Deliverables
An enzyme engineering programme, combined with newly developed activity assays, will be run to generate insight into structure-function relationships and to, eventually, create better industrial biocatalysts.
Generated mutant enzymes will have been tested as improved alternatives in quorum sensing.
By X-ray diffraction, high resolution structures will be determined.
Identification and functional characterisation of new cofactor-independent oxygenases within the tautomerase superfamily, with special focus on hypothetical oxygenases for which the genomic context gives clues about their physiological function.
Crystal structures of mutant oxygenases will be obtained with the aim to better understand mechanistic properties of such oxygenases.
Biotechnological exploration of flavin-dependent monooxygenases for the production of indigoid dyes and application testing for developing a novel process for (in-situ) textile dyeing.
Production of the generated heme-dependent monoxygeneases for formulation as lyophilisates in 96 well plates to develop novel metabolite test kits for the hydroxylation of heterocyclic compounds which are key intermediates for active pharmaceutical intermediates (API) synthesis
Using the generated monooxygenases, biocatalytic conversions of heterocycles will be performed, and the formed products will be isolated and characterized.
The work will focus on eukaryotic flavin-containing monooxygenases that are essential for the detoxification of xenobiotics. The target enzymes will be produced and purified using protein chromatography.
Novel heterocycle hydroxylating heme-dependent monooxygenases: novel enzymes will be identified by in silico and functional screening of plant and fungal transcriptomes.
Investigation of three heme-dependent monooxygenases for the conversion of heterocyclic compounds. Through modelling and random mutagenesis positions will be identified and mutated to elucidate on the molecular level structure-function relationships.
Laboratory data will be used to optimise processes in real (pilot-scale) industrial bioreactors, where process conditions such as how oxygen availability affects operational LPMO performance and the overall outcome of biomass conversion processes will be assessed.
The studies monooxygenases will be subjected to multi-site mutagenesis. Three libraries will be prepared.
Elucidation of crystal structures of eukaryotic flavin-containing monooxygenases and analysis of the structural featues in order to understand enzyme-based catalysis.
In-depth mechanistic and structural studies of a cofactor-independent monooxygenase from the tautomerase superfamily that catalyses the oxidative cleavage of HPP.
Generate knowledge-based focussed enzyme mutant libraries
Insight into the mechanism of oxygen activation by the copper active site of LPMOs will be gained and a wholly new assay for the activity of new LPMOs will be developed based on Surface-Enhanced Raman spectroscopy (SERS) of polysaccharide-coated nanoparticles.
Screening of the generated libraries will result in discovery of improved variants. This directed evolution approach will focus on generating and studying engineered enzymes optimized for conversion of indole-related and pharmaceutical compounds.
Knowledge-based design of copper complexes will be tested as mimics of LPMOs.
The developed enzymes will be tested under industrially relevant conditions. The performance will be compared with existing traditional methods.
Isolation of new cofactor-independent dioxygenases from the alpha/beta hydrolase fold superfamily active toward the Pseudomonas quinolone signal (PQS), exploiting natural genetic diversity; and functional characterisation of PQS-cleaving enzymes, with a focus on substrate specificity and affinity, catalytic efficiency, and applicability to interfere with quorum sensing.
Using technology of SSB, mutant libraries will be prepared and transfered to the relevant partners.
Mutants of LPMOs will be prepared and checked for activity.
For identifying ESR candidates, OXYTRAIN vacancies advertised.
Doctoral degrees will be awarded.
A conference on “Mechanistic and applied aspects of oxygenases” will be organized.
An OXYTRAIN website will be built and launched
Publications
Author(s): Andrea N. Fabara, Marco W. Fraaije
Published in: Applied Microbiology and Biotechnology, Issue 104/3, 2020, Page(s) 925-933, ISSN 0175-7598
DOI: 10.1007/s00253-019-10292-5
Author(s): Sandra C. Wullich, Alba Arranz San Martín, Susanne Fetzner
Published in: Applied and Environmental Microbiology, Issue 86/9, 2020, Page(s) 1-12, ISSN 0099-2240
DOI: 10.1128/aem.00279-20
Author(s): Gustavo de Almeida Santos, Gaurao V. Dhoke, Mehdi D. Davari, Anna Joëlle Ruff, Ulrich Schwaneberg
Published in: International Journal of Molecular Sciences, Issue 20/13, 2019, Page(s) 3353, ISSN 1422-0067
DOI: 10.3390/ijms20133353
Author(s): Gaston Courtade, Luisa Ciano, Alessandro Paradisi, Peter J. Lindley, Zarah Forsberg, Morten Sørlie, Reinhard Wimmer, Gideon J. Davies, Vincent G. H. Eijsink, Paul H. Walton, Finn L. Aachmann
Published in: Proceedings of the National Academy of Sciences, Issue 117/32, 2020, Page(s) 19178-19189, ISSN 0027-8424
DOI: 10.1073/pnas.2004277117
Author(s): Gautier Bailleul, Callum R. Nicoll, María Laura Mascotti, Andrea Mattevi, Marco W. Fraaije
Published in: Journal of Biological Chemistry, Issue 296, 2021, Page(s) 100221, ISSN 0021-9258
DOI: 10.1074/jbc.ra120.016297
Author(s): F. Calderaro, M. Keser, M. Akeroyd, L. E. Bevers, V. G. H. Eijsink, A. Várnai, M. A. van den Berg
Published in: Biotechnology for Biofuels, Issue 13/1, 2020, ISSN 1754-6834
DOI: 10.1186/s13068-020-01836-3
Author(s): Nico D. Fessner
Published in: ChemCatChem, 2019, ISSN 1867-3880
DOI: 10.1002/cctc.201801829
Author(s): Nikola Lončar, Filippo Fiorentini, Gautier Bailleul, Simone Savino, Elvira Romero, Andrea Mattevi, Marco W. Fraaije
Published in: Applied Microbiology and Biotechnology, Issue 103/4, 2019, Page(s) 1755-1764, ISSN 0175-7598
DOI: 10.1007/s00253-018-09579-w
Author(s): Callum R. Nicoll, Gautier Bailleul, Filippo Fiorentini, María Laura Mascotti, Marco W. Fraaije, Andrea Mattevi
Published in: Nature Structural & Molecular Biology, Issue 27/1, 2020, Page(s) 14-24, ISSN 1545-9993
DOI: 10.1038/s41594-019-0347-2
Author(s): Marie-Cathérine Sigmund, Gerrit J. Poelarends
Published in: Nature Catalysis, Issue 3/9, 2020, Page(s) 690-702, ISSN 2520-1158
DOI: 10.1038/s41929-020-00507-8