Abstract
1,6-hexamethylenediamine (HMD) is an important precursor for nylon-66 material synthesis, while research on the bioproduction of HMD has been relatively scarce in scientific literature. As concerns about climate change, environmental pollution, and the depletion of fossil fuel reserves continue to grow, the significance of producing fundamental chemicals from renewable sources is becoming increasingly prominent. In recent investigations, the bioproduction of HMD from adipic acid has been reported but with lingering challenges concerning costly raw materials and low yields. Here, we have undertaken the reconstruction of the HMD synthetic pathway within Escherichia coli, which was constituted with l-lysine α-oxidase (Raip), LeuABCD, α-ketoacid decarboxylase (KivD), and transaminases (Vfl), leveraging a carbon chain extension module and a metabolic pathway of transaminase-decarboxylase cascade catalysis within the strain WD20, which successfully produce 46.7 ± 2.0 mg/L HMD. To increase the cascade activity and create a higher tolerance to external environmental disturbance for l-lysine to convert into HMD, another two enzymes d-alanine aminotransferase (Dat) and alpha-ketoacid decarboxylase (KdcA) were introduced into WD21 to provide flux flexibility for α-ketoacid metabolization, which was named "Smart-net metabolic engineering" in our research, and high-efficiency synthesis of HMD utilizing l-lysine as the substrate has been successfully achieved. Finally, we established a + 1C bioconversion multienzyme cascade catalyzing up to 65% conversion of l-lysine to HMD. Notably, our fermentation process yielded an impressive 213.5 ± 8.7 mg/L, representing the highest reported yield to date for the bioproduction of HMD from l-lysine.