Metabolic engineering for 4-aminophenylalanine production from lignocellulosic biomass by recombinant Escherichia coli

Abstract

To synthesize a high-performance biopolyimide bioplastic from lignocellulosic feedstock, Escherichia coli was metabolically engineered to produce 4-amino-L-phenylalanine (4APhe) as a diamine monomer. A high-biomass sorghum cultivar was used as the model lignocellulosic feedstock, and the enzymatic hydrolysate was used as a substrate for 4APhe production in fed-batch culture. When using the ldh mutant strain, HKE6027, 4APhe production from glucose was increased by over three-fold compared to the parent strain. This increase was due to the disruption of biosynthetic pathways that produce either acetate or lactate as by-products. Comparative metabolomic analysis revealed increased flux in both the pentose phosphate and shikimate pathways in HKE6027, resulting in the highest yields. However, 5.7 g L⁻¹ of 4APhe was produced from enzymatic hydrolysis by HKE6027, which was 24% lower than that obtained from glucose. The concentrations of 14 potential fermentation inhibitors present in the enzymatic hydrolysate were determined, and their inhibitory effects on both cell growth and 4APhe production were examined. The addition of groups of potential inhibitors present in the enzymatic hydrolysate of sorghum bagasse showed that benzaldehyde- and cinnamic acid derivatives inhibited 4APhe fermentation at low concentrations (half-maximum inhibitor concentration IC₅₀ = 16 mg L⁻¹), whereas furfural and 5-hydroxymethylfurfural, which are well-known fermentation inhibitors, inhibited 4APhe fermentation at relatively high concentrations (IC₅₀ = 640 mg L⁻¹). These results provide insight into the design of metabolic pathways tailored for the utilization of lignocellulosic biomass to produce aromatic compounds through the shikimate pathway.