The HotZyme Newsletter

Transcription

1 Issue The HotZyme Newsletter SYSTEMATIC SCREENING FOR NOVEL HYDROLASES FROM HOT ENVIRONMENTS From the Editor Dear readers, Welcome to the 13th and the last edition of the HotZyme Newsletter. HotZyme project will come to an end in March Project partners successfully concluded the work as initially proposed and the latest results obtained are reported in details in all deliverables that have been submitted to the reviewers during the 48 th month of the project. In the last quarter of the project, the HotZyme team successfully hosted a symposium in Haifa, Israel and the entire consortium met for the last time for a final annual meeting in Copenhagen, Denmark. In this Issue Highlights from HotZyme Symposium: Discovery of Extremophilic Novel Enzymes with Application for Industrial Biotechnology HotZyme final consortium meeting Last deliverables and what s next On behalf of the HotZyme consortium, we thank you for your support and interest in our project. Haimo Gilbert (EuCRF) Editor, the HotZyme Newsletter Website: For more information about the project, its participating partners and events, please visit the project official website at: Contribution: What s your team up to? Please share the breakthroughs and other news with us by writing to haimo@eu- crf.net Forward this Do you know anyone who might be interested in receiving this quarterly newsletter? Please forward this PDF on and spread the word. Also, please send the e- mail address to haimo@eu- crf.net Contact: EuCRF - European Centre for Research & Financing. 2 Hamarva Street, Binyamina 30500; ISRAEL; Tel ; haimo@eu- crf.net

2 Issue HotZyme Symposium Highlights The HotZyme project and its consortium are committed to the training of young scientists working in the discovery, characterisation and application of extremophilic enzymes. Therefore as one of the last organised project events, the HotZyme consortium sponsored a symposium for young scientists where the issues dealt in the project and related topics are presented and discussed by the project experts, external leading specialists and the young scientists (mainly PhD students and post- doctoral fellows) themselves. This 2- day FREE event was held on 9-10 February 2015 in Hotel Nof, in the highly attractive city of Haifa, Israel. The title of the symposium was: Discovery of Extremophilic Novel Enzymes with Application for Industrial Biotechnology. At the very beginning of the symposium (9th February), HotZyme partner Prof. Jennifer Littlechild made an introduction that presented the project and the work done within its framework and its achievements and future perspectives. The opening talk of the symposium was presented by Prof. Aharon Oren of The Hebrew University of Jerusalem, entitled: "From Lot's wife to hypersaline biotechnology: Diversity, physiology and exploitation of life at high salt concentrations". This was followed by the presentation of HotZyme scientist Prof. Jenny Littlechild (University of Exeter), under the title: Thermophilic Enzymes with Applications in Industrial Biocatalysis. HotZyme partner Dr. Georgios Skretas, (National Technical University of Athens), followed with his talk about Discovery and characterisation of two thermostable hydrolases from HotZyme metagenomes. After dedicated time devoted to informal meetings of participating research students with consortium members & guest scientists, followed by lunch, Prof. Yoram Gerchman, (University of Haifa) presented his talk under the title: "Phyllosphere Bacteria as a Source for Novel Methanol Tolerance Lipase(s)". A local Technion PhD student (of Prof. Ayelet Fishman), Mr Adi Dror, delivered a talk under the title of A Thermophilic Lipase as a Platform for Evolving a Methanol Stable Biocatalyst, which was followed by the presentation of HotZyme partner Prof. Elizaveta Bonch- Osmolovskaya, entitled Thermophilic Prokaryotes with Hydrolytic Activities: New and Familiar Names. The first day of the symposium was concluded with Prof. Yuval Shoham (Technion), whose talk was about Structure, Function and Design of Glycoside Hydrolases: Breaking and Making One of the Most Stable Bonds in Nature. This was

3 Issue followed by another informal meetings and discussion session which led to a dinner in which both HotZyme members and guest scientists had participated. The next day of the symposium (10th February) was dedicated to presentations of HotZyme partners Dr. Roland Wohlgemuth of Sigma Aldrich ( Solving Enzyme - Substrate Puzzles Towards Sustainable Manufacturing Routes ), Dr. Zalan Szabo of MicroDish ("HotZyme SME partner Microdish - development of devices for microbial culturing and enzyme screening") and finally, after yet another session of informal meetings and talks, Dr. Ilya Kublanov of IMMI ( Search for Novel Glycosidases from Thermophilc Microorganisms ). Poster Presentations: A strong emphasis was placed on the active participation of research students, both in the oral presentations and through the exposition of posters pertaining to their work. Such posters have been placed at the venue where all participants could examine and discuss them. To ensure interaction with the audience, refreshment breaks and dedicated informal meetings for discussion were scheduled in the programme, so that members of the audience would have the opportunity to ask questions of experts and presenters and enhance their scientific networking. Guest speakers presentation summaries: * Professor Aharon Oren1 - Topic: "From Lot's wife to hypersaline biotechnology: Diversity, physiology and exploitation of life at high salt concentrations" The Dead Sea, the north arm of Great Salt Lake, Utah, crystallizer ponds of solar salterns worldwide and other salt- saturated aquatic environments are populated by a surprising diversity of microorganisms. Halophiles able to grow up to the highest salinities can be found in each of the three major divisions of life: the Archaea (the order Halobacteriales), the Bacteria (e.g. Salinibacter (Bacteroidetes), and the Eukarya (unicellular green algae of the genus Dunaliella). Due to the often high community densities and the presence of carotenoid and other pigments, communities of halophiles often impart conspicuous colors to their environment. Also physiologically 1 Dept. of Plant and Environmental Sciences, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel

4 Issue the halophiles are a diverse group as two fundamentally different strategies are known that enable microorganisms to thrive at high salt concentrations. The strategy chosen by the Halobacteriales and by Salinibacter is based on the accumulation of molar concentrations of KCl within the cells and adaptation of the entire intracellular enzymatic machinery to function at high salt. On the other hand, most halophilic and halotolerant members of the Bacteria and the eukaryotic microorganisms exclude salts to a large extent and instead they synthesize and/or accumulate organic osmotic solutes. Some of the biotechnological applications of halophiles are directly derived from these salt adaptation strategies. Thus, ectoine and hydroxyectoine, widespread as osmotic compatible solutes among the Bacteria, are now industrially produced and used in a variety of biomedical and other applications. Bacteriorhodopsin, the light- driven proton pump of Halobacterium, has found different uses e.g. as a material for optical information recording. The abundant production of β- carotene has made Dunaliella a favorite source of the pigment for the health food industry. A large number of other applications has been proposed for enzymes and for other compounds produced by different halophilic microorganisms, but except for the above- mentioned success stories of halophilic biotechnology these are not yet commercially exploited. * Professor Yoram Gerchman 2 - Topic: " From Olive Phyllosphere to Methanol Tolerant Lipases " Lipases are enzymes that catalyze the hydrolysis of fats with many industrial applications. One much sought- after application is the production of biodiesel from waste fats, but this application is hampered by the scarcity of methanol resistance lipases, currently coming from handful of organisms. Plant and microorganisms maintain a complex biological interaction - many microorganisms reside near and inside the root area (rhizosphere), inside the plant tissue (entophytes) and on the above- ground surfaces (phyllosphere). To- date, the majority of research held in phyllosphere microbiology focused on study of specific plant pathogen or microbial community structure, but from industrial point of view, this habitat is still a "Newfoundland". Given the roughness of this environment (dryness and long exposure to UV) there is much interest in exploring this complex ecological system as a potential source of enzymes for the biotechnology industry. In our research, we are utilizing the phyllosphere bacterial population as a source for lipase producing bacterial species, searching to methanol producing lipases. In our screening, over 25% of the bacteria isolated expressed lipases capable of digesting long chain fatty acids triglycerides, and at- least one of the isolates produced a lipase active in the presence of up to 60% methanol. These findings demonstrate that this special habitat can be a useful source for industrial applicable enzymes. * Adi Dror 3 - Topic: "A Thermophilic Lipase as a Platform for Evolving a Methanol Stable Biocatalyst" Enzymes ability to catalyze reactions in non- natural environments of organic solvents has opened new opportunities for enzyme- based industrial processes. An example for such a process is the enzymatic production of biodiesel by transesterification of oil and an alcohol that involves lipases as catalysts in a non- aqueous environment. Methanol is utilized frequently as the alcohol in the reaction and the limited stability of lipases in high methanol concentrations hinders industrial implementation of the process. In this research we applied random and rational protein engineering approaches to evolve a lipase for enhanced stability in methanol in order to improve its performance in biodiesel production. We have chosen to work with an unexplored lipase from a thermophilic bacterium, Geobacillus stearothermophilus T6 because a positive correlation between enzyme thermostability and stability in organic solvents was reported in the literature. 2 University of Haifa in Oranim, Haifa, Israel 3 Dept. of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa, Israel; co-authors: Margarita Kanteev, Shalev Gihaz, and Ayelet Fishman

5 Issue We found three single mutations that significantly enhanced the lipase stability in 70% methanol compare to the wild type enzyme. The combination of the mutations to a triple variant, H86Y/A269T/R374W, resulted in an additive stabilizing effect in methanol, elevated thermostability, and improved methanolysis activity of both soybean and waste oils. Insights from crystal structures of variants and the wild type enzyme revealed that all three mutations were situated on the surface and enabled the formation of new hydrogen bonds to other residues and to structural water molecules. * Professor Yuval Shoham4 - Topic: Structure, Function and Design of Glycoside Hydrolases: Breaking and Making One of the Most Stable Bonds in Nature The degradation of cellulose and hemicellulose (the main components of the plant cell wall) is a pivotal step in the carbon cycle on earth. This process is mediated mainly by microorganisms that are found either free in nature or as part of the digestive tracts of higher animals. Glycoside hydrolases (EC ) are a widespread group of enzymes that hydrolyse the glycosidic bond between two or more carbohydrates or between a carbohydrate and a non- carbohydrate moiety. Glycoside hydrolases catalyze the hydrolysis of the glycosidic bond with a remarkable rate reaching kcat /kuncat of over This catalysis is achieved without the use of any cofactor or metal but by simply applying highly tuned acid base catalysis utilizing two amino acid residues of the enzyme: a general acid (proton donor) and a nucleophile/base. In this talk, different experimental approaches will be presented for elucidating the catalytic mechanism and the substrate binding forces of glycoside hydrolases and how these enzymes can be converted to perform the reverse reaction of making (rather than breaking) a glycosidic bond. HotZyme Final Annual Meeting The HotZyme project comes to an end on March 31st. To conclude the remaining work, the HotZyme partners have been actively involved in finalizing the set of deliverables that are planned for M48 and submitting all the 4 Dept. of Biotechnology and Food Engineering, Technion. Refernces: Hövel et al., (2003) EMBO J. 22: ; Zolotnitsky et al., (2004) PNAS 101: ; Shallom et al., (2005) Biochemistry 44: ; Ben-David et al., (2007) ChemBioChem 8: ; Ben-David et al (2007) Chemistry & Biology 15: ; Lansky et al., (2014) Acta Cryst D70: ; Dann et al., (2014) Acta Cryst F70,

6 Issue remaining reports for the commission to review. After working together for the past 4 years, the entire HotZyme consortium congregated for the last time in Copenhagen, Denmark for a 2- day annual meeting on the th of March. Besides concluding current project business and each partner presenting a final summary report, another goal for this meeting was to provide the partners a face- to- face opportunity to discuss and plan any potential future collaboration and project ideas. Remaining deliverables to be submitted by project end (Month 48): D6.5 - Crystallographic analysis of selected proteins: Crystallographic analysis will be performed for selected proteins and the structures will be reported. D6.6 - Production of protein variants by rational design based on modeling and structural analysis: Protein variants will be produced by rational design based on modeling and structural analysis. D9.1 - Interest groups and interests: Identifying and mapping interest groups and their respective interests. These will include such commercial / industrial parties and groupings, political local, national and regional parties and groupings, including regulatory and policy- making parties, etc. For each identifiable party its potential and actual interests will be mapped and indicated. D9.2 - Policy- related consideration: The report will analyse the various consideration that pertain to socio- political decision processes that would potentially influence such decisions affecting the potential uses of the project results, like heritage consideration that might affect possible restrictions on use or environmental vs. employment consideration pertaining to industries to which the project result are related. D9.3 - Potential markets and use: In conjunction with the commercialisation consideration, potential markets, clientele, users, etc. will be mapped so as to determine the potential market developmental trends and directions, with reference to the various socio- economic considerations that might affect such market developmental patterns and direction. Thank you for your support! As the project comes to an end, on behalf of the entire HotZyme Consortium, we'd like to thank you for your interest and support in our project and its achievements. We hope you will continue to follow the work and academic contributions of our HotZyme scientist and partners. We welcome any interest for future collaborations. Thank you! The HotZyme consortium

7 Issue HotZyme Partners: Copenhagen University Microdish BV Norwegian Geotechnical Institute Winogradsky Institute of Microbiology (RAS) National Research Council of Italy National Technical University of Athens EuCRF European Centre for Research & Financing European Commission Scientific Officer Dr. Herman VAN MELLAERT This is a newsletter distributed by the HotZyme project consortium to those interested in our EU FP7 coordination action project on Systematic Screening for Novel Hydrolases from Hot Environments. HotZyme (GA: ) is a project supported by the 7th Framework Programme for Research and Technological Development (FP7) of the European Union. The research project is a large scale integrating project collaborative project - on the systematic screening for novel hydrolases from hot environments.