CHARACTERIZATION AND EVALUATION OF A PTA (PHOSPHOTRANSACETYLASE) NEGATIVE MUTANT OF ESCHERICHIA-COLI HB101 AS PRODUCTION HOST OF FOREIGN LIPASE

Abstract

In order to evaluate the pta(phosphotransacetylase) (-) mutant of Escherichia coli as a potential host of foreign lipase expression, the pta(-) mutant HB101 was constructed for the purpose of blocking the acetate synthetic pathway. Since acetate is known as a major inhibitory by-product of cell growth and foreign protein production, the growth characteristics and expression kinetics of the microbial lipase of the pta(-) E. coli mutant were investigated. The growth rate was considerably decreased (about 30%) when grown on M9 minimal media containing glucose, mannose or glycerol. Growth retardation was not observed when a gluconeogenic carbon source (acetate, malate or succinate) was utilized. It should be noted that the growth rate of the mutant was enhanced (about 20%) in modified M9 media including a gluconeogenic carbon source and NZ-amine. Growth inhibition of the ptcr(-) mutant by menadione, a representative redox-cycling drug, was more pronounced than that of the parental type of E. coli. Furthermore, the inhibition effect was more pronounced in glucose minimal medium, whereas the menadione sensitivity was not observed when a gluconeogenic carbon source was used as a sole carbon source or the lactate dehydrogenase gene from Lactobacillus casei was introduced in the pta(-) mutant. Therefore, it is suggested that the growth deficiency of the pta(-) mutant is closely related to the intracellular redox balance. When the pseudomonad lipase was expressed in the pta(-) mutant, a comparable expression rate and yield to the parental type strain was observed. High-cell-density culture of the mutant was easy to achieve even under the fluctuating conditions of residual glucose concentration.