Metabolic Engineering of <i>Escherichia coli</i> for Efficient <scp>l</scp>-Isoleucine Production based on the Citramalate Pathway

l-isoleucine, a value-added branched-chain amino acid, has been widely used in the food, feed, and pharmaceutical industries. However, the production efficiency of l-isoleucine is relatively low due to the complex and inefficient biosynthetic network of the canonical threonine pathway. Here, we report the exploitation of a concise citramalate pathway for the development of an efficient l-isoleucine producer. First, chassis strain and key genes were screened for establishment of the citramalate pathway. Subsequently, a citramalate importer was identified and applied to enhance citramalate's utilization efficiency. Finally, a plasmid-free high-l-isoleucine producer was developed by enhancement of l-isoleucine efflux, optimization of rate-limiting gene expression, and introduction of a nonoxidative glycolysis pathway. Fed-batch fermentation of the final strain in a 10-L bioreactor produced 56.6 g/L l-isoleucine with a productivity of 1.66 g/L/h, which is the highest l-isoleucine titer and productivity reported. This study paves the way for construction of efficient microbial cell factories for production of l-isoleucine and related derivatives based on the citramalate pathway.