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

Physiological adaptations of yeasts living in cold environments and their potential applications

Artículo de publicación ISIYeasts, 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.Fondecyt 1130333 Instituto Antartico Chileno (INACH, Chile) RT_07-1

Purification and characterization of a novel cold adapted fungal glucoamylase

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 Km and kcat 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

Amplicon-metagenomic analysis of fungi from antarctic terrestrial habitats

© 2017 Baeza, Barahona, Alcaíno and Cifuentes. 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 cluste

Modeling the interfacial interactions between CrtS and CrtR from xanthophyllomyces dendrorhous, a P450 system involved in astaxanthin production

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 Xanthophyl

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.Comision Nacional de Investigacion Cientifica y Tecnologica (CONICYT), Fondecyt grant no. 1130333

Identification and characterization of yeasts isolated from sedimentary rocks of Union Glacier at the Antarctica

© 2016, Springer Japan. 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., Sporidiob

Screening and characterization of amylase and cellulase activities in psychrotolerant yeasts

Artículo de publicación ISIBackground: 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 degrees C, except for Rhodotorula glacialis that showed elevated activity at 10-22 degrees C. In general, cellulase activity was high until pH 6.2 and between 22-37 degrees C, while the sample from Mrakia blollopis showed high activity at 4-22 degrees 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 degrees 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 degrees C. In this sense, these yeasts are interesting candidates for industrial processes that require lower temperatures.FONDECYT 113033

Identification and characterization of yeasts isolated from the South Shetland Islands and the Antarctic Peninsula

© 2016, Springer-Verlag Berlin Heidelberg. Antarctica is considered one of the most extreme environments on Earth because of its low temperatures, dryness, high incidence of solar radiation and low nutrient availability. Nevertheless, microorganisms including yeast have successfully colonized Antarctica; however, little is known about Antarctic yeast. In this study, cultivable yeast from soil samples collected from several islands of the South Shetland archipelago and Antarctic Peninsula were identified and characterized at different levels. Most yeasts were psychrotolerant and belonged to eleven genera, with the majority belonging to the Cryptococcus genus. Most yeasts were able to oxidize dextrin, α-d-glucose, sucrose and d-trehalose and to assimilate turanose, d-xylose, dextrin, d-trehalose, α-d-glucose and salicin. Evaluation of twelve hydrolytic enzymes revealed that yeast isolates displayed four to seven different enzyme activities, with lipase, alkaline phosphatase and invertas

Characterization of the cytochrome P450 monooxygenase genes (P450ome) from the carotenogenic yeast Xanthophyllomyces dendrorhous

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 oxygenbinding 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.FONDECYT, 116020