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Abstract Background Antarctica has been successfully colonized by microorganisms despite presenting adverse conditions for life such as low temperatures, high solar radiation, low nutrient availability and dryness. Although these “cold-loving” microorganisms are recognized as primarily responsible for nutrient and organic matter recycling/mineralization, the yeasts, in particular, remain poorly characterized and understood. The aim of this work was to study the yeast microbiota in soil and water samples collected on King George Island. Results A high number of yeast isolates was obtained from 34 soil and 14 water samples. Molecular analyses based on rDNA sequences revealed 22 yeast species belonging to 12 genera, with Mrakia and Cryptococcus genera containing the highest species diversity. The species Sporidiobolus salmonicolor was by far the most ubiquitous, being identified in 24 isolates from 13 different samples. Most of the yeasts were psychrotolerant and ranged widely in their ability to assimilate carbon sources (consuming from 1 to 27 of the 29 carbon sources tested). All species displayed at least 1 of the 8 extracellular enzyme activities tested. Lipase, amylase and esterase activity dominated, while chitinase and xylanase were less common. Two yeasts identified as Leuconeurospora sp. and Dioszegia fristingensis displayed 6 enzyme activities. Conclusions A high diversity of yeasts was isolated in this work including undescribed species and species not previously isolated from the Antarctic region, including Wickerhamomyces anomalus, which has not been isolated from cold regions in general. The diversity of extracellular enzyme activities, and hence the variety of compounds that the yeasts may degrade or transform, suggests an important nutrient recycling role of microorganisms in this region. These yeasts are of potential use in industrial applications requiring high enzyme activities at low temperatures.

Abstract Background Xanthophyllomyces dendrorhous is a basidiomycetous yeast that synthesizes astaxanthin, which is a carotenoid with a great biotechnological impact. The ergosterol and carotenoid synthesis pathways are derived from the mevalonate pathway, and in both pathways, cytochrome P450 enzymes are involved. Results In this study, we isolated and described the X. dendrorhous CYP61 gene, which encodes a cytochrome P450 involved in ergosterol biosynthesis. This gene is composed of nine exons and encodes a 526 amino acid polypeptide that shares significant percentages of identity and similitude with the C22-sterol desaturase, CYP61, from other fungi. Mutants derived from different parental strains were obtained by disrupting the CYP61 gene with an antibiotic selection marker. These mutants were not able to produce ergosterol and accumulated ergosta-5,8,22-trien-3-ol and ergosta-5,8-dien-3-ol. Interestingly, all of the mutants had a more intense red color phenotype than their respective parental strains. The carotenoid composition was qualitatively and quantitatively analyzed by RP-HPLC, revealing that the carotenoid content was higher in the mutant strains without major changes in their composition. The expression of the HMGR gene, which encodes an enzyme involved in the mevalonate pathway (3-hydroxy-3-methylglutaryl-CoA reductase), was analyzed by RT-qPCR showing that its transcript levels are higher in the CYP61 mutants. Conclusions These results suggest that in X. dendrorhous, ergosterol regulates HMGR gene expression by a negative feedback mechanism and in this way; it contributes in the regulation of the carotenoid biosynthesis.

Abstract Background 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. Results In this work, we showed that glucose caused transcriptional repression of the three genes involved in the synthesis of astaxanthin from geranylgeranyl pyrophosphate in X. dendrorhous, which correlates with a complete inhibition of pigment synthesis. Strikingly, this regulatory response was completely altered in mutant strains that are incapable of synthesizing astaxanthin. However, we found that addition of ethanol caused the induction of crtYB and crtS gene expression and promoted de novo synthesis of carotenoids. The induction of carotenogenesis was noticeable as early as 24 h after ethanol addition. Conclusion For the first time, we demonstrated that carbon source-dependent regulation of astaxanthin biosynthesis in X. dendrorhous involves changes at the transcriptional level. Such regulatory mechanism provides an explanation for the strong and early inhibitory effect of glucose on the biosynthesis of this carotenoid.

Abstract Background The yeast Xanthophyllomyces dendrorhous is used for the microbiological production of the antioxidant carotenoid astaxanthin. In this study, we established an optimal protocol for protein extraction and performed the first proteomic analysis of the strain ATCC 24230. Protein profiles before and during the induction of carotenogenesis were determined by two-dimensional polyacrylamide gel electrophoresis and proteins were identified by mass spectrometry. Results Among the approximately 600 observed protein spots, 131 non-redundant proteins were identified. Proteomic analyses allowed us to identify 50 differentially expressed proteins that fall into several classes with distinct expression patterns. These analyses demonstrated that enzymes related to acetyl-CoA synthesis were more abundant prior to carotenogenesis. Later, redox- and stress-related proteins were up-regulated during the induction of carotenogenesis. For the carotenoid biosynthetic enzymes mevalonate kinase and phytoene/squalene synthase, we observed higher abundance during induction and/or accumulation of carotenoids. In addition, classical antioxidant enzymes, such as catalase, glutathione peroxidase and the cytosolic superoxide dismutases, were not identified. Conclusions Our results provide an overview of potentially important carotenogenesis-related proteins, among which are proteins involved in carbohydrate and lipid biosynthetic pathways as well as several redox- and stress-related proteins. In addition, these results might indicate that X. dendrorhous accumulates astaxanthin under aerobic conditions to scavenge the reactive oxygen species (ROS) generated during metabolism.

Abstract 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 synthesizes astaxanthin, a high-value carotenoid with biotechnological relevance in the nutraceutical and aquaculture industries. However, enhancing carotenoid production through strain engineering remains an ongoing challenge. Recent studies have demonstrated that carotenogenesis in X. dendrorhous is regulated by the SREBP pathway, which includes the transcription factor Sre1, particularly in the mevalonate pathway that also produces precursors used for ergosterol synthesis. In this study, we explored a novel approach to enhance carotenoid synthesis by replacing the native crtE promoter, which drives geranylgeranyl pyrophosphate synthesis (the step where carotenogenesis diverges from ergosterol biosynthesis), with the promoter of the HMGS gene, which encodes 3-hydroxy-3-methylglutaryl-CoA synthase from the mevalonate pathway. The impact of this substitution was evaluated in two mutant strains that already overproduce carotenoids due to the presence of an active Sre1 transcription factor: CBS.cyp61-, which does not produce ergosterol and strain CBS.SRE1N.FLAG, which constitutively expresses the active form of Sre1. Wild-type strain CBS6938 was used as a control. Our results showed that this modification increased the crtE transcript levels more than threefold and fourfold in CBS.cyp61 - .pHMGS/crtE and CBS.SRE1N.FLAG.pHMGS/crtE, respectively, resulting in 1.43-fold and 1.22-fold increases in carotenoid production. In contrast, this modification did not produce significant changes in the wild-type strain, which lacks the active Sre1 transcription factor under the same culture conditions. This study highlights the potential of promoter substitution strategies involving genes regulated by Sre1 to enhance carotenoid production, specifically in strains where the SREBP pathway is activated, offering a promising avenue for strain improvement in industrial applications., (© 2024. The Author(s).)

Codon usage bias (CUB) has been described in viruses, prokaryotes, and eukaryotes and has been linked to several cellular and environmental factors, such as the organism's growth temperature, gene expression levels, and regulation of protein synthesis and folding. Most of the studies in this area have been conducted in bacteria and higher eukaryotes, in some cases with different results. In this study, a comparative analysis of CUB in yeasts isolated from cold and template environments was performed in order to evaluate the correlation of CUB with yeast optimal temperature of growth (OTG), gene expression levels, cellular function, and structure of encoded proteins. Among the main findings, highly expressed ORFs tend to have a more similar CUB within and between yeasts, and a direct correlation between codons ending in C and expression level was generally found. A low correspondence between CUB and OTG was observed, with an inverse correlation for some codons ending in C. The clustering of yeasts based on their CUB partially aligns with their OTG, being more consistent for yeasts with lower OTG. In most yeasts, the abundance of preferred codons was generally lower at the 5' end of ORFs, higher in segments encoding beta strand, lower in segments encoding extracellular and transmembrane regions, and higher in "translation" and "energy metabolism" pathways, especially in highly expressed ORFs. Based on our findings, it is suggested that the abundance and distribution of preferred and non-preferred codons along mRNAs contribute to proper protein folding and functionality by regulating protein synthesis rates, becoming a more important factor under conditions that require faster protein synthesis in yeasts., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Baeza, Sepulveda, Cifuentes and Alcaíno.)

The sterol regulatory element-binding protein (SREBP) pathway is an integral cellular mechanism that regulates lipid homeostasis, in which transcriptional activator SREBPs regulate the expression of various genes. In the carotenogenic yeast Xanthophyllomyces dendrorhous , Sre1 (the yeast SREBP homolog) regulates lipid biosynthesis and carotenogenesis, among other processes. Despite the characterization of several components of the SREBP pathway across various eukaryotes, the specific elements of this pathway in X. dendrorhous remain largely unknown. This study aimed to explore the potential regulatory mechanisms of the SREBP pathway in X. dendrorhous using the strain CBS. cyp61 - as a model, which is known to have Sre1 in its active state under standard culture conditions, resulting in a carotenoid-overproducing phenotype. This strain was subjected to random mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (NTG), followed by a screening methodology that focused on identifying mutants with altered Sre1 activation phenotypes. Single-nucleotide polymorphism (SNP) analysis of 20 selected mutants detected 5439 single-nucleotide variants (SNVs), narrowing them down to 1327 SNPs of interest after a series of filters. Classification based on SNP impact identified 116 candidate genes, including 49 genes with high impact and 68 genes with deleterious moderate-impact mutations. BLAST, InterProScan, and gene ontology enrichment analyses highlighted 25 genes as potential participants in regulating Sre1 in X. dendrorhous . The key findings of this study include the identification of genes potentially encoding proteins involved in protein import/export to the nucleus, sterol biosynthesis, the ubiquitin-proteasome system, protein regulatory activities such as deacetylases, a subset of kinases and proteases, as well as transcription factors that could be influential in SREBP regulation. These findings are expected to significantly contribute to the current understanding of the intricate regulation of the transcription factor Sre1 in X. dendrorhous , providing valuable groundwork for future research and potential biotechnological applications.

The Phaffia rhodozyma UCD 67-385 genome harbors a 7873 bp cluster containing DDGS , OMT, and ATPG , encoding 2-desmethy-4-deoxygadusol synthase, O-methyl transferase, and ATP-grasp ligase, respectively, of the mycosporine glutaminol (MG) biosynthesis pathway. Homozygous deletion mutants of the entire cluster, single-gene mutants, and the Δ ddgs -/- ;Δ omt -/- and Δ omt -/- ;Δ atpg -/- double-gene mutants did not produce mycosporines. However, Δ atpg -/- accumulated the intermediate 4-deoxygadusol. Heterologous expression of the DDGS and OMT or DDGS , OMT, and ATPG cDNAs in Saccharomyces cerevisiae led to 4-deoxygadusol or MG production, respectively. Genetic integration of the complete cluster into the genome of the non-mycosporine-producing CBS 6938 wild-type strain resulted in a transgenic strain (CBS 6938_MYC) that produced MG and mycosporine glutaminol glucoside. These results indicate the function of DDGS , OMT, and ATPG in the mycosporine biosynthesis pathway. The transcription factor gene mutants Δ mig1 -/- , Δcyc8 -/- , and Δ opi1 -/- showed upregulation, Δ rox1 -/- and Δ skn7 -/- showed downregulation, and Δ tup6 -/- and Δ yap6 -/- showed no effect on mycosporinogenesis in glucose-containing medium. Finally, comparative analysis of the cluster sequences in several P. rhodozyma strains and the four newly described species of the genus showed the phylogenetic relationship of the P. rhodozyma strains and their differentiation from the other species of the genus Phaffia .

Xanthophyllomyces dendrorhous is a natural source of astaxanthin and mycosporines. This yeast has been isolated from high and cold mountainous regions around the world, and the production of these secondary metabolites may be a survival strategy against the stress conditions present in its environment. Biosynthesis of astaxanthin is regulated by catabolic repression through the interaction between MIG1 and corepressor CYC8-TUP1. To evaluate the role of the stress-associated transcription factors SKN7, ROX1, and YAP6, we employed an omic and phenotypic approach. Null mutants were constructed and grown in two fermentable carbon sources. The yeast proteome and transcriptome were quantified by iTRAQ and RNA-seq, respectively. The total carotenoid, sterol, and mycosporine contents were determined and compared to the wild-type strain. Each mutant strain showed significant metabolic changes compared to the wild type that were correlated to its phenotype. In a metabolic context, the principal pathways affected were glycolysis/gluconeogenesis, the pentose phosphate (PP) pathway, and the citrate (TCA) cycle. Additionally, fatty acid synthesis was affected. The absence of ROX1 generated a significant decline in carotenoid production. In contrast, a rise in mycosporine and sterol synthesis was shown in the absence of the transcription factors SKN7 and YAP6, respectively.

A variety of long-term stress conditions may exist in fish cultivation, some of which are so severe that fish can no longer reestablish homeostasis. In teleost fish, the brain and gastrointestinal tract integrate signals that include the perception of stress factors regulating physiological responses, such as social stress by fish population density, where peripheral and central signals, such as peptide hormones, are the main regulators. Therefore, we proposed in this study to analyze the effect of different stock densities (SD) in the gene expression of brain neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP), together with the gastrointestinal peptide hormones leptin (Lep), vasointestinal peptide (VIP), and protachykinin-1 (Prk-1) in Salmo salar post-smolt. The coding sequence of S. salar VIP and Prk-1 precursors were firstly cloned and characterized. Then, the mRNA expression of these genes, together with the NPY, Lep, and CGRP genes, were evaluated in post-smolts kept at 11 Kg/m 3 , 20 Kg/m 3 , and 40 Kg/m 3 . At 14 days of culture, the brain CGRP and liver leptin mRNA levels increased three and tenfold in the post-smolt salmons kept at the highest SD, respectively. The high levels of leptin were kept during all the fish culture experiments. In addition, the highest expression of intestine VIP mRNA was obtained on Day 21 in the group of 40 Kg/m 3 returning to baseline on Day 40. In terms of stress biochemical parameters, cortisol levels were increased in the 20 Kg/m 3 and 40 Kg/m 3 groups on Day 40 and were the highest in the 20 Kg/m 3 group on Day 14. This study provides new insight into the gastrointestinal signals that could be affected by chronic stress induced by high stock density in fish farming. Thus, the expression of these peptide hormones could be used as molecular markers to improve production practices in fish aquaculture.

Cytochrome P450s (P450s) are heme-containing proteins involved in several cellular functions, including biosynthesis of steroidal hormones, detoxification of xenobiotic compounds, among others. Damage response protein 1 (Dap1) has been described as a positive regulator of P450s through protein-protein interactions in organisms such as Schizosaccharomyces pombe. Three P450s in the carotenogenic yeast Xanthophyllomyces dendrorhous have thus far been characterized: Cyp51 and Cyp61, which are involved in ergosterol biosynthesis, and CrtS (astaxanthin synthase), which is involved in biosynthesis of the carotenoid astaxanthin. In this work, we describe the X. dendrorhous DAP1 gene, deletion of which affected yeast pigmentation by decreasing the astaxanthin fraction and increasing the β-carotene (a substrate of CrtS) fraction, which is consistent with the known role of CrtS. We found that the proportion of ergosterol was also decreased in the Δdap1 mutant. However, even though the fractions of the end products of these two pathways (the synthesis of carotenoids and sterols) were decreased in the Δdap1 mutant, the transcript levels of genes from the P450 systems involved were higher than those in the wild-type strain. We demonstrate that Dap1 coimmunoprecipitates with these three P450s, suggesting that Dap1 interacts with these three proteins. We propose that Dap1 regulates the synthesis of astaxanthin and ergosterol in X. dendrorhous, probably by regulating the P450s involved in both biosynthetic pathways at the protein level. This work suggests a new role for Dap1 in the regulation of carotenoid biosynthesis in X. dendrorhous., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2021. The Author(s).)

Type II interferon gamma (IFNγ) is a pleiotropic cytokine capable of modulating the innate and adaptive immune responses which has been widely characterized in several teleost families. In fish, IFNγ stimulates the expression of cytokines and chemokines associated with the pro-inflammatory response and enhances the production of nitrogen and oxygen reactive species in phagocytic cells. This work studied the effect of IFNγ on the expression of cell-surface markers on splenocytes of Atlantic salmon ( Salmo salar ). In vitro results showed that subpopulations of mononuclear splenocytes cultured for 15 days were capable of increasing gene expression and protein availability of cell-surface markers such as CD80/86, CD83 and MHC II, after being stimulated with recombinant IFNγ. These results were observed for subpopulations with characteristics associated with monocytes (51%), and features that could be related to lymphocytes (46.3%). In addition, a decrease in the expression of zbtb46 was detected in IFNγ-stimulated splenocytes. Finally, the expression of IFNγ and cell-surface markers was assessed in Atlantic salmon under field conditions. In vivo results showed that the expression of ifnγ increased simultaneously with the up-regulation of cd80/86 , cd83 and mhcii during a natural outbreak of Piscirickettsia salmonis . Overall, the results obtained in this study allow us to propose IFNγ as a candidate molecule to stimulate the phenotypic progression of a small population of immune cells, which will increase antigen presenting cells markers. Thereby, modulatory strategies using IFNγ may generate a robust and coordinated immune response in fish against pathogens that affect aquaculture., Competing Interests: JA was employed by the company AquaAdvise. CS was employed by the company Salmones Camanchaca. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Morales-Lange, Ramírez-Cepeda, Schmitt, Guzmán, Lagos, Øverland, Wong-Benito, Imarai, Fuentes, Boltaña, Alcaíno, Soto and Mercado.)

Xanthophyllomyces dendrorhous is a basidiomycete yeast that produces carotenoids, mainly astaxanthin. Astaxanthin is an organic pigment of commercial interest due to its antioxidant and coloring properties. X. dendrorhous has a functional SREBP pathway, and the Sre1 protein is the SREBP homolog in this yeast. However, how sterol regulatory element (Sre)1 promotes the biosynthesis of sterols and carotenoids in X. dendrorhous is unknown. In this work, comparative RNA-sequencing analysis between modified X. dendrorhous strains that have an active Sre1 protein and the WT was performed to identify Sre1-dependent genes. In addition, Sre1 direct target genes were identified through ChIP combined with lambda exonuclease digestion (ChIP-exo) assays. SRE motifs were detected in the promoter regions of several Sre1 direct target genes and were consistent with the SREs described in other yeast species. Sre1 directly regulates genes related to ergosterol biosynthesis as well as genes related to the mevalonate (MVA) pathway, which synthesizes the building blocks of isoprenoids, including carotenoids. Two carotenogenic genes, crtE and crtR , were also identified as Sre1 direct target genes. Thus, carotenogenesis in X. dendrorhous is regulated by Sre1 through the regulation of the MVA pathway and the regulation of the crtE and crtR genes. As the crtR gene encodes a cytochrome P450 reductase, Sre1 regulates pathways that include cytochrome P450 enzymes, such as the biosynthesis of carotenoids and sterols. These results demonstrate that Sre1 is a sterol master regulator that is conserved in X. dendrorhous ., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 Gómez et al.)

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.Δ erg3 and CBS.Δ erg4 did not produce ergosterol; CBS.Δ erg3 primarily accumulated episterol, and CBS.Δ erg4 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.Δ erg4 , but the transcription levels were still 10-fold lower than in strain CBS. cyp61 - . Both CBS.Δ erg3 and CBS.Δ erg4 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., (Copyright © 2020 Venegas, Barahona, González, Sepúlveda, Zúñiga, Baeza, Cifuentes and Alcaíno.)

Xanthophyllomyces dendrorhous is a carotenogenic yeast with a singular metabolic capacity to produce astaxanthin, a valuable antioxidant pigment. This yeast can assimilate several carbon sources and sustain fermentation even under aerobic conditions. Since astaxanthin biosynthesis is affected by the carbon source, the study of carotenogenesis regulatory mechanisms is key for improving astaxanthin yield in X. dendrorhous This study aimed to elucidate the regulation of the metabolism of different carbon sources and the phenomenon of catabolic repression in this yeast. To this end, protein and transcript levels were quantified by iTRAQ (isobaric tags for relative and absolute quantification) and transcriptomic sequencing (RNA-seq) in the wild-type strain under conditions of glucose, maltose, or succinate treatment and in the mutant strains for genes MIG1 , CYC8 , and TUP1 under conditions of glucose treatment. Alternative carbon sources such as maltose and succinate affected the relative abundances of 14% of the wild-type proteins, which were mainly grouped into the carbohydrate metabolism category, with the glycolysis/gluconeogenesis and citrate cycle pathways being the most highly represented pathways. Each mutant strain showed significant proteomic profile changes, affecting approximately 2% of the total proteins identified, compared to the wild-type strain under glucose treatment conditions. Similarly to the results seen with the alternative carbon sources, the changes in the mutant strains mainly affected carbohydrate metabolism, with glycolysis/gluconeogenesis and the pentose phosphate and citrate cycle pathways being the most highly represented pathways. Our results showed convergence between carbon assimilation and catabolic repression in the strains studied. Interestingly, indications of cooperative, opposing, and overlapping processes during catabolic regulation were found. We also identified target proteins of the regulatory processes, reinforcing the likelihood of catabolic repression at the posttranscriptional level. IMPORTANCE The conditions affecting catabolic regulation in X. dendrorhous are complex and suggest the presence of an alternative mechanism of regulation. The repressors Mig1, Cyc8, and Tup1 are essential elements for the regulation of the use of glucose and other carbon sources. All play different roles but, depending on the growth conditions, can work in convergent, synergistic, and complementary ways to use carbon sources and to regulate other targets for yeast metabolism. Our results reinforced the belief that further studies in X. dendrorhous are needed to clarify a specific regulatory mechanism at the domain level of the repressors as well as its relationship with those of other metabolic repressors, i.e., the stress response, to elucidate carotenogenic regulation at the transcriptomic and proteomic levels in this yeast., (Copyright © 2020 Martinez-Moya et al.)

Xanthophyllomyces dendrorhous is a basidiomycete yeast known as a natural producer of astaxanthin, a carotenoid of commercial interest because of its antioxidant properties. Recent studies indicated that X. dendrorhous has a functional SREBP pathway involved in the regulation of isoprenoid compound biosynthesis, which includes ergosterol and carotenoids. SREBP is a major regulator of sterol metabolism and homeostasis in mammals; characterization in fungi also provides information about its role in the hypoxia adaptation response and virulence. SREBP protease processing is required to activate SREBP pathway functions in fungi. Here, we identified and described the STP1 gene, which encodes a metallopeptidase of the M50 family involved in the proteolytic activation of the transcription factor Sre1 of the SREBP pathway, in X. dendrorhous We assessed STP1 function in Δ stp1 strains derived from the wild-type and a mutant of ergosterol biosynthesis that overproduces carotenoids and sterols. Bioinformatic analysis of the deduced protein predicted the presence of characteristic features identified in homologs from mammals and fungi. The Δ stp1 mutation decreased yeast growth in the presence of azole drugs and reduced transcript levels of Sre1-dependent genes. This mutation also negatively affected the carotenoid- and sterol-overproducing phenotype. Western blot analysis demonstrated that Sre1 was activated in the yeast ergosterol biosynthesis mutant and that the Δ stp1 mutation introduced in this strain prevented Sre1 proteolytic activation. Overall, our results demonstrate that STP1 encodes a metallopeptidase involved in proteolytic activation of Sre1 in X. dendrorhous , contributing to our understanding of fungal SREBP pathways., (Copyright © 2020 Gómez et al.)

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 .

Background: 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., Results: 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., Conclusions: 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.

Background: 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., Results: 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%)., Conclusions: 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.

Double-stranded RNA (dsRNA) molecules are widely found in yeasts and filamentous fungi. It has been suggested that these molecules may play an important role in the evolution of eukaryote genomes and could be a valuable tool in yeast typing. The characterization of these extrachromosomal genetic elements is usually a laborious process, especially when trying to analyze a large number of samples. In this chapter, we describe a simple method to isolate dsRNA elements from yeasts using low amounts of starting material and their application to different Xanthophyllomyces dendrorhous strains and other psychrotolerant carotenogenic yeasts. Furthermore, the methodologies for enzymatic and hybridization characterizations and quantification of relative dsRNA abundance are detailed.

The use of enzymes in diverse industries has increased substantially over past decades, creating a well-established and growing global market. Currently, the use of enzymes that work better at ambient or lower temperatures in order to decrease the temperatures of production processes is desirable. There is thus a continuous search for enzymes in cold environments, especially from microbial sources, with amylases, proteases, lipases and, cellulases being the most studied. Other enzymes, such as glucose oxidase (GOD), invertase (Inv), and alkaline phosphatase (ALP), also have a high potential for application, but have been much less studied in microorganisms living in cold-environments. In this work, secretion of these three enzymes by Antarctic yeast species was analyzed, and five, three, and five species were found to produce extracellular GOD, Inv, and ALP, respectively. The major producers of GOD, Inv, and ALP were Goffeauzyma gastrica, Wickerhamomyces anomalus , and Dioszegia sp., respectively, from which the enzymes were purified and characterized. Contrary to what was expected, the highest GOD and Inv activities were found at 64°C and 60°C, respectively, and at 47°C for ALP. However, the three enzymes maintained a significant percentage of activity at lower temperatures, especially ALP that kept a 67 and 43% of activity at 10°C and 4°C, respectively.

In cold environments such as polar regions, microorganisms play important ecological roles, and most of our knowledge about them comes from studies of cultivable microorganisms. Metagenomic technologies are powerful tools that can give a more comprehensive assessment of microbial communities, and the amplification of rDNA followed by next-generation sequencing has given good results in studies aimed particularly at environmental microorganisms. Culture-independent studies of microbiota in terrestrial habitats of Antarctica, which is considered the driest, coldest climate on Earth, are increasing and indicate that micro-diversity is much higher than previously thought. In this work, the microbial diversity of terrestrial habitats including eight islands of the South Shetland Archipelago, two islands on the Antarctic Peninsula and Union Glacier, was studied by amplicon-metagenome analysis. Molecular analysis of the studied localities clustered together the islands of the South Shetland Archipelago, except Greenwich Island, and separated them from the Litchfield and Lagotellerie islands and Union Glacier, which is in agreement with the latitudinal difference among them. Among fungi, 87 genera and 123 species were found, of which species belonging to 37 fungal genera not previously cultivated from Antarctica were detected. Phylogenetic analysis, including the closest BLAST-hit sequences, clustered fungi in 11 classes being the most represented Lecanoromycetes and Eurotiomycetes.

Background: The cytochromes P450 (P450s) are a large superfamily of heme-containing monooxygenases involved in the oxidative metabolism of an enormous diversity of substrates. These enzymes require electrons for their activity, and the electrons are supplied by NAD(P)H through a P450 electron donor system, which is generally a cytochrome P450 reductase (CPR). The yeast Xanthophyllomyces dendrorhous has evolved an exclusive P450-CPR system that specializes in the synthesis of astaxanthin, a carotenoid with commercial potential. For this reason, the aim of this work was to identify and characterize other potential P450 genes in the genome of this yeast using a bioinformatic approach., Results: Thirteen potential P450-encoding genes were identified, and the analysis of their deduced proteins allowed them to be classified in ten different families: CYP51, CYP61, CYP5139 (with three members), CYP549A, CYP5491, CYP5492 (with two members), CYP5493, CYP53, CYP5494 and CYP5495. Structural analyses of the X. dendrorhous P450 proteins showed that all of them have a predicted transmembrane region at their N-terminus and have the conserved domains characteristic of the P450s, including the heme-binding region (FxxGxRxCxG); the PER domain, with the characteristic signature for fungi (PxRW); the ExxR motif in the K-helix region and the oxygen-binding domain (OBD) (AGxDTT); also, the characteristic secondary structure elements of all the P450 proteins were identified. The possible functions of these P450s include primary, secondary and xenobiotic metabolism reactions such as sterol biosynthesis, carotenoid synthesis and aromatic compound degradation., Conclusions: The carotenogenic yeast X. dendrorhous has thirteen P450-encoding genes having potential functions in primary, secondary and xenobiotic metabolism reactions, including some genes of great interest for fatty acid hydroxylation and aromatic compound degradation. These findings established a basis for future studies about the role of P450s in the carotenogenic yeast X. dendrorhous and their potential biotechnological applications.

Background: Amylases are used in various industrial processes and a key requirement for the efficiency of these processes is the use of enzymes with high catalytic activity at ambient temperature. Unfortunately, most amylases isolated from bacteria and filamentous fungi have optimal activity above 45 °C and low pH. For example, the most commonly used industrial glucoamylases, a type of amylase that degrades starch to glucose, are produced by Aspergillus strains displaying optimal activities at 45-60 °C. Thus, isolating new amylases with optimal activity at ambient temperature is essential for improving industrial processes. In this report, a glucoamylase secreted by the cold-adapted yeast Tetracladium sp. was isolated and biochemically characterized., Results: The effects of physicochemical parameters on enzyme activity were analyzed, and pH and temperature were found to be key factors modulating the glucoamylase activity. The optimal conditions for enzyme activity were 30 °C and pH 6.0, and the K m and k cat using soluble starch as substrate were 4.5 g/L and 45 min -1 , respectively. Possible amylase or glucoamylase encoding genes were identified, and their transcript levels using glucose or soluble starch as the sole carbon source were analyzed. Transcription levels were highest in medium supplemented with soluble starch for the potential glucoamylase encoding gene. Comparison of the structural model of the identified Tetracladium sp. glucoamylase with the solved structure of the Hypocrea jecorina glucoamylase revealed unique structural features that may explain the thermal lability of the glucoamylase from Tetracladium sp., Conclusion: The glucoamylase secreted by Tetracladium sp. is a novel cold-adapted enzyme and its properties should render this enzyme suitable for use in industrial processes that require cold-active amylases, such as biofuel production.

Background: The basidiomycetous yeast Xanthophyllomyces dendrorhous has been described as a potential biofactory for terpenoid-derived compounds due to its ability to synthesize astaxanthin. Functional knowledge of the genes involved in terpenoid synthesis would create opportunities to enhance carotenoid production. A thiolase enzyme catalyzes the first step in terpenoid synthesis., Results: Two potential thiolase-encoding genes were found in the yeast genome; bioinformatically, one was identified as an acetyl-CoA C-acetyltransferase (ERG10), and the other was identified as a 3-ketoacyl Co-A thiolase (POT1). Heterologous complementation assays in Saccharomyces cerevisiae showed that the ERG10 gene from X. dendrorhous could complement the lack of the endogenous ERG10 gene in S. cerevisiae, thereby allowing cellular growth and sterol synthesis. X. dendrorhous heterozygous mutants for each gene were created, and a homozygous POT1 mutant was also obtained. This mutant exhibited changes in pigment composition and higher ERG10 transcript levels than the wild type strain., Conclusions: The results support the notion that the ERG10 gene in X. dendrorhous is a functional acetyl-CoA C-acetyltransferase essential for the synthesis of mevalonate in yeast. The POT1 gene would encode a functional 3-ketoacyl Co-A thiolase that is non-essential for cell growth, but its mutation indirectly affects pigment production.

Background: The yeast Xanthophyllomyces dendrorhous produces carotenoids of commercial interest, including astaxanthin and β-carotene. Although carotenogenesis in this yeast and the expression profiles of the genes controlling this pathway are known, the mechanisms regulating this process remain poorly understood. Several studies have demonstrated that glucose represses carotenogenesis in X. dendrorhous, suggesting that this pathway could be regulated by catabolic repression. Catabolic repression is a highly conserved regulatory mechanism in eukaryotes and has been widely studied in Saccharomyces cerevisiae. Glucose-dependent repression is mainly observed at the transcriptional level and depends on the DNA-binding regulator Mig1, which recruits the co-repressor complex Cyc8-Tup1, which then represses the expression of target genes. In this work, we studied the regulation of carotenogenesis by catabolic repression in X. dendrorhous, focusing on the role of the co-repressor complex Cyc8-Tup1., Results: The X. dendrorhous CYC8 and TUP1 genes were identified, and their functions were demonstrated by heterologous complementation in S. cerevisiae. In addition, cyc8 - and tup1 - mutant strains of X. dendrorhous were obtained, and both mutations were shown to prevent the glucose-dependent repression of carotenogenesis in X. dendrorhous, increasing the carotenoid production in both mutant strains. Furthermore, the effects of glucose on the transcript levels of genes involved in carotenogenesis differed between the mutant strains and wild-type X. dendrorhous, particularly for genes involved in the synthesis of carotenoid precursors, such as HMGR, idi and FPS. Additionally, transcriptomic analyses showed that cyc8 - and tup1 - mutations affected the expression of over 250 genes in X. dendrorhous., Conclusions: The CYC8 and TUP1 genes are functional in X. dendrorhous, and their gene products are involved in catabolic repression and carotenogenesis regulation. This study presents the first report involving the participation of Cyc8 and Tup1 in carotenogenesis regulation in yeast.

The red yeast X. dendrorhous is one of the few natural sources of astaxanthin, a carotenoid used in aquaculture for salmonid fish pigmentation and in the cosmetic and pharmaceutical industries for its antioxidant properties. Genetic control of carotenogenesis is well characterized in this yeast; however, little is known about the regulation of the carotenogenesis process. Several lines of evidence have suggested that carotenogenesis is regulated by catabolic repression, and the aim of this work was to identify and functionally characterize the X. dendrorhous MIG1 gene encoding the catabolic repressor Mig1, which mediates transcriptional glucose-dependent repression in other yeasts and fungi. The identified gene encodes a protein of 863 amino acids that demonstrates the characteristic conserved features of Mig1 proteins, and binds in vitro to DNA fragments containing Mig1 boxes. Gene functionality was demonstrated by heterologous complementation in a S. cerevisiae mig1- strain; several aspects of catabolic repression were restored by the X. dendrorhous MIG1 gene. Additionally, a X. dendrorhous mig1- mutant was constructed and demonstrated a higher carotenoid content than the wild-type strain. Most important, the mig1- mutation alleviated the glucose-mediated repression of carotenogenesis in X. dendrorhous: the addition of glucose to mig1- and wild-type cultures promoted the growth of both strains, but carotenoid synthesis was observed only in the mutant strain. Transcriptomic and RT-qPCR analyses revealed that several genes were differentially expressed between X. dendrorhous mig1- and the wild-type strain when cultured with glucose as the sole carbon source. The results obtained in this study demonstrate that catabolic repression in X. dendrorhous is an active process in which the identified MIG1 gene product plays a central role in the regulation of several biological processes, including carotenogenesis., Competing Interests: The authors have declared that no competing interests exist.

The study of the yeasts that inhabit cold environments, such as Antarctica, is an active field of investigation oriented toward understanding their ecological roles in these ecosystems. In a great part, the interest in cold-adapted yeasts is due to several industrial and biotechnological applications that have been described for them. The aim of this work was to isolate and identify yeasts from sedimentary rock samples collected at the Union Glacier, Antarctica. Furthermore, the yeasts were physiologically characterized, including the production of metabolites of biotechnological interest. The yeasts isolated that were identified at the molecular level belonged to genera Collophora (1 isolate), Cryptococcus (2 isolates), Sporidiobolus (4 isolates), Sporobolomyces (1 isolate) and Torrubiella (2 isolates). The majority of yeasts were basidiomycetous and psychrotolerant. By cross-test assays for anti-yeast activity, it was determined that Collophora sp., Sporidiobolus salmonicolor, and Sporobolomyces roseus secreted a protein factor that kills Sporidiobolus metaroseus. The colored yeasts Sp. salmonicolor, Sp. metaroseus and Collophora sp. produced several carotenoid pigments that were identified as 2,3 dihydroxy-γ-carotene, -carotene, 4-ketotorulene, torulene β-cryptoxanthin and spirilloxanthin. Concerning analysis of mycosporines, these metabolites were only found in the yeasts Torrubiella sp. and Cryptococcus sp. T11-10-1. Furthermore, the yeasts were evaluated for the production of extracellular hydrolytic activities. Of the twelve activities analyzed, alkaline phosphatase, invertase, gelatinase, cellulase, amylase, and protease enzyme activities were detected. The yeasts Cryptococcus sp. T11-10-1 and Sporidiobolus metaroseus showed the highest number of different enzyme activities.

Background: Amylases and cellulases have great potential for application in industries such as food, detergent, laundry, textile, baking and biofuels. A common requirement in these fields is to reduce the temperatures of the processes, leading to a continuous search for microorganisms that secrete cold-active amylases and cellulases. Psychrotolerant yeasts are good candidates because they inhabit cold-environments. In this work, we analyzed the ability of yeasts isolated from the Antarctic region to grow on starch or carboxymethylcellulose, and their potential extracellular amylases and cellulases., Result: All tested yeasts were able to grow with soluble starch or carboxymethylcellulose as the sole carbon source; however, not all of them produced ethanol by fermentation of these carbon sources. For the majority of the yeast species, the extracellular amylase or cellulase activity was higher when cultured in medium supplemented with glucose rather than with soluble starch or carboxymethylcellulose. Additionally, higher amylase activities were observed when tested at pH 5.4 and 6.2, and at 30-37 °C, except for Rhodotorula glacialis that showed elevated activity at 10-22 °C. In general, cellulase activity was high until pH 6.2 and between 22-37 °C, while the sample from Mrakia blollopis showed high activity at 4-22 °C. Peptide mass fingerprinting analysis of a potential amylase from Tetracladium sp. of about 70 kDa, showed several peptides with positive matches with glucoamylases from other fungi., Conclusions: Almost all yeast species showed extracellular amylase or cellulase activity, and an inducing effect by the respective substrate was observed in a minor number of yeasts. These enzymatic activities were higher at 30 °C in most yeast, with highest amylase and cellulase activity in Tetracladium sp. and M. gelida, respectively. However, Rh. glacialis and M. blollopis displayed high amylase or cellulase activity, respectively, under 22 °C. In this sense, these yeasts are interesting candidates for industrial processes that require lower temperatures.

Carotenoids are naturally occurring red, orange and yellow pigments that are synthesized by plants and some microorganisms and fulfill many important physiological functions. This chapter describes the distribution of carotenoid in microorganisms, including bacteria, archaea, microalgae, filamentous fungi and yeasts. We will also focus on their functional aspects and applications, such as their nutritional value, their benefits for human and animal health and their potential protection against free radicals. The central metabolic pathway leading to the synthesis of carotenoids is described as the three following principal steps: (i) the synthesis of isopentenyl pyrophosphate and the formation of dimethylallyl pyrophosphate, (ii) the synthesis of geranylgeranyl pyrophosphate and (iii) the synthesis of carotenoids per se, highlighting the differences that have been found in several carotenogenic organisms and providing an evolutionary perspective. Finally, as an example, the synthesis of the xanthophyll astaxanthin is discussed.

Yeasts colonizing the Antarctic region are exposed to a high ultraviolet radiation evolving mechanisms to minimize the UV radiation damages, such as the production of UV-absorbing or antioxidant compounds like carotenoid pigments and mycosporines. Ergosterol has also been suggested to play a role in this response. These compounds are also economically attractive for several industries such as pharmaceutical and food, leading to a continuous search for biological sources of them. In this work, the UV-C radiation tolerance of yeast species isolated from the sub-Antarctic region and their production of carotenoids, mycosporines, and ergosterol were evaluated. Dioszegia sp., Leuconeurospora sp. (T27Cd2), Rhodotorula laryngis, Rhodotorula mucilaginosa, and Cryptococcus gastricus showed the highest UV-C radiation tolerance. The yeasts with the highest content of carotenoids were Dioszegia sp. (OHK torulene), Rh. laryngis (torulene and lycopene), Rh. mucilaginosa, (torulene, gamma carotene, and lycopene), and Cr. gastricus (2-gamma carotene). Probable mycosporine molecules and biosynthesis intermediates were found in Rh. laryngis, Dioszegia sp., Mrakia sp., Le. creatinivora, and Leuconeurospora sp. (T27Cd2). Ergosterol was the only sterol detected in all yeasts, and M. robertii and Le. creatinivora showed amounts higher than 4 mg g−1. Although there was not a well-defined relation between UV-C tolerance and the production of these three kinds of compounds, the majority of the yeasts with lower amounts of carotenoids showed lower UV-C tolerance. Dioszegia sp., M. robertii, and Le. creatinivora were the greatest producers of carotenoids, ergosterol, and mycosporines, respectively, representing good candidates for future studies intended to increase their production for large-scale applications.

The eukaryotic microsomal cytochrome P450 systems consist of a cytochrome P450 enzyme (P450) and a cytochrome P450 redox partner, which generally is a cytochrome P450 reductase (CPR) that supplies electrons from NADPH. However, alternative electron donors may exist such as cytochrome b5 reductase and cytochrome b5 (CBR and CYB5, respectively) via, which is NADH-dependent and are also anchored to the endoplasmic reticulum. In the carotenogenic yeast Xanthophyllomyces dendrorhous, three P450-encoding genes have been described: crtS is involved in carotenogenesis and the CYP51 and CYP61 genes are both implicated in ergosterol biosynthesis. This yeast has a single CPR (encoded by the crtR gene), and a crtR- mutant does not produce astaxanthin. Considering that this mutant is viable, the existence of alternative cytochrome P450 electron donors like CBR and CYB5 could operate in this yeast. The aim of this work was to characterize the X. dendrorhous CBR encoding gene and to study its involvement in P450 reactions in ergosterol and carotenoid biosynthesis. Two CBRs genes were identified (CBR.1 and CBR.2), and deletion mutants were constructed. The two mutants and the wild-type strain showed similar sterol production, with ergosterol being the main sterol produced. The crtR- mutant strain produced a lower proportion of ergosterol than did the parental strain. These results indicate that even though one of the two CBR genes could be involved in ergosterol biosynthesis, crtR complements their absence in the cbr- mutant strains, at least for ergosterol production. The higher NADH-dependent cytochrome c reductase activity together with the higher transcript levels of CBR.1 and CYB5 in the crtR- mutant as well as the lower NADH-dependent activity in CBS-cbr.1- strongly suggest that CBR.1-CYB5 via participates as an alternative electron donor pathway for P450 enzymes involved in ergosterol biosynthesis in X. dendrorhous.

Yeasts, widely distributed across the Earth, have successfully colonized cold environments despite their adverse conditions for life. Lower eukaryotes play important ecological roles, contributing to nutrient recycling and organic matter mineralization. Yeasts have developed physiological adaptations to optimize their metabolism in low-temperature environments, which affect the rates of biochemical reactions and membrane fluidity. Decreased saturation of fatty acids helps maintain membrane fluidity at low temperatures and the production of compounds that inhibit ice crystallization, such as antifreeze proteins, helps microorganisms survive at temperatures around the freezing point of water. Furthermore, the production of hydrolytic extracellular enzymes active at low temperatures allows consumption of available carbon sources. Beyond their ecological importance, interest in psychrophilic yeasts has increased because of their biotechnological potential and industrial uses. Long-chain polyunsaturated fatty acids have beneficial effects on human health, and antifreeze proteins are attractive for food industries to maintain texture in food preserved at low temperatures. Furthermore, extracellular cold-active enzymes display unusual substrate specificities with higher catalytic efficiency at low temperatures than their mesophilic counterparts, making them attractive for industrial processes requiring high enzymatic activity at low temperatures. In this minireview, we describe the physiological adaptations of several psychrophilic yeasts and their possible biotechnological applications.

Background: Xanthophyllomyces dendrorhous is a basidiomycetous yeast that synthesizes astaxanthin, a carotenoid with great biotechnological impact. The ergosterol and carotenoid synthetic pathways derive from the mevalonate pathway and involve cytochrome P450 enzymes. Among these enzymes, the CYP51 family, which is involved in ergosterol biosynthesis, is one of the most remarkable that has C14-demethylase activity., Results: In this study, the CYP51 gene from X. dendrorhous was isolated and its function was analyzed. The gene is composed of ten exons and encodes a predicted 550 amino acid polypeptide that exhibits conserved cytochrome P450 structural characteristics and shares significant identity with the sterol C14-demethylase from other fungi. The functionality of this gene was confirmed by heterologous complementation in S. cerevisiae. Furthermore, a CYP51 gene mutation in X. dendrorhous reduced sterol production by approximately 40% and enhanced total carotenoid production by approximately 90% compared to the wild-type strain after 48 and 120 h of culture, respectively. Additionally, the CYP51 gene mutation in X. dendrorhous increased HMGR (hydroxy-methylglutaryl-CoA reductase, involved in the mevalonate pathway) and crtR (cytochrome P450 reductase) transcript levels, which could be associated with reduced ergosterol production., Conclusions: These results suggest that the CYP51 gene identified in X. dendrorhous encodes a functional sterol C14-demethylase that is involved in ergosterol biosynthesis.

Background: Synonymous codons are used differentially in organisms from the three domains of life, a phenomenon referred to as codon usage bias. In addition, codon pair bias, particularly in the 3' codon context, has also been described in several organisms and is associated with the accuracy and rate of translation. An improved understanding of both types of bias is important for the optimization of heterologous protein expression, particularly in biotechnologically important organisms, such as the yeast Xanthophyllomyces dendrorhous, a promising bioresource for the carotenoid astaxanthin. Using genomic and transcriptomic data, the codon usage and codon context biases of X. dendrorhous open reading frames (ORFs) were analyzed to determine their expression levels, GC% and sequence lengths. X. dendrorhous totiviral ORFs were also included in these analyses., Results: A total of 1,695 X. dendrorhous ORFs were identified through comparison with sequences in multiple databases, and the intron-exon structures of these sequences were determined. Although there were important expression variations among the ORFs under the studied conditions (different phases of growth and available carbon sources), most of these sequences were highly expressed under at least one of the analyzed conditions. Independent of the culture conditions, the highly expressed genes showed a strong bias in both codon usage and the 3' context, with a minor association with the GC% and no relationship to the sequence length. The codon usage and codon-pair bias of the totiviral ORFs were highly variable with no similarities to the host ORFs., Conclusions: There is a direct relation between the level of gene expression and codon usage and 3' context bias in X. dendrorhous, which is more evident for ORFs that are expressed at the highest levels under the studied conditions. However, there is no direct relation between the totiviral ORF biases and the host ORFs.

Background: Astaxanthin is a potent antioxidant with increasing biotechnological interest. In Xanthophyllomyces dendrorhous, a natural source of this pigment, carotenogenesis is a complex process regulated through several mechanisms, including the carbon source. X. dendrorhous produces more astaxanthin when grown on a non-fermentable carbon source, while decreased astaxanthin production is observed in the presence of high glucose concentrations. In the present study, we used a comparative proteomic and metabolomic analysis to characterize the yeast response when cultured in minimal medium supplemented with glucose (fermentable) or succinate (non-fermentable)., Results: A total of 329 proteins were identified from the proteomic profiles, and most of these proteins were associated with carotenogenesis, lipid and carbohydrate metabolism, and redox and stress responses. The metabolite profiles revealed 92 metabolites primarily associated with glycolysis, the tricarboxylic acid cycle, amino acids, organic acids, sugars and phosphates. We determined the abundance of proteins and metabolites of the central pathways of yeast metabolism and examined the influence of these molecules on carotenogenesis. Similar to previous proteomic-stress response studies, we observed modulation of abundance from several redox, stress response, carbohydrate and lipid enzymes. Additionally, the accumulation of trehalose, absence of key ROS response enzymes, an increased abundance of the metabolites of the pentose phosphate pathway and tricarboxylic acid cycle suggested an association between the accumulation of astaxanthin and oxidative stress in the yeast. Moreover, we observed the increased abundance of late carotenogenesis enzymes during astaxanthin accumulation under succinate growth conditions., Conclusions: The use of succinate as a carbon source in X. dendrorhous cultures increases the availability of acetyl-CoA for the astaxanthin production compared with glucose, likely reflecting the positive regulation of metabolic enzymes of the tricarboxylic acid and glyoxylate cycles. The high metabolite level generated in this pathway could increase the cellular respiration rate, producing reactive oxygen species, which induces carotenogenesis.

Two dsRNAs of estimated lengths of 5 (L1) and 3.7 (L2) kpb are commonly found in strains of the basidiomycetous yeast Xanthophyllomyces dendrorhous, and the presence of virus-like particles (VLPs) have been described in some strains. Recently, two putative totiviruses (XdV-L1A and XdV-L1B) were identified from L1 dsRNA and one (XdV-L2) from L2 dsRNA in the strain UCD 67-385. In some strains, there are smaller dsRNAs (0.9-1.4 kb) that probable are satellite elements. In this work, the VLPs from several strains of X. dendrorhous, which differ in their dsRNAs content, were separated by sucrose gradient and characterized in relation to the dsRNAs and proteins that compose them. It was found that all types of dsRNAs were encapsidated into VLPs, supporting the hypothesis that the smaller dsRNAs are satellite molecules. A main protein of approx. 76 or 37 kDa composed the virions that only have the L1-dsRNA or L2-dsRNA, respectively. In the strain UCD 67-385, these both proteins were identified as viral capsid protein (CP), allow to confirm the gag predicted ORFs in XdV-L1A, XdV-L1B, and XdV-L2, with CPs of 76.6, 76.2, and 38.8 kDa, respectively. Analysis of predicted structures of CPs of XdV-L1A and XdV-L1B, showed high similitudes with the CPs of ScV-L-A and other totiviruses.

Antarctic microorganisms have developed different strategies to live in their environments, including modifications to their membrane components to regulate fluidity and the production of photoprotective metabolites such as carotenoids. Three yeast colonies (ANCH01, ANCH06 and ANCH08) were isolated from soil samples collected at King George Island, which according to their rDNA sequence analyses, were determined to be Xanthophyllomyces dendrorhous. This yeast is of biotechnological interest, because it can synthesize astaxanthin as its main carotenoid, which is a powerful antioxidant pigment used in aquaculture. Then, the aim of this work was to characterize the ANCH isolates at their molecular and phenotypic level. The isolates did not display any differences in their rDNA and COX1 gene nucleotide sequences. However, ANCH01 produces approximately sixfold more astaxanthin than other wild type strains. Moreover, even though ANCH06 and ANCH08 produce astaxanthin, their main carotenoid was β-carotene. In contrast to other X. dendrorhous strains, the ANCH isolates did not produce mycosporines. Finally, the ANCH isolates had a higher proportion of polyunsaturated fatty acids than other wild type strains. In conclusion, the reported X. dendrorhous isolates are phenotypically different from other wild type strains, including characteristics that could make them more resistant and better able to inhabit their original habitat, which may also have biotechnological potential.

The yeast Xanthophyllomyces dendrorhous synthesizes the carotenoid astaxanthin, which has applications in biotechnology because of its antioxidant and pigmentation properties. However, wild-type strains produce too low amounts of carotenoids to be industrially competitive. Considering this background, it is indispensable to understand how the synthesis of astaxanthin is controlled and regulated in this yeast. In this work, the steps leading to the synthesis of the carotenoid precursor geranylgeranyl pyrophosphate (GGPP, C20) in X. dendrorhous from isopentenyl pyrophosphate (IPP, C5) and dimethylallyl pyrophosphate (DMAPP, C5) was characterized. Two prenyl transferase encoding genes, FPS and crtE, were expressed in E. coli. The enzymatic assays using recombinant E. coli protein extracts demonstrated that FPS and crtE encode a farnesyl pyrophosphate (FPP, C15) synthase and a GGPP-synthase, respectively. X. dendrorhous FPP-synthase produces geranyl pyrophosphate (GPP, C10) from IPP and DMAPP and FPP from IPP and GPP, while the X. dendrorhous GGPP-synthase utilizes only FPP and IPP as substrates to produce GGPP. Additionally, the FPS and crtE genes were over-expressed in X. dendrorhous, resulting in an increase of the total carotenoid production. Because the parental strain is diploid, the deletion of one of the alleles of these genes did not affect the total carotenoid production, but the composition was significantly altered. These results suggest that the over-expression of these genes might provoke a higher carbon flux towards carotenogenesis, most likely involving an earlier formation of a carotenogenic enzyme complex. Conversely, the lower carbon flux towards carotenogenesis in the deletion mutants might delay or lead to a partial formation of a carotenogenic enzyme complex, which could explain the accumulation of astaxanthin carotenoid precursors in these mutants. In conclusion, the FPS and the crtE genes represent good candidates to manipulate to favor carotenoid biosynthesis in X. dendrorhous.

Background: Xanthophyllomyces dendrorhous is a basidiomycetous yeast that is relevant to biotechnology, as it can synthesize the carotenoid astaxanthin. However, the astaxanthin levels produced by wild-type strains are low. Although different approaches for promoting increased astaxanthin production have been attempted, no commercially competitive results have been obtained thus far. A promising alternative to facilitate the production of carotenoids in this yeast involves the use of genetic modification. However, a major limitation is the few available molecular tools to manipulate X. dendrorhous., Results: In this work, the DNA assembler methodology that was previously described in Saccharomyces cerevisiae was successfully applied to assemble DNA fragments in vivo and integrate these fragments into the genome of X. dendrorhous by homologous recombination in only one transformation event. Using this method, the gene encoding astaxanthin synthase (crtS) was overexpressed in X. dendrorhous and a higher level of astaxanthin was produced., Conclusions: This methodology could be used to easily and rapidly overexpress individual genes or combinations of genes simultaneously in X. dendrorhous, eliminating numerous steps involved in conventional cloning methods.

Xanthophyllomyces dendrorhous is a natural source of astaxanthin, a carotenoid widely used in the food industry. In this yeast, astaxanthin is synthesized from β-carotene by a cytochrome P450, CrtS, which depends on CrtR, the four-domain cytochrome P450 reductase (CPR). Although Saccharomyces cerevisiae has an endogenous CPR (ScCPR), expression of CrtS does not result in astaxanthin production unless it is coexpressed with CrtR. Assuming that CrtS could interact with the FMN-binding domain of either CrtR or ScCPR (XdFMNbd and ScFMNbd, respectively), the aim of this work was to identify possible interaction differences between these alternative complexes by protein modeling and short molecular dynamics simulations. Considering the recently proposed membrane orientation of a mammalian P450, our CrtS-CrtR model predicts that both N-terminal ends stand adjacent to the membrane plane, allowing their anchoring. Compared with the possible interface between CrtS and both FMNbd, the Xanthophyllomyces system appears to be stabilized by more saline bridges.

Background: 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., Results: In this work, we showed that glucose caused transcriptional repression of the three genes involved in the synthesis of astaxanthin from geranylgeranyl pyrophosphate in X. dendrorhous, which correlates with a complete inhibition of pigment synthesis. Strikingly, this regulatory response was completely altered in mutant strains that are incapable of synthesizing astaxanthin. However, we found that addition of ethanol caused the induction of crtYB and crtS gene expression and promoted de novo synthesis of carotenoids. The induction of carotenogenesis was noticeable as early as 24 h after ethanol addition., Conclusion: For the first time, we demonstrated that carbon source-dependent regulation of astaxanthin biosynthesis in X. dendrorhous involves changes at the transcriptional level. Such regulatory mechanism provides an explanation for the strong and early inhibitory effect of glucose on the biosynthesis of this carotenoid.

Background: The yeast Xanthophyllomyces dendrorhous is used for the microbiological production of the antioxidant carotenoid astaxanthin. In this study, we established an optimal protocol for protein extraction and performed the first proteomic analysis of the strain ATCC 24230. Protein profiles before and during the induction of carotenogenesis were determined by two-dimensional polyacrylamide gel electrophoresis and proteins were identified by mass spectrometry., Results: Among the approximately 600 observed protein spots, 131 non-redundant proteins were identified. Proteomic analyses allowed us to identify 50 differentially expressed proteins that fall into several classes with distinct expression patterns. These analyses demonstrated that enzymes related to acetyl-CoA synthesis were more abundant prior to carotenogenesis. Later, redox- and stress-related proteins were up-regulated during the induction of carotenogenesis. For the carotenoid biosynthetic enzymes mevalonate kinase and phytoene/squalene synthase, we observed higher abundance during induction and/or accumulation of carotenoids. In addition, classical antioxidant enzymes, such as catalase, glutathione peroxidase and the cytosolic superoxide dismutases, were not identified., Conclusions: Our results provide an overview of potentially important carotenogenesis-related proteins, among which are proteins involved in carbohydrate and lipid biosynthetic pathways as well as several redox- and stress-related proteins. In addition, these results might indicate that X. dendrorhous accumulates astaxanthin under aerobic conditions to scavenge the reactive oxygen species (ROS) generated during metabolism.

Xanthophyllomyces dendrorhous is a basidiomycetous yeast of considerable biotechnological interest because it synthesizes astaxanthin as its main carotenoid. The carotenoid production increases when it is grown using nonfermentable compounds as the sole carbon source. This work analyzes the expression of the carotenogenic genes and their relationship with the amount and types of carotenoids produced when X. dendrorhous is grown using a nonfermentable (succinate) or a fermentable carbon source (glucose). When X. dendrorhous is grown in succinate, carotenoid production is approximately three times higher than when it is grown in glucose. Moreover, carotenoid biosynthesis occurs at the start of the growth cycle when X. dendrorhous is grown in succinate, whereas when it is grown in glucose, carotenoids are produced at the end of the exponential phase. Additionally, we observed that some carotenogenic genes, such as alternative transcripts of crtYB and crtI, are differentially expressed when the yeast is grown in these carbon sources; other genes, such as crtS, exhibit a similar pattern of expression. Our data indicate that transcriptional regulation is not sufficient to explain the differences in carotenoid production between the two culture conditions, indicating that additional regulatory mechanisms may be operating in the carotenogenic pathway of X. dendrorhous., (FEMS Yeast Research © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. No claim to original Spanish government works.)