:Folia Miorobiol. 23, 194---197 (1978)

Effect of Malonate and Maleate on Growth and Brefeldin A Formation in Curvularia lunata C. T. SAM* Department of Pharmacy, University of Sydney, Sydney, New South Wales, Australia 2006

Received July 10, 1977

ABSTRACT. Malonate exerted a stronger inhibitory effect on brefeldin A production than on myeelia growth in cultures of Curvularia lunata especially at inhibitory levels of 100 to 200 rnM. The extent of 200 inM malonate inhibition of growth and brefeldin A production was greater in cultures treated with malonate prior to inoculation t h a n those treated following 5 days after inoculation. Maleate at levels of 40 to 220 mM activated brefeldin A formation in cultures though exerting variable effects on myeelia growth.

Brefeldin A is a maerolide antibiotic originally isolated from a strain of Curvularia lunata by Coombe and co-workers (1968). The biosynthesis of brefeldin A in C. lunata was found to involve a uniform head-tail incorporation of 1-x4C-acetate (Coombe et al., 1967). Tracer studies using submerged cultures of Penicillium cyaneum and 9-14C-palmitate led Bu'Lock and Clay (1969) to suggest t h a t the resultant labelling pattern in brefeldin A could arise principally from the direct incorporation of an intact labelled palminate molecule. Nutrient-limited studies by Sam (1977) revealed that omission of inorganic phosphate in replacement cultures of C. lunata activated the production of the antibiotic. F u r t h e r studies by Sam (1978) indicated t h a t the growth and production of brefeldin A were affected by the incorporation of crude extracts of brefeldin A into the media prior to inoculation. Malonie acid at the 40 m ~ level inhibited the induced synthesis of cellulase by lactose in cultures of Trichoderma lignorum (Lobanok and Ivlicheva, 1971). However, Rolinson (1954) reported t h a t addition of 200 mM malonate to a culture medium at p H 6.0 or higher exerted no effect on penicillin production or respiration by washed mycelial suspensions of Penicillium chrysogenum. The present investigation was undertaken to ascertain the effects of malonate and maleate on mycelial growth a n d brefeldin A production. MATERIALS AND METHODS

Organism and cultivation. An isolate of Curvularia lunata (BO~D) was obtained from the collection in the Department of Botany, University of Sydney, Australia. The complete culture medium for competitive studies was comprised of 2 ~/o glucose, 0.5 ~ bactopeptone and trace amounts of inorganic salts. The pH of the medium was adjusted to a final value of 5.4 with K O H prior to inoculation (Sam, 1977). Except in the isolated case of 200 m ~ malonate-treated cultures, malonate and * Present address: 2-V, Lorong Delima Sembilan, Island Glades, Penang, Malaysia.

1978

E F F E C T OF M A L O N A T E A N D M A L E A T E ON C. L U N A T A

tgS

TA~L~ I. The effect of m a l o n a t e on mycelial growth a n d brefeldin A production in Curvularia lunata

Malonate concentration mM

Mycelial dry weight rng/ml

50 100 150 200 200a Control b Control c

6.4 6.9 6.5 6.6 3.1 7.8 8.8

Culture b r o t h ghlcose mg/inl

brefeldin A

0.42 0.81 0.85 1.42 11.10 0.12 0.57

80 21 10.5 13.8 0.6 120 31

~g/ml

aMalonate added prior to inoculation; cultivation for 15 days. bCultivation for 20 days. eCultivation for 15 days.

maleate were individually added in duplicate to the cultures five days following inoculation. The malonate-treated and maleate-treated cultures were incubated at 28 ~ in a stationary manner for a further 15 days and 10 days, respectively, whereas the duplicate set of 200 mM malonate-added cultures having malonate added prior to inoculation was incubated for 15 days under the same conditions. Malonate and maleate, in the form of potassium salts, were dissolved in aqueous media and adjusted to a final p H value of 5.4 with HC1. Determination of brefeldin A. The culture broths were extracted with diethyl ether and brefeldin A was determined isothermally at 210 ~ according to Sam (1977) using a gas-liquid chromatograph fitted with a flame ionization detector and a glass column packed with the silicone elastomer SE-30 on chromosorb W, 80/100 mesh. An acetone solution of phenyl salicylate was used as an internal standard. Glucose was determined b y the glucose oxidase method of Huggett and Nixon (1957) with slight modifications (Sam, 1977). RESULTS

Effect of malonate The addition of increasing levels of malonate to 5-day-old cultures of C. lunata resulted in a significant drop in the level of brefeldin A on the 20th d a y of cultivation (Table I). TABLE II. The effect of m a l e a t e on mycelial growth a n d brefeldin A production in Curvularia lunata

Maleateconcentration mM

Mycelial d r y weight mg/ml

40 90 130 170 220 Control

12.1 9.1 7.9 7.1 7.9 8.8

Culture broth pH

glucose mg/ml

brefcldin A ~tg/ml

6.3 6.0 5.8 5.7 5.0 4.0

0.31 1.00 1.25 1.23 1.34 0.57

68 84 75 67 50 31

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C . T . SAM

Vol. 23

There was little difference in the brefeldin A levels of these 150 and 200 m ~ malonate-treated cultures. However, the brefeldin A levels of the 50 mM and 150 mM malonate-treated cultures were 67 ~/o and 8.7 ~/o respectively, of that of the control. Glucose assimilation was high in all these cultures and the minimum mycelial yield was no less than 82 % of the control. In contrast, the 200 m ~ malonate-treated cultures with malonate added to the media prior to inoculation produced extremely low brefeldin A and mycelial yields.

Effect of maleate The addition of increasing levels of maleate to 5-day-old cultures resulted in high yields of brefeldin A on the 15th d a y of cultivation (Table II). The level of brefeldin A present in the 90 mM maleate-treated cultures was optimal and amounted to about 2.7 times that of the control. Cultures treated with 40 mM to 170 m~1 maleate exhibited decreased mycelial development b u t the 220 mM maleate-treated cultures has a higher mycelial yield than the 170 mM one. Moreover, the 220 mM maleate-treated cultures produced a minimum brefeldin A yield, amounting to 160 ~ of the control. Glucose utilization in all these cultures and the control was extensive and showed insignificant differences. The broth p H values decreased with increasing levels of maleate though these values were still higher than the control. DISCUSSION

The formation of brefeldin A in comparison to mycelial development was more sensitive to the increased levels of malonate and this could result in low metabolic pool(s) for antibiotic formation (Table I). Malonate (as a structural analogue of succinate) in the event of assimilation of an appropriate level, is usually decarboxylated to acetate prior to cellular utilization. Thus the significantly more intense inhibition of brefeldin A production than mycelial growth coupled with the high glucose uptake suggests t h a t any assimilated malonate could not possibly be utilized for antibiotic production or cellular multiplication and that an alternative route for glucose metabolism was p r o b a b l y in operation with metabolites derived therefrom being utilized preferentially for mycelial growth. In contrast, a separate s t u d y b y Kitada and F u k i m b a r a (1970) revealed that malonate at 0.1 to 10 m ~ levels exhibited an almost linear relationship in the inhibition of kojic acid formation and the assimilation of glucose from the medium. It is also apparent that cultures having malonate added prior to inoculation were more sensitive to malonate than those having malonate added on the 5th day of cultivation. The findings in Table I I suggest that at the levels of maleate used, especially at 40 and 90 mM, the cultures had apparently utilized some of it for mycelial growth and possibly for brefeldin A production, resulting in elevated broth p H values. Maleate (as the cis-isomer of fumarate) could function as a competitive inhibitor of fumarase, a sulfhydryl reagent or a chelator of cations. It seems t h a t at the higher (130 to 220 raM) maleate levels, mycelial growth becomes slightly sensitive although the brefeldin A levels were still higher than the control. REFERENCES B u ' L o c K J. D., CLAY P. T.: F a t t y acid cyclization in the b i o s y n t h e s i s of brefeldin A: a now route to some fungal metabolites. Chem. Commun. 25, 237 (1969). COO•BE R. G., F o s s P. S., WATSON T. R . : The biosynthesis o f brcfeldin A. Chem. Commun. 23, 1229 (1967). Coo~BE R. G., JACOBS J. J., WATSON T. R . : Constituents of some C~rvularia species. Au~CraL J . Chem. 21, 783 (1968).

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E F F E C T OF MALONATE AND MALEATE ON C. LU1VATA

197

HUOG~.~rT A. St. G., NIxo~r D. A.: Use of glucose oxidase, peroxidase and o-dianisidine in determination of blood and urine glucose. Lancet 273, 369 (1957). KITADA M., FUKIY,BARA T. : Kojic acid fermentation: Effect of metabolic inhibitors, Hakko Kogaku Zaoshi 48, 676 (1970). LO:BANOKA. G., IVLICItEVAA. M, : Inhibition of induced synthesis of cellulase by some organic oompound~, Vestnik Akad. NauIr Beloru~. S.S.R., Set. Biol. Nauk 2, 70 (1971). RoLINSO~ G. N.: Effect of certain enzyme inhibitors on respiration and on penieillLu formation by Penicillium chrysogenum. J. Gen. Microbiol. 11, 412 (1954). SAM C. T.: Effect of nutrient variations on growth and brofeldin A formation in Gurvularia lunata. ~olia Microbiol. 22, 43 (1977). SA~ C. T.: Effect of brefeldin A and aetinomycin D on culture growth and brefeldin A yield in Curvularia lunata. Folia Microbiol. 23, 133 (1978).

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Effect of malonate and maleate on growth and brefeldin A formation in Curvularia lunata.

:Folia Miorobiol. 23, 194---197 (1978) Effect of Malonate and Maleate on Growth and Brefeldin A Formation in Curvularia lunata C. T. SAM* Department...
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