Ruth C, Zuellig T, Mellitzer A, Weis R, Looser V, Kovar K, Glieder A
Syst Synth Biol. 2010 Sep;4(3):181-91. doi: 10.1007/s11693-010-9057-0. Epub 2010 May 29.
Natural tools for recombinant protein production show technological limitations. Available natural promoters for gene expression in Pichia pastoris are either constitutive, weak or require the use of undesirable substances or procedures for induction. Here we show the application of deletion variants based on the well known methanol inducible AOX1 promoter and small synthetic promoters, where cis-acting elements were fused to core promoter fragments. They enable differently regulated target protein expression and at the same time to replace methanol induction by a glucose or glycerol feeding strategy. Trypsinogen, the precursor of the serine protease trypsin, was expressed using these different promoters. Depending on the applied promoter the production window (i.e. the time of increasing product concentration) changed significantly. In fedbatch processes trypsinogen yields before induction with methanol were up to 10 times higher if variants of the AOX1 promoter were applied. In addition, the starting point of autoproteolytic product degradation can be predetermined by the promoter choice.
用于重组蛋白生产的天然工具存在技术局限性。毕赤酵母中现有的用于基因表达的天然启动子要么是组成型的、较弱的，要么需要使用不良物质或程序进行诱导。在此，我们展示了基于著名的甲醇诱导型AOX1启动子和小型合成启动子的缺失变体的应用，其中顺式作用元件与核心启动子片段融合。它们能够实现对靶蛋白表达的不同调控，同时通过葡萄糖或甘油补料策略取代甲醇诱导。使用这些不同的启动子表达了丝氨酸蛋白酶胰蛋白酶的前体胰蛋白酶原。根据所应用的启动子，生产窗口（即产物浓度增加的时间）发生了显著变化。在补料分批培养过程中，如果应用AOX1启动子的变体，甲醇诱导前的胰蛋白酶原产量可提高至10倍。此外，自催化产物降解的起始点可通过启动子的选择预先确定。
