Formaldehyde (FALD) is a volatile and highly toxic compound widely used in industry and a major environmental
pollutant due to its genotoxic and carcinogenic effects. Developing efficient methods to convert formaldehyde
into value-added, non-toxic products is essential for both environmental protection and chemical
sustainability. In this study, we present a biocatalytic cascade for the selective enzymatic conversion of formaldehyde
into enantiopure L-glyceraldehyde, a high-value chiral C3 compound. The system employs a structurally
engineered fructose-6-phosphate aldolase (GaFSA) from Gilliamella apicola, which catalyzes
carbon–carbon bond formation via aldol condensation between glycolaldehyde (GALD) and formaldehyde.
However, this system included a substantial portion of D-threose as a byproduct. By identifying Ser166 and
Val203 as critical determinants of regioselectivity, structure-guided mutagenesis (S166R/V203S) suppressed Dthreose
formation and achieved >93 % selectivity under mild aqueous conditions. To avoid external GALD
supplementation, the engineered GaFSA was coupled with an optimized glyoxylate carboligase from E. coli
(EcGCL), enabling in situ GALD production from formaldehyde. This one-pot enzymatic cascade reached a
conversion efficiency of ~94 % from 25 mM FALD at pH 7.5 and 40 ◦C, with minimal byproducts. The reaction
proceeds entirely in water, under ambient pressure, without toxic reagents or organic solvents, requiring only
natural cofactors for EcGCL activity. This work offers a sustainable enzymatic platform for formaldehyde
detoxification and valorization, enabling selective C1-to-C3 upgrading and supporting greener chemical
manufacturing.