Effects of Random Mutagenesis and In Vivo Selection on the Specificity and Stability of a Thermozyme

Factors that give enzymes stability, activity, and substrate recognition result from the combination of few weak molecular interactions, which can be difficult to study through rational protein engineering approaches. We used irrational random mutagenesis and in vivo selection to test if a β-glycosidase from the thermoacidophile Saccharolobus solfataricus (Ssβ-gly) could complement an Escherichia coli strain unable to grow on lactose. The triple mutant of Ssβ-gly (S26L, P171L, and A235V) was more active than the wild type at 85 °C, inactivated at this temperature almost 300-fold quicker, and showed a 2-fold higher kcat on galactosides. The three mutations, which were far from the active site, were analyzed to test their effect at the structural level. Improved activity on galactosides was induced by the mutations. The S26L and P171L mutations destabilized the enzyme through the removal of a hydrogen bond and increased flexibility of the peptide backbone, respectively. However, the flexibility added by S26L mutation improved the activity at T > 60 °C. This study shows that random mutagenesis and biological selection allowed the identification of residues that are critical in determining thermal activity, stability, and substrate recognitio

Transcript Regulation of the Recoded Archaeal α-L-Fucosidase In Vivo

Genetic decoding is flexible, due to programmed deviation of the ribosomes from standard translational rules, globally termed “recoding”. In Archaea, recoding has been unequivocally determined only for termination codon readthrough events that regulate the incorporation of the unusual amino acids selenocysteine and pyrrolysine, and for −1 programmed frameshifting that allow the expression of a fully functional α-l-fucosidase in the crenarchaeon Saccharolobus solfataricus, in which several functional interrupted genes have been identified. Increasing evidence suggests that the flexibility of the genetic code decoding could provide an evolutionary advantage in extreme conditions, therefore, the identification and study of interrupted genes in extremophilic Archaea could be important from an astrobiological point of view, providing new information on the origin and evolution of the genetic code and on the limits of life on Earth. In order to shed some light on the mechanism of programmed −1 frameshifting in Archaea, here we report, for the first time, on the analysis of the transcription of this recoded archaeal α-l-fucosidase and of its full-length mutant in different growth conditions in vivo. We found that only the wild type mRNA significantly increased in S. solfataricus after cold shock and in cells grown in minimal medium containing hydrolyzed xyloglucan as carbon source. Our results indicated that the increased level of fucA mRNA cannot be explained by transcript up-regulation alone. A different mechanism related to translation efficiency is discusse

The Italian National Project of Astrobiology-Life in Space-Origin, Presence, Persistence of Life in Space, from Molecules to Extremophiles

The \u2018\u2018Life in Space\u2019\u2019 project was funded in the wake of the Italian Space Agency\u2019s proposal for the development of a network of institutions and laboratories conceived to implement Italian participation in space astrobiology experiments

Identification and characterization of glycoside hydrolases for biotechnological applications

The importance of carbohydrates in several biological processes is directly mirrored in a wide number of biotechnological applications, based mainly on glycoside hydrolases (GHs), including conversion of agricultural byproducts in fermentable sugars for the bioethanol production, the use of these biocatalysts in the formulation of laundry detergents and in food industry (e.g. hydrolysis of lactose and preparation of HFCS). Moreover, GHs are used also in several biomedical approaches, as the production of universal blood , the enzyme replacement therapy for the treatment of lysosomal storage diseases and as alterative to the chemical synthesis of therapeutic biomolecules as heparin and galactooligosaccharides. The development of a new class of enzyme, the glycosynthases (GS), obtained by mutating glycoside hydrolases, represent reliable alternative for the chemo-enzymatic synthesis of oligosaccharides. Here, the key of this approach is to cancel the hydrolytic activity of the enzyme by site-directed mutagenesis, but maintaining intact the structure of the active site. Therefore, by using substrate donor and certain reaction conditions, the engineered enzymes are able to synthesize products in quantitative yield. This thesis is directed to the identification and characterization of glycosyl hydrolase for biotechnological applications and was divided in two different parts. The first part (Chapters I and II) is aimed to the application of glycosidases in the oligosaccharides synthesis. In particular, I adressed my work to the study and characterization of the catalytic mechanism of a beta-galactosidase from the moderate thermophile Alicyclobacillus acidocaldarius (Aaβgal) for the development of a new β-galactosynthase (Chapter I). Moreover I have characterized a new alpha-galactosynthase from the hyperthermophile Thermotoga maritima (TmGalA D327G) (Chapter II) to validate the approach based on the use of beta-glycosyl azide donors recently proposed. The second part of this thesis (Chapters III and IV) is dedicated to the characterization in detail of two new glycoside hydrolases: an alpha-glycosidase SSO1353 and an alpha-mannosidase (Ss-alpha-Man) both from the hyperthermophilic crenarchaeon Sulfolobus solfataricus. The characterization of SSO1353 was directed to the study of the catalytic mechanism and in particular to the identification of nucleophile and acid/base residues. Differently, the study of Ss-alpha-Man activity was directed to the analysis of its substrate specificity toward glycoconjugates and glycoproteins

Metagenomics of hyperthermophilic environments: Biodiversity and biotechnology

The field of thermophilic microbiology was born in the late 1970s with the pioneering work of Brock (Thermophiles biodiversity, ecology, and evolution. Springer, Boston, pp. 1–9, 2001) and dramatically expanded through the ’80s with the isolation of hyperthermophiles by Stetter (FEMS Microbiol Rev 18:149–158, 1996). The development of SSU rRNA phylogenetics revealed the complexity and diversity of prokaryotic phylotypes on biotopes widely differing in extreme conditions (e.g. spanning gradients of pH between 0 and 10 and temperatures from 60 °C to over 120 °C, respectively). Sites of volcanic activity all over the Earth’s surface and under the sea provide a variety of different environments for extremophilic microorganisms. Hot springs populated by hyperthermophiles (Topt > 65 °C), the majority of which belonging to the domain of Archaea, are very diverse and some of them show combinations of other extreme conditions, for example, acidic, alkaline, high pressure, and high concentrations of salts and heavy metals (Cowan et al. in Curr Opin Microbiol 25:97–102, 2015). Archaea inhabiting hot springs are considered to be the closest living descendants of the earliest living forms on Earth and their study provide insights into the origin and evolution of life (Woese et al. in Proc Natl Acad Sci USA 87:4576–4579, 1990; Olsen et al. in J Bacteriol 176:1–6, 1994). As with all studies of environmental microbiology, our understanding of the function of (hyper)thermophilic microbial consortia has lagged substantially behind. However, recent advances in ‘omics’ technologies, particularly within a system biology context, have made significant progresses into the prediction of in situ functionality (Cowan et al. in Curr Opin Microbiol 25:97–102, 2015). Most extremophilic microorganisms are recalcitrant to cultivation-based approaches (Amann et al. in Microbiol Rev 59:143–69, 1995; Lorenz et al. in Curr Opin Biotechnol 13:572–577, 2002); therefore, culture-independent metagenomic strategies are promising approaches to assess the phylogenetic composition and functional potential of microbial communities living in extreme environments (López-López et al. in Life 3:308–320, 2013). In addition, these approaches implement tremendously the access to enzymes from (hyper)thermophilic microorganisms that have important potential applications in several biotechnological processes. We report here on the state-of-the-art of the metagenomic surveys of different hot springs (T > 65 °C) (Table 5.1) and on the recent advance in the discovery of new hyperthermostable biocatalysts of biotechnological interest from metagenomic studies of these extreme environments

Thermophilic glycosynthases for oligosaccharides synthesis

Glycosynthases are engineered glycoside hydrolases that in suitable reaction conditions promote the synthesis of oligosaccharides with exquisite stereoselectivity and enhanced regioselectivity, if compared to traditional chemical methods. This approach was demonstrated to be successful in a number of cases including β-glycosynthases acting at the termini or within an oligosaccharide chain (exo- and endo-glycosynthases, respectively) and, more recently, α-glycosynthases. This led to the production of a vast repertoire of products that include poly- and oligosaccharides, glycoconjugates, and glycopeptides. These molecules can be used as ligands of glycoside hydrolases, for the characterization of therapeutic enzymes, and as leads of drugs for the pharmaceutical industry. In this panorama, hyperthermophilic organisms, which thrive at temperatures as high as 80°C, which usually impede the growth of other living forms, have been used in the development of interesting novel glycosynthases. In fact, the extreme stability of these catalysts to extremes of pH and high concentrations of organics has allowed the exploration of novel reaction conditions, revealing new avenues for enzyme-catalyzed oligosaccharide synthesis

Metagenomics of microbial and viral life in terrestrial geothermal environments

Geothermally heated regions of Earth, such as terrestrial volcanic areas (fumaroles, hot springs, and geysers) and deep-sea hydrothermal vents, represent a variety of different environments populated by extremophilic archaeal and bacterial microorganisms. Since most of these microbes thriving in such harsh biotopes, they are often recalcitrant to cultivation; therefore, ecological, physiological and phylogenetic studies of these microbial populations have been hampered for a long time. More recently, culture-independent methodologies coupled with the fast development of next generation sequencing technologies as well as with the continuous advances in computational biology, have allowed the production of large amounts of metagenomic data. Specifically, these approaches have assessed the phylogenetic composition and functional potential of microbial consortia thriving within these habitats, shedding light on how extreme physico-chemical conditions and biological interactions have shaped such microbial communities. Metagenomics allowed to better understand that the exposure to an extreme range of selective pressures in such severe environments, accounts for genomic flexibility and metabolic versatility of microbial and viral communities, and makes extreme- and hyper-thermophiles suitable for bioprospecting purposes, representing an interesting source for novel thermostable proteins that can be potentially used in several industrial processes

Archaea as a Model System for Molecular Biology and Biotechnology

Archaea represents the third domain of life, displaying a closer relationship with eukaryotes than bacteria. These microorganisms are valuable model systems for molecular biology and biotechnology. In fact, nowadays, methanogens, halophiles, thermophilic euryarchaeota, and crenarchaeota are the four groups of archaea for which genetic systems have been well established, making them suitable as model systems and allowing for the increasing study of archaeal genes’ functions. Furthermore, thermophiles are used to explore several aspects of archaeal biology, such as stress responses, DNA replication and repair, transcription, translation and its regulation mechanisms, CRISPR systems, and carbon and energy metabolism. Extremophilic archaea also represent a valuable source of new biomolecules for biological and biotechnological applications, and there is growing interest in the development of engineered strains. In this review, we report on some of the most important aspects of the use of archaea as a model system for genetic evolution, the development of genetic tools, and their application for the elucidation of the basal molecular mechanisms in this domain of life. Furthermore, an overview on the discovery of new enzymes of biotechnological interest from archaea thriving in extreme environments is reported