Exploiting the natural diversity of the yeast Scheffersomyces stipitis for improved second generation biofuel production

The impacts of global warming necessitate an urgent transition away from fossil fuels, with biorefineries playing a crucial role via their conversion of biomass into renewable fuels, power, and valuable chemicals. Biofuels are particularly essential for decarbonizing the transport sector, one of the major contributors to greenhouse gas emissions. Second-generation biofuels, such as bioethanol produced from lignocellulosic biomass avoid using edible feedstocks but require pre-treatment to release fermentable hexose and pentose sugars. This process creates inhibitory conditions that hinder the growth of fermenting microorganisms, and thus reduces potential ethanol yields. The yeast Scheffersomyces stipitis is a promising microorganism to use for second generation bioethanol production due to its natural ability to ferment pentose and hexose sugars. However, industrial applications of S. stipitis have been limited by its low ethanol tolerance and susceptibility to pre-treatment inhibitors. Additionally, S. stipitis primarily uses non-homologous end joining to repair DNA damage. This complicates the ability to use CRISPR-Cas9 for precise genetic manipulation and strain engineering. S. stipitis demonstrates significant genomic plasticity, facilitating its adaptation to harsh environmental conditions. Therefore, this project investigated this potential by screening various natural isolates for unique phenotypes and their resistance to industrially relevant conditions. Natural isolate Y-11543 was identified to exhibit a novel filamentous phenotype that enhanced its resistance via biofilm formation. Whole genome sequencing of Y-11543 enabled comparisons to the reference genome and highlighted repetitive DNA sequences as significant drivers of genomic plasticity between the strains. Variations in Tps5-like retrotransposons altered the centromere structure of Y-11543, which was hypothesized to affect kinetochore formation. This resulted in abnormal chromosome segregation that likely contributed to the filamentous phenotype of Y-11543. Temporal control of Cas9 to increase homology directed repair via the CRISPR-LINEAR system enabled precise genetic manipulation in S. stipitis. Modification of CRISPR-LINEAR via the incorporation of an antibiotic selection marker and construction of plasmids by Golden Gate assembly facilitated this work. A nonsense mutation in GCN4 was identified as a potential genetic driver behind the filamentous phenotype of Y-11543. Gcn4 was shown to induce pseudohyphae morphogenesis in response to amino acid starvation via the GCN response. However, use of CRISPRLINEAR to fix the nonsense mutation in Y-11543 did not resolve its filamentous phenotype. This study highlighted the benefits of genomic plasticity in S. stipitis, leading to natural isolates with unique phenotypes and enhanced stress resistance. Several mechanisms were identified to contribute to the filamentous phenotype of Y-11543. However, further research is required to elucidate the mechanism(s) responsible, and to determine how this phenotype affects the fermentation rate and ethanol yields of Y-11543.