毕赤酵母中Tet-on诱导系统的开发与优化

Development and Optimization of A Tet -on Inducible System in Komagataella phaffii

摘要:

基因表达精准调控对于高效能细胞工厂构建至关重要，基于非营养源的小分子诱导系统在基因表达调控具有广泛的应用潜力。该研究在巴斯德毕赤酵母中开发了基于强力霉素响应的Tet-on诱导系统，并进一步对该系统的各个元件进行优化。通过筛选合适的核心启动子序列、优化控制rtTA表达的启动子、转录激活结构域优化以及构建rtTA突变体，最终得到一系列的可响应强力霉素的诱导型启动子。其中基于rtTA-V10的Tet-on诱导系统拥有较低的本底水平泄露（荧光值为野生型菌株的1.05倍），而在添加强力霉素后，荧光强度的动态范围达到了52.76倍。而基于rtTA-VPR的Tet-on诱导系统在各种碳源条件下都表现出良好的表达强度，甲醇条件下达到了PAOX1的111.16%，在甘油、乙醇、葡萄糖条件下达到了PGCW14的110.65%、106.05%、112.24%。通过添加不同浓度的诱导剂即可以实现基因的精细化调控，这为毕赤酵母代谢工程和合成生物学领域中基因表达精准调控提供有力的遗传操作工具。

Abstract:

Precise regulation of gene expression is crucial for the construction of high-performance cell factories, and non-nutrient source-based small molecule inducible system has a wide range of application potential in the regulation of gene expression. In this study, a Tet-on inducible system based on doxycycline response was developed in Komagataella phaffii and every elements of the system were further optimized. A series of inducible promoters that can respond to doxycycline were eventually obtained by screening appropriate core promoter sequences, optimizing the promoters that regulate rtTA expression, optimizing the transcriptional activation domains, and creating rtTA mutants. Among them, the Tet-on inducible system based on rtTA-V10 had a low background level leakage (fluorescence value was 1.05 times that of wild-type strains), and a dynamic range of fluorescence intensity reached 52.76 times after the addition of doxycycline. The rtTA-VPR-based Tet-on inducible system showed good expression intensity under various carbon source conditions, reaching 111.16% of PAOX1 under methanol condition, and 110.65%, 106.05%, 112.24% of PGCW14 under the glycerol, ethanol and glucose condition, respectively. Precise gene regulation can be achieved by adding different concentrations of inducers, which provides a powerful genetic manipulation tool for precise gene expression regulation in the fields of Komagataella phaffii metabolic engineering and synthetic biology.