• Creating a Drug Sensitive Strain of Pichia Pastoris by Deleting Putative Multi-Drug Transport Protein Transcription Factors

      Jones, Preston Dimitri; Department of Chemistry and Physics (Augusta University, 2015-05)
      Commonly known as baker’s yeast, Saccharomyces cerevisiae is a strain of yeast that has been extensively studied genetically. In S. cerevisiae, the expression of multi-drug transport proteins (MDTPs) is found to be under the control of transcription factors, PDR1 and PDR3. Deletion of these genes in S. cerevisiae leads to decreased expression of MDTPs and decreased efficiency in drug export. Mutant strains of this yeast can be used in experiments involving the introduction of drugs into the yeast. Many experiments require a drug-protein interaction, and examining the results of this interaction is the subject of many genetic studies (1). These studies often involve the purification of the protein of interest after drug manipulation has occurred. Pichia pastoris is a better strain of yeast to use in these experiments because it grows to higher cell densities in fermentation than S. cerevisiae, providing more protein to work with. The goal of this project is to create a drug sensitive strain of P.pastoris by deletion of transcription factors that are homologous to those already characterized in S. cerevisiae. Putative MDTP transcription factors in P.pastoris have been determined via a blast search comparing the P. pastoris genome to S. cerevisiae. The results found three candidate genes, 0203, 0233, and 0322 that matched with the PDR1 and PDR3 genes in S.cerevisiae (2). We hypothesize that knocking out one or more of these genes will cause decreased expression of MDTPs in our mutant strain. Using homologous recombination and two selectable markers (ability to synthesize histidine and resistance to the toxin G418), we have successfully knocked out all 3 of these genes individually and have created two double knockout strains (0233-0322 and 0203-0233). Drug sensitivity assays in which we grew the mutant strains on plates with doxorubicin or camptothecin showed no enhancement in drug sensitivity (all strains were still able to grow when incubated with the toxin). Because we cannot measure the expression of MDTPs directly, we use this assay to indirectly relate the growth of the yeast in the presence of a drug to expression of MDTPs. The continued growth of our mutant yeast strains leads us to believe that all three genes must be deleted in a single strain to cause reduced MDTP expression. It is also possible that our deletion had an effect that was immeasurable by a growth assay.