Driving an in vitro multienzymatic cascade of laminaribiose biosynthesis from non-food cellulose with balancing the precursor supply

High-valued disaccharide laminaribiose, derived from two glucose units, is widely used in the pharmaceutical, cosmetic and food industries. The technology for laminaribiose synthesis from non-food feedstock cellulose instead of sucrose, glucose and starch was developed. First, we investigated a two-enzyme cascade converting cellobiose to laminaribiose by cellobiose phosphorylase and laminaribiose phosphorylase and 0.47 mol/mol cellobiose yield was obtained with cellobiose. An in vitro enzymatic cascade composed of cellobiohydrolase, cellodextrin phosphorylase, cellobiose phosphorylase and laminaribiose phosphorylase was designed to synthesize laminaribiose from cellulose. Laminaribiose production reached 2.35 and 1.23 g/L in pathways I & II, respectively. Furthermore, another multienzyme cascade was designed to eliminate the imbalance of glucose and glucose-1-p supply and laminaribiose yield finally reached 72% of that yield from cellobiose. This in vitro enzymatic cascades for laminaribiose biosynthesis not only widen the utilization of the non-food cellulose, but also provide a model that improves the economy of lignocellulosic biorefineries.