Metabolic engineering of Escherichia coli based on adaptive evolution and omics technology for highly efficient l-valine production under oxygen-limited conditions

In industrial production, the yield of desired targets derived from carbon sources is frequently diminished by the competitive influence of cellular metabolism within microbial cell factories. The bioproduction of l-valine exemplifies a classic process that is confronted with such a dilemma, substantially hindering its economic industrial-scale production. In this study, we aim to engineer a cell factory capable of efficiently synthesizing l-valine with high yield by minimizing the consumption of its precursor pyruvate through the TCA cycle under oxygen-limited conditions. Metabolic engineering-based adaptive laboratory evolution (ALE) under oxygen-limited conditions resulted in the development of an evolved strain ALE2-40 with better cell growth and enhanced l-valine yield. Through comparative omics analysis and validation experiments, it was uncovered that during the ALE process, both pyruvate dehydrogenase activity and NADH availability were significantly improved. Moreover, beneficial targets have the potential to contribute to the NADH and ATP pools, thereby further promoting l-valine synthesis. Based on these results, reverse engineering of the evolved strain ALE2-40 was further conducted. Ultimately, the final strain VAL19 demonstrated remarkable performance, achieving an impressive l-valine titer of 93.7 g/L within 28 h in a 5-L bioreactor under oxygen-limited conditions, with a remarkable yield of 60.4% from glucose-equivalent to 92.9% of the theoretical yield-and a productivity of 3.35 g/L/h. These results set a new benchmark for the fermentative production of l-valine, with the highest yield and productivity reported so far.