CORDIS

For the selective and effective incorporation of oxygen into biomolecules, various oxygenating enzymes have evolved in nature. The chemistry feasible with these biocatalysts (oxygenases) is unrivaled when compared with conventional chemical methods. Therefore, these enzymes are promising tools in biotechnology, as the industrial use of oxygenases could facilitate environmentally friendly processes to replace harsh chemical processes. Although significant development and research has been carried out in recent years, the exploitation of oxygenases is still in its infancy. For a large part, this is due to a relatively poor availability of industrially suitable oxygenases and methodologies for applying them. To fully exploit the catalytic power of oxygenases, a higher level of knowledge on these enzymes is needed, while also technical aspects have to be solved.
The main goal of the OXYTRAIN is to boost the development and exploitation of oxygenating enzymes in industrial applications. For that, well-trained researchers are indispensable, as are new tools and approaches for the generation of industrially applicable enzymes. The European Training Network OXYTRAIN, is a joint academic/non-academic training initiative that provides an inter-disciplinary innovative research and training programme for early-stage researchers (ESRs) to satisfy the need for knowledge and skills to produce and apply oxidative enzymes. OXYTRAIN provides a training network for 12 ESRs divided in four work packages (WP), corresponding to the major oxygenase classes: flavin-dependent monooxygenases (WP1), heme-dependent monooxygenases (WP2), copper-dependent monooxygenases (WP3) and cofactor-independent oxygenases (WP4).
The three main research objectives are:
- Integrating structural and mechanistic insights into oxygenase-driven oxygen activation and subsequent substrate oxygenation;
- Generating a collection of industrially applicable oxygenases by design and engineering of oxygenases using state of-the-art protein engineering methodologies;
- Developing innovative methods for efficient usage of oxygenases in research and industry.

WP1: Flavin-dependent monooxygenases
A thermostable microbial Flavin-containing Monooxygenase from Nitrincola lacisaponensis (NiFMO) was characterized and its activity on different substrates was elucidated. Mutants of NiFMO were also prepared and expressed, and further tests are being performed. Another project focused on creating ancestral FMOs has also been investigated. The genes of the ancestral FMOs were produced, cloned and expressed in E.coli strains. The proteins were purified and kinetics measurements and structural elucidation of these ancestral FMOs are being conducted (ESR1, ESR2). Different mutants of microbial flavin-containing monooxygenase (mFMO) were expressed, purified and tested for immobilization. Confocal Raman spectroscopy was used to characterize indigo. A fused enzyme to produce indigo was expressed purified and future work will be done to characterize it (ESR3).
WP2: Heme-dependent monooxygenases
Reliable methods for screening libraries in a high throughput platform for P450 BM3 have been developed. Diversity generation methods were employed, yielding two improved variants. Several amino acids positions in heme-dependent monooxygenases structure were also identified (ESR4). The means for a successful discovery of novel P450s had been established, as well as the necessary plasmid tool to express target enzymes. Different loss-of-function strains of the yeast Komagataella phaffii have been generated and these new platform will be used for new enzyme discovery (ESR5). New amino acid positions in the CYP3A4 enzyme have been identified as potential targets for mutagenesis. Site saturation mutagenesis is being performed to improve testosterone bioconversion while gaining knowledge on structure-function relationship of the CYP3A4 enzyme (ESR6).
W3: Copper-dependent monooxygenases
A protocol to produce and purify a LPMO with fungal origin was established, and the optimal reaction conditions were explored. The reaction variables for different substrates have been determined by two methodologies (ESR7). Confocal Raman spectroscopy was performed on natural structures of crystalline cellulose isolated from celery (Apium graveolens) collenchyma. This will serve as a natural substrate for new activity assays of LPMOs (ESR8). A library of several LPMOs was produced. The activity of these LPMOs was studied using a model cellulose substrate and a pretreated cornstover feedstock. The most active LPMOs were selected and purification was scaled up. These LPMOs will be characterized and the degradation of biomass evaluated in larger scale bioreactors (ESR9).
W4: Cofactor-independent oxygenases
Three 4HPP-like molecules were prepared and characterized as potential substrates/inhibitors of RhCC. The native RhCC crystal structure was determined, and crystals of the RhCC homologue Rhop3 were obtained. Experiments to obtain enzyme-ligand complex structures are in progress (ESR10). Twelve bacterial proteins were selected on basis of amino acid sequence similarity with the 4-HPP monooxygenase RhCC from Rhodococcus opacus. The activity of the proteins was tested, where one showed high-level activity in 4-HPP conversion, and six others exhibited low-level activity. The proteins were screened for activity towards 4-HPP derivatives, and two showed significant tautomerase activity (ESR11). Putative PQS dioxygenases have been expressed in E. coli. Catalytic efficiency and physical and proteolytic stability were determined. The catalytic activity and stability of proteins of fungal origin will be tested (ESR12).

The innovative aspects of the Oxytrain program aims at the development of new generic tools for the analysis of monooxygenases, and insights into critical enzymatic steps, which together will transform and expand the industrial application of these enzymes. Some of these innovative aspects are listed below.
WP1: New tailor-made monooxygenases will be generated for use as biocatalysts in biotechnological applications (ESR1); New structural insights into enzyme-catalysed oxygenation reactions will be obtained (ESR2); New in situ indigoid dyeing process and new bio-catalytic indigoid production process will be developed (ESR3).
WP2: A heme-containing monooxygenase toolbox for the conversion of heterocycles will be developed through exploring the potential of simultaneous site-saturation mutagenesis (ESR4); New tailor-made monooxygenases will be discovered and re-engineered for use as robust whole cell oxygenation biocatalysts in biotechnological applications (ESR5); A first kit for regio- and stereoselective heterocycle activation will be generated (ESR6).
WP3: Novel experimental tools for LPMO characterisation and generation of LPMO variants with improved application properties (ESR7); Use of novel biophysical methods for discovering and understanding highly active LPMOs (ESR8); New and improved monooxygenases, leading to more robust industrial applications (ESR9).
WP4: Detailed insights into how a cofactor-independent HPP oxygenase functions and for what type of conversions it can be employed (ESR10); Structure and biological function of new cofactor-independent oxygenases (ESR11); New cofactor-less oxygenases to interfere with quorum sensing and thus virulence of P. aeruginosa (ESR12).