Highly efficient production of Clostridium cellulolyticum H10 D-psicose 3-epimerase in Bacillus subtilis and use of these cells to produce D-psicose.

BACKGROUND: D-Psicose 3-epimerase (DPEase) catalyzes the isomerization of D-fructose to the rare sugar D-psicose, which may help prevent obesity, reduce blood sugar and blood fat, and inhibit intra-abdominal fat accumulation.

RESULTS: In this study, the DPEase of Clostridium cellulolyticum H10 was expressed in the food-grade host Bacillus subtilis. Optimization of the culture medium during shake-flask experiments yielded a DPEase activity of 314 U/mL. The optimal medium included 20 g/L peptone, 15 g/L corn steep powder, 5 g/L glycerol, and 1 mM Ca2+ . Controlling the carbon source concentration was important because elevated concentrations can result in catabolite metabolic suppression (CCR). To avoid CCR and increase DPEase expression, we developed a fed-batch strategy in a 3.6-L fermenter. We altered the ratio of carbon source to nitrogen source (C/N) in the feeding medium and employed a constant feeding rate (6 g/L/h). This strategy improved the DPEase activity to 2246 U/mL (7.8 g/L), which is almost 15 times higher than that observed in the original shake-flask cultures. Finally, we used the DPEase-expressing B. subtilis cells to produce D-psicose from D-fructose, and a 28% conversion yield was achieved with these cells, demonstrating their potential use in D-psicose production.

CONCLUSIONS: This is the first report to enhance recombinant DPEase production in B. subtilis using efficient and convenient fermentation strategy, and the DPEase yield is three times higher than the highest yield reported to date. The recombinant B. subtilis cells were further used in the efficient synthesis of D-psicose. This study provides a basis for the industrial production of D-psicose.