Simultaneous Fermentation Of Pentose And Hexose For D-lactic Acid By Metabolically Engineered Escherichia Coli

D-lactic acid is widely used in agriculture, medicine and chemical industry as an important chiral intermediate as well as a substrate for polylactic acid. The industrial production for D-lactic acid is mainly by microorganisms fermentation, and using cheap lignocellulose raw material to produce high optical purity of D-lactic acid is a hotspot of current researches. Regarding to current circumstance of fermentation production, few microbes can use pentose derived from lignocellulose hydrolysate; and there is carbon catabolite repression in fermentation by mixed surgars. In order to solve these problems,the strain could use pentose and hexose simultaneously for D-lactic acid production was engineered. The research content and results are as follows:(1) To construct a strain for homofermentative prodution of D-lactic acid,.an ethanologenic Escherichia coli strain RM10 which could use xylose efficiently was reengineered, by deleting the alcohol dehydrogenase gene(adh E) and integrating the D-lactic dehydrogenase gene(ldh A) instead. The result strain E.coli DX02 produced84.3 g/L D-lactic acid with a yield of 90.97% in 120 h in LB medium containing 10%xylose. When fermented in LB medium containing 10% glucose, 85.6 g/L D-lactic acid was produced with a yield of 90.1% in 28 h. When fermented in LB medium containing5% glucose and 5% xylose, E.coli DX02 used xylose only after the exhaustion of glucose. During the 114 h fermentation, 50 g/L glucose and 32 g/L xylose were used;66.02 g/L D-lactic acid was produced with a yield of 80.51%.(2) In order to reduce the carbon catabolite repression in co-fermentation by mixed surgars, the strain E.coli DX03 was constructed by deleting a glucose transmembrance transporter gene pts G by Red homologous recombination technique. The strain E.coli DX03 could utilize both glucose and xylose simultaneously and completely for fermentation of 10% mixed sugars(5% glucose and 5% xylose), the production of D-lactic acid reaching 83.04 g/L; xylose utilization ratio, xylose consumption rate and production of D-lactic acid was improved by 56.25%, 29.41%,25.86%, respectively,compared to strain E.coli DX02.Fermentations of strain E.coli DX03 with xylose and other monose in different media were also tested and evaluated. E.coli DX03 showed the strongest fermentativeability with glucose, while much weaker with xylose or L-arabinose.When using mixed sugars, E.coli DX03 had relative higher production and productivity of lactic acid and in LB medium than NBS and modified NBS medium. Considering of productive cost,modified NBS medium was more suitable for industrial of D-lactic acid.