Sarovich, Derek (2007). CLONING AND HETEROLOGOUS EXPRESSION OF ANTITUMOUR ANTIBIOTIC GENE CLUSTERS PhD Thesis, School of Molecular and Microbial Sciences , University of Queensland.

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Author Sarovich, Derek
School, Centre or Institute School of Molecular and Microbial Sciences
Institution University of Queensland
Publication date 2007
Thesis type PhD Thesis
Supervisor Dr John Pemberton
Abstract/Summary The indolocarbazoles are a significant group of naturally occurring antitumour antibiotics well known to medical science. They possess a range of activities including anticancer, immunosuppressive and anti-hypertension properties. In 2000, it was discovered that the core of all indolocarbazoles was being synthesised by a highly conserved enzyme, named indolocarbazole synthase (ICS). The first indolocarbazole to be isolated and characterized was the strikingly purple pigmented violacein from Chromobacterium violaceum. Following the isolation of violacein, several other indolocarbazoles have been isolated including rebeccamycin and the well-known staurosporine. In the current study, a number of genetic tools were developed for the screening, isolation, characterisation and expression of the indolocarbazole gene clusters, with wider applicability for other multi-gene synthesis pathways. Due to the conserved nature of the ICS present in all indolocarbazole clusters, a PCR-based screening method was designed to detect the ICS gene. The assay was successful in detecting the presence of the ICS in both the violacein and rebeccamycin gene clusters. A second PCRbased screening method was designed that allowed distinction between the violacein cluster and other indolocarbazole clusters. The PCR-based assays designed in this work have potential utility for screening environmental isolates and DNA libraries for the presence of known or novel indolocarbazole clusters. The current vectors used for the isolation of indolocarbazoles and other multigene pathways are inadequate for a number of reasons. Firstly, cloning vectors encoding the synthesis of toxic metabolites are often unstable in the Escherichia coli host. Secondly, most vectors possess a limited host range and cannot be transferred to heterologous hosts other than E. coli for screening purposes. Therefore, the current study describes the construction of a number of novel vectors that facilitated the cloning of both single and multi-gene phenotypes in the E. coli host. One of these vectors, pPSX, is a 14-kilobase (kb) vector derived from the IncW plasmid pR388. pPSX is highly stable in E. coli in the absence of antibiotic selection, even while expressing the toxic indolocarbazole antitumour antibiotic violacein. The incorporation of the lambda cos sequence enables construction of cosmid libraries with inserts ranging from 24 to 36kb. The inclusion of a lacZ’ gene fragment and the multiple cloning site (MCS) from pUC18 allows blue-white screening. pPSX cosmids can be extracted from the host cell in large amounts using commercial plasmid extraction kits, facilitating downstream analysis and sequencing of cloned DNA. Like pR388, pPSX has a broad host range and can be transferred from E. coli to a wide variety of heterologous hosts by either electroporation or mobilisation. While it shows less stability in non-E. coli hosts without antibiotic selection, heterologous host strains such as Rhodobacter sphaeroides and Pseudomonas stutzeri will maintain the plasmid under antibiotic selection to allow screening of expressed inserts. pPSX provides the benefits of large insert sizes with high stability to allow cloning of chemotherapeutic gene clusters in E. coli and a range of other heterologous hosts. In addition to pPSX, a PCR product (amplicon) cloning vector, pPSY, was designed to directly clone amplicons using the commercial cloning kit, pGEM®- T-Easy. The pPSY vector is able to clone and express single gene phenotypes from both Gram-negative and Gram-positive bacteria. Like pPSX, pPSY is completely stable in the E. coli host and is easily transferred to a range of heterologous hosts, including P. stutzeri and R. sphaeroides. Finally, the P. stutzeri host was altered to allow the transfer of DNA libraries from E. coli. To facilitate this transfer, the native restriction system of the P. stutzeri host was removed. This restrictionless mutant showed dramatic increases (1000x) in electroporation frequency of plasmid DNA. Using this strain pPSX cosmid clone banks, constructed in E. coli, can be moved into P. stutzeri for screening purposes. In addition, pPSX has the potential to be used as a bacterial artificial chromosome (BAC) vector, allowing direct cloning of inserts of up to 300kb into either E. coli or P. stutzeri. It is envisaged that the vectors and strains developed in this study will have wide applicability in cloning and heterologous expression of single and multiple gene phenotypes from both Gram-negative and Gram-positive bacteria. These vectors and strains can be used for initial cloning and downstream analysis of gene pathways, which may not be possible using currently available vectors. Of particular interest are the many indolocarbazole antitumour antibiotic gene clusters and other chemotherapeutic gene clusters yet to be discovered within the genetically diverse streptomycetes.

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Created: Fri, 21 Nov 2008, 16:20:58 EST