biostudies-literatureFu GTianjin Synthetic Biotechnology Innovation Capacity Improvement ProjectNational Key R&amp;amp;D Program of Chinae2119980119https://www.ebi.ac.uk/biostudies/studies/S-EPMC8931375biostudies-literatureUnknown119(11)SignificanceA gene regulatory system is an important tool for the engineering of biosynthetic pathways of organisms. Here, we report the development of an inducible-ON/OFF regulatory system using a <i>malO</i> operator as a key element. We identified and modulated sequence, position, numbers, and spacing distance of <i>malO</i> operators, generating a series of activating or repressive promoters with tunable strength. The stringency and robustness are both guaranteed in this system, a maximal induction factor of 790-fold was achieved, and nine proteins from different organisms were expressed with high yields. This system can be utilized as a gene switch, promoter enhancer, or metabolic valve in synthetic biology applications. This operator-based engineering strategy can be employed for developing similar regulatory systems in different microorganisms.Proceedings of the National Academy of Sciences of the United States of AmericaAn operator-based expression toolkit for <i>Bacillus subtilis</i> enables fine-tuning of gene expression and biosynthetic pathway regulation.PMC8931375TSBICIP-KJGG-011-022020YFA0907800Li YZhang DLee SYLi DFu GYue JfalseAn operator-based expression toolkit for <i>Bacillus subtilis</i> enables fine-tuning of gene expression and biosynthetic pathway regulation.SignificanceA gene regulatory system is an important tool for the engineering of biosynthetic pathways of organisms. Here, we report the development of an inducible-ON/OFF regulatory system using a <i>malO</i> operator as a key element. We identified and modulated sequence, position, numbers, and spacing distance of <i>malO</i> operators, generating a series of activating or repressive promoters with tunable strength. The stringency and robustness are both guaranteed in this system, a maximal induction factor of 790-fold was achieved, and nine proteins from different organisms were expressed with high yields. This system can be utilized as a gene switch, promoter enhancer, or metabolic valve in synthetic biology applications. This operator-based engineering strategy can be employed for developing similar regulatory systems in different microorganisms.2022-01-01T00:00:00Z2022 Mar2025-04-04T20:23:41.467Z2025-04-04T20:23:41.467ZS-EPMC89313753526322410.1073/pnas.2119980119