La nueva Biotecnología apuesta por la vinificación

La OTRI del Centro Tecnológico Nacional de la Conserva y Alimentación junto con la OTT del Consejo Superior de Investigaciones Científicas, colaboran en el Proyecto AGROCSIC, el cual fue aprobado por el Ministerio de Ciencia y Tecnología y financiado por el Ministerio de Educación y Ciencia. El objetivo principal de esta nueva actuación es estudiar las distintas líneas de trabajo de los Centros del CSIC relacionadas con la alimentación, para transferir sus resultados al sector industrial.Desde el punto de vista microbiológico, la transformación del mosto de uva en vino o vinificación es un proceso complejo en el que intervienen distintos microorganismos, y donde las levaduras, principalmente Saccharomyces cerevisiae, juegan el papel más destacado. Durante la vinificación las levaduras utilizan los azúcares y otros componentes del mosto para su crecimiento, produciendo etanol, anhídrido carbónico, y en menor medida otros compuestos responsables de la composición química y las cualidades sensoriales del vino. Desde un punto de vista bioquímico, el vino puede considerarse como un producto de la transformación enzimática del mosto de uva.En las últimas décadas, coincidiendo con el avance de la Biotecnología, se han desarrollado nuevas técnicas de vinificación, que incluyen, entre otras, la utilización de cepas seleccionadas de S. cerevisiae (para normalizar la microbiota inicial y dar lugar a fermentaciones homogéneas año tras año), y el empleo de enzimas (para solucionar problemas puntuales del proceso y mejorar la calidad del producto final). El uso de levaduras seleccionadas, capaces de conducir la fermentación alcohólica e imponerse al resto de levaduras presentes, abre la posibilidad de aplicar técnicas de ingeniería genética a la levadura vínica para obtener nuevas cepas capaces de producir, a lo largo de la fermentación, las enzimas de interés en enología cuya adición se realiza de forma regular. Asimismo, las técnicas de ADN recombinante se están aplicando a la vid, donde se están produciendo importantes avances (como por ejemplo la producción de plantas resistentes a ciertas enfermedades víricas y fúngicas) y a los microorganismos productores de los preparados enzimáticos comerciales de uso en enología (por ejemplo para conseguir mayores rendimientos y preparados más específicos).La información que las autoras aportan a esta revisión es fruto del desarrollo de varios proyectos financiados por la Comisión Interministerial de Ciencia y Tecnología (CICYT) entre los que se incluyen los proyectos en curso AGL2002-01906, AGL2003-01295/ALI y AGL2004-00978/ALI.Peer reviewe

De novo production of six key grape aroma monoterpenes by a geraniol synthase-engineered S. cerevisiae wine strain

[Background] Monoterpenes are important contributors to grape and wine aroma. Moreover, certain monoterpenes have been shown to display health benefits with antimicrobial, anti-inflammatory, anticancer or hypotensive properties amongst others. The aim of this study was to construct self-aromatizing wine yeasts to overproduce de novo these plant metabolites in wines.[Results] Expression of the Ocimum basilicum (sweet basil) geraniol synthase (GES) gene in a Saccharomyces cerevisiae wine strain substantially changed the terpene profile of wine produced from a non-aromatic grape variety. Under microvinification conditions, and without compromising other fermentative traits, the recombinant yeast excreted geraniol de novo at an amount (~750 μg/L) well exceeding (>10-fold) its threshold for olfactory perception and also exceeding the quantities present in wines obtained from highly aromatic Muscat grapes. Interestingly, geraniol was further metabolized by yeast enzymes to additional monoterpenes and esters: citronellol, linalool, nerol, citronellyl acetate and geranyl acetate, resulting in a total monoterpene concentration (~1,558 μg/L) 230-fold greater than that of the control. We also found that monoterpene profiles of wines derived from mixed fermentations were found to be determined by the composition of the initial yeast inocula suggesting the feasibility of producing ‘à la carte’ wines having predetermined monoterpene contents.[Conclusions] Geraniol synthase-engineered yeasts demonstrate potential in the development of monoterpene enhanced wines.This work was funded by the Spanish MINECO/FEDER Grants CSD2007-0063 and AGL2011-29925. We also acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). E. Pardo was financially supported by JCI2007-123/733 and JAEDOC086-FSE contracts. J. Rico was the recipient of a FPI (Formación de Personal de Investigador) pre-doctoral fellowship from the Comisión Interministerial de Ciencia y Tecnología (CICYT).Peer Reviewe

Transcriptional analysis of the lichenase-like gene cel12A of the filamentous fungus Stachybotrys atra BP-A and its relevance for lignocellulose depolymerization

To rationally optimize the production of industrial enzymes by molecular means requires previous knowledge of the regulatory circuits controlling the expression of the corresponding genes. The genus Stachybotrys is an outstanding producer of cellulose-degrading enzymes. Previous studies isolated and characterized the lichenase-like/non-typical cellulase Cel12A of S. atra (AKA S. chartarum) belonging to glycosyl hydrolase family 12 (GH12). In this study, we used RT-qPCR to determine the pattern of expression of cel12A under different carbon sources and initial ambient pH. Among the carbon sources examined, rice straw triggered a greater increase in the expression of cel12A than 1% lactose or 0.1% glucose, indicating specific induction by rice straw. In contrast, cel12A was repressed in the presence of glucose even when combined with this inducer. The proximity of 2 adjacent 5′-CTGGGGTCTGGGG-3′ CreA consensus target sites to the translational start site of cel12A strongly suggests that the carbon catabolite repression observed is directly mediated by CreA. Ambient pH did not have a significant effect on cel12A expression. These findings present new knowledge on transcriptional regulatory networks in Stachybotrys associated with cellulose/hemicellulose depolymerization. Rational engineering of CreA to remove CCR could constitute a novel strategy for improving the production of Cel12A

L-Rhamnose induction of Aspergillus nidulans α-L-rhamnosidase genes is glucose repressed via a CreA-independent mechanism acting at the level of inducer uptake

<p>Abstract</p> <p>Background</p> <p>Little is known about the structure and regulation of fungal α-L-rhamnosidase genes despite increasing interest in the biotechnological potential of the enzymes that they encode. Whilst the paradigmatic filamentous fungus <it>Aspergillus nidulans </it>growing on L-rhamnose produces an α-L-rhamnosidase suitable for oenological applications, at least eight genes encoding putative α-L-rhamnosidases have been found in its genome. In the current work we have identified the gene (<it>rhaE</it>) encoding the former activity, and characterization of its expression has revealed a novel regulatory mechanism. A shared pattern of expression has also been observed for a second α-L-rhamnosidase gene, (AN10277/<it>rhaA</it>).</p> <p>Results</p> <p>Amino acid sequence data for the oenological α-L-rhamnosidase were determined using MALDI-TOF mass spectrometry and correspond to the amino acid sequence deduced from AN7151 (<it>rhaE</it>). The cDNA of <it>rhaE </it>was expressed in <it>Saccharomyces cerevisiae </it>and yielded <it>p</it>NP-rhamnohydrolase activity. Phylogenetic analysis has revealed this eukaryotic α-L-rhamnosidase to be the first such enzyme found to be more closely related to bacterial rhamnosidases than other α-L-rhamnosidases of fungal origin. Northern analyses of diverse <it>A. nidulans </it>strains cultivated under different growth conditions indicate that <it>rhaA </it>and <it>rhaE </it>are induced by L-rhamnose and repressed by D-glucose as well as other carbon sources, some of which are considered to be non-repressive growth substrates. Interestingly, the transcriptional repression is independent of the wide domain carbon catabolite repressor CreA. Gene induction and glucose repression of these <it>rha </it>genes correlate with the uptake, or lack of it, of the inducing carbon source L-rhamnose, suggesting a prominent role for inducer exclusion in repression.</p> <p>Conclusions</p> <p>The <it>A. nidulans rhaE </it>gene encodes an α-L-rhamnosidase phylogenetically distant to those described in filamentous fungi, and its expression is regulated by a novel CreA-independent mechanism. The identification of <it>rhaE </it>and the characterization of its regulation will facilitate the design of strategies to overproduce the encoded enzyme - or homologs from other fungi - for industrial applications. Moreover, <it>A. nidulans </it>α-L-rhamnosidase encoding genes could serve as prototypes for fungal genes coding for plant cell wall degrading enzymes regulated by a novel mechanism of CCR.</p

Protein-DNA interactions in the promoter region of the Phycomyces carB and carRA genes correlate with the kinetics of their mRNA accumulation in response to light.

[EN] Carotene biosynthesis in Phycomyces is photoinducible and carried out by phytoene dehydrogenase (encoded by carB) and a bifunctional enzyme possessing lycopene cyclase and phytoene synthase activities (carRA). A light pulse followed by periods of darkness produced similar biphasic responses in the expression of the carB and carRA genes, indicating their coordinated regulation. Specific binding complexes were formed between the carB-carRA intergenic region and protein extracts from wild type mycelia grown in the dark or 8min after irradiation. These two conditions correspond to the points at which the expression of both genes is minimal, suggesting that these binding complexes are involved in the down-regulation of photocarotenogenesis in Phycomyces. Protein extracts from carotene mutants failed to form the dark retardation complex, suggesting a role of these genes in the regulation of photocarotenogenesis. In contrast, protein extracts from phototropic mutants formed dark retardation complexes identical to that of the wild type.Junta de Castilla y León; Spanish Ministerio de Educación y Ciencia/FEDE

Intralabyrinthine schwannoma. A case report

Introduction and objectives: The intralabyrinthine schwannoma is a rare benign tumor, with extradural slow growth, located primarily in the membranous labyrinth. Through a clinical case, we present the characteristics of its evolution, we try to offer a physio-pathological explanation of symptoms and audio-vestibular findings and finally, we describe the therapeutic options. Description: We present a 59 year old woman, beginning with non-specific symptoms of the left ear and then concerned because of a hearing loss in that ear. To confirm that it is a non-justified unilateral hearing loss , MRI was requested and it shows the presence of a small intravestibular tumor of 2-3 mm which we will review periodically. Discussion: The origin and the prevalence of intralabyrinthine schwannomas are unknown. Even though the clinic is confused with other frequent otological processes, the implementation of MRI would increase the number of diagnosed cases. Depending on the patient, their symptoms, evolution and tumor characteristics, the type of treatment is decided. Conclusion: Intralabyrinthine schwannoma should be part of the differential diagnosis in patients with unilateral audio-vestibular symptoms and request a high resolution MRI to obtain the definitive diagnosis. Presenting an excellent prognosis, the observation is considered the best treatment option.Introducción y objetivos: El schwannoma intralaberíntico es un tumor benigno muy infrecuente, de lento crecimiento extradural, localizado primariamente en el laberinto membranoso. A través de un caso clínico presentamos las características de su evolución, intentamos ofrecer una explicación fisiopatológica de la sintomatología y de los hallazgos audio-vestibulares y describimos finalmente, las diferentes opciones terapéuticas. Descripción: Se trata de una mujer de 59 años, que comienza con síntomas inespecíficos del oído izquierdo a los que posteriormente se añade una pérdida auditiva. Al confirmar que se trata de una hipoacusia unilateral no justificada, solicitamos una RM que pone de manifiesto la presencia de un pequeño tumor de 2-3 mm intravestibular, al que revisaremos periódicamente. Discusión: El origen y la prevalencia del schwannoma intralaberíntico son desconocidos. Aunque la clínica se confunde con la de otros procesos otológicos más frecuentes, la realización de RM incrementaría el número de casos diagnosticados. En función del paciente, de su sintomatología, de la evolución y de las características tumorales, se decide el tipo de tratamiento Conclusión: El schwannoma intralaberíntico debe formar parte del diagnóstico diferencial en pacientes con síntomas audio-vestibulares unilaterales y solicitar una RM de alta resolución para obtener el diagnóstico definitivo. Presentando un pronóstico excelente, la observación es considerada como la mejor opción terapéutica

Retropharyngeal tendinitis, brachial plexopathy and Horner's syndrome from an anomalous cervical posture

[ES] Introducción y objetivo: Presentamos el caso clínico de un paciente que, a consecuencia de una mala postura cervical, manifiesta una sintomatología y exploración compatibles con tendinitis retrofaríngea, síndrome de Horner y plexopatía braquial del mismo lado. No habiendo encontrado en la literatura una descripción similar, analizamos las características y posible fisiopatología de esta rara asociación. Caso clínico: Tras consumir en exceso alcohol y drogas, un hombre joven se queda dormido, inmóvil durante horas, con la cabeza inclinada sobre el hombro izquierdo. Al despertar acude a urgencias por presentar un intenso dolor de nuca y pérdida de sensibilidad en el cuello y miembro superior derecho. Posteriormente refiere dolor de garganta. La exploración clínica, analítica y radiológica permite el diagnóstico de síndrome de Horner, plexopatía braquial y tendinitis retrofaríngea del lado derecho. Discusión: Son pocos los casos publicados en los que una mala postura desencadene una tendinitis retrofaríngea, una plexopatía braquial o un síndrome de Horner y el único conocido por nosotros donde la extensión cervical forzada sea la causa de esta triple asociación. Las alteraciones neurológicas se diagnosticaron a través de la exploración clínica y radiológica, pero la tendinitis retrofaríngea ofrece una sintomatología y una exploración que puede confundirse con otras patologías de mayor transcendencia. Conclusión: Creemos que la postura produjo un estiramiento del “músculo longus colli”, del plexo braquial y de las fibras cervicales simpáticas preganglionares, desencadenando este cuadro clínico, que nunca hemos visto anteriormente descrito en la literatura. [EN] Introduction and objective: We present a clinical case of a patient who, because of an incorrect cervical posture, displayed symptoms and examination compatible with retropharyngeal tendinitis, Horner's syndrome and brachial plexopathy all on the same side. We have not seen a similar case described in the literature. The characteristics and possible physiopathology of this rare association is analysed. Case report: After excessive alcohol and drug consumption, a young man fell asleep and lay immobile for hours with his head bent towards his left shoulder. Upon awakening, he went to emergency services due to severe pain in the nape of his neck and loss of feeling in his neck and upper right arm. He later reported severe throat pain. Physical examination, analytical tests and x-rays yielded the diagnosis of retropharyngeal tendinitis, Horner's syndrome and brachial plexopathy of the right side. Discussion: There are few published cases where poor posture triggers a tendinitis retropharyngeal, brachial plexopathy or Horner Syndrome and the only one we know, where forced cervical extension causes the association of the three pictures. Neurological alterations were quickly diagnosed through clinical and radiological examination, but the retropharyngeal tendinitis offers a symptomatology and exploration which can be confused with other pathologies of greater importance. Conclusions: We feel the patient's posture stretched the “longus colli muscle”, brachial plexus and preganglionic sympathetic fibres, triggering this associated condition, which we have not seen before in the literature

Expansion of Signal Transduction Pathways in Fungi by Extensive Genome Duplication

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Enhanced glycosyl hydrolase production in Aspergillus nidulans using transcription factor engineering approaches

[Background] Engineered fungi are attractive platforms for the production of plant cell wall hydrolytic enzymes which, among other biotechnological applications, are required for the efficient conversion of biomass to glucose and other fermentable sugars. As a fungal model system, Aspergillus nidulans provides genetic tools that are of relevance in this context and potentially applicable to industrially important filamentous fungi. The goal of this study is to assess the utility of A. nidulans as a host for recombinant protein production.[Results] We have successfully applied a transcription factor engineering approach to improve the efficiency of the A. nidulans xylanolytic XlnR-xln p expression system. Specifically, endo-1,4-β-xylanases and an α-L-rhamnosidase were chosen as representatives of endogenous and heterologous glycosyl hydrolases involved in plant cell wall deconstruction. By deregulating the expression of the xylanolytic transcriptional activator XlnR and modulating the activity of the pH regulator PacC we improved protein production and reduced production times. Xylanase activity was about 200-fold greater in gpdA p::xlnR strains compared to controls 4 hours after transfer to inducing conditions, and 10-fold greater after 24 hours. Remarkably, 75% of the xylanase activity was present in the engineered strains within 4 hours. Engineering XlnR expression also had a considerable impact on foreign protein production, especially when the promoter of the 'acidic' xlnB gene was used to express the transgene. α-L-rhamnosidase activity in xlnB p::rhaA, gpdA p::xlnR strains was about 19-fold greater than that of controls 72 hours after transfer to xylan (about 85% of the total activity produced), and 10-fold greater at later times (120 hours). The performance of these strains was further enhanced by impeding the proteolytic activation of PacC; introduction of the palA1 allele in xlnB p::rhaA, gpdA p::xlnR strains resulted in an additional 2.7-fold increase in α-L-rhamnosidase activity by 48 hours (about 87% of the total activity produced) and a 1.7-fold increase at later times.[Conclusions] Our results show that the XlnR-xln p expression system is a valuable tool for manipulating the production of plant cell wall degrading enzymes in A. nidulans and establish the biotechnological potential of the transcription factors XlnR and PacC to boost and control the strength of xylanolytic promoters.This work was supported by the Spanish Ministerio de Ciencia e Innovación/FEDER (grant numbers AGL2002-01906, AGL2011-29925, given to MO, and CSD2007-0063/Consolider-Ingenio]. JAT was the recipient of a pre-doctoral fellowship (FPI, Formación de Personal de Investigador) from the Comisión Interministerial de Ciencia y Tecnología given to MO. The group also participates in the EC COST Action FA0907_BIOFLAVOURPeer Reviewe