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Background: The genetic engineering of antibiotic-producing Streptomyces strains is an approach that became a successful methodology in developing new natural polyketide derivatives. Glycosyltransferases are important biosynthetic enzymes that link sugar moieties to aglycones, which often derive from polyketides. Biological activity is frequently generated along with this process. Here we report the use of glycosyltransferase genes isolated from the landomycin biosynthetic gene cluster to create hybrid landomycin/urdamycin oligosaccharide antibiotics. Results: Production of several novel urdamycin derivatives by a mutant of Streptomyces fradiae Tu2717 has been achieved in a combinatorial biosynthetic approach using glycosyltransferase genes from the landomycin producer Streptomyces cyanogenus S136. For the generation of gene cassettes useful for combinatorial biosynthesis experiments new vectors named pMUNI, pMUNII and pMUNIII were constructed. These vectors facilitate the construction of gene combinations taking advantage of the compatible Mun I and Eco RI restriction sites. Conclusions: The high-yielding production of novel oligosaccharide antibiotics using glycosyltransferase gene cassettes generated in a very convenient way proves that glycosyltransferases can be flexible towards the alcohol substrate. In addition, our results indicate that LanGT1 from S. cyanogenus S136 is a D-olivosyltransferase, whereas LanGT4 is a L-rhodinosyltransferase.

In Streptomyces fradiae, calcium ions induce alterations in intensity and specificity of the secondary metabolism and stimulate aerial mycelium formation and sporulation. Using in vitro labeling, we demonstrate that in S. fradiae in the late exponential growth phosphorylation of 65-kDa membrane-associated protein is also influenced by Ca(2+) added exogenously. Calcium ions at physiological concentration stimulate intensive Ca(2+)-dependent phosphorylation of 65-kDa protein at multiple sites on serine, threonine, and tyrosine residues. Assay of protein kinases in situ demonstrated in the fraction of membrane-associated proteins the presence of two autophosphorylating protein serine/threonine kinases with molecular masses of 127 kDa and 65 kDa. Autophosphorylation of both proteins is also Ca(2+)-dependent.

The polyketide aglycone, tylactone (protylonolide), does not normally accumulate during tylosin production in Streptomyces fradiae, suggesting that the capacity of the organism to glycosylate tylactone exceeds the capacity for polyketide synthesis. Consistent with this model, tylosin yields were significantly increased (due to bioconversion of the added material) when exogenous tylactone was added to fermentations. However, tylosin yield improvements were also observed (albeit at lower levels) in solvent controls to which dimethylsulfoxide (DMSO) was added. At least in part, the latter effect resulted from stimulation of polyketide metabolism by DMSO. This was revealed when the solvent was added to fermentations containing the tylA mutant, S. fradiae GS14, which normally accumulates copious quantities of tylactone. Journal of Industrial Microbiology & Biotechnology (2001) 27, 46–51.

The tylosin-biosynthetic (tyl) gene cluster occupies about 1% of the genome of Streptomycesfradiae and includes at least 43 open reading frames. In addition to structural genes required for tylosin production, the tyl cluster contains three resistance determinants and several regulatory genes. Tylosin production is evidently controlled by pathway-specific and pleiotropic regulators with the likely involvement of y-butyrolactone signalling factors. Accumulation of the polyketide aglycone is controlled by glycosylated macrolides and optimal performance of the complex polyketide synthase enzyme requires the activity of an editing thioesterase.

The ddcA gene from Streptomyces fradiae, which is located adjacent to the left edge of the tylosin biosynthetic cluster, has been cloned and sequenced. DNA sequence analysis revealed an ORF of 1194 bp that encodes a product of 42.6 kDa. This protein showed significant similarity to the extracellular endopeptidase with beta-lactamase activity encoded by the adp gene from Bacillus cereus and to PBPs (DD-carboxypeptidases and DD-endopeptidases) and beta-lactamases. Moreover, it contains three characteristic motifs conserved in PBPs and beta-lactamases, including an essential serine residue in the active centre and a putative leader peptide. Heterologous expression of the ddcA gene in Streptomyces lividans demonstrated the presence in the transformants of an extracellular beta-lactamase active against penicillin G, ampicillin and the chromogenic cephalosporin nitrocefin.

Background: Streptomyces fradiae is the principal producer of urdamycin A. The antibiotic consists of a polyketide-derived aglycone, which is glycosylated with four sugar components, 2× D-olivose (first and last sugar of a C-glycosidically bound trisaccharide chain at the 9-position), and 2× L-rhodinose (in the middle of the trisaccharide chain and at the 12b-position). Limited information is available about both the biosynthesis of D-olivose and L-rhodinose and the influence of the concentration of both sugars on urdamycin biosynthesis. Results: To further investigate urdamycin biosynthesis, a 5.4 kb section of the urdamycin biosynthetic gene cluster was sequenced. Five new open reading frames (ORFs) ( urdZ3 , urdQ , urdR , urdS , urdT ) could be identified each one showing significant homology to deoxysugar biosynthetic genes. We inactivated four of these newly allocated ORFs ( urdZ3 , urdQ , urdR , urdS ) as well as urdZ1 , a previously found putative deoxysugar biosynthetic gene. Inactivation of urdZ3 , urdQ and urdZ1 prevented the mutant strains from producing L-rhodinose resulting in the accumulation of mainly urdamycinone B. Inactivation of urdR led to the formation of the novel urdamycin M, which carries a C-glycosidically attached D-rhodinose at the 9-position. The novel urdamycins N and O were detected after overexpression of urdGT1c in two different chromosomal urdGT1c deletion mutants. The mutants lacking urdS and urdQ accumulated various known diketopiperazines. Conclusions: Analysis of deoxysugar biosynthetic genes of the urdamycin biosynthetic gene cluster revealed a widely common biosynthetic pathway leading to D-olivose and L-rhodinose. Several enzymes responsible for specific steps of this pathway could be assigned. The pathway had to be modified compared to earlier suggestions. Two glycosyltransferases normally involved in the C-glycosyltransfer of D-olivose at the 9-position (UrdGT2) and in conversion of 100-2 to urdamycin G (UrdGT1c) show relaxed substrate specificity for their activated deoxysugar co-substrate and their alcohol substrate, respectively. They can transfer activated D-rhodinose (instead of D-olivose) to the 9-position, and attach L-rhodinose to the 4A-position normally occupied by a D-olivose unit, respectively.

To examine what causes increased viscosity in culture broth in Streptomyces fradiae culture, various natural nitrogen sources were investigated. Extracellular protease activity increased with culture time and decomposed the natural nitrogen source into amino acids. In the case of gluten meal, after a culture time of 5 d, concentrations of glutamic acid and aspartic acid had increased to 600 and 200 mg/L, respectively, which were about 3- and 2-fold as high as levels in cultures under similar conditions using Pharmamedia. For various amino acids tested, the addition of glutamic acid or aspartic acid mixture to the culture medium raised the apparent viscosity to its highest demonstrated value, 260 mPa.s after 5 d of culture, which was 3-fold higher than without amino acids. Consumption of the decomposed glutamic acid and aspartic acid was dependent on the activities of glutamate dehydrogenase and aspartate aminotransferase, respectively. When ammonium ion was used as the nitrogen source, cell concentration reached 1.75 g/L measured as an intracellular nucleic acid concentration, which was about 2.3-fold higher than that with any other natural nitrogen source. However, apparent viscosity was only 75 mPa.s, a value one-third that of the amino acid mixture, and 70% of the pellets were bigger than 1.2 x 10(4) microm(2). In the case of gluten meal or the amino acid mixture, pellets bigger than 1.2 x 10(4) microm(2) comprised only 8%. This demonstrates that consumption of some amino acids affected the formation of filamentous morphology, which caused an increase in the apparent viscosity of the culture broth, and the apparent viscosity was not caused by the mycelial concentration but the mycelial morphology.

In Streptomyces fradiae, calcium ions induce alterations in intensity and specificity of the secondary metabolism and stimulate sporulation. Using in vivo labeling, we demonstrate that in S. fradiae phosphorylation of some proteins are also influenced by Ca2+ added exogenously. Calcium ions at physiological concentration increase phosphorylation of multiple proteins on serine/threonine residues and suppress modification of a 140-kDa protein on tyrosine residues. Assay of protein kinases in situ demonstrated that Ca2+-induced differences in the pattern of protein phosphorylation in vivo are accompanied by Ca2+-dependent cessation of autophosphorylation of 140-kDa tyrosine kinase and by increased autophosphorylation of three serine/threonine kinases with molecular masses of 127, 65, and 31.5 kDa.

The tlrB gene from Streptomyces fradiae has been cloned and used to construct bifunctional Streptomyces-Escherichia coli shuttle vectors carrying the antibiotic resistance genes to kanamycin-neomycin, thiostrepton and tylosin as selection markers. In the same way, the tlrB gene was subcloned in plasmids including the apramycin resistance gene and the oriT sequence from the plasmid pSET152 to facilitate conjugation of Streptomyces spores. The usefulness of the tlrB gene as tylosin resistance marker was ascertained in Streptomyces lividans, Streptomyces parvulus and Streptomyces coelicolor, but not in Streptomyces clavuligerus. The tlrB gene constitutes a useful selection marker when high-frequency of conjugation/transformation is not required or as secondary marker in recombinant Streptomyces species where thiostrepton and kanamycin have been utilized for primary selection.

The biological activity of polyketide antibiotics is often strongly dependent on the presence and type of deoxysugar residues attached to the aglycone core. A system is described here, based on the erythromycin-producing strain of Saccharopolyspora erythraea, for detection of hybrid glycoside formation, and this system has been used to demonstrate that an amino sugar characteristic of 14-membered macrolides (D-desosamine) can be efficiently attached to a 16-membered aglycone substrate. First, the S. erythraea mutant strain DM was created by deletion of both eryBV and eryCIII genes encoding the respective ery glycosyltransferase genes. The glycosyltransferase OleG2 from Streptomyces antibioticus, which transfers L-oleandrose, has recently been shown to transfer rhamnose to the oxygen at C-3 of erythronolide B and 6-deoxyerythronolide B. In full accordance with this finding, when oleG2 was expressed in S. erythraea DM, 3-O-rhamnosyl-erythronolide B and 3-O-rhamnosyl-6-deoxyerythronolide B were produced. Having thus validated the expression system, endogenous aglycone production was prevented by deletion of the polyketide synthase (eryA) genes from S. erythraea DM, creating the triple mutant SGT2. To examine the ability of the mycaminosyltransferase TylM2 from Streptomyces fradiae to utilise a different amino sugar, tylM2 was integrated into S. erythraea SGT2, and the resulting strain was fed with the 16-membered aglycone tylactone, the normal TylM2 substrate. A new hybrid glycoside was isolated in good yield and characterized as 5-O-desosaminyl-tylactone, indicating that TylM2 may be a useful glycosyltransferase for combinatorial biosynthesis. 5-O-glucosyl-tylactone was also obtained, showing that endogenous activated sugars and glycosyltransferases compete for aglycone in these cells.

Urdamycin A, the principal product of Streptomyces fradiae Tu2717, is an angucycline-type antibiotic and anticancer agent containing C-glycosidically linked d-olivose. To extend knowledge of the biosynthesis of urdamycin A the authors have cloned further parts of the urdamycin biosynthetic gene cluster. Three new ORFs (urdK, urdJ and urdO) were identified on a 3·35 kb fragment, and seven new ORFs (urdL, urdM, urdJ2, urdZ1, urdGT2, urdG and urdH) on an 8·05 kb fragment. The deduced products of these genes show similarities to transporters (urdJ and urdJ2), regulatory genes (urdK), reductases (urdO), cyclases (urdL) and deoxysugar biosynthetic genes (urdG, urdH and urdZ1). The product of urdM shows striking sequence similarity to oxygenases (N-terminal sequence) as well as reductases (C-terminal sequence), and the deduced amino acid sequence of urdGT2 resembles those of glycosyltransferases. To determine the function of urdM and urdGT2, targeted gene inactivation experiments were performed. The resulting urdM deletion mutant strains accumulated predominantly rabelomycin, indicating that UrdM is involved in oxygenation at position 12b of urdamycin A. A mutant in which urdGT2 had been deleted produced urdamycin I, urdamycin J and urdamycin K instead of urdamycin A. Urdamycins I, J and K are tetracyclic angucyclinones lacking a C–C connected deoxysugar moiety. Therefore UrdGT2 must catalyse the earliest glycosyltransfer step in the urdamycin biosynthetic pathway, theC-glycosyltransfer of one NDP-d-olivose.

The tylCK region of the Streptomyces fradiae genome was sequenced, revealing an incomplete set of five tylC genes encoding all-but-one of the enzymes involved in the biosynthesis of mycarose. The latter is a 6-deoxyhexose sugar required during production of the macrolide antibiotic, tylosin. The missing mycarose-biosynthetic gene, tylCVI, was found about 50 kb distant from its functional partners, on the other side of the tylG (polyketide synthase) gene complex. Mutational analysis, involving targeted gene transplacement, was employed to confirm the functions of specific genes, including tylCVI. Particularly interesting was the similarity between the tylosin-biosynthetic mycarosyltransferase enzyme, TylCV, and proteins of the macrolide glycosyltransferase (MGT) family that inactivate macrolides via glycosylation of attached sugar residues and are involved in resistance and/or antibiotic efflux. The arrangement of genes within the ‘mycarose cluster’ would allow their expression as two short operons with divergent, and perhaps co-regulated, promoters. Whether displacement of tylCVI relative to the other tylC genes provides additional regulatory opportunities remains to be established.

A targeted search for glycosyltransferase (GT) encoding genes in the gene cluster of the urdamycin A producer Streptomyces fradiae Tu2717 resulted in the discovery of urdGT2, a GT encoding gene located approximately 7 kb downstream of the minimal polyketide synthase (PKS) encoding genes. Subsequent inactivation of this gene created a mutant strain, which produces completely different metabolites than the wild-type strain, consisting of the three new urdamycins I, J, and K. Their structures provide new insight into the important C-glycosyl-transfer step of the urdamycin biosynthetic pathway. The structures indicate that the corresponding gene product UrdGT2 catalyzes the C-glycosyl transfer of activated d-olivose to an angucyclinone precursor, which already bears the angular 12b-OH group. The structures of the new urdamycins could not have arisen without the involvement of substrate flexible post-PKS modifying genes, i.e., glycosyltransferases and oxidoreductases. This work proves that targeted gene disrup...

The tylE-J region of the tylosin-biosynthetic gene cluster of Streptomyces fradiae contains six open reading frames. The products of tylJ and tylD are nucleoside diphospho (NDP)-deoxyhexose 3-epimerase and NDP-deoxyhexose 4-ketoreductase, respectively, involved in the synthesis of NDP-6-deoxyallose from NDP-4-keto, 6-deoxyglucose. After incorporation of deoxyallose at C23-OH of the polyketide lactone, tylosin biosynthesis is completed by the products of tylE and tylF, which convert the deoxyallosyl moiety to mycinose via bis-O-methylation at 2-OH and 3-OH, respectively. Hydroxylation of the polyketide lactone at C23 is catalysed by the cytochrome P450 enzyme, TylHl. The product of tylHll is a ferredoxin of unknown specificity that could conceivably act together with TylHl.

Background: The polyketide lactone tylactone is produced in Streptomycesfradiae by the TyIG complex of five multifunctional proteins. As with other type I polyketide synthases, the enzyme catalysing the final elongation step (TyIGV) possesses an integral thioesterase domain that is believed to be responsible for chain termination and ring closure to form tylactone, which is then glycosylated to yield tylosin. In common with other macrolide producers, S. fradiae also possesses an additional thioesterase gene ( orf5 ) located within the cluster of antibiotic biosynthetic genes. The function of the Orf5 protein is addressed here. Results: Disruption of orf5 reduced antibiotic accumulation in S. fradiae by atleast 85%. Under such circumstances, the strain accumulated desmycosin (demycarosyl-tylosin) due to a downstream polar effect on the expression of orf6 , which encodes a mycarose biosynthetic enzyme. High levels of desmycosin production were restored in the disrupted strain by complementation with intact orf5 , or with the corresponding thioesterase gene, nbmB , from S. narbonensis , but not with DNA encoding the integral thioesterase domain of TyIGV. Conclusions: Polyketide metabolism in S. fradiae is strongly dependent on thethioesterase activity encoded by orf5 ( tylO ). It is proposed that the TyIG complex might operate with a significant error frequency and be prone to blockage with aberrant polyketides. A putative editing activity associated with TyIO might be essential to unblock the polyketide synthase complex and thereby promote antibiotic accumulation.

The effect of the upstream region of the β-lactamase structural gene was studied on the production of β-lactamase from Streptomyces fradiae Y59. In contrast to the case in S. cacaoi, β-lactamase activity was weakly enhanced by the presence of the upstream 5-kb DNA fragment but was not induced by 7-ACA. The sequence analysis of this region revealed a couple of putative regulatory proteins, although their actual functions were not clear. It was therefore suggested that at least one of the ORFs might be involved in the weak enhancement of β-lactamase gene expression of S. fradiae Y59

The tlrB gene, which confers inducible resistance to a range of macrolide antibiotics including biosynthetic precursors of tylosin, was isolated and sequenced. In the genome of Streptomyces fradiae, it lies between pbp, which encodes a putative penicillin-binding protein, and tylN, encoding a glycosyltransferase involved in tylosin biosynthesis. The TlrB protein was produced in E. coli as a fusion to MalE. The fusion protein, but not MalE alone, inactivates macrolides in the presence of S-adenosyl-methionine (SAM) but the modified product(s) has not been characterised.

(1R,2S)-Epoxypropylphosphonic acid, also known as fosfomycin, is a clinically utilized, highly effective antibiotic that has low toxicity and is effective against methicillin and vancomycin resistant pathogens. Seminal pioneering studies by Seto and Hammerschmidt using genetic techniques complemented by feeding studies with isotopically labeled precursors suggested the biosynthetic pathway for fosfomycin shown in Scheme 1A. Yet, the mechanism of an unprecedented methyl transfer step in the biosynthesis of fosfomycin has remained unresolved. We recently identified the complete biosynthetic cluster for this natural product from Streptomyces fradiae and achieved heterologous production in S. lividans. Here we use genetic and chemical complementation studies to show that fomC, a gene with a previously unknown role in fosfomycin biosynthesis, is absolutely required for heterologous production. These results in combination with bioinformatics analysis lead to a radically different mechanism for the methyltransferase encoded by fom3. In the currently accepted biosynthetic pathway for fosfomycin (Scheme 1A), PEP is converted to phosphonopyruvate (PnPy) by PEP mutase (Fom1), followed by decarboxylation by Fom2 to produce phosphonoacetaldehyde (PnAA). In the subsequent step, Fom3 has been proposed to catalyze transfer of a methyl anion from methylcobalamin (MeCbl) to PnAA to form 2-hydroxypropylphosphonate (HPP) (Scheme 2). In the final step, HPP is oxidized by Fom4 to form the epoxide of fosfomycin. The proposed mechanism of the methyl transfer reaction catalyzed by Fom3 is fundamentally different from the mechanisms for all known MeCbl dependent methyltransferases in which methylation takes place via nucleophilic attack by the substrate at the methyl group of MeCbl. Intrigued by the apparent mechanistic paradox in how Fom3 would utilize B12 for methyl transfer, we initiated a reinvestigation of the pathway. Several genes besides fom1–4 have been suggested to be part of the biosynthetic cluster and were labeled fomA–F. FomA and FomB provide resistance to fosfomycin via phosphorylation. We recently proposed a function for fomD and determined that fomE,F are not involved directly in fosfomycin production. No function has yet been proposed for fomC. Seto and coworkers showed that a mutant strain with a block in the vitamin B12 biosynthetic pathway could not produce fosfomycin, but did convert HPP to fosfomycin. In addition C-labeled MeCbl fed to this blocked mutant resulted in Clabeled fosfomycin. Based on these results, it was proposed that MeCbl was the methyl donor. However, no intermediates could be isolated from the mutants in which either the B12 pathway 15 or the putative methyltransferase Fom3 was disrupted and neither PnAA nor aminoethylphosphonate (AEP) could be converted to fosfomycin in S. lividans expressing Fom3 and Fom4, whereas HPP was successfully converted. Thus, although the pathway in

The most potent chemical mutagens commonly used for yield enhancement in streptomycetes induce a limited spectrum of base-pair substitutions, heavily dominated by GC to AT transitions. The AT to CG transversion pathway complements the GC to AT pathway, but no strong mutagens with this specificity are available. However, mutT mutations in Escherichia coli enhance spontaneous AT to CG transversions about 1000-fold, so such mutations in streptomycetes could be very beneficial for random mutagenesis. Rate-limiting steps in secondary metabolite biosynthesis, on the other hand, can be best addressed by cloning and insertion of extra copies of the appropriate gene(s) into a neutral genomic site. This approach has been used successfully to improve the production of tylosin and pristinomycin. Transposon mutagenesis can be used to identify and clone neutral genomic sites, as demonstrated in Streptomyces fradiae to improve tylosin yields. Transposon mutagenesis can also be used to activate or enhance the transcription of genes important for secondary metabolite production, and to identify and clone both positive and negative regulatory elements. In some cases it may be advantageous to amplify the complete biosynthetic pathway for secondary metabolite production. In Streptomyces rimosus, a highly productive oxytetracycline producer was shown to contain the complete biosynthetic pathway near the end of the linear chromosome, and on a hybrid linear plasmid that had undergone recombination with the chromosome. This concept of linear replicon fusion might be applicable to other secondary metabolite pathway genes that are located near one end of the linear chromosome.

Tylosin production in the culture of Streptomyces fradiae T1558 was improved by decreasing the apparent viscosity by means of a newly designed medium. When a medium containing 15 g/ l Pharmamedia (a proprietary nutrient derived from cotton seed) and 25 g/ l gluten meal was used in a 3- l air-lift bioreactor, the tylosin concentration and apparent viscosity of the broth were 2.1 g/ l and 190 cP, on day 10 with preferential filamentous growth, while the DO level could hardly be controlled at an appropriate level during fermentation because of the high apparent viscosity. However, when a newly designed medium containing 10 g/ l Pharmamedia and 10 g/ l gluten meal was used, the tylosin production and apparent viscosity of the broth were 7.4 g/ l and 50 cP, on day 10 with preferential pellet growth and appropriate control of DO level. The decrease in the apparent viscosity which was dependent on morphological change, was effective in increasing the tylosin productivity in an air-lift bioreactor through the improvement of mass transfer.

To investigate why more tylosin was produced when Streptomyces fradiae T1558 was cultured in a rapeseed oil medium than in a glucose or starch medium, we measured the activity of methylmalonyl-CoA carboxyltransferase (EC 2.1.3.1) and intracellular propionic acid. The activity of the enzyme, which catalyzes the formation of the precursor of tylosin, protylonolide, was 0.19 U/mg protein in 5 days of culture in rapeseed oil medium, which was 2.5- and 1.3-fold that with the glucose or starch medium, respectively. The intracellular propionic acid concentration was 1.2 g/g of dry weight, which was 4.3- and 2.1-fold that with the glucose or starch medium, respectively. The addition of propionic acid increased tylosin production in batch culture: when 0.2 g/l (final concentration) propionic acid was added to the glucose medium, 3.8 g/l tylosin was produced in 10 days of culture, 4.7-fold the amount without propionic acid. These findings suggest that in glucose medium, intracellular propionic acid is a limiting factor because of the low activity of methylmalonyl-CoA carboxyltransferase of the tylosin biosynthesis pathway.

In addition to methionine, aspartate may also induce sporulation in the Streptomyces fradiae St3110 mutant that requires methionine for sporulation but not for growth. Partially purified aspartokinase (EC 2.7.2.4.) of the mutant was tested for feed-back control by methionine, threonine, and lysine. Methionine or threonine alone did not have any significant effect but they had a concerted inhibitory effect together that was further increased by lysine. The threonine-lysine combination also caused inhibition of the enzyme activity, while lysine alone activated the enzyme. Methionine was also found to partially repress the aspartokinase activity to about one third of the control. The sporulating aerial mycelia induced by aspartate were lacking the characteristic sporulation pigmentation present in the wild type or after induction of spores by methionine. No significant difference in heat resistance between the two types of the spores was detected. The number of spores produced by aspartate was only about one fourth of that after induction by methionine. The data may indicate a role of aspartokinase control by methionine in restoring the normal sporulation, although in addition to this methionine may act by a different mechanism, as well.

Streptomyces fradiaeproduces several extracellular proteases and many of these are inducible. An 8.8 kb DNA fragment ofStreptomyces fradiaecloned on pIJ699 caused increased protease activity inStreptomyces lividans.Clones carrying this recombinant plasmid showed a significant delay in sporulation. A protein of 18 kDa was purified from the extracellular proteins secreted by the host carrying the recombinant plasmid. Further characterization showed that this protease is a metalloprotease.

Several transposons have been developed from the streptomycete insertion sequence IS493. They have broad host specificity in Streptomyces species and insert relatively randomly into a consensus target sequence of gNCaNTgNNy. Collectively, they have specialized features that facilitate the following: cloning of DNA flanking insertions; physical mapping of insertions; construction of highly stable mutants; and efficient construction of mutant libraries. All of the transposons can be introduced into streptomycetes by conjugation from E. coli, and can be delivered by curing the temperature sensitive delivery plasmid. Tn5099 was used to physically map genes involved in daptomycin and red pigment production in Streptomyces roseosporus, and to clone daptomycin biosynthetic genes. Tn5099 was also used in Streptomyces fradiae to identify and clone a neutral genomic site for the insertion of a second copy of the tylF gene. Recombinants containing two copies of the tylF gene carried out the no rmally rate limiting conversion of macrocin to tylosin very efficiently, thus causing substantial increases in tylosin yield.

The intrastructure of mycelial pellets of Streptomyces fradiae , which produces tylosin, was visualized following labeling with fluorescein isothiocyanate (FITC) and propidium iodide (PI) using confocal scanning laser microscopy. A PI-labeled inactive core was present inside the mycelial pellet in both fermentor and air-lift reactor cultures. The thickness of the active mycelial layer on the pellet surface was calculated from a density sliced image to be 60 and 68 μm respectively, in the fermentor and in air-lift reactor cultures. Using image analysis, the active mycelial concentration of pellets in the fermentor culture was predicted to be 2.1 times higher than that in the air-lift reactor culture. The tylosin production rate in the fermentor reached 0.78 g/ l /d, which was 2.5-fold that in the air-lift reactor culture. These results indicate that the higher tylosin production rate in the fermentor culture was due to the higher active mycelial concentration in the fermentor compared to that in the air-lift reactor.

The mechanism of 2-deoxy-scyllo-inosose synthase reaction, a carbocycle formation step from D-glucose-6-phosphate in the biosynthesis of the 2-deoxystreptamine aglycon of clinically important aminocyclitol antibiotics, was investigated with a partially purified enzyme from butirosin-producing Bacillus circulans SANK 72073. Nonlabeled and double-labeled D-[4-2H, 3-18O]glucose-6-phosphate were used for cross-over experiment, and the oxime-TMS ether derivative of the 2-deoxy-scyllo-inosose product was analyzed by GC-MS. The deuterium label at C-4 of the substrate appeared to be retained at C-6 of the inosose product without scrambling of the double-labeled isotopes. Since the transient reduction of NAD+ cofactor was proved to be essential in the 2-deoxy-scyllo-inosose reaction, the hydride abstraction and returning appeared to take place within the same glucose molecule. The observed kinetic isotope effect was estimated to be kH/kD=2.4. These results strongly suggest that this carbocycle formation is catalyzed by a single 2-deoxy-scyllo-inosose synthase enzyme with catalytic requirement of NAD+, the mechanism of which appears to be resembled closely to the 2-deoxy-scyllo-inosose synthase in the Streptomyces fradiae.

Morphological change during culture of the tylosin producer Streptomyces fradiae under various shear conditions was investigated using an image analysis system. The morphology was classified as filament, entangled filament, or pellet by measuring the mycelial area and convex perimeter. At fermentor agitation rates of 400 rpm and higher, filament and entangled filament morphology comprised more than 50%. However, in cultures at agitation rates lower than 400 rpm, pellet morphology increased gradually and until it comprised more than 50%; in an air-lift reactor, the pellet ratio reached 80% after 160-h culture. The average areas of pellets and mycelia in the air-lift reactor were respectively 100- and 20-fold those under high shear condition. Both pellet area and mycelial morphology changed during the culture period according to the shear conditions. Among the various shear conditions, tylosin production and rapeseed oil consumption were lowest in the airlift fermentor culture with against in the fermentor at 400 rpm. From the viewpoint of tylosin production, the optimal mycelial area that gave the maximum production rate under various shear conditions was around 4 × 10 3 μm 2 . When the mycelia and pellet areas exceeded 4 × 10 3 μm 2 , the tylosin production rate decreased drastically.

The production of neomycin by the actinomycete Streptomyces fradiae, under semi-solid-state fermentation conditions was the main subject of this study. Two supports (nylon sponge and orange peelings) were tested in order to determine the most suitable one for the production of neomycin by the above-mentioned microorganism. Nylon sponge led to the highest neomycin production, reaching a maximum value of 13,903 μg/mL on the 10th day of cultivation. As a control, the same experiment was performed under submerged fermentation (SmF) conditions, without solid support. Here the production of neomycin by S. fradiae was about 55-fold lower (i.e. 250 μg/mL) than that obtained for SSF.

A tylosin-producer, Streptomyces fradiae, was transformed with plasmids carrying genes from Streptomyces thermotolerans that are involved in acyl modification of macrolide antibiotics. A transformant with pMAB3, in which macrolide 4"-O-acyltransferase gene (acyB1) and its regulatory gene (acyB2) are subcloned, produced several types of 4"-O-acyltylosins. A transformant with pAB11 delta EH containing macrolide 3-O-acyltransferase gene (acyA) in addition to the above two genes produced 3-O-acetyltylosin and 3-O-acetyl-4"-O-acyltylosins. Among the products of the latter transformant, 3-O-acetyl-4"-O-isovaleryltylosin (AIV) was detected as a minor component. When L-leucine, a precursor of isovaleryl-CoA, was added to the medium at the late stage of the fermentation, AIV content among the total macrolides increased ten-fold and AIV became a main product. This fact suggests that a high level of endogenous isovaleryl-CoA may be essential for the selective production of AIV by S. fradiae carrying pAB11 delta EH.

The macrolide antibiotic, tylosin (Ty), is produced by Streptomyces fradiae. Two resistance determinants (tlrA, synonym ermSF, and tlrD) conferring resistance to macrolide, lincosamide and streptogramin B type (MLS) antibiotics were previously isolated from this strain, and their products shown to methylate 23S ribosomal RNA (rRNA) at a common site, thereby rendering the ribosomes MLS resistant. However, the TlrA and TlrD proteins differ in their action; the former dimethylates, and the latter monomethylates, the target nucleotide. Here, 2.2 kb of DNA from the tylLM region of the tylosin biosynthetic gene cluster of S. fradiae has been sequenced and shown to encompass tlrD. Comparison of the sequences of tlrA and tlrD (and of their deduced products) with those of related ('erm-type') genes from other actinomycetes suggests that the combined presence of tlrA and tlrD in S. fradiae is not the result of recent gene duplication.

Tylosin production in flask and jar fermentor culture of Streptomyces fradiae using seven different vegetable oils as sole carbon sources was investigated. Among the vegetable oils, rapeseed oil was the best carbon source for tylosin production. When the initial rapeseed oil concentration was 60 g/l, the maximum tylosin concentration reached was 7.0 g/l, which was about 1.6- and 7-fold that reached using starch and glucose, respectively, at the same initial concentration. Although oil consumption increased with increasing agitation rate, the highest tylosin yield was obtained using an agitation rate of 400 rpm. This indicates that in the case of using an insoluble carbon source, use of an appropriate agitation rate is important for efficient production of tylosin.

We explored transposition in Streptomyces fradiae (Sf) as a means to insert a second copy of the tylF gene to improve tylosin (Ty) production. Transposons Tn5096 and Tn5099 transposed relatively randomly in Sf, and many of the insertions caused no deleterious effects on Ty production yields. Tn5098, a derivative of Tn5096 containing tylF and tylJ genes, recombined into the chromosome into the tyl gene cluster and transposition was not observed. However, following the tagging of a neutral site (NS) by Tn5099 transposition, tylF was effectively inserted into the NS by homologous recombination (transposon exchange). Recombinants obtained by transposon exchange produced higher yields of Ty.

Rat pancreatic secretory trypsin inhibitor-I (PSTI-I) and -II (PSTI-II) were expressed in Saccharomyces cerevisiae AH22 under the control of the PHO5 promoter. Both proteins were secreted into the culture broth and purified by affinity chromatography using a genetically engineered mutant Streptomyces fradiae trypsin-like enzyme as a ligand. Rat PSTI-I was purified to homogeneity by one-step affinity chromatography, but the PSTI-II fraction contained two molecular mass forms (6.2 kDa and 5.6 kDa) after affinity chromatography. N-terminal amino acid sequencing revealed that the larger molecular mass protein was intact rat PSTI-II and five amino acid residues at its N-terminal had been truncated to the smaller form. The N-terminal amino acid sequences, amino acid compositions and trypsin inhibitory activities of recombinant rat PSTI-I and -II were identical to those of natural PSTIs. Moreover, the pancreatic exocrine secretion activity of recombinant rat PSTI-I in vivo also agreed well with that of natural PSTI-I.

Fradcarbazoles A-C (1-3), three new indolocarbazoles, were isolated from a mutant strain of the marine-derived actinomycete Streptomyces fradiae 007M135. Their structures were established by spectroscopic analysis, quantum chemical calculation, CD spectra, and chemical transformation. Fradcarbazole A (1) possessed a unique skeleton consisting of a staurosporine core, a thiazole ring, and an indole fragment. Compounds 1-3 displayed significant cytotoxicity against HL-60, K562, A-549, and BEL-7402 cell lines and inhibitory effects on the kinase PKC-α with IC(50) values of 0.001-4.58 μM.

Among Streptomyces fradiae parent strain (NRRL 2702), aspartate auxotroph strain (SMF 305), and revenant strain (SMF 306), the revertant strain is the highest producer of tylosin and showed different repression patterns of tylosin production by ammonium ion from the parent strain. These results were elucidated by the facts that the revertant strain was superior to the parent or auxotroph strain in the biosynthesis of glutamine synthetase involved in ammonium assimilation and in the biosynthesis of threonine dehydratase (TDT) involved in providing precursors necessary for tylosin production, and ammonium ion inhibited the activity of TDT purified from the parent strain more than that of TDT from the revertant strain. TDT from the parent strain has been purified by DEAE cellulose, hydroxyapatite, Mono Q HR 5/5, and reversed-phase Protein C 4 chromatography. The molecular mass was 60 kDa by SDS-PAGE and 240 kDa by gel filtration. The N-terminal amino acid sequence of TDT was NH 3 -E-A-T-G-P-L-T-T-E-S-G-A-P-V. The activity of TDT was allosterically activated by adenosine monophosphate.

SUMMARY A second NAD-dependent valine dehydrogenase (VDH) of Streptomyces fradiae was detected and purified to homogeneity by affinity chromatography on Reactive-Blue 2 Sepharose followed by gel filtration and Mono Q fast protein liquid chromatography. The relative molecular masses of the native enzyme and its subunits were determined to be 80000 and 41000, respectively, indicating that the enzyme is a homodimer. The enzyme was the only active VDH in S. fradiae; its activity was significantly induced by L-valine, but was repressed by ammonia. Among branched- and straight-chain amino acids that serve as enzyme substrates, L-2-aminobutyrate and L-valine are preferred. Significant activities were found with deamino-NAD+ and 3-pyridinealdehyde-NAD-. The molecular and catalytic properties of the enzyme distinguish it from the enzyme previously purified, and thus indirectly indicate the existence of two VDHs in S. fradiae.

Activity of valine dehydrogenases (VDH) from Streptomyces aureofaciens and S. fradiae is strongly induced by L-valine even in the presence of 25mM NH4 +. When added into 16 h-old cultures growing with 100mM NH4 +, L-valine induced the synthesis of VDH. The results indicate that Streptomyces can utilize L-valine in the presence of NH4 +, and the induction of VDH activity by L-valine is not repressed by NH4 +.

Streptomyces fradiae was cultivated in both an air-lift bioreactor and a jar-fermentor with various agitation rates from 200 to 800 rpm to investigate differences in neomycin production between the two reactors. Final neomycin concentrations in the jar-fermentor operated at 600 rpm and the air-lift bioreactor were 3.19 and 1.39 g/l, respectively. On the other hand, levels of soybean oil consumption in the two reactors were 25.9 and 9.4 g/l, respectively. Shear stress due to mechanical agitation caused changes in the morphology of mycelia and influenced neomycin production. The morphological changes of the mycelia in the jar-fermentor caused the viscosity of the culture broth to decrease by half, and soybean oil consumption and fatty acid uptake rate to increase 3- and 1.8-fold, respectively, in comparison with those of the air-lift bioreactor. The product yield coefficient determined from the level of soybean oil consumption in the air-lift bioreactor was similar to that of the jar-fermentor at 600 rpm, but the neomycin yield was less than one-half. In the case of the jar-fermentor, the yield increased with increasing agitation rate and was maximum at 600 rpm. To maximize neomycin production in S. fradiae cultures using soybean oil as sole carbon source, it was necessary to provide a degree of shear stress to the mycelia and to optimize liquid mixing. In an air-lift bioreactor, the soybean oil consumption may be suppressed due to a low degree of liquid mixing.

The beta-lactamase gene from Streptomyces fradiae Y59 was cloned and sequenced. To determine which amino acid residues are critical in binding activity to blue dextran, chimera beta-lactamases were constructed and their binding abilities were determined. The results suggested that blue dextran binding may depend more on overall conformation of about two-thirds of the beta-lactamase molecule from the N terminus than on the primary structure.

Kerase, a serine protease from Streptomyces fradiae, was immobilized on porous glass (SIKUG®) by covalent attachment, through amino groups on the enzyme. Modifications of four lysine residues (44·4% of the accessible or superficial amino groups) results in a loss of 6·5% of the enzymic activity. After immobilization, the optimal reaction pH changed from a range of 7·5–8·5 to 9–10. The immobilized protease was stable in a broad pH range, 6–12, while the soluble protease was irreversibly denaturated at alkaline pHs (pH > 8). The optimal reaction temperature was displaced from 55 to 65°C, showing a higher thermal stability of the immobilized enzyme. Kerase immobilized onto porous glass was stable for at least 28 days, working in a repeated-batch process of three cycles per day, with an activity loss of 22·1 ± 3·1%.

The trypsin-like enzyme from Streptomyces fradiae (SFT) is one of the extracellular proteases secreted by gram-positive bacteria. Since the primary structure of SFT is still unknown, a gene encoding SFT was cloned from a S. fradiae genomic library using an amplified polymerase chain reaction product within the SFT gene as a probe. The nucleotide sequence of the cloned fragment revealed that the gene encoded an open reading frame with 259 amino acids. The 38 N-terminal amino acids resemble a typical prokaryotic signal peptide, but the predicted signal sequence cleavage site suggests the existence of a very short, four-amino-acid prosequence. The mature SFT consists of 221 amino acids with a molecular weight of 22900. SFT was expressed in Streptomyces lividans 1326 using pIJ702 as a vector, and the secreted protein was purified from culture supernatant by soybean trypsin inhibitor-affinity chromatography. The N-terminal amino acid sequence and molecular weight of the protein were identical to those of natural SFT, indicating correct processing by S. lividans 1326. Also, the amino acid composition of the recombinant SFT agreed with that of natural SFT and that deduced from the nucleotide sequence. Comparison of the amino acid sequence of SFT and other trypsins of microbial and mammalian origins revealed that SFT exhibits 85% identity to Streptomyces griseus trypsin (SGT), but low identity to Saccharopolyspora erythraea trypsin (SET) (38%) and to bovine trypsin (35%). The sequence alignment shows that the catalytic triad, the substrate-binding site and six cysteine residues are highly conserved. We found amino acid substitutions between SFT and SGT in regions involved in substrate specificity and sequence differences between Streptomyces and mammalian trypsins in substrate-binding regions.

Marine actinomycetes have become sources of great interest to natural product chemistry due to their new chemical entities and bioactive metabolites. Since April 2010, we have screened actinobacteria from five sites that represent different ecosystems of Segara Anakan lagoon. In this present study we focus on specific isolates, K-2C which covers 1) actinomycetes identification based on morphology observation and 16S rRNA gene; 2) fermentation and isolation of pigment; 3) structure determination of pigment; and 4) hydrolytic enzymes characterization; Methodologies relevant to the studies were implemented accordingly. The results indicated that K-2C was likely Streptomyces fradiae strain RSU15, and the best fermentation medium should contain starch and casein with 21 days of incubation. The isolate has extracellular as well as intracellular pigments. Isolated pigments gave purple color with λmax of 529.00 nm. The pigment was structurally characterized. Interestingly, Streptomyces K-2C was able to produce potential hydrolytic enzymes such as amylase, cellulase, protease, lipase, urease, and nitrate reductase.

Capoamycin-type antibiotics (2–5) and polyene acids (6, 7) were isolated from marine Streptomyces fradiae strain PTZ0025. Their structures were established by extensive nuclear magnetic resonance (NMR) and high resolution electron spray ionization mass spectroscopy (HRESIMS) analyses and chemical degradation. Compounds 3, 4, 6, 7 were found to be new and named as fradimycins A (3) and B (4), and fradic acids A (6) and B (7). Compounds 3–5 showed in vitro antimicrobial activity against Staphylococcus aureus with a minimal inhibitory concentration (MIC) of 2.0 to 6.0 μg/mL. Interestingly, Compounds 3–5 also significantly inhibited cell growth of colon cancer and glioma with IC50 values ranging from 0.13 to 6.46 μM. Fradimycin B (4), the most active compound, was further determined to arrest cell cycle and induce apoptosis in tumor cells. The results indicated that fradimycin B (4) arrested the cell cycle at the G0/G1 phase and induced apoptosis and necrosis in colon cancer and glioma cells. Taken together, the results demonstrated that the marine natural products 3–5, particularly fradimycin B (4), possessed potent antimicrobial and antitumor activities.

Fosfomycin is a wide-spectrum antibiotic that is used clinically to treat acute cystitis in the United States. The compound is produced by several strains of streptomycetes and pseudomonads. We sequenced the biosynthetic gene cluster responsible for fosfomycin production in Pseudomonas syringae PB-5123. Surprisingly, the biosynthetic pathway in this organism is very different from that in Streptomyces fradiae and Streptomyces wedmorensis . The pathways share the first and last steps, involving conversion of phosphoenolpyruvate to phosphonopyruvate (PnPy) and 2-hydroxypropylphosphonate (2-HPP) to fosfomycin, respectively, but the enzymes converting PnPy to 2-HPP are different. The genome of P. syringae PB-5123 lacks a gene encoding the PnPy decarboxylase found in the Streptomyces strains. Instead, it contains a gene coding for a citrate synthase-like enzyme, Psf2, homologous to the proteins that add an acetyl group to PnPy in the biosynthesis of FR-900098 and phosphinothricin. Heterologous expression and purification of Psf2 followed by activity assays confirmed the proposed activity of Psf2. Furthermore, heterologous production of fosfomycin in Pseudomonas aeruginosa from a fosmid encoding the fosfomycin biosynthetic cluster from P. syringae PB-5123 confirmed that the gene cluster is functional. Therefore, two different pathways have evolved to produce this highly potent antimicrobial agent.

The antibacterial study revealed that the isolated marine soil actinomycetes by crowded plate method and was identified by MIDI Advance technology (gas chromatographic) method and Thus, the microbes were identified according to their fatty acid sequences. The actinomycetes species were identified as Streptomyces rimosus, Streptomyces fradiae, and Streptomyces griseoflavus and the antibacterial assay was carried out by well diffusion method. Out of these only Streptomyces rimosus having high antimicrobial potential against Gram positive as well as Gram negative bacteria. In Gram positive Streptomyces aureus exhibits higher sensitivity followed by Bacillus subtilis where as gram negative bacteria E.Coli was extremely sensitive to the metabolites followed by Pseudomonas aeruginosa.

as chromatography - mass spectrometry has proved that the only by-product, resulted from cholesterol decomposition in the reaction mixtures containing the enzyme and cholesterol (as substrate), was 4-cholesten-3-one. By using the high performance liquid chromatography (HPLC), it was found that the highest amount of this product in the reaction mixtures occurred when Streptomyces fradiae was exposed to 210 mW Nd-YAG laser delivered for 8 min in presence 0.5 mg/ml chlorophyllin as photosensitizer. Purification of cholesterol oxidase, recovered from non-irradiated and laser-irradiated photosensitized culture of S. fradiae, was performed through fractional precipitation with ammonium sulfate and by applying DEAE-cellulose column followed by Sephadex G-200 column chromatography. The specific activity of the enzyme recovered from radiated culture (11.21 units/mg) was significantly higher than that recovered from non-irradiated one (7.45 units/mg). The purity of the enzymes was judged by the appearance of only one band for each enzyme using SDS-polyacrylamide gel electrophoresis. The molecular weights of the enzymes recovered from non-irradiated and laser-irradiated cultures were 55.0 and 52.1 kDa, respectively. In in vitro experiment, a decrease in the total and free human serum blood cholesterol contents by 38.79 and 80.90% was induced in the case of pure enzyme treatment recovered from irradiated culture as compared to 16.36 and 59.43% in the case of the corresponding non-irradiated control, respectively. Experimental data indicated that injection of white chickens with cholesterase enzyme recovered from laser-irradiated culture of S. fradiae, resulted in a remarkable serum and egg yolk cholesterol reduction reaching 27.2 and 19.4% as compared to 19.4 and 12.7% for the enzyme recovered from non-irradiated culture, respectively. Neither mortalities nor disease symptoms occurred within all birds by the end of one month observation. Key words: Cholesterol degradation, Nd-YAG laser, photosensitizer, Streptomyces fradiae.