A probiotic engineering bacterium with high yield of d-pantothenic acid and application thereof in d-pantothenic acid production and preparation of probiotic additive

Abstract

The application discloses a high-yield D-pantothenic acid probiotic engineering bacterium and application of the bacterium in D-pantothenic acid production and preparation of a probiotic additive, wherein the probiotic engineering bacterium is constructed by replacing the promoters of panBCD gene cluster, alsS gene, ilvD gene, panE2 gene and aspB gene in the genome of Bacillus subtilis ATCC 23857 with a P43 promoter, replacing a UTR sequence in front of the panBCD gene with a UTR m , knocking out one of pdhA gene or spo0A gene, and simultaneously knocking out ilvA gene and aspA gene. The probiotic engineering bacterium reduces the demand for a substrate beta-alanine in a D-pantothenic acid synthesis process, and realizes the synthesis of the D-pantothenic acid without precursor addition. The probiotic engineering bacterium constructed in the application can also be applied to feed, so as to achieve the effect of promoting the growth and development of animals, for example, the weight increase of zebra fish.

Description

(I) Technical Field

[0001] This invention relates to a biotechnology-based probiotic strain that produces high levels of D-pantothenic acid and its application in the production of D-pantothenic acid and the preparation of probiotic additives. (II) Background Technology

[0002] D-Pantothenic acid (also known as vitamin B5) is mainly synthesized by microorganisms and plants in nature. It is an important precursor to acetyl-CoA and is also essential for the synthesis of steroids from fatty acids in the body. It is indispensable in the metabolic regulation of carbohydrates, proteins, and fatty acids. Currently, vitamin B5 and its derivatives are widely used as feed additives in agriculture, livestock, and aquaculture. Appropriate addition of vitamin B5 can prevent stunted growth in livestock and improve their adaptability. Meanwhile, the domestic and international demand for vitamin B5 production is enormous. Compared with the traditional chemical methods of vitamin B5 production in industry, providing a greener, safer, and more efficient biological method for the production of vitamin B5 is of great value.

[0003] Bacillus subtilis is one of the most important microbial species in the field of biotechnology. It is a "Generally Recognized As Safe (GRAS)" production strain and can be used in the fermentation production of food and pharmaceuticals. As a probiotic, Bacillus subtilis metabolizes and proliferates in the animal gut, regulating intestinal flora, improving digestive capacity, and enhancing immunity and disease resistance. In industrial production, Bacillus subtilis is an ideal strain for constructing microbial chassis cells due to its clear genetic background, lack of exotoxin production, absence of significant codon bias, and ease of gene manipulation, making it suitable for large-scale industrial production.

[0004] In recent years, with the development of genetic engineering and the bio-industry, the use of probiotics that produce nutrients as feed products or production strains has gradually become an important trend. However, the biological production of pantothenic acid, an important vitamin, has problems such as the need for precursor addition, limited yield, and engineered probiotics being unsuitable for feed application. (III) Summary of the Invention

[0005] The purpose of this invention is to provide a probiotic engineered strain that produces high levels of D-pantothenic acid and its application in D-pantothenic acid production and the preparation of probiotic additives. This invention selects *Bacillus subtilis* as a substrate bacterium to overexpress the pantothenic acid synthesis gene and introduces the UTR... m This invention further enhances pantothenic acid synthesis and optimizes the pyruvate metabolic pathway, thereby achieving pantothenic acid synthesis in Bacillus subtilis without the addition of precursors. Furthermore, feeding zebrafish with the probiotics constructed in this invention as a feed ingredient resulted in increased body weight.

[0006] The technical solution adopted in this invention is:

[0007] This invention provides a probiotic engineered strain that produces high levels of D-pantothenic acid. The probiotic engineered strain uses Bacillus subtilis ATCC 23857 as the substrate bacteria, with the promoters of the panBCD gene cluster, alsS gene, ilvD gene, panE2 gene, and aspB gene in its genome replaced by the P43 promoter, and the pre-UTR sequence of the panBCD gene replaced by the UTR sequence. m The probiotic strain described above was constructed by knocking out the pdhA or spo0A gene, and simultaneously knocking out the ilvA and aspA genes, and is denoted as B. subtilis MX-3. The UTR... m The nucleotide sequence is shown in SEQ ID NO.1.

[0008] Preferably, the nucleotide sequence of the P43 promoter is shown in SEQ ID NO.2.

[0009] Preferably, the panBCD gene cluster contains the following gene IDs: panB (GeneID: 939032), panC (GeneID: 939030), panD (GeneID: 939033); alsS (GeneID: 936852); ilvA (GeneID: 939093); ilvD (GeneID: 939084); panE2 (GeneID: 936782); aspB (GeneID: 939037); pdhA (GeneID: 936005); spo0A (GeneID: 938655); and aspA (GeneID: 938721).

[0010] Preferably, the probiotic engineered bacteria that produce high levels of D-pantothenic acid are constructed according to the following method:

[0011] (1) Using B. subtilis ATCC 23857 as the chassis strain, the promoter of the panBCD gene cluster in the genome was replaced with the P43 promoter using the B. subtilis Cre / loxP gene editing system to obtain the recombinant strain B. subtilisATCC23857 / P43-panBCD, denoted as B. subtilis X-1;

[0012] (2) Using the B.subtilis Cre / loxP gene editing system, the ilvA gene in the B.subtilis X-1 genome was knocked out to obtain B.subtilis X-1 / ΔilvA, which is denoted as B.subtilis X-2;

[0013] (3) Using the B.subtilis Cre / loxP gene editing system, the promoters of the alsS and ilvD genes in the B.subtilis X-2 genome were replaced with the P43 promoter to obtain B.subtilis X-2 / P43-alsS-P43-ilvD, denoted as B.subtilis X-3;

[0014] (4) Using the B.subtilis Cre / loxP gene editing system, the panE2 gene promoter in the B.subtilis X-3 genome was replaced with the P43 promoter to obtain B.subtilis X-3 / P43-panE2, denoted as B.subtilisMX-1;

[0015] (5) Using the B. subtilis Cre / loxP gene editing system, the pre-UTR sequence of the panBCD gene in the B. subtilis MX-1 genome was replaced with the UTR. m MX-1 / UTR m -panBCD, denoted as B.subtilis MX-2;

[0016] (6) Using the B. subtilis Cre / loxP gene editing system, the pdhA gene in the B. subtilis MX-2 genome was knocked out to obtain the recombinant strain B. subtilis MX-2ΔpdhA; or the spo0A gene in the B. subtilis MX-2 genome was knocked out to obtain the recombinant strain B. subtilis MX-2Δspo0A;

[0017] (7) Using the B. subtilis Cre / loxP gene editing system, the aspA gene in the genome of B. subtilis MX-2ΔpdhA or B. subtilis MX-2Δspo0A was knocked out to obtain recombinant strains B. subtilis MX-2ΔpdhAΔaspA or B. subtilis MX-2Δspo0AΔaspA;

[0018] (8) Using the B.subtilis Cre / loxP gene editing system, the aspB gene promoter in the B.subtilis MX-2ΔpdhAΔaspA genome was replaced with the P43 promoter to obtain the recombinant strain B.subtilis MX-2ΔpdhAΔaspA / P43-aspB, denoted as B.subtilis MX-3.

[0019] This invention utilizes the strong promoter P43 to activate D-pantothenic acid metabolism-related genes (alsS, ilvD, panE2, and panBCD gene cluster) in *B. subtilis* ATCC 23857, thereby enhancing the expression of 3-methyl-2-hydroxymethyltransferase, dihydroxy acid dehydratase, lactate synthase, ketopantolysin reductase, aspartate decarboxylase, and pantolysin-β-alanine ligase in the D-pantothenic acid metabolic pathway. Simultaneously, knocking out the ilvA gene weakens branched-chain amino acid synthesis, resulting in the preliminarily D-pantothenic acid-producing probiotic *B. subtilis* MX-1. A UTR region sequence with a high translation rate is introduced. m To further enhance the translation levels of aspartate decarboxylase, hydroxymethyltransferase, and pantothenic acid synthase, a probiotic engineered bacterium, *B. subtilis* MX-2, with an earlier and stronger ability to produce D-pantothenic acid was obtained. In *B. subtilis*, pantothenic acid synthesis is a non-linear pathway involving two main modules: pantothenic acid synthesis and β-alanine synthesis. Constructing a pantothenic acid-producing probiotic without precursor addition requires further enhancement of β-alanine synthesis in *B. subtilis*. Based on *B. subtilis* MX-2, the *pdhA* gene or the *spo0A* gene was knocked out of the genome. The former weakens acetyl-CoA synthesis and inhibits early cell growth, while the latter eliminates the ability of *B. subtilis* to produce spores. Both methods enabled pantothenic acid production without precursor addition, thus obtaining strains *B. subtilis* MX-2ΔpdhA and *B. subtilis* MX-2Δspo0A. Subsequently, based on *B. subtilis* MX-2ΔpdhA or *B. subtilis* MX-2Δspo0A, the aspA gene in the genome was knocked out to block the metabolic pathway of aspartic acid and fumarate, thereby increasing the accumulation of β-alanine. Finally, the promoter of the aspB gene in the genome was replaced by the P43 promoter to promote the synthesis of aspartic acid from oxaloacetate, further increasing the supply of β-alanine, ultimately resulting in a *B. subtilis* probiotic engineered strain that produces pantothenic acid without the need for precursor addition. In this invention, the synthesis pathways of pantothenic acid and β-alanine were modified respectively, eliminating the need for any exogenous precursor addition in the fermentation broth, thus achieving the goal of microbial metabolism to synthesize D-pantothenic acid without the need for precursor addition.

[0020] This invention also relates to the application of the probiotic engineered bacteria with high D-pantothenic acid production in the preparation of D-pantothenic acid by microbial fermentation. The application is as follows: the probiotic engineered bacteria strain is inoculated into a fermentation medium and fermented at 37-45℃ and 150-250rpm for 48 to 120 hours. After fermentation, the fermentation broth is filtered, and the filtrate is separated and purified to obtain D-pantothenic acid.

[0021] Preferably, the fermentation medium is composed of the following: 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 extract, 1.5 g / L calcium carbonate, and 1 ml / L trace element solution in deionized water, with the pH adjusted to neutral every 4 hours; the trace element solution is composed of: 10 g / L calcium chloride, 10 g / L ferrous sulfate heptahydrate, 1 g / L zinc sulfate heptahydrate, 0.2 g / L copper sulfate, and 0.02 g / L cobalt chloride heptahydrate in deionized water.

[0022] Preferably, the probiotic engineered bacteria are cultured in a fermenter according to the following steps: the probiotic engineered bacteria are inoculated into a fermenter containing fermenter culture medium, and fermented at 35-45℃, 150-800 rpm, and an aeration rate of 0.5-3.8 V / V·min (preferably 41℃, 500 rpm, and an aeration rate of 1.2 V / V·min), with dissolved oxygen controlled above 20%; when the initial glucose in the fermentation broth is consumed, the automatic feeding system is activated, and feeding medium is added at a flow rate of 0-15 mL / min (preferably 8 mL / min), and the pH is adjusted with 20% ammonia water to a pH between 6.0 and 7.5 (preferably 6.8); fermentation continues for a total time of 48-120 h (preferably 120 h) to obtain a fermentation broth containing D-pantothenic acid;

[0023] The fermentation tank culture medium consists of: glucose 40 g / L, glycerol 10 g / L, ammonium sulfate 10 g / L, KH2PO4 0.8 g / L, MgSO4 0.5 g / L, yeast extract 5 g / L, soybean peptone 10 g / L, and 1 mL / L trace element solution. The solvent is deionized water, and the pH value is natural.

[0024] The feed culture medium consists of: 500 g / L glucose, 10 g / L soybean peptone, 5 g / L yeast extract, 8 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 10 g / L ammonium sulfate, and 1 mL / L trace element solution, dissolved in deionized water.

[0025] The trace element solution consists of 10 g / L CaCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.20 g / L CuSO4, and 0.02 g / L NiCl2·7H2O, with deionized water as the solvent.

[0026] The probiotic engineered bacteria usually need to undergo pre-culture before fermentation, that is, the probiotic engineered bacteria are inoculated in LB medium and cultured at 37°C for 12 hours, and then inoculated into the fermentation medium at a volume concentration of 4-10%.

[0027] This invention also relates to the application of the high-D-pantothenic acid-producing probiotic engineered bacteria in the preparation of probiotic additives. The application involves: inoculating the probiotic engineered bacteria into LB liquid medium and culturing at 37°C for 12 hours; then transferring the bacteria to LB liquid medium at a volume concentration of 4% and culturing overnight at 37°C and 200 rpm in a shaker; centrifuging the culture medium, resuspending the precipitate in PBS buffer and washing 2-3 times, then resuspending in PBS to obtain the probiotic additive; the probiotic additive contains at least 9 × 10⁻⁶ bacterial cells. 10 indivual.

[0028] The present invention also provides a probiotic feed prepared from the probiotic additive, wherein the probiotic feed is prepared by mixing the probiotic additive with feed at a volume ratio of 1:1, for example, the probiotic additive is mixed evenly with brine shrimp larvae feed at a volume ratio of 1:1, which is used to improve the intestinal flora of zebrafish, significantly improve the utilization rate of feed by zebrafish, and thus promote the growth and development of zebrafish.

[0029] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0030] 1. This invention enhances the expression and translation of the alss, ilvD, panE2, and panBCD gene clusters on the *B. subtilis* genome by using the P43 promoter derived from plasmid pWBUC01 and a highly efficient UTR sequence. This strengthens the expression of key enzymes involved in D-pantothenic acid synthesis in *B. subtilis*. Simultaneously, it knocks out the ilvA and aspA genes on the genome and employs an innovative modification route involving the knockout of the pdhA or spoA gene and overexpression of the aspB gene. This yields probiotic engineered bacteria, reducing the demand for the substrate β-alanine in D-pantothenic acid synthesis and achieving precursor-free D-pantothenic acid synthesis. Currently, β-alanine accounts for more than 50% of the price of calcium pantothenate. Precursor-free D-pantothenic acid synthesis can save significant costs associated with precursors and subsequent separation, demonstrating enormous industrial potential.

[0031] 2. This invention improves the fermentation medium of the constructed probiotic engineered bacteria and optimizes the fermentation conditions, ultimately increasing the D-pantothenic acid yield from below the detection limit to 3.32 g / L without precursor addition compared to the starting strain. This is currently the highest level of D-pantothenic acid production without substrate addition using the probiotic Bacillus subtilis. Furthermore, compared to other strains, Bacillus subtilis, being a probiotic, is safer, does not have the endotoxin production issues associated with Escherichia coli, and is more conducive to high-density fermentation, thus having a wider range of applications in industry and daily life.

[0032] 3. The probiotic engineered bacteria constructed in this invention can also be applied to feed to promote animal growth and development, such as the weight gain of zebrafish. (iv) Description of the attached drawings

[0033] Figure 1 The probiotic OD constructed in Example 1 600 And the D-pantothenic acid content in the fermentation broth supernatant.

[0034] Figure 2 UTR used in Example 2 m Functional validation of strong gene expression.

[0035] Figure 3 The probiotic OD constructed in Example 3 600 And the D-pantothenic acid content in the fermentation broth supernatant.

[0036] Figure 4 Construction of probiotic OD in Example 4 600 And the D-pantothenic acid content in the fermentation broth supernatant.

[0037] Figure 5 The probiotic OD constructed in Example 5 600 And the D-pantothenic acid content in a 1.3-L fermenter.

[0038] Figure 6 The probiotic OD constructed in Example 6 600 And the D-pantothenic acid content in a 5-L fermenter.

[0039] Figure 7 Example 7 illustrates the in vivo application of probiotic engineered bacteria in animals. (V) Detailed Implementation

[0040] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0041] The substrate bacterium Bacillus subtilis 168 of this invention is Bacillus subtilis ATCC 23857.

[0042] The composition of LB liquid medium is: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0, with water as the solvent.

[0043] The fermentation medium consisted of the following: 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 extract, 1.5 g / L calcium carbonate, and 1 ml / L trace element solution. The solvent was deionized water, and the pH was adjusted to neutral every 4 hours. The trace element solution consisted of: 10 g / L calcium chloride, 10 g / L ferrous sulfate heptahydrate, 1 g / L zinc sulfate heptahydrate, 0.2 g / L copper sulfate, and 0.02 g / L cobalt chloride heptahydrate. The solvent was deionized water.

[0044] Table 1: Genes involved in gene editing and corresponding pathways

[0045] panB 939032 Pantothenic acid synthesis panC 939030 Pantothenic acid synthesis panD 939033 Pantothenic acid synthesis panE2 936782 Synthesis of pantothenic acid ilvD 939084 Synthesis of α-ketoisovaleric acid ilvA 939093 Isoleucine synthesis alsS 936852 Acetolactate synthesis pdhA 936005 Acetyl-CoA Synthesis spo0A 938655 Spore formation aspA 938721 Aspartic acid synthesis aspB 939037 Aspartic acid synthesis B. subtilis 168 genome NC_000964 Bacillus subtilis subsp.subtilis str.168complete genome

[0046] Table 2: Primers

[0047]

[0048]

[0049] In the examples, unless otherwise specified, the final concentration of bleomycin in the culture medium is 20 mg / L, the final concentration of spectinomycin in the culture medium is 50 mg / L, and the final concentration of erythromycin in the culture medium is 10 mg / L.

[0050] SEQ ID NO.1

[0051] gtatattaga aaggaggaat atata.

[0052] SEQ ID NO.2

[0053]

[0054] Example 1: Construction of probiotic engineered strain B. subtilis MX-1 and shake-flask fermentation

[0055] Using Bacillus subtilis 168 as the substrate bacterium, the original promoter of the panBCD gene cluster was first replaced with the P43 promoter (nucleotide sequence shown in SEQ ID NO.2) using fusion PCR and homologous recombination technology to enhance pantothenic acid synthesis. Subsequently, the ilvA gene in the genome was knocked out using the B. subtilis Cre / loxP gene editing system, weakening the metabolic branching pathway. Then, the promoters of the alsS and ilvD genes in the genome were replaced with the P43 promoter using fusion PCR and homologous recombination technology, further increasing the expression of acetolactate synthase and α-ketoisovalerate synthase in the substrate bacterium strain. Finally, the panE2 gene promoter in the genome was replaced with the P43 promoter using the B. subtilis Cre / loxP gene editing system to improve pantothenic acid production capacity. The specific operational steps are as follows:

[0056] (1) Construction of the spec-P43-RBS, Zeo, and Z-P43-RBS sequences: Using plasmid p7S6P43 (F.Ceroni, A.Boo, S.Furini, TEGorochowski, O.Borkowski, YNLadak, ARAwan, C.Gilbert, G.Stan, T.Ellis, Burden-driven feedback control of gene expression, Nat. Methods 15(2018)387–393.) as a template, PCR cloning was performed using primers P1 and P2 to obtain the spec-P43-RBS sequence; p7Z6P43 (F.Ceroni, A.Boo, S.Furini, TEGorochowski, O.Borkowski, YNLadak, ARAwan, C.Gilbert, G.Stan, T.Ellis, Burden-driven feedback control of gene expression, Nat. Methods Using plasmid p7Z6P43 as a template, PCR cloning was performed with primers P3 and P5 to obtain the Zeo sequence fragment; using plasmid p7Z6P43 as a template, PCR cloning was performed with primers P3 and P4 to obtain the Zeo-P43-RBS sequence fragment. All products were verified to be correct and purified and preserved.

[0057] (2) Construction of panBCD upstream and downstream sequence fragments: Using the B. subtilis 168 genome as a template, PCR cloning was performed using primers P6 and P7 to obtain the panBCD upstream sequence fragment, and PCR cloning was performed using primers P8 and P9 to obtain the panBCD downstream sequence fragment. All products were verified to be correct and purified and preserved.

[0058] (3) Construction of upstream and downstream ilvA sequence fragments: Using the B. subtilis 168 genome as a template, PCR cloning was performed using primers P10 and P11 to obtain the upstream ilvA sequence fragment, and PCR cloning was performed using primers P12 and P13 to obtain the downstream ilvA sequence fragment. All products were verified to be correct and purified and preserved.

[0059] (4) Construction of upstream and downstream alsS sequence fragments: Using the B. subtilis 168 genome as a template, PCR cloning was performed using primers P14 and P15 to obtain the upstream alsS sequence fragment, and PCR cloning was performed using primers P16 and P17 to obtain the downstream alsS sequence fragment. All products were verified to be correct and purified and preserved.

[0060] (5) Construction of upstream and downstream ilvD sequence fragments: Using the B. subtilis 168 genome as a template, PCR cloning was performed using primers P18 and P19 to obtain the upstream ilvD sequence fragment, and PCR cloning was performed using primers P20 and P21 to obtain the downstream ilvD sequence fragment. All products were verified to be correct and purified and preserved.

[0061] (6) Construction of the upstream gene and downstream sequence fragment of panE2: Using B. subtilis 168 as a template, PCR cloning was performed using primers P22 and P23 to obtain the upstream sequence fragment of panE2, and PCR cloning was performed using primers P24 and P25 to obtain the downstream sequence fragment of panE2. All PCR products were purified and preserved after verification.

[0062] (7) Using P1 and P9 as primers, the spec-P43-RBS sequence fragment, the upstream sequence fragment of panBCD, and the downstream sequence fragment of panBCD were PCR fused into a U-spec-P43-panBCD-D sequence fragment. The fusion fragment was transformed into Bacillus subtilis 168. After screening for spectinomycin resistance on LB plates, the transformants were verified to be correct by PCR. The spectinomycin resistance gene integrated into the genome was eliminated using Cre / loxP gene recombination technology to obtain the engineered bacterium Bacillus subtilis ATCC23857 / P43-panBCD, denoted as B. subtilis X-1.

[0063] (8) Using P3 and P13 as primers, the Zeo sequence fragment, the upstream gene fragment of ilvA, and the downstream sequence fragment of ilvA were PCR-fused into a U-Zeo-ilvA-D sequence fragment. The fusion fragment was transformed into B. subtilis X-1. After bleomycin resistance was screened on LB plates, the transformants were verified to be correct by PCR. The bleomycin resistance gene integrated into the genome was eliminated using Cre / loxP gene recombination technology to obtain engineered bacteria X-1 / ΔilvA, denoted as B. subtilis X-2.

[0064] (9) Using P1 and P17 as primers, the spec-P43-RBS sequence fragment, the upstream alsS sequence fragment, and the downstream alsS sequence fragment were PCR-fused into a U-spec-P43-alsS-D sequence fragment. The fusion fragment was transformed into B. subtilis X-2, and the transformants were selected by spectinomycin resistance LB plates. After the transformants were verified to be correct by PCR, the spectinomycin resistance gene integrated into the genome was eliminated using Cre / loxP gene recombination technology to obtain engineered bacteria X-2 / P43-alsS.

[0065] (10) Using P3 and P21 as primers, the Zeo-P43-RBS sequence fragment, the upstream ilvD sequence fragment, and the downstream ilvD sequence fragment were PCR-fused into the U-Zeo-P43-ilvD-D sequence fragment. The fusion fragment was transformed into the engineered strain X-2 / P43-alsS. After bleomycin resistance screening on LB plates, the transformants were verified to be correct by PCR. The bleomycin resistance gene integrated into the genome was eliminated using Cre / loxP gene recombination technology to obtain the engineered strain X-2 / P43-alsS-P43-ilvD, denoted as B. subtilis X-3.

[0066] (11) Using P3 and P27 as primers, the Zeo-P43-RBS sequence fragment, the upstream sequence fragment of panE2, and the downstream sequence fragment of panE2 were PCR fused into the U-Zeo-P43-panE2-D sequence fragment. The fusion fragment was transformed into B. subtilis X-3, and the transformants were selected by bleomycin resistance LB plates. After the transformants were verified to be correct by PCR, the bleomycin resistance gene integrated into the genome was eliminated by Cre / loxP gene recombination technology to obtain engineered strain X-3 / P43-panE2, denoted as B. subtilisMX-1.

[0067] (12) Engineered strains B. subtilis 168, B. subtilis X-1, B. subtilis X-2, B. subtilis X-3, and B. subtilis MX-1 were inoculated into LB liquid medium and cultured overnight at 37°C and 200 rpm. The above seed culture was then transferred at a volume concentration of 4% to 250 ml shake flasks containing 50 ml of fermentation medium (with 2 g / L β-alanine added) and fermented at 41°C and 200 rpm for 48 h. The OD600 of the fermentation broth was measured, and the content of D-pantothenic acid in the fermentation broth was detected by high performance liquid chromatography. The results are as follows: Figure 1 As shown.

[0068] Depend on Figure 1 It was found that, compared with *B. subtilis* 168, the engineered strain *B. subtilis* X-1, obtained by enhancing the expression level of the panBCD gene cluster, showed an increase in D-pantothenic acid production from undetectable to 0.14 g / L upon the addition of the precursor β-alanine. Simultaneously, by deleting the ilvA gene from engineered strain *B. subtilis* X-1, engineered strain *B. subtilis* X-2 was obtained, further increasing D-pantothenic acid production to 0.18 g / L upon the addition of the precursor β-alanine. Subsequently, by overexpressing the alsS and ilvD genes in engineered strain *B. subtilis* X-2, engineered strain *B. subtilis* X-3 was obtained, further increasing D-pantothenic acid production to 0.32 g / L upon the addition of the precursor β-alanine. Finally, by overexpressing the panE2 gene on the engineered strain B. subtilis X-3, the engineered strain B. subtilis MX-1 was obtained, which increased the D-pantothenic acid production to 0.42 g / L when the precursor β-alanine was added. This indicates that the alss gene, ilvD gene, and panE2 gene all play important roles in the synthesis of pantothenic acid by the saccharidobacterium B. subtilis168.

[0069] Example 2: UTR in B. subtilis m Functional validation of sequence-mediated gene expression enhancement

[0070] To verify the UTR in B.subtilis m The sequence (nucleotide sequence as shown in SEQ ID NO.1) has the ability to enhance gene expression. Using plasmid pWBUC01-egfp (Shen, MJ, Chen, ZY, Mao, XD, Wang, L., Liang, JY, Huo, QY, et al. (2018). Two different restriction-modification systems for degrading exogenous DNA in Paenibacillus polymyxa. Biochem. Bioph. Res. Co 504, 927–932.) as a vector, the UTR sequence after promoter P43 was replaced with the UTR sequence. m The sequence was used to construct the plasmid pWBUC01-UTR. m -egfp. The B. subtilis two-step transformation method was used to transform plasmid pWBUC01-egfp with plasmid pWBUC01-UTR. m -egfp was transformed into B. subtilis 168 to obtain strains B. subtilis 168 / pWBUC01-egfp and B. subtilis 168 / pWBUC01-UTR. m -egfp.

[0071] Further selection of strains B. subtilis 168, B. subtilis 168 / pWBUC01-egfp and B. subtilis168 / pWBUC01-UTR m -egfp single colonies were transferred to 5 ml LB broth in test tubes and incubated overnight at 37°C in a shaker. The culture was then inoculated at a 1% (v / v) concentration into 250 ml shake flasks containing 50 ml of basic salt medium, and incubated at 37°C and 200 rpm for 16 h. The bacterial count and fluorescence intensity were measured every 2 h during this period. Results are shown below. Figure 2 As shown.

[0072] The basic salt culture medium consists of the following components: 14.0 g of K2HPO4·3H2O, 5.2 g of KH2PO4, 2 g of (NH4)2SO4, 0.3 g of MgSO4, 1 g of peptone, and 10 g of glucose per liter of ultrapure water.

[0073] Figure 2 This represents the relative fluorescence intensity at different times for the corresponding strains. Figure 2It can be seen that B. subtilis 168, B. subtilis 168 / pWBUC01-egfp and B. subtilis 168 / pWBUC01-UTR m The growth of the three strains of -egfp did not differ significantly, and the replacement of UTR... m The sequence slowed the growth of the strain, but the cell mass became uniform over time as the strain reached a plateau. The fluorescence intensity of the wild-type strain remained at a very low level (reaching 136.26 RFU at 16 h), while expression of the egfp gene on a plasmid resulted in a stronger fluorescence intensity (reaching 13715 RFU at 16 h). Further UTR replacement... m The fluorescence intensity of the sequence strain continued to increase significantly, reaching 33963 RFU after 16 hours of culture. This indicates that the UTR in *B. subtilis* is significantly elevated. m Sequences have the function of enhancing gene expression.

[0074] Example 3: Construction of effective engineered strain B. subtilis MX-2 and shake-flask fermentation

[0075] Increased levels of both transcription and translation can promote the expression of the target protein, thereby increasing the yield of the target product. Based on the principle described in Example 1 of increasing pantothenic acid production by replacing the original promoter of the panBCD gene cluster with a strong promoter, this paper further replaces the non-coding region sequence of the transcribed mRNA to improve stability and thus promote the translation rate, introducing a new UTR sequence. m To promote the production of pantothenic acid. The specific operation is as follows:

[0076] (1) Construction of the S-P43-UTR sequence fragment: Using plasmid p7S6P43 as a template, PCR cloning was performed using primers P28 and P29 to obtain the spec sequence fragment; using plasmid pWBUC01 (Chinese Patent CN107760705B) as a template, PCR cloning was performed using primers P30 and P31 to obtain the P43-UTR. m Sequence fragment; using P26 and P29 as primers, the spec sequence fragment and P43-UTR... m PCR fusion of sequence fragments into spec-P43-UTR m Sequence fragments were obtained, and the products were verified to be correct, purified, and preserved.

[0077] (2) Construction of panBCD upstream sequence fragment and panB sequence fragment: Using the B. subtilis 168 genome as a template, PCR cloning was performed using primers P26 and P27 to obtain the panBCD upstream sequence fragment, and PCR cloning was performed using primers P32 and P33 to obtain the panB sequence fragment. All products were verified to be correct and purified and preserved.

[0078] (3) Using P30 and P33 as primers, spec-P43-UTR m The sequence fragment, the upstream sequence fragment of panBCD, and the panB sequence fragment were PCR fused into U-spec-P43-UTR. m -panBCD-D sequence fragment, obtained product to verify correctness, and purified and preserved.

[0079] (4) The above U-spec-P43-UTR was converted using the B. subtilis method. m The -panBCD-D sequence fragment was transformed into *B. subtilis* MX-1 strain, and homologous recombinant transformants were obtained by screening on solid LB agar plates containing 100 mg / L spectinomycin. A subset of transformants (3–9) were selected for PCR verification. Strains with correct sequences were selected and subjected to antibiotic resistance elimination. The spectinomycin-resistant strain was preserved to obtain the engineered strain MX-1 / P43-UTR. m -panBCD, denoted as MX-2.

[0080] (5) Engineered strains B. subtilis MX-1 and B. subtilis MX-2 were inoculated into LB liquid medium and cultured overnight at 37°C and 200 rpm. The above seed culture was transferred at a volume concentration of 4% to a 250 ml shake flask containing 50 ml of fermentation medium (with or without 2 g / L β-alanine) and fermented at 41°C and 200 rpm for 72 h. The OD600 and the content of D-pantothenic acid in the fermentation broth were determined by high performance liquid chromatography. The results are as follows: Figure 3 As shown.

[0081] Depend on Figure 3 It was found that the engineered bacterium *B. subtilis* MX-1 achieved the highest D-pantothenic acid production (0.42 g / L) after 72 hours with the addition of the precursor β-alanine, but the D-pantothenic acid production was only 0.13 g / L without the addition of the precursor β-alanine. This was achieved by replacing the non-coding region before the panBCD gene cluster in the engineered bacterium *B. subtilis* MX-1 with a UTR. mThe engineered strain *B. subtilis* MX-2 was successfully constructed. With the addition of the precursor β-alanine, *B. subtilis* MX-2 achieved a D-pantothenic acid yield of 0.62 g / L after 72 hours. Furthermore, with the addition of β-alanine, the D-pantothenic acid yield of *B. subtilis* MX-2 reached 0.58 g / L after 48 hours, approaching its 72-hour yield. This indicates that compared to *B. subtilis* MX-1, *B. subtilis* MX-2 can produce pantothenic acid faster and at a higher rate. This provides an effective solution to the problem of pantothenic acid production typically occurring in the later stages and having a long fermentation cycle.

[0082] Example 4: Construction and shake-flask fermentation of engineered strains for pantothenic acid production without precursor addition

[0083] The pantothenic acid production in Examples 1 and 3 above requires the addition of the precursor β-alanine. However, pantothenic acid production without precursor addition has higher economic value and practical significance. Therefore, this study was conducted based on the engineered bacterium *B. subtilis* MX-2. To achieve the goal of producing pantothenic acid without precursor addition, the tricarboxylic acid cycle was weakened and spore formation was eliminated. The specific operations are as follows:

[0084] (1) Construction of the U-erm-pdhA-D sequence fragment: Using plasmid pRN5101 (Zhang, W., Ding, Y., Yao, L., Liu, K., and Du, B. (2013). Construction of gene knock-out system for Paenibacillus polymyxa SC2. Acta Microbiol. Sin. 53, 1258–1266.) as a template, PCR cloning was performed using primers P34 and P35 to obtain the erm sequence fragment; using the B. subtilis 168 genome as a template, PCR cloning was performed using primers P36 and P37 to obtain the upstream sequence fragment of pdhA; using B. subtilis Using the 168 genome as a template, PCR cloning was performed using primers P38 and P39 to obtain the downstream sequence fragment of pdhA. Using primers P34 and P39, the erm sequence fragment, the upstream sequence fragment of pdhA, and the downstream sequence fragment of pdhA were PCR fused into a U-erm-pdhA-D sequence fragment. All products were verified to be correct and purified and preserved.

[0085] (2) Construction of the U-zeo-spo0A-D sequence fragment: Using plasmid pMDZ as a template, PCR cloning was performed using primers P40 and P41 to obtain the zeo′ sequence fragment; using the B. subtilis 168 genome as a template, PCR cloning was performed using primers P42 and P43 to obtain the spo0A upstream sequence fragment; using the B. subtilis 168 genome as a template, PCR cloning was performed using primers P44 and P45 to obtain the spo0A downstream sequence fragment; using primers P46 and P47, the zeo′ sequence fragment, the spo0A upstream sequence fragment, and the spo0A downstream sequence fragment were PCR fused into the U-zeo′-spo0A-D sequence fragment. All products were verified to be correct and purified and preserved.

[0086] (3) The above U-erm-pdhA-D sequence fragment was transformed into B. subtilis MX-2 strain using the B. subtilis transformation method. Homologous recombinant transformants were obtained by screening on solid LB plates containing erythromycin. A portion of the transformants (3-9) were selected for PCR verification. Strains with correct sequences were selected and their resistance was eliminated. The strains with eliminated erythromycin resistance were preserved to obtain engineered strain MX-2 / ΔpdhA, denoted as MX-2ΔpdhA.

[0087] (4) The above U-zeo′-spo0A-D sequence fragment was transformed into B. subtilis MX-2 strain using the B. subtilis transformation method. Homologous recombinant transformants were obtained by screening on solid LB plates containing bleomycin. A portion of the transformants (3-9) were selected for PCR verification. Strains with correct sequences were selected and their resistance was eliminated. The strains with eliminated bleomycin resistance were preserved to obtain engineered strain MX-2 / Δspo0A, denoted as MX-2Δspo0A.

[0088] (5) Construction of the U-zeo-aspA-D sequence fragment: Using plasmid pMDZ as a template, PCR cloning was performed using primers P40 and P41 to obtain the zeo′ sequence fragment; using the B. subtilis 168 genome as a template, PCR cloning was performed using primers P46 and P47 to obtain the aspA upstream sequence fragment; using the B. subtilis 168 genome as a template, PCR cloning was performed using primers P48 and P49 to obtain the aspA downstream sequence fragment; using primers P46 and P49, the zeo sequence fragment, the aspA upstream sequence fragment, and the aspA downstream sequence fragment were PCR fused into the U-zeo′-aspA-D sequence fragment. All products were verified to be correct and purified and preserved.

[0089] (6) The above U-zeo′-aspA-D sequence fragment was transformed into B. subtilis MX-2 / ΔpdhA strain using the B. subtilis transformation method. Homologous recombinant transformants were obtained by screening on solid LB plates containing bleomycin. A portion of the transformants (3-9) were selected for PCR verification. Strains with correct sequences were selected and their resistance was eliminated. The bleomycin-resistant strain was preserved to obtain the engineered strain B. subtilis MX-2ΔpdhAΔaspA.

[0090] (7) Construction of the U-spec-P43-aspB-D sequence fragment: Using plasmid p7S6P43 as a template, PCR cloning was performed using primers P1 and P2 to obtain the spec-P43-RBS sequence fragment; using the B. subtilis 168 genome as a template, PCR cloning was performed using primers P50 and P51 to obtain the aspB upstream sequence fragment; using the B. subtilis 168 genome as a template, PCR cloning was performed using primers P52 and P53 to obtain the aspB downstream sequence fragment; using primers P50 and P53, the spec-P43-RBS sequence fragment, the aspB upstream sequence fragment, and the aspB downstream sequence fragment were PCR fused into the U-spec-P43-aspB-D sequence fragment. All products were verified to be correct and purified and preserved.

[0091] (8) The above U-spec-P43-aspB-D sequence fragment was transformed into B. subtilis MX-2ΔpdhAΔaspA strain using the B. subtilis transformation method. Homologous recombinant transformants were obtained by screening on solid LB plates containing spectinomycin. A portion of the transformants (3-9) were selected for PCR verification. Strains with correct sequences were selected and their resistance was eliminated. The strains with eliminated spectinomycin resistance were preserved to obtain the engineered strain B. subtilis MX-2ΔpdhAΔaspA / P43-AspB, denoted as MX-2ΔpdhAΔaspA / P43-AspB, and also denoted as B. subtilis MX-3.

[0092] (9) The engineered strains B. subtilis MX-2, B. subtilis MX-2 / ΔpdhA, B. subtilis MX-2 / Δspo0A, B. subtilis MX-2ΔpdhAΔaspA, and B. subtilis MX-2ΔpdhAΔaspA / P43-AspB were inoculated into LB liquid medium and cultured overnight at 37°C and 200 rpm. The above seed culture was transferred at a 4% inoculation rate to a 250 ml shake flask containing 50 ml of fermentation medium (without β-alanine) and fermented at 41°C and 200 rpm for 72 h. The OD600 and the D-pantothenic acid content in the fermentation broth were determined by high performance liquid chromatography (HPLC). The results are as follows: Figure 4 As shown.

[0093] Depend on Figure 4 It was found that the pantothenic acid production of the engineered bacterium *B. subtilis* MX-2 was only 0.13 g / L without the addition of the precursor β-alanine. By knocking out the *pdhA* and *spo0A* genes from the genome of *B. subtilis* MX-2, engineered bacteria *B. subtilis* MX-2 / ΔpdhA and *B. subtilis* MX-2 / Δspo0A were constructed. Although the pantothenic acid production of engineered bacterium *B. subtilis* MX-2 / ΔpdhA was low in the first 24 hours (0.03 g / L), its pantothenic acid production was higher than that of *B. subtilis* MX-2 / Δspo0A in the later stages of fermentation (0.17 g / L, 0.33 g / L, and 0.38 g / L at 48 h, 72 h, and 96 h, respectively), reaching 0.19 g / L, 0.40 g / L, and 0.45 g / L at 48 h, 72 h, and 96 h, respectively. Subsequently, the aspA gene was knocked out of the B. subtilis MX-2 / ΔpdhA genome to obtain the engineered strain B. subtilis MX-2ΔpdhAΔaspA. The pantothenic acid yield was further increased to 0.57 g / L after 96 h without precursor addition. Furthermore, the aspB gene was overexpressed in the genome of the engineered strain MX-2ΔpdhAΔaspA to construct the engineered strain B. subtilis MX-2ΔpdhAΔaspA / P43-AspB. This strain showed a further increase in fermentation yield compared to the probiotic MX-2ΔpdhAΔaspA (0.61 g / L). Based on this, it is speculated that β-alanine has reached saturation in the precursor equilibrium of pantothenic acid synthesis, and the insufficiency of pantothenic acid has become the limiting factor for pantothenic acid production in the current engineered strain. Further improvements in the pantothenic acid synthesis pathway are needed to further increase pantothenic acid yield.

[0094] Example 5: Precursor addition fermentation culture of engineered strain B. subtilis MX-3 in a 1.3L fermenter

[0095] (1) Preparation of seed culture: Pick a fresh single colony of B. subtilis MX-3 from the plate and put it into LB test tube medium. Incubate overnight at 37°C and 200 rpm to prepare the seed culture in the shake flask. Inoculate 4% of the culture into 50 mL of LB shake flask medium and incubate overnight at 37°C and 200 rpm to prepare the seed culture for the upper tank.

[0096] (2) Fed-feed fermentation culture of B. subtilis MX-3: The seed culture from the upper tank was inoculated at a volume concentration of 5% into a fermenter containing 400 mL of fermentation medium containing 2 g / L β-alanine. Fermentation was carried out at 41℃, 500 rpm, and an aeration rate of 1 V / V·min, with dissolved oxygen controlled above 20%. When the initial glucose in the fermentation broth was consumed, the automatic feeding system was activated, and feed medium containing 20 g / L β-alanine was added at a flow rate of 8 mL / min. The pH was adjusted with 20% ammonia to be between 6.5 and 7.5. Fermentation continued until the total time was 84 h, and the concentration of D-pantothenic acid in the fermentation broth was measured. HPLC analysis showed that the final concentration of D-pantothenic acid in the fermentation broth was 7.14 g / L. Figure 5 As shown.

[0097] The fermenter culture medium consists of: glucose 40 g / L, glycerol 10 g / L, ammonium sulfate 10 g / L, KH2PO4 0.8 g / L, MgSO4 0.5 g / L, yeast extract 5 g / L, soybean peptone 10 g / L, and a 1 mL / L trace element solution in deionized water with a natural pH. The trace element solution consists of: 10 g / L CaCl2, 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.

[0098] Feeding medium: 450 g / L glucose, 50 g / L glycerol, 10 g / L yeast extract, 8 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 10 g / L ammonium sulfate, dissolved in deionized water.

[0099] Example 6: Precursor-free fermentation culture of engineered strain B. subtilis MX-3 in a 5L fermenter

[0100] (1) Preparation of seed culture: Pick a fresh single colony of B. subtilis MX-3 from the plate and put it into LB test tube medium. Incubate overnight at 37°C and 200 rpm to prepare the seed culture in the shake flask. Inoculate 4% of the culture into 50 mL of LB shake flask medium and incubate overnight at 37°C and 200 rpm to prepare the seed culture for the upper tank.

[0101] (2) Fed-feed fermentation culture of B. subtilis MX-3: The seed culture from the upper tank was inoculated into a fermenter containing 2L of fermentation medium at a volume concentration of 10%. Fermentation was carried out at 41℃, 500rpm, and an aeration rate of 1.2V / V·min, with dissolved oxygen controlled above 20%. When the initial glucose in the fermentation broth was consumed, the automatic feeding system was activated, and feed medium was added at a flow rate of 8mL / min. The pH was adjusted with 20% ammonia to be between 6.5 and 7.5. Fermentation continued until the total time was 120h, and the concentration of D-pantothenic acid in the fermentation broth was measured. HPLC analysis showed that the final concentration of D-pantothenic acid in the fermentation broth was 3.32g / L. Figure 6 As shown.

[0102] The fermenter culture medium consists of the following components: glucose 40 g / L, glycerol 10 g / L, ammonium sulfate 10 g / L, KH2PO4 0.8 g / L, MgSO4 0.5 g / L, yeast extract 5 g / L, soybean peptone 10 g / L, and a 1 mL / L trace element solution in deionized water with a natural pH. The trace element solution consists of: 10 g / L CaCl2, 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.

[0103] Feeding medium: 500 g / L glucose, 10 g / L soybean peptone, 5 g / L yeast extract, 8 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 10 g / L ammonium sulfate, and 1 mL / L trace element solution, dissolved in deionized water.

[0104] Example 7: In vivo application of engineered bacteria in animals

[0105] The engineered bacteria B. subtilis MX-3 was mixed into zebrafish feed as a probiotic additive to regulate and improve the intestinal flora of zebrafish, improve their digestive capacity, enhance their immunity and disease resistance, and thus promote the growth and development of juvenile zebrafish.

[0106] (1) Probiotic culture and probiotic feed preparation: Engineered B. subtilis MX-3 was inoculated into a 250ml shake flask containing 50ml LB liquid medium and cultured overnight at 37℃ and 200rpm. The entire culture was centrifuged and resuspended in 20-30ml PBS buffer, washing 2-3 times. After the final centrifugation, the supernatant was discarded, and the bacteria were resuspended in 1ml PBS to prepare a concentrated probiotic solution with a cell count of 9.0 × 10⁻⁶ cells / mL. 10One. The concentrated probiotic solution was mixed evenly with brine shrimp larvae feed at a 1:1 volume ratio to obtain the probiotic feed. Under the same conditions, the concentrated probiotic solution prepared by replacing B. subtilis MX-3 with B. subtilis 168 served as a control.

[0107] (2) Zebrafish rearing: 40-100 healthy zebrafish of uniform weight were randomly divided into two groups. The experimental group was fed probiotic feed, while the control group was fed a mixture of PBS buffer and brine shrimp feed at a 1:1 volume ratio. During the experiment, the zebrafish were fed 2% of their body weight daily, and weighed weekly to adjust the feeding amount. Excrement was removed daily, and half of the water was changed every two days to ensure good water quality. The experimental water was tap water that had been aerated for one day, with a water temperature of around 28℃, dissolved oxygen levels greater than 5 mg / L, and natural light. The rearing period was two weeks.

[0108] (3) Sample collection and zebrafish weight measurement: After the experiment, the fish were fasted for one day. 15 zebrafish were randomly selected from each tank and their total weight was weighed. The fish were then returned to the tank. The operation was repeated 6 times. At the same time, the fish in each tank were counted and the average body weight and weight gain rate were calculated.

[0109] like Figure 7 The results show the effect of probiotic diet on zebrafish weight. During the two-week culture period, the average weight gain of zebrafish in the control group was 12.28%. Adding concentrated probiotic solution prepared from B. subtilis 168 to the diet increased the average weight gain to 15.33%. When concentrated probiotic solution prepared from B. subtilis MX-3 was added instead, the average weight gain further increased to 21.06%. These results indicate that adding concentrated probiotic solution prepared from B. subtilis MX-3 to the diet can significantly improve the intestinal flora of zebrafish, significantly improve feed utilization, and thus promote zebrafish growth and development.

Claims

1. A probiotic engineered bacterium that produces high levels of D-pantothenic acid, characterized in that, The probiotic strain is Bacillus subtilis (Bacillus subtilis) Bacillus subtilis ATCC 23857 is a chassis bacterium, and its genome contains... panBCD Gene clusters alsS Gene, ilvD Gene, panE2 Gene, aspB The promoters of all genes were replaced with the P43 promoter. panBCD Pre-UTR sequence replacement with UTR m Knockout pdhA Genes, and knockout ilvA Genes and aspA Genes were used to construct the probiotic strains described above; the UTR m The nucleotide sequence is shown in SEQ ID NO.

1.

2. The probiotic engineered bacteria with high D-pantothenic acid production as described in claim 1, characterized in that, The nucleotide sequence of the P43 promoter is shown in SEQ ID NO.

2.

3. The probiotic engineered bacteria with high D-pantothenic acid production as described in claim 1, characterized in that, The probiotic engineered bacteria that produce high levels of D-pantothenic acid were constructed using the following method: (1) with B. subtilis ATCC 23857 is a chassis strain, utilizing B. subtilis Cre / loxP Gene editing systems can insert gene-editing molecules into the genome. panBCD The promoter of the gene cluster was replaced with the P43 promoter to obtain the recombinant strain. B. subtilis ATCC23857 / P43- panBCD , recorded as B. subtilis X-1; (2) Utilization B. subtilis Cre / loxP Gene editing systems will B. subtilis X-1 genome ilvA Gene knockout, resulting in B. subtilis X-1 / ΔilvA, Recorded as B. subtilis X-2; (3) Utilization B. subtilis Cre / loxP Gene editing systems will B. subtilis X-2 genome alsS Genes and ilvD The gene promoter was replaced with the P43 promoter, resulting in B. subtilis X-2 / P43- alsS -P43- ilvD , recorded as B. subtilis X-3; (4) Utilization B. subtilis Cre / loxP Gene editing systems will B. subtilis X-3 genome panE2 The gene promoter was replaced with the P43 promoter, resulting in B. subtilis X-3 / P43- panE2 , recorded as B. subtilis MX-1; (5) Utilization B. subtilis Cre / loxP Gene editing systems will B. subtilis In the MX-1 genome panBCD Pre-UTR sequence replacement with UTR m MX-1 / UTR m - panBCD, Recorded as B. subtilis MX-2; (6) Utilization B. subtilis Cre / loxP Gene editing systems will B. subtilis In the MX-2 genome pdhA Gene knockout yields recombinant strains B. subtilis MX-2 ΔpdhA ; (7) Utilization B. subtilis Cre / loxP Gene editing systems will B. subtilis MX-2 ΔpdhA In the genome aspA Gene knockout yields recombinant strains B. subtilis MX-2 ΔpdhAΔaspA ; (8) Utilization B. subtilis Cre / loxP Gene editing systems will B. subtilis MX-2 ΔpdhAΔaspA In the genome aspB The gene promoter was replaced with the P43 promoter to obtain a recombinant strain. B. subtilis MX-2 ΔpdhAΔ aspA / P43- aspB , recorded as B. subtilis MX-3.

4. The application of the probiotic engineered bacteria with high D-pantothenic acid production as described in claim 1 in the microbial fermentation preparation of D-pantothenic acid.

5. The application as described in claim 4, characterized in that, The application is as follows: the probiotic engineered bacteria strain is inoculated into a fermentation medium and fermented at 37~45℃ and 150~250 rpm for 48 to 120 h. After fermentation, the fermentation broth is filtered, and the filtrate is separated and purified to obtain D-pantothenic acid. The fermentation medium consists of the following components: 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 extract, 1.5 g / L calcium carbonate, and 1 ml / L trace element solution in deionized water. The pH is adjusted to neutral every 4 h. The trace element solution consists of: 10 g / L calcium chloride, 10 g / L ferrous sulfate heptahydrate, 1 g / L zinc sulfate heptahydrate, 0.2 g / L copper sulfate, and 0.02 g / L cobalt chloride heptahydrate in deionized water.

6. The application as described in claim 5, characterized in that, The probiotic engineered bacteria were cultured in a fermenter according to the following steps: the probiotic engineered bacteria were inoculated into a fermenter containing fermentation culture medium and fermented at 35-45℃, 150-800 rpm, and an aeration rate of 0.5-3.8 V / V∙min, with dissolved oxygen controlled above 20%; when the initial glucose in the fermentation broth was consumed, the automatic feeding system was activated, and feeding medium was added at a flow rate of 8 mL / min, and the pH was adjusted to between 6.0 and 7.5 with 20% ammonia water; fermentation continued until the total time was 48-120 h, to obtain a fermentation broth containing D-pantothenic acid; The fermentation tank culture medium consists of: glucose 20-60 g / L, glycerol 1-20 g / L, ammonium sulfate 1-16 g / L, KH2PO4 1-1.2 g / L, MgSO4 1-1.0 g / L, yeast extract 1-20 g / L, soybean peptone 1-20 g / L, and 1-3 mL / L trace element solution. The solvent is deionized water, and the pH value is natural. The feed culture medium consists of: 400-800 g / L glucose, 1-20 g / L soybean peptone, 1-15 g / L yeast extract, 1-15 g / L potassium dihydrogen phosphate, 1-3 g / L magnesium sulfate, 1-20 g / L ammonium sulfate, and 1-3 mL / L trace element solution, dissolved in deionized water. The trace element solution consists of 10 g / L CaCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.20 g / L CuSO4, and 0.02 g / L NiCl2·7H2O, with deionized water as the solvent.

7. The application as described in claim 5 or 6, characterized in that, Before fermentation, the probiotic engineered bacteria were first inoculated into LB medium and cultured at 37°C for 12 h. Then, they were inoculated into the fermentation medium at a volume concentration of 4-10% and cultured.

8. The use of the genetically engineered bacteria with high D-pantothenic acid production as described in claim 1 in the preparation of probiotic additives.

9. The application as described in claim 8, characterized in that, The application is as follows: the probiotic engineered bacteria are inoculated into LB liquid medium and cultured at 37°C for 12 h. Then, they are transferred to LB liquid medium at a volume concentration of 4% and cultured overnight at 37°C and 200 rpm in a shaker. The culture medium is centrifuged, and the precipitate is resuspended in PBS buffer and washed 2-3 times, then resuspended in PBS to obtain the probiotic additive. The probiotic additive contains no less than 9 × 10⁻⁶ bacteria. 10 indivual.

10. A probiotic feed prepared from the probiotic additive of claim 8, characterized in that, The probiotic feed is made by mixing probiotic additives with feed at a volume ratio of 1:1.

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