Towards an enzyme cascade synthesis of the bulk chemical acrylic acid.

Acrylic acid is a bulk chemical predominantly produced via fossil-based processes. Here, we repurpose and engineer a thiamine diphosphate (ThDP)-dependent enzyme to catalyze direct H 2 PO 4 - elimination from dihydroxyacetone phosphate (DHAP) for acrylic acid synthesis. Quantum chemical calculations reveal the catalytic mechanism involving isomerization, enol-ThDP intermediate formation, H 2 PO 4 - elimination, and hydrolysis. Using an automated high-throughput screening platform, we perform directed evolution and obtain a 13.6-fold improved variant, designated acrylic acid synthase (AAS). We establish an enzymatic methanol-to-acrylic acid (MAAP) pathway, achieving a titer of 4.3 g L-1, a productivity of 268.9 mg L-1 h-1, and 99.5% conversion. This work establishes a high-efficiency biomanufacturing paradigm for bulk acrylic acid, and expands the catalytic repertoire of ThDP-dependent enzymes for sustainable one-carbon conversion.