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.

Xanthophyllomyces dendrorhous is a basidiomycete yeast that synthesizes carotenoids, mainly astaxanthin, which are of great commercial interest. Currently, there are many unknown aspects related to regulatory mechanisms on the synthesis of carotenoids in this yeast. Our recent studies showed that changes in sterol levels and composition resulted in upregulation of genes in the mevalonate pathway required for the synthesis of carotenoid precursors, leading to increased production of these pigments. Sterol Regulatory Element-Binding Proteins (SREBP), called Sre1 in yeast, are conserved transcriptional regulators of sterol homeostasis and other cellular processes. Given the results linking sterols and carotenoids, we investigated the role of SREBP in sterol and carotenoid synthesis in X. dendrorhous. In this study, we present the identification and functional characterization of the X. dendrorhous SRE1 gene, which encodes the transcription factor Sre1. The deduced protein has the characteristic features of SREBP/Sre1 and binds to consensus DNA sequences in vitro. RNA-seq analysis and chromatin-immunoprecipitation experiments showed that genes of the mevalonate pathway and ergosterol biosynthesis are directly regulated by Sre1. The sre1^- mutation reduced sterol and carotenoid production in X. dendrorhous, and expression of the Sre1 N-terminal domain (Sre1N) increased carotenoid production more than twofold compared to wild-type. Overall, our results indicate that in X. dendrorhous transcriptional regulation of genes in the mevalonate pathway control production of the isoprenoid derivatives, carotenoids and sterol. Our results provide new insights into the conserved regulatory functions of SREBP/Sre1 and identify pointing to the SREBP pathway as a potential target to enhance carotenoid production in X. dendrorhous.

Melissa Gómez, Sebastián Campusano, María Soledad Gutiérrez, Dionisia Sepúlveda, Salvador Barahona, Marcelo Baeza, Victor Cifuentes, Jennifer Alcaíno

Xanthophyllomyces dendrorhous synthesizes astaxanthin, a carotenoid used in aquaculture. Astaxanthin is synthesized from metabolites of the mevalonate pathway, which are also precursors for sterols biosynthesis. The interruption of the CYP61 gene, which is involved in the synthesis of ergosterol (mutant CBS.cyp61^–), resulted in a phenotype that overproduces carotenoids due to the activation of the SREBP pathway. In this work, we constructed other mutants of ergosterol biosynthesis in this yeast to evaluate whether they have the same phenotype as mutant CBS.cyp61^–. By bioinformatic analysis, the ERG3 and ERG4 genes of X. dendrorhous were identified, and each gene was deleted in the wild-type strain. Mutants CBS.Δerg3 and CBS.Δerg4 did not produce ergosterol; CBS.Δerg3 primarily accumulated episterol, and CBS.Δerg4 primarily accumulated ergosta-5,7,22,24(28)-tetraenol. The transcription levels of the HMGS gene of the mevalonate pathway were evaluated by RT-qPCR, which showed a slight increase in CBS.Δerg4, but the transcription levels were still 10-fold lower than in strain CBS.cyp61^–. Both CBS.Δerg3 and CBS.Δerg4 did not overproduce carotenoids, even though they do not produce ergosterol. Thus, the results of this study indicate that the absence of ergosterol does not activate the SREBP pathway in X. dendrorhous, but rather it depends on other alterations in sterol composition.

Xanthophyllomyces dendrorhous is a basidiomycete yeast that naturally produces the red–orange carotenoid astaxanthin, which has remarkable antioxidant properties. The biosynthesis of carotenoids and sterols share some common elements that have been studied in X. dendrorhous. For example, their synthesis requires metabolites derived from the mevalonate pathway and in both specific pathways, cytochrome P450 enzymes are involved that share a single cytochrome P450 reductase, CrtR, which is essential for astaxanthin biosynthesis, but is replaceable for ergosterol biosynthesis. Research on the regulation of carotenoid biosynthesis is still limited in X. dendrorhous; however, it is known that the Sterol Regulatory Element-Binding Protein (SREBP) pathway, which is a conserved regulatory pathway involved in the control of lipid metabolism, also regulates carotenoid production in X. dendrorhous. This review addresses the similarities and differences that have been observed between mammal and fungal SREBP pathways and what it is known about this pathway regarding the regulation of the production of carotenoids and sterols in X. dendrorhous.