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ASSEMBLING NRPS MODULES IN E. COLI TO ESTABLISH A PLATFORM
FOR
RATIONAL DESIGN OF BIOLOGICALLY ACTIVE COMPOUNDS
by
Alex Praseuth
____________________________________________________________________
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PHARMACOLOGY AND PHARMACEUTICAL SCIENCES)
August 2008
Copyright 2008 Alex Praseuth
Object Description
| Title | Assembling NRPS modules in e. coli to establish a platform for rational design of biologically active compounds |
| Author | Praseuth, Alex |
| Author email | praseuth@gmail.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Pharmaceutical Sciences |
| School | School of Pharmacy |
| Date defended/completed | 2008-06-06 |
| Date submitted | 2008 |
| Restricted until | Unrestricted |
| Date published | 2008-07-02 |
| Advisor (committee chair) | Wang, Clay |
| Advisor (committee member) |
Haworth, Ian Roberts, Richard Neamati, Nouri |
| Abstract | Nonribosomal peptides (NRPs) are synthesized by modular mega–enzyme called NRP synthetase (NRPS) that catalyze a peptide bond forming reaction using natural amino acid as substrate. The majority of compounds from this class exhibit crucial biological activities such as antibiotic, immunosuppressive, antiviral, and antitumor activities. However, several of these sought-after natural products are often difficult to isolate in adequate amounts from its natural sources due to low production levels or the producing organism's stringent need for expensive and atypical culture apparatus. As we know, enzymes capable of synthesizing these natural products including essential or nonessential secondary metabolites are encoded by biosynthetic genes located on either chromosomal or plasmid DNA.; An alternative and straightforward approach to obtain these compounds is to express the entire biosynthetic gene cluster for an NRPS responsible for producing the intact molecule using E. coli as a heterologous host was developed in our laboratory. Production of triostin A (2) using small shake flask was optimized to provide a quantitative yield rivaling levels isolated from S. triostinicus. Addition of QXC, the priming unit for 2, helped to increase its production and identify the bottleneck in our system. Knock-down of two M-domain in Ecm7 provided modest amounts of 8, an unnatural natural product. A novel compound, 9, was also identified from our culture which will require additional characterization.; This study has ascertained a novel platform for creating a peptide library by means of premeditated engineering of biosynthetic domain(s) to provide a customized macromolecule. |
| Keyword | NRPS |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Type | texts |
| Legacy record ID | usctheses-m1317 |
| Rights | Praseuth, Alex |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | http://www.usc.edu/isd/libraries/services/ask_a_librarian/email/ |
| Filename | etd-Praseuth-20080702 |
| Archival file | uscthesesreloadpub_Volume29/etd-Praseuth-20080702.pdf |
Description
| Title | Page 1 |
| Full text | ASSEMBLING NRPS MODULES IN E. COLI TO ESTABLISH A PLATFORM FOR RATIONAL DESIGN OF BIOLOGICALLY ACTIVE COMPOUNDS by Alex Praseuth ____________________________________________________________________ A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (PHARMACOLOGY AND PHARMACEUTICAL SCIENCES) August 2008 Copyright 2008 Alex Praseuth |
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