Cloning and characterization of the

Among non-conventional yeasts of industrial interest, the dimorphic oleaginous yeast Yarrowia lipolytica appears as one of the most attractive for a large range of white biotechnology applications, from heterologous proteins secretion to cell factories process development. The past, present and potential applications of wild-type, traditionally improved or genetically modified Yarrowia lipolytica strains will be resumed, together with the wide array of molecular tools now available to genetically engineer and metabolically remodel this yeast. The present review will also provide a detailed description of Yarrowia lipolytica strains and highlight the natural biodiversity of this yeast, a subject little touched upon in most previous reviews. This work intends to fill this gap by retracing the genealogy of the main Yarrowia lipolytica strains of industrial interest, by illustrating the search for new genetic backgrounds and by providing data about the main publicly available strains in yeast collections worldwide. At last, it will focus on exemplifying how advances in engineering tools can leverage a better biotechnological exploitation of the natural biodiversity of Yarrowia lipolytica and of other yeasts from the Yarrowia clade.

Yarrowia lipolytica Strains and Their Biotechnological Applications: How Natural Biodiversity and Metabolic Engineering Could Contribute to Cell Factories Improvement.

Coproducts of algae and yeast-derived single cell oils: A critical review of their role in improving biorefinery sustainability.

A total of 11 yeast strains of Yarrowia lipolytica, Metschnikowia sp., Rhodotorula sp. and Rhodosporidium toruloides were grown under nitrogen-limited conditions with crude glycerol employed as substrate in shake flasks, presenting interesting dry cell weight (DCW) production. Three of these strains belonging to Metschnikowia sp. accumulated significant quantities of endopolysaccharides (i.e. the strain V.V.-D4 produced 11.0 g/L of endopolysaccharides, with polysaccharides in DCW ≈ 63% w/w). A total of six Y. lipolytica strains produced either citric acid or mannitol. Most of the screened yeasts presented somehow elevated lipid and polysaccharides in DCW values at the early steps of growth despite nitrogen appearance in the fermentation medium. Lipid in DCW values decreased as growth proceeded. R. toruloides DSM 4444 cultivated on media presenting higher glycerol concentrations presented interesting lipid-accumulating capacities (maximum lipid = 12.5 g/L, maximum lipid in DCW = 43.0-46.0% w/w, conversion yield on glycerol = 0.16 g/g). Replacement of crude glycerol by xylose resulted in somehow decreased lipid accumulation. In xylose/glycerol mixtures, xylose was more rapidly assimilated from glycerol. R. toruloides total lipids were mainly composed of triacylglycerols. Total cellular fatty acid composition on xylose presented some differences compared with that on glycerol. Cellular lipids contained mainly oleic and palmitic acid.

Production of added-value microbial metabolites during growth of yeast strains on media composed of biodiesel-derived crude glycerol and glycerol/xylose blends.

Crude glycerol (CG) and hemicellulose hydrolysate (HH) are low—value side-products of biodiesel transesterification and pulp—and paper industry or lignocellulosic ethanol production, respectively, which can be converted to microbial lipids by oleaginous yeasts. This study aimed to test the ability of oleaginous yeasts to utilise CG and HH and mixtures of them as carbon source. Eleven out of 27 tested strains of oleaginous yeast species were able to grow in plate tests on CG as sole carbon source. Among them, only one ascomycetous strain, belonging to Lipomyces starkeyi, was identified, the other 10 strains were Rhodotorula spec. When yeasts were cultivated in mixed CG/ HH medium, we observed an activation of glycerol conversion in the Rhodotorula strains, but not in L. starkeyi. Two strains—Rhodotorula toruloides CBS 14 and Rhodotorula glutinis CBS 3044 were further tested in controlled fermentations in bioreactors in different mixtures of CG and HH. The highest measured average biomass and lipid concentration were achieved with R. toruloides in 10% HH medium mixed with 55 g/L CG—19.4 g/L and 10.6 g/L, respectively, with a lipid yield of 0.25 g lipids per consumed g of carbon source. Fatty acid composition was similar to other R. toruloides strains and comparable to that of vegetable oils. There were big strain differences in the ability to convert CG to lipids, as only few of the tested strains were able to grow. Lipid production rates and yields showed that mixing GC and HH have a stimulating effect on lipid accumulation in R. toruloides and R. glutinis resulting in shortened fermentation time to reach maximum lipid concentration, which provides a new perspective on converting these low-value compounds to microbial lipids.

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Microbial lipid production from crude glycerol and hemicellulosic hydrolysate with oleaginous yeasts

Aims To assess the ability of various newly isolated or belonging in official collections yeast strains to convert biodiesel-derived glycerol (Gly) into added-value compounds. Methods and results Ten newly isolated yeast strains belonging to Debaryomyces sp., Naganishia uzbekistanensis, Rhodotorula sp. and Yarrowia lipolytica, isolated from fishes, metabolized Gly under nitrogen limitation. The aim of the study was to identify potential newly isolated microbial candidates that could produce single-cell oil (SCO), endopolysaccharides and polyols when these micro-organisms were grown on biodiesel-derived Gly. As controls producing SCO and endopolysaccharides were the strains Rhodotorula glutinis NRRL YB-252 and Cryptococcus curvatus NRRL Y-1511. At initial Gly (Gly0 ) ≈40 g l-1 , most strains presented remarkable dry cell weight (DCW) production, whereas Y. lipolytica and Debaryomyces sp. produced non-negligible quantities of mannitol and arabitol (Ara). Five strains were further cultivated at increasing Gly0 concentrations. Rhodotorula glutinis NRRL YB-252 produced 7·2 g l-1 of lipid (lipid in DCW value ≈38% w/w), whereas Debaryomyces sp. FMCC Y69 in batch-bioreactor experiment with Gly0 ≈80 g l-1 , produced 30-33 g l-1 of DCW and ~30 g l-1 of Ara. At shake-flasks with Gly0 ≈125 g l-1 , Ara of ~48 g l-1 (conversion yield of polyol on Gly consumed ≈0·62 g g-1 ) was achieved. Cellular lipids of all yeasts contained in variable concentrations oleic, palmitic, stearic and linoleic acids. Conclusions Newly isolated, food-derived and non-previously studied yeast isolates converted biodiesel-derived Gly into several added-value metabolites. Significance and impact of the study Alternative ways of crude Gly valorization through yeast fermentations were provided and added-value compounds were synthesized.

Isolation, identification and screening of yeasts towards their ability to assimilate biodiesel-derived crude glycerol: microbial production of polyols, endopolysaccharides and lipid.

Dimitrios Antoniou

Papers

Isolation, identification and screening of yeasts towards their ability to assimilate biodiesel-derived crude glycerol: microbial production of polyols, endopolysaccharides and lipid.

Production of added-value microbial metabolites during growth of yeast strains on media composed of biodiesel-derived crude glycerol and glycerol/xylose blends.