Mapping the nonribosomal specificity code through promiscuity-guided A-domain engineering

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Nonribosomal peptide synthetases (NRPSs) are crucial for assembling diverse bioactive peptides.
• Predicting NRPS products is possible, but designing specific NRPSs remains challenging due to unknown multispecificity.

Purpose of the Study:

• To investigate and engineer the specificity of nonribosomal peptide synthetase adenylation domains.
• To understand the role of multispecificity in NRPS evolution and design.

Main Methods:

• Utilized a hydroxamate assay (HAMA) to assess multispecificity in mutant libraries of the SrfAC adenylation domain.
• Employed full randomization of 15 active site residues and computational modeling to analyze specificity divergence.

Main Results:

• Identified specific mutations with significant impact on NRPS specificity, demonstrating remarkable evolvability.
• Statistical analysis revealed three key sequence positions influencing specificity, offering a guide for NRPS engineering.
• Developed a multispecific SrfAC version, showcasing functional flexibility.

Conclusions:

• Promiscuity in adenylation domains drives neofunctionalization, a process that can be mimicked for laboratory design of valuable peptides.
• The findings provide a roadmap for engineering NRPS specificity and designing novel peptide therapeutics or industrial compounds.