Stable High-Copy-Number Integration of Aspergillus oryzae α-AMYLASE cDNA in an Industrial Baker's Yeast Strain

Abstract

The Aspergillus oryzae alpha-amylase cDNA was placed under the control of the Saccharomyces cerevisiae actin promoter (pACT1) and introduced into the ribosomal DNA locus of an industrial baker's yeast strain. To obtain a strain eligible for commercial use, we constructed an integrative cassette lacking bacterial DNA sequences but containing the alpha-amylase cDNA and ribosomal DNA sequences to target the integration to this locus. High-copy-number integrants were obtained including a defective TRP1d 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 alpha-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 alpha-amylase to the flour, reducing allergenic work-related symptoms due to this enzyme.

Autocloning and Amplification of LIP2 in Yarrowia lipolytica

“…These vectors are the first of their kind for Y. lipolytica and are the third example of this type. Such integrative cassettes devoid of bacterial DNA which could be amplified have also been used for the expression of Aspergillus oryzae ␣-amylase in an industrial baker's yeast strain (29) and for the expression of monellin in Candida utilis (17).…”

Auxotrophic Yeast Strains in Fundamental and Applied Research

“…An additional advantage of these "defective" markers is that they may avoid protein-burden effects (39) due to massive overexpression of the marker gene. Both LEU2d and TRP1d markers have been used successfully to obtain high copy numbers of plasmid-borne as well as integrated expression cassettes (29,30).…”

Construction of Amylolytic Industrial Strains of Saccharomyces cerevisiae for Improved Ethanol Production from Raw Starch

“…Residual starch was determined to be 5% (w/v) after 3 days of fermentation and 3.6% (w/v) after 6 days of fermentation, respectively. This result indicates that the increase in the activity of glucoamylase via rDNA integration of the GA1 genes results in the improvements of raw starch degradation to glucose and of ethanol fermentation (Nieto et al, 1999). Yamada et al (2010) has reported that a tetraploid strain of S. cerevisiae that secretes Streptococcus bovis α -amylase to hydrolyze raw starch and Rhizopus oryzae glucoamylase produced 70 g ethanol/L after 3 days of fermentation.…”

“…These values were 1.2-times and 1.9-times higher than the respective activities of the parental strain (Kim et al, 2011). The additional multicopy rDNA-integration of the GA1 or AMY genes can be correlated with the high-level expression of the corresponding genes (Nieto et al, 1999;Choi et al, 2002). The increase in activities of glucoamylase in addition to α-amylase is necessary for the more complete degradation of raw starch into glucose units, and consequently, more efficient ethanol production (Shigechi et al, 2004;Sun et al, 2010).…”

“…The operating conditions were 65℃, water mobile phase, flow rate of 0.6 ml/min, and ethanol concentrations were monitored using a refractive index detector (Waters). Conversion used: 1% (v/v) ethanol is equivalent to (Nieto et al, 1999). The integrative cassette (YIpBA2rD)-harboring yeast was named ATCC 9763/YIpδAGSAδ/YIpBA2rD.…”