CYP98A22, a phenolic ester 3’-hydroxylase specialized in the synthesis of chlorogenic acid, as a new tool for enhancing the furanocoumarin concentration in Ruta graveolens

Abstract

BackgroundFuranocoumarins are molecules with proven therapeutic properties and are produced in only a small number of medicinal plant species such as Ruta graveolens. In vivo, these molecules play a protective role against phytophageous insect attack. Furanocoumarins are members of the phenylpropanoids family, and their biosynthetic pathway is initiated from p-coumaroyl coA. The enzymes belonging to the CYP98A cytochrome P450 family have been widely described as being aromatic meta-hydroxylases of various substrates, such as p-coumaroyl ester derivatives, and are involved in the synthesis of coumarins such as scopoletin. In furanocoumarin-producing plants, these enzymes catalyze the step directly downstream of the junction with the furanocoumarin biosynthetic pathway and might indirectly impact their synthesis.ResultsIn this work, we describe the cloning and functional characterization of the first CYP98A encoding gene isolated from R. graveolens. Using Nicotiana benthamiana as a heterologous expression system, we have demonstrated that this enzyme adds a 3-OH to p-coumaroyl ester derivatives but is more efficient to convert p-coumaroyl quinate into chlorogenic acid than to metabolize p-coumaroyl shikimate. Plants exposed to UV-B stress showed an enhanced expression level of the corresponding gene. The R. graveolens cyp98a22 open reading frame and the orthologous Arabidopsis thaliana cyp98a3 open reading frame were overexpressed in stable transgenic Ruta plants. Both plant series were analyzed for their production of scopoletin and furanocoumarin. A detailed analysis indicates that both genes enhance the production of furanocoumarins but that CYP98A22, unlike CYP98A3, doesn’t affect the synthesis of scopoletin.ConclusionsThe overexpression of CYP98A22 positively impacts the concentration of furanocoumarins in R. graveolens. This gene is therefore a valuable tool to engineer plants with improved therapeutical values that might also be more resistant to phytophageous insects.

A coumarin‐specific prenyltransferase catalyzes the crucial biosynthetic reaction for furanocoumarin formation in parsley

“…To solve this problem, N. benthamiana leaves were infiltrated with a mixture of Agrobacterium tumefaciens LBA4404 transformed with pBIN–PcPT and A. tumefaciens C5851 containing pBIN61–P19, to express parsley PT, as described by Karamat et al . (). Three days post‐infiltration, the leaves were further infiltrated with a mixture of 18 coumarins/furanocoumarins (Figure S2), which provided an in vivo screening method to detect prenylation of these coumarin molecules.…”

“…Nicotiana benthamiana plants were used for infiltration experiments as described by Voinnet et al . (), with some modifications (Karamat et al ., ). Bacterial strains were individually plated on YEB medium (5.0 g L −1 sucrose, 5.0 g L −1 peptone, 5.0 g L −1 beef extract, 1.0 g L −1 yeast extract, 0.049 g L −1 MgSO 4 ·7 H 2 O, 10 g L −1 agar, pH adjusted to 7.2) in the presence of antibiotics (100 mg L −1 rifampicine and 50 mg L −1 kanamycine), and incubated at 30°C for 2 days.…”

Cloning and Characterization ofLonicera japonicap-Coumaroyl Ester 3-Hydroxylase Which Is Involved in the Biosynthesis of Chlorogenic Acid

“…Its kinetics favored the metabolism of shikimate ester rather than that of quinate ester, reminiscent of the behavior of CYP98A3 of A. thaliana 18) and CYP98A49 of globe artichoke. 19) CYP98A35 (of coffee), on the other hand, is capable of metabolizing p-coumaroylquinate and p-coumaroylshikimate at the same level of efficiency, 4) while CYP98A22 of R. graveolens 20) and a carrot enzyme prefer the quinate ester as substrate. 21) Some CYP98 genes are expressed constitutively, while others, particularly those involved in the stress response are inducible.…”

From Bioreactor to Entire Plants

“…Concerning field production, plant breeding and agronomical practises (fertilization, crop protection) can be optimized also but environmental culture conditions cannot be highly easily modulated. Overexpression of transgenes (Gravot et al, 2004;Karamat et al, 2012;Larbat et al, 2007;Lièvre et al, 2005), multiple expression of transgenes (Bourgaud et al, 2006;Naqvi et al, 2010), gene silencing presenting an emphasis on RNA interference, transcription factors as a powerful tool for the engineering of biosynthetic pathways, the importance of novel gene promoters and optimization, together with the involvement of compartmentalization and transport are all various ways for modulating SMs synthesis (Zarate, 2010). In non-axenic conditions, growth improvement can be obtained using viable bacteria such as PGPR that may lead to faster growth and sometimes higher SM levels in the plant biomass ( Van de Mortel et al, 2012).…”