A Scaffoldomics Platform for Modular In Vivo Enzyme Colocalisation and Its Application to Naringenin Biosynthesis.

Insufficient product yield remains a major bottleneck in the development of microbial cell factories for fine chemical production. In vivo enzyme colocalisation on synthetic scaffolds has emerged as a promising strategy to enhance metabolic efficiency, yet current approaches are often labour-intensive and inefficient. Here, we present the development of a 'scaffoldomics' platform, a generic synthetic framework designed to enable combinatorial scaffolding of biosynthetic pathways for the generation of diverse multi-enzyme complexes. This system enables the assembly of up to four pathway enzymes onto a protein scaffold. As a proof of concept, the platform was applied to the biosynthesis of naringenin, a key flavonoid intermediate. Integration of a chromosomally encoded naringenin biosensor in Escherichia coli allowed for real-time detection and pathway evaluation. The biosensor response curve was established and confirmed functional naringenin production. Moreover, comparative experiments demonstrated that the addition of a scaffold to docking enzymes significantly enhanced yield up to a ~19-fold, indicating a positive colocalisation effect. These results highlight the utility of the scaffoldomics platform as a powerful tool for further efficient combinatorial design, construction, and optimisation of biosynthetic pathways in synthetic biology.