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Ammonium salts are the principle nitrogen sources which have been reported to interfere with antibiotic production. The importance of this effect indicates that it is

not limited to any particular type of antibiotic or microorganism. Although a possible

indirect effect of pH has not always been ruled out, the repression of enzymes involved in antibiotic biosynthesis seems to be quite a common effect of ammonium

ions.

In cephalosporins biosynthesis by S. clavuligerus, ammonium ions cause a concentration dependent reduction of production. This interference was observed when ammonium was used as the only nitrogen source or in combination with amino acids, like asparagine, that supported good production (Aharonowitz and Demain, 1979; Brana et al, 1985). In order to distinguish between possible repressive or inhibitory effects of ammonium, the ability of the cells to produce cephalosporins in resting cell systems was assessed.

In this technique, washed cells previously grown in fermentation medium are incubated in buffer with the addition of a protein synthesis inhibitor. Therefore, the

initial rate of antibiotic production in this system is a reflection of the antibiotic

biosynthesis machinery existent in the cells at the moment of harvest. Resting cell productivity in S. clavuligerus was observed to decrease if the cells had been previously grown in the presence of ammonium salts, suggesting that antibiotic synthesis was repressed by ammonium ions. On the other hand, addition of

ammonium salts to the resting cell systems did not immediately decrease their initial productivity, indicating a lack of direct inhibition by ammonium ions. Similar studies

with erythromycin producer S. erythreus have also indicated a repressive, but not an inhibitory, effect of ammonium ions on antibiotic production (Flore and Sanchez, 1985). Clavulanic acid synthesis by resting cells of S. clavuligerus was also decreased by ammonium ions (Romero et al, 1984).

INTRODUCTION Nitrogen in Microbial Process

However, in this case, since protein synthesis was not inhibited as part of the experimental design, it is not clear whether repression, inhibition, or both was

involved. The production of tylosin by Streptomyces fradiae is another example of an ammonium-sensitive process that has been studied in detail (Omura and Tanaka,

1985). Decreased production was observed when ammonium salts were added to the

medium, especially at early times in the fermentation. Tylosin does not contain

nitrogen in its aglycone, but labelling studies indicated that the lower fatty acids used as precursors originated from several amino acids whose metabolism was decreased

by ammonium ions (Masuma et al, 1983; Omura et al, 1984). At least two primary metabolism enzymes, valine dehydrogenase (Omura et al, 1983) and threonine deaminase (Omura and Tanaka, 1985), were repressed by ammonium ions in parallel

with the decline of protylonolide titres. Omura and Tanaka (1985) also found that ammonium ions interfered with the incorporation of succinate into protylonolide, indicating that the control is not restricted to the metabolism of nitrogen-containing compounds.

Vinning et a l (1985) studied the production of chloramphenical by Streptomyces venezuelae and showed that the mechanism by which ammonium ions depress the production of chloramphenical is less clear. Ammonium ions did not seem to repress

arylamine synthetase, the initial enzyme in the pathway of chloramphenical biosynthesis, and the onset of production was not necessarily linked to ammonium

depletion in the cultures (Shapiro and Vining, 1984). Supplementation with ammonium ions of cultures already engaged in antibiotic production caused an abrupt

interruption in the biosynthesis of chloramphenical. However, resumption of

production occurred in the presence of ammonium ions, independent of the amount added, suggesting that neither repression nor inhibition of chloramphenical pathway

enzymes was the cause of the interruption. A metabolic reorganisation in response to the sudden availability of ammonium ions that would affect precursor pools was

proposed as an explanation of this effect (Shapiro and Vining, 1985).

INTRODUCTION Nitrogen in Microbiai Process

The example from nourseothricin production by S. noursei showed that ammonium ions concentration was among the effectors regulating the alternation between two

metabolic states. At high ammonium concentrations, catabolism of amino acids and

repression of anabolic NADP-dependent glutamate dehydrogenase occurred along

with low nourseothricin production.

A predominance of anabolism with high glutamate dehydrogenase activity and

antibiotic synthesis characterised the alternative metabolic state occurring in the

presence of low ammonium concentrations. A number of other factors were also implicated in the inter-conversion between both types of metabolism (Grafe et al, 1981). Addition of o-aminobenzoic acid increased the NADH/NAD ratio in the mycelium (Grafe et al, 1978), repressed formation of cytochrome a (Grafe et al,

1980), decreased amino acid transport and catabolism, and switched the cell into the metabolic state characterised by high antibiotic production. A complex model of regulation was proposed (Grafe et al, 1981) in which ammonium ions would interfere with nourseothricin synthesis at the levels of expression of secondary metabolism and/or the formation of antibiotic precursors.

The sensitivity of different antibiotic biosynthetic processes to ammonium

concentration depends on the antibiotic, the producing microorganism, and the experimental conditions. Cephamycin C production in batch cultures of S. clavuligerus took place during growth with ammonium as nitrogen sources and stopped when ammonium was depleted (Brana et al, 1985; Lubbe et al, 1985). But in S. cattleya, another cephamycin C producer, the synthesis of this antibiotic began only after ammonium consumption had ceased (Bushell and Fryday, 1983). Nanaomycin

synthesis by S. rosa subsp. notoensis was hindered by high ammonium concentrations, but the maintenance of a relatively low level of ammonium ion (10 mM) was

necessary for optimal production (Tanaka et al, 1984). But in the case of streptomycin production by S. griseus, ammonium was thought to allow an accumulation of glutamate during growth, with the subsequent abundance of amino

donors for antibiotic biosynthesis during the production phase (Inoue et a l, 1983).

INTRODUCTION Nitrogen in Microbial Process

However, the possible interactions between the ammonium supply and the rest of the nutrients employed should be taken into account. Growth-limiting conditions may be

obtained with a suitable carbon source in the presence of ammonium, overcoming

totally or partially the interference of the nitrogen source (Chatteijee et al, 1983). The concentrations of phosphate or other salts may also modulate the response of antibiotic

production to ammonium levels (Kuratsu et al, 1983; Young et al, 1985).