Direct Pathway Synthesis and Editing (DiPaSE): A One-Pot DNA Assembly Method for Accurate and Efficient Refactoring of High-GC Biosynthetic Gene Clusters

Natural products (NPs) produced by actinobacteria, particularly <i>Streptomyces</i> species, represent a rich source of bioactive compounds and have yielded many clinically important compounds. Actinobacterial genomes are characterized by high GC content and typically harbor 20-40 biosynthetic gene clusters (BGCs) per genome, which encode diverse NPs such as polyketides, peptides, and glycosides. CRISPR/Cas-based genome editing has emerged as a promising tool to activate silent BGCs and engineer NP biosynthesis. However, the efficiency of multiplex editing drastically decreases as the number of targeted sites increases. Here, we report a novel one-pot DNA assembly method, termed direct pathway synthesis and editing (DiPaSE), for the efficient synthesis and multiplex editing of long, high-GC BGCs. DiPaSE accurately assembles multiple high-GC DNA fragments up to 60 kb and enables simultaneous deletions and insertions within a target BGC without compromising the assembly efficiency. Using this approach, we identified functions of previously uncharacterized genes in the aureothin BGC and significantly enhanced the titer of the corresponding NP. The workflow employs conventional polymerase chain reaction, type IIP restriction enzymes, commercially available DNA assembly reagents, and <i>Escherichia coli</i>, providing a simple, cost-effective, and broadly applicable platform for genome mining, BGC refactoring, and rational design of artificial biosynthetic pathways.