Novel insights into siderophore formation in myxobacteria

The myxochelins are catecholate-type siderophores produced by a number of myxobacterial strains, and their corresponding biosynthetic gene clusters have been identified in Stigmatella aurantiaca Sg a15, and Sorangium cellulosum So ce56; the latter being presented in this work. Biochemical and genetic studies described here further clarify myxochelin biosynthesis. In addition to the myxochelin A biosynthetic complex, the aminotransferase MxcL is required in order to form myxochelin B, starting from 2,3-dihydroxy benzoic acid and L-lysine. Additionally, the substrate specificity of the myxochelin A biosynthetic complex was analyzed in vitro; this led to the formation of novel myxochelin derivatives. Furthermore, MxcD was over-expressed and its function as an active isochorismic acid synthase in Escherichia coli was verified by complementation studies, as was activity in vitro. The organization of the myxochelin gene cluster of S. cellulosum So ce56 was compared to that of the Sg a15 gene cluster. The comparison revealed that although the organization of the biosynthetic genes is completely different, the biosynthesis is most probably extremely similar.     

SorF: a glycosyltransferase with promiscuous donor substrate specificity in vitro

Glycosylations are well-established steps in numerous biosynthetic pathways, and the attached sugar moieties often influence the specificity or pharmacology of the modified compounds. The sorangicins belong to the polyketide family of natural products, and exhibit antibiotic activity through inhibition of bacterial RNA polymerase. We have identified the sorangicin biosynthetic gene cluster in the producing myxobacterium Sorangium cellulosum So ce12. Within the cluster, sorF encodes a putative glycosyltransferase. To determine its function in sorangicin biosynthesis, SorF was heterologously expressed as a fusion protein in Escherichia coli. After purification by affinity chromatography, SorF was found to glucosylate sorangicin A in vitro, utilizing UDP-alpha-D-glucose as the natural donor substrate. Additionally, SorF showed high flexibility towards further UDP- and dTDP-sugars and was able to transfer several other sugar moieties-alpha-D-galactose, alpha-D-xylose, beta-L-rhamnose, and 6-deoxy-4-keto-alpha-D-glucose-onto the aglycon. SorF is therefore one of the rare glycosyltransferases able to transfer both D- and L-sugars, and could thus be used to generate novel sorangiosides.     

Characterization of the Gene Cluster CYP264B1‐geoA from Sorangium cellulosum So ce56: Biosynthesis of (+)‐Eremophilene and Its Hydroxylation

Terpenoids can be found in almost all forms of life; however, the biosynthesis of bacterial terpenoids has not been intensively studied. This study reports the identification and functional characterization of the gene cluster CYP264B1–geoA from Sorangium cellulosum So ce56. Expression of the enzymes and synthesis of their products for NMR analysis and X‐ray diffraction were carried out by employing an Escherichia coli whole‐cell conversion system that provides the geoA substrate farnesyl pyrophosphate through simultaneous overexpression of the mevalonate pathway genes. The geoA product was identified as a novel sesquiterpene, and assigned NMR signals unambiguously proved that geoA is an (+)‐eremophilene synthase. The very tight binding of (+)‐eremophilene (～0.40 μM ), which is also available in S. cellulosum So ce56, and its oxidation by CYP264B1 suggest that the CYP264B1–geoA gene cluster is required for the biosynthesis of (+)‐eremophilene derivatives.     

Reporter-Guided Transposon Mutant Selection for Activation of Silent Gene Clusters in Burkholderia thailandensis

Most natural product biosynthetic gene clusters that can be observed bioinformatically are silent. This insight has prompted the development of several methodologies for inducing their expression. One of the more recent methods, termed reporter-guided mutant selection (RGMS), entails creation of a library of mutants that is then screened for the desired phenotype via reporter gene expression. Herein, we apply a similar approach to Burkholderia thailandensis and, using transposon mutagenesis, mutagenize three strains, each carrying a fluorescent reporter in the malleilactone (mal), capistruin (cap), or an unidentified ribosomal peptide (tomm) gene cluster. We show that even a small library of <500 mutants can be used to induce expression of each cluster. We also explore the mechanism of activation and find that inhibition of pyrimidine biosynthesis is linked to the induction of the mal cluster. Both a transposon insertion into pyrF as well as small-molecule-mediated inhibition of PyrF trigger malleilactone biosynthesis. Our results pave the way toward the broad application of RGMS and related approaches to Burkholderia spp.     

Cloning and Heterologous Expression of the Aurachin RE Biosynthesis Gene Cluster Afford a New Cytochrome P450 for Quinoline N‐Hydroxylation

Aurachin RE is a prenylated quinoline antibiotic that was first isolated from the genus Rhodococcus. It shows potent antibacterial activity against a variety of Gram‐positive bacteria. Here we have identified a minimal biosynthesis gene cluster for aurachin RE in Rhodococcus erythropolis JCM 6824 by using random transposon mutagenesis and heterologous production. The Rhodococcus aurachin (rau) gene cluster consists of genes encoding cytochrome P450 (rauA), prenyltransferase, polyketide synthase, and farnesyl pyrophosphate synthase, as well as others including genes involved in regulation and transport. Markerless gene disruption of rauA resulted in the complete loss of aurachin RE production and in the accumulation of a new aurachin derivative lacking the N‐hydroxy group. When the recombinant RauA was expressed in Escherichia coli, it catalyzed N‐hydroxylation of the derivative to form aurachin RE. This study establishes the biosynthetic pathway of aurachin RE and provides experimental evidence for the role of P450 RauA in catalyzing N‐hydroxylation of the quinoline ring, which is indispensable for the antibacterial activity of aurachin RE.     

Characterization of an Orphan Type III Polyketide Synthase Conserved in Uncultivated “Entotheonella” Sponge Symbionts

Uncultivated bacterial symbionts from the candidate genus “Entotheonella” have been shown to produce diverse natural products previously attributed to their sponge hosts. In addition to these known compounds, “Entotheonella” genomes contain rich sets of biosynthetic gene clusters that lack identified natural products. Among these is a small type III polyketide synthase (PKS) cluster, one of only three clusters present in all known “Entotheonella” genomes. This conserved “Entotheonella” PKS (cep) cluster encodes the type III PKS CepA and the putative methyltransferase CepB. Herein, the characterization of CepA as an enzyme involved in phenolic lipid biosynthesis is reported. In vitro analysis showed a specificity for alkyl starter substrates and the production of tri- and tetraketide pyrones and tetraketide resorcinols. The conserved distribution of the cep cluster suggests an important role for the phenolic lipid polyketides produced in “Entotheonella” variants.     

A Novel Cold-Adapted Lipase from Sorangium cellulosum Strain So0157-2: Gene Cloning, Expression, and Enzymatic Characterization

Genome sequencing of cellulolytic myxobacterium Sorangium cellulosum reveals many open-reading frames (ORFs) encoding various degradation enzymes with low sequence similarity to those reported, but none of them has been characterized. In this paper, a predicted lipase gene (lipA) was cloned from S. cellulosum strain So0157-2 and characterized. lipA is 981-bp in size, encoding a polypeptide of 326 amino acids that contains the pentapeptide (GHSMG) and catalytic triad residues (Ser114, Asp250 and His284). Searching in the GenBank database shows that the LipA protein has only the 30% maximal identity to a human monoglyceride lipase. The novel lipA gene was expressed in Escherichia coli BL21 and the recombinant protein (r-LipA) was purified using Ni-NTA affinity chromatography. The enzyme hydrolyzed the p-nitrophenyl (pNP) esters of short or medium chain fatty acids (≤C(10)), and the maximal activity was on pNP acetate. The r- LipA is a cold-adapted lipase, with high enzymatic activity in a wide range of temperature and pH values. At 4 °C and 30 °C, the K(m) values of r-LipA on pNP acetate are 0.037 ± 0.001 and 0.174 ± 0.006 mM, respectively. Higher pH and temperature conditions promoted hydrolytic activity toward the pNP esters with longer chain fatty acids. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents. The results suggest that the r-LipA protein has some new characteristics potentially promising for industrial applications and S. cellulosum is an intriguing resource for lipase screening.     

Reassembly of anthramycin biosynthetic gene cluster by using recombinogenic cassettes

The reassembly and heterologous expression of complete gene clusters in shuttle vectors has enabled investigations of several large biosynthetic pathways in recent years. With a gene cluster in a mobile construct, the interrogation of gene functions from both culturable and nonculturable organisms is greatly accelerated and large pathway engineering efforts can be executed to produce "new" natural products. However, the genetic manipulation of complete natural product biosynthetic gene clusters is often complicated by their sheer size (10-200 kbp), which makes standard restriction/ligation-based methods impracticable. To circumvent these problems, alternative recombinogenic methods, which depend on engineered homology-based recombination have recently arisen as a powerful alternative. Here, we describe a new general technique that can be used to reconstruct large biosynthetic pathways from overlapping cosmids by retrofitting each cosmid with a "recombinogenic cassette" that contains a shared homologous element and orthogonal antibiotic markers. We employed this technique to reconstruct the anthramycin biosynthetic gene cluster of the thermotolerant actinomycete Streptomyces refuineus, from two >30 kbp cosmids into a single cosmid and integrate it into the genome of Streptomyces lividans. Anthramycin production in the heterologous Streptomyces host confirmed the integrity of the reconstructed pathway and validated the proposed boundaries of the gene cluster. Notably, anthramycin production by recombinant S. lividans was seen only during growth at high temperature--a property also shown by the natural host. This work provides tools to engineer the anthramycin biosynthetic pathway and to explore the connection between anthramycin production and growth at elevated temperatures.     

New Sesquiterpene Oxidations with CYP260A1 and CYP264B1 from Sorangium cellulosum So ce56

Sesquiterpenes are natural products derived from the common precursor farnesyl pyrophosphate (FPP) but are highly diverse in structure and function. Cytochrome P450 enzymes (P450s) exhibit the unique ability to introduce molecular oxygen into non‐activated C?H bonds. In plant biosynthetic pathways, P450s commonly derivatize sesquiterpene hydrocarbons. However, the potential of bacterial P450s for terpene derivatization is still underinvestigated. This work compares the substrate specificities and regioselectivities of the sesquiterpene hydroxylases CYP260A1 and CYP264B1 from myxobacterium Sorangium cellulosum So ce56. Four tested substrate classes (eremophilanes, humulanes, caryophyllanes, and cedranes) were converted by both P450s. The achievable variety of oxidations is demonstrated on the model substrates (+)‐nootkatone and zerumbone. Increasing the number of functionally investigated P450s, this study represents a step towards the selective derivatization of sesquiterpenes.     

Biosynthesis of Rubellins in Ramularia collo-cygni—Genetic Basis and Pathway Proposition

The important disease Ramularia leaf spot of barley is caused by the fungus Ramularia collo-cygni. The disease causes yield and quality losses as a result of a decrease in photosynthesis efficiency due to the appearance of necrotic spots on the leaf surface. The development of these typical Ramularia leaf spot symptoms is thought to be linked with the release of phytotoxic secondary metabolites called rubellins in the host. However, to date, neither the biosynthetic pathways leading to the production of these metabolites nor their exact role in disease development are known. Using a combined in silico genetic and biochemistry approach, we interrogated the genome of R. collo-cygni to identify a putative rubellin biosynthetic gene cluster. Here we report the identification of a gene cluster containing homologues of genes involved in the biosynthesis of related anthraquinone metabolites in closely related fungi. A putative pathway to rubellin biosynthesis involving the genes located on the candidate cluster is also proposed.     

Cloning and Characterization of the Ravidomycin and Chrysomycin Biosynthetic Gene Clusters

The gene clusters responsible for the biosynthesis of two antitumor antibiotics, ravidomycin and chrysomycin, have been cloned from Streptomyces ravidus and Streptomyces albaduncus, respectively. Sequencing of the 33.28 kb DNA region of the cosmid cosRav32 and the 34.65 kb DNA region of cosChry1‐1 and cosChryF2 revealed 36 and 35 open reading frames (ORFs), respectively, harboring tandem sets of type II polyketide synthase (PKS) genes, D ‐ravidosamine and D ‐virenose biosynthetic genes, post‐PKS tailoring genes, regulatory genes, and genes of unknown function. The isolated ravidomycin gene cluster was confirmed to be involved in ravidomycin biosynthesis through the production of a new analogue of ravidomycin along with anticipated pathway intermediates and biosynthetic shunt products upon heterologous expression of the cosmid, cosRav32, in Streptomyces lividans TK24. The identity of the cluster was further verified through cross complementation of gilvocarcin V (GV) mutants. Similarly, the chrysomycin gene cluster was demonstrated to be indirectly involved in chrysomycin biosynthesis through cross‐complementation of gilvocarcin mutants deficient in the oxygenases GilOII, GilOIII, and GilOIV with the respective chrysomycin monooxygenase homologues. The ravidomycin glycosyltransferase (RavGT) appears to be able to transfer both amino‐ and neutral sugars, exemplified through the structurally distinct 6‐membered D ‐ravidosamine and 5‐membered D ‐fucofuranose, to the coumarin‐based polyketide derived backbone. These results expand the library of biosynthetic genes involved in the biosyntheses of gilvocarcin class compounds that can be used to generate novel analogues through combinatorial biosynthesis.     

The mildiomycin biosynthesis: initial steps for sequential generation of 5-hydroxymethylcytidine 5'-monophosphate and 5-hydroxymethylcytosine in Streptoverticillium rimofaciens ZJU5119

Mildiomycin (MIL) is a peptidyl nucleoside antibiotic with strong activity against powdery mildew disease of plants. We have cloned the MIL biosynthetic gene cluster in Streptoverticillum rimofaciens ZJU5119 and shown that this organism also produces the related antifungal compound, deshydroxymethyl mildiomycin (dHM-MIL). A cosmid genomic library was screened for a putative nucleotide hydrolase gene that is related to blsM from the blasticidin S cluster. Six cosmids were identified that contained a 3.5 kb DNA fragment that harbors a homologue of blsM. The sequence of the fragment revealed two open-reading frames that are likely to function in MIL formation: milA is a CMP hydroxymethylase gene and milB is the homologue of the CMP hydrolase gene blsM. Insertional disruption of milA abolished the production of MIL but not dHM-MIL, whereas a milB knockout strain did not produce either of the peptidyl nucleosides. Recombinant MilA was produced in E. coli and shown to specifically introduce a C-5 hydroxymethyl group on CMP, but it did not accept cytosine or dCMP as a substrate. MilB was also expressed and purified from E. coli and shown to efficiently hydrolyze both hydroxymethyl-CMP (HMCMP) and could accept CMP as an alternative substrate. The ratio of free HMC and cytosine released by MilB was ca. 9:1 in in vitro assays, and is consistent with the higher levels of MIL compared to dHM-MIL that are produced by Streptoverticillum rimofaciens.     

Identifying the Biosynthetic Gene Cluster for Triacsins with an N-Hydroxytriazene Moiety

Triacsins are a family of natural products having in common an N-hydroxytriazene moiety not found in any other known secondary metabolites. Though many studies have examined the biological activity of triacsins in lipid metabolism, their biosynthesis has remained unknown. Here we report the identification of the triacsin biosynthetic gene cluster in Streptomyces aureofaciens ATCC 31442. Bioinformatic analysis of the gene cluster led to the discovery of the tacrolimus producer Streptomyces tsukubaensis NRRL 18488 as a new triacsin producer. In addition to targeted gene disruption to identify necessary genes for triacsin production, stable isotope feeding was performed in vivo to advance the understanding of N-hydroxytriazene biosynthesis.     

Acyltransferase Domain Exchange between Two Independent Type I Polyketide Synthases in the Same Producer Strain of Macrolide Antibiotics

Streptomyces graminofaciens A-8890 produces two macrolide antibiotics, FD-891 and virustomycin A, both of which show significant biological activity. In this study, we identified the virustomycin A biosynthetic gene cluster, which encodes type I polyketide synthases (PKSs), ethylmalonyl-CoA biosynthetic enzymes, methoxymalony-acyl carrier protein biosynthetic enzymes, and post-PKS modification enzymes. Next, we demonstrated that the acyltransferase domain can be exchanged between the Vsm PKSs and the PKSs involved in FD-891 biosynthesis (Gfs PKSs), without any supply problems of the unique extender units. We exchanged the malonyltransferase domain in the loading module of Gfs PKS with the ethylmalonyltransferase domain and the methoxymalonyltransferase domain of Vsm PKSs. Consequently, the expected two-carbon-elongated analog 26-ethyl-FD-891 was successfully produced with a titer comparable to FD-891 production by the wild type; however, exchange with the methoxymalonyltransferase domain did not produce any FD-891 analogs. Furthermore, 26-ethyl-FD-891 showed potent cytotoxic activity against HeLa cells, like natural FD-891.     

Identification and Characterization of the Cuevaene A Biosynthetic Gene Cluster in Streptomyces sp. LZ35

Genome sequence analysis of Streptomyces sp. LZ35 has revealed a large number of secondary metabolite pathways, including one encoded in an orphan type I polyketide synthase gene cluster that contains a putative chorismatase/3‐hydroxybenzoate synthase gene. Mutagenesis and comparative metabolic profiling led to the identification of cuevaene A as the metabolic product of the gene cluster, thus making it the first 3‐HBA containing polyketide biosynthetic gene cluster described to date. Cuv10 was proven to be responsible for the conversion of chorismate into 3‐HBA; Cuv18 is speculated to be responsible for the 6‐hydroxylation of 3‐HBA during polyketide chain elongation. Additionally, several pathway‐specific regulatory factors that affect the production of cuevaene A were identified. Our results indicate that targeted inactivation of a gene followed by comparative metabolic profiling is a useful approach to identify and characterize cryptic biosynthetic gene clusters.     

Direct Cloning, Genetic Engineering, and Heterologous Expression of the Syringolin Biosynthetic Gene Cluster in E. coli through Red/ET Recombineering

The reconstruction of a natural product biosynthetic pathway from bacteria in a vector and subsequent heterologous expression in a technically amenable microbial system represents an efficient alternative to empirical traditional methods for functional discovery, yield improvement, and genetic engineering to produce "unnatural" derivatives. However, the traditional cloning procedure based on genomic library construction and screening are complicated due to the large size (>10 kb) of most biosynthetic pathways. Here, we describe the direct cloning of a partial syringolin biosynthetic gene cluster (sylCDE, 19 kb) from a digested genomic DNA mixture of Pseudomonas syringae into a plasmid in which sylCDE is under the control of an inducible promoter by one step linear-plus-linear homologous recombination (LLHR) in Escherichia coli. After expression in E. coli GB05-MtaA, two new syringolin derivatives were discovered. The complete syringolin gene cluster was assembled by addition of sylAB and exchange of a synthetic bidirectional promoter against the native promoter to drive sylB and sylC expression by using Red/ET recombineering. The varying production distribution of syringolin derivatives showed the different efficiencies of native and synthetic promoters in E. coli. The successful reconstitution and expression of the syringolin biosynthetic pathway shows that Red/ET recombineering is an efficient tool to clone and engineer secondary metabolite biosynthetic pathways.     

Identification of the Tirandamycin Biosynthetic Gene Cluster from Streptomyces sp. 307‐9

The structurally intriguing bicyclic ketal moiety of tirandamycin is common to several acyl‐tetramic acid antibiotics, and is a key determinant of biological activity. We have identified the tirandamycin biosynthetic gene cluster from the environmental marine isolate Streptomyces sp. 307–9, thus providing the first genetic insight into the biosynthesis of this natural product scaffold. Sequence analysis revealed a hybrid polyketide synthase–nonribosomal peptide synthetase gene cluster with a colinear domain organization, which is entirely consistent with the core structure of the tirandamycins. We also identified genes within the cluster that encode candidate tailoring enzymes for elaboration and modification of the bicyclic ketal system. Disruption of tamI, which encodes a presumed cytochrome P450, led to a mutant strain deficient in production of late stage tirandamycins that instead accumulated tirandamycin C, an intermediate devoid of any post assembly‐line oxidative modifications.     

Five‐Membered Cyclitol Phosphate Formation by a myo‐Inositol Phosphate Synthase Orthologue in the Biosynthesis of the Carbocyclic Nucleoside Antibiotic Aristeromycin

Aristeromycin is a unique carbocyclic nucleoside antibiotic produced by Streptomyces citricolor. In order to elucidate its intriguing carbocyclic formation, we used a genome‐mining approach to identify the responsible enzyme. In silico screening with known cyclitol synthases involved in primary metabolism, such as myo‐inositol‐1‐phosphate synthase (MIPS) and dehydroqunate synthase (DHQS), identified a unique MIPS orthologue (Ari2) encoded in the genome of S. citricolor. Heterologous expression of the gene cluster containing ari2 with a cosmid vector in Streptomyces albus resulted in the production of aristeromycin, thus indicating that the cloned DNA region (37.5 kb) with 33 open reading frames contains its biosynthetic gene cluster. We verified that Ari2 catalyzes the formation of a novel five‐membered cyclitol phosphate from d ‐fructose 6‐phosphate (F6P) with NAD⁺ as a cofactor. This provides insight into cyclitol phosphate synthase as a member of the MIPS family of enzymes. A biosynthetic pathway to aristeromycin is proposed based on bioinformatics analysis of the gene cluster.