Acetylhydroxy acid synthase mutants and their applications

Abstract

This invention provides an acetylhydroxyl synthase mutant and its applications. This invention achieves a mutation in acetylhydroxyl synthase by modifying the ilvN gene derived from Corynebacterium, resulting in a mutant containing ilvN. V25I The mutant strain exhibited enhanced valine production compared to the unmodified strain, while its ability to produce the byproduct isoleucine decreased, ultimately leading to increased valine yield. This invention provides a reference for constructing production strains that produce branched-chain amino acids such as valine, leucine, and isoleucine, as well as derivatives derived from them.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to acetylhydroxy acid synthase mutants and their applications. Background Technology

[0002] Branched-chain amino acids (BCAAs) include valine, leucine, and isoleucine. L-valine, chemically known as L-α-aminoisovaleric acid, has the molecular formula C5H12H2O. 11 NO2 has a relative molecular mass of 117.15. L-valine is a white crystalline or crystalline powder, odorless, and bitter in taste. Its solubility in water is 88.5 g / L at 25°C and 96.2 g / L at 50°C. It is insoluble in cold ethanol, ether, and acetone. Its isoelectric point is 5.96, and its melting point is 315°C.

[0003] L-valine is one of the eight essential amino acids for the human body. Due to its unique structure and function, it plays a particularly important role in human metabolism. L-valine has wide applications in the pharmaceutical, food, and feed industries. In the pharmaceutical industry, L-valine is used as a major component of amino acid infusions and comprehensive amino acid preparations, and can be used to treat liver failure and central nervous system dysfunction. In the food industry, L-valine is used as a food additive, nutritional supplement, and flavoring agent. L-valine is also used in amino acid functional beverages and sports drinks, which have effects such as muscle building, strengthening liver function, and reducing muscle fatigue. In the feed industry, L-valine plays an important role in promoting milk secretion from animal mammary glands.

[0004] Currently, there are three main methods for producing L-valine: extraction, chemical synthesis, and microbial fermentation. Extraction and chemical synthesis are difficult to scale up industrially due to limitations in raw material sources, high production costs, and environmental pollution. Microbial fermentation, on the other hand, offers advantages such as low raw material costs, mild reaction conditions, and ease of large-scale production, making it the most common method for L-valine production. However, the fermentation performance of current L-valine-producing strains remains relatively poor, and the high content of the byproduct leucine results in a low conversion rate, making it difficult to meet the demands of large-scale industrial production. Summary of the Invention

[0005] The purpose of this invention is to provide acetylhydroxy acid synthase mutants and their applications.

[0006] Another objective of this invention is to provide novel microorganisms capable of efficiently producing branched-chain amino acids and their derivatives.

[0007] In order to achieve the objectives of the present invention, in a first aspect, the present invention provides an acetylhydroxyl synthase mutant, said mutant comprising a mutation in the 25th amino acid of acetylhydroxyl synthase from V to I.

[0008] In this invention, the reference sequence number of acetylhydroxy acid synthase on NCBI is WP_003861429.1.

[0009] In a second aspect, the present invention provides a nucleic acid molecule encoding the mutant or a biological material containing the nucleic acid molecule, the biological material including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria or transgenic cell lines.

[0010] Thirdly, the present invention provides any of the following applications of the nucleic acid molecule or the biological material:

[0011] (1) Used for the fermentation production of branched-chain amino acids and their derivatives;

[0012] (2) Used to increase the fermentation yield of branched-chain amino acids and their derivatives;

[0013] (3) Used to construct genetically engineered bacteria that produce branched-chain amino acids and their derivatives.

[0014] The branched-chain amino acids include valine, leucine, isoleucine, etc.

[0015] Fourthly, the present invention provides a method for constructing strains that produce branched-chain amino acids and their derivatives, by using genetic engineering techniques to introduce mutations into the microbial genome so that the encoded acetylhydroxyl synthase contains the V25I mutation site.

[0016] Furthermore, the method also includes enhancing the ppc and / or gndA genes in the strain (preferably ppc enhancement, or simultaneous enhancement of ppc and gndA); wherein the reference sequence numbers of the ppc and gndA genes on NCBI are CEY17_RS08480 and CEY17_RS07800, respectively.

[0017] The enhancement method is selected from the following 1) to 6), or any combination thereof:

[0018] 1) Enhancement is achieved by introducing a plasmid containing the gene;

[0019] 2) Enhanced by increasing the copy number of the aforementioned genes on the chromosome;

[0020] 3) Enhancement is achieved by altering the promoter sequence of the aforementioned genes on the chromosome;

[0021] 4) Enhancement is achieved by operatively linking a strong promoter to the gene;

[0022] 5) Enhancement through the introduction of enhancers;

[0023] 6) Enhancement is achieved by using genes or alleles that encode the corresponding enzymes or proteins with high activity.

[0024] Preferably, the enhancement method is to replace the original promoter of the ppc and / or gndA gene with a strong promoter.

[0025] The strong promoter can be selected from Ptac, Ptac, Ptrc, Psod, or Ptuf.

[0026] The microorganism is preferably Corynebacterium, such as Corynebacterium glutamicum, Corynebacterium pekinense, Breviabacterium flavum, or Escherichia coli, and more preferably Corynebacterium glutamicum MHZ-1012-3, see CN201911370732.9.

[0027] Fifthly, the present invention provides strains that produce branched-chain amino acids and their derivatives, constructed according to the method described herein.

[0028] Sixthly, the present invention provides a method for producing branched-chain amino acids and their derivatives, the method comprising the following steps:

[0029] 1) Cultivate the strain to obtain a culture of the microorganism;

[0030] 2) Collect the branched-chain amino acids and their derivatives obtained from the culture obtained in step 1).

[0031] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0032] The acetylhydroxy acid synthase mutant strain 3-ilvN provided by this invention V25I The valine accumulation reached 9.8 g / L, which is 2.2 g / L higher than the valine accumulation of the starting strain MHZ-1012-3 (7.6 g / L), representing an increase of 28.9%. The accumulation of the byproduct isoleucine was 1.3 g / L, which is 40.9% lower than the starting strain MHZ-1012-3. There were no significant changes in leucine and cell OD.

[0033] Meanwhile, in the acetylhydroxyl synthase mutant ilvN V25I Based on this, the addition of PPC enhancement, or the simultaneous enhancement of PPC and GNDA, significantly increased the accumulation of valine, reaching 11.6 g / L and 13.2 g / L, respectively, with increases of 18.4% and 34.7%, respectively.

[0034] The acetylhydroxy acid synthase mutant ilvN provided by this invention V25I and containing ilvN V25I The mutant strain has a significant positive effect on the yield of the main product valine and a significant negative effect on the yield of the byproduct isoleucine, providing a reference for the construction of production strains that produce branched-chain amino acids such as valine, leucine, and isoleucine, as well as derivatives based on them.

[0035] The above-mentioned mutation sites can be applied to Corynebacterium glutamicum, Corynebacterium pekinense, Breviabacterium flavum, or Escherichia coli, but are not limited to the above-mentioned species. For example, Bacillus subtilis can also be used to produce branched-chain amino acids such as valine, leucine, and isoleucine or their derivatives. Detailed Implementation

[0036] The present invention aims to provide a method for producing branched-chain amino acids using microorganisms, and a novel microorganism capable of producing branched-chain amino acids with high efficiency.

[0037] Research has found that modifying the acetylhydroxy acid synthase of Corynebacterium glutamicum or Escherichia coli enables microorganisms to produce valine efficiently and reduces the content of the byproduct isoleucine, thus successfully creating a new microorganism capable of producing valine efficiently.

[0038] The present invention adopts the following technical solution:

[0039] The present invention provides a Corynebacterium, wherein the 25th amino acid of the acetylhydroxy acid synthase (NCBI reference sequence number WP_003861429.1) encoded by the ilvN gene (NCBI reference sequence number CEY17_RS06890) is mutated from valine (V) to isoleucine (I).

[0040] In this invention, the Corynebacterium is Corynebacterium glutamicum, Corynebacterium pekinense, or Breviabacterium flavum.

[0041] The starting strain MHZ-1012-3 of this invention is *Corynebacterium glutamicum*, and its construction method is described in CN201911370732.9. This strain is obtained by mutating the first base of the coding region of the α-isopropylmalate synthase gene leuA of the starting strain MHZ-1012-2 from A to G. Strain MHZ-1012-2 can be found in CN201611250330.1.

[0042] The ilvN gene encodes acetylhydroxy acid synthase, which is the first enzyme in the biosynthesis of branched-chain amino acids and a key enzyme in the biosynthesis of branched-chain amino acids. It catalyzes the formation of acetyllactic acid from two molecules of pyruvate (acetyllactic acid is a precursor of valine and leucine), and also catalyzes the formation of α-acetylhydroxybutyrate from α-ketobutyrate and pyruvate (α-acetylhydroxybutyrate is a precursor of isoleucine).

[0043] This invention modifies the ilvN gene from Corynebacterium to mutate acetylhydroxy acid synthase, thereby enhancing the ability of the microorganism to produce valine compared to the unmodified strain, reducing its ability to produce the byproduct isoleucine, and ultimately increasing the yield of valine.

[0044] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2001), or as recommended by the manufacturer's instructions.

[0045] The construction method of the strain of the present invention is not limited in the order of the steps. Anyone skilled in the art who achieves the purpose of the present invention by following the disclosure of the present invention is within the protection scope of the present invention.

[0046] The strains used in this invention are named as follows: 3-ilvN V25I 3-ilvN V25I / ppc、3-ilvN V25I / ppc / gndA etc. are used for ease of description, but should not be construed as limiting the present invention.

[0047] The primer sequences used in the following examples are shown in Table 1.

[0048] Table 1

[0049]

[0050]

[0051] Example 1 Construction of acetylhydroxy acid synthase mutant strain

[0052] Starting with MHZ-1012-3, the ilvN gene in MHZ-1012-3 was mutated to encode the acetylhydroxyl synthase mutant gene of SEQ ID NO:2, thus constructing the acetylhydroxyl synthase mutant strain 3-ilvN. V25I The specific construction method is as follows:

[0053] 1. Plasmid pK18mobsacB-ilvN V25I Construction

[0054] Using Phusion superfidelity polymerase (New England BioLabs), with the genome of the starting strain MHZ-1012-3 as a template, and ilvN V25I -UP-1F / ilvN V25I Using UP-1R as primers, recombinant fragment UP-1 was prepared and then... V25I -DN-2F / ilvN V25I Using DN-2R as primers, recombinant fragment DN-1 was prepared; using the genome of Corynebacterium glutamicum type strain ATCC13032 as a template, ilvN V25I -1F / ilvN V25I -1R was used as a primer to prepare the recombinant fragment ilvN V25I Using plasmid pk18-mob-sacB as a template, and ilvN V25I -pk18-3F / ilvN V25I -pk18-3R was used as a primer to obtain fragment pk18-1, which was purified using an agarose gel extraction kit (Tiangen). The reaction was then carried out according to the Gibson assembly kit configuration system, as shown in Table 2.

[0055] Table 2 Gibson Assembly Reaction System

[0056] Components UP-1 DN-1 <![CDATA[ilvN V25I ]]> pk18-1 CE Buffer CE Exnase sterile water Volume / μL 1 1 1 2 4 2 9

[0057] The prepared reaction mixture was incubated at 37°C for 30 min. 10 μL of the incubator was then transformed into Trans1T1 competent cells (TransGen Biotech). Single clones were picked, and colony PCR was used to confirm the correct insertion fragment. Further enzyme digestion confirmed the presence of a positive clone containing the pK18mobsacB fragment. Finally, the plasmid was sent to Genewiz Biotechnology Co., Ltd. for sequencing. The correctly sequenced plasmid was named pK18mobsacB-ilvN. V25I .

[0058] 2. Acetylhydroxy acid synthase mutant strain 3-ilvN V25I Construction

[0059] The recombinant plasmid pK18mobsacB-ilvN obtained in step 1 is used V25I Transformed into the starting strain MHZ-1012-3, recombinants were selected on selective medium containing 15 mg / L kanamycin. The culture temperature was 30°C, and the culture was inverted. The selected transformants were cultured overnight in ordinary liquid brain heart extract medium at 30°C with shaking at 220 rpm. During this culture, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange. The culture was then serially diluted (10⁻⁶ m² / 4 ... -2 Continuous dilution to 10 -4 The diluted solution was spread onto ordinary solid brain and heart extract medium containing 10% sucrose and incubated at 33°C for 48 hours. Transformants grown on this medium were identified. The target sequence was amplified by PCR, and nucleotide sequencing analysis was performed to obtain the target mutant strain, named 3-ilvN. V25I .

[0060] Example 2 Construction of ppc gene enhanced mutant strain

[0061] The acetylhydroxyl synthase mutant strain 3-ilvN constructed in Example 1 V25I Furthermore, the ppc gene with Ptac as the promoter was introduced at site cg1507 to enhance ppc gene expression. The specific method is as follows:

[0062] 1. Construction of plasmid pK18mobsacB-ppc

[0063] Using the genome of the starting strain MHZ-1012-3 as a template, the upper homologous arm recombinant fragment UP4 was prepared using PI-ppc-1f / PI-ppc-1r as primers; the ppc gene and terminator recombinant fragment PPC was prepared using PI-ppc-2f / PI-ppc-2 as primers; and the lower homologous arm recombinant fragment DN4 was prepared using PI-ppc-4f / I-ppc-4r as primers. Using plasmid pXMJ19 as a template, the tac promoter recombinant fragment Ptac was prepared using PI-ppc-3f / PI-ppc-3r as primers; and using plasmid pK18-mob-sacB as a template, the recombinant fragment pk18-4 was prepared using PI-pK18-F / PI-pK18-R as primers. All fragments were purified using an agarose gel extraction kit (Tiangen), and then reacted according to the Gibson assembly kit configuration. The reaction system is shown in Table 3.

[0064] Table 3 Gibson Assembly Reaction System

[0065] Components UP4 PPC DN4 Ptac pk18-4 CE Buffer CE Exnase sterile water Volume / μL 1 1 1 1 2 4 2 8

[0066] The prepared reaction system was incubated at 37℃ for 30 min. 10 μL was then transformed into Trans1T1 competent cells (TransGen Biotech). Single clones were picked, and colony PCR was used to confirm that the inserted fragment was correct. Further enzyme digestion was used to identify positive clones with the fragment inserted into pK18mobsacB. Finally, the plasmid was sent to Genewiz Biotechnology Co., Ltd. for sequencing. The obtained correctly sequenced plasmid was named pK18mobsacB-ppc.

[0067] 2. Construction of PPC gene-enhanced mutant bacteria

[0068] The recombinant plasmid pK18mobsacB-ppc obtained in step 1 was transformed into the acetylhydroxy acid synthase mutant strain 3-ilvN constructed in Example 1. V25I In this study, recombinants were selected on selective medium containing 15 mg / L kanamycin. The culture temperature was 30°C, and the culture was inverted. The selected transformants were then cultured overnight in standard liquid brain heart extract medium at 30°C with shaking at 220 rpm on a rotary shaker. During this culture, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange. The culture was then serially diluted (10⁻⁶ mcg / L). -2 Continuous dilution to 10 -4 The diluted solution was spread onto ordinary solid brain and heart extract medium containing 10% sucrose and incubated at 33°C for 48 hours. Transformants grown on this medium were identified. The target sequence was amplified by PCR, and nucleotide sequencing analysis was performed to obtain the target mutant strain, named 3-ilvN. V25I / ppc.

[0069] Example 3 Construction of gndA gene enhanced mutant strain

[0070] The mutant strain 3-ilvN constructed in Example 2 V25I In the / ppc file, the original promoter of the gndA gene is replaced with the strong promoter Ptac to enhance the expression of the gndA gene. The specific method is as follows:

[0071] 1. Construction of plasmid pK18mobsacB-gndA

[0072] Using the genome of the originating strain MHZ-1012-3 as a template, the upper homologous arm recombination fragment UP5 was prepared using PI-gndA-1f / PI-gndA-1r primers, and the lower homologous arm recombination fragment DN5 was prepared using PI-gndA-3f / PI-gndA-3r primers. Using plasmid pXMJ19 as a template, the tac promoter recombination fragment Ptac was prepared using PI-gndA-2f / PI-gndA-2r primers. The three recombination fragments were fused using overlap PCR, and the fused fragment was ligated to the pK18-mob-sacB vector using the BamHI / EcoRI restriction site. 10 μL of the ligation fragment was then transformed into Trans1T1 competent cells (TransGen). Biotech selected single clones, confirmed the correct insertion fragment by colony PCR, and further identified positive clones with the pK18mobsacB fragment insertion by enzyme digestion. Finally, the plasmid was sent to Genewiz Biotechnology Co., Ltd. for sequencing, and the obtained correctly sequenced plasmid was named pK18mobsacB-gndA.

[0073] 2. Construction of gndA gene-enhanced mutant bacteria

[0074] The recombinant plasmid pK18mobsacB-gndA obtained in step 1 was transformed into the mutant strain 3-ilvN constructed in Example 2. V25I In / ppc, recombinants were selected on selective medium containing 15 mg / L kanamycin. The culture temperature was 30°C, and the culture was inverted. The selected transformants were cultured overnight in ordinary liquid brain heart extract medium at 30°C with shaking at 220 rpm. During this culture, the transformants underwent a second recombination, removing the vector sequence from the genome through gene exchange. The culture was then serially diluted (10⁻⁶ mcg / mL). -2 Continuous dilution to 10 -4 The diluted solution was spread onto ordinary solid brain and heart extract medium containing 10% sucrose and incubated at 33°C for 48 hours. Transformants grown on this medium were identified. The target sequence was amplified by PCR, and nucleotide sequencing analysis was performed to obtain the target mutant strain, named 3-ilvN. V25I / ppc / gndA.

[0075] Example 4: Valine production by shake-flask fermentation of Corynebacterium glutamicum

[0076] 1. Culture medium

[0077] Seed culture medium: 15 g / L soybean meal extract, 20 g / L glucose, 7 g / L ammonium sulfate, 0.5 g / L magnesium sulfate, 1 g / L potassium dihydrogen phosphate, 1 g / L dipotassium hydrogen phosphate, 2 g / L urea, balance water, pH 7.2.

[0078] Fermentation medium: 15 g / L soybean meal extract, 20 g / L glucose, 7 g / L ammonium sulfate, 0.5 g / L magnesium sulfate, 1 g / L potassium dihydrogen phosphate, 1 g / L dipotassium hydrogen phosphate, 2 g / L urea, 15 μg / L vitamin B3, 100 μg / L vitamin B1·HCl, balance water, pH 7.2.

[0079] 2. Shake-flask fermentation

[0080] (1) Seed culture: Pick one loop of slant seeds and inoculate them into a 500 mL Erlenmeyer flask containing 50 mL of seed culture medium. Culture at 30 °C and 220 r / min for 10-12 h with shaking.

[0081] (2) Fermentation culture: 5 mL of seed culture was inoculated into a 500 mL Erlenmeyer flask containing 50 mL of fermentation culture medium and cultured at 30 °C and 220 r / min for 72 h.

[0082] (3) Centrifuge 1 mL of fermentation broth (12000 rpm, 2 min), collect the supernatant, and use HPLC to detect L-valine, leucine and isoleucine in the fermentation broth. Detect the OD value at 562 nm by spectrophotometry. The results are shown in Table 4.

[0083] Table 4 Fermentation Results

[0084] MHZ-1012-3 <![CDATA[3-ilvN V25I ]]> <![CDATA[3-ilvN V25I / ppc]]> <![CDATA[3-ilvN V25I / ppc / gndA]]> Valine g / L 7.6 9.8* 11.6* 13.2* Isoleucine g / L 2.2 1.3* 1.3* 1.5* Leucine g / L 1.0 1.0 1.1 1.2* OD (562nm) 54.8 55.3 54.1 56.8

[0085] Note: * indicates a significant difference compared to the control (P < 0.01).

[0086] The results showed that the valine accumulation of the starting strain MHZ-1012-3 was 7.6 g / L, while the acetylhydroxy acid synthase mutant strain 3-ilvN provided by this invention... V25I The valine accumulation reached 9.8 g / L, an increase of 2.2 g / L, representing a 28.9% increase. The accumulation of the byproduct isoleucine was 1.3 g / L, a decrease of 40.9% compared to the original strain MHZ-1012-3. There were no significant changes in leucine and cell OD.

[0087] Therefore, the acetylhydroxy acid synthase mutant ilvN provided by the present invention can be seen. V25I and containing ilvN V25I The mutant strain has a significant positive effect on the yield of the main product valine and a significant negative effect on the yield of the byproduct isoleucine. This provides a reference for the construction of production strains that produce branched-chain amino acids such as valine, leucine, and isoleucine, as well as derivatives based on them.

[0088] Meanwhile, in the acetylhydroxyl synthase mutant ilvN V25IBased on the existing PPC enhancement, or simultaneous enhancement of PPC and GNDA, the accumulation of valine significantly increased, reaching 11.6 g / L and 13.2 g / L, respectively, representing increases of 18.4% and 34.7%. This indicates that the acetylhydroxyl synthase mutant ilvN... V25I The combination of PPC enhancement and simultaneous enhancement of PPC and GNDA has a significant positive effect, providing a reference for the construction of production strains that produce branched-chain amino acids such as valine, leucine, and isoleucine, as well as derivatives based on these precursors.

[0089] The sequence is described as follows:

[0090] The nucleotide sequence of the wild-type ilvN gene of Corynebacterium glutamicum is shown in SEQ ID NO:1.

[0091] Corynebacterium glutamicum ilvN V25I The nucleotide sequence of the mutant is shown in SEQ ID NO:2.

[0092] The amino acid sequence of wild-type acetylhydroxy acid synthase from Corynebacterium glutamicum is shown in SEQ ID NO:3.

[0093] Corynebacterium glutamate acetylhydroxyl synthase mutant ilvN V25I The amino acid sequence is shown in SEQ ID NO:4.

[0094] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. sequence list <110> Langfang Meihua Biotechnology Development Co., Ltd. <120> Acetylhydroxy acid synthase mutants and their applications <130> KHP221115361.2 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 519 <212> DNA <213> Corynebacterium glutamicum <400> 1 atggctaatt ctgacgtcac ccgccacatc ctgtccgtac tcgttcagga cgtagacgga 60 atcatttccc gcgtatcagg tatgttcacc cgacgcgcat tcaacctcgt gtccctcgtg 120 tctgcaaaga ccgaaacaca cggcatcaac cgcatcacgg ttgttgtcga cgccgacgag 180 ctcaacattg agcagatcac caagcagctc aacaagctga tccccgtgct caaagtcgtg 240 cgacttgatg aagagaccac tatcgcccgc gcaatcatgc tggttaaggt ctctgcggac 300 agcaccaacc gtccgcagat cgtcgacgcc gcgaacatct tccgcgcccg agtcgtcgac 360 gtggctccag actctgtggt tattgaatcc acaggcaccc caggcaagct ccgcgcactg 420 cttgacgtga tggaaccatt cggaatccgc gaactgatcc aatccggaca gattgcactc 480 aaccgcggtc cgaagaccat ggctccggcc aagatctaa 519 <210> 2 <211> 519 <212> DNA <213> Corynebacterium glutamicum <400> 2 atggctaatt ctgacgtcac ccgccacatc ctgtccgtac tcgttcagga cgtagacgga 60 atcatttccc gcatatcagg tatgttcacc cgacgcgcat tcaacctcgt gtccctcgtg 120 tctgcaaga ccgaaacaca cggcatcaac cgcatcacgg tgttgtcga cgccgacgag 180 ctcacattg agcagatcac caagcagctc aacaagctga tccccgtgct caaagtcgtg 240 cgacttgatg agagaccac tatcgcccgc gcaatcatgc tggttaggt ctctgcggac 300 agcaccaacc gtccgcagat cgtcgacgcc gcgaacatct tccgcgccg agtcgtcgac 360 gtggctccag actctgtggt tattgaatcc acaggcaccc caggcaagct ccgcgcactg 420 cttgacgtga tggaaccatt cggaatccgc gaactgatcc aatccggaca gattgcactc 480 aaccgcggtc cgaagaccat ggctccggcc aagatctaa 519 <210> 3 <211> 172 <212> PRT <213> Corynebacterium glutamicum <400> 3 Met Ala Asn Ser Asp Val Thr Arg His Ile Leu Ser Val Leu Val Gln 1 5 10 15 Asp Val Asp Gly Ile With Arg Val Serving Gly Met Phe Thr Arg Arg 20 25 30 Ala Phe Asn Leu Val Ser Leu Val Ser Ala Lys Thr Glu Thr His Gly 35 40 45 Ile Asn Arg Ile Thr Val Val Val Asp Ala Asp Glu Leu Asn Ile Glu 50 55 60 Gln Ile Thr Lys Gln Leu Asn Lys Leu Ile Pro Val Leu Lys Val Val 65 70 75 80 Arg Leu Asp Glu Glu Thr Thr Ile Ala Arg Ala Ile Met Leu Val Lys 85 90 95 Val Ser Ala Asp Ser Thr Asn Arg Pro Gln Ile Val Asp Ala Ala Asn 100 105 110 Ile Phe Arg Ala Arg Val Val Asp Val Ala Pro Asp Ser Val Val Ile 115 120 125 Glu Ser Thr Gly Thr Pro Gly Lys Leu Arg Ala Leu Leu Asp Val Met 130 135 140 Glu Pro Phe Gly Ile Arg Glu Leu Ile Gln Ser Gly Gln Ile Ala Leu 145 150 155 160 Asn Arg Gly Pro Lys Thr Met Ala Pro Ala Lys Ile 165 170 <210> 4 <211> 172 <212> PRT <213> Corynebacterium glutamicum <400> 4 Met Ala Asn Ser Asp Val Thr Arg His Ile Leu Ser Val Leu Val Gln 1 5 10 15 Asp Val Asp Gly Ile Ile Ser Arg Ile Ser Gly Met Phe Thr Arg Arg 20 25 30 Ala Phe Asn Leu Val Ser Leu Val Ser Ala Lys Thr Glu Thr His Gly 35 40 45 Ile Asn Arg Ile Thr Val Val Val Asp Ala Asp Glu Leu Asn Ile Glu 50 55 60 Gln Ile Thr Lys Gln Leu Asn Lys Leu Ile Pro Val Leu Lys Val Val 65 70 75 80 Arg Leu Asp Glu Glu Thr Thr Ile Ala Arg Ala Ile Met Leu Val Lys 85 90 95 Val Ser Ala Asp Ser Thr Asn Arg Pro Gln Ile Val Asp Ala Ala Asn 100 105 110 Ile Phe Arg Ala Arg Val Val Asp Val Ala Pro Asp Ser Val Val Ile 115 120 125 Glu Ser Thr Gly Thr Pro Gly Lys Leu Arg Ala Leu Leu Asp Val Met 130 135 140 Glu Pro Phe Gly Ile Arg Glu Leu Ile Gln Ser Gly Gln Ile Ala Leu 145 150 155 160 Asn Arg Gly Pro Lys Thr Met Ala Pro Ala Lys Ile 165 170

Claims

1. An acetylhydroxy acid synthase mutant, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

4.

2. A nucleic acid molecule encoding the mutant of claim 1 or biological material containing said nucleic acid molecule; The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, or engineered bacteria.

3. Any of the following applications of the nucleic acid molecule or the biological material described in claim 2: (1) Used for the fermentation production of branched-chain amino acids; (2) Used to increase the fermentation yield of branched-chain amino acids; (3) Genetically engineered bacteria used to construct branched-chain amino acid-producing bacteria; in, The branched-chain amino acid is valine.

4. A method for constructing a strain that produces branched-chain amino acids, characterized in that, Using genetic engineering techniques, mutations were introduced into the acetylhydroxyl synthase gene in the microbial genome, so that the amino acid sequence of the acetylhydroxyl synthase it encodes is shown in SEQ ID NO:4; The branched-chain amino acid is valine.

5. The method according to claim 4, characterized in that, The method further includes enhancing the ppc and / or gndA genes in the strain; wherein the reference sequence numbers of the ppc and gndA genes on NCBI are CEY17_RS08480 and CEY17_RS07800, respectively. The enhancement method is selected from the following 1) to 4), or an optional combination thereof: 1) Enhancement is achieved by introducing a plasmid containing the gene; 2) Enhanced by increasing the copy number of the aforementioned genes on the chromosome; 3) Enhancement is achieved by operatively linking a strong promoter to the gene; 4) Enhancement is achieved by introducing enhancers.

6. The method according to claim 5, characterized in that, Replace the original promoters of the ppc and / or gndA genes with strong promoters; The strong promoter is selected from Ptac, Ptrc, Psod, or Ptuf.

7. The method according to any one of claims 4-6, characterized in that, The microorganism in question is Corynebacterium.

8. The method according to claim 7, characterized in that, The Corynebacterium is Corynebacterium glutamicum ( Corynebacterium glutamicum Corynebacterium pingeri () Corynebacterium pekinense ).

9. A strain producing branched-chain amino acids, constructed according to any one of claims 4-8; in, The branched-chain amino acid is valine.

10. A method for producing branched-chain amino acids, characterized in that, The method includes the following steps: 1) Cultivate the strain according to claim 9 to obtain a culture of the microorganism; 2) Collect the branched-chain amino acids produced from the culture obtained in step 1); The branched-chain amino acid is valine.

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