Construction Of E.coli Engineering Bacteria Using Mixed Sugar To Produce L-lactic Acid And Its Fermentation Of Wheat Straw

L-lactic acid is one of the important organic acids and is widely used in many fields.When the strain uses the mixed sugar in the hydrolyzed solution of wheat straw,most microorganisms cannot use the xylose in the hydrolyzed solution.The reason is Carbon catabolite repression(CRR).This paper uses Red homologous recombination technology to knock out xylose.In addition to the genes galP,glK,manX,and nag A related to glucose transport in E.coli WL210,an engineered Escherichia coli bacteria that can efficiently use the hydrolysate of wheat straw to ferment and produce L-lactic acid was constructed:(1)E.coli WL210 was an engineered bacteria that can use glucose and xylose to produce L-lactic acid.Using WL210 as the starting strain,the galP gene was successfully knocked out by the RED gene knockout technology to construct E.coli WL220;the glK gene was knocked out on the basis of E.coli WL220 to construct WL240;the manX gene was knocked out on the basis of E.coli WL240,Construct WL260;knock out the nag A gene on the basis of E.coli WL260,construct E.coli JH2705.(2)When fermenting with a glucose concentration of 60 g/L,the glucose consumption rates of WL210,WL220,WL240,WL260,and JH2705 were 3.92 g(L·h)^-1,3.67 g(L·H)^-1,3.33 g(L·h)^-1,2.58 g(L·h)^-1,2.25 g(L·h)^-1,compare WL210,WL220,WL240,WL260,JH2705 glucose consumption rate respecti Verely Decrease6.03%,14.02%,34.03%,42.04%.It showed that the knockout of galP,glK,manX and nagBAC genes can reduce the rate of glucose consumption by the strain.(3)When fermenting with 6%xylose as a carbon source,the consumption rate of xylose for WL210,WL220,WL240,WL260,and JH2705 were 0.65 g(L·h)^-1,0.70 g(L·h)^-1,0.75 g(L·h)^-1,0.76 g(L·h)^-1,0.80 g(L·h)^-1,compared with WL210,WL220,WL240,WL260,JH2705 xylose The consumption rate increased by 8.02%,14.97%,17.03%,and 22.97%respecti Verely.It showed that the knockout of galP,glK,manX,nagBAC genes can increase the utilization rate of xylose by the strain.(4)When using 3%glucose and 3%xylose as the fermentation carbon source,in terms of the glucose consumption rate,compared with the starting E.coli WL210,WL220,WL240,WL260,JH2705,the glucose consumption rate was reduced by 4.01%,23.92%,32.04%,respecti Verely.36.01%.In terms of the consumption rate of xylose,compared with the starting E.coli WL210,the consumption rate of xylose of WL220,WL240,WL260,and JH2705 increased by 3.00%,13.01%,21.97%,49.01%,respecti Verely.In terms of lactic acid production intensity,compared with the E.coli WL210,the lactic acid production intensity of WL220,WL240,WL260,JH2705increased by 7.01%,11.02%,14.92%,27.00%,respecti Verely.From the perspecti Vere of bacterial growth,the maximum OD₆₀₀ of WL210 was 7.13,the maximum OD₆₀₀ of WL220 was 7.2,the maximum OD₆₀₀ of WL240 was 7.58,the maximum OD₆₀₀ of WL260 was 8.28,and the maximum OD₆₀₀ of JH2705 was 8.28.The maximum OD Veralues of WL220,WL240,WL260,and JH2705 were 1.01 times,1.06 times,1.10times,and 1.16 times the maximum OD Veralues of WL210,respecti Verely.In terms of sugar and acid con Verersion rate,compared with the starting bacteria WL210,WL220,WL240,WL260,JH2705,the sugar acid con Verersion rate increased by 5.30%,2.50%,3.70%,6.01%.It showed that after knocking out galP,glK,manX,and nagBAC,it was beneficial to impro Vere the utilization ability of E.coli to use xylose in the mixed sugar,shorten the fermentation time,and correspondingly increased the production intensity of lactic acid and increased the sugar-acid con Verersion rate.(5)In the 7 L fermenter,when the wheat straw hydrolysate was used as the carbon source for fermentation,the glucose consumption rate is reduced by 14.28%,25.00%,33.33%,compared with WL210,WL220,WL240,WL260,and JH2705,respecti Verely.35.08%.From the perspecti Vere of xylose consumption rate,compared with WL210,the xylose consumption rate of WL220,WL240,WL260,JH2705 increased by 8.33%,18.22%,18.98%,and 30.00%,respecti Verely.Compared with WL210,the lactic acid production rate of WL220,WL240,WL260,JH2705 increased by 8.62%,20.23%,22.90%,23.14%,respecti Verely.It showed that after knocking out the four genes galP,glK,manX,and nagBAC,it was beneficial to impro Vere the utilization ability of E.coli to use xylose in the hydrolysate of wheat straw,the glucose effect was weakened,the fermentation time was shortened,the lactic acid production rate was increased,and the sugar and acid con Verersion was impro Vered.The rate was impro Vered,and the six-carbon sugar and fi Vere-carbon sugar can be used simultaneously and efficiently,which was not significantly different from the mixed sugar.