Protocolos de microalgas de la Red RENUWAL

Publicação digitalRESUMEN: Las microalgas pueden ser utilizadas para la recuperación de nutrientes contenidos en diversos efluentes, contribuyendo de esta forma a la mejora de la sostenibilidad de multitud de procesos. Sin embargo, para poder conseguir procesos eficientes es necesario evaluar con detalle cada aplicación en función de los efluentes que se van a procesar. Dependiendo del proceso, la biomasa algal obtenida puede tener interés para su aplicación en distintos sectores y productos finales con diferente valor añadido, i.e., farmacéutico, cosmético, agroalimentario-ganadero-acuícola, ambiental, químico o incluso energético. En el año 2019, varios grupos de investigación pertenecientes a diferentes países iberoamericanos junto con 6 empresas que trabajan con microalgas se unieron para solicitar al programa de Redes de la CYTED (Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo; https://www.cyted.org/es/cyted) la creación de una red temática para el tratamiento de efluentes con microalgas. Las Redes Temáticas que subvenciona este organismo, son asociaciones de grupos de investigación y desarrollo (I+D) de entidades públicas o privadas y empresas de los países miembros del Programa CYTED, cuyas actividades científicas o tecnológicas están relacionadas dentro de un ámbito común de interés y enmarcadas en una de las Áreas del Programa. En nuestro caso, la red se solicitó por el área industrial. La red RENUWAL 320RT0005 fue concedida en esta convocatoria del 2019 para iniciarse en el año 2020 durante un periodo de 4 años que, con motivo de la Pandemia, se ha extendido hasta el 2024 (https://www.cyted.org/es/renuwal).N/

Functional differentiation of 3-ketosteroid Δ1-dehydrogenase isozymes in Rhodococcus ruber strain Chol-4

Background: 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

New insights into the genome of Rhodococcus ruber strain Chol-4

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

Metabolic engineering of Rhodococcus ruber Chol-4: A cell factory for testosterone production

Rhodococcus 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

Xerotolerance: a new property in Exiguobacterium Genus

The 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

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

Analysis of intermediates of steroid transformations in resting cells by thin-layer chromatography (TLC)

Thin-layer chromatography (TLC) is a useful and convenient method for the analysis of steroids due to: its simple sample preparation, low time-consuming process, high sensitivity, low equipment investment and capacity to work on many samples simultaneously. Here we describe a TLC easy protocol very useful to analyze steroid molecules derived from a biotransformation carried out in wild-type and mutant resting cells of Rhodococcus ruber strain Chol-4. Following this protocol, we were able to detect the presence or the absence of some well-known intermediates of cholesterol catabolism in Rhodococcus, namely AD, ADD, and 9OHAD