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.

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.

Tolerance to Ultraviolet Radiation of Psychrotolerant Yeasts and Analysis of Their Carotenoid, Mycosporine, and Ergosterol Content.

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.

Identification and functional characterization of the CYP51 gene from the yeast Xanthophyllomyces dendrorhous that is involved in ergosterol biosynthesis.

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.

Enhancement of carotenoid production by disrupting the C22-sterol desaturase gene (CYP61) in Xanthophyllomyces dendrorhous.

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.

Occurrence of killer yeast strains in industrial and clinical yeast isolates

The secretion of proteinaceous toxins is a widespread characteristic in environmental and laboratory yeast isolates, a phenomenon called "killer system". The killer phenotype (K+) can be encoded by extrachromosomal genetic elements (EGEs) as double stranded DNA or RNA molecules (dsDNA, dsRNA) or in nuclear genes. The spectrum of action and the activity of killer toxins are influenced by temperature, salinity and pH of media. In the present work we determined the existence of K+ in a collection of S. cerevisiae and P. anómala yeasts isolated from environmental, industrial and clinical sources. The assays were performed in strains belonging to three yeast genera used as sensitive cells and under a wide range of pH and temperatures. Approximately 51 percent of isolates tested showed toxicity against at least one sensitive yeast strain under the conditions tested. The K+ P. anómala isolates showed a wide spectrum of action and two of them had toxic activity against strains of the three yeast genera assayed, including C. albicans strains. In all S. cerevisiae K+ isolates an extrachromosomal dsRNA molecule (4.2 Kb) was observed, contrary to P. anómala K+ isolates, which do not possess any EGEs. The K+ phenotype is produced by an exported protein factor and the kinetics of killer activity production was similar in all isolates with high activity in the log phase of growth, decaying in the stationary phase.

Genomic organization of the structural genes controlling the astaxanthin biosynthesis pathway of Xanthophyllomyces dendrorhous

The cloning and nucleotide sequence of the genes (idi, crtE, crtYB, crtl and crtS) controlling the astaxanthin biosynthesis pathway of the wild-type ATCC 24230 strain of Xanthophyllomyces dendrorhous in their genomic and cDNA version were obtained. The idi, crtE, crtYB, crtl and crtS genes were cloned, as fragments of 10.9, 11.5, 15.8, 5.9 and 4 kb respectively. The nucleotide sequence data analysis indicates that the idi, crtE, crtYB, crtl and crtS genes have 4, 8,4, 11, and 17 introns and 5, 9, 5, 12 and 18 exons respectively. In addition, a highly efficient site-directed mutagenesis system was developed by transformation by integration, followed by mitotic recombination (the double recombinant method). Heterozygote idi (idi+ / idi-::hph), crtE (crtE+ / crtE -::hph), crtYB (crtYB + / crtYB -::hph), crtI (crtI+ / crtI-::hph) and crtS (crtS +/crtS -::hph) and homozygote mutants crtYB (crtYB -::hph/crtYB -::hph), crtI (crtI -::hph/crtI -::hph) and crtS (crtS -::hph / crtS -::hph) were constructed. All the heterozygote mutants have a pale phenotype and produce less carotenoids than the wild-type strain. The genetic analysis of the crtYB, crtl and crtS loci in the wild-type, heterozygote, and homozygote give evidence of the diploid constitution of ATCC 24230 strains. In addition, the cloning of a truncated form of the crtYB that lacks 153 amino acids of the N-terminal region derived from alternatively spliced mRNA was obtained. Their heterologous expression in Escherichia coli carrying the carotenogenic cluster of Erwinia uredovora result in trans-complementation and give evidence of its functionality in this bacterium, maintaining its phytoene synthase activity but not the lycopene cyclase activity.

Cooperative uptake of microcin E492 by receptors FepA, Fiu, and Cir and inhibition by the siderophore enterochelin and its dimeric and trimeric hydrolysis products.

Microcin E492 uptake by FepA, Fiu, and Cir is cooperative, with FepA being the main receptor. No TonB-mediated interaction with the ferric catecholate receptors is needed for microcin to exert action at the cytoplasmic membrane. Microcin E492 uptake by the receptors is inhibited by the dimer and trimer of dihydroxybenzoylserine.

Genetic polymorphism of clinical and environmental strains of Pichia anomala

In this work 20 clinical and 3 environmental yeast isolates were characterized by classical morphological and physiological methods, as well as molecular methods based on PCR of the ITS1-5.8S rDNA-ITS2 region. The characteristic morphology and biochemical profiles observed in these samples correspond to those described for the Pichia genera, more specifically to P. anomala. The profiles of susceptibility to five antifungal drugs were determined by two broth dilution methods. The results obtained by both methods were comparable and showed that clinical isolates presented more resistance to azoles, amphotericin B, and 5-fluorocytosine, than environmental ones did. By amplification and sequencing of internal transcribed spacers (ITS1 and ITS2) and the ribosomal 5.8S DNA, the yeast samples were divided into four groups, where the strains within each group had the same sequence. Of the analyzed yeast isolates, 78 por percent were identified as Pichia anomala. Using RAPD analysis with seven different Operon primers, polymorphism was observed within the four groups. Our study highlights the growing importance of P. anomala in fungemic episodes in premature neonates. Furthermore, the methodologies used provide a powerful tool to identify and determine differences in similar strains of this yeast.