Bacillus licheniformis cells that can be stably and repeatedly used for the conversion and synthesis of D-psicose.

Abstract

The present invention provides a Bacillus licheniformis cell that can be stably and repeatedly used for the conversion synthesis of D-psicose, which belongs to the field of biotechnology. D-psicose is a functional sugar with high health value, and is expected to be applied in the fields of food, medicine, health care, etc. The Bacillus licheniformis cell of the present invention expresses recombinant D-psicose-3-epimerase with high efficiency, and can convert and produce D-psicose using a high concentration of fructose as a substrate. The whole cell catalyst has a conversion rate of 30% or more after 10 repeated uses. Compared with the pure enzyme or disposable whole cell conversion method, the synthesis of D-psicose by this technology has significant advantages such as low catalyst usage cost and easy separation and purification.

Description

[Technical Field]

[0001] This invention relates to Bacillus licheniformis cells that can be stably and repeatedly used for the conversion synthesis of D-psicose, and belongs to the field of biotechnology. [Background technology]

[0002] D-psicose is a naturally occurring sugar found in extremely small quantities. Its sweetness reaches 70% of that of sucrose, and it has a refreshing taste with no bitterness. Furthermore, because D-psicose is either not metabolized by the body or is metabolized very little, it is considered the most promising substitute for sucrose and is expected to have applications in fields such as food, medicine, and healthcare.

[0003] D-psicose is difficult and inefficient to synthesize chemically. Biological conversion is becoming the primary method for D-psicose synthesis due to its advantages, including mild reaction conditions, fewer by-products, simple purification steps, and environmental friendliness. Currently, D-psicose bioconversion mainly involves expressing D-psicose-3-epimerase in host cells such as Escherichia coli, Corynebacterium glutamicum, or Bacillus subtilis, obtaining pure enzyme through cell disruption, and converting it to D-fructose as a substrate to synthesize D-psicose. In this production method, the pure enzyme used as a catalyst can only be used once, which makes product separation inconvenient. Currently, there are few reports of D-psicose synthesis using whole-cell conversion, and the host cells mainly used are still Escherichia coli, Corynebacterium glutamicum, and Bacillus subtilis. Whole-cell catalysts do not require the step of disrupting cells after fermentation, and the catalyst can be easily separated from the product by centrifugation or filtration. However, E. coli, Corynebacterium glutamicum, and herbaceous bacteria cells are easily decomposed at catalytic temperatures of 60-70°C, and even if the cells are recovered, they are extremely susceptible to phage infection during the process. According to literature reports, Bacillus subtilis exhibits a serious autolysis phenomenon.For example, as stated in the article by Regamey and Karamat et al., "However, there is a problem with cell autolysis in fermentation cultures of B. subtilis," "As mentioned in the article by Regamey and Karamat et al., after B. subtilis was subjected to thermal excitation at 50 °C, prophage spβ was induced, and many cells underwent autolysis," or in the paper Ren, K.; Wang, Q.; Hu, M.; Chen, Y.; Xing, R.; You, J.; Xu, M.; Zhang, X.; Rao, Z. Research Progress on the Effect of Autolysis to Bacillus subtilis Fermentation Bioprocess. Fermentation2022, 8, 685., Bacillus subtilis exhibits autolysis, and the problem of autolysis becomes particularly serious in high-temperature (50°C) environments. Therefore, the reuse of the entire cell catalyst is difficult.

[0004] Bacillus licheniformis is a widely used production host for food enzyme preparations and important nutritional chemicals, and its products are certified by the FDA as "generally regarded as safe" (GRAS). Furthermore, this strain is a typical thermostable microorganism, capable of growth at 50°C, and its cultured cells exhibit excellent stability at 60-70°C, with very few phage infection problems. Engineered bacteria constructed using Bacillus licheniformis can be stably and repeatedly used for the conversion synthesis of D-psicose, offering significant advantages in terms of production efficiency and cost compared to conventional methods. [Overview of the Initiative]

[0005] The first object of the present invention is to provide a gene encoding D-psicose-3-epimerase, the nucleotide sequence of which is indicated by SEQ ID NO.1.

[0006] A second object of the present invention is to provide a vector for expressing the above-mentioned gene.

[0007] In one embodiment, the vector is pHY300-PLK.

[0008] A third object of the present invention is to provide cells carrying the above gene or vector.

[0009] In one embodiment, the cells are Bacillus licheniformis.

[0010] In one embodiment, the Bacillus licheniformis includes Bacillus licheniformis CICIM B1341.

[0011] A fourth object of the present invention is to provide recombinant Bacillus licheniformis carrying the gene encoding D-psicose-3-epimerase.

[0012] In one embodiment, the D-psicose-3-epimerase gene is located in promoter P lan Its expression is initiated by this.

[0013] In one embodiment, the D-psicose-3-epimerase gene is integrated into the amyL site of the amylase-coding gene in the genome of Bacillus licheniformis.

[0014] In one embodiment, the promoter P lan The nucleotide sequence is shown in SEQ ID NO.2.

[0015] In one embodiment, the nucleotide sequence of the amylase-coding gene is indicated by SEQ ID NO.4.

[0016] In one embodiment, the expression vector of the recombinant Bacillus licheniformis is pHY300-PLK.

[0017] In one embodiment, the recombinant Bacillus licheniformis uses Bacillus licheniformis CICIM B1341 as the host.

[0018] In one embodiment, the Bacillus licheniformis CICIM B1341 is obtained from a natural strain preserved in the China National Platform for Industrial Microbial Resources.

[0019] The fifth object of the present invention is to provide a whole cell catalyst containing the above recombinant Bacillus licheniformis.

[0020] The sixth object of the present invention is to fuse the D-psicose-3-epimerase gene a1 with the promoter P lan and the terminator ter to obtain an integration fragment, ligate the integration fragment to the pHY300-PLK vector to obtain the recombinant plasmid pHY300-P lan -a1, introduce the recombinant plasmid into Bacillus licheniformis, and through subculture, integrate the expression frame of the D-psicose-3-epimerase gene into the site of the amylase-encoding gene amyL in the genome of Bacillus licheniformis, and provide a method for constructing the above recombinant Bacillus licheniformis.

[0021] In one embodiment, the nucleotide sequence of the promoter P lan is shown by SEQ ID NO.2, and the nucleotide sequence of the terminator ter is shown by SEQ ID NO.3.

[0022] In one embodiment, the nucleotide sequence of the amylase-encoding gene is shown by SEQ ID NO.4.

[0023] The seventh object of the present invention is to provide a method for synthesizing D-psicose by whole-cell conversion, which uses D-fructose as a substrate, adds the wet cells of the recombinant Bacillus licheniformis or the whole-cell catalyst, and reacts at 50-70 °C for 5-10 h.

[0024] In one embodiment, after the reaction is completed, the recombinant Bacillus licheniformis or the whole-cell catalyst is recovered and repeatedly used, added to a reaction system using D-fructose as a substrate, and D-psicose is synthesized.

[0025] In one embodiment, the number of times of repeated use is 10 or more.

[0026] In one embodiment, the recombinant Bacillus licheniformis or the whole-cell catalyst is inoculated into a seed medium, cultured at 37 °C - 42 °C for 12 - 24 h, then transferred to a fermentation medium at an inoculation amount of 1% - 5% by volume, and cultured at 37 °C - 42 °C for 24 - 36 h to collect wet cells.

[0027] In one embodiment, the OD 600 value of the wet cells is 50 - 100.

[0028] In one embodiment, the seed medium contains 8 - 12 g / L of tryptone, 4 - 6 g / L of yeast powder, and 8 - 12 g / L of NaCl.

[0029] In one embodiment, the fermentation medium contains 20 - 70 g / L of sucrose, 10 - 30 g / L of cottonseed protein, 9.12 g / L of K2HPO4·3H2O, 1.36 g / L of KH2PO4, and 10 g / L of (NH4)2HPO4, and the initial pH is 7.5.

[0030] The present invention also provides the use of the above gene, or the above vector, or the above cell, or the above recombinant Bacillus licheniformis, or the above whole-cell catalyst in the production of a product containing D-psicose.

[0031] Compared with the prior art, the progressive and positive effects of the present invention are as follows.

[0032] 1. The present invention uses Bacillus licheniformis, a food-safe microorganism, as a starting strain, and utilizes the excellent high-temperature tolerance of its cells to protect the D-psicose-3-epimerase expressed in the cells during a catalytic process. The recombinant Bacillus licheniformis according to the present invention can grow at 37-42°C.

[0033] 2. The present invention constructs a gene encoding D-psicose-3-epimerase, whose nucleotide sequence is indicated by SEQ ID NO.1, in Bacillus licheniformis, and obtains a whole-cell catalyst that can repeat 10 fermentation reactions at a temperature of 60°C with a conversion rate of 30% or more. The technical solution of the present invention has a significant advantage in terms of manufacturing cost and efficiency compared to other whole-cell catalyst methods that are prone to pure enzymatic conversion due to the disposability of catalysts or to cell destruction. [Brief explanation of the drawing]

[0034] [Figure 1] This involves mapping and verifying the enzymatic digestion of the recombinant plasmid pHY300-Plan-a1. [Figure 2] This is a map of recombinant plasmid pMA5-a1. [Figure 3] This is the result of the HPLC analysis of the reaction solution. [Figure 4] This is a whole-cell catalyst synthesis of D-psicose using 10 batches of repeated applications. [Figure 5] This is a single-ceramic membrane continuous bioconversion apparatus. [Modes for carrying out the invention]

[0035] The present invention will be further described below with reference to the drawings and specific examples of the specification, but the examples are not intended to limit the present invention in any way. Unless otherwise specified, the reagents and materials used in the following examples are either commercially available or can be prepared by known methods.

[0036] Method according to the following embodiment 1. Method for measuring D-psicose: The catalytic reaction system was centrifuged at 13,000 r / min for 20 minutes, the supernatant was diluted 2-fold with anhydrous ethanol, allowed to stand at 4°C for 2 hours, then filtered through a 0.22 μM aqueous film, and D-psicose was detected by high-performance liquid chromatography.

[0037] HPLC detection conditions Column: 250 mm * 4.6 μm Polyamino HILIC; Mobile phase: Acetonitrile:Water = 75:25; Detector: Differential detector; Flow rate: 1 mL / min; Column temperature: 40°C; Differential detector cell temperature: 40°C.

[0038] 2. Method for measuring the enzyme activity of D-psicose-3-epimerase: 1 mL of fermentation broth sample is centrifuged at 12000 r / min for 5 minutes, the precipitate is repeatedly washed with phosphate buffer (50 mmol / L, pH 7.5), centrifuged three times, the cell precipitate is diluted to an appropriate concentration, and then 1 mL of the final volume is added with a fructose substrate solution prepared with HEPEs buffer (50 mmol / L, pH 7.5) (fructose mass concentration is 90 g / L, Co 2+ Mn 2+ The solution was resuspended to a concentration of 1 mmol / L, incubated at 60°C for 10 minutes, boiled for 5 minutes to kill the enzyme, then centrifuged at 12000 r / min for 5 minutes. The supernatant was passed through a 0.22 μm filter membrane and D-psicose was analyzed under HPLC conditions.

[0039] Definition of enzyme activity: 1 mg of fructose within 1 hour D-Psychocolate The enzyme activity required to convert to is defined as 1 U.

[0040] Plasmids and strains related to the following examples Bacillus licheniformis: CICIM B1341, stored in this laboratory, available for purchase from the Center for Industrial Microbial Resources and Information of China Higher Education University, Jiangnan University.

[0041] Culture medium according to the following examples Seed culture medium: 10 g / L tryptone, 5 g / L yeast powder, 10 g / L NaCl.

[0042] Fermentation medium: 70 g / L sucrose, 30 g / L cottonseed protein, 9.12 g / L K2HPO4·3H2O, 1.36 g / L KH2PO4, 10 g / L (NH4)2HPO4; initial pH is 7.5.

[0043] Example 1: Construction of a recombinant Bacillus licheniformis strain overexpressing the D-psicose-3-epimerase gene. (1) Knock out the amylase-coding gene amyL and express the D-psicose-3-epimerase gene in combination.

[0044] An a1 gene fragment expression cassette, obtained by fusion of the promoter and terminator through codon optimization, was used as a template to amplify the gene, yielding an expression cassette containing the a1 gene fragment whose nucleotide sequence is indicated by SEQ ID NO. 1. The amplification primers are as follows:

[0045] Upstream primer: 5-GCGCGGATCCATGAAGCACGGTATCTATTA-3 (BamHI), SEQ ID NO.5 Downstream primer: 5-CCGGAAGCTTGGAGTGTTTGTGACATTCTA-3 (HindIII), SEQ ID NO.6 PCR conditions: Denaturation at 94°C for 2 min, denaturation at 98°C for 30 s, annealing at 50°C for 30 s, and extension at 68°C for 1 min.

[0046] Using two restriction enzymes, Bam HI and Hind III, the expression cassette containing the a1 gene fragment obtained by PCR amplification was digested with enzymes at 37 °C and ligated at 16 °C using T4 ligase. The resulting ligation product was transformed into Escherichia coli DH5α and screened on a seed medium plate with ampicillin resistance. Transformants were selected for plasmid extraction, enzyme digestion verification, and gene sequencing. As shown in Figure 1, the recombinant plasmid with the correct sequence was designated as pHY300-P lan -a1.

[0047] The recombinant plasmid pHY300-P lan -a1 was extracted from Escherichia coli. After adding the homologous arm of the amylase-encoding gene amyL according to the method described in Li, Y.; Jin, K.; Zhang, L.; Ding, Z.; Gu, Z.; Shi, G. Development of an Inducible Secretory Expression System in Bacillus licheniformis Based on an Engineered Xylose Operon. Journal of Agricultural and Food Chemistry 2018, 66, 9456-9464., it was introduced into Bacillus licheniformis to obtain recombinant Bacillus licheniformis BLA1 in which the a1 gene fragment expression cassette was integrated into the site of the amylase-encoding gene amyL.

[0048] (2) Free expression of the D-psicose-3-epimerase gene An a1 gene fragment expression cassette, obtained by fusion of the promoter and terminator through codon optimization, was used as a template to amplify the gene, yielding an expression cassette containing the a1 gene fragment whose nucleotide sequence is indicated by SEQ ID NO. 1. The amplification primers are as follows:

[0049] Upstream primer: 5-GCGCGGATCCATGAAGCACGGTATCTATTA-3 (BamHI), SEQ ID NO.5 Downstream primer: 5-CCGGCTAGCTGGAGTGTTTGTGACATTCTA-3 (H ind III), SEQ ID NO.6 PCR conditions: Denaturation at 94°C for 2 minutes, denaturation at 98°C for 30 seconds, annealing at 50°C for 30 seconds, and extension at 68°C for 1 minute.

[0050] Using two restriction enzymes, BamHI and H ind III, an expression cassette containing the a1 gene fragment obtained by PCR amplification with the pMA5 vector (derived from Xu, Yinbiao, et al. "Unraveling the specific regulation of the shikimate pathway for tyrosine accumulation in Bacillus licheniformis." Journal of Industrial Microbiology and Biotechnology 46.8 (2019): 1047-1059) was enzymatically cleaved at 37°C, ligated at 16°C using T4 ligase, and the resulting ligation product was transformed into E. coli DH5a. The transformed cells were screened on an ampicillin-resistant seed medium plate, and plasmid extraction, enzymatic cleavage verification, and gene sequencing were performed. As shown in Figure 2, the recombinant plasmid whose sequence was determined to be accurate was named pMA5-a1.

[0051] Recombinant plasmid pMA5-a1 was extracted from Escherichia coli and introduced into Bacillus licheniformis according to the method described in Li, Y.; Jin, K.; Zhang, L.; Ding, Z.; Gu, Z.; Shi, G. Development of an Inducible Secretory Expression System in Bacillus licheniformis Based on an Engineered Xylose Operon. Journal of Agricultural and Food Chemistry 2018, 66, 9456-9464. to obtain recombinant Bacillus licheniformis BLA2 in which the a1 gene fragment expression cassette was freely expressed.

[0052] (3) Knock out the protease coding gene aprE and express the D-psicose-3-epimerase gene in combination.

[0053] An a1 gene fragment expression cassette, obtained by fusion of the promoter and terminator through codon optimization, was used as a template to amplify the gene, yielding an expression cassette containing the a1 gene fragment whose nucleotide sequence is indicated by SEQ ID NO. 1. The amplification primers are as follows:

[0054] Upstream primer: 5-GCGCGGATCCATGAAGCACGGTATCTATTA-3 (BamHI), SEQ ID NO.5 Downstream primer: 5-CCGGAAGCTTGGAGTGTTTGTGACATTCTA-3 (HindIII), SEQ ID NO.6 PCR conditions: Denaturation at 94°C for 2 minutes, denaturation at 98°C for 30 seconds, annealing at 50°C for 30 seconds, and extension at 68°C for 1 minute.

[0055] Using two restriction enzymes, Bam HI and Hind III, an expression cassette containing the a1 gene fragment obtained by PCR amplification with the pHY300-PLK vector (derived from Li, Y.; Jin, K.; Zhang, L.; Ding, Z.; Gu, Z.; Shi, G. Development of an Inducible Secretory Expression System in Bacillus licheniformis Based on an Engineered Xylose Operon. Journal of Agricultural and Food Chemistry 2018, 66, 9456-9464.) was enzymatically cleaved at 37°C, ligated at 16°C using T4 ligase, and the resulting ligation product was transformed into E. coli DH5a. Screening was performed on an ampicillin-resistant seed medium plate, transformants were selected, plasmid extraction was performed, enzymatic cleavage verification was verified, and gene sequencing was determined. As shown in Figure 1, recombinant plasmids whose sequences were determined to be accurate were named pHY300-P lan I named it -a1.

[0056] Recombinant plasmid pHY300-P from E. coli lan -a1 was extracted and, following the method described in Li, Y.; Jin, K.; Zhang, L.; Ding, Z.; Gu, Z.; Shi, G. Development of an Inducible Secretory Expression System in Bacillus licheniformisBased on an Engineered Xylose Operon. Journal of Agricultural and Food Chemistry 2018, 66, 9456-9464., homologous arms of the protease-coding gene aprE were added, and then introduced into Bacillus licheniformis to obtain recombinant Bacillus licheniformis BLA3 in which the a1 gene fragment expression cassette was incorporated at the site of the protease-coding gene aprE.

[0057] Example 2: Culture and enzyme production of each recombinant bacterial cell. Recombinant Bacillus licheniformis BLA1, BLA2, and BLA3 prepared in Example 1 were inoculated into fermentation medium, respectively, and cultured at 37°C. After 72 hours, samples were collected and cell OD was measured. 600 The product concentration was also detected. Table 1 shows the OD after fermentation culture of each recombinant microorganism. 600 The values ​​and enzyme activity yields were shown. The results indicated that the enzyme activity when the D-psicose-3-epimerase gene was integrated into the amylase-coding gene amyL site was significantly higher than the enzyme activity when it was expressed freely or when the D-psicose-3-epimerase gene was integrated into the amylase-coding gene aprE site.

[0058] Table 1 Cell concentration and enzyme activity of recombinant bacteria at various temperatures TIFF0007846707000001.tif43170 Example 3: Culture and enzyme production of recombinant bacterial cells BLA1 under various temperature conditions Recombinant Bacillus licheniformis BLA1, constructed in Example 1, was inoculated into fermentation medium and cultured at 37°C and 42°C, respectively. Samples were collected on days 3 and 4, and the cellular OD (Optical Dioxide Disorder) was measured. 600 The product concentration was also detected. Table 2 shows the OD of bacterial cells at 37°C and 42°C. 600 This shows that, in the case of 72h fermentation culture, the cell volume was higher under 37°C conditions, and in the case of 96h culture, the cell volume was higher under 42°C conditions. However, under the same temperature conditions, the cell volume on day 4 was higher than on day 3, and the growth trends of the two were the same, but the magnitude of the increase in cell volume differed, suggesting that the increase in cell volume was even greater under 42°C conditions. In the case of 72h fermentation culture, enzyme activity was higher under 37°C conditions.

[0059] Table 2 Cell concentration and enzyme activity of recombinant bacteria at various temperatures TIFF0007846707000002.tif35170 Example 4: Reusable whole cell conversion (1) Filtering of cells by centrifugation Recombinant Bacillus licheniformis BLA1 prepared in Example 1 was inoculated into seed medium and cultured at 37°C for 12-24 hours. Then, it was transferred to fermentation medium at an inoculation rate of 1%-5% (v / v) and cultured at 37°C for 24-36 hours to obtain 1 mL of fermentation broth. Cellular OD was performed using 100 μL of this broth. 600 The values ​​were measured, the remaining fermentation broth was centrifuged at 12000 r / min at 4°C for 20 minutes, the supernatant was discarded, the precipitate was washed once with 10 mM PB buffer, centrifuged again, the supernatant was discarded, and the cells were resuspended in the corresponding volume of PB buffer. 600 The cell suspension was added to a 500 mM HEPEs buffer (containing 5 mM CoCl2 and 5 mM MnCl2) at pH 7.5 with a volume ratio of fructose solution to HEPEs buffer of 10:3, and an equal volume of fructose solution with an initial concentration of 400 g / L. The reaction was carried out in a metal bath at 60°C for 6 hours. After the reaction was complete, the mixture was centrifuged at 12000 r / min for 20 minutes, and the precipitate was reused as the whole cell catalyst. An equal volume of anhydrous ethanol was added to the supernatant, and the mixture was allowed to stand at 4°C for at least 2 hours. The mixture was then centrifuged at 12000 r / min for 20 minutes, the supernatant was diluted 50-fold, filtered to 200 μL in a lining tube using a water filter tip, and placed in a liquid phase detection bottle for liquid phase detection. As a result, as shown in Figure 3, an analytical peak for D-psicose was present in the reaction mixture, with a D-psicose yield of 124.4 g / L and a conversion rate of 31.4%.

[0060] The same batch of cell catalysts was reused 10 times. As a result, the conversion rate remained above 30% even after 10 reuses of the cell catalysts (Figure 4).

[0061] (2) Cell filtration by microfiltration To continuously produce D-psicose using Bacillus licheniformis, a pilot-scale continuous bioconversion apparatus (Figure 5) was assembled by combining equipment such as ceramic membranes, pumps, a constant-temperature bath, and a reaction vessel, simulating industrial production. Here, the configuration of the circulation unit was based on that of industrial production, and the pressure the cells experienced during the production process was above the industrial production level.

[0062] Cellular cells that continuously produced D-psicose were subjected to a cyclic treatment using ceramic membrane microfiltration to test the stability and catalytic activity of the cells, with a total of 10 microfiltration cycles performed.

[0063] Table 3. Cell stability tests and data recording by serial microfiltration. TIFF0007846707000003.tif131170 As a result, it was revealed that recombinant Bacillus licheniformis BLA1 constructed in Example 1 exhibited high cellular stability and low degradation in the circulating catalyst of the reaction apparatus. The cell retention rate after 10 lot cycles was 96.7%, and the enzyme activity retention rate per cell was 97%, enabling true industrial production.

[0064] Comparative Example 1 The gene encoding D-psicose-3-epimerase, whose nucleotide sequence is indicated by SEQ ID NO.7, was constructed in Bacillus licheniformis using the same method as in Example 1 to obtain recombinant Bacillus licheniformis BLA7. When D-psicose was produced using the whole-cell conversion method shown in Example 3, the conversion rate of recombinant Bacillus licheniformis BLA7 at the same concentration was only 13.2%, while the conversion rate of recombinant Bacillus licheniformis BLA1 was 2.3 times higher.

[0065] Comparative Example 2 The gene encoding D-psicose-3-epimerase, whose nucleotide sequence is indicated by SEQ ID NO. 8, was constructed in Bacillus licheniformis using the same method as in Example 1 to obtain recombinant Bacillus licheniformis BLA8. When D-psicose was produced using the whole-cell conversion method shown in Example 3, the conversion rate of recombinant Bacillus licheniformis BLA8 at the same concentration was only 11.7%, while the conversion rate of recombinant Bacillus licheniformis BLA1 was 2.6 times higher.

[0066] Comparative Example 3 The gene encoding D-psicose-3-epimerase, whose nucleotide sequence is indicated by SEQ ID NO.9, was constructed in Bacillus licheniformis using the same method as in Example 1 to obtain recombinant Bacillus licheniformis BLA9. When D-psicose was produced using the whole-cell conversion method shown in Example 3, the conversion rate of recombinant Bacillus licheniformis BLA9 at the same concentration was only 8.3%, while the conversion rate of recombinant Bacillus licheniformis BLA1 was 3.7 times higher.

[0067] Although the present invention has been disclosed in better embodiments as described above, this does not limit the invention, and any modifications and modifications can be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be as defined in the claims.

Claims

1. Recombinant Bacillus licheniformis, characterized in that Bacillus licheniformis CICIM B1341 is used as the host, and a gene encoding D-psicose-3-epimerase, whose nucleotide sequence is SEQ ID NO. 1, is incorporated into the amylase-coding gene amyL in the genome of Bacillus licheniformis.

2. A whole-cell catalyst for activating a reaction to synthesize D-psicose using D-fructose as a substrate, characterized in that it contains the recombinant Bacillus licheniformis described in Claim 1.

3. A method for synthesizing D-psicose by whole-cell conversion, characterized by using D-fructose as a substrate, adding recombinant Bacillus licheniformis as described in claim 1 or the whole-cell catalyst as described in claim 2, and reacting at 50 to 70°C for 5 to 10 hours.

4. The method according to claim 3, characterized in that, after the reaction is complete, the recombinant Bacillus licheniformis or the whole cell catalyst is recovered by centrifugation or filtration, reused, and added to a reaction system using D-fructose as a substrate to synthesize D-psicose.

5. Use of recombinant Bacillus licheniformis according to claim 1, or the whole cell catalyst according to claim 2, in the production of a D-psicose-containing product.

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