CN109402098B

CN109402098B - Threonine aldolase, mutant and application of mutant in preparation of substituted phenylserine derivative

Info

Publication number: CN109402098B
Application number: CN201811310893.4A
Authority: CN
Inventors: 王喆明; 张荣珍; 谭昊; 李利宏
Original assignee: Hangzhou Fazheluo Biotechnology Co ltd
Current assignee: Hangzhou Fazheluo Biotechnology Co ltd
Priority date: 2018-11-06
Filing date: 2018-11-06
Publication date: 2021-09-17
Anticipated expiration: 2038-11-06
Also published as: CN109402098A; CN113481188B; CN113481188A

Abstract

The invention discloses threonine aldolase, a mutant, a coding gene of the threonine aldolase and the mutant, a recombinant vector constructed by the coding gene, a recombinant genetic engineering bacterium obtained by transforming the recombinant vector, and application of the threonine aldolase and the mutant in preparation of 2-/3-/4-substituted phenylserine derivatives. The invention takes threonine aldolase and mutant as biocatalyst, takes 2-/3-/4-substituted benzaldehyde as substrate, takes 5-pyridoxal phosphate as coenzyme, takes glycine/glycine ester as auxiliary substrate, and carries out enzyme catalytic reaction in proper conditions and medium to separate and prepare a series of phenylserine derivatives with different substituents, the total yield range of the method is 76-99%, the ee value of the product is more than 99%, and the de value range is 70-99%.

Description

Threonine aldolase, mutant and application of mutant in preparation of substituted phenylserine derivative

Technical Field

The invention relates to the technical field of biology, in particular to threonine aldolase, a mutant and application thereof in preparation of 2-/3-/4-substituted phenylserine derivatives.

Background

The 2-/3-/4-substituted phenylserine derivative is an important pharmaceutical intermediate and chemical intermediate, particularly 4-methylsulfonylphenylserine, widely used for preparation of human antibiotics, poultry and livestock antibiotics and the like, and represents drugs such as chloramphenicol, thiamphenicol, florfenicol and the like.

The synthetic method of the 2-/3-/4-substituted phenylserine derivative mainly comprises a chemical synthetic method and an enzyme catalysis method. The chemical synthesis method is mainly characterized in that chiral 2-/3-/4-substituted phenylserine derivatives are obtained by addition reaction, esterification reaction and chemical resolution method, although the chemical method is simple to operate, a large amount of water, heavy metal and virulent hydrogen sulfide are used in the reaction process, and a large amount of optical isomers are used as byproducts, so that the method does not conform to the atomic economy and environmental protection policies advocated and promoted by the current and future countries. The enzyme catalysis method has the advantages of mild conditions, small water consumption (only one tenth of that of the chemical method), no heavy metal pollution, no use of highly toxic chemicals, high chiral selection and the like, and has good application prospect.

Threonine aldolase has very wide application potential, and can catalyze and synthesize chiral pharmaceutical key intermediate beta-hydroxy-alpha-amino acid. However, the stability of threonine aldolase is poor, and the chiral selectivity, especially the chiral selectivity of beta-hydroxy is insufficient, so that the application of the threonine aldolase in chiral synthesis is limited.

Disclosure of Invention

The invention aims to provide threonine aldolase, mutant and application thereof in preparing 2-/3-/4-substituted phenylserine derivatives so as to overcome the defects of the prior art.

The invention adopts the following technical scheme:

in a first aspect, the present invention provides a threonine aldolase selected from Pseudomonas (Pseudomonas putida) having an amino acid sequence of SEQ ID NO:2, the gene sequence is SEQ ID NO:1 is shown in the specification; selected from the group consisting of Bacillus crescentus (Caulobacter creescens) having an amino acid sequence of SEQ ID NO:19, the gene sequence is SEQ ID NO: 88; selected from Thermotoga maritima (Thermotoga maritima), the amino acid sequence of which is SEQ ID NO: 23 is shown; selected from Listeria monocytogenes (Listeria monocytogenes) having an amino acid sequence of SEQ ID NO: shown at 24; selected from Escherichia coli (Escherichia coli) having an amino acid sequence of SEQ ID NO: 25 is shown; selected from Aeromonas jannaschii (Aeromonas jandaei) and the amino acid sequence of which is SEQ ID NO: 26 is shown; selected from Saccharomyces cerevisiae (Saccharomyces cerevisiae) with an amino acid sequence of SEQ ID NO: 27 is shown; selected from Ashbya gossypii (Ashbya gossypii) and the amino acid sequence of which is SEQ ID NO: 28 is shown; selected from Shewanella baumannii (Shewanella loihica), the amino acid sequence of which is SEQ ID NO: 29 is shown; is selected from Aeromonas veronii (Aeromonas veronii), and the amino acid sequence of the Aeromonas veronii is SEQ ID NO: 30 is shown in the figure; is selected from thioredoxin (Geobacter sulfuridunduns), and the amino acid sequence of the thioredoxin is SEQ ID NO: shown at 31.

In a second aspect, the present invention provides a threonine aldolase mutant, which is a mutation at four sites of threonine aldolase derived from different strains, wherein the mutation is a single-point mutation, a double-point mutation, a three-point mutation or a four-point mutation, and the mutation sites and the original amino acids are listed as follows:

threonine aldolase	First mutation point	Second mutation point	Third mutation point	Fourth mutation Point
					Ps_LTA	N17	H94	S182	T212
Cc_LTA	N14	H91	A178	T206
					Ec_LTA	T8	H83	A168	S196
Lm_LTA	---	H98	A178	Y206
					Tm_LTA	T8	H83	A170	S198
Aj_LTA	T10	H85	A170	S198
					Sc_LTA	T21	H97	A184	S212
Ag_LTA	T22	H98	A185	S213
					Sl_LTA	T8	H83	A168	S196
Av_LTA	T10	H85	A170	S198
					Gs_LTA	T10	H85	A170	S198

The corresponding amino acids that are replaced are as follows: the first mutation point N/T is replaced by Q, the second mutation point H is replaced by W/P/F/V/A/S, the third mutation point S/A is replaced by Y, and the fourth mutation point T/S/Y is replaced by I; wherein, threonine aldolases derived from different strains are defined as follows: ps LTA-threonine aldolase derived from Pseudomonas putida, Cc LTA-threonine aldolase derived from Bacillus crescentus, Ec LTA-threonine aldolase derived from Escherichia coli, Lm LTA-threonine aldolase derived from Listeria monocytogenes, Tm-LTA-threonine aldolase derived from Thermotoga maritima, Aj LTA-threonine aldolase derived from Aeromonas jandara, Sc LTA-threonine aldolase derived from Saccharomyces cerevisiae, Ag LTA-threonine aldolase derived from Aeromonas gossypii, Ashbya stenotrophaya, and Slavi LTA-threonine aldolase derived from Aeromonas sobria, gs _ LTA is derived from threonine aldolase of Geobacter sulfuricum (Geobacter sulfuriduccuns); wherein, the abbreviations for amino acids have the following meanings: n-asparagine, T-threonine, Q-glutamine, H-histidine, W-tryptophan, P-proline, F-phenylalanine, V-valine, A-alanine, S-serine, Y-tyrosine, I-isoleucine.

Further, a mutant of threonine aldolase selected from a mutant of threonine aldolase of Pseudomonas (Pseudomonas putida) having an amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, the gene sequence is SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, the sequence shown in SEQ ID NO: 4, the mutant shown in SEQ ID NO:2 to tyrosine from serine 182 of the amino acid sequence shown in SEQ ID NO: 6, the mutant shown in SEQ ID NO:2, the 94 th histidine of the amino acid sequence shown in SEQ ID NO: the mutant shown in the SEQ ID NO:2 to the amino acid sequence shown in SEQ ID NO: 10 is the mutant shown in SEQ ID NO:2 to isoleucine from threonine 212 of the amino acid sequence shown in seq id no; a threonine aldolase mutant selected from bacillus crescentus having an amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38; a mutant of threonine aldolase selected from Thermotoga maritima (Thermotoga maritima), having an amino acid sequence of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 is shown; a mutant of threonine aldolase selected from Listeria monocytogenes (Listeria monocytogenes) having an amino acid sequence of SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48 is shown; a mutant of threonine aldolase selected from Escherichia coli (Escherichia coli) having an amino acid sequence of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: shown at 53; a mutant of threonine aldolase selected from Aeromonas jannaschii (Aeromonas jandaei) having an amino acid sequence of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: shown at 58; a mutant of threonine aldolase selected from Saccharomyces cerevisiae (Saccharomyces cerevisiae) having an amino acid sequence of SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63; a threonine aldolase mutant selected from Ashbya gossypii (Ashbya gossypii) having an amino acid sequence of SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68; a mutant of threonine aldolase selected from Shewanella loihica (Shewanella loihica) having an amino acid sequence of SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73; a mutant of threonine aldolase selected from Aeromonas veronii (Aeromonas veronii) having an amino acid sequence of SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, respectively; a mutant of threonine aldolase selected from Geobacter sulfurreducens, having an amino acid sequence of SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, respectively.

In a third aspect, the present invention provides a gene encoding the above threonine aldolase and a mutant threonine aldolase.

In a fourth aspect, the present invention provides recombinant vectors constructed from the above-described encoding genes, which can be constructed by ligating the nucleotide sequences of the threonine aldolase and threonine aldolase mutants of the present invention to various vectors by methods conventional in the art. The vector may be any of various vectors conventionally used in the art, such as various plasmid vectors and the like.

In a fifth aspect, the present invention provides a recombinant genetically engineered bacterium obtained by transforming the above recombinant vector, which can be obtained by transforming the recombinant expression vector of the present invention into a host microorganism. The host microorganism may be any of various host microorganisms conventionally used in the art, as long as the recombinant expression vector can stably self-replicate and the carried threonine aldolase, threonine aldolase mutant gene of the present invention can be efficiently expressed, such as Escherichia coli, Bacillus subtilis, Corynebacterium, yeast, etc.

In a sixth aspect, the invention provides the use of the threonine aldolase and threonine aldolase mutant as described above for preparing 2-/3-/4-substituted phenylserine derivatives.

Further, using threonine aldolase or a mutant of threonine aldolase as a catalyst, using 2-/3-/4-substituted benzaldehyde as a substrate, using 5-pyridoxal phosphate as a coenzyme, using glycine/glycine ester as a co-substrate, carrying out enzyme catalysis in a reaction medium under the conditions of controlling temperature and stirring speed, and after the reaction is finished, separating and purifying to obtain the 2-/3-/4-substituted phenylserine derivative.

Further, the substrate is one of the following: benzaldehyde, 2-chlorobenzaldehyde, 2-fluorobenzaldehyde, 2-bromobenzaldehyde, 2-nitrobenzaldehyde, 2-methylsulfonylbenzaldehyde, 2-hydroxybenzaldehyde, 3-chlorobenzaldehyde, 3-fluorobenzaldehyde, 3-bromobenzaldehyde, 3-nitrobenzaldehyde, 3-methylsulfonylbenzaldehyde, 3-hydroxybenzaldehyde, 4-chlorobenzaldehyde, 4-fluorobenzaldehyde, 4-bromobenzaldehyde, 4-nitrobenzaldehyde, 4-methylsulfonylbenzaldehyde; the co-substrate is one of the following: l-glycine, L-glycine hydrochloride, L-glycine methyl ester hydrochloride, L-glycine ethyl ester hydrochloride, N-Boc-L-glycine ethyl ester, N-Z-L-glycine ethyl ester, N-Z-L-glycine methyl ester; the reaction medium is one of the following: a phosphoric acid buffer system with pH of 8-9, a water-ethanol system, a water-methanol system, a methanol system and an ethanol system.

Further, the dosage of the enzyme is 50-1000mg/L, the concentration of the substrate is 1-100g/L, the concentration of glycine/glycine ester is 5-200g/L, the concentration of pyridoxal 5-phosphate is 0.01-0.2g/L, the temperature is controlled at 25-45 ℃, and the stirring speed is 70-300 rpm.

The invention has the beneficial effects that:

1. the threonine aldolase mutants with good stability and high chiral selectivity are screened out by performing site-directed mutagenesis on threonine aldolases from different bacterial strains, and are successfully used for synthesizing 2-/3-/4-substituted phenylserine derivatives with high chiral purity, so that the excellent application prospect is shown.

2. The invention takes threonine aldolase and mutant as biocatalyst, takes 2-/3-/4-substituted benzaldehyde as substrate, takes 5-pyridoxal phosphate as coenzyme, takes glycine/glycine ester as auxiliary substrate, and carries out enzyme catalytic reaction in proper conditions and medium to separate and prepare a series of phenylserine derivatives with different substituents, the total yield range of the method is 76-99%, the ee value of the product is more than 99%, and the de value range is 70-99%.

Drawings

FIG. 1 is an HPLC chromatogram of example 19.

FIG. 2 is a nuclear magnetic spectrum of example 20.

Detailed Description

The invention is explained in more detail below with reference to exemplary embodiments and the accompanying drawings. The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1: amplification of threonine Aldolase Gene PSALD

Based on the threonine aldolase gene information from Pseudomonas putida recorded in Genebank (the gene sequence is shown in SEQ ID NO: 1), total genomic DNA of the cells was extracted using a nucleic acid rapid extraction apparatus, and PCR amplification was performed using the genomic DNA as a template and primers 1(ATGAATGGTGAAA CCAGCCG) and 2 (TTAACGTTCCTGGGTGCGAT). PCR reaction system (total volume 50. mu.L) 10 Xpfu DNA Polymerase Buffer 5. mu.L, 10mM dNTPmix (2.5 mM each of dATP, dCTP, dGTP and dTTP) 1. mu.L, 50. mu.M each of clone primer 1 and primer 2 1. mu.L, genomic DNA L. mu.L, Pfu DNA Polymerase 1. mu.L, and nucleic acid-free water 40. mu.L.

The PCR reaction conditions of a BioRad PCR instrument are pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, tempering at 65 ℃ for 45s, extension at 72 ℃ for 1min for 30 cycles, and extension at 72 ℃ for 10 min. The PCR reaction solution was subjected to electrophoresis on 0.9% agarose gel, and the fragment was recovered and purified by cutting the gel to obtain threonine aldolase gene PSALD.

Example 2: amplification of threonine Aldolase Gene CCALD

Based on the recorded information on the threonine aldolase gene from Bacillus crescentus (expressed as SEQ ID NO: 88) from Genebank, the total genomic DNA of the cells was extracted using a nucleic acid rapid extraction apparatus, and PCR amplification was performed using the genomic DNA as a template and primers 3(ATGACCCAFACCGCGCCCCGCTA) and 4 (CTAAGCCACTCGCTTCAGCGCC). PCR reaction system (total volume 50. mu.L) 10 Xpfu DNA Polymerase Buffer 5. mu.L, 10mM dNTPmix (2.5 mM each of dATP, dCTP, dGTP and dTTP) 1. mu.L, 50. mu.M each of clone primer 1 and primer 2 1. mu.L, genomic DNA L. mu.L, Pfu DNA Polymerase 1. mu.L, and nucleic acid-free water 40. mu.L.

The PCR reaction conditions of a BioRad PCR instrument are pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, tempering at 65 ℃ for 45s, extension at 72 ℃ for 1min for 30 cycles, and extension at 72 ℃ for 10 min. The PCR reaction solution was subjected to electrophoresis on 0.9% agarose gel, and the fragment was recovered and purified by cutting the gel to obtain threonine aldolase gene CCALD.

Example 3: construction of recombinant Escherichia coli for threonine aldolase PSALD

One, the acquisition of the recombinant plasmid pET28a-PSALD

Obtaining PSALD gene sequence, treating amplified fragment with Nde I and Xho I restriction enzyme (TaKaRa) after sequencing, and using T₄DNA ligase (TaKaRa) this fragment was ligated with a commercial vector pET28a treated with the same restriction enzyme to construct an expression vector pET28a-PSALD。

Coli BL21(DE3) transformed by recombinant plasmid

To 100. mu.L of E.coli BL21(DE3) competent cell suspension per tube was added 10. mu.L of the LPCR product, gently mixed, and allowed to stand in an ice bath for 30 min. Transferring into 42 deg.C water bath, and thermally shocking for 90 s. Transfer quickly to ice bath to cool for 3 min. 700. mu.L of LB liquid medium was added to each tube, and incubated at 37 ℃ for 1 hour with a shaker at 100 rpm. Centrifuging the cultured bacterial solution at 3,000rpm for 2min, discarding supernatant 700 μ L, mixing the rest bacterial solution, and spreading to a medium containing 50 μ g/mL^-1Kanamycin sulfate on LB plate, 37 degrees C inverted culture overnight.

Selection of positive clones: 4 clones were picked up and transferred to a cell containing 5mL of a DNA containing 50. mu.g.mL^-1The cells were cultured in LB medium containing kanamycin sulfate at 37 ℃ for 8 hours, and plasmids were extracted using Mini-Plasmid Rapid Isolation Kit (Bio-technology technologies, Inc., Boda, Beijing). 20 μ L of plasmid was taken out and submitted to Shanghai Biotech sequencing. Verify correct e.coli BL21(DE3)/pET28 a-PSALD.

Example 4: construction of recombinant Escherichia coli of threonine aldolase gene CCALD

One, the acquisition of the recombinant plasmid pET28a-CCALD

Obtaining CCALD gene sequence, treating amplified fragments with Nde I and Xho I restriction enzymes (TaKaRa) after sequencing, and using T₄DNA ligase (TaKaRa) this fragment was ligated with a commercial vector pET28a treated with the same restriction enzyme to construct an expression vector pET28 a-CCALD.

Coli BL21(DE3) transformed by recombinant plasmid

Selection of positive clones: 4 clones were picked up and transferred to a cell containing 5mL of a DNA containing 50. mu.g.mL^-1The cells were cultured in LB medium containing kanamycin sulfate at 37 ℃ for 8 hours, and plasmids were extracted using Mini-Plasmid Rapid Isolation Kit (Bio-technology technologies, Inc., Boda, Beijing). 20 μ L of plasmid was taken out and submitted to Shanghai Biotech sequencing. Verify correct e.coli BL21(DE3)/pET28 a-CCALD.

Example 5: amplification of expression mutant PSALD (S182Y), PSALD (H94P), PSALD (N17Q), PSALD (T212I) genes

Synthesizing two-end primers, wherein the sequences are respectively shown as SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18, respectively. :

the mutation is introduced by a PCR method, which comprises the following steps: and (3) PCR reaction system: ddH₂O22. mu.L, Premix PrimeSTAR 25. mu.L, forward primer (20. mu.M) 1. mu.L, reverse primer (20. mu.M) 1. mu.L, plasmid DNA 1. mu.L.

And (3) PCR reaction: pre-denaturation at 98 ℃ for 30 s; 30 cycles of 98 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 7 min; extension at 72 ℃ for 10 min. PSALD is used as a template, and PCR reaction is carried out by using primers to obtain a mutant full-length gene. The DNA fragment was purified using 3S Spin Agarose Gel DNA Purification Kit (Kyoho Biotech Co., Ltd.).

The purified gene PCR product was template digested with Dpn I enzyme:

enzyme digestion system: PCR product 30. mu.L, 10 XBuffer 5. mu.L, Dpn I1.5. mu.L, ddH₂O make up the system to 50. mu.L.

After 2h of digestion, the DNA fragment was purified using the 3S Spin Agarose Gel DNA Purification Kit (Kyoho Biotech Co., Ltd.).

The PCR reaction solution was subjected to 0.9% agarose gel electrophoresis, and the fragment was recovered and purified by cutting the gel, to thereby obtain threonine aldolase mutant genes PSALD (S182Y), PSALD (H94P), PSALD (N17Q) and PSALD (T212I).

Example 6: amplification of expression mutant CCALD (A178Y), CCALD (H91S) genes

Synthesizing two-end primers, wherein the sequences are respectively shown as SEQ ID NO: 84. SEQ ID NO: 85. SEQ ID NO: 86. SEQ ID NO: 87, respectively.

And (3) PCR reaction: pre-denaturation at 98 ℃ for 30 s; 30 cycles of 98 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 7 min; extension at 72 ℃ for 10 min. And (3) carrying out PCR reaction by using CCALD as a template and using a primer to obtain a mutant full-length gene. The DNA fragment was purified using 3S Spin Agarose Gel DNA Purification Kit (Kyoho Biotech Co., Ltd.).

The purified gene PCR product was template digested with Dpn I enzyme:

The PCR reaction solution was subjected to 0.9% agarose gel electrophoresis, and the fragment was recovered and purified by gel cutting to obtain threonine aldolase mutant genes CCALD (A178Y) and CCALD (H91S).

Example 7: construction of recombinant E.coli expressing mutants S182Y, H94P, N17Q, T212I

One, recombinant plasmid pET28a-PSALD (S182Y), pET28a-PSALD (H94P), pET28a-PSALD (N17Q), pET28a-PSALD (T212I) obtained

The mutant gene sequence was obtained, and the amplified fragment was treated with Nde I and Xho I restriction enzymes (TaKaRa) after sequencing and ligated with a commercial vector pET28a treated with the same restriction enzymes using T4DNA ligase (TaKaRa) to construct expression vectors pET28a-PSALD (S182Y), pET28a-PSALD (H94P), pET28a-PSALD (N17Q), pET28a-PSALD (T212I).

Coli BL21(DE3) transformed by recombinant plasmid

Add 10. mu.L of LPCR product to 100. mu.L E.coli BL21(DE3) competent cell suspension per tube, gentlyMixing, and standing in ice bath for 30 min. Transferring into 42 deg.C water bath, and thermally shocking for 90 s. Transfer quickly to ice bath to cool for 3 min. 700. mu.L of LB liquid medium was added to each tube, and incubated at 37 ℃ for 1 hour with a shaker at 100 rpm. Centrifuging the cultured bacterial solution at 3,000rpm for 2min, discarding supernatant 700 μ L, mixing the rest bacterial solution, and spreading to a medium containing 50 μ g/mL^-1Kanamycin sulfate on LB plate, 37 degrees C inverted culture overnight.

Selection of positive clones: 4 clones were picked up and transferred to a cell containing 5mL of a DNA containing 50. mu.g.mL^-1The cells were cultured in LB medium containing kanamycin sulfate at 37 ℃ for 8 hours, and plasmids were extracted using Mini-Plasmid Rapid Isolation Kit (Bio-technology technologies, Inc., Boda, Beijing). 20 μ L of plasmid was taken out and submitted to Shanghai Biotech sequencing. The amino acid sequences of threonine aldolase PSALD expressed by E.coli BL21(DE3)/pET28a-PSALD (S182Y), E.coli BL21(DE3)/pET28a-PSALD (H94P), E.coli BL21(DE3)/pET28a-PSALD (N17Q), E.coli BL21(DE3)/pET28a-PSALD (T212I) were verified to be correct, and serine at position 182 was mutated to tyrosine, histidine at position 94 to proline, asparagine at position 17 to glutamine, and threonine at position 212 to isoleucine, relative to the sequence shown in SEQ ID NO: 2.

Example 8: construction of recombinant E.coli expressing mutants A178Y, H91S

One, the obtainment of recombinant plasmids pET28a-CCALD (A178Y), pET28a-CCALD (H91S)

The mutant gene sequences were obtained, and the amplified fragments were treated with Nde I and Xho I restriction enzymes (TaKaRa) after sequencing and ligated with a commercial vector pET28a treated with the same restriction enzymes using T4DNA ligase (TaKaRa) to construct expression vectors pET28a-CCALD (A178Y) and pET28a-CCALD (H91S).

Coli BL21(DE3) transformed by recombinant plasmid

To 100. mu.L of E.coli BL21(DE3) competent cell suspension per tube was added 10. mu.L of the LPCR product, gently mixed, and allowed to stand in an ice bath for 30 min. Transferring into 42 deg.C water bath, and thermally shocking for 90 s. Transfer quickly to ice bath to cool for 3 min. 700. mu.L of LB liquid medium was added to each tube at 37 ℃ 10Incubate at 0rpm for 1 h. Centrifuging the cultured bacterial solution at 3,000rpm for 2min, discarding supernatant 700 μ L, mixing the rest bacterial solution, and spreading to a medium containing 50 μ g/mL^-1Kanamycin sulfate on LB plate, 37 degrees C inverted culture overnight.

Selection of positive clones: 4 clones were picked up and transferred to a cell containing 5mL of a DNA containing 50. mu.g.mL^-1The cells were cultured in LB medium containing kanamycin sulfate at 37 ℃ for 8 hours, and plasmids were extracted using Mini-Plasmid Rapid Isolation Kit (Bio-technology technologies, Inc., Boda, Beijing). 20 μ L of plasmid was taken out and submitted to Shanghai Biotech sequencing. The amino acid sequences of threonine aldolase CCALD expressed by E.coli BL21(DE3)/pET28a-CCALD (A178Y) and E.coli BL21(DE3)/pET28a-CCALD (H91S) were verified to have the alanine at position 178 mutated to tyrosine and the histidine at position 91 mutated to serine, respectively, relative to the sequence shown in SEQ ID NO: 19.

Example 9: construction of recombinant Bacillus subtilis expressing mutants S182Y, H94P, N17Q and T212I

One, the obtainment of recombinant plasmids pHCMC04-PSALD (S182Y), pHCMC04-PSALD (H94P), pHCMC04-PSALD (N17Q), pHCMC04-PSALD (T212I)

Obtaining the mutant gene sequence, processing the amplified fragment by Kpn I and Sma I restriction enzyme (TaKaRa) after sequencing, and using T₄The fragment was ligated with commercial vector pHCMC04 treated with the same restriction enzyme using DNA ligase (TaKaRa) to construct expression vectors pHCMC04-PSALD (S182Y), pHCMC04-PSALD (H94P), pHCMC04-PSALD (N17Q), pHCMC04-PSALD (T212I).

Secondly, transforming the recombinant plasmid into Bacillus subtilis SCK6

The bacillus subtilis SCK6 is scratched to a T2 culture medium solid plate containing ampicillin resistance, a single colony is selected and inoculated in 5ml of LB liquid culture medium, shaking culture is carried out at 37 ℃ overnight, 500 mu L of culture is taken and transferred to 10ml of LB liquid culture medium (OD600 is diluted to about 1), xylose with the final concentration of 1% (w/v) is added, shaking culture is carried out at 37 ℃ for 2 hours, glycerol with the final concentration of 10% is added, 500 mu L of each tube is subpackaged and then frozen at-70 ℃, and the obtained competent cell is obtained.

Mu.g of plasmid DNA was added to 500. mu.L of competent cells, cultured with shaking at 37 ℃ for 1.5 hours, plated with ampicillin-resistant T2 plate, and examined for transformation after standing overnight at 37 ℃.

Selection of positive clones: selecting a single colony from a transformation plate, inoculating the single colony in 5ml LB liquid culture medium, carrying out shaking culture at 37 ℃ overnight, taking 3ml of culture, centrifugally collecting thalli, carrying out resuspension by 150 mu L of TE, adding lysozyme, then carrying out incubation for 1 hour at 37 ℃, adding SDS and proteinase K with final concentration of 0.5% (w/v), carrying out inversion mixing, carrying out incubation at 37 ℃ until mixed liquid is clear, adding 300 mu L of solution TE, carrying out inversion mixing, carrying out centrifugation, collecting supernatant, adsorbing and purifying by using a silicon matrix column in an Omega small-amount plasmid extraction kit, and eluting by using 50 mu LTE to obtain plasmid DNA. 20 μ L of plasmid was taken out and submitted to Shanghai Biotech sequencing. The amino acid sequences of threonine aldolase PSALD (T212I) verified to be correctly expressed, namely Bacillus subtilis SCK6/pHCMC04-PSALD (S182Y), Bacillus subtilis SCK6/pHCMC04-PSALD (H94P), Bacillus subtilis SCK6/pHCMC04-PSALD (N17Q), Bacillus subtilis SCK6/pHCMC04-PSALD, have serine at position 182 changed into tyrosine, histidine at position 94 changed into proline, asparagine at position 17 changed into glutamine, and threonine at position 212 changed into isoleucine, compared with the sequence shown in SEQ ID NO: 2.

Example 10: construction of recombinant Corynebacterium glutamicum expressing mutants S182Y, H94P, N17Q and T212I

One, obtaining of recombinant plasmids pUC19-PSALD (S182Y), pUC19-PSALD (H94P), pUC19-PSALD (N17Q), pUC19-PSALD (T212I)

Obtaining mutant gene sequence, processing amplified fragment with Hind I and BamH I restriction enzyme (TaKaRa) after sequencing, and using T₄The fragment was ligated with a commercial vector pUC19 treated with the same restriction enzyme (TaKaRa) to construct expression vectors pUC19-PSALD (S182Y), pUC19-PSALD (H94P), pUC19-PSALD (N17Q) and pUC19-PSALD (T212I).

Secondly, the recombinant plasmid is transformed into Corynebacterium glutamicum C.glutamicum TK26₀₂₅₁

Picking fresh plates of C.glutamicum TK26₀₂₅₁Single colonyInoculating into 2mL liquid LB culture medium containing 0.5% glucose, and culturing at 30 deg.C on rotary shaker at 250r/min for 12-14 h. Inoculating to 50mL liquid LB medium containing 3% glycine and 0.1% Tween-80 at an inoculation amount of about 1%, and culturing at 30 deg.C until cell OD₆₀₀Up to 0.4. After the cell culture is finished, the bacterial liquid is iced for 15min, and then the thalli are collected by centrifugation (5500g, 20min, 4 ℃). The cells were suspended in 250mL of ice buffer (10% w/v glycerol, 8mmol/L, Tris, pH 7.4) and collected by centrifugation (4 ℃, 5500r/min, 20 min). The cells were washed 4 times with 100mL of pre-cooled 10% glycerol. Finally, the cells were resuspended in 0.2mL of 10% glycerol and dispensed into 1.5mL centrifuge tubes at 50. mu.L/tube. The treated cells can be used directly for electrotransformation or stored in a refrigerator at-70 ℃ for later use.

And (3) electric conversion: freezing and thawing 50 μ L of Corynebacterium glutamicum competent cells for 1 time, adding 5 μ L of plasmid DNA to 50 μ L of competent cells, mixing, transferring the mixture to a 0.2mm electric rotor, opening the electric rotor for 5min in ice bath, and setting parameters of 1.5-3.0kV, 200Q and 25 μ F. Wiping off the condensed water outside the electric rotating cup, putting the electric rotating cup into an electric shock instrument, and starting electric pulse to the cells. After the pulse was completed, the electric rotor was removed as soon as possible and 1mL of SOC medium was added. Mixing, transferring into EP tube, and standing at 30 deg.C for 1 hr.

Selection of positive clones: 100-300 mu L of bacterial liquid is taken to be coated on an ampicillin resistance plate containing 10 mu g/ml for inverted culture at 30 ℃, after bacterial growth, a single colony is picked and inoculated on an LB culture medium of 3 percent glycine with 20 mu g/ml, and cultured overnight at 30 ℃, and the quality-improving enzyme is cut and verified. 20 μ L of plasmid was taken out and submitted to Shanghai Biotech sequencing. Verify correct C.glutamcum TK26₀₂₅₁The amino acid sequences of threonine aldolase PSALD expressed by pUC19-PSALD (S182Y), pUC19-PSALD (H94P), pUC19-PSALD (N17Q), pUC19-PSALD (T212I) were mutated to tyrosine at position 182, proline at position 94, glutamine at position 17, and isoleucine at position 212, respectively, with respect to the sequence shown in SEQ ID NO. 2.

Example 11: construction of recombinant Saccharomyces cerevisiae expressing mutants S182Y, H94P, N17Q, T212I

One, recombinant plasmid pESC-URA-PSALD (S182Y), pESC-URA-PSALD (H94P), pESC-URA-PSALD (N17Q), pESC-URA-PSALD (T212I) obtained

Obtaining the mutant gene sequence, processing the amplified fragment by using BamH I and Kpn I restriction enzyme (TaKaRa) after sequencing, and using T₄The fragment was ligated with a commercial vector pESC-URA treated with the same restriction enzyme using DNA ligase (TaKaRa) to construct expression vectors pESC-URA-PSALD (S182Y), pESC-URA-PSALD (H94P), pESC-URA-PSALD (N17Q), and pESC-URA-PSALD (T212I).

Secondly, the Saccharomyces cerevisiae YPH501 is transformed by the recombinant plasmid

Thawing the saccharomyces cerevisiae YPH501 competent cells in an ice bath, and placing for later use; 1 μ g of the recombinant vectors pESC-URA-PSALD (S182Y), pESC-URA-PSALD (H94P), pESC-URA-PSALD (N17Q), pESC-URA-PSALD (T212I) and 100 μ g of linear salmon sperm were added to the thawed competent cells; adding 300 mu L of LTE/PEG-3350 (40%), reversing the upside down and mixing evenly; culturing at 30 deg.C for 30 min; thermally shocking at 42 deg.C for 15 min; adding 500 μ L preheated YPD liquid culture medium, and culturing at 30 deg.C for 2 hr; and (3) coating the bacterial liquid on an uracil-deficient SD-U plate culture medium according to a gradient, and culturing for 2-3 d at 30 ℃.

After transformation, the bacterial liquid is coated on YPD culture medium containing ampicillin antibiotic for screening, and positive transformant is obtained. The empty vector pAUR123 was transformed into yeast competent cells containing the empty vector pESC-URA in the same manner. Colony PCR verification is carried out on the transformant, and the system is prepared as 2.4.3, and the program is as follows: 94 ℃ for 5 min; 94 ℃, 40 s; at 58 ℃ for 50 s; 72 ℃ for 2 min; (ii) Rep 30; 72 ℃ for 10 min; hold, 4 ℃. After the PCR was completed, the amplification result was checked by electrophoresis on 1% agarose gel.

Selection of positive clones: 100-300 mu L of bacterial liquid is taken to be coated on a Cm resistance plate containing 10 mu g/ml for inverted culture at 30 ℃, after the bacterial colony grows out, a single bacterial colony is picked and inoculated on an LB culture medium of 3 percent glycine with 20 mu g/ml, the culture is carried out overnight at 30 ℃, and the quality-improving enzyme cutting is carried out for verification. 20 μ L of plasmid was taken out and submitted to Shanghai Biotech sequencing. The amino acid sequences of threonine aldolase PSALD expressed by YPH501/pESC-PSALD (S182Y), YPH501/pESC-PSALD (H94P), YPH501/pESC-PSALD (N17Q), and YPH501/pESC-PSALD (T212I) were verified to be correct in that serine at position 182 was mutated to tyrosine, histidine at position 94 was mutated to proline, asparagine at position 17 was mutated to glutamine, and threonine at position 212 was mutated to isoleucine, respectively, with respect to the sequence shown in SEQ ID NO: 2.

Example 12: induced expression culture of recombinant Escherichia coli

The obtained E.coli BL21(DE3)/pET28a-PSALD (S182Y), E.coli BL21(DE3)/pET28a-PSALD (H94P), E.coli BL21(DE3)/pET28a-PSALD (N17Q), E.coli BL21(DE3)/pET28a-PSALD (T212I), E.coli BL21(DE3)/pET28a-CCALD (A178Y), E.coli BL21(DE3)/pET28a-CCALD (H91S) and control group BL21(pET-28a empty vector transformant) were used as negative controls and inoculated into 5mL of transformants containing 50. mu.g.mL^-1Kanamycin sulfate in LB liquid medium, at 37 degrees, 200rpm shaking culture overnight. 10mL of the culture medium was inoculated to 1L of a medium containing 50. mu.g/mL^-1Culturing in LB liquid medium containing kanamycin sulfate at 37 deg.C under shaking at 200rpm to OD₆₀₀About 0.6 to about 0.8. isopropyl-BETA-D-thiogalactoside was added to the culture at a final concentration of 0.5mM, and induction culture was carried out at 25 ℃ for 12 hours. The cells were collected, dissolved in 20mM phosphate buffer (pH 7.0), and sonicated for 30min with a working time of 2s and a pause time of 3 s. The disrupted cells were mixed at 12,000rpm for 40 min. Taking supernatant and sediment respectively, and detecting protein expression by SDS-PAGE.

Example 13: induced expression culture of recombinant bacillus subtilis

The obtained Bacillus subtilis containing 4 genes and a control group SCK6(pHCMC04 empty vector transformant) were used as negative controls, inoculated into a test tube containing 5ml of LB liquid medium, and cultured overnight with shaking at 37 ℃. Transferring 2ml of each culture on the next day into a 250ml shake flask containing 50ml of 5YC liquid culture medium, performing shaking culture at 33 ℃ for 8 hours, sampling, centrifuging, collecting supernatant, adding isopropyl-BETA-D-thiogalactoside with final concentration of 1mM for induction, continuing culturing for 48 hours, and sampling, centrifuging and collecting culture supernatant every 8 hours. The medium in the above shake flask experiments contained 25. mu.g/ml chloramphenicol. Adding 20 μ l culture supernatant into 5 μ l 4 × loading buffer solution, mixing, treating with boiling water bath for 5min, loading 20 μ l, concentrating gel concentration 5%, separating gel concentration 17%, inner tank is cathode buffer solution, and outer tank is anode buffer solution during electrophoresis.

Example 14: induced expression culture of recombinant corynebacterium glutamicum

The obtained Corynebacterium glutamicum containing 4 genes and YPH501(pUC19 empty vector) of the control group were inoculated into 20mL of seed medium and cultured on a shaker at 30 ℃ and 200 r/min. OD of bacterial liquid₆₀₀When the inoculum size is 1.0%, the seed solution is inoculated to a fermentation medium at 5%, and cultured on a shaker at 30 ℃ and 200 r/min. When OD is reached₆₀₀When the concentration of isopropyl-BETA-D-thiogalactoside is 3.5, adding isopropyl-BETA-D-thiogalactoside to make the concentration of isopropyl-BETA-D-thiogalactoside in the culture medium reach 1mM, and culturing at 28 deg.C and 200r/min for 36 hr. 30mL of the culture medium was centrifuged in a mini centrifuge at 12000r/min at 4 ℃ for 10 min. The cells were resuspended in 30mL of sodium acetate buffer (50mmol/L, pH 6.0). The cell suspension was sonicated for 30 min. Cell debris was removed by centrifugation at 12000r/min for 30min at 4 ℃. mu.L of the supernatant was added to 9. mu.L of 5xSDS-PAGE electrophoresis buffer and mixed well. Boiling at 100 deg.C for 10min, and storing at-20 deg.C until protein electrophoresis analysis.

Example 15: induced expression culture of recombinant saccharomyces cerevisiae

The obtained yeast engineering bacteria containing 4 genes and a reference group saccharomyces cerevisiae YPH501(pESC-URA empty vector) are simultaneously inoculated in a YPD liquid culture medium, after overnight culture, bacterial liquid is added until a new culture medium adjusts the OD600nm value to be 0.5, shaking culture is carried out for 3d at the temperature of 30 ℃, and galactose is added for inducing expression for 1 d; collecting yeast culture solution, centrifuging at 6000rpm at 4 deg.C for 10min, removing supernatant, and collecting thallus precipitate; freezing and thawing the thallus precipitate with liquid nitrogen, adding cell lysate into the thallus precipitate, and mixing; crushing the bacterial liquid by using a cell crushing instrument under the conditions of 25KHz, 10s work/interval and 20 min; centrifuging at 4 ℃ and collecting the supernatant to obtain a crude enzyme sample of the saccharomyces cerevisiae.

Example 16: purification of recombinant proteins S182Y, H94P, N17Q, T212I, A178Y and H91S (expression of recombinant E.coli BL21(DE3) system)

Purification of recombinant proteins S182Y, H94P, N17Q, T212I, a178Y, H91S using His-Trap HP affinity column:

single colonies of the positive clones were picked and inoculated into 5mL of a medium containing 50. mu.g.mL^-1Kanamycin sulfate in LB liquid medium, at 37 degrees, 200rpm shaking culture overnight. 10mL of the culture medium was inoculated to 1L of a medium containing 50. mu.g/mL^-1Culturing in LB liquid medium containing kanamycin sulfate at 37 deg.C under shaking at 200rpm to OD₆₀₀About 0.6 to about 0.8. 0.5mM of isopropyl-BETA-D-thiogalactoside as an inducer was added to the culture, and the induction culture was carried out at a culture temperature of 25 ℃ for 12 hours. The cultured recombinant E.coli cells were collected after centrifugation at 12,000rpm for 10min and three-time washing with physiological saline.

2g of wet cells were weighed, added with an appropriate amount of 20mM Tris, 150mM NaCl (pH8.0) buffer to suspend the cells, and subjected to ultrasonication in an ice bath (2 s for work, 3s apart, 30min for work time). The supernatant was collected as a crude enzyme solution by centrifugation at 12,000rpm for 30min at 4 ℃. Purifying the crude enzyme solution by His-Trap affinity chromatography produced by GE company, and ultrafiltering and desalting the pure enzyme solution for specifically converting 2-/3-/4-substituted benzaldehyde.

Example 17: determination of specific enzyme Activity before and after mutation

Determination of the mutant enzyme Activity:

threonine aldolase catalyzes the cleavage of threonine to glycine and acetaldehyde in the presence of a cofactor (pyridoxal-5-phosphate, PLP), acetaldehyde being reduced by alcohol dehydrogenase in the presence of NADH, NAD⁺The production of (A) causes a decrease in absorbance at 340nm, and therefore the activity of the phenylserine aldolase can be measured by measuring the change in absorbance at 340nm during the reaction.

Standard conditions for enzyme activity assay: the total reaction volume was 250. mu.L, 100mM Hepes-NaOH buffer (pH8.0), 50uM PLP, 50mM L-threonine, 0.5mM NADH, 30U alcohol dehydrogenase were added, the mixture was incubated at 30 ℃ for 2min, and the change in absorbance at 340nm was scanned after adding a suitable amount of pure enzyme. Protein content was determined by the Bradford method using bovine serum albumin BSA as a standard. Enzyme activity is defined as: under the above conditions, the amount of enzyme catalyzing 1. mu. mol of NADH per minute is defined as one unit U.

The calculation formula of the enzyme activity is as follows: enzyme activity (U) ═ EW × V × 103/(6220 × 0.3)

Calculation formula of specific activity: specific activity (U mg-1) ═ enzyme activity (U)/protein amount (mg)

Wherein, EW: change in absorbance at 340nm within 1 min; v: volume of reaction solution (mL); 6220: molar extinction coefficient (L mol)^-1cm^-1) (ii) a 0.3: optical path distance (cm).

TABLE 1 specific enzyme Activity of different mutants

The specific enzyme activity of the obtained mutant is obviously improved compared with that of the wild enzyme by carrying out site-directed mutagenesis on the wild enzyme.

Example 18: catalyzing 4-methylsulfonyl-benzaldehyde and glycine to generate L-syn-beta-4-methylsulfonyl phenyl serine

Reaction system 2ml pH 8.0: KH (Perkin Elmer)₂PO₄(50mM) buffer, glycine (1M), 4-methylsulfonylbenzaldehyde (100mM), PLP (240. mu.M), 2mg of enzyme (S182Y). The reaction was stopped at 30 ℃ for 1h at 200rpm by adjusting the pH to 2 with 1M HCl.

Example 19: HPLC detection of L-syn-beta-4-methylsulfonylphenyl serine

The reaction system was derivatized with OPA/NAC and detected with Agilent 1260.

Liquid phase conditions: purospher STAR RP18(250mM, 5um) C18 column buffer pH8.0 (50mM KH)₂PO₄)/CH₃CN 81:19, flow rate 0.8ml/min, run time 20min, temperature 40 ℃. The reaction in example 18 was examined to determine the HPLC analytical yield of 82%, ee value>99% and de 70%, as shown in FIG. 1.

Example 20:¹H-NMR detection of L-syn-beta-4-methylsulfonylphenyl serine

The reaction solution after termination is centrifuged at 5000rmp, the supernatant is filtered through a 0.2 μm filter membrane, the filtrate is dried at a set temperature of 55 ℃ and a pressure of 40mbar on a rotary evaporator, and the collected solid is sent to a nuclear magnetic resonance spectrometer for detection. The deuterated solvent is deuterium oxide, and the detection instrument is a Bruker AVANCE III 400MHz nuclear magnetic resonance spectrometer. The nuclear magnetism verifies that the product structure is correct, and the diastereoisomer of the product accords with the detection result of HPLC, as shown in figure 2.

Example 21: catalyzing 3-bromobenzaldehyde and glycine to generate L-syn-beta-3-bromophenylserine

Reaction system 2ml pH 8.0: KH (Perkin Elmer)₂PO₄(50mM) buffer, glycine (1M), 3-bromobenzaldehyde (100mM), PLP (240. mu.M), 2mg of enzyme (S182Y)). The reaction was stopped at 30 ℃ for 1h at 200rpm by adjusting the pH to 2 with 1M HCl. HPLC analysis yield 81%, ee value>99% and de value 90%.

Example 22: catalyzing 4-bromobenzaldehyde and glycine to generate L-syn-beta-4-bromophenylserine

Reaction system 2ml pH 8.0: KH (Perkin Elmer)₂PO₄(50mM) buffer, glycine (1M), 4-bromobenzaldehyde (100mM), PLP (240. mu.M), 2mg of enzyme (S182Y). The reaction was stopped at 30 ℃ for 1h at 200rpm by adjusting the pH to 2 with 1M HCl. HPLC analysis yield 79%, ee value>99% and de value 70%.

Example 23: catalyzing 2-nitrobenzaldehyde and glycine to generate L-syn-beta-2-nitrophenylserine

Reaction system 2ml pH 8.0: KH (Perkin Elmer)₂PO₄(50mM) buffer, glycine (1M), 2-nitrobenzaldehyde (100mM), PLP (240. mu.M), 2mg of enzyme (A178Y). The reaction was stopped at 30 ℃ for 1h at 200rpm by adjusting the pH to 2 with 1M HCl. HPLC analysis yield 99%, ee value>99% and de 85%.

Example 24: catalyzing 3-nitrobenzaldehyde and glycine to generate L-syn-beta-3-nitrophenylserine

Reaction system 2ml pH 8.0: KH (Perkin Elmer)₂PO₄(50mM) buffer, glycine (1M), 3-nitrobenzaldehyde (100mM), PLP (240. mu.M), 2mg of enzyme (A178Y). The reaction was stopped at 30 ℃ for 1h at 200rpm by adjusting the pH to 2 with 1M HCl. HPLC analysis yield 83%, ee value>99% and de 77%.

Example 25: catalyzing 4-nitrobenzaldehyde and glycine to generate L-syn-beta-4-nitrophenylserine

Reaction system 2ml pH 8.0: KH (Perkin Elmer)₂PO₄(50mM) buffer, glycine (1M), 4-nitrobenzaldehyde (100mM), PLP (240. mu.M), 2mg of enzyme (A178Y). The reaction was stopped at 30 ℃ for 1h at 200rpm by adjusting the pH to 2 with 1M HCl. HPLC analysis yield 89%, ee value>99% and de value 90%.

Example 26: catalyzing 4-methylsulfonylbenzaldehyde and glycine to generate L-syn-beta-4-methylsulfonylphenylserine

Reaction system 2ml pH 8.0: KH (Perkin Elmer)₂PO₄(50mM) buffer, glycine (1M), 4-methylsulfonylbenzaldehyde (100mM), PLP (240. mu.M), 2mg of enzyme (A178Y). The reaction was stopped at 30 ℃ for 1h at 200rpm by adjusting the pH to 2 with 1M HCl. HPLC analysis yield 91%, ee value>99% and de 92%.

Example 27: catalyzing 3-hydroxybenzaldehyde and glycine to generate L-syn-beta-3-hydroxyphenyl serine

Reaction system 2ml pH 8.0: KH (Perkin Elmer)₂PO₄(50mM) buffer, glycine (1M), 3-hydroxybenzaldehyde (100mM), PLP (240. mu.M), 2mg enzyme (H91S). The reaction was stopped at 30 ℃ for 1h at 200rpm by adjusting the pH to 2 with 1M HCl. HPLC analysis yield 76%, ee value>99% and de 71%.

Sequence listing

<110> Wang Ji Ming

HANGZHOU FAZHELUO BIOTECHNOLOGY Co.,Ltd.

<120> threonine aldolase, mutant and application thereof in preparation of substituted phenylserine derivative

<130> 1

<160> 88

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1074

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<213> Pseudomonas threonine aldolase Gene (Artificial sequence)

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atgaatggtg aaaccagccg tccgccggca ctgggtttta gcagcgataa tattgcaggt 60

gcctctccgg aagtggcaca ggcactggtt aaacattctt caggtcaggc aggtccgtat 120

ggtaccgatg aactgaccgc acaggttaaa cgtaaatttt gtgaaatttt tgaacgtgat 180

gttgaagtgt ttctggttcc gaccggcacc gccgcaaatg cactgtgtct gagcgcaatg 240

accccgccgt ggggtaatat ttattgtcat ccggcaagcc atattaataa tgatgaatgt 300

ggtgcaccgg aattcttttc taatggtgca aaactgatga ccgttgatgg tccggccgca 360

aaactggata ttgttcgtct gcgcgaacgc acccgtgaaa aagttggtga tgttcatacc 420

acacagccgg catgtgttag tattacccag gcgaccgaag ttggtagcat ttataccctg 480

gatgaaattg aagcgattgg tgatgtttgt aaaagcagta gcctgggtct gcatatggat 540

ggtagccgtt ttgcgaatgc actggtgagc ctgggttgca gtccggcaga aatgacctgg 600

aaagcgggcg ttgatgcgct gtcttttggc gcaaccaaaa atggtgttct ggcagcagaa 660

gcaattgttc tgtttaatac cagcctggca accgaaatga gctatcgtcg taaacgcgca 720

ggtcatctga gcagtaaaat gcgttttctg agtgcacaga ttgatgccta tctgaccgat 780

gatctgtggc tgcgtaatgc gcgtaaagcc aatgccgcag cgcagcgtct ggcgcagggt 840

ctggaaggtc tgggtggtgt tgaagtgctg ggtggtaccg aagcaaatat tctgttttgt 900

cgcctggata gcgccatgat tgatgccctg ctgaaagccg gttttggttt ttatcatgat 960

cgttggggtc cgaatgtggt tcgttttgtt accagctttg ccaccaccgc agaagatgtg 1020

gatcatctgc tgaatcaggt tcgtctggca gcagatcgca cccaggaacg ttaa 1074

<210> 2

<211> 357

<212> PRT

<213> Pseudomonas threonine Aldolase amino acids (Artificial sequence)

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Met Asn Gly Glu Thr Ser Arg Pro Pro Ala Leu Gly Phe Ser Ser Asp

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Asn Ile Ala Gly Ala Ser Pro Glu Val Ala Gln Ala Leu Val Lys His

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Ser Ser Gly Gln Ala Gly Pro Tyr Gly Thr Asp Glu Leu Thr Ala Gln

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Val Lys Arg Lys Phe Cys Glu Ile Phe Glu Arg Asp Val Glu Val Phe

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Leu Val Pro Thr Gly Thr Ala Ala Asn Ala Leu Cys Leu Ser Ala Met

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Thr Pro Pro Trp Gly Asn Ile Tyr Cys His Pro Ala Ser His Ile Asn

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Asn Asp Glu Cys Gly Ala Pro Glu Phe Phe Ser Asn Gly Ala Lys Leu

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Met Thr Val Asp Gly Pro Ala Ala Lys Leu Asp Ile Val Arg Leu Arg

115 120 125

Glu Arg Thr Arg Glu Lys Val Gly Asp Val His Thr Thr Gln Pro Ala

130 135 140

Cys Val Ser Ile Thr Gln Ala Thr Glu Val Gly Ser Ile Tyr Thr Leu

145 150 155 160

Asp Glu Ile Glu Ala Ile Gly Asp Val Cys Lys Ser Ser Ser Leu Gly

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Leu His Met Asp Gly Ser Arg Phe Ala Asn Ala Leu Val Ser Leu Gly

180 185 190

Cys Ser Pro Ala Glu Met Thr Trp Lys Ala Gly Val Asp Ala Leu Ser

195 200 205

Phe Gly Ala Thr Lys Asn Gly Val Leu Ala Ala Glu Ala Ile Val Leu

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Phe Asn Thr Ser Leu Ala Thr Glu Met Ser Tyr Arg Arg Lys Arg Ala

225 230 235 240

Gly His Leu Ser Ser Lys Met Arg Phe Leu Ser Ala Gln Ile Asp Ala

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Tyr Leu Thr Asp Asp Leu Trp Leu Arg Asn Ala Arg Lys Ala Asn Ala

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Ala Ala Gln Arg Leu Ala Gln Gly Leu Glu Gly Leu Gly Gly Val Glu

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Val Leu Gly Gly Thr Glu Ala Asn Ile Leu Phe Cys Arg Leu Asp Ser

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Ala Met Ile Asp Ala Leu Leu Lys Ala Gly Phe Gly Phe Tyr His Asp

305 310 315 320

Arg Trp Gly Pro Asn Val Val Arg Phe Val Thr Ser Phe Ala Thr Thr

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Ala Glu Asp Val Asp His Leu Leu Asn Gln Val Arg Leu Ala Ala Asp

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Arg Thr Gln Glu Arg

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<210> 3

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<213> Pseudomonas threonine aldolase gene mutation (Artificial sequence)

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atgaatggtg aaaccagccg tccgccggca ctgggtttta gcagcgataa tattgcaggt 60

gcctctccgg aagtggcaca ggcactggtt aaacattctt caggtcaggc aggtccgtat 120

ggtaccgatg aactgaccgc acaggttaaa cgtaaatttt gtgaaatttt tgaacgtgat 180

gttgaagtgt ttctggttcc gaccggcacc gccgcaaatg cactgtgtct gagcgcaatg 240

accccgccgt ggggtaatat ttattgtcat ccggcaagcc atattaataa tgatgaatgt 300

ggtgcaccgg aattcttttc taatggtgca aaactgatga ccgttgatgg tccggccgca 360

aaactggata ttgttcgtct gcgcgaacgc acccgtgaaa aagttggtga tgttcatacc 420

acacagccgg catgtgttag tattacccag gcgaccgaag ttggtagcat ttataccctg 480

gatgaaattg aagcgattgg tgatgtttgt aaaagcagta gcctgggtct gcatatggat 540

ggttaccgtt ttgcgaatgc actggtgagc ctgggttgca gtccggcaga aatgacctgg 600

aaagcgggcg ttgatgcgct gtcttttggc gcaaccaaaa atggtgttct ggcagcagaa 660

gcaattgttc tgtttaatac cagcctggca accgaaatga gctatcgtcg taaacgcgca 720

ggtcatctga gcagtaaaat gcgttttctg agtgcacaga ttgatgccta tctgaccgat 780

gatctgtggc tgcgtaatgc gcgtaaagcc aatgccgcag cgcagcgtct ggcgcagggt 840

ctggaaggtc tgggtggtgt tgaagtgctg ggtggtaccg aagcaaatat tctgttttgt 900

cgcctggata gcgccatgat tgatgccctg ctgaaagccg gttttggttt ttatcatgat 960

cgttggggtc cgaatgtggt tcgttttgtt accagctttg ccaccaccgc agaagatgtg 1020

gatcatctgc tgaatcaggt tcgtctggca gcagatcgca cccaggaacg ttaa 1074

<210> 4

<211> 357

<212> PRT

<213> Pseudomonas threonine aldolase amino acid mutation (Artificial sequence)

<400> 4

Met Asn Gly Glu Thr Ser Arg Pro Pro Ala Leu Gly Phe Ser Ser Asp

1 5 10 15

Asn Ile Ala Gly Ala Ser Pro Glu Val Ala Gln Ala Leu Val Lys His

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Ser Ser Gly Gln Ala Gly Pro Tyr Gly Thr Asp Glu Leu Thr Ala Gln

35 40 45

Val Lys Arg Lys Phe Cys Glu Ile Phe Glu Arg Asp Val Glu Val Phe

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Leu Val Pro Thr Gly Thr Ala Ala Asn Ala Leu Cys Leu Ser Ala Met

65 70 75 80

Thr Pro Pro Trp Gly Asn Ile Tyr Cys His Pro Ala Ser His Ile Asn

85 90 95

Asn Asp Glu Cys Gly Ala Pro Glu Phe Phe Ser Asn Gly Ala Lys Leu

100 105 110

Met Thr Val Asp Gly Pro Ala Ala Lys Leu Asp Ile Val Arg Leu Arg

115 120 125

Glu Arg Thr Arg Glu Lys Val Gly Asp Val His Thr Thr Gln Pro Ala

130 135 140

Cys Val Ser Ile Thr Gln Ala Thr Glu Val Gly Ser Ile Tyr Thr Leu

145 150 155 160

Asp Glu Ile Glu Ala Ile Gly Asp Val Cys Lys Ser Ser Ser Leu Gly

165 170 175

Leu His Met Asp Gly Tyr Arg Phe Ala Asn Ala Leu Val Ser Leu Gly

180 185 190

Cys Ser Pro Ala Glu Met Thr Trp Lys Ala Gly Val Asp Ala Leu Ser

195 200 205

Phe Gly Ala Thr Lys Asn Gly Val Leu Ala Ala Glu Ala Ile Val Leu

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Phe Asn Thr Ser Leu Ala Thr Glu Met Ser Tyr Arg Arg Lys Arg Ala

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Gly His Leu Ser Ser Lys Met Arg Phe Leu Ser Ala Gln Ile Asp Ala

245 250 255

Tyr Leu Thr Asp Asp Leu Trp Leu Arg Asn Ala Arg Lys Ala Asn Ala

260 265 270

Ala Ala Gln Arg Leu Ala Gln Gly Leu Glu Gly Leu Gly Gly Val Glu

275 280 285

Val Leu Gly Gly Thr Glu Ala Asn Ile Leu Phe Cys Arg Leu Asp Ser

290 295 300

Ala Met Ile Asp Ala Leu Leu Lys Ala Gly Phe Gly Phe Tyr His Asp

305 310 315 320

Arg Trp Gly Pro Asn Val Val Arg Phe Val Thr Ser Phe Ala Thr Thr

325 330 335

Ala Glu Asp Val Asp His Leu Leu Asn Gln Val Arg Leu Ala Ala Asp

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Arg Thr Gln Glu Arg

355

<210> 5

<211> 1074

<212> DNA

<213> Pseudomonas threonine aldolase gene mutation (Artificial sequence)

<400> 5

atgaatggtg aaaccagccg tccgccggca ctgggtttta gcagcgataa tattgcaggt 60

gcctctccgg aagtggcaca ggcactggtt aaacattctt caggtcaggc aggtccgtat 120

ggtaccgatg aactgaccgc acaggttaaa cgtaaatttt gtgaaatttt tgaacgtgat 180

gttgaagtgt ttctggttcc gaccggcacc gccgcaaatg cactgtgtct gagcgcaatg 240

accccgccgt ggggtaatat ttattgtcat ccggcaagcc ctattaataa tgatgaatgt 300

ggtgcaccgg aattcttttc taatggtgca aaactgatga ccgttgatgg tccggccgca 360

aaactggata ttgttcgtct gcgcgaacgc acccgtgaaa aagttggtga tgttcatacc 420

acacagccgg catgtgttag tattacccag gcgaccgaag ttggtagcat ttataccctg 480

gatgaaattg aagcgattgg tgatgtttgt aaaagcagta gcctgggtct gcatatggat 540

ggtagccgtt ttgcgaatgc actggtgagc ctgggttgca gtccggcaga aatgacctgg 600

aaagcgggcg ttgatgcgct gtcttttggc gcaaccaaaa atggtgttct ggcagcagaa 660

gcaattgttc tgtttaatac cagcctggca accgaaatga gctatcgtcg taaacgcgca 720

ggtcatctga gcagtaaaat gcgttttctg agtgcacaga ttgatgccta tctgaccgat 780

gatctgtggc tgcgtaatgc gcgtaaagcc aatgccgcag cgcagcgtct ggcgcagggt 840

ctggaaggtc tgggtggtgt tgaagtgctg ggtggtaccg aagcaaatat tctgttttgt 900

cgcctggata gcgccatgat tgatgccctg ctgaaagccg gttttggttt ttatcatgat 960

cgttggggtc cgaatgtggt tcgttttgtt accagctttg ccaccaccgc agaagatgtg 1020

gatcatctgc tgaatcaggt tcgtctggca gcagatcgca cccaggaacg ttaa 1074

<210> 6

<211> 357

<212> PRT

<213> Pseudomonas threonine aldolase amino acid mutation (Artificial sequence)

<400> 6

Met Asn Gly Glu Thr Ser Arg Pro Pro Ala Leu Gly Phe Ser Ser Asp

1 5 10 15

Asn Ile Ala Gly Ala Ser Pro Glu Val Ala Gln Ala Leu Val Lys His

20 25 30

Ser Ser Gly Gln Ala Gly Pro Tyr Gly Thr Asp Glu Leu Thr Ala Gln

35 40 45

Val Lys Arg Lys Phe Cys Glu Ile Phe Glu Arg Asp Val Glu Val Phe

50 55 60

Leu Val Pro Thr Gly Thr Ala Ala Asn Ala Leu Cys Leu Ser Ala Met

65 70 75 80

Thr Pro Pro Trp Gly Asn Ile Tyr Cys His Pro Ala Ser Pro Ile Asn

85 90 95

Asn Asp Glu Cys Gly Ala Pro Glu Phe Phe Ser Asn Gly Ala Lys Leu

100 105 110

Met Thr Val Asp Gly Pro Ala Ala Lys Leu Asp Ile Val Arg Leu Arg

115 120 125

Glu Arg Thr Arg Glu Lys Val Gly Asp Val His Thr Thr Gln Pro Ala

130 135 140

Cys Val Ser Ile Thr Gln Ala Thr Glu Val Gly Ser Ile Tyr Thr Leu

145 150 155 160

Asp Glu Ile Glu Ala Ile Gly Asp Val Cys Lys Ser Ser Ser Leu Gly

165 170 175

Leu His Met Asp Gly Ser Arg Phe Ala Asn Ala Leu Val Ser Leu Gly

180 185 190

Cys Ser Pro Ala Glu Met Thr Trp Lys Ala Gly Val Asp Ala Leu Ser

195 200 205

Phe Gly Ala Thr Lys Asn Gly Val Leu Ala Ala Glu Ala Ile Val Leu

210 215 220

Phe Asn Thr Ser Leu Ala Thr Glu Met Ser Tyr Arg Arg Lys Arg Ala

225 230 235 240

Gly His Leu Ser Ser Lys Met Arg Phe Leu Ser Ala Gln Ile Asp Ala

245 250 255

Tyr Leu Thr Asp Asp Leu Trp Leu Arg Asn Ala Arg Lys Ala Asn Ala

260 265 270

Ala Ala Gln Arg Leu Ala Gln Gly Leu Glu Gly Leu Gly Gly Val Glu

275 280 285

Val Leu Gly Gly Thr Glu Ala Asn Ile Leu Phe Cys Arg Leu Asp Ser

290 295 300

Ala Met Ile Asp Ala Leu Leu Lys Ala Gly Phe Gly Phe Tyr His Asp

305 310 315 320

Arg Trp Gly Pro Asn Val Val Arg Phe Val Thr Ser Phe Ala Thr Thr

325 330 335

Ala Glu Asp Val Asp His Leu Leu Asn Gln Val Arg Leu Ala Ala Asp

340 345 350

Arg Thr Gln Glu Arg

355

<210> 7

<211> 1074

<212> DNA

<213> Pseudomonas threonine aldolase gene mutation (Artificial sequence)

<400> 7

atgaatggtg aaaccagccg tccgccggca ctgggtttta gcagcgatca gattgcaggt 60

gcctctccgg aagtggcaca ggcactggtt aaacattctt caggtcaggc aggtccgtat 120

ggtaccgatg aactgaccgc acaggttaaa cgtaaatttt gtgaaatttt tgaacgtgat 180

gttgaagtgt ttctggttcc gaccggcacc gccgcaaatg cactgtgtct gagcgcaatg 240

accccgccgt ggggtaatat ttattgtcat ccggcaagcc atattaataa tgatgaatgt 300

ggtgcaccgg aattcttttc taatggtgca aaactgatga ccgttgatgg tccggccgca 360

aaactggata ttgttcgtct gcgcgaacgc acccgtgaaa aagttggtga tgttcatacc 420

acacagccgg catgtgttag tattacccag gcgaccgaag ttggtagcat ttataccctg 480

gatgaaattg aagcgattgg tgatgtttgt aaaagcagta gcctgggtct gcatatggat 540

ggtagccgtt ttgcgaatgc actggtgagc ctgggttgca gtccggcaga aatgacctgg 600

aaagcgggcg ttgatgcgct gtcttttggc gcaaccaaaa atggtgttct ggcagcagaa 660

gcaattgttc tgtttaatac cagcctggca accgaaatga gctatcgtcg taaacgcgca 720

ggtcatctga gcagtaaaat gcgttttctg agtgcacaga ttgatgccta tctgaccgat 780

gatctgtggc tgcgtaatgc gcgtaaagcc aatgccgcag cgcagcgtct ggcgcagggt 840

ctggaaggtc tgggtggtgt tgaagtgctg ggtggtaccg aagcaaatat tctgttttgt 900

cgcctggata gcgccatgat tgatgccctg ctgaaagccg gttttggttt ttatcatgat 960

cgttggggtc cgaatgtggt tcgttttgtt accagctttg ccaccaccgc agaagatgtg 1020

gatcatctgc tgaatcaggt tcgtctggca gcagatcgca cccaggaacg ttaa 1074

<210> 8

<211> 357

<212> PRT

<213> Pseudomonas threonine aldolase amino acid mutation (Artificial sequence)

<400> 8

Met Asn Gly Glu Thr Ser Arg Pro Pro Ala Leu Gly Phe Ser Ser Asp

1 5 10 15

Gln Ile Ala Gly Ala Ser Pro Glu Val Ala Gln Ala Leu Val Lys His

20 25 30

Ser Ser Gly Gln Ala Gly Pro Tyr Gly Thr Asp Glu Leu Thr Ala Gln

35 40 45

Val Lys Arg Lys Phe Cys Glu Ile Phe Glu Arg Asp Val Glu Val Phe

50 55 60

Leu Val Pro Thr Gly Thr Ala Ala Asn Ala Leu Cys Leu Ser Ala Met

65 70 75 80

Thr Pro Pro Trp Gly Asn Ile Tyr Cys His Pro Ala Ser His Ile Asn

85 90 95

Asn Asp Glu Cys Gly Ala Pro Glu Phe Phe Ser Asn Gly Ala Lys Leu

100 105 110

Met Thr Val Asp Gly Pro Ala Ala Lys Leu Asp Ile Val Arg Leu Arg

115 120 125

Glu Arg Thr Arg Glu Lys Val Gly Asp Val His Thr Thr Gln Pro Ala

130 135 140

Cys Val Ser Ile Thr Gln Ala Thr Glu Val Gly Ser Ile Tyr Thr Leu

145 150 155 160

Asp Glu Ile Glu Ala Ile Gly Asp Val Cys Lys Ser Ser Ser Leu Gly

165 170 175

Leu His Met Asp Gly Ser Arg Phe Ala Asn Ala Leu Val Ser Leu Gly

180 185 190

Cys Ser Pro Ala Glu Met Thr Trp Lys Ala Gly Val Asp Ala Leu Ser

195 200 205

Phe Gly Ala Thr Lys Asn Gly Val Leu Ala Ala Glu Ala Ile Val Leu

210 215 220

Phe Asn Thr Ser Leu Ala Thr Glu Met Ser Tyr Arg Arg Lys Arg Ala

225 230 235 240

Gly His Leu Ser Ser Lys Met Arg Phe Leu Ser Ala Gln Ile Asp Ala

245 250 255

Tyr Leu Thr Asp Asp Leu Trp Leu Arg Asn Ala Arg Lys Ala Asn Ala

260 265 270

Ala Ala Gln Arg Leu Ala Gln Gly Leu Glu Gly Leu Gly Gly Val Glu

275 280 285

Val Leu Gly Gly Thr Glu Ala Asn Ile Leu Phe Cys Arg Leu Asp Ser

290 295 300

Ala Met Ile Asp Ala Leu Leu Lys Ala Gly Phe Gly Phe Tyr His Asp

305 310 315 320

Arg Trp Gly Pro Asn Val Val Arg Phe Val Thr Ser Phe Ala Thr Thr

325 330 335

Ala Glu Asp Val Asp His Leu Leu Asn Gln Val Arg Leu Ala Ala Asp

340 345 350

Arg Thr Gln Glu Arg

355

<210> 9

<211> 1074

<212> DNA

<213> Pseudomonas threonine aldolase gene mutation (Artificial sequence)

<400> 9

atgaatggtg aaaccagccg tccgccggca ctgggtttta gcagcgataa tattgcaggt 60

gcctctccgg aagtggcaca ggcactggtt aaacattctt caggtcaggc aggtccgtat 120

ggtaccgatg aactgaccgc acaggttaaa cgtaaatttt gtgaaatttt tgaacgtgat 180

gttgaagtgt ttctggttcc gaccggcacc gccgcaaatg cactgtgtct gagcgcaatg 240

accccgccgt ggggtaatat ttattgtcat ccggcaagcc atattaataa tgatgaatgt 300

ggtgcaccgg aattcttttc taatggtgca aaactgatga ccgttgatgg tccggccgca 360

aaactggata ttgttcgtct gcgcgaacgc acccgtgaaa aagttggtga tgttcatacc 420

acacagccgg catgtgttag tattacccag gcgaccgaag ttggtagcat ttataccctg 480

gatgaaattg aagcgattgg tgatgtttgt aaaagcagta gcctgggtct gcatatggat 540

ggtagccgtt ttgcgaatgc actggtgagc ctgggttgca gtccggcaga aatgacctgg 600

aaagcgggcg ttgatgcgct gtcttttggc gcaatcaaaa atggtgttct ggcagcagaa 660

gcaattgttc tgtttaatac cagcctggca accgaaatga gctatcgtcg taaacgcgca 720

ggtcatctga gcagtaaaat gcgttttctg agtgcacaga ttgatgccta tctgaccgat 780

gatctgtggc tgcgtaatgc gcgtaaagcc aatgccgcag cgcagcgtct ggcgcagggt 840

ctggaaggtc tgggtggtgt tgaagtgctg ggtggtaccg aagcaaatat tctgttttgt 900

cgcctggata gcgccatgat tgatgccctg ctgaaagccg gttttggttt ttatcatgat 960

cgttggggtc cgaatgtggt tcgttttgtt accagctttg ccaccaccgc agaagatgtg 1020

gatcatctgc tgaatcaggt tcgtctggca gcagatcgca cccaggaacg ttaa 1074

<210> 10

<211> 357

<212> PRT

<213> Pseudomonas threonine aldolase amino acid mutation (Artificial sequence)

<400> 10

Met Asn Gly Glu Thr Ser Arg Pro Pro Ala Leu Gly Phe Ser Ser Asp

1 5 10 15

Asn Ile Ala Gly Ala Ser Pro Glu Val Ala Gln Ala Leu Val Lys His

20 25 30

Ser Ser Gly Gln Ala Gly Pro Tyr Gly Thr Asp Glu Leu Thr Ala Gln

35 40 45

Val Lys Arg Lys Phe Cys Glu Ile Phe Glu Arg Asp Val Glu Val Phe

50 55 60

Leu Val Pro Thr Gly Thr Ala Ala Asn Ala Leu Cys Leu Ser Ala Met

65 70 75 80

Thr Pro Pro Trp Gly Asn Ile Tyr Cys His Pro Ala Ser His Ile Asn

85 90 95

Asn Asp Glu Cys Gly Ala Pro Glu Phe Phe Ser Asn Gly Ala Lys Leu

100 105 110

Met Thr Val Asp Gly Pro Ala Ala Lys Leu Asp Ile Val Arg Leu Arg

115 120 125

Glu Arg Thr Arg Glu Lys Val Gly Asp Val His Thr Thr Gln Pro Ala

130 135 140

Cys Val Ser Ile Thr Gln Ala Thr Glu Val Gly Ser Ile Tyr Thr Leu

145 150 155 160

Asp Glu Ile Glu Ala Ile Gly Asp Val Cys Lys Ser Ser Ser Leu Gly

165 170 175

Leu His Met Asp Gly Ser Arg Phe Ala Asn Ala Leu Val Ser Leu Gly

180 185 190

Cys Ser Pro Ala Glu Met Thr Trp Lys Ala Gly Val Asp Ala Leu Ser

195 200 205

Phe Gly Ala Ile Lys Asn Gly Val Leu Ala Ala Glu Ala Ile Val Leu

210 215 220

Phe Asn Thr Ser Leu Ala Thr Glu Met Ser Tyr Arg Arg Lys Arg Ala

225 230 235 240

Gly His Leu Ser Ser Lys Met Arg Phe Leu Ser Ala Gln Ile Asp Ala

245 250 255

Tyr Leu Thr Asp Asp Leu Trp Leu Arg Asn Ala Arg Lys Ala Asn Ala

260 265 270

Ala Ala Gln Arg Leu Ala Gln Gly Leu Glu Gly Leu Gly Gly Val Glu

275 280 285

Val Leu Gly Gly Thr Glu Ala Asn Ile Leu Phe Cys Arg Leu Asp Ser

290 295 300

Ala Met Ile Asp Ala Leu Leu Lys Ala Gly Phe Gly Phe Tyr His Asp

305 310 315 320

Arg Trp Gly Pro Asn Val Val Arg Phe Val Thr Ser Phe Ala Thr Thr

325 330 335

Ala Glu Asp Val Asp His Leu Leu Asn Gln Val Arg Leu Ala Ala Asp

340 345 350

Arg Thr Gln Glu Arg

355

<210> 11

<211> 25

<212> DNA

<213> primer (Artificial sequence)

<400> 11

atggatggtt accgttttgc gaatg 25

<210> 12

<211> 26

<212> DNA

<213> primer (Artificial sequence)

<400> 12

gcattcgcaa aacggtaacc atccat 26

<210> 13

<211> 27

<212> DNA

<213> primer (Artificial sequence)

<400> 13

atttattgtc atccggcaag ccctatt 27

<210> 14

<211> 29

<212> DNA

<213> primer (Artificial sequence)

<400> 14

gcaccacatt catcattatt aatagggct 29

<210> 15

<211> 24

<212> DNA

<213> primer (Artificial sequence)

<400> 15

agcagcgatc agattgcagg tgcc 24

<210> 16

<211> 23

<212> DNA

<213> primer (Artificial sequence)

<400> 16

ggcacctgca atctgatcgc tgc 23

<210> 17

<211> 29

<212> DNA

<213> primer (Artificial sequence)

<400> 17

tttggcgcaa tcaaaaatgg tgttctggc 29

<210> 18

<211> 27

<212> DNA

<213> primer (Artificial sequence)

<400> 18

acaccatttt tgattgcgcc aaaagac 27

<210> 19

<211> 343

<212> PRT

<213> threonine aldolase amino acid (artificial sequence) of Caulobacter crescentus

<400> 19

Met Thr Gln Thr Ala Pro Arg Tyr Asp Phe Ala Ser Asp Asn Val Ala

1 5 10 15

Gly Ala Met Pro Glu Val Met Glu Ala Leu Ile Ala Ala Asn Ala Gly

20 25 30

Thr Ala Ser Gly Tyr Gly Thr Asp His Val Ser Arg Ala Ala Ala Asp

35 40 45

Arg Ile Arg Ala Ala Leu Asp Ala Asp Ala Gln Val Arg Phe Thr Ala

50 55 60

Ser Gly Thr Ala Ala Asn Ala Phe Ala Leu Thr Leu Leu Ala Gln Pro

65 70 75 80

His Glu Ala Val Leu Ala His Glu His Ala His Ile Cys Thr Asp Glu

85 90 95

Thr Gly Ala Pro Gly Phe Phe Gly Gln Gly Val Gly Leu Ile Gly Leu

100 105 110

Pro Gly Ala Ser Gly Lys Met Glu Leu Ala Ala Leu Glu Ala Ala Leu

115 120 125

Ala Gln Pro Asp Val Ser Tyr Arg Gln Pro Ala Ala Ala Leu Ser Leu

130 135 140

Thr Thr Ala Thr Glu Tyr Gly Thr Val Tyr Ser Glu Asp His Leu Arg

145 150 155 160

Ala Leu Ile Ala Pro Val Lys Ala Lys Gly Tyr Gly Val His Leu Asp

165 170 175

Gly Ala Arg Leu Ala Asn Ala Val Ala Gly Gly Phe Asp Leu Lys Ser

180 185 190

Ile Ala Lys Met Gly Val Asp Ile Leu Val Met Gly Gly Thr Lys Ala

195 200 205

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

210 215 220

Lys Arg Leu Asp Ala Arg Leu Lys His Ala Gly Gln Leu Ile Ser Lys

225 230 235 240

Gly Arg Phe Leu Ala Ala Pro Trp Leu Gly Leu Leu Gly Glu Asn Gly

245 250 255

Gln Thr Ala Pro Trp Ala Ala Arg Ala Ala His Ala Asn Ala Met Ala

260 265 270

Gln Lys Leu Ala Ala Leu Met Pro Val Pro Ile Lys His Pro Val Glu

275 280 285

Ala Asn Gly Ile Phe Val Glu Met Asp Glu Leu Ala Leu Glu Arg Leu

290 295 300

Arg Gly Glu Gly Trp Phe Val Tyr Arg Phe Leu Asp Gly Thr Val Arg

305 310 315 320

Phe Met Cys Ser Trp Ala Thr Thr Pro Glu Met Val Glu Asp Leu Gly

325 330 335

Ala Ala Leu Lys Arg Val Ala

340

<210> 20

<211> 343

<212> PRT

<213> threonine aldolase amino acid mutation of Caulobacter crescentus (artificial sequence)

<400> 20

Met Thr Gln Thr Ala Pro Arg Tyr Asp Phe Ala Ser Asp Asn Val Ala

1 5 10 15

Gly Ala Met Pro Glu Val Met Glu Ala Leu Ile Ala Ala Asn Ala Gly

20 25 30

Thr Ala Ser Gly Tyr Gly Thr Asp His Val Ser Arg Ala Ala Ala Asp

35 40 45

Arg Ile Arg Ala Ala Leu Asp Ala Asp Ala Gln Val Arg Phe Thr Ala

50 55 60

Ser Gly Thr Ala Ala Asn Ala Phe Ala Leu Thr Leu Leu Ala Gln Pro

65 70 75 80

His Glu Ala Val Leu Ala His Glu His Ala His Ile Cys Thr Tyr Thr

85 90 95

Thr Gly Ala Pro Gly Phe Phe Gly Gln Gly Val Gly Leu Ile Gly Leu

100 105 110

Pro Gly Ala Ser Gly Lys Met Glu Leu Ala Ala Leu Glu Ala Ala Leu

115 120 125

Ala Gln Pro Asp Val Ser Tyr Arg Gln Pro Ala Ala Ala Leu Ser Leu

130 135 140

Thr Thr Ala Thr Glu Tyr Gly Thr Val Tyr Ser Glu Asp His Leu Arg

145 150 155 160

Ala Leu Ile Ala Pro Val Lys Ala Lys Gly Tyr Gly Val His Leu Asp

165 170 175

Gly Ala Arg Leu Ala Asn Ala Val Ala Gly Gly Phe Asp Leu Lys Ser

180 185 190

Ile Ala Lys Met Gly Val Asp Ile Leu Val Met Gly Gly Thr Lys Ala

195 200 205

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

210 215 220

Lys Arg Leu Asp Ala Arg Leu Lys His Ala Gly Gln Leu Ile Ser Lys

225 230 235 240

Gly Arg Phe Leu Ala Ala Pro Trp Leu Gly Leu Leu Gly Glu Asn Gly

245 250 255

Gln Thr Ala Pro Trp Ala Ala Arg Ala Ala His Ala Asn Ala Met Ala

260 265 270

Gln Lys Leu Ala Ala Leu Met Pro Val Pro Ile Lys His Pro Val Glu

275 280 285

Ala Asn Gly Ile Phe Val Glu Met Asp Glu Leu Ala Leu Glu Arg Leu

290 295 300

Arg Gly Glu Gly Trp Phe Val Tyr Arg Phe Leu Asp Gly Thr Val Arg

305 310 315 320

Phe Met Cys Ser Trp Ala Thr Thr Pro Glu Met Val Glu Asp Leu Gly

325 330 335

Ala Ala Leu Lys Arg Val Ala

340

<210> 21

<211> 343

<212> PRT

<400> 21

Met Thr Gln Thr Ala Pro Arg Tyr Asp Phe Ala Ser Asp Asn Val Ala

1 5 10 15

Gly Ala Met Pro Glu Val Met Glu Ala Leu Ile Ala Ala Asn Ala Gly

20 25 30

Thr Ala Ser Gly Tyr Gly Thr Asp His Val Ser Arg Ala Ala Ala Asp

35 40 45

Arg Ile Arg Ala Ala Leu Asp Ala Asp Ala Gln Val Arg Phe Thr Ala

50 55 60

Ser Gly Thr Ala Ala Asn Ala Phe Ala Leu Thr Leu Leu Ala Gln Pro

65 70 75 80

His Glu Ala Val Leu Ala His Glu His Ala His Ile Cys Thr Leu Glu

85 90 95

Thr Gly Ala Pro Gly Phe Phe Gly Gln Gly Val Gly Leu Ile Gly Leu

100 105 110

Pro Gly Ala Ser Gly Lys Met Glu Leu Ala Ala Leu Glu Ala Ala Leu

115 120 125

Ala Gln Pro Asp Val Ser Tyr Arg Gln Pro Ala Ala Ala Leu Ser Leu

130 135 140

Thr Thr Ala Thr Glu Tyr Gly Thr Val Tyr Ser Glu Asp His Leu Arg

145 150 155 160

Ala Leu Ile Ala Pro Val Lys Ala Lys Gly Tyr Gly Val His Leu Asp

165 170 175

Gly Ala Arg Leu Ala Asn Ala Val Ala Gly Gly Phe Asp Leu Lys Ser

180 185 190

Ile Ala Lys Met Gly Val Asp Ile Leu Val Met Gly Gly Thr Lys Ala

195 200 205

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

210 215 220

Lys Arg Leu Asp Ala Arg Leu Lys His Ala Gly Gln Leu Ile Ser Lys

225 230 235 240

Gly Arg Phe Leu Ala Ala Pro Trp Leu Gly Leu Leu Gly Glu Asn Gly

245 250 255

Gln Thr Ala Pro Trp Ala Ala Arg Ala Ala His Ala Asn Ala Met Ala

260 265 270

Gln Lys Leu Ala Ala Leu Met Pro Val Pro Ile Lys His Pro Val Glu

275 280 285

Ala Asn Gly Ile Phe Val Glu Met Asp Glu Leu Ala Leu Glu Arg Leu

290 295 300

Arg Gly Glu Gly Trp Phe Val Tyr Arg Phe Leu Asp Gly Thr Val Arg

305 310 315 320

Phe Met Cys Ser Trp Ala Thr Thr Pro Glu Met Val Glu Asp Leu Gly

325 330 335

Ala Ala Leu Lys Arg Val Ala

340

<210> 22

<211> 343

<212> PRT

<400> 22

Met Thr Gln Thr Ala Pro Arg Tyr Asp Phe Ala Ser Asp Asn Val Ala

1 5 10 15

Gly Ala Met Pro Glu Val Met Glu Ala Leu Ile Ala Ala Asn Ala Gly

20 25 30

Thr Ala Ser Gly Tyr Gly Thr Asp His Val Ser Arg Ala Ala Ala Asp

35 40 45

Arg Ile Arg Ala Ala Leu Asp Ala Asp Ala Gln Val Arg Phe Thr Ala

50 55 60

Ser Gly Thr Ala Ala Asn Ala Phe Ala Leu Thr Leu Leu Ala Gln Pro

65 70 75 80

His Glu Ala Val Leu Ala His Glu His Ala His Ile Cys Thr Asp Glu

85 90 95

Thr Gly Ala Pro Gly Phe Phe Gly Gln Gly Val Gly Leu Ile Gly Leu

100 105 110

Pro Gly Ala Ser Gly Lys Met Glu Leu Ala Ala Leu Glu Ala Ala Leu

115 120 125

Ala Gln Pro Asp Val Ser Tyr Arg Gln Pro Ala Ala Ala Leu Ser Leu

130 135 140

Thr Thr Ala Thr Glu Tyr Gly Thr Val Tyr Ser Glu Asp His Leu Arg

145 150 155 160

Ala Leu Ile Ala Pro Val Lys Ala Lys Gly Tyr Gly Val His Leu Glu

165 170 175

Gly Ala Arg Leu Ala Asn Ala Val Ala Gly Gly Phe Asp Leu Lys Ser

180 185 190

Ile Ala Lys Met Gly Val Asp Ile Leu Val Met Gly Gly Thr Lys Ala

195 200 205

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

210 215 220

Lys Arg Leu Asp Ala Arg Leu Lys His Ala Gly Gln Leu Ile Ser Lys

225 230 235 240

Gly Arg Phe Leu Ala Ala Pro Trp Leu Gly Leu Leu Gly Glu Asn Gly

245 250 255

Gln Thr Ala Pro Trp Ala Ala Arg Ala Ala His Ala Asn Ala Met Ala

260 265 270

Gln Lys Leu Ala Ala Leu Met Pro Val Pro Ile Lys His Pro Val Glu

275 280 285

Ala Asn Gly Ile Phe Val Glu Met Asp Glu Leu Ala Leu Glu Arg Leu

290 295 300

Arg Gly Glu Gly Trp Phe Val Tyr Arg Phe Leu Asp Gly Thr Val Arg

305 310 315 320

Phe Met Cys Ser Trp Ala Thr Thr Pro Glu Met Val Glu Asp Leu Gly

325 330 335

Ala Ala Leu Lys Arg Val Ala

340

<210> 23

<211> 343

<212> PRT

<213> Thermotoga maritima threonine aldolase amino acid (Artificial sequence)

<400> 23

Met Ile Asp Leu Arg Ser Asp Thr Val Thr Lys Pro Thr Glu Glu Met

1 5 10 15

Arg Lys Ala Met Ala Gln Ala Glu Val Gly Asp Asp Val Tyr Gly Glu

20 25 30

Asp Pro Thr Ile Asn Glu Leu Glu Arg Leu Ala Ala Glu Thr Phe Gly

35 40 45

Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Gly Asn Gln Val

50 55 60

Ser Ile Met Ala His Thr Gln Arg Gly Asp Glu Val Ile Leu Glu Ala

65 70 75 80

Asp Ser His Ile Phe Trp Tyr Glu Val Gly Ala Met Ala Val Leu Ser

85 90 95

Gly Val Met Pro His Pro Val Pro Gly Lys Asn Gly Ala Met Asp Pro

100 105 110

Asp Asp Val Arg Lys Ala Ile Arg Pro Arg Asn Ile His Phe Pro Arg

115 120 125

Thr Ser Leu Ile Ala Ile Glu Asn Thr His Asn Arg Ser Gly Gly Arg

130 135 140

Val Val Pro Leu Glu Asn Ile Lys Glu Ile Cys Thr Ile Ala Lys Glu

145 150 155 160

His Gly Ile Asn Val His Ile Asp Gly Ala Arg Ile Phe Asn Ala Ser

165 170 175

Ile Ala Ser Gly Val Pro Val Lys Glu Tyr Ala Gly Tyr Ala Asp Ser

180 185 190

Val Met Phe Cys Leu Ser Lys Gly Leu Cys Ala Pro Val Gly Ser Val

195 200 205

Val Val Gly Asp Arg Asp Phe Ile Glu Arg Ala Arg Lys Ala Arg Lys

210 215 220

Met Leu Gly Gly Gly Met Arg Gln Ala Gly Val Leu Ala Ala Ala Gly

225 230 235 240

Ile Ile Ala Leu Thr Lys Met Val Asp Arg Leu Lys Glu Asp His Glu

245 250 255

Asn Ala Arg Phe Leu Ala Leu Lys Leu Lys Glu Ile Gly Tyr Ser Val

260 265 270

Asn Pro Glu Asp Val Lys Thr Asn Met Val Ile Leu Arg Thr Asp Asn

275 280 285

Leu Lys Val Asn Ala His Gly Phe Ile Glu Ala Leu Arg Asn Ser Gly

290 295 300

Val Leu Ala Asn Ala Val Ser Asp Thr Glu Ile Arg Leu Val Thr His

305 310 315 320

Lys Asp Val Ser Arg Asn Asp Ile Glu Glu Ala Leu Asn Ile Phe Glu

325 330 335

Lys Leu Phe Arg Lys Phe Ser

340

<210> 24

<211> 358

<212> PRT

<213> Listeria monocytogenes threonine aldolase amino acid (artificial sequence)

<400> 24

Met Thr Asn Thr Leu Lys Thr Ser Tyr Gln Lys Thr Pro Tyr Lys Leu

1 5 10 15

Gly Gly Asn Gly Pro Arg Asn Val Gly Val Leu Thr Glu Ala Leu Gln

20 25 30

Asn Ile Asp Asp Asn Leu Glu Ser Asp Ile Tyr Gly Asn Gly Ala Val

35 40 45

Ile Glu Asp Phe Glu Thr Lys Ile Ala Lys Ile Leu Gly Lys Gln Ser

50 55 60

Ala Val Phe Phe Pro Ser Gly Thr Met Ala Gln Gln Ile Ala Leu Arg

65 70 75 80

Ile Trp Ala Asp Arg Lys Glu Asn Arg Arg Val Ala Tyr His Pro Leu

85 90 95

Ser His Leu Glu Ile His Glu Gln Asp Gly Leu Lys Glu Leu Gln Gln

100 105 110

Ile Thr Pro Leu Leu Leu Gly Thr Ala Asn Gln Leu Leu Thr Ile Asp

115 120 125

Asp Ile Lys Ser Leu Arg Glu Pro Val Ser Ser Val Leu Ile Glu Leu

130 135 140

Pro Gln Arg Glu Ile Gly Gly Gln Leu Pro Ala Phe Glu Glu Leu Glu

145 150 155 160

Lys Ile Ser Glu Tyr Cys His Glu Gln Gly Ile Ser Leu His Leu Asp

165 170 175

Gly Ala Arg Leu Trp Glu Ile Thr Pro Phe Tyr Gln Lys Ser Ala Glu

180 185 190

Glu Ile Cys Ala Leu Phe Asp Ser Val Tyr Val Ser Phe Tyr Lys Gly

195 200 205

Ile Gly Gly Ile Ala Gly Ala Ile Leu Ala Gly Asn Asp Asp Phe Val

210 215 220

Gln Glu Ala Lys Ile Trp Lys Arg Arg Tyr Gly Gly Asp Leu Ile Ser

225 230 235 240

Leu Tyr Pro Tyr Ile Leu Ser Ala Asp Tyr Tyr Phe Glu Lys Arg Ile

245 250 255

Gly Lys Met Ala Glu Tyr Phe Glu Ala Ala Lys Gly Leu Ala Glu Arg

260 265 270

Phe Asn Ser Cys Ser Gly Val Lys Thr Val Pro Glu Val Pro Val Ser

275 280 285

Asn Met Phe His Val Tyr Phe Glu Asn Ser Ala Asp Glu Ile Gly Ala

290 295 300

Ile Leu Thr Lys Ile Gln Asp Glu Thr Gly Val Gly Ile Ser Gly Tyr

305 310 315 320

Leu Gln Glu Lys Ser Ala Asp Val Cys Ala Phe Glu Val Ser Val Gly

325 330 335

Asp Ala Phe Ala Glu Ile Pro Ala Lys Asn Leu Glu Leu Val Phe Arg

340 345 350

Cys Leu Glu Lys Glu Leu

355

<210> 25

<211> 333

<212> PRT

<213> threonine aldolase amino acid (artificial sequence) of Escherichia coli

<400> 25

Met Ile Asp Leu Arg Ser Asp Thr Val Thr Arg Pro Ser Arg Ala Met

1 5 10 15

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

20 25 30

Asp Pro Thr Val Asn Ala Leu Gln Asp Tyr Ala Ala Glu Leu Ser Gly

35 40 45

Lys Glu Ala Ala Ile Phe Leu Pro Thr Gly Thr Gln Ala Asn Leu Val

50 55 60

Ala Leu Leu Ser His Cys Glu Arg Gly Glu Glu Tyr Ile Val Gly Gln

65 70 75 80

Ala Ala His Asn Tyr Leu Phe Glu Ala Gly Gly Ala Ala Val Leu Gly

85 90 95

Ser Ile Gln Pro Gln Pro Ile Asp Ala Ala Ala Asp Gly Thr Leu Pro

100 105 110

Leu Asp Lys Val Ala Met Lys Ile Lys Pro Asp Asp Ile His Phe Ala

115 120 125

Arg Thr Lys Leu Leu Ser Leu Glu Asn Thr His Asn Gly Lys Val Leu

130 135 140

Pro Arg Glu Tyr Leu Lys Glu Ala Trp Glu Phe Thr Arg Lys Arg Asn

145 150 155 160

Leu Ala Leu His Val Asp Gly Ala Arg Ile Phe Asn Ala Val Val Ala

165 170 175

Tyr Gly Cys Glu Leu Lys Glu Ile Thr Gln Tyr Cys Asp Ser Phe Thr

180 185 190

Ile Cys Leu Ser Lys Gly Leu Gly Thr Pro Val Gly Ser Leu Leu Val

195 200 205

Gly Asn Arg Asp Tyr Ile Lys Arg Ala Ile Arg Trp Arg Lys Met Thr

210 215 220

Gly Gly Gly Met Arg Gln Ser Gly Ile Leu Ala Ala Ala Gly Met Tyr

225 230 235 240

Ala Leu Lys Asn Asn Val Ala Arg Leu Gln Glu Asp His Asp Asn Thr

245 250 255

Ala Trp Met Ala Glu Gln Leu Arg Glu Ala Gly Ala Asp Val Met Arg

260 265 270

Gln Asp Thr Asn Met Leu Phe Val Arg Val Gly Glu Glu Asn Ala Ala

275 280 285

Ala Leu Gly Glu Tyr Met Lys Ala Arg Asn Val Leu Ile Asn Ala Ser

290 295 300

Pro Ile Val Arg Leu Val Thr His Leu Asp Val Ser Arg Ala Gln Leu

305 310 315 320

Ala Glu Val Ala Ala His Trp Arg Ala Phe Leu Ala Arg

325 330

<210> 26

<211> 338

<212> PRT

<213> Aeromonas simplicifolia threonine aldolase amino acid (artificial sequence)

<400> 26

Met Arg Tyr Ile Asp Leu Arg Ser Asp Thr Val Thr Gln Pro Thr Asp

1 5 10 15

Ala Met Arg Gln Cys Met Leu His Ala Glu Val Gly Asp Asp Val Tyr

20 25 30

Gly Glu Asp Pro Gly Val Asn Ala Leu Glu Ala Tyr Gly Ala Asp Leu

35 40 45

Leu Gly Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn

50 55 60

Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Val Leu

65 70 75 80

Gly Ser Ala Ala His Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val

85 90 95

Leu Gly Ser Val Ala Leu Gln Pro Val Pro Met Gln Ala Asp Gly Ser

100 105 110

Leu Ala Leu Ala Asp Val Arg Ala Ala Ile Ala Pro Asp Asp Val His

115 120 125

Phe Thr Pro Thr Arg Leu Val Cys Leu Glu Asn Thr His Asn Gly Lys

130 135 140

Val Leu Pro Leu Pro Tyr Leu Arg Glu Met Arg Glu Leu Val Asp Glu

145 150 155 160

His Gly Leu Gln Leu His Leu Asp Gly Ala Arg Leu Phe Asn Ala Val

165 170 175

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

180 185 190

Val Ser Ile Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu

195 200 205

Leu Val Gly Ser His Ala Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys

210 215 220

Met Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly

225 230 235 240

Leu Phe Ala Leu Gln Gln His Val Val Arg Leu Ala Asp Asp His Arg

245 250 255

Arg Ala Arg Gln Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Arg

260 265 270

Leu Asp Leu Ala Gln Val Gln Thr Asn Met Val Phe Leu Gln Leu Thr

275 280 285

Ser Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Ala Arg Gly Ile

290 295 300

Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile

305 310 315 320

His Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu

325 330 335

Gly Ala

<210> 27

<211> 387

<212> PRT

<213> threonine aldolase amino acid (artificial sequence) of Saccharomyces cerevisiae

<400> 27

Met Thr Glu Phe Glu Leu Pro Pro Lys Tyr Ile Thr Ala Ala Asn Asp

1 5 10 15

Leu Arg Ser Asp Thr Phe Thr Thr Pro Thr Ala Glu Met Met Glu Ala

20 25 30

Ala Leu Glu Ala Ser Ile Gly Asp Ala Val Tyr Gly Glu Asp Val Asp

35 40 45

Thr Val Arg Leu Glu Gln Thr Val Ala Arg Met Ala Gly Lys Glu Ala

50 55 60

Gly Leu Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Ile Arg

65 70 75 80

Thr His Leu Met Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg Ala

85 90 95

His Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln Ala

100 105 110

Met Val Val Pro Val Val Pro Ser Asn Gly Asp Tyr Leu Thr Leu Glu

115 120 125

Asp Ile Lys Ser His Tyr Val Pro Asp Asp Gly Asp Ile His Gly Ala

130 135 140

Pro Thr Arg Leu Ile Ser Leu Glu Asn Thr Leu His Gly Ile Val Tyr

145 150 155 160

Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn Gly

165 170 175

Leu Lys Leu His Cys Asp Gly Ala Arg Ile Trp Asn Ala Ala Ala Gln

180 185 190

Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Ile Phe Asp Ser Ile Ser

195 200 205

Ile Cys Leu Ser Lys Ser Met Gly Ala Pro Ile Gly Ser Val Leu Val

210 215 220

Gly Asn Leu Lys Phe Val Lys Lys Ala Thr His Phe Arg Lys Gln Gln

225 230 235 240

Gly Gly Gly Ile Arg Gln Ser Gly Met Met Ala Arg Met Ala Leu Val

245 250 255

Asn Ile Asn Asn Asp Trp Lys Ser Gln Leu Leu Tyr Ser His Ser Leu

260 265 270

Ala His Glu Leu Ala Glu Tyr Cys Glu Ala Lys Gly Ile Pro Leu Glu

275 280 285

Ser Pro Ala Asp Thr Asn Phe Val Phe Ile Asn Leu Lys Ala Ala Arg

290 295 300

Met Asp Pro Asp Val Leu Val Lys Lys Gly Leu Lys Tyr Asn Val Lys

305 310 315 320

Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Val Thr Arg Asp Thr

325 330 335

Leu Glu Lys Val Lys Leu Ala Ile Ser Glu Ala Phe Asp Tyr Ala Lys

340 345 350

Glu His Pro Phe Asp Cys Asn Gly Pro Thr Gln Ile Tyr Arg Ser Glu

355 360 365

Ser Thr Glu Val Asp Val Asp Gly Asn Ala Ile Arg Glu Ile Lys Thr

370 375 380

Tyr Lys Tyr

385

<210> 28

<211> 382

<212> PRT

<213> threonine aldolase amino acid (artificial sequence) of Ashbya gossypii

<400> 28

Met Asn Gln Asp Met Glu Leu Pro Glu Ala Tyr Thr Ser Ala Ser Asn

1 5 10 15

Asp Phe Arg Ser Asp Thr Phe Thr Thr Pro Thr Arg Glu Met Ile Glu

20 25 30

Ala Ala Leu Thr Ala Thr Ile Gly Asp Ala Val Tyr Gln Glu Asp Ile

35 40 45

Asp Thr Leu Lys Leu Glu Gln His Val Ala Lys Leu Ala Gly Met Glu

50 55 60

Ala Gly Met Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Leu

65 70 75 80

Arg Thr His Leu Thr Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg

85 90 95

Ala His Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln

100 105 110

Ala Met Val Thr Pro Val Ile Pro Ser Asn Gly Asn Tyr Leu Thr Leu

115 120 125

Glu Asp Ile Lys Lys His Tyr Ile Pro Asp Asp Gly Asp Ile His Gly

130 135 140

Ala Pro Thr Lys Val Ile Ser Leu Glu Asn Thr Leu His Gly Ile Ile

145 150 155 160

His Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn

165 170 175

Asp Leu Arg Leu His Cys Asp Gly Ala Arg Ile Trp Asn Ala Ser Ala

180 185 190

Glu Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Leu Phe Asp Ser Ile

195 200 205

Ser Ile Cys Leu Ser Lys Ser Met Gly Ala Pro Met Gly Ser Ile Leu

210 215 220

Val Gly Ser His Lys Phe Ile Lys Lys Ala Asn His Phe Arg Lys Gln

225 230 235 240

Gln Gly Gly Gly Val Arg Gln Ser Gly Met Met Cys Lys Met Ala Met

245 250 255

Val Ala Ile Gln Gly Asp Trp Lys Gly Lys Met Arg Arg Ser His Arg

260 265 270

Met Ala His Glu Leu Ala Arg Phe Cys Ala Glu His Gly Ile Pro Leu

275 280 285

Glu Ser Pro Ala Asp Thr Asn Phe Val Phe Leu Asp Leu Gln Lys Ser

290 295 300

Lys Met Asn Pro Asp Val Leu Val Lys Lys Ser Leu Lys Tyr Gly Cys

305 310 315 320

Lys Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Ile Ser Glu Glu

325 330 335

Ser Leu Glu Lys Ile Lys Gln Ala Ile Leu Glu Ala Phe Glu Tyr Ser

340 345 350

Lys Lys Asn Pro Tyr Asp Glu Asn Gly Pro Thr Lys Ile Tyr Arg Ser

355 360 365

Glu Ser Ala Asp Ala Val Gly Glu Ile Lys Thr Tyr Lys Tyr

370 375 380

<210> 29

<211> 335

<212> PRT

<213> threonine aldolase amino acid (artificial sequence) of Shewanella baumannii

<400> 29

Met Ile Asp Phe Arg Ser Asp Thr Val Thr Gln Pro Thr Ala Ala Met

1 5 10 15

Arg Arg Ala Met Ala Asp Ala Gln Val Gly Asp Asp Val Tyr Gly Asp

20 25 30

Asp Pro Ser Val Asn Arg Leu Glu Ala Met Ala Ala Glu Arg Phe Gly

35 40 45

Phe Asp Ser Ala Leu Phe Thr Ser Ser Gly Thr Gln Ala Asn Leu Leu

50 55 60

Ala Leu Met Ser His Cys Asp Arg Gly Asp Glu Tyr Leu Cys Gly Gln

65 70 75 80

Gln Ala His Asn Tyr Lys Phe Glu Gly Gly Gly Ala Ala Val Leu Gly

85 90 95

Ser Ile Gln Pro Gln Pro Leu Thr Asn Gln Pro Asp Gly Ser Ile Leu

100 105 110

Leu Ser Asp Ile Glu Ala Ala Ile Lys Pro Asp Asp Phe His Phe Ala

115 120 125

Arg Thr Arg Leu Leu Ser Leu Glu Asn Thr Ile Gly Gly Lys Val Leu

130 135 140

Pro Gln Ser Tyr Leu Ala Glu Ala Gln Ala Leu Ala Phe Asn Lys Arg

145 150 155 160

Leu Lys Ile His Leu Asp Gly Ala Arg Ile Ala Asn Ala Ala Val Ala

165 170 175

His Asn Leu Asp Ile Ala Asp Ile Thr Gln Tyr Phe Asp Ser Val Ser

180 185 190

Ile Cys Leu Ser Lys Gly Leu Cys Ala Pro Val Gly Ser Leu Leu Leu

195 200 205

Gly Asp Glu Arg Leu Ile Asn Lys Ala Arg Arg Trp Arg Lys Met Leu

210 215 220

Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Val Ala Gly Glu Ile

225 230 235 240

Ala Leu Asn Glu Gln Val Ser Arg Leu Ala Val Asp His Glu Asn Ala

245 250 255

Arg Tyr Leu Ala Glu Gln Leu Ser Cys Leu Asp Glu Phe Glu Val Asp

260 265 270

Leu Gly Glu Val Gln Thr Asn Met Leu Phe Ala Arg Val Val Glu Gly

275 280 285

Val Ala Ile Asp Lys Leu Ala Thr Ser Leu Lys Ala Ser Gly Ile Leu

290 295 300

Ile Ser Pro Gly Lys Thr Leu Arg Met Val Thr His Ala Asp Ile Arg

305 310 315 320

Leu Glu Asp Ile Asp Leu Phe Ile Asp Lys Leu Lys Ser Leu Leu

325 330 335

<210> 30

<211> 339

<212> PRT

<213> Aeromonas veronii threonine aldolase amino acid (Artificial sequence)

<400> 30

Met Arg Tyr Ile Asp Leu Arg Ser Asp Thr Val Thr Gln Pro Thr Asp

1 5 10 15

Ala Met Arg Gln Ala Met Leu His Ala Glu Val Gly Asp Asp Val Tyr

20 25 30

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

35 40 45

Leu Gly Lys Gln Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn

50 55 60

Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Ile Leu

65 70 75 80

Gly Asn Ala Ala His Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val

85 90 95

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

100 105 110

Leu Ala Phe Asp Asp Ile Lys Ala Ala Leu Ala Pro Asp Asp Ala His

115 120 125

Phe Val Gln Thr Arg Leu Ile Cys Leu Glu Asn Thr His Asn Gly Lys

130 135 140

Val Leu Pro Leu Ser Tyr Leu Gln Glu Met Gly Thr Phe Val Ala Glu

145 150 155 160

Arg Gly Leu Lys Leu His Leu Asp Gly Ala Arg Leu Phe Asn Ala Ala

165 170 175

Val Ala Ser Glu Thr Pro Val Ala Val Ile Ala Ala Pro Phe Asp Ser

180 185 190

Ile Ser Ile Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu

195 200 205

Leu Val Gly Ser His Asp Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys

210 215 220

Met Leu Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly

225 230 235 240

Leu Phe Ala Leu Glu Gln His Val Thr Arg Leu Ala Asp Asp His Arg

245 250 255

Arg Ala Lys Arg Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Glu

260 265 270

Leu Asp Leu Ser Leu Val Gln Ser Asn Met Val Phe Leu Arg Leu Arg

275 280 285

Glu Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Glu Arg Gly Ile

290 295 300

Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile

305 310 315 320

Asn Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu

325 330 335

Gly Ala Arg

<210> 31

<211> 348

<212> PRT

<213> threonine aldolase amino acid (artificial sequence) of sulfur-reducing Geobacillus

<400> 31

Met Asn Ile Ile Asp Leu Arg Ser Asp Thr Val Thr Arg Pro Ser Pro

1 5 10 15

Ala Met Arg Ala Ala Met Ala Gly Ala Glu Val Gly Asp Asp Val Tyr

20 25 30

Gly Glu Asp Pro Thr Val Asn Arg Leu Glu Glu Leu Gly Ala Ala Thr

35 40 45

Leu Gly Thr Glu Ala Ala Leu Phe Thr Ala Ser Gly Thr Gln Ala Asn

50 55 60

Leu Leu Ala Leu Leu Ala His Cys Arg Arg Gly Asp Glu Tyr Ile Ala

65 70 75 80

Gly Gln Thr Ala His Cys Tyr Arg Tyr Glu Gly Gly Gly Ala Ala Ala

85 90 95

Leu Gly Gly Ile Gln Pro Gln Pro Leu Glu Val Glu Pro Asp Gly Thr

100 105 110

Leu Asp Leu Ser Ala Val Ala Ala Ala Ile Lys Pro Asp Asp Leu His

115 120 125

Phe Ala Arg Thr Arg Leu Leu Cys Leu Glu Asn Thr His Ala Gly Arg

130 135 140

Val Leu Pro Leu Asp Tyr Leu Ala Arg Ala Arg Arg Leu Cys Asn Glu

145 150 155 160

Gln Ser Leu Gly Leu His Met Asp Gly Ala Arg Ile Phe Asn Ala Ala

165 170 175

Val Lys Leu Gly Val Pro Val Arg Glu Ile Ala Gly His Val Asp Ser

180 185 190

Val Ser Val Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu

195 200 205

Leu Cys Gly Gly Arg Glu Phe Val Ala Thr Ala Arg Arg Trp Arg Lys

210 215 220

Ala Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Ala Ala Gly

225 230 235 240

Ile Leu Ala Leu Thr Glu Asn Val Asp Arg Leu Ala Glu Asp His Ala

245 250 255

Asn Ala Arg Arg Leu Ala Glu Gly Leu Ala Ala Ile Pro Gly Leu Gly

260 265 270

Leu Asp Pro Ser Gln Val Gln Thr Asn Met Val Phe Leu Cys Leu Pro

275 280 285

Leu Arg Thr Ala Asp Arg Leu Ala Ser Phe Leu Asn Glu Gln Gly Ile

290 295 300

Leu Ile Ser Gly Arg Glu Ser Ile Arg Leu Val Thr His Leu Asp Val

305 310 315 320

Thr Ser Ser Asp Val Glu Arg Val Ile Ala Ala Phe Gly Ala Phe Phe

325 330 335

Ala Gly His Gly Glu Met Gln Pro Gly Ala Ser Ser

340 345

<210> 32

<211> 343

<212> PRT

<400> 32

Met Thr Gln Thr Ala Pro Arg Tyr Asp Phe Ala Ser Asp Thr Val Ala

1 5 10 15

Gly Ala Met Pro Glu Val Met Glu Ala Leu Ile Ala Ala Asn Ala Gly

20 25 30

Thr Ala Ser Gly Tyr Gly Thr Asp His Val Ser Arg Ala Ala Ala Asp

35 40 45

Arg Ile Arg Ala Ala Leu Asp Ala Asp Ala Gln Val Arg Phe Thr Ala

50 55 60

Ser Gly Thr Ala Ala Asn Ala Phe Ala Leu Thr Leu Leu Ala Gln Pro

65 70 75 80

His Glu Ala Val Leu Ala His Glu His Ala His Ile Cys Thr Asp Glu

85 90 95

Thr Gly Ala Pro Gly Phe Phe Gly Gln Gly Val Gly Leu Ile Gly Leu

100 105 110

Pro Gly Ala Ser Gly Lys Met Glu Leu Ala Ala Leu Glu Ala Ala Leu

115 120 125

Ala Gln Pro Asp Val Ser Tyr Arg Gln Pro Ala Ala Ala Leu Ser Leu

130 135 140

Thr Thr Ala Thr Glu Tyr Gly Thr Val Tyr Ser Glu Asp His Leu Arg

145 150 155 160

Ala Leu Ile Ala Pro Val Lys Ala Lys Gly Tyr Gly Val His Leu Asp

165 170 175

Gly Ala Arg Leu Ala Asn Ala Val Ala Gly Gly Phe Asp Leu Lys Ser

180 185 190

Ile Ala Lys Met Gly Val Asp Ile Leu Val Met Gly Gly Thr Lys Ala

195 200 205

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

210 215 220

Lys Arg Leu Asp Ala Arg Leu Lys His Ala Gly Gln Leu Ile Ser Lys

225 230 235 240

Gly Arg Phe Leu Ala Ala Pro Trp Leu Gly Leu Leu Gly Glu Asn Gly

245 250 255

Gln Thr Ala Pro Trp Ala Ala Arg Ala Ala His Ala Asn Ala Met Ala

260 265 270

Gln Lys Leu Ala Ala Leu Met Pro Val Pro Ile Lys His Pro Val Glu

275 280 285

Ala Asn Gly Ile Phe Val Glu Met Asp Glu Leu Ala Leu Glu Arg Leu

290 295 300

Arg Gly Glu Gly Trp Phe Val Tyr Arg Phe Leu Asp Gly Thr Val Arg

305 310 315 320

Phe Met Cys Ser Trp Ala Thr Thr Pro Glu Met Val Glu Asp Leu Gly

325 330 335

Ala Ala Leu Lys Arg Val Ala

340

<210> 33

<211> 343

<212> PRT

<400> 33

Met Thr Gln Thr Ala Pro Arg Tyr Asp Phe Ala Ser Asp Gln Val Ala

1 5 10 15

Gly Ala Met Pro Glu Val Met Glu Ala Leu Ile Ala Ala Asn Ala Gly

20 25 30

Thr Ala Ser Gly Tyr Gly Thr Asp His Val Ser Arg Ala Ala Ala Asp

35 40 45

Arg Ile Arg Ala Ala Leu Asp Ala Asp Ala Gln Val Arg Phe Thr Ala

50 55 60

Ser Gly Thr Ala Ala Asn Ala Phe Ala Leu Thr Leu Leu Ala Gln Pro

65 70 75 80

His Glu Ala Val Leu Ala His Glu His Ala His Ile Cys Thr Asp Glu

85 90 95

Thr Gly Ala Pro Gly Phe Phe Gly Gln Gly Val Gly Leu Ile Gly Leu

100 105 110

Pro Gly Ala Ser Gly Lys Met Glu Leu Ala Ala Leu Glu Ala Ala Leu

115 120 125

Ala Gln Pro Asp Val Ser Tyr Arg Gln Pro Ala Ala Ala Leu Ser Leu

130 135 140

Thr Thr Ala Thr Glu Tyr Gly Thr Val Tyr Ser Glu Asp His Leu Arg

145 150 155 160

Ala Leu Ile Ala Pro Val Lys Ala Lys Gly Tyr Gly Val His Leu Asp

165 170 175

Gly Ala Arg Leu Ala Asn Ala Val Ala Gly Gly Phe Asp Leu Lys Ser

180 185 190

Ile Ala Lys Met Gly Val Asp Ile Leu Val Met Gly Gly Thr Lys Ala

195 200 205

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

210 215 220

Lys Arg Leu Asp Ala Arg Leu Lys His Ala Gly Gln Leu Ile Ser Lys

225 230 235 240

Gly Arg Phe Leu Ala Ala Pro Trp Leu Gly Leu Leu Gly Glu Asn Gly

245 250 255

Gln Thr Ala Pro Trp Ala Ala Arg Ala Ala His Ala Asn Ala Met Ala

260 265 270

Gln Lys Leu Ala Ala Leu Met Pro Val Pro Ile Lys His Pro Val Glu

275 280 285

Ala Asn Gly Ile Phe Val Glu Met Asp Glu Leu Ala Leu Glu Arg Leu

290 295 300

Arg Gly Glu Gly Trp Phe Val Tyr Arg Phe Leu Asp Gly Thr Val Arg

305 310 315 320

Phe Met Cys Ser Trp Ala Thr Thr Pro Glu Met Val Glu Asp Leu Gly

325 330 335

Ala Ala Leu Lys Arg Val Ala

340

<210> 34

<211> 343

<212> PRT

<400> 34

Met Thr Gln Thr Ala Pro Arg Tyr Asp Phe Ala Ser Asp Asn Val Ala

1 5 10 15

Gly Ala Met Pro Glu Val Met Glu Ala Leu Ile Ala Ala Asn Ala Gly

20 25 30

Thr Ala Ser Gly Tyr Gly Thr Asp His Val Ser Arg Ala Ala Ala Asp

35 40 45

Arg Ile Arg Ala Ala Leu Asp Ala Asp Ala Gln Val Arg Phe Thr Ala

50 55 60

Ser Gly Thr Ala Ala Asn Ala Phe Ala Leu Thr Leu Leu Ala Gln Pro

65 70 75 80

His Glu Ala Val Leu Ala His Glu His Ala Trp Ile Cys Thr Asp Glu

85 90 95

Thr Gly Ala Pro Gly Phe Phe Gly Gln Gly Val Gly Leu Ile Gly Leu

100 105 110

Pro Gly Ala Ser Gly Lys Met Glu Leu Ala Ala Leu Glu Ala Ala Leu

115 120 125

Ala Gln Pro Asp Val Ser Tyr Arg Gln Pro Ala Ala Ala Leu Ser Leu

130 135 140

Thr Thr Ala Thr Glu Tyr Gly Thr Val Tyr Ser Glu Asp His Leu Arg

145 150 155 160

Ala Leu Ile Ala Pro Val Lys Ala Lys Gly Tyr Gly Val His Leu Asp

165 170 175

Gly Ala Arg Leu Ala Asn Ala Val Ala Gly Gly Phe Asp Leu Lys Ser

180 185 190

Ile Ala Lys Met Gly Val Asp Ile Leu Val Met Gly Gly Thr Lys Ala

195 200 205

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

210 215 220

Lys Arg Leu Asp Ala Arg Leu Lys His Ala Gly Gln Leu Ile Ser Lys

225 230 235 240

Gly Arg Phe Leu Ala Ala Pro Trp Leu Gly Leu Leu Gly Glu Asn Gly

245 250 255

Gln Thr Ala Pro Trp Ala Ala Arg Ala Ala His Ala Asn Ala Met Ala

260 265 270

Gln Lys Leu Ala Ala Leu Met Pro Val Pro Ile Lys His Pro Val Glu

275 280 285

Ala Asn Gly Ile Phe Val Glu Met Asp Glu Leu Ala Leu Glu Arg Leu

290 295 300

Arg Gly Glu Gly Trp Phe Val Tyr Arg Phe Leu Asp Gly Thr Val Arg

305 310 315 320

Phe Met Cys Ser Trp Ala Thr Thr Pro Glu Met Val Glu Asp Leu Gly

325 330 335

Ala Ala Leu Lys Arg Val Ala

340

<210> 35

<211> 343

<212> PRT

<400> 35

Met Thr Gln Thr Ala Pro Arg Tyr Asp Phe Ala Ser Asp Asn Val Ala

1 5 10 15

Gly Ala Met Pro Glu Val Met Glu Ala Leu Ile Ala Ala Asn Ala Gly

20 25 30

Thr Ala Ser Gly Tyr Gly Thr Asp His Val Ser Arg Ala Ala Ala Asp

35 40 45

Arg Ile Arg Ala Ala Leu Asp Ala Asp Ala Gln Val Arg Phe Thr Ala

50 55 60

Ser Gly Thr Ala Ala Asn Ala Phe Ala Leu Thr Leu Leu Ala Gln Pro

65 70 75 80

His Glu Ala Val Leu Ala His Glu His Ala Pro Ile Cys Thr Asp Glu

85 90 95

Thr Gly Ala Pro Gly Phe Phe Gly Gln Gly Val Gly Leu Ile Gly Leu

100 105 110

Pro Gly Ala Ser Gly Lys Met Glu Leu Ala Ala Leu Glu Ala Ala Leu

115 120 125

Ala Gln Pro Asp Val Ser Tyr Arg Gln Pro Ala Ala Ala Leu Ser Leu

130 135 140

Thr Thr Ala Thr Glu Tyr Gly Thr Val Tyr Ser Glu Asp His Leu Arg

145 150 155 160

Ala Leu Ile Ala Pro Val Lys Ala Lys Gly Tyr Gly Val His Leu Asp

165 170 175

Gly Ala Arg Leu Ala Asn Ala Val Ala Gly Gly Phe Asp Leu Lys Ser

180 185 190

Ile Ala Lys Met Gly Val Asp Ile Leu Val Met Gly Gly Thr Lys Ala

195 200 205

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

210 215 220

Lys Arg Leu Asp Ala Arg Leu Lys His Ala Gly Gln Leu Ile Ser Lys

225 230 235 240

Gly Arg Phe Leu Ala Ala Pro Trp Leu Gly Leu Leu Gly Glu Asn Gly

245 250 255

Gln Thr Ala Pro Trp Ala Ala Arg Ala Ala His Ala Asn Ala Met Ala

260 265 270

Gln Lys Leu Ala Ala Leu Met Pro Val Pro Ile Lys His Pro Val Glu

275 280 285

Ala Asn Gly Ile Phe Val Glu Met Asp Glu Leu Ala Leu Glu Arg Leu

290 295 300

Arg Gly Glu Gly Trp Phe Val Tyr Arg Phe Leu Asp Gly Thr Val Arg

305 310 315 320

Phe Met Cys Ser Trp Ala Thr Thr Pro Glu Met Val Glu Asp Leu Gly

325 330 335

Ala Ala Leu Lys Arg Val Ala

340

<210> 36

<211> 343

<212> PRT

<400> 36

Met Thr Gln Thr Ala Pro Arg Tyr Asp Phe Ala Ser Asp Asn Val Ala

1 5 10 15

Gly Ala Met Pro Glu Val Met Glu Ala Leu Ile Ala Ala Asn Ala Gly

20 25 30

Thr Ala Ser Gly Tyr Gly Thr Asp His Val Ser Arg Ala Ala Ala Asp

35 40 45

Arg Ile Arg Ala Ala Leu Asp Ala Asp Ala Gln Val Arg Phe Thr Ala

50 55 60

Ser Gly Thr Ala Ala Asn Ala Phe Ala Leu Thr Leu Leu Ala Gln Pro

65 70 75 80

His Glu Ala Val Leu Ala His Glu His Ala His Ile Cys Thr Asp Glu

85 90 95

Thr Gly Ala Pro Gly Phe Phe Gly Gln Gly Val Gly Leu Ile Gly Leu

100 105 110

Pro Gly Ala Ser Gly Lys Met Glu Leu Ala Ala Leu Glu Ala Ala Leu

115 120 125

Ala Gln Pro Asp Val Ser Tyr Arg Gln Pro Ala Ala Ala Leu Ser Leu

130 135 140

Thr Thr Ala Thr Glu Tyr Gly Thr Val Tyr Ser Glu Asp His Leu Arg

145 150 155 160

Ala Leu Ile Ala Pro Val Lys Ala Lys Gly Tyr Gly Val His Leu Asp

165 170 175

Gly Tyr Arg Leu Ala Asn Ala Val Ala Gly Gly Phe Asp Leu Lys Ser

180 185 190

Ile Ala Lys Met Gly Val Asp Ile Leu Val Met Gly Gly Thr Lys Ala

195 200 205

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

210 215 220

Lys Arg Leu Asp Ala Arg Leu Lys His Ala Gly Gln Leu Ile Ser Lys

225 230 235 240

Gly Arg Phe Leu Ala Ala Pro Trp Leu Gly Leu Leu Gly Glu Asn Gly

245 250 255

Gln Thr Ala Pro Trp Ala Ala Arg Ala Ala His Ala Asn Ala Met Ala

260 265 270

Gln Lys Leu Ala Ala Leu Met Pro Val Pro Ile Lys His Pro Val Glu

275 280 285

Ala Asn Gly Ile Phe Val Glu Met Asp Glu Leu Ala Leu Glu Arg Leu

290 295 300

Arg Gly Glu Gly Trp Phe Val Tyr Arg Phe Leu Asp Gly Thr Val Arg

305 310 315 320

Phe Met Cys Ser Trp Ala Thr Thr Pro Glu Met Val Glu Asp Leu Gly

325 330 335

Ala Ala Leu Lys Arg Val Ala

340

<210> 37

<211> 343

<212> PRT

<400> 37

Met Thr Gln Thr Ala Pro Arg Tyr Asp Phe Ala Ser Asp Asn Val Ala

1 5 10 15

Gly Ala Met Pro Glu Val Met Glu Ala Leu Ile Ala Ala Asn Ala Gly

20 25 30

Thr Ala Ser Gly Tyr Gly Thr Asp His Val Ser Arg Ala Ala Ala Asp

35 40 45

Arg Ile Arg Ala Ala Leu Asp Ala Asp Ala Gln Val Arg Phe Thr Ala

50 55 60

Ser Gly Thr Ala Ala Asn Ala Phe Ala Leu Thr Leu Leu Ala Gln Pro

65 70 75 80

His Glu Ala Val Leu Ala His Glu His Ala His Ile Cys Thr Asp Glu

85 90 95

Thr Gly Ala Pro Gly Phe Phe Gly Gln Gly Val Gly Leu Ile Gly Leu

100 105 110

Pro Gly Ala Ser Gly Lys Met Glu Leu Ala Ala Leu Glu Ala Ala Leu

115 120 125

Ala Gln Pro Asp Val Ser Tyr Arg Gln Pro Ala Ala Ala Leu Ser Leu

130 135 140

Thr Thr Ala Thr Glu Tyr Gly Thr Val Tyr Ser Glu Asp His Leu Arg

145 150 155 160

Ala Leu Ile Ala Pro Val Lys Ala Lys Gly Tyr Gly Val His Leu Asp

165 170 175

Gly Ala Arg Leu Ala Asn Ala Val Ala Gly Gly Phe Asp Leu Lys Ser

180 185 190

Ile Ala Lys Met Gly Val Asp Ile Leu Val Met Gly Gly Ile Lys Ala

195 200 205

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

210 215 220

Lys Arg Leu Asp Ala Arg Leu Lys His Ala Gly Gln Leu Ile Ser Lys

225 230 235 240

Gly Arg Phe Leu Ala Ala Pro Trp Leu Gly Leu Leu Gly Glu Asn Gly

245 250 255

Gln Thr Ala Pro Trp Ala Ala Arg Ala Ala His Ala Asn Ala Met Ala

260 265 270

Gln Lys Leu Ala Ala Leu Met Pro Val Pro Ile Lys His Pro Val Glu

275 280 285

Ala Asn Gly Ile Phe Val Glu Met Asp Glu Leu Ala Leu Glu Arg Leu

290 295 300

Arg Gly Glu Gly Trp Phe Val Tyr Arg Phe Leu Asp Gly Thr Val Arg

305 310 315 320

Phe Met Cys Ser Trp Ala Thr Thr Pro Glu Met Val Glu Asp Leu Gly

325 330 335

Ala Ala Leu Lys Arg Val Ala

340

<210> 38

<211> 343

<212> PRT

<400> 38

Met Thr Gln Thr Ala Pro Arg Tyr Asp Phe Ala Ser Asp Asn Val Ala

1 5 10 15

Gly Ala Met Pro Glu Val Met Glu Ala Leu Ile Ala Ala Asn Ala Gly

20 25 30

Thr Ala Ser Gly Tyr Gly Thr Asp His Val Ser Arg Ala Ala Ala Asp

35 40 45

Arg Ile Arg Ala Ala Leu Asp Ala Asp Ala Gln Val Arg Phe Thr Ala

50 55 60

Ser Gly Thr Ala Ala Asn Ala Phe Ala Leu Thr Leu Leu Ala Gln Pro

65 70 75 80

His Glu Ala Val Leu Ala His Glu His Ala His Ile Cys Thr Asp Glu

85 90 95

Thr Gly Ala Pro Gly Phe Phe Gly Gln Gly Val Gly Leu Ile Gly Leu

100 105 110

Pro Gly Ala Ser Gly Lys Met Glu Leu Ala Ala Leu Glu Ala Ala Leu

115 120 125

Ala Gln Pro Asp Val Ser Tyr Arg Gln Pro Ala Ala Ala Leu Ser Leu

130 135 140

Thr Thr Ala Thr Glu Tyr Gly Thr Val Tyr Ser Glu Asp His Leu Arg

145 150 155 160

Ala Leu Ile Ala Pro Val Lys Ala Lys Gly Tyr Gly Val His Leu Asp

165 170 175

Gly Ala Arg Leu Ala Asn Ala Val Ala Gly Gly Phe Asp Leu Lys Ser

180 185 190

Ile Ala Lys Met Gly Val Asp Ile Leu Val Met Gly Gly Ser Lys Ala

195 200 205

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

210 215 220

Lys Arg Leu Asp Ala Arg Leu Lys His Ala Gly Gln Leu Ile Ser Lys

225 230 235 240

Gly Arg Phe Leu Ala Ala Pro Trp Leu Gly Leu Leu Gly Glu Asn Gly

245 250 255

Gln Thr Ala Pro Trp Ala Ala Arg Ala Ala His Ala Asn Ala Met Ala

260 265 270

Gln Lys Leu Ala Ala Leu Met Pro Val Pro Ile Lys His Pro Val Glu

275 280 285

Ala Asn Gly Ile Phe Val Glu Met Asp Glu Leu Ala Leu Glu Arg Leu

290 295 300

Arg Gly Glu Gly Trp Phe Val Tyr Arg Phe Leu Asp Gly Thr Val Arg

305 310 315 320

Phe Met Cys Ser Trp Ala Thr Thr Pro Glu Met Val Glu Asp Leu Gly

325 330 335

Ala Ala Leu Lys Arg Val Ala

340

<210> 39

<211> 343

<212> PRT

<213> threonine aldolase amino acid mutation (artificial sequence) of Thermotoga maritima

<400> 39

Met Ile Asp Leu Arg Ser Asp Gln Val Thr Lys Pro Thr Glu Glu Met

1 5 10 15

Arg Lys Ala Met Ala Gln Ala Glu Val Gly Asp Asp Val Tyr Gly Glu

20 25 30

Asp Pro Thr Ile Asn Glu Leu Glu Arg Leu Ala Ala Glu Thr Phe Gly

35 40 45

Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Gly Asn Gln Val

50 55 60

Ser Ile Met Ala His Thr Gln Arg Gly Asp Glu Val Ile Leu Glu Ala

65 70 75 80

Asp Ser His Ile Phe Trp Tyr Glu Val Gly Ala Met Ala Val Leu Ser

85 90 95

Gly Val Met Pro His Pro Val Pro Gly Lys Asn Gly Ala Met Asp Pro

100 105 110

Asp Asp Val Arg Lys Ala Ile Arg Pro Arg Asn Ile His Phe Pro Arg

115 120 125

Thr Ser Leu Ile Ala Ile Glu Asn Thr His Asn Arg Ser Gly Gly Arg

130 135 140

Val Val Pro Leu Glu Asn Ile Lys Glu Ile Cys Thr Ile Ala Lys Glu

145 150 155 160

His Gly Ile Asn Val His Ile Asp Gly Ala Arg Ile Phe Asn Ala Ser

165 170 175

Ile Ala Ser Gly Val Pro Val Lys Glu Tyr Ala Gly Tyr Ala Asp Ser

180 185 190

Val Met Phe Cys Leu Ser Lys Gly Leu Cys Ala Pro Val Gly Ser Val

195 200 205

Val Val Gly Asp Arg Asp Phe Ile Glu Arg Ala Arg Lys Ala Arg Lys

210 215 220

Met Leu Gly Gly Gly Met Arg Gln Ala Gly Val Leu Ala Ala Ala Gly

225 230 235 240

Ile Ile Ala Leu Thr Lys Met Val Asp Arg Leu Lys Glu Asp His Glu

245 250 255

Asn Ala Arg Phe Leu Ala Leu Lys Leu Lys Glu Ile Gly Tyr Ser Val

260 265 270

Asn Pro Glu Asp Val Lys Thr Asn Met Val Ile Leu Arg Thr Asp Asn

275 280 285

Leu Lys Val Asn Ala His Gly Phe Ile Glu Ala Leu Arg Asn Ser Gly

290 295 300

Val Leu Ala Asn Ala Val Ser Asp Thr Glu Ile Arg Leu Val Thr His

305 310 315 320

Lys Asp Val Ser Arg Asn Asp Ile Glu Glu Ala Leu Asn Ile Phe Glu

325 330 335

Lys Leu Phe Arg Lys Phe Ser

340

<210> 40

<211> 343

<212> PRT

<400> 40

Met Ile Asp Leu Arg Ser Asp Thr Val Thr Lys Pro Thr Glu Glu Met

1 5 10 15

Arg Lys Ala Met Ala Gln Ala Glu Val Gly Asp Asp Val Tyr Gly Glu

20 25 30

Asp Pro Thr Ile Asn Glu Leu Glu Arg Leu Ala Ala Glu Thr Phe Gly

35 40 45

Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Gly Asn Gln Val

50 55 60

Ser Ile Met Ala His Thr Gln Arg Gly Asp Glu Val Ile Leu Glu Ala

65 70 75 80

Asp Ser Trp Ile Phe Trp Tyr Glu Val Gly Ala Met Ala Val Leu Ser

85 90 95

Gly Val Met Pro His Pro Val Pro Gly Lys Asn Gly Ala Met Asp Pro

100 105 110

Asp Asp Val Arg Lys Ala Ile Arg Pro Arg Asn Ile His Phe Pro Arg

115 120 125

Thr Ser Leu Ile Ala Ile Glu Asn Thr His Asn Arg Ser Gly Gly Arg

130 135 140

Val Val Pro Leu Glu Asn Ile Lys Glu Ile Cys Thr Ile Ala Lys Glu

145 150 155 160

His Gly Ile Asn Val His Ile Asp Gly Ala Arg Ile Phe Asn Ala Ser

165 170 175

Ile Ala Ser Gly Val Pro Val Lys Glu Tyr Ala Gly Tyr Ala Asp Ser

180 185 190

Val Met Phe Cys Leu Ser Lys Gly Leu Cys Ala Pro Val Gly Ser Val

195 200 205

Val Val Gly Asp Arg Asp Phe Ile Glu Arg Ala Arg Lys Ala Arg Lys

210 215 220

Met Leu Gly Gly Gly Met Arg Gln Ala Gly Val Leu Ala Ala Ala Gly

225 230 235 240

Ile Ile Ala Leu Thr Lys Met Val Asp Arg Leu Lys Glu Asp His Glu

245 250 255

Asn Ala Arg Phe Leu Ala Leu Lys Leu Lys Glu Ile Gly Tyr Ser Val

260 265 270

Asn Pro Glu Asp Val Lys Thr Asn Met Val Ile Leu Arg Thr Asp Asn

275 280 285

Leu Lys Val Asn Ala His Gly Phe Ile Glu Ala Leu Arg Asn Ser Gly

290 295 300

Val Leu Ala Asn Ala Val Ser Asp Thr Glu Ile Arg Leu Val Thr His

305 310 315 320

Lys Asp Val Ser Arg Asn Asp Ile Glu Glu Ala Leu Asn Ile Phe Glu

325 330 335

Lys Leu Phe Arg Lys Phe Ser

340

<210> 41

<211> 343

<212> PRT

<400> 41

Met Ile Asp Leu Arg Ser Asp Thr Val Thr Lys Pro Thr Glu Glu Met

1 5 10 15

Arg Lys Ala Met Ala Gln Ala Glu Val Gly Asp Asp Val Tyr Gly Glu

20 25 30

Asp Pro Thr Ile Asn Glu Leu Glu Arg Leu Ala Ala Glu Thr Phe Gly

35 40 45

Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Gly Asn Gln Val

50 55 60

Ser Ile Met Ala His Thr Gln Arg Gly Asp Glu Val Ile Leu Glu Ala

65 70 75 80

Asp Ser Pro Ile Phe Trp Tyr Glu Val Gly Ala Met Ala Val Leu Ser

85 90 95

Gly Val Met Pro His Pro Val Pro Gly Lys Asn Gly Ala Met Asp Pro

100 105 110

Asp Asp Val Arg Lys Ala Ile Arg Pro Arg Asn Ile His Phe Pro Arg

115 120 125

Thr Ser Leu Ile Ala Ile Glu Asn Thr His Asn Arg Ser Gly Gly Arg

130 135 140

Val Val Pro Leu Glu Asn Ile Lys Glu Ile Cys Thr Ile Ala Lys Glu

145 150 155 160

His Gly Ile Asn Val His Ile Asp Gly Ala Arg Ile Phe Asn Ala Ser

165 170 175

Ile Ala Ser Gly Val Pro Val Lys Glu Tyr Ala Gly Tyr Ala Asp Ser

180 185 190

Val Met Phe Cys Leu Ser Lys Gly Leu Cys Ala Pro Val Gly Ser Val

195 200 205

Val Val Gly Asp Arg Asp Phe Ile Glu Arg Ala Arg Lys Ala Arg Lys

210 215 220

Met Leu Gly Gly Gly Met Arg Gln Ala Gly Val Leu Ala Ala Ala Gly

225 230 235 240

Ile Ile Ala Leu Thr Lys Met Val Asp Arg Leu Lys Glu Asp His Glu

245 250 255

Asn Ala Arg Phe Leu Ala Leu Lys Leu Lys Glu Ile Gly Tyr Ser Val

260 265 270

Asn Pro Glu Asp Val Lys Thr Asn Met Val Ile Leu Arg Thr Asp Asn

275 280 285

Leu Lys Val Asn Ala His Gly Phe Ile Glu Ala Leu Arg Asn Ser Gly

290 295 300

Val Leu Ala Asn Ala Val Ser Asp Thr Glu Ile Arg Leu Val Thr His

305 310 315 320

Lys Asp Val Ser Arg Asn Asp Ile Glu Glu Ala Leu Asn Ile Phe Glu

325 330 335

Lys Leu Phe Arg Lys Phe Ser

340

<210> 42

<211> 343

<212> PRT

<400> 42

Met Ile Asp Leu Arg Ser Asp Thr Val Thr Lys Pro Thr Glu Glu Met

1 5 10 15

Arg Lys Ala Met Ala Gln Ala Glu Val Gly Asp Asp Val Tyr Gly Glu

20 25 30

Asp Pro Thr Ile Asn Glu Leu Glu Arg Leu Ala Ala Glu Thr Phe Gly

35 40 45

Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Gly Asn Gln Val

50 55 60

Ser Ile Met Ala His Thr Gln Arg Gly Asp Glu Val Ile Leu Glu Ala

65 70 75 80

Asp Ser His Ile Phe Trp Tyr Glu Val Gly Ala Met Ala Val Leu Ser

85 90 95

Gly Val Met Pro His Pro Val Pro Gly Lys Asn Gly Ala Met Asp Pro

100 105 110

Asp Asp Val Arg Lys Ala Ile Arg Pro Arg Asn Ile His Phe Pro Arg

115 120 125

Thr Ser Leu Ile Ala Ile Glu Asn Thr His Asn Arg Ser Gly Gly Arg

130 135 140

Val Val Pro Leu Glu Asn Ile Lys Glu Ile Cys Thr Ile Ala Lys Glu

145 150 155 160

His Gly Ile Asn Val His Ile Asp Gly Tyr Arg Ile Phe Asn Ala Ser

165 170 175

Ile Ala Ser Gly Val Pro Val Lys Glu Tyr Ala Gly Tyr Ala Asp Ser

180 185 190

Val Met Phe Cys Leu Ser Lys Gly Leu Cys Ala Pro Val Gly Ser Val

195 200 205

Val Val Gly Asp Arg Asp Phe Ile Glu Arg Ala Arg Lys Ala Arg Lys

210 215 220

Met Leu Gly Gly Gly Met Arg Gln Ala Gly Val Leu Ala Ala Ala Gly

225 230 235 240

Ile Ile Ala Leu Thr Lys Met Val Asp Arg Leu Lys Glu Asp His Glu

245 250 255

Asn Ala Arg Phe Leu Ala Leu Lys Leu Lys Glu Ile Gly Tyr Ser Val

260 265 270

Asn Pro Glu Asp Val Lys Thr Asn Met Val Ile Leu Arg Thr Asp Asn

275 280 285

Leu Lys Val Asn Ala His Gly Phe Ile Glu Ala Leu Arg Asn Ser Gly

290 295 300

Val Leu Ala Asn Ala Val Ser Asp Thr Glu Ile Arg Leu Val Thr His

305 310 315 320

Lys Asp Val Ser Arg Asn Asp Ile Glu Glu Ala Leu Asn Ile Phe Glu

325 330 335

Lys Leu Phe Arg Lys Phe Ser

340

<210> 43

<211> 343

<212> PRT

<400> 43

Met Ile Asp Leu Arg Ser Asp Thr Val Thr Lys Pro Thr Glu Glu Met

1 5 10 15

Arg Lys Ala Met Ala Gln Ala Glu Val Gly Asp Asp Val Tyr Gly Glu

20 25 30

Asp Pro Thr Ile Asn Glu Leu Glu Arg Leu Ala Ala Glu Thr Phe Gly

35 40 45

Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Gly Asn Gln Val

50 55 60

Ser Ile Met Ala His Thr Gln Arg Gly Asp Glu Val Ile Leu Glu Ala

65 70 75 80

Asp Ser His Ile Phe Trp Tyr Glu Val Gly Ala Met Ala Val Leu Ser

85 90 95

Gly Val Met Pro His Pro Val Pro Gly Lys Asn Gly Ala Met Asp Pro

100 105 110

Asp Asp Val Arg Lys Ala Ile Arg Pro Arg Asn Ile His Phe Pro Arg

115 120 125

Thr Ser Leu Ile Ala Ile Glu Asn Thr His Asn Arg Ser Gly Gly Arg

130 135 140

Val Val Pro Leu Glu Asn Ile Lys Glu Ile Cys Thr Ile Ala Lys Glu

145 150 155 160

His Gly Ile Asn Val His Ile Asp Gly Ala Arg Ile Phe Asn Ala Ser

165 170 175

Ile Ala Ser Gly Val Pro Val Lys Glu Tyr Ala Gly Tyr Ala Asp Ser

180 185 190

Val Met Phe Cys Leu Ile Lys Gly Leu Cys Ala Pro Val Gly Ser Val

195 200 205

Val Val Gly Asp Arg Asp Phe Ile Glu Arg Ala Arg Lys Ala Arg Lys

210 215 220

Met Leu Gly Gly Gly Met Arg Gln Ala Gly Val Leu Ala Ala Ala Gly

225 230 235 240

Ile Ile Ala Leu Thr Lys Met Val Asp Arg Leu Lys Glu Asp His Glu

245 250 255

Asn Ala Arg Phe Leu Ala Leu Lys Leu Lys Glu Ile Gly Tyr Ser Val

260 265 270

Asn Pro Glu Asp Val Lys Thr Asn Met Val Ile Leu Arg Thr Asp Asn

275 280 285

Leu Lys Val Asn Ala His Gly Phe Ile Glu Ala Leu Arg Asn Ser Gly

290 295 300

Val Leu Ala Asn Ala Val Ser Asp Thr Glu Ile Arg Leu Val Thr His

305 310 315 320

Lys Asp Val Ser Arg Asn Asp Ile Glu Glu Ala Leu Asn Ile Phe Glu

325 330 335

Lys Leu Phe Arg Lys Phe Ser

340

<210> 44

<211> 358

<212> PRT

<213> Listeria monocytogenes threonine aldolase amino acid mutation (artificial sequence)

<400> 44

Met Thr Asn Thr Leu Lys Thr Ser Tyr Gln Lys Thr Pro Tyr Lys Leu

1 5 10 15

Gly Gly Asn Gly Pro Arg Asn Val Gly Val Leu Thr Glu Ala Leu Gln

20 25 30

Asn Ile Asp Asp Asn Leu Glu Ser Asp Ile Tyr Gly Asn Gly Ala Val

35 40 45

Ile Glu Asp Phe Glu Thr Lys Ile Ala Lys Ile Leu Gly Lys Gln Ser

50 55 60

Ala Val Phe Phe Pro Ser Gly Thr Met Ala Gln Gln Ile Ala Leu Arg

65 70 75 80

Ile Trp Ala Asp Arg Lys Glu Asn Arg Arg Val Ala Tyr His Pro Leu

85 90 95

Ser Trp Leu Glu Ile His Glu Gln Asp Gly Leu Lys Glu Leu Gln Gln

100 105 110

Ile Thr Pro Leu Leu Leu Gly Thr Ala Asn Gln Leu Leu Thr Ile Asp

115 120 125

Asp Ile Lys Ser Leu Arg Glu Pro Val Ser Ser Val Leu Ile Glu Leu

130 135 140

Pro Gln Arg Glu Ile Gly Gly Gln Leu Pro Ala Phe Glu Glu Leu Glu

145 150 155 160

Lys Ile Ser Glu Tyr Cys His Glu Gln Gly Ile Ser Leu His Leu Asp

165 170 175

Gly Ala Arg Leu Trp Glu Ile Thr Pro Phe Tyr Gln Lys Ser Ala Glu

180 185 190

Glu Ile Cys Ala Leu Phe Asp Ser Val Tyr Val Ser Phe Tyr Lys Gly

195 200 205

Ile Gly Gly Ile Ala Gly Ala Ile Leu Ala Gly Asn Asp Asp Phe Val

210 215 220

Gln Glu Ala Lys Ile Trp Lys Arg Arg Tyr Gly Gly Asp Leu Ile Ser

225 230 235 240

Leu Tyr Pro Tyr Ile Leu Ser Ala Asp Tyr Tyr Phe Glu Lys Arg Ile

245 250 255

Gly Lys Met Ala Glu Tyr Phe Glu Ala Ala Lys Gly Leu Ala Glu Arg

260 265 270

Phe Asn Ser Cys Ser Gly Val Lys Thr Val Pro Glu Val Pro Val Ser

275 280 285

Asn Met Phe His Val Tyr Phe Glu Asn Ser Ala Asp Glu Ile Gly Ala

290 295 300

Ile Leu Thr Lys Ile Gln Asp Glu Thr Gly Val Gly Ile Ser Gly Tyr

305 310 315 320

Leu Gln Glu Lys Ser Ala Asp Val Cys Ala Phe Glu Val Ser Val Gly

325 330 335

Asp Ala Phe Ala Glu Ile Pro Ala Lys Asn Leu Glu Leu Val Phe Arg

340 345 350

Cys Leu Glu Lys Glu Leu

355

<210> 45

<211> 358

<212> PRT

<400> 45

Met Thr Asn Thr Leu Lys Thr Ser Tyr Gln Lys Thr Pro Tyr Lys Leu

1 5 10 15

Gly Gly Asn Gly Pro Arg Asn Val Gly Val Leu Thr Glu Ala Leu Gln

20 25 30

Asn Ile Asp Asp Asn Leu Glu Ser Asp Ile Tyr Gly Asn Gly Ala Val

35 40 45

Ile Glu Asp Phe Glu Thr Lys Ile Ala Lys Ile Leu Gly Lys Gln Ser

50 55 60

Ala Val Phe Phe Pro Ser Gly Thr Met Ala Gln Gln Ile Ala Leu Arg

65 70 75 80

Ile Trp Ala Asp Arg Lys Glu Asn Arg Arg Val Ala Tyr His Pro Leu

85 90 95

Ser Pro Leu Glu Ile His Glu Gln Asp Gly Leu Lys Glu Leu Gln Gln

100 105 110

Ile Thr Pro Leu Leu Leu Gly Thr Ala Asn Gln Leu Leu Thr Ile Asp

115 120 125

Asp Ile Lys Ser Leu Arg Glu Pro Val Ser Ser Val Leu Ile Glu Leu

130 135 140

Pro Gln Arg Glu Ile Gly Gly Gln Leu Pro Ala Phe Glu Glu Leu Glu

145 150 155 160

Lys Ile Ser Glu Tyr Cys His Glu Gln Gly Ile Ser Leu His Leu Asp

165 170 175

Gly Ala Arg Leu Trp Glu Ile Thr Pro Phe Tyr Gln Lys Ser Ala Glu

180 185 190

Glu Ile Cys Ala Leu Phe Asp Ser Val Tyr Val Ser Phe Tyr Lys Gly

195 200 205

Ile Gly Gly Ile Ala Gly Ala Ile Leu Ala Gly Asn Asp Asp Phe Val

210 215 220

Gln Glu Ala Lys Ile Trp Lys Arg Arg Tyr Gly Gly Asp Leu Ile Ser

225 230 235 240

Leu Tyr Pro Tyr Ile Leu Ser Ala Asp Tyr Tyr Phe Glu Lys Arg Ile

245 250 255

Gly Lys Met Ala Glu Tyr Phe Glu Ala Ala Lys Gly Leu Ala Glu Arg

260 265 270

Phe Asn Ser Cys Ser Gly Val Lys Thr Val Pro Glu Val Pro Val Ser

275 280 285

Asn Met Phe His Val Tyr Phe Glu Asn Ser Ala Asp Glu Ile Gly Ala

290 295 300

Ile Leu Thr Lys Ile Gln Asp Glu Thr Gly Val Gly Ile Ser Gly Tyr

305 310 315 320

Leu Gln Glu Lys Ser Ala Asp Val Cys Ala Phe Glu Val Ser Val Gly

325 330 335

Asp Ala Phe Ala Glu Ile Pro Ala Lys Asn Leu Glu Leu Val Phe Arg

340 345 350

Cys Leu Glu Lys Glu Leu

355

<210> 46

<211> 358

<212> PRT

<400> 46

Met Thr Asn Thr Leu Lys Thr Ser Tyr Gln Lys Thr Pro Tyr Lys Leu

1 5 10 15

Gly Gly Asn Gly Pro Arg Asn Val Gly Val Leu Thr Glu Ala Leu Gln

20 25 30

Asn Ile Asp Asp Asn Leu Glu Ser Asp Ile Tyr Gly Asn Gly Ala Val

35 40 45

Ile Glu Asp Phe Glu Thr Lys Ile Ala Lys Ile Leu Gly Lys Gln Ser

50 55 60

Ala Val Phe Phe Pro Ser Gly Thr Met Ala Gln Gln Ile Ala Leu Arg

65 70 75 80

Ile Trp Ala Asp Arg Lys Glu Asn Arg Arg Val Ala Tyr His Pro Leu

85 90 95

Ser His Leu Glu Ile His Glu Gln Asp Gly Leu Lys Glu Leu Gln Gln

100 105 110

Ile Thr Pro Leu Leu Leu Gly Thr Ala Asn Gln Leu Leu Thr Ile Asp

115 120 125

Asp Ile Lys Ser Leu Arg Glu Pro Val Ser Ser Val Leu Ile Glu Leu

130 135 140

Pro Gln Arg Glu Ile Gly Gly Gln Leu Pro Ala Phe Glu Glu Leu Glu

145 150 155 160

Lys Ile Ser Glu Tyr Cys His Glu Gln Gly Ile Ser Leu His Leu Asp

165 170 175

Gly Tyr Arg Leu Trp Glu Ile Thr Pro Phe Tyr Gln Lys Ser Ala Glu

180 185 190

Glu Ile Cys Ala Leu Phe Asp Ser Val Tyr Val Ser Phe Tyr Lys Gly

195 200 205

Ile Gly Gly Ile Ala Gly Ala Ile Leu Ala Gly Asn Asp Asp Phe Val

210 215 220

Gln Glu Ala Lys Ile Trp Lys Arg Arg Tyr Gly Gly Asp Leu Ile Ser

225 230 235 240

Leu Tyr Pro Tyr Ile Leu Ser Ala Asp Tyr Tyr Phe Glu Lys Arg Ile

245 250 255

Gly Lys Met Ala Glu Tyr Phe Glu Ala Ala Lys Gly Leu Ala Glu Arg

260 265 270

Phe Asn Ser Cys Ser Gly Val Lys Thr Val Pro Glu Val Pro Val Ser

275 280 285

Asn Met Phe His Val Tyr Phe Glu Asn Ser Ala Asp Glu Ile Gly Ala

290 295 300

Ile Leu Thr Lys Ile Gln Asp Glu Thr Gly Val Gly Ile Ser Gly Tyr

305 310 315 320

Leu Gln Glu Lys Ser Ala Asp Val Cys Ala Phe Glu Val Ser Val Gly

325 330 335

Asp Ala Phe Ala Glu Ile Pro Ala Lys Asn Leu Glu Leu Val Phe Arg

340 345 350

Cys Leu Glu Lys Glu Leu

355

<210> 47

<211> 358

<212> PRT

<400> 47

Met Thr Asn Thr Leu Lys Thr Ser Tyr Gln Lys Thr Pro Tyr Lys Leu

1 5 10 15

Gly Gly Asn Gly Pro Arg Asn Val Gly Val Leu Thr Glu Ala Leu Gln

20 25 30

Asn Ile Asp Asp Asn Leu Glu Ser Asp Ile Tyr Gly Asn Gly Ala Val

35 40 45

Ile Glu Asp Phe Glu Thr Lys Ile Ala Lys Ile Leu Gly Lys Gln Ser

50 55 60

Ala Val Phe Phe Pro Ser Gly Thr Met Ala Gln Gln Ile Ala Leu Arg

65 70 75 80

Ile Trp Ala Asp Arg Lys Glu Asn Arg Arg Val Ala Tyr His Pro Leu

85 90 95

Ser His Leu Glu Ile His Glu Gln Asp Gly Leu Lys Glu Leu Gln Gln

100 105 110

Ile Thr Pro Leu Leu Leu Gly Thr Ala Asn Gln Leu Leu Thr Ile Asp

115 120 125

Asp Ile Lys Ser Leu Arg Glu Pro Val Ser Ser Val Leu Ile Glu Leu

130 135 140

Pro Gln Arg Glu Ile Gly Gly Gln Leu Pro Ala Phe Glu Glu Leu Glu

145 150 155 160

Lys Ile Ser Glu Tyr Cys His Glu Gln Gly Ile Ser Leu His Leu Asp

165 170 175

Gly Ala Arg Leu Trp Glu Ile Thr Pro Phe Tyr Gln Lys Ser Ala Glu

180 185 190

Glu Ile Cys Ala Leu Phe Asp Ser Val Tyr Val Ser Phe Ile Lys Gly

195 200 205

Ile Gly Gly Ile Ala Gly Ala Ile Leu Ala Gly Asn Asp Asp Phe Val

210 215 220

Gln Glu Ala Lys Ile Trp Lys Arg Arg Tyr Gly Gly Asp Leu Ile Ser

225 230 235 240

Leu Tyr Pro Tyr Ile Leu Ser Ala Asp Tyr Tyr Phe Glu Lys Arg Ile

245 250 255

Gly Lys Met Ala Glu Tyr Phe Glu Ala Ala Lys Gly Leu Ala Glu Arg

260 265 270

Phe Asn Ser Cys Ser Gly Val Lys Thr Val Pro Glu Val Pro Val Ser

275 280 285

Asn Met Phe His Val Tyr Phe Glu Asn Ser Ala Asp Glu Ile Gly Ala

290 295 300

Ile Leu Thr Lys Ile Gln Asp Glu Thr Gly Val Gly Ile Ser Gly Tyr

305 310 315 320

Leu Gln Glu Lys Ser Ala Asp Val Cys Ala Phe Glu Val Ser Val Gly

325 330 335

Asp Ala Phe Ala Glu Ile Pro Ala Lys Asn Leu Glu Leu Val Phe Arg

340 345 350

Cys Leu Glu Lys Glu Leu

355

<210> 48

<211> 358

<212> PRT

<400> 48

Met Thr Asn Thr Leu Lys Thr Ser Tyr Gln Lys Thr Pro Tyr Lys Leu

1 5 10 15

Gly Gly Asn Gly Pro Arg Asn Val Gly Val Leu Thr Glu Ala Leu Gln

20 25 30

Asn Ile Asp Asp Asn Leu Glu Ser Asp Ile Tyr Gly Asn Gly Ala Val

35 40 45

Ile Glu Asp Phe Glu Thr Lys Ile Ala Lys Ile Leu Gly Lys Gln Ser

50 55 60

Ala Val Phe Phe Pro Ser Gly Thr Met Ala Gln Gln Ile Ala Leu Arg

65 70 75 80

Ile Trp Ala Asp Arg Lys Glu Asn Arg Arg Val Ala Tyr His Pro Leu

85 90 95

Ser His Leu Glu Ile His Glu Gln Asp Gly Leu Lys Glu Leu Gln Gln

100 105 110

Ile Thr Pro Leu Leu Leu Gly Thr Ala Asn Gln Leu Leu Thr Ile Asp

115 120 125

Asp Ile Lys Ser Leu Arg Glu Pro Val Ser Ser Val Leu Ile Glu Leu

130 135 140

Pro Gln Arg Glu Ile Gly Gly Gln Leu Pro Ala Phe Glu Glu Leu Glu

145 150 155 160

Lys Ile Ser Glu Tyr Cys His Glu Gln Gly Ile Ser Leu His Leu Asp

165 170 175

Gly Ala Arg Leu Trp Glu Ile Thr Pro Phe Tyr Gln Lys Ser Ala Glu

180 185 190

Glu Ile Cys Ala Leu Phe Asp Ser Val Tyr Val Ser Phe Ser Lys Gly

195 200 205

Ile Gly Gly Ile Ala Gly Ala Ile Leu Ala Gly Asn Asp Asp Phe Val

210 215 220

Gln Glu Ala Lys Ile Trp Lys Arg Arg Tyr Gly Gly Asp Leu Ile Ser

225 230 235 240

Leu Tyr Pro Tyr Ile Leu Ser Ala Asp Tyr Tyr Phe Glu Lys Arg Ile

245 250 255

Gly Lys Met Ala Glu Tyr Phe Glu Ala Ala Lys Gly Leu Ala Glu Arg

260 265 270

Phe Asn Ser Cys Ser Gly Val Lys Thr Val Pro Glu Val Pro Val Ser

275 280 285

Asn Met Phe His Val Tyr Phe Glu Asn Ser Ala Asp Glu Ile Gly Ala

290 295 300

Ile Leu Thr Lys Ile Gln Asp Glu Thr Gly Val Gly Ile Ser Gly Tyr

305 310 315 320

Leu Gln Glu Lys Ser Ala Asp Val Cys Ala Phe Glu Val Ser Val Gly

325 330 335

Asp Ala Phe Ala Glu Ile Pro Ala Lys Asn Leu Glu Leu Val Phe Arg

340 345 350

Cys Leu Glu Lys Glu Leu

355

<210> 49

<211> 333

<212> PRT

<213> threonine aldolase amino acid mutation in Escherichia coli (Artificial sequence)

<400> 49

Met Ile Asp Leu Arg Ser Asp Gln Val Thr Arg Pro Ser Arg Ala Met

1 5 10 15

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

20 25 30

Asp Pro Thr Val Asn Ala Leu Gln Asp Tyr Ala Ala Glu Leu Ser Gly

35 40 45

Lys Glu Ala Ala Ile Phe Leu Pro Thr Gly Thr Gln Ala Asn Leu Val

50 55 60

Ala Leu Leu Ser His Cys Glu Arg Gly Glu Glu Tyr Ile Val Gly Gln

65 70 75 80

Ala Ala His Asn Tyr Leu Phe Glu Ala Gly Gly Ala Ala Val Leu Gly

85 90 95

Ser Ile Gln Pro Gln Pro Ile Asp Ala Ala Ala Asp Gly Thr Leu Pro

100 105 110

Leu Asp Lys Val Ala Met Lys Ile Lys Pro Asp Asp Ile His Phe Ala

115 120 125

Arg Thr Lys Leu Leu Ser Leu Glu Asn Thr His Asn Gly Lys Val Leu

130 135 140

Pro Arg Glu Tyr Leu Lys Glu Ala Trp Glu Phe Thr Arg Lys Arg Asn

145 150 155 160

Leu Ala Leu His Val Asp Gly Ala Arg Ile Phe Asn Ala Val Val Ala

165 170 175

Tyr Gly Cys Glu Leu Lys Glu Ile Thr Gln Tyr Cys Asp Ser Phe Thr

180 185 190

Ile Cys Leu Ser Lys Gly Leu Gly Thr Pro Val Gly Ser Leu Leu Val

195 200 205

Gly Asn Arg Asp Tyr Ile Lys Arg Ala Ile Arg Trp Arg Lys Met Thr

210 215 220

Gly Gly Gly Met Arg Gln Ser Gly Ile Leu Ala Ala Ala Gly Met Tyr

225 230 235 240

Ala Leu Lys Asn Asn Val Ala Arg Leu Gln Glu Asp His Asp Asn Thr

245 250 255

Ala Trp Met Ala Glu Gln Leu Arg Glu Ala Gly Ala Asp Val Met Arg

260 265 270

Gln Asp Thr Asn Met Leu Phe Val Arg Val Gly Glu Glu Asn Ala Ala

275 280 285

Ala Leu Gly Glu Tyr Met Lys Ala Arg Asn Val Leu Ile Asn Ala Ser

290 295 300

Pro Ile Val Arg Leu Val Thr His Leu Asp Val Ser Arg Ala Gln Leu

305 310 315 320

Ala Glu Val Ala Ala His Trp Arg Ala Phe Leu Ala Arg

325 330

<210> 50

<211> 333

<212> PRT

<400> 50

Met Ile Asp Leu Arg Ser Asp Thr Val Thr Arg Pro Ser Arg Ala Met

1 5 10 15

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

20 25 30

Asp Pro Thr Val Asn Ala Leu Gln Asp Tyr Ala Ala Glu Leu Ser Gly

35 40 45

Lys Glu Ala Ala Ile Phe Leu Pro Thr Gly Thr Gln Ala Asn Leu Val

50 55 60

Ala Leu Leu Ser His Cys Glu Arg Gly Glu Glu Tyr Ile Val Gly Gln

65 70 75 80

Ala Ala Trp Asn Tyr Leu Phe Glu Ala Gly Gly Ala Ala Val Leu Gly

85 90 95

Ser Ile Gln Pro Gln Pro Ile Asp Ala Ala Ala Asp Gly Thr Leu Pro

100 105 110

Leu Asp Lys Val Ala Met Lys Ile Lys Pro Asp Asp Ile His Phe Ala

115 120 125

Arg Thr Lys Leu Leu Ser Leu Glu Asn Thr His Asn Gly Lys Val Leu

130 135 140

Pro Arg Glu Tyr Leu Lys Glu Ala Trp Glu Phe Thr Arg Lys Arg Asn

145 150 155 160

Leu Ala Leu His Val Asp Gly Ala Arg Ile Phe Asn Ala Val Val Ala

165 170 175

Tyr Gly Cys Glu Leu Lys Glu Ile Thr Gln Tyr Cys Asp Ser Phe Thr

180 185 190

Ile Cys Leu Ser Lys Gly Leu Gly Thr Pro Val Gly Ser Leu Leu Val

195 200 205

Gly Asn Arg Asp Tyr Ile Lys Arg Ala Ile Arg Trp Arg Lys Met Thr

210 215 220

Gly Gly Gly Met Arg Gln Ser Gly Ile Leu Ala Ala Ala Gly Met Tyr

225 230 235 240

Ala Leu Lys Asn Asn Val Ala Arg Leu Gln Glu Asp His Asp Asn Thr

245 250 255

Ala Trp Met Ala Glu Gln Leu Arg Glu Ala Gly Ala Asp Val Met Arg

260 265 270

Gln Asp Thr Asn Met Leu Phe Val Arg Val Gly Glu Glu Asn Ala Ala

275 280 285

Ala Leu Gly Glu Tyr Met Lys Ala Arg Asn Val Leu Ile Asn Ala Ser

290 295 300

Pro Ile Val Arg Leu Val Thr His Leu Asp Val Ser Arg Ala Gln Leu

305 310 315 320

Ala Glu Val Ala Ala His Trp Arg Ala Phe Leu Ala Arg

325 330

<210> 51

<211> 333

<212> PRT

<400> 51

Met Ile Asp Leu Arg Ser Asp Thr Val Thr Arg Pro Ser Arg Ala Met

1 5 10 15

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

20 25 30

Asp Pro Thr Val Asn Ala Leu Gln Asp Tyr Ala Ala Glu Leu Ser Gly

35 40 45

Lys Glu Ala Ala Ile Phe Leu Pro Thr Gly Thr Gln Ala Asn Leu Val

50 55 60

Ala Leu Leu Ser His Cys Glu Arg Gly Glu Glu Tyr Ile Val Gly Gln

65 70 75 80

Ala Ala Pro Asn Tyr Leu Phe Glu Ala Gly Gly Ala Ala Val Leu Gly

85 90 95

Ser Ile Gln Pro Gln Pro Ile Asp Ala Ala Ala Asp Gly Thr Leu Pro

100 105 110

Leu Asp Lys Val Ala Met Lys Ile Lys Pro Asp Asp Ile His Phe Ala

115 120 125

Arg Thr Lys Leu Leu Ser Leu Glu Asn Thr His Asn Gly Lys Val Leu

130 135 140

Pro Arg Glu Tyr Leu Lys Glu Ala Trp Glu Phe Thr Arg Lys Arg Asn

145 150 155 160

Leu Ala Leu His Val Asp Gly Ala Arg Ile Phe Asn Ala Val Val Ala

165 170 175

Tyr Gly Cys Glu Leu Lys Glu Ile Thr Gln Tyr Cys Asp Ser Phe Thr

180 185 190

Ile Cys Leu Ser Lys Gly Leu Gly Thr Pro Val Gly Ser Leu Leu Val

195 200 205

Gly Asn Arg Asp Tyr Ile Lys Arg Ala Ile Arg Trp Arg Lys Met Thr

210 215 220

Gly Gly Gly Met Arg Gln Ser Gly Ile Leu Ala Ala Ala Gly Met Tyr

225 230 235 240

Ala Leu Lys Asn Asn Val Ala Arg Leu Gln Glu Asp His Asp Asn Thr

245 250 255

Ala Trp Met Ala Glu Gln Leu Arg Glu Ala Gly Ala Asp Val Met Arg

260 265 270

Gln Asp Thr Asn Met Leu Phe Val Arg Val Gly Glu Glu Asn Ala Ala

275 280 285

Ala Leu Gly Glu Tyr Met Lys Ala Arg Asn Val Leu Ile Asn Ala Ser

290 295 300

Pro Ile Val Arg Leu Val Thr His Leu Asp Val Ser Arg Ala Gln Leu

305 310 315 320

Ala Glu Val Ala Ala His Trp Arg Ala Phe Leu Ala Arg

325 330

<210> 52

<211> 333

<212> PRT

<400> 52

Met Ile Asp Leu Arg Ser Asp Thr Val Thr Arg Pro Ser Arg Ala Met

1 5 10 15

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

20 25 30

Asp Pro Thr Val Asn Ala Leu Gln Asp Tyr Ala Ala Glu Leu Ser Gly

35 40 45

Lys Glu Ala Ala Ile Phe Leu Pro Thr Gly Thr Gln Ala Asn Leu Val

50 55 60

Ala Leu Leu Ser His Cys Glu Arg Gly Glu Glu Tyr Ile Val Gly Gln

65 70 75 80

Ala Ala His Asn Tyr Leu Phe Glu Ala Gly Gly Ala Ala Val Leu Gly

85 90 95

Ser Ile Gln Pro Gln Pro Ile Asp Ala Ala Ala Asp Gly Thr Leu Pro

100 105 110

Leu Asp Lys Val Ala Met Lys Ile Lys Pro Asp Asp Ile His Phe Ala

115 120 125

Arg Thr Lys Leu Leu Ser Leu Glu Asn Thr His Asn Gly Lys Val Leu

130 135 140

Pro Arg Glu Tyr Leu Lys Glu Ala Trp Glu Phe Thr Arg Lys Arg Asn

145 150 155 160

Leu Ala Leu His Val Asp Gly Tyr Arg Ile Phe Asn Ala Val Val Ala

165 170 175

Tyr Gly Cys Glu Leu Lys Glu Ile Thr Gln Tyr Cys Asp Ser Phe Thr

180 185 190

Ile Cys Leu Ser Lys Gly Leu Gly Thr Pro Val Gly Ser Leu Leu Val

195 200 205

Gly Asn Arg Asp Tyr Ile Lys Arg Ala Ile Arg Trp Arg Lys Met Thr

210 215 220

Gly Gly Gly Met Arg Gln Ser Gly Ile Leu Ala Ala Ala Gly Met Tyr

225 230 235 240

Ala Leu Lys Asn Asn Val Ala Arg Leu Gln Glu Asp His Asp Asn Thr

245 250 255

Ala Trp Met Ala Glu Gln Leu Arg Glu Ala Gly Ala Asp Val Met Arg

260 265 270

Gln Asp Thr Asn Met Leu Phe Val Arg Val Gly Glu Glu Asn Ala Ala

275 280 285

Ala Leu Gly Glu Tyr Met Lys Ala Arg Asn Val Leu Ile Asn Ala Ser

290 295 300

Pro Ile Val Arg Leu Val Thr His Leu Asp Val Ser Arg Ala Gln Leu

305 310 315 320

Ala Glu Val Ala Ala His Trp Arg Ala Phe Leu Ala Arg

325 330

<210> 53

<211> 333

<212> PRT

<400> 53

Met Ile Asp Leu Arg Ser Asp Thr Val Thr Arg Pro Ser Arg Ala Met

1 5 10 15

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

20 25 30

Asp Pro Thr Val Asn Ala Leu Gln Asp Tyr Ala Ala Glu Leu Ser Gly

35 40 45

Lys Glu Ala Ala Ile Phe Leu Pro Thr Gly Thr Gln Ala Asn Leu Val

50 55 60

Ala Leu Leu Ser His Cys Glu Arg Gly Glu Glu Tyr Ile Val Gly Gln

65 70 75 80

Ala Ala His Asn Tyr Leu Phe Glu Ala Gly Gly Ala Ala Val Leu Gly

85 90 95

Ser Ile Gln Pro Gln Pro Ile Asp Ala Ala Ala Asp Gly Thr Leu Pro

100 105 110

Leu Asp Lys Val Ala Met Lys Ile Lys Pro Asp Asp Ile His Phe Ala

115 120 125

Arg Thr Lys Leu Leu Ser Leu Glu Asn Thr His Asn Gly Lys Val Leu

130 135 140

Pro Arg Glu Tyr Leu Lys Glu Ala Trp Glu Phe Thr Arg Lys Arg Asn

145 150 155 160

Leu Ala Leu His Val Asp Gly Ala Arg Ile Phe Asn Ala Val Val Ala

165 170 175

Tyr Gly Cys Glu Leu Lys Glu Ile Thr Gln Tyr Cys Asp Ser Phe Thr

180 185 190

Ile Cys Leu Ile Lys Gly Leu Gly Thr Pro Val Gly Ser Leu Leu Val

195 200 205

Gly Asn Arg Asp Tyr Ile Lys Arg Ala Ile Arg Trp Arg Lys Met Thr

210 215 220

Gly Gly Gly Met Arg Gln Ser Gly Ile Leu Ala Ala Ala Gly Met Tyr

225 230 235 240

Ala Leu Lys Asn Asn Val Ala Arg Leu Gln Glu Asp His Asp Asn Thr

245 250 255

Ala Trp Met Ala Glu Gln Leu Arg Glu Ala Gly Ala Asp Val Met Arg

260 265 270

Gln Asp Thr Asn Met Leu Phe Val Arg Val Gly Glu Glu Asn Ala Ala

275 280 285

Ala Leu Gly Glu Tyr Met Lys Ala Arg Asn Val Leu Ile Asn Ala Ser

290 295 300

Pro Ile Val Arg Leu Val Thr His Leu Asp Val Ser Arg Ala Gln Leu

305 310 315 320

Ala Glu Val Ala Ala His Trp Arg Ala Phe Leu Ala Arg

325 330

<210> 54

<211> 338

<212> PRT

<213> Aeromonas simplicifolia threonine aldolase amino acid mutation (artificial sequence)

<400> 54

Met Arg Tyr Ile Asp Leu Arg Ser Asp Gln Val Thr Gln Pro Thr Asp

1 5 10 15

Ala Met Arg Gln Cys Met Leu His Ala Glu Val Gly Asp Asp Val Tyr

20 25 30

Gly Glu Asp Pro Gly Val Asn Ala Leu Glu Ala Tyr Gly Ala Asp Leu

35 40 45

Leu Gly Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn

50 55 60

Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Val Leu

65 70 75 80

Gly Ser Ala Ala His Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val

85 90 95

Leu Gly Ser Val Ala Leu Gln Pro Val Pro Met Gln Ala Asp Gly Ser

100 105 110

Leu Ala Leu Ala Asp Val Arg Ala Ala Ile Ala Pro Asp Asp Val His

115 120 125

Phe Thr Pro Thr Arg Leu Val Cys Leu Glu Asn Thr His Asn Gly Lys

130 135 140

Val Leu Pro Leu Pro Tyr Leu Arg Glu Met Arg Glu Leu Val Asp Glu

145 150 155 160

His Gly Leu Gln Leu His Leu Asp Gly Ala Arg Leu Phe Asn Ala Val

165 170 175

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

180 185 190

Val Ser Ile Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu

195 200 205

Leu Val Gly Ser His Ala Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys

210 215 220

Met Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly

225 230 235 240

Leu Phe Ala Leu Gln Gln His Val Val Arg Leu Ala Asp Asp His Arg

245 250 255

Arg Ala Arg Gln Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Arg

260 265 270

Leu Asp Leu Ala Gln Val Gln Thr Asn Met Val Phe Leu Gln Leu Thr

275 280 285

Ser Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Ala Arg Gly Ile

290 295 300

Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile

305 310 315 320

His Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu

325 330 335

Gly Ala

<210> 55

<211> 338

<212> PRT

<400> 55

Met Arg Tyr Ile Asp Leu Arg Ser Asp Thr Val Thr Gln Pro Thr Asp

1 5 10 15

Ala Met Arg Gln Cys Met Leu His Ala Glu Val Gly Asp Asp Val Tyr

20 25 30

Gly Glu Asp Pro Gly Val Asn Ala Leu Glu Ala Tyr Gly Ala Asp Leu

35 40 45

Leu Gly Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn

50 55 60

Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Val Leu

65 70 75 80

Gly Ser Ala Ala Trp Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val

85 90 95

Leu Gly Ser Val Ala Leu Gln Pro Val Pro Met Gln Ala Asp Gly Ser

100 105 110

Leu Ala Leu Ala Asp Val Arg Ala Ala Ile Ala Pro Asp Asp Val His

115 120 125

Phe Thr Pro Thr Arg Leu Val Cys Leu Glu Asn Thr His Asn Gly Lys

130 135 140

Val Leu Pro Leu Pro Tyr Leu Arg Glu Met Arg Glu Leu Val Asp Glu

145 150 155 160

His Gly Leu Gln Leu His Leu Asp Gly Ala Arg Leu Phe Asn Ala Val

165 170 175

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

180 185 190

Val Ser Ile Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu

195 200 205

Leu Val Gly Ser His Ala Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys

210 215 220

Met Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly

225 230 235 240

Leu Phe Ala Leu Gln Gln His Val Val Arg Leu Ala Asp Asp His Arg

245 250 255

Arg Ala Arg Gln Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Arg

260 265 270

Leu Asp Leu Ala Gln Val Gln Thr Asn Met Val Phe Leu Gln Leu Thr

275 280 285

Ser Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Ala Arg Gly Ile

290 295 300

Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile

305 310 315 320

His Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu

325 330 335

Gly Ala

<210> 56

<211> 338

<212> PRT

<400> 56

Met Arg Tyr Ile Asp Leu Arg Ser Asp Thr Val Thr Gln Pro Thr Asp

1 5 10 15

Ala Met Arg Gln Cys Met Leu His Ala Glu Val Gly Asp Asp Val Tyr

20 25 30

Gly Glu Asp Pro Gly Val Asn Ala Leu Glu Ala Tyr Gly Ala Asp Leu

35 40 45

Leu Gly Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn

50 55 60

Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Val Leu

65 70 75 80

Gly Ser Ala Ala Pro Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val

85 90 95

Leu Gly Ser Val Ala Leu Gln Pro Val Pro Met Gln Ala Asp Gly Ser

100 105 110

Leu Ala Leu Ala Asp Val Arg Ala Ala Ile Ala Pro Asp Asp Val His

115 120 125

Phe Thr Pro Thr Arg Leu Val Cys Leu Glu Asn Thr His Asn Gly Lys

130 135 140

Val Leu Pro Leu Pro Tyr Leu Arg Glu Met Arg Glu Leu Val Asp Glu

145 150 155 160

His Gly Leu Gln Leu His Leu Asp Gly Ala Arg Leu Phe Asn Ala Val

165 170 175

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

180 185 190

Val Ser Ile Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu

195 200 205

Leu Val Gly Ser His Ala Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys

210 215 220

Met Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly

225 230 235 240

Leu Phe Ala Leu Gln Gln His Val Val Arg Leu Ala Asp Asp His Arg

245 250 255

Arg Ala Arg Gln Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Arg

260 265 270

Leu Asp Leu Ala Gln Val Gln Thr Asn Met Val Phe Leu Gln Leu Thr

275 280 285

Ser Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Ala Arg Gly Ile

290 295 300

Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile

305 310 315 320

His Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu

325 330 335

Gly Ala

<210> 57

<211> 338

<212> PRT

<400> 57

Met Arg Tyr Ile Asp Leu Arg Ser Asp Thr Val Thr Gln Pro Thr Asp

1 5 10 15

Ala Met Arg Gln Cys Met Leu His Ala Glu Val Gly Asp Asp Val Tyr

20 25 30

Gly Glu Asp Pro Gly Val Asn Ala Leu Glu Ala Tyr Gly Ala Asp Leu

35 40 45

Leu Gly Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn

50 55 60

Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Val Leu

65 70 75 80

Gly Ser Ala Ala His Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val

85 90 95

Leu Gly Ser Val Ala Leu Gln Pro Val Pro Met Gln Ala Asp Gly Ser

100 105 110

Leu Ala Leu Ala Asp Val Arg Ala Ala Ile Ala Pro Asp Asp Val His

115 120 125

Phe Thr Pro Thr Arg Leu Val Cys Leu Glu Asn Thr His Asn Gly Lys

130 135 140

Val Leu Pro Leu Pro Tyr Leu Arg Glu Met Arg Glu Leu Val Asp Glu

145 150 155 160

His Gly Leu Gln Leu His Leu Asp Gly Tyr Arg Leu Phe Asn Ala Val

165 170 175

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

180 185 190

Val Ser Ile Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu

195 200 205

Leu Val Gly Ser His Ala Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys

210 215 220

Met Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly

225 230 235 240

Leu Phe Ala Leu Gln Gln His Val Val Arg Leu Ala Asp Asp His Arg

245 250 255

Arg Ala Arg Gln Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Arg

260 265 270

Leu Asp Leu Ala Gln Val Gln Thr Asn Met Val Phe Leu Gln Leu Thr

275 280 285

Ser Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Ala Arg Gly Ile

290 295 300

Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile

305 310 315 320

His Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu

325 330 335

Gly Ala

<210> 58

<211> 338

<212> PRT

<400> 58

Met Arg Tyr Ile Asp Leu Arg Ser Asp Thr Val Thr Gln Pro Thr Asp

1 5 10 15

Ala Met Arg Gln Cys Met Leu His Ala Glu Val Gly Asp Asp Val Tyr

20 25 30

Gly Glu Asp Pro Gly Val Asn Ala Leu Glu Ala Tyr Gly Ala Asp Leu

35 40 45

Leu Gly Lys Glu Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn

50 55 60

Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Val Leu

65 70 75 80

Gly Ser Ala Ala His Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val

85 90 95

Leu Gly Ser Val Ala Leu Gln Pro Val Pro Met Gln Ala Asp Gly Ser

100 105 110

Leu Ala Leu Ala Asp Val Arg Ala Ala Ile Ala Pro Asp Asp Val His

115 120 125

Phe Thr Pro Thr Arg Leu Val Cys Leu Glu Asn Thr His Asn Gly Lys

130 135 140

Val Leu Pro Leu Pro Tyr Leu Arg Glu Met Arg Glu Leu Val Asp Glu

145 150 155 160

His Gly Leu Gln Leu His Leu Asp Gly Ala Arg Leu Phe Asn Ala Val

165 170 175

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

180 185 190

Val Ser Ile Cys Leu Ile Lys Gly Leu Gly Ala Pro Val Gly Ser Leu

195 200 205

Leu Val Gly Ser His Ala Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys

210 215 220

Met Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly

225 230 235 240

Leu Phe Ala Leu Gln Gln His Val Val Arg Leu Ala Asp Asp His Arg

245 250 255

Arg Ala Arg Gln Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Arg

260 265 270

Leu Asp Leu Ala Gln Val Gln Thr Asn Met Val Phe Leu Gln Leu Thr

275 280 285

Ser Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Ala Arg Gly Ile

290 295 300

Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile

305 310 315 320

His Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu

325 330 335

Gly Ala

<210> 59

<211> 387

<212> PRT

<213> threonine aldolase amino acid mutation (artificial sequence) of Saccharomyces cerevisiae

<400> 59

Met Thr Glu Phe Glu Leu Pro Pro Lys Tyr Ile Thr Ala Ala Asn Asp

1 5 10 15

Leu Arg Ser Asp Gln Phe Thr Thr Pro Thr Ala Glu Met Met Glu Ala

20 25 30

Ala Leu Glu Ala Ser Ile Gly Asp Ala Val Tyr Gly Glu Asp Val Asp

35 40 45

Thr Val Arg Leu Glu Gln Thr Val Ala Arg Met Ala Gly Lys Glu Ala

50 55 60

Gly Leu Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Ile Arg

65 70 75 80

Thr His Leu Met Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg Ala

85 90 95

His Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln Ala

100 105 110

Met Val Val Pro Val Val Pro Ser Asn Gly Asp Tyr Leu Thr Leu Glu

115 120 125

Asp Ile Lys Ser His Tyr Val Pro Asp Asp Gly Asp Ile His Gly Ala

130 135 140

Pro Thr Arg Leu Ile Ser Leu Glu Asn Thr Leu His Gly Ile Val Tyr

145 150 155 160

Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn Gly

165 170 175

Leu Lys Leu His Cys Asp Gly Ala Arg Ile Trp Asn Ala Ala Ala Gln

180 185 190

Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Ile Phe Asp Ser Ile Ser

195 200 205

Ile Cys Leu Ser Lys Ser Met Gly Ala Pro Ile Gly Ser Val Leu Val

210 215 220

Gly Asn Leu Lys Phe Val Lys Lys Ala Thr His Phe Arg Lys Gln Gln

225 230 235 240

Gly Gly Gly Ile Arg Gln Ser Gly Met Met Ala Arg Met Ala Leu Val

245 250 255

Asn Ile Asn Asn Asp Trp Lys Ser Gln Leu Leu Tyr Ser His Ser Leu

260 265 270

Ala His Glu Leu Ala Glu Tyr Cys Glu Ala Lys Gly Ile Pro Leu Glu

275 280 285

Ser Pro Ala Asp Thr Asn Phe Val Phe Ile Asn Leu Lys Ala Ala Arg

290 295 300

Met Asp Pro Asp Val Leu Val Lys Lys Gly Leu Lys Tyr Asn Val Lys

305 310 315 320

Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Val Thr Arg Asp Thr

325 330 335

Leu Glu Lys Val Lys Leu Ala Ile Ser Glu Ala Phe Asp Tyr Ala Lys

340 345 350

Glu His Pro Phe Asp Cys Asn Gly Pro Thr Gln Ile Tyr Arg Ser Glu

355 360 365

Ser Thr Glu Val Asp Val Asp Gly Asn Ala Ile Arg Glu Ile Lys Thr

370 375 380

Tyr Lys Tyr

385

<210> 60

<211> 387

<212> PRT

<400> 60

Met Thr Glu Phe Glu Leu Pro Pro Lys Tyr Ile Thr Ala Ala Asn Asp

1 5 10 15

Leu Arg Ser Asp Thr Phe Thr Thr Pro Thr Ala Glu Met Met Glu Ala

20 25 30

Ala Leu Glu Ala Ser Ile Gly Asp Ala Val Tyr Gly Glu Asp Val Asp

35 40 45

Thr Val Arg Leu Glu Gln Thr Val Ala Arg Met Ala Gly Lys Glu Ala

50 55 60

Gly Leu Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Ile Arg

65 70 75 80

Thr His Leu Met Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg Ala

85 90 95

Trp Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln Ala

100 105 110

Met Val Val Pro Val Val Pro Ser Asn Gly Asp Tyr Leu Thr Leu Glu

115 120 125

Asp Ile Lys Ser His Tyr Val Pro Asp Asp Gly Asp Ile His Gly Ala

130 135 140

Pro Thr Arg Leu Ile Ser Leu Glu Asn Thr Leu His Gly Ile Val Tyr

145 150 155 160

Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn Gly

165 170 175

Leu Lys Leu His Cys Asp Gly Ala Arg Ile Trp Asn Ala Ala Ala Gln

180 185 190

Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Ile Phe Asp Ser Ile Ser

195 200 205

Ile Cys Leu Ser Lys Ser Met Gly Ala Pro Ile Gly Ser Val Leu Val

210 215 220

Gly Asn Leu Lys Phe Val Lys Lys Ala Thr His Phe Arg Lys Gln Gln

225 230 235 240

Gly Gly Gly Ile Arg Gln Ser Gly Met Met Ala Arg Met Ala Leu Val

245 250 255

Asn Ile Asn Asn Asp Trp Lys Ser Gln Leu Leu Tyr Ser His Ser Leu

260 265 270

Ala His Glu Leu Ala Glu Tyr Cys Glu Ala Lys Gly Ile Pro Leu Glu

275 280 285

Ser Pro Ala Asp Thr Asn Phe Val Phe Ile Asn Leu Lys Ala Ala Arg

290 295 300

Met Asp Pro Asp Val Leu Val Lys Lys Gly Leu Lys Tyr Asn Val Lys

305 310 315 320

Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Val Thr Arg Asp Thr

325 330 335

Leu Glu Lys Val Lys Leu Ala Ile Ser Glu Ala Phe Asp Tyr Ala Lys

340 345 350

Glu His Pro Phe Asp Cys Asn Gly Pro Thr Gln Ile Tyr Arg Ser Glu

355 360 365

Ser Thr Glu Val Asp Val Asp Gly Asn Ala Ile Arg Glu Ile Lys Thr

370 375 380

Tyr Lys Tyr

385

<210> 61

<211> 387

<212> PRT

<400> 61

Met Thr Glu Phe Glu Leu Pro Pro Lys Tyr Ile Thr Ala Ala Asn Asp

1 5 10 15

Leu Arg Ser Asp Thr Phe Thr Thr Pro Thr Ala Glu Met Met Glu Ala

20 25 30

Ala Leu Glu Ala Ser Ile Gly Asp Ala Val Tyr Gly Glu Asp Val Asp

35 40 45

Thr Val Arg Leu Glu Gln Thr Val Ala Arg Met Ala Gly Lys Glu Ala

50 55 60

Gly Leu Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Ile Arg

65 70 75 80

Thr His Leu Met Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg Ala

85 90 95

Pro Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln Ala

100 105 110

Met Val Val Pro Val Val Pro Ser Asn Gly Asp Tyr Leu Thr Leu Glu

115 120 125

Asp Ile Lys Ser His Tyr Val Pro Asp Asp Gly Asp Ile His Gly Ala

130 135 140

Pro Thr Arg Leu Ile Ser Leu Glu Asn Thr Leu His Gly Ile Val Tyr

145 150 155 160

Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn Gly

165 170 175

Leu Lys Leu His Cys Asp Gly Ala Arg Ile Trp Asn Ala Ala Ala Gln

180 185 190

Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Ile Phe Asp Ser Ile Ser

195 200 205

Ile Cys Leu Ser Lys Ser Met Gly Ala Pro Ile Gly Ser Val Leu Val

210 215 220

Gly Asn Leu Lys Phe Val Lys Lys Ala Thr His Phe Arg Lys Gln Gln

225 230 235 240

Gly Gly Gly Ile Arg Gln Ser Gly Met Met Ala Arg Met Ala Leu Val

245 250 255

Asn Ile Asn Asn Asp Trp Lys Ser Gln Leu Leu Tyr Ser His Ser Leu

260 265 270

Ala His Glu Leu Ala Glu Tyr Cys Glu Ala Lys Gly Ile Pro Leu Glu

275 280 285

Ser Pro Ala Asp Thr Asn Phe Val Phe Ile Asn Leu Lys Ala Ala Arg

290 295 300

Met Asp Pro Asp Val Leu Val Lys Lys Gly Leu Lys Tyr Asn Val Lys

305 310 315 320

Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Val Thr Arg Asp Thr

325 330 335

Leu Glu Lys Val Lys Leu Ala Ile Ser Glu Ala Phe Asp Tyr Ala Lys

340 345 350

Glu His Pro Phe Asp Cys Asn Gly Pro Thr Gln Ile Tyr Arg Ser Glu

355 360 365

Ser Thr Glu Val Asp Val Asp Gly Asn Ala Ile Arg Glu Ile Lys Thr

370 375 380

Tyr Lys Tyr

385

<210> 62

<211> 387

<212> PRT

<400> 62

Met Thr Glu Phe Glu Leu Pro Pro Lys Tyr Ile Thr Ala Ala Asn Asp

1 5 10 15

Leu Arg Ser Asp Thr Phe Thr Thr Pro Thr Ala Glu Met Met Glu Ala

20 25 30

Ala Leu Glu Ala Ser Ile Gly Asp Ala Val Tyr Gly Glu Asp Val Asp

35 40 45

Thr Val Arg Leu Glu Gln Thr Val Ala Arg Met Ala Gly Lys Glu Ala

50 55 60

Gly Leu Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Ile Arg

65 70 75 80

Thr His Leu Met Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg Ala

85 90 95

His Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln Ala

100 105 110

Met Val Val Pro Val Val Pro Ser Asn Gly Asp Tyr Leu Thr Leu Glu

115 120 125

Asp Ile Lys Ser His Tyr Val Pro Asp Asp Gly Asp Ile His Gly Ala

130 135 140

Pro Thr Arg Leu Ile Ser Leu Glu Asn Thr Leu His Gly Ile Val Tyr

145 150 155 160

Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn Gly

165 170 175

Leu Lys Leu His Cys Asp Gly Tyr Arg Ile Trp Asn Ala Ala Ala Gln

180 185 190

Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Ile Phe Asp Ser Ile Ser

195 200 205

Ile Cys Leu Ser Lys Ser Met Gly Ala Pro Ile Gly Ser Val Leu Val

210 215 220

Gly Asn Leu Lys Phe Val Lys Lys Ala Thr His Phe Arg Lys Gln Gln

225 230 235 240

Gly Gly Gly Ile Arg Gln Ser Gly Met Met Ala Arg Met Ala Leu Val

245 250 255

Asn Ile Asn Asn Asp Trp Lys Ser Gln Leu Leu Tyr Ser His Ser Leu

260 265 270

Ala His Glu Leu Ala Glu Tyr Cys Glu Ala Lys Gly Ile Pro Leu Glu

275 280 285

Ser Pro Ala Asp Thr Asn Phe Val Phe Ile Asn Leu Lys Ala Ala Arg

290 295 300

Met Asp Pro Asp Val Leu Val Lys Lys Gly Leu Lys Tyr Asn Val Lys

305 310 315 320

Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Val Thr Arg Asp Thr

325 330 335

Leu Glu Lys Val Lys Leu Ala Ile Ser Glu Ala Phe Asp Tyr Ala Lys

340 345 350

Glu His Pro Phe Asp Cys Asn Gly Pro Thr Gln Ile Tyr Arg Ser Glu

355 360 365

Ser Thr Glu Val Asp Val Asp Gly Asn Ala Ile Arg Glu Ile Lys Thr

370 375 380

Tyr Lys Tyr

385

<210> 63

<211> 387

<212> PRT

<400> 63

Met Thr Glu Phe Glu Leu Pro Pro Lys Tyr Ile Thr Ala Ala Asn Asp

1 5 10 15

Leu Arg Ser Asp Thr Phe Thr Thr Pro Thr Ala Glu Met Met Glu Ala

20 25 30

Ala Leu Glu Ala Ser Ile Gly Asp Ala Val Tyr Gly Glu Asp Val Asp

35 40 45

Thr Val Arg Leu Glu Gln Thr Val Ala Arg Met Ala Gly Lys Glu Ala

50 55 60

Gly Leu Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Ile Arg

65 70 75 80

Thr His Leu Met Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg Ala

85 90 95

His Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln Ala

100 105 110

Met Val Val Pro Val Val Pro Ser Asn Gly Asp Tyr Leu Thr Leu Glu

115 120 125

Asp Ile Lys Ser His Tyr Val Pro Asp Asp Gly Asp Ile His Gly Ala

130 135 140

Pro Thr Arg Leu Ile Ser Leu Glu Asn Thr Leu His Gly Ile Val Tyr

145 150 155 160

Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn Gly

165 170 175

Leu Lys Leu His Cys Asp Gly Ala Arg Ile Trp Asn Ala Ala Ala Gln

180 185 190

Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Ile Phe Asp Ser Ile Ser

195 200 205

Ile Cys Leu Ile Lys Ser Met Gly Ala Pro Ile Gly Ser Val Leu Val

210 215 220

Gly Asn Leu Lys Phe Val Lys Lys Ala Thr His Phe Arg Lys Gln Gln

225 230 235 240

Gly Gly Gly Ile Arg Gln Ser Gly Met Met Ala Arg Met Ala Leu Val

245 250 255

Asn Ile Asn Asn Asp Trp Lys Ser Gln Leu Leu Tyr Ser His Ser Leu

260 265 270

Ala His Glu Leu Ala Glu Tyr Cys Glu Ala Lys Gly Ile Pro Leu Glu

275 280 285

Ser Pro Ala Asp Thr Asn Phe Val Phe Ile Asn Leu Lys Ala Ala Arg

290 295 300

Met Asp Pro Asp Val Leu Val Lys Lys Gly Leu Lys Tyr Asn Val Lys

305 310 315 320

Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Val Thr Arg Asp Thr

325 330 335

Leu Glu Lys Val Lys Leu Ala Ile Ser Glu Ala Phe Asp Tyr Ala Lys

340 345 350

Glu His Pro Phe Asp Cys Asn Gly Pro Thr Gln Ile Tyr Arg Ser Glu

355 360 365

Ser Thr Glu Val Asp Val Asp Gly Asn Ala Ile Arg Glu Ile Lys Thr

370 375 380

Tyr Lys Tyr

385

<210> 64

<211> 382

<212> PRT

<213> threonine aldolase amino acid mutation (artificial sequence) of Ashbya gossypii

<400> 64

Met Asn Gln Asp Met Glu Leu Pro Glu Ala Tyr Thr Ser Ala Ser Asn

1 5 10 15

Asp Phe Arg Ser Asp Gln Phe Thr Thr Pro Thr Arg Glu Met Ile Glu

20 25 30

Ala Ala Leu Thr Ala Thr Ile Gly Asp Ala Val Tyr Gln Glu Asp Ile

35 40 45

Asp Thr Leu Lys Leu Glu Gln His Val Ala Lys Leu Ala Gly Met Glu

50 55 60

Ala Gly Met Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Leu

65 70 75 80

Arg Thr His Leu Thr Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg

85 90 95

Ala His Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln

100 105 110

Ala Met Val Thr Pro Val Ile Pro Ser Asn Gly Asn Tyr Leu Thr Leu

115 120 125

Glu Asp Ile Lys Lys His Tyr Ile Pro Asp Asp Gly Asp Ile His Gly

130 135 140

Ala Pro Thr Lys Val Ile Ser Leu Glu Asn Thr Leu His Gly Ile Ile

145 150 155 160

His Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn

165 170 175

Asp Leu Arg Leu His Cys Asp Gly Ala Arg Ile Trp Asn Ala Ser Ala

180 185 190

Glu Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Leu Phe Asp Ser Ile

195 200 205

Ser Ile Cys Leu Ser Lys Ser Met Gly Ala Pro Met Gly Ser Ile Leu

210 215 220

Val Gly Ser His Lys Phe Ile Lys Lys Ala Asn His Phe Arg Lys Gln

225 230 235 240

Gln Gly Gly Gly Val Arg Gln Ser Gly Met Met Cys Lys Met Ala Met

245 250 255

Val Ala Ile Gln Gly Asp Trp Lys Gly Lys Met Arg Arg Ser His Arg

260 265 270

Met Ala His Glu Leu Ala Arg Phe Cys Ala Glu His Gly Ile Pro Leu

275 280 285

Glu Ser Pro Ala Asp Thr Asn Phe Val Phe Leu Asp Leu Gln Lys Ser

290 295 300

Lys Met Asn Pro Asp Val Leu Val Lys Lys Ser Leu Lys Tyr Gly Cys

305 310 315 320

Lys Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Ile Ser Glu Glu

325 330 335

Ser Leu Glu Lys Ile Lys Gln Ala Ile Leu Glu Ala Phe Glu Tyr Ser

340 345 350

Lys Lys Asn Pro Tyr Asp Glu Asn Gly Pro Thr Lys Ile Tyr Arg Ser

355 360 365

Glu Ser Ala Asp Ala Val Gly Glu Ile Lys Thr Tyr Lys Tyr

370 375 380

<210> 65

<211> 382

<212> PRT

<400> 65

Met Asn Gln Asp Met Glu Leu Pro Glu Ala Tyr Thr Ser Ala Ser Asn

1 5 10 15

Asp Phe Arg Ser Asp Thr Phe Thr Thr Pro Thr Arg Glu Met Ile Glu

20 25 30

Ala Ala Leu Thr Ala Thr Ile Gly Asp Ala Val Tyr Gln Glu Asp Ile

35 40 45

Asp Thr Leu Lys Leu Glu Gln His Val Ala Lys Leu Ala Gly Met Glu

50 55 60

Ala Gly Met Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Leu

65 70 75 80

Arg Thr His Leu Thr Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg

85 90 95

Ala Trp Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln

100 105 110

Ala Met Val Thr Pro Val Ile Pro Ser Asn Gly Asn Tyr Leu Thr Leu

115 120 125

Glu Asp Ile Lys Lys His Tyr Ile Pro Asp Asp Gly Asp Ile His Gly

130 135 140

Ala Pro Thr Lys Val Ile Ser Leu Glu Asn Thr Leu His Gly Ile Ile

145 150 155 160

His Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn

165 170 175

Asp Leu Arg Leu His Cys Asp Gly Ala Arg Ile Trp Asn Ala Ser Ala

180 185 190

Glu Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Leu Phe Asp Ser Ile

195 200 205

Ser Ile Cys Leu Ser Lys Ser Met Gly Ala Pro Met Gly Ser Ile Leu

210 215 220

Val Gly Ser His Lys Phe Ile Lys Lys Ala Asn His Phe Arg Lys Gln

225 230 235 240

Gln Gly Gly Gly Val Arg Gln Ser Gly Met Met Cys Lys Met Ala Met

245 250 255

Val Ala Ile Gln Gly Asp Trp Lys Gly Lys Met Arg Arg Ser His Arg

260 265 270

Met Ala His Glu Leu Ala Arg Phe Cys Ala Glu His Gly Ile Pro Leu

275 280 285

Glu Ser Pro Ala Asp Thr Asn Phe Val Phe Leu Asp Leu Gln Lys Ser

290 295 300

Lys Met Asn Pro Asp Val Leu Val Lys Lys Ser Leu Lys Tyr Gly Cys

305 310 315 320

Lys Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Ile Ser Glu Glu

325 330 335

Ser Leu Glu Lys Ile Lys Gln Ala Ile Leu Glu Ala Phe Glu Tyr Ser

340 345 350

Lys Lys Asn Pro Tyr Asp Glu Asn Gly Pro Thr Lys Ile Tyr Arg Ser

355 360 365

Glu Ser Ala Asp Ala Val Gly Glu Ile Lys Thr Tyr Lys Tyr

370 375 380

<210> 66

<211> 382

<212> PRT

<400> 66

Met Asn Gln Asp Met Glu Leu Pro Glu Ala Tyr Thr Ser Ala Ser Asn

1 5 10 15

Asp Phe Arg Ser Asp Thr Phe Thr Thr Pro Thr Arg Glu Met Ile Glu

20 25 30

Ala Ala Leu Thr Ala Thr Ile Gly Asp Ala Val Tyr Gln Glu Asp Ile

35 40 45

Asp Thr Leu Lys Leu Glu Gln His Val Ala Lys Leu Ala Gly Met Glu

50 55 60

Ala Gly Met Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Leu

65 70 75 80

Arg Thr His Leu Thr Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg

85 90 95

Ala Pro Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln

100 105 110

Ala Met Val Thr Pro Val Ile Pro Ser Asn Gly Asn Tyr Leu Thr Leu

115 120 125

Glu Asp Ile Lys Lys His Tyr Ile Pro Asp Asp Gly Asp Ile His Gly

130 135 140

Ala Pro Thr Lys Val Ile Ser Leu Glu Asn Thr Leu His Gly Ile Ile

145 150 155 160

His Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn

165 170 175

Asp Leu Arg Leu His Cys Asp Gly Ala Arg Ile Trp Asn Ala Ser Ala

180 185 190

Glu Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Leu Phe Asp Ser Ile

195 200 205

Ser Ile Cys Leu Ser Lys Ser Met Gly Ala Pro Met Gly Ser Ile Leu

210 215 220

Val Gly Ser His Lys Phe Ile Lys Lys Ala Asn His Phe Arg Lys Gln

225 230 235 240

Gln Gly Gly Gly Val Arg Gln Ser Gly Met Met Cys Lys Met Ala Met

245 250 255

Val Ala Ile Gln Gly Asp Trp Lys Gly Lys Met Arg Arg Ser His Arg

260 265 270

Met Ala His Glu Leu Ala Arg Phe Cys Ala Glu His Gly Ile Pro Leu

275 280 285

Glu Ser Pro Ala Asp Thr Asn Phe Val Phe Leu Asp Leu Gln Lys Ser

290 295 300

Lys Met Asn Pro Asp Val Leu Val Lys Lys Ser Leu Lys Tyr Gly Cys

305 310 315 320

Lys Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Ile Ser Glu Glu

325 330 335

Ser Leu Glu Lys Ile Lys Gln Ala Ile Leu Glu Ala Phe Glu Tyr Ser

340 345 350

Lys Lys Asn Pro Tyr Asp Glu Asn Gly Pro Thr Lys Ile Tyr Arg Ser

355 360 365

Glu Ser Ala Asp Ala Val Gly Glu Ile Lys Thr Tyr Lys Tyr

370 375 380

<210> 67

<211> 382

<212> PRT

<400> 67

Met Asn Gln Asp Met Glu Leu Pro Glu Ala Tyr Thr Ser Ala Ser Asn

1 5 10 15

Asp Phe Arg Ser Asp Thr Phe Thr Thr Pro Thr Arg Glu Met Ile Glu

20 25 30

Ala Ala Leu Thr Ala Thr Ile Gly Asp Ala Val Tyr Gln Glu Asp Ile

35 40 45

Asp Thr Leu Lys Leu Glu Gln His Val Ala Lys Leu Ala Gly Met Glu

50 55 60

Ala Gly Met Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Leu

65 70 75 80

Arg Thr His Leu Thr Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg

85 90 95

Ala His Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln

100 105 110

Ala Met Val Thr Pro Val Ile Pro Ser Asn Gly Asn Tyr Leu Thr Leu

115 120 125

Glu Asp Ile Lys Lys His Tyr Ile Pro Asp Asp Gly Asp Ile His Gly

130 135 140

Ala Pro Thr Lys Val Ile Ser Leu Glu Asn Thr Leu His Gly Ile Ile

145 150 155 160

His Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn

165 170 175

Asp Leu Arg Leu His Cys Asp Gly Tyr Arg Ile Trp Asn Ala Ser Ala

180 185 190

Glu Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Leu Phe Asp Ser Ile

195 200 205

Ser Ile Cys Leu Ser Lys Ser Met Gly Ala Pro Met Gly Ser Ile Leu

210 215 220

Val Gly Ser His Lys Phe Ile Lys Lys Ala Asn His Phe Arg Lys Gln

225 230 235 240

Gln Gly Gly Gly Val Arg Gln Ser Gly Met Met Cys Lys Met Ala Met

245 250 255

Val Ala Ile Gln Gly Asp Trp Lys Gly Lys Met Arg Arg Ser His Arg

260 265 270

Met Ala His Glu Leu Ala Arg Phe Cys Ala Glu His Gly Ile Pro Leu

275 280 285

Glu Ser Pro Ala Asp Thr Asn Phe Val Phe Leu Asp Leu Gln Lys Ser

290 295 300

Lys Met Asn Pro Asp Val Leu Val Lys Lys Ser Leu Lys Tyr Gly Cys

305 310 315 320

Lys Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Ile Ser Glu Glu

325 330 335

Ser Leu Glu Lys Ile Lys Gln Ala Ile Leu Glu Ala Phe Glu Tyr Ser

340 345 350

Lys Lys Asn Pro Tyr Asp Glu Asn Gly Pro Thr Lys Ile Tyr Arg Ser

355 360 365

Glu Ser Ala Asp Ala Val Gly Glu Ile Lys Thr Tyr Lys Tyr

370 375 380

<210> 68

<211> 382

<212> PRT

<400> 68

Met Asn Gln Asp Met Glu Leu Pro Glu Ala Tyr Thr Ser Ala Ser Asn

1 5 10 15

Asp Phe Arg Ser Asp Thr Phe Thr Thr Pro Thr Arg Glu Met Ile Glu

20 25 30

Ala Ala Leu Thr Ala Thr Ile Gly Asp Ala Val Tyr Gln Glu Asp Ile

35 40 45

Asp Thr Leu Lys Leu Glu Gln His Val Ala Lys Leu Ala Gly Met Glu

50 55 60

Ala Gly Met Phe Cys Val Ser Gly Thr Leu Ser Asn Gln Ile Ala Leu

65 70 75 80

Arg Thr His Leu Thr Gln Pro Pro Tyr Ser Ile Leu Cys Asp Tyr Arg

85 90 95

Ala His Val Tyr Thr His Glu Ala Ala Gly Leu Ala Ile Leu Ser Gln

100 105 110

Ala Met Val Thr Pro Val Ile Pro Ser Asn Gly Asn Tyr Leu Thr Leu

115 120 125

Glu Asp Ile Lys Lys His Tyr Ile Pro Asp Asp Gly Asp Ile His Gly

130 135 140

Ala Pro Thr Lys Val Ile Ser Leu Glu Asn Thr Leu His Gly Ile Ile

145 150 155 160

His Pro Leu Glu Glu Leu Val Arg Ile Lys Ala Trp Cys Met Glu Asn

165 170 175

Asp Leu Arg Leu His Cys Asp Gly Ala Arg Ile Trp Asn Ala Ser Ala

180 185 190

Glu Ser Gly Val Pro Leu Lys Gln Tyr Gly Glu Leu Phe Asp Ser Ile

195 200 205

Ser Ile Cys Leu Ile Lys Ser Met Gly Ala Pro Met Gly Ser Ile Leu

210 215 220

Val Gly Ser His Lys Phe Ile Lys Lys Ala Asn His Phe Arg Lys Gln

225 230 235 240

Gln Gly Gly Gly Val Arg Gln Ser Gly Met Met Cys Lys Met Ala Met

245 250 255

Val Ala Ile Gln Gly Asp Trp Lys Gly Lys Met Arg Arg Ser His Arg

260 265 270

Met Ala His Glu Leu Ala Arg Phe Cys Ala Glu His Gly Ile Pro Leu

275 280 285

Glu Ser Pro Ala Asp Thr Asn Phe Val Phe Leu Asp Leu Gln Lys Ser

290 295 300

Lys Met Asn Pro Asp Val Leu Val Lys Lys Ser Leu Lys Tyr Gly Cys

305 310 315 320

Lys Leu Met Gly Gly Arg Val Ser Phe His Tyr Gln Ile Ser Glu Glu

325 330 335

Ser Leu Glu Lys Ile Lys Gln Ala Ile Leu Glu Ala Phe Glu Tyr Ser

340 345 350

Lys Lys Asn Pro Tyr Asp Glu Asn Gly Pro Thr Lys Ile Tyr Arg Ser

355 360 365

Glu Ser Ala Asp Ala Val Gly Glu Ile Lys Thr Tyr Lys Tyr

370 375 380

<210> 69

<211> 335

<212> PRT

<213> threonine aldolase amino acid mutation (artificial sequence) of Shewanella baumannii

<400> 69

Met Ile Asp Phe Arg Ser Asp Gln Val Thr Gln Pro Thr Ala Ala Met

1 5 10 15

Arg Arg Ala Met Ala Asp Ala Gln Val Gly Asp Asp Val Tyr Gly Asp

20 25 30

Asp Pro Ser Val Asn Arg Leu Glu Ala Met Ala Ala Glu Arg Phe Gly

35 40 45

Phe Asp Ser Ala Leu Phe Thr Ser Ser Gly Thr Gln Ala Asn Leu Leu

50 55 60

Ala Leu Met Ser His Cys Asp Arg Gly Asp Glu Tyr Leu Cys Gly Gln

65 70 75 80

Gln Ala His Asn Tyr Lys Phe Glu Gly Gly Gly Ala Ala Val Leu Gly

85 90 95

Ser Ile Gln Pro Gln Pro Leu Thr Asn Gln Pro Asp Gly Ser Ile Leu

100 105 110

Leu Ser Asp Ile Glu Ala Ala Ile Lys Pro Asp Asp Phe His Phe Ala

115 120 125

Arg Thr Arg Leu Leu Ser Leu Glu Asn Thr Ile Gly Gly Lys Val Leu

130 135 140

Pro Gln Ser Tyr Leu Ala Glu Ala Gln Ala Leu Ala Phe Asn Lys Arg

145 150 155 160

Leu Lys Ile His Leu Asp Gly Ala Arg Ile Ala Asn Ala Ala Val Ala

165 170 175

His Asn Leu Asp Ile Ala Asp Ile Thr Gln Tyr Phe Asp Ser Val Ser

180 185 190

Ile Cys Leu Ser Lys Gly Leu Cys Ala Pro Val Gly Ser Leu Leu Leu

195 200 205

Gly Asp Glu Arg Leu Ile Asn Lys Ala Arg Arg Trp Arg Lys Met Leu

210 215 220

Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Val Ala Gly Glu Ile

225 230 235 240

Ala Leu Asn Glu Gln Val Ser Arg Leu Ala Val Asp His Glu Asn Ala

245 250 255

Arg Tyr Leu Ala Glu Gln Leu Ser Cys Leu Asp Glu Phe Glu Val Asp

260 265 270

Leu Gly Glu Val Gln Thr Asn Met Leu Phe Ala Arg Val Val Glu Gly

275 280 285

Val Ala Ile Asp Lys Leu Ala Thr Ser Leu Lys Ala Ser Gly Ile Leu

290 295 300

Ile Ser Pro Gly Lys Thr Leu Arg Met Val Thr His Ala Asp Ile Arg

305 310 315 320

Leu Glu Asp Ile Asp Leu Phe Ile Asp Lys Leu Lys Ser Leu Leu

325 330 335

<210> 70

<211> 335

<212> PRT

<400> 70

Met Ile Asp Phe Arg Ser Asp Thr Val Thr Gln Pro Thr Ala Ala Met

1 5 10 15

Arg Arg Ala Met Ala Asp Ala Gln Val Gly Asp Asp Val Tyr Gly Asp

20 25 30

Asp Pro Ser Val Asn Arg Leu Glu Ala Met Ala Ala Glu Arg Phe Gly

35 40 45

Phe Asp Ser Ala Leu Phe Thr Ser Ser Gly Thr Gln Ala Asn Leu Leu

50 55 60

Ala Leu Met Ser His Cys Asp Arg Gly Asp Glu Tyr Leu Cys Gly Gln

65 70 75 80

Gln Ala Trp Asn Tyr Lys Phe Glu Gly Gly Gly Ala Ala Val Leu Gly

85 90 95

Ser Ile Gln Pro Gln Pro Leu Thr Asn Gln Pro Asp Gly Ser Ile Leu

100 105 110

Leu Ser Asp Ile Glu Ala Ala Ile Lys Pro Asp Asp Phe His Phe Ala

115 120 125

Arg Thr Arg Leu Leu Ser Leu Glu Asn Thr Ile Gly Gly Lys Val Leu

130 135 140

Pro Gln Ser Tyr Leu Ala Glu Ala Gln Ala Leu Ala Phe Asn Lys Arg

145 150 155 160

Leu Lys Ile His Leu Asp Gly Ala Arg Ile Ala Asn Ala Ala Val Ala

165 170 175

His Asn Leu Asp Ile Ala Asp Ile Thr Gln Tyr Phe Asp Ser Val Ser

180 185 190

Ile Cys Leu Ser Lys Gly Leu Cys Ala Pro Val Gly Ser Leu Leu Leu

195 200 205

Gly Asp Glu Arg Leu Ile Asn Lys Ala Arg Arg Trp Arg Lys Met Leu

210 215 220

Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Val Ala Gly Glu Ile

225 230 235 240

Ala Leu Asn Glu Gln Val Ser Arg Leu Ala Val Asp His Glu Asn Ala

245 250 255

Arg Tyr Leu Ala Glu Gln Leu Ser Cys Leu Asp Glu Phe Glu Val Asp

260 265 270

Leu Gly Glu Val Gln Thr Asn Met Leu Phe Ala Arg Val Val Glu Gly

275 280 285

Val Ala Ile Asp Lys Leu Ala Thr Ser Leu Lys Ala Ser Gly Ile Leu

290 295 300

Ile Ser Pro Gly Lys Thr Leu Arg Met Val Thr His Ala Asp Ile Arg

305 310 315 320

Leu Glu Asp Ile Asp Leu Phe Ile Asp Lys Leu Lys Ser Leu Leu

325 330 335

<210> 71

<211> 335

<212> PRT

<400> 71

Met Ile Asp Phe Arg Ser Asp Thr Val Thr Gln Pro Thr Ala Ala Met

1 5 10 15

Arg Arg Ala Met Ala Asp Ala Gln Val Gly Asp Asp Val Tyr Gly Asp

20 25 30

Asp Pro Ser Val Asn Arg Leu Glu Ala Met Ala Ala Glu Arg Phe Gly

35 40 45

Phe Asp Ser Ala Leu Phe Thr Ser Ser Gly Thr Gln Ala Asn Leu Leu

50 55 60

Ala Leu Met Ser His Cys Asp Arg Gly Asp Glu Tyr Leu Cys Gly Gln

65 70 75 80

Gln Ala Pro Asn Tyr Lys Phe Glu Gly Gly Gly Ala Ala Val Leu Gly

85 90 95

Ser Ile Gln Pro Gln Pro Leu Thr Asn Gln Pro Asp Gly Ser Ile Leu

100 105 110

Leu Ser Asp Ile Glu Ala Ala Ile Lys Pro Asp Asp Phe His Phe Ala

115 120 125

Arg Thr Arg Leu Leu Ser Leu Glu Asn Thr Ile Gly Gly Lys Val Leu

130 135 140

Pro Gln Ser Tyr Leu Ala Glu Ala Gln Ala Leu Ala Phe Asn Lys Arg

145 150 155 160

Leu Lys Ile His Leu Asp Gly Ala Arg Ile Ala Asn Ala Ala Val Ala

165 170 175

His Asn Leu Asp Ile Ala Asp Ile Thr Gln Tyr Phe Asp Ser Val Ser

180 185 190

Ile Cys Leu Ser Lys Gly Leu Cys Ala Pro Val Gly Ser Leu Leu Leu

195 200 205

Gly Asp Glu Arg Leu Ile Asn Lys Ala Arg Arg Trp Arg Lys Met Leu

210 215 220

Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Val Ala Gly Glu Ile

225 230 235 240

Ala Leu Asn Glu Gln Val Ser Arg Leu Ala Val Asp His Glu Asn Ala

245 250 255

Arg Tyr Leu Ala Glu Gln Leu Ser Cys Leu Asp Glu Phe Glu Val Asp

260 265 270

Leu Gly Glu Val Gln Thr Asn Met Leu Phe Ala Arg Val Val Glu Gly

275 280 285

Val Ala Ile Asp Lys Leu Ala Thr Ser Leu Lys Ala Ser Gly Ile Leu

290 295 300

Ile Ser Pro Gly Lys Thr Leu Arg Met Val Thr His Ala Asp Ile Arg

305 310 315 320

Leu Glu Asp Ile Asp Leu Phe Ile Asp Lys Leu Lys Ser Leu Leu

325 330 335

<210> 72

<211> 335

<212> PRT

<400> 72

Met Ile Asp Phe Arg Ser Asp Thr Val Thr Gln Pro Thr Ala Ala Met

1 5 10 15

Arg Arg Ala Met Ala Asp Ala Gln Val Gly Asp Asp Val Tyr Gly Asp

20 25 30

Asp Pro Ser Val Asn Arg Leu Glu Ala Met Ala Ala Glu Arg Phe Gly

35 40 45

Phe Asp Ser Ala Leu Phe Thr Ser Ser Gly Thr Gln Ala Asn Leu Leu

50 55 60

Ala Leu Met Ser His Cys Asp Arg Gly Asp Glu Tyr Leu Cys Gly Gln

65 70 75 80

Gln Ala His Asn Tyr Lys Phe Glu Gly Gly Gly Ala Ala Val Leu Gly

85 90 95

Ser Ile Gln Pro Gln Pro Leu Thr Asn Gln Pro Asp Gly Ser Ile Leu

100 105 110

Leu Ser Asp Ile Glu Ala Ala Ile Lys Pro Asp Asp Phe His Phe Ala

115 120 125

Arg Thr Arg Leu Leu Ser Leu Glu Asn Thr Ile Gly Gly Lys Val Leu

130 135 140

Pro Gln Ser Tyr Leu Ala Glu Ala Gln Ala Leu Ala Phe Asn Lys Arg

145 150 155 160

Leu Lys Ile His Leu Asp Gly Tyr Arg Ile Ala Asn Ala Ala Val Ala

165 170 175

His Asn Leu Asp Ile Ala Asp Ile Thr Gln Tyr Phe Asp Ser Val Ser

180 185 190

Ile Cys Leu Ser Lys Gly Leu Cys Ala Pro Val Gly Ser Leu Leu Leu

195 200 205

Gly Asp Glu Arg Leu Ile Asn Lys Ala Arg Arg Trp Arg Lys Met Leu

210 215 220

Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Val Ala Gly Glu Ile

225 230 235 240

Ala Leu Asn Glu Gln Val Ser Arg Leu Ala Val Asp His Glu Asn Ala

245 250 255

Arg Tyr Leu Ala Glu Gln Leu Ser Cys Leu Asp Glu Phe Glu Val Asp

260 265 270

Leu Gly Glu Val Gln Thr Asn Met Leu Phe Ala Arg Val Val Glu Gly

275 280 285

Val Ala Ile Asp Lys Leu Ala Thr Ser Leu Lys Ala Ser Gly Ile Leu

290 295 300

Ile Ser Pro Gly Lys Thr Leu Arg Met Val Thr His Ala Asp Ile Arg

305 310 315 320

Leu Glu Asp Ile Asp Leu Phe Ile Asp Lys Leu Lys Ser Leu Leu

325 330 335

<210> 73

<211> 335

<212> PRT

<400> 73

Met Ile Asp Phe Arg Ser Asp Thr Val Thr Gln Pro Thr Ala Ala Met

1 5 10 15

Arg Arg Ala Met Ala Asp Ala Gln Val Gly Asp Asp Val Tyr Gly Asp

20 25 30

Asp Pro Ser Val Asn Arg Leu Glu Ala Met Ala Ala Glu Arg Phe Gly

35 40 45

Phe Asp Ser Ala Leu Phe Thr Ser Ser Gly Thr Gln Ala Asn Leu Leu

50 55 60

Ala Leu Met Ser His Cys Asp Arg Gly Asp Glu Tyr Leu Cys Gly Gln

65 70 75 80

Gln Ala His Asn Tyr Lys Phe Glu Gly Gly Gly Ala Ala Val Leu Gly

85 90 95

Ser Ile Gln Pro Gln Pro Leu Thr Asn Gln Pro Asp Gly Ser Ile Leu

100 105 110

Leu Ser Asp Ile Glu Ala Ala Ile Lys Pro Asp Asp Phe His Phe Ala

115 120 125

Arg Thr Arg Leu Leu Ser Leu Glu Asn Thr Ile Gly Gly Lys Val Leu

130 135 140

Pro Gln Ser Tyr Leu Ala Glu Ala Gln Ala Leu Ala Phe Asn Lys Arg

145 150 155 160

Leu Lys Ile His Leu Asp Gly Ala Arg Ile Ala Asn Ala Ala Val Ala

165 170 175

His Asn Leu Asp Ile Ala Asp Ile Thr Gln Tyr Phe Asp Ser Val Ser

180 185 190

Ile Cys Leu Ile Lys Gly Leu Cys Ala Pro Val Gly Ser Leu Leu Leu

195 200 205

Gly Asp Glu Arg Leu Ile Asn Lys Ala Arg Arg Trp Arg Lys Met Leu

210 215 220

Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Val Ala Gly Glu Ile

225 230 235 240

Ala Leu Asn Glu Gln Val Ser Arg Leu Ala Val Asp His Glu Asn Ala

245 250 255

Arg Tyr Leu Ala Glu Gln Leu Ser Cys Leu Asp Glu Phe Glu Val Asp

260 265 270

Leu Gly Glu Val Gln Thr Asn Met Leu Phe Ala Arg Val Val Glu Gly

275 280 285

Val Ala Ile Asp Lys Leu Ala Thr Ser Leu Lys Ala Ser Gly Ile Leu

290 295 300

Ile Ser Pro Gly Lys Thr Leu Arg Met Val Thr His Ala Asp Ile Arg

305 310 315 320

Leu Glu Asp Ile Asp Leu Phe Ile Asp Lys Leu Lys Ser Leu Leu

325 330 335

<210> 74

<211> 339

<212> PRT

<213> amino acid mutation (artificial sequence) of threonine aldolase from Aeromonas veronii

<400> 74

Met Arg Tyr Ile Asp Leu Arg Ser Asp Gln Val Thr Gln Pro Thr Asp

1 5 10 15

Ala Met Arg Gln Ala Met Leu His Ala Glu Val Gly Asp Asp Val Tyr

20 25 30

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

35 40 45

Leu Gly Lys Gln Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn

50 55 60

Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Ile Leu

65 70 75 80

Gly Asn Ala Ala His Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val

85 90 95

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

100 105 110

Leu Ala Phe Asp Asp Ile Lys Ala Ala Leu Ala Pro Asp Asp Ala His

115 120 125

Phe Val Gln Thr Arg Leu Ile Cys Leu Glu Asn Thr His Asn Gly Lys

130 135 140

Val Leu Pro Leu Ser Tyr Leu Gln Glu Met Gly Thr Phe Val Ala Glu

145 150 155 160

Arg Gly Leu Lys Leu His Leu Asp Gly Ala Arg Leu Phe Asn Ala Ala

165 170 175

Val Ala Ser Glu Thr Pro Val Ala Val Ile Ala Ala Pro Phe Asp Ser

180 185 190

Ile Ser Ile Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu

195 200 205

Leu Val Gly Ser His Asp Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys

210 215 220

Met Leu Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly

225 230 235 240

Leu Phe Ala Leu Glu Gln His Val Thr Arg Leu Ala Asp Asp His Arg

245 250 255

Arg Ala Lys Arg Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Glu

260 265 270

Leu Asp Leu Ser Leu Val Gln Ser Asn Met Val Phe Leu Arg Leu Arg

275 280 285

Glu Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Glu Arg Gly Ile

290 295 300

Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile

305 310 315 320

Asn Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu

325 330 335

Gly Ala Arg

<210> 75

<211> 339

<212> PRT

<400> 75

Met Arg Tyr Ile Asp Leu Arg Ser Asp Thr Val Thr Gln Pro Thr Asp

1 5 10 15

Ala Met Arg Gln Ala Met Leu His Ala Glu Val Gly Asp Asp Val Tyr

20 25 30

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

35 40 45

Leu Gly Lys Gln Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn

50 55 60

Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Ile Leu

65 70 75 80

Gly Asn Ala Ala Trp Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val

85 90 95

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

100 105 110

Leu Ala Phe Asp Asp Ile Lys Ala Ala Leu Ala Pro Asp Asp Ala His

115 120 125

Phe Val Gln Thr Arg Leu Ile Cys Leu Glu Asn Thr His Asn Gly Lys

130 135 140

Val Leu Pro Leu Ser Tyr Leu Gln Glu Met Gly Thr Phe Val Ala Glu

145 150 155 160

Arg Gly Leu Lys Leu His Leu Asp Gly Ala Arg Leu Phe Asn Ala Ala

165 170 175

Val Ala Ser Glu Thr Pro Val Ala Val Ile Ala Ala Pro Phe Asp Ser

180 185 190

Ile Ser Ile Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu

195 200 205

Leu Val Gly Ser His Asp Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys

210 215 220

Met Leu Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly

225 230 235 240

Leu Phe Ala Leu Glu Gln His Val Thr Arg Leu Ala Asp Asp His Arg

245 250 255

Arg Ala Lys Arg Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Glu

260 265 270

Leu Asp Leu Ser Leu Val Gln Ser Asn Met Val Phe Leu Arg Leu Arg

275 280 285

Glu Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Glu Arg Gly Ile

290 295 300

Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile

305 310 315 320

Asn Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu

325 330 335

Gly Ala Arg

<210> 76

<211> 339

<212> PRT

<400> 76

Met Arg Tyr Ile Asp Leu Arg Ser Asp Thr Val Thr Gln Pro Thr Asp

1 5 10 15

Ala Met Arg Gln Ala Met Leu His Ala Glu Val Gly Asp Asp Val Tyr

20 25 30

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

35 40 45

Leu Gly Lys Gln Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn

50 55 60

Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Ile Leu

65 70 75 80

Gly Asn Ala Ala Pro Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val

85 90 95

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

100 105 110

Leu Ala Phe Asp Asp Ile Lys Ala Ala Leu Ala Pro Asp Asp Ala His

115 120 125

Phe Val Gln Thr Arg Leu Ile Cys Leu Glu Asn Thr His Asn Gly Lys

130 135 140

Val Leu Pro Leu Ser Tyr Leu Gln Glu Met Gly Thr Phe Val Ala Glu

145 150 155 160

Arg Gly Leu Lys Leu His Leu Asp Gly Ala Arg Leu Phe Asn Ala Ala

165 170 175

Val Ala Ser Glu Thr Pro Val Ala Val Ile Ala Ala Pro Phe Asp Ser

180 185 190

Ile Ser Ile Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu

195 200 205

Leu Val Gly Ser His Asp Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys

210 215 220

Met Leu Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly

225 230 235 240

Leu Phe Ala Leu Glu Gln His Val Thr Arg Leu Ala Asp Asp His Arg

245 250 255

Arg Ala Lys Arg Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Glu

260 265 270

Leu Asp Leu Ser Leu Val Gln Ser Asn Met Val Phe Leu Arg Leu Arg

275 280 285

Glu Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Glu Arg Gly Ile

290 295 300

Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile

305 310 315 320

Asn Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu

325 330 335

Gly Ala Arg

<210> 77

<211> 339

<212> PRT

<400> 77

Met Arg Tyr Ile Asp Leu Arg Ser Asp Thr Val Thr Gln Pro Thr Asp

1 5 10 15

Ala Met Arg Gln Ala Met Leu His Ala Glu Val Gly Asp Asp Val Tyr

20 25 30

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

35 40 45

Leu Gly Lys Gln Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn

50 55 60

Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Ile Leu

65 70 75 80

Gly Asn Ala Ala His Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val

85 90 95

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

100 105 110

Leu Ala Phe Asp Asp Ile Lys Ala Ala Leu Ala Pro Asp Asp Ala His

115 120 125

Phe Val Gln Thr Arg Leu Ile Cys Leu Glu Asn Thr His Asn Gly Lys

130 135 140

Val Leu Pro Leu Ser Tyr Leu Gln Glu Met Gly Thr Phe Val Ala Glu

145 150 155 160

Arg Gly Leu Lys Leu His Leu Asp Gly Tyr Arg Leu Phe Asn Ala Ala

165 170 175

Val Ala Ser Glu Thr Pro Val Ala Val Ile Ala Ala Pro Phe Asp Ser

180 185 190

Ile Ser Ile Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu

195 200 205

Leu Val Gly Ser His Asp Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys

210 215 220

Met Leu Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly

225 230 235 240

Leu Phe Ala Leu Glu Gln His Val Thr Arg Leu Ala Asp Asp His Arg

245 250 255

Arg Ala Lys Arg Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Glu

260 265 270

Leu Asp Leu Ser Leu Val Gln Ser Asn Met Val Phe Leu Arg Leu Arg

275 280 285

Glu Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Glu Arg Gly Ile

290 295 300

Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile

305 310 315 320

Asn Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu

325 330 335

Gly Ala Arg

<210> 78

<211> 339

<212> PRT

<400> 78

Met Arg Tyr Ile Asp Leu Arg Ser Asp Thr Val Thr Gln Pro Thr Asp

1 5 10 15

Ala Met Arg Gln Ala Met Leu His Ala Glu Val Gly Asp Asp Val Tyr

20 25 30

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

35 40 45

Leu Gly Lys Gln Ala Ala Leu Phe Val Pro Ser Gly Thr Met Ser Asn

50 55 60

Leu Leu Ala Val Met Ser His Cys Gln Arg Gly Glu Gly Ala Ile Leu

65 70 75 80

Gly Asn Ala Ala His Ile Tyr Arg Tyr Glu Ala Gln Gly Ser Ala Val

85 90 95

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

100 105 110

Leu Ala Phe Asp Asp Ile Lys Ala Ala Leu Ala Pro Asp Asp Ala His

115 120 125

Phe Val Gln Thr Arg Leu Ile Cys Leu Glu Asn Thr His Asn Gly Lys

130 135 140

Val Leu Pro Leu Ser Tyr Leu Gln Glu Met Gly Thr Phe Val Ala Glu

145 150 155 160

Arg Gly Leu Lys Leu His Leu Asp Gly Ala Arg Leu Phe Asn Ala Ala

165 170 175

Val Ala Ser Glu Thr Pro Val Ala Val Ile Ala Ala Pro Phe Asp Ser

180 185 190

Ile Ser Ile Cys Leu Ile Lys Gly Leu Gly Ala Pro Val Gly Ser Leu

195 200 205

Leu Val Gly Ser His Asp Phe Ile Ala Arg Ala Arg Arg Leu Arg Lys

210 215 220

Met Leu Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Gln Ala Gly

225 230 235 240

Leu Phe Ala Leu Glu Gln His Val Thr Arg Leu Ala Asp Asp His Arg

245 250 255

Arg Ala Lys Arg Leu Ala Glu Gly Leu Ala Ala Leu Pro Gly Ile Glu

260 265 270

Leu Asp Leu Ser Leu Val Gln Ser Asn Met Val Phe Leu Arg Leu Arg

275 280 285

Glu Gly Glu Ser Ala Pro Leu Leu Ala Phe Met Lys Glu Arg Gly Ile

290 295 300

Leu Phe Ser Gly Tyr Gly Glu Leu Arg Leu Val Thr His Leu Gln Ile

305 310 315 320

Asn Asp Asp Asp Ile Glu Glu Val Ile Asp Ala Phe Thr Glu Tyr Leu

325 330 335

Gly Ala Arg

<210> 79

<211> 348

<212> PRT

<213> threonine aldolase amino acid mutation (artificial sequence) of sulfur-reducing Geobacillus

<400> 79

Met Asn Ile Ile Asp Leu Arg Ser Asp Gln Val Thr Arg Pro Ser Pro

1 5 10 15

Ala Met Arg Ala Ala Met Ala Gly Ala Glu Val Gly Asp Asp Val Tyr

20 25 30

Gly Glu Asp Pro Thr Val Asn Arg Leu Glu Glu Leu Gly Ala Ala Thr

35 40 45

Leu Gly Thr Glu Ala Ala Leu Phe Thr Ala Ser Gly Thr Gln Ala Asn

50 55 60

Leu Leu Ala Leu Leu Ala His Cys Arg Arg Gly Asp Glu Tyr Ile Ala

65 70 75 80

Gly Gln Thr Ala His Cys Tyr Arg Tyr Glu Gly Gly Gly Ala Ala Ala

85 90 95

Leu Gly Gly Ile Gln Pro Gln Pro Leu Glu Val Glu Pro Asp Gly Thr

100 105 110

Leu Asp Leu Ser Ala Val Ala Ala Ala Ile Lys Pro Asp Asp Leu His

115 120 125

Phe Ala Arg Thr Arg Leu Leu Cys Leu Glu Asn Thr His Ala Gly Arg

130 135 140

Val Leu Pro Leu Asp Tyr Leu Ala Arg Ala Arg Arg Leu Cys Asn Glu

145 150 155 160

Gln Ser Leu Gly Leu His Met Asp Gly Ala Arg Ile Phe Asn Ala Ala

165 170 175

Val Lys Leu Gly Val Pro Val Arg Glu Ile Ala Gly His Val Asp Ser

180 185 190

Val Ser Val Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu

195 200 205

Leu Cys Gly Gly Arg Glu Phe Val Ala Thr Ala Arg Arg Trp Arg Lys

210 215 220

Ala Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Ala Ala Gly

225 230 235 240

Ile Leu Ala Leu Thr Glu Asn Val Asp Arg Leu Ala Glu Asp His Ala

245 250 255

Asn Ala Arg Arg Leu Ala Glu Gly Leu Ala Ala Ile Pro Gly Leu Gly

260 265 270

Leu Asp Pro Ser Gln Val Gln Thr Asn Met Val Phe Leu Cys Leu Pro

275 280 285

Leu Arg Thr Ala Asp Arg Leu Ala Ser Phe Leu Asn Glu Gln Gly Ile

290 295 300

Leu Ile Ser Gly Arg Glu Ser Ile Arg Leu Val Thr His Leu Asp Val

305 310 315 320

Thr Ser Ser Asp Val Glu Arg Val Ile Ala Ala Phe Gly Ala Phe Phe

325 330 335

Ala Gly His Gly Glu Met Gln Pro Gly Ala Ser Ser

340 345

<210> 80

<211> 348

<212> PRT

<400> 80

Met Asn Ile Ile Asp Leu Arg Ser Asp Thr Val Thr Arg Pro Ser Pro

1 5 10 15

Ala Met Arg Ala Ala Met Ala Gly Ala Glu Val Gly Asp Asp Val Tyr

20 25 30

Gly Glu Asp Pro Thr Val Asn Arg Leu Glu Glu Leu Gly Ala Ala Thr

35 40 45

Leu Gly Thr Glu Ala Ala Leu Phe Thr Ala Ser Gly Thr Gln Ala Asn

50 55 60

Leu Leu Ala Leu Leu Ala His Cys Arg Arg Gly Asp Glu Tyr Ile Ala

65 70 75 80

Gly Gln Thr Ala Trp Cys Tyr Arg Tyr Glu Gly Gly Gly Ala Ala Ala

85 90 95

Leu Gly Gly Ile Gln Pro Gln Pro Leu Glu Val Glu Pro Asp Gly Thr

100 105 110

Leu Asp Leu Ser Ala Val Ala Ala Ala Ile Lys Pro Asp Asp Leu His

115 120 125

Phe Ala Arg Thr Arg Leu Leu Cys Leu Glu Asn Thr His Ala Gly Arg

130 135 140

Val Leu Pro Leu Asp Tyr Leu Ala Arg Ala Arg Arg Leu Cys Asn Glu

145 150 155 160

Gln Ser Leu Gly Leu His Met Asp Gly Ala Arg Ile Phe Asn Ala Ala

165 170 175

Val Lys Leu Gly Val Pro Val Arg Glu Ile Ala Gly His Val Asp Ser

180 185 190

Val Ser Val Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu

195 200 205

Leu Cys Gly Gly Arg Glu Phe Val Ala Thr Ala Arg Arg Trp Arg Lys

210 215 220

Ala Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Ala Ala Gly

225 230 235 240

Ile Leu Ala Leu Thr Glu Asn Val Asp Arg Leu Ala Glu Asp His Ala

245 250 255

Asn Ala Arg Arg Leu Ala Glu Gly Leu Ala Ala Ile Pro Gly Leu Gly

260 265 270

Leu Asp Pro Ser Gln Val Gln Thr Asn Met Val Phe Leu Cys Leu Pro

275 280 285

Leu Arg Thr Ala Asp Arg Leu Ala Ser Phe Leu Asn Glu Gln Gly Ile

290 295 300

Leu Ile Ser Gly Arg Glu Ser Ile Arg Leu Val Thr His Leu Asp Val

305 310 315 320

Thr Ser Ser Asp Val Glu Arg Val Ile Ala Ala Phe Gly Ala Phe Phe

325 330 335

Ala Gly His Gly Glu Met Gln Pro Gly Ala Ser Ser

340 345

<210> 81

<211> 348

<212> PRT

<400> 81

Met Asn Ile Ile Asp Leu Arg Ser Asp Thr Val Thr Arg Pro Ser Pro

1 5 10 15

Ala Met Arg Ala Ala Met Ala Gly Ala Glu Val Gly Asp Asp Val Tyr

20 25 30

Gly Glu Asp Pro Thr Val Asn Arg Leu Glu Glu Leu Gly Ala Ala Thr

35 40 45

Leu Gly Thr Glu Ala Ala Leu Phe Thr Ala Ser Gly Thr Gln Ala Asn

50 55 60

Leu Leu Ala Leu Leu Ala His Cys Arg Arg Gly Asp Glu Tyr Ile Ala

65 70 75 80

Gly Gln Thr Ala Pro Cys Tyr Arg Tyr Glu Gly Gly Gly Ala Ala Ala

85 90 95

Leu Gly Gly Ile Gln Pro Gln Pro Leu Glu Val Glu Pro Asp Gly Thr

100 105 110

Leu Asp Leu Ser Ala Val Ala Ala Ala Ile Lys Pro Asp Asp Leu His

115 120 125

Phe Ala Arg Thr Arg Leu Leu Cys Leu Glu Asn Thr His Ala Gly Arg

130 135 140

Val Leu Pro Leu Asp Tyr Leu Ala Arg Ala Arg Arg Leu Cys Asn Glu

145 150 155 160

Gln Ser Leu Gly Leu His Met Asp Gly Ala Arg Ile Phe Asn Ala Ala

165 170 175

Val Lys Leu Gly Val Pro Val Arg Glu Ile Ala Gly His Val Asp Ser

180 185 190

Val Ser Val Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu

195 200 205

Leu Cys Gly Gly Arg Glu Phe Val Ala Thr Ala Arg Arg Trp Arg Lys

210 215 220

Ala Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Ala Ala Gly

225 230 235 240

Ile Leu Ala Leu Thr Glu Asn Val Asp Arg Leu Ala Glu Asp His Ala

245 250 255

Asn Ala Arg Arg Leu Ala Glu Gly Leu Ala Ala Ile Pro Gly Leu Gly

260 265 270

Leu Asp Pro Ser Gln Val Gln Thr Asn Met Val Phe Leu Cys Leu Pro

275 280 285

Leu Arg Thr Ala Asp Arg Leu Ala Ser Phe Leu Asn Glu Gln Gly Ile

290 295 300

Leu Ile Ser Gly Arg Glu Ser Ile Arg Leu Val Thr His Leu Asp Val

305 310 315 320

Thr Ser Ser Asp Val Glu Arg Val Ile Ala Ala Phe Gly Ala Phe Phe

325 330 335

Ala Gly His Gly Glu Met Gln Pro Gly Ala Ser Ser

340 345

<210> 82

<211> 348

<212> PRT

<400> 82

Met Asn Ile Ile Asp Leu Arg Ser Asp Thr Val Thr Arg Pro Ser Pro

1 5 10 15

Ala Met Arg Ala Ala Met Ala Gly Ala Glu Val Gly Asp Asp Val Tyr

20 25 30

Gly Glu Asp Pro Thr Val Asn Arg Leu Glu Glu Leu Gly Ala Ala Thr

35 40 45

Leu Gly Thr Glu Ala Ala Leu Phe Thr Ala Ser Gly Thr Gln Ala Asn

50 55 60

Leu Leu Ala Leu Leu Ala His Cys Arg Arg Gly Asp Glu Tyr Ile Ala

65 70 75 80

Gly Gln Thr Ala His Cys Tyr Arg Tyr Glu Gly Gly Gly Ala Ala Ala

85 90 95

Leu Gly Gly Ile Gln Pro Gln Pro Leu Glu Val Glu Pro Asp Gly Thr

100 105 110

Leu Asp Leu Ser Ala Val Ala Ala Ala Ile Lys Pro Asp Asp Leu His

115 120 125

Phe Ala Arg Thr Arg Leu Leu Cys Leu Glu Asn Thr His Ala Gly Arg

130 135 140

Val Leu Pro Leu Asp Tyr Leu Ala Arg Ala Arg Arg Leu Cys Asn Glu

145 150 155 160

Gln Ser Leu Gly Leu His Met Asp Gly Tyr Arg Ile Phe Asn Ala Ala

165 170 175

Val Lys Leu Gly Val Pro Val Arg Glu Ile Ala Gly His Val Asp Ser

180 185 190

Val Ser Val Cys Leu Ser Lys Gly Leu Gly Ala Pro Val Gly Ser Leu

195 200 205

Leu Cys Gly Gly Arg Glu Phe Val Ala Thr Ala Arg Arg Trp Arg Lys

210 215 220

Ala Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Ala Ala Gly

225 230 235 240

Ile Leu Ala Leu Thr Glu Asn Val Asp Arg Leu Ala Glu Asp His Ala

245 250 255

Asn Ala Arg Arg Leu Ala Glu Gly Leu Ala Ala Ile Pro Gly Leu Gly

260 265 270

Leu Asp Pro Ser Gln Val Gln Thr Asn Met Val Phe Leu Cys Leu Pro

275 280 285

Leu Arg Thr Ala Asp Arg Leu Ala Ser Phe Leu Asn Glu Gln Gly Ile

290 295 300

Leu Ile Ser Gly Arg Glu Ser Ile Arg Leu Val Thr His Leu Asp Val

305 310 315 320

Thr Ser Ser Asp Val Glu Arg Val Ile Ala Ala Phe Gly Ala Phe Phe

325 330 335

Ala Gly His Gly Glu Met Gln Pro Gly Ala Ser Ser

340 345

<210> 83

<211> 348

<212> PRT

<400> 83

Met Asn Ile Ile Asp Leu Arg Ser Asp Thr Val Thr Arg Pro Ser Pro

1 5 10 15

Ala Met Arg Ala Ala Met Ala Gly Ala Glu Val Gly Asp Asp Val Tyr

20 25 30

Gly Glu Asp Pro Thr Val Asn Arg Leu Glu Glu Leu Gly Ala Ala Thr

35 40 45

Leu Gly Thr Glu Ala Ala Leu Phe Thr Ala Ser Gly Thr Gln Ala Asn

50 55 60

Leu Leu Ala Leu Leu Ala His Cys Arg Arg Gly Asp Glu Tyr Ile Ala

65 70 75 80

Gly Gln Thr Ala His Cys Tyr Arg Tyr Glu Gly Gly Gly Ala Ala Ala

85 90 95

Leu Gly Gly Ile Gln Pro Gln Pro Leu Glu Val Glu Pro Asp Gly Thr

100 105 110

Leu Asp Leu Ser Ala Val Ala Ala Ala Ile Lys Pro Asp Asp Leu His

115 120 125

Phe Ala Arg Thr Arg Leu Leu Cys Leu Glu Asn Thr His Ala Gly Arg

130 135 140

Val Leu Pro Leu Asp Tyr Leu Ala Arg Ala Arg Arg Leu Cys Asn Glu

145 150 155 160

Gln Ser Leu Gly Leu His Met Asp Gly Ala Arg Ile Phe Asn Ala Ala

165 170 175

Val Lys Leu Gly Val Pro Val Arg Glu Ile Ala Gly His Val Asp Ser

180 185 190

Val Ser Val Cys Leu Ile Lys Gly Leu Gly Ala Pro Val Gly Ser Leu

195 200 205

Leu Cys Gly Gly Arg Glu Phe Val Ala Thr Ala Arg Arg Trp Arg Lys

210 215 220

Ala Val Gly Gly Gly Met Arg Gln Ala Gly Ile Leu Ala Ala Ala Gly

225 230 235 240

Ile Leu Ala Leu Thr Glu Asn Val Asp Arg Leu Ala Glu Asp His Ala

245 250 255

Asn Ala Arg Arg Leu Ala Glu Gly Leu Ala Ala Ile Pro Gly Leu Gly

260 265 270

Leu Asp Pro Ser Gln Val Gln Thr Asn Met Val Phe Leu Cys Leu Pro

275 280 285

Leu Arg Thr Ala Asp Arg Leu Ala Ser Phe Leu Asn Glu Gln Gly Ile

290 295 300

Leu Ile Ser Gly Arg Glu Ser Ile Arg Leu Val Thr His Leu Asp Val

305 310 315 320

Thr Ser Ser Asp Val Glu Arg Val Ile Ala Ala Phe Gly Ala Phe Phe

325 330 335

Ala Gly His Gly Glu Met Gln Pro Gly Ala Ser Ser

340 345

<210> 84

<211> 24

<212> DNA

<213> primer (Artificial sequence)

<400> 84

ctggacggct accggctggc caat 24

<210> 85

<211> 24

<212> DNA

<213> primer (Artificial sequence)

<400> 85

attggccagc cggtagccgt ccag 24

<210> 86

<211> 22

<212> DNA

<213> primer (Artificial sequence)

<400> 86

agcacgccag catctgcacg ga 22

<210> 87

<211> 22

<212> DNA

<213> primer (Artificial sequence)

<400> 87

tccgtgcaga tgctggcgtg ct 22

<210> 88

<211> 1032

<212> DNA

<213> threonine aldolase gene (artificial sequence) of Bacillus crescentus

<400> 88

atgacccaga ccgcgccccg ctacgatttc gcctccgaca acgtcgccgg cgccatgccc 60

gaggtgatgg aggcgttgat cgcggccaat gcggggacgg cctcgggcta cgggaccgac 120

catgtcagcc gtgccgccgc cgatcgcatc cgcgcggcgc tggacgccga cgcgcaggtg 180

cggttcacag cctcgggcac ggcggccaac gccttcgccc tgaccctgct ggcccagccg 240

cacgaggcgg tgctggccca cgagcacgcc cacatctgca cggacgagac cggcgcgccc 300

ggcttctttg gccagggcgt cggcctgatc ggcttgccgg gcgcatcggg caagatggag 360

ttggcggccc tggaggcggc gctggcccag ccggacgtct cgtaccgcca gccggcggcg 420

gccttgtcgc tgaccaccgc caccgagtac ggcacggtct attcggaaga ccacctgcgg 480

gccctgatcg cgccggtgaa ggccaagggc tatggcgtcc acctggacgg cgcgcggctg 540

gccaatgcgg tcgcgggggg ctttgacctg aaaagcatcg ccaagatggg cgtcgacatc 600

ctggtgatgg gcggcaccaa ggccggctcg acgcccaccg aggccgtggt gttcctgaac 660

cccgaccacg ccaaacgcct ggacgcgcgc ctcaagcacg ccggccagct gatctcgaag 720

ggacgcttcc tggccgcgcc gtggctgggc ctgctgggcg agaacggtca gaccgccccc 780

tgggccgccc gcgccgccca cgccaacgcc atggcgcaga agctggccgc gctgatgccc 840

gtcccgatca agcacccggt cgaggccaac ggcatcttcg tcgagatgga cgaacttgcc 900

ctggaacgcc tgcgcggcga aggctggttc gtctatcgct tcctggacgg cacggtgcgc 960

ttcatgtgct cgtgggccac gacgcccgag atggtcgagg atctgggcgc ggcgctgaag 1020

cgagtggctt ag 1032

Claims

1. Threonine aldolase mutant characterized in that it is selected from Pseudomonas (Pseudomonas sp.) (I.)Pseudomonas putida) The mutant of threonine aldolase of (1), whose amino acid sequence is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10.

2. a gene encoding the threonine aldolase mutant as described in claim 1.

3. A recombinant vector constructed from the coding gene of claim 2.

4. A recombinant genetically engineered bacterium transformed with the recombinant vector of claim 3.

5. Use of the threonine aldolase mutant as described in claim 1 for producing a phenylserine derivative substituted at the 2-/3-/4-position.

6. The use according to claim 5, characterized in that the 2-/3-/4-substituted phenylserine derivative is obtained by carrying out the enzymatic catalysis in a reaction medium under the conditions of controlled temperature and stirring speed by using a threonine aldolase mutant as a catalyst, 2-/3-/4-substituted benzaldehyde as a substrate, pyridoxal-5-phosphate as a coenzyme and glycine/glycine as a co-substrate, and after the reaction is finished, separating and purifying.

7. The use according to claim 6, wherein the substrate is one of: benzaldehyde, 2-chlorobenzaldehyde, 2-fluorobenzaldehyde, 2-bromobenzaldehyde, 2-nitrobenzaldehyde, 2-methylsulfonylbenzaldehyde, 2-hydroxybenzaldehyde, 3-chlorobenzaldehyde, 3-fluorobenzaldehyde, 3-bromobenzaldehyde, 3-nitrobenzaldehyde, 3-methylsulfonylbenzaldehyde, 3-hydroxybenzaldehyde, 4-chlorobenzaldehyde, 4-fluorobenzaldehyde, 4-bromobenzaldehyde, 4-nitrobenzaldehyde, 4-methylsulfonylbenzaldehyde; the co-substrate is one of the following: l-glycine, L-glycine hydrochloride, L-glycine methyl ester hydrochloride, L-glycine ethyl ester hydrochloride, N-Boc-L-glycine ethyl ester, N-Z-L-glycine ethyl ester, N-Z-L-glycine methyl ester; the reaction medium is one of the following: a phosphoric acid buffer system with pH of 8-9, a water-ethanol system, a water-methanol system, a methanol system and an ethanol system.

8. The use according to claim 6, wherein the enzyme is used in an amount of 50 to 1000mg/L, the substrate concentration is 1 to 100g/L, the glycine/glycine ester concentration is 5 to 200g/L, the pyridoxal 5-phosphate concentration is 0.01 to 0.2g/L, the temperature is controlled at 25 to 45 ℃ and the stirring speed is 70 to 300 rpm.