The contribution of the transcription factors Crz1 and Rim101 to the response to alkalinization in Komagataella phaffii
In the industrially relevant yeast Komagataella phaffii, as in other yeasts, alkalinization of the medium results in fast and extensive transcriptional remodeling. This response has been useful to design novel methanol-free, regulatable platforms for heterologous protein expression. However, its molecular bases are fully unknown. Crz1 and Rim101/PacC are transcription factors known to contribute to the alkaline pH transcriptional responses in many fungi. We identified here the transcription factors Crz1 and Rim101 in K. phaffii and used CRISPR/Cas9 techniques to create single and double mutant strains. Phenotypic characterization shows that crz1 cells are strongly sensitive to Ca2+ ions, mildly sensitive to alkaline pH and hypertolerant to SDS. In contrast, rim101 cells are markedly hypersensitive to Na+ and Li+ ions, to alkaline pH and to SDS. RNA-seq transcriptomic analysis revealed that mutation of RIM101 affects 55% of genes whose expression is altered at pH 8.0, whereas lack of Crz1 alters only 11% of these. Thirty-eight genes are co-regulated by both Crz1 and Rim101. The PMC1 vacuolar Ca+2-ATPase is regulated by Crz1, whereas the ENA2 Na+-ATPase, the PHO89 and PHO84 phosphate transporters, and diverse genes involved in iron homeostasis are Rim101-dependent. These effects were confirmed by proteomic analyses after 3 h of alkalinization. Therefore, while Rim101 appears as a major player in the transcriptional response to alkalinization, nearly 40% of the alkaline pH transcriptional response in K. phaffii is controlled still unknown pathways. Overall design: Comparison of mRNA levels by RNA-Seq between Komagataella phaffii yeast cells grown  on YP-glycerol at pH 5.5 (reference) and 8.0 (alkaline pH) for wild-type GS115 strain and cell mutated for PAS_chr3_0625 (RIM101) and PAS_chr2-2_0202 (CRZ1) transcription factors. Samples were taken after 0, 15, 30 and 60 min  upon alkalinization of the medium.