| As food additive and functional amino acid,L-alanine,produced mainly through microbial fermentation,is widely used in many fields such as food and medicine.The glucose effect in mixed sugars utilization is the key obstacle to the development of fermentatve production of L-alanine while using low-cost rice straw as raw material.In this study in order to repress the glucose effect,genes pts G and mgl B?key glucose transporter genes?were knocked out by Red homologous recombination from E.coli JH-B2,to construct an engineered strain with higher utilizing efficiency of mixed sugars for L-alanine prodcution..?1?E.coli JH-B2??frd BC,?adh E,?pta,?dad X,?ldh A,?pfl B::ala D?can produce L-alanine from glucose and xylose.E.coli JH-B3 was constructed by knocking out pts G form E.coli JH-B2,followed by the knock out of mgl B gene to construct E.coli JH-B6.?2?With 100 g·L?1glucose as carbon source in a 7 L fermentor,the rate of glucose consumption of JH-B3 was 2.37 g·L?1·h?1,13.82%lower than that of JH-B2;the rate of glucose consumption of JH-B6 was 2.09 g·L?1·h?1,11.81%and 24.00%lower than those of JH-B3 and JH-B2 respectively,which indicates the glucose consumption was reduced because of the deletion of pts G and mgl B genes.With 100g·L?1xylose as carbon source in a 7 L fermentor,the rate of xylose consumption of JH-B3 and JH-B2 were almost identical;the rate of xylose consumption of JH-B6 was0.92 g·L?1h·?1,about 66%higher than those of JH-B3 and JH-B2.These results indicate that the deletion of pts G alone has no efficient effect on xylose consumption,but the double deletion of pts G and mgl B could increase the xylose consumption.With the mixed sugar(50 g·L?1glucose and 50 g·L?1xylose)as carbon sources in a 7 L fermentor,both JH-B6 and JH-B3 consumed glucose and xylose simultaneously.For JH-B3,rate of glucose consumption was 1.04 g·L?1·h?1,49.76%lower than that of JH-B2;rate of xylose consumption of JH-B3 was 0.30 g·L?1·h?1,66.67%higher than that of JH-B2,which indicated that the knockout of the pts G gene can greatly reduce the rates of the strains consuming glucose and improve the rates of strains consuming xylose in mixed sugar,resulting in the alleviated glucose effect.For JH-B6,the glucose consuming rate was 0.92 g·L?1·h?1,10.58%and 55.56%lower than those of JH-B3 and JH-B2 respectively;the consuming xylose rate was 0.49 g·L?1·h?1,63.33%and172.22%higher than those of JH-B3 and JH-B2 respectively,indicating that the deletion of mgl B genes can further reduce the rates of strains consuming glucose and improve the rates of strains consuming xylose in mixed sugar,which can further alleviate glucose effect.?3?With the rice straw hydrolysate as carbon sources in a 7 L fermentor,both JH-B6 and JH-B3 simultaneously consumed glucose and xylose.For JH-B3,the glucose consuming rate was 0.92 g·L?1·h?1,52.08%lower than that of JH-B2;the xylose consuming rate was 0.20 g·L?1·h?1,66.67%higher than that of JH-B2.For JH-B6,The glucose consuming rate was 0.81 g·L?1?1,11.96%and 57.81%lower than those of JH-B3 and JH-B2;the xylose consuming rate was 0.35 g·L?1·h?1,30.00%%and116.67%higher than those of JH-B3 and JH-B2.The result indicated that the deletion of pts G and mgl B genes can also reduce the rates of strains consuming glucose and improved the rates of strains consuming xylose in rice straw hydrolysate,which can lead to mitigated glucose effect.The fermentation efficiency in the hydrolysate was slightly lower than that in mixed sugar.While for JH-B6,In the hydrolysate,although the glucose and xylose consuming rates were 11.96%and 46.94%lower than that in the mixed sugar,respectively,but the yield was as high as 88.88%,not significantly different from that in mixed sugar,which indicated the ability of JH-B6 to convert reducing sugar into L-alanine is still good.In this study,the pts G and mgl B genes?related to glucose transporting?were knocked out,resulting in the glucose effect alleviated when E.coli fermented L-alanine with rice straw hydrolysate,which provides research basis for producing L-alanine with rice straw as the raw material. |
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