Abstract：
BACKGROUND: 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-cPAOX1 was 70 % stronger than the constitutive promoter PGAP, with L/D ratio = 77. The light-repressive promoter PGAP-pLRE was strictly suppressed by light, with expression capacity comparable with PGAP 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.
CONCLUSIONS: The newly designed light-regulated transcription and translation systems offer innovative tools that optimize the application of P. pastoris in biotechnology and synthetic biology.
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-cPAOX1 was 70 % stronger than the constitutive promoter PGAP, with L/D ratio = 77. The light-repressive promoter PGAP-pLRE was strictly suppressed by light, with expression capacity comparable with PGAP 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.
CONCLUSIONS: The newly designed light-regulated transcription and translation systems offer innovative tools that optimize the application of P. pastoris in biotechnology and synthetic biology.
摘要：
背景：P.pastoris是有效生物合成异源蛋白和小分子的常见宿主。基因转录和蛋白质合成的精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，自然或工程，在发酵安全性或表达能力方面存在缺陷。使用化学诱导剂通常会增加产品纯化过程的复杂性，但是除了启动子之外，还没有其他控制良好的蛋白质合成系统。
目的：本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应对上述挑战。
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域连接来设计反式作用因子。然后通过嵌合设计顺式元件（光响应元件，LRE)和内源启动子。反式因子/LRE对的各种配置，以及不同的LRE位置和拷贝数进行了最佳启动子性能测试。除了转录,通过“稀有密码子制动”设计构建了一个光抑制翻译系统。稀有密码子被故意用来作为蛋白质合成过程中的刹车，通过相应的pLRE-tRNA表达的光调节变化来打开和关闭。
结果：如GFP所示，光诱导启动子4pLRE-cPAOX1比组成型启动子PGAP强70%，L/D比=77。光抑制启动子PGAP-pLRE被光严格抑制，在黑暗中的表达能力与PGAP相当。至于光压抑的翻译系统，“三重制动”设计成功地消除了泄漏，并实现了对蛋白质合成的轻度抑制，而对mRNA表达没有任何影响。
结论：新设计的光调节转录和翻译系统提供了创新工具，可优化巴斯德毕赤酵母在生物技术和合成生物学中的应用。
目的：本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应对上述挑战。
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域连接来设计反式作用因子。然后通过嵌合设计顺式元件（光响应元件，LRE)和内源启动子。反式因子/LRE对的各种配置，以及不同的LRE位置和拷贝数进行了最佳启动子性能测试。除了转录,通过“稀有密码子制动”设计构建了一个光抑制翻译系统。稀有密码子被故意用来作为蛋白质合成过程中的刹车，通过相应的pLRE-tRNA表达的光调节变化来打开和关闭。
结果：如GFP所示，光诱导启动子4pLRE-cPAOX1比组成型启动子PGAP强70%，L/D比=77。光抑制启动子PGAP-pLRE被光严格抑制，在黑暗中的表达能力与PGAP相当。至于光压抑的翻译系统，“三重制动”设计成功地消除了泄漏，并实现了对蛋白质合成的轻度抑制，而对mRNA表达没有任何影响。
结论：新设计的光调节转录和翻译系统提供了创新工具，可优化巴斯德毕赤酵母在生物技术和合成生物学中的应用。
