Genetically engineered bacterium for high-yield production of d-pantothenic acid, and preparation method therefor and use thereof

Abstract

Provided are a genetically engineered bacterium for high-yield production of D-pantothenic acid (D-PA), and a preparation method therefor and a use thereof. The D-PA production yield of the engineered bacterium is significantly improved by means of the following actions: overexpressing acetolactate synthases IlvB and IlvN for pyruvate diversion while deleting a transcription attenuation region and mutating a valine feedback inhibition site; overexpressing ketol-acid reductoisomerase IlvC and dihydroxy acid dehydratase IlvD, and altering the coenzyme preference of IlvC from NADPH to NADH to unlock the upstream pathway for D-PA synthesis; overexpressing PanB and overexpressing a serine-glycine transport system to promote the synthesis of 5,10-methylenetetrahydrofolate; at the same time, enhancing the expression of ketopantoic acid reductase PanE; and finally overexpressing pantothenate synthetase PanC and increasing the content of cofactor ATP required by PanC. By means of the integrated enhancement of the above multiple modules, the provided engineered bacterium achieves improved cell growth due to balanced cofactors and increased D-PA.

Description

A genetically engineered bacterium that produces high levels of D-pantothenic acid, its preparation method, and its applications. Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and in particular to a genetically engineered bacterium that produces high levels of D-pantothenic acid, its preparation method, and its applications. Background Technology

[0002] Metabolic engineering aims to maximize the production of high-value-added compounds by using microorganisms as cellular factories. A key research focus in biosynthetic product production is how to construct these cellular factories, achieve high yields and conversion rates under effective regulation, and maximize carbon transfer to product synthesis while ensuring normal strain growth and minimizing carbon source waste. Furthermore, product yield and sugar conversion rate are standards for achieving industrial-scale production; therefore, methods that directly enhance product synthesis and improve product accumulation are among the primary operations in current metabolic engineering modifications.

[0003] Pantothenic acid (D-PA), also known as vitamin B5, is a component of coenzyme A and plays a crucial role in important biochemical reactions such as energy metabolism and the citric acid cycle. Currently, D-PA for industrial applications is mainly produced through a chemical method involving the condensation of D-pantolactone and β-alanine in methanol or ethanol. However, the esters produced using chemical separation or enzymatic catalysis involve toxic chemical reagents and expensive separation methods. With the continuous development of genetic engineering and synthetic biology, the production of D-PA using environmentally friendly, inexpensive, and simple biosynthetic methods has attracted widespread attention from scholars. However, current biological methods for producing D-PA still have drawbacks, such as unstable fermentation processes and low yields.

[0004] Escherichia coli, as an industrial microorganism, has advantages such as a clear genetic background and simple operation, making it one of the ideal substrates for most fermentation production processes. Summary of the Invention

[0005] Currently, biological methods for producing D-pantothenic acid suffer from low yields and unstable fermentation. To address these technical problems, this invention provides a genetically engineered bacterium that produces high levels of D-pantothenic acid, along with its preparation method and applications.

[0006] The specific technical solution of the present invention is as follows: Firstly, the present invention provides a genetically engineered bacterium that produces high levels of D-pantothenic acid. The genome of the engineered bacterium includes: a strongly expressed ilvBN gene, wherein the ilvN gene encodes a protein with amino acid mutated at position 20 (glycine) to aspartic acid, amino acid 21 (valine) to aspartic acid, and amino acid 22 (methionine) to phenylalanine; a strongly expressed ilvC gene, wherein the ilvC gene encodes a protein with amino acid mutated at position 67 (leucine) to glutamic acid, amino acid 68 (arginine) to phenylalanine, and amino acid 75 (lysine) to glutamic acid; a strongly expressed ilvD gene; a strongly expressed panB gene; a strongly expressed serABC gene; a strongly expressed glyA gene; a strongly expressed panE gene; a strongly expressed panC gene, wherein the start codon of the panC gene is replaced with ATG; and a knocked-out amn gene.

[0007] The engineered bacteria provided by this invention, compared with the chassis bacteria, showed a 68.1% increase in D-pantothenic acid titer during shake-flask fermentation, reaching 6.44 g / L. The engineered bacteria provided by this invention significantly increased D-PA production through the following mechanisms: overexpression of pyruvate-splitting acetolactate synthases IlvB and IlvN, while deleting the transcriptional attenuation region and mutating the valine feedback inhibition site; overexpression of keto-alcohol reductase IlvC and dihydroxy acid dehydratase IlvD, and changing the coenzyme preference of IlvC from NADPH to NADH, thus opening up the upstream pathway for D-PA synthesis; overexpression of PanB and the serine-glycine transport system to promote the synthesis of 5,10-methylenetetrahydrofolate; enhanced expression of keto-pantothenic acid reductase PanE; and finally, overexpression of pantothenic acid synthase PanC and increased ATP content required for PanC. The engineered bacteria provided by this invention, through the overall enhancement of the above-mentioned multi-module, have improved cell growth due to the balance of cofactors and the increase of D-PA, and the D-PA production has reached 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 a spore-forming bacterium, and mutating the ilvN gene in the ilvBN gene such that the amino acid glycine at position 20 of the ilvN gene-encoded protein is mutated to aspartic acid, the amino acid valine at position 21 is mutated to aspartic acid, and the amino acid methionine at position 22 is mutated to phenylalanine; (2) enhancing the expression of the ilvC gene in a spore-forming bacterium, and mutating the ilvC gene such that the amino acid leucine at position 67 of the ilvC gene-encoded protein 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) enhancing the expression of the ilvD gene in a spore-forming bacterium; (4) enhancing the expression of the panB gene in a spore-forming bacterium; (5) enhancing the expression of the serABC gene in a spore-forming bacterium; (6) enhancing the expression of the glyA gene in a spore-forming bacterium; (7) enhancing the expression of the panE gene in a spore-forming bacterium; (8) Enhance the expression of the panC gene in the sclerotium and replace the start codon of the panC gene with ATG; (9) Knock out the amn gene in the sclerotium.

[0009] The purpose of this invention is to utilize 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, thereby driving carbon flux to the D-PA main pathway. This achieves increased yield and conversion rate solely through overexpression of the product synthesis pathway, and demonstrates the application of this technology in the microbial fermentation preparation of D-pantothenic acid.

[0010] In the above construction method, the functions of each step are as follows: (1) Overexpressing pyruvate into the first gene cluster ilvBN in the D-PA main pathway, catalyzing the synthesis of acetolactate, and promoting the synthesis of D-pantothenic acid precursor D-pantolysinic acid; (2) Overexpressing ketool acid reductase IlvC, catalyzing the synthesis of isovaleric acid dihydroxy ester, and simultaneously modifying the cofactor dependence of IlvC, changing it from NADPH to NADH; (3) Overexpressing dihydroxy acid dehydratase IlvD, converting the generated isovaleric acid dihydroxy ester into α-ketoisovalerate; (4) Overexpressing ketopantolysinic acid hydroxymethyltransferase PanB, converting α-ketoisovalerate into (5) Overexpression of phosphoglycerate dehydrogenases 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 ketopantoacid reductase PanE to catalyze the formation of pantothenic acid from ketopantoacid; (8) Overexpression of pantothenic acid synthase PanC to condense pantothenic acid with β-alanine to form D-pantothenic acid; (9) Knockout of AMP nucleoside enzyme Amn to reduce nucleotide degradation and retain ATP content to promote the D-PA main pathway.

[0011] Through the interaction of the above steps, this invention ultimately constructs a genetically engineered bacterium with excellent D-PA yield. This invention utilizes the entire D-PA overexpression pathway and its coenzyme cycle supply, significantly increasing D-PA production without significant inhibition of cell growth, providing a new approach for the industrial production of D-PA.

[0012] Preferably, the bacteria in the substrate are Escherichia coli.

[0013] Further preferred, the genotype of the *E. coli* strain is: *E. coli* W3110 derivative, *Trc-panCpanEpanB / ilvC / ilvG*. * / ΔavtA / ilvE * / coaA * / ΔpoxB / Δpta / ΔplfB / ΔldhA / Trc-pykA / ilvN* / ilvH* / 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 cytomegalovirus genome is replaced.

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

[0016] Preferably, in steps (2), (3), (4), and (6), the enhanced expression is: replacing the original promoter of the gene to be enhanced with a Trc promoter, and then replacing the corresponding in situ gene in the cytomegalovirus genome.

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

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

[0019] Preferably, in step (1), the ilvN gene is mutated by changing codon GGC at position 20 to GAU, codon GUA at position 21 to GAC, and codon AUG at position 22 to UUC.

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

[0021] Thirdly, the present invention provides the application of the genetically engineered bacteria constructed by the above-described 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 promotes the production of D-PA by the interaction of the following operations: (1) Overexpression of pyruvate enters the first gene cluster ilvBN in the main pathway of D-PA, catalyzes the synthesis of acetolactate, relieves the transcriptional attenuation inhibition of the ilvBN operon, and promotes the synthesis of D-pantothenic acid precursor D-pantothenic acid; (2) Overexpression of keto-alcoholic acid reductase IlvC, catalyzes the synthesis of isovaleric acid dihydroxy ester, and at the same time modifies the cofactor dependence of IlvC, changing it from NADPH to NADH; (3) Overexpression of dihydroxy acid dehydratase IlvD, converting the generated isovaleric acid dihydroxy ester into α-keto-isovalerate; (4) Overexpression of keto-pantothenic acid hydroxymethyltransferase PanB, converting α-keto-isovalerate into α-keto-isovalerate. (5) Overexpression of phosphoglycerate dehydrogenases SerA, B, and C of the serine-glycine transport system and serine hydroxymethyltransferase GlyA promotes the synthesis of 5,10-methylenetetrahydrofolate; (6) Overexpression of ketopantoacid reductase PanE catalyzes the generation of pantothenic acid from ketopantoacid; (7) Overexpression of pantothenic acid synthase PanC condenses pantothenic acid with β-alanine to form D-pantothenic acid; (8) Knockout of AMP nucleoside enzyme Amn reduces nucleotide degradation and preserves ATP content to promote the D-PA main pathway. This invention utilizes the entire D-PA pathway and its coenzyme cycle supply through overexpression to greatly increase D-PA production without significant inhibition of cell growth, providing a new approach for the industrial production of D-PA. Attached Figure Description

[0023] Figure 1 is a schematic diagram of the D-pantothenic acid metabolic pathway; Figure 2 is the OD of SA-D1. 600 And changes in D-pantothenic acid potency; Figure 3 shows the OD of SA-D2. 600 And changes in D-pantothenic acid potency; Figure 4 shows the OD of SA-D3. 600 And changes in D-pantothenic acid potency; Figure 5 shows the OD of SA-D4. 600 And changes in D-pantothenic acid potency; Figure 6 shows the OD of SA-D5. 600 And changes in D-pantothenic acid potency; Figure 7 shows the OD of SA-D6. 600 And changes in D-pantothenic acid potency; Figure 8 shows the OD of SA-D7. 600 And changes in D-pantothenic acid potency; Figure 9 shows the OD of SA-D8.600 And changes in D-pantothenic acid potency; Figure 10 shows the OD of SA-D9. 600 And changes in D-pantothenic acid potency. Detailed Implementation

[0024] The present invention will be further described below with reference to embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

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

[0026] Table 1. Genes involved in gene editing and corresponding pathways. The primer sequence information involved in the embodiments of the present invention is shown in Table 2.

[0027] Table 2 Primer sequences In this embodiment of the 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 this invention was deposited at the Coli Genetic Stock Center (CGSC) of Yale University on August 5, 1975, with accession number CGSC#4474, and has been disclosed in patents US2009 / 0298135A1 and US2010 / 0248311A1.

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

[0030] Example 2 SA-D1 (DPA-SA derivative, P) WT -ilvBN::Ptrc-ilvBN DDF Construction and shake-flask fermentation of DPA-SA (E. coli W3110 derivative, Trc-panCpanEpanB / ilvC / ilvG) * / ΔavtA / ilvE * / coaA * Using strain ( / △poxB / Δpta / ΔplfB / ΔldhA / Trc-pykA / ilvN* / ilvH* / spoT* / Trc-spoT* / Trc-lpd / Trc-ilvD / ΔlacI / Trc-alaS / Trc-ilvD / ptsG::Trc-glk,galP) as the starting strain, CRISPR-Cas9-mediated gene editing technology was used. Through gene knockout, the original promoter of the ilvBN gene was replaced with the Trc promoter, enhancing ilvBN gene expression. Then, the operon of the in situ ilvBN gene on the genome of the engineered strain DPA-SA was replaced with the ilvBN gene linked to the Trc promoter. Simultaneously, the G20D V21D M22F site of the ilvN gene was mutated, resulting in 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 lactate was enhanced. Specifically, the mutations in the ilvN gene were: codon 20 GGC changed to GAU, codon 21 GUA changed to GAC, and codon 22 AUG changed to UUC.

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

[0032] (2) Construct pTarget-Ptrc-ilvBN DDFPlasmids: Using the genome of the D-PA engineered 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, and ilvBN-down was amplified with primers ilvBN-down-F and ilvBN-down-R to obtain ilvBN-down. The pTarget-ilvBN plasmid was amplified with primers PT-line-F and PT-line-R to obtain pTarget-line-ilvBN. DNA fragments were recovered using a Clean up kit; according to... The (One-step clone kit, Vazyme Biotech, Nanjing, China) manual links pTarget-line-ilvBN, ilvBN-up, and ilvBN-down together, and the pTarget-Ptrc-ilvBN was verified by sequencing. DDF Plasmid.

[0033] (3) The pCas plasmid (Addgene Plasmid#62225) was introduced into DPA-SA, and a single clone was transferred into an LB tube containing 0.05 mg / L kanamycin and cultured overnight at 30°C; then, 1% (v / v) was inoculated into a 250 mL shake flask containing 50 mL of LB medium, and 500 μl of 1 mol / L L-arabinose was added. The mixture was cultured at 150 rpm and 30°C until OD500. 600 Cells were collected by centrifugation at 4000 rpm and 4℃ for 10 min to prepare electrocompetent cells.

[0034] (4) Mix 200 ng of pTarget-pfkB plasmid with 100 μL of electroporation competent cells, transfer to a pre-cooled 2 mm electroporation cuvette, incubate on ice for 1 min, and then electroporate using a MicroPluser. TM The cells were transformed by electroporation using BIO-RAD. Immediately after electroporation, 1 mL of LB medium was added and gently aspirated, then transferred to a 1.5 mL centrifuge tube. After recovery at 30°C for 2-3 hours, the cells were plated onto LB plates containing 0.05 mg / L kanamycin and 0.05 mg / L spectinomycin. The plates were incubated upside down at 37°C for 12-16 hours. Colony PCR was performed using pfkB-VF and pfkB-VR primers for verification. If a fragment of about 1500 bp could be successfully cloned, it would prove that the colony was a DPA-SA derivative, ΔpfkB positive.

[0035] (5) Plasmid elimination: Positive single colonies were picked and inoculated into LB tubes containing 1 mM IPTG and 0.05 mg / L kanamycin, and incubated overnight at 30°C. The next day, the bacterial culture was streaked onto LB agar containing 0.05 mg / L kanamycin and incubated at 30°C for 24 h. Single colonies were picked and streaked onto LB agar containing 0.05 mg / L spectinomycin. The pTarget-pfkB plasmid of single colonies that could not be eliminated on LB agar containing 0.05 mg / L spectinomycin was eliminated. Single colonies with successful elimination of pTarget-pfkB plasmid were picked into LB tubes and incubated overnight at 37°C. The next day, the bacterial culture was streaked onto LB agar and incubated at 37°C for 12 h. Single colonies were picked and streaked onto LB agar containing 0.05 mg / L kanamycin. The pCas plasmid of single colonies that could not be eliminated on LB agar containing 0.05 mg / L kanamycin was eliminated. Finally, plasmid-free DPA-SA derivative, ΔpfkB was obtained.

[0036] (6) DPA-SA derivative, P WT -ilvBN::Ptrc-ilvBN DDF Using DPA-SA as the control group, 10 mL of each culture was inoculated into LB medium and cultured at 37℃ and 200 rpm as a pre-culture. After 8-12 h, 1 mL of the pre-culture was inoculated into a 500 mL shake flask containing 50 mL of MS medium, and then fermented in a constant temperature shaker at 30℃ and 200 rpm for 48 h. After fermentation, 1 mL of the fermentation broth was taken to determine the OD. 600 Take 1 mL of fermentation broth, centrifuge at 12000 rpm for 3 min at room temperature, dilute the fermentation supernatant 10 times, and perform HPLC analysis according to Example 1. OD 600 The D-pantothenic acid content in the fermentation broth supernatant is shown in Figure 2.

[0037] As shown in Figure 2, overexpression of the ilvBN gene cluster in the DPA-SA genome has a certain inhibitory effect on cell growth, and cell OD... 600 The D-PA titer decreased from 4.23 to 4.16. This was because the promoter regulatory region of the ilvBN operon may bind to IHF and regulate the expression level of genes within the operon. Deleting this region can improve the expression of this gene cluster to some extent. The IlvN mutation site, which resists valine feedback inhibition, was also introduced into this gene cluster, thus ultimately making the D-PA titer reach 4.29 g / L.

[0038] The LB medium consisted of 10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl, with deionized water as the solvent and a natural pH value.

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

[0040] Example 3 SA-D2 (SA-D1 derivative, P) WT -ilvC::Ptrc-ilvC EFE Construction and shake-flask fermentation: Using SA-D1 as the starting strain, CRISPR-Cas9-mediated gene editing technology was employed. The original promoter of the ilvC gene was replaced with a Trc promoter to enhance ilvC gene expression. Then, the in situ ilvC gene on the engineered SA-D1 genome was replaced with an ilvC gene linked to the Trc promoter. Simultaneously, a mutation was performed at the L67E R68F K75E site of the ilvC gene, resulting in Ptrc-ilvC. EFE The cofactor preference shifted from NADPH to NADH, reducing the excessive consumption of reducing power H during bacterial growth, balancing the NADPH / NADH ratio, and benefiting cell growth and metabolism. Specifically, the ilvC gene underwent the following mutations: codon CUG at position 67 changed to GAA, codon CGU at position 68 changed to UUU, and codon AAG at position 75 changed to GAA.

[0041] (1) Construction of pT-ilvC plasmid: Using pTarget F plasmid (Addgene Plasmid#62226) as a template, PCR amplification was performed using ilvC-sgRNA-F / ilvC-sgRNA-R as primers. The PCR product was digested with Dpn I at 37℃ for 3 h, and then transformed into E.coli DH5α. The plasmid was screened by spectrozin plate and sequenced to verify that the correct pT-ilvC plasmid was obtained, which was used for subsequent ligation of ilvC-up and ilvC-down.

[0042] (2) Construct pTarget-Ptrc-ilvC EFEPlasmids: Using the genome of the D-PA engineered strain DPAN16 produced in the original laboratory as a template, ilvC-up was amplified using ilvC-up-F and ilvC-up-R primers to obtain ilvC-up, and ilvC-down was amplified using ilvC-down-F and ilvC-down-R primers to obtain ilvC-down. The pT-ilvC plasmid was amplified using PT-line-F and PT-line-R primers to obtain pTarget-line-ilvC. DNA fragments were recovered using a Clean up kit; according to... The (One-step clone kit, Vazyme Biotech, Nanjing, China) manual links pTarget-line-ilvC, ilvC-up, and ilvC-down together, and verifies the result using sequencing to obtain pTarget-Ptrc-ilvC. EFE Plasmid.

[0043] (3) The pCas plasmid (Addgene Plasmid#62225) was introduced into the SA-D1 competent cells obtained in Example 2 (5). The SA-D1 competent cells were prepared in the same way as 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 in Example 2(5), and plasmid-free SA-D2 is obtained.

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

[0047] As shown in the figure, mutation and overexpression of ilvC in the SA-D1 genome leads to increased cellular OD. 600 The D-pantothenic acid titer decreased from 4.16 to 4.10, and then remained almost unchanged at 4.33. This indicates that the mutant ilvC gene is closely related to D-PA production and cell growth. Both deficiency and excess will negatively inhibit the production of the strain. Therefore, considering the feasibility of subsequent strategies, since the ilvC gene has been overexpressed to saturation, this step will be retained and the gene will not be modified again. Furthermore, the growth rate of the strain will be gradually increased to ensure sufficient cell volume for D-PA production.

[0048] Example 4 SA-D3 (SA-D2 derivative, P) WTThe construction and shake-flask fermentation of -ilvD::Ptrc-ilvD) started with SA-D2 as the starting strain. Using CRISPR-Cas9-mediated gene editing technology, the original promoter of the ilvD gene was replaced with the Trc promoter to enhance the expression of the ilvD gene. Then, the in situ ilvD gene on the genome of the engineered strain SA-D2 was replaced with the ilvD gene linked with the Trc promoter. The dihydroxy acid dehydratase IlvD is the last enzyme upstream of the D-PA synthesis pathway. It converts the generated isovaleric acid dihydroxy ester into α-ketoisovalerate, which is then transferred to the downstream pathway.

[0049] (1) Construction of pT-ilvD plasmid: Using pTarget F plasmid (Addgene Plasmid#62226) as a template, PCR amplification was performed using ilvD-sgRNA-F / ilvD-sgRNA-R as primers. The PCR product was digested with Dpn I at 37℃ for 3 h, and then transformed into E.coli DH5α. The plasmid was screened by spectrozin plate and sequenced to verify that the correct pT-ilvD plasmid was obtained, which was then used 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 ilvD-up-F and ilvD-up-R primers to obtain ilvD-up, and ilvD-down was amplified using ilvD-down-F and ilvD-down-R primers to obtain ilvD-down. The pT-ilvD plasmid was amplified using PT-line-F and PT-line-R primers to obtain pTarget-line-ilvD. DNA fragments were recovered using a Clean up kit; according to... The instructions for the (One-step clone kit, Vazyme Biotech, Nanjing, China) link pTarget-line-ilvD, ilvD-up, and ilvD-down together, and the pTarget-Ptrc-ilvD plasmid is 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 SA-D2 competent cells were prepared in the same way as in 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 method is the same as in Example 2(5) to obtain plasmid-free SA-D3.

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

[0055] As shown in Figure 4, overexpression of the ilvD gene in the SA-D2 genome leads to the introduction of carbon metabolic flux into the D-PA synthesis pathway, and similarly, cellular OD... 600 No significant changes were observed, but the D-pantothenic acid (DPA) titer increased from 4.33 g / L to 4.48 g / L. The combined action of acetolactate synthase and dihydroxy acid dehydratase greatly enhanced the DPA titer, demonstrating that the upstream pathway has been successfully established. However, the current growth rate is too slow, which has adverse effects on gene manipulation and fermentation. Further optimization of the downstream pathway will be conducted to address this issue.

[0056] In this embodiment, SA-D1 was used 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. Then, the in situ ilvD gene on the genome of the engineered strain SA-D2 was replaced with the ilvD gene linked to the Trc promoter to obtain the control strain SA-VSD3. The specific steps are as described in (1) to (6) for the construction of SA-D3. The results showed that the D-pantothenic acid titer of SA-VSD3 was 4.34 g / L, which was not effectively improved compared to 4.29 g / L of SA-D1. However, the D-pantothenic acid titer of SA-D3 was increased from 4.29 g / L to 4.48 g / L compared to SA-D1. This illustrates the importance of the enhanced expression and mutation of the ilvC gene in the construction of SA-D2 in this embodiment.

[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 from Corynebacterium glutamicum with the Trc promoter through gene knockout, thereby enhancing the expression of the panB gene. Then, the panB gene linked to the Trc promoter was used to replace the in situ panB gene on the genome of the engineered strain SA-D3, catalyzing the synthesis of downstream D-PA pathway and maximizing the diversion of α-ketoisovalerate carbon flux.

[0058] (1) Construction of pT-EcpanB plasmid: Using pTarget F plasmid (Addgene Plasmid#62226) as a template, PCR amplification was performed using EcpanB-sgRNA-F / EcpanB-sgRNA-R as primers. The PCR product was digested with Dpn I at 37℃ for 3 h, and then transformed into E.coli DH5α. The plasmid was screened with spectrozin plates and sequenced to verify that the correct pT-EcpanB plasmid was obtained, 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 was obtained by amplification with primers EcpanB-up-F and EcpanB-up-R, EcpanB-down was obtained by amplification with primers EcpanB-down-F and EcpanB-down-R, and Ptrc-CgpanB was obtained by amplification with primers CgpanB-F and CgpanB-R. pTarget-line-EcpanB was obtained by amplification of the pT-EcpanB plasmid using primers PT-line-F and PT-line-R. DNA fragments were recovered using a Clean up kit; according to... The (One-step clone kit, Vazyme Biotech, Nanjing, China) manual links pTarget-line-EcpanB, EcpanB-up, EcpanB-down, and Ptrc-CgpanB together, and the pTarget-Ptrc-CgpanB plasmid is obtained by sequencing verification.

[0060] (3) The pCas plasmid (Addgene Plasmid#62225) was introduced into the SA-D3 competent cells obtained in Example 4 (5). The SA-D3 competent cells were prepared in the same way as in 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 in Example 2(5), and plasmid-free SA-D4 is obtained.

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

[0064] As shown in the figure, overexpression of Ptrc-CgpanB in the SA-D4 genome reduces cellular OD. 600 PanB, a key enzyme in the entire D-PA synthesis pathway, has increased from 4.09 to 4.26. Reports have shown that overexpression of the panB gene from Corynebacterium glutamicum can effectively promote bacterial growth and increase D-PA titer. Currently, the D-pantothenic acid titer has increased from 4.48 g / L to 4.71 g / L, which undoubtedly lays the foundation for subsequent modifications. Considering that the full efficacy of PanB requires the assistance 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, CRISPR-Cas9-mediated gene editing technology was used to link the Trc promoter to the serABC gene cluster, making the Trc promoter the promoter of the serABC gene cluster. Then, the yeep pseudogene on the genome of the engineered strain SA-D4 was replaced with the serABC gene cluster linked to the Trc promoter, 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 was used as primers for PCR amplification. The PCR product was digested with Dpn I at 37℃ for 3 h and then transformed into E.coli DH5α. The plasmid was screened with spectrozin plate and sequenced to verify that the correct pT-yeep plasmid was obtained, which was used for subsequent ligation of yeep-up / yeep-down.

[0067] (2) Construction of pTarget-Ptrc-serABC plasmid: Using the E. coli W3110 genome as a template, yeep-up was obtained by amplification with yeep-up-F and yeep-up-R primers, yeep-down was obtained by amplification with yeep-down-F and yeep-down-R primers, and Ptrc-serABC was obtained by amplification with serABC-F and serABC-R primers. pTarget-line-yeep was obtained by amplification of the pT-yeep plasmid with PT-line-F and PT-line-R primers. DNA fragments were recovered using a Clean up kit; according to... The instructions for the (One-step clone kit, Vazyme Biotech, Nanjing, China) link pTarget-line-yeep, yeep-up, yeep-down, and Ptrc-serABC together, and the pTarget-Ptrc-serABC plasmid is obtained by sequencing verification.

[0068] (3) The pCas plasmid (Addgene Plasmid#62225) was introduced into the SA-D4 competent cells obtained in Example 5 (5). The SA-D4 competent cells were prepared in the same way as in 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 in Example 2 (5), and plasmid-free SA-D5 is obtained.

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

[0072] As shown in the figure, overexpression of the serABC gene cluster in the SA-D4 genome leads to increased cellular OD. 600 The decrease from 4.26 to 4.19 indicates that the overexpression of L-serine led to uneven amino acid distribution within the bacteria, which also indirectly proves that L-serine has a certain toxic effect on cell growth. Although the increase in D-pantothenic acid titer from 4.71 g / L to 4.77 g / L was not significant, it also proves that the 5,10-methylenetetrahydrofolate produced by the intracellular serine-glycine transport system alone is insufficient to meet the needs 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 The construction and shake-flask fermentation of (-glyA::Ptrc-glyA) started with SA-D5 as the starting strain. Using CRISPR-Cas9-mediated gene editing technology, the in situ glyA gene on the genome of the engineered strain SA-D5 was replaced with the glyA gene carrying the Trc promoter, which promoted the conversion of L-serine to L-glycine and the conversion of tetrahydrofolate to 5,10-methylenetetrahydrofolate.

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

[0075] (2) Construction of pTarget-Ptrc-glyA plasmid: Using the genome of *M. motile fermentum* as a template, glyA-up was obtained by amplification with primers glyA-up-F and glyA-up-R, Ptrc-glyA was obtained by amplification with primers glyA-F and glyA-R, glyA-down was obtained by amplification with primers glyA-down-F and glyA-down-R, and pTarget-line-glyA was obtained by amplification of pT-glyA plasmid with primers PT-line-F and PT-line-R. DNA fragments were recovered using a Clean up kit; according to... (One-step clone kit, Vazyme Biotech, Nanjing, China) The instructions link pTarget-line-glyA, glyA-up, Ptrc-glyA, and glyA-down together, and the pTarget-Ptrc-glyA plasmid is obtained by sequencing verification.

[0076] (3) The pCas plasmid (Addgene Plasmid#62225) was introduced into the SA-D5 competent cells obtained in Example 6 (5). The SA-D5 competent cells were prepared in the same way as in 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 in Example 2 (5), and plasmid-free SA-D6 is obtained.

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

[0080] As shown in the figure, overexpression of glyA in the SA-D5 genome leads to increased cellular OD. 600 The concentration of 5,10-methylenetetrahydrofolate (5,10-methylenetetrahydrofolate) increased slightly from 4.19 to 4.23. Considering the consumption of L-serine and the production of L-glycine, the intracellular amino acid toxicity decreased. The D-pantothenic acid titer increased from 4.77 g / L to 4.86 g / L, demonstrating that the excessive supply of 5,10-methylenetetrahydrofolate significantly increased PanB activity. Looking solely at the glyA gene, it converts L-serine to L-glycine while releasing 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. Then, the panE gene linked with the Trc promoter was used to replace the in situ yjiV gene on the genome of the engineered strain SA-D6, thereby promoting the synthesis of D-pantolytic acid catalyzed by ketopantolytic acid.

[0082] (1) Construction of pT-yjiV plasmid: Using pTarget F plasmid (Addgene Plasmid#62226) as a template, PCR amplification was performed using yjiV-sgRNA-F / yjiV-sgRNA-R as primers. The PCR product was digested with Dpn I at 37℃ for 3 h, and then transformed into E.coli DH5α. The plasmid was screened by spectrozin plate and sequenced to verify that the correct pT-yjiV plasmid was obtained, 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 *M. motile fermentum* as a template, yjiV-up was amplified using yjiV-up-F and yjiV-up-R primers, Ptrc-panE was amplified using panE-F and panE-R primers, yjiV-down was amplified using yjiV-down-F and yjiV-down-R primers, and pTarget-line-yjiV was amplified using PT-line-F and PT-line-R primers. DNA fragments were recovered using a Clean up kit; according to... The instructions for the (One-step clone kit, Vazyme Biotech, Nanjing, China) link pTarget-line-yjiV, yjiV-up, Ptrc-panE, and yjiV-down together, and the pTarget-Ptrc-panE plasmid is obtained by sequencing verification.

[0084] (3) The pCas plasmid (Addgene Plasmid#62225) was introduced into the SA-P6 competent cells obtained in Example 7 (5). The SA-P6 competent cells were prepared in the same way as in 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 in Example 2(5), and plasmid-free SA-D7 is obtained.

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

[0088] As shown in the figure, overexpression of panE in the SA-D6 genome leads to increased cellular OD. 600 Almost unchanged, while the D-pantothenic acid potency increased from 4.86 g / L to 4.93 g / L, resulting in a slight increase in yield. Similar to ilvC, PanE-catalyzed synthesis of D-pantothenic acid from ketopantothenic acid requires the participation of NADPH, and overexpression can have a certain negative impact on cell growth. Therefore, only one copy was added to SA-D6 as a preparatory step to accumulate D-pantothenic acid for D-pantothenic acid synthesis.

[0089] Example 9 SA-D8 (SA-D7 derivative, P) WT -panC::Ptrc-panC T*AThe construction and shake-flask fermentation of the strain SA-D8 were carried out using CRISPR-Cas9-mediated gene editing technology. The original panC gene on the SA-D8 genome was replaced by the panC gene, which is regulated by the pTrc99A Trc promoter, through gene knockout. 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 final step of the D-PA synthesis pathway, the condensation of D-pantothenic acid and β-alanine, to form D-pantothenic acid.

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

[0091] (2) Construct pTarget-Ptrc-panC T*A Plasmids: Using the E. coli W3110 genome as a template, panC-up was obtained by amplification with panC-up-F and panC-up-R primers, and Ptrc-panC was obtained by amplification with panC-F and panC-R primers. T*A panC3-down was amplified using panC-down-F and panC-down-R primers. pTarget-line-panC was amplified using pT-panC plasmid with PT-line-F and PT-line-R primers. DNA fragments were recovered using a Clean Up kit. (One-step clone kit, Vazyme Biotech, Nanjing, China) The instruction manual mentions pTarget-line-panC, panC-up, and Ptrc-panC. T*A Linked with panC-down, pTarget-Ptrc-panC was obtained through sequencing validation. T*A Plasmid.

[0092] (3) The pCas plasmid (Addgene Plasmid#62225) was introduced into the SA-P7 competent cells obtained in Example 8 (5). The SA-D7 competent cells were prepared in the same way 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 in Example 2 (5), and plasmid-free SA-D8 is obtained.

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

[0096] As shown in the figure, overexpression of panC in the SA-D7 genome leads to increased cellular OD. 600 The growth factor increased from 4.25 to 4.36, indicating a further improvement in D-PA synthesis, while the D-pantothenic acid titer increased from 4.93 g / L to 5.10 g / L. PanC, as the final enzyme in the D-PA synthesis pathway, plays a crucial role in the condensation of D-pantothenic acid and β-alanine. Therefore, its expression level determines whether D-pantothenic acid can be converted to D-pantothenic acid to the maximum extent. Furthermore, although PanC requires one ATP molecule as a cofactor donor, which may slightly reduce cell growth, the synthesized D-pantothenic acid can promote partial cell recovery, compensating for this deficiency. This allowed the strain to gradually recover to the growth state of the substrate 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, which encodes nucleotide degrading enzymes, in the SA-D8 genome. This made it difficult for ATP to be degraded into ADP and AMP, and accumulated the cofactors required by PanC.

[0098] (7) Construction of pT-amn plasmid: Using pTarget F plasmid (Addgene Plasmid#62226) as a template, PCR amplification was performed using amn-sgRNA-F / amn-sgRNA-R primers. The PCR product was digested with Dpn I at 37℃ for 3 h, and then transformed into E.coli DH5α. The plasmid was screened using spectrozin plates and sequenced to verify that the correct pT-amn plasmid was obtained, which was then used for subsequent ligation of amn-up and amn-down.

[0099] (8) Construction of pTarget-amn plasmid: Using the genome of *M. motile fermentum* as a template, amplification was performed using primers amn-up-F and amn-up-R to obtain amn-up, and amplification was performed using primers amn-r to obtain pta. Amplification was performed using primers amn-down-F and amn-down-R to obtain amn-down. The pT-amn plasmid was amplified using primers PT-line-F and PT-line-R to obtain pTarget-line-amn. DNA fragments were recovered using a Clean up kit; according to... The instructions for the (One-step clone kit, Vazyme Biotech, Nanjing, China) link pTarget-line-amn, amn-up, and amn-down together, and the pTarget-amn plasmid is obtained by sequencing verification.

[0100] (9) The pCas plasmid (Addgene Plasmid#62225) was introduced into the SA-D8 competent cells obtained in Example 9 (5). The SA-D8 competent cells were prepared in the same way as in 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 method is the same as in Example 2 (5) to obtain plasmid-free SA-D9.

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

[0104] As shown in the figure, over-knockout of amn in the SA-D8 genome reduces cellular OD. 600 Almost no change was observed, while the D-pantothenic acid titer increased from 5.10 g / L to a final 5.17 g / L, indicating a significant increase in D-pantothenic acid production. Although amn increased ATP levels and contributed to the sustained effect of PanC, the reduction in ADP and AMP may lead to a decrease in nucleotides, and under global cellular regulation, OD... 600 There were no significant changes. In terms of D-PA potency, SA-D9 achieved overexpression of the entire D-PA synthesis pathway and supply of cofactors, and achieved multi-module modification to enhance the metabolism of D-PA through the main pathway. Thus, this invention achieves high-yield D-PA production by optimizing metabolism through the main pathway alone.

[0105] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0106] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope 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: The expression of the ilvBN gene was enhanced, and the 20th amino acid glycine of the ilvN gene-encoded protein was mutated to aspartic acid, the 21st amino acid valine was mutated to aspartic acid, and the 22nd amino acid methionine was mutated to phenylalanine. The expression of the ilvC gene was enhanced, and the amino acid 67 leucine was mutated to glutamic acid, the amino acid 68 arginine was mutated to phenylalanine, and the amino acid 75 lysine was mutated to glutamic acid in the protein encoded by the ilvC gene. Enhanced expression of the ilvD gene; Enhanced expression of the panB gene; Enhanced expression of the serABC gene; Enhanced expression of the glyA gene; Enhanced expression of the panE gene; Enhanced expression of the panC gene, with the start codon of the panC gene replaced by ATG; Knock out the amn gene.

2. A method for constructing a genetically engineered bacterium that produces high levels of D-pantothenic acid, characterized in that: Includes the following steps: (1) Enhance the expression of the ilvBN gene in the ilvBN gene, and mutate the ilvN gene in the ilvBN gene so that the 20th amino acid glycine is mutated to aspartic acid, the 21st amino acid valine is mutated to aspartic acid and the 22nd amino acid methionine is mutated to phenylalanine. (2) Enhance the expression of the ilvC gene in the diatoms and mutate the ilvC gene so that the amino acid leucine at position 67 of the ilvC gene encoded protein 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 Chameleonella bacteria; (4) Enhance the expression of the panB gene in the scutellaria baicalensis; (5) Enhanced expression of the serABC gene in Chameleon bacteria; (6) Enhance the expression of the glyA gene in Chameleon bacteria; (7) Enhance the expression of the panE gene in Chameleonella; (8) Enhance the expression of the panC gene in the sclerotium, and replace the start codon of the panC gene with ATG; (9) Knock out the amn gene in the basal bacteria.

3. The construction method as described in claim 2, characterized in that: The bacteria on the chassis are Escherichia coli.

4. The construction method as described in claim 3, characterized in that: The genotype of the *E. coli* strain is: *E. coli* W3110 derivative. Trc-panCpanEpanB / ilvC / ilvG * / ΔavtA / ilvE * / coaA * / △poxB / Δpta / ΔplfB / ΔldhA / Trc-pykA / ilvN* / ilvH* / spoT* / Trc-spoT* / Trc-lpd / Trc-ilvD / ΔlacI / Trc-alaS / Trc-ilvD / ptsG::Trc-glk,galP。 5. The construction method as described in claim 3 or 4, characterized in that: 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 cytomegalovirus genome is replaced.

6. The construction method as described in claim 3 or 4, characterized in that: In steps (2), (3), (4), and (6), the enhanced expression is: replacing the original promoter of the gene to be enhanced with a Trc promoter, and then replacing the corresponding in situ gene in the cytomegalovirus genome; In steps (5) and (7), the enhanced expression is: replacing the original promoter of the gene to be enhanced with a Trc promoter, and then replacing the pseudogene in the in situ genome of the basal bacteria.

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

8. The construction method as described in claim 3 or 4, characterized in that: In step (1), the ilvN gene is mutated by changing codon GGC at position 20 to GAU, codon GUA at position 21 to GAC, and codon AUG at position 22 to UUC.

9. The construction method as described in claim 3 or 4, characterized in that: In step (2), the ilvC gene is mutated by changing the codon CUG at position 67 of the ilvC gene-encoded protein to GAA, the codon CGU at position 68 to UUU, and the codon AAG at position 75 to GAA.

10. The use of the genetically engineered bacteria as described in claim 1, or the genetically engineered bacteria 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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