A study on the physiology of growth and product formation in Saccharopolyspora species.

An unknown Saccharopolyspora species which displayed fungicidal activity was selected as a representative of the genus. The antifungal activity could not be repeated in liquid cultures after the initial screen despite several different approaches. A defined medium was developed. However, biomass yields were low (< lg/1) and antifungal activity was not detected. Good growth was observed in nutrient broth (NB) and on nutrient agar (NA). The maximum specific growth rate in NB was 0.25h-1. Growth on NA (measured using image analysis) conformed to the linear growth law with a mean radial growth rate of 0.0327mm/h. Fungicidal activity was measured on solid media, this was presumably due to antibiotic production, although an active compound could not be isolated. A mutation program yielded only non producing mutants despite the mutants having a range of growth rates. A known Saccharopolyspora species, Saccharopolyspora erythraea, was then adopted as a representative of the genus for the remainder of the study. Defined media were developed to permit growth limitation by a range of nutrients. A comparison of several approaches to calculate the maximum specific growth rate was made (ranging from 0.119 to 0.148h-1 in carbon limited media, 0.093 to 0.130h-1 in nitrogen limited, and 0.143 to 0.165h-1 in phosphate limited). Curve fitting provided an accurate series of specific growth rate estimates. The control mechanisms involved in the biosynthesis of erythromycin by Saccharopolyspora erythraea were investigated. By culturing this organism in a range of conditions, and analysing the effects on various growth parameters, nutrient specific patterns of control were observed. Increasing biomass and increasing antibiotic titres occurred concurrently in media when growth was carbon (C), nitrogen (N), and phosphate (P) limited. Generally, when antibiotic titre parallels growth, it is assumed that the product is growth linked. However, further analysis contradicted this. The peaks of specific growth rates and specific erythromycin production rates, obtained by curve fitting, were clearly temporally separated in carbon and phosphate limited conditions indicating non-growth linked production of erythromycin. Growth associated production was confirmed in nitrogen limited conditions. Phosphate limitation supported higher antibiotic yields than the other limitations (Yp/x max mg/g = 6.4 in CL1, 18.5 in NL1, and 28.8 in PL1). Erythromycin production was sensitive to ammonium and to glucose during phosphate limitation. However, this sensitivity to glucose was not obvious in the nitrate limited medium, suggesting different control mechanisms may affect the growth linked and non-growth linked production of erythromycin. Investigations into the regulation of the initiation of erythromycin production indicated that energy charge was not obviously involved, as it appeared to be related to the specific growth rate rather than antibiotic production. The rate of protein synthesis appeared to be a strong candidate for the initiator of antibiotic biosynthesis. Chemostat culture confirmed that erythromycin production was growth linked in nitrate limited conditions. However, the kinetics were more complicated as both biomass and antibiotic decreased with increasing dilution rates. The data conformed to the model proposed by Pirt (1975) for non-competitive inhibition by a growth linked product. A modelling package generated estimates of parameters involved (ki = 1.2mg/1, ks = 0.017g/l, micro max = 0.062h-1 and Yp/s = 0.0019).