Biosynthesis Of Mannitol In A Whole-Cell Biotransformation With Recombinant E.Coli And The Utility Of Inulin As Substrate

Mannitol is a naturally six-carbon sugar polyol that is widely used in the medicine, food and pharmaceutical industry, as well as in chemical industry. At present, mannitol is produced by natural extraction, catalytic hydrogenation, and biological processes. Biological processes have several advantages, such as complete conversion of fructose to mannitol, absence of side products, moderate production conditions and no requirement of highly purified substrates.In this paper, a whole-cell biotransfornation systerm was constructed using the biology convertion. The whole-cell catalysis is a biological catalysis between fermentation and extraction enzyme catalysis, this method can be produced chitosan and nucleotides through the coupling of genetic engineering strains. Restriction-free (RF-clone) was used to modified the mannitol dehydrogenase (MDH) gene, which is removed the Nde I restriction site. The mdh gene without Nde I was expressed normolly through analysis by SDS-PAGE and measurement the activity of MDH. Fructose reduction to mannitol is catalyzed by an NADH-linked MDH. Due to the price of NADH is high, Formate dedydrogenase (FDH) was functionally co-expressed. FDH generates the NADH used for D-fructose reduction by dehydrogenation of formate to carbon. A whole-cell biotransformation system for the conversion of D-fructose to D-mannitol is debeloped in E. coli by constructing a recombinant oxidation/reduction cycle. This recombinant E.coli cells (BL21/pET23b-fdh-mdh-N) were able to yield D-mannitol (5.23g/L) from D-fructose after8h. The introduction of a glucose facilitator (GLF) gene, encoding the glucose facilitator protein of Zymomonas mobilis, allowed the cells to efficiently take up D-fructose. Resting cells of this E.coli strain BL21/pET23b-fdh-mdh-N pZY507-glf produced15.34g/L mannitol in8h. This study first developed the method using inulin as substrate to product D-mannitol. The results showed that the productivity of mannitol is58%, that may provide a reference for use the cheap material, Jerusalem artichoke or frisee as the substrates to product the mannitol.These results show that the biotransformation can product mannitol in lab, which might be useful references for commercializing mannitol production.