Construction of Escherichia coli L-isoleucine cell factories based on propionate pathway

Isoleucine, an essential branched-chain amino acid with broad applications in food, pharmaceuticals, and feed, is predominantly produced via the microbial threonine pathway, which suffers from catalytic complexity and regulatory inefficiency. The propionate pathway offers a streamlined alternative but remains unexplored for L-isoleucine biosynthesis. Here, we engineered Escherichia coli to establish the first propionate pathway-based L-isoleucine cell factory. Critical enzymes—propionyl-CoA synthase (PCS), propionyl-CoA transferase (PCT), and α-ketobutyrate synthase (OBS)—were identified for converting propionate to α-ketobutyrate. Key genes (prpE from Salmonella, pctcP from Gibberella, pctcN from Clostridium propionicum, and nifJ from Moorella thermoacetica) were integrated with the propionate transporter (prpP) and carbonic anhydrase (can) to enhance substrate utilization. ILE-5a, derived from E. coli BW25113 with deletions in brnQ, livJ, and livK, and containing specific insertions, was further modified to create ILE-5b with an additional deletion in yjiP. Plasmid-based expression in these strains, ILE-5a and ILE-5b, yielded top producers ILE-5a-P10 and ILE-5b-P10, which achieved L-isoleucine titers of 304 mg/L and 235 mg/L, respectively, in shake-flask fermentation using glucose and propionate as carbon sources. To stabilize production, the optimal gene set (pctN, nifJ, prpP, can) was genomically integrated via a transposon-encoded CRISPR-Cas system, generating mutants ILE-5a-P11 and ILE-5b-P11. Response surface methodology-optimized medium and 3-L fed-batch fermentation further elevated titers to 1.13 g/L (ILE-5a-P11) and 11.33 g/L (ILE-5b-P11). This study pioneers the propionate pathway for efficient L-isoleucine production, demonstrating its industrial potential through systematic metabolic engineering and process optimization.To our knowledge, this is the first study to design and demonstrate an E. coli platform for isoleucine production that simultaneously leverages the glutamate and propionate precursors.