Enzyme and cofactor engineering to increase d-xylonate dehydratase activity for improved d-1,2,4-butanetriol production from d-xylose.

d-1,2,4-Butanetriol (BTO), a C4 platform compound, is widely used in fields such as military and pharmaceuticals. Biosynthesis of d-1,2,4-BTO from lignocellulose-derived d-xylose presents a promising production route. However, the low catalytic activity of d-xylonate dehydratase leading to the accumulation of d-xylonic acid remains a key bottleneck for the efficient production of d-1,2,4-BTO. In this study, we aimed to enhance the catalytic activity of d-xylonate dehydratase through an integrated enzyme and cofactor engineering approach. Firstly, we evolved the d-xylonate dehydratase YjhG by using both random mutagenesis and site-directed saturation mutagenesis. Among the generated variants, YjhG showed an 1.82-fold increase in d-xylonic acid consumption compared to the wild-type enzyme. When introduced into the producing strain, this variant increased d-1,2,4-BTO production by 1.34-fold compared to the original strain. Further enhancement was achieved by modifying the iron-sulfur [Fe-S] cluster synthesis system, which was critical for d-xylonate dehydratase activity. We systematically evaluated three [Fe-S] assembly systems, including SUF (encoded by ), ISC (encoded by ), and CSD (encoded by ). Comparative analysis revealed that the overexpression of SUF system conferred the highest catalytic efficiency of YjhG. The recombinant strain of BT-YjhG-SUF produced 10.36 g/L of d-1,2,4-BTO from d-xylose, achieving a molar yield of 73.6 %, which was 1.88-fold that of the original strain. This study provided a robust foundation for high-efficiency d-1,2,4-BTO production through enzyme and cofactor engineering.