OVEREXPRESSION AND SECRETION OF CELLULOLYTIC ENZYMES BY DELTA-SEQUENCE-MEDIATED MULTICOPY INTEGRATION OF HETEROLOGOUS DNA-SEQUENCES INTO THE CHROMOSOMES OF SACCHAROMYCES-CEREVISIAE

Saccharomyces cerevisiae transformants which secrete high levels of cellulolytic enzymes, with chromosome-integrated multicopies of heterologous DNA sequences encoding the cellulolytic enzymes were constructed. An expression construct of beta-glucosidase and carboxymethyl cellulase directed by the GAP promoter was integrated into the chromosomes of the haploid S. cerevisiae using the a sequence-mediated integration system. Southern blot analysis of the chromosomes prepared from various integrants and separated by pulse-field gel electrophoresis demonstrated that the integration occurred mainly in a particular chromosome and the copy number of the integration was variable. The amount of enzymes secreted by the transformants correlated with the copy number of integration. For each enzyme, the highest activity was about 1.4-fold that produced by the transformant harboring the same expression cassette on a YEp-type plasmid. The delta-integrated exogenous DNA was mitotically stable in rich medium. A haploid double transformant which coexpresses and secretes beta-glucosidase and carboxymethyl cellulase was further constructed by genetic crossing of the haploid transformant that produces a high level of the enzyme, followed by meiotic segregation of the resulting diploid strain. The haploid double transformant, but neither of the single transformant, could grow on a plate containing carboxymethyl cellulose as a sole carbon source. It is suggested that the delta-sequence-mediated integration system is a very useful means for the genetic engineering of yeast, especially when overproduction and secretion of multiple heterologous enzymes are desired.