Plant natural products comprise a wide group of heterogeneous compounds with potent biological activities. The 2-pyrone moiety (i.e. six-membered unsaturated lactone) is present in many valuable compounds covering a broad spectrum of biological activities such as antimicrobial (Schinkovitz et al., 2003, Fairlamb et al., 2004, Ismaiel and Ali, 2017), antitumor (Suzuki et al., 1997, Kondoh et al., 1998), antiparasitic (Tempone et al., 2017), antioxidant (Zhao et al., 2017), or anti-inflammatory properties (Witaicenis et al., 2014, Liaw et al., 2015). 2-Pyrones contain an important lead core, i.e. they are privileged moieties for bioactivity, including specifically biocides such as fungicides and anti-oomycotics (Altomare et al., 2000, Igarashi et al., 2000, Koskela et al., 2011, Altomare et al., 2004, Chattapadhyay and Dureja, 2006). Among plant pathogens, fungi have a prominent impact on yields, both qualitatively and quantitatively (Oerke, 2006). The substantial plant damage by invasive fungal infections can be reduced by protective measures such as the application of fungicidal agents. Synthetic agents have been very successful for plant disease control (Russell, 2006). However, considering the alarming rate of the development of the (multiple) resistance to systemic fungicides and the lack of anti-oomycotics, a more detailed research into new bioactive compounds is necessary (Oerke, 2006, Kretschmer et al., 2006). Especially natural or nature-inspired substances are in demand as they combine higher acceptance by both consumers and authorities (Otto et al., 2016, Bette et al., 2015).

Plant biogenic routes leading to 2-pyrones proceed through the common phenylpropanoid pathway using coenzyme A (CoA)-ligated substrates (Kai et al., 2006, Vogt, 2010). Particularly, a group of type III polyketide synthases known as 2-pyrone synthases (2PS) are specific for the biosynthesis of various 2-pyrones (Scheme 1). Such enzymes perform complex sequential reactions involving two decarboxylative condensations of acetate units derived from malonyl-CoA with the starter substrate followed by intramolecular cyclization of the linear polyketide to e.g. 4-hydroxy-6-methyl-2-pyrone (if acetyl-CoA is used as a starter unit) (Helariutta et al., 1995). The latter compound is an important precursor for the fungicides gerberin and parasorboside from the ornamental plant Gerbera x hybrida (Koskela et al., 2011; Eckermann et al., 1998).

The common biosynthesis of benzo-2-pyrones (i.e. coumarins) in plants occurs through ortho-hydroxylation of the benzene ring of cinnamoyl-CoAs (Scheme 1) catalyzed by 2-oxoglutarate dependent dioxygenases (2OGD) (Kai et al., 2008). In Arabidopsis thaliana, two isoforms of 2OGD feruloyl-CoA 6’-hydroxylases called AtF6’H1 and AtF6’H2 are known (Kai et al., 2008). So far, in vitro studies on the functional characterization of these enzymes were mainly focused on a few selected substrates (i.e. feruloyl-CoA (5), cinnamoyl-CoA (7), 4-coumaroyl-CoA (8) and caffeoyl-CoA (11) in Fig. 1A). The 2OGD superfamily represents important non-heme metabolic enzymes involved in various oxidative reactions (Islam et al., 2018). In these reactions, 2-oxoglutarate and molecular oxygen act as co-substrates, and Fe2+ is the catalytic metal ion for the oxidation of a substrate, producing succinate and carbon dioxide as by-products. The ortho-hydroxylation of cinnamoyl-CoA substrates is coupled with trans-cis isomerization followed by lactonization yielding the coumarin core structure (cf. scopoletin (6) in Scheme 1). The latter reactions occur partially spontaneously but are also catalyzed by coumarin synthase (COSY) in plant cells (Vanholme et al., 2019).

Previously it was described that a remarkably diverse range of phenolic compounds could be generated in vitro by application of promiscuous plant enzymes (Kim et al., 2009, Dippe et al., 2019). Considering complications associated with the isolation of 2-pyrones from natural sources (McGlacken and Fairlamb, 2005) or multistep chemical synthesis in organic solvents commonly involving halogenated reagents and protective groups (Fairlamb et al., 2004, Lee, 2015, Zhang et al., 2007, Brandenburg et al., 2020, Douglas et al., 1999), the present study aimed to develop a generic framework for the environmentally friendly production of natural but also new-to-nature 2-pyrone analogues by utilizing the catalytic flexibility of 2PS and 2OGD enzymes. Therefore, we studied the in vitro activity of four recombinant plant enzymes using a library of twenty acyl-CoA thioesters.