Janse B J, Pretorius I S
Department of Microbiology, University of Stellenbosch, South Africa.
Curr Genet. 1993 Jul-Aug;24(1-2):32-7. doi: 10.1007/BF00324662.
A 3800-base pair (bp) DNA fragment encoding the mature pullulanase from Klebsiella pneumoniae was inserted between two different yeast expression-secretion cassettes and an yeast gene terminator. These cassettes were cloned into an yeast centromeric plasmid YCplacIII and transformed into laboratory strains of Saccharomyces cerevisiae. Transcription initiation signals were derived from the mating pheromone alpha-factor (MF alpha 1p) and alcohol dehydrogenase (ADC1p) gene promoters. Secretion of pullulanase was directed by the leader sequence of the yeast mating pheromone alpha-factor (MF alpha 1s). Transcription termination was effected by the yeast tryptophan synthase gene terminator (TRP5T). Southernblot analysis confirmed the presence of pulA in transformed yeasts and Northern-blot analysis revealed the presence of PUL1 mRNA. A pullulan agarose assay indicated the extracellular production of biologically active pullulanase by S. cerevisiae.
将一段编码肺炎克雷伯菌成熟支链淀粉酶的3800碱基对（bp）DNA片段插入两个不同的酵母表达-分泌盒和一个酵母基因终止子之间。这些盒被克隆到酵母着丝粒质粒YCplacIII中，并转化到酿酒酵母的实验室菌株中。转录起始信号来自交配信息素α-因子（MFα1p）和乙醇脱氢酶（ADC1p）基因启动子。支链淀粉酶的分泌由酵母交配信息素α-因子（MFα1s）的前导序列引导。转录终止由酵母色氨酸合成酶基因终止子（TRP5T）完成。Southern印迹分析证实转化酵母中存在pulA，Northern印迹分析显示存在PUL1 mRNA。支链淀粉酶琼脂糖测定表明酿酒酵母可在细胞外产生具有生物活性的支链淀粉酶。
