(12b) Synthetic Biology Enabled Refactoring of Cryptic Fungal Gene Clusters for Natural Product Discovery

In recent years, the exponential increase in genome sequence data has revealed a myriad of secondary metabolite gene clusters across all domains of life for which the cognate product is unknown.  The presence of these so-called “cryptic” gene clusters suggests that many organisms possess the potential to produce a far greater diversity of natural products than have ever been detected in a laboratory setting.  Many methods have been developed to unlock this potential in the native host, including screening of varied culture conditions, co-culture with different competitive species, over-expression of putative pathway-specific regulators, and addition of epigenetic factors.  However, the results of these approaches vary on a case-by-case basis, and are difficult or even impossible to predict a priori.  As a result, it is our goal to develop a robust method for natural product discovery based on complete refactoring of the target cryptic pathway and heterologous expression in a well-characterized, highly tractable host.  Our current focus is on the refactoring of fungal polyketide synthase (PKS) clusters and their expression in Saccharomyces cerevisiae.  One such cluster has been identified from Penicillium marneffei, a pathogenic fungus containing 25 PKS genes but only one known polyketide metabolite.  The target cluster contains two PKS genes with ~40 % amino acid identity to those of multiple known resorcylic acid lactone (RAL) gene clusters.  RALs are a class of macrocyclic compounds that possess a wide range of bioactivities, from antibacterials and antifungals to kinase inhibitors.  While multiple fungal RALs have previously been identified, the three tailoring genes found in the target cluster do not match those of any other known cluster, suggesting that the target cluster encodes a novel member of the RAL family.  Using the DNA assembler method, we have rapidly removed the introns from each gene in the cluster and constructed an expression vector with each gene placed under a characterized constitutive promoter.  We have also constructed a set of control vectors with individual genes disrupted.  Transcription of the refactored heterologous genes in S. cerevisiae has been confirmed by qRT-PCR.  A novel product was detected by HPLC-MS/MS and its structural characterization is currently in progress.