Phosphoenolpyruvate carboxylase mutant with synergistic yield-increasing effect and application thereof in l-amino acid fermentation production

Abstract

The application discloses a phosphoenolpyruvate carboxylase mutant with a synergistic yield-increasing effect and application thereof in L-amino acid fermentation production, and belongs to the field of genetic engineering and molecular biology. The phosphoenolpyruvate carboxylase mutant comprises one or more mutations of T457R, I495Q, F619V, C665F, N814D and Y835F on the basis of a wild type; the mutant is applied to typical aspartic acid group L-amino acid production strains such as L-lysine, L-threonine and L-arginine, can effectively remodel the central carbon metabolic pathway of the strain, strengthen the synthesis and supply of key oxaloacetate precursors, relieve the metabolic flow bottleneck mediated by the wild type PPC, can significantly improve the fermentation yield and product conversion rate of the target L-amino acid, can optimize the fermentation growth characteristics of the strain, shorten the fermentation cycle, reduce the fermentation production cost, and greatly improve the economic benefits of industrialized fermentation production.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and molecular biology, and specifically relates to a phosphoenolpyruvate carboxylase mutant with synergistic yield-increasing effect and its application in L-amino acid fermentation production. Background Technology

[0002] L-Lysine, L-threonine, and L-arginine are important industrial amino acids, widely used in the food, feed, pharmaceutical, and fermentation industries. Microbial fermentation is one of the main industrial methods for producing these amino acids, with *Escherichia coli* being widely used due to its clear genetic background and mature metabolic modification techniques.

[0003] In microbial metabolic networks, oxaloacetate (OAA) is a crucial intermediate linking glycolysis, the tricarboxylic acid (TCA) cycle, and the aspartate family metabolic pathway. Its supply capacity directly influences the synthesis efficiency of various aspartate family amino acids, including L-lysine, L-threonine, L-methionine, and L-isoleucine. Oxaloacetate not only carries the carbon skeleton's flow distribution but also determines the balance between reducing power (NADH / NADPH) and energy (ATP) in anabolic and catabolitic processes. Phosphoenolpyruvate carboxylase (PEPC / PPC) catalyzes the reaction of phosphoenolpyruvate (PEP) with bicarbonate (HCO3-). - The reaction involves carboxylation to produce oxaloacetic acid and release inorganic phosphate. This reaction is an important reabsorption pathway for CO2 fixation within microorganisms, and its core significance lies in: (1) Carbon capture and refixation: PPC fixes HCO3 - (The hydrated form of CO2) introduces inorganic carbon into the organic metabolic network, significantly enhancing the supply capacity of oxaloacetate without consuming additional ATP. This process is strategically significant for reducing carbon loss (preventing PEP from flowing to pyruvate or acetic acid) and improving carbon yield.

[0004] (2) Enhance the replenishment flux of the TCA cycle: When the intermediates of the TCA cycle are consumed in large quantities for amino acid synthesis, the replenishment reaction catalyzed by PPC can efficiently replenish oxaloacetic acid, maintain the cycle operation, and thus ensure the continuous generation of energy and the stable supply of precursor substances.

[0005] (3) Flexible regulation of CO2 response and metabolism: During fermentation, the ability of PPC to fix CO2 is affected by HCO3-. - Complex regulation of concentration, pH value, and product accumulation. Improving the affinity and fixation efficiency of PPC for CO2 can enable cells to more effectively capture CO2 released from the environment or respiratory metabolism during high-yield fermentation, thus achieving carbon reuse.

[0006] Existing research and patent publications have shown that site-directed mutagenesis, directed evolution, or domain substitution of PPC / PEPC can effectively relieve natural feedback inhibition, improve catalytic efficiency, and thus enhance the accumulation of target compounds (such as L-aspartic acid, L-lysine, L-threonine, and L-arginine) using oxaloacetic acid as a precursor. However, existing mutants still have the following shortcomings: ① High sensitivity to feedback inhibition: They remain sensitive to inhibition of end products or intermediate metabolites such as aspartic acid and malic acid, leading to a significant decrease in enzyme activity during the high-yield stage; ② Limited substrate utilization efficiency: They are sensitive to CO2 / HCO3... - The apparent affinity is low, and the fixation capacity cannot be fully utilized under the CO2 partial pressure of conventional fermentation; ③ Insufficient adaptability of product system: Different amino acid synthesis pathways have different requirements for OAA, ATP, reducing power and carbon flux, and the same mutant is difficult to perform well in multiple fermentation systems such as lysine, threonine and arginine at the same time.

[0007] Therefore, especially in applications using *E. coli* as a host and targeting the industrial fermentation production of amino acids such as L-lysine, L-threonine, and L-arginine, there is an urgent need to obtain PPC mutants with superior performance. Developing and applying such novel PPC mutants is expected to significantly improve the fermentation levels of L-lysine, L-threonine, and L-arginine, while simultaneously laying the enzyme engineering foundation for achieving low-carbon, high-yield biomanufacturing routes. Summary of the Invention

[0008] The present invention aims to provide a phosphoenolpyruvate carboxylase mutant, a polynucleotide encoding the mutant, an expression vector containing the polynucleotide, a host cell containing the expression vector, and a method for increasing the fermentation yield of L-amino acids using the mutant.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a phosphoenolpyruvate carboxylase mutant, wherein the phosphoenolpyruvate carboxylase mutant has one or more amino acid mutations at positions 457, 495, 619, 665, 814, and 835, based on the amino acid sequence corresponding to the parental phosphoenolpyruvate carboxylase, or has one or more amino acid mutations at amino acid residues at equivalent positions in the parental phosphoenolpyruvate carboxylase; the amino acid sequence of the parental phosphoenolpyruvate carboxylase has at least 85% identity with the amino acid shown in SEQ ID NO.1 and has phosphoenolpyruvate carboxylase activity.

[0010] Wild-type sequence: SEQ ID NO:1 MNEQYSALRSNVSMLGKVLGETIKDALGEHILERVETIRKLSKSSRAGNDANRQELLTTLQNLSNDELLPVARAFSQFLNLANTAEQYHSISPKGEAASNPEVIARTLRK LKNQPELSEDTIKKAVESLSLELVLTAHPTEITRRTLIHKMVEVNACLKQLDNKDIADYEHNQLMRRLRQLIAQSWHTDEIRKLRPSPVDEAKWGFAVVENSLWQGVPNY LRELNEQLEENLGYKLPVEFVPVRFTSWMGGDRDGNPNVTADITRHVLLLSRWKATDLFLKDIQVLVSELSMVEATPELLALVGEEGAAEPYRYLMKNLRSRLMATQAWL EARLKGEELPKPEGLLTQNEELWEPLYACYQSLQACGMGIIANGDLLDTLRRVKCFGVPLVRIDIRQESTRHTEALGELTRYLGIGDYESWSEADKQAFLIRELNSKRPLL PRNWQPSAETREVLDTCQVIAEAPQGSIAAYVISMAKTPSDVLAVHLLLKEAGIGFAMPVAPLFETLDDLNNANDVMTQLLNIDWYRGLIQGKQMVMIGYSDSAKDAGVM AASWAQYQAQDALIKTCEKAGIELTLFHGRGGSIGRGGAPAHAALLSQPPGSLKGGLRVTEQGEMIRFKYGLPEITVSSLSLYTGAILEANLLPPPEPKESWRRIMDELSV ISCDVYRGYVRENKDFVPYFRSATPEQELGKLPLGSRPAKRRPTGGVESLRAIPWIFAWTQNRLMLPAWLGAGTALQKVVEDGKQSELEAMCRDWPFFSTRLGMLEMVFA KADLWLAEYYDQRLVDKALWPLGKELRNLQEEDIKVVLAIANDSHLMADLPWIAESIQLRNIYTDPLNVLQAELLHRSRQAEKEGQEPDPRVEQALMVTIAGIAAGMRNTG In one embodiment of the present invention, the mutation corresponds to the amino acid sequence shown in SEQ ID NO.1. The 457th amino acid is mutated to K or H; preferably, it is mutated to R. The 495th amino acid is mutated to N or E; preferably, it is mutated to Q. The amino acid at position 619 is mutated to either I or L; preferably, it is mutated to V. The 665th amino acid is mutated to Y or W; preferably, it is mutated to F. The 814th amino acid is mutated to either E or N; preferably, it is mutated to D. The 835th amino acid is mutated to Y or H; preferably, it is mutated to F.

[0011] In one embodiment of the present invention, the mutant is, corresponding to the amino acid sequence shown in SEQ ID NO.1, the phosphoenolpyruvate carboxylase mutant at least simultaneously possesses any of the following mutations equivalent to or corresponding to the parental phosphoenolpyruvate carboxylase mutant: T457R, I495Q, F619V, C665F, N814D, Y835F, T457R / F619V, T457R / C665F, I495Q / F619V, I49 5Q / C665F, T457R / N814D, T457R / Y835F, I495Q / N814D, I495Q / Y835F, F619V / N814D, F 619V / Y835F, C665F / N814D, C665F / Y835F, T457R / F619V / N814D, I495Q / C665F / Y835F.

[0012] In one embodiment of the present invention, the mutant is obtained by performing any of the following mutations on the wild-type enzyme corresponding to the amino acid sequence shown in SEQ ID NO.1: The threonine at position 457 of the wild-type enzyme was mutated to arginine and named T457R. The isoleucine at position 495 of the wild-type enzyme was mutated to glutamine and named I495Q; The phenylalanine at position 619 of the wild-type enzyme was mutated to valine, and named F619V. The cysteine ​​at position 665 of the wild-type enzyme was mutated to phenylalanine and named C665F. The asparagine at position 814 of the wild-type enzyme was mutated to aspartic acid and named N814D. The tyrosine residue at position 835 of the wild-type enzyme was mutated to phenylalanine and named Y835F. The wild-type enzyme was mutated from threonine at position 457 to arginine, and from phenylalanine at position 619 to valine, and named T457R / F619V. The wild-type enzyme was mutated from threonine at position 457 to arginine, and from cysteine ​​at position 665 to phenylalanine, and named T457R / C665F. The isoleucine at position 495 of the wild-type enzyme was mutated to glutamine, and the phenylalanine at position 619 was mutated to valine, named I495Q / F619V. The isoleucine at position 495 of the wild-type enzyme was mutated to glutamine, and the cysteine ​​at position 665 was mutated to phenylalanine, named I495Q / C665F. The wild-type enzyme was mutated from threonine at position 457 to arginine, and from asparagine at position 814 to aspartic acid, and named T457R / N814D. The wild-type enzyme was mutated from threonine at position 457 to arginine, and from tyrosine at position 835 to phenylalanine, and named T457R / Y835F. The isoleucine at position 495 of the wild-type enzyme was mutated to glutamine, and the asparagine at position 814 was mutated to aspartic acid, named I495Q / N814D. The isoleucine at position 495 of the wild-type enzyme was mutated to glutamine, and the tyrosine at position 835 was mutated to phenylalanine, named I495Q / Y835F. The wild-type enzyme was mutated from phenylalanine at position 619 to valine, and from asparagine at position 814 to aspartic acid, and named F619V / N814D. The wild-type enzyme was mutated from phenylalanine at position 619 to valine, and from tyrosine at position 835 to phenylalanine, and named F619V / Y835F. The cysteine ​​at position 665 of the wild-type enzyme was mutated to phenylalanine, and the asparagine at position 814 was mutated to aspartic acid, named C665F / N814D. The cysteine ​​at position 665 of the wild-type enzyme was mutated to phenylalanine, and the tyrosine at position 835 was mutated to phenylalanine. The enzyme was named C665F / Y835F. The wild-type enzyme was mutated by changing threonine at position 457 to arginine, phenylalanine at position 619 to valine, and asparagine at position 814 to aspartic acid. It was named T457R / F619V / N814D. The isoleucine at position 495 of the wild-type enzyme was mutated to glutamine, while the cysteine ​​at position 665 was mutated to phenylalanine, and the tyrosine at position 835 was mutated to phenylalanine. The enzyme was named I495Q / C665F / Y835F.

[0013] T457R / F619V / N814D Amino Acid Sequence (SEQ ID NO.3): MNEQYSALRSNVSMLGKVLGETIKDALGEHILERVETIRKLSKSSRAGNDANRQELLTTLQNLSNDELLPVARAFSQFLNLANTAEQYHSISPKGEAASNPEVIARTLRKLKNQPELSEDTIKKAVESLSLELVLTAHPTEITRRTLIHKMVEVNACLKQLDNKDIADYEHNQLMRRLRQLIAQSWHTDEIRKLRPSPVDEAKWGFAVVENSLWQGVPNYLRELNEQLEENLGYKLPVEFVPVRFTSWMGGDRDGNPNVTADITRHVLLLSRWKATDLFLKDIQVLVSELSMVEATPELLALVGEEGAAEPYRYLMKNLRSRLMATQAWLEARLKGEELPKPEGLLTQNEELWEPLYACYQSLQACGMGIIANGDLLDTLRRVKCFGVPLVRIDIRQESTRHTEALGELTRYLGIGDYESWSEADKQAFLIRELNSKRPLLPRNWQPSAETREVLDRCQVIAEAPQGSIAAYVISMAKTPSDVLAVHLLLKEAGIGFAMPVAPLFETLDDLNNANDVMTQLLNIDWYRGLIQGKQMVMIGYSDSAKDAGVMAASWAQYQAQDALIKTCEKAGIELTLFHGRGGSIGRGGAPAHAALLSQPPGSLKGGLRVTEQGEMIRVKYGLPEITVSSLSLYTGAILEANLLPPPEPKESWRRIMDELSVISCDVYRGYVRENKDFVPYFRSATPEQELGKLPLGSRPAKRRPTGGVESLRAIPWIFAWTQNRLMLPAWLGAGTALQKVVEDGKQSELEAMCRDWPFFSTRLGMLEMVFAKADLWLAEYYDQRLVDKALWPLGKELRNLQEEDIKVVLAIADDSHLMADLPWIAESIQLRNIYTDPLNVLQAELLHRSRQAEKEGQEPDPRVEQALMVTIAGIAAGMRNTG In one embodiment of the present invention, the enzyme containing at least 85% sequence identity and having phosphoenolpyruvate carboxylase activity as the parent enzyme shown in SEQ ID NO.1 can be derived from: Erwinia、Klebsiella、Mangrovibacter、Tenebrionibacter / Tenebrionicola group、 unclassified Erwinia、Affinibrenneria salicis、Biostraticola tofi、Brenneria goodwinii、Cedecea colo、Chimaeribacter arupi、Citrobacter braakii、Dickeya dadantii、Dickeya oryzae、Edaphovirga cremea、Enterobacillus tribolii、 Enterobacter hormaechei、Enterobacterales bacterium、Enterobacteriaceae bacterium ESL0689、Erwinia sp. HR93、Erwinia sp. OLSSP12、Erwinia sp. V71、 Escherichia coli .

[0014] Optionally, the amino acid sequence of the phosphoenolpyruvate carboxylase is registered at WP_034935286.1, WP_138362857.1, WP_036115866.1, WP_238714932.1, WP_056233714.1, WP_150436508.1, WP_131866617.1, WP_390253167.1, WP_167614716.1, WP_101858209.1, S Any one of UX71733.1, WP_226054296.1, GAB7195701.1, WP_411704556.1, WP_115460055.1, WP_328711892.1, MDN6633159.1, MDF7679377.1, WP_322836539.1, WP_099740117.1, WP_437614329.1, and WP_054623263.1, compared with the amino acid of SEQ ID NO.1, has the following percentages: 83%, 89.4%, 87%, 88%, 81%, 83%, 81%, 81%, 87%, 82%, 93%, 81%, 82%, 83%, 82%, 89%, 83%, 85%, 87%, 82%, 83%, and 97%, respectively.

[0015] The present invention also provides a polynucleotide encoding the phosphoenolpyruvate carboxylase mutant.

[0016] The present invention also provides a recombinant vector carrying the above-mentioned polynucleotides.

[0017] In one embodiment of the present invention, the vector is selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR / Cas9 vectors, or viral vectors.

[0018] The present invention also provides a recombinant cell carrying the above-mentioned recombinant vector or expressing the above-mentioned phosphoenolpyruvate carboxylase mutant.

[0019] In one embodiment of the present invention, the recombinant cells are bacteria or fungi as host cells.

[0020] In one embodiment of the present invention, the host cell is an L-lysine, L-threonine, or L-arginine producing strain, and the strain is an engineered Escherichia coli.

[0021] In one embodiment of the present invention, the host cell is Escherichia coli, Corynebacterium glutamicum, Lactobacillus brevis, Flavobacterium flavomarginata, or Corynebacterium pekinensis.

[0022] In one embodiment of the present invention, the host cell includes, but is not limited to, E. coli CCTCCNO:M2019435 E. coli NRRLB-12185, E. coli GDMCC NO.1.318 E. coli CCTCC NO: M2022562 E. coli CGMCC NO:25404 E. coli CCTCC NO: M2026397 E. coli CGMCC NO:25402.

[0023] In one embodiment of the invention, the cell carries the aforementioned polynucleotide or the aforementioned vector, or expresses the aforementioned phosphoenolpyruvate carboxylase mutant. The cell may refer to a single cell, a cell line, or a cell culture. As used herein, "cell" includes its progeny, which may not be identical to the primary cell due to natural, accidental, or intentional mutations, and may differ morphologically and / or in genomic DNA. The cell may be a natural cell or a transformant.

[0024] The present invention also provides a method for synthesizing polynucleotides using the above-mentioned phosphoenolpyruvate carboxylase mutant; wherein the synthesis method includes in vitro transcription synthesis, isothermal amplification, or transcription-mediated amplification.

[0025] In one embodiment of the present invention, the synthesis method includes catalytic synthesis of polynucleotides using an enzyme system containing the phosphoenolpyruvate carboxylase mutant or whole-cell catalytic synthesis of polynucleotides using the aforementioned cells.

[0026] The present invention also provides a recombinant enzyme catalyst containing the above-mentioned phosphoenolpyruvate carboxylase, which is any one of the following forms: (1) Culture recombinant expression transformants containing phosphoenolpyruvate carboxylase mutants and isolate transformant cells containing recombinant phosphoenolpyruvate carboxylase mutant enzymes; (2) Culture recombinant expression transformants containing phosphoenolpyruvate carboxylase mutant, isolate transformant cells containing recombinant phosphoenolpyruvate carboxylase mutant enzyme, and break the transformant cells containing the recombinant phosphoenolpyruvate carboxylase mutant enzyme to obtain cell lysate. (3) Cultivate recombinant expression transformants containing phosphoenolpyruvate carboxylase mutants, isolate transformant cells containing recombinant phosphoenolpyruvate carboxylase mutant enzymes, break the transformant cells containing the recombinant phosphoenolpyruvate carboxylase mutant enzymes, obtain cell lysate, and freeze-dry the cell lysate of the recombinant phosphoenolpyruvate carboxylase mutant enzymes to obtain lyophilized enzyme powder.

[0027] The present invention also provides a method for improving at least one performance of phosphoenolpyruvate carboxylase, wherein the performance is selected from enzyme activity, substrate affinity and / or catalytic efficiency, and reduced sensitivity to feedback inhibition. The method comprises that the phosphoenolpyruvate carboxylase mutant has one or more amino acid mutations at positions 457, 495, 619, 665, 814, and 835, based on the amino acid sequence corresponding to the parental phosphoenolpyruvate carboxylase, or has one or more amino acid mutations at amino acid residues at equivalent positions in the parental phosphoenolpyruvate carboxylase; the amino acid sequence of the parental phosphoenolpyruvate carboxylase has at least 85% identity with the amino acid shown in SEQ ID NO. 1 and has phosphoenolpyruvate carboxylase activity.

[0028] The performance characteristics are selected from ① reducing inhibition of end products or intermediate metabolites such as aspartic acid and malic acid, i.e., improving tolerance to end products or intermediate metabolites such as aspartic acid and malic acid; ② improving substrate utilization efficiency: for CO2 / HCO3 - The apparent affinity is improved, and the fixation ability is fully utilized under the CO2 partial pressure of conventional fermentation; ③ Improve the adaptability of the product system: the same mutant can perform well in multiple fermentation systems such as lysine, threonine, and arginine.

[0029] In one embodiment of the present invention, corresponding to the amino acid sequence shown in SEQ ID NO.1, the phosphoenolpyruvate carboxylase mutant at least simultaneously possesses any of the following mutations equivalent to or corresponding to the parental phosphoenolpyruvate carboxylase mutant: T457R, I495Q, F619V, C665F, N814D, Y835F, T457R / F619V, T457R / C665F, I495Q / F61 9V, I495Q / C665F, T457R / N814D, T457R / Y835F, I495Q / N814D, I495Q / Y835F, F619 V / N814D, F619V / Y835F, C665F / N814D, C665F / Y835F, T457R / F619V / N814D, I495Q / C665F / Y835F.

[0030] The present invention also provides a polynucleotide synthesis kit comprising the above-mentioned phosphoenolpyruvate carboxylase mutant.

[0031] The present invention also provides an engineered Escherichia coli strain expressing the above-mentioned phosphoenolpyruvate carboxylase mutant.

[0032] In one embodiment of the present invention, the engineered Escherichia coli is constructed by cloning the gene encoding the phosphoenolpyruvate carboxylase into an expression vector and transforming it into Escherichia coli host cells.

[0033] In one embodiment of the present invention, the expression vector is selected from pTrc99a.

[0034] In one embodiment of the present invention, the vector is selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR / Cas9 vectors, or viral vectors.

[0035] In one embodiment of the present invention, the host is an L-lysine, L-threonine, or L-arginine producing strain.

[0036] In one embodiment of the present invention, the Escherichia coli host is selected from... E. coli Lys-1 (CCTCCNO:M2019435), E. coli Lys-2 ( E. coli NRRLB-12185) E. coli Lys-3( E. coli GDMCC NO. 1.318) E. coli Thr-1 (CCTCC NO: M2022562) E. coliThr-2 (CGMCC NO:25404) E. coli Arg-1 (CCTCC NO: M2026397) E. coli Arg-2 (CGMCC NO:25402).

[0037] The E. coli Lys-1 is Escherichia coli FMME lys, accession number CCTCC NO:M2019435, is described in the text of Chinese invention patent application with publication number CN110964670A; The E. coli Lys-2 is Escherichia coli NRRLB-12185 was purchased from the Agricultural Research Service Culture Collection (NRRL). The E. coli Lys-3 is Escherichia coli GDMCC NO.1.318 was purchased from GDMCC.

[0038] The E. coli Thr-1 is E. coli FMME AA5, the Escherichia coli FMME-AA5 with accession number CCTCC NO: M2022562, is described in the text of Chinese invention patent with publication number CN114990013B; The E. coli Thr-2 is Escherichia coli. Escherichia coli YP0158, with accession number CGMCCNo.25404, is recorded in the text of Chinese invention patent with publication number CN116515878A; The E. coli Arg-1 is Escherichia coli. Escherichia coli MMEC-Arg1.0; The Escherichia coli MMEC-Arg1.0 has the accession number CCTCC NO: M 2026397 and is described in the text of Chinese invention patent with publication number CN122081251A; The E. coli Arg-2 is an Escherichia coli bacteria. Escherichia coli YP004-8, with accession number CGMCCNo.25402, is recorded in the text of Chinese invention patent with publication number CN119082065B.

[0039] The present invention also provides a method for increasing the production of L-amino acids in Escherichia coli, wherein the method comprises expressing the above-mentioned phosphoenolpyruvate carboxylase mutant in Escherichia coli.

[0040] In one embodiment of the present invention, the L-amino acid is selected from at least one of L-lysine, L-threonine and L-arginine.

[0041] In one embodiment of the present invention, the Escherichia coli host is selected from... Escherichia coli BL21(DE3), Escherichia coli BL21 Escherichia coli Rosetta (DE3) Escherichia coli C41(DE3) Escherichia coli C43 (DE3) Escherichia coli JM109 Escherichia coli DH5α or its derivative strains.

[0042] In one embodiment of the present invention, the Escherichia coli host includes, but is not limited to, those mentioned above. E. coli CCTCCNO:M2019435 E. coli NRRLB-12185, E. coli GDMCC NO.1.318 E. coli CCTCC NO: M2022562 E. coli CGMCC NO:25404 E. coli CCTCC NO: M2026397 E. coli CGMCC NO:25402.

[0043] The present invention also provides a method for preparing L-amino acids, comprising inoculating the above-mentioned recombinant cells or the above-mentioned engineered Escherichia coli into a culture medium for fermentation culture to obtain a fermentation system, and obtaining the target L-amino acid from the fermentation system.

[0044] In one embodiment of the present invention, the L-amino acid is selected from at least one of L-lysine, L-threonine and L-arginine.

[0045] In one embodiment of the present invention, when the L-amino acid is L-lysine, the engineered Escherichia coli is an L-lysine-producing strain, and the host cell includes, but is not limited to, L-lysine-producing strains. E. coli Lys-1 (CCTCCNO:M2019435), E. coli Lys-2 ( E. coli NRRLB-12185); E. coli Lys-3( E. coli GDMCC NO.1.318); the engineered Escherichia coli is... E. coliLys-1, E. coli Lys-2 or E. coli Lys-3 cells were used as chassis cells, and pTrc99a was used as the expression vector to express any of the following mutants: T457R, I495Q, F619V, C665F, N814D, Y835F, T457R / F619V, T457R / C665F, I495Q / F619V, I495Q / C665F, T457R / N814D, T457R / Y835F, I495Q / N814D, I495Q / Y835F, F619V / N814D, F619V / Y835F, C665F / N814D, C665F / Y835F, T457R / F619V / N814D, I495Q / C665F / Y835F; The method is as follows: Escherichia coli engineered bacterial seed culture is inoculated into the fermentation medium at an inoculum rate of 10-15% (v / v). The fermentation temperature is 35-37 °C, the initial stirring speed is 350-400 r / min, and sterile air is introduced. During the process, dissolved oxygen is maintained at 30%-40% by adjusting the stirring speed and aeration rate. The pH is maintained between 6.70 and 6.80 by adding ammonia water, the residual sugar concentration is controlled at 1-2 g / L by adding glucose, and the ammonium ion concentration is controlled at 0.1-0.4 g / L by adding ammonium sulfate. The carbon-nitrogen ratio during fermentation is controlled between 1:1 and 1:2 by adjusting the feeding strategy to increase L-lysine yield and reduce by-product accumulation. The fermentation time is 36 h.

[0046] In one embodiment of the present invention, when the L-amino acid is L-threonine, the engineered Escherichia coli is an L-threonine-producing strain, and the host cell includes, but is not limited to, L-threonine-producing strains. E. coli Thr-1 (CCTCC NO: M2022562), E. coli Thr-2 (CGMCC NO.25404); the engineered Escherichia coli is... E. coli Thr-1 or E. coliThr-2 cells were used as chassis cells, and pTrc99a was used as the expression vector to express any of the following mutants: T457R, I495Q, F619V, C665F, N814D, Y835F, T457R / F619V, T457R / C665F, I495Q / F619V, I495Q / C665F, T457R / N814D, T457R / Y835F, I495Q / N814D, I495Q / Y835F, F619V / N814D, F619V / Y835F, C665F / N814D, C665F / Y835F, T457R / F619V / N814D, I495Q / C665F / Y835F; The method is as follows: Escherichia coli engineered bacterial seed liquid is inoculated into fermentation medium at an inoculation rate of 10-15% (v / v), fermentation temperature is 35-37 ℃, initial rotation speed is 300-350 r / min, dissolved oxygen is maintained at 30%-50% by adjusting stirring speed and aeration rate, pH is maintained at 6.9-7.0 by adding ammonia water, and residual sugar concentration is controlled at 1-2 g / L by adjusting glucose flow rate. Fermentation ends after 38 h; the feed medium is preferably 800 g / L glucose.

[0047] In one embodiment of the present invention, when the L-amino acid is L-arginine, the engineered Escherichia coli is an L-arginine-producing strain, and the host cell includes, but is not limited to, L-arginine-producing strains. E. coli Arg-1 (CCTCC NO: M2026397) E. coli Arg-2 (CGMCC NO.25402); the engineered Escherichia coli is... E. coli Arg-1 or E. coli Arg-2 cells were used as the chassis cells, and pTrc99a was used as the expression vector to express any of the following mutants: T457R, I495Q, F619V, C665F, N814D, Y835F, T457R / F619V, T457R / C665F, I495Q / F619V, I495Q / C665F, T457R / N814D, T457R / Y835F, I495Q / N814D, I495Q / Y835F, F619V / N814D, F619V / Y835F, C665F / N814D, C665F / Y835F, T457R / F619V / N814D, I495Q / C665F / Y835F; The method is as follows: Escherichia coli engineered bacterial seed culture is inoculated into the fermentation medium at an inoculation rate of 16-18% (v / v). The fermentation temperature is 35-37 °C. The pH is maintained at 7.3-7.4 by adding NH4OH. Dissolved oxygen is maintained at approximately 30% by adjusting the stirring speed to 400-1000 r / min, the aeration rate to 2-3 L / min, and the tank pressure. The initial glucose concentration is controlled at 15 g / L. When the glucose concentration is below 1 g / L, an 800 g / L glucose solution is added to maintain it at 0.1-1 g / L. When NH4OH... + When the concentration is below 0.2 g / L, 500 g / L of ammonium sulfate is added, and glucose and ammonium sulfate are added in a fixed ratio of 4:1. The fermentation time is 48 h.

[0048] The present invention also provides the application of phosphoenolpyruvate carboxylase mutant, or the above-mentioned polynucleotide, or the above-mentioned recombinant expression vector, or the above-mentioned recombinant cell, or the above-mentioned recombinant enzyme catalyst, or the above-mentioned engineered Escherichia coli in the preparation of L-lysine, L-threonine or L-arginine.

[0049] The present invention also provides the use of phosphoenolpyruvate carboxylase mutant, or the above-mentioned polynucleotide, or the above-mentioned recombinant expression vector, or the above-mentioned recombinant cell, or the above-mentioned recombinant enzyme catalyst, or the above-mentioned engineered Escherichia coli in the preparation of products containing L-lysine, L-threonine or L-arginine.

[0050] The present invention also provides the application of phosphoenolpyruvate carboxylase mutant, or the above-mentioned polynucleotide, or the above-mentioned recombinant expression vector, or the above-mentioned recombinant cell, or the above-mentioned recombinant enzyme catalyst, or the above-mentioned engineered Escherichia coli in improving the fermentation yield of L-lysine, L-threonine or L-arginine by genetically engineered bacteria.

[0051] Beneficial effects Compared to the natural wild-type phosphoenolpyruvate carboxylase (PPC), the PPC mutant constructed in this invention overcomes the inherent performance defects of the wild-type enzyme in microbial metabolic regulation through specific amino acid site modification. It has significant technical advantages in terms of enzymatic characteristics, substrate adaptability, strain fermentation application, and metabolic engineering modification. At the same time, the construction strategy of this mutant has good universality and promotion value. The specific technical benefits are as follows: (1) Significantly reduces product feedback inhibition sensitivity and improves enzyme catalytic stability: Wild-type PPC is susceptible to feedback inhibition by downstream amino acid metabolites, and its catalytic activity continues to decline with the accumulation of fermentation products, severely restricting the efficient conduct of metabolic reactions. The PPC mutant obtained by this invention can effectively weaken the negative regulatory effect of downstream metabolites on enzyme activity, reduce the sensitivity of feedback inhibition response, and maintain stable catalytic activity in fermentation systems with high product accumulation, effectively breaking through the metabolic regulation bottleneck of wild-type enzymes and ensuring the continuous and efficient conduct of carboxylation reactions.

[0052] (2) Optimized substrate binding capacity and improved substrate utilization: Based on site-directed mutagenesis, the PPC mutant has an optimized protein spatial conformation, and the adaptability and specificity of the substrate binding pocket are significantly improved. This can effectively enhance the recognition and binding efficiency of the core substrate phosphoenolpyruvate, reduce substrate waste, and improve substrate conversion efficiency. Compared with wild-type PPC, the mutant can drive substrate-directed conversion more efficiently in complex intracellular metabolic environments, thus optimizing the efficiency of basal metabolic reactions.

[0053] (3) Significantly improves the fermentation production performance of L-amino acid engineered strains: Applying the PPC mutant of this invention to typical aspartic acid group L-amino acid producing strains such as L-lysine, L-threonine, and L-arginine can effectively reshape the central carbon metabolism pathway of the strains, enhance the supply of key precursor substances for oxaloacetate synthesis, and alleviate the metabolic flux bottleneck mediated by wild-type PPC. It can significantly improve the fermentation yield and product conversion rate of target L-amino acids, while optimizing the fermentation growth characteristics of the strains, shortening the fermentation cycle, reducing fermentation production costs, and greatly improving the economic benefits of industrial fermentation production.

[0054] (4) Possesses broad-spectrum metabolic engineering application value and universal construction characteristics: The PPC mutant of this invention can precisely regulate the carbon metabolic flux allocation of key metabolic nodes of oxaloacetate in microbial cells, and is an efficient molecular tool for optimizing the central carbon metabolic network and reshaping amino acid synthesis pathways. More importantly, the mutant modification strategy of this invention is not limited to a single strain or a single amino acid synthesis system, and has strong universality. It can be widely adapted to various industrial microbial hosts such as Escherichia coli and Corynebacterium. It can be specifically applied to the construction and modification of various L-amino acid engineered strains with oxaloacetate as a key precursor. It breaks through the technical limitations of the traditional wild-type PPC modification scheme with narrow application scope and single targeting, and provides a universal and efficient modification idea and technical carrier for the targeted breeding of various high-yield strains of aspartic acid family and derived L-amino acids. Detailed Implementation

[0055] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are one or more implementations of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0056] To facilitate understanding of this invention, some of the terms used in this invention are explained below. Unless otherwise expressly defined, the following terms have the following meanings in this application.

[0057] Some definitions or terms involved in this invention: Performance enhancement: The term "performance enhancement" refers to characteristics associated with a variant that is improved relative to the parent. Such performance enhancement includes, but is not limited to, one or more of the following: enzyme activity, substrate affinity, enzyme kinetic parameters, product tolerance, product yield, product productivity, reduced sensitivity to feedback inhibition, and improved substrate utilization.

[0058] Corresponding to: As used herein, the term "corresponding to" refers to the manner in which a specific amino acid in a sequence is identified (where a specific amino acid sequence is referenced). For example, for the purposes of this invention, when referring to a specific amino acid position, a person skilled in the art can compare another amino acid sequence with the referenced amino acid sequence to determine which specific amino acid might be of interest in the other amino acid sequence. Alternative alignment methods can be used, and such methods are well known to those skilled in the art.

[0059] Mutant: As used herein, when referring to variations of the invention, the terms "mutant," "peptide variant," "peptide," or "phosphoenolpyruvate carboxylase mutant" mean a polypeptide having phosphoenolpyruvate carboxylase activity and containing altered (i.e., substitution, insertion, and / or deletion) positions relative to the "parent" phosphoenolpyruvate carboxylase. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position. In describing variations of the invention, the nomenclature described below has been adapted for ease of reference. Accepted IUPAC single-letter or three-letter amino acid abbreviations are used. Substitution: For amino acid substitution, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, mutating threonine at position 457 of the wild-type enzyme to arginine is represented as "T457R." Multiple mutations are separated by a symbol (" / "), for example, "T457R / C665F" means that the threonine at position 457 of the wild-type enzyme is mutated to arginine, and the cysteine ​​at position 665 is mutated to phenylalanine.

[0060] Amino acid abbreviations: Alanine (Ala); Cysteine ​​(Cys); Aspartic acid (D Asp); Glutamic acid (Glu); Phenylalanine (Phe); Glycine (Gly); Histidine (His); Isoleucine (Ile); Lysine (Lys); Leucine (L) Methionine (M), Met; Asparagine (N), Asn; Proline (P), Pro; Glutamine (Q), Gln; Arginine (R), Arg; Serine (S), Ser; Threonine (T), Thr; Valine (V), Val; Tryptophan (W), Trp; Tyrosine (Y), Tyr.

[0061] Parental or parental phosphoenolpyruvate carboxylase: As used herein, the term "parental" phosphoenolpyruvate carboxylase refers to a phosphoenolpyruvate carboxylase modified to produce a mutant of the phosphoenolpyruvate carboxylase of the present invention. The term also refers to a polypeptide to which the mutant of the present invention is compared. The parent can be a naturally occurring (wild-type) polypeptide, or it can be, or even a variant thereof, prepared by any suitable means. For example, the parental protein can be a variant of a naturally occurring polypeptide whose amino acid sequence has been modified or altered. Thus, the parental phosphoenolpyruvate carboxylase may have one or more (or one or more) amino acid substitutions, deletions, and / or insertions. Thus, the parental phosphoenolpyruvate carboxylase can be a variant of the parental phosphoenolpyruvate carboxylase. The parent can also be an allelic variant, which is a polypeptide encoded by any of two or more alternative forms of a gene occupying the same chromosomal locus.

[0062] Wild-type enzyme: When referring to an amino acid or nucleic acid sequence, the term "wild-type" means that the amino acid or nucleic acid sequence is a naturally occurring or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance found in nature (e.g., a protein, amino acid, or nucleic acid sequence). Conversely, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant nucleic acid and protein sequences produced in a laboratory, or modifications of wild-type sequences). When the parent enzyme is not a variant enzyme, the terms "wild-type enzyme" and "parent enzyme" are used interchangeably.

[0063] Sequence identity: The degree of association between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity".

[0064] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48:443-453) is used to determine sequence identity between two amino acid sequences. This algorithm is implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Trends in Genetics] 16:276-277) (preferably version 5.0.0 or later). The parameters used can be a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of "longest identity" marked by Needle (obtained using the -nobrief option) is used as the identity percentage and calculated as follows: (identical residues x 100) / (alignment length - total number of vacancies in the alignment).

[0065] Alternatively, the parameters used can be a vacancy open penalty of 10, a vacancy extension penalty of 0.5, and EDNAFULL (the EMBOSS version of NCBI NUC4.4) to replace the matrix. The output of "Longest Identity" marked with Needle (obtained using the -nobrief option) is used as the identity percentage and calculated as follows: (identical deoxyribonucleotides × 100) / (alignment length – total number of gaps in the alignment).

[0066] Expression: As used in this article, “expression” refers to any step involving variant generation, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0067] Expression vector: As used herein, the term “expression vector” refers to a linear or circular DNA molecule that contains a polynucleotide encoding a variant and is operatively linked to a control sequence that provides for its expression.

[0068] Host cell: The term "host cell" means any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "host cell" encompasses any offspring of a parent cell that differs from the parent cell due to mutations occurring during replication, along with recombinant host cells, isolated host cells (e.g., isolated recombinant host cells), and heterologous host cells.

[0069] The host cell can be any cell useful in the recombinant production of the phosphoenolpyruvate carboxylase mutant, such as prokaryotic or eukaryotic cells.

[0070] Recombination: When used to refer to cells, nucleic acids, proteins, or vectors, the term "recombination" means that the cell has been modified from its natural state. Thus, for example, recombinant cells express genes not found in the natural (non-recombinant) form of the cell, or express natural genes at different levels or under different conditions compared to those found in nature. The difference between recombinant nucleic acids and their natural sequences lies in the operative linking of one or more nucleotides and / or a foreign sequence (e.g., a foreign promoter in an expression vector). The difference between recombinant proteins and their natural sequences may lie in the fusion of one or more amino acids and / or a foreign sequence. A vector containing nucleic acids encoding a polypeptide is a recombinant vector. The term "recombination" is synonymous with "genetically modified" and "transgenic."

[0071] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0072] It should be noted that the abbreviations of amino acid residues in this article follow the general definition in this field, but in some instances, the full name is still indicated in parentheses after the abbreviation. In this article, "amino acid residue" and "amino acid" can be used interchangeably. The amino acid sequence in this article starts from the N-terminus.

[0073] In some optional embodiments, the present invention provides a phosphoenolpyruvate carboxylase mutant, wherein the phosphoenolpyruvate carboxylase mutant has one or more amino acid mutations at positions 457, 495, 619, 665, 814, and 835, based on the amino acid sequence corresponding to the parental phosphoenolpyruvate carboxylase, or has one or more amino acid mutations at amino acid residues at equivalent positions in the parental phosphoenolpyruvate carboxylase; the amino acid sequence of the parental phosphoenolpyruvate carboxylase has at least 85% identity with the amino acid shown in SEQ ID NO.1 and has phosphoenolpyruvate carboxylase activity.

[0074] In some optional embodiments, the mutant is, corresponding to the amino acid sequence shown in SEQ ID NO.1, the phosphoenolpyruvate carboxylase mutant having at least simultaneously any of the following mutations equivalent to or corresponding to the parental phosphoenolpyruvate carboxylase mutant: T457R, I495Q, F619V, C665F, N814D, Y835F, T457R / F619V, T457R / C665F, I495Q / F619V, I495Q / C665F, T457R / N814D, T457R / Y835F, I495Q / N814D, I495Q / Y835F, F619V / N814D, F619V / Y835F, C665F / N814D, C665F / Y835F. T457R / F619V / N814D, I495Q / C665F / Y835F.

[0075] In some alternative embodiments, the present invention also provides a polynucleotide encoding the above-described phosphoenolpyruvate carboxylase mutant.

[0076] In this document, "polynucleotide" refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases. Polynucleotides encode the aforementioned phosphoenolpyruvate carboxylase mutant, optionally encoding either the sense or antisense strand. Polynucleotides can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

[0077] In some alternative embodiments, the present invention also provides a vector carrying a polynucleotide encoding the aforementioned phosphoenolpyruvate carboxylase mutant. The vector is known to those skilled in the art and includes, but is not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses. Optionally, the vector of the present invention contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0078] In some alternative embodiments, the present invention also provides a cell carrying the aforementioned polynucleotide or the aforementioned vector, or expressing the aforementioned phosphoenolpyruvate carboxylase mutant. The cell may refer to a single cell, a cell line, or a cell culture. As used herein, "cell" includes its progeny, which may not be identical to the primary cell due to natural, accidental, or intentional mutations, and may differ morphologically and / or in genomic DNA from the primary cell. The cell may be a natural cell or a transformant.

[0079] In some alternative embodiments, the present invention also provides a method for synthesizing a polynucleotide using the phosphoenolpyruvate carboxylase mutant of the present invention. This method of polynucleotide synthesis includes a synthesis reaction catalyzed by an enzyme system comprising the aforementioned phosphoenolpyruvate carboxylase mutant. The synthesis method includes in vitro transcription (IVT), isothermal amplification, such as sequence-specific nucleic acid-based amplification (NASBA), or transcription-mediated amplification (TMA). In some optional embodiments, the present invention also provides a kit for polynucleotide synthesis comprising the aforementioned phosphoenolpyruvate carboxylase mutant. It is understood that the kit for polynucleotide synthesis provided by the present invention may also comprise other detection reagents or consumables acceptable in the art, and those skilled in the art can select appropriate reagents and / or consumables based on information well known in the art and described in various general and more specific textbooks, references, process manuals, product instructions, standard documents, and equipment manuals. Specific optional reagents and consumables in the kit include, but are not limited to, one or more of the following: buffer solutions, salts, metal ions, dNTPs, enzymes, primers, probes, fluorescent dyes, luminescent substrates, controls, quality control products, and calibrators.

[0080] The term "wild-type PPC" in this article refers to naturally occurring phosphoenolpyruvate carboxylase that has not been artificially modified.

[0081] In some embodiments of the present invention, the wild-type PPC is preferably a phosphoenolpyruvate carboxylase derived from *Escherichia coli*, and its amino acid sequence is preferably the sequence shown in SEQ ID NO. 1. Unless otherwise specified, the mutation site numbers involved in this application are based on the amino acid sequence shown in SEQ ID NO. 1.

[0082] The term "PPC mutant" in this article refers to a protein that, relative to wild-type PPC, has one or more amino acid residues replaced at the corresponding site, but still retains phosphoenolpyruvate carboxylase activity.

[0083] The term "the Xth amino acid corresponding to the amino acid sequence shown in SEQ ID NO.1" in this document refers to the amino acid residue that corresponds to the corresponding position in SEQ ID NO.1 after sequence alignment. It should be understood that, without affecting protein function, if the target protein introduces a tag sequence, a linker peptide sequence, or has a small amount of additional sequences that do not affect the correspondence, the amino acid residue that corresponds to the corresponding position in SEQ ID NO.1 after alignment still falls within the scope of the "corresponding site" described in this application.

[0084] The term "reduced feedback inhibition sensitivity" in this document refers to a PPC mutant exhibiting a higher relative enzyme activity than the corresponding wild-type PPC under the same inhibitor type and concentration. In some embodiments, the inhibitor is preferably L-aspartic acid. In this application, any mutant exhibiting higher residual enzyme activity, lower inhibition level, or better inhibition tolerance relative to the wild type under feedback inhibition conditions is considered to possess the characteristic of "reduced feedback inhibition sensitivity."

[0085] The term "improved substrate utilization" in this document refers to the improved utilization of the substrate phosphoenolpyruvate by PPC mutants compared to wild-type PPC. This improvement may manifest as an increase in substrate affinity, a decrease in apparent Km, an increase in catalytic efficiency, an increase in Vmax, an increase in reaction rate per unit amount of enzyme, or at least one other enzymatic indicator that characterizes enhanced substrate utilization. Unless otherwise specified, "improved substrate utilization" as used in this application is not limited to a change in a single enzymatic parameter.

[0086] The term "recombinant microorganism" in this document refers to microorganisms obtained by artificially introducing exogenous polynucleotides, recombinant expression vectors, or related genetic elements. In this invention, the recombinant microorganism is preferably a microorganism containing a polynucleotide encoding the PPC mutant of this invention or a recombinant expression vector containing such polynucleotide. Preferably, the recombinant microorganism is *Escherichia coli*. In some embodiments, the recombinant microorganism may further contain other metabolic engineering modifications that facilitate L-amino acid synthesis.

[0087] The term "fermentation production of L-amino acids" in this document refers to the process of accumulating target L-amino acids in a fermentation system by culturing recombinant microorganisms containing the PPC mutant under suitable culture conditions. The target L-amino acids are preferably selected from one or more of L-lysine, L-threonine, and L-arginine. "Fermentation production" includes product accumulation under shake flask culture conditions, as well as product accumulation under batch culture, fed-batch culture, or continuous culture conditions in fermenters.

[0088] It should be understood that the above-described terms are for illustrative purposes only and are not intended to unnecessarily limit the scope of protection of this invention. Without departing from the essence of this invention, those skilled in the art can provide reasonable interpretations of the relevant terms within the scope of their ordinary understanding.

[0089] The present invention will be further described below with reference to embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. All equivalent substitutions, simple modifications or improvements made based on the disclosure of the present invention should fall within the scope of protection of the present invention.

[0090] Unless otherwise specified, the raw materials, reagents, culture media and instruments used in this invention can be conventional products in the field; unless otherwise specified, the molecular biological operations, strain cultivation, fermentation and detection methods used in this invention are conventional methods in the field.

[0091] In this invention, the wild-type phosphoenolpyruvate carboxylase (PPC) is preferably derived from *Escherichia coli*, and its amino acid sequence is preferably the sequence shown in SEQ ID NO. 1. The PPC mutant is obtained by introducing one or more mutations from T457R, I495Q, F619V, C665F, N814D, and Y835F based on the amino acid sequence corresponding to SEQ ID NO. 1. Preferably, the PPC mutant simultaneously contains the T457R, F619V, and N814D mutations.

[0092] The culture media involved in the following examples: LB liquid medium for culturing Escherichia coli: 10 g / L NaCl, 5 g / L yeast extract, 10 g / L peptone.

[0093] LB solid medium for culturing Escherichia coli: 10 g / L NaCl, 5 g / L yeast extract, 10 g / L peptone, 20 g / L agar powder.

[0094] Escherichia coli L-lysine seed culture medium: secondary seed culture medium (g·L) -1 ): Corn steep liquor powder 7.62, hair hydrolysate 5, glucose 10, betaine 5, biotin 2, (NH4)2SO4 14.4, KH2PO4 6, MgSO4·7H2O 3.2, FeSO4 0.02, MnSO4 0.02, ZnSO4 0.08, CuSO4 0.04.

[0095] Escherichia coli L-lysine fermentation medium: 20 g / L glucose, 30 g / L beet molasses, 10 g / L corn steep liquor powder, 3 g / L betaine, 1.5 g / L MgSO4, 20 mg / L FeSO4, 450 mg / L threonine, 400 mg / L methionine, 0.6 mg / L biotin, pH adjusted to 7.2 with ammonia.

[0096] Escherichia coli L-threonine seed culture medium: 5 g / L yeast extract, 10 g / L peptone, 3 g / L NaCl, 1 g / L KH2PO4, 0.72 g / L MgSO4·7H2O, 10 g / L glucose, and NaOH solid adjusted to pH 7.0.

[0097] Escherichia coli L-threonine fermentation medium: 30 g / L glucose, 5 g / L cane molasses, 8 g / L corn steep liquor, 1 mg / L MnSO4·H2O, 1.2 mg / L FeSO4·7H2O, 0.8 g / L MgSO4·7H2O, 0.5 g / L KH2PO4, 0.8 mg / L biotin, 0.4 mg / L vitamin B1, 3 mL / L antifoaming agent, and ammonia water to adjust the pH to 7.00.

[0098] Escherichia coli L-arginine seed culture medium: 25 g / L glucose, 5 g / L yeast extract, 4 g / L peptone, 1 g / L (NH4)2SO4, 3 g / L K2HPO4, 3.0 g / L MgSO4·7H2O, 0.02 g / L FeSO4·7H2O, 0.10 g / L MnSO4·7H2O, 2 mg / L vitamin B1, 2 mg / L vitamin B3, 4 mg / L vitamin B5, 4 mg / L vitamin B7, pH adjusted to 7.0-7.2 with NaOH.

[0099] Escherichia coli L-arginine fermentation medium: 15 g / L glucose, 3 g / L yeast extract, 4 g / L succinate, 10 g / L (NH4)2SO4, 1.5 g / L beet molasses, 0.6 g / L betaine, 3 g / L K2HPO4, 2 g / L MgSO4·7H2O, 0.02 g / L FeSO4·7H2O, 0.02 g / L MnSO4·7H2O, 2 mg / L vitamin B1, 2 mg / L vitamin B3, 2 mg / L vitamin B5, 2 mg / L vitamin B7.

[0100] NDG liquid medium: 1.5 g / L glucose, 5 g / L yeast extract, 7.5 g / L beef extract powder, 10 g / L peptone, 2.5 g / L NaCl.

[0101] NDG solid medium: 1.5 g / L glucose, 5 g / L yeast extract, 7.5 g / L beef extract, 10 g / L peptone, 2.5 g / L NaCl, 18 g / L agar powder.

[0102] The amino acid content detection methods involved in the following examples are as follows: Determination of threonine products: Fermentation broth pretreatment: After centrifuging the fermentation broth at 12000 rpm for 10 min, take the supernatant and filter it through a 0.22 μm filter membrane.

[0103] High-performance liquid chromatography (HPLC) was used to determine the L-threonine content in the fermentation broth: Mobile phase A was a 0.01 mol / L KH₂PO₄ solution, adjusted to pH 5.3 with KOH; Mobile phase B was a mixture of acetonitrile, methanol, and mobile phase A in a volume ratio of 5:3:1, adjusted to pH 5.3 with acetic acid. An Aglient ZORBAX SB-Aq column (250 × 4.6 mm, 5 µm) was used. Pre-column online derivatization was employed with OPA as the derivatizing agent. Gradient elution was performed at a column temperature of 35 ℃ and a flow rate of 1.0 mL / min. The detection wavelength was UV 254 nm; FLD: excitation wavelength 330 nm / emission wavelength 465 nm.

[0104] Determination of lysine products: Fermentation broth pretreatment: Take 1 mL of fermentation broth, centrifuge at 12,000 r / min for 2 min, and dilute the supernatant to a suitable concentration. Derivatize L-lysine using diethyl ethoxymethylene malonate (DEEMM). The derivatization system consisted of 4.5 μL DEEMM, 70.5 μL distilled water, 150 μL methanol, and 450 μL borate buffer. The derivatized mixture was filtered through a 0.22 μm filter.

[0105] High performance liquid chromatography (HPLC) was used to determine the L-lysine content in the fermentation broth: An Agilent-C18 column (250 mm × 4.6 mm, 5 μm) was used at a column temperature of 35 ℃ and a flow rate of 1 mL / min. The mobile phase consisted of 100% acetonitrile (A) and 25 mmol / L sodium acetate (B) at pH 4.80.

[0106] Determination of arginine products: After centrifuging the fermentation broth at 12000 rpm for 10 min, the supernatant was collected and filtered through a 0.22 μm filter membrane. The L-arginine content in the fermentation broth was determined by high-performance liquid chromatography (HPLC): 2,4-dinitrofluorobenzene (DNFB) was pre-derivatized using a ZORBAX Eclipse AAA analytical column (4.6 × 150 mm, 5 μm, Agilent Technologies).

[0107] Methods for calculating yield: Yield = Mass of L-amino acids in fermentation broth at the end of fermentation / Mass of glucose consumed during fermentation, i.e.: Y K / G =M K / M G Among them, Y K / G M represents the yield of L-amino acids. K M represents the mass of L-amino acids in the fermentation broth at the end of fermentation. GThis indicates the mass of glucose consumed during fermentation.

[0108] Example 1: Rational Design and Vector Construction of PPC Mutants This embodiment provides a method for the rational design and vector construction of PPC mutants, the steps of which are as follows: (1) Design of mutation sites: Wild-type Escherichia coli ppc Using genes as templates, and combining PPC protein sequence analysis, structural region analysis, and functional site optimization requirements, 456, 457, 495, 617, 619, 665, 810, 814, 835, and 839 were selected as modification sites. Among them, 456, 457, and 495 were used to regulate the enzyme's response to feedback inhibition; 617, 619, and 665 were used to improve substrate binding or substrate channel-related performance; and 810, 814, 835, and 839 were used to improve protein conformational stability or overall catalytic performance.

[0109] (2) Obtaining the mutant gene and constructing the recombinant plasmid: Contains wild type ppc Genetic E. coli Using MG1655 as a template, site-directed mutagenesis primers were designed targeting the mutation site (as shown in Table 1). PCR amplification was used to introduce the corresponding base substitutions to obtain samples containing the stated mutation site. ppc Gene fragments. After the PCR reaction, the amplification products were purified, and homologous recombination was used to separate the mutated gene fragments. ppc The gene was inserted into the expression vector pTrc99a; the double mutation was achieved by using an expression vector containing a single mutation as a template, and employing the primers shown in Table 1, using PCR amplification to introduce the corresponding base substitutions, thereby obtaining a gene containing the aforementioned mutation site. ppc Gene fragments. After the PCR reaction, the amplification products were purified, and homologous recombination was used to separate the mutated gene fragments. ppc It was prepared by inserting the gene into an expression vector containing a single mutation.

[0110] wild type ppc The gene is as follows (SEQ ID NO.2): At the same time, constructing a system containing wild-type ppc The control expression vector for the gene, as well as the recombinant expression vectors containing single-point mutants and double-point mutants respectively, are provided for subsequent comparisons.

[0111] Table 1: Primer Design

[0112] Example 2: Enzymatic characterization of PPC mutants This embodiment provides a method for preparing a PPC mutant and characterizing its corresponding enzymatic properties, with the following steps: (1) Protein expression and preparation: Recombinant vectors encoding wild-type PPC and its mutants (T457R, I495Q, F619V, C665F, N814D, Y835F, or combinations thereof) were obtained according to the method in Example 1 (the specific implementation method is the same as in Example 1, except that the vector was adjusted to the expression vector pET-28a(+)). When ligating to the expression vector, a 6×His tag with the amino acid sequence HHHHHH was introduced at the N-terminus of the target protein (wild-type PPC and its mutants), followed by a flexible linker peptide (GGGGS) to reduce the impact on protein structure and function; recombinant vectors were prepared respectively: pET-28a(+)-D456A, pET-28a(+)-T457R, pET-28a(+)-I495Q, pET-28a(+)-I 617P, pET-28a(+)-F619V, pET-28a(+)-C665F, pET-28a(+)-L810A, pET-28a (+)-N814D, pET-28a(+)-Y835F, pET-28a(+)-L839D, pET-28a(+)-T457R / F6 19V, pET-28a(+)-T457R / C665F, pET-28a(+)-I495Q / F619V, pET-28a(+)-I4 95Q / C665F, pET-28a(+)-T457R / N814D, pET-28a(+)-T457R / Y835F, pET-28a (+)-I495Q / N814D, pET-28a(+)-I495Q / Y835F, pET-28a(+)-F619V / N814D, p ET-28a(+)-F619V / Y835F, pET-28a(+)-C665F / N814D, pET-28a(+)-C665F / Y 835F, pET-28a(+)-T457R / F619V / N814D, pET-28a(+)-I495Q / C665F / Y835F; The constructed recombinant vectors were respectively converted to E. coli Single colonies of BL21(DE3) competent cells were picked and inoculated into LB liquid medium containing ampicillin, and cultured at 37°C and 200 rpm until the bacterial culture reached OD. 600 The concentration was increased to 0.6-0.8. Isopropyl-β-D-thiogalactoside (IPTG) was added to the culture system to a final concentration of 0.1-0.5 mM. The culture temperature was then lowered to 16℃, and expression was induced for another 12-16 h. After induction, the culture medium was centrifuged at 8000 rpm for 10 min at 4℃ to collect the cells. The cells were resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 5 mM MgCl2), and lysed using sonication. The cells were then centrifuged at 12000 rpm for 30 min at 4℃, and the supernatant was collected as crude enzyme solution. The following solutions were prepared: Crude enzyme solutions of wild-type phosphoenolpyruvate carboxylase (WT), and mutants: D456A, T457R, I495Q, I617P, F619V, C665F, L810A, N814D, Y835F, L839D, T457R / F619V, T457R / C665F, I495Q / F619V, and I495Q / C6 Crude enzyme solution of 65F, crude enzyme solution of T457R / N814D, crude enzyme solution of T457R / Y835F, crude enzyme solution of I495Q / N814D, crude enzyme solution of I495Q / Y835F, crude enzyme solution of F619V / N814D, crude enzyme solution of F619V / Y835F, crude enzyme solution of C665F / N814D, crude enzyme solution of C665F / Y835F, crude enzyme solution of T457R / F619V / N814D, crude enzyme solution of I495Q / C665F / Y835F.

[0113] (2) Enzyme activity assay: The activity of phosphoenolpyruvate carboxylase (PPC) was determined by a coupled enzyme method. The reaction system (1 mL) included: 50 mM Tris-HCl buffer (pH 8.0), 5 mM MgCl2, 10 mM NaHCO3, 2 mM phosphoenolpyruvate (PEP), 0.2 mM NADH, 2 U malate dehydrogenase, and appropriate amount of deionized water to the final volume. After pre-equilibration at 30℃, 50 μL of crude enzyme solution was added. The rate of decrease of NADH absorbance (ΔA) was continuously monitored at 340 nm using a UV spectrophotometer. 340 / min).

[0114] Enzyme activity is defined as the amount of enzyme required to catalyze the consumption of 1 μmol NADH per minute under the above conditions, which is one enzyme activity unit (U).

[0115] The enzyme activity results are shown in Table 2 below. The mutants at positions 456, 617, 810, and 839 showed decreased enzyme activity, while the other mutants all achieved increased phosphoenolpyruvate carboxylase (PPC) activity. Table 2: Enzyme activity assays of mutant and wild-type enzymes

[0116] (3) Determination of enzyme kinetic parameters The kinetic parameters of phosphoenolpyruvate carboxylase (PPC) were detected, and the results are shown in Table 3. Table 3: Comparison of enzyme kinetic parameters between wild-type PPC and different PPC mutants

[0117] The results showed that, compared with wild-type phosphoenolpyruvate carboxylase (PPC), mutants at positions 456, 617, 810, and 839 exhibited decreased substrate utilization performance, while other mutants showed improved substrate utilization performance. The preferred combination mutants showed better substrate affinity and / or catalytic efficiency.

[0118] (4) Sensitivity test for feedback inhibition: To evaluate the response characteristics of the performance-enhancing PPC mutant to feedback inhibition, different concentrations of the feedback-inhibiting metabolite L-aspartate (0 mM, 0.2 mM, 0.4 mM) were added to the reaction system for enzyme activity assay, and the relative enzyme activity under different L-aspartate concentrations was measured. Relative enzyme activity = A Asp / A0×100%, where A0 represents the initial enzyme activity without inhibitor, A Asp The values ​​indicate the enzyme activity after the addition of L-aspartic acid. The results are shown in Table 4. Table 4: Comparison of relative enzyme activities of wild-type PPC and mutants at different aspartate concentrations

[0119] The results showed that the enzyme activity of wild-type PPC decreased significantly in the presence of inhibitors, while the mutant described in this invention could still maintain high enzyme activity under the same conditions, indicating that its sensitivity to feedback inhibition was reduced.

[0120] The combined mutant containing the three mutations T457R, F619V and N814D exhibits higher residual enzyme activity under the presence of feedback inhibition.

[0121] Example 3: Construction of an amino acid-producing engineered strain This embodiment provides a method for constructing and fermenting an amino acid-producing engineered strain, the steps of which are as follows: (1) Host bacterial preparation: Preserved in the laboratory E. coli Lys-1 (CCTCCNO:M2019435), E. coli Lys-2 ( E. coli NRRLB-12185); E. coli Lys-3( E. coli GDMCC NO.1.318) was used as a lysine host strain in laboratory-preserved strains. E. coli Thr-1 (CCTCC NO: M2022562), E. coli Thr-2 (CGMCC NO.25404), as a threonine host strain, was preserved in the laboratory. E. coli Arg-1 (CCTCC NO: M2026397) E. coli Arg-2 (CGMCC NO.25402) is the arginine host strain.

[0122] (2) Construction of recombinant strains: The preferred performance-enhanced expression vectors constructed in Example 1 (pTrc99a-PPC, pTrc99a-T457R, pTrc99a-I495Q, pTrc99a-F619V, pTrc99a-C665F, pTrc99a-N814D, pTrc99a-Y835F, pTrc99a-T457R / F619V, pTrc99a-T457R / C665F, pTrc99a-I495Q / F619V, pTrc99a-I495Q / C665F, pTrc99a-T457R / N814D, pTrc99a-T457R / Y835F, pTrc99a-I495Q ...T457R / Y835F, pTrc99a-I495Q / N814D, pTrc99a-T457R / Y835F, pTrc99a-T The strains pTrc99a-I495Q / Y835F, pTrc99a-F619V / N814D, pTrc99a-F619V / Y835F, pTrc99a-C665F / N814D, pTrc99a-C665F / Y835F, pTrc99a-T457R / F619V / N814D, and pTrc99a-I495Q / C665F / Y835F were transformed into different host bacteria expressing different amounts of L-lysine, L-threonine, and L-arginine in step (1) to obtain recombinant engineered strains expressing wild-type PPC and its mutants (genotypes are shown in Table 5). After ampicillin resistance screening and colony PCR verification, the final engineered strains were obtained. Among them, the engineered strain expressing the three-point combination mutant T457R / A619V / N814D was preferred as the target strain of this invention.

[0123] Table 5: Recombinant engineered strains with PPC introduced

[0124] Example 4: Application of PPC mutant in L-lysine-producing strains The 27 L-lysine-producing strains containing the wild-type and mutant expression vectors of the PPC of the present invention, prepared in Example 3, were: Lys-1-WT, Lys-1-PPC(T457R), Lys-1-PPC(F619V), Lys-1-PPC(N814D), Lys-1-PPC(T457R / F619V), Lys-1-PPC(F619V / N814D), Lys-1-PPC(T457R / N814D), Lys-1-PPC(T457R / F619V / N814D), Lys-1-PPC(I495Q / C665F / Y835F), Lys-2-WT, Lys-2-PPC(I495Q), Lys-2-PPC(C665F), Lys-2-P PC(Y835F), Lys-2-PPC(I495Q / C665F), Lys-2-PPC(C665F / Y835F), Lys-2-PPC(I495 Q / Y835F), Lys-2-PPC (T457R / F619V / N814D), Lys-2-PPC (I495Q / C665F / Y835F), Lys Fermentation verification was performed using Lys-3-WT, Lys-3-PPC(T457R / Y835F), Lys-3-PPC(I495Q / N814D), Lys-3-PPC(C665F / N814D), Lys-3-PPC(T457R / F619V / N814D), and Lys-3-PPC(I495Q / C665F / Y835F).

[0125] (1) Seed culture: Take 200 μL of glycerol culture and place it in an Erlenmeyer flask containing 50 mL of LB medium. After culturing at 37 ℃ and 200 r / min for 8 h, take 1 mL of the culture and inoculate it into 500 mL of seed culture medium containing 50 mL of medium. Culturing at 37 ℃ and 200 r / min for 5 h will yield the seed culture.

[0126] (2) 5 L fermenter fermentation: The initial fermentation volume (fermentation medium) was 2 L. The seed culture obtained in step (1) was inoculated into the fermentation medium at an inoculation rate of 15% (v / v). The fermentation temperature was 37 ℃, the initial rotation speed was 400 r / min, and sterile air was introduced. During the process, dissolved oxygen was maintained at 30%-40% by adjusting the stirring speed and aeration rate. The pH was maintained between 6.70 and 6.80 by adding ammonia water. The residual sugar concentration was controlled at 1-2 g / L by adding glucose water, and the ammonium ion concentration was controlled at 0.1-0.4 g / L by adding ammonium sulfate water. The carbon-nitrogen ratio during the fermentation process was controlled between 1:1 and 1:2 by adjusting the feeding strategy to increase the yield of L-lysine and reduce the accumulation of by-products. The fermentation time was 36 h.

[0127] The results are shown in Table 6: Table 6: Comparison of 5 L fermentation performance of PPC mutants in L-lysine-producing strains

[0128] The results show that: The three mutations are most effective: Compared with the control strain Lys-1-WT, the strains containing PPC mutants Lys-1-PPC (T457R / F619V / N814D) and Lys-1-PPC (I495Q / C665F / Y835F) showed superior production performance in L-lysine fermentation, with yield, productivity, and production intensity increased by 26.5%, 7.1%, and 27.0%, and 23.4%, 2.4%, and 23.6%, respectively.

[0129] Compared with the control strain Lys-2-WT, the strains Lys-2-PPC (T457R / F619V / N814D) and Lys-2-PPC (I495Q / C665F / Y835F) containing the PPC triple mutant showed superior production performance in L-lysine fermentation, with yield, productivity, and production intensity increased by 24.7%, 6.3%, and 25.2%, and by 22.3%, 2.1%, and 22.9%, respectively.

[0130] Compared with the control strain Lys-3-WT, the strains containing the PPC mutant Lys-3-PPC (T457R / F619V / N814D) and Lys-3-PPC (I495Q / C665F / Y835F) showed superior production performance in L-lysine fermentation, with yield, productivity, and production intensity increased by 11.1%, 10.2%, and 10.7%, and by 9.3%, 6.1%, and 9.05%, respectively.

[0131] Example 5: Application of PPC mutant in L-threonine producing strains The 18 L-threonine engineered strains (Thr-1-WT, Thr-1-PPC(T457R), Thr-1-PPC(F619V), Thr-1-PPC(N814D), Thr-1-PPC(T457R / F619V), Thr-1-PPC(F619V / N814D), Thr-1-PPC(T457R / N814D), Thr-1-PPC(T457R / F619V / N814D), Thr-1-PPC(I)) containing the wild-type and mutant expression vectors of the present invention prepared in Example 3 were used. Fermentation verification was performed using 495Q / C665F / Y835F, Thr-2-WT, Thr-2-PPC(I495Q), Thr-2-PPC(C665F), Thr-2-PPC(Y835F), Thr-2-PPC(T457R / Y835F), Thr-2-PPC(I495Q / N814D), Thr-2-PPC(C665F / N814D), Thr-2-PPC(T457R / F619V / N814D), and Thr-2-PPC(I495Q / C665F / Y835F).

[0132] (1) Seed culture: Using an inoculation loop, scoop the bacterial culture from the glycerol storage tube and streak it onto an LB solid slant. Incubate at 37°C for 12–14 h, then rinse the slant with 5 mL of sterile water and dilute to OD. 600 The concentration was 3.0. 1 mL was inoculated into 500 mL of seed culture medium with a volume of 50 mL, and cultured at 37 °C and 200 r / min for 5 h to prepare the seed solution.

[0133] (2) Fermentation in a 5 L fermenter: A 5 L fully automatic stirred fermenter was used with an initial liquid volume of 1.5 L. The seed liquid obtained in step (1) was inoculated into the fermentation medium at an inoculation rate of 15% (v / v). The fermentation temperature was 37 ℃ and the initial rotation speed was 300 r / min. During the process, dissolved oxygen was maintained at 30%-50% by adjusting the stirring speed and aeration rate. The pH was maintained at around 7.0 by adding ammonia water. The residual sugar concentration was controlled at 1-2 g / L by adjusting the glucose flow rate. Fermentation ended after 38 h. The preferred feed medium was 800 g / L glucose.

[0134] The results are shown in Table 7: Table 7: Comparison of 5 L fermentation performance of PPC mutants in L-threonine-producing strains

[0135] The results show that: Compared with the control strain Thr-1-WT, the strains Thr-1-PPC (T457R / F619V / N814D) and Thr-1-PPC (I495Q / C665F / Y835F) containing PPC mutants showed superior production performance in L-threonine fermentation, with yield, productivity, and production intensity increased by 9.8%, 7.3%, and 10.0%, and by 8.1%, 4.9%, and 8.1%, respectively.

[0136] Compared with the control strain Thr-2-WT, the strains Thr-2-PPC (T457R / F619V / N814D) and Thr-2-PPC (I495Q / C665F / Y835F) containing PPC mutants showed superior production performance in L-threonine fermentation, with yield, productivity, and production intensity increased by 8.1%, 6.8%, and 8.4%, and 7.1%, 6.8%, and 7.4%, respectively.

[0137] Example 6: Application of PPC mutant in L-arginine producing strains Eighteen L-arginine-producing strains (Arg-1-WT, Arg-1-PPC(T457R), Arg-1-PPC(F619V), Arg-1-PPC(N814D), Arg-1-PPC(T457R / F619V), Arg-1-PPC(F619V / N814D), Arg-1-PPC(T457R / N814D), Arg-1-PPC(T457R / F619V / N814D), Arg-1-PPC(I)) containing the wild-type and mutant expression vectors of the present invention, prepared in Example 3, were used. Fermentation verification was performed on Arg-2-WT, Arg-2-PPC(I495Q), Arg-2-PPC(C665F), Arg-2-PPC(Y835F), Arg-2-PPC(T457R / Y835F), Arg-2-PPC(I495Q / N814D), Arg-2-PPC(C665F / N814D), Arg-2-PPC(T457R / F619V / N814D), and Arg-2-PPC(I495Q / C665F / Y835F).

[0138] (1) Seed culture: Take 1 mL of bacterial solution from the glycerol tube and inoculate it into a 100 mL shake flask containing 30 mL of liquid NDG medium. Incubate in a rotary incubator. Culture conditions: 37 ℃, 250 rpm, 4-6 h. When OD... 600When the OD value reaches 2.0-3.0, the bacterial strain activation culture is complete. Subsequently, the bacterial solution is transferred from liquid NDG medium to NDG plates and incubated at 37 ℃ for 12 h. Then, the colonies on each plate are thoroughly rinsed with 5 mL of physiological saline to ensure the OD value of the bacterial solution after washing is within acceptable limits. 600 The value was controlled within the range of 3.0-4.0. Finally, 1 mL of the washed bacterial solution was inoculated into a 500 mL shake flask containing 100 mL of liquid seed culture medium, and cultured further in a rotary incubator. Culture conditions: 37 ℃, 250 rpm, 3-4 h; when OD... 600 When the value reaches 2.0-3.0, the seed culture of the strain is complete.

[0139] (2) Fermentation in a 7.5 L fermenter: A 7.5 L fermenter was used with an initial liquid volume of 2.4 L and an inoculum size of 16% (v / v). The fermentation temperature was 37 °C. The pH was maintained at 7.3 by adding NH4OH, and the dissolved oxygen was maintained at approximately 30% by adjusting the stirring speed (400-1000 r / min), aeration rate (2-3 L / min), and tank pressure. The initial glucose concentration was controlled at 15 g / L. When the glucose concentration fell below 1 g / L, an 800 g / L glucose solution was added to maintain the concentration at 0.1-1 g / L. When NH4OH was added... + When the concentration is below 0.2 g / L, 500 g / L of ammonium sulfate is added, and glucose and ammonium sulfate are added in a fixed ratio of 4:1. The fermentation time is 48 h.

[0140] The results are shown in Table 8: Table 8: Comparison of 7.5 L fermentation performance of PPC mutants in L-arginine-producing strains

[0141] The results show that: Compared with the control strain Arg-1-WT, the PPC mutant strains Arg-1-PPC (T457R / F619V / N814D) and Arg-1-PPC (I495Q / C665F / Y835F) exhibited superior production performance in L-arginine fermentation. Specifically, Arg-1-PPC (T457R / F619V / N814D) showed increased yield, productivity, and production intensity by 18.4%, 14.3%, and 18.1%, respectively; while Arg-1-PPC (I495Q / C665F / Y835F) showed increased yield, productivity, and production intensity by 17.5%, 9.5%, and 18.1%, respectively.

[0142] Compared with the control strain Arg-2-WT, the PPC mutant strains Arg-2-PPC (T457R / F619V / N814D) and Arg-2-PPC (I495Q / C665F / Y835F) also exhibited superior production performance in L-arginine fermentation. Specifically, Arg-2-PPC (T457R / F619V / N814D) showed increased yield, productivity, and production intensity by 12.5%, 7.7%, and 12.1%, respectively; while Arg-2-PPC (I495Q / C665F / Y835F) showed increased yield, productivity, and production intensity by 10.4%, 3.8%, and 10.1%, respectively.

[0143] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A phosphoenolpyruvate carboxylase mutant, characterized in that, The phosphoenolpyruvate carboxylase mutant is based on the amino acid sequence corresponding to the parental phosphoenolpyruvate carboxylase, with one or more amino acid mutations at positions T457, I495, F619, C665, N814, and Y835, or with one or more amino acid mutations at amino acid residues at equivalent positions in the parental phosphoenolpyruvate carboxylase; the amino acid sequence of the parental phosphoenolpyruvate carboxylase has at least 85% identity with the amino acid shown in SEQ ID NO.1 and has phosphoenolpyruvate carboxylase activity.

2. The phosphoenolpyruvate carboxylase mutant according to claim 1, characterized in that, The mutation corresponds to the amino acid sequence shown in SEQ ID NO.

1. The 457th amino acid is mutated to R, K, or H; preferably, it is mutated to R. The 495th amino acid is mutated to: Q, N, or E; preferably, it is mutated to: Q; The 619th amino acid is mutated to: V, I, or L; preferably, it is mutated to: V; The 665th amino acid is mutated to: F, Y, or W; preferably, it is mutated to: F; The 814th amino acid is mutated to D, E, or N; preferably, it is mutated to D. The 835th amino acid is mutated to: F, Y or H; preferably, it is mutated to: F.

3. The phosphoenolpyruvate carboxylase mutant according to claim 1, characterized in that, The mutant is defined as follows: corresponding to the amino acid sequence shown in SEQ ID NO.1, the phosphoenolpyruvate carboxylase mutant contains at least one of the following mutations equivalent to or corresponding to the parental phosphoenolpyruvate carboxylase mutant: T457R, I495Q, F619V, C665F, N814D, Y835F, T457R / F619V, T457R / C665F, I495Q / F61 9V, I495Q / C665F, T457R / N814D, T457R / Y835F, I495Q / N814D, I495Q / Y835F, F619 V / N814D, F619V / Y835F, C665F / N814D, C665F / Y835F, T457R / F619V / N814D, I495Q / C665F / Y835F.

4. A polynucleotide encoding a phosphoenolpyruvate carboxylase mutant according to any one of claims 1 to 3.

5. A recombinant vector carrying the polynucleotide of claim 4.

6. The recombinant vector according to claim 5, characterized in that, The vector is selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR / Cas9 vectors, or viral vectors; Preferably, the expression vector is selected from the pTrc99a vector.

7. A recombinant cell carrying the recombinant vector of claim 5 or 6 or expressing the phosphoenolpyruvate carboxylase mutant of any one of claims 1 to 3.

8. The recombinant cell according to claim 7, characterized in that, The recombinant cells are bacteria or fungi as host cells; Preferably, the host cell is an L-lysine, L-threonine, or L-arginine producing strain; Preferably, the host cells include, but are not limited to, Escherichia coli, Corynebacterium glutamicum, Lactobacillus brevis, Bacillus flavus, or Corynebacterium pingeri; Preferably, the host cell includes, but is not limited to, E. coli CCTCCNO:M2019435 E. coli NRRLB-12185, E. coli GDMCC NO.1.318 E. coli CCTCC NO: M2022562 E. coli CGMCC NO:25404 E. coli CCTCC NO: M2026397 E. coli CGMCC NO:25402.

9. A method for synthesizing a polynucleotide, characterized in that, Synthesized using the phosphoenolpyruvate carboxylase mutant according to any one of claims 1 to 3; wherein the synthesis method includes in vitro transcription synthesis, isothermal amplification, or transcription-mediated amplification.

10. A recombinant enzyme catalyst containing the phosphoenolpyruvate carboxylase according to any one of claims 1 to 3, characterized in that, It is any of the following forms: (1) Culture recombinant expression transformants containing phosphoenolpyruvate carboxylase mutants and isolate transformant cells containing recombinant phosphoenolpyruvate carboxylase mutant enzymes; (2) Culture recombinant expression transformants containing phosphoenolpyruvate carboxylase mutant, isolate transformant cells containing recombinant phosphoenolpyruvate carboxylase mutant enzyme, and break the transformant cells containing the recombinant phosphoenolpyruvate carboxylase mutant enzyme to obtain cell lysate. (3) Cultivate recombinant expression transformants containing phosphoenolpyruvate carboxylase mutants, isolate transformant cells containing recombinant phosphoenolpyruvate carboxylase mutant enzymes, break the transformant cells containing the recombinant phosphoenolpyruvate carboxylase mutant enzymes, obtain cell lysate, and freeze-dry the cell lysate of the recombinant phosphoenolpyruvate carboxylase mutant enzymes to obtain lyophilized enzyme powder.

11. A method for improving at least one property of phosphoenolpyruvate carboxylase, said property being selected from enzyme activity, substrate affinity and / or catalytic efficiency, and reduced sensitivity to feedback inhibition of the product or substrate, characterized in that, The method is as follows: the phosphoenolpyruvate carboxylase mutant is based on the amino acid sequence corresponding to the parental phosphoenolpyruvate carboxylase, with one or more amino acid mutations at positions T457, I495, F619, C665, N814, and Y835, or with one or more amino acid mutations at amino acid residues at equivalent positions in the parental phosphoenolpyruvate carboxylase; the amino acid sequence of the parental phosphoenolpyruvate carboxylase has at least 85% identity with the amino acid shown in SEQ ID NO.1 and has phosphoenolpyruvate carboxylase activity.

12. The method according to claim 11, characterized in that, Corresponding to the amino acid sequence shown in SEQ ID NO.1, the phosphoenolpyruvate carboxylase mutant at least simultaneously possesses any of the following mutations equivalent to or corresponding to the parental phosphoenolpyruvate carboxylase mutant: T457R, I495Q, F619V, C665F, N814D, Y835F, T457R / F619V, T457R / C665F, I495Q / F61 9V, I495Q / C665F, T457R / N814D, T457R / Y835F, I495Q / N814D, I495Q / Y835F, F619 V / N814D, F619V / Y835F, C665F / N814D, C665F / Y835F, T457R / F619V / N814D, I495Q / C665F / Y835F.

13. A polynucleotide synthesis kit, characterized in that, It includes the phosphoenolpyruvate carboxylase mutant according to any one of claims 1 to 3.

14. An engineered strain of *Escherichia coli*, characterized in that, The engineered Escherichia coli strain expressed the phosphoenolpyruvate carboxylase mutant according to any one of claims 1 to 3.

15. The engineered Escherichia coli strain according to claim 14, characterized in that, The engineered Escherichia coli strain was constructed by cloning the gene encoding the phosphoenolpyruvate carboxylase into an expression vector and transforming it into Escherichia coli host cells. Preferably, the expression vector is selected from the pTrc99a vector; Preferably, the host is an L-lysine, L-threonine, or L-arginine producing strain; Preferably, the host includes, but is not limited to, E. coli CCTCCNO:M2019435 E. coli NRRLB-12185, E. coli GDMCC NO.1.318 E. coli CCTCC NO: M2022562 E. coli CGMCC NO:25404 E. coli CCTCC NO: M2026397 E. coli CGMCC NO:25402.

16. A method for increasing the yield of L-amino acids in Escherichia coli, characterized in that, The method involves expressing the phosphoenolpyruvate carboxylase mutant of any one of claims 1 to 3 in Escherichia coli; Preferably, the L-amino acid is selected from at least one of L-lysine, L-threonine, and L-arginine.

17. The method according to claim 16, characterized in that, The *E. coli* host cells used for protein expression preparation are selected from... Escherichia coli BL21 (DE3) Escherichia coli BL21 Escherichia coli Rosetta (DE3) Escherichia coli C41 (DE3) Escherichia coli C43 (DE3) Escherichia coli JM109 Escherichia coli DH5α or its derivative strains.

18. The method according to claim 16, characterized in that, The E. coli host includes, but is not limited to, E. coli CCTCCNO:M2019435 E. coli NRRLB-12185, E. coli GDMCC NO.1.318 E. coli CCTCC NO: M2022562 E. coli CGMCC NO:25404 E. coli CCTCC NO: M2026397 E. coli CGMCC NO:25402.

19. A method for preparing L-amino acids, characterized in that, The method involves inoculating the recombinant cells of claim 7 or 8, or the engineered Escherichia coli of claim 14 or 15, into a culture medium for fermentation to obtain a fermentation system, and obtaining the target L-amino acid from the fermentation system. Preferably, the L-amino acid is selected from at least one of L-lysine, L-threonine, and L-arginine.

20. The use of the mutant according to any one of claims 1-3, or the polynucleotide according to claim 4, or the recombinant expression vector according to claim 5 or 6, or the recombinant cell according to claim 7 or 8, or the recombinase catalyst according to claim 10, or the engineered Escherichia coli according to claim 14 or 15 in the preparation of L-lysine, L-threonine or L-arginine or in increasing the fermentation yield of L-lysine, L-threonine or L-arginine by genetically engineered bacteria.

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