bioreactor studies of heterologous protein production by ...

The commoniy used markers are the genes that can confer fesistance to antibiotics.
Such gens an incorporated into plasrnids and die resulting vectors are transformed into
host ce& Antibiotics-containing seleaive media are now used to eliminate plasnid-fiee
cells whik stably retaining the tfansformed mils. As many antibiotics that are lethal to
bacteria are not lethal to yeasts, the use of antibiotics is resained by the
avaüability of antibiotics. GenetiQn G418, an aminoglycosid~ has been found to be
effective against yeasts. This antibiotic is inactivated by the enzyme arninoglycoside
phospho-transferaseerase3'(I) that is coded by the bacteriai uansposon Tn601. Yeast
Pansformed with a plasmid carrying Tn601 becomes mistant to G418 at concentrations
pater than 150 (Jimenez and Davies, 1980). Other seiectabIe markers have been
used to confer nsistance to antibiotics such as methomxate (Zhu et ai.. 1985; Rouf et al.,
1996) and chlorarnphenicol (Hadfield et al., 1986). However, the use of antibiotics is not
free of pidaiis. Antibiotics are expensive, and their presence cornplicares product
recovery. Sometimes, for example in wastewater treatment with recombinants (Bryers
and Huang, 1995). use of antibiotics may be totally impractical Another selectable marku
is the CLIP1 gene that impm nesistance to high levels of copper. This gene has ben
cloned and characterized by Bun et al. (1984); it offers a potentiaily useful alternative to
antibio tics,
The genes coding for enzymes in the amino acid biosynthetic pathway are &O
commonly used for selection markers. Two such markers are LEU2 or ZRPL Host
mutants for leucine and tryptophan auxotrophy are easily selected. The plasmid canying