54

Incorporation of Linoleic Acid and Its Conversion to y-Linolenic Acid in Fungi Yasushi Karnisakaa, *, Toshihiro Yokochia, Toro N a k a h a r a a and O s a m u Suzukib aBiological Chemistry Division and bOrganic Chemistry Division, National Chemical Laboratory for Industry, Higashi 1-1, Tsukuba, Ibaraki, 305, Japan

The incorporation of [1-14C]linoleic acid {LA) into lipids of Mortierella ramanniana var. angulispora was studied to determine which lipid classes participated in the h6-desaturation of [1-14C]LA. [1-14C]LA was rapidly taken up into fungal cells and esterified into various lipids. Comparison of the profile of [1-14C]LA incorporation between fungal cells at the exponential growth phase and the s t a t i o n a r y g r o w t h p h a s e s h o w e d t h a t [1-14C]LA incorporation into most lipids--except for triacylglycerol {TG} and phosphatidylcholine { P C ) were greatly reduced at the stationary growth phase. Desaturation of [1-14C]LA into ),-linolenic acid (GLA) readily occurred at the exponential growth phase, but was greatly decreased at the stationary growth phase. Moreover, pulse-chase experiments revealed that the radiolabel incorporated into phosphatidylserine (PS) and PC rapidly t u r n e d over, while t h a t in T G and diacylglycerol {DG) accumulated after the 4 hr chase. In addition to the change of the radiolabel in individual lipids, the content of radiolabeled GLA converted from [1-14C]LA varied with individual lipids. In phospholipids such as PC, phosphatidylethanolamine (PE) and PS, radiolabeled GLA rapidly increased after 1 hr and then decreased after 4 hr. On the other hand, a gradual increase in radiolabeled GLA until 4 hr was observed in TG. These results suggest t h a t LA, which has been esterified into phospholipids such as PC, P E and PS, is readily desaturated to GLA, which is then transferred to TG. These differences in the fate of GLA derived from LA between phospholipids and neutral lipids m a y be reflected in the G L A c o n t e n t in the individual lipids. Lipids 25, 54-60 (1990).

which produces a-linolenic acid in plants (8,9}. Thus, the desaturation systems which produce polyunsaturated fatty acids have been shown to use several types of f a t t y acid derivatives as substrates depending on the organism. Recently, we found t h a t the filamentous fungus, MortiereUa genus, proliferated well under high glucose concentrations and efficiently produced GLA I10,11}. Thus, we tried to examine the synthesis of GLA from LA and the regulation of GLA synthesis. Since little is known about the h6-desaturation of LA to GLA in fungi, the comparison with h6-desaturase previously described in mammals is interesting. In a previous paper (12), we described the effects of metal ions in culture media and of temperature on the GLA content in Mortierella ramanniana var. angulispora. The results suggested t h a t these factors had different effects on the GLA content of polar lipids and neutral lipids in this fungus. However, the content of GLA was affected not only by the h6-desaturation, but also by the acylation or degradation of GLA. In the p r e s e n t p a p e r we e x a m i n e t h e i n c o r p o r a t i o n of [1-14C]LA into individual lipids and its conversion into GLA. The process of 56-desaturation is discussed from the distribution of ~4C-labeled GLA among various lipid classes.

MATERIALS AND METHODS

Materials. [1-14C]stearic acid (59 mCi/mmol), [l-14C]oleic acid (59 mCi/mmol} and [1-14C]LA {59 mCi/ mmoll were obtained from New England Nuclear Corporation (Boston, MA}. Unlabeled stearic acid, oleic acid, LA and GLA were purchased from Sigma Chemical Co. (St Louis, MO}. Silica gel G thin-layer chromatography (TLC} plates were obtained from Merck (DarmThough f a t t y acid desaturation systems in eucaryotic stadt, Federal Republic of Germany}, and KC18 (remicroorganisms such as yeasts and fungi have been versed phase} TLC plates were acquired from Whatstudied for a long time {1}, the biosynthesis of polyun- man (Maidstone, U.K.}. All solvents were of reagent saturated f a t t y acids is poorly understood. One of the grade. Microorganisms and culture conditions. Mortierella f a t t y acid desaturases participating in the biosynthesis of polyunsaturated f a t t y acids, h6-desaturase, was ramanniana var. angulispora (IFO 8187} was obtained purified to homogeneity from rat liver microsomes and from the culture collection of the Institute of Fermenit was shown that the purified t6-desaturase acted on tation (Osaka, Japan}. The fungi were maintained on a the CoA derivative of linoleic acid {LA) to form ),- yeast-extract, m a l t - e x t r a c t agar medium. The liquid linolenic acid {GLA} (2). On the other hand, the desatu- medium contained glucose, inorganic salts and vitaration of phospholipid-linked oleic acid has been re- mins as described previously (12}. Incorporation of 14C-labeled compounds into fungal ported to occur in yeasts ~3,4), fungi (5), algae {6) and higher plants (7}. Moreover, it has been suggested that lipidS. One ml of fungal cell culture grown in rotary glycolipid-linked LA is a substrate of hl5-desaturase, shakers 1180 rpm) at 30~ for one day, when cells were at the exponential growth phase, were incubated with 3.4 ~M (0.2 t~Ci/ml} [1-14C]fatty acids at 30~ for 1-6 *To whom correspondence should be addressed. hr. In some experiments, fungal cells which were at the Abbreviations: DG, diacylglycerol; MG, monoacylglycerol; FFA, stationary growth phase and precultured for eight days, free fatty acid; GL, glycolipid; GLA, y-linolenic acid; LA, linoleic were incubated with 14C-labeled compounds. After inacid; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphati- cubation, the fungal cells were cooled on ice and washed dylserine; SE, sterol ester; TG, triacylglycerol; TLC, thin-layer with 1 ml of 0.1 M phosphate buffer (pH 6.0) by centrifugation /1000 g, 5 min} to remove 14C-labeled comchromatography. LIPIDS,Vol, 25, No, 1 (1990)

55 FATTY ACID METABOLISM IN FUNGI 1. The incorporation profile of [1-14C]LA was changed when fungal cells at different phases of growth were incubated with [1-14C]LA. In fungal cells at the exponential growth phase, [1-14C]LA was taken up into fungal cells so rapidly that the radioactivity of the free fatty acid (FFA) fraction was very high at zero time, and gradually decreased afterward. Corresponding to the decrease in [14C]FFA, an increase in 14C-incorporation into triacylglycerol {TG) occurred. On the other hand, 14C-incorporation into phosphatidylcholine (PC) or phosphatidylethanolamine (PE) reached a plateau early in the incubation period and decreased at later incubation times. Incorporation of radioactivity into phosphatidylserine (PS) was the most transient, and it reached a plateau after 1 hr. In fungal cells, at the stationary growth phase, the incorporation of [1-14C]LA into most lipid classes was reduced in comparison with fungal cells at the exponential growth phase. However, the degree of reduction was different for each lipid class. Incorporation of radioactivity into TG or PC was not affected by the growth phase, though a difference in ~4C-incorporation into TG was found at longer incubation times. In the same e x p e r i m e n t , the d i s t r i b u t i o n of [14C]fatty acids in the total lipids is shown in Figure 2. In fungal cells, at the exponential growth phase, amounts of [14C]GLA formed increased linearly for the first 4 hr, and reached a plateau after a 6-hr incubation period, whereas amounts of [1-14C]LA reached a maximum after 2 hr, and gradually decreased afterward. The ratio of [14C]GLA to the total ~4C-label taken up into the fungal cells reached 15-20% after 6 hr. In fungal cells at the stationary growth .phase, the synthesis of radiolabeled GLA was greatly reduced (5% of total 14C-label after 6 hr), even though the reduction of [14C]LA was taken into consideration. Thus, it seemed that differences in ~4C-incorporation patterns between the fungal cells at two growth phases were correlated with the conversion of [1-~4C]LA into [1-14C]GLA. To further examine the pathway of [1-14C]LA incorporation and its conversion, we performed a pulsechase experiment in fungal cells at the exponential phase of growth. Figure 3 shows the ~4C-incorporation into various lipid classes after a 10-min or 60-min pulse. The amounts of radiolabel in TG, diacylglycerol (DG) and glycolipid (GL) increased after the chase, which meant that [14C]LA or [14C]GLA of these lipids had a tendency to be accumulated. On the other hand, radiolabels in phospholipids were more exchangeable. The amount of radiolabel in PS decreased more rapidly than that in other phospholipids, and this was similar to the decrease of radiolabel in FFA. The results obtained from the 10-min and 60-min pulse experiments showed a similar tendency, though the 60-min pulse made the amounts of individual lipid classes less interchangeable after the chase. The distribution of [~4C]fatty acids in the total lipids after the chase is shown in Figure 4. The amounts of radiolabel in GLA were increased greatly after the chase, while the amounts of [~4C]LA decreased after the chase. These results suggested that GLA produced from LA was likely to RESULTS be stored and not further metabolized into other fatty Incorporation of [1-14C]LA into fungal lipids. [1-14C]LA acid derivatives. was incorporated into fungal lipids as shown in Figure Fatty acid composition of various lipid classes in pounds not taken up i n t o the fungal cells. For the pulse-chase experiments, fungal cells were incubated with [1-14C]LA for 10 or 60 min, and then washed twice with a liquid medium containing 3.4 t~M unlabeled LA. After the washing, these cells were suspended in the same medium and chased with 3.4 gM unlabeled LA, which was the same amount as labeled LA. Extraction and analysis of lipids. Lipids were extracted from 1 ml of fungal cell suspension with 3 ml of chloroform/methanol {l:2,v/v). After 1 hr, 1 ml of chloroform and 1 ml of 0.1 M phosphate buffer were added. The upper aqueous layer was washed twice with 1 ml of chloroform, and the lower chloroform layers were collected and evaporated to dryness under N 2. The extracted lipids were analyzed for the distribution of radioactivity in individual fatty acids and individual neutral lipid classes and polar lipid classes. For fatty acid analysis, extracted lipids were transmethylated and the resultant fatty acid methyl esters were separated by reversed phase TLC on KC18 plates with acetonitrile/acetic acid (200:1, v/v). Neutral lipid classes were separated by TLC on Silica gel 60 plates with benzene/diethyl ether/ethanol/NH 3 (50:40:2:0.5, by vol) as the first solvent, and hexane/dimethyl ether (94:6, by vol) as the second solvent. Polar lipid classes were separated by TLC on Silica gel 60 plates with chloroform/ acetone/methanol/acetic acid/H20 150:20:10:10:5, by vol). When necessary, two-dimensional TLC was performed for checking 14C-labeled TLC fractions of polar lipids. Chloroform/methanol/H20 (65:25:4, v/v/v} was used as the developing solvent in the second dimension. 14CLabeled spots were detected by autoradiography and scraped into scintillation vials. Radioactivity was determined with a Beckman liquid scintillation system {model LS1701) with automatic quenching correction. In some experiments, 14C-labeled fractions of neutral or polar lipids detected by autoradiography were scraped off and extracted with chloroform/methanol {2:1, by vol). Then, the extracted neutral and polar lipid classes were transmethylated for analysis of their fatty acids. The mass fatty acid composition of various lipid classes was analyzed by gas liquid chromatography as described previously (12). For quantifying the amounts of the fatty acids in individual lipid classes, heptadecanoic acid was added as the internal standard. To confirm the identity of 14C-labeled incubation products such as GLA, the position of the first double bond from the carboxyl end in 1-14C-labeled products was determined using the permanganate-periodate procedure (13). The resultant 14C-labeled dibasic acids were separated by. TLC on Silica gel 60 plates with xylene/ phenol/n-butanol/formic acid/water (70:30:10:8:2, by vol) (14). Since adipic acid which contained 6 carbon atoms, was obtained from a 14C-labeled incubation product corresponding to GLA, GLA was shown to be synthesized from [l-14C]LA and [1-14C]oleicacid. Other methods. The dry cell weight and total lipid content were measured by weight as described previously (12}.

LIPIDS,Vol. 25, No, 1 (1990)

56 Y. KAMISAKA ETAL.

A

B

10

10

&

Z-

.,, ~.o-..~........
5

5 0 U >,

E I

0

0

~E ,1 o

1

F

I

I

I

0

>, > U 0

:6 rr

0.5

0.5

0

0

I

I

I

2

4

6 Incubation

0

0 time

2

4

6

(hr)

FIG. 1. Incorporation of [1-14C]LAinto fungal lipids. [1-14C]LAwas incubated with fungal cells at the exponential growth phase (A) [total lipids/dry cell weight (w/w) = 15%], or at the stationary growth phase (B) [total lipids/dry cell weight (w/w) = 50%]. [1J4C]LA was incorporated into TG (9 FFA (A), DG ([:3),PC (e), PE (A), PS (I) and GL (V). The GL fraction was composed of a major unidentified glycolipid and several minor fractions. Radiolabeled PI, PA, SE and MG were also detected separately, but at lower levels. The incorporation of [1-14C]LA at zero time may occur while the fungal cells were washed according to "Materials and Methods". When lipids were rapidly extracted by chloroform/methanol solution without washing at zero time, no [1-14C]LA incorporation into individual lipids was observed. Values are means of duplicates for a typical experiment of several independent ones.

fungal cells at various growth phases. To determine the difference in the [1-14C]LA incorporation into the fungal lipids at different growth phases, the f a t t y acid composition of various lipid classes in the fungal cells was analyzed. As shown in Table 1, some similarities in f a t t y acid compositions a m o n g the various lipid classes were observed. TG was the major fungal lipid, especially at longer culture time, and its f a t t y acid composition, which was similar to that of DG, was mostly unchanged during culture. F F A contained more saturated f a t t y acids, probably reflecting de novo f a t t y acid synthesis. Polar lipids were divided into two groups from the point of the f a t t y acid composition. One included PC and PE, which contained smaller amounts LIPIDS,Vol, 25, No, ] (]990)

of palmitic acid and larger amounts of L A and GLA. The other included PS, P I and GL, which contained larger amounts of palmitic acid and relatively smaller amounts of LA and GLA. Though PS contained large amounts of G L A only in the one day culture, a decrease in the G L A content in PS was seen at longer culture time.

Incorporation of [1-14C]stearic acid or [1-14C]oleic acid into fungal lipids. The incorporation of f a t t y acids other than LA was also examined. Table 2 shows the c o n v e r s i o n s of i n c o r p o r a t e d [1-14C]stearic acid, [1-14C]oleic acid and [1-14C]LA. Compared with stearic acid, which was mostly unchanged, oleic acid was actively converted to other f a t t y acids. It was notable

57 FATTY ACID METABOLISM IN FUNGI in TG also increased after 4 hr, the increase in [14C]GLA in T G was likely to reflect t r a n s f e r from the polar lipids, especially PC and PS. On the other hand, the content of [14C]GLA in F F A was very low, and the amount of [14C]GLA also remained unchanged. Thus, the desaturation process from LA to GLA probably involved f a t t y acid esterification into certain phospholipids.

20

Y: I0

DISCUSSION

-g " O..

U "ID

E I

0

7

|

!

O X

3

D 2 "6

eO

O

~3 r (X

1

0.~..0. ..... ~

1D". . . . . .

0--

i

I

I

2

4

6

Incubation time ( h r ) FIG. 2. Conversion of [1-14C]LA into [1-14C]GLA. After the incubation of [1-14C]LA with fungal cells at the exponential phase of growth ( ) or at the stationary phase of growth (. . . . ), the extracted lipids were transmethylated, and the amounts of radioactive LA {O) and GLA tO) were determined. Values are means of duplicates for a typical experiment of several independent ones.

that the desaturation of [1-~4C]oleic acid was sequential, that is, [14C]GLA rather than [14C]LA was accumulated after the [1-~4C]oleic acid incorporation.

Conversion of [1-mC]LA to [1-14C]GLA in fungal cells. To elucidate the role of substrates used in the conversion of LA to GLA, the distribution of [~4C]fatty acids in various lipid classes after the incorporation of [1-14C]LA was examined. As shown in the lower panel of Figure 5, [14C]GLA was present in the polar lipid fractions at the early incubation time. Moreover, the upper panel of Figure 5 shows that the ratio of [14C]GLA to the incorporated radioactivity was high in the polar lipid fractions from the beginning of the incubation. Among the polar lipids, the content of [14C]GLA in PS reached 60%. However, the amounts of [14C]GLA in the polar lipid fractions, especially in PC and PS, decreased after 4 hr, when the amounts of [~4C]GLA in T G rapidly increased. Since the content of [~4C]GLA

The process of aerobic desaturation has been well analyzed, mainly through the studies of oleic acid desaturation {15). However, knowledge about the desaturation of polyunsaturated f a t t y acids still remains limited with respect to the desaturation mechanisms involved. Though the A6-desaturase, which catalyzed the synthesis of GLA from LA, was purified to homogeneity in mammalian cells, little was known about this enzyme in microorganisms or plants. Recently, the synthesis of GLA was examined in microsomes of plant seeds 116,17) and the desaturation process from LA to GLA in plants was found to be different from t h a t of mammalian cells, i.e., the h6-desaturase purified from rat liver used linoleoyl-CoA as a substrate, while the plant h6-desaturase used as a substrate the linoleoyl moiety linked to phospholipid. In the present study, we studied the incorporation of [1-14C]LA into lipids of Mortierella genus to examine which lipid classes participated in 56-desaturation. First, we did a comparison of LA metabolism between fungal cells at the exponential growth phase and the stationary growth phase. In fungal cells at the stationary growth phase, the conversion of [1-14C]LA into GLA was reduced compared with fungal cells at the exponential growth phase {Figure 2). The decrease of the conversion of [1-14C]LA into GLA was accompanied by decreases in ~4C-incorporation into most lipid classes, but ~4C-incorporation into PC or T G was unchanged regardless of growth phase tFig. 1). In pulse-chase experiments, radiolabels derived from [1-14C]LA turned over at different rates depending on the lipid class tFig. 3). The radioactive f a t t y acids in phospholipids turned over fast, whereas those in neutral lipids had a t e n d e n c y to be more stable. F u r t h e r m o r e , Figure 5 shows that the radioactive GLA formed from LA first recurred in phospholipids, such as PC, PE and PS, and later accumulated in T G as it decreased in phospholipids. In view of the results mentioned above, we postulated t h a t the synthesis of radioactive GLA first occurred in the course of [1-14C]LA incorporation into certain phospholipids, and that the synthesized radioactive GLA was then transferred into neutral lipids, mainly TG. As shown in Table 1, the mass content of GLA in phospholipids such as PC, PE and PS was high and varied during cell growth, while it was low in neutral lipids and remained almost constant. Thus, the difference in the metabolism of LA in phospholipids and neutral lipids may be reflected in the mass contents of GLA. These differences might also provide the clue to explain the different effects of metal ions and temperature on the GLA content of polar and neutral lipids as described previously (12). LIPIDS,Vol. 25, No, 1(1990)

58 Y. KAMISAKAE T A L . B 60 min pulse

A 10min pulse 400 I

400

300

300

200

200

100

100

A

v

.,w.,

0 n} OC

0 0

2

4

0 L ~ ' - - ~ 0 2

4

Chase time ( h r ) FIG. 3. Distribution of radioactivity in fungal lipids after pulse-chase with [1-14C]LA. After 10-min pulse (A} or 60 min pulse {B) with [1-14C]LA, incorporation of radioactivity into TG (O), FFA {A), DG (El), PC (e), PE (A), PS (I) and GL (T) was chased with unlabeled LA. Values are expressed as percentages of radioactivities incorporated into individual lipids during pulse (means of duplicates}. The absolute values during the 10-rain and 60-rain pulse are in I)PM X 10-3 oI the fatty acid incorporated/rag dry cell weight, 10-min pulse: TG, 24.0; FFA, 78.8; DG, 1.0; PC, 30.0; PE, 11.8; PS, 10.1; GL, 1.4; and 60-rain pulse: TG, 42.0; FFA, 41.2; DG, 1.4; PC, 29.9; PE, 15.2; PS, 6.3; and GL, 2.4.

Though formed GLA is found to exist preferentially in phospholipids in the early incubation time when GLA is actively formed by the h6-desaturase, it is still unclear which derivative is a substrate of the h6-desaturase, i.e., whether GLA formed as thioester derivative (either CoA or acyl carrier protein derivative) and is preferentially incorporated into phospholipids, or whether the h6-desaturase directly acts on phospholipid-linked LA to form GLA. The latter case has been reported for the t9-desaturase in some microorganisms and higher plants (3-7). The former case involves the regulatory systems which allow the desaturation products to be transacylated to specific lipids. In mammalian cells, the selective incorporation of specific fatty acids, such as eicosanoid precursor fatty acids into specific lipids, has been reported (18,19). LIPIDS,Vol, 25, No. 1(1990)

Thus, it is possible that the specific acylation systems are involved in the LA and GLA incorporation into the specific lipids in this fungus. These fatty acids may be incorporated into phospholipids either by the deacylation-reacylation pathway (20) or by d e n o v o synthesis, which would first form PA by acylation of glycerol-3-phosphate or 1-acyl-glycerol-3-phosphate (8). Since these acylation processes have been thought to be catalyzed by separate acyltransferases (21), the cooperation of these acyltransferases with the A6-desaturase may be responsible for the distribution of GLA among individual lipids. Besides the LA incorporation, oleic acid seemed to be sequentially desaturated into GLA, which agrees with the results obtained from the plant seeds (16). Since the amounts of GLA formed from oleic acid were

59 FATTY ACID M E T A B O L I S M IN F U N G I

300

60

c

E~ 0

u ~

200

30

.J

(D 0 .~,

~

10

g,E

s

o "U

,~ 100

I0

;

0

I

I

2

4

o

0

4 TG

014 FFA

01 PC

014 PE

014 PS

(hr)

FIG. 5. Distribution of [14C]GLA derived from [1-14C]LA in major lipid classes. The distribution of [14C]GLA at zero time was considered to be due to the same reason as mentioned in Figure 1. GLA content in upper column represents [14C]GLA/[14C]LA + [14C]GLA. Values are means of triplicates.

Chase time ( h r ) FIG. 4. Distribution of radioactivity in fatty acids after pulsechase with [1-14C]LA. After 10-rain pulse ( ) or 60-rain pulse (. . . . ) with [1A4C]LA, distribution of radioactivity in LA (O) or GLA ( e ) was chased with unlabeled LA. Values are expressed as percentages of radioactivities distributed after pulse (means of duplicates}. The absolute values incorporated during the 10rain and 60-min pulse are in D P M X 10 -3 of the f a t t y acid incorporated/rag dry cell weight, 10-min pulse: LA, 151.1; GLA, 12.4; and 60-rain pulse: LA, 86.6; GLA, 20.5.

almost equal to the amounts of G L A formed from LA, the possibility that LA formed from oleic acid enter pools different from those of LA taken up from culture must be considered. Among filamentous fungi, several studies on the desaturation of u n s a t u r a t e d f a t t y acids have been reported in Neurospora crassa (22, 23), although the molecular nature of the desaturases still remains unclear.

TABLE 1 F a t t y Acid Composition of Major Lipid Classes in M o r t i e r e i l a r a m a n n i a n a var. a n g u l i s p o r a a Culture time (day}

F a t t y acid composition {%)

Lipids

Acyl content (nmol/mg dry cell weight}

16:0

18:0

18:1

18:2

18:3 b

TG DG SE FFA PC PE PS GL PI

251.2 11.4 8.1 4.3 26.5 37.1 9.3 10.1 4.4

30.4 34.0 20.6 42.1 19.9 16.7 43.7 34.5 47.0

8.6 9.9 1.7 17.1 2.6 1.8 2.9 2.5 2.0

46.3 41.7 42.9 23.0 17.0 32.9 15.3 30.6 17.6

7.1 8.9 10.2 1.9 27.1 18.3 11.5 19.6 21.1

7.6 5.6 12.6 4.4 27.6 27.7 24.1 6.4 7.5

TG DG SE FFA PC PE PS GL PI

1034.3 62.3 13.0 4.8 13.6 14.9 3.5 6.9 5.6

27.9 30.4 15.4 28.4 6.1 11.2 37.2 38.4 40.5

4.8 6.2 2.6 20.7 0.9 1.0 4.0 1.6 2.6

51.7 49.4 39.5 30.3 51.0 52.4 40.6 41.8 39.7

9.2 9.1 22.8 3.9 26.3 14.8 5.0 9.2 8.9

6.0 5.3 10.7 5.7 11.1 18.1 5.3 1.3 3.4

aValues are means of triplicates. bGLA.

LIPIDS, Vol, 25, No, 1 (1990)

60

Y. KAMISAKA E T A L . TABLE 2 Incorporation and Conversion of Other [1-14C]Fatty Acids a

Incorporated fatty acid [1A4C]stearic acid

[1A4C]oleic acid

[1A4C]linoleicacid

Time (hr) 0 1 4

18:0 97.0 94.7 87.9

0 1 4 0 1 4

2.5 3.8 3.5 0.2 0.4 1.0

[14C]fatty acid (%) 18:1 18:2 1.5 1.0 2.8 1.5 6.8 3.1 89.7 78.7 72.7 1.0 1.4 2.0

1.3 3.2 7.9 96.3 90.8 82.7

18:3b 0.5 0.7 2.2

Total [14C] (DPM • 10 -4) 18.0 23.5 23.6

6.5 14.2 15.9 2.6 7.4 14.3

8.8 22.1 23.5 14.1 17.3 22.3

aValues are means of duplicates. [1-14C]fatty acids were incubated with fungal cells at the exponential growth phase. bGLA.

MortiereUa genus, which synthesizes large a m o u n t s of G L A i n s t e a d of a-linolenic acid f o u n d i n N e u r o s p o r a crassa c a n be e x p e c t e d to h a v e a n active h 6 - d e s a t u r a s e s y s t e m , a l t h o u g h n o b i o c h e m i c a l or g e n e t i c i n s i g h t s h a v e b e e n g a i n e d so far. I n a d d i t i o n to G L A , some species of the Mortierella g e n u s p r o d u c e d large a m o u n t s of a r a c h i d o n i c acid (24,25) a n d e i c o s a p e n t a e n o i c acid (26). T h u s , the d e s a t u r a t i o n s y s t e m s for p r o d u c i n g polyu n s a t u r a t e d f a t t y acids in t h e Mortierella g e n u s are of g r e a t i n t e r e s t . T h e r e s u l t s o b t a i n e d in Mortierella g e n u s s u g g e s t t h a t L A , w h i c h is i n c o r p o r a t e d i n t o some p h o s p h o l i p i d s , s u c h as P S a n d P E , has a tend e n c y to be d e s a t u r a t e d i n t o G L A , a n d t h e G L A prod u c e d is g r a d u a l l y a c c u m u l a t e d m a i n l y i n T G f r o m p h o s p h o l i p i d s . Since t h i s f u n g u s is u s e d i n t h e product i o n of lipids (10), t h e s e f i n d i n g s o n t h e role of individu a l lipids i n the h 6 - d e s a t u r a t i o n m a y p r o v i d e a s t r a t e g y to i m p r o v e t h e f a t t y acid c o m p o s i t i o n of this fungus.

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