Identification of a gene cluster that directs putrebactin biosynthesis in Shewanella species: PubC catalyzes cyclodimerization of N-hydroxy-N-succinylputrescine

Putrebactin is a dihydroxamate iron chelator produced by the metabolically versatile marine bacterium Shewanella putrefaciens. It is a macrocyclic dimer of N-hydroxy-N-succinyl-putrescine (HSP) and is structurally related to desferrioxamine E, which is a macrocyclic trimer of N-hydroxy-N-succinyl-cadaverine (HSC). We recently showed that DesD, a member of the NIS synthetase superfamily, catalyzes the key step in desferrioxamine E biosynthesis: ATP-dependent trimerisation and macrocylization of HSC. Here we report identification of a conserved gene cluster in the sequenced genomes of several Shewanella species, including Shewanella putrefaciens, which is hypothesized to direct putrebactin biosynthesis from putrescine, succinyl-CoA and molecular oxygen. The pubC gene within this gene cluster encodes a protein with 65% similarity to DesD. We overexpressed pubC from Shewanella species MR-4 and MR-7 in E. coli. The resulting His6-PubC fusion proteins were purified by Ni-NTA affinity and gel filtration chromatography. The recombinant proteins were shown to catalyze ATP-dependent cyclodimerization of HSP to form putrebactin. The uncyclized dimer of HSP pre-putrebactin was shown to be an intermediate in the conversion of two molecules of HSP to putrebactin. The data indicate that pre-putrebactin is converted to putrebactin via PubC-catalyzed activation of the carboxyl group by adenylation, followed by PubC-catalyzed nucleophilic attack of the amino group on the carbonyl carbon of the acyl adenylate. This mechanism for macrocycle formation is very different from the mechanism involved in the biosynthesis of many other macrocyclic natural products, where already-activated acyl thioesters are converted by thioesterase domains of polyketide synthases and nonribosomal peptide synthetases to macrocycles via covalent enzyme bound intermediates. The results of this study demonstrate that two closely related enzymes, PubC and DesD, catalyze specific cyclodimerization and cyclotrimerization reactions, respectively, of structurally similar substrates, raising intriguing questions regarding the molecular mechanism of specificity.     

NMR confirmation that tryptophan dehydrogenation occurs with syn stereochemistry during the biosynthesis of CDA in Streptomyces coelicolor

Doubly labeled (2'S,3'R)-[3'-2H1,13C1]-tryptophan was fed to the Trp-His auxotrophic Streptomyces coelicolor strain WH101. Mass spectrometry showed single and double incorporation of the labeled Trp into the calcium-dependent lipopeptide antibiotic (CDA4a). From 13C NMR spectroscopy, it was apparent that the C3'-signal of the (Z)-2',3'-dehydrotryptophan (position 11 in CDA4) was a 1:1:1 triplet indicating that the deuterium atom in the pro-R position of the methylene group is retained during Trp-oxidation. This provides definitive proof that Trp dehydrogenation occurs through the loss of the 2' and pro-3'S hydrogen atoms with overall syn stereochemistry.     

Divalent Transition‐Metal‐Ion Stress Induces Prodigiosin Biosynthesis in Streptomyces coelicolor M145: Formation of Coeligiosins

The bacterium Streptomyces coelicolor M145 reacts to transition‐metal‐ion stress with myriad growth responses, leading to different phenotypes. In particular, in the presence of Co²⁺ ions (0.7 mM ) S. coelicolor consistently produced a red phenotype. This phenotype, when compared to the wild type, differed strongly in its production of volatile compounds as well as high molecular weight secondary metabolites. LC‐MS analysis revealed that in the red phenotype the production of the prodigiosins, undecylprodigiosin and streptorubin B, was strongly induced and, in addition, several intense signals appeared in the LC‐MS chromatogram. Using LC‐MS/MS and NMR spectroscopy, two new prodigiosin derivatives were identified, that is, coeligiosin A and B, which contained an additional undecylpyrrolyl side chain attached to the central carbon of the tripyrrole ring system of undecylprodigiosin or streptorubin B. This example demonstrates that environmental factors such as heavy metal ion stress can not only induce the production of otherwise not observed metabolites from so called sleeping genes but alter the products from well‐studied biosynthetic pathways.     

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.     

Biochemistry and molecular genetics of the biosynthesis of the earthy odorant methylisoborneol in Streptomyces coelicolor

Methylisoborneol (2) is a volatile organic compound produced by a wide variety of Actinomycete soil organisms, myxobacteria, and cyanobacteria. It has an unusually low odor threshold and, together with geosmin, is responsible for the characteristic smell of moist soil as well as unpleasant taste and odor episodes associated with public water supplies and contamination of various foodstuffs, including fish, wine, and beer. Despite considerable interest in detection and remediation of methylisoborneol, the biosynthesis of this methylated monoterpene has been obscure. In Streptomyces coelicolor, the sco7700 and sco7701 genes are shown to correspond to a two-gene operon responsible for methylisoborneol biosynthesis. Both genes have been amplified by PCR and the resulting DNA has been cloned and expressed in Escherichia coli. Incubation of recombinant SCO7701 protein, annotated as a possible C-methyltransferase, with geranyl diphosphate (1) and S-adenosylmethionine gave the previously unknown compound, (E)-2-methylgeranyl diphosphate (3). Incubation of 3 in the presence of Mg2+ with recombinant SCO7700, previously annotated only as a possible metal-binding protein or terpenoid synthase, resulted in the formation of 2-methylisoborneol (2). The steady-state kinetic parameters for both biochemical reactions have been determined. Incubation of geranyl diphosphate and S-adenosylmethionine with a mixture of both SCO7700 and SCO7701 resulted in formation of methylisoborneol (2). Cyclization of 2-methylgeranyl diphosphate (3) to methylisoborneol (2) likely involves the intermediacy of 2-methyllinalyl diphosphate.     

Structure,biosynthetic origin,and engineered biosynthesis of calcium-dependent antibiotics from Streptomyces coelicolor

The calcium-dependent antibiotic (CDA), from Streptomyces coelicolor, is an acidic lipopeptide comprising an N-terminal 2,3-epoxyhexanoyl fatty acid side chain and several nonproteinogenic amino acid residues. S. coelicolor grown on solid media was shown to produce several previously uncharacterized peptides with C-terminal Z-dehydrotryptophan residues. The CDA biosynthetic gene cluster contains open reading frames encoding nonribosomal peptide synthetases, fatty acid synthases, and enzymes involved in precursor supply and tailoring of the nascent peptide. On the basis of protein sequence similarity and chemical reasoning, the biosynthesis of CDA is rationalized. Deletion of SCO3229 (hmaS), a putative 4-hydroxymandelic acid synthase-encoding gene, abolishes CDA production. The exogenous supply of 4-hydroxymandelate, 4-hydroxyphenylglyoxylate, or 4-hydroxyphenylglycine re-establishes CDA production by the DeltahmaS mutant. Feeding analogs of these precursors to the mutant resulted in the directed biosynthesis of novel lipopeptides with modified arylglycine residues.     

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 an 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.     

Spectroscopic characterization of a high-potential lipo-cupredoxin found in Streptomyces coelicolor

For many streptomycetes, a distinct dependence on the "bioavailability" of copper ions for their morphological development has been reported. Analysis of the Streptomyces coelicolor genome reveals a number of gene products encoding for putative copper-binding proteins. One of these appears as an unusual copper-binding protein with a lipoprotein signal sequence and a cupredoxin-like domain harboring a putative Type-1 copper-binding motif. Cloning of this gene from S. coelicolor and subsequent heterologous expression in Escherichia coli has allowed for a thorough spectroscopic interrogation of this putative copper-binding protein. Optical and electron paramagnetic resonance spectroscopies have confirmed the presence of a "classic" Type-1 copper site with the axial ligand to the copper a methionine. Paramagnetic NMR spectroscopy on both the native Cu(II) form and Co(II)-substituted protein has yielded active-site structural information, which on comparison with that of other cupredoxin active sites reveals metal-ligand interactions most similar to the "classic" Type-1 copper site found in the amicyanin family of cupredoxins. Despite this high structural similarity, the Cu(II)/(I) midpoint potential of the S. coelicolor protein is an unprecedented +605 mV vs normal hydrogen electrode at neutral pH (amicyanin approximately +250 mV), with no active-site protonation of the N-terminal His ligand observed. Suggestions for the physiological role/function of this high-potential cupredoxin are discussed.     

Analysis of the prodiginine biosynthesis gene cluster of Streptomyces coelicolor A3(2): new mechanisms for chain initiation and termination in modular multienzymes

BACKGROUND: Prodiginines are a large family of pigmented oligopyrrole antibiotics with medicinal potential as immunosuppressants and antitumour agents that are produced by several actinomycetes and other eubacteria. Recently, a gene cluster in Streptomyces coelicolor encoding the biosynthesis of undecylprodiginine and butyl-meta-cycloheptylprodiginine has been sequenced. RESULTS: Using sequence comparisons, functions have been assigned to the majority of the genes in the cluster, several of which encode homologues of enzymes involved in polyketide, non-ribosomal peptide, and fatty acid biosynthesis. Based on these assignments, a complete pathway for undecylprodiginine and butyl-meta-cycloheptylprodiginine biosynthesis in S. coelicolor has been deduced. Gene knockout experiments have confirmed the deduced roles of some of the genes in the cluster. CONCLUSIONS: The analysis presented provides a framework for a general understanding of the genetics and biochemistry of prodiginine biosynthesis, which should stimulate rational approaches to the engineered biosynthesis of novel prodiginines with improved immunosuppressant or antitumour activities. In addition, new mechanisms for chain initiation and termination catalysed by hitherto unobserved domains in modular multienzyme systems have been deduced.     

Multiple regulatory genes in the tylosin biosynthetic cluster of Streptomyces fradiae.

BACKGROUND: The macrolide antibiotic tylosin is composed of a polyketide lactone substituted with three deoxyhexose sugars. In order to produce tylosin efficiently, Streptomyces fradiae presumably requires control mechanisms that balance the yields of the constituent metabolic pathways together with switches that allow for temporal regulation of antibiotic production. In addition to possible metabolic feedback and/or other signalling devices, such control probably involves interplay between specific regulatory proteins. Prior to the present work, however, no candidate regulatory gene(s) had been identified in S. fradiae. RESULTS: DNA sequencing has shown that the tylosin biosynthetic gene cluster, within which four open reading frames utilise the rare TTA codon, contains at least five candidate regulatory genes, one of which (tylP) encodes a gamma-butyrolactone signal receptor for which tylQ is a probable target. Two other genes (tylS and tylT) encode pathway-specific regulatory proteins of the Streptomyces antibiotic regulatory protein (SARP) family and a fifth, tylR, has been shown by mutational analysis to control various aspects of tylosin production. CONCLUSIONS: The tyl genes of S. fradiae include the richest collection of regulators yet encountered in a single antibiotic biosynthetic gene cluster. Control of tylosin biosynthesis is now amenable to detailed study, and manipulation of these various regulatory genes is likely to influence yields in tylosin-production fermentations.     

The gene cluster for fluorometabolite biosynthesis in Streptomyces cattleya: a thioesterase confers resistance to fluoroacetyl-coenzyme A

A genomic library of Streptomyces cattleya was screened to isolate a gene cluster encoding enzymes responsible for the production of fluorine-containing metabolites. In addition to the previously described fluorinase FlA which catalyzes the formation of 5'-fluoro-5'-deoxyadenosine from S-adenosylmethionine and fluoride, 11 other putative open reading frames have been identified. Three of the proteins encoded by these genes have been characterized. FlB was determined to be the second enzyme in the pathway, catalyzing the phosphorolytic cleavage of 5'-fluoro-5'-deoxyadenosine to produce 5-fluoro-5-deoxy-D-ribose-1-phosphate. The enzyme FlI was found to be an S-adenosylhomocysteine hydrolase, which may act to relieve S-adenosylhomocysteine inhibition of the fluorinase. Finally, flK encodes a thioesterase which catalyzes the selective breakdown of fluoroacetyl-CoA but not acetyl-CoA, suggesting that it provides the producing strain with a mechanism for resistance to fluoroacetate.     

Elucidation of the Streptomyces coelicolor pathway to 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde, an intermediate in prodiginine biosynthesis

The biosynthetic pathway to 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde (MBC), a key intermediate in the biosynthesis of prodiginine antibiotics in Streptomyces coelicolor, has been elucidated using a combination of gene replacements and feeding experiments with chemically synthesised MBC and a synthetic analogue of a pathway intermediate.     

A gene cluster from a marine Streptomyces encoding the biosynthesis of the aromatic spiroketal polyketide griseorhodin A

The telomerase inhibitor griseorhodin A is probably the most heavily oxidized bacterial polyketide known and features a unique epoxyspiroketal moiety crucial for its activity. To gain insight into which tailoring enzymes generate this pharmacophore, we have cloned and fully sequenced the griseorhodin biosynthesis gene cluster. Among other unusual features, this aromatic polyketide synthase (PKS) system encodes an unprecedented number of functionally diverse oxidoreductases, which are involved in the oxidative modification of a polyaromatic tridecaketide precursor by cleavage of three carbon-carbon bonds. The cluster was highly unstable on a variety of shuttle plasmids but could finally be functionally expressed in its entirety in Streptomyces lividans using a novel integrative cosmid vector. The availability of the tailoring system now opens up the possibility of engineering nonnatural biosynthetic pathways yielding novel pharmacologically active analogs with a similar pharmacophore.     

Insights about the biosynthesis of the avermectin deoxysugar L-oleandrose through heterologous expression of Streptomyces avermitilis deoxysugar genes in Streptomyces lividans.

BACKGROUND: The avermectins, produced by Streptomyces avermitilis, are potent anthelminthic agents with a polyketide-derived macrolide skeleton linked to a disaccharide composed of two alpha-linked L-oleandrose units. Eight contiguous genes, avrBCDEFGHI (also called aveBI-BVIII), are located within the avermectin-producing gene cluster and have previously been mapped to the biosynthesis and attachment of thymidinediphospho-oleandrose to the avermectin aglycone. This gene cassette provides a convenient way to study the biosynthesis of 2,6-dideoxysugars, namely that of L-oleandrose, and to explore ways to alter the biosynthesis and structures of the avermectins by combinatorial biosynthesis. RESULTS: A Streptomyces lividans strain harboring a single plasmid with the avrBCDEFGHI genes in which avrBEDC and avrIHGF were expressed under control of the actI and actIII promoters, respectively, correctly glycosylated exogenous avermectin A1a aglycone with identical oleandrose units to yield avermectin A1a. Modified versions of this minimal gene set produced novel mono- and disaccharide avermectins. The results provide further insight into the biosynthesis of L-oleandrose. CONCLUSIONS: The plasmid-based reconstruction of the avr deoxysugar genes for expression in a heterologous system combined with biotransformation has led to new information about the mechanism of 2,6-deoxysugar biosynthesis. The structures of the di-demethyldeoxysugar avermectins accumulated indicate that in the oleandrose pathway the stereochemistry at C-3 is ultimately determined by the 3-O-methyltransferase and not by the 3-ketoreductase or a possible 3,5-epimerase. The AvrF protein is therefore a 5-epimerase and not a 3,5-epimerase. The ability of the AvrB (mono-)glycosyltransferase to accommodate different deoxysugar intermediates is evident from the structures of the novel avermectins produced.     

A complete gene cluster from Streptomyces nanchangensis NS3226 encoding biosynthesis of the polyether ionophore nanchangmycin

The PKS genes for biosynthesis of the polyether nanchangmycin are organized to encode two sets of proteins (six and seven ORFs, respectively), but are separated by independent ORFs that encode an epimerase, epoxidase, and epoxide hydrolase, and, notably, an independent ACP. One of the PKS modules lacks a corresponding ACP. We propose that the process of oxidative cyclization to form the polyether structure occurs when the polyketide chain is still anchored on the independent ACP before release. 4-O-methyl-L-rhodinose biosynthesis and its transglycosylation involve four putative genes, and regulation of nanchangmycin biosynthesis seems to involve activation as well as repression. In-frame deletion of a KR6 domain generated the nanchangmycin aglycone with loss of 4-O-methyl-L-rhodinose and antibacterial activity, in agreement with the assignments of the PKS domains catalyzing specific biosynthetic steps.     

Genome mining in Streptomyces coelicolor: molecular cloning and characterization of a new sesquiterpene synthase

The terpene synthase encoded by the SCO5222 (SC7E4.19) gene of Streptomyces coelicolor was cloned by PCR and expressed in Escherichia coli as an N-terminal-His6-tag protein. Incubation of the recombinant protein, SCO5222p, with farnesyl diphosphate (1, FPP) in the presence of Mg(II) gave a new sesquiterpene, (+)-epi-isozizaene (2), whose structure and stereochemistry were determined by a combination of 1H, 13C, COSY, HMQC, HMBC, and NOESY NMR. The steady-state kinetic parameters were kcat 0.049 +/- 0.001 s-1 and a Km (FPP) of 147 +/- 14 nM. Individual incubations of recombinant epi-isozizaene synthase with [1,1-2H2]FPP (1a), (1R)-[1-2H]-FPP (1b), and (1S)-[1-2H]-FPP (1c) and NMR analysis of the resulting deuterated epi-isozizaenes supported an isomerization-cyclization-rearrangement mechanism involving the intermediacy of (3R)-nerolidyl diphosphate (3).     

Insights into polyether biosynthesis from analysis of the nigericin biosynthetic gene cluster in Streptomyces sp. DSM4137

Nigericin was among the first polyether ionophores to be discovered, but its biosynthesis remains obscure. The biosynthetic gene cluster for nigericin has been serendipitously cloned from Streptomyces sp. DSM4137, and deletion of this gene cluster abolished the production of both nigericin and the closely related metabolite abierixin. Detailed comparison of the nigericin biosynthetic genes with their counterparts in the biosynthetic clusters for other polyketides has prompted a significant revision of the proposed common pathway for polyether biosynthesis. In particular, we present evidence that in nigericin, nanchangmycin, and monensin, an unusual ketosynthase-like protein, KSX, transfers the initially formed linear polyketide chain to a discrete acyl carrier protein, ACPX, for oxidative cyclization. Consistent with this, deletion of either monACPX or monKSX from the monensin gene cluster effectively abolished monensin A biosynthesis.     

Amphotericin biosynthesis in Streptomyces nodosus: deductions from analysis of polyketide synthase and late genes.

BACKGROUND: The polyene macrolide amphotericin B is produced by Streptomyces nodosus ATCC14899. Amphotericin B is a potent antifungal antibiotic and has activity against some viruses, protozoans and prions. Treatment of systemic fungal infections with amphotericin B is complicated by its low water-solubility and side effects which include severe nephrotoxicity. Analogues with improved properties could be generated by manipulating amphotericin biosynthetic genes in S. nodosus. RESULTS: A large polyketide synthase gene cluster was cloned from total cellular DNA of S. nodosus. Nucleotide sequence analysis of 113193 bp of this region revealed six large polyketide synthase genes as well as genes for two cytochrome P450 enzymes, two ABC transporter proteins, and genes involved in biosynthesis and attachment of mycosamine. Phage KC515-mediated gene disruption was used to show that this region is involved in amphotericin production. CONCLUSIONS: The availability of these genes and the development of a method for gene disruption and replacement in S. nodosus should allow production of novel amphotericins. A panel of analogues could lead to identification of derivatives with increased solubility, improved biological activity and reduced toxicity.     

A gene cluster encoding resistomycin biosynthesis in Streptomyces resistomycificus; exploring polyketide cyclization beyond linear and angucyclic patterns

Resistomycin is a pentacyclic polyketide metabolite of Streptomyces resistomycificus that exhibits a variety of pharmacologically relevant properties. While virtually all bacterial aromatic polyketides can be grouped into linear and angular polyphenols, resistomycin has a unique "discoid" ring system. We have successfully identified the entire gene cluster encoding resistomycin biosynthesis by heterologously expressing a pooled cosmid library and screening for the fluorescence of the metabolite produced. The rem gene cluster exhibits several unusual features of the type II PKS involved, most remarkably a putative MCAT with highest homology to AT domains from modular PKSs. In addition, we provide the first insight into the molecular basis of a unique mode of cyclization giving rise to a discoid polyketide.     

Identification of 5-fluoro-5-deoxy-D-ribose-1-phosphate as an intermediate in fluorometabolite biosynthesis in Streptomyces cattleya

5'-Fluoro-5'-deoxy-D-ribose-1-phosphate (FDRP) is identified as a biosynthetic intermediate during fluorometabolite biosynthesis in Streptomyces cattleya.