Valencene oxidase CYP706M1 from Alaska cedar (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 Plant Research International, Wageningen University and Research Centre, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands 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 b c

a r t i c l e

i n f o

Article history: Received 26 November 2013 Revised 21 January 2014 Accepted 21 January 2014 Available online 11 February 2014 Edited by Peter Brzezinski Keywords: Sesquiterpene Cytochrome P450 Alaska cedar (+)-Valencene (+)-Nootkatone Callitropsis nootkatensis

a b s t r a c t (+)-Nootkatone is a natural sesquiterpene ketone used in grapefruit and citrus flavour compositions. It occurs in small amounts in grapefruit and is a major component of Alaska cedar (Callitropsis nootkatensis) heartwood essential oil. Upon co-expression of candidate cytochrome P450 enzymes from Alaska cedar in yeast with a valencene synthase, a C. nootkatensis valencene oxidase (CnVO) was identified to produce trans-nootkatol and (+)-nootkatone. Formation of (+)-nootkatone was 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.

1. Introduction (+)-Nootkatone is an important oxidized sesquiterpene, which is found in grapefruit flavedo [1], and was originally identified in the heartwood of the Alaska cedar, Callitropsis nootkatensis [2]. Its flavour characteristics have been described as grapefruit, citrus, orange and butter [3] and it has a low odor threshold [4], which renders (+)-nootkatone highly interesting for flavour and fragrance uses. The plant probably benefits from the presence of nootkatone because it is active against insects and it shows a repellent activity against termites [5]. The use of nootkatone as a potent tick repellent has also been reported [6–8]. The biosynthesis of (+)-nootkatone in plants has not yet been fully elucidated. Its first dedicated step is the formation of (+)-valencene from farnesyl pyrophosphate by a valencene synthase. Abbreviations: CnVO, Callitropsis nootkatensis valencene oxidase; CnVS, Callitropsis nootkatensis valencene synthase; EST, expressed sequence tag ⇑ Corresponding author. Address: Plant Research International, PO Box 619, 6708 PD Wageningen, The Netherlands. Fax: +31 0317 418094. E-mail address: [email protected] (J. Beekwilder).

Valencene synthases have been described in citrus species [9] and recently also in C. nootkatensis [10]. The enzymatic steps from (+)-valencene to (+)-nootkatone have not yet been described, however a 2-step enzymatic conversion of (+)-valencene has been proposed, via a regioselective allylic hydroxylation of the 2-position of (+)-valencene to nootkatol, followed by the oxidation to (+)-nootkatone (Fig. 1) [11]. Both steps could be catalysed by a single multifunctional cytochrome P450 enzyme, or involve an oxidation step to nootkatol by a cytochrome P450, followed by an alcohol dehydrogenase activity to yield (+)-nootkatone. Several microbial enzymes that catalyse the formation of either nootkatol or/and (+)-nootkatone from (+)-valencene have been described. Enzymatic conversion of (+)-valencene was demonstrated for a fungal dioxygenase from Pleurotus sapidus [12] and engineered bacterial cytochrome P450cam from Pseudomonas putida and P450BM3 from Bacillus megaterium [13,14]. For plants, cytochrome P450 enzymes from the CYP71 family were probed for (+)-valencene oxidising activity. The premnaspirodiene oxygenase CYP71D55 from henbane (Hyoscyamus muticus) was shown to catalyse oxidation of (+)-valencene, primarily to nootkatol in vitro [15].

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- or cis-nootkatol intermediates, followed by the oxidation to (+)-nootkatone.

CYP71D51v2 from tobacco (Nicotiana tabacum) was reported to oxidise (+)-valencene predominantly to trans-nootkatol [16]. Coexpression of a chicory (Cichorium intybus) valencene oxidase CYP71AV8 in yeast with valencene synthase, lead to de novo synthesis of trans-nootkatol and small quantities of (+)-nootkatone [17]. However, neither henbane nor chicory nor tobacco contain (+)-nootkatone. C. nootkatensis, Alaska cedar, also known as Nootka cypress is native to the Pacific Northwest coast of North America. The heartwood essential oil contains carvacrol (35%), nootkatene (17%) and (+)-nootkatone (20%) [18], and has been shown to have insecticidal, acaricidal and antimicrobial properties [8,19,20]. The chemical composition of the oil remains stable for several decades in dead C. nootkatensis trees rendering the wood highly decay-resistant [21]. Recently we described the cloning of valencene synthase from C. nootkatensis heartwood by a cDNA sequencing approach [10]. In the current study, we describe the identification of a novel cytochrome P450 enzyme in C. nootkatensis heartwood that catalyses the oxidation of (+)-valencene. This is the first description of a valencene oxidase from a plant that naturally synthesizes (+)-nootkatone. 2. Materials and methods 2.1. Cloning of cytochrome P450 candidates from C. nootkatensis cDNA library An expressed sequence tag (EST) database of a cDNA library derived from the C. nootkatensis heartwood [10] was examined for sequences with high homology to the following terpene oxidases: premnaspirodiene oxygenase from H. muticus [15], amorphadiene mono-oxygenase from Artemisia annua [22,23], (+)-delta-cadinene-8-hydroxylase from Gossypium arboreum [24], 5-epi-aristolochene-1,3-dihydroxylase form N. tabacum [25], (2)-4S-Limonene-3-hydroxylase from Mentha x piperita [26] and Catharanthus roseus geraniol 10-hydroxylase [27]. 108 contigs were identified with homology to cytochrome P450 enzymes and RACE PCR (Clontech) strategy was used to amplify the 50 -region of all candidate genes. The sequence of all 108 PCR fragments was obtained by DETT sequencing (GE healthcare) and assembled using SeqMan Pro v.9.0.4 software (DNASTAR). Several of the 108 contigs belonged to the same open reading frame, resulting in a total of 60 cytochrome P450 candidate genes. The P450 candidates were amplified from the C. nootkatensis 30 -RACE cDNA library [10] using a specific forward primer at the start codon region containing a NotI restriction site (forward primer for the valencene oxidase: SC077_ATGnot: 50 -tagcggccgcATGGACATGAGCACAATAT

GGTACTACTGGGT-30 ; NotI restriction site underlined) and a universal reverse primer (UPMshortPac: 50 -tcttaattaaCTAATACGACTC ACTATAGGGC-30 ; PacI restriction site underlined) using Phusion DNA polymerase (Finnzymes, Finland). The PCR conditions were as follows: initial denaturation of 45 s at 98 °C was followed by thirty PCR cycles of 10 s at 98 °C, 20 s at 68 °C and 2 min at 72 °C and a final extension of 5 min at 72 °C. The final concentration of PCR reagents was 1x Phusion HF Buffer (Finnzymes), 200 lM dNTPs, 200 nM primers and 0.02 U/lL Phusion DNA polymerase (Finnzymes). C. nootkatensis P450 candidates were cloned into the yeast expression vector pYEDP60 [28] which was modified to contain NotI and PacI restriction sites at the polylinker. The C. nootkatensis valencene synthase (CnVS) was cloned into the pYES3/CT yeast expression vector (Invitrogen) [10]. The cloning of the chicory germacrene A synthase [29] and A. annua amorpha-4,11-diene synthase [30] into pYEDP80 and into pYES3/CT vectors was described previously [17]. 2.2. In vivo activity screening of C. nootkatensis P450 candidates for (+)-valencene oxidation in yeast Candidate C. nootkatensis P450 enzymes were co-transformed with CnVS into yeast strain WAT11 [31] using standard protocols [32]. The recombinant yeast colonies were selected on solid Synthetic dextrose minimal medium (SD medium: 0.67% Difco yeast nitrogen base medium without amino acids, 2% D-glucose, 2% agar) supplemented with amino acids, but omitting L-tryptophan, adenine sulphate and uracil for auxotrophic selection. The WAT11 yeast strain transformed with only CnVS or empty pYES3/ct and pYEDP60 plasmids were used as negative controls in induction experiments. Gene expression was induced in the Synthetic galactose minimal medium (SG medium: 0.67% Difco yeast nitrogen base medium without amino acids, 2% D-galactose) supplemented with amino acids, but without L-tryptophan, adenine sulphate and uracil for auxotrophic selection. A single yeast colony was inoculated in 5 ml of SG medium and grown overnight at 30 °C at 300 rpm. The cultures were diluted until the optical density (OD600) of 0.05 in 50 mL of SG medium and incubated at 200 rpm at 30 °C. The cultures were overlaid with 5 mL of n-dodecane at the OD600 of 0.8 to 1 and cultivation was continued at 30 °C and 200 rpm for 3 days. Subsequently, the n-dodecane layer was separated by a glass separation funnel from the yeast cultures, diluted 3-fold in ethyl acetate, dried using anhydrous Na2SO4 and used for GC–MS analysis. This led to the identification of the C. nootkatensis valencene 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 GenBank database under accession number JX518290. The sequence was deposited in the cytochrome P450 classification database [33] and was assigned the name CYP706M1.

concentration of these sesquiterpenes was calculated from the total ion current (TIC) chromatogram peak area by comparison to a standard curve prepared by measuring a dilution series of authentic standards with a known concentration.

2.3. Phylogenetic analysis

3. Results

The deduced protein sequence of CnVO was compared to other (+)-valencene oxidising plant cytochrome P450s from family CYP71 (C. intybus CYP71AV8, H. muticus CYP71D55 and N. tabacum CYP71D51v2), CYP706B1 from G. arboreum and uncharacterised members of CYP706 family encoded in the genomes of Vitis vinifera, Arabidopsis thaliana, Oryza sativa and Populus trichocarpa, which were retrieved from the Cytochrome P450 Homepage (http://drnelson.uthsc.edu/CytochromeP450.html). The sequence of A. thaliana CYP85A1, a brassinosteroid-6-oxidase, was used as an outgroup since it belongs to clan 85 of plant cytochrome P450 enzymes [34]. Multiple protein sequence alignments were performed using the ClustalW algorithm using ClustalX 2.1 software [35]. Bootstrap N-J trees were generated by the ClustalX 2.1 software with a 1000 replicates of bootstrap analysis and exported as a Phyllip format tree. The phylogeny was visualized using the Figtree v1.3.1. software.

3.1. Identification of the C. nootkatensis valencene oxidase

2.4. In vivo production of (+)-nootkatone Flask fermentation with the WAT11 yeast strain containing CnVS and CnVO was repeated in triplicates, however the n-dodecane layer was omitted. After a three-day fermentation 50 mL of yeast culture was extracted with 25 mL of ethyl acetate. 15 mL of ethyl acetate was retrieved from the yeast culture by glass funnel separation and evaporated under a nitrogen flow to reduce the volume to 2.5 mL. The samples were dried using anhydrous Na2SO4 and analysed by GC–MS. 2.5. Microsome preparation and in vitro enzyme assay pYEDP60 plasmid containing CnVO was transformed into WAT11 strain and colonies were selected on SD medium omitting adenine sulphate and uracil for auxotrophic selection. Microsomes from yeast cultures were prepared as previously published [28,36]. Microsomal preparations from WAT11 cultures containing empty pYEDP60 plasmid were used as negative controls. Enzyme assays 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. Terpenes 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.

A cDNA sequence library of C. nootkatensis heartwood containing sequence fragments of 34,700 contigs [10] was screened for cytochrome P450 genes. In total 108 contigs were found that showed homology to the cytochrome P450 family. The contig length varied between 105 and 1717 bp, with an average of 412 bp. A homology search did not yield candidates with deduced amino-acid sequence identity above 40% to the previously characterised terpene oxidases [15,22,24–27]. The position of the start codon from the open reading frames tagged by each of these 108 contigs was determined by a 50 RACE strategy. After connecting overlapping contigs, 60 cytochrome P450 candidate genes were cloned into a yeast expression vector [28]. The activity of the enzymes of the C. nootkatensis P450 collection on (+)-valencene was assessed in yeast. Each P450 enzyme was co-expressed with the valencene synthase from C. nootkatensis (CnVS) [10]. Clones were expressed for 3 days in a biphasic medium, using in situ extraction of sesquiterpenes by a layer of n-dodecane, to prevent possible toxic effects of produced trans-nootkatol or (+)-nootkatone on yeast [16]. Presence of (+)-valencene and oxidized (+)-valencene products was monitored by GC–MS analysis of the n-dodecane layer. One positive yeast strain was identified, for which two novel oxidised products were detected in the n-dodecane layer (Fig. 2). The major product was identified as trans-nootkatol and the minor product was identified as (+)-nootkatone by comparison to authentic standards. This enzyme was named C. nootkatensis valencene oxidase, CnVO. The protein sequence of the CnVO was analysed and compared to sequences of other characterised sesquiterpene and monoterpene oxidases. The sequence showed low amino-acid identity with previously characterised plant cytochrome P450s that oxidise (+)-valencene. Amino acididentity to C. intybus valencene oxidase CYP71AV8, N. tabacum valencene oxidase CYP71D51v2 and H. muticus premnaspirodiene oxygenase CYP71D55 was found to be 28%, 29% and 30%, respectively (Supplemental Fig. 1). The highest level of amino acid

2.6. GC–MS analysis Analytes from 1 lL samples were separated using a gas chromatograph (5890 series II, Hewlett-Packard) equipped with a 30 m  0.25 mm, 0.25 mm film thickness column (ZB-5, Phenomenex) 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 after 1 min to 300 °C at a rate of 10 °C/min and held for 5 min at 300 °C. The GC was coupled to a mass-selective detector (model 5972A, Hewlett-Packard). Compounds were identified by comparison of mass spectra and retention times with those of the authentic standards of (+)-nootkatone (Fluka), trans-nootkatol (Isobionics), cis-nootkatol (Isobionics) and (+)-valencene (Fluka). The absolute

Fig. 2. Production of trans-nootkatol and (+)-nootkatone in the n-dodecane layer by CnVO. GC chromatogram of the n-dodecane layer from yeast fermentations using WAT11 yeast strains expressing the C. nootkatensis valencene synthase (CnVS) and 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 oxidation product of (+)-valencene in the n-dodecane overlay of strain CnVS/CnVO (peak 1). (+)-Nootkatone was formed as a minor product (peak 2). The mass 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-hydroxylase from G. arboreum, which belongs to cytochrome P450 family CYP706. CnVO was classified into a new cytochrome P450 subfamily as CYP706M1. 3.2. In vivo production of (+)-nootkatone and activity of CnVO on different sesquiterpene substrates CnVO was further examined for (+)-nootkatone formation. To this end, flask fermentation of WAT 11 yeast expressing the valencene synthase CnVS in combination with CnVO was performed in the absence of a n-dodecane layer. The produced terpenes were extracted using ethyl acetate after the fermentation was finished. No sesquiterpenes were detected in the ethyl acetate extract of the WAT11 strain containing an empty plasmid, and also not in the culture expressing solely the valencene synthase, indicating that all produced (+)-valencene had evaporated from the culture. In the ethylacetate extract of the CnVS/CnVO yeast culture, (+)-nootkatone was detected (Fig. 3), at a concentration of 144 ± 10 lg/L yeast culture (Table 1).

The activity of CnVO on other sesquiterpene substrates was tested in yeast strains expressing amorphadiene synthase from Artemisia annua [30] or germacrene A synthase from chicory [29]. Analysis of the n-dodecane layer learned that amorphadiene was not converted by CnVO. Germacrene A was partially converted into a product with m/z = 220. This product did not match germacra1(10),4,11(13)-trien-12-ol or germacra-1(10),4,11 (13)-trien-12al, as deduced from comparison to authentic standards. These are the oxidation products of the germacrene A oxidases that hydroxylate germacrene A on the 12-position [17,37]. This indicates that CnVO does not oxidise germacrene A in position 12 and therefore has regio-selectivity on germacrene A that differs from germacrene A oxidases. 3.3. CnVO produces (+)-nootkatone from (+)-valencene in vitro In order to further characterize the product spectrum of CnVO, microsomal preparations were produced from yeast expressing CnVO and WAT11-empty vector control yeast cultures. In vitro 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 (lg/L yeast culture)

trans-Nootkatol (lg/L yeast culture)

(+)-Nootkatone (lg/L yeast culture)

In situ extraction with n-dodecane

CnVO + CnVS CnVS

1305 ± 117 1266 ± 24

116 ± 44 ND

3±1 ND

Ethyl acetate extraction after fermentation

CnVO + CnVS CnVS

ND ND

ND ND

144 ± 10 ND

CnVO = C. nootkatensis valencene oxidase, CnVS = C. nootkatensis valencene synthase, ND = not detected.

K. Cankar et al. / FEBS Letters 588 (2014) 1001–1007

Fig. 4. Oxidation of (+)-valencene by CnVO in vitro. The oxidation products of (+)valencene are shown for enzyme reactions employing microsomes of WAT11 yeast strain expressing C. nootkatensis valencene oxidase (CnVO), compared to the microsomes prepared from the control yeast strain transformed with an empty pYEDP60 plasmid (ctrl). Formation of trans-nootkatol (peak 1) and (+)-nootkatone (peak 2) was detected.

concentration of 200 lM. The actual dissolved concentration of (+)valencene in these assays was as low as 0.5–1.0 lM (in 40 mM KPi buffer containing 2% DMSO) determined by measuring turbidity in the UV-VIS spectrum caused by undissolved (+)-valencene. In addition to this the solubility was measured by putting empty dialysis tubing in 200 lM (+)-valencene solutions. Once (+)-valencene is consumed, new (+)-valencene will dissolve. For this reason, assays were foremost qualitative. In microsomal assays with (+)-valencene and CnVO microsomes, (+)-nootkatone was found as a predominant peak (82% of total products), in addition to a minor peak of trans-nootkatol (18%) (Fig. 4). 4. Discussion The primary significance of the present work is the identification of a valencene oxidase from the (+)-nootkatone producing tree C. nootkatensis. Based on the protein sequence CnVO belongs to the cytochrome p450 class of CYP706 and is the first member of a new cytochrome P450 subfamily CYP706M (Fig. 5). One enzyme of the CYP706 family has been functionally characterised previously, namely the CYP706B1, (+)-delta-cadinene-8-hydroxylase from G. arboreum [24]. This enzyme is involved in the early steps of biosyn-

1005

thesis of sesquiterpenoid gossypol and catalyses the hydroxylation of (+)-delta-cadinene to 8-hydroxy-(+)-delta-cadinene. Interestingly, the other plant P450s with valencene oxidase activity e.g. C. intybus valencene oxidase CYP71AV8, H. muticus premnaspirodiene 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 family have been identified by homology screening methods [16,17,37]. Genomes of plants may contain several members of the CYP706 family, for example in the Arabidopsis and grapevine genomes 7 and 9 cyp706 genes were identified, respectively [34]. None of these have been functionally characterised. It would be of interest to explore their function, especially with respect to terpene biosynthesis. Substrate specificity of terpene oxidases can be high, as shown for the A. annua amorphadiene oxidase [22,37]. On the other hand, the germacrene A oxidases from the Asteraceae family were shown to be able to oxidise several sesquiterpenes, namely amorphadiene, germacrene A and (+)-valencene [17,37]. CnVO showed a limited substrate promiscuity, and did not oxidise amorphadiene, but showed a partial oxidation of germacrene A. The activity in yeast and in vitro indicates that CYP706M1 catalyzes both the conversion from (+)-valencene to trans-nootkatol, and the conversion from nootkatol to (+)-nootkatone. Multi-step oxidation reactions were previously shown for sesquiterpene oxidases. Amorphadiene oxidase from A. annua CYP71AV1 catalyses the oxidation of amorphadiene to artemisinic alcohol, aldehyde and acid [22]. Similarly, germacrene A oxidases from different species of the Asteraceae family convert germacrene A through an alcohol and aldehyde to germacrene A acid [37]. CnVO catalyses complete oxidation of (+)-valencene to (+)-nootkatone while C. intybus valencene oxidase CYP71AV8, H. muticus premnaspirodiene oxygenase CYP71D55 and N. tabacum valencene oxidase CYP71D51v2, form predominantly trans-nootkatol [15–17]. The 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 expressing the chicory valencene oxidase CYP71AV8 in the same yeast platform only minor production of (+)-nootkatone was observed in vivo [17]. As illustrated here, the use of a biphasic fermentation system may not be desirable for (+)-nootkatone production, since (+)-valencene and trans-nootkatol are sequestered in the n-dodecane layer and may thereby be unavailable for full oxidation to (+)-nootkatone. When using biphasic fermentation, the yield of sesquiterpenes was much higher, compared to single-phase fermentation (1.4 mg/L vs. 0.14 mg/L; Table 1). However, while in the biphasic system the majority of valencene was not oxidised and some trans-nootkatol was observed, the monophasic system yielded almost exclusively (+)-nootkatone as a product. Apparently, in the monophasic system all (+)-valencene that was first converted to trans-nootkatol, is further processed to (+)-nootkatone by CnVO. Efficient systems to produce oxidised terpenoids have been developed in yeast [23]. Upon expression of CnVO with the CnVS in WAT11 yeast strain 144 ± 10 lg/L of (+)-nootkatone was produced after a three-day flask fermentation. Thereby a yeast expressing CnVO in combination with a valencene synthase and ATR1 is the first microbial production system for (+)-nootkatone from a simple carbon source. In comparison to other (+)-valencene oxidizing enzymes, very little intermediates or side-products are produced. This possibly relates to CnVO being the first valencene oxidase isolated from a plant that naturally produces (+)-nootkatone. Acknowledgements Francel Verstappen is acknowledged for assistance with GC–MS analysis. We would like to thank Dr. Cinzia Bertea for her suggestions on the microsomal preparation. We thank Dr. David Nelson for cytochrome P450 family determination of CnVO. This work has been supported by the Dutch Ministry of Economic Affairs, through ACTS IBOS Grant 053.63.322. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2014.01. 061. References [1] Delrio, J.A., Ortuno, A., Garciapuig, D., Porras, I., Garcialidon, A. and Sabater, F. (1992) Variations of nootkatone and valencene levels during the development of grapefruit. J. Agric. Food Chem. 40, 1488–1490. [2] Erdtman, H. and Hirose, Y. (1962) Chemistry of natural order Cupressales. 46 structure of Nootkatone. Acta Chim. Scand. 16, 1311–1314. [3] Burdock, G.A. (2002) Fenaroli’s Handbook of Flavor Ingredients, CRC Press, London. [4] Haring, H.G., Boelens, H., Vanderge, A. and Rijkens, F. (1972) Olfactory studies on enantiomeric eremophilane sesquiterpenoids. J. Agric. Food Chem. 20, 1018–1021. [5] Zhu, B.C.R., Henderson, G., Chen, F., Maistrello, L. and Laine, R.A. (2001) Nootkatone is a repellent for Formosan subterranean termite (Coptotermes formosanus). J. Chem. Ecol. 27, 523–531. [6] Dolan, M.C. et al. (2009) Ability of two natural products, nootkatone and carvacrol, to suppress Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) in a Lyme disease endemic area of New Jersey. J. Econ. Entomol. 102, 2316–2324. [7] Flor-Weiler, L.B., Behle, R.W. and Stafford, K.C. (2011) Susceptibility of four tick species, Amblyomma americanum, Dermacentor variabilis, Ixodes scapularis, and Rhipicephalus sanguineus (Acari: Ixodidae), to nootkatone from essential oil of grapefruit. J. Med. Entomol. 48, 322–326. [8] Panella, N.A., Dolan, M.C., Karchesy, J.J., Xiong, Y.P., Peralta-Cruz, J., Khasawneh, M., Montenieri, J.A. and Maupin, G.O. (2005) Use of novel compounds for pest control: Insecticidal and acaricidal activity of essential oil components from heartwood of Alaska yellow cedar. J. Med. Entomol. 42, 352–358.

[9] Sharon-Asa, L. et al. (2003) Citrus fruit flavor and aroma biosynthesis: isolation, functional characterization, and developmental regulation of Cstps1, a key gene in the production of the sesquiterpene aroma compound valencene. Plant J. 36, 664–674. [10] Beekwilder, J. et al. (2014) Valencene synthase from the heartwood of Nootka cypress (Callitropsis nootkatensis) for biotechnological production of valencene. Plant Biotechnol. J. 12, 174–182. [11] Fraatz, M.A., Berger, R.G. and Zorn, H. (2009) Nootkatone-a biotechnological challenge. Appl. Microbiol. Biotechnol. 83, 35–41. [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 valencene to nootkatone. J. Mol. Catal. B: Enzym. 61, 202–207. [13] Sowden, R.J., Yasmin, S., Rees, N.H., Bell, S.G. and Wong, L.L. (2005) Biotransformation of the sesquiterpene (+)-valencene by cytochrome P450cam and P450BM-3. Org. Biomol. Chem. 3, 57–64. [14] Seifert, A., Vomund, S., Grohmann, K., Kriening, S., Urlacher, V.B., Laschat, S. and Pleiss, J. (2009) Rational design of a minimal and highly enriched CYP102A1 mutant library with improved regio-, stereo- and chemoselectivity. Chembiochem 10, 853–861. [15] Takahashi, S., Yeo, Y.S., Zhao, Y., O’Maille, P.E., Greenhagen, B.T., Noel, J.P., Coates, R.M. and Chappell, J. (2007) Functional characterization of premnaspirodiene oxygenase, a cytochrome P450 catalyzing regio- and stereo-specific hydroxylations of diverse sesquiterpene substrates. J. Biol. Chem. 282, 31744–31754. [16] Gavira, C., Hofer, R., Lesot, A., Lambert, F., Zucca, J. and Werck-Reichhart, D. (2013) Challenges and pitfalls of P450-dependent (+)-valencene bioconversion by Saccharomyces cerevisiae. Metab. Eng. 18, 25–35. [17] Cankar, K., van Houwelingen, A., Bosch, D., Sonke, T., Bouwmeester, H. and Beekwilder, J. (2011) A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene. FEBS Lett. 585, 178–182. [18] Khasawneh, M.A., Xiong, Y.P., Peralta-Cruz, J. and Karchesy, J.J. (2011) Biologically important eremophilane sesquiterpenes from Alaska cedar heartwood essential oil and their semi-synthetic derivatives. Molecules 16, 4775–4785. [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. [20] Johnston, W.H., Karchesy, J.J., Constantine, G.H. and Craig, A.M. (2001) Antimicrobial activity of some Pacific Northwest woods against anaerobic bacteria and yeast. Phytother. Res. 15, 586–588. [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 80 years or more in southeast Alaska. J. Chem. Ecol. 31, 2653–2670. [22] Teoh, K.H., Polichuk, D.R., Reed, D.W., Nowak, G. and Covello, P.S. (2006) Artemisia annua L. (Asteraceae) trichome-specific cDNAs reveal CYP71AV1, a cytochrome P450 with a key role in the biosynthesis of the antimalarial 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. [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 biosynthesis. Plant J. 28, 95–104. [25] Ralston, L., Kwon, S.T., Schoenbeck, M., Ralston, J., Schenk, D.J., Coates, R.M. and 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. [26] Lupien, S., Karp, F., Wildung, M. and Croteau, R. (1999) Regiospecific cytochrome P450 limonene hydroxylases from mint (Mentha) species: cDNA isolation, characterization, and functional expression of (-)-4S-limonene-3hydroxylase and (-)-4S-limonene-6-hydroxylase. Arch. Biochem. Biophys. 368, 181–192. [27] Collu, G., Unver, N., Peltenburg-Looman, A.M., van der Heijden, R., Verpoorte, R. and Memelink, J. (2001) Geraniol 10-hydroxylase, a cytochrome P450 enzyme involved in terpenoid indole alkaloid biosynthesis. FEBS Lett. 508, 215–220. [28] Pompon, D., Louerat, B., Bronine, A. and Urban, P. (1996) Yeast expression of animal and plant P450s in optimized redox environments. Methods Enzymol. 272, 51–64. [29] Bouwmeester, H.J., Kodde, J., Verstappen, F.W.A., Altug, I.G., de Kraker, J.W. and Wallaart, T.E. (2002) Isolation and characterization of two germacrene A synthase cDNA clones from chicory. Plant Physiol. 129, 134–144. [30] Wallaart, T.E., Bouwmeester, H.J., Hille, J., Poppinga, L. and Maijers, N.C.A. (2001) Amorpha-4,11-diene synthase: cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin. Planta 212, 460–465. [31] Urban, P., Mignotte, C., Kazmaier, M., Delorme, F. and Pompon, D. (1997) Cloning, yeast expression, and characterization of the coupling of two distantly related Arabidopsis thaliana NADPH-Cytochrome P450 reductases with P450 CYP73A5. J. Biol. Chem. 272, 19176–19186. [32] Gietz, R.D. and Woods, R.A. (2002) Transformation of yeast by lithium acetate/ single-stranded carrier DNA/polyethylene glycol method. Guide to Yeast Genetics Mol. Cell Biol. Pt. B 350, 87–96. [33] Nelson, D.R. (2009) The Cytochrome P450 homepage. Hum. Genomics 4, 59– 65.

K. Cankar et al. / FEBS Letters 588 (2014) 1001–1007 [34] Nelson, D. and Werck-Reichhart, D. (2011) A P450-centric view of plant evolution. Plant J. 66, 194–211. [35] Larkin, M.A. et al. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948. [36] Bertea, C.M., Schalk, M., Karp, F., Maffei, M. and Croteau, R. (2001) Demonstration that menthofuran synthase of mint (Mentha) is a cytochrome

1007

P450 monooxygenase: cloning, functional expression, and characterization of the responsible gene. Arch. Biochem. Biophys. 390, 279–286. [37] Nguyen, D.T., Gopfert, J.C., Ikezawa, N., MacNevin, G., Kathiresan, M., Conrad, J., Spring, O. and Ro, D.K. (2010) Biochemical conservation and evolution of germacrene A oxidase in Asteraceae. J. Biol. Chem. 285, 16588–16598.