Active-site residue, domain and module swaps in modular polyketide synthases

Area of Science:

• Biochemistry
• Synthetic Biology
• Molecular Biology

Background:

• Modular polyketide synthases (PKSs) are large enzyme complexes responsible for producing a diverse array of natural products.
• Acyltransferase (AT) domains within PKSs dictate the selection of extender units during polyketide chain assembly.
• Understanding AT domain specificity is crucial for engineering PKSs to produce novel compounds.

Purpose of the Study:

• To investigate the role of a specific sequence motif within AT domains in determining extender unit selection.
• To engineer the bimodular PKS DEBS1-TE to alter its substrate specificity.
• To develop a versatile cassette system for constructing hybrid modular PKSs.

Main Methods:

• Sequence analysis of multiple acyltransferase (AT) domains from various PKSs.
• Site-directed mutagenesis of the identified motif in the DEBS1-TE PKS from Saccharopolyspora erythraea.
• Construction and characterization of hybrid PKSs using a novel cassette system based on the tylosin PKS from Streptomyces fradiae.

Main Results:

• A conserved short sequence motif in AT domains was correlated with the type of extender unit incorporated.
• Alteration of this motif in DEBS1-TE resulted in the production of triketide lactones with acetate units instead of the expected propionate units.
• A functional cassette system was established, enabling domain and module swaps within modular PKSs.

Conclusions:

• The identified sequence motif is a key determinant of extender unit selection in AT domains.
• Engineering this motif provides a strategy for redirecting PKS product formation.
• The developed cassette system offers a powerful tool for the combinatorial biosynthesis of complex polyketides.