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Callitropsis Nootkatensis) FEBS Letters 588 (2014) 1001–1007 journal homepage: www.FEBSLetters.org Valencene oxidase CYP706M1 from Alaska cedar (Callitropsis nootkatensis) Katarina Cankar a,b, Adèle van Houwelingen b, Miriam Goedbloed a, Rokus Renirie c, René M. de Jong d, ⇑ Harro Bouwmeester a, Dirk Bosch b, Theo Sonke e, Jules Beekwilder b, a Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PD Wageningen, The Netherlands b Plant Research International, Wageningen University and Research Centre, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands c Division of Molecular and Computational Toxicology, Free University of Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands d DSM Biotechnology Center, Alexander Fleminglaan 1, 2613 AX Delft, The Netherlands e Isobionics, Urmonderbaan 22, 6167 RD Geleen, The Netherlands article info abstract Article history: (+)-Nootkatone is a natural sesquiterpene ketone used in grapefruit and citrus flavour compositions. Received 26 November 2013 It occurs in small amounts in grapefruit and is a major component of Alaska cedar (Callitropsis noot- Revised 21 January 2014 katensis) heartwood essential oil. Upon co-expression of candidate cytochrome P450 enzymes from Accepted 21 January 2014 Alaska cedar in yeast with a valencene synthase, a C. nootkatensis valencene oxidase (CnVO) was Available online 11 February 2014 identified to produce trans-nootkatol and (+)-nootkatone. Formation of (+)-nootkatone was Edited by Peter Brzezinski detected at 144 ± 10 lg/L yeast culture. CnVO belongs to a new subfamily of the CYP706 family of cytochrome P450 oxidases. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Sesquiterpene Cytochrome P450 Alaska cedar (+)-Valencene (+)-Nootkatone Callitropsis nootkatensis 1. Introduction Valencene synthases have been described in citrus species [9] and recently also in C. nootkatensis [10]. The enzymatic steps from (+)-Nootkatone is an important oxidized sesquiterpene, which (+)-valencene to (+)-nootkatone have not yet been described, how- is found in grapefruit flavedo [1], and was originally identified in ever a 2-step enzymatic conversion of (+)-valencene has been pro- the heartwood of the Alaska cedar, Callitropsis nootkatensis [2]. Its posed, via a regioselective allylic hydroxylation of the 2-position of flavour characteristics have been described as grapefruit, citrus, (+)-valencene to nootkatol, followed by the oxidation to (+)-nootk- orange and butter [3] and it has a low odor threshold [4], which atone (Fig. 1) [11]. Both steps could be catalysed by a single multi- renders (+)-nootkatone highly interesting for flavour and fragrance functional cytochrome P450 enzyme, or involve an oxidation step uses. The plant probably benefits from the presence of nootkatone to nootkatol by a cytochrome P450, followed by an alcohol dehy- because it is active against insects and it shows a repellent activity drogenase activity to yield (+)-nootkatone. against termites [5]. The use of nootkatone as a potent tick repel- Several microbial enzymes that catalyse the formation of either lent has also been reported [6–8]. nootkatol or/and (+)-nootkatone from (+)-valencene have been de- The biosynthesis of (+)-nootkatone in plants has not yet been scribed. Enzymatic conversion of (+)-valencene was demonstrated fully elucidated. Its first dedicated step is the formation of (+)-val- for a fungal dioxygenase from Pleurotus sapidus [12] and engi- encene from farnesyl pyrophosphate by a valencene synthase. neered bacterial cytochrome P450cam from Pseudomonas putida and P450BM3 from Bacillus megaterium [13,14]. For plants, cyto- chrome P450 enzymes from the CYP71 family were probed for Abbreviations: CnVO, Callitropsis nootkatensis valencene oxidase; CnVS, (+)-valencene oxidising activity. The premnaspirodiene oxygenase Callitropsis nootkatensis valencene synthase; EST, expressed sequence tag ⇑ Corresponding author. Address: Plant Research International, PO Box 619, 6708 CYP71D55 from henbane (Hyoscyamus muticus) was shown to cata- PD Wageningen, The Netherlands. Fax: +31 0317 418094. lyse oxidation of (+)-valencene, primarily to nootkatol in vitro [15]. E-mail address: [email protected] (J. Beekwilder). http://dx.doi.org/10.1016/j.febslet.2014.01.061 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 1002 K. Cankar et al. / FEBS Letters 588 (2014) 1001–1007 Fig. 1. Proposed biosynthetic pathway of (+)-nootkatone. In the first enzymatic reaction (+)-valencene formation from farnesyl pyrophosphate (FPP) is catalysed by a valencene synthase. The 2-step enzymatic conversion of (+)-valencene to (+)-nootkatone is proposed to proceed via a hydroxylation at the 2-position of (+)-valencene yielding trans-orcis-nootkatol intermediates, followed by the oxidation to (+)-nootkatone. CYP71D51v2 from tobacco (Nicotiana tabacum) was reported to GGTACTACTGGGT-30; NotI restriction site underlined) and a uni- oxidise (+)-valencene predominantly to trans-nootkatol [16]. Co- versal reverse primer (UPMshortPac: 50-tcttaattaaCTAATACGACTC expression of a chicory (Cichorium intybus) valencene oxidase CY- ACTATAGGGC-30; PacI restriction site underlined) using Phusion P71AV8 in yeast with valencene synthase, lead to de novo synthesis DNA polymerase (Finnzymes, Finland). The PCR conditions were as of trans-nootkatol and small quantities of (+)-nootkatone [17]. follows: initial denaturation of 45 s at 98 °C was followed by thirty However, neither henbane nor chicory nor tobacco contain PCR cycles of 10 s at 98 °C, 20 s at 68 °Cand2minat72°C and a final (+)-nootkatone. extension of 5 min at 72 °C. The final concentration of PCR reagents C. nootkatensis, Alaska cedar, also known as Nootka cypress is was 1x Phusion HF Buffer (Finnzymes), 200 lM dNTPs, 200 nM prim- native to the Pacific Northwest coast of North America. The heart- ers and 0.02 U/lL Phusion DNA polymerase (Finnzymes). wood essential oil contains carvacrol (35%), nootkatene (17%) and C. nootkatensis P450 candidates were cloned into the yeast (+)-nootkatone (20%) [18], and has been shown to have insecti- expression vector pYEDP60 [28] which was modified to contain cidal, acaricidal and antimicrobial properties [8,19,20]. The chem- NotI and PacI restriction sites at the polylinker. The C. nootkatensis ical composition of the oil remains stable for several decades in valencene synthase (CnVS) was cloned into the pYES3/CT yeast dead C. nootkatensis trees rendering the wood highly decay-resis- expression vector (Invitrogen) [10]. The cloning of the chicory ger- tant [21]. Recently we described the cloning of valencene synthase macrene A synthase [29] and A. annua amorpha-4,11-diene syn- from C. nootkatensis heartwood by a cDNA sequencing approach thase [30] into pYEDP80 and into pYES3/CT vectors was [10]. In the current study, we describe the identification of a novel described previously [17]. cytochrome P450 enzyme in C. nootkatensis heartwood that catal- yses the oxidation of (+)-valencene. This is the first description of 2.2. In vivo activity screening of C. nootkatensis P450 candidates for a valencene oxidase from a plant that naturally synthesizes (+)-valencene oxidation in yeast (+)-nootkatone. Candidate C. nootkatensis P450 enzymes were co-transformed 2. Materials and methods with CnVS into yeast strain WAT11 [31] using standard protocols [32]. The recombinant yeast colonies were selected on solid Syn- 2.1. Cloning of cytochrome P450 candidates from C. nootkatensis cDNA thetic dextrose minimal medium (SD medium: 0.67% Difco yeast library nitrogen base medium without amino acids, 2% D-glucose, 2% agar) supplemented with amino acids, but omitting L-tryptophan, ade- An expressed sequence tag (EST) database of a cDNA library nine sulphate and uracil for auxotrophic selection. The WAT11 derived from the C. nootkatensis heartwood [10] was examined yeast strain transformed with only CnVS or empty pYES3/ct and for sequences with high homology to the following terpene pYEDP60 plasmids were used as negative controls in induction oxidases: premnaspirodiene oxygenase from H. muticus [15], experiments. amorphadiene mono-oxygenase from Artemisia annua [22,23], Gene expression was induced in the Synthetic galactose mini- (+)-delta-cadinene-8-hydroxylase from Gossypium arboreum [24], mal medium (SG medium: 0.67% Difco yeast nitrogen base med- 5-epi-aristolochene-1,3-dihydroxylase form N. tabacum [25], ium without amino acids, 2% D-galactose) supplemented with (2)-4S-Limonene-3-hydroxylase from Mentha x piperita [26] and amino acids, but without L-tryptophan, adenine sulphate and uracil Catharanthus roseus geraniol 10-hydroxylase [27]. 108 contigs for auxotrophic selection. A single yeast colony was inoculated in were identified with homology to cytochrome P450 enzymes and 5 ml of SG medium and grown overnight at 30 °C at 300 rpm. RACE PCR (Clontech) strategy was used to amplify the 50-region The cultures were diluted until the optical density (OD600)of of all candidate genes. The sequence of all 108 PCR fragments 0.05 in 50 mL of SG medium and incubated at 200 rpm at 30 °C. was obtained by DETT sequencing (GE healthcare) and assembled The cultures were overlaid with 5 mL of n-dodecane at the OD600 using SeqMan Pro v.9.0.4 software (DNASTAR). Several of the 108 of 0.8 to 1 and cultivation was continued at 30 °C and 200 rpm contigs belonged to the same open reading frame, resulting in a to- for 3 days. Subsequently, the n-dodecane
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