One-pot sustainable synthesis of glucosylglycerate from starch and glycerol through artificial in vitro enzymatic cascade.

Glucosylglycerate (R-2-O-α-D-glucopyranosyl-glycerate, GG) is a negatively charged compatible solution with versatile functions. Here, an artificial in vitro enzymatic cascade was designed to feasibly and sustainably produce GG from affordable starch and glycerol. First, Spirochaeta thermophila glucosylglycerate phosphorylase (GGP) was carefully selected because of its excellent heterologous expression, specific activity, and thermostability. The optimized two-enzyme cascade, consisting of alpha-glucan phosphorylase (αGP) and GGP, achieved a remarkable 81 % conversion rate from maltodextrin and D-glycerate. Scaling up this cascade resulted in a practical concentration of 58 g/L GG with a 62 % conversion rate based on the added D-glycerate. Additionally, the production of GG from inexpensive starch and glycerol in one-pot using artificial four-enzyme cascade was successfully implemented, which integrates alditol oxidase and catalase with αGP and GGP. Collectively, this sustainable enzymatic cascade demonstrates the feasibility of the practical synthesis of GG and has the potential to produce other glycosides using the phosphorylase-and-phosphorylase paradigm.

Sustainable cellobionic acid biosynthesis from starch via artificial in vitro synthetic enzymatic biosystem.

Cellobionic acid (CBA), a kind of aldobionic acid, offers potential applications in the fields of pharmaceutical, cosmetic, food, and chemical industry. To tackle the high cost of the substrate cellobiose in CBA production using quinoprotein glucose dehydrogenase, this study developed a coenzyme-free and phosphate-balanced in vitro synthetic enzymatic biosystem (ivSEBS) to enable the sustainable CBA synthesis from cost-effective starch in one-pot via the CBA-synthesis module and gluconic acid-supply module. The metabolic fluxes of this artificial biosystem were strengthened using design-build-test-analysis strategy, which involved exquisite pathway design, meticulous enzyme selection, module validation and integration, and optimization of the key enzyme dosage. Under the optimized conditions, a remarkable concentration of 6.2 g/L CBA was achieved from initial 10 g/L maltodextrin with a starch-to-CBA molar conversion rate of 60 %. Taking into account that the biosystem simultaneously accumulated 3.6 g/L of gluconic acid, the maltodextrin utilization rate was calculated to be 93.3 %. Furthermore, a straightforward scaling-up process was performed to evaluate the industrial potential of this enzymatic biosystem, resulting in a yield of 21.2 g/L CBA from 50 g/L maltodextrin. This study presents an artificial ivSEBS for sustainable production of CBA from inexpensive starch, demonstrating an alternative paradigm for biomanufacturing of other aldobionic acids.