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.

Saez-Jimenez, V., Scrima, S., Lambrughi, M., Papaleo, E., Mapelli, V., Engqvist, M., et al. (2022). Directed Evolution of (R)-2-Hydroxyglutarate Dehydrogenase Improves 2-Oxoadipate Reduction by 2 Orders of Magnitude. ACS SYNTHETIC BIOLOGY [10.1021/acssynbio.2c00162].

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

Mapelli, Valeria
Membro del Collaboration Group
;
2022

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.
Si
Articolo in rivista - Articolo scientifico
Scientifica
(R)-2-hydroxyacid dehydrogenase; (R)-2-hydroxyadipate; adipic acid; protein engineering; random mutagenesis; saturation mutagenesis
English
2022 Aug 8, Online ahead of print
Saez-Jimenez, V., Scrima, S., Lambrughi, M., Papaleo, E., Mapelli, V., Engqvist, M., et al. (2022). Directed Evolution of (R)-2-Hydroxyglutarate Dehydrogenase Improves 2-Oxoadipate Reduction by 2 Orders of Magnitude. ACS SYNTHETIC BIOLOGY [10.1021/acssynbio.2c00162].
Saez-Jimenez, V; Scrima, S; Lambrughi, M; Papaleo, E; Mapelli, V; Engqvist, M; Olsson, L
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10281/389909
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