Regulation of spiramycin synthesis in Streptomyces ambofaciens: effects of glucose and inorganic phosphate.

The production of the 16-membered macrolide antibiotic, spiramycin, in Streptomyces ambofaciens is inhibited by glucose, 2-deoxyglucose and inorganic phosphate. The role of intracellular ATP content and phosphorylated metabolites as common regulating signals of both glucose and phosphate inhibitory effects is discussed. Two enzymatic targets of the effect of phosphate on spiramycin biosynthesis were studied. Valine dehydrogenase, the first enzyme of valine catabolism (supplier of aglycone spiramycin precursors), and alkaline phosphatase, which cleaves phosphorylated intermediates, were repressed in the presence of excess phosphate.

Glycerol effect on spiramycin production and valine catabolism in Streptomyces ambofaciens.

Spiramycin production by Streptomyces ambofaciens in a chemically defined medium, with valine as nitrogen source, was controlled by the nature and the concentration of the carbon source. The production of this antibiotic was better in dextrins than in glycerol-containing medium. The negative effect of glycerol could be attributed in part to an excess of energy and a high specific growth rate. The intracellular ATP content, at the start of spiramycin production, was twofold higher in glycerol than in dextrin-containing medium. Increasing the initial concentrations of glycerol led to an increase in the specific growth rate and a drop in spiramycin production. Comparison between glycerol and a protein synthesis inhibitor effects and the use of resting cell systems (RCS) proved that glycerol exerted both inhibitory and repressive actions on spiramycin production independently from the growth. At the enzymatic level, glycerol interfered with valine catabolism by repressing partially valine dehydrogenase (VDH) and alpha-ketoisovalerate dehydrogenase (KIVDH), generator of spiramycin aglycone precursors.

Regulation of valine catabolism by ammonium in Streptomyces ambofaciens, producer of spiramycin.

In Streptomyces ambofaciens, valine favored spiramycin biosynthesis by supplying aglycone precursors. The kinetics of valine consumption and isobutyrate production showed that isobutyrate accumulated in the cell during the growth phase, was excreted in the stationary phase, and then was reassimilated during spiramycin production. When valine was in excess, its deamination led to high ammonium excretion and to a significant drop in spiramycin production. We demonstrated that ammonium ions were the cause of the negative effect. Addition of a chelator agent, Ca3(PO4)2, improved spiramycin production by sixfold. In contrast, addition of ammonium, between 0 and 48 h, severely reduced spiramycin production. The negative effect of ammonium was reversed by addition of a catabolic intermediate of valine, isobutyrate. In addition to stimulating the specific growth rate, ammonium ions slowed down valine catabolism: the specific valine uptake rate, excretion, and reassimilation of isobutyrate were lowered by the pulse of ammonium. Our study showed that in addition to valine dehydrogenase, which provided the nitrogen necessary to the cell, ammonium ions repressed ketoisovalerate dehydrogenase, which introduced valine as carbon, energy, and aglycone precursor sources. However, valine dehydrogenase and ketoisovalerate dehydrogenase did not constitute the principal enzymatic targets of the negative effect of ammonium in spiramycin production.

Physiology and genetics of antibiotic production and resistance.

Actinomycetes have the genetic capability to synthesize many different biologically active secondary metabolites and of these compounds, antibiotics predominate in therapeutic and commercial importance. Intensive research often centres on the use of molecular techniques to investigate the physiology and genetics of antibiotic biosynthesis with a view to improving production. The isolation of clones of Streptomyces hygroscopicus, the producer of geldanamycin, which synthesizes geldanamycin in S. lividans, is reported. Molecular approaches using genes for elongation factors (tuf) were used in attempts to increase the fermentation yield of kirromycin, whilst probes for aphD and sph, genes for streptomycin phosphotransferases, were used to gather information on streptomycin genes in soil. Actinomycete populations in soil and earthworms may help in developing a strategy for discovering additional antimicrobials in soil. The relationship of proline metabolism to the secondary metabolite undecylprodigiosin and the carbon regulation of spiramycin biosynthesis in S. ambofaciens is also reported.