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Title: A new polyketide synthase in Saccharopolyspora erythraea
Author: Boakes, S.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2002
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Amino acid and DNA sequence comparisons of modular PKSs have shown that ketosynthase (KS) domains are highly conserved. Degenerate primers were designed and used in polymerase chain reactions (PCRs) to amplify the DNA encoding these enzymatic domains from a range of streptomycete genomic templates. Analysis of the PCR products did not only identify a number of known KS domains but included a number of unknown KSs. Using one of these products as a probe to screen a genomic library, a new PKS gene cluster has been identified in Saccharopolyspora erythraea. This new PKS gene cluster, named BOA, has been sequenced. Sequence analysis and database comparisons have led to the elucidation of the modular arrangement of this new cluster and annotation of the flanking genes. Based upon the modular arrangement of this new PKS a putative polyketide product has been proposed. In an effort to overexpress this new PKS and isolate the putative polyketide product a number of compounds that demonstrate bioactivity towards Gram-positive organisms have been discovered. One of these compounds has been isolated and its structure determined via nuclear magnetic resonance (NMR). Results from NMR analysis suggest that this bioactive compound is a tetrapeptide that functions as an ion-chelating siderophore. To confirm the function of domains and modules from the new PKS a range of hybrid PKSs were engineered. These synthases were constructed by substituting modules or domains from the well-studied DEBS 1-TE system with components from the new PKS. In a number of cases expression of these synthases resulted in production of the expected polyketide products. The results confirm the assigned function of the respective domain or module from the BOA PKS and demonstrate the potential use of these components in future engineering of hybrid synthases. A newly discovered gene from S. erythraea that encodes for red-pigment production has been used to develop a novel series of promoter probe plasmids.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available