Enzyme activities in oleaginous yeasts accumulating and utilizing exogenous or endogenous lipids

The activities of ATP: citrate lyase (ACL; EC 4. 1. 3. 8), carnitine acetyltransferase (CAT; EC 2. 3. 1. 7), NADP+-dependent isocitrate dehydrogenase (ICDH; EC 1. 1. 1. 42), isocitrate lyase (ICL; EC 4. 1. 3. 1) and malic enzyme (malate dehydrogenase; EC 1. 1. 1. 40) were measured in four oleaginous yeasts, Candida curvata D, Trichosporon cutaneum and two strains of Rhodosporidium toruloides, grown either to accumulate lipid, or to utilize their own lipid reserves or an exogenous supply of lipid. During lipid utilization, activities of ACL and malic enzyme diminished to low levels; CAT and ICL increased considerably in activity and ICDH activity was slightly increased but catalase (EC 1. 11. 1. 6) diminished in activity in both strains of R. toruloides. In all cases, yeasts utilizing exogenous lipid showed greater changes in enzyme activities than cells utilizing their endogenous reserves. Electron micrographs of Candida curvata D showed proliferation of peroxisomes in starved cells utilizing their own lipid reserve though peroxisomes were more in evidence when the yeast had been grown on exogenous lipid. In Lipomyces starkeyi, which shows only minimal utilization of its stored lipid and furthermore cannot grow on exogenous lipid, only the occasional peroxisome was seen when cells were starved of carbon.     

Pathways to acetyl-CoA formation in Candida albicans

The supply of acetyl units from the mitochondrion to the cytosol of Candida albicans appears to be dependent only upon the activity of carnitine acetyltransferase (CAT). The enzyme ATP:citrate lyase (ACL), the major source of acetyl units in oleaginous yeasts, is absent from C. albicans in both the mycelial and yeast forms. There appears to be no other active translocation of acetate or acetyl groups except via the action of carnitine acetyltransferase.     

尼罗红染色法筛选产油酵母及定量检测胞内油脂含量的研究

【目的】建立产油酵母筛选以及胞内油脂含量测定的简便方法。【方法】利用尼罗红与胞内油脂成分结合后在紫外光照射下发出荧光且荧光强弱与油脂含量相关的原理。通过在添加尼罗红的培养基中培养酵母,并观察菌落荧光的方法对385株深海酵母进行产油脂菌株筛选,利用26S rDNA D1/D2区序列分析方法对筛选获得的产油酵母菌株进行鉴定,并以其中的一株高产油脂酵母(2A00015)为试验菌株,建立了一套尼罗红染色快速测定油脂含量的方法。【结果】获得22株产油酵母,其中油脂含量最高可达62.9%,经分子鉴定后显示这22株酵母分别属于(Candida viswanathii)、近平滑假丝酵母(Candidaparapsilosis)、粘质红酵母(Rhodotorula mucilaginosa)、汉逊德巴利酵母(Debaryomyceshansenii)、季也蒙毕赤酵母(Pichia guilliermondii)以及Rhodosporidium paludigenum酵母。尼罗红染色快速测定油脂含量方法的最佳检测条件为:菌悬液OD600小于1.2,尼罗红浓度0.5 mg/L,染色时间5 min,激发波长488 nm,发射波长570 nm。该测定方法得到相对荧光强度与称重法得到油脂含量呈正相关性,R2=0.9637。     

Simultaneous utilization of glucose and xylose byCandida curvata D in continuous culture

Summary Of ten, mainly oleaginous, yeasts examined for the ability to use glucose and xylose simultaneously, only one,Candida curvata D, was found which could do so. This yeast was examined further in a single-stage chemostat wherein it produced similar biomass yields, lipid contents and fatty acids on glucose plus xylose mixed in varying proportions. This oleaginous yeast would therefore be capable of growing on hydrolysed wood and straw wastes as a potential source of single cell oil.     

Yeasts from sub-Antarctic region: biodiversity,enzymatic activities and their potential as oleaginous microorganisms

Various microbial groups are well known to produce a range of extracellular enzymes and other secondary metabolites. However, the occurrence and importance of investment in such activities have received relatively limited attention in studies of Antarctic soil microbiota. Sixty-one yeasts strains were isolated from King George Island, Antarctica which were characterized physiologically and identified at the molecular level using the D1/D2 region of rDNA. Fifty-eight yeasts (belonging to the genera Cryptococcus, Leucosporidiella, Rhodotorula, Guehomyces, Candida, Metschnikowia and Debaryomyces) were screened for extracellular amylolytic, proteolytic, esterasic, pectinolytic, inulolytic xylanolytic and cellulolytic activities at low and moderate temperatures. Esterase activity was the most common enzymatic activity expressed by the yeast isolates regardless the assay temperature and inulinase was the second most common enzymatic activity. No cellulolytic activity was detected. One yeast identified as Guehomyces pullulans (8E) showed significant activity across six of seven enzymes types tested. Twenty-eight yeast isolates were classified as oleaginous, being the isolate 8E the strain that accumulated the highest levels of saponifiable lipids (42 %).     

Genetic enhancement of fatty acid synthesis by targeting rat liver ATP:citrate lyase into plastids of tobacco

ATP:citrate lyase (ACL) catalyzes the conversion of citrate to acetyl-coenzyme A (CoA) and oxaloacetate and is a key enzyme for lipid accumulation in mammals and oleaginous yeasts and fungi. To investigate whether heterologous ACL genes can be targeted and imported into the plastids of plants, a gene encoding a fusion protein of the rat liver ACL with the transit peptide for the small subunit of ribulose bisphosphate carboxylase was constructed and introduced into the genome of tobacco. This was sufficient to provide import of the heterologous protein into the plastids. In vitro assays of ACL in isolated plastids showed that the enzyme was active and synthesized acetyl-CoA. Overexpression of the rat ACL gene led to up to a 4-fold increase in the total ACL activity; this increased the amount of fatty acids by 16% but did not cause any major change in the fatty acid profile. Therefore, increasing the availability of acetyl-CoA as a substrate for acetyl-CoA carboxylase and subsequent reactions of fatty acid synthetase has a slightly beneficial effect on the overall rate of lipid synthesis in plants.     

Eighteen new oleaginous yeast species

Of 1600 known species of yeasts, about 70 are known to be oleaginous, defined as being able to accumulate over 20 % intracellular lipids. These yeasts have value for fundamental and applied research. A survey of yeasts from the Phaff Yeast Culture Collection, University of California Davis was performed to identify additional oleaginous species within the Basidiomycota phylum. Fifty-nine strains belonging to 34 species were grown in lipid inducing media, and total cell mass, lipid yield and 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 species were not previously reported to be oleaginous. Triacylglycerol profiles were suitable for biodiesel production. These results greatly expand the number of known oleaginous yeast species, and reveal the wealth of natural diversity of triacylglycerol profiles within wild-type oleaginous Basidiomycetes.     

Candida zeylanoides as a new yeast model for lipid metabolism studies: effect of nitrogen sources on fatty acid accumulation

Lipid homeostasis is well-known in oleaginous yeasts, but there are few non-oleaginous yeast models apart from Saccharomyces cerevisiae. We are proposing the non-oleaginous yeast Candida zeylanoides QU 33 as model. The aim of this study was to investigate the influence of the carbon/nitrogen ratio and the type of nitrogen source upon oil accumulation by this yeast grown on shake flask cultures. The maximum biomass was obtained in yeast extract (2.39?±?0.19 g/l), followed by peptone (2.24?±?0.05 g/l), while the highest content of microbial oil (0.35?±?0.01 g/l) and the maximum lipid yield (15.63 %) were achieved with peptone. Oleic acid was the predominant cellular fatty acid in all culture media (>32.23 %), followed by linoleic (>15.79 %) and palmitic acids (>13.47 %). The highest lipid yield using glucose and peptone was obtained at the C/N ratio of 200:1.     

A comparison of phospholipase activity, cellular adherence and pathogenicity of yeasts

Phospholipase A and lysophospholipase activities were measured in the culture fluid and in the blastospores of Candida albicans. When phospholipase activity was measured in six yeasts (four strains of C. albicans and a single strain each of Candida parapsilosis and Saccharomyces cerevisiae) a correlation was found between this activity and two potential parameters of pathogenicity. The C. albicans isolates which adhered most strongly to buccal epithelial cells and were most pathogenic in mice had the highest phospholipase activities. Non-pathogenic yeasts, including C. albicans isolates which did not adhere and did not kill mice, had lower phospholipase activities.     

Identification of oleaginous yeast strains able to accumulate high intracellular lipids when cultivated in alkaline pretreated corn stover

Microbial oil is a potential alternative to food/plant-derived biodiesel fuel. Our previous screening studies identified a wide range of oleaginous yeast species, using a defined laboratory medium known to stimulate lipid accumulation. In this study, the ability of these yeasts to grow and accumulate lipids was further investigated in synthetic hydrolysate (SynH) and authentic ammonia fiber expansion (AFEX?)-pretreated corn stover hydrolysate (ACSH). Most yeast strains tested were able to accumulate lipids in SynH, but only a few were able to grow and accumulate lipids in ACSH medium. Cryptococcus humicola UCDFST 10-1004 was able to accumulate as high as 15.5 g/L lipids, out of a total of 36 g/L cellular biomass when grown in ACSH, with a cellular lipid content of 40 % of cell dry weight. This lipid production is among the highest reported values for oleaginous yeasts grown in authentic hydrolysate. Preculturing in SynH media with xylose as sole carbon source enabled yeasts to assimilate both glucose and xylose more efficiently in the subsequent hydrolysate medium. This study demonstrates that ACSH is a suitable medium for certain oleaginous yeasts to convert lignocellullosic sugars to triacylglycerols for production of biodiesel and other valuable oleochemicals.     

Fatty acids from oleaginous yeasts and yeast-like fungi and their potential applications

Purpose: Oleaginous yeasts, fatty acids biosynthesis and regulation in the oleaginous yeasts and the fatty acids from the oleaginous yeasts and their applications are reviewed in this article.

Results: Oleaginous yeasts such as Rhodosporidium toruloides, Yarrowia lipolytica, Rhodotorula mucilaginosa, and Aureobasidium melanogenum, which can accumulate over 50% lipid of their cell dry weight, have many advantages over other oleaginous microorganisms. The fatty acids from the oleaginous yeasts have many potential applications. Many oleaginous yeasts have now been genetically modified to over-produce fatty acids and their derivatives. The most important features of the oleaginous yeasts are that they have special enzymatic systems for enhanced biosynthesis and regulation of fatty acids in their lipid particles. Recently, some oleaginous yeasts such as R. toruloides have been found to have a unique fatty acids synthetase and other oleaginous yeasts such as A. melanogenum have a unique highly reducing polyketide synthase (HR-PKS) involved in the biosynthesis of hydroxyl fatty acids.

Conclusions: It is necessary to further enhance lipid biosynthesis using metabolic engineering and explore new applications of fatty acids in biotechnology.     

Diversity and characterization of cultivable oleaginous yeasts isolated from mangrove forests

A total of 198 yeasts were isolated from 140 samples collected from 7 mangrove forests in 4 provinces of Thailand, and were found to belong to 30 genera, 45 described species and at least 12 undescribed species based on their 26S rRNA (D1/D2 domain) gene sequence. The most prevalent species was Candida tropicalis, followed by Candida pseudolambica and Rhodosporidium paludigena. Lipid accumulation, as determined by Nile red staining, of the isolated yeasts revealed that 69 and 18 strains were positive and strongly positive, respectively, while quantitative analysis of the intracellular lipid accumulated in the latter indicated that 10 of these strains, Pseudozyma tsukubaensis (YWT7-2 and YWT7-3), Rhodotorula sphaerocarpa (YWW6-1 and SFL14-1SF), Saitozyma podzolica (YWT1-1, NS3-3 and NS10-2), Prototheca zopfii var. hydrocarbonea OMS6-1 and Prototheca sp. (YMTW3-1 and YMTS5-2), were oleaginous. In this study we found that under nitrogen depletion condition (155 C/N ratio) Pseudozyma tsukubaensis YWT7-2 accumulated the highest level of intracellular lipid at 32.4% (w/w, dry cell weight), with a broadly similar fatty acid composition to that in palm oil.     

A biochemical explanation for lipid accumulation in Candida 107 and other oleaginous micro-organisms.

The biochemical explanation for lipid accumulation was investigated principally in Candida 107 and, for comparison, in the non-oleaginous yeast Candida utilis. There were no significant differences between these two yeasts in their control of glucose uptake; in both yeasts, the rates of glucose uptake were independent of the growth rate and were higher in carbon-limited chemostat cultures than in nitrogen-limited cultures. There was no lipid turnover in either yeast, as judged from [14C]acetate uptake and subsequent loss of 14C from the lipid of steady-state chemostat cultures. Acetyl-CoA carboxylase from both yeasts was similar in most characteristics except that from Candida 107 was activated by citrate (40% activation at 1 mM). The enzyme from Candida 107 was relatively unstable and, when isolated from nitrogen-limited (lipid-accumulating) cultures, was accompanied by a low molecular weight inhibitor. The reason for lipid accumulation is attributed to the decrease in the intracellular concentration of AMP as cultures become depleted of nitrogen. As the NAD+-dependent isocitrate dehydrogenase of Candida 107, but not C. utilis, requires AMP for activity, the metabolism of citrate through the tricarboxylic acid cycle in the mitochondria becomes arrested. In Candida 107, but not in C. utilis, there is an active ATP:citrate lyase which converts the accumulating citrate, when it passes into the cytosol, into acetyl-CoA and oxaloacetate. The former product is then available for fatty acid biosynthesis which is stimulated by the high ATP concentration within the cells, by the activation of acetyl-CoA carboxylase by citrate and by the provision of NADPH generated as oxaloacetate is converted via malate to pyruvate. Similar characteristics were evident in oleaginous strains of Rhodotorula glutinis and Mucor circinelloides but not in non-oleaginous representatives of these species.     

Comparative Proteomics Profile of Lipid-Cumulating Oleaginous Yeast: An iTRAQ-Coupled 2-D LC-MS/MS Analysis

Accumulation of intracellular lipid in oleaginous yeast cells has been studied for providing an alternative supply for energy, biofuel. Numerous studies have been conducted on increasing lipid content in oleaginous yeasts. However, few explore the mechanism of the high lipid accumulation ability of oleaginous yeast strains at the proteomics level. In this study, a time-course comparative proteomics analysis was introduced to compare the non-oleaginous yeast Saccharomyces cerevisiae, with two oleaginous yeast strains, Cryptococcus albidus and Rhodosporidium toruloides at different lipid accumulation stages. Two dimensional LC-MS/MS approach has been applied for protein profiling together with isobaric tag for relative and absolute quantitation (iTRAQ) labelling method. 132 proteins were identified when three yeast strains were all at early lipid accumulation stage; 122 and 116 proteins were found respectively within cells of three strains collected at middle and late lipid accumulation stages. Significantly up-regulation or down-regulation of proteins were experienced among comparison. Essential proteins correlated to lipid synthesis and regulation were detected. Our approach provides valuable indication and better understanding for lipid accumulation mechanism from proteomics level and would further contribute to genetic engineering of oleaginous yeasts.     

Modeling lipid accumulation in oleaginous fungi in chemostat cultures. II: Validation of the chemostat model using yeast culture data from literature

A model that predicts cell growth, lipid accumulation and substrate consumption of oleaginous fungi in chemostat cultures (Meeuwse et al. in Bioproc Biosyst Eng. doi:, 2011) was validated using 12 published data sets for chemostat cultures of oleaginous yeasts and one published data set for a poly-hydroxyalkanoate accumulating bacterial species. The model could describe all data sets well with only minor modifications that do not affect the key assumptions, i.e. (1) oleaginous yeasts and fungi give the highest priority to C-source utilization for maintenance, second priority to growth and third priority to lipid accumulation, and (2) oleaginous yeasts and fungi have a growth rate independent maximum specific lipid production rate. The analysis of all data showed that the maximum specific lipid production rate is in most cases very close to the specific production rate of membrane and other functional lipids for cells growing at their maximum specific growth rate. The limiting factor suggested by Ykema et al. (in Biotechnol Bioeng 34:1268–1276, 1989), i.e. the maximum glucose uptake rate, did not give good predictions of the maximum lipid production rate.     

The presence of Candida spp and rods of Enterobacteriaceae family in the vaginal swab specimens

The aim of the present work was to determine the presence and quantity of yeasts (Candida) and rods of Enterobacteriaceae family in the ontocenosis of vagina in women. Also, we aimed at finding which species of Enterobacteriaceae accompany the yeasts. Furthermore we aimed at determining the etiological factor (Candida yeasts or Enterobacteriaceae rods) causing identical clinical symptoms such as burn, itch, leucorrhea. Vagina secretion collected from 649 patients constituted the material for examination Candida. In 137 patients (which constituted 21.1%) Candida yeasts were found (group I). These patients were additionally divided into 2 subgroups (I a and I b). Group I a consisted of 92 patients (14.2%) who exhibited the presence of Candida without the accompanying rods of Enterobacteriaceae and group I b consisting of 45 patients (6.9%), in whom Candida was found together with the rods. 171 patients (26.3%) were classified to group II, who had only Enterobacteriaceae rods in the vagina. Among Candida yeasts, the most common species were C. albicans, C. krusei, C. tropicalis. The most common rods of Enterobacteriaceae family were E. coli and P. mirabilis. The bacterium which most frequently accompanied the yeasts was E. coli. Considering the fact that patients in group I and patients in group II reported similar problems, the role of the etiological factor in the vaginal inflammatory processes should be analyzed.     

Characterization of oleaginous yeasts revealed two novel species: Trichosporon cacaoliposimilis sp. nov. and Trichosporon oleaginosus sp. nov

Two new species in the anamorphic basidiomycetous genus Trichosporon (Tremellomycetes, Agaricomycotina) were uncovered in a DNA sequence-based molecular analysis of oleaginous yeasts maintained in the ATCC Mycology Collection. One yeast is named as Trichosporon cacaoliposimilis sp. nov. for its capability of synthesizing and accumulating a large amount of lipids having a composition equivalent to that of natural cacao butter. The type strain is ATCC 20505(T), originally deposited as Trichosporon sp. The other can use food industry wastes and agricultural byproducts as the substrate for growth and accumulation of a high level of oil and accordingly is named Trichosporon oleaginosus sp. nov. The type strain is ATCC 20509(T), previously identified as Cryptococcus curvatus. Molecular phylogenetic analyses indicate that T. cacaoliposimilis is a novel taxon in the Gracile clade of the genus, close to T. gracile and T. dulcitum, and that T. oleaginosus belongs to the Cutaneum clade, with T. jirovecii as the closest sister taxon. Other oleaginous yeasts were identified as new strains of known taxa, T. insectorum, Candida orthopsilosis and C. palmioleophila.     

Biomass yields and energetic yields of oleaginous yeasts in batch culture

This article provides the analysis on biomass yields and energetic yields of the oleaginous yeasts. The biomass yields of the oleaginous yeasts are consistently lower than nonoleaginous microorganisms, whereas their energetic yields are higher. Data inconsistencies of literature are explained by the variation of energy contents of oleaginous yeasts.     

Yeasts associated with fresh and frozen pulps of Brazilian tropical fruits

The occurrence of yeasts on ripe fruits and frozen pulps of pitanga (Eugenia uniflora L), mangaba (Hancornia speciosa Gom.), umbu (Spondias tuberosa Avr. Cam.), and acerola (Malpighia glaba L) was verified. The incidence of proteolytic, pectinolytic, and mycocinogenic yeasts on these communities was also determined. A total of 480 colonies was isolated and grouped in 405 different strains. These corresponded to 42 ascomycetous and 28 basidiomycetous species. Candida sorbosivorans, Pseudozyma antarctica, C. spandovensis-like, C. spandovensis, Kloeckera apis, C. parapsilosis, Rhodotorula graminis, Kluyveromyces marxianus, Cryptococcus laurentii, Metchnikowia sp (isolated only from pitanga ripe fruits), Issatchenkia occidentalis and C. krusei (isolated only from mangaba frozen pulps), were the most frequent species. The yeast communities from pitanga ripe fruits exhibited the highest frequency of species, followed by communities from acerola ripe fruits and mangaba frozen pulps. Yeast communities from frozen pulp and ripe fruits of umbu had the lowest number of species. Except the yeasts from pitanga, yeast communities from frozen pulp exhibited higher number of yeasts than ripe fruit communities. Mycocinogenic yeasts were found in all of the substrates studied except in communities from umbu ripe fruits and pitanga frozen pulps. Most of the yeasts found to produce mycocins were basidiomycetes and included P. antarctica, Cryptococcus albidus, C. bhutanensis-like, R. graminis and R. mucilaginosa-like from pitanga ripe fruits as well as black yeasts from pitanga and acerola ripe fruits. The umbu frozen pulps community had the highest frequency of proteolytic species. Yeasts able to hydrolyse casein at pH 5.0 represented 38.5% of the species isolated. Thirty-seven percent of yeast isolates were able to hydrolyse casein at pH 7.0. Pectinolytic yeasts were found in all of the communities studied, excepted for those of umbu frozen pulps. The highest frequency of pectinolytic activity was found in mangaba frozen pulp communities. Around 30% of all isolates produced pectinases. The ability to split arbutin was observed in all communities ranging from 8% in yeasts from pitanga frozen pulps to 40.6% in acerola ripe fruit communities. Among 432 species tested, 125 were active for beta-glucosidase production, and Kloeckera apis, P. antarctica, C. sorbosivorans, and C. spandovensis-like were the most active species.     

Occurrence of Yeasts in Cloacae of Migratory Birds

Several species of yeast have been reported as pathogens in humans based on increases in immunodeficiency syndromes and as a result of immunosuppressant chemotherapy in cancer treatment. Domestic and wild birds are known to act as carriers of human pathogenic fungi. To gain additional information on the yeasts present in the cloacae of some species of migratory birds, 421 wild birds (24.39% out of 1726 birds caught in Romania, Hungary and Bulgaria) were sampled with the permission of the local judicial authority. The state of conservation of the birds (i.e. post-mortem alterations, colour of the mucosae etc.), along with their age and sex were determined. Samples were collected directly from the cloacae and cultured, and colonies were identified in each positive sample. Yeasts were isolated from 15.7% of the animals sampled, with the highest percentage found in coots (Fulica atra −58.8%) and the lowest in quails (Coturnix coturnix −1.7%). A total of 131 isolates belonging to 15 species of yeast were identified. Rhodotorula rubra was the yeast with the highest number of isolates (28.2%), followed by Cryptococcus albidus (18.4%), Candida albicans (9.2%), Trichosporon cutaneum (8.4%), Candida guilliermondii (6.1%), Candida tropicalis (6.1%) and other species. The present study represents the first survey on the occurrence of yeasts in the cloacae of migratory birds. The prevalence and species of yeasts isolated is discussed on the basis of the ecology, diet, and habitat of the birds.