Modulating Fatty Acid Epoxidation vs Hydroxylation by Peroxygenase Enzyme

Researchers from the Group of Biotechnology for Lignocellulosic Biomass at the CIB, in collaboration with the Group of Lignocellulosic Materials of Industrial Interest (IRNAS, CSIC, Seville), have been able to modulate for the first time the catalytic activity of a fungal peroxygenase by rational design, using an Escherichia coli expression system developed by the group. The results of this work have been published in ACS Catalysis and were obtained in the frame of the SusBind (https://susbind.eu) European project, whose objective is to substitute petrol-based and potentially toxic binder constituents by biobased compounds in the manufacture of boards and furniture (in collaboration with IKEA, among other partners).

Unspecific peroxygenases (UPOs) are fungal secreted counterparts of the cytochrome P450 monooxygenases present in most living cells. Although phylogenetically unrelated, both enzyme types are able to perform selective oxygenation reactions. However, peroxygenases present several key advantages over P450s for biotechnological applications, being much more stable and requiring only cheap peroxide as electron acceptor and oxygen source, in contrast to whole-cell reaction setups necessary with P450s.

Hydroxylation and epoxidation are among the main reactions catalyzed by UPOs. Recently, some fungal UPOs, such as the enzyme produced by the basidiomycete Marasmius rotula (MroUPO), have been found to epoxidize unsaturated lipids, a reaction that could be the alternative of choice in the production of industrial fatty acid epoxides, of interest for the production of a variety of chemicals and intermediates, including adhesive and binder components, among others.

In this work, the authors heterologously expressed MroUPO as an active enzyme in E. coli. Then, guided by computational simulations of substrate diffusion and binding at the crystal-structure active site, they were able to engineer rMroUPO to tune its epoxidizing vs hydroxylating activities on mono- and polyunsaturated (18-carbon) fatty acids, an important aspect to produce reactive compounds of industrial interest.

The results open the door for further protein engineering studies on other reactions of interest catalyzed by this and other fungal peroxygenases comprising an outstanding family of promising biocatalysts.

SusBind is funded by BBI-JU (www.bbi-europe.eu) under grant agreement nº 792063; the JU receives funding from H2020 and the Biobased Industries Consortium.

Reference: Modulating Fatty Acid Epoxidation vs Hydroxylation in a Fungal Peroxygenase. Carro J, González-Benjumea A, Fernández-Fueyo E, Aranda C, Guallar V, Gutiérrez A, Martínez AT [2019] . ACS Catal. 9: 6234-6242. https://pubs.acs.org/doi/10.1021/acscatal.9b01454