Genetically engineered bacterium of high-yield D-pantothenic acid as well as preparation method and application of genetically engineered bacterium

Abstract

The invention relates to the technical field of microbial fermentation, and discloses a genetically engineered bacterium of high-yield D-pantothenic acid as well as a preparation method and application of the genetically engineered bacterium. The D-PA yield of the engineering bacteria is greatly improved through the following effects that acetolactate synthase IlvB and IlvN shunted by pyruvic acid are subjected to overexpression, meanwhile, a transcription attenuation region is deleted, and valine feedback inhibition sites are mutated; the method comprises the following steps: overexpressing keto-alcohol acid reductoisomerase IlvC and dihydroxy acid dehydratase IlvD, and converting the IlvC coenzyme preference from NADPH into NADH, so as to break through the upstream of D-PA synthesis; overexpression of PanB is carried out, overexpression of a serine-glycine transport system is carried out, and synthesis of 5, 10-methylenetetrahydrofolic acid is promoted; meanwhile, the expression of ketopantoic acid reductase PanE is enhanced; and finally, over-expressing pantothenic acid synthetase PanC, and increasing the content of a cofactor ATP required by the PanC. According to the engineering bacteria provided by the invention, through overall reinforcement of the multiple modules, cell growth is improved due to balance of cofactors and increase of D-PA, and the yield of D-PA reaches an unprecedented height.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to a genetic engineering bacterium with high production of D-pantothenic acid and a preparation method and application thereof. Background Art

[0002] Metabolic engineering aims to maximize the production of high value-added compounds using microorganisms as cell factories. How to build a cell factory, achieve high yield and high conversion rate under effective regulation, and how to transfer carbon flow to product synthesis as much as possible while ensuring the normal growth of the strain, and reduce the waste of carbon sources, are all major research focuses for biosynthetic production of products. In addition, product yield and sugar conversion rate are the standards for whether industrial products are achieved. Intuitively strengthening the method of product synthesis and enhancing product accumulation is one of the primary operations of metabolic engineering transformation.

[0003] Pantothenic acid (D-PA), also known as vitamin B5, is a component of coenzyme A and plays an important role in important biochemical reactions such as energy metabolism and the citric acid cycle. At present, D-PA for industrial applications is mainly produced by chemical methods using the condensation of D-pantoic acid lactone and β-alanine in methanol or ethanol. However, the use of chemical separation or enzyme-catalyzed esters involves toxic chemical reagents and expensive separation methods. With the continuous development of genetic engineering and synthetic biology, the production of D-PA using biosynthetic methods that are environmentally friendly, cheap and simple has attracted widespread attention from scholars. However, there are still defects in the current production of D-PA using biological methods, such as unstable fermentation process and low yield.

[0004] As an industrial microorganism, Escherichia coli has advantages such as clear genetic background and simple operation, and is one of the ideal chassis for most fermentation production. Summary of the invention

[0005] At present, the biological production of D-pantothenic acid has low yield and unstable fermentation. In order to solve the above technical problems, the present invention provides a genetic engineering bacterium with high yield of D-pantothenic acid and its preparation method and application.

[0006] The specific technical scheme of the present invention is: In a first aspect, the present invention provides a genetically engineered bacterium that produces high levels of D-pantothenic acid. The genome of the engineered bacterium includes: an ilvBN gene that is expressed with enhanced expression, and the amino acid glycine at position 20 of the protein encoded by the ilvN gene is mutated to aspartic acid, the amino acid valine at position 21 is mutated to aspartic acid, and the methionine at position 22 is mutated to phenylalanine; An ilvC gene with enhanced expression, wherein the amino acid leucine at position 67 of the protein encoded by the ilvC gene is mutated to glutamic acid, the amino acid arginine at position 68 is mutated to phenylalanine, and the amino acid lysine at position 75 is mutated to glutamic acid; Enhanced expression of the ilvD gene; Enhanced expression of panB gene; Enhanced expression of serABC genes; Enhanced expression of glyA gene; Enhanced expression of the panE gene; Strengthen the expression of the panC gene, and replace the start codon of the panC gene with ATG; Knock out the amn gene.

[0007] In the engineering bacteria provided by the present invention, compared with the bottom plate bacteria, the titer of D-pantothenic acid in the shake flask fermentation is increased by 68.1%, reaching 6.44g / L. The engineering bacteria provided by the present invention greatly improves the D-PA production of the engineering bacteria through the following actions: overexpressing the acetolactate synthase IlvB and IlvN of the pyruvate diversion, deleting the transcription attenuation zone, and mutating the valine feedback inhibition site; overexpressing the ketol acid reductoisomerase IlvC and the dihydroxy acid dehydratase IlvD, and converting the IlvC coenzyme preference from NADPH to NADH, opening up the upstream of D-PA synthesis; overexpressing PanB, overexpressing the serine-glycine transport system, and promoting the synthesis of 5,10-methylenetetrahydrofolate; at the same time, strengthening the expression of the ketopantoate reductase PanE; finally, overexpressing the pantothenate synthase PanC, and increasing the content of the cofactor ATP required by PanC. The engineered bacteria provided by the present invention are comprehensively strengthened through the above-mentioned multiple modules, and cell growth is improved due to the balance of cofactors and the increase of D-PA, and the D-PA yield reaches an unprecedented level.

[0008] In a second aspect, the present invention provides a method for constructing a genetically engineered bacterium that produces high levels of D-pantothenic acid, comprising the following steps: (1) enhancing the expression of the ilvBN gene in the chassis bacteria, and mutating the ilvN gene in the ilvBN gene, so that the amino acid glycine at position 20 of the protein encoded by the ilvN gene is mutated to aspartic acid, the amino acid valine at position 21 is mutated to aspartic acid, and the methionine at position 22 is mutated to phenylalanine; (2) enhancing the expression of the ilvC gene in the bottom plate bacteria, and mutating the ilvC gene so that the amino acid leucine at position 67 of the protein encoded by the ilvC gene is mutated to glutamic acid, the amino acid arginine at position 68 is mutated to phenylalanine, and the amino acid lysine at position 75 is mutated to glutamic acid; (3) Enhanced expression of the ilvD gene in the chassis bacteria; (4) Enhanced expression of the panB gene in the chassis bacteria; (5) Enhanced expression of serABC genes in chassis bacteria; (6) Enhanced expression of the glyA gene in the chassis bacteria; (7) Enhanced expression of the panE gene in the chassis bacteria; (8) enhancing the expression of the panC gene in the chassis bacteria, and replacing the start codon of the panC gene with ATG; (9) Knock out the amn gene in the chassis bacteria.

[0009] The purpose of the present invention is to use rational design and CRISPR-Cas9 gene editing technology to further enhance the flux of supersaturated D-PA main pathway metabolism on the basis of strains with improved sugar uptake rate, push the carbon flow to the D-PA main pathway, and achieve the improvement of yield and conversion rate only by overexpression of product synthesis pathway, and embody the application of this technology in the preparation of D-pantothenic acid by microbial fermentation.

[0010] In the above construction method, the functions of each step are as follows: (1) overexpressing the first gene cluster ilvBN of the main pathway of pyruvate entering D-PA, catalyzing the synthesis of acetolactate, and promoting the synthesis of D-pantothenic acid precursor D-pantoic acid; (2) overexpressing the ketol-acid reductoisomerase IlvC, catalyzing the synthesis of dihydroxy isovalerate, and at the same time transforming the cofactor dependence of IlvC from NADPH to NADH; (3) overexpressing the dihydroxy acid dehydratase IlvD, converting the generated dihydroxy isovalerate into α-ketoisovalerate; (4) overexpressing the ketopantoic acid hydroxymethyltransferase PanB, converting α-ketoisovalerate into Ketopantoate; (5) Overexpression of phosphoglycerate dehydrogenase SerA, B, and C of the serine-glycine transport system to promote the synthesis of 5,10-methylenetetrahydrofolate; (6) Overexpression of serine hydroxymethyltransferase GlyA to promote the synthesis of 5,10-methylenetetrahydrofolate; (7) Overexpression of ketopantoate reductase PanE to catalyze the conversion of ketopantoate to pantoate; (8) Overexpression of pantothenate synthase PanC to condense pantoate with β-alanine to form D-pantothenate; (9) Knockout of AMP nucleosidase Amn to reduce the degradation of nucleotides and retain the ATP content to promote the D-PA main pathway.

[0011] The present invention finally constructs a genetically engineered bacterium with excellent D-PA production through the interaction of the above steps. The present invention utilizes the entire pathway of overexpression of D-PA and the supply of its coenzyme cycle to greatly increase the D-PA production, and there is no obvious inhibition of cell growth, which provides a way of thinking for the industrial production of D-PA.

[0012] Preferably, the bottom plate bacteria is Escherichia coli.

[0013] Further preferably, the genotype of the bottom plate bacteria is: E. coli W3110 derivative, Trc-panCpanEpanB / ilvC / ilvG * / ΔavtA / ilvE * / coaA * / ΔpoxB / Δpta / ΔplfB / ΔldhA / Trc-pykA / ilvN* / ilv H* / spoT* / Trc-spoT* / Trc-lpd / Trc-ilvD / ΔlacI / Trc-alaS / Trc-ilvD / ptsG::Trc-glk,galP.

[0014] Preferably, in step (1), the enhanced expression is: after replacing the original promoter of the ilvBN gene with the Trc promoter, the operon of the in situ ilvBN gene in the genome of the chassis bacteria is replaced.

[0015] By relieving the transcriptional attenuation inhibition of the ilvBN operon, the synthesis of D-pantothenic acid precursor D-pantoic acid is further promoted.

[0016] Preferably, in step (2), step (3), step (4) and step (6), the enhanced expression is: after replacing the original promoter of the gene to be enhanced with the Trc promoter, the corresponding in situ gene in the genome of the chassis bacteria is replaced.

[0017] Preferably, in step (5) and step (7), the enhanced expression is: replacing the original promoter of the gene to be enhanced with the Trc promoter, and then replacing the pseudogene in the in situ genome of the chassis bacteria.

[0018] Preferably, in step (4), the panB gene is derived from Corynebacterium glutamicum.

[0019] Preferably, in step (1), the ilvN gene mutation is: the codon GGC at position 20 of the protein encoded by the ilvN gene is changed to GAU, the codon GUA at position 21 is changed to GAC, and the codon AUG at position 22 is changed to UUC.

[0020] Preferably, in step (2), the ilvC gene is mutated by changing the codon CUG at position 67 of the protein encoded by the ilvC gene to GAA, the codon CGU at position 68 to UUU, and the codon AAG at position 75 to GAA.

[0021] In a third aspect, the present invention provides the use of the genetically engineered bacteria constructed by the above construction method in the preparation of D-pantothenic acid.

[0022] Compared with the prior art, the present invention has the following technical effects: The present invention greatly improves the yield of D-PA through the interaction of the following operations: (1) overexpressing the first gene cluster ilvBN of the main pathway of pyruvate entering D-PA, catalyzing the synthesis of acetolactate, relieving the transcriptional attenuation inhibition of the ilvBN operon, and promoting the synthesis of D-pantothenic acid precursor D-pantoic acid; (2) overexpressing the ketol-acid reductoisomerase IlvC, catalyzing the synthesis of dihydroxy isovaleric acid ester, and at the same time transforming the cofactor dependence of IlvC from NADPH to NADH; (3) overexpressing the dihydroxy acid dehydratase IlvD, converting the generated dihydroxy isovaleric acid ester into α-ketoisovaleric acid; (4) overexpressing the dihydroxy acid dehydratase IlvD, and converting the generated dihydroxy isovaleric acid ester into α-ketoisovaleric acid; (5) Overexpression of phosphoglycerate dehydrogenase SerA, B, C and serine hydroxymethyltransferase GlyA of the serine-glycine transport system to promote the synthesis of 5,10-methylenetetrahydrofolate; (6) Overexpression of ketopantoate reductase PanE to catalyze ketopantoate to generate pantoate; (7) Overexpression of pantothenate synthase PanC to condense pantoate with β-alanine to form D-pantothenate; (8) Knockout of AMP nucleosidase Amn to reduce nucleotide degradation and retain ATP content to promote the main D-PA pathway. The present invention uses the entire pathway of overexpression of D-PA and the supply of its coenzyme cycle to greatly increase the yield of D-PA, and there is no obvious inhibition of cell growth, which provides a way of thinking for the industrial production of D-PA. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the metabolic pathway of D-pantothenic acid; Figure 2 OD of SA-D1 600 and changes in D-pantothenic acid titer; Figure 3 OD of SA-D2 600 and changes in D-pantothenic acid titer; Figure 4 OD for SA-D3 600 and changes in D-pantothenic acid titer; Figure 5 OD of SA-D4 600 and changes in D-pantothenic acid titer; Figure 6 OD for SA-D5 600 and changes in D-pantothenic acid titer; Figure 7 OD of SA-D6 600 and changes in D-pantothenic acid titer; Figure 8 OD for SA-D7 600 and changes in D-pantothenic acid titer; Fig. 9 OD for SA-D8600 and changes in D-pantothenic acid titer; Fig.10 OD for SA-D9 600 and changes in D-pantothenic acid titer. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should belong to the scope of protection of the present invention.

[0025] Figure 1 Schematic diagram of D-pantothenic acid metabolic pathway. The genes and corresponding pathways involved in gene editing in the embodiment of the present invention are shown in Table 1. Among them, the accession number is an identifier used to uniquely identify the gene in the Entrez Gene database of the National Center for Biotechnology Information (NCBI) of the United States.

[0026] Table 1 Genes involved in gene editing and corresponding pathways Gene name Accession Number Involved pathways ilvB 7437257 Acetolactate synthase catalyzes the production of acetolactate from pyruvate ilv 948183 Acetohydroxyacid synthase subunit I, catalyzes the production of acetolactate from pyruvate together with ilvB ilvC 7438941 Catalytic synthesis of dihydroxy isovalerate from acetolactate ilvD 7437141 Conversion of dihydroxyisovalerate to α-ketoisovalerate ecpanB 7435565 Catalyzes the synthesis of α-ketoisovalerate into ketopantoate cgpanB 3345597 Catalyzes the synthesis of α-ketoisovalerate into ketopantoate panE 945065 Catalyzes the formation of pantoate from ketopantoate panC 7436477 Condensation of pantothenic acid with β-alanine to form D-pantothenic acid serABC 7438763 Convert 3-phosphoglycerate to L-serine glyA 7439470 Catalyzes the formation of glycine from serine, simultaneously releasing 5,10-methylenetetrahydrofolate amn 946508 ATP degradation pathway AMP nucleosidase Yj 7438149 Pseudogene cluster yeep 7439610 Pseudogene cluster The primer sequence information involved in the embodiments of the present invention is shown in Table 2.

[0027] Table 2 Primer sequences In the example of the present invention, the final concentration of kanamycin in the culture medium is 0.05 mg / L, and the final concentration of spectinomycin in the culture medium is 0.05 mg / L.

[0028] The parent strain E. coli W3110 of the present invention is from the Coli Genetic Stock Center of Yale University, with a deposit date of August 5, 1975 and a deposit number of CGSC#4474, and has been disclosed in patents US2009 / 0298135 A1 and US2010 / 0248311A1.

[0029] Example 1D - HPLC determination of pantothenic acid content The detection method is as follows: Chromatographic conditions: C 18Column (250 × 4.6 mm, particle size 5 μm, Agilent Technologies Co., Santa Clara, CA, USA), detection wavelength: 200 nm, column temperature: 30 °C; Sample treatment: Dilute the sample with ultrapure water to keep the D-pantothenic acid content between 0.05 g / L and 0.40 g / L; Mobile phase: acetonitrile / water / phosphoric acid (volume ratio: 50 / 949 / 1); Data collection time: 18 minutes.

[0030] Example 2SA-D1 (DPA-SA derivative, P WT -ilvBN::Ptrc-ilvBN DDF ) and shake flask fermentation with DPA-SA (E. coli W3110 derivative, Trc-panCpanEpanB / ilvC / ilvG * / ΔavtA / ilvE * / coaA * / △poxB / Δpta / ΔplfB / ΔldhA / Trc-pykA / ilvN* / ilv H* / spoT* / Trc-spoT* / Trc-lpd / Trc-ilvD / ΔlacI / Trc-alaS / Trc-ilvD / ptsG::Trc-glk,galP) was used as the starting strain, and CRISPR-Cas9-mediated gene editing technology was used to replace the original promoter of the ilvBN gene with the Trc promoter through gene knockout to enhance the expression of the ilvBN gene. Then, the ilvBN gene connected to the Trc promoter replaced the operon of the in situ ilvBN gene on the genome of the engineered bacteria DPA-SA, and the G20D V21D M22F site of the ilvN gene was mutated, i.e., Ptrc-ilvBN DDF , while obtaining a mutant resistant to valine feedback inhibition, the ilvBN transcriptional attenuation region was deleted, and the conversion of pyruvate to acetate and lactate was enhanced. Among them, the mutations of the ilvN gene were: the codon GGC at position 20 was changed to GAU, the codon GUA at position 21 was changed to GAC, and the codon AUG at position 22 was changed to UUC.

[0031] (1) Construction of pT-ilvBN plasmid: pTarget F plasmid (Addgene Plasmid #62226) was used as a template and ilvBN-sgRNA-F / ilvBN-sgRNA-R as primers for PCR amplification. The PCR product was digested with Dpn I at 37°C for 3 h and then transformed into E. coli DH5α. The plasmid was screened on spectinomycin plates and sequenced to verify the correct pT-ilvBN plasmid, which was used for subsequent ligation of ilvBN-up and ilvBN-down.

[0032] (2) Construction of pTarget-Ptrc-ilvBN DDF Plasmid: Using the genome of the D-PA engineering strain DPAN16 produced in the original laboratory as a template, ilvBN-up was amplified with primers ilvBN-up-F and ilvBN-up-R to obtain ilvBN-up, ilvBN-down-F and ilvBN-down-R were amplified with primers to obtain ilvBN-down, and pTarget-ilvBN plasmid was amplified with primers PT-line-F and PT-line-R to obtain pTarget-line-ilvBN, and the Clean up kit was used to recover the DNA fragments; according to (One stepclone kit, Vazyme Biotech, Nanjing, China) instructions to connect pTarget-line-ilvBN, ilvBN-up and ilvBN-down together, and obtain pTarget-Ptrc-ilvBN by sequencing verification. DDF Plasmid.

[0033] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into DPA-SA, and the single clone was transferred to an LB test tube containing 0.05 mg / L kanamycin and cultured at 30°C overnight; then, the inoculum was inoculated into a 250 mL shake flask containing 50 mL LB medium at a volume concentration of 1%, and 500 μl 1 mol / L L-arabinose was added, and the culture was carried out at 150 rpm and 30°C until the OD 600 0.4~0.6; 4000rpm, 4℃ centrifugation for 10min to collect cells and prepare electroporation competent cells.

[0034] (4) Take 200 ng of pTarget-pfkB plasmid and mix it with 100 μL of electroporated competent cells, transfer it into a pre-cooled 2 mm electroporation cup, place it on ice for 1 min, and use an electroporator (MicroPluser TM, BIO-RAD) for electroporation transformation. After the electroporation is completed, 1 mL of LB medium is immediately added and gently aspirated, and the plate is transferred to a 1.5 mL centrifuge tube. After resuscitation at 30°C for 2-3 hours, it is coated with LB plates containing 0.05 mg / L kanamycin and 0.05 mg / L spectinomycin, and inverted culture is carried out at 37°C for 12-16 hours. Colony PCR verification is performed using pfkB-VF and pfkB-VR as primers. If a fragment of about 1500 bp can be successfully cloned, it proves to be a DPA-SAderivative, ΔpfkB-positive colony.

[0035] (5) Plasmid elimination: Pick a single positive colony and inoculate it into an LB test tube containing 1 mM IPTG and 0.05 mg / L kanamycin, culture it at 30°C overnight, streak the next day on an LB plate containing 0.05 mg / L kanamycin, culture it at 30°C for 24 h, pick a single colony and streak it onto an LB plate containing 0.05 mg / L spectinomycin, if the single colony cannot be on the LB plate containing 0.05 mg / L spectinomycin, its pTarget-pfkB plasmid is successfully eliminated, pick a single colony from which the pTarget-pfkB plasmid is successfully eliminated, place it in an LB test tube, culture it at 37°C overnight, streak the next day on an LB plate, culture it at 37°C for 12 h, pick a single colony and streak it onto an LB plate containing 0.05 mg / L kanamycin, if the single colony cannot be on the LB plate containing 0.05 mg / L kanamycin, its pCas plasmid is successfully eliminated, and finally obtain a plasmid-free DPA-SA derivative, ΔpfkB.

[0036] (6) DPA-SA derivative, P WT -ilvBN::Ptrc-ilvBN DDF , with DPA-SA as the control group, inoculated into 10 mL of LB medium, and cultured at 37°C and 200 rpm as pre-culture; after 8-12 hours, inoculated 1 mL of pre-culture into a 500 mL shake flask containing 50 mL of MS medium, and then fermented in a constant temperature shaker at 30°C and 200 rpm for 48 hours; after the fermentation, took 1 mL of the fermentation liquid to measure the OD 600 1 mL of fermentation broth was taken and centrifuged at 12000 rpm for 3 min at room temperature. The fermentation supernatant was diluted 10 times and HPLC was performed according to Example 1. OD 600 and the D-pantothenic acid content in the fermentation supernatant. Figure 2 shown.

[0037] Depend on Figure 2 It can be seen that overexpression of the ilvBN gene cluster in the DPA-SA genome has a certain inhibitory effect on cell growth, and the cell OD 600The DPA-SA value dropped from 4.23 to 4.16. Since the promoter regulatory region of the ilvBN operon may bind to IHF and regulate the expression level of genes in the operon, deleting this region can increase the expression of the gene cluster to a certain extent. The IlvN mutation site that resists valine feedback inhibition is also introduced into the gene cluster, which ultimately makes the D-PA titer reach 4.29g / L.

[0038] The composition of LB medium is: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, the solvent is deionized water, and the pH value is natural.

[0039] The composition of MS culture medium is: glucose 20g / L, ammonium sulfate 16g / L, KH2PO4 0.8g / L, MgSO4 0.5g / L, yeast extract 2g / L, β-alanine 2.5g / L, CaCO3 10g / L (sterilized separately), 1mL / L trace element solution, the solvent is deionized water, and the pH value is natural; the composition of trace element solution: 10g / L CuCl2, 10g / L FeSO4·7H2O, 1g / L ZnSO4·7H2O, 0.20g / L CuSO4, 0.02g / L NiCl2·7H2O, the solvent is deionized water.

[0040] Example 3 SA-D2 (SA-D1 derivative, P WT -ilvC::Ptrc-ilvC EFE ) was constructed and shake flask fermented with SA-D1 as the starting strain. CRISPR-Cas9-mediated gene editing technology was used to replace the original promoter of the ilvC gene with the Trc promoter to enhance the expression of the ilvC gene. The in situ ilvC gene on the genome of the engineered bacteria SA-D1 was then replaced with the ilvC gene connected to the Trc promoter. At the same time, the L67E R68F K75E site of the ilvC gene was mutated to form Ptrc-ilvC. EFE , the cofactor preference changes from NAPDH to NADH, reducing the excessive consumption of reducing power H for bacterial growth, balancing the ratio of NADPH / NADH, and facilitating cell growth and metabolic needs. Among them, the mutations of the ilvC gene are: the codon CUG at position 67 changes to GAA, the codon CGU at position 68 changes to UUU, and the codon AAG at position 75 changes to GAA.

[0041] (1) Construction of pT-ilvC plasmid: pTarget F plasmid (Addgene Plasmid #62226) was used as a template and ilvC-sgRNA-F / ilvC-sgRNA-R as primers for PCR amplification. The PCR product was digested with Dpn I at 37°C for 3 h and then transformed into E. coli DH5α. The product was screened on a spectinomycin plate and sequenced to obtain the correct pT-ilvC plasmid for subsequent ligation of ilvC-up and ilvC-down.

[0042] (2) Construction of pTarget-Ptrc-ilvC EFE Plasmid: Using the genome of the D-PA engineering strain DPAN16 produced in the original laboratory as a template, ilvC-up was amplified with primers ilvC-up-F and ilvC-up-R to obtain ilvC-down, ilvC-down was amplified with primers ilvC-down-F and ilvC-down-R to obtain ilvC-down, pT-ilvC plasmid was amplified with primers PT-line-F and PT-line-R to obtain pTarget-line-ilvC, and the Clean up kit was used to recover the DNA fragments; according to pTarget-line-ilvC, ilvC-up and ilvC-down were connected together according to the instructions of the One step cloning kit (VazymeBiotech, Nanjing, China) and pTarget-Ptrc-ilvC was obtained by sequencing verification. EFE Plasmid.

[0043] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the SA-D1 competent cell obtained in Example 2 (5). The preparation method of SA-D1 competent cell was the same as that in Example 2 (3).

[0044] (4) SA-D2 positive colonies were constructed using the same method as in Example 2 (4).

[0045] (5) Plasmid elimination: The implementation method is the same as Example 2 (5) to obtain plasmid-free SA-D2.

[0046] (6) The constructed SA-D2 engineered strain was used as a control group with the SA-D1 constructed in Example 2, and shake flask tests and detection were performed according to the method of Example 2 (6). 600 and the D-pantothenic acid content in the fermentation supernatant. Figure 3 shown.

[0047] As shown in the figure, ilvC was mutated and overexpressed in the SA-D1 genome, and the cell OD 600It decreased from 4.16 to 4.10 again, and the D-pantothenic acid titer became 4.33, which was almost unchanged. It can be seen that the mutant ilvC gene has an inseparable connection with D-PA production and cell growth. Lack or surplus will have negative feedback inhibition on strain production. Therefore, considering the feasibility of subsequent strategies, the ilvC gene has been overexpressed to saturation. This step will be retained and no changes will be made to this gene. In addition, the growth rate of the strain will be gradually increased to ensure sufficient bacterial volume to produce D-PA.

[0048] Example 4 SA-D3 (SA-D2 derivative, P WT -ilvD::Ptrc-ilvD) was constructed and shake flask fermented with SA-D2 as the starting strain. CRISPR-Cas9-mediated gene editing technology was used to replace the original promoter of the ilvD gene with the Trc promoter to enhance the expression of the ilvD gene. The in situ ilvD gene on the genome of the engineered bacterium SA-D2 was then replaced with the ilvD gene connected to the Trc promoter. The dihydroxyacid dehydratase IlvD is the last enzyme upstream of the D-PA synthesis pathway, which converts the generated isovaleric acid dihydroxy ester into α-ketoisovaleric acid and then enters the downstream pathway.

[0049] (1) Construction of pT-ilvD plasmid: pTarget F plasmid (Addgene Plasmid #62226) was used as a template and ilvD-sgRNA-F / ilvD-sgRNA-R as primers for PCR amplification. The PCR product was digested with Dpn I at 37°C for 3 h and then transformed into E. coli DH5α. The product was screened on a spectinomycin plate and sequenced to obtain the correct pT-ilvD plasmid for subsequent ligation of ilvD-up / ilvD-down.

[0050] (2) Construction of pTarget-Ptrc-ilvD plasmid: Using the E. coli W3110 genome as a template, ilvD-up was amplified using primers ilvD-up-F and ilvD-up-R, ilvD-down was amplified using primers ilvD-down-F and ilvD-down-R, and pT-ilvD plasmid was amplified using primers PT-line-F and PT-line-R to obtain pTarget-line-ilvD. DNA fragments were recovered using a Cleanup kit; pTarget-line-ilvD, ilvD-up and ilvD-down were connected together according to the instruction manual of the kit (One step cloning kit, Vazyme Biotech, Nanjing, China), and the pTarget-Ptrc-ilvD plasmid was obtained by sequencing verification.

[0051] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the SA-D2 competent cells obtained in Example 3 (5). The preparation method of SA-D2 competent cells was the same as that of Example 2 (3).

[0052] (4) SA-D3 positive colonies were constructed using the same method as in Example 2 (4).

[0053] (5) Plasmid elimination: The implementation method is the same as Example 2 (5) to obtain plasmid-free SA-D3.

[0054] (6) The constructed SA-D3 engineered strain was used as a control group with the SA-D2 constructed in Example 3, and shake flask tests and detection were performed according to the method of Example 2 (6). 600 and the D-pantothenic acid content in the fermentation supernatant. Figure 4 shown.

[0055] Depend on Figure 4 It can be seen that the ilvD gene is overexpressed in the SA-D2 genome, and the carbon metabolism flow is further introduced into the D-PA synthesis pathway. 600 No significant changes occurred, while the titer of D-pantothenic acid increased from 4.33g / L to 4.48g / L. The combined action of acetolactate synthase and dihydroxyacid dehydratase greatly increased the titer of D-PA, proving that the upstream pathway has been opened, but the current growth rate is too slow, which has side effects on genetic manipulation and fermentation. Subsequent downstream pathways will be further optimized for this.

[0056] This example also uses SA-D1 as the starting strain, and uses CRISPR-Cas9-mediated gene editing technology to replace the original promoter of the ilvD gene with the Trc promoter, thereby enhancing the expression of the ilvD gene. Then, the ilvD gene connected to the Trc promoter replaces the in situ ilvD gene on the genome of the engineered bacterium SA-D2, thereby obtaining the comparative strain SA-VSD3. The specific steps refer to the construction of SA-D3 in (1) to (6). The results show that the D-pantothenic acid titer of SA-VSD3 is 4.34 g / L, which is not effectively improved compared to 4.29 g / L of SA-D1, while SA-D3 is improved from 4.29 g / L to 4.48 g / L compared to SA-D1, which illustrates the importance of enhanced expression and mutation of the ilvC gene in the construction of SA-D2 in this example.

[0057] Example 5 Construction and shake flask fermentation of SA-D4 (SA-D3 derivative, EcpanB::Ptrc-CgpanB) Using SA-D3 as the starting strain, CRISPR-Cas9-mediated gene editing technology was used to replace the original promoter of the panB gene derived from Corynebacterium glutamicum with the Trc promoter through the gene knockout method to enhance the expression of the panB gene. Then, the panB gene connected to the Trc promoter was used to replace the in situ panB gene on the genome of the engineered bacterium SA-D3, catalyzing the synthesis of the downstream pathway of D-PA and maximally diverting the carbon flow of α-ketoisovalerate.

[0058] (1) Construction of pT-EcpanB plasmid: pTarget F plasmid (Addgene Plasmid #62226) was used as a template and EcpanB-sgRNA-F / EcpanB-sgRNA-R as primers for PCR amplification. The PCR product was digested with Dpn I at 37°C for 3 h and then transformed into E. coli DH5α. The plasmid was screened on a spectinomycin plate and sequenced to verify the correct pT-EcpanB plasmid, which was used for subsequent ligation of EcpanB-up, EcpanB-down and Ptrc-CgpanB.

[0059] (2) Construction of pTarget-Ptrc-CgpanB plasmid: Using the E. coli W3110 genome as a template, EcpanB-up-F and EcpanB-up-R were used as primers to amplify EcpanB-up, EcpanB-down-F and EcpanB-down-R were used as primers to amplify EcpanB-down, and CgpanB-F and CgpanB-R were used as primers to amplify Ptrc-CgpanB. pT-EcpanB plasmid was amplified using PT-line-F and PT-line-R as primers to obtain pTarget-line-EcpanB, and the DNA fragment was recovered using the Clean up kit; pTarget-line-EcpanB, EcpanB-up, EcpanB-down and Ptrc-CgpanB were connected together according to the instruction manual of (One step cloning kit, Vazyme Biotech, Nanjing, China) and the pTarget-Ptrc-CgpanB plasmid was obtained by sequencing verification.

[0060] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the SA-D3 competent cell obtained in Example 4 (5). The preparation method of SA-D3 competent cell was the same as that of Example 2 (3).

[0061] (4) SA-D4 positive colonies were constructed using the same method as in Example 2 (4).

[0062] (5) Plasmid elimination: The implementation method is the same as Example 2 (5) to obtain plasmid-free SA-D4.

[0063] (6) The constructed SA-D4 engineered strain was used as a control group with the SA-D3 constructed in Example 4, and the shake flask test and detection were carried out according to the method of Example 2 (6). 600 and the D-pantothenic acid content in the fermentation supernatant. Figure 5 shown.

[0064] As shown in the figure, overexpression of Ptrc-CgpanB in the SA-D4 genome increased the cell OD 600 From 4.09 to 4.26, PanB is the key enzyme in the entire D-PA synthesis pathway. It has been reported that overexpression of the panB gene from Corynebacterium glutamicum can effectively promote bacterial growth and increase the titer of D-PA. At present, the titer of D-pantothenic acid has increased from 4.48g / L to 4.71g / L, which undoubtedly lays the foundation for subsequent transformation. Considering that the full efficacy of PanB requires the help of the cofactor 5,10-methylenetetrahydrofolate, the content of the cofactor will be further increased.

[0065] Example 6 Construction and shake flask fermentation of SA-D5 (SA-D4 derivative, yeep::Ptrc-serABC) Using SA-D4 as the starting strain, the Trc promoter was connected to the serABC gene cluster using CRISPR-Cas9-mediated gene editing technology, so that the Trc promoter served as the promoter of the serABC gene cluster, and then the serABC gene cluster connected to the Trc promoter was used to replace the yeep pseudogene on the genome of the engineered bacterium SA-D4, overexpressing the serine-glycine transport system and promoting the synthesis of 5,10-methylenetetrahydrofolate.

[0066] (1) Construction of pT-yeep plasmid: pTarget F plasmid (Addgene Plasmid #62226) was used as a template and yeep-sgRNA-F / yeep-sgRNA-R as primers for PCR amplification. The PCR product was digested with Dpn I at 37°C for 3 h and then transformed into E. coli DH5α. The product was screened on a spectral plate and sequenced to verify the correct pT-yeep plasmid for subsequent yeep-up / yeep-down connection.

[0067] (2) Construction of pTarget-Ptrc-serABC plasmid: Using the E. coli W3110 genome as a template, yeep-up-F and yeep-up-R were used as primers to amplify yeep-up, yeep-down-F and yeep-down-R were used as primers to amplify yeep-down, and serABC-F and serABC-R were used as primers to amplify Ptrc-serABC. pT-yeep plasmid was amplified using PT-line-F and PT-line-R as primers to obtain pTarget-line-yeep, and the DNA fragment was recovered using the Clean up kit; pTarget-line-yeep, yeep-up, yeep-down and Ptrc-serABC were connected together according to the instruction manual of (One step cloning kit, Vazyme Biotech, Nanjing, China) and the pTarget-Ptrc-serABC plasmid was obtained by sequencing verification.

[0068] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the SA-D4 competent cell obtained in Example 5 (5). The preparation method of SA-D4 competent cell was the same as that of Example 2 (3).

[0069] (4) SA-D5 positive colonies were constructed using the same method as in Example 2 (4).

[0070] (5) Plasmid elimination: The implementation method is the same as Example 2 (5) to obtain plasmid-free SA-D5.

[0071] (6) The constructed SA-D5 engineered strain was used as a control group with the SA-D4 constructed in Example 5, and shake flask tests and detection were performed according to the method of Example 2 (6). 600 and the D-pantothenic acid content in the fermentation supernatant. Figure 6 shown.

[0072] As can be seen from the figure, the serABC gene cluster is overexpressed in the SA-D4 genome, and the cell OD 600 It decreased from 4.26 to 4.19. The overexpression of L-serine led to uneven distribution of amino acids in the bacteria, which also indirectly proved that L-serine had a certain toxic effect on cell growth. The D-pantothenic acid titer increased from 4.71g / L to 4.77g / L. Although the increase was not obvious, it also proved that 5,10-methylenetetrahydrofolate generated by the intracellular serine-glycine transport system alone was not enough to maintain the demand of PanB. Only by opening up the 5,10-methylenetetrahydrofolate conversion pathway can the cofactor be effectively supplied to PanB.

[0073] Example 7 SA-D6 (SA-D5 derivative, P WT Using SA-D5 as the starting strain and CRISPR-Cas9-mediated gene editing technology, the glyA gene carrying the Trc promoter was used to replace the in situ glyA gene on the genome of the engineered bacterium SA-D5, promoting the conversion of L-serine to L-glycine and at the same time converting tetrahydrofolate to 5,10-methylenetetrahydrofolate.

[0074] (1) Construction of pT-glyA plasmid: Using the E. coli W3110 genome as a template, glyA-up-F and glyA-up-R were used as primers to amplify glyA-up, glyA-down-F and glyA-down-R were used as primers to amplify glyA-down, and glyA-F and glyA-R were used as primers to amplify Ptrc-glyA. pT-glyA plasmid was amplified using PT-line-F and PT-line-R as primers to obtain pTarget-line-glyA, and the DNA fragment was recovered using a Clean up kit; pTarget-line-glyA, glyA-up, glyA-down and Ptrc-glyA were connected together according to the instruction manual of (One step clone kit, Vazyme Biotech, Nanjing, China) and the pTarget-Ptrc-glyA plasmid was obtained by sequencing verification.

[0075] (2) Construction of pTarget-Ptrc-glyA plasmid: Using the genome of Zymomonas mobilis as a template, glyA-up-F and glyA-up-R were used as primers to amplify glyA-up, glyA-F and glyA-R were used as primers to amplify Ptrc-glyA, glyA-down-F and glyA-down-R were used as primers to amplify glyA-down, pT-glyA plasmid was amplified using PT-line-F and PT-line-R as primers to obtain pTarget-line-glyA, and the DNA fragment was recovered using a Clean up kit; pTarget-line-glyA, glyA-up, Ptrc-glyA and glyA-down were connected together according to the instruction manual of Onestep clone kit (Vazyme Biotech, Nanjing, China), and the pTarget-Ptrc-glyA plasmid was obtained by sequencing verification.

[0076] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the SA-D5 competent cell obtained in Example 6 (5). The preparation method of SA-D5 competent cell was the same as that of Example 2 (3).

[0077] (4) SA-D6 positive colonies were constructed using the same method as in Example 2 (4).

[0078] (5) Plasmid elimination: The implementation method is the same as Example 2 (5) to obtain plasmid-free SA-D6.

[0079] (6) The constructed SA-D6 engineered strain was used as a control group with the SA-D5 constructed in Example 6, and shake flask tests and detection were performed according to the method of Example 2 (6). 600 and the D-pantothenic acid content in the fermentation supernatant. Figure 7 shown.

[0080] As shown in the figure, overexpression of glyA in the SA-D5 genome increased the cell OD 600 It increased slightly from 4.19 to 4.23. Considering the consumption of L-serine and the generation of L-glycine, the toxicity of amino acids in cells was reduced, and the titer of D-pantothenic acid increased from 4.77g / L to 4.86g / L, proving that the excessive supply of 5,10-methylenetetrahydrofolate greatly increased the activity of PanB. From the glyA gene alone, it converts L-serine into L-glycine and releases 5,10-methylenetetrahydrofolate. Although L-glycine can also decompose to generate 5,10-methylenetetrahydrofolate, the environmental harm caused by the generated NH3 and CO2 far outweighs the benefits.

[0081] Example 8 Construction and shake flask fermentation of SA-D7 (SA-D6 derivative, yjiV::Ptrc-panE) Using SA-D6 as the starting strain, CRISPR-Cas9-mediated gene editing technology was used to replace the original promoter of the panE gene with the Trc promoter to enhance the expression of the panE gene. The panE gene connected to the Trc promoter was then used to replace the in situ yjiV gene on the genome of the engineered bacterium SA-D6, thereby promoting the catalytic synthesis of D-pantoic acid by ketopantoic acid.

[0082] (1) Construction of pT-yjiV plasmid: pTarget F plasmid (Addgene Plasmid #62226) was used as a template and yjiV-sgRNA-F / yjiV-sgRNA-R as primers for PCR amplification. The PCR product was digested with Dpn I at 37°C for 3 h and then transformed into E. coli DH5α. The plasmid was screened on a spectinomycin plate and sequenced to verify the correct pT-yjiV plasmid, which was used for subsequent ligation of yjiV-up, Ptrc-panE, and yjiV-down.

[0083] (2) Construction of pTarget-Ptrc-panE plasmid: Using the genome of Zymomonas mobilis as a template, yjiV-up was amplified using primers yjiV-up-F and yjiV-up-R, Ptrc-panE was amplified using primers panE-F and panE-R, yjiV-down was amplified using primers yjiV-down-F and yjiV-down-R, pT-yjiV plasmid was amplified using primers PT-line-F and PT-line-R to obtain pTarget-line-yjiV, and the DNA fragment was recovered using a Clean up kit; pTarget-line-yjiV, yjiV-up, Ptrc-panE and yjiV-down were connected together according to the instruction manual of Onestep cloning kit (Vazyme Biotech, Nanjing, China), and the pTarget-Ptrc-panE plasmid was obtained by sequencing verification.

[0084] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the SA-P6 competent cell obtained in Example 7 (5). The preparation method of SA-P6 competent cell was the same as that of Example 2 (3).

[0085] (4) SA-D7 positive colonies were constructed using the same method as in Example 2 (4).

[0086] (5) Plasmid elimination: The implementation method is the same as Example 2 (5) to obtain plasmid-free SA-D7.

[0087] (6) The constructed SA-D7 engineered strain was used as a control group with the SA-D6 constructed in Example 7, and the shake flask test and detection were carried out according to the method of Example 2 (6). 600 and the D-pantothenic acid content in the fermentation supernatant. Figure 8 shown.

[0088] As can be seen from the figure, overexpression of panE in the SA-D6 genome increased the cell OD 600The expression of D-pantothenic acid was almost unchanged, while the titer of D-pantothenic acid increased from 4.86 g / L to 4.93 g / L, and the yield increased slightly. Similar to ilvC, PanE promotes the catalytic synthesis of D-pantothenic acid from ketopantoic acid and requires the participation of NADPH, and overexpression will have a certain negative impact on cell growth. Therefore, only one copy is added to SA-D6 as a subsequent step to accumulate D-pantothenic acid and synthesize D-pantothenic acid.

[0089] Example 9SA-D8 (SA-D7 derivative, P WT -panC::Ptrc-panC T*A ) was constructed and fermented in shake flasks with SA-D8 as the starting strain. CRISPR-Cas9-mediated gene editing technology was used to replace the original gene panC on the genome of strain SA-D8 by the panC gene regulated by the Trc promoter of pTrc99A through the gene knockout method. At the same time, the initial codon of panC was changed from TTG to ATG, which increased the expression level of PanC and promoted the condensation of D-pantothenic acid and β-alanine in the last step of the D-PA synthesis pathway to form D-pantothenic acid.

[0090] (1) Construction of pT-panC plasmid: pTarget F plasmid (Addgene Plasmid #62226) was used as a template and panC-sgRNA-F / panC-sgRNA-R as primers for PCR amplification. The PCR product was digested with Dpn I at 37°C for 3 h and then transformed into E. coli DH5α. The plasmid was screened on a spectaclease plate and sequenced to verify the correct pT-panC plasmid for subsequent ligation of panC-up, Ptrc-panC T*A and panC-down.

[0091] (2) Construction of pTarget-Ptrc-panC T*A Plasmid: Using the E. coli W3110 genome as a template, panC-up-F and panC-up-R were used as primers to amplify panC-up, and panC-F and panC-R were used as primers to amplify Ptrc-panC T*A , panC-down-F and panC-down-R were used as primers to amplify panC3-down, pT-panC plasmid was amplified with PT-line-F and PT-line-R as primers to obtain pTarget-line-panC, and the Clean up kit was used to recover the DNA fragments; according to (One step cloning kit, Vazyme Biotech, Nanjing, China) instructions to pTarget-line-panC, panC-up, Ptrc-panC T*A and panC-down, and the pTarget-Ptrc-panC was obtained by sequencing verification. T*A Plasmid.

[0092] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the SA-P7 competent cell obtained in Example 8 (5). The SA-D7 competent cell was prepared in the same manner as in Example 2 (3).

[0093] (4) SA-D8 positive colonies were constructed using the same method as in Example 2 (4).

[0094] (5) Plasmid elimination: The implementation method is the same as Example 2 (5) to obtain plasmid-free SA-D8.

[0095] (6) The constructed SA-D8 engineered strain was used as a control group with the SA-D7 constructed in Example 8, and shake flask tests and detection were performed according to the method of Example 2 (6). 600 and the D-pantothenic acid content in the fermentation supernatant. Fig. 9 shown.

[0096] As shown in the figure, panC was overexpressed in the SA-D7 genome, and the cell OD 600 From 4.25 to 4.36, the growth was further improved due to the synthesis of D-PA, and the D-pantothenic acid titer increased from 4.93g / L to 5.10g / L. PanC, as the last enzyme in the D-PA synthesis pathway, plays a role in condensing D-pantothenic acid and β-alanine. Therefore, whether its expression level is sufficient determines whether D-pantothenic acid can be converted to D-pantothenic acid to the greatest extent. In addition, since PanC requires a portion of ATP as a cofactor donor, although it may reduce the cell growth rate, the synthesized D-pantothenic acid can promote the recovery of some cells and make up for this defect, which also allows the strain to gradually return to the growth state of the chassis strain.

[0097] Example 10 Construction and shake flask fermentation of SA-D9 (SA-D8 derivative, Δamn) Using SA-D8 as the starting strain, CRISPR-Cas9-mediated gene editing technology was used to knock out the Amn gene encoding the nucleotide degrading enzyme on the genome of the strain SA-D8 through a gene knockout method, thereby making ATP less likely to be degraded into ADP and AMP, and accumulating the cofactors required for PanC.

[0098] (7) Construction of pT-amn plasmid: pTarget F plasmid (Addgene Plasmid #62226) was used as a template and amn-sgRNA-F / amn-sgRNA-R as primers for PCR amplification. The PCR product was digested with Dpn I at 37°C for 3 h and then transformed into E. coli DH5α. The plasmid was screened on a spectinomycin plate and sequenced to verify the correct pT-amn plasmid for subsequent connection of amn-up and amn-down.

[0099] (8) Construction of pTarget-amn plasmid: Using the genome of Zymomonas mobilis as a template, amn-up-F and amn-up-R were used as primers to amplify amn-up and amn-R were used as primers to amplify pta, amn-down-F and amn-down-R were used as primers to amplify amn-down, and pT-amn plasmid was amplified using PT-line-F and PT-line-R as primers to obtain pTarget-line-amn. The DNA fragments were recovered using a Clean up kit; pTarget-line-amn, amn-up and amn-down were connected together according to the instruction manual of the kit (One step cloning kit, Vazyme Biotech, Nanjing, China), and the pTarget-amn plasmid was obtained by sequencing verification.

[0100] (9) The pCas plasmid (Addgene Plasmid #62225) was introduced into the SA-D8 competent cell obtained in Example 9 (5). The preparation method of SA-D8 competent cell was the same as that of Example 2 (3).

[0101] (10) SA-D9 positive colonies were constructed using the same method as in Example 2 (4).

[0102] (11) Plasmid elimination: The implementation method is the same as Example 2 (5) to obtain plasmid-free SA-D9.

[0103] (12) The constructed SA-D9 engineered strain was used as a control group with the SA-P8 constructed in Example 9, and shake flask tests and detection were performed according to the method of Example 2 (6). 600 and the D-pantothenic acid content in the fermentation supernatant. Fig.10 shown.

[0104] As can be seen from the figure, in the SA-D8 genome, amn was knocked out and the cell OD 600There was almost no change, while the D-pantothenic acid titer increased from 5.10 g / L to the final 5.17 g / L, and the D-pantothenic acid production increased significantly. Although amn increases the ATP content and has a certain function in the sustained effect of PanC, the reduction of ADP and AMP may lead to a reduction in nucleotides, and under the global regulation of the cell, OD 600 From the perspective of the potency of D-PA, SA-D9 achieves overexpression of the entire D-PA synthesis pathway and the supply of cofactors, and achieves multi-module transformation to enhance the promotion of D-PA main pathway metabolism. Therefore, the present invention achieves high D-PA production by optimizing metabolism through the main pathway alone.

[0105] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0106] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A genetically engineered bacterium that produces high levels of D-pantothenic acid, characterized in that: The genome of the engineered bacteria includes: Strengthen the expression ilvB Genes, and ilvN The amino acid glycine at position 20 of the gene-encoded protein mutated to aspartic acid, the amino acid valine at position 21 mutated to aspartic acid, and the methionine at position 22 mutated to phenylalanine; Strengthen the expression ilvC Genes, and ilvC The amino acid leucine at position 67 of the gene-encoded protein mutated to glutamic acid, the amino acid arginine at position 68 mutated to phenylalanine, and the lysine at position 75 mutated to glutamic acid; Strengthen the expression ilvD Gene; Strengthen the expression panB Gene; Strengthen the expression serABC Gene; Strengthen the expression glyA Gene; Strengthen the expression panE Gene; Strengthen the expression panC gene, and the panC The start codon of the gene was replaced with ATG; Knockout amn Gene.

2. A method for constructing a genetically engineered bacterium that produces high levels of D-pantothenic acid, characterized in that: The following steps are involved: (1) Enhanced expression in chassis bacteria ilvB gene, and ilvB In the genes ilv Gene mutation, ilvN The amino acid glycine at position 20 of the gene-encoded protein mutated to aspartic acid, the amino acid valine at position 21 mutated to aspartic acid, and the methionine at position 22 mutated to phenylalanine; (2) Enhanced expression in chassis bacteria ilvC gene, and ilvC Gene mutation, ilvC The amino acid leucine at position 67 of the gene-encoded protein mutated to glutamic acid, the amino acid arginine at position 68 mutated to phenylalanine, and the lysine at position 75 mutated to glutamic acid; (3) Enhanced expression in chassis bacteria ilvD Gene; (4) Enhanced expression in chassis bacteria panB Gene; (5) Enhanced expression in chassis bacteria serABC Gene; (6) Enhanced expression in chassis bacteria glyA Gene; (7) Enhanced expression in chassis bacteria panE Gene; (8) Enhanced expression in chassis bacteria panC gene, and the panC The start codon of the gene was replaced with ATG; (9) Knockout in chassis bacteria amn Gene.

3. The construction method according to claim 2, characterized in that: The bottom plate bacteria is Escherichia coli.

4. The construction method according to claim 3, characterized in that: The genotype of the bottom plate bacteria is: E. coli W3110derivative, Trc- panCpanEpanB / ilvC / ilvG * / Δ aV / ilvE * / a * / △ poxB / Δ pta / Δ pf / Δ oeLh / Trc- pykA / ilv * / iV * / spoT * / Trc- spoT * / Trc- lpd / Trc- ilvD / Δ lacI / Trc- aL / Trc- ilvD / ptsG ::Trc- glk , galP .

5. The construction method according to claim 3 or 4, characterized in that: In step (1), the strengthening expression is: ilvB After the original promoter of the gene was replaced with the Trc promoter, the in situ ilvB Operon of genes.

6. The construction method according to claim 3 or 4, characterized in that: In step (2), step (3), step (4), and step (6), the enhanced expression is: replacing the original promoter of the gene to be enhanced with the Trc promoter, and then replacing the corresponding in situ gene in the genome of the chassis bacteria; In step (5) and step (7), the enhanced expression is as follows: after replacing the original promoter of the gene to be enhanced with the Trc promoter, the pseudogene in the in situ genome of the chassis bacteria is replaced.

7. The construction method according to claim 3 or 4, characterized in that: In step (4), the panB The gene is derived from Corynebacterium glutamicum.

8. The construction method according to claim 3 or 4, characterized in that: In step (1), the ilvN Gene mutation: ilvN The codon GGC at position 20 of the gene encoding the protein is changed to GAU, the codon GUA at position 21 is changed to GAC, and the codon AUG at position 22 is changed to UUC.

9. The construction method according to claim 3 or 4, characterized in that: In step (2), the ilvC Gene mutation: ilvC The codon CUG at position 67 of the gene encoding the protein was changed to GAA, the codon CGU at position 68 was changed to UUU, and the codon AAG at position 75 was changed to GAA.

10. Use of the genetically engineered bacterium according to claim 1, or the genetically engineered bacterium constructed by the construction method according to any one of claims 2 to 9 in the preparation of D-pantothenic acid.

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