High-throughput engineering of glycolaldehyde synthase for enhanced formaldehyde conversion to C2 and C3 products via mass spectrometry-based screening.

One-carbon (C1) biomanufacturing offers a sustainable route for producing value-added chemicals. Glycolaldehyde synthase (GALS), a thiamine pyrophosphate (TPP)-dependent enzyme engineered from benzoylformate decarboxylase of Pseudomonas putida, catalyzes the condensation of formaldehyde (FALD) to yield the C2 and C3 platform chemicals glycolaldehyde (GALD) and dihydroxyacetone (DHA). However, its industrial application is constrained by low catalytic efficiency and the lack of high-throughput screening (HTS) methods. This chapter presents a comprehensive HTS workflow for engineering GALS variants. We first detail the construction of an optimized Escherichia coli chassis (BL21(DE3)-Δ6) via CRISPR/Cas9-mediated deletion of six endogenous reductase genes to minimize competing FALD consumption. Next, we present the development of a mass spectrometry (MS)-based assay using 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) derivatization coupled with RapidFire-MS/MS for simultaneous quantification of GALD and DHA. Integrating this MS assay with a robotic biofoundry enables automated screening of site-saturation mutagenesis (SSM) libraries at a throughput of ∼10 s per sample. Finally, we establish validation protocols for candidate variants, including enzyme purification and kinetic characterization using microplate-based colorimetric assays. The established methodology is readily adaptable to the engineering of diverse aldehyde-producing TPP-dependent enzymes.