Starter unit flexibility for engineered product synthesis by the nonreducing polyketide synthase PksA

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Nonreducing polyketide synthases (NR-PKSs) are large enzymes crucial for polyketide biosynthesis.
• Their unique starter unit:acyl-carrier protein (ACP) transacylase (SAT) domain selects the initial acyl group.
• PksA, an NR-PKS, synthesizes aflatoxin B1 using a hexanoyl starter from a yeast-like fatty acid synthase (FAS).

Purpose of the Study:

• To investigate the substrate specificity of the PksA NR-PKS.
• To explore the potential of PksA to initiate programmed polyketide synthesis with diverse starter units.
• To define the predictable synthesis of unnatural polyketide products by NR-PKSs.

Main Methods:

• Enzymatic assays using PksA with various acyl-CoA starter units.
• Analysis of polyketide products using mass spectrometry and NMR.
• Site-directed mutagenesis of PksA active site cysteine residues.

Main Results:

• PksA successfully accepted and processed a range of starter units, producing predicted naphthopyrone products.
• Analysis revealed domain-specific tolerance trends for alternative starter units.
• Natural and unnatural variants of the active site cysteine supported biosynthesis, suggesting alternative substrate loading mechanisms.

Conclusions:

• NR-PKSs exhibit significant substrate plasticity, allowing for the synthesis of diverse polyketide structures.
• The PksA enzyme's domain tolerance provides a framework for engineering novel polyketide products.
• Understanding these mechanisms facilitates the predictable biosynthesis of unnatural compounds using NR-PKSs.