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Updated: Jun 19, 2026

Standardized Modular Assembly of Polycistronic Operons with Modular Cloning (MoClo) using the In-Cloning toolkit
Published on: September 2, 2025
5'-End Translationalization: Iterative Assembly of Leaderless Polycistronic Amplifiers for Context-Independent
Manman Sun1,2,3, Bin Ye1,3, Yimeng Zhao1,3
1Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, China.
Abstract:
Corynebacterium glutamicum is a crucial food-grade (GRAS) bacterial chassis widely utilized for the industrial production of amino acids and nutraceuticals. However, the efficient production of recombinant proteins and secondary metabolites in this host remains limited by context dependence and low translational efficiency. To overcome this, we introduce a 5'-end translationalization strategy. By repurposing passive 5' untranslated regions (5'UTRs) into actively translated fore-cistrons, we converted conventional monocistronic designs into context-independent, leaderless polycistronic designs (PCDs). This assembly of concatenated fore-cistrons functions as a translational amplifier, largely decoupling protein output from mRNA abundance. We validated this platform by optimizing two biomanufacturing paradigms: achieving a 4.07-fold enhanced secretion of OmlA, a porcine vaccine antigen, and boosting biosynthesis of the food-grade pigment indigoidine to 1.20 g/L (a 7.33-fold increase over baselines). Together, this framework establishes a versatile, portable toolkit to overcome translational bottlenecks, enabling robust hyperproduction of recombinant proteins and engineered metabolites in biotechnology.
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