Type III Polyketide Synthases: Discovery, Characterization, and Engineering
Pitel, Sheryl Beth Rubin
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Permalink
https://hdl.handle.net/2142/72138
Description
Title
Type III Polyketide Synthases: Discovery, Characterization, and Engineering
Author(s)
Pitel, Sheryl Beth Rubin
Issue Date
2009
Doctoral Committee Chair(s)
Zhao, Huimin
Department of Study
Chemical and Biomolecular Engineering
Discipline
Chemical Engineering
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Engineering, Chemical
Abstract
The polyketides are a diverse group of natural products with important applications in medicine and industry. Industry, especially the pharmaceutical industry, is under pressure to deliver "greener" chemical syntheses that are less environmentally damaging and incorporate renewable resources. There exists potential to replace current chemical syntheses of natural product or natural product-inspired pharmaceuticals with biological syntheses using enzymes. Polyketide biosynthesis is a particularly attractive target, as polyketide-derived products alone comprise 20% of the top-selling pharmaceuticals.
This thesis describes our efforts to enable biosynthesis of polyketides via the discovery, characterization, and engineering of Type III PKS enzymes. Type III PKSs produce a wide array of aromatic structures in spite of their structural simplicity. Their products are often bioactive, and several compounds from plant Type III PKSs are under study for their health benefits. Herein we mined scientific literature and genomic data to discover these enzymes, and applied heterologous protein expression, protein crystallization, and protein and metabolic engineering strategies to understand and improve their properties.
The enzyme PhlD was identified from scientific literature as a Type III PKS. We hypothesized that PhlD may be a phloroglucinol synthase, and observed phloroglucinol production when PhlD was expressed heterologously in E. coli. Protein and metabolic engineering were applied to improve phloroglucinol production, and achieved approximately four-fold increase in productivity to date. The enzyme ORAS from Neurospora crassa was initially identified as a putative Type III PKS in the sequenced genome of its host. We characterized the activity of ORAS and the crystal structure was solved and analyzed in collaboration with the laboratory of Professor Satish Nair at the University of Illinois, Urbana. Finally, we developed a screening system for the discovery of new Type III PKSs from medicinal plants. We created and validated a PCR-based screen for new Type III PKS enzymes from plants and also present preliminary data from screening the plants Eucalyptus camaldulensis and Eucalyptus robusta using this system.
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