Cloning of catBCIJFD genes for catechol degradation into chromosomal pobA and genetic stability of the recombinant Acinetobacter calcoaceticus.

Abstract

A possible obstacle in the development of hybrid strains of Acinetobacter calcoaceticus by the introduction of a metabolic pathway into the chromosome is genetic instability of the resulting recombinant strains. Therefore, the possibility that the pobA gene can be used as a chromosomal cloning site where the transposed genes can be maintained and expressed, was explored in this study. For this purpose, two model hybrid strains of A. calcoaceticus were created, in which a DNA fragment carrying catBCIJFD genes for catabolic degradation of catechol was inserted into pobA in opposite directions of each other, and their genetic stabilities were experimentally examined. Our data demonstrated that the stability of the genes neighboring the insertions depends on the orientations of the insertions. Also, the data further indicated that the functional metabolic pathways introduced into pobA can be expressed successfully as far as the insertion is engineered in an appropriate way. Concurrently, it was proposed that the pobA can be used as a chromosomal cloning site, and that introduction of an useful metabolic pathway into pobA may offer considerable promise to the construction of a hybrid strain with improved metabolic capabilities.