Synthetic translational coupling system for accurate and predictable polycistronic gene expression control in bacteria.

Precise and predictable genetic elements are required to address various issues, such as suboptimal metabolic flux or imbalanced protein assembly caused by the inadequate control of polycistronic gene expression in bacteria. Here, we devised a synthetic biopart based on the translational coupling to control polycistronic gene expression. This module links the translation of genes within a polycistronic mRNA, maintaining their expression ratios regardless of coding sequences, transcription rate, and upstream gene translation rate. By engineering the Shine-Dalgarno sequences within these synthetic bioparts, we adjusted the expression ratios of polycistronic genes. We created 41 bioparts with varied relative expression ratios, ranging from 0.03 to 0.92, enabling precise control of pathway enzyme gene expression in a polycistronic manner. This led to up to a 7.6-fold increase in the production of valuable biochemicals such as 3-hydroxypropionic acid, poly(3-hydroxybutyrate), and lycopene. Our work provides genetic regulatory modules for precise and predictable polycistronic gene expression, facilitating efficient protein assembly, biosynthetic gene cluster expression, and pathway optimization.