Adnan Ahmetagic (2011). STABLE HIGH LEVEL EXPRESSION OF HETEROLOGOUS GENE CLUSTERS ENCODING ANTITUMOUR ANTIBIOTICS IN ESCHERICHIA COLI K12 PhD Thesis, School of Chemistry & Molecular Biosciences, The University of Queensland.

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Author Adnan Ahmetagic
School, Centre or Institute School of Chemistry & Molecular Biosciences
Institution The University of Queensland
Publication date 2011
Thesis type PhD Thesis
Supervisor Dr John Pemberton
Total pages 183
Total colour pages 29
Total black and white pages 154
Language eng
Subjects 0604 Genetics
0605 Microbiology
Abstract/Summary The process of introducing and expressing foreign genetic material in a bacterial host organism has been used by the biotechnology and pharmaceutical industries for the production of large quantities of drugs, amino acids, proteins, enzymes and fuels amongst other things. This heterologous gene expression has evolved to a point where it is now possible to design and construct new biological functions and systems not found in nature spurring the age of synthetic biology. The research presented in this thesis describes methods to obtain Escherichia coli K12 strains that demonstrate stable, high level heterologous expression of genes and gene clusters from a range of both Gram-positive and Gram-negative bacteria. 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 overproduce 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 overproduced 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 overproducing strain E. coli K12 JM109 and the overproducing plasmid pPSX-vioABCDE opv-1, a stable hyper producing strain (E. coli K12 JM109 pPSX-vioABCDE opv-1) was constructed. Two additional stable vectors, pPSX10 and pPSX20, were constructed to facilitate subcloning and functional analysis studies. Using a variety of antibiotics, it was found that nine separate isolates of spontaneous antibiotic resistant mutants of E. coli K12 pPSX-vioABCDE overproduce the anti-tumour antibiotic violacein. Subsequent analysis showed that seven of these mutations occurred on the plasmid pPSX-vioABCDE. The other two overproducing strains carried spontaneous chromosomal mutations to lincomycin and kanamycin. The kanamycin resistant mutant of E. coli K12 DH10B (AA23) and a lincomycin resistant mutant of E. coli K12 LE392 (AA24) increased the synthesis of violacein. The plasmid pPSX-vioABCDE opv-1 contains a violacein overproduction (opv-1) mutation which when introduced into either E. coli K12 AA23 or AA24, resulted in a hyper-production of violacein. Remarkably, E. coli K12 AA23 pPSX-vioABCDE opv-1 produced 41 times the normal level of violacein. In addition, both E. coli K12 AA23 and E. coli K12 AA24 demonstrated an increase in expression of an alpha amylase gene from S. lividans and the urease gene cluster from Klebsiella oxytoca. These results suggest that selection of antibiotic resistant mutants can increase heterologous gene expression in E. coli K12. Additionally, the increased expression is a general effect applicable to genes and gene clusters cloned into E. coli K12 from both Gram-positive and Gram-negative bacteria. The next step was to assess gene expression in a single strain of E. coli K12 carrying multiple antibiotic resistance mutations. Using spectrophotometric analysis, it was found that a triple antibiotic resistant (kanamycin, lincomycin and rifampicin) strain of E. coli K12 DH5α (AA25) produced 19 times more violacein than the original strain. This overproduction phenotype extended to an alpha amylase gene from S. lividans and a urease gene cluster from K. oxytoca. The combination of multiple antibiotic resistance mutations in E. coli K12 demonstrates the utility these strains may have on increasing heterologous gene expression and secondary metabolite production. These techniques can be applied to improve protein production and the screening capabilities of this, and potentially other bacteria. Previous studies have revealed that violacein has activity against bacteria, viruses, cancer and parasites. Since many important diseases such as malaria are caused by protozoal parasites, E. coli K12 strains engineered to produce varying levels of violacein in this current study were used to develop a simple, quantitative assay of anti-parasitic activity. A bactivorous predatory protozoan isolate, Colpoda sp. A4, was isolated from soil and tested for its ability to ‘graze’ on various violacein producing strains of E. coli K12. A grazing assay was developed based on protozoan “plaque” formation. As expected, the more violacein the E. coli K12 strain produced, the more resistant it was to predation by the protozoa. In addition, this simple predation assay was used to detect other anti-parasitic molecules, namely pentachlorophenol and erythromycin. These results suggest that these techniques may be used in future experiments to detect the expression of anti-parasitic molecules produced by recombinant E. coli K12.
Keyword E. coli
gene expression
antibiotic resistance
stable cloning
Additional Notes 31, 44, 51, 53, 59, 61-62, 71-73, 77, 79-81, 88, 99, 101, 103, 107, 117, 121, 123-124, 134, 136, 138, 144, 148-149.

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Created: Tue, 24 Apr 2012, 15:56:47 EST by Adnan Ahmetagic on behalf of Library - Information Access Service