Genome sequencing and oenologically relevant traits of the Uruguayan native yeast Issatchenkia terricola

Issatchenkia terricola 0621 is a non-Saccharomyces yeast strain isolated from Tannat grapes from Uruguayan vineyards; it stands out for its ability to produce high levels of β-glucosidase activity, which contributes to the aromatic complexity of wines. To delve into the potential oenological applications of this strain, its high-quality genome was obtained and explored, allowing the main central carbon and nitrogen metabolic pathways to be reconstructed. I. terricola is able to utilise glycerol as the sole carbon source in a way that has not previously been described for yeasts. The genes of the fermentome and those involved in stress resistance during winemaking were also identified, and differences were found when compared to S. cerevisiae, which may explain why I. terricola is unable to complete fermentation. The pathways responsible for natural aroma synthesis were also reconstructed, and the production of aromatic acids, alcohols, esters, acetates and lactones was verified experimentally. Finally, sequences encoding for β-glucosidases, in addition to the previously characterised one, were identified in the genome. The work presented here lays the groundwork for experimental research focused on the dissection of the metabolism of a native non-Saccharomyces strain and its application for oenological and biotechnological purposes.ANII: FMV_1_2017_1_13657

Informe final del proyecto: Obtención de una cepa de Saccharomyces cerevisiae productora de una beta-glucosidasa de Issatchenkia terricola y explotación del genoma de esta levadura nativa para la identificación de nuevas enzimas con potencial aplicación en enología

El desarrollo del aroma del vino depende en gran parte de la existencia durante la elaboración, de enzimas (glicosidasas) capaces de actuar eficientemente sobre los sustratos glicosídicos existentes, generando compuestos volátiles. Estudios previos de nuestro grupo con enzimas aisladas de la microbiota de viñedos uruguayos, demostraron que una beta-glucosidasa de la cepa Issatchenkia terrícola presenta propiedades muy promisorias en condiciones enológicas y se destaca por impartir características aromáticas propias a los vinos locales. Sin embargo, los bajos niveles producidos por la cepa autóctona constituyen una limitante para la manipulación y posible aplicación biotecnológica de dicha glucosidasa. Con el objetivo de clonarla y expresarla en Saccharomyces cerevsiae con mayor rendimiento, nos encontramos actualmente focalizados en obtener la secuencia de dicha glucosidasa mediante el diseño de cebadores degenerados, dado que no disponemos aún del genoma de I. terrícola. La cepa generada será utilizada en ensayos de microvinificaciones y análisis químico y sensorial de aromas de los vinos obtenidos. Complementariamente, se propone avanzar en la caracterización molecular mediante secuenciación masiva del genoma de I. terrícola. Esto permitirá identificar la presencia de otros genes codificantes para beta-glucosidasas así como otras glicosidasas. Asimismo, la interpretación del genoma permitirá identificar otras actividades enzimáticas con potencial interés biotecnológico. El proyecto implica el diseño y uso racional del potencial existente en la microbiota nativa enológica, integrando conocimientos desde un enfoque multidisciplinario desde las áreas de bioquímica, biología molecular, genómica y química de aromas. Los resultados podrían generar productos potencialmente transferibles a la industria enológica.Agencia Nacional de Investigación e Innovació

Evaluation of SYBR Green real time PCR for detecting SARS-CoV-2 from clinical samples

The pandemic caused by SARS-CoV-2 has triggered an extraordinary collapse of healthcare systems and hundred thousand of deaths worldwide. Following the declaration of the outbreak as a Public Health Emergency of International Concern by the World Health Organization (WHO) on January 30th, 2020, it has become imperative to develop diagnostic tools to reliably detect the virus in infected patients. Several methods based on real time reverse transcription polymerase chain reaction (RT-qPCR) for the detection of SARS-CoV-2 genomic RNA have been developed. In addition, these methods have been recommended by the WHO for laboratory diagnosis. Since most of these protocols are based on the use of fluorogenic probes and one-step reagents (cDNA synthesis followed by PCR amplification in the same tube), these techniques can be difficult to perform given the limited supply of reagents in low- and middle-income countries. In order to develop an inexpensive SARS-CoV-2 detection protocol using available resources we evaluated the SYBR Green based detection of SARS-CoV-2 to establish a suitable assay. To do so, we adapted one of the WHO recommended TaqMan-based one-step real time PCR protocols (from the University of Hong Kong) to SYBR Green. Our results indicate that SYBR-Green detection of ORF1b-nsp14 target represents a reliable cost-effective alternative to increase the testing capacity.ANII: FCE_1_2019_1_156157ANII: FCE_1_2019_1_15593

Early transcontinental import of SARS-CoV-2 variant of concern 202012/01 (B.1.1.7) from Europe to Uruguay

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant B.1.1.7 causes a more transmissible and apparently more severe disease. We report its early introduction from Europe to South America from a traveler who arrived in Uruguay from the United Kingdom, even before B.1.1.7 was recognized as a variant of concern. This highlights the risk of introduction of SARS-CoV-2 variants despite strict contingency protocols and underscores the need of improving real-time surveillance worldwide.ANII: POS_NAC_2018_1_15149

Emergence and spread of a B.1.1.28-derived P.6 lineage with Q675H and Q677H spike mutations in Uruguay

Uruguay controlled the viral dissemination during the first nine months of the SARS-CoV-2 pandemic. Unfortunately, towards the end of 2020, the number of daily new cases exponentially increased. Herein, we analyzed the country-wide genetic diversity of SARS-CoV-2 between November 2020 and April 2021. We identified that the most prevalent viral variant during the first epidemic wave in Uruguay (December 2020–February 2021) was a B.1.1.28 sublineage carrying Spike mutations Q675H + Q677H, now designated as P.6, followed by lineages P.2 and P.7. P.6 probably arose around November 2020, in Montevideo, Uruguay’s capital department, and rapidly spread to other departments, with evidence of further local transmission clusters; it also spread sporadically to the USA and Spain. The more efficient dissemination of lineage P.6 with respect to P.2 and P.7 and the presence of mutations (Q675H and Q677H) in the proximity of the key cleavage site at the S1/S2 boundary suggest that P.6 may be more transmissible than other lineages co-circulating in Uruguay. Although P.6 was replaced by the variant of concern (VOC) P.1 as the predominant lineage in Uruguay since April 2021, the monitoring of the concurrent emergence of Q675H + Q677H in VOCs should be of worldwide interest

Real-Time genomic surveillance for SARS-CoV-2 variants of concern, Uruguay

We developed a genomic surveillance program for realtime monitoring of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) in Uruguay. We report on a PCR method for SARSCoV- 2 VOCs, the surveillance workflow, and multiple independent introductions and community transmission of the SARS-CoV-2 P.1 VOC in Uruguay

Superfamilia SCP/TAPS de Mesocestoides corti : contribución a la dilucidación del rol de estas proteínas durante el desarrollo estrobilar

Tribunal: Dra. Cecilia Fernández, Dra. Adriana Esteves, Dra. Verónica Fernánde

Informe final del proyecto: Obtención de una cepa de Saccharomyces cerevisiae productora de una beta-glucosidasa de Issatchenkia terricola y explotación del genoma de esta levadura nativa para la identificación de nuevas enzimas con potencial aplicación en enología

El desarrollo del aroma del vino depende en gran parte de la existencia durante la elaboración, de enzimas (glicosidasas) capaces de actuar eficientemente sobre los sustratos glicosídicos existentes, generando compuestos volátiles. Estudios previos de nuestro grupo con enzimas aisladas de la microbiota de viñedos uruguayos, demostraron que una beta-glucosidasa de la cepa Issatchenkia terrícola presenta propiedades muy promisorias en condiciones enológicas y se destaca por impartir características aromáticas propias a los vinos locales. Sin embargo, los bajos niveles producidos por la cepa autóctona constituyen una limitante para la manipulación y posible aplicación biotecnológica de dicha glucosidasa. Con el objetivo de clonarla y expresarla en Saccharomyces cerevsiae con mayor rendimiento, nos encontramos actualmente focalizados en obtener la secuencia de dicha glucosidasa mediante el diseño de cebadores degenerados, dado que no disponemos aún del genoma de I. terrícola. La cepa generada será utilizada en ensayos de microvinificaciones y análisis químico y sensorial de aromas de los vinos obtenidos. Complementariamente, se propone avanzar en la caracterización molecular mediante secuenciación masiva del genoma de I. terrícola. Esto permitirá identificar la presencia de otros genes codificantes para beta-glucosidasas así como otras glicosidasas. Asimismo, la interpretación del genoma permitirá identificar otras actividades enzimáticas con potencial interés biotecnológico. El proyecto implica el diseño y uso racional del potencial existente en la microbiota nativa enológica, integrando conocimientos desde un enfoque multidisciplinario desde las áreas de bioquímica, biología molecular, genómica y química de aromas. Los resultados podrían generar productos potencialmente transferibles a la industria enológica.Agencia Nacional de Investigación e Innovació

Adaptive Radiation of the Flukes of the Family Fasciolidae Inferred from Genome-Wide Comparisons of Key Species.

Liver and intestinal flukes of the family Fasciolidae cause zoonotic food-borne infections that impact both agriculture and human health throughout the world. Their evolutionary history and the genetic basis underlying their phenotypic and ecological diversity are not well understood. To close that knowledge gap, we compared the whole genomes of Fasciola hepatica, Fasciola gigantica, and Fasciolopsis buski and determined that the split between Fasciolopsis and Fasciola took place similar to 90 Ma in the late Cretaceous period, and that between 65 and 50 Ma an intermediate host switch and a shift from intestinal to hepatic habitats occurred in the Fasciola lineage. The rapid climatic and ecological changes occurring during this period may have contributed to the adaptive radiation of these flukes. Expansion of cathepsins, fatty-acid-binding proteins, protein disulfide-isomerases, and molecular chaperones in the genus Fasciola highlights the significance of excretory-secretory proteins in these liver-dwelling flukes. Fasciola hepatica and Fasciola gigantica diverged similar to 5 Ma near the Miocene-Pliocene boundary that coincides with reduced faunal exchange between Africa and Eurasia. Severe decrease in the effective population size similar to 10ka in Fasciola is consistent with a founder effect associated with its recent global spread through ruminant domestication. G-protein-coupled receptors may have key roles in adaptation of physiology and behavior to new ecological niches. This study has provided novel insights about the genome evolution of these important pathogens, has generated genomic resources to enable development of improved interventions and diagnosis, and has laid a solid foundation for genomic epidemiology to trace drug resistance and to aid surveillance