Practical oxyfunctionalisation biocatalysts by engineering monooxygenases into peroxyzymes.

Project Information

PeroxyZyme

Grant agreement ID: 101054658

DOI

10.3030/101054658

EC signature date 1 June 2022

Start date 1 August 2022

End date 31 July 2027

Funded under

• European Research Council (ERC)

Total cost

€ 2 500 000,00

EU contribution

€ 2 500 000,00

Coordinated by

TECHNISCHE UNIVERSITEIT DELFT
Netherlands

Periodic Reporting for period 1 - PeroxyZyme (Practical oxyfunctionalisation biocatalysts by engineering monooxygenases into peroxyzymes.)

Reporting period: 2022-08-01 to 2025-01-31

Summary of the context and overall objectives of the project

The project PeroxyZymes is set in the context of the ongoing demand for more environmentally benign methods in chemical synthesis, an area of increasing global concern due to the environmental and safety challenges associated with traditional chemical processes. A central theme of the project is selective oxyfunctionalisation, a highly sought-after transformation in the synthesis of various chemical compounds due to its potential to introduce oxygen into molecules in a controlled and specific manner. This capability is crucial for producing a wide range of chemicals, from pharmaceuticals to fine chemicals, with high precision and minimal waste.

To achieve selective oxyfunctionalisation, enzymes stand out as promising catalysts, particularly monooxygenases, which are among the most potent due to their ability to catalyze reactions with exceptional specificity and efficiency. However, the industrial application of monooxygenases is limited by two major hurdles: the complexity of their electron transport chains and the inherent "oxygen dilemma" associated with their function. These challenges lead to inefficient catalysis and increase the complexity of implementing monooxygenases in large-scale chemical processes.

The PeroxyZymes project aims to overcome these obstacles by transforming monooxygenases into peroxygenases. By engineering these enzymes, we can bypass the need for complex electron transport chains and eliminate the oxygen dilemma, allowing for a more direct and efficient method of oxygenation. This innovation has the potential to unlock the use of these biocatalysts on an industrial scale, providing a sustainable alternative to conventional chemical synthesis methods.

The expected impact of the PeroxyZymes project is significant. By enabling more efficient and selective oxyfunctionalisation, this research will contribute to reducing the environmental footprint of chemical manufacturing, aligning with green chemistry principles. Moreover, the advances made in enzyme engineering will open new possibilities for the sustainable production of complex molecules, driving innovation in industries ranging from pharmaceuticals to agrochemicals. The broader implication of this work is the development of greener, safer industrial processes that reduce the reliance on toxic reagents and harsh reaction conditions.

In projects like PeroxyZymes, the integration of social sciences and humanities plays an important role in assessing the societal and ethical implications of adopting these biotechnological innovations. Understanding the societal acceptance of enzyme-based technologies and addressing concerns related to sustainability and ethical manufacturing practices will ensure that the outcomes of the project are not only technically viable but also socially responsible and aligned with broader societal values. By integrating these perspectives, the project aims to foster a more holistic approach to environmental innovation, ensuring its benefits are widely recognized and accepted.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Engineering of Heme- and Non-Heme Monooxygenases:
Significant effort was devoted to transforming monooxygenases into functional peroxygenases by reengineering their catalytic mechanisms. For heme-based monooxygenases, modifications were made to alter their active sites, enabling them to utilize hydrogen peroxide directly as an oxygen donor, bypassing the need for external electron donors or complex electron transport chains. Similarly, non-heme monooxygenases were engineered to enhance their oxidative capabilities and to reduce reliance on oxygen activation mechanisms.

Engineering of Unspecific Peroxygenases (UPOs):
Building on the success of naturally occurring UPOs, the project further optimized existing UPOs through protein engineering. This work aimed to enhance their catalytic efficiency, stability under industrial conditions, and substrate scope. These improvements were achieved through site-directed mutagenesis and directed evolution approaches, leading to UPO variants with higher activity, better selectivity, and improved tolerance to operational conditions such as high hydrogen peroxide concentrations.

Demonstrating Practical Applicability:
The project placed a strong emphasis on demonstrating the practical utility of engineered peroxygenases in real-world applications. This involved integrating the developed enzymes into industrially relevant oxyfunctionalisation reactions, proving their ability to function efficiently at scale. Several case studies were carried out, including the synthesis of pharmaceutical intermediates and fine chemicals, showing that these enzymes could perform selective oxygenation reactions with high yield and specificity.

Expanding Product Scope:
To broaden the range of potential industrial applications, the team expanded the substrate and product scope of the engineered peroxygenases. This included identifying new target molecules for oxyfunctionalisation and optimizing enzyme-substrate interactions to ensure high activity across a wider range of chemical compounds. The successful expansion of the substrate scope highlights the versatility of peroxygenases for different chemical transformations, from simple molecules to complex, multifunctional substrates.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Selective oxyfunctionalizations hold significant potential to make the chemical industry more sustainable. The current state-of-the-art in biocatalysis relies largely on cofactor-dependent monooxygenases, which have limited feasibility for industrial application in producing low to moderate-value products. The PeroxyZymes project addresses this limitation by developing simple H2O2-dependent enzymes.

One approach focuses on advancing known peroxygenases into viable industrial catalysts through optimization of reaction conditions. Another strategy involves repurposing monooxygenases to function with H2O2 as a co-substrate.

Monooxygenases to be converted into PeroxyZymes