Stable high level expression of the violacein indolocarbazole anti-tumour gene cluster and the Streptomyces lividans amyA gene in E. coli K12

Ahmetagic, Adnan and Pemberton, John M. (2010) Stable high level expression of the violacein indolocarbazole anti-tumour gene cluster and the Streptomyces lividans amyA gene in E. coli K12. Plasmid, 63 2: 79-85. doi:10.1016/j.plasmid.2009.11.004

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Author Ahmetagic, Adnan
Pemberton, John M.
Title Stable high level expression of the violacein indolocarbazole anti-tumour gene cluster and the Streptomyces lividans amyA gene in E. coli K12
Formatted title
Stable high level expression of the violacein indolocarbazole anti-tumour gene cluster and the Streptomyces lividans amyA gene in E. coli K12
Journal name Plasmid   Check publisher's open access policy
ISSN 0147-619X
1095-9890
Publication date 2010-03
Year available 2009
Sub-type Article (original research)
DOI 10.1016/j.plasmid.2009.11.004
Volume 63
Issue 2
Start page 79
End page 85
Total pages 7
Editor D. K. Chattoraj
Place of publication New York, NY, U.S.A.
Publisher Academic Press
Collection year 2010
Language eng
Subject C1
060501 Bacteriology
970106 Expanding Knowledge in the Biological Sciences
0604 Genetics
Formatted abstract
Previous studies showed that when pPSX-vioABCDE was used to transform E. coli K12 DH5α the strain retained the plasmid even after 100 generations of unselected growth but produced a low level of the anti-tumour antibiotic violacein. Markedly higher levels of violacein synthesis were obtained from E. coli K12 DH5α pUC18-vioABCDE and Sphingomonas sp. JMP4092 pPSX-vioABCDE. Unfortunately, both strains were extremely unstable regardless of presence or absence of antibiotic selection to retain the plasmid. The current study was undertaken to determine if strains of E. coli K12 could be isolated which stably over produce violacein. When a range of E. coli K12 strains were transformed with pPSX-vioABCDE, most produced small amounts of violacein. However, a small number of related strains of E. coli K12 JM101, JM105 and JM109 not only over-produced violacein, but also maintained the high stability. In addition, E. coli K12 JM109 strongly expressed an alpha amylase gene (amyA) from Streptomyces lividans indicating that the S. lividans amyA promoter is highly active in E. coli K12 JM109. In another set of experiments, a violacein overproduction mutation (opv-1) of the plasmid pPSX-vioABCDE was isolated which enabled E. coli K12 DH5α to overproduce violacein while retaining high stability. The plasmid pPSX-vioABCDE opv-1 possesses a single base pair deletion in the promoter region of the violacein operon. By combining the over producing strain E. coli K12 JM109 and the over producing plasmid pPSX-vioABCDE opv-1, a stable hyper producing strain (E. coli K12 JM109 pPSX-vioABCDE opv-1) was constructed. Finally, two additional stable vectors, pPSX10 and pPSX20, were constructed to facilitate subcloning and functional analysis studies.
© 2009 Elsevier Inc.
Keyword E.Coli K12
Stable cloning
Expression
Streptomycete
Violacein
Staurosporine
Escherichia-coli
Chromobacterium-violaceum
Heterologous expression
Biological-activity
Natural-products
Biosynthesis
Cloning
Derivatives
Transformation
Construction
Q-Index Code C1
Q-Index Status Confirmed Code
Additional Notes Available online 3 December 2009.

 
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Created: Mon, 22 Feb 2010, 09:25:50 EST by Jennifer Falknau on behalf of School of Chemistry & Molecular Biosciences