Cells of the methylotrophic yeast Ogataea parapolymorpha have two genes encoding low-affinity phosphate transporters: PHO87, encoding the plasma membrane transporter, and PHO91, encoding a protein, which is homologous to the Saccharomyces cerevisiae vacuolar membrane transporter. Earlier, we reported that inactivation of PHO91 in O. parapolymorpha interferes with methanol utilization due to the lack of activity of methanol oxidase encoded by the MOX gene. In this work, we showed that this defect was completely suppressed by inactivating the PHO87 gene or introducing additional copies of the MOX gene into the cell. The PHO91 gene knockout decreased the level of long-chained polyphosphates only in methanol-grown cells, but not in glucose-grown cells. This effect remained even in the strain with extra copies of MOX, which rescues the ability of the mutant to grow on methanol. In contrast, the PHO87 gene knockout changed the levels of short-chained and long-chained polyphosphates in both methanol- and glucose-grown cells. Inactivation of PHO91 did not change vanadate resistance, while inactivation of PHO87 increased this resistance. Our data suggest that in O. parapolymorpha, Pho87 and Pho91 transporters have different roles in inorganic polyphosphate metabolism and adaptation to methanol consumption.

Bach Institute of Biochemistry Federal Research Center Fundamentals of Biotechnology of the Russian Academy of Sciences Moscow Russian Federation

Federal Research Center Pushchino Scientific Center for Biological Research Institute for Biological Instrumentation Russian Academy of Sciences Pushchino Russian Federation

Federal Research Center Pushchino Scientific Center for Biological Research Skryabin Institute of Biochemistry and Physiology of Microorganisms Russian Academy of Sciences Pushchino Russian Federation

See more in PubMed

Achbergerová L, Nahálka J (2011) Polyphosphate - an ancient energy source and active metabolic regulator. Microb Cell Fact 10:63. https://doi.org/10.1186/1475-2859-10-63 PubMed DOI PMC

Agaphonov MO, Trushkina PM, Sohn JH, Choi ES, Rhee SK, Ter-Avanesyan MD (1999) Vectors for rapid selection of integrants with different plasmid copy numbers in the yeast Hansenula polymorpha DL1. Yeast 15(7):541–551. https://doi.org/10.1002/(SICI)1097-0061(199905) PubMed DOI

Albi T, Serrano A (2016) Inorganic polyphosphate in the microbial world. Emerging roles for a multifaceted biopolymer. World J Microbiol Biotechnol 32(2). https://doi.org/10.1007/s11274-015-1983-2

Andreeva N, Ryazanova L, Zvonarev A, Trilisenko L, Kulakovskaya T, Eldarov M (2018) Inorganic polyphosphate in methylotrophic yeasts. Apply Microbiol Biotechnol 102:5235–5244. https://doi.org/10.1007/s00253-018-9008-3 DOI

Andreeva N, Ledova L, Ryazanova L, Tomashevsky A, Kulakovskaya T, Eldarov M (2019) Ppn2 endopolyphosphatase overexpressed in Saccharomyces cerevisiae: Comparison with Ppn1, Ppx1, and Ddp1 polyphosphatases. Biochimie 163:101–107. https://doi.org/10.1016/j.biochi.2019.06.001 PubMed DOI

Auesukaree C, Homma T, Kaneko Y, Harashima S (2003) Transcriptional regulation of phosphate-responsive genes in low-affinity phosphate-transporter-defective mutants in Saccharomyces cerevisiae. Biochem Biophys Res Commun 306(4):843–850. https://doi.org/10.1016/s0006-291x(03)01068-4 PubMed DOI

Azevedo C, Saiardi A (2017) Eukaryotic phosphate homeostasis: the inositol pyrophosphate perspective. Trends Biochem Sci 42(3):219–231. https://doi.org/10.1016/j.tibs.2016.10.008 PubMed DOI

Bond C (2007). Freeze-drying of yeast cultures. In: Day JG, Stacey GN (eds) Cryopreservation and Freeze-Drying Protocols. Meth Mol Biol 368: 368:99–107, Humana Press. https://doi.org/10.1007/978-1-59745-362-2_6

Choi J, Rajagopa A, Xu YF, Rabinowitz JD, O’Shea EK (2017) A systematic genetic screen for genes involved in sensing inorganic phosphate availability in Saccharomyces cerevisiae. PLoS ONE 12(5):e0176085. https://doi.org/10.1371/journal.pone.0176085 ] PubMed DOI PMC

Eskes E, Deprez MA, Wilms T, Winderickx J (2018) pH homeostasis in yeast; the phosphate perspective. Curr Genet 64(1):155–161. https://doi.org/10.1007/s00294-017-0743-2 ] PubMed DOI

Farofonova V, Andreeva N, Kulakovskaya E, Karginov A, Agaphonov M, Kulakovskaya T (2023) Multiple effects of the PHO91 gene knockout in Ogataea parapolymorpha. Folia Microbiol (Praha) 68(4):587–593. https://doi.org/10.1007/s12223-023-01039-x ] PubMed DOI

Ghillebert R, Swinnen E, De Snijder P, Smets B, Winderickx J (2011) Differential roles for the low-affinity phosphate transporters Pho87 and Pho90 in Saccharomyces cerevisiae. Biochem J 434(2):243–251. https://doi.org/10.1042/BJ20101118 ] PubMed DOI

Gruzman MB, Titorenko VI, Ashin VV, Lusta KA, Trotsenko YA (1996) Multiple molecular forms of alcohol oxidase from the methylotrophic yeast Pichia methanolica. Biochemistry (Moscow) 61 (12): 1537–1544.

Gupta R, Laxman S (2021) Cycles, sources, and sinks: conceptualizing how phosphate balance modulates carbon flux using yeast metabolic networks. Elife 10:e63341. https://doi.org/10.7554/eLife.63341 PubMed DOI PMC

Hürlimann HC, Stadler-Waibel M, Werner TP, Freimoser FM (2007) Pho91 is a vacuolar phosphate transporter that regulates phosphate and polyphosphate metabolism in Saccharomyces cerevisiae. Mol Biol Cell 18 (11): 4438–4445. https://doi.org/10.1091/mbc.e07-05-0457

Karginov AV, Fokina AV, Kang HA, Kalebina TS, Sabirzyanova TA, Ter-Avanesyan MD, Agaphonov MO (2018) Dissection of differential vanadate sensitivity in two Ogataea species links protein glycosylation and phosphate transport regulation. Sci Rep 8:16428. https://doi.org/10.1038/s41598-018-34888-5 > PubMed DOI PMC

Karginov AV, Alexandrov AI, Kushnirov VV, Agaphonov MO (2021) Perturbations in the heme and siroheme biosynthesis pathways causing accumulation of fluorescent free base porphyrins and auxotrophy in Ogataea yeasts. J Fungi (Basel) 7(10):884. https://doi.org/10.3390/jof7100884 PubMed DOI

Mani S, Nachiappan V (2022) Lack of low-affinity phosphate transporter pho91 alters lipid metabolism in yeast Saccharomyces cerevisiae. Indian J Sci Technol 15, Issue(43) : 2282–2289. https://doi.org/10.17485/IJST/v15i43.1264

Mouillon JM, Persson BL (2006) New aspects on phosphate sensing and signaling in Saccharomyces cerevisiae. FEMS Yeast Res 6(2):171–176. https://doi.org/10.1111/j.1567-1364.2006.00036.x PubMed DOI

Nicolay K, Veenhuis M, Douma AC, Harder W (1987) A 31P NMR study of the internal pH of yeast peroxisomes. Arch Microbiol 147(1):37–41. https://doi.org/10.1007/BF00492902 PubMed DOI

Pinson B, Merle M, Franconi JM, Daignan-Fornier B (2004) Low affinity orthophosphate carriers regulate PHO gene expression independently of internal orthophosphate concentration in Saccharomyces cerevisiae. J Biol Chem 279(34):35273–35280. https://doi.org/10.1074/jbc.M405398200 PubMed DOI

Potapenko E, Cordeiro CD, Huang G, Storey M, Wittwer C, Dutta AK, Jessen HJ, Starai VJ, Docampo R (2018) 5-Diphosphoinositol pentakisphosphate (5-IP PubMed DOI PMC

Preston RA, Murphy RF, Jones EW (1989) Assay of vacuolar pH in yeast and identification of acidification-defective mutants. Proc Natl Acad Sci U S A 86(18):7027–7031. https://doi.org/10.1073/pnas.86.18.7027 PubMed DOI PMC

Rao NN, Gómez-García MR, Kornberg A (2009) Inorganic polyphosphate: essential for growth and survival. Annu Rev Biochem 78(1):605–647. https://doi.org/10.1146/annurev.biochem.77.083007.093039

Rubin GM (1973) The nucleotide sequence of Saccharomyces cerevisiae 5.8 S ribosomal ribonucleic acid. J Biol Chem 11:3860–3875 DOI

Rumjantsev AM, Bondareva OV, Padkina MV, Sambuk EV (2014) Effect of nitrogen source and inorganic phosphate concentration on methanol utilization and PEX genes expression in Pichia pastoris. Sci World J 2014:743615. https://doi.org/10.1155/20h14/743615 DOI

Sulter GJ, Harder W, Veenhuis M (1993) Structural and functional aspects of peroxisomal membranes in yeasts. FEMS Microbiol Rev 11(4):285–296. https://doi.org/10.1111/j.1574-6976.1993.tb00002.x

Van der Klei IJ, Yurimoto H, Sakai Y, Veenhuis M (2006) The significance of peroxisomes in methanol metabolism in methylotrophic yeast. Biochim Biophys Acta 1763(12):1453–1462. https://doi.org/10.1016/j.bbamcr.2006.07.016 PubMed DOI

Waterham HR, Keizer-Gunnink I, Goodman JM, Harder W, Veenhuis M (1990) Immunocytochemical evidence for the acidic nature of peroxisomes in methylotrophic yeasts. FEBS Lett 262(1):17–19. https://doi.org/10.1016/0014-5793(90)80142-6 PubMed DOI

Yurimoto Y, Yasuyoshi Sakai Y, Kato N (2002) Chapter 5. Methanol Metabolism. In: Gellissen, G. (Eds.) Hansenula polymorpha: Biology and Applications. Wiley‐VCH, Verlag GmbH  https://doi.org/10.1002/3527602356.ch5

Zhou Y, Yuikawa N, Nakatsuka H, Maekawa H, Harashima S, Nakanishi Y, Kaneko Y (2016) Core regulatory components of the PHO pathway are conserved in the methylotrophic yeast Hansenula polymorpha. Curr Genet 62(3):595–605. https://doi.org/10.1007/s00294-016-0565-7 PubMed DOI PMC