CN111454971A

CN111454971A - Method for improving conversion rate of chiral amine

Info

Publication number: CN111454971A
Application number: CN202010209530.2A
Authority: CN
Inventors: 宋浩; 赵妍淑; 张金华
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2020-03-23
Filing date: 2020-03-23
Publication date: 2020-07-28
Anticipated expiration: 2040-03-23
Also published as: CN111454971B

Abstract

The invention discloses a method for improving conversion rate of chiral amine, which comprises the steps of synthesizing and amplifying optimized ArR-omega TA nucleotide sequences containing enzyme cutting sites, constructing P2A 4-ArR-omega TA, P2A4-FA and P2A4-ldh, respectively introducing FA which is a gene for expressing formate acetyltransferase into escherichia coli to obtain recombinant bacteria 1, FA and 6, fermenting to obtain recombinant bacteria 1, FA and 6 fermentation liquor, centrifuging to obtain recombinant bacteria 1, FA and 6 fermentation liquor sediment, respectively preparing FA crude enzyme liquid and ldh crude enzyme liquid, re-suspending the recombinant bacteria 1 fermentation liquor sediment by PBS, adding P L P, NAD, re-suspending⁺Reacting; adding FA crude enzyme solution, ldh crude enzyme solution, formate dehydrogenase, D-alanine, substrate ketone and CoA, adding cosolvent, reacting, terminating, extracting, and drying. Method of the inventionRemoving the byproduct pyruvic acid and improving the conversion rate of chiral amine. Adding formate dehydrogenase to recover coenzyme I.

Description

Method for improving conversion rate of chiral amine

Technical Field

The invention belongs to the field of chemistry, and particularly relates to a method for improving the conversion rate of chiral amine.

Background

In recent years, enantiomerically pure amines have played a crucial role in the chemical, pharmaceutical and agrochemical industries. Chiral amines are widely used in therapeutic drugs such as neurologic drugs, cardiovascular drugs, hypotensive drugs, anti-infective drugs, and vaccines. At present, chemical methods, biological resolution methods and biological asymmetric synthesis methods are the main methods for preparing chiral amines. The asymmetric synthesis method has little pollution to the environment, and the theoretical yield can reach 100 percent, so the method is a hot point of research. In the asymmetric synthesis of chiral amines using transaminases, the transamination reaction is a reversible reaction and the byproduct pyruvate prevents the reaction from proceeding toward the production of the amine. At present, a coupling system of lactate dehydrogenase and glucose dehydrogenase is mainly used for eliminating the inhibition effect of byproduct pyruvic acid and regenerating cofactors so as to improve the conversion rate of chiral amine. In the reaction for asymmetric synthesis of chiral amine by using transaminase, no report has been made on improvement of conversion rate of chiral amine by coupling system of formate acetyltransferase and lactate dehydrogenase.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for improving the conversion rate of chiral amine.

The technical scheme of the invention is summarized as follows:

a method for increasing the conversion rate of chiral amine comprises the following steps:

(1) in vitro synthesis and amplification of genes:

the amino acid sequence of an (R) -selective ω -transaminase derived from arthrobacter sp, abbreviated as: ArR- ω TA; the amino acid sequence of the ArR-omega TA is shown in SEQ ID NO. 1;

the amino acid sequence of ArR- ω TA is determined at the JCAT site by entering: avoiding restriction sites of XbaI, NdeI, SpeI and HindIII as conditions to obtain an optimized ArR-omega TA nucleotide sequence;

adding an XbaI restriction site and a HindIII restriction site before and after the optimized ArR-omega TA nucleotide sequence to obtain an optimized ArR-omega TA nucleotide sequence containing the restriction sites, wherein the optimized ArR-omega TA nucleotide sequence containing the restriction sites is shown as SEQ ID NO. 2;

(2) connecting the optimized ArR-omega TA nucleotide sequence containing the enzyme cleavage site obtained in the step (1) to a p2A4 vector to obtain p2A 4-ArR-omega TA; expressing the nucleotide sequence of the formate acetyltransferase by using FA, and connecting the FA into a p2A4 vector to obtain p2A 4-FA; ligating the nucleotide sequence of ldh into the p2A4 vector to give p2A 4-ldh;

the nucleotide sequence of the p2A4 vector is shown as SEQ ID NO. 18;

the nucleotide sequence of ldh is shown as SEQ ID NO. 7;

(3) p2A 4-ArR-omega TA; p2A 4-FA; introducing p2A4-ldh into Escherichia coli (E.coli MG1655) respectively to obtain recombinant bacterium 1, recombinant bacterium FA and recombinant bacterium 6;

(4) respectively fermenting the recombinant bacteria 1, the recombinant bacteria FA and the recombinant bacteria 6 to obtain recombinant bacteria 1 fermentation liquor, recombinant bacteria FA fermentation liquor and recombinant bacteria 6 fermentation liquor;

(5) respectively centrifuging, collecting and washing the fermentation liquor obtained in the step (4) to obtain a recombinant bacterium 1 fermentation liquor precipitate, a recombinant bacterium FA fermentation liquor precipitate and a recombinant bacterium 6 fermentation liquor precipitate;

(6) respectively re-suspending cells of the recombinant bacteria FA fermentation liquor sediment obtained in the step (5) and the recombinant bacteria 6 fermentation liquor sediment by PBS and carrying out ultrasonic crushing treatment; obtaining FA crude enzyme solution and ldh crude enzyme solution;

(7) re-suspending the fermentation liquid sediment (ArR-omega TA whole cells) of the recombinant bacteria 1 obtained in the step (5) by PBS to obtain a mixed liquid, adding the mixed liquid into P L P, NAD⁺Reacting for 20-40 minutes at 25-40 ℃ and 180-220 rpm; adding FA crude enzyme solution, ldh crude enzyme solution, formate dehydrogenase, D-alanine, substrate ketone and CoA to obtain reaction solution, adding cosolvent, reacting at 25-40 deg.C and 180-220rpm for 12-24 hr, adding NADH to terminate the reaction, extracting with ethyl acetate, adding Na into the extracted organic phase₂SO₄Drying, and detecting by gas chromatography.

The FA is ybiw, pflB, pflD or tdcE, the nucleotide sequence of ybiw is shown as SEQ ID NO.3, the nucleotide sequence of pflB is shown as SEQ ID NO.4, the nucleotide sequence of pflD is shown as SEQ ID NO.5, and the nucleotide sequence of tdcE is shown as SEQ ID NO. 6.

Step (7) is preferably: and (3) resuspending the fermentation liquor sediment of the recombinant bacteria 1 obtained in the step (5) by using PBS (phosphate buffer solution) with the pH value of 7.5 and the concentration of 100mM to obtain a mixed liquor, wherein the volume of the PBS is the volume of the PBS1/7 of the volume of the fermentation liquid of the recombinant bacterium 1 obtained in the step (4), taking 1m L mixed liquid according to the proportion, adding 1.5mM P L P and 1mM NAD⁺Reacting for 20-40 minutes at 25-40 ℃ and 180-220 rpm; then adding 10U of FA crude enzyme solution, 10U of ldh crude enzyme solution, 10U of formate dehydrogenase, D-alanine with the final concentration of 250mM, substrate ketone with the final concentration of 25mM and CoA with the final concentration of 0.1mM to obtain a reaction solution, adding DMSO with the volume of 15-60% of the reaction solution, reacting for 12-24 hours at the temperature of 25-40 ℃, 180-220rpm, adding NADH with the final concentration of 10M to terminate the reaction, extracting with ethyl acetate twice, adding Na into the organic phase after extraction₂SO₄Drying, and detecting by gas chromatography.

The ketone is preferably 2-pentanone, cyclohexanone or benzylacetone, and other ketones may be used.

The invention has the advantages that:

compared with the reported method for eliminating the byproduct pyruvic acid by one way, the method removes the byproduct pyruvic acid and can improve the conversion rate of the chiral amine. Meanwhile, the introduction of formate dehydrogenase can be used for recovering the cofactor reduced coenzyme I.

Drawings

FIG. 1 shows the ArR- ω TA/TdcE/FDH/L DH coupling system.

FIG. 2 shows the enzyme activities of ybiw, pflB, pflD, tdcE.

FIG. 3 shows the conversion of 2-pentanone, benzylacetone, cyclohexanone in the ArR- ω TA/TdcE/FDH/L DH coupling system.

Detailed Description

P L P pyridoxal phosphate

NAD⁺: coenzyme I

CoA: coenzyme A

NADH: reduced coenzyme I

DMSO dimethyl sulfoxide

The present invention will be further described with reference to the following examples.

Primers ybiw-F and ybiw-R were designed, ybiw was amplified from genomic DNA of E.coli MG1655 by PCR;

primers pflB-F and pflB-R were designed, and pflB was amplified from genomic DNA of E.coli MG1655 by PCR;

primers pflD-F and pflD-R were designed, and pflD was amplified from genomic DNA of E.coli MG1655 by PCR;

primers tdcE-F and tdcE-R are designed, and tdcE is amplified from the genome DNA of the Escherichia coli MG1655 through PCR;

primers ldh-F and ldh-R were designed and ldh was amplified from genomic DNA of E.coli MG1655 by PCR;

the nucleotide sequence of ybiw is shown in SEQ ID NO.3,

the nucleotide sequence of pflB is shown in SEQ ID NO.4,

the nucleotide sequence of pflD is shown in SEQ ID NO.5,

the nucleotide sequence of tdcE is shown in SEQ ID NO.6,

the nucleotide sequence of ldh is shown as SEQ ID NO.7,

the nucleotide sequence of ybiw-F is shown as SEQ ID NO.8, the nucleotide sequence of ybiw-R is shown as SEQ ID NO.9,

the nucleotide sequence of pflB-F is shown in SEQ ID NO.10, the nucleotide sequence of pflB-R is shown in SEQ ID NO.11,

the nucleotide sequence of pflD-F is shown in SEQ ID NO.12, the nucleotide sequence of pflD-R is shown in SEQ ID NO.13,

the nucleotide sequence of tdcE-F is shown as SEQ ID NO.14, the nucleotide sequence of tdcE-R is shown as SEQ ID NO.15,

the nucleotide sequence of ldh-F is shown as SEQ ID NO.16, and the nucleotide sequence of ldh-R is shown as SEQ ID NO. 17.

Example 1

(1) in vitro synthesis and amplification of genes:

(2) connecting the optimized ArR-omega TA nucleotide sequence containing the enzyme cleavage site obtained in the step (1) to a p2A4 vector to obtain p2A 4-ArR-omega TA; expressing a nucleotide sequence of formate acetyltransferase (FA is tdcE) by using tdcE, and connecting the tdcE into a p2A4 vector to obtain p2A 4-tdcE; ligating the nucleotide sequence of ldh into the p2A4 vector to give p2A 4-ldh;

the nucleotide sequence of the p2A4 vector is shown as SEQ ID NO. 18;

(3) p2A 4-ArR-omega TA; p2A 4-tdcE; introducing p2A4-ldh into Escherichia coli (E.coli MG1655) respectively to obtain recombinant bacteria 1, recombinant bacteria 5 and recombinant bacteria 6;

(4) respectively fermenting the recombinant bacteria 1, the recombinant bacteria 5 and the recombinant bacteria 6 to obtain recombinant bacteria 1 fermentation liquor, recombinant bacteria 5 fermentation liquor and recombinant bacteria 6 fermentation liquor;

and (3) fermentation process:

recombinant bacterium 1, recombinant bacterium 5 and recombinant bacterium 6 were cultured in TB liquid plus glucose medium (10 g glucose in 1 liter of TB liquid medium) containing (100. mu.g/ml ampicillin) respectively at 37 ℃ and 220rpm for 12 hours, the culture was transferred at a ratio of 1:100 into a 250m L shake flask containing 50m L TB medium and grown at 37 ℃, Isopropylthiogalactoside (IPTG) was added to give a final concentration of 1mM when the optical density at 600nm (OD600) reached about 0.6 and grown at 25 ℃ and 200rpm for 16 hours.

(5) Respectively centrifuging the fermentation liquor obtained in the step (4) at 4 ℃ and 6500rpm, collecting precipitates, washing and washing the precipitates with 0.01M PBS (pH is 7.5), and obtaining recombinant bacterium 1 fermentation liquor precipitates, recombinant bacterium 5 fermentation liquor precipitates and recombinant bacterium 6 fermentation liquor precipitates;

(6) respectively resuspending the recombinant bacterium 5 fermentation liquor sediment and the recombinant bacterium 6 fermentation liquor sediment obtained in the step (5) by PBS (100mM, pH 7.5) and carrying out ultrasonic disruption treatment, wherein the pulse is 5s, the interval is 10s, and the total working time is 20 minutes; obtaining a recombinant bacteria 5 crude enzyme solution (namely tdcE crude enzyme solution) and ldh crude enzyme solution;

(7) re-suspending the recombinant bacterium 1 fermentation liquid precipitate obtained in the step (5) by PBS (the volume of which is 1/7 of the volume of the recombinant bacterium 1 fermentation liquid obtained in the step (4) and the pH value of which is 7.5 mM) and the concentration of which is 100mM to obtain a mixed liquid, taking 1m L mixed liquid, adding P L P with the final concentration of 1.5mM and NAD with the final concentration of 1mM to obtain a mixed liquid⁺Reacting at 30 ℃ and 180rpm for 30 minutes, adding 10U of tdcE crude enzyme solution, 10U of ldh crude enzyme solution, 10U of formate dehydrogenase, 250mM D-alanine, 25mM substrate 2-pentanone and 0.1mM CoA to obtain a reaction solution, adding 30% DMSO by volume of the reaction solution, reacting at 30 ℃ and 180rpm for 24 hours, adding 10M NADH to terminate the reaction, extracting with ethyl acetate, extracting twice, each time adding 500 mu L, adding Na to the organic phase after extraction₂SO₄Drying, and detecting by gas chromatography.

Gas chromatography was carried out using TG-5MS column (30m × 0.32.32 mm × 0.25.25 μm; Thermo Fisher scientific) under the conditions of sample injector temperature of 250 ℃, constant flow of 1.8 ml/min, temperature program of 40 ℃/min hold of 2 min, 80 ℃/rate of 5 ℃/min hold of 2 min, 250 ℃/rate of 20 ℃/min hold of 10 min.

See fig. 1, 2 and 3.

Benzyl acetone and cyclohexanone were used instead of 2-pentanone in this example, respectively, and the conversion rates in the ArR- ω TA/TdcE/FDH/L DH coupling system are shown in FIG. 3.

Example 2

(1) same as example 1, step (1);

(2) replacing tdcE in example 1 with ybiw, the same as in step (2) of example 1;

(3) p2A 4-ArR-omega TA; p2A 4-ybiw; introducing p2A4-ldh into Escherichia coli (E.coli MG1655) respectively to obtain recombinant bacterium 1, recombinant bacterium 2 and recombinant bacterium 6;

(4) respectively fermenting the recombinant bacteria 1, the recombinant bacteria 2 and the recombinant bacteria 6 to obtain recombinant bacteria 1 fermentation liquor, recombinant bacteria 2 fermentation liquor and recombinant bacteria 6 fermentation liquor;

the fermentation process was the same as in example 1;

(5) respectively centrifuging the fermentation liquor obtained in the step (4) at 4 ℃ and 6500rpm, collecting precipitates, washing and washing the precipitates with 0.01M PBS (pH 7.5), and obtaining recombinant bacterium 1 fermentation liquor precipitates, recombinant bacterium 2 fermentation liquor precipitates and recombinant bacterium 6 fermentation liquor precipitates;

(6) respectively re-suspending the recombinant bacterium 2 fermentation liquor sediment and the recombinant bacterium 6 fermentation liquor sediment obtained in the step (5) by using PBS (100mM, PH 7.5,) for cell re-suspension treatment, carrying out ultrasonic disruption treatment for 5s pulse and 10s interval, wherein the total working time is 20 minutes; obtaining a recombinant bacteria 2 crude enzyme solution (namely ybiw crude enzyme solution) and ldh crude enzyme solution;

(7) re-suspending the recombinant bacterium 1 fermentation liquid precipitate obtained in the step (5) by PBS (the volume of which is 1/7 of the volume of the recombinant bacterium 1 fermentation liquid obtained in the step (4) and the pH value of which is 7.5 mM) and the concentration of which is 100mM to obtain a mixed liquid, taking 1m L mixed liquid, adding P L P with the final concentration of 1.5mM and NAD with the final concentration of 1mM to obtain a mixed liquid⁺Reacting at 40 ℃ and 180rpm for 40 minutes, adding 10U ybiw crude enzyme solution, 10U ldh crude enzyme solution, 10U formate dehydrogenase, 250mM D-alanine, 25mM cyclohexanone as substrate and 0.1mM CoA to obtain a reaction solution, adding 15% by volume of DMSO as a cosolvent, reacting at 40 ℃ and 180rpm for 12 hours, adding 10M NADH to terminate the reaction, extracting with ethyl acetate twice, adding 500 mu L each time, adding Na to the organic phase after extraction₂SO₄Drying, and detecting by gas chromatography.

Example 3

(1) same as example 1, step (1);

(2) the step (2) of example 1 was followed by replacing tdcE in example 1 with pflB;

(3) p2A 4-ArR-omega TA; p2A 4-pflB; introducing p2A4-ldh into Escherichia coli (E.coli MG1655) respectively to obtain recombinant bacteria 1, recombinant bacteria 3 and recombinant bacteria 6;

(4) respectively fermenting the recombinant bacteria 1, the recombinant bacteria 3 and the recombinant bacteria 6 to obtain recombinant bacteria 1 fermentation liquor, recombinant bacteria 3 fermentation liquor and recombinant bacteria 6 fermentation liquor;

the fermentation process was the same as in example 1;

(5) respectively centrifuging the fermentation liquor obtained in the step (4) at 4 ℃ and 6500rpm, collecting precipitates, washing and washing the precipitates with 0.01M PBS (pH 7.5) to obtain recombinant bacterium 1 fermentation liquor precipitates, recombinant bacterium 3 fermentation liquor precipitates and recombinant bacterium 6 fermentation liquor precipitates;

(6) respectively re-suspending the recombinant bacterium 3 fermentation liquor sediment and the recombinant bacterium 6 fermentation liquor sediment obtained in the step (5) by using PBS (100mM, PH 7.5,) for cell re-suspension treatment, carrying out ultrasonic disruption treatment for 5s pulse and 10s interval, wherein the total working time is 20 minutes; obtaining a recombinant bacterium 3 crude enzyme solution (namely pflB crude enzyme solution) and ldh crude enzyme solution;

(7) re-suspending the recombinant bacterium 1 fermentation liquid precipitate obtained in the step (5) by PBS (the volume of which is 1/7 of the volume of the recombinant bacterium 1 fermentation liquid obtained in the step (4) and the pH value of which is 7.5 mM) and the concentration of which is 100mM to obtain a mixed liquid, taking 1m L mixed liquid, adding P L P with the final concentration of 1.5mM and NAD with the final concentration of 1mM to obtain a mixed liquid⁺Reacting at 40 ℃ and 220rpm for 20 minutes, adding 10U of pflB crude enzyme solution, 10U of ldh crude enzyme solution, 10U of formate dehydrogenase, 250mM D-alanine, 25mM benzyl acetone and 0.1mM CoA to obtain a reaction solution, adding 60% DMSO based on the volume of the reaction solution, reacting at 40 ℃ and 220rpm for 12 hours, adding 10M NADH to terminate the reaction, extracting with ethyl acetate, extracting twice, each adding 500 mu L, adding Na to the organic phase after extraction₂SO₄Drying, and detecting by gas chromatography.

Example 4

(1) same as example 1, step (1);

(2) replacing tdcE in example 1 with pflD, otherwise the same as in step (2) of example 1;

(3) p2A 4-ArR-omega TA; p2A 4-pflD; introducing p2A4-ldh into Escherichia coli (E.coli MG1655) respectively to obtain recombinant bacteria 1, recombinant bacteria 4 and recombinant bacteria 6;

(4) respectively fermenting the recombinant bacteria 1, the recombinant bacteria 4 and the recombinant bacteria 6 to obtain recombinant bacteria 1 fermentation liquor, recombinant bacteria 4 fermentation liquor and recombinant bacteria 6 fermentation liquor;

the fermentation process was the same as in example 1;

(5) respectively centrifuging the fermentation liquor obtained in the step (4) at 4 ℃ and 6500rpm, collecting precipitates, washing and washing the precipitates with 0.01M PBS (pH 7.5), and obtaining recombinant bacterium 1 fermentation liquor precipitates, recombinant bacterium 4 fermentation liquor precipitates and recombinant bacterium 6 fermentation liquor precipitates;

(6) respectively re-suspending the recombinant bacterium 4 fermentation liquor sediment and the recombinant bacterium 6 fermentation liquor sediment obtained in the step (5) by using PBS (100mM, PH 7.5,) for cell re-suspension treatment, carrying out ultrasonic disruption treatment for 5s pulse and 10s interval, wherein the total working time is 20 minutes; obtaining crude enzyme solution (namely pflD crude enzyme solution) of recombinant bacteria 4 and ldh crude enzyme solution;

(7) re-suspending the recombinant bacterium 1 fermentation liquid precipitate obtained in the step (5) by PBS (the volume of which is 1/7 of the volume of the recombinant bacterium 1 fermentation liquid obtained in the step (4) and the pH value of which is 7.5 mM) and the concentration of which is 100mM to obtain a mixed liquid, taking 1m L mixed liquid, adding P L P with the final concentration of 1.5mM and NAD with the final concentration of 1mM to obtain a mixed liquid⁺Reacting at 25 ℃ and 180rpm for 40 minutes, adding 10U of pflD crude enzyme solution, 10U of ldh crude enzyme solution, 10U of formate dehydrogenase, 250mM D-alanine, 25mM substrate 2-pentanone and 0.1mM CoA to obtain a reaction solution, adding 15% DMSO by volume of the reaction solution, reacting at 25 ℃ and 180rpm for 12 hours, adding 10M NADH to terminate the reaction, extracting with ethyl acetate, extracting twice, each adding 500 mu L, adding Na to the organic phase after extraction₂SO₄Drying, and detecting by gas chromatography.

Sequence listing

<110> Tianjin university

<120> method for improving conversion rate of chiral amine

<160>18

<170>SIPOSequenceListing 1.0

<210>1

<211>330

<212>PRT

<213> Arthrobacter sp

<400>1

Met Ala Phe Ser Ala Asp Thr Ser Glu Ile Val Tyr Thr His Asp Thr

1 5 10 15

Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn

20 25 30

Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro

35 40 45

Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Tyr Leu His Ser Asp

50 55 60

Val Thr Tyr Thr Val Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu

65 70 75 80

Asp Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Met Arg Ile

85 90 95

Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu

100 105 110

Val Ala Lys Thr Glu Leu Arg Glu Ala Phe Val Ser Val Ser Ile Thr

115 120 125

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

130 135 140

Pro Gln Val Tyr Met Tyr Ala Val Pro Tyr Gln Trp Ile Val Pro Phe

145 150 155 160

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

165 170 175

Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp

180 185 190

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

195 200 205

Ala Pro Leu Leu Leu Asp Gly Asp Gly Leu Leu Ala Glu Gly Ser Gly

210 215 220

Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg

225 230 235 240

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

245 250 255

Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Leu Ala Glu Leu

260 265 270

Leu Asp Ala Asp Glu Val Leu Gly Cys Thr Thr Ala Gly Gly Val Trp

275 280 285

Pro Phe Val Ser Val Asp Gly Asn Pro Ile Ser Asp Gly Val Pro Gly

290 295 300

Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu

305 310 315 320

Ser Ser Ser Leu Leu Thr Pro Val Gln Tyr

325 330

<210>2

<211>1001

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

catatggctt tctctgctga cacctctgaa atcgtttaca cccacgacac cggtctggac 60

tacatcacct actctgacta cgaactggac ccggctaacc cgctggctgg tggtgctgct 120

tggatcgaag gtgctttcgt tccgccgtct gaagctcgta tctctatctt cgaccagggt 180

tacctgcact ctgacgttac ctacaccgtt ttccacgttt ggaacggtaa cgctttccgt 240

ctggacgacc acatcgaacg tctgttctct aacgctgaat ctatgcgtat catcccgccg 300

ctgacccagg acgaagttaa agaaatcgct ctggaactgg ttgctaaaac cgaactgcgt 360

gaagcgtttg tatctgtaag catcacccgt ggttactctt ctaccccggg tgaacgtgac 420

atcaccaaac accgtccgca ggtttacatg tacgctgttc cgtaccagtg gatcgttccg 480

ttcgaccgta tccgtgacgg tgttcacgct atggttgctc agtctgttcg tcgtaccccg 540

cgttcttcta tcgacccgca ggttaaaaac ttccagtggg gtgacctgat ccgtgctgtt 600

caggaaaccc acgacagggg cttcgaagct ccgctcctgc tggacggtga cggtctgctg 660

gctgaaggtt ctggtttcaa cgttgttgtt atcaaagacg gtgttgttcg ttctccgggt 720

cgtgctgctc tgccgggtat cacccgtaaa accgttctgg aaatcgctga atctctgggt 780

cacgaagcta tcctggctga catcaccctg gctgaactgc tggacgctga cgaagttctg 840

ggttgcacca cagctggcgg tgtctggccg ttcgtctctg ttgacggtaa cccgatctct 900

gacggtgttc cgggtccggt tacccagtct atcatccgtc gttactggga actgaacgtt 960

gaatcttctt ctctgctgac cccggttcag tactaaagct t 1001

<210>3

<211>2454

<212>DNA

<213> Escherichia coli (Escherichia coli)

<400>3

ggaattccat atgatgacca cactgaaact ggacacgctc agcgaccgca ttaaagcgca 60

caaaaatgcg ctggtgcata ttgtgaaacc gccagtctgt accgagcgcg cgcagcacta 120

taccgagatg tatcaacaac atctcgataa gccgatcccg gtacgtcgcg cgctggcact 180

ggcgcatcac ctggcgaatc gcaccatctg gatcaaacac gatgagttga tcattggcaa 240

ccaggcaagc gaagttcgcg ccgcgccgat cttcccggaa tatactgtct cgtggatcga 300

aaaagagatt gatgatctgg cagatcgtcc cggtgctggc tttgcggtga gcgaagagaa 360

caaacgcgtt ctgcatgaag tgtgcccgtg gtggcgcggt cagaccgtac aggatcgctg 420

ctacggcatg tttaccgatg agcaaaaagg tctgctggcg accggaatca ttaaagcgga 480

aggcaatatg acctccggcg atgcgcacct ggcggtgaat ttcccgctgc tgctggaaaa 540

agggcttgat ggtctgcgcg aggaagtagc ggaacgtcgc tcgcgcatca acctgacggt 600

gctggaagat ttacacggtg agcaattcct gaaagcgatt gatatcgtgc tggtggcagt 660

cagtgaacac attgaacgtt tcgctgccct ggcgcgtgaa atggccgcga ccgaaacccg 720

cgaaagccgt cgcgatgaac tgctggcgat ggcagaaaac tgcgatctta tcgcccacca 780

gccgccgcag actttctggc aggcgctgca actgtgttac ttcatccagt tgattttgca 840

gatcgaatct aacggtcact cagtatcgtt tggtcgtatg gaccagtatc tctacccgta 900

ctatcgccgc gacgttgaac tcaaccagac gctggatcgc gaacacgcca tcgagatgct 960

gcatagctgc tggctgaaac tgctggaagt gaacaagatc cgctccggct cacactcaaa 1020

agcctctgcg ggaagtccgc tgtatcagaa cgtcactatt ggcgggcaaa atctggttga 1080

tggtcaacca atggacgcgg tgaatccact ctcttacgcg atcctcgaat cctgcggtcg 1140

cctgcgttcc actcagccta acctcagcgt gcgttaccat gcaggaatga gcaacgattt 1200

cctcgacgcc tgcgtacagg tgatccgttg cggcttcggg atgccggcgt tcaacaacga 1260

cgaaatcgtg atcccggaat ttattaaact cggtattgaa ccgcaggacg cttatgacta 1320

cgcagcgatt ggttgtatag aaaccgccgt cggtggcaaa tggggctatc gctgtaccgg 1380

catgagcttt atcaacttcg cccgcgtgat gctggcggcg ctggaaggcg ggcatgatgc 1440

caccagcggc aaagtgttcc tgccacaaga aaaagcgttg tcggcaggta acttcaacaa 1500

cttcgatgaa gtgatggacg cgtgggatac gcaaatccgt tactacaccc gcaaatcaat 1560

cgaaatcgaa tatgtcgtcg acaccatgct ggaagagaac gtgcacgata ttctctgctc 1620

ggcgctggtg gatgactgta ttgagcgagc gaaaagtatc aagcaaggcg gcgcgaaata 1680

tgactgggtt tctggcctgc aggtcggcat tgccaacctc ggcaacagcc tggcggcagt 1740

gaagaaactg gtgtttgaac aaggtgcgat tggtcagcaa cagcttgctg ccgcactggc 1800

agatgacttc gacggcctga ctcacgagca gctgcgtcag cggctgatta acggtgcgcc 1860

gaagtacggc aacgacgatg atactgtcga tacgctgctg gctcgcgctt atcagaccta 1920

tatcgacgaa ctgaaacagt accataatcc gcgctacggt cgtggtccgg ttggcggcaa 1980

ctattacgcg ggtacgtcat caatctccgc taacgtaccg tttggcgcgc agactatggc 2040

aacaccggac gggcgtaaag cccacacccc gctggcagaa ggcgcaagcc cggcctccgg 2100

tactgaccat cttggcccta ctgcggtcat tggctcagtg ggtaaactgc ctacggcagc 2160

gattctcggc ggcgtgttgc tcaaccagaa actgaatccg gcaacgctgg agaacgaatc 2220

tgacaagcag aaactgatga tcctgctgcg taccttcttt gaagtgcata aaggctggca 2280

tattcagtac aacatcgttt cccgcgaaac gctgctggat gcgaaaaaac atcccgatca 2340

gtatcgcgat ctggtagtgc gtgtcgcggg ctattccgcg ttcttcaccg cgctctctcc 2400

agacgctcag gacgatatca tcgcccgtac tgaacatatg ctgtaaacta gtcc 2454

<210>4

<211>2322

<212>DNA

<213> Escherichia coli (Escherichia coli)

<400>4

ggaattccat atgcatcacc atcatcacca catgtccgag cttaatgaaa agttagccac 60

agcctgggaa ggttttacca aaggtgactg gcagaatgaa gtaaacgtcc gtgacttcat 120

tcagaaaaac tacactccgt acgagggtga cgagtccttc ctggctggcg ctactgaagc 180

gaccaccacc ctgtgggaca aagtaatgga aggcgttaaa ctggaaaacc gcactcacgc 240

gccagttgac tttgacaccg ctgttgcttc caccatcacc tctcacgacg ctggctacat 300

caacaagcag cttgagaaaa tcgttggtct gcagactgaa gctccgctga aacgtgctct 360

tatcccgttc ggtggtatca aaatgatcga aggttcctgc aaagcgtaca accgcgaact 420

ggatccgatg atcaaaaaaa tcttcactga ataccgtaaa actcacaacc agggcgtgtt 480

cgacgtttac actccggaca tcctgcgttg ccgtaaatct ggtgttctga ccggtctgcc 540

agatgcatat ggccgtggcc gtatcatcgg tgactaccgt cgcgttgcgc tgtacggtat 600

cgactacctg atgaaagaca aactggcaca gttcacttct ctgcaggctg atctggaaaa 660

cggcgtaaac ctggaacaga ctatccgtct gcgcgaagaa atcgctgaac agcaccgcgc 720

tctgggtcag atgaaagaaa tggctgcgaa atacggctac gacatctctg gtccggctac 780

caacgctcag gaagctatcc agtggactta cttcggctac ctggctgctg ttaagtctca 840

gaacggtgct gcaatgtcct tcggtcgtac ctccaccttc ctggatgtgt acatcgaacg 900

tgacctgaaa gctggcaaga tcaccgaaca agaagcgcag gaaatggttg accacctggt 960

catgaaactg cgtatggttc gcttcctgcg tactccggaa tacgatgaac tgttctctgg 1020

cgacccgatc tgggcaaccg aatctatcgg tggtatgggc ctcgacggtc gtaccctggt 1080

taccaaaaac agcttccgtt tcctgaacac cctgtacacc atgggtccgt ctccggaacc 1140

gaacatgacc attctgtggt ctgaaaaact gccgctgaac ttcaagaaat tcgccgctaa 1200

agtgtccatc gacacctctt ctctgcagta tgagaacgat gacctgatgc gtccggactt 1260

caacaacgat gactacgcta ttgcttgctg cgtaagcccg atgatcgttg gtaaacaaat 1320

gcagttcttc ggtgcgcgtg caaacctggc gaaaaccatg ctgtacgcaa tcaacggcgg 1380

cgttgacgaa aaactgaaaa tgcaggttgg tccgaagtct gaaccgatca aaggcgatgt 1440

cctgaactat gatgaagtga tggagcgcat ggatcacttc atggactggc tggctaaaca 1500

gtacatcact gcactgaaca tcatccacta catgcacgac aagtacagct acgaagcctc 1560

tctgatggcg ctgcacgacc gtgacgttat ccgcaccatg gcgtgtggta tcgctggtct 1620

gtccgttgct gctgactccc tgtctgcaat caaatatgcg aaagttaaac cgattcgtga 1680

cgaagacggt ctggctatcg acttcgaaat cgaaggcgaa tacccgcagt ttggtaacaa 1740

tgatccgcgt gtagatgacc tggctgttga cctggtagaa cgtttcatga agaaaattca 1800

gaaactgcac acctaccgtg acgctatccc gactcagtct gttctgacca tcacttctaa 1860

cgttgtgtat ggtaagaaaa cgggtaacac cccagacggt cgtcgtgctg gcgcgccgtt 1920

cggaccgggt gctaacccga tgcacggtcg tgaccagaaa ggtgcagtag cctctctgac 1980

ttccgttgct aaactgccgt ttgcttacgc taaagatggt atctcctaca ccttctctat 2040

cgttccgaac gcactgggta aagacgacga agttcgtaag accaacctgg ctggtctgat 2100

ggatggttac ttccaccacg aagcatccat cgaaggtggt cagcacctga acgttaacgt 2160

gatgaaccgt gaaatgctgc tcgacgcgat ggaaaacccg gaaaaatatc cgcagctgac 2220

catccgtgta tctggctacg cagtacgttt caactcgctg actaaagaac agcagcagga 2280

cgttattact cgtaccttca ctcaatctat gtaaactagt cc 2322

<210>5

<211>2319

<212>DNA

<213> Escherichia coli (Escherichia coli)

<400>5

ggaattccat atgatgacga atcgtatctc tcgcctcaaa actgcactgt ttgccaatac 60

ccgtgaaatc tcgctggagc gggcgctgct ttataccgcc agccatcggc aaaccgaagg 120

cgaaccggtg atattgcgcc gggcgaaagc aacagcgtat atccttgaac atgttgaaat 180

ttcgattcgt gatgaagaac tgattgccgg taaccgcacc gtaaaaccgc gcgccgggat 240

tatgtcgccg gaaatggacc cttactggct gctgaaagag ctggatcaat tcccgacgcg 300

tccgcaggac cgctttgcta tcagcgaaga agataaacgt atctaccgtg aagagttgtt 360

cccgtactgg gaaaaacgtt cgatgaaaga tttcatcaac gggcagatga cagatgaagt 420

aaaagccgcg accaacacgc agattttcag catcaaccag acggataaag gccaggggca 480

cattattatt gattacccac gcctgttgaa tcacgggctg ggtgagctgg tggcacagat 540

gcagcaacat tgtcagcaac agccggagaa tcacttttat caggccgcgt tgttactgct 600

ggaagcctcg cagaaacaca ttttgcgtta cgccgaactg gcggaaacga tggcggcaaa 660

ctgcacagat gcccagcgtc gcgaagagct gctgactatt gcagagatct cccgccataa 720

cgcgcaacat aagccgcaga cgttctggca ggcgtgccag ttattctggt acatgaacat 780

cattctgcaa tacgaatcca acgccagttc gctatcgttg gggcgcttcg accagtatat 840

gttgccgttc tatcagacat cattaaccca gggcgaagat gcggcgttcc tgaaagaact 900

gctcgaatct ttatgggtga aatgcaacga catcgtgctg ttgcgctcca ccagtagcgc 960

gcgttatttc gccggtttcc cgaccggcta taccgcactg ctcggcgggt taaccgagaa 1020

cggacgtagc gcggtgaacg tgctttcgtt cctttgcctt gacgcctatc aaagcgtgca 1080

attaccgcaa ccgaacctcg gcgtgcgcac taacgccttg atcgacacgc cgttcctgat 1140

gaaaaccgcc gaaaccattc gcttcggtac cggtattccg caaatcttta acgatgaagt 1200

ggtggtgcca gcgttcctca accgtggcgt ttcgctggaa gatgcgcgcg actattccgt 1260

agtgggctgt gtggaattat ctattcccgg cagaacctac ggcttgcatg acatcgcgat 1320

gtttaatctg ctgaaagtga tggaaatctg cctgcatgaa aatgaaggca atgctgcgct 1380

gacttatgaa ggtttactgg agcagatccg cgccaagatc agccactaca tcaccctgat 1440

ggttgagggc agcaatattt gtgatatcgg ccatcgcgac tgggcacctg taccgctgct 1500

ctcatcgttt atcagcgatt gtctggaaaa aggccgcgat attaccgatg gcggcgcgcg 1560

ttataacttc tccggcgtac aggggatcgg tatcgccaac ctgagcgatt ctctccatgc 1620

gttgaaaggg atggtttttg agcaacagcg tttaagtttt gacgaattgc tgtcggtatt 1680

aaaagccaac ttcgcaacgc cagaaggcga aaaagtccgc gctcgcttaa ttaaccgctt 1740

cgagaaatac ggtaacgata tcgacgaggt ggataacatc agcgccgaac tgttgcgcca 1800

ctactgcaaa gaagtggaaa aataccagaa cccgcgcggc ggctacttca cgccgggatc 1860

gtataccgtt tctgctcacg tcccgttggg atcggtggtt ggcgcgacgc cagacggtcg 1920

ttttgccgga gaacagctgg cagacggcgg cttgtcaccc atgctgggcc aggacgcaca 1980

agggccaaca gcggtactga agtcagtcag taagctcgat aacacgctgc tgtctaacgg 2040

tacgttgctg aacgtgaaat tcactccggc gaccctggaa ggtgaagcag gattacgcaa 2100

actggccgac ttcttacggg cgtttaccca gcttaagtta caacatattc agtttaacgt 2160

ggtgaacgcc gacacgttgc gggaagcgca acagcgccca caagattatg ccgggctggt 2220

ggtgcgcgtt gccggataca gcgccttctt tgtcgaactg tcgaaggaga tccaggatga 2280

catcatccgc cggacagcgc atcagctgta aactagtcc2319

<210>6

<211>2316

<212>DNA

<213> Escherichia coli (Escherichia coli)

<400>6

ggaattccat atgatgaagg tagatattga taccagcgat aagctgtacg ccgacgcatg 60

gcttggcttt aaaggtacgg actggaaaaa cgaaattaat gtccgcgatt ttattcaaca 120

taactataca ccgtatgaag gcgatgaatc tttcctcgcc gaagcgacgc ctgccaccac 180

ggaattgtgg gaaaaagtaa tggaaggcat ccgtatcgaa aatgcaaccc acgcgccggt 240

tgatttcgat accaatattg ccaccacaat taccgctcat gatgcgggat atattaacca 300

gccgctggaa aaaattgttg gcctgcaaac ggatgcgccg ttgaaacgtg cgctacaccc 360

gttcggtggc attaatatga ttaaaagttc attccacgcc tatggccgag aaatggacag 420

tgaatttgaa tatctgttta ccgatctgcg taaaacccat aaccagggcg tatttgatgt 480

ttactcaccg gatatgctgc gctgccgtaa atctggcgtg ctgaccggtt taccagatgg 540

ctatggccgt gggcgcatta tcggtgacta tcgccgcgta gcgctgtatg gcatcagtta 600

tctggtacgt gaacgcgaac tgcaatttgc cgatctccag tctcgtctgg aaaaaggcga 660

ggatctggaa gccaccatcc gtctgcgtga ggagctggca gagcatcgtc atgcgctgtt 720

gcagattcag gaaatggcgg cgaaatatgg ctttgatatc tctcgcccgg cgcagaatgc 780

gcaggaagcg gtgcagtggc tctacttcgc ttatctggcg gcagtgaaat cgcaaaatgg 840

cggcgcgatg tcgctgggcc gcacggcatc gttcctcgat atctacattg agcgcgactt 900

taaagctggc gtactcaatg agcagcaggc acaggaactg atcgatcact tcatcatgaa 960

gatccgtatg gtacgcttcc tgcgtacacc ggaatttgat tcgctgttct ccggcgaccc 1020

aatctgggcg acggaagtga tcggcgggat ggggctggac ggtcgtacgc tggtgaccaa 1080

aaactccttc cgctatttgc acaccctgca cactatgggg ccggcaccgg aacctaacct 1140

gaccattctt tggtcggaag aattaccgat tgccttcaaa aaatatgccg cgcaggtgtc 1200

gatcgtcacc tcttccttgc agtatgaaaa tgacgatctg atgcgtactg acttcaacag 1260

cgacgattac gcgattgcct gctgcgtcag cccaatggtg attggtaagc aaatgcagtt 1320

ctttggtgca cgcgctaacc tggcgaaaac gctgctctac gcaattaacg gcggggtgga 1380

cgagaagctg aagattcagg tcgggccgaa aacagcaccg ctgatggacg acgtgctgga 1440

ttacgacaaa gtgatggaca gcctcgatca cttcatggac tggctggcgg tgcagtacat 1500

cagcgcgctg aatatcattc actacatgca cgacaagtac agctacgaag cttcgctgat 1560

ggcgctgcac gatcgtgatg tctatcgcac tatggcatgc ggcatcgcgg gcctgtcggt 1620

ggcgacggac tccctgtctg ccatcaaata tgcccgcgtg aaaccaatcc gtgacgaaaa 1680

cggcctggcg gtggactttg aaatcgacgg tgaatatccg cagtacggca acaacgacga 1740

gcgcgtagac agcattgcct gcgacctggt tgaacgcttt atgaagaaaa ttaaagcgct 1800

gccaacctat cgcaacgccg tccctaccca gtcgattctg actatcactt ctaacgtggt 1860

gtacggccag aaaaccggta atacgccgga cggtcgtcgc gccggaacac cgttcgcgcc 1920

gggcgctaac ccgatgcatg gtcgtgaccg caaaggtgcc gtggcctcat tgacgtcggt 1980

ggcgaaactg ccgttcacctacgccaaaga tgggatctcg tacaccttct caatcgttcc 2040

tgcggcgctg ggcaaagaag atccagtacg taaaaccaac cttgtcggcc tgctggatgg 2100

gtatttccac cacgaagcgg atgtcgaagg cggtcaacac ctcaacgtca acgtaatgaa 2160

tcgggaaatg ctgctggatg ccatcgagca cccggaaaaa tatcctaacc tgacaatccg 2220

tgtctctggc tacgccgtgc gcttcaacgc actgacccgt gaacagcaac aggatgttat 2280

ttcacgtacc tttacccagg cgctctgaac tagtcc 2316

<210>7

<211>990

<212>DNA

<213> Escherichia coli (Escherichia coli)

<400>7

atgaaactcg ccgtttatag cacaaaacag tacgacaaga agtacctgca acaggtgaac 60

gagtcctttg gctttgagct ggaatttttt gactttctgc tgacggaaaa aaccgctaaa 120

actgccaatg gctgcgaagc ggtatgtatt ttcgtaaacg atgacggcag ccgcccggtg 180

ctggaagagc tgaaaaagca cggcgttaaa tatatcgccc tgcgctgtgc cggtttcaat 240

aacgtcgacc ttgacgcggc aaaagaactg gggctgaaag tagtccgtgt tccagcctat 300

gatccagagg ccgttgctga acacgccatc ggtatgatga tgacgctgaa ccgccgtatt 360

caccgcgcgt atcagcgtac ccgtgatgct aacttctctc tggaaggtct gaccggcttt 420

actatgtatg gcaaaacggc aggcgttatc ggtaccggta aaatcggtgt ggcgatgctg 480

cgcattctga aaggttttgg tatgcgtctg ctggcgttcg atccgtatcc aagtgcagcg 540

gcgctggaac tcggtgtgga gtatgtcgat ctgccaaccc tgttctctga atcagacgtt 600

atctctctgc actgcccgct gacaccggaa aactatcatc tgttgaacga agccgccttc 660

gaacagatga aaaatggcgt gatgatcgtc aataccagtc gcggtgcatt gattgattct 720

caggcagcaa ttgaagcgct gaaaaatcag aaaattggtt cgttgggtat ggacgtgtat 780

gagaacgaac gcgatctatt ctttgaagat aaatccaacg acgtgatcca ggatgacgta 840

ttccgtcgcc tgtctgcctg ccacaacgtg ctgtttaccg ggcaccaggc attcctgaca 900

gcagaagctc tgaccagtat ttctcagact acgctgcaaa acttaagcaa tctggaaaaa 960

ggcgaaacct gcccgaacga actggtttaa 990

<210>8

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

ggaattccat atgatgacca cac 23

<210>9

<211>30

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

ggactagttt acagcatatg ttcagtacgg 30

<210>10

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

ggaattccat atgtccgagc tta 23

<210>11

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

ggactagtta catagattga gtgaagg 27

<210>12

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

ggaattccat atgacgaatc gtatc 25

<210>13

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

ggactagtta cagctgatgc gct 23

<210>14

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

ggccgtccat atgaaggtag atattga 27

<210>15

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

ggactagttc agagcgcctg ggtaa 25

<210>16

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>16

ggaattccat atgatgaaac tcgc 24

<210>17

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>17

actagtttaa accagttcgt tcggg 25

<210>18

<211>2167

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>18

ggatccttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 60

gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 120

tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca 180

ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 240

ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 300

ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 360

aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 420

cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 480

gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 540

ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaagct 600

tgaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 660

agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 720

atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 780

cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 840

actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 900

aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 960

cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 1020

ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 1080

cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 1140

ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 1200

cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 1260

ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 1320

tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 1380

ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 1440

aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 1500

ataacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 1560

gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 1620

ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 1680

catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 1740

atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta 1800

taaaaatagg cgtatcacga ggcagaattt cagataaaaa aaatccttag ctttcgctaa 1860

ggatgatttc tggaattcgc ggccgcttct agagtactag tagcggccgc tgcagtccgg 1920

caaaaaaggg caaggtgtca ccaccctgcc ctttttcttt aaaaccgaaa agattacttc 1980

gcgttatgca ggcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 2040

cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 2100

gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 2160

tggcgtt 2167

Claims

1. A method for improving the conversion rate of chiral amine is characterized by comprising the following steps:

(1) in vitro synthesis and amplification of genes:

the nucleotide sequence of the p2A4 vector is shown as SEQ ID NO. 18;

the nucleotide sequence of ldh is shown as SEQ ID NO. 7;

(3) p2A 4-ArR-omega TA; p2A 4-FA; introducing the p2A4-ldh into escherichia coli respectively to obtain a recombinant bacterium 1, a recombinant bacterium FA and a recombinant bacterium 6;

(6) respectively carrying out cell resuspension treatment on the recombinant bacterium FA fermentation liquor sediment obtained in the step (5) and the recombinant bacterium 6 fermentation liquor sediment by using PBS (phosphate buffer solution) and carrying out ultrasonic disruption treatment; obtaining FA crude enzyme solution and ldh crude enzyme solution;

(7) re-suspending the recombinant bacterium 1 fermentation liquid precipitate obtained in the step (5) by PBS to obtain a mixed solution, adding P L P, NAD into the mixed solution⁺Reacting for 20-40 minutes at 25-40 ℃ and 180-220 rpm; adding FA crude enzyme solution, ldh crude enzyme solution, formate dehydrogenase, D-alanine, substrate ketone and CoA to obtain reaction solution, adding cosolvent, reacting at 25-40 deg.C and 180-220rpm for 12-24 hr, adding NADH to terminate the reaction, extracting with ethyl acetate, adding Na into the extracted organic phase₂SO₄Drying, and detecting by gas chromatography.

2. The method for improving the conversion rate of chiral amine according to claim 1, wherein the FA is ybiw, pflB, pflD or tdcE, the nucleotide sequence of ybiw is shown as SEQ ID No.3, the nucleotide sequence of pflB is shown as SEQ ID No.4, the nucleotide sequence of pflD is shown as SEQ ID No.5, and the nucleotide sequence of tdcE is shown as SEQ ID No. 6.

3. The method for improving the conversion rate of chiral amine according to claim 1, wherein the step (7) is that the recombinant bacterium 1 fermentation liquid precipitate obtained in the step (5) is resuspended in PBS with a pH of 7.5 and a concentration of 100mM to obtain a mixed solution, the volume of the PBS is 1/7 of the volume of the recombinant bacterium 1 fermentation liquid obtained in the step (4), 1m L of the mixed solution is proportionally added with P L P with a final concentration of 1.5mM and NAD with a final concentration of 1mM⁺Reacting for 20-40 minutes at 25-40 ℃ and 180-220 rpm; then adding 10U of FA crude enzyme solution, 10U of ldh crude enzyme solution, 10U of formate dehydrogenase, D-alanine with the final concentration of 250mM, substrate ketone with the final concentration of 25mM and CoA with the final concentration of 0.1mM to obtain a reaction solution, adding DMSO with the volume of 15-60% of the reaction solution, reacting for 12-24 hours at the temperature of 25-40 ℃, 180-220rpm, adding NADH with the final concentration of 10M to terminate the reaction, extracting with ethyl acetate twice, adding Na into the organic phase after extraction₂SO₄Drying, and detecting by gas chromatography.

4. The method according to claim 1 or 3, wherein the ketone is 2-pentanone, cyclohexanone or benzyl acetone.