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A Multi-Layer-Controlled Strategy for Cloning and Expression of Toxin Genes in Escherichia coli.

ABSTRACT: Molecular cloning and controlled expression remain challenging when the target gene encodes a protein that is toxic to the host. We developed a set of multi-layer control systems to enable cloning of genes encoding proteins known to be highly toxic in Escherichia coli and other bacteria. The different multi-layer control systems combine a promoter-operator system on a transcriptional level with a riboswitch for translational control. Additionally, replicational control is ensured by using a strain that reduces the plasmid copy number. The use of weaker promoters (such as P_BAD or PfdeA) in combination with the effective theophylline riboswitch is essential for cloning genes that encode notoriously toxic proteins that directly target translation and transcription. Controlled overexpression is possible, allowing the system to be used for evaluating in vivo effects of the toxin. Systems with a stronger promoter can be used for successful overexpression and purification of the desired protein but are limited to toxins that are more moderate and do not interfere with their own production.

SUBMITTER: Vandierendonck J

PROVIDER: S-EPMC10467106 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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A Multi-Layer-Controlled Strategy for Cloning and Expression of Toxin Genes in <i>Escherichia coli</i>.

Vandierendonck Jessie J Girardin Yana Y De Bruyn Pieter P De Greve Henri H Loris Remy R

Toxins 20230818 8

Molecular cloning and controlled expression remain challenging when the target gene encodes a protein that is toxic to the host. We developed a set of multi-layer control systems to enable cloning of genes encoding proteins known to be highly toxic in <i>Escherichia coli</i> and other bacteria. The different multi-layer control systems combine a promoter-operator system on a transcriptional level with a riboswitch for translational control. Additionally, replicational control is ensured by using ...[more]

PMID: 37624265

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Project description:This report describes the isolation and preliminary characterization of a 5.0-kilobase-pair (kbp) EcoRI DNA restriction fragment carrying the catBCDE genes from Acinetobacter calcoaceticus. The respective genes encode enzymes that catalyze four consecutive reactions in the catechol branch of the beta-ketoadipate pathway: catB, muconate lactonizing enzyme (EC 5.5.1.1); catC, muconolactone isomerase (EC 5.3.3.4); catD, beta-ketoadipate enol-lactone hydrolase (EC 3.1.1.24); and catE, beta-ketoadipate succinyl-coenzyme A transferase (EC 2.8.3.6). In A. calcoaceticus, pcaDE genes encode products with the same enzyme activities as those encoded by the respective catDE genes. In Pseudomonas putida, the requirements for both catDE and pcaDE genes are met by a single set of genes, designated pcaDE. A P. putida mutant with a dysfunctional pcaE gene was used to select a recombinant pKT230 plasmid carrying the 5.0-kbp EcoRI restriction fragment containing the A. calcoaceticus catE structural gene. The recombinant plasmid, pAN1, complemented P. putida mutants with lesions in catB, catC, pcaD, and pcaE genes; the complemented activities were expressed constitutively in the recombinant P. putida strains. After introduction into Escherichia coli, the pAN1 plasmid expressed the activities constitutively but at much lower levels that those found in the P. putida transformants or in fully induced cultures of A. calcoaceticus or P. putida. When placed under the control of a lac promoter on a recombinant pUC13 plasmid in E. coli, the A. calcoaceticus restriction fragment expressed catBCDE activities at levels severalfold higher than those found in fully induced cultures of A. calcoaceticus. Thus there is no translational barrier to expression of the A. calcoaceticus genes at high levels in E. coli. The genetic origin of the cloned catBCDE genes was demonstrated by the fact that the 5.0-kbp EcoRI restriction fragment hybridized with a corresponding fragment from wild-type A. calcoaceticus DNA. This fragment was missing in DNA from an A. calcoaceticus mutant in which the cat genes had been removed by deletion. The properties of the cloned fragment demonstrate physical linkage of the catBCDE genes and suggest that they are coordinately transcribed.

Dataset Information

A Multi-Layer-Controlled Strategy for Cloning and Expression of Toxin Genes in Escherichia coli.

Publications

A Multi-Layer-Controlled Strategy for Cloning and Expression of Toxin Genes in <i>Escherichia coli</i>.

Similar Datasets

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