The antibiotic compound pleuromutilin, produced by Clitopilus passeckerianus, has antibiotic activity against some clinically important bacteria, including MRSA. However yield from the native producer is low and there is no derivative compound available with oral or intravenal bioavailability. The identification of the pleuromutilin gene cluster in Clitopilus passeckerianus allows for molecular biology techniques to be used to attempt to resolve these issues. This investigation has sought to help ameliorate the problems with pleuromutilin via three experimental approaches. Gene silencing in Cl. passeckeriqnus was used to attempt to generate intermediate compounds from the pleuromutilin pathway. Although several strains transformed with an antisense Acyltransferase (ATF) produced no pleuromutilin, no other. intermediatesaccumulated. Chemical analysis revealed the accumulation of a novel sesquiterpene compound, pilobarbatriol. QRT+PCR was used to determine the impact of ATF silencing on the transcription of other genes in the cluster. This approach revealed that the initial two genes in the pathway, the Geranylgeranyl diphosphate synthase (GGS) and the Cyclase, were not transcriptionally active in ATF silenced lines preventing, accumulation of intermediate compounds. This approach demonstrated the viability of antisense silencing in Cl. passeckerianus and its potential for metabolite engineering. Heterologous expression in Coprinopsis cinerea was then attempted with the transfer of the pleuromutilin biosynthetic cluster in a genomic configuration. Co. cinerea transformants were generated with the entire gene cluster; however bioassay analysis revealed no antibiotic activity. RTPCR determined that all but two genes, Cyclase and p450-3, were being transcribed in Co. cinerea. Promoter-GFP analysis showed that the Cyclase and p450-3 promoter regions were active in Co. cinerea and therefore not preventing transcription. Transformation with cDNA versions of the two genes led to the transcription of p450-3. The successful expression of six of seven genes in Co . . cinerea may suggest that this approach would perhaps be effective for gene clusters smaller than that of the pleuromutilin biosynthetic gene cluster but not the pleuromutilin gene cluster itself. Expression of genes from the pleuromutilin gene cluster was subsequently attempted in the ascomycete fungus Aspergillus oryzae. On transformation with a GGS expression plasmid, a compound with similar characteristics to GGPP was found to be accumulating. Subsequently A. oryzae was transformed with both the GGS and Cyclase expression constructs and transcripts were detected for both genes. Chemical characterisation revealed the accumulation of compounds in transformed strains which were absent in controls suggesting that these two genes are generating products in A. oryzae, demonstrating this approach is appropriate for the heterologous production of secondary metabolites.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:665173 |
| Date | January 2014 |
| Creators | Hayes, Patrick |
| Publisher | University of Bristol |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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