Creating New Access Points for Strategic Synthase Engineering: Ethane Thioester Analogs as Truncated Coenzyme A Building Blocks Unlock Expanded Acyl Carrier Protein Loading Capabilities

Document Type

Journal Article

Role

Author

Published In

Biochemistry

Haverford Libraries Support

APC Waiver - American Chemical Society

Publication Date

8-15-2026

Abstract

Strategic engineering of natural product biosynthetic pathways through the incorporation of alternative, tunable carbon-based building blocks represents a promising approach for accessing medicinally relevant molecules. However, efforts toward this goal have been hindered by the substrate specificity of component enzymes. In type I and type II fatty acid synthases (FASs) and polyketide synthases (PKSs), the acyltransferase (AT) selects a specific malonyl-based coenzyme A (CoA) building block and transfers it onto the acyl carrier protein (ACP) for subsequent processing. Inspired by the observation that some ACPs can bypass the AT and “self-acylate”, we herein explored the tolerance of FAS and PKS ACPs to load both a variety of CoA substrates and ethane thioester (ET) analogs serving as truncated CoA building blocks. We observe that the Escherichia coli (E. coli) AT, FabD, can load and transfer methylmalonyl-CoA (mm-CoA) and malonyl-CoA (m-CoA) onto three ACPs: the type II Streptomyces coelicolor actinorhodin PKS ACP (ActACP), the E. coli type II FAS ACP (AcpP), and the type I Saccharopolyspora erythraea 6-deoxyerythronolide B PKS ACP6 (DEBS ACP6). Synthesized ET analogs of mm-CoA and m-CoA were loaded onto all three ACPs through FabD-assisted acylation. Additionally, both in the presence and absence of FabD, ACPs could be acylated with ET analogs of fluoromalonyl-, succinyl-, and glutaryl- building blocks. Overall, this work pushes the limits of ACP substrate loading, revealing new complexity in carbon-based building block selection and establishing foundations for novel routes toward diverse functional group incorporation in FAS/PKS biosynthetic pathways.

Keywords

Bacteria, Biosynthesis, Acyls, Post-translational modification, Organic reactions

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