Background: The yeast Xanthophyllomyces dendrorhous synthesizes astaxanthin, a carotenoid with high commercial interest. The proposed biosynthetic route in this organism is isopentenyl-pyrophosphate (IPP) → geranyleranyl pyrophosphate (GGPP) → phytoene → lycopene → β-carotene → astaxanthin. Recently, it has been published that the conversion of β-carotene into astaxanthin requires only one enzyme, astaxanthin synthase or CrtS, encoded by crtS gene. This enzyme belongs to the cytochrome P450 protein family. Results: In this work, a crtR gene was isolated from X. dendrorhous yeast, which encodes a cytochrome P450 reductase (CPR) that provides CrtS with the necessary electrons for substrate oxygenation. We determined the structural organization of the crtR gene and its location in the yeast electrophoretic karyotype. Two transformants, CBSTr and T13, were obtained by deleting the crtR gene and inserting a hygromycin B resistance cassette. The carotenoid composition of the transformants was altered in relation to the wild type strain. CBSTr forms yellow colonies because it is unable to produce astaxanthin, hence accumulating β-carotene. T13 forms pale colonies because its astaxanthin content is reduced and its β-carotene content is increased. Conclusion: In addition to the crtS gene, X. dendrorhous requires a novel gene, crtR, for the conversion of β-carotene to astaxanthin.

The yeast Xanthophyllomyces dendrorhous is one of the most promising and economically attractive natural sources of astaxanthin. The biosynthesis of this valuable carotenoid is a complex process for which the regulatory mechanisms remain mostly unknown. Several studies have shown a strong correlation between the carbon source present in the medium and the amount of pigments synthesized. Carotenoid production is especially low when high glucose concentrations are used in the medium, while a significant increase is observed with non-fermentable carbon sources. However, the molecular basis of this phenomenon has not been established.

Pectinolytic enzymes, which are used in several industries, especially in the clarification process during wine and fruit juice production, represent approximately 10% of the global enzyme market. To prevent the proliferation of undesired microorganisms, to retain labile and volatile flavor compounds, and to save energy, the current trend is to perform this process at low temperatures. However, the commercially available pectinases are highly active at temperatures approximately 50 °C and poorly active at temperatures below 35 °C, which is the reason why there is a constant search for cold-active pectinases. In preliminary studies, pectinolytic activity was detected in cold-adapted yeasts and yeast-like microorganisms isolated from Antarctica. The aim of the present work was to characterize pectinases secreted by these microorganisms and to express the best candidate in Pichia pastoris. Degradation of pectin by extracellular protein extracellular extracts obtained from 12 yeast cultures were assayed in plates at 4 °C to 37 °C and pH from 5.4 to 7.0, obtaining positive results in samples obtained from Dioszegia sp., Phenoliferia glacialis and Tetracladium sp. An enzyme was purified from Tetracladium sp., analyzed by peptide mass fingerprinting and compared to genome and transcriptome data from the same microorganism. Thus, the encoding gene was identified corresponding to a polygalacturonase-encoding gene. The enzyme was expressed in Pichia pastoris, and the recombinant polygalacturonase displayed higher activity at 15 °C than a mesophilic counterpart. Extracellular pectinase activity was found in three yeast and yeast-like microorganisms from which the highest activity was displayed by Tetracladium sp., and the enzyme was identified as a polygalacturonase. The recombinant polygalacturonase produced in P. pastoris showed high activity at 15 °C, representing an attractive candidate to be applied in clarification processes in the production of fermented beverages and fruit juices.

Microorganisms have evolved a number of mechanisms to thrive in cold environments, including the production of antifreeze proteins, high levels of polyunsaturated fatty acids, and ergosterol. In this work, several yeast species isolated from Antarctica were analyzed with respect to their freeze-thaw tolerance and production of the three abovementioned compounds, which may also have economic importance. The freeze-thaw tolerance of yeasts was widely variable among species, and a clear correlation with the production of any of the abovementioned compounds was not observed. Antifreeze proteins that were partially purified from Goffeauzyma gastrica maintained their antifreeze activities after several freeze-thaw cycles. A relatively high volumetric production of ergosterol was observed in the yeasts Vishniacozyma victoriae, G. gastrica and Leucosporidium creatinivorum, i.e., 19, 19 and 16 mg l− 1, respectively. In addition, a high percentage of linoleic acid with respect to total fatty acids was observed in V. victoriae (10%), Wickerhamomyces anomalus (12%) and G. gastrica (13%), and a high percentage of alpha linoleic acid was observed in L. creatinivorum (3.3%). Given these results, the abovementioned yeasts are good candidates to be evaluated for use in the production of antifreeze proteins, fatty acids, and ergosterol at the industrial scale.