Abstract

The goal of this three year proposal is to develop E. coli as an efficient heterologous expression system for the DNA bisintercalator triostin. A non-ribosomally synthesized peptide, triostin is a known antibiotic and anticancer agent. We will test the hypothesis that by metabolically engineering the E. coli host, modifying the plasmid vector, and altering the triostin gene, triostin and its analogs could be biosynthesized. There are two specific aims for this research, (1) Metabolically engineer E. coli to produce triostin. (2) Modify the triostin NRPS to produce analogs with altered sequence specificity and selectivity. Preliminary results. We have identified a four module non-ribosomal peptide synthetase that contains adenylation domains specific for Serine, Alanine, Cysteine, Valine amino acids (the four amino acids found in triostin.) The significance of this research is that it will (a) improve the mechanistic understanding of the assembly of C2-symmetric non-ribosomal peptides, (b) provide a general host for the expression of diverse non-ribosomal peptide synthetase genes.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM075857-02
Application #
7125549
Study Section
Special Emphasis Panel (ZRG1-BCMB-R (50))
Program Officer
Schwab, John M
Project Start
2005-09-23
Project End
2008-07-31
Budget Start
2006-08-01
Budget End
2007-07-31
Support Year
2
Fiscal Year
2006
Total Cost
$278,547
Indirect Cost

  Institution

Name
University of Southern California
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90089

  Publications

Li, Angela Ying-Jian; Lin, Her Helen; Kuo, Ching-Ying et al. (2011) High-mobility group A2 protein modulates hTERT transcription to promote tumorigenesis. Mol Cell Biol 31:2605-17
Watanabe, Kenji; Hotta, Kinya; Praseuth, Alex P et al. (2009) Rationally engineered total biosynthesis of a synthetic analogue of a natural quinomycin depsipeptide in Escherichia coli. Chembiochem 10:1965-8
Watanabe, Kenji; Hotta, Kinya; Nakaya, Mino et al. (2009) Escherichia coli allows efficient modular incorporation of newly isolated quinomycin biosynthetic enzyme into echinomycin biosynthetic pathway for rational design and synthesis of potent antibiotic unnatural natural product. J Am Chem Soc 131:9347-53
Wang, Clay C C; Chiang, Yi-Ming; Kuo, Po-Lin et al. (2008) Norsolorinic acid from Aspergillus nidulans inhibits the proliferation of human breast adenocarcinoma MCF-7 cells via Fas-mediated pathway. Basic Clin Pharmacol Toxicol 102:491-7
Wang, Clay C C; Chiang, Yi-Ming; Kuo, Po-Lin et al. (2008) Norsolorinic acid inhibits proliferation of T24 human bladder cancer cells by arresting the cell cycle at the G0/G1 phase and inducing a Fas/membrane-bound Fas ligand-mediated apoptotic pathway. Clin Exp Pharmacol Physiol 35:1301-8
Praseuth, Alex P; Wang, Clay C C; Watanabe, Kenji et al. (2008) Complete sequence of biosynthetic gene cluster responsible for producing triostin A and evaluation of quinomycin-type antibiotics from Streptomyces triostinicus. Biotechnol Prog 24:1226-31
Chiang, Yi-Ming; Szewczyk, Edyta; Nayak, Tania et al. (2008) Molecular genetic mining of the Aspergillus secondary metabolome: discovery of the emericellamide biosynthetic pathway. Chem Biol 15:527-32
Marques, Michael A; Citron, Diane M; Wang, Clay C (2007) Development of Tyrocidine A analogues with improved antibacterial activity. Bioorg Med Chem 15:6667-77
Ma, Suzanne M; Zhan, Jixun; Watanabe, Kenji et al. (2007) Enzymatic synthesis of aromatic polyketides using PKS4 from Gibberella fujikuroi. J Am Chem Soc 129:10642-3
Zhang, Wenjun; Watanabe, Kenji; Wang, Clay C C et al. (2007) Investigation of early tailoring reactions in the oxytetracycline biosynthetic pathway. J Biol Chem 282:25717-25

Showing the most recent 10 out of 12 publications