Development of an autoregulated protein expression system in Escherichia coli

Research project P 14556 Autoregulated protein expression in E. coli Diethard MATTANOVICH 09.10.2000 The overexpression of recombinant proteins in Escherichia coli exerts a metabolic burden on the host cell, which is manifested by stress signals. Among other consequences, this stress leads to an inhibition of cell division and finally the cessation of product formation. For a stable and reproducible biotechnological production process, a highly productive expression system that does not overburden the host organism is of utmost interest. The aim of this project is the construction of an autoregulated expression system that is able to down-regulate the expression rate in reaction to the stress signal guanosine tetraphosphate. For this purpose it is intended to test the efficacy of the ribosomal RNA B promoter P I as the basis of a stress regulated recombinant protein production system.

Bacteria are one of the most important groups of organisms for the biotechnological production of proteins. The production of these proteins often exerts a strong stress on the cells, so that cell metabolism and growth are impaired, and the productivity is negatively affected. The aim of this project was to develop a cellular control system that adapts the production rate of a desired protein to the stress status of the cell by utilising genetic switches that react to a typical stress signal of bacterial cells. Different strains were constructed and compared with conventional strains by measuring growth, product formation and the stress status of the cells, as assessed by different stress signals. In order to achieve first data in the suitability of the novel systems, the best strains were also cultivated in the laboratory scale under conditions similar to an industrial production. The project yielded novel basic scientific knowledge on interrelations in the metabolic regulation of bacterial cells under stress conditions, as well as applied scientific approaches for the optimisation of biotechnological production systems.