Glucose-6-phosphate dehydrogenase mutant, l-lysine production strain and construction method and application thereof

Abstract

The application provides a glucose-6-phosphate dehydrogenase mutant, an L-lysine production strain and a construction method and application thereof. The glucose-6-phosphate dehydrogenase mutant comprises any one or a combination of the following mutations relative to wild-type glucose-6-phosphate dehydrogenase of Corynebacterium glutamicum: 1) T351D; and 2) N96E. The application proves that the above-mentioned site mutation can effectively improve the L-lysine synthesis capacity, and provides an important gene target and engineering strategy for constructing an industrial strain for efficiently producing L-lysine.

Description

Technical Field

[0001] This invention relates to the fields of metabolic engineering and genetic engineering, and in particular to a glucose-6-phosphate dehydrogenase mutant, an L-lysine-producing strain expressing the mutant, its construction method, and its application. Background Technology

[0002] L-Lysine is one of the eight essential amino acids that cannot be synthesized by the human and animal bodies, and it has broad application prospects in food fortification, feed additives, and medicine. With the continuous growth of global demand for amino acids, the construction of high-yield and stable L-lysine industrial strains has become a research hotspot in the field of bio-fermentation.

[0003] Currently, the main bacterial strains used industrially for the production of L-lysine include Corynebacterium glutamicum (Bacillus glutamicum). Corynebacterium glutamicum ) and Escherichia coli ( Escherichia coli Corynebacterium glutamicum, a classic Gram-positive producing strain, boasts advantages such as high safety and strong tolerance; Escherichia coli, on the other hand, is widely used in genetic engineering due to its clear genetic background and ease of manipulation. Breeding techniques for L-lysine-producing strains have evolved from traditional mutagenesis to precision genetic engineering. Early studies often employed physicochemical mutagens to treat parental strains, combined with auxotrophic or analogue resistance screening, to obtain high-yielding mutants with feedback inhibition relief characteristics. However, traditional mutagenesis methods suffer from problems such as strong randomness in mutations and difficulty in stably inheriting desirable traits.

[0004] With the development of metabolic engineering and synthetic biology, the modification of L-lysine-producing strains is gradually moving towards rational design. Engineered strains constructed using the aforementioned strategies, with optimized fed-batch fermentation processes, can achieve L-lysine yields exceeding 260 g / L and a sugar-acid conversion rate of 80%. Optimization at the fermentation process level is also crucial for L-lysine production, including selection of culture medium components, optimization of nitrogen sources, dissolved oxygen control, and feeding strategies, all of which can effectively improve cell biomass and product synthesis efficiency. The significant advancements in microbial metabolic engineering technology have enabled microbial fermentation to overcome the limitations of traditional methods, while also offering advantages such as low raw material costs, mild reaction conditions, high fermentation yields, environmental friendliness, high economic benefits, and ease of large-scale production, making it one of the most promising production methods.

[0005] zwf The gene encodes glucose-6-phosphate dehydrogenase (G6PD), a key rate-limiting enzyme in the first step of the pentose phosphate pathway (PPP) oxidation process. Its core function is to convert NADP into glucose. +As a cofactor, it catalyzes the oxidation of glucose-6-phosphate to gluconolactone-6-phosphate, simultaneously generating reduced coenzyme NADPH. This reaction is one of the main sources of intracellular NADPH supply. Studies have shown that overexpression of zwf in Corynebacterium glutamicum increases the flux of the pentose phosphate pathway by 15%, thereby increasing NADPH supply and ultimately increasing L-lysine production; while knockout... zwf This leads to a significant decrease in NADPH levels (approximately 89.5%), and a corresponding decrease in L-lysine production of 87.69%. Furthermore, through analysis of... zwf Introducing point mutations into genes can enhance the enzyme's response to NADP. + This enhances the affinity for lysine and reduces feedback inhibition from metabolites such as ATP, further optimizing L-lysine synthesis. Therefore, zwf It is a key metabolic engineering target that regulates intracellular NADPH supply and thus affects L-lysine production.

[0006] In recent years, with the development of synthetic biology and systems metabolic engineering, the efficiency of fermentation for L-lysine production has been continuously improved, and it has firmly become the mainstream method in industrial production. Therefore, constructing a high-yielding L-lysine engineered strain with a clear genetic background, optimized metabolic network, and stable performance is a core technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] In one aspect, the present invention provides a glucose-6-phosphate dehydrogenase (G6PD) mutant that, relative to wild-type Corynebacterium glutamicum glucose-6-phosphate dehydrogenase, comprises any one or a combination of the following mutations: 1) T351D; and 2) N96E.

[0008] In some embodiments, the G6PD mutant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the wild-type G6PD of Corynebacterium glutamicum.

[0009] In some embodiments, the amino acid sequence of the wild-type G6PD is shown in SEQ ID NO: 2.

[0010] In some embodiments, the G6PD mutant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, 6, or 8, and retains G6PD enzyme activity.

[0011] In some embodiments, the amino acid sequence of the G6PD mutant is shown as SEQ ID NO: 4, 6 or 8.

[0012] On the other hand, the present invention provides biomaterials related to the G6PD mutant described in the above-mentioned technical solutions, which are any of the following: (1) The nucleic acid molecule encoded by the G6PD mutant; (2) An expression cassette encoding a nucleic acid molecule as described in (1); (3) A recombinant vector comprising (1) the nucleic acid molecule or (2) the expression cassette; (4) A host cell comprising (1) the encoding nucleic acid molecule, (2) the expression cassette or (3) the recombinant vector.

[0013] In some implementations, the host cell is a prokaryote or a eukaryote.

[0014] In some embodiments, the host cell is an L-lysine-producing bacterium.

[0015] In some implementations, the host cell includes Escherichia coli, Corynebacterium, or Bacillus.

[0016] In some implementations, the host cell is a corynebacterium.

[0017] In some embodiments, the host cell is Corynebacterium glutamicum.

[0018] In some embodiments, the nucleotide sequence encoding the nucleic acid molecule is shown as SEQ ID NO: 3, 5 or 7.

[0019] In some embodiments, the encoding nucleic acid has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 3, 5, or 7.

[0020] On the other hand, the present invention provides the application of the above-mentioned G6PD mutant or biological material in the construction of L-lysine producing strains.

[0021] On the other hand, the present invention provides an L-lysine-producing strain comprising the above-mentioned biological material or expressing the above-mentioned G6PD mutant.

[0022] In some embodiments, the L-lysine-producing strain is a prokaryote or a eukaryote.

[0023] In some embodiments, the L-lysine-producing strains include Escherichia coli, Corynebacterium, or Bacillus.

[0024] In some embodiments, the L-lysine-producing strain is Corynebacterium.

[0025] In some embodiments, the L-lysine-producing strain is Corynebacterium glutamicum.

[0026] In some embodiments, the Corynebacterium glutamicum includes ATCC13032 and its derived industrial strains.

[0027] In some embodiments, the Corynebacterium glutamicum integrates the coding nucleic acid molecule of the aforementioned G6PD mutant via homologous recombination.

[0028] On the other hand, the present invention provides a method for constructing an L-lysine-producing strain, comprising integrating the coding nucleic acid molecule of the above-mentioned G6PD mutant into the genome of Corynebacterium glutamicum.

[0029] In some implementations, the encoding nucleic acid molecule is integrated into the genome of Corynebacterium glutamicum via homologous recombination.

[0030] On the other hand, the present invention provides the application of the above-mentioned G6PD mutant, biomaterial, or L-lysine-producing strain in the preparation or production of L-lysine.

[0031] On the other hand, the present invention provides the application of the above-mentioned G6PD mutant, biomaterial, or L-lysine-producing strain in increasing L-lysine production.

[0032] On the other hand, the present invention provides a method for preparing L-lysine, comprising culturing the aforementioned host cells or L-lysine-producing strains and isolating the generated L-lysine.

[0033] Shake-flask fermentation experiments showed that the L-lysine yield of strains expressing the above mutants was higher than that of the control strains. The optimal expression of the mutants resulted in an L-lysine yield of approximately 35.59 g / L, which was about 6.6 g / L higher than that of Corynebacterium glutamicum expressing wild-type G6PD. Furthermore, performance verification in a 5 L fermentation tank showed a yield of up to 191.0 g / L, significantly higher than that of the control strain. This invention demonstrates that the above-mentioned site mutations can effectively enhance L-lysine synthesis capacity, providing important gene targets and engineering strategies for constructing industrial strains that efficiently produce L-lysine. Detailed Implementation

[0034] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0035] Mention glucose-6-phosphate dehydrogenase (G6PD, encoded by the gene is...) zwf When using the "mutant" (such as the G6PD mutant or...), the "mutant" used should be... zwf A mutant is defined as a protein with a modified amino acid sequence compared to the wild-type G6PD, such as the substitution, deletion, and / or insertion of one or more amino acids. When referring to a strain, a mutant (e.g., a mutant strain) means that the strain can express the aforementioned G6PD mutant and / or includes the coding nucleic acid molecule of the G6PD mutant. For amino acid substitution, in this document, it refers to the replacement of one amino acid (called the original amino acid) at a specific position in the amino acid sequence with another amino acid (the substituted amino acid). For example, if the amino acid residue at position 351 of the wild-type protein is changed from threonine (T) to aspartic acid (D), an amino acid substitution can be considered to exist in the mutant protein. For amino acid substitution, the following nomenclature is used in this document: original amino acid, position, and substituted amino acid, and the amino acid name uses the single-letter abbreviations for amino acids as defined by IUPAC. For the example described above, it can be represented as T351D.

[0036] The wild type used in this article zwf The gene sequence can be derived from Corynebacterium glutamicum, and the corresponding wild-type G6PD amino acid sequence is shown in SEQ ID NO: 2. When describing specific amino acid mutation sites, this wild-type amino acid sequence is used as a reference.

[0037] 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.

[0038] 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.

[0039] The term "recombinant vector" as used in this article refers to linear or circular nucleic acid molecular constructs that contain the coding schemes provided in this article. zwf The mutant polynucleotide, operably linked with additional nucleotides for its expression, constitutes an expression cassette. The expression vector also includes host replication capability, typically conferred by the origin of replication, and / or a selection gene that aids in the identification of the transformant. Typically, recombinant vectors used in recombinant DNA technology are in the form of "plasmids," which are circular double-stranded DNA loops.

[0040] The term "host cell" as used in this article refers to a cell that can be introduced with the aforementioned coding nucleic acid molecules, expression cassettes, or recombinant vectors, such as *Corynebacterium glutamicum* or *Escherichia coli*. 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. The host cell includes 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.

[0041] 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).

[0042] 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 at 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.

[0043] 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.

[0044] 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.

[0045] The term "approximately" refers to an approximate range that a person skilled in the art can reasonably understand regarding the stated value after taking into account factors such as measurement error, experimental error, instrument error, and process fluctuations. In some embodiments, it refers to a range within ±2%; in other embodiments, it refers to a range within ±1%.

[0046] This invention is based on Corynebacterium glutamicum. zwf The original sequence was used to design rational mutations to obtain amino acid coding sequences for two effective mutation sites related to zwf. Through gene editing, the coding sequences of the wild-type sequence, the mutant coding gene, and the combination of mutant genes were integrated into the genome of *Corynebacterium glutamicum* Clys-66-2 (refer to Chinese patent application CN122629036A), resulting in four new strains: Clys-77-1 (WT), Clys-77-2 (T351D), Clys-77-3 (N96E), and Clys-77-4 (T351D+N96E). Compared with the unmutated strain Clys-77-1 (WT), the three new strains Clys-77-2 (T351D), Clys-77-3 (N96E), and Clys-77-4 (T351D+N96E) showed significantly increased L-lysine production, indicating that the two mutation sites in the zwf gene have a significant effect on L-lysine production. The new strain is plasmid-free, has no growth defects, requires no induction, and possesses advantages such as good genetic stability and high fermentation yield, making it an excellent strain for stable L-lysine production. This strain efficiently synthesizes L-lysine de novo using glucose as a substrate. Shake-flask fermentation experiments showed that its L-lysine yield was higher than the control strain throughout a 40-hour fermentation period. The optimal mutant achieved an L-lysine yield of approximately 35.59 g / L, comparable to that of the wild-type strain. zwf The yield of Corynebacterium glutamicum increased by about 6.6 g / L compared to the control strain; after 40 h of fermentation in a 5L tank, the L-lysine yield of the mutant strain exceeded 180 g / L, which was also significantly higher than that of the control strain.

[0047] The technical solution of the present invention will be further described below with reference to embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are merely exemplary and do not constitute a limitation on the scope of the present invention. It should be noted that, unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art.

[0048] Example The original used in the embodiments zwf (EC:1.1.1.49) sequence is Corynebacterium glutamicum ( Corynebacterium glutamicum Wild type in the host.

[0049] The gene editing method used is based on the literature (Wang T, Li Y, Li J, et al . An update of thesuicide plasmid-mediated genome editing system in Corynebacterium glutamicum (This was performed in *Microbial Biotechnology*, 2019, 12: 907-919). The technical terms used in the following examples, such as gene integration and homologous recombination, are explained in that article.

[0050] Example 1: Construction of Corynebacterium glutamicum Clys-66-2 The construction process was carried out with reference to Chinese patent application CN122629036A (application number: CN202611129907.7, invention title: diaminopimelic acid decarboxylase mutant, lysine-producing strain and its construction method and application, publication date: 2026.08.25), the entire text of which is incorporated herein by reference. A brief description of the construction process is as follows.

[0051] 1.1 Construction of Corynebacterium glutamicum Clys-35 By starting strain C . glutamicum Introduced on the ATCC 13032 genome hom (V59A) mutant, overexpressing non-lysine-repressed... lysC (T311I); Knockout lysI Genes, overexpressing genes in the lysine synthesis pathway aspB , asd , lysA and efflux proteins lysE It was obtained by overexpressing the gene pyc(G1A) in the precursor pathway. The specific construction method of Corynebacterium glutamicum Clys-35 is described in Chinese Patent Publication CN121931063A (Application No.: 202610396310.2, Invention Title: A Nicotinamide Nucleotide Transhydrogenase Mutant and Its Application, Publication Date: 2026.04.28), which is incorporated herein by reference in its entirety.

[0052] 1.2 Construction of Corynebacterium glutamicum Clys-45-4 It was obtained by further introducing the coding sequence of the glyceraldehyde-3-phosphate dehydrogenase mutant gapN (G419K) into the genome of Corynebacterium glutamicum Clys-35.

[0053] 1.3 Construction of Corynebacterium glutamicum Clys-66-2 It was obtained by further introducing the mutant lysA (C57R) of diaminopimelic acid decarboxylase (lysA) into the genome of Corynebacterium glutamicum Clys-45-4.

[0054] Example 2 Mutation of protein sequence 2.1 Rational design of protein sequences using PROSS The core principle of PROSS protein mutation design is to significantly improve protein stability and expressibility through multi-site co-mutation, constrained by the evolutionary information of the natural protein sequence and combined with structure-energy calculations, without destroying functional sites. The overall design philosophy of PROSS is not to "change function," but to repair and optimize the stability (thermal stability, solubility, and expression level) of natural proteins. Its basic assumption is that natural evolution has already screened out the key residues required for function, while stability still has room for optimization. Therefore, PROSS adopts a dual constraint strategy of "evolutionary conservation + structure-energy minimization."

[0055] Based on the above design strategy, the input comes from... C.glutamicum The amino acid sequence encoded by the wild-type zwf gene in ATCC13032 was used to identify mutations that could potentially enhance enzyme activity.

[0056] 2.2 Fireprot for predicting enzyme thermostability FireProt is a computational tool for rationally designing protein thermal stability. The website allows for Consensus mutations and Energy low-risk mutations. Consensus mutations involve replacing a specific site in the target protein with the amino acid that appears most frequently in multiple sequence alignment (MSA). Amino acids retained by most homologous proteins during evolution are often more stable. Energy low-risk mutations, based on protein 3D structure and energy function calculations, screen for mutations that theoretically reduce free energy (ΔΔG < 0) and are less likely to cause structural conflicts. These mutations exhibit a stabilizing tendency but are unlikely to cause misfolding or loss of function. Based on these two types of mutations, the potential point mutations obtained in section 2.1 are further screened to improve... zwf The most likely mutations for the activity of the gene-encoded glucose-6-phosphate dehydrogenase are T351D and N96E.

[0057] Example 3 Integration zwf and zwf Preparation of mutant gene strains In order to make the original zwf and zwf The coding sequence of the point mutation gene was inserted into *Corynebacterium glutamicum* Clys-66-2 to obtain strains Clys-77-1 (WT), Clys-77-2 (T351D), Clys-77-3 (N96E), and Clys-77-4 (T351D+N96E), respectively, and vectors containing the target mutation fragment were constructed. Specifically, wild-type strains were obtained by PCR using wild-type ATCC13032 genomic DNA as a template. zwf Sequence and mutant sequence. The PCR reaction conditions are as follows: first, denaturation at 95°C for 5 minutes; then 29 cycles, each cycle including denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds; finally, extension at 72°C for 5 minutes. A 1605 bp PCR product sequence of wild-type unmutated zwf was obtained using primers zwf-F and zwf-R. Amplification was then performed using zwf-F and T351D-2, T351D-1 and zwf-R respectively to obtain upstream and downstream fragments of the T351D mutation site. A 1605 bp PCR product sequence of zwf(T351D) mutant was then obtained using zwf-F and zwf-R. Amplification was then performed using zwf-F and N96E-2, N96E-1 and zwf-R respectively to obtain upstream and downstream fragments of the N96E mutation site. A 1605 bp PCR product sequence of wild-type unmutated zwf was then obtained using zwf-F and zwf-R. The PCR product sequence of the zwf(N96E) mutation was obtained by amplification using zwf-F and N96E-2, N96E-1 and T351D-2, and T351D-1 and zwf-R, respectively, to obtain upstream and downstream fragments of the N96E and T351D mutation sites. Then, the PCR product sequence of the zwf(N96E+T351D) mutation was obtained by amplification using zwf-F and zwf-R. The product was recovered using a gel extraction kit to obtain the target fragment. The upstream homologous arm was amplified using primers Up-F and Up-R, and the downstream homologous arm was amplified using primers Down-F and Down-R (all primers are shown in Table 1).

[0058] Wild type obtained zwf Fragments, fragments with different mutation sites, and fragments with different combinations of mutation sites were ligated to upstream and downstream homologous arms using a linear vector with the seamless cloning enzyme pk18, and then transformed into *E. coli* Top10. After transforming the prepared gene into *E. coli* Top10, transformed strains were selected in LB medium containing kanamycin. Using a plasmid extraction kit, plasmid DNA was obtained from the correct Top10 strains, yielding the vectors pk18-zwf(T351D), pk18-zwf(N96E), and pk18-zwf(T351D+N96E) containing the introduced mutations, as well as the unmutated vector pk18-zwf.

[0059] The vectors pk18-zwf(T351D), pk18-zwf(N96E), pk18-zwf(T351D+N96E), and pk18-zwf were electroporated into the genome of *Corynebacterium glutamicum* Clys-66-2 constructed in Example 1 via homologous recombination on the chromosome. Strains that had inserted the vectors into the chromosome via homologous sequence recombination were selected in a kanamycin-containing medium. Using primers CX1 and CX2, PCR was performed on *Corynebacterium glutamicum* transformants that had undergone secondary recombination as templates to identify whether they carried the designed mutation sites. Sequencing of the PCR products showed that the sequences were consistent with the corresponding sequences in the designed mutant sequences zwf(T351D), zwf(N96E), and zwf(T351D+N96E). The recombinant zwf mutant strains were named Corynebacterium glutamicum Clys-77-2 (T351D), Clys-77-3 (N96E), and Clys-77-4 (T351D+N96E), while the strain carrying the original zwf sequence was named Corynebacterium glutamicum Clys-77-1 (WT).

[0060] Table 1: Primer sequences used in the experiment Example 4: Identification of L-Lysine Production Capacity 4.1 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.

[0061] 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.

[0062] 4.2 Seed activation and culture Clys-77-1, Clys-77-2, Clys-77-3 and Clys-77-4 bacterial suspensions were inoculated into the preservation tubes and evenly spread onto the activation slant. The mixture was incubated at 32°C for 12 h, then transferred to the activation slant and incubated for another 10 h. Finally, the mixture was transferred to a shaker containing 5 mL of seed culture medium for seed culture.

[0063] 4.3 Fermentation culture: The seed culture was inoculated at a rate of 15% into 500 mL Erlenmeyer flasks containing fermentation medium (final volume 30 mL). The flasks were sealed with nine layers of gauze and incubated at 37°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 40 h, and no antibiotics or inducers were added during the fermentation process. After 40 h of shake-flask fermentation, the concentration of L-lysine in the fermentation broth was analyzed by HPLC. The analytical concentrations of L-lysine are shown in Table 2.

[0064] Table 2 Results of shake-flask fermentation 4.4 5 L tank fermentation: Seed culture tank: 2 L seed culture medium, inoculated with 10 mL seed liquid, pH 7.20-7.40, temperature 34℃, dissolved oxygen 30-50% self-controlled, tank pressure 0.03-0.05 MPa; OD600×100≥0.40, transfer fermentation.

[0065] Fermentation culture: Seed culture was transferred for fermentation at a volume ratio of 30% (V:V). Fermentation broth was added to the initial fermentation tank to a volume of 2 L. The temperature was 36℃, pH 7.20-7.60, rotation speed 200-1000 rpm, tank pressure 0.03-0.1 MPa, aeration ratio 0.2-3, inoculum size 30%, residual sugar 0.5-1.0%, ammonia nitrogen 0.1-0.2%. Ammonium sulfate was added initially, and ammonia nitrogen was controlled at 0.1-0.15%. The fed-batch volume ratio was 100 parts sugar to 25 parts ammonium sulfate. Ammonium sulfate solution: 200 g ammonium sulfate was weighed and diluted to 450 mL. After 40 h of fermentation, the concentration of L-lysine in the fermentation broth was analyzed by HPLC. The analytical concentration and conversion rate of L-lysine are shown in Table 3.

[0066] Table 3. Fermentation results in 5 L tanks In this embodiment, a recombinant strain was constructed by introducing the mutant zwf(T351D) gene into Corynebacterium glutamicum using gene editing technology. Subsequent fermentation potential experiments showed that the L-lysine yield of this recombinant strain was further increased compared to the control, and no significant difference in conversion rate was found in a 5 L fermenter. This result confirms that the T351D, N96E, and combined mutations of both can effectively improve the L-lysine production efficiency of the strain.

[0067] This invention obtains three mutants, T351D, N96E, and T351D+N96E, by rationally designing mutations in glucose-6-phosphate dehydrogenase, which significantly increase the L-lysine yield of L-lysine-producing strains. Based on this, gene editing technology is used to modify these mutants... zwf The gene mutation fragment was integrated into the genome of the starting strain *Corynebacterium glutamicum* Clys-66-2 via homologous recombination, successfully constructing recombinant strains Clys-77-2(T351D), Clys-77-3(N96E), and Clys-77-4(T351D+N96E). Shake-flask fermentation experiments showed that the L-lysine yield of these mutant strains was higher than that of the control strain, with the optimal mutant reaching an L-lysine yield of approximately 35.59 g / L, comparable to that of the wild-type strain. wf The yield of *Corynebacterium glutamicum* Clys-66-2 was increased by approximately 6.6 g / L compared to the control strain. Furthermore, in a 5 L fermentation performance verification, the highest yield reached 191.0 g / L, significantly higher than the control strain. This invention confirms... zwf Mutations at the aforementioned sites in the gene can effectively enhance the synthesis capacity of L-lysine, providing important gene targets and engineering strategies for constructing industrial strains that efficiently produce L-lysine.

[0068] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Improvements and modifications such as strain modification based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.

[0069] The relevant nucleic acid and amino acid sequences involved in this article are as follows.

[0070] original C. glutamicum ATCC13032 bacteria zwf sequence: zwf Gene coding sequence (SEQ ID NO: 1): zwf Amino acid sequence corresponding to the gene (SEQ ID NO: 2): mstnttpsswtnplrdpqdkrlpriagpsgmvifgvtgdlarkkllpaiydlanrgllppgfslvgygrrewskedfekyvrdaasagartefrenvwerlaegmefvrgnfdddaafdnlaatlkridktrgtagnwayylsippdsftavchqlersgmaesteeawrrviiekpfghnlesahelnqlvnavfpessvfridhylgketvqnilalrfanqlfeplwnsnyvdhvqitmaediglggragyydgigaardviqnhliqllalvameepisfvpaqlqaekikvlsatkpcypldktsargqyaagwqgselvkglreedgfnpesttetfaactleitsrrwagvpfylrtgkrlgrrvteiavvfkdaphqpfdgdmtvslgqnaivirvqpdegvlirfgskvpgsamevrdvnmdfsysesfteespeayerlildalldesslfptneevelswkildpileawdadgepedypagtwgpksademlsrnghtwrrp Gene sequence corresponding to the mutated zwf(T351D) (SEQ ID NO: 3): Amino acid sequence corresponding to mutated zwf(T351D) (SEQ ID NO: 4): mstnttpsswtnplrdpqdkrlpriagpsgmvifgvtgdlarkkllpaiydlanrgllppgfslvgygrrewskedfekyvrdaasagartefrenvwerlaegmefvrgnfdddaafdnlaatlkridktrgtagnwayylsippdsftavchqlersgmaesteeawrrviiekpfghnlesahelnqlvnavfpessvfridhylgketvqnilalrfanqlfeplwnsnyvdhvqitmaediglggragyydgigaardviqnhliqllalvameepisfvpaqlqaekikvlsatkpcypldktsargqyaagwqgselvkglreedgfnpesttetfaactleidsrrwagvpfylrtgkrlgrrvteiavvfkdaphqpfdgdmtvslgqnaivirvqpdegvlirfgskvpgsamevrdvnmdfsysesfteespeayerlildalldesslfptneevelswkildpileawdadgepedypagtwgpksademlsrnghtwrrp Gene sequence corresponding to mutated zwf(N96E) (SEQ ID NO: 5): Amino acid sequence corresponding to mutated zwf(N96E) (SEQ ID NO: 6): mstnttpsswtnplrdpqdkrlpriagpsgmvifgvtgdlarkkllpaiydlanrgllppgfslvgygrrewskedfekyvrdaasagartefreevwerlaegmefvrgnfdddaafdnlaatlkridktrgtagnwayylsippdsftavchqlersgmaesteeawrrviiekpfghnlesahelnqlvnavfpessvfridhylgketvqnilalrfanqlfeplwnsnyvdhvqitmaediglggragyydgigaardviqnhliqllalvameepisfvpaqlqaekikvlsatkpcypldktsargqyaagwqgselvkglreedgfnpesttetfaactleitsrrwagvpfylrtgkrlgrrvteiavvfkdaphqpfdgdmtvslgqnaivirvqpdegvlirfgskvpgsamevrdvnmdfsysesfteespeayerlildalldesslfptneevelswkildpileawdadgepedypagtwgpksademlsrnghtwrrp Gene sequence corresponding to mutated zwf(T351D+N96E) (SEQ ID NO: 7): The amino acid sequence corresponding to the mutated zwf(T351D+N96E) (SEQ ID NO: 8): mstnttpsswtnplrdpqdkrlpriagpsgmvifgvtgdlarkkllpaiydlanrgllppgfslvgygrrewskedfekyvrdaasagartefreevwerlaegmefvrgnfdddaafdnlaatlkri dktrgtagnwayylsippdsftavchqlersgmaesteeawrrviiekpfghnlesahelnqlvnavfpessvfridhylgketvqnilalrfanqlfeplwnsnyvdhvqitmaediglggragyydg igaardviqnhliqllalvameepisfvpaqlqaekikvlsatkpcypldktsargqyaagwqgselvkglreedgfnpesttetfaactleidsrrwagvpfylrtgkrlgrrvteiavvfkdaphq pfdgdmtvslgqnaivirvqpdegvlirfgskvpgsamevrdvnmdfsysesfteespeayerlildalldesslfptneevelswkildpileawdadgepedypagtwgpksademlsrnghtwrrp The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A glucose-6-phosphate dehydrogenase mutant, characterized in that, The amino acid sequence is shown in SEQ ID NO: 4, 6 or 8.

2. Biomaterials related to the glucose-6-phosphate dehydrogenase mutant of claim 1, characterized in that, It can be any of the following: (1) The nucleic acid molecule encoded by the glucose-6-phosphate dehydrogenase mutant; (2) An expression cassette encoding a nucleic acid molecule as described in (1); (3) A recombinant vector comprising (1) the nucleic acid molecule or (2) the expression cassette; (4) A host cell comprising (1) the encoding nucleic acid molecule, (2) the expression cassette or (3) the recombinant vector.

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: 3, 5 or 7.

4. The biomaterial as described in claim 2 or 3, characterized in that, The host cell is Corynebacterium glutamicum.

5. An L-lysine-producing strain, characterized in that, Includes the biological material of claim 2 or 3 or expressing the glucose-6-phosphate dehydrogenase mutant of claim 1, wherein the biological material is the encoding nucleic acid molecule, expression cassette or recombinant vector.

6. The L-lysine-producing strain as described in claim 5, characterized in that, The L-lysine-producing strain is Corynebacterium glutamicum.

7. The application of the glucose-6-phosphate dehydrogenase mutant of claim 1 or the biomaterial of any one of claims 2-4 in the construction of L-lysine-producing strains, characterized in that, The L-lysine-producing strain is Corynebacterium glutamicum.

8. The use of the glucose-6-phosphate dehydrogenase mutant of claim 1, the biomaterial of any one of claims 2-4, or the L-lysine-producing strain of claim 5 or 6 in the production of L-lysine.

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