Bacillus subtilis for producing D-psicose and application thereof

Abstract

The invention relates to bacillus subtilis for producing D-psicose and application of the bacillus subtilis, and belongs to the technical field of microorganisms. The invention discloses a recombinant bacillus subtilis obtained by performing gene editing by using a CRISPR / Cpf1 system, a D-psicose 6-phosphoric acid 3-epimerase coding gene A6PE, a glucokinase coding gene glck and a glucose transporter coding gene glf are integrally expressed in a starting bacillus subtilis genome, and a 6-phosphofructokinase coding gene pfkA is knocked out, so that the recombinant bacillus subtilis is obtained. A metabolic pathway for producing D-psicose by taking glucose as a substrate is constructed in bacillus subtilis, and the production efficiency is improved in a form of enhancing glucose uptake and conversion capacity, so that the yield of D-psicose of the constructed recombinant bacillus subtilis at a 250 mL shake flask fermentation level reaches 23 g / L. The bacillus subtilis for producing D-psicose provided by the invention has the advantages of directional regulation and control of metabolic flux, high substrate utilization rate, low production cost and the like, and is suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a D-psicose-producing Bacillus subtilis and applications thereof. Background Art

[0002] D-allulose (D-psicose) is a rare six-carbon ketose and the diastereomer corresponding to the third carbon position of D-fructose. Its sweetness is 70% of that of sucrose but its calories are only about 0.4kcal / g (significantly lower than the 4kcal / g of sucrose). It is present in trace amounts in figs, raisins, maple syrup and some fermented foods. D-psicose has the functions of low calories, high sweetness, inhibiting fat accumulation, protecting nerves, and inhibiting parasite growth. It is a potential anthelmintic. The main application areas of D-psicose are the food and pharmaceutical industries. In the United States, D-psicose was recognized by the FDA as a "generally recognized as safe (GRAS) ingredient" in 2015. In 2020, the FDA announced that D-psicose would be excluded from the "added sugar" and "total sugar" labels.

[0003] At present, the main methods for producing D-psicose are chemical synthesis, biosynthesis (enzyme method) and microbial fermentation. In the chemical synthesis method, D-psicose is prepared from glucose or fructose through chemical epimerization, refined chromatography separation, concentration and crystallization to produce D-psicose crystals. The chemical synthesis method has problems such as poor economic efficiency, serious environmental pollution and insufficient stereoselectivity, and cannot become the mainstream method for preparing D-psicose. There are several biosynthesis methods: (1) Using epimerase to catalyze the conversion of D-fructose into D-psicose is the first choice for industrial production. Ketose 3-epimerase mainly includes D-tagatose 3-epimerase (DTE) and D-psicose 3-epimerase (DPE). Ketose 3-epimerase has a highly conserved active center and key amino acid residues with similar characteristics. The optimal reaction temperature range of most DTE and DPE is 40-70℃ and the optimal reaction pH is 7.5-9. However, this method has low enzyme catalytic efficiency and reversible reaction, and the by-product fructose is difficult to separate and purify. (2) Isoamylase (IA) catalyzes the conversion of starch to dextrin, which is then converted to maltose by amyloglucosidase (AMG). Maltose is then phosphorylated by polyphosphate glucokinase (PPGK) to produce glucose-6-phosphate (G6P), which is then converted to fructose-6-phosphate (F6P) by phosphoglucose isomerase (PGI). D-psicose 6-phosphate epimerase (A6PE) converts F6P to D-psicose 6-phosphate (A6P), and finally, D-psicose 6-phosphate phosphatase (A6PP) dephosphorylates A6P to produce D-psicose. While this in vitro method for synthesizing D-psicose has high conversion efficiency, it requires multiple enzymes and purification, making it difficult to scale up for industrial production. Furthermore, the reaction may require the addition of substances such as ATP.

[0004] Microbial fermentation methods currently use Bacillus subtilis as a starting strain. By integrating relevant genes and deleting competing branch genes, a synthetic pathway for D-psicose synthesis using glycerol as a substrate has been constructed in Bacillus subtilis. This allows for fermentation of D-psicose using glycerol as a substrate. Bacillus subtilis is a common intestinal probiotic that effectively prevents pathogenic bacteria from colonizing the intestine. Bacillus subtilis can utilize proteins, various sugars, and starch for growth and reproduction. Due to its safety, Bacillus subtilis has been designated as a GRAS (Generally Recognized as Safe) strain by the US Food and Drug Administration (FDA). Currently, Bacillus subtilis has been developed as a host for expressing ketose 3-epimerase, which catalyzes the synthesis of D-psicose using fructose as a substrate using whole cells. Bacillus subtilis has also been used as a starting strain, with modifications enabling the synthesis of D-psicose using glycerol as the sole carbon source. However, the whole-cell catalytic synthesis of D-psicose using fructose as a substrate or the fermentation synthesis of D-psicose using glycerol as a substrate is subject to problems such as expensive raw materials and inconvenient industrial applications. Therefore, to further leverage the characteristics of Bacillus subtilis as a food-safe strain and reduce production costs, a de novo synthesis pathway for D-psicose can be developed to produce D-psicose using recombinant Bacillus subtilis using inexpensive carbon sources as raw materials. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a D-psicose-producing Bacillus subtilis and its application. The D-psicose 6-phosphate 3-epimerase encoding gene A6PE, the glucokinase gene glck, and the glucose transporter encoding gene glf are integrated and expressed in the Bacillus subtilis genome, and the 6-phosphofructokinase encoding gene pfkA is knocked out. A metabolic pathway for producing D-psicose using glucose as a substrate is constructed in Bacillus subtilis, and production efficiency is improved by enhancing the glucose uptake and conversion capacity.

[0006] A first object of the present invention is to provide a D-psicose-producing Bacillus subtilis strain, wherein the genome of the Bacillus subtilis strain is integrated with and expresses the D-psicose 6-phosphate 3-epimerase encoding gene A6PE, the glucokinase encoding gene glck, and the glucose facilitated diffusion transporter encoding gene glf, while the 6-phosphofructokinase encoding gene pfkA is knocked out.

[0007] Furthermore, the gene A6PE is inserted between the ybbU gene and the alkA gene in the Bacillus subtilis genome;

[0008] The gene glck is inserted between the pycA gene and the ctaA gene in the Bacillus subtilis genome;

[0009] The gene glf is inserted between the yqiG gene and the spo0A gene in the Bacillus subtilis genome.

[0010] Furthermore, the crRNA sequence targeting the integration expression site of gene A6PE is shown in SEQ ID NO.17.

[0011] Furthermore, the crRNA sequence targeting the integration expression site of the gene glck is shown in SEQ ID NO.19.

[0012] Furthermore, the crRNA sequence targeting the integration expression site of the gene glf is shown in SEQ ID NO.20.

[0013] Furthermore, the D-psicose 6-phosphate phosphatase encoding gene A6PP is integrated and expressed in the genome of the Bacillus subtilis.

[0014] Furthermore, the gene A6PP is inserted between the ydjC gene and the gutR gene in the Bacillus subtilis genome.

[0015] Furthermore, the crRNA sequence targeting the integration expression site of gene A6PP is shown in SEQ ID NO.18.

[0016] Furthermore, the nucleotide sequence of the A6PE gene is shown in SEQ ID NO.2.

[0017] Furthermore, the nucleotide sequence of the A6PP gene is shown in SEQ ID NO.7.

[0018] Furthermore, the Bacillus subtilis is Bacillus subtilis 168.

[0019] Furthermore, the A6PE gene is driven by the promoter P veg Express.

[0020] Furthermore, the promoter P veg The nucleotide sequence is shown in SEQ ID NO.14.

[0021] Furthermore, the glck gene and the glf gene are driven by the promoter P spovG Express.

[0022] Furthermore, the promoter P spovG The nucleotide sequence is shown in SEQ ID NO.15.

[0023] Furthermore, the Bacillus subtilis uses the CRISPR / Cpf1 system for gene editing.

[0024] Furthermore, the gene editing comprises the following steps:

[0025] Step S1, transforming the first recombinant plasmid expressing Cpf1 protein into Bacillus subtilis competent cells;

[0026] Step S2, constructing a second recombinant plasmid, wherein the second recombinant plasmid contains an A6PE gene expression cassette and a corresponding crRNA sequence, a glck gene expression cassette and a corresponding crRNA sequence, a glf gene expression cassette and a corresponding crRNA sequence, and a pfkA gene knockout cassette and a corresponding crRNA sequence;

[0027] Step S3: transforming the second recombinant plasmid into the Bacillus subtilis competent cells obtained in step S1 to obtain recombinant Bacillus subtilis.

[0028] A second object of the present invention is to provide a bacterial agent comprising the above-mentioned Bacillus subtilis.

[0029] The third object of the present invention is to provide use of the above-mentioned Bacillus subtilis or the above-mentioned bacterial agent in the production of D-psicose.

[0030] A fourth object of the present invention is to provide a method for producing D-psicose, comprising adding the above-mentioned Bacillus subtilis or the above-mentioned bacterial agent to a fermentation system using glucose as a substrate.

[0031] Furthermore, the method includes inoculating the activated Bacillus subtilis into a seed culture medium to obtain a seed liquid, and inoculating the seed liquid into a fermentation culture medium to ferment to obtain D-psicose.

[0032] Furthermore, the seed solution is cultured for 10-12 hours.

[0033] Furthermore, the fermentation time is 60-84 hours.

[0034] Beneficial effects of the present invention:

[0035] The present invention integrates the A6PE, glck, and glf genes into the Bacillus subtilis genome and knocks out the pfkA gene, redirecting carbon metabolism flux toward the D-psicose synthesis pathway, reducing byproduct accumulation and significantly improving the yield of the target product. The construction process of Bacillus subtilis uses the CRISPR / Cpf1 system to achieve multi-gene integration and knockout, shortening the strain construction cycle and improving the success rate. The constructed Bacillus subtilis uses inexpensive glucose as a substrate and, combined with a high-conversion strain, reduces raw material and energy costs, making it suitable for large-scale fermentation production. A yield of 23 g / L of psicose can be obtained in 60-84 hours of horizontal fermentation on 250 mL of flask, demonstrating strong process stability and ease of scale-up. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0037] Figure 1 Schematic diagram of the synthetic pathway for producing D-psicose by the recombinant Bacillus subtilis of the present invention;

[0038] Figure 2 Schematic diagram of the D-psicose production of each recombinant Bacillus subtilis in Example 1 of the present invention;

[0039] Figure 3 Schematic diagram of the D-psicose production of each recombinant Bacillus subtilis in Example 2 of the present invention;

[0040] Figure 4 Schematic diagram of the D-psicose production of each recombinant Bacillus subtilis in Example 3 of the present invention. DETAILED DESCRIPTION

[0041] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0042] The materials involved in the embodiments are as follows:

[0043] Bacillus subtilis gene editing plasmids pHT-XCR6 and pcrF19NM: laboratory collection, available through the molecular cloud plasmid sharing platform, accession numbers MC_0068418 and MC_0101256, respectively).

[0044] Liquid LB medium (g / L): peptone 10, yeast powder 5, NaCl 10.

[0045] Seed culture medium (g / L): peptone 10, yeast powder 5, NaCl 10.

[0046] Shake flask fermentation medium (g / L): peptone 12, yeast powder 24, K2HPO4·3H2O 12.5, KH2PO4 2.5, glucose 90.

[0047] The culture method involved in the embodiment is as follows:

[0048] After activation in LB medium, the recombinant Bacillus subtilis was inoculated into a liquid seed culture medium at 37°C, 220 rpm, for 10-12 hours. The seed solution was then inoculated into a fermentation medium at a 5% inoculum rate and incubated at 37°C, 220 rpm, for 72 hours.

[0049] The detection method involved in the embodiment is as follows:

[0050] High performance liquid chromatography (HPLC) system (Agilent Technologies 1260 series) was used for detection. TM Sugar-Pak TM The concentration of D-psicose in the fermentation broth was determined using a chromatographic column. The HPLC detector was a differential detector, the detection temperature of the chromatographic column was set at 70°C, the mobile phase was ultrapure water, and the detection flow rate was 0.5 mL / min.

[0051] Example 1: Integrated expression of D-psicose 6-phosphate 3-epimerase in Bacillus subtilis

[0052] The Bacillus subtilis gene editing plasmid pHT-XCR6 was transformed into Bacillus subtilis competent cells, and positive clones containing the target plasmid were obtained by chloramphenicol resistance screening. The positive single clones were prepared into Bacillus subtilis competent cells containing the plasmid pHT-XCR6.

[0053] According to the genome information of Bacillus subtilis 168 (purchased from the American Type Culture Collection, ATCC No. 27370) published in the National Center for Biotechnology Information (NCBI), the A6PE gene integration site was determined to be between the ybbU gene and the alkA gene, and the constitutive promoter P veg (nucleotide sequence as shown in SEQ ID NO.14) and A6PE gene sequences from different sources (nucleotide sequence of EcA6PE as shown in SEQ ID NO.1, nucleotide sequence of PeA6PE as shown in SEQ ID NO.2, nucleotide sequence of EuA6PE as shown in SEQ ID NO.3, nucleotide sequence of RuA6PE as shown in SEQ ID NO.4) to construct different A6PE integration expression cassettes P veg -EcA6PE、P veg -PeA6PE、P veg -EuA6PE and P veg -RuA6PE.

[0054] The constructed different A6PE integration expression cassettes were connected to the plasmid pcrF19-S1 (crRNA sequence containing integration site, crRNA sequence as shown in SEQ ID NO.17) to obtain recombinant plasmids pcrF19-S1-EcA6PE, pcrF19-S1-PeA6PE, pcrF19-S1-EuA6PE, pcrF19-S1-RuA6PE. The four recombinant plasmids were respectively transferred into Bacillus subtilis competent cells containing pHT-XCR6 plasmid, and Bacillus subtilis CRISPR / Cpf1 genome editing was performed. By chloramphenicol and kanamycin double resistance screening and colony PCR verification, it was confirmed that recombinant Bacillus subtilis was obtained. A single colony of a positive clone was inoculated into liquid LB medium and cultured at 50°C and 220 rpm for 10-12 hours to eliminate the plasmids pcrF19-S1-EcA6PE, pcrF19-S1-PeA6PE, pcrF19-S1-EuA6PE, and pcrF19-S1-RuA6PE, thereby obtaining recombinant Bacillus subtilis BS1-BS4 with A6PE gene integration. The D-psicose production of the recombinant Bacillus subtilis BS1-BS4 was tested according to the above-mentioned culture and detection methods. The results are as follows: Figure 2 As shown in the figure, it can be seen that the introduction of PeA6PE into the Bacillus subtilis genome significantly increased the production of D-psicose.

[0055] Example 2: Integrated expression of D-psicose 6-phosphate phosphatase in Bacillus subtilis

[0056] According to the genome information of Bacillus subtilis 168 published on NCBI, the site for integrating the A6PP gene was determined to be between the ydjC gene and the gutR gene, and the constitutive promoter P spovG The A6PP integration expression cassettes P were constructed by sequencing different A6PP genes (the nucleotide sequence of BsA6PP is shown in SEQ ID NO. 5, the nucleotide sequence of EcA6PP is shown in SEQ ID NO. 6, the nucleotide sequence of PlaA6PP is shown in SEQ ID NO. 7, and the nucleotide sequence of SeA6PP is shown in SEQ ID NO. 8). spovG -BsA6PP、P spovG -EcA6PP、P spovG -PlaA6PP、P spovG -SeA6PP).

[0057] The constructed different A6PP integration expression cassettes were connected to the plasmid pcrF19-S2 (crRNA sequence containing the integration site, the crRNA sequence is shown in SEQ ID NO.18) to obtain recombinant plasmids pcrF19-S2-BsA6PP, pcrF19-S2-EcA6PP, pcrF19-S2-PlaA6PP, pcrF19-S2-SeA6PP. Four different recombinant plasmids were transferred into Bacillus subtilis competent cells containing pHT-XCR6 plasmid. By chloramphenicol and kanamycin double resistance screening and colony PCR verification, it was confirmed that recombinant Bacillus subtilis was obtained. The positive clone single colony was inoculated into liquid LB medium, placed at 50 ° C, and cultured under the conditions of 220 rpm for 10-12 h to eliminate the recombinant plasmid, and recombinant Bacillus subtilis BS01-BS04 with completed A6PP gene integration was obtained. The D-psicose production of recombinant Bacillus subtilis BS1-BS4 was detected according to the above-mentioned culture method and detection method. The results are as follows: Figure 2 As shown, it can be seen that the introduction of A6PP into the genome of Bacillus subtilis did not enable Bacillus subtilis to acquire the ability to produce D-psicose.

[0058] The lytic proteins malE and NusA were connected upstream of the gene SeA6PP (the nucleotide sequence of malE-SeA6PP is shown in SEQ ID NO.9, and the nucleotide sequence of NusA-SeA6PP is shown in SEQ ID NO.10). Recombinant plasmids pcrF19-S2-malE-SeA6PP and pcrF19-S2-NusA-SeA6PP were obtained according to the above method and transferred into Bacillus subtilis competent cells containing the pHT-XCR6 plasmid. Recombinant Bacillus subtilis BS05 and BS06 were obtained through dual resistance screening, colony PCR verification, and plasmid elimination. The D-psicose production of recombinant Bacillus subtilis BS1-BS4 was tested according to the above culture and detection methods, and the results are shown in Figure 2. Figure 2 As shown, the introduction of solubilizing protein also failed to enable Bacillus subtilis to acquire the ability to produce D-psicose.

[0059] Example 3: Combined Expression of D-psicose 6-phosphate 3-epimerase and D-psicose 6-phosphate phosphatase in Bacillus subtilis

[0060] The recombinant plasmids pcrF19-S1-EcA6PE, pcrF19-S1-PeA6PE, pcrF19-S1-EuA6PE, and pcrF19-S1-RuA6PE containing different A6PE gene expression cassettes constructed in Example 1 were respectively integrated into the competent cells of BS01-BS06 strains containing the pHT-XCR6 plasmid. By chloramphenicol and kanamycin double resistance screening and colony PCR verification, it was confirmed that recombinant Bacillus subtilis was obtained. The positive clone single colony was inoculated into liquid LB medium and placed at 50 ° C, 220 rpm and cultured for 10-12 h to eliminate the plasmid, and recombinant Bacillus subtilis BS11-BS16, BS21-BS26, BS31-BS36, and BS41-BS46 with completed A6PP gene integration were obtained. The D-psicose production of recombinant Bacillus subtilis BS11-BS16, BS21-BS26, BS31-BS36, and BS41-BS46 was detected according to the above-mentioned culture and detection methods. The results are as follows: Figure 2 As shown, it can be seen that the D-psicose production of Bacillus subtilis BS31-BS36 is significantly improved compared with the other recombinant Bacillus subtilis, which is consistent with the experimental results of Example 1. Therefore, the recombinant Bacillus subtilis BS3 expressing PeA6PE was selected in subsequent examples.

[0061] Example 4: Enhancement of D-psicose metabolic pathway in Bacillus subtilis

[0062] According to the genomic information of Bacillus subtilis 168 published on NCBI, the upstream and downstream gene sequences of the pfkA gene (nucleotide sequence as shown in SEQ ID NO.11) were determined to construct the pfkA knockout cassette. The constructed pfkA knockout cassette was connected to the plasmid pcrF19NM (sequence as shown in SEQ ID NO.16) to obtain the pcrF19-pfkA plasmid. The pcrF19-pfkA plasmid was transferred into the competent cells of the BS3 strain containing the pHT-XCR6 plasmid. By chloramphenicol and kanamycin double resistance screening and colony PCR verification, it was confirmed that the recombinant Bacillus subtilis was obtained. The positive clone single colony was inoculated into liquid LB medium and placed at 50 ° C, 220 rpm to culture for 10-12h to eliminate the plasmid pcrF19-pfkA, and the recombinant Bacillus subtilis BS3P with the pfkA gene knocked out was obtained. The D-psicose production of recombinant Bacillus subtilis BS3P was detected according to the above-mentioned culture and detection methods. The results are as follows: Figure 3 As shown, the D-psicose production of Bacillus subtilis BS3P was significantly improved from 300 mg / L to 11 g / L.

[0063] Example 5: Improvement of Glucose Uptake and Conversion Capacity of Bacillus subtilis

[0064] The plasmid pHT-XCR6 was transformed into Bacillus subtilis competent cells, and positive clones containing the target plasmid were obtained by chloramphenicol resistance screening. The positive single clones were prepared into Bacillus subtilis competent cells containing the plasmid pHT-XCR6.

[0065] According to the genome information of Bacillus subtilis 168 published on NCBI, the site of integration of glck gene was determined to be located between pycA gene and ctaA gene in the genome of Bacillus subtilis, and adjacent to the constitutive promoter P spovG and the glck gene sequence of Bacillus subtilis 168 strain (nucleotide sequence shown in SEQ ID NO.12) to construct the glck integration expression cassette P spovG -glck.

[0066] The constructed glck gene integration expression cassette is connected to plasmid pcrF19-S4 (crRNA sequence containing integration site, crRNA sequence as shown in SEQ ID NO.19) to obtain recombinant plasmid pcrF19-S4-glck. The pcrF19-S4-glck plasmid is transferred into BS3P strain competent cells containing pHT-XCR6 plasmid. By chloramphenicol and kanamycin double resistance screening and colony PCR verification, it is confirmed that recombinant Bacillus subtilis is obtained. The positive clone single colony is inoculated into liquid LB medium, placed at 50 ° C, and cultured under the conditions of 220 rpm for 10-12h to eliminate plasmid pcrF19-S4-glck, and the recombinant Bacillus subtilis BS3P-glck with completed glck gene integration is obtained.

[0067] According to the genome information of Bacillus subtilis 168 published on NCBI, the site for integration of the glf gene was determined to be located between the yqiG gene and the spo0A gene in the Bacillus subtilis genome, and to be closely related to the constitutive promoter P spovG The glf integration expression cassette was constructed by using the glucose facilitated diffusion transporter glf gene sequence of Zymomonas mobilis (nucleotide sequence shown in SEQ ID NO.13).

[0068] The constructed glf gene integration expression cassette is connected to the plasmid pcrF19-S6 (crRNA sequence containing integration site, crRNA sequence is as shown in SEQ ID NO.20) to obtain pcrF19-S6-glf plasmid, and the pcrF19-S6-glf plasmid is transferred into BS3P strain competent cells containing pHT-XCR6 plasmid. By chloramphenicol and kanamycin double resistance screening and colony PCR verification, it is confirmed that recombinant Bacillus subtilis is obtained. The positive clone single colony is inoculated into liquid LB medium, placed at 50 DEG C, and cultured under the conditions of 220rpm for 10-12h to eliminate plasmid pcrF19-S6-glf, and the recombinant Bacillus subtilis BS3P-glf having completed glf gene integration is obtained.

[0069] The recombinant plasmid pcrF19-S6-glf, containing the glf gene integration expression cassette, was integrated into competent cells of the BS3P-glck strain containing the pHT-XCR6 plasmid. Chloramphenicol and kanamycin resistance screening and colony PCR verification confirmed the generation of recombinant Bacillus subtilis. A single colony of a positive clone was inoculated into liquid LB medium and cultured at 50°C, 220 rpm, for 10-12 hours to eliminate the pcrF19-S6-glf plasmid, thereby obtaining recombinant Bacillus subtilis BS3P-glck-glf with complete glf gene integration.

[0070] The D-psicose production of recombinant Bacillus subtilis BS3P-glck, BS3P-glf and BS3P-glck-glf was detected according to the above-mentioned culture and detection methods. The results are as follows: Figure 4 As shown in the results, expressing either the glck or glf gene alone in recombinant Bacillus subtilis has limited effect on increasing D-psicose production and may even reduce yield. However, expressing both the glck and glf genes simultaneously in Bacillus subtilis, significantly increasing D-psicose production to 23 g / L, can significantly boost glucose uptake and conversion.

[0071] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A Bacillus subtilis for producing D-psicose, characterized in that: The genome of the Bacillus subtilis is integrated with and expressed with the D-psicose 6-phosphate 3-epimerase encoding gene A6PE, the glucokinase encoding gene glck, and the glucose facilitated diffusion transporter encoding gene glf, and the 6-phosphofructokinase encoding gene pfkA is knocked out.

2. The Bacillus subtilis according to claim 1, wherein: The gene A6PE is inserted between the ybbU gene and the alkA gene in the Bacillus subtilis genome; The gene glck is inserted between the pycA gene and the ctaA gene in the Bacillus subtilis genome; The gene glf is inserted between the yqiG gene and the spo0A gene in the Bacillus subtilis genome.

3. The Bacillus subtilis according to claim 1, wherein: The D-psicose 6-phosphate phosphatase encoding gene A6PP is integrated and expressed in the genome of the Bacillus subtilis.

4. The Bacillus subtilis according to claim 1, wherein: The nucleotide sequence of the A6PE gene is shown in SEQ ID NO.

2.

5. The Bacillus subtilis according to claim 1, wherein: The Bacillus subtilis is Bacillus subtilis 168.

6. The Bacillus subtilis according to claim 1, wherein: A6PE gene is regulated by promoter P veg Express.

7. The Bacillus subtilis according to claim 1, wherein: The glck and glf genes are driven by promoter P spovG Express.

8. The Bacillus subtilis according to claim 1, wherein: The Bacillus subtilis is gene-edited using the CRISPR / Cpf1 system.

9. The Bacillus subtilis according to claim 8, characterized in that The gene editing comprises the following steps: Step S1, transforming the first recombinant plasmid expressing Cpf1 protein into Bacillus subtilis competent cells; Step S2, constructing a second recombinant plasmid, wherein the second recombinant plasmid contains an A6PE gene expression cassette and a corresponding crRNA sequence, a glck gene expression cassette and a corresponding crRNA sequence, a glf gene expression cassette and a corresponding crRNA sequence, and a pfkA gene knockout cassette and a corresponding crRNA sequence; Step S3: transforming the second recombinant plasmid into the Bacillus subtilis competent cells obtained in step S1 to obtain recombinant Bacillus subtilis.

10. A bacterial agent comprising the Bacillus subtilis according to any one of claims 1 to 9.

11. Use of the Bacillus subtilis according to any one of claims 1 to 9 or the bacterial agent according to claim 10 in producing D-psicose.

12. A method for producing D-psicose, characterized in that: Glucose is used as a substrate in a fermentation system, and the Bacillus subtilis according to any one of claims 1 to 9 or the bacterial agent according to claim 10 is added.

13. The method according to claim 12, wherein: The steps include activating the Bacillus subtilis and inoculating it into a seed culture medium to obtain seed liquid, and inoculating the seed liquid into a fermentation culture medium to ferment and obtain D-psicose.

14. The method according to claim 13, wherein: The culturing time of the seed solution is 10-12 hours.

15. The method according to claim 13, wherein: The fermentation time is 60-84 hours.

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