Production Of D-mannitol By Metabolically Engineered Escherichiacoli

D-Mannitol, as a functional sugar alcohol, is widely used in pharmaceuticals, food, and chemistry. Production of D-mannitol by metabolically engineered strains received growing attentions for its safer and environment-friendly route and higher specificity. Escherichia coli, which possesses the maturest genetic manipulation methods and has been generally accepted as an excellent platform for production of biologically based chemicals, is regarded as an excellent host for D-mannitol production.In this study, a genetically stable Escherichia coli strain Mtl-002for D-mannitol production was constructed by integrating mannitol dehydrogenase (mdh) and fructose permease (fupL) genes of Leuconostoc pseudomesenteroides ATCC12291into chromosome of E. coli ATCC8739and inactivating other fermentation pathways. Mtl-002could produce1.15mmol/L D-mannitol.Then, the phosphotransferase systems (PTS) was inactivated and glucose and fructose were transported into cells using galactose permease (GalP) and fructose transporter (FupL) and the constructed strain was named as Mtl-003. Using mineral salts medium with glucose and fructose as carbon sources,1.21mmol/L D-mannitol was produced by Mtl-003after anaerobic fermentation for6days.Based on the coupling of cell growth and D-mannitol production, metabolic evolution was used to improve D-mannitol production. After evolution for80generations, the productity of D-mannitol was4.4mmol/L, the specific activity of MDH was0.38U/mg pro.the encoding gene of MDH was mutated to D301Y(g901t) and the resulted strain was kept and named as Mtl-004.To improve the ability for fructose transport further, the regulated glucose facilitator (glf)gene was integrated into Mtl-004to obtain strain Mtl-005which could produce6.22mmol/L D-mannitol.To transform glucose to D-mannitol directly, a glucose isomerase (aGI) gene of Actinoplanes missouriensis were integrated into strain Mtl-005. aGI was regulated by different strength promoter and one of the strains named Mtl-009could produce the highest yield of D-mannitol was6.44mmol/Lwhen fermented using glucose as the sole carbon sources.Through integrating the gene of D-mannitol dehydrogenase, fructose transporter and glucose isomerase for the production of D-mannitol into the chromosome of Escherichia coli, a genetically stable Escherichia coli strain,which could produce D-mannitol by using glucose as the sole carbon sources,was obtained and the highest yield of D-mannitol was6.44mmol/L...