Mass Spectrometric Screening for Improving Enzymatic Conversion of Formaldehyde into C2 and C3 Products

One-carbon biomanufacturing offers a sustainable route for producing value-added chemicals. Glycolaldehyde synthase (GALS), an engineered enzyme from <i>Pseudomonas putida</i>, catalyzes a key step by condensing formaldehyde (FALD) into carbohydrate molecules, such as glycolaldehyde (GALD) and dihydroxyacetone (DHA). However, its industrial application is limited by low catalytic efficiency and lack of high-throughput screening methods. Here, we developed a mass spectrometry (MS)-based assay for simultaneous detection and quantification of GALD and DHA products from whole-cell FALD biotransformation by GALS-expressing <i>Escherichia coli</i>. Integrating this MS assay with a robotic biofoundry, we created and screened site-directed mutagenesis libraries targeting seven key residues of GALS, achieving a throughput of ∼10 s per sample. Several improved mutants were successfully isolated, including one with a 3.7-fold increase in <i>k</i>_cat for GALD production and another with a 5-fold reduction in <i>K</i>_m for DHA production, compared to the wild type GALS. Molecular dynamics simulations were applied to understand the mutational impact on substrate binding and product specificity. This high-throughput workflow may be extended to engineer other enzymes for one-carbon feedstock utilization.