Role of the general stress response during strong overexpression of a heterologous gene in Escherichia coli.

The strong overexpression of heterologous genes in Escherichia coli often leads to inhibition of cell growth, ribosome destruction, loss of culturability, and induction of stress responses, such as a heat shock-like response. Here we demonstrate that the general stress response, which is connected to the stress response regulator sigmas (sigma38, rpoS gene product), is suppressed during strong overproduction of a heterologous alpha-glucosidase. The mRNA levels of the rpoS and osmY stress genes drastically decrease after induction of the strong overexpression system. It is shown that an rpoS mutation causes a significant loss of cell viability after induction of the expression system. Furthermore, it is demonstrated that an E. coli c/pP mutant, which could be suggested to improve heterologous protein production, is not a good production host if a tac-promoter is used to control the expression of the recombinant gene. Data from this study suggest that the overexpression of the alpha-glucosidase was greatly decreased by sigma factor competition in the clpP mutant, due to the increased sigmas level in this mutant background.

Screening, nucleotide sequence, and biochemical characterization of an esterase from Pseudomonas fluorescens with high activity towards lactones.

A genomic library of Pseudomonas fluorescens DSM 50106 in a lambdaRESIII phage vector was screened in Escherichia coli K-12 for esterase activity by using alpha-naphthyl acetate and Fast Blue RR. A 3.2-kb DNA fragment was subcloned from an esterase-positive clone and completely sequenced. Esterase EstF1 was encoded by a 999-bp open reading frame (ORF) and exhibited significant amino acid sequence identity with members of the serine hydrolase family. The deduced amino acid sequences of two other C-terminal truncated ORFs exhibited homology to a cyclohexanone monooxygenase and an alkane hydroxylase. However, esterase activity was not induced by growing of P. fluorescens DSM 50106 in the presence of several cyclic ketones. The esterase gene was fused to a His tag and expressed in E. coli. The gene product was purified by zinc ion affinity chromatography and characterized. Detergents had to be added for purification, indicating that the enzyme was membrane bound or membrane associated. The optimum pH of the purified enzyme was 7.5, and the optimum temperature was 43 degreesC. The showed highest purified enzyme activities towards lactones. The activity increased from gamma-butyrolactone (18.1 U/mg) to epsilon-caprolactone (21.8 U/mg) to delta-valerolactone (36.5 U/mg). The activities towards the aliphatic esters were significantly lower; the only exception was the activity toward ethyl caprylate, which was the preferred substrate.