Citrate synthase mutant, valine-producing strain, and construction method and application thereof

Abstract

The application provides a citrate synthase mutant, a valine-producing strain and a construction method and application thereof. The serine (Ser) at the 243rd position of the citrate synthase mutant is mutated into tyrosine (Tyr), the glycine (Gly) at the 315th position is mutated into isoleucine (Ile), the arginine (Arg) at the 325th position is mutated into proline (Pro), and the asparagine (Asn) at the 370th position is mutated into tyrosine (Tyr), which are used for constructing a valine-producing strain. The new strain does not contain plasmids, has no growth defects, and does not need to be induced, and has the advantages of good genetic stability, high fermentation conversion rate and the like, and is an excellent strain capable of stably producing valine. The strain efficiently synthesizes valine from scratch with glucose as a substrate, and the yield of valine is significantly improved.

Description

Technical Field

[0001] This invention relates to the fields of metabolic engineering and genetic engineering, and in particular to a citrate synthase mutant, a valine-producing strain expressing the citrate synthase mutant, its construction method, and its application. Background Technology

[0002] L-valine, chemically known as 2-amino-3-methylbutyric acid, is an important branched-chain amino acid (BCAA) that plays a crucial role in protein synthesis, muscle metabolism, tissue repair, and energy supply. As an essential component of human and animal nutrition, L-valine is widely used in food fortification, feed additives, pharmaceuticals, and cosmetics. Furthermore, L-valine is also an important pharmaceutical and chemical intermediate for the synthesis of antibiotics, antihypertensive drugs, and herbicides, with continuously growing market demand. In recent years, with the rapid development of metabolic engineering and synthetic biology technologies, constructing efficient, green, and economical L-valine production systems has become a research hotspot in the industry.

[0003] Currently, the main methods for producing L-valine include chemical synthesis, direct extraction, and microbial fermentation. Among these, chemical synthesis and direct extraction suffer from significant drawbacks such as limited raw materials, demanding reaction conditions, low product optical purity, and environmental pollution, and have been gradually phased out. In contrast, microbial fermentation, with its advantages of low raw material costs, mild reaction conditions, high product purity, environmental friendliness, and ease of large-scale production, has become the mainstream solution for the industrial production of L-valine. Corynebacterium glutamicum, due to its clear genetic background, well-defined metabolic pathway, high food safety, and strong tolerance, is widely used in the industrial production of L-valine. Notably, citrate synthase, as the rate-limiting enzyme of the tricarboxylic acid cycle, plays a crucial role in maintaining intracellular energy and redox balance by regulating the distribution of carbon metabolic flux between the tricarboxylic acid cycle and the L-valine synthesis pathway, thus becoming a potential target for optimizing L-valine production.

[0004] Citrate synthase (CS) is the first rate-limiting enzyme in the tricarboxylic acid cycle (TCA), catalyzing the condensation of acetyl-CoA and oxaloacetate to produce citrate, providing energy and metabolic precursors for cells. In producing strains such as Corynebacterium glutamicum, citrate synthase directly affects the allocation of carbon metabolic flux in the TCA cycle, thereby influencing the accumulation of target products such as L-valine. In recent years, optimizing the allocation of carbon metabolic flux between the TCA cycle and the L-valine synthesis pathway through the rational design of citrate synthase mutants has become an important strategy in the field of metabolic engineering, and related citrate synthase mutants and their applications in L-valine production have been reported. However, research on constructing highly efficient L-valine-producing strains using citrate synthase mutants is still relatively limited and requires further investigation. Summary of the Invention

[0005] In one aspect, the present invention provides a citrate synthase mutant comprising one or more of the following amino acid substitutions relative to wild-type Corynebacterium glutamicum citrate synthase: 1) Replace the serine (Ser) at position 243 with tyrosine (Tyr); 2) Replace the glycine (Gly) at position 315 with isoleucine (Ile); 3) Replace the arginine (Arg) at position 325 with proline (Pro); 4) Replace the asparagine (Asn) at position 370 with tyrosine (Tyr).

[0006] In some embodiments, the citrate synthase mutant comprises the following amino acid substitutions relative to the wild-type citrate synthase of Corynebacterium glutamicum: 1) Replace the serine (Ser) at position 243 with tyrosine (Tyr); 2) Replace the glycine (Gly) at position 315 with isoleucine (Ile); 3) Replace the arginine (Arg) at position 325 with proline (Pro); 4) Replace the asparagine (Asn) at position 370 with tyrosine (Tyr).

[0007] In some embodiments, the citrate synthase mutant has at least 80%, 85%, 90%, or 95% amino acid sequence identity with the wild-type citrate synthase of Corynebacterium glutamicum.

[0008] In some embodiments, the citrate synthase mutant comprises the following amino acid sequence: MFERDIVATDNNKAVLHYPGGEFEMDIIEASEGNNGVVLGKMLSETGLITFDPGYVSTGSTESKITYIDGDAGILRYRGYDIADLAENATFNEVSYLLINGELPTPDELHKFNDEIRHHTLLDEDFKSQFNVFPRDAHPMATLASSVNILSTYYQDQLNPLDEAQLDKATVRLMAKVPMLAAYAHRARKGAPYMYPDNSLNARENF LRMMFGYPTEPYEIDPIMVKALDKLLILHADHEQNCYTSTVRMIGSAQANMFVSIAGGINALSGPLHGGANQAVLEMLEDIKSNHGGDATEFMNKVKNKEDGVRLMGFIHRVYKNYD PPAAIVKETAHEILEHLGGDDLLDLAIKLEEIALADDYFISRKLYPYVDFYTGLIYRAMGFPTDFFTVLFAIGRLPGWIAHYREQLGAAGNKINRPRQVYTGNESRKLVPREER (SEQ ID NO.1, gltA-12).

[0009] In some embodiments, the citrate synthase mutant has an amino acid sequence that is at least 80%, 85%, 90%, or 95% sequence identical to the amino acid sequence shown in SEQ ID NO.1.

[0010] On the other hand, the present invention provides biomaterials related to citrate synthase mutants as described in the above-mentioned technical solutions, which are any of the following: (1) The nucleic acid molecule encoded by the citrate synthase mutant; (2) Includes the expression cassette encoding the nucleic acid molecule described in (1); (3) A recombinant vector comprising the nucleic acid molecule encoded by (1) or the expression cassette by (2).

[0011] gltA-12 ).

[0012] In some embodiments, the encoding nucleic acid molecule encodes the above-mentioned citrate synthase mutant and has at least 80%, 85%, 90%, or 95% sequence identity with the nucleotide sequence shown in SEQ ID NO. 2.

[0013] On the other hand, the present invention provides a valine-producing strain, comprising the biological material described in the above technical solution or expressing the citrate synthase mutant described in the above technical solution.

[0014] In some embodiments, the valine-producing strain is Corynebacterium glutamicum.

[0015] In some embodiments, the valine-producing strain is Corynebacterium glutamicum ATCC 13032 or ATCC13869.

[0016] In some embodiments, the *Corynebacterium glutamicum* in its gltA The gene locus is recombined and integrated with the coding nucleic acid molecule of the citrate synthase mutant described in the above technical solution.

[0017] On the other hand, the present invention provides a method for preparing an L-valine-producing strain, comprising integrating the coding nucleic acid molecule of the above-mentioned citrate synthase mutant into the genome of Corynebacterium glutamicum, wherein the nucleotide sequence of the coding nucleic acid molecule is shown in SEQ ID NO.2.

[0018] In some embodiments, the encoding nucleic acid molecule is integrated into the genome of Corynebacterium glutamicum. gltA Gene loci.

[0019] On the other hand, the present invention provides the application of the above-mentioned citrate synthase mutant, biomaterial, or L-valine-producing strain in the preparation or production of L-valine.

[0020] On the other hand, the present invention provides a method for preparing L-valine, comprising culturing the aforementioned host cells or L-valine-producing strains and isolating the generated L-valine. Detailed Implementation

[0021] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0022] When referring to citrate synthase, the term "mutant" (e.g., citrate synthase mutant) refers to a modified amino acid sequence compared to wild-type citrate synthase, such as the substitution, deletion, and / or insertion of one or more amino acids; when referring to a strain, the term "mutant" (e.g., mutant strain) refers to a strain that can express the aforementioned citrate synthase mutant and / or includes the coding nucleic acid molecules of the aforementioned citrate synthase mutant.

[0023] gltA In describing specific amino acid mutation sites, this paper uses the amino acid sequence of the wild-type citrate synthase as a reference.

[0024] As used in this article, "encoding nucleic acid molecule" refers to a nucleic acid molecule that follows the rules of the genetic code, directs the synthesis of a specific protein or polypeptide chain, and determines its amino acid sequence. Encoding nucleic acid molecules can be DNA, cDNA, or RNA. For DNA-encoding nucleic acid molecules, their boundaries are typically determined by open reading frames (ORFs), which usually begin with the ATG start codon or alternative start codons such as GTG and TTG, and end with a stop codon such as TAA, TAG, and TGA.

[0025] The term "expression cassette" as used in this article refers to a DNA molecule or fragment thereof, containing the essential elements required for gene expression. In other words, it is the smallest functional unit capable of driving the transcription and translation of a target gene (such as a nucleic acid-coding molecule). Typically, an expression cassette includes at least a promoter, a target gene, and a termination signal sequence.

[0026] As used herein, "recombinant vector" refers to a linear or circular nucleic acid molecule construct containing a polynucleotide encoding a citrate synthase mutant as provided herein, and said polynucleotide being operatively linked to additional nucleotides provided for its expression, thus forming an expression cassette. The expression vector also includes host replication capability, typically conferred by the origin of replication, and / or carries selection genes that aid in the identification of transformants. Typically, recombinant vectors used in recombinant DNA technology are in the form of "plasmids," i.e., circular double-stranded DNA loops.

[0027] In this article, "host cell" refers to a cell that can be introduced with the aforementioned coding nucleic acid molecules, expression cassettes, or recombinant vectors, such as *Corynebacterium glutamicum*. After the introduction of these coding nucleic acid molecules, expression cassettes, or recombinant vectors (which may be located outside the host cell genome or integrated into the host cell genome), the host cell can express the protein or polypeptide encoded by that coding nucleic acid molecule. Host cells include the progeny of the host cell, and the progeny may not necessarily be completely identical to the original parent cell due to natural, accidental, or intentional mutations.

[0028] The term “amino acid sequence” as used in this article is synonymous with the terms “polypeptide,” “protein,” and “peptide” and is used interchangeably. A standard single-letter or three-letter code for the amino acid residues is used, where the amino acid sequence is presented with a standard amino-to-carboxyl terminus orientation (i.e., N→C).

[0029] When referring to amino acid or nucleotide sequences, the term "sequence identity" (also known as "sequence uniformity") refers to the degree of similarity between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), typically expressed as a percentage. Commonly used sequence alignment algorithms or software include DANMAN, CLUSTALW, MAFFT, BLAST, MUSCLE, etc. For the purposes of this invention, the publicly available alignment software BLAST (available from https: / / www.ncbi.nlm.nih.gov / ) can be used to obtain the optimal sequence alignment and calculate the sequence identity between the two amino acid or nucleotide sequences using default settings.

[0030] As used in this article, “including,” “contains,” and “has” mean “including but not limited to,” but also refers to situations consisting only of the listed elements.

[0031] The term "or" refers to a single element among the listed optional elements, unless the context explicitly indicates otherwise. The term "and / or" refers to any one, any two, any three, any more, or all of the listed optional elements.

[0032] The valine-producing strain provided by this invention uses the citrate synthase (gltA) sequence from *Corynebacterium glutamicum* ATCC 13032 as a template and constructs a random mutant library using error-prone PCR with universal primers gltA-F and gltA-R. The mutants in this library are then integrated into the genome of *Corynebacterium glutamicum* C2val-4, obtained previously, to obtain a mutant strain library. Fermentation performance screening revealed that the strain integrating the specific advantageous mutants exhibited slightly higher valine yield and significantly higher conversion rate than the control strain C2val-4. Further research was conducted on *Corynebacterium glutamicum* (… Corynebacterium glutamicum ATCC 13869 verification revealed that the mutant was functionally stable. The new strain does not contain plasmids, has no growth defects, requires no induction, and has advantages such as good genetic stability and high fermentation yield. It is an excellent strain for stable production of valine. The strain efficiently synthesizes valine de novo using glucose as a substrate. After 36 hours of shake-flask fermentation, the valine conversion rate can reach as high as 20.49%.

[0033] To further illustrate the present invention, the technical solutions of the present invention will be described in detail below with reference to specific embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] The strain C2val-4 used in the examples was obtained in our laboratory from wild-type Corynebacterium glutamicum (… Corynebacterium glutamicum The valine strain was obtained through engineering modification based on ATCC 13032, with a valine yield of 10.2 g / L (for specific modification methods, please refer to the preparation process of strain Cval-4 in Chinese Patent Publication CN121737078A, which is incorporated herein by reference in its entirety). The validation strain was *Corynebacterium glutamicum* (…). Corynebacterium glutamicum The engineered strain C9val-2, derived from ATCC 13869 through engineering modifications, produces approximately 6.5 g / L of valine.

[0035] The gene editing method used was based on the literature (Wang T, Li Y, Li J, et al. An update of the suicide plasmid-mediated genome editing system in Corynebacterium glutamicum). Microbial Biotechnology (2019, 12: 907-919.). The technical terms such as gene integration and homologous recombination used in the following examples are explained in this article.

[0036] Example 1 Construction of C9val-2 engineered strain 1.1 Genes ilvB and ilvN Construction of point mutant strains Corynebacterium glutamicum ( Corynebacterium glutamicum Using the ATCC 13869 genome as a template, PCR amplification of the upstream homologous arm was performed using primer BN-UF / R, and PCR amplification of the downstream homologous arm was performed using primer BN-DF / R. Two PCR amplifications were performed using primers ilvB(A138V)-R2, ilvB(A138V)-F2, ilvB(A138V)-F1, and ilvB(A138V)-R1. ilvB Using the recovered PCR products containing the mutation as templates, overlap PCR was performed with primers ilvB(A138V)-R2 and ilvB(A138V)-F1 to obtain the complete gene after point mutation. ilvB (A138V) gene fragment. PCR amplification was performed using primers ilvN(202122)-F and ilvN(202122)-R. ilvN Genes were modified by introducing G20D, I21D, and I22F mutations using primers, resulting in mutated genes. ilvN (202122) gene fragment (primer sequences are shown in Table 1).

[0037] Table 1 Primer Sequences fragment ilvB (A138V) ilvN (202122) Homologous arms were ligated to the pk18 linear vector (pK18mobSacB, purchased from Miaoling Plasmid Platform, catalog number P0100) using a seamless cloning enzyme, and then transformed into E. coli Top10 (purchased from Shanghai Angyu Biotechnology Co., Ltd., catalog number: G6015). After transforming the prepared vector into E. coli Top10, the transformed strains were selected in LB medium containing kanamycin. Plasmid DNA was obtained from the correct Top10 using a plasmid extraction kit, which yielded the vector used to construct pk18-ilvB(A138V)-ilvN(202122).

[0038] The plasmid pk18-ilvB(A138V)-ilvN(202122) was electroporated into Corynebacterium glutamicum ( Corynebacterium glutamicum ATCC 13869 was electroporated into competent cells. After electroporation, the cells were plated onto BHI plates containing kanamycin resistance. Transformants that grew on the plates were verified by colony PCR using primers pK18F and pK18R. Single colonies with single-exchange were inoculated into BHI shakers containing 10 μg / mL kanamycin resistance and cultured at 32°C for 12 h. The colonies were then diluted 300 times with BHIS resuscitation solution and plated onto BHI plates containing sucrose. Single colonies that grew on the plates were then spotted onto BHI plates containing both sucrose and kanamycin resistance. Colonies that did not grow on BHI plates containing kanamycin resistance (i.e., the resistance gene was removed) but grew well on BHI plates containing sucrose were verified by colony PCR using identification primers GBN-F / R. The fragment size obtained was consistent with the theoretical value after agarose gel electrophoresis. Positive transformants were screened to obtain strain C9Val1.

[0039] 1.2 Genes ilvB (A138V) ilvN Construction of strains with (G20D, I21D, I22F) strong promoters Corynebacterium glutamicum ( Corynebacterium glutamicum Using the ATCC13869 genome as a template, PCR amplification was performed using primers Ptuf-F / R to obtain the promoter sequence fragment tuf; the upstream homologous arm was amplified using primers BN-Ptuf-UF / R; and the downstream homologous arm was amplified using primers BN-Ptuf-DF / R.

[0040] The tuf fragment was ligated to the upstream and downstream homologous arms using a seamless cloning enzyme in a pk18 linear vector (pK18mobSacB, purchased from Miaoling Plasmid Platform, catalog number P0100), and then transformed into *E. coli* Top10 (purchased from Shanghai Angyu Biotechnology Co., Ltd., catalog number: G6015). After transforming the prepared vector into *E. coli* Top10, the transformed strains were selected in LB medium containing kanamycin. Plasmid DNA was obtained from the correct Top10 using a plasmid extraction kit, yielding the DNA used to construct the pk18-Ptuf (BN) vector.

[0041] The pk18-Ptuf(BN) plasmid was electroporated into C9Val1 electrocompetent cells. After electroporation, the cells were plated on BHI plates containing kanamycin resistance. Transformants grown on the plates were verified by colony PCR using primers pK18F and pK18R. Single-exchange colonies were inoculated into BHI shakers containing 10 μg / mL kanamycin resistance and cultured at 32°C for 12 h. The colonies were then diluted 300-fold with BHIS resuscitation solution and plated onto cells containing sucrose. The bacteria were grown on BHI plates; single colonies grown on the plates were then spotted onto BHI plates containing sucrose and kanamycin resistance. Using the identification primer G-Ptuf(BN)-F / R, colonies that did not grow on the kanamycin-resistant BHI plates (i.e., the resistance gene was removed) but grew well on the sucrose-containing BHI plates were verified by colony PCR. The fragment size obtained by agarose gel electrophoresis was consistent with the theoretical value, and positive transformants were screened to obtain strain C9Val2.

[0042] 1.3 Fermentation potential test of strains C9Val1 and C9Val2 1.3.1 Culture medium Seed culture medium: Yeast powder 8.0 g / L, peptone 3.0 g / L, KH2PO4 3.0 g / L, V B1 V B2 V B3 V B5 V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O 0.5 g / L, the remainder is water.

[0043] Fermentation medium: Glucose 80.0 g / L, corn flour 15.0 g / L, glutamic acid 2.0 g / L, KH2PO4 2.3 g / L, MgSO4·7H2O 1.5 g / L, FeSO4·7H2O 10 mg / L, V B1 V B2 V B3 VB5 V B12 1 mg / L each, V H 0.1 mg / L, phenol red 2%, the remainder is water.

[0044] 1.3.2 Seed activation and culture: The bacterial suspensions of C9Val1 and C9Val2 inoculated in the preservation tubes were evenly spread onto the slant of the activation medium and incubated at 32°C for 12 h. The culture was then transferred to the slant of the activation medium and incubated for another 10 h. Finally, the culture was transferred to a shaker containing 5 mL of seed medium for seed culture.

[0045] 1.3.3 Fermentation culture: The seed culture was inoculated at a rate of 15% into 500 mL Erlenmeyer flasks containing fermentation medium (final volume 35 mL). The flasks were sealed and incubated at 32°C with shaking at 220 rpm. During fermentation, the pH was maintained at 7.0-7.2 by supplementing with urea (phenol red was used as an indicator; the fermentation broth turning yellow indicated acidity, at which point urea was added). The fermentation period was 36 h, and no antibiotics or inducers were added during the fermentation process. After 36 h of shake-flask fermentation, the concentration of L-valine in the fermentation broth was analyzed by HPLC. The L-valine concentrations are shown in Table 2.

[0046] Table 2. Valine concentration in fermentation broth Example 2 Construction of mutant libraries and preparation of strains 2.1 Construction of random mutant libraries To obtain a dominant mutant of citrate synthase, a strain derived from Corynebacterium glutamicum ( Corynebacterium glutamicum ATCC 13032 gltA The gene was used as a mutation template, and a random mutant library was constructed using error-prone PCR with universal primers gltA-F and gltA-R. The sequence of universal primers gltA-F is ggaagagtttttttccgaacaaatatgtttgaaagggatatcgtggctactg (SEQ ID NO.35), and the sequence of gltA-R is gttgaacgagaatcatccgctaaatttagcgctcctcgcgagg (SEQ ID NO.36). The random mutation reaction system is shown in Table 3. When constructing the random mutant library, the mutation rate was controlled to be around 1% by changing the amount of template added, annealing temperature, and the amount of other components in the PCR system. Agarose gel electrophoresis results showed that the error-prone PCR bands were the correct size, and sequencing results showed that the mutation rate met the requirements. The PCR products were purified using a DNA purification kit (TIANGEN, China) to obtain... gltA A library of random mutant genes.

[0047] Table 3 Random Mutation Response System 2.2 Preparation of strains containing random mutants In order to gltA A gene mutant was inserted into the engineered strain C2val-4 to construct a vector containing the target mutant fragment. Specifically, the target mutant fragment was inserted into the engineered strain C2val-4. gltA For the gene locus, primers gltA-UF / R were designed to amplify the upstream homologous arm, and primer gltA-DF / R was designed to amplify the downstream homologous arm. The PCR reaction conditions were as follows: first, denaturation at 95℃ for 5 minutes; then 29 cycles were performed, each cycle including denaturation at 95℃ for 30 seconds, annealing at 58℃ for 30 seconds, and extension at 72℃ for 150 seconds; finally, extension at 72℃ for 5 minutes. (The primers used are shown in Table 2).

[0048] The mutant fragment was ligated to the upstream and downstream homologous arms using a seamless cloning enzyme in a pk18 linear vector (pK18mobSacB, purchased from Miaoling Plasmid Platform, catalog number P0100), and then transformed into *E. coli* Top10 (purchased from Shanghai Angyu Biotechnology Co., Ltd., catalog number: G6015). After transforming the prepared vector into *E. coli* Top10, the transformed strains were selected in LB medium containing kanamycin. Plasmid DNA was obtained from the correct Top10 strains using a plasmid extraction kit, yielding the pk18-gltA vector containing the mutant.

[0049] The pk18-gltA plasmid was electroporated into C2val-4 electroporation competent cells. After electroporation, the cells were plated on BHI plates containing kanamycin resistance. Transformants growing on these plates were validated by colony PCR using primers pK18F and pK18R. Single-crossover colonies were inoculated into BHI shakers containing 10 μg / mL kanamycin resistance and cultured at 32°C for 12 h. The colonies were then diluted 300-fold with BHIS resuscitation buffer and plated onto BHI plates containing sucrose. Single colonies growing on these plates were then used to spot BHI plates containing both sucrose and kanamycin resistance. Colony PCR was performed on colonies that did not grow on kanamycin-resistant BHI plates (i.e., those with the resistance gene removed) but grew well on sucrose BHI plates using identification primers G-gltA-F / R. The resulting fragments, when detected by agarose gel electrophoresis, were consistent with theoretical values. Positive transformants were screened, thus obtaining a strain library containing multiple random mutants.

[0050] Preparation of the activation culture medium used: Glucose 2.0 g / L, peptone 10.0 g / L, yeast powder 5.0 g / L, sodium chloride 2.5 g / L, KH2PO4 1.0 g / L, MgSO4 0.2 g / L, agar powder 2%, dissolved in water, then adjusted to pH 7.0-7.2 with sodium hydroxide, and brought to a final volume of 500 mL. Sterilized in a high-pressure steam oven at 121℃ for 20 min.

[0051] Example 3 Evaluation of dominant mutants and screening of strains To select mutant strains with increased L-valine production capacity compared to the engineered strain C2val-4, fermentation potential tests were performed on strains from a library of random mutant strains.

[0052] 3.1 Culture medium Seed culture medium: Yeast powder 8.0 g / L, peptone 3.0 g / L, KH2PO4 3.0 g / L, V B1 V B2 V B3 V B5 V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O 0.5 g / L, the remainder is water.

[0053] Fermentation medium: Glucose 80.0 g / L, corn flour 15.0 g / L, glutamic acid 2.0 g / L, KH2PO4 2.3 g / L, MgSO4·7H2O 1.5 g / L, FeSO4·7H2O 10 mg / L, V B1 V B2 V B3 V B5 V B12 1 mg / L each, V H 0.1 mg / L, phenol red 2%, the remainder is water.

[0054] 3.2 Seed activation and culture: The C2val-4 inoculated in the preservation tube and the library strain prepared in Example 2 were evenly spread on the slant of the activation medium and cultured at 32°C for 12 h. The culture was then transferred to the activation medium slant and cultured for another 10 h. Finally, the culture was transferred to a shaker containing 5 mL of seed medium for seed culture.

[0055] 3.3 Fermentation Culture: Inoculate the seed culture at a rate of 15% into 500 mL Erlenmeyer flasks containing fermentation medium (final volume 35 mL), seal the flasks, and incubate at 32℃ with shaking at 220 r / min. During fermentation, maintain the pH at 7.0-7.2 by adding urea (using phenol red as an indicator; when the fermentation broth turns yellow, it is considered acidic, and urea should be added). The fermentation period is 36 h, and no antibiotics or inducers are added during fermentation. After 36 h of shake-flask fermentation, the concentration of L-valine in the fermentation broth is analyzed by HPLC, and the sugar-acid conversion rate is calculated using the following formula based on the fermentation broth volume and residual sugar. The L-valine concentration, conversion rate, and cell OD are shown in Table 4. Table 4. L-valine concentration, sugar-acid conversion rate, and OD in fermentation broth （36h） Based on the results shown in Table 4, it was found that C2val-gltA-12 slightly increased valine production compared to the engineered strain C2val-4, while significantly improving the conversion rate. The strain C2val-gltA-12, which showed the greatest increase in valine production compared to the engineered strain C2val-4, was selected for subsequent experiments. The results of this example confirm that introducing the gltA dominant mutant can effectively improve the valine conversion rate of the strain.

[0056] Example 4 Identifying mutations through gene sequencing The mutant gene of strain C2val-gltA-12, which exhibits high valine production, was sequenced and compared with that of its originating strain C2val-4. gltA Gene comparisons were conducted. The results revealed a mutant in strain C2val-gltA-12 with enhanced valine production capacity (… gltA-12 A specific location in the ORF region of a gene contains multiple nucleotide sequence mutations.

[0057] Specifically, it was discovered gltA-12 and gltA The gene underwent multiple site mutations, and the specific locations of the resulting amino acid mutations and changes are shown in Table 5.

[0058] Table 5 gltA-12 The encoded protein is relatively gltA Mutation location and amino acid changes in encoded proteins Example 5 mutant gltA-12 Functional verification To further verify the mutant gltA-12 The function of introducing this mutant into strain C9val-2 was utilized. Specifically, targeting... gltA-12 The mutant was amplified using primers gltA-UF / R for the upstream homologous arm and gltA-DF / R for the downstream homologous arm. Using primers gltA-F / R, and with the Cval-gltA-12 strain genome as a template, amplification was performed... gltA- 12 Fragment. The PCR reaction conditions are as follows: first, denature at 95℃ for 5 minutes; then perform 29 cycles, each cycle including denaturation at 95℃ for 30 seconds, annealing at 58℃ for 30 seconds, and extension at 72℃ for 150 seconds; finally, extend at 72℃ for 5 minutes. (Primers used are the same as in 1.2).

[0059] The mutant fragment was ligated to the upstream and downstream homologous arms using a seamless cloning enzyme in the pk18 linear vector (pK18mobSacB, purchased from Miaoling Plasmid Platform, catalog number P0100), and then transformed into *E. coli* Top10 (purchased from Shanghai Angyu Biotechnology Co., Ltd., catalog number: G6015). After transforming the prepared vector into *E. coli* Top10, the transformed strains were selected in LB medium containing kanamycin. Plasmid DNA was obtained from the correct Top10 using a plasmid extraction kit, yielding the pk18-gltA-12 vector containing the specific mutant.

[0060] pk18-gltA-12 was electroporated into C9val-2. After electroporation, the transformed cells were plated on BHI plates containing kanamycin resistance. Transformants growing on these plates were validated by colony PCR using primers pK18F and pK18R. Single colonies with single-exchange were inoculated into BHI shakers containing 10 μg / mL kanamycin resistance and cultured at 32°C for 12 h. The colonies were then diluted 300-fold with BHIS resuscitation buffer and plated onto BHI plates containing sucrose. Single colonies growing on these plates were then spotted onto BHI plates containing both sucrose and kanamycin resistance. Colony PCR was performed using primers G-gltA-F and G-gltA-R to validate colonies that did not grow on kanamycin-resistant BHI plates (i.e., those with the resistance gene removed) but grew well on sucrose BHI plates. Agarose gel electrophoresis showed fragment sizes consistent with theoretical values, and positive transformants were obtained, leading to the strain C9val-gltA-12. The fermentation potential of strain C9val-gltA-12 was tested (using the same method as in Example 3). The L-valine concentration, conversion rate, and cell OD are shown in Table 6.

[0061] Table 6. L-valine concentration, sugar-acid conversion rate, and OD in fermentation broth （36h） Based on the results shown in Table 6, it was found that compared with strain C9val-2, C9val-gltA-12 slightly increased valine production while significantly improving the conversion rate, and strain growth was not affected. The results of this example confirm that the dominant mutant gltA-12 can effectively improve the valine conversion rate of different chassis strains and has a positive effect on yield.

[0062] In summary, this invention employs random mutation technology to... gltA Gene sequences were randomly mutagenized to construct a random mutant library. Mutants from this library were integrated into the genome of engineered strain C2val-4 to obtain a mutant strain library. Fermentation performance screening revealed that strains integrating specific advantageous mutants exhibited significantly higher valine production compared to non-mutated strains. Further integration of this mutant into another chassis strain, C9val-2, for fermentation validation showed that the mutant positively promoted valine production in both chassis strains.

[0063] This invention utilizes error-prone PCR technology to randomly mutate the citrate synthase encoding gene, constructs a mutant library, and screens for a citrate synthase mutant that significantly improves the conversion rate of valine production. This mutant is then introduced into engineered strains to further enhance the conversion rate and yield of L-valine. This strategy provides a new approach for constructing highly efficient L-valine industrial production strains, possessing significant practical value and broad application prospects.

[0064] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A citrate synthase mutant, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1.

2. A biomaterial related to the citrate synthase mutant of claim 1, characterized in that, It can be any of the following: (1) The nucleic acid molecule encoded by the citrate synthase mutant; (2) An expression cassette encoding a nucleic acid molecule as described in (1); (3) A recombinant vector comprising the nucleic acid molecule encoded by (1) or the expression cassette by (2).

3. The biomaterial as described in claim 2, characterized in that, The nucleotide sequence encoding the nucleic acid molecule is shown in SEQ ID NO.

2.

4. A valine-producing strain, characterized in that, The valine-producing strain is Corynebacterium glutamicum, including the biological material described in claim 2 or 3 or expressing the citrate synthase mutant described in claim 1.

5. The valine-producing strain according to claim 4, characterized in that, The Corynebacterium glutamicum in its gltA The gene locus recombination and integration includes the encoding nucleic acid molecule of the citrate synthase mutant as described in claim 1.

6. A method for preparing an L-valine-producing strain, characterized in that, This includes integrating the coding nucleic acid molecule of the citrate synthase mutant of claim 1 into the genome of Corynebacterium glutamicum.

7. The method as described in claim 6, characterized in that, The nucleotide sequence encoding the nucleic acid molecule is shown in SEQ ID NO.

2.

8. The method as described in claim 7, characterized in that, The encoded nucleic acid molecule was recombined and integrated into the genome of Corynebacterium glutamicum. gltA Gene loci.

9. The use of the citrate synthase mutant of claim 1, or the biomaterial of claim 2 or 3, or the valine-producing strain of claim 4 or 5 in the preparation or production of L-valine.

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