Investigating genetic code change and killer plasmids of Saccharomycopsis yeasts

Budding yeasts represent a broad genetic diversity spanning 400 million years. They have undergone the only known case of eukaryotic nuclear genome sense-to-sense genetic code change. Despite their diverse biology, and open questions, very few species are investigated at the molecular and genomic level. In this thesis, I outline our efforts to understand the genetic code change undergone by a group of species within the Saccharomycopsis genus. In chapter 2, I outline a novel CRISPR editing method I developed in S. capsularis to perform reverse genetics; I use bioinformatics and proteomics to determine if there are rare phenotypic states in which these yeasts may switch their genetic code, which I could not detect. I present the argument that the ancestral tRNA-Leu(CAG) gene of Saccharomycopsis species, which inexplicably exists after millions of years despite not being used, seemingly cannot be used during translation and must have a non-translational role. In chapter 3, I elaborate my efforts to sequence cryptic extrachromosomal elements of these obscure yeast species, which have only been described electrophoretically thus far. These elements, unique to yeast, are associated with killer toxins that are anticodon nucleases and which have been implicated in driving the unprecedented genetic code change we observe only in yeasts. In chapter 4, I list my contributions to several WGS projects, wherein long read data was used to generate highly contiguous genome assemblies, often the representative genome of an understudied species outside the Saccharomycopsidaceae. I briefly discuss the benefits of dual-assembly approaches, combining long and short reads, to get extremely high-quality genome assemblies. In chapter 5, the discussion, I conclude with remarks of the major new methods we have enabled in Saccharomycopsis yeasts (e.g. use of CRISPR, genome assemblies, etc.), and the diversity of the extrachromosomal cytoplasmic elements that may still be at large.