Biosynthesis and identification of volatiles released by the myxobacterium Stigmatella aurantiaca

The volatiles released by agar plate cultures of two strains of the myxobacterium Stigmatella aurantiaca (strains Sg a15 and DW4/3-1) were collected in a closed-loop stripping apparatus (CLSA) and analyzed by GC-MS. Large numbers of substances from different compound classes (ketones, esters, lactones, terpenes, and sulfur and nitrogen compounds) were identified; several of them are reported from natural sources for the first time. The volatiles 2-methyltridecan-4-one (17), its isomer 3-methyltridecan-4-one (20), and the higher homologue 2-methyltetradecan-4-one (18) were identified in the extracts of both strains and were synthesized. In addition, strain Sg a15 produced 2,12-dimethyltridecan-4-one (19), 2-methyltridec-2-en-4-one (23), and a series of phenyl ketones, among them 1-phenyldecan-1-one (14) and 9-methyl-1-phenyldecan-1-one (16), whereas strain DW4/3-1 emitted traces of 10-methylundecan-2-one (21). The biosynthesis of 14 and 16 was examined in feeding experiments with deuterated precursors carried out on agar plate cultures. The leucine-derived starter unit isovalerate was shown to be incorporated into 16, as was phenylalanine-derived benzoic acid into both 14 and 16. The results point to formation both of the phenyl ketones and of the structurally related aliphatic ketones through an unusual head-to-head coupling between a starter unit such as benzoyl-CoA and a fatty acyl-CoA, followed by decarboxylation.     

A biosynthetic pathway to isovaleryl-CoA in myxobacteria: the involvement of the mevalonate pathway

A biosynthetic shunt pathway branching from the mevalonate pathway and providing starter units for branched-chain fatty acid and secondary metabolite biosynthesis has been identified in strains of the myxobacterium Stigmatella aurantiaca. This pathway is upregulated when the branched-chain alpha-keto acid dehydrogenase gene (bkd) is inactivated, thus impairing the normal branched-chain amino acid degradation process. We previously proposed that, in this pathway, isovaleryl-CoA is derived from 3,3-dimethylacrylyl-CoA (DMA-CoA). Here we show that DMA-CoA is an isomerization product of 3-methylbut-3-enoyl-CoA (3MB-CoA). This compound is directly derived from 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) by a decarboxylation/ dehydration reaction resembling the conversion of mevalonate 5-diphosphate to isopentenyl diphosphate. Incubation of cell-free extracts of a bkd mutant with HMG-CoA gave product(s) with the molecular mass of 3MB-CoA or DMA-CoA. The shunt pathway most likely also operates reversibly and provides an alternative source for the monomers of isoprenoid biosynthesis in myxobacteria that utilize L-leucine as precursor.     

Identification of a napsamycin biosynthesis gene cluster by genome mining

Napsamycins are potent inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan biosynthesis, and are classified as uridylpeptide antibiotics. They comprise an N-methyl diaminobutyric acid, an ureido group, a methionine and two non-proteinogenic aromatic amino acid residues in a peptide backbone that is linked to a 5'-amino-3'-deoxyuridine by an unusual enamide bond. The napsamycin gene cluster was identified in Streptomyces sp. DSM5940 by using PCR probes from a putative uridylpeptide biosynthetic cluster found in S. roseosporus NRRL15998 by genome mining. Annotation revealed 29 hypothetical genes encoding for resistance, regulation and biosynthesis of the napsamycins. Analysis of the gene cluster indicated that the peptide core structure is assembled by a nonlinear non-ribosomal peptide synthetase (NRPS)-like mechanism that involves several discrete single or didomain proteins. Some genes could be assigned, for example, to the synthesis of the N-methyl diaminobutyric acid, to the generation of m-tyrosine and to the reduction of the uracil moiety. The heterologous expression of the gene cluster in Streptomyces coelicolor M1154 resulted in the production of napsamycins and mureidomycins as demonstrated by LC-ESI-MS and MS/MS analysis. The napsamycin gene cluster provides a molecular basis for the detailed study of the biosynthesis of this class of structurally unusual compounds.     

AuaA, a membrane-bound farnesyltransferase from Stigmatella aurantiaca, catalyzes the prenylation of 2-methyl-4-hydroxyquinoline in the biosynthesis of aurachins

Aurachins are quinoline alkaloids isolated from the myxobacterium Stigmatella aurantiaca. They are substituted with an isoprenoid side chain and act as potent inhibitors in the electron transport chain. A biosynthetic gene cluster that contains at least five genes (auaA-auaE) has been identified for aurachin biosynthesis. In this study, auaA, the gene encoding a putative prenyltransferase of 326 amino acids, was cloned and overexpressed in Escherichia coli. Biochemical investigations showed that AuaA catalyzes the prenylation of 2-methyl-4-hydroxyquinoline in the presence of farnesyl diphosphate (FPP), thereby resulting in the formation of aurachin D. The hydroxyl group at position C4 of the quinoline ring is essential for an acceptance by AuaA; this was concluded by testing 18 quinoline derivatives or analogues with AuaA and FPP. (1) H NMR and HR-EI-MS analyses of six isolated enzyme products revealed the presence of a farnesyl moiety at position C3 of the quinoline ring. K(M) values of 43 and 270 μM were determined for FPP and 2-methyl-4-hydroxyquinoline, respectively. Like other known membrane-bound prenyltransferases, the reaction catalyzed by AuaA is dependent on the presence of metal ions such as Mg(2+) , Mn(2+) and Co(2+) , although no typical (N/D)DXXD binding motif was found in the sequence.     

Discovery of Six Ramoplanin Family Gene Clusters and the Lipoglycodepsipeptide Chersinamycin**

Ramoplanins and enduracidins are peptidoglycan lipid intermediate II-binding lipodepsipeptides with broad-spectrum activity against methicillin- and vancomycin-resistant Gram-positive pathogens. Targeted genome mining using probes from conserved sequences within the ramoplanin/enduracidin biosynthetic gene clusters (BGCs) was used to identify six microorganisms with BGCs predicted to produce unique lipodepsipeptide congeners of ramoplanin and enduracidin. Fermentation of Micromonospora chersina yielded a novel lipoglycodepsipeptide, called chersinamycin, which exhibited good antibiotic activity against Gram-positive bacteria (1–2 μg/mL) similar to the ramoplanins and enduracidins. The covalent structure of chersinamycin was determined by NMR spectroscopy and tandem mass spectrometry in conjunction with chemical degradation studies. These six new BGCs and isolation of a new antimicrobial peptide provide much-needed tools to investigate the fundamental aspects of lipodepsipeptide biosynthesis and to facilitate efforts to produce novel antibiotics capable of combating antibiotic-resistant infections.     

Unraveling the biosynthesis of penicillenols by genome mining polyketide synthase and nonribosomal peptide synthetase gene clusters in Penicillium citrinum

Penicillenols belong to the family of tetramic acids with anticancer and antibacterial activities. Here, we report the discovery of the biosynthetic gene cluster (pnc) for penicillenol A₁ and E in Penicillium citrinum ATCC9849 by genome mining. We discover the pnc cluster based on the results of gene deletions in P. citrinum and gene cluster heterologous expression in Aspergillus nidulans. We also propose the assembly line of the polyketide synthase module in PncA with the reduction by PncB provides a highly reduce polyketide chain to be further linked with an l -threonine molecule and released from PncA to produce penicillenol E. Further formation of penicillenol A₁ requires the N-methylation of tetramic acid group by PncC. Our work deepens the understanding of the biosynthetic logic for N-methylated tetramic acids and contributes to the discovery of new penicillenols by genome mining.     

In vivo mutational analysis of the mupirocin gene cluster reveals labile points in the biosynthetic pathway: the "leaky hosepipe" mechanism

A common feature of the mupirocin and other gene clusters of the AT-less polyketide synthase (PKS) family of metabolites is the introduction of carbon branches by a gene cassette that contains a beta-hydroxy-beta-methylglutaryl CoA synthase (HMC) homologue and acyl carrier protein (ACP), ketosynthase (KS) and two crotonase superfamily homologues. In vivo studies of Pseudomonas fluorescens strains in which any of these components have been mutated reveal a common phenotype in which the two major isolable metabolites are the truncated hexaketide mupirocin H and the tetraketide mupiric acid. The structure of the latter has been confirmed by stereoselective synthesis. Mupiric acid is also the major metabolite arising from inactivation of the ketoreductase (KR) domain of module 4 of the modular PKS. A number of other mutations in the tailoring region of the mupirocin gene cluster also result in production of both mupirocin H and mupiric acid. To explain this common phenotype we propose a mechanistic rationale in which both mupirocin H and mupiric acid represent the products of selective and spontaneous release from labile points in the pathway that occur at significant levels when mutations block the pathway either close to or distant from the labile points.     

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.     

Characterization of a single gene cluster responsible for methylpendolmycin and pendolmycin biosynthesis in the deep sea bacterium Marinactinospora thermotolerans

The nine-membered indolactam antibiotics belong to a small group of antibiotics showing broad biological activities. However, the in vivo genetic engineering of compounds of this type has not been performed. Here we report the identification of a single gene cluster responsible for the biosynthesis of methylpendolmycin and pendolmycin, two members of this family of antibiotics, from the deep sea bacterium Marinactinospora thermotolerans SCSIO 00652. Bioinformatics analysis and gene inactivation, coupled with metabolite characterization, reveal that MpnB, a nonribosomal peptide synthetase, MpnC, a cytochrome P450, and MpnD, a prenyltransferase, are sufficient to catalyze the biosynthesis of the two antibiotics from L-Ile (or L-Val), L-Trp, and methionine. MpnD is the first identified enzyme that transfers a C5 prenyl unit in a reverse manner to the C-7 position of a Trp-derived natural product.     

Core Steps to the Azaphilone Family of Fungal Natural Products

Azaphilones are a family of polyketide-based fungal natural products that exhibit interesting and useful bioactivities. This minireview explores the literature on various characterised azaphilone biosynthetic pathways, which allows for a proposed consensus scheme for the production of the core azaphilone structure, as well as identifying early diversification steps during azaphilone biosynthesis. A consensus understanding of the core enzymatic steps towards a particular family of fungal natural products can aid in genome-mining experiments. Genome mining for novel fungal natural products is a powerful technique for both exploring chemical space and providing new insights into fungal natural product pathways.     

Biosynthesis of Sinapigladioside,an Antifungal Isothiocyanate from Burkholderia Symbionts

Sinapigladioside is a rare isothiocyanate-bearing natural product from beetle-associated bacteria (Burkholderia gladioli) that might protect beetle offspring against entomopathogenic fungi. The biosynthetic origin of sinapigladioside has been elusive, and little is known about bacterial isothiocyanate biosynthesis in general. On the basis of stable-isotope labeling, bioinformatics, and mutagenesis, we identified the sinapigladioside biosynthesis gene cluster in the symbiont and found that an isonitrile synthase plays a key role in the biosynthetic pathway. Genome mining and network analyses indicate that related gene clusters are distributed across various bacterial phyla including producers of both nitriles and isothiocyanates. Our findings support a model for bacterial isothiocyanate biosynthesis by sulfur transfer into isonitrile precursors.     

Strategies for the Discovery of New Natural Products by Genome Mining

New drugs from silent gene clusters : Analysis of genome sequence data has identified numerous “cryptic” gene clusters encoding novel natural product biosynthetic assembly lines; this suggests that many new bioactive metabolites remain to be discovered, even in extensively investigated organisms. Several related and complementary strategies for identifying the products of these clusters have emerged recently and revitalized the search for novel bioactive natural products.

     

Production of the tubulin destabilizer disorazol in Sorangium cellulosum: biosynthetic machinery and regulatory genes

Myxobacteria show a high potential for the production of natural compounds that exhibit a wide variety of antibiotic, antifungal, and cytotoxic activities. The genus Sorangium is of special biotechnological interest because it produces almost half of the secondary metabolites isolated from these microorganisms. We describe a transposon-mutagenesis approach to identifying the disorazol biosynthetic gene cluster in Sorangium cellulosum So ce12, a producer of multiple natural products. In addition to the highly effective disorazol-type tubulin destabilizers, S. cellulosum So ce12 produces sorangicins, potent eubacterial RNA polymerase inhibitors, bactericidal sorangiolides, and the antifungal chivosazoles. To obtain a transposon library of sufficient size suitable for the identification of the presumed biosynthetic gene clusters, an efficient transformation method was developed. We present here the first electroporation protocol for a strain of the genus Sorangium. The transposon library was screened for disorazol-negative mutants. This approach led to the identification of the corresponding trans-acyltransferase core biosynthetic gene cluster together with a region in the chromosome that is likely to be involved in disorazol biosynthesis. A third region in the genome harbors another gene that is presumed to be involved in the regulation of disorazol production. A detailed analysis of the biosynthetic and regulatory genes is presented in this paper.     

A gene cluster for prenylated naphthoquinone and prenylated phenazine biosynthesis in Streptomyces cinnamonensis DSM 1042

Streptomyces cinnamonensis DSM 1042 produces two classes of secondary metabolites of mixed isoprenoid/nonisoprenoid origin: the polyketide-isoprenoid compound furanonaphthoquinone I (FNQ I) and several prenylated phenazines, predominantly endophenazine A. We now report the cloning and sequence analysis of a 55 kb gene cluster required for the biosynthesis of these compounds. Several inactivation experiments confirmed the involvement of this gene cluster in the biosynthesis of FNQ I and endophenazine A. The six identified genes for endophenazine biosynthesis showed close similarity to phenazine biosynthetic genes from Pseudomonas. Of the 28 open reading frames identified in the adjacent FNQ I cluster, 13 showed close similarity to genes contained in the cluster for furaquinocin-a structurally similar metabolite from another Streptomyces strain. These genes included a type III polyketide synthase sequence, a momA-like monooxygenase gene, and two cloQ-like prenyltransferase genes designated fnq26 and fnq28. Inactivation experiments confirmed the involvement of fnq26 in FNQ I biosynthesis, whereas no change in secondary-metabolite formation was observed after fnq28 inactivation. The FNQ I cluster contains a contiguous group of five genes, which together encode all the enzymatic functions required for the recycling of S-adenosylhomocysteine (SAH) to S-adenosylmethionine (SAM). Two SAM-dependent methyltransferases are encoded within the cluster. Inactivation experiments showed that fnq9 is responsible for the 7-O-methylation and fnq27 for the 6-C-methylation reaction in FNQ I biosynthesis.     

Discovery of the Tiancilactone Antibiotics by Genome Mining of Atypical Bacterial Type II Diterpene Synthases

Although genome mining has advanced the identification, discovery, and study of microbial natural products, the discovery of bacterial diterpenoids continues to lag behind. Herein, we report the identification of 66 putative producers of novel bacterial diterpenoids, and the discovery of the tiancilactone (TNL) family of antibiotics, by genome mining of type II diterpene synthases that do not possess the canonical DXDD motif. The TNLs, which are broad‐spectrum antibiotics with moderate activities, are produced by both Streptomyces sp. CB03234 and Streptomyces sp. CB03238 and feature a highly functionalized diterpenoid skeleton that is further decorated with chloroanthranilate and γ‐butyrolactone moieties. Genetic manipulation of the tnl gene cluster resulted in TNL congeners, which provided insights into their biosynthesis and structure–activity relationships. This work highlights the biosynthetic potential that bacteria possess to produce diterpenoids and should inspire continued efforts to discover terpenoid natural products from bacteria.