Sydor, Paulina K. (2010) Elucidation of the prodiginine biosynthetic pathway in Streptomyces coelicolor A3(2). PhD thesis, University of Warwick.

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Abstract

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 (PhD)
Subjects:	Q Science > QD Chemistry R Medicine > RM Therapeutics. Pharmacology
Library of Congress Subject Headings (LCSH):	Antibiotics -- Synthesis, Antibiotic-producing organisms, Streptomyces
Official Date:	February 2010
Dates:	Date Event February 2010 Submitted
Institution:	University of Warwick
Theses Department:	Department of Chemistry
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Challis, Gregory L.
Sponsors:	University of Warwick
Extent:	xx, 225 leaves : ill., charts
Language:	eng

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