A gene-encoded aldehyde tag repurposed from RiPP cyclophane-forming pathway

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Gene-encoded aldehyde tag technology is crucial for bioorthogonal chemistry and biotechnology.
• Existing aldehyde tags, like (C/S)x(P/A)xR, have limitations or lack orthogonality.
• The rSAM cyclophane synthase SjiB is known for its role in triceptide biosynthesis.

Purpose of the Study:

• To utilize the promiscuous SjiB enzyme as a dedicated and efficient formylglycine synthase.
• To introduce a novel, biosynthetically orthogonal aldehyde tag sequence (YQSSI).
• To validate the SjiB/YQSSI system for protein labeling applications.

Main Methods:

• Engineering the rSAM enzyme SjiB for formylglycine synthesis.
• Designing and synthesizing the novel YQSSI aldehyde tag sequence.
• Validating the system through fluorescent labeling of model proteins.

Main Results:

• SjiB was successfully repurposed as a highly efficient formylglycine synthase.
• The YQSSI aldehyde tag sequence demonstrated biosynthetic orthogonality to known tags.
• The SjiB/YQSSI system proved effective for fluorescent protein labeling.

Conclusions:

• The SjiB/YQSSI system represents a novel and efficient tool for bioorthogonal protein labeling.
• This new aldehyde tag expands the toolkit for chemical biology and biotechnological applications.
• The system's orthogonality offers advantages for multiplexed labeling strategies.