Identification and Heterologous Expression of the Chaxamycin Biosynthesis Gene Cluster from Streptomyces leeuwenhoekii

Streptomyces leeuwenhoekii, isolated from the hyperarid Atacama Desert, produces the new ansamycin-like compounds chaxamycins A to D, which possess potent antibacterial activity and moderate antiproliferative activity. We report the development of genetic tools to manipulate S. leeuwenhoekii and the identification and partial characterization of the 80.2-kb chaxamycin biosynthesis gene cluster, which was achieved by both mutational analysis in the natural producer and heterologous expression in Streptomyces coelicolor A3(2) strain M1152. Restoration of chaxamycin production in a nonproducing ΔcxmK mutant (cxmK encodes 3-amino-5-hydroxybenzoic acid [AHBA] synthase) was achieved by supplementing the growth medium with AHBA, suggesting that mutasynthesis may be a viable approach for the generation of novel chaxamycin derivatives.     

Cloning and Heterologous Expression of the Thioviridamide Biosynthesis Gene Cluster from Streptomyces olivoviridis

Thioviridamide is a unique peptide antibiotic containing five thioamide bonds from Streptomyces olivoviridis. Draft genome sequencing revealed a gene (the tvaA gene) encoding the thioviridamide precursor peptide. The thioviridamide biosynthesis gene cluster was identified by heterologous production of thioviridamide in Streptomyces lividans.     

链霉菌Streptomyces tenebrarius H6中与抗生素有关的糖生物合成基因的克隆

链霉菌S.tenebrarius H6产生多种氨基糖甙类抗生素,主要有阿普霉素、妥普霉素及卡那霉素B,其中阿普霉素因含有8碳糖的一种特殊结构令人注目,它的抗菌谱广,特别是对革兰氏阴性菌有较强的抗菌活性,不容易产生耐药性,对已有的耐药菌产生的氨基糖苷转移酶等失活酶仍有抵抗力.主要用于牛、猪、鸡等的大肠杆菌、沙门氏菌和支原体所引起的白痢、腹泻和肺炎等疾病.迄今有关八碳糖生物合成基因簇的研究在国内外尚无报道,在该菌株开展有关糖合成代谢基因的研究有着一定的意义.     

Multiplexed Integrating Plasmids for Engineering of the Erythromycin Gene Cluster for Expression in Streptomyces spp. and Combinatorial Biosynthesis

Bacteria in the genus Streptomyces and its close relatives are prolific producers of secondary metabolites with antibiotic activity. Genome sequencing of these bacteria has revealed a rich source of potentially new antibiotic pathways, whose products have never been observed. Moreover, these new pathways can provide novel genes that could be used in combinatorial biosynthesis approaches to generate unnatural analogues of existing antibiotics. We explore here the use of multiple orthologous integrating plasmid systems, based on the int/attP loci from phages TG1, SV1, and ϕBT1, to express the polyketide synthase (PKS) for erythromycin in a heterologous Streptomyces host. Streptomyces strains containing the three polyketide synthase genes eryAI, eryAII, and eryAIII expressed from three different integrated plasmids produced the aglycone intermediate, 6-deoxyerythronolide B (6-dEB). A further pair of integrating plasmids, both derived from the ϕC31 int/attP locus, were constructed carrying a gene cassette for glycosylation of the aglycone intermediates, with or without the tailoring gene, eryF, required for the synthesis of erythronolide B (EB). Liquid chromatography-mass spectrometry of the metabolites indicated the production of angolosaminyl-6-dEB and angolosaminyl-EB. The advantages of using multiplexed integrating plasmids for engineering expression and for combinatorial biosynthesis were demonstrated.     

圈卷产色链霉菌尼可霉素生物合成基因-sanK的克隆及功能研究

利用染色体步移策略,以尼可霉素生物合成相关的基因片段为探针,从圈卷产色链霉菌中克隆到了一个大约１０ｋｂ的ＤＮＡ片段。对其中１．８ｋｂ的ＰｖｕⅡ－ＳａｃⅡ片段进行了序列分析,结果表明：此片段中含有一个具有１１７０个核苷酸的完整开放阅读框,起始密码子为４４７位的ＡＴＧ,终止密码子为１６１４位的ＴＧＡ,推测其编码一个３８９个氨基酸的蛋白质产物。利用ＢＬＡＳＴＸ程序进行了分析揭示,此基因编码一个肌氨酸单体     

Development of a Genetic System for Combinatorial Biosynthesis of Lipopeptides in Streptomyces fradiae and Heterologous Expression of the A54145 Biosynthesis Gene Cluster

A54145 factors are calcium-dependent lipopeptide antibiotics produced by Streptomyces fradiae NRRL 18160. A54145 is structurally related to the clinically important daptomycin, and as such may be a useful scaffold for the development of a novel lipopeptide antibiotic. We developed methods to genetically manipulate S. fradiae by deletion mutagenesis and conjugal transfer of plasmids from Escherichia coli. Cloning the complete pathway on a bacterial artificial chromosome (BAC) vector and the construction of ectopic trans-complementation with plasmids utilizing the φC31 or φBT1 site-specific integration system allowed manipulation of A54145 biosynthesis. The BAC clone pDA2002 was shown to harbor the complete A54145 biosynthesis gene cluster by heterologous expression in Streptomyces ambofaciens and Streptomyces roseosporus strains in yields of >100 mg/liter. S. fradiae mutants defective in LptI methyltransferase function were constructed, and they produced only A54145 factors containing glutamic acid (Glu₁₂), at the expense of factors containing 3-methyl-glutamic acid (3mGlu₁₂). This provided a practical route to produce high levels of pure Glu₁₂-containing lipopeptides. A suite of mutant strains and plasmids was created for combinatorial biosynthesis efforts focused on modifying the A54145 peptide backbone to generate a compound with daptomycin antibacterial activity and activity in Streptococcus pneumoniae pulmonary infections.The calcium-dependent cyclic acidic lipodepsipeptide antibiotics were first reported in the 1980s and 1990s (8). These include A21978C, produced by Streptomyces roseosporus (17, 18), calcium-dependent antibiotic (CDA), produced by Streptomyces coelicolor (26), and A54145, produced by Streptomyces fradiae NRRL 18160 (11, 12, 23). A21978C (Fig. ​(Fig.1)1) has been of particular interest because the N-decanoyl lipid tail derivative of the A21978C peptide is daptomycin (8), which is approved for the treatment of complicated skin and skin structure infections caused by Gram-positive bacteria (2) and for bacteremia and right-sided endocarditis caused by Staphylococcus aureus, including strains resistant to methicillin (MRSA) (21). Daptomycin lacks efficacy in community-acquired pneumonia (CAP) infections, even though it is very active in vitro against the predominant pathogen, Streptococcus pneumoniae (8, 43). In vitro studies have shown that daptomycin becomes sequestered in bovine pulmonary surfactant, most likely in the lipid component, and has decreased antibacterial potency against Gram-positive pathogens (46); this may be a significant factor contributing to the poor clinical efficacy in CAP. Attempts to improve the efficacy of daptomycin through chemical modifications of the lipid side chain or additions to the δ-amino group of ornithine (Orn₆) (reviewed in reference 8), or by molecular engineering of peptide assembly (4, 13, 25, 37-39), have not generated a lead molecule with sufficient in vivo efficacy in a mouse pneumonia model for S. pneumoniae.Open in a separate windowFIG. 1.Structures of the lipopeptide antibiotics and NRPS protein subunit relationships. (Top) A54145 factors normally produced by S. fradiae. Note that factors A, A₁, D, and F have Glu at position 12, and factors B, B₁, C, and E have 3mGlu at position 12. (Bottom) A21978C factors normally produced by S. rosesosporus and daptomycin.A54145 factors share a number of features in common with daptomycin, but they differ at several amino acid positions (Fig. ​(Fig.1).1). The most biologically active A54145 factors against S. aureus contain four modified amino acids, l-hydroxy-Asn₂ (hAsn₂), sarcosine₅ (Sar₅), l-methoxy-Asp₉ (mOAsp₉), and l-3-methyl-Glu₁₂ (3mGlu₁₂) (14). During a standard fermentation, multiple A54145 factors are produced as the result of natural variation at position 12 (3mGlu or Glu), at position 13 (Ile or Val) and at the lipid tail attached to the peptide core. The A54145 factors A, A₁, and D (collectively designated the A-core) have the identical peptide containing Glu₁₂ and Ile₁₃ but have different lipid tails, whereas factors B, B₁, and E (the B-core) contain 3mGlu₁₂ and Ile₁₃. During fermentation of S. fradiae, factor A accumulates as a major component but plateaus early, and factor B₁ accumulates preferentially late in the fermentation (11, 12). In studies at Eli Lilly and Company, it was shown that the B-core factors were slightly more potent antibiotics, but factor B was substantially more toxic than its Glu₁₂-containing counterpart, factor A₁ (14).During the development of molecular engineering approaches to modify daptomycin biosynthesis, the genes for A54145 lipopeptide biosynthesis (lpt) were cloned and sequenced to provide nonribosomal peptide synthetase (NRPS) modules and subunits to exchange with those of daptomycin (37). Since some of the A54145 A-core factors were shown to be much less inhibited by bovine surfactant than daptomycin (40), the A54145 A-core lipopeptides should be useful starting points for both chemical and molecular engineering modification studies. We initiated a program to develop molecular genetics methods, with plasmids and host cloning strains to facilitate molecular engineering of A54145 biosynthesis in S. fradiae.In this report, we describe the engineering of a bacterial artificial chromosome (BAC) containing the A54145 biosynthesis genes by using λ-Red-mediated recombination in Escherichia coli and expression of the A54145 biosynthesis pathway in heterologous streptomycetes. The development of S. fradiae strains deleted for multiple A54145 genes and the construction of plasmid vectors with conjugation and site-specific integration functions for ectopic expression of sets of A54145 biosynthesis genes in S. fradiae and combinatorial biosynthesis (40) are discussed. This genetic system was used to generate a strain with deletion of lptI, a gene that encodes a methyltransferase involved in the biosynthesis of 3mGlu₁₂, and the mutant produced the desired A-core lipopeptides containing Glu₁₂, which are important starting materials for medicinal chemistry approaches to produce novel lipopeptides.     

Cloning and Characterization of a Gene Cluster for Hatomarubigin Biosynthesis in Streptomyces sp. Strain 2238-SVT4

Streptomyces sp. strain 2238-SVT4 produces hatomarubigins A, B, C, and D, which belong to the angucycline family. Among them, hatomarubigin D has a unique dimeric structure with a methylene linkage. PCR using aromatase and cyclase gene-specific primers identified the hrb gene cluster for angucycline biosynthesis in Streptomyces sp. 2238-SVT4. The cluster consisted of 30 open reading frames, including those for the minimal polyketide synthase, ketoreductase, aromatase, cyclase, O-methyltransferase, oxidoreductase, and oxygenase genes. Expression of a part of the gene cluster containing hrbR1 to hrbX in Streptomyces lividans TK23 resulted in the production of hatomarubigins A, B, and C. Hatomarubigin D was obtained from the conversion of hatomarubigin C by a purified enzyme encoded by hrbY, among the remaining genes.The angucycline antibiotics are a large group of naturally occurring aromatic polyketides of microbial origin (11, 15). They exhibit a wide range of biological activities, which include antibacterial, antiviral, antitumor, enzyme inhibitory, and platelet aggregation inhibitory effects. Although all the members contain a benz[a]anthraquinone skeleton of decaketide origin, their structural diversity is very broad and they have a wide variety of oxidation states. Hatomarubigins A, B, C, and D (Fig. ​(Fig.1)1) belong to the angucycline family and reverse colchicine resistance in multidrug-resistant tumor cells (8). Among them, hatomarubigin D is a unique hatomarubigin C dimer with a methylene linkage. Such a dimer has not been reported previously, and little is known about the mechanism of the methylene bridge formation between two aromatic rings. In this study, a gene cluster for hatomarubigin biosynthesis was identified in Streptomyces sp. strain 2238-SVT4, and a part of the gene cluster was expressed in Streptomyces lividans to produce the hatomarubigins.Open in a separate windowFIG. 1.Structures of angucycline antibiotics.     

Characterization of the Gene Cluster Responsible for Cylindrospermopsin Biosynthesis

Toxic cyanobacterial blooms cause economic losses and pose significant public health threats on a global scale. Characterization of the gene cluster for the biosynthesis of the cyanobacterial toxin cylindrospermopsin (cyr) in Cylindrospermopsis raciborskii AWT205 is described, and the complete biosynthetic pathway is proposed. The cyr gene cluster spans 43 kb and is comprised of 15 open reading frames containing genes required for the biosynthesis, regulation, and export of the toxin. Biosynthesis is initiated via an amidinotransfer onto glycine followed by five polyketide extensions and subsequent reductions, and rings are formed via Michael additions in a stepwise manner. The uracil ring is formed by a novel pyrimidine biosynthesis mechanism and tailoring reactions, including sulfation and hydroxylation that complete biosynthesis. These findings enable the design of toxic strain-specific probes and allow the future study of the regulation and biological role of cylindrospermopsin.     

Analysis of the Mycosamine Biosynthesis and Attachment Genes in the Nystatin Biosynthetic Gene Cluster of Streptomyces noursei ATCC 11455

The polyene macrolide antibiotic nystatin produced by Streptomyces noursei contains a deoxyaminosugar mycosamine moiety attached to the C-19 carbon of the macrolactone ring through the β-glycosidic bond. The nystatin biosynthetic gene cluster contains three genes, nysDI, nysDII, and nysDIII, encoding enzymes with presumed roles in mycosamine biosynthesis and attachment as glycosyltransferase, aminotransferase, and GDP-mannose dehydratase, respectively. In the present study, the functions of these three genes were analyzed. The recombinant NysDIII protein was expressed in Escherichia coli and purified, and its in vitro GDP-mannose dehydratase activity was demonstrated. The nysDI and nysDII genes were inactivated individually in S. noursei, and analyses of the resulting mutants showed that both genes produced nystatinolide and 10-deoxynystatinolide as major products. Expression of the nysDI and nysDII genes in trans in the respective mutants partially restored nystatin biosynthesis in both cases, supporting the predicted roles of these two genes in mycosamine biosynthesis and attachment. Both antifungal and hemolytic activities of the purified nystatinolides were shown to be strongly reduced compared to those of nystatin, confirming the importance of the mycosamine moiety for the biological activity of nystatin.     

Structure and Function of sanV: A Gene Involved in Nikkomycin Biosynthesis of Streptomyces ansochromogenes

A 6.3-kb BamHI-BglII DNA fragment was cloned from cos20 by using chromosome walking strategy. It was partially sequenced with the result that there is a possible ORF of 1272 nucleotides. The ORF designated sanV was deposited in GenBank under accession no. AF469955. Database search indicated that the deduced protein of sanV shows 28% identity and 44% similarity over 405 amino acid residues to the large component (E) of glutamate mutases from Clostridium cochlearium. Gene disruption was performed to study the function of sanV. It was found that sanV disruptants exhibited much poorer inhibition to the test strain than the wild-type S. ansochromogenes 7100. Furthermore, HPLC analysis indicated that the sanV disruptants almost did not produce nikkomycins X and Z, whereas they accumulated new nikkomycins O_x and O_z, which revealed that sanV is an important gene involved in the biosynthesis of the peptidyl moiety of nikkomycins. Received: 13 June 2002 / Accepted: 27 July 2002     

Glycerol Utilization Gene Cluster in Streptomyces clavuligerus

The Streptomyces clavuligerus ATCC 27064 glycerol cluster gylR-glpF1K1D1 is induced by glycerol but is not affected by glucose. S. clavuligerus growth and clavulanic acid production are stimulated by glycerol, but this does not occur in a glpK1-deleted mutant. Amplification of glpK1D1 results in transformants yielding larger amounts of clavulanic acid in the wild-type strain and in overproducer S. clavuligerus Gap15-7-30 or S. clavuligerus ΔrelA strains.Streptomyces clavuligerus ATCC 27064 produces the β-lactamase inhibitor clavulanic acid (CA). This compound is formed from arginine (17) and the three-carbon molecule glyceraldehyde-3-phosphate (6) which are condensed by the carboxyethylarginine synthase, the first enzyme of the pathway, encoded by ceaS2. Mutants disrupted in this gene do not produce CA in tryptic soy broth or starch-asparagine (SA) medium but form moderate amounts of CA in glycerol-supplemented media, probably due to glycerol utilization through the duplicated CeaS1 carboxyethylarginine synthase (10).The role of d-glyceraldehyde-3-phosphate as a CA precursor was further supported by the construction of a glyceraldehyde-3-phosphate dehydrogenase (gap1) mutant of S. clavuligerus, which produces 180 to 210% CA in comparison to the wild-type strain due to higher availability of the glyceraldehyde-3-phosphate precursor (9).Genes for glycerol utilization in Streptomyces coelicolor form an operon, gylCABX (15, 16), containing a gene for a putative glycerol transporter, a glycerol kinase, a glycerol-3-phosphate dehydrogenase, and a gene of unknown function. They are preceded by gylR (5), which encodes a glycerol-inducible repressor controlling both gylR and the gylCABX operon. Glycerol induction and glucose catabolite repression of the glp genes are thought to be directly related to the GylR protein in S. coelicolor (5).Due to the importance of the glycerol flow for CA production, we decided to analyze the glycerol-utilizing gene cluster of S. clavuligerus.     

Cloning, Function, and Expression of sanS: A Gene Essential for Nikkomycin Biosynthesis of Streptomyces ansochromogenes

A 2-kb SmaI DNA fragment was cloned from the cosmid library of Streptomyces ansochromogenes. This DNA fragment contains a complete open reading frame which is 1275 bp in length, designated sanS (GenBank accession no. AF322179). The deduced SanS protein consists of 424 amino acids and belongs to a superfamily of enzymes with an unusual ATP-grasp fold. The disruption and complementation of sanS indicated that sanS is essential for nikkomycin biosynthesis in Streptomyces ansochromogenes. The sanS gene was subcloned into expression vector pET23b and overexpressed in E. coli BL21 (DE3). The protein was then purified and showed ATPase activity.     

Cloning, Sequencing, and Function of sanF: A Gene Involved in Nikkomycin Biosynthesis of Streptomyces ansochromogenes

A 111-bp DNA fragment related to nikkomycin biosynthesis of Streptomyces ansochromogenes 7100 was obtained with the method of reverse genetics. Then, a 2.2-kb DNA fragment was cloned from the DNA library of S. ansochromogenes 7100 by using the 111-bp fragment as a probe. Sequence analysis showed that the fragment contains one complete open reading frame (ORF) that encodes a 219-amino acid (aa) protein, and this gene was designated sanF (GenBank Accession No. AF223971). The function of the sanF gene was studied by a strategy of gene disruption, and the resulting sanF mutants lost the ability to synthesize biologically active nikkomycin, indicating that sanF is essential for nikkomycin biosynthesis. Received: 17 April 2000 / Accepted: 23 May 2000     

Disruption of the ste22 Gene Encoding a Glycosyltransferase and Its Function in Biosynthesis of Ebosin in Streptomyces sp. 139

Streptomyces sp.139 produces an exopolysaccharide (EPS) designated Ebosin with remarkable anti-rheumatic arthritis activity in vivo. The ste (Streptomyces eps) gene cluster required for Ebosin biosynthesis has been identified. According to similarities with other proteins in the database, ste22 shows high homology with glycosyltransferases originated from different microorganisms. In this study, the ste22 gene was disrupted by double crossover via homologous recombination. The EPS produced by the mutant strain Streptomyces sp.139 (ste22⁻) has a different monosaccharide composition profile in comparison with that of Ebosin. This derivative of Ebosin retained the original antagonistic activity of IL-1R in vitro but lost the bioactivities of anti-inflammation and pain relief in vivo.     

Aflatoxin Biosynthesis Cluster Gene cypA Is Required for G Aflatoxin Formation

Aspergillus flavus isolates produce only aflatoxins B1 and B2, while Aspergillus parasiticus and Aspergillus nomius produce aflatoxins B1, B2, G1, and G2. Sequence comparison of the aflatoxin biosynthesis pathway gene cluster upstream from the polyketide synthase gene, pksA, revealed that A. flavus isolates are missing portions of genes (cypA and norB) predicted to encode, respectively, a cytochrome P450 monooxygenase and an aryl alcohol dehydrogenase. Insertional disruption of cypA in A. parasiticus yielded transformants that lack the ability to produce G aflatoxins but not B aflatoxins. The enzyme encoded by cypA has highest amino acid identity to Gibberella zeae Tri4 (38%), a P450 monooxygenase previously shown to be involved in trichodiene epoxidation. The substrate for CypA may be an intermediate formed by oxidative cleavage of the A ring of O-methylsterigmatocystin by OrdA, the P450 monooxygenase required for formation of aflatoxins B1 and B2.     

Widespread Occurrence and Lateral Transfer of the Cyanobactin Biosynthesis Gene Cluster in Cyanobacteria

Cyanobactins are small cyclic peptides produced by cyanobacteria. Here we demonstrate the widespread but sporadic occurrence of the cyanobactin biosynthetic pathway. We detected a cyanobactin biosynthetic gene in 48 of the 132 strains included in this study. Our results suggest that cyanobactin biosynthetic genes have a complex evolutionary history in cyanobacteria punctuated by a series of ancient horizontal gene transfer events.