The research part of Oxytrain was divided into four distinct work packages that each focused on a specific monooxygenase class. Each WP contained three ESRs that performed the respective research. The main findings of each WP are summarized below.
WP1 - Flavin-dependent monooxygenases. A bacterial FMO was discovered and found to represent an interesting enzyme. As part of the search for a biocatalytic process for producing one of the most used dyes, indigo, it could be demonstrated that FMOs developed in Oxytrain could be used to convert tryptophan, a natural compound, into indigo. Ancestral Sequence Reconstruction was used to generate new monooxygenases, and proved to be a powerful tool for the reconstruction of ancient mammalian FMOs that were biochemically and structurally characterized. The obtained results provide profound insight into their evolution and function. Moreover, this approach enables the development enzymes that are more amenable to protein crystallization, opening avenues towards the biotechnological design of stable proteins out of ancestral FMOs. These can be optimized further by approaches such as directed evolution.
WP2 - Heme-dependent monooxygenases.The activity of three heme-dependent monooxygenases towards the conversion of benzo-1,4-dioxane and indole was successfully optimized, different screening and quantification methods were established and applied using different platforms. Overall, the successful bioprospecting for eukaryotic P450s was achieved. A high activity strain, expressing wild-type CYP3A4 was discovered during screening of a rational design library. A hydroxylation kit was successfully developed and presented to customers at the Convention on Pharmaceutical Ingredients (CPhI).
WP3 - Copper-dependent monooxygenases. Lytic polysaccharide monooxygenases (LPMOs) capable of catalyzing peroxygenase reactions with high efficiency were identified and produced in active forms. It was demonstrated that orientation-dependent Raman spectroscopy can be used to quickly monitor polysaccharide degradation by LPMOs. Overall, the results contributed to an improved understanding of a specific LPMO both biochemically as well as its performance-wise under industrially relevant conditions.
WP4 - Cofactor-independent oxygenases. The substrate scope of the 4-HPP monooxygenases seems to be limited to 4-HPP and no other oxygenation substrates could be elucidated, making them unattractive enzymes for biotechnological applications. As alternative, our efforts focused on the development of an effective cofactor-independent peroxygenase. The peroxygenase activity of the tautomerase superfamily member 4-oxalocrotonate tautomerase (4-OT) was enhanced by several rounds of directed evolution. A 4-OT variant was developed that uniquely possesses both aldol condensation and peroxygenase activity, allowing the two-step enzymatic cascade synthesis of highly enantioenriched epoxy-aldehydes from simpler building blocks. Furthermore, new cofactor-independent dioxygenases from the alpha/beta hydrolase fold superfamily active toward the bacterial quinolone signal molecule were discovered. The respective oxygenases were studied in detail, revealing new insights into the molecular functioning of these specialized enzymes.
In addition to the four research-oriented WPs, Oxytrain included WP5 that concerned the training of the ESRs.
WP5 - Training events. Secondments - Each ESR worked on their individual research project that was performed at the local host institute. Additionally, the ESRs participated in secondments within the network. All ESRs successfully completed their secondments, although several were postponed or cancelled because of the COVID19 crisis. Training schools - As part of WP5, four training schools were scheduled during the course of the project. These were completed successfully. ESR conference - The ESRs organized in year 4 of Oxytrain an international scientific conference in close collaboration with their supervisors. This conference was called the Symposium on Industrial Applications of Redox Biocatalysts and would be held on May 6 2020 in Milan, Italy. Because of the rising number of COVID19 infections throughout Europe in March 2020 and Italy in particular it was decided to cancel the Oxytrain symposium.
