CN117165549A - Cystathionine-gamma-synthase mutant and application thereof - Google Patents

Abstract

The present invention relates to a highly active cystathionine-γ-synthase mutant and its application. In order to overcome the problem of low L-methionine synthesis efficiency caused by low cystathionine-γ-synthase activity, the present invention will provide a highly active cystathionine-γ-synthase mutant, and Enzymes for L‑methionine biosynthesis. The cystathionine-γ-synthase mutant MetB ^M is obtained by mutating Q5R, F87I, E136G, V231E, F321V and A346T on the basis of wild-type cystathionine-γ-synthase derived from Escherichia coli W3110. The Km and Kcat of the enzyme are 0.13mmol/L and 138.2s ^‑1 respectively, the Kcat/Km is 1063.1 (mmol/L) ^‑1 S ^‑1 , and the specific activity is 195.3U/mg. Substrate binding efficiency and enzyme activity are higher than wild-type MetB. The cystathionine-γ-synthase mutant was applied to the synthesis of L-methionine, which increased its yield 11 times and can be widely used in the production of L-methionine.

Description

A kind of cystathionine-γ-synthase mutant and its application

Technical areas:

The invention relates to a highly active cystathionine-γ-synthase mutant and its application, and belongs to the field of metabolic engineering.

Background technique:

L-methionine, also known as "L-methionine", is the only sulfur-containing amino acid among the eight essential amino acids. It belongs to Asparagus together with L-threonine, L-isoleucine and L-lysine. Acid family of amino acids. L-methionine plays an important role in many body functions. In addition to its role in protein biosynthesis, it is also involved in the transfer of methyl groups, phosphorus metabolism, and the bioavailability of selenium and zinc. L-methionine can also be used directly to treat medical conditions such as allergies and rheumatic fever. However, L-methionine cannot be synthesized by itself in the body of higher animals and needs to be ingested from the outside. Today, most industrially produced L-methionine is added to animal feed to promote animal growth and reproduction and reduce production costs. It is precisely because of the special biological and chemical activities of L-methionine and its derivatives that they are widely used in food, medicine, agriculture, cosmetics, feed additives and other industries. At present, L-methionine is mainly synthesized through chemical methods, but there are problems such as toxic synthetic raw materials, high energy consumption, harsh reaction conditions, complex extraction processes, and environmental pollution. In contrast, microbial fermentation has low production costs, mild conditions, and is green and environmentally friendly. In recent years, this method has been widely used to produce various amino acids.

In the L-methionine biosynthetic pathway, cystathionine-γ-synthase encoded by metB is a key enzyme. This enzyme catalyzes L-cysteine and O-succinyl-homoserine to generate cystathionine and succinic acid. Cystathionine forms L-homocysteine under the action of β-cystathionase, and then L-homocysteine forms L-methionine under the catalysis of homocysteine methyltransferase. Insufficient cystathionine-γ-synthase activity limits the efficient synthesis of L-methionine to a great extent.

Contents of the invention:

In order to overcome the problem of low L-methionine synthesis efficiency caused by low cystathionine-γ-synthase activity, the present invention will provide a highly active cystathionine-γ-synthase mutant, and Enzyme used in L-methionine biosynthesis.

One of the technical solutions of the present invention to solve the above problems is to provide a highly active cystathionine-γ-synthase mutant MetB ^M , the amino acid sequence of which is shown in SEQ ID NO. 1, and the cystathionine-γ-synthase mutant MetB M The enzyme mutant MetB ^M is based on the wild-type cystathionine-γ-synthase derived from Escherichia coli W3110 shown in SEQ ID NO. 3, with Q5R, F87I, E136G, V231E, F321V and A346T mutations. get.

The second technical solution provided by the present invention is the encoding gene of the cystathionine-γ-synthase mutant MetB ^M ;

Further, the encoding gene of the cystathionine-γ-synthase mutant is metB ^M , and the nucleotide sequence is shown in the sequence list SEQ ID NO. 2.

The third technical solution provided by the present invention is the application of the cystathionine-γ-synthase mutant MetB ^M , especially in the production of L-methionine.

Beneficial effects:

The cystathionine-γ-synthase MetB ^M encoded by the metB ^M gene of the present invention has the following characteristics: the Km and Kcat of the enzyme are 0.13mmol/L and 138.2s ^-1 respectively, and the Kcat/Km is 1063.1 (mmol/L) ^-1 S ^-1 , specific activity is 195.3U/mg. Substrate binding efficiency and enzyme activity are higher than wild-type MetB.

Picture description:

Figure 1 Schematic diagram of the screening principle of highly active cystathionine-γ-synthase mutants;

Figure 2 Strain biomass.

Detailed ways:

In order to make the purpose, technical solutions and advantages of this patent more clear, this patent will be further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described here are only used to explain the patent and are not intended to limit the invention.

In the present invention, the screening process of the mutant MetB ^M is as follows:

Error-prone PCR was used to obtain a metB mutant library of the cystathionine-γ-synthase encoding gene, which was connected to pUC19 to obtain a recombinant plasmid library containing metB mutants (named pUC-metBs). Then, Escherichia coli W3110 was used as the starting strain, and its cystathionine-γ-synthase encoding gene metB and succinyl-CoA encoding gene sucCD were sequentially deleted (strain MET02 was obtained). The pUC-metBs library was transformed into MET02 and spread on solid selection medium. Larger colonies were picked and inoculated into 96-well cell culture plates in liquid screening medium. The fast-growing strain was selected, the plasmid was extracted, and the gene encoding the cystathionine-γ-synthase mutant was amplified by PCR. The PCR product was recovered and sequenced, and it was found that the enzyme had the following amino acid mutations relative to the wild-type cystathionine-γ-synthase from E. coli W3110: Q5R, F87I, E136G, V231E, F321V, and A346T.

The following definitions are used in this invention:

1. Nomenclature of amino acids and DNA nucleic acid sequences

Use the accepted IUPAC nomenclature for amino acid residues in three-letter/single-letter code format. DNA nucleic acid sequences adopt the recognized IUPAC nomenclature.

2. Identification of cystathionine-γ-synthase mutants

“Original amino acid + position + substituted amino acid” is used to represent the mutated amino acids in cystathionine-γ-synthase mutants. For example, Q5R means that the amino acid at position 5 is replaced by Arg from Gln of wild-type cystathionine-γ-synthase. Q5 means that the amino acid at position 5 is Gln. The number of the position corresponds to the wild-type cystathionine in SEQ ID NO.3. Amino acid sequence number of ether-gamma-synthase.

In the present invention, metB represents the wild-type cystathionine-γ-synthase synthase encoding gene (shown in SEQ ID NO. 4), and MetB represents the wild-type cystathionine-γ-synthase (shown in SEQ ID NO. 3). ); metB ^M is a cystathionine-γ-synthase mutant gene (shown in SEQ ID NO. 2); MetB ^M is a cystathionine-γ-synthase mutant (shown in SEQ ID NO. 1). The comparison of amino acids before and after mutation is as follows:

cystathionine-gamma-synthase amino acids MetB Q5, F87, E136, V231, F321, A346 ^M Q5R, F87I, E136G, V231E, F321V, A346T

The cystathionine-γ-synthase mutant MetB ^M has the following enzymatic properties: Km, Kcat and specific activity are 0.13mmol/L, 138.2s ^-1 and 195.3U/mg respectively, Kcat/Km is 1063.1 ( mmol/L) ^-1 S ^-1 .

Part of the genes or amino acid sequences involved in the present invention are as follows:

Cystathionine-γ-synthase synthase mutant MetB ^M , SEQ ID NO. 1:

MTRKRATIAVRSGLNDDEQYGCVVPPIHLSSTYNFTGFNEPRAHDYSRRGNPTRDVVQRALAELEGGAGAVLTNTGMSAIHLVTTVILKPGDLLVAPHDCYGGSYRLFDSLAKRGCYRVLFVDQGDEQALRAALAGKPKLVLVESPSNPLLRVVDIAKICHLAREVGAVSVVDNTFLSPALQNPLALGADLVLHSCTKYLNGHSDVVAG VVIAKDPDVVTELAWWANNIGETGGAFDSYLLLRGLRTLVPRMELAQRNAQAIVKYLQTQPLVKKLYHPSLPENQGHEIAARQQKGFGAMLSFELDGDEQTLRRFLGGLSLVTLAESLGGVESLISHAATMTHAGMTPEARAAAGISETLLRISTGIEDGEDLIADLENGFRAANKG.

Cystathionine-γ-synthase mutant gene metB ^M , SEQ ID NO.2:

ATGACGCGTAAACGGGCCACCATCGCAGTGCGTAGCGGGTTAAATGACGACGAACAGTATGGTTGCGTTGTCCCACCGATCCATCTTTCCAGCACCTATAACTTTACCGGATTTAATGAACCGCGCGCGCATGATTACTCGCGTCGCGGCAACCCAACGCGCGATGTGGTTCAGCGTGCGCTGGCAGAACTGGAAGGTGGTGCTGGTGCAGTACTTACTAATACCGGCATGTCCGCGATTCACCTGGTAACGACCGT CATTTTGAAACCTGGCGATCTGCTGGTTGCGCCGCACGACTGCTACGGCGGTAGCTATCGCCTGTTCGACAGTCTGGCGAAACGCGGTTGCTATCGCGTGTTGTTTGTTGATCAAGGCGATGAACAGGCATTACGGGCAGCGCTGGCAGGAAAACCCAAACTGGTACTGGTAGAAAGCCCAAGTAATCCATTGTTACGCGTCGTGGATATTGCGAAAATCTGCCATCTGGCAAGGGAAGTCGGGGCGGTGAGCGTGGT GGATAACACCTTCTTAAGCCCGGCATTACAAAATCCGCTGGCATTAGGTGCCGATCTGGTGTTGCATTCATGCACGAAATATCTGAACGGTCACTCAGACGTAGTGGCCGGCGTGGTGATTGCTAAAGACCCGGACGTTGTCACTGAACTGGCCTGGTGGGCAAACAATATTGGCGAGACGGGGCGCGTTTGACAGCTATCTGCTGCTACGTGGGTTGCGAACGCTGGTGCCGCGTATGGAGCTGGCGCAGCGCAACG CGCAGGCGATTGTGAAATACCTGCAAACCCAGCCGTTGGTGAAAAAAACTGTATCACCCGTCGTTGCCGGAAAATCAGGGGCATGAAATTGCCGCGCGCCAGCAAAAAGGCTTTGGCGCAATGTTGAGTTTTGAACTGGATGGCGATGAGCAGACGCTGCGTCGTTTCCTGGGCGGGCTGTCGTTGGTTACGCTGGCGGAATCATTAGGGGGAGTGGAAAGTTTAATCTCTCACGCCGCAACCATGACACATGCAGGCAT GACACCAGAAGCGCGTGCTGCCGCCGGGATCTCCGAGACGCTGCTGCGTATCTCCACCGGTATTGAAGATGGCGAAGATTTAATTGCCGACCTGGAAAATGGCTTCCGGGCTGCAAACAAGGGGTAA.

Wild-type cystathionine-γ-synthase MetB, SEQ ID NO.3:

MTRKQATIAVRSGLNDDEQYGCVVPPIHLSSTYNFTGFNEPRAHDYSRRGNPTRDVVQRALAELEGGAGAVLTNTGMSAIHLVTTVFLKPGDLLVAPHDCYGGSYRLFDSLAKRGCYRVLFVDQGDEQALRAALAEKPKLVLVESPSNPLLRVVDIAKICHLAREVGAVSVVDNTFLSPALQNPLALGADLVLHSCTKYLNGHSDVVAG VVIAKDPDVVTELAWWANNIGVTGGAFDSYLLLRGLRTLVPRMELAQRNAQAIVKYLQTQPLVKKLYHPSLPENQGHEIAARQQKGFGAMLSFELDGDEQTLRRFLGGLSLFTLAESLGGVESLISHAATMTHAGMAPEARAAAGISETLLRISTGIEDGEDLIADLENGFRAANKG.

Wild-type cystathionine-γ-synthase synthetase encoding gene metB, SEQ ID NO.4:

ATGACGCGTAAACAGGCCACCATCGCAGTGCGTAGCGGGTTAAATGACGACGAACAGTATGGTTGCGTTGTCCCACCGATCCATCTTTCCAGCACCTATAACTTTACCGGATTTAATGAACCGCGCGCGCATGATTACTCGCGTCGCGGCAACCCAACGCGCGATGTGGTTCAGCGTGCGCTGGCAGAACTGGAAGGTGGTGCTGGTGCAGTACTTACTAATACCGGCATGTCCGCGATTCACCTGGTAACGACCGT CTTTTTGAAACCTGGCGATCTGCTGGTTGCGCCGCACGACTGCTACGGCGGTAGCTATCGCCTGTTCGACAGTCTGGCGAAACGCGGTTGCTATCGCGTGTTGTTTGTTGATCAAGGCGATGAACAGGCATTACGGGCAGCGCTGGCAGAAAAACCCAAACTGGTACTGGTAGAAAGCCCAAGTAATCCATTGTTACGCGTCGTGGATATTGCGAAAATCTGCCATCTGGCAAGGGAAGTCGGGGCGGTGAGCGTGG TGGATAACACCTTCTTAAGCCCGGCATTACAAAATCCGCTGGCATTAGGTGCCGATCTGGTGTTGCATTCATGCACGAAATATCTGAACGGTCACTCAGACGTAGTGGCCGGCGTGGTGATTGCTAAAGACCCGGACGTTGTCACTGAACTGGCCTGGTGGGCAAACAATATTGGCGTGACGGGCGGCGCGTTTGACAGCTATCTGCTGCTACGTGGGTTGCGAACGCTGGTGCCGCGTATGGAGCTGGCGCAGCGCAA CGCGCAGGCGATTGTGAAATACCTGCAAACCCAGCCGTTGGTGAAAAAAACTGTATCACCCGTCGTTGCCGGAAAATCAGGGGCATGAAATTGCCGCGCGCCAGCAAAAAGGCTTTGGCGCAATGTTGAGTTTTGAACTGGATGGCGATGAGCAGACGCTGCGTCGTTTCCTGGGCGGGCTGTCGTTGTTTACGCTGGCGGAATCATTAGGGGGAGTGGAAAGTTTAATCTCTCACGCCGCAACCATGACACATGCAGG CATGGCACCAGAAGCGCGTGCTGCCGCCGGGATCTCCGAGACGCTGCTGCGTATCTCCACCGGTATTGAAGATGGCGAAGATTTAATTGCCGACCTGGAAAATGGCTTCCGGGCTGCAAACAAGGGGTAA.

The present invention will be further explained below through specific examples.

Example 1: Construction of metB knockout strain METO1

(1) Construction of overlapping fragments U _metB -D _metB

Using the wild-type E. coli W3110 genome as a template, primers metB-1/metB-2 and metB-3/metB-4 were used to amplify the upstream and downstream homology arms of metB, and then overlapping PCR was used to obtain the fusion of the upstream and downstream homology arms of metB. Fragment U _metB -D _metB .

(2)Construction of pGRB-metB plasmid

The gRNA 20bp forward and reverse sequences pG-metB-1/pG-metB-2 were designed and synthesized based on the metB sequence. After annealing, the two were connected to Plasmid pGRB was transformed into E.coli DH5α, screened in LB solid medium containing 100 μg/mL ampicillin, and sequenced to identify the recombinant plasmid pGRB-metB.

(3) Construction of metB knockout strain MET01

The recombinant plasmid pGRB-metB and the fusion fragment U _metB -D _metB were electrotransformed into E.coliW3110 competent cells containing the pREDcas9 plasmid, and after recovery, spread on LB solid culture containing 100 μg/mL spectinomycin and ampicillin. Incubate at 32°C overnight. The next day, colony PCR was used to identify the colonies using primers metB-1/metB-4, and positive transformants were screened. Activate the transformant, add arabinose at a final concentration of 0.2 mmol/L, and culture with shaking at 32°C overnight to cause the loss of pGRB-metB; then culture with shaking at 42°C overnight to lose the pREDcas9 plasmid and obtain the metB knockout strain MET01.

Example 2: Construction of sucCD knockout strain MET02

As shown in Figure 1, succinyl-CoA synthase encoded by sucCD catalyzes succinate-CoA to generate succinate. Therefore, after knocking out the sucCD gene of strain MET01, the strain cannot grow on the selection medium due to succinic acid deficiency. Cystathionine-γ-synthase catalyzes L-cysteine and O-succinyl-homoserine to generate cystathionine and succinic acid. Therefore, if metB is restored in the MET01 strain with the sucCD gene knocked out, it can produce TCA. The cycle provides succinic acid and the strain resumes growth. And the higher the cystathionine-γ-synthase activity, the greater the amount of succinic acid produced and the faster the strain grows. Using this principle, cystathionine-γ-synthase mutants with high activity are obtained by screening strains that grow quickly (large colonies).

(1) Construction of overlapping fragment U _sucCD -D _sucCD

Using the wild-type E. coli W3110 genome as a template, primers sucCD-1/sucCD-2 and sucCD-3/sucCD-4 were used to amplify the upstream and downstream homology arms of sucCD, and then overlapping PCR was used to obtain sucCD upstream and downstream homology arm fusion. Fragment U _sucCD -D _sucCD .

(2) Construction of pGRB-sucCD plasmid

The gRNA 20bp forward and reverse sequences pG-sucCD-1/pG-sucCD-2 were designed and synthesized based on the sucCD sequence. After annealing, the two were connected using the recombination kit ClonExpress IIOne Step Cloning Kit (Nanjing Novozan Medical Technology Co., Ltd.) Plasmid pGRB was transformed into E.coli DH5α, screened in LB solid medium containing 100 μg/mL ampicillin, and sequenced to identify the recombinant plasmid pGRB-sucCD.

(3) Construction of sucCD knockout strain MET02

The recombinant plasmid pGRB-sucCD and the fusion fragment U _sucCD -D _sucCD were electrotransformed into MET01 competent cells containing the pREDcas9 plasmid. After recovery, they were spread on LB solid culture containing 100 μg/mL spectinomycin and ampicillin at 32°C. Incubate at constant temperature overnight. The next day, colony PCR identification was performed using primers sucCD-1/sucCD-4 to screen positive transformants. Activate the transformant, add arabinose at a final concentration of 0.2 mmol/L, and culture with shaking at 32°C overnight to cause the loss of pGRB-sucCD; then culture with shaking at 42°C overnight to lose the pREDcas9 plasmid and obtain strain MET02.

Example 3: Screening of metB ^M

The wild-type E. coli W3110 genome was used as a template, and the primers ER-1/ER-2 were used to perform error-prone PCR (ready-to-use error-prone PCR kit, Beijing Tianenze Gene Technology Co., Ltd.) to amplify the metB mutant library. The error-prone PCR reaction system is 30 μL: 3 μL error-prone PCR Mix, 3 μL dNTP, 3 μL 5mmol/LMnCl ₂ , 1 μL template, 10 μL each of ER-1 and ER-2, 1 μL Taq DNA polymerase, and deionized water to make up to 30 μL. PCR conditions were: 94°C for 3 min, 1 cycle; 94°C for 1 min s, 45°C for 90 s, and 72°C for 1 min, 30 cycles.

The amplified metB mutant was then recombinantly ligated with the expression plasmid pUC19 (digested by Hind III) to obtain a recombinant plasmid library containing the metB mutant (named pUC-metBs).

pUC-metBs was transformed into MET02 sensor cells. MET02 transformed with pUC plasmid containing wild-type metB (named pUC-metB) was used as a control (named CK). After recovery, the cells were spread on solid screening medium containing 100 μg/mL ampicillin and cultured at a constant temperature of 37°C. The next day, 480 larger single colonies on the plate were picked and transferred to a 96-well cell culture plate in a liquid selection medium containing ampicillin (100 μg/mL), and cultured with shaking at 37°C for 24 hours. Use a microplate reader to measure OD ₆₀₀ . Select the 20 strains with the highest OD ₆₀₀ (named M1-M20 respectively), inoculate them into shake tubes containing 5 mL liquid screening medium at an inoculum volume of 1% for re-screening, and use a spectrophotometer after shaking culture at 37°C for 24 hours. OD ₆₀₀ was measured and the results are shown in Figure 2. Among them, M4 had the highest OD ₆₀₀ value (1.89), which was 1.17 times higher than the control strain.

The plasmid in M4 was extracted and its mutant metB ^M was sequenced (the plasmid was named pUC-metB ^M ). The nucleotide sequence result is shown in SEQ ID NO.2, and the corresponding amino acid sequence is shown in SEQ ID NO.1.

This enzyme was found to have the following amino acid mutations relative to wild-type cystathionine-γ-synthase from Escherichia coli W3110: Q5R, F87I, E136G, V231E, F321V, A346T.

Solid screening medium: glucose 10g/L, MgSO ₄ 0.24g/L, KH ₂ PO ₄ 2.5g/L, (NH ₄ ) ₂ SO ₄ 5g/L, FeSO ₄ 2g/L, agar 20g/L, deionized 1000mL of water, pH 6.5-7.0.

Liquid screening medium: glucose 10g/L, MgSO ₄ 0.24g/L, KH ₂ PO ₄ 2.5g/L, (NH ₄ ) ₂ SO ₄ 5g/L, FeSO ₄ 2g/L, deionized water 1000mL, pH 6.5 -7.0.

Example 4: Analysis of enzymatic properties of cystathionine-γ-synthase

Using pUC-metB and pUC-metB ^M as templates respectively, primers CGS-1 and CGS-2 were used to PCR amplify metB and metB ^M. After electrophoresis recovery, the recombination kit ClonExpress IIOne Step Cloning Kit (Nanjing Novozan Medical Technology Co., Ltd. ) was connected to the expression vector pET-28a digested by Sac I to obtain plasmids p28-metB and p28-metB ^M . They were transformed into E. coli BL21 (DE3) competent cells to obtain recombinant strains DE-CGS and DE-CGS ^M respectively.

DE-CGS and DE-CGS ^M seed cultures were inoculated into LB liquid medium respectively, and expression was induced with 0.1 mmol/LIPTG for 4 h. Take 1 mL of the culture and centrifuge it at 10,000 g for 1 min at 4°C to collect the bacterial cells. Wash the bacterial pellet three times with 1 mL of buffer (200 mmol/LTris-HCl, pH 8.1) and then resuspend in 1 mL of buffer. Use an ultrasonic crusher to ultrasonically crush the above-mentioned bacterial suspension. The working conditions are: power 350W, working time 5sec, interval 10sec, 5 cycles, and operate on ice. The broken liquid was centrifuged at 8000 g and 4°C, and the supernatant was taken, and Ni-NTA affinity chromatography was used to separate and purify the recombinant cystathionine-γ-synthase MetB and its mutant MetB ^M .

Take an appropriate amount of recombinant enzyme solution and measure its enzyme activity. The reaction conditions are: 50mmol/L Tris-HCl solution (pH7.8), containing 20mmol/L O-succinyl-homoserine, 0.025-20mmol/L L-cysteine (L-cysteine when measuring enzyme activity amino acid concentration is 20mmol/L), 10μL cystathionine-γ-synthase solution, 20μmol/L pyridoxal phosphate. After incubating at 25°C for 30 min, the reaction was terminated with acetone. L-cysteine consumption was measured using high-performance liquid chromatography. Enzyme activity is defined as: the amount of enzyme required to convert 1 μmol L-cysteine in 1 minute under the conditions of 25°C and pH 7.8 is one enzyme activity unit (U).

Detection of L-cysteine in the reaction solution: The reaction solution was centrifuged at 8000×g for 10 minutes, the supernatant was taken and diluted 10 times with deionized water, and 0.8% (V/V) 2,4-dinitrofluoride was used. Benzene was used to derivatize the reaction solution, and high-performance liquid chromatography was used to determine the L-cysteine content. The detection conditions were: Agilent C18 (150 mm × 4.6 mm, 5 μm), using acetonitrile/sodium acetate binary gradient elution, and the column The temperature is 33℃ and the detection wavelength is 360nm.

The enzymatic reaction rates of MetB and MetB ^M were measured under different L-cysteine concentrations, and the double reciprocal method was used to calculate the Km value of the enzyme; at the same time, the enzyme activity was measured when the L-cysteine concentration was 20mmol/L. See the table below for specific results.

Km Kcat Kcat/Km Specific enzyme activity (U/mg) MetB 0.25mmol/L 111.9s ^-1 447.6(mmol/L) ^-1 S ^-1 ) 158.1 ^M 0.13mmol/L 138.2s ^-1 1063.1(mmol/L) ^-1 S ^-1 195.3

From the above results, it can be seen that the Km and Kcat of MetB ^M are somewhat different from those of MetB. The smaller the Km, the stronger the substrate specificity; the larger the Kcat, the faster the enzyme converts the substrate. The specific activity of MetB ^M is 23.5% higher than that of MetB. It can be seen that the affinity and catalytic efficiency of the cystathionine-γ-synthase mutant obtained in the present invention to the substrate are higher than those of the wild-type cystathionine-γ-synthase.

Example 5: Application of highly active cystathionine-γ-synthase in the synthesis of L-methionine

(1) Use pUC-metB and pUC-metB ^M as templates, use primers pS-1/pS-2 to amplify metB and metB ^M respectively, and use the recombination kit ClonExpress IIOne Step Cloning Kit (Nanjing Novozan Medical Technology Co., Ltd. Company) was connected to the BamHI-digested plasmid pSTV28 to obtain plasmids pSTV-metB and pSTV-metB ^M . pSTV-metB and pSTV-metB ^M were transformed into E. coli W3110, respectively, to obtain recombinant strains LMT-1 and LMT-2.

(2) Inoculate LMT-1 and LMT-2 into 30 mL of basic medium respectively, and culture them with shaking at 37°C and 220 rpm shaker for 24 hours.

The basic culture ingredients are: glucose 15g/L, MgSO ₄ 0.3g/L, KH ₂ PO ₄ 2g/L, (NH ₄ ) ₂ SO ₄ 4g/L, MgSO ₄ 1g/L, FeSO ₄ ·7H ₂ O 10mg/ L, MnSO ₄ 10mg/L, deionized water 1000mL, pH 6.5-7.0.

(3) Detection of L-methionine in fermentation broth

The fermentation broth was centrifuged at 8000 × g for 10 minutes, the supernatant was taken and diluted with deionized water, and 0.8% (V/V) 2,4-dinitrofluorobenzene was used for derivatization of the fermentation broth, and high-performance liquid chromatography was used for determination. For L-methionine content, the detection conditions are: Agilent C18 (150mm×4.6mm, 5μm), using acetonitrile/sodium acetate binary gradient elution, the column temperature is 33°C, and the detection wavelength is 360nm.

The L-methionine yields of LMT-1 and LMT-2 are 0.02g/L and 0.25g/L respectively, indicating that the cystathionine-γ-synthase mutant MetB ^M obtained in the present invention synthesizes L-methionine. Acid capacity was significantly improved relative to the wild type.

List of primer sequences used in the examples of the present invention:

The above-mentioned embodiments only express several implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. It should be noted that for those of ordinary skill in the art, several modifications, combinations and improvements can be made to each of the above embodiments without departing from the concept of this patent, and these all fall within the protection scope of this patent. Therefore, the scope of protection of this patent should be determined by the claims.

Claims (5)

1. The cystathionine-gamma-synthase mutant is characterized in that the amino acid sequence of the cystathionine-gamma-synthase is shown as SEQ ID NO. 1.

2. A gene encoding the cystathionine-gamma-synthase mutant according to claim 1.

3. The coding gene as claimed in claim 2, wherein the nucleotide sequence is shown in the sequence table SEQ ID NO. 2.

4. Use of the cystathionine- γ -synthase mutant according to claim 1.

5. The use according to claim 4, wherein the cystathionine- γ -synthase mutant is used for producing L-methionine.