Final Report Summary - MAMBA (Marine Metagenomics for New Biotechnological Applications) Executive Summary:MAMBA stands for “Marine Metagenomics for New Biotechnological Applications”, a small collaborative project to mine for and use of new microbial activities, in particular for targeted production of fine chemicals, antioxidants and anti-cancer drugs. This Project builds up on the previous efforts of European Framework Programs and National research initiatives for exploiting the catalytic activities of marine microorganisms and microbial communities and to gain the new knowledge on the mechanisms of survival of living organisms in extreme environments. Project Context and Objectives:Context: The urge for the integrated approach in metagenomics. The majority of current biotechnological applications are of microbial origin, and it is widely appreciated that the microbial world contains by far the greatest fraction of biodiversity in the biosphere, so it is the microbes that will deliver the greater part of enzyme diversity and the majority of new applications. It is generally accepted that marine microbial communities account for more than 80% of life on Earth, and have an indispensable role in primary energy and carbon cycling, and therefore, marine biochemical and chemo-diversity is considered to be a major target for the prospecting for new enzymes and natural products. However, the well-known feature of microbes – that the majority cannot be cultivated – limits application of the traditional means of enzyme discovery. The anticipated rich enzymatic pickings from the uncultured microbes have stimulated the development of new genomics-based discovery approaches, the so-called “metagenomics”. The MAMBA aimed at the mining of enzymes and metabolic pathways from extremophilic marine organisms and metagenomes from microbial communities from peculiar marine environments and consequent funnelling the new enzymatic reactions and processes towards new biotechnological applications. The project was build up on the scientific and technological excellence of individual academic and industrial partners, and beyond that, on application of the state-of-the-art technologies for archiving, molecular activity screening, protein structure elucidation, enzyme engineering and directed evolution and establishing new biotechnological processes including biocatalysis for the synthesis of fine chemicals. Marine sampling hotspots to produce the metagenomic resources for their further exploration, and microorganisms with recently sequenced genomes covered the whole diversity of marine microbial life at its limits (hypersaline, low and high temperature, high pressure and low water activity conditions, etc.), which is of great interest to explore the new mechanisms determining the limits to life in the biosphere, but is also of significant relevance for discovery of new industrially relevant enzymatic processes. The central approach of the enzyme discovery was based on the “Activity First” principle, allowing to directly access metabolic reactions of biotechnological relevance. This approach contrasts with the massive sequencing and consequent “genome/metagenome gazing” approach which can only reveal those genes encoding the enzymes of already known protein families. Individual enzymes interacting with the substrates were identified, and in case they are new, hyperexpressed, crystallized, and their structures were elucidated. Consequently, the most promising candidates were scored against the batteries of chiral and non-activated substrates of relevance for biocatalysis and their ability to perform in water-free systems was evaluated. Designed and/or directed evolution methods were conducted to improve the performance and specificity of the enzymes. A comprehensive bioinformatic survey throughout the whole tree of cellular life revealed and suggested new candidates homologous to the discovered new proteins, from other organisms cloned and assayed. The implementation of the set of new enzymes in the biotechnological processes was conducted in a strong alliance with competent industrial partners as well as increase in the performance and specificity of the new biotechnological processes compared to the existing ones, and establishing a new platform encompassing the whole set of technologies including enzyme discovery, bioinformatics, and enzyme engineering to further explore the yet undiscovered marine protein diversity space.Project Results:- Successful sampling of more than 30 sites has been accomplished in four cruises 2009-2012 to provide essential resources for downstream analyses. Among sampled sites, molecular survey including the whole metagenome sequencing of the new Thetis Lake was performed by to establish key biochemical pathways such as sulphur respiration, autotrophic CO2 fixation and methanogenesis and the taxonomic composition of microbial communities. - The number of different samples i.e. Kryos, brine-seawater interface layers; Medee basin interface layer; Matapan Deep; enrichments from some of these samples; Rimicaris exoculata deep sea shrimp’s gill chamber and gut epibiontic microbial communities, two marine hydrocarbon-degrading strains, two strains of archaea and strain of the UV-resistant marine bacterium, have been sequenced. - In total, 27 expression libraries from above genomic and metagenomics resources have been constructed. For screening of metagenomic libraries more than 20 different established assays and 4 new methods (covering three general activities, namely hydrolase, oxidoreductase and hydrogenase activities plus two PCR-based and in silico methods for isolating PKS clusters) have been successfully implemented, to score the activity against 4 million clones, from which more than 1.100 clones (fosmids and phagemids) to encode enzymatic activities, have been identified.- The microbial strain collections have been subjected to the screening for the UVB-resistant isolates for the forthcoming in-depth studies of the proteome response to the irradiation and oxidative stresses. Two bacterial strains exhibited a high resistance, with a positive response following the three treatments, Ruegeria sp. and Erythrobacter sp.- The study on the vertical distribution of ammonia-oxidizing Crenarchaeota at offshore site in Southern Tyrrhenian Sea through metaproteomics coupled with labeled carbon measurements provided a clear proof of chemolithoautotrophic physiology of bathypelagic crenarchaeota, suggested the fifth pathway of inorganic carbon fixation via 4-hydroxybutyryl-CoA dehydratase (4-hbd) to play a pivotal role in the carbon turnover and suggested that this numerically predominant group of crenarchaea drive the sink for inorganic C of a global importance.- From more than 600 shortlisted protein targets from isolates and metagenomic libraries established from marine microorganisms around 300 proteins have already been cloned, expressed and purified. More than 60 proteins were crystallized, and 20 protein crystal structures have been resolved and additionally 24 structural 3D models produced.- The MAMBA consortium has more than doubled the number of structures of cold-adapted proteins available in public databases and provided so far the largest set of protein structures from a single cold-adapted organism. - A number of shuttle vectors for heterologous expression of metagenomic DNA have been constructed and successfully tested with a number of model enzymes. This allows to significantly expand the list of host organisms for heterologous expression and to use the hosts like Bacilllus subtilis, Rhodobacter capsulatus and Pseudomonas putida for screening of metagenomic libraries for activities or expression of individual enzymes.- In frames of the project, 27 peer-reviewed papers have been published in high-impact journals, including the Nature Communications, ISME Journal, Environmental Microbiology, Biochemical Journal, CHEMBIOCHEM and Applied and Environmental Microbiology. One patent application filed and licensed, with few more applications pending.- The resources generated in frames MAMBA (new genes, enzymes, reactions/processes) pose a great potential for the further industrial exploitation. Potential Impact:MAMBA is a unique project with a much-focused program and excellent research groups involved, each of which has an outstanding scientific level. MAMBA has produced a number of unique protocols and know-hows, in particular those related to the establishment of an innovative screening technology patented and licensed by Reactomix S.L. The company provides the screening platform dubbed "Enzymolome", as a service to a number of clients from academia and companies, including the world's largest producers of industrial enzymes and the world leading suppliers of reagents for Life Sciences. Apart from its obvious utility in discovery of new enzymatic catalysts (with a range of applications, from drugs’ synthesis to food/feed modification and from household detergents to biofuels production), this screening platform has in particular been useful for functional elucidation of unknown proteins (and potential drug targets) in the important human pathogen H. pylori. The interest expressed by a number of companies seeking for use the resources generated in frames of MAMBA (in particular, hydrolases (glycoside hydrolases, lipases, etc.), lyases, transferases and oxidoreductases), underpins its significant value for industry. Regarding the impact on research and innovation in Europe and worldwide, MAMBA consortium has evidently been successful by producing within its lifetime more than two dozens of high impact peer-reviewed scientific publications with more to be published in coming few years. It is worth mentioning that the experience gained in frames of this project became a solid background of successful grant applications at the EU level (e.g. Kill-Spill, MACUMBA, MICROB3) and to the National funding agencies in the areas of Biotechnology, Environment and Synthetic Biology. The resources produced in frames of MAMBA and the expertise built up in the partner labs are expected to significantly contribute to the building a European knowledge-based Bio-Economy.List of Websites:MAMBA website has been established and is accessible at the URL is: http://mamba.bangor.ac.uk It consists of the public and of the restricted-use domains. The public domain represents Project summary, Concept, Objectives, Participants, and Publications, the latter section is updated every six months. It has a link to webinar and project Partners’ presentations. It also provides a link to the contacts for third parties with potential interests for getting ad hoc access to the materials and services.
