Fatty acid composition of cold-adapted carotenogenic basidiomycetous yeasts

D. Libkind^1*; M. T. Arts², M. Van Broock¹

¹ Laboratorio de Microbiología Aplicada y Biotecnología, INIBIOMA: Universidad Nacional del Comahue - CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas, Bariloche, Río Negro, Argentina;
² National Water Research Institute, Environment Canada, 867 Lakeshore Road, Burlington, Ontario, L7R 4A6, Canada.

*Correspondence. E-mail: libkind@crub.uncoma.edu.ar

ABSTRACT

We studied fatty acids (FAs) profiles in six carotenoid-producing yeast species isolated from temperate aquatic environments in Patagonia. Total FAs ranged from 2 to 15% of dry biomass. Linoleic, oleic, palmitic and α-linolenic acids were the major FAs constituents, which accounted for as much as 40%, 34%, 13% and 9% of total FAs, respectively. The proportion of each FA varied markedly depending on the taxonomic affiliation of the yeast species and on the culture media used. The high percentage of polyunsaturated fatty acids (PUFAs) found in Patagonian yeasts, in comparison to other yeasts, is indicative of their cold-adapted metabolism. Our results suggest that Patagonian yeasts may be considered an interesting source of essential PUFAs.

Key words: Native yeasts; Patagonia; Carotenoids; Lipids; Fatty acids.

RESUMEN

Composición de ácidos grasos de levaduras carotenogénicas. Se estudiaron los ácidos grasos de seis especies de levaduras productoras de carotenoides aisladas de ambientes acuáticos templados de la Patagonia. El contenido total de ácidos grasos fluctuó entre 2 y 15% de la biomasa seca. Los ácidos linoleico, oleico, palmítico y α-linolénico fueron los mayoritarios y contribuyeron en un 40%, 34%, 13% y 9% al total de ácidos grasos, respectivamente. La proporción de cada ácido graso varió de modo considerable según la filiación taxonómica de la especie y el medio de cultivo utilizado. El alto porcentaje de ácidos grasos poliinsaturados (PUFA) observado en las levaduras patagónicas, en comparación con levaduras de otros orígenes, es indicativo de un metabolismo adaptado al frío. Nuestros resultados sugieren que las levaduras patagónicas pueden ser consideradas una fuente interesante de PUFA esenciales.

Palabras clave: Levaduras nativas; Patagonia; Carotenoides; Lípidos; Ácidos grasos.

The search for unconventional sources of naturallyoccurring fatty acids (FAs) is of increasing interest given their well publicized nutritional, physiological and pharmacological functions. It is now possible to produce specific oils on an industrial scale, using lipid-producing microorganisms ⁽⁸⁾. Yeast species can accumulate high levels of intracellular lipids ⁽³⁾. Most of the lipids produced by oleaginous yeasts are long-chain FAs comparable to conventional vegetable oils ⁽⁹⁾, and may include polyunsaturated fatty acids (PUFAs). This is of significant interest because there is ample evidence that a subset of the PUFAs, termed essential fatty acids (EFAs), plays crucial physiological and nutritional roles in animals and humans ⁽¹²⁾.
Many oleaginous yeasts also accumulate carotenoid pigments, and are therefore called red yeasts. Carotenoids are generally localized in lipid droplets in these microorganisms and also in other fungi ⁽²⁾. Red yeasts are a diverse group of unrelated organisms (mostly Basidiomycota) and the majority of the known species are distributed in four taxonomic groups (Orders): the Sporidiobolales and Cystobasidiales/ Erythrobasidiales of the Pucciniomycotina, and the Cystofilobasidiales and Tremellales of the Hymenomycotina ⁽¹⁰⁾.
Numerous studies have dealt with FA production by red yeasts ^{(1, 7, 13)}, however, they have only investigated a limited number of red yeast species within the Sporidiobolales. Thus, the aim of our research was to study FA production in a set of recently isolated yeast strains from temperate aquatic environments in Patagonia. We chose representative species of the four major red yeast groups and thus FA synthesis was also discussed from a taxonomic point of view. Finally, it was also of interest to assess PUFA production by these coldadapted yeasts.
Six pigmented yeast strains, isolated from Patagonian ultra oligotrophic aquatic environments, were selected for FA composition studies (Table 1), as described in Libkind et al. ⁽⁴⁾. Strain identification was performed by combining conventional and molecular techniques ⁽⁴⁾. Cultures were maintained as described in Libkind et al. ⁽⁴⁾.

Table 1.
Yeast strains studied, taxonomic placement and other relevant information

Yeasts were grown in 250 ml Erlenmeyer flasks containing 50 ml of one of the two following media: MYP (same as above but without agar) and minimal medium salts (MMS), composed of 10.0 g/l glucose, 2.0 g/l (NH₄)₂SO₄, 2.0 g/l KH₂PO₄, 0.5 g/l MgSO₄·7H₂O, 0.1 g/l CaCl₂·2H₂O, and 1.0 g/l yeast extract (initial pH 5). Inocula consisted of 5% v/v of 36 h cultures using the same propagation media and culture conditions. Inoculated flasks were incubated in an INNOVA 4000 rotary incubator at 21 °C and 200 rpm. After 72 h, yeast biomass was harvested by centrifugation, washed twice with distilled water and lyophilized in a HETO (Dry Winner) freeze drier. Samples were kept under a nitrogen atmosphere, at -20 °C until further processing. Cell dry mass was determined after drying at 105 °C until constant weight.
Fatty acid methyl esters (FAMEs) were obtained in a three-step process: extraction, derivatization, and quantification on a gas chromatograph (GC) following the procedures outlined in Schlechtriem et al. ⁽¹¹⁾. FAME concentrations were quantified on a Hewlett Packard 6890 GC configured as in Schlechtriem et al. ⁽¹¹⁾. A 37-component FAME standard (Supelco #47885-U) was used to identify and quantify (4-point calibration curves) FAMEs in the samples (unknown) i.e. by comparing their retention times to those of the FAME standard. Results were reported as μg FAME/mg dry weight fungal tissue or as proportions (% contribution of any individual FA to total measured FAs). Statistical analysis of FA values between the two culture media was carried out using the Student's t-test.
Six different basidiomycetous yeast species were studied, distributed among the four taxonomic groups that include the majority of known carotenogenic yeasts (Table 1), four of which corresponded to novel taxa ⁽⁴⁾. All seven species accumulated FAs when grown on the two experimental semi-synthetic media, though with considerable qualitative and quantitative differences. Total lipids, as a percentage of dry biomass, ranged from 2.2 to 14.7% in Rhodotorula minuta and Sporobolomyces patagonicus, respectively (Table 2). These values are similar to those found in the literature for closely related species ⁽⁷⁾ and yeasts in general ⁽³⁾. MYP stimulated FA accumulation (p < 0.05) in the three Sporidiobolales species, while members of the other 3 groups showed higher, though not statistically significant, FA percentages in MMS (p = 0.225).

Table 2.
Fatty acid profiles of six Patagonian carotenogenic yeast species using two different culture media.

Saturated fatty acids (SAFAs), FAs with one double bond (MUFAs) and PUFAs were detected in yeast tissues in average proportions of 16%, 34% and 50% of total FAs, respectively. Omega-3 and omega-6 PUFAs were accumulated in average proportions of ~9% and ~40%, respectively. We also observed an influence of culture media on SAFA, MUFA and PUFA proportions. Significant higher values of SAFAs (p < 0.001) were obtained when MYP was employed as culture medium. PUFA values showed also a slight increase in MYP (p = 0.111). MMS medium stimulated the production of MUFAs (p = 0.023) in all yeast species.
The analysis of FA composition in all 6 yeasts, revealed the prevalence of four major FA constituents in red yeasts: linoleic acid (18:2n-6, in an average proportion of 40% of total FA), oleic acid (18:1n-9, ~34%), palmitic acid (16:0, ~13%) and α-linolenic acid (18:3n-3, ~9%). Figures 1A and B show the distribution of these FA among red yeasts as a function of the culture media used. These results correspond to those already reported for other yeasts ^{(3, 7, 13, 15)}. However, it is worth noting that Patagonian yeasts had a higher percentage of PUFAs (particularly linoleic and α-linolenic acids) in comparison to yeasts of different origin. Native strains (especially those from groups other than Sporidiobolales) produced in general, PUFA values > 50% of total FAs; whereas, in most yeasts, the sum of linoleic and α-linolenic acids rarely exceed 30% ^{(7, 15)}. Similarly to our results, yeast species isolated from Antarctic ecosystems ⁽¹³⁾ and species considered psycrophilic ⁽⁶⁾ were reported as being enriched by PUFAs. Thus, Patagonian strains, had FA profiles typical of cold-adapted yeasts. This observation is in agreement with the low annual average temperature (< 10 °C) registered at the region of isolation. Furthermore, it can be hypothesized that intracellular carotenoids in yeasts may account for at least one of several, lipid-based, protection mechanisms against oxidative damage ⁽¹⁴⁾ and that red yeasts should be capable of accumulating high quantities of PUFAs. Most of the yeasts studied in the present work, showed high levels of intracellular carotenoid pigments (200-300 μg/g) ⁽⁵⁾, which, in addition to their adaptation to cold environments, is a probable explanation for the high proportion of PUFAs observed.

Figure 1.
Relative composition of four major fatty acids found in carotenogenic yeasts. Relative percentages of the four major fatty acids (> 5% on a dry weight basis; as a proportion of total identified FAMEs) found in carotenogenic yeasts grown in Malt Yeast extract Peptone (MYP; A) and Minimum Medium Salt (MMS; B). Rh.: Rhodotorula, Sp.: Sporobolomyces, S.: Sporidiobolus, Cyst.: Cystofilobasidium.

The production of additional FAs at trace concentrations (< 2% of total FA) was also observed. Stearic (18:0), undecaenoic (11:0), palmitoleic (16:1n7), heptadecaenoic (17:0) and elaidic (18:1n9t) acids were detected in all yeasts, while myristic (14:0), pentadecaenoic (15:0), linoleic (18:2n6c), arachidonic (20:0), γ-linolenic (18:3n6), heneicosanoeic (21:0), cis-11,14-eicosadienoic (20:2), eicosatrienoic (20:3n3), erucic (22:1n9) and nervonic (24:1n9) acids were also found in a few cases. S. patagonicus accumulated the broadest variety of different FAs while Sporidiobolus longiusculus produced the longest FAs (C22 and C24).
We observed that different species showed different behaviors depending on their phylogenetic position. For example, Sporidiobolales yeasts showed lower quantities of omega-6 PUFAs either in MYP (p < 0.001) or MMS (p < 0.05), and higher amounts of MUFAs and lower of PUFAs were observed in MMS broth (p < 0.05). These results indicate an heterogeneous response to the culture media used depending on the taxonomic affiliation of the yeast species. This information is useful for optimization purposes since not all taxonomic groups will respond similarly to changes in culture conditions. Furthermore, we speculate that Sporidiobolales yeasts, which are those that have been typically studied as sources of FAs, may not necessarily be the best choice for PUFA production because the other groups of carotenogenic yeasts examined here produced higher PUFA proportions and demonstrated a greater range of plasticity in their FA profiles in response to the different culture media used. However, we stress that this conclusion must be considered tentative both because the limited number of species and the relatively narrow range of culture conditions herein examined. We conclude that these Patagonian yeast species can be considered an interesting source of carotenoid pigments, PUFAs and essential lipids; and that a more thorough assessment of their potential utilization as a biotechnological supply of these compounds should be carried out.

Acknowledgements: This work was partially funded by the Universidad Nacional del Comahue, Consejo Nacional de Investigaciones Científicas y Tecnológicas, CONICET (Project PIP 6536) & Agencia Nacional de Investigaciones Científicas, ANPCyT (Projects PICTR2003-00528 and PICT2004-22200).

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Recibido: 25/04/08
Aceptado: 19/08/08.