Enzyme engineering for optimizing biosynthesis of 2,4-dihydroxybutyric acid via the synthetic threose-dependent...

Ethylene glycol is a potential feedstock for next-generation biorefineries, as it can be derived from both plastic waste and carbon dioxide. The synthetic D-threose-dependent glycolaldehyde assimilation (STEGA) pathway, which is orthogonal to central metabolism of Escherichia coli, was recently constructed for the biosynthesis of the platform chemical 2,4-dihydroxybutyric acid (DHB) from ethylene glycol (EG). However, the performance of this pathway was limited by the low catalytic efficiency of key enzymes. Therefore, in the present study, structure-guided semi-rational engineering was employed to improve the activities of D-threose aldolase and D-threose dehydrogenase, originally provided by the fructose-6-phosphate aldolase L107Y:A129G mutant (Ec.FsaA^TA) from E. coli and the promiscuous D-threo-1-aldose dehydrogenase Pc.TadH from Paraburkholderia caryophylli, respectively. The substrate specificity of Ec.FsaA^TA was improved by replacing Arg134 by either isoleucine, methionine or valine which effectively eliminated activity of the mutant enzyme toward phosphorylated substrates while retaining homo-aldol condensation activity toward glycolaldehyde for D-threose formation. In-vivo implementation of the improved aldolase within the STEGA pathway reduced off-target glycolaldehyde flux, as demonstrated by ¹³C-carbon tracing experiments. Furthermore, the engineered Pc.TadH A24G:F58L double mutant exhibited an 11-fold increase in specificity constant (k_cat/K_m) on D-threose compared to the wild-type enzyme. As a consequence of these improvements, co-expression of both engineered enzymes in the STEGA pathway significantly enhanced EG-to-DHB bioconversion, achieving a 68% increase in final DHB titer (5.2 ± 0.11 mM) and a 23% improvement in carbon yield (0.16 ± 0.003 Cmol/Cmol).