AN AUTOSELECTION SYSTEM IN RECOMBINANT KLUYVEROMYCES-LACTIS ENHANCES CLONED GENE STABILITY AND PROVIDES FREEDOM IN MEDIUM SELECTION

An autoselection system for increasing plasmid stability in Kluyveromy ces lactis, based on the blockage of the pyrimidine de novo and salvag e pathways, was investigated. In a manner analogous to that used in Sa ccharomyces cerevisiae, a putative ''fur 1'' mutation was selected in a uraA K. lactis strain using 5-fluorouracil and 5-fluorocytosine plat es. Survival of the mutant required expression of a plasmid-borne URA3 gene regardless of the culture medium employed, verifying the efficac y of this autoselection system in K. lactis. The expression of heterol ogous invertase, encoded by the S. cerevisiae SUC2 gene, was studied d uring long-term sequential batch cultures (70 generations) in complex yeast/peptone/glucose medium. The fur1 mutant successfully retained th e plasmid; invertase specific activity remained above 90% of the initi al level. Furthermore, no mutation reversion was observed. In contrast , for the control non-furl strain, only 4% of the cells retained the p lasmid after 70 generations, and invertase specific activity dropped t o less than 10% of the initial level. Experiments comparing growth and activity in different media indicated the potential for improving pro ductivity through medium enrichment using this autoselection system.