(228cf) Scaleable Assembly and Characterization of Fungal Natural Product Gene Clusters

Fungi have been an important source of medically relevant natural products (NPs) since the inception of biochemical engineering in the 1940s. Recent advances in DNA sequencing have revealed that the biosynthetic potential of fungal genomes is much deeper than early investigators realized. However, due to difficulties of culturing and genetically engineering many fungi, and the fact that many NP biosynthetic gene clusters (BGCs) are not expressed under laboratory conditions, progress to experimentally characterize the products of these gene clusters has been slow.

Here we describe the realization of a pipeline for the high-throughput characterization of fungal NP biosynthetic gene clusters. Our pipeline encompasses:

• bioinformatic tools for identification and prioritization of BGCs, and tools for sequence editing (e.g. intron removal) for improved expression in S. cerevisiae

• an assembly platform that combines commercial gene synthesis and yeast homologous recombination for the cost-effective construction of large (> 20 kb) DNAs
• high-throughput untargeted LC-MS based metabolomics screening of recombinant yeast cultures for the rapid detection and prioritization of potential novel compounds

With this pipeline, we successfully detected potentially novel NPs in four out of six previously unstudied fungal BGCs, and have structurally characterized a subset of the BGC-associated compounds. Our pipeline shows how high-throughput synthetic biology tools can facilitate the rapid discovery and biosynthetic production of complex chemical scaffolds of potential pharmaceutical relevance.