Reconstitution of Orphan Fungal Polyketide Gene Clusters in Saccharomyces Cerevisiae

Natural products (also known as secondary metabolites) broadly refer to organic compounds formed in living organisms, but are usually taken to mean small molecule metabolites that are not required for growth and development. More than half of FDA-approved antibiotics and anticancer drugs are natural products (NPs) or their derivatives. In particular, fungi produce a wide range of bioactive compounds such as penicillin, whose discovery kicked off modern era of antibiotic discovery, and lovastatin, a cholesterol-control drug that generated billions of dollars in annual sales.
The landscape of fungal natural products has been changing since the explosion of genomic data. Many NP gene clusters can now be readily identified based on homology, but no associated NP could be identified as being produced by the organism, and often the genes were not even expressed. Those biosynthetic pathways whose secondary metabolites are unknown are defined as â??orphanâ? gene clusters. The thousands of orphan fungal gene clusters computationally identified may encode a wealth of unknown natural products that may exhibit attractive bioactivities.
To explore various NP clusters, researchers have heterologously expressed them in various model organisms, among which S. cerevisiae has emerged as a powerful host. Our project aims to explore some of the thousands of orphan fungal gene clusters using S. cerevisiae as a host to heterologous express the fungal gene clusters. We hypothesize that more natural product clusters can be explored by bypassing expression in native hosts, and instead expressing the predicted gene clusters in the heterologous host, Saccharomyces cerevisiae. We explore two orphan clusters: one in Aspergillus fumigatus, a common pathogenic fungal strain that causes disease in immune-comprised patients. The other is in Trichoderma virens, an opportunistic avirulent fungal species that protect many plants from various pathogens.
We designed and purchased codon-optimized synthetic DNA fragments and will assemble them into our yeast expression system. Once the genetic tools are constructed, we will access protein expression via western blotting and use LC/MS to determine the molecular formula of products from the pathway. NMR structural characterization will be performed subsequently. We will also determine the structure of secondary metabolic intermediates and elucidate the order of reactions to understand the function and specificity of the enzymes involved.
Our work could provide an alternative traditional natural product chemistry to access natural product and a platform for detailed enzymatic and biochemical studies on the function and specificity of enzymes involved in natural product biosynthesis.

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