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Industrial Applications of Marine Enzymes: Innovative screening and expression platforms to discover and use the functional protein diversity from the sea

Periodic Reporting for period 3 - INMARE (Industrial Applications of Marine Enzymes: Innovative screening and expression platforms to discover and use the functional protein diversity from the sea)

Période du rapport: 2018-04-01 au 2019-03-31

Problem/issue addressed:
It is widely appreciated that marine environments represent a largely untapped potential for industrial applications. 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 industry.

Importance for the Society:
Project aimed at facilitating the enhancement, 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 .
• Identification of new lead products and prototypes and delivery of new biocatalytic processes within the project lifetime.
Horizon 2020-funded project INMARE (634496) is the consortium of 25 European and Canadian academic and industrial partners with the total budget of EUR 6 M. Within four years (2015-2019) it has produced following significant results.

New resources for bioprospecting.
INMARE consortium has established substantial resources for bioprospecting of new metabolites for medical applications and enzymes for applications in biocatalysis, production of fine chemicals and drugs and for bioremediation. 14 geographic locations were sampled for eDNA extraction, setting up enrichment cultures with substrates of interest and for isolation of microorganisms. A total of 53 metagenomic libraries have been established with about 600 microbial isolates successfully cultivated.

New screening and expression tools.
Application of new or improved naïve and sequence-based screens, as well as metatranscriptomics and cultivation techniques allowed to detect enzymes with activities of interest. INMARE has created one of the biggest collections of genome- and metagenome-derived enzymes of marine origin. Screening of ca. 1.5 million clones has resulted in identification of 1150 enzymes of which 1040 are available in ready-to-use expression systems.

Large-scale enzyme activity profiling.
INMARE has produced bio-catalytic data for a large number of enzymes using few hundred target molecules, including those of industrial relevance.
445 enzymes were expressed as soluble proteins, 393 purified in active form and 330 fully characterized. 24 enzyme mutants were engineered of which 16 were with improved catalytic performance. 55 different immobilized preparations were established and tested in biocatalytic processes. 26 crystal structures of 18 enzymes were resolved. The comparative analysis of bio-catalytic data revealed that prominent “frequent hitters” (or enzymes with outstanding broad substrate profile) have a moderate probability to occur (13% of the total enzymes tested), which is independent on the activity level of the enzyme.

Bioinformatic tools for enzyme discovery and biodiversity assessment.
Biosynthetic gene clusters and potential single gene-encoded enzymes have been identified in metagenomes and genomes and proposed for functional analysis. Families of genomic and environmental “unknowns” from ~140 million genes have been predicted. 476 enzyme targets were identified using genome mining approach. A multi-layered computational approach was developed, which puts in context the functions of unknown genes opening the door for new biodiscovery. INMARE has established first tools to predict promiscuity and for engineering enzymes with multiple active sites.

Small-scale process development and assessment.
Small-scale reactors were designed, built and used to assess biocatalyst performance with selected enzymes. The subset of selected 15 INMARE Enzymes were produced with high yields (up to 30% total protein). We designed and tested at least 5 different immobilisation techniques to prepare 6 improved biocatalysts identified as having the best catalytic performance. Bioprocesses selected for scale-up have been evaluated regarding product yields and conditions using number of target molecules and conversions of industrial interest. production at 50L scale was achieved for several strains for the production of pharmaceutical compounds.

Scale-up and applications.
Identified enzymes with extended substrate promiscuity, two hydrolases and four transferases, have been produced in 20 and 30 L batch fermentations and tested for special features, applications and critical parameters. The full design of industrial scale processes (upstream and downstream, evaluation of utilities and costs estimation), nanoparticles-immobilisation based enzyme preparation assessments and large-scale heterologous expression of a transferase (process development and validation), have been concluded.
Milligrams of a bacterial physiologically active pigment, > 100 mg of the new patented anti-tumour polyketide and about 400 g of another anti-cancer polyketide were obtained from 50-500 L fermentations.

Dissemination and impact
INMARE consortium has established and carried out:
• the INMARE website available at http://www.inmare-h2020.eu
• two publications on the project in a popular (non-scientific) magazine and a blog
• 105 peer-reviewed papers and book chapters;
• 104 contributions to the conferences and symposia;
• CLIB2021 Forum event on 27 April 2016, Düsseldorf: “Innovative Biocatalysis: New Enzymes, Processes and Products Enabled by (marine) Biotechnology);
• INMARE Symposium on Metagenomics and Bioprospecting”, April 5, 2017, Hamburg
• INMARE Symposium “The Access, Use and Circulation of Biogenetic Resources: The Nagoya Protocol and Implementing Measures”. September 11-12, 2018 London School of Economics, London.
• Scientific Seminar “Biodiversity and Applications of Marine Microorganisms and Enzymes” March 5, 2019 Catholic University Valencia, Spain
• Legacy document, policy brief available at INMARE website http://www.inmare-h2020.eu and a short movie on INMARE philosophy and achievements available in YouTube https://www.youtube.com/watch?v=2wie1wiUKfs
Within its duration, the INMARE project has fully achieved its goals. The project has and will 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;
• 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

INMARE Project has produced four patent applications and one start-up company.