The Effects of Temperature on Growth and Production of the Antibiotic Granaticin by a Thermotolerant Streptomycete

Abstract

The synthesis of granaticin, a polyketide-derived antibiotic synthesized as a secondary metabolite by Streptomyces thermoviolaceus strain NCIB 10076, was studied at different growth temperatures. Quantitative measurements of the antibiotic made during batch fermentations showed that the yield was greatest at 45 "C, whereas the rate of synthesis was most rapid at 37 "C. The timing of the appearance of granaticin in culture could not be assigned to any particular phase of growth or to de-repression due to depletion of any particular nutrient. However, at all temperatures, appearance of the antibiotic coincided with a rise in ammoniacal nitrogen presumably due to deamination of glutamate, the carbon source for growth. We have previously shown that production of the antibiotic is pH sensitive and that some carbon sources result in higher titres than others. This paper examines the effect of temperature on the physiology of growth and on antibiotic production in more detail under conditions that also allow an exact measurement of granaticin yield. I N T R O D U C T I O NTaxonomic studies have revealed that many Streptomyces are able to grow at 45 "C (Williams et al., 1983). However until recently there were relatively few reports concerning the ability of species from this group to grow at temperatures in excess of 50 "C. Goodfellow et al. (1987) surveyed 50 thermophilic streptomycetes from a range of habitats which they were able to assign to a number of clusters in a numerical phenetic study that identified some features of their physiology and metabolic versatility. Such work is valuable in view of the commercial advantages of thermophilic species. These include faster reaction times and reduced cooling costs for large fermentations which make the thermophilic streptomycetes potentially useful vehicles for antibiotic production at high temperatures. Streptomyces thermoviolaceus is a particularly useful model system for such studies because it grows over a temperature range of around 25 to 58 "C (Edwards & Ball, 1987) and produces granaticin, a pigmented and pH sensitive antibiotic (Maehr et al., 1979) which is synthesized via the polyketide pathway (Arnone et al., 1979; Snipes et al., 1979). Snipes et al. (1979), using 13C-labelled acetate and glucose, showed that granaticin is produced by mixed biogenesis from eight acetate units and one dideoxyglucose moiety. This agrees with other radiolabelling work that showed polyketide antibiotics to be acetate derived (Floss et al., 1985). Using an lSO2 atmosphere, Floss et al. (1985) also demonstrated that no molecular oxygen was incorporated into the molecule, indicating a direct cyclization of the polyacetyl chain rather than a classical hydroxylation using molecular oxygen, followed by a dehydration to the lactone. METHODSOrganism and culture conditions. Streptomyces thermoviolaceus subsp. thermoviolaceus NCIB 10076 was maintained on complex solid medium that contained (g 1-l): glucose, 4; malt extract, 10; yeast extract, 4; and after adjusting to pH 7.2, agar, 20....

The effects of temperature, pH and growth rate on secondary metabolism in Streptomyces thermoviolaceus grown in a chemostat

“…Therefore the major processes associated with the phase of secondary metabolism in S. thermoviolaceus consist of antibiotic synthesis and protein and protease secretion. These occur at a time when nutrients in growth media are still in excess and therefore when growth is still occurring (James & Edwards, 1989). This observation is interesting in the light of the more usual finding that secondary metabolites are synthesized only in 'stationary phase' cultures or in cultures that have extremely low growth rates (e.g.…”

“…This organism grows between 25 and 57 "C despite evidence that indicates that some of the reactions of energy production are thermolabile at temperatures upwards of 50 "C (Edwards & Ball, 1987). S. thermoviolaceus utilizes a wide range of carbon sources for growth and many of these also support antibiotic synthesis at temperatures between 30 and 55 "C (James & Edwards, 1988), but optimally at 45 "C (James & Edwards, 1989). At 45 "C, growth in batch culture is biphasic, antibiotic synthesis commencing at the point of inflexion and continuing during the second, slower growth phase.…”

Identification and Characterization of Antibiotic-Producing Actinomycetes Isolates

“…These occur at a time when nutrients in growth media are still in excess and growth is still continuing (James and Edwards, 1989). It has also been suggested that the protein secretion is invariably linked to the production of graniticin antibiotic in Streptomyces thermoviolaceus.…”

Thermostable extracellular peroxidases from Streptomyces thermoviolaceus

“…Its physiology has been well studied in relation t o production of the pigmented antibiotic granaticin (James & Edwards, 1989), and 5'. tbermoviolaceu.r is a good producer of extracellular enzymes such as xylanases (Tsujibo e t al., 1992) and proteases (James e t al., 1991).…”