Sammendrag
One task of the Centre for Digital Life Norway project INBioPharm is to develop optimized Streptomyces Superhost strains for heterologous production of new bioactive compounds by using Systems and Synthetic Biology approaches. One important experimental input to this task will be the generation of high-resolution quantitative metabolite profiles of the Streptomyces host production strains. In this study, wild type strain M145 of Streptomyces coelicolor A3(2), its mutant strain M1146 with the deleted biosynthetic gene clusters producing actinorhodin, undecylprodigiosin, CPK and CDA and M1152 having introduced point mutation into rpoB, were used for complete profiling of the intracellular primary metabolite pools.
Intracellular metabolite pools were quantified using several MS/MS based methods; i.e. two UPLC-MS/MS methods for amino acids and organic acids, a capIC-MS/MS method for nucleotides, sugar phosphate and other phosphometabolites.
Fermentation data were used for mass balance, growth and production kinetics analysis. SSBM medium coupled with either L-glutamate or phosphate limitation triggered antibiotics production. Contrary to phosphate limitation, nitrogen (L-glutamate) limitation triggered the stringent stress response as observed by a sharp decline in the CO2 production and biomass concentration. The consumption of L-glutamate in broth reflected in the depletion of intracellular L-glutamate and the corresponding decrease in intracellular glutamine and α-Ketoglutarate, an entry point of L-glutamate into metabolic pathway. 6-phosphogluconic acid, first metabolite of pentose phosphate pathway (PPP) was found to rise with biomass generation showing increased PPP activity during growth phase. A higher activity of PPP ensures a continued supply of NADPH and precursor metabolites for highly dividing cells. The energy charge ratio was higher during mid-exponential and antibiotic production phase in comparison of stationary phase.
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