Regulatory mechanism of gene expression depending on themethanol concentration in the yeast used for heterologous protein
production
Hiroya YURIMOTO
Graduate School of Agriculture, Kyoto University
Research objective
Methanol-utilizing yeasts (methylotrophic yeasts) with strong methanol-induced genepromoters are widely used as hosts for heterologous protein production. The expression levelsof methanol-induced genes change in response to the methanol concentration, but not in aconcentration-dependent manner. The levels increase in the presence of 0.001–0.1% methanol,
but decrease in the presence of methanol concentrations greater than 0.1%. We defined thisphenomenon as “concentration-regulated methanol induction (CRMI)” and we are currently
elucidating its molecular mechanism. Previously, we identified Wsc family proteins(KpWsc1/KpWsc3) as cell surface sensors that participate in the methanol-sensing mechanismin the methylotrophic yeast Komagataella phaffii (Pichia pastoris), and we reported that thedeletion of both Wsc genes impairs CRMI.1) However, it is unclear how the methanol
concentration information is transmitted to transcriptional regulators. To develop aheterologous protein-producing host that maximizes the potential of methylotrophic yeasts,
we aimed to elucidate the molecular mechanisms underlying CRMI and its signal transduction
pathway.
Methods
Among the transcriptional activators involved in methanol-induced gene expression,
KpMxr1, which is a homolog of Saccharomyces cerevisiae Adr1, was suggested to bephosphorylated. Therefore, we analyzed the phosphorylation status of KpMxr1 at various
methanol concentrations. We previously found that the serine residues of KpMxr1 weredephosphorylated under methanol culture conditions, and the threonine residues werephosphorylated in a methanol-concentration-dependent manner. In this study, we performed
liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of KpMxr1 purified
from cells cultured in the presence of glucose or methanol. We generated alanine-substituted
mutants of serine and threonine residues that were predicted to be phosphorylated andanalyzed their phosphorylation state and the AOX1 expression level at various methanol
concentrations.
To elucidate the signal transduction pathway to KpMxr1, constitutively active mutants of

the kinases KpPkc1 and KpMpk1, whichfunction downstream of the Wsc family
proteins, were expressed under the controlof the Cu2+-inducible CUP1 promoter. We
investigated AOX1 expression levels andthe phosphorylation status of KpMxr1.
Results
The LC-MS/MS analysis revealed thatKpMxr1 had numerous phosphorylation

Fig. 1．Phosphorylation of KpMxr1-FLAG
sites. As most of the phosphorylatedamino acid residues were in the 1–230 amino acid region, Phos-tag sodium dodecyl sulfate1-230polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed on the strain
expressing KpMxr11-230-FLAG. We found that KpMxr1 was highly phosphorylated underglucose culture conditions and dephosphorylated under methanol culture conditions. We also
found that the phosphorylation state of KpMxr1 changed with the methanol concentration (Fig.
1). Based on the LC-MS/MS results, the putative phosphorylated serine and threonine residues
were replaced by alanine residues. We constructed strains expressing the KpMxr1 SA mutant(S110A/S111A) and TA mutant (T121A/T124A/T125A/ T128A/T131A). In these strains, theresponse to low concentrations of methanol decreased. In the TA mutant strain, the expression
peak of AOX1 shifted to a higher methanol concentration. These results revealed that strict
control of the phosphorylation of serine and threonine residues in KpMxr1 is important for
CRMI in K. phaffii.
Among the kinases in the cell wall integrity (CWI) signaling pathway, overexpression ofKpPkc1R390P, a constitutively active mutant of KpPkc1, decreased the AOX1 transcript levels.
However, overexpression of the constitutively active mutant KpMkk1S313P did not affect AOX1transcript levels. Furthermore, the phosphorylation level of KpMxr11-230-FLAG and thetranscript level of AOX1 changed owing to the difference in the induction level of KpPkc1R390P(Fig. 2).
Conclusion
Based on the results described above, the methanol concentration information fromKpWsc1/KpWac3 is transmitted through an unknown pathway independent of the MAP kinasecascade that branches out from KpPkc1 to KpMxr1, and the phosphorylation status of KpMxr1
depends on the activation level of KpPkc1. Finally, we propose a CRMI pathway from Wsc
family proteins to KpMxr1 (Fig. 3).2)

Fig. 2．Transcript level of AOX1 (A) and phosphorylation of KpMxr11-230-FLAG (B) incells expressing KpPkc1R390P

Fig. 3．Regulation of KpMxr1 phosphorylation by the CRMI pathway

References
1) Ohsawa, S., Yurimoto, H., and Sakai, Y. (2017) Novel function of Wsc proteins as amethanol-sensing machinery in the yeast Pichia pastoris. Mol. Microbiol. 104: 349363.
2) Inoue, K., Ohsawa, S., Ito, S., Yurimoto, H., and Sakai, Y. (2022) Phosphoregulationof the transcription factor Mxr1 plays a crucial role in the concentration-regulated
methanol induction in Komagataella phaffii. Mol. Microbiol. 118: 683-697.