The transcriptional response to environmental alkalization involves both Komagataella phaffii Pho4 transcription...

Pho4 is a fungal transcription factor essential for the response to phosphate (Pi) starvation in many fungi and is usually encoded by a single gene. However, we recently reported that the methylotrophic yeast Komagataella phaffii encodes two functional Pho4 proteins, named Pho4(A) and Pho4(B), and that only Pho4(B) was required for the transcriptional response to Pi limitation. Pho4 is also important in many yeasts for adaptation to an alkaline environment. We evaluate here the participation of Pho4(A) and Pho4(B) in the transcriptional response to alkaline pH.

We show that the expression of nearly one-hundred genes is altered in a pho4(A) pho(B) mutant. In contrast to Pi starvation, Pho4(A) becomes important under high pH stress for the short-time expression of genes required for Pi homeostasis, such as PHO89, VTC1, VTC2, VTC4 and PHO4(B), suggesting that both transcription factors respond to different signals. Integration of the transcriptional data with that obtained from crz1 or rim101 mutants shows that nearly 80 genes are regulated by at least two of these transcription factors. Thus, we show that induction of PHO89 by Pi starvation or high pH is fully abolished in both double pho4(A) pho4(B) and single rim101 mutants, and that mutation of CRZ1 and RIM101 partially eliminates the sensitivity to high pH of the pho4(A) pho4(B) strain, revealing unexpected genetic interactions between these mutations. Finally, we identify an alkali-sensitive region in the PHO89 promoter and demonstrate that its duplication further increases the promoter's response to alkalinization.

The transcription factor Pho4(A) is not required for the transcriptional response to Pi starvation, but it contributes to the response to alkalinization. Gene expression remodeling upon alkalinization in K. phaffii is controlled by diverse signaling pathways, including Pho4, Crz1 and Rim101 as well as other components still to uncover. Detailed knowledge of the signaling mechanisms triggered upon alkalinization and promoter mapping of the genes responsive to high pH will be useful tools for the generation of alkaline pH-regulated hybrid synthetic promoters in K. phaffii with improved features.