Construction and Diversification of Natural Product Biosynthetic Gene Clusters at High Efficiency and Accuracy

Biosynthetic gene clusters (BGCs) encode the biosynthesis of natural products, which serve as the foundation for therapeutics such as antibiotics, anticancer agents, antifungals, and immunosuppressants. The vast majority of the BGCs remain uncharacterized due to lack of expression or inability to cultivate the native host, making refactoring and expression of BGCs in optimized hosts a prerequisite for genome-based drug discovery. Transformation-associated recombination (TAR) cloning and Gibson assembly are error prone due to the use of homologous recombination. Here, we present a BGC cloning and refactoring strategy based on a hierarchical Golden Gate Assembly (GGA), which enables systematic pathway engineering and mutagenesis with unprecedented accuracy and efficiency. We constructed the 23 kb actinorhodin (ACT) BGC and 23 mutant derivatives with either one of the <i>act</i> genes inactivated, within the same experiment and with 100% efficiency. Introduction of the BGCs in the ACT-nonproducer <i>Streptomyces coelicolor</i> M1152 revealed that nine genes are essential for ACT production, while inactivation of others led to significant rewiring of the biosynthetic pathway. Global Natural Products Social (GNPS) molecular networking thereby revealed a surprisingly large number of unidentified molecules, significantly expanding the chemical space associated with ACT biosynthesis. Additionally, we refactored the <i>act</i> cluster through promoter engineering and evaluated expression outcomes across multiple <i>Streptomyces</i> strains. Together, our work establishes a GGA-based platform for BGC construction, refactoring, and functional dissection, accelerating synthetic-biology-driven natural product discovery.