Genetic instability and tryptophan productivity in continuous culture of recombinant escherichia coli harboring trp-operon plasmid

Abstract

For the analysis of genetic instability and productivity of plasmid coded product in a recombinant cell fermentation system, a continuous culture was performed using a mutant of E.coli W3110 $\triangle$trp LD trp R$^{-ts}$ tna (pCRT185) harboring trp-operon. The host cell with the trp repression system possesses the resistance against feedback regulation and the temperature induction of derepression. In a batch culture, temperature were determined to be $37\,^\circ\!C$ for the optimal cell growth with repression of trp-operon (permissive temperature) and $42\,^\circ\!C$ for the maximum expression of trp-operon accompanied by derepression (nonpermissive temperature), respectively. The activity of tryptophanase was found to be 430 units/g-cell at $42\,^\circ\!C$ which was 3 times higher than that obtained at $37\,^\circ\!C$. It was also found that addition of anthranilic acid (0.5 g/l) to the medium increased the tryptophan production 11 times comparing to without its addition. In a continuous culture, specific tryptophan productivity was increased with time elapsed at a early phase and then decreased gradually due to the loss of plasmid from the host cells and rapid increase of plasmid-free cells. It was also decreased with increase of dilution rate. At dilution rate, 0.075 hr$^{-1}$, specific tryptophan productivity was found to be about 10 mg/g-cell-hr in the derepressed state, representing about thirty times increase over that found in the repressed state. From the kinetic analysis of plasmid instability in a continuous culture, it was found that the rate constant of plasmid loss was a order of magnitude of $10^{-5}$ and the increase of plasmid-free cells during the culture was mainly caused by the difference of specific growth rate between plasmid-free cells and plasmid-harboring cells rather than by the rate of plasmid loss.