Metabolic Engineering of Escherichia coli for Fumarate Production from Ethanol.

Ethanol is a widely available and inexpensive commodity chemical, and can be utilized by microorganisms with high carbon yield, making it a potential alternative carbon source in fermentation. In this study, fumarate, a versatile platform chemical, was biosynthesized from ethanol via partial tricarboxylic acid (TCA) cycle and glyoxylate shunt in engineered Escherichia coli strain. To address growth deficiency of E. coli on ethanol, adaptive laboratory evolution was performed, resulting in evolved strains with improved growth and enhanced fumarate production. Knockout of citrate lyase and increased adenylate cyclase activity significantly contributed to growth improvement. Furthermore, gluconeogenesis and glyoxylate shunt were further transcriptionally activated in the evolved strain. Finally, the optimized strain Q6926 produced 31.03 mM fumarate from ethanol in fed-batch fermentation. This work represents the first demonstration of growth-coupled fumarate bioproduction from ethanol, demonstrating the feasibility of using ethanol as a sustainable feedstock for the biosynthesis of TCA cycle-derived chemicals.