Kinetic analysis of temperature-sensitive gene expression in a recombinant E. coli using two-stage continuous culture system

Abstract

For the kinetic analysis and for the stable maintenance of plasmid in a recombinant cell culture system, twostage continuous culture was performed using a mutant of $\underline{E.}$ $\underline{coli}$ W3110 $\underline{trp}$LD $\underline{trp}R^{ts}$ $tna^-$/pCRT185 carrying trp operon. In the first stage at 37$^\circ$C, the plasmid was stably maintained for more than 200 hrs because the cloned genes were not expressed at this repressed condition. In the second stage at 42$^\circ$C, the cloned genes were expressed highly and the plasmid stability was maintained at 100\% as far as the plasmid stability in the first stage was maintained. As a result of the stable maintenance of plasmid in the second stage, the specific production rate of L-tryptophan, $q_p$, was maintained constantly in second stage. The $q_p$ was influenced mainly by the specific growth rate of cell population in the second stage, $\mu_2$, and was the highest (4.2mg L-tryptophan/g-cell$\cdot$hr) at $\mu_2 = 0.011 hr^{-1}$. The plasmid content was increased from 0.05 to 0.4 mg-DNA/g-cell as the $\mu_2$ was decreased from 0.277 to 0.011 $hr^{-1}$. But the expression efficiency of the plasmid-coded gene was increased from 11 to 77 mg-L-tryptophan/mg-DNA$\cdot$hr as the $\mu_2$ was increased from 0.011 to 0.277 $hr^{-1}$. The relative content of plasmid-coded tryptophan synthetase enzyme per cell was decreased from 100\% to 46\% as the $\mu_2$ was increased from 0.011 to 0.277 $hr^{-1}$.