d-Integration technique and efficient heterologous expression in yeasts tailored for bioethanol production

Bioethanol production from biomass could be economically feasible using the Consolidated Bioprocessing (CBP) approach in which a Saccharomyces cerevisiae yeast efficiently hydrolyses and ferments biomass into ethanol. S. cerevisiae lacks any hydrolytic activity necessary for direct utilization of biomass and its proper manipulation would contribute to the CBP production of ethanol. Multi-copy integrations are the most suitable methods for expressing foreign genes in S. cerevisiae. However, the number of integrated genes is probably not as important as the need for high-level expression, mainly affected by the impact of codon bias on the translation of heterologous proteins and by their improper folding. These problems may be overcome using codon-optimized genes. This work reports on the construction of new delta integrative plasmids for the overexpression of codon-optimized amylolytic genes into wild type S. cerevisiae strains with proper fermentative traits. The synthetic sgaI gene, glucoamylase from Aspergillus awamori, was successfully integrated into S. cerevisiae strains. The mitotically stable transformants, showing high expression levels, produced amylolytic activities and promising ethanol yields from both soluble and raw starch. The adopted constructing strategy, resulting in yeasts suitable for the CBP of starchy industrial residues, proved effective and is currently applied to other amylolytic and cellulolytic genes for their efficient overexpression in S. cerevisiae.