Insights into an Unusual Nonribosomal Peptide Synthetase Biosynthesis: IDENTIFICATION AND CHARACTERIZATION OF THE GE81112 BIOSYNTHETIC GENE CLUSTER*

The GE81112 tetrapeptides (1–3) represent a structurally unique class of antibiotics, acting as specific inhibitors of prokaryotic protein synthesis. Here we report the cloning and sequencing of the GE81112 biosynthetic gene cluster from Streptomyces sp. L-49973 and the development of a genetic manipulation system for Streptomyces sp. L-49973. The biosynthetic gene cluster for the tetrapeptide antibiotic GE81112 (getA-N) was identified within a 61.7-kb region comprising 29 open reading frames (open reading frames), 14 of which were assigned to the biosynthetic gene cluster. Sequence analysis revealed the GE81112 cluster to consist of six nonribosomal peptide synthetase (NRPS) genes encoding incomplete di-domain NRPS modules and a single free standing NRPS domain as well as genes encoding other biosynthetic and modifying proteins. The involvement of the cloned gene cluster in GE81112 biosynthesis was confirmed by inactivating the NRPS gene getE resulting in a GE81112 production abolished mutant. In addition, we characterized the NRPS A-domains from the pathway by expression in Escherichia coli and in vitro enzymatic assays. The previously unknown stereochemistry of most chiral centers in GE81112 was established from a combined chemical and biosynthetic approach. Taken together, these findings have allowed us to propose a rational model for GE81112 biosynthesis. The results further open the door to developing new derivatives of these promising antibiotic compounds by genetic engineering.     

The small MbtH-like protein encoded by an internal gene of the balhimycin biosynthetic gene cluster is not required for glycopeptide production

The balhimycin biosynthetic gene cluster of the glycopeptide producer Amycolatopsis balhimycina includes a gene (orf1) with unknown function. orf1 shows high similarity to the mbtH gene from Mycobacterium tuberculosis. In almost all nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters, we could identify a small mbtH-like gene whose function in peptide biosynthesis is not known. The mbtH-like gene is always colocalized with the NRPS genes; however, it does not have a specific position in the gene cluster. In all glycopeptide biosynthetic gene clusters the orf1-like gene is always located downstream of the gene encoding the last module of the NRPS. We inactivated the orf1 gene in A. balhimycina by generating a deletion mutant. The balhimycin production is not affected in the orf1-deletion mutant and is indistinguishable from that of the wild type. For the first time, we show that the inactivation of an mbtH-like gene does not impair the biosynthesis of a nonribosomal peptide.     

炭团木霉中非核糖体多肽基因NRPS1的功能研究

[目的] 木霉属真菌是应用最为广泛和潜力最大的生防真菌,其产生的典型化合物哌珀霉素（peptaibols）类抗生素在生物防治中发挥重要作用。本研究采用基因组挖掘技术（genome mining）发现炭团木霉（Trichoderma hypoxylon）的潜在哌珀霉素生物合成基因簇及对病原菌的防治作用。[方法] 生物信息学分析预测合成哌珀霉素的基因簇,利用Quick-change技术构建基因骨架敲除盒,通过PEG介导的原生质体转化方法获得敲除突变株,通过平板对峙法和菌丝生长毒力实验验证该基因簇对炭团木霉生物活性的影响。[结果] 基因挖掘鉴定一个非核糖体多肽合成酶（nonribosomal peptide synthetases,NRPS）可能合成哌珀霉素类抗生素,命名为NRPS1,对该基因进行部分敲除,成功获得3株NRPS1缺失突变株。对峙实验表明,突变株对寄生曲霉（Aspergillus parasiticus）、尖孢镰刀菌（Fusarium oxysporum）、黑白轮枝菌（Verticillium alboatrum）等9株植物病原真菌的抑制作用与野生株相比显著下降,且突变株的粗提物的抑菌活性明显弱于野生型。[结论] NRPS1是一个潜在的哌珀霉素合成基因,该基因在宿主与病原真菌对抗过程中起关键作用,该研究为炭团木霉哌珀霉素结构解析及生物防治机理研究奠定了基础。     

Avermectin: biochemical and molecular basis of its biosynthesis and regulation

Avermectin and its analogues, produced by Streptomyces avermitilis, are major commercial antiparasitic agents in the field of animal health, agriculture, and human infections. They are 16-membered pentacyclic lactone compounds derived from polyketide and linked to a disaccharide of the methylated deoxysugar l-oleandrose. Labeling studies, analyses of the biosynthetically blocked mutants, and the identification of the avermectin gene cluster allows characterization of most of the biosynthetic pathway. Recent completion of S. avermitilis genome sequencing is also expected to help in revealing the precise biosynthetic sequence and the complicated regulatory mechanism for avermectin biosynthesis, which has been long-awaited to be elucidated. The well characterized avermectin biosynthetic pathway and availability of S. avermitilis genome information in combination with the recent development of combinatorial biosynthesis should allow us to redesign more potent avermectin analogues and to engineer S. avermitilis as a more efficient host for the production of important commercial analogues.     

Function of MbtH homologs in nonribosomal peptide biosynthesis and applications in secondary metabolite discovery

Mycobacterium tuberculosis encodes mycobactin, a peptide siderophore that is biosynthesized by a nonribosomal peptide synthetase (NRPS) mechanism. Within the mycobactin biosynthetic gene cluster is a gene that encodes a 71-amino-acid protein MbtH. Many other NRPS gene clusters harbor mbtH homologs, and recent genetic, biochemical, and structural studies have begun to shed light on the function(s) of these proteins. In some cases, MbtH-like proteins are required for biosynthesis of their cognate peptides, and non-cognate MbtH-like proteins have been shown to be partially complementary. Biochemical studies revealed that certain MbtH-like proteins participate in tight binding to NRPS proteins containing adenylation (A) domains where they stimulate adenylation reactions. Expression of MbtH-like proteins is important for a number of applications, including optimal production of native and genetically engineered secondary metabolites produced by mechanisms that employ NRPS enzymes. They also may serve as beacons to identify gifted actinomycetes and possibly other bacteria that encode multiple functional NRPS pathways for discovery of novel secondary metabolites by genome mining.     

Draft genome sequence of Streptomyces coelicoflavus ZG0656 reveals the putative biosynthetic gene cluster of acarviostatin family α-amylase inhibitors

Aims: The aims of this study are to obtain the draft genome sequence of Streptomyces coelicoflavus ZG0656, which produces novel acarviostatin family α‐amylase inhibitors, and then to reveal the putative acarviostatin‐related gene cluster and the biosynthetic pathway. Methods and Results: The draft genome sequence of S. coelicoflavus ZG0656 was generated using a shotgun approach employing a combination of 454 and Solexa sequencing technologies. Genome analysis revealed a putative gene cluster for acarviostatin biosynthesis, termed sct‐cluster. The cluster contains 13 acarviostatin synthetic genes, six transporter genes, four starch degrading or transglycosylation enzyme genes and two regulator genes. On the basis of bioinformatic analysis, we proposed a putative biosynthetic pathway of acarviostatins. The intracellular steps produce a structural core, acarviostatin I00‐7‐P, and the extracellular assemblies lead to diverse acarviostatin end products. Conclusions: The draft genome sequence of S. coelicoflavus ZG0656 revealed the putative biosynthetic gene cluster of acarviostatins and a putative pathway of acarviostatin production. Significance and Impact of the Study: To our knowledge, S. coelicoflavus ZG0656 is the first strain in this species for which a genome sequence has been reported. The analysis of sct‐cluster provided important insights into the biosynthesis of acarviostatins. This work will be a platform for producing novel variants and yield improvement.     

非核糖体多肽Surugamides生物合成基因簇镶嵌式结构的解析

【目的】多肽化合物Surugamides(sgm)生物合成基因簇包含4个非核糖体多肽合酶(NRPS)基因surA–D,负责2个NRPS生物合成途径。已有报道确认surA基因与SurugamideA产物相关,而surB基因与sgm F产物相关,但对surC和surD基因功能的归属尚没有实验证据。本工作拟在之前研究的基础上进一步确认surA和surD负责Surugamide A产物生物合成,为基因工程改造Surugamides生物合成途径以及研究其NRPS蛋白之间的识别机制提供理论依据。【方法】从海绵中分离放线菌并通过16S rRNA基因序列比对分析其分类单元。通过在线数据库antiSMASH分析基因组序列,发现天然产物生物合成基因簇。通过UPLC-Q-TOF-MS和~(13)CNMR鉴定化合物结构。把构建完成的同源重组双交换质粒导入链霉菌宿主后筛选基因缺失或替换突变株。【结果】从胄甲海绵来源链霉菌S.albidoflavus LHW3101基因组中发现了Surugamides生物合成基因簇,确认了该菌株发酵产物中的化合物sgmA和sgm F。构建了surB和surC基因同时缺失的突变株RJ9,发现RJ9不再产sgm F而仍然产Surugamide A。在缺失突变surB和surC基因的同时在surD基因前引入了组成型强启动子ermEp*,结果发现RJ9产SurugamideA水平是野生型菌株的约2倍。【结论】确认了surB和surC基因与sgmA产物无关。在surD基因前引入强启动子后显著提高了SurugamideA的产量,提示surD基因与sgmA产物相关,结合已报到surA基因与Surugamide A产物相关的证据,进一步确认了surA和surD基因负责Surugamide A生物合成的推论。     

Identification and engineering of the cytochalasin gene cluster from Aspergillus clavatus NRRL 1

Cytochalasins are a group of fungal secondary metabolites with diverse structures and bioactivities, including cytochalasin E produced by Aspergillus clavatus, which is a potent anti-angiogenic agent. Here, we report the identification and characterization of the cytochalasin gene cluster from A. clavatus NRRL 1. As a producer of cytochalasin E and K, the genome of A. clavatus was analyzed and the ∼30 kb ccs gene cluster was identified based on the presence of a polyketide synthase–nonribosomal peptide synthetases (PKS–NRPS) and a putative Baeyer–Villiger monooxygenase (BVMO). Deletion of the central PKS–NRPS gene, ccsA, abolished the production of cytochalasin E and K, confirming the association between the natural products and the gene cluster. Based on bioinformatic analysis, a putative biosynthetic pathway is proposed. Furthermore, overexpression of the pathway specific regulator ccsR elevated the titer of cytochalasin E from 25 mg/L to 175 mg/L. Our results not only shed light on the biosynthesis of cytochalasins, but also provided genetic tools for increasing and engineering the production.     

A Complex Ergovaline Gene Cluster in Epichloë Endophytes of Grasses

Clavicipitaceous fungal endophytes of the genera Epichloë and Neotyphodium form symbioses with grasses of the subfamily Pooideae, in which they can synthesize an array of bioprotective alkaloids. Some strains produce the ergopeptine alkaloid ergovaline, which is implicated in livestock toxicoses caused by ingestion of endophyte-infected grasses. Cloning and analysis of a nonribosomal peptide synthetase (NRPS) gene from Neotyphodium lolii revealed a putative gene cluster for ergovaline biosynthesis containing a single-module NRPS gene, lpsB, and other genes orthologous to genes in the ergopeptine gene cluster of Claviceps purpurea and the clavine cluster of Aspergillus fumigatus. Despite conservation of gene sequence, gene order is substantially different between the N. lolii, C. purpurea, and A. fumigatus ergot alkaloid gene clusters. Southern analysis indicated that the N. lolii cluster was linked with previously identified ergovaline biosynthetic genes dmaW and lpsA. The ergovaline genes are closely associated with transposon relics, including retrotransposons and autonomous and nonautonomous DNA transposons. All genes in the cluster were highly expressed in planta, but expression was very low or undetectable in mycelia from axenic culture. This work provides a genetic foundation for elucidating biochemical steps in the ergovaline pathway, the ecological role of individual ergot alkaloid compounds, and the regulation of their synthesis in planta.     

The lipopeptide antibiotic A54145 biosynthetic gene cluster from Streptomyces fradiae

Ca(2+)-dependent cyclic lipodepsipeptides are an emerging class of antibiotics for the treatment of infections caused by Gram-positive pathogens. These compounds are synthesized by nonribosomal peptide synthetase (NRPS) complexes encoded by large gene clusters. The gene cluster encoding biosynthetic pathway enzymes for the Streptomyces fradiae A54145 NRP was cloned from a cosmid library and characterized. Four NRPS-encoding genes, responsible for subunits of the synthetase, as well as genes for accessory functions such as acylation, methylation and hydroxylation, were identified by sequence analysis in a 127 kb region of DNA that appears to be located subterminally in the bacterial chromosome. Deduced epimerase domain-encoding sequences within the NRPS genes indicated a D: -stereochemistry for Glu, Lys and Asn residues, as observed for positionally analogous residues in two related compounds, daptomycin, and the calcium-dependent antibiotic (CDA) produced by Streptomyces roseosporus and Streptomyces coelicolor, respectively. A comparison of the structure and the biosynthetic gene cluster of A54145 with those of the related peptides showed many similarities. This information may contribute to the design of experiments to address both fundamental and applied questions in lipopeptide biosynthesis, engineering and drug development.     

Elucidation of the complete ferrichrome A biosynthetic pathway in Ustilago maydis

Iron is an important element for many essential processes in living organisms. To acquire iron, the basidiomycete Ustilago maydis synthesizes the iron‐chelating siderophores ferrichrome and ferrichrome A. The chemical structures of these siderophores have been elucidated long time ago but so far only two enzymes involved in their biosynthesis have been described. Sid1, an ornithine monoxygenase, is needed for the biosynthesis of both siderophores, and Sid2, a non‐ribosomal peptide synthetase (NRPS), is involved in ferrichrome generation. In this work we identified four novel enzymes, Fer3, Fer4, Fer5 and Hcs1, involved in ferrichrome A biosynthesis in U. maydis. By HPLC‐MS analysis of siderophore accumulation in culture supernatants of deletion strains, we show that Fer3, an NRPS, Fer4, an enoyl‐coenzyme A (CoA)‐hydratase, and Fer5, an acylase, are required for ferrichrome A production. We demonstrate by conditional expression of the hydroxymethyl glutaryl (HMG)‐CoA synthase Hcs1 in U. maydis that HMG‐CoA is an essential precursor for ferrichrome A. In addition, we heterologously expressed and purified Hcs1, Fer4 and Fer5, and demonstrated the enzymatic activities by in vitro experiments. Thus, we describe the first complete fungal siderophore biosynthetic pathway by functionally characterizing four novel genes responsible for ferrichrome A biosynthesis in U. maydis.     

Chemical variation from the neoantimycin depsipeptide assembly line

Here we report the biosynthetic pathway for the neoantimycin and present three novel neoantimycin analogues, neoantimycin D (1), E (2) and F (3), from this assembly system from Streptoverticillium orinoci. Identification of these novel neoantimycin variants was achieved by selective MS/MS interrogation of natural product extracts using diagnostic fragments of the known neoantimycins. Their structures, including the absolute configurations, were elucidated using a combination of NMR experiments, detailed MS/MS experiments and the advanced Marfey’s method. The biosynthetic pathway of neoantimycin was dissected by genome sequencing data analysis for the first time, which includes a hybrid nonribosomal peptide synthetase (NRPS) and polyketide synthetase (PKS) assembly lines.     

Identification and characterization of the pyridomycin biosynthetic gene cluster of Streptomyces pyridomyceticus NRRL B-2517

Pyridomycin is a structurally unique antimycobacterial cyclodepsipeptide containing rare 3-(3-pyridyl)-l-alanine and 2-hydroxy-3-methylpent-2-enoic acid moieties. The biosynthetic gene cluster for pyridomycin has been cloned and identified from Streptomyces pyridomyceticus NRRL B-2517. Sequence analysis of a 42.5-kb DNA region revealed 26 putative open reading frames, including two nonribosomal peptide synthetase (NRPS) genes and a polyketide synthase gene. A special feature is the presence of a polyketide synthase-type ketoreductase domain embedded in an NRPS. Furthermore, we showed that PyrA functioned as an NRPS adenylation domain that activates 3-hydroxypicolinic acid and transfers it to a discrete peptidyl carrier protein, PyrU, which functions as a loading module that initiates pyridomycin biosynthesis in vivo and in vitro. PyrA could also activate other aromatic acids, generating three pyridomycin analogues in vivo.     

Structures and biosynthesis of 12-membered macrocyclic depsipeptides from Streptomyces sp. ML55

Two novel depsipeptides (1–2) were isolated from Streptomyces sp. ML55 together with two known analogues (3–4). Their structures were elucidated using a combination of NMR experiments, as well as detailed MS/MS experiments. The biosynthetic pathway of isolated compounds was dissected by genome sequencing data analysis for a hybrid nonribosomal peptide synthetase (NRPS) and polyketide synthetase (PKS) assembly line.     

Characterization of the saframycin A gene cluster from Streptomyces lavendulae NRRL 11002 revealing a nonribosomal peptide synthetase system for assembling the unusual tetrapeptidyl skeleton in an iterative manner

Saframycin A (SFM-A), produced by Streptomyces lavendulae NRRL 11002, belongs to the tetrahydroisoquinoline family of antibiotics, and its core is structurally similar to the core of ecteinascidin 743, which is a highly potent antitumor drug isolated from a marine tunicate. In this study, the biosynthetic gene cluster for SFM-A was cloned and localized to a 62-kb contiguous DNA region. Sequence analysis revealed 30 genes that constitute the SFM-A gene cluster, encoding an unusual nonribosomal peptide synthetase (NRPS) system and tailoring enzymes and regulatory and resistance proteins. The results of substrate prediction and in vitro characterization of the adenylation specificities of this NRPS system support the hypothesis that the last module acts in an iterative manner to form a tetrapeptidyl intermediate and that the colinearity rule does not apply. Although this mechanism is different from those proposed for the SFM-A analogs SFM-Mx1 and safracin B (SAC-B), based on the high similarity of these systems, it is likely they share a common mechanism of biosynthesis as we describe here. Construction of the biosynthetic pathway of SFM-Y3, an aminated SFM-A, was achieved in the SAC-B producer (Pseudomonas fluorescens). These findings not only shed new insight on tetrahydroisoquinoline biosynthesis but also demonstrate the feasibility of engineering microorganisms to generate structurally more complex and biologically more active analogs by combinatorial biosynthesis.     

Turnerbactin,a Novel Triscatecholate Siderophore from the Shipworm Endosymbiont Teredinibacter turnerae T7901

Shipworms are marine bivalve mollusks (Family Teredinidae) that use wood for shelter and food. They harbor a group of closely related, yet phylogenetically distinct, bacterial endosymbionts in bacteriocytes located in the gills. This endosymbiotic community is believed to support the host''s nutrition in multiple ways, through the production of cellulolytic enzymes and the fixation of nitrogen. The genome of the shipworm endosymbiont Teredinibacter turnerae T7901 was recently sequenced and in addition to the potential for cellulolytic enzymes and diazotrophy, the genome also revealed a rich potential for secondary metabolites. With nine distinct biosynthetic gene clusters, nearly 7% of the genome is dedicated to secondary metabolites. Bioinformatic analyses predict that one of the gene clusters is responsible for the production of a catecholate siderophore. Here we describe this gene cluster in detail and present the siderophore product from this cluster. Genes similar to the entCEBA genes of enterobactin biosynthesis involved in the production and activation of dihydroxybenzoic acid (DHB) are present in this cluster, as well as a two-module non-ribosomal peptide synthetase (NRPS). A novel triscatecholate siderophore, turnerbactin, was isolated from the supernatant of iron-limited T. turnerae T7901 cultures. Turnerbactin is a trimer of N-(2,3-DHB)-L-Orn-L-Ser with the three monomeric units linked by Ser ester linkages. A monomer, dimer, dehydrated dimer, and dehydrated trimer of 2,3-DHB-L-Orn-L-Ser were also found in the supernatant. A link between the gene cluster and siderophore product was made by constructing a NRPS mutant, TtAH03. Siderophores could not be detected in cultures of TtAH03 by HPLC analysis and Fe-binding activity of culture supernatant was significantly reduced. Regulation of the pathway by iron is supported by identification of putative Fur box sequences and observation of increased Fe-binding activity under iron restriction. Evidence of a turnerbactin fragment was found in shipworm extracts, suggesting the production of turnerbactin in the symbiosis.     

Distribution of NRPS gene families within the Neotyphodium/Epichloë complex

Neotyphodium and Epichloë spp are closely related asexual and sexual endophytic fungi, respectively, that form mutualistic associations with cool season grasses of the subfamily Pooideae. The endophytes confer a number of advantages to their hosts, but also can cause animal toxicoses and these effects are, in many cases, due to the production of fungal secondary metabolites. In filamentous fungi, secondary metabolite genes are commonly clustered and, for those pathways involved in non-ribosomal peptide synthesis, a non-ribosomal peptide synthetase (NRPS) gene is always found as a key component of the cluster. Members of this gene family encode large multifunctional enzymes that synthesize a diverse range of bioactive compounds and in numerous cases have been shown to serve as pathogenicity or virulence factors, in addition to suggested roles in niche adaptation. We have used a degenerate PCR approach to identify members of the NRPS gene family from symbiotic fungi of the Neotyphodium/Epichloë complex, and have shown that collectively, at least 12 NRPS genes exist within the genomes examined. This suggests that secondary metabolites are important during the life cycles of these fungi with their hosts. Indeed, both the ergovaline and peramine biosynthetic pathways, which confer competitive abilities to Neotyphodium and Epichloë symbioses, contain NRPS genes at their core. The distribution of these genes among different Neotyphodium/Epichloë lineages suggests that a common ancestor contributed most of the complement of NRPS genes, which have been either retained or lost during the evolution of these fungi.