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.     

Genome Mining‐Directed Activation of a Silent Angucycline Biosynthetic Gene Cluster in Streptomyces chattanoogensis

Genomic sequencing of actinomycetes has revealed the presence of numerous gene clusters seemingly capable of natural product biosynthesis, yet most clusters are cryptic under laboratory conditions. Bioinformatics analysis of the completely sequenced genome of Streptomyces chattanoogensis L10 (CGMCC 2644) revealed a silent angucycline biosynthetic gene cluster. The overexpression of a pathway‐specific activator gene under the constitutive ermE* promoter successfully triggered the expression of the angucycline biosynthetic genes. Two novel members of the angucycline antibiotic family, chattamycins A and B, were further isolated and elucidated. Biological activity assays demonstrated that chattamycin B possesses good antitumor activities against human cancer cell lines and moderate antibacterial activities. The results presented here provide a feasible method to activate silent angucycline biosynthetic gene clusters to discover potential new drug leads.     

Generation of New Complestatin Analogues by Heterologous Expression of the Complestatin Biosynthetic Gene Cluster from Streptomyces chartreusis AN1542

The heterologous expression of the biosynthetic gene cluster (BGC) of natural products enables the production of complex metabolites in a well‐characterized host, and facilitates the generation of novel analogues by the manipulation of the genes. However, the BGCs of glycopeptides such as vancomycin, teicoplanin, and complestatin are usually too large to be directly cloned into a single cosmid. Here, we describe the heterologous expression of the complestatin BGC. The 54.5 kb cluster was fully reconstituted from two overlapping cosmids into one cosmid by λ‐RED recombination‐mediated assembly. Heterologous expression of the assembled gene cluster in Streptomyces lividans TK24 resulted in the production of complestatin. Deletion of cytochrome P450 monooxygenase genes (open reading frames 10 and 11) and heterologous expression of the modified clusters led to the production of two new monocyclic and linear derivatives, complestatins M55 and S56.     

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.     

Genomics-Guided Efficient Identification of 2,5-Diketopiperazine Derivatives from Actinobacteria

Secondary metabolites derived from microorganism constitute an important part of natural products. Mining of the microbial genomes revealed a large number of uncharacterized biosynthetic gene clusters, indicating their greater potential to synthetize specialized or secondary metabolites (SMs) than identified by classic fermentation and isolation approaches. Various bioinformatics tools have been developed to analyze and identify such gene clusters, thus accelerating significantly the mining process. Heterologous expression of an individual biosynthetic gene cluster has been proven as an efficient way to activate the genes and identify the encoded metabolites that cannot be detected under normal laboratory cultivation conditions. Herein, we describe a concept of genomics-guided approach by performing genome mining and heterologous expression to uncover novel CDPS-derived DKPs and functionally characterize novel tailoring enzymes embedded in the biosynthetic pathways. Recent works focused on the identification of the nucleobase-related and dimeric DKPs are also presented.     

Exploration and mining of the bacterial terpenome

Tens of thousands of terpenoids are present in both terrestrial and marine plants, as well as fungi. In the last 5-10 years, however, it has become evident that terpenes are also produced by numerous bacteria, especially soil-dwelling Gram-positive organisms such as Streptomyces and other Actinomycetes. Although some microbial terpenes, such as geosmin, the degraded sesquiterpene responsible for the smell of moist soil, the characteristic odor of the earth itself, have been known for over 100 years, few terpenoids have been identified by classical structure- or activity-guided screening of bacterial culture extracts. In fact, the majority of cyclic terpenes from bacterial species have only recently been uncovered by the newly developed techniques of "genome mining". In this new paradigm for biochemical discovery, bacterial genome sequences are first analyzed with powerful bioinformatic tools, such as the BLASTP program or Profile Hidden Markov models, to screen for and identify conserved protein sequences harboring a characteristic set of universally conserved functional domains typical of all terpene synthases. Of particular importance is the presence of variants of two universally conserved domains, the aspartate-rich DDXX(D/E) motif and the NSE/DTE triad, (N/D)DXX(S/T)XX(K/R)(D/E). Both domains have been implicated in the binding of the essential divalent cation, typically Mg(2+), that is required for cyclization of the universal acyclic terpene precursors, such as farnesyl and geranyl diphosphate. The low level of overall sequence similarity among terpene synthases, however, has so far precluded any simple correlation of protein sequence with the structure of the cyclized terpene product. The actual biochemical function of a cryptic bacterial (or indeed any) terpene synthase must therefore be determined by direct experiment. Two common approaches are (i) incubation of the expressed recombinant protein with acyclic allylic diphosphate substrates and identification of the resultant terpene hydrocarbon or alcohol and (ii) in vivo expression in engineered bacterial hosts that can support the production of terpene metabolites. One of the most attractive features of the coordinated application of genome mining and biochemical characterization is that the discovery of natural products is directly coupled to the simultaneous discovery and exploitation of the responsible biosynthetic genes and enzymes. Bacterial genome mining has proved highly rewarding scientifically, already uncovering more than a dozen newly identified cyclic terpenes (many of them unique to bacteria), as well as several novel cyclization mechanisms. Moreover, bioinformatic analysis has identified more than 120 presumptive genes for bacterial terpene synthases that are now ripe for exploration. In this Account, we review a particularly rich vein we have mined in the genomes of two model Actinomycetes, Streptomyces coelicolor and Streptomyces avermitilis, from which the entire set of terpenoid biosynthetic genes and pathways have now been elucidated. In addition, studies of terpenoid biosynthetic gene clusters have revealed a wealth of previously unknown oxidative enzymes, including cytochromes P450, non-heme iron-dependent dioxygenases, and flavin monooxygenases. We have shown that these enzymes catalyze a variety of unusual biochemical reactions, including two-step ketonization of methylene groups, desaturation-epoxidation of secondary methyl groups, and pathway-specific Baeyer-Villiger oxidations of cyclic ketones.     

Unveiling Two Consecutive Hydroxylations: Mechanisms of Aromatic Hydroxylations Catalyzed by Flavin-Dependent Monooxygenases for the Biosynthesis of Actinorhodin and Related Antibiotics

Flavin-dependent monooxygenases are ubiquitous in living systems and are classified into single- or two-component systems. Actinorhodin, produced by Streptomyces coelicolor, is a representative polycyclic polyketide that is hydroxylated through the action of the two-component ActVA-5/ActVB hydroxylase system. These homologous systems are widely distributed in bacteria, but their reaction mechanisms remain unclear. This in vitro investigation has provided chemical proof of two consecutive hydroxylations via hydroxynaphthalene intermediates involved in actinorhodin biosynthesis. The ActVA-5 oxygenase component catalyzed a stepwise dihydroxylation of the substrate, whereas the ActVB flavin reductase not only supplied a reduced cofactor, but also regulated the quinone–hydroquinone interconversion of an intermediate. Our study provides clues for understanding the general biosynthetic mechanisms of highly functionalized aromatic natural products with structural diversity.     

New Insights into the Glycosylation Steps in the Biosynthesis of Sch47554 and Sch47555

Sch47554 and Sch47555 are antifungal compounds from Streptomyces sp. SCC‐2136. The availability of the biosynthetic gene cluster made it possible to track genes that encode biosynthetic enzymes responsible for the structural features of these two angucyclines. Sugar moieties play important roles in the biological activities of many natural products. An investigation into glycosyltransferases (GTs) might potentially help to diversify pharmaceutically significant drugs through combinatorial biosynthesis. Sequence analysis indicates that SchS7 is a putative C‐GT, whereas SchS9 and SchS10 are proposed to be O‐GTs. In this study, the roles of these three GTs in the biosynthesis of Sch47554 and Sch47555 are characterized. Coexpression of the aglycone and sugar biosynthetic genes with schS7 in Streptomyces lividans K4 resulted in the production of C‐glycosylated rabelomycin, which revealed that SchS7 attached a d ‐amicetose moiety to the aglycone core structure at the C‐9 position. Gene inactivation studies revealed that subsequent glycosylation steps took place in a sequential manner, in which SchS9 first attached either an l ‐aculose or l ‐amicetose moiety to 4′‐OH of the C‐glycosylated aglycone, then SchS10 transferred an l ‐aculose moiety to 3‐OH of the angucycline core.     

Rapid Reconstitution of Biosynthetic Machinery for Fungal Metabolites in Aspergillus oryzae: Total Biosynthesis of Aflatrem

Reconstitution of the biosynthetic machinery for fungal secondary metabolites in Aspergillus oryzae provides an opportunity both for stepwise determination of the biosynthetic pathways and the total biosynthesis of fungal natural products. However, to maximize the utility of the reconstitution system, a simple and rapid strategy for the introduction of heterologous genes into A. oryzae is required. In this study, we demonstrated an effective method for introducing multiple genes involved in the biosynthesis of fungal metabolites by using the expression vectors pUARA2 and pUSA2, each of which contains two cloning sites. The successful introduction of all the aflatrem biosynthetic genes (seven genes in total) after two rounds of transformation enabled the total biosynthesis of aflatrem. This rapid reconstitution strategy will facilitate the functional analysis of the biosynthetic machinery of fungal metabolites.     

Epoxyquinone formation catalyzed by a two-component flavin-dependent monooxygenase involved in biosynthesis of the antibiotic actinorhodin

The biosynthetic gene cluster of the aromatic polyketide antibiotic actinorhodin (ACT) in Streptomyces coelicolor A3(2) carries a pair of genes, actVA-ORF5 and actVB, that encode a two-component flavin-dependent monooxygenase (FMO). Our previous studies have demonstrated that the ActVA-ORF5/ActVB system functions as a quinone-forming C-6 oxygenase in ACT biosynthesis. Furthermore, we found that this enzyme system exhibits an additional oxygenation activity with dihydrokalafungin (DHK), a proposed intermediate in the ACT biosynthetic pathway, and generates two reaction products. These compounds were revealed to be monooxygenated derivatives of kalafungin, which is spontaneously formed through oxidative lactonization of DHK. Their absolute structures were elucidated from their NMR spectroscopic data and by computer modeling and X-ray crystallography as (5S,14R)-epoxykalafungin and (5R,14S)-epoxykalafungin, demonstrating an additional epoxyquinone-forming activity of the ActVA-ORF5/ActVB system in vitro.     

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.     

Structural Diversification of Macrolactones by Substrate‐Flexible Cytochrome P450 Monooxygenases

The substrate flexibilities of several cytochrome P450 monooxygenases involved in macrolide biosynthesis were investigated to test their potential for the generation of novel macrolides. PikC hydroxylase in the pikromycin producer Streptomyces venezuelae accepted oleandomycin as an alternative substrate and introduced a hydroxy group at the C‐4 position, which is different from the intrinsic C‐12 hydroxylation position in the natural substrate. This is the first report of C‐4 hydroxylation activity of cytochrome P450 monooxygenase involved in the biosynthesis of 14‐membered macrolides. EryF hydroxylase from the erythromycin biosynthetic pathway of Saccharopolyspora erythraea and OleP oxidase from the oleandomycin biosynthetic pathway of Streptomyces antibioticus also showed a certain degree of plasticity towards alternative substrates. In particular, EryF and OleP were found to oxidize a 12‐membered macrolactone as an alternative substrate. These results demonstrate the potential usefulness of these enzymes to diversify macrolactones by post‐PKS oxidations.     

The l-Alanosine Gene Cluster Encodes a Pathway for Diazeniumdiolate Biosynthesis

N-Nitroso-containing natural products are bioactive metabolites with antibacterial and anticancer properties. In particular, compounds containing the diazeniumdiolate (N-nitrosohydroxylamine) group display a wide range of bioactivities ranging from cytotoxicity to metal chelation. Despite the importance of this structural motif, knowledge of its biosynthesis is limited. Herein we describe the discovery of a biosynthetic gene cluster in Streptomyces alanosinicus ATCC 15710 responsible for producing the diazeniumdiolate natural product l -alanosine. Gene disruption and stable isotope feeding experiments identified essential biosynthetic genes and revealed the source of the N-nitroso group. Additional biochemical characterization of the biosynthetic enzymes revealed that the non-proteinogenic amino acid l -2,3-diaminopropionic acid (l -Dap) is synthesized and loaded onto a free-standing peptidyl carrier protein (PCP) domain in l -alanosine biosynthesis, which we propose may be a mechanism of handling unstable intermediates generated en route to the diazeniumdiolate. These discoveries will facilitate efforts to determine the biochemistry of diazeniumdiolate formation.     

Definition of the common and divergent steps in carbapenem β-lactam antibiotic biosynthesis

Approximately 50 naturally occurring carbapenem β-lactam antibiotics are known. All but one of these have been isolated from Streptomyces species and are disubstituted structural variants of a simple core that is synthesized by Pectobacterium carotovorum (Erwinia carotovora), a phylogenetically distant plant pathogen. While the biosynthesis of the simple carbapenem, (5R)-carbapen-2-em-3-carboxylic acid, is impressively efficient requiring only three enzymes, CarA, CarB and CarC, the formation of thienamycin, one of the former group of metabolites from Streptomyces, is markedly more complex. Despite their phylogenetic separation, bioinformatic analysis of the encoding gene clusters suggests that the two pathways could be related. Here we demonstrate with gene swapping, stereochemical and kinetics experiments that CarB and CarA and their S. cattleya orthologues, ThnE and ThnM, respectively, are functionally and stereochemically equivalent, although their catalytic efficiencies differ. The biosynthetic pathways, therefore, to thienamycin, and likely to the other disubstituted carbapenems, and to the simplest carbapenem, (5R)-carbapen-2-em-3-carboxylic acid, are initiated in the same manner, but share only two common steps before diverging.     

Characterization of the biosynthesis gene cluster for alkyl-O-dihydrogeranyl-methoxyhydroquinones in Actinoplanes missouriensis

A polyketide biosynthesis gene cluster (agq) was found on the genome of a rare actinomycete, Actinoplanes missouriensis. Streptomyces lividans expressing agqA encoding a type III polyketide synthase produced alkylresorcinols mainly from C(16-17) fatty acids. Heterologous expression of the agq genes in S. lividans indicated the function of cognate polyketide modification enzymes; a monooxygenase AgqB hydroxylates the alkylresorcinols to yield 6-alkyl-2-hydroxyhydroquinones, a methyltransferase AgqC catalyzes O-methylation of the alkyl-hydroxyhydroquinones to yield 6-alkyl-2-methoxyhydroquinones, and a UbiA-like prenyltransferase AgqD attaches a prenyl group to the C-4 hydroxy group of the alkyl-methoxyhydroquinones to yield 6-alkyl-4-O-geranyl-2-methoxyhydroquinones and 6-alkyl-4-O-dihydrofarnesyl-2-methoxyhydroquinones derived from C(16-17) fatty acids. In contrast, A. missouriensis was found to produce 6-alkyl-4-O-dihydrogeranyl-2-methoxyhydroquinones derived from C(16-18) fatty acids by the function of the agq gene cluster. All of these prenylated phenolic lipids were novel compounds.     

Biosynthesis of Hygrocins,Antitumor Naphthoquinone Ansamycins Produced by Streptomyces sp. LZ35

Hygrocins are naphthoquinone ansamycins with significant antitumor activities. Here, we report the identification and characterization of the hygrocin biosynthetic gene cluster (hgc) in Streptomyces sp. LZ35. A biosynthetic pathway is proposed based on bioinformatics analysis of the hgc genes and intermediates accumulated in selected gene disruption mutants. One of the steps during the biosynthesis of hygrocins is a Baeyer–Villiger oxidation between C5 and C6, catalyzed by luciferase‐like monooxygenase homologue Hgc3. Hgc3 represents the founding member of a previously uncharacterized family of enzymes acting as Baeyer–Villiger monooxygenases.     

Analysis of the tetronomycin gene cluster: insights into the biosynthesis of a polyether tetronate antibiotic

The biosynthetic gene cluster for tetronomycin (TMN), a polyether ionophoric antibiotic that contains four different types of ring, including the distinctive tetronic acid moiety, has been cloned from Streptomyces sp. NRRL11266. The sequenced tmn locus (113 234 bp) contains six modular polyketide synthase (PKS) genes and a further 27 open-reading frames. Based on sequence comparison to related biosynthetic gene clusters, the majority of these can be assigned a plausible role in TMN biosynthesis. The identity of the cluster, and the requirement for a number of individual genes, especially those hypothesised to contribute a glycerate unit to the formation of the tetronate ring, were confirmed by specific gene disruption. However, two large genes that are predicted to encode together a multifunctional PKS of a highly unusual type seem not to be involved in this pathway since deletion of one of them did not alter tetronomycin production. Unlike previously characterised polyether PKS systems, oxidative cyclisation appears to take place on the modular PKS rather than after transfer to a separate carrier protein, while tetronate ring formation and concomitant chain release share common mechanistic features with spirotetronate biosynthesis.