Person:
Guevara Acosta, Flor Govinda

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First Name
Flor Govinda
Last Name
Guevara Acosta
URI
https://hdl.handle.net/20.500.14352/74397
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Ciencias Biológicas
Department
Bioquímica y Biología Molecular
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    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 María
    Background: 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.
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    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 María
    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.