Design and construction of light-regulated gene transcription and protein translation systems in yeast P. Pastoris,Journal of Advanced Research

当前位置： X-MOL 学术 › J. Adv. Res. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

Design and construction of light-regulated gene transcription and protein translation systems in yeast P. Pastoris
Journal of Advanced Research ( IF 11.4 ) Pub Date : 2024-08-06 , DOI: 10.1016/j.jare.2024.08.008
Siyu Zhang ₁ , Jiazhen Zhang ₁ , Ru Lin ₁ , Chaoyu Lu ₁ , Bohao Fang ₁ , Jiacheng Shi ₁ , Tianyi Jiang ₂ , Mian Zhou ₁

Affiliation

Introduction

P. pastoris is a common host for effective biosynthesis of heterologous proteins as well as small molecules. Accurate regulation of gene transcription and protein synthesis is necessary to coordinate synthetic gene circuits and optimize cellular energy distribution. Traditional methanol or other inducible promoters, natural or engineered, have defects in either fermentation safety or expression capacity. The utilization of chemical inducers typically adds complexity to the product purification process, but there is no other well-controlled protein synthesis system than promoters yet.

Objective

The study aimed to address the aforementioned challenges by constructing light-regulated gene transcription and protein translation systems with excellent expression capacity and light sensitivity.

Methods

Trans-acting factors were designed by linking the N. crassa blue-light sensor WC-1 with the activation domain of endogenous transcription factors. Light inducible or repressive promoters were then constructed through chimeric design of cis-elements (light-responsive elements, LREs) and endogenous promoters. Various configurations of trans-acting factor/LRE pairs, along with different LRE positions and copy numbers were tested for optimal promoter performance. In addition to transcription, a light-repressive translation system was constructed through the “rare codon brake” design. Rare codons were deliberately utilized to serve as brakes during protein synthesis, which were switched on and off through the light-regulated changes in the expression of the corresponding pLRE-tRNA.

Results

As demonstrated with GFP, the light-inducible promoter 4pLRE-cP_AOX1 was 70 % stronger than the constitutive promoter P_GAP, with L/D ratio = 77. The light-repressive promoter P_GAP-pLRE was strictly suppressed by light, with expression capacity comparable with P_GAP in darkness. As for the light-repressive translation system, the “triple brake” design successfully eliminated leakage and achieved light repression on protein synthesis without any impact on mRNA expression.

Conclusion

The newly designed light-regulated transcription and translation systems offer innovative tools that optimize the application of P. pastoris in biotechnology and synthetic biology.

中文翻译：

酵母 P. Pastoris 中光调控基因转录和蛋白质翻译系统的设计和构建

介绍

P. pastoris 是异源蛋白和小分子有效生物合成的常见宿主。基因转录和蛋白质合成的准确调控对于协调合成基因回路和优化细胞能量分布是必要的。传统的甲醇或其他诱导型启动子，无论是天然的还是工程的，在发酵安全性或表达能力方面都存在缺陷。化学诱导剂的使用通常会增加产品纯化过程的复杂性，但除了启动子之外，还没有其他控制良好的蛋白质合成系统。

目的

该研究旨在通过构建具有优异表达能力和光敏感性的光调节基因转录和蛋白质翻译系统来解决上述挑战。

方法

通过将 N. crassa 蓝光传感器 WC-1 与内源性转录因子的激活结构域联系起来来设计反式作用因子。然后通过顺式元件（光响应元件，LRE）和内源性启动子的嵌合设计构建光诱导或抑制性启动子。测试了反式作用因子/LRE 对的各种配置，以及不同的 LRE 位置和拷贝数，以实现最佳启动子性能。除了转录之外，还通过 “rare codon brake” 设计构建了光抑制翻译系统。罕见密码子被特意用于蛋白质合成过程中的制动器，通过相应 pLRE-tRNA 表达的光调节变化来打开和关闭。

结果

正如 GFP 所证明的那样，光诱导启动子 4pLRE-cP_AOX1 比组成型启动子 P_GAP 强 70%，L/D 比 = 77。光抑制启动子 P_GAP-pLRE 被光严格抑制，在黑暗中的表达能力与 P_GAP 相当。对于光抑制翻译系统，“triple brake”设计成功消除了泄漏，实现了对蛋白质合成的轻抑制，对 mRNA 表达没有任何影响。