Tailored UPRE2 variants for dynamic gene regulation in yeast

IF 9.1 1区综合性期刊 Q1 MULTIDISCIPLINARY SCIENCES

Proceedings of the National Academy of Sciences of the United States of America Pub Date : 2024-04-30 DOI:10.1073/pnas.2315729121

Chufan Xiao, Xiufang Liu, Yuyang Pan, Yanling Li, Ling Qin, Zhibo Yan, Yunzi Feng, Mouming Zhao, Mingtao Huang

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引用次数: 0

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 HAC1 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.

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为酵母动态基因调控量身定制的 UPRE2 变体

遗传元素是合成生物学的基础，是重要的组成部分。它们可以对宿主细胞进行编程，从而高效生产有价值的化学品和重组蛋白质。未折叠蛋白反应（UPR）是一种应激途径，其中转录因子 Hac1 与启动子上游的未折叠蛋白反应元件（UPRE）相互作用，以恢复内质网（ER）的平衡。在这里，我们创建了一个 UPRE2 突变体（UPRE2m）文库。经过几轮筛选，我们发现了许多具有更强响应性和更宽动态范围的元件。最活跃的元件 m84 的反应活性是原生 UPRE2 的 3.72 倍。这些强效元件用途广泛，可与各种启动子兼容。HAC1的过表达增强了应激信号转导，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的结合亲和力更高。这揭示了Hac1-UPRE2m相互作用的机制。重要的是，应用 UPRE2m 对目标基因进行调控能有效提高重组蛋白产量和天然产物合成量。这些遗传元件为动态调控酵母细胞工厂的基因表达提供了宝贵的工具。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Proceedings of the National Academy of Sciences of the United States of America 综合性期刊-综合性期刊

CiteScore

19.00

自引率

0.90%

发文量

3575

审稿时长

2.5 months

期刊介绍： The Proceedings of the National Academy of Sciences (PNAS), a peer-reviewed journal of the National Academy of Sciences (NAS), serves as an authoritative source for high-impact, original research across the biological, physical, and social sciences. With a global scope, the journal welcomes submissions from researchers worldwide, making it an inclusive platform for advancing scientific knowledge.