A stable and high-yielding engineered bacterium for D-pantothenic acid production, its construction method and application

Abstract

This invention relates to the field of genetic engineering technology, and discloses an engineered bacterium that stably produces high levels of D-pantothenic acid, its construction method, and its applications. This invention utilizes metabolic engineering techniques to modify key genes in its metabolic pathway. Specifically, by enhancing the expression of the ysaA gene, the methylenetetrahydrofolate (MTF) biosynthesis pathway is modified, increasing the MTF level, which leads to an increase in the level of ketopantothenic acid, a key intermediate in the pantothenic acid biosynthesis pathway, thereby increasing the yield of D-pantothenic acid. Simultaneously, knocking out the spoIVB and / or spoIIIE genes in the genome reduces the energy consumption and material waste associated with spore formation, promotes the generation of secondary metabolites, and further increases the synthesis of D-pantothenic acid. Additionally, knocking out the prophage1 (alkA-ybdO), prophage3 (ydiM-ydjJ), and prophage6 (yobB-yobO) regions in the genome reduces genomic redundancy, improves the metabolic efficiency and stability of the strain, and thus effectively increases the synthesis of D-pantothenic acid.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to an engineered bacterium that stably produces high levels of D-pantothenic acid, its construction method, and its applications. Background Technology

[0002] Pantothenic acid, also known as vitamin B5, is a water-soluble vitamin. It is widely found in various foods, such as meats (chicken, beef, fish), whole grains, nuts, leafy green vegetables, and whole grains. Vitamin B5 promotes cell metabolism, helps repair and regenerate damaged cells, thus maintaining the health of skin, hair, and other tissues; it participates in the synthesis of neurotransmitters, such as acetylcholine, helping to maintain the normal function and health of the nervous system; and it is a component of coenzyme A, promoting the metabolism of carbohydrates, fats, and proteins, providing energy for the body. This function is crucial for maintaining normal physiological functions. Acetyl-CoA participates in the synthesis of acetylcholine and acetylglucosamine, as well as the biosynthesis of steroidal compounds. It has oxidizing effects on fatty acids, pyruvate, α-ketoglutarate, and acetaldehyde. Malonyl-CoA plays an important role in the biosynthesis of fatty acids. Acyl-CoA derivatives participate in the synthesis of triglycerides and phospholipids. Therefore, pantothenic acid functions in all cellular metabolisms through coenzyme A. Pantothenic acid exists in two configurations, D and L, but only the D-form (D-PA) is biologically active.

[0003] Currently, methods for producing D-pantothenic acid include physical-induced crystallization, chemical resolution, and microbial methods. The physical-induced crystallization method is a mature process. The main steps involve condensing calcified β-alanine with D,L-pantolactone to obtain a mixed solution of D,L-calcium pantothenate, then adding D-calcium pantothenate seed crystals to induce crystallization and resolve D-calcium pantothenate. However, it can only produce calcium pantothenate and cannot be used for the production of other pantothenic acid derivatives. The chemical resolution method involves adding chemical resolving agents such as quinine, chloramphenicol, and ephedrine to the racemic D,L-pantolactone to obtain D-pantolactone, which is then reacted to yield D-calcium pantothenate. This is currently the most important synthetic method, but it generates significant amounts of waste, is expensive, difficult to separate, and causes severe environmental pollution, making sustainable development impossible.

[0004] With the development of genetic engineering technology, the production of D-pantothenic acid using microorganisms has received increasing attention due to its advantages. Bio-fermentation production of D-pantothenic acid utilizes inexpensive industrial raw materials such as glucose, and the product is obtained through the body's own metabolic reactions. By rationally utilizing microorganisms in D-pantothenic acid production, not only can the quality of pantothenic acid resolution be guaranteed, but reaction costs can also be reduced. Compared to chemical methods, biological methods have advantages such as being environmentally friendly, having a shorter process route, producing superior product quality, and exhibiting good stability and mixability. However, currently, bio-fermentation production of D-pantothenic acid still faces problems such as low fermentation yield and fermentation instability. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an engineered bacterium that stably and efficiently produces high levels of D-pantothenic acid. This invention utilizes metabolic engineering techniques to modify key genes in its metabolic pathway. Specifically, by enhancing the expression of the ysaA gene, the methylenetetrahydrofolate (MTF) biosynthesis pathway is modified. Increasing MTF levels leads to an increase in the level of ketopantothenic acid, a key intermediate in the pantothenic acid biosynthesis pathway, thereby increasing D-pantothenic acid production. Simultaneously, knocking out the spoIVB and / or spoIIIE genes in the genome reduces energy consumption and material waste during spore formation, promotes the generation of secondary metabolites, and further increases D-pantothenic acid synthesis. Additionally, knocking out the prophage1 (alkA-ybdO), prophage3 (ydiM-ydjJ), and prophage6 (yobB-yobO) regions in the genome reduces genomic redundancy, improves the strain's metabolic efficiency and stability, and further increases D-pantothenic acid synthesis.

[0006] The specific technical solution of this invention is as follows:

[0007] In a first aspect, the present invention provides a stable, high-yielding engineered bacterium for producing D-pantothenic acid, wherein the engineered bacterium overexpresses the ysaA gene, knocks out the spoIVB or / and spoIIIE genes in the genome, and knocks out the prophage1 (alkA-ybdO), prophage3 (ydiM-ydiJ), and prophage6 (yoaM-yobO) regions.

[0008] Pantothenic acid is produced from α-ketoisovalerate and L-aspartic acid through a four-step enzymatic reaction. Finally, under the catalysis of pantothenic acid synthase, ATP provides energy to connect β-alanine and pantothenic acid to form pantothenic acid.

[0009] This invention utilizes metabolic engineering techniques to modify key genes in the metabolic pathway. Specifically, by enhancing the expression of the ysaA gene, the methylenetetrahydrofolate (MTF) biosynthesis pathway is modified, increasing MTF levels. This leads to an increase in the level of ketopantolytic acid, a key intermediate in the pantothenic acid biosynthesis pathway, thereby increasing D-pantothenic acid production. Simultaneously, knocking out the spoIVB and / or spoIIIE genes in the genome reduces energy consumption and material waste during spore formation, promotes the generation of secondary metabolites, and further increases D-pantothenic acid synthesis. Additionally, knocking out the prophage1 (alkA-ybdO), prophage3 (ydiM-ydjJ), and prophage6 (yobB-yobO) regions in the genome reduces genomic redundancy, improves the metabolic efficiency and stability of the strain, and further increases D-pantothenic acid synthesis.

[0010] As a preferred choice, the substrate fungus is Bacillus subtilis 168.

[0011] Preferably, the ysaA gene sequence is shown in SEQ ID No. 2, the spoIVB gene sequence is shown in SEQ ID No. 4, and the spoIIIE gene sequence is shown in SEQ ID No. 5.

[0012] Secondly, the present invention provides a method for constructing the above-mentioned engineered bacteria that stably and highly produce D-pantothenic acid, characterized by comprising the following steps:

[0013] Step S1: Enhance the expression of the ysaA gene in the Chameleonella genome;

[0014] Step S2: Knock out the spoIVB and / or spoIIIE genes;

[0015] Step S3: Knock out the regions prophage1(alkA-ybdO), prophage3(ydiM-ydiJ), and prophage6(yoaM-yobO).

[0016] Preferably, the substrate bacterium is Bacillus subtilis 168.

[0017] Preferably, the ysaA gene sequence is shown in SEQ ID No. 2, the spoIVB gene sequence is shown in SEQ ID No. 4, and the spoIIIE gene sequence is shown in SEQ ID No. 5.

[0018] As a preferred method, the expression of the ysaA gene in the cytomegalovirus genome is to replace the promoter of the ysaA gene with the P43 promoter.

[0019] Preferably, the gene sequence of the P43 promoter is shown in SEQ ID No. 1.

[0020] Thirdly, the present invention provides the application of the above-mentioned engineered bacteria in the production of D-pantothenic acid.

[0021] Compared with the prior art, the present invention has the following technical effects:

[0022] This invention uses *Bacillus subtilis* as the substrate bacterium and employs metabolic engineering techniques to modify key genes in its metabolic pathways. Specifically, by enhancing the expression of the ysaA gene, the methylenetetrahydrofolate (MTF) biosynthesis pathway is modified, increasing MTF levels. This leads to an increase in the level of ketopantothenic acid, a key intermediate in the pantothenic acid biosynthesis pathway, thereby increasing D-pantothenic acid production. Simultaneously, knocking out the spoIVB and / or spoIIIE genes in the genome reduces energy consumption and material waste during spore formation, promotes the generation of secondary metabolites, and further increases D-pantothenic acid synthesis. Additionally, knocking out the prophage1 (alkA-ybdO), prophage3 (ydiM-ydjJ), and prophage6 (yobB-yobO) regions in the genome reduces genomic redundancy, improves the metabolic efficiency and stability of the strain, and further increases D-pantothenic acid synthesis. Based on this, a genetically engineered Bacillus subtilis strain with high D-pantothenic acid production was constructed, which improved its growth status and stability in the fermentation preparation of D-pantothenic acid, thus enabling efficient production of D-pantothenic acid. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the D-pantothenic acid metabolic pathway modification sites of the present invention;

[0024] Figure 2 The OD of the engineered bacteria constructed in Example 2 600 And the results of D-pantothenic acid content in fermentation broth supernatant;

[0025] Figure 3 The OD of the engineered bacteria constructed in Example 3 600 And the results of D-pantothenic acid content in fermentation broth supernatant;

[0026] Figure 4 The OD of the engineered bacteria constructed in Example 4 600 And the results of D-pantothenic acid content in fermentation broth supernatant;

[0027] Figure 5 The OD of the engineered bacteria constructed in Example 5 600 And the results of D-pantothenic acid content in fermentation broth supernatant;

[0028] Figure 6 OD of the engineered bacteria constructed in Example 6 600 And the results of D-pantothenic acid content in fermentation broth supernatant;

[0029] Figure 7 OD of the engineered bacteria constructed in Example 7 600 And the results of D-pantothenic acid content in fermentation broth supernatant;

[0030] Figure 8OD of the engineered bacteria constructed in Example 8 600 The results of D-pantothenic acid content in the fermentation broth supernatant are shown in the figure. Detailed Implementation

[0031] 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.

[0032] In this embodiment of the invention, the genotype B. subtilis 168P43-panB panC panD panE ilvC ilvD serC aspC was used as the substrate bacteria for experiments to verify the technical solution of the invention. This genotype strain uses Bacillus subtilis 168 as the substrate bacteria, and its construction method includes the following steps:

[0033] (1) Using Bacillus subtilis as the substrate bacteria, the Cre / loxP gene editing system was employed.

[0034] The promoter of the panB gene in its genome was replaced with the P43 promoter to obtain the engineered bacterium Bacillus subtilis (P43-panB);

[0035] (2) The promoter of the panC gene in the genome of engineered bacterium Bacillus subtilis (P43-panB) was replaced with the P43 promoter using the Cre / loxP gene editing system to obtain engineered bacterium Bacillus subtilis (P43-panBpanC).

[0036] (3) The promoter of the panD gene in the genome of engineered bacterium Bacillus subtilis (P43-panBpanC) was replaced with the P43 promoter using the Cre / loxP gene editing system to obtain engineered bacterium Bacillus subtilis (P43-panBpanC panD);

[0037] (4) The promoter of the panE gene in the genome of engineered bacterium Bacillus subtilis (P43-panBpanCpanD) was replaced with the P43 promoter using the Cre / loxP gene editing system to obtain engineered bacterium Bacillus subtilis (P43-panBpanC panD panE).

[0038] (5) The promoter of the ilvC gene in the genome of engineered bacterium Bacillus subtilis (P43-panBpanCpanDpanE) was replaced with the P43 promoter using the Cre / loxP gene editing system to obtain engineered bacterium Bacillus subtilis (P43-panBpanCpanDpanEilvC);

[0039] (6) The promoter of the ilvD gene in the genome of engineered bacterium Bacillus subtilis (P43-panBpanCpanDpanEilvC) was replaced with the P43 promoter using the Cre / loxP gene editing system to obtain engineered bacterium Bacillus subtilis (P43-panBpanCpanDpanEilvCilvD).

[0040] (7) The promoter of the serC gene in the genome of engineered bacterium Bacillus subtilis (P43-panBpanCpanDpanEilvCilvD) was replaced with the P43 promoter using the Cre / loxP gene editing system to obtain engineered bacterium Bacillus subtilis (P43-panBpanCpanDpanEilvCilvDserC);

[0041] (8) Using the Cre / loxP gene editing system, the promoter of the aspC gene in the genome of the engineered bacterium Bacillus subtilis (P43-panBpanCpanDpanEilvCilvDserC) was replaced with the P43 promoter to obtain the engineered bacterium Bacillus subtilis (P43-panBpanCpanDpanEilvCilvDserCaspC), which is the chassis strain DPA8 (B. subtilis ATCC 6633 (P43-panBpanCpanDpanEilvCilvDserCaspC) of this invention.

[0042] The D-pantothenic acid metabolic pathway and the modified sites of this invention are described in [link to invention]. Figure 1 The genes and corresponding pathways involved in this invention and its embodiments are shown in Table 1.

[0043] Table 1. Genes involved in gene editing and corresponding pathways.

[0044]

[0045] In Table 1, the ysaA gene sequence is shown in SEQ ID No. 2, the spo0A gene sequence is shown in SEQ ID No. 3, the spoIVB gene sequence is shown in SEQ ID No. 4, and the spoIIIE gene sequence is shown in SEQ ID No. 5.

[0046] The primer information involved in the embodiments of this invention is shown in Table 2.

[0047] Table 2 Primer sequences

[0048]

[0049]

[0050] In this embodiment of the invention, unless otherwise specified, the final concentration of bleomycin in the culture medium is 20 mg / L, the final concentration of kanamycin in the culture medium is 50 mg / L, and the final concentration of IPTG in the culture medium is 0.2 mmol / L.

[0051] Example 1: Determination of D-pantothenic acid content

[0052] The detection method is as follows:

[0053] Chromatographic conditions: C18 column (250×4.6mm, particle size 5μm, Agilent), detection wavelength 200nm, column temperature 30℃;

[0054] Sample preparation: Dilute the sample with ultrapure water to maintain the D-pantothenic acid content between 0.5 g / L and 1.0 g / L;

[0055] Mobile phase: Acetonitrile:water:phosphoric acid = 50:949:1 (volume ratio);

[0056] Data acquisition time: 15 minutes.

[0057] Example 2: Construction of strain DPA8 (P43-ysaA) and shake-flask fermentation. Using DPA8 (B. subtilis 168P43-panBpanC panD panE ilvC ilvD serC aspC) as the starting strain, Cre / loxP gene editing technology was employed. The original promoter of the ysaA gene was replaced in the genome with the P43 promoter derived from pP43NMK (nucleotide sequence shown in SEQ ID NO.1), enhancing the expression intensity of the ysaA gene and thus increasing the yield of D-pantothenic acid. The specific steps are as follows:

[0058] (1) Construction of the upstream sequence of the target gene ysaA: Using the genome of Bacillus subtilis DPA8 (B. subtilis 168P43-panBpanC panD panE ilvC ilvD serC aspC) as a template, PCR amplification was performed using ysaA-LF and ysaA-LR as primers. After amplification, the product was verified by gel electrophoresis, cleaned up and purified for later use.

[0059] (2) Construction of lox71-zeo-lox66 sequence: Using plasmid p7z6 as template, PCR amplification was performed using ysaA-zeo-F and ysaA-zeo-R as primers. After amplification, the product was verified by gel running, cleaned up and purified for later use.

[0060] (3) Construction of P43 promoter sequence: Using plasmid pP43NMK as template, PCR amplification was performed using ysaA-P43-F and ysaA-P43-R as primers. After amplification, the product was verified by gel running, cleaned up and purified for later use.

[0061] (4) Construction of the target gene ysaA sequence: Using the genome of Bacillus subtilis DPA8 (B. subtilis 168P43-panBpanC panD panE ilvC ilvD serC aspC) as a template, PCR amplification was performed using ysaA-F and ysaA-R as primers. After amplification, the product was verified by gel electrophoresis, cleaned up and purified for later use.

[0062] (5) Using fusion PCR technology, the upstream of the target gene, the lox71-zeo-lox66, the P43 promoter, and the ysaA target gene sequence were fused to obtain a promoter replacement frame. The promoter replacement frame was transformed into Bacillus subtilis DPA8 (B. subtilis 168P43-panB panC panD panE ilvC ilvD serC aspC). Bleomycin-resistant LB plates were used for selection. After PCR verification, the transformants were transformed into plasmid PDG148. The transformants carrying the PDG148 plasmid were inoculated into a solution containing 0.2 mmol / L... In IPTG-containing liquid LB medium, the culture was carried out for 24 h to express cyclization recombinase to promote recombination at lox71 and lox66 sites, and the bleomycin resistance gene was recovered. Then, 10 μL of the culture medium was inoculated into 10 mL of fresh LB liquid medium and cultured at 50 °C and 220 rpm for 10 h with shaking. The culture was then streaked onto antibiotic-free LB solid plates. Using a 10 μL pipette tip, the colonies grown on the antibiotic-free LB solid plates were spotted onto bleomycin, kanamycin, and antibiotic-free LB solid plates, respectively. The strain that could only grow on antibiotic-free solid plates was the engineered strain DPA8 (P43-ysaA) with the PDG148 plasmid eliminated.

[0063] (6) The DPA8 (P43-ysaA) strain, with DPA8 (B. subtilis 168P43-panB panC panD panEilvC ilvD serC aspC) as the control group, was inoculated into 10 mL of LB liquid medium and cultured overnight at 37°C and 200 rpm. 1 mL of the pre-culture was then inoculated into a 500 mL shake flask containing 50 mL of fermentation medium, and cultured continuously at 30°C and 300 rpm for 48 h. After fermentation, 1 mL of the fermentation broth was centrifuged at 12000 rpm for 3 min at room temperature. The supernatant was diluted 5 times and analyzed by HPLC according to Example 1. OD 600 and the D-pantothenic acid content in the fermentation broth supernatant, such as Figure 2 As shown.

[0064] Depend on Figure 2 It is evident that replacing the ysaA gene promoter in the genome increased the production of D-pantothenic acid from 2.65 g / L to 2.93 g / L. The reason for this is that enhanced expression of the ysaA gene increased the supply of one-carbon units, and increased MTF levels can lead to increased levels of ketopantolytic acid, a key intermediate in the pantothenic acid biosynthesis pathway, which is beneficial to the synthesis of D-pantothenic acid in Bacillus subtilis.

[0065] 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.

[0066] The fermentation medium consists of the following components: glucose 20 g / L, ammonium sulfate 16 g / L, potassium dihydrogen phosphate 0.8 g / L, magnesium sulfate 0.5 g / L, yeast extract 2 g / L, and a 1 mL / L trace element solution in deionized water. The pH is 6-7. 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.

[0067] Example 3: Construction of strain DPA8 (P43-ysaA△spo0A) and shake-flask fermentation

[0068] Using the DPA8(P43-ysaA) strain constructed in Example 2 as the starting strain, the spo0A gene in the genome was knocked out using Cre / loxP gene editing technology to reduce the energy consumption for spore formation. The specific steps are as follows:

[0069] (1) Construction of upstream sequence of target gene spo0A: Using the DPA8 (P43-ysaA) genome as a template, PCR amplification was performed using spo0A-LF and spo0A-LR as primers. After amplification, the product was verified by gel electrophoresis, cleaned up and purified for later use.

[0070] (2) Construction of lox71-zeo-lox66 sequence: Using plasmid p7z6 as template, PCR amplification was performed using spo0A-zeo-F and spo0A-zeo-R as primers. After amplification, the product was verified by gel running, cleaned up and purified for later use.

[0071] (3) Construction of the target gene spo0A sequence: Using the DPA8 (P43-ysaA) genome as a template, PCR amplification was performed using spo0A-F and spo0A-R as primers. After amplification, the product was verified by gel electrophoresis, cleaned up and purified for later use.

[0072] (4) Using fusion PCR technology, the upstream, lox71-zeo-lox66, and spo0A target gene sequences were fused to obtain a promoter substitution frame. The promoter substitution frame was transformed into DPA8(P43-ysaA), and the transformants were screened using bleomycin-resistant LB plates. After the transformants were verified to be correct by PCR, they were transformed into plasmid PDG148. The transformants carrying plasmid PDG148 were inoculated into a solution containing 0.2 mmol / L... In IPTG-containing liquid LB medium, the culture was carried out for 24 h to express cyclization recombinase to promote recombination at lox71 and lox66 sites, and the bleomycin resistance gene was recovered. Then, 10 μL of the culture medium was inoculated into 10 mL of fresh LB liquid medium and cultured at 50 °C and 220 rpm for 10 h with shaking. The culture was then streaked onto antibiotic-free LB solid plates. Using a 10 μL pipette tip, the colonies grown on the antibiotic-free LB solid plates were spotted onto bleomycin, kanamycin, and antibiotic-free LB solid plates, respectively. The strain that could only grow on antibiotic-free solid plates was the engineered strain DPA8 (P43-ysaA△spo0A) with the PDG148 plasmid eliminated.

[0073] (5) Using DPA8(P43-ysaA△spo0A) strain as a control, 10 mL of each strain was inoculated into LB liquid medium and cultured overnight at 37°C and 200 rpm. 1 mL of the pre-culture was then inoculated into a 500 mL shake flask containing 40 mL of fermentation medium, and cultured continuously at 37°C and 300 rpm for 48 h. After fermentation, 1 mL of the fermentation broth was centrifuged at 12000 rpm for 3 min at room temperature. The supernatant was diluted 5 times and analyzed by HPLC according to Example 1. OD 600 and the D-pantothenic acid content in the fermentation broth supernatant, such as Figure 3 As shown.

[0074] Depend on Figure 3 It is evident that knocking out the spo0A gene in the genome reduced the yield of D-pantothenic acid from 2.88 g / L to 1.88 g / L. Further analysis suggests that the reason for this is that the growth rate of strains with the spo0A gene knocked out increased, which, under fermentation conditions, affected the distribution of intracellular nutrients and metabolic flux, thus hindering the synthesis of D-pantothenic acid in Bacillus subtilis.

[0075] Example 4: Construction of an effective strain DPA8 (P43-ysaA△spoIVB) and shake-flask fermentation. Using the DPA8 (P43-ysaA) strain constructed in Example 2 as the starting strain, the spoIVB gene in the genome was knocked out using Cre / loxP gene editing technology, blocking the formation of mature spores and thus increasing the yield of D-pantothenic acid. The specific steps are as follows:

[0076] (1) Construction of upstream sequence of target gene spoIVB: Using the DPA8 (P43-ysaA) genome as a template, PCR amplification was performed using spoIVB-LF and spoIVB-LR as primers. After amplification, the product was verified by gel electrophoresis, cleaned up and purified for later use.

[0077] (2) Construction of lox71-zeo-lox66 sequence: Using plasmid p7z6 as template, PCR amplification was performed using spoIVB-zeo-F and spoIVB-zeo-R as primers. After amplification, the product was verified by gel running, cleaned up and purified for later use.

[0078] (3) Construction of the target gene spoIVB sequence: Using the DPA8 (P43-ysaA) genome as a template, PCR amplification was performed using spoIVB-F and spoIVB-R as primers. After amplification, the product was verified by gel electrophoresis, cleaned up and purified for later use.

[0079] (4) Using fusion PCR technology, the upstream, lox71-zeo-lox66, and spoIVB target gene sequences were fused to obtain a promoter substitution frame. This frame was then transformed into DPA8 (P43-ysaA), and the transformants were screened using bleomycin-resistant LB plates. After PCR verification, the transformants were transformed into plasmid PDG148. Transformants carrying the PDG148 plasmid were inoculated into liquid LB containing 0.2 mmol / L IPTG and cultured for 24 h to express cyclization recombinase to promote lox71-zeo-lox66 expression. Recombination at the x71 and lox66 sites yielded the bleomycin resistance gene. Then, 10 μL of the culture medium was inoculated into 10 mL of fresh LB liquid medium and cultured at 50°C and 220 rpm for 10 h with shaking. The culture was then streaked onto antibiotic-free LB agar plates. Colonies grown on the antibiotic-free LB agar plates were spotted with a 10 μL pipette tip onto bleomycin, kanamycin, and antibiotic-free LB agar plates, respectively. Only those colonies that grew on antibiotic-free agar plates were identified as the engineered DPA8 (P43-ysaA△spoIVB) strain with the PDG148 plasmid eliminated.

[0080] (5) The DPA8(P43-ysaA△spoIVB) strain, with DPA8(P43-ysaA) as the control group, was inoculated into 10 mL of LB liquid medium and cultured overnight at 37°C and 200 rpm. 1 mL of the pre-culture was then inoculated into a 500 mL shake flask containing 50 mL of fermentation medium, and cultured continuously at 37°C and 200 rpm for 48 h. After fermentation, 1 mL of the fermentation broth was centrifuged at 12000 rpm for 3 min at room temperature. The supernatant was diluted 5 times and analyzed by HPLC according to Example 1. OD 600and the D-pantothenic acid content in the fermentation broth supernatant, such as Figure 4 As shown.

[0081] Depend on Figure 4 It is evident that knocking out spoIVB in the genome increased the production of D-pantothenic acid from 2.91 g / L to 3.13 g / L. This indicates that knocking out the spoIVB gene can promote cell growth and block the formation of mature spores, thereby benefiting the synthesis of D-pantothenic acid in Bacillus subtilis.

[0082] Example 5: Construction of an effective strain DPA8 (P43-ysaA△spoIIIE) and shake-flask fermentation. Using the DPA8 (P43-ysaA) strain constructed in Example 2 as the starting strain, Cre / loxP gene editing technology was used to knock out the spoIIIE gene in the genome, inhibiting spore formation and thus increasing the yield of D-pantothenic acid. The specific steps are as follows:

[0083] (1) Construction of upstream sequence of target gene spoIIIE: Using the DPA8 (P43-ysaA) genome as a template, PCR amplification was performed using spoIIIE-LF and spoIIIE-LR as primers. After amplification, the product was verified by gel electrophoresis, cleaned up and purified for later use.

[0084] (2) Construction of lox71-zeo-lox66 sequence: Using plasmid p7z6 as template, PCR amplification was performed using spoIIIE-zeo-F and spoIIIE-zeo-R as primers. After amplification, the product was verified by gel running, cleaned up and purified for later use.

[0085] (3) Construction of the target gene spoIIIE sequence: Using the DPA8 (P43-ysaA) genome as a template, PCR amplification was performed using spoIIIE-F and spoIIIE-R as primers. After amplification, the product was verified by gel running, cleaned up and purified for later use.

[0086] (4) Using fusion PCR technology, the upstream, lox71-zeo-lox66, and spoIIIE target gene sequences were fused to obtain a promoter substitution frame. The promoter substitution frame was transformed into DPA8(P43-ysaA), and the transformants were screened using bleomycin-resistant LB plates. After the transformants were verified to be correct by PCR, they were transformed into plasmid PDG148. The transformants carrying plasmid PDG148 were inoculated into a solution containing 0.2 mmol / L... In IPTG-containing liquid LB medium, the cells were cultured for 24 hours to express cyclization recombinase to promote recombination at lox71 and lox66 sites, and the bleomycin resistance gene was recovered. Then, 10 μL of the culture was inoculated into 10 mL of fresh LB liquid medium and cultured at 50°C and 220 rpm for 10 hours with shaking. The culture was then streaked onto antibiotic-free LB agar plates. Using a 10 μL pipette tip, colonies grown on the antibiotic-free LB agar plates were spotted onto bleomycin, kanamycin, and antibiotic-free LB agar plates, respectively. Only colonies that grew on antibiotic-free agar plates were considered engineered to have the PDG148 plasmid eliminated.

[0087] DPA8(P43-ysaAΔspoIIIE) strain.

[0088] (5) The DPA8(P43-ysaA△spoIIIE) strain, with DPA8(P43-ysaA) as the control group, was inoculated into 10 mL of LB liquid medium and cultured overnight at 37°C and 200 rpm. 1 mL of the pre-culture was then inoculated into a 500 mL shake flask containing 50 mL of fermentation medium, and cultured continuously at 37°C and 200 rpm for 48 h. After fermentation, 1 mL of the fermentation broth was centrifuged at 12000 rpm for 3 min at room temperature. The supernatant was diluted 5 times and analyzed by HPLC according to Example 1. OD 600 The D-pantothenic acid content in 0 and fermentation broth supernatant is as follows Figure 5 As shown.

[0089] Depend on Figure 5 It is evident that knocking out spoIIIE from the genome increases the production of D-pantothenic acid from 2.90 g / L to 3.23 g / L. Knocking out the spoIIIE gene can inhibit spore formation without affecting the function of the strain itself, thus benefiting the synthesis of D-pantothenic acid in Bacillus subtilis.

[0090] Example 6: Construction of an effective strain DPA8 (P43-ysaA△spoIIIE△prophahe1) and shake-flask fermentation. Using the DPA8 (P43-ysaA△spoIIIE) strain constructed in Example 5 as the starting strain, Cre / loxP gene editing technology was used to knock out the prophage1 region (alkA-ydbO) in the genome, reducing genomic redundancy and thus increasing the yield of D-pantothenic acid. The specific steps are as follows:

[0091] (1) Construction of the upstream sequence of prophage1 in the target region: Using the DPA8 (P43-ysaA△spoIIIE) genome as a template, PCR amplification was performed using prophage1-LF and prophage1-LR as primers. After amplification, the product was verified by gel running, cleaned up and purified for later use.

[0092] (2) Construction of lox71-zeo-lox66 sequence: Using plasmid p7z6 as template, PCR amplification was performed using prophage1-zeo-F and prophage1-zeo-R as primers. After amplification, the product was verified by gel running, cleaned up and purified for later use.

[0093] (3) Construction of the target region prophage1 sequence: Using the DPA8 (P43-ysaA△spoIIIE) genome as a template, PCR amplification was performed using prophage1-F and prophage1-R as primers. After amplification, the product was verified by gel running, cleaned up and purified for later use.

[0094] (4) The upstream, lox71-zeo-lox66, and prophage1 target gene sequences were fused using fusion PCR to obtain a promoter substitution frame. The promoter substitution frame was transformed into DPA8 (P43-ysaA△spoIIIE), and the transformants were screened using bleomycin-resistant LB plates. After the transformants were verified to be correct by PCR, they were transformed into plasmid PDG148. The transformants carrying plasmid PDG148 were inoculated into a solution containing 0.2 mmol / L... In IPTG-containing liquid LB medium, the culture was carried out for 24 h to express cyclization recombinase to promote recombination at lox71 and lox66 sites, and the bleomycin resistance gene was recovered. Then, 10 μL of the culture medium was inoculated into 10 mL of fresh LB liquid medium and cultured at 50 °C and 220 rpm for 10 h with shaking. The culture was then streaked onto antibiotic-free LB solid plates. Using a 10 μL pipette tip, the colonies grown on the antibiotic-free LB solid plates were spotted onto bleomycin, kanamycin, and antibiotic-free LB solid plates, respectively. Only the colonies that could grow on antibiotic-free solid plates were the engineered strain DPA8 (P43-ysaA△spoIIIE△prophahe1) with the PDG148 plasmid eliminated.

[0095] (5) Using DPA8(P43-ysaA△spoIIIE△prophahe1) strain as a control, 10 mL of each strain was inoculated into LB liquid medium and cultured overnight at 37°C and 200 rpm. 1 mL of the pre-culture was then inoculated into a 500 mL shake flask containing 50 mL of fermentation medium, and cultured continuously at 37°C and 200 rpm for 48 h. After fermentation, 1 mL of the fermentation broth was centrifuged at 12000 rpm for 3 min at room temperature. The supernatant was diluted 5 times and analyzed by HPLC according to Example 1. OD 600 and the D-pantothenic acid content in the fermentation broth supernatant, such as Figure 6 As shown.

[0096] Depend on Figure 6 It is evident that knocking out the prophahe1 region in the genome increased the production of D-pantothenic acid from 3.23 g / L to 3.38 g / L. Knocking out the prophahe1 region in the genome can reduce genomic redundancy and improve the metabolic efficiency of the strain, thereby benefiting the synthesis of D-pantothenic acid in Bacillus subtilis.

[0097] Example 7: Construction of effective strain DPA8 (P43-ysaA△spoIIIE△prophahe1△prophahe3) and shake-flask fermentation.

[0098] Using the DPA8 (P43-ysaA△spoIIIE△prophahe1) strain constructed in Example 6 as the starting strain, the prophahe3 region in the genome was knocked out using Cre / loxP gene editing technology, further reducing genomic redundancy and thus increasing the yield of D-pantothenic acid. The specific steps are as follows:

[0099] (1) Construction of upstream sequence of target gene prophahe3: Using the DPA8(P43-ysaA△spoIIIE△prophahe1) genome as a template, PCR amplification was performed using prophahe3-LF and prophahe3-LR as primers. After amplification, the product was verified by gel running, cleaned up and purified for later use.

[0100] (2) Construction of lox71-zeo-lox66 sequence: Using plasmid p7z6 as template, PCR amplification was performed using prophahe3-zeo-F and prophahe3-zeo-R as primers. After amplification, the product was verified by gel running, cleaned up and purified for later use.

[0101] (3) Construction of the target gene prophahe3 sequence: Using the DPA8 (P43-ysaA△spoIIIE△prophahe1) genome as a template, PCR amplification was performed using prophahe3-F and prophahe3-R as primers. After amplification, the product was verified by gel running, cleaned up and purified for later use.

[0102] (4) Using fusion PCR technology, the upstream, lox71-zeo-lox66, and prophahe3 target gene sequences were fused to obtain a promoter substitution frame. This frame was then transformed into DPA8 (P43-ysaA△spoIIIE△prophahe1). Transformants were screened using bleomycin-resistant LB plates. After PCR verification, the transformants were transformed into plasmid PDG148. Transformants carrying PDG148 plasmid were inoculated into liquid LB containing 0.2 mmol / L IPTG and cultured for 24 h to express cyclization recombinase to promote lox71-zeo-lox66. Recombination at the x71 and lox66 sites yielded the bleomycin resistance gene. Then, 10 μL of the culture medium was inoculated into 10 mL of fresh LB liquid medium and cultured at 50°C and 220 rpm for 10 h with shaking. The culture was then streaked onto antibiotic-free LB agar plates. Using a 10 μL pipette tip, colonies grown on antibiotic-free LB agar plates were spotted onto bleomycin, kanamycin, and antibiotic-free LB agar plates, respectively. Only colonies that grew on antibiotic-free agar plates were identified as the engineered strain DPA8 (P43-ysaA△spoIIIE△prophahe1△prophahe3) with the PDG148 plasmid eliminated.

[0103] (5) The DPA8 (P43-ysaA△spoIIIE△prophahe1△prophahe3) strain was used as...

[0104] DPA8 (P43-ysaA△spoIIIE△prophahe1) served as the control group. Each culture was inoculated into 10 mL of LB liquid medium and cultured overnight at 37°C and 200 rpm. 1 mL of the pre-culture was then inoculated into a 500 mL shake flask containing 50 mL of fermentation medium, and continuously cultured at 30°C and 200 rpm for 48 h. After fermentation, 1 mL of the fermentation broth was centrifuged at 12000 rpm for 3 min at room temperature. The supernatant was diluted 5 times and analyzed by HPLC according to Example 1. OD 600 and the D-pantothenic acid content in the fermentation broth supernatant, such as Figure 7 As shown.

[0105] Depend on Figure 7It is evident that knocking out the prophahe3 region in the genome increased the production of D-pantothenic acid from 3.36 g / L to 3.49 g / L. The reason for this is that genome simplification enhanced the OD600 of the strain after fermentation, which is beneficial for cell growth and thus promotes the synthesis of D-pantothenic acid in Bacillus subtilis.

[0106] Example 8 describes the construction of an effective strain DPA8 (P43-ysaA△spoIIIE△prophahe1△prophahe3△prophahe6) and shake-flask fermentation. Using the DPA8 (P43-ysaA△spoIIIE△prophahe1△prophahe3) strain constructed in Example 8 as the starting strain, Cre / loxP gene editing technology was used to knock out the prophahe6 region in the genome, reducing genomic redundancy and thus increasing the yield of D-pantothenic acid. The specific steps are as follows:

[0107] (1) Construction of upstream sequence of target gene prophahe6: Using the DPA8 (P43-ysaA△spoIIIE△prophahe1△prophahe3) genome as a template, PCR amplification was performed using prophahe6-LF and prophahe6-LR as primers. After amplification, the product was verified by gel electrophoresis, cleaned up and purified for later use.

[0108] (2) Construction of lox71-zeo-lox66 sequence: Using plasmid p7z6 as template, PCR amplification was performed using prophahe6-zeo-F and prophahe6-zeo-R as primers. After amplification, the product was verified by gel running, cleaned up and purified for later use.

[0109] (3) Construction of the target region prophahe6 sequence: Using the DPA8 (P43-ysaA△spoIIIE△prophahe1△prophahe3) genome as a template, PCR amplification was performed using prophahe6-F and prophahe6-R as primers. After amplification, the product was verified by gel running, cleaned up and purified for later use.

[0110] (4) Using fusion PCR technology, the upstream, lox71-zeo-lox66, and prophahe6 target gene sequences were fused to obtain the promoter substitution frame, which was then transformed into...

[0111] In DPA8 (P43-ysaA△spoIIIE△prophahe1△prophahe3), bleomycin-resistant LB agar plates were used for selection. Transformants, after PCR verification, were transformed into plasmid PDG148. Transformants carrying the PDG148 plasmid were inoculated into liquid LB containing 0.2 mmol / L LIPTG and cultured for 24 h to express cyclization recombinase to promote recombination at lox71 and lox66 sites, thus recovering the bleomycin resistance gene. Then, 10 μL of the culture was inoculated into 10 mL of fresh LB liquid medium and cultured at 50℃ and 220 rpm for 10 h with shaking. The culture was then streaked onto antibiotic-free LB agar plates. Colonies grown on antibiotic-free LB agar plates were spotted using a 10 μL pipette tip onto bleomycin, kanamycin, and antibiotic-free LB agar plates, respectively. Only colonies growing on antibiotic-free agar plates were considered engineered strains with the PDG148 plasmid eliminated.

[0112] DPA8(P43-ysaA△spoIIIE△prophahe1△prophahe3△prophahe6).

[0113] (5) The DPA8 (P43-ysaA△spoIIIE△prophahe1△prophahe3△prophahe6) strain, with DPA8 (P43-ysaA△spoIIIE△prophahe1△prophahe3) as the control group, was inoculated into 10 mL of LB liquid medium and cultured overnight at 37°C and 200 rpm. 1 mL of the pre-culture was then inoculated into a 500 mL shake flask containing 50 mL of fermentation medium, and cultured continuously at 30°C and 200 rpm for 48 h. After fermentation, 1 mL of the fermentation broth was centrifuged at 12000 rpm for 3 min at room temperature. The supernatant was diluted 5 times and analyzed by HPLC according to Example 1. OD 600 and the D-pantothenic acid content in the fermentation broth supernatant, such as Figure 8 As shown.

[0114] Depend on Figure 8 It is evident that knocking out the prophahe6 region in the genome increased the yield of D-pantothenic acid from 3.46 g / L to 3.57 g / L. The reason for this is that the continuous simplification of the genome improved cell growth and fermentation stability, thus favoring the synthesis of D-pantothenic acid in Bacillus subtilis.

[0115] Therefore, as can be seen from Examples 2-8, by replacing the original promoter of the ysaA gene with the P43 promoter and RBS sequence derived from pP43NMK, the level of MTF in the methylenetetrahydrofolate (MTF) biosynthesis pathway can be increased by modifying the MTF biosynthesis pathway, thereby increasing the level of ketopantolytic acid, a key intermediate in the pantothenic acid biosynthesis pathway, and thus increasing the yield of D-pantothenic acid. At the same time, knocking out the spoIVB and spoIIIE genes in the genome to produce spores eliminates the energy consumption and material waste of spore formation, promotes the generation of secondary metabolites, and thus increases the synthesis of D-pantothenic acid. However, although knocking out the spo0A gene can eliminate the energy consumption of spore formation, it will lead to a decrease in yield. In addition, knocking out the prophage1 (alkA-ybdO), prophage3 (ydiM-ydjJ), and prophage6 (yobB-yobO) regions in the genome reduces genomic redundancy, which can improve the metabolic efficiency and stability of the strain and increase the yield of D-pantothenic acid. Based on the above, as described in Example 8, the present invention can construct a high-yielding Bacillus subtilis genetically engineered bacterium DPA8 (P43-ysaA△spoIIIE△prophahe1△prophahe3△prophahe6).

[0116] 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.

[0117] 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 stable, high-yielding engineered bacterium for producing D-pantothenic acid, characterized in that: In the engineered bacteria, the ysaA gene is overexpressed, the spoIVB and spoIIIE genes in the genome are knocked out, and the prophage1, prophage3, and prophage6 regions are knocked out. Among them, the ysaA gene sequence is shown in SEQ ID No. 2, the spoIVB gene sequence is shown in SEQ ID No. 4, and the spoIIIE gene sequence is shown in SEQ ID No. 5; The method for knocking out prophage1 is as follows: using Bacillus subtilis 168 P43-panB panC panD panEilvC ilvD serC aspC ysaA△spoIIIE as a template, PCR amplification is performed using prophage1-LF and prophage1-LR as primers to obtain the upstream homologous arm of prophage1; using Bacillus subtilis 168 P43-panB panCpanD panE ilvC ilvD serC aspC Using ysaA△spoIIIE as a template, PCR amplification was performed with prophage1-F and prophage1-R as primers to obtain the downstream homologous arm of prophage1; using plasmid p7z6 as a template, PCR amplification was performed with prophage1-zeo-F and prophage1-zeo-R as primers to obtain the resistance selection cassette of prophage1; the upstream homologous arm of prophage1, the downstream homologous arm of prophage1, and the resistance selection cassette of prophage1 were fused by PCR to obtain the promoter replacement frame; the promoter replacement frame was transformed into Bacillus subtilis 168, replacing the original prophage1 segment on the Bacillus subtilis genome, thus completing the knockout of prophage1, resulting in Bacillus subtilis 168 P43-panB panC panD panE ilvC ilvDserC aspC ysaA△spoIIIE△prophahe1; The method for knocking out prophage3 is as follows: using *Bacillus subtilis 168 P43-panB panC panD panEilvC ilvD serC aspC ysaA△spoIIIE△prophahe1* as a template, PCR amplification is performed using prophage3-LF and prophage3-LR as primers to obtain the upstream homologous arm of prophage3; using *Bacillus subtilis168 P43-panB panC panD panE ilvC ilvD serC aspC* as a template, PCR amplification is performed to obtain the upstream homologous arm of prophage3; using *Bacillus subtilis168 P43-panB panC panD panE ilvC ilvD serC aspC* as a template, PCR amplification is performed to obtain the upstream homologous arm of prophage3; Using ysaA△spoIIIE△prophahe1 as a template, PCR amplification was performed with prophage3-F and prophage3-R as primers to obtain the downstream homologous arm of prophage3; using plasmid p7z6 as a template, PCR amplification was performed with prophage3-zeo-F and prophage3-zeo-R as primers to obtain the resistance selection cassette of prophage3; the upstream homologous arm of prophage3, the downstream homologous arm of prophage3, and the resistance selection cassette of prophage3 were fused by PCR to obtain the promoter replacement frame; the promoter replacement frame was transformed into Bacillus subtilis 168 to replace the original prophage3 segment on the Bacillus subtilis genome, thus completing the knockout of prophage3 and obtaining Bacillus subtilis 168 P43-panB panC panD panE ilvC ilvD serC aspC ysaA△spoIIIE△prophahe1△prophahe3; The method for knocking out prophage6 is as follows: using Bacillus subtilis 168 P43-panB panC panD panEilvC ilvD serC aspC ysaA△spoIIIE△prophahe1△prophahe3 as a template, PCR amplification is performed using prophage6-LF and prophage6-LR as primers to obtain the upstream homologous arm of prophage6; using Bacillus subtilis168 P43-panB panC panD panE ilvC ilvD serC aspC Using ysaA△spoIIIE△prophahe1△prophahe3 as templates, PCR amplification was performed with prophage6-F and prophage6-R as primers to obtain the downstream homologous arm of prophage6; using plasmid p7z6 as templates, PCR amplification was performed with prophage6-zeo-F and prophage6-zeo-R as primers to obtain the resistance selection cassette of prophage6; the upstream homologous arm of prophage6, the downstream homologous arm of prophage6, and the resistance selection cassette of prophage6 were fused by PCR to obtain the promoter replacement frame; the promoter replacement frame was transformed into Bacillus subtilis 168 P43-panB panC panD panE ilvC ilvD serC aspC ysaA△spoIIIE△prophahe1△prophahe3△prophahe6; Primers prophage1-LF, prophage1-LR, prophage1-F, prophage1-R, prophage1-zeo-F, prophage1-zeo-R, prophage3-LF, prophage3-LR, prophage3-F, proph The sequences of age3-R, prophage3-zeo-F, prophage3-zeo-R, prophage6-LF, prophage6-LR, prophage6-F, prophage6-R, prophage6-zeo-F, prophage6-zeo-R are as follows: 。 2. The method for constructing engineered bacteria as described in claim 1, characterized in that: Includes the following steps: Step S1: Enhance the expression of the ysaA gene in the genome of Bacillus subtilis, the ysaA gene sequence of which is shown in SEQ ID No.2, and the Bacillus subtilis is Bacillus subtilis 168; Step S2: Knock out the spoIVB and spoIIIE genes. The spoIVB gene sequence is shown in SEQ ID No. 4, and the spoIIIE gene sequence is shown in SEQ ID No.

5. Step S3: Remove regions prophage1, prophage3, and prophage6; The method for knocking out prophage1 is as follows: using Bacillus subtilis 168 P43-panB panC panD panEilvC ilvD serC aspC ysaA△spoIIIE as a template, PCR amplification is performed using prophage1-LF and prophage1-LR as primers to obtain the upstream homologous arm of prophage1; using Bacillus subtilis 168 P43-panB panCpanD panE ilvC ilvD serC aspC Using ysaA△spoIIIE as a template, PCR amplification was performed with prophage1-F and prophage1-R as primers to obtain the downstream homologous arm of prophage1; using plasmid p7z6 as a template, PCR amplification was performed with prophage1-zeo-F and prophage1-zeo-R as primers to obtain the resistance selection cassette of prophage1; the upstream homologous arm of prophage1, the downstream homologous arm of prophage1, and the resistance selection cassette of prophage1 were fused by PCR to obtain the promoter replacement frame; the promoter replacement frame was transformed into Bacillus subtilis 168, replacing the original prophage1 segment on the Bacillus subtilis genome, thus completing the knockout of prophage1, resulting in Bacillus subtilis 168 P43-panB panC panD panE ilvC ilvDserC aspC ysaA△spoIIIE△prophahe1; The method for knocking out prophage3 is as follows: using *Bacillus subtilis 168 P43-panB panC panD panEilvC ilvD serC aspC ysaA△spoIIIE△prophahe1* as a template, PCR amplification is performed using prophage3-LF and prophage3-LR as primers to obtain the upstream homologous arm of prophage3; using *Bacillus subtilis168 P43-panB panC panD panE ilvC ilvD serC aspC* as a template, PCR amplification is performed to obtain the upstream homologous arm of prophage3; using *Bacillus subtilis168 P43-panB panC panD panE ilvC ilvD serC aspC* as a template, PCR amplification is performed to obtain the upstream homologous arm of prophage3; Using ysaA△spoIIIE△prophahe1 as a template, PCR amplification was performed with prophage3-F and prophage3-R as primers to obtain the downstream homologous arm of prophage3; using plasmid p7z6 as a template, PCR amplification was performed with prophage3-zeo-F and prophage3-zeo-R as primers to obtain the resistance selection cassette of prophage3; the upstream homologous arm of prophage3, the downstream homologous arm of prophage3, and the resistance selection cassette of prophage3 were fused by PCR to obtain the promoter replacement frame; the promoter replacement frame was transformed into Bacillus subtilis 168 to replace the original prophage3 segment on the Bacillus subtilis genome, thus completing the knockout of prophage3 and obtaining Bacillus subtilis 168 P43-panB panC panD panE ilvC ilvD serC aspC ysaA△spoIIIE△prophahe1△prophahe3; The method for knocking out prophage6 is as follows: using Bacillus subtilis 168 P43-panB panC panD panEilvC ilvD serC aspC ysaA△spoIIIE△prophahe1△prophahe3 as a template, PCR amplification is performed using prophage6-LF and prophage6-LR as primers to obtain the upstream homologous arm of prophage6; using Bacillus subtilis168 P43-panB panC panD panE ilvC ilvD serC aspC Using ysaA△spoIIIE△prophahe1△prophahe3 as templates, PCR amplification was performed with prophage6-F and prophage6-R as primers to obtain the downstream homologous arm of prophage6; using plasmid p7z6 as templates, PCR amplification was performed with prophage6-zeo-F and prophage6-zeo-R as primers to obtain the resistance selection cassette of prophage6; the upstream homologous arm of prophage6, the downstream homologous arm of prophage6, and the resistance selection cassette of prophage6 were fused by PCR to obtain the promoter replacement frame; the promoter replacement frame was transformed into Bacillus subtilis 168 P43-panB panC panD panE ilvC ilvD serC aspC ysaA△spoIIIE△prophahe1△prophahe3△prophahe6; Primers prophage1-LF, prophage1-LR, prophage1-F, prophage1-R, prophage1-zeo-F, prophage1-zeo-R, prophage3-LF, prophage3-LR, prophage3-F, proph The sequences of age3-R, prophage3-zeo-F, prophage3-zeo-R, prophage6-LF, prophage6-LR, prophage6-F, prophage6-R, prophage6-zeo-F, prophage6-zeo-R are as follows: 。 3. The construction method as described in claim 2, characterized in that: The gene sequence of the P43 promoter is shown in SEQ ID No.

1.

4. The application of the engineered bacteria as described in claim 1 or the engineered bacteria constructed by the construction method according to any one of claims 2 to 3 in the production of D-pantothenic acid.

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