Multi-layered transcriptional and post-translational fine-tuning of metabolic pathways for overproduction of L-valine

L-valine is an essential amino acid widely used in the food, pharmaceutical, and animal feed industries. Currently, engineering microbial cell factories to produce L-valine from low-cost feedstocks has emerged as a leading strategy. However, there is still a lack of an L-valine-producing strain that simultaneously exhibits high titer, high yield, and high productivity. The metabolic engineering strategies reported for L-valine biosynthesis primarily rely on conventional unidimensional, transcriptional-level modifications, which limit fine-tuning and do not provide comprehensive, multi-layered regulation. In this study, we constructed an Escherichia coli hyperproducer of L-valine by multiplexed transcriptional and post-translational fine-tuning of metabolic pathways. Initially, the transcriptional repression in L-valine synthetic pathway was eliminated by using promoter engineering strategy. Then, the “push-pull-inhibit” and transport engineering strategies were used to improve L-valine accumulation, achieving a flask fermentation titer of 21.6 g/L and a productivity of 0.45 g/L/h. Subsequently, we rationally designed membraneless organelles (MLOs) to enable spatial regulation of metabolic biosynthesis, which enhanced the targeted recruitment of dihydroxy-acid dehydratase and branched-chain amino acid aminotransferase. This spatial reorganization led to a 95.6 % increase in productivity, reaching 0.88 g/L/h. Finally, the best-performing strain produced 90.6 g/L L-valine in a 3-L bioreactor at 28 h, with a yield of 0.48 g/g glucose and a productivity of 3.24 g/L/h. To the best of our knowledge, this represents the highest L-valine productivity achieved to date. Our strategy provides a practical and effective approach for advancing microbial amino acid biosynthesis by multi-layered transcriptional and post-translational regulation of metabolic pathways.