantiomerically enriched aryl (S)-amine containing phosphorus, a process for producing an enantiomerically enriched (S)-amino acid containing phosphorus, more preferably a process for producing an enantiomerically enriched (S)-α-amino acid containing phosphorus, even more preferably a process for producing an enantiomerically enriched branched (S)-α-amino acid containing phosphorus, an aromatic (S)-α-amino acid containing phosphorus or an aromatic (S)-α-amino acid containing phosphorus with a substituted phenyl group, even more preferably a process for producing an enantiomerically enriched (S)-α-amino acid containing phosphorus, even further more preferably a process for producing an enantiomerically enriched (S)-α-amino acid containing methyl-substituted phosphorus, and most preferably a process for producing an enantiomerically enriched (S)-glufosinate.

[0290] Another preferred embodiment of the process according to the invention for producing a composition comprising an enantiomerically enriched (S)-amine is a process for producing a composition comprising an (S)-amine with an enantiomeric excess (ee) of at least 20%, more preferably at least 40%, even more preferably at least 60%, even more preferably at least 80%, even further more preferably at least 90%, particularly preferably at least 94%, most preferably at least 96% or particularly preferably at least 98%.

[0291] Regarding the preferred embodiments of the amine acceptor molecules provided in step a) and the preferred embodiments of the amounts provided, and the preferred embodiments of the amine donor molecules provided in step b) and the amounts provided, as defined above for the amine acceptor molecules applicable to step a) and the amine donor molecules applicable to step b) respectively in a process for producing a composition comprising an (S)-amine in excess over its (respective) (R)-amine enantiomer. However, it is understood that in the case where the amine donor molecules provided in step b) in the process for producing a composition comprising an (S)-amine in excess over its (respective) (R)-amine enantiomer are chiral molecules, an enantiomeric mixture comprising the (S)-enantiomer of the amine donor is provided, preferably a racemic mixture of the amine donor is provided. If it is economically desirable and feasible, the chiral amine donor may preferably be provided in a mixture in which the (S)-enantiomer is in enantiomeric excess, more preferably the amine donor may be provided in the form of a composition comprising the (S)-enantiomer in high enantiomeric excess, where in this case high enantiomeric excess means an enantiomeric excess of at least 30%, more preferably at least 40%, even more preferably at least 60%, even more preferably at least 80%, even further more preferably at least 90%, particularly preferably at least 94%, most preferably at least 96% or particularly preferably at least 98%.

[0292] Regarding solutions, aqueous solutions, aqueous solutions containing organic solvents, buffer systems, pH values and / or temperatures, the form of the protein (crude cell extract, partially purified protein, purified protein, protein present in the form of components of live or non-live host cells, (further) processed host cells, spray-dried host cells), the amount of the protein, and the presence and amount of PLP in step c) of the process according to the invention for producing amines, as defined above for being applicable to step c) of a process for producing a composition comprising an (S)-amine in excess over its (respective) (R)-amine enantiomer.

[0293] Regarding the preferred embodiments of step d) of the process according to the invention for producing amines, as defined above for being applicable to step d) of a process for producing a composition comprising an (S)-amine in excess over its (respective) (R)-amine enantiomer.

[0294] Except as defined for step d) of the process according to the invention for the production of amines, preferably, a composition comprising an (S)-amine with an enantiomeric excess of at least 40%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even further more preferably at least 95%, particularly preferably at least 97%, most preferably at least 98% or particularly preferably at least 99% is obtained in step d) of a process for the production of a composition comprising an (S)-amine with an excess of the (S)-amine over its (respective) (R)-amine enantiomer.

[0295] The protein according to the invention can also be used in a process for reducing or eliminating stereoisomers from a composition comprising (R)- and (S)-amine enantiomers. When reducing or eliminating stereoisomers from a composition comprising (R)- and (S)-amine enantiomers, the reaction catalyzed by the protein according to the invention follows the general equation (Ia). Compared to the reaction for synthesizing amines (see equation (I)), in the reaction for reducing or eliminating stereoisomers in a composition comprising (R)- and (S)-amines, it can be seen that the amino donor and the amino acceptor are exchanged with each other (see equation (Ia)). The advantage of the reaction according to equation (Ia) is that a specific stereoisomer can be enriched in a composition comprising different stereoisomers, or in other words, a specific stereoisomer can be removed from the composition, which is sometimes also referred to in the art as resolving an enantiomeric mixture. In the case of producing compounds by chemical synthesis, these methods are particularly important, as chemical synthesis usually results in a racemic mixture. For reasons of process economy or other reasons, such chemical synthesis of compounds may be the desired production process. However, separating the enantiomers produced by chemical synthesis may be difficult, expensive or even impossible. The protein according to the invention can be used to selectively remove stereoisomers from such racemic mixtures produced by chemical synthesis.

[0296] Accordingly, a further embodiment of the invention relates to a process for reducing the amount of amine enantiomers in a composition comprising an (R)-amine and an (S)-amine, which comprises the following steps

[0297] a) providing a composition comprising (R)- and (S)-amine enantiomers;

[0298] b) providing an amine acceptor molecule;

[0299] c) contacting the composition provided in step a) and the amine acceptor provided in step b) with the protein according to the invention;

[0300] d) optionally, obtaining a composition in which the amount of amine enantiomers is reduced compared to the amount present in the composition provided in step a).

[0301] In methods for reducing the amount of amine enantiomers in a composition comprising (R)-amine and (S)-amine, it is not determined how many structurally different (R)-amine and (S)-amine molecules are present in the composition provided in step a) of each of these methods, provided that there is at least one (S)-amine and one (R)-amine molecule.

[0302] The composition comprising (R)- and (S)-amine provided in step a) of the method for reducing the amount of amine enantiomers in a composition comprising (R)- and (S)-amine comprises at least one (R)-amine and at least one (S)-amine, wherein at least one (R)-amine and at least one (S)-amine can be stereoisomers of the same molecule or at least one (R)-amine and at least one (S)-amine can be stereoisomers of structurally different molecules.

[0303] A preferred embodiment of the method for reducing the amount of amine enantiomers in a composition comprising (R)-amine and (S)-amine is a method for reducing the amount of enantiomers of aliphatic amines (including but not limited to linear, branched or cyclic alkylamines, enamines, alkynes), or a method for reducing the amount of enantiomers of arylamines, or a method for reducing the amount of enantiomers of amino acids, more preferably a method for reducing the amount of enantiomers of α-amino acids, even more preferably a method for reducing the amount of enantiomers of branched-chain α-amino acids, aromatic α-amino acids or aromatic α-amino acids containing a substituted phenyl, and most preferably a method for reducing the amount of enantiomers of an amino acid selected from the group consisting of norvaline, leucine, phenylalanine or tyrosine.

[0304] For ω-TA variants according to the invention comprising further amino acid modifications, the method according to the invention for reducing the amount of amine enantiomers in a composition comprising (R)- and (S)-amine is preferably a method for reducing the amount of enantiomers of aliphatic amines containing phosphorus (including but not limited to linear, branched or cyclic alkylamines, enamines, alkynes containing phosphorus), or a method for reducing the amount of enantiomers of arylamines containing phosphorus, or a method for reducing the amount of enantiomers of amino acids containing phosphorus, more preferably a method for reducing the amount of enantiomers of α-amino acids containing phosphorus, even more preferably for reducing the amount of enantiomers of branched-chain α-amino acids containing phosphorus, aromatic α-amino acids containing phosphorus or aromatic α-amino acids containing a substituted phenyl and containing phosphorus, even more preferably a method for reducing the amount of enantiomers of α-amino acids containing substituted phosphorus, and even further more preferably a method for reducing the amount of enantiomers of α-amino acids containing methyl-substituted phosphorus, and most preferably a method for reducing the amount of enantiomers of glufosinate.

[0305] Regarding the solution, aqueous solution, aqueous solution containing an organic solvent, buffer system, pH value and / or temperature, form of the protein (crude cell extract, partially purified protein, purified protein, protein present in the form of components of live or inactivated host cells, (further) processed host cells, spray-dried host cells), amount of the protein, and the presence and amount of PLP in step c) of the process according to the invention for producing amines, as defined above for step c) of the process applicable for reducing the amount of amine enantiomers in a composition comprising (R)-amine and (S)-amine.

[0306] Regarding the preferred embodiment of step d) of the process according to the invention for producing amines, as defined above for step d) of the process applicable for reducing the amount of amine enantiomers in a composition comprising (R)-amine and (S)-amine.

[0307] The protein according to the invention can in particular be used in a process for reducing the amount of the (S)-enantiomer or substantially or almost completely eliminating the (S)-enantiomer in a composition comprising (R)-amine and (S)-amine, thereby producing a composition in which the (R)-amine is present in enantiomeric excess. Each reaction catalyzed in the process for producing enantiomerically enriched or almost enantiomerically pure amines by (S)-selective ω-TA can be described by the general equation (II) R 1 -CH((S,R)-NH2)-R 2 +R 3 -CO-R 4 →R 1 -CO-R 2 +R 3 -CH((R)-NH2)-R 4

[0308] Many compounds with biological activity (such as drugs, active compounds used in agriculture, dietary supplements, feed additives, etc.) exist in the form of enantiomers. In the vast majority of cases, only one enantiomer exhibits the desired biological activity, while the other enantiomer is inactive and often shows adverse side effects. Nowadays, many compounds with biological activity and used as drugs, in agriculture, as dietary supplements or feed additives (for example, amino acids) can only be produced by chemical synthesis or only under economically viable conditions, and the drawback is that these compounds are only provided in the form of a racemic mixture. The protein according to the present invention has the advantage that the amount of (S)-amine can be partially, significantly or almost completely removed from such a racemic mixture, with the effect that a composition containing the biologically active enantiomer or precursor is obtained, which is used in the production process of the biologically active enantiomer, or contains the biologically active enantiomer or its precursor in the composition, and the composition contains almost no inactive enantiomer. This reduces the side effects of drugs, agricultural products or products containing dietary supplements or feed additives.

[0309] In a preferred embodiment, the method for reducing the amount of amine enantiomers in a composition containing (R)-amine and (S)-amine enantiomers is a method for reducing the amount of (S)-amine enantiomers in a composition containing (R)-amine and (S)-amine, which comprises the following steps

[0310] a) Providing a composition containing (R)-amine and (S)-amine;

[0311] b) Providing an amine receptor molecule;

[0312] c) Contacting the composition provided in step a) and the amine receptor molecule provided in step b) with the protein according to the present invention;

[0313] d) Optionally, obtaining a composition in which the amount of (S)-amine enantiomers is reduced compared to the amount present in the composition provided in step a).

[0314] A preferred embodiment of the method for reducing the amount of the (S)-amine enantiomer in a composition comprising (R)- and (S)-amine enantiomers is a method for reducing the amount of an aliphatic (S)-amine (including but not limited to linear, branched or cyclic alkan(S)-amines, alken(S)-amines, alkyn(S)-amines), or a method for reducing the amount of an aryl (S)-amine, or a method for reducing the amount of an (S)-amino acid, more preferably a method for reducing the amount of an (S)-α-amino acid, even more preferably a method for reducing the amount of a branched-chain (S)-α-amino acid, an aromatic (S)-α-amino acid or an aromatic (S)-α-amino acid comprising a substituted phenyl, and most preferably a method for reducing the amount of an amino acid selected from: (S)-norvaline, (S)-leucine, (S)-phenylalanine or (S)-tyrosine.

[0315] For ω-TA variants according to the invention comprising further amino acid modifications, the method according to the invention for reducing the amount of the (S)-amine enantiomer in a composition comprising (R)- and (S)-amines is preferably a method for reducing the amount of an aliphatic (S)-amine comprising phosphorus (including but not limited to linear, branched or cyclic alkan(S)-amines, alken(S)-amines, alkyn(S)-amines comprising phosphorus), or a method for reducing the amount of an aryl (S)-amine comprising phosphorus, or a method for reducing the amount of an (S)-amino acid comprising phosphorus, more preferably a method for reducing the amount of an (S)-α-amino acid comprising phosphorus, even more preferably a method for reducing the amount of a branched-chain (S)-α-amino acid comprising phosphorus, an aromatic (S)-α-amino acid comprising phosphorus or an aromatic (S)-α-amino acid comprising a substituted phenyl and comprising phosphorus, even more preferably a method for reducing the amount of an (S)-α-amino acid comprising substituted phosphorus, and even more preferably a method for reducing the amount of an (S)-α-amino acid comprising methyl-substituted phosphorus, and most preferably a method for reducing the amount of (S)-glufosinate.

[0316] Preferably, for a method for reducing the amount of an amine enantiomer in a composition comprising an (R)-amine and an (S)-amine or for a method for reducing the amount of the (S)-amine enantiomer in a composition comprising an (R)-amine and an (S)-amine, the composition comprising an (R)-amine and an (S)-amine provided in step a) is an (R)- and / or (S)-amine selected from the group consisting of: aliphatic (R)- and (S)-amines (including but not limited to linear, branched or cyclic alkane (R)- and (S)-amines, alkene (R)- and (S)-amines, alkyne (R)- and (S)-amines), or aryl (R)- and (S)-amines, or (R)- and (S)-amino acids, more preferably (R)- and (S)-α-amino acids, even more preferably branched-chain (R)- and (S)-α-amino acids, aromatic (R)- and (S)-α-amino acids or aromatic (R)- and (S)-α-amino acids comprising a substituted phenyl, most preferably the amino acids (R)- and (S)-norvaline, (R)- and (S)-leucine, (R)- and (S)-phenylalanine or (R)- and (S)-tyrosine.

[0317] For an ω-TA variant according to the invention comprising further amino acid modifications, preferably, for a method for reducing the amount of an amine enantiomer in a composition comprising an (R)-amine and an (S)-amine or for a method for reducing the amount of the (S)-amine enantiomer in a composition comprising an (R)-amine and an (S)-amine, the composition comprising an (R)-amine and an (S)-amine provided in step a) is an (R)- and / or (S)-amine selected from the group consisting of: aliphatic (R)- and (S)-amines comprising phosphorus (including but not limited to linear, branched or cyclic alkane (R)- and (S)-amines, alkene (R)- and (S)-amines, alkyne (R)- and (S)-amines comprising phosphorus), or aryl (R)- and (S)-amines comprising phosphorus, or (R)- and (S)-amino acids comprising phosphorus, more preferably (R)- and (S)-α-amino acids comprising phosphorus, even more preferably branched-chain (R)- and (S)-α-amino acids comprising phosphorus, aromatic (R)- and (S)-α-amino acids comprising phosphorus or aromatic (R)- and (S)-α-amino acids comprising a substituted phenyl and comprising phosphorus, even more preferably (R)- and (S)-α-amino acids comprising a substituted phosphorus, even further more preferably (R)- and (S)-α-amino acids comprising a methyl-substituted phosphorus, most preferably (R)- and (S)-glufosinate.

[0318] More preferably, for the method of reducing the amount of amine enantiomers in a composition containing (R)-amine and (S)-amine or for the method of reducing the amount of (S)-amine enantiomers in a composition containing (R)-amine and (S)-amine, the composition containing (R)-amine and (S)-amine provided in step a) is a composition containing (R)- and / or (S)-amine of the same molecule, more preferably it contains (R)- and (S)-amine which are enantiomers representing a single compound selected from the group consisting of the following compounds: aliphatic (R)- and (S)-amines (including but not limited to linear, branched or cyclic alkane (R)- and (S)-amines, alkene (R)- and (S)-amines, alkyne (R)- and (S)-amines), or aryl (R)- and (S)-amines, or (R)- and (S)-amino acids, more preferably (R)- and (S)-α-amino acids, even more preferably branched-chain (R)- and (S)-α-amino acids, aromatic (R)- and (S)-α-amino acids or aromatic (R)- and (S)-α-amino acids containing a substituted phenyl group, most preferably the amino acids (R)- and (S)-norvaline, (R)- and (S)-leucine, (R)- and (S)-phenylalanine or (R)- and (S)-tyrosine.

[0319] For ω-TA variants according to the invention containing further amino acid modifications, preferably, for the method of reducing the amount of amine enantiomers in a composition containing (R)-amine and (S)-amine or for the method of reducing the amount of (S)-amine enantiomers in a composition containing (R)-amine and (S)-amine, the composition containing (R)-amine and (S)-amine provided in step a) is a composition containing (R)- and / or (S)-amine of the same molecule, more preferably it contains (R)- and (S)-amine which are enantiomers representing a single compound selected from the group consisting of the following compounds: aliphatic (R)- and (S)-amines containing phosphorus (including but not limited to linear, branched or cyclic alkane (R)- and (S)-amines containing phosphorus, alkene (R)- and (S)-amines containing phosphorus, alkyne (R)- and (S)-amines containing phosphorus), or aryl (R)- and (S)-amines containing phosphorus, or (R)- and (S)-amino acids containing phosphorus, more preferably (R)- and (S)-α-amino acids containing phosphorus, even more preferably branched-chain (R)- and (S)-α-amino acids containing phosphorus, aromatic (R)- and (S)-α-amino acids containing phosphorus or aromatic (R)- and (S)-α-amino acids containing a substituted phenyl group and containing phosphorus, even more preferably (R)- and (S)-α-amino acids containing a substituted phosphorus, even further more preferably (R)- and (S)-α-amino acids containing a methyl-substituted phosphorus, most preferably (R)- and (S)-glufosinate.

[0320] Preferably, for a method of reducing the amount of amine enantiomers in a composition comprising (R)-amine and (S)-amine or for a method of reducing the amount of (S)-amine enantiomer in a composition comprising (R)-amine and (S)-amine, the amine acceptor molecule provided in step b) is a molecule whose structure corresponds to the structure of the amine donor molecule provided in step b) of the method for producing an amine as described above, except that, in addition, the amine groups of those molecules described above as amine donor molecules in step b) of the method for producing an amine are replaced by carbonyl groups. For example, the amine group of isopropylamine as provided by the amine donor molecule in step b) of the method for producing an amine is replaced by a carbonyl group, resulting in the corresponding amine acceptor molecule in step b) of a method for reducing the amount of amine enantiomers in a composition comprising (R)-amine and (S)-amine or for a method of reducing the amount of (S)-amine enantiomer in a composition comprising (R)-amine and (S)-amine.

[0321] The most preferred amine acceptor molecule provided in step b) of a method for reducing the amount of amine enantiomers in a composition comprising (R)-amine and (S)-amine or for a method of reducing the amount of (S)-amine enantiomer in a composition comprising (R)-amine and (S)-amine is acetone.

[0322] Regarding the solution, aqueous solution, aqueous solution containing an organic solvent, buffer system, pH value and / or temperature, form of the protein (crude cell extract, partially purified protein, purified protein, protein present in the form of components of live or non-live host cells, (further) processed host cells, spray-dried host cells), amount of the protein, and the presence and amount of PLP in step c) of the method according to the invention for producing an amine, those defined above are applicable to step c) of a method for reducing the amount of (S)-amine enantiomer in a composition comprising (R)-amine and (S)-amine.

[0323] Regarding the preferred embodiment of step d) of the method according to the invention for producing an amine, those defined above are applicable to step d) of a method for reducing the amount of (S)-amine enantiomer in a composition comprising (R)-amine and (S)-amine.

[0324] A further embodiment of the present invention is the use of the protein according to the present invention for producing an amine (preferably for producing (S)-amine).

[0325] The use of the protein according to the present invention for reducing the amount of an amine (preferably the amount of (S)-amine) in an enantiomer mixture is also an embodiment of the present invention.

[0326] The use of a nucleic acid molecule according to the invention for expressing a protein according to the invention in a host cell according to the invention is also an embodiment of the invention.

[0327] Another embodiment of the invention relates to the use of a nucleic acid molecule according to the invention, a recombinant nucleic acid molecule according to the invention, a plasmid according to the invention or a vector according to the invention for transforming or genetically modifying a host cell according to the invention, or for producing a protein according to the invention.

[0328] The use of a host cell according to the invention for producing an amine or for reducing the amount of an amine (preferably, the amount of the (S)-amine in an enantiomeric mixture) is also an embodiment of the invention.

[0329] Sequence Listing

[0330] Throughout the application, nucleotide and amino acid abbreviations are used according to the following IUPAC codes:

[0331]

[0332] Table 3

[0333] To distinguish between amino acids and nucleotides, the uppercase nucleotide code abbreviations given in the above table are written in lowercase form herein.

[0334]

[0335]

[0336] Table 4

[0337] The use of codons follows the so-called "universal genetic code" in the following table, where "t" is replaced by "u" in ribonucleic acid (RNA) sequences. "TLC" represents the three-letter code of an amino acid and "SLC" represents the single-letter code of an amino acid.

[0338]

[0339]

[0340]

[0341] Table 5

[0342] SEQ ID NO 1: A nucleic acid sequence encoding an ω - transaminase (ω - TA) from Bacillus megaterium obtained by reverse - translating the amino acid sequence shown in SEQ ID NO 3, wherein the reverse - translation follows the translation principle due to the degeneracy of the universal genetic code. Before the stop codon at positions 1450 to 1452, nucleotides encoding 6 His amino acids are inserted into the sequence from Bacillus megaterium at positions 1432 to 1449.

[0343] SEQ ID NO 2: A nucleic acid sequence encoding an ω - TA from Bacillus megaterium having the amino acid sequence shown in SEQ ID NO 3. Before the stop codon at positions 1450 to 1452, nucleotides encoding 6 His amino acids are inserted into the sequence from Bacillus megaterium at positions 1432 to 1449.

[0344] SEQ ID NO 3: The amino acid sequence of ω - TA from Bacillus megaterium derived from the GenPept (PDB) accession number 5G09_A. The indicated amino acids are encoded by the nucleic acid sequences shown in SEQ ID NO 1 and 2. 6 His amino acids are inserted into the sequence from Bacillus megaterium at positions 478 to 483 by a sequence - modification method.

[0345] SEQ ID NO 4: A nucleic acid sequence encoding an ω - TA from Arthrobacter obtained by reverse - translating the amino acid sequence shown in SEQ ID NO 6, wherein the reverse - translation follows the translation principle due to the degeneracy of the universal genetic code.

[0346] SEQ ID NO 5: A nucleic acid sequence encoding an ω - TA from Arthrobacter having the amino acid sequence shown in SEQ ID NO 6. Before the stop codon at positions 1456 to 1458, nucleotides encoding 6 His amino acids are inserted into the sequence from Arthrobacter at positions 1438 to 1455.

[0347] SEQ ID NO 6: The amino acid sequence of ω - TA from Arthrobacter derived from the GenPept (PDB) accession number 5G2P_A. The indicated amino acids are encoded by the nucleic acid sequences shown in SEQ ID NO 4 and 5. 6 His amino acids are inserted into the sequence from Arthrobacter at positions 480 to 485 by a sequence - modification method.

[0348] SEQ ID NO 7: A nucleic acid sequence encoding an ω - TA from Bacillus sp. (soil 76801D1) obtained by reverse - translating the amino acid sequence shown in SEQ ID NO 9, wherein the reverse - translation follows the translation principle due to the degeneracy of the universal genetic code.

[0349] SEQ ID NO 8: Nucleic acid sequence of ω-TA from Bacillus sp. (soil 76801D1) derived from GenBank accession number LMTA01000079.1.

[0350] SEQ ID NO 9: Amino acid sequence of ω-TA from Bacillus sp. (soil 76801D1) derived from GenPept (PDB) accession number KRF52528.1. The indicated amino acids are encoded by the nucleic acid sequences shown in SEQ ID NOs 7 and 8 as described above.

[0351] SEQ ID NO 10: Nucleic acid sequence encoding a mutant ω-TA from Arthrobacter sp. obtained by back-translating the amino acid sequence shown in SEQ ID NO 12, wherein the back-translation follows the translation principle due to the degeneracy of the universal genetic code.

[0352] SEQ ID NO 11: Nucleic acid sequence encoding a mutant ω-TA variant from Arthrobacter sp. having the amino acid sequence shown in SEQ ID NO 12. The sequence may be derived from SEQ ID NO 15 in WO 2006 / 063336 A2.

[0353] SEQ ID NO 12: Amino acid sequence of a mutant ω-TA from Arthrobacter sp. that may be derived from SEQ ID NO 16 in WO 2006 / 06336 A2. The indicated amino acids are encoded by the nucleic acid sequences shown in SEQ ID NOs 11 and 12 as described above.

[0354] SEQ ID NO 13: Nucleic acid sequence encoding the wild-type ω-TA from Arthrobacter sp. obtained by back-translating the amino acid sequence shown in SEQ ID NO 15, wherein the back-translation follows the translation principle due to the degeneracy of the universal genetic code.

[0355] SEQ ID NO 14: Nucleic acid sequence encoding the wild-type ω-TA from Arthrobacter sp. having the amino acid sequence shown in SEQ ID NO 15. The sequence may be derived from SEQ ID NO 1 in WO 2006 / 063336 A2.

[0356] SEQ ID NO 15: Amino acid sequence of the wild-type ω-TA from Arthrobacter sp. that may be derived from SEQ ID NO 2 in WO 2006 / 06336 A2. The indicated amino acids are encoded by the nucleic acid sequences shown in SEQ ID NOs 13 and 14 as described above.

[0357] SEQ ID NO 16: A nucleic acid sequence encoding an improved ω-TA obtained by back-translating the amino acid sequence shown in SEQ ID NO 18, wherein the back-translation follows the translation principle due to the degeneracy of the universal genetic code.

[0358] SEQ ID NO 17: A nucleic acid sequence encoding an improved ω-TA having the amino acid sequence shown in SEQ ID NO 18.

[0359] SEQ ID NO 18: The amino acid sequence of an improved ω-TA, wherein improvements are obtained by amino acid substitutions as compared to the amino acid sequences from Bacillus megaterium shown in SEQ ID NOs 3 and 9 and as compared to the amino acid sequences from Arthrobacter spp. shown in SEQ ID NOs 6, 12, and 15.

[0360] SEQ ID NO 19: The nucleic acid coding sequence of the D-amino acid oxidase (DAO1) gene from Rhodotorula toruloides (synonym: Rhodotorula gracilis).

[0361] SEQ ID NO 20: The amino acid sequence of a protein having D-amino acid oxidase (DAO1) activity obtained from the coding sequence shown in SEQ ID NO 19.

[0362] SEQ ID NO 21: The nucleic acid coding sequence of a D-amino acid oxidase (DAO1) gene variant from Rhodotorula toruloides, which contains nucleotide substitutions (replacements) in the codons identified by the nucleotides at positions 160 - 162, and at positions 172 - 174, and at positions 637 - 639 as compared to the nucleic acid sequence from Rhodotorula toruloides.

[0363] SEQ ID NO 22: The amino acid sequence of a protein having D-amino acid oxidase activity obtained from the coding sequence shown in SEQ ID NO 21. As compared to positions 54, 58, and 213 of the nucleic acid sequence of Rhodotorula toruloides and as compared to the amino acid sequence shown in SEQ ID NO 21, the amino acid sequence contains amino acid substitutions (replacements) and is thus the amino acid sequence of a DAAO variant (mutant).

[0364] SEQ ID NO 23: The nucleic acid coding sequence encoding the catalase gene from Listeria seeligeri.

[0365] SEQ ID NO 24: The amino acid sequence of a protein with catalase activity obtained from the coding sequence shown in SEQ ID NO 23.

[0366] SEQ ID NO 25: The nucleic acid sequence encoding a protein with catalase activity having the amino acid sequence shown in SEQ ID NO 24.

[0367] SEQ ID NO 26: Figure 1 The nucleic acid sequence of the genetic element called "lac operon" in.

[0368] SEQ ID NO 27: Figure 1 The nucleic acid sequence of the genetic element called "Trc promoter" in.

[0369] SEQ ID NO 28: Figure 1 The nucleic acid sequence of the genetic element called "rrnB" in.

[0370] SEQ ID NO 29: Figure 1 The nucleic acid sequence of the genetic element called "cistron" in.

[0371] SEQ ID NO 30: Figure 1 The nucleic acid sequence of the genetic element called "rrnB terminator" in. Description of the Drawings

[0372] Figure 1 : A plasmid map showing the genetic elements for expressing proteins with DAAO, ω-TA, and catalase activities, which are from a single operon as a tricistronic RNA. Abbreviation explanations of the regulatory genetic elements involved in the transcription and translation of the tricistronic RNA:

[0373] lac operon: Ullmann, 2001, Encyclopedia of Life Sciences, John Wiley & Sons, Ltd, ISBN: 9780470015902; Ullmann, 2009, Encyclopedia of Life Sciences (ELS), John Wiley & Sons, Ltd: Chichester. DOI: 10.1002 / 9780470015902.a0000849.pub2; Composed of the nucleic acid sequence shown in SEQ ID NO26.

[0374] Trc promoter: A synthetic promoter derived from the E. coli trp and lacUV5 promoters (Brosius et al., 1985, J Biol Chem 260, 3539–3541); consisting of the nucleic acid sequence shown in SEQ ID NO 27.

[0375] rrnB: A RhoI-independent transcription termination signal (Pfeiffer & Hartmann, 1997, J Mol Biol. 265(4) 385-393; Orosz et al., 1991, Eur J Biochem. 201(3), 653-659); consisting of the nucleic acid sequence shown in SEQ ID NO 28.

[0376] t7 enhancer: A transcriptional enhancer sequence from the t7 gene (sequence used: ttaacttta).

[0377] RBS1: Ribosome binding site (sequence: gaggt).

[0378] Cistron: A transcription termination sequence; consisting of the nucleic acid sequence shown in SEQ ID NO 29.

[0379] RBS2: Ribosome binding site (sequence used: aaggag).

[0380] boxA: A transcription antitermination sequence (sequence used: tgctctttaacaa).

[0381] Cistron: A synthetic cistron consisting of the nucleic acid sequence shown in SEQ ID NO 29.

[0382] rrnB terminator: A transcription termination signal: consisting of the nucleic acid sequence shown in SEQ ID NO 30.

[0383] T2 terminator: A translation termination signal (Orosz et al., 1991, Eur J Biochem. 201(3), 653-659).

[0384] Figure 2 : Shows the production of (S)-norvaline by amination of 2-oxovaline catalyzed by the wild-type ω-TA protein from Arthrobacter sp. with the amino acid sequence shown in SEQ ID NO 6 or from Bacillus megaterium with the amino acid sequence shown in SEQ ID NO 3, compared to the ω-TA variant with the amino acid sequence shown in SEQ ID NO 18.

[0385] Figure 3: Shows the production of (S)-leucine by amination of 4-methyl-2-oxo-valine catalyzed by the wild-type ω-TA protein from Arthrobacter sp. having the amino acid sequence shown in SEQ ID NO 6 or from Bacillus megaterium having the amino acid sequence shown in SEQ ID NO 3, compared to the ω-TA variant having the amino acid sequence shown in SEQ ID NO 18.

[0386] Figure 4 : Shows the production of (S)-phenylalanine by amination of phenylpyruvic acid catalyzed by the wild-type ω-TA protein from Arthrobacter sp. having the amino acid sequence shown in SEQ ID NO 6 or from Bacillus megaterium having the amino acid sequence shown in SEQ ID NO 3, compared to the ω-TA variant having the amino acid sequence shown in SEQ ID NO 18.

[0387] Figure 5 : Shows the production of (S)-tyrosine by amination of p-hydroxyphenylpyruvic acid catalyzed by the wild-type ω-TA protein from Arthrobacter sp. having the amino acid sequence shown in SEQ ID NO 6 or from Bacillus megaterium having the amino acid sequence shown in SEQ ID NO 3, compared to the ω-TA variant having the amino acid sequence shown in SEQ ID NO 18. Detailed Description

[0388] General Method

[0389] 1. Production of ω-TA variants and ω-TA variants with further amino acid modifications

[0390] The known nucleotide sequences described herein encoding the proteins with ω-TA activity described herein were synthesized by the service provider Eurofins Genomics GmbH (Eurofins Genomics GmbH, Anzinger Str. 7a, 85560 Ebersberg, Germany).

[0391] Nucleotide substitutions (replacements) are introduced into the nucleic acid sequences shown in SEQ ID Nos 2, 5, 8, 11, 14. Such substitutions can be made in the nucleic acid sequences encoding the reference polypeptides by any method suitable for substituting nucleotides in nucleic acid sequences. These methods are widely described in the literature and are well known to those skilled in the art in the corresponding order. Several molecular biology methods can be used to effect the corresponding nucleotide substitutions. Useful methods for preparing the mutant nucleic acid sequences and the corresponding proteins according to the invention include site-directed mutagenesis of the codons encoding one or more preselected amino acids so as to change the selected codons in such a way that they encode different amino acids. Methods for obtaining such site-directed mutations are well known to those skilled in the art and are widely described in the literature (in particular: Directed Mutagenesis: A Practical Approach, 1991, Edited by M.J. McPHERSON, IRL PRESS), or methods where commercially available kits can be applied (e.g., the QUIKCHANGE Lightning Mutagenesis Kit from Qiagen or Stratagene). After site-directed mutagenesis, the nucleic acids are transformed into the Escherichia coli strain MG1655. Cells containing the mutant polypeptides with favorable bioconversion yields are selected by using appropriate screening methods. Suitable screening methods are described under items 4 and 7 of the "General Methods" herein. The mutant nucleic acid sequences encoding the improved polypeptides are subjected to sequence verification. Methods for sequence verification are well known to those skilled in the art and are widely described in the literature (e.g., Sambrook and Russell (2012) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)). TM Lightning Mutagenesis Kit). After site-directed mutagenesis, the nucleic acids are transformed into the Escherichia coli strain MG1655. Cells containing the mutant polypeptides with favorable bioconversion yields are selected by using appropriate screening methods. Suitable screening methods are described under items 4 and 7 of the "General Methods" herein. The mutant nucleic acid sequences encoding the improved polypeptides are subjected to sequence verification. Methods for sequence verification are well known to those skilled in the art and are widely described in the literature (e.g., Sambrook and Russell (2012) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)).

[0392] 2. Expression Vectors / Host Cells for ω-TA Variants

[0393] The nucleic acid sequences encoding wild-type ω-TA (SEQ ID Nos 2, 5, 8, 14) or known ω-TA containing mutations (SEQ ID NO 11) or ω-TA variants as described herein are cloned into the commercially available pET22B vector (Merck KGaA, Frankfurter Str 250, 64293 Darmstadt, Germany) and expressed in Escherichia coli strain BL21DE3 cells.

[0394] 3. Expression of ω-TA Variants

[0395] A pre-culture of the Escherichia coli strain BL21DE3 containing the pET22B vector, into which the corresponding nucleic acid sequence encoding the ω-TA variant has been introduced, was cultured overnight at 37 °C on a rotary shaker at 180 rpm in 20 ml of LB medium supplemented with carbenicillin. Expression of the ω-TA protein was carried out by transferring the pre-culture into a flask containing 250 ml of LB medium supplemented with carbenicillin. After culturing at 37 °C on a rotary shaker at 180 rpm until an OD (optical density) between 0.6 - 0.8 was reached, expression of the ω-TA protein was induced by adding 0.5 mM IPTG (final concentration). The induced cell culture was incubated at 20 °C with shaking at 180 rpm for 20 h. Enzyme purification was carried out using the Ni-NTA Fast Start kit from Qiagen (Qiagen GmbH, QiagenStrasse 1, 40724 Hilden) according to the manufacturer's protocol.

[0396] 4. Activity assay of the ωTA variant in the presence of an amine acceptor and an amine donor

[0397] To 40 μl of triethanolamine buffer (a 200 mM solution in deionized water, pH = 9.0), 10 μl of pyridoxal phosphate (a 10 mM solution in deionized water) and 10 μl of amine donor (a 2 M solution in deionized water, adjusted to pH = 9.0 by adding aqueous HCl) were added at room temperature. Subsequently, 20 μl of amine acceptor (a 100 mM solution in deionized water) was added (if the amine acceptor is not soluble in water, a proportional amount of DMSO was added). Finally, 20 μl of transaminase (1.5 mg / ml) was added at room temperature, and the mixture was incubated at 40 °C at 800 rpm on a rotary shaker for 6 - 7 h. Aliquots were taken at different time intervals during the reaction and analyzed by HPLC to monitor the transamination reaction.

[0398] 5. Expression vector / host cell for ω-TA variants with further amino acid modifications.

[0399] Activity assays of ω-TA variants with further amino acid modifications were carried out by using a method involving two reaction steps.

[0400] The first reaction step (Step 1) produces the amine acceptor of ω-TA. This step is catalyzed by D-amino acid oxidase (DAAO or DAO, EC 1.4.3.3). DAAO is a flavoprotein containing flavin adenine dinucleotide (FAD) and can catalyze the oxidative deamination reaction of D-amino acids with oxygen according to the following general equation (III) to produce the corresponding 2-oxo acid, as well as hydrogen peroxide and ammonia:

[0401] α-D-amino acid + H2O + O2 → α-2-oxo carboxylic acid + NH3 + H2O2

[0402] The protein with DAAO activity for producing α-2-oxo carboxylic acid in the first reaction step is a DAAO variant of the DAO1 protein from Rhodosporidium toruloides. The coding nucleic acid sequence of the wild-type DAO1 protein from Rhodosporidium toruloides can be derived from GenBank accession number U6006.1 (shown as SEQ ID NO 19), and the corresponding amino acid sequence encoded by the nucleic acid sequence shown as SEQ ID NO 19 can be derived from UniProt accession number P80324 (shown as SEQ ID NO 20). The DAAO variant used herein was disclosed as mutant Ac305 in WO 2017 / 151573 (page 36, Table 1). Compared with SEQ ID NO 20, mutant Ac305 contains amino acid substitutions (replacements) at positions 54, 58, and 213. In mutant Ac305, the amino acid N at position 54 in SEQ ID NO 20 is replaced by C, the amino acid F at position 58 in SEQ ID NO 20 is replaced by H, and the amino acid M at position 213 in SEQ ID NO 20 is replaced by S. The amino acid sequence of mutant Ac305 is shown in SEQ ID NO 22. The corresponding nucleic acid sequence encoding the protein with the amino acid sequence shown in SEQ ID NO 22 is shown in SEQ ID NO 21. The reaction of step 1 is catalyzed by a protein with DAAO activity having the amino acid sequence shown in SEQ ID NO 22.

[0403] In the second reaction step (step 2), the α-2-oxo carboxylic acid produced by the protein with DAAO activity in step 1 is converted into an amino acid by a protein with ω-TA activity in the presence of an amine donor according to the general equation (I).

[0404] It can be clearly seen from the description of step 1 by the general equation (III) that the conversion of D-amino acid to keto acid catalyzed by the protein with DAAO activity produces hydrogen peroxide (H2O2). It may be desirable to remove H2O2, but the removal of H2O2 is not required in every case. In combination with the present invention, the removal of H2O2 is accomplished by adding a protein with catalase activity.

[0405] The protein with catalase activity (EC 1.11.1.6; hydrogen peroxide: hydrogen peroxide oxidoreductase) is well known in the art and catalyzes the conversion of hydrogen peroxide (H2O2) into water (H2O) and oxygen (O2) according to the following general equation (IV):

[0406] 2H2O2 → O2 + 2H2O

[0407] The amino acid sequence of the protein having catalase activity from Listeria seeligeri for removing H2O2 is shown in SEQ ID NO 24 and can be derived from GenePept accession number WP_012986600.1. SEQ ID NO 23 (derivable from GenBank accession number NC_013891.1) shows the nucleic acid coding sequence of the catalase protein from Listeria seeligeri having the amino acid sequence shown in SEQ ID NO 24. SEQ ID NO 25 is also a nucleic acid sequence encoding a catalase protein having the amino acid sequence shown in SEQ ID NO 24. Compared with the nucleic acid sequence shown in SEQ ID NO 23, the codons of the nucleic acid sequence shown in SEQ ID NO 25 have been changed to be suitable for codon usage in Escherichia coli.

[0408] To produce the proteins having DAAO activity, ω-TA activity, and catalase activity, the nucleic acid sequences encoding the corresponding three proteins were cloned into an E. coli expression vector in such a way that all three proteins were transcribed as a polycistronic RNA from a single operon by the trc-promoter (a hybrid promoter consisting of sequences derived from the trp- and lacUV5-promoters). The gene order transcribed from the promoter was DAAO (SEQ ID NO 21) -> a nucleic acid molecule encoding a variant of ω-TA with further amino acid modifications as described above herein -> catalase (SEQ ID NO 25). SEQ ID NO 21 was translationally fused at its 5′-end to the nucleic acid sequence encoding the amino acid sequence M A R I R L. The expression vector used was based on pSE420 (description and sequence from: Addgene, 75 Sidney St, Suite 550A, Cambridge, MA 02139; https: / / www.addgene.org / vector-database / 4064 / or from Thermo Fisher Scientific (Invitrogen), Thermo Fisher Scientific Inc. 168 Third Avenue, Waltham, MA 02451 USA, https: / / www.thermofisher.com / search / results?query =pSE420&focusarea ). The genetic elements were introduced into the modified pSE420 vector by commonly known methods. The relevant genetic elements present in the expression vector used are as shown in Figure 1 . To express the three enzymes, the expression vector was transferred into Escherichia coli strain MG1655 cells.

[0409] 6. Expression of ω-TA variants containing further amino acid modifications

[0410] The ω-TA variants containing further amino acid modifications were cloned into the tricistronic expression vector described above in item 5 of "General Methods" and expressed in Escherichia coli strain MG1655 cells. For this purpose, a 20 ml pre-culture in LB medium supplemented with kanamycin was cultured overnight in a shake flask at 37 °C at 180 rpm on a rotary shaker. The expression of ωTA protein was carried out by transferring the pre-culture to a flask containing 200 ml of LB medium supplemented with kanamycin. The expression of ωTA protein was induced after reaching an OD of 0.6 - 0.8 by adding 1 mM IPTG (final concentration). The induced cell culture was incubated with shaking at 180 rpm at 20 °C for 20 h. For harvesting, the cell culture was centrifuged at 8000 g for 15 minutes at 4 °C, and the obtained cell pellet was stored at -80 °C until freeze-drying or spray-drying.

[0411] 7. Activity detection of ω-TA variants with further amino acid modifications

[0412] In a 1-liter temperature-adjustable glass double-layer reactor equipped with a mechanical stirrer, an O2-gas inlet tube, and a pH control dosing device, 268 ml of a 50 w% aqueous solution of racemic (R,S)-glufosinate ammonium salt (corresponding to 160.8 g of racemic glufosinate ammonium salt) was added. An aqueous solution of 2 M isopropylamine was added through the pH control dosing unit under mechanical stirring (250 rpm) until a pH of 9.0 was reached. During the entire reaction, the pH was kept constant by controlling the addition of the 2 M aqueous isopropylamine solution. The reactor was heated to an internal temperature of 35 °C.

[0413] In a beaker, 8 g of spray-dried Escherichia coli strain MG1655 cells, 200 mg of pyridoxal phosphate, 2 ml of polypropylene glycol (P 2000), and 138 ml of deionized water were mixed, and the cells contained Figure 1 the expression vector described in, which expressed wild-type ω-TA protein and ω-TA variants with further amino acid modifications. At 35 °C, under stirring (250 rpm), this mixture was added to the glass reactor. Through the O2-gas inlet tube, oxygen was introduced into the reaction mixture at a flow rate of 0.1 l / min. The mixture was stirred for 24 h, and the progress of the reaction was monitored by taking aliquots at different time intervals during the reaction and analyzing them by HPLC. Subsequently, the oxygen feed and the isopropylamine feed were stopped, and the reaction mixture was denatured at 90 °C under stirring (250 rpm) for 30 min. The remaining mixture was cooled to room temperature.

[0414] 8. Detection of amines produced by ω-TA

[0415] A) Analysis of the transamination products (S)-norvaline, (S)-leucine, (S)-tyrosine, and (S)-glufosinate-ammonium

[0416] The progress of the transamination reaction was monitored by HPLC analysis. The HPLC methodology used for this operation was based on the article published by Davankov et al. (1980, Chromatographia 13(11), 677–685).

[0417] Specifically, the following HPLC parameters were used:

[0418] Column: Phenomenex Chirex 3126(D) – penicillamine 150*4,6 mm (Catalog No.: 00F-3126-E0)

[0419] Flow rate: 1 ml / min

[0420] Eluent: A) Deionized water + 0.5 g / L CuSO4 (v / v)

[0421] B) Methanol

[0422] A:B = 90:10 (isocratic)

[0423] Detector: DAD 230 nm

[0424] Oven: 30 °C

[0425] Run time: 15 min

[0426] B) Analysis of the transamination product L-phenylalanine:

[0427] The progress of the transamination reaction was monitored by HPLC analysis. Specifically, the following HPLC parameters were used:

[0428] Column: Phenomenex Prodigy 3μm ODS-3 100A 100*4 mm (Catalog No.: 00D-4222-D0)

[0429] Flow rate: 2 ml / min

[0430] Eluent: A) Acetonitrile

[0431] B) Deionized water

[0432] Gradient from A:B = 5:95 to A:B = 95:5 in 7 min

[0433] Detector: VWD1 A, 210 nm

[0434] Oven: 40 °C

[0435] Running time: 9 min

[0436] 9. Spray drying of cells

[0437] The spray drying experiment was carried out in a laboratory (laboratory scale) spray dryer with a maximum input temperature of 220 °C. The dryer uses compressed air or nitrogen at 200 - 800 l / h (liters per hour) at 5 - 8 bar. The maximum air flow can reach 35 m 3 / h (meters 3 / hour).

[0438] To dry the bacterial cell mass in a flask culture or fermentation material (i.e., a total volume of 1 liter), the broth was concentrated tenfold (10x) by centrifugation and resuspended in the culture supernatant obtained after centrifugation to a final volume of 100 ml. The obtained concentrate needs to be suitable for pumping and should be continuously mixed by a magnetic stirrer. Using an air flow of 500 l / h, the aspirator was set to 100%, and the liquid was applied to a 0.7 mm nozzle. The typical product flow rate was 10 ml / min, and the average inlet temperature applied was -145 °C and the outlet temperature was 85 °C. The dried biomass was weighed and used for biotransformation experiments at a ratio of g / l.

[0439] Examples

[0440] 1. Conversion of 2-oxovaleric acid to (S)-norvaline

[0441] Express and purify the wild-type ω-TA protein from Arthrobacter sp. with the amino acid sequence shown in SEQ ID NO 6, or from Bacillus megaterium with the amino acid sequence shown in SEQ ID NO 3, or the ω-TA variant with the amino acid sequence shown in SEQ ID NO 18 as described in item 3 of the "General Method".

[0442] To 25 μl of triethanolamine buffer in deionized water (a 200 mM solution in deionized water, pH = 9.0), 10 μl of pyridoxal phosphate (PLP) in deionized water (a 10 mM solution in deionized water) and 10 μl of isopropylamine in deionized water (a 2 M solution in deionized water, adjusted to pH = 9.0 by adding aqueous HCl solution) were added at room temperature. Subsequently, 20 μl of 2-oxovaleric acid (a 100 mM solution in deionized water) was added. Finally, 35 μl of a solution containing 1.5 mg / ml of each ω-TA protein was added at room temperature, and the mixture was incubated at 40 °C at 800 rpm on a rotary shaker for 6 h. As described in item 8 of the "General Method", aliquots were taken at different time intervals during the reaction and analyzed by HPLC to monitor the transamination reaction.

[0443] Table 6 shows the results obtained with the ω-TA variant having the amino acid sequence shown in SEQ ID NO 18 compared to those of the wild-type proteins from Arthrobacter sp. having the amino acid sequence shown in SEQ ID NO 6 and from Bacillus megaterium having the amino acid sequence shown in SEQ ID NO 3. The results are also shown in Figure 2 .

[0444]

[0445] Table 6

[0446] Explanation of Table 6:

[0447] "Time", measured in hours (h), represents the time elapsed since the start of the reaction.

[0448] "mAU*s" is the abbreviation for milli (m) absorbance (A) unit (U) multiplied by (*) seconds (s); the standard unit describing the area under the peak in an HPLC chromatogram. The higher the area under the peak, the higher the amount of the corresponding product.

[0449] From Table 6 and Figure 2 it can be concluded that the production of (S)-norvaline from 2-oxovaleric acid in the reaction catalyzed by the ω-TA variant is faster than the reactions catalyzed by the wild-type proteins from Arthrobacter sp. and Bacillus megaterium. In addition, compared to the reactions catalyzed by the wild-type proteins from Arthrobacter sp. and Bacillus megaterium, in the reaction catalyzed by the ω-TA variant, (S)-norvaline reaches its maximum amount significantly earlier during the reaction.

[0450] 2. Conversion of 4-methyl-2-oxovaleric acid to (S)-leucine

[0451] Express and purify the wild-type ω-TA protein from Arthrobacter sp. having the amino acid sequence shown in SEQ ID NO 3, or from Bacillus megaterium having the amino acid sequence shown in SEQ ID NO 6, or the ω-TA variant having the amino acid sequence shown in SEQ ID NO 18 as described in item 3 of the "General Method".

[0452] To 40 μl of triethanolamine buffer (a solution of 200 mM in deionized water, pH = 9.0), 10 μl of pyridoxal phosphate (a solution of 10 mM in deionized water) and 10 μl of isopropylamine (a solution of 2 M in deionized water, adjusted to pH = 9.0 by adding aqueous HCl solution) were added at room temperature. Subsequently, 20 μl of 4-methyl-2-oxovaleric acid (a solution of 100 mM in deionized water) was added. Finally, 20 μl of a solution containing 1.5 mg / ml of each ω-TA protein was added at room temperature, and the mixture was incubated at 40 °C at 800 rpm on a rotary shaker for 6 h. Aliquots were taken at different time intervals during the reaction and analyzed by HPLC to monitor the transamination reaction as described in item 8 of the "General Method".

[0453] Table 7 shows the results obtained with the ω-TA variant having the amino acid sequence shown in SEQ ID NO 18 compared to those of the wild-type proteins from Arthrobacter sp. having the amino acid sequence shown in SEQ ID NO 3 and from Bacillus megaterium having the amino acid sequence shown in SEQ ID NO 6. The results are also shown in Figure 3 in.

[0454]

[0455] Table 7

[0456] Explanation of Table 7: See the explanation of Table 6

[0457] From Table 7 and Figure 3 it can be concluded that the wild-type enzymes from Arthrobacter sp. and Bacillus megaterium cannot produce (S)-leucine by amination of 4-methyl-2-oxovaleric acid, while the ω-TA variant produces (S)-leucine quite efficiently.

[0458] 3. Conversion of phenylpyruvic acid to (S)-phenylalanine

[0459] The wild-type ω-TA protein from Arthrobacter sp. having the amino acid sequence shown in SEQ ID NO 3, or from Bacillus megaterium having the amino acid sequence shown in SEQ ID NO 6, or the ω-TA variant having the amino acid sequence shown in SEQ ID NO 18 was expressed and purified as described in item 3 of the "General Method".

[0460] To 40 μl of triethanolamine buffer (a solution of 200 mM in deionized water, pH = 9.0), 10 μl of pyridoxal phosphate (a solution of 10 mM in deionized water) and 10 μl of isopropylamine (a solution of 2 M in deionized water, adjusted to pH = 9.0 by adding aqueous HCl solution) were added at room temperature. Subsequently, 20 μl of phenylpyruvic acid (a 100 mM phenylpyruvic acid solution) in DMSO / deionized water in a ratio of 1:1 was added. Finally, 20 μl of a solution containing 1.5 mg / ml of each ω-TA protein was added at room temperature, and the mixture was incubated at 40 °C at 800 rpm on a rotary shaker for 6 h. Aliquots were taken at different time intervals during the reaction and analyzed by HPLC to monitor the transamination reaction as described in item 8 of the "General Method".

[0461] Table 8 shows the results obtained with the ω-TA variant having the amino acid sequence shown in SEQ ID NO 18 compared to those of the wild-type proteins from Arthrobacter sp. having the amino acid sequence shown in SEQ ID NO 3 and from Bacillus megaterium having the amino acid sequence shown in SEQ ID NO 6. The results are also shown in Figure 4 in.

[0462]

[0463] Table 8

[0464] Explanation of Table 8: See the explanation of Table 6

[0465] From Table 8 and Figure 4 it can be concluded that compared to the amount of (S)-phenylalanine produced by the ω-TA variant, the wild-type enzyme from Arthrobacter sp. cannot produce (S)-phenylalanine from phenylpyruvic acid, and the wild-type enzyme from Bacillus megaterium produces (S)-phenylalanine very slowly and in a lower amount.

[0466] 4. Conversion of p-hydroxyphenylpyruvic acid to (S)-tyrosine:

[0467] The wild-type ω-TA protein from Arthrobacter sp. having the amino acid sequence shown in SEQ ID NO 3, or from Bacillus megaterium having the amino acid sequence shown in SEQ ID NO 6, or the ω-TA variant having the amino acid sequence shown in SEQ ID NO 18 was expressed and purified as described in item 3 of the "General Method".

[0468] To 40 μl of triethanolamine buffer (a solution of 200 mM in deionized water, pH = 9.0), 10 μl of pyridoxal phosphate (a solution of 10 mM in deionized water) and 10 μl of isopropylamine (a solution of 2 M in deionized water, adjusted to pH = 9.0 by adding aqueous HCl) were added at room temperature. Subsequently, 20 μl of p-hydroxyphenylpyruvic acid (100 mM p-hydroxyphenylpyruvic acid solution) in DMSO / deionized water at a ratio of 1:1 was added. Finally, 20 μl of a solution containing 1.5 mg / ml of each ω-TA protein was added at room temperature, and the mixture was incubated at 40 °C at 800 rpm on a rotary shaker for 6 h. Aliquots were taken at different time intervals during the reaction and analyzed by HPLC to monitor the transamination reaction as described in item 8 of the "General Method".

[0469] Table 9 shows the results obtained with the ω-TA variant having the amino acid sequence shown in SEQ ID NO 18 compared to those of the wild-type proteins from Arthrobacter sp. having the amino acid sequence shown in SEQ ID NO 3 and from Bacillus megaterium having the amino acid sequence shown in SEQ ID NO 6. The results are also shown in Figure 5 in.

[0470]

[0471] Table 9

[0472] Explanation of Table 9: See the explanation of Table 6

[0473] From Table 9 and Figure 5 it can be concluded that the wild-type enzymes from Arthrobacter sp. and Bacillus megaterium cannot produce (S)-tyrosine by amination of p-hydroxyphenylpyruvic acid, while the ω-TA variant produces (S)-tyrosine quite efficiently.

[0474] 5. Production of (S)-glufosinate from 4-[hydroxy(methyl)phosphinyl]-2-oxobutanoic acid by ω-TA variants containing further amino acid modifications

[0475] As described in item 6 of the "General Method", the ω-TA variant having the amino acid sequence shown in SEQ ID NO 18 and the ω-TA variant containing further amino acid modifications as described in Table 2 herein are co-expressed with a protein having DAAO activity and a protein having catalase activity (see item 5 of the "General Method"), and then spray-dried as described in item 9 of the "General Method". DAAO produces 4-[hydroxy(methyl)phosphoryloxy]-2-oxobutanoic acid through the deamination of (R)-glufosinate. Subsequently, using 4-[hydroxy(methyl)phosphoryloxy]-2-oxobutanoic acid as an amino acceptor through the ω-TA variant with further amino acid modifications, and it is converted into (S)-glufosinate in the amination reaction. The activity of the ω-TA variant having the amino acid sequence shown in SEQ ID NO 18 and the ω-TA variant containing further amino acid modifications as described in Table 2 herein is detected according to the detection described under item 7 of the "General Method". After 5 h from the start of the reaction, as described in item 8 of the "General Method", the amount of (S)-glufosinate produced in each reaction is determined by HPLC analysis to monitor the transamination reaction.

[0476] Table 10 shows the amount of (S)-glufosinate (S-GA) produced by each ω-TA variant containing further amino acid modifications and the amount of the ω-TA variant having the amino acid sequence shown in SEQ ID NO 18.

[0477]

[0478] Table 10

[0479] Explanation of Table 10:

[0480] To identify the amino acid changes, the numbers in the first column identify the amino acid positions in the amino acid sequence shown in SEQ ID NO 18. The characters that appear before the numbers identify the amino acids present at each position in the amino acid sequence shown in SEQ ID NO 18. The characters that appear after the numbers identify the amino acids present at each position in the amino acid sequence of the ω-TA variant with further amino acid modifications. Compared with the amino acid sequence shown in SEQ ID NO 18, the two numbers (each with a character before and after) given in the rows of the same column identify two simultaneous amino acid substitutions (replacements).

[0481] It can be seen from Table 10 that compared with the ω-TA variant having the amino acid sequence shown in SEQ ID NO 18, the ω-TA variant containing further amino acid modifications produces more (S)-glufosinate.

Claims

1. A protein having ω-TA activity, which consists of the amino acid sequence from position 1 to 476 as shown in SEQ ID NO 18.

2. The protein according to claim 1, which is selected from the following: a) The protein according to claim 1, except that the amino acid at position 166 is G and the amino acid at position 327 is Q; b) The protein according to claim 1, except that the amino acid at position 327 is Q and the amino acid at position 384 is S; c) The protein according to claim 1, except that the amino acid at position 326 is Q and the amino acid at position 327 is Q; d) The protein according to claim 1, except that the amino acid at position 327 is Q; e) The protein according to claim 1, except that the amino acid at position 326 is F and the amino acid at position 327 is Q; f) The protein according to claim 1, except that the amino acid at position 327 is C; g) The protein according to claim 1, except that the amino acid at position 327 is I; h) The protein according to claim 1, except that the amino acid at position 327 is M; i) The protein according to claim 1, except that the amino acid at position 164 is Y; j) The protein according to claim 1, except that the amino acid at position 164 is S; k) The protein according to claim 1, except that the amino acid at position 327 is V; l) The protein according to claim 1, except that the amino acid at position 409 is R; m) The protein according to claim 1, except that the amino acid at position 327 is S; n) The protein according to claim 1, except that the amino acid at position 271 is I; o) The protein according to claim 1, except that the amino acid at position 329 is G; p) The protein according to claim 1, except that the amino acid at position 409 is P; q) The protein according to claim 1, except that the amino acid at position 414 is M; r) The protein according to claim 1, except that the amino acid at position 165 is K; s) The protein according to claim 1, except that the amino acid at position 414 is R; t) The protein according to claim 1, except that the amino acid at position 414 is H; u) The protein according to claim 1, except that the amino acid at position 165 is C; v) The protein according to claim 1, except that the amino acid at position 164 is C; w) The protein according to claim 1, except that the amino acid at position 409 is K.

3. A nucleic acid molecule, which encodes the protein according to claim 1 or 2.

4. The nucleic acid molecule according to claim 3, which encodes a protein having ω-TA activity, and is selected from the following: a) A nucleic acid molecule consisting of the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 17; b) A nucleic acid molecule encoding a protein consisting of the amino acid sequence from position 1 to 476 in the amino acid sequence shown in SEQ ID NO 18; c) A nucleic acid molecule derived from the nucleic acid molecule defined in a) or b) due to the degeneracy of codons; d) A nucleic acid molecule consisting of the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 16.

5. The nucleic acid molecule according to claim 3 or claim 4, which encodes a protein having ω-TA activity and is selected from the following: a) A nucleic acid molecule consisting of the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 16 or SEQ ID NO 17, except that the codon at nucleotide positions 496 to 498 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence ggn and the codon at nucleotide positions 979 to 981 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence car; b) A nucleic acid molecule consisting of the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 16 or SEQ ID NO 17, except that the codon at nucleotide positions 979 to 981 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence car and the codon at nucleotide positions 1150 to 1152 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence wsn; c) A nucleic acid molecule consisting of the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 16 or SEQ ID NO 17, except that the codon at nucleotide positions 976 to 978 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence car and the codon at nucleotide positions 979 to 981 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence car; d) A nucleic acid molecule consisting of the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 16 or SEQ ID NO 17, except that the codon at nucleotide positions 979 to 981 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence car; e) A nucleic acid molecule consisting of the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 16 or SEQ ID NO 17, except that the codon at nucleotide positions 976 to 978 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence tty and the codon at nucleotide positions 979 to 981 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence car; f) A nucleic acid molecule consisting of the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 16 or SEQ ID NO 17, except that the codon at nucleotide positions 979 to 981 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence ath; g) A nucleic acid molecule consisting of the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 16 or SEQ ID NO 17, except that the codon at nucleotide positions 979 to 981 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence atg; h) A nucleic acid molecule consisting of the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 16 or SEQ ID NO 17, except that the codon at nucleotide positions 490 to 492 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence tay; i) A nucleic acid molecule consisting of the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 16 or SEQ ID NO 17, except that the codon at nucleotide positions 490 to 492 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence wsn; j) A nucleic acid molecule consisting of the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 16 or SEQ ID NO 17, except that the codon at nucleotide positions 979 to 981 in SEQ ID NO16 or SEQ ID NO 17 has the nucleotide sequence gtn; k) A nucleic acid molecule consisting of the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 16 or SEQ ID NO 17, except that the codon at nucleotide positions 1225 to 1227 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence mgn; l) A nucleic acid molecule consisting of the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 16 or SEQ ID NO 17, except that the codon at nucleotide positions 979 to 981 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence wsn; m) A nucleic acid molecule consisting of the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 16 or SEQ ID NO 17, except that the codon at nucleotide positions 811 to 813 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence ath; n) A nucleic acid molecule consisting of the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 16 or SEQ ID NO 17, except that the codon at nucleotide positions 985 to 987 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence ggn; o) A nucleic acid molecule consisting of the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 16 or SEQ ID NO 17, except that the codon at nucleotide positions 1225 to 1227 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence ccn; p) A nucleic acid molecule consisting of the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 16 or SEQ ID NO 17, except that the codon at nucleotide positions 1240 to 1242 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence atg; q) A nucleic acid molecule consisting of the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 16 or SEQ ID NO 17, except that the codon at nucleotide positions 493 to 495 in SEQ ID NO16 or SEQ ID NO 17 has the nucleotide sequence aar; r) A nucleic acid molecule consisting of the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 16 or SEQ ID NO 17, except that the codon at nucleotide positions 1240 to 1242 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence mgn; s) A nucleic acid molecule consisting of the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 16 or SEQ ID NO 17, except that the codon at nucleotide positions 1240 to 1242 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence cay; t) A nucleic acid molecule consisting of the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 16 or SEQ ID NO 17, except that the codon at nucleotide positions 493 to 495 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence tgy; u) A nucleic acid molecule consisting of the nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO 16 or SEQ ID NO 17, except that the codon at nucleotide positions 490 to 492 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence tgy; v) A nucleic acid molecule consisting of a nucleic acid sequence from position 1 to 1428 in the nucleic acid sequence shown in SEQ ID NO: 16 or SEQ ID NO: 17, except that the codon at nucleotide positions 1225 to 1227 in SEQ ID NO: 16 or SEQ ID NO: 17 has the nucleotide sequence aar.

6. A recombinant nucleic acid molecule comprising the nucleic acid molecule according to any one of claims 3 to 5.

7. The recombinant nucleic acid molecule according to claim 6, wherein the recombinant nucleic acid molecule is a vector or plasmid.

8. A host cell comprising the protein according to claim 1 or 2, or comprising the nucleic acid molecule according to any one of claims 3 to 5, or comprising the recombinant nucleic acid molecule according to claim 6 or claim 7.

9. A method for producing an amine, comprising the following steps: a) Providing an amine acceptor molecule; b) Providing an amine donor molecule; c) Contacting the amine acceptor molecule provided in step a) and the amine donor molecule provided in step b) with the protein according to claim 1 or 2.

10. A method for reducing the amount of amine enantiomers in a composition comprising (R)- and (S)-amine enantiomers, comprising the following steps: a) Providing a composition comprising (R)- and (S)-amine enantiomers; b) Providing an amine acceptor molecule; c) Contacting the composition provided in step a) and the amine acceptor provided in step b) with the protein according to claim 1 or 2.

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