The Tryptophan Operator Acts as a Portable Downstream Enhancer for Recombinant Protein Expression in Escherichia coli

Downstream enhancer-like regulatory elements remain largely unexplored in prokaryotes. The tryptophan operator sequence (trpO), an 18-bp near-palindromic element from the Escherichia coli trpEDCBA operon, is conventionally defined as the binding site for the TrpR repressor, which blocks transcription initiation. Here, we report a previously unrecognized function of trpO: it can serve as a portable, orientation-dependent downstream enhancer that increases recombinant protein expression in E. coli. Inserting trpO downstream of multiple constitutive promoters increased eGFP reporter expression, with the reverse orientation (retrpO) conferring the most prominent enhancement, yielding a 1.56-fold expression increase. Adding L-threonine further raised expression to 3.98-fold. After screening plasmid replicons and host strains, the combination of the CloDF13 replicon and W3110 host, together with L-threonine supplementation, achieved a cumulative 9.57-fold enhancement in protein expression. Together, these findings challenge the long-standing notion that trpO functions exclusively as a negative regulatory element and establish trpO as a portable tool for increasing protein yields in E. coli.