ABSTRACT
BACKGROUND: Iterative type II polyketide synthases (PKSs) produce polyketide chains of variable but defined length from a specific starter unit and a number of extender units. They also specify the initial regiospecific folding and cyclization pattern of nascent polyketides either through the action of a cyclase (CYC) subunit or through the combined action of site-specific ketoreductase (KR) and CYC subunits. Additional CYCs and other modifications may be necessary to produce linear aromatic polyketides. The principles of the assembly of the linear aromatic polyketides, several of which are medically important, are well understood, but it is not clear whether the assembly of the angular aromatic (angucyclic) polyketides follows the same rules. RESULTS: We performed an in vivo evaluation of the subunits of the PKS responsible for the production of the angucyclic polyketide jadomycin (jad), in comparison with their counterparts from the daunorubicin (dps) and tetracenomycin (tcm) PKSs which produce linear aromatic polyketides. No matter which minimal PKS was used to produce the initial polyketide chain, the JadD and DpsF CYCs produced the same two polyketides, in the same ratio; neither product was angularly fused. The set of jadABCED PKS plus putative jadl CYC genes behaved similarly. Furthermore, no angular polyketides were isolated when the entire set of jad PKS enzymes and Jadl or the jad minimal PKS, Jadl and the TcmN CYC were present. The DpsE KR was able to reduce decaketides but not octaketides; in contrast, the KRs from the jad PKS (JadE) or the actinorhodin PKS (ActIII) could reduce octaketide chains, giving three distinct products. CONCLUSIONS: It appears that the biosynthesis of angucyclic polyketides cannot be simply accomplished by expressing the known PKS subunits from artificial gene cassettes under the control of a non-native promoter. The characteristic structure of the angucycline ring system may arise from a kinked precursor during later cyclization reactions involving additional, but so far unknown, components of the extended decaketide PKS. Our results also suggest that some KRs have a minimal chain length requirement and that CYC enzymes may act aberrantly as first-ring aromatases that are unable to perform all of the sequential cyclization steps. Both of these characteristics may limit the widespread application of CYC or KR enzymes in the synthesis of novel polyketides.
Subject(s)
Alcohol Oxidoreductases/metabolism , Anti-Bacterial Agents/biosynthesis , Bacterial Proteins , Multienzyme Complexes/metabolism , Alcohol Oxidoreductases/genetics , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Cloning, Molecular , Cyclization , Gene Expression , Genes, Bacterial , Isoquinolines/chemistry , Isoquinolines/metabolism , Isoquinolines/pharmacology , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Structure , Multienzyme Complexes/genetics , Plasmids , Streptomyces/enzymology , Streptomyces/geneticsABSTRACT
Evidence now being obtained through nucleotide (nt) sequence analysis supports the concept that secondary metabolism has arisen by modification of existing primary metabolic reactions. Although amino acid sequence identity deduced from nt sequences of genes encoding proteins from related primary and secondary metabolic pathways is sufficient to indicate a common ancestry, the match is often better when genes in different rather than in the same species are compared. The information so far available suggests that gene transfer between organisms has been an important factor in the evolution of secondary metabolism. Many secondary pathways may be of relatively ancient origin and they may have arisen only infrequently. Much subsequent elaboration of the pathways has probably taken place after their acquisition by other species and so has been influenced by a variety of selective conditions. The characteristic diversity of secondary metabolites and their functions can be accounted for by the random manner in which the pathways initially evolved and have subsequently been exploited.
Subject(s)
Anti-Bacterial Agents/metabolism , Biological Evolution , Genes, Bacterial , Anti-Bacterial Agents/biosynthesis , LactamsABSTRACT
Genes involved in the biosynthesis of p-aminobenzoic acid (PABA) in Streptomyces lividans 1326 were cloned in pBR322 by complementing a pabB mutant of Escherichia coli. A 2.7-kb BamHI-SstI fragment of the cloned DNA complemented pabA and pabB mutations in both E. coli and S. lividans; complementation in S. lividans was accompanied by integration of the recombinant plasmid into the host chromosome. The nucleotide (nt) sequence of the 2.7-kb fragment contained two open reading frames, the deduced amino acid sequences of which were similar to those of pabA and pabB products from other bacteria. The nt sequences indicated that pabA and pabB are closely linked in S. lividans and supported cloning evidence that the genes are expressed from a promoter with features resembling those of most E. coli promoters.
Subject(s)
Genes, Bacterial , Streptomyces/enzymology , Transaminases/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Bacterial , Genetic Complementation Test , Molecular Sequence Data , Open Reading Frames , Streptomyces/genetics , Transaminases/metabolismABSTRACT
A second regulatory gene (jadR(1)) is located immediately upstream of the putative repressor gene (jadR(2)) in the jad cluster for biosynthesis of the antibiotic jadomycin B in Streptomyces venezuelae ISP5230. It encodes a 234-amino acid polypeptide with a sequence resembling those of response regulator proteins in two-component control systems. Features in the conserved C-terminal domain of JadR(1) place the protein in the OmpR-PhoB subfamily of response regulators. In mutants where jadR(1) was deleted or disrupted, jadomycin B was not produced, implying that the gene has an essential role in biosynthesis of the antibiotic. Cloning jadR(1) from S. venezuelae in pJV73A, and introducing additional copies of the gene into the wild-type parent by plasmid transformation gave unstable strains with pJV73A integrated into the chromosome. The transformants initially showed increased production of jadomycin B but gave lower titers as excess copies of jadR(1) were lost; mature cultures stabilized with a wild-type level of antibiotic production. The mutant from which jadR(1) had been deleted could not be transformed with pJV73A. Altering the composition of jadR genes in the chromosome by integration of vectors carrying intact and disrupted copies of jadR(1) and jadR(2) provided evidence that the two genes form a regulatory pair different in function from previously reported two-component systems controlling antibiotic biosynthesis in streptomycetes.
Subject(s)
Genes, Regulator/genetics , Isoquinolines/metabolism , Streptomyces/genetics , Alleles , Amino Acid Sequence , Base Sequence , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Gene Dosage , Genes, Regulator/physiology , Molecular Sequence Data , Mutation , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Streptomyces/metabolismABSTRACT
The isopenicillin N synthetase (IPNS) gene from Streptomyces clavuligerus was isolated from an Escherichia coli plasmid library of S. clavuligerus genomic DNA fragments using a 44-mer mixed oligodeoxynucleotide probe. The nucleotide sequence of a 3-kb region of the cloned fragment from the plasmid, pBL1, was determined and analysis of the sequence showed an open reading frame that could encode a protein of 329 amino acids with an Mr of 36,917. When the S. clavuligerus DNA from pBL1 was introduced into an IPNS-deficient mutant of S. clavuligerus on the Streptomyces vector pIJ941, the recombinant plasmid was able to complement the mutation and restore IPNS activity. The protein coding region of the S. clavuligerus IPNS gene shows about 63% and 62% similarity to the Cephalosporium acremonium and Penicillium chrysogenum IPNS nucleotide sequences, respectively, and the predicted amino acid sequence of the encoded protein showed about 56% similarity to both fungal sequences.
Subject(s)
Bacterial Proteins/genetics , Enzymes/genetics , Genes, Bacterial , Oxidoreductases , Streptomyces/genetics , Acremonium/genetics , Amino Acid Sequence , Base Sequence , Fungal Proteins/genetics , Genes , Molecular Sequence Data , Penicillium chrysogenum/genetics , Recombinant Proteins/genetics , Sequence Homology, Nucleic Acid , Streptomyces/enzymologyABSTRACT
Some of the accumulated information on the physiology and nutritional control of antibiotic production in actinomycetes can now be related to recent discoveries in the field of actinomycete molecular biology. This review focuses on aspects of genetic and metabolic control of antibiotic biosynthesis. It surveys some well established principles in the relationship between primary and secondary metabolism, and summarizes briefly the areas where progress is being made in elucidating the molecular organization of regulatory systems underlying this relationship.
ABSTRACT
The interaction between growth and secondary metabolism develops from physiological responses of the producer organism to its environment. Nutrients are channelled into primary growth processes or into secondary processes such as antibiotic biosynthesis by a variety of metabolic controls, the nature of which has been extensively studied in organisms producing beta-lactam antibiotics via the tripeptide, delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine. In the following article we review the early stages of beta-lactam biosynthesis in fungi and actinomycetes, keeping in mind the regulation of primary pathways that provide the amino acid precursors of this group of antibiotics, as well as the regulation of the secondary pathway itself. Of special importance to organisms engaging in secondary metabolism are the control mechanisms that suppress the nonessential process during rapid growth but allow secondary metabolic genes to be expressed and resources to be diverted when environmental factors generate the appropriate biochemical signals.
ABSTRACT
Media and culture conditions were developed for experiments on the physiology of secondary metabolism in Streptomyces coelicolor A3(2). Well dispersed mycelial growth was obtained in a buffered starch-glutamate-salts medium; a high (5%) starch concentration and addition of glass beads aided dispersal. Under the conditions developed, production of actinorhodin was suppressed during trophophase growth and began abruptly near the growth maximum.
Subject(s)
Anti-Bacterial Agents/biosynthesis , Streptomyces/metabolism , Anthraquinones/metabolism , Culture Media , Streptomyces/growth & developmentABSTRACT
When genomic DNA fragments from Streptomyces venezuelae ISP5230 were probed at moderate stringency with recA from Mycobacterium tuberculosis, a 2.0-kb SmaI fragment was identified. The fragment was isolated by cloning a BamHI digest of S. venezuelae DNA in pHJL400 and screening the plasmids in Escherichia coli by Southern hybridization using a sib-selection technique. Sequencing the hybridizing region located an open reading frame encoding 377 amino acids. Its deduced amino acid sequence resembled that of recA genes from other bacteria. The cloned S. venezuelae gene conferred partial resistance to ethyl methanesulfonate when expressed in E. coli from the lacZ promoter.
Subject(s)
Genes, Bacterial/genetics , Rec A Recombinases/genetics , Streptomyces/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Bacterial/analysis , Escherichia coli/drug effects , Escherichia coli/genetics , Ethyl Methanesulfonate/pharmacology , Molecular Sequence Data , Open Reading Frames/genetics , Rec A Recombinases/chemistry , Restriction Mapping , Sequence Alignment , Sequence Analysis, DNA , Streptomyces/chemistryABSTRACT
Fragments of genomic DNA from Streptomyces venezuelae ISP5230 were cloned in the Escherichia coli expression vector pTZ18R and the plasmids were used to transform E. coli JA194 (trpE). The transformants included a prototrophic strain containing a recombinant plasmid, pDQ181, with an approximately 6.8-kb insert. Subcloning located the trpE-complementing DNA in a 2.4-kb segment. Transformation of E. coli ED23 (lacking both trpE and trpG functions) with plasmids containing the 2.4-kb DNA segment gave prototrophic strains exhibiting both the ASI and ASII activities of anthranilate synthetase. The results indicated that trpE and trpG are clustered in S. venezuelae. Regions hybridizing to the pDQ181 insert were present in the genomic DNA of other streptomycetes.
Subject(s)
Anthranilate Synthase/genetics , Genes, Bacterial , Streptomyces/genetics , Anthranilate Synthase/metabolism , Blotting, Southern , Cloning, Molecular , Escherichia coli/genetics , Restriction Mapping , Streptomyces/enzymology , Transformation, Bacterial , Tryptophan/geneticsABSTRACT
LC-MS and LC-MS/MS analyses indicated that an enzyme responsible for inactivating the antibiotic etamycin is specific for streptogramins and acts on both type B-I and B-II streptogramin subgroups. No enzymatic activity was detected for other cyclodepsipeptides such as surfactins and viscosin. It was demonstrated using analogs of etamycin that the picolinyl moiety is essential to obtain enzyme-generated ring-opened compounds. Because the picolinyl moiety is also essential for the biological activity of streptogramins, it is proposed that this residue is a distinctive topographic feature in the binding of this group of antibiotics to enzyme active sites.
Subject(s)
Streptomyces/enzymology , Virginiamycin/metabolism , Chromatography, Liquid , Magnetic Resonance Spectroscopy , Mass Spectrometry , Substrate Specificity , Virginiamycin/chemistryABSTRACT
The intracellular α-aminoadipic acid pool in Streptomyces glavuligerus mycelium growing in a starch-peptone medium decreased during the late exponential and stationary phases when cephamycin was being produced; however, the amino acid accumulated extracellularly. Although the specific activity of lysine É-aminotransferase (LAT) decreased during this period, there was no indication that the extracellular α-aminoadipic acid functioned as a precursor reserve for synthesis of the ß-lactam antibiotic. Measurement of LAT activity in cultures grown in defined media with starch and various nitrogen sources indicated that the enzyme was synthesized preferentially only during early growth. In its insensitivity to induction by a precursor, and in its susceptibility to carbon catabolite repression, LAT behaved as a secondary metabolic pathway enzyme. Unexpectedly, however, the enzyme increased in specific activity when cultures were supplemented with excess phosphate. Unlike LAT, cadeverine aminotransferase was inducible by lysine or cadaverine and insensitive to phosphate; its features were consistent with a role in the catabolism of lysine by S. clavuligerus.
ABSTRACT
Cultures of Streptomyces lincolnensis accumulated 3-methylthioacrylic acid in amounts directly related to the concentration of methionine in the medium. The metabolite was labeled by L-(methyl-14C) but not by DL-(carboxyl-14C) methionine, indicating biosynthesis from the amino acid with loss of the carboxyl group. S. lincolnensis mycelium contained sufficient peroxidase activity to catalyse oxidative decarboxylation of L-methionine to 3-methylthiopropionamide as the initial step of a biosynthetic sequence. The enzyme, partially purified by ammonium sulfate precipitation, chromatography on a DEAE-cellulose column ang gel filtration, had a molecular weight of approximately 350,000, a pH optimum of 6.0, with o-dianisidine as electron donor and a Km value of 7.5 x 10(-4) M with respect to hydrogen peroxide. Cultures of S. lincolnensis supplemented with 3-(methyl-14C) methylthiopropionic acid gave labeled 3-methylthioacrylic acid. However, 3-(methyl-14C) methylthiopropionamide did not label the metabolite, suggesting that the first intermediate in the pathway may be the keto acid, which is then oxidatively decarboxylated to 3-methylthiopropionic acid.
Subject(s)
Acrylates/metabolism , Methacrylates/metabolism , Methionine/metabolism , Streptomyces/metabolism , Acrylates/isolation & purification , Fungal Proteins/analysis , Peroxidases/isolation & purification , Streptomyces/enzymology , Time FactorsABSTRACT
Cynodontin, emodin, endocrocin and secalonic acids A, E and G have been isolated from five strains of Pyrenochaeta terrestis. Aspergillus aculeatus produces emodin, endocrocin and secalonic acids B, D and F. No cynodontin was detected. Isolation of emodin in small amounts supports previous evidence that it is an intermediate in secalonic acid biosynthesis. Isolation of cynodontin and endocrocin, which are co-produced with secalonic acids in other organisms, suggests that these compounds are formed by a common branching pathway. A natural isolate of P. terrestris contained variant strains which produced different relative amounts of secalonic acids A, E and G. From the combinations of secalonic acids produced in organisms so far examined it is concluded that precursor tetrahydroxanthone units or formed in pairs differing in stereo-chemistry only at C-5 or at the trans-invariant C-6 : C-10a positions. A possible biosynthetic pathway is discussed.
Subject(s)
Anthraquinones/isolation & purification , Anti-Bacterial Agents/biosynthesis , Aspergillus/metabolism , Emodin/isolation & purification , Mitosporic Fungi/metabolism , Xanthenes/isolation & purification , Xanthones , Acetylation , Anthracenes/isolation & purification , Anti-Bacterial Agents/isolation & purification , Culture Media , FermentationABSTRACT
Cultures of Streptomyces griseus grown under phosphate-limiting conditions produced a complex of green products. Three of these were separated from the mixture and characterized. One was identified as viridomycin A, the ferrous chelate of 4-hydroxy-3-nitrosobenzaldehyde; the second (actinoviridin A) was the corresponding carboxylic acid chelate and the third (viridomycin E) was a hybrid chelate containing both the aldehyde and acid ligands. Only two out of nine strains of S. griseus examined produced viridomycins and the ligands were biosynthesized only in media from which phosphate had been exhausted. Optimization of the production medium showed that fructose and alanine were the most favorable carbon and nitrogen sources and that relatively high concentrations of ferrous ions were necessary. The results suggest that viridomycins are not produced by S. griseus as iron scavengers in response to iron deficiency but as secondary metabolites that are stabilized adventitiously in the broth by metal ion chelation.
Subject(s)
Anti-Bacterial Agents/biosynthesis , Pigments, Biological/biosynthesis , Streptomyces/metabolism , Carbon/metabolism , Chemical Phenomena , Chemistry, Physical , Culture Media , Iron/metabolism , Nitrogen/metabolism , Nitroso Compounds/biosynthesis , Spectrum AnalysisABSTRACT
A new compound isolated from cultures of Aspergillus candidus Link is shown to be 3-hydroxyterphenyllin. The structure was assigned by comparing the 1H and 13C nmr spectra of the metabolite and its acetate derivative with those of terphenyllin and terphenyllin triacetate.
Subject(s)
Aspergillus/metabolism , Terphenyl Compounds/isolation & purification , Chemical Phenomena , Chemistry , Magnetic Resonance SpectroscopyABSTRACT
The proton noise decoupled 13C nuclear magnetic resonance spectrum of tetracycline hydrochloride prepared from Streptomyces aureofaciens cultures supplemented with [1-13C]acetate and [2-13C]acetate showed enrichment of nine alternating ring carbons. In addition, a small enrichment of the carboxamide carbon by [1-13C]acetate was observed. The labelling patterns clearly demonstrated the polyketide origin of the tetracyclic nucleus. The 13C nuclear magnetic resonance spectrum of tetracycline hydrochloride derived from [1,2-13C]acetate showed all 18 ring carbons as doublets with coupling constants appropriate for the incorporation of nine intact two-carbon precursors, confirming that head-to-tail condensation of C2 units had occurred. Absence of bond scission within the C2 units and a low level of uncoupled 13C in the carboxamide substituent indicated that when the organism is supplemented with acetate, malonyl coenzyme A used for tetracycline biosynthesis is formed by direct carboxylation of acetyl coenzyme A.
Subject(s)
Acyl Coenzyme A/metabolism , Malonyl Coenzyme A/metabolism , Streptomyces aureofaciens/metabolism , Tetracycline/biosynthesis , Acetates/metabolism , Carbon Isotopes , Carbon Radioisotopes , Magnetic Resonance SpectroscopyABSTRACT
Bikaverin obtained by supplementing cultures of Fusarium oxysporum with singly and doubly 13C labeled acetate was enriched by approximately 0.5 atom percent with the 13C isotope. At this low enrichment 13C NMR spectra of samples labeled from (1-13C)- and (2-13C) acetate did not show, unequivocally, the pattern of isotopic incorporation. Small sample size, poor solubility and difficulties in the assignment of resonances also restricted the amount of information thacetate. The difficulty was overcome by using 13C homonuclear single-frequency decoupling in conjunction with 1H heteronuclear decoupling to locate bonded 13C-13C pairs. The carbon skeleton of bikaverin was shown to be biosynthesized entirely by condensation of acetate units and the pattern of assembly was established.
Subject(s)
Anti-Bacterial Agents/biosynthesis , Fusarium/metabolism , Acetates/metabolism , Carbon Isotopes , Chemical Phenomena , Chemistry , Magnetic Resonance Spectroscopy , Methionine/metabolismABSTRACT
Cultures of actinomycete strain WRAT-210 produced a dark red crystalline metabolite which was named texazone. Spectroscopic evidence suggested that the structure of texazone is 2-(N-methylamino)-3H-phenoxazin-3-one-8-carboxylic acid. The structure was confirmed by chemical synthesis through oxidative dimerization of ethyl 3-amino-4-hydroxybenzoate with 2-(N-methylamino)phenol and subsequent hydrolysis of the resultant phenoxazinone ester.
Subject(s)
Actinomyces/metabolism , Anti-Bacterial Agents/biosynthesis , Oxazines/biosynthesis , Indicators and Reagents , Magnetic Resonance Spectroscopy , Mass Spectrometry , Oxazines/isolation & purification , SpectrophotometryABSTRACT
Streptomyces griseoviridus produces in addition to etamycin several related compounds which can be separated by partition chromatography. One of these has been characterized by amino acid analysis and mass spectrometry and shown to have the same structure as etamycin except for replacement of the hydroxyproline residue by proline. Evidence was obtained for additional congeners similarly related to etamycin by amino acid exchange. The relative proportions of such congeners produced by S. griseoviridus depends upon the medium in which the culture is grown. Certain amino acids support good yields of the metabolites and the culture appears to be steered towards the synthesis of congeners containing such amino acids.