Leaderless Bicistronic Design for Precise and Reliable Control of Gene Expression in Corynebacterium Glutamicum

In synthetic biology, the precise control of gene expression
is
challenging due to the limited orthogonality of expression elements.
Here, to address this issue and improve the reusability of genetic
elements, we developed a bicistronic expression cassette in Corynebacterium glutamicum based on a leaderless
promoter lacking a 5′UTR. The created leaderless bicistronic
design (BCD) significantly improved the orthogonality of expression
elements across different genes of interest. We also explored the
importance of the fore-cistron and SD motif in maintaining the strength
of leaderless BCDs. Additionally, we established a library containing
55,901 fore-cistrons and demonstrated that the regulatory range of
gene expression in leaderless BCDs can be broader by modifying the
fore-cistron sequence. This study provides a novel synthetic biology
tool based on leaderless BCD for fine-tuning gene expression in C. glutamicum using fore-cistrons. Moreover, the
strategy developed here can also be applied to improve the performance
of other leaderless promoters in other bacteria.