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

[Image: see text] 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.