Dihydroxy acid dehydratase mutant, recombinant microorganism and application thereof

Abstract

The application relates to the technical field of microbial engineering, and particularly discloses a dihydroxy acid dehydratase mutant, a recombinant microorganism thereof and application. The dihydroxy acid dehydratase mutant takes the amino acid sequence of wild-type dihydroxy acid dehydratase as a reference sequence, and contains a mutation that the 237th alanine is substituted by lysine, arginine, histidine or proline. The fermentation bacteria with the mutant of the application have obviously improved L-valine production capacity, high yield and few by-products. The application provides a new high-efficiency production mode for fermentative production of L-valine.

Description

Technical Field

[0001] This invention relates to the field of microbial engineering technology, and more specifically, to a dihydroxy acid dehydratase mutant, its recombinant microorganism, and its 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 a dihydroxy acid dehydratase mutant that can improve the fermentation performance of L-valine strains, as well as recombinant microorganisms containing it and their applications.

[0006] The specific technical solution of the present invention is as follows:

[0007] A dihydroxy acid dehydratase mutant, with the amino acid sequence of wild-type dihydroxy acid dehydratase as a reference sequence, wherein the dihydroxy acid dehydratase mutant contains a mutation in which alanine (A) at position 237 is replaced by lysine (K), arginine (R), histidine (H) or proline (P).

[0008] This invention has found that when dihydroxy acid dehydratase is mutated in a specific way, the ability of recombinant microorganisms containing the mutant to produce L-valine is enhanced, while the ability to produce the byproduct isoleucine is reduced, which is beneficial to improving the efficiency of industrial production of valine.

[0009] Preferably, the amino acid sequence of the dihydroxy acid dehydratase mutant is shown in SEQ ID NO.12.

[0010] The present invention also provides a DNA molecule with the wild-type ilvD gene as a reference sequence, wherein the DNA molecule contains a mutation at bases 709-711 where GCC is mutated to AAG, CGC, CAC or CCC.

[0011] Preferably, the nucleotide sequence of the DNA molecule is as shown in SEQ ID NO.10.

[0012] The sequence of the wild-type ilvD gene is shown in SEQ ID NO.9.

[0013] The present invention also provides a recombinant microorganism expressing the above-mentioned dihydroxy acid dehydratase mutant.

[0014] Preferably, the recombinant microorganism also expresses an acetylhydroxy acid synthase mutant and / or an acetylhydroxy acid isomer reductase mutant;

[0015] The acetylhydroxy acid synthase mutant uses the amino acid sequence of wild-type acetylhydroxy acid synthase as a reference sequence and contains a mutation at amino acid position 25 where valine (V) is changed to isoleucine (I) (as shown in SEQ ID No. 4).

[0016] The acetylhydroxy acid isomer reductase mutant uses the amino acid sequence of wild-type acetylhydroxy acid isomer reductase as a reference sequence and contains a mutation at amino acid position 90, in which isoleucine (I) is mutated to serine (S) (as shown in SEQ ID No. 8).

[0017] The inventors conducted in-depth research to solve the above problems and discovered that modifying Corynebacterium acetylhydroxy acid synthase and / or acetylhydroxy acid isomer reductase and / or dihydroxy acid dehydratase can enable microorganisms to produce valine efficiently and reduce the content of the byproduct isoleucine, thereby successfully creating a new microorganism capable of producing valine efficiently.

[0018] Specifically, preferably, the Corynebacterium provided by this invention has an amino acid mutation at position 25 of the acetylhydroxy acid synthase (reference sequence number WP_003861429.1 on NCBI), encoded by the ilvN gene (reference sequence number CEY17_RS06890 on NCBI), which is mutated from valine (V) to isoleucine (I). The acetylhydroxy acid synthase encoded by the ilvN gene is the first enzyme in branched-chain amino acid biosynthesis and a key enzyme in it. 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).

[0019] The Corynebacterium of this invention has an acetylhydroxy acid isomer reductase (NCBI reference sequence number WP_003854117.1) encoded by the ilvC gene (NCBI reference sequence number CEY17_RS06895) in its cells, in which the 90th amino acid is mutated from isoleucine (I) to serine (S). The acetylhydroxy acid isomer reductase encoded by the ilvC gene is 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). + ).

[0020] In the Corynebacterium of the present invention, the 237th amino acid of the dihydroxy acid dehydratase (reference sequence number WP_003854128.1 on NCBI) encoded by the ilvD gene (reference sequence number CEY17_RS06870 on NCBI) is mutated from alanine (A) to lysine (K), arginine (R), histidine (H) or proline (P).

[0021] Preferably, the ppc gene of the recombinant microorganism of the present invention is enhanced, or ppc and gndA are enhanced simultaneously;

[0022] More preferably, the enhancement of the ppc gene is achieved by introducing the ppc gene with Ptac as the promoter at cg1507 of the starting strain.

[0023] Enhancement of the gndA gene is achieved by replacing the original promoter of the gndA gene with the strong promoter Ptac.

[0024] Further preferably, the present invention further modifies the ppc and / or gndA genes of the above-mentioned valine-producing Corynebacterium to obtain engineered bacteria with further enhanced valine production. The reference sequence numbers of the ppc and gndA genes on NCBI are CEY17_RS08480 and CEY17_RS07800, respectively.

[0025] In this invention, the starting strain of the recombinant microorganism is Corynebacterium glutamicum, Corynebacterium pekinense, Breviabacterium flavum, or Escherichia coli. Preferably, the starting strain of the recombinant microorganism is Corynebacterium glutamicum.

[0026] The above-mentioned mutation sites can be applied to Corynebacterium glutamicum, Corynebacterium pingeri, Corynebacterium flavum, or Escherichia coli, but are not limited to Corynebacterium glutamicum, Corynebacterium pingeri, Corynebacterium flavum, or Escherichia coli. They can also be applied to Bacillus subtilis and other bacteria for the production of branched-chain amino acids such as valine, leucine, and isoleucine or their derivatives.

[0027] The present invention further provides any of the following applications of the above-mentioned recombinant microorganisms:

[0028] (1) Application in the fermentation production of L-valine and its derivatives;

[0029] (2) Application in microbial genetic breeding for the production of L-valine and its derivatives;

[0030] (3) Application in increasing the yield of L-valine and its derivatives in fermentation production;

[0031] (4) Application in reducing the formation of isoleucine by-product during the fermentation production of L-valine and its derivatives.

[0032] The present invention also provides a method for producing L-valine, which includes a step of fermentation culture with recombinant microorganisms as described above.

[0033] The beneficial effects of this invention are at least as follows:

[0034] This invention achieves a mutation in dihydroxy acid dehydratase (preferably combined with specific modification of the ilvD gene from Corynebacterium glutamicum) by specifically modifying the ilvD gene (preferably combined with specific modification of the ilvN gene and / or ilvC gene from Corynebacterium glutamicum), thereby enhancing the ability of the microorganism to produce valine compared to the unmodified strain, reducing the ability to produce the byproduct isoleucine, and ultimately increasing the yield of valine.

[0035] The fermentation strain possessing the mutant of this invention exhibits a significantly enhanced ability to produce L-valine, with high yield and low byproduct production. This invention provides a new, highly efficient method for the fermentation production of L-valine. Detailed Implementation

[0036] The preferred embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and intent. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Where specific techniques or conditions are not specified in the following examples, they are performed according to the techniques or conditions described in the literature in the art, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0037] The primer names and sequences involved in the embodiments of the present invention are shown in Table 1.

[0038] Table 1 Primer sequences (SEQ ID No. 13-54)

[0039]

[0040]

[0041] The starting strain of this invention, MHZ-1012-3, is *Corynebacterium glutamicum*, and its construction method is described in Chinese Patent 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 Chinese Patent CN201611250330.1.

[0042] Example 1 Construction of acetylhydroxy acid synthase mutant strain

[0043] Starting with strain MHZ-1012-3, the ilvN gene in MHZ-1012-3 (wild-type ilvN nucleotide sequence as shown in SEQ ID NO.1, wild-type acetylhydroxyl synthase amino acid sequence as shown in SEQ ID NO.3) was mutated into the gene encoding the acetylhydroxyl synthase mutant ilvN (SEQ ID NO.2). V25I The amino acid sequence is shown in SEQ ID NO.4. An acetylhydroxy acid synthase mutant strain was constructed using the following method:

[0044] 1. Plasmid pK18mobsacB-ilvN V25I Construction

[0045] 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.

[0046] Table 2 Gibson Assembly Reaction System

[0047] 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

[0048] 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 .

[0049] 2. Construction of acetylhydroxy acid synthase mutant strains

[0050] 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.

[0051] Example 2 Construction of acetylhydroxy acid isomer reductase mutant strain

[0052] Starting with strain MHZ-1012-3, the ilvC gene in MHZ-1012-3 (wild-type ilvC nucleotide sequence as shown in SEQ ID NO.5, wild-type acetylhydroxy acid isomer reductase amino acid sequence as shown in SEQ ID NO.7) was mutated into the gene encoding the acetylhydroxy acid isomer reductase mutant ilvC (SEQ ID NO.6). I90S The amino acid sequence is shown in SEQ ID NO.8. An acetylhydroxy acid isomer reductase mutant strain was constructed using the following method:

[0053] 1. Plasmid pK18mobsacB-ilvC I90S Construction

[0054] 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.

[0055] Table 3 Gibson Assembly Reaction System

[0056] Components UP-1 DN-1 pk18-1 CE Buffer CE Exnase sterile water Volume / μL 1 1 2 4 2 10

[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-ilvC. I90S .

[0058] 2. Construction of acetylhydroxy acid isomer reductase mutant strain

[0059] The recombinant plasmid pK18mobsacB-ilvC obtained by the method described in 1 above. I90S 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 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.

[0060] Example 3: Construction of a superimposed strain of acetylhydroxy acid isomer reductase mutants

[0061] 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 of SEQ ID NO.6, thus constructing an acetylhydroxy acid isomer reductase mutant superimposed strain. The specific construction method is as follows:

[0062] The recombinant plasmid pK18mobsacB-ilvC constructed using the method described in Example 2 above. I90STransformed 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.

[0063] Example 4 Construction of ppc gene mutant strain

[0064] In the acetylhydroxy acid isomer reductase superimposed mutant strain MHZ-1012-35 constructed by the method described in Example 3 above, the ppc gene with Ptac as the promoter was further introduced at site cg1507 to enhance the expression of the ppc gene. The specific method is as follows:

[0065] 1. Construction of plasmid pK18mobsacB-ppc

[0066] 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-2r 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. 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 4.

[0067] Table 4 Gibson Assembly Reaction System

[0068] Components UP4 PPC DN4 Ptac pk18-4 CE Buffer CE Exnase sterile water Volume / μL 1 1 1 1 2 4 2 8

[0069] 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.

[0070] 2. Construction of PPC gene-enhanced mutant bacteria

[0071] 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.

[0072] Example 5 Construction of gndA gene enhanced mutant strain

[0073] In the strain MHZ-1012-37 constructed using 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:

[0074] 1. Construction of plasmid pK18mobsacB-gndA

[0075] 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.

[0076] 2. Construction of gndA gene-enhanced mutant bacteria

[0077] 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.

[0078] Example 6 Construction of dihydroxy acid dehydratase mutant strain

[0079] Using MHZ-1012-3 as the starting strain, the ilvD gene in MHZ-1012-3 (wild-type ilvD nucleotide sequence as shown in SEQ ID NO. 9, wild-type dihydroxy acid dehydratase amino acid sequence as shown in SEQ ID NO. 11) was mutated into the gene encoding the dihydroxy acid dehydratase mutant ilvD (SEQ ID NO. 10). A237KThe amino acid sequence is shown in SEQ ID NO.12. A dihydroxy acid dehydratase mutant strain was constructed using the following method:

[0080] 1. Plasmid pK18mobsacB-ilvD A237K Construction

[0081] Using Phusion superfidelity polymerase (New England BioLabs), with the genome of the starting strain MHZ-1012-3 as a template, and ilvD A237K -UP-1F / ilvD A237K Using UP-1R as primers, recombinant fragment UP-1 was prepared, and ilvD was used as the primer. A237K -DN-2F / ilvD A237K Using DN-2R as primers, recombinant fragment DN-1 was prepared; using plasmid pk18-mob-sacB as a template, and ilvD A237K -pk18-3F / ilvD A237K -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 5.

[0082] Table 5 Gibson Assembly Reaction System

[0083] Components UP-1 DN-1 pk18-1 CE Buffer CE Exnase sterile water Volume / μL 1 1 2 4 2 10

[0084] 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-ilvD. A237K .

[0085] 2. Construction of mutant strains

[0086] The recombinant plasmid pK18mobsacB-ilvD obtained by the method described in section 1 above. A237K 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 -4The 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-1012-51.

[0087] 3. Using the same methods described in 1 and 2 above, the alanine at position 237 of the wild-type dihydroxy acid dehydratase was mutated to other amino acids similar to lysine: arginine (R), histidine (H) or proline (P), and mutant strains were constructed and named MHZ-1012-52, MHZ-1012-53 and MHZ-1012-54, respectively.

[0088] The primers used were replaced with ilvD accordingly. A237R -UP-1R、ilvD A237R -DN-2F、ilvD A237H -UP-1R、ilvD A237H -DN-2F、ilvD A237P -UP-1R、ilvD A237P -DN-2F.

[0089] Example 7 Construction of a superimposed strain of dihydroxy acid dehydratase mutants

[0090] Using MHZ-1012-31, MHZ-1012-35, and MHZ-1012-38 as starting strains, respectively, the recombinant plasmid pK18mobsacB-ilvD constructed by the method described in Example 6 was applied. A237K Transformed into the starting strain, 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. 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 culture 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 strains, named MHZ-1012-58, MHZ-1012-59, and MHZ-1012-60.

[0091] Example 8: Valine production by shake-flask fermentation of Corynebacterium glutamicum.

[0092] 1. Culture medium

[0093] 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.

[0094] 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.

[0095] 2. Shake-flask fermentation

[0096] (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.

[0097] (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.

[0098] (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 6.

[0099] In Table 6, strain A is a strain that mutates the ilvN gene in Corynebacterium ATCC14067 to the gene shown in SEQ ID NO.2; strain B is a strain that mutates the ilvC gene in Corynebacterium ATCC14067 to the gene shown in SEQ ID NO.6; strain C is a strain that mutates the ilvN gene in Corynebacterium ATCC14067 to the gene shown in SEQ ID NO.2 and the ilvC gene to the gene shown in SEQ ID NO.6; strain D is a strain that mutates the ilvD gene in Corynebacterium ATCC14067 to the gene shown in SEQ ID NO.10; and strain E is a strain that mutates the ilvN gene in Corynebacterium ATCC14067 to the gene shown in SEQ ID NO.2, the ilvC gene to the gene shown in SEQ ID NO.6, and the ilvD gene to the gene shown in SEQ ID NO.10.

[0100] Table 6 Fermentation Results

[0101]

[0102] Note: * indicates a significant difference compared to the respective origin strain (P < 0.01).

[0103] The results showed 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.

[0104] Furthermore, the superimposed mutant ilvC I90S The valine accumulation of the obtained acetylhydroxy acid isomer reductase mutant superimposed strain MHZ-1012-35 reached 10.5 g / L, which is 0.6 g / L higher than that of the original strain MHZ-1012-31, representing an increase of 6%. The byproduct isoleucine did not increase further, and there were no significant changes in leucine and cell OD.

[0105] 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.

[0106] 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 15.2% 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.

[0107] The valine accumulation of the dihydroxy acid dehydratase mutant strain MHZ-1012-51 provided by this invention reached 8.2 g / L, which is 0.7 g / L higher than the original strain MHZ-1012-3, representing an increase of 9.3%. The effect is better than strains MHZ-1012-52, MHZ-1012-53 and MHZ-1012-54, indicating that the optimal amino acid for dihydroxy acid dehydratase is to mutate from alanine to lysine, followed by proline, arginine and histidine.

[0108] The valine accumulation in the superimposed strains of dihydroxy acid dehydratase mutation MHZ-1012-58, MHZ-1012-59, and MHZ-1012-60 were 10.8 g / L, 11.9 g / L, and 15.3 g / L, respectively, which were 0.9 g / L, 1.4 g / L, and 1.5 g / L higher than the original strain before superposition, representing increases of 9.1%, 13.3%, and 10.9%, respectively. The byproducts isoleucine in strain MHZ-1012-60 were reduced by 0.3 g / L and leucine by 0.2 g / L compared to the original strain MHZ-1012-38, and the bacterial growth was normal.

[0109] Therefore, the acetylhydroxy acid synthase mutant ilvN provided by the present invention can be seen. V25I Acetylhydroxy acid isomer reductase mutant ilvC I90S dihydroxy acid dehydratase mutant ilvD A237K 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. The superposition of these mutants also showed a positive effect. This provides a reference for the construction of production strains that produce valine, leucine, isoleucine, and other three-branched amino acids, as well as derivatives derived from them.

[0110] The construction of the strain of the present invention is not limited in terms of the order of steps. Any person skilled in the art who achieves the purpose of the present invention by following the disclosure of the present invention shall fall within the protection scope of the present invention.

[0111] 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. A dihydroxy-acid dehydratase mutant, characterized in that, The mutation is substitution of alanine at position 237 with lysine or proline, based on the amino acid sequence of wild-type dihydroxy acid dehydratase, wherein the amino acid sequence of the wild-type dihydroxy acid dehydratase is shown as SEQ ID NO.

11.

2. A DNA molecule, characterized in that, On the basis of the nucleotide sequence of the wild type ilvD The mutation is that the bases at positions 709-711 are changed from GCC to AAG or CCC. The nucleotide sequence of the wild type ilvD The nucleotide sequence of the wild type gene is shown as SEQ ID NO.

9.

3. A recombinant microorganism, characterized in that, The recombinant microorganism expresses the dihydroxy acid dehydratase mutant of claim 1.

4. The recombinant microorganism of claim 3, wherein, The recombinant microorganism further expresses an acetohydroxy acid synthase mutant and / or an acetohydroxy acid isomeroreductase mutant; The amino acid sequence of the acetohydroxy acid synthase mutant is shown as SEQ ID NO. 4; The amino acid sequence of the acetohydroxy acid isomeroreductase mutant is shown as SEQ ID NO.

8.

5. The recombinant microorganism of claim 3 or 4, characterized in that, The ppc gene of the recombinant microorganism is enhanced, or ppc and gndA are simultaneously enhanced; the starting strain of the recombinant microorganism is Corynebacterium glutamicum (ATCC 13869) Corynebacterium glutamicum ).

6. The recombinant microorganism of claim 5, wherein, The enhancement of the ppc gene is achieved by introducing a ppc gene with a Ptac promoter at cg1507 of the starting strain; The enhancement of the gndA gene is achieved by replacing the original promoter of the gndA gene with a strong promoter Ptac.

7. Use of the recombinant microorganism of any one of claims 3-6 in any one of the following: (1) in the fermentation production of L-valine; (2) in the genetic breeding of microorganisms for the production of L-valine; (3) in the improvement of the yield of fermentation production of L-valine.

8. A method for producing L-valine, characterized by, A process comprising the step of fermentation culture with the recombinant microorganism as claimed in any one of claims 3-6.

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