Reengineering and Discovery of Nonribosomal Peptide Synthetases

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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2011

Author dc:creator

• Evans, Bradley S.

Contributors dc:contributor

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

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

Statement dc:rights

• copyright 2010 Bradley S. Evans

Language dc:language

en

Handle dc:identifier

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

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/18572