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

Abstract

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. Bicistronmodified 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 builtin 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 balanced, and the yield of a target metabolite can be further improved.

Increased Production of Riboflavin by Coordinated Expression of Multiple Genes in Operons in Bacillus subtilis

“…However, the excessive increase of gene expression leads to the problems of slow cell growth and low utilization of intermediate metabolites. In recent years, synthetic biology methods have been used to fine-tune gene expression to balance intracellular metabolic flow and thus maximize the yield of target products . The engineered strain with a high riboflavin titer was successfully constructed by the TIGR approach, which confirmed the application potential of synthetic biology in the riboflavin industry.…”

Synthetic Biology Toolkits and Metabolic Engineering Applied in Corynebacterium glutamicum for Biomanufacturing

“…In addition, the RBS engineering was widely used to regulate the gene translation and related enzyme activity, to increase the yield of the target product in C. glutamicum . Bicistron-modified RBSs were developed in C. glutamicum to minimize the context dependency of RBSs, which has been applied to regulate the arginine synthesis pathway . Additionally, the sequence between RBS and the start codon of downstream genes also affected the expression of target genes significantly …”

“…28 Bicistronmodified RBSs were developed in C. glutamicum to minimize the context dependency of RBSs, which has been applied to regulate the arginine synthesis pathway. 29 Additionally, the sequence between RBS and the start codon of downstream genes also affected the expression of target genes significantly. 30 Except these traditional methods, the CRISPR interference (CRISPRi) technology has been widely used to regulate the expression of target genes.…”

Enhancing heterologous expression of a key enzyme for the biosynthesis of 2′‐fucosyllactose

“…25 Despite the excellent expression enabled by the integration of various modules, it remains challenging to design a one-for-all method that can accommodate the effective heterologous expression of proteins with different properties. 26,27 In the case of expression of ⊍-1,2-FUTs, it is still of great importance to specifically assemble gene expression regulation elements with high cooperative and minimum cell harmful actions to finely ameliorate its expression.…”

“…For example, studies have used codon optimization, culture conditions improvement, molecular chaperone and fusion tag to express the VenB and VenC, which were commonly regarded as hard‐characterized P450 enzymes 25 . Despite the excellent expression enabled by the integration of various modules, it remains challenging to design a one‐for‐all method that can accommodate the effective heterologous expression of proteins with different properties 26,27 . In the case of expression of α‐1,2‐FUTs, it is still of great importance to specifically assemble gene expression regulation elements with high cooperative and minimum cell harmful actions to finely ameliorate its expression.…”