Signaling pathways contributing to the transcriptional response to alkalinization in Komagataella phaffii: role of the transcription factors Crz1 and Rim101

Promoters able to respond to moderate alkalinization in the industrially relevant yeast Komagataella phaffii have been proposed as a tool for the design of novel methanol-free platforms for heterologous protein expression. However, the molecular bases of such response are unknown. We identify here the transcription factors Crz1 and Rim101 in K. phaffii and use CRISPR/Cas9 techniques to create single and double mutant strains. While crz1 cells are strongly sensitive to Ca²⁺ ions, mildly sensitive to alkaline pH and hypertolerant to SDS, rim101 cells are markedly hypersensitive to Na⁺ and Li⁺ ions, to alkaline pH and to SDS. RNA-seq analysis revealed that mutation of RIM101 affects 55% of alkaline pH-responsive genes, whereas lack of Crz1 alters only 11% of these. Thirty-eight genes are co-regulated by both Crz1 and Rim101. The PMC1 vacuolar Ca²⁺-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, Rim101 appears as a major player in the transcriptional response to alkalinization and, together with Crz1, they account for nearly 60% of the alkaline pH transcriptional response in K. phaffii.