Promoter library designed for fine-tuned gene expression in Pichia pastoris.
Promoter library designed for fine-tuned gene expression in Pichia pastoris.
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DOI：
10.1093/nar/gkn369
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发表时间：
2008-07
影响因子：
14.9
通讯作者：
Glieder A
中科院分区：
文献类型：
--
作者：
Hartner FS;Ruth C;Langenegger D;Johnson SN;Hyka P;Lin-Cereghino GP;Lin-Cereghino J;Kovar K;Cregg JM;Glieder A
尽管经常用作蛋白质生产宿主，但只有一组有限的启动子可用于驱动巴斯德毕赤酵母中重组蛋白质的表达。通常需要对基因表达进行微调，以最大限度地提高产品产量和质量。然而，对于有效的基于知识的工程，更好地了解启动子功能是必不可少的。因此，我们通过删除和复制 AOX1 启动子 (PAOX1) 序列中推定的转录因子结合位点来创建启动子文库。第一个文库最初的活性范围为野生型启动子活性的 ∼6% 至 >160%。在对使用绿色荧光蛋白（GFP）变体的启动子库进行表征后，新的调控工具箱成功地应用于“真实案例”，即工业酶的表达。在抑制、去抑制和诱导条件下对文库进行表征，显示至少 12 个顺式作用元件参与 PAOX1 驱动的高水平表达。基于该缺失分析，通过将顺式作用元件与基础启动子组合构建了新型短人工启动子变体。除了提高异源蛋白质生产的产量和质量外，新的 PAOX1 合成启动子文库还构成了一个基本工具箱，可用于微调代谢工程中的基因表达以及合成生物学中蛋白质表达的顺序诱导。
Although frequently used as protein production host, there is only a limited set of promoters available to drive the expression of recombinant proteins in Pichia pastoris. Fine-tuning of gene expression is often needed to maximize product yield and quality. However, for efficient knowledge-based engineering, a better understanding of promoter function is indispensable. Consequently, we created a promoter library by deletion and duplication of putative transcription factor-binding sites within the AOX1 promoter (PAOX1) sequence. This first library initially spanned an activity range between ∼6% and >160% of the wild-type promoter activity. After characterization of the promoter library employing a green fluorescent protein (GFP) variant, the new regulatory toolbox was successfully utilized in a ‘real case’, i.e. the expression of industrial enzymes. Characterization of the library under repressing, derepressing and inducing conditions displayed at least 12 cis-acting elements involved in PAOX1-driven high-level expression. Based on this deletion analysis, novel short artificial promoter variants were constructed by combining cis-acting elements with basal promoter. In addition to improving yields and quality of heterologous protein production, the new PAOX1 synthetic promoter library constitutes a basic toolbox to fine-tune gene expression in metabolic engineering and sequential induction of protein expression in synthetic biology.
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影响因子：
3.5
作者：
GODECKE, S;ECKART, M;HOLLENBERG, CP
通讯作者：
HOLLENBERG, CP
影响因子：
9.9
作者：
Cox, Robert Sidney 3rd;Surette, Michael G;Elowitz, Michael B
通讯作者：
Elowitz, Michael B
影响因子：
5
作者：
Ahn, Jungoh;Hong, Jiyeon;Lee, Hongweon
通讯作者：
Lee, Hongweon
影响因子：
3.5
作者：
Griffith, DA;Delipala, C;Oesterhelt, D
通讯作者：
Oesterhelt, D
影响因子：
5.8
作者：
Cartharius, K;Frech, K;Werner, T
通讯作者：
Werner, T