Use this URL to cite or link to this record in EThOS:
Title: Chemical and biological investigation of iterative PKS-NRPS programming during aspyridone and tenellin biosynthesis
Author: Wasil, Zahida
ISNI:       0000 0004 5358 8311
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2014
Availability of Full Text:
Access from EThOS:
Hybrid polyketide-nonribosomal peptides are one of the important classes of fungal natural products. The aim of this work was to investigate the programming behaviour of the iterative enzymes of PKS-NRPS and delineate biosynthetic steps of PKS-NRPS compounds. This work involves isolation and structural elucidation of compounds obtained from heterologous expression of selected tenellin 87, desmethyl bassianin 88 . and aspyridone 84 PKS-NRPS genes in Aspergillus oryzae (M-2-3). Transformants obtained from gene silencing in the fungus Beauveria bassiana strain 110.25 were also chemically analysed. The tenS gene encodes a hybrid polyketide synthase nonribosomal-peptide synthetase (PKS-NRPS) in tenellin 87 and requires trans-acting ER encoding gene tenC for correct programming of tenellin compounds. Silencing of enoyl reductase gene tenC in B. bassiana 110.25 by P amyB was unsuccessful because variation of the carbon source in the original TPM media failed to produce tenellin compounds. We isolated two new A. 07yzae wild type compounds 146 and 147 from A. oryzae tenSPKS-dmbC clone but did not observe the anticipated product of TENSPKS expressed in A. oryzae without the NRPS portion. Aspyridone A 84 and aspyridone B 226 were reported as final products of a putative PKS-NRPS gene cluster in Aspergillus nidulans. We carried out heterologous expression of aspyridone PKS-NRPS encoding gene apdA and enoyl reductase encoding gene apdC in different combinations with aspyridone tailoring genes in A. oryzae. The heterologous expression produced eight different compounds in addition to aspyridone A 84. Our study showed: that the cytochrome P450 ApdE catalyses oxidative ring expansion; tetramic acid benzylic hydroxylation and an exclusive dephenylation activity; that ApdB, a second cytochrome P450 enzyme in the apd cluster catalyses an unusual N-hydroxylation of dephenylated 2-pyridones; that ApdC enoyl reductase perform different stereoselectivities during different cycles of polyketide chain synthesis. We did not observe any apparent role for ApdD and ApdG and our heterologous expression of apd genes did not produce aspyridone B 226, one of the previously reported final products of the apd pathway. This study unveiled the high biosynthetic potential of the apd pathway and diverse chemical capabilities of cYtochrome P450 enzymes in this fungus.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available