A macrolide 3-O-acyltransferase gene from the midecamycin-producing species Streptomyces mycarofaciens.

The Streptomyces mycarofaciens mdmB gene encodes a 3-O-acyltransferase that catalyzes the addition of acetyl and propionyl groups to position 3 of the lactone ring in 16-member macrolide antibiotics like midecamycin and spiramycin. A putative O-methyltransferase gene (mdmC) is immediately downstream of mdmB, and both of these genes are closely linked to the mdmA midecamycin resistance gene.     

多烯大环内酯类抗生素生物合成基因簇中调控因子的研究进展

多烯大环内酯类抗生素具有良好的抗真菌活性,广泛应用于医疗卫生、食品加工和农业生产领域。随着高通量测序技术和生物信息学技术的发展,越来越多的链霉菌抗生素生物合成基因簇被发现和鉴定,调控因子作为生物合成基因簇中的重要组成部分,在庞大复杂的调控网络中起着至关重要的作用。本文总结了链霉菌中重要的调控因子类型,综述了多烯大环内酯类抗生素生物合成基因簇中调控因子的生物学功能、结合位点、作用机制等研究进展,并展望了后续研究工作。     

Glycosylation of macrolide antibiotics. Purification and kinetic studies of a macrolide glycosyltransferase from Streptomyces antibioticus

The oleD gene has been identified in the oleandomycin producer Streptomyces antibioticus and it codes a macrolide glycosyltransferase that is able to transfer a glucose moiety from UDP-glucose (UDP-Glc) to many macrolides. The glycosyltransferase coded by the oleD gene has been purified 371-fold from a Streptomyces lividans clone expressing this protein. The reaction product was isolated, and its structure determined by NMR spectroscopy. The kinetic mechanism of the reaction was analyzed using the macrolide antibiotic lankamycin (LK) as substrate. The reaction operates via a compulsory order mechanism. This has been shown by steady-state kinetic studies and by isotopic exchange reactions at equilibrium. LK binds first to the enzyme, followed by UDP-glucose. A ternary complex is thus formed prior to transfer of glucose. UDP is then released, followed by the glycosylated lankamycin (GS-LK). A pH study of the reaction was performed to determine values for the molecular pK values, suggesting possible amino acid residues involved in the catalytic process.     

Characterization of the P450 monooxygenase NysL, responsible for C-10 hydroxylation during biosynthesis of the polyene macrolide antibiotic nystatin in Streptomyces noursei

The nysL gene, encoding a putative P450 monooxygenase, was identified in the nystatin biosynthetic gene cluster of Streptomyces noursei. Although it has been proposed that NysL is responsible for hydroxylation of the nystatin precursor, experimental evidence for this activity was lacking. The nysL gene was inactivated in S. noursei by gene replacement, and the resulting mutant was shown to produce 10-deoxynystatin. Purification and an in vitro activity assay for 10-deoxynystatin demonstrated its antifungal activity being equal to that of nystatin. The NysL protein was expressed heterologously in Escherichia coli as a His-tagged protein and used in an enzyme assay with 10-deoxynystatin as a substrate. The results obtained clearly demonstrated that NysL is a hydroxylase responsible for the post-polyketide synthase modification of 10-deoxynystatin at position C-10. Kinetic studies with the purified recombinant enzyme allowed determination of K(m) and k(cat) and revealed no inhibition of recombinant NysL by either the substrate or the product. These studies open the possibility for in vitro evolution of NysL aimed at changing its specificity, thereby providing new opportunities for engineered biosynthesis of novel nystatin analogues hydroxylated at alternative positions of the macrolactone ring.     

A Sorangium cellulosum (myxobacterium) gene cluster for the biosynthesis of the macrolide antibiotic soraphen A: cloning, characterization, and homology to polyketide synthase genes from actinomycetes.

A 40-kb region of DNA from Sorangium cellulosum So ce26, which contains polyketide synthase (PKS) genes for synthesis of the antifungal macrolide antibiotic soraphen A, was cloned. These genes were detected by homology to Streptomyces violaceoruber genes encoding components of granaticin PKS, thus extending this powerful technique for the identification of bacterial PKS genes, which has so far been applied only to actinomycetes, to the gram-negative myxobacteria. Functional analysis by gene disruption has indicated that about 32 kb of contiguous DNA of the cloned region contains genes involved in soraphen A biosynthesis. The nucleotide sequence of a 6.4-kb DNA fragment, derived from the region with homology to granaticin PKS genes, was determined. Analysis of this sequence has revealed the presence of a single large open reading frame beginning and ending outside the 6.4-kb fragment. The deduced amino acid sequence indicates the presence of a domain with a high level of similarity to beta-ketoacyl synthases that are involved in polyketide synthesis. Other domains with high levels of similarity to regions of known polyketide biosynthetic functions were identified, including those for acyl transferase, acyl carrier protein, ketoreductase, and dehydratase. We present data which indicate that soraphen A biosynthesis is catalyzed by large, multifunctional enzymes analogous to other bacterial PKSs of type I.     

Combinatorial biosynthesis and antibacterial evaluation of glycosylated derivatives of 12-membered macrolide antibiotic YC-17

Expression plasmids carrying different deoxysugar biosynthetic gene cassettes and the gene encoding a substrate-flexible glycosyltransferase DesVII were constructed and introduced into Streptomyces venezuelae YJ003 mutant strain bearing a deletion of a desosamine biosynthetic (des) gene cluster. The resulting recombinants produced macrolide antibiotic YC-17 analogs possessing unnatural sugars replacing native d-desosamine. These metabolites were isolated and further purified using chromatographic techniques and their structures were determined as d-quinovosyl-10-deoxymethynolide, l-rhamnosyl-10-deoxymethynolide, l-olivosyl-10-deoxymethynolide, and d-boivinosyl-10-deoxymethynolide on the basis of 1D and 2D NMR and MS analyses and the stereochemistry of sugars was confirmed using coupling constant values and NOE correlations. Their antibacterial activities were evaluated in vitro against erythromycin-susceptible and -resistant Enterococcus faecium and Staphylococcus aureus. Substitution with l-rhamnose displayed better antibacterial activity than parent compound YC-17 containing native sugar d-desosamine. The present study on relationships between chemical structures and antibacterial activities could be useful in generation of novel advanced antibiotics utilizing combinatorial biosynthesis approach.     

Characterization of Renibacterium salmoninarum with reduced susceptibility to macrolide antibiotics by a standardized antibiotic susceptibility test

Three cohorts of juvenile and subadult Chinook salmon Oncorhynchus tshawytscha received multiple treatments with macrolide antibiotics for bacterial kidney disease (BKD) during rearing in a captive broodstock program. A total of 77 mortalities among the cohorts were screened for Renibacterium salmoninarum, the etiologic agent of BKD, by agar culture from kidney, and isolates from 7 fish were suitable for growth testing in the presence of macrolide antibiotics. The minimum inhibitory concentration (MIC) of erythromycin and azithromycin was determined by a modification of the standardized broth assay using defined medium. The American Type Culture Collection (ATCC) type strain 33209 exhibited a MIC of 0.008 microg m(-1) to either erythromycin or azithromycin. Isolates from 3 fish displayed MICs identical to the MICs for the ATCC type strain 33209. In contrast, isolates from 4 fish exhibited higher MICs, ranging between 0.125 and 0.250 microg ml(-1) for erythromycin and between 0.016 and 0.031 microg ml(-1) for azithromycin. Sequence analysis of the mutational hotspots for macrolide resistance in the 23S rDNA gene and the open reading frames of ribosomal proteins L4 and L22 found identical sequences among all isolates, indicating that the phenotype was not due to mutations associated with the drug-binding site of 23S rRNA. These results are the first report of R. salmoninarum with reduced susceptibility to macrolide antibiotics isolated from fish receiving multiple antibiotic treatments.     

Inversion of the anomeric configuration of the transferred sugar during inactivation of the macrolide antibiotic oleandomycin catalyzed by a macrolide glycosyltransferase

Macrolides are a group of antibiotics structurally characterized by a macrocyclic lactone to which one or several deoxy-sugar moieties are attached. The sugar moieties are transferred to the different aglycones by glycosyltransferases (GTF). The OleI GTF of an oleandomycin producer, Streptomyces antibioticus, catalyzes the inactivation of this macrolide by glycosylation. The product of this reaction was isolated and its structure elucidated. The donor substrate of the reaction was UDP-alpha-D-glucose, but the reaction product showed a beta-glycosidic linkage. The inversion of the anomeric configuration of the transferred sugar and other data about the kinetics of the reaction and primary structure analysis of several GTFs are compatible with a reaction mechanism involving a single nucleophilic substitution at the sugar anomeric carbon in the catalytic center of the enzyme.     

Characterization of an endophytic whorl-forming Streptomyces from Catharanthus roseus stems producing polyene macrolide antibiotic

An endophytic whorl-forming Streptomyces sp. designated as TS3RO having antifungal activity against a large number of fungal pathogens, including Sclerotinia sclerotiorum, Rhizoctonia solani, Colletotrichum gloeosporioides, Cryphonectria parasitica, Fusarium oxysporum, Pyrenophora tritici-repentis, Epidermophyton floccosum, and Trichophyton rubrum, was isolated from surface-sterilized Catharanthus roseus stems. Preliminary identification showed that Streptomyces cinnamoneus subsp. sparsus was its closest related species. However, strain TS3RO could readily be distinguished from this species using a combination of phenotypic properties, 16S rDNA sequence similarity, and phylogenetic analyses. Thus, the whorl-forming Streptomyces sp. strain TS3RO is likely a new subspecies within the Streptomyces cinnamoneus group. Direct bioautography on a thin-layer chromatography plate with Cladosporium cucumerinum was conducted throughout the purification steps for bioassay-guided isolation of the active antifungal compounds from the crude extract. Structural elucidation of the isolated bioactive compound was obtained via LC-MS spectrometry, UV-visible spectra, and nuclear magnetic resonance data. It revealed that fungichromin, a known methylpentaene macrolide antibiotic, was the main antifungal component of TS3RO strain, as shown by thin-layer chromatography bioautography. This is the first report of an endophytic whorl-forming Streptomyces isolated from the medically important plant Catharanthus roseus.     

Alirinomycin C--a novel macrolide antibiotic from Streptomyces felleus VIZR 8]

The culture of Streptomyces felleus VIZP 8 producing new macrolide antibiotic alirinomycin C was isolated in the screening of new insecticide substances. Antibiotic was related to the carbomycin-cirramycin group. Physico-chemical and biological properties investigation and identification were performed in comparison with other antibiotics of the same group, described in literature.     

Amino acid catabolism and antibiotic synthesis: valine is a source of precursors for macrolide biosynthesis in Streptomyces ambofaciens and Streptomyces fradiae.

Targeted inactivation of the valine (branched-chain amino acid) dehydrogenase gene (vdh) was used to study the role of valine catabolism in the production of tylosin in Streptomyces fradiae and spiramycin in Streptomyces ambofaciens. The deduced products of the vdh genes, cloned and sequenced from S. fradiae C373.1 and S. ambofaciens ATCC 15154, are approximately 80% identical over all 363 amino acids and 96% identical over a span of the first N-terminal 107 amino acids, respectively, to the deduced product of the Streptomyces coelicolor vdh gene. The organization of the regions flanking the vdh genes is the same in all three species. Inactivation of the genomic copy of the vdh gene in S. fradiae and S. ambofaciens by insertion of a hygromycin resistance (hyg) gene caused loss of the valine dehydrogenase (Vdh) activity, and thus only one enzyme is responsible for the Vdh activity in these organisms. Analysis of the culture broth by bioassay revealed that the vdh::hyg mutants produce an approximately sixfold-lower level of tylosin and an approximately fourfold-lower level of spiramycin than the wild-type S. fradiae and S. ambofaciens strains, while maintaining essentially identical growth in a defined minimal medium with either 25 mM ammonium ion or 0.05% asparagine as the nitrogen source. The addition of the valine catabolite, propionate or isobutyrate, and introduction of the wild-type vdh gene back to each vdh::hyg mutant reversed the negative effect of the vdh::hyg mutation on spiramycin and tylosin production. These data show that the catabolism of valine is a major source of fatty acid precursors for macrolide biosynthesis under defined growth conditions and imply that amino acid catabolism is a vital source of certain antibiotic precursors in actinomycetes.     

Solution NMR structures of the polyene macrolide antibiotic filipin III

The solution structure of filipin III, an antifungal polyene macrolide biosynthesized by Streptomyces filipinensis and widely used for the detection and the quantitation of cholesterol in biomembranes, has been calculated with a set of geometrical restraints derived from 1H NMR in DMSO-d(6) at 25 degrees C. Filipin III appears as a rod-shaped molecule of 18 A length. Its amphiphilic structure is made of an all-syn 1,3-polyol motif, stabilized by intramolecular hydrogen bonds on one side, and a conjugated pentaene moiety on the other side of the molecule. The overall shape is comparable to cholesterol, and the molecular structure of filipin III affords a first molecular basis to the comprehensive understanding of the interactions possible in the filipin III-cholesterol complex which is still unknown at the atomic resolution.     

Biosynthesis of the macrolide antibiotic chlorothricin: basic building blocks

The biosynthesis of chlorothricin (I), a macrolide antibiotic isolated from Streptomyces antibioticus Tü 99, has been studied by feeding experiments with 14C- and 3H-labeled precursors. Acetate and propionate, but not methionine and mevalonate, were incorporated into the macrocylic aglycone of the antibiotic. Glucose and the various carbon atoms of tyrosine, except the carboxyl carbon, also contributed label to the aglycone. Glucose also seems to be a specific precursor of the 2-deoxyrhamnose moiety, probably via a process involving a hydrogen shift from C-4 to C-6 of the hexose. The substituted 6-methylsalicylic acid moiety seems to be derived from acetate and one O-methyl group provided by methionine; shikimic acid is not incorporated.     

Organization of the biosynthetic gene cluster for the polyketide antitumor macrolide, pladienolide, in Streptomyces platensis Mer-11107

Pladienolides are novel 12-membered macrolides produced by Streptomyces platensis Mer-11107. They show strong antitumor activity and are a potential lead in the search for novel antitumor agents. We sequenced the 65-kb region covering the biosynthetic gene cluster, and found four polyketide synthase genes (pldAI-pldAIV) composed of 11 modules, three genes involved in post-modifications (pldB-D), and a luxR-family regulatory gene (pldR). The thioesterase domain of pldAIV was more dissimilar to that of polyketide synthase systems synthesizing 12/14-membered macrolide polyketides than to that of systems synthesizing other cyclic polyketides. The pldB gene was identified as a 6-hydroxylase belonging to a cytochrome P450 of the CYP107 family. This was clarified by a disruption experiment on pldB, in which the disruptant produced 6-dehydroxy pladienolide B. Two genes located downstream of pldB, designated pldC and pldD, are thought to be a probable genes for 7-O-acetylase and 18, 19-epoxydase respectively.     

螺旋霉素3-O-酰基转移酶基因的删除和主要产生螺旋霉素组分Ⅰ菌株的获得

螺旋霉素(SP)为16元环大环内酯类抗生素,含有螺旋霉素Ⅰ、Ⅱ和Ⅲ个组分,其结构的差异为16元内酯环的C3上分别连接羟基(SPⅠ)、乙酰基(SPⅡ)和丙酰基(SPⅢ);SPⅡ和SPⅢ是在相同的3-O-酰基转移酶催化下SPⅠ进一步酰化的产物。SPⅠ、SPⅡ和SPⅢ在生物学活性方面无大差异。为简化螺旋霉素组分,便于今后对其结构进行进一步改造,根据碳霉素和麦迪霉素生物合成中的3-O-酰基转移酶序列,设计了简并性PCR引物,并采用SON-PCR(single oligonucleotide nested PCR)方法,从螺旋霉素产生菌S.spiramyceticus F21中进行特异性扩增,获得了螺旋霉素3-O-酰基转移酶基因(sspA)及其侧翼序列,共约4.3kb(其中的3457nt DNA序列已被Genbank收录,DQ642742)。采用DNA同源双交换技术对S.spiramyceticus F21中的sspA进行了删除。对螺旋霉素原株和sspA缺失变株进行发酵产物提取和HPLC分析表明:原株中SPⅠ、SPⅡ和SPⅢ的相对含量分别为7.8%、67%和25%,变株中则分别为72%、18%和9.6%;变株主要组分为SPⅠ。螺旋霉素sspA缺失变株的获得为螺旋霉素组分简化及其衍生物的结构改造奠定了基础。     

Cloning vectors, mutagenesis, and gene disruption (ermR) for the erythromycin-producing bacterium Aeromicrobium erythreum.

Genetic systems for study of Aeromicrobium erythreum, a gram-positive, G + C-rich (72%) bacterium with the capacity for erythromycin biosynthesis, are described. High-copy-number plasmids suitable as gene cloning vectors include derivatives of the Streptomyces plasmids pIJ101, pVE1, and pJV1. pIJ101 derivatives with missense substitutions at the rep gene BamHI site do not replicate in A. erythreum. Ethyl methanesulfonate treatment generated several amino acid auxotrophs and non-erythromycin-producing (Ery-) strains. Using the Ery- strain AR1807 as a recipient for plasmid-directed integrative recombination, the chromosomal ermR gene (encoding 23S rRNA methyltransferase) was disrupted. Phenotypic characterizations demonstrated that ermR is the sole determinant of macrolide antibiotic resistance in A. erythreum.     

Cloning vectors, mutagenesis, and gene disruption (ermR) for the erythromycin-producing bacterium Aeromicrobium erythreum.

Genetic systems for study of Aeromicrobium erythreum, a gram-positive, G + C-rich (72%) bacterium with the capacity for erythromycin biosynthesis, are described. High-copy-number plasmids suitable as gene cloning vectors include derivatives of the Streptomyces plasmids pIJ101, pVE1, and pJV1. pIJ101 derivatives with missense substitutions at the rep gene BamHI site do not replicate in A. erythreum. Ethyl methanesulfonate treatment generated several amino acid auxotrophs and non-erythromycin-producing (Ery-) strains. Using the Ery- strain AR1807 as a recipient for plasmid-directed integrative recombination, the chromosomal ermR gene (encoding 23S rRNA methyltransferase) was disrupted. Phenotypic characterizations demonstrated that ermR is the sole determinant of macrolide antibiotic resistance in A. erythreum.     

The 1.92-A structure of Streptomyces coelicolor A3(2) CYP154C1. A new monooxygenase that functionalizes macrolide ring systems

Evolutionary links between cytochrome P450 monooxygenases, a superfamily of extraordinarily divergent heme-thiolate proteins catalyzing a wide array of NADPH/NADH- and O(2)-dependent reactions, are becoming better understood because of availability of an increasing number of fully sequenced genomes. Among other reactions, P450s catalyze the site-specific oxidation of the precursors to macrolide antibiotics in the genus Streptomyces introducing regiochemical diversity into the macrolide ring system, thereby significantly increasing antibiotic activity. Developing effective uses for Streptomyces enzymes in biosynthetic processes and bioremediation requires identification and engineering of additional monooxygenases with activities toward a diverse array of small molecules. To elucidate the molecular basis for substrate specificity of oxidative enzymes toward macrolide antibiotics, the x-ray structure of CYP154C1 from Streptomyces coelicolor A3(2) was determined (Protein Data Bank code ). Relocation of certain common P450 secondary structure elements, along with a novel structural feature involving an additional beta-strand transforming the five-stranded beta-sheet into a six-stranded variant, creates an open cleft-shaped substrate-binding site between the two P450 domains. High sequence similarity to macrolide monooxygenases from other microbial species translates into catalytic activity of CYP154C1 toward both 12- and 14-membered ring macrolactones in vitro.