In Vitro Cell-free Synthesis Of Laminaribiose From Lignocellulose Biomass

Lignocellulosic biomass,one of the most abundant renewable resources in the world,is degraded to fermentable sugars by cellulase or microorganism,and further servered as sustainable feedstock for biosynthesis of high value-added products in biorefining industry after.With the outbreak of Covid-19 pandemic,the crisis on food gradually appears over the world,and brings new challenge on Biorefinery technology using starch and other edible resources as raw materials.Utilization and conversion of lignocellulose biomass for biosynthesis of high value-added products is promising and expected.Laminaribiose is a reducing glucobiose connected by β-1,3 glycosidic bonds.Laminaribiose can be used as a precursor of hyaluronic acid in the pharmaceutical and cosmetic industries,and also can be used as a health food additive because of probiotic effects.However,the limited supply of natural products causes the high price of laminaribiose.Therefore,it is significant to develop a low-cost and environment-friendly technology for efficient biosynthesis of laminaribiose.With the development of synthetic biology,in vitro cell-free synthesis has been attracted the attention of scientists.In vitro cell-free synthesis is easy-used,simple in industrialized production process and can achieve many reactions that are difficult to be achieved in the cell.In this work,we designed an enzymatic catalytic system that uses in vitro cell-free synthesis to produce laminaribiose from cellulose.In this system,we mainly divided into two modules for research.They are the first module for producing cellobiose with cellulose as the substrate and the second module for synthesizing laminaribiose with cellobiose as the substrate.The most active cellobiohydrolase,CBHIK183A(EC 3.2.1.91)and the most effective substrate,regenerating amorphous cellulose(RAC)were firstly screened out.The yield of cellobiose produced by CBHIK183A from RAC reach up to 55%.Then,the generated cellobiose was used to produce laminaribiose.We chose cellobiose phosphorylase(CBP,EC 2.4.1.20)and laminaribiose phosphorylase(LBP,EC 2.4.1.31)to convert cellobiose into laminaribiose.The maximum conversion rate from cellobiose to laminaribiose was 49.3%.Based on above results,multienzyme cascades system composed of CBHI,CBP and LBP for biosynthesis laminaribiose from cellulose was successfully developed,which only requires three readily available enzymes and substrate instead of additional coenzymes and ATP.After optimizing the conditions,2.53 g/L laminaribiose was obtained from 10 g/L RAC.The conversion rate reached 25.3%(g laminaribiose/g RAC).In addition,when adding 30 mM glucose-1-phosphate(G-1-P)during the reaction,the laminaribiose production reached 3.56 g/L.The conversion rate of laminaribiose increased to 35.6%based on the cellulose.In the process of exploration,we also found a new synthetic pathway for laminaribiose.We used cellodextrin phosphorylase(CDP,EC 2.4.1.49),CBP and LBP to catalyze cellodextrin through triple-enzyme cascade catalysis,and finally successfully obtained laminaribiose.In this way,0.23 g/L laminaribiose was obtained from 10 g/L cellodextrin.In vitro cell-free synthesis used in this study not only constructs an efficient utilization of non-food biomass for producing laminaribiose and greatly reduce the production cost of laminaribiose,but also provide a new idea for the utilization of lignocellulose for biosynthesis of other high-valued disaccharides.