From shake-flask, three photos were taken from each sample. Each given .... To sum it up, we can state that above a critical production rate the cephalosporin-C.
BIOTECHNOLOGY LETTERS
Volmne 18 No.6 (June 1996) p.701-706 Received as revised 15th April.
CEPHALOSPORIN-C PRODUCTION, MORPHOLOGY AND ALTERNATIVE RESPIRATION OF ACREMONIUM CHR YSOGENUM IN GLUCOSE-LIMITED CHEMOSTAT Levente Karaffa*, Erzs6bet S~indor, J6zsef Kozma and Attila Szentirmai Dept. Microbiology and Biotechnology, Kossuth University of Sciences, P.O.Box. 63, Debrecen, H-4010, Hungary
Summary: In this paper, the connection between morphology, cephalosporin-C production and alternative respiration ofAcremonmm chlysogemtm is examined. As demonstrated by chemostat experiments, the ratio of the filamentous and the yeast-like forms depended on the growth rate. The yeast-like form, but not the filamentous tbrm exhibited cyanide-resistant alternative respiration. As a consequence, the yeast-like form was regarded to be more suitable for antibiotic overproduction. Introduction:
The filamentous mould Acremonmm chrysogemml is the main producer of the cephalosporin-C antibiotic. The fungus shows different morphological forms in submerged cultures: hyphae, conidia, metabolically inactive arthrospores and wide, swollen hyphae fragments named yeast-like forms (Bartoshevich et al, 1990). While the wild strains form conidia predominantly, the strains with high and medium cephalosporin-C productivity tend to differentiate into yeast-like form (Bartoshevich et al, 1985). That the maximum rate of cephalosporin-C production coincides with the conversion of slender hyphal filaments to swollen hyphal fragments (Huber and Nash, 1971). Furthermore, it has also been demonstrated, that the antibiotic-producing potential of the yeast-like form is considerably higher than that of any other cell types with different morphology and they are appearing at the second half of fermentation (Bartoshevich et al, 1990). Previous data indicate, that growth rate might be a crucial factor in the changes of morphology and antibiotic production. In the case of another 13-1actam producer mould, Penic'illium chlysogenmn, it was shown, that the steady-state penicillin concentration (unit m1-1) rose as the reciprocal of the specific growth rate (Pirt and Righelato, 1967), while the specific penicillin production rate (unit mgl,h l) was independent of it.
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Importantly, Acrenloniun~ chrysogeuunz possesses a cyanide-resistant alternative respiration path, which is branching out at the level of Coenzyme-Q, and hence, it bypasses complex III and 1V in the cytochrome pathway without producing ATP (Rich and Moore, 1976). Therefore, the alternative path is resistant to the inhibitors of complex III and IV, but it can be blocked selectively by aromatic hydroxamic acids, like salicylic-hidroxamate (SHAM; Schonbaum et al, 1971). The alternative route is essential in every system, where the ATP production due to the regeneration of reduced coenzymes inhibits the metabolite overproduction (Kubicek et al, 1980, Jeppsson et al, 1995). This was found to be the case in Acremonium chrysogenum as well (Kozma et al, 1992). In complex batch cultures the antibiotic production rate was found to be proportional to the intensity of the alternative respiration, and it was also demonstrated, that the addition of SHAM e.g. the selective inhibition of the alternative route - immediately stopped cephalosporin-C biosynthesis (Kozma and Karaffa, 1996). In this paper, the connection between cephalosporin-C production and alternative respiration was further investigated as a function of the specific growth rate and of the morphological changes in cultures. Materials & Methods:
In present study, Acremouium chrysogeuum ATCC 46117 (synonym: Cephalosporium acremonium W 532553) was grown on a New Brunswick orbital shaker at 28 °C, 250 rpm in a medium containing 5g/l CaCO3, 10g/1 peptone, 26.8 g/1 yeast extract and 24 g/1 malt extract. Fermentation media were inoculated with 10 % three day old seed culture. The minimal fermentation medium used in chemostat experiments contained 2 % glucose as a carbon source, 0.5 % KH2PO4, Na2HPO4, K2SO4 and DL-methionine, 0.3 % carbamide, 1.5 % (NH4)2SO4, 0.04 % CaC12, and 0.2 % trace element solution, which contained 1.9 % MnSO4-1H20, 1% FeSO4, 1.25 % ZnSO4 and CuSO4. The medium was supplemented with 1 % corn steep solid extract, which contained important trace elements and growth factors (Asz~ir Starch Factory, Asz~.r, Hungary). Glucose, CaC12 and MgSO4 were sterilised separately. Chemostat experiments were performed in a Richter Inel (Hungary) jar with one liter total and 750 mt useful volume. Aeration was carried out by sparging air at 0.5 bar and 40 1 min1, The impeller speed (700 rpm) was adjusted to maintain the dissolved 02 concentration above 50 percent of saturation, which is known to be the critical oxygen concentration for cephalosporin-C biosynthesis (Scheidegger et al, 1984). The temperature was controlled at 28°C. No external pH control was needed, as the pH measured in the outflow medium remained between 6.6-6.7. Polypropylene glycol 2000 was used as anti foam agent, which was injected into the culture media twice a day through a Sartorius membrane filter. The onset of steady-state in the fermenter was established if no changes in the cell mass could be observed in three successive samples taken over a period of three residence times. Batch fermentations were performed in 80 ml aliquots of complex medium prepared by the description of Scheidegger et al. (1984) in 500 nil Erlenmeyer flasks.
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Respiration was measured in an oxygraphic cell, using a Clark-type polarographi¢ oxygen electrode, at 28°C. The ratio of the cytochrome and alternative respiration was determinated by the method of Bahr and Bonner (1973). Potassium cyanide (1 mM) and salicylhidroxamic,-acid (SHAM, 5-20 raM) were used as inhibitors of the cytochrome and alternative respirations, respectively. Both the capacity and activity values of alternative respiration were determined. The first term is the oxygen consumption in the presence of KCN, while activity refers to the m vivo contribution of the alternative path to total respiration, and can be calculated as the ratio of SHAM-resistant respiration to total respiration (McIntosh, 1994). The oxygen consumption in the presence of both KCN and SHAM is called residual respiration. It is caused by cytosolie oxygenrequiring processes (Moiler et al, 1985), and was taken into consideration at each measurement. The physiological effect of SHAM was tested by the addition of the inhibitor into the chemostat in a concentration of 10 raM. Cephalosporin-C (CP.C) production was measured by on a Zorbax C18 reverse phase column (Rocklands Inc., Chadds Ford, PA, U.S.A), which was eluated with 14 mM Na2HPO4, 10.3 mM tetra butyl-ammonium-hydrogen-sulphate, 5% methanol; pH=6.5. Respiration and CP.C. production rates are given on dry cell weight basis. For mycelial dry weight determination 10 ml aliquots of the culture media were filtered through a Sartorius glass wool filter (SM 134) under vacuum and the mycelia were dried in an oven at 80°C until constant weight. The cell morphology was studied on photomicrographs taken with a phase contrast lnicroscope. Negatives were analysed with a microfilm reader, and the length and the diameter of each cell were measured. If the length:diameter ratio was less than or equal to 2, the cell was considered a yeast-like one. For statistical analysis at least three samples were taken at each generation, and the reproducibility of the results was estimated by standard error values. For observing morphological changes, ten samples were taken from each chemostat run, and three photos were taken from each sample. From shake-flask, three photos were taken from each sample. Each given data is the result of more than one hundred measured cells. All chemicals were of analytical grade and were purchased from the Sigma-Chemical Co., Sigma-Aldrich Kff, Budapest, Hungary Results and Discussion:
Chemostat cultures were grown batchwise for 36 hours after inoculation. At an early stage of continuous cultivations, oscillations occurred in the specific biomass production of cultures (Brown, 1990) therefore measurements were started only after 10 residence times. No significant changes could be observed in the specific biomass production of cultures even at P=10 %, similar values were obtained independently of the growth rate. It is worth noting that no washing-out occurred even at a dilution rate of D=0.131 h1, when the culture generation time was only about 5 hour long. The fact that the specific biomass production was independent of the dilution rate shows that the growth of a filamentous mould - similarly to bacteria - also tbllows tile exponential law in chemostat, and that the growth was not limited by the lack of oxygen (Pi~,1957).
703
Physiological activities of cultures were characterised by specific respiration rates (Qoz). These values did not show any difference at P=I0% either, although the non-specific oxygen demand of the cultures was proportional to the growth rate. This might be a surprising phenomenon as one would expect respiration rate to be a function of growth rate. However, the respiration rate of Penicillium chrysogenum was also found to be independent of growth rate above a critical value, which was less than half of its ~t,,,~, (Mason and Righelato, 1975). A similar situation can be supposed in this case as well. Unlike total respiration, the alternative respiration was inversely proportional to the dilution rate. Capacity values showed significant differences even at P=0.1% (Fig. 1). The SHAM-sensitivity of the respiration, which reflected the activity of alternative path to the overall respiration, was also inversely proportional to the dilution rate (Fig.2). Between the 5 and 20 mM SHAM-concentration, the inhibitory effect increased with increasing concentration. Residual respiration was 8-10 percent of the total respiration at all measurements. 12
50 45 ~ 40 35 =~ 30
~. 10 "7
":
8
=
6 ~,
20 ~ 15 10 5 0
4
2 0.131
0.101
0.131
(I.067 0.037
Dilution rate (h1) Fig. 1. Capacity of alternative respiration at different dilution rates in chemostats. Bars indicate standard deviation at P=0.05.
0.101 0.067 0.037 Dilution rate (11-~)
Fig. 2. Salicylic-hidroxamate (SHAM) - sensitivity in percentage of total respiration in chemostats (OndVl[~, 5n~l [ ] ,lOmM ~R~,20mM[ ] ).
The morphology of cells also exhibited differences at different dilution rates. The proportion of yeast-like form in cultures with higher dilution rates was much lower (Fig. 3.), therefore cultures dominated by yeast-like cells had a higher proportion of alternative respiration. In contrast, the specific cephalosporin-C production rate (0.55 _+ 0.02 mg CP.C g-1 hour-1 ) was found constant at all dilution rates studied, so it seemed to be independent of the morphology and of the alternative respiration as well (Fig.4.).
704
0.8 a= 0.7--
tO0 90 80
.~ =
"7
0.6--
70
T
-~
0.5-
60 50 40 30
o)
~5
"1"
0.4-
0.30.2-
20
O.lO,
lO o o.131
0.131
O.lOl 0 . 0 6 7 0.037 Dilution rate (h -~)
Fig. 3. Proportion of yeast-like forms at different dilution rates in chemostats. Bars indicate standard deviations at P=5%. Each data represents over one hundred measured ceils.
0.101
0.067
0.037
Dilution rate (h "~) Fig. 4. Specific production rate of cephalospofin-C (CP.C) at differentdilution rates in chemostats. Bars indicate standard error at P=-5%.
It was reported earlier that in batch fermentations on complete medium with good CP.C. production (2.5-3 g 11) the respiration of cultures was found to be highly cyanide-resistant and the inhibition of the alternative path by SHAM immediately stopped antibiotic synthesis, e.g. production rate declined to zero (Kozma and Karaffa, 1996). In contrast, in minimal medium in chemostats where CP.C. production was 10o
only 0.55 mg g-i h-i no such effect could be
80
observed at any dilution rates studied and the
70 60 , 5(1
same cephalosporin-C production rates could
~
be measured before and after the addition of SHAM. In complex medium, morphology
o
2(1 10 0
could be characterised with an increasing !
I
~
q
t
2(1 40 60 80 100 Ferlnentation time (hours)
rate of yeast-like cell formation in the production phase (Fig. 5.). At the begining of batch cultivation, the high percent of
Fig.5. Proportion of yeast-like forms during shakeflask fermentation. Each data represents over one hundred measured cclls. Bars indicate standard deviation at P=5%.
yeast-like form can be attributed to the inoculum culture, which exhibits yeast-like lnorphology before the inoculation of the main culture
705
Up to this point, it was proven by chemostat cultures that the slower growth rate (e.g. lower dilution rate) involves higher proportion of yeast-like cells and alternative respiration as well. However, the dependence of cephalosporin-C biosynthesis on alternative respiration and morphology was completely different in complete and in minimal medium. This contradiction might be interpreted by the ratio of the produced antibiotic and mycelia. In the chemostat, the cell metabolism was hardly influenced by the biosynthesis of the small amount of cephalosporin-C, which was about 2 percent of the biomass produced. On the other hand, when the fungus was cultivated on a complete medium and the cephalosporin-C production was significantly higher (about 20 percent of the biomass), the biosynthesis needed the alternative respiration, regarding that the cytochrome-dependent respiration could not completely regenerate the reduced coenzymes alone. To sum it up, we can state that above a critical production rate the cephalosporin-C biosynthesis needs the operation of the alternative respiration. This path seems to be an obligate feature of the yeast-like, but not of the filamentous form. We suggest, therefore, that the possession of this path makes the yeast-like cells potentially suitable for high cephalosporin-C production. References:
Bahr, J.T., Bonner, WD. (1973): J. Biol. Chem. 248, 3446-50 Bartoshevich, Yu. E.; Zaslavskaya, P.L.; Novak, M.J.; Yudina, OD.: (1990): 3". Basic Microbiol. 30, 5, 313-320 Brown, A.(1990): Fed-batch and continuous culture. In:Fermentation: a practical approach. B. McNeil and L. M. Harvey, eds. pp. 113-130. Oxford University Press. Kozma, J.; Lucas, L.; Schiiger[, K. (1993): Appl. Microbiol. Biotechnol. 40, 463-465 Kozma, J.; Karaffa, L. (1996): J. Biotechnology (in press). Kubicek, C. P.; Zehentgruber, O.; El Kalak, H.; R6hr, M. (1980): Eur. J. AppHedMicrobiol. Biotechnol. 9, 101 - 105. Jeppsson, H.; Alexander, NS.; Hahn-Hagerdal, B. (1995): Appl. Env. Microbiol. 61, 7, 2596260b. Mason, H.; Righelato, R. C. (1975): J. Appl. Chem. Biotechnol. 26, 145-152 (1976). Moiler, I. M.; Berczi, A.; van der Plas, L.; Lambers, H.(1988): Plautphysiol. 72, 642-49. Nash, C.H.; Huber, F.M.(1971): Applied Microbiology, 22, 1, 6-10. Pitt (1957): J. Geu. Microbiol. 16, 59-70 Pitt, S. J.; Righelato, R. C. (1967): Applied Microbiology, 15, 6, 1284-1290. Rich, P.R.; Moore, A.L. (1976): FEB5: Letters, 65, 3,339-344. Scheidegger, A.; Ki~enzi,M. T.; Ntiesch, J.: (1984): The J. Autibiotics 37, 522- 531. Schonbaum, G.R.; Bonner, W.D.; Storey, B.T.; Bahr, J.T.: (1971): PlantPhysioL 47, 124-128. Telesnina, G. N.; Bartoshevich, Yu. E.; Krakhmaleva, I. N.; Petyushenko, R.M.; Sazykin, Yu. O.; Navaskin, S. M.: (1990): J. BasicMicrobiol. 30, 4-8.
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