Inorganic phosphate self-sufficient whole-cell biocatalysts containing two co-expressed phosphorylases facilitate cellobiose production

Cellobiose, a natural disaccharide, attracts extensive attention as a potential functional food/feed additive. In this study, we present an inorganic phosphate (Pi) self-sufficient biotransformation system to produce cellobiose by co-expressing sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP). The Bifidobacterium adolescentis SP (BASP) and Cellvibrio gilvus CBP (CGCBP) were co-expressed in Escherichia coli. Escherichia coli cells containing BASP and CGCBP were used as whole-cell catalysts to convert sucrose and glucose to cellobiose. The effects of reaction pH, temperature, Pi concentration, and substrate concentration were investigated. In the optimum biotransformation conditions, 800 mM cellobiose was produced from 1.0 M sucrose, 1.0 M glucose, and 50 mM Pi, within 12 hr. The by-product fructose and residual substrate (sucrose and glucose) were efficiently removed by treatment with yeast, to help purify the product cellobiose. The wider applicability of this Pi self-sufficiency strategy was demonstrated in the production of laminaribiose by co-expressing SP and laminaribiose phosphorylase. This study suggests that the Pi self-sufficiency strategy through co-expressing two phosphorylases has the advantage of great flexibility for enhanced production of cellobiose (or laminaribiose).