Reengineering and Discovery of Nonribosomal Peptide Synthetases

Evans, Bradley S.

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Description

Title

Reengineering and Discovery of Nonribosomal Peptide Synthetases

Author(s)

Evans, Bradley S.

Issue Date

2011-01-21T22:46:57Z

Director of Research (if dissertation) or Advisor (if thesis)

Kelleher, Neil L.

Doctoral Committee Chair(s)

Kelleher, Neil L.

Committee Member(s)

• van der Donk, Wilfred A.
• Gerlt, John A.
• Zhao, Huimin

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• nonribosomal peptide synthetases
• directed evolution
• high-throughput screening
• antibiotics
• Fourier-transform mass spectrometry
• proteomics
• natural products

Abstract

Nonribosomal peptides are an important class of natural products including the life-saving antibiotics penicillin and vancomycin and the deadly microcystins and cereulide. This important category of natural products is thus a focus of many research labs around the world, including the Kelleher lab. These often complex peptides are assembled by large enzymes having multiple active sites for activation, peptide bond formation as well as chemical tailoring. The peptides are assembled on nonribosomal peptide synthetase (NRPS) assembly lines covalently tethered to the synthetases throughout biosynthesis. Mass spectrometry is an especially well suited tool for analyzing both the products of NRPS pathways and the machinery that produces them. A review of NRPS enzymology and contemporary mass spectrometric methods for their analysis is given in Chapter 1. The work described in Chapter 2 for the first time brings to bear the power of mass spectrometry and directed evolution against the challenge of reengineering NRPSs. A collaborative effort between members of the Kelleher lab that led to the development of a natural product discovery platform based upon proteomic analysis is described in Chapter 3. The first discovery, sequencing and characterization of a novel NRPS biosynthetic gene cluster using this new proteomics based platform in the Kelleher lab is described in Chapter 4. In summary, this thesis is founded on and extends upon the pioneering work of in vitro characterization of NRPSs performed in the Kelleher lab over the last decade and paves the way for an explosion of future discoveries using these and yet to be developed techniques.

Graduation Semester

2010-12

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http://hdl.handle.net/2142/18572

Copyright and License Information

copyright 2010 Bradley S. Evans

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