Applied Microbiology and Biotechnology

, Volume 100, Issue 21, pp 9393–9405 | Cite as

Metabolic engineering of the oleaginous yeast Rhodosporidium toruloides IFO0880 for lipid overproduction during high-density fermentation

  • Shuyan Zhang
  • Masakazu Ito
  • Jeffrey M. Skerker
  • Adam P. Arkin
  • Christopher V. RaoEmail author
Bioenergy and biofuels


Natural lipids can be used to make biodiesel and many other value-added compounds. In this work, we explored a number of different metabolic engineering strategies for increasing lipid production in the oleaginous yeast Rhodosporidium toruloides IFO0880. These included increasing the expression of enzymes involved in different aspects of lipid biosynthesis—malic enzyme (ME), pyruvate carboxylase (PYC1), glycerol-3-P dehydrogenase (GPD), and stearoyl-CoA desaturase (SCD)—and deleting the gene PEX10, required for peroxisome biogenesis. Only malic enzyme and stearoyl-CoA desaturase, when overexpressed, were found to significantly increase lipid production. Only stearoyl-CoA desaturase, when overexpressed, further increased lipid production in a strain previously engineered to overexpress acetyl-CoA carboxylase (ACC1) and diacylglycerol acyltransferase (DGA1). Our best strain produced 27.4 g/L lipid with an average productivity of 0.31 g/L/h during batch growth on glucose and 89.4 g/L lipid with an average productivity of 0.61 g/L/h during fed-batch growth on glucose. These results further establish R. toruloides as a platform organism for the production of lipids and potentially other lipid-derived compounds from sugars.


Oleaginous yeast Lipids Metabolic engineering Rhodosporidium toruloides 


Compliance with ethical standards


This work was supported by the Energy Biosciences Institute Grants OO7G02 (A.P.A) and OO3G18 (C.V.R.). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2016_7815_MOESM1_ESM.pdf (2.1 mb)
ESM 1 (PDF 2184 kb)


  1. Abbott EP, Ianiri G, Castoria R, Idnurm A (2013) Overcoming recalcitrant transformation and gene manipulation in Pucciniomycotina yeasts. Appl Microbiol Biotechnol 97(1):283–295. doi: 10.1007/s00253-012-4561-7 CrossRefPubMedGoogle Scholar
  2. Ageitos JM, Vallejo JA, Veiga-Crespo P, Villa TG (2011) Oily yeasts as oleaginous cell factories. Appl Microbiol Biotechnol 90(4):1219–1227. doi: 10.1007/s00253-011-3200-z CrossRefPubMedGoogle Scholar
  3. Beopoulos A, Cescut J, Haddouche R, Uribelarrea JL, Molina-Jouve C, Nicaud JM (2009) Yarrowia lipolytica as a model for bio-oil production. Prog Lipid Res 48(6):375–387. doi: 10.1016/j.plipres.2009.08.005 CrossRefPubMedGoogle Scholar
  4. Blazeck J, Liu L, Knight R, Alper HS (2013) Heterologous production of pentane in the oleaginous yeast Yarrowia lipolytica. J Biotechnol 165(3–4):184–194. doi: 10.1016/j.jbiotec.2013.04.003 CrossRefPubMedGoogle Scholar
  5. Blazeck J, Hill A, Liu L, Knight R, Miller J, Pan A, Otoupal P, Alper HS (2014) Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production. Nat Commun 5:3131. doi: 10.1038/ncomms4131 CrossRefPubMedGoogle Scholar
  6. Bommareddy RR, Sabra W, Maheshwari G, Zeng AP (2015) Metabolic network analysis and experimental study of lipid production in Rhodosporidium toruloides grown on single and mixed substrates. Microb Cell Factories 14:36. doi: 10.1186/s12934-015-0217-5 CrossRefGoogle Scholar
  7. Camoes F, Islinger M, Guimaraes SC, Kilaru S, Schuster M, Godinho LF, Steinberg G, Schrader M (2015) New insights into the peroxisomal protein inventory: acyl-CoA oxidases and -dehydrogenases are an ancient feature of peroxisomes. Biochim Biophys Acta 1853(1):111–125. doi: 10.1016/j.bbamcr.2014.10.005 CrossRefPubMedGoogle Scholar
  8. Chen L, Zhang J, Chen WN (2014) Engineering the Saccharomyces cerevisiae beta-oxidation pathway to increase medium chain fatty acid production as potential biofuel. PLoS One 9(1):e84853. doi: 10.1371/journal.pone.0084853 CrossRefPubMedPubMedCentralGoogle Scholar
  9. Chilton MD, Currier TC, Farrand SK, Bendich AJ, Gordon MP, Nester EW (1974) Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors. Proc Natl Acad Sci U S A 71(9):3672–3676CrossRefPubMedPubMedCentralGoogle Scholar
  10. Dulermo T, Nicaud JM (2011) Involvement of the G3P shuttle and beta-oxidation pathway in the control of TAG synthesis and lipid accumulation in Yarrowia lipolytica. Metab Eng 13(5):482–491. doi: 10.1016/j.ymben.2011.05.002 CrossRefPubMedGoogle Scholar
  11. Fillet S, Gibert J, Suarez B, Lara A, Ronchel C, Adrio JL (2015) Fatty alcohols production by oleaginous yeast. J Ind Microbiol Biotechnol 42(11):1463–1472. doi: 10.1007/s10295-015-1674-x CrossRefPubMedPubMedCentralGoogle Scholar
  12. Flowers MT, Ntambi JM (2008) Role of stearoyl-coenzyme A desaturase in regulating lipid metabolism. Curr Opin Lipidol 19(3):248–256. doi: 10.1097/MOL.0b013e3282f9b54d CrossRefPubMedPubMedCentralGoogle Scholar
  13. Folch J, Lees M, Stanley GHS (1957) A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226(1):497–509PubMedGoogle Scholar
  14. Freitag J, Ast J, Bolker M (2012) Cryptic peroxisomal targeting via alternative splicing and stop codon read-through in fungi. Nature 485(7399):522–525. doi: 10.1038/nature11051 CrossRefPubMedGoogle Scholar
  15. Friedlander J, Tsakraklides V, Kamineni A, Greenhagen EH, Consiglio AL, MacEwen K, Crabtree DV, Afshar J, Nugent RL, Hamilton MA, Joe Shaw A, South CR, Stephanopoulos G, Brevnova EE (2016) Engineering of a high lipid producing Yarrowia lipolytica strain. Biotechnol Biofuels 9:77. doi: 10.1186/s13068-016-0492-3 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Fu TJ, Seeman NC (1993) DNA double-crossover molecules. Biochemistry 32(13):3211–3220CrossRefPubMedGoogle Scholar
  17. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5):343–345. doi: 10.1038/nmeth.1318 CrossRefPubMedGoogle Scholar
  18. Granger LM, Perlot P, Goma G, Pareilleux A (1993) Effect of various nutrient limitations on fatty-acid production by Rhodotorula glutinis. Appl Microbiol Biotechnol 38(6):784–789CrossRefGoogle Scholar
  19. Hill J, Nelson E, Tilman D, Polasky S, Tiffany D (2006) Environmental, economic, and energetic costs and benefits of biodiesel and ethanol biofuels. Proc Natl Acad Sci U S A 103(30):11206–11210. doi: 10.1073/pnas.0604600103 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Hiltunen JK, Mursula AM, Rottensteiner H, Wierenga RK, Kastaniotis AJ, Gurvitz A (2003) The biochemistry of peroxisomal beta-oxidation in the yeast Saccharomyces cerevisiae. FEMS Microbiol Rev 27(1):35–64. doi: 10.1016/S0168-6445(03)00017-2 CrossRefPubMedGoogle Scholar
  21. Koh CMJ, Liu YB, Moehninsi, Du MG, Ji LH (2014) Molecular characterization of KU70 and KU80 homologues and exploitation of a KU70-deficient mutant for improving gene deletion frequency in Rhodosporidium toruloides. BMC Microbiol 14. doi: 10.1186/1471-2180-14-50
  22. Kretschmer M, Klose J, Kronstad JW (2012) Defects in mitochondrial and peroxisomal beta-oxidation influence virulence in the maize pathogen Ustilago maydis. Eukaryot Cell 11(8):1055–1066. doi: 10.1128/EC.00129-12 CrossRefPubMedPubMedCentralGoogle Scholar
  23. Kretzschmar A, Otto C, Holz M, Werner S, Hubner L, Barth G (2013) Increased homologous integration frequency in Yarrowia lipolytica strains defective in non-homologous end-joining. Curr Genet 59(1–2):63–72. doi: 10.1007/s00294-013-0389-7 CrossRefPubMedGoogle Scholar
  24. Lane S, Zhang S, Wei N, Rao C, Jin YS (2015) Development and physiological characterization of cellobiose-consuming Yarrowia lipolytica. Biotechnol Bioeng 112(5):1012–1022. doi: 10.1002/bit.25499 CrossRefPubMedGoogle Scholar
  25. Ledesma-Amaro R, Nicaud JM (2016) Yarrowia lipolytica as a biotechnological chassis to produce usual and unusual fatty acids. Prog Lipid Res 61:40–50. doi: 10.1016/j.plipres.2015.12.001 CrossRefPubMedGoogle Scholar
  26. Lepage G, Roy CC (1986) Direct transesterification of all classes of lipids in a one-step reaction. J Lipid Res 27(1):114–120PubMedGoogle Scholar
  27. Li YH, Zhao ZB, Bai FW (2007) High-density cultivation of oleaginous yeast Rhodosporidium toruloides Y4 in fed-batch culture. Enzym Microb Technol 41(3):312–317. doi: 10.1016/j.enzmictec.2007.02.008 CrossRefGoogle Scholar
  28. Li Z, Sun H, Mo X, Li X, Xu B, Tian P (2013) Overexpression of malic enzyme (ME) of Mucor circinelloides improved lipid accumulation in engineered Rhodotorula glutinis. Appl Microbiol Biotechnol 97(11):4927–4936. doi: 10.1007/s00253-012-4571-5 CrossRefPubMedGoogle Scholar
  29. Liang MH, Jiang JG (2013) Advancing oleaginous microorganisms to produce lipid via metabolic engineering technology. Prog Lipid Res 52(4):395–408. doi: 10.1016/j.plipres.2013.05.002 CrossRefPubMedGoogle Scholar
  30. Lin X, Wang Y, Zhang S, Zhu Z, Zhou YJ, Yang F, Sun W, Wang X, Zhao ZK (2014) Functional integration of multiple genes into the genome of the oleaginous yeast Rhodosporidium toruloides. FEMS Yeast Res 14(4):547–555. doi: 10.1111/1567-1364.12140 CrossRefPubMedGoogle Scholar
  31. Liu Y, Koh CM, Sun L, Hlaing MM, Du M, Peng N, Ji L (2013) Characterization of glyceraldehyde-3-phosphate dehydrogenase gene RtGPD1 and development of genetic transformation method by dominant selection in oleaginous yeast Rhodosporidium toruloides. Appl Microbiol Biotechnol 97(2):719–729. doi: 10.1007/s00253-012-4223-9 CrossRefPubMedGoogle Scholar
  32. Liu L, Pan A, Spofford C, Zhou N, Alper HS (2015) An evolutionary metabolic engineering approach for enhancing lipogenesis in Yarrowia lipolytica. Metab Eng 29:36–45. doi: 10.1016/j.ymben.2015.02.003 CrossRefPubMedGoogle Scholar
  33. Lustig AJ (1999) The Kudos of non-homologous end-joining. Nat Genet 23(2):130–131. doi: 10.1038/13755 CrossRefPubMedGoogle Scholar
  34. Madzak C (2015) Yarrowia lipolytica: recent achievements in heterologous protein expression and pathway engineering. Appl Microbiol Biotechnol 99(11):4559–4577. doi: 10.1007/s00253-015-6624-z CrossRefPubMedGoogle Scholar
  35. Ohlrogge J, Browse J (1995) Lipid biosynthesis. Plant Cell 7(7):957–970. doi: 10.1105/Tpc.7.7.957 CrossRefPubMedPubMedCentralGoogle Scholar
  36. Papanikolaou S, Aggelis G (2011) Lipids of oleaginous yeasts. Part I: biochemistry of single cell oil production. Eur J Lipid Sci Technol 113(8):1031–1051. doi: 10.1002/ejlt.201100014 CrossRefGoogle Scholar
  37. Papanikolaou S, Beopoulos A, Koletti A, Thevenieau F, Koutinas AA, Nicaud JM, Aggelis G (2013) Importance of the methyl-citrate cycle on glycerol metabolism in the yeast Yarrowia lipolytica. J Biotechnol 168(4):303–314CrossRefPubMedGoogle Scholar
  38. Qiao K, Imam Abidi SH, Liu H, Zhang H, Chakraborty S, Watson N, Kumaran Ajikumar P, Stephanopoulos G (2015) Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica. Metab Eng 29:56–65. doi: 10.1016/j.ymben.2015.02.005 CrossRefPubMedGoogle Scholar
  39. Ratledge C (1987) Lipid biotechnology—a wonderland for the microbial physiologist. Journal of the American Oil Chemists Society 64(12):1647–1656. doi: 10.1007/Bf02542498 CrossRefGoogle Scholar
  40. Ratledge C (2014) The role of malic enzyme as the provider of NADPH in oleaginous microorganisms: a reappraisal and unsolved problems. Biotechnol Lett 36(8):1557–1568. doi: 10.1007/s10529-014-1532-3 CrossRefPubMedGoogle Scholar
  41. Ratledge C, Wynn JP (2002) The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Adv Appl Microbiol 51:1–51. doi: 10.1016/S0065-2164(02)51000-5 CrossRefPubMedGoogle Scholar
  42. Richard GF, Kerrest A, Lafontaine I, Dujon B (2005) Comparative genomics of hemiascomycete yeasts: genes involved in DNA replication, repair, and recombination. Mol Biol Evol 22(4):1011–1023. doi: 10.1093/molbev/msi083 CrossRefPubMedGoogle Scholar
  43. Rutter CD, Zhang S, Rao CV (2015) Engineering Yarrowia lipolytica for production of medium-chain fatty acids. Appl Microbiol Biotechnol 99(17):7359–7368. doi: 10.1007/s00253-015-6764-1 CrossRefPubMedGoogle Scholar
  44. Ryu S, Hipp J, Trinh CT (2015) Activating and elucidating metabolism of complex sugars in Yarrowia lipolytica. Appl Environ Microbiol 82(4):1334–1345. doi: 10.1128/AEM.03582-15 CrossRefPubMedGoogle Scholar
  45. Silverman AM, Qiao K, Xu P, Stephanopoulos G (2016) Functional overexpression and characterization of lipogenesis-related genes in the oleaginous yeast Yarrowia lipolytica. Appl Microbiol Biotechnol. doi: 10.1007/s00253-016-7376-0 PubMedGoogle Scholar
  46. Sitepu I, Selby T, Lin T, Zhu S, Boundy-Mills K (2014) Carbon source utilization and inhibitor tolerance of 45 oleaginous yeast species. J Ind Microbiol Biotechnol 41(7):1061–1070. doi: 10.1007/s10295-014-1447-y CrossRefPubMedPubMedCentralGoogle Scholar
  47. Slininger PJ, Dien BS, Kurtzman CP, Moser BR, Bakota EL, Thompson SR, O’Bryan PJ, Cotta MA, Balan V, Jin M, Sousa LD, Dale BE (2016) Comparative lipid production by oleaginous yeasts in hydrolyzates of lignocellulosic biomass and process strategy for high titers. Biotechnol Bioeng. doi: 10.1002/bit.25928 PubMedGoogle Scholar
  48. Tai M, Stephanopoulos G (2013) Engineering the push and pull of lipid biosynthesis in oleaginous yeast Yarrowia lipolytica for biofuel production. Metab Eng 15:1–9. doi: 10.1016/j.ymben.2012.08.007 CrossRefPubMedGoogle Scholar
  49. Verbeke J, Beopoulos A, Nicaud JM (2013) Efficient homologous recombination with short length flanking fragments in Ku70 deficient Yarrowia lipolytica strains. Biotechnol Lett 35(4):571–576. doi: 10.1007/s10529-012-1107-0 CrossRefPubMedGoogle Scholar
  50. Wasylenko TM, Ahn WS, Stephanopoulos G (2015) The oxidative pentose phosphate pathway is the primary source of NADPH for lipid overproduction from glucose in Yarrowia lipolytica. Metab Eng 30:27–39. doi: 10.1016/j.ymben.2015.02.007 CrossRefPubMedGoogle Scholar
  51. Wiebe MG, Koivuranta K, Penttila M, Ruohonen L (2012) Lipid production in batch and fed-batch cultures of Rhodosporidium toruloides from 5 and 6 carbon carbohydrates. BMC Biotechnol 12:26. doi: 10.1186/1472-6750-12-26 CrossRefPubMedPubMedCentralGoogle Scholar
  52. Winans SC, Kerstetter RA, Nester EW (1988) Transcriptional regulation of the virA and virG genes of Agrobacterium tumefaciens. J Bacteriol 170(9):4047–4054PubMedPubMedCentralGoogle Scholar
  53. Wu SG, Hu CM, Jin GJ, Zhao X, Zhao ZK (2010) Phosphate-limitation mediated lipid production by Rhodosporidium toruloides. Bioresour Technol 101(15):6124–6129. doi: 10.1016/j.biortech.2010.02.111 CrossRefPubMedGoogle Scholar
  54. Wu SG, Zhao X, Shen HW, Wang QA, Zhao ZBK (2011) Microbial lipid production by Rhodosporidium toruloides under sulfate-limited conditions. Bioresour Technol 102(2):1803–1807. doi: 10.1016/j.biortech.2010.09.033 CrossRefPubMedGoogle Scholar
  55. Xue Z, Sharpe PL, Hong SP, Yadav NS, Xie D, Short DR, Damude HG, Rupert RA, Seip JE, Wang J, Pollak DW, Bostick MW, Bosak MD, Macool DJ, Hollerbach DH, Zhang H, Arcilla DM, Bledsoe SA, Croker K, McCord EF, Tyreus BD, Jackson EN, Zhu Q (2013) Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica. Nat Biotechnol 31(8):734–740. doi: 10.1038/nbt.2622 CrossRefPubMedGoogle Scholar
  56. Zhang Y, Adams IP, Ratledge C (2007) Malic enzyme: the controlling activity for lipid production? Overexpression of malic enzyme in Mucor circinelloides leads to a 2.5-fold increase in lipid accumulation. Microbiology 153(Pt 7):2013–2025. doi: 10.1099/mic.0.2006/002683-0 CrossRefPubMedGoogle Scholar
  57. Zhang H, Zhang L, Chen H, Chen YQ, Ratledge C, Song Y, Chen W (2013) Regulatory properties of malic enzyme in the oleaginous yeast, Yarrowia lipolytica, and its non-involvement in lipid accumulation. Biotechnol Lett 35(12):2091–2098. doi: 10.1007/s10529-013-1302-7 CrossRefPubMedGoogle Scholar
  58. Zhang S, Skerker JM, Rutter CD, Maurer MJ, Arkin AP, Rao CV (2016) Engineering Rhodosporidium toruloides for increased lipid production. Biotechnol Bioeng 113(5):1056–1066. doi: 10.1002/bit.25864 CrossRefPubMedGoogle Scholar
  59. Zhao X, Hu C, Wu S, Shen H, Zhao ZK (2011) Lipid production by Rhodosporidium toruloides Y4 using different substrate feeding strategies. J Ind Microbiol Biotechnol 38(5):627–632. doi: 10.1007/s10295-010-0808-4 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Shuyan Zhang
    • 1
  • Masakazu Ito
    • 2
    • 3
  • Jeffrey M. Skerker
    • 2
    • 3
  • Adam P. Arkin
    • 2
    • 3
  • Christopher V. Rao
    • 1
    Email author
  1. 1.Department of Chemical and Biomolecular EngineeringUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  2. 2.Department of BioengineeringUniversity of CaliforniaBerkeleyUSA
  3. 3.Environmental Genomics and Systems Biology DivisionLawrence Berkeley National LaboratoryBerkeleyUSA

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