T7 RNA聚合酶
枯草芽孢杆菌
生物
清脆的
质粒
基因簇
基因
抄写(语言学)
发起人
合成生物学
RNA聚合酶
基因表达调控
基因表达
计算生物学
大肠杆菌
生物化学
遗传学
细菌
噬菌体
语言学
哲学
作者
Yaokang Wu,Yang Li,Shouxin Zhang,Yanfeng Liu,Jianghua Li,Guocheng Du,Xueqin Lv,Long Liu
标识
DOI:10.1021/acssynbio.3c00331
摘要
Bacillus subtilis is a generally recognized as safe microorganism that is widely used for protein expression and chemical production, but has a limited number of genetic regulatory components compared with the Gram-negative model microorganism Escherichia coli. In this study, a two-module plug-and-play T7-based optimized output strategy for transcription (T7-BOOST) systems with low leakage expression and a wide dynamic range was constructed based on the inducible promoters Phy-spank and PxylA. The first T7 RNA polymerase-driven module was seamlessly integrated into the genome based on the CRISPR/Cpf1 system, while the second expression control module was introduced into low, medium, and high copy plasmids for characterization. As a proof of concept, the T7-BOOST systems were successfully employed for whole-cell catalysis production of γ-aminobutyric acid (109.8 g/L with a 98.0% conversion rate), expression of human αS1 casein and human lactoferrin, and regulation of exogenous lycopene biosynthetic gene cluster and endogenous riboflavin biosynthetic gene cluster. Overall, the T7-BOOST system serves as a stringent, controllable, and effective tool for regulating gene expression in B. subtilis.
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