Integration of metabolic and evolutionary processes to construct efficient xylose-utilizing strain of Zymomonas mobilis for lignocellulosic ethanol production

Xylose is a major component of lignocellulose, and it is crucial to enable microorganisms the capability of efficient xylose utilization for economical lignocellulosic biochemical production. <i>Zymomonas mobilis</i> is a natural ethanologenic bacterium with xylose-utilizing recombinant strains engineered for commercial lignocellulosic ethanol production. However, low efficiency of xylose utilization is still the major hurdle for lignocellulosic biochemical production, especially in the lignocellulosic hydrolysate containing inhibitory compounds and mixed sugars of glucose and xylose. In this study, xylose-utilization capability of a xylose-utilizing recombinant strain 8b-S38 of <i>Z. mobilis</i> was further improved by evaluating the effects of different xylose metabolic pathways, xylose isomerases, and xylose transporters. The results demonstrated that xylose isomerase pathway is still the most efficient one in <i>Z. mobilis</i> among the xylose metabolic pathways examined, and the introduction of xylose isomerase (XI) such as StrXI and PanXI to increase its copy numbers can improve xylose utilization. In addition, the introduction of effective xylose transporter such as high-affinity xylose transporter XylFGH and the glucose facilitator mutant Glf^A165M-K458I promoted xylose transportation and subsequent utilization. A recombinant strain S67SPGlf^m was constructed combining rational engineering of xylose isomerases and transporters and semi-rational adaptation, which exhibited enhanced xylose utilization capability in lignocellulosic hydrolysate. This study illustrated that the construction of robust and efficient industrial strains with complex phenotypes requires a combination of different strategies such as metabolic engineering and adaptive laboratory evolution, which also provided biological parts such as xylose isomerases and xylose transporters to help design and construct microbial cell factories for efficient xylose utilization in the future.