Estrategias didácticas para la enseñanza de la biología molecular y la biotecnología, en estudiantes de educación media

El proyecto tuvo como objetivo contribuir a la formación científica, desarrollo de habilidades y destrezas experimentales en estudiantes de educación media, mediante la realización de módulos experimentales en biología molecular y en biotecnología. Los estudiantes, a través de la metodología científica, resolvieron la situación problema de aislar, clonar y expresar un gen de interés industrial en una levadura. Para ello, manipularon y caracterizaron, citológica y bioquímicamente, los microorganismos empleados en el proyecto. Luego, aislaron, amplificaron y secuenciaron el gen que codifica por la enzima alfa-acetolactato descarboxilasa, a partir de un plásmido bacteriano. Posteriormente, clonaron el gen en un vector de expresión dual y, realizaron ensayos de transformación genética en levadura y, evaluaron la actividad enzimática, mediante ensayo in vitro

Regulation of carotenogenesis in the red yeast Xanthophyllomyces dendrorhous: the role of the transcriptional co-repressor complex Cyc8–Tup1 involved in catabolic repression

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.FONDECYT 1140504 and by a graduate scholarships CONICYT 21110252 to PC and MECESUP 2-UCH0604 to NBPeer Reviewe

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. 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.FONDECYT 1160202 and by graduate scholarships CONICYT 21130708 to MSG and CONICYT 2117061

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.dendrorhousSRE1 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

Identification and analysis of metabolite production with biotechnological potential in Xanthophyllomyces dendrorhous isolates

Artículo de publicación ISIAbstract 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 b-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.CONICY

Molecular Characterization and Functional Analysis of Cytochrome b5 Reductase (CBR) Encoding Genes from the Carotenogenic Yeast Xanthophyllomyces dendrorhous.

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

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

Functional characterization of the Xanthophyllomyces dendrorhous farnesyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase encoding genes that are involved in the synthesis of isoprenoid precursors.

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