State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
Appl Environ Microbiol. 2011 Jun;77(11):3600-8. doi: 10.1128/AEM.02843-10. Epub 2011 Apr 15.
A library of engineered promoters of various strengths is a useful genetic tool that enables the fine-tuning and precise control of gene expression across a continuum of broad expression levels. The methylotrophic yeast Pichia pastoris is a well-established expression host with a large academic and industrial user base. To facilitate manipulation of gene expression spanning a wide dynamic range in P. pastoris, we created a functional promoter library through mutagenesis of the constitutive GAP promoter. Using yeast-enhanced green fluorescent protein (yEGFP) as the reporter, 33 mutants were chosen to form the functional promoter library. The 33 mutants spanned an activity range between ∼0.6% and 19.6-fold of the wild-type promoter activity with an almost linear fluorescence intensity distribution. After an extensive characterization of the library, the broader applicability of the results obtained with the yEGFP reporter was confirmed using two additional reporters (β-galactosidase and methionine adenosyltransferase [MAT]) at the transcription and enzyme activity levels. Furthermore, the utility of the promoter library was tested by investigating the influence of heterologous MAT gene expression levels on cell growth and S-adenosylmethionine (SAM) production. The extensive characterization of the promoter strength enabled identification of the optimal MAT activity (around 1.05 U/mg of protein) to obtain maximal volumetric SAM production. The promoter library permits precise control of gene expression and quantitative assessment that correlates gene expression level with physiologic parameters. Thus, it is a useful toolbox for both basic and applied research in P. pastoris.
一个具有不同强度的工程启动子库是一种有用的遗传工具，它可以在广泛的表达水平范围内对基因表达进行微调，并进行精确控制。甲醇营养酵母巴斯德毕赤酵母是一种经过充分验证的表达宿主，拥有庞大的学术和工业用户群体。为了方便在巴斯德毕赤酵母中实现跨越广泛动态范围的基因表达调控，我们通过对组成型 GAP 启动子进行诱变，创建了一个功能性启动子库。使用酵母增强型绿色荧光蛋白（yEGFP）作为报告基因，选择了 33 个突变体来形成功能性启动子库。这 33 个突变体的活性范围约为野生型启动子活性的 0.6%至 19.6 倍，荧光强度分布几乎呈线性。对文库进行了广泛的表征后，使用另外两个报告基因（β-半乳糖苷酶和甲硫氨酸腺苷转移酶 [MAT]）在转录和酶活性水平上进一步证实了用 yEGFP 报告基因获得的结果具有更广泛的适用性。此外，通过研究异源 MAT 基因表达水平对细胞生长和 S-腺苷甲硫氨酸（SAM）生产的影响，测试了启动子文库的实用性。对启动子强度的广泛表征使我们能够确定最佳的 MAT 活性（约 1.05 U/mg 蛋白），以获得最大的体积 SAM 产量。该启动子文库允许对基因表达进行精确控制和定量评估，将基因表达水平与生理参数相关联。因此，它是巴斯德毕赤酵母基础研究和应用研究的有用工具包。
