Evolution-guided engineering of trans-acyltransferase polyketide synthases.

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Bacterial multimodular polyketide synthases (PKSs) are large enzymes producing valuable natural products.
• Engineering PKSs allows for the diversification of polyketide structures, but challenges remain, especially with trans-acyltransferase (trans-AT) PKSs.

Purpose of the Study:

• To identify a reliable method for engineering trans-AT PKSs.
• To create diverse, functional engineered trans-AT PKSs for generating novel polyketides.

Main Methods:

• Analysis of amino acid coevolution in trans-AT PKSs to find a conserved module site.
• Insertion and deletion of PKS parts at the identified site to create engineered enzymes.
• Testing the functionality of 22 engineered trans-AT PKSs in different bacterial producers.

Main Results:

• A common module site was identified through amino acid coevolution analysis.
• Successful engineering of 22 trans-AT PKSs by inserting and deleting diverse PKS parts.
• Demonstrated broad applicability of the engineering approach across different pathways and bacterial hosts.

Conclusions:

• The identified module site provides a versatile platform for engineering trans-AT PKSs.
• This method facilitates the generation of complex, designer polyketides with high success rates.
• The approach has broad implications for natural product drug discovery and synthetic biology.