Department of Biotechnology, University of Natural Resources and Life Sciences Vienna (BOKU), Muthgasse 18, 1190, Vienna, Austria.
Microb Cell Fact. 2013 Jan 24;12:6. doi: 10.1186/1475-2859-12-6.
Repressible promoters are a useful tool for down-regulating the expression of genes, especially those that affect cell viability, in order to study cell physiology. They are also popular in biotechnological processes, like heterologous protein production.
Here we present five novel repressible Pichia pastoris promoters of different strength: PSER1, PMET3, PTHR1, PPIS1 and PTHI11. eGFP was expressed under the control of each of these promoters and its fluorescence could be successfully decreased in liquid culture by adding different supplements. We also expressed the essential genes with different native promoter strength, ERO1 and PDI1, under the control of two of the novel promoters. In our experiments, a clear down-regulation of both repressible promoters on transcriptional level could be achieved. Compared to the transcript levels of these two genes when expressed under the control of their native promoters, only ERO1 was significantly down-regulated.
Our results show that all of the novel promoters can be used for repression of genes in liquid culture. We also came to the conclusion that the choice of the repressible promoter is of particular importance. For a successful repression experiment it is crucial that the native promoter of a gene and the repressible promoter in its non-repressed state are of similar strength.
抑制性启动子是下调基因表达的有用工具，特别是那些影响细胞活力的基因，以便研究细胞生理学。它们在生物技术过程中也很受欢迎，如异源蛋白生产。
在这里，我们提出了五个新的、不同强度的毕赤酵母可抑制启动子：PSER1、PMET3、PTHR1、PPIS1 和 PTHI11。在这些启动子的控制下表达了 eGFP，并且可以通过添加不同的补充剂在液体培养中成功降低其荧光。我们还在两种新型启动子的控制下表达了具有不同天然启动子强度的必需基因ERO1 和 PDI1。在我们的实验中，在转录水平上可以清楚地抑制两种可抑制启动子。与这两个基因在其天然启动子控制下表达的转录水平相比，只有 ERO1 被显著下调。
我们的结果表明，所有新型启动子都可用于液体培养中基因的抑制。我们还得出结论，选择可抑制的启动子尤为重要。对于成功的抑制实验，关键是基因的天然启动子和其非抑制状态下的可抑制启动子具有相似的强度。
