Rational protein engineering of branched-chain α-keto acid decarboxylase for enhanced 3-hydroxypropionic acid production in Escherichia coli

Wang, C.; Olsthoorn, R.R.C.L.; Groot, H.J.M. de (2026)

Article / Letter to editor

The oxaloacetate (OAA) pathway represents a promising biosynthetic route to produce 3-hydroxypropionic acid (3-HP), comprising two steps: the decarboxylation of OAA to malonic semialdehyde, followed by its reduction to 3-HP. A thiamine diphosphate (ThDP)-dependent α-keto acid decarboxylase was identified as a potential bottleneck in this pathway due to its low catalytic efficiency toward the non-natural substrate OAA. In this study, rational protein engineering is employed to enhance the catalytic efficiency of KdcA. By rearranging the interaction network within the enzyme's binding pocket, variants S286R and S286K are developed, exhibiting 4.6-fold and 6.2-fold increases in activity, respectively, compared to wild-type KdcA (WT). Further reduction of the binding pocket volume leads to the creation of enhanced variants S286K/V461I/M538Y and S286K/F381W/V461I/M538Y, which display significantly lower K_m values (6.6 and 6.0 mM, respectively) relative to those of WT (K_m > 20 mM), along with up to about 120-fold increases in catalytic efficiency (k_cat/K_m). When the variant S286K/V461I/M538Y is integrated into Escherichia coli (E. coli), 3-HP production reaches 1.6 ± 0.2 mM in shake flask cultures. This study demonstrates the effectiveness of protein engineering in overcoming enzymatic bottlenecks to improve biochemical production.

• All authors: Wang, C.; Olsthoorn, R.R.C.L.; Groot, H.J.M. de
• Date: 2026-03-17
• Journal: ACS Synthetic Biology
• Advanced Publication: Yes
• DOI: doi:10.1021/acssynbio.5c00889
• Link: https://pubs.acs.org/doi/10.1021/acssynbio.5c00889