1,2,4-Butanetriol is a crucial fine chemical intermediate for energetic materials, high-value-added chemicals, and pharmaceuticals; however, its microbial biosynthesis pathway is often hindered by intermediate accumulation and imbalanced metabolic flux. To overcome these challenges, Candida tropicalis was employed as the chassis, and a synergistic strategy integrating protein scaffold-driven enzyme colocalization with key enzyme engineering was developed to optimize the pathway from xylose. Stage-specific repression of xylose dehydrogenase (XylB) confirmed the growth inhibition caused by xylonate. To alleviate substrate channeling limitations, a spatial colocalization strategy was employed by fusing the key enzymes XylB, xylonate dehydratase (XylD), and α-keto-acid decarboxylase (KdcA) with scaffold ligands and modulating the protein scaffold GBDx-SH3y-PDZz domain ratio to an optimal 1:3:1, enabling their programmable assembly into a highly efficient multienzyme complex. Subsequently, deletion of mig1 enhanced the xylose utilization efficiency, while pathway efficiency was further optimized via synergistic enzyme engineering, which included overexpressing the ligand-fused XylD and KdcA mutants. This integrated approach raised the 1,2,4-butanetriol titer to 2.1 g/L in shake flasks and 11.18 g/L in a 5 L bioreactor. This study validates the effectiveness and broad application prospects of integrating spatial colocalization of metabolic pathways with metabolic engineering in enhancing the performance of complex metabolic pathways.
Wei et al. (Tue,) studied this question.