Metabolic modelling of stringent response in recombinant Escherichia coli

摘要

Recombinant protein production in Escherichia coli often derive cellular stress responsescharacterized by several biochemical reactions, most of which controlled at the genetic level.Drainage of precursors and energy often results in nutrient starvation, especially amino aciddeprivation inducing the cellular stringent response. The production of a specific nucleotide,ppGpp, catalyzed by the enzyme RelA, is the primary signalling and initiating event in thestringent response, when an uncharged tRNA attaches to a ribosome. This nucleotideinteracts with RNA polymerase to control its activity in a promoter-selective mechanism,which ultimately leads to the inhibition of synthesis of stable RNAs (rRNAs and tRNAs),which results in the decrease of ribosome concentration and, therefore comprising the overallprotein synthesis machinery. Moreover, this stress response also leads to the activation ofsynthesis of specific mRNAs coding for proteins involved in proteolysis and other stressfactors.To gain a better understanding of the dynamics of the stringent response, a mathematicalmodel was developed. Here we propose an hybrid modelling approach based on cooperationbetween kinetics-based dynamic model and FBA-based static model. FBA simulation wasused to incorporate valuable data in the kinetic model of the E. coli stringent response, i.e.amino acid synthesis. The model integrates the main cellular events involved in stringentresponse: the amino acids biosynthesis and the individual tRNA charging reactions, detectionof uncharged tRNA by the ribosome and consequently activation of the enzyme relA,ultimately leading to the formation of ppGpp. The effect of ppGpp on the control oftranscription rate is also predicted.