A New Phenolic Acid Decarboxylase from the Brown-Rot Fungus Neolentinus lepideus Natively Decarboxylates Biosourced Sinapic Acid into Canolol, a Bioactive Phenolic Compound

Elise Odinot; Alexandra Bisotto-Mignot; Toinou Frezouls; Bastien Bissaro; David Navarro; Eric Record; Frédéric Cadoret; Annick Doan; Didier Chevret; Frédéric Fine; Anne Lomascolo

doi:10.3390/bioengineering11020181

A New Phenolic Acid Decarboxylase from the Brown-Rot Fungus Neolentinus lepideus Natively Decarboxylates Biosourced Sinapic Acid into Canolol, a Bioactive Phenolic Compound

Bioengineering (Basel). 2024 Feb 14;11(2):181. doi: 10.3390/bioengineering11020181.

Affiliations

¹ OléoInnov, 19 Rue du Musée, F-13001 Marseille, France.
² INRAE, Aix-Marseille Université, UMR1163 BBF Fungal Biodiversity and Biotechnology, 163 Avenue de Luminy, F-13009 Marseille, France.
³ INRAE, UMR1319 MICALIS Institute, PAPPSO, Domaine de Vilvert, F-78350 Jouy-en-Josas, France.
⁴ TERRES INOVIA, Parc Industriel, 11 Rue Monge, F-33600 Pessac, France.

Abstract

Rapeseed meal (RSM) is a cheap, abundant and renewable feedstock, whose biorefinery is a current challenge for the sustainability of the oilseed sector. RSM is rich in sinapic acid (SA), a p-hydroxycinnamic acid that can be decarboxylated into canolol (2,6-dimethoxy-4-vinylphenol), a valuable bioactive compound. Microbial phenolic acid decarboxylases (PADs), mainly described for the non-oxidative decarboxylation of ferulic and p-coumaric acids, remain very poorly documented to date, for SA decarboxylation. The species Neolentinus lepideus has previously been shown to biotransform SA into canolol in vivo, but the enzyme responsible for bioconversion of the acid has never been characterized. In this study, we purified and characterized a new PAD from the canolol-overproducing strain N. lepideus BRFM15. Proteomic analysis highlighted a sole PAD-type protein sequence in the intracellular proteome of the strain. The native enzyme (NlePAD) displayed an unusual outstanding activity for decarboxylating SA (V_max of 600 U.mg^-1, k_cat of 6.3 s^-1 and k_cat/K_M of 1.6 s^-1.mM^-1). We showed that NlePAD (a homodimer of 2 × 22 kDa) is fully active in a pH range of 5.5-7.5 and a temperature range of 30-55 °C, with optima of pH 6-6.5 and 37-45 °C, and is highly stable at 4 °C and pH 6-8. Relative ratios of specific activities on ferulic, sinapic, p-coumaric and caffeic acids, respectively, were 100:24.9:13.4:3.9. The enzyme demonstrated in vitro effectiveness as a biocatalyst for the synthesis of canolol in aqueous medium from commercial SA, with a molar yield of 92%. Then, we developed processes to biotransform naturally-occurring SA from RSM into canolol by combining the complementary potentialities of an Aspergillus niger feruloyl esterase type-A, which is able to release free SA from the raw meal by hydrolyzing its conjugated forms, and NlePAD, in aqueous medium and mild conditions. NlePAD decarboxylation of biobased SA led to an overall yield of 1.6-3.8 mg canolol per gram of initial meal. Besides being the first characterization of a fungal PAD able to decarboxylate SA, this report shows that NlePAD is very promising as new biotechnological tool to generate biobased vinylphenols of industrial interest (especially canolol) as valuable platform chemicals for health, nutrition, cosmetics and green chemistry.

Keywords: 4-vinylguaiacol; Neolentinus lepideus; biorefinery process; canolol; ferulic acid; phenolic acid decarboxylase; rapeseed meal; sinapic acid.

Grants and funding

This work was initially funded by the Technical Centre for Oilseed Crops, Grain Legumes, and Industrial Hemp (TERRES INOVIA, Pessac, France) and the Inter-Branch Organization for Vegetable Oils and Proteins (TERRES UNIVIA, Paris, France) in the framework of the Oléochampi4 project, and further funded by the OléoInnov company in the framework of the Ecocanolol project.