CN117946991A - A-isopropyl malic acid synthase mutant and application thereof - Google Patents

Abstract

The invention relates to the technical field of bioengineering, in particular to an a-isopropyl malic acid synthase mutant and application thereof. The mutant is obtained by mutating the 246 th position of the alpha-isopropyl malic acid synthase from leucine to other amino acids. After the 246 th amino acid of the microorganism a-isopropyl malate synthase is mutated into other amino acids, the efficiency of lysine production is obviously improved, and particularly, the improvement range is larger when the microorganism a-isopropyl malate synthase is mutated into methionine, valine or proline. Further research of the invention shows that the editing of dihydroxy-acid dehydratase, acetohydroxy-acid isomerase reductase, acetohydroxy-acid synthase, gndA gene or ppc gene can further improve the yield of branched-chain amino acid produced by microorganism on the basis of the microorganism with the mutation, which has important significance in the field of amino acid production.

Description

A-isopropyl malic acid synthase mutant and application thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to an a-isopropyl malic acid synthase mutant and application thereof.

Background

Branched-chain amino acids (branch chain amino acid, BCAA) include valine, leucine, and isoleucine. Wherein L-valine (L-valine) has a chemical name of L-alpha-aminoisovaleric acid, a molecular formula of C ₅H₁₁NO₂ and a relative molecular mass of 117.15. L-valine is white crystal or crystalline powder, odorless, bitter in taste, and has a solubility of 88.5g/L in water at 25deg.C, a solubility of 96.2g/L at 50deg.C, insolubility in cold ethanol, diethyl ether, and acetone, an isoelectric point of 5.96, and a melting point of 315 deg.C.

L-valine is one of eight essential amino acids of the human body and has a particularly important position in human life metabolism due to its special structure and function. L-valine can be widely applied to the pharmaceutical industry, the food industry, the feed industry and the like. Wherein, in the pharmaceutical industry, L-valine can be used as the main component of amino acid transfusion and comprehensive amino acid preparations, and can treat liver failure and central nervous system dysfunction. In the food industry, L-valine is useful as a food additive, a nutritional supplement, a flavoring agent, and the like. L-valine can also be used as amino acid functional beverage and athlete beverage, and has effects of forming muscle, strengthening liver function, relieving muscle fatigue, etc. In the feed industry, L-valine has an important promoting effect on the milk secretion of mammary tissue of animals.

At present, the production method of L-valine mainly comprises three steps: extraction, chemical synthesis, and microbial fermentation. The extraction method and the chemical synthesis method have the problems of limited raw material sources, high production cost, environmental pollution and the like, so that the industrialized production is difficult to realize. The microbial fermentation method for producing L-valine has the advantages of low raw material cost, mild reaction conditions, easy realization of large-scale production and the like, and is the most main method for producing L-valine at present. However, the fermentation performance of the L-valine strain is still poor at present, and the content of the byproduct leucine is high, so that the conversion rate is still low, and the requirement of large-scale industrial production is difficult to meet.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides an a-isopropyl malate synthase mutant and application thereof.

In a first aspect, the invention provides a mutant of a-isopropyl malate synthase, which is obtained by mutating position 246 of a-isopropyl malate synthase from leucine to another amino acid.

Further, the a-isopropyl malate synthase comprises an amino acid sequence as shown in SEQ ID NO.1.

Further, the mutant is characterized in that the 246 th site of the alpha-isopropyl malate synthase is mutated from leucine into methionine, valine or proline. The amino acid sequences corresponding to the three mutants are SEQ ID NO.3-5.

The invention further provides nucleic acids encoding the mutants, the corresponding nucleotide sequences being SEQ ID NO.6-8.

In a second aspect, the invention provides a recombinant microorganism in which the position 246 of the a-isopropylmalate synthase is mutated from leucine to one or more of methionine, valine or proline.

Further, the a-isopropyl malate synthase comprises an amino acid sequence as shown in SEQ ID NO.1.

Further, the coding gene of the alpha-isopropyl malate synthase comprises a nucleotide sequence shown as SEQ ID NO. 2.

Further, the recombinant microorganism may be subjected to any one or more of the following editing modes:

i) Mutation of the ilvN protein from valine to isoleucine at position 25;

ii) the ilvC protein is mutated from isoleucine to serine at position 90;

iii) Increasing the expression level of the ppc gene;

iv) increasing the expression level of gndA genes;

v) mutation of the ilvD protein at position 237 from alanine to lysine.

Wherein the level of the gene is increased in iii) and iv), the expression level of the gene can be increased by replacing a strong promoter, increasing the copy number or by means of a mutation of the gene, for example by replacing the promoters of the ppc gene and gndA gene with Ptac.

Further, the ilvD protein has reference sequence number WP_003854128.1 on NCBI and the nucleotide sequence has reference sequence number CEY17_RS06870 on NCBI.

Further, the ilvC protein has a reference sequence number wp_003854117.1 on NCBI and the nucleotide sequence has a reference sequence number cey17_rs06895 on NCBI.

Further, the ilvN protein has reference sequence number WP_003861429.1 on NCBI and the nucleotide sequence has reference sequence number CEY17_RS06890 on NCBI.

Further, the ppc, gndA genes have reference sequence numbers CEY17_RS08480, CEY17_RS07800, respectively, at NCBI.

Further, the recombinant microorganism is Corynebacterium glutamicum, corynebacterium beijing, brevibacterium flavum or Escherichia coli.

The invention further provides the use of said mutant or said nucleic acid for increasing the ability of a microorganism to produce an amino acid or derivative thereof.

Further, the microorganism is Corynebacterium glutamicum, corynebacterium beijing, brevibacterium flavum or Escherichia coli.

Further, the amino acid is a branched chain amino acid; valine is preferred.

The present invention further provides a method for improving the ability of a microorganism to produce an amino acid or derivative thereof, comprising:

Mutating leucine to one or more of methionine, valine or proline at position 246 of a-isopropyl malate synthase in said microorganism.

Further, any one of the following editing modes is also included:

i) Mutation of the ilvN protein from valine to isoleucine at position 25;

ii) the ilvC protein is mutated from isoleucine to serine at position 90;

iii) Increasing the expression level of the ppc gene;

iv) increasing the expression level of gndA genes;

v) mutation of the ilvD protein at position 237 from alanine to lysine.

The invention has the following beneficial effects:

The invention discovers that the mutant of the alpha-isopropyl malic acid synthase, namely the 246 th amino acid of the alpha-isopropyl malic acid synthase is mutated into other amino acids, can obviously improve the capability of producing branched-chain amino acids by microorganisms, and particularly improves the effect yield greatly when the mutant is mutated into methionine, valine or proline. The invention further carries out editing of dihydroxy-acid dehydratase, acetohydroxy-acid isomerase reductase, acetohydroxy-acid synthase, gndA gene or ppc gene on the basis of the mutant, can further improve the yield of branched-chain amino acid produced by microorganisms, and has important significance in the field of improving the yield of branched-chain amino acid.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention.

The names and sequences of the primers involved in the examples are shown in Table 1.

Table 1 primers involved in the examples

The invention discloses a corynebacterium glutamicum which is an initial strain MHZ-1012-3, the construction method of the corynebacterium glutamicum is shown in China patent CN201911370732.9, the corynebacterium glutamicum is obtained by mutating 1 st base of a coding region of an a-isopropyl malate synthase gene leuA of the initial strain MHZ-1012-2 from A to G. MHZ-1012-2 is preserved in China general microbiological culture Collection center (CGMCC) of China Committee for culture Collection of microorganisms (including China) for 11 and 30 days in 2016, the preservation center is the China national academy of sciences of China, including the Korean area North Star, including the West way No.1, no. 3, and the preservation number is CGMCC No.13406, which is shown in China patent CN201611250330.1.

Example 1: construction of acetohydroxyacid synthase mutant strains

The application takes MHZ-1012-3 as an initial strain, and changes the ilvN gene (the nucleotide sequence is shown as SEQ ID NO. 9) in MHZ-1012-3 into a gene for coding an acetohydroxy acid synthase mutant of SEQ ID NO.10, and correspondingly, changes the amino acid sequence from SEQ ID NO.11 into SEQ ID NO.12 to construct an acetohydroxy acid synthase mutant strain, and the specific construction method is as follows.

1. Construction of plasmid pK18mobsacB-ilvN ^V25I

Preparing a recombinant fragment UP-1 by using Phusion super fidelity polymerase (NEW ENGLAND BioLabs), taking a genome of a starting strain MHZ-1012-3 as a template, taking ilvN ^V25I-UP-1F/ilvN^V25I -UP-1R as a primer, and preparing a recombinant fragment DN-1 by taking ilvN ^V25I-DN-2F/ilvN^V25I -DN-2R as a primer; preparing a recombinant fragment ilvN ^V25I by taking a genome of a corynebacterium glutamicum model strain ATCC13032 as a template and ilvN ^V25I-1F/ilvN^V25I -1R as a primer; the fragment pk18-1 was obtained using the plasmid pk18-mob-sacB as a template and ilvN ^V25I-pk18-3F/ilvN^V25I -pk18-3R as a primer, purified by agarose gel recovery kit (Tiangen), and then reacted according to Jeep assembling kit configuration system, the reaction system being shown in Table 2.

TABLE 2 Jeep Assembly reaction System

Component (A)	UP-1	DN-1	ilvNV25I	pk18-1	CE Buffer	CE Exnase	Sterile water
								Volume/. Mu.L	1	1	1	2	4	2	9

The prepared reaction system is reacted for 30min at 37 ℃, 10 mu L of Trans1T1 competent cells are sucked (TransGen Biotech), monoclonal is selected, the inserted fragments are identified to be correct through colony PCR, positive clones of the fragments inserted into pK18mobsacB are obtained through further enzyme digestion identification, finally plasmids are sent to Jin Weizhi biotechnology Co-Ltd for sequencing, and the obtained plasmid with correct sequencing is named pK18mobsacB-ilvN ^V25I.

2. Construction of acetohydroxyacid synthase mutant strains

The recombinant plasmid pK18mobsacB-ilvN ^V25I obtained by the construction of the method described in 1 above was transferred into the starting strain MHZ-1012-3, and the crossover recombinant was selected on a selection medium containing 15mg/L kanamycin. The temperature of the culture was 30℃and the culture was inverted. The transformants obtained by screening were cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a rotary shaking table. The culture was serially diluted (10 ^-2 serial diluted to 10 ^-4) and the diluted solution was spread on a normal solid brain heart infusion medium containing 10% sucrose and incubated at 33℃for 48h. Transformants grown on this medium were identified. The target mutant strain, designated MHZ-1012-31, was obtained by PCR amplification of the target sequence and nucleotide sequencing analysis.

Example 2: construction of acetohydroxy acid isomerase mutant strains

Taking MHZ-1012-3 as an initial strain, mutating ilvC gene (with a nucleotide sequence of SEQ ID NO. 13) in MHZ-1012-3 into a gene for encoding an acetohydroxy acid isomerase reductase mutant of SEQ ID NO.14, and mutating the corresponding amino acid sequence from SEQ ID NO.15 to SEQ ID NO.16 to construct the acetohydroxy acid isomerase mutant strain.

1. Construction of plasmid pK18mobsacB-ilvC ^I90S

Preparing a recombinant fragment UP-1 by using Phusion super fidelity polymerase (NEW ENGLAND BioLabs), taking a genome of a starting strain MHZ-1012-3 as a template, taking ilvC ^I90S-UP-1F/ilvC^I90S -UP-1R as a primer, and preparing a recombinant fragment DN-1 by taking ilvC ^I90S-DN-2F/ilvC^I90S -DN-2R as a primer; the fragment pk18-1 was obtained using the plasmid pk18-mob-sacB as a template and ilvC ^I90S-pk18-3F/ilvC^I90S -pk18-3R as a primer, purified by agarose gel recovery kit (Tiangen), and then reacted according to Jeep assembling kit configuration system, the reaction system being shown in Table 3.

TABLE 3 Jeep Assembly reaction System

The prepared reaction system is reacted for 30min at 37 ℃, 10 mu L of Trans1T1 competent cells are sucked (TransGen Biotech), monoclonal is selected, the inserted fragments are identified to be correct through colony PCR, positive clones of the fragments inserted into pK18mobsacB are obtained through further enzyme digestion identification, finally plasmids are sent to Jin Weizhi biotechnology Co-Ltd for sequencing, and the obtained plasmid with correct sequencing is named pK18mobsacB-ilvC ^I90S.

2. Construction of acetohydroxy acid isomerase mutant strains

The recombinant plasmid pK18mobsacB-ilvC ^I90S obtained by the construction of the method described in 1 above was transferred into the starting strain MHZ-1012-3, and the crossover recombinant was selected on a selection medium containing 15mg/L kanamycin. The temperature of the culture was 30℃and the culture was inverted. The transformants obtained by screening were cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a rotary shaking table. The culture was serially diluted (10 ^-2 serial diluted to 10 ^-4) and the diluted solution was spread on a normal solid brain heart infusion medium containing 10% sucrose and incubated at 33℃for 48h. Transformants grown on this medium were identified. The target mutant strain, designated MHZ-1012-33, was obtained by PCR amplification of the target sequence and nucleotide sequencing analysis.

Example 3: construction of acetohydroxy acid isomerase-reductase mutant strains

The method for constructing the acetohydroxy acid isomerase mutant strain by taking MHZ-1012-31 as an initial strain and mutating ilvC gene in MHZ-1012-31 into a gene for coding the acetohydroxy acid isomerase mutant of SEQ ID NO.14 comprises the following specific construction method.

The recombinant plasmid pK18mobsacB-ilvC ^I90S constructed as described in example 2 above was transferred into the starting strain MHZ-1012-31 and the crossover recombinants were selected on selection medium containing 15mg/L kanamycin. The temperature of the culture was 30℃and the culture was inverted. The transformants obtained by screening were cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a rotary shaking table. The culture was serially diluted (10 ^-2 serial diluted to 10 ^-4) and the diluted solution was spread on a normal solid brain heart infusion medium containing 10% sucrose and incubated at 33℃for 48h. Transformants grown on this medium were identified. The target mutant strain, named MHZ-1012-35, was obtained by PCR amplification of the target sequence and nucleotide sequencing analysis.

Example 4: construction of ppc Gene mutant strains

In the acetohydroxy acid isomerase mutant strain MHZ-1012-35 constructed as described in example 3 above, a ppc gene using Ptac as a promoter was further introduced at a site cg1507 to enhance expression of the ppc gene as follows.

1. Construction of plasmid pK18mobsacB-ppc

Preparing an upper homologous arm recombinant fragment UP4 by taking a genome of a starting strain MHZ-1012-35 as a template, taking PI-PPC-1f/PI-PPC-1r as a primer, preparing a PPC gene and a terminator recombinant fragment PPC by taking PI-PPC-2f/PI-PPC-2r as primers, and preparing a lower homologous arm recombinant fragment DN4 by taking PI-PPC-4f/I-PPC-4r as primers; preparing a recombinant fragment Ptac of the tac promoter by taking plasmid pXMJ19 as a template and PI-ppc-3f/PI-ppc-3r as a primer; the recombinant fragment pK18-4 was prepared using plasmid pK18-mob-sacB as a template and PI-pK18-F/PI-pK18-R as a primer, purified by agarose gel recovery kit (Tiangen), and then reacted according to Jeep assembly kit configuration system, the reaction system is shown in Table 4.

TABLE 4 Jeep Assembly reaction System

Component (A)	UP4	PPC	DN4	Ptac	pk18-4	CE Buffer	CE Exnase	Sterile water
									Volume/. Mu.L	1	1	1	1	2	4	2	8

The prepared reaction system is reacted for 30min at 37 ℃, 10 mu L of Trans1T1 competent cells are sucked (TransGen Biotech), monoclonal is selected, the inserted fragments are identified to be correct through colony PCR, positive clones of the fragments inserted into pK18mobsacB are obtained through further enzyme digestion identification, finally plasmids are sent to Jin Weizhi biotechnology Co, sequencing is carried out, and the obtained plasmid with correct sequencing is named pK18mobsacB-ppc.

2. Construction of ppc Gene-enhanced mutant

The recombinant plasmid pK18mobsacB-ppc obtained by the construction of the method described in the above 1 was transferred into the strain MHZ-1012-35, and the crossover recombinant was selected on a selection medium containing 15mg/L kanamycin. The temperature of the culture was 30℃and the culture was inverted. The transformants obtained by screening were cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a rotary shaking table. The culture was serially diluted (10 ^-2 serial diluted to 10 ^-4) and the diluted solution was spread on a normal solid brain heart infusion medium containing 10% sucrose and incubated at 33℃for 48h. Transformants grown on this medium were identified. The target sequence was amplified by PCR and analyzed by nucleotide sequencing to obtain the target mutant strain designated MHZ-1012-37.

Example 5: construction of gndA Gene-enhanced mutant Strain

In the strain MHZ-1012-37 constructed by the method described in example 4 above, the original promoter of gndA gene was replaced with a strong promoter Ptac to enhance the expression of gndA gene, as follows.

1. Construction of plasmid pK18mobsacB-gndA

The genome of the initial strain MHZ-1012-3 is used as a template, PI-gndA-1f/PI-gndA-1r is used as a primer to prepare an upper homologous arm recombinant fragment UP5, and PI-gndA-3f/PI-gndA-3r is used as a primer to prepare a lower homologous arm recombinant fragment DN5; preparing a recombinant fragment Ptac of the tac promoter by taking plasmid pXMJ19 as a template and PI-gndA-2f/PI-gndA-2r as a primer; 3 recombinant fragments are fused by utilizing overlap PCR, the fusion fragment is connected with a pK18-mob-sacB vector by utilizing an enzyme cutting site BamHI/EcoRI, 10 mu L of transformation Trans1T1 competent cells (TransGen Biotech) are extracted, a monoclonal is selected, the correctness of the inserted fragments is identified by colony PCR, positive clones of the fragments inserted into the pK18mobsacB are obtained by further enzyme cutting identification, finally, plasmids are sent to Jin Weizhi biotechnology company for sequencing, and the plasmid with the accuracy of sequencing is named as pK18mobsacB-gndA.

2. Construction of gndA Gene-enhanced mutant

The recombinant plasmid pK18mobsacB-gndA obtained by the construction of the method described in the above 1 was transferred into the strain MHZ-1012-37, and the crossover recombinant was selected on a selection medium containing 15mg/L kanamycin. The temperature of the culture was 30℃and the culture was inverted. The transformants obtained by screening were cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a rotary shaking table. During this culture, a second recombination of the transformant takes place and the vector sequence is removed from the genome by gene exchange. The culture was serially diluted (10 ^-2 serial diluted to 10 ^-4) and the diluted solution was spread on a normal solid brain heart infusion medium containing 10% sucrose and incubated at 33℃for 48h. Transformants grown on this medium were identified. The target sequence was amplified by PCR and analyzed by nucleotide sequencing to obtain the target mutant strain designated MHZ-1012-38.

Example 6: construction of dihydroxy-acid dehydratase mutant Strain

Taking MHZ-1012-38 as an initial strain, mutating ilvD gene (nucleotide sequence is shown as SEQ ID NO. 17) in MHZ-1012-38 into gene for encoding dihydroxy-acid dehydratase mutant of SEQ ID NO.18, and mutating corresponding amino acid sequence from SEQ ID NO.19 to SEQ ID NO.20 to construct dihydroxy-acid dehydratase mutant strain, wherein the specific construction method is as follows.

1. Construction of plasmid pK18mobsacB-ilvD ^A237K

Preparing a recombinant fragment UP-1 by using Phusion super fidelity polymerase (NEW ENGLAND BioLabs), taking a genome of a starting strain MHZ-1012-38 as a template, taking ilvD ^A237K-UP-1F/ilvD^A237K -UP-1R as a primer, and preparing a recombinant fragment DN-1 by taking ilvD ^A237K-DN-2F/ilvD^A237K -DN-2R as a primer; the fragment pk18-1 was obtained using the plasmid pk18-mob-sacB as a template and ilvD ^A237K-pk18-3F/ilvD^A237K -pk18-3R as a primer, purified by agarose gel recovery kit (Tiangen), and then reacted according to Jeep assembling kit configuration system, the reaction system being shown in Table 5.

TABLE 5 Jeep Assembly reaction System

Component (A)	UP-1	DN-1	pk18-1	CE Buffer	CE Exnase	Sterile water
							Volume/. Mu.L	1	1	2	4	2	10

The prepared reaction system is reacted for 30min at 37 ℃, 10 mu L of Trans1T1 competent cells are sucked (TransGen Biotech), monoclonal is selected, the inserted fragments are identified to be correct through colony PCR, positive clones of the fragments inserted into pK18mobsacB are obtained through further enzyme digestion identification, finally plasmids are sent to Jin Weizhi biotechnology Co-Ltd for sequencing, and the obtained plasmid with correct sequencing is named pK18mobsacB-ilvD ^A237K.

2. Construction of mutant strains

The recombinant plasmid pK18mobsacB-ilvD ^A237K obtained by the construction of the method described in 1 above was transferred into the starting strain MHZ-1012-38, and the crossover recombinant was selected on a selection medium containing 15mg/L kanamycin. The temperature of the culture was 30℃and the culture was inverted. The transformants obtained by screening were cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a rotary shaking table. The culture was serially diluted (10 ^-2 serial diluted to 10 ^-4) and the diluted solution was spread on a normal solid brain heart infusion medium containing 10% sucrose and incubated at 33℃for 48h. Transformants grown on this medium were identified. The target mutant strain, designated MHZ-1012-63, was obtained by PCR amplification of the target sequence and nucleotide sequencing analysis.

Example 7: construction of mutant strains of a-isopropyl malate synthase

The method for constructing the alpha-isopropyl malate synthase mutant strain comprises the steps of taking MHZ-1012-3 as an initial strain, mutating a leuA gene (a nucleotide sequence is shown as SEQ ID NO. 2) in the MHZ-1012-3 into a gene for encoding SEQ ID NO. 6.

1. Construction of plasmid pK18mobsacB-leuA ^L246M

Preparing a recombinant fragment UP-1 by using Phusion super fidelity polymerase (NEW ENGLAND BioLabs), taking a genome of a starting strain MHZ-1012-3 as a template, using leuA ^L246M-UP-1F/leuA^L246M -UP-1R as a primer, and preparing a recombinant fragment DN-1 by using leuA ^L246M-DN-2F/leuA^L246M -DN-2R as a primer; the fragment pk18-1 was obtained by using the plasmid pk18-mob-sacB as a template and leuA ^L246M-pk18-3F/leuA^L246M -pk18-3R as a primer, purified by agarose gel recovery kit (Tiangen), and then reacted according to Jeep assembling kit configuration system, the reaction system being shown in Table 6.

TABLE 6 Jeep assembling reaction System

Component (A)	UP-1	DN-1	pk18-1	CE Buffer	CE Exnase	Sterile water
							Volume/. Mu.L	1	1	2	4	2	10

The prepared reaction system is reacted for 30min at 37 ℃, 10 mu L of Trans1T1 competent cells are sucked (TransGen Biotech), monoclonal is selected, the inserted fragments are identified to be correct through colony PCR, positive clones of the fragments inserted into pK18mobsacB are obtained through further enzyme digestion identification, finally plasmids are sent to Jin Weizhi biotechnology Co-Ltd for sequencing, and the obtained plasmid with correct sequencing is named pK18mobsacB-leuA ^L246M.

2. Construction of mutant strains

The recombinant plasmid pK18mobsacB-leuA ^L246M obtained by the construction of the above 1 was transferred into the starting strain MHZ-1012-3, and the crossover recombinant was selected on a selection medium containing 15mg/L kanamycin. The temperature of the culture was 30℃and the culture was inverted. The transformants obtained by screening were cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a rotary shaking table. The culture was serially diluted (10 ^-2 serial diluted to 10 ^-4) and the diluted solution was spread on a normal solid brain heart infusion medium containing 10% sucrose and incubated at 33℃for 48h. Transformants grown on this medium were identified. The target mutant strain, designated MHZ-1012-81, was obtained by PCR amplification of the target sequence and nucleotide sequencing analysis.

3. The mutant strains were constructed and obtained as MHZ-1012-82 and MHZ-1012-83 by mutating leucine at position 246 of a-isopropyl malate synthase to other amino acids such as valine (V) and proline (P) in the same manner as described in 1 and 2 above.

Example 8: construction of a mutant variant A-isopropyl Malate synthase strains

The recombinant plasmid pK18mobsacB-leuA ^L246M constructed as described in example 7 was transferred into the starting strain using MHZ-1012-31, MHZ-1012-35, MHZ-1012-38 and MHZ-1012-63 as the starting strain, respectively, and the crossover recombinant was selected on a selection medium containing 15mg/L kanamycin. The temperature of the culture was 30℃and the culture was inverted. The transformants obtained by screening were cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a rotary shaking table. The culture was serially diluted (10 ^-2 serial diluted to 10 ^-4) and the diluted solution was spread on a normal solid brain heart infusion medium containing 10% sucrose and incubated at 33℃for 48h. Transformants grown on this medium were identified. The target mutant strain is obtained by PCR amplification of target sequence and nucleotide sequencing analysis, and is named MHZ-1012-91, MHZ-1012-92, MHZ-1012-93 and MHZ-1012-94.

Example 9: production of valine by corynebacterium glutamicum shake flask fermentation

1. Culture medium

Seed culture medium: 15g/L of soybean meal extract, 20g/L of glucose, 7g/L of ammonium sulfate, 0.5g/L of magnesium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 2g/L of urea and the balance of water, and pH7.2.

Fermentation medium: 15g/L of soybean meal extract, 20g/L of glucose, 7g/L of ammonium sulfate, 0.5g/L of magnesium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 2g/L of urea, 15 mug/L of VB3, 100 mug/L of VB1 and HCl and the balance of water, and pH7.2.

2. Shaking flask fermentation

(1) Seed culture: the slant seed 1 loop is selected, inoculated into a 500mL triangular flask filled with 50mL of seed culture medium, and cultured for 10-12h at 30 ℃ under 220r/min in a shaking way.

(2) Fermentation culture: 5mL of the seed solution was inoculated into a 500mL Erlenmeyer flask containing 50mL of the fermentation medium, and cultured at 30℃under shaking at 220r/min for 72 hours.

(3) 1ML of the fermentation broth was centrifuged (12000 rpm,2 min), and the supernatant was collected, and the fermentation broth was assayed for L-valine, leucine and isoleucine by HPLC, and the OD at 562nm was determined spectrophotometrically, and the results are shown in Table 3.

In Table 7, strain A is a strain in which ilvN gene in Corynebacterium ATCC14067 was mutated to encode SEQ ID NO. 10; strain B is a strain which mutates the ilvC gene in Corynebacterium ATCC14067 to a strain encoding SEQ ID NO.14; strain C is a strain in which the ilvN gene in Corynebacterium ATCC14067 is mutated to encode SEQ ID NO.10 and the ilvC gene is mutated to encode SEQ ID NO.14; strain D is a strain in which the ilvD gene in Corynebacterium ATCC14067 has been mutated to encode SEQ ID NO. 18; the strain E is a strain in which ilvN gene in corynebacterium ATCC14067 is mutated to code for SEQ ID NO.10, ilvC gene is mutated to code for SEQ ID NO.14 and ilvD gene is mutated to code for SEQ ID NO. 18; the strain F is a strain in which ilvN gene in corynebacterium ATCC14067 is mutated to code SEQ ID NO.10, ilvC gene is mutated to code SEQ ID NO.14, ilvD gene is mutated to code SEQ ID NO.18, and leuA gene is mutated to code SEQ ID NO. 6; strain G, H, K is a strain obtained by mutating the leuA gene of Corynebacterium ATCC14067 to the strains encoding SEQ ID No.6, 7, 8, respectively.

TABLE 7 branched chain amino acid yield in fermentation products

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Description: * The results are shown to be significantly different (P < 0.01) from the results of the comparison with the respective starting bacteria.

The result shows that the valine accumulation of the starting strain MHZ-1012-3 is 7.5g/L, the valine accumulation of the acetohydroxy acid synthase mutant strain MHZ-1012-31 provided by the invention reaches 9.9g/L, the valine accumulation is increased by 2.4g/L, the lifting amplitude reaches 32%, the isoleucine accumulation of a byproduct is 1.5g/L, the valine accumulation is reduced by 34.8% compared with the bacterial strain MHZ-1012-3, and leucine and bacterial OD have no obvious change.

Furthermore, the accumulation of valine of the acetohydroxy acid isomerase reductase mutant strain MHZ-1012-35 obtained by superposing the mutant ilvC ^I90S reaches 10.5g/L, the accumulation of valine is increased by 0.6g/L compared with that of the strain MHZ-1012-31, the lifting amplitude is 6%, the byproduct isoleucine is not further increased, and leucine and thalli OD are not obviously changed.

Further, the accumulation amount of valine of the strain MHZ-1012-38 obtained by superposing mutants ppc and gndA reaches 13.8g/L, the accumulation amount of valine is increased by 3.3g/L compared with that of the strain MHZ-1012-35, the lifting amplitude is 31.4%, and the byproduct isoleucine is not obviously increased and the leucine and the bacterial OD are not obviously changed.

Further, the accumulation amount of valine of the mutant strain MHZ-1012-63 obtained by superposing the mutant ilvD ^A237K reaches 15.3g/L, the accumulation amount is increased by 1.5g/L compared with that of the developed strain MHZ-1012-38, the lifting amplitude is 10.9%, the byproduct isoleucine is reduced by 0.3g/L, the leucine is reduced by 0.2g/L, and the bacterial OD is reduced by 1.4.

Further, the accumulation amount of valine of the mutant strain MHZ-1012-94 obtained by superposing the mutant leuA ^L246M reaches 18.1g/L, the accumulation amount is increased by 2.8g/L compared with that of the developed strain MHZ-1012-63, the lifting amplitude is 18.3%, the byproduct isoleucine is reduced by 0.2g/L, the leucine is reduced by 0.5g/L, and the bacterial OD is not changed obviously.

Therefore, the acetohydroxy acid synthase mutant ilvN ^V25I, the acetohydroxy acid isomerism reductase mutant ilvC ^I90S, the dihydroxy acid dehydratase mutant ilvD ^A237K and the a-isopropyl malate synthase mutant leuA ^L246M and mutant strains thereof have obvious positive effects on the yield of a main product valine, have certain negative effects on the yield of isoleucine or leucine, and provide references for the construction of production strains for producing three-branched-chain amino acids such as valine, leucine, isoleucine and the like and derivatives taking the three-branched-chain amino acids as precursors.

Meanwhile, on the basis of the acetohydroxy acid synthase mutant ilvN ^V25I and the acetohydroxy acid isomerous reductase mutant ilvC ^I90S, the stacking ppc is enhanced, or the ppc and gndA are enhanced simultaneously, the accumulation amount of valine is obviously increased to 12.1g/L and 13.8g/L respectively, and the improvement amplitude is 15.2% and 31.4% respectively. From this, it can be seen that acetohydroxy acid synthase mutant ilvN ^V25I and acetohydroxy acid isomerase mutant ilvC ^I90S, which are enhanced with ppc or both ppc and gndA, have remarkable positive effects, and provide references for the construction of production strains for producing three-branched-chain amino acids such as valine, leucine and isoleucine, and derivatives using the same as precursors.

In addition, in acetohydroxy acid synthase mutant ilvN ^V25I and acetohydroxy acid isomeroreductase mutant ilvC ^I90S; and ppc enhancement, or both ppc and gndA enhancement, on its basis; and further superimposing the dihydroxy-acid dehydratase mutant ilvD ^A237K on the substrate, further superimposing the a-isopropyl malate synthase and then further increasing the valine yield, and further decreasing the yield of the byproduct isoleucine.

The order of the steps of the construction of the strain of the present invention is not limited, and it is within the scope of the present invention for a person skilled in the art to achieve the object of the present invention according to the present disclosure.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. A mutant of a-isopropyl malate synthase, wherein the mutant is obtained by mutating position 246 of the a-isopropyl malate synthase from leucine to another amino acid.

2. The mutant according to claim 1, wherein the a-isopropyl malate synthase comprises an amino acid sequence as shown in SEQ ID No. 1.

3. The mutant according to claim 1 or 2, wherein the mutant is a mutation of leucine at position 246 of a-isopropylmalate synthase to methionine, valine or proline.

4. A nucleic acid for encoding the mutant according to any one of claims 1 to 3.

5. A recombinant microorganism, wherein the position 246 of a-isopropylmalate synthase in the recombinant microorganism is mutated from leucine to one or more of methionine, valine, or proline.

6. The recombinant microorganism according to claim 5, wherein the a-isopropyl malate synthase comprises an amino acid sequence as shown in SEQ ID No. 1.

7. The microorganism of claim 5 or 6, wherein the recombinant microorganism further comprises any one or more of the following mutations:

i) Mutation of the ilvN protein from valine to isoleucine at position 25;

ii) the ilvC protein is mutated from isoleucine to serine at position 90;

iii) Increasing the expression level of the ppc gene;

iv) increasing the expression level of gndA genes;

v) mutation of the ilvD protein at position 237 from alanine to lysine.

8. The recombinant microorganism according to claim 5 or 6, wherein the recombinant microorganism is corynebacterium glutamicum, corynebacterium beijing, brevibacterium flavum, or escherichia coli.

9. A method for increasing the ability of a microorganism to produce branched-chain amino acids comprising:

Mutating leucine to one or more of methionine, valine or proline at position 246 of a-isopropyl malate synthase in said microorganism.

10. Use of a mutant according to any one of claims 1 to 3 or a nucleic acid according to claim 4 for increasing the ability of a microorganism to produce an amino acid or derivative thereof.