Advertisement

Protocols for Monitoring Growth and Lipid Accumulation in Oleaginous Yeasts

  • Jean-Marc NicaudEmail author
  • Anne-Marie Crutz-Le Coq
  • Tristan Rossignol
  • Nicolas Morin
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Oleaginous yeasts can synthesize and store lipids up to 20% of their dry weight and have emerged as resources of choice for biotechnological applications, such as bio-lipid production. The number of species and mutant libraries consequently available for screening is exponentially growing. Cultivation strategies and growth media for bio-lipid production need to be optimized to accelerate screening and identification of production strains. In this chapter we describe methods for high-throughput cell growth in 96 microtiter plates in various media including opaque broth by using a fluorescent reporter, carbon/nitrogen ratio determination for optimal lipid accumulation, and in vivo real-time detection of lipid accumulation using a neutral lipid fluorescent dye. We provide examples using two well-established oleaginous yeasts, Yarrowia lipolytica and Rhodosporidium toruloides. These methods can be extended to other oleaginous yeast species for high-throughput screening of bio-lipid accumulation.

Keywords:

Lipid accumulation Oleaginous yeast Rhodosporidium Single cell oil Yarrowia 

References

  1. 1.
    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 48:375–387CrossRefPubMedGoogle Scholar
  2. 2.
    Ratledge C, Wynn JP (2002) The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Adv Appl Microbiol 51:1–51CrossRefPubMedGoogle Scholar
  3. 3.
    Thevenieau F, Nicaud JM (2013) Microorganisms as sources of oils. OCL 60:D603CrossRefGoogle Scholar
  4. 4.
    Nicaud JM (2012) Yarrowia lipolytica. Yeast 29:409–418CrossRefPubMedGoogle Scholar
  5. 5.
    Dujon B, Sherman D, Fischer G et al (2004) Genome evolution in yeasts. Nature 430:35–44CrossRefPubMedGoogle Scholar
  6. 6.
    Le Dall MT, Nicaud JM, Gaillardin C (1994) Multiple-copy integration in the yeast Yarrowia lipolytica. Curr Genet 26:38–44CrossRefPubMedGoogle Scholar
  7. 7.
    Pignede G, Wang HJ, Fudalej F, Seman M, Gaillardin C, Nicaud JM (2000) Autocloning and amplification of LIP2 in Yarrowia lipolytica. Appl Environ Microbiol 66:3283–3289CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Fickers P, Le Dall MT, Gaillardin C, Thonart P, Nicaud JM (2003) New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica. J Microbiol Methods 55:727–737CrossRefPubMedGoogle Scholar
  9. 9.
    Loira N, Dulermo T, Nicaud JM, Sherman DJ (2012) A genome-scale metabolic model of the lipid-accumulating yeast Yarrowia lipolytica. BMC Syst Biol 6:35CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Pan P, Hua Q (2012) Reconstruction and in silico analysis of metabolic network for an oleaginous yeast, Yarrowia lipolytica. PLoS One 7:e51535CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Kumar S, Kushwaha H, Bachhawat AK, Raghava GP, Ganesan K (2012) Genome sequence of the oleaginous red yeast Rhodosporidium toruloides MTCC 457. Eukaryot Cell 11:1083–1084CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Zhu Z, Zhang S, Liu H et al (2012) A multi-omic map of the lipid-producing yeast Rhodosporidium toruloides. Nat Commun 3:1112CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Lin X, Wang Y, Zhang S et al (2014) Functional integration of multiple genes into the genome of the oleaginous yeast Rhodosporidium toruloides. FEMS Yeast Res. doi: 10.1111/1567-1364.12140 Google Scholar
  14. 14.
    Abbott EP, Ianiri G, Castoria R, Idnurm A (2013) Overcoming recalcitrant transformation and gene manipulation in Pucciniomycotina yeasts. Appl Microbiol Biotechnol 97:283–295CrossRefPubMedGoogle Scholar
  15. 15.
    R-Core-Team (2014) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, AustriaGoogle Scholar
  16. 16.
    Piatkevich KD, Efremenko EN, Verkhusha VV, Varfolomeev DD (2010) Red fluorescent proteins and their properties. Russ Chem Rev 79:243–258CrossRefGoogle Scholar
  17. 17.
    Matz MV, Fradkov AF, Labas YA et al (1999) Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol 17:969–973CrossRefPubMedGoogle Scholar
  18. 18.
    Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909CrossRefPubMedGoogle Scholar
  19. 19.
    Baird GS, Zacharias DA, Tsien RY (2000) Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci U S A 97:11984–11989CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Knop M, Barr F, Riedel CG, Heckel T, Reichel C (2002) Improved version of the red fluorescent protein (drFP583/DsRed/RFP). Biotechniques 33:592, 594, 596–598 passimGoogle Scholar
  21. 21.
    Janke C, Magiera MM, Rathfelder N et al (2004) A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21:947–962CrossRefPubMedGoogle Scholar
  22. 22.
    Bozaquel-Morais BL, Madeira JB, Maya-Monteiro CM, Masuda CA, Montero-Lomeli M (2010) A new fluorescence-based method identifies protein phosphatases regulating lipid droplet metabolism. PLoS One 5:e13692CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Springer, New YorkCrossRefGoogle Scholar
  24. 24.
    Beopoulos A, Haddouche R, Kabran P, Dulermo T, Chardot T, Nicaud JM (2012) Identification and characterization of DGA2, an acyltransferase of the DGAT1 acyl-CoA:diacylglycerol acyltransferase family in the oleaginous yeast Yarrowia lipolytica. New insights into the storage lipid metabolism of oleaginous yeasts. Appl Microbiol Biotechnol 93:1523–1537CrossRefPubMedGoogle Scholar
  25. 25.
    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 13:482–491CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Jean-Marc Nicaud
    • 2
    • 1
    Email author
  • Anne-Marie Crutz-Le Coq
    • 2
    • 1
  • Tristan Rossignol
    • 2
    • 1
  • Nicolas Morin
    • 2
    • 1
  1. 1.AgroParisTechJouy-en-JosasFrance
  2. 2.INRAJouy-en-JosasFrance

Personalised recommendations