Periodic Reporting for period 2 - OXYTRAIN (Harnessing the power of enzymatic oxygen activation)
Reporting period: 2019-01-01 to 2021-03-31
Many molecules important to our daily lives are obtained through oxidation reactions. These reactions are industrially important but are performed in a chemical manner at a high temperature, involving toxic organic solvents and expensive metal catalysts. It is therefore desirable that these reactions are replaced by “greener” variants, thereby fulfilling the need for environmentally friendly and sustainable alternatives. For example, oxygenases have emerged as promising tools for a number of industrial oxidative processes owing to their chemical versatility. The catalytic use of enzymes in chemical conversions is known as biocatalysis. Oxygenases are a group of enzymes that carry out the regio-, stereo- and chemoselective introduction of oxygen into a large range of organic molecules under mild conditions. The industrial use of oxygenases is still lagging behind, despite their staggering potential in chemical transformations. This is mainly due to a relatively poor availability of industrially suitable biocatalysts and methodology to apply these enzymes. To exploit the catalytic power of oxygenases, the Oxytrain network was established, which was aimed at improving the knowledge on oxygenases and their use in industrial applications. This required well-trained researchers from different backgrounds as well as new tools and approaches for the generation of industrially applicable oxygenases. The Oxytrain network comprised: different leading academic institutions, various companies, that included key players in textile industry, enzyme production, application, and evaluation and preparation of enzyme mutant libraries as well as partner organizations with expertise in enzyme production and monooxygenase-based applications, biorefining of lignocellulose, enzyme evaluation in industrial conditions, industrial microbes and bioprocesses and chemical synthesis and analysis.
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.
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.
Oxytrain aimed at improving the knowledge on oxygenases and their exploitation in industrial applications. This required well-trained researchers from different backgrounds as well as new tools and approaches for the generation of industrially applicable oxygenases. From the start, Oxytrain comprised a vehicle for providing an innovative research and training programme for early-stage researchers. Based on the first ESRs having successfully obtained their PhD and the good prospects for the other ESRs to do the same, it can be concluded that Oxytrain was indeed a fruitful training network. In fact, the ESRs that finished their PhD continue their career in biotechnology-related research, indicating that the work has prepared them for taking up a role in future research positions. Moreover, the numerous scientific publications and novel biocatalytic technology generated by the Oxytrain consortium emphasize that a primary goal of Oxytrain, the development and exploitation of oxygenating enzymes in industrial applications, is successfully accomplished.