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Title: Quantitative microbial physiology of Streptomyces coelicolor A3(2)
Author: Davidson, Anne Ogilvie
ISNI:       0000 0001 3407 4321
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1992
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Production of secondary metabolites by Streptomyces requires the use of primary metabolites as precursors. A relationship therefore exists between primary and secondary metabolism which involves diversion of the flow (flux) of these precursors from primary processes to secondary metabolism. Deregulation of control of these fluxes may allow the flux to secondary metabolism to be increased and, ultimately, improve productivity. Attempts to identify enzymes involved in regulation of such fluxes have included the use of "reverse genetics", which has resulted in purification (and cloning of the genes) of several enzymes of the primary metabolic pathways of Streptomyces coelicolor. Quantitation of the fluxes to biomass and to antibiotic production would identify additional enzymes of the central metabolic pathways which are involved in diverting these fluxes from primary to secondary metabolism. A straightforward method for quantifying fluxes is that described for Escherichia coli by W.H. Holms, which requires derivation of the composition of the micro-organism and knowledge of the central metabolic and biosynthetic pathways. Assuming that primary metabolism of Streptomyces was similar to that of E. coli, this method was considered a suitable starting point to quantify the fluxes through S. coelicolor to biomass and to the polyketide antibiotic actinorhodin. Therefore, a requirement for this was to derive compositional data for S. coelicolor. In a minimal medium suitable for the calculation of carbon fluxes (NMM-J; containing a mechanical dispersing agent), S. coelicolor grew in a semi-pelleted fashion in flask and fermenter cultures and exhibited variability of production of both biomass and actinorhodin. Biomass yields were low in comparison to those reported by other workers using different defined media containing carbon-based dispersing agents. Production of actinorhodin occurred in NMM-J when carbon, nitrogen and phosphate were in excess. Dispersed growth of S. coelicolor was obtained by continuous cultivation in a phosphate- limited chemostat (in Jena, Germany); single hyphae grew from spores at low dilution rates and branches appeared, grew longer and became entangled as the dilution rate increased. Actinorhodin and undecylprodigiosin were produced simultaneously during the transient period between steady states at each dilution rate, possibly because of metabolic imbalances. A yellow pigment was synthesized during each steady state. Compositional analyses of S. coelicolor involved measurement of the macromolecular, monomeric and elemental contents. An approximate macromolecular composition was determined to be 53% protein, 14% RNA and 6% DNA; the extent of irreproducibility of growth of S. coelicolor in NMM-J was reflected in the macromolecular compositions which were variable. The approximate contents of nucleic acids were similar to those reported for E. coli at a slow growth rate {i.e., a doubling time of 100 minutes). However, the content of protein was low. Other components were possibly responsible for the lower protein content of the streptomycete in comparison to the enterobacterium. The monomeric composition was determined from the nucleic acid and lipid content and also by direct measurement of amino acids. The amino acid composition showed some relationship to the high (approximately 70%) GC bias of the streptomycete DNA. The elemental composition of S. coelicolor, which had a high oxygen content, was similar to those of other Streptomyces analyzed. This may have been the consequence of the obligatory aerobic growth of Streptomyces. The monomeric composition of S. coelicolor was used to determine the fluxes to biomass using the method of Holms (1986). The fluxes were greatest via pyruvate and least via triose phosphate. Expression as carbon fluxes showed that the highest proportion of the total input flux was to CO2 (approximately 70%), reflecting the low yields of biomass. This theoretical prediction was supported experimentally by measurement of CO2 produced during a single fermentation. This may suggest the requirement for a high maintenance energy. The carbon flux to actinorhodin was less than 2% of the total input, measurement of which was likely to be lost in experimental error. Therefore, production of actinorhodin by wild type strains of S. coelicolor may not be suitable for identification of enzymes involved in regulation of secondary metabolism. Fluxes to biomass and actinorhodin production were compared, assuming organic carbon excreted by S. coelicolor was derived from a-ketoglutarate and pyruvate. An increase in flux through phosphoeno/pyruvate (PEPC) was determined which had been reported for stationary phase cells of Streptomyces sp. C5. Increased activity of PEPC in aging mycelia of S. coelicolor had also been observed in this laboratory. It is therefore proposed that PEPC should be concentrated on as a possible candidate enzyme in regulation of production of secondary metabolites.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Microbiology