THE KIL GENE OF THE COLE1 PLASMID OF ESCHERICHIA-COLI CONTROLLED BY AGROWTH-PHASE-DEPENDENT PROMOTER MEDIATES THE SECRETION OF A HETEROLOGOUS PERIPLASMIC PROTEIN DURING THE STATIONARY-PHASE

Heterologous gene products produced by Escherichia coli cells can be e xported into the culture medium by the action of the kil gene of the C olE1 plasmid, which encodes a bacterial release protein. The kil gene was fused with the stationary-phase promoter of the fic gene of E. col i, and a secretion cassette (Kil-Km cassette) containing the regulated kil gene, the Km-resistance gene, and multiple cloning sites for the integration of target genes was constructed. Using the gene for beta-g lucanase (bgl) as a target gene, it was shown that the protein produce d was only secreted into the medium during the stationary phase. Quasi -lysis and lethality were not observed. The primary effect of the indu ction of the kil gene was the overproduction of beta-glucanase. The to tal amount produced per milliliter of bacterial culture was almost thr eefold higher than that of the corresponding Kil(-) control. The prote in pattern of periplasm and culture medium was analyzed before and aft er induction of the kil gene expression, indicating that the release o f periplasmic proteins is semiselective. This secretion system is the first to use a growth-phase-regulated promoter for the expression of t he kil gene.