2 NOVEL GENE-EXPRESSION SYSTEMS BASED ON THE YEASTS SCHWANNIOMYCES-OCCIDENTALIS AND PICHIA-STIPITIS

Two non-Saccharomyces yeasts have been developed as hosts for heterolo gous gene expression. The celD gene from Clostridium thermocellum, enc oding a heat-stable cellulase, served as the test sequence. The first system is based on the amylolytic species Schwanniomyces occidentalis, the second on the xylolytic species Pichia stipitis. The systems comp rise auxotrophic host strains (trp5 in the case of S. occidentalis; tr p5-10, his3 in the case of P. stipitis) and suitable transformation ve ctors. Vector components consist of an S. occidentalis-derived autonom ously replicating sequence (SwARS) and the Saccharomyces cerevisiae-de rived TRP5 sequence for plasmid propagation and selection in the yeast hosts, an ori and an ampicillin-resistance sequence for propagation a nd selection in a bacterial host. A range of vectors has been engineer ed employing different promoter elements for heterologous gene express ion control in both species. Homologous elements derived from highly e xpressed genes of the respective hosts appeared to be of superior qual ity: in the case of S, occidentalis that of the GAM1 gene, in the case of P. stipitis that of the XYL1 gene. Further elements tested are the S. cerevisiae-derived ADH1 and PDC1 promoter sequences.