Applied and basic aspects of sulfite metabolism in Saccharomyces cerevisiae Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/p2676x55m

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  • In an effort to understand the basis for sulfite detoxification in S. cerevisiae, the functions of two genes were analyzed. SSU1, which encodes a plasma membrane protein, was found to be required for efficient sulfite efflux. FZFl-4, a dominant allele of a transcriptional activator of SSUl, was also found to be involved in efficient sulfite efflux. Analysis of an SSUl promoter-lacZ fusion showed that FZFl-4 conferred sulfite resistance through hyperactivation of SSUl. Efflux assays in cells expressing multicopy SSUl or FZFl-4 suggested that Ssulp specifically mediates efflux of the free form of sulfite. Sulfite resistance, mediated by either FZFl-4 or multicopy SSUl, was found to be a useful marker for selecting transformants of industrial and laboratory strains of S. cerevisiae. FZFl-4 was found to be more efficient than multicopy SSUl, and in the case of the laboratory strains, was found to be about half as efficient a selectable marker as URA3. Sulfite transport was studied to clarify the mechanism of sulfite uptake in S. cerevisiae. The kinetics of uptake were saturable, indicating a carrier-mediated process. Uptake was significantly reduced in cells pretreated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) or 2,4-dinitrophenol (DNP), both of which dissipate proton gradients. Extracellular alkalization was observed during sulfite uptake. These findings suggest that an anionic form of sulfite, HSO₃, is taken up by carrier-mediated proton symport. As an alternative to costly disposal of spent cherry brine, a sulfite-containing waste stream generated during maraschino cherry processing, brine was tested as a substrate for ethanol production by S. cerevisiae. Initially, the toxic level of sulfite in brine was reduced by raising brine pH to 8.5 with Ca(OH)₂ to precipitate calcium sulfite. Because the alkalization was found to result in a 10-fold reduction of phosphorus, brine was subsequently titrated with phosphoric acid to pH 6.0 prior to inoculation with S. cerevisiae. All strains of S. cerevisiae tested were able to efficiently ferment all lots of Ca(OH)₂-treated and phosphorus-enriched brine.
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