Engineering bacterium capable of stably producing D-pantothenic acid at high yield as well as construction method and application of engineering bacterium

Abstract

The invention relates to the technical field of genetic engineering, and discloses an engineering bacterium capable of stably producing D-pantothenic acid at high yield as well as a construction method and application of the engineering bacterium. According to the invention, key genes in a metabolic pathway are modified through a metabolic engineering technology, and particularly, the level of MTF is improved by enhancing expression of a ysaA gene and modifying a biosynthetic pathway of methylenetetrahydrofolic acid (MTF), so that the level of ketopantoic acid serving as a key intermediate in the biosynthetic pathway of pantothenic acid is improved, and the yield of D-pantothenic acid is improved. Meanwhile, spoIVB or / and spoIIIE genes for producing spores in the genome are knocked out, so that energy consumption and material waste caused by spore formation are reduced, generation of secondary metabolites is promoted, and synthesis of D-pantothenic acid is increased; besides, the regions of prophage1 (alkA-ybdO), prophage3 (ydiM-ydjJ) and prophage6 (yobB-yobO) in the genome are knocked out, so that the redundancy of the genome is reduced, the metabolic efficiency and stability of the strain are improved, and the synthesis of D-pantothenic acid is effectively increased.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and in particular to an engineering bacterium capable of stably and highly producing D-pantothenic acid, and a construction method and application thereof. Background Art

[0002] Pantothenic acid, also known as vitamin B5, is a water-soluble vitamin. It is widely found in various foods, such as meat (chicken, beef, fish), whole-wheat foods, nuts, green leafy vegetables, and whole grains. Vitamin B5 can promote cell metabolism, help repair and regenerate damaged cells, and thus maintain the health of tissues such as skin and hair; participate in the synthesis of neurotransmitters, such as acetylcholine, which helps maintain the normal function and health of the nervous system; it is a component of coenzyme A, which can promote the metabolism of carbohydrates, fats, and proteins, and provide energy for the human body. This function is essential for maintaining the normal physiological functions of the body. Acetyl-CoA participates in the synthesis of acetylcholine, acetylglucosamine, and the biosynthesis of steroid compounds. It has an oxidizing effect on fatty acids, pyruvate, α-ketoglutaric acid, 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 plays a role in all cellular metabolism through coenzyme A. Pantothenic acid exists in two forms: D-form and L-form, but only the D-form (D-PA) is biologically active.

[0003] At present, the production methods of D-pantothenic acid include physical induced crystallization, chemical splitting and microbial methods. The physical induced crystallization process is mature, and the main steps are to condense calcified β-alanine with D,L-pantothenic acid lactone to obtain a D,L-pantothenic acid calcium mixed solution, and add D-pantothenic acid calcium seed to induce crystallization and split D-pantothenic acid calcium. However, it can only produce calcium pantothenate and cannot be used for the production of other pantothenic acid derivatives. The chemical splitting method is to add quinine, chloramphenicol and ephedrine and other chemical splitting reagents to the D,L-pantothenic acid lactone racemate to split D-pantothenic acid lactone, and then react to obtain D-pantothenic acid calcium. It is the most important synthesis method at present, but it consumes a lot, is expensive and difficult to separate, and has serious environmental pollution, and cannot achieve sustainable development.

[0004] With the development of genetic engineering technology, the use of microorganisms to produce D-pantothenic acid has attracted widespread attention due to its advantages. The use of biological fermentation to produce D-pantothenic acid products can use cheap industrial raw materials such as glucose and obtain D-pantothenic acid products through the metabolic reaction of the organism itself. By rationally using microorganisms to produce D-pantothenic acid, it can not only ensure the quality of pantothenic acid splitting but also reduce the reaction cost. Compared with chemical methods, biological methods have the advantages of being green and environmentally friendly, short process routes, excellent product quality, and good stability and miscibility. However, at present, the production of D-pantothenic acid by biological fermentation still has problems such as low fermentation yield and unstable fermentation. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides an engineered bacterium with a stable and high yield of D-pantothenic acid. The present invention transforms the key genes in its metabolic pathway through metabolic engineering technology. Specifically, by enhancing the expression of the ysaA gene, the methylenetetrahydrofolate (MTF) biosynthetic pathway is modified to improve the level of MTF, which can lead to an increase in the level of ketopantoic acid, a key intermediate in the pantothenic acid biosynthetic pathway, thereby increasing the yield of D-pantothenic acid. At the same time, the spoIVB or / and spoIIIE genes that produce spores in the genome are knocked out to reduce the energy consumption and material waste of spore formation, promote the generation of secondary metabolites, and thus increase the synthesis of D-pantothenic acid; in addition, the prophage1 (alkA-ybdO), prophage3 (ydiM-ydjJ), and prophage6 (yobB-yobO) regions in the genome are knocked out to reduce the redundancy of the genome, improve the metabolic efficiency and stability of the strain, and thus increase the synthesis of D-pantothenic acid.

[0006] The specific technical scheme of the present invention is: In the first aspect, the present invention provides an engineered bacterium that stably and highly produces D-pantothenic acid, in which the ysaA gene is overexpressed, the spoIVB and / or spoIIIE genes in the genome are knocked out, and the prophage1 (alkA-ybdO), prophage3 (ydiM-ydiJ), and prophage6 (yoaM-yobO) regions are knocked out.

[0007] Pantothenic acid is produced by a four-step enzymatic reaction of α-ketoisovalerate and L-aspartate. Finally, ATP provides energy to connect β-alanine and pantothenic acid to produce pantothenic acid under the catalysis of pantothenate synthase.

[0008] The present invention transforms key genes in its metabolic pathway through metabolic engineering technology. Specifically, by enhancing the expression of the ysaA gene, the methylenetetrahydrofolate (MTF) biosynthesis pathway is modified to improve the level of MTF, which can lead to an increase in the level of ketopantoic acid, a key intermediate in the pantothenic acid biosynthesis pathway, thereby increasing the yield of D-pantothenic acid. At the same time, the spoIVB or / and spoIIIE gene for spore production in the genome is knocked out to reduce the energy consumption and material waste of spore formation, promote the generation of secondary metabolites, and thus increase the synthesis of D-pantothenic acid; in addition, the prophage1 (alkA-ybdO), prophage3 (ydiM-ydjJ), and prophage6 (yobB-yobO) regions in the genome are knocked out to reduce the redundancy of the genome, improve the metabolic efficiency and stability of the strain, and thus increase the synthesis of D-pantothenic acid.

[0009] Preferably, the bottom bacteria is Bacillus subtilis 168.

[0010] Preferably, the ysaA gene sequence is shown as SEQ ID No.2, the spoIVB gene sequence is shown as SEQ ID No.4, and the spoIIIE gene sequence is shown as SEQ ID No.5.

[0011] In a second aspect, the present invention provides a method for constructing the above-mentioned engineering bacteria that stably and highly produces D-pantothenic acid, characterized in that it comprises the following steps: Step S1: enhancing the expression of the ysaA gene in the genome of the fungus; Step S2: knocking out spoIVB and / or spoIIIE genes; Step S3: Knock out the prophage1 (alkA-ybdO), prophage3 (ydiM-ydiJ), and prophage6 (yoaM-yobO) regions.

[0012] Preferably, the bottom plate bacteria is Bacillus subtilis 168.

[0013] Preferably, the ysaA gene sequence is shown as SEQ ID No.2, the spoIVB gene sequence is shown as SEQ ID No.4, and the spoIIIE gene sequence is shown as SEQ ID No.5.

[0014] Preferably, the method for enhancing the expression of the ysaA gene in the genome of the chassis bacteria is: replacing the promoter of the ysaA gene with the P43 promoter.

[0015] Preferably, the gene sequence of the P43 promoter is shown as SEQ ID No.1.

[0016] In a third aspect, the present invention provides the use of the above-mentioned engineered bacteria in the production of D-pantothenic acid.

[0017] Compared with the prior art, the present invention has the following technical effects: The present invention uses Bacillus subtilis as the base bacteria, and transforms the key genes in its metabolic pathway through metabolic engineering technology. Specifically, by enhancing the expression of the ysaA gene, the methylenetetrahydrofolate (MTF) biosynthesis pathway is modified to improve the level of MTF, which can lead to an increase in the level of ketopantoic acid, a key intermediate in the pantothenic acid biosynthesis pathway, thereby increasing the yield of D-pantothenic acid. At the same time, the spoIVB or / and spoIIIE genes that produce spores in the genome are knocked out to reduce the energy consumption and material waste of spore formation, promote the generation of secondary metabolites, and thus increase the synthesis of D-pantothenic acid; in addition, the prophage1 (alkA-ybdO), prophage3 (ydiM-ydjJ), and prophage6 (yobB-yobO) regions in the genome are knocked out to reduce the redundancy of the genome, improve the metabolic efficiency and stability of the strain, and thus increase the synthesis of D-pantothenic acid. Based on this, a genetically engineered Bacillus subtilis strain with high production of D-pantothenic acid was constructed, which improved its growth state and stability in the fermentation preparation of D-pantothenic acid and efficiently produced D-pantothenic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the modification site of the D-pantothenic acid metabolic pathway of the present invention; Figure 2 The OD of the engineered bacteria constructed in Example 2 600 And the result graph of D-pantothenic acid content in the fermentation broth supernatant; Figure 3 The OD of the engineered bacteria constructed in Example 3 600 And the result graph of D-pantothenic acid content in the fermentation broth supernatant; Figure 4 The OD of the engineered bacteria constructed in Example 4 600 And the result graph of D-pantothenic acid content in the fermentation broth supernatant; Figure 5 The OD of the engineered bacteria constructed in Example 5 600 And the result graph of D-pantothenic acid content in the fermentation broth supernatant; Figure 6 The OD of the engineered bacteria constructed in Example 6 600 And the result graph of D-pantothenic acid content in the fermentation broth supernatant; Figure 7 The OD of the engineered bacteria constructed in Example 7 600 And the result graph of D-pantothenic acid content in the fermentation broth supernatant; Figure 8 The OD of the engineered bacteria constructed in Example 8 600 And the D-pantothenic acid content in the fermentation broth supernatant. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should belong to the scope of protection of the present invention.

[0020] The present invention uses a genotype of B.subtilis 168P43-panB panC panD panE ilvCilvD serC aspC as the base strain to conduct experiments and verify the technical solution of the present invention. The genotype strain uses Bacillus subtilis 168 as the base strain, and its construction method includes the following steps: (1) Using Bacillus subtilis as the substrate, the Cre / loxP gene editing system was used The promoter of the panB gene in its genome was replaced with the P43 promoter to obtain the engineered bacterium Bacillus subtilis (P43-panB); (2) Using the Cre / loxP gene editing system, the promoter of the panC gene in the genome of the engineered bacterium Bacillus subtilis (P43-panB) was replaced with the P43 promoter to obtain the engineered bacterium Bacillus subtilis (P43-panBpanC); (3) Using the Cre / loxP gene editing system, the promoter of the panD gene in the genome of the engineered bacterium Bacillus subtilis (P43-panBpanC) was replaced with the P43 promoter to obtain the engineered bacterium Bacillus subtilis (P43-panBpanC panD); (4) Using the Cre / loxP gene editing system, the promoter of the panE gene in the genome of the engineered bacterium Bacillus subtilis (P43-panBpanCpanD) was replaced with the P43 promoter to obtain the engineered bacterium Bacillus subtilis (P43-panBpanC panD panE); (5) Using the Cre / loxP gene editing system, the promoter of the ilvC gene in the genome of the engineered bacterium Bacillus subtilis (P43-panBpanCpanDpanE) was replaced with the P43 promoter to obtain the engineered bacterium Bacillus subtilis (P43-panBpanCpanDpanEilvC); (6) Using the Cre / loxP gene editing system, the promoter of the ilvD gene in the genome of the engineered bacterium Bacillus subtilis (P43-panBpanCpanDpanEilvC) was replaced with the P43 promoter to obtain the engineered bacterium Bacillus subtilis (P43-panBpanCpanDpanEilvCilvD); (7) Using the Cre / loxP gene editing system, the promoter of the serC gene in the genome of the engineered bacterium Bacillus subtilis (P43-panBpanCpanDpanEilvCilvD) was replaced with the P43 promoter to obtain the engineered bacterium Bacillus subtilis (P43-panBpanCpanDpanEilvCilvDserC); (8) The Cre / loxP gene editing system was used to replace the promoter of the aspC gene in the genome of the engineered bacterium Bacillus subtilis (P43-panBpanCpanDpanEilvCilvDserC) with the P43 promoter to obtain the engineered bacterium Bacillus subtilis (P43-panBpanCpanDpanEilvCilvDserCaspC), which is the chassis strain DPA8 (B. subtilis ATCC 6633 (P43-panBpanCpanD panEilvCilvDserCaspC) of the present invention.

[0021] D-pantothenic acid metabolic pathway and the modification site of the present invention are shown in Figure 1 The genes and corresponding pathways involved in the present invention and the examples are shown in Table 1.

[0022] Table 1 Genes involved in gene editing and corresponding pathways In Table 1, the ysaA gene sequence is shown as SEQ ID No.2, the spo0A gene sequence is shown as SEQ ID No.3, the spoIVB gene sequence is shown as SEQ ID No.4, and the spoIIIE gene sequence is shown as SEQ ID No.5.

[0023] The primer information involved in the examples of the present invention is shown in Table 2.

[0024] Table 2 Primer sequences In the examples of the present 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.

[0025] Example 1D - Determination of pantothenic acid content The detection method is as follows: Chromatographic conditions: C18 column (250 × 4.6 mm, particle size 5 μm, Agilent), detection wavelength 200 nm, column temperature 30 °C; Sample treatment: Dilute the sample with ultrapure water to keep the D-pantothenic acid content between 0.5 g / L and 1.0 g / L; Mobile phase: acetonitrile: water: phosphoric acid = = 50:949:1 (volume ratio); Data collection time: 15 minutes.

[0026] 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, using Cre / loxP gene editing technology, the P43 promoter derived from pP43NMK (nucleotide sequence as shown in SEQ ID NO.1) was used to replace the original promoter of the ysaA gene on the genome, thereby enhancing the expression intensity of the ysaA gene and thereby increasing the yield of D-pantothenic acid. The specific steps are as follows: (1) Construction of the upstream sequence of the target gene ysaA: The genome of Bacillus subtilis DPA8 (B. subtilis 168P43-panBpanC panD panE ilvC ilvD serC aspC) was used as a template and PCR amplification was performed with ysaA-LF and ysaA-LR as primers. After amplification, the product was run on agarose gel for verification, cleaned up, purified and stored for later use.

[0027] (2) Construction of lox71-zeo-lox66 sequence: Plasmid p7z6 was used as a template and ysaA-zeo-F and ysaA-zeo-R were used as primers for PCR amplification. After amplification, the product was run on agarose gel for verification, cleaned up, purified and stored for later use.

[0028] (3) Construction of P43 promoter sequence: Plasmid pP43NMK was used as a template and ysaA-P43-F and ysaA-P43-R were used as primers for PCR amplification. After amplification, the product was run on agarose gel for verification, cleaned up, purified and stored for later use.

[0029] (4) Construction of the target gene ysaA sequence: The genome of Bacillus subtilis DPA8 (B. subtilis 168P43-panBpanC panD panE ilvC ilvD serC aspC) was used as a template and PCR amplification was performed with ysaA-F and ysaA-R as primers. After amplification, the product was run on agarose gel for verification, cleaned up, purified and stored for later use.

[0030] (5) The upstream of the target gene, lox71-zeo-lox66, P43 promoter and ysaA target gene sequences were fused by fusion PCR technology to obtain a promoter replacement frame, which was transformed into Bacillus subtilis DPA8 (B. subtilis 168P43-panB panC panD panE ilvC ilvD serC aspC), and screened using bleomycin-resistant LB plates. After the transformants were verified to be correct by PCR, they were transferred into plasmid PDG148; the transformants carrying the PDG148 plasmid were inoculated in a medium containing 0.2 mmol / L The cells were cultured in liquid LB containing IPTG for 24 h to express the circular recombinase that promoted the recombination of lox71 and lox66 sites and to recover the bleomycin resistance gene. After that, 10 μL of the culture solution was inoculated into 10 mL of fresh LB liquid culture medium. After shaking culture at 50°C and 220 rpm for 10 h, the cells were streaked onto a non-resistant LB solid plate. The colonies grown on the non-resistant LB solid plate were spotted onto bleomycin, kanamycin and non-resistant LB solid plates with a 10 μL pipette tip. The strain that could only grow on the non-resistant solid plate was the engineered strain DPA8 (P43-ysaA) that eliminated the PDG148 plasmid.

[0031] (6) The DPA8 (P43-ysaA) strain and the DPA8 (B. subtilis 168P43-panB panC panD panEilvC ilvD serC aspC) control group were inoculated into 10 mL of LB liquid culture medium and cultured overnight at 37°C and 200 rpm. 1 mL of the preculture was inoculated into a 500 mL shake flask containing 50 mL of fermentation medium, and then cultured continuously at 30°C and 300 rpm for 48 h. After the fermentation was completed, 1 mL of the fermentation broth was centrifuged at 12000 rpm at room temperature for 3 min, the supernatant of the fermentation broth was diluted 5 times, and HPLC detection was performed according to Example 1. The OD 600and the D-pantothenic acid content in the fermentation supernatant. Figure 2 shown.

[0032] Depend on Figure 2 It can be seen that by replacing the ysaA gene promoter in the genome, the production of D-pantothenic acid increased from 2.65g / L to 2.93g / L. The reason for this is that enhancing the expression of the ysaA gene increased the supply of one-carbon units. Increasing the level of MTF can lead to an increase in the level of ketopantothenic acid, a key intermediate in the pantothenic acid biosynthesis pathway, which is beneficial to the synthesis of D-pantothenic acid in Bacillus subtilis.

[0033] The LB medium consists of 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, the solvent is deionized water, and the pH value is natural.

[0034] The fermentation medium composition is as follows: 20 g / L glucose, 16 g / L ammonium sulfate, 0.8 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 2 g / L yeast powder, 1 mL / L trace element solution, the solvent is deionized water, the pH value is 6-7, the trace element solution composition is: 10 g / L CuCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.20 g / L CuSO4, 0.02 g / L NiCl2·7H2O, the solvent is deionized water.

[0035] Example 3 Construction of strain DPA8 (P43-ysaAΔspo0A) and shake flask fermentation Using DPA8 (P43-ysaA) constructed in Example 2 as the starting strain, the Cre / loxP gene editing technology was used to knock out the spo0A gene in the genome to reduce the energy consumption of spore formation. The specific steps are as follows: (1) Construction of upstream sequence of target gene spo0A: Using DPA8 (P43-ysaA) genome as template and spo0A-LF and spo0A-LR as primers, PCR amplification was performed. After amplification, the product was run on gel for verification, cleaned up, purified and stored for later use.

[0036] (2) Construction of lox71-zeo-lox66 sequence: Plasmid p7z6 was used as a template and spo0A-zeo-F and spo0A-zeo-R were used as primers for PCR amplification. After amplification, the product was run on agarose gel for verification, cleaned up, purified and stored for later use.

[0037] (3) Construction of the target gene spo0A sequence: The DPA8 (P43-ysaA) genome was used as a template and spo0A-F and spo0A-R were used as primers for PCR amplification. After amplification, the product was run on agarose gel for verification, cleaned up, purified and stored for later use.

[0038] (4) By using fusion PCR technology, the upstream of the target gene, lox71-zeo-lox66 and spo0A target gene sequences were fused to obtain a promoter replacement frame, which was transformed into DPA8 (P43-ysaA) and screened using a bleomycin-resistant LB plate. After the transformants were verified to be correct by PCR, they were transferred into plasmid PDG148; the transformants carrying the PDG148 plasmid were inoculated in a medium containing 0.2 mmol / L The cells were cultured in liquid LB containing IPTG for 24 h to express the circular recombinase that promoted the recombination of lox71 and lox66 sites and to recover the bleomycin resistance gene. After that, 10 μL of the culture solution was inoculated into 10 mL of fresh LB liquid culture medium. After shaking culture at 50°C and 220 rpm for 10 h, the cells were streaked onto a non-resistant LB solid plate. The colonies grown on the non-resistant LB solid plate were spotted onto bleomycin, kanamycin and non-resistant LB solid plates with a 10 μL pipette tip. The strain that could only grow on the non-resistant solid plate was the engineered strain DPA8 (P43-ysaA△spo0A) that eliminated the PDG148 plasmid.

[0039] (5) The DPA8 (P43-ysaA △ spo0A) strain, with DPA8 (P43-ysaA) as the control group, was inoculated into 10 mL of LB liquid culture medium, cultured overnight at 37°C and 200 rpm, and 1 mL of the preculture was inoculated into a 500 mL shake flask containing 40 mL of fermentation medium, and then cultured continuously for 48 h at 37°C and 300 rpm; after the fermentation was completed, 1 mL of the fermentation broth was centrifuged at 12000 rpm at room temperature for 3 min, the supernatant of the fermentation broth was diluted 5 times, and HPLC detection was performed according to Example 1, and OD 600 and the D-pantothenic acid content in the fermentation supernatant. Figure 3 shown.

[0040] Depend on Figure 3 It can be seen that when the spo0A gene was knocked out of the genome, the production of D-pantothenic acid decreased from 2.88g / L to 1.88g / L. Further analysis showed that the principle is that the growth rate of the strain with the spo0A gene knocked out increased, which affected the distribution and metabolic flux of intracellular nutrients under fermentation conditions, thus being unfavorable for the synthesis of D-pantothenic acid in Bacillus subtilis.

[0041] Example 4 Construction of effective strain DPA8 (P43-ysaA △ spoIVB) and shake flask fermentation Using DPA8 (P43-ysaA) constructed in Example 2 as the starting strain, the Cre / loxP gene editing technology was used to knock out the spoIVB gene in the genome, blocking the formation of mature spores, thereby increasing the yield of D-pantothenic acid. The specific steps are as follows: (1) Construction of the upstream sequence of the target gene spoIVB: The DPA8 (P43-ysaA) genome was used as a template and spoIVB-LF and spoIVB-LR were used as primers for PCR amplification. After amplification, the product was run on agarose gel for verification, cleaned up, purified and stored for later use.

[0042] (2) Construction of lox71-zeo-lox66 sequence: Plasmid p7z6 was used as a template and spoIVB-zeo-F and spoIVB-zeo-R were used as primers for PCR amplification. After amplification, the product was run on agarose gel for verification, cleaned up, purified and stored for later use.

[0043] (3) Construction of the target gene spoIVB sequence: The DPA8 (P43-ysaA) genome was used as a template and spoIVB-F and spoIVB-R were used as primers for PCR amplification. After amplification, the product was run on agarose gel for verification, cleaned up, purified and stored for later use.

[0044] (4) By using fusion PCR technology, the upstream of the target gene, lox71-zeo-lox66 and spoIVB target gene sequences were fused to obtain a promoter replacement frame, which was transformed into DPA8 (P43-ysaA) and screened using a bleomycin-resistant LB plate. After the transformants were verified to be correct by PCR, they were transferred into the plasmid PDG148; the transformants carrying the PDG148 plasmid were inoculated in liquid LB containing 0.2 mmol / L IPTG and cultured for 24 h to express the cyclization recombinase to promote lo The x71 site was recombined with the lox66 site to recover the bleomycin resistance gene; then, 10 μL of the culture solution was inoculated into 10 mL of fresh LB liquid culture medium, and after shaking culture at 50°C and 220 rpm for 10 hours, it was streaked onto an LB solid plate without resistance; the colonies grown on the LB solid plate without resistance were spotted on bleomycin, kanamycin and LB solid plates without resistance with a 10 μL pipette tip, and only the colonies that could grow on the solid plates without resistance were the engineered DPA8 (P43-ysaA△spoIVB) strains that had eliminated the PDG148 plasmid.

[0045] (5) The DPA8 (P43-ysaA △ spoIVB) strain, with DPA8 (P43-ysaA) as the control group, was inoculated into 10 mL of LB liquid culture medium, cultured overnight at 37°C and 200 rpm, and 1 mL of the preculture was inoculated into a 500 mL shake flask containing 50 mL of fermentation medium, and then cultured continuously for 48 h at 37°C and 200 rpm. After the fermentation was completed, 1 mL of the fermentation broth was centrifuged at 12000 rpm at room temperature for 3 min, the supernatant of the fermentation broth was diluted 5 times, and HPLC detection was performed according to Example 1, and OD 600and the D-pantothenic acid content in the fermentation supernatant. Figure 4 shown.

[0046] Depend on Figure 4 It can be seen that when spoIVB was knocked out of the genome, the production of D-pantothenic acid increased from 2.91 g / L to 3.13 g / L, which shows that knocking out the spoIVB gene can promote cell growth and block the formation of mature spores, thereby facilitating the synthesis of D-pantothenic acid in Bacillus subtilis.

[0047] Example 5 Construction of effective strain DPA8 (P43-ysaA△spoIIIE) and shake flask fermentation Using DPA8 (P43-ysaA) constructed in Example 2 as the starting strain, the Cre / loxP gene editing technology was used to knock out the spoIIIE gene in the genome, so that spore formation was inhibited, thereby increasing the yield of D-pantothenic acid. The specific steps are as follows: (1) Construction of the upstream sequence of the target gene spoIIIE: Using the DPA8 (P43-ysaA) genome as a template and spoIIIE-LF and spoIIIE-LR as primers, PCR amplification was performed. After amplification, the product was run on agarose gel for verification, cleaned up, purified and stored for later use.

[0048] (2) Construction of lox71-zeo-lox66 sequence: Plasmid p7z6 was used as a template and spoIIIE-zeo-F and spoIIIE-zeo-R were used as primers for PCR amplification. After amplification, the product was run on agarose gel for verification, cleaned up, purified and stored for later use.

[0049] (3) Construction of the target gene spoIIIE sequence: The DPA8 (P43-ysaA) genome was used as a template and spoIIIE-F and spoIIIE-R were used as primers for PCR amplification. After amplification, the product was run on agarose gel for verification, cleaned up, purified and stored for later use.

[0050] (4) By using fusion PCR technology, the upstream of the target gene, lox71-zeo-lox66 and spoIIIE target gene sequences were fused to obtain a promoter replacement frame, which was transformed into DPA8 (P43-ysaA) and screened using a bleomycin-resistant LB plate. After the transformants were verified to be correct by PCR, they were transferred into plasmid PDG148; the transformants carrying the PDG148 plasmid were inoculated in a medium containing 0.2 mmol / L IPTG liquid LB, culture for 24 hours to express circular recombinase to promote the recombination of lox71 site and lox66 site, and recover the bleomycin resistance gene; then, take 10 μL culture solution and inoculate it into 10 mL fresh LB liquid medium, shake and culture at 50 ° C and 220 rpm for 10 hours, and then streak it on the non-resistance LB solid plate; use a 10 μL pipette tip to spot the colonies grown on the non-resistance LB solid plate on bleomycin, kanamycin and non-resistance LB solid plates respectively, and only those that can grow on the non-resistance solid plate are the engineering to eliminate the PDG148 plasmid DPA8(P43-ysaAΔspoIIIE) strain.

[0051] (5) The DPA8 (P43-ysaA △ spoIIIE) strain, with DPA8 (P43-ysaA) as the control group, was inoculated into 10 mL of LB liquid culture medium, cultured overnight at 37°C and 200 rpm, and 1 mL of the preculture was inoculated into a 500 mL shake flask containing 50 mL of fermentation medium, and then cultured continuously for 48 h at 37°C and 200 rpm. After the fermentation was completed, 1 mL of the fermentation broth was centrifuged at 12000 rpm at room temperature for 3 min, the supernatant of the fermentation broth was diluted 5 times, and HPLC detection was performed according to Example 1, and OD 600 0 and the D-pantothenic acid content in the fermentation supernatant. Figure 5 shown.

[0052] Depend on Figure 5 It can be seen that when spoIIIE was knocked out of the genome, the production of D-pantothenic acid increased from 2.90g / L to 3.23g / L. Knocking out the spoIIIE gene can inhibit spore formation without affecting the function of the strain itself, thereby facilitating the synthesis of D-pantothenic acid in Bacillus subtilis.

[0053] Example 6 Construction of effective strain DPA8 (P43-ysaA△spoIIIE△prophahe1) and shake flask fermentation Using DPA8 (P43-ysaA△spoIIIE) constructed in Example 5 as the starting strain, the Cre / loxP gene editing technology was used to knock out the prophage1 region (alkA-ydbO) in the genome to reduce the redundancy of the genome, thereby increasing the yield of D-pantothenic acid. The specific steps are as follows: (1) Construction of the upstream sequence of the target region prophage1: Using the DPA8 (P43-ysaA△spoIIIE) genome as a template and prophage1-LF and prophage1-LR as primers, PCR amplification was performed. After amplification, the product was run on agarose gel for verification, cleaned up, purified and stored for later use.

[0054] (2) Construction of lox71-zeo-lox66 sequence: Plasmid p7z6 was used as a template and prophage1-zeo-F and prophage1-zeo-R were used as primers for PCR amplification. After amplification, the product was run on agarose gel for verification, cleaned up, purified and stored for later use.

[0055] (3) Construction of the target region prophage1 sequence: Using the DPA8 (P43-ysaA△spoIIIE) genome as a template, PCR amplification was performed with prophage1-F and prophage1-R as primers. After amplification, the product was run on agarose gel for verification, cleaned up, purified and stored for later use.

[0056] (4) The upstream of the target gene, lox71-zeo-lox66 and the prophage1 target gene sequence were fused by fusion PCR technology to obtain a promoter replacement frame, which was transformed into DPA8 (P43-ysaA△spoIIIE) and screened using bleomycin-resistant LB plates. After the transformants were verified to be correct by PCR, they were transferred into plasmid PDG148; the transformants carrying the PDG148 plasmid were inoculated in a medium containing 0.2 mmol / L The cells were cultured in liquid LB containing IPTG for 24 h to express the circular recombinase that promoted the recombination of lox71 and lox66 sites and to recover the bleomycin resistance gene. After that, 10 μL of the culture solution was inoculated into 10 mL of fresh LB liquid culture medium and cultured at 50°C and 220 rpm for 10 h before being streaked onto an LB solid plate without resistance. The colonies grown on the LB solid plate without resistance were spotted onto bleomycin, kanamycin and LB solid plates without resistance using a 10 μL pipette tip. Only the colonies that could grow on the solid plate without resistance were the engineered strains DPA8 (P43-ysaA△spoIIIE△prophahe1) that eliminated the PDG148 plasmid.

[0057] (5) The DPA8 (P43-ysaA△spoIIIE△prophahe1) strain, with DPA8 (P43-ysaA△spoIIIE) as the control group, was inoculated into 10 mL of LB liquid culture medium, cultured overnight at 37°C and 200 rpm, and 1 mL of the preculture was inoculated into a 500 mL shake flask containing 50 mL of fermentation medium, and then cultured continuously for 48 h at 37°C and 200 rpm; after the fermentation was completed, 1 mL of the fermentation broth was taken and centrifuged at 12000 rpm at room temperature for 3 min, the supernatant of the fermentation broth was diluted 5 times, and HPLC detection was performed according to Example 1, and OD 600 and the D-pantothenic acid content in the fermentation supernatant. Figure 6 shown.

[0058] Depend on Figure 6 It can be seen that by knocking out the prophahe1 region of the genome, the production of D-pantothenic acid increased from 3.23 g / L to 3.38 g / L. Knocking out the prophahe1 region of the genome can reduce the redundancy of the genome and improve the metabolic efficiency of the strain, which is beneficial to the synthesis of D-pantothenic acid in Bacillus subtilis.

[0059] Example 7 Construction of effective strain DPA8 (P43-ysaA△spoIIIE△prophahe1△prophahe3) and shake flask fermentation.

[0060] Using DPA8 (P43-ysaA△spoIIIE△prophahe1) constructed in Example 6 as the starting strain, the prophahe3 region in the genome was knocked out using Cre / loxP gene editing technology to further reduce the redundancy of the genome and thereby increase the production of D-pantothenic acid. The specific steps are as follows: (1) Construction of upstream sequence of target gene prophahe3: Using DPA8 (P43-ysaA△spoIIIE△prophahe1) genome as template, PCR amplification was performed with prophahe3-LF and prophahe3-LR as primers. After amplification, the product was run on gel for verification, cleaned up, purified and stored for later use.

[0061] (2) Construction of lox71-zeo-lox66 sequence: Plasmid p7z6 was used as a template and prophahe3-zeo-F and prophahe3-zeo-R were used as primers for PCR amplification. After amplification, the product was run on agarose gel for verification, cleaned up, purified and stored for later use.

[0062] (3) Construction of the target gene prophahe3 sequence: The DPA8 (P43-ysaA△spoIIIE△prophahe1) genome was used as a template and prophahe3-F and prophahe3-R were used as primers for PCR amplification. After amplification, the product was run on agarose gel for verification, cleaned up, purified and stored for later use.

[0063] (4) By using fusion PCR technology, the upstream of the target gene, lox71-zeo-lox66 and prophahe3 target gene sequences were fused to obtain a promoter replacement frame, which was transformed into DPA8 (P43-ysaA△spoIIIE△prophahe1) and screened using a bleomycin-resistant LB plate. After the transformants were verified to be correct by PCR, they were transferred into the plasmid PDG148; the transformants carrying the PDG148 plasmid were inoculated in liquid LB containing 0.2 mmol / L PTG and cultured for 24 h to express the circular recombinase to promote lo The x71 site was recombined with the lox66 site to recover the bleomycin resistance gene; then, 10 μL of the culture solution was inoculated into 10 mL of fresh LB liquid culture medium, and after shaking culture at 50°C and 220 rpm for 10 hours, it was streaked on a non-resistant LB solid plate; the colonies grown on the non-resistant LB solid plate were spotted on bleomycin, kanamycin and non-resistant LB solid plates with a 10 μL pipette tip, and only the colonies that could grow on the non-resistant solid plates were the engineered strains DPA8 (P43-ysaA△spoIIIE△prophahe1△prophahe3) that had eliminated the PDG148 plasmid.

[0064] (5) DPA8 (P43-ysaA△spoIIIE△prophahe1△prophahe3) strain was used to DPA8 (P43-ysaA△spoIIIE△prophahe1) was used as the control group, and was inoculated into 10 mL of LB liquid culture medium, cultured overnight at 37°C and 200 rpm, and 1 mL of the preculture was inoculated into a 500 mL shake flask containing 50 mL of fermentation medium, and then cultured continuously for 48 h at 30°C and 200 rpm; after the fermentation was completed, 1 mL of the fermentation broth was centrifuged at 12000 rpm at room temperature for 3 min, the supernatant of the fermentation broth was diluted 5 times, and HPLC detection was performed according to Example 1, and OD 600 and the D-pantothenic acid content in the fermentation supernatant. Figure 7 shown.

[0065] Depend on Figure 7It can be seen that the production of D-pantothenic acid increased from 3.36 g / L to 3.49 g / L by knocking out the prophahe3 region of the genome. The reason for this is that the OD600 of the strain after fermentation was improved by genome simplification, which is beneficial to cell growth and thus to the synthesis of D-pantothenic acid in Bacillus subtilis.

[0066] Example 8 Construction of effective strain DPA8 (P43-ysaA△spoIIIE△prophahe1△prophahe3△prophahe6) and shake flask fermentation Using DPA8 (P43-ysaA△spoIIIE△prophahe1△prophahe3) constructed in Example 8 as the starting strain, the Cre / loxP gene editing technology was used to knock out the prophahe6 region in the genome to reduce the redundancy of the genome, thereby increasing the yield of D-pantothenic acid. The specific steps are as follows: (1) Construction of upstream sequence of target gene prophahe6: Using DPA8 (P43-ysaA△spoIIIE△prophahe1△prophahe3) genome as template, PCR amplification was performed with prophahe6-LF and prophahe6-LR as primers. After amplification, the product was run on gel for verification, cleaned up, purified and stored for later use.

[0067] (2) Construction of lox71-zeo-lox66 sequence: plasmid p7z6 was used as template and prophahe6-zeo-F and prophahe6-zeo-R were used as primers for PCR amplification. After amplification, the product was run on agarose gel for verification, cleaned up, purified and stored for later use.

[0068] (3) Construction of the target region prophahe6 sequence: Using the DPA8 (P43-ysaA△spoIIIE△prophahe1△prophahe3) genome as a template, PCR amplification was performed with prophahe6-F and prophahe6-R as primers. After amplification, the product was run on agarose gel for verification, cleaned up, purified and stored for later use.

[0069] (4) By using fusion PCR technology, the upstream of the target gene, lox71-zeo-lox66 and prophahe6 target gene sequences were fused to obtain a promoter replacement frame, and the promoter replacement frame was transformed into DPA8 (P43-ysaA△spoIIIE△prophahe1△prophahe3) was screened using a bleomycin-resistant LB plate, and after the transformants were verified to be correct by PCR, plasmid PDG148 was transferred; the transformants carrying the PDG148 plasmid were inoculated in liquid LB containing 0.2mmol / LIPTG, and cultured for 24h to express the circular recombinase to promote the recombination of the lox71 site and the lox66 site, and the bleomycin resistance gene was recovered; then, 10μL of the culture solution was inoculated in 10mL of fresh LB liquid culture medium, and after shaking culture at 50℃ and 220rpm for 10h, it was streaked on a non-resistant LB solid plate; the colonies grown on the non-resistant LB solid plate were spotted on bleomycin, kanamycin and non-resistant LB solid plates with a 10μL pipette tip, and only those that could grow on the non-resistant solid plate were engineered strains that eliminated the PDG148 plasmid. DPA8(P43-ysaA△spoIIIE△prophahe1△prophahe3△prophahe6).

[0070] (5) The DPA8 (P43-ysaA△spoIIIE△prophahe1△prophahe3△prophahe6) strain and the DPA8 (P43-ysaA△spoIIIE△prophahe1△prophahe3) control group were inoculated into 10 mL of LB liquid culture medium, cultured overnight at 37°C and 200 rpm, and 1 mL of the preculture was inoculated into a 500 mL shake flask containing 50 mL of fermentation medium, and then cultured continuously for 48 h at 30°C and 200 rpm; after the fermentation was completed, 1 mL of the fermentation broth was taken and centrifuged at 12000 rpm at room temperature for 3 min, the supernatant of the fermentation broth was diluted 5 times, and HPLC detection was performed according to Example 1, and OD 600 and the D-pantothenic acid content in the fermentation supernatant. Figure 8 shown.

[0071] Depend on Figure 8 It can be seen that the production of D-pantothenic acid increased from 3.46 g / L to 3.57 g / L by knocking out the prophahe6 region of the genome. The reason for this is that in the process of continuous genome simplification, the growth state and fermentation stability of the cells have been improved, which is conducive to the synthesis of D-pantothenic acid in Bacillus subtilis.

[0072] Therefore, in summary, it can be seen from Examples 2 to 8 that by replacing the original promoter of the ysaA gene with the P43 promoter and RBS sequence derived from pP43NMK, increasing the level of methylenetetrahydrofolate (MTF) by modifying the biosynthetic pathway of methylenetetrahydrofolate (MTF) can lead to an increase in the level of ketopantoic acid, a key intermediate in the pantothenic acid biosynthetic pathway, thereby increasing the yield of D-pantothenic acid; at the same time, the spoIVB and spoIIIE genes for spore production in the genome are knocked out respectively, the energy consumption and material waste of spore formation are eliminated, the generation of secondary metabolites is promoted, and the synthesis of D-pantothenic acid is increased. However, although the knockout of the spo0A gene can eliminate the energy consumption of spore formation, the knockout of the spo0A gene will lead to a decrease in yield; in addition, the prophage1 (alkA-ybdO), prophage3 (ydiM-ydjJ), and prophage6 (yobB-yobO) regions in the genome are knocked out to reduce the redundancy of the genome, 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 Bacillus subtilis genetically engineered bacterium DPA8 (P43-ysaA△spoIIIE△prophahe1△prophahe3△prophahe6) with high production of D-pantothenic acid.

[0073] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0074] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. An engineered bacterium that stably and highly produces D-pantothenic acid, characterized in that: In the engineered bacteria, the ysaA gene is overexpressed, the spoIVB and / or spoIIIE genes in the genome are knocked out, and the prophage1 (alkA-ybdO), prophage3 (ydiM-ydiJ), and prophage6 (yoaM-yobO) regions are knocked out.

2. The engineered bacteria according to claim 1, characterized in that: The bottom fungus is Bacillus subtilis 168.

3. The engineered bacteria according to claim 1, characterized in that: 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.

4. The method for constructing an engineered bacterium according to any one of claims 1 to 3, characterized in that: The following steps are involved: Step S1: enhancing the expression of the ysaA gene in the genome of the fungus; Step S2: knocking out spoIVB and / or spoIIIE genes; Step S3: Knock out the prophage1 (alkA-ybdO), prophage3 (ydiM-ydiJ), and prophage6 (yoaM-yobO) regions.

5. The construction method according to claim 4, characterized in that: The bottom plate bacteria is Bacillus subtilis 168.

6. The construction method according to claim 4, characterized in that: 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.

7. The construction method according to claim 4, characterized in that: The method for enhancing the expression of the ysaA gene in the genome of the chassis bacteria is: replacing the promoter of the ysaA gene with the P43 promoter.

8. The construction method according to claim 7, characterized in that: The gene sequence of the P43 promoter is shown in SEQ ID No.

1.

9. Use of the engineered bacteria according to any one of claims 1 to 3 or the engineered bacteria constructed by the construction method according to any one of claims 4 to 8 in the production of D-pantothenic acid.

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