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.

The e cient utilization of both glucose and xylose, the two most abundant sugars in biomass hydrolysates, is one of the main objectives of biofermentation with lignocellulosic materials. The utilization of xylose is commonly inhibited by glucose, which is known as glucose catabolite repression (GCR). Here we report a GCR-based dynamic control (GCR-DC) strategy aiming at a better co-utilization of glucose and xylose, by decoupling the cell growth and biosynthesis of ribo avin as a product. Using the thermophilic strain Geobacillus thermoglucosidasius DSM2542 as a host, we constructed extra ribo avin biosynthetic pathways that were activated by xylose but not glucose. The engineered strains showed a two-stage fermentation process. In the rst stage, glucose was preferentially used for cell growth and no production of ribo avin was observed, while in the second stage where glucose was nearly depleted, xylose was effectively utilized for ribo avin biosynthesis. Using the corn cob hydrolysate as a carbon source, the optimized ribo avin yields of strains DSM2542-DCall-MSS (full pathway dynamic control strategy) and DSM2542-DCrib (single module dynamic control strategy) were 5.26 and 2.26 folds higher than that of the control strain DSM2542 Rib-Gtg constitutively producing ribo avin, respectively. This GCR-DC strategy should also be applicable to the construction of cell factories that can e ciently use natural carbon sources with multiple sugar components for the production of high-value chemicals in future.