Two at once: simultaneous increased production of astaxanthin and mycosporines in a single batch culture using a Phaffia rhodozyma mutant strain.

Phaffia rhodozyma is a basidiomycetous yeast characterized by its production of the carotenoid pigment astaxanthin, which holds high commercial value for its significance in aquaculture, cosmetics and as nutraceutics, and the UV-B-absorbing compound mycosporine-glutaminol-glucoside (MGG), which is of great biotechnological relevance for its incorporation into natural sunscreens. However, the industrial exploitation has been limited to the production of astaxanthin in small quantities. On the other hand, the accumulation of MGG in P. rhodozyma was recently reported and could add value to the simultaneous production of both metabolites. In this work, we obtain a mutant strain that overproduces both compounds, furthermore we determined how the accumulation of each is affected by the carbon-to-nitrogen ratio and six biotic and abiotic factors. The mutant obtained produces 159% more astaxanthin (470.1 µg g-1) and 220% more MGG (57.9 mg g-1) than the parental strain (295.8 µg g-1 and 26.2 mg g-1 respectively). Furthermore, we establish that the carotenoids accumulate during the exponential growth phase while MGG accumulates during the stationary phase. The carbon-to-nitrogen ratio affects each metabolite differently, high ratios favoring carotenoid accumulation while low ratios favoring MGG accumulation. Finally, the accumulation of both metabolites is stimulated only by photosynthetically active radiation and low concentrations of hydrogen peroxide. The mutant strain obtained is the first hyper-productive mutant capable of accumulating high concentrations of MGG and astaxanthin described to date. The characterization of how both compounds accumulate during growth and the factors that stimulate their accumulation, are the first steps toward the future commercial exploitation of strains for the simultaneous production of two biotechnologically important metabolites.

Fatty acid composition of cold-adapted carotenogenic basidiomycetous yeasts.

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 alpha-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.

Rhodotorula mucilaginosa, a carotenoid producing yeast strain from a Patagonian high-altitude lake.

The red yeast Rhodotorula mucilaginosa strain CRUB 0138 (previously identified as R. lactosa) was isolated from a high-altitude Patagonian Lake Toncek (1700 m a.s.l.), and assigned with mucilaginosa species. Its biochemical, physiological and molecular features were assessed and compared to R. mucilaginosa PYCC 5166 type strain using a polyphasic approach; in addition, biomass and carotenoid pigment production at different C/N ratios were determined in an incubator shaker. Phenetic characterization by means of 70 current physiological tests including assimilation of aldaric acids and aromatic compounds, and also the ability to grow with amino acids as sole carbon sources, was carried out. According to numerical taxonomy calculations, similarity indexes between R. mucilaginosa CRUB 0138 and PYCC 5166 type strain were 0.86 and 0.77, corresponding to a complete set of physiological tests and MSP-PCR (Mini/Micro Satellite Primed PCR; (GTG)5, M13 and (GAC)5 primers were employed) fingerprinting. Killer activity against 2 native strains, Rhodosporidium kratochvilovae and R. mucilaginosa was detected. Maximum biomass-glucose conversion efficiency (87%) and maximum carotenoid yield (2.32 mg/L) were obtained at C/N = 5 in culture medium containing 10 and 40 g/L glucose, respectively. Different C/N ratios did not influence carotenoid pigment production but low C/N enhanced biomass yield.

Screening and typing of Patagonian wine yeasts for glycosidase activities.

AIMS: The purpose of this study was to select autochthonous glycosidase producer yeasts with potential use in industrial production of Patagonian red wines. METHODS AND RESULTS: The study was carried out in oenological autochthonous yeasts from Comahue region (Argentinean North Patagonia). A set of screenable yeast phenotypic characteristics indicative of their potential usefulness in more aromatic red wine production was defined and tested in both, Saccharomyces and non-Saccharomyces populations. Twelve isolates showing six different glycosidase phenotypes were selected and they were characterized at species and strain levels using molecular methods. A close correlation between molecular and phenotypic characteristics was observed. Five strains belonging to Candida guilliermondii, C. pulcherrima and Kloeckera apiculata with highest constitutive beta-glucosidase activity levels without anthocyanase activity were discriminated. Some of them also showed constitutive beta-xylosidase and inductive alpha-rhamnosidase activities. CONCLUSIONS: The extension of the selection of oenological yeast to non-Saccharomyces species provided strains possessing novel and interesting oenological characteristics which could have significant implications in the production of more aromatic young red wine. SIGNIFICANCE AND IMPACT OF THE STUDY: As these non-Saccharomyces are indigenous to wine, they can be used in mixed starters at the beginning or as pure cultures at the end fermentation to contribute in enhancing the wine nuance that is typical of this specific area.

Saccharomyces cerevisiae wine yeast populations in a cold region in Argentinean Patagonia. A study at different fermentation scales.

AIMS: To study the diversity and dynamics of indigenous Saccharomyces wine populations during Malbec spontaneous fermentation, a representative Patagonian red wine, at both industrial and laboratory scale. METHODS AND RESULTS: Two molecular techniques, including restriction fragment length polymorphism of mitochondrial (mt) DNA and polymorphism of amplified delta interspersed element sequences, were used for characterization of indigenous yeasts at strain level. The mtDNA restriction patterns showed the major discriminative power; however, by combining the two molecular approaches it was possible to distinguish a larger number of strains and, therefore, draw more representative conclusions about yeast diversity. Although a great diversity of wild Saccharomyces cerevisiae strains was observed, only nine represented more than half of the total Saccharomyces yeast biota analysed; five of these were common and took over the Malbec must fermentation in both vinifications. CONCLUSIONS: Many different indigenous S. cerevisiae strains were identified; nevertheless, the dominant strains in both industrial and laboratory vinification processes were just a few and the same. SIGNIFICANCE AND IMPACT OF THE STUDY: Small-scale fermentation appears to be a valuable tool in winemaking, one especially helpful in evaluating microbiological aspects of as well as possible interactions between inoculated selected strains and native strains.

Killer behaviour in wild wine yeasts associated with Merlot and Malbec type musts spontaneously fermented from northwestern Patagonia (Argentina).

The occurrence of killer wine yeasts in Comahue Region (Patagonia, Argentina) was studied. Wild wine yeasts were isolated from spontaneously fermenting Merlot and Malbec type musts. Out of 135 isolates analyzed 37% were sensitive to some well characterized killer toxins as K1-K10 and did not show killer activity (sensitive phenotype, S), 21% showed neutral phenotype (N) and 42% demonstrated killer activity (killer phenotype, K). All but two killer strains, identified as Candida pulcherrima and Kluyveromyces marxianus, were Saccharomyces cerevisiae. Additionally, all killer strains were sensitive to some killer reference strains, showing a killer-sensitive phenotype (KS); neither Saccharomyces or non-Saccharomyces wild yeasts were phenotype killer-resistant (KR). The incidence of the killer character varied with respect to fermentation stage and grape variety, increasing throughout fermentation (13-55% to 36-90%). Irrespective of grape must type, the neutral and sensitive yeasts were ever predominant at initial stages of fermentation. All but six neutral strains, identified as Saccharomyces cerevisiae, were Kloeckera apiculata.