Activating cryptic biosynthetic gene cluster through a CRISPR–Cas12a-mediated direct cloning approach

Abstract

Direct cloning of biosynthetic gene clusters (BGCs) from microbial genomes facilitates natural product-based drug discovery. Here, by combining Cas12a and the advanced features of bacterial artificial chromosome library construction, we developed a fast yet efficient in vitro platform for directly capturing large BGCs, named CAT-FISHING (CRISPR/Cas12a-mediated fast direct biosynthetic gene cluster cloning). As demonstrations, several large BGCs from different actinomycetal genomic DNA samples were efficiently captured by CAT-FISHING, the largest of which was 145 kb with 75% GC content. Furthermore, the directly cloned, 110 kb long, cryptic polyketide encoding BGC from Micromonospora sp. 181 was then heterologously expressed in a Streptomyces chassis. It turned out to be a new macrolactam compound, marinolactam A, which showed promising anticancer activity. Our results indicate that CAT-FISHING is a powerful method for complicated BGC cloning, and we believe that it would be an important asset to the entire community of natural product-based drug discovery.

Taxonomic Positions and Secondary Metabolite-Biosynthetic Gene Clusters of Akazaoxime- and Levantilide-Producers

“…T1pks-5 encoded five PKSs. These PKSs showed high similarities to those in the marinolactam-BGC ( mrl ) [ 33 ]. Their domain organization was identical to that of mrl except for the presence of a DH domain in the first module of MrlB, which is absent in that of TPA0907_35890.…”

Training old dogs to do new tricks: A general multi-pronged activation approach for natural product discovery in Actinomycetes

“…In the current landscape of increasingly accessible, rapidly improving automation 39 and genetic tools, our approach provides the blueprint for a streamlined, accelerated, and scalable strategy to interrogate cryptic BGCs in native strain collections, circumventing the time-consuming traditional requirements of precise engineering, refactoring, and assembly. 14,15,40,41 Moving forward, we envision that such a strategy together with high throughput data analytics, will serve as key enabling technologies to drive actionable insights enabling the utilization of Nature's full chemical repertoire.…”

Assigning the stereochemical structures of aurantinin A and B with the assistance of biosynthetic investigations

“…Along with the rapidly increasing knowledge of natural product biosynthesis, several common guidelines about the links between catalytic enzymes and natural product structures have been compiled and employed in structural elucidation and revision. [29][30][31][32] For example, by virtue of comprehensive mechanistic investigation of PKSs, the stereochemistry of polyketides can be elicited by analyses on KR and DH domains, which was applied in the absolute configuration assignment of neaumycin B, marinolactam A, gargantulides, [33][34][35] and in structural revision of azalomycins. 36 In addition, the empirical rule associated with the stereoselective biosynthesis logic of fungal highly reducing PKSs was established recently, which aided in the structural revision of prothermolide as well as the stereochemical assignment of cubensic acid, sporminarin A, malaysic acid, and roselipin 1A.…”