Whole-cell catalytic production of ethylene glycol from C1 compounds using engineered glycolaldehyde synthase

Abstract

Glycolaldehyde synthase (GALS), a ThDP-dependent aldolase, is a promising catalyst for C–C bond formation. However, its practical application is limited by its low substrate affinity and catalytic efficiency. Here, we used a semi-rational design approach based on tunnel engineering to enhance the catalytic efficiency of GALS. Compared with the GALS, variant GALS-M3 exhibited a significant 15.97-fold increase in catalytic efficiency, reaching 153.3 M⁻¹ s⁻¹, while preserving high product selectivity. Molecular dynamics simulations demonstrated that the mutation improved the tunnel state, thereby increasing substrate affinity. Recombinant E. coli cells expressing GALS-M3 and lactaldehyde reductase fuco could produce 0.78 g L⁻¹ (12.57 mM) ethylene glycol from formaldehyde, with a bioconversion efficiency of 62.83%. Our research methods underscore the value of targeting inactive center sites for improving the catalytic efficiency of GALS and its homologous enzymes. The application of GALS variants in the green biosynthesis of ethylene glycol from C1 compounds was also demonstrated.