Isolation and characterization of the epothilone biosynthetic gene cluster from Sorangium cellulosum

The epothilone biosynthetic gene cluster was isolated from Sorangium cellulosum strain SMP44. The gene cluster contains seven genes and spans approx. 56kb. The genes encoding the PKS, epoA, epoC, epoD, epoE, and epoF, are divided into nine modules. The EpoB protein is a non-ribosomal peptide synthetase (NRPS) that catalyzes formation of the thiazole found in the epothilones. EpoK is a P450 enzyme responsible for the epoxidation of epothilones C and D to epothilones A and B, respectively. EpoK was expressed in Escherichia coli, and the purified protein was shown to convert epothilone D to epothilone B in vitro.     

Isolation and characterization of the tobramycin biosynthetic gene cluster from Streptomyces tenebrarius

The biosynthetic gene cluster for tobramycin, a 2-deoxystreptamine-containing aminoglycoside antibiotic, was isolated from Streptomyces tenebrarius ATCC 17920. A genomic library of S. tenebrarius was constructed, and a cosmid, pST51, was isolated by the probes based on the core regions of 2-deoxy-scyllo-inosose (DOI) synthase, and L-glutamine:DOI aminotransferase and L-glutamine:scyllo-inosose aminotransferase. Sequencing of 33.9 kb revealed 24 open reading frames (ORFs) including putative tobramycin biosynthetic genes. We demonstrated that one of these ORFs, tbmA, encodes DOI synthase by in vitro enzyme assay of the purified protein. The catalytic residues of TbmA and dehydroquinate synthase were studied by homology modeling. The gene cluster found is likely to be involved in the biosynthesis of tobramycin.     

Molecular characterization and analysis of the biosynthetic gene cluster for the azoxy antibiotic valanimycin

Streptomyces viridifaciens MG456-hF10 produces the antibiotic valanimycin, a naturally occurring azoxy compound. Valanimycin is known to be derived from valine and serine with the intermediacy of isobutylamine and isobutylhydroxylamine, but little is known about the stages in the pathway leading to the formation of the azoxy group. In previous studies, a cosmid containing S. viridifaciens DNA was isolated that conferred valanimycin production upon Strepto-myces lividans TK24. Subcloning of DNA from the valanimycin-producing cosmid has led to the identi-fication of a 22 kb segment of DNA sufficient to allow valanimycin production in S. lividans TK24. Sequencing of this DNA segment and the surrounding DNA revealed the presence of 20 genes. Gene disruption experiments defined the boundaries of the valanimycin gene cluster, which appears to contain 14 genes. The cluster includes an amino acid decar-boxylase gene (vlmD), a valanimycin resistance gene (vlmF ), at least two regulatory genes (vlmE, vlmI ), two genes encoding a flavin monooxygenase (vlmH, vlmR), a seryl tRNA synthetase gene (vlmL ) and seven genes of unknown function. Overproduction and characterization of VlmD demonstrated that it catalyses the decarboxylation of l-valine. An unusual feature of the valanimycin gene cluster is that four genes involved in branched amino acid biosynthesis are located near its 5' end.     

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.     

Heterologous expression of the diazaquinomycin biosynthetic gene cluster

Journal of Industrial Microbiology & Biotechnology - Members of the diazaquinomycin class of natural products have shown potent and selective activity against Mycobacterium tuberculosis....     

Lessons from the rifamycin biosynthetic gene cluster.

There is currently intense interest in unravelling the modus operandi of type I modular polyketide synthases in order to lay the ground work for their use in the combinatorial biosynthesis of new bioactive molecules. Much of our knowledge is derived from studies on 6-deoxyerythronolide B (DEBS), the enzyme assembling the polyketide backbone of erythromycin. Work on the rifamycin polyketide synthase has revealed a number of features that differ from those seen with DEBS.     

Sequence analysis of porothramycin biosynthetic gene cluster

The biosynthetic gene cluster of porothramycin, a sequence-selective DNA alkylating compound, was identified in the genome of producing strain Streptomyces albus subsp. albus (ATCC 39897) and sequentially characterized. A 39.7 kb long DNA region contains 27 putative genes, 18 of them revealing high similarity with homologous genes from biosynthetic gene cluster of closely related pyrrolobenzodiazepine (PBD) compound anthramycin. However, considering the structures of both compounds, the number of differences in the gene composition of compared biosynthetic gene clusters was unexpectedly high, indicating participation of alternative enzymes in biosynthesis of both porothramycin precursors, anthranilate, and branched L-proline derivative. Based on the sequence analysis of putative NRPS modules Por20 and Por21, we suppose that in porothramycin biosynthesis, the methylation of anthranilate unit occurs prior to the condensation reaction, while modifications of branched proline derivative, oxidation, and dimethylation of the side chain occur on already condensed PBD core. Corresponding two specific methyltransferase encoding genes por26 and por25 were identified in the porothramycin gene cluster. Surprisingly, also methyltransferase gene por18 homologous to orf19 from anthramycin biosynthesis was detected in porothramycin gene cluster even though the appropriate biosynthetic step is missing, as suggested by ultra high-performance liquid chromatography-diode array detection-mass spectrometry (UHPLC-DAD-MS) analysis of the product in the S. albus culture broth.     

Biochemical characterization of VlmL, a Seryl-tRNA synthetase encoded by the valanimycin biosynthetic gene cluster

Previous studies have shown that the valanimycin producer Streptomyces viridifaciens contains two genes encoding proteins that are similar to seryl-tRNA synthetases (SerRSs). One of these proteins (SvsR) is presumed to function in protein biosynthesis, because it exhibits a high degree of similarity to the single SerRS of Streptomyces coelicolor. The second protein (VlmL), which exhibits a low similarity to the S. coelicolor SerRS, is hypothesized to play a role in valanimycin biosynthesis, because the vlmL gene resides within the valanimycin biosynthetic gene cluster. To investigate the role of VlmL in valanimycin biosynthesis, VlmL and SvsR have been overproduced in soluble form in Escherichia coli, and the biochemical properties of both proteins have been analyzed and compared. Both proteins were found to catalyze a serine-dependent exchange of 32P-labeled pyrophosphate into ATP and to aminoacylate total E. coli tRNA with L-serine. Kinetic parameters for the two enzymes show that SvsR is catalytically more efficient than VlmL. The results of these experiments suggest that the role of VlmL in valanimycin biosynthesis is to produce seryl-tRNA, which is then utilized for a subsequent step in the biosynthetic pathway. Orthologs of VlmL were identified in two other actinomycetes species that also contain orthologs of the S. coelicolor SerRS. The significance of these findings is herein discussed.     

福林霉素生物合成基因簇的组装和异源表达

[目的]本研究旨在以来源于林可链霉菌NRRL2936中的nosokomycinB2的生物合成基因簇(noso-BGC)为基础,组装获得完整的福林霉素生物合成基因簇(pho-BGC),再利用异源表达策略,激活pho-BGC的表达并通过底盘宿主的优选实现福林霉素发酵产量的提升.[方法]首先,在林可霉素基因簇缺失突变株JCK...     

Utilization of the methoxymalonyl-acyl carrier protein biosynthesis locus for cloning of the tautomycin biosynthetic gene cluster from Streptomyces spiroverticillatus

Tautomycin (TTM), a potent protein phosphatase inhibitor, consists of a polyketide chain containing a spiroketal moiety and an acyl chain bearing a dialkylmaleic anhydride structure. PCR using degenerate primers was used to clone genes from Streptomyces spiroverticillatus for formation of the methoxymalonyl-acyl carrier protein. This locus was found to contain five genes (ttmC, ttmA, ttmD, ttmB, and ttmE), one of which was used as a probe to clone the 110-kb TTM biosynthetic gene cluster. The involvement of the ttmA gene in TTM biosynthesis was confirmed by gene inactivation and mutation complementation experiments.     

Cloning and characterization of the histidine biosynthetic gene cluster of Streptomyces coelicolor A3(2)

Biochemical and genetic data indicate that in Streptomyces coelicolor A3(2) the majority of the genes involved in the biosynthesis of histidine are clustered in a small region of the chromosome [Carere et al., Mol. Gen. Genet. 123 (1973) 219-224; Russi et al., Mol. Gen. Genet. 123 (1973) 225-232]. To investigate the structural organization and the regulation of these genes, we have constructed genomic libraries from S. coelicolor A3(2) in pUC vectors. Recombinant clones were isolated by complementation of an Escherichia coli hisBd auxotroph. A recombinant plasmid containing a 3.4-kb fragment of genomic DNA was further characterized. When cloned in the plasmid vector, pIJ699, this fragment was able to complement S. coelicolor A3(2) hisB mutants. Overlapping clones spanning a 15-kb genomic region were isolated by screening other libraries with labeled DNA fragments obtained from the first clone. Derivative clones were able to complement mutations in four different cistrons of the his cluster of S. coelicolor A3(2). Nucleotide sequence analysis of a 4-kb region allowed the identification of five ORFs which showed significant homology with the his gene products of E. coli. The order of the genes in S. coelicolor A3(2) (5'--hisD-hisC-hisBd-hisH-hisA-3') is the same as in the his operon of E. coli.     

The bialaphos biosynthetic genes ofStreptomyces hygroscopicus: Molecular cloning and characterization of the gene cluster

Summary We have isolated and studied the organization ofStreptomyces hygroscopicus genes responsible for the biosynthesis of the antibiotic herbicide bialaphos. Bialaphos production genes were cloned from genomic DNA using a plasmid vector (pIJ702). Three plasmids were isolated which restored productivity toS. hygroscopicus mutants blocked at different steps of the biosynthetic pathway. Subcloning experiments using other nonproducing mutants showed that four additional bialaphos production genes were also contained on these plasmids. A gene conferring resistance to bialaphos, which was independently cloned using the plasmid vector pIJ61, and an antibiotic-sensitive host (S. lividans), was also linked to the production genes. Cosmids were isolated which defined the location of these genes in a 16 kb cluster.     

Cloning a part of the ochratoxin A biosynthetic gene cluster of Penicillium nordicum and characterization of the ochratoxin polyketide synthase gene

Penicillium nordicum is a fungal species able to produce high amounts of ochratoxin A. A 10kb genomic DNA fragment of P. nordicumn has been cloned which carries three long open reading frames. One open reading frame (otapksPN) has homology to fungal polyketide synthases. The second open reading frame (npsPN) has homology to non-ribosomal peptide synthetases and the third open reading frame (aspPN) has homology to fungal alkaline serine proteinases. The non-ribosomal peptide synthetase and the polyketide synthase are convergently transcribed. Interestingly, the polyketide synthase can be identified by PCR only in P. nordicum strains and not in the related species Penicillium verrucosum or in ochratoxigenic Aspergillus species, indicating that the ochratoxin polyketide synthases are different in the important ochratoxigenic species. In contrast, the non-ribosomal peptide synthetase can be identified in P. nordicum and P. verrucosum, but not in other species. An inactivation of the polyketide synthase resulted in strains with abolished capacity to produce ochratoxin A. Expression of the polyketide synthase correlates with ochratoxin A biosynthesis.     

Molecular cloning and characterization of a 2-deoxystreptamine biosynthetic gene cluster in gentamicin-producing Micromonospora echinospora ATCC15835

The organization of the 2-deoxystreptamine (DOS) biosynthetic gene cluster of Micromonospora echinospora has been determined. Sequencing of a 14.04 kb-region revealed twelve open reading frames (ORFs): four putative DOS biosynthetic genes (gtmA, B, C, and D), five amino sugars biosynthetic genes (gtmE, G, H, I, and gacB), two aminoglycoside resistance genes (gtmF and J) as well as a hypothetical ORF (gacA). One of the putative DOS biosynthetic genes, gtmA, was expressed in Escherichia coli, and the purified protein was shown to convert glucose-6-phosphate (G-6-P) to 2-deoxy-scyllo-inosose (DOI), a key step in DOS biosynthesis. In addition gtmJ was expressed in Streptomyces lividans and shown to confer gentamicin resistance. Thus gtmA and gtmJ are implicated in the biosynthesis of gentamicin and in resistance to it, respectively.