Project description:The purpose of this experiment was to identify the genes bound by Ndt80 in P. pastoris when constitutively expressed. Ndt80 was tagged with c-myc, the native Ndt80 promoter was replaced with the E. gossypii Tef1 promoter and the protein was immunoprecipitated with a c-myc antibody. Cells were grown unti log-phase in YPD. Each experiment was repeated twice and sequenced on an Illumina HiSeq 2500.
Project description:The purpose of this experiment was to identify the genes bound by Ndt80 in S. cerevisiae when constitutively expressed, for the native Ndt80 as well as a heterologous Ndt80 from Pichia pastoris. For the ChIPs on the native Ndt80s, Ndt80 was tagged with c-myc; for the heterologous ChIP P. pastoris Ndt80 was integrated into the S. cerevisiae genome and tagged with c-myc. In both cases, the native Ndt80 promoter was also replaced with an A. gossypii Gal1 promoter and the protein was immunoprecipitated with a c-myc antibody. Cells were grown overnight in SRaffinose until log-phase, then in SRaffinose + 1.7% Galactose for 5 hours. Each experiment was repeated twice and sequenced on an Illumina HiSeq 4000.
Project description:The purpose of this experiment was to identify the genes bound by Ndt80 in K. lactis when constitutively expressed. Ndt80 was tagged with c-myc, the native Ndt80 promoter was replaced with the K. lactis Gal1 promoter and the protein was immunoprecipitated with a c-myc antibody. Cells were grown overnight in SRaffinose until log-phase, then in SRaffinose + 1.7% Galactose for 5 hours. Each experiment was repeated twice and sequenced on an Illumina HiSeq 4000.
Project description:The purpose of this experiment was to identify the genes bound by the two Ndt80 paralogs in S. stipitis when constitutively expressed. Both paralogs were tagged with c-myc, the native Ndt80 promoter was replaced with the E. gossypii Tef1 promoter and the protein was immunoprecipitated with a c-myc antibody. Cells were grown unti log-phase in YPD. Each experiment was repeated twice and sequenced on an Illumina HiSeq 2500.
Project description:We investigated gene expression during the bench-scale batch fermentation phase, glycerol feed phase, glycerol-methanol mixture feed (GM) phase, and at different time points following methanol induction using RNA-Seq. We report that the addition of the GM phase may help to alleviate the adverse effects of methanol addition (alone) on P. pastoris cells. Secondly, enhanced upregulation of the mitogen-activated protein kinase (MAPK) signaling pathway was observed in P. pastoris following methanol induction. The MAPK signaling pathway may be related to P. pastoris cell growth, and may regulate the AOX1 promoter via regulatory factors activated by methanol-mediated stimulation. Thirdly, the unfolded protein response (UPR) and ER-associated degradation (ERAD) pathways were not significantly upregulated during the methanol induction period. These results imply that the presence of unfolded or misfolded phytase protein did not represent a serious problem in our study. Finally, the upregulation of the autophagy pathway during the methanol induction phase may be related to the degradation of damaged peroxisomes but not to the production of phytase. This work describes the metabolic characteristics of P. pastoris during heterologous protein production under high-cell-density fed-batch cultivation. We believe that the results of this study will aid further in-depth studies of P. pastoris protein expression, regulation, and secretory mechanisms.