Production of clovamide and its analogues inSaccharomyces cerevisiaeandLactococcus lactis

Abstract

Clovamide and its analogues are N‐hydroxycinnamoyl‐L‐amino acids (HAA) that exhibit antioxidant activities. For environmental and economic reasons, biological synthesis of these plant‐derived metabolites has garnered interest. In this study, we exploited HDT1, a BAHD acyltransferase recently isolated from red clover, for the production of clovamide and derivatives in S. cerevisiae and L. lactis. HDT1 catalyses the transfer of hydroxycinnamoyl‐coenzyme A (CoA) onto aromatic amino acids. Therefore, by heterologously co‐expressing HDT1 with 4‐coumarate:CoA ligase (4CL), we succeeded in the biological production of clovamide and more than 20 other HAA, including halogenated ones, upon feeding the engineered micro‐organisms with various combinations of cinnamates and amino acids. To the best of our knowledge, this is the first report on the biological synthesis of HAA and, more generally, on the synthesis of plant‐derived antioxidant phenolic compounds in L. lactis. The production of these health beneficial metabolites in Generally Recognized As Safe (GRAS) micro‐organisms such as S. cerevisiae and L. lactis provides new options for their delivery as therapeutics. Significance and Impact of the Study N‐hydroxycinnamoyl‐L‐amino acids such as clovamide are bioactive plant‐derived phenolic compounds with health beneficial effects. Relying on chemical synthesis or direct extraction from plant sources for the supply of these valuable molecules poses challenges to environmental sustainability. As an alternative route, this work demonstrates the potential for biological synthesis of N‐hydroxycinnamoyl‐L‐amino acids using engineered microbial hosts such as Saccharomyces cerevisiae and Lactococcus lactis. Besides being more eco‐friendly, this approach should also provide more structurally diverse compounds and offer new methods for their delivery to the human body.

Clovamide, a Hydroxycinnamic Acid Amide, Is a Resistance Factor Against Phytophthora spp. in Theobroma cacao

“…Furthermore, increased clovamide content in food may provide human health benefits due to its antiinflammatory and neuroprotective properties (Park et al, 2007(Park et al, , 2017Fallarini et al, 2009;Zeng et al, 2011;Kolodziejczyk-Czepas et al, 2017;Tsunoda et al, 2018). The recent cloning and characterization of a hydroxycinnamoyl-CoA:L-DOPA hydroxycinnamoyl transferases (HDT) from red clover (Trifolium pratense) capable of catalyzing the formation of clovamide from caffeoyl-CoA and L-DOPA (Sullivan and Bonawitz, 2018;Bouchez et al, 2019) means it is now conceivable to transfer clovamide biosynthesis into other crops via genetic engineering.…”

“…The isobaric monodeoxyclovamides coumaroyl-DOPA and caffeoyl-tyrosine could not be differentiated by MS/MS fragments, since two features in the LC-MS/MS dataset had all four diagnostic MS/MS fragments for both compounds (see Supplementary Data File 2). The two compounds were therefore annotated based on relative retention times for the two compounds reported by (Bouchez et al, 2019).…”

Theobromacacao Criollo var. Beans: Biological Properties and Chemical Profile

“…Compound 10 was putatively identified as N-caffeoyl 3-hydroxytyrosine (clovamide), which was previously reported as a constituent in cocoa beans and hulls [25]. According to Bouchez et al [26], the deprotonated molecular ion at m/z 358.0929, through CO-Cα' bond cleavage, gave the ions at m/z 222.0396, which could correspond to the 4-((2,5-dioxooxazolidin-4-yl)methyl)-2-hydroxyphenolate and 135.0448 (base peak). This latter, together with the fragment ions at m/z 178.0503, and 161.0237, as well as those at m/z 134.6163, and 133.0301 confirmed the caffeoyl identity of the hydroxycinnamoyl moiety ( Figure 2B).…”

Red Clover HDT, a BAHD Hydroxycinnamoyl-Coenzyme A:L-3,4-Dihydroxyphenylalanine (L-DOPA) Hydroxycinnamoyl Transferase That Synthesizes Clovamide and Other N-Hydroxycinnamoyl-Aromatic Amino Acid Amides

“…In all cases, product formation required the presence of the recombinant protein, as control extracts of E. coli transformed with pET28 lacking an HDT insert failed to form these amide products when incubated with hydroxycinnamoyl-CoA donor and acceptor substrates. In addition to the above aromatic amino acids, L-Trp and L-Leu were also tested as acceptors as it had been demonstrated that HDT1 substrate specificity might extend to these amino acids as well based on HDT1 expression in a novel yeast system (Bouchez et al, 2019). Although not all amino acids were tested as acceptors, no product was detected when arginine was used as an acceptor.…”