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Title: Investigating programming and production of fungal polyketide synthases
Author: Koziol, Magdalena D.
ISNI:       0000 0004 5919 716X
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2014
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Fungal natural products are secondary metabolites produced by complex biosynthetic pathways, many of which include the activities of multidomain polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) megasynthases. This thesis reports investigations of the intrinsic programming of PKS-NRPS hybrid enzymes and of the reconstruction of polyketide biosynthetic pathways by heterologous expression in Aspergillus oryzae. Rational domain swaps were performed between megasynthases involved in the synthesis of structurally-related 2-pyridones. Swaps between tenellin and desmethylbassianin synthases (TENS and DMBS) from closely related isolates of Beauveria bassiana confirmed that the C-methyltransferase domain controls methylation pattern and the ketoreductase domain contributes to chain-length programming; the hybrid enzymes produced were highly active. Hybrid enzymes from more distant swaps had reduced activity but showed potential for the production of chimaeric compounds. Exchanges between even more distantly-related enzymes resulted in complete loss of enzyme activity. Attempts to reconstruct the militarinone A biosynthetic pathway in A. oryzae met more success when MILS was co-expressed with enzymes from the DMB pathway than with its cognate enzymes. In contrast, heterologous expression of five genes from Aspergillus terreus resulted in full reconstruction of the lovastatin biosynthetic pathway. However, efficient lovastatin production was found to require additional co-expression of the thioesterase responsible for releasing the lovastatin nonaketide from the nonaketide synthase. Curiously, co-expression of the subset of enzymes required for synthesis of lovastatin intermediates was unproductive. The genome of Phoma sp. C2932 was sequenced and analysed to identify the squalestatin biosynthetic gene cluster. The putative squalestatin hexaketide synthase (SQHKS) expressed in isolation in A. oryzae did not produce novel compounds. SQHKS employs an unusual starter unit, the production of which is likely to be governed by other genes identified in the cluster. Co-expression of SQHKS with phenylalanine ammonia lyase and an AMP-dependent Co-A ligase, in the presence of cinnamic acid, yielded novel compounds, albeit in insufficient yield to confirm a structure related to squalestatin hexaketide. The results of lovastatin-production experiments suggest that co-expression of additional genes from the squalestatin pathway may improve product yields.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available