Abstract

L-valine is an essential amino acid for animal nutrition. Ideally, it can be produced from D-glucose through homotypic L-valine fermentation in a growth-coupled manner. To date, no known microorganism, native or engineered, can grow on D-glucose and ammonia anaerobically with L-valine as the sole product. Here, we direct the metabolic flux through a reinforced L-valine synthetic pathway by blocking mixed-acid fermentation and L-alanine synthesis reactions to create an NADH driving force in Escherichia coli . We further evolve the engineered strain to debottleneck growth constraints by anaerobic growth rescue. The resulting evolved hyper-valine producer converts D-glucose in a 320 m 3 reactor to 83.6 g/L L-valine within 60 h, reaching a yield of 0.55 g/g glucose (85% of the theoretical maximum). Through reverse engineering, we identify that more than a 10-fold improvement in anaerobic growth and L-valine production rate arises from the amplified L-valine synthetic pathway, the additional electron sinks and reprogramming of global regulation. Together, we changed the way of L-valine production into homotypic L-valine fermentation and demonstrate how E. coli variants adapted their metabolic activities and transcriptional regulation to boost fitness in an anoxic condition, with L-valine synthesis serving as the primary NADH-consuming pathway.