Construction of a Food Grade Recombinant Bacillus subtilis Based on Replicative Plasmids with an Auxotrophic Marker for Biotransformation of D-Fructose to D-Allulose

A food grade recombinant Bacillus subtilis that produces D-psicose 3-epimerase (DPEase; EC 5.1.3.30) was constructed by transforming a replicative multicopy plasmid with a D-alanine racemase gene marker into B. subtilis 1A751 with the D-alanine racemase gene knocked out. The DPEase was expressed in B. subtilis without antibiotic resistance genes and without adding antibiotics during fermentation. Whole cells of the food grade recombinant B. subtilis were used to biotransform D-fructose to D-allulose. The two tandem promoters, including the HpaII and P43 promoters, increased expression levels compared to the use of one promoter, HpaII. For large-scale D-allulose production, the optimal enzyme dose was 40 enzyme activity units of dry cells per gram of D-fructose, which produced a 28.5% turnover yield in 60 min. The recombinant plasmid exhibited stability over 100 generations. This food grade recombinant B. subtilis may be used for large-scale D-allulose production in the food industry.