Nonactin biosynthesis: the product of nonS catalyzes the formation of the furan ring of nonactic acid.
ABSTRACT: Nonactin is the parent compound of a group of ionophore antibiotics, known as the macrotetrolides, produced by Streptomyces griseus subsp. griseus ETH A7796. Nonactin is a significant compound because of its inhibitory effects on the P170 glycoprotein-mediated efflux of chemotherapeutic agents in multiple-drug-resistant cancer cells. Nonactin is also significant in that it is a highly atypical polyketide. Very little is presently known about the genes of the nonactin biosynthesis cluster. In this paper we describe our efforts to establish a connection between the product of a gene from the nonactin biosynthesis cluster and a known biochemical transformation in nonactin biosynthesis. Nonactate synthase is the enzyme which catalyzes the formation of nonactic acid from an acyclic precursor in nonactin biosynthesis. We have synthesized the substrate for this enzyme and have detected the in vitro cyclization activity of the substrate in cell-free preparations of S. griseus subsp. griseus ETH A7796. Previous studies by R. Plater and J. A. Robinson (Gene 112:117-122, 1992) had suggested, based on sequence homology, that the product of a partial open reading frame found close to the tetranactin resistance gene of S. griseus could be the nonactate synthase. We have therefore cloned, sequenced, and heterologously expressed this full gene (nonS), and we have shown that the gene product, NonS, does indeed catalyze the formation of the furan ring of nonactic acid as hypothesized.
Project description:The macrotetrolides are a family of cyclic polyethers derived from tetramerization, in a stereospecific fashion, of the enantiomeric nonactic acid (NA) and its homologs. Isotope labeling experiments established that NA is of polyketide origin, and biochemical investigations demonstrated that 2-methyl-6,8-dihydroxynon-2E-enoic acid can be converted into NA by a cell-free preparation from Streptomyces lividans that expresses nonS. These results lead to the hypothesis that macrotetrolide biosynthesis involves a pair of enantiospecific polyketide pathways. In this work, a 55-kb contiguous DNA region was cloned from Streptomyces griseus DSM40695, a 6.3-kb fragment of which was sequenced to reveal five open reading frames, including the previously reported nonR and nonS genes. Inactivation of nonS in vivo completely abolished macrotetrolide production. Complementation of the nonS mutant by the expression of nonS in trans fully restored its macrotetrolide production ability, with a distribution of individual macrotetrolides similar to that for the wild-type producer. In contrast, fermentation of the nonS mutant in the presence of exogenous (+/-)-NA resulted in the production of nonactin, monactin, and dinactin but not in the production of trinactin and tetranactin. These results prove the direct involvement of nonS in macrotetrolide biosynthesis. The difference in macrotetrolide production between in vivo complementation of the nonS mutant by the plasmid-borne nonS gene and fermentation of the nonS mutant in the presence of exogenously added (+/-)-NA suggests that NonS catalyzes the formation of (-)-NA and its homologs, supporting the existence of a pair of enantiospecific polyketide pathways for macrotetrolide biosynthesis in S. griseus. The latter should provide a model that can be used to study the mechanism by which polyketide synthase controls stereochemistry during polyketide biosynthesis.
Project description:Nonactin is a polyketide antibiotic produced by Streptomyces griseus ETH A7796 and is an ionophore that is selective for K(+) ions. It is a cyclic tetraester generated from two monomers of (+)-nonactic acid and two of (-)-nonactic acid, arranged (+)-(-)-(+)-(-) so that nonactin has S4 symmetry and is achiral. To understand why achiral nonactin is the naturally generated diastereoisomer, we generated two alternate diastereoisomers of nonactin, one prepared solely from (+)-nonactic acid and one prepared solely from (-)-nonactic acid, referred to here as 'all-(+)-nonactin' and 'all-(-)-nonactin', respectively. Both non-natural diastereoisomers were 500-fold less active against gram positive organisms than nonactin confirming that the natural stereochemistry is necessary for biological activity. We used isothermal calorimetry to obtain the K(a), DeltaG, DeltaH, and DeltaS of formation for the K(+), Na(+), and NH(4)(+) complexes of nonactin and all-(-)-nonactin; the natural diastereoisomer bound K(+) 880-fold better than all-(-)-nonactin. A picrate partitioning assay confirmed that all-(-)-nonactin, unlike nonactin, could not partition K(+) ions into organic solvent. To complement the thermodynamic data we used a simple model system to show that K(+) transport was facilitated by nonactin but not by all-(-)-nonactin. Modeling of the K(+) complexes of nonactin and all-(-)-nonactin suggested that poor steric interactions in the latter complex precluded tight binding to K(+). Overall, the data show that both enantiomers of nonactic acid are needed for the formation of a nonactin diastereoisomer that can act as an ionophore and has antibacterial activity.
Project description:A 65-kb region of DNA from Streptomyces viridochromogenes Tü57, containing genes encoding proteins involved in the biosynthesis of avilamycins, was isolated. The DNA sequence of a 6.4-kb fragment from this region revealed four open reading frames (ORF1 to ORF4), three of which are fully contained within the sequenced fragment. The deduced amino acid sequence of AviM, encoded by ORF2, shows 37% identity to a 6-methylsalicylic acid synthase from Penicillium patulum. Cultures of S. lividans TK24 and S. coelicolor CH999 containing plasmids with ORF2 on a 5.5-kb PstI fragment were able to produce orsellinic acid, an unreduced version of 6-methylsalicylic acid. The amino acid sequence encoded by ORF3 (AviD) is 62% identical to that of StrD, a dTDP-glucose synthase from S. griseus. The deduced amino acid sequence of AviE, encoded by ORF4, shows 55% identity to a dTDP-glucose dehydratase (StrE) from S. griseus. Gene insertional inactivation experiments of aviE abolished avilamycin production, indicating the involvement of aviE in the biosynthesis of avilamycins.
Project description:A gene, pcbC, encoding the isopenicillin N synthase of Streptomyces griseus NRRL 3851, has been cloned in a 6.4-kb Bg/II DNA fragment and located in an internal 1.55-kb PvuII segment by hybridization with the Penicillium chrysogenum pcbC gene. Hybridization studies revealed the presence of homologous sequences in the DNAs of several Streptomyces strains and Nocardia lactamdurans. The S. griseus pcbC gene was not expressed in Streptomyces lividans but was expressed in Streptomyces clavuligerus and complemented a mutation, nce2, that impaired isopenicillin N synthase and cephamycin biosynthesis. The pcbC gene contained an open reading frame of 990 nucleotides that encodes a protein of 329 amino acids with a deduced Mr of 37,371. The isopenicillin N synthase formed after expression of the pcbC gene in the S. clavuligerus nce2 mutant strain was found to have an Mr of 38,000 by gel filtration. A protein of about 38 kDa was observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of extracts of a transformant of the nce2 mutant strain; this protein was absent from the untransformed mutant strain. The G+C content of the pcbC gene was 63.6%, and the strongly biased codon usage was typical of that of Streptomyces strains. A transcription initiation site was found 44 nucleotides upstream of the ATG translation initiation triplet. A transcript of 1.1 kb was observed in the donor S. griseus strain and also in the S. clavuligerus nce2 mutant strain transformed with the pcbC gene, suggesting that it is transcribed as a monocistronic mRNA.
Project description:Zincophorin is a polyketide antibiotic that possesses potent activity against Gram-positive bacteria, including human pathogens. While a number of total syntheses of this highly functionalized natural product were reported since its initial discovery, the genetic basis for the biosynthesis of zincophorin has remained unclear. In this study, the co-linearity inherent to polyketide pathways was used to identify the zincophorin biosynthesis gene cluster in the genome of the natural producer Streptomyces griseus HKI 0741. Interestingly, the same locus is fully conserved in the streptomycin-producing actinomycete S. griseus IFO 13350, suggesting that the latter bacterium is also capable of zincophorin biosynthesis. Biological profiling of zincophorin revealed a dose-dependent inhibition of the Gram-positive bacterium Streptococcus pneumoniae. The antibacterial effect, however, is accompanied by cytotoxicity. Antibiotic and cytotoxic activities were completely abolished upon esterification of the carboxylic acid group in zincophorin.
Project description:Albomycin (ABM), also known as grisein, is a sulfur-containing metabolite produced by Streptomyces griseus ATCC 700974. Genes predicted to be involved in the biosynthesis of ABM and ABM-like molecules are found in the genomes of other actinomycetes. ABM has potent antibacterial activity, and as a result, many attempts have been made to develop ABM into a drug since the last century. Although the productivity of S. griseus can be increased with random mutagenesis methods, understanding of Streptomyces sulfur amino acid (SAA) metabolism, which supplies a precursor for ABM biosynthesis, could lead to improved and stable production. We previously characterized the gene cluster (abm) in the genome-sequenced S. griseus strain and proposed that the sulfur atom of ABM is derived from either cysteine (Cys) or homocysteine (Hcy). The gene product, AbmD, appears to be an important link between primary and secondary sulfur metabolic pathways. Here, we show that propargylglycine or iron supplementation in growth media increased ABM production by significantly changing the relative concentrations of intracellular Cys and Hcy. An SAA metabolic network of S. griseus was constructed. Pathways toward increasing Hcy were shown to positively impact ABM production. The abmD gene and five genes that increased the Hcy/Cys ratio were assembled downstream of hrdBp promoter sequences and integrated into the chromosome for overexpression. The ABM titer of one engineered strain, SCAK3, in a chemically defined medium was consistently improved to levels ?400% of the wild type. Finally, we analyzed the production and growth of SCAK3 in shake flasks for further process development.
Project description:Streptomyces phage Nanodon is a temperate double-stranded DNA Siphoviridae belonging to cluster BD1. It was isolated from soil collected in Kilauea, HI, using Streptomyces griseus subsp. griseus as a host.
Project description:The Streptomyces bacteriophage Abt2graduatex2 is a double-stranded DNA (dsDNA) Siphoviridae isolated from soil collected in Baltimore, MD, and harvested using Streptomyces griseus subsp. griseus Abt2graduatex2, a cluster BG phage, encodes an HicA-like toxin.
Project description:A-74528 is a recently discovered natural product of Streptomyces sp. SANK 61196 that inhibits 2',5'-oligoadenylate phosphodiesterase (2'-PDE), a key regulatory enzyme of the interferon pathway. Inhibition of 2'-PDE by A-74528 reduces viral replication, and therefore shows promise as a new type of antiviral drug. The complete A-74528 gene cluster, comprising 29 open reading frames, was cloned and sequenced, and shown to possess a type II polyketide synthase (PKS) at its core. Its identity was confirmed by analysis of a mutant generated by targeted disruption of a PKS gene, and by functional expression in a heterologous Streptomyces host. Remarkably, it showed exceptional end-to-end sequence identity to the gene cluster responsible for biosynthesis of fredericamycin A, a structurally unrelated antitumor antibiotic with a distinct mode of action. Whereas the fredericamycin producing strain, Streptomyces griseus, produced undetectable quantities of A-74528, the A-74528 gene cluster was capable of producing both antibiotics. The biosynthetic roles of three genes, including one that represents the only qualitative difference between the two gene clusters, were investigated by targeted gene disruption. The implications for the evolution of antibiotics with different biological activities from the same gene cluster are discussed.
Project description:Streptomyces griseus DSM 2608 produces bafilomycin, an antifungal plecomacrolide antibiotic. We cloned and sequenced an 87.4-kb region, including a polyketide synthase (PKS) region, methoxymalonate genes, flavensomycinate genes, and other putative regulatory genes. The 58.5kb of PKS region consisting 12 PKS modules arranged in five different PKS genes, was assumed to be responsible for the biosynthesis of plecomacrolide backbone including 16-membered macrocyclic lactone. All the modules showed high similarities with typical type I PKS genes. However, the starting module of PKS gene was confirmed to be specific for isobutyrate by sequence comparison of an acyltransferase domain. In downstream of PKS region, the genes for methoxymalonate biosynthesis were located, among which a gene for FkbH-like protein was assumed to play an important role in the production of methoxymalonyl-CoA from glyceryl-CoA. Further the genes encoding flavensomycinyl-ACP biosynthesis for the post-PKS tailoring were also found in the upstream of PKS region. By gene disruption experiments of a dehydratase domain of module 12 and an FkbH-like protein, this gene cluster was confirmed to be involved in the biosynthesis of bafilomycin.