Identification and characterization of the ficellomycin biosynthesis gene cluster from Streptomyces ficellus

Applied Microbiology and Biotechnology - Ficellomycin is a peptide-like antibiotic which exhibits potent in vitro activity against Penicillium oxalicum and Staphylococcus aureus, even against...     

Identification and characterization of a new exopolysaccharide biosynthesis gene cluster from Streptomyces

We report the identification and characterization of the ste (Streptomyces eps) gene cluster of Streptomyces sp. 139 required for exopolysaccharide (EPS) biosynthesis. This report is the first genetic work on polysaccharide production in Streptomyces. To investigate the gene cluster involved in exopolysaccharide 139A biosynthesis, degenerate primers were designed to polymerase chain reaction amplify an internal fragment of the priming glycosyltransferase gene that catalyzes the first step in exopolysaccharide biosynthesis. Screening of a genomic library of Streptomyces sp. 139 with this polymerase chain reaction product as probe allowed the isolation of a ste gene cluster containing 22 open reading frames similar to polysaccharide biosynthesis genes of other bacterial species. Involvement of the ste gene cluster in exopolysaccharide biosynthesis was confirmed by disrupting the priming glycosyltransferase gene in Streptomyces sp. 139 to generate non-exopolysaccharide-producing mutants.     

Identification of the gene cluster involved in muraymycin biosynthesis from Streptomyces sp. NRRL 30471

Muraymycin, a potent translocase I inhibitor with clinical potential, is produced by Streptomyces sp. NRRL 30471. The structure of muraymycin is highly unusual and contains the hexahydro-2-imino-4-pyrimidylglycyl moiety (epicapreomycidine) and an ureido bond. Here we report the identification of the muraymycin gene cluster from Streptomyces sp. NRRL 30471. Sequencing analysis of a 43.4-kb contiguous region revealed 33 ORFs, 26 of which were proposed to be involved in muraymycin biosynthesis. Independent targeted inactivation of mur16 and mur17 directly abolished muraymycin production, demonstrating the role of the genes essential for muraymycin biosynthesis. These data provide insights into the molecular mechanisms for muraymycin biosynthesis, and lay a foundation for the generation of muraymycin derivatives with enhanced bioactivity via the strategies of combinatorial biosynthesis.     

Identification of the gene encoding an N-acetylpuromycin N-acetylhydrolase in the puromycin biosynthetic gene cluster from Streptomyces alboniger.

The biologically inactive compound N-acetylpuromycin is the last intermediate of the puromycin antibiotic biosynthetic pathway in Streptomyces alboniger. Culture filtrates from either this organism or Streptomyces lividans transformants harboring the puromycin biosynthetic gene cluster cloned in low-copy-number cosmids contained an enzymic activity which hydrolyzes N-acetylpuromycin to produce the active antibiotic. A gene encoding the deacetylase enzyme was located at one end of this cluster, subcloned in a 2.5-kb DNA fragment, and expressed from a high-copy-number plasmid in S. lividans.     

High-level expression in Streptomyces lividans 66 of a gene encoding Streptomyces subtilisin inhibitor from Streptomyces albogriseolus S-3253

A secretory expression system for Streptomyces subtilisin inhibitor (SSI) was established in a heterologous host, Streptomyces lividans 66, by introducing the 1.8-kbp BglII/SalI fragment containing SSI gene into the Streptomyces multicopy vector, pIJ 702. The expression of SSI did not depend on the orientation of the 1.8-kbp BglII/SalI fragment or on the promoter for tyrosinase gene (mel) in pIJ 702, which suggested that this fragment also carries the SSI promoter. The expressed SSI in S.lividans 66 was secreted into the culture medium in a large amount, as observed with the original strain, S. albogriseolus S-3253. Amino acid sequence analysis showed that the SSI secreted from S. lividans 66 contained three additional amino acid residues in the NH2-terminal region. The inhibitory activity toward subtilisin BPN' and the antigenic activity of the SSI secreted from S. lividans 66 were found to be identical with those of authentic SSI.     

Identification and expression of the gene encoding phosphonopyruvate decarboxylase of Streptomyces hygroscopicus

The first step of C-P compound biosynthesis is a C-P bond formation reaction catalyzed by phosphoenolpyruvate phosphomutase, but this reaction favors the cleavage of the C-P bond. This C-P bond forming reaction is driven by the following reaction catalyzed by phosphonopyruvate (PnPy) decarboxylase. We have cloned and sequenced the gene (bcpC) encoding PnPy decarboxylase, a key enzyme of C-P compound biosynthesis, from the bialaphos (BA) producing microorganism Streptomyces hygroscopicus by complementation methods using Streptomyces wedmorensis NP-7, which is a mutant of a fosfomycin producing strain deficient in this step. The location of this gene in the BA biosynthetic gene cluster was determined by using the expression system in Streptomyces lividans. DNA sequencing of this gene revealed a 1203-bp open reading frame encoding a polypeptide of 401 amino acids.     

Identification of the herboxidiene biosynthetic gene cluster in Streptomyces chromofuscus ATCC 49982

The 53-kb biosynthetic gene cluster for the novel anticholesterol natural product herboxidiene was identified in Streptomyces chromofuscus ATCC 49982 by genome sequencing and gene inactivation. In addition to herboxidiene, a biosynthetic intermediate, 18-deoxy-herboxidiene, was also isolated from the fermentation broth of S. chromofuscus ATCC 49982 as a minor metabolite.     

A gene cluster for biosynthesis of kanamycin from Streptomyces kanamyceticus: comparison with gentamicin biosynthetic gene cluster

Gene clusters for the biosynthesis of kanamycin (Km) and gentamicin (Gm) were isolated from the genomic libraries of Streptomyces kanamyceticus and Micromonospora echinospora, respectively. The sequencing of the 47 kb-region of S. kanamyceticus genomic DNA revealed 40 putative open reading frames (ORFs) encoding Km biosynthetic proteins, regulatory proteins, and resistance and transport proteins. Similarly, the sequencing of 32.6 kb genomic DNA of M. echinospora revealed a Gm biosynthetic gene cluster flanked by resistant genes. Biosynthetic pathways for the formation of Km were proposed by the comparative study of biosynthetic genes. Out of 12 putative Km biosynthetic genes, kanA was expressed in Escherichia coli and determined its function as a 2-deoxy-scyllo-inosose synthase. Furthermore, the acetylations of aminoglycoside-aminocyclitols (AmAcs) by Km acetyltransferase (KanM) were also demonstrated. The acetylated derivatives completely lost their antibacterial activities against Bacillus subtilis. The comparative genetic studies of Gm, Km, tobramycin (Tm), and butirosin (Bn) reveal their similar biosynthetic routes and provide a framework for the further biosynthetic studies.     

A gene cluster inChlorobium vibrioforme encoding the first enzymes of chlorophyll biosynthesis

A cloned 5.8-kb genomic fragment of the green sulfur bacteriumChlorobium vibrioforme encodes the genes for three enzymes catalyzing early steps in the biosynthetic pathway of tetrapyrroles, common to chlorophyll and heme. ThehemA, hemC andhemD genes encode the enzymes glutamyl tRNA dehydrogenase, porphobilinogen deaminase and uroporphyrinogen III synthase, respectively. The cloned genes were expressed in transformedEscherichia coli orSalmonella typhimurium and conferred autotrophy on the respective auxotrophs. Activities of the enzymes encoded by the cloned genes were demonstrated in vitro, with cell extracts obtained from the transformed enterobacteria. The proximity of these genes indicates that they form a cluster inChlorobium vibrioforme, while in most other organisms they appear to be scattered. The presence of this cluster may imply coordinate regulation of the genes involved and they may constitute an operon.     

Cloning and characterization of the polyether salinomycin biosynthesis gene cluster of Streptomyces albus XM211

Salinomycin is widely used in animal husbandry as a food additive due to its antibacterial and anticoccidial activities. However, its biosynthesis had only been studied by feeding experiments with isotope-labeled precursors. A strategy with degenerate primers based on the polyether-specific epoxidase sequences was successfully developed to clone the salinomycin gene cluster. Using this strategy, a putative epoxidase gene, slnC, was cloned from the salinomycin producer Streptomyces albus XM211. The targeted replacement of slnC and subsequent trans-complementation proved its involvement in salinomycin biosynthesis. A 127-kb DNA region containing slnC was sequenced, including genes for polyketide assembly and release, oxidative cyclization, modification, export, and regulation. In order to gain insight into the salinomycin biosynthesis mechanism, 13 gene replacements and deletions were conducted. Including slnC, 7 genes were identified as essential for salinomycin biosynthesis and putatively responsible for polyketide chain release, oxidative cyclization, modification, and regulation. Moreover, 6 genes were found to be relevant to salinomycin biosynthesis and possibly involved in precursor supply, removal of aberrant extender units, and regulation. Sequence analysis and a series of gene replacements suggest a proposed pathway for the biosynthesis of salinomycin. The information presented here expands the understanding of polyether biosynthesis mechanisms and paves the way for targeted engineering of salinomycin activity and productivity.     

The ribostamycin biosynthetic gene cluster in Streptomyces ribosidificus: comparison with butirosin biosynthesis

A cluster of genes for ribostamycin (Rbm) biosynthesis was isolated from Streptomyces ribosidificus ATCC 21294. Sequencing of 31.892 kb of the genomic DNA of S. ribosidificus revealed 26 open reading frames (ORFs) encoding putative Rbm biosynthetic genes as well as resistance and other genes. One of ten putative Rbm biosynthetic genes, rbmA, was expressed in S. lividans TK24, and shown to encode 2-deoxy-scyllo-inosose (DOI) synthase. Acetylation of various aminoglycoside-aminocyclitol (AmAcs) by RbmI confirmed it to be an aminoglycoside 3-N-acetyltransferase. Comparison of the genetic control of ribostamycin and butirosin biosynthesis pointed to a common biosynthetic route for these compounds, despite the considerable differences between them in genetic organization.     

Chain initiation in the leinamycin-producing hybrid nonribosomal peptide/polyketide synthetase from Streptomyces atroolivaceus S-140. Discrete, monofunctional adenylation enzyme and peptidyl carrier protein that directly load D-alanine

Nonribosomal peptide natural products are biosynthesized from amino acid precursors by nonribosomal peptide synthetases (NRPSs), which are organized into modules. For a typical NRPS initiation module, an adenylation (A) domain activates an amino acid and installs it onto a peptidyl carrier protein (PCP) domain as a thioester; an elongation module, which has a condensation (C) domain located between every consecutive pair of A and PCP domains, catalyzes the formation of the peptide bond between the upstream aminoacyl/peptidyl-S-PCP and the free amino group of the downstream aminoacyl-S-PCP. D-amino acid constituents in peptide natural products usually arise from the L-enantiomers through the action of integral epimerization (E) domains of an NRPS. The biosynthetic gene cluster for leinamycin, a hybrid nonribosomal peptide/polyketide containing a D-alanine moiety, does not encode a typical NRPS initiation module with the expected A-PCP-E domains; instead, it has only an A protein (LnmQ) and a PCP (LnmP), both of which are encoded by separate genes. Here we show the results of biochemical experiments as follows: (i) we demonstrate that LnmQ directly activates D-alanine as D-alaninyl-AMP and installs it onto LnmP to generate a D-alaninyl-S-PCP intermediate; (ii) we confirm that aminoacylation of LnmP by LnmQ in trans is the result of specific communication between the separate A and PCP proteins; and (iii) we reveal that leinamycin production can be improved by supplementation of exogenous D-alanine in the fermentation broth of Streptomyces atroolivaceous S-140. These findings unveil an unprecedented NRPS initiation module structure that is characterized by a discrete D-alanine-specific A protein and a PCP.     

Isolation, characterization, and heterologous expression of the biosynthesis gene cluster for the antitumor anthracycline steffimycin

The biosynthetic gene cluster for the aromatic polyketide steffimycin of the anthracycline family has been cloned and characterized from Streptomyces steffisburgensis NRRL 3193. Sequence analysis of a 42.8-kbp DNA region revealed the presence of 36 open reading frames (ORFs) (one of them incomplete), 24 of which, spanning 26.5 kb, are probably involved in steffimycin biosynthesis. They code for all the activities required for polyketide biosynthesis, tailoring, regulation, and resistance but show no evidence of genes involved in L-rhamnose biosynthesis. The involvement of the cluster in steffimycin biosynthesis was confirmed by expression of a region of about 15 kb containing 15 ORFS, 11 of them forming part of the cluster, in the heterologous host Streptomyces albus, allowing the isolation of a biosynthetic intermediate. In addition, the expression in S. albus of the entire cluster, contained in a region of 34.8 kb, combined with the expression of plasmid pRHAM, directing the biosynthesis of L-rhamnose, led to the production of steffimycin. Inactivation of the stfX gene, coding for a putative cyclase, revealed that this enzymatic activity participates in the cyclization of the fourth ring, making the final steps in the biosynthesis of the steffimycin aglycon similar to those in the biosynthesis of jadomycin or rabelomycin. Inactivation of the stfG gene, coding for a putative glycosyltransferase involved in the attachment of L-rhamnose, allowed the production of a new compound corresponding to the steffimycin aglycon compound also observed in S. albus upon expression of the entire cluster.     

Self-cloning in Streptomyces griseus of an str gene cluster for streptomycin biosynthesis and streptomycin resistance.

An str gene cluster containing at least four genes (strR, strA, strB, and strC) involved in streptomycin biosynthesis or streptomycin resistance or both was self-cloned in Streptomyces griseus by using plasmid pOA154. The strA gene was verified to encode streptomycin 6-phosphotransferase, a streptomycin resistance factor in S. griseus, by examining the gene product expressed in Escherichia coli. The other three genes were determined by complementation tests with streptomycin-nonproducing mutants whose biochemical lesions were clearly identified. strR complemented streptomycin-sensitive mutant SM196 which exhibited impaired activity of both streptomycin 6-phosphotransferase and amidinotransferase (one of the streptomycin biosynthetic enzymes) due to a regulatory mutation; strB complemented strain SD141, which was specifically deficient in amidinotransferase; and strC complemented strain SD245, which was deficient in linkage between streptidine 6-phosphate and dihydrostreptose. By deletion analysis of plasmids with appropriate restriction endonucleases, the order of the four genes was determined to be strR-strA-strB-strC. Transformation of S. griseus with plasmids carrying both strR and strB genes enhanced amidinotransferase activity in the transformed cells. Based on the gene dosage effect and the biological characteristics of the mutants complemented by strR and strB, it was concluded that strB encodes amidinotransferase and strR encodes a positive effector required for the full expression of strA and strB genes. Furthermore, it was found that amplification of a specific 0.7-kilobase region of the cloned DNA on a plasmid inhibited streptomycin biosynthesis of the transformants. This DNA region might contain a regulatory apparatus that participates in the control of streptomycin biosynthesis.     

Identification and cloning of the gene involved in the final step of chlortetracycline biosynthesis in Streptomyces aureofaciens

For chlortetracycline biosynthesis in Streptomyces aureofaciens, the final reduction step is essential to give an antibiotic activity to its intermediate, which is catalyzed by tetracycline dehydrogenase with 7,8-dedimethyl-8-hydroxy-5-deazariboflavin (FO) as a cofactor. We identified and cloned the gene, which is essential for the biosynthesis of 6-demethyltetracycline and participates in the final step of its biosynthesis, from the genomic DNA of the 6-demethyltetracycline producer S. aureofaciens HP77. DNA sequence analysis revealed that the gene (tchA) had an open reading frame of 455 amino acids with an estimated molecular mass of 48.1 kDa. Southern hybridization analysis revealed that the tchA gene was located external to the chlortetracycline biosynthetic gene cluster in the genome. A conserved domain search of protein sequence databases indicated that TchA showed a similarity to FbiB, which is involved in the modification of FO in Mycobacterium bovis.     

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 beta-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.