(246f) Rational Design of Enoate Reductase for Bio-Nylon Production from Renewable Feedstocks

Adipic acid is a key industrial intermediate of dicarboxylic acid, mostly applied as a precursor for the production of resins, nylons, lubricants, and plasticizers. The commercial production of adipic acid utilizes petroleum-derived benzene, which results in significant greenhouse gas emissions.^{1, 2} Developing a more sustainable production strategy is therefore crucial to minimizing its negative environmental impact. The transformation of renewable feedstocks into adipic acid has been attempted using enzyme discovery and metabolic engineering procedures.^3-6 One of the most promising non-natural synthetic pathways is the muconic acid route, which requires a following catalytic hydrogenation step using chemical catalysts to produce adipic acid.⁷ The discovery of enoate reductases that reduce C=C bonds of α, β-unsaturated chemicals, using NAD(P)H as the source of an electron, could replace the chemical hydrogenation step and construct the adipic acid biosynthetic pathway.^{5, 6} Enoate reductase is a family of flavoenzymes and composed of several domains including FMN and FAD binding domains that are connected by a 4Fe-4S cluster, and an NADH binding domain.⁸ However, the mechanism of enoate reductase-catalyzed hydrogenation of carboxylic acids has been poorly understood to date. Aliphatic carboxylic acids such as muconic acid is a non-native substrate for enoate reductases that generally favor aromatic carboxylic acids such as cinnamic acid. The improved activity of enoate reductase towards the reduction of muconic acid is then required to enhance the production of adipic acid biosynthetic pathway. Rational design of enoate reductases by site-directed mutagenesis provides a strategy for improving the activity of the enzyme. Since the substrate binding pocket of enoate reductase (FMN binding domain) consists of negatively charged amino acids, the binding/activity with aliphatic carboxylic acids can be improved by introducing positively charged amino acids such as lysine, arginine and histidine at certain positions. For this purpose, we considered 19 positions in the substrate binding pocket of enoate reductase from Bacillus coagulans and generated 89 variants. The variants were expressed in strains including E. coli BL21 (DE3) delta-iscR and E. coli LOBSTR. The activity of variants is evaluated for the conversion of muconic acid to adipic acid in vivo under anaerobic conditions and compared to WT activity. Desired variants will be selected for further characterization. This work will demonstrate whether mutations at the enzyme substrate binding pocket can improve the catalytic activity of enoate reductase towards the formation of adipic acid from muconic acid. These data, combined with related studies, will provide crucial information to better understand the characteristics of the enzyme active sites and the catalytic mechanism of enoate reductase.

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