Diaminopimelate decarboxylase mutant, lysine-producing strain, and construction method and application thereof

Abstract

The application provides a diaminopimelic acid decarboxylase mutant, a lysine production strain, a construction method and application thereof. A cysteine (Cys) at the 57th position of diaminopimelic acid decarboxylase is replaced by arginine (Arg) to construct a lysine production strain. The constructed lysine production strain can effectively improve the L-lysine yield and sugar acid conversion rate.

Description

Technical Field

[0001] This invention relates to the fields of metabolic engineering and genetic engineering, and in particular to a diaminopimelic acid decarboxylase mutant, a lysine-producing strain expressing the diaminopimelic acid decarboxylase 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 (… Corynebacterium glutamic ) and Escherichia coli ( Escherichia coli Corynebacterium glutamicum, a classic Gram-positive lysine-producing strain, possesses 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 lysine-producing strains have evolved from traditional mutagenesis to precision genetic engineering. Early studies often used physicochemical mutagens to treat parental strains, combined with auxotrophic or analog 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 lysine-producing strains has gradually moved 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 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 the most promising production method.

[0005] In recent years, with the development of synthetic biology and systems metabolic engineering, the efficiency of lysine production via fermentation has been continuously improved, and it has firmly become the mainstream method in industrial production. Therefore, constructing a high-yield lysine-producing 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

[0006] In one aspect, the present invention provides a diaminopimelic acid decarboxylase mutant that, relative to the wild-type diaminopimelic acid decarboxylase of Corynebacterium glutamicum, includes an amino acid substitution: the cysteine ​​(Cys) at position 57 is replaced with arginine (Arg).

[0007] In some embodiments, the diaminopimelic acid decarboxylase mutant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% amino acid sequence identity with the wild-type diaminopimelic acid decarboxylase of Corynebacterium glutamicum.

[0008] In some embodiments, the amino acid sequence of the wild-type diaminopimelic acid decarboxylase is shown in SEQ ID NO: 2.

[0009] In some embodiments, the diaminopimelic acid decarboxylase mutant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, and retains diaminopimelic acid decarboxylase activity, wherein the amino acid corresponding to position 57 of the wild-type protein is arginine.

[0010] In some embodiments, the amino acid sequence of the diaminopimelic acid decarboxylase mutant is shown in SEQ ID NO: 4.

[0011] On the other hand, the present invention provides biomaterials related to the diaminopimelic acid decarboxylase mutant described in the above-mentioned technical solutions, which are any of the following: (1) The nucleic acid molecule encoded by the diaminopimelic acid decarboxylase 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).

[0012] In some embodiments, the nucleotide sequence encoding the nucleic acid molecule is shown in SEQ ID NO: 3.

[0013] 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.

[0014] On the other hand, the present invention provides the application of the above-mentioned diaminopimelic acid decarboxylase mutant or biological material in the construction of lysine-producing strains.

[0015] On the other hand, the present invention provides a lysine-producing strain comprising the above-mentioned biological material or a mutant expressing the above-mentioned diaminopimelic acid decarboxylase.

[0016] In some embodiments, the lysine-producing strain is Corynebacterium glutamicum.

[0017] In some embodiments, the Corynebacterium glutamicum includes ATCC13032 and its derived industrial strains.

[0018] In some embodiments, the *Corynebacterium glutamicum* in its pyk The intergenic spacer region between the gene and its downstream genes is recombined and integrated with the nucleic acid molecule encoded by the diaminopimelic acid decarboxylase mutant described in the above technical solution.

[0019] 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 diaminopimelic acid decarboxylase mutant into the genome of Corynebacterium glutamicum.

[0020] In some embodiments, the encoding nucleic acid molecule is integrated into the genome of Corynebacterium glutamicum. pyk The intergenic space between a gene and its downstream genes.

[0021] On the other hand, the present invention provides the application of the above-mentioned diaminopimelic acid decarboxylase mutant, biomaterial, or L-lysine-producing strain in the preparation or production of L-lysine.

[0022] On the other hand, the present invention provides the application of the above-mentioned diaminopimelic acid decarboxylase mutant, biomaterial, or L-lysine producing strain in improving L-lysine yield or sugar-acid conversion rate.

[0023] 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.

[0024] The lysine-producing strain provided by this invention uses a chromosome-integrated expression mutant LysA (C57R). While no growth defects were observed, it improved the sugar-acid conversion rate during L-lysine fermentation. Fermentation validation in a 5 L tank showed a sugar-acid conversion rate of 70.2 g / g after 40 hours. It also significantly increased L-lysine yield, reaching as high as 29.01 g / L after 40 hours of shake-flask fermentation. Compared to the wild-type lysA strain, lysine yield increased by approximately 2.03 g / L, and the sugar-acid conversion rate increased by approximately 5%, providing a novel protein engineering strategy for constructing efficient industrial lysine-producing strains. Detailed Implementation

[0025] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0026] When referring to diaminopimelic acid decarboxylase, the term "mutant" (e.g., diaminopimelic acid decarboxylase mutant) refers to a modified amino acid sequence compared to the wild-type diaminopimelic acid decarboxylase mutant, such as the substitution, deletion, and / or insertion of one or more amino acids. When referring to a strain, "mutant" (e.g., mutant strain) refers to a strain that expresses the aforementioned diaminopimelic acid decarboxylase mutant and / or includes the coding nucleic acid molecules of the aforementioned diaminopimelic acid decarboxylase mutant. For amino acid substitutions, the following nomenclature is used here: 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 C57R.

[0027] The wild-type diaminopimelic acid decarboxylase used in this article can be derived from Corynebacterium glutamicum, whose amino acid sequence is shown in SEQ ID NO: 2, and the corresponding nucleotide sequence encoding the nucleic acid molecule is shown in SEQ ID NO: 1.

[0028] In describing specific amino acid mutation sites, this paper uses the amino acid sequence of the wild-type diaminopimelic acid decarboxylase as a reference.

[0029] 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.

[0030] 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.

[0031] As used herein, "recombinant vector" refers to a linear or circular nucleic acid molecule construct containing a polynucleotide encoding a diaminopimelic acid decarboxylase mutant, such as that provided herein, and said polynucleotide is 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.

[0032] 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.

[0033] 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).

[0034] 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.

[0035] 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.

[0036] 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.

[0037] This invention focuses on the diaminopimelic acid decarboxylase (hereinafter referred to as lysA protein) derived from Corynebacterium glutamicum ATCC 13032. First, the protein was rationally designed using PROSS, and then its thermal stability was predicted using FireProt. The candidate mutation site C57R was screened and obtained. The engineered strain expressing this mutant was constructed using gene editing technology, which improved lysine yield and sugar-acid conversion rate.

[0038] This invention first involves the rational design of a mutant, C57R, to mutate diaminopimelic acid decarboxylase (lysA). Experimental verification yielded a mutant C57R that improves the sugar-acid conversion rate in lysine-producing strains. It was confirmed that expressing this mutant in *Corynebacterium glutamicum* significantly increases lysine yield and conversion rate. Based on this, gene editing technology was used to integrate the wild-type and mutant lysA gene fragments into the genomes of the starting strains *Corynebacterium glutamicum* Clys-45-4 and Clys-35 via homologous recombination, successfully constructing recombinant strains Clys-66-2 and Clys-55. Shake-flask fermentation experiments showed that the mutant strain Clys-66-1 (C57R) produced higher lysine than the control strain Clys-66-1 (WT), with the mutant reaching 29.01 g / L. This was 2.03 g / L higher than the yield of the non-mutated strain *Corynebacterium glutamicum* Clys-66-1 (WT) in shake flasks, and significantly higher than the non-mutated lysA strain. While the yield difference between the two strains was not significant in a 5 L fermenter, the mutant strain showed a 5% increase in sugar-acid conversion rate compared to the wild-type strain, indicating that the mutant has a significant effect on improving lysine conversion. Similar results were observed with the Clys-55 strain. Shake-flask fermentation experiments showed that the mutant strain Clys-55 (C57R) produced higher lysine than the control strain Clys-55 (WT), reaching 22.53 g / L. This was 2.41 g / L higher than the yield of the non-mutated strain *Corynebacterium glutamicum* Clys-55 (WT), and significantly higher than the non-mutated lysA strain. This invention confirms that the mutation at the aforementioned site in the lysA gene can effectively improve the efficiency of L-lysine synthesis, providing an important gene target and engineering strategy for constructing an industrial strain that efficiently produces L-lysine.

[0039] 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.

[0040] The original lysA (EC:4.1.1.20) sequence used in the examples is as follows: C.glutamicum Wild type in the host of ATCC13032.

[0041] The gene editing method used is based on the literature (Wang T, Li Y, Li J, and others . An update of thesuicide 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.

[0042] Example 1: Preparation of Corynebacterium glutamicum Clys-35 and Clys-45-4 1.1 Corynebacterium glutamicum Clys-35 By starting strain C. glutamicum The ATCC 13032 genome was modified by introducing the hom(V59A) mutant and overexpressing the non-lysine-repressing lysC (T311I); the lysI gene was knocked out and genes involved in the lysine synthesis pathway were overexpressed. aspB , asd , lysA and efflux proteins lysE It was obtained by overexpressing the gene pyc(G1A) in the precursor pathway. The specific preparation method of Corynebacterium glutamicum Clys-35 is described in Chinese Patent Publication CN121931063A (Application No.: 202610396310.2, Publication Date: 2026.04.28, Invention Title: A Nicotinamide Nucleotide Transhydrogenase Mutant and Its Application), which is incorporated herein by reference in its entirety.

[0043] 1.2 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.

[0044] The specific preparation process is as follows. The encoding nucleotide sequence of the gene synthesis mutant (GenScript Biotechnology Co., Ltd.) was used as a DNA template for PCR. The PCR reaction conditions were as follows: first, denaturation at 95℃ for 5 minutes; then 29 cycles, each cycle including denaturation at 95℃ for 30 seconds, annealing at 58℃ for 30 seconds, and extension at 72℃ for 30 seconds; finally, extension at 72℃ for 5 minutes. A 1425 bp PCR product was obtained using primers gapN-F and gapN-R, and the product was recovered using a gel extraction kit to obtain the target fragment. The upstream homologous arm was amplified using primers Up-F1 and Up-R1, and the downstream homologous arm was amplified using primers Down-F1 and Down-R1 (all primers are shown in Table 1). The obtained amplified fragment was ligated to the upstream and downstream homologous arms using the seamless cloning enzyme pk18 linear vector, and then transformed into E. coli Top10. After the prepared gene was transformed into E. coli Top10, the transformed strains were selected in LB medium containing kanamycin. Using a plasmid extraction kit, plasmid DNA was obtained from the correct Top 10, yielding the pk18-gapN(G419K) vector containing the introduced mutation. The pk18-gapN(G419K) vector was electroporated into *Corynebacterium glutamicum* Clys-35 via homologous recombination on the chromosome. Strains that had inserted the vector into the chromosome via homologous sequence recombination were selected in BHI medium containing kanamycin. PCR was performed using primers CX3 and CX4 on *Corynebacterium glutamicum* transformants that had undergone two recombinations as templates to identify whether they carried the designed mutation site. Sequencing of the PCR product confirmed that the sequence matched the designed mutant sequence gapN(G419K). The strain with the recombinant gapN mutation was named *Corynebacterium glutamicum* Clys-45-4.

[0045] Table 1 Primer Sequences 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 without destroying functional sites, using natural protein sequence evolutionary information as a constraint and combining structural energy calculation. PROSS's overall design philosophy: PROSS's goal is not to "change function", but to repair and optimize the stability (thermal stability, solubility, expression level) of natural proteins; Its basic assumption is that natural evolution has already screened out the key residues required for function, while there is still room for optimization in stability. Therefore, PROSS adopts a dual constraint strategy of "evolutionary conservation + structural energy minimization".

[0046] Based on the above design strategy, the input comes from... C.glutamicum The wild-type sequence of ATCC13032 was used to identify a mutation that could potentially enhance enzyme activity.

[0047] 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. A Consensus mutation involves 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 were further screened, resulting in C57R, one of the mutations with a high probability of increasing enzyme activity.

[0048] In the following examples, the introduction of lysA and lysA(C57R) into Corynebacterium glutamicum Clys-45-4 and Clys-35 using gene editing technology yielded new strains, named Clys-66-1(WT), Clys-66-2(C57R), and Clys-55-1(WT), Clys-55-2(C57R), respectively, and fermentation potential tests were conducted.

[0049] Example 3: Preparation and lysine production capacity identification of Corynebacterium glutamicum strains Clys-66-1(WT), Clys-66-2(C57R), and Clys-55-1(WT), Clys-55-2(C57R) incorporating lysA and the mutant lysA(C57R). 3.1 Preparation of strains expressing lysA and lysA(C57R) mutant genes integrated into chromosomes To insert the original lysA and lysA site mutant gene coding sequences into Corynebacterium glutamicum Clys-45-4 and Clys-35, respectively, to obtain Clys-66-1(WT), Clys-66-2(C57R), and Clys-55-1(WT), Clys-55-2(C57R) strains, vectors containing the target mutant fragments were constructed. Specifically, wild-type lysA sequences and mutant sequences (GenScript Biotechnology Co., Ltd.) were synthesized, and the obtained wild-type and mutant lysA sequences were used as templates for PCR. The PCR reaction conditions were 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 1338 bp PCR product was obtained using primers lysA-F and lysA-R. The product was then 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 2 below).

[0050] The wild-type lysA fragment and the fragment containing the C57R mutation site, along with their upstream and downstream homologous arms, were seamlessly assembled into a linearized pk18 vector, which was then transformed into *E. coli* Top10. After the prepared gene was transformed into *E. coli* Top10, 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-lysA(C57R) vector containing the introduced mutation and the unmutated pk18-lysA vector.

[0051] The vectors pk18-lysA(C57R) and pk18-lysA were electroporated into *Corynebacterium glutamicum* Clys-45-4 and Clys-35 via homologous recombination on chromosomes. The vectors were inserted into the gene pyk and downstream intergenic spacer region (pykT::Psod-lysA) via homologous sequence recombination in kanamycin-containing medium. PCR was performed using primers CX1 and CX2 on *Corynebacterium glutamicum* transformants that had undergone secondary recombination as templates to identify whether they carried the designed mutation site. Sequencing of the PCR products showed that the sequences matched the designed mutant sequence lysA(C57R). The strains with the recombinant lysA mutation were named *Corynebacterium glutamicum* Clys-66-2(C57R) and Clys-55-2(C57R), and the strains carrying the original lysA sequence were named *Corynebacterium glutamicum* Clys-66-1 (WT) and Clys-55-1 (WT).

[0052] Table 2 Primer sequences used in the experiment 3.2 Identification of Lysine Production Capacity 3.2.1 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] 3.2.2 Seed activation and culture: Inoculate the bacterial suspensions of Corynebacterium glutamicum Clys-66-1(WT), Clys-66-2(C57R), and Clys-55-1(WT), Clys-55-2(C57R) into the preservation tubes and spread them evenly onto the activation slant. Incubate at 32°C for 12 h, transfer to the activation slant and continue incubation for 10 h, then transfer to a shaker containing 5 mL of seed culture medium for seed culture.

[0055] 3.2.3 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 cycle was 40 hours, and no antibiotics or inducers were added during the fermentation process. Each strain was replicated three times. After 40 hours of shake-flask fermentation, the concentration of L-lysine was analyzed using HPLC, and the analytical concentrations of L-lysine are shown in Table 3.

[0056] Table 3. Results of L-lysine yield obtained from 40 h shake-flask fermentation. * Significant differences were observed compared to their respective wild-type lysA strains (WT). p < 0.05.

[0057] 3.2.4 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.

[0058] Fermentation culture: Seed culture was transferred for fermentation at a volume ratio of 30% (V:V). Fermentation broth was added to the initial fermenter to a volume of 2 L. The temperature was 36℃, pH 7.20-7.60, rotation speed 200-1100 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 thiamine. Ammonium sulfate solution: 200 g ammonium sulfate was weighed and diluted to 45 mL. Each strain was repeated three times. After 40 h of fermentation, the concentration of L-lysine was analyzed by HPLC. The average concentration and conversion rate of L-lysine are shown in Table 4.

[0059] Table 4.5 Results of L-lysine yield and sugar-acid conversion rate in L-tank fermentation * Significantly different from the wild-type lysA strain (WT). p < 0.05.

[0060] In this embodiment, a recombinant strain was constructed by introducing the mutant lysA (C57R) gene into Corynebacterium glutamicum using gene editing technology. Subsequent fermentation potential experiments showed that the lysine production of this recombinant strain was further increased compared to the control, and the conversion rate was significantly improved in a 5 L fermenter. In shake-flask fermentation experiments, compared to the wild-type LysA-expressing strain Clys-55-1 (WT), the recombinant strain Clys-55-2 (C57R) showed an increase of approximately 2.4 g / L in L-lysine production, an increase exceeding 10%.

[0061] The recombinant strain showed an approximately 5% increase in sugar-acid conversion and an increase in lysine production of approximately 2.03 g / L (shake flask) and 2.4 g / L, respectively, indicating that the C57R site mutation can improve the catalytic efficiency of LysA, thereby increasing the L-lysine production efficiency. This result confirms that the site mutation of the lysA(C57R) enzyme can effectively improve the lysine production efficiency of the strain.

[0062] 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.

[0063] This article covers some of the nucleic acid and amino acid sequences: original C. glutamicum ATCC13032 bacteria lysA sequence: lysA Gene coding sequence: 1 atggctacag ttgaaaattt caatgaactt cccgcacacg tatggccacg caatgccgtg 61 cgccaagaag acggcgttgt caccgtcgct ggtgtgcctc tgcctgacct cgctgaagaa 121 tacggaaccc cactgttcgt agtcgacgag gacgatttcc gttcccgctg tcgcgacatg 181 gctaccgcat tcggtggacc aggcaatgtg cactacgcat ctaaagcgtt cctgaccaag 241 accattgcac gttgggttga tgaagagggg ctggcactgg acattgcatc catcaacgaa 301 ctgggcattg ccctggccgc tggtttcccc gccagccgta tcaccgcgca cggcaacaac 361 aaaggcgtag agttcctgcg cgcgttggtt caaaacggtg tgggacacgt ggtgctggac 421 tccgcacagg aactagaact gttggattac gttgccgctg gtgaaggcaa gattcaggac 481 gtgttgatcc gcgtaaagcc aggcatcgaa gcacacacc acgagttcat cgccactagc 541 cacgaagacc agaagttcgg attctccctg gcatccggtt ccgcattcga agcagcaaaa 601 gccgccaaca acgcagaaaa cctgaacctg gttggcctgc actgccacgt tggttcccag 661 gtgttcgacg ccgaaggctt caagctggca gcagaacgcg tgttgggcct gtactcacag 721 atccacagcg aactgggcgt tgcccttcct gaactggatc tcggtggcgg atacggcatt 781 841 901 ggccgcgcta tcgcaggccc ctccaccgtg accatctacg aagtcggcac caccaaagac 961 gtccacgtag agacgacaa aacccgccgt tacatcgccg tggacggagg catgtccgac 1021 aacatccgcc cagcactcta cggctccgaa tacgacgccc gcgtagtatcccgcttcgcc 1081 gaaggacc cagtaagcac ccgcatcgtg ggctcccact gcgaatccggcgatatcctg 1141 atcaacgatg aaatctaccc atctgacatc accagcggcg acttccttgcactcgcagcc 1201 accggcgcat actgctacgc catgagctcc cgctacaacg ccttcacacggcccgccgtc 1261 gtgtccgtcc gcgctggcag ctcccgcctc atgctgcgcc gcgaaacgctcgacgacatc 1321 ctctcactag aggcataa (SEQ ID NO: 1) lysA Amino acid sequence corresponding to the gene: MATVENFNELPAHVWPRNAVRQEDGVVTVAGVPLPDLAEEYGTPLFVVDEDDFRSRCRDMATAFGGPGNVHYASKAFLTKTIARWVDEEGLALDIASINELGIALAAGFPASRITAHGNNKGVEFLRALVQNGVGHVVLDSAQELELLDYVAAGEGKIQDVLIRVKPGIEAHTHEFIATSHEDQKFGFSLASGSAFEAAKAANNAENLNLVGLHCHVGSQVFDAEGFKLAAERVLGLYSQIHSELGVALPELDLGGGYGIAYTAAEEPLNVAEVASDLLTAVGKMAAELGIDAPTVLVEPGRAIAGPSTVTIYEVGTTKDVHVDDDKTRRYIAVDGGMSDNIRPALYGSEYDARVVSRFAEGDPVSTRIVGSHCESGDILINDEIYPSDITSGDFLALAATGAYCYAMSSRYNAFTRPAVVSVRAGSSRLMLRRETLDDILSLEA (SEQ ID NO: 2) Gene sequence corresponding to the mutated lysA(C57R): 1 atggctacag ttgaaaattt caatgaactt cccgcacacg tatggccacg caatgccgtg 61 cgccaagaag acggcgttgt caccgtcgct ggtgtgcctc tgcctgacct cgctgaagaa 121 tacggaaccc cactgttcgt agtcgacgag gacgatttcc gttcccgc c g tcgcgacatg 181 gctaccgcat tcggtggacc aggcaatgtg cactacgcat ctaaagcgtt cctgaccaag 241 accattgcac gttgggttga tgaagagggg ctggcactgg acattgcatc catcaacgaa 301 ctgggcattg ccctggccgc tggtttcccc gccagccgta tcaccgcgca cggcaacaac 361 aaaggcgtag agttcctgcg cgcgttggtt caaaacggtg tgggacacgt ggtgctggac 421 tccgcacagg aactagaact gttggattac gttgccgctg gtgaaggcaa gattcaggac 481 gtgttgatcc gcgtaaagcc aggcatcgaa gcacacaccc acgagttcat cgccactagc 541 cacgaagacc agaagttcgg attctccctg gcatccggtt ccgcattcga agcagcaaaa 601 gccgccaaca acgcagaaa cctgaacctg gttggcctgc actgccacgt tggttcccag 661 gtgttcgacg ccgaaggctt caagctggca gcagaacgcg tgttgggcct gtactcacag 721 atccacagcg aactgggcgt tgcccttcct gaactggatc tcggtggcgg atacggcatt 781 gcctataccg cagctgaaga accactcaac gtcgcagaag ttgcctccga cctgctcacc 841 gcagtcggaa aaatggcagc ggaactaggc atcgacgcac caaccgtgct tgttgagccc 901 ggccgcgcta tcgcaggccc ctccaccgtg accatctacg aagtcggcac caccaaagac 961 gtccacgtag acgacgacaa aacccgccgt tacatcgccg tggacggagg catgtccgac 1021 aacatccgcc cagcactcta cggctccgaa tacgacgccc gcgtagtatcccgcttcgcc 1081 gaaggagacc cagtaagcac ccgcatcgtg ggctcccact gcgaatccggcgatatcctg 1141 atcaacgatg aaatctaccc atctgacatc accagcggcg acttccttgcactcgcagcc 1201 accggcgcat actgctacgc catgagctcc cgctacaacg ccttcacacggcccgccgtc 1261 gtgtccgtcc gcgctggcag ctcccgcctc atgctgcgcc gcgaaacgctcgacgacatc ctctcactag aggcataa (SEQ ID NO: 3) Amino acid sequence corresponding to the mutated lysA(C57R): MATVENFNELPAHVWPRNAVRQEDGVVTVAGVPLPDLAEEYGTPLFVVDEDDFRSR RRDMATAFGGPGNVHYASKAFLTKTIARWVDEEGLALDIASINELGIALAAGFPASRITAHGNNKGVEFLRALVQNGVGHVVLDSAQELELLDYVAAGEGKIQDVLIRVKPGIEAHTHEFIATSHEDQKFGFSLASGSAFEAAKAANNAENLNLVGLHCHVGSQVFDAEGFKLAAERVLGLYSQIHSELGVALPELDLGGGYGIAYTAAEEPLNVAEVASDLLTAVGKMAAELGIDAPTVLVEPGRAIAGPSTVTIYEVGTTKDVHVDDDKTRRYIAVDGGMSDNIRPALYGSEYDARVVSRFAEGDPVSTRIVGSHCESGDILINDEIYPSDITSGDFLALAATGAYCYAMSSRYNAFTRPAVVSVRAGSSRLMLRRETLDDILSLEA (SEQ ID NO: 4) Coding nucleotide sequence of mutant gapN(G419K): Amino acid sequence of mutated gapN(G419K): MTKQYKNYVNGEWKLSENEIKIYEPASGAELGSVPAMSTEEVDYVYASAKKAQPAWRSLSYIERAAYLHKVADILMRDKEKIGAVLSKEVAKGYKSAVSEVVRTAEIINYAAEGLRMEGEVLEGGSFEAASKKIAVVRREPVGLVLAISPFNYPVNLAGSKIAPALIAGNVIAFKPPTQGSISGLLLAEAFAEAGLPAGVFNTITGRGSEIGDYIVEHQAVNFINFTGSTGIGERIGKMAGMRPIMLELGGKDSAIVLEDADLELTAKNIIAGAFGYSGQRCTAVKRVLVMESVADELVEKIREKVLALTIGNPEDDADITPLIDTKSADYVEGLINDANDKGAALTEIKREGNLICPILFDKVTTDMRLAWEEPFGPVLPIIRVTSVEEAIEISNKSEYGLQASIFTNDFPRAFKIAEQLEVGTVHINNKTQRGTDNFPFLGAKKSGAGIQGVKYSIEAMTTVKSVFDIK(SEQ ID NO: 22)

Claims

1. A diaminopimelic acid decarboxylase mutant, characterized in that, The amino acid sequence of the diaminopimelic acid decarboxylase mutant is shown in SEQ ID NO:

4.

2. Biomaterials related to the diaminopimelic acid decarboxylase mutant of claim 1, characterized in that, It can be any of the following: (1) The nucleic acid molecule encoded by the diaminopimelic acid decarboxylase 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).

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

3.

4. A lysine-producing strain, characterized in that, Includes the biological material as described in claim 2 or 3, or the mutant expressing the diaminopimelic acid decarboxylase as described in claim 1.

5. The lysine-producing strain as described in claim 4, characterized in that, The lysine-producing strain is Corynebacterium glutamicum.

6. The lysine-producing strain as described in claim 5, characterized in that, The genome of Corynebacterium glutamicum pyk The recombination and integration of the intergenic spacer region between the gene and its downstream genes comprises the nucleic acid molecule encoded by the diaminopimelic acid decarboxylase mutant as described in claim 1.

7. A method for constructing an L-lysine-producing strain, characterized in that, This includes integrating the encoding nucleic acid molecule of the diaminopimelic acid decarboxylase mutant of claim 1 into the genome of Corynebacterium glutamicum.

8. The application of the diaminopimelic acid decarboxylase mutant of claim 1, or the biomaterial of claim 2 or 3, or the lysine-producing strain of any one of claims 4-6 in improving L-lysine yield or sugar-acid conversion rate.

9. The use of the diaminopimelic acid decarboxylase mutant of claim 1, or the biomaterial of claim 2 or 3, or the lysine-producing strain of any one of claims 4-6 in the preparation or production of L-lysine.

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