Protein engineering of formolase for the synthesis of 1,3-dihydroxyacetone from formaldehyde.

Formaldehyde is an attractive one-carbon building block for sustainable biomanufacturing, but efficient conversion of formaldehyde into higher-value chemicals remains challenging. Formolases, a class of engineered thiamine pyrophosphate-dependent enzymes, catalyze the condensation of formaldehyde to produce glycolaldehyde or 1,3-dihydroxyacetone (DHA), yet their catalytic performance is often insufficient for practical application. This chapter presents a workflow for engineering a benzoylformate decarboxylase (BFD)-derived formolase through a channel-modulating helix strategy. The protocol covers target identification by homologous structural motion analysis and substrate-channel mapping, construction of site-saturation mutagenesis libraries, high-throughput screening in 96-well plates under low-formaldehyde conditions (20 mM), and biochemical characterization of positive variants. Detailed procedures are provided for primer design, library construction, culture and expression in microplates, DHA detection, and kinetic evaluation. This workflow offers a practical platform for improving formolase performance and may be adapted for engineering other channel-dependent enzymes.