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

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

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

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.EThOS - Electronic Theses Online ServiceUniversity of WarwickGBUnited Kingdo

Structural basis for chain release from the enacyloxin polyketide synthase

Modular polyketide synthases and nonribosomal peptide synthetases are molecular assembly lines consisting of several multienzyme subunits that undergo dynamic self-assembly to form a functional mega-complex. N- and C-terminal docking domains are usually responsible for mediating interactions between subunits. Here we show that communication between two nonribosomal peptide synthetase subunits responsible for chain release from the enacyloxin polyketide synthase, which assembles an antibiotic with promising activity against Acinetobacter baumannii, is mediated by an intrinsically disordered short linear motif and a ß-hairpin docking domain. The structures, interactions and dynamics of these subunits are characterised using several complementary biophysical techniques, providing extensive insights into binding and catalysis. Bioinformatics analyses reveal that short linear motif/ß-hairpin docking domain pairs mediate subunit interactions in numerous nonribosomal peptide and hybrid polyketide-nonribosomal peptide synthetases, including those responsible for assembling several important drugs. Short linear motifs and ß-hairpin docking domains from heterologous systems are shown to interact productively, highlighting the potential of such interfaces as tools for biosynthetic engineering

Stereochemical elucidation of streptorubin B

Streptorubin B is a structurally remarkable mem-ber of the prodiginine group of antibiotics produced by several actinobacteria, including the model organism Streptomyces coelicolor A3(2). Transannular strain within the pyrrolophane structure of this molecule causes restricted rotation that gives rise to the possibility of (diastereomeric) atropisomers. Neither the relative nor the absolute stereochemistry of streptorubin B is known. NOESY NMR experiments were used to define the relative stereochemistry of the major atropisomer of streptorubin B·HCl in solution as anti. We exploited this finding together with our knowledge of streptorubin B biosynthesis in S. coelicolor to determine the absolute stereochemistry of the anti atropisomer. 2-Undecylpyrrole stereoselectively labeled with deuterium at C-4′ was synthesized and fed to a mutant of S. coelicolor, which was unable to produce streptorubin B because it was blocked in 2-undecylpyrrole biosynthesis, and in which the genes responsible for the last two steps of streptorubin B biosynthesis were overexpressed. 1H and 2H NMR analysis of the stereoselectively deuterium-labeled streptorubin B·HCl produced by this mutasynthesis strategy allowed us to assign the absolute stereochemistry of the major (anti) atropisomer as 7′S. HPLC analyses of streptorubin B isolated from S. coelicolor on a homochiral stationary phase and comparisons with streptorubin B derived from an enantioselective synthesis showed that the natural product consists of an approximately 88:7:5 mixture of the (7′S, anti), (7′S, syn), and (7′R, anti) stereoisomers

Role and substrate specificity of the Streptomyces coelicolor RedH enzyme in undecylprodiginine biosynthesis

The function of RedH from Streptomyces coelicolor as an enzyme that catalyses the condensation of 4-methoxy- 2,2'- bipyrrole-5- carboxaldehyde (MBC) and 2-undecylpyrrole to form the natural product undecylprodiginine has been experimentally proven, and the substrate specificity of RedH has been probed in vivo by examining its ability to condense chemically-synthesised MBC analogues with 2-undecylpyrrole to afford undecylprodiginine analogues

A dual transacylation mechanism for polyketide synthase chain release in enacyloxin antibiotic biosynthesis.

Polyketide synthases assemble diverse natural products with numerous important applications. The thioester intermediates in polyketide assembly are covalently tethered to acyl carrier protein domains of the synthase. Several mechanisms for polyketide chain release are known, contributing to natural product structural diversification. Here, we report a dual transacylation mechanism for chain release from the enacyloxin polyketide synthase, which assembles an antibiotic with promising activity against Acinetobacter baumannii. A non-elongating ketosynthase domain transfers the polyketide chain from the final acyl carrier protein domain of the synthase to a separate carrier protein, and a non-ribosomal peptide synthetase condensation domain condenses it with (1S,3R,4S)-3,4-dihydroxycyclohexane carboxylic acid. Molecular dissection of this process reveals that non-elongating ketosynthase domain-mediated transacylation circumvents the inability of the condensation domain to recognize the acyl carrier protein domain. Several 3,4-dihydroxycyclohexane carboxylic acid analogues can be employed for chain release, suggesting a promising strategy for producing enacyloxin analogues

Elucidation of the Streptomyces coelicolor pathway to 2-undecylpyrrole, a key intermediate in undecylprodiginine and streptorubin B biosynthesis

The red gene cluster of Streptomyces coelicolor directs production of undecylprodiginine. Here we report that this gene cluster also directs production of streptorubin B and show that 2-undecylpyrrole (UP) is an intermediate in the biosynthesis of undecylprodiginine and streptorubin B. The redPQRKL genes are involved in UP biosynthesis. RedL and RedK are proposed to generate UP from dodecanoic acid or a derivative. A redK(-) mutant produces a hydroxylated undecylprodiginine derivative, whereas redL(-) and redK(-) mutants require addition of chemically synthesized UP for production of undecylprodiginine and streptorubin B. Fatty acid biosynthetic enzymes can provide dodecanoic acid, but efficient and selective prodiginine biosynthesis requires RedPQR Deletion of redP, redQ, or redR leads to an 80%-95% decrease in production of undecylprodiginine and and array of prodiginine analogs with varying alkyl chains. In a redR(-) mutant, the ratio of these can be altered in a logical manner by feeding various fatty acids

A dual transacylation mechanism for polyketide synthase chain release in enacyloxin antibiotic biosynthesis.

Polyketide synthases assemble diverse natural products with numerous important applications. The thioester intermediates in polyketide assembly are covalently tethered to acyl carrier protein domains of the synthase. Several mechanisms for polyketide chain release are known, contributing to natural product structural diversification. Here, we report a dual transacylation mechanism for chain release from the enacyloxin polyketide synthase, which assembles an antibiotic with promising activity against Acinetobacter baumannii. A non-elongating ketosynthase domain transfers the polyketide chain from the final acyl carrier protein domain of the synthase to a separate carrier protein, and a non-ribosomal peptide synthetase condensation domain condenses it with (1S,3R,4S)-3,4-dihydroxycyclohexane carboxylic acid. Molecular dissection of this process reveals that non-elongating ketosynthase domain-mediated transacylation circumvents the inability of the condensation domain to recognize the acyl carrier protein domain. Several 3,4-dihydroxycyclohexane carboxylic acid analogues can be employed for chain release, suggesting a promising strategy for producing enacyloxin analogues

Molecular basis for control of antibiotic production by a bacterial hormone

Actinobacteria produce numerous antibiotics and other specialized metabolites that have important applications in medicine and agriculture1. Diffusible hormones frequently control the production of such metabolites by binding TetR family transcriptional repressors (TFTRs), but the molecular basis for this remains unclear2. The production of methylenomycin antibiotics in Streptomyces coelicolor A3(2) is initiated by the binding of 2-alkyl-4-hydroxymethylfuran-3-carboxylic acid (AHFCA) hormones to the TFTR MmfR3. Here we report the X-ray crystal structure of an MmfR–AHFCA complex, establishing the structural basis for hormone recognition. We also elucidate the mechanism for DNA release upon hormone binding through the single-particle cryo-electron microscopy structure of an MmfR–operator complex. DNA binding and release assays with MmfR mutants and synthetic AHFCA analogues define the role of individual amino acid residues and hormone functional groups in ligand recognition and DNA release. These findings will facilitate the exploitation of actinobacterial hormones and their associated TFTRs in synthetic biology and in the discovery of new antibiotics