A novel engineered malonyl-CoA synthetase from Astragalus mongholicus with improved catalytic performance.

Malonyl-CoA is a key precursor for the biosynthesis of numerous active natural products. Malonyl-CoA synthetases (MatBs) provide a direct route to malonyl-CoA from malonic acid and CoA. However, the limited availability of plant MatBs with high catalytic efficiency restricts their application in biocatalysis. In this study, we identified a novel malonyl-CoA synthetase AmMatB from Astragalus mongholicus. Biochemical assays confirmed that recombinant AmMatB directly catalyzes malonyl-CoA formation from malonic acid and CoA, while also accepting methylmalonate as a substrate. To enhance its utility, we engineered a truncated variant lacking a predicted N-terminal mitochondrial transit peptide. This modification markedly improved catalytic efficiency by increasing substrate affinity, reducing the Km value for malonic acid from 317 μM to 182.5 μM. Furthermore, homology modeling, molecular docking, and site-directed mutagenesis elucidated the pivotal roles of residues C212, V218, F241, R277, S307, and K524 in substrate binding and product stabilization. This study not only provides an efficient engineered enzyme for malonyl-CoA production but also offers new structural insights to facilitate the rational design of plant MatBs for biocatalysis.