| Summary: | Abstract Background l-Leucine is a high-value amino acid with promising applications in the medicine and feed industries. However, the complex metabolic network and intracellular redox imbalance in fermentative microbes limit their efficient biosynthesis of l-leucine. Results In this study, we applied rational metabolic engineering and a dynamic regulation strategy to construct a plasmid-free, non-auxotrophic Escherichia coli strain that overproduces l-leucine. First, the l-leucine biosynthesis pathway was strengthened through multi-step rational metabolic engineering. Then, a cooperative cofactor utilization strategy was designed to ensure redox balance for l-leucine production. Finally, to further improve the l-leucine yield, a toggle switch for dynamically controlling sucAB expression was applied to accurately regulate the tricarboxylic acid cycle and the carbon flux toward l-leucine biosynthesis. Strain LEU27 produced up to 55 g/L of l-leucine, with a yield of 0.23 g/g glucose. Conclusions The combination of strategies can be applied to the development of microbial platforms that produce l-leucine and its derivatives.
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