Reassessment of IRES Element Activity in Komagataella phaffii Using a Standardized Bicistronic System: Cryptic Promoter Activity Mimics Cap-Independent Translation

Internal ribosome entry site (IRES) elements have been proposed for constructing polycistronic mRNAs in Komagataella phaffii, but their reported activity is inconsistent and has not been evaluated under standardized conditions. Here, a standardized bicistronic EGFP-IRES-mKate2 system was developed in which the cap-dependent first cistron (EGFP) reports promoter activity, and translation of the second (mKate2) should depend on the IRES. The RhPV, TEV, PVY, and Saccharomyces cerevisiae GPR1 elements were assessed under PAOX1 and PTHI11, with EMCV and HCV as negative controls. EGFP tracked promoter induction and repression, whereas mKate2 remained at background for GPR1, EMCV, and HCV; for RhPV, TEV, and PVY it was low, promoter-independent, and persisted after promoter deletion. By RT-qPCR, the transcript was intact across both cistrons, except in the RhPV construct, where its 3′ region was depleted. The residual synthesis therefore reflected cryptic promoter activity below 1% of that of PAOX1, not IRES-dependent initiation. None of the elements directed detectable cap-independent translation above 0.4% of cap-dependent expression of the same ORF, limiting their use for polycistronic expression. The system, the first to place IRES activity in K. phaffii on a quantitative scale, distinguishes IRES-dependent translation from cryptic transcription and can facilitate the search for functional elements.