Analysis of the Yarrowia lipolytica proteome reveals subtle variations in expression levels between lipogenic and non-lipogenic conditions.

Oleaginous yeasts have the ability to store greater than 20% of their mass as neutral lipids, in the form of triacylglycerides. The ATP citrate lyase is thought to play a key role in triacylglyceride synthesis, but the relationship between expression levels of this and other related enzymes is not well understood in the role of total lipid accumulation conferring the oleaginous phenotype. We conducted comparative proteomic analyses with the oleaginous yeast, Yarrowia lipolytica, grown in either nitrogen-sufficient rich media or nitrogen-limited minimal media. Total proteins extracted from cells collected during logarithmic and late stationary growth phases were analyzed by 1D liquid chromatography, followed by mass spectroscopy. The ATP citrate lyase enzyme was expressed at similar concentrations in both conditions, in both logarithmic and stationary phase, but many upstream and downstream enzymes showed drastically different expression levels. In non-lipogenic conditions, several pyruvate enzymes were expressed at higher concentration. These enzymes, especially the pyruvate decarboxylase and pyruvate dehydrogenase, may be regulating carbon flux away from central metabolism and reducing the amount of citrate being produced in the mitochondria. While crucial for the oleaginous phenotype, the constitutively expressed ATP citrate lyase appears to cleave citrate in response to carbon flux upstream from other enzymes creating the oleaginous phenotype.

Lipid production in the under-characterized oleaginous yeasts, Rhodosporidium babjevae and Rhodosporidium diobovatum, from biodiesel-derived waste glycerol.

The growth, lipid production, and carbon use efficiency of two oleaginous yeasts, Rhodosporidium babjevae and Rhodosporidium diobovatum, were compared under nitrogen-limiting conditions with glycerol as the carbon source. Final biomass concentrations of R. babjevae and R. diobovatum were 9.4±0.80g/L and 12.0±0.82g/L, respectively, after 120h of growth. Final lipid accumulation in for R. babjevae and R. diobovatum were 34.9±3.0% cell dry weight (cdw) and 63.7±4.5% cdw, respectively. Biomass production increased to 9.9±0.2g/L for R. babjevae and 14.1±0.2g/L for R. diobovatum in medium containing biodiesel-derived waste glycerol, but lipid accumulation decreased in both species. In a 7L bioreactor with biodiesel-derived glycerol as carbon source, R. diobovatum produced 13.6±0.4g/L biomass and accumulated lipids to 50.7±2.2% cdw, matching flask experiments.

Oleaginous yeasts for biodiesel: current and future trends in biology and production.

Production of biodiesel from edible plant oils is quickly expanding worldwide to fill a need for renewable, environmentally-friendly liquid transportation fuels. Due to concerns over use of edible commodities for fuels, production of biodiesel from non-edible oils including microbial oils is being developed. Microalgae biodiesel is approaching commercial viability, but has some inherent limitations such as requirements for sunlight. While yeast oils have been studied for decades, recent years have seen significant developments including discovery of new oleaginous yeast species and strains, greater understanding of the metabolic pathways that determine oleaginicity, optimization of cultivation processes for conversion of various types of waste plant biomass to oil using oleaginous yeasts, and development of strains with enhanced oil production. This review examines aspects of oleaginous yeasts not covered in depth in other recent reviews. Topics include the history of oleaginous yeast research, especially advances in the early 20th century; the phylogenetic diversity of oleaginous species, beyond the few species commonly studied; and physiological characteristics that should be considered when choosing yeast species and strains to be utilized for conversion of a given type of plant biomass to oleochemicals. Standardized terms are proposed for units that describe yeast cell mass and lipid production.

Growth and neutral lipid synthesis by Yarrowia lipolytica on various carbon substrates under nutrient-sufficient and nutrient-limited conditions.

Growth and TAG production by Yarrowia lipolytica were compared for cells cultured in nitrogen-complete medium containing waste glycerol derived from biodiesel production, as well as pure glycerol, dextrose, or canola oil as the carbon sources. Growth and TAG production were also analyzed for Y. lipolytica cells cultured in nitrogen-limited media containing either pure glycerol or glycerol plus dextrose. Significantly greater amounts of TAGs were synthesized by Y. lipolytica cultured in minimal media compared to rich media (approximately 3-fold on dry weight basis when grown on glycerol). Cultures in minimal medium containing glycerol yielded 31% TAGs on a dry cell weight (dcw) basis, while cultures in minimal medium containing glycerol plus dextrose produced 38% TAGs (dcw), with glycerol consumption favored over dextrose consumption. Our results suggest that Y. lipolytica could serve as a source of TAGs for biodiesel production using crude waste glycerol generated by biodiesel synthesis.

Manipulation of culture conditions alters lipid content and fatty acid profiles of a wide variety of known and new oleaginous yeast species.

Oleaginous yeasts have been studied for oleochemical production for over 80 years. Only a few species have been studied intensely. To expand the diversity of oleaginous yeasts available for lipid research, we surveyed a broad diversity of yeasts with indicators of oleaginicity including known oleaginous clades, and buoyancy. Sixty-nine strains representing 17 genera and 50 species were screened for lipid production. Yeasts belonged to Ascomycota families, Basidiomycota orders, and the yeast-like algal genus Prototheca. Total intracellular lipids and fatty acid composition were determined under different incubation times and nitrogen availability. Thirteen new oleaginous yeast species were discovered, representing multiple ascomycete and basidiomycete clades. Nitrogen starvation generally increased intracellular lipid content. The fatty acid profiles varied with the growth conditions regardless of taxonomic affiliation. The dominant fatty acids were oleic acid, palmitic acid, linoleic acid, and stearic acid. Yeasts and culture conditions that produced fatty acids appropriate for biodiesel were identified.