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Genetically controlled cell lysis in the yeast Saccharomyces cerevisiae
Author(s) -
Zhang Nianshu,
Gardner David C. J.,
Oliver Stephen G.,
Stateva Lubomira I.
Publication year - 1999
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(19990905)64:5<607::aid-bit11>3.0.co;2-0
Subject(s) - lysis , chemistry , cell , biology , biochemistry
The cell wall of the yeast Saccharomyces cerevisiae is a tough, rigid structure, which presents a significant barrier to the release of native or recombinant proteins from this biotechnologically important organism. There is hence a need to develop inexpensive and efficient methods of lysing yeast cells in order to release their intracellular contents. To develop such a method, a tightly regulated promoter, p MET3, has been used to control three genes involved in cell wall biogenesis: PDE2, SRB1/PSA1, and PKC1. Two of these regulation cassettes, p MET3‐SRB1/PSA1 and p MET3‐PKC1, have been integrated at the chromosomal loci of the respective genes in order to overcome problems of plasmid instability. Although repression of PDE2 did not cause cell 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 p MET3‐SRB1/PSA1 and p MET3‐PKC1 cassettes in place of SRB1/PSA1 and PKC1, was constructed and found to permit the efficient release of both homologous and heterologous proteins. © 1999 John Wiley & Sons, Inc., Biotechnol Bioeng 64: 607–615, 1999.