Plotka M.,University of Gdansk |
Kaczorowska A.-K.,University of Gdansk |
Stefanska A.,University of Gdansk |
Morzywolek A.,University of Gdansk |
And 10 more authors.
Applied and Environmental Microbiology | Year: 2014
In this study, we present the discovery and characterization of a highly thermostable endolysin from bacteriophage Ph2119 infecting Thermus strain MAT2119 isolated from geothermal areas in Iceland. Nucleotide sequence analysis of the 16S rRNA gene affiliated the strain with the species Thermus scotoductus. Bioinformatics analysis has allowed identification in the genome of phage 2119 of an open reading frame (468 bp in length) coding for a 155-amino-acid basic protein with an Mr of 17,555. Ph2119 endolysin does not resemble any known thermophilic phage lytic enzymes. Instead, it has conserved amino acid residues (His30, Tyr58, His132, and Cys140) that form a Zn2+ binding site characteristic of T3 and T7 lysozymes, as well as eukaryotic peptidoglycan recognition proteins, which directly bind to, but also may destroy, bacterial peptidoglycan. The purified enzyme shows high lytic activity toward thermophiles, i.e., T. scotoductus (100%), Thermus thermophilus (100%), and Thermus flavus (99%), and also, to a lesser extent, toward mesophilic Gram-negative bacteria, i.e., Escherichia coli (34%), Serratia marcescens (28%), Pseudomonas fluorescens (13%), and Salmonella enterica serovar Panama (10%). The enzyme has shown no activity against a number of Gram-positive bacteria analyzed, with the exception of Deinococcus radiodurans (25%) and Bacillus cereus (15%). Ph2119 endolysin was found to be highly thermostable: it retains approximately 87% of its lytic activity after 6 h of incubation at 95°C. The optimum temperature range for the enzyme activity is 50°C to 78°C. The enzyme exhibits lytic activity in the pH range of 6 to 10 (maximum at pH 7.5 to 8.0) and is also active in the presence of up to 500 mM NaCl. © 2014, American Society for Microbiology.
Agency: European Commission | Branch: H2020 | Program: RIA | Phase: BIOTEC-6-2015 | Award Amount: 7.96M | Year: 2016
Biological sequence diversity in nowhere as apparent as in the vast sequence space of viral genomes. The Virus-X project will specifically explore the outer realms of this diversity by targeting the virosphere of selected microbial ecosystems and investigate the encoded functional variety of viral gene products. The project is driven by the expected large innovation value and unique properties of viral proteins, previously demonstrated by the many virally-derived DNA and RNA processing enzymes used in biotechnology. Concomitantly, the project will advance our understanding of important aspects of ecology in terms of viral diversity, ecosystem dynamics and virus-host interplay. Last but not least, due to the inherent challenges in gene annotation, functional assignments and other virus-specific technical obstacles of viral metagenomics, the Virus-X project specifically addresses these challenges using innovative measures in all parts of the discovery and analysis pipeline, from sampling difficult extreme biotopes, through sequencing and innovative bioinformatics to efficient production of enzymes for molecular biotechnology. Virus-X will advance the metagenomic tool-box significantly and our capabilities for future exploitation of viral biological diversity, the largest unexplored genetic reservoir on Earth.
PubMed | Matis, Adam Mickiewicz University, Prokazyme ehf, University of Gdansk and 5 more.
Type: Journal Article | Journal: PloS one | Year: 2015
Phage vB_Tsc2631 infects the extremophilic bacterium Thermus scotoductus MAT2631 and uses the Ts2631 endolysin for the release of its progeny. The Ts2631 endolysin is the first endolysin from thermophilic bacteriophage with an experimentally validated catalytic site. In silico analysis and computational modelling of the Ts2631 endolysin structure revealed a conserved Zn2+ binding site (His30, Tyr58, His131 and Cys139) similar to Zn2+ binding site of eukaryotic peptidoglycan recognition proteins (PGRPs). We have shown that the Ts2631 endolysin lytic activity is dependent on divalent metal ions (Zn2+ and Ca2+). The Ts2631 endolysin substitution variants H30N, Y58F, H131N and C139S dramatically lost their antimicrobial activity, providing evidence for the role of the aforementioned residues in the lytic activity of the enzyme. The enzyme has proven to be not only thermoresistant, retaining 64.8% of its initial activity after 2 h at 95C, but also highly thermodynamically stable (Tm = 99.82C, Hcal = 4.58 10(4) cal mol(-1)). Substitutions of histidine residues (H30N and H131N) and a cysteine residue (C139S) resulted in variants aggregating at temperatures 75C, indicating a significant role of these residues in enzyme thermostability. The substrate spectrum of the Ts2631 endolysin included extremophiles of the genus Thermus but also Gram-negative mesophiles, such as Escherichia coli, Salmonella panama, Pseudomonas fluorescens and Serratia marcescens. The broad substrate spectrum and high thermostability of this endolysin makes it a good candidate for use as an antimicrobial agent to combat Gram-negative pathogens.
Agency: European Commission | Branch: FP7 | Program: CP-FP | Phase: KBBE.2010.3.3-01 | Award Amount: 3.55M | Year: 2011
The aim of the AMYLOMICS project is to develop novel, robust enzymes for the starch and carbohydrate industries. The novel enzymes should enable the formation of new primary products, such as oligosaccharides of defined sizes, composition and degree of branching, new types of linkages, cyclic or more complex polysaccharides and an increased digestive resistance, as well as secondary sugar derivatives such as substituted starches, rare sugars or novel isomers. Fundamental to the success of the project will be the development of an efficient metagenomic platform technology for enzyme screening based on massive parallel 454 sequencing and microarray sequence capture. This platform will enable genome walking of complex metagenomic DNA and greatly facilitate the access to the largely unexplored wealth of genes in the environment. The starch industry is the most developed sector of the polysaccharide industry and European companies play a leading role in the world market. The industry is in a constant need for a range of robust enzymes that can be used for the synthesis, fractionation and/or modification of carbohydrates. It actively searches for sustainable and more economical alternatives to existing techniques, both for the production of novel higher value products and for the improvement of older processes. The metagenomic mining platform developed in the project is expected to provide a large number of robust thermophilic starch and carbohydrate modifying enzymes and lead to new and improved biocatalytic process technologies. Lead users of the projects results will be companies like the project partners Roquette Frres, a world leader in starch processing, Roche Molecular Systems, a leading providers of new tools, technologies and services in the genomic industry, and SME companies like Prokazyme who through the improvement of sequence based metagenomic bioprospecting platform can expand their product range of speciality products.
Stefanska A.,University of Gdansk |
Kaczorowska A.-K.,University of Gdansk |
Plotka M.,University of Gdansk |
Fridjonsson O.H.,Matis ohf |
And 6 more authors.
Journal of Biotechnology | Year: 2014
The recA gene of newly discovered Thermus thermophilus MAT72 phage Tt72 (Myoviridae) was cloned and overexpressed in Escherichia coli. The 1020-bp gene codes for a 339-amino-acid polypeptide with an Mr of 38,155 which shows 38.7% positional identity to the E. coli RecA protein. When expressed in E. coli, the Tt72 recA gene did not confer the ability to complement the ultraviolet light (254nm) sensitivity of an E. coli recA mutant. Tt72 RecA protein has been purified with good yield to catalytic and electrophoretic homogeneity using a three-step chromatography procedure. Biochemical characterization indicated that the protein can pair and promote ATP-dependent strand exchange reaction resulting in formation of a heteroduplex DNA at 60°C under conditions otherwise optimal for E. coli RecA. When the Tt72 RecA protein was included in a standard PCR-based DNA amplification reaction, the specificity of the PCR assays was significantly improved by eliminating non-specific products. © 2014 Elsevier B.V.
Hjorleifsdottir S.,Matis ohf |
Aevarsson A.,Prokazyme ehf |
Hreggvidsson G.O.,Matis ohf |
Hreggvidsson G.O.,University of Iceland |
And 2 more authors.
Extremophiles | Year: 2014
Several bacteriophages that infect different strains of the thermophilic bacterium Rhodothermus marinus were isolated and their infection pattern was studied. One phage, named RM378 was cultivated and characterized. The RM378 genome was also sequenced and analyzed. The phage was grouped as a member of the Myoviridae family with A2 morphology. It had a moderately elongated head, with dimensions of 85 and 95 nm between opposite apices and a 150 nm long tail, attached with a connector to the head. RM378 showed a virulent behavior that followed a lytic cycle of infection. It routinely gave lysates with 1011 pfu/ml, and sometimes reached titers as high as 1013 pfu/ml. The titer remained stable up to 65 °C but the phage lost viability when incubated at higher temperatures. Heating for 30 min at 96 °C lowered the titer by 104. The RM378 genome consisted of ds DNA of 129.908 bp with a GC ratio of 42.0 % and contained about 120 ORFs. A few structural proteins, such as the major head protein corresponding to the gp23 in T4, could be identified. Only 29 gene products as probable homologs to other proteins of known function could be predicted, with most showing only low similarity to known proteins in other bacteriophages. These and other studies based on sequence analysis of a large number of phage genomes showed RM378 to be distantly related to all other known T4-like phages. © 2013 Springer Japan.
Agency: European Commission | Branch: FP7 | Program: CP-TP | Phase: KBBE.2012.3.2-01 | Award Amount: 8.15M | Year: 2012
Marine organisms, in particular sponges and their associated microorganisms, are an inexhaustible source of novel bioactive (lead) compounds for biomedical application. Industrial exploitation of this natural resource using traditional approaches is, however, hampered, with a few exceptions, by unsolvable supply problems - despite of numerous efforts in the past. Therefore, there is, very likely, only one way: to start from the genes encoding the bioproducts, or their biosynthetic pathways, to sustainably obtain the active molecules in sufficient amounts. The aim of the presented industry-driven integrating project is to combine the knowledge in marine genomics, chemogenetics and advanced chemistry to produce recombinantly prepared novel secondary metabolite (lead) compounds and analogous from them, as well as pharmacologically active peptides, and to bring them up to the pre-clinical, and hopefully also to the clinical studies. This ambitious approach is based on breakthrough discoveries and the results of previous successful EU projects of members of the applying consortium, including European leaders (or worldwide leaders) in marine (sponge) genomics, metagenomics (polyketide synthase clusters), combinatorial biosynthesis and marine natural product chemistry/structure elucidation. This multidisciplinary project, driven by high-tech genomics-based SMEs with dedicated interest in bringing marine-biotechnology-derived products to the market, will also involve the discovery and sustainable production of bioactive molecules from hitherto unexploited extreme environments, such as hydrothermal vents and deep-sea sources, and the expression/scale-up of unique enzymes/proteins of biomedical and biotechnological interest. The molecular-biology-based strategies developed in this project for a sustainable exploitation of aquatic molecular biodiversity will further strengthen the international position and effectiveness of European (SME-based) blue biotechnology industry.
Agency: European Commission | Branch: FP7 | Program: CP-TP | Phase: KBBE.2012.3.2-01 | Award Amount: 9.93M | Year: 2012
SeaBioTech is a 48-month project designed and driven by SMEs to create innovative marine biodiscovery pipelines as a means to convert the potential of marine biotechnology into novel industrial products for the pharmaceutical (human and aquaculture), cosmetic, functional food and industrial chemistry sectors. SeaBioTech will reduce barriers to successful industrial exploitation of marine biodiversity for companies more accustomed to terrestrial biotechnology. SeaBioTech directly addresses five key challenges to remove bottlenecks in the marine biodiscovery pipeline, leading to (1) improvements in the quality of marine resources available for biotechnological exploitation, (2) improvement in technical aspects of the biodiscovery pipeline to shorten time to market, and (3) developing sustainable modes of supply of raw materials for industry. The two last challenges centre on enabling activities to enhance the marine biodiscovery process: first, clarification of legal aspects to facilitate access to marine resources, their sustainable use, and their secure exploitation; second, to create an improved framework for access to marine biotechnology data and research materials. To achieve its goals, SeaBioTech brings together complementary and world-leading experts, integrating biology, genomics, natural product chemistry, bioactivity testing, industrial bioprocessing, legal aspects, market analysis and knowledge exchange. The expertise assembled within the consortium reflects the industry-defined needs, from the SME partners initial definition of market and product opportunities to their ultimate proof-of-concept demonstration activities. SeaBioTech will have significant impact on research and technology, on innovation, on European competitiveness and on economic growth. It will provide a model to accelerate the development of European biotechnology into a world leading position.
Agency: European Commission | Branch: FP7 | Program: BSG-SME | Phase: SME-2011-1 | Award Amount: 1.25M | Year: 2011
The main objective of the EXGENOMES project is to develop new and improved thermostable enzymes for use, as reagents, in large-scale DNA synthesis and/or that can act on unnatural components such as in LNA (Locked Nucleic Acids). The target source for the new enzymes is a range of self-replicating mobile genetic elements (phages, plasmids and transposons) from thermophilic bacteria. Increased understanding of self-replication in many mobile genetic elements, such as phi29, has now made the commercial development of new self-priming & strand-displacing polymerases and other enzymes, much more plausible. A number of candidate enzymes, such as a new transposon-coded Thermus DNA polymerase are at hand for this project in the thermophilic bacteria & phage genome bank at Matis. Nucleic acids based technologies now underpin a large and fast-growing industry, including research, diagnostics and pharmaceutical production. Thermophilic enzymes have played a key role in this development, as polymerases (DNA and RNA), ligases, nucleases, reverse transcriptases, polynucleotide kinases, lysozymes and more, are of great importance in the research industry today. The partner SMEs are all active players in this area from bioprospecting (Prokazyme), laboratory distribution (A&A Biotechnologies), LNA manufacture & diagnostics (Exiqon) to DNA vaccine production (Touchlight Genetics). Together with the highly competent RTD partners the consortium is well positioned to implement the project according to its goals. The successful development of new thermostable polymerases and other enzymes with the desired properties would have a substantial impact on strengthening the current market status of the SME partners, resulting in growth in income and employment.
Agency: European Commission | Branch: FP7 | Program: BSG-SME | Phase: SME-2012-1 | Award Amount: 1.51M | Year: 2012
The main objective of the TASTE project is to develop flavour ingredients from edible seaweeds (Ascophyllum nodosum, Saccharina latissima, and Fucus vesiculosus) with the potential to replace sodium in food products. This can be done through two options, namely flavour enhancing properties or mineral content. Health authorities worldwide have recommended reducing salt in processed foods in order to reduce the risk of high blood pressure. Salt, i.e. sodium chloride, is a recognised flavour potentiator. Thus, the reduction of salt in food leads to reduced flavour besides a lack of salty taste. Seaweeds have a naturally salty taste being abundant in minerals like potassium, magnesium besides sodium. This salty taste improves the flavour profile of foodstuffs. In addition, some seaweeds contain a range of potential flavour components that can naturally enhance the flavour of the food. Mild processing can release potential flavour components like proteins, amino acids and reducing sugars. In particular, the proteinaceous compounds that are present in the seaweeds may be responsible for enhancing flavour characteristics (e.g. umami, meaty and roasted) in addition to providing textural mouth feel. The aim of the project is therefore to produce flavour-active building blocks from seaweeds by applying suitable processing and to develop flavour ingredients with these for application in different salt-reduced foods. By doing so, this project offers innovative processing solutions, new healthy flavour ingredients and novel approaches to meeting salt reduction targets to a group of SMEs in the food sector. This consortium is well positioned to implement the project according to its objective. Successful development of different flavour ingredients, with the properties to replace sodium in food products, will have a great impact on strengthening the current market status of the SME partners, resulting in growth in income and employment.