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Title: Investigating secondary metabolism in Fusarium sacchari
Author: Munawar, Asifa
ISNI:       0000 0004 2718 6810
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2011
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Initial observations on the study organism suggested that its identification as Colletotrichum falcatum was incorrect. Further morphological and molecular studies identified the organism as Fusarium sacchari. When grown on a range of culture media F sacchari produced a diverse array of secondary metabolites, several of which were purified and identified. They included known compounds such as fusaric acid and bikaverin. The structure of one of several unknown compounds was determined; the novel compound, fusarachromene, was produced by F sacchari on Czapek Dox medium. Fusarachromene is not a polyketide and did not show any antibacterial or antifungal activity. Degenerate primers based on conserved segments of polyketide synthase (PKS) genes were used to amplify segments of the genome of F sacchari. Sequencing of one PCR product identified it as a fragment of a non-ribosomal peptide synthetase (NRPS) rather than a PKS gene. F. sacchari genomic libraries were constructed and screened with homologous probes generated from the PCR products. Mapping and partial sequencing of selected clones yielded complete gene sequences for a PKS (designated FSP 1) and a hybrid PKS-NRPS (designated FSPN1), together with the almost complete sequence of an NRPS gene (designated FSN 1). The missing 3' end sequence of the FSN 1 gene was obtained by RT-PCR. FSN1 encodes a 4707 amino acid NRPS consisting of three complete adenylation, thiolation and condensation domains followed by two additional thiolation and condensation domains repeats. This structure is similar to that of the ferricrocin synthetase of Aspergillus nidulans. Upon heterologous expression in Aspergillus oryzae, FSN1 was shown to direct the synthesis of ferrirhodin, a siderophore that was found to be present in culture filtrates of F sacchari. No prior report of a ferrirhodin synthetase has been found in the literature. FSP 1 encodes a 2591 amino acid PKS consisting of keto synthase, acyl transferase, C- methyl transferase, enoyl reductase, ketoreductase and acyl carrier protein domains. Heterologous expression of FSP 1 in A. oryzae was deemed unsuccessful as no novel metabolites were detected. FSPN1 encodes a 4074 amino acid protein with PKS domains (keto synthase, acyl transferase C-methyl transferase, ketoreductase and acyl carrier protein) and NRPS domains (condensation, adenylation, thiolation and reductase). The heterologous expression of FSPN1 in A. oryzae yielded a number of new LC-MS peaks. The molecular masses of all but one of these peaks were far too small to correspond to that of the expected metabolite. Further functional analysis of FSPN1 may require eo- expression with other members of its gene cluster.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available