APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1981, p. 187-191 0099-2240/81/080187-05$02.

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Vol. 42, No. 2

Regio-Selective 10-Hydroxylation of Patchoulol, Sesquiterpene, by Pithomyces Species

YASUJI SUHARA, SAYURI ITOH, MAYUMI OGAWA, KAZUTERU YOKOSE, TOYOAKI SAWADA, TAKASHI SANO, RIEKO NINOMIYA, AND HIROMI B. MARUYAMA* Department ofMicrobiology and Chemotherapy, Nippon Roche Research Center, Kajiwara, Kamakura-247, Japan
Received 16 October 1980/Accepted 1 May 1981

Of some 350 microorganisms screened, four strains of Pithomyces species were found to carry out regio-selective hydroxylation of patchoulol, a sesquiterpene, to 10-hydroxypatchoulol: Pithomyces sp. NRJ201, P. chartarum NRJ210, and, to a lesser extent, P. cynodontis ATCC 26150 and P. atro-olivaceus IFO 6651 were found to catalyze this reaction. A method has been developed by which 10hydroxypatchoulol was obtained in 25 to 45% yields in 1- to 5-liter fermentation jars at 2 to 4 g of patchoulol per liter and isolated as pure material in 30% yields.

Regio- or enantioselective reactions have been of considerable importance in organic syntheses. Microbes are well known to carry out such reactions, as illustrated by the oxidation of various steroids, some of which are produced by microbes on an industrial scale (2, 10). In contrast, only a few reports have been published on the microbial regio-selective reactions of terpenes (8). A recent example describes the selective hydroxylation of patchoulol [Fig. 1, I; 3, 4, 4afl, 5, 6fl, 7, 8, 8a-octahydro-4a, 8al?, 9, 9-tetramethyl- 1,6-methanonaphthalen- 1,8(2H) -ol], a sesquiterpene isolated from patchouli oil (essential oil fragrance from Pogostemon cablin Benth.) at C-10 by rabbits and dogs as reported by Luu et al. (9). We have screened soil microorganisms capable of such a regio-specific transformation and found that a few Pithomyces strains converted this substrate to 10-hydroxypatchoulol (Fig. 1, II) in a high yield. Other fungi, including Penicillium, Paecilomyces, and Phoma strains, also carry out this reaction as reported elsewhere (A. Fujiwara, M. Tazoe, Y. Shiomi, and M. Fujiwara, Abstr. VIth Int. Ferm. Symp., 1980, F-20.9, p. 176). This reaction may provide an economic production of norpatchoulenol (9; Fig. 1, IV), an expensive fragrance (12). This paper describes this hydroxylation process by a few strains of Pithomyces. (Portions of this work were presented at the 10th annual meeting of the Pharmaceutical Society of Japan [Abstr. Annu. Meet. Pharm. Soc. Jpn. 100th, Tokyo, Japan, p. 272, 1980].) MATERIALS AND METHODS Microorganisms used. Pithomyces sacchari ATCC 24323, P. cynodontis ATCC 26150, P. chartarum ATCC 22637, and P. atro-olivaceus IFO 6651

were used. In addition, two strains of this genus (Pithomyces sp. NRJ201-p-2f [NRJ201] and P. chartarum NRJ210-p-lf [NRJ210]), selected from 313 cultures, were isolated from soil samples collected in Aichi and Nagano Prefectures, Japan, by the enrichment technique with 0.01 g of patchoulol per ml as the sole carbon source and gave 10-hydroxypatchoulol in more than 20% yield. Only traces of 10-hydroxypatchoulol were produced by 105 other fungal isolates. None of the bacterial strains carried out the reaction. Cultures were maintained on an agar slant containing medium G (see below) at room temperature, transferred monthly or lyophilized in 10% skimmed milk. Storage at -20 or -196C caused a loss of the transforming activity, but the cell viability kept well. Culture conditions and media for patchoulol transformation. Medium F contained 5% glucose, 0.05% NaNO3, 1.0% polypeptone, and trace metals. Medium G consisted of 1% glucose, 1% com steep liquor, and 0.1% peptone; medium Ro 422, which was used for screening, contained 5.0% glucose, 0.125% corn steep liquor, 0.2% NaNO3, 0.05% MgSO4.7H2O, 0.1% K2HPO4, 0.05% KCI, and FeSO4.7H20. A 100-ml portion of the medium in a 500-ml Erlenmeyer flask with baffles was inoculated with the spores or with 5 ml of a 24- to 72-h seed culture. After 1 to 5 days of incubation (270C, 180 strokes per min), 200 to 800 mg of patchoulol (suspended in 10 ml of 20% Tween 80) was added to each flask. The incubation was continued for an additional 168 h, and a sample was assayed by gas chromatography. Scale-up fermentations were carried out in 1- and 5-liter jar fermentors (cylinder-propeller stirring type, MJ-5-6; Marubishi Rika) under the conditions found optimal in the shaken flasks. Assay for patchoulol and 10-hydroxypatchoulol. The analysis was carried out by gas chromatographic methods with Shimazu GC-4CM(PF). A sample (1.5 ml) of the broth was diluted with 4.0 ml of water, extracted with 4.0 ml of ethyl acetate containing 0.40 mg of methylstearate per ml as the internal standard, and centrifuged. The extract (4 pi) was assayed on a column (2 m by 3-mm inner diameter) packed 187

188

SUHARA ET AL.
HO
Pithomyces sp.

APPL. ENVIRON. MICROBIOL.

HO@

02,PtO2

HO+4

Pb(OAc)4HO

PATCHOULOL

ii 10-HYDROXY

'H20H

ICOOH
NORPATCHOULENOL

-PATCHOULOL

FIG. 1. Overall process for norpatchoulenol (IV) production from patchoulol (I). with 5% SE-30 on Chromosorb W (DMCS) (carrier, helium at 30 ml/min; injection and detector temperature, 2600C; temperature at column, 2300C; detection, FID). Retention times of patchoulol, 10-hydroxypatchoulol, and the internal standards were 2.3, 4.4 and 6.0 min, respectively. The peak-height ratio was converted to the concentration value by reference to the calibration curves (range: 0.01 to 0.7 mg/ml for patchoulol, 0.02 to 1.5 mg/ml for 10-hydroxypatchoulol). Merck F254 silica gel plates were used for qualitative determinations (benzene-acetone, 5:2) and visualized by anisaldehyde-sulfuric acid (Rf values of patchoulol and 10-hydroxypatchoulol were 0.63 and 0.34, respectively). Chemicals. Patchoulol and 10-hydroxypatchoulol were provided by M. Montavon, F. Hoffmann-La Roche AG, Basel, Switzerland. TABLE 1. Hydroxylation ofpatchoulol with Pithomyces strains

SubStrain
concn

% l0-Hydroxypatchoulol pro- Other

duced at: 72-96 h 168 h 0 6
prodUCta

(mg/ml)
Pithomyces atro-olivaceus IFO 6651 Pithomyces sacchari ATCC 24323 Pithomyces cynodontis ATCC 26150 Pithomyces sp. NRJ201 Pithomyces chartarum ATCC 22637 Pithomyces chartarum NRJ210
2

2
2 2 5.6

3
15 0

19
40 0

+
+

17

20

RESULTS Biotransformation of patchoulol by Pithomyces species. When patchoulol (Fig. 1, I) (1, 3, 11) was added to the 72-h suspensions of six Pithomyces strains (final concentration of 2.0 and 5.6 g/liter) and incubated for an additional 5 to 7 days, it was converted to 10-hydroxypatchoulol and to minor products (Table 1). The soil isolate, P. chartarum NRJ210, gave a yield of 10-hydroxypatchoulol of up to 20%. ATCC 22637 of the same species produced no detectable amount of 10-hydroxypatchoulol but converted more than 50% of patchoulol to other mono- and dihydroxylated products (by thinlayer chromatography), the structures of which are under investigation. Similar products consisting of three to six distinct derivatives of patchoulol were detected in the broths of P. sacchari and P. cynodontis. Characteristics of the hydroxylation. 10Hydroxylation of patchoulol by the two most efficient strains (Pithomyces sp. NRJ201 and P. chartarum NRJ210) was further studied in flask fermentations containing medium G or F (2 to 8 g of patchoulol per liter added to 4-day-old mycelium). Figure 2 shows the time course of the hydroxylation at various substrate concentrations. Pithomyces sp. NRJ201 gave the highest yield (40 to 45%) at 2 g/liter of patchoulol, but the yield decreased inversely with the increasing substrate concentration. P. chartarum NRJ210 yielded as much as 25% of 10-hydroxypatchoulol within the range of 2 to 6 g of sub-

strate per liter (Fig. 2b). At 2 g/liter, P. chartarum NRJ210 degraded the formed 10-hydroxypatchoulol upon an extended incubation (7 days), whereas Pithomyces sp. NRJ201 did not. In repeated trials, the deviation range of the yield in 3 to 5days of fermentation by the strain NRJ201 was 40 to 50% (200 mg of patchoulol/ 100 ml, 180 strokes per min, 100 ml of medium F per 500-ml baffled flask). The reproducible gas chromatography pattern of the products is shown in Fig. 3. Figure 4 shows the effect of timing of the patchoulol addition on the yield of 10-hydroxypatchoulol. Due to a weak antifungal activity of patchoulol against Pithomyces strains, the addition of patchoulol at 0 h resulted in only a faint growth and hence no production of 10hydroxypatchoulol. Full growth was needed before the patchoulol addition for rapid production of 10-hydroxypatchoulol, although the final yield with NRJ201 was not so much affected by the timing of the patchoulol addition. Thus, in the case of 5% seed inoculation, the addition after 2 to 3 days preculture gave the best production of 10-hydroxypatchoulol in the fermentation period of 4 to 5 days. The maximal production was also influenced by the medium composition, especially by glucose concentration. When the glucose concentration in medium G was reduced from 5 to 1%, the initiation of 10-hydroxypatchoulol production was shortened to 3 days, whereas 10-hydroxypatchoulol, once produced, tended to decrease more rapidly after reaching the maximum.

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MICROBIAL HYDROXYLATION OF A SESQUITERPENE

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5 6 7 8

DAYS AFTER SUBSTRATE ADDITION

FIG. 2. Time course of 10-hydroxypatchoulol production from patchoulol by Pithomyces sp. NRJ201 (a) and P. chartarum NRJ210 (b). Medium F was used as substrate at concentrations of 2 glliter (-), 4 glliter (0), 6 glliter (*), and 8 glliter (A).

RETENTION TIME (MIN.)

FIG. 3. Gas chromatography assay for broth containing both 10-hydroxypatchoulol and patchoulol. See text for details.

Isolation of 10-hydroxypatchoulol. To isolate 10-hydroxypatchoulol, 5 ml of a seed culture of Pithomyces sp. NRJ201 was inoculated in 500-ml flasks containing 100 ml of medium G, incubated for 48 h (at which time 200 mg of patchoulol in 10 ml of saline containing 20% Tween 80 was added to each of 100 flasks), and the conversion was continued for an additional 114 h. The combined fermentation broths were extracted twice with 10 liters of dichloromethane, and the extracts were evaporated under reduced pressure. The residue was subjected to column chromatography separation on silica gel (Wako gel C-200, 5.3 by 87 cm). The material was eluted with benzene-ethyl methyl ketone (10:1), monitored by thin-layer chromatography, and collected in 15-ml fractions. Fractions 421 through 830 were combined, evaporated under reduced pressure, and gave a crystalline residue. Recrystaization from benzene-hexane (1:1) afforded 5.11 g of 10-hydroxypatchoulol as colorless needles. The mother liquor was combined with oils, obtained from fractions 357 through 420 and 831 through 1,040, which contained 10hydroxypatchoulol and small amounts of other hydroxylated products, and the solvent was evaporated. The resulting material was subjected to column chromatography on silica gel (Wako gel C-300, 3.3 by 61 cm), and the column was eluted with benzene-acetone (15:1). Fractions 71 through 120 were combined and evaporated under reduced pressure and the recrystallized residue from benzene-hexane (1:1) yielded an additional 1.79 g of 10-hydroxypatchoulol as colorless needles. The combined yield was 6.90 g (32.1% yield). Hydroxylation scale-up. A detailed examination of the effect of media composition, anti-

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APPL. ENVIRON. MICROBIOL.

tation. The overall yield by the isolation procedure in Fig. 5a was 30%. Thus, starting with 10 liters of culture fed with 20 g of patchoulol, 6.45 g of 10-hydroxypatchoulol was obtained as colorless crystal: melting point = 1040C; [a]D' = -119.5C (c = 1.0 in CHC13); proton magnetic resonance (in C0013): 83.45 (d, J = 7H2, 2H).

-I
z
0

DISCUSSION In 1973, Teisseire (12) found that the fragrant principle in patchouli oil is norpatchoulenol (Fig. 1, IV) but that its content in the oil amounted

30
a 20 ~~ADDITION
PATCHOULOL

.8
\7

100

IN10C_UXATIONN

L5
2
3

1 _
3
5

DAYS AFTER SUBSTRATE ADDITION

0.

FIG. 4. Effect oftiming of the substrate addition to patchoulol on the production of 10-hydroxypatchoulol. Addition of patchoulol at days 1 (0), 2 (A), 3 (A), and 4 (0) after the inoculation. Substrate concentrations were 2 glliter (Pithomyces sp. NRJ 201) (a) and 6 glliter (P. chartarum NRJ210) (b). Other conditions were the same as described in legend to Fig. 3.

00
0

0.6

PATCHOU ADDITION

L01

0.4-

foam, initial pH, aeration, agitation, and tem0.2perature on the hydroxylation of patchoulol to 10-hydroxypatchoulol was made and the followC 2 3 4 1 ing conditions were found optimal: (i) a culture 5 6 7 DAYS AFTER SUBSTRATE ADDITION medium of 1.0% corn steep liquor, 2.0% glucose, 0.5% peptone, 0.2% NaNO3, 0.05% FeSO4. 7H20, FIG. 5. Time course of 10-hydroxypatchoulol pro0.1% K2HPO4 (pH 5.8), 400 ml per 1-liter and 3 liters per 5-liter jars; (ii) inoculum size of 5% duction in 5-liter jar fermentation with Pithomyces and P. NRJ210 (b) at g/ seed; (iii) a culture period before the substrate sp. NRJ201 (a)various chartarum concentrations 2(b). liter (a) and substrate addition of about 30 h; (iv) a substrate of 2 g/ Modified medium, Ro 422 contained 2%o glucose, 0.5% liter (final) in 20% Tween 80; (v) aeration at 0.5 polypeptone, 1% corn steep liquor, 0.2%o NaNO3, 0.05% volumes of air/volume of medium per min; (vi) MgSO4. 7H20, 0.1% K2HPO4, and 0.05% FeSO4. 7H20 agitation at 300 rpm; (vii) temperature at 24 to in a total volume of 3 liters. Other conditions: (a) 270C; (viii) pH, no control; and (ix) Shin-etsu aeration, 0.5 volume of air per volume of medium per Silicone KM-70 antifoam at 0.25 to 0.5%. Under min; agitation, 300 rpm; temperature, 26.5 0.50C; these conditions, Pithomyces NRJ201 gave con- pH, no control and (b) aeration, 0.5 volume of air per volume of medium per agitation, stantly a 35 to 45% (1-liter jar) and a 25 to 35% perature, 26.5 0.5C; min; no control;300 rpm; tempH, (5-liter jar) yields (Fig. 5a) of 10-hydroxypatch- 48 h; preculture, 31 h, inoculum size,seed culture, 5%. (a) 10oulol from 2 g of patchoulol per liter, whereas P. hydroxypatchoulol (0) and patchoulol (0) were prochartarum NRJ210 produced 0.8 g of 10-hy- duced. (b) 10-Hydroxypatchoulol was produced at the droxypatchoulol per liter from 4 g of patchoulol substrate concentrations of 2 glliter (0), 4 glliter (0), and 8 glliter (A). per liter (15 to 22% yield) in 5-liter jar fermen-

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MICROBIAL HYDROXYLATION OF A SESQUITERPENE

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only to 0.3% or less. Our study was undertaken to develop a practical synthesis of norpatchoulenol from patchoulol, the major component in the oil. Since the conversion of 10-hydroxypatchoulol to norpatchoulenol via the intermediate (Fig. 1, III) is easily attained by chemical methods (9), we focused on the selective conversion of patchoulol to 10-hydroxypatchoulol, which is practically impossible to obtain by chemical methods. The study led to the establishment of a microbial process which yields 35 to 50% of 10-hydroxypatchoulol from patchoulol in 1- and 5-liter fermentation jars. Although rabbit and dog liver homogenates are reported to perform it (9; German patent 2,529,603, February 1976; Swiss application 10,676/74, August 1974; U.S. patent 4,055,594, October 1977), this microbial hydroxylation is the only practical method for obtaining 10-hydroxypatchoulol. Hydroxylation of a-kessyl alcohol (4), cyperotundone (5), quaioxide (6), and liguloxide (7) by Cunninghamella, Corticium, Mucor, and Streptomyces species, respectively, has been reported and contributed to elucidate the stereochemistry of these terpenes. Our findings demonstrate that Pithomyces species are also able to hydroxylate these kinds of compounds and that they can be used to prepare the desired product as a result of regio-selective hydroxylation. Whether this selectivity can be ascribed to the intrinsic nature of a particular hydroxylase or is due to the ratio of more than several enzymes of different regio-specificities or to the conditional regulation of regio-nonspecific hydroxylase(s) is not known at present. The enzymatic system appears to be noninducible, since the addition of patchoulol as an inducer did not cause any increase of 10-hydroxypatchoulol. As in the case of other sesquiterpene hydroxylations (4, 6, 7), the strains found positive by our screening were exclusively fungi, and bacteria were ineffective in this respect. Strain NRJ210 was identified as a variety of P. chartarum by its conidiophore morphology

and by other taxonomic properties. Its hydroxylating ability differs greatly from the type culture strain of P. chartarum ATCC 22637 (Table 1). Strain NRJ201 resembled P. cynodontis but could be differentiated from it, thus indicating a novel taxon. Taxonomic details will be reported elsewhere.
LITERATURE CITED
1. Buchi, G., and R. E. Erickson. 1956. Terpenes. V. The structure of patchouly alcohol. J. Am. Chem. Soc. 78: 1262-1263. 2. Charney, W., and H. L. Herzog. 1967. Microbial transformations of steroids. Academic Press, Inc., New York. 3. Hefendehl, F. W. 1977. Die analytische Bewertung von Patchouliol. Seifen Oele Fette Wachse 103:159-161. 4. Hikino, H., Y. Tokuoka, Y. Hikino, and T. Takemoto. 1968. Biochemical syntheses. I. Microbial transformation of a-kessyl alcohol to kessyl glycol and kessane-2,B, 7-diol. Tetrahedron 24:3147-3152. 5. Hikino, H., K. Aota, Y. Tokuoka, and T. Takemoto. 1968. Biochemical syntheses. II. Microbial transformation of cyperotundone to sygeonol and isopatchoul-4en-3-on-8a-ol. Chem. Pharm. Bull. (Tokyo) 16:10881090. 6. Ishii, H., T. Tozyo, M. Nakaamura, and H. Minato. 1970. Studies on sesquiterpenoids. XIX. Structure and absolute configuration of liguoxide, liguloxidol and liguloxidol acetate. Tetrahedron 26:2911-2918. 7. Ishii, H., T. Tozyo, 1W. Nakamura, and E. Yunke. 1972. Microbial transformation of sesquiterpenoids. V. Preparation of 1-epiliguloxide, the sixth stereoisomer of guaioxide. Chem. Pharm. Bull. (Tokyo) 20:203-205. 8. Kieslich, K. 1976. Microbial transformations of non-steroid cyclic compounds, p. 56. George Thieme Publishers, Stuttgart. 9. Luu, B., G. Ourisson, and P. Teisseire. 1975. Hydroxylation of patchoulol by rabbits. Hemi-synthesis of norpatchoulenol, the odour carrier of patchouli oil. Tetrahedron Lett., p. 2211-2214. 10. Peterson, D. H. 1963. Microbial transformations of steroids and their application to the preparation of hormones and derivatives, p. 537-606. In C. Rainbow and A. H. Rose (ed.), Biochemistry of industrial microorganisms. Academic Press, Inc., London. 11. Senich, v. Y., G. Z. Schisdhkov, and B. V. Artem'ev. 1976. On the content of patchouli alcohol in patchouli oil samples obtained by extraction. Izv. Vyssh. Uchebn. Zaved. Pishch. Tekhnol. 1:4445. 12. Teisseire, P. 1973. Chemistry of patchouli oil. Riv. Ital. Essenze Profumi Piante Offic. Aromi Saponi Cosmet. Aerosol 55:572-592.