Bacterioferritin: a key iron storage modulator that affects strain growth and butenyl-spinosyn biosynthesis in Saccharopolyspora pogona.

BACKGROUND: Butenyl-spinosyn, produced by Saccharopolyspora pogona, is a promising biopesticide due to excellent insecticidal activity and broad pesticidal spectrum. Bacterioferritin (Bfr, encoded by bfr) regulates the storage and utilization of iron, which is essential for the growth and metabolism of microorganisms. However, the effect of Bfr on the growth and butenyl-spinosyn biosynthesis in S. pogona has not been explored. RESULTS: Here, we found that the storage of intracellular iron influenced butenyl-spinosyn biosynthesis and the stress resistance of S. pogona, which was regulated by Bfr. The overexpression of bfr increased the production of butenyl-spinosyn by 3.14-fold and enhanced the tolerance of S. pogona to iron toxicity and oxidative damage, while the knockout of bfr had the opposite effects. Based on the quantitative proteomics analysis and experimental verification, the inner mechanism of these phenomena was explored. Overexpression of bfr enhanced the iron storage capacity of the strain, which activated polyketide synthase genes and enhanced the supply of acyl-CoA precursors to improve butenyl-spinosyn biosynthesis. In addition, it induced the oxidative stress response to improve the stress resistance of S. pogona. CONCLUSION: Our work reveals the role of Bfr in increasing the yield of butenyl-spinosyn and enhancing the stress resistance of S. pogona, and provides insights into its enhancement on secondary metabolism, which provides a reference for optimizing the production of secondary metabolites in actinomycetes.

Metabolomics analysis of the effect of dissolved oxygen on spinosad production by Saccharopolyspora spinosa.

Spinosad, a universal bio-pesticide, is obtained from the soil actinomycete Saccharopolyspora spinosa. Dissolved oxygen, an important contributing factor in aerobic microbial fermentation, however, is not always available in sufficient amounts. To alleviate oxygen limitation in spinosad production, three different oxygen vectors, namely oleic acid, toluene, and n-dodecane, were added into early fermentation. Results indicated that n-dodecane was the optimal oxygen vector. Spinosad yield was increased by 44.2% compared to that in the control group in the presence of 0.5% n-dodecane, added after 120 h of incubation. Yields of the test group reached 6.52 mg/g dry cell weight (DCW), while that of the control group was limited to 4.52 mg/g DCW. Metabolomics analysis by gas chromatography coupled to mass spectrometry was performed to demonstrate the metabolism mechanism in the presence and absence of oxygen vector. In total, 78 principal intracellular metabolites in S. spinosa were detected and quantified in the presence and absence of n-dodecane. Levels of some metabolites that were related to the tricarboxylic acid cycle and pentose phosphate pathway varied significantly. Aspartic acid and glucose-1-phosphate levels varied significantly and contributed most in the distinction of the fermentation conditions and phases. The above findings give new insights into the improvement and the metabolomic characteristics of industrial spinosad production.

Enhanced oxygen transfer rate and bioprocess yield by using magnetite nanoparticles in fermentation media of erythromycin.

BACKGROUND: Magnetite nanoparticles have widespread biomedical applications. In the aerobic bioprocesses, oxygen is a limiting factor for the microbial metabolic rate; hence a high availability of oxygen in the medium is crucial for high fermentation productivity. This study aimed to examine the effect of using magnetite nanoparticles on oxygen transfer rate in erythromycin fermentation culture. METHODS: Magnetite nanoparticles were synthetized through co-precipitation method. After observing the enhanced oxygen transfer rate in deionized water enriched with magnetite nanoparticles, these nanoparticles were used in the media of by Saccharopolyspora erythraea growth to explore their impact on erythromycin fermentation titer. Treatments comprised different concentrations of magnetite nanoparticles, (0, 0.005, 0.02 v/v). RESULTS: In the medium containing 0.02 v/v magnetite nanoparticles, KLa was determined to be 1.89 time higher than that in magnetite nanoparticle-free broth. An improved 2.25 time higher erythromycin titer was obtained in presence of 0.02 v/v nanoparticles. CONCLUSIONS: Our results, demonstrate the potential of magnetite nanoparticles for enhancing the productivity of aerobic pharmaceutical bioprocesses.

Biochemical parameters of Saccharopolyspora erythraea during feeding ammonium sulphate in erythromycin biosynthesis phase.

The physiology of feeding ammonium sulphate in erythromycin biosynthesis phase of Saccharopolyspora erythraea on the regulation of erythromycin A (Er-A) biosynthesis was investigated in 50 L fermenter. At an optimal feeding ammonium sulphate rate of 0.03 g/L per h, the maximal Er-A production was 8281 U/mL at 174 h of growth, which was increased by 26.3% in comparison with the control (6557 U/mL at 173 h). Changes in cell metabolic response of actinomycete were observed, i.e. there was a drastic increase in the level of carbon dioxide evolution rate and oxygen consumption. Assays of the key enzyme activities and organic acids of S. erythraea and amino acids in culture broth revealed that cell metabolism was enhanced by ammonium assimilation, which might depend on the glutamate transamination pathway. The enhancement of cell metabolism induced an increase of the pool of TCA cycle and the metabolic flux of erythromycin biosynthesis. In general, ammonium assimilation in the erythromycin biosynthesis phase of S. erythraea exerted a significant impact on the carbon metabolism and formation of precursors of the process for dramatic regulation of secondary metabolites biosynthesis.

Significant decrease of broth viscosity and glucose consumption in erythromycin fermentation by dynamic regulation of ammonium sulfate and phosphate.

In this study, the effects of nitrogen sources on broth viscosity and glucose consumption in erythromycin fermentation were investigated. By controlling ammonium sulfate concentration, broth viscosity and glucose consumption were decreased by 18.2% and 61.6%, respectively, whereas erythromycin biosynthesis was little affected. Furthermore, erythromycin A production was increased by 8.7% still with characteristics of low broth viscosity and glucose consumption through the rational regulations of phosphate salt, soybean meal and ammonium sulfate. It was found that ammonium sulfate could effectively control proteinase activity, which was correlated with the utilization of soybean meal as well as cell growth. The pollets formation contributed much to the decrease of broth viscosity. The accumulation of extracellular propionate and succinate under the new regulation strategy indicated that higher propanol consumption might increase the concentration of methylmalonyl-CoA and propionyl-CoA and thus could increase the flux leading to erythromycin A.

A novel isoquinoline alkaloid, DD-carboxypeptidase inhibitor, with antibacterial activity isolated from Streptomyces sp. 8812. Part I: Taxonomy, fermentation, isolation and biological activities.

A novel isoquinoline alkaloid of molecular formula C10H9NO4, labeled JS-1, was isolated from the culture broth of Streptomyces sp. 8812. It was purified by acetone protein precipitation from the culture supernatant, followed by anion exchange and C18 RP HPLC columns. JS-1 is an inhibitor of exocellular DD-carboxypeptidases/transpeptidases (DD-peptidases) 64-575 II from Saccharopolyspora erythraea 64-575 II, and R39 from Actinomadura R39. JS-1 exhibits activity against Gram-negative bacteria, such as Bordetella bronchiseptica, Stenotrophomonas maltophilia, Proteus vulgaris, P. mirabilis, Burkholderia cepacia and Acinetobacter baumanii, with MIC values 10-160 microg ml(-1), and against Gram-positive bacteria, such as Staphylococcus aureus, with MIC values 40-206 microg ml(-1).

Improvement of Saccharopolyspora spinosa and the kinetic analysis for spinosad production.

In this paper, a new spinosad-producing mutant UV-42-13 was obtained by employing rhamnose and sodium propionate resistant selection strategies in series with UV irradiation. Spinosad production of the mutant was 125.3 mg/L, improved 285.5% compared with that of the wild strain (32.5 mg/L).The results of experiment on tolerance of propyl alcohol addition showed that the tolerant ability to precursor was higher. The precursor-resistant ability of the mutant improved through tolerance experiment by adding propyl alcohol, and the spinosad production was greatly increased. The kinetic models for biomass, substrate consumption, and spinosad production of mutant strain and wild strain were studied by conducting batch fermentation in the shaking flask. The result showed that the kinetic models could describe the fermentation process of spinosad producing well.

[Antioxidant effect on the growth of Saccharopolyspora erythraea and biosynthesis of erythromycins].

Oxidative stress in the mycelium of submerged culture of S. erythraea, an organism producing erythromycins, was observed. The stress could be eliminated by addition of antioxidants, such as butyloxyanisole, alpha-tocopherol, cytochrome C to the culture. In the presence of the antioxidants the mycelium lysis decreased, the level of the decrease being higher in the medium preferably containing carbohydrates vs. the medium mainly containing lipids (soybean oil). The antioxidants had no specific effect on the biosynthesis of erythromycins.

Enhancing of erythromycin production by Saccharopolyspora erythraea with common and uncommon oils.

The enhancing effect of various concentrations of 18 oils and a silicon antifoam agent on erythromycin production by Saccharopolyspora erythraea was evaluated in a complex medium containing soybean flour and dextrin as the main substrates. The oils used consisted of sunflower, pistachio, cottonseed, melon seed, water melon seed, lard, corn, olive, soybean, hazelnut, rapeseed, sesame, shark, safflower, coconut, walnut, black cherry kernel and grape seed oils. The biomass, erythromycin, dextrin and oil concentrations and the pH value were measured. Also, the kinds and frequencies of fatty acids in the oils were determined. The productivity of erythromycin in the oil-containing media was higher than that of the control medium. However, oil was not suitable as a main carbon source for erythromycin production by S. erythraea. The highest titer of erythromycin was produced in medium containing 55 g/l black cherry kernel oil (4.5 g/l). The titers of erythromycin in the other media were also recorded, with this result: black cherry kernel > water melon seed > melon seed > walnut > rapeseed > soybean > (corn = sesame) > (olive = pistachio = lard = sunflower) > (hazelnut = cotton seed) > grape seed > (shark = safflower = coconut). In media containing various oils, the hyphae of S. erythraea were longer and remained in a vegetative form after 8 days, while in the control medium, spores were formed and hyphae were lysed.

Analysis of an 8.1-kb DNA fragment contiguous with the erythromycin gene cluster of Saccharopolyspora erythraea in the eryCI-flanking region.

An 8.1-kb region of the Saccharopolyspora erythraea genome, significant for its contiguity to the known genes of the erythromycin biosynthetic gene cluster, was mutationally analyzed and its DNA sequence was determined. The region lies immediately adjacent to eryCI. The newly characterized region is notable for a large, 3.0-kb segment, predicted not to be translated, followed by four probable genes: an acetyltransferase gene, a protease inhibitor gene, a methyltransferase gene, and a transposase gene. Because the probable functions of the genes in this region are not required for erythromycin biosynthesis or resistance and because a deletion of a 6.0-kb portion of this region had no effect on erythromycin biosynthesis, this region marks the outside boundary of the erythromycin gene cluster. Therefore, eryCI represents the end of the cluster. These results complete the analysis of the erythromycin gene cluster and eliminate the possibility that additional sought-after pathway-specific structural or regulatory genes might be found within or adjacent to the cluster.

[Isolation and characterization of Saccharopolyspora erythraea mutants, resistant to chloramphenicol]. / Poluchenie i kharakteristika mutantov Saccharopolyspora erythraea, ustoichivykh k khloramfenikolu.

Formation of chloramphenicol resistant (CMr) spontaneous and nitroso-ethyl-urea-induced mutants of S.erythraea, an organism producing erythromycin, was studied. The mutants differed by the level of the chloramphenicol resistance (10 to 40 micrograms/ml). Part of the chloramphenicol resistance mutations had a pleiotropic pattern. 63.8 per cent of the CMr mutants was characterized by the growth thermosensitivity and 18.9 per cent was characterized by the absence of the melanin production property. The antibiotic potency of 56 per cent of the CMr mutants was higher than that of the initial strain. In some of the CMr mutants the property of resistance to other antibiotics was changed. A higher resistance of some mutants to chloramphenicol correlated with their increased lincomycin resistance. It was shown that chloramphenicol induced resistance to kanamycin in S.erythraea 5. Such a property for the resistance induction was preserved in the spontaneous low CMr mutants while the high CMr mutants isolated after the culture exposure to the mutagen lacked it. The CMr S.erythraea phenotype was genetically instable. Two different amplifying DNA sequences (18 and 70-78 kb in size) were detected in the CMr mutant genome. The chloramphenicol resistance mutation was localized in the S.erythraea chromosome region restricted by markers ura1 and met1.

[Affinity of exocellular DD-carboxypeptidase/transpeptidase from Saccharopolyspora erythraea PZH TZ-575 to beta-lactam compounds]. / Powinowactwo zewnatrzkomórkowej DD-karboksypeptydazy/transpeptydazy szczepu Saccharopolyspora erythraea PZH TZ 64-575 do zwiazków beta-laktamowych.

The DD-carboxypeptidase/transpeptidases (DD-peptidases) involved in bacterial cell wall metabolism, catalyse the attack of C-terminal D-alanyl-D-alanine peptide bond of the peptydoglycan precursor. These enzymes are inactivated by beta-lactam antibiotics. DD-peptidase from Saccharopolyspora erythraea PZH TZ 64-575 was purified by the use of DEAE-cellulose, Sephadex G-100, Q-Sepharose resins and FPLC (Mono Q). After each step the effluent was concentrated by Amicon ultrafiltration. The purified enzyme showed DD-carboxypeptidase specific activity of 50.9 U/mg. The enzyme exhibited high affinity to beta-lactam compounds e.g. cefamandole, cefapirin, cefradin 1.5-2.6 x 10(-8) M. It was used to screen strains from the Culture Collection of the National Institute of Hygiene in Warsaw for the production of DD-peptidase inhibitors.

[The isolation and characteristics of mutants of the Saccharopolyspora erythraea strain resistant to thiostrepton]. / Poluchenie i kharakteristika mutantov shtamma Saccharopolyspora erythraea, ustoichivykh k tiostreptonu.

The formation of thiostreptone resistant spontaneous and nitrosoguanidine-induced mutants in the erythromycin-producing organism Saccharopolyspora erythraea was investigated. The investigated collection of the mutants was heterogeneous by the level of the thiostreptone resistance (2.5 to 20 micrograms/ml). The thiostreptone resistance mutations had a pleiotropic effect: 17 per cent of the mutants was characterized by the growth thermosensitivity and 26 and 5.8 per cent of the mutants were characterized by loss of the ability to form melanine and aerial mycelium respectively. Such phenotypes were most frequent in the mutants resistant to low concentrations of thiostreptone (2 to 5 micrograms/ml). The absolute majority of the isolated thiostreptone resistant mutants was unstable and formed both the antibiotic resistant and the antibiotic sensitive clones. The greatest portion of the strains with high antibiotic activity (20 per cent) was detected among the S. erythraea spontaneous mutants on the medium with 2.5 micrograms/ ml of thiostreptone. It was shown that the instability of the high antibiotic activity in the mutants was associated with loss of the thiostreptone resistance property.

[Localization of rifampicin resistance determinants on genetic map of Saccharopolyspora erythraea]. / Lokalizatsiia determinantov ustolchivosti k rifampitsinu na geneticheskoi karte saccharopolyspora erythraea.

Genetic mapping of 2 mutations designated as rif5 and rif8 determining different levels of rifampicin resistance in Saccharopolyspora erythraea, an organism producing erythromycin, was performed. The mutations were inherited as chromosomal markers and localized in the same region of the chromosome restricted by ura1 and met1 loci. No close linking of rif5 and rif8 with any of the auxotrophic property markers in S. erythraea was detected. There were observed differences in the indices of correlation between the inheritance of rif8, rif5 and the chromosomal markers between which they were located. The data indicated that mutations rif5 and rif8 probably occurred in different genes.

The use of PCR to isolate a putative ABC transporter from Saccharopolyspora erythraea.

A gene (ertX) encoding a putative ABC transporter was cloned from the erythromycin producer Saccharopolyspora erythraea, using PCR. The primers were based on regions of homology from ABC transporters which confer resistance to macrolide antibiotics. While ertX encodes a protein with a strong degree of similarity to other macrolide ABC transporters from streptomycetes and staphylococci, it did not confer resistance to erythromycin, tylosin, spiramycin, oleandomycin, josamcin, chalcomycin or midecamycin when subcloned into sensitive streptomycete hosts. Southern blot analysis suggested that ertX did not constitute part of the erythromycin gene cluster as identified to date.

The mRNA for the 23S rRNA methylase encoded by the ermE gene of Saccharopolyspora erythraea is translated in the absence of a conventional ribosome-binding site.

Transcriptional analysis of the ermE gene of Saccharopolyspora erythraea, which confers resistance to erythromycin by N6-dimethylation of 23S rRNA and which is expressed from two promoters, ermEp1 and ermEp2, revealed a complex regulatory region in which transcription is initiated in a divergent and overlapping manner. Two promoters (eryC1p1 and eryC1p2) were identified for the divergently transcribed erythromycin biosynthetic gene eryC1, which plays a role in the formation of desosamine or its attachment to the macrolide ring. Transcription from eryC1p2 starts at the same position as that of ermEp1, but on the opposite strand of the DNA helix, suggesting co-ordinate regulation of genes for erythromycin production and resistance. ermEp1 initiates transcription at, and one nucleotide before, the ermE translational start codon. Site-directed and deletion mutagenesis, combined with immunochemical analysis, demonstrated that the ermEp1 transcript is translated in the absence of a conventional ribosome-binding site to give rise to the full-length 23S rRNA methylase. Deletion of the -35 region of ermEp1 reduced, but did not abolish, promoter activity, reminiscent of the 'extended -10' class of bacterial promoters which, like ermEp1, possess TGN motifs immediately upstream of their -10 regions and which initiate transcription seven nucleotides downstream of the -10 region.

Microbial conversion of avermectins by Saccharopolyspora erythraea: glycosylation at C-4' and C-4''.

Avermectins and ivermectins are glycosylated at C-4'' and C-4' by both growing and resting cells of Saccharopolyspora erythraea. The reaction is catalyzed by a glycosyltransferase which is constitutive. The enzyme uses UDP-glucose as the glycosyl donor and avermectin or ivermectin mono- and disaccharides as acceptors. Avermectin and ivermectin aglycones and erythromycin A are not substrates for the enzyme.

Characterization of two different types of resistance genes among producers of fortimicin-group antibiotics.

Fortimicin-A (FTM-A; astromicin)-resistance genes (fmr genes) isolated from six producers of the FTM-group of antibiotics were analysed. These genes could be classified into two types by the resistance profiles to aminoglycoside antibiotics and by their DNA homologies. Three genes, fmrT from the istamycin producer Streptomyces tenjimariensis ATCC 31603, fmrS from the sannamycin producer Streptomyces sannanensis IFO 14239 and fmrH from the sporaricin producer Saccharopolyspora hirsuta ATCC 20501, conferred resistance to FTM-A, kanamycin (Km) and neomycin B (Nm-B), but not to gentamicin (Gm). The other three genes, fmrO from the FTM-A producer Micromonospora olivasterospora ATCC 21819, fmrM from the antibiotic SF-2052 producer Micromonospora sp. SF-2098 (ATCC 31580) and fmrD from the dactimicin producer Dactylosporangium matsuzakiense ATCC 31570, conferred resistance to FTM-A, Km and Gm, but not to Nm-B. No DNA homology was detected between the two types of the resistance genes in Southern-blot analysis. The present results revealed that, in spite of the similarity of their biosynthesis genes, there are at least two different types of resistance genes among the FTM-group antibiotic producers.

[Effect of the methods of sterilization on the quality of nutrient media and on the level of antibiotic production]. / Vliianie sposobov sterilizatsii na kachestvo pitatel'nykh sred i na uroven' obrazovaniia antibiotikov.

It was shown that conditions for heat sterilization of nutrient media for biosynthesis of antibiotics had an impact on their biochemical composition and activity of a fermentation broth in production of penicillin and erythromycin. The temperature of 120 degrees C and the time of 25 minutes proved to be optimal for sterilization of the media in regard to both preservation of their biochemical composition and providing of the maximum antibiotic productivity on the one hand and maintenance of the sterility during the fermentation process on the other hand.