Kauniolide synthase is a P450 with unusual hydroxylation and cyclization-elimination activity

Abstract

Guaianolides are an important class of sesquiterpene lactones with unique biological and pharmaceutical properties. They have been postulated to be derived from germacranolides, but for years no progress has been made in the elucidation of their biosynthesis that requires an unknown cyclization mechanism. Here we demonstrate the isolation and characterization of a cytochrome P450 from feverfew (Tanacetum parthenium), kauniolide synthase. Kauniolide synthase catalyses the formation of the guaianolide kauniolide from the germacranolide substrate costunolide. Unlike most cytochrome P450s, kauniolide synthase combines stereoselective hydroxylation of costunolide at the C3 position, with water elimination, cyclization and regioselective deprotonation. This unique mechanism of action is supported by in silico modelling and docking experiments. The full kauniolide biosynthesis pathway is reconstructed in the heterologous hosts Nicotiana benthamiana and yeast, paving the way for biotechnological production of guaianolide-type sesquiterpene lactones.

Traps and Pitfalls—Unspecific Reactions in Metabolic Engineering of Sesquiterpenoid Pathways

“…One well-observed example is as the rearrangement reaction from a germacrene to a eudesmane backbone. This acid-induced rearrangement converts, for instance, germacrene A acid (10) to α-costic acid (11), β-costic acid (12), and γ-costic acid (13) [9,10,30] with the double bond positions ∆3→4 (f), ∆4→15 (g) or ∆4→5 (h). The subsequent introduction of water (i) resulting in ilicic acid (14) has also been reported [9,10], likely neutralizing a carbocationic intermediate.…”

“…In the case of a yeast expression system, culture and extraction conditions can be altered to differentiate between specific and unspecific enzyme products. For instance, Nguyen et al (2010) could show an increase of the specific enzyme product germacrene A acid (10) and a decrease of the rearrangement products costic acids and ilicic acid (11)(12)(13)(14) by comparing extracts from buffered versus unbuffered yeast cultures on a GC-MS and a LC-MS system [10]. Co-elution of acid-induced isomers on GC-MS can be overcome by the use of chiral columns which allow the separation of α-costic acid (11) and β-costic acid (12) [10].…”

“…The elucidation and metabolic engineering of their biosynthetic pathways have made significant progress in the past decades, as many sesquiterpenes are of commercial interest as fragrances [1], biodiesel [2] or pharmaceuticals [3,4]. The reconstruction of sesquiterpenoid metabolic pathways has been carried out in various prokaryotic and eukaryotic host models such as Escherichia coli [5,6], Saccharomyces cervisiae [4,[7][8][9][10][11][12], Nicotiana benthamiana [7,8,[13][14][15][16] and Physcomitrella patens [17,18]. The analysis of sesquiterpenes is usually performed by gas or liquid chromatography coupled with mass spectrometry, depending on the volatility of the compound.…”

“…In a second step, the intermediate is often modified by cytochrome P450 enzymes that introduce oxygen into the core backbone [20,21]. Cytochrome P450 enzymes have been reported to introduce hydroxy-, epoxy-, acid-and estergroups [7][8][9][10]13] and the conversion from a germacrene to a guaiane backbone [12]. One of the challenges when expressing biosynthetic enzymes of sesquiterpenoid pathways is the differentiation of the direct enzyme product and artificial products that arise from unspecific subsequent reactions.…”

Substrate promiscuity of enzymes from the sesquiterpene biosynthetic pathways from Artemisia annua and Tanacetum parthenium allows for novel combinatorial sesquiterpene production

“…TpPTS (KC954155), Tp3β-hydroxylase (KC954153) and TpKLS (MF197558) were previously cloned into the pESC-Ura vector under the control of the galactose-inducible promoter by addition of BamHI and NotI restriction sites (Liu et al, 2018(Liu et al, , 2014. Microsomal fraction isolation was performed according to the method described by Liu et al (2018).…”