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Engineering expanded spliceosome function in Saccharomyces cerevisiae


Nature has long been a source of chemical diversity and its untapped potential is a major resource for solving our medical and energy problems. In particular, filamentous fungi have been prolific producers of medicinal compounds in the fight against human disease. Therefore, there has been interest in leveraging the advances in genomics to discover new fungal biosynthetic pathways that yield novel bioactive compounds. Heterologous expression of biosynthetic genes in model organisms will be increasingly necessary for high-throughput exploration of this genomic sequence space. Unfortunately, fungal genes contain many non-coding introns, which are difficult to manually annotate or predict in silico. Additionally, it is not possible to obtain intron-free cDNA from uncultivable species or transcriptionally silent gene clusters. This intron problem magnifies as the number of genomes increases and it risks derailing heterologous expression of this new genetic data. Unfortunately, the native spliceosome of the commonly used model eukaryote Saccharomyces cerevisiae cannot remove introns from distant fungi.

In this thesis, I will describe my efforts to engineer S. cerevisiae with expanded spliceosome functionality. I identified two failure modes that prevent splicing of an intron from Aspergillus fumigatus. This led to the generation of a chimeric yeast-fungal BranchBinding Protein that has enhanced specificity for an intron containing a fungal branchpoint site. Expression of this mutant protein enabled a 2-fold improvement in splicing of an intron with a suboptimal branchpoint site. Additionally, we identified multiple synergistic splicing factor mutations with mBBP, YHC1-D36A and downregulation of IFH1, that enabled a 1.6-fold improvement of splicing of the A. fumigatus intron. Additional studies modifying the U2 small nuclear RNA as well splicing proofreaders PRP5, PRP16, and PRP28 will be described, highlighting the drawbacks of these approaches. This study is the first demonstration of improved splicing of an Aspergillus intron through spliceosome engineering in S. cerevisiae. Using the tools, methodologies, and yeast strains provided by this work, the spliceosome can be engineered with new function, broadening the scope of how synthetic biology will be used to enhance heterologous expression in diverse research fields, such as in the elucidation of the splicing code and in natural products discovery.

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