Systematic Engineering of Escherichia coli to Enhance 1,6‐Hexamethylenediamine Biosynthesis and Mitigate Byproduct 1,5‐Pentanediamine

1,6-Hexamethylenediamine (HMD), a key nylon 6,6 intermediate, is traditionally derived from fossil feedstocks, demanding sustainable alternatives. Despite the great potential of the L-lysine-based carbon chain elongation system for biosynthetic HMD, its practical application is hampered by low catalytic efficiency. In this study, systematic engineering strategies were developed to overcome these limitations: site-saturation mutagenesis of 3-isopropylmalate dehydratase (LeuCD) yielded a dual-site mutant with a 2.13-fold higher HMD biosynthesis. Synergistic integration of NAD⁺ synthase overexpression, pyridoxal 5'-phosphate supplementation, optimized ammonia donors, and fed-batch fermentation markedly boosted HMD biosynthesis. Specifically, continuous feeding of glucose and L-lysine resulted in an HMD titer of 1835.35 ± 14.64 mg/L, with a productivity of 25.48 mg/L/h, representing over a 7.15-fold increase versus shake flask fermentation. Finally, a novel dual-cell module further enhanced the HMD titer, increased the molar yield of L-lysine to HMD to 24.78%, and reduced the byproduct 1,5-pentanediamine (PDA) to 22.22% of the original level. This work establishes a feasible, efficient, and sustainable HMD biomanufacturing process, addressing the critical issue of substrate competition in multi-step biosynthetic pathways.