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

In synthetic biology, the precise control of gene expressionischallenging due to the limited orthogonality of expression elements.Here, to address this issue and improve the reusability of geneticelements, we developed a bicistronic expression cassette in Corynebacterium glutamicum based on a leaderlesspromoter lacking a 5 & PRIME;UTR. The created leaderless bicistronicdesign (BCD) significantly improved the orthogonality of expressionelements across different genes of interest. We also explored theimportance of the fore-cistron and SD motif in maintaining the strengthof leaderless BCDs. Additionally, we established a library containing55,901 fore-cistrons and demonstrated that the regulatory range ofgene expression in leaderless BCDs can be broader by modifying thefore-cistron sequence. This study provides a novel synthetic biologytool based on leaderless BCD for fine-tuning gene expression in C. glutamicum using fore-cistrons. Moreover, thestrategy developed here can also be applied to improve the performanceof other leaderless promoters in other bacteria.

Automated summary

In this study, a bicistronic expression cassette based on a leaderless promoter was developed in Corynebacterium glutamicum, which improved the orthogonality and regulatory range of gene expression. The importance of the fore-cistron and SD motif in maintaining the strength of leaderless bicistronic designs was also explored.