Adaptive evolution of pentose-fermenting Zymomonas mobilis strains for enhanced ability to convert biomass sugars for ethanol production

To utilize C5 sugars in the lignocellulosic biomass for production of ethanol and other chemicals, we have metabolically engineered Z. mobilis to utilize the C5 sugars (i.e. D-xylose and L-arabinose) by introducing the respective pentose pathways into the organism thus enabling its ability to convert C5 sugars derived from pretreated lignocellulosic biomass in addition to glucose to etahnol. However, effective utilization of both C6 and C5 sugars and maximization of the product yield by the recombinant organisms met challenges when using biomass hydrolysates due to many inhibitors existed in the hydrolysate. Adaptive evolution is a powerful tool harnessing the cell's abilities to optimize its survival in toxic environment, complementing rational approaches in strain development. We showed that through selective adaptation in presence of the major inhibitor, acetate, as well as continuous adaptation on dilute acid pretreated corn stover hydrolysate the evolved recombinant Z. mobilis strains greatly improved its ability to utilize xylose with increased ethanol production. Furthermore, we constructed knockout and overexpression libraries of Z. mobilis to allow great gene diversity and applied the evolution approach to select enhanced properties to tolerate another inhibitor, furfural as well as on hydrolysate, we successfully identified several key genes that conferred the toxicity towards the inhibitors in the hydrolysates.