Engineering Promoter Architecture in Oleaginous Yeast Yarrowia lipolytica
Engineering Promoter Architecture in Oleaginous Yeast Yarrowia lipolytica
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DOI：
10.1021/acssynbio.5b00100
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发表时间：
2016-03-01
影响因子：
4.7
通讯作者：
Blenner, Mark A.
中科院分区：
文献类型：
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作者：
Hussain, Murtaza Shabbir;Gambill, Lauren;Blenner, Mark A.
真核启动子具有复杂的结构，以控制基因转录的强度和时间，从起始位点跨越多达数千个碱基。这种复杂性使得真菌中启动子的合理微调难以预测;然而，这种非常相同的复杂性使得工程化启动子强度的多种可能策略成为可能。在这里，我们研究了启动子架构的产油酵母，解脂耶氏酵母。虽然最近的研究主要集中在上游激活序列上，但我们系统地检查了真菌启动子中常见的各种成分。在这里，我们研究了几个启动子组件，包括上游激活序列，近端启动子序列，核心启动子，和TATA盒在自主复制的表达质粒和整合到基因组中。我们的研究结果表明，启动子的强度可以微调，通过工程的TATA盒序列，核心启动子，和上游激活序列。此外，我们确定了一个以前未报道的油酸响应转录增强的XPR 2上游激活序列，这说明了真菌启动子的复杂性。本文设计的启动子为Y. lipolytica的启动子，并提供可用于工程化其它非模型真菌系统的启动子工程化策略。
Eukaryotic promoters have a complex architecture to control both the strength and timing of gene transcription spanning up to thousands of bases from the initiation site. This complexity makes rational fine-tuning of promoters in fungi difficult to predict; however, this very same complexity enables multiple possible strategies for engineering promoter strength. Here, we studied promoter architecture in the oleaginous yeast, Yarrowia lipolytica. While recent studies have focused on upstream activating sequences, we systematically examined various components common in fungal promoters. Here, we examine several promoter components including upstream activating sequences, proximal promoter sequences, core promoters, and the TATA box in autonomously replicating expression plasmids and integrated into the genome. Our findings show that promoter strength can be fine-tuned through the engineering of the TATA box sequence, core promoter, and upstream activating sequences. Additionally, we identified a previously unreported oleic acid responsive transcription enhancement in the XPR2 upstream activating sequences, which illustrates the complexity of fungal promoters. The promoters engineered here provide new genetic tools for metabolic engineering in Y. lipolytica and provide promoter engineering strategies that may be useful in engineering other non-model fungal systems.