Harnessing the power of enzymatic oxygen activation

Resultado final

Engineered LPMOs with improved properties

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 interference

Generated mutant enzymes will have been tested as improved alternatives in quorum sensing.

High-resolution crystal structures of HPP oxygenase complexed with substrate or inhibitors

By X-ray diffraction, high resolution structures will be determined.

Cofactor-independent oxygenases with few new functions identified within the tautomerase superfamily

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.

High-resolution crystal structure of the most promising tautomerase superfamily oxygenase

Crystal 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 monooxygenases

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.

Development of a heterocycle hydroxylation kit

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

Isolated and characterised hydroxylated heterocycles in multi mg to g scale

Using the generated monooxygenases, biocatalytic conversions of heterocycles will be performed, and the formed products will be isolated and characterized.

Flavin-dependent monooxygenases produced and purified

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.

Identification of at least 2, preferably 5 new hydroxylating heme-dependent monooxygenases

Novel 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 monooxygenases

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.

Protocol for evaluating (improved) monooxygenases for industrially relevant conversions

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.

Three simultaneous saturation mutagenesis libraries per monooxygenase

The studies monooxygenases will be subjected to multi-site mutagenesis. Three libraries will be prepared.

Crystal structures of flavin-dependent monooxygenases

Elucidation 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 oxygenase

In-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 prepared

Generate knowledge-based focussed enzyme mutant libraries

A SERS-based method for the assaying of LPMO activity on a range of polysaccharide substrates

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.

Identification of improved flavin-dependent monooxygenases

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.

A range of new small-molecule copper complexes which can mimic LPMOs

Knowledge-based design of copper complexes will be tested as mimics of LPMOs.

Evaluation of engineered flavin-dependent monooxygenases in industrial settings

The 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 characterised

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.

Generation and transfer of six SSB mutant libraries to the RWTH/TUG partners for screening

Using technology of SSB, mutant libraries will be prepared and transfered to the relevant partners.

Libraries of LPMO mutants prepared

Mutants of LPMOs will be prepared and checked for activity.

OXYTRAIN vacancies advertised

For identifying ESR candidates, OXYTRAIN vacancies advertised.

Awards of doctoral degrees

Doctoral degrees will be awarded.

Conference organisation

A conference on “Mechanistic and applied aspects of oxygenases” will be organized.

Project Website

An OXYTRAIN website will be built and launched

Publicaciones

An overview of microbial indigo-forming enzymes

Autores: Andrea N. Fabara, Marco W. Fraaije
Publicado en: Applied Microbiology and Biotechnology, Edición 104/3, 2020, Página(s) 925-933, ISSN 0175-7598
Editor: Springer Verlag
DOI: 10.1007/s00253-019-10292-5

An α/β-Hydrolase Fold Subfamily Comprising Pseudomonas Quinolone Signal-Cleaving Dioxygenases

Autores: Sandra C. Wullich, Alba Arranz San Martín, Susanne Fetzner
Publicado en: Applied and Environmental Microbiology, Edición 86/9, 2020, Página(s) 1-12, ISSN 0099-2240
Editor: American Society for Microbiology
DOI: 10.1128/aem.00279-20

Directed Evolution of P450 BM3 towards Functionalization of Aromatic O-Heterocycles

Autores: Gustavo de Almeida Santos, Gaurao V. Dhoke, Mehdi D. Davari, Anna Joëlle Ruff, Ulrich Schwaneberg
Publicado en: International Journal of Molecular Sciences, Edición 20/13, 2019, Página(s) 3353, ISSN 1422-0067
Editor: Multidisciplinary Digital Publishing Institute (MDPI)
DOI: 10.3390/ijms20133353

Mechanistic basis of substrate–O 2 coupling within a chitin-active lytic polysaccharide monooxygenase: An integrated NMR/EPR study

Autores: 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
Publicado en: Proceedings of the National Academy of Sciences, Edición 117/32, 2020, Página(s) 19178-19189, ISSN 0027-8424
Editor: National Academy of Sciences
DOI: 10.1073/pnas.2004277117

Ancestral reconstruction of mammalian FMO1 enables structural determination, revealing unique features that explain its catalytic properties

Autores: Gautier Bailleul, Callum R. Nicoll, María Laura Mascotti, Andrea Mattevi, Marco W. Fraaije
Publicado en: Journal of Biological Chemistry, Edición 296, 2021, Página(s) 100221, ISSN 0021-9258
Editor: American Society for Biochemistry and Molecular Biology Inc.
DOI: 10.1074/jbc.ra120.016297

Characterization of an AA9 LPMO from Thielavia australiensis, TausLPMO9B, under industrially relevant lignocellulose saccharification conditions

Autores: F. Calderaro, M. Keser, M. Akeroyd, L. E. Bevers, V. G. H. Eijsink, A. Várnai, M. A. van den Berg
Publicado en: Biotechnology for Biofuels, Edición 13/1, 2020, ISSN 1754-6834
Editor: BMC (part of Springer Nature)
DOI: 10.1186/s13068-020-01836-3

P450 Monooxygenases Enable Rapid Late-Stage Diversification of Natural Products via C−H Bond Activation

Autores: Nico D. Fessner
Publicado en: ChemCatChem, 2019, ISSN 1867-3880
Editor: Wiley - VCH Verlag GmbH & CO. KGaA
DOI: 10.1002/cctc.201801829

Characterization of a thermostable flavin-containing monooxygenase from Nitrincola lacisaponensis (NiFMO)

Autores: Nikola Lončar, Filippo Fiorentini, Gautier Bailleul, Simone Savino, Elvira Romero, Andrea Mattevi, Marco W. Fraaije
Publicado en: Applied Microbiology and Biotechnology, Edición 103/4, 2019, Página(s) 1755-1764, ISSN 0175-7598
Editor: Springer Verlag
DOI: 10.1007/s00253-018-09579-w

Ancestral-sequence reconstruction unveils the structural basis of function in mammalian FMOs

Autores: Callum R. Nicoll, Gautier Bailleul, Filippo Fiorentini, María Laura Mascotti, Marco W. Fraaije, Andrea Mattevi
Publicado en: Nature Structural & Molecular Biology, Edición 27/1, 2020, Página(s) 14-24, ISSN 1545-9993
Editor: Nature Publishing Group
DOI: 10.1038/s41594-019-0347-2

Current state and future perspectives of engineered and artificial peroxygenases for the oxyfunctionalization of organic molecules

Autores: Marie-Cathérine Sigmund, Gerrit J. Poelarends
Publicado en: Nature Catalysis, Edición 3/9, 2020, Página(s) 690-702, ISSN 2520-1158
Editor: Springer Nature
DOI: 10.1038/s41929-020-00507-8

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