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Polyketide Synthase Engineering via Catalytic Domain Exchanges

  • Author(s): Eng, Clara Hoifung
  • Advisor(s): Keasling, Jay D
  • et al.
Abstract

Polyketides comprise a structurally diverse class of natural products that includes many highly effective pharmaceuticals. As such, the development of techniques to produce polyketide analogs with potentially improved pharmacokinetic or toxicological properties is a research area of great interest. The modular nature of PKSs has also motivated researchers to explore methods of employing engineered PKSs for the biological synthesis of industrially relevant small molecules, in addition to natural product drugs.

We utilize in vitro enzyme studies to elucidate the biosynthetic potential of polyketide synthases (PKSs), the large, multisubunit enzymes responsible for polyketide production, and further leverage the insights gained from benchtop experiments to inform alterations to culture conditions that enable the production of non-natural natural product analogs.

In addition, we report progress developing methods to harness our understanding of the modular structure of PKSs to reliably exchange ketoreductase (KR) domains between PKS systems. The importance of stereochemistry in determining biological activity makes an ability to understand and engineer KR domains, which dictate the stereochemistry at many of the stereocenters in polyketide products, essential to the objective of effectively exploiting PKSs for combinatorial biosynthesis. Our work to identify heuristics that facilitate reliable KR domain exchanges furnished a novel library of PKS variants harboring different KR domains, further enabling studies that provide insight into how KR domains function in the context of a PKS module.

Finally, we build on the knowledge gleaned from KR domain exchange experiments to develop more general strategies for PKS engineering. A limited theoretical understanding of the determinants of PKS fold and function poses a substantial barrier to the design of active variants, and identifying strategies to reliably construct functional PKS chimeras remains an active area of research. We develop and formalize a paradigm for the design of PKS chimeras, and implement a computational platform to streamline and simplify the process of designing experiments to test strategies for PKS engineering. Finally, we discuss the implications of this system for enabling the effective use of PKSs in synthetic biology applications.

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