The effects of TORC signal interference on lipogenesis in the oleaginous yeast Trichosporon oleaginosus
Tóm tắt
Oleaginous organisms are a promising, renewable source of single cell oil. Lipid accumulation is mainly induced by limitation of nutrients such as nitrogen, phosphorus or sulfur. The oleaginous yeast Trichosporon oleaginosus accumulates up to 70% w/w lipid under nitrogen stress, while cultivation in non-limiting media only yields 9% w/w lipid. Uncoupling growth from lipid accumulation is key for the industrial process applicability of oleaginous yeasts. This study evaluates the effects of rapamycin on TOR specific signaling pathways associated with lipogenesis in Trichosporon oleaginosus for the first time. Supplementation of rapamycin to nutrient rich cultivation medium led to an increase in lipid yield of up to 38% g/L. This effect plateaued at 40 μM rapamycin. Interestingly, the fatty acid spectrum resembled that observed with cultivation under nitrogen limitation. Significant changes in growth characteristics included a 19% increase in maximum cell density and a 12% higher maximum growth rate. T. oleaginosus only has one Tor gene much like the oleaginous yeast Rhodosporidium toruloides. Consequently, we analyzed the effect of rapamycin on T. oleaginosus specific TORC signaling using bioinformatic methodologies. We confirm, that target of rapamycin complex 1 (TORC1) is involved in control of lipid production and cell proliferation in T. oleaginosus and present a homology based signaling network. Signaling of lipid induction by TORC1 and response to carbon depletion to this complex appear to be conserved, whereas response to nitrogen limitation and autophagy are not. This work serves as a basis for further investigation regarding the control and induction of lipid accumulation in oil yeasts.
Tài liệu tham khảo
Lu C, Napier JA, Clemente TE, Cahoon EB. New frontiers in oilseed biotechnology: meeting the global demand for vegetable oils for food, feed, biofuel, and industrial applications. Curr Opin Biotechnol. 2011;22:252–9.
Pfleger BF, Gossing M, Nielsen J. Metabolic engineering strategies for microbial synthesis of oleochemicals. Metab Eng. 2015;29:1–11.
Fritsche UR, Sims REH, Monti A. Direct and indirect land-use competition issues for energy crops and their sustainable production - an overview. Biofuels Bioprod Biorefin. 2010;4:692–704. John Wiley & Sons, Ltd.
Papanikolaou S, Aggelis G. Lipids of oleaginous yeasts. Part I: Biochemistry of single cell oil production. Eur J Lipid Sci Technol. 2011;113:1031–51.
Ageitos JM, Vallejo JA, Veiga-Crespo P, Villa TG. Oily yeasts as oleaginous cell factories. Appl Microbiol Biotechnol. 2011;90:1219–27.
de Virgilio C, Loewith R. The TOR signalling network from yeast to man. Int J Biochem Cell Biol. 2006;38:1476–81.
Aggarwal D, Fernandez ML, Soliman GA. Rapamycin, an mTOR inhibitor, disrupts triglyceride metabolism in guinea pigs. Metabolism. 2006;55:794–802.
Madeira JB, Masuda CA, Maya-Monteiro CM, Matos GS, Montero-Lomelí M, Bozaquel-Morais BL. TORC1 inhibition induces lipid droplet replenishment in yeast. Mol Cell Biol. 2015;35:737–46.
Mukaida S, Ogawa T, Ohishi K, Tanizawa Y, Ohta D, Arita M. The effect of rapamycin on biodiesel-producing protist Euglena gracilis. Biosci Biotechnol Biochem. 2016;80:1223–9.
Görner C, Redai V, Bracharz F, Schrepfer P, Garbe D, Brück T. Genetic engineering and production of modified fatty acids by the non-conventional oleaginous yeast Trichosporon oleaginosus ATCC 20509. Green Chem. 2016;18:2037–46. The Royal Society of Chemistry.
Folch J, Lees M, Sloane SGH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957;226:497–509.
Griffiths MJ, van Hille RP, Harrison STL. Selection of direct transesterification as the preferred method for assay of fatty acid content of microalgae. Lipids. 2010;45:1053–60.
Sitepu IR, Ignatia L, Franz AK, Wong DM, Faulina SA, Tsui M, et al. An improved high-throughput Nile red fluorescence assay for estimating intracellular lipids in a variety of yeast species. J Microbiol Methods. 2012;91:321–8.
R Core Team. R: A language and environment for statistical computing. Vienna: R Foundation for Statistical Computing. 2013. http://www.R-project.org/. ISBN 3-900051-07-0.
Kahm M, Hasenbrink G, Lichtenberg-frate H, Ludwig J, Kschischo M. Grofit: fitting biological growth curves. J Stat Softw. 2010;33:1–21.
Kourist R, Bracharz F, Lorenzen J, Kracht ON, Chovatia M, Daum C, et al. Genomics and transcriptomics analyses of the oil-accumulating basidiomycete yeast Trichosporon oleaginosus: Insights into substrate utilization and alternative evolutionary trajectories of fungal mating systems. MBio. 2015;6:1–13.
Rumin J, Bonnefond H, Saint-Jean B, Rouxel C, Sciandra A, Bernard O, et al. The use of fluorescent Nile red and BODIPY for lipid measurement in microalgae. Biotechnol Biofuels. 2015;8:42.
Brown NF, Stefanovic-Racic M, Sipula IJ, Perdomo G. The mammalian target of rapamycin regulates lipid metabolism in primary cultures of rat hepatocytes. Metabolism. 2007;56:1500–7.
Richards FJ. A flexible growth function for empirical use. J Exp Bot. 1959;10:290–301.
Hedbacker K, Carlson M. SNF1/AMPK pathways in yeast. Front Biosci. 2008;13:2408–20.
Zhang M, Galdieri L, Vancura A. The yeast AMPK homolog SNF1 regulates acetyl coenzyme A homeostasis and histone acetylation. Mol Cell Biol. 2013;33:4701–17.
Wang R, Levine B. Autophagy in cellular growth control. FEBS Lett. 2010;584:1417–26.
Huang J, Brendan DM. The TSC1–TSC2 complex: a molecular switchboard controlling cell growth. Biochem J. 2009;290:1717–21.
Jacinto E. What controls TOR? IUBMB Life. 2008;60:483–96.
Choi J, Chen J, Schreiber SL, Clardy J. Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP. Science. 1996;273:239–42.
Chantaravisoot N, Tamanoi F. mTOR signaling and human cancer. Struct. Funct. Regul. TOR complexes from Yeasts to Mamm. Part B. Cambridge: Academic Press; 2010. p. 301–16.
Cybulski N, Hall MN. TOR complex 2: a signaling pathway of its own. Trends Biochem Sci. 2009;34:620–7. Elsevier Ltd.
Zhang L, Liu N, Ma X, Jiang L. The transcriptional control machinery as well as the cell wall integrity and its regulation are involved in the detoxification of the organic solvent dimethyl sulfoxide in Saccharomyces cerevisiae. FEMS Yeast Res. 2013;13:200–18.
Chen Y, Klionsky DJ. The regulation of autophagy - unanswered questions. J Cell Sci. 2011;124:161–70.
Laor D, Cohen A, Kupiec M, Weisman R. TORC1 regulates developmental responses to nitrogen stress via regulation of the GATA transcription factor Gaf1. MBio Am Soc Microbiol. 2015;6:e00959–15.
Yanagida M, Ikai N, Shimanuki M, Sajiki K. Nutrient limitations alter cell division control and chromosome segregation through growth-related kinases and phosphatases. Philos Trans R Soc Lond B Biol Sci. 2011;366:3508–20. The Royal Society.
Matsuo T, Otsubo Y, Urano J, Tamanoi F, Yamamoto M. Loss of the TOR kinase Tor2 mimics nitrogen starvation and activates the sexual development pathway in fission yeast. Mol Cell Biol. 2007;27:3154–64.
Weisman R, Cohen A, Gasser SM. TORC2--a new player in genome stability. EMBO Mol Med. 2014;6:995–1002. EMBO Press.
Ikai N, Nakazawa N, Hayashi T, Yanagida M. The reverse, but coordinated, roles of Tor2 (TORC1) and Tor1 (TORC2) kinases for growth, cell cycle and separase-mediated mitosis in Schizosaccharomyces pombe. Open Biol. 2011;1:110007.
Pic A, Lim FL, Ross SJ, Veal EA, Johnson AL, Sultan MR, et al. The forkhead protein Fkh2 is a component of the yeast cell cycle transcription factor SFF. EMBO J. 2000;19:3750–61. EMBO Press.
Hollenhorst PC, Bose ME, Mielke MR, Müller U, Fox CA. Forkhead genes in transcriptional silencing, cell morphology and the cell cycle: overlapping and distinct functions for FKH1 and FKH2 in Saccharomyces cerevisiae. Genetics. 2000;154:1533–48.
Du W, Forte GM, Smith D, Petersen J. Phosphorylation of the amino-terminus of the AGC kinase Gad8 prevents its interaction with TORC2. Open Biol. 2016;6:150189.
Lee H, Khanal Lamichhane A, Garraffo HM, Kwon-Chung KJ, Chang YC. Involvement of PDK1, PKC and TOR signalling pathways in basal fluconazole tolerance in Cryptococcus neoformans. Mol Microbiol. 2012;84:130–46. NIH Public Access.
Gaubitz C, Oliveira TM, Prouteau M, Leitner A, Karuppasamy M, Konstantinidou G, et al. Molecular basis of the rapamycin insensitivity of target of rapamycin complex 2. Mol Cell. 2015;58:977–88.
Sarbassov DD, Ali SM, Sengupta S, Sheen J-H, Hsu PP, Bagley AF, et al. Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol Cell. 2006;22:159–68. Elsevier.