Work

Directed Evolution of Branched-Chain α-Keto Acid Decarboxylase for 3-Hydroxypropionic Acid Production in Escherichia coli via Oxaloacetate

Year: 2025

Type: article

Abstract: High Resolution Image Download MS PowerPoint Slide 3-Hydroxypropionic acid (3-HP) serves as a crucial platform chemical with diverse applications across various industries. In this study, the oxaloacetate pathway was utilized for 3-HP production. This pathway involves the decarboxylation of oxaloacetate into malonic semialdehyde, catalyzed by branched-chain α-keto acid decarboxylase (KdcA), which is subsequently reduced to 3-HP by dehydrogenases. Directed evolution of KdcA was carried out to enhance its catalytic efficiency toward oxaloacetate, resulting in a KdcA M8 mutant with the following substitutions: S286R, S287T, F381H, F382P, L534S, L535F, M538T, and G539F. Compared to wild-type (WT) KdcA, KdcA M8 exhibits a lower K M value toward oxaloacetate ( K M = 1.15 mM vs K M > 25 mM). Among these mutations, the single mutants S286R and S287T exhibited 5.5-fold and 1.3-fold increased activities, respectively. Instead of WT KdcA, the KdcA M8 mutant was integrated into Escherichia coli ( E. coli ) BL21 strain, resulting in the production of 3-HP at a concentration of 0.11 mM. To further improve 3-HP production, two dehydrogenases were compared for the downstream conversion of malonic semialdehyde into 3-HP, and two carboxylases were explored to enhance the upstream precursor supply of oxaloacetate. Additionally, the growth conditions were optimized. Finally, a nonnatural oxaloacetate pathway was successfully engineered in the E. coli BL21 strain, achieving a 3-HP titer of approximately 0.71 mM from glucose. This work illustrates that protein engineering is a powerful tool for modulating flux in the target pathway and holds promise for the future development of the oxaloacetate pathway to improve the 3-HP yield.

Source: ACS Synthetic Biology

DOI: 10.1021/acssynbio.5c00267

Authors: Chuang Wang, René C. L. Olsthoorn, Huub J. M. de Groot

Institution: Leiden University

Language: English

Topic: Microbial Metabolic Engineering and Bioproduction

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Estimated APC paid: $4,500

Funder: China Scholarship Council