Heterologous expression, characterization and applications of metagenome- and genome-sourced enzymes.

Microorganisms encompass the largest resource of metabolic and genetic diversity encountered on Earth. This unparalleled biodiversity risks remaining inaccessible since the major part of the microbiota is unculturable by traditional methods. Metagenomics (i.e. the culture-independent analysis of the genetic complement of an entire habitat) represents a promising and innovative tool for the exploitation of the biotechnological potential encrypted in microbial communities. The present PhD dissertation has been developed in the frame of the FP7 European project MetaExplore, whose aim was the identification through metagenomics of novel biocatalysts involved in the biodegradation of recalcitrant natural molecules, such as chitinases, enzymes with many industrial and environmental applications. The first part of the dissertation focuses on the heterologous production in Escherichia coli and biochemical, functional and structural characterisation of two metagenome-sourced chitinases, Chi18H8 and 53D1. The enzymes are endowed with innovative and valuable features, making them interesting candidates for the biocontrol of plant pathogenic fungi and for the sustainable and environment-friendly treatment and valorisation of seafood wastes, respectively. The second section describes the employment of the Gram-positive bacteria Streptomyces spp. as alternative expression platforms for the production of two proteins, VanYn and Chi18H8. Finally, in the third section the possibility to employ chitinolytic enzymes as alternatives to traditional chemical-based insect pesticides is investigated

Heterologous expression, characterization and applications of metagenome- and genome-sourced enzymes.

Microorganisms encompass the largest resource of metabolic and genetic diversity encountered on Earth. This unparalleled biodiversity risks remaining inaccessible since the major part of the microbiota is unculturable by traditional methods. Metagenomics (i.e. the culture-independent analysis of the genetic complement of an entire habitat) represents a promising and innovative tool for the exploitation of the biotechnological potential encrypted in microbial communities. The present PhD dissertation has been developed in the frame of the FP7 European project MetaExplore, whose aim was the identification through metagenomics of novel biocatalysts involved in the biodegradation of recalcitrant natural molecules, such as chitinases, enzymes with many industrial and environmental applications. The first part of the dissertation focuses on the heterologous production in Escherichia coli and biochemical, functional and structural characterisation of two metagenome-sourced chitinases, Chi18H8 and 53D1. The enzymes are endowed with innovative and valuable features, making them interesting candidates for the biocontrol of plant pathogenic fungi and for the sustainable and environment-friendly treatment and valorisation of seafood wastes, respectively. The second section describes the employment of the Gram-positive bacteria Streptomyces spp. as alternative expression platforms for the production of two proteins, VanYn and Chi18H8. Finally, in the third section the possibility to employ chitinolytic enzymes as alternatives to traditional chemical-based insect pesticides is investigated

Heterologous expression, characterization and applications of metagenome- and genome-sourced enzymes.

Microorganisms encompass the largest resource of metabolic and genetic diversity encountered on Earth. This unparalleled biodiversity risks remaining inaccessible since the major part of the microbiota is unculturable by traditional methods. Metagenomics (i.e. the culture-independent analysis of the genetic complement of an entire habitat) represents a promising and innovative tool for the exploitation of the biotechnological potential encrypted in microbial communities. The present PhD dissertation has been developed in the frame of the FP7 European project MetaExplore, whose aim was the identification through metagenomics of novel biocatalysts involved in the biodegradation of recalcitrant natural molecules, such as chitinases, enzymes with many industrial and environmental applications. The first part of the dissertation focuses on the heterologous production in Escherichia coli and biochemical, functional and structural characterisation of two metagenome-sourced chitinases, Chi18H8 and 53D1. The enzymes are endowed with innovative and valuable features, making them interesting candidates for the biocontrol of plant pathogenic fungi and for the sustainable and environment-friendly treatment and valorisation of seafood wastes, respectively. The second section describes the employment of the Gram-positive bacteria Streptomyces spp. as alternative expression platforms for the production of two proteins, VanYn and Chi18H8. Finally, in the third section the possibility to employ chitinolytic enzymes as alternatives to traditional chemical-based insect pesticides is investigated

Heterologous expression, characterization and applications of metagenome- and genome-sourced enzymes.

Microorganisms encompass the largest resource of metabolic and genetic diversity encountered on Earth. This unparalleled biodiversity risks remaining inaccessible since the major part of the microbiota is unculturable by traditional methods. Metagenomics (i.e. the culture-independent analysis of the genetic complement of an entire habitat) represents a promising and innovative tool for the exploitation of the biotechnological potential encrypted in microbial communities. The present PhD dissertation has been developed in the frame of the FP7 European project MetaExplore, whose aim was the identification through metagenomics of novel biocatalysts involved in the biodegradation of recalcitrant natural molecules, such as chitinases, enzymes with many industrial and environmental applications. The first part of the dissertation focuses on the heterologous production in Escherichia coli and biochemical, functional and structural characterisation of two metagenome-sourced chitinases, Chi18H8 and 53D1. The enzymes are endowed with innovative and valuable features, making them interesting candidates for the biocontrol of plant pathogenic fungi and for the sustainable and environment-friendly treatment and valorisation of seafood wastes, respectively. The second section describes the employment of the Gram-positive bacteria Streptomyces spp. as alternative expression platforms for the production of two proteins, VanYn and Chi18H8. Finally, in the third section the possibility to employ chitinolytic enzymes as alternatives to traditional chemical-based insect pesticides is investigated

Heterogeneous A40926 self-resistance profile in nonomuraea gerenzanensis population informs strain improvement

5noopenNonomuraea gerenzanensis ATCC 39727 produces the glycopeptide antibiotic A40926, which is the natural precursor of the semi-synthetic, last-resort drug dalbavancin. To reduce the cost of dalbavancin production, it is mandatory to improve the productivity of the producing strain. Here, we report that the exposure of N. gerenzanensis wild-type population to sub-inhibitory concentrations of A40926 led to the isolation of differently resistant phenotypes to which a diverse A40926 productivity was associated. The most resistant population (G, grand colonies) represented at least the 20% of the colonies growing on 2 µg/mL of A40926. It showed a stable phenotype after sub-culturing and a homogeneous profile of self-resistance to A40926 in population analysis profile (PAP) experiments. The less resistant population (P, petit) was represented by slow-growing colonies to which a lower A40926 productivity was associated. At bioreactor scale, the G variant produced twice more than the wild-type (ca. 400 mg/L A40926 versus less than 200 mg/L, respectively), paving the way for a rational strain improvement based on the selection of increasingly self-resistant colonies.openElisa Binda, Francesca Berini, Flavia Marinelli, Adriana Bava, Fabrizio BeltramettiBinda, Elisa; Berini, Francesca; Marinelli, Flavia; Bava, Adriana; Beltrametti, Fabrizi

Genetics Behind the Glycosylation Patterns in the Biosynthesis of Dalbaheptides

Glycopeptide antibiotics are valuable natural metabolites endowed with different pharmacological properties. Among them are dalbaheptides, used to treat different infections caused by multidrug-resistant Gram-positive pathogens. Dalbaheptides are produced by soil-dwelling high G-C Gram-positive actinobacteria. Their biosynthetic pathways are encoded within large biosynthetic gene clusters. A non-ribosomally synthesized heptapeptide aglycone is the common scaffold for all dalbaheptides. Different enzymatic tailoring steps, including glycosylation, are further involved in decorating it. Glycosylation of dalbaheptides is crucial conferring them specific biological activities. It is achieved by a plethora of glycosyltransferases, encoded within the corresponding biosynthetic gene clusters, able to install different sugar residues. These sugars might originate from primary metabolism, or, alternatively, their biosynthesis might be encoded within the biosynthetic gene clusters. Already installed monosaccharides might be further enzymatically modified or work as substrates for additional glycosylation. In the current minireview, we cover recent updates concerning the genetics and enzymology behind the glycosylation of dalbaheptides, building a detailed and consecutive picture of this process and of its biological evolution. A thorough understanding of how glycosyltransferases function in dalbaheptide biosynthesis might open new ways to use them in chemo-enzymes synthesis and/or in combinatorial biosynthesis for building novel glycosylated antibiotics

A Bombyx mori Infection Model for Screening Antibiotics against Staphylococcus epidermidis

The increasing number of microorganisms that are resistant to antibiotics is prompting the development of new antimicrobial compounds and strategies to fight bacterial infections. The use of insects to screen and test new drugs is increasingly considered a promising tool to accelerate the discovery phase and limit the use of mammalians. In this study, we used for the first time the silkworm, Bombyx mori, as an in vivo infection model to test the efficacy of three glycopeptide antibiotics (GPAs), against the nosocomial pathogen Staphylococcus epidermidis. To reproduce the human physiological temperature, the bacterial infection was performed at 37 °C and it was monitored over time by evaluating the survival rate of the larvae, as well the response of immunological markers (i.e., activity of hemocytes, activation of the prophenoloxidase system, and lysozyme activity). All the three GPAs tested (vancomycin, teicoplanin, and dalbavancin) were effective in curing infected larvae, significantly reducing their mortality and blocking the activation of the immune system. These results corroborate the use of this silkworm infection model for the in vivo studies of antimicrobial molecules active against staphylococci

Biocatalyzed synthesis of glycostructures with anti-infective activity

Molecules containing carbohydrate moieties play essential roles in fighting a variety of bacterial and viral infections. Consequently, the design of new carbohydrate-containing drugs or vaccines has attracted great attention in recent years as means to target several infectious diseases. Conventional methods to produce these compounds face numerous challenges because their current production technology is based on chemical synthesis, which often requires several steps and uses environmentally unfriendly reactants, contaminant solvents, and inefficient protocols. The search for sustainable processes such as the use of biocatalysts and eco-friendly solvents is of vital importance. Therefore, their use in a variety of reactions leading to the production of pharmaceuticals has increased exponentially in the last years, fueled by recent advances in protein engineering, enzyme directed evolution, combinatorial biosynthesis, immobilization techniques, and flow biocatalysis. In glycochemistry and glycobiology, enzymes belonging to the families of glycosidases, glycosyltransferases (Gtfs), lipases, and, in the case of nucleoside and nucleotide analogues, also nucleoside phosphorylases (NPs) are the preferred choices as catalysts. In this Account, on the basis of our expertise, we will discuss the recent biocatalytic and sustainable approaches that have been employed to synthesize carbohydrate-based drugs, ranging from antiviral nucleosides and nucleotides to antibiotics with antibacterial activity and glycoconjugates such as neoglycoproteins (glycovaccines, GCVs) and glycodendrimers that are considered as very promising tools against viral and bacterial infections. In the first section, we will report the use of NPs and N-deoxyribosyltransferases for the development of transglycosylation processes aimed at the synthesis of nucleoside analogues with antiviral activity. The use of deoxyribonucleoside kinases and hydrolases for the modification of the sugar moiety of nucleosides has been widely investigated. Next, we will describe the results obtained using enzymes for the chemoenzymatic synthesis of glycoconjugates such as GCVs and glycodendrimers with antibacterial and antiviral activity. In this context, the search for efficient enzymatic syntheses represents an excellent strategy to produce structure-defined antigenic or immunogenic oligosaccharide analogues with high purity. Lipases, glycosidases, and Gtfs have been used for their preparation. Interestingly, many authors have proposed the use Gtfs originating from the biosynthesis of natural glycosylated antibiotics such as glycopeptides, macrolides, and aminoglycosides. These have been used in the chemoenzymatic semisynthesis of novel antibiotic derivatives by modification of the sugar moiety linked to their complex scaffold. These contributions will be described in the last section of this review because of their relevance in the fight against the spreading phenomenon of antibiotic resistance. In this context, the pioneering in vivo synthesis of novel derivatives obtained by genetic manipulation of producer strains (combinatorial biosynthesis) will be shortly described as well. All of these strategies provide a useful and environmentally friendly synthetic toolbox. Likewise, the field represents an illustrative example of how biocatalysis can contribute to the sustainable development of complex glycan-based therapies and how problems derived from the integration of natural tools in synthetic pathways can be efficiently tackled to afford high yields and selectivity. The use of enzymatic synthesis is becoming a reality in the pharmaceutical industry and in drug discovery to rapidly afford collections of new antibacterial or antiviral molecules with improved specificity and better metabolic stability

Antimicrobial activity of nanoconjugated glycopeptide antibiotics and their effect on Staphylococcus aureus biofilm

In the era of antimicrobial resistance, the use of nanoconjugated antibiotics is regarded as a promising approach for preventing and fighting infections caused by resistant bacteria, including those exacerbated by the formation of difficult-to-treat bacterial biofilms. Thanks to their biocompatibility and magnetic properties, iron oxide nanoparticles (IONPs) are particularly attractive as antibiotic carriers for the targeting therapy. IONPs can direct conjugated antibiotics to infection sites by the use of an external magnet, facilitating tissue penetration and disturbing biofilm formation. As a consequence of antibiotic localization, a decrease in its administration dosage might be possible, reducing the side effects to non-targeted organs and the risk of antibiotic resistance spread in the commensal microbiota. Here, we prepared nanoformulations of the ‘last-resort’ glycopeptides teicoplanin and vancomycin by conjugating them to IONPs via surface functionalization with (3-aminopropyl) triethoxysilane (APTES). These superparamagnetic NP-TEICO and NP-VANCO were chemically stable and NP-TEICO (better than NP-VANCO) conserved the typical spectrum of antimicrobial activity of glycopeptide antibiotics, being effective against a panel of staphylococci and enterococci, including clinical isolates and resistant strains. By a combination of different methodological approaches, we proved that NP-TEICO and, although to a lesser extent, NP-VANCO were effective in reducing biofilm formation by three methicillin-sensitive or resistant Staphylococcus aureus strains. Moreover, when attracted and concentrated by the action of an external magnet, NP-TEICO exerted a localized inhibitory effect on S. aureus biofilm formation at low antibiotic concentration. Finally, we proved that the conjugation of glycopeptide antibiotics to IONPs reduced their intrinsic cytotoxicity toward a human cell line

Identification and Characterization of a Novel Plasmid-Encoded Laccase-Like Multicopper Oxidase from Ochrobactrum sp. BF15 Isolated from an On-Farm Bio-Purification System

Research background. In recent decades, laccases (p-diphenol-dioxygen oxidoreductases; EC 1.10.3.2) have attracted the attention of researchers due to their wide range of biotechnological and industrial applications. Laccases can oxidize a variety of organic and inorganic compounds, making them suitable as biocatalysts in biotechnological processes. Even though the most traditionally used laccases in the industry are of fungal origin, bacterial laccases have shown an enormous potential given their ability to act on several substrates and in multiple conditions. The present study aims to characterize a plasmid-encoded laccase-like multicopper oxidase (LMCO) from Ochrobactrum sp. BF15, a bacterial strain previously isolated from polluted soil. Experimental approach. We used in silico profile hidden Markov models to identify novel laccase-like genes in Ochrobactrum sp. BF15. For laccase characterization, we performed heterologous expression in Escherichia coli, purification and activity measurement on typical laccase substrates. Results and conclusions. Profile hidden Markov models allowed us to identify a novel LMCO, named Lac80. In silico analysis of Lac80 revealed the presence of three conserved copper oxidase domains characteristic of three-domain laccases. We successfully expressed Lac80 heterologously in E. coli, allowing us to purify the protein for further activity evaluation. Of thirteen typical laccase substrates tested, Lac80 showed lower activity on 2,2\u2019-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), pyrocatechol, pyrogallol and vanillic acid, and higher activity on 2,6-dimethoxyphenol. Novelty and scientific contribution. Our results show Lac80 as a promising laccase for use in industrial applications. The present work shows the relevance of bacterial laccases and highlights the importance of environmental plasmids as valuable sources of new genes encoding enzymes with potential use in biotechnological processes