Expanding the σ54-dependent transcription process with orthogonal designs

Area of Science:

• Synthetic Biology
• Molecular Biology
• Microbiology

Background:

• Orthogonal gene expression is crucial for predictable synthetic biology.
• Bacterial sigma factors (σ factors) regulate gene transcription, with σ54 having a distinct recognition pattern.
• σ54-dependent promoters require bacterial enhancer-binding proteins (bEBPs) for activation.

Purpose of the Study:

• To engineer orthogonal transcription systems based on the σ54 factor for synthetic biology applications.
• To identify novel σ54 variants with distinct promoter specificities and high mutual orthogonality.
• To demonstrate the transferability and applicability of these systems in diverse bacterial hosts.

Main Methods:

• Knowledge-based screening and rewiring of the RpoN box in σ54.
• Identification and characterization of σ54 variants (R456H, R456Y, R456L) and their cognate promoters.
• Testing the orthogonality and transferability of the engineered systems in multiple bacterial species.

Main Results:

• Three sets of orthogonal σ54-based expression systems were identified (σ54-R456H, R456Y, R456L).
• These systems exhibit distinct promoter preferences and high mutual orthogonality with each other and native σ54.
• The orthogonality was successfully demonstrated in three non-model bacteria, showing transferability.

Conclusions:

• Engineered σ54 factors provide a robust platform for orthogonal gene expression in bacteria.
• These systems enable the orthogonal control of downstream gene expression in response to signals.
• The developed orthogonal transcription systems expand the toolkit for synthetic biology, enabling reliable and diversified gene expression.