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 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 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 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--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 layer was separated by tal of 60 cytochrome P450 candidate genes. The P450 candidates a glass separation funnel from the yeast cultures, diluted 3-fold were amplified from the C. nootkatensis 30-RACE cDNA library in ethyl acetate, dried using anhydrous Na2SO4 and used for [10] using a specific forward primer at the start codon region con- GC–MS analysis. taining a NotI restriction site (forward primer for the valencene This led to the identification of the C. nootkatensis valencene oxidase: SC077_ATGnot: 50-tagcggccgcATGGACATGAGCACAATAT oxidase (CnVO). The full length CnVO reading frame was analysed K. Cankar et al. / FEBS Letters 588 (2014) 1001–1007 1003 by DETT sequencing (GE healthcare) and was deposited in the Gen- concentration of these was calculated from the to- Bank database under accession number JX518290. The sequence tal ion current (TIC) chromatogram peak area by comparison to a was deposited in the cytochrome P450 classification database standard curve prepared by measuring a dilution series of authen- [33] and was assigned the name CYP706M1. tic standards with a known concentration.

2.3. Phylogenetic analysis 3. Results

The deduced protein sequence of CnVO was compared to other 3.1. Identification of the C. nootkatensis valencene oxidase (+)-valencene oxidising plant cytochrome P450s from family CYP71 (C. intybus CYP71AV8, H. muticus CYP71D55 and N. tabacum A cDNA sequence library of C. nootkatensis heartwood contain- CYP71D51v2), CYP706B1 from G. arboreum and uncharacterised ing sequence fragments of 34,700 contigs [10] was screened for members of CYP706 family encoded in the genomes of Vitis vinif- cytochrome P450 genes. In total 108 contigs were found that era, Arabidopsis thaliana, Oryza sativa and Populus trichocarpa, showed homology to the cytochrome P450 family. The contig which were retrieved from the Cytochrome P450 Homepage length varied between 105 and 1717 bp, with an average of (http://drnelson.uthsc.edu/CytochromeP450.html). The sequence 412 bp. A homology search did not yield candidates with deduced of A. thaliana CYP85A1, a brassinosteroid-6-oxidase, was used as amino-acid sequence identity above 40% to the previously charac- an outgroup since it belongs to clan 85 of plant cytochrome P450 terised terpene oxidases [15,22,24–27]. The position of the start enzymes [34]. Multiple protein sequence alignments were per- codon from the open reading frames tagged by each of these 108 formed using the ClustalW algorithm using ClustalX 2.1 software contigs was determined by a 50RACE strategy. After connecting [35]. Bootstrap N-J trees were generated by the ClustalX 2.1 soft- overlapping contigs, 60 cytochrome P450 candidate genes were ware with a 1000 replicates of bootstrap analysis and exported cloned into a yeast expression vector [28]. as a Phyllip format tree. The phylogeny was visualized using the The activity of the enzymes of the C. nootkatensis P450 collec- Figtree v1.3.1. software. tion on (+)-valencene was assessed in yeast. Each P450 enzyme was co-expressed with the valencene synthase from C. nootkatensis 2.4. In vivo production of (+)-nootkatone (CnVS) [10]. Clones were expressed for 3 days in a biphasic med- ium, using in situ extraction of sesquiterpenes by a layer of n-dode- Flask fermentation with the WAT11 yeast strain containing cane, to prevent possible toxic effects of produced trans-nootkatol CnVS and CnVO was repeated in triplicates, however the n-dode- or (+)-nootkatone on yeast [16]. Presence of (+)-valencene and oxi- cane layer was omitted. After a three-day fermentation 50 mL of dized (+)-valencene products was monitored by GC–MS analysis of yeast culture was extracted with 25 mL of ethyl acetate. 15 mL of the n-dodecane layer. One positive yeast strain was identified, for ethyl acetate was retrieved from the yeast culture by glass funnel which two novel oxidised products were detected in the n-dode- separation and evaporated under a nitrogen flow to reduce the vol- cane layer (Fig. 2). The major product was identified as trans-noo- ume to 2.5 mL. The samples were dried using anhydrous Na2SO4 tkatol and the minor product was identified as (+)-nootkatone by and analysed by GC–MS. comparison to authentic standards. This enzyme was named C. nootkatensis valencene oxidase, CnVO. The protein sequence of 2.5. Microsome preparation and in vitro enzyme assay the CnVO was analysed and compared to sequences of other char- acterised sesquiterpene and monoterpene oxidases. The sequence pYEDP60 plasmid containing CnVO was transformed into showed low amino-acid identity with previously characterised WAT11 strain and colonies were selected on SD medium omitting plant cytochrome P450s that oxidise (+)-valencene. Amino acid- adenine sulphate and uracil for auxotrophic selection. Microsomes identity to C. intybus valencene oxidase CYP71AV8, N. tabacum val- from yeast cultures were prepared as previously published [28,36]. encene oxidase CYP71D51v2 and H. muticus premnaspirodiene Microsomal preparations from WAT11 cultures containing empty oxygenase CYP71D55 was found to be 28%, 29% and 30%, pYEDP60 plasmid were used as negative controls. Enzyme assays respectively (Supplemental Fig. 1). The highest level of amino acid were conducted in a total volume of 500 lL, containing 40 mM KPi buffer (pH = 7.5), 200 lM (+)-valencene (Fluka), 2% DMSO and 80 lL of microsomal preparation in a 1.5 mL glass vial. The enzymatic reaction was started by the addition of 2 mM NADPH. The reactions were incubated for 2.5 h at 25 °C at 250 rpm. Terp- enes were extracted with 1.5 mL of ethyl acetate and three-fold concentrated under nitrogen flow. The samples were dried using anhydrous Na2SO4 and used for GC–MS analysis.

2.6. GC–MS analysis

Analytes from 1 lL samples were separated using a gas chro- matograph (5890 series II, Hewlett-Packard) equipped with a 30 m 0.25 mm, 0.25 mm film thickness column (ZB-5, Phenome- nex) using helium as carrier gas at a flow rate of 1 mL/min. The injector was used in splitless mode with the inlet temperature set to 250 °C. The initial oven temperature of 45 °C was increased Fig. 2. Production of trans-nootkatol and (+)-nootkatone in the n-dodecane layer by after 1 min to 300 °C at a rate of 10 °C/min and held for 5 min at CnVO. GC chromatogram of the n-dodecane layer from yeast fermentations using 300 °C. The GC was coupled to a mass-selective detector (model WAT11 yeast strains expressing the C. nootkatensis valencene synthase (CnVS) and 5972A, Hewlett-Packard). Compounds were identified by compari- both the C. nootkatensis valencene synthase and valencene oxidase (CnVS/CnVO) is compared to the empty vector control. Trans-nootkatol was found as a predominant son of mass spectra and retention times with those of the authentic oxidation product of (+)-valencene in the n-dodecane overlay of strain CnVS/CnVO standards of (+)-nootkatone (Fluka), trans-nootkatol (Isobionics), (peak 1). (+)-Nootkatone was formed as a minor product (peak 2). The mass cis-nootkatol (Isobionics) and (+)-valencene (Fluka). The absolute spectrum of trans-nootkatol is shown in the insert. 1004 K. Cankar et al. / FEBS Letters 588 (2014) 1001–1007 identity (40%) was found with the (+)-delta-cadinene-8-hydroxy- The activity of CnVO on other sesquiterpene substrates was lase from G. arboreum, which belongs to cytochrome P450 family tested in yeast strains expressing amorphadiene synthase from CYP706. CnVO was classified into a new cytochrome P450 subfam- Artemisia annua [30] or germacrene A synthase from chicory [29]. ily as CYP706M1. Analysis of the n-dodecane layer learned that amorphadiene was not converted by CnVO. Germacrene A was partially converted into 3.2. In vivo production of (+)-nootkatone and activity of CnVO on a product with m/z = 220. This product did not match germacra- different sesquiterpene substrates 1(10),4,11(13)-trien-12-ol or germacra-1(10),4,11 (13)-trien-12- al, as deduced from comparison to authentic standards. These are CnVO was further examined for (+)-nootkatone formation. To the oxidation products of the germacrene A oxidases that hydrox- this end, flask fermentation of WAT 11 yeast expressing the valen- ylate germacrene A on the 12-position [17,37]. This indicates that cene synthase CnVS in combination with CnVO was performed in CnVO does not oxidise germacrene A in position 12 and therefore the absence of a n-dodecane layer. The produced were ex- has regio-selectivity on germacrene A that differs from germacrene tracted using ethyl acetate after the fermentation was finished. No A oxidases. sesquiterpenes were detected in the ethyl acetate extract of the WAT11 strain containing an empty plasmid, and also not in the 3.3. CnVO produces (+)-nootkatone from (+)-valencene in vitro culture expressing solely the valencene synthase, indicating that all produced (+)-valencene had evaporated from the culture. In In order to further characterize the product spectrum of CnVO, the ethylacetate extract of the CnVS/CnVO yeast culture, (+)-nootk- microsomal preparations were produced from yeast expressing atone was detected (Fig. 3), at a concentration of 144 ± 10 lg/L CnVO and WAT11-empty vector control yeast cultures. In vitro yeast culture (Table 1). activity was tested by adding (+)-valencene to yield a total

Fig. 3. Detection of (+)-valencene oxidation products in the ethyl acetate extract of yeast cultures. (A) GC chromatograms of ethyl acetate extracts of WAT11 yeast cultures expressing C. nootkatensis valencene synthase (CnVS) or co-expressing the valencene synthase with C. nootkatensis valencene oxidase (CnVS/CnVO) are compared to the empty vector control. (+)-Nootkatone (peak 2) was found to be the predominant product upon expression of CnVO. In addition, an oxidation product of germacrene A (peak 1), a side product of valencene synthase, was detected. (B) Comparison of the mass spectrum of (+)-nootkatone produced by the CnVO and the authentic standard of (+)- nootkatone (Fluka).

Table 1 Quantification of sesquiterpenes produced by CnVO in yeast fermentations.

Expressed genes (+)-Valencene trans-Nootkatol (+)-Nootkatone (lg/L yeast culture) (lg/L yeast culture) (lg/L yeast culture) In situ extraction with n-dodecane CnVO + CnVS 1305 ± 117 116 ± 44 3 ± 1 CnVS 1266 ± 24 ND ND Ethyl acetate extraction after fermentation CnVO + CnVS ND ND 144 ± 10 CnVS ND ND ND

CnVO = C. nootkatensis valencene oxidase, CnVS = C. nootkatensis valencene synthase, ND = not detected. K. Cankar et al. / FEBS Letters 588 (2014) 1001–1007 1005

thesis of sesquiterpenoid gossypol and catalyses the hydroxylation of (+)-delta-cadinene to 8-hydroxy-(+)-delta-cadinene. Interest- ingly, the other plant P450s with valencene oxidase activity e.g. C. intybus valencene oxidase CYP71AV8, H. muticus premnaspirodi- ene oxygenase CYP71D55 and N. tabacum valencene oxidase CYP71D51v2 belong to another cytochrome P450 family, CYP71. Members of the family CYP71 have been shown to be involved in terpene biosynthesis several times, and relevant members of this Fig. 4. Oxidation of (+)-valencene by CnVO in vitro. The oxidation products of (+)- family have been identified by homology screening methods valencene are shown for enzyme reactions employing microsomes of WAT11 yeast [16,17,37]. Genomes of plants may contain several members of strain expressing C. nootkatensis valencene oxidase (CnVO), compared to the the CYP706 family, for example in the Arabidopsis and grapevine microsomes prepared from the control yeast strain transformed with an empty genomes 7 and 9 cyp706 genes were identified, respectively [34]. pYEDP60 plasmid (ctrl). Formation of trans-nootkatol (peak 1) and (+)-nootkatone (peak 2) was detected. None of these have been functionally characterised. It would be of interest to explore their function, especially with respect to ter- concentration of 200 lM. The actual dissolved concentration of (+)- pene biosynthesis. valencene in these assays was as low as 0.5–1.0 lM (in 40 mM KPi Substrate specificity of terpene oxidases can be high, as shown buffer containing 2% DMSO) determined by measuring turbidity in for the A. annua amorphadiene oxidase [22,37]. On the other hand, the UV-VIS spectrum caused by undissolved (+)-valencene. In addi- the germacrene A oxidases from the Asteraceae family were shown tion to this the solubility was measured by putting empty dialysis to be able to oxidise several sesquiterpenes, namely amorphadiene, tubing in 200 lM (+)-valencene solutions. Once (+)-valencene is germacrene A and (+)-valencene [17,37]. CnVO showed a limited consumed, new (+)-valencene will dissolve. For this reason, assays substrate promiscuity, and did not oxidise amorphadiene, but were foremost qualitative. In microsomal assays with (+)-valen- showed a partial oxidation of germacrene A. cene and CnVO microsomes, (+)-nootkatone was found as a pre- The activity in yeast and in vitro indicates that CYP706M1 cata- dominant peak (82% of total products), in addition to a minor lyzes both the conversion from (+)-valencene to trans-nootkatol, peak of trans-nootkatol (18%) (Fig. 4). and the conversion from nootkatol to (+)-nootkatone. Multi-step oxidation reactions were previously shown for sesquiterpene oxi- 4. Discussion dases. Amorphadiene oxidase from A. annua CYP71AV1 catalyses the oxidation of amorphadiene to artemisinic alcohol, aldehyde The primary significance of the present work is the identifica- and acid [22]. Similarly, germacrene A oxidases from different spe- tion of a valencene oxidase from the (+)-nootkatone producing tree cies of the Asteraceae family convert germacrene A through an C. nootkatensis. Based on the protein sequence CnVO belongs to the alcohol and aldehyde to germacrene A acid [37]. CnVO catalyses cytochrome p450 class of CYP706 and is the first member of a new complete oxidation of (+)-valencene to (+)-nootkatone while C. cytochrome P450 subfamily CYP706M (Fig. 5). One enzyme of the intybus valencene oxidase CYP71AV8, H. muticus premnaspirodiene CYP706 family has been functionally characterised previously, oxygenase CYP71D55 and N. tabacum valencene oxidase namely the CYP706B1, (+)-delta-cadinene-8-hydroxylase from G. CYP71D51v2, form predominantly trans-nootkatol [15–17]. The arboreum [24]. This enzyme is involved in the early steps of biosyn- second oxidation step may also result from unspecific alcohol

Fig. 5. Phylogenetic tree of the cytochrome CYP706 family. The protein sequence of CnVO (Cn_CYP706M1) was compared to putative members of CYP706 family encoded in the genomes of Vitis vinifera (Vv_CYP706C7, Vv_CYP706C8, Vv_CYP706G1, Vv_CYP706G2, Vv_CYP706G3, Vv_CYP706H1, Vv_CYP706J1, Vv_CYP706J3, Vv_CYP706J5), Arabidopsis thaliana (At_CYP706A1, At_CYP706A2, At_CYP706A3, At_CYP706A4, At_CYP706A5, At_CYP706A6, At_CYP706A7), Oryza sativa (Os_CYP706C1, Os_CYP706C2, Os_CYP706C3, Os_CYP706C4), Populus trichocarpa (Pt_CYP706B3, Pt_CYP706C5, Pt_CYP706C6, Pt_CYP706D1, Pt_CYP706D2) and (+)-delta-cadinene-8-hydroxylase from G. arboreum (Ga_CYP706B1). For comparison (+)-valencene oxidising plant cytochrome P450s from family CYP71 (C. intybus Ci_CYP71AV8, H. muticus Hm_CYP71D55 and N. tabacum Nt_CYP71D51v2) are displayed. The (+)-valencene oxidising enzymes are boxed. 1006 K. Cankar et al. / FEBS Letters 588 (2014) 1001–1007 dehydrogenase activity present in yeast. However, when express- [9] Sharon-Asa, L. et al. (2003) Citrus fruit flavor and aroma biosynthesis: ing the chicory valencene oxidase CYP71AV8 in the same yeast isolation, functional characterization, and developmental regulation of Cstps1, a key gene in the production of the sesquiterpene aroma compound platform only minor production of (+)-nootkatone was observed valencene. Plant J. 36, 664–674. in vivo [17]. [10] Beekwilder, J. et al. (2014) Valencene synthase from the heartwood of Nootka As illustrated here, the use of a biphasic fermentation system cypress (Callitropsis nootkatensis) for biotechnological production of valencene. Plant Biotechnol. J. 12, 174–182. may not be desirable for (+)-nootkatone production, since (+)-val- [11] Fraatz, M.A., Berger, R.G. and Zorn, H. (2009) Nootkatone-a biotechnological encene and trans-nootkatol are sequestered in the n-dodecane challenge. Appl. Microbiol. Biotechnol. 83, 35–41. layer and may thereby be unavailable for full oxidation to (+)-noo- [12] Fraatz, M.A., Riemer, S.J.L., Stober, R., Kaspera, R., Nimtz, M., Berger, R.G. and Zorn, H. (2009) A novel oxygenase from Pleurotus sapidus transforms tkatone. When using biphasic fermentation, the yield of sesquiter- valencene to nootkatone. J. Mol. Catal. B: Enzym. 61, 202–207. penes was much higher, compared to single-phase fermentation [13] Sowden, R.J., Yasmin, S., Rees, N.H., Bell, S.G. and Wong, L.L. (2005) (1.4 mg/L vs. 0.14 mg/L; Table 1). However, while in the biphasic Biotransformation of the sesquiterpene (+)-valencene by cytochrome P450cam and P450BM-3. Org. Biomol. Chem. 3, 57–64. system the majority of valencene was not oxidised and some [14] Seifert, A., Vomund, S., Grohmann, K., Kriening, S., Urlacher, V.B., Laschat, S. trans-nootkatol was observed, the monophasic system yielded al- and Pleiss, J. (2009) Rational design of a minimal and highly enriched most exclusively (+)-nootkatone as a product. Apparently, in the CYP102A1 mutant library with improved regio-, stereo- and chemoselectivity. monophasic system all (+)-valencene that was first converted to Chembiochem 10, 853–861. [15] Takahashi, S., Yeo, Y.S., Zhao, Y., O’Maille, P.E., Greenhagen, B.T., Noel, J.P., trans-nootkatol, is further processed to (+)-nootkatone by CnVO. Coates, R.M. and Chappell, J. (2007) Functional characterization of Efficient systems to produce oxidised terpenoids have been premnaspirodiene oxygenase, a cytochrome P450 catalyzing regio- and developed in yeast [23]. Upon expression of CnVO with the CnVS stereo-specific hydroxylations of diverse sesquiterpene substrates. J. Biol. Chem. 282, 31744–31754. in WAT11 yeast strain 144 ± 10 lg/L of (+)-nootkatone was pro- [16] Gavira, C., Hofer, R., Lesot, A., Lambert, F., Zucca, J. and Werck-Reichhart, D. duced after a three-day flask fermentation. Thereby a yeast (2013) Challenges and pitfalls of P450-dependent (+)-valencene bioconversion expressing CnVO in combination with a valencene synthase and by Saccharomyces cerevisiae. Metab. Eng. 18, 25–35. [17] Cankar, K., van Houwelingen, A., Bosch, D., Sonke, T., Bouwmeester, H. and ATR1 is the first microbial production system for (+)-nootkatone Beekwilder, J. (2011) A chicory cytochrome P450 mono-oxygenase CYP71AV8 from a simple carbon source. In comparison to other (+)-valencene for the oxidation of (+)-valencene. FEBS Lett. 585, 178–182. oxidizing enzymes, very little intermediates or side-products [18] Khasawneh, M.A., Xiong, Y.P., Peralta-Cruz, J. and Karchesy, J.J. (2011) Biologically important eremophilane sesquiterpenes from Alaska cedar are produced. This possibly relates to CnVO being the first heartwood essential oil and their semi-synthetic derivatives. Molecules 16, valencene oxidase isolated from a plant that naturally produces 4775–4785. (+)-nootkatone. [19] Dietrich, G., Dolan, M.C., Peralta-Cruz, J., Schmidt, J., Piesman, J., Eisen, R.J. and Karchesy, J.J. (2006) Repellent activity of fractioned compounds from Chamaecyparis nootkatensis essential oil against nymphal Ixodes scapularis (Acari: Ixodidae). J. Med. Entomol. 43, 957–961. Acknowledgements [20] Johnston, W.H., Karchesy, J.J., Constantine, G.H. and Craig, A.M. (2001) Antimicrobial activity of some Pacific Northwest woods against anaerobic Francel Verstappen is acknowledged for assistance with GC–MS bacteria and yeast. Phytother. Res. 15, 586–588. analysis. We would like to thank Dr. Cinzia Bertea for her sugges- [21] Kelsey, R.G., Hennon, P.E., Huso, M. and Karchesy, J.J. (2005) Changes in heartwood chemistry of dead yellow-cedar trees that remain standing for tions on the microsomal preparation. We thank Dr. David Nelson 80 years or more in southeast Alaska. J. Chem. Ecol. 31, 2653–2670. for cytochrome P450 family determination of CnVO. This work [22] Teoh, K.H., Polichuk, D.R., Reed, D.W., Nowak, G. and Covello, P.S. (2006) has been supported by the Dutch Ministry of Economic Affairs, Artemisia annua L. (Asteraceae) trichome-specific cDNAs reveal CYP71AV1, a cytochrome P450 with a key role in the biosynthesis of the antimalarial through ACTS IBOS Grant 053.63.322. sesquiterpene lactone artemisinin. FEBS Lett. 580, 1411–1416. [23] Ro, D.K. et al. (2006) Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440, 940–943. Appendix A. Supplementary data [24] Luo, P., Wang, Y.H., Wang, G.D., Essenberg, M. and Chen, X.Y. (2001) Molecular cloning and functional identification of (+)-delta-cadinene-8-hydroxylase, a cytochrome P450 mono-oxygenase (CYP706B1) of cotton sesquiterpene Supplementary data associated with this article can be found, in biosynthesis. Plant J. 28, 95–104. the online version, at http://dx.doi.org/10.1016/j.febslet.2014.01. [25] Ralston, L., Kwon, S.T., Schoenbeck, M., Ralston, J., Schenk, D.J., Coates, R.M. and 061. Chappell, J. (2001) Cloning, heterologous expression, and functional characterization of 5-epi-aristolochene-1,3-dihydroxylase from tobacco (Nicotiana tabacum). Arch. Biochem. Biophys. 393, 222–235. References [26] Lupien, S., Karp, F., Wildung, M. and Croteau, R. (1999) Regiospecific cytochrome P450 limonene hydroxylases from mint (Mentha) species: cDNA [1] Delrio, J.A., Ortuno, A., Garciapuig, D., Porras, I., Garcialidon, A. and Sabater, F. isolation, characterization, and functional expression of (-)-4S-limonene-3- (1992) Variations of nootkatone and valencene levels during the development hydroxylase and (-)-4S-limonene-6-hydroxylase. Arch. Biochem. Biophys. of grapefruit. J. Agric. Food Chem. 40, 1488–1490. 368, 181–192. [2] Erdtman, H. and Hirose, Y. (1962) Chemistry of natural order Cupressales. 46 [27] Collu, G., Unver, N., Peltenburg-Looman, A.M., van der Heijden, R., Verpoorte, structure of Nootkatone. Acta Chim. Scand. 16, 1311–1314. R. and Memelink, J. (2001) Geraniol 10-hydroxylase, a cytochrome P450 [3] Burdock, G.A. (2002) Fenaroli’s Handbook of Flavor Ingredients, CRC Press, enzyme involved in terpenoid indole alkaloid biosynthesis. FEBS Lett. 508, London. 215–220. [4] Haring, H.G., Boelens, H., Vanderge, A. and Rijkens, F. (1972) Olfactory studies [28] Pompon, D., Louerat, B., Bronine, A. and Urban, P. (1996) Yeast expression of on enantiomeric eremophilane sesquiterpenoids. J. Agric. Food Chem. 20, animal and plant P450s in optimized redox environments. Methods Enzymol. 1018–1021. 272, 51–64. [5] Zhu, B.C.R., Henderson, G., Chen, F., Maistrello, L. and Laine, R.A. (2001) [29] Bouwmeester, H.J., Kodde, J., Verstappen, F.W.A., Altug, I.G., de Kraker, J.W. and Nootkatone is a repellent for Formosan subterranean termite (Coptotermes Wallaart, T.E. (2002) Isolation and characterization of two germacrene A formosanus). J. Chem. Ecol. 27, 523–531. synthase cDNA clones from chicory. Plant Physiol. 129, 134–144. [6] Dolan, M.C. et al. (2009) Ability of two natural products, nootkatone and [30] Wallaart, T.E., Bouwmeester, H.J., Hille, J., Poppinga, L. and Maijers, N.C.A. carvacrol, to suppress Ixodes scapularis and Amblyomma americanum (Acari: (2001) Amorpha-4,11-diene synthase: cloning and functional expression of a Ixodidae) in a Lyme disease endemic area of New Jersey. J. Econ. Entomol. 102, key enzyme in the biosynthetic pathway of the novel antimalarial drug 2316–2324. artemisinin. Planta 212, 460–465. [7] Flor-Weiler, L.B., Behle, R.W. and Stafford, K.C. (2011) Susceptibility of four tick [31] Urban, P., Mignotte, C., Kazmaier, M., Delorme, F. and Pompon, D. (1997) species, Amblyomma americanum, Dermacentor variabilis, Ixodes scapularis, and Cloning, yeast expression, and characterization of the coupling of two Rhipicephalus sanguineus (Acari: Ixodidae), to nootkatone from essential oil of distantly related Arabidopsis thaliana NADPH-Cytochrome P450 reductases grapefruit. J. Med. Entomol. 48, 322–326. with P450 CYP73A5. J. Biol. Chem. 272, 19176–19186. [8] Panella, N.A., Dolan, M.C., Karchesy, J.J., Xiong, Y.P., Peralta-Cruz, J., [32] Gietz, R.D. and Woods, R.A. (2002) Transformation of yeast by lithium acetate/ Khasawneh, M., Montenieri, J.A. and Maupin, G.O. (2005) Use of novel single-stranded carrier DNA/polyethylene glycol method. Guide to Yeast compounds for pest control: Insecticidal and acaricidal activity of essential Genetics Mol. Cell Biol. Pt. B 350, 87–96. oil components from heartwood of Alaska yellow cedar. J. Med. Entomol. 42, [33] Nelson, D.R. (2009) The Cytochrome P450 homepage. Hum. Genomics 4, 59– 352–358. 65. K. Cankar et al. / FEBS Letters 588 (2014) 1001–1007 1007

[34] Nelson, D. and Werck-Reichhart, D. (2011) A P450-centric view of plant P450 monooxygenase: cloning, functional expression, and characterization of evolution. Plant J. 66, 194–211. the responsible gene. Arch. Biochem. Biophys. 390, 279–286. [35] Larkin, M.A. et al. (2007) Clustal W and Clustal X version 2.0. Bioinformatics [37] Nguyen, D.T., Gopfert, J.C., Ikezawa, N., MacNevin, G., Kathiresan, M., Conrad, J., 23, 2947–2948. Spring, O. and Ro, D.K. (2010) Biochemical conservation and evolution of [36] Bertea, C.M., Schalk, M., Karp, F., Maffei, M. and Croteau, R. (2001) germacrene A oxidase in Asteraceae. J. Biol. Chem. 285, 16588–16598. Demonstration that menthofuran synthase of mint (Mentha) is a cytochrome