Formation of farnesyl oleate and three other farnesyl fatty acid esters by cell-free extracts from Botryococcus braunii B race

Pergamon

0031-9422(94)EOllS-9

Phptochemtnry.

Vol

36, No.

5. pp.

1203

1207.

1994

Copyright :c 1994 Elvwer Science Ltd PnnlcdI” G,,l Bnlrun. All qhts reserved 00319422,‘94 57.00+000

FORMATION OF FARNESYL OLEATE AND THREE OTHER FARNESYL FATTY ACID ESTERS BY CELL-FREE EXTRACTS FROM BOTRYOCOCCUS BRAUNZI B RACE HIT~SHI

INOUE,

TATSUMI

KORENAGA,

HIROSHI

SAGAMI,

KYOZO Institute

for Chemical

Reaction

TANETOSHI

KOYAMA,

HIROSHI SUGIYAMA

and

OGURA

Science, Tohoku University, 2-1-I Katahira, Aobaku. Sendai, 980, Japan (Recebed in revised form 6 January 1994)

Key Word Index-Botryococcus

braunii;

Chlorophyceae;

alga; farnesol; fatty acid esters.

Farnesyl oleate (3,7,1 l-trimethyl-2,6,lO-dodecatrienyl9-octadecenoate), farnesyl palmitate (3,7,1 l-trimethyl-2,6,10-dodecatrienyl hexadecanoate), farnesyl palmitoleate (3,7,1 I-trimethyl-2,6,10-dodecatrienyl9-hexadecenoate) and farnesyl linolenate (3.7.1 I-trimethyl-2,6,10-dodecatrienyl9,12,15-octadecatrienoate) were obtained by incubating farnesol with fractions from homogenized cells of Botryococcus braunii B race. Farnesyl oleate was also found among metabolites derived from mevalonic acid in feeding experiments with the alga. Abstract-

INTRODUCTION

RESULTS AND DISCIJ!33IOK

The green colonial microalga Botryococcus hraunii is well-known for its unusually high content of hydrocarbons. On the basis of the chemical structures of the hydrocarbons they produce, they have been classified into three races. The A race produces straight-chain hydrocarbons, alkadienes and trienes, whilst the B race yields isoprenoid hydrocarbons termed botryococcenes [l] and the L race algae synthesize lycopadiene (a tetraterpenoid hydrocarbon) [2]. For the biosynthesis of the hydrocarbons, it has been shown that the direct precursor of non-isoprenoid hydrocarbons of the A race is oleic acid [3]. By analysing the results of feeding experiments with stereospecifically ‘Hor ‘H-labelled farnesol, Huang and Poulter [4] have proposed that botryococcenes of the B race are biosynthesized through presqualene diphosphate, which is well-established as a key precursor of squalene biosynthesis. It is noteworthy that farnesol is efficiently incorporated into these irregular isoprenoids. This implies the occurrence of enzymes which are responsible for conversion of farnesol into farnesyl diphosphate. However, the possibility cannot be excluded that botryococcenes are biosynthesized from farnesol by a way not involving farnesyl diphosphate. In an attempt to clarify these problems, we followed up in detail the conversion of radiolabelled farnesol incubated with fractions from homogenized cells of the algae. During this study, it was found that new products less polar than farnesol were formed in abundance. This paper is concerned with the identification of these products.

Formation

of products

derived from

[ 1-‘4Clfarnesol

After l&12 days ofcultivation aerated with CO, (5%)enriched air, cells of the Berkeley strain [5] of B. hraunii B race were harvested by filtration. When the cells were homogenized with buffer and centrifuged at 1200 9. the mixture was separated into three layers, top layer, supernatant layer and sediment. The top layer was turbid and floating on the supernatant layer. A mixture of the top layer and the supernatant is referred to as ‘crude extract’. [ 1-‘4C]FarnesoI was incubated with the crude extract for 70 hr at 25’. and the products extracted with hexane. TLC analysis of the hexane extract showed that 10.8% of farnesol was converted to less polar products than farnesol, although formation of labelled botryoccene was not observed. A large amount of endogenous hydrocarbons associated with the top layer was removed by silica gel column chromatography. The radioactive products less polar than farnesol were roughly separated

1203

RCOO

1 2 3 4

R R R R

= I = =

CH3(CH2),4 CH,(CH&CH=CH(CH2)7 CH3(CH2)sCH=CH(CH& CH3(CH2CH=CH)3CH2(CH2)(1

Farnesyl Table

2. NMR spectral

Assignment

“C (6)

‘H [a. J (Hz)]

COSY cross-peaks

DEPT

1 2 3 4-7, 12-15 8, 11 9, 10 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

173.9 34.4 25.0 29.1-29.8* 27.2, 27.2 129.8, 30.0 31.7 22.7 14.1 61.2 116.4 142.1 39.5 26.2 123.6 135.5 39.7 26.7 124.3 131.3 25.7 16.5 16.0 17.7

2.30 1.62 1.28 2.00 5.34 1.28 1.28 0.88 4.59 5.34

_

C

1.62 2.30. 1.28 1.62, 2.00 1.28, 5.34 2.00

CH, CH, CH, CH,

3. NMR spectral

(ZH, f, 7.7) (ZH, m) (16H. m) (4H, m) (2H, m) (2H, m) (ZH, m) (3H, I, 7.0) (2H, d. 7.0) (I H, m)

1

13C(6) 174.0

CH CH, CH, Me

0.88

1.28 5.34, 1.71 4.59, 1.71

CH, CH C

2.06 (2H, m) 2.12 (2H. m) 5.10 (IH, m)

2.12 2.06, 5.10. 1.6Ot 2.12. 1.60

CH, CH, CH C

2.00 (2H. m) 2.06 (2H. m) 5.09 (IH, m)

2.06 2.00, 5.09. 1.68t. 1.6Ot 2.06, 1.68, 1.60

1.68 1.71 1.60 1.60

5.09, 5.34, 5.10, 5.09,

CH, CH, CH C Me Me Me Me

data for 1 (600 MHz,

(3H. (3H, (3H, (3H.

s) s) s) s)

2.06t 4.59 2.12t 2.06t

moiety.

centrifugation

of homogenized

cells. Although

the forma-

of four fatty acid esters of farnesol was observed under in vitro conditions, only farnesyl oleate was clearly detected among the metabolites of mevalonic acid. The radioactivities associated with the other three esters were too low to indicate significant incorporation from mevalonic acid (data not shown). The substrate specificity of the enzyme responsible for the formation of fatty acid ester is not stringent as shown by in vitro experiments, but the in uiuo esterification might be oriented to discriminate the oleoyl donor from the other acyl ones. In view of the unusually abundant production of the irregular triterpenoid hydrocarbons, botryococcenes, it is an attractive hypothesis that the fatty acid ester of farnesol serves as storage of the farnesyl moiety required for botryococcene biosynthesis. tion

CDCl,) Assignment

1205

data for 2 (600 MHz. CDCI,)

*29.1, 29.1, 29.2, 29.3, 29.5, 29.7, 29.8. t Weak coupling. Cl -Cl 8; oleoyl moiety, Cl9- C33; farnesyl

Table

hraunii

esters from Botryococcus

‘H [&J(Hz)I -

2 3 4-13 14 15 16 17 18 19

34.4 25.0 29.2-29.7. 31.9 22.7 14.1 61.2 116.4 142.1

2.30 1.62 1.25 1.25 1.25 0.88 4.59 5.34 _

(2H, t, 7.6) (2H. m) (20H, m) (2H, m) (2H, m) (3H. t, 7.0) (2H. d, 7.1) (I H, m)

20 21 22 23 24 25 26 27 28 29 30 31

39.5 26.2 123.6 135.5 39.7 26.7 124.3 131.3 25.7 16.2 16.0 17.7

2.06 (2H, m) 2.12 (2H. m) 5.10 (IH, m) 1.98 (2H, f, 7.7) 2.06 (2H. m) 5.09 (IH, m) 1.68 1.71 1.60 1.60

(3H, (3H, (3H, (3H,

s) s) s) s)

+29.2, 29.3, 29.4, 29.5, 29.6, 29.7, 29.7, 29.7. Cl-C16; palmitoleoyl moiety. Cl7-C31; famesyl moiety.

EXPERIMENTAL

General. ‘H and “CNMR spectra were recorded using residual CHCI, as int. standard S = 7.26 and 77.0, respectively. Both high-resolution MS and EI-GCMS analyses were carried out with a I m OVl column. Adsorbent for CC (0.8 x 10 cm) was silica gel (23&400 mesh). Prep. TLC was carried out on silica gel (2 mm thick) with benzene-EtOAc (9: 1). Reverse-phase radioHPLC was carried out using 2 columns (Waters 8Cl810 p. 0.8 x IO cm and YMC R-ODS-5,0.5 x 50 cm)

H.

1206 Table

INCNIE

4. NMR spectral data for 3 (600 MHz, CDCI,)

Assignment 1 2 3 4-7. 12. 13 8, II 9. JO 14 15 16 I7 18 I9 20 21 22 23 24 25 26 27 28 29 30 31

‘-‘C (6) _*

‘H [&J (Hz)] -_.

34.4 25.0 [email protected] 27.2. 27.2 129.8. 130.0 31.2 22.7 14.1 61.2 116.4 *

2.30 1.62 1.30 2.01 5.34 1.30 1.30 0.88 4.59 5.34

39.5 26.2 123.6

2.06 (2H, m) 2. I2 (2H. m) 5.10 (IH. m)

(2H. I, 7.6) (2H, m) (12H. hr m) (4H, m) (2H. m) (2H. hr m) (2H, hr m) (3H. I, 7.0) (2H, d. 7.4) (IH. m)

* 39.7 26.7 124.3 .* 25.7 165 16.0 17.7

1.98 (ZH, m) 2.06 (2H, m) 5.09 (I H, m) _. 1.68 1.71 1.60 1.60

(3H. (3H. (3H. (3H.

s) s) s) .sl

*Too long machine time was required In order to gel quarterly carbon signals. t29.0. 29.1. 29.1, 29.2, 29 7. 29.7. Cl-C16, palmitolcoyl moiety. C17-C?I; farncsyl moiety.

in series. Elution was performed with MeCN-Et,0 (4: 1) at a flow rate of0.5 ml min.. ’ under a pressure of 17 kg cm-‘. Normal-phase radio-HPLC was performed using 2 columns (Waters 8Sl10, 0.8 x IO cm and YMC R-SIL-5, 0.5 x 50 cm) connected in series; hexane was used at a flow rate of 0.5 ml min- ’ under a pressure of I7 kg cm ’ unless otherwise described. [l‘JC]lsopcntenyl diphosphate (specific activity, 53 Ci acid (specific activity mmol ‘) and [5-3H]mevalonic 50.2 Ci mmol- ‘) were products of Amersham. Geranyl diphosphate was the same prepn as used in our previous work [9]. Culture conditions. Algae were grown in a modified CHU I3 medium [IO] of 4-fold strength containing the following components (mg 1.’ ’ of deionized H,O): KNO, (2100), K,HPO, (80). MgS0,+.7H,O (200). CaCI,. 2H,O (107). ferric citrate (20), CoCI, (0.04). H,BO, (2X6), MnCI,. 4H,O (1.81), ZnSO,. 7H20 (0.22). CuSO,. 5H,O (0.08), Na,MoO, (0.042). The pH was adjusted to 7 with KOH before autoclaving. The culture vessels (1.5 I flat flasks) were thermostated at 25” on a H,O bath and continuously illuminated at 59 W rn--’ with incandescent lamps. Sterile air enriched with 5% CO, was bubbled through the bottom of the vessels at a rate of 5- 7 I hr ‘. Harvest and preporurion qj’ cell-jiiee extracts. After I@ I2 days of cultivation, algae were harvested by filtra-

connected

t’l a/.

Table

5. NMR spectral data for 4 (600 MHI. C‘DCl,)

Asslgnmenl

“C(h)

I 2 3 4- 7 8 9 10 II, I4 I?. I3 15 16 17 I8 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

173.9 34.4 25.0 29.1 29.1’ 27.2 130.3 127.7 25.5, 25.6 128.3. 128 3 127.1 1320 20.5 14.3 61.2 116.4 142.1 39.5 26.2 123.6 135.4 39.7 26.7 124.3 131.3 25.7 16.5 16.0 17.7

‘H Lci.J(Hzl)

2.30 1.62 1.31 2.05 5.39 5.33 2.81 5.37 5.32 5.39 2.08 0.98 4.59 5 34

(2H. (21~. (XH. (ZH, (1 H, ( 1H, (4H. (2H. (I H. (I H. (?I~. (ZH. (2H. (1 H.

r. 7.6) m) m) ml ,,I)+ m)t hr m) m)t ,,I)+ m)+ m) I. 7.5) d. 7.1) m)t

2.06 (2H. m) 2.12 (‘H. m) 5.10 (1H. m) I.98 (2H. 1. 7.7) 2.06 (2H. m) S.09 (I H. m) I.68 1.71 1.60 1.60

(3H. (3H. (3H. (3H,

s) .\I s) s)

‘29.1. 29.2, 29.6. 29.7. Whemical shifts were determined referring lo CH COSY cross-peaks as ‘H NMR paka are overlapped In this region. Cl-Cl6; linolenyl moiety. Cl9 C33: farnesyl moiety.

tion through nylon cloth (30pm mesh) on a Biichner funnel with slight suction and stored at -40. until used. The yield was ca 8 g I _ ’ on a wet wt basis. The crude extracts were prcpd as follows. Freeze-dried algae (60 g) were homogenized in sea-sand with a mortar and pestle in 90 ml of0. I M Pi-buffer (pH 6.8) containing I mM EDTA and 10% (v/v) glycerol. The homogenates were centrifuged at 1200 y for I5 min at 0 and the supernatant containing the top layer was collected. [ I-‘4C]Farne.sol was enzymatically synthesized by incubating [ I-‘4C]isopentcnyl diphosphate and geranyl diphosphate with partially purified farnesyl diphosphate synthase [EC 2.5. I. IO] of Babllus .~rc~urorkermophilus overexpressed in Escherichia co/i cells [9]. Radiolabclled farnesyl diphosphate thus formed was hydrolysed with calf intestine alkaline phosphatase to give [I-‘“Clfarnesol. The specific activity of the radioactive farnesol was adjusted to 50 mCi mol- ’ by adding non-labelled farnesol before use. Production and purificurion o/‘jhr:~~ ucid esters of [I “Clfnrnesol. The incubation mlxt. contained in a final

Farnesyl

esters from Botryococcus

vol. of 320 ml, 90 mg [1-‘4C]famesol, 20 mM MgCI,, 20 mM MnCI,, 80 mg BSA, 0.5% (w/v) Triton X-100 and 64 ml crude extract. The mixt. was incubated at 25’ for 70 hr, then extracted with 320 ml hexane. The solvent was removed under red. pres. to yield a deeply coloured oil. The oil was loaded on to a silica gel column and the column washed with 12 column vols of hexane in order to remove non-labelled hydrocarbons associated with the crude extract. The column was then eluted with 20 column vols of hexane-isoPrOH (19.9:O.l) and the radioactive products collected. After the solvent was removed under red. pres., the radioactive products were analysed by silica gel prep. TLC. The distribution of radioactivity was detected with a radioscanner, and products less polar than farnesol were extracted from the TLC plate with Et,O. The solvent was removed under a N, stream and the radioactive products fractionated and purified by reverse-phase radio-HPLC and normal-phase radioHPLC. Feeding experiments with [5-3H]meoalonic acid. [S-‘HIMevalonic acid (150 @i) was fed to a 500 ml culture of the alga inoculated 2 days before feeding. After 15 days of cultivation under the conditions mentioned above (except for the air flow rate, 2&40 mlmin-‘), the culture was filtered through nylon cloth (30 pm mesh) and the cells on the filter lyophilized to dryness to yield 2.60 g of dry biomass. Radioactive products were extracted twice with 30 ml of hexane for 1 hr. The solvent was removed under red. pres. and the remaining oil was loaded on to a silica gel column. In order to obtain frs which possibly contain the products having the same polarities as those of the fatty acid esters, the column was first washed with 10 column vols of hexane to remove hydrocarbons and then eluted with 24 column vols of hexane-isoProH (19.9:O.l). The products thus obtained were combined and co-

hraunii

1207

injected with [1-‘4C]famesyl oleate using reversephase radio-HPLC and normal-phase radio-HPLC (hexane-isoProH (99: 1) with a flow rate of 0.5 ml min- ‘). Acknowledgements-We

would like to thank Dr Hiroaki Iwamoto of Meiji University for providing the Berkeley strain of the algae and generously assisting us in establishment of the culture in our laboratory. We also wish to thank Dr Masako Ueno of Tohoku University for NMR analysis.

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111, 2713. 5. Wolf, F. R., Nonomura, A. M. and Bassham, J. A. (1985) J. Phycol. 21, 388. 6. Laureillard, J., Largeau, C. and Casadevall, E. (1988) Phytochemistry 21, 2095. 7. Derenne, S., Largeau, C., Casadevall, E. and Sellier, N. (1990) Phytochemistry 29, 2 187. 8. Sindelar, P., Chojnacki, T. and Valtersoon, C. (1992) J. Biol. Chem. 267, 20594. 9. Koyama, T., Obata, S., Osabe, M., Takeshita, A.,

Yokoyama, K., Uchida, M., Nishino, T. and Ogura, K. (1993) J. Biochem. 113, 355. 10. Brown, A. C. and Knights, B. A. (1969) Phytochemistry 8, 543.