Fine-Tuning Multi-Gene Clusters via Well-Characterized Gene Expression Regulatory Elements: Case Study of the Arginine Synthesis Pathway in C. glutamicum

作者：Yanting Duan, Weiji Zhai, Weijia Liu, Xiaomei Zhang, Xiaomei Zhang, Jin‐Song Shi, Xiaojuan Zhang, Xiaojuan Zhang, Zhenghong Xu · 发表于：ACS Synthetic Biology · 年份：2020 · DOI：10.1021/acssynbio.0c00405 · 被引用次数：38 · 研究领域：Microbial Metabolic Engineering and Bioproduction、Viral Infectious Diseases and Gene Expression in Insects、RNA and protein synthesis mechanisms

Promoters and ribosome binding sites (RBSs) are routinely applied in gene expression regulation, but their orthogonality and combinatorial effects have not yet been systematically studied in Corynebacterium glutamicum. Here, 17 core promoters and 29 RBSs in C. glutamicum were characterized, which exhibited 470-fold and 430-fold in transcriptional and translational activity, respectively. By comparing the expression of two reporter genes regulated by multiple RBSs, the RBS efficacy showed significant dependence on the gene context, besides the RBSs’ strength, reflecting the poor orthogonality of RBSs. Bicistron-modified RBS (referred as bc-RBS) was adapted to C. glutamicum, which improved RBS reliability. By coupling a series of promoters with RBSs/bc-RBSs, a much broader regulation range that spanned 4 orders of magnitude was observed compared with that of a sole element, and the contribution to gene expression of RBS was more than that of promoter. Finally, promoters and RBSs were applied as built-in elements to fine-tune the gene cluster in the arginine synthesis pathway in C. glutamicum . Compared with the original strain, more arginine (1.61-fold) or citrulline (2.35-fold) was accumulated in a 7 L bioreactor by strains with the gene expression regulation system rationally engineered. We demonstrated that, via combination of well-characterized gene elements, and overall consideration for both transcription and translation, the biosynthesis pathway can be effectively balanc...