Orthogonal Ribosome–Based Growth-Decoupled Strategy for Enhanced Microbial Protein Production

Abstract

Efficiently balancing cellular resources between growth and recombinant protein production remains challenging. Strong T7 promoter systems boost expression but deplete ATP, amino acids, and ribosomes, causing growth slowdown, lower protein quality, and metabolic stress. We employed an orthogonal ribosome (O-ribosome) system to separate ribosomes for growth from those for target protein synthesis. Engineered 16S rRNA anti-Shine–Dalgarno sequences enable O-ribosomes to translate only cognate mRNAs, reducing cellular burden and enhancing recombinant protein yield. To overcome their lower translation efficiency, we strengthened ribosomebinding sites, introduced 16S rRNA mutations to improve assembly, and used small RNAs to partially repress host 16S rRNA, thereby expanding the O-ribosome pool.This growth-decoupled strategy increased protein yield, alleviated metabolic stress, and offers a versatile platform for scalable microbial protein production.