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
P
hy‑spank
and P
xylA
. 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.