The aim of the OxyLipids project is to produce bio-based compounds, to substitute crude-based compounds, from selective oxygenation of vegetable lipids using computationally-designed enzymes with peroxygenase activity. Therefore, OxyLipids aims to contribute to the objectives of both the green (promoting the use of renewable raw materials and clean industrial technologies) and the digital (promoting bio-intelligent industrial manufacturing) transitions, and to the synergy between both.

The project will start with data mining by the Barcelona Supercomputing Center (BSC, subproject 3) using sequenced genomes and other sources where over 4000 peroxygenase-type genes can be identified. Then, the molecular structures of representative sequences will be obtained using the AlphaFold software that will permit BSC to generate hundreds of accurate predictions in very short time. This machine learning approach (Nature 596, 583-589, 2021) represents an unprecedented advance in protein science and will allow us to overcome the scarcity of crystal structures, as a main bottleneck in computational modeling of these enzymes. The above information will permit BSC (i) to identify new peroxygenase types and "de novo" design peroxygenase enzymes from databases, and (ii) to engineer the molecular structures of peroxygenases for their lipid substrates.

In both cases, the sequences will be transferred to the CSIC Center of Biological Research "Margarita Salas" (CIB, subproject 1) for converting them into active enzymes (note that only a handful of peroxygenase enzymes are currently available). Most known peroxygenase genes are of fungal origin and their heterologous expression represents a new challenge to be addressed. Although alternatives will be also evaluated, we plan to focus on Escherichia coli expression as fusion proteins, or coexpression, with molecular chaperones or foldases, two options never assayed before for these enzymes, to obtain soluble and active peroxygenases. The same method will be used to express the new and designed sequences received from BSC, and the variants obtained by site-directed mutagenesis at CIB, which will be all characterized (in terms of activity and stability) before transferring them for experimental evaluation in selective oxygenations.

The latter studies, to be carried out at the CSIC Institute of Natural Resources and Agrobiology of Sevilla (IRNAS, subproject 2), will focus on the enzymatic epoxidation of unsaturated fatty acids from vegetable oils yielding reactive epoxides, but will also analyze their hydroxylated and dicarboxylic derivatives. Epoxidized lipids are of interest for the chemical industry, including bio-based adhesives as an alternative to formaldehyde-based adhesives, while the latter derivatives could substitute crude-based building blocks in the synthesis of plastic polymers. To attain results of industrial interest, important parameters of the peroxygenase reactions (especially substrate loading and peroxide concentration) will be optimized, and the products will be chemically characterized to exploit the desired regioselectivity and stereoselectivity of the different enzymes (a third challenge in OxyLipids).

Thanks to successive iterations in the above experimental and computational studies (i) the enzyme selectivity yielding bio-based chemicals will be progressively increased, and (ii) a digital platform for enzyme mining and engineering will be developed.

Project TED2021-129264B funded by MCIN/AEI/10.13039/501100011033 and by the European Union NextGenerationEU/PRTR.

Funded by MCIN/AEI/10.13039/501100011033 and by the European Union NextGenerationEU/PRTR