European J Appl Mictobiol Biotechnol(1981) 11".

131- 132

Microbiologyand

t plied

B techndogy
9 Spfinger-Veflag. 1981

The Effect of Phosphate on Flavinogenesis in Eremothecium ashbyii

H. B. Mehta and V. V. Modi
Department of Microbiology, Faculty of Science, M. S. University of Baroda, Baroda - 390 002, India

Decreasing and decreased duction

the phosphate

concentration

in

riboflavin,

a pseudo-secondary (Madia et al. 1976)

metabolite, 1970)

in

the medium resulted

in an increased ashbyii.

riboflavin (FMN) pro-

As h b 7 ~
E. ashbyi~

(Kaplan and Demain

and in

flavin mononueleotide transition

has been repor-

in Eremothecium

There was also

ted . Here, we report on the effect of phosphate on flavinogenesis and growth of E. ashbzii. (Mehta et con-

a morphological

from thin mycelial

forms to swollen cells. The medium described previously The influence of phosphate metabolite synthesis, especially of antibiotics The temperature has been reviewed dependence on secondary the production (Martin 1977). al. 1972) centrations. from culture Riboflavin was used with varying phosphate

and F~q were seperated at 450 nm (Fazekas

filtrates by passing through sepha-

of the formation of

dex G-15 column and estimated and Kokai 1971).

,IL

1.6
9 A

4O

60 ~" t20z \0.8

u 20

.~
=

2
Fig. I. Effect of varying flavin (i - 9 specific

T
growth

JO

Log Phosphate cmM)

concentrations of phosphate on total extracellular to FMN flavins (A-A). CO - 0), Ribofil(O - O) and the ratio of riboflavin different phosphate

flavin and flavin mononucleotide trates of E. a s h b y i ~ g r o w n

(FMN) were separated by gel chromatography

of culture

in medium containing

concentrations

132
As shown in Fig. i, maximum flavin production occurred at a concentration (io5.10-3~4) of phosphate which is suboptimal for growth. Increasing the phosphate concentration in the medium from O.O015 ~4 to 150 mM increased the growth by 70% but decreased the accumulation of flavins in the broth by 60%. However, at the same time the value of total flavin expressed in terms of growth (specific flavins) was decreased by 80%. This probably indicated reduced flavinogenic efficiency of cells grown in the high phosphate (150 mM). It was interesting to note that under the same condition, the ratio of riboflavin to F~Z was lowered nearly seven times. This was because the amount of F 89 increased by two fold whereas that of riboflavin, expressed in terms of mg. dry wt., was decreased by 78%. The inhibition of riboflavin production could be correlated with the phosphate inhibition of GTP cyclohydrolase which catalyzes the first step in riboflavin biosynthesis (Mehta and

H.B. Mehta and V. V. Modi: Flavinogenesis inEremothecium a~hbyii

Modi, submitted for publication). It seems, therefore that in cells grown in high phosphate concentrations, F~q formation is favoured and/ or a part of the riboflavin formed is converted into Fi~q. Medium containing high concentrations (150 mM) of phosphate supported uniformly spread mycelial growth. Low phosphate concentrations supported only relatively scanty and granular growth. Microscopic examination of E. ashbyii grown in high phosphate showed thin long mycelia with cytoplasmic polyphosphate granules (Fig. 2a). Typical swollen forms become predominant (Fig. 2b) in E. ashb~ii grown in low phosphate. The polyphosphate granules were m o r e
or

less absent.

A number of mechanisms have been postulated to explain the effect of phosphate on secondary metabolism (Martin 1977). As the industrial fermentation of flavin coenzymes may be preferred to riboflavin due to their direct applicability and demand, it will be worth exploiting nutritional or other factors to increase F~Z production by the industrially important and highly flavinogenic mold E. ashbyii. Acknowledgements. edged. References Fazekas AG, Kokai K (1971) Extraction, purifiThe helpful discussion

with Dr. H. S. Chhatpar is gratefully acknowl-

cation and separation of tissue flavins for spectrophotometric determination. In: McCormick DB, Wright LD (eds)Methods in enzymology, vol 18-B. Academic Press, New York London, pp 385398 Kaplan L, Demain AL (1970) Nutritional studies

on riboflavin overproduction by A. gossypii. In:Ahearn DS (ed) Recent trends in yeast research. Georgia State University Press, Atlanta, pp 137-159 Madia ~M, Mattoo AK, Modi VV (1976) Enzymic constitution, ribitol formation and flavinogenesis in E. ashbyii. Ind J Exp Biol 14:

b
F ~ . 2a and b. The morphological transitions in E . ashbyi~ in response to phosphate. Cells grown in (~) High phosphate (150 n~) and (b) low phosphate (0.O015 raM). The magnification was 430. E . a s h b y i i ceils grown in the presence of high (150 mM) and low (O.OO15 m94) phosphate were stained with Albert's modified stain. Photographs of wet mounts were taken with a Zeiss Ikon Camera and Carlzeiss p h o t o m i c r o s c o p e .

680-683 Martin JF (1977) Control of antibiotic synthesis by phosphate. In: Ghose TK, Fiechter A, Blakegrough N (eds) Advances in biochemical engineering, vol 6. Springer, Berlin Heidelberg, New York, pp 105-127 Mehta SU, Mattoo AK, Modi VV (1972) Ribitol and flavinogenesis in E. ashbyii. Biochem J 130: 159-166 Received January 14, 1980