Extremophiles are microorganisms that populate habitats considered inhospitable from an anthropocentric point of view and are able to tolerate harsh conditions such as high temperatures, extreme pHs, high concentrations of salts, toxic organic substances, and/or heavy metals. These microorganisms have been broadly studied in the last 30 years and represent precious sources of biomolecules and bioprocesses for many biotechnological applications; in this context, scientific efforts have been focused on the employment of extremophilic microbes and their metabolic pathways to develop biomonitoring and bioremediation strategies to face environmental pollution, as well as to improve biorefineries for the conversion of biomasses into various chemical compounds. This review gives an overview on the peculiar metabolic features of certain extremophilic microorganisms, with a main focus on thermophiles, which make them attractive for biotechnological applications in the field of environmental remediation; moreover, it sheds light on updated genetic systems (also those based on the CRISPR-Cas tool), which expand the potentialities of these microorganisms to be genetically manipulated for various biotechnological purposes.

We here describe the discovery of an unknown protein by using a proteomic approach with a functionally related protein as bait. Remarkably, we successfully obtained a novel type of enzyme through the interaction with a transcription regulator controlling the expression of this enzyme.

In the bio-based era, cellulolytic and hemicellulolytic enzymes are biocatalysts used in many industrial processes, playing a key role in the conversion of recalcitrant lignocellulosic waste biomasses. In this context, many thermophilic microorganisms are considered as convenient sources of carbohydrate-active enzymes (CAZymes). In this work, a functional genomic annotation of Alicyclobacillus mali FL18, a recently discovered thermo-acidophilic microorganism, showed a wide reservoir of putative CAZymes. Among them, a novel enzyme belonging to the family 9 of glycosyl hydrolases (GHs), named AmCel9, was identified; in-depth in silico analyses highlighted that AmCel9 shares general features with other GH9 members. The synthetic gene was expressed in Escherichia coli and the recombinant protein was purified and characterized. The monomeric enzyme has an optimal catalytic activity at pH 6.0 and has comparable activity at temperatures ranging from 40 °C to 70 °C. It also has a broad substrate specificity, a typical behavior of multifunctional cellulases; the best activity is displayed on β-1,4 linked glucans. Very interestingly, AmCel9 also hydrolyses filter paper and microcrystalline cellulose. This work gives new insights into the properties of a new thermophilic multifunctional GH9 enzyme, that looks a promising biocatalyst for the deconstruction of lignocellulose.

Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to man. In a previous study, we discovered an arsenic-responsive transcriptional regulator in the thermophilic bacterium Thermus thermophilus HB27 (TtSmtB). Here, we characterize the arsenic resistance system of T. thermophilus in more detail. We employed TtSmtB-based pull-down assays with protein extracts from cultures treated with arsenate and arsenite to obtain an S-adenosylmethionine (SAM)-dependent arsenite methyltransferase (TtArsM). In vivo and in vitro analyses were performed to shed light on this new component of the arsenic resistance network and its peculiar catalytic mechanism. Heterologous expression of TtarsM in Escherichia coli resulted in arsenite detoxification at mesophilic temperatures. Although TtArsM does not contain a canonical arsenite binding site, the purified protein does catalyse SAM-dependent arsenite methylation. In addition, in vitro analyses confirmed the unique interaction between TtArsM and TtSmtB. Next, a highly efficient ThermoCas9-based genome-editing tool was developed to delete the TtArsM-encoding gene on the T. thermophilus genome, and to confirm its involvement in the arsenite detoxification system. Finally, the TtarsX efflux pump gene in the T. thermophilus ΔTtarsM genome was substituted by a gene, encoding a stabilised yellow fluorescent protein (sYFP), to create a sensitive genome-based bioreporter system for the detection of arsenic ions.