Capítulo IV Resultados
3. Modelo Universitario Minerva Ejes transversales del MUM
3.4 Uso de un segundo idioma
The nitrogen regulation of erythromycin biosynthesis in S. erythraea, formerly Streptomyces erythreus (Labeda, 1987), has also been studied. Many studies were focused intensely on the effect of nitrogen source on erythromycin production rather than on the growth of this organism in particular. The relationship between growth
and the production of erythromycin can be reversed by the nature and the concentration of the medium ingredients (Trilli et al, 1987; McDermott et al, 1993).
INTRODUCTION Nitrogen in Microbial Process
Cultures of this microorganism, when grown with glycine as nitrogen source, produce
more antibiotic than cells grown in the presence of ammonium ions (Smith et al, 1962). Erythromycin formation decreased with increasing ammonium concentrations
present in the medium (Flores and Sanchez, 1985). Total inhibition of synthesis was
obtained with 100 mM ammonium chloride but medium pH and culture growth were
not significantly affected. A similar effect was obtained with ammonium nitrate or
ammonium sulphate indicating that ammonium ions probably repressed the formation of antibiotic. Ammonium repression of erythromycin biosynthesis is also supported
by the fact that chloramphenicol decreased antibiotic production in a similar fashion,
when it was added to the culture after erythromycin production had started. This phenomenon of nitrogen repression has been reported to affect many catabolic enzymes, including histidase and urease, where enzyme concentration is dependent on
the ammonium ion concentration in the medium (Tyler, 1978).
In relation to specific growth rate and specific product formation, the rate of product formation was found to increase with increasing growth rate, indicating that antibiotic production firom S. erythraeus was growth-linked (Trilli et a/., 1987). Higher initial concentrations of ammonium sulphate reduced the final erythromycin production yield to the benefit of a biomass accretion, as the cell pellets were abundant at the onset of
the erythromycin production. On the other hand, high concentrations of ammonium nitrate favoured erythromycin yield, which was doubled. The ammonium assimilatory
pathways could be involved in the regulation of erythromycin production (Potvin and Peringer, 1994a), but it suggests a different role for nitrate as a nitrogen source.
Generally, ammonium ions are assimilated via the glutamate dehydrogenase (GDH) to
form glutamate (high concentration) or through glutamine synthetase (GS) to form
glutamine (low concentration) (Shapio, 1989) Under non-limiting nutritional
conditions erythromycin production was growth associated with ammonium sulphate as the nitrogen source, but was growth-dissociated with ammonium nitrate. It was
suggested that the residual nitrate level might be the key regulatory element. A sufficient residual amount of nitrate is necessary to induce glutamine synthetase (GS);
otherwise, nitrate could then become a complementary nitrogen source.
INTRODUCTION Nitrogen in Microbial Process
A nitrate-induced glutamine synthetase pathway was postulated which was active
according to the nature and initial concentration of the ammonium salt (Potvin and Peringer, 1994b).
Ammonium assimilation in S. erythraea was mediated by the glutamine synthetase (GS) / glutamate synthase (GOGAT) pathway depending on the concentration of ammonium in the culture medium. Glutamine synthetase (GS) formation was
repressed by high ammonium concentrations, and this led to a rapid loss of its activity after ammonium addition. On the other hand, the glutamine synthetase (GS) activity
was found high upon the use of glutamic acid as the nitrogen source, while there was
no erythromycin production. Glutamate synthase (GOGAT) was form independently of the nitrogen sources used, but glutamate dehydrogenase (GDH) was presented when ammonium was added as a nitrogen source, and it was NADPH-dependent. It was suggested that there was no correlation between the levels of nitrogen assimilating
enzymes and erythromycin biosynthesis in this microorganism (Flores and Sanchez, 1989).
The macrolide antibiotic erythromycin produced by S. erythraea consists of the lactone ring which requires one propionate and six methylmalonate molecules as precursors for its biosynthesis (Roberts, et al, 1993). These compound could have multiple metabolic origins in actinomycetes; catabolism of odd-numbered fatty acids, reduction of acrylate, rearrangement of succinyl-coenzyme A (CoA) and catabolism of
methionine, threonine or valine. The latter two processes are likely to be the primary routes to 2-methylmalonyl-CoA and propionyl-CoA under typical growth conditions
(Tang era/., 1994).
The previous evidence obtained by Hsieh and Kolatukudy (1994) indicates that the
main way to form propionyl-CoA is through the decarboxylation of methylmalonyl- CoA reaction catalysed by methylmalonyl-CoA decarboxylase. On the other hand,
methylmalonyl-CoA could be obtained by isomerization of succinyl-CoA via
methylmalonyl-CoA mutase activity or by corboxylation of propionyl-CoA using propionyl-CoA carboxylase. Both activities have been detected in S. erythraea (Hanaiti and Kolattukudy, 1984; Hanaiti and Kolattukudy, 1982), but the
INTRODUCTION Nitrogen in Microbial Process
erythromycin production in this microorganism did not require propionyl-CoA carboxylase activity (Donadio et al, 1996). Thus, succinyl-CoA becomes an important intermediary and regulation of erythromycin precursor synthesis which
should affect methylmalonyl and propionyl-CoA pools.
Bermudez et a/.(1998) have determined the activity profiles of methylmalonyl-CoA mutase, methylmalonyl-CoA decarboxylase and NADP^-isocitrate dehydrogenase (located in TCA cycle) in Saccharopolyspora erythraea CA340, where their pathway and locations are shown in Figure 1.4. They found that methylmalonyl-CoA mutase and isocitrate dehydrogenase activities were associated with growth, while maximal
activity of methylmalonyl-CoA decarboxylase was observed during the stationary
phase. Isocitrate dehydrogenase synthesis was stimulated by glucose and repressed by ammonium ions. Glucose decreased the synthesis of methyhnalonyl-CoA mutase, whereas methylmalonyl-CoA decarboxylse production was not affected by carbon or nitrogen sources. This suggests that the negative effect of glucose or ammonium ions on erythromycin production could be due, in part, to lower pools of succinyl-CoA and methylmalonyl-CoA.
Isocitrate dehydrogenase
I
TCA Cycle |Methionine
Threonine
V a lm e — ^ Propionyl-CoA
Odd-number fatty acid
Carboxylase Acrylate Decarboxylase
i
Succinyl-CoA Mutase R-Methylmalonyl-CoA ^ Epimerase S-Methylmalonyl-CoA ^ Erythromycin -4-Figure 1.4 Routes of formation of propionyl-CoA and methylmalonyl-CoA reported in antibiotic-producing actinomycetes (Bermudez et al, 1998).
EXPERIM ENTAL PR O G R A M M E S S. erythraea S tu d ie s