Unraveling the Molecular Basis of Mycosporine Biosynthesis in Fungi.

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 Δddgs-/-;Δomt-/- and Δomt-/-;Δatpg-/- double-gene mutants did not produce mycosporines. However, Δatpg-/- 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 Δmig1-/-, Δcyc8-/-, and Δopi1-/- showed upregulation, Δrox1-/- and Δskn7-/- showed downregulation, and Δtup6-/- and Δyap6-/- 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.

A genomic approach to analyze the cold adaptation of yeasts isolated from Italian Alps.

Microorganisms including yeasts are responsible for mineralization of organic matter in cold regions, and their characterization is critical to elucidate the ecology of such environments on Earth. Strategies developed by yeasts to survive in cold environments have been increasingly studied in the last years and applied to different biotechnological applications, but their knowledge is still limited. Microbial adaptations to cold include the synthesis of cryoprotective compounds, as well as the presence of a high number of genes encoding the synthesis of proteins/enzymes characterized by a reduced proline content and highly flexible and large catalytic active sites. This study is a comparative genomic study on the adaptations of yeasts isolated from the Italian Alps, considering their growth kinetics. The optimal temperature for growth (OTG), growth rate (Gr), and draft genome sizes considerably varied (OTG, 10°C-20°C; Gr, 0.071-0.0726; genomes, 20.7-21.5 Mpb; %GC, 50.9-61.5). A direct relationship was observed between calculated protein flexibilities and OTG, but not for Gr. Putative genes encoding for cold stress response were found, as well as high numbers of genes encoding for general, oxidative, and osmotic stresses. The cold response genes found in the studied yeasts play roles in cell membrane adaptation, compatible solute accumulation, RNA structure changes, and protein folding, i.e., dihydrolipoamide dehydrogenase, glycogen synthase, omega-6 fatty acid, stearoyl-CoA desaturase, ATP-dependent RNA helicase, and elongation of very-long-chain fatty acids. A redundancy for several putative genes was found, higher for P-loop containing nucleoside triphosphate hydrolase, alpha/beta hydrolase, armadillo repeat-containing proteins, and the major facilitator superfamily protein. Hundreds of thousands of small open reading frames (SmORFs) were found in all studied yeasts, especially in Phenoliferia glacialis. Gene clusters encoding for the synthesis of secondary metabolites such as terpene, non-ribosomal peptide, and type III polyketide were predicted in four, three, and two studied yeasts, respectively.

Role of ROX1, SKN7, and YAP6 Stress Transcription Factors in the Production of Secondary Metabolites in Xanthophyllomyces dendrorhous.

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.

Response to Cold: A Comparative Transcriptomic Analysis in Eight Cold-Adapted Yeasts.

Microorganisms have evolved to colonize all biospheres, including extremely cold environments, facing several stressor conditions, mainly low/freezing temperatures. In general, terms, the strategies developed by cold-adapted microorganisms include the synthesis of cryoprotectant and stress-protectant molecules, cold-active proteins, especially enzymes, and membrane fluidity regulation. The strategy could differ among microorganisms and concerns the characteristics of the cold environment of the microorganism, such as seasonal temperature changes. Microorganisms can develop strategies to grow efficiently at low temperatures or tolerate them and grow under favorable conditions. These differences can be found among the same kind of microorganisms and from the same cold habitat. In this work, eight cold-adapted yeasts isolated from King George Island, subAntarctic region, which differ in their growth properties, were studied about their response to low temperatures at the transcriptomic level. Sixteen ORFeomes were assembled and used for gene prediction and functional annotation, determination of gene expression changes, protein flexibilities of translated genes, and codon usage bias. Putative genes related to the response to all main kinds of stress were found. The total number of differentially expressed genes was related to the temperature variation that each yeast faced. The findings from multiple comparative analyses among yeasts based on gene expression changes and protein flexibility by cellular functions and codon usage bias raise significant differences in response to cold among the studied Antarctic yeasts. The way a yeast responds to temperature change appears to be more related to its optimal temperature for growth (OTG) than growth velocity. Yeasts with higher OTG prepare to downregulate their metabolism to enter the dormancy stage. In comparison, yeasts with lower OTG perform minor adjustments to make their metabolism adequate and maintain their growth at lower temperatures.

Damage response protein 1 (Dap1) functions in the synthesis of carotenoids and sterols in Xanthophyllomyces dendrorhous.

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 ß-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.

The SREBP (Sterol Regulatory Element-Binding Protein) pathway: a regulatory bridge between carotenogenesis and sterol biosynthesis in the carotenogenic yeast Xanthophyllomyces dendrorhous.

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.

Identification of Stress-Related Genes and a Comparative Analysis of the Amino Acid Compositions of Translated Coding Sequences Based on Draft Genome Sequences of Antarctic Yeasts.

Microorganisms inhabiting cold environments have evolved strategies to tolerate and thrive in those extreme conditions, mainly the low temperature that slow down reaction rates. Among described molecular and metabolic adaptations to enable functioning in the cold, there is the synthesis of cold-active proteins/enzymes. In bacterial cold-active proteins, reduced proline content and highly flexible and larger catalytic active sites than mesophylls counterparts have been described. However, beyond the low temperature, microorganisms' physiological requirements may differ according to their growth velocities, influencing their global protein compositions. This hypothesis was tested in this work using eight cold-adapted yeasts isolated from Antarctica, for which their growth parameters were measured and their draft genomes determined and bioinformatically analyzed. The optimal temperature for yeasts' growth ranged from 10 to 22°C, and yeasts having similar or same optimal temperature for growth displayed significative different growth rates. The sizes of the draft genomes ranged from 10.7 (Tetracladium sp.) to 30.7 Mb (Leucosporidium creatinivorum), and the GC contents from 37 (Candida sake) to 60% (L. creatinivorum). Putative genes related to various kinds of stress were identified and were especially numerous for oxidative and cold stress responses. The putative proteins were classified according to predicted cellular function and subcellular localization. The amino acid composition was compared among yeasts considering their optimal temperature for growth and growth rates. In several groups of predicted proteins, correlations were observed between their contents of flexible amino acids and both the yeasts' optimal temperatures for growth and their growth rates. In general, the contents of flexible amino acids were higher in yeasts growing more rapidly as their optimal temperature for growth was lower. The contents of flexible amino acids became lower among yeasts with higher optimal temperatures for growth as their growth rates increased.

The SREBP (Sterol Regulatory Element-Binding Protein) pathway: a regulatory bridge between carotenogenesis and sterol biosynthesis in the carotenogenic yeast Xanthophyllomyces dendrorhous

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.

Sterol regulatory element-binding protein Sre1 regulates carotenogenesis in the red yeast Xanthophyllomyces dendrorhous.

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.

Isolation of Three Lycorine Type Alkaloids from Rhodolirium speciosum (Herb.) Ravenna Using pH-Zone-Refinement Centrifugal Partition Chromatography and Their Acetylcholinesterase Inhibitory Activities.

Preparative separation of three lycorine type alkaloids from Rhodolirum speciosum (Amaryllidaceae) was successfully carried out using pH-zone-refinement centrifugal partition chromatography (CPC) using the solvent system methyl-tert-butyl ether/acetonitrile/water (4:1:5, v/v/v) in descending mode. Using this system, Alkaloid 1 (165.7 mg, 88.2%, purity), 2 (60.1 mg, 97.7% purity) and 3 (12.3 mg, 84.4% purity) were obtained in one step. For structure elucidation, the pure alkaloids were subjected to spectroscopy analysis using nuclear magnetic resonance experiments (1H-NMR, 13C-NMR) and gas chromatography coupled with mass spectrometry (GC-MS). Alkaloids 1, 2, and 3 were identified as 1-O-acetyl-5,6-dehydrolycorine, 1-O-acetyl-lycorine, and 1,2-O-diacetyl-5,6-dehydrolycorine, respectively. The acetylcholinesterase inhibitory activity of these alkaloids was IC50 151.1 µg/mL, IC50 203.5 µg/mL, IC50 470.0 µg/mL, and IC50 17.1 µg/mL, respectively.

Phenotypic Analysis of Mutants of Ergosterol Biosynthesis Genes (ERG3 and ERG4) in the Red Yeast Xanthophyllomyces dendrorhous.

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.

Convergence between Regulation of Carbon Utilization and Catabolic Repression in Xanthophyllomyces dendrorhous.

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.

In vitro micropropagation and alkaloids analysis by GC-MS of Chilean Amaryllidaceae plants: Rhodophiala pratensis.

INTRODUCTION: Plants from Amaryllidaceae family are of interest since they produce a particular type of alkaloid useful for the treatment of neurodegenerative diseases of the central nervous system, such as Galanthamine. Given the low content of these secondary metabolites in the plant, it is necessary to study mechanisms to increase the productivity of them. OBJECTIVE: To obtain fast qualitative and quantitative analysis of the alkaloids and extend the understanding of biosynthesis and metabolism in these kinds of plants. Furthermore, establish a reliable, simple and fast analytical method for the in vitro callus culture of vegetative organs for Rhodophiala pratensis species. METHODS: The alkaloids composition of the callus culture of R. pratensis were analysed by gas chromatography coupled with mass spectrometry (GC-MS). RESULTS: A methodology for the qualitative and quantitative analysis of the alkaloids present in fresh callus culture of this wild plant species was established. The analysis showed alternation in the alkaloids type ratio and number of compounds between wild bulbs, in vitro bulbs and callus. It was possible to identify 24 alkaloids from a pool of 60 signals whose fragmentation pattern corresponds to the alkaloids of Amaryllidaceae plants. Together with the aforementioned, the amount and type of alkaloid present in the plant material obtained by in vitro culture of R. pratensis was determined in the same way. The results show the high biosynthetic potential of in vitro grown bulbs and callus tissue that are able to produce significant amounts of pharmacologically relevant alkaloids from R. pratensis in various proportions that depend on the culture conditions such as supplementation with growth substances. The in vitro grown bulbs produce an alkaloidal extract that contain a 52.6% w/w of alkaloids. CONCLUSION: This study allowed the alkaloid content in callus culture of R. pratensis to be found by means of GC-MS. These results allowed a relationship between the type of growth regulator and the type of alkaloids found to be established. Finally, we can say that the results achieved to state that the production of alkaloids using different combinations of growth regulators could be directed during in vitro micropropagation from provided plant material.

Metallopeptidase Stp1 activates the transcription factor Sre1 in the carotenogenic yeast Xanthophyllomyces dendrorhous.

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 Δstp1 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 Δstp1 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 Δstp1 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.

Neuroprotective activity of isoquinoline alkaloids from of Chilean Amaryllidaceae plants against oxidative stress-induced cytotoxicity on human neuroblastoma SH-SY5Y cells and mouse hippocampal slice culture.

In this study we evaluate the chemical composition and neuroprotective effects of alkaloid fractions of the Amaryllidaceae species Rhodophiala pratensis, Rhodolirium speciosum, Phycella australis and Phaedranassa lehmannii. Gas chromatography-mass spectrometry (GC/MS) enable the identification of 41 known alkaloids. Rhodolirium speciosum and Rhodophiala pratensis were the most active extracts against acetylcholinesterase (AChE), with IC50 values of 35.22 and 38.13 µg/mL, respectively. The protective effect of these extracts on human neuroblastoma cells (SH-SY5Y) subjected to mitochondrial oxidative stress with rotenone/oligomycin A (R/O) and toxicity promoted by okadaic acid (OA) was evaluated. Only Phycella australis and Rhodophiala pratensis at 0.75 and 1.5 µg/mL, tend to reverse the cell death induced by R/O by around 12%. In OA assay, alkaloid fractions of Phycella Australis and Phaedranassa lehmannii displayed a concentration-dependent (0.375-3.0 µg/mL) effect with a maximum neuroprotective response of 78% and 84%, respectively. Afterwards, neuroprotective effects of Phycella australis (3 and 6 µg/mL) in mouse hippocampal slices stressed with oxygen glucose deprivation/reoxygenation (OGD/R), shown a protection greater than 14%. Finally, Phycella Australis (6 µg/mL) reverted the cell viability from 65% to 90% in slices treated with OA, representing a protection of 25% attributable to the alkaloids of this species.

Characterization of gelatinase produced by Antarctic Mrakia sp.

In the present study, 20 psychrotolerant yeast species isolated from the soils of King George Island in the sub-Antarctic region were evaluated for the production of extracellular gelatinase, an enzyme with high potential for applications in diverse areas, such as food and medicine. The production of extracellular gelatinase was confirmed in the yeasts Metschnikowia sp., Leucosporidium fragarium, and Mrakia sp., the last one being the yeast in which the highest gelatinase activity was detected. The enzyme was purified from cultures of Mrakia sp., and the effect of different physical-chemical factors on its activity was determined. The gelatinase produced by Mrakia sp. would correspond to a protein of relative molecular weight (rMW) 37,000, which displayed the highest activity at 36°C, pH 7.0, 10 mM CaCl 2 , and 5 mM ZnSO 4 .

Sterol Regulatory Element-Binding Protein (Sre1) Promotes the Synthesis of Carotenoids and Sterols in Xanthophyllomyces dendrorhous.

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.

Pectinase secreted by psychrotolerant fungi: identification, molecular characterization and heterologous expression of a cold-active polygalacturonase from Tetracladium sp.

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.

Isolation and Characterization of Extrachromosomal Double-Stranded RNA Elements from Carotenogenic Yeasts.

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.

Antarctic yeasts: analysis of their freeze-thaw tolerance and production of antifreeze proteins, fatty acids and ergosterol.

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.