Actividades enzimáticas de terribacillus SP. AE2B 122 con aplicaciones medioambientales

La búsqueda de nuevas fuentes de energías alternativas, sostenibles y renovables se ha convertido en un área de investigación de gran importancia en los últimos años, debido, entre otras razones, a la previsión de que el petróleo se agote en las próximas décadas. En este sentido, el Biodiésel es una alternativa prometedora al gasóleo, debido a una serie de ventajas respecto al Diésel derivado del petróleo. Actualmente el proceso de producción de Biodiésel que se utiliza a nivel industrial es la metanólisis de aceites (transesterificación) utilizando catalizadores alcalinos (KOH y NaOH). Este proceso de producción no es económicamente rentable, lo que ha llevado a que el desarrollo de nuevas tecnologías alternativas para la producción de Biodiésel, innovadoras, competitivas y respetuosas con el medio ambiente constituya uno de los principales objetivos de los últimos trabajos de investigación. Una de las líneas de investigación más importantes en esta área consiste en el uso de lipasas para la producción de Biodiésel, permitiendo que la “biotecnología enzimática blanca” se introduzca en el sector industrial energético. En este contexto, la primera parte de este trabajo de Tesis Doctoral se centra en la utilización de las lipasas como catalizadores en la producción de Biodiésel, debido a la capacidad de estas enzimas para realizar reacciones de transestereficación de aceites en medios no acuosos. Siguiendo esta línea, se desarrolló un método rápido de selección de microorganismos productores de Biodiésel en una almazara de Écija, basándonos tanto en sus actividades hidrolíticas como en sus actividades transesterificadoras. De los 1.016 microorganismos analizados, dos bacterias presentaron los mejores rendimientos en la producción de FAEs tras la transesterificación de aceite de girasol. Estas bacterias resultaron pertenecer al mismo género, el género Terribacillus, el cual nunca antes había sido descrito como productor de Biodiésel, por lo que se seleccionó una de estas cepas, Terribacillus sp. AE2B 122, para posteriores análisis genéticos y bioquímicos. Adicionalmente, se comprobó mediante cromatografía de gases que el producto de la transesterificación, usando los microorganismos analizados, difería del Biodiésel convencional ya que integraba el glicerol en su composición como MG, lo que podría simplificar el procesamiento de este Biofuel. Se obtuvo la secuencia completa del genoma de Terribacillus sp. AE2B 122 y tras el análisis bioinformático de este genoma se identificaron 8 lipasas/esterasas. De ellas, Tel7 es la única de las candidatas que ha mostrado cierta actividad transesterificadora frente a pNPP. El uso de microorganismos en la biorremediación de metales pesados constituye otra área de gran interés en biotecnología ambiental. Siguiendo la línea de este trabajo en cuanto a la búsqueda de nuevas actividades de microorganismos de interés ambiental de esta tesis, la segunda parte se desarrolla en el contexto de la contaminación ambiental por metales pesados. En primer lugar, se realizó el estudio in silico de los genes presentes en el genoma de Terribacillus sp. AE2B 122 relacionados con la resistencia a metales pesados y metaloides. Para realizar este análisis se utilizaron herramientas bioinformáticas (BLAST y EXPASY) y se realizaron ensayos de resistencia de la bacteria a estos metales en medio líquido. De los metales analizados, nos centramos en el estudio de la resistencia a arsénico al comprobar que Terribacillus sp. AE2B 122 exhibía una disposición singular de los genes homólogos a los genes responsables de la resistencia a arsénico en su genoma. Los genes de resistencia a arsénico suelen co-transcribirse formando un operón, siendo generalmente la combinación mínima de genes arsRBC (ej.: E. coli, P. fluorescens, Staphylococcus). En el caso de Terribacillus sp. AE2B 122, el gen homólogo para la reductasa de As(V) arsC, no se localiza en el genoma junto a ninguno de los otros genes responsables de la resistencia a arsénico (arsB y arsR). El análisis de la expresión de estos genes mediante RT-qPCR, mostró que la expresión de uno de los genes homólogos a arsB y uno de los homólogos a arsR, que se localizan contiguos en el genoma de la bacteria, era inducible por As(V). Además, se llevaron a cabo estudios de complementación heteróloga del mutante E. coli AW3110 (ΔarsRBC::cam) con los genes homólogos a arsB y arsC de Terribacillus sp. AE2B 122 y se comprobó que la sensibilidad a As(III) del mutante se complementaba con uno de los genes homólogos de la bomba de As(III) arsB. La complementación de la sensibilidad a As(V) de E. coli AW3110 no fue tan clara, pudiendo deberse a que ninguna de las enzimas reductasas analizadas funcionen como tal, lo que podría significar que la resistencia a As(V) de la bacteria estudiada se deba a otro mecanismo de resistencia no relacionado con la reducción, como la eliminación directa del As(V) sin reducir, su unión a glutatión o chaperonas en el espacio intracelular o respuesta a estrés oxidativo

Development of the “Applied Proteomics” Concept for Biotechnology Applications in Microalgae: Example of the Proteome Data in Nannochloropsis gaditana

Most of the marine ecosystems on our planet are still unknown. Among these ecosystems, microalgae act as a baseline due to their role as primary producers. The estimated millions of species of these microorganisms represent an almost infinite source of potentially active biocomponents offering unlimited biotechnology applications. This review considers current research in microalgae using the “omics” approach, which today is probably the most important biotechnology tool. These techniques enable us to obtain a large volume of data from a single experiment. The specific focus of this review is proteomics as a technique capable of generating a large volume of interesting information in a single proteomics assay, and particularly the concept of applied proteomics. As an example, this concept has been applied to the study of Nannochloropsis gaditana, in which proteomics data generated are transformed into information of high commercial value by identifying proteins with direct applications in the biomedical and agri-food fields, such as the protein designated UCA01 which presents antitumor activity, obtained from N. gaditana

Selection and characterization of biofuel-producing environmental bacteria isolated from vegetable oil-rich wastes

Fossil fuels are consumed so rapidly that it is expected that the planet resources will be soon exhausted. Therefore, it is imperative to develop alternative and inexpensive new technologies to produce sustainable fuels, for example biodiesel. In addition to hydrolytic and esterification reactions, lipases are capable of performing transesterification reactions useful for the production of biodiesel. However selection of the lipases capable of performing transesterification reactions is not easy and consequently very few biodiesel producing lipases are currently available. In this work we first isolated 1,016 lipolytic microorganisms by a qualitative plate assay. In a second step, lipolytic bacteria were analyzed using a colorimetric assay to detect the transesterification activity. Thirty of the initial lipolytic strains were selected for further characterization. Phylogenetic analysis revealed that 23 of the bacterial isolates were Gram negative and 7 were Gram positive, belonging to different clades. Biofuel production was analyzed and quantified by gas chromatography and revealed that 5 of the isolates produced biofuel with yields higher than 80% at benchtop scale. Chemical and viscosity analysis of the produced biofuel revealed that it differed from biodiesel. This bacterial-derived biofuel does not require any further downstream processing and it can be used directly in engines. The freeze-dried bacterial culture supernatants could be used at least five times for biofuel production without diminishing their activity. Therefore, these 5 isolates represent excellent candidates for testing biofuel production at industrial scale

Proteomic study of the membrane components of signalling cascades of Botrytis cinerea controlled by phosphorylation

Protein phosphorylation and membrane proteins play an important role in the infection of plants by phytopathogenic fungi, given their involvement in signal transduction cascades. Botrytis cinerea is a well-studied necrotrophic fungus taken as a model organism in fungal plant pathology, given its broad host range and adverse economic impact. To elucidate relevant events during infection, several proteomics analyses have been performed in B. cinerea, but they cover only 10% of the total proteins predicted in the genome database of this fungus. To increase coverage, we analysed by LC-MS/MS the first-reported overlapped proteome in phytopathogenic fungi, the “phosphomembranome” of B. cinerea, combining the two most important signal transduction subproteomes. Of the 1112 membrane-associated phosphoproteins identified, 64 and 243 were classified as exclusively identified or overexpressed under glucose and deproteinized tomato cell wall conditions, respectively. Seven proteins were found under both conditions, but these presented a specific phosphorylation pattern, so they were considered as exclusively identified or overexpressed proteins. From bioinformatics analysis, those differences in the membrane-associated phosphoproteins composition were associated with various processes, including pyruvate metabolism, unfolded protein response, oxidative stress response, autophagy and cell death. Our results suggest these proteins play a significant role in the B. cinerea pathogenic cycl

An arsRB resistance operon confers tolerance to arsenite in the environmental isolate Terribacillus sp. AE2B 122

Terribacillus sp. AE2B 122 is an environmental strain isolated from olive-oil agroindustry wastes. This strain displays resistance to arsenic, one of the most ubiquitous carcinogens found in nature. Terribacillus sp. AE2B 122 possesses an unusual ars operon, consisting of the transcriptional regulator (arsR) and arsenite efflux pump (arsB) but no adjacent arsenate reductase (arsC) locus. Expression of arsR and arsB was induced when Terribacillus was exposed to sub-lethal concentrations of arsenate. Heterologous expression of the arsB homologue in Escherichia coliarsRBC demonstrated that it conferred resistance to arsenite and reduced the accumulation of arsenic inside the cells. Two members of the arsC-like family (Te3384 and Te2854) found in the Terribacillus genome were not induced by arsenic, but their heterologous expression in E. coli Delta arsC and Delta arsRBC increased the accumulation of arsenic in both strains. We found that both Te3384 and Te2854 slightly increased resistance to arsenate in E. coli Delta arsC and Delta arsRBC, possibly by chelation of arsenic or by increasing the resistance to oxidative stress. Finally, arsenic speciation assays suggest that Terribacillus is incapable of arsenate reduction, in agreement with the lack of an arsC homologue in the genome

An arsRB resistance operon confers tolerance to arsenite in the environmental isolate Terribacillus sp. AE2B 122

Terribacillus sp. AE2B 122 is an environmental strain isolated from olive-oil agroindustry wastes. This strain displays resistance to arsenic, one of the most ubiquitous carcinogens found in nature. Terribacillus sp. AE2B 122 possesses an unusual ars operon, consisting of the transcriptional regulator (arsR) and arsenite efflux pump (arsB) but no adjacent arsenate reductase (arsC) locus. Expression of arsR and arsB was induced when Terribacillus was exposed to sub-lethal concentrations of arsenate. Heterologous expression of the arsB homologue in Escherichia coli ∆arsRBC demonstrated that it conferred resistance to arsenite and reduced the accumulation of arsenic inside the cells. Two members of the arsC-like family (Te3384 and Te2854) found in the Terribacillus genome were not induced by arsenic, but their heterologous expression in E. coli ∆arsC and ∆arsRBC increased the accumulation of arsenic in both strains. We found that both Te3384 and Te2854 slightly increased resistance to arsenate in E. coli ∆arsC and ∆arsRBC, possibly by chelation of arsenic or by increasing the resistance to oxidative stress. Finally, arsenic speciation assays suggest that Terribacillus is incapable of arsenate reduction, in agreement with the lack of an arsC homologue in the genome

A Straightforward Access to New Families of Lipophilic Polyphenols by Using Lipolytic Bacteria

The chemical synthesis of new lipophilic polyphenols with improved properties presents technical difficulties. Here we describe the selection, isolation and identification of lipolytic bacteria from food-processing industrial wastes, and their use for tailoring a new set of com- pounds with great interest in the food industry. These bacteria were employed to produce lipolytic supernatants, which were applied without further purification as biocatalysts in the chemoselective and regioselective synthesis of lipophilic partially acetylated phenolic com- pounds derived from olive polyphenols. The chemoselectivity of polyphenols acylation/dea- cylation was analyzed, revealing the preference of the lipases for phenolic hydroxyl groups and phenolic esters. In addition, the alcoholysis of peracetylated 3,4-dihydroxyphenylglycol resulted in a series of lipophilic 2-alkoxy-2-(3,4-dihydroxyphenyl)ethyl acetate through an unexpected lipase-mediated etherification at the benzylic position. These new compounds are more lipophilic and retained their antioxidant properties. This approach can provide access to unprecedented derivatives of 3,4-dihydroxyphenylglycol with improved propertiesJunta de Andalucía P08-NMR-3515, P11-CVI-7427 MO, FQM134 y BIO-213European Regional Development Fund (FEDER

Development of New Antiproliferative Compound against Human Tumor Cells from the Marine Microalgae Nannochloropsis gaditana by Applied Proteomics

Proteomics is a crucial tool for unravelling the molecular dynamics of essential biological processes, becoming a pivotal technique for basic and applied research. Diverse bioinformatic tools are required to manage and explore the huge amount of information obtained from a single proteomics experiment. Thus, functional annotation and protein-protein interactions are evaluated in depth leading to the biological conclusions that best fit the proteomic response in the system under study. To gain insight into potential applications of the identified proteins, a novel approach named "Applied Proteomics" has been developed by comparing the obtained protein information with the existing patents database. The development of massive sequencing technology and mass spectrometry (MS/MS) improvements has allowed the application of proteomics nonmodel microorganisms, which have been deeply described as a novel source of metabolites. Between them, Nannochloropsis gaditana has been pointed out as an alternative source of biomolecules. Recently, our research group has reported the first complete proteome analysis of this microalga, which was analysed using the applied proteomics concept with the identification of 488 proteins with potential industrial applications. To validate our approach, we selected the UCA01 protein from the prohibitin family. The recombinant version of this protein showed antiproliferative activity against two tumor cell lines, Caco2 (colon adenocarcinoma) and HepG-2 (hepatocellular carcinoma), proving that proteome data have been transformed into relevant biotechnological information. From Nannochloropsis gaditana has been developed a new tool against cancer-the protein named UCA01. This protein has selective effects inhibiting the growth of tumor cells, but does not show any effect on control cells. This approach describes the first practical approach to transform proteome information in a potential industrial application, named "applied proteomics". It is based on a novel bioalgorithm, which is able to identify proteins with potential industrial applications. From hundreds of proteins described in the proteome of N. gaditana, the bioalgorithm identified over 400 proteins with potential uses; one of them was selected as UCA01, "in vitro" and its potential was demonstrated against cancer. This approach has great potential, but the applications are potentially numerous and undefined

Cepa microbiana Terribacillus SP-AE2B 122 con capacidad para llevar a cabo reacciones de transesterificación y usos de la misma

Cepa Microbiana Terribacillus SP. AE2B 122 con capacidad para llevar a cabo reacciones de transesterificación y usos de la misma.La presente invención se refiere a una cepa bacteriana del género Terribacillus, denominada Terribacillus sp. AE2B 122 con ca