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  • Periodic Reporting for period 1 - INMARE (Industrial Applications of Marine Enzymes: Innovative screening and expression platforms to discover and use the functional protein diversity from the sea)

INMARE Report Summary

Project ID: 634486

Periodic Reporting for period 1 - INMARE (Industrial Applications of Marine Enzymes: Innovative screening and expression platforms to discover and use the functional protein diversity from the sea)

Reporting period: 2015-04-01 to 2016-09-30

Summary of the context and overall objectives of the project

Problem/issue to be addressed:
It is widely appreciated that marine environments represent a largely untapped potential for industrial enzymes. For the last decade, a significant number of projects including those funded by the EU, have been carried out to target the novel enzymes by using innovative technologies and screening approaches. However, so far only a very small fraction of marine enzymes could make it to the industrial biocatalysis. INMARE Consortium enhances the pathways from the discovery to the industrial applications of new marine enzymes and bioactives for targeted production of fine chemicals, drugs and in environmental clean-up.

Importance for the Society:
Project will facilitate the enhancing the competitiveness and sustainability of European industry sectors through increase in efficiency in the enzyme identification-to-market success rate, bring broad societal benefits by facilitating the development of novel, improved or more economic and eco-friendly end-products and processes and contributing to realising the objectives of European policy initiatives, such as the EU Blue Growth Strategy and EU Strategy for Key Enabling Technologies.

Overall objectives:
• Streamlining and shortening pipelines of enzyme and bioactives’ discovery towards industrial applications by increasing the value of enzyme collections.
• Identification of new lead products and prototypes and delivery of new biocatalytic processes within the project lifetime.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

New resources for bioprospecting. In order to establish project resources we have successfully sampled and processed sample materials from different unique microbial biodiversity hotspots from various geographical locations with extreme physical chemical features, including St. Margherita di Savoia (the largest salterns in Europe), Arctic Mid-Ocean Ridge, Aeolian Islands hydrothermal plumes, Eastern Mediterranean deep-sea MgCl2-rich brine lakes, Danish highly alkaline sites (chalk cliffs) and Antarctica subglacial lake Enigma and brines entrapped in pack ice. Furthermore, we established a number of enrichment cultures from various marine resources with industrially relevant substrates and successfully cultivated a number of new isolates, extracted high quality DNA for direct sequencing and library production and constructed 15 new metagenomic libraries. Selected resources, including pre-existing ones, have been distributed among the INMARE Partners focused on certain screening and expression platform.

New screening and expression tools. Multiple and complementary in vitro expression platforms to overcome host-related expression and screening problems have been established. Improved screening and cultivation tools have been developed. The ongoing screening of metagenomic libraries and strain collections with approx. 1.5 million clones/isolates has so far resulted in more than 1,000 positive hits with activities of interest. Extensive substrate profiling (by use of colorimetric, fluorimetric and metabolomics platforms) has allowed a panoramic view of substrate promiscuity, used to select, and subsequently sequence and identify the genes of interest. A few hundreds of enzymes with activities of interest are available in suitable expression systems.

Large-scale activity profiling. A customized and broad library of substrates was designed to examine the degree of substrate ambiguity, and to investigate what defines enzymes’ capability of binding to multiple substrates. Substrate profiling, established for different types of enzymatic activities, has allowed assignment of functions to a number of previously unannotated enzymes, as well as expanded our fundamental understanding on sequence space and substrate specificity profile relationships. It has furthermore allowed discovery of versatile biocatalysts whose in-depth analysis and potential application in biotransformation are being explored. A number of enzyme targets with outstanding properties of interest for biotech applications have been identified.

Bioinformatic tools for enzyme discovery and biodiversity assessment.
Assessing the biodiversity in the source environments, taxonomic profiling and assembly pipelines have been successfully deployed and used to determine the microbial composition of two salterns and three submarine hydrothermal sites. The assemblies of sequenced fosmid and phage clones, isolates and enrichment cultures provided an input into sequence-based enzyme identification approach, for subsequent candidate selection, cloning, expression and characterisation. Biosynthetic gene clusters and potential single gene-encoded enzymes have been identified in metagenomes and genomes and proposed for functional analysis. Generation of families of genomic unknowns (hypothetical proteins in sequenced genomes) and environmental unknowns (any reference in public databases) from ~140 million ORFs have been predicted from three of the largest publicly available marine metagenomes.

Small-scale process development and assessment. Small-scale reactors were designed, built and used to assess biocatalyst performance with selected enzymes. Biochemical properties of wild and engineered enzymes, such as inulinases, esterases and lipases, polyketide and no ribosomal synthetases are other interesting enzymes for new biopharmaceutical bioactives used in free and/or immobilized form, have been studied. The best reaction conditions for whole cells and cells extracts acting as b

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Within its first 18 months, the INMARE project has achieved a significant progress towards its goals. The project will hence contribute to:
• strengthening the single market through European harmonization of legal guidelines and IPR approaches, in our case, using valuable marine microbial diversity and through improving scientific research communication infrastructures;
• helping with the introduction of a novel industrial innovation policy by forming innovative links between researchers and the industrial biotechnology sector, one of the key technologies mentioned (also s. below);
• making natural sciences and engineering more interesting disciplines, including technological and skill- intensive activities, as well as applied research and SME aspects.
• bringing broad societal benefits by facilitating the development of novel, improved or more economic and eco-friendly end-products and processes

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