Eighteen New Oleaginous Yeast Species

J Ind Microbiol Biotechnol (2016) 43:887–900 DOI 10.1007/s10295-016-1765-3 BIOENERGY/BIOFUELS/BIOCHEMICALS Eighteen new oleaginous yeast species Luis A. Garay1 · Irnayuli R. Sitepu1,2 · Tomas Cajka3 · Idelia Chandra1 · Sandy Shi1 · Ting Lin1 · J. Bruce German4 · Oliver Fiehn3,5 · Kyria L. Boundy‑Mills1 Received: 15 February 2016 / Accepted: 28 March 2016 / Published online: 12 April 2016 © Society for Industrial Microbiology and Biotechnology 2016 Abstract Of 1600 known species of yeasts, about 70 are species were not previously reported to be oleaginous. Tria- known to be oleaginous, defined as being able to accu- cylglycerol profiles were suitable for biodiesel production. mulate over 20 % intracellular lipids. These yeasts have These results greatly expand the number of known oleagi- value for fundamental and applied research. A survey of nous yeast species, and reveal the wealth of natural diver- yeasts from the Phaff Yeast Culture Collection, University sity of triacylglycerol profiles within wild-type oleaginous of California Davis was performed to identify additional Basidiomycetes. oleaginous species within the Basidiomycota phylum. Fifty-nine strains belonging to 34 species were grown in Keywords Oleaginous yeast · Triacylglycerol · lipid inducing media, and total cell mass, lipid yield and Basidiomycete · Intracellular lipid · Biodiesel triacylglycerol profiles were determined. Thirty-two species accumulated at least 20 % lipid and 25 species accumulated over 40 % lipid by dry weight. Eighteen of these Introduction The term oleaginous is used to describe microorganisms L. A. Garay, I. R. Sitepu and T. Cajka contributed equally to the capable of accumulating at least 20 % intracellular oil realization of the manuscript and are co-first authors. by dry weight [47, 50]. Lipid synthesis and intracellular lipid accumulation in oleaginous microorganisms Electronic supplementary material The online version of this article (doi:10.1007/s10295-016-1765-3) contains supplementary including yeasts have been studied for many decades to material, which is available to authorized users. understand basic principles of oil synthesis and accumulation [1, 16, 29, 51, 52] and for development of tech- * Kyria L. Boundy‑Mills nologies to produce oleochemicals [10, 14, 15, 22, 48, [email protected] 49, 57, 67]. 1 Phaff Yeast Culture Collection, Department of Food Science Environmental conditions that trigger intracellular accu- and Technology, University of California, One Shields Ave, mulation of triacylglycerol (TG) in oleaginous yeasts, such Davis, CA 95616‑8598, USA as nutrient deprivation, have been identified and examined 2 Bioentrepreneurship Department, Indonesia International (reviewed in [57]). It has been known for many decades Institute for Life Sciences, Jalan Pulo Mas Barat Kav. 88, that when a nutrient such as nitrogen is depleted from the East Jakarta, DKI Jakarta 13210, Indonesia culture medium, a major shift in metabolism occurs, pro- 3 Metabolomics, UC Davis Genome Center, University tein synthesis is depressed, and fatty acid synthesis and of California Davis, 451 Health Sciences Drive, Davis, CA 95616, USA accumulation activated. Genetic and metabolic factors responsible for this shift have been studied in selected ole- 4 Department of Food Science and Technology, University of California, One Shields Ave, Davis, CA 95616, USA aginous yeast species, particularly in ascomycetous genera Lipomyces [33] and Yarrowia [7]. Historically, ascomyce- 5 Biochemistry Department, Faculty of Science, King Abdulaziz University, PO Box 80203, Jeddah 21589, tous yeasts have been favored because literature is more Saudi Arabia extensive, and genetic tools are available. 1 3 888 J Ind Microbiol Biotechnol (2016) 43:887–900 However, recent years have seen an increase in investi- Materials and methods gation of basidiomycetous oleaginous yeasts. For example the genome of basidiomycetous yeast Rhodosporidium tor- Chemicals and yeast strains uloides was sequenced, and genetic tools are being devel- oped [30, 32, 35, 64, 69]. The metabolic and genetic factors All chemicals were of analytical grade, except glucose (cat- that are responsible for increased fatty acid synthesis and alog number S25295B, Fisher Science Education Pennsyl- lipid accumulation in oleaginous species have been identi- vania, USA) which was technical grade. fied in some yeast species [69]. For example, the presence Yeast strains used in this study are listed in Table 1. of two acyl carrier proteins in the R. toruloides genome Saccharomyces cerevisiae UCDFST 61-351 was used as a suggests improved fatty acid synthase efficiency. Addition- negative control, because it has been reported that S. cerevi- ally, oleaginous yeasts display particular types of perilipin- siae wild strains are not oleaginous [54]. The yeast R. toru- like proteins (such as MPL1), which are key for lipid drop- loides UCDFST 67-52 was included as a positive control, let formation [8, 66]. since its oleaginous behavior has been highly studied [69]. Some taxonomic groups, such as ascomycetous genera Yeasts were obtained from the Phaff Yeast Culture Collec- Lipomyces and basidiomycetous genera Myxozyma, con- tion, University of California Davis, and are available for tain almost exclusively species that are high in lipid. Other research purposes (http://phaffcollection.ucdavis.edu). All groups, such as the Cryptococcus curvatus clade, contain yeasts were identified to species by ribosomal sequencing high lipid species amidst low lipid relatives. Furthermore, using standard methods [17, 19] (data not shown). The tax- oil accumulation can be highly variable among strains of onomic term “aff.” within the yeast species indicates that a given species [55, 56]. The broad taxonomic distribution the strain belongs to a novel, undescribed species that is of oleaginous yeasts suggests that multiple mechanisms of most closely related to the indicated species. high oil accumulation may have evolved in different clades. Identification of additional yeast species, and strains within Cultivation methods a species, that are capable of accumulating high intracellu- 1 1 lar lipids is therefore desirable to broaden the field of can- Medium A [63] with 50 g L− glucose, 0.1 g L− cal- 1 1 didate yeasts to be used in fundamental and applied studies. cium chloride, 0.5 g L− NH4Cl, 1.5 g L− yeast extract, 1 1 1 One of the most important needs for the future of biodiesel 7.0 g L− KH2PO4, 5 g L− Na2HPO4 2H2O, 1.5 g L− 1 · 1 is to identify new feedstocks with higher oil content [20], MgSO4 7H2O, 0.08 g L− FeCl3 6H2O, 10.0 mg L− · 1 · 1 preferably not competing with food [4]. The identification ZnSO4 7H2O, 0.07 mg L− MnSO4 H2O, 0.1 mg L− · 1 · of new high lipid oleaginous yeast is among the first steps CuSO4, and 0.063 mg L− Co(NO3)2 was used without [3] towards developing yeast based biodiesel technology in pH adjustment at all times. The starting pH of the medium agreement with the aforementioned need. Furthermore, the was 6.5. The estimated molar carbon to nitrogen ratio was metabolic foundation of oleaginicity is also shared in the 60.2:1, within the appropriate range for lipid accumulation production of other high value lipid classes, such as phos- [40]. pholipids [16], terpenoids, steroids [41], and other mol- Strains were revived from cryopreserved stocks and ecules containing non polar moieties, such as glycolipids. streaked on potato dextrose agar plates as described [56]. The common currency in the first metabolic steps towards Inocula from plates less than 7 days old were prepared production of these molecules is the creation of a robust by suspending a loopful of cells ( 0.2 g) in 5 mL deion- ≈ cytosolic acetyl coenzyme A pool [12], that can be con- ized sterile water at room temperature and transferring verted to fatty acids and further downstream to triacylglyc- 0.5 mL of cell suspension into 50 mL bioreaction conical erol and phospholipids, or to mevalonate and further down tubes (part number 229475, Celltreat Scientific Products, to the different carotenoid and steroid backbones [34]. Shirley, MA, USA) containing 9.5 mL of medium A. The The purpose of this study was to identify additional tubes were incubated at 200 rpm in a rotary shaker incuba- basidiomycetous yeasts that are capable of accumulating tor (Series 25, New Brunswick Scientific Co., Edison, NJ, high lipid levels. Taxonomic order Sporidiobolales was pri- USA) at 24 °C for 24 h. Five mL of the inocula were used marily targeted because numerous high lipid species reside to inoculate 95 mL of medium A in 500 mL baffled shake in this clade, including several species recently shown to be flasks covered with foam stoppers (part number L800-D, oleaginous [43, 55–57]. Members of this clade have useful Identi-Plugs®, Jaece Industrie, Inc., NY, USA) to increase characteristics for biotechnological development such as aeration. Cultures were incubated in a rotary shaker incuba- ability to utilize pentoses [31] and tolerate growth inhibi- tor (Series 25, New Brunswick Scientific Co., Edison, NJ, tors such as furans [59] or ionic liquids [60]. USA) at 24 °C for 7 days. 1 3 J IndMicrobiolBiotechnol(2016)43:887–900 Table 1 Taxonomic placement and intracellular lipid content of yeast strains used in this study 1 1 Phaff collection strain ID Species Source location and habitat Cell mass production, g L− % oil by dry weight Lipid output, g L− Genbank accession numbers Phylum Basidiomycota, class Microbotryomycetes, order Sporidiobolales UCDFST 04-877 (n 3) Rhodosporidium babjevae Olive fly, Davis, CA, USA 15.4 0.4 62.4 0.7 9.6 0.2 KU609429, KU609487 = ± ± ± UCDFST 05-775 (n 3) Rhodosporidium babjevae Dry sap of olive tree, Win- 14.8 0.2 57.9 5.1 8.6 0.7 KU609430, KU609488 = ters, CA, USA ± ± ± UCDFST 67-458 (n 2) Rhodosporidium babjevae Exudate of Betula ermani, 13.4 0.1 51.8 0.4 7.0 0.1 KU609433, KU609491 = Mt. Fuji, Japan ± ± ± UCDFST 68-916.1 (n 2) Rhodosporidium babjevae Insect frass in Alnus sp.

Load more