Adaptive Laboratory Evolution and Metabolic Engineering of Zymomonas mobilis for Bioethanol Production Using Molasses

Molasses with abundant sugars is widely used for bioethanol production. Although the ethanologenic bacterium Zymomonas mobilis can use glucose, fructose, and sucrose for ethanol production, levan production from sucrose reduces the ethanol yield of molasses fermentation. To increase ethanol production from sucrose-rich molasses, Z. mobilis was adapted in molasses, sucrose, and fructose in parallel. Adaptation in fructose is the most effective route to generate an evolved strain F74 with improved molasses utilization, which is majorly due to a G99S mutation in Glf for enhanced fructose import. Subsequent sacB deletion and sacC overexpression in F74 to divert sucrose metabolism from levan production to ethanol production further enhanced ethanol productivity 28.6% to 1.35 g/L/h. The efficient utilization of molasses by diverting sucrose metabolic flux through adaptation and genome engineering not only generated an excellent ethanol producer using molasses but also provided the strategy for developing microbial cell factories.