Biosynthetic Incorporation of Non-native Aryl Acid Building Blocks into Peptide Products Using Engineered Adenylation...

Area of Science:

• Biochemistry and Molecular Biology
• Metabolic Engineering
• Natural Product Synthesis

Background:

• Nonribosomal peptides (NRPs) are diverse secondary metabolites with therapeutic potential, biosynthesized by nonribosomal peptide synthetases (NRPSs).
• Aryl acids are key building blocks contributing to the structural variety of NRPs.
• Previous studies engineered the EntE adenylation domain to accept monosubstituted benzoic acids.

Purpose of the Study:

• To investigate the substrate scope of a previously engineered EntE mutant (N235G) with various disubstituted benzoic acid derivatives.
• To elucidate the structural basis for substrate recognition by the mutant EntE enzyme.
• To demonstrate the utility of engineered NRPS enzymes in producing novel aryl acid-containing peptides.

Main Methods:

• Site-directed mutagenesis of the EntE adenylation domain (Asn-to-Gly substitution at position 235).
• Enzymatic assays using various substituted benzoic acid derivatives and nonhydrolyzable aryl-AMP analogues.
• Structural analysis of mutant EntE (N235G) complexed with substrate analogues.
• Coupling of engineered EntE mutants with enterobactin and vibriobactin biosynthetic enzymes.

Main Results:

• The mutant EntE (N235G) successfully accommodated diverse disubstituted benzoic acids (halogen, methyl, methoxy, nitro, cyano) and a 3-alkyne substituted benzoic acid.
• Structural studies revealed that the enlarged substrate-binding pocket of mutant EntE accommodates bulky and functionalized aryl acids.
• Engineered NRPS pathways produced peptides containing 3-hydroxybenzoic acid, salicylic acid, and 3-bromo-2-fluorobenzoic acid.

Conclusions:

• The engineered EntE (N235G) mutant exhibits an expanded substrate specificity for aryl acid activation.
• Enlargement of the substrate-binding pocket is crucial for accommodating diverse aryl acid structures.
• NRPS pathway engineering offers a powerful strategy for generating novel aryl acid-containing metabolites with potential therapeutic applications.