01whole.pdf (3.13 MB)
Exploring natural yeast diversity for high protein secretion
thesisposted on 2022-03-28, 23:01 authored by John Timmins
Ethanol is seen as a sustainable biofuel, reducing reliance on non-sustainable fossil fuels. Cellulose is an abundant component in agricultural waste and is composed of glucose, linked by β-1,4-glycosidic bonds. If this waste biomass could be utilised as a potential source of glucose for fermentation to ethanol, it would lower production costs. However, cellulose is recalcitrant to enzymatic hydrolysis, requiring high enzyme levels for efficient sugar liberation. In this project, the natural variation of divergent Saccharomyces cerevisiae isolates was utilised to identify high heterologous cellobiohydrolase secreting strains. In preparation for quantitative trait loci identification, 18 haploid S. cerevisiae strains were generated from these isolates. Expression cassettes were constructed to allow the single copy integration and expression of two closely related cellobiohydrolase genes from Rasamsonia emersonii (Te) and Trichoderma reesei (Tr), and the ability of each of the 18 haploid strains to secrete the two cellobiohydrolases was evaluated. There were distinct secretion phenotypes between different strains secreting the same CBH1, with up to a 5-fold difference between the lowest and highest secretors. These high secretion phenotypes resulted in the identification of different strains efficient at secreting either the Te or Tr CBH1. Strains with superior secretion characteristics were combined with reference strains, and representative high secreting progeny were selected for downstream beneficial allele identification.