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Sterol regulatory element-binding proteins (SREBPs) regulate lipid homeostasis and coordinate sterol metabolism and carotenogenesis in the astaxanthin-producing yeast Phaffia rhodozyma. While Sre1, the SREBP ortholog, and the site-2 protease Stp1 have been identified as essential components of this pathway in P. rhodozyma, additional factors involved in Sre1 processing or regulation remain unknown. In Aspergillus species, a signal peptide peptidase contributes to the activation of the SREBP ortholog, raising the possibility of a similar role in this yeast. In this work, we identified and characterized the P. rhodozyma signal peptide peptidase (SppA) homolog. Sequence analysis, domain prediction, and phylogenetic analyses supported its classification within the SPP family of intramembrane aspartyl proteases. To evaluate its functional role, ΔsppA mutants were constructed in genetic backgrounds with constitutive Sre1 activity, including the cyp61− mutant and a strain expressing the active form of Sre1 (Sre1N). Deletion of SPPA did not alter sensitivity to clotrimazole or cobalt chloride, nor affect pigmentation, indicating that SppA is not required for Sre1 activation in P. rhodozyma. Transcriptomic analyses further showed that expression of SRE1 and of its known target genes remained unchanged upon SPPA deletion. Interestingly, the loss of SppA in the Sre1N background caused marked downregulation of genes associated with protein refolding and unfolded protein binding. In agreement with these transcriptional changes, the Sre1NΔsppA strain displayed increased sensitivity to dithiothreitol. These findings suggest that, although SppA is not involved in Sre1 activation in P. rhodozyma, it may play a role in protein stress-related processes. Future studies will be required to define the molecular mechanisms underlying this role and its integration with protein homeostasis networks.

Sterol regulatory element-binding proteins (SREBPs) regulate lipid homeostasis in mammals via sequential activation by the site-1 (S1P) and site-2 (S2P) proteases. In the yeast Phaffia rhodozyma, homologs of SREBP (Sre1) and S2P (Stp1) were identified, with Sre1 cleaved by Stp1 and involved in the regulation of sterol and carotenoid biosynthesis. Additional regulatory roles of S2P have been described in other organisms, but such functions remain unexplored in P. rhodozyma, a question addressed in this study. Transcriptomic analyses of Δsre1, Δstp1, and Δsre1Δstp1 mutants were performed in both wild-type and Sre1-activated conditions. Potential genes regulated by Stp1 independently of Sre1 were identified, and their cellular roles were determined by KEGG mapping and Gene Ontology classification. As expected, most transcriptional changes in Δstp1 mutants reflected Sre1-mediated regulation. Notably, a subset of genes displayed differential expression independently of Sre1. These genes were linked to diverse aspects of cellular homeostasis, including metabolism, protein folding, ER stress response, and ribosome biogenesis. The transcriptomic analysis suggests that Stp1 regulates gene expression beyond the Sre1 transcription factor in P. rhodozyma, providing a framework for future studies to confirm and further explore these functions.

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.

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.

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.

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.

The primary carotenoid synthesized by Xanthophyllomyces dendrorhous is astaxanthin, which is used as a feed additive in aquaculture. Cell growth kinetics and carotenoid production were correlated with the mRNA levels of the idi, crtE, crtYB, crtI, crtS and crtR genes, and the changes in gene sequence between the wild-type and a carotenoid overproducer XR4 mutant strain were identified. At the late stationary phase, the total carotenoid content in XR4 was fivefold higher than that of the wild-type strain. Additionally, the mRNA levels of crtE and crtS increased during the XR4 growth and were three times higher than the wild-type strain in the late stationary phase. Moreover, the nucleotide sequences of crtYB, crtI and crtR exhibited differences between the strains. Both the higher crtE and crtS transcript levels and the crtYB, crtI and crtR mutations can, at least in part, act to up-regulate the carotenoid biosynthesis pathway in the XR4 strain.

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.

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.

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.