Introduction

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The dimorphic, non-conventional yeast Yarrowia lipolytica, belonging to the Ascomyceta phylum, was first isolated in 1960s from lipid-rich materials, hence the name “lipolytica”. The organism was classified and reclassified a number of times, first as Candida lipolytica, then Endomycopsis lipolytica, Saccharomycopsis lipolytica and finally Yarrowia lipolytica¹. The figure shows Y. lipolytica cells under a microscope. The magnification factor is 100x.

In recent years Y. Lipolytica has received increased attention from researchers, as studies have found it to possess great potential for producing industrial enzymes and pharmaceutical proteins Biotechnological applications of Yarrowia lipolytica: Past, present and future. This potential is a result of several advantages Y. Lipolytica has over the conventional yeast S. cerevisiae. Y. Lipolytica prefer secreting proteins through the co-transcription pathway and does so very efficiently², it does not exhibit hyperglycosylation as S. cerevisiae³. Y. Lipolytica has also been shown to exhibit excellent characteristics for the production of value-added chemicals such as a long range of organic acids and polyols Biotechnological applications of Yarrowia lipolytica: Past, present and future, and the recent introduction of several genome-scale models for Y. Lipolytica will most likely lead to more processes utilizing the chassis for production. Perhaps the most important advantage for using Y. Lipolytica over S. cerevisiae, to our project at least, is the broad substrate utilization range of Y. Lipolytica. Y. Lipolytica is known to naturally utilize alcohols (especially glycerol), acetate and hydrophobic substrates (eg. alkanes, fatty acids and oils) as carbon sources⁴. This has naturally lead to Y. Lipolytica becoming a model organism for several metabolic pathways, especially fatty acid transport and metabolism, and single cell oil (SCO) accumulation. Y. Lipolytica has even been shown to exhibit enhanced growth on mixed substrates Yarrowia lipolytica as an oleaginous cell factory platform for production of fatty acid-based biofuel and bioproducts, which renders it ideal for utilization of industrial waste streams due to the highly diverse content of these. These findings had us believe that we had found an excellent candidate chassis for our project. The table shows a comparison of the substrate range of Y. Lipolytica W29 and S. cerevisiae CEN.PK113-7D.

	Y. Lipolytica	S. cerevisiae
Sediment from canola oil production	µ = 0.31	None
Glycerol from Perstop	µ = 0.27	None
Glycerol from Emmelev	µ = 0.45	None
Glycerol from Daka	µ = 0.31	None
Molasses from Dansukker	µ = 0.42*	µ = 0.47

* it should be noted that the molasses was autoclaved thus degrading some of the sucrose content. This growth might not be possible to replicate with untreated molasses.

As seen in the table Y. Lipolytica is able to grow on all the waste sources we tested, while S. cerevisiae is only able to grow on molasses.

Methods

Each growth experiment (for Y. Lipolytica and S. cerevisiae) is conducted according to the following:

Minimal medium is produced as stated by Mhairi Workman⁵ using a C-source concentration of 20 g/L was used all for growth experiments.

The cells were grown overnight in YPD medium, and prepared by spinning down and washed twice. The preculture was then used as inoculum for minimal medium (substituents) to a final concentration of 0,001 (OD600). The cultivations were carried out in a cytometer (brand) shaking 900 rpm at 30 degrees celsius. Cultures were grown, shaked and measured in 48 well microtitre plates (Cellstar). Measurements was carried out by a Hamilton Robot, (cpe201, Hamilton industries) connected to a BioTek spectrophotometer (See protocols here). OD600 Measurements were taken every 2 hours until the cultures reached stationary phase, and data was analysed using R-studio.

Strains	Genotype	Comment/source
Y. Lipolytica	Wildtype	Parent strain to our laboratory bug, PO1f
S. cerevisiae	CEN.PK113-7D	Derived from parental strains ENY.WA-1A and MC996A, and is popular for use in systems biology studies

Outline of proces

We performed growth experiments on an array of pure C-sources to get a baseline of Y. Lipolytica growth patterns starting to determine the substrate range. In these experiments we observed the following growth rates or lack of growth.

	Y. Lipolytica	S. cerevisiae
Glucose	µ = 0.24	µ = 0.19
Fructose	µ = 0.23	µ = 0.426
Glycerol	µ = 0.27	None
Canola oil	µ = 0.08	None
Sucrose	None	µ = 0.396
Maltose	None	Growth7
Xylose	None	None8
Arabinose	None	None8
Starch	None	None

Section 4

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Section 5

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Section 2.1

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Section 2.2

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Section 2.3

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Section 6

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Section 7

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References

1. Barth and Gaillardin, 1996
2. (Domínguez et al., 1998)
3. (Shusta et al. 1998)
4. Barth 2013
5. (Mhairi Workman, Philippe Holt, 2013)
6. H. Shafaghat, G.D. Najafpour. Growth Kinetics and Ethanol Productivity of Saccharomyces cerevisiae PTCC 24860 on Varius Carbon Sources. ISSN 1818-4952
7. Jansen, M. L. A., Daran-Lapujade, P., de Winde, J. H., Piper, M. D. W., & Pronk, J. T. (2004). Prolonged Maltose-Limited Cultivation of Saccharomyces cerevisiae Selects for Cells with Improved Maltose Affinity and Hypersensitivity. Applied and Environmental Microbiology, 70(4), 1956–1963. http://doi.org/10.1128/AEM.70.4.1956-1963.2004
8. Wisselink, H. W., Toirkens, M. J., del Rosario Franco Berriel, M., Winkler, A. A., van Dijken, J. P., Pronk, J. T., & van Maris, A. J. A. (2007). Engineering of Saccharomyces cerevisiae for Efficient Anaerobic Alcoholic Fermentation of l-Arabinose . Applied and Environmental Microbiology, 73(15), 4881–4891. http://doi.org/10.1128/AEM.00177-07