65 results about "Phosphoenolpyruvic acid" patented technology

The invention discloses construction and application of an L-malic acid producing issatchenkia orientalis strain, and belongs to the technical field of microorganisms and the technical field of microbial fermentation and metabolic engineering. Through a metabolic engineering means, a cytoplasm metabolic pathway of an issatchenkia orientalis strain is transformed, and a pyruvate decarboxylase coding gene, glycerol dehydrogenase and lactic dehydrogenase are knocked out; a pyruvate carboxylase gene derived from aspergillus flavus, a malate dehydrogenase gene derived from corynebacterium glutamicum and phosphoenolpyruvate carboxylase derived from escherichia coli are screened and integrated, so that a cytoplasm rTCA cycle synthesis pathway is enhanced; inserting a dicarboxylic acid external transporter gene from schizosaccharomyces cerevisiae at a neutral site GD21, and knocking out endogenous dicarboxylic acid internal transporter; the L-malic acid yield of the modified yeast strain in the fermentation process is remarkably increased and reaches 140.16 g / L. The engineering strain provided by the invention has good industrial application potential in efficient L-malic acid production.

The invention provides recombinant escherichia coli for de novo synthesis of anisic acid. At least one of the following improvements is carried out on an escherichia coli starting strain: (1) overexpression of O-methyltransferase PSA-OMT1 from Pleuromyces sapidus, (2) overexpression of O-methyltransferase PSA-OMT1 from Pleuromyces sapidus, and (3) overexpression of O-methyltransferase PSA-OMT1 from Pleuromyces sapidus; the method comprises the following steps: carrying out overexpression on chorismate lyase UbiC derived from E.coli MG1655 or Providencia rustiana, and carrying out the overexpression on the chorismate lyase UbiC derived from E.coli MG1655 or Providencia rustiana; (2) carrying out overexpression on a 3-deoxy-D-arabic-heptanone acid 7-phosphate (DAHP) synthase feedback inhibition mutant AroGD146N derived from E.coli MG1655 on the basis of the step (1); and (3) overexpressing synthase PpsA derived from phosphoenolpyruvate (PEP) on the basis of the step (2), and knocking out a pykF gene for coding pyruvate kinase I. Therefore, in the fermentation process of the recombinant escherichia coli, the accumulation of the anisic acid in the fermentation liquor can be realized, the maximum anisic acid shake flask yield of the recombinant escherichia coli can reach 284.9 mg / L, and a foundation is laid for the subsequent construction of high-yield anisic acid engineering bacteria.

The present invention relates to the field of bioengineering technology and provides a ppc mutant and its application in the fermentative production of L-valine. By performing point mutations on the ppc gene, the expression activity of phosphoenolpyruvate carboxylase is reduced, the competition of the bypass pathway is weakened, the supply of pyruvate precursors is enhanced, and the production capacity and biomass of the valine engineering strain are further improved. It can be seen from the fermentation results that this method can significantly increase the yield of valine, from the original 1.3 g / L to 1.49 g / L, with a 14.6% increase in yield. Therefore, the present invention has application value.

The present application relates to a mutant gene encoding phosphoenolpyruvate carboxylase containing a mutant 5' untranslated region (5'UTR), a polyhydroxyalkanoate (PHA)-producing microorganism containing the gene, and a PHA production method using the same.

The invention discloses a genetically engineered bacterium for high yield of L-alanine as well as a construction method and application of the genetically engineered bacterium, a dominant strain is obtained by knocking out an alanine racemase gene dadX and integrating coding sequences of alanine dehydrogenase from different sources, and the shake flask yield is 10.8 g / L; the activity of a corresponding mutant M18L / E75A / A78S / I266V is 4.3 times that of a wild type; 6 by-product related genes are knocked out, meanwhile, a multi-copy strain integrated with alanine dehydrogenase is constructed, and the yield of E.coli A-LB6 in a 5L fermentation tank is increased to 133.3 g / L. Through a dynamic regulation and control strategy, an oxygen-sensitive promoter Pomp * is adopted to replace a phosphoenolpyruvate carboxylase promoter, so that succinic acid byproducts are reduced by 85.8%, and the yield reaches 143.5 g / L after 48 hours of aerobic and anaerobic two-stage fermentation. And finally, by regulating and controlling the ATP synthetase gene cluster atp promoter, the yield of L-alanine in a 50 L fermentation tank reaches 151.3 g / L, and the saccharic acid conversion rate reaches 95.2%. The combination of polygene knockout and dynamic metabolism regulation effectively enhances the biosynthesis ability of alanine, and provides an efficient engineering strain for industrial production.

The invention discloses a construction method of an escherichia coli mutant for producing succinic acid by fermentation of a synthetic medium. The method comprises the following steps: firstly, knocking out a lactic dehydrogenase gene ldhA, a pyruvate formate lyase gene pflB, a ptsG gene responsible for encoding a phosphotransferase system EIIBC protein, an ethanol dehydrogenase gene adhE, an acetokinase-phosphate transacetylase gene ackA-pta, and a ptsG gene responsible for encoding a phosphotransferase system EIIBC protein in escherichia coli; a phosphoenolpyruvate carboxykinase gene pck from bacillus subtilis is integrated at an SS9 safety site of a strain to obtain escherichia coli ESC6 with high succinic acid yield; and mutating one or more loci in one or more genes of a glucose-transcriptional inhibition factor gene mlc, a nitrate response regulatory factor gene narL and a cyclic adenylate receptor protein gene crp to obtain the escherichia coli mutant capable of producing succinic acid by fermentation of a synthetic culture medium, wherein the one or more loci in one or more genes of the glucose-transcriptional inhibition factor gene mlc, the nitrate response regulatory factor gene narL and the cyclic adenylate receptor protein gene crp are mutated. The strain can be fermented in a synthetic medium to produce succinic acid, so that the fermentation cost is greatly reduced, and the strain has a very wide application prospect.

The invention discloses a modified aureobasidium pullulans strain based on reduction of malic acid pathway consumption and application of the modified aureobasidium pullulans strain. Gene for coding NADPH dependent malic enzyme and phosphoenolpyruvate carboxykinase in an aureobasidium pullulans genome is directionally knocked out. The transformation blocks a key consumption branch for synthesis and accumulation of the cytoplasmic malic acid, and effectively guides a central carbon metabolic flow to be more efficiently guided to a target product. Experimental results show that the polymalic acid yield and the saccharic acid conversion rate of the double-knockout engineering strain D3N5-delta3617 / deltapepck in fermentation are remarkably improved compared with those of an original strain, and the biomass is also increased. The invention provides a high-performance strain with important application value for industrial efficient fermentation production of polymalic acid.

The present disclosure relates to a genetically modified microorganism satisfying some of predetermined conditions. The predetermined conditions include: (I) succinate dehydrogenase activity or fumarate reductase activity being reduced or inactivated relative to a wild-type microorganism; (II) lactate dehydrogenase activity being reduced or inactivated relative to the wild-type microorganism; (III) the genetically modified microorganism having modified phosphoenolpyruvate carboxylase activity showing resistance to feedback inhibition by aspartic acid in wild-type phosphoenolpyruvate carboxylase activity, or exogenous phosphoenolpyruvate carboxylase activity having higher resistance to feedback inhibition by aspartic acid than that of the wild-type phosphoenolpyruvate carboxylase activity shown by the wild-type microorganism; and (IV) pyruvate:quinone oxidoreductase being reduced or inactivated relative to the wild-type microorganism.

The invention provides recombinant Escherichia coli for synthesizing heme and derivatives thereof, and the recombinant Escherichia coli is used for knocking out genes such as pyruvate oxidase poxB, pyruvate formate lyase pflB and porphyrinoperoxidase yfeX and enhancing genes such as phosphoenolpyruvate carboxylase ppc, glycerol transporter glpF and 3-phosphoglycerol dehydrogenase glpD, so that the heme and the derivatives thereof can be synthesized. After genes such as exogenous 5-aminolevulinic acid synthase hemA and ferrous chelating enzyme hemH are introduced, the heme and the derivatives thereof can be synthesized by taking glycerol as a raw material, and the heme has an application prospect in the field of biological manufacturing.

Recombinant microorganism in which a gene encoding a transcriptional repressor of the glycine cleavage system, pyruvate formate lyase or phosphoglycerate dehydrogenase, is attenuated or deleted from a host microorganism with a formic acid assimilation pathway, a ppsR gene encoding a phosphoenolpyruvate synthase regulatory protein, or a purT gene encoding a phosphoribosylglycinamide formyltransferase, is attenuated or deleted, a gcvTHP gene, consisting of the gcvT gene, the gcvH gene and the gcvP gene, which encodes an enzyme involved in a glycine cleavage system reaction in which host microorganism with the formic acid assimilation pathway is highly expressed, and a gene encoding formate tetrahydrofolate ligase, methenyl tetrahydrofolate cyclohydrolase or methylene tetrahydrofolate dehydrogenase is introduced into the host microorganism via the formic acid assimilation pathway.

Disclosed are genetically engineered Schizosaccharomyces pombe strains capable of fermenting glucose to produce malic acid. The S. pombe strains produce an exogenous malate dehydrogenase (MDH) enzyme and optionally produce an exogenous pyruvate carboxylase (PYC) enzyme or an exogenous phosphoenolpyruvate carboxylase (PPC) enzyme. Additionally, the S. pombe strains may be further engineered to produce a fumarase selectively located in mitochondria and / or have an inactivated mitochondrial citrate transporter, so that the resulting S. pombe strains are capable of producing malic acid as the predominant C4 dicarboxylic acid. Also disclosed are methods of producing malic acid with the S. pombe strains.

The invention provides an application of a CsPPC3 gene in regulation and control of carbon and nitrogen metabolism of cucumber fruits, a major gene CsPPC3 for coding phosphoenolpyruvate carboxylase (PEPC) in carbon fixation of the cucumber fruits is taken as a research target, an overexpression and gene knockout transgenic plant is constructed, the effect of the CsPPC3 gene in carbon fixation and carbon and nitrogen metabolism of photosynthesis of the fruits is researched, and the CsPPC3 gene is applied to regulation and control of carbon and nitrogen metabolism of the cucumber fruits. It is found that the amount of CO2 released by respiration of a transgenic plant overexpressed by the CsPPC3 gene is reduced, the CO2 refixation capacity is enhanced, the single fruit weight of the fruit is increased, the content of malic acid, soluble sugar and starch is increased, and the content of amino acid is reduced, while the amount of CO2 released by respiration of a transgenic plant knocked out by the CsPPC3 gene is increased, and the single fruit weight of the fruit is reduced. Meanwhile, the contents of malic acid, soluble sugar and amino acid are reduced, the starch content is increased, and the CO2 refixing capacity is reduced. The method disclosed by the invention has practical guiding significance for cultivating transgenic plants with high CO2 fixing efficiency.

The invention belongs to the field of microorganisms, and particularly relates to lactobacillus paracasei with multi-matrix fermentation capacity and application of the lactobacillus paracasei. The lactobacillus paracasei is lactobacillus paracasei Shannan.LPA-972, and the preservation number of the lactobacillus paracasei Shannan.LPA-972 is CCTCC (China Center For Type Culture Collection) NO: M 20242026. The lactobacillus paracasei is lactobacillus paracasei Shannan.LPA-972. The lactobacillus paracasei is related to a phosphoenolpyruvate-glycotransferase system carried by the lactobacillus paracasei. When the fermentation agent containing lactobacillus paracasei, lactobacillus delbrueckii subsp. Lactis and streptococcus thermophilus subsp. Salivarius with a specific viable count ratio is used for fermenting plant milk and animal milk, broad-spectrum adaptability of animal milk raw materials and plant milk raw materials is achieved, meanwhile, the nutrition and sensory quality of fermentation products are remarkably improved, the problem of multi-matrix fermentation can be effectively solved, and the production cost is reduced. And the process cost and complexity of a dairy product production end are effectively reduced.

The application belongs to the technical field of biology, and discloses a strain for efficiently fixing carbon dioxide and improving succinic acid conversion rate and a construction method thereof. The application discloses a genetically engineered bacterium, which is obtained by overexpressing carbonic anhydrase genes, phosphoenolpyruvate carboxylase genes and phosphoenolpyruvate carboxykinase genes in a succinic acid-producing strain. ‑ The carbonic anhydrase can enhance the conversion efficiency of CO2 to HCO3 , and the phosphoenolpyruvate carboxylase and the phosphoenolpyruvate carboxykinase can realize efficient fixation of C3 and CO2 into C4 intermediates. Experiments prove that the genetically engineered bacterium can effectively produce succinic acid by using glucose, and has a relatively high sugar acid conversion rate (up to 79.80%).

The application provides a pyroglutamic acid transaminase mutant and application thereof, the mutant is obtained by mutation on the basis of wild-type pyroglutamic acid transaminase, and specific mutation includes at least one of the following sites: (1) the 13th isoleucine is mutated into arginine; (2) the 35th valine is mutated into arginine; (3) the 183rd asparagine is mutated into aspartic acid; and the mutant is suitable for industrial enzymatic production of L-aspartic acid and the process of amino acids taking L-aspartic acid as a precursor; especially, the I13R / V35R / N183D mutant obtained by modification has high positive catalytic activity and strict direction selectivity, and completely loses the reverse reaction activity of decomposing L-aspartic acid; the mutant is overexpressed with phosphoenolpyruvate carboxylase in L-threonine production strain THRS-6, and a high-efficiency L-threonine synthesis metabolic pathway is constructed, and the yield of L-threonine is significantly improved when glucose is used as a substrate.

The present application relates to the technical field of genetic engineering, and particularly relates to a phosphoenolpyruvate carboxylase mutant and application thereof. The phosphoenolpyruvate carboxylase mutant is obtained by a plurality of mutations in the amino acid sequence of the phosphoenolpyruvate carboxylase. The present application provides a plurality of mutation sites of the phosphoenolpyruvate carboxylase, and further finds that after the mutation of the sites of the phosphoenolpyruvate carboxylase in the strain, the yield of amino acids such as glutamic acid, glutamine and proline is significantly improved, which has important significance in the field of improving the strain to produce glutamic acid and its derivatives.

The invention discloses a phosphoenolpyruvate carboxylase mutant and application thereof, which is characterized in that an enzyme with an amino acid sequence as shown in SEQ ID NO.1 is used as a parent, and the 935th site or 155th site of the parent is mutated. The thermal stability of the phosphoenolpyruvate carboxylase is improved.

The invention relates to the technical field of bioengineering, and provides a ppc mutant and application thereof in fermentation production of L-valine. According to the invention, the ppc gene is subjected to point mutation, so that the expression activity of phosphoenolpyruvate carboxylase is reduced, bypass pathway competition is weakened, pyruvic acid precursor supply is enhanced, and the production capacity and biomass of the valine engineering strain are further improved. Fermentation results show that the method can obviously improve the yield of valine, the yield is improved from 1.3 g / L to 1.49 g / L, and the yield is improved by 14.6%. Therefore, the method has application value.

The invention relates to the technical field of microorganisms, and particularly discloses a phosphoenolpyruvate carboxykinase mutant and application thereof in lysine production. The research finds that the 377th amino acid of phosphoenolpyruvate carboxykinase is mutated from proline to serine, so that the yield of lysine produced by zymophyte can be increased, and therefore, the phosphoenolpyruvate carboxykinase mutant is provided, which takes an amino acid sequence as shown in SEQ ID NO.7 or 9 as a reference sequence; the phosphoenolpyruvate carboxykinase mutant contains a 377th amino acid mutation which is mutated from proline to serine. According to the phosphoenolpyruvate carboxykinase mutant provided by the invention, the yield of lysine fermentation can be increased, and a new effective method is provided for lysine production.

The present application provides a recombinant E. coli for synthesizing heme and derivatives thereof, wherein the recombinant E. coli is knocked out of genes such as pyruvate oxidase poxB, pyruvate formate-lyase pflB and protoporphyrinogen peroxidase yfeX, and meanwhile, the genes such as phosphopyruvate carboxylase ppc, glycerol transporter glpF and 3-phosphoglycerate dehydrogenase glpD are enhanced, and the genes such as 5-aminovaleryl-CoA synthase hemA and ferrous chelatase hemH are introduced, so that the recombinant E. coli can synthesize heme and derivatives thereof by using glycerol as a raw material, and has application prospects in the field of biological manufacturing.

The present application relates to a kind of transcription factor-based phosphoenolpyruvate biosensor and its application, belong to the field of biotechnology.The present application utilizes the natural response mechanism of phosphoenolpyruvate and LysR type transcription factor CbbR, and the biosensor of phosphoenolpyruvate response is constructed, its main element includes CbbR protein, CbbR protein specific promoter and fluorescent reporter gene, using fluorescence signal intensity to characterize phosphoenolpyruvate concentration, combined with RBS sequence screening, sigma factor regulation and promoter engineering, the sensitivity and dynamic range of biosensor are significantly improved, so that biosensor shows excellent time resolution, can monitor the dynamic change of intracellular phosphoenolpyruvate level in real time, provide new ideas and means for the construction and optimization of biological dynamic monitoring tool.

The present invention relates to the technical field of microorganisms, and provides a phosphoenolpyruvate carboxykinase mutant and an application thereof in the production of lysine. Studies have found that mutating an amino acid at position 377 of phosphoenolpyruvate carboxykinase from proline to serine increases yield of lysine produced by a fermentation strain. Therefore, provided is a phosphoenolpyruvate carboxykinase mutant, wherein an amino acid sequence shown in SEQ ID NO. 7 or 9 is used as a reference sequence, and the phosphoenolpyruvate carboxykinase mutant comprises a mutation in which the amino acid at position 377 is mutated from proline to serine. The provided phosphoenolpyruvate carboxykinase mutant increases the fermentation yield of lysine and provides an effective new method for lysine production.

The invention discloses a construction method and application of an engineering bacterium for increasing the yield of alpha-ketoglutaric acid. Wild E.coli.W3110 serves as a chassis strain, lactic dehydrogenase ldhA, pyruvate dehydrogenase poxB and formic acid C-acetyltransferase pflB are knocked out firstly, and the flow direction of pyruvic acid to lactic acid, acetic acid and formic acid is blocked; meanwhile, the promoter PBBaJ23109 is used for replacing a natural promoter of the sucA gene, so that alpha-ketoglutaric acid dehydrogenase is weakened, and the decomposition way of alpha-ketoglutaric acid is weakened; the phosphoenolpyruvate carboxylase ppc is overexpressed, and the flow direction of phosphoenolpyruvate to oxaloacetic acid is enhanced; isocitrate dehydrogenase icd from escherichia coli is overexpressed, so that synthesis of a product is promoted; besides, pyruvate dehydrogenase derived from saccharomyces cerevisiae is introduced and coded by genes PDB1, PDA1 and LAT1, so that an important precursor, namely pyruvic acid, is promoted to flow to acetyl coenzyme A; the engineering strain with high yield of alpha-ketoglutaric acid is finally obtained, the yield of 75.1 g / L can be achieved in a 5L fermentation tank through a fed-batch fermentation method for 40 hours, and the conversion rate is 39.6%.

The invention belongs to the field of molecular biology, and particularly relates to a construction method and application of a phosphoenolpyruvate synthase T419D mutation site. The mutation site is obtained by mutating threonine at the 419th site of phosphoenolpyruvate synthase of an escherichia coli wild type into aspartic acid, the sensitivity of an escherichia coli mutant strain with the mutation site to nalidixic acid, mitomycin C and polymyxin sulfate B is remarkably improved, and the mutation site can be used for improving the sensitivity of bacteria to drugs.

The invention discloses a construction method and application of escherichia coli genetically engineered bacteria for producing malic acid by synergistically utilizing CO2, and belongs to the technical field of bioengineering. According to the invention, an L-malic acid chassis strain MG1655W (DE3) constructed by a gene editing technology in a laboratory is used as an initial strain, a lactic dehydrogenase encoding gene ldhA and a phosphate acetyltransferase encoding gene pta in escherichia coli are knocked out on the basis, the production paths of lactic acid and formic acid are blocked, and the yield of lactic acid and formic acid is increased. The method comprises the following steps of: firstly, amplifying a phosphoenolpyruvate carboxylase gene ppc and a CO2 transporter gene CA, constructing a recombinant plasmid, introducing the recombinant plasmid pACYCDuet-1-CA-ppc into an expression host MG1655W (DE3) delta ldhA delta pta, and screening to obtain the engineering bacterium MG1655W (DE3) delta ldhA delta pta-CA-ppc by overexpressing the ppc gene and the CA gene and improving the CO2 immobilization and concentration capability of escherichia coli. The obtained engineering bacterium is subjected to cell proliferation under an aerobic condition and malic acid accumulation under an oxygen limiting condition, the final malic acid yield is increased to 140-212%, and the engineering bacterium has important industrial application value.

The present disclosure relates to genetically engineered bacteria for producing D-pantothenic acid, a construction method thereof, and application of the genetically engineered bacteria in preparation of the D-pantothenic acid through microbial fermentation. Mainly, (1) carbon flux is further directed toward the synthesis of the D-pantothenic acid by increasing the copy number of key genes in the synthesis pathway of pantothenic acid in an Escherichia coli (E. Coli) organism; and (2) in order to further enhance the carbon flux to direct toward the synthesis of the D-pantothenic acid, glycolysis front-end genes are activated by weakening a nitrogen limitation-negative regulatory transcription factor to drive the carbon flow, phosphoenolpyruvic acid is saved, the central carbon flow entering TCA is reduced, and a plasma-free and antibiotic-free genetically engineered bacterium strain for producing D-pantothenic acid is obtained. Finally, D-pantothenic acid shake flask titer is increased by 87.2% compared with an original strain, which reaches 5.43 g / L; and fermentation is performed with a 5 L fermentation tank for 84 hours, and the yield of the D-pantothenic acid can reach 94.2 g / L.

The application discloses a construction method and application of recombinant bacteria for efficiently converting glucose to produce tetrahydropyrimidine, and the recombinant bacteria are constructed according to a biosynthesis pathway of tetrahydropyrimidine and a carbon metabolic flow distribution of balanced phosphoenolpyruvic acid to oxaloacetic acid and pyruvic acid, and the recombinant bacteria strain directly produces tetrahydropyrimidine by fermentation with glucose as a substrate; 36-53 g / L of tetrahydropyrimidine can be accumulated in a 56-hour fermentation liquor, the yield per cell reaches 1.21-1.91 g / g DCW, and the glucose molar conversion rate reaches 0.31-0.38 mol / mol, and the yield, the yield and the conversion rate are all higher than those of an existing process; compared with an existing fermentation method and a whole-cell catalysis method for producing tetrahydropyrimidine salt, the recombinant bacteria provided by the application have the advantages of low raw material cost, simple culture condition, small equipment loss, short fermentation period, simple operation, no need to collect bacterial bodies to extract enzyme liquor and high tetrahydropyrimidine production efficiency.

The invention discloses a method for improving the yield of glutamic acid of corynebacterium glutamicum through metabolic engineering transformation, and belongs to the field of metabolic engineering and synthetic biology. According to the corynebacterium glutamicum provided by the invention, the expression of glutamate dehydrogenase and glutamate synthase is enhanced, isocitrate lyase is knocked out, and the carbon flow of glyoxylic acid circulation is weakened; the expression of transcription factors RosR, NAD kinase, phosphoenolpyruvate carboxylase, phosphofructokinase and pyruvate kinase is enhanced, the yield of glutamic acid is further increased, and finally the fermentation process is optimized. When the recombinant corynebacterium glutamicum provided by the invention is used for fed-batch fermentation in a 5L fermentation tank for 32 hours, the yield of glutamic acid reaches 230.57 g / L, and the sugar-acid conversion rate is 76.2%.

The application specifically relates to a method for preparing glutathione by immobilized enzyme and application. The method for preparing glutathione provided by the application comprises synthesizing glutathione by using saccharomyces cerevisiae haploid ascospores as a fixed enzyme catalyst to catalyze substrate reaction, using an ATP in-situ regeneration reaction system of phosphoenolpyruvate as an energy supply system, the saccharomyces cerevisiae haploid ascospores are knocked out or inactivated in combination of one or more than two of cell wall beta-glucan synthesis genes, and GSH1 genes, GSH2 genes and PYK1 genes are co-expressed. The saccharomyces cerevisiae haploid ascospores for simultaneously producing GSH1, GSH2 and pyruvate kinase are used as a fixed enzyme catalyst to catalyze synthesis of glutathione, three enzymes no longer need to be added externally, raw material cost is greatly reduced, the obtained product is easy to purify, and the prepared glutathione is high in purity.

The present invention relates to a genetically engineered bacterium capable of producing D-pantothenic acid, a method for constructing it, and the use of this recombinant bacterium in the production of D-pantothenic acid by microbial fermentation. The main components of this invention are: (1) increasing the copy number of key genes in the pantothenic acid synthesis pathway in Escherichia coli to further direct carbon flux toward D-pantothenic acid synthesis; (2) attenuating nitrogen-limiting negative regulatory transcription factors and activating front-end genes of glycolysis to increase carbon flux, conserving phosphoenolpyruvate and reducing central carbon flux into the TCA cycle, thereby obtaining a recombinant bacterial strain for D-pantothenic acid production without the addition of plasmids or antibiotics. Finally, the shake flask titer of D-pantothenic acid was increased by 87.2% compared to the original strain, reaching 5.43 g / L. After 84 hours of fermentation in a 5-L fermentation tank, D-pantothenic acid production reached 94.2 g / L.