Functional Hybrid Natural Product Synthases by Tracking Acyl Carrier Protein Binding and Conformational Dynamics

PROJECT SUMMARY
Microorganisms produce molecules of vast structural and functional diversity. In particular, the polyketide class
of natural products holds a profound track record for being repurposed as medicinally relevant molecules.
Polyketides are natively produced by multi-enzyme assemblies called synthases. Within each polyketide
synthase (PKS) an acyl carrier protein (ACP) plays the central role of transferring and presenting molecular
building blocks and intermediates to its team of enzymatic partners. The strategic redesign of PKSs presents
an exciting and sustainable route to access new antibiotics and anticancer agents; However, the success of
any redesign approach hinges on a thorough understanding of how ACPs interact with different substrates and
enzymes during the biosynthetic process. In particular, how ACPs select their molecular building blocks is a
foundational question that if answered could enable the strategic engineering of PKSs to incorporate desired
structures at targeted locations on the polyketide product. The goal of this study is to uncover the molecular
ground rules for why some ACPs strictly accept a single substrate through an acyl transferase (AT) facilitated
exchange whereas others can bypass the gate-keeping AT and ‘self-acylate’ with a broader range of
substrates. This will be accomplished by 1) connecting ACP self-acylation ability to ACP sequence and
secondary structure, 2) characterizing ACP conformational dynamics and substrate scope, and 3) engineering
ACPs to display modified acylation properties.
Innovative methods, such as site-specific vibrational spectroscopy, will be used to connect fast ACP
conformational dynamics to acylation properties, providing unprecedented temporal insights. The approach to
PKS engineering is novel in that critical ACP-protein interactions, which when disrupted often lead to system
failure, will be maintained. Thus, these studies directly address the limited substrate scope of native polyketide
biosynthetic pathways as an alternative route to unlocking access to novel polyketides. Over 30 undergraduate
students will engage in this work at the chemistry-biology interface through independent research projects and
course-based undergraduate research experiences, thereby expanding the impact of the proposed research
into training the next generation of critical thinkers and innovators.

Grant Number: 2R15GM120704-03
NIH Institute/Center: NIH
Principal Investigator: Louise Charkoudian