bioreactor studies of heterologous protein production by ...

CEtAPTER 3
MATERIALS AND METHODS
3.1. Recombinant S. cerevisiae and Cuiture Media
3.1.1. Microorganism and Plasmid
The recombinant S. cerevisiae saaui used is C468IpGAC9 (ATCC 20690), which secretes
glucoamylase into the extraceiiular medium (Nunberg et al, 1988). The soain con-
the hybrid piasmid pGAC9. The piasmid pGAC9 contains a portion of the yeast 2p
plasmid (2p micron cinle), a DNA fragment h m the El coü piasmid pBR322, a DNA
fragment (LEU2 gene) brn S. cerevisicre that encodes the LEU2 gene product (leucine),
and a section of a glucoamyk gene hm Aspergillus awamori. under control of the
yeast enolase 1 promoter and teminator. A plasmid map for pGAC9 is illustrated in
Appendix A. The yeast S& C468 (host ceii) is haploid S. cerevisiae with auxotrophic
markers for leucine and histidine and cames a mutation (mal) blocking the utiiization of
maltose as clubon source. Therefore, the host ceil C468 is complementary to the leucine
prototrophy by inserthg the selectable marker (LEU2) into the expression plasrnid and the
presence of the glucoamylase gene on the plasnid dows the host cell to grow on maltose.
This recombinant yeast was selececi for its Wty to excrete an enzyme, md the 2p-based
shuttie vector, pGAC9, has features common to many yeast vectors and thus selves as a
good mode1 organism.