Expression of chloramphenicol acetyltransferase gene in escherichia coli by zymomonas mobilis DNA fragments

Abstract

This research was carried out for the purpose of developing shuttle vectors which can express foreigns genes efficiently in both Zymomonas mobilis and Eschericha coli. The initial approach was done by isolating and charaterizing Z. mobilis DNA fragments having promoter activity in E. coli. For this, a promoter screening vector, pCMT215 was constructed by ligating pMT21 with chloramphenicol acetyltransferase (CAT) gene from pYEJ001. The pMT21 is a plasmid containing -lactamase gene and multiple cloning sites without nonspecific binding with E. coli RNA polymerase. A promoterless CAT structrual gene was available from pYEJ001. Fragments of Z. mobilis DNA were inserted in pCMT215 and a gene library was prepared in E. coli. Fourteen clones out of this library showed resistance to chloramphenicol ranging from 30 ug/ml to 750 ug/ml of broth. The recombinant plasmids isolated from the Cm transformatns contained inserts with the size of 170-1800 base pairs. Among fourteen clones, five clones were selected and their nucleotide sequencies were analysed adn their bases for transcription initiation were determined. The Z. mobilis DNA fragments examined share many features described for the promoter regions such as partial sequence homology with -35 and -10 region of E. coli consensus promoters, AT rich regions, poly A``s or T``s stretches and palindromic sequences. The positions of transcriptional initiation appeared at morethan one site spaced over by 4 to 170 base pairs in E. coli. All the fourteen clones have a potential to be developed as a shuttle expression vector. Basic informations on the requirements in sequences of the transcription and translation in Z. mobilis will be also available.