Connector-enabled integration of Golden Gate Assembly and yeast recombination for streamlined multigene pathway...

Rewiring the metabolic flux for efficient microbial conversion requires robust, scalable gene assembly. However, conventional gene assembly approaches are labor-intensive, highly experience-dependent, and require extensive expertise to ensure reproducibility and efficiency. Even with advanced automation platforms such as biofoundries, assembling gene arrays with multiple transcriptional units (TUs) remains challenging. In this study, we present Efficient Modular Gene Assembly (EffiModular), an integrated in vitro and in vivo gene assembly platform compatible with automated workflows. EffiModular enables the assembly of up to eight TUs with 80% efficiency in a single transformation. Integrated into a biofoundry workflow, it enabled the construction of 120 distinct yeast strains with varying levels of expression of the β-carotene biosynthesis genes within 3 days. Compared with conventional approaches, it significantly reduces procedural complexity, minimizes reliance on operator expertise, and accelerates workflow timelines. These features establish EffiModular as a next-generation gene assembly platform for scalable, reproducible gene assembly in biofoundry-based genetic engineering.