CN117586998A - Dihydroxy-acid dehydratase mutant, recombinant microorganism thereof and application thereof - Google Patents
Dihydroxy-acid dehydratase mutant, recombinant microorganism thereof and application thereof Download PDFInfo
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- CN117586998A CN117586998A CN202210977534.4A CN202210977534A CN117586998A CN 117586998 A CN117586998 A CN 117586998A CN 202210977534 A CN202210977534 A CN 202210977534A CN 117586998 A CN117586998 A CN 117586998A
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- 108700016168 Dihydroxy-acid dehydratases Proteins 0.000 title claims abstract description 33
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- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims description 24
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1022—Transferases (2.) transferring aldehyde or ketonic groups (2.2)
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01086—Ketol-acid reductoisomerase (1.1.1.86)
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- C12Y202/00—Transferases transferring aldehyde or ketonic groups (2.2)
- C12Y202/01—Transketolases and transaldolases (2.2.1)
- C12Y202/01006—Acetolactate synthase (2.2.1.6)
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- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/01—Hydro-lyases (4.2.1)
- C12Y402/01009—Dihydroxy-acid dehydratase (4.2.1.9), i.e. acetohydroxyacid dehydratase
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Abstract
The invention relates to the technical field of microbial engineering, and particularly discloses a dihydroxy-acid dehydratase mutant, recombinant microorganisms thereof and application thereof. The dihydroxy-acid dehydratase mutant of the present invention uses the amino acid sequence of wild dihydroxy-acid dehydratase as a reference sequence, and the dihydroxy-acid dehydratase mutant contains a mutation in which alanine at position 237 is replaced with lysine, arginine, histidine or proline. The zymophyte with the mutant has obviously improved L-valine production capacity, high yield and less byproducts. The invention provides a novel high-efficiency production mode for producing L-valine by fermentation.
Description
Technical Field
The invention relates to the technical field of microbial engineering, in particular to a dihydroxy-acid dehydratase mutant, recombinant microorganism and application thereof.
Background
Branched-chain amino acids (branch chain amino acid, BCAA) include valine, leucine and isoleucine. Wherein L-valine (L-valine) has chemical name of L-alpha-aminoisovaleric acid and molecular formula of C 5 H 11 NO 2 The relative molecular mass was 117.15. L-valine is white crystal or crystalline powder, odorless, bitter in taste, and has a solubility of 88.5g/L in water at 25deg.C, a solubility of 96.2g/L at 50deg.C, insolubility in cold ethanol, diethyl ether, and acetone, an isoelectric point of 5.96, and a melting point of 315 deg.C.
L-valine is one of eight essential amino acids of the human body and has a particularly important position in human life metabolism due to its special structure and function. L-valine can be widely applied to the pharmaceutical industry, the food industry, the feed industry and the like. Wherein, in the pharmaceutical industry, L-valine can be used as the main component of amino acid transfusion and comprehensive amino acid preparations, and can treat liver failure and central nervous system dysfunction. In the food industry, L-valine is useful as a food additive, a nutritional supplement, a flavoring agent, and the like. L-valine can also be used as amino acid functional beverage and athlete beverage, and has effects of forming muscle, strengthening liver function, relieving muscle fatigue, etc. In the feed industry, L-valine has an important promoting effect on the milk secretion of mammary tissue of animals.
At present, the production method of L-valine mainly comprises three steps: extraction, chemical synthesis, and microbial fermentation. The extraction method and the chemical synthesis method have the problems of limited raw material sources, high production cost, environmental pollution and the like, so that the industrialized production is difficult to realize. The microbial fermentation method for producing L-valine has the advantages of low raw material cost, mild reaction conditions, easy realization of large-scale production and the like, and is the most main method for producing L-valine at present. However, the fermentation performance of the L-valine strain is still poor at present, and the content of the byproduct leucine is high, so that the conversion rate is still low, and the requirement of large-scale industrial production is difficult to meet.
Disclosure of Invention
The invention aims to provide a dihydroxy-acid dehydratase mutant capable of improving fermentation performance of L-valine strain, recombinant microorganism containing the dihydroxy-acid dehydratase mutant and application of the dihydroxy-acid dehydratase mutant.
The specific technical scheme of the invention is as follows:
a dihydroxy-acid dehydratase mutant which comprises a mutation wherein alanine (a) at position 237 is substituted with lysine (K), arginine (R), histidine (H) or proline (P) using the amino acid sequence of a wild-type dihydroxy-acid dehydratase as a reference sequence.
According to the research of the invention, when dihydroxy-acid dehydratase is subjected to specific mutation, the L-valine production capacity of recombinant microorganisms containing the mutant can be improved, the isoleucine production capacity of a byproduct is reduced, and the industrial valine production efficiency is improved.
Preferably, the amino acid sequence of the dihydroxy-acid dehydratase mutant is shown in SEQ ID NO. 12.
The present invention also provides a DNA molecule having a wild-type ilvD gene as a reference sequence, said DNA molecule comprising a mutation of bases 709-711 from GCC to AAG, CGC, CAC or CCC.
Preferably, the nucleotide sequence of the DNA molecule is shown in SEQ ID NO. 10.
The sequence of the wild type ilvD gene can be shown as SEQ ID NO. 9.
The invention also provides a recombinant microorganism which expresses the dihydroxy-acid dehydratase mutant.
Preferably, the recombinant microorganism further expresses an acetohydroxyacid synthase mutant and/or an acetohydroxyacid isomerase mutant;
the acetohydroxy acid synthase mutant takes the amino acid sequence of wild acetohydroxy acid synthase as a reference sequence, and contains a mutation (shown as SEQ ID No. 4) that the 25 th amino acid is mutated from valine (V) to isoleucine (I);
the acetohydroxy acid isomerase mutant takes the amino acid sequence of wild acetohydroxy acid isomerase reductase as a reference sequence, and contains mutation of 90 th amino acid from isoleucine (I) to serine (S) (shown as SEQ ID No. 8).
The present inventors have conducted intensive studies to solve the above problems, and have found that acetohydroxy acid synthase and/or acetohydroxy acid isomerase reductase and/or dihydroxy acid dehydratase of a modified coryneform bacterium can enable a microorganism to produce valine with high efficiency and reduce the content of isoleucine as a by-product, thereby successfully creating a novel microorganism capable of producing valine with high efficiency.
Specifically, preferably, the present invention provides a coryneform bacterium in which amino acid 25 of acetohydroxyacid synthase (reference sequence number wp_003861429.1 on NCBI) encoded by ilvN gene (reference sequence number cey17_rs06890 on NCBI) is mutated from valine (V) to isoleucine (I). The acetohydroxy acid synthase encoded by the ilvN gene is the first enzyme in branched-chain amino acid biosynthesis and is also a key enzyme in branched-chain amino acid biosynthesis, catalyzing two molecules of pyruvic acid to produce acetolactate (acetolactate is a precursor of valine and leucine) and also catalyzing a-ketobutyric acid and pyruvic acid to produce a-acetohydroxybutyric acid (a-acetohydroxybutyric acid is a precursor of isoleucine).
The coryneform bacterium of the present invention has an amino acid 90 of an acetohydroxyacid isomeroreductase (reference sequence number WP_003854117.1 at NCBI) encoded by ilvC gene (reference sequence number CEY17_RS06895 at NCBI) mutated from isoleucine (I) to serine (S). The acetohydroxy acid isomerase encoded by ilvC gene is an important enzyme in the biosynthesis of branched-chain amino acids and catalyzes the formation of 1 molecule of alpha-acetolactate or alpha-acetohydroxybutyrate into 1 molecule of alpha-dihydroxyisovalerate or alpha, beta-dimethylolvalerate (alpha-dihydroxyisovalerate is a precursor of valine and leucine and alpha, beta-dimethylolvalerate is a precursor of isoleucine) while digesting 1 molecule of reducing power (reduced nicotinamide adenine dinucleotide phosphate, NADPH) to produce 1 molecule of Nicotinamide Adenine Dinucleotide Phosphate (NADP) + )。
The coryneform bacterium of the present invention has an intracellular mutation of amino acid 237 of dihydroxy-acid dehydratase (reference sequence number WP_003854128.1 at NCBI) encoded by ilvD gene (reference sequence number CEY17_RS06870 at NCBI) from alanine (A) to lysine (K), arginine (R), histidine (H) or proline (P).
Preferably, the ppc gene of the recombinant microorganism of the invention is enhanced, or both ppc and gndA are enhanced;
more preferably, enhancement of the ppc gene is achieved by introducing a Ptac promoter ppc gene at cg1507 of the starting strain;
enhancement of the gndA gene is achieved by replacing the original promoter of the gndA gene with a strong promoter Ptac.
Still preferably, the present invention further provides an engineering bacterium in which the ppc and/or gndA genes of the valine-producing coryneform bacterium are modified to further improve valine production. Wherein, the reference sequence numbers of ppc and gndA genes on NCBI are CEY17_RS08480 and CEY17_RS07800 respectively.
The starting strain of the recombinant microorganism in the invention is Corynebacterium glutamicum (Corynebacterium glutamicum), corynebacterium beijing (Corynebacterium pekinense), brevibacterium flavum (Breviabacterium flavum) or Escherichia coli. Preferably, the starting strain of the recombinant microorganism is Corynebacterium glutamicum.
The mutation site can be applied to corynebacterium glutamicum, corynebacterium beijing, brevibacterium flavum or escherichia coli, but is not limited to corynebacterium glutamicum, corynebacterium beijing, brevibacterium flavum or escherichia coli, and can also be applied to bacillus subtilis and the like, and is used for producing valine, leucine, isoleucine and other branched-chain amino acids or derivatives thereof.
The invention further provides an application of any one of the recombinant microorganisms as follows:
(1) The application in the fermentation production of L-valine and derivatives thereof;
(2) Use in genetic breeding of microorganisms for producing L-valine and derivatives thereof;
(3) The application of the method in improving the yield of L-valine and derivatives thereof produced by fermentation;
(4) The use of reducing the formation of by-product isoleucine in the fermentative production of L-valine and derivatives thereof.
The present invention also provides a process for producing L-valine, comprising the step of fermentation culture with a recombinant microorganism as described above.
The invention has the advantages that:
the present invention realizes mutation of dihydroxyacid dehydratase (preferably acetohydroxy acid synthase and/or acetohydroxy acid isomerase reductase) by specific modification of ilvD gene derived from Corynebacterium glutamicum (preferably specific modification of ilvN gene and/or ilvC gene derived from Corynebacterium glutamicum), so that valine production ability of the microorganism is enhanced as compared with that of unmodified strain, isoleucine production ability of byproduct is reduced, and valine yield is finally improved.
The zymophyte with the mutant has obviously improved L-valine production capacity, high yield and less byproducts. The invention provides a novel high-efficiency production mode for producing L-valine by fermentation.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. The following examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the product specifications. The reagents or equipment used were conventional products available for purchase through regular channels, with no manufacturer noted.
The names and sequences of the primers involved in the examples of the present invention are shown in Table 1.
TABLE 1 primer sequences (SEQ ID No. 13-54)
The invention discloses a corynebacterium glutamicum which is an initial strain MHZ-1012-3, the construction method of the corynebacterium glutamicum is shown in China patent CN201911370732.9, the corynebacterium glutamicum is obtained by mutating 1 st base of a coding region of an a-isopropyl malate synthase gene leuA of the initial strain MHZ-1012-2 from A to G. MHZ-1012-2 is preserved in China general microbiological culture Collection center (CGMCC) of China Commission for culture Collection of microorganisms (CGMCC) at 11 and 30 days of 2016, the preservation center is the North Silu No.1, 3 of the Chaoyang area of Beijing, and the preservation number is CGMCC No.13406, see Chinese patent CN201611250330.1.
EXAMPLE 1 construction of acetohydroxyacid synthase mutant Strain
Taking MHZ-1012-3 as an initial strain, mutating ilvN gene (wild type ilvN nucleotide sequence is shown as SEQ ID NO.1, wild type acetohydroxy acid synthase amino acid sequence is shown as SEQ ID NO. 3) in MHZ-1012-3 into gene (acetohydroxy acid synthase mutant ilvN) of acetohydroxy acid synthase mutant encoding SEQ ID NO.2 V25I The amino acid sequence is shown as SEQ ID NO. 4), and the acetohydroxy acid synthase mutant strain is constructed by the following specific construction method:
1. plasmid pK18mobsacB-ilvN V25I Construction of (3)
The genome of the initial strain MHZ-1012-3 is used as a template and ilvN is used as a template by utilizing Phusion super fidelity polymerase (New England BioLabs) V25I -UP-1F/ilvN V25I Preparation of recombinant fragment UP-1 with ilvN using UP-1R as primer V25I -DN-2F/ilvN V25I Preparing a recombinant fragment DN-1 by taking DN-2R as a primer; the genome of Corynebacterium glutamicum model strain ATCC13032 was used as a template, ilvN was used V25I -1F/ilvN V25I Preparation of recombinant fragment ilvN using-1R as primer V25I The method comprises the steps of carrying out a first treatment on the surface of the The plasmid pk18-mob-sacB was used as a template, ilvN was used V25I -pk18-3F/ilvN V25I The fragment pk18-1 was obtained by using pk18-3R as a primer, purified by agarose gel recovery kit (Tiangen) and then reacted according to Jeep's kit configuration system, the reaction system being shown in Table 2.
TABLE 2 Jeep Assembly reaction System
| Component (A) | UP-1 | DN-1 | ilvN V25I | pk18-1 | CE Buffer | CE Exnase | Sterile water |
| Volume/. Mu.L | 1 | 1 | 1 | 2 | 4 | 2 | 9 |
Reaction to be formulatedThe system was reacted at 37℃for 30min, 10. Mu.L of transformed Trans1T1 competent cells (TransGen Biotech) were aspirated, the single clone was picked up, the correct inserted fragment was identified by colony PCR, the positive clone with the fragment inserted into pK18mobsacB was further identified by restriction enzyme, and finally the plasmid was sent to Jin Weizhi Biotech Co., ltd. For sequencing, and the plasmid with the correct sequencing was named pK18mobsacB-ilvN V25I 。
2. Construction of acetohydroxyacid synthase mutant strains
Recombinant plasmid pK18mobsacB-ilvN obtained by constructing the method of 1 above V25I The transformant was transferred into the starting strain MHZ-1012-3, and the crossover recombinant was selected on a selection medium containing 15mg/L kanamycin. The temperature of the culture was 30℃and the culture was inverted. The transformants obtained by screening were cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a rotary shaking table. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution is coated on a common solid brain heart infusion medium containing 10% sucrose, and is subjected to stationary culture at 33 ℃ for 48 hours. Transformants grown on this medium were identified. The target mutant strain, designated MHZ-1012-31, was obtained by PCR amplification of the target sequence and nucleotide sequencing analysis.
EXAMPLE 2 construction of acetohydroxy acid isomerase mutant strains
Taking MHZ-1012-3 as an initial strain, mutating ilvC gene (wild type ilvC nucleotide sequence is shown as SEQ ID NO.5, and wild type acetohydroxy acid isomerase reductase amino acid sequence is shown as SEQ ID NO. 7) in MHZ-1012-3 into a gene (acetohydroxy acid isomerase mutant ilvC) encoding acetohydroxy acid isomerase mutant of SEQ ID NO.6 I90S The amino acid sequence is shown as SEQ ID NO. 8), and the acetohydroxy acid isomerase reductase mutant strain is constructed by the following specific construction method:
1. plasmid pK18mobsacB-ilvC I90S Construction of (3)
The genome of the initial strain MHZ-1012-3 is used as a template and ilvC is used by Phusion super fidelity polymerase (New England BioLabs) I90S -UP-1F/ilvC I90S Preparation of recombinant by using-UP-1R as primerFragment UP-1, ilvC I90S -DN-2F/ilvC I90S Preparing a recombinant fragment DN-1 by taking DN-2R as a primer; the plasmid pk18-mob-sacB was used as a template, ilvC was used I90S -pk18-3F/ilvC I90S The fragment pk18-1 was obtained by using pk18-3R as a primer, purified by agarose gel recovery kit (Tiangen) and then reacted according to Jeep's kit configuration system, the reaction system being shown in Table 3.
TABLE 3 Jeep Assembly reaction System
| Component (A) | UP-1 | DN-1 | pk18-1 | CE Buffer | CE Exnase | Sterile water |
| Volume/. Mu.L | 1 | 1 | 2 | 4 | 2 | 10 |
The prepared reaction system was reacted at 37℃for 30min, and 10. Mu.L of the transformed Trans1T1 competent cells (TransGen B)iotech), selecting a monoclonal, identifying the correct inserted fragment by colony PCR, further performing enzyme digestion to identify a positive clone of the inserted fragment pK18mobsacB, finally sending the plasmid to Jin Weizhi Biotech Co., ltd for sequencing, and naming the obtained plasmid with correct sequencing as pK18mobsacB-ilvC I90S 。
2. Construction of acetohydroxy acid isomerase mutant strains
Recombinant plasmid pK18mobsacB-ilvC obtained by constructing the method of 1 above I90S The transformant was transferred into the starting strain MHZ-1012-3, and the crossover recombinant was selected on a selection medium containing 15mg/L kanamycin. The temperature of the culture was 30℃and the culture was inverted. The transformants obtained by screening were cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a rotary shaking table. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution is coated on a common solid brain heart infusion medium containing 10% sucrose, and is subjected to stationary culture at 33 ℃ for 48 hours. Transformants grown on this medium were identified. The target mutant strain, designated MHZ-1012-33, was obtained by PCR amplification of the target sequence and nucleotide sequencing analysis.
EXAMPLE 3 construction of Acetylhydroxy acid isomerase mutant strains
Taking MHZ-1012-31 as an initial strain, mutating ilvC gene in MHZ-1012-31 into a gene for encoding acetohydroxy acid isomerase mutant of SEQ ID NO.6, and constructing an acetohydroxy acid isomerase mutant superposition strain, wherein the specific construction method is as follows:
recombinant plasmid pK18mobsacB-ilvC constructed as described in example 2 above I90S The recombinant was transferred into the starting strain MHZ-1012-31, and the crossover recombinant was selected on a selection medium containing 15mg/L kanamycin. The temperature of the culture was 30℃and the culture was inverted. The transformants obtained by screening were cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a rotary shaking table. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution is coated on a common solid brain heart infusion culture medium containing 10% sucrose, and is calm at 33deg.CCulturing for 48h. Transformants grown on this medium were identified. The target mutant strain, named MHZ-1012-35, was obtained by PCR amplification of the target sequence and nucleotide sequencing analysis.
EXAMPLE 4 construction of ppc Gene mutant strains
In the acetohydroxy acid isomerase-superimposing mutant strain MHZ-1012-35 constructed as described in the above example 3, a ppc gene using Ptac as a promoter was further introduced at a site cg1507 to enhance the expression of the ppc gene, specifically by the following method:
1. construction of plasmid pK18mobsacB-ppc
Preparing an upper homologous arm recombinant fragment UP4 by taking a genome of a starting strain MHZ-1012-35 as a template, taking PI-PPC-1f/PI-PPC-1r as a primer, preparing a PPC gene and a terminator recombinant fragment PPC by taking PI-PPC-2f/PI-PPC-2r as primers, and preparing a lower homologous arm recombinant fragment DN4 by taking PI-PPC-4f/I-PPC-4r as primers; preparing a recombinant fragment Ptac of the tac promoter by taking plasmid pXMJ19 as a template and PI-ppc-3f/PI-ppc-3r as a primer; the recombinant fragment pK18-4 was prepared using plasmid pK18-mob-sacB as a template and PI-pK18-F/PI-pK18-R as a primer, purified by agarose gel recovery kit (Tiangen), and then reacted according to Jeep assembly kit configuration system, the reaction system being shown in Table 4.
TABLE 4 Jeep Assembly reaction System
| Component (A) | UP4 | PPC | DN4 | Ptac | pk18-4 | CE Buffer | CE Exnase | Sterile water |
| Volume/. Mu.L | 1 | 1 | 1 | 1 | 2 | 4 | 2 | 8 |
The prepared reaction system was reacted at 37℃for 30min, 10. Mu.L of transformed Trans1T1 competent cells (TransGen Biotech) were extracted, the single clone was picked up, the correct inserted fragment was identified by colony PCR, the positive clone of the inserted fragment into pK18mobsacB was further identified by digestion, and finally the plasmid was sent to Jin Weizhi Biotech Co., ltd. For sequencing, and the plasmid with the correct sequencing was named pK18mobsacB-ppc.
2. Construction of ppc Gene-enhanced mutant
The recombinant plasmid pK18mobsacB-ppc obtained by the construction of the method described in the above 1 was transferred into the strain MHZ-1012-35, and the crossover recombinant was selected on a selection medium containing 15mg/L kanamycin. The temperature of the culture was 30℃and the culture was inverted. The transformants obtained by screening were cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a rotary shaking table. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution is coated on a common solid brain heart infusion medium containing 10% sucroseAnd (3) standing and culturing at 33 ℃ for 48 hours. Transformants grown on this medium were identified. The target sequence was amplified by PCR and analyzed by nucleotide sequencing to obtain the target mutant strain designated MHZ-1012-37.
EXAMPLE 5 construction of gndA Gene-enhanced mutant strains
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 a strong promoter Ptac to enhance the expression of the gndA gene, and the specific method is as follows:
1. construction of plasmid pK18mobsacB-gndA
Preparing an upper homologous arm recombinant fragment UP5 by taking a genome of a starting strain MHZ-1012-3 as a template, taking PI-gndA-1f/PI-gndA-1r as a primer, and preparing a lower homologous arm recombinant fragment DN5 by taking PI-gndA-3f/PI-gndA-3r as a primer; preparing a recombinant fragment Ptac of the tac promoter by taking plasmid pXMJ19 as a template and PI-gndA-2f/PI-gndA-2r as a primer; 3 recombinant fragments are fused by utilizing overlap PCR, the fusion fragment is connected with a pK18-mob-sacB vector by utilizing an enzyme cutting site BamHI/EcoRI, 10 mu L of transformation Trans1T1 competent cells (TransGen Biotech) are extracted, a monoclonal is selected, the correct inserted fragments are identified by colony PCR, positive clones of the inserted fragments into the pK18mobsacB are obtained by further enzyme cutting identification, finally, plasmids are sent to Jin Weizhi biotechnology Co for sequencing, and the obtained plasmid with correct sequencing is named as pK18mobsacB-gndA.
2. Construction of gndA Gene-enhanced mutant
The recombinant plasmid pK18mobsacB-gndA obtained by the construction of the method described in the above 1 was transferred into the strain MHZ-1012-37, and the crossover recombinant was selected on a selection medium containing 15mg/L kanamycin. The temperature of the culture was 30℃and the culture was inverted. The transformants obtained by screening were cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a rotary shaking table. During this culture, a second recombination of the transformant takes place and the vector sequence is removed from the genome by gene exchange. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution is coated on a common solid brain heart infusion medium containing 10% sucroseAnd (3) standing and culturing at 33 ℃ for 48 hours. Transformants grown on this medium were identified. The target sequence was amplified by PCR and analyzed by nucleotide sequencing to obtain the target mutant strain designated MHZ-1012-38.
EXAMPLE 6 construction of dihydroxy-acid dehydratase mutant Strain
Taking MHZ-1012-3 as an initial strain, mutating ilvD gene (wild type ilvD nucleotide sequence is shown as SEQ ID NO.9, and wild type dihydroxy-acid dehydratase amino acid sequence is shown as SEQ ID NO. 11) in MHZ-1012-3 into gene (dihydroxy-acid dehydratase mutant ilvD) for encoding dihydroxy-acid dehydratase mutant of SEQ ID NO.10 A237K The amino acid sequence is shown as SEQ ID NO. 12), and the dihydroxy-acid dehydratase mutant strain is constructed by the following specific construction method:
1. plasmid pK18mobsacB-ilvD A237K Construction of (3)
The genome of the initial strain MHZ-1012-3 is used as a template and ilvD is used by Phusion super fidelity polymerase (New England BioLabs) A237K -UP-1F/ilvD A237K Preparation of recombinant fragment UP-1 with ilvD using UP-1R as primer A237K -DN-2F/ilvD A237K Preparing a recombinant fragment DN-1 by taking DN-2R as a primer; the plasmid pk18-mob-sacB was used as a template and ilvD was used A237K -pk18-3F/ilvD A237K The fragment pk18-1 was obtained by using pk18-3R as a primer, purified by agarose gel recovery kit (Tiangen) and then reacted according to Jeep's kit configuration system, the reaction system being shown in Table 5.
TABLE 5 Jeep Assembly reaction System
| Component (A) | UP-1 | DN-1 | pk18-1 | CE Buffer | CE Exnase | Sterile water |
| Volume/. Mu.L | 1 | 1 | 2 | 4 | 2 | 10 |
The prepared reaction system is reacted for 30min at 37 ℃, 10 mu L of Trans1T1 competent cells (TransGen Biotech) are sucked, monoclonal is selected, the inserted fragments are identified to be correct through colony PCR, positive clones of the fragments inserted into pK18mobsacB are obtained through further enzyme digestion identification, finally plasmids are sent to Jin Weizhi Biotech company for sequencing, and the obtained plasmid with correct sequencing is named pK18mobsacB-ilvD A237K 。
2. Construction of mutant strains
Recombinant plasmid pK18mobsacB-ilvD obtained by constructing the method of 1 above A237K The transformant was transferred into the starting strain MHZ-1012-3, and the crossover recombinant was selected on a selection medium containing 15mg/L kanamycin. The temperature of the culture was 30℃and the culture was inverted. The transformants obtained by screening were cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a rotary shaking table. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution is coated on a common solid brain heart infusion medium containing 10% sucrose, and is subjected to stationary culture at 33 ℃ for 48 hours. Transformants grown on this medium were identified. Nucleotide sequencing analysis by PCR amplification of the sequence of interestThe desired mutant strain, designated MHZ-1012-51, was obtained.
3. Using the same method as described in 1 and 2 above, alanine at position 237 of the wild-type dihydroxy-acid dehydratase was mutated to another amino acid similar to lysine: arginine (R), histidine (H) or proline (P), the mutant strains were constructed and obtained and named MHZ-1012-52, MHZ-1012-53 and MHZ-1012-54, respectively.
Wherein the primers used are replaced correspondingly by ilvD A237R -UP-1R、ilvD A237R -DN-2F、ilvD A237H -UP-1R、ilvD A237H -DN-2F、ilvD A237P -UP-1R、ilvD A237P -DN-2F。
EXAMPLE 7 construction of dihydroxy-acid dehydratase mutant strains
Recombinant plasmids pK18mobsacB-ilvD constructed as described in example 6 above were prepared using MHZ-1012-31, MHZ-1012-35 and MHZ-1012-38 as starting strains, respectively A237K The crossover recombinants were selected on selection medium containing 15mg/L kanamycin, transferred into the starting strain. The temperature of the culture was 30℃and the culture was inverted. The transformants obtained by screening were cultured overnight in a common liquid brain heart infusion medium at a temperature of 30℃and shaking culture at 220rpm with a rotary shaking table. The cultures were serially diluted in gradient (10 -2 Serial dilution to 10 -4 ) The diluted solution is coated on a common solid brain heart infusion medium containing 10% sucrose, and is subjected to stationary culture at 33 ℃ for 48 hours. Transformants grown on this medium were identified. The target mutant strain is obtained through PCR amplification of target sequence and nucleotide sequencing analysis, and is named MHZ-1012-58, MHZ-1012-59 and MHZ-1012-60.
Example 8 production of valine by Corynebacterium glutamicum shake flask fermentation
1. Culture medium
Seed culture medium: 15g/L of soybean meal extract, 20g/L of glucose, 7g/L of ammonium sulfate, 0.5g/L of magnesium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 2g/L of urea and the balance of water, and pH7.2.
Fermentation medium: 15g/L of soybean meal extract, 20g/L of glucose, 7g/L of ammonium sulfate, 0.5g/L of magnesium sulfate, 1g/L of monopotassium phosphate, 1g/L of dipotassium phosphate, 2g/L of urea, 15 mug/L of VB3, 100 mug/L of VB1 and HCl and the balance of water, and pH7.2.
2. Shaking flask fermentation
(1) Seed culture: the slant seed 1 loop is selected, inoculated into a 500mL triangular flask filled with 50mL of seed culture medium, and cultured for 10-12h at 30 ℃ under 220r/min in a shaking way.
(2) Fermentation culture: 5mL of the seed solution was inoculated into a 500mL Erlenmeyer flask containing 50mL of the fermentation medium, and cultured at 30℃under shaking at 220r/min for 72 hours.
(3) 1mL of the fermentation broth was centrifuged (12000 rpm,2 min), and the supernatant was collected, and the fermentation broth was assayed for L-valine, leucine and isoleucine by HPLC, and the OD at 562nm was determined spectrophotometrically, and the results are shown in Table 6.
In Table 6, strain A is a strain obtained by mutating ilvN gene in Corynebacterium ATCC14067 to the gene shown in SEQ ID NO. 2; the strain B is a strain obtained by mutating ilvC gene in corynebacterium ATCC14067 into a gene shown in SEQ ID NO. 6; the strain C is obtained by mutating ilvN gene in corynebacterium ATCC14067 to a gene shown in SEQ ID NO.2 and mutating ilvC gene to a gene shown in SEQ ID NO. 6; the strain D is a strain obtained by mutating ilvD gene in corynebacterium ATCC14067 into a gene shown in SEQ ID NO. 10; the strain E is a strain in which the ilvN gene in Corynebacterium ATCC14067 is mutated to the gene shown in SEQ ID NO.2, the ilvC gene is mutated to the gene shown in SEQ ID NO.6, and the ilvD gene is mutated to the gene shown in SEQ ID NO. 10.
TABLE 6 fermentation results
Description: * Indicating a significant difference (P < 0.01) compared to the respective starting bacteria.
The result shows that the valine accumulation of the starting strain MHZ-1012-3 is 7.5g/L, the valine accumulation of the acetohydroxy acid synthase mutant strain MHZ-1012-31 provided by the invention reaches 9.9g/L, the valine accumulation is increased by 2.4g/L, the lifting amplitude reaches 32%, the isoleucine accumulation of a byproduct is 1.5g/L, the valine accumulation is reduced by 34.8% compared with the bacterial strain MHZ-1012-3, and leucine and bacterial OD have no obvious change.
Further, the superposition mutant ilvC I90S The accumulation amount of valine of the obtained acetohydroxy acid isomerism reductase mutant superposition strain MHZ-1012-35 reaches 10.5g/L, the accumulation amount of valine is increased by 0.6g/L compared with that of the strain MHZ-1012-31, the lifting amplitude is 6%, the byproduct isoleucine is not further increased, and leucine and thalli OD are not obviously changed.
From this, it can be seen that the acetohydroxy acid synthase mutant ilvN provided by the present invention V25I And acetohydroxy acid isomerate reductase mutant ilvC I90S The mutant strain thereof has obvious positive effect on the yield of valine as a main product, has obvious negative effect on the yield of isoleucine as a byproduct, and provides reference for the construction of the production strain for producing three-branched-chain amino acids such as valine, leucine, isoleucine and the like and derivatives taking the mutant strain as precursors.
Meanwhile, in acetohydroxy acid synthase mutant ilvN V25I And acetohydroxy acid isomerate reductase mutant ilvC I90S Based on the above, the enhancement of ppc or the simultaneous enhancement of ppc and gndA is carried out, the accumulation amount of valine is obviously increased to 12.1g/L and 13.8g/L respectively, and the improvement amplitude is respectively 15.2% and 31.4%. From this, it can be seen that acetohydroxy acid synthase mutant ilvN V25I And acetohydroxy acid isomerate reductase mutant ilvC I90S The modified amino acid has obvious positive effect when added with the enhancement of ppc or the simultaneous enhancement and superposition of ppc and gndA, and provides reference for the construction of production strains for producing valine, leucine, isoleucine and other three-branched-chain amino acids and derivatives taking the amino acids as precursors.
The accumulation amount of valine of the dihydroxy-acid dehydratase mutant strain MHZ-1012-51 reaches 8.2g/L, the accumulation amount of valine is increased by 0.7g/L compared with that of the strain MHZ-1012-3, the lifting amplitude is 9.3%, the effect is better than that of the strains MHZ-1012-52, MHZ-1012-53 and MHZ-1012-54, and the situation that the 237 th amino acid of the dihydroxy-acid dehydratase is mutated from alanine to lysine optimally is shown, and then proline, arginine and histidine are sequentially carried out.
The accumulation of valine of the dihydroxy-acid dehydratase mutant superposition strain MHZ-1012-58, MHZ-1012-59 and MHZ-1012-60 is sequentially 10.8g/L, 11.9g/L and 15.3g/L, which are sequentially increased by 0.9g/L, 1.4g/L and 1.5g/L compared with the initial strain before superposition, the increase amplitude is respectively 9.1%, 13.3% and 10.9%, the byproduct isoleucine of the strain MHZ-1012-60 is reduced by 0.3g/L, the leucine is reduced by 0.2g/L, and the bacterial growth is normal.
From this, it can be seen that the acetohydroxy acid synthase mutant ilvN provided by the present invention V25I Acetohydroxy acid isomerism reductase mutant ilvC I90S Dihydroxy-acid dehydratase mutant ilvD A237K And mutant strains thereof have a remarkable positive effect on the yield of valine as a main product and a remarkable negative effect on the yield of isoleucine as a by-product. The superposition of the above mutants also shows positive effects. Provides reference for the construction of the production strain which takes the amino acid with three branched chains such as valine, leucine, isoleucine and the like as the precursor.
The order of the steps of the construction of the strain of the present invention is not limited, and it is within the scope of the present invention for a person skilled in the art to achieve the object of the present invention according to the present disclosure.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. A dihydroxy-acid dehydratase mutant comprising a mutation wherein alanine at position 237 is replaced with lysine, arginine, histidine, or proline, using the amino acid sequence of a wild-type dihydroxy-acid dehydratase as a reference sequence.
2. The dihydroxy-acid dehydratase mutant of claim 1 having an amino acid sequence as shown in SEQ ID No. 12.
3. A DNA molecule comprising a mutation of bases 709-711 from GCC to AAG, CGC, CAC or CCC, using the wild-type ilvD gene as a reference sequence.
4. A DNA molecule according to claim 3, characterized in that the nucleotide sequence of said DNA molecule is shown in SEQ ID No. 10.
5. A recombinant microorganism expressing the dihydroxy-acid dehydratase mutant of claim 1 or 2.
6. The recombinant microorganism according to claim 5, wherein the recombinant microorganism further expresses an acetohydroxyacid synthase mutant and/or an acetohydroxyacid isomerase mutant;
the acetohydroxy acid synthase mutant takes the amino acid sequence of wild acetohydroxy acid synthase as a reference sequence, and contains a mutation of the 25 th amino acid from valine to isoleucine;
the acetohydroxy acid isomerase mutant takes the amino acid sequence of wild acetohydroxy acid isomerase reductase as a reference sequence and contains a mutation of 90 th amino acid from isoleucine to serine.
7. The recombinant microorganism according to claim 5 or 6, characterized in that the ppc gene of the recombinant microorganism is enhanced, or both ppc and gndA are enhanced;
preferably, enhancement of the ppc gene is achieved by introducing a Ptac promoter ppc gene at cg1507 of the starting strain;
enhancement of the gndA gene is achieved by replacing the original promoter of the gndA gene with a strong promoter Ptac.
8. The recombinant microorganism according to any of claims 5 to 7, characterized in that the starting strain of the recombinant microorganism is corynebacterium glutamicum (Corynebacterium glutamicum), corynebacterium beijing (Corynebacterium pekinense), brevibacterium flavum (Breviabacterium flavum) or Escherichia coli.
9. Use of a recombinant microorganism according to any one of claims 5-8 for any one of the following:
(1) The application in the fermentation production of L-valine and derivatives thereof;
(2) Use in genetic breeding of microorganisms for producing L-valine and derivatives thereof;
(3) The application of the method in improving the yield of L-valine and derivatives thereof produced by fermentation;
(4) The use of reducing the formation of by-product isoleucine in the fermentative production of L-valine and derivatives thereof.
10. A process for producing L-valine, comprising the step of fermentation culturing with a recombinant microorganism according to any one of claims 5 to 8.
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