Expanding Extender Substrate Selection for Unnatural Polyketide Biosynthesis by Acyltransferase Domain Exchange...

Area of Science:

• Biochemistry
• Synthetic Biology
• Enzymology

Background:

• Modular polyketide synthases (PKSs) are crucial for synthesizing diverse polyketide natural products.
• PKS modules typically incorporate malonyl-CoA or methylmalonyl-CoA, but natural diversity arises from various malonyl-CoA analogues.
• Acyltransferase (AT) domains dictate the extender substrate specificity within PKS modules.

Purpose of the Study:

• To engineer PKS modules for altered extender substrate specificity by exchanging AT domains.
• To explore the biosynthesis of novel polyketides using engineered PKSs and diverse extender substrates.
• To develop a computational method for predicting AT substrate range.

Main Methods:

• Exchanged acyltransferase (AT) domains in modular polyketide synthases (PKSs) to alter extender substrate specificity.
• Performed in vitro polyketide biosynthesis reactions using wild-type and engineered PKSs with 14 different extender substrates.
• Developed a computational workflow to predict AT substrate range based on active site volumes.

Main Results:

• Generated 13 structurally distinct polyketides, including several novel compounds, from approximately 200 in vitro reactions.
• Engineered PKSs produced target polyketides over 100-fold higher in some cases compared to wild-type PKS.
• Unusual AT domains preferentially incorporated rare extender substrates similar in size or slightly larger than natural substrates, rather than malonyl-CoA or methylmalonyl-CoA.

Conclusions:

• Acyltransferase (AT) domain exchange is a viable strategy for engineering PKSs to produce novel polyketides in vitro.
• Understanding the substrate specificity of rare AT domains expands the toolbox for polyketide biosynthesis.
• The developed computational workflow aids in selecting appropriate AT domains for PKS engineering and novel chemical synthesis.