Functional analysis of Mpk1-mediated cell wall integrity signaling pathway in the thermotolerant methylotrophic yeast...

Understanding the characteristics and regulation mechanisms of cell wall integrity (CWI) in yeast is important not only for basic research but also in biotechnological applications. We found significantly different CWIs in two representative strains of the thermotolerant methylotrophic yeast Hansenula polymorpha. Compared to the A16 strain (classified as Ogataea polymorpha), the DL1-L strain (classified as Ogataea parapolymorpha) has a thinner cell wall that was found to be more fragile following long-term cultivation and more sensitive to zymolyase. To gain a deeper insight into this difference, we compared the characteristics of the Mpk1pmediated CWI signaling pathway in the two strains. While a DL1-L mutant deficient in Mpk1p (mpk1Δ) showed severe growth retardation at both normal and high growth temperatures and in the presence of cell-wall disrupting agents, the A16 mpk1Δ mutant displayed only a mild defect in cell growth. Sorbitol effect on rescuing growth retardation was different in the two mpk1Δ strains, which could partly be ascribed to subtle differences in the activation of HOG pathway. Among the cell wall disruptors evaluated, only caffeine clearly increased phosphorylation of Mpk1p in DL1-L, but not in A16. A transcriptome analysis of the DL1-L strain revealed that caffeine significantly increased the expression of a subset of cell-wall related genes in an Mpk1p-dependent manner, but not the expected Rlm1-target genes. Taken together, our data support an essential role for Mpk1p in maintaining CWI in H. polymorpha, although the requirement for Mpk1p and its regulation under diverse stress conditions varies depending on the strain background.