Elucidation of the prodiginine biosynthetic pathway in Streptomyces coelicolor A3(2)
Sydor, Paulina K. (2010) Elucidation of the prodiginine biosynthetic pathway in Streptomyces coelicolor A3(2). PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b2339930~S15
The prodiginine antibiotics are produced by eubacteria, in particular members of the actinomycete family. Interest in this group of compounds has been stimulated by their antitumour, immunosuppressant and antimalarial activities at non-toxic levels. Streptomyces coelicolor A3(2) produces two prodiginines: undecylprodiginine and its carbocyclic derivative streptorubin B, which are both derived from the two intermediates 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde (MBC) and 2-undecylpyrrole (2-UP). The red gene cluster of S. coelicolor contains 23 genes responsible for prodiginine biosynthesis. PCR-targeting was used to generate rapid in-frame deletions or replacements of several genes in the S. coelicolor red cluster. Using this method redI, redJ, redK, the A domain encoding region of redL, redT and redV were disrupted. Prodiginine production by these mutants was analysed by LC-MS allowing roles for the genes investigated to be hypothesised. A major focus was investigating the function of RedH (proposed to catalyse the condensation of 2-UP and MBC) and RedG (proposed to be responsible for the oxidative carbocyclisation of undecylprodiginine to form streptorubin B) by genetic complementation of existing mutants and heterologous expression of the genes in S. venezuelae coupled with feeding of synthetic MBC and 2-UP. The results of these experiments clearly defined the roles of RedH in the condensation of MBC and 2-UP and RedG in the oxidative carbocyclisation of undecylprodiginine. Streptomyces longispororuber is known to produce undecylprodiginine (like S. coelicolor) and a carbocyclic undecylprodiginine derivative called metacycloprodigiosin (streptorubin A), which contains a 12-membered carbocycle instead of the 10-membered carbocycle of streptorubin B. A S. longispororuber fosmid library was constructed, from which a clone containing a previously identified redG orthologue was isolated and partially sequenced. Expression of the S. longispororuber redG orthologue in the S. coelicolor redG mutant resulted in production of metacycloprodigiosin instead of streptorubin B showing that RedG and its S. longispororuber orthologue catalyse carbocyclisation reactions during prodiginine biosynthesis. Another aim of the work was to investigate redU, a gene from the red cluster that encodes a phosphopantetheinyl transferase (PPTase). PPTases are responsible for post-translational modification of acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs). A pre-existing redU mutant and two newly constructed mutants lacking PPTases encoded elsewhere in the S. coelicolor genome were analysed to investigate the role of PPTases in S. coelicolor metabolite biosynthesis. Production of prodiginines, actinorhodins, methylenomycins, calcium dependent antibiotics, coelichelin and grey spore pigment was investigated as ACPs and PCPs are involved in biosynthesis of these compounds. Different specific PPtases were found to be required to modify the ACP/PCP domains/proteins in the biosynthesis of these metabolites.
|Item Type:||Thesis or Dissertation (PhD)|
|Subjects:||Q Science > QD Chemistry
R Medicine > RM Therapeutics. Pharmacology
|Library of Congress Subject Headings (LCSH):||Antibiotics -- Synthesis, Antibiotic-producing organisms, Streptomyces|
|Institution:||University of Warwick|
|Theses Department:||Department of Chemistry|
|Supervisor(s)/Advisor:||Challis, Gregory L.|
|Sponsors:||University of Warwick|
|Extent:||xx, 225 leaves : ill., charts|
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