δ-Integration of endo/exo-glucanase and β-glucosidase genes into the yeast chromosomes for direct conversion of cellulose to ethanol

For the direct conversion of cellulose to ethanol, recombinant yeast having a full set of genes for the cellulolytic enzymes was developed. Using the delta-sequences of the Ty1 retrotransposon as target sites for homologous recombination, heterologous genes of endo/exo-glucanase and beta-glucosidase were integrated into the chromosomes of Saccharomyces cerevisiae. The number of both integrated genes was found to be approximately 44. This newly constructed yeast, S. cerevisiae L2612 delta GC, successfully expressed and secreted the cellulolytic enzymes. Expression levels of cellulolytic enzymes, cell growth, and ethanol production in cellulose-containing media were significantly increased in comparison with plasmid-based expression. Maintenance of the integrated genes was also perfect up to 50 generations even in nonselectable media. (C) 1999 Elsevier Science Inc. All rights reserved.