Development of a self-flocculating Zymomonas mobilis biocatalyst for efficient bioethanol production from lignocellulosic biomass

Flocculation represents a promising biological strategy to enhance biomass recovery and improve process efficiency in bioethanol production. In this study, we engineered a self-flocculating strain of Zymomonas mobilis, designated ZAM2, by inactivating the EAL domain (phosphodiesterase region) of the regulatory gene ZMO_1055. This targeted genetic modification led to enhanced cell aggregation and improved stress tolerance against common fermentation inhibitors, including acetic acid, furfural, and vanillin, which are typically generated during lignocellulosic biomass pretreatment. Compared to its parental strain ZM401, ZAM2 accumulated higher levels of intracellular c-di-GMP, exhibited reduced PDE activity, and upregulated cellulose biosynthesis genes. These changes contributed to enlarged floc size and superior fermentation performance. Notably, ZAM2 completed glucose consumption and ethanol production more rapidly than ZM401 under inhibitor-rich conditions, resulting in increased ethanol productivity. Our results demonstrate a rational genetic strategy for enhancing microbial robustness and process efficiency, offering a viable approach for sustainable, high-gravity bioethanol fermentation from lignocellulosic feedstocks.