sb3c00331_si_001.pdf (1.31 MB)
Efficient Protein Expression and Biosynthetic Gene Cluster Regulation in Bacillus subtilis Driven by a T7-BOOST System
journal contributionposted on 2023-10-27, 05:29 authored by Yaokang Wu, Yang Li, Yuting Zhang, Yanfeng Liu, Jianghua Li, Guocheng Du, Xueqin Lv, Long Liu
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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