APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1993, p. 1336-13410099-2240/93/051336-06$02.00/0Copyright C 1993, American Society for Microbiology

Novel Allylic Oxidation of ot-Cedrene to sec-Cedrenol by a

Rhodococcus StrainHIROFUMI TAKIGAWA,1* HIROMI KUBOTA,' HIROSHI SONOHARA,1 MITSUYOSHI OKUDA,'

SHIGEYOSHI TANAKA,2 YOSHIAKI FUJIKURA,2 AND SUSUMU ITO'

Tochigi Research Laboratories ofKao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi 321-34,1 andWakayama Research Laboratonies ofKao Corporation, 1334 Minato, Wakayama 640,2Japan

Received 10 September 1992/Accepted 8 February 1993

A bacterial strain, designated KSM-7358, that can use of-cedrene for growth was isolated. The strain was

identified as a member of the genus Rhodococcus and catalyzed the novel allylic oxidation of a-cedreneregiospecifically to produce (R)-10-hydroxycedrene (sec-cedrenol) with a very high yield. ai-Curcumene was

also produced as a possible metabolite of sec-cedrenol. A possible pathway for the microbial conversion ofa-cedrene to sec-cedrenol and a-curcumene is proposed.

Monoterpenoids, sesquiterpenoids, and related com-

pounds are major components of some fragrances and fla-vors and are also important precursors of certain pharma-ceuticals. Many researchers have therefore examined themicrobial conversion of these terpenes to other compounds,possibly because of difficulties encountered in the chemicalsynthesis of useful products from terpenes. For example,Bhattacharyya and coworkers reported that D-a-pinene andcyclohexene were hydroxylated by a strain of Aspergillusniger to D-cis-verbenol (18) and (+)-2-cyclohexene-1-ol (3),respectively. Such allylic oxidation has also been demon-strated by Tabenkin et al. (21), who used strains ofA. nigerand Streptomyces aureofaciens to convert cinerone to cin-erolone. In addition, microbial conversion aimed at theproduction of compounds that mimic natural flavors hasbeen demonstrated with ionones (16, 25), a-damascone (19),and cinnamic acid (7) as starting materials.

ca-Cedrene is a major component of cedar wood oil whichis used widely as a perfume and as a precursor of syntheticperfumes for toiletry and cosmetic products. Abraham andcoworkers reported that Corynespora cassicola DSM 62474and Rhodococcus rhodochrous (formerly Mycobacteriumrhodochrous) ATCC 999 converted a-cedrene to a variety ofminor products, such as 3-hydroxy- and 12-hydroxy-a-ce-drenes (1) and cedrenone and 10-methoxy-a-cedrene (11),respectively. The formation of such products from a-ce-drene by Beauveria sulfurescens ATCC 7159 was also re-

ported by Lamare et al. (12, 13). However, the biotransfor-mations of a-cedrene reported thus far require a longcultivation time, and the yields of products have beenextremely low.

Recently, we isolated a strain that could hydroxylateca-cedrene to sec-cedrenol regiospecifically with a very highyield. The present study was aimed at a taxonomic descrip-tion of the isolate and the optimization of the oxidationreaction by resting cells of the organism. To our knowledge,this is the first report of the microbial allylic oxidation ofa-cedrene.

MATERIALS AND METHODSCulture media. SCD medium (Nihon Pharmaceutical Co.,

Ltd., Tokyo, Japan) contained 1.7% (wt/vol) casein peptone,

* Corresponding author.

0.3% (wt/vol) soybean peptone, 0.25% (wt/vol) K2HPO4,0.5% (wt/vol) NaCl, and deionized water. GP medium (Ni-hon Pharmaceutical Co., Ltd.) contained 2% (wt/vol) glu-cose, 0.5% (wt/vol) casein peptone, 0.2% (wt/vol) yeastextract, 0.1% (wtlVol) KH2PO4, 0.05% (wt/vol) MgSO4-7H20, and deionized water. P medium contained 0.5%(wt/vol) a-cedrene, 0.35% (wt/vol) NH4NO3, 0.071% (wt/vol) Na2SO4, 0.001% (wt/vol) FeCl3 6H20, 0.0017% (wt/vol) MgCl2. 6H20, 0.01% (wt/vol) CaCl2- 2H20, and 50mM phosphate buffer (pH 7.0). F medium (16) contained 5%(wt/vol) sucrose, 0.1% (wt/vol) NaNO3, 0.1% (wt/vol) K2HPO4, 0.05% (wt/vol) KCl, 0.05% (wt/Vol) MgSO4 7H20,0.1% (wt/vol) yeast extract (Difco), and deionized water (pH7.0). Media were solidified with 1.5% Bacto-Agar (Difco)when necessary.

Organisms and screening methods. The following proce-dures were used to screen microorganisms for those thatcould transform a-cedrene. We first tested 3,969 unidentifiedn-hexadecane-utilizing bacteria in our culture collection (22)for their ability to grow on a-cedrene as the sole source ofcarbon and energy. We also tested a number of bacterial andfungal strains purchased from the American Type CultureCollection, Rockville, Md.; the Institute of Fermentation,Osaka, Japan; the Japan Collection of Microorganisms,Wako, Japan; and the Institute of Applied Microbiology,University of Tokyo, Tokyo, Japan. One loopful of bacterialcells grown on an SCD agar slant was streaked on plates ofagar-solidified synthetic P medium that contained 0.5%(wt/vol) a-cedrene. GP agar slants were used for fungi. Aftera 2- to 7-day incubation at 30°C, the growth in the presenceof a-cedrene was examined visually on the plates. Then theability of these test organisms to transform a-cedrene was

examined in an appropriate liquid medium as follows. Oneloopful of bacterial cells grown on an SCD agar slant wasinoculated into 10-ml volumes of SCD medium in test tubes(20 by 2.5 cm), and the cells were cultured with shaking ona reciprocal shaker for 2 days at 30°C. For fungi, a loopful ofmycelia from a GP agar slant was inoculated into 100-mlvolumes of F medium in 500-ml Erlenmeyer flasks andcultured on a rotary shaker (120 rpm) for 2 days at 30°C. Toeach of the cultures, at-cedrene was added at 0.5% (wt/vol),and incubation was continued for a further 2 to 7 days at30°C. The spent media obtained were extracted with anequal volume of n-hexane, and the products in the extractswere examined by gas chromatography (GC).

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RHODOCOCCUS ALLYLIC OXIDATION OF a-CEDRENE 1337

We identified one isolate, designated KSM-7358, that useda-cedrene as the sole source of carbon and energy andproduced sec-cedrenol from a-cedrene in quantity. Thisstrain was originally isolated, as follows, from a soil samplecollected in Wakayama, Japan (22). A spoonful (0.5 g) of thesoil sample was suspended in 10 ml of saline. The suspensionwas mixed well and then spread on an agar plate thatcontained 1% (wt/vol) n-hexadecane, 1% (wt/vol) (NH4)2HPO4, 0.2% (wtlvol) K2HPO4, 0.03% (wt/Vol) MgSO4.7H2O, 0.001% (wt/vol) FeSO4. 7H20, 0.0008% (wt/vol)ZnSO4- 7H20, 0.0008% (wt/vol) MnSO4- 7H20, 0.02% (wt/vol) yeast extract (Difco), and deionized water (pH 7.0).After incubation at 30°C for 2 days, one of the colonies thatappeared on the plate was picked up as an n-alkane utilizerand placed in our culture collection.Chemotaxonomic identification of the isolated strain. Sug-

ars and cross-linked amino acid in the cell wall were char-acterized by the method of Lechevalier and Lechevalier(15), and the N-acyl residue was identified by the method ofUchida and Aida (23). R erythropolis IFO 12320, Mycobac-teriumphlei IFO 13160, and Arthrobacter citreus IFO 12957were used as reference strains for the experiments. Themenaquinone composition was determined by reverse-phasehigh-performance liquid chromatography (HPLC) (5), withNocardia asteroides JCM 6043 [(MK-8(H4)] and R equiJCM 1311 [(MK-8(H2)] as reference strains. Other taxo-nomic characteristics were examined by the procedure ofLechevalier (14).

Conversion of a-cedrene by suspensions of resting cells. Oneloopful of cells grown on an SCD agar slant was inoculatedinto 100 ml of SCD medium in a 500-ml Sakaguchi flask (aculture flask with "shoulders"), and cultures were incu-bated, with shaking, on a reciprocal shaker (120 rpm). Aftercultivation at 30°C for 2 days, cells were harvested bycentrifugation at 12,000 x g for 10 min. The resulting cellpellet was washed with chilled 50 mM phosphate buffer (pH7.0), and the cells were suspended in the same buffer suchthat theA600 of the suspension was between 0.3 and 0.5 aftera 1:100 dilution. Portions (50 ml) of the standardized suspen-sion of cells were placed in 500-ml Erlenmeyer flasks thateach contained 0.25 g of a-cedrene, and the flasks wereincubated at 30°C for an appropriate number of days on arotary shaker (120 rpm). Products and residual substratewere isolated from the spent media by extraction with anequal volume of n-hexane, and compounds were quantifiedby GC. For the preparation of a large amount of cells, a5-liter jar fermentor (L. E. Marubishi Co., Ltd., Tokyo,Japan) was used. Cells were propagated in SCD medium at30°C for 20 to 24 h (working volume, 2.5 liters; aeration rate,1 volume velocity per min agitation, 400 rpm; pH, controlledat 7 by addition of 5 N HCl).GC. Substrates and products were analyzed on an HP5880

gas chromatograph (Hewlett-Packard) equipped with a flameionization detector and a capillary column (25 m by 0.25 mm)coated with Carbowax 20M (helium as carrier gas, at a flowrate of 25 ml/min; Gasukurokogyo, Tokyo, Japan). Thetemperature was programmed isothermally at 80°C for thefirst 10 min and then raised from 80 to 150°C at 2°C/min,raised from 150 to 220°C at 10°C/min, and finally held at220°C for 5 min.

Spectroscopy and MS. The following spectroscopes andspectrometer were used: a Hitachi model 270-30 infrared(IR) spectroscope for recording infrared spectra; a JOELGX-400 nuclear magnetic resonance (NMR) spectroscope(400 MHz) for recording 13C-NMR and 1H-NMR spectra;

and a Finnigan MAT 1020 mass spectrometer for performinghigh-resolution mass spectrometry (MS).

Chemicals and preparation of authentic compounds. a-Ce-drene was prepared by dehydration of cedrol isolated fromcedar wood oil (T. Hasegawa Co., Tokyo, Japan). Whitecrystalline cedrol (700 g), isolated from cedar wood oil byfractional distillation, was added to 500 g of acetic acid thatcontained 7 g of FeCl3. The mixture was stirred at 80°C for1 h, and then the solvent layer was withdrawn and washedwith an equal volume of a saturated solution of NaHCO3.The washed solvent layer was distilled to yield 513.3 g ofproduct (boiling point, 102 to 104°C at 6 mm Hg). It consistedof 97.5% a-cedrene, as judged by GC. Cedrane was preparedby the conventional method from a-cedrene by hydrogena-tion with palladium, and cedrenone was prepared by pyridi-nium chlorochromate oxidation of the sec-cedrenol pro-duced by isolate KSM-7358.The diastereomers of sec-cedrenol were synthesized from

cedrenone as follows. Tetrahydrofuran (7 ml) containing 50mg of cedrenone was added to 10 ml of tetrahydrofuran thatcontained 11.5 mg of LiAlH4. The mixture was stirred atroom temperature for 30 min, and then the products wereisolated by column chromatography on silica gel and then byHPLC (in 6% ethyl acetate in n-hexane). Two diastereomerswere obtained at a ratio of 19:74, as determined quantita-tively by GC. Famesene, which consists of a- and p-forms,was purified by rectification (reflux ratio of 20) from acommercial product obtained from Kuraray Co., Tokyo,Japan.

RESULTS

Survey of a-cedrene-transforming strains. A total of 4,022microorganisms were tested for their ability to transforma-cedrene. The microorganisms used included 41 standardstrains (18 bacteria, 10 yeasts, and 13 molds), 12 unidentifiedmolds, and 3,969 bacterial strains that had been isolatedfrom soils as utilizers of n-hexadecane in our laboratory (22).Some of these bacteria and fungi were found to utilize andconvert a-cedrene to unidentified products, but the yields ofproduct were extremely low under our growth conditions.Bacillus pumilus ATCC 6631, known to hydroxylate ciner-one at the allylic position (21), and R. rhodochrous ATCC999, known to convert a-cedrene to cedrenone (11), didnot generate any detectable products from a-cedrene underour conditions. In addition, Beauvaria bassiana ATCC13144, Cladosporium resinae ATCC 22712, Cunninghamellablakesleeana ATCC 8688a, and Penicillium adametzi ATCC10407 (4), which are known to hydroxylate and hydratecostunolide to generate various minor products, such ascolartin and dihydrocostunolide, all failed to grow on or formany detectable products from a-cedrene in F medium.

Identification of the a-cedrene-utilizing strain KSM-7358.Among the test organisms, only one strain, designatedKSM-7358, was found to use a-cedrene as the sole source ofcarbon and energy in synthetic P medium and to producelarge amounts of unidentified products from a-cedrene, asrevealed by GC. The organism was an aerobic, gram-positive, immotile bacterium, which formed substrate myce-lia and produced neither aerial mycelia nor conidia. The cellwall contained arabinose and galactose, meso-diami-nopimelic acid as the cross-linked amino acid, and theglycolyl type of N-acyl residue. In addition, MK-8(H2) wasdetected as the major menaquinone. From these morpholog-ical and chemotaxonomic results (14), we classified thisorganism as a member of the genus Rhodococcus, rather

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TABLE 1. Taxonomic characteristics of Rhodococcusstrain KSM-7358

Characteristic Value

Morphological characteristicsGram staining .......................... +Size (,um) .......................... 0.8-1.0 by 1.0-12Fragmentation of substratemycelium ................... ....... +

Aerial mycelium produced............... -Conidia formed .......................... -

Mobility .......................... -

Acid-fastness .......................... -

Anaerobic growth (N2 atmosphere) ......a-Growth temperature ('C) .................... 10-37 (25-30)bGrowth pH .......................... 3-10 (5-9)bAcid from glucose (OF test). +cMajor menaquinone ..........................MK-8(H2)Cell wall components

Sugar.......................... Arabinose, galactoseCross-linked amino acid ..................meso-Diaminopimelic acidN-Acyl residue .......................... Glycolyl type

Utilization of carbon sourcedn-Decane.......................... -

n-Hexadecane .......................... +n-Octadecane .......................... +Pristane ............ .............. +Famesene ............... ........... +d-Limonene .......................... -

Ethylbenzene .......................... -

p-Cymene .......................... -

a-Cedrene ............... ........... +Cedrane .......................... -

Cedrol .......... ................ -Caryophyllene .......................... +

aGrowth was observed under stationary conditions.b Optimum ranges are given in parentheses.c Positive with Andrade indicator but negative with Bromo Thymol Blue

after a 7-day incubation.d Added, each at 0.5% (wt/vol), to P medium.

than as a member of the genus Mycobacterium or Nocardia.The major taxonomic properties of strain KSM-7358 aresummarized in Table 1. It seemed likely that the organismwas related to R. erythropolis and R. rhodochrous, althoughit was positive for utilization of inositol and negative forgrowth at 40'C. Rhodococcus sp. strain KSM-7358 was ableto grow on caryophyllene, one of the sesquiterpenes; onn-alkanes, such as n-hexadecane and n-octadecane; and onbranched-chain hydrocarbons, such as pristane and farne-sene. Cedrol, cedrane, d-limonene, and alkylbenzenes, suchas ethylbenzene and p-cymene, all failed to support thegrowth of the isolate.

Identification of products generated from a-cedrene. WhenRhodococcus sp. strain KSM-7358 was grown for 4 days inP medium that contained 0.5% a-cedrene, four metabolites(a through d) were found in the medium, as judged by GC(Fig. 1).Compound b (0.21 g) was purified from 1.6 g of the

n-hexane extract of a 6-day-old culture by HPLC on InertsilPREP Sil (GL Sciences, Tokyo, Japan) with n-hexane as theelution solvent, and it was 99.4% pure as determined by GC.Compound b was deduced to be ot-curcumene from thefollowing data obtained from 'H-NMR, 13C-NMR, and IRspectroscopy and MS: 13C-NMR bc (CDCl3), 17.6, 20.9, 22.5(1-C), 25.6, 26.2 (11-C), 38.5 (8-C), 37.1 (10-C), 124.6 (12-C),126.8, 128.9, 131.2, 135.0 (2-C), 144.5 (5-C); 1H-NMR bH(CDCl3), 1.21 (d, 3H, 9-CH3, J = 6.9Hz), 1.51 (s, 3H,

0 20 40 60

Retention time ( min)FIG. 1. Gas chromatogram of products generated from a-ce-

drene by growing cells of Rhodococcus sp. strain KSM-7358. Theorganism was grown for 4 days, with shaking, in P medium placed ina 500-ml Erlenmeyer flask that contained 0.5% a-cedrene. Residualsubstrate (S) and products (a, b, c, and d) were extracted withn-hexane and detected by GC.

14-CH3), 1.66 (s, 3H, 15-CH3), 1.5 to 1.7 (m, 2H, 10-CH2),1.8 to 1.9 (m, 2H, 11-CH2), 2.28 (s, 3H, 1-CH3), 2.63 (m, 1H,8-CH), 5.09 (t, 1H, 12-CH,J = 7.7Hz), 7.05 (br s, 4H, phenyl3-H, 4-H, 6-H, 7-H); MS m/z, 202 (M+), 145, 132, 119 (basepeak), 105, 91, 83, 69, 55, 41; IR (KBr), 3,130, 3,000(aromatic H), 2,960 (methyl H), 2,900, 1,535 (aromatic H),1,475 (aromatic H), and 1,400 cm-'. These spectral data forcompound b are consistent with those for ao-curcumenereported in the literature (9).Compound d was the almost exclusive product generated

by resting cells of Rhodococcus sp. strain KSM-7358 (Fig.2). About 50 g of the n-hexane extract was applied to acolumn of silica gel (400 g; Wakogel C-200; Wako PureChemicals, Tokyo, Japan), and the column was washed with2 liters of n-hexane and then with 2 liters of 2.5% ethylacetate in n-hexane. The material eluted with 5% ethylacetate in n-hexane was concentrated to yield approximately20 g of white crystals of compound d. The material wasrecrystallized in n-hexane at -5'C, and the purity of thecrystals reached 99.2%, as determined by GC. Compound dwas deduced to be sec-cedrenol from the following dataobtained from 'H-NMR, 13C-NMR, and IR spectra and fromMS: 13C-NMR 5c (CDCl3), 14.8 (12-C), 23.6, 24.9, 25.6,28.4, 33.7, 35.1, 38.4, 45.5 (6-C), 51.8, 54.4 (5-C), 60.0 (1-C),72.2 (10-C), 124.0 (9-C), 144.0 (8-C); 'H-NMR 8H (CDCl3),0.78 to 1.0 (9H, 12-CH3, 13-CH3, 14-CH3), 1.2 to 1.5 (m,

S

a c

d

0 20 40 60

Retention time (min)FIG. 2. Gas chromatogram of products generated from a-ce-

drene by resting cells of Rhodococcus sp. strain KSM-7358. Prop-agation of cells, reaction conditions, and quantification of productsand residual substrate were described in Materials and Methods.Results were obtained after incubation for 4 days.

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Cultivation time (days)FIG. 3. Time course of the conversion of a-cedrene to sec-

cedrenol and a-curcumene by Rhodococcus sp. strain KSM-7358 inP medium. The organism was cultured with shaking in 5.0-mlaliquots of P medium in test tubes that contained 0.5% oa-cedrene. Atspecific times, products and residual substrate in each test tube wereexamined by GC. A, oa-cedrene; 0, sec-cedrenol; *, a-curcumene.

6H), 1.67 (s, 3H, 15-CH3), 1.5 to 1.9 (m, 6H), 3.7 (br s, 1H,10-CH), 5.3 (br s, 1H, 9-H); MS m/z, 220 (M+), 205, 177, 163,159, 152, 135 (base peak), 121, 109, 95, 81, 69, 55, 41; IR(KBr), 3,390 (hydroxyl H), 2,970 (methyl H), 2,920, 1,490,1,460, 1,400, and 1,000 (hydroxyl H) cm-1. In general,during LiAlH4 reduction of cyclic ketones, attack by alumi-num hydride occurs preferentially from the less stericallyhindered side (exo attack) to generate a sterically hinderedalcohol (endo alcohol), as reported by House (8). From thereported stereochemistry of ao-cedrene (20), when we con-sider the chemical synthesis of sec-cedrenol from ce-drenone, the main product must be (S)-10-hydroxycedrene,and this compound had a retention time of 39.5 min duringGC [the minor product is (R)-10-hydroxycedrene, with aretention time of 42.1 min]. The ratio of (R)-cedrenol to(S)-cedrenol was 19:74 (wt/wt), as determined by GC (seeMaterials and Methods). sec-Cedrenol produced by ourstrain was detected as a single peak on GC with a retentiontime of 42.1 min. Therefore, we concluded that the productwas exclusively in the (R)-configuration, namely, (R)-10-hydroxycedrene. To confirm the structure of compound d,we converted it chemically to cedrenone. A 20-ml volume ofa solution of dichloromethane that contained 0.3 g of purifiedcompound d was added dropwise to a 20-ml solution ofdichloromethane that contained 0.6 g of pyridinium chloro-chromate. After the mixture was stirred at 25°C for 1 h, itwas applied to a column of silica gel (30 g of Wakogel C-200)and eluted with dichloromethane, and the eluate was dried invacuo to yield 275 mg of white powder. All spectral data forthe synthesized product were identical to those for ce-drenone reported elsewhere (6).From the retention time (40.7 min) of the synthesized

cedrenone during GC, the minor peak, compound c, in Fig.1 is suggested to correspond to cedrenone. Peak a, near thepeak of the substrate a-cedrene (peak S), has not yet beenidentified.

Figure 3 shows the time course for the conversion ofa-cedrene to sec-cedrenol and ot-curcumene during growthof cells in P medium in test tubes. The level of sec-cedrenolincreased, in parallel with a decrease in the amount ofsubstrate cedrene, and reached a maximum (60 mg/liter)after 5 days. a-Curcumene was produced in only very smallamounts during the first 3 days of the incubation, but its level

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Reaction time ( hrs)FIG. 4. Production of sec-cedrenol from a-cedrene by resting

cells of Rhodococcus sp. strain KSM-7358 in a jar fermentor. A1.4-liter volume of a suspension of resting cells (A6. = 30) in 50 mMphosphate buffer (pH 7.0) was placed in a 5-liter jar fermentor. Thejar was operated under optimum conditions (30°C, 400 rpm, 1 wm,working volume of 1.4 liter), and the culture was sampled occasion-ally for determination of levels of a-cedrene (A) and sec-cedrenol(-). At specific times (arrows), 10 ml of the broth was withdrawnfrom and 7 g of a-cedrene was added to the reaction mixture.

was maximal (15 mg/liter) after 5 days. The levels of bothproducts then decreased gradually, while the level of a-ce-drene also continued to decrease. A trace amount of ce-drenone (<2 mg/liter) was detected after 3 days (data notshown). It appeared that sec-cedrenol was an intermediatemetabolite in the synthesis of a-curcumene and that the twoproducts accumulated transiently under our growth condi-tions. The maximum yield of cells seemed to be obtainedafter a 5-day incubation, but growth could not be monitoredaccurately by measuring turbidity or by counting viable cellson plates of solidified P or SCD medium. When test tubescontaining culture were stationary, cells floated in the oil-water (a-cedrene-water) interphase, and they could not beharvested by centrifugation (20,000 x g), even in the pres-ence of a number of synthetic nonionic surfactants withdifferent hydrophile-lipophile balance values, which wereadded to decrease the affinity of the cells for a-cedrene.Moreover, cells grew on the oily substrate as solid clumps ofdifferent sizes. By contrast, the cells grew normally in SCDmedium, so we used SCD-grown cells for further experi-ments.

Production of sec-cedrenol by resting cells in a jar fermen-tor. When the initial concentration of a-cedrene was 0.5%(wt/vol), a maximum conversion yield of sec-cedrenol (over65% [wt/vol]) was generated by resting cells of this organismin a 500-ml Erlenmeyer flask. In the presence of excesssubstrate (more than 0.5% [wt/vol]), the yield of sec-cedre-nol tended to be lower. To avoid the negative effect ofa-cedrene and to increase the final yield of sec-cedrenol,a-cedrene was added intermittently to a suspension of rest-ing cells in a 5-liter jar (Fig. 4). When the a-cedrene addedhad been almost consumed, 7 g of a-cedrene was added tothe jar, and this procedure was repeated four times. After140 h of incubation, a total of approximately 24 g ofsec-cedrenol was obtained from 35 g of a-cedrene. During

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FIG. 5. Possible pathway for the formation of sec-cedrenol and a-curcumene from a-cedrene by Rhodococcus sp. strain KSM-7358.

such an incubation, neither a-curcumene nor cedrenoneaccumulated in the reaction mixture (data not shown).

DISCUSSIONWe have isolated a microorganism, Rhodococcus sp.

strain KSM-7358, that can convert a-cedrene to sec-cedre-nol [(R)-10-hydroxycedrene] and a-curcumene with highyields, when either resting cells or growing cells of theorganism are used. This strain is closely related to R.erythropolis and R. rhodochrous. sec-Cedrenol and a-cur-cumene have not been reported to data as microbial prod-ucts, although prim-cedrenol, rather than sec-cedrenol, andao-curcumene have been found in cedar wood oil (2) andlavender oil (10), respectively. No microorganisms that cangrow on cyclic sesquiterpenes have been reported to date.Our isolate, Rhodococcus sp. strain KSM-7358, is the firstidentified microorganism that can use sesquiterpenes, suchas a-cedrene and caryophyllene, for growth. It can utilizethese sesquiterpenes in the absence of specific growth fac-tors.The enzyme system(s) involved in the allylic oxidation of

a-cedrene by Rhodococcus sp. strain KSM-7358 appears notto be inducible, since the addition of a-cedrene to culturesdid not change the rate of conversion of a-cedrene tosec-cedrenol by resting cells (data not shown). Conversionof a-cedrene to sec-cedrenol (and a-curcumene) by restingcells is not stoichiometric (Fig. 4), and some of the substratemay be dissimilated and used as a source of the energyrequired for the allylic oxidation reaction. It is possible thata mixed-type oxygenation, which requires oxygen andNAD(P)H, is involved in the mechanism of allylic oxidationof a-cedrene. NAD(P)H may be produced during catabolismof ot-cedrene in the cells.Abraham and coworkers (1, 11) reported that R. rhodo-

chrous ATCC 999 converted ot-cedrene to cedrenone and10-methoxy-a-cedrene with very low yields (<1%), but theydid not describe the reaction conditions or provide spectral

data for the products. To confirm their studies, we tried totransform a-cedrene by using R. rhodochrous ATCC 999under both growing and resting conditions, but we failed todetect such products. Cedrenone could be formed as thefinal product from sec-cedrenol by R. rhodochrous ATCC999. There also remains the possibility that the formation ofcedrenone preceded that of sec-cedrenol in cells of Rhodo-coccus sp. strain KSM-7358, but we have obtained noexperimental results that support this possibility (Fig. 1 to 3).Some microorganisms other than Rhodococcus spp. whichare able to transform a-cedrene and its related compoundshave been found. Wang et al. (24) reported that 3-hydrox-ycedrol was the main oxidation product generated fromcedrol by A. niger ATCC 9142, and Abraham et al. (1)demonstrated the hydroxylation of cedrol at C-2 and C-12 byfive different strains, Rhizopus stolonifer CBS 38252, Strep-tomyces bikiniensis IFO 13350, Verticillium tenerum DSM63545, Streptoverticillium reticuli DSM 40776, and Coryne-spora cassiicola DSM 62474. The latter group also reported,with respect to the oxidation of cedrol and a-cedrene, that C.cassicola DSM 62474 hydroxylated the respective C-3 andC-12 atoms of these compounds in addition to catalyzing theallylic hydroxylation at the C-15 of a-cedrene.From the results of the present study, we suggest a

possible pathway for the formation of sec-cedrenol, ce-drenone, and a-curcumene from a-cedrene by Rhodococcussp. strain KSM-7358, as shown in Fig. 5. a-Cedrene may firstbe allylically oxidized to sec-cedrenol (or cedrenone), whichis then converted to ot-curcumene via repeated Wagner-Meerwein rearrangements of the product of allylic oxidation.In the first step of the proposed oxidation of a-cedrene,Rhodococcus sp. strain KSM-7358 may recognize the dou-ble-bond structure of a-cedrene because this organism can-not attack cedrane. A similar regiospecificity has beenreported for B. bassiana ATCC 7157: this strain can oxidizeot-cedrene both at C-3 and C-15 and at C-12 and C-15 butcannot oxidize cedrane (12). The regiospecific oxidation of

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RHODOCOCCUS ALLYLIC OXIDATION OF a-CEDRENE 1341

squalene has been reported with a Nocardia strain whichcannot attack squalane (17).

Recently, we found that sec-cedrenol has several biologi-cal activities, such as vasodilator and antihistamine activities(22a). Current work is focusing on further optimization of theproduction of sec-cedrenol and on the evaluation of thiscompound for possible medical applications.

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2. Arctander, S. 1960. Monographs on raw materials, p. 139-147. InPerfume and flavor materials of natural origin. Elizabeth, N.J.

3. Bhattacharyya, P. K., and K. Ganapathy. 1965. Microbiologicaltransformations of terpenes. VI. Studies on the mechanism ofsome fungal hydroxylation reactions with the aid of modelsystems. Indian J. Biochem. 2:137-145.

4. Clark, A. M., and C. D. Hufford. 1978. Microbial transforma-tions of the sesquiterpene lactone costunolide. J. Chem. Soc.Perkin Trans. I. 1978:3022-3028.

5. Collins, M. D. 1982. A note on the separation of natural mixturesof bacterial menaquinones using reverse-phase high-perfor-mance liquid chromatography. J. Appl. Bacteriol. 52:457-460.

6. Grantham, P. J., and A. G. Douglas. 1980. The nature and originof sesquiterpenoids in some tertiary fossil resins. Geochim.Cosmochim. Acta 44:1801.

7. Hilton, M. D., and W. J. Cain. 1990. Bioconversion of cinnamicacid to acetophenone by a pseudomonad: microbial production ofa natural flavor compound. Appl. Environ. Microbiol. 56:623-627.

8. House, H. 0. 1972. Metal hydride reductions and related reac-tions, p. 59-63. In R. Breslow (ed.), Modem synthetic reac-tions, 2nd ed. W. A. Benjamin Inc., Menlo Park, Calif.

9. Judith, S., R. Zilenovsld, and S. S. Hall. 1981. Selective synthe-sis of either complex aromatic alkenes or (1,4-cyclohexadienyl)alkenes by tandem arylation-multistep reduction of ot,13,y,8-unsaturated ketones. J. Org. Chem. 46:4139-4142.

10. Kaiser, R., and D. Lamparsky. 1983. New carbonyl compoundsin the high-boiling fraction of lavender oil. Second communica-tion. Helv. Chim. Acta 66:1843-1849.

11. Kieslich, K., W. R. Abraham, B. Stumpf, B. Thede, and P.Washausen. 1986. Transformations of terpenoids, p. 367-394. InProgress in essential oil research. Walter de Gruyter & Co.,Berlin.

12. Lamare, V., J. D. Fourneron, and R. Furstoss. 1987. Microbialtransformations. IX. Biohydroxylation of a-cedrene and cedrol.Synthesis of an odoriferous minor component of cedar woodessential oil. Tetrahedron Lett. 28:6269-6272.

13. Lamare, V., and R. Furstoss. 1990. Bioconversion of sesquiter-penes. Tetrahedron 46:4109-4132.

14. Lechevalier, H. A. 1986. Nocardioforms, p. 1458-1506. InP. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.),Bergey's manual of systematic bacteriology, vol. 2. The Wil-liams & Wilkins Co., Baltimore.

15. Lechevalier, M. P., and H. A. Lechevalier. 1970. Chemicalcomposition as a criterion in the classification of aerobic acti-nomycetes. Int. J. Syst. Bacteriol. 20:435-443.

16. Mikami, Y., Y. Fukunaga, M. Arita, and T. Kisaki. 1981.Microbial transformation of P-ionone and 13-methylionone.Appl. Environ. Microbiol. 41:610-617.

17. Nakajima, K., A. Sato, T. Misono, and T. lida. 1981. Microbialoxidation of the isoprenoid hydrocarbon squalene. NipponNogeikagaku Kaishi 55:1187-1195.

18. Preme, B. R., and P. K. Bhattacharyya. 1962. Microbiologicaltransformations of terpenes. II. Transformations of a-pinene.Appl. Environ. Microbiol. 10:524-528.

19. Schoch, E., I. Benda, and P. Schreier. 1991. Bioconversion ofa-damascone by Botrytis cinerea. Appl. Environ. Microbiol.57:15-18.

20. Stork, G., and F. H. Clarke, Jr. 1955. The total synthesis ofcedrol and cedrene. J. Am. Chem. Soc. 77:1072-1073.

21. Tabenkin, B., R. A. Lemahiev, R. A., J. Berger, and R. W.Kierstead. 1969. Microbiological hydroxylation of cinerone tocinerolone. Appl. Environ. Microbiol. 17:714-717.

22. Takeuchi, K., K. Koike, and S. Ito. 1990. Production of cis-unsaturated hydrocarbons by a strain of Rhodococcus in re-peated batch culture with a phase-inversion, hollow-fiber sys-tem. J. Biotechnol. 14:179-186.

22a.Takigawa, H., and S. Ito. Unpublished results.23. Uchida, K., and K. Aida. 1977. Acyl type of bacterial cell wall:

its simple identification by colorimetric method. J. Gen. Appl.Microbiol. 23:249-260.

24. Wang, K. C., L. Y. Ho, and Y. S. Cheng. 1972. Microbialoxidation of terpene. I. Hydroxylation of cedrol. J. Chin.Biochem. Soc. 1:53-55.

25. Yamasaki, Y., Y. Hayashi, M. Arita, T. Hieda, and Y. Mikami.1988. Microbial conversion of a-ionone, a-methylionone, anda-isomethylionone. Appl. Environ. Microbiol. 54:2354-2360.

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