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 ce llulolytic enzymes, with chromosome-integrated multicopies of heterolo gous DNA sequences encoding the cellulolytic enzymes were constructed. An expression construct of beta-glucosidase and carboxymethyl cellula se directed by the GAP promoter was integrated into the chromosomes of the haploid S. cerevisiae using the a sequence-mediated integration s ystem. Southern blot analysis of the chromosomes prepared from various integrants and separated by pulse-field gel electrophoresis demonstra ted that the integration occurred mainly in a particular chromosome an d the copy number of the integration was variable. The amount of enzym es secreted by the transformants correlated with the copy number of in tegration. For each enzyme, the highest activity was about 1.4-fold th at produced by the transformant harboring the same expression cassette on a YEp-type plasmid. The delta-integrated exogenous DNA was mitotic ally stable in rich medium. A haploid double transformant which coexpr esses and secretes beta-glucosidase and carboxymethyl cellulase was fu rther 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 c arboxymethyl cellulose as a sole carbon source. It is suggested that t he delta-sequence-mediated integration system is a very useful means f or the genetic engineering of yeast, especially when overproduction an d secretion of multiple heterologous enzymes are desired.