Candicidin biosynthesis in <Emphasis Type="Italic">Streptomyces griseus</Emphasis>

The biosynthesis of the aromatic polyene macrolide antibiotic candicidin, produced by Streptomyces griseus IMRU 3570, begins with a p-aminobenzoic acid (PABA) molecule which is activated to PABA-CoA and used as starter for the head-to-tail condensation of four propionate and 14 acetate units to produce a polyketide molecule to which the deoxysugar mycosamine is attached. Using the gene coding for the PABA synthase ( pabAB) from S. griseusIMRU 3570 as the probe, a 205-kb region of continuous DNA from the S. griseus chromosome was isolated and partially sequenced. Some of the genes possibly involved in the biosynthesis of candicidin were identified including part of the modular polyketide synthase (PKS), genes for thioesterase, deoxysugar biosynthesis, modification, transport, and regulatory proteins. The regulatory mechanisms involved in the production of candicidin, such as phosphate regulation, were studied using internal probes for some of the genes involved in the biosynthesis of the three moieties of candicidin (PKS, aromatic moiety and amino sugar). mRNAs specific for these genes were detected only in the production medium (SPG) but not in the SPG medium supplemented with phosphate or in the inoculum medium, indicating that phosphate represses the expression of genes involved in candicidin biosynthesis. The modular architecture of the candicidin PKS and the availability of the PKSs involved in the biosynthesis of three polyene antibiotics (pimaricin, nystatin, and amphotericin B) shall make possible the creation of new, less toxic and more active polyene antibiotics through combinatorial biosynthesis and targeted mutagenesis.     

Regulation by aromatic amino acids of the biosynthesis of candicidin by Streptomyces griseus

The biosynthesis by Streptomyces griseus of candicidin, an aromatic polyene macrolide antibiotic, was inhibited by L-tryptophan, L-phenylalanine and, to a lesser degree, by L-tyrosine. A mixture of the three aromatic amino acids inhibited candicidin biosynthesis to a greater extent than did each amino acid separately. L-Tryptophan strongly inhibited the incorporation of the labelled precursors propionate or 4-aminobenzoic acid into candicidin. Incorporation of propionate into candicidin was 50% inhibited by 2.5 mM-tryptophan. Inhibition by tryptophan did not require protein synthesis as the same effect was observed in cells in which protein synthesis was prevented by chloramphenicol. The inhibitory effect of L-tryptophan was partially reversed by exogenous 4-aminobenzoic acid suggesting that this effect is exerted at the level of 4-aminobenzoic acid synthase.     

Origin of p-aminobenzoic acid from chorismic rather than iso-chorismic acid in Enterobacter aerogenes and Streptomyces species

Enzyme extracts from Enterobacter aerogenes (62-1), Streptomyces aminophilus, and Streptomyces coelicolor were used to investigate the biosynthesis of p-aminobenzoic acid. The enzyme preparations from E. aerogenes and S. aminophilus contained both p-aminobenzoate synthase and iso-chorismate synthase activity, and were able to convert both chorismic and iso-chorismic acid to p-aminobenzoic acid. The apparent KM for chorismic acid was, however, significantly lower than that for iso-chorismic acid, while the Vmax was identical for both substrates in both enzyme systems. The enzyme preparations from S. coelicolor did not contain iso-chorismate synthase activity and p-aminobenzoic acid synthesis took place in this system from chorismic acid only. It is concluded that iso-chorismic acid is not an obligatory intermediate in p-aminobenzoic acid biosynthesis in these organisms.     

Cloning and characterization of a phosphate-regulated promoter involved in phosphate control of candicidin biosynthesis

Phosphate strongly repressed the formation of p-aminobenzoic acid (PABA) synthase, an enzyme involved in candicidin biosynthesis. Expression in Streptomyces lividans of the pabS gene (encoding PABA synthase) of Streptomyces griseus is repressed by phosphate at concentrations above 0.1 mM. However, expression of the pabS gene in Escherichia coli is not regulated by phosphate. Phosphate control of the expression of the pabS gene was observed in all plasmids containing the original 4.5-kb BamHI fragment, whereas no phosphate regulation was found when an upstream 1-kb fragment that carries the pabS promoter was deleted. Using the promoter-probe plasmid pIJ424, a '114-bp' promoter was cloned. Expression of the promoterless kanamycin phosphotransferase gene when fused to the '114-bp' promoter was strongly reduced by phosphate (90% at 5 mM concentration). The '114-bp' promoter has been sequenced and the first transcribed nucleotide identified by S1 mapping. The '114-bp' fragment is A + T-rich (54%), as compared to the Streptomyces genome (70-73% GC). The presence of a phosphate control sequence (pcs) in the upstream region of the pabS gene is proposed.     

Use of a cloned gene involved in candicidin production to discover new polyene producer Streptomyces strains

A p-aminobenzoic synthase gene (pabS) from Streptomyces griseus IMRU 3570 involved in candicidin production was used as probe to find new aromatic polyene producing Streptomyces strains. The pab gene hybridizes with 6 out of 16 Streptomyces strains, and those strains which hybridize turned out to be polyene producers. Such strains were never before described as polyene producers.     

Characterization and regulation of anthranilate synthetase from a chloramphenicol-producing streptomycete.

In Streptomyces sp. 3022a, anthranilate synthetase is composed of two non-identical subunits. The major subunit (molecular weight, 72,000) converts chorismic acid to anthranilic acid, using ammonia as the source of the amino group. The smaller subunit (molecular weight 28,000 to 29,000) confers on the enzyme the ability to use glutamine instead of ammonia as a substrate. In this study, reactivity with glutamine reached its maximum at pH 7.2 to 7.6, whereas that with ammonia increased linearly through pH 9.0 without reaching a maximum. Activity was increased and stabilized by adding glutamine and magnesium chloride to the buffer system. Both activities of the enzyme were inhibited by anthranilic acid and by tryptophan. Synthesis was repressed by histidine, anthranilic acid, tryptophan, and p-aminobenzoic acid. When activity was repressed by anthranilic acid and by tryptophan, there was a concomitant increase in the activity of arylamine synthetase, an enzyme involved in chloramphenicol production. Stimulating arylamine synthetase, however, did not increase antibiotic synthesis.     

Isolation,taxonomic and fermentation studies on a new strain of Streptomyces arenae var ukrainiana producing a tetraene antibiotic

A Streptomyces strain UK10 was isolated from Ukrainian soil and identified by taxonomical studies as Streptomyces arenae var ukrainiana. HA-2-91 was isolated from the biomass of S. arenae var ukrainiana and is supposedly a polyene macrolide antibiotic belonging to the tetraene group. HA-2-91 showed promising antifungal activity (in vitro) against yeasts and filamentous fungi, including plant pathogens and dermatophytes and was found to be less toxic in mice than nystatin and rimocidin.     

Streptomycin sensitivity of ribosomes isolated from a streptomycin-producing Streptomyces griseus.

The streptomycin sensitivity of ribosomes derived from a streptomycin-producing Streptomyces griseus was examined in a polyuridylic acid directed 14C-phenylalanine incorporating system. In order to get reproducible results it is essential to use cell-free extracts which do not inactivate streptomycin. This condition can be fulfilled by the combination of washed ribosomes of the streptomycin-producing strain and the 110 000 g supernatant of the streptomycin-nonproducing variant of S. griseus, because the streptomycin-phosphorylating activity can be washed out from ribosomes of younger streptomycin-producing cultures, and the streptomycin-nonproducing S. griseus does not have any streptomycin-inactivating capacity. In this amino acid polymerizing system the ribosomes of the streptomycin-producing strain were as sensitive to streptomycin as the ribosomes of the nonproducing variant or of Escherichia coli.     

Biochemistry and genetics of chloramphenicol production

In Streptomyces venezuelae, chloramphenicol is derived by an unusual diversion of chorismate, the branchpoint intermediate of the pathway involved in the biosynthesis of aromatic amino acids. In the chloramphenicol-producing organism, the DAHP synthetase was neither feedback inhibited nor repressed. Chorismate mutase was not repressed or inhibited by the intermediates or end-products of the shikimate-chorismate pathway. However, anthranilate synthetase and prephenate dehydratase are feedback inhibited by tryptophan and phenylalanine, respectively. During growth, when primary metabolism is not perfectly coordinated, decreasing demand for aromatic amino acids results in shunting of chorismate towards chloramphenicol biosynthesis.The endogenous synthesis of chloramphenicol produced by Streptomyces venezuelae is inhibited by the increasing concentration of chloramphenicol in the medium. Arylamine synthetase, the first enzyme involved in chloramphenicol biosynthesis, is repressed by the secreted chloramphenicol, by dl-p-aminophenylalanine and l-threo-p-aminophenylserinol. The excess intracellular chorismate pool is diverted to other aromatic shunt metabolites if biosynthesis of chloramphenicol is inhibited. There appears to be a glutamine binding protein subunit which is shared by several enzymes involved in amination of the aromatic ring of chorismate.Chloramphenicol producing organism also inactivated intracellular chloramphenicol. However, the resistance of the streptomycetes is due to inducible impermeability of the organism to chloramphenicol during antibiotic production. Streptomyces venezuelae is sensitive to chloramphenicol when it is not engaged in antibiotic production. The resistance to and production of chloramphenicol are induced simultaneously.A linkage map for 17 marker loci using Streptomyces venezuelae has been constructed. Restriction enzyme map of a plasmid from the chloramphenicol-producing streptomycetes has also been developed. The role of the plasmid in chloramphenicol biosynthesis and the life-cycle of the Streptomyces venezuelae is not yet understood.     

Biosynthesis and phosphate control of candicidin byStreptomyces acrimycini JI2236: effect of amplification of thepabAB gene

Summary Biosynthesis of candicidin byStreptomyces acrimycini JI2236 was strongly inhibited by phosphate.p-Aminobenzoic acid (PABA) synthase activity, required for the synthesis of PABA, a candicindin precursor, was reduced by 72% in cells grown in medium supplemented with 7.5 mM phosphate. Hybridization studies showed that the DNA region ofS. acrimycini carrying thepabAB gene (encoding PABA synthase) is very similar to the homologous region ofS. griseus 3570.S. acrimycini was easily transformed with plasmids containing thepabAB gene ofS. griseus. Four transformants were studied in detail; three of the transformants synthesized higher levels of PABA synthase and two transformants produced more candicidin than control cultures transformed with pIJ699. The fourth transformant was unable to synthesize the antibiotic. Formation of PABA synthase and candicidin production was equally sensitive to phosphate regulation in transformants with thepabAB than in the untransformedS. acrimycini strain.     

Regulatory control of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthetase in Streptomyces antibioticus

Streptomyces antibioticus possesses a tryptophan-inhibitable 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthetase whose synthesis is also repressed by L-tryptophan. Studies of the DAHP synthetase obtained by ammonium sulfate fractionation of a crude extract derived from S. Antibioticus revealed that the enzymic activity was only partially inhibited by tryptophan. Inhibition of the DAHP synthetase activity was strongly pH dependent at values below 7.0. A number of tryptophan analogues was noted to inhibit the enzyme; by contrast, other aromatic amino acid end products failed to affect DAHP synthetase activity. Chorismic acid, a key intermediate in aromatic amino acid biosynthesis, was ineffective as an inhibitor when used alone; however, if supplied with L-tryptophan, a further reduction of DAHP synthetase activity (15--25%) was routinely observed.     

Purification and characterization of a soybean flour-inducible ferredoxin reductase of Streptomyces griseus.

We have purified an NADH-dependent ferredoxin reductase from crude extracts of Streptomyces griseus cells grown in soybean flour-enriched medium. The purified protein has a molecular weight of 60,000 as determined by sodium dodecyl sulfate gel electrophoresis. The enzyme requires Mg2+ ion for catalytic activity in reconstituted assays, and its spectral properties resemble those of many other flavin adenine dinucleotide-containing flavoproteins. A relatively large number of hydrophobic amino acid residues are found by amino acid analysis, and beginning with residue 7, a consensus flavin adenine dinucleotide binding sequence, GXGXXGXXXA, is revealed in this protein. In the presence of NADH, the ferredoxin reductase reduces various electron acceptors such as cytochrome c, potassium ferricyanide, dichlorophenolindophenol, and nitroblue tetrazolium. However, only cytochrome c reduction by the ferredoxin reductase is enhanced by the addition of ferredoxin. In the presence of NADH, S. griseus ferredoxin and cytochrome P-450soy, the ferredoxin reductase mediates O dealkylation of 7-ethoxycoumarin.     

Isolation of a chitinase overproducing mutant of Streptomyces peucetius defective in daunorubicin biosynthesis

Streptomyces peucetius, producer of the antitumor anthracycline antibiotic daunorubicin, was mutagenized, and mutants defective in daunorubicin biosynthesis were screened. One mutant (SPVI), which failed to produce daunorubicin, was found to overproduce an extracellular chitinase. Time course analyses of chitinase production and of the extracellular protein profile showed that the increase in activity is due to increased synthesis of the enzyme protein. The production of chitinase in SPVI was repressed by glucose as in the case of wild-type S. peucetius. PFGE analysis of VspI restriction fragments of S. peucetius and SPVI showed that there was no major alteration in the mutant genome. The hybridization pattern of S. peucetius and SPVI genomic DNA digested with various restriction enzymes was identical when probed with dnrUVJI genes of the S. peucetius daunorubicin cluster and chiA of Streptomyces lividans 66. The possible step affected in the daunorubicin biosynthetic pathway could be a polyketide synthase, since aklanonic acid, the earliest detectable intermediate in the daunorubicin pathway, was not synthesized in SPVI.     

Characterization, expression in Streptomyces lividans, and processing of the amylase of Streptomyces griseus IMRU 3570: two different amylases are derived from the same gene by an intracellular processing mechanism.

Extracellular amylase in Streptomyces lividans was undetectable in starch-supplemented medium. However, S. lividans produced fivefold-higher levels of amylase than Streptomyces griseus IMRU 3570 when transformed with the S. griseus amy gene. Two major proteins of 57 and 50 kDa with amylase activity accumulated in the culture broths of the donor S. griseus and S. lividans transformed with the amy gene. Both proteins were also present in protoplast lysates in the same relative proportion; they gave a positive reaction with antibodies against the 57-kDa amylase. They did not differ in substrate specificity or enzyme kinetics. The two amylases were purified to homogeneity by a two-step procedure. Both proteins showed the same amino-terminal sequence of amino acids, suggesting that both proteins are derived from the same gene. The deduced signal peptide has 28 amino acids with two positively charged arginines near the amino-terminal end. When an internal NcoI fragment was removed from the amy gene, the resulting S. lividans transformants did not synthesize any of the two amylase proteins and showed no reaction in immunoblotting. Formation of the 50-kDa protein was observed when pure 57-kDa amylase was treated with supernatants of protoplast lysates but not when it was treated with membrane preparations, indicating that the native 57-kDa amylase could be processed intracellularly.     

Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2).

A strain of Streptomyces lividans, TK24, was found to produce a pigmented antibiotic, actinorhodin, although S. lividans normally does not produce this antibiotic. Genetic analyses revealed that a streptomycin-resistant mutation str-6 in strain TK24 is responsible for induction of antibiotic synthesis. DNA sequencing showed that str-6 is a point mutation in the rpsL gene encoding ribosomal protein S12, changing Lys-88 to Glu. Gene replacement experiments with the Lys88-->Glu str allele demonstrated unambiguously that the str mutation is alone responsible for the activation of actinorhodin production observed. In contrast, the strA1 mutation, a genetic marker frequently used for crosses, did not restore actinorhodin production and was found to result in an amino acid alteration of Lys-43 to Asn. Induction of actinorhodin production was also detected in strain TK21, which does not harbor the str-6 mutation, when cells were incubated with sufficient streptomycin or tetracycline to reduce the cell's growth rate, and 40 and 3% of streptomycin- or tetracycline-resistant mutants, respectively, derived from strain TK21 produced actinorhodin. Streptomycin-resistant mutations also blocked the inhibitory effects of relA and brgA mutations on antibiotic production, aerial mycelium formation or both. These str mutations changed Lys-88 to Glu or Arg and Arg-86 to His in ribosomal protein S12. The decrease in streptomycin production in relC mutants in Streptomyces griseus could also be abolished completely by introducing streptomycin-resistant mutations, although the impairment in antibiotic production due to bldA (in Streptomyces coelicolor) or afs mutations (in S. griseus) was not eliminated. These results indicate that the onset and extent of secondary metabolism in Streptomyces spp. is significantly controlled by the translational machinery.     

Molecular cloning and nucleotide sequence of Streptomyces griseus trypsin gene.

Streptomyces griseus trypsin (E.C. 3.4.21.4) is one of the major extracellular proteinase, which is secreted by S. griseus. The gene encoding S. griseus trypsin was isolated from a S. griseus genomic library by using a synthetic oligonucleotide probe. Fragments containing the gene for S. griseus trypsin were characterized by hybridization and demonstration of proteolytic activity in S. lividans. Deduced amino acid sequence from the nucleotide sequence suggests that S. griseus trypsin is produced as a precursor, consisting of three portions; an amino-terminal pre sequence (32 amino acid residues), a pro sequence (4 residues), and the mature trypsin. The S. griseus trypsin consists of 223 amino acids with a computed molecular weight of 23,112. The existence of proline at the pro and mature junction suggests that the processing of S. griseus trypsin is non-autocatalytic.     

Detection and changes of nucleotide pyrophosphotransferase activity of Streptomyces griseus strains in submerged culture.

Nucleotide pyrophosphotransferase (NPT) activity of two Streptomyces griseus strains was studied in submerged culture during their life cycle. NPT activity could be detected only in the culture filtrate but not in the membrane fraction or in cell extract of the sporulating (streptomycin-non-producing) S. griseus No. 45-H. No enzyme could be detected in the non-sporulating (streptomycin-producing) S. griseus No 52--1 cultures.