Genetically controlled cell lysis in the yeast Saccharomyces cerevisiae

The cell wall of the yeast Saccharomyces cerevisiae is a tough, rigid struc ture, which presents a significant barrier to the release of native or reco mbinant proteins from this biotechnologically important organism. There is hence a need to develop inexpensive and efficient methods of lysing yeast c ells in order to release their intracellular contents. To develop such a me thod, a tightly regulated promoter, pMET3, has been used to control three g enes involved in cell wall biogenesis: PDE2, SRB1/PSA1, and PKC1. Two of th ese regulation cassettes, pMET3-SRB1/PSA1 and pMET3-PKC1, have been integra ted at the chromosomal loci of the respective genes in order to overcome pr oblems of plasmid instability. Although repression of PDE2 did not cause ce ll lysis, cells depleted of Srb1p/Psa1p gradually lost their viability and integrity, releasing about 10% of total protein into the medium. Repression of PKC1 led to extensive cell lysis, accompanied by the release of 45% of cellular protein into the medium. A double mutant, carrying both pMET3-SRB1 /PSA1 and pMET3-PKC1 cassettes in place of SRB1/PSA1 and PKC1, was construc ted and found to permit the efficient release of both homologous and hetero logous proteins. (C) 1999 John Wiley & Sons, Inc.