Selection of Heterologous Protein-Producing Strains in Yarrowia lipolytica

  • Paul Soudier
  • Macarena Larroude
  • Ewelina Celińska
  • Tristan Rossignol
  • Jean-Marc NicaudEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1923)


Yarrowia lipolytica has emerged as an alternative expression system for heterologous protein production and enzyme evolution. Several different expression systems dedicated for this species have been developed, ranging from the simple cloning of expression vectors to recently developed high-throughput methodologies using efficient cloning and assembly such as Gateway and Golden Gate strategies. The latter strategies, due to their modular character, enable multiple vector construction and the construction of expression cassettes containing different genes or a gene under different promoters of various strengths.

Here, we present the Golden Gate cloning strategy for the construction of multiple expression cassettes, the transformation into Y. lipolytica, and the selection of efficient enzyme-producing strains using an insect alpha-amylase as a reporter detected via a thermal cycler-based microassay.

Key words

Yarrowia lipolytica Heterologous protein Secretion Golden Gate Gateway Gene assembly Inducible promoter Targeting sequence High-throughput techniques 



The experiments conducted by EC were financially supported by the Polish Ministry of Sciences and Higher Education (Project No IP2015 011074).


  1. 1.
    Madzak C (2015) Yarrowia lipolytica: recent achievements in heterologous protein expression and pathway engineering. Appl Microbiol Biotechnol 99:4559–4577. Scholar
  2. 2.
    Madzak C, Beckerich J-M (2013) Heterologous protein expression and secretion in Yarrowia lipolytica. In: Barth G (ed) Yarrowia lipolytica: biotechnological applications. Springer Berlin Heidelberg, Berlin, Heidelberg, pp 1–76. Scholar
  3. 3.
    Le Dall MT, Nicaud JM, Gaillardin C (1994) Multiple-copy integration in the yeast Yarrowia lipolytica. Curr Genet 26(1):38–44CrossRefGoogle Scholar
  4. 4.
    Pignede G, Wang H-J, Fudalej F, Seman M, Gaillardin C, Nicaud J-M (2000) Autocloning and amplification of LIP2 in Yarrowia lipolytica. Appl Environ Microbiol 66(8):3283–3289. Scholar
  5. 5.
    Nicaud J-M, Madzak C, van den Broek P, Gysler C, Duboc P, Niederberger P, Gaillardin C (2002) Protein expression and secretion in the yeast Yarrowia lipolytica. FEMS Yeast Res 2(3):371–379. Scholar
  6. 6.
    Ogrydziak DM, Nicaud JM (2012) Characterization of Yarrowia lipolytica XPR2 multi-copy strains over-producing alkaline extracellular protease—a system for rapidly increasing secretory pathway cargo loads. FEMS Yeast Res 12(8):938–948. Scholar
  7. 7.
    Madzak C, Treton B, Blanchin-Roland S (2000) Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica. J Mol Microbiol Biotechnol 2(2):207–216PubMedGoogle Scholar
  8. 8.
    Bordes F, Fudalej F, Dossat V, Nicaud JM, Marty A (2007) A new recombinant protein expression system for high-throughput screening in the yeast Yarrowia lipolytica. J Microbiol Methods 70(3):493–502CrossRefGoogle Scholar
  9. 9.
    Juretzek T, Le Dall M-T, Mauersberger S, Gaillardin C, Barth G, Nicaud J-M (2001) Vectors for gene expression and amplification in the yeast Yarrowia lipolytica. Yeast 18(2):97–113.<97::aid-yea652>;2-uCrossRefPubMedGoogle Scholar
  10. 10.
    Barth G, Gaillardin C (1996) The dimorphic fungus Yarrowia lipolytica. In: Wolf K (ed) Genetics, biochemistry and molecular biology of non conventional yeasts. Springer Verlag, Heidelberg, pp 313–388Google Scholar
  11. 11.
    Müller S, Sandal T, Kamp-Hansen P, Dalbøge H (1998) Comparison of expression systems in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha, Klyveromyces lactis, Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica. Yeast 14(14):1267–1283.<1267::aid-yea327>;2-2CrossRefPubMedGoogle Scholar
  12. 12.
    Sumita T, Iida T, Yamagami S, Horiuchi H, Takagi M, Ohta A (2002) YlALK1 encoding the cytochrome P450ALK1 in Yarrowia lipolytica is transcriptionally induced by n-alkane through two distinct cis-elements on its promoter. Biochem Biophys Res Commun 294(5):1071–1078. Scholar
  13. 13.
    Trassaert M, Vandermies M, Carly F, Denies O, Thomas S, Fickers P, Nicaud J-M (2017) New inducible promoter for gene expression and synthetic biology in Yarrowia lipolytica. Microb Cell Factories 16:141. Scholar
  14. 14.
    Carly F, Vandermies M, Telek S, Steels S, Thomas S, Nicaud J-M, Fickers P (2017) Enhancing erythritol productivity in Yarrowia lipolytica using metabolic engineering. Metab Eng 42:19–24. Scholar
  15. 15.
    Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) Golden Gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4(5):e5553. Scholar
  16. 16.
    Celinska E, Ledesma-Amaro R, Larroude M, Rossignol T, Pauthenier C, Nicaud JM (2017) Golden Gate Assembly system dedicated to complex pathway manipulation in Yarrowia lipolytica. Microb Biotechnol 10(2):450–455. Scholar
  17. 17.
    Celińska E, Borkowska M, Białas W (2016) Evaluation of heterologous alpha-amylase production in two expression platforms dedicated for Yarrowia lipolytica—commercial Po1g-pYLSC (php4d) and custom-made A18-pYLTEF (pTEF). Yeast 33(5):165–181. Scholar
  18. 18.
    Celińska E, Borkowska M, Białas W (2016) Evaluation of a recombinant insect-derived amylase performance in simultaneous saccharification and fermentation process with industrial yeasts. Appl Microbiol Biotechnol 100(6):2693–2707. Scholar
  19. 19.
    Celińska E, Białas W, Borkowska M, Grajek W (2015) Cloning, expression, and purification of insect (Sitophilus oryzae) alpha-amylase, able to digest granular starch, in Yarrowia lipolytica host. Appl Microbiol Biotechnol 99(6):2727–2739. Scholar
  20. 20.
    Leplat C, Nicaud J-M, Rossignol T (2015) High-throughput transformation method for Yarrowia lipolytica mutant library screening. FEMS Yeast Res 15(6). Scholar
  21. 21.
    Chen DC, Beckerich JM, Gaillardin C (1997) One-step transformation of the dimorphic yeast Yarrowia lipolytica. Appl Microbiol Biotechnol 48(2):232–235. Scholar
  22. 22.
    Back A, Rossignol T, Krier F, Nicaud J-M, Dhulster P (2016) High-throughput fermentation screening for the yeast Yarrowia lipolytica with real-time monitoring of biomass and lipid production. Microb Cell Factories 15(1):147. Scholar
  23. 23.
    Beneyton T, Thomas S, Griffiths AD, Nicaud J-M, Drevelle A, Rossignol T (2017) Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica. Microb Cell Factories 16(1):18. Scholar
  24. 24.
    Ledesma-Amaro R, Dulermo T, Nicaud J (2015) Engineering Yarrowia lipolytica to produce biodiesel from raw starch. Biotechnol Biofuels 8(1):148CrossRefGoogle Scholar
  25. 25.
    Dulermo R, Brunel F, Dulermo T, Ledesma-Amaro R, Vion J, Trassaert M, Thomas S, Nicaud J-M, Leplat C (2017) Using a vector pool containing variable-strength promoters to optimize protein production in Yarrowia lipolytica. Microb Cell Factories 16(1):31. Scholar
  26. 26.
    Borkowska M, Białas W, Kubiak M, Celinska E (2017) Micro-assays for determination of amylolytic activity using a thermalcycler: miniaturization of Somogyi-Nelson and Starch-Iodine Tests. To be publishedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Paul Soudier
    • 1
  • Macarena Larroude
    • 1
  • Ewelina Celińska
    • 2
  • Tristan Rossignol
    • 1
  • Jean-Marc Nicaud
    • 1
    Email author
  1. 1.Micalis Institute, INRA, AgroParisTechUniversité Paris-SaclayJouy-en-JosasFrance
  2. 2.Department of Biotechnology and Food MicrobiologyPoznan University of Life SciencesPoznanPoland

Personalised recommendations