Directed Evolution of (R)-2-Hydroxyglutarate Dehydrogenase Improves 2-Oxoadipate Reduction by 2 Orders of Magnitude

Abstract

Pathway engineering is commonly employed to improve the production of various metabolites but may incur in bottlenecks due to the low catalytic activity of a particular reaction step. The reduction of 2-oxoadipate to ( R )-2-hydroxyadipate is a key reaction in metabolic pathways that exploit 2-oxoadipate conversion via α-reduction to produce adipic acid, an industrially important platform chemical. Here, we engineered ( R )-2-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans (Hgdh) with the aim of improving 2-oxoadipate reduction. Using a combination of computational analysis, saturation mutagenesis, and random mutagenesis, three mutant variants with a 100-fold higher catalytic efficiency were obtained. As revealed by rational analysis of the mutations found in the variants, this improvement could be ascribed to a general synergistic effect where mutation A206V played a key role since it boosted the enzyme’s activity by 4.8-fold. The Hgdh variants with increased activity toward 2-oxoadipate generated within this study pave the way for the bio-based production of adipic acid.

Metabolic Engineering for Effective Synthesis of 2-Hydroxyadipate

“…In order to further improve the titer and yield of 2-hydroxyadipate, more metabolic engineering optimizations can be tested and applied, including the regulation of the TCA cycle and related genes at the transcription and translation levels, rational design of key enzymes, omics analysis, etc. For example, Lisbeth Olsson et al have obtained HgdH mutants with a 100-fold increase in catalytic efficiency through computational analysis, saturation mutagenesis, and random mutagenesis . These mutants could be used to construct a 2-hydroxyadipate biosynthesis pathway that promotes carbon flux into 2-hydroxyadipate synthesis.…”

“…For example, Lisbeth Olsson et al have obtained HgdH mutants with a 100-fold increase in catalytic efficiency through computational analysis, saturation mutagenesis, and random mutagenesis. 42 These mutants could be used to construct a 2hydroxyadipate biosynthesis pathway that promotes carbon flux into 2-hydroxyadipate synthesis. In addition, the conversion of 2-hydroxyadipate to adipic acid is the key step in the pathway with L-lysine as a precursor.…”

Microbial alcohol dehydrogenases: recent developments and applications in asymmetric synthesis

“…The metabolic pathway for adipic acid biosynthesis from ( S )-lysine involves the reduction of 2-oxoadipate to ( R )-2-hydroxyadipate. 111 A ( R )-2-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans (Hgdh) identified for this reduction was engineered based on FuncLib-predicted amino acid sites and FoldX-based Δ G calculations. Site saturation mutagenesis (SSM) on the predicted amino acid positions provided V11K or V11M with higher activity than the wild type.…”