Effects of a recombinant gene product and growth conditions on plasmid stability in pectinolytic Escherichia coli cells.

Abstract

The genes encoding pectin methylesterase (pme) and pectate lyase (pel) from Bacteroides thetaiotaomicron were previously cloned in Escherichia coli. In the absence of selective pressure the recombinant vectors harbouring a functional pel gene were rapidly lost. This instability was due to a toxic effect of the pel gene product when overproduced and was closely related (1) to a decrease of the growth rate, and (2) to the impossibility of transforming different strains of E. coli with the recombinant plasmids harbouring a functional pel gene. When the expression level of the pel gene was reduced and the tet gene partially deleted, the stability was greatly improved. The export of pectate lyase in the extracellular medium was significantly enhanced in the presence of glycine with a positive effect on plasmid stability for low concentrations. Furthermore, using a factorial design at two levels, the effects of tetracycline, ampicillin, glucose and magnesium on pBT4 stability were quantified.