fabH deletion increases DHA productionin Escherichia coli expressing Pfa genes

Background: Some marine bacteria, such as Moritella marina, produce the nutraceutical docosahexaenoic acid (DHA) thanks to a specific enzymatic complex called Pfa synthase. Escherichia coli heterologously expressing the pfa gene cluster from M. marina also produces DHA. The aim of this study was to find genetic or metabolic conditions to increase DHA production in E. coli. Results: First, we analysed the effect of the antibiotic cerulenin, showing that DHA production increased twofold. Then, we tested a series of single gene knockout mutations affecting fatty acid biosynthesis, in order to optimize the synthesis of DHA. The most effective mutant, fabH, showed a threefold increase compared to wild type strain. The combination of cerulenin inhibition and fabH deletion rendered a 6.5-fold improvement compared to control strain. Both strategies seem to have the same mechanism of action, in which fatty acid synthesis via the canonical pathway (fab pathway) is affected in its first catalytic step, which allows the substrates to be used by the heterologous pathway to synthesize DHA. Conclusions: DHA-producing E. coli strain that carries a fabH gene deletion boosts DHA production by tuning down the competing canonical biosynthesis pathway. Our approach can be used for optimization of DHA production in different organisms.Funding: The work in the FdlC and GM laboratories was financed by the Spanish Ministry of Economy, Industry and Competitiveness Grant BFU2014-55534-C2

Fuentes orales y archivos: Modelos de Syllabus de clases prácticas para Grados de Historia, Historia del Arte y Arqueología

Este documento contiene la memoria final del proyecto de innovación docente nº. 11 de la convocatoria para el curso académico 2022-2023. En ella se contiene modelos de clases prácticas para aquellos profesores de los Grados de Historia, Historia del Arte o Arqueología que pudieran impartir asignaturas de dichos Grados.Depto. de Historia Moderna y ContemporáneaFac. de Geografía e HistoriaFALSEsubmitte

InnoCOVID-19: Primer congreso multidisciplinar Innocampus

[EN] The main objective of the educational innovation project Innocampus Explora is to show the existing interrelationship between the different scientific and technical degrees of Burjassot-Paterna campus of the University of Valencia. In this paper we present the InnoCOVID-19 Congress, activity organized during the 2020-21 academic year by the work team, made up of students and professors from all the Campus faculties.The objective of this conference, developed in virtual format, was to present the multidisciplinary projects that were formed on campus in response to the health crisis caused by the coronavirus SARS-CoV-2. This activity contributes to a quality transversal training for all participating students.[ES] El proyecto de innovación educativa Innocampus Explora tiene como objetivo principal mostrar la interrelación existente entre los diferentes grados científicos y técnicos del campus de Burjassot-Paterna de la Universitat de València. En este artículo presentamos el congreso InnoCOVID-19, la actividad organizada durante el curso académico 2020-21 por el equipo de trabajo, integrado por estudiantes y profesores de todas las facultades y escuelas. El objetivo de este congreso, desarrollado en formato virtual, ha sido presentar los proyectos multidisciplinares que se han formado en el campus en respuesta a la crisis sanitaria provocada por el coronavirus SARS-CoV-2. Esta actividad contribuye a una formación transversal de calidad para todos los estudiantes participantes.Proyecto UV-SFPIE PIC-1363315 financiado por el Servei de Formació Permanent i Innovació Educativa de la Universitat de València.Amorós Hernández, L.; Blas Medina, A.; Cervera Sanz, M.; Cosme Llópez, E.; García Gil, R.; García Lázaro, S.; García Robles, I.... (2021). InnoCOVID-19: Primer congreso multidisciplinar Innocampus. En IN-RED 2021: VII Congreso de Innovación Edicativa y Docencia en Red. Editorial Universitat Politècnica de València. 1171-1182. https://doi.org/10.4995/INRED2021.2021.13449OCS1171118

Innocampus Explora: una aproximación multidisciplinar a la problemática ambiental

[ES] Presentamos las actividades del proyecto de innovación Innocampus Explora desarrollado en el campus de Burjassot-Paterna de la Universitat de València y cuyo objetivo principal es mostrar la interrelación existente entre los diferentes grados científicos y técnicos del campus. En la presente anualidad, el equipo de trabajo integrado por estudiantes y profesores de todos las facultades y escuelas del campus de Burjassot-Paterna, ha desarrollado actividades en torno a la problemática medioambiental. Una visión transversal e interdisciplinar de los problemas de los usos del plástico y de la energía nuclear que enlaza con varios de los Objetivos de Desarrollo Sostenible (ODS) dictados por Naciones Unidas. Con el desarrollo de este proyecto contribuimos a una formación transversal de calidad para todos los estudiantes participantes.[EN] We present the activities of the Innocampus Explora innovation project developed on the Burjassot-Paterna campus of the Universitat de València and whose main objective is to show the interrelation between the different scientific and technical degrees on campus. In this year, the work team made up of students and professors from all the faculties and schools of the Burjassot-Paterna campus, have carried out activities around environmental issues. A cross-sectional and interdisciplinary vision of the problems of the uses of plastic and nuclear energy that link with several of the Sustainable Development Goals (SDGs) dictated by the United Nations. With the development of this project we contribute to quality transversal training for all participating students.Moros Gregorio, J.; Quílez Asensio, A.; Jimenez Romero, D.; Blas Medina, A.; Giménez Escamilla, I.; Amorós Hernández, L.; Giner, L.... (2021). Innocampus Explora: una aproximación multidisciplinar a la problemática ambiental. En IN-RED 2020: VI Congreso de Innovación Educativa y Docencia en Red. Editorial Universitat Politècnica de València. 1003-1014. https://doi.org/10.4995/INRED2020.2020.11996OCS1003101

Optimization of docosahexaenoic acid production by heterologous expression in Escherichia coli of pfa gene cluster from Moritella marina

Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid, whose usage has risen in the last few years due to its benefits on human health. The main goal of this project is the sustainable production of DHA with a better quality and cheaper production in microorganisms. This molecule is found naturally in many marine bacteria because it is needed to increase the fluidity of cell membrane as a cold-resistance mechanism adaptation. A fatty acid synthase coded by a 25-Kbp PKS-like gene cluster (pfaABCD) converts malonyl-CoA into DHA. Heterologous expression of Moritella marina pfaABCD gene cluster in Escherichia coli produced a 5% DHA of total fatty acids at 15ºC, but no DHA was found in cells grown at 25ºC. Gene expression analysis by RT-qPCR showed a decrease in pfaA, pfaC and pfaD when cultured at higher temperatures, which could partially indicate a cold-mediated gene expression regulation. pfaE increased expression by T7 promoter induction with IPTG does not increase the DHA production, meaning PPTase activity is not a limiting factor in the process. In addition, a diacylglycerol acyl transferase from Thermomonospora curvata (tDGAT) was co-expressed with pfa gene cluster in order to divert DHA from the membrane to triacylglycerols (TAGs) and avoid an increase of membrane fluidity at high temperatures. Results showed a correct formation of TAGs but no DHA was detected at 25ºC. Further studies are needed to determine DGAT ability to incorporate DHA into TAG.Máster en Biología Molecular y Biomedicin

Optimización de la producción heteróloga de ácido docosahexaenoico en E. coli

RESUMEN: Algunas bacterias marinas, como Moritella marina, son capaces de producir ácido docosahexaenoico (DHA) gracias a un complejo enzimático llamado complejo Pfa. Este complejo expresado en Escherichia coli es capaz de producir DHA. El objetivo de esta tesis es encontrar las condiciones genéticas y metabólicas para mejorar la producción de DHA en E. coli u otros organismos. Primeramente, se estudió el patrón de expresión de los promotores nativos, que fueron reemplazados por promotores fuertes e inducibles con el objetivo de incrementar la expresión de los genes, y finalmente de DHA. En segundo lugar, se estudió la alteración de los flujos metabólicos y se optimizó la disponibilidad de sustratos mediante la adición del inhibidor cerulenina, y la deleción del gen fabH. En tercer lugar, se implementaron distintos mecanismos de acumulación del producto final. Por último, se mejoró el crecimiento de E. coli a bajas temperaturas mediante el uso de chaperoninas psicrófilas con el objetivo de restaurar la actividad chaperona. Todas estas aproximaciones han incrementado la eficiencia final del proceso de síntesis de DHA y otros ácidos grasos poliinsaturados.ABSTRACT: Some marine bacteria, such as Moritella marina, are able to produce certain amounts of the nutraceutical docosahexaenoic acid (DHA) thanks to a specific enzymatic complex called Pfa synthase. Moreover, Escherichia coli heterologously expressing this pfa gene cluster from M. marina is also able to produce DHA. The aim of this study was to find genetic or metabolic conditions to improve DHA production in E. coli or any other microorganism. Firstly, we studied native promoter expression pattern in E.coli and replaced these promoters by inducible pBAD system in order to increase the production of the final product. Secondly, we altered the canonical carbon flux in E.coli to improve the availability of substrates for Pfa complex by two strategies: using the exogenous compound cerulenin, and deleting the main initiation enzyme of a competing pathway. Both strategies exploit a substrate competition mechanism between the native fatty acid synthase from E.coli and the heterologous Pfa complex from M.marina. Finally, we improved E.coli growth at low temperature by introducing two psychrophilic chaperonins, Cpn10 and Cpn60 from Oleispira antarctica, in order to improve protein folding. These approaches have increased the overall efficiency of this process, and could be used for biotechnological optimization in different organisms to synthesize DHA or other polyunsaturated fatty acids.El presente trabajo ha sido realizado entre el Departamento de Biología Molecular de la Universidad de Cantabria y el Departamento de Microbiología y Genómica del Instituto de Biomedicina y Biotecnología de Cantabria, bajo la dirección del Dr. Fernando de la Cruz Calahorra y el Dr. Gabriel Moncalián Montes, gracias a un contrato de la Universidad de Cantabria asociado a los proyectos “Ingeniería Genética de Escherichia coli para mejorar la producción de lípidos ricos en ácidos Grasos omega-3” (TF16-XX-003 - SODERCAN/FEDER) y “Producción de ácidos grasos omega-3 a partir de microorganismos” (2014) en colaboración con la empresa Biomar Microbial Technologies

Heterologous synthesis of docosahexaenoic acid in Escherichia coli

Resumen del trabajo presentado en el 41 Congreso de la Sociedad Española de Bioquímica y Biología Molecular SEBBM, celebrado en Santander (España) del 10 al 13 de septiembre de 2018.Some marine bacteria, such as Moritella marina, are able to produce certain amounts of the nutraceutical docosahexaenoic acid (DHA) thanks to a specific enzymatic complex called Pfa synthase. Moreover, E.coli heterologously expressing this pfa gene cluster from M. marina is also able to produce DHA [1]. The aim of this study was to find genetic or metabolic conditions to improve DHA production in E. coli or any other microorganism. Firstly, we studied the expression of Pfa promoters in E. coli and replaced these promoters by inducible pBAD promoters in order to increase the production of the final product. Secondly, we altered the canonical carbon flux in E. coli to improve the availability of substrates for Pfa complex by two strategies: using the exogenous compound cerulenin, and deleting a initiation enzyme of a competing pathway, FabH [2]. Both strategies exploit a substrate competition mechanism between the native fatty acid synthase from E. coli and the heterologous Pfa complex from M. marina. Finally, we improved E. coli growth at low temperature by introducing two psychrophilic chaperonins, Cpn10 and Cpn60 from Oleispira antarctica [3], in order to improve protein folding. These approaches have increased the overall efficiency of DHA synthesis in E. coli, and could be used for biotechnological optimization in different organisms to synthesize DHA or other polyunsaturated fatty acids

A gene deletion improves docosahexaenoic acid production in Escherichia coli expressing Pfa genes

Resumen del póster presentado al XL Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Barcelona del 23 al 26 de octubre de 2017.Some marine bacteria, such as Moritella marina, are able to produce certain amounts of the nutraceutical docosahexaenoic acid (DHA) thanks to a specifi c enzymatic complex called Pfa synthase. Moreover, Escherichia coli heterologously expressing this pfa gene cluster from M. marina is also able to produce DHA. The aim of this study was to find genetic or metabolic conditions to improve DHA production in E. coli or any other microorganism. First, we studied the eff ect of the antibiotic cerulenin on DHA production, which increased by 2-fold the DHA content of the cells. Then, we tested a series of single gene knock-out strains in order to alter the metabolic routes to optimize the biosynthesis of DHA. The best mutant strain carried the deletion of a specific gene, and showed a 3-fold increase compared to wild type strain. The combination of both cerulenin inhibition and this gene deletion rendered 12.75±1.21 mg DHA/g DCW, a 6.5-fold improvement compared to control strain. Furthermore, both strategies seem to have the same mechanism of action, in which the fatty acid synthesis in E. coli is affected in its fi rst catalytic step and allows the substrates to be used by the heterologous pathway to synthesize DHA. This data could be used for the biotechnological optimization in different organisms of DHA production and other synthetic pathways using the same competing substrate.Peer reviewe

In vitro regeneration of two Populus hybrid clones. The role of pectin domains in cell processes underlying shoot organogenesis induction

[EN] An efficient plant regeneration protocol has been established for two hybrid clones of Populus deltoides, MC (P. x euramericana) and UNAL (P. x interamericana). The culture of internode segments on MS medium with 0.5 μM α-Napthalene acetic acid (NAA) and 4 μM N6 -Benzyladenine for 7 weeks –2 in absence of activated charcoal and 5 in its presence—gave the highest frequency of shoot regeneration (100% for MC and 82% for UNAL). Good results also were achieved using leaves as explants, but only if they remained 5 weeks in the first medium. The 100% of regenerated shoots longer than 2 cm were rooted on ½ MS medium, independently of the addition of 0.1 μM NAA. Nevertheless, shoots developed better-formed roots in NAA-free medium, which had a positive effect in the acclimatization of plants. To know the cellular processes underlying in vitro shoot organogenesis, a histological study was made in UNAL internodes. Results revealed that in vitro culture caused swelling around the cut off zones in all explants, but only those undergoing organogenesis formed proliferation centers under subepidermal cells, which lead to bud primordia. Moreover, in vivo tissues and explants with different in vitro response showed different immunolabelling patterns when they were treated with fluorescent-monoclonal antibodies against several pectin-polysaccharides of the cell wall. Results allow us to point homogalactouronan (HG) with high degree of methyl-esterification and α-1,5-L- arabinan side chains of ramnogalactouronan-I as markers for cell division and growth, and HG with low degree of methyl-esterification as a marker for bud primordia initiation.S