Mutants of enzymes used in branched-chain amino acid biosynthesis and methods of making and using same
By mutating and modifying the genes of acetylhydroxy acid synthase and acetylhydroxy acid isomer reductase, the yield of L-valine was increased and the production of the byproduct isoleucine was reduced, solving the problem of low efficiency in the microbial fermentation method for producing L-valine and realizing efficient industrial production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- MEIHUA BIOTECH LANGFANG CO LTD
- Filing Date
- 2022-08-12
- Publication Date
- 2026-06-09
AI Technical Summary
Existing microbial fermentation methods for producing L-valine have low conversion rates and high leucine content as a byproduct, making it difficult to meet the needs of large-scale industrial production.
By mutating the acetylhydroxy acid synthase encoded by the ilvN gene and the acetylhydroxy acid isomer reductase encoded by the ilvC gene, mutants were formed and expressed in Corynebacterium glutamicum. Combined with the modification of the ppc and gndA genes, their expression was enhanced, increasing the production of L-valine and reducing the production of the byproduct isoleucine.
It significantly increased the yield of L-valine, reduced the production of the byproduct isoleucine, and improved the efficiency of L-valine production by microbial fermentation, meeting the needs of large-scale industrial production.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of bioengineering technology, specifically to mutants of enzymes used in the biosynthesis of branched-chain amino acids, their construction methods, and 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] To overcome the shortcomings of existing technologies, the present invention aims to provide a method for producing the branched-chain amino acid L-valine using microorganisms, and a novel microorganism capable of producing L-valine with high efficiency. Specifically, the present invention aims to provide an L-valine-producing strain with high L-valine production and low production of byproducts leucine and isoleucine.
[0006] In a first aspect, the present invention provides a mutant of an enzyme used in the biosynthesis of branched-chain amino acids, the mutant comprising: the 25th amino acid of the wild-type acetylhydroxyl synthase encoded by the ilvN gene is mutated from valine V to isoleucine I, forming an acetylhydroxyl synthase mutant;
[0007] And / or, the 90th amino acid of the wild-type acetylhydroxy acid isomer reductase encoded by the ilvC gene is mutated from isoleucine I to serine S, forming an acetylhydroxy acid isomer reductase mutant.
[0008] The ilvN gene (NCBI reference sequence number CEY17_RS06890) encodes acetylhydroxy acid synthase (NCBI reference sequence number WP_003861429.1), which is the first enzyme in branched-chain amino acid biosynthesis and a key enzyme in branched-chain amino acid biosynthesis. It catalyzes the formation of acetolactate from two molecules of pyruvate (acetolactate 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).
[0009] The ilvC gene (NCBI reference sequence number CEY17_RS06895) encodes acetylhydroxy acid isomer reductase (NCBI reference sequence number WP_003854117.1), an important enzyme in the biosynthesis of branched-chain amino acids. It catalyzes the conversion of one molecule of α-acetolactate or α-acetylhydroxybutyrate to one molecule of α-dihydroxyisovalerate or α,β-dihydroxymethylvalerate (α-dihydroxyisovalerate is a precursor of valine and leucine, and α,β-dihydroxymethylvalerate is a precursor of isoleucine), while simultaneously digesting one molecule of reducing power (reduced nicotinamide adenine dinucleotide phosphate, NADPH) to produce one molecule of nicotinamide adenine dinucleotide phosphate (NADP+).
[0010] In the mutant of the enzyme used for branched-chain amino acid biosynthesis provided by the present invention, the amino acid sequence of the wild-type acetylhydroxy acid synthase is shown in SEQ ID NO.3, and the amino acid sequence of the acetylhydroxy acid synthase mutant is shown in SEQ ID NO.4.
[0011] In the mutant of the enzyme used for branched-chain amino acid biosynthesis provided by the present invention, the nucleotide sequence of the gene encoding the acetylhydroxy acid synthase mutant is shown in SEQ ID NO.2.
[0012] In the mutant of the enzyme used for branched-chain amino acid biosynthesis provided by the present invention, the amino acid sequence of the wild-type acetylhydroxy acid isomer reductase is shown in SEQ ID NO.7; the amino acid sequence of the mutant acetylhydroxy acid isomer reductase is shown in SEQ ID NO.8.
[0013] In the mutant of the enzyme used for branched-chain amino acid biosynthesis provided by the present invention, the nucleotide sequence of the gene encoding the acetylhydroxy acid isomer reductase mutant is as shown in SEQ ID NO.6.
[0014] Secondly, the present invention provides a biological material containing a mutant of the enzyme used in the biosynthesis of the aforementioned branched-chain amino acids.
[0015] Furthermore, the ppc and / or gndA genes in the biological material described in this invention are modified to enhance the expression of the ppc and / or gndA genes.
[0016] The reference sequence numbers for the ppc and gndA genes on NCBI are CEY17_RS08480 and CEY17_RS07800, respectively.
[0017] Specifically, the biological material provided by the present invention is microorganisms, and the original strains of the microorganisms include: Corynebacterium glutamicum, Corynebacterium pingeri, Bacillus flavus, Escherichia coli or Bacillus subtilis;
[0018] Preferably, the original strain is obtained by mutating the first base of the leuA coding region of the α-isopropylmalate synthase gene of the strain with accession number CGMCC No. 13406 from A to G.
[0019] The starting strain of this invention, MHZ-1012-3, is *Corynebacterium glutamicum*, and its construction method is described in patent document 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. MHZ-1012-2 was deposited on November 30, 2016, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 13406, as described in patent document CN201611250330.1.
[0020] This invention modifies the ilvN and / or ilvC genes from Corynebacterium glutamicum to mutate acetylhydroxy acid synthase and / or acetylhydroxy acid isomer reductase, 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.
[0021] Based on the understanding of those skilled in the art, this invention seeks to protect mutants of the enzymes used in the biosynthesis of branched-chain amino acids or the use of the aforementioned biological materials in the biosynthesis of branched-chain amino acids or their derivatives.
[0022] The application of mutants of the enzymes used in the above-mentioned branched-chain amino acid biosynthesis or the above-mentioned biological materials in increasing L-valine production while reducing the production of leucine or isoleucine in the L-valine biosynthesis process.
[0023] The beneficial effects of this invention are as follows:
[0024] The acetylhydroxy acid synthase mutant strain MHZ-1012-31 provided by this invention has a valine accumulation of 9.9 g / L, which is 2.4 g / L higher than the original strain MHZ-1012-3, representing an increase of 32%. The accumulation of the byproduct isoleucine is 1.5 g / L, which is 34.8% lower than the original strain MHZ-1012-3. There are no significant changes in leucine and cell OD.
[0025] The valine isoreductase mutant strain MHZ-1012-35 provided by this invention has a valine accumulation of 10.5 g / L, which is 3.0 g / L higher than the original strain MHZ-1012-3, representing an increase of 40%. The byproduct isoleucine did not increase further, and the isoleucine accumulation was 1.5 g / L, which is 34.8% lower than the original strain MHZ-1012-3. Leucine increased by 0.2 g / L, representing an increase of 22.2%. There was no significant change in bacterial OD.
[0026] Therefore, the acetylhydroxy acid synthase mutant ilvN provided by the present invention can be seen. V25I and acetylhydroxy acid isomer reductase mutant ilvC I90S The strain and its mutant strains showed 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.
[0027] Meanwhile, in the acetylhydroxyl synthase mutant ilvN V25I and acetylhydroxy acid isomer reductase mutant ilvC I90S Based on the existing PPC enhancement, or simultaneous enhancement of PPC and GNDA, the accumulation of valine significantly increased, reaching 12.1 g / L and 13.8 g / L, respectively, representing increases of 13.2% and 31.4%. This indicates that the acetylhydroxyl synthase mutant ilvN... V25I and acetylhydroxy acid isomer reductase mutant ilvC I90S 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 three-branched amino acids such as valine, leucine, and isoleucine, as well as derivatives based on these precursors. Detailed Implementation
[0028] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.
[0029] The primer names and sequences involved in the embodiments are shown in Table 1.
[0030] Table 1 Primers
[0031]
[0032]
[0033] Example 1 Construction of acetylhydroxy acid synthase mutant strain
[0034] The starting strain MHZ-1012-3 in this embodiment is *Corynebacterium glutamicum*, and its construction method is described in patent document 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. MHZ-1012-2 was deposited on November 30, 2016, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 13406, as described in patent document CN201611250330.1.
[0035] Using MHZ-1012-3 as the starting strain, the ilvN gene in MHZ-1012-3 was mutated to encode the acetylhydroxy acid synthase mutant gene of SEQ ID NO.2, and an acetylhydroxy acid synthase mutant strain was constructed. The specific construction method is as follows.
[0036] 1. Plasmid pK18mobsacB-ilvN V25I Construction
[0037] 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 ilvNV25I -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.
[0038] Table 2 Gibson Assembly Reaction System
[0039] 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
[0040] 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 .
[0041] 2. Construction of acetylhydroxy acid synthase mutant strains
[0042] The recombinant plasmid pK18mobsacB-ilvN obtained by the method described in 1 above. V25I Transformed into the starting strain MHZ-1012-3, recombinant mutants 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 on a rotary shaker. The culture was then serially diluted (10⁻⁶ oz / 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 MHZ-1012-31.
[0043] In this embodiment, the wild-type acetylhydroxy acid synthase strain was used as the starting strain. The ilvN gene in MHZ-1012-3 was mutated to the gene encoding the acetylhydroxy acid synthase mutant of SEQ ID NO.2, and the acetylhydroxy acid synthase mutant strain was constructed. The specific construction method is as follows.
[0044] Example 2 Construction of acetylhydroxy acid isomer reductase mutant strain
[0045] Using MHZ-1012-3 as the starting strain, the ilvC gene in MHZ-1012-3 was mutated to encode the acetylhydroxy acid isomer reductase mutant gene of SEQ ID NO.6, and an acetylhydroxy acid isomer reductase mutant strain was constructed. The specific construction method is as follows.
[0046] 1. Plasmid pK18mobsacB-ilvC I90S Construction
[0047] Using Phusion superfidelity polymerase (New England BioLabs), with the genome of the starting strain MHZ-1012-3 as a template, and ilvC I90S -UP-1F / ilvC I90S Using UP-1R as primers, recombinant fragment UP-1 was prepared, and ilvC was used as the primer. I90S -DN-2F / ilvC I90S Using DN-2R as primers, recombinant fragment DN-1 was prepared; using plasmid pk18-mob-sacB as a template, and ilvC... I90S -pk18-3F / ilvC I90S -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 3.
[0048] Table 3 Gibson Assembly Reaction System
[0049] Components UP-1 DN-1 pk18-1 CE Buffer CE Exnase sterile water Volume / μL 1 1 2 4 2 10
[0050] 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-ilvC. I90S .
[0051] 2. Construction of acetylhydroxy acid isomer reductase mutant strain
[0052] The recombinant plasmid pK18mobsacB-ilvC obtained by the method described in 1 above. I90STransformed into the starting strain MHZ-1012-3, recombinant mutants 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 on a rotary shaker. The culture was then serially diluted (10⁻⁶ oz / mL). -2 Continuous dilution to 10 -4 The diluted solution was spread onto ordinary solid brain 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 MHZ-1012-33.
[0053] Example 3: Construction of a superimposed strain of acetylhydroxy acid isomer reductase mutants
[0054] Using MHZ-1012-31 as the starting strain, the ilvC gene in MHZ-1012-31 was mutated to encode the acetylhydroxy acid isomer reductase mutant gene of SEQ ID NO.6, and an acetylhydroxy acid isomer reductase mutant strain was constructed. The specific construction method is as follows.
[0055] The recombinant plasmid pK18mobsacB-ilvC constructed using the method described in Example 2 above. I90S Transformed into the starting strain MHZ-1012-31, recombinant mutants 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 on a rotary shaker. The culture was then serially diluted (10⁻⁶ oz / mL). -2 Continuous dilution to 10 -4 The diluted solution was spread onto ordinary solid brain 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 MHZ-1012-35.
[0056] Example 4: Construction of PPC gene mutant strain
[0057] 1. Construction of plasmid pK18mobsacB-ppc
[0058] Using the genome of the starting strain MHZ-1012-35 as a template, the upper homologous arm recombination fragment UP4 was prepared using PI-ppc-1f / PI-ppc-1r as primers; the ppc gene and terminator recombination fragment PPC was prepared using PI-ppc-2f / PI-ppc-2 as primers; and the lower homologous arm recombination fragment DN4 was prepared using PI-ppc-4f / I-ppc-4r as primers. Using plasmid pXMJ19 as a template, the tac promoter recombination fragment Ptac was prepared using PI-ppc-3f / PI-ppc-3r as primers; and using plasmid pK18-mob-sacB as a template, the recombination fragment pk18-4 was prepared using PI-pK18-F / PI-pK18-R as primers. The 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 4.
[0059] Table 4 Gibson Assembly Reaction System
[0060] Components UP4 PPC DN4 Ptac pk18-4 CE Buffer CE Exnase sterile water Volume / μL 1 1 1 1 2 4 2 8
[0061] 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.
[0062] 2. Construction of PPC gene-enhanced mutant bacteria
[0063] The recombinant plasmid pK18mobsacB-ppc obtained by the method described in section 1 above was transformed into strain MHZ-1012-35, and exchange recombinants were selected on selective medium containing 15 mg / L kanamycin. The culture temperature was 30°C, and the culture was inverted. The screened transformants were cultured overnight in ordinary liquid brain heart extract medium at 30°C with shaking on a rotary shaker at 220 rpm. The culture was then serially diluted (10⁻⁶ oz / mL). -2 Continuous dilution to 10 -4 The diluted solution was spread onto ordinary solid brain 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 analyzed by nucleotide sequencing, and the resulting mutant strain was named MHZ-1012-37.
[0064] Example 5: Construction of a gndA gene-enhanced mutant strain
[0065] In the strain MHZ-1012-37 constructed by the method described in Example 4 above, the original promoter of the gndA gene was replaced with the strong promoter Ptac to enhance the expression of the gndA gene. The specific method is as follows.
[0066] 1. Construction of plasmid pK18mobsacB-gndA
[0067] Using the genome of the originating strain MHZ-1012-37 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.
[0068] 2. Construction of gndA gene-enhanced mutant bacteria
[0069] The recombinant plasmid pK18mobsacB-gndA obtained by the method described in section 1 above was transformed into strain MHZ-1012-37, and exchange 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 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 serially diluted (10⁻⁶ oz / mL). -2 Continuous dilution to 10 -4 The diluted solution was spread onto ordinary solid brain 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 MHZ-1012-38.
[0070] Example 6: Valine production by shake-flask fermentation of Corynebacterium glutamicum
[0071] 1. Culture medium
[0072] 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.
[0073] 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.
[0074] 2. Shake-flask fermentation
[0075] (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.
[0076] (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.
[0077] (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 of different examples are shown in Table 5.
[0078] In Table 5, strain A is a strain in which the ilvN gene in Corynebacterium ATCC14067 is mutated to encode SEQ ID NO.2; strain B is a strain in which the ilvC gene in Corynebacterium ATCC14067 is mutated to encode SEQ ID NO.6; and strain C is a strain in which the ilvN gene in Corynebacterium ATCC14067 is mutated to encode SEQ ID NO.2 and the ilvC gene is mutated to encode SEQ ID NO.6.
[0079] Table 5 Fermentation Results
[0080]
[0081] Note: * indicates a significant difference compared to the control (P < 0.01).
[0082] Table 5 shows that the valine accumulation of the starting strain MHZ-1012-3 was 7.5 g / L, while the valine accumulation of the acetylhydroxy acid synthase mutant strain MHZ-1012-31 provided by this invention reached 9.9 g / L, an increase of 2.4 g / L, representing a 32% increase. The accumulation of the byproduct isoleucine was 1.5 g / L, a decrease of 34.8% compared to the starting strain MHZ-1012-3. There were no significant changes in leucine and cell OD.
[0083] Furthermore, the superimposed mutant ilvC I90S The valine accumulation of the obtained acetylhydroxy acid isomer reductase mutant strain MHZ-1012-35 reached 10.5 g / L, which is 3.0 g / L higher than that of the original strain MHZ-1012-3, representing an increase of 40%. The byproduct isoleucine did not increase further, and the accumulation of isoleucine was 1.5 g / L, which is 34.8% lower than that of the original strain MHZ-1012-3. Leucine increased by 0.2 g / L, representing an increase of 22.2%. There was no significant change in cell OD.
[0084] Therefore, the acetylhydroxy acid synthase mutant ilvN provided by the present invention can be seen. V25I and acetylhydroxy acid isomer reductase mutant ilvC I90S The mutant strains showed 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 three-branched amino acids such as valine, leucine, and isoleucine, as well as derivatives based on them.
[0085] Meanwhile, in the acetylhydroxyl synthase mutant ilvN V25I and acetylhydroxy acid isomer reductase mutant ilvC I90S Based on the existing PPC enhancement, or simultaneous enhancement of PPC and GNDA, the accumulation of valine significantly increased, reaching 12.1 g / L and 13.8 g / L, respectively, representing increases of 13.2% and 31.4%. This indicates that the acetylhydroxyl synthase mutant ilvN... V25I and acetylhydroxy acid isomer reductase mutant ilvC I90S 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 three-branched amino acids such as valine, leucine, and isoleucine, as well as derivatives based on these precursors.
[0086] 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.
Claims
1. An acetylhydroxy acid isomer reductase mutant, characterized in that, The amino acid sequence of the acetylhydroxy acid isomer reductase mutant is shown in SEQ ID NO.
8.
2. The acetylhydroxy acid isomer reductase mutant according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the acetylhydroxy acid isomer reductase mutant is shown in SEQ ID NO.
6.
3. A biomaterial, characterized in that, Contains the acetylhydroxy acid isoreductase mutant as described in claim 1 or 2.
4. The biomaterial according to claim 3, characterized in that, The ppc gene in the biological material was modified to enhance its expression. The biological material is a microorganism, and the original strain of the microorganism is Corynebacterium glutamicum.
5. The biomaterial according to claim 4, characterized in that, The original strain was derived from the α-isopropylmalate synthase gene of strain with accession number CGMCC No. 13406. leuA It is obtained by mutating the first base of the coding region from A to G.
6. The use of the acetylhydroxy acid isomer reductase mutant according to claim 1 or 2 or the biomaterial according to any one of claims 3-5 in the biosynthesis of L-valine.