230 BIOCHIMICA ET BIOPHYSICA ACTA

THE OCCURRENCE OF y-LINOLENIC ACID IN FUNGI

ROBERT SHAW

U&ever Research Laboratory, Colworth House, Sharnbvook. Bedfordshire (Great Britain)

(Received June 3rd. 1964)

SUMMARY

I. The fatty acids from fat extracted from 31 species of fungi artificially cult- ured on fat-free media have been analysed by gas-liquid chromatography.

2. One or more examples were taken from four orders of phycomycetes, seven orders of ascomycetes and five orders of basidiomycetes. y-Linolenic (octadeca-6,9,12- enoic) acid was found only in the phycomycetes.

3. All nine species of phycomycetes studied, including six species of the order Mucorales, contained y-linolenic acid, but no a-linolenic acid.

4. No y-linolenic acid was detected in the ascomycetes and basidiomycetes. Many members of these classes yielded a-linolenic acid.

5. The phylogenetic significance of these results is discussed.

INTRODUCTION

In 1919, HEIDUSHKA AND LUFT~ reported that the seed oil of Oenofhera biennis (the Evening Primrose) yielded what appeared to be an isomer of the commonly occurring a-linolenic acid (octadeca-9,r2,rg-trienoic acid). They named the new isomer y-linolenic acid. EIBNER ef al.* suggested in 1927 that y-linolenic acid was octadecad,g,r;?-trienoic acid. RILEY* used more modem techniques to confirm this Structure in 1949, showing it to be ah-cis configuration.

y-Linolenic acid has also been found in Oenothera lamarckiana4 but as a smaller proportion than in 0. biennis’. There is no a-linolenic acid in oil from mature Oeno- thera seeds.

y-Linolenic acid has recently been found in seed-oils of hop, hemp‘ and mem- bers of the order Boraginaceae’.

By contrast, y-linolenic acid probably occurs widely in animals, although it is not present in more than trace amounts in mammalsa. Feeding experiments and experiments in w&oe-lr have implicated y-linolenic acid as a participant in an “es- sential fatty acid” pathway in rats, involving conversion of linoleic to arachidonic acid, via CoA esters of y-linolenic and homoy-linolenic (eicosa-8,rr,r4-trienoic) acids.

In 1948 BERNHARD AND ALBRECHT~~ detected y-linolenic acid in the abundant

Biochim. Biophys. Acta, g8 (x965) 2p-237

y-LINOLENIC ACID IN FUNGI 231

mycelial fat of the phycomycete mould Phy ces fatty acids being y-linol&ii: acid”. NW”““ _ 9 czimbsd that the strncture of Tzx

blak-m&ramq up to 16% of the

y-linolenic acid from P. b~~kes~e~n~s was identical with that from Oenothera seed oil,

y-Linolenic acid from both sources has been used experimentally to fulfll the “essential

fatty acid” requirements of rats, radio-labelling showing conversion to arachidonic

acids+. Recently, y-linolenic acid has ‘been found in the lipids of phytoflagellates of

several different ordersl’sl*, and in four out of five ciliate protozoa examined by

ERWIN AND BLOCH~$. In view of the detection of y-linolenic acid in these lowly plants and animals,

it was of interest to see what isomer of linolenic acid was present in fungi other than

P. blakesleeanzcs. Unlike seed oils, comparatively few fungal fats have been analysed,

and only the publications of BERNHARD and his co-workersW* appertain to phy-

comycetes. Hence to determine the pattern of the occurrence of various polyenoic

acids, it was necessary to analyse the fatty acids of a wide range of fungi.

Fungi were grown on fat-free media to avoid possible incorporation of ready-

syuthesised fatty acids into the lipids, as can happen in protozoapo. The occurrence

of a particular fatty acid in an organism where no oppo~unity for incorporation of

ready-synthesised fatty acids exists, is taken as showing genetic ability to synthesise

that fatty acid. ERWIN AND BLOCH 18*lg were able to suggest evolutionary routes by study of the comparative biochemistry of the synthesis of polyenoic acids in protozoa. It was hoped that study of the fatty acids of fungi might similarly throw light on the phylogenetic relationship of the classes of fungi to one another, and to their possible ancestry.

All the phycomycetes examined contained @iolenic acid, but-no a-linolenic acid. With ascomycetes and basidiomycetes, the reverse was the case. These results are consistent with the implication of earlier published results*l-81, that the com- position of the fatty acids from the mycelia of ascomycetes and basidiomycetes is not unlike that of seed oils, the linolenic isomer, if present, being or-linolenic acid*+-“.

A fourth class of fungi, the myxomycetes or slime-moulds, was not covered in this survey. DAVIDOFF AND KORN sB detected no linolenic acid in Dictyoste&m dis- cvidewz. If this is true of other myxomycetes, then in that respect this class of fungi resembles bacteriass.

EXPERIMENTAL

Culture methods Named cultures were obtained from the Commonwealth Mycological Institute,

and were grown up on roe-ml volumes of media in zso-ml erlemneyer flasks. Satis-

factory growth of all species was obtained on medium of the following composition : 40 g glucose, 20 g maltose, IO g Evans’ bacte~olo~~l peptone, 5 g potassium dihy- drogen phosphate, I g magnesium sulphate, I ml of trace element solution, made up to I 1 with tap water. The trace element solution contained per I, 5 g ferric sulphate, I g zinc sulphate, 0.5 g cupric sulphate, 0.1 g ammonium molybdate, 0.1 g manganese sulphate, 0.1 g boric acid and 0.1 g calcium hydroxide. Both shaking and stationary culture at 25’ were used, and whichever type of culture gave best yield of mycelium was harvested, after autoclaving to prevent possible spore dissemination. Mycelia

232 R. SHAW

were partly dried in a warm air current (55-60”) for 3 h, residual moisture (about 5% of the weight of the mycelium) being removed in vaclto.

Dried mycelia were powdered by ball-milling for 2 min. The powder was extracted for 8 h in a Soxhlet thimble in a Tate tube, using ethanol-benzene (257 : 594, v/v) solvent, 50 ml per g of mycelium. After extraction the solvent was reduced to 2-3 ml by distillation, and solvent removal completed on a Coleman rotary eva- porator 8*.

Diethyl ether was added to this residue. Ether-insolubles were separated and ether removed on a rotary evaporator. The ether-soluble residue was refluxed for I h with 0.6 M ethanolic KOH (IO ml per g residue). After removal of ethanol, the soaps produced were dissolved in water and titrated with HCl to pH 2.0. The precipitated fatty acids were extracted exhaustively with diethyl ether and again, final solvent removal was achieved on a rotary evaporator.

Analysis of fatty acids Unlike many positional isomers of long-chain polyenoic acid, y-linolenic acid

can be distin~shed from a-linolenic acid by gas-liquid c~~mato~aphy. After me- thylation with diazomethane, fatty acids were analysed on an adipate column at 197” (compare ref. 39, which gave the following retention times relative to stearic acid: oleic, 1.15; linoleic, 1.31; y-linolenic, 1.54; a-linolenic acid, I.$.

More precise data on the position of double bonds in unsaturated fatty acids was obtained by the permanganate-periodate oxidation method of VON RUDLOFF’*, used in conjunction with gas-liquid chromatography which enabled the method to be drastically scaleddown. The resultant short-chain acids were analysed without me- thylation on a succinate column, the dioic acids after methylation on an adipate column.

RESULTS

Table I shows the fatty acid composition of the fat of nine phycomycetes, six of which belong to the ubiquitous order Mucorales. All contain y-linolenic acid, and none contain &nolenic acid. On gas-liquid chromatographical analysis, fatty acids from ~~t~~~~ ~eba~~an~~ included three components which did not occur in the mixtures from other phycomycetes. Two of these had retention times which coin- cided with those of authentic samples of eicosatrienoic acid and of arachidonic acid, respectively, The retention time of the third approximated to that of docosatrienoic acid. These three acids contributed 0.6, 4.3 and 6.6% respectively, to the total fatty acids from P. &baryanHm. Analysis after bromination of the mixture confirmed that all three were unsaturated acids.

The figures for the relative composition of C,, and CrB acids for P. blakesleea#ecs compare well with the results of BERNRARD d al.‘“, but as Table I shows, in com- parison with this earlier analysis by paper chromatography, no C,,-C,, acids were detected by gas-liquid chromatographical analysis, in the strain of P. blakesleeanus (CM1 21156) studied here.

Tables II and III show the fatty acid analysis for some ascomycetes and basi-

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yLINOLENIC ACID IN FUNGI 235

diomycetes respectively. None containy-h&e& acid. Those that display linolenic acid have the a-isomer. hr the case of Bof$& &&ti, U&I amounts to 41.7%, but in few other cases does it exceed 10% of the fatty acids. The fats of several ascomycetes contain no linolenic acid at all: of these P~o?utffa domes&cum is unusual in that just four fatty acids make up over 99% of the total. Few of the fats are more than 30% saturated: the notable exception is that of Botryosfikaeria ribis. Nevertheless, the relative composition of the fatty acids of the ascomycetes show more variation from species to species than do those of the basidiomycetes, which are all rather similar to one another.

Figures for the fatty acid composition of the fat of four of the ascomycetes listed in Table II have been published previously a6-aS. None of these authors used gas-liquid chromatography. There is some variation between these earlier results and those in Table II, especially in the relative composition of the unsaturated fatty acids. This may be due to differences in the strain studied, or may be due to different growth conditions employed. Temperature a’1, pH 88 and composition of media** affect the relative fatty acid composition of fungal fat, though there is no evidence that qual- itative composition is affected by environmental factors. Although the presence of ricinoleic acid has been consistently reported in Claticefis @r@urea *l~*a+O, a member of the Hypocreales, this fatty acid could not be detected in three other members of this order which were studied in this survey.

DISCUSSION

Regarding the function of the naturally occurring isomers of linolenic acid, it has been suggested%‘* that a-linolenic acid is associated with chloroplasts and a plant-like mode of life. Conversely, there is some evidence that y-linolenic and arachi- donic acids are characteristic of subjects of the animal kingdom. Certain primitive protists which display animal and plant-like characteristics, are able to synthesise both a-linolenic and y-linolenic acids 87. In primitive animals which lack the enzyme system to convert y-linolenic to arachidonic acid, ERWIN AND BLOCH~* suggest that y-linolenic acid fulfils a similar function to that of arachidonic acid in higher animals.

Just what this function is remains unknown, but clearly the molecular structure of the isomer of linolenic acid is important to the animal, as a-linolenic will not sub- stitute for y-linolenic acid. In the case of the phycomycetes however, there is no evidence so far that the particular isomeric form of the linolenic acid is of physiological importance. All the phycomycetes studied yielded a considerable amount of lipid. Globules of storage oil were visible in the mycelium. The proportion of y-linolenic acid in the triglyceride fraction was very similar to that in the total extractable lipids”‘. It is likely, therefore, y-linolenic acid was mainly present in storage lipids. The physiologically significant properties of a storage oil depend considerably on the relative proportions of fatty acids of differing degrees of saturation which go into its make up, trienes having a particularly marked effect. As a triene, y-linolenic acid would not be markedly less effective than a-linolenic acid in this unexacting role.

There may thus be a physiological analogy between the occurrence of y-linolenic acidin phycomycetes and in Oenothera, hop, hemp and members of the Boraginaceae. However, the phylogenetic significance of the common occurrence of y-linolenic acid in phycomycetes is quite different.

Biochim. Biofihys. Acta, 98 (1965) zp-237

236 R. SHAW

Whilst it would seem likely that the presence of y-linolenic acid in the seed oils of a very few flowering plants is a phylogenetic idiosyncracy, and that the molecular structure of this fatty acid is of little significance in its function as an energy store in the seed, it is impossible to assert that the occurrence of y-linolenic acid in primi- tiveorganismsisso insignificant. If the structure of cc-linolenic acid has a fundamental function in photosynthetic plants, and if y-linolenic acid is of equal importance to the animal-like mode of life, then it may be that a-linolenic acid in non-photosynthetic higher fungi, and y-linolenic acid in non-motile phycomycetes, is vestigial. That is to say, the genetic ability to biosynthesise these fatty acids persists even though the function of these fatty acids is relegated to one where precise molecular structure is of little significance, such as energy-storage.

No phycomycete was found to contain a-linolenic acid, and y-linolenic acid has not been found in ascomycetes or basidiomycetes, here or previously. This is probably the most fundamental biochemical difference between the phycomycetes and the other fungi yet observed. This difference is so marked that, bearing in mind pub- lished data on the fatty acid composition of other primitive organisms as recently summarised by ERWIN AND BLOCHIS, it would seem that the evolutionary pathway leading to the phycomycetes was quite different from that for the higher fungi. It is widely assumed that fungi were evolved from algae-like forms. The composition of the fatty acids of the ascomycetes and basidiomycetes, and of the few algae studied so far, does not contradict this theory. However, the common occur- rence of y-linolenic acid in phycomycetes, as in protozoa, means that the matter must remain in question, at least until members of more orders of algae have been examined. In particular, we need to know more about the lipids of those algae which morphologically most closely resemble phycomycetes, such as certain members of the order Siphonales 44.

ACKNOWLEDGEMENTS

The able technical assistance of Mrs. J. CLARKE, Mr. T. P. COULTATE and Mr. J. K. MESSENGER is gratefully acknowledged.

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y_LINOLENIC ACID IN FUNGI 237

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