利用即插即用载体和液相色谱-质谱联用筛选技术促进米曲霉天然产物发现的策略。
Strategy of employing plug-and-play vectors and LC-MS screening to facilitate the discovery of natural products using Aspergillus oryzae.
作者信息
Shi Hanliang, Lin Beibei, Zheng Mengmeng, Gan Fengyu, Lin Zhi, Xin Xiujuan, Zhao Jian, Qu Xudong, An Faliang
机构信息
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Mei Long Road, Shanghai, 200237, China.
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
出版信息
Bioresour Bioprocess. 2025 Jan 8;12(1):2. doi: 10.1186/s40643-024-00833-w.
Aspergillus oryzae is a widely used host for heterologous expression of fungal natural products. However, the vectors previously developed are not convenient for use and screening positive transformants by PCR and fermentation is time- and effort-consuming. Hence, three plug-and-play vectors were developed here for multi-gene expression and liquid chromatography mass spectrometry detection was introduced to screen positive transformants. Using rug BGC for verification, we demonstrated that the vectors we developed perform well and liquid chromatography mass spectrometry detection is feasible to screen positive transformants. For deleterious gene expression, PxyrA rather than PamyB was employed. Utilizing the toolkit described here to express natural products, dozen days can be saved.
米曲霉是一种广泛用于真菌天然产物异源表达的宿主。然而，先前开发的载体使用起来不方便，通过聚合酶链式反应（PCR）筛选阳性转化体且发酵既耗时又费力。因此，本文开发了三种即插即用型载体用于多基因表达，并引入液相色谱 - 质谱检测来筛选阳性转化体。使用rug生物合成基因簇（BGC）进行验证，我们证明了所开发的载体性能良好，并且液相色谱 - 质谱检测对于筛选阳性转化体是可行的。对于有害基因的表达，使用的是木聚糖酶A启动子（PxyrA）而非淀粉酶B启动子（PamyB）。利用本文所述的工具包来表达天然产物，可以节省十天时间。
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