Construction And Optimization Of(-)-Vibo-Quercitol Production Via An In Vitro Synthetic Enzymatic Biosystem

(-)-vibo-Quercitol is a deoxyinositol(1L-1,2,4/3,5-cyclohexanepentol)that naturally occurs in oak species,honeydew honey,wines aged in oak barrels,and Gymnema sylvestre.As a result of its glucose-like structure,it has been introduced as an alternative chiral building block in the synthesis of bioactive compounds to control diabetes.In this study,an ATP-free in vitro synthetic enzymatic biosystem comprised of five enzymes was constructed to produce(-)-vibo-quercitol from maltodextrin in one-pot.This pathway is comprised of five enzymes,includes a-glucan phosphorylase,phosphoglucomutase,2-deoxy-scyllo-inosose synthase,(-)-vibo-quercitol 1-dehydrogenase and formate dehydrogenase(including one enzyme for NADH regeneration).All the reconbinant enzymes could be expressed well in E.coli BL21(DE3)or Bacillus subtilis after optimization of the expression conditions.The working temperature was set at 37? for transformation of maltodextrin into(-)-vibo-quercitol in one pot after considering the characteristic of enzymes.In order to achieve efficient conversion of the substrate to products via in vitro pathway,the biocatalysis conditions of(-)-vibo-quercitol production from maltodextrin were optimized.The best reaction conditions were the optimal ratio of aGP:PGM:DOIS:QUDH:FDH was 2:2:2:3:2,the concentration of NAD+was 2 mmol/L,the concentration of phosphate was 20 mmol/L and the concentration of formate was 70 mmol/L.In addition,isoamylase(IA)and 4-glucanotransferase(4GT)were added to the reaction system to increase the utilization of the substrate maltodextrin.After optimization of reaction conditions,7.6 g/L(-)-vibo-quercitol was produced from 10 g/L maltodextrin with a product yield of 77%.Finally,to obtain a higher production of(-)-vibo-quercitol,the concentation of maltodextrin was increased to 50 g/L.As a result,the yield of(-)-vibo-quercitol increased to 25.3 g/L,and the product yield was 51.4%%.This study describes an in vitro synthetic enzymatic biosystem that converts maltodextrin to(-)-vibo-quercitol,and has great potential in industrial scale production.and their application in the production of potential pharmaceuticals.