Optimized Genetic Tools Allow the Biosynthesis of Glycocin F and Analogues Designed To Test the Roles of gcc Cluster Genes in Bacteriocin Production

Abstract

The entire 7-gene cluster for the diglycosylated bacteriocin glycocin F (GccF), including the natural promoters responsible for gcc gene expression, has been ligated into the Escherichia coli -lactic acid bacteria (LAB) shuttle vector pRV610 to produce the easily modifiable 11.2-kbp plasmid pRV610 gcc for the efficient production of glycocin F analogues. In contrast to the refactoring approach, chemical synthesis, or chemoenzymatic synthesis, all of which have been successfully used to probe glycocin structure and function, this plasmid can also be used to probe in vivo the evolutionary constraints on glycocin scaffolds and their processing by the maturation pathway machinery, thus increasing understanding of the enzymes involved, the order in which they act, and how they are regulated.