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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
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 48:375–387
Ratledge C, Wynn JP (2002) The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Adv Appl Microbiol 51:1–51
Thevenieau F, Nicaud JM (2013) Microorganisms as sources of oils. OCL 60:D603
Nicaud JM (2012) Yarrowia lipolytica. Yeast 29:409–418
Dujon B, Sherman D, Fischer G et al (2004) Genome evolution in yeasts. Nature 430:35–44
Le Dall MT, Nicaud JM, Gaillardin C (1994) Multiple-copy integration in the yeast Yarrowia lipolytica. Curr Genet 26:38–44
Pignede G, Wang HJ, Fudalej F, Seman M, Gaillardin C, Nicaud JM (2000) Autocloning and amplification of LIP2 in Yarrowia lipolytica. Appl Environ Microbiol 66:3283–3289
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 55:727–737
Loira N, Dulermo T, Nicaud JM, Sherman DJ (2012) A genome-scale metabolic model of the lipid-accumulating yeast Yarrowia lipolytica. BMC Syst Biol 6:35
Pan P, Hua Q (2012) Reconstruction and in silico analysis of metabolic network for an oleaginous yeast, Yarrowia lipolytica. PLoS One 7:e51535
Kumar S, Kushwaha H, Bachhawat AK, Raghava GP, Ganesan K (2012) Genome sequence of the oleaginous red yeast Rhodosporidium toruloides MTCC 457. Eukaryot Cell 11:1083–1084
Zhu Z, Zhang S, Liu H et al (2012) A multi-omic map of the lipid-producing yeast Rhodosporidium toruloides. Nat Commun 3:1112
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
Abbott EP, Ianiri G, Castoria R, Idnurm A (2013) Overcoming recalcitrant transformation and gene manipulation in Pucciniomycotina yeasts. Appl Microbiol Biotechnol 97:283–295
R-Core-Team (2014) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria
Piatkevich KD, Efremenko EN, Verkhusha VV, Varfolomeev DD (2010) Red fluorescent proteins and their properties. Russ Chem Rev 79:243–258
Matz MV, Fradkov AF, Labas YA et al (1999) Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol 17:969–973
Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909
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 97:11984–11989
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 passim
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 21:947–962
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:e13692
Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Springer, New York
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 93:1523–1537
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 13:482–491
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer-Verlag Berlin Heidelberg
About this protocol
Cite this protocol
Nicaud, JM., Coq, AM.CL., Rossignol, T., Morin, N. (2014). Protocols for Monitoring Growth and Lipid Accumulation in Oleaginous Yeasts. In: McGenity, T., Timmis, K., Nogales, B. (eds) Hydrocarbon and Lipid Microbiology Protocols. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8623_2014_40
Download citation
DOI: https://doi.org/10.1007/8623_2014_40
Published:
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-662-53113-6
Online ISBN: 978-3-662-53115-0
eBook Packages: Springer Protocols