Development and Use of Host-Vector Systems for the Characterization of Lactococcal Expression Signals

摘要

Recent research on the genetics of lactococci mainly focuses on the identification and characterization of important traits for dairying, such as proteolysis, lactose fermentation, and phage insensitivity. Since lactic streptococci are GRAS (generally regarded as safe) organisms, they may be used in an advantageous way for the production of proteins not related to dairying. The thesis describes the construction of cloning-, promoter screening- and terminator screening-vectors. In order to clone directly in L. lactis subsp. lactis, a protoplast transformation system is described in which liposomes are introduced to enhance the transformation efficiency, which enabled direct gene cloning in L. lactis. Since the vectors constructed can replicate in B. subtilis as well as in L. lactis, an intergeneric protoplast fusion system was developed, enabling the shuttling of the broad host range screening- and cloning-vectors from the one host to the other without the need for plasmid isolation. The putative promoters of the genes involved in proteinase synthesis (prtP) and maturation (prtM) are characterized with respect to their transcription initiation sites and their capacity to express the cat-86 gene in strain MG1363 and B. subtilis.