Identification of 8-amino-2,5,7-trihydroxynaphthalene-1,4-dione, a novel intermediate in the biosynthesis of Streptomyces meroterpenoids

Furaquinocin is a natural polyketide-isoprenoid hybrid (meroterpenoid) produced by Streptomyces sp. strain KO-3988. All of the fur genes required for furaquinocin biosynthesis have been cloned, and the heterologous production of furaquinocin has been demonstrated in Streptomyces albus. Here, we report the identification of 8-amino-2,5,7-trihydroxynaphthalene-1,4-dione (8-amino-flaviolin) produced by the S. albus heterologous expression of the three contiguous genes encoding type III polyketide synthase (Fur1), monooxygenase (Fur2), and aminotransferase (Fur3) in the furaquinocin biosynthetic gene cluster. An S. albus transformant (S. albus/pWHM-Fur2_del3) harboring the fur gene cluster and lacking the fur3 gene did not produce furaquinocin, whereas furaquinocin production was restored when 8-amino-flaviolin was added to the culture medium of S. albus/pWHM-Fur2_del3. These results demonstrate that Fur3 aminotransferase is essential for furaquinocin biosynthesis and that 8-amino-flaviolin is an early-stage intermediate in furaquinocin biosynthesis. We propose that the biosynthetic pathway generating 8-amino-flaviolin is the common route for the biosynthesis of Streptomyces meroterpenoids.     

Chemoenzymatic syntheses of prenylated aromatic small molecules using Streptomyces prenyltransferases with relaxed substrate specificities

NphB is a soluble prenyltransferase from Streptomyces sp. strain CL190 that attaches a geranyl group to a 1,3,6,8-tetrahydroxynaphthalene-derived polyketide during the biosynthesis of anti-oxidant naphterpin. Here we report multiple chemoenzymatic syntheses of various prenylated compounds from aromatic substrates including flavonoids using two prenyltransferases NphB and SCO7190, a NphB homolog from Streptomyces coelicolor A3(2), as biocatalysts. NphB catalyzes carbon-carbon-based and carbon-oxygen-based geranylation of a diverse collection of hydroxyl-containing aromatic acceptors. Thus, this simple method using the prenyltransferases can be used to explore novel prenylated aromatic compounds with biological activities. Kinetic studies with NphB reveal that the prenylation reaction follows a sequential ordered mechanism.     

Catalytic Reactions of the Homogentisate Prenyl Transferase Involved in Plastoquinone-9 Biosynthesis

Homogentisate solanesyl transferase (HST) catalyzes the prenylation and decarboxylation of homogentisate to form 2-methyl-6-solanesyl-1,4-benzoquinol, the first intermediate in plastoquinone-9 biosynthesis. In vitro, HST from Spinacia oleracea L., Arabidopsis thaliana, and Chlamydomonas reinhardtii were all found to use not only solanesyl diphosphate but also short chain prenyl diphosphates of 10–20 carbon atoms as prenyl donors. Surprisingly, with these donors, prenyl transfer was largely decoupled from decarboxylation, and thus the major products were 6-prenyl-1,4-benzoquinol-2-methylcarboxylates rather than the expected 2-methyl-6-prenyl-1,4-benzoquinols. The 6-prenyl-1,4-benzoquinol-2-methylcarboxylates were not substrates for HST-catalyzed decarboxylation, and the enzyme kinetics associated with forming these products appeared quite distinct from those for 2-methyl-6-prenyl-1,4-benzoquinol formation in respect of catalytic rate, substrate K_m value, and the pattern of inhibition by haloxydine, a molecule that appeared to act as a dead end mimic of homogentisate. These observations were reconciled into a simple model for the HST mechanism. Here, prenyl diphosphate binds to HST to form at least two alternative complexes that go on to react differently with homogentisate and prenylate it either with or without it first being decarboxylated. It is supposed that solanesyl diphosphate binds tightly and preferentially in the mode that compels prenylation with decarboxylation.     

Valine dehydrogenase from Streptomyces albus: gene cloning, heterologous expression and identification of active site by site-directed mutagenesis

A gene encoding valine dehydrogenase (Vdh) has been cloned from Streptomyces albus, a salinomycin producer, and expressed in Escherichia coli. The S. albus Vdh is composed of 364 amino acids that showed high homology with several other amino acid dehydrogenases as well as Vdhs from Streptomyces spp. and leucine and phenylalanine dehydrogenases (Ldh and Pdh) from Bacillus spp. A protein of 38 kDa, corresponding to the approximate mass of the predicted S. albus Vdh product (38.4 kDa) exhibiting specific Vdh activity, was observed when the S. albus vdh gene was overexpressed in E. coli under the controlled T7 promoter and was subsequently purified to homogeneity. Among branched- and straight-chain amino acids, L-valine and L-alpha-aminobutyrate were the preferred substrates for the enzyme. Lys-79 and Lys-91 of S. albus Vdh were highly conserved in the corresponding region of NAD(P)(+)-dependent amino acid dehydrogenase sequences. To elucidate the functional roles of the lysyl residues, the Lys residues have individually been replaced with Ala by site-directed mutagenesis. Kinetic analyses of the Lys-79 and Lys-91-mutated enzymes revealed that they are involved in the substrate binding site and catalysis, respectively, analogous to the corresponding residues in the homologous Ldh and Pdh.     

Deoxysugar methylation during biosynthesis of the antitumor polyketide elloramycin by Streptomyces olivaceus. Characterization of three methyltransferase genes

The anthracycline-like polyketide drug elloramycin is produced by Streptomyces olivaceus Tü2353. Elloramycin has antibacterial activity against Gram-positive bacteria and also exhibits antitumor activity. From a cosmid clone (cos16F4) containing part of the elloramycin biosynthesis gene cluster, three genes (elmMI, elmMII, and elmMIII) have been cloned. Sequence analysis and data base comparison showed that their deduced products resembled S-adenosylmethionine-dependent O-methyltransferases. The genes were individually expressed in Streptomyces albus and also coexpressed with genes involved in the biosynthesis of l-rhamnose, the 6-deoxysugar attached to the elloramycin aglycon. The resulting recombinant strains were used to biotransform three different elloramycin-type compounds: l-rhamnosyl-tetracenomycin C, l-olivosyl-tetracenomycin C, and l-oleandrosyl-tetracenomycin, which differ in their 2'-, 3'-, and 4'-substituents of the sugar moieties. When only the three methyltransferase-encoding genes elmMI, elmMII, and elmMIII were individually expressed in S. albus, the methylating activity of the three methyltransferases was also assayed in vitro using various externally added glycosylated substrates. From the combined results of all of these experiments, it is proposed that methyltransferases ElmMI, ElmMII, and ElmMIII are involved in the biosynthesis of the permethylated l-rhamnose moiety of elloramycin. ElmMI, ElmMII, and ElmMIII are responsible for the consecutive methylation of the hydroxy groups at the 2'-, 3'-, and 4'-position, respectively, after the sugar moiety has been attached to the aglycon.     

Generation of hybrid elloramycin analogs by combinatorial biosynthesis using genes from anthracycline-type and macrolide biosynthetic pathways

Elloramycin and oleandomycin are two polyketide compounds produced by Streptomyces olivaceus Tü2353 and Streptomyces antibioticus ATCC11891, respectively. Elloramycin is an anthracycline-like antitumor drug and oleandomycin a macrolide antibiotic. Expression in S. albus of a cosmid (cos16F4) containing part of the elloramycin biosynthetic gene cluster produced the elloramycin non-glycosylated intermediate 8-demethyl-tetracenomycin C. Several plasmid constructs harboring different gene combinations of L-oleandrose (neutral 2,6-dideoxyhexose attached to the macrolide antibiotic oleandomycin) biosynthetic genes of S. antibioticus that direct the biosynthesis of L-olivose, L-oleandrose and L-rhamnose were coexpressed with cos16F4 in S. albus. Three new hybrid elloramycin analogs were produced by these recombinant strains through combinatorial biosynthesis, containing elloramycinone or 12a-demethyl-elloramycinone (= 8-demethyl-tetracenomycin C) as aglycone moiety encoded by S. olivaceus genes and different sugar moieties, coded by the S. antibioticus genes. Among them is L-olivose, which is here described for the first time as a sugar moiety of a natural product.     

Structure and action of the C-C bond-forming glycosyltransferase UrdGT2 involved in the biosynthesis of the antibiotic urdamycin

The glycosyltransferase UrdGT2 from Streptomyces fradiae catalyzes the formation of a glycosidic C-C bond between a polyketide aglycone and D-olivose. The enyzme was expressed in Escherichia coli, purified and crystallized. Its structure was established by X-ray diffraction at 1.9 A resolution. It is the first structure of a C-glycosyltransferase. UrdGT2 belongs to the structural family GT-B of the glycosyltransferases and is likely to form a C(2)-symmetric dimer in solution. The binding structures of donor and acceptor substrates in five structurally homologous enzymes provided a clear and consistent guide for the substrate-binding structure in UrdGT2. The modeled substrate locations suggest the deeply buried Asp137 as the activator for C-C bond formation and explain the reaction. The putative model can be used to design mutations that change the substrate specificity. Such mutants are of great interest in overcoming the increasing danger of antibiotic resistance.     

Geranyl diphosphate:4-hydroxybenzoate geranyltransferase from Lithospermum erythrorhizon. Cloning and characterization of a ket enzyme in shikonin biosynthesis.

Two cDNAs encoding geranyl diphosphate:4-hy- droxybenzoate 3-geranyltransferase were isolated from Lithospermum erythrorhizon by nested PCR using the conserved amino acid sequences among polyprenyl- transferases for ubiquinone biosynthesis. They were functionally expressed in yeast COQ2 disruptant and showed a strict substrate specificity for geranyl diphosphate as the prenyl donor, in contrast to ubiquinone biosynthetic enzymes, suggesting that they are involved in the biosynthesis of shikonin, a naphthoquinone secondary metabolite. Regulation of their expression by various culture conditions coincided with that of geranyltransferase activity and the secondary metabolites biosynthesized via this enzyme. This is the first established plant prenyltransferase that transfers the prenyl chain to an aromatic substrate.     

Peptide inhibitors of Streptomyces dd-carboxypeptidases

1. Peptides that inhibit the dd-carboxypeptidases from Streptomyces strains albus G and R61 were synthesized. They are close analogues of the substrates of these enzymes. The enzymes from albus G and R61 strains are in general inhibited by the same peptides, but the enzyme from strain R39 differs considerably. 2. The two C-terminal residues of the peptide substrates and inhibitors appear to be mainly responsible for the initial binding of the substrate to the enzymes from albus G and R61 strains. The side chain in the third residue from the C-terminus seems critical in inducing catalytic activity. 3. Experimental evidence is presented suggesting that the amide bond linking the two C-terminal residues has a cis configuration when bound to the enzymes from strains albus G and R61. 4. The peptide inhibitors are not antibiotics against the same micro-organisms.     

Insertional inactivation of mtrX and mtrY genes from the mithramycin gene cluster affects production and growth of the producer organism Streptomyces argillaceus

Mithramycin is an antitumor aromatic polyketide synthesized by Streptomyces argillaceus. Two genes (mtrX and mtrY) of the mithramycin gene cluster were inactivated by gene replacement. Inactivation of mtrX, that encodes an ABC excision nuclease system for DNA repair, produced a mutant that was affected in the normal rate of growth. Expression of mtrX in Streptomyces albus in a multicopy plasmid vector conferred a low increase in resistance to mithramycin. Inactivation of mtrY, that encodes a protein of unknown function, produced a 50% decrease in mithramycin biosynthesis. When mtrY was expressed in the wild-type S. argillaceus in a multicopy plasmid, this caused about 47% increase in the levels of mithramycin production. It is proposed that mtrX and mtrY could code for a secondary defense mechanism and a mithramycin regulatory element, respectively.     

A hydroxylase-like gene product contributes to synthesis of a polyketide spore pigment in Streptomyces halstedii.

A gene, schC, adjacent to the sch gene cluster encoding the biosynthesis of a polyketide spore pigment in Streptomyces halstedii was sequenced. Its deduced product resembled flavin adenine nucleotide-containing hydroxylases involved in the biosynthesis of polycyclic aromatic polyketide antibiotics and in catabolic pathways of aromatic compounds. When schC was disrupted, the normally green spores of S. halstedii became lilac. An schC-like gene was located in an equivalent position next to a large gene cluster (whiE) known to determine spore pigment in Streptomyces coelicolor A3(2).     

Cloning of modular type I polyketide synthase genes from salinomycin producing strain of Streptomyces albus

Cloning of polyether polyketide synthase (PKS) genes for salinomycin biosynthesis was attempted from Streptomyces albus. Seven beta-ketoacyl synthase (KS) core regions were obtained by PCR amplification using primers designed based on the conserved KS domains of type I PKSs. Using the KS fragment as a probe, screening of an S. albus genomic DNA library was carried out by colony hybridization. From the positive cosmid clone isolated, a 4.5-kbBamHI fragment was subcloned and sequenced. It showed high homology with bacterial type I PKSs and was deduced to code for KS, malonyl transferase, and ketoreductase motifs. By gene disruption with this 4.5-kb BamHI fragment, the cloned gene was shown to be a part of the salinomycin biosynthetic gene cluster of S. albus.     

A new substrate specificity for acyl transferase domains of the ascomycin polyketide synthase in Streptomyces hygroscopicus.

Ascomycin (FK520) is a structurally complex macrolide with immunosuppressant activity produced by Streptomyces hygroscopicus. The biosynthetic origin of C12-C15 and the two methoxy groups at C13 and C15 has been unclear. It was previously shown that acetate is not incorporated into C12-C15 of the macrolactone ring. Here, the acyl transferase (AT) of domain 8 in the ascomycin polyketide synthase was replaced with heterologous ATs by double homologous recombination. When AT8 was replaced with methylmalonyl-CoA-specific AT domains, the strains produced 13-methyl-13-desmethoxyascomycin, whereas when AT8 was replaced with a malonyl-specific domain, the strains produced 13-desmethoxyascomycin. These data show that ascomycin AT8 does not use malonyl- or methylmalonyl-CoA as a substrate in its native context. Therefore, AT8 must be specific for a substrate bearing oxygen on the alpha carbon. Feeding experiments showed that [(13)C]glycerol is incorporated into C12-C15 of ascomycin, indicating that both modules 7 and 8 of the polyketide synthase use an extender unit that can be derived from glycerol. When AT6 of the 6-deoxyerythronolide B synthase gene was replaced with ascomycin AT8 and the engineered gene was expressed in Streptomyces lividans, the strain produced 6-deoxyerythronolide B and 2-demethyl-6-deoxyerythronolide B. Therefore, although neither malonyl-CoA nor methylmalonyl-CoA is a substrate for ascomycin AT8 in its native context, both are substrates in the foreign context of the 6-deoxyerythronolide B synthase. Thus, we have demonstrated a new specificity for an AT domain in the ascomycin polyketide synthase and present evidence that specificity can be affected by context.     

Alteration of the synthesis of the Clp ATP-dependent protease affects morphological and physiological differentiation in Streptomyces

The genes of Streptomyces coelicolor A3(2) encoding catalytic subunits (ClpP) and regulatory subunits (ClpX and ClpC) of the ATP-dependent protease family Clp were cloned, mapped and characterized. S. coelicolor contains at least two clpP genes, clpP1 and clpP2, located in tandem upstream from the clpX gene, and at least two unlinked clpC genes. Disruption of the clpP1 gene in S. lividans and S. coelicolor blocks differentiation at the substrate mycelium step. Overexpression of clpP1 and clpP2 accelerates aerial mycelium formation in S. lividans, S. albus and S. coelicolor. Overproduction of ClpX accelerates actinorhodin production in S. coelicolor and activates its production in S. lividans.     

Detergent-dependent kinetics of truncated Plasmodium falciparum dihydroorotate dehydrogenase

The survival of the malaria parasite Plasmodium falciparum is dependent upon the de novo biosynthesis of pyrimidines. P. falciparum dihydroorotate dehydrogenase (PfDHODH) catalyzes the fourth step in this pathway in an FMN-dependent reaction. The full-length enzyme is associated with the inner mitochondrial membrane, where ubiquinone (CoQ) serves as the terminal electron acceptor. The lipophilic nature of the co-substrate suggests that electron transfer to CoQ occurs at the two-dimensional lipid-solution interface. Here we show that PfDHODH associates with liposomes even in the absence of the N-terminal transmembrane-spanning domain. The association of a series of ubiquinone substrates with detergent micelles was studied by isothermal titration calorimetry, and the data reveal that CoQ analogs with long decyl (CoQ(D)) or geranyl (CoQ(2)) tails partition into detergent micelles, whereas that with a short prenyl tail (CoQ(1)) remains in solution. PfDHODH-catalyzed reduction of CoQ(D) and CoQ(2), but not CoQ(1), is stimulated as detergent concentrations (Tween 80 or Triton X-100) are increased up to their critical micelle concentrations, beyond which activity declines. Steady-state kinetic data acquired for the reaction with CoQ(D) and CoQ(2) in substrate-detergent mixed micelles fit well to a surface dilution kinetic model. In contrast, the data for CoQ(1) as a substrate were well described by solution steady-state kinetics. Our results suggest that the partitioning of lipophilic ubiquinone analogues into detergent micelles needs to be an important consideration in the kinetic analysis of enzymes that utilize these substrates.     

Production of novel antioxidative prenyl naphthalen-ols by combinational bioconversion with dioxygenase PhnA1A2A3A4 and prenyltransferase NphB or SCO7190

We performed combinational bioconversion of substituted naphthalenes with PhnA1A2A3A4 (an aromatic dihydroxylating dioxygenase from marine bacterium Cycloclasticus sp. strain A5) and prenyltransferase NphB (geranyltransferase from Streptomyces sp. strain CL190) or SCO7190 (dimethylallyltransferase from Streptomyces coelicolor A3(2)) to produce prenyl naphthalen-ols. Using 2-methylnaphthalene, 1-methoxynaphthalene, and 1-ethoxynaphthalene as the starting substrates, 10 novel prenyl naphthalen-ols were produced by combinational bioconversion. These novel prenyl naphthalen-ols each showed potent antioxidative activity against a rat brain homogenate model. 2-(2,3-Dihydroxyphenyl)-5,7-dihydroxy-chromen-4-one (2',3'-dihydroxychrysin) generated with another aromatic dihydroxylating dioxygenase and subsequent dehydrogenase was also geranylated at the C-5'-carbon by the action of NphB.     

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.