Anda di halaman 1dari 5

APPLIED

AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1981, p. 187-191

Vol.42,No. 2

0099-2240/81/080187-05$02.00/0

Regio-Selective 10-Hydroxylation ofPatchoulol, a 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, fourstrainsofPithomyces species were foundtocarry outregio-selectivehydroxylation ofpatchoulol, a sesquiterpene, to

10-hydroxypatchoulol: Pithomycessp. 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 10- hydroxypatchoulol was obtained in 25 to 45% yields in 1- to 5-literfermentation jarsat 2 to 4 g ofpatchoulolper literand isolated as pure material in 30% yields.

Regio-orenantioselectivereactionshavebeen

ofconsiderableimportanceinorganicsyntheses. Microbes are well known to carry out such re-

actions,asillustratedbytheoxidationofvarious steroids, some of which are produced by mi-

crobeson an industrialscale (2,10).Incontrast, only a few reports have been published on the microbial regio-selective reactions of terpenes (8). A recent example describes the selective hydroxylation ofpatchoulol [Fig. 1,I;3,4,4afl, 5, 6fl, 7, 8, 8a-octahydro-4a, 8al?, 9, 9-tetra- methyl-1,6-methanonaphthalen-1,8(2H)-ol], a

were used. In addition, two strains ofthis genus (Pi- thomyces sp. NRJ201-p-2f [NRJ201] and P. char-

tarum NRJ210-p-lf[NRJ210]),selectedfrom313cul-

tures, were isolated from soil samples collected in Aichi and Nagano Prefectures, Japan, by the enrich- ment techniquewith0.01 gofpatchoulolperml asthe

sole carbon source and gave 10-hydroxypatchoulol in

more than20% yield.Onlytraces of10-hydroxypatch-

oulolwereproducedby105otherfungalisolates.None

ofthe bacterial strains carried out the reaction. Cul- tures were maintained on an agar slant containing medium G (see below) at room temperature, trans-

ferred monthly or lyophilized in 10% skimmed milk.

Storage at -20 or -196°C caused a lossofthe trans-

  • sesquiterpeneisolatedfrompatchoulioil(essen- formingactivity,butthe cellviabilitykeptwell. Culture conditions and media for patchoulol

tial oil fragrance from Pogostemon cablin Benth.) atC-10byrabbitsand dogsasreported

by Luu et al. (9).We have screened soilmicro-

transformation. Medium F contained 5% glucose,

0.05% NaNO3, 1.0% polypeptone, and trace metals.

  • organismscapableofsucharegio-specifictrans- Medium G consisted of 1% glucose, 1% com steep liquor, and 0.1% peptone; medium Ro 422, which was

formation and found that a few Pithomyces

strains converted this substrate to 10-hydroxy-

patchoulol (Fig. 1, II) in a high yield. Other

fungi,includingPenicillium, Paecilomyces, and

Phoma strains, also carry out this reaction as

reported elsewhere (A. Fujiwara, M. Tazoe, Y. Shiomi,andM. Fujiwara,Abstr. VIthInt.Ferm. Symp., 1980, F-20.9, p. 176). This reaction may providean economic productionofnorpatchou- lenol(9;Fig. 1,IV),an expensivefragrance (12).

Thispaper describesthishydroxylationprocess

by afew strainsofPithomyces.

(Portions ofthis work were presented at the 10th annual meeting ofthe Pharmaceutical So- cietyofJapan [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. char-

tarum ATCC 22637,

and P. atro-olivaceus IFO 6651

usedforscreening,contained5.0%glucose,0.125%corn

steep liquor, 0.2% NaNO3, 0.05%

MgSO4.7H2O, 0.1%

K2HPO4, 0.05% KCI, and FeSO4.7H20. A 100-mlpor-

tionofthemedium ina 500-mlErlenmeyerflaskwith baffleswas inoculatedwith the sporesor with 5 ml of

a 24- to 72-h seed culture. After 1 to 5 days ofincu- bation (270C, 180 strokes per min), 200 to 800 mg of

patchoulol (suspendedin 10ml of20% Tween 80) was

addedtoeachflask.The incubationwas continuedfor

an additional 168 h,and a

samplewas

assayedbygas

carried

chromatography. Scale-upfermentationswere

outin 1- and

5-literjarfermentors

stirringtype,MJ-5-6;Marubishi

(cylinder-propeller

Rika)

under the con-

ditionsfound optimalinthe

Assay for

patchoulol

shakenflasks.

and

10-hydroxypatch-

bygas

chromato-

oulol. The analysiswas carriedout

graphicmethods

with

ple (1.5 ml)

of

Shimazu GC-4CM(PF). A sam-

the broth was diluted with 4.0 ml of

water,extractedwith4.0mlofethylacetatecontaining

0.40mg

dard, and

ofmethylstearateperml

as

centrifuged.

The extract

by 3-mm inner

on a column (2 m

theinternalstan-

(4pi)

was

diameter)

assayed

packed

187

  • 188 SUHARA ET AL.

APPL. ENVIRON. MICROBIOL.

HO

Pithomyces sp.

PATCHOULOL

HO@

ii

02,PtO2

'H20H

10-HYDROXY

-PATCHOULOL

HO+4

Pb(OAc)4 HO

»

ICOOH

NOR-

PATCHOULENOL

FIG. 1. Overallprocessfornorpatchoulenol (IV)productionfrompatchoulol (I).

with 5% SE-30 on Chromosorb W (DMCS) (carrier,

helium at30ml/min; injectionand detectortempera-

ture,2600C;temperature atcolumn,2300C;detection, FID). Retention times of patchoulol, 10-hydroxy- patchoulol, 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 1.5mg/ml for 10-hydroxypatchou-

lol).

MerckF254silicagelplateswereusedforqualitative

determinations (benzene-acetone, 5:2) and visualized by anisaldehyde-sulfuric acid (Rfvaluesofpatchoulol and 10-hydroxypatchoulol were 0.63 and 0.34,respec- tively).

Chemicals. Patchoulol and

10-hydroxypatchoulol

F. Hoffmann-La

were provided by M. Montavon,

Roche AG, Basel,Switzerland.

TABLE 1. Hydroxylationofpatchoulolwith

Pithomycesstrains

Strain

Sub-

concn

%

l0-Hydroxy- Other

patchoulolpro-

duced at:

prod-

(mg/ml) 72-96h 168h

UCta

Pithomyces atro-oliva-

ceusIFO 6651

Pithomyces sacchari

ATCC 24323

Pithomyces cynodontis

ATCC 26150

Pithomycessp.NRJ201

Pithomyces chartarum

ATCC 22637

Pithomyces chartarum

NRJ210

2

2

2

2

2

5.6

0

0

3

15

0

17

6

0

19

40

0

20

±

+

+

+

±

+

RESULTS

Biotransformation of patchoulol by Pi-

thomyces species. When patchoulol (Fig. 1,I) (1, 3, 11) was added to the 72-h suspensions of

sixPithomycesstrains(finalconcentrationof2.0

strate per liter (Fig. 2b). At 2 g/liter, P. char-

tarum NRJ210 degradedtheformed 10-hydrox-

ypatchoulol upon an extended incubation (7 days),whereas Pithomyces sp.NRJ201 didnot. In repeated trials, the deviation range of the

yieldin3to5daysoffermentationbythestrain

and 5.6 g/liter) and incubated for an additional

NRJ201 was 40 to 50% (200 mg

ofpatchoulol/

  • 5to7days,itwasconvertedto10-hydroxypatch- 100 ml, 180 strokes per min, 100 ml ofmedium

ouloland to minor products (Table 1).

The soilisolate,P. chartarum NRJ210, gave

a yield of 10-hydroxypatchoulol of up to 20%.

ATCC 22637 ofthe same species produced no

Fper500-mlbaffledflask).Thereproduciblegas

chromatography pattern of the products is shown inFig.3.

Figure 4 shows the effect of timing of the

detectableamount of10-hydroxypatchoulolbut

convertedmore than50% ofpatchoulolto other mono- and dihydroxylated products (by thin-

layerchromatography), the structures ofwhich are under investigation. Similar products con- sisting of three to six distinct derivatives of

patchoulol were detected in the broths of P.

sacchariand P. cynodontis. Characteristics ofthe hydroxylation. 10-

Hydroxylation of patchoulol by the two most efficientstrains (Pithomycessp.NRJ201 andP. chartarumNRJ210) wasfurtherstudiedinflask 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 concen- trations.Pithomycessp.NRJ201 gave thehigh- est yield (40 to 45%) at 2 g/liter ofpatchoulol,

but the yield decreased inversely with the in- creasing substrate concentration. P. chartarum NRJ210 yieldedasmuch as 25% of10-hydroxy- patchoulol within the range of2 to 6 g ofsub-

patchouloladdition on the yieldof10-hydroxy-

patchoulol.Due toaweak antifungalactivityof

patchoulol against Pithomyces strains, the ad-

dition of patchoulol at 0 h resulted in only a faint growth and hence no production of 10- hydroxypatchoulol. Fullgrowth was needed be- forethepatchouloladditionforrapidproduction

of 10-hydroxypatchoulol, although the final

yieldwithNRJ201 was notsomuch affectedby

the timing ofthe patchoulol addition. Thus, in the case of 5% seed inoculation, the addition after 2 to 3 days preculture gave the best pro- duction of10-hydroxypatchoulol inthe fermen-

tationperiodof4 to 5 days.

The maximal

production was also influenced

by the medium composition, especially by glu-

coseconcentration.Whentheglucoseconcentra-

tioninmedium G wasreducedfrom 5to 1%,the

initiation of 10-hydroxypatchoulol production

was shortened to 3 days, whereas 10-hydroxy-

patchoulol, once produced, tended to decrease

more

rapidlyafterreachingthe maximum.

VOL. 42,1981

MICROBIAL HYDROXYLATION OF A SESQUITERPENE

189

5

6

7

8

3

DAYS AFTER SUBSTRATE ADDITION

FIG. 2. Time course of10-hydroxypatchoulolproduction

and P. chartarum NRJ210 (b).Medium F was used as

from patchoulol by Pithomyces sp. NRJ201 (a)

substrate at concentrations of2glliter (-), 4glliter

(0),6glliter(*),and8glliter(A).

O

2

4

6

8

RETENTION TIME (MIN.)

FIG. 3. Gas chromatography assay for broth con- taining both 10-hydroxypatchoulol and patchoulol.

See textfordetails.

Isolationof10-hydroxypatchoulol.Toiso-

late10-hydroxypatchoulol,5mlofaseedculture

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 of100 flasks), and

the conversion was continued for an additional

  • 114 h. The combined fermentation broths were

extracted twice with 10 liters of dichlorometh-

ane, and the extracts were evaporated under

reduced pressure. The residue was subjected to

column chromatographyseparationonsilicagel (Wako gel C-200, 5.3 by 87 cm). The material was eluted with benzene-ethyl methyl ketone

(10:1),monitoredbythin-layerchromatography,

and collected in 15-ml fractions. Fractions 421

through 830 were combined, evaporated under

reducedpressure,andgaveacrystallineresidue.

Recrystaization from benzene-hexane (1:1) af- forded 5.11 g of 10-hydroxypatchoulol as color-

less needles. The mother liquor was combined with oils, obtained from fractions 357 through

  • 420 and 831 through 1,040,which contained 10-

hydroxypatchoulol and small amounts ofother

hydroxylated products,

evaporated. The

jected to

and the solvent was

resulting material was sub-

column chromatography on silica gel

cm),and thecolumn

(WakogelC-300,3.3by61

was eluted

with benzene-acetone (15:1). Frac-

tions 71 through 120 were combined and evap-

orated under reduced pressure and the recrys- tallized residue from benzene-hexane (1:1)

yieldedan additional1.79gof10-hydroxypatch-

oulol as colorless needles. The combined

was 6.90 g (32.1%yield).

Hydroxylation

nation ofthe

effect

scale-up.

yield

A detailed exami-

ofmedia composition, anti-

  • 190 SUHARA ET AL.

-I

z

0

J

0

APPL. ENVIRON. MICROBIOL.

tation. The overallyieldby theisolation proce-

dure in Fig. 5a was 30%. Thus, startingwith 10

litersofculturefedwith 20 g ofpatchoulol, 6.45

gof10-hydroxypatchoulolwasobtainedascolor-

less crystal: melting point = 1040C; [a]D' = -119.5°C (c = 1.0 in CHC13); proton magnetic

resonance (inC0013): 83.45 (d,J = 7H2, 2H).

DISCUSSION

In 1973,Teisseire (12)foundthatthefragrant

principleinpatchoulioilisnorpatchoulenol(Fig.

1, IV) but that its content in the oil amounted

_

3

5

7

9

DAYS AFTER SUBSTRATE ADDITION

FIG. 4. Effectoftimingofthesubstrate addition to

patchoulol on theproduction of10-hydroxypatchou- lol. Addition ofpatchoulol at days 1 (0), 2 (A), 3 (A), and 4 (0) after the inoculation. Substrate centrations were 2 glliter (Pithomyces sp. NRJ 201)

con-

(a) and 6 glliter (P. chartarum NRJ210) (b). Other

conditions were the same as described in legend to

Fig. 3.

foam, initial pH, aeration, agitation, and tem- perature on the hydroxylation of patchoulol to 10-hydroxypatchoulol was made and the follow- ing conditions were found optimal: (i) a culture

medium of 1.0% corn steep liquor, 2.0% glucose,

0.5% peptone, 0.2% NaNO3, 0.05% FeSO4.7H20, 0.1% K2HPO4 (pH 5.8),400 ml per 1-liter and 3

liters per 5-liter jars; (ii) inoculum size of 5% seed; (iii) a culture period before the substrate

addition of about 30 h; (iv) a substrate of 2 g/

liter (final) in 20% Tween 80; (v) aeration at 0.5

volumes ofair/volume ofmedium per min; (vi)

agitation at 300 rpm; (vii) temperature at 24 to

270C; (viii) pH, no control; and (ix) Shin-etsu

Silicone KM-70 antifoam at 0.25 to 0.5%. Under

these conditions,PithomycesNRJ201 gave con- stantly a 35 to 45% (1-literjar) and a 25 to 35% (5-literjar) yields (Fig. 5a) of 10-hydroxypatch-

oulolfrom 2g ofpatchoulolper liter,whereasP. chartarum NRJ210 produced 0.8 g of 10-hy-

droxypatchoulol per literfrom 4 g ofpatchoulol

per liter (15 to 22% yield) in 5-literjar fermen-

 

30

.8

a 20

PATCHOULOL

~~ADDITION

\7

6

 

IN10C_UXATIONN

 

L5

0

10-

 

1

0

1

2

3

4

5

0.

00

b

0

0.6

PATCHOU L01

ADDITION

0.4-

0.2-

C 1

2

3

4

5

6

7

DAYS AFTER SUBSTRATE ADDITION

FIG. 5. Time course of10-hydroxypatchoulolpro-

duction in 5-literjar

fermentation with Pithomyces

sp. NRJ201 (a)and

P. chartarum NRJ210 (b)at2g/

liter (a) and various substrate concentrations (b).

Modifiedmedium,

polypeptone, 1%

Ro 422contained2%oglucose, 0.5%

cornsteepliquor,0.2%oNaNO3, 0.05%

MgSO4. 7H20, 0.1%K2HPO4, and 0.05%FeSO4. 7H20

in a total volume of 3 liters. Other conditions: (a) aeration, 0.5volume ofairper volume ofmediumper

min; agitation, 300 rpm; temperature, 26.5 ±

0.50C;

pH, no controland (b)aeration, 0.5volume ofairper volume ofmediumper min; agitation, 300 rpm; tem-

perature, 26.5 ± 0.5°C;pH,

48 h; preculture,

no control; seed culture,

  • 31 h, inoculum size, 5%. (a) 10-

hydroxypatchoulol (0) andpatchoulol (0) werepro-

duced. (b)10-Hydroxypatchoulolwasproducedatthe

substrate concentrations of 2

(0),and 8glliter(A).

glliter (0), 4 glliter

VOL. 42,1981

MICROBIAL HYDROXYLATION OF A SESQUITERPENE

191

only to 0.3% or less. Our study was undertaken

to develop a practical synthesis ofnorpatchou-

lenol from patchoulol, the major component in

the oil. Since the conversion of 10-hydroxy-

patchoulol to norpatchoulenol via the interme- diate (Fig. 1,III) iseasilyattained by chemical

methods (9),we focusedontheselectiveconver-

sion of patchoulol to 10-hydroxypatchoulol, which is practically impossible to obtain by chemical methods. The study led to the estab- lishment ofa microbialprocess which yields 35

to50%of10-hydroxypatchoulolfrompatchoulol

in 1- and 5-liter fermentation jars. Although rabbit and dog liver homogenates are reported to perform it (9;German patent 2,529,603, Feb-

ruary 1976; Swiss application 10,676/74,August 1974; U.S. patent 4,055,594, October 1977), this

microbial hydroxylation is the only practical

method forobtaining 10-hydroxypatchoulol.

Hydroxylation ofa-kessyl alcohol (4), cyper-

otundone (5), quaioxide (6), and liguloxide (7)

by Cunninghamella, Corticium, Mucor, and

Streptomycesspecies,respectively, has beenre- ported and contributed to elucidate the stereo- chemistry ofthese terpenes. Our findings dem- onstratethatPithomycesspeciesarealsoableto hydroxylate these kindsofcompounds and that theycan beusedtoprepare thedesiredproduct

as a result of regio-selective hydroxylation.

Whether this selectivity

can be ascribed to the

intrinsicnature ofaparticularhydroxylaseoris

duetotheratioofmore thanseveralenzymes of differentregio-specificitiesor to the conditional

regulationofregio-nonspecifichydroxylase(s) is

not known at present. The enzymatic system

appearstobenoninducible,sincetheadditionof patchoulol as an inducer didnot cause any in-

crease of 10-hydroxypatchoulol. As in the case ofother sesquiterpene hydroxylations (4, 6, 7), thestrainsfoundpositivebyour screeningwere

exclusively fungi, and bacteria were ineffective inthisrespect. Strain NRJ210 was identified as a variety of P. chartarum by its conidiophore morphology

and by other taxonomic properties. Itshydrox-

ylatingabilitydiffersgreatlyfrom the type cul- turestrainofP. chartarum ATCC 22637 (Table 1).StrainNRJ201 resembled P. cynodontisbut couldbe differentiatedfromit,thusindicatinga noveltaxon. Taxonomic detailswillbereported elsewhere.

LITERATURE CITED

  • 1. Buchi, G., and R. E. Erickson. 1956. Terpenes. V. The structure ofpatchouly alcohol. J. Am. Chem. Soc. 78:

1262-1263.

  • 2. Charney, W., and H. L. Herzog. 1967. Microbial trans- formationsofsteroids.AcademicPress,Inc.,NewYork.

  • 3. Hefendehl, F. W. 1977. Die analytische Bewertung von Patchouliol. SeifenOele FetteWachse 103:159-161.

  • 4. Hikino,H.,Y.Tokuoka,Y.Hikino,andT.Takemoto.

 

1968.

Biochemical syntheses. I. Microbial transforma-

tionofa-kessylalcoholtokessylglycolandkessane-2,B,

7-diol.Tetrahedron24:3147-3152.

  • 5. Hikino, H., K. Aota, Y. Tokuoka, and T. Takemoto.

 

1968.

Biochemical syntheses. II.Microbial transforma-

tion of cyperotundone to sygeonol and isopatchoul-4- en-3-on-8a-ol. Chem. Pharm. Bull. (Tokyo) 16:1088-

1090.

  • 6. Ishii, H., T. Tozyo, M. Nakaamura, and H. Minato.

 

1970.

Studieson sesquiterpenoids. XIX. Structure and

absolute configuration ofliguoxide, liguloxidol and lig- uloxidolacetate. Tetrahedron 26:2911-2918.

  • 7. Ishii,H.,T.Tozyo,1W.Nakamura, andE.Yunke. 1972.

 

Microbial transformationofsesquiterpenoids. V. Prep- aration of 1-epiliguloxide, the sixth stereoisomer of guaioxide. Chem. Pharm. Bull. (Tokyo) 20:203-205.

  • 8. Kieslich, K. 1976. Microbial transformations ofnon-ste- roid cyclic compounds, p. 56. George Thieme Publish-

 

ers,Stuttgart.

9.

Luu, B.,G. Ourisson, and P. Teisseire. 1975. Hydrox-

 

ylationofpatchoulolbyrabbits.Hemi-synthesisofnor-

patchoulenol, the odour carrierofpatchouli oil.Tetra- hedron Lett.,p.2211-2214.

  • 10. Peterson, D. H. 1963. Microbial transformations ofste- roids and their application to the preparation ofhor- mones and derivatives, p. 537-606. In C. Rainbow and A. H. Rose (ed.), Biochemistry of industrial microor-

 

ganisms. Academic Press,Inc.,London.

  • 11. Senich, v.Y.,G. Z.Schisdhkov, and B. V. Artem'ev.

 

1976.

On the content ofpatchouli alcohol in patchouli

oilsamplesobtainedbyextraction. Izv.Vyssh.Uchebn. Zaved. Pishch. Tekhnol. 1:4445.

  • 12. Teisseire, P. 1973. Chemistry ofpatchouli oil.Riv. Ital.

Essenze Profumi Piante Offic. Aromi Saponi Cosmet.

Aerosol 55:572-592.