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.
Evaluation of most promising PQS-cleaving dioxygenases for use in quorum sensing interferenceGenerated mutant enzymes will have been tested as improved alternatives in quorum sensing.
High-resolution crystal structures of HPP oxygenase complexed with substrate or inhibitorsBy X-ray diffraction, high resolution structures will be determined.
Cofactor-independent oxygenases with few new functions identified within the tautomerase superfamilyIdentification 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.
High-resolution crystal structure of the most promising tautomerase superfamily oxygenaseCrystal structures of mutant oxygenases will be obtained with the aim to better understand mechanistic properties of such oxygenases.
Effective biotechnological process for producing indigoid compounds using flavin-dependent monooxygenasesBiotechnological exploration of flavin-dependent monooxygenases for the production of indigoid dyes and application testing for developing a novel process for (in-situ) textile dyeing.
Development of a heterocycle hydroxylation kitProduction 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
Isolated and characterised hydroxylated heterocycles in multi mg to g scaleUsing the generated monooxygenases, biocatalytic conversions of heterocycles will be performed, and the formed products will be isolated and characterized.
Flavin-dependent monooxygenases produced and purifiedThe 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.
Identification of at least 2, preferably 5 new hydroxylating heme-dependent monooxygenasesNovel heterocycle hydroxylating heme-dependent monooxygenases: novel enzymes will be identified by in silico and functional screening of plant and fungal transcriptomes.
Identification of amino acid positions that govern selectivity of three heme-dependent monooxygenasesInvestigation 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.
Protocol for evaluating (improved) monooxygenases for industrially relevant conversionsLaboratory 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.
Three simultaneous saturation mutagenesis libraries per monooxygenaseThe studies monooxygenases will be subjected to multi-site mutagenesis. Three libraries will be prepared.
Crystal structures of flavin-dependent monooxygenasesElucidation of crystal structures of eukaryotic flavin-containing monooxygenases and analysis of the structural featues in order to understand enzyme-based catalysis.
Identification of substrates and inhibitors of HPP oxygenaseIn-depth mechanistic and structural studies of a cofactor-independent monooxygenase from the tautomerase superfamily that catalyses the oxidative cleavage of HPP.
Libraries of enzyme mutants preparedGenerate knowledge-based focussed enzyme mutant libraries
A SERS-based method for the assaying of LPMO activity on a range of polysaccharide substratesInsight 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.
Identification of improved flavin-dependent monooxygenasesScreening 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.
A range of new small-molecule copper complexes which can mimic LPMOsKnowledge-based design of copper complexes will be tested as mimics of LPMOs.
Evaluation of engineered flavin-dependent monooxygenases in industrial settingsThe developed enzymes will be tested under industrially relevant conditions. The performance will be compared with existing traditional methods.
PQS-cleaving cofactor-independent dioxygenases purified and functionally characterisedIsolation 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.
Libraries of LPMO mutants preparedMutants of LPMOs will be prepared and checked for activity.
For identifying ESR candidates, OXYTRAIN vacancies advertised.
Awards of doctoral degreesDoctoral degrees will be awarded.
Conference organisationA 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
Publisher:
Springer Verlag
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
Publisher:
American Society for Microbiology
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
Publisher:
Multidisciplinary Digital Publishing Institute (MDPI)
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
Publisher:
National Academy of Sciences
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
Publisher:
American Society for Biochemistry and Molecular Biology Inc.
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
Publisher:
BMC (part of Springer Nature)
DOI:
10.1186/s13068-020-01836-3
Author(s):
Nico D. Fessner
Published in:
ChemCatChem, 2019, ISSN 1867-3880
Publisher:
Wiley - VCH Verlag GmbH & CO. KGaA
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
Publisher:
Springer Verlag
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
Publisher:
Nature Publishing Group
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
Publisher:
Springer Nature
DOI:
10.1038/s41929-020-00507-8
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