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PNNL study of metabolic processes paves way to optimize lipids production in yeast Y. lipolytica

Lipid-derived biofuels have been proposed as a promising substitute for fossil fuels. The oleaginous ascomycete (sac fungus) yeast Yarrowia lipolytica accumulates large amounts of lipids and has potential as a biofuel producing organism; however, little is known about the key biological processes involved. To address this gap in knowledge, a recent study by a team from the Pacific Northwest National Laboratory (PNNL) identified and characterized major pathways involved in lipid accumulation from glucose in Y. lipolytica.

This study builds a platform for efforts to engineer the yeast to optimize lipid accumulation and maximize the yield of carbon-based products. Because lipids from Y. lipolytica have chemical properties similar to those of diesel fuel, they can be readily used as biodiesel using current vehicles and existing infrastructure at gas stations. Thus, harnessing lipids from Y. lipolytica could represent a practical approach for transitioning more quickly to a biofuel-based energy system.

Yarrowia lipolytica
Yarrowia lipolytica is a dimorphic ascomycete; in common media, the fungus grows as a mixture of yeast-like and short mycelial cells (the vegetative part of a fungus, comprising branching, threadlike hyphae).
A 2002 study (Ruiz-Herrera and Sentandreu) proposed that pH is the most important factor regulating the dimorphic transition. Mycelium formation was maximal at pH near neutrality and decreased as pH was lowered to become almost null at pH 3.
A 2014 study (Bellou et al.) concluded that dissolved oxygen concentration (DOC) should be considered as the major factor affecting yeast morphology. When growth occurred at low or zero DOC the mycelial and/or pseudomycelial forms predominated over the yeast form independently of the carbon and nitrogen sources used.

The researchers from EMSL, the Environmental Molecular Sciences Laboratory, at PNNL and their PNNL colleagues used a combination of metabolomic and lipidomic profiling approaches as well as microscopic techniques to identify and characterize the key pathways involved in de novo lipid accumulation from glucose in batch cultured, wild-type Y. lipolytica.

They found lipids accumulated rapidly and peaked at 48 hours during the five-day experiment, concurrent with altered amino acid metabolism. Accumulation of lipids to their maximum level occurred when cells began to synthesize amino acids in the presence of excess glucose—the major carbon source in the culture medium. However, the highest proportion of a biofuel-friendly lipid occurred at 24 hours, suggesting the biofuel quality of the lipids was highest prior to peak lipid accumulation.

By 72 hours, the cells with depleted glucose levels began to make thicker cell walls, possibly to protect themselves until they could find more favorable environmental conditions. From the perspective of a bioengineer interested in converting glucose input into lipid output, the development of a thicker cell wall under starvation conditions represents wasted carbon that would otherwise be used for lipid production. Therefore, genes involved in cell wall synthesis could be promising targets to improve the efficiency of lipid producing yeast strains.

For analysis of the lipids, the team took a portion of the lipids extract and converted them to fatty acid methyl esters (FAMEs). The bulk of the isolated FAMEs were C16:0, C16:1, C18:0, C18:1 and C18:2 chains, as previously found for wild-type Y. lipolytica. At lower, but still detectable levels are saturated C20:0, C22:0 and C24:0 chains. Saturated C16:0 and C18:0 chains generally have good fuel stability, lower NOx emission, higher ignition quality but lower cold flow properties than conventional diesel making them poor for cold weather, while C18:2 chains may be too unstable for use as a fuel.

Image
Transmission electron microscopy shows cell wall thickening during lipid accumulation. Both (A) confocal laser scanning microscopy and TEM show thickened cell wall morphology in later time points. Cells were stained for cell wall (blue) and lipid bodies (red) in (A). CW indicates the cell wall in (B). Pomraning et al. Click to enlarge.

The monounsaturated C16:1 and 18:1 chains have a cetane number above the lower acceptable limits in the US and EU and exhibit biofuel properties that balance stability, emissions, ignition quality and cold weather flow.

We found the highest concentration of C18:1 FAMEs after 48 h of aerobic growth in shake flasks, however, at 24 h, C18:1 chains make up a higher fraction of the total FAMEs. This indicates that the biofuel quality of the lipids was highest prior to peak lipid accumulation; the main difference in FAME data between 24 and 48 h being the increase in C18:2 FAMEs.

—Pomraning et al.

Taken together, the findings provide important insights into optimal timing and nutrient conditions for harnessing lipids from yeast. Moreover, the new comprehensive dataset describing lipid accumulation in Y. lipolytica will enable more detailed experiments to provide specific genetic targets for future metabolic engineering efforts.

In summary, we have used a variety of techniques to characterize lipid accumulation in the oleaginous yeast Y. lipolytica metabolically and microscopically. In our growth conditions we observed complex changes in intra- and extracellular metabolite levels during batch culture and correlate these with microscopically observed cellular features. Substantial effort has been applied in the past few years to understanding the nature of and engineering oleaginous yeast, but to date only limited work has been done to characterize lipid accumulation metabolically. Here we provide a comprehensive dataset describing lipid accumulation in Y. lipolytica that will enable more detailed experiments to understand the oleaginous nature of Yarrowia and provide genetic targets for future metabolic engineering efforts.

—Pomarang et al.

Resources

  • Pomraning KR, SW Wei, SA Karagiosis, YM Kim, AC Dohnalkova, BW Arey, EL Bredeweg, G Orr, TO Metz, and SE Baker (2015) “Comprehensive Metabolomic, Lipidomic and Microscopic Profiling of Yarrowia lipolytica during Lipid Accumulation Identifies Targets for Increased Lipogenesis.” PLoS One 10(4): e0123188 doi: 10.1371/journal.pone.0123188

  • Bellou S, Makri A, Triantaphyllidou IE, Papanikolaou S, Aggelis G. (2014) “Morphological and metabolic shifts of Yarrowia lipolytica induced by alteration of the dissolved oxygen concentration in the growth environment.” Microbiology 160(Pt 4):807-17. doi: 10.1099/mic.0.074302-0

  • José Ruiz-Herrera, Rafael Sentandreu (2002) “Different effectors of dimorphism in Yarrowia lipolytica” Archives of Microbiology Volume 178, Issue 6, pp 477-483 doi: 10.1007/s00203-002-0478-3

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