Construction Of D-lactic Acid-producing Escherichia Coli Strains By Knockout Of Sugar Transport And Metabolism Genes

Microbial fermentation using mixed sugar has a catabolism repressive effect,which greatly reduces the fermentation efficiency.To improve the D-lactic production efficiency,E.coli JH13 and E.coli JH18 were chosen for gene knockout by RED recombination technology.Xylose-deficient JH1303 strain and mixed sugars co-utilization JH27 strain are constructed.The research contents and results are as follows:?1?JH13 is an engineering bacteria producing D-lactic acid efficiently with xylose,knocked out pyruvate formate lyase?foc A-pfl B?,fumaric acid reductase?frd ABCD?,acetic acid kinase?ack A?,alcohol dehydrogenase?adh E?and the nuclease gene?rng HSR2?.JH13 was used to construct a xylose-deficient JH1303 strain,knocked out xylose transporter genes xyl FGH,xyl E and xylose metabolism gene xyl A sequentially.Compared to JH13,JH1303's glucose consumption rate is almost unchanged using 6%glucose as carbon source for fermentation.While with 6%xylose fermentation,the xylose consumption rate decreased from 0.626 g?L·h?^-1 to 0 g?L·h?^-1.The results suggest that the knockout of the three genes xyl FGH,xyl E and xyl A has no effect on the glucose consumption rate,and has an additive effect on the reduction of the xylose consumption rate completely blocking the use of xylose.When fermentated with mixed sugars of 3%glucose and 3%xylose,the glucose consumption rate increased from 1.695 g?L·h?^-1 to2.278 g?L·h?^-1,and the sugar-acid conversion rate increased from 79.37%to 87.3%.E.coli JH1303 as sole-glucose comsuption strain,would be used for mixed fermentation with sole-xylose strains which would avoid the glucose effect while using mixed sugars as carbon sources.It would improve the fermentation production efficiency.?2?E.coli JH18 is a xylose-efficient production D-lactic acid strain,knocked out of?pts G?and methylgalactoside binding protein gene?mgl B?on the basis of E.coli JH13.JH18 was used to construct a mixed sugars co-utilization strain JH27 by sequentially knockout of Galactose transporter gene gal P,Glucokinase gene gl K,Mannose transporter man XYZ,Acetylglucosamine transporter nag ABC.Compared to E.coli JH13,when using 6%glucose as a carbon source for fermentation,glucose consumption rate of JH27 decreased from 4.05 g?L·h?^-1to 2.275 g?L·h?^-1;when fermentation was carried out with 6%xylose as the carbon source,the xylose consumption rate of JH27increased from 0.64 g?L·h?^-1 to 0.8 g?L·h?^-1.These suggest that the knockout of the four genes gal P,gl K,man XYZ,and nag ABC reduces the consumption rate of glucose and increases the consumption rate of xylose.When using mixed sugars of 3%glucose and 3%xylose,lactic acid production intensity of E.coli JH27 increased from 0.73 g?L·h?^-1 to 1.0 g?L·h?^-1.The conversion rate of sugar acid was increased from 84.5 g?L·h?^-1 to 86.1 g?L·h?^-1,and the fermentation period was shortened from 72 h to 54 h.The repressive effect of carbon metabolism of E.coli JH27 is obviously weakened,and the production intensity of lactic acid and the conversion rate of sugar and acid are increased.It can efficiently use mixed sugar fermentation to produce D-lactic acid and improve the fermentation production efficiency.