Aventis Pharma, Process Development Biotechnology, 13 quai Jules Guesde, 94400 Vitry sur Seine, France1
Author for correspondence: François Voelker. Tel: +33 1 55 71 31 06. Fax: +33 1 55 71 33 52. e-mail: francois.voelker{at}aventis.com
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ABSTRACT |
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Keywords: Streptomyces pristinaespiralis, streptogramin, nitrogen regulation, ammonium
Abbreviations: ADH, alanine dehydrogenase; AT, alanine transaminase; BCDH, branched-chain keto-acid dehydrogenase; GDH, glutamate dehydrogenase; GOGAT, glutamate synthase; GS, glutamine synthetase; PI, type I pristinamycin; PII, type II pristinamycin
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INTRODUCTION |
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However, this enzyme is not present in all Streptomyces species (Brana et al., 1986 ) and an alternative pathway involving alanine dehydrogenase (ADH) has been proposed in the same high ammonium conditions (Novak et al., 1992
). In this case, the synthesis of glutamate would require the function of an alanine transaminase (AT) and would yield the same global reaction.
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When the level of ammonium is low, the GS-GOGAT pathway is used for the synthesis of glutamate.
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When utilized as source of nitrogen, amino acids can generate ammonium or glutamate (Fig. 2). Generally, the first step in the utilization of amino acids is the partition of the molecule into the amino group on one side and the carbon backbone on the other side. This can be done either by deamination of the amino acid, yielding free ammonium, or by transfer of the amino group to an acceptor, generally 2-oxoglutarate giving glutamate (Untrau et al., 1994
). In addition, the existence of two GSs in many Streptomyces species (Behrmann et al., 1990
; Kumada et al., 1990
) and the possible occurrence of two GDHs (NAD-dependent and NADP-dependent) that could function in either the assimilative or degradative direction (Vancurova et al., 1989
) has been shown. In S. pristinaespiralis, the occurrence of enzymes of central nitrogen metabolism has not yet been investigated.
On an industrial scale, culture media for antibiotic production generally contain complex nitrogen sources such as soybean meal. These sources have been selected for their ability to sustain high antibiotic titres and this property is supposed to be linked to the slow release of nitrogenous components during the course of the fermentation. More generally, several studies have shown that nitrogen assimilation is crucial for the regulation of antibiotic production but the mechanisms involved have not yet been unravelled. In addition, there is experimental evidence for repression of antibiotic production exerted by some nitrogen sources and especially ammonium (Aharonowitz, 1980 ; Brana & Demain, 1988
).
In this study, we investigate the regulation of pristinamycin production by nitrogen using different nitrogen sources and synthetic culture media to control the supply and exhaustion of the major nutrient sources. The nitrogen sources were chosen from among inorganic (nitrate, ammonium) or organic (amino acids) compounds. They represent three main pathways of nitrogen assimilation, either by nitrate reduction, direct incorporation of ammonium into central nitrogen metabolism or catabolism of amino acids (Fig. 2). All of the growth experiments were carried out under controlled conditions in fermenters to prevent unwanted side effects such as oxygen limitation or pH effects.
We report the kinetics of growth and antibiotic production by S. pristinaespiralis when supplied with these different nitrogen sources, and characterize the different physiological phases occurring during the fermentations. Finally, the regulation of the production of pristinamycins by nitrogen in S. pristinaespiralis is discussed.
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METHODS |
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Media and culture conditions.
Synthetic medium used as seed medium had the following composition (per litre of distilled water): 20 g sucrose, 0·3 g MgSO4.7H2O, 5 g (NH4)2SO4, 0·75 g K2HPO4, 1 ml trace metal solution, 40 g MOPS. The pH was adjusted to 6·8 before autoclaving for 20 min at 110 °C. Trace metal solution consisted of (per litre of distilled water): 45 g Na2EDTA.2H2O, 11 g CaCl2.2H2O, 7 g FeSO4.7H2O, 2 g MnCl2.4H2O, 2 g ZnSO4.7H2O, 0·4 g CuSO4.5H2O, 0·4 g CoCl2.6H2O. Synthetic medium used as production medium contained (per litre of distilled water): 40 g glucose, 0·3 g MgSO4.7H2O, 1·2 g K2HPO4, 1 ml trace metal solution. The nitrogen source was varied as described in the text. These media were sterilized for 20 min at 110 °C. Cultures were initiated by inoculating 0·5 ml of frozen mycelia into a 300 ml baffled Erlenmeyer flask containing 40 ml seed medium. The culture was incubated for 26 h at 27 °C on a rotary shaker at 325 r.p.m. Forty millilitres of the grown seed culture was then inoculated into a 2 l fermenter containing 1·2 l production medium. The pH and dissolved oxygen level were monitored using Ingold specific electrodes, and the pH was maintained at 6·8 by the automatic addition of 3 M NaOH and 3 M HCl. Temperature was regulated at 27 °C and an aeration rate of 1 vessel volume per minute (1 v.v.m.) was employed. The agitation rate was adjusted to keep the dissolved oxygen level above 30% saturation with a starting rate of 800 r.p.m.
Analytical procedures.
For the estimation of biomass dry weight, 10 ml samples of culture were centrifuged (at 6000 g for 10 min) in preweighed tubes, the pellet was washed twice with distilled water and the tube plus pellet was dried at 110 °C for 48 h. The first supernatant was kept for further analysis of the extracellular medium after filtration through 0·22 µm filters (Millipore).
Amino acid analyses of culture supernatants were performed with an amino acid analysis system from Applied Biosystems following the standard procedures. The system consisted of a model 420A derivatizer for pre-column derivatization of amino acids coupled to a model 130A separation system. Norleucine was used as internal standard.
The glucose, glycerol and 2-ketoisovalerate concentrations in culture supernatants were measured using an HPLC method. Separation was achieved at 56 °C on an Aminex HPX-87H column. The column was eluted with a mobile phase of 0·005 M sulfuric acid at a flow rate of 0·8 ml min-1. Column effluent was monitored using a differential refractometer (Shimadzu RID-6A).
Assays of residual phosphate and nitrate in culture supernatants were carried out by ion exchange chromatography using a Dionex DX 120 HPLC system and employing the standard operating procedures for assay of anions.
The concentration of ammonium in culture supernatants was determined with an Orion ammonium electrode model 95-12 with ammonium chloride as standard in conjunction with a digital ion analyser model from Kent.
Extraction, separation and quantitative analysis of pristinamycins were done as follows. One vol. culture broth was mixed with 2 vols HPLC mobile phase (see below) and shaken at 325 r.p.m. for 20 min. The mixture was filtered through a paper filter and the filtrate was analysed by HPLC. PIA, PIIA and PIIB were separated at 23 °C on a Nucleosil C8 column eluted at a flow rate of 1 ml min-1 with a mobile phase consisting of 37% (v/v) 0·1 M potassium phosphate buffer pH 3·0 and 63% (v/v) acetonitrile. Pristinamycins were detected and quantitated by their absorbance at 260 nm.
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RESULTS AND DISCUSSION |
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Metabolic transitions linked to nutrient exhaustions
In this study, synthetic culture media were designed in order to prevent the exhaustion of glucose as the carbon and energy source during the course of fermentation, ensuring at the same time enough carbon for antibiotic synthesis. Phosphate was provided so as to be the limiting nutrient, as confirmed in Fig. 6. Phosphate was always the first major nutrient (out of carbon, nitrogen and phosphorus sources) to be exhausted in the different media, the time of exhaustion corresponding to the maximum respiration rate and to the minimum dissolved oxygen value (Fig. 6
). However, biomass accretion and uptake of carbon and nitrogen sources persisted after phosphate depletion. Increase in biomass after phosphate exhaustion was recorded in all of the media (up to 40% in the medium with glutamate), except in the one with valine. Owing to reserves of phosphate, growth can continue but at a slower rate after exhaustion of phosphate from the medium (Wanner & Egli, 1990
). Phosphate storage materials can include polyphosphates, RNA or teichoic acids in Gram-positive organisms (Mundry & Kuhn, 1991
). The occurrence of teichoic acids serving as cell wall polymers has been demonstrated in Streptomyces by Naumova et al. (1980)
. It is only after the exhaustion of these reserves of phosphate that the growth of the cell really stopped.
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With glutamate as the nitrogen source, a thorough look at the concentration of nutrients in the extracellular medium after phosphate depletion allowed the reconstruction of the different metabolic phases (Fig. 7). The first phase, immediately after phosphate exhaustion, was characterized by a large release of both ammonium and glycerol (Fig. 7
). Ammonium originated from the catabolism of glutamate (the sole nitrogen source) that was still consumed at a high rate after phosphate exhaustion. However, at this time, glutamate seemed to be consumed primarily as a carbon source because of the excretion of ammonium. This would mean that 2-oxoglutarate, the product of deamination of glutamate, was utilized preferentially as the other carbon sources, probably to replenish the tricarboxylic acid cycle in a time of energy deprivation. Consequently, glycerol accumulated in the medium as a by-product. The second phase was initiated by the exhaustion of the amino acid, and the exhaustion of the keto-acid as carbon source must have occurred more or less simultaneously (Fig. 7
). This time corresponded to the peak in the concentration of ammonium. The ammonium was then reutilized by the strain to satisfy its nitrogen needs until its exhaustion in the medium. The residual carbon sources were then glycerol and glucose, and both were used simultaneously. Interestingly, we did not observe any repression of glycerol consumption by glucose as has been observed in other Streptomyces spp. (Hodgson, 1982
). This kinetic profile was observed in two fermentations with different initial amounts of glutamate as the sole nitrogen source (15 vs 13 g l-1) in Fig. 7
. For the fermentation with a higher initial amount of glutamate, more residual glutamate (5 vs 2 g l-1) was present at the time of phosphate depletion and consequently ammonium was excreted in larger amounts (280 vs 160 mg l-1) before exhaustion of glutamate. That meant that more keto-acid was obtained, sparing the utilization of glycerol. Consequently, the level of glycerol released was higher (1230 vs 715 mg l-1). Similar excretion and consumption profiles were observed with ammonium and glycerol in the medium with alanine (Fig. 6c
).
In conclusion, on the basis of the events occurring after growth limitation by phosphate, and especially on the basis of the products excreted, it is possible to distinguish three groups of media. These groups are the same as those already identified according to the utilization of nitrogen sources during growth. The first group, where the production of glycerol was low and where no production of ammonium was observed, corresponded to media with mineral nitrogen sources, and the second group, with a higher level of glycerol production and excretion of ammonium, corresponded to media where amino acids were used as nitrogen sources. The medium with valine was an exceptional case because of the excretion of 2-ketoisovalerate during growth.
Characteristics of the production of pristinamycin
The typical kinetics of production of pristinamycins in synthetic media are shown in Fig. 7. The production phase lasted for less than 10 h and the maximum titres were always below 100 mg l-1. Type II pristinamycins (A and B) represented the major fraction of the total pristinamycins (above 80%), the remainder being pristinamycin IA (see Fig. 1
). Type II pristinamycins (but not type I pristinamycins) are subject to degradation in the fermentation broth (unpublished results). Therefore, one peak of production and then a slow decrease in the pristinamycin titre were observed (Fig. 7
). In Table 2
, maximum production levels are given together with the biomass at the time of maximum production. Their ratio gives a tentative yield of pristinamycins per unit of biomass. Results for media containing valine have been omitted because of the absence of production throughout the course of the fermentation. In the medium with nitrate as nitrogen source, production occurred very early during growth with a resulting low amount of biomass (1·5 g l-1). For media with ammonium, alanine or glutamate, production took place during the stationary phase, after phosphate exhaustion. The yield of pristinamycins per unit of biomass was very similar for all of the media investigated. Regardless of nitrogen source, high biomass production was a necessary condition to obtain a good level of pristinamycin production. This could reflect a limited amount of precursors of pristinamycin per unit of biomass in these media. Apparently, the limitation cannot be alleviated by changing the nitrogen source.
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Possible regulation of production of pristinamycin
The production of antibiotics can take place only if three conditions are fulfilled. First, it is necessary to have the cell factories to synthesize the molecule, i.e. enough biomass; secondly, building blocks, i.e. precursors, have to be present; and thirdly, the workers, i.e. the enzymes to process these building blocks into antibiotics, should be present and active. Specific regulatory factors of antibiotic production have also been reported in the literature. A-factor and virginiae butanolides, compounds with a gamma-butyrolactone structure, have been shown to be involved in the regulatory cascade leading to streptomycin and virginiamycin production respectively (Hara & Beppu, 1982 ; Yamada et al., 1987
). Although the occurrence of similar compounds is known in S. pristinaespiralis cultures (Paquet et al., 1992
), their possible role in the onset of pristinamycin production is not discussed in this paper. In our experiments, the amount of biomass seems to be important only for the quantity of pristinamycin produced and had no influence on the onset of production. Consequently, the absence of production or the delay observed with some nitrogen sources could result either from a shortage of precursors or from a negative regulation of the enzymes involved in pristinamycin biosynthesis.
The absence of production of pristinamycin throughout the fermentation with valine as the sole nitrogen source can be explained by the specificity of the catabolism of valine in this strain. The first steps of branched-chain amino acid catabolism are well established in Streptomyces (Zhang et al., 1999 ; Fig. 8
). In the case of S. pristinaespiralis, the branched-chain keto-acid dehydrogenase (BCDH) enzyme is negatively regulated in a medium with valine, as demonstrated by the excretion of 2-ketoisovalerate. Hafner et al. (1991)
demonstrated that a functional BCDH is mandatory for the synthesis of avermectins because the starters originate exclusively from the degradation of either isoleucine or valine (Fig. 8
). A BCDH- mutant is unable to produce avermectin unless supplied with isobutyrate or methylbutyrate. The same characteristics have been demonstrated for type II pristinamycin synthesis, which shares the same starters, 2-methylbutyryl-CoA or isobutyryl-CoA, with avermectin biosynthesis. The inhibition or repression of BCDH could be explained in two ways. Nitrogen regulation mediated by excess ammonium generated by the degradation of valine can be proposed. It has been reported by Lounes et al. (1995)
that ammonium repressed BCDH in Streptomyces ambofaciens. However, in our work, ammonium was not likely to accumulate because valine was consumed at the same rate as biomass was formed and ammonium was never excreted in the medium throughout the course of the fermentation (Fig. 6d
). The hypothesis of carbon catabolic regulation mediated by glucose is more appealing. According to our results, glucose was utilized as a preferred carbon source compared to the carbon backbone of valine by S. pristinaespiralis. The target of this possible catabolic regulation obviously cannot be the first step of valine catabolism which provides nitrogen to the cell, but would be the second step catalysed by BCDH, preventing any further catabolism of the carbon backbone of valine.
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The various kinetics of pristinamycin production observed in media differing only in the nitrogen source suggest a strong influence of nitrogen metabolism on the production of antibiotics, as already outlined by several authors (Gräfe, 1982 ; Shapiro, 1989
). Occurrence of early production with nitrate as sole nitrogen source compared to late production with either ammonium or some amino acids (alanine and glutamate) could be the result of the absence of regulation by the nitrogen source in the first case and of a negative action in the other cases. This hypothesis is supported by the experiments where production was delayed when using higher initial amounts of ammonium or glutamate as nitrogen source. Nitrogen catabolic regulation has been documented for production of several antibiotics. A negative action of ammonium has already been observed, especially for macrolides (Tanaka et al., 1986
) and beta-lactams (Brana et al., 1985
; Castro et al., 1985
). The direct involvement of an amino acid has been shown in the mechanism of nitrogen catabolic repression of actinomycin D production by glutamate (Foster & Katz, 1981
). The preferred utilization of ammonium over nitrate as nitrogen source is evidence for the occurrence of a mechanism of nitrogen catabolite regulation. Moreover, nitrate assimilation is tightly controlled in order not to generate excess ammonium. Therefore, absence of catabolic repression mediated by ammonium is likely to occur in this case, allowing early production of pristinamycin. The hypothesis of ammonium catabolic regulation is also sustainable in the media with ammonium as sole nitrogen source where the high initial concentrations will prevent pristinamycin production and where the consumption of ammonium will determine the time of onset of production. However, extracellular ammonium was not always totally exhausted at that time. This could be an indication that there is a lower threshold level that triggers production. As for an organic nitrogen source, it is difficult to distinguish between regulation exerted either by the amino acid per se or by ammonium generated by the catabolism of this amino acid. During growth, amino acids were used in the first place as nitrogen sources and in this case no ammonium was excreted. High intracellular levels of ammonium were then not likely to be produced. After phosphate exhaustion, amino acids were used preferentially as carbon source, almost certainly by direct deamination because ammonium was excreted in the extracellular medium. In this case, the probability of excess intracellular ammonium was high. However, whereas pristinamycin production always started after total exhaustion of the amino acids, residual ammonium was still present in the medium at that time. This would once again sustain the hypothesis of a lower threshold level if ammonium regulation is involved. Nitrogen regulation could also be linked, not specifically to catabolic repression, but to the general nitrogen status of the cell. When intracellular ammonium is high, it is generally recognized that nitrogen assimilation in Streptomyces proceeds either by the GDH pathway or by the ADH pathway. However, these dehydrogenases exhibit high affinity constant values and cannot be responsible for assimilation of ammonium when the intracellular level falls down to a threshold value. In this case, the GS-GOGAT pathway takes precedence for assimilation of ammonium (Paress & Streicher, 1985
; Fisher & Wray, 1989
). It is striking that the conditions under which the GS-GOGAT pathway is known to operate are also the conditions of pristinamycin production in our experiments (nitrate-containing medium or medium with low ammonium). Therefore, the system responsible for the switch between the different assimilation pathways could also be triggering the production of antibiotics.
Concluding remarks
In this study, the conditions for controlled culture of S. pristinaespiralis in synthetic media with several single nitrogen sources and using phosphate as limiting nutrient were established. For each case, the main physiological phases of the culture were identified and their features characterized. This knowledge of the patterns of utilization of carbon and nitrogen sources was a prerequisite for the study of the influence of nitrogen metabolism on pristinamycin production. The growth of S. pristinaespiralis is well supported by various organic and inorganic nitrogen sources. Pristinamycin production is independent of the nitrogen source supplied in terms of specific production. However, the nature and the amount of the nitrogen source are both critical in determining the onset of antibiotic production. The results of this study suggest that nitrogen catabolic repression of pristinamycin production is taking place. Moreover, ammonium, either directly supplied as a nitrogen source or originating from the breakdown of amino acids, may play a central role in this negative regulation. Difficulties were encountered during this study in using amino acids as nitrogen sources. Amino acids are sources of carbon as well as nitrogen catabolites, and both are able to exert a regulatory effect. Regulation of pristinamycin production by carbon rather than by nitrogen catabolites is therefore not excluded when using amino acids. In addition, some amino acids have to be considered as direct precursors for antibiotic synthesis and not only as nutrient sources. This study represents the first step toward an understanding of the determinants of pristinamycin production. It will be interesting to investigate the occurrence of nitrogen catabolic repression under different culture conditions, especially under nitrogen limitation. Moreover, to elucidate the mechanism of nitrogen regulation of pristinamycin production, enzymic studies are needed.
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ACKNOWLEDGEMENTS |
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Received 23 January 2001;
revised 2 May 2001;
accepted 22 May 2001.
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