Engineering of fructose-6-phosphate aldolase for one-carbon conversion to mannitol in a designed biotransformation system

Developing artificial synthetic pathways for converting one-carbon compounds into value-added chemicals represents a promising strategy for carbon-neutral manufacturing. Enzymatic C–C bond formation plays a central role in carbon-chain extension and structural diversification. Fructose-6-phosphate aldolase (FSA) has been employed in in vitro multienzyme systems for producing starch and sugars from methanol. However, its atomistic catalytic mechanism has remained unclear, limiting rational enzyme engineering. Here, we elucidate the aldol reaction mechanism between dihydroxyacetone (DHA) and glyceraldehyde-3-phosphate (GALP) catalyzed by FSA using QM/MM calculations, identifying the Schiff-base/iminium formation step as the rate-limiting step in the aldol condensation. Guided by this mechanism, we engineered FSA variants with a 34-fold increase in catalytic efficiency compared with the wildtype. We further integrated the engineered FSA into a designed in vitro cascade converting methanol to mannitol, achieving a high yield of 88%. Collectively, these mechanistic insights and improved biocatalysts expand the toolkit for green enzymatic C–C bond formation and one-carbon utilization.