Unsaturated fatty acid metabolism in Neurospora

Unsaturated Fatty Acid M e t a b o l i s m in N e u r o s p o r a 1 Joseph Lein2, Theresa A. Puglisi and Patricia S. Lein From the Department of Zoology, Syracuse University, Syracuse, New York Received February 19, 1953

INTRODUCTION The findings that unsaturated fatty acid deficiencies can be produced in the rat (1), mouse (2), dog (3), and chicken (4) indicate that such fatty acids play an essential metabolic role in these organisms. The further demonstration that certain microorganisms require for growth unsaturated fatty acids (5-10) suggests that the metabolic role of these acids is a very fundamental one. The higher and lower organisms do display some differences in the types of fatty acids that can prevent the deficiency. The microorganisms respond to oleic acid as well as to the more unsaturated acids (11, 12), while the mammalian and avian species respond only to the more unsaturated acids, linoleic, linolenic, and arachidonic acids (13, 14). It is interesting to note that the normal development of the flour moth is dependent on linoleic or linolenic acid but not on oleic acid (15). If it is assumed that oleic acid is the precursor of the more unsaturated acids then presumably the higher organisms lack the enzyme system necessary for the conversion of oleic to linoleic acid. With the finding (16) that a requirement for unsaturated f a t t y acid could be produced by gene mutation in Neurospora crassa, it was hoped that such mutants would be the material of choice for the study of the route of synthesis of these compounds. Accordingly an attempt was made to obtain more mutants of this type. These mutants were also studied to see if they displayed some of the relationships postulated to exist between unsaturated f a t t y acid requirement and pyridoxine (17), biotin (12, 7), and aspartic acid (18). 1 This work was generously supported by a grant from the Williams-Waterman Fund of the Research Corporation. Present address: Dept. of Microbiology, Bristol Laboratories Inc., Syracuse, N.Y. 434

UNSATURATED ~'ATTY ACID METABOLISM

435

~/[ETHODS

Isolation of Mutants The mutants were isolated using the technique of Lein, Mitchell, and ttoulahan (19). The medium into which the presumed mutant types were placed contained in most cases 25 rag.% each of oleic, linoleic, and linolenic acids and 100 rag.% of polyoxyethylene sorbitan monostearate (Tween 60). The general manipulative techniques used were those described by Beadle and T a t u m (20). Nine separate mutant searches were carried out, and these yielded seven mutants which grew in unsaturated f a t t y acids and not in unsupplemented minimal medium. Of these seven mutants at least five represented independent occurrences since they were isolated in different searches.

Materials and Procedures The oleic, linoleie, and linolenic acids used were obtained from the Hormel Foundation and were of very high purity. The saturated acids were obtained from Eastman Kodak Company and were used as received except for the myristic, palmitic, and stearic acids which were recrystallized from ethanol. A highly purified sample of methyl arachidonate was obtained from Dr. J. B. Brown. It had an iodine value of 314. The f a t t y acids were added in emulsion form. One per cent emulsions were made using a Waring blendor and then diluted to yield the desired concentrations. The solid compounds were first melted and then emulsified in water whose ternperature was above their melting points. In tests involving arachidonic acid a somewhat different technique of testing was used. The methyl arachidonate was tested as both the methyl ester and the hydrolyzed product. Mild conditions of hydrolysis were employed because of the lability of the arachidonic acid, and the effectiveness of the hydrolysis procedure was determined by treating methyl oleate in the same way. Hydrolysis was carried out by reacting the f a t t y acid esters with 10% K 0 H in 95% ethanol for 3 hr. at 25°C. The solution was neutralized before adding to the sterile medium in the flasks. Since the f a t t y acid esters and the hydrolytic products were in alcoholic solutions, no further sterilization was necessary. This avoided heating the labile arachidonic acid. For the growth studies 125-ml. flasks containing 20 ml. of minimal medium supplemented with the growth factor were used. These were sterilized by autoclaving at 15 lb. for 10 min. and then inoculated with one drop of a conidial suspension. The inoculated flasks were incubated at 25°C. for 120 hr.; the myeelial mats were removed, pressed out, dried at 100°C., and weighed. For the genetic studies, crosses were carried out on Westergaard's medium (21) and ascospores dissected as described [)y Beadle and T a t u m (20). Heterocaryotic studies were carried out in liquid medium in flasks. I:~ESULTS

A s a r e s u l t of t h e m u t a n t searches, s e v e n m u t a n t s d e s i g n a t e d as S l l , $33, $72, $82, $83, $92, a n d $93 w e r e i s o l a t e d . M u t a n t s $82 a n d $83

436

LEIN~ PUGLISI A N D

LEIN

were isolated in the same search and $92 and $93 in another. Genetic crosses of the mutants to wild type indicated that the fatty acid requirements segregated as if they were associated with a single gene mutation in each ease. Growth studies were carried out with these mutants in both the presence and absence of Tween 60. This compound was used since it markedly decreased the toxicity of the various fatty acids towards Neurospora (16, 22). The fatty acids were added in three different concentrations: 10, 5, and 1 rag./20 ml. medium. When Tween 60 was used it was added in a concentration of 20 rag./20 ml. medium. In the ease of araehidonie acid, results are presented for both the methyl ester and the hydrolyzed ester. Parallel experiments with methyl oleate indicated that the hydrolytic procedure used was an adequate one. These products were tested using mutants Sll, $83, and $92. Experiments were also carried out with these preparations using wild type 7A. These demonstrated that toxicity did not account for the araehidonic acid's inability to enable growth. It was found that acetic, butyric, caproie, eaprylie, eaprie, laurie, myristic, palmitie, and stearie acids did not enable any of the mutants to grow either in the presence or absence of Tween 60. In the presence of either oleie, linoleie, or linolenic acid, however, all mutants grew except $72. Table I lists the growth of these mutants. Mutant $72 will be considered later. Growth occurred in the presence of Tween 60 at all concentrations of the unsaturated acids except arachidonie acid. A marked inhibition of growth was obtained in the absence of Tween 60 in the ease of linolenie and linoleie acids demonstrating the toxicity of these compounds as well as their ability to serve as growth factors. The data also show that none of the concentrations of methyl arachidonate or araehidonie acid used enabled the mutants to grow. This result cannot be attributed to the toxicity of the compounds, for at the 5 and 1 mg. level, wild type 7A grew readily. Furthermore the results of the hydrolyzed methyl oleate indicate that the hydrolytic conditions were sufficient to hydrolyze the esters, and incomplete hydrolysis cannot account for the lack of growth. It appears that araehidonie acid is not effective in enabling the fatty acid mutants to grow. This is surprising in ~¢iew of the findings that araehidonie acid is able to relieve the fatty acid deficiency symptoms of animals (23). Mutant $72 behaved quite differently from the other mutants. Unlike the others it has only a partial block and this is apparently between lino-

437

UNSATURATED FATTY ACID METABOLISM TABLE I

Growth of Mutants in Various Concentrations of Unsaturated Fatty Acids at Five Days Fatty acid

Mycelial growth, rag. Concn,

Tween 60 Sll

None

0 -

01eic

Linoleie

Linolenic

II

1 5 10 1 5 10 1 5 10 1 5 10

1

Hydrolyzed methyl arachidonate

1 g 10 1 5 10

S92 I $93 4 0

+ ÷ +

8 11 16 27 41 67

8 11 13 26 38 65

47 51 60 70 60 86

8 J3 16 35 49 95

+ + +

1 0 0 53 22 14

1 0 0 .7 8 :3

27 51 54 50 60 54

0 0 0 27 26 18

+ + ÷

5 0 0 33 30 8

6 0 0 [6 ]5 9

55 24 3 51 61 35

10 0 0 28 43 17

+ ÷ +

0 0 0 0 0 0

0 0 0 0 0 0

+ ÷ +

0 0 0 0 0 0

0 0 0 0 0 0

1

5 10 1 5 10

$83 0 0

÷

1 5 10 5 10

Methyl araehidonate

$82

438

LEIN~ PUGLISI AND LEIN

leic a n d linolenic acids. T h i s is shown in T a b l e I I w h e r e d a t a a r e pres e n t e d for t h e g r o w t h of $72 after 2, 3, a n d 4 d a y s on m i n i m a l m e d i u m a n d oleic, linoleic, a n d linolenic a c i d - s u p p l e m e n t e d m e d i a . T h e g r o w t h s h o u l d b e c o m p a r e d w i t h t h a t o b t a i n e d w i t h 7 A wild t y p e on m i n i m a l m e d i u m . I t will be seen f r o m t h e d a t a t h a t $72 does grow on m i n i m a l med i u m b u t it t a k e s longer to s t a r t t h a n wild t y p e a n d t h e g r o w t h does n o t a t t a i n t h e wild t y p e levels in t h e p e r i o d t e s t e d . S u p p l e m e n t i n g t h e med i u m w i t h oleic or linoleic acid does n o t i m p r o v e t h e growth. H o w e v e r , linolenie acid does m a r k e d l y increase t h e g r o w t h d e s p i t e t h e fact t h a t TABLE I I Growth of $72 and 7A Wild Type in Minimal Medium at Different Periods of Time and the Effect of Unsaturated Fatty Acids on the Growth of $72

Supplement

Conch.

Stralu

Mycelialgrowth,rag. 2-day

lay

4-day

7A $72

38 0

1 5

67 33

mg./ZOml.

None

Oleie acid

0.1 1.0 10.0

$72 $72 $72

0 0 0

3 2 3

26 29 34

Linoleic acid

0.1 1.0 10.0

$72 $72 872

0 1 0

9 13 1

49 54 13

Linolenic acid

0.1 1.0 10.0

$72 $72 $72

12 9 0

51 47 2

65 58 35

it is t h e m o s t toxic of t h e f a t t y acids used. T h e results are consistent w i t h t h e h y p o t h e s i s t h a t $72 has a p a r t i a l b l o c k b e t w e e n linoleic a n d linolenie acids. An attempt was made to determine if $72 accumulates a fatty acid as a result of the partial block. Wild type 7A was grown in 500 ml. of minimal medium in a Fernbach flask. $72 was grown both in 500 ml. of minimal medium and 500 ml. of minimal medium supplemented with 2.5 rag. linolenic acid. After 6 days at 25°C., the mycelia were removed, washed, lyophilized to dryness, and weighed. The mycelia were ground up and extracted by refluxing for 20 min. with 50 ml. of 3:1 ethyl ether-ethanol mixture. Aliquots of the filtrate were titrated with 0.01 N

UNSATURATED FATTY ACID METABOLISM

439

KOH in ethanol and corrected for the blank to determine the free acid content. The filtrate was then evaporated to a small volume and saponified with 10% alcoholic KOH at 56°C. for 1.5 hr. The mixture was acidified, water was added, and the fatty acids were extracted into petroleum ether. The petroleum ether was washed three times with water, dried with CaSO4, and aliquots were titrated. The results obtained indicated that there is no accumulation of either free or esterified fatty acid by $72 grown in linolenic acid. Thus, with wild type the free fatty acid content was 0.084 mequiv./g, while $72 contained 0.071. The total fatty acids in wild type was 0.138 mequiv./g, while the corresponding figure for $72 was 0.108. Presumably the linoleic acid that might accumulate is removed by being converted into other compounds. One such compound has been previously shown to be acetate (21). In view of the fact that certain microorganisms can substitute biotin for their oleie acid requirement, an experiment was designed to determine if high concentrations of biotin would substitute for the unsaturated f a t t y acids in the Neurospora mutants. Biotin is the only growth factor required by the wild type. I t was found that a thousandfold increase in biotin concentration over that found in minimal medium did not enable mutant S11 to grow in the absence of unsaturated fatty acid. The converse experiment was also carried out to see if oleic acid can substitute for biotin in wild type. In order to remove all vestiges of biotin, 0.5 ml. of fresh egg white was added to each of the flasks. This contained ample avidin to effectively remove the contaminating biotin in the medium. I t was found that oleic acid cannot substitute for biotin in either wild type 7A or m u t a n t S 11. In View of the fact that Broquist and Snell (18) reported that for some organisms both unsaturated f a t t y acid and aspartic acid were required to substitute for biotin, an experiment was carried out similar to that described above except that 25 mg. of DL-aspartic acid was used in addition to the oleic acid. No growth was found, however, in the absence of biotin. An attempt was also made to determine if pyridoxine exerted a sparing action on the requirement for f a t t y acid by m u t a n t S l l . Amounts of pyridoxine varying from 0.001 to i mg. were given to the mutants grown on different quantities of oleic acid, but no sparing action was found. From the results obtained it appears that the f a t t y acid requirement is independent of biotin, aspartic acid, and pyridoxine. The unsaturated • f a t t y acids must therefore play an essential metabolic role aside from that involving these substances. Genetic studies were carried out on the mutants to determine if the same or different genes had been mutated in the different mutants iso-

440

LEIN~ PUGLISI AND LEIN

lated. Both crossing experiments and heterocaryotic experiments indicated that mutants SII, $33, $83,892, and $93 were allelie, that is, the same gene had been mutated in these mutants. Mutant $72 proved to be non-allelic to these by both tests. Thus the genetic studies confirmed the growth studies. The mutants which behaved alike had the same gene mutated while $72, whose behavior was different, had another gene mutated.

DISCUSSION It has been generally assumed that oleie acid arises from stearic acid by dehydrogenation [(24), also see review by Breuseh (25)]. The mutants of Neurospora that were obtained indicated that, in this form at least, the unsaturated acids are synthesized by a pathway distinct from that leading to the synthesis of the saturated acids. Thus, four separate occurrences of mutants blocked before oleic acid were observed. If oleic acid is synthesized from stearic acid, then it is a great coincidence that these mutants blocked between stearic and oleic acids were obtained. It might be argued that perhaps the selection method was such that it specifically overlooked mutants that might be blocked before oleic acid and which would grow in stearie acid. This is thought improbable, however, since stearie and palmitic acids are not at all toxic to Neurospora (22). If stearic acid is the precursor of oleie acid then such a distribution of mutants would be expected to occur only if stearic acid is an essential metabolite. There is no evidence for this being the case. It is thought that the most reasonable interpretation of the distribution of mutants would be to assume that the unsaturated fatty acids are synthesized through a pathway in which the saturated acids do not directly participate. The fact that four independent occurrences of allelie mutants were obtained indicates that the immediate precursor of oleic acid is some as yet unknown but essential metabolite. It is thought to be essential because this would account for the selection method yielding the large proportion of allelic mutants. Since the precursor was not present in the selection medium, non-allelic mutants which would grow on it were automatically excluded. M u t a n t $72 establishes the likely route of synthesis as oleic to linoleic to linolenie acid since unlike the other mutants it responded to linolenie but not ]inoleic acid. Whether these acids in themselves are essential or whether they are synthesized into other acids of a high degree of unsaturation which are essential cannot be determined with certainty yet. The

UNSATURATED FATTY ACID METABOLISM

441

negative results obtained with arachidonic acid can be construed as indicating that the synthesi~ of this compound is not the essential reaction involving the 18-carbon unsaturated acids in Neurospora.The fact that arachidonie acid does not enable the mutants to grow points to a major difference between unsaturated fatty acid metabolism in Neurosporaand the higher vertebrates. It also makes it probable that the fatty acid deficiency symptoms produced in higher organisms may not be associated with the particular metabolic deficiency found to exist in the fatty acidrequiring microorganisms. The interpretation of the relationships outlined above is schematically presented in Fig. 1. Sll $33 $82 $93 *

> -----)

J

Vertebrates

r i

i i

i J

,

X i i

$72

oleic acid

' ,

~, linoleic acid

--+ linolenie acid

----->

FIG. 1. P a t h w a y of synthesis of u n s a t u r a t e d f a t t y acids a n d location of t h e genetic blocks. SUMMARY

Five independent occurrences of Neurospora mutants requiring unsaturated fatty acids were found. Four of these were allelic and grew in the presence of either oleic, linoleic, or linolenic acid. The remaining mutant which was not allelic was partially blocked in the conversion of linoleic to linolenic acid. Biotin, aspartic acid, or pyridoxine did not affect the requirement for unsaturated fatty acids in these mutants. The mutants tested did not respond to arachidonic acid. The results were interpreted as indicating that the unsaturated fatty acids are synthesized through a route distinct from that of the saturated acids and that they are synthesized in the order of oleic to linoleic to linolenie acid. REFERENCES 1. BURR, G. O., AND BURR, M. NI., J . Biol. Chem. 82,345 (1929). 2. WHITE, E. A., FoY, J. R., AXD CE~ECEDO, L. R., Proc. Soc. Exptl. Biol. Med. 54, 301 (1943). 3. HANSEN, A. E., AND WIESE, H. F., Proc. Soc. Exptl. Biol. Med. 52,205 (1943). 4. REISER, R., J. N u t r i t i o n 42, 319 (1950).

442 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

LEIN~ PUGLISI AND LEIN ]-IUTNER, S. H., ]. Bacteriol. 43, 629 (1942). HUTCHINGS, B. L., AND BOGGIANO,E., J. Biol. Chem. 169,229 (1947). WILLIAMS,V. R., AND FIEGER, A. E., J. Biol. Chem. 170,399 (1947). WILLIAMS,W. L., BROQUIST, H. P., AND SNELL, E. E., J. Biol. Chem. 170, 619 (1947). SHELL, G. M., THOMA, 1:~. W., AND PETERSON, W. I-I., Arch. Biochem. 20, 227 (1949). KITAY, E., AND SNELL, E. E., J. Bacteriol. 60, 49 (1950). WILLIAMS,V. R., AND FIEGER, E. A., J. Biol. Chem. 177,739 (1949). AXELROD,A. E., MITZ, M., AND HOFMANN,K.~ J. Biol. Chem. 175,265 (i948). BVHH, G. O., BVRH, M. M., AND MILLER, E. S., J. Biol. Chem. 97, 1 (1932). REISER, R., AND GIBSON, B., J. Nutrition 42,325 (1950). FRAENKEL, G., AND BLEWETT, M., Biochem. J. (London) 40, xxii (1946). LEIN, J., AND LEIN, P. S., J. Bacteriol. 58,595 (1949). QUAC~:~NBUSg,F.: AND STEENBOCK, H., J. Nutrition 24,393 (1942). BHOQUIST, H. P., AND SNELL, ]~. E., J. Biol. Chem. 188,431 (1951). LEIN, J., MITCHELL, H. K., AND HOULAHAN, M. B., Proc. Natl. Acad. Sci. U. S. 34, 435 (1948). BEADLE, G. W., AND TATUM, E. L., Am. J. Botany 32,678 (1945). WESTERGAARD,M., AND MITCHELL, H. R., Am. J. Botany 34, 573 (1947). LEIN, J., AND LEIN, P. S., J. Bacteriol. 60, 185 (1950). HOLMAN,R. T., AND TAYLOR,W. S., Arch. Biochem. 29,295 (1950). :BERNHARD, K., AND ALBRECHT, H., Helv. Physiol. Acta 6, 277 (1948). BREUSC~, F. L., Advances in Enzymol. 8, 343 (1948).