Engineered suppressor tRNAs enable precise translational control of genetic circuits in E. coli

Precise and orthogonal regulation of genetic circuits is a central challenge in synthetic biology, particularly at the translational level where tools remain scarce. Here, we address this by engineering suppressor transfer RNAs (sup-tRNAs) charged with canonical amino acids to enable programmable nonsense mutation readthrough in Escherichia coli. Screening of 20 variants revealed a clear sup-tRNA design rule: readthrough efficiency is dictated by the similarity of the native tRNA anticodon to amber codon (CUA), as it preserves native aaRS interactions. We then demonstrate the power of this tool for advanced genetic circuit engineering. First, in a LacI-based biosensor, sup-tRNA regulation reduced background leakage by >77% and increased the induction dynamic range by 4.3-fold (from 6.67 to 28.68). Second, by dynamically balancing glycolytic flux through targeted pykA and pykF regulation, we increased the titer of N-acetylneuraminic acid by 66% (from 5.33 to 8.82 g/l) without compromising cell growth. Our work establishes engineered cAA-charged sup-tRNAs as a versatile, efficient, and cost-effective platform for precision translational control within genetic circuits, opening new avenues for biosensor optimization and metabolic engineering.