Engineering the Spliceosome of Saccharomyces Cerevisiae to Splice Heterologous Introns from Distant Fungi

As filamentous fungi are prolific producers of valuable compounds in the fight against human disease, there has been renewed interest in leveraging the recent advances in genomics to discover biosynthetic pathways that yield new chemical scaffolds. The explosion of genomic sequence data has revealed a wealth of uncharacterized natural product gene clusters in microbial species and it is essential to develop new tools to rapidly decode and translate this genetic information in order to discover new bioactive compounds. Fungal gene clusters contain many non-coding introns, which are difficult to manually annotate or predict in silico. If the gene cluster is active in the native producer, intron-free cDNA can be obtained, but neither silent gene clusters nor gene clusters in uncultivable species can be interrogated by this approach. Further, the native spliceosome of the model eukaryote Saccharomyces cerevisiae cannot remove introns from distant fungi, thus hampering natural product characterization efforts. Two naturally-occurring introns were used in this study: the second MATa1 intron from S. cerevisiae and the PES1 intron from Aspergillus fumigatus. Using a prototrophic selection marker as a splicing reporter, intron sequence motifs from PES1 were identified that abolish splicing in yeast. A chromosomally-integrated splicing reporter was developed for the rapid, cloning-free construction of intron variants and subsequent characterization of splicing activity via dosage-dependent copper selection. Through the mutation of specific splicing factors in S. cerevisiae, the intron sequence motifs from A. fumigatus are identified by the spliceosome and intron splicing is restored. A yeast strain capable of removing the heterologous introns in gene clusters from different fungal species will be a valuable synthetic biology tool for the discovery of new chemical compounds and potential therapeutics.