Jesús F. Aparicio

A complex multienzyme system encoded by five polyketide synthase genes is involved in the biosynthesis of the 26-membered polyene macrolide pimaricin in Streptomyces natalensis

BACKGROUND Polyene macrolides are a class of large macrocyclic polyketides that interact with membrane sterols, having antibiotic activity against fungi but not bacteria. Their rings include a chromophore of 3-7 conjugated double bonds which constitute the distinct polyene structure. Pimaricin is an archetype polyene, important in the food industry as a preservative to prevent mould contamination of foods, produced by Streptomyces natalensis. We set out to clone, sequence and analyse the gene cluster responsible for the biosynthesis of this tetraene. RESULTS A large cluster of 16 open reading frames spanning 84985 bp of the S. natalensis genome has been sequenced and found to encode 13 homologous sets of enzyme activities (modules) of a polyketide synthase (PKS) distributed within five giant multienzyme proteins (PIMS0-PIMS4). The total of 60 constituent active sites, 25 of them on a single enzyme (PIMS2), make this an exceptional multienzyme system. Eleven additional genes appear to govern modification of the polyketide-derived framework and export. Disruption of the genes encoding the PKS abolished pimaricin production. CONCLUSIONS The overall architecture of the PKS gene cluster responsible for the biosynthesis of the 26-membered polyene macrolide pimaricin has been determined. Eleven additional tailoring genes have been cloned and analysed. The availability of the PKS cluster will facilitate the generation of designer pimaricins by combinatorial biosynthesis approaches. This work represents the extensive description of a second polyene macrolide biosynthetic gene cluster after the one for the antifungal nystatin.

The Biosynthetic Gene Cluster for the 26-Membered Ring Polyene Macrolide Pimaricin A NEW POLYKETIDE SYNTHASE ORGANIZATION ENCODED BY TWO SUBCLUSTERS SEPARATED BY FUNCTIONALIZATION GENES

The biosynthetic gene cluster for the 26-membered ring of the polyene macrolide pimaricin extends for about 110 kilobase pairs of contiguous DNA in the genome of Streptomyces natalensis. Two sets of polyketide synthase (PKS) genes are separated by a group of small polyketide-functionalizing genes. Two of the polyketide synthase genes, pimS0 and pimS1, have been fully sequenced and disrupted proving the involvement of each of these genes in pimaricin biosynthesis. The pimS0 gene encodes a relatively small acetate-activating PKS (∼193 kDa) that appears to work as a loading protein which “presents” the starter unit to the second PKS subunit. The pimS1 gene encodes a giant multienzyme (∼710 kDa) harboring 15 activities responsible for the first four cycles of chain elongation in pimaricin biosynthesis, resulting in formation of the polyene chromophore.

Engineered biosynthesis of novel polyenes: a pimaricin derivative produced by targeted gene disruption in Streptomyces natalensis

BACKGROUND The post-polyketide synthase biosynthetic tailoring of polyene macrolides usually involves oxidations catalysed by cytochrome P450 monooxygenases (P450s). Although members from this class of enzymes are common in macrolide biosynthetic gene clusters, their specificities vary considerably toward the substrates utilised and the positions of the hydroxyl functions introduced. In addition, some of them may yield epoxide groups. Therefore, the identification of novel macrolide monooxygenases with activities toward alternative substrates, particularly epoxidases, is a fundamental aspect of the growing field of combinatorial biosynthesis. The specific alteration of these activities should constitute a further source of novel analogues. We investigated this possibility by directed inactivation of one of the P450s belonging to the biosynthetic gene cluster of an archetype polyene, pimaricin. RESULTS A recombinant mutant of the pimaricin-producing actinomycete Streptomyces natalensis produced a novel pimaricin derivative, 4,5-deepoxypimaricin, as a major product. This biologically active product resulted from the phage-mediated targeted disruption of the gene pimD, which encodes the cytochrome P450 epoxidase that converts deepoxypimaricin into pimaricin. The 4,5-deepoxypimaricin has been identified by mass spectrometry and nuclear magnetic resonance following high-performance liquid chromatography purification. CONCLUSIONS We have demonstrated that PimD is the epoxidase responsible for the conversion of 4,5-deepoxypimaricin to pimaricin in S. natalensis. The metabolite accumulated by the recombinant mutant, in which the epoxidase has been knocked out, constitutes the first designer polyene obtained by targeted manipulation of a polyene biosynthetic gene cluster. This novel epoxidase could prove to be valuable for the introduction of epoxy substituents into designer macrolides.

Identification of PimR as a Positive Regulator of Pimaricin Biosynthesis in Streptomyces natalensis

Sequencing of the DNA region on the left fringe of the pimaricin gene cluster revealed the presence of a 3.6-kb gene, pimR, whose deduced product (1,198 amino acid residues) was found to have amino acid sequence homology with bacterial regulatory proteins. Database comparisons revealed that PimR represents the archetype of a new class of regulators, combining a Streptomyces antibiotic regulatory protein (SARP)-like N-terminal section with a C-terminal half homologous to guanylate cyclases and large ATP-binding regulators of the LuxR family. Gene replacement of pimR from Streptomyces natalensis chromosome results in a complete loss of pimaricin production, suggesting that PimR is a positive regulator of pimaricin biosynthesis. Gene expression analysis by reverse transcriptase PCR (RT-PCR) of the pimaricin gene cluster revealed that S. natalensis DeltaPimR shows no expression at all of the cholesterol oxidase-encoding gene pimE, and very low level transcription of the remaining genes of the cluster except for the mutant pimR gene, thus demonstrating that this regulator activates the transcription of all the genes belonging to the pimaricin gene cluster but not its own transcription.

Characterization of the polyene macrolide P450 epoxidase from Streptomyces natalensis that converts de-epoxypimaricin into pimaricin

The biosynthesis of the antifungal agent pimaricin by Streptomyces natalensis has been proposed to involve a cytochrome P450 encoded by the gene pimD. Pimaricin is derived from its immediate precursor de-epoxypimaricin by epoxidation of the C-4-C-5 double bond on the macrolactone ring. We have overproduced PimD with a N-terminal His6 affinity tag in Escherichia coli and purified the enzyme for kinetic analysis. The protein showed a reduced CO-difference spectrum with a Soret maximum at 450 nm, indicating that it is a cytochrome P450. Purified PimD was shown to catalyse the in vitro C-4-C-5 epoxidation of 4,5-de-epoxypimaricin to pimaricin. The enzyme was dependent on NADPH for activity with optimal pH at 7.5, and the temperature optimum was 30 degrees C. The kcat value for the epoxidation of de-epoxypimaricin was similar to the values reported for other macrolide oxidases. Enzyme activity was inhibited at high substrate concentration. This is the first time that a polyene macrolide P450 mono-oxygenase has been expressed heterologously and studied. The unique specificity of this epoxidase should be useful for the oxidative modification of novel polyene macrolide antibiotics.

Molecular Control of Polyene Macrolide Biosynthesis DIRECT BINDING OF THE REGULATOR PimM TO EIGHT PROMOTERS OF PIMARICIN GENES AND IDENTIFICATION OF BINDING BOXES

Control of polyene macrolide production in Streptomyces natalensis is mediated by the transcriptional activator PimM. This regulator, which combines an N-terminal PAS domain with a C-terminal helix-turn-helix motif, is highly conserved among polyene biosynthetic gene clusters. PimM, truncated forms of the protein without the PAS domain (PimMΔPAS), and forms containing just the DNA-binding domain (DBD) (PimMDBD) were overexpressed in Escherichia coli as GST-fused proteins. GST-PimM binds directly to eight promoters of the pimaricin cluster, as demonstrated by electrophoretic mobility shift assays. Assays with truncated forms of the protein revealed that the PAS domain does not mediate specificity or the distinct recognition of target genes, which rely on the DBD domain, but significantly reduces binding affinity up to 500-fold. Transcription start points were identified by 5′-rapid amplification of cDNA ends, and the binding regions of PimMDBD were investigated by DNase I protection studies. In all cases, binding took place covering the −35 hexamer box of each promoter, suggesting an interaction of PimM and RNA polymerase to cause transcription activation. Information content analysis of the 16 sequences protected in target promoters was used to deduce the structure of the PimM-binding site. This site displays dyad symmetry, spans 14 nucleotides, and adjusts to the consensus TVGGGAWWTCCCBA. Experimental validation of this binding site was performed by using synthetic DNA duplexes. Binding of PimM to the promoter region of one of the polyketide synthase genes from the Streptomyces nodosus amphotericin cluster containing the consensus binding site was also observed, thus proving the applicability of the findings reported here to other antifungal polyketides.

Functional conservation of PAS–LuxR transcriptional regulators in polyene macrolide biosynthesis

Control of polyene macrolide production in Streptomyces natalensis is mediated by the PAS-LuxR transcriptional activator PimM. Expression of target genes in this strain is positively regulated by binding of the regulator to 14-nucleotide sites showing dyad symmetry, and overlapping the -35 element of each promoter. These sequences have been found in the upstream regions of genes belonging to different polyene biosynthetic gene clusters. All the sequences in the amphotericin, nystatin, and filipin clusters were cloned and the binding of PimM to all of them has been shown by electrophoretic mobility shift assays. The precise binding regions were investigated by DNaseI protection studies. Results indicated that PAS-luxR regulators share the same regulatory pattern in different polyene-producing strains, these genes being responsible for polyketide chain construction, and when available, the genes for sugar dehydration and attachment, and the ABC transporters, the targets for regulation. Information content analysis of the 24 sequences protected in target promoters was used to refine the information-based model of the binding site. This site now spans 16 nucleotides and adjusts to the consensus CTVGGGAWWTCCCBAG. Gene complementation of S. natalensis ΔpimM with a single copy of heterologous regulators of the PAS/LuxR class integrated into the chromosome, such as amphRIV, nysRIV, or pteF, restored antifungal production, thus proving the functional conservation of these regulators. Introduction of a single copy of pimM into the amphotericin producing strain Streptomyces nodosus, or into the filipin producing strain S. avermitilis, boosted the production of both polyenes, thus indicating that the expression of the PAS-LuxR regulator constitutes a bottleneck in the biosynthesis of the antifungal, and also that these regulators are fully exchangeable. This work is the first report of a general mechanism regulating polyene production.

Structure of Cytochrome P450 PimD Suggests Epoxidation of the Polyene Macrolide Pimaricin Occurs via a Hydroperoxoferric Intermediate

We present the X-ray structure of PimD, both substrate-free and in complex with 4,5-desepoxypimaricin. PimD is a cytochrome P450 monooxygenase with native epoxidase activity that is critical in the biosynthesis of the polyene macrolide antibiotic pimaricin. Intervention in this secondary metabolic pathway could advance the development of drugs with improved pharmacologic properties. Epoxidation by P450 typically includes formation of a charge-transfer complex between an oxoferryl pi-cation radical species (Compound I) and the olefin pi-bond as the initial intermediate. Catalytic and structural evidence presented here suggest that epoxidation of 4,5-desepoxypimaricin proceeds via a hydroperoxoferric intermediate (Compound 0). The oxygen atom of Compound 0 distal to the heme iron may insert into the double bond of the substrate to make an epoxide ring. Stereoelectronic features of the putative transition state suggest substrate-assisted proton delivery.

Microbial cholesterol oxidases: bioconversion enzymes or signal proteins?

Cholesterol oxidases (3beta-hydroxysterol oxidases; EC 1.1.3.6), serve as catalysts for the initial step in the degradation of cholesterol, and probably other natural sterols, that are used as carbon sources for growth of different bacteria. Because of their suitability for attacking cholesterol they have been widely used for the quantification of cholesterol in clinical and food specimens. Cholesterol oxidase has also found application as a probe for membrane structure, as an insecticide, and has been implicated in bacterial pathogenesis. Recently, we have found that a Streptomyces cholesterol oxidase is required for the biosynthesis of the antifungal polyene pimaricin, apparently acting as an antifungal sensor. Here we describe our current understanding of these fascinating enzymes.

PimT, an amino acid exporter controls polyene production via secretion of the quorum sensing pimaricin-inducer PI-factor in Streptomyces natalensis

BackgroundPolyenes represent a major class of antifungal agents characterised by the presence of a series of conjugated double bonds in their planar hydroxylated macrolide ring structure. Despite their general interest, very little is known about the factors that modulate their biosynthesis. Among these factors, we have recently discovered a new inducing compound (PI-factor) in the pimaricin producer Streptomyces natalensis, which elicits polyene production in a manner characteristic of quorum sensing. Here, we describe the involvement of an amino-acid exporter from S. natalensis in modulating the expression of pimaricin biosynthetic genes via secretion of the quorum-sensing pimaricin-inducer PI-factor.ResultsAdjacent to the pimaricin gene cluster lies a member of the RhtB family of amino-acid exporters. Gene deletion and complementation experiments provided evidence for a role for PimT in the export of L-homoserine, L-serine, and L-homoserine lactone. Expression of the gene was shown to be induced by homoserine and by the quorum-sensing pimaricin-inducer PI-factor. Interestingly, the mutant displayed 65% loss of pimaricin production, and also 50% decrease in the production of PI, indicating that PimT is used as PI-factor exporter, and suggesting that the effect in antifungal production might be due to limited secretion of the inducer.ConclusionThis report describes the involvement of an amino acid exporter (encoded by pimT in the vicinity of the pimaricin cluster) in modulating the expression of antibiotic biosynthetic genes via secretion of the quorum-sensing pimaricin-inducer PI-factor. The discovery of the participation of amino acid exporters in a signal transduction cascade for the production of polyene macrolides is unexpected, and represents an important step forward towards understanding the regulatory network for polyene regulation. Additionally, this finding constitutes the first detailed characterization of an amino-acid exporter in an Actinomycete, and to our knowledge, the first evidence for the implication of this type of exporters in quorum sensing.