Reprogramming Nonribosomal Peptide Synthetases for Site-Specific Insertion of α-Hydroxy Acids

Area of Science:

• Biotechnology and Synthetic Biology
• Biochemistry
• Enzyme Engineering

Background:

• High-throughput engineering offers potential for sustainable production of pharmaceutical natural product analogs.
• Nonribosomal peptide synthetases (NRPS) are key enzymes in natural product biosynthesis.
• Gatekeeper adenylation domains (A-domains) control substrate specificity in NRPS.

Purpose of the Study:

• To engineer the substrate specificity of NRPS gatekeeper A-domains.
• To switch specificity from α-amino acids to α-hydroxy acids.
• To produce novel peptide and depsipeptide analogs.

Main Methods:

• Combinatorial mutagenesis and yeast cell surface display were employed.
• Engineered A-domains were selected for altered substrate specificity.
• The modified domains were integrated into a pathway context for production.

Main Results:

• Single-round engineering successfully switched A-domain specificity from α-amino acids to α-hydroxy acids.
• Engineered domains efficiently produced α-hydroxy acid-containing linear peptides and cyclic depsipeptides.
• The study provides insights into protein discrimination between amino and hydroxy groups.

Conclusions:

• Engineered NRPS A-domains can be readily repurposed for novel metabolite synthesis.
• This approach enables predictable tuning of peptide metabolite activities.
• The method facilitates sustainable production of pharmaceutically relevant compounds.