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Elucidation of the prodiginine biosynthetic pathway in Streptomyces coelicolor A3(2)

By Paulina K. Sydor

Abstract

The prodiginine antibiotics are produced by eubacteria, in particular members of\ud the actinomycete family. Interest in this group of compounds has been stimulated by\ud their antitumour, immunosuppressant and antimalarial activities at non-toxic levels.\ud Streptomyces coelicolor A3(2) produces two prodiginines: undecylprodiginine and its\ud carbocyclic derivative streptorubin B, which are both derived from the two intermediates\ud 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde (MBC) and 2-undecylpyrrole (2-UP).\ud The red gene cluster of S. coelicolor contains 23 genes responsible for prodiginine\ud biosynthesis.\ud PCR-targeting was used to generate rapid in-frame deletions or replacements of\ud several genes in the S. coelicolor red cluster. Using this method redI, redJ, redK, the A\ud domain encoding region of redL, redT and redV were disrupted. Prodiginine production\ud by these mutants was analysed by LC-MS allowing roles for the genes investigated to be\ud hypothesised. A major focus was investigating the function of RedH (proposed to\ud catalyse the condensation of 2-UP and MBC) and RedG (proposed to be responsible for\ud the oxidative carbocyclisation of undecylprodiginine to form streptorubin B) by genetic\ud complementation of existing mutants and heterologous expression of the genes in S.\ud venezuelae coupled with feeding of synthetic MBC and 2-UP. The results of these\ud experiments clearly defined the roles of RedH in the condensation of MBC and 2-UP\ud and RedG in the oxidative carbocyclisation of undecylprodiginine.\ud Streptomyces longispororuber is known to produce undecylprodiginine (like S.\ud coelicolor) and a carbocyclic undecylprodiginine derivative called metacycloprodigiosin (streptorubin A), which contains a 12-membered carbocycle instead of the 10-membered\ud carbocycle of streptorubin B. A S. longispororuber fosmid library was constructed, from\ud which a clone containing a previously identified redG orthologue was isolated and\ud partially sequenced. Expression of the S. longispororuber redG orthologue in the S.\ud coelicolor redG mutant resulted in production of metacycloprodigiosin instead of\ud streptorubin B showing that RedG and its S. longispororuber orthologue catalyse\ud carbocyclisation reactions during prodiginine biosynthesis.\ud Another aim of the work was to investigate redU, a gene from the red cluster\ud that encodes a phosphopantetheinyl transferase (PPTase). PPTases are responsible for\ud post-translational modification of acyl carrier proteins (ACPs) and peptidyl carrier\ud proteins (PCPs). A pre-existing redU mutant and two newly constructed mutants lacking\ud PPTases encoded elsewhere in the S. coelicolor genome were analysed to investigate the\ud role of PPTases in S. coelicolor metabolite biosynthesis. Production of prodiginines,\ud actinorhodins, methylenomycins, calcium dependent antibiotics, coelichelin and grey\ud spore pigment was investigated as ACPs and PCPs are involved in biosynthesis of these\ud compounds. Different specific PPtases were found to be required to modify the\ud ACP/PCP domains/proteins in the biosynthesis of these metabolites

Topics: QD, RM
OAI identifier: oai:wrap.warwick.ac.uk:3794

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