High-yield production of L-valine in engineered Escherichia coli by a novel two-stage fermentation.

L-valine is an essential amino acid and an important amino acid in the food and feed industry. The relatively low titer and low fermentation yield currently limit the large-scale application of L-valine. Here, we constructed a chromosomally engineered Escherichia coli to efficiently produce L-valine. First, the synthetic pathway of L-valine was enhanced by heterologous introduction of a feedback-resistant acetolactate acid synthase from Bacillus subtilis and overexpression of other two enzymes in the L-valine synthetic pathway. For efficient efflux of L-valine, an exporter from Corynebacterium glutamicum was subsequently introduced. Next, the precursor pyruvate pool was increased by knockout of GTP pyrophosphokinase and introduction of a ppGpp 3'-pyrophosphohydrolase mutant to facilitate the glucose uptake process. Finally, in order to improve the redox cofactor balance, acetohydroxy acid isomeroreductase was replaced by a NADH-preferring mutant, and branched-chain amino acid aminotransferase was replaced by leucine dehydrogenase from Bacillus subtilis. Redox cofactor balance enabled the strain to synthesize L-valine under oxygen-limiting condition, significantly increasing the yield in the presence of glucose. Two-stage fed-batch fermentation of the final strain in a 5 L bioreactor produced 84 g/L L-valine with a yield and productivity of 0.41 g/g glucose and 2.33 g/L/h, respectively. To the best of our knowledge, this is the highest L-valine titer and yield ever reported in E. coli. The systems metabolic engineering strategy described here will be useful for future engineering of E. coli strains for the industrial production of L-valine and related products.