Abstract
Backgroundd-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.ResultsIn 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.ConclusionsThis 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.
Highlights
The International Society of Rare Sugars first proposed the widespread application of rare sugars in human nutrition and health at its first academic conference in 2001
Zhang et al produced d-psicose by using pretreated Rhodobacter sphaeroides cells [9] and Shin et al increased the production of food-grade d-tagatose by pretreating and immobilizing Corynebacterium glutamicum cells [10]
Bacterial strain and vectors The codon-optimized gene encoding the d-psicose 3-epimerase (DPEase) from Clostridium cellulolyticum H10 was synthesized to Generay Co., Ltd (Shanghai, China)
Summary
The International Society of Rare Sugars first proposed the widespread application of rare sugars in human nutrition and health at its first academic conference in 2001. There are currently 34 species of rare sugar or sugar alcohol under consideration, including d-psicose, d-tagatose, d-allose, and xylitol. D-Psicose, an epimer of d-fructose at the C3 position, is 70% as sweet as sucrose, but its caloric d-Psicose is rarely found in nature, so most of the available material is produced by enzymatic synthesis. Su et al Microb Cell Fact (2018) 17:188 configuration of the hydroxyl group at the C3 position (Fig. 1). This reaction is reversible, and it usually achieves a maximum ratio of d-psicose:d-fructose between 20 and 33% [8]. Zhang et al produced d-psicose by using pretreated Rhodobacter sphaeroides cells [9] and Shin et al increased the production of food-grade d-tagatose by pretreating and immobilizing Corynebacterium glutamicum cells [10]
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