Rational Design of pPsiTacI Promoter Variants Exhibiting Differential PsiR Binding Strengths

Authors:
  • DPDingmao Pan
    Dingmao Pan
  • SZSicheng Zhao
    Sicheng Zhao
  • HDHui Ding
    Hui Ding
  • XZXiaofei Zhao
    Xiaofei Zhao
  • LWLeilei Wang
    Leilei Wang
  • HWHongguang Wang
    Hongguang Wang
  • DTDan Tan
    Dan Tan
  • XLXiaoyun Lu
    Xiaoyun Lu
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Abstract
Native transcription factor (TF)-promoter modules generally function as fixed regulatory units, limiting the quantitative programming of transcriptional outputs required for synthetic biology applications. Here, we present a structure-guided promoter engineering strategy that converts the D-allulose-responsive PsiR-pPsiTacI module into a quantitatively tunable promoter library without modifying the transcription factor itself. Candidate promoter variants were rationally designed through AlphaFold3-based protein–DNA docking and molecular dynamics simulations to modulate PsiR–DNA binding affinity. Experimental characterization using both Pepper RNA and sfGFP reporters demonstrated that the engineered promoters generated graded transcriptional outputs while preserving the native regulatory mechanism. Quantitative dose-response modeling further revealed distinct promoter response functions, spanning different maximal outputs, induction thresholds, dynamic ranges, and activation kinetics. These experimentally calibrated response profiles were consistent with computational predictions of protein-DNA binding energetics, establishing an interpretable sequence-to-structure-to-function relationship. Collectively, this work transforms a native TF-promoter module into a reusable library of programmable regulatory elements with quantitatively defined response functions. The proposed structure-guided strategy provides a general framework for engineering inducible promoters with predictable regulatory behaviors and expands the toolkit for dynamic gene expression control, pathway optimization, and synthetic regulatory circuit design.

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