Understanding thioamitide biosynthesis using pathway engineering and untargeted metabolomics

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Thioamitides are a complex family of ribosomally synthesized and post-translationally modified peptides (RiPPs) with promising anti-tumor properties.
• Despite identified biosynthetic gene clusters (BGCs), thioamitide biosynthesis remains poorly understood, hindering their potential applications.

Purpose of the Study:

• To elucidate the biosynthetic pathway of thiostreptamide S4 by engineering its BGC in a heterologous host.
• To identify key enzymes and intermediates involved in thioamitide biosynthesis.
• To leverage biosynthetic insights for discovering and engineering new RiPPs.

Main Methods:

• Engineering of the thiostreptamide S4 BGC in a heterologous expression system.
• Untargeted metabolomics to analyze pathway intermediates.
• Targeted mutations of the precursor peptide and identified enzymes.
• Bioinformatic analysis of RiPP-like BGCs.

Main Results:

• Detailed elucidation of the thiostreptamide S4 biosynthetic pathway.
• Identification of a novel protein essential for amino acid dehydration, showing homology to plant pathogen effector HopA1.
• Demonstration of heterologous expression enabling biosynthetic studies.

Conclusions:

• Heterologous expression and metabolomics are powerful tools for understanding complex RiPP biosynthesis.
• The identified dehydration enzyme expands the known repertoire of RiPP-modifying enzymes.
• This work provides a foundation for the discovery and engineering of novel thioamitides and other RiPPs.