Formaldehyde aldolases: Key enzymes in C-C bond formation and C1 assimilation

The conversion of C1 compounds such as CO₂, formic acid, formaldehyde (FALD), and methanol into longer-chain molecules depends on efficient C-C bond formation. Enzymatic strategies offer sustainable routes for such transformations, but their application remains limited by insufficient stability, specificity, and/or activity. A critical step here is the enzymatic condensation of FALD, which is promising building block due to its reactivity. This review discusses the catalytic mechanism, application, and limitation of three classes of aldolases that catalyze C-C bond formation using formaldehyde as a donor and/or acceptor (here called FALD aldolases): ThDP-dependent enzymes (Benzaldehyde lyases (BAL), benzoylformate decarboxylases (BFD), and glyoxylate carboligases (GCL)), class I aldolases (Fructose-6-phosphate aldolases (FSA) and Deoxyribose-5-phosphate aldolases (DERA)), and class II aldolases (2-keto-3-deoxy-l-rhamnonate aldolase YafU). Recent advances of engineering these enzymes addressed key bottlenecks, enhancing enzyme performance and enabling more robust pathway design. These developments were critically evaluated emphasizing both the potential and challenges of using FALD aldolases for efficient and sustainable C1 biomanufacturing, with the aim of guiding future proteins engineering campaigns.