Novel Bioproduction of 1,6-Hexamethylenediamine from l-Lysine Based on an Artificial One-Carbon Elongation Cycle

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.

Figure 1

(A) Engineered artificial iterative carbon-chain-extension cycle to produce HMD. (B) Synthetic operons for gene expression. (1) Synthetic operon for protein overexpression to drive the carbon flux toward 2K6AC. (2) Synthetic operon carrying mutations of LeuA (LeuA^#) for protein overexpression to drive the carbon flux toward 2K6AC. (3) Synthetic operon for protein overexpression for decarboxylase and transaminase. Raip, l-lysine α oxidase; LeuA, α-isopropylmalate synthase; LeuA*, LeuA mutants; LeuA^#, LeuA with H97L/S139G/G462D mutations; LeuB, 3-isopropylmalate dehydrogenase; LeuC, 3-isopropylmalate dehydratase; LeuD, 3-isopropylmalate dehydratase; KdcA, alpha-ketoacid decarboxylase; Dat, d-amino acid transaminase; Vfl, pyruvate transaminase; and KivD, α-ketoacid decarboxylase.

Figure 2

(A) LC–MS confirmation of NNSCAAs (DAP, HMD, and DAHP) biosynthesis by strain WD21. (B) HPLC results of DAP, HMD, and DAHP from fermentation broth. Samples were derived with phenyl isothiocyanate for LC–MS analysis. Strain WD21 is the strain BL21(DE3) harboring plasmids pIVC2 and pET21aRKD. DAP, 1,5-diaminopentane; HMD, 1,6-hexamethylenediamine; and DAHP, 1,7-diaminoheptane.

Figure 3

NNSCAA synthesis by engineered strain WD21 in a 250 mL flask. The cells were grown in 50 mL of LB supplemented with 100 μg/mL ampicillin, 50 μg/mL kanamycin, 0.5 mM of IPTG, 5 g/L l-lysine, 1.0 mM MgSO₄, and 0.5 mM ThDP at 37 °C with 250 rpm orbital shaking. Strain WD21 is strain BL21(DE3) plus plasmids pIVC2 and pET21aRKD. The total diamines conclude DAP, HMD, and DAHP. DAP, 1,5-diaminopentane; HMD, 1,6-hexamethylenediamine; and DAHP, 1,7-diaminoheptane. Each experiment was conducted at least three times to ensure reliability and data are presented as mean values ± SD.

Figure 4

(A) Synthesis of NNSCAAs from l-lysine was carried out using a variety of NAD⁺ concentrations. (B) Manufacturing process of NNSCAAs from l-lysine involved conducting reactions with varying levels of d-alanine. Each assay mixture included 2.0 mM acetyl-CoA, 1.0 μM Raip, 20.0 μM LeuA^#, 4.0 μM LeuB, 2.0 μM LeuC, 2.0 μM LeuD, 5.0 μM KdcA, 2.0 μM Dat, 5.0 μM KivD, 2.0 μM Vfl, 2.5 mM l-lysine, 1.0 mM MgCl₂, 1.0 mM TCEP, and 0.5 mM ThDP. Reactions incubated for 8 h at 37 °C. Each experiment was conducted at least three times to ensure reliability and data are presented as mean values ± SD.

Figure 5

LC–MS confirmation of 6-aminohexanal and 2,7-diaminoheptanoic acid biosynthesis by strains. (A) Mass spectrum results of 6-aminohexanal from fermentation broth by strain WD21. Strain WD21 is strain BL21(DE3) plus plasmids pIVC2 and pET21aRKD. (B) Mass spectrum results of 2,7-diaminoheptanoic acid from fermentation broth by strain WD20. Strain WD20 is strain BL21(DE3) plus plasmids pIVC2 and pET21aRVK.

Figure 6

Effect of different amine donors on HMD yield. The yield of HMD was measured individually when Gln was added alone, d-ala was added alone, and Gln and d-ala were both added. All experiments were performed a minimum of three independent sets. Each experiment was conducted at least three times to ensure reliability, and data are presented as mean values ± SD.