Nieto A, Prieto J A, Sanz P
Departamento de Biotecnología, Instituto Agroquímica y Tecnología de los Alimentos, Consejo Superior de Investigaciones Científicas, P.O. Box 73, 46100 Burjassot, Valencia, Spain.
Biotechnol Prog. 1999 May-Jun;15(3):459-66. doi: 10.1021/bp9900256.
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.
米曲霉α-淀粉酶cDNA置于酿酒酵母肌动蛋白启动子（pACT1）的控制下，并导入工业面包酵母菌株的核糖体DNA位点。为了获得符合商业用途的菌株，我们构建了一个不含细菌DNA序列但含有α-淀粉酶cDNA和核糖体DNA序列的整合盒，以便将整合靶向该位点。获得了高拷贝数整合体，包括整合盒中缺陷的TRP1d启动子。我们选择了一个转化体Rib-AMY（CECT10872），即使在非选择性培养基中生长200代后，其多整合序列仍保持稳定。该转化体还表达并分泌高水平的α-淀粉酶。用该菌株制作的面包比用对照菌株制作的面包体积更大、密度更低、面包心更软。Rib-AMY转化体在延缓面包变硬方面也很有用。这种新菌株满足了商业利用的所有要求，应该会减少或消除在面粉中添加外源α-淀粉酶的需求，减少因这种酶引起的与过敏相关的工作症状。
