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定制化的 UPRE2 变体用于酵母中的动态基因调控。

Tailored UPRE2 variants for dynamic gene regulation in yeast.

机构信息

School of Food Science and Engineering, South China University of Technology, Guangzhou 510641, China.

出版信息

Proc Natl Acad Sci U S A. 2024 May 7;121(19):e2315729121. doi: 10.1073/pnas.2315729121. Epub 2024 Apr 30.

Abstract

Genetic elements are foundational in synthetic biology serving as vital building blocks. They enable programming host cells for efficient production of valuable chemicals and recombinant proteins. The unfolded protein response (UPR) is a stress pathway in which the transcription factor Hac1 interacts with the upstream unfolded protein response element (UPRE) of the promoter to restore endoplasmic reticulum (ER) homeostasis. Here, we created a UPRE2 mutant (UPRE2m) library. Several rounds of screening identified many elements with enhanced responsiveness and a wider dynamic range. The most active element m84 displayed a response activity 3.72 times higher than the native UPRE2. These potent elements are versatile and compatible with various promoters. Overexpression of enhanced stress signal transduction, expanding the signal output range of UPRE2m. Through molecular modeling and site-directed mutagenesis, we pinpointed the DNA-binding residue Lys60 in Hac1(Hac1-K60). We also confirmed that UPRE2m exhibited a higher binding affinity to Hac1. This shed light on the mechanism underlying the Hac1-UPRE2m interaction. Importantly, applying UPRE2m for target gene regulation effectively increased both recombinant protein production and natural product synthesis. These genetic elements provide valuable tools for dynamically regulating gene expression in yeast cell factories.

摘要

遗传元件是合成生物学的基础,它们是至关重要的构建模块。它们使宿主细胞能够有效地生产有价值的化学物质和重组蛋白。未折叠蛋白反应 (UPR) 是一种应激途径,其中转录因子 Hac1 与启动子的上游未折叠蛋白反应元件 (UPRE) 相互作用,以恢复内质网 (ER) 的稳态。在这里,我们创建了一个 UPRE2 突变体 (UPRE2m) 文库。经过几轮筛选,确定了许多具有增强响应性和更宽动态范围的元件。最活跃的元件 m84 比天然 UPRE2 的响应活性高 3.72 倍。这些有效的元件用途广泛,与各种启动子兼容。增强的应激信号转导的过表达,扩大了 UPRE2m 的信号输出范围。通过分子建模和定点突变,我们确定了 Hac1(Hac1-K60)中的 DNA 结合残基 Lys60。我们还证实 UPRE2m 与 Hac1 具有更高的结合亲和力。这揭示了 Hac1-UPRE2m 相互作用的机制。重要的是,应用 UPRE2m 对靶基因进行调控,有效地提高了重组蛋白的生产和天然产物的合成。这些遗传元件为在酵母细胞工厂中动态调节基因表达提供了有价值的工具。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f4e8/11087760/d5bb3dd983f1/pnas.2315729121fig01.jpg

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