Development of biotechnological tools in cyanobacteria

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, leída el 20-10-2022Las cianobacterias se encuentran entre los organismos vivos más antiguos del planeta y se pueden hallar en todos los ecosistemas de la Tierra, desde océanos, ríos y lagos de agua dulce hasta fuentes termales y desiertos. Aparte de su papel ecológico en los ciclos globales del carbono y el nitrógeno, las cianobacterias se están convirtiendo en un chasis cada vez más atractivo en la producción biotecnológica de compuestos de alto valor añadido, pues los requerimientos para su crecimiento, luz y CO2, son mínimos, gratuitos y abundantes. Si bien diversas cianobacterias, sobre todo Synechocystis spp. y Synechococcus spp., han sido ya caracterizadas para su aplicación industrial, muchas de ellas crecen en condiciones muy concretas, principalmente en condiciones controladas de laboratorio. Por este motivo, continuamente se buscan nuevas cianobacterias que puedan utilizarse como chasis o bien aporten genes de interés en biotecnología. Por lo tanto, es importante estudiar la diversidad de cianobacterias para poder desarrollar su potencial para beneficio humano. Por otro lado, aunque cada vez se describen más, las herramientas moleculares de las que se dispone para modificar el metabolismo de las cianobacterias, aún son limitadas en comparación con las desarrolladas para otros microorganismos de uso industrial y biotecnológico...Cyanobacteria are among the oldest living organisms on the planet, and they can be found in every ecosystem on Earth, from freshwater oceans, rivers and lakes to hot springs and deserts. Apart from their ecological role in the global carbon and nitrogen cycles, cyanobacteria are becoming an increasingly attractive chassis in the biotechnological production of high value-added compounds since the requirements for their growth, light, and CO2, are minimal, costless, and abundant.Although various cyanobacteria, notably Synechocystis spp. and Synechococcus spp., have already been characterized for their industrial application, many of them grow in very specific conditions, mainly under controlled laboratory conditions. For this reason, new cyanobacteria that can be used as chassis or provide genes of interest in biotechnology are continually being sought. Therefore, it is important to study the diversity of cyanobacteria in order to develop their potential for human benefit. On the other hand, as molecular tools available to modify the metabolism of cyanobacteria are still limited compared to those described for other industrially or biotechnologically-used microorganisms, there is a need for developing new and more versatile biotechnological tools for them...Fac. de Ciencias BiológicasTRUEunpu

Suppression of NYVAC infection in hela cells requires RNase L but is independent of protein kinase R activity

Protein kinase R (PKR) and RNase L are host cell components that function to contain viral spread after infections. In this study, we analyzed the role of both proteins in the abortive infection of human HeLa cells with the poxvirus strain NYVAC, for which an inhibition of viral A27L and B5R gene expression is described. Specifically, the translation of these viral genes is independent of PKR activation, but their expression is dependent on the RNase L activityThis work was supported by grants from the Spanish Ministry of Health (FIS2011-00127) and Bayer Group Grants4Grants (2013-08-0982 to S.G., SAF2008-02036 to M.E., and AGL2010-15495 to D.R.

ISGylation controls exosome secretion by promoting lysosomal degradation of MVB proteins

Exosomes are vesicles secreted to the extracellular environment through fusion with the plasma membrane of specific endosomes called multivesicular bodies (MVB) and mediate cell-to-cell communication in many biological processes. Posttranslational modifications are involved in the sorting of specific proteins into exosomes. Here we identify ISGylation as a ubiquitin-like modification that controls exosome release. ISGylation induction decreases MVB numbers and impairs exosome secretion. Using ISG15-knockout mice and mice expressing the enzymatically inactive form of the de-ISGylase USP18, we demonstrate in vitro and in vivo that ISG15 conjugation regulates exosome secretion. ISG15 conjugation triggers MVB co-localization with lysosomes and promotes the aggregation and degradation of MVB proteins. Accordingly, inhibition of lysosomal function or autophagy restores exosome secretion. Specifically, ISGylation of the MVB protein TSG101 induces its aggregation and degradation, being sufficient to impair exosome secretion. These results identify ISGylation as a novel ubiquitin-like modifier in the control of exosome production.We thank Dr K. Knobeloch, Dr A. Garcia-Sastre and Dr M.A. Alonso for providing reagents, and Dr S. Bartlett for assistance with English editing. C.E. is thankful to electron microscopy facility (campus Casanova), CCiT-University of Barcelona. This study was supported by grants SAF2014-55579-R from the Spanish Ministry of Economy and Competitiveness, INDISNET-S2011/BMD-2332 from the Comunidad de Madrid, Cardiovascular Network RD12-0042-0056 and PIE13/00041 from Instituto de Salud Carlos III (Fondo de Investigacion Sanitaria del Instituto de Salud Carlos III and co-funding by Fondo Europeo de Desarrollo Regional FEDER), ERC-2011-AdG 294340-GENTRIS and COST-Action BM1202 to F.S.-M.; grant SAF2014-54623-R, FIS grant PI11/00127 (Fondo de Investigacion Sanitaria del Instituto de Salud Carlos III and Ministry of Health of Spain, State secretary of R+D and FEDER/FSE) and Bayer Group Grants4Grants (ID 2013-08-0982) to S.G.; and grant BFU2015-66785-P from the Spanish Ministry of Economy and Competitiveness to C.E.; Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). C.V.-B. was supported by FPU programme (Spanish Ministry of Education). M. M. is supported by MS14/00219 from Instituto de Salud Carlos III.S

Volume 279, February 2024, 127572

16 p.-4 fig.-4 tab.The filamentous cyanobacterium Limnospira platensis, formerly known as Arthrospira platensis or spirulina, is one of the most commercially important species of microalgae. Due to its high nutritional value, pharmacological and industrial applications it is extensively cultivated on a large commercial scale. Despite its widespread use, its precise manipulation is still under development due to the lack of effective genetic protocols. Genetic transformation of Limnospira has been attempted but the methods reported have not been generally reproducible in other laboratories. Knowledge of the transformation defense mechanisms is essential for understanding its physiology and for broadening their applications. With the aim to understand more about the genetic defenses of L. platensis, in this work we have identified the restriction-modification and CRISPR-Cas systems and we have cloned and characterized thirteen methylases. In parallel, we have also characterized the methylome and orphan methyltransferases using genome-wide analysis of DNA methylation patterns and RNA-seq. The identification and characterization of these enzymes will be a valuable resource to know how this strain avoids being genetically manipulated and for further genomics studies.This work was supported by projects S2013/ABI-2783 (INSPIRA1-CM), S2018/BAA-4532 (ALGATEC-CM) from “Comunidad de Madrid /ESF-ERDF”; RTI2018–094399-A-I00 (SETH) from the Spanish Ministry of Economy and Competitivity; RobExplode PID2019-108458RB-I00 (AEI/10.13039/501100011033) and by Sycosys TED2021–130689B-C33 from Spanish Ministry of Science and Innovation (MICINN) grants.Peer reviewe

Further Studies on the 3-Ketosteroid 9α-Hydroxylase of Rhodococcus ruber Chol-4, a Rieske Oxygenase of the Steroid Degradation Pathway

The 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

SEVA-Cpf1, a CRISPR-Cas12a vector for genome editing in cyanobacteria

Background 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.Introducción: Las cianobacterias son autótrofos fotosintéticos con alta plasticidad metabólica y capacidad para crecer utilizando CO2 y luz solar, lo que hace que tengan un tremendo potencial en aplicaciones industriales. La tecnología CRISPR se ha utilizado en la edición genética de cianobacterias en procesos de ingeniería metabólica. A pesar de los avances significativos en la edición del genoma en varias cepas de cianobacterias, la falta de herramientas genéticas adecuadas sigue siendo un factor limitante en comparación con otras especies de laboratorio modelo. Entre las limitaciones, una de las principales es contar con un banco plásmidos versátiles que puedan facilitar el uso de la tecnología CRISPR. Resultados: En este estudio se desarrollaron varios vectores CRISPR-Cpf1 basados en los plásmidos SEVA (Figura 1). La colección SEVA son vectores con elementos modulares que se pueden intercambiar y modificar llamados cargos (p. ej. Origen de replicación, resistencia, etc.). Como primer paso se probaron plásmidos con un origen de replicación RSF1010, de amplio espectro. Estos plásmidos fueron capaces de transformar no solo cianobacterias modelo si no también una cianobacteria extremófila, Chroococcidiopsis sp. Una vez verificada la capacidad de transformación, se construyeron vectores SEVA con los elementos del sistema CRISPR-Cpf1 y se demostró que la capacidad de transformación se mantenía en todas las cepas de cianobacterias probadas, también se pudo transformar naturalmente Synechosystis sp. Finalmente, como prueba de concepto se procedió a la deleción sin marca del gen nblA en Synechocystis sp. PCC 6803, demostrando que los plásmidos SEVA basados en la tecnología CRISPR-Cpf1 construidos en este trabajo se pueden usar para la modificación genética de cianobacterias. Conclusiones: En este estudio se han desarrollados vectores de edición genética mediante el sistema CRISPR-Cpf1 basados en los plásmidos SEVA. De acuerdo a los resultados obtenidos estos vectores son de fácil manipulación en el laboratorio, capaces de transformar por conjugación o naturalmente diferentes especies de cianobacterias, además de presentar una tasa alta de eliminación del plásmido para futuras ediciones. Todo esto hace que los plásmidos obtenidos sean una herramienta de gran utilidad con una amplia gama de aplicaciones que pueden ir desde la ingeniería metabólica y estudios de ciencia básica.Comunidad de MadridUniversidad Complutense de MadridFondo para el Desarrollo Regional Europeo (FEDER)Fondo Social Europeo (FSE)Depto. de Bioquímica y Biología MolecularFac. de Ciencias BiológicasTRUEpu

Primera caracterización de aislamientos cultivables de Chroococcidiopsis extremófila a partir de un panel solar

Introduction: 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.MINECOCOMUNIDAD DE MADRIDEuropean Social Fund and the European Regional Development FundDepto. de Bioquímica y Biología MolecularFac. de Ciencias QuímicasTRUEpu

Desarrollo de un consorcio sintético Synechococcus elongatus-Azohydromonas lata para la producción de bioplásticos a partir de luz y CO2

1 p.Actualmente, las cianobacterias están atrayendo gran atención en aplicaciones biotecnológicas como la producción de bioplásticos debido a que los requisitos para su crecimiento son mínimos. No obstante, su uso implica la optimización genética del metabolismo para lograr la sobreproducción del compuesto de interés. Además, un enfoque de monocultivo generalmente requiere una extensa investigación y desarrollo para optimizar cada cepa. Por todo ello, los estudios biotecnológicos más recientes apuntan al uso de consorcios microbianos sintéticos como una estrategia más eficaz para resolver biotransformaciones complejas.En este trabajo, se ha desarrollado un consorcio impulsado por la luz y CO2 formado por la cianobacteria Synechococcus elongatus y la bacteria heterotófica Azohydromonas lata para producir bioplásticos.En nuestro consorcio sintético, la cianobacteria Synechococcus elongatus SBG363 se utilizó para producir sacarosa a partir de CO2 y luz solar. La sacarosa se secreta al medio de cultivo y permite el crecimiento de A. lata, que acumula PHA. Previo al co-cultivo, optimizamos las condiciones de crecimiento y producción de bioplásticos por A. lata en el medio de cianobacterias BG11, utilizando diferentes concentraciones de sacarosa.Descubrimos que un medio con mayor concentración de fosfato era suficiente para un crecimiento robusto y una buena producción de PHA. Nuestros resultados indican una buena compatibilidad de los miembros del consorcio en el mismo medio de cultivo, mostrando que el consorcio sintético S. elongatus - A. lata es una herramienta prometedora para la bioproducción de compuestos de interés a partir de CO2.ALGATEC-CM (P2018/BAA-4532)Peer reviewe

ISG15 governs mitochondrial function in macrophages following vaccinia virus infection.

The interferon (IFN)-stimulated gene 15 (ISG15) encodes one of the most abundant proteins induced by interferon, and its expression is associated with antiviral immunity. To identify protein components implicated in IFN and ISG15 signaling, we compared the proteomes of ISG15-/- and ISG15+/+ bone marrow derived macrophages (BMDM) after vaccinia virus (VACV) infection. The results of this analysis revealed that mitochondrial dysfunction and oxidative phosphorylation (OXPHOS) were pathways altered in ISG15-/- BMDM treated with IFN. Mitochondrial respiration, Adenosine triphosphate (ATP) and reactive oxygen species (ROS) production was higher in ISG15+/+ BMDM than in ISG15-/- BMDM following IFN treatment, indicating the involvement of ISG15-dependent mechanisms. An additional consequence of ISG15 depletion was a significant change in macrophage polarization. Although infected ISG15-/- macrophages showed a robust proinflammatory cytokine expression pattern typical of an M1 phenotype, a clear blockade of nitric oxide (NO) production and arginase-1 activation was detected. Accordingly, following IFN treatment, NO release was higher in ISG15+/+ macrophages than in ISG15-/- macrophages concomitant with a decrease in viral titer. Thus, ISG15-/- macrophages were permissive for VACV replication following IFN treatment. In conclusion, our results demonstrate that ISG15 governs the dynamic functionality of mitochondria, specifically, OXPHOS and mitophagy, broadening its physiological role as an antiviral agent