Untitled

Abstract

The gene encoding Schwanniomyces occidentalis alpha-amylase (AMY) was introduced into the chromosomal delta sequences of an industrial strain of Saccharomyces cerevisiae. To obtain a strain suitable for commercial use, an delta-integrative cassette devoid of bacterial DNA sequences was constructed that contains the AMY gene and aureobasidin A resistance gene (AUR1-C) as the selection marker. The AMY gene was expressed under the control of the alcohol dehydrogenase gene promoter (ADC1p). The alpha-amylase activity of Sacc. cerevisiae transformed with this integrative cassette was 6 times higher than that of Sch. occidentalis. The transformants (integrants) were mitotically stable after 100 generations in nonselective medium.

Natural Saccharomyces cerevisiae Strain Reveals Peculiar Genomic Traits for Starch-to-Bioethanol Production: the Design of an Amylolytic Consolidated Bioprocessing Yeast

“…Despite the large employment of S. cerevisiae in biotechnological research, only CBP yeasts with limited amylolytic activity are currently employed on an industrial scale. The genome engineering for amylase expression in industrial S. cerevisiae strains has already been reported by integration of heterologous genes at delta sequences of the Ty retrotransposon (Cho et al, 1999;Kang et al, 2003;Favaro et al, 2010Favaro et al, , 2015Cripwell et al, 2019a) or ribosomal DNA (Lopes et al, 1989(Lopes et al, , 1996Nieto et al, 1999;Choi et al, 2002;Liao et al, 2012). Although these strategies are known as very efficient in S. cerevisiae because of the native homologous recombination machinery, the inserts often result in long tandem repeats at one location leading to genome instability and unstable phenotypes.…”