bioreactor studies of heterologous protein production by ...

LEU2 or TRPI is cloned into the auxotmphic mutant. Plasmid retention is then oecessary
for Sunnvai of the auxotmph if the medmm is devoid of esse- leucine or ûyptophaa.
This systern quires the use of a defined seiective growth medium, a limitaiion that ir
overcome in autoselective systems. The latter combines a host mutant that lacks a gene
that is essential for suMvai with a plasmid vector carrying the essential gene. Thus,
without the plasmici, the mutant œU dies regafdless of the culture medium and conditions-
S. cerevisiae autoselective strains with mutation in the W, FURI, and URID gens
have been obtained through sequential isolation (Napp and Da Silva, 1993). These
mutations effectively block both the pyrimidine biosynthetic and salvage pathways, and, in
combination, are lethal to the host. Therefore, a plasmid carrying a URA3 gene is essential
for sumival, and nonselective media cm be employed without the risk of plasmid loss
(Napp and Da Silva, 1993). A more advanced autoselection system was desnibed by Lee
and Hassan (1988). In this case, the selection pnssun was imposed by utüituig the yeast
killer toxin-irnrnunity complementary DNA (cDNA) inserted into a plasmid. Thus, the
presence of such a piasmid vector confered not only host irnmmity but also the selective
agent (killer toxin) which was secreted hm the ceils into the culture medium. The
plasmid-carrying celis survived because of the Unmunity to the kiner toxin conferred by the
plasmids while the plasmid-h tells perished due to the killu toxin- Lee and Hassan
(1988) nansformed the piasmid (pYTI6û-ADHI) containing the yeast lriIIer toxin-
immunity cDNA into a leucine-histidine mutant (AH22) and showed that the phsrnid was
extremely stable (100% Mer cek). This system has the potentid to operate in any strains
which are initidy sensitive to killer toxin and in any medium As autoselection systems