CONTROLLED-EXPRESSION VECTOR FOR PSEUDOMO NAS BACTERIA INVOLVING REGULATORY ELEMENTS OF TRPIBA GENES OF PSEUDOMONAS-PUTIDA

A bireplicon controlled-expression vector pPS10 was developed based, o n trpIBA genes of Pseudomonas putida. It is a low-copy-number vector i n Pseudomonas bacteria, and a high-copy-number vector in Escherichia c oli. The vector is 10.4 kilobase pairs (kb), determines resistance to kanamycin, carries a replicon of cryptic Pseudomonas pMK1 plasmid; a p BR322 replicon; the par locus of pMT2 plasmid; and the trpI gene of P. putida, which encodes the activator protein and the promoter Pba of t rpBA genes. Expression of the promoter is induced by the TrpI protein activator and the precursor of tryptophan, indole-3-glycerolphosphate (InGP). InGP is an unstable compound, and its accumulation in bacteria l cells is ensured by using trpE mutants grown in the presence of anth ranilate; no InGP is produced among trpE mutants on the media suppleme nted with tryptophan. As shown in the pheA gene of E. coli, the expres sion of genetic material cloned under control of the Pba promoter into the pPS10 vector, may be enhanced more than 70-fold in cells of Pseud omonas under conditions of InGP accumulation.