Centro de Biotecnologia Molecular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF 70910-900, Brazil.
J Ind Microbiol Biotechnol. 2012 Nov;39(11):1673-83. doi: 10.1007/s10295-012-1170-5. Epub 2012 Aug 15.
Used for millennia to produce beverages and food, Saccharomyces cerevisiae also became a workhorse in the production of biofuels, most notably bioethanol. Yeast strains have acquired distinct characteristics that are the result of evolutionary adaptation to the stresses of industrial ethanol production. JP1 is a dominant industrial S. cerevisiae strain isolated from a sugarcane mill and is becoming increasingly popular for bioethanol production in Brazil. In this work, we carried out the genetic characterization of this strain and developed a set of tools to permit its genetic manipulation. Using flow cytometry, mating type, and sporulation analysis, we verified that JP1 is diploid and homothallic. Vectors with dominant selective markers for G418, hygromycin B, zeocin, and ρ-fluoro-DL-phenylalanine were used to successfully transform JP1 cells. Also, an auxotrophic ura3 mutant strain of JP1 was created by gene disruption using integration cassettes with dominant markers flanked by loxP sites. Marker excision was accomplished by the Cre/loxP system. The resulting auxotrophic strain was successfully transformed with an episomal vector that allowed green fluorescent protein expression.
千百年来，酿酒酵母不仅用于生产饮料和食品，还成为生物燃料生产的主要工具，尤其是生物乙醇。酵母菌株具有独特的特性，这是它们对工业乙醇生产压力进行进化适应的结果。JP1 是一种从甘蔗厂分离出来的优势工业酿酒酵母菌株，在巴西的生物乙醇生产中越来越受欢迎。在这项工作中，我们对该菌株进行了遗传特征分析，并开发了一套工具来允许对其进行遗传操作。我们使用流式细胞术、交配型和孢子形成分析验证了 JP1 是二倍体和同型接合的。带有 G418、潮霉素 B、zeocin 和 ρ-氟-DL-苯丙氨酸显性选择性标记的载体被成功用于转化 JP1 细胞。此外，还通过带有loxP 位点侧翼的显性标记整合盒对 JP1 的营养缺陷型 ura3 突变菌株进行基因破坏，创建了一个营养缺陷型菌株。通过 Cre/loxP 系统实现了标记的切除。然后，用一个能够表达绿色荧光蛋白的附加型载体成功转化了这个营养缺陷型菌株。
