Precursor balancing by reprogramming pyruvate synthesis for high-yield l-isoleucine production in engineered...

l-isoleucine (l-Ile) is an essential branched-chain amino acid with broad applications in food, animal feed, and pharmaceutical industries. Although the microbial fermentation method for l-Ile synthesis has been developed, the yield is far below the theoretical yield. This study aims to enhance the l-Ile yield from glucose by balancing the two direct precursors, 2-oxobutyrate and pyruvate. First, a plasmid-free l-threonine (l-Thr) synthesis module was constructed to enhance the supply of 2-oxobutyrate. Then, the l-isoleucine synthesis module was optimized by modifying transport systems and balancing the expression levels of key enzymes. Next, a critical strategy was raised for balancing the ratio of pyruvate to 2-oxobutyrate by deleting pykFA and leveraging the glucose PTS transport system. This strategy partitions phosphoenolpyruvate, directing one molecule to pyruvate and another to 2-oxobutyrate, resulting in the high-yield l-isoleucine production. Additionally, attenuation of the TCA cycle flux minimized pyruvate loss, while the introduction of leucine dehydrogenase addressed the glutamate deficiency. The engineered strain achieved a high yield of 0.67 mol l-Ile/mol glucose with a productivity of 1.72 g/L/h. Meanwhile, the byproduct l-valine (l-Val) was also significantly reduced by balancing the 2-oxobutyrate/pyruvate ratio, thereby facilitating the downstream purification of l-isoleucine. These integrated strategies provide a platform for developing cell factories dedicated to producing l-isoleucine and its derivatives.