Biocatalytic Conversion of Caprolactam to 1,6-Hexanediamine: A Novel Route for Nylon Monomer Production

As a key monomer for nylon-66 production, 1,6-hexanediamine (HMD) has traditionally been synthesized via chemical routes relying on highly toxic hydrocyanic acid and complex multistep processes. In this study, we report an in vivo biosynthetic system for HMD production from caprolactam (CPL), leveraging enzyme mining, enzyme engineering, and microbial consortia construction. The engineered biosynthetic pathway comprises six enzymes. The amidohydrolase 4270/4271 was identified for its efficient hydrolysis of CPL to 6-aminocaproic acid (ACA). To overcome the kinetic bottleneck posed by the carboxylic acid reductase (CAR) toward ACA, multiround engineering yielded a quadruple variant (L342E/L284T/S986A/D987N), which demonstrated a 5.3-fold improvement over the wild type. Molecular dynamics simulations revealed that enhanced polarity in the adenylation (A) domain stabilizes substrate adenylation, while polarity redistribution and reduced steric hindrance in the reductase (R) domain facilitate optimal hydride transfer geometry. Through systematic optimization of the enzymatic cascade, a biosynthetic system consisting of different cell modules (dubbed MCPL-ACA3_MACA-AH24_MAH-HMD1) was successfully constructed. This developed biosynthetic system achieved a product concentration of 43.5 mM HMD with a yield of 43.5% from 100 mM CPL, representing the highest reported in vivo product concentration to date.