Screening of high glucose tolerant Escherichia coli for L-valine fermentation by autonomous evolutionary mutation

L-valine holds wide-ranging applications in medicine, food, feed, and various industrial sectors. Escherichia coli, a pivotal strain in industrial L-valine production, features a concise fermentation period and a well-defined genetic background. This study focuses on mismatch repair genes (mutH, mutL, mutS, and recG) and genes associated with mutagenesis (dinB, rpoS, rpoD, and recA), employing a high-glucose adaptive culture in conjunction with metabolic modifications to systematically screen for superior phenotypes. This approach enhances the spontaneous survival rate of stress cells and facilitates the enrichment of positive mutations. Leveraging a multi-fragment seamless recombination technique, we successfully assembled the ilvBN, ilvC, ilvE, and ilvD pathway enzyme genes, transforming E. coli from a non-producer into a proficient L-valine producer capable of generating up to 6.62 g/L. Through a synergistic application of self-evolution engineering and metabolic engineering strategies, the engineered E. coli strain exhibited significantly enhanced tolerance and demonstrated heightened accumulation of L-valine. KEY POINTS: • The innovation centered on mutated genes and mismatch repair genes • By integrating modification with adaptive culture, a superior phenotype was attained • Double plasmids expressing enzymes for L-valine production in E. coli were obtained.