Expression Of D-psicose 3-epimerase In Bacillus Subtilis

As a low-calorie functional sugar,D-allulose is a good substitute for sucrose and is recognized as the fourth-generation natural sweetener.D-psicose 3-epimerase（DPE）can specifically catalyze the conversion of D-fructose to D-allulose,which is the key enzyme for the production of D-allulose in industry.In this study,DPE from Clostridium scindens ATCC35704 was recombined and expressed in Bacillus subtilis,and its promoter and ribosomal binding site（RBS）were optimized at the transcriptional and translational levels.Then,the expression of DPE was further improved by shaking flask fermentation and 3 L tank fermentation at the level of fermentation engineering.Finally,the application of recombinant strain whole cells in the preparation of D-allulose was studied.This study laid the foundation and provided theoretical support for the preparation of D-allulose in industrial production.The main results are as follows:（1）The recombinant expression plasmid of DPE in host B.subtilis WB800 was constructed.In order to improve the expression of DPE,the differences of DPE expression in recombinant strains mediated by 9 single promoters P_{amy E}、P_{apr E}、P_{gsi B}、P_hag、P_{Hpa II}、P_{npr E}、P₄₃、P_{sig X} and P_{ylb P}were investigated.After shaking flask fermentation,it was found that the single promoter with the best expression effect was P_hag,and the enzyme activity of the corresponding recombinant strain was 19.62 U/m L,followed by the single promoters P_{ylb P} and P₄₃,with the corresponding enzyme activities of 16.62 U/m L and 15.05 U/m L,respectively.The single promoters P_hag,P_{ylb P} and P₄₃ were connected in pairs/repeated tandem to construct 9 tandem promoters Phag-P43、PP43-hag、Pylb P-hag、Phag-ylb P、Pylb P-P43、PP43-ylb P、PP43-P43、Pylb P-ylb P and Phag-_hag.After shaking flask fermentation,the best tandem promoter was P_{ylb P-ylb P},with enzyme activity of 17.95 U/m L,slightly lower than that of the single promoter P_hag.（2）To improve the translational levels of DPE,15 recombinant mutants were constructed by mutating part of the RBS sequence of single promoter P_hag.The experimental data showed that the enzyme activity of B.subtilis WB800/p P43NMK-hag-R4-DPE was the highest at 25.39U/m L,which was 29.4%higher than that of the recombinant strain before RBS sequence optimization.（3）Two non-resistant expression plasmids PUB-hag-R4-DPE-dal and p P43NMK-hag-R4-DPE-dal were constructed by using D-alanine racemase gene instead of antibiotic resistance gene as screening markers.The plasmids were transferred into the D-alanine deficient host strain B.subtilis 1A751,which was constructed in the early stage of the laboratory.After culturing,the enzyme activity of the two recombinant strains reached 24.72 U/m L and 19.13U/m L,respectively.（4）The effect of the composition of the culture medium on the production of DPE by the recombinant strain was investigated at the shaking flask level.The results showed that the optimized medium contains 15 g/L glucose,16.1 g/L soybean peptone,20 g/L yeast extract,8g/L Na Cl,1 g/L Na₂HPO₄·12H₂O and 1 g/L Mg SO₄·7H₂O,the enzyme activity increased to33.91 U/m L,41%higher than that of the pre-optimized medium.Subsequently,the fermentation conditions of the recombinant strains B.subtilis WB800/p P43NMK-hag-R4-DPE were further optimized in a 3 L tank.The results showed that when the p H was not adjusted,and the feeding rate was 40 m L/h,DPE had the highest fermentation enzyme activity,which was 714.84 U/m L.（5）To explore the effects of different reaction conditions on the whole-cell catalytic production of D-allulose by DPE-producing recombinant strains.The optimum reaction conditions were as follows:when the concentration of substrate D-fructose was 45%（w/w）,the reaction temperature was 55°C,the amount of enzyme was 30 U/g,the p H was adjusted to 7.5,and the rotational speed was 200r/min the conversion rate of D-allulose could be as high as28.45%.When the reaction went on for 8 h,the whole reaction reached equilibrium.