TY - JOUR
T1 - Directed Evolution of (R)-2-Hydroxyglutarate Dehydrogenase Improves 2-Oxoadipate Reduction by 2 Orders of Magnitude
AU - Saez-Jimenez, Veronica
AU - Scrima, Simone
AU - Lambrughi, Matteo
AU - Papaleo, Elena
AU - Mapelli, Valeria
AU - Engqvist, Martin K.M.
AU - Olsson, Lisbeth
N1 - Publisher Copyright:
© 2022 The Authors. Published by American Chemical Society.
PY - 2022
Y1 - 2022
N2 - 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.
AB - 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.
KW - (R)-2-hydroxyacid dehydrogenase
KW - (R)-2-hydroxyadipate
KW - Adipic acid
KW - Protein engineering
KW - Random mutagenesis
KW - Saturation mutagenesis
U2 - 10.1021/acssynbio.2c00162
DO - 10.1021/acssynbio.2c00162
M3 - Journal article
C2 - 35939387
AN - SCOPUS:85136156357
SN - 2161-5063
VL - 11
SP - 2779
EP - 2790
JO - ACS Synthetic Biology
JF - ACS Synthetic Biology
IS - 8
ER -