INCREASED ACTIVITY OF A MODEL HETEROLOGOUS PROTEIN IN SACCHAROMYCES-CEREVISIAE STRAINS WITH REDUCED VACUOLAR PROTEINASES

Strains of Saccharomyces cerevisiae with reduced activity of the four major vacuolar proteinases were constructed and used as an expression system for a model heterologous gene product (beta-galactosidase from Escherichia coli). The vacuolar proteinases were inactivated by mutati on within the structural genes encoding proteinase A (PRA1), proteinas e B (PRB1), carboxypeptidase Y (PRC1) and carboxypeptidase S (CPS1). S trains were constructed with mutations in one or more of these structu ral genes. Having constructed the strains, the E. coli beta-galactosid ase (lacZ) gene was introduced by transformation. Batch cultures of ea ch strain were grown and the activity of beta-galactosidase measured. An assessment of the effect of the loss of specific proteinases on the heterologous gene product was then made. The results indicated that s trains with reduced vacuolar proteinase activity showed as much as 173 % higher beta-galactosidase activity than a strain with wild-type prot einase activity carrying the lacZ gene. The most productive strains of all were those with reduced carboxypeptidase activity and/or reduced proteinase A activity. At first sight the inclusion of a pra1 mutation and/or the prc1 and cps1 mutations would appear worthwhile for signif icantly enhanced expression of a heterologous gene product in yeast. H owever this conclusion is too simplistic: each heterologous protein wi ll require a host specifically ''tailored'' to ensure optimum expressi on.