Efficient exploration of terpenoid biosynthetic gene clusters in filamentous fungi

Terpenoids, critical components of human medicine, are the largest family of natural products. Fungi are an important source of terpenoids, but many of the corresponding biosynthetic gene clusters (BGCs) are silent in laboratory conditions. Strategies such as homologous activation and heterologous expression were usually used to active a single cluster, making them low efficiency. Here we developed an automated and high-throughput (auto-HTP) biofoundry workflow using Aspergillus oryzae as a chassis that enables efficient genome mining, characterization of BGCs and identification of bioactive fungal terpenoids. We simultaneously refactored 39 BGCs into 208 engineered strains, producing 185 distinct terpenoids. An anti-inflammatory screen returned the sesterterpenoid mangicol J; re-examination of our engineered strains revealed the likely biosynthetic pathway. Finally, we optimized the mevalonate pathway in A. oryzae to provide a more efficient chassis for overproduction of terpenoids. The auto-HTP biofoundry workflow together with the optimized A. oryzae chassis can accelerate the discovery and development of terpenoid natural products.