Precise l-threonine-to-l-isoleucine pathway regulation for engineering high-efficiency whole-cell biocatalysts

L-Isoleucine (L-Ile), a critical branched-chain amino acid with diverse applications in food, pharmaceutical, and cosmetic industries, is difficult to produce efficiently at scale in microbial systems due to metabolic bottlenecks and cofactor limitations. This study metabolically engineered Escherichia coli BL21(DE3) to develop a whole-cell biocatalyst for efficient L-Ile biosynthesis. Key strategies included screening acetohydroxy acid synthase (AHAS) isoenzymes, identifying ilvGM -encoded AHAS II as the optimal enzyme, relieving feedback inhibition of ilvA (encoding L-threonine dehydratase) through mutant screening, and optimizing genetic circuits (promoter tuning, plasmid copy number). Dual-precursor supplementation revealed L-threonine as a critical factor for suppressing L-valine byproduct. Fed-batch fermentation in a 5 L bioreactor achieved a peak molar conversion rate of 98.4%, yielding 40.1 g/L L-Ile within 36 h. The mass conversion rate (L-Ile/glucose) achieved 0.36 g/g and the production efficiency achieved 1.11 g/L/h, demonstrating the feasibility of whole-cell catalysis. This work provides a robust framework for industrial L-Ile production and transferable strategies for branched-chain amino acid pathway optimization. • Escherichia coli was engineered to enhance L-isoleucine production. • Feedback-inhibited ilvGM and ilvA eco L447F/L451A boost conversion efficiency. • Genetic circuit engineering optimizes L-threonine to L-isoleucine biosynthesis. • Cofactor specificity remodeling via ilvC cge S34G/L47E/R48F enhances L-isoleucine production. • Whole-cell biocatalysis achieves 98.4% L-threonine to L-isoleucine conversion.