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Fungal genomes carry many gene clusters seemingly capable of natural product biosynthesis, yet most clusters remain silent. This places a major constraint on the conventional approach of cloning these genes in more amenable heterologous host for the natural product biosynthesis. One way to overcome this difficulty is to activate the silent gene clusters within the context of the target fungus. Here, we successfully activated a silent polyketide biosynthetic gene cluster in Aspergillus oryzae by overexpressing a transcriptional regulator found within the cluster from a plasmid. This strategy allowed us to isolate a new polyketide product and to efficiently decipher its biosynthetic pathway. Through this exercise, we also discovered unexpected activities of the biosynthetic enzymes found in the cluster. These results indicate that our approach would be valuable for isolating novel natural products and engineering analogues of comparable, if not more potent, bioactivity.

Fungal genome sequencing has revealed many genes coding for biosynthetic enzymes, including polyketide synthases and nonribosomal peptide synthetases. However, characterizing these enzymes and identifying the compounds they synthesize remains a challenge, whether the genes are expressed in their original hosts or in more tractable heterologous hosts, such as yeast. Here, we developed a streamlined method for isolating biosynthetic genes from fungal sources and producing bioactive molecules in an engineered Saccharomyces cerevisiae host strain. We used overlap extension PCR and yeast homologous recombination to clone desired fungal polyketide synthase or a nonribosomal peptide synthetase genes (5-20 kb) into a yeast expression vector quickly and efficiently. This approach was used successfully to clone five polyketide synthases and one nonribosomal peptide synthetase, from various fungal species. Subsequent detailed chemical characterizations of the resulting natural products identified six polyketide and two nonribosomal peptide products, one of which was a new compound. Our system should facilitate investigating uncharacterized fungal biosynthetic genes, identifying novel natural products, and rationally engineering biosynthetic pathways for the production of enzyme analogues possessing modified bioactivity.