A cell-free system for 1,6-hexanediamine synthesis using bulk chemical caprolactam as the feedstock.

Biomanufacturing, with its advantages of mildness, low carbon emissions, and renewability, drives the frontier of green chemistry and sustainable development. However, complex metabolic pathways and intracellular resource competition frequently limit the efficiency and yield of metabolic engineering approaches, particularly for non-natural compounds such as 1,6-hexanediamine (1,6-HMD), an important precursor for nylon 66. To address these challenges, we developed a cell-free biocatalytic system employing 6-aminocaproic acid (6-ACA), derived from low-cost caprolactam, together with optimized enzyme assembly and cofactor regeneration. As a result, the 1,6-hexanediamine titer was enhanced by 511-fold, reaching a record-high level of 10.35 g/L with a conversion rate of 97.4%. The system was further extended to the production of C7-C10 α,ω-diamines, achieving high-level titers. In addition, the cell-free biocatalytic system retained considerable activity after 7 days of storage at 4 °C, with a 25.9% reduction in titer. This cell-free biocatalysis overcame the inherent limitations of biological systems and preserved enzyme activity, demonstrating the substantial potential for efficient diamine synthesis and indicating broad applicability for future practical implementations.