Stable High-Copy-Number Integration of Aspergillus oryzae (x-AMYLASE cDNA in an Industrial Bakers Yeast Strain

The Aspergillus oryzae a-amylase cDNA was placed under the control of the Saccharomyces cerevisiae actin promoter (pACTI) and introduced into the ribosomal DNA locus of an industrial bakerʹs yeast strain. To obtain a strain eligible for commercial use, we constructed an integrative cassette lacking bacterial DNA sequences but containing the a-amylase cDNA and ribosomal DNA sequences to target the integration to this locus. High-copy-number integrants were obtained including a defective TRPId promoter in the integrative cassette. We selected one transformant, Rib-AMY (CECT10872), in which the multi-integrated sequences were stable even after 200 generations of growth in nonselective medium. This transformant also expressed and secreted high levels of a-amylase. Bread made with this strain had a higher volume, lower density, and softer crumbs than bread made with a control strain. The Rib-AMY transformant also was useful in retarding bread firming. This new strain fulfills all the requirements for commercial utilization and should reduce or eliminate the requirement for addition of exogenous a-amylase to the flour, reducing allergenic work-related symptoms due to this enzyme.