Person: Navarro Llorens, Juana María
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First Name
Juana María
Last Name
Navarro Llorens
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Ciencias Químicas
Department
Bioquímica y Biología Molecular
Area
Bioquímica y Biología Molecular
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11 results
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Item Functional differentiation of 3-ketosteroid Δ1-dehydrogenase isozymes in Rhodococcus ruber strain Chol-4(Microbial Cell Factories, 2017) Guevara, Govinda; Fernández de las Heras, Laura; Perera González, Jesús; Navarro Llorens, Juana MaríaBackground: The Rhodococcus ruber strain Chol-4 genome contains at least three putative 3-ketosteroid Δ1 - dehydrogenase ORFs (kstD1, kstD2 and kstD3) that code for flavoenzymes involved in the steroid ring degradation. The aim of this work is the functional characterization of these enzymes prior to the developing of different biotechnological applications. Results: The three R. ruber KstD enzymes have different substrate profiles. KstD1 shows preference for 9OHAD and testosterone, followed by progesterone, deoxy corticosterone AD and, finally, 4-BNC, corticosterone and 19OHAD. KstD2 shows maximum preference for progesterone followed by 5α-Tes, DOC, AD testosterone, 4-BNC and lastly 19OHAD, corticosterone and 9OHAD. KstD3 preference is for saturated steroid substrates (5α-Tes) followed by progesterone and DOC. A preliminary attempt to model the catalytic pocket of the KstD proteins revealed some structural differences probably related to their catalytic differences. The expression of kstD genes has been studied by RT-PCR and RT-qPCR. All the kstD genes are transcribed under all the conditions assayed, although an additional induction in cholesterol and AD could be observed for kstD1 and in cholesterol for kstD3. Co-transcription of some correlative genes could be stated. The transcription initiation signals have been searched, both in silico and in vivo. Putative promoters in the intergenic regions upstream the kstD1, kstD2 and kstD3 genes were identified and probed in an apramycin-promoter-test vector, leading to the functional evidence of those R. ruber kstD promoters. Conclusions: At least three putative 3-ketosteroid Δ1 -dehydrogenase ORFs (kstD1, kstD2 and kstD3) have been identified and functionally confirmed in R. ruber strain Chol-4. KstD1 and KstD2 display a wide range of substrate preferences regarding to well-known intermediaries of the cholesterol degradation pathway (9OHAD and AD) and other steroid compounds. KstD3 shows a narrower substrate range with a preference for saturated substrates. KstDs differences in their catalytic properties was somehow related to structural differences revealed by a preliminary structural modelling. Transcription of R. ruber kstD genes is driven from specific promoters. The three genes are constitutively transcribed, although an additional induction is observed in kstD1 and kstD3. These enzymes have a wide versatility and allow a fine tuning-up of the KstD cellular activity.- Project number: 140
Item Estrategias de flipped learning en fundamentos de ingeniería genética(2019) Navarro Llorens, Juana María; Lorente Pérez, María del Mar; Sánchez Torralba, Antonio; Blázquez Ortiz, Cristina; Ranz Valdecasa, María Regina; López Conejo, María Teresa; Guevara Acosta, Flor GovindaLa asignatura de Fundamentos de Ingeniería genética del grado de Biología resulta muy árida en su actual formulación. Este proyecto pretende darle la vuelta para que sea el alumno quien construya el conocimiento y participe en el aprendizaje. Item New insights into the genome of Rhodococcus ruber strain Chol-4(BMC Genomics, 2019) Guevara Acosta, Flor Govinda; Castillo López, María; Alonso, Sergio; Perera González, Julián; Navarro Llorens, Juana MaríaBackground: Rhodococcus ruber strain Chol-4, a strain isolated from a sewage sludge sample, is able to grow in minimal medium supplemented with several compounds, showing a broad catabolic capacity. We have previously determined its genome sequence but a more comprehensive study of their metabolic capacities was necessary to fully unravel its potential for biotechnological applications. Results: In this work, the genome of R. ruber strain Chol-4 has been re-sequenced, revised, annotated and compared to other bacterial genomes in order to investigate the metabolic capabilities of this microorganism. The analysis of the data suggests that R. ruber Chol-4 contains several putative metabolic clusters of biotechnological interest, particularly those involved on steroid and aromatic compounds catabolism. To demonstrate some of its putative metabolic abilities, R. ruber has been cultured in minimal media containing compounds belonging to several of the predicted metabolic pathways. Moreover, mutants were built to test the naphtalen and protocatechuate predicted catabolic gene clusters. Conclusions: The genomic analysis and experimental data presented in this work confirm the metabolic potential of R. ruber strain Chol-4. This strain is an interesting model bacterium due to its biodegradation capabilities. The results obtained in this work will facilitate the application of this strain as a biotechnological tool.Item SEVA-Cpf1, a CRISPR-Cas12a vector for genome editing in cyanobacteria(Microbial Cell Factories, 2022) Baldanta Callejo, Sara; Guevara Acosta, Flor Govinda; Navarro Llorens, Juana MaríaBackground Cyanobacteria are photosynthetic autotrophs that have tremendous potential for fundamental research and industrial applications due to their high metabolic plasticity and ability to grow using CO2 and sunlight. CRISPR technology using Cas9 and Cpf1 has been applied to different cyanobacteria for genome manipulations and metabolic engineering. Despite significant advances with genome editing in several cyanobacteria strains, the lack of proper genetic toolboxes is still a limiting factor compared to other model laboratory species. Among the limitations, it is essential to have versatile plasmids that could ease the benchwork when using CRISPR technology. Results In the present study, several CRISPR-Cpf1 vectors were developed for genetic manipulations in cyanobacteria using SEVA plasmids. SEVA collection is based on modular vectors that enable the exchangeability of diverse elements (e.g. origins of replication and antibiotic selection markers) and the combination with many cargo sequences for varied end-applications. Firstly, using SEVA vectors containing the broad host range RSF1010 origin we demonstrated that these vectors are replicative not only in model cyanobacteria but also in a new cyanobacterium specie, Chroococcidiopsis sp., which is different from those previously published. Then, we constructed SEVA vectors by harbouring CRISPR elements and showed that they can be easily assimilated not only by conjugation, but also by natural transformation. Finally, we used our SEVA-Cpf1 tools to delete the nblA gene in Synechocystis sp. PCC 6803, demonstrating that our plasmids can be applied for CRISPR-based genome editing technology. Conclusions The results of this study provide new CRISPR-based vectors based on the SEVA (Standard European Vector Architecture) collection that can improve editing processes using the Cpf1 nuclease in cyanobacteria.- Project number: 306
Item La comunidad del anillo IGGIA: construyendo redes de mentoría en Ingeniería Genética mediante gamificación, internacionalización y accesibilidad(2022) Sánchez Torralba, Antonio; Benítez Prian, Mario; Blázquez Ortiz, Cristina; Bruñén Alfaro, Francisco; Cañadas Benito, Olga; García de la Camacha Selgas, Nuria; García-Fojeda García-Valdecasas, Belén; González Miranda, David; Guevara Acosta, Govinda; López Conejo, María Teresa; Lorente Pérez, María del Mar; Mateo Mendoza, Jorge Mario; Nogués Vera, Laura; Raisman, Andrea; Ranz Valdecasa, María Regina; Ruiz Ortega, Marta; Sánchez-Escalonilla Relea, Jose Luis; Sánchez Velasco, Teresa; Toledo Marcos, Juan; Velasco Díez, Guillermo; Navarro Llorens, Juana María Item Metabolic engineering of Rhodococcus ruber Chol-4: A cell factory for testosterone production(Plos One, 2019) Guevara Acosta, Flor Govinda; Olortegui Flores, Yamileth; Fernández de las Heras, Laura; Perera González, Julián; Navarro Llorens, Juana MaríaRhodococcus ruber Chol-4 is a potent steroid degrader that has a great potential as a biotechnological tool. As proof of concept, this work presents testosterone production from 4- androstene-3,17-dione by tailoring innate catabolic enzymes of the steroid catabolism inside the strain. A R. ruber quadruple mutant was constructed in order to avoid the breakage of the steroid nucleus. At the same time, an inducible expression vector for this strain was developed. The 17-ketoreductase gene from the fungus Cochliobolus lunatus was cloned and overexpressed in this vector. The engineered strain was able to produce testosterone from 4-androstene-3,17-dione using glucose for cofactor regeneration with a molar conversion of 61%. It is important to note that 91% of the testosterone was secreted outside the cell after 3 days of cell biotransformation. The results support the idea that Rhodococcus ruber Chol-4 can be metabolically engineered and can be used for the production of steroid intermediates.Item Further Studies on the 3-Ketosteroid 9α-Hydroxylase of Rhodococcus ruber Chol-4, a Rieske Oxygenase of the Steroid Degradation Pathway(Microorganisms, 2021) Baldanta Callejo, Sara; Navarro Llorens, Juana María; Guevara Acosta, Flor GovindaThe biochemistry and genetics of the bacterial steroid catabolism have been extensively studied during the last years and their findings have been essential to the development of biotechnological applications. For instance, metabolic engineering of the steroid-eater strains has allowed to obtain intermediaries of industrial value. However, there are still some drawbacks that must be overcome, such as the redundancy of the steroid catabolism genes in the genome and a better knowledge of its genetic regulation. KshABs and KstDs are key enzymes involved in the aerobic breakage of the steroid nucleus. Rhodococcus ruber Chol-4 contains three kshAs genes, a single kshB gene and three kstDs genes within its genome. In the present work, the growth of R. ruber ΔkshA strains was evaluated on different steroids substrates; the promoter regions of these genes were analyzed; and their expression was followed by qRT-PCR in both wild type and ksh mutants. Additionally, the transcription level of the kstDs genes was studied in the ksh mutants. The results show that KshA2B and KshA1B are involved in AD metabolism, while KshA3B and KshA1B contribute to the cholesterol metabolism in R. ruber. In the kshA single mutants, expression of the remaining kshA and kstD genes is re-organized to survive on the steroid substrate. These data give insight into the fine regulation of steroid genes when several isoforms are present.- Project number: 112
Item Estrategias multimedia para el aprendizaje en el laboratorio integrado de bioquímica y biología molecular I.(2016) Sánchez Torralba, Antonio; Feito Castellano, María Jose; Arroyo Sánchez, Miguel; Saborido Modia, Ana; Navarro Llorens, Juana MaríaEste proyecto persigue fomentar la adquisición de conocimientos y habilidades en el laboratorio de Bioquímica combinando estrategias multimedia con el aprendizaje cooperativo centrado en el alumno y haciendo hincapié en la adaptación al inglés de los contenidos. Para ello se proponen tres estrategias didácticas complementarias para potenciar el aprendizaje autónomo del alumno en la asignatura: adaptación de parte de los contenidos de la materia al inglés, visualización de vídeos docentes elaborados para este proyecto (preparación de tampones, preparación de diluciones y realización de tablas de pipeteo) así como cuestiones test relacionados con el vídeo y la realización de un congreso en donde se presentan los trabajos realizados por los alumnos en formato póster o vídeo. Item Xerotolerance: a new property in Exiguobacterium Genus(Microorganisms, 2021) Castillo López, María; Galán, Beatriz; Carmona, Manuel; Navarro Llorens, Juana María; Peretó, Juli; Porcar, Manuel; Getino, Luis; Olivera, Elías R.; Luengo, José M.; García, José Luis; Castro Ruiz, LauraThe highly xerotolerant bacterium classified as Exiguobacterium sp. Helios isolated from a solar panel in Spain showed a close relationship to Exiguobacterium sibiricum 255-15 isolated from Siberian permafrost. Xerotolerance has not been previously described as a characteristic of the extremely diverse Exiguobacterium genus, but both strains Helios and 255-15 showed higher xerotolerance than that described in the reference xerotolerant model strain Deinococcus radiodurans. Significant changes observed in the cell morphology after their desiccation suggests that the structure of cellular surface plays an important role in xerotolerance. Apart from its remarkable resistance to desiccation, Exiguobacterium sp. Helios strain shows several polyextremophilic characteristics that make it a promising chassis for biotechnological applications. Exiguobacterium sp. Helios cells produce nanoparticles of selenium in the presence of selenite linked to its resistance mechanism. Using the Lactobacillus plasmid pRCR12 that harbors a cherry marker, we have developed a transformation protocol for Exiguobacterium sp. Helios strain, being the first time that a bacterium of Exiguobacterium genus has been genetically modified. The comparison of Exiguobacterium sp. Helios and E. sibiricum 255-15 genomes revealed several interesting similarities and differences. Both strains contain a complete set of competence-related DNA transformation genes, suggesting that they might have natural competence, and an incomplete set of genes involved in sporulation; moreover, these strains not produce spores, suggesting that these genes might be involved in xerotolerance.Item First characterization of cultivable extremophile Chroococcidiopsis isolates from a solar panel(Frontiers in Microbiology, 2023) Baldanta Callejo, Sara; Arnal, Raquel; Blanco-Rivero, Amaya; Guevara Acosta, Flor Govinda; Navarro Llorens, Juana MaríaIntroduction: Microorganisms colonize a wide range of natural and artificial environments. Even though most of them are unculturable in laboratory conditions, some ecosystems are ideal niches for bioprospecting extremophiles with unique properties. Up today, there are few reports concerning microbial communities found on solar panels, a widespread, artificial, extreme habitat. Microorganisms found in this habitat belong to drought-, heat- and radiation-adapted genera, including fungi, bacteria, and cyanobacteria. Methods: Here we isolated and identified several cyanobacteria from a solar panel. Then, some strains isolated were characterizated for their resistance to desiccation, UV-C exposition, and their growth on a range of temperature, pH, NaCl concentration or diverse carbon and nitrogen sources. Finally, gene transfer to these isolates was evaluated using several SEVA plasmids with different replicons to assess their potential in biotechnological applications. Results and discussion: This study presents the first identification and characterization of cultivable extremophile cyanobacteria from a solar panel in Valencia, Spain. The isolates are members of the genera Chroococcidiopsis, Leptolyngbya, Myxacorys, and Oculatella all genera with species commonly isolated from deserts and arid regions. Four of the isolates were selected, all of them Chroococcidiopsis, and characterized. Our results showed that all Chroococcidiopsis isolates chosen were resistant up to a year of desiccation, viable after exposition to high doses of UV-C, and capable of being transformed. Our findings revealed that a solar panel is a useful ecological niche in searching for extremophilic cyanobacteria to further study the desiccation and UV-tolerance mechanisms. We conclude that these cyanobacteria can be modified and exploited as candidates for biotechnological purposes, including astrobiology applications.