FOOD-GRADE CLONING AND EXPRESSION SYSTEM FOR LACTOCOCCUS-LACTIS

A versatile set of cloning and expression vectors has been developed f or application in self-cloning and other genetic modifications of Lact ococcus lactis, The expression vectors were equipped with the controll ed and strong lacA promoter of the lactococcal lactose operon, In addi tion, the transcriptional terminator of the aminopeptidase N gene, pep N, was inserted, which in some cases increased the genetic stabilities of the vectors and the cloned DNA, The small, 0.3-kb lacF gene encodi ng the soluble carrier enzyme IIA(Lac) was used as a dominant selectio n marker in the plasmid free L. lactis strain NZ3000 carrying an in-fr ame deletion of the chromosomal lacF gene, Lactose-utilizing transform ants were easily selected on lactose indicator plates at high frequenc ies and showed a copy number of approximately 50 plasmids per cell, Al l vectors were stably maintained in the lacF strain NZ3000 when grown on lactose, and only the high-level expression vectors showed some ins tability when their host was grown on glucose-containing medium, The a pplication potentials of the expression vectors carrying the lacF mark er were determined by cloning of the promoterless Escherichia coli gus A reporter gene under control of the lacA promoter followed by analysi s of its expression, While in one of the vectors this resulted in a pr omoter-down mutation in the -10 region of the lacA promoter, in other vectors high-level and controlled expression of the gusA gene was obse rved.