Engineered genetic devices and gene circuits can reprogram cellular behavior towards different applications in synthetic biology domains. One of the main barriers is the lack of genetic regulatory elements with ideal characteristics in different organisms.   

In contrast to the most popular gram-negative model organism Escherichia coli, only a few elements with ideal strength, stringency and wide dynamic range were exploited in the gram-positive model bacterium Bacillus subtilis (B. subtilis). Meanwhile, multiple types of genetic elements have to be employed to meet the requirements of different genes and synthetic pathways for strength or stringency, increasing the system complexity and cell burden, making these engineered strain less viable for industrial applications. 

Nine promoter mutants harboring malO operator with drastically improved activities were achieved by semi-rational mutagenesis. Based on these mutants, the inducible activated/repressive gene regulation system MATE-ON/OFF was established by modulating the sequence, position or number of the malO operator in promoter.  

Moreover, the carbon catabolite repression (CCR) effect was fully alleviated in the presence of 20 g/L glucose by engineering both the promoter element and host cell. The induction fold of MATE system was also improved to 790-fold without compromising the robustness by intercalating riboswitch elements to promoters in MATE system.  

To prove the robustness of MATE system, nine heterologous proteins from mammals, insects and bacteria were over-expressed as examples, and the average expression level of intracellular or secreted proteins could achieve 60% or 80% of host cellular proteins respectively, which is comparable with the widely used pET/T7 expression system in Escherichia coli. The homogeneity and reproducibility of MATE system was also well-proved by flow cytometry analysis. The cell morphology gene divIVA and violacein biosynthetic pathway which is toxic for the host cell were chosen as examples to prove the stringency in applications of gene functions and pathway regulations.  

A new strategy was then provided in bacteria to exploit operator as a promoter enhancer to inducible modify the activities of bacterial native promoters with negligible influence on their architecture and minimal burden on host cell.  

Furthermore, riboflavin and violacein biosynthetic pathways were regulated by MATE-ON/OFF dual controlling device simultaneously to optimize the titer of chemical products. As a result, the potential of utilizing MATE system as gene switch, promoter enhancer or metabolic valve in synthetic biology applications was well-verified. 

In summary, a universal gene expression system that combine both robust and stringency was established in B. subtilis, which has a great potential in industrial protein production and synthetic biology applications. This work will offer a new path ahead in the development of more flexible and orthogonal operator-based genetics devices and components, as well as address various biological challenges and boost the applications of B. subtilis.