Efficient Expression Of D-psicose 3-epimerase In Bacillus Subtilis And Its Application

Sucrose, a traditional sweetener, has many shortcomings including high calorie and high blood sugar. Excessive intake of sucrose may lead to diabetes, obesity, cardiovascular disease and other chronic diseases. Therefore, new-type functional sweeteners with low-calorie have become a research focus in the international food science field. D-allulose, a newly discovered functional sweetener, is one of rare sugars with 70% relative sweetness of sucrose but less than 10% of the energy of sucrose. In addition, D-allulose has been confirmed to have excellent physiological functions, such as prevention of dental caries, having the capacity to decrease blood sugar level, prevention and improvement of diabetes, and suppression of the accumulation of body fat. In 2011, D-allulose was approved as Generally Recognized as Safe(GRAS) based on the laws and regulations of the U.S. Food and Drug Administration(FDA) and has been granted permission for application in food and medical industries as an ingredient.D-allulose is commonly prepared using an enzymatic method, where D-psicose 3-epimerase(DPEase) enzyme is involved. DPEase can efficiently catalyze D-fructose epimerization to produce D-allulose. However, the safety in the usage of genetically engineered bacteria and antibiotics resistance have become global issues. The selection of safe hosts and construction of engineering strains without antibiotic resistance genes are the development direction of future research. In the present study, the gene encoding DPEase from Clostridium scindens ATCC 35704 was used for this research project. The recombinant Bacillus subtilis expressing DPEase free of antibiotic resistance genes was constructed. The main contents and results of this study are as follows:(1) The B. subtilis 1A751(dal-), deficient in biosynthesis of D-alanine was constructed using the method combining the Cre/lox specific recombination system and overlap extension PCR. The auxotrophic strain could not grow up in the common medium without addition of D-alanine. A simple-promoter plasmid pUB-dpe-dal and dual-promoter plasmid pUB-P43dpe-dal were constructed in B. subtilis using the “simple cloning” method described elsewhere. In this study, the D-alanine racemase gene(dal) was used as a selection marker. The dal gene on the replicative plasmids could complement a chromosomal deletion of dal gene and provide selective pressure for maintenance of the plasmids. The enzyme activity of the recombinant strain 1A751/pUB-P43dpe-dal was 6 U/mL, which was higher than that of the recombinant strain 1A751/pUB-dpe-dal(4.5 U/mL). Whole-cell reactions were adopted for the production of D-allulose. 750 g/L of D-fructose and 2 g/L of enzyme powder produced 178 g/L of D-allulose in 1 h.(2) The promoter P43 from B. subtilis was fused with the dpe gene using the overlap extension PCR. The generating P43-dpe expression cassette and the lox71-spc-lox66 cassette were together integrated into the amyE locus in the chromosome of B. subtilis 1A751. Finally, the antibiotic resistance gene, spc gene was knocked out by the Cre/lox system, generating the recombinant B. subtilis expressing DPEase based on chromosomal integration free of antibiotic resistance genes. In addition, the recombinant strain 1A751/P43-dpe exhibited excellent stability.Isocaudamer sites were introduced at both ends of the P43-dpe expression cassette. The recombinant integration plasmids pDG-ndpe(n=1, 2, 3), which contained one, two, or three copy numbers of P43-dpe cassette, were constructed using the isocaudamer strategy. Then, one, two, and three consecutive P43-dpe tandem repeats were integrated into the chromosome of B. subtilis and the antibiotic resistance gene was also knocked out, generating the recombinant B. subtilis 1A751-nDPE(n=1, 2, 3). The enzyme activity of the recombinant strain 1A751-3DPE was about 0.75 U/mL, which was 2.2-fold higher than that of the 1A751-1DPE strain. So, multiple copies of dpe gene improved the expression of DPEase, thereby increasing DPEase activity. The recombinant strain 1A751-3DPE exhibited the highest specific activity(80 U/g cells) and total enzyme activity(2000 U/L) in the SOC medium among the selected media. Under the optimal conditions, 8 g/L of freeze-dried enzyme powder could convert 20% D-fructose(300 g/L) into D-allulose after 1 h.(3) The dpe gene was fused with the spore coat protein coding gene CotZ. Then, the fusion gene CotZ-CL-dpe was integrated into the chromosome of B. subtilis WB800, using the trpC gene as a selection marker. In addition, the DPEase was fused at the C-terminus of the CotZ protein, via an !-helix linker peptide, which assisted folding of DPEase protein and stabilized its structure. The formation of spores was induced by the exhaustion method in Difco sporulation medium(DSM). The specific activity of spores distinctly decreased after protease treatment, which demonstrated that the DPEase was located on the surface of spores. In addition, the optimal temperature and pH of spore surface-displayed DPEase was 55°C and 7.5-8.0, respectively. The half-life of spore surface-displayed DPEase was 170 min at 60°C. The optimal conditions for producing D-allulose was 500 g/L of D-fructose added with 30 g/L of the recombinant spores. D-allulose yield was 7.1 g/L/h with a 17% conversion after 12 h. Moreover, the spores exhibited excellent reusability. After five cycles of utilization, the specific enzyme activity of spore surface-displayed DPEase remained unchanged, while the yield of D-allulose dropped to 60% of the original value.