title: IS2-mediated re-arrangement of the promoter sequence suppresses metabolic burden of the recombinant plasmid.
abstract: Recombinant plasmid pKA18 of the high expression bacterial system for penicillin amidase ('penicillin G acylase') bears the 3' end region of IS2 element. The IS2 sequence replaces the -35 region of promoter of pga and extends up to TAGTAT box at position -10 of the promoter region. It therefore forms a hybrid promoter of pga ppgaHT. A natural promoter ppgaWT was not detected on any recombinant plasmid isolated from recombinant strains of Escherichia coli constitutively producing penicillin amidase. PCR fragments carrying both types of promoters were cloned into the promoter-probe vector pET2 to compare their transcriptional activity: the activity of ppgaWT was 5x higher than that of ppgaHT. The same nucleotide "G" localized 28 nucleotides upstream of the translation start point was identified as the respective transcription start point of both mRNAs. An attempt was made to place the pga gene cloned on a plasmid under the control of the natural promoter: not a single clone expressing penicillin amidase was found among 150 transformants. High transcriptional activity of the natural promoter together with high pga gene dosage could result in a deleterious metabolic burden of the periplasmic enzyme.
authors.affiliation: Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague, Czechia.
authors.forename: R
authors.initials: R
authors.lastname: Valesová
authors.forename: V
authors.initials: V
authors.lastname: Stepánek
authors.forename: B
authors.initials: B
authors.lastname: Vecerek
authors.forename: P
authors.initials: P
authors.lastname: Kyslík
mesh_terms: D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D015342:DNA Probes; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D010399:Penicillin Amidase / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D014158:Transcription, Genetic
chemical_list: D015342:DNA Probes; D004251:DNA Transposable Elements; D011994:Recombinant Proteins; D015500:Chloramphenicol O-Acetyltransferase; D010399:Penicillin Amidase