Construction Of Glucose Transporter-deficient Escherichia Coli Bacteria And Its D-lactic Acid Fermentation

D-lactic acid as an important chiral intermediates and a raw materials for the synthesis of polylactic acid,widely used in agriculture,chemical and other fields.At present,the emerging research trend is the development of cheap lignocellulosic raw materials,However,the problem is that Escherichia coli has Catabolite repression on the use of mixed carbon source of lignocellulose hydrolysates.In order to solve these problems,this paper intends to knock out the glucose transporter-related genes mglB and galP fromEcoli JH15(?ptsG)by genetic engineering method,and construct two strains of Escherichia coli that can simultaneously use five-carbon sugar and six-carbon sugar to produce D-lactic acid.The results are as follows:(1)JH13 is an engineering bacterium capable of producing D-lactic acid efficiently with xylose,which knocks out pyruvate formate lyase(focA-pflB),fumaric acid reductase(frdABCD),acetic acid kinase(ackA),alcohol dehydrogenase(adhE),Part of the nuclease gene(rng HSR2).JH15(?ptsG),which was fermented to produce D-lactic acid by mixed sugar,was constructed by JH13,and JH15 was used as the starting strain to knock out the methylgalactoside binding protein gene(mgl B),which is named JH18.And the galactose transporter gene(galP)was further knocked out on the basis of E.coli JH18,named E.coli JH20(2)Knockout mglB gene and galP gene can indeed reduce thespeed of glucose into the cell.JH18 glucose consumption rate was 2.56 g/L when fermented with 10% glucose as carbon source in LB medium,which was 22.2% and 26.9% lower than JH15 and JH13,respectively.The glucose consumption rate of JH20 was 2.37 g/L,Which was 28% and 34.9% lower than JH15 and JH13,respectively.(3)The knockout of mglB and galP genes had a significant effect on xylose consumption rate.JH18 xylose consumption rate was 1.58 g/L when fermented with 10% xylose as a carbon source in LB medium,which was twice as fast as JH15 and JH13,and the consumption rate of JH20 xylose was 1.2 g/L,which was 51.9% higher than that of JH15 and JH13.(4)The deletion of the mgl B and galP genes could reduced the glucose effect and increased the rate of xylose utilization in the mixed carbon source on the basis of JH15.JH18 glucose consumption rate was 1.1 g/L when fermented with 10% mixed sugar(5% glucose and 5% xylose)as a carbon source in LB medium,which was 19.7% and 53.4% lower than that of JH15 and JH13,respectively.The xylose sugar consumption rate was 0.83 g/L,which was 69.8% and 160.7% higher than that of JH15 and JH13,respectively.The glucose consumption rate of JH20 was 0.85 g/L,which was 38% and 63.7% lower than that of JH15 and JH13,respectively.The xylose sugar consumption rate was 0.48 g/L,which was 72.1%and 164.3% higher than that of JH15 and JH13.(5)In the low-mixed sugar(1% glucose and 1% xylose)environment,the mglB gene had a greater effect on the decrease in glucose consumption rate than the ptsG gene.JH18 consumed 12 hours of fermentation,glucose consumption rate is 0.86 g/L,xylose consumption rate is0.79 g/L;JH15 consumed 24 hours of fermentation,glucose consumption rate is 1.62 g/L,xylose consumption rateis0.58 g/L.