Metabolic potential of E. coli related to foreign protein expression and evaluation of acetate(-) mutant as production host

Abstract

A metabolic potential of genetically engineered E. coli as expressed by rate and yield of foreign protein production was studied. The lipase from Pseudomonas fluorescens and human lymphotoxin (hlymphotoxin) were produced in the different physiological states of E. coli. Whereas the lipase synthesis controlled by tac promoter was continued for about 4 h after IPTG induction, the hlymphotoxin controlled by T7 promoter was synthesized for about 2 h after IPTG induction. The duration of synthetic phase of two proteins were irrespective of expression rates and yields, which were manipulated by using α-methyl glucose (α-MG), a competitive inhibitor of glucose. Therefore the expression rate after induction would determine the final expression yield of foreign lipase and hlymphotoxin in E. coli. By measuring the specific oxygen uptake rate ($qO_2$), specific $CO_2$ evolution rate ($qCO_2$), specific glucose uptake rate (qs) and intracellular protease level, the limited duration of expression phase was found to be caused by the metabolic stress due to rapid and massive production of foreign protein. The expression periods of both proteins were considerably reduced to less than a half of normal period of each protein by the addition of menadione, a redox cycling drug, seemingly due to the acceleration of energetic flow of host cells after induction. In contrast, the productive phases of both proteins were extended to 8 and 5 h, respectively, in glucose-starved condition although the final expression level was much lower than that in glucose-surplus condition. Temperature shock on the cell and foreign protein expression also controled the synthetic duration of both proteins. Based on these observations, it could be suggested that E. coli exhibits the restrained capacity of foreign protein production because of the metabolic stress due to the explosive synthesis of foreign protein under the strong promoters. In order to evaluate the acetate (-) mutant of E. coli as a poten...