Corynebacterium glutamicum strain capable of efficiently producing d-pantothenic acid, construction method therefor, and use thereof

Abstract

The present invention relates to the technical field of microbial metabolic engineering, and in particular to a Corynebacterium glutamicum strain capable of efficiently producing D-pantothenic acid, a construction method therefor, and the use thereof. The construction method specifically comprises the following steps: (1) using wild-type Corynebacterium glutamicum ATCC 13032 as a chassis, attenuating ilvA and performing mutation expression so as to construct DPA1; (2) knocking out pyruvate quinone oxidoreductase and inserting a panBCE gene expression cassette into same so as to construct DPA2; (3) introducing into the DPA2 an acetohydroxy acid synthase, an acetolactate isomeroreductase and a 2-hydroxyacid dehydratase which are overexpressed by using a pEC-xk99E overexpression plasmid and derived from Escherichia coli W3110 and Bacillus subtilis 168, and are endogenous and are obtained by screening; and (4) under the control of a strong promoter, subjecting the selected ilvBNCD gene to tandem overexpression by means of pEC-xk99E to construct DPA3, so that by enhancing the driving force in a principle synthesis pathway of pantothenic acid and upstream pathways, the Corynebacterium glutamicum strain capable of efficiently producing D-pantothenic acid is obtained. The strain provided by the present invention can use carbon sources to produce D-pantothenic acid with a high yield and high conversion rate.

Description

A highly efficient Corynebacterium glutamicum producing D-pantothenic acid, its construction method and application Technical Field

[0001] This invention relates to the field of microbial metabolic engineering technology, and in particular to a highly efficient Corynebacterium glutamicum producing D-pantothenic acid, its construction method, and its application. Background Technology

[0002] Pantothenic acid (D-Pantothenic Acid), chemically named α,γ-dihydroxy-β, also known as pantothenic acid or vitamin B5, has a molecular weight of 219.24. As a precursor to coenzyme A (CoA) and acyl carrier protein (ACP) in the body, D-Pantothenic acid indirectly participates in the metabolism of carbohydrates, proteins, and fats. Therefore, it is widely used as an additive in the pharmaceutical, food, and feed industries.

[0003] Currently, there are three production methods: 1. Chemical production method: In 1940, Ringrose et al. first synthesized D-calcium pantothenate through chemical synthesis. This method first chemically synthesizes the precursors of D-pantothenic acid, D / L-pantothenic acid lactone and β-alanine. D / L-pantothenic acid lactone is mainly synthesized using the Sriller method. β-alanine is mainly prepared chemically from intermediates such as acrylonitrile, β-aminopropionitrile, and succinimide. D-calcium pantothenate can then be synthesized via two routes: one is the chiral resolution of D / L-pantothenic acid lactone to obtain D-pantothenic acid lactone, which then reacts directly with calcium β-aminopropionate to obtain D-calcium pantothenate; the other is the direct reaction of D / L-pantothenic acid lactone with calcium β-aminopropionate to generate racemic calcium pantothenate, which is then resolved by crystallization-induced separation to obtain D-calcium pantothenate. 2. Enzymatic production method: Enzymatic production has advantages such as mild reaction conditions and low environmental pollution. Currently, the industrially implemented technology route involves the selective hydrolysis of D-pantolactone by microbial enzymes to separate D / L-pantolactone. This primarily utilizes D-pantolactone hydrolases to hydrolyze D / L-pantolactone into D-pantolactone, which is then further hydrolyzed into D-pantothenic acid by D-pantolactone hydrolases. The resulting D-pantothenic acid is then separated and condensed with calcium β-aminopropionate to obtain D-pantothenic acid. 3. Microbial fermentation: Microbial fermentation utilizes natural multi-enzyme systems or metabolically modified synthetic pathways within microorganisms to synthesize the desired product from inexpensive raw materials. Significant progress has been made in this area, but for industrial production, the production capacity of engineered strains still needs improvement. Suitable strains are needed to increase yield and productivity. Therefore, improving the production performance of engineered D-pantothenic acid bacteria is a goal for those skilled in the art.

[0004] Currently, the global annual production of D-calcium pantothenate is approximately 75 tons, while the annual demand is 100 tons, resulting in a supply shortage. Major domestic producers of D-calcium pantothenate include Zhejiang Hangzhou Xinfu Pharmaceutical Co., Ltd. and Shandong Huachen, while international manufacturers include BASF (Germany) and DSM (Netherlands). As of 2019, China's annual D-calcium pantothenate production reached approximately 24,000 tons, with a value of about US$1.08 billion, representing a 27% increase year-on-year. This demonstrates the high market demand and development potential of D-calcium pantothenate. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems of low yield and low conversion rate of D-pantothenic acid producing strains in the fermentation production of D-pantothenic acid in the prior art, and to provide a high-efficiency D-pantothenic acid producing strain of Corynebacterium glutamicum, its construction method and application.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions: In the first aspect, the present invention provides a high-efficiency D-pantothenic acid production strain of Corynebacterium glutamicum, a construction method and application, specifically including the following steps: (1) Using wild-type Corynebacterium glutamicum ATCC 13032 as a chassis, weakening ilvA and mutating expression to construct DPA1; (2) Knocking out pyruvate quinone oxidoreductase (pqo) and inserting a panBCE gene expression cassette into it to construct DPA2; (3) Introducing into DPA2 using the pEC-xk99E overexpression plasmid to screen for expression of Escherichia coli W3110, Bacillus subtilis 168, and endogenous acetylhydroxy acid synthase (AHAS, ilvBN), acetyllactate isomerase (AHAIR, ilvC) and 2-hydroxy acid dehydratase (DHAD, ilvD); (4) The screened ilvBNCD gene was overexpressed in tandem by pEC-xk99E under the control of a strong promoter to construct DPA3, thereby strengthening the main biosynthetic pathway of pantothenic acid and the upstream pathway, thus obtaining the Corynebacterium glutamicum that produces D-pantothenic acid efficiently.

[0007] This invention constructs DPA1 by using wild-type Corynebacterium glutamicum ATCC 13032 as a chassis, weakening ilvA, and performing mutations. To improve the production performance of the engineered D-pantothenic acid strain, the carbon flow in the main biosynthetic pathway can be pulled to the product. This invention knocks out pyruvate quinone oxidoreductase (pqo), enriches the pyruvate pool, reduces carbon metabolic flow loss, and inserts a panBCE gene expression cassette to direct more carbon metabolic flow towards D-pantothenic acid synthesis. To further enhance carbon metabolic flow, Escherichia coli W3110, Bacillus subtilis 168, and endogenous acetylhydroxy acid synthase, acetyllactate isomerase, and 2-hydroxy acid dehydratase are overexpressed in DPA2. The selected ilvBNCD gene is then expressed in tandem to strengthen the downstream pull of the pantothenic acid main biosynthetic pathway. Through the tandem expression of these effective modified sites, this invention successfully obtained a Corynebacterium glutamicum strain that produces D-pantothenic acid efficiently.

[0008] The genes encoding acetylhydroxylase, acetyllactate isomerase, and 2-hydroxylase are typically represented as ilvBN, ilvC, and ilvD. These gene sequences can be obtained from the public database of the National Center for Biotechnology Information (NCBI). For example, ilvBN has GenBank ID 3345149, ilvC has GenBank ID NC_000964.3, and ilvD has GenBank ID NC_000913.3.

[0009] Preferably, the start codon of ilvA in the wild-type Corynebacterium glutamicum ATCC 13032 is changed from ATG to GTG.

[0010] Preferably, panB, panC, and panE are all integrated into the pqo gene locus.

[0011] Preferably, panB and panC, integrated into the pqo gene locus, are derived from Corynebacterium glutamicum ATCC 13032, and panE is derived from Escherichia coli W3110; ilvBN is derived from Corynebacterium glutamicum ATCC 13032, ilvC is derived from Bacillus subtilis 168, and ilvD is derived from Escherichia coli W3110.

[0012] Preferably, the panB, panC, panE, ilvBN, ilvC, and ilvD genes are expressed by strong promoters.

[0013] Preferably, the strong promoter is Ptuf, whose nucleotide sequence is SEQ ID NO.1.

[0014] Secondly, a highly efficient Corynebacterium glutamicum for producing D-pantothenic acid is prepared by any of the methods described above.

[0015] Thirdly, the present invention provides an application of Corynebacterium glutamicum in the fermentation production of D-pantothenic acid.

[0016] Preferably, the fermentation production process employs an electroporation method.

[0017] Preferably, the application involves inoculating the Corynebacterium glutamicum into a fermentation medium, fermenting it under suitable conditions, and then separating and purifying the culture broth to obtain D-pantothenic acid.

[0018] In this invention, *Corynebacterium glutamicum* was inoculated into a fermentation medium at a 2% inoculum and fermented at 30°C and 180–220 rpm. The culture broth was then separated and purified to obtain D-pantothenic acid. The final concentration of the fermentation medium (g) was: glucose 30, corn steep liquor 25, ammonium sulfate 15, ammonium acetate 15, urea 2, sodium citrate 2, K₂HPO₄·3H₂O 1.3, MgSO₄·H₂O 0.5, L-isoleucine 0.06, MnSO₄·H₂O 0.01, and biotin 1 x 10⁻⁶. -4 Thiamine 2x10 -4 CaCO3 30, pH 7.0-7.2. Sterilize at 115℃ for 10 min.

[0019] The present invention involves slant activation and seed culture before fermentation. The seed culture is then inoculated into the fermentation medium. The slant activation method is as follows: Corynebacterium glutamicum is inoculated onto an LB agar plate and cultured overnight at 30°C to obtain slant cells. The seed culture method is as follows: a single colony of the slant cells is picked and inoculated into LB liquid medium and cultured overnight at 30°C and 180–200 rpm to obtain a seed culture. The seed culture is then inoculated into the fermentation medium at a volume concentration of 2%.

[0020] The present invention has the following beneficial effects: (1) The modified Corynebacterium glutamicum provided by the present invention can better utilize carbon sources such as glucose to produce D-pantothenic acid compared with wild type and chassis strain. The strain with plasmid has good production performance, with high yield and high conversion rate. (2) The strain with the best performance obtained after modification by the present invention has a significantly improved level of D-pantothenic acid production by fermentation compared with the original strain DPA2. The level of the strain in shake flask increased from 0.01g / L to 2.35g / L. (3) The Corynebacterium glutamicum provided by the present invention has important industrial application value. Attached Figure Description

[0021] Figure 1 is a schematic diagram of the metabolic modification of the DPA-producing strain.

[0022] Figure 2 shows a schematic diagram of the shake-flask fermentation performance of strains WT, DPA1, DPA2, and DPA3.

[0023] Figure 3 shows the fermentation test of DPA3 in a 5L fermenter. Detailed Implementation

[0024] The present invention will be further described below with reference to specific 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] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0026] Unless otherwise specified, the experimental materials used in the following examples are all conventional biochemical reagents.

[0027] The term "enhancement" refers to increasing the activity of an enzyme encoded by a corresponding polynucleotide. This can be achieved by increasing the copy number of the gene or by replacing the expression regulatory sequence of that gene in the genome (such as promoter substitution). The term "weakening" refers to decreasing the activity of an enzyme encoded by a corresponding polynucleotide. This can be achieved by replacing the expression regulatory sequence of that gene in the genome (such as non-coding region sequence substitution).

[0028] LB liquid medium consists of 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride, in deionized water at a natural pH. LB plates are made by adding agar to LB liquid medium to a final concentration of 2 g / L.

[0029] BHIS+Gly medium: brain-heart infusion medium 71g / L, D-sorbitol 37g / L, Gly 40g / L, solvent is tap water, pH value is natural.

[0030] LBS solid culture medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 100 g / L sucrose, 4 g / L agar, solvent: tap water, pH: natural. Example 1: Weakening ilvA

[0031] Using wild-type Corynebacterium glutamicum ATCC 13032 as the starting strain, the ilvA gene was weakened using CRISPR / Cpf1 gene editing technology mediated by the editing plasmid pJYS3, changing its start codon from ATG to GTG, and DPA1 was constructed. The GenBank ID of ilvA is Cgl2332.

[0032] Using Corynebacterium glutamicum ATCC 13032 as a template, the upstream homologous arm of ilvA (1000 bp) was obtained by PCR using primers 1 and 2, and the downstream homologous arm of ilvA (GTG) (1000 bp) was obtained by PCR using primers 3 and 4. The PCR reaction conditions were as follows: 98℃ for 8 min; 98℃ for 30 s, 56℃ for 30 s, 72℃ for 45 s, for 32 cycles. After confirmation by nucleic acid gel electrophoresis, the obtained PCR stock solution was purified by gel extraction using the Qingke Gel Extraction Kit, and the concentration was determined by Nanoparticles. The purified fragments were then stored at -20℃ for later use.

[0033] E. coli carrying the pJYS3 empty plasmid were first streaked onto LB solid medium supplemented with kanamycin for 12-16 hours. Single colonies were then picked and cultured on LB liquid medium supplemented with kanamycin at 37°C for 12-16 hours. 2-4 mL of fresh bacterial culture was collected, and plasmids were extracted according to the instructions of the Qingke Plasmid Mini-Prep Kit. The concentration of the extracted plasmids was determined using NANO assay and stored at -20°C for later use.

[0034] Using the diluted pJYS3 vector as a template, linearized pJYS3 vector was obtained by PCR using primers 5 and 6. The PCR reaction conditions were as follows: 95℃ for 5 min; 95℃ for 30 s, 56℃ for 30 s, 72℃ for 5 min and 15 s, for 32 cycles. After verification by nucleic acid electrophoresis, DpnI was added and the sample was heated at 37℃ for 30 min to eliminate the template. After determining the backbone concentration using nanoparticles, the sample was stored at -20℃ for later use.

[0035] The obtained ilvA upstream and downstream homologous arms and the linearized pJYS3 vector were used for multi-fragment ligation using C115. The reaction system and conditions are as follows:

[0036] After reacting at 50℃ for 30 min, the ligation product can be obtained. The ligation product is transformed into E. coli DH5α competent cells and cultured overnight at 37℃. After successful colony PCR verification, the recombinant plasmid pJYS3-ilvA can be obtained by picking bacteria and inoculating test tubes and extracting the plasmid using a plasmid extraction kit. 10 μL of the plasmid is sent for sequencing and the remaining plasmid is stored at -20℃.

[0037] The correctly sequenced plasmid was introduced into the PAM site via PCR using primers 7 and 8. The PCR reaction conditions were as follows: 95℃ for 5 min; 95℃ for 30 s, 56℃ for 30 s, 72℃ for 6 min, for 32 cycles. After confirmation by nucleic acid gel electrophoresis, the obtained PCR stock solution was purified using the Chinco gel extraction kit, and the concentration was determined by nanoparticles. The purified fragment was directly transferred into E. coli DH5α competent cells and cultured overnight at 37℃. After successful colony PCR verification using primers 9 and 10, the recombinant plasmid pJYS3-ilvA(GTG) was obtained by picking bacteria and inoculating into test tubes, and then extracting the plasmid using a plasmid extraction kit. 10 μL of the plasmid was sent for sequencing, and the remaining plasmid was stored at -20℃.

[0038] Preparation of electrocompetent cells for wild-type Corynebacterium glutamicum: A single colony of wild-type Corynebacterium glutamicum was picked and inoculated into a small shaker containing 10 mL of LB medium. The culture was incubated overnight at 30°C and 200 rpm. 1 mL of the overnight culture was transferred to a 250 mL Erlenmeyer flask containing 40 mL of LB medium and incubated at 30°C and 200 rpm for 6 hours until the OD of the culture was reached. 600 Greater than 0.8. Place the cultured bacterial solution on ice to cool for about 20 minutes, pour into 50mL centrifuge tubes, centrifuge at 5000rpm for 10min at 4℃, remove the supernatant in a clean bench and retain the bacterial cells, add about 20mL of pre-sterilized and pre-cooled 10% glycerol solution, resuspend in an ice-water bath, centrifuge at 5000rpm for 5min at 4℃, repeat three times, add 1mL of pre-cooled 10% glycerol, resuspend in an ice-water bath, aliquot into 1.5mL sterile centrifuge tubes (100μL per tube), reserve one tube for later use, and store the rest at -80℃.

[0039] Plasmid pJYS3-ilvA(GTG) was introduced into DPA1: 5 μL of pJYS3-ilvA(GTG) was added to electrotransformed competent DPA1 cells and gently mixed by pipetting. The cells were incubated on ice for 5 min, followed by two electroporations at 1.8 kV. Then, 1 mL of preheated 46℃ LB medium was added, and the cells were immediately heat-shocked at 46℃ for 6 min. The cells were then incubated at 30℃ for 200 rpm for 2 h. The resulting culture was plated on Kansei resistant LB plates and incubated at 30℃ for 40 h. ilvA was verified by colony PCR using primers 11 and 12. The PCR products of the correctly sequenced strains were selected and named DPA1.

[0040] Plasmid removal: Select colonies with correct sequencing and inoculate them into 10 mL of antibiotic-free LB medium and incubate for 18 hours. Then, streak the bacterial solution onto antibiotic-free medium and incubate at 30°C for 36–48 hours. Select single colonies to replicate on Kan resistant and antibiotic-free LB solid medium. Select strains that grow on antibiotic-free plates but not on Kan resistant plates, indicating successful plasmid removal. Preserve the bacteria for later use.

[0041] Fermentation of strain DPA1: Using the wild-type strain as a control, both DPA1 and DPA2 were inoculated into 10 mL of LB medium and cultured at 30°C and 200 rpm to prepare the seed culture. After 12 h, 1 mL of the seed culture was inoculated into a 500 mL shake flask containing 50 mL of fermentation medium, and then cultured at 30°C and 180 rpm until the cell concentration reached OD200. 600 When the concentration of iodine (I0.8-1.0) was reached, IPTG was added to a final concentration of 0.1 mM, and the culture was continued for 48 h. After fermentation, 1 mL of fermentation broth was centrifuged at 12,000 rpm for 2 min at room temperature, and the supernatant was collected for HPLC analysis. Another 1 mL of fermentation broth was used to determine the biomass OD0.05. 600 HPLC detection: (1) Chromatographic conditions: C18 column (250×4.6mm, particle size 5μm, Agilent Technologies Co, Santa Clara, CA, USA); detection wavelength: 200nm; column temperature: 30℃; (2) Sample preparation: dilute the sample with ultrapure water to maintain the D-pantothenic acid content at 0.05-0.40g / L; (3) Mobile phase: acetonitrile / water / phosphoric acid (volume ratio 50 / 949 / 1); (4) Data acquisition time: 15min. The D-pantothenic acid detection in the following examples all used this method. Example 2 Knockout of pyruvate quinone oxidoreductase (pqo) and integration of its site panBCE

[0042] Using DPA1 as the starting strain, the pqo gene was knocked out using homologous recombination gene editing technology mediated by shuttle plasmid pk18mobsacB, and a panBCE expression cassette was simultaneously knocked in. The GenBank IDs for panB, panC, and panE are: panB (WP_011013400.1), panC (WP_011013399.1), and panE (NP_389394.1).

[0043] Using Corynebacterium glutamicum ATCC 13032 as a template, the upstream homologous arm of pqo (500 bp) was obtained by PCR using primers 13 and 14, and the downstream homologous arm of pqo (500 bp) was obtained by PCR using primers 15 and 16. The PCR reaction conditions were as follows: 98℃ for 8 min; 98℃ for 30 s, 56℃ for 30 s, 72℃ for 45 s, for 32 cycles. The panBC gene fragment (2000 bp) was obtained by PCR using primers 17 and 18. The PCR reaction conditions were as follows: 98℃ for 8 min; 98℃ for 30 s, 56℃ for 30 s, 72℃ for 1 min 30 s, for 32 cycles. Using *Escherichia coli* W3110 as a template, a 1000 bp panE gene fragment was obtained by PCR using primers 19 and 20. The PCR reaction conditions were as follows: 98℃ for 5 min; 98℃ for 30 s, 56℃ for 30 s, 72℃ for 45 s, for 32 cycles. After confirmation by nucleic acid gel electrophoresis, the obtained PCR stock solution was purified by gel extraction using the Qingke Gel Extraction Kit, and the concentration was determined by nanoparticles. The purified fragment was then stored at -20℃ for later use.

[0044] E. coli carrying the empty plasmid pk18mobsacB was streaked onto LB solid medium supplemented with kanamycin for 12-16 hours. Single colonies were then picked and cultured on LB liquid medium supplemented with kanamycin at 37°C for 12-16 hours. 2-4 mL of fresh bacterial culture was collected, and plasmids were extracted according to the instructions of the Qingke Plasmid Mini-Prep Kit. The concentration of the extracted plasmids was determined using NANO assay and stored at -20°C for later use.

[0045] The diluted pk18mobsacB vector was used as a template, and linearized pk18mobsacB vector was obtained by PCR using primers 21 and 22. The PCR reaction conditions were as follows: 95℃ for 5 min; 95℃ for 30 s, 56℃ for 30 s, 72℃ for 5 min 15 s, for 32 cycles. After verification by nucleic acid electrophoresis, DpnI was added and the sample was heated at 37℃ for 30 min to eliminate the template. After determining the backbone concentration using nanoparticles, the sample was stored at -20℃ for later use.

[0046] The obtained pqo upstream and downstream homologous arms, panBC, panE, and linearized pk18mobsacB vector were used for multi-fragment ligation using C115. The reaction system and conditions are as follows:

[0047] After reacting at 50℃ for 30 min, the ligation product can be obtained. The ligation product is transformed into E. coli DH5α competent cells and cultured overnight at 37℃. After successful colony PCR verification, the recombinant plasmid pK18mobsacB-Δpqo::panBCE can be obtained by picking bacteria and inoculating test tubes, and extracting the plasmid using the plasmid extraction kit according to Example 1. 10 μL of the plasmid is sent for sequencing and the remaining plasmid is stored at -20℃.

[0048] Preparation of DPA1 electrocompetent cells: A single DPA1 colony was picked and inoculated into a small shake flask containing 10 mL of LB medium. The culture was incubated overnight at 30°C and 200 rpm. 1 mL of the overnight culture was transferred to a 250 mL Erlenmeyer flask containing 40 mL of LB medium and incubated at 30°C and 200 rpm for 6 hours until the OD of the culture was reached. 600 Greater than 0.8. Place the cultured bacterial solution on ice to cool for about 20 minutes, pour into 50mL centrifuge tubes, centrifuge at 5000rpm for 10min at 4℃, remove the supernatant in a clean bench and retain the bacterial cells, add about 20mL of pre-sterilized and pre-cooled 10% glycerol solution, resuspend in an ice-water bath, centrifuge at 5000rpm for 5min at 4℃, repeat three times, add 1mL of pre-cooled 10% glycerol, resuspend in an ice-water bath, aliquot into 1.5mL sterile centrifuge tubes (100μL per tube), reserve one tube for later use, and store the rest at -80℃.

[0049] Plasmid pK18mobsacB-Δpqo::panBCE was introduced into DPA1: 5 μL of pK18mobsacB-Δpqo::panBCE was added to electrotransfer competent cells DPA1 and gently mixed by pipetting. The mixture was placed in an ice bath for 5 min, followed by two electroshocks at 1.8 kV. Then, 1 mL of preheated 46℃ LB medium was added, and the mixture was immediately heat-shocked at 46℃ for 6 min. The mixture was then incubated at 30℃ and 200 rpm for 2 h. The mixture was then plated on Kans resistant LB plates (single exchange screening) and incubated at 30℃ for 40 h. Single colonies were picked and inoculated into 10 mL of liquid Kan resistant LB medium and cultured for 12 hours. Then, 1% of the colony was transferred to 10 mL of antibiotic-free LB medium and cultured for relaxation for 12 hours. After that, the bacterial culture was streaked onto LBS solid medium for a second screening (double exchange screening). Single colonies were picked from LBS medium and imprinted on Kan resistant and antibiotic-free LB solid medium. Strains that grew on antibiotic-free plates but not on Kan resistant plates were selected as templates. Colony PCR with primers 23 and 24 was used to verify whether pqo was knocked out and whether panBCE was successfully integrated. Strains with correct bands were selected, inoculated into 10 mL of LB liquid medium, cultured overnight at 30°C and 200 rpm, and then sent for sequencing. The correctly sequenced strain was named DPA2.

[0050] Fermentation of strain DPA2: Using DPA1 as a control strain, DPA1 and DPA2 were inoculated into 10 mL of LB medium and cultured at 30℃ and 200 rpm to prepare the seed culture. After 12 h, 1 mL of the seed culture was inoculated into a 500 mL shake flask containing 50 mL of fermentation medium, and then cultured at 30℃ and 180 rpm until the cell concentration reached OD200. 600 When the concentration of iodine (I0.8-1.0) is reached, IPTG is added to a final concentration of 0.1 mM, and the culture is continued for 48 h. After fermentation, 1 mL of fermentation broth is centrifuged at 12000 rpm for 2 min at room temperature, and the supernatant is collected for HPLC analysis. Another 1 mL of fermentation broth is used to determine the biomass OD0.05. 600 HPLC detection: (1) Chromatographic conditions: C18 column (250×4.6mm, particle size 5μm, Agilent Technologies Co, Santa Clara, CA, USA); detection wavelength: 200nm; column temperature: 30℃; (2) Sample preparation: dilute the sample with ultrapure water to maintain the D-pantothenic acid content at 0.05-0.40g / L; (3) Mobile phase: acetonitrile / water / phosphoric acid (volume ratio 50 / 949 / 1); (4) Data acquisition time: 15min. The D-pantothenic acid detection in the following examples all used this method. Example 3: Screening expression of ilvBN gene

[0051] To enhance the upstream pull of pantothenic acid and direct more carbon flow to the D-pantothenic acid main synthesis pathway, the pEC-xk99E overexpression plasmid was introduced into Corynebacterium glutamicum DPA2 strain to screen for expression from Escherichia coli W3110, Bacillus subtilis 168, endogenous acetylhydroxyl synthase (AHAS, ilvBN), and an acetylhydroxyl synthase mutant from Corynebacterium glutamicum ATCC 13032 itself. This resulted in the highly efficient Corynebacterium glutamicum producing D-pantothenic acid, which was then applied to the fermentation production of D-pantothenic acid.

[0052] The acetylhydroxy acid synthase (AHAS, ilvBN) derived from Escherichia coli W3110, Bacillus subtilis 168, and Corynebacterium glutamicum ATCC 13032 and its mutants was overexpressed using pEC-xk99E via the strong promoter Ptuf.

[0053] Using the diluted pEC-xk99E vector (plasmid from laboratory storage) as a template, and primers 25 and 26, a series of pEC-xk99E expression vector backbones capable of expressing the ilvBN gene were constructed by PCR. The PCR reaction conditions were as follows: 95℃ for 5 min; 95℃ for 30 s, 56℃ for 30 s, 72℃ for 5 min 15 s, for 32 cycles. After confirming the presence of a bright and clear target band by gel electrophoresis, DpnI was added and the sample was heated at 37℃ for 30 min to eliminate the template. The backbone concentration was then measured using nanoparticles, and the sample was stored at -20℃ for later use.

[0054] Subsequently, based on the ilvBN gene of *Corynebacterium glutamicum* ATCC 13032 published on NCBI, the endogenous ilvBN sequence of *Corynebacterium glutamicum* was amplified using primers 27 and 28. The PCR reaction conditions were as follows: 98℃ for 10 min; 98℃ for 30 s, 56℃ for 30 s, 72℃ for 1 min 30 s, for 32 cycles; 72℃ for 5 min. The *Escherichia coli* and *Bacillus subtilis* ilvBN sequences were amplified using primers 29 / 30 and 31 / 32 in the same manner. The PCR products were purified using a purification kit.

[0055] The Ptuf strong promoter was introduced by PCR using linearized pEC-xk99E as a template and primers 33 and 34. The PCR reaction conditions were as follows: 95℃ for 5 min; 95℃ for 30 s, 56℃ for 30 s, 72℃ for 5 min 30 s, for 32 cycles. The PCR product was purified using a purification kit.

[0056] The process for preparing competent *E. coli* cells by chemical transformation is as follows: A single colony of *E. coli* is picked and inoculated into a test tube containing 5 mL of LB medium. The culture is incubated overnight at 37°C and 200 rpm using a shaker. 1 mL of the overnight culture is transferred to a 250 mL Erlenmeyer flask containing 40 mL of LB medium and incubated at 37°C and 200 rpm for 1-2 hours until the bacterial growth rate reaches OD (overflow retardation). 600 The culture temperature should be between 0.4 and 0.6. Cool the cultured bacterial suspension on ice for approximately 10 minutes, then pour it into 50 mL centrifuge tubes. Centrifuge at 5500 rpm for 5 minutes at 4°C. Remove the supernatant in a clean bench, retaining the bacterial cells. Add approximately 40 mL of pre-sterilized and pre-chilled 0.1 MCaCl2 solution, resuspend in an ice-water bath, and incubate on ice for 30 minutes. Centrifuge at 6000 rpm for 5 minutes, then add 1 mL of pre-chilled solution containing 0.1 MCaCl2 and 15% glycerol. Resuspend in an ice-water bath, and aliquot into 1.5 mL sterile centrifuge tubes (100 μL per tube). Reserve one tube for later use; store the rest at -80°C.

[0057] The linearized pEC-xk99E vector with the Ptuf strong promoter and the obtained ilvBN gene fragment were ligated using C115. The reaction conditions were as follows: 50℃ for 30 min. One competent E. coli cell was taken and placed on ice. The reaction product was added to the competent cell in a clean bench and placed on ice for 5 min. After heat shock at 42℃ for 1 min and on ice for 3 min, 700 μL LLB medium was added. After incubation at 37℃ for 1 h, the cell was removed and centrifuged at 5000 rpm for 2 min. 600 μL LLB medium was discarded, and the remaining medium was mixed by pipetting and spread onto LB solid medium (Kan resistance). The cell was incubated overnight at 37℃.

[0058] Single colonies were selected as templates and colony PCR was performed using primers 35 and 36 for verification. After confirming the correct bands, the colonies were sent to the company for sequencing. If the sequencing was successful, the colonies were shaken again to extract plasmids. Plasmid extraction was performed using a plasmid extraction kit. The remaining expression vectors were constructed in the same manner.

[0059] The preparation of competent Corynebacterium glutamicum cells by electroporation is as follows: A single colony of Corynebacterium glutamicum is picked and inoculated into a small shaker containing 10 mL of LB medium. The culture is incubated overnight at 30°C and 200 rpm. 1 mL of the overnight culture is then transferred to a 250 mL Erlenmeyer flask containing 40 mL of LB medium and incubated at 30°C and 200 rpm for 6 hours until the OD value of the culture reaches the target value. 600 Greater than 0.8. Cool the cultured bacterial suspension on ice for approximately 20 minutes, then pour into 50 mL centrifuge tubes. Centrifuge at 4°C, 5000 rpm for 10 min. Remove the supernatant in a clean bench, retaining the bacterial cells. Add approximately 20 mL of pre-sterilized and pre-chilled 10% glycerol solution, resuspend in an ice-water bath, and centrifuge at 4°C, 5000 rpm for 5 min. Repeat three times. Add 1 mL of pre-chilled 10% glycerol, resuspend in an ice-water bath, and aliquot into 1.5 mL sterile centrifuge tubes (100 μL per tube). Reserve one tube for later use; store the rest at -80°C. The electroporation process is as follows: Take one electroporation competent cell and place it on ice. Add 3 μL of overexpression vector to a clean bench, gently mix with a pipette, and incubate on ice for 5 min. Transfer to a pre-chilled 2 mm electroporation cuvette using a pipette, and incubate on ice for 1 min. Wipe the outside of the electroporation cuvette dry with a paper towel and place it in the electroporator. Electroporate at 1.8 kV. Add 1 mL of pre-cooled LB medium to the groove of the electroporation cup in a clean bench. Tilt the electroporation cup and aspirate all the bacterial culture from the mouth of the cup, transferring it to a 2 mL sterile EP tube. Recover at 30°C and 200 rpm for 3 h. Spread 60 μL onto LB solid medium (Kan / 2 resistant) and incubate at 30°C for 36-72 h. Example 4: Screening expression of the ilvC gene.

[0060] To enhance the downstream driving force of pantothenic acid and direct more carbon flow to the D-pantothenic acid main synthesis pathway, the pEC-xk99E overexpression plasmid was introduced into Corynebacterium glutamicum DPA2 strain to screen for expression of Escherichia coli W3110, Bacillus subtilis 168, and endogenous acetyllactone isomerase (AHAIR, ilvC). The resulting Corynebacterium glutamicum producing strain with high efficiency in D-pantothenic acid production was applied to the fermentation production of D-pantothenic acid.

[0061] The acetyl-lactate isomerase (AHAIR, ilvC) derived from Escherichia coli W3110, Bacillus subtilis 168, and Corynebacterium glutamicum ATCC 13032 was overexpressed using pEC-xk99E via the strong promoter Ptuf.

[0062] Using the diluted pEC-xk99E vector (plasmid from laboratory storage) as a template, and primers 25 and 26, a series of pEC-xk99E expression vector backbones capable of expressing the ilvC gene were constructed by PCR. The PCR reaction conditions were as follows: 95℃ for 5 min; 95℃ for 30 s, 56℃ for 30 s, 72℃ for 5 min 15 s, for 32 cycles. After confirming the presence of a bright and clear target band by gel electrophoresis, DpnI was added and the sample was heated at 37℃ for 30 min to eliminate the template. The backbone concentration was then measured using nanoparticles, and the sample was stored at -20℃ for later use.

[0063] Subsequently, based on the ilvC gene of *Corynebacterium glutamicum* ATCC 13032 published on NCBI, the endogenous ilvC sequence of *Corynebacterium glutamicum* was amplified using primers 37 and 38. The PCR reaction conditions were as follows: 98℃ for 10 min; 98℃ for 30 s, 56℃ for 30 s, 72℃ for 1 min 30 s, for 32 cycles; 72℃ for 5 min. The *Escherichia coli* and *Bacillus subtilis* ilvC sequences were amplified using primers 39 / 40 and 41 / 42 in the same manner. The PCR products were purified using a purification kit.

[0064] The Ptuf strong promoter was introduced by PCR using linearized pEC-xk99E as a template and primers 33 and 34. The PCR reaction conditions were as follows: 95℃ for 5 min; 95℃ for 30 s, 56℃ for 30 s, 72℃ for 5 min 30 s, for 32 cycles. The PCR product was purified using a purification kit.

[0065] The process for preparing competent *E. coli* cells by chemical transformation is as follows: A single colony of *E. coli* is picked and inoculated into a test tube containing 5 mL of LB medium. The culture is incubated overnight at 37°C and 200 rpm using a shaker. 1 mL of the overnight culture is transferred to a 250 mL Erlenmeyer flask containing 40 mL of LB medium and incubated at 37°C and 200 rpm for 1-2 hours until the bacterial growth rate reaches OD (overflow retardation). 600 The culture temperature should be between 0.4 and 0.6. Cool the cultured bacterial suspension on ice for approximately 10 minutes, then pour it into 50 mL centrifuge tubes. Centrifuge at 5500 rpm for 5 minutes at 4°C. Remove the supernatant in a clean bench, retaining the bacterial cells. Add approximately 40 mL of pre-sterilized and pre-chilled 0.1 MCaCl2 solution, resuspend in an ice-water bath, and incubate on ice for 30 minutes. Centrifuge at 6000 rpm for 5 minutes, then add 1 mL of pre-chilled solution containing 0.1 MCaCl2 and 15% glycerol. Resuspend in an ice-water bath, and aliquot into 1.5 mL sterile centrifuge tubes (100 μL per tube). Reserve one tube for later use; store the rest at -80°C.

[0066] The linearized pEC-xk99E vector with the Ptuf strong promoter and the obtained ilvC gene fragment were ligated using C115. The reaction conditions were as follows: 50℃ for 30 min. One competent E. coli cell was taken and placed on ice. The reaction product was added to the competent cell in a clean bench and placed on ice for 5 min. After heat shock at 42℃ for 1 min and on ice for 3 min, 700 μL LLB medium was added. After incubation at 37℃ for 1 h, the cell was removed and centrifuged at 5000 rpm for 2 min. 600 μL LLB medium was discarded, and the remaining medium was mixed by pipetting and spread onto LB solid medium (Kan resistance). The cell was incubated overnight at 37℃.

[0067] Single colonies were selected as templates and colony PCR was performed using primers 35 and 36 for verification. After confirming the correct bands, the colonies were sent to the company for sequencing. If the sequencing was successful, the colonies were shaken again to extract plasmids. Plasmid extraction was performed using a plasmid extraction kit. The remaining expression vectors were constructed in the same manner.

[0068] The preparation of competent Corynebacterium glutamicum cells by electroporation is as follows: A single colony of Corynebacterium glutamicum is picked and inoculated into a small shaker containing 10 mL of LB medium. The culture is incubated overnight at 30°C and 200 rpm. 1 mL of the overnight culture is then transferred to a 250 mL Erlenmeyer flask containing 40 mL of LB medium and incubated at 30°C and 200 rpm for 6 hours until the OD value of the culture reaches the target value. 600Greater than 0.8. Cool the cultured bacterial suspension on ice for approximately 20 minutes, then pour into 50 mL centrifuge tubes. Centrifuge at 4°C, 5000 rpm for 10 min. Remove the supernatant in a clean bench, retaining the bacterial cells. Add approximately 20 mL of pre-sterilized and pre-chilled 10% glycerol solution, resuspend in an ice-water bath, and centrifuge at 4°C, 5000 rpm for 5 min. Repeat three times. Add 1 mL of pre-chilled 10% glycerol, resuspend in an ice-water bath, and aliquot into 1.5 mL sterile centrifuge tubes (100 μL per tube). Reserve one tube for later use; store the rest at -80°C. The electroporation process is as follows: Take one electroporation competent cell and place it on ice. Add 3 μL of overexpression vector to a clean bench, gently mix with a pipette, and incubate on ice for 5 min. Transfer to a pre-chilled 2 mm electroporation cuvette using a pipette, and incubate on ice for 1 min. Wipe the outside of the electroporation cuvette dry with a paper towel and place it in the electroporator. Electroporate at 1.8 kV. Add 1 mL of pre-cooled LB medium to the groove of the electroporation cup in a clean bench. Tilt the electroporation cup and aspirate all the bacterial culture from the mouth of the cup, transferring it to a 2 mL sterile EP tube. Recover at 30°C and 200 rpm for 3 h. Spread 60 μL onto LB solid medium (Kan / 2 resistant) and incubate at 30°C for 36-72 h. Example 5: Screening expression of the ilvD gene.

[0069] To enhance the downstream driving force of pantothenic acid and direct more carbon flow to the D-pantothenic acid main synthesis pathway, the pEC-xk99E overexpression plasmid was introduced into Corynebacterium glutamicum DPA2 strain to screen for expression of Escherichia coli W3110, Bacillus subtilis 168, and endogenous 2-hydroxy acid dehydratase (DHAD, ilvD). The resulting high-efficiency Corynebacterium glutamicum producing strain for D-pantothenic acid production was then applied to the fermentation production of D-pantothenic acid.

[0070] The 2-hydroxy acid dehydratase (DHAD, ilvD) derived from Escherichia coli W3110, Bacillus subtilis 168, and Corynebacterium glutamicum ATCC 13032 was overexpressed using pEC-xk99E via the strong promoter Ptuf.

[0071] Using the diluted pEC-xk99E vector (plasmid from laboratory storage) as a template, and primers 25 and 26, a series of pEC-xk99E expression vector backbones capable of expressing the ilvD gene were constructed by PCR. The PCR reaction conditions were as follows: 95℃ for 5 min; 95℃ for 30 s, 56℃ for 30 s, 72℃ for 5 min 15 s, for 32 cycles. After confirming the presence of a bright and clear target band by gel electrophoresis, DpnI was added and the sample was heated at 37℃ for 30 min to eliminate the template. The backbone concentration was then measured using nanoparticles, and the sample was stored at -20℃ for later use.

[0072] Subsequently, based on the ilvD gene of *Corynebacterium glutamicum* ATCC 13032 published on NCBI, the endogenous ilvD sequence of *Corynebacterium glutamicum* was amplified using primers 43 and 44. The PCR reaction conditions were as follows: 98℃ for 10 min; 98℃ for 30 s, 56℃ for 30 s, 72℃ for 1 min 30 s, for 32 cycles; 72℃ for 5 min. The *Escherichia coli* ilvD sequence and the *Bacillus subtilis* ilvD sequence were amplified using primers 45 / 46 and 47 / 48 in the same manner. The PCR products were purified using a purification kit.

[0073] The Ptuf strong promoter was introduced by PCR using linearized pEC-xk99E as a template and primers 33 and 34. The PCR reaction conditions were as follows: 95℃ for 5 min; 95℃ for 30 s, 56℃ for 30 s, 72℃ for 5 min 30 s, for 32 cycles. The PCR product was purified using a purification kit.

[0074] The process for preparing competent *E. coli* cells by chemical transformation is as follows: A single colony of *E. coli* is picked and inoculated into a test tube containing 5 mL of LB medium. The colony is incubated overnight at 37°C and 200 rpm using a shaker. 1 mL of the overnight culture is transferred to a 250 mL Erlenmeyer flask containing 40 mL of LB medium. The flask is incubated at 37°C and 200 rpm for 1-2 hours, until the OD600 is between 0.4 and 0.6. The cultured bacterial solution is cooled on ice for about 10 minutes, then poured into a 50 mL centrifuge tube. The tube is centrifuged at 5500 rpm for 5 minutes at 4°C. The supernatant is removed in a clean bench, retaining the bacterial cells. Approximately 40 mL of pre-sterilized and pre-cooled 0.1 M CaCl2 solution is added, and the cells are resuspended in an ice-water bath for 30 minutes. After centrifuging at 6000 rpm for 5 min, add 1 mL of pre-cooled solution containing 0.1 M CaCl2 and 15% glycerol. Resuspend in an ice-water bath and aliquot into 1.5 mL sterile centrifuge tubes (100 μL per tube). Reserve one tube for later use and store the rest at -80 °C.

[0075] The linearized pEC-xk99E vector with the Ptuf strong promoter and the obtained ilvD gene fragment were ligated using C115. The reaction conditions were as follows: 50℃ for 30 min. One competent E. coli cell was taken and placed on ice. The reaction product was added to the competent cell in a clean bench and placed on ice for 5 min. After heat shock at 42℃ for 1 min and on ice for 3 min, 700 μL LLB medium was added. After incubation at 37℃ for 1 h, the cell was removed and centrifuged at 5000 rpm for 2 min. 600 μL LLB medium was discarded, and the remaining medium was mixed by pipetting and spread onto LB solid medium (Kan resistance). The cell was incubated at 37℃ overnight.

[0076] Single colonies were selected as templates and colony PCR was performed using primers 35 and 36 for verification. After confirming the correct bands, the colonies were sent to the company for sequencing. If the sequencing was successful, the colonies were shaken again to extract plasmids. Plasmid extraction was performed using a plasmid extraction kit. The remaining expression vectors were constructed in the same manner.

[0077] The preparation of competent Corynebacterium glutamicum cells by electroporation is as follows: A single colony of Corynebacterium glutamicum is picked and inoculated into a small shaker containing 10 mL of LB medium. The culture is incubated overnight at 30°C and 200 rpm. 1 mL of the overnight culture is then transferred to a 250 mL Erlenmeyer flask containing 40 mL of LB medium and incubated at 30°C and 200 rpm for 6 hours until the OD value of the culture reaches the target value. 60 0 > 0.8. Cool the cultured bacterial suspension on ice for about 20 minutes, then pour it into 50mL centrifuge tubes. Centrifuge at 4°C, 5000rpm for 10 minutes. Remove the supernatant in a clean bench, retaining the bacterial cells. Add about 20mL of pre-sterilized and pre-cooled 10% glycerol solution, resuspend in an ice-water bath, and centrifuge at 4°C, 5000rpm for 5 minutes. Repeat three times. Add 1mL of pre-cooled 10% glycerol, resuspend in an ice-water bath, and aliquot into 1.5mL sterile centrifuge tubes (100μL per tube). Reserve one tube for later use, and store the rest at -80°C.

[0078] The electroporation process is as follows: Take one electroporation competent cell and place it on ice. Add 3 μL of overexpression vector to a clean bench, gently mix with a pipette, and incubate on ice for 5 min. Transfer the mixture to a pre-chilled 2 mm electroporation cuvette and incubate on ice for 1 min. Wipe the outside of the electroporation cuvette dry with a paper towel and place it in the electroporator, using 1.8 kV for electroporation. Add 1 mL of pre-chilled LB medium to the groove of the electroporation cuvette in a clean bench, tilt the cuvette, and aspirate all the bacterial culture from the mouth of the cuvette, transferring it to a 2 mL sterile EP tube. Recover at 30°C and 200 rpm for 3 h. Spread 60 μL onto LB solid medium (Kan / 2 resistant) and incubate at 30°C for 36-72 h. Example 6: Tandem expression of the ilvBNCD gene

[0079] To enhance the downstream pull of the strain and direct more metabolic flux toward D-pantothenic acid synthesis, the ilvBN, ilvC, and ilvD genes screened by shake-flask fermentation were expressed in tandem using pEC-xk99E.

[0080] Using *Corynebacterium glutamicum* ATCC 13032 as a template, primers 49 and 50 were used to construct a PCR vector expressing the target gene ilvBN; using *Bacillus subtilis* 168 as a template, primers 51 and 52 were used to construct a PCR vector expressing the target gene ilvC; and using *Escherichia coli* W3110 as a template, primers 53 and 54 were used to construct a PCR vector expressing the target gene ilvD. The PCR reaction conditions, vector construction process, and electroporation process were the same as in Example 1. Table 1 Primer List Example 7: Shake-flask fermentation experiment of strains with weakened ilvA gene. Table 2: D-pantothenic acid yield of each genotype strain during shake-flask fermentation.

[0081] Table 2 shows that the metabolically modified Corynebacterium glutamicum strains, due to their ability to produce and accumulate D-pantothenic acid extracellularly, are better able to utilize carbon sources such as glucose for D-pantothenic acid production compared to the wild type. The modified strain with the best performance increased the D-pantothenic acid production level from almost none to 0.1 g / L compared to the original strain. Example 8: Shake-flask fermentation experiment of integrated expression of the panBCE gene. Table 3: D-pantothenic acid production of each genotype strain during shake-flask fermentation.

[0082] Table 3 shows that the metabolically modified *Corynebacterium glutamicum* strain, due to its ability to produce and accumulate D-pantothenic acid extracellularly, can utilize carbon sources such as glucose for D-pantothenic acid production better than the wild type. The modified strain with the best performance increased the level of D-pantothenic acid produced during fermentation from 0.1 g / L to 0.3 g / L compared to the original strain. Example 9: Screening and Tandem Expression of the ilvBNCD Gene in Shake-Flavor Fermentation Experiment

[0083] The stability of the constructed D-pantothenic acid-producing strain was tested. The strain was streaked onto LB agar plates and incubated overnight at 30°C. A single colony was picked and inoculated into 10 mL of LB medium, incubated at 30°C for 14 h, and then 1 mL of the culture was inoculated into a 500 mL Erlenmeyer flask containing 50 mL of fermentation medium (g·L⁻¹). -1The final concentration composition is: glucose 30, corn steep liquor 25, ammonium sulfate 15, ammonium acetate 15, urea 2, sodium citrate 2, K2HPO4·3H2O 1.3, MgSO4·7H2O 0.5, L-isoleucine 0.06, MnSO4·H2O 0.01, biotin 1x10 -4 Thiamine 2x10 -4 CaCO3 30, pH 7.0-7.2. Sterilize at 115℃ for 10 min. Table 4: D-pantothenic acid yield of each genotype strain during shake-flask fermentation.

[0084] As shown in Table 4, the metabolically modified Corynebacterium glutamicum strain, due to its ability to produce and accumulate D-pantothenic acid extracellularly, can utilize carbon sources such as glucose for D-pantothenic acid production better than the wild type. The modified strain with the best performance increased the level of D-pantothenic acid produced during fermentation from 0.3 g / L to 0.94 g / L compared to the original strain. Example 10: Screening and tandem expression of the ilvBNCD gene; fermentation test in a 5L fermenter.

[0085] The optimal strain DPA3, selected for shake-flask testing, was used to test its D-pantothenic acid production performance in a 5L fermenter. The strain was streaked onto LB agar plates and incubated overnight at 30°C. A single colony was picked and inoculated into 10ml of LB medium, incubated at 30°C for 14 hours. Then, 1ml of the shake-flask culture was inoculated into a 500ml Erlenmeyer flask containing 100ml of seed culture (LB liquid) and incubated at 200rpm at 30°C for 14 hours. 200ml of the seed culture was then inoculated into a 5L fermenter (BIOTECH-5JG) containing 1.5L of fermentation medium. Feed-batch culture was added, and the fermentation cycle was 50-100 hours. During fermentation, the glucose concentration was maintained at 1-5g / L through feed-batch culture. The dissolved oxygen (DO) level was controlled at approximately 20% during fermentation using a stirred-coupled dissolved oxygen (DO) mode. The stirring speed was maintained at 300–600 rpm, and the aeration rate at 1–2 vvm. The fermentation temperature was controlled at 30℃, and the pH was adjusted to 6.8 ± 0.2 using 50% ammonia. The fermentation medium consisted of: glucose 30g, corn steep liquor 25g, ammonium sulfate 15g, ammonium acetate 15g, urea 2g, sodium citrate 2g, K₂HPO₄·3H₂O 1.3g, MgSO₄·7H₂O 0.5g, L-isoleucine 0.06g, MnSO₄·H₂O 0.01g, and biotin 1x10g. -4 Thiamine 2x10 -4CaCO3 30, pH 7.0-7.2. Sterilize at 115℃ for 10 min. The fermentation curve during the fermentation process is shown in Figure 3. According to Figure 3, after 80 h, the yield of D-pantothenic acid is 11.9 g / L, and the sugar-acid conversion rate is 0.109 g / g.

Claims

1. A method for constructing Corynebacterium glutamicum for efficient production of D-pantothenic acid, characterized in that, Specifically, the steps include the following: (1) Using wild-type Corynebacterium glutamicum ATCC 13032 as a chassis, ilvA was weakened and DPA1 was constructed by mutation expression; (2) Knock out pyruvate quinone oxidoreductase and insert a panBCE gene expression cassette into it to construct DPA2; (3) The pEC-xk99E overexpression plasmid was introduced into DPA2 to screen for the expression of Escherichia coli W3110, Bacillus subtilis 168, as well as endogenous acetylhydroxy acid synthase, acetyl lactate isomerase and 2-hydroxy acid dehydratase. (4) The screened ilvBNCD gene was overexpressed in tandem by pEC-xk99E under the control of a strong promoter to construct DPA3, thereby strengthening the main biosynthetic pathway of pantothenic acid and the upstream pathway, thus obtaining the Corynebacterium glutamicum that produces D-pantothenic acid efficiently.

2. The method for constructing Corynebacterium glutamicum for efficient production of D-pantothenic acid as described in claim 1, characterized in that, In wild-type Corynebacterium glutamicum ATCC 13032, the start codon of ilvA is changed from ATG to GTG.

3. The method for constructing Corynebacterium glutamicum for efficient D-pantothenic acid production as described in claim 1, characterized in that, panB, panC, and panE are all integrated into the pyruvate quinone oxidoreductase gene site.

4. The method for constructing Corynebacterium glutamicum for efficient D-pantothenic acid production as described in claim 3, characterized in that, The panB and panC integrated into the pqo gene locus are derived from Corynebacterium glutamicum ATCC 13032, and panE is derived from Escherichia coli W3110; the ilvBN is derived from Corynebacterium glutamicum ATCC 13032, the ilvC is derived from Bacillus subtilis 168, and the ilvD is derived from Escherichia coli W3110.

5. The method for constructing Corynebacterium glutamicum for efficient D-pantothenic acid production as described in claim 1, characterized in that, panB, panC, panE, ilvBN, ilvC, and ilvD are all expressed by strong promoters.

6. The method for constructing Corynebacterium glutamicum for efficient D-pantothenic acid production as described in claim 5, characterized in that, The strong promoter is Ptuf, and its nucleotide sequence is SEQ ID NO.

1.

7. A highly efficient Corynebacterium glutamicum for the production of D-pantothenic acid, characterized in that, It is prepared by any one of the methods in claims 1-6.

8. The application of Corynebacterium glutamicum as described in claim 7 in the fermentation production of D-pantothenic acid.

9. The application as described in claim 8, characterized in that, The fermentation process employs an electroporation method.

10. The application as described in claim 9, characterized in that, The application involves inoculating the aforementioned Corynebacterium glutamicum into a fermentation medium, fermenting and culturing it under suitable conditions, and then separating and purifying the culture broth to obtain D-pantothenic acid.

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