NUCLEIC ACIDS ENCODING IMPROVED TRANSAMINASE PROTEINS

Abstract

The present invention concerns proteins having improved omega-transaminase (ω-TA) activity, nucleic acid molecules encoding respective proteins having improved ω-TA activity and methods for stereo selective synthesis of chiral amines and amino acids or increasing of chiral amines isomers in enantiomer mixtures.

Claims

1. A protein having the activity of an ω-transaminase (ω-TA), wherein the protein is selected from the group consisting of: a) proteins comprising the amino acid sequence from positions 1 to 477 as shown under SEQ ID NO 3 apart from that the amino acid at position 25 is different from F and the amino acid at position 64 is different from L and the amino acid at position 88 is different from T and the amino acid at position 157 is different from T and the amino acid at position 165 is different from R and the amino acid at position 169 is different from V and the amino acid at position 174 is different from E and the amino acid at position 187 is different from S and the amino acid at position 197 is different from M and the amino acid at position 239 is different from S and the amino acid at position 327 is different from S and the amino acid at position 328 is different from V and the amino acid at position 384 is different from Y and the amino acid at position 389 is different from I and the amino acid at position 391 is different from D and the amino acid at position 396 is different from K and the amino acid at position 410 is different from H and the amino acid at position 414 is different from P; b) proteins comprising the amino acid sequence from positions 1 to 479 as shown under SEQ ID NO 6 apart from that the amino acid at position 25 is different from F and the amino acid at position 64 is different from L and the amino acid at position 88 is different from T and the amino acid at position 157 is different from T and the amino acid at position 165 is different from R and the amino acid at position 169 is different from V and the amino acid at position 174 is different from E and the amino acid at position 187 is different from S and the amino acid at position 197 is different from T and the amino acid at position 239 is different from S and the amino acid at position 327 is different from S and the amino acid at position 328 is different from V and the amino acid at position 384 is different from Y and the amino acid at position 389 is different from I and the amino acid at position 391 is different from D and the amino acid at position 396 is different from K and the amino acid at position 410 is different from H and the amino acid at position 414 is different from P; c) proteins comprising the amino acid sequence from positions 1 to 476 as shown under SEQ ID NO 9 apart from that the amino acid at position 25 is different from F and the amino acid at position 64 is different from L and the amino acid at position 88 is different from T and the amino acid at position 157 is different from T and the amino acid at position 165 is different from R and the amino acid at position 169 is different from V and the amino acid at position 174 is different from E and the amino acid at position 187 is different from S and the amino acid at position 197 is different from M and the amino acid at position 239 is different from S and the amino acid at position 327 is different from S and the amino acid at position 328 is different from V and the amino acid at position 384 is different from Y and the amino acid at position 389 is different from I and the amino acid at position 391 is different from D and the amino acid at position 396 is different from K and the amino acid at position 410 is different from H and the amino acid at position 414 is different from P; d) proteins comprising the amino acid sequence from positions 1 to 476 as shown under SEQ ID NO 12 apart from that the amino acid at position 25 is different from F and the amino acid at position 64 is different from L and the amino acid at position 88 is different from T and the amino acid at position 157 is different from T and the amino acid at position 165 is different from R and the amino acid at position 169 is different from V and the amino acid at position 174 and the amino acid at position 187 is different from S is different from E and the amino acid at position 197 is different from T the amino acid at position 239 is different from S and the amino acid at position 327 is different from S and the amino acid at position 328 is different from V and the amino acid at position 384 is different from Y and the amino acid at position 389 is different from I and the amino acid at position 391 is different from D and the amino acid at position 396 is different from K and the amino acid at position 410 is different from H and the amino acid at position 414 is different from P; e) proteins comprising the amino acid sequence from positions 1 to 476 as shown under SEQ ID NO 15 apart from that the amino acid at position 25 is different from F and the amino acid at position 64 is different from L and the amino acid at position 88 is different from T and the amino acid at position 157 is different from T and the amino acid at position 165 is different from R and the amino acid at position 169 is different from V and the amino acid at position 174 and the amino acid at position 187 is different from S is different from E and the amino acid at position 197 is different from M and the amino acid at position 239 is different from S and the amino acid at position 327 is different from S and the amino acid at position 328 is different from V and the amino acid at position 384 is different from Y and the amino acid at position 389 is different from I and the amino acid at position 391 is different from D and the amino acid at position 396 is different from K and the amino acid at position 410 is different from H and the amino acid at position 414 is different from P; and f) proteins having an amino acid sequence having at least 60%, preferably 70%, more preferably 80%, further more preferably 90%, even more preferably 95%, even further more preferably 96%, particular preferably 97%, most preferably 98% or especially preferably 99% identity with any of the amino acid sequence's shown under a), b), c), d), e) or f), given that in each case the amino acid corresponding to position 25 is different from F and the amino acid corresponding to position 64 is different from L and the amino acid corresponding to 88 is different from T and the amino acid corresponding to position 157 is different from T and the amino acid corresponding to position 165 is different from R and the amino acid corresponding to position 169 is different from V and the amino acid corresponding to position 174 is different from E and the amino acid corresponding to position 187 is different from S and the amino acid corresponding to position 197 is different from T or M and the amino acid corresponding to position 239 is different from S and the amino acid corresponding to position 327 is different from S and the amino acid corresponding to position 328 is different from V and the amino acid corresponding to position 384 is different from Y and the amino acid corresponding to position 389 is different from I and the amino acid corresponding to position 391 is different from D and the amino acid corresponding to position 396 is different from K and the amino acid corresponding to position 410 is different from H and the amino acid corresponding to position 414 is different from P.

2. The protein according to claim 1 selected from the group consisting of: a) proteins comprising the amino acid sequence as defined in claim 1, section a) apart from that in addition the amino acid at position 2 is different from S and the amino acid at position 48 is different from D and the amino acid at position 164 is different from Y and the amino acid at position 202 is different from D and the amino acid at position 205 is different from L and the amino acid at position 242 is different from A and the amino acid at position 245 is different from A and the amino acid at position 311 is different from L and the amino acid at position 353 is different from F and the amino acid at position 359 is different from D and the amino acid at position 424 is different from K and the amino acid at position 475 is different from A and the amino acid at position 476 is different from L and the amino acid at position 477 is deleted; b) proteins comprising the amino acid sequence as defined in claim 1, section b) apart from that in addition the amino acid at position 46 is different from T and the amino acid at position 60 is different from C and the amino acid at position 185 is different from C and the amino acid at position 186 is different from S and the amino acid at position 195 is different from S and the amino acid at position 205 is different from Y and the amino acid at position 252 is different from V and the amino acid at position 268 is different from S and the amino acid at position 409 is different from R and the amino acid at position 436 is different from A and the amino acids at positions 477 and 478 and 479 are deleted; c) proteins comprising the amino acid sequence as defined in claim 1, section c) apart from that in addition the amino acid at position 2 is different from S and the amino acid at position 48 is different from D and the amino acid at position 69 is different from P and the amino acid at position 90 is different from S and the amino acid at position 164 is different from Y and the amino acid at position 242 is different from A and the amino acid at position 245 is different from A and the amino acid at position 268 is different from T and the amino acid at position 311 is different from L and the amino acid at position 318 is different from E and the amino acid at position 322 is different from R and the amino acid at position 353 is different from S and the amino acid at position 424 is different from K and the amino acid at position 452 is different from E; d) proteins comprising the amino acid sequence as defined in claim 1, section d) apart from that in addition the amino acid at position 46 is different from T and the amino acid at position 60 is different from C and the amino acid at position 185 is different from C and the amino acid at position 186 is different from C and the amino acid at position 195 is different from S and the amino acid at position 205 is different from Y and the amino acid at position 252 is different from V and the amino acid at position 268 is different from S and the amino acid at position 409 is different from R and the amino acid at position 436 is different from A; e) proteins comprising the amino acid sequence as defined in claim 1, section d) apart from that in addition the amino acid at position 48 is different from D and the amino acid at position 164 is different from Y and the amino acid at position 242 is different from A and the amino acid at position 245 is different from A and the amino acid at position 255 is different from F and the amino acid at position 424 is different from K; and f) proteins having an amino acid sequence having at least 60% identity with any of the amino acid sequences as defined under a), b), c), d) or e) given that each amino acid position as defined under a), b), c), d) or e), respectively, is also present at the corresponding amino acid position in the amino acid sequences of the protein sequence being at least 60% identical to the amino acid sequence as defined in each of a), b), c), d) or e).

3. The protein according to claim 1, further defined as selected from the group consisting of: a) proteins comprising the amino acid sequence from positions 1 to 476 as shown under SEQ ID NO 18; b) proteins having an amino acid sequence having at least 60% identity with the amino acid sequence from positions 1 to 476 as shown under SEQ ID NO 18 given 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 in SEQ ID NO 18 represent those amino acids shown at the respective positions in the amino acid sequence shown under SEQ ID NO 18; and c) proteins having an amino acid sequence having at least 60% identity with the amino acid sequence from positions 1 to 476 as shown under SEQ ID NO 18 given that the amino acids corresponding to positions 2, 25, 46, 48, 60, 64, 69, 88, 90, 157, 164, 165, 169, 174, 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, 476 and 477 in SEQ ID NO 18 represent those amino acids shown at the respective positions in the amino acid sequence shown under SEQ ID NO 18.

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

5. The protein according to claim 4 selected from the group consisting of: a) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from that the amino acid S at position 166 in SEQ ID NO 18 is substituted by G and the amino acid T at position 327 in SEQ ID NO 18 is substituted by Q; b) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from that the amino acid T at position 327 in SEQ ID NO 18 is substituted by Q and the amino acid C at position 384 in SEQ ID NO 18 is substituted by S; c) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from that the amino acid E at position 326 in SEQ ID NO 18 is substituted by Q and the amino acid T at position 327 in SEQ ID NO 18 is substituted by Q; d) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from that the amino acid T at position 327 in SEQ ID NO 18 is substituted by Q; e) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from hat the amino acid E at position 326 in SEQ ID NO 18 is substituted by F and the amino acid T at position 327 in SEQ ID NO 18 is substituted by Q; f) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from that the amino acid T at position 327 in SEQ ID NO 18 is substituted by C; g) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from that the amino acid T at position 327 in SEQ ID NO 18 is substituted by I; h) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from that the amino acid T at position 327 in SEQ ID NO 18 is substituted by M; i) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from that the amino acid F at position 164 in SEQ ID NO 18 is substituted by Y; j) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from that the amino acid F at position 164 in SEQ ID NO 18 is substituted by S; k) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from that the amino acid T at position 327 in SEQ ID NO 18 is substituted by V; l) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from that the amino acid T at position 409 in SEQ ID NO 18 is substituted by R; m) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from that the amino acid T at position 327 in SEQ ID NO 18 is substituted by S; n) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from that the amino acid V at position 271 in SEQ ID NO 18 is substituted by I; o) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from that the amino acid S at position 329 in SEQ ID NO 18 is substituted by G; p) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from that the amino acid T at position 409 in SEQ ID NO 18 is substituted by P; q) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from that the amino acid L at position 414 in SEQ ID NO 18 is substituted by M; r) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from that the amino acid Q at position 165 in SEQ ID NO 18 is substituted by K; s) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from that the amino acid L at position 414 in SEQ ID NO 18 is substituted by R; t) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from that the amino acid L at position 414 in SEQ ID NO 18 is substituted by H; u) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from that the amino acid Q at position 165 in SEQ ID NO 18 is substituted by C; v) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from that the amino acid T at position 327 in SEQ ID NO 18 is substituted by V; w) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from that the amino acid F at position 164 in SEQ ID NO 18 is substituted by C; x) proteins having the amino acid sequence from positions 1 to 476 in the amino acid sequence shown under SEQ ID NO 18, apart from that the amino acid T at position 409 in SEQ ID NO 18 is substituted by K; and y) proteins having an amino acid sequence having at least 60%, identity with any of the amino acid sequences as defined under 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) given that each amino acid position as defined under 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), respectively, is also present at the corresponding amino acid position in the amino acid sequences of the protein sequence having at least 60% identity with any of the amino acid sequences as defined under 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).

6. A nucleic acid molecule encoding a protein according to claim 1.

7. A nucleic acid molecule according to claim 6, further defined as encoding a protein having the activity of an ω-TA selected from the group consisting of: a) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequence as shown under SEQ ID NO 17; b) nucleic acid molecules encoding a protein comprising the amino acid sequence from position 1 to 476 in the amino acid sequence as shown under SEQ ID NO 18; c) nucleic acid molecules having at least 60% identity with the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequence shown under SEQ ID NO 17, given that the codon corresponding to nucleotide positions 73 to 75 in SEQ ID NO 17 has the nucleotide sequence mgn and the codon corresponding to nucleotide positions 190 to192 in SEQ ID NO 17 has the nucleotide sequence ath and the codon corresponding to nucleotide positions 262 to 264 in SEQ ID NO 17 has the nucleotide sequence gcn and the codon corresponding to nucleotide positions 469 to 471 in SEQ ID NO 17 has the nucleotide sequence gcn and the codon corresponding to nucleotide positions 493 to 495 in SEQ ID NO 17 has the nucleotide sequence mgn and the codon corresponding to nucleotide positions 505 to 507 in SEQ ID NO 17 has the nucleotide sequence gcn and the codon corresponding to nucleotide positions 520 to 522 in SEQ ID NO 17 has the nucleotide sequence ggn and the codon corresponding to nucleotide positions 589 to 591 in SEQ ID NO 17 has the nucleotide sequence gcn and the codon corresponding to nucleotide positions 559 to 561 in SEQ ID NO 17 has the nucleotide sequence aay and the codon corresponding to nucleotide positions 715 to 717 in SEQ ID NO 17 has the nucleotide sequence ccn and the codon corresponding to nucleotide positions 979 to 981 in SEQ ID NO 17 has the nucleotide sequence acn and the codon at nucleotide positions 982 to 984 in SEQ ID NO 17 has the nucleotide sequence ggn and the codon corresponding to nucleotide positions 1150 to 1152 in SEQ ID NO 17 has the nucleotide sequence tgy and the codon corresponding to nucleotide positions 1165 to 1167 in SEQ ID NO 17 has the nucleotide sequence ytn and the codon corresponding to nucleotide positions 1171 to 1173 in SEQ ID NO 17 has the nucleotide sequence gar and the codon corresponding to nucleotide positions 1186 to 1188 in SEQ ID NO 17 has the nucleotide sequence gar and 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; d) nucleic acid molecules having at least 60% identity with the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequence shown under SEQ ID NO 17, given that the codon corresponding to nucleotide positions 4 to 6 in SEQ ID NO 17 has the nucleotide sequence ggn and the codon corresponding to nucleotide positions 73 to 75 in SEQ ID NO 17 has the nucleotide sequence mgn and the codon the codon corresponding to nucleotide positions 136 to 138 in SEQ ID NO 17 has the nucleotide sequence atg and the codon the codon corresponding to nucleotide positions 142 144 in SEQ ID NO 17 has the nucleotide sequence ggn and the codon corresponding to nucleotide positions 178 to 180 in SEQ ID NO 17 has the nucleotide sequence tay and the codon corresponding to nucleotide positions 190 to 192 in SEQ ID NO 17 has the nucleotide sequence ath and the codon corresponding to nucleotide positions 205 to 207 in SEQ ID NO 17 has the nucleotide sequence car and the codon corresponding to nucleotide positions 262 to 264 in SEQ ID NO 17 has the nucleotide sequence gcn and the codon corresponding to nucleotide positions 268 to 270 in SEQ ID NO 17 has the nucleotide sequence gcn and the codon corresponding to nucleotide positions 469 to 471 in SEQ ID NO 17 has the nucleotide sequence gcn and the codon corresponding to nucleotide positions 490 to 492 in SEQ ID NO 17 has the nucleotide sequence tty and the codon corresponding to nucleotide positions 493 to 495 in SEQ ID NO 17 has the nucleotide sequence car and the codon corresponding to nucleotide positions 505 to 507 in SEQ ID NO 17 has the nucleotide sequence gcn and the codon corresponding to nucleotide positions 520 to 522 in SEQ ID NO 17 has the nucleotide sequence ggn and the codon corresponding to nucleotide positions 553 to 555 in SEQ ID NO 17 has the nucleotide sequence tay and the codon corresponding to nucleotide positions 556 to 558 in SEQ ID NO 17 has the nucleotide sequence aay and the codon corresponding to nucleotide positions 559 to 561 in SEQ ID NO 17 has the nucleotide sequence aay and the codon corresponding to nucleotide positions 583 to 585 in SEQ ID NO 17 has the nucleotide sequence ccn and the codon corresponding to nucleotide positions 589 to 591 in SEQ ID NO 17 has the nucleotide sequence gcn and the codon corresponding to nucleotide positions 604 to 606 in SEQ ID NO 17 has the nucleotide sequence aay and the codon corresponding to nucleotide positions 613 to 615 in SEQ ID NO 17 has the nucleotide sequence tgy and the codon corresponding to nucleotide positions 715 to 717 in SEQ ID NO 17 has the nucleotide sequence ccn and the codon corresponding to nucleotide positions 724 to 726 in SEQ ID NO 17 has the nucleotide sequence gtn and the codon corresponding to nucleotide positions 733 to 735 in SEQ ID NO 17 has the nucleotide sequence acn and the codon corresponding to nucleotide positions 754 to 756 in SEQ ID NO 17 has the nucleotide sequence ath and the codon corresponding to nucleotide positions 763 to 765 in SEQ ID NO 17 has the nucleotide sequence ath and the codon corresponding to nucleotide positions 802 to 804 in SEQ ID NO 17 has the nucleotide sequence aay and the codon corresponding to nucleotide positions 931 to 933 in SEQ ID NO 17 has the nucleotide sequence gtn and the codon corresponding to nucleotide positions 952 to 954 in SEQ ID NO 17 has the nucleotide sequence gcn and the codon corresponding to nucleotide positions 964 to 966 in SEQ ID NO 17 has the nucleotide sequence aar and the codon corresponding to nucleotide positions 979 to 981 in SEQ ID NO 17 has the nucleotide sequence acn and the codon corresponding to nucleotide positions 982 to 984 in SEQ ID NO 17 has the nucleotide sequence ggn and the codon corresponding to nucleotide positions 1057 to 1059 in SEQ ID NO 17 has the nucleotide sequence ytn and the codon corresponding to nucleotide positions 1075 to 1077 in SEQ ID NO 17 has the nucleotide sequence aay and the codon corresponding to nucleotide positions 1150 to 1152 in SEQ ID NO 17 has the nucleotide sequence tay and the codon corresponding to nucleotide positions 1165 to 1167 in SEQ ID NO 17 has the nucleotide sequence ytn and the codon corresponding to nucleotide positions 1171 to 1173 in SEQ ID NO 17 has the nucleotide sequence gar and the codon corresponding to nucleotide positions 1186 to 1188 in SEQ ID NO 17 has the nucleotide sequence gar and the codon corresponding to nucleotide positions 1225 to 1227 in SEQ ID NO 17 has the nucleotide sequence acn and 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 and the codon corresponding to nucleotide positions 1270 to 1272 in SEQ ID NO 17 has the nucleotide sequence gar and 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; e) nucleic acid molecules hybridizing with the complementary strand of the nucleic acid molecules defined under a), b), c) or d), given that the codon corresponding to nucleotide positions 73 to 75 in SEQ ID NO 17 has the nucleotide sequence mgn and the codon corresponding to nucleotide positions 190 to192 in SEQ ID NO 17 has the nucleotide sequence ath and the codon corresponding to nucleotide positions 262 to 264 in SEQ ID NO 17 has the nucleotide sequence gcn and the codon corresponding to nucleotide positions 469 to 471 in SEQ ID NO 17 has the nucleotide sequence gcn and the codon corresponding to nucleotide positions 493 to 495 in SEQ ID NO 17 has the nucleotide sequence mgn and the codon corresponding to nucleotide positions 505 to 507 in SEQ ID NO 17 has the nucleotide sequence gcn and the codon corresponding to nucleotide positions 520 to 522 in SEQ ID NO 17 has the nucleotide sequence ggn and the codon corresponding to nucleotide positions 559 to 561 in SEQ ID NO 17 has the nucleotide sequence aay and the codon corresponding to nucleotide positions 715 to 717 in SEQ ID NO 17 has the nucleotide sequence ccn and the codon corresponding to nucleotide positions 979 to 981 in SEQ ID NO 17 has the nucleotide sequence acn and the codon at nucleotide positions 982 to 984 in SEQ ID NO 17 has the nucleotide sequence ggn and the codon corresponding to nucleotide positions 1150 to 1152 in SEQ ID NO 17 has the nucleotide sequence tgy and the codon corresponding to nucleotide positions 1165 to 1167 in SEQ ID NO 17 has the nucleotide sequence ytn and the codon corresponding to nucleotide positions 1171 to 1173 in SEQ ID NO 17 has the nucleotide sequence gar and the codon corresponding to nucleotide positions 1186 to 1188 in SEQ ID NO 17 has the nucleotide sequence gar and 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; f) nucleic acid molecules hybridizing with the complemantary strand of the nucleic acid molecules defined under a), b), c) or d) given that the codon corresponding to nucleotide positions 4 to 6 in SEQ ID NO 17 has the nucleotide sequence ggn and the codon corresponding to nucleotide positions 73 to 75 in SEQ ID NO 17 has the nucleotide sequence mgn and the codon the codon corresponding to nucleotide positions 136 to 138 in SEQ ID NO 17 has the nucleotide sequence atg and the codon the codon corresponding to nucleotide positions 142 144 in SEQ ID NO 17 has the nucleotide sequence ggn and the codon corresponding to nucleotide positions 178 to 180 in SEQ ID NO 17 has the nucleotide sequence tay and the codon corresponding to nucleotide positions 190 to 192 in SEQ ID NO 17 has the nucleotide sequence ath and the codon corresponding to nucleotide positions 205 to 207 in SEQ ID NO 17 has the nucleotide sequence car and the codon corresponding to nucleotide positions 262 to 264 in SEQ ID NO 17 has the nucleotide sequence gcn and the codon corresponding to nucleotide positions 268 to 270 in SEQ ID NO 17 has the nucleotide sequence gcn and the codon corresponding to nucleotide positions 469 to 471 in SEQ ID NO 17 has the nucleotide sequence gcn and the codon corresponding to nucleotide positions 490 to 492 in SEQ ID NO 17 has the nucleotide sequence tty and the codon corresponding to nucleotide positions 493 to 495 in SEQ ID NO 17 has the nucleotide sequence car and the codon corresponding to nucleotide positions 505 to 507 in SEQ ID NO 17 has the nucleotide sequence gcn and the codon corresponding to nucleotide positions 520 to 522 in SEQ ID NO 17 has the nucleotide sequence ggn and the codon corresponding to nucleotide positions 553 to 555 in SEQ ID NO 17 has the nucleotide sequence tay and the codon corresponding to nucleotide positions 556 to 558 in SEQ ID NO 17 has the nucleotide sequence aay and the codon corresponding to nucleotide positions 559 to 561 in SEQ ID NO 17 has the nucleotide sequence aay and the codon corresponding to nucleotide positions 583 to 585 in SEQ ID NO 17 has the nucleotide sequence ccn and the codon corresponding to nucleotide positions 589 to 591 in SEQ ID NO 17 has the nucleotide sequence gcn and the codon corresponding to nucleotide positions 604 to 606 in SEQ ID NO 17 has the nucleotide sequence aay and the codon corresponding to nucleotide positions 613 to 615 in SEQ ID NO 17 has the nucleotide sequence tgy and the codon corresponding to nucleotide positions 715 to 717 in SEQ ID NO 17 has the nucleotide sequence ccn and the codon corresponding to nucleotide positions 724 to 726 in SEQ ID NO 17 has the nucleotide sequence gtn and the codon corresponding to nucleotide positions 733 to 735 in SEQ ID NO 17 has the nucleotide sequence acn and the codon corresponding to nucleotide positions 754 to 756 in SEQ ID NO 17 has the nucleotide sequence ath and the codon corresponding to nucleotide positions 763 to 765 in SEQ ID NO 17 has the nucleotide sequence ath and the codon corresponding to nucleotide positions 802 to 804 in SEQ ID NO 17 has the nucleotide sequence aay and the codon corresponding to nucleotide positions 931 to 933 in SEQ ID NO 17 has the nucleotide sequence gtn and the codon corresponding to nucleotide positions 952 to 954 in SEQ ID NO 17 has the nucleotide sequence gcn and the codon corresponding to nucleotide positions 964 to 966 in SEQ ID NO 17 has the nucleotide sequence aar and the codon corresponding to nucleotide positions 979 to 981 in SEQ ID NO 17 has the nucleotide sequence acn and the codon corresponding to nucleotide positions 982 to 984 in SEQ ID NO 17 has the nucleotide sequence ggn and the codon corresponding to nucleotide positions 1057 to 1059 in SEQ ID NO 17 has the nucleotide sequence ytn and the codon corresponding to nucleotide positions 1075 to 1077 in SEQ ID NO 17 has the nucleotide sequence aay and the codon corresponding to nucleotide positions 1150 to 1152 in SEQ ID NO 17 has the nucleotide sequence tay and the codon corresponding to nucleotide positions 1165 to 1167 in SEQ ID NO 17 has the nucleotide sequence ytn and the codon corresponding to nucleotide positions 1171 to 1173 in SEQ ID NO 17 has the nucleotide sequence gar and the codon corresponding to nucleotide positions 1186 to 1188 in SEQ ID NO 17 has the nucleotide sequence gar and the codon corresponding to nucleotide positions 1225 to 1227 in SEQ ID NO 17 has the nucleotide sequence acn and 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 and the codon corresponding to nucleotide positions 1270 to 1272 in SEQ ID NO 17 has the nucleotide sequence gar and 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; g) nucleic acid molecules deviating from the nucleic acid molecules defined under a), b), c), d), e) or f) due to degeneracy of the genetic code; h) nucleic acid molecules encoding a protein having at least 60% identity with the amino acid sequence from positions 1 to 476 as shown under SEQ ID NO 18 given that the amino acids corresponding to positions 25, 64, 88, 157, 165, 169, 174, 187, 239, 327, 328, 384, 389, 391, 396, 410 and 414 in SEQ ID NO 18 represent those amino acids shown at the respective positions in the amino acid sequence shown under SEQ ID NO 18; i) nucleic acid molecules encoding a protein having at least 60% identity with the amino acid sequence from positions 1 to 476 as shown under SEQ ID NO 18 given 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 in SEQ ID NO 18 represent those amino acids shown at the respective positions in the amino acid sequence shown under SEQ ID NO 18; and j) nucleic acid molecules comprising the nucleic acid sequence from positions from 1 to 1428 in the nucleic acid sequence as shown under SEQ ID NO 16.

8. A nucleic acid molecule according to claim 6 encoding a protein having the activity of an ω-TA selected from the group consisting of: a) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from 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 position at nucleotide positions 979 to 981 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence car; b) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from 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) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from 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 position at nucleotide positions 979 to 981 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence car; d) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from 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) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from 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) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from that the codon at nucleotide positions 979 to 981 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence car; g) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from that the codon at nucleotide positions 979 to 981 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence ath; h) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from that the codon at nucleotide positions 979 to 981 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence atg; i) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from that the codon at nucleotide positions 490 to 492 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence tay; j) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from that the codon at nucleotide positions 490 to 492 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence wsn; k) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from that the codon at nucleotide positions 979 to 981 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence gtn; l) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from that the codon at nucleotide positions 1225 to 1227 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence mgn; m) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from that the codon at nucleotide positions 979 to 981 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence wsn; n) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from that the codon at nucleotide positions 811 to 813 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence ath; o) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from that the codon at nucleotide positions 985 to 987 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence ggn; p) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from that the codon at nucleotide positions 1225 to 1227 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence ccn; q) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from that the codon at nucleotide positions 1240 to 1242 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence atg; r) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from that the codon at nucleotide positions 493 to 495 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence aar; s) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from that the codon at nucleotide positions 1240 to 1242 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence mgn; t) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from that the codon at nucleotide positions 1240 to 1242 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence cay; u) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from that the codon at nucleotide positions 493 to 495 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence tgy; v) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from that the codon at nucleotide positions 979 to 981 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence gtn; w) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from that the codon at nucleotide positions 490 to 492 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence tgy; x) nucleic acid molecules comprising the nucleic acid sequence from positions 1 to 1428 in the nucleic acid sequences as shown under SEQ ID NO 16 or SEQ ID NO 17, apart from that the codon at nucleotide positions 1225 to 1227 in SEQ ID NO 16 or SEQ ID NO 17 has the nucleotide sequence aar; and y) nucleic acid molecules having a nucleic acid sequence having at least 60% identity with any of the nucleic acid sequences as defined under 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) given that each codon nucleotide sequence as defined under 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), respectively, is also present at the corresponding codon nucleotide position in the nucleic acid sequences having at least 60% identity with any of the nucleic acid sequences as defined under 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).

9. A recombinant nucleic acid molecule comprising a nucleic acid molecule according to claim 6.

10. The recombinant nucleic acid molecule according to claim 9, wherein the recombinant nucleic acid molecule is a vector or a plasmid.

11. A host cell comprising a protein according to claim 1.

12. A method for the production of an amine comprising the steps of: 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 a protein according to claim 1.

13. A method for decreasing the amount an amine enantiomer in a composition comprising (R)- and (S)-amine enantiomers, comprising the steps of 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 a protein according to claim 1.

14. A host cell comprising a nucleic acid molecule according to claim 6.

15. A host cell comprising a recombinant nucleic acid molecule according to claim 9.

Description

DESCRIPTION OF THE FIGURES

[0353] FIG. 1: Plasmid map showing the genetic elements used for the expression of proteins having the activity of a DAAO, ω-TA and catalase from a single operon as tri-cistronic RNA. Explanations to abbreviations of regulatory genetic elements involved in transcription and translation of the tri-cistronic RNA:

[0354] lac operator: 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; consisting of the nucleic acid sequence shown under SEQ ID NO 26.

[0355] Trc promotor: 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 under SEQ ID NO 27.

[0356] rrnB: 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 under SEQ ID NO 28.

[0357] t7 enhancer: Transcription enhancing sequence from the t7 gene. (Sequence used: ttaacttta).

[0358] RBS1: Ribosome binding site (Sequence: gaggt).

[0359] cistron: Transcription termination sequence; consisting of the nucleic acid sequence shown under SEQ ID NO 29.

[0360] RBS2: Ribosome binding site (Sequence used: aaggag).

[0361] boxA: Transcriptional anti-termination sequence (Sequence used: tgctctttaacaa).

[0362] cistron: Synthetic cistron consisting of the nucleic acid sequence shown under SEQ ID NO 29.

[0363] rrnB terminator: Transcription termination signal; consisting of the nucleic acid sequence shown under SEQ ID NO 30.

[0364] T2 terminator: Translation termination signal (Orosz et al., 1991, Eur J Biochem. 201(3), 653-659).

[0365] FIG. 2: Presents the production of (S)-norvaline by amination of 2-oxovaleric acid ctalysed by wild-type ω-TA proteins from Arthrobacter sp. having the amino acid sequence shown under SEQ ID NO 6 or from Bacillus megaterium having the amino acid sequence shown under SEQ ID NO 3 compared to ω-TA variants having the amino acid sequence shown under SEQ ID NO 18.

[0366] FIG. 3: Presents the production of (S)-leucine by amination of 4-methyl-2-oxo-valeric acid ctalysed by wild-type ω-TA proteins from Arthrobacter sp. having the amino acid sequence shown under SEQ ID NO 6 or from Bacillus megaterium having the amino acid sequence shown under SEQ ID NO 3 compared to ω-TA variants having the amino acid sequence shown under SEQ ID NO 18.

[0367] FIG. 4: Presents the production of (S)-phenylalnine by amination of phenylpyruvic acid ctalysed by wild-type ω-TA proteins from Arthrobacter sp. having the amino acid sequence shown under SEQ ID NO 6 or from Bacillus megaterium having the amino acid sequence shown under SEQ ID NO 3 compared to ω-TA variants having the amino acid sequence shown under SEQ ID NO 18.

[0368] FIG. 5: Presents the production of (S)-tyrosine by amination of p-hydroxyphenylpyruvic acid ctalysed by wild-type ω-TA proteins from Arthrobacter sp. having the amino acid sequence shown under SEQ ID NO 6 or from Bacillus megaterium having the amino acid sequence shown under SEQ ID NO 3 compared to ω-TA variants having the amino acid sequence shown under SEQ ID NO 18.

GENERAL METHODS

[0369] 1. Production of ω-TA Variants and ω-TA Variants having Further Amino Acid Modifications

[0370] Known nucleotide sequences described herein encoding proteins having the activity of an ω-TA described herein were synthesized by the service provider by Eurofins Genomics GmbH (Eurofins Genomics GmbH, Anzinger Str. 7a, 85560 Ebersberg, Germany).

[0371] Into the nucleic acid sequences shown under SEQ ID Nos 2, 5, 8, 11, 14 nucleotide substitutions (replacements) were introduced. The replacement can be effected in the nucleic acid sequences which encode the reference polypeptide by any means which is appropriate for replacing nucleotides in nucleic acid sequences. Those methods are widely described in the literature and well known to the skilled person in the respective sequence. Several molecular biological methods can be used to achieve respective nucleotide replacements. A useful method for preparing a mutated nucleic acid sequence according to the invention and the corresponding protein comprises carrying out site-directed mutagenesis on codons encoding one or more amino acids which are selected in advance, thereby changing the selected codons in a way that they code for different amino acids. The methods for obtaining these site-directed mutations are well known to the skilled person and widely described in the literature (in particular: Directed Mutagenesis: A Practical Approach, 1991, Edited by M. J. McPHERSON, IRL PRESS), or are methods for which it is possible to employ commercial kits (for example the QUIKCHANGE™ lightening mutagenesis kit from Qiagen or Stratagene). After site-directed mutagenesis, nucleic acids were transformed into the Escherichia coli starin MG1655. The cells which contain a mutated polypeptide with advantageous biotransformation yields were selected by using an appropriate screening method. Appropriate screening methods are described herein under “General Methods”, items 4 and 7. Mutated nucleic acid sequences coding for improved polypeptides were sequence verified. The methods for sequence verification are well known to the skilled person and widely described in the literature (for example Sambrook and Russell (2012) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)).

[0372] 2. Expression Vectors/Host Cells for ω-TA Variants

[0373] Nucleic acid sequences encoding wild-type ω-TAs (SEQ ID Nos 2, 5, 8, 14) or known ω-TAs comprising mutations (SEQ ID NO 11) or ω-TA variants as described herein were cloned into the commercial pET22B vector (Merck KGaA, Frankfurter Str 250, 64293 Darmstadt, Germany) and expressed in Escherichia coli strain BL21 DE3 cells.

[0374] 3. Expression of ω-TA Variants

[0375] A pre-culture of Escherichia coli strain BL21 DE3 comprising the pET22B vector into which the respective nucleic acid sequence encoding ω-TA variants was introduced were grown in flasks containing 20 ml LB-Medium supplemented with carbenicillin at 37° C. on a rotary shaker at 180 rpm overnight. Expression of the coTA proteins was performed by transferring the pre-culture into flasks containing 250 ml LB-Medium supplemented with carbenicillin. Expression of the ω-TA proteins was induced by the addition of 0.5 mM IPTG (final concentration) after an OD (optical density) of between 0.6-0.8 was reached by growth at 37° C. on a rotary shaker at 180 rpm. The induced cell culture was incubated at 20° C. for 20 h at 180 rpm shaking. Purification of enzymes was performed using the Ni-NTA Fast Start Kit of Qiagen (Qiagen GmbH, Qiagen Strasse 1, 40724 Hilden) according to the manufacturers protocol.

[0376] 4. Activity Test for ωTA Variants in Presence of Amine Acceptors and Amine Donors

[0377] To 40 μl triethanolamine buffer (200 mM solution in deionized water, pH=9,0), 10 μl pyridoxal phosphate (10 mM solution in deionized water) and 10 μl of the amino donor (2 M solution in deionized water, adjusted to pH=9,0 by addition of aqueous HCl) was added at room temperature. Subsequently, 20 μl of the amino acceptor (100 mM solution in deionized water) was added (if the amino acceptor is not soluble in water, proportional DMSO is added). Finally, 20 μl of the transaminase enzyme (1,5 mg/ml) was added at room temperature and the mixture was incubated at 40° C. on a rotary shaker at 800 rpm for 6-7 h. The transamination reaction was monitored by HPLC-analysis of aliquots taken at various time intervals during the reaction.

[0378] 5. Expression Vectors/Host Cells Used for ω-TA Variants having Further Amino Acid Modifications

[0379] The activity test for ω-TA variants having further amino acid modifications was performed by using a method comprising two reaction steps.

[0380] The first reaction step (step 1) produces an amine acceptor for ω-TAs. The step is catalyzed by a D-amino acid oxidase (DAAO or DAO, EC 1.4.3.3). DAAOs are flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyse the oxidative deamination of D-amino acids with oxygen to generate the corresponding 2-oxo acids along with hydrogen peroxide and ammonia according to the following general equation (III):

α-D-amino acid+H.sub.2O+O.sub.2.fwdarw.α-2-oxo carboxylic acid+NH.sub.3+H.sub.2O.sub.2

[0381] The protein having the activity of a DAAO used for the production of α-2-oxo carboxylic acids in the first reaction step was a DAAO variant of the DAO1 protein from Rhodosporidium toruloides. The coding nucleic acid sequence of the wild-type DAO1 protein from Rhodosporidium toruloides is derivable from GenBank acc. No. U6006.1 (shown under SEQ ID NO 19) and the corresponding amino acid sequence encoded by the nucleic acid sequence shown under SEQ ID NO 19 is derivable from UniProt acc. No. P80324 (shown under SEQ ID NO 20). The DAAO variant used herein is disclosed in WO 201 7/1 51 573 as Mutant Ac305 (page 36, Table 1). Compared to SEQ ID NO 20 Mutant Ac305 comprises amino acid substations (replacements) at positions 54 and 58 and 213. In Mutant Ac305 the amino acid N at position 54 in SEQ ID NO 20 is substituted (replaced) by C and the amino acid F at position 58 in SEQ ID NO 20 is substituted (replaced) by H and the amino acid M at position 213 in SEQ ID NO 20 is substituted (replaced) by S. The amino acid sequence of Mutant Ac305 is shown under SEQ ID NO 22. A respective nucleic acid sequence encoding the protein having the amino acid sequence shown under SEQ ID NO 22 is shown under SEQ ID NO 21. The reaction of step 1 was catalyzed by a protein having the activity of a DAAO having the amino acid sequence shown under SEQ ID NO 22.

[0382] In a second reaction step (step 2) the α-2-oxo carboxylic acid produced by a protein having the activity of a DAAO) in step 1 is converted in presence of an amine donor by a protein having ω-TA activity into an amino acid according the general equation (I).

[0383] As becomes clear from description of step 1 by general equation (III), conversion of D-amino acids into keto acids catalysed by proteins having the activity of a DAAO generates hydrogen peroxide (H.sub.2O2). Removal of H.sub.2O.sub.2 may be desired, but is not necessarily needed under every circumstance. Removal of H.sub.2O.sub.2 was accomplished in connection with the present invention by adding a protein having the activity of a catalase.

[0384] Proteins having the activity of a catalase (EC 1.11.1.6; hydrogen-peroxide:hydrogen-peroxide oxidoreductase) are known in the art and catalyze the conversion of hydrogen peroxide (H.sub.2O.sub.2) into water (H.sub.2O) and oxygen (O.sub.2) according to following general equation (IV):

2H.sub.2O.sub.2>O.sub.2+2H.sub.2O

[0385] The amino acid sequence of a protein having the activity of a catalase from Listeria seeligeri used for removal of H.sub.2O.sub.2 is shown under SEQ ID NO 24 and derivable under GenePept accession no. WP_012986600.1. SEQ ID NO 23 (derivable under GenBank accession no. NC_013891.1) shows the nucleic acid coding sequence from Listeria seeligeri for the catalase protein having the amino acid sequence as shown under SEQ ID NO 24. SEQ ID NO 25 is a nucleic acid sequence also encoding the catalase protein having the amino acid sequence shown under SEQ ID NO 24. Compared to the nucleic acid sequence shown under SEQ ID VNO 23 codons of the nucleic acid sequence shown under SEQ ID NO 25 have been adapted to the codon usage of Escherichia coli.

[0386] For producing proteins having the activity of a DAAO, proteins having the activity of an ω-TA and proteins having the activity of a catalase, nucleic acid sequences encoding the respective three proteins were cloned into an E. coli expression vector in a manner that all three proteins were transcribed from a single operon as tri-cistronic RNA from the trc-promoter (a hybrid promoter composed of sequences originating from the trp- and the lacUV5-promoter). The order of the genes in respect to transcription from the promoter was DAAO (SEQ ID NO 21).fwdarw.nucleic acid molecules encoding ω-TA variants comprising further amino acid modifications (as described herein above).fwdarw.catalase (SEQ ID NO 25). SEQ ID NO 21 was translationally fused at its 5′-end with a nucleic acid sequence encoding the amino acid sequence M A R I R L. The expression vector used is based on pSE420 (description and sequences derivable from: Addgene, 75 Sidney St, Suite 550A, Cambridge, Mass. 02139; https://www.addgene.org/vector-database/4064/ or from Thermo Fisher Scientific (Invitrogen), Thermo Fisher Scientific Inc. 168 Third Avenue, Waltham, Mass. 02451 USA, https://www.thermofisher.com/search/results?query=pSE420&focusarea). Genetic elements were introduced into a modified pSE420 vector by means of commonly known methods. The relevant genetic elements present in the expression vector used are shown in FIG. 1. For expression of the three enzymes he expression vector was transferred Escherichia coli strain MG1655 cells.

[0387] 6. Expression of ω-TA Variants Comprising Further Amino Acid Modifications

[0388] ω-TA variants comprising further amino acid modifications were cloned into the tri-cistronic expression vector described above under “General Methods”, item 5 and expressed in Escherichia coli strain MG1655 cells. For this, a 20 ml pre-culture in LB-Medium supplemented with kanamycin was grown overnight in a shake flask at 37° C. on a rotary shaker at 180 rpm. Expression of the ωTA proteins was performed by transferring the pre-culture into flasks containing 200 ml LB-Medium supplemented with kanamycin. Expression of the ωTA proteins was induced after an OD of between 0.6-0.8 was reached by the addition of 1 mM IPTG (final concentration). The induced cell culture was incubated at 20° C. for 20 h at 180 rpm shaking. For harvest, the cell culture was centrifuged at 8000 g at 4° C. for 15 minutes and the obtained cell pellet was stored at −80° C. until freeze drying or spray drying.

[0389] 7. Activity Test of ω-TA Variants Having Further Amino Acid Modifications

[0390] In a one litre temperature-adjustable glass double jacketed reactor equipped with a mechanical stirrer, an O.sub.2-gas inlet tube and a pH-controlled dosing unit, 268 ml of an aqueous 50 w % racemic (R,S)-glufosinate ammonium solution (corresponds to 160,8 g racemic glufosinate ammonium) was added. Via the pH-controlled dosing unit an aqueous 2 M iso-propylamine solution was added under mechanical stirring (250 rpm) until a pH=9,0 was reached. The pH is kept constant during the entire reaction time by controlled addition of the aqueous 2 M iso-propylamine solution. The reactor is heated to 35° C. internal temperature.

[0391] In a beaker, 8 g of spray-dried Escherichia coli strain MG1655 cells containing the expression vector described in FIG. 1, expressing wild-type ω-TA proteins and ω-TA variants having further amino acid modifications, 200 mg pyridoxal phosphate, 2 ml polypropylene glycole (P 2000) and 138 ml deionized water were mixed. This mixture was added to the glass reactor under stirring (250 rpm) at 35° C. Via the O.sub.2-gas inlet tube, oxygen gas was bubbled through the reaction mixture at a flow rate ofr 0,1 l/min. The mixture was stirred for 24 h and the reaction progress was monitored via HPLC-analysis of aliquots taken at various time intervals during the reaction. Afterwards, the oxygen gas feed as well as the iso-propylamine feed was stopped and the reaction mixture was denatured at 90° C. for 30 min under stirring (250 rpm). The residual mixture was cooled down to room temperature.

[0392] 8. Detection of Amines Produced by ω-TAs

[0393] A) Analysis of Transamination Products (S)-norvaline, (S)-leucine, (S)-tyrosine and (S)-glufosinate Ammonium

[0394] The course of the transamination reaction was monitored via HPLC analysis. The HPLC methodology used in this work is based on the publication of Davankov et al. (1980, Chromatographia 13(11), 677-685).

[0395] Specifically, the following HPLC parameters were used:

[0396] Column: Phenomenex Chirex 3126 (D)—penicillamine 150*4,6 mm (Cat.: 00F-3126-E0)

[0397] Flow rate: 1 ml/min

[0398] Eluents: A) deionized water+0,5 g/L CuSO4 (v/v)

[0399] B) methanol

[0400] A:B=90:10 (isocratic)

[0401] Detector: DAD 230 nm

[0402] Oven: 30° C.

[0403] Runtime: 15 min

[0404] B) Analysis of Transamination Product L-Phenylalanine:

[0405] The course of the transamination reaction was monitored via HPLC analysis. Specifically, the following HPLC parameters were used:

[0406] Column: Phenomenex Prodigy 3 μm ODS-3 100A 100*4 mm (Cat.: 00D-4222-D0)

[0407] Flow rate: 2 ml/min

[0408] Eluents: A) acetonitrile

[0409] B) deionized water

[0410] Gradient from A:B=5:95 to A:B=95:5 within 7 min

[0411] Detector: VWD1 A, 210 nm

[0412] Oven: 40° C.

[0413] Run time: 9 min

[0414] 9. Spray Drying of Cells

[0415] Spray drying experiments have been performed in a laboratory (Lab scale) spray dryer with a maximum temperature input of 220° C. The dryer uses either compressed air or nitrogen with 200-800 l/h (litre/hour) under 5-8 bar. The maximum airflow can be reached with 35 m3/h (meter3/hour).

[0416] In order to dry bacterial cell mass from either flask grown culture or fermented material (i.e. 1 litre total volume) the broth has been ten times (10×) concentrated by centrifugation and re-suspended to a final volume of 100 ml in culture supernatant obtained after cemntrifugation.

[0417] The obtained concentrate needs to be suitable for pumping and should be constantly mixed by a magnetic stirrer. The liquid was applied to a 0.7 mm nozzle using an airflow of 500 l/h with the aspirator set to 100%. The typical product flow was 10 ml/min. and the applied temperatures averaged for the inlet −145° C. and the outlet 85° C. The subsequent dried biomass has been 5 weighed and used in g/l scale for the biotransformation experiments.

EXAMPLES

[0418] 1. Conversion of 2-Oxovaleric Acid to (S)-Norvaline

[0419] Wild-type ω-TA proteins from Arthrobacter sp. having the amino acid sequence shown under SEQ ID NO 6 or from Bacillus megaterium having the amino acid sequence shown under SEQ ID NO 3 or ω-TA variants having the amino acid sequence shown under SEQ ID NO 18 were expressed and purified as described under “General Methods”, item 3.

[0420] To 25 μl triethanolamine buffer in deionized water (200 mM solution in deionized water, pH=9.0), 10 μl of pyridoxal phosphate (PLP) in deionized water (10 mM solution in deionized water) and 10 μl of iso-propylamine in deionized water (2 M solution in deionized water, adjusted to pH=9.0 by addition of aqueous HCl) was added at room temperature. Subsequently, 20 μl of 2-oxovaleric acid (100 mM solution in deionized water) was added. Finally, 35 μl of a solution comprising 1.5 mg/ml of the respective ω-TA protein was added at room temperature and the mixture was incubated at 40° C. on a rotary shaker at 800 rpm for 6 h. The transamination reaction was monitored by HPLC-analysis of aliquots taken at different time intervals during the reaction as described under “General Methods” item 8.

[0421] Table 6 presents the results obtained for an ω-TA variant having the amino acid sequence shown under SEQ ID NO 18 compared to those of wild-type proteins from Arthrobacter sp. having the amino acid sequence shown under SEQ ID NO 6 and Bacillus megaterium having the amino acid sequence shown under SEQ ID NO 3. The results are also shown in FIG. 2.

TABLE-US-00006 TABLE 6 Area amine [mAU * s] time Area amine [mAU * s] Bacillus Arthrobacter sp. (SEQ ID Area amine [mAU * s] ω-TA [h] megaterium (SEQ ID NO 3) NO 6) variant (SEQ ID NO 18) 0 0 0 0 1 4691 1551 6096 2 5871 2486 8727 3 7508 3946 8868 5 8500 5333 9014 6 8463 5973 8573

[0422] Description of Table 6:

[0423] “time” measured in hours (h) indicates the time elapsed since the reaction was started.

[0424] “mAU*s” is the abbreviation for milli (m) absorbance (A) units (U) multiplied (*) with seconds (s); a standard unit describing the area under a peak in a HPLC chromatogram. The higher the area under a peak, the higher is the amount of the respective product.

[0425] It is derivable form Table 6 and FIG. 2, that in the reaction catalyzed by the ω-TA variant production of (S)-norvaline from 2-oxovaleric acid proceeds faster than in the reactions catalyzed by wild-type proteins from Arthrobacter sp. and Bacillus megaterium. In addition, the maximum amount of (S)-norvaline produced during the reaction is reached significantly earlier in the reaction catalyzed by the ω-TA variant compared to reactions catalyzed by wild-type proteins from Arthrobacter sp. and Bacillus megaterium.

[0426] 2. Conversion of 4-methyl-2-oxo-valeric Acid to (S)-Leucine

[0427] Wild-type ω-TA proteins from Arthrobacter sp. having the amino acid sequence shown under SEQ ID NO 3 or from Bacillus megaterium having the amino acid sequence shown under SEQ ID NO 6 or ω-TA variants having the amino acid sequence shown under SEQ ID NO 18 were expressed and purified as described under “General Methods”, item 3.

[0428] To 40 μl triethanolamine buffer (200 mM solution in deionized water, pH=9,0), 10 μl pyridoxal phosphate (10 mM solution in deionized water) and 10 μl of iso-propylamine (2 M solution in deionized water, adjusted to pH=9.0 by addition of aqueous HCl) was added at room temperature. Subsequently, 20 μl of 4-methyl-2-oxo-valeric acid (100 mM solution in deionized water) was added. Finally, 20 μl of a solution comprising 1.5 mg/ml of the respective ω-TA protein was added at room temperature and the mixture was incubated at 40° C. on a rotary shaker at 800 rpm for 6 h. The transamination reaction was monitored by HPLC-analysis of aliquots taken at different time intervals during the reaction as described under “General Methods” item 8.

[0429] Table 7 presents the results obtained for an ω-TA variant having the amino acid sequence shown under SEQ ID NO 18 compared to those of wild-type proteins from Arthrobacter sp. having the amino acid sequence shown under SEQ ID NO 3 and Bacillus megaterium having the amino acid sequence shown under SEQ ID NO 6. The results are also shown in FIG. 3.

TABLE-US-00007 TABLE 7 Area amine [mAU * s] time Area amine [mAU * s] Bacillus Arthrobacter sp. (SEQ ID Area amine [mAU * s] ω-TA [h] megaterium (SEQ ID NO 3) NO 6) variant (SEQ ID NO 18) 0 0 0 0 1 0 0 6971 2 0 0 7954 3 0 0 8052 4 0 0 7975 5 0 0 8267 6 0 0 8077

[0430] Description of Table 7: See description of Table 6

[0431] It is derivable from Table 7 and FIG. 3 that the wild-type enzymes from Arthrobacter sp. and Bacillus megaterium do not produce (S)-leucine by amination of 4-methyl-2-oxo-valeric acid, whereas the ω-TA variant does produce (S)-leucine quite efficiently.

[0432] 3. Conversion of Phenylpyruvic Acid to (S)-Phenylalanine

[0433] Wild-type ω-TA proteins from Arthrobacter sp. having the amino acid sequence shown under SEQ ID NO 3 or from Bacillus megaterium having the amino acid sequence shown under SEQ ID NO 6 or ω-TA variants having the amino acid sequence shown under SEQ ID NO 18 were expressed and purified as described under “General Methods”, item 3.

[0434] To 40 μl triethanolamine buffer (200 mM solution in deionized water, pH=9.0), 10 μl pyridoxal phosphate (10 mM solution in deionized water) and 10 μl of iso-propylamine (2 M solution in deionized water, adjusted to pH=9.0 by addition of aqueous HCl) was added at room temperature. Subsequently, 20 μl of phenylpyruvic acid in DMSO/deionized water in a ratio 1:1 (100 mM phenylpyruvic acid solution) was added. Finally, 20 μl of a solution comprising 1.5 mg/ml of the respective ω-TA protein was added at room temperature and the mixture was incubated at 40° C. on a rotary shaker at 800 rpm for 6 h. The transamination reaction was monitored by HPLC-analysis of aliquots taken at different time intervals during the reaction as described under “General Methods” item 8.

[0435] Table 8 presents the results obtained for an ω-TA variant having the amino acid sequence shown under SEQ ID NO 18 compared to those of wild-type proteins from Arthrobacter sp. having the amino acid sequence shown under SEQ ID NO 3 and Bacillus megaterium having the amino acid sequence shown under SEQ ID NO 6. The results are also shown in FIG. 4.

TABLE-US-00008 TABLE 8 Area amine [mAU * s] time Area amine [mAU * s] Bacillus Arthrobacter sp. (SEQ ID Area amine [mAU * s] ω-TA [h] megaterium SEQ ID NO 6) NO 6) variant (SEQ ID NO 18) 0 0 0 0 1 0 0 3271 2 245 0 5613 3 338 0 7056 4 403 0 7845 5 481 0 8772 6 540 0 10022

[0436] Description of Table 8: See description of Table 6

[0437] It is derivable from Table 8 and FIG. 4 that the wild-type enzyme from Arthrobacter sp. does not produce (S)-phenylalanine from phenylpyruvic acid, the wild-type enzyme from Bacillus megaterium produces (S)-phenylalanine very slowly and in low amounts compared to the amount of (S)-phenylalanine produced by the ω-TA variant.

[0438] 4. Conversion of p-hydroxyphenylpyruvic Acid to (S)-Tyrosine:

[0439] Wild-type ω-TA proteins from Arthrobacter sp. having the amino acid sequence shown under SEQ ID NO 3 or from Bacillus megaterium having the amino acid sequence shown under SEQ ID NO 6 or ω-TA variants having the amino acid sequence shown under SEQ ID NO 18 were expressed and purified as described under “General Methods”, item 3.

[0440] To 40 μl triethanolamine buffer (200 mM solution in deionized water, pH=9,0), 10 μl pyridoxal phosphate (10 mM solution in deionized water) and 10 μl of iso-propylamine (2 M solution in deionized water, adjusted to pH=9.0 by addition of aqueous HCl) was added at room temperature. Subsequently, 20 μl of p-hydroxyphenylpyruvic acid in DMSO/deionized water in a ratio 1:1 (100 mM p-hydroxyphenylpyruvic acid solution) was added. Finally, 20 μl of a solution comprising 1.5 mg/ml of the respective ω-TA protein was added at room temperature and the mixture was incubated at 40° C. on a rotary shaker at 800 rpm for 6 h. The transamination reaction was monitored by HPLC-analysis of aliquots taken at different time intervals during the reaction as described under “General Methods” item 8.

[0441] Table 9 presents the results obtained for an ω-TA variant having the amino acid sequence shown under SEQ ID NO 18 compared to those of wild-type proteins from Arthrobacter sp. having the amino acid sequence shown under SEQ ID NO 3 and Bacillus megaterium having the amino acid sequence shown under SEQ ID NO 6. The results are also shown in FIG. 5.

TABLE-US-00009 TABLE 9 Area amine [mAU * s] time Area amine [mAU * s] Bacillus Arthrobacter sp. (SEQ ID Area amine [mAU * s] ω-TA [h] megaterium (SEQ ID NO 3) NO 6) variant (SEQ ID NO 18) 0 0 0 0 1 0 0 3723 2 0 0 4902 3 0 0 5891 4 0 0 6450 5 0 0 8129 6 0 0 8912

[0442] Description of Table 9: See description of Table 6

[0443] It is derivable from Table 9 and FIG. 5 that the wild-type enzymes from Arthrobacter sp. and Bacillus megaterium do not produce (S)-tyrosine by amination p-hydroxyphenylpyruvic acid, whereas the ω-TA variant does produce (S)-tyrosine very efficiently.

[0444] 5. Production of (S)-Glufosinate from 4-[hydroxy(methyl)phosphoryl]-2-Oxobutanoic Acid by ω-TA Variants Comprising Further Amino Acid Modifications

[0445] ω-TA variants having the amino acid sequence shown under SEQ ID NO 18 and ω-TA variants comprising further amino acid modifications as described herein in Table 2 were expressed together with the proteins having the activity of a DAAO and having the activity of a catalase (see “General Methods”, item 5) as described under “General Methods”, item 6, followed by spray drying as described under “General Methods”, item 9. DAAO produces 4-[hydroxy(methyl)phosphoryl]-2-oxobutanoic acid by deamination of (R)-glufosinate. 4-[hydroxy(methyl)phosphoryl]-2-oxobutanoic acid is subsequently used by ω-TA variants having further amino acid modifications as amino acceptor and converted in an amination reaction into (S)-glufosinate. Activity tests of ω-TA variants having the amino acid sequence shown under SEQ ID NO 18 and ω-TA variants comprising further amino acid modifications as described herein in Table 2 were performed according to the test described under “General Methods, item 7. The transamination reaction was monitored by HPLC-analysis as described under “General Methods” item 8 by determining the amount of (S)-glufosinate produced in each reaction 5 h after the reaction was started.

[0446] Table 10 presents the amount of the amount of (S)-glufosinate (S-GA) produced by each of the ω-TA variants comprising further amino acid modifications and the amount produced by the ω-TA variant having the amino acid sequence shown under SEQ ID NO 18.

TABLE-US-00010 TABLE 10 Mutations introduced in S-GA respect to the amino acid produced sequence shown under in 5 h SEQ ID NO 18 [g/l] T327Q, S166G 20.01 T327Q, C384S 19.91 T327Q, E326Q 19.66 T327Q 19.01 T327Q, E326F 18.92 T327C 18.53 T327I 17.97 T327M 17.92 F164Y 17.71 F164S 16.9 T327V 16.82 T409R 16.66 T327S 16.17 V271I 15.47 S329G 15.31 T409P 15.25 L414M 15.14 Q165K 15.09 L414R 14.42 L414H 14.32 Q165C 14.23 T327V 14.02 F164C 13.87 T409K 12.82 None 12.76

[0447] Description of Table 10:

[0448] For identification of amino acid changes, numbers in column 1 identify the amino acid position in the amino acid sequence shown under SEQ ID NO 18. The character appearing before the number identifies the amino acid present at the respective position in the amino acid sequence shown under SEQ ID NO 18. The character appearing after the number identifies the amino acid present at the respective position in amino acid sequences of ω-TA variants comprising further amino acid modifications. Two numbers given in the same row of column , each with a character appearing before and after the number, identifies two simultaneous amino acid substitutions (replacements) compared to the amino acid sequence shown under SEQ ID NO 18.

[0449] It is derivable from Table 10, that ω-TA variants comprising further amino acid modifications produce more (S)-glufosinate compared to ω-TA variants having the amino acid sequence shown under SEQ ID NO 18.