Genetic manipulation of Kluyveromyces lactis linear DNA plasmids: gene targeting and plasmid shuffles

Genetic manipulation of yeast linear DNA plasmids, particularly of k1 and k 2 from the non-conventional dairy yeast Kluyveromyces ces lactis, has been advanced by the recent establishment of DNA transformation-mediated one-ste p gene disruption and allele replacement techniques. These methods provide the basis for a strategy for the functional analysis of plasmid genes and D NA elements. By use of double selection regimens, these single-gene procedu res have been extended to effect disruption of individual genes on plasmid k2 and transplacement of a functional copy onto plasmid k1, resulting in th e production of yeast strains with an altered plasmid composition. This cyt oplasmic gene shuffle system facilitates the introduction of specifically m odified alleles into k1 or k2 in order to study the function, expression (f rom UCS promoters) and regulation of cytoplasmic linear plasmid genes. Addi tionally, identification, characterization and localization of plasmid gene products of interest are made possible by shuffling GFP-, epitope- or affi nity purification-tagged alleles between k2 and k1. The gene shuffle approa ch can also be used for vector development and heterologous protein express ion in order to exploit the biotechnical potential of the K. lactis k1/k2, system in yeast cell factory research. (C) 1999 Federation of European Micr obiological Societies. Published by Elsevier Science B.V. All rights reserv ed.