Protein Scaffold-Driven Enzyme Colocalization Combined with Glucose Derepression in Candida tropicalis Enables High-Level 1,2,4-Butanetriol Production.

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 [GBD]x-[SH3]y-[PDZ]z 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.