EFFICIENT SYSTEM FOR GENETIC-MODIFICATION OF LACTIC BACTERIA - CONSTRUCTION OF FOOD GRADE STRAINS

The construction of recombinant strains of lactic bacteria has become an important objective for many researchers. New strains may resolve s ome of the pre-existing problems in industrial fermentation, and may o ffer new approaches to meeting medical and pharmaceutical needs. Such an approach is limited by technical problems of manipulation of the la ctic bacteria. We have therefore focused on the methodology for the is olation of recombinant strains. Here, we describe the development of a broad host range thermosensitive (Ts) vector, pG+host, which allows e fficient integration of DNA by single and double crossing over into th e chromosome of a variety of Gram-positive bacteria. pG+host has been adapted with an origin of transfer which allows it to be mobilized in trans by a conjugative helper plasmid derived from broad host range pl asmid pIP501. We describe a method, using pG+host, which permits the c onstruction of food grade recombinant strains, ie, the integration of foreign DNA (or other modifications) without leaving a selectable mark er behind. Using this method, 1-40 % of a bacterial population underwe nt replacement recombination in the absence of selection for the repla cing DNA. With selection, 50-98 % of chromosomal replacements were sel ected. Possible desirable features of recombinant lactic strains are d iscussed.