초록
The metabolic control of l-phenylalanine producing Escherichia coli cells was studied in a fed-batch process with glycerol as carbon source. To this end, metabolic perturbation experiments were performed in parallel to the operating process with cells harvested at two process times of interest: at the beginning of the l-phenylalanine production phase and at the end of the process with decreasing l-phenylalanine production. Data from the parallelised short-term perturbation experiments (19min) was used for the evaluation of microbial metabolome, fluxome and thermokinetics. This data was to determine local elasticity coefficients and subsequently the global flux control coefficients. Through the application of this methodical setup, several metabolic reactions were identified which control carbon flux towards l-phenylalanine: reactions that consume and produce phosphoenolpyruvate, while enzymes catalysing reactions of the glycerol metabolism were identified as being highly relevant for l-phenylalanine production. A significant change in metabolic control was detected by comparing the flux control coefficients during the two process phases. At decreasing l-phenylalanine production metabolic control appeared to be redirected towards a limiting fructose-6-phosphate and phosphoenolpyruvate supply. In all, due to the combination of an experimental approach and the framework of metabolic control analysis, improved knowledge about the metabolic network was generated within one single experiment.