Cloning and expression of Acinetobacter calcoaceticus catBCDE genes in Pseudomonas putida and Escherichia coli.

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