Efecto de la contaminación crónica, factores geoquímicos y bioestimulación en el catabolismo de hidrocarburos en ambientes marinos contaminados

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 19 de febrero de 201

Estimating the success of enzyme bioprospecting through metagenomics: current status and future trends

Recent reports have suggested that the establishment of industrially relevant enzyme collections from environmental genomes has become a routine procedure. Across the studies assessed, a mean number of approximately 44 active clones were obtained in an average size of approximately 53 000 clones tested using naie screening protocols. This number could be significantly increased in shorter times when novel metagenome enzyme sequences obtained by direct sequencing are selected and subjected to high-throughput expression for subsequent production and characterization. The pre-screening of clone libraries by naive screens followed by the pyrosequencing of the inserts allowed for a 106-fold increase in the success rate of identifying genes encoding enzymes of interest. However, a much longer time, usually on the order of years, is needed from the time of enzyme identification to the establishment of an industrial process. If the hit frequency for the identification of enzymes performing at high turnover rates under real application conditions could be increased while still covering a high natural diversity, the very expensive and time-consuming enzyme optimization phase would likely be significantly shortened. At this point, it is important to review the current knowledge about the success of fine-tuned naie-and sequence-based screening protocols for enzyme selection and to describe the environments worldwide that have already been subjected to enzyme screen programmes through metagenomic tools. Here, we provide such estimations and suggest the current challenges and future actions needed before environmental enzymes can be successfully introduced into the market

Biodiversity for biocatalysis: A review of the α/β-hydrolase fold superfamily of esterases-lipases discovered in metagenomes

Review Article.Natural biodiversity undoubtedly inspires biocatalysis research and innovation. Biotransformations of interest also inspire the search for appropriate biocatalysts in nature. Indeed, natural genetic resources have been found to support the hydrolysis and synthesis of not only common but also unusual synthetic scaffolds. The emerging tool of metagenomics has the advantage of allowing straightforward identification of activity directly applicable as biocatalysis. However, new enzymes must not only have outstanding properties in terms of performance but also other properties superior to those of well-established commercial preparations in order to successfully replace the latter. Esterases (EST) and lipases (LIP) from the α/β-hydrolase fold superfamily are among the enzymes primarily used in biocatalysis. Accordingly, they have been extensively examined with metagenomics. Here we provided an updated (October 2015) overview of sequence and functional data sets of 288 EST–LIP enzymes with validated functions that have been isolated in metagenomes and (mostly partially) characterized. Through sequence, biochemical, and reactivity analyses, we attempted to understand the phenomenon of variability and versatility within this group of enzymes and to implement this knowledge to identify sequences encoding EST–LIP which may be useful for biocatalysis. We found that the diversity of described EST–LIP polypeptides was not dominated by a particular type of protein or highly similar clusters of proteins but rather by diverse nonredundant sequences. Purified EST–LIP exhibited a wide temperature activity range of 10–85 °C, although a preferred bias for a mesophilic temperature range (35–40 °C) was observed. At least 60% of the total characterized metagenomics-derived EST–LIP showed outstanding properties in terms of stability (solvent tolerance) and reactivity (selectivity and substrate profile), which are the features of interest in biocatalysis. We hope that, in the future, the search for and utilization of sequences similar to those already encoded and characterized EST–LIP enzymes from metagenomes may be of interest for promoting unresolved biotransformations in the chemical industry. Some examples are discussed in this review.The authors gratefully acknowledge the financial support provided by the European Community project MAMBA (FP7-KBBE-2008-226977), MAGIC-PAH (FP7-KBBE-2009-245226), ULIXES (FP7-KBBE-2010-266473), MicroB3 (FP7-OCEAN.2011-2-287589), KILL-SPILL (FP7-KBBE-2012-312139) and Royal Society UK-Russia Exchange Grant (IE130218). We thank EU Horizon 2020 Program for the support of the Project INMARE H2020-BG-2014-2634486. This work was further funded by grants BIO2011-25012,PCIN-2014-107 and BIO2014-54494-R from the Spanish Ministry of Economy and Competitiveness. The present investigation was funded by the Spanish Ministry of Economy and Competitiveness, the UK Biotechnology and Biological Sciences Research Council (BBSRC) and the German Federal Ministry of Education and Research (BMBF) within the ERA NET-IB2 program, grant number ERA-IB-14-030. The authors gratefully acknowledge the financial support provided by the European Regional Development Fund (ERDF).Peer reviewe

Proteome cold-shock response in the extremely acidophilic archaeon, Cuniculiplasma divulgatum

© 2020 by the authors.The archaeon Cuniculiplasma divulgatum is ubiquitous in acidic environments with low-to-moderate temperatures. However, molecular mechanisms underlying its ability to thrive at lower temperatures remain unexplored. Using mass spectrometry (MS)-based proteomics, we analysed the effect of short-term (3 h) exposure to cold. The C. divulgatum genome encodes 2016 protein-coding genes, from which 819 proteins were identified in the cells grown under optimal conditions. In line with the peptidolytic lifestyle of C. divulgatum, its intracellular proteome revealed the abundance of proteases, ABC transporters and cytochrome C oxidase. From 747 quantifiable polypeptides, the levels of 582 proteins showed no change after the cold shock, whereas 104 proteins were upregulated suggesting that they might be contributing to cold adaptation. The highest increase in expression appeared in low-abundance (0.001–0.005 fmol%) proteins for polypeptides’ hydrolysis (metal-dependent hydrolase), oxidation of amino acids (FAD-dependent oxidoreductase), pyrimidine biosynthesis (aspartate carbamoyltransferase regulatory chain proteins), citrate cycle (2-oxoacid ferredoxin oxidoreductase) and ATP production (V type ATP synthase). Importantly, the cold shock induced a substantial increase (6% and 9%) in expression of the most-abundant proteins, thermosome beta subunit and glutamate dehydrogenase. This study has outlined potential mechanisms of environmental fitness of Cuniculiplasma spp. allowing them to colonise acidic settings at low/moderate temperatures.We acknowledge the support of the Centre for Environmental Biotechnology Project (CEB) part-funded by the European Regional Development Fund (ERDF) through the Welsh Government. R.B. and B.P. acknowledge also the support of the Supercomputing Wales project, which is part-funded by the European Regional Development Fund (ERDF) via the Welsh Government.Peer reviewe

Microbial Diversity of a Disused Copper Mine Site (Parys Mountain, UK), Dominated by Intensive Eukaryotic Filamentous Growth

The Parys Mountain copper mine (Wales, UK) contains a wide range of discrete environmental microniches with various physicochemical conditions that shape microbial community composition. Our aim was to assess the microbial community in the sediments and overlying water column in an acidic mine drainage (AMD) site containing abundant filamentous biogenic growth via application of a combination of chemical analysis and taxonomic profiling using 16S rRNA gene amplicon sequencing. Our results were then compared to previously studied sites at Parys Mt. Overall, the sediment microbiome showed a dominance of bacteria over archaea, particularly those belonging to Proteobacteria (genera Acidiphilium and Acidisphaera), Acidobacteriota (subgroup 1), Chloroflexota (AD3 cluster), Nitrospirota (Leptospirillum) and the uncultured Planctomycetota/CPIa-3 termite group. Archaea were only present in the sediment in small quantities, being represented by the Terrestrial Miscellaneous Euryarchaeota Group (TMEG), Thermoplasmatales and Ca. Micrarchaeota (Ca. Micracaldota). Bacteria, mostly of the genera Acidiphilium and Leptospirillum, also dominated within the filamentous streamers while archaea were largely absent. This study found pH and dissolved solutes to be the most important parameters correlating with relative proportions of bacteria to archaea in an AMD environment and revealed the abundance patterns of native acidophilic prokaryotes inhabiting Parys Mt sites and their niche specificities

Structure and evolutionary trace-assisted screening of a residue swapping the substrate ambiguity and chiral specificity in an esterase

11 pags., 6figs., 3 pags.Our understanding of enzymes with high substrate ambiguity remains limited because their large active sites allow substrate docking freedom to an extent that seems incompatible with stereospecificity. One possibility is that some of these enzymes evolved a set of evolutionarily fitted sequence positions that stringently allow switching substrate ambiguity and chiral specificity. To explore this hypothesis, we targeted for mutation a serine ester hydrolase (EH) that exhibits an impressive 71-substrate repertoire but is not stereospecific (e.e. 50%). We used structural actions and the computational evolutionary trace method to explore specificity-swapping sequence positions and hypothesized that position I244 was critical. Driven by evolutionary action analysis, this position was substituted to leucine, which together with isoleucine appears to be the amino acid most commonly present in the closest homologous sequences (max. identity, ca. 67.1%), and to phenylalanine, which appears in distant homologues. While the I244L mutation did not have any functional consequences, the I244F mutation allowed the esterase to maintain a remarkable 53-substrate range while gaining stereospecificity properties (e.e. 99.99%). These data support the possibility that some enzymes evolve sequence positions that control the substrate scope and stereospecificity. Such residues, which can be evolutionarily screened, may serve as starting points for further designing substrate-ambiguous, yet chiral-specific, enzymes that are greatly appreciated in biotechnology and synthetic chemistry.MF acknowledges the grant ‘INMARE’ from the EuropeanUnion’s Horizon 2020 (grant agreement no. 634486), the grantsPCIN-2017-078 (within the Marine Biotechnology ERA-NET) and BIO2017-85522-R from the Ministerio de Economía, Industria y Competitividad, Agencia Estatal de Investigación (AEI), Fondo Eur-opeo de Desarrollo Regional (FEDER) and the European Union (EU),and the grant 2020AEP061 from the Agencia Estatal CSIC. J.S-A.acknowledges grant PID2019-105838RB-C33 from the Ministeriode Ciencia e Innovación, Agencia Estatal de Investigación (AEI),Fondo Europeo de Desarrollo Regional (FEDER) and the EuropeanUnion (EU). P.N.G. acknowledges the support of the Era-Net IB Pro-ject MetaCat funded through UK Biotechnology and BiologicalSciences Research Council (BBSRC), grant No. BB/M029085/1, andthe Centre for Environmental Biotechnology Project, co-fundedby European Regional Development Fund (ERDF) via the WelshGovernment (WEFO); R.B. acknowledges the Supercomputing Wales project, co-funded by ERDF via WEFO. OL and PK were sup-ported by the National Institutes of Health (NIH) grants 5R01AG061105, 5R01GM066099, and 5R01GM079656. C. Coscolínthanks the Ministerio de Economía y Competitividad and FEDER fora PhD fellowship (Grant BES-2015-073829). Staff of the Synchrotron Radiation Source at Alba (Barcelona, Spain) for assistance at the BL13-XALOC beamlin