Selective production of epothilone B by heterologous expression of propionyl-CoA synthetase in Sorangium cellulosum

The metabolic engineering of epothilones, as secondary metabolites, was investigated using Sorangium cellulosum to achieve the selective production of epothilone B, a potent anticancer agent. Thus, the propionyl-CoA synthetase gene (prpE) from Ralstonia solanacearum was heterologously expressed in S. cellulosum to increase the production of epothilone B. Propionyl-CoA synthetase converts propionate into propionyl-CoA, a potent precursor of epothilone B. The recombinant S. cellulosum containing the prpE gene exhibited a significant increase in the resolution of epothilones B/A, with an epothilone B to A ratio of 127 to 1, which was 100 times higher than that of the wild-type cells, demonstrating its potential use for the selective production of epothilone B.     

Production of the glycopeptide antibiotic A40926 by<Emphasis Type="Italic"> Nonomuraea</Emphasis> sp. ATCC 39727: influence of medium composition in batch fermentation

Nonomuraea sp. ATCC 39727 is a novel actinomycete species and the producer of A40926, a glycopeptide antibiotic structurally similar to teichoplanin. In the present study, a defined minimal medium was designed for Nonomuraea fermentation. The influence of initial phosphate, glucose and ammonium concentrations on antibiotic productivity was investigated in batch fermentation and the effect of glucose limitation was studied in fed-batch fermentation. It was found that low initial concentrations of phosphate and ammonium are beneficial for A40926 production and that productivity is not enhanced during glucose limitation. Furthermore, the initiation of A40926 production was not governed by residual ammonium and phosphate concentrations, although the level of these nutrients strongly influenced A40926 production rates and final titers. Electronic Publication     

Isolation of Sorangium cellulosum carrying epothilone gene clusters

Epothilone and its analogs are a potent new class of anticancer compounds produced by myxobacteria. Thus, in an effort to identify new myxobacterial strains producing epothilone and its analogs, cellulose-degrading myxobacteria were isolated from Korean soils, and 13 strains carrying epothilone biosynthetic gene homologs were screened using a polymerase chain reaction. A migration assay revealed that Sorangium cellulosum KYC3013, 3016, 3017, and 3018 all produced microtubule-stabilizing compounds, and an LCMS/ MS analysis showed that S. cellulosum KYC3013 synthesized epothilone A.     

Characterisation,genome size and genetic manipulation of the myxobacterium <Emphasis Type="Italic">Sorangium cellulosum</Emphasis> So ce56

In this study, Sorangium cellulosum So ce56 was phenotypically and genotypically analysed in order to evaluate whether this strain can be used in a comprehensive genome project as a representative of the secondary metabolite-producing myxobacteria. In contrast to many other strains of S. cellulosum, strain So ce56 was found to have various advantageous features, including fast and homogeneous growth in submerged cultures and the ability to complete its morphological differentiation cycle on agar, even when the inoculant originates from a liquid culture. Two groups of secondary metabolites isolated from the culture broth were identified, the polyketides etnangien and chivosazole. The presence of polyketide synthase-encoding genes in the genome of strain So ce56 was demonstrated via PCR. The phenotypic classification was confirmed by comparison of 16S rDNA sequences which showed that S. cellulosum So ce56 clusters within a separate lineage together with S. cellulosum ATCC 25531 and the epothilone producer S. cellulosum So ce90. The genome of S. cellulosum So ce56 belongs to the largest bacterial genomes described so far. It is estimated to be 12.2 Mb in size, by pulsed-field gel electrophoresis. In order to demonstrate that S. cellulosum So ce56 is a convenient strain for molecular biological studies, a genetic manipulation system was developed. Using triparental mating, polyketide synthase-encoding genes were inactivated, leading to chivosazole-negative mutants.     

培养基组成对纤维堆壤菌产埃博霉素的影响

埃博霉素(Epothilone)是粘细菌纤维堆壤菌(Sorangium cellulosum)产生的具有抗肿瘤活性的次级代谢产物。为了提高埃博霉素A和B的产量,以及B/A的比率,以G52培养基为基础培养基,研究了黄豆粉、酵母粉、酪蛋白胨和丙酸钠对纤维堆壤菌产埃博霉素的影响。结果表明:低脂黄豆粉和酪蛋白胨作为氮源,同时加入6.25 mmol/L丙酸钠作为前体物质,埃博霉素A和B的产量分别比原始条件平均提高1.47倍和2.88倍,B/A的比率比原始条件平均提高了1.97倍。     

Myxobacteria: proficient producers of novel natural products with various biological activities--past and future biotechnological aspects with the focus on the genus Sorangium

Myxobacteria are gram-negative bacteria which are most noted for their ability to form fruiting bodies upon starvation. Within the last two decades, they increasingly gained attention as producers of natural products with biological activity. Here, recent and future biotechnological research on certain key myxobacteria and on their ability to produce natural products is reviewed with the focus on the production of myxovirescin, soraphen and epothilone. Aspects of product improvement and yield as well as statistics regarding secondary metabolite formation are discussed. Future research will deal with the exploitation of the biosynthetic potential of the myxobacteria, for example via the isolation of new myxobacterial species with different physiological properties. Additionally, the genetic potential of myxobacteria to form natural products can be exploited by the identification and activation of biosynthetic gene clusters. These can be found frequently within their genomes, which is shown by the analysis of the unfinished genomes of Myxococcus xanthus and Sorangium cellulosum. The current status of the S. cellulosum functional genome project with model strain So ce56 is discussed.     

Gene expression during development of Myxococcus xanthus. Analysis of the genes for protein S

Protein S is an abundant spore coat protein produced during fruiting body formation (development) of the bacterium Myxococcus xanthus. We have cloned the DNA which codes for protein S and have found that this DNA hybridizes to three protein S RNA species from developmental cells but does not hybridize to RNA from vegetative cells. The half-life of protein S RNA was found to be unusually long, about 38 minutes, which, at least in part, accounts for the high level of protein S synthesis observed during development. Hybridization of restriction fragments from cloned M. xanthus DNA to the developmental RNAs enabled us to show that M. xanthus has two directly repeated genes for protein S (gene 1 and gene 2) which are separated by about 10(3) base-pairs on the bacterial chromosome. To study the expression of the protein S genes in M. xanthus, eight M. xanthus strains were isolated with Tn5 insertions at various positions in the DNA which codes for protein S. The strains which contained insertions in gene 1 or between gene 1 and gene 2 synthesized all three protein S RNA species and exhibited normal levels of protein S on spores. In contrast, M. xanthus strains exhibited normal levels of protein S on spores. In contrast, M. xanthus strains with insertions in gene 2 had no detectable protein S on spores and lacked protein S RNA. Thus, gene 2 is responsible for most if not all of the production of protein S during M. xanthus development. M. xanthus strains containing insertions in gene 1, gene 2 or both genes, were found to aggregate and sporulate normally even though strains bearing insertions in gene 2 contained no detectable protein S. We examined the expression of gene 1 in more detail by constructing a fusion between the lacZ gene of Escherichia coli and the N-terminal portion of protein S gene 1 of M. xanthus. The expression of beta-galactosidase activity in an M. xanthus strain containing the gene fusion was shown to be under developmental control. This result suggests that gene 1 is also expressed during development although apparently at a much lower level than gene 2.     

Nutrient effects on anthracycline production by Streptomyces peucetius in a defined medium

A defined medium was developed for Streptomyces peucetius that optimally contained 0.5 mM magnesium, 1 mM phosphate, 75-125 mM glucose, 10 mM nitrate, and microelements. Poorer results were obtained with nitrite, aspartate, or ammonia as sole nitrogen sources. Other carbon sources which supported best growth and highest anthracycline titers were fructose, maltose, and soluble starch. In each case, substantial residual carbon remained at the end of 6 days, suggesting a lack of catabolite repression by the carbohydrate carbon sources on anthracycline biosynthesis. Studies involving limiting and nonlimiting concentrations of glucose supplemented with arabinose, a poorly utilizable carbon source, indicated that high carbon concentrations were not necessary for osmotic stabilization. Inorganic phosphate was found to have an inhibitory effect on anthracycline production. Furthermore, when cultures at early stages of anthracycline production were spiked with inorganic phosphate, a delay in further anthracycline production resulted until the added phosphate was depleted. A 10% inoculum of stationary phase cells yielded the best growth and most consistent anthracycline production. Spectrophotometric analyses at 495 nm of chloroform--methanol-extracted material were also found to be useful for the determination of total anthracyclines in culture extracts.     

Mutation and a high-throughput screening method for improving the production of Epothilones of <Emphasis Type="Italic">Sorangium</Emphasis>

The epothilones are highly promising prospective anticancer agents that are produced by the myxobacterium Sorangium cellulosum. We mutated the epothilone producing S. cellulosum strain So0157-2 to improve the production of epothilones. For evaluation in high-throughput of a large number of mutants, we developed a simple microtiter method for primary screening. Using the classical UV-mutation method plus selection pressures, the production capacity was increased about 0.5 approximately 2.5 times the starting strain. The mutants with higher production and different phenotypes were further subjected to recursive protoplast fusions and the fusants products were screened under multi-selection pressure. Furthermore, the production was greatly increased by the genome shuffling. For epothilone B, the production of one fusant was increased about 130 times compared to the starting strain, increasing from 0.8 mg l(-1) to 104 mg l(-1).     

Nitrogen repression of fumonisin B1 biosynthesis in Gibberella fujikuroi

Fumonisins are a group of structurally related mycotoxins produced by Gibberella fujikuroi. The fungus produced fumonisin B1 (FB1) as early as 18 hour in a defined medium containing 1.25 mM or 2.5 mM ammonium phosphate, whereas fumonisin B1 production was repressed for 75 hour and 125 hour when mycelia were resuspended in media containing ammonium phosphate at 10 mM or 20 mM, respectively. Although total fumonisin B1 production was greater in resuspension cultures grown in higher concentrations of ammonium phosphate, the accumulation was independent of the inoculum size and carbon/nitrogen ratio. The addition of ammonium phosphate to cracked corn cultures also repressed fumonisin B1 production by 97%, and persisted for at least three weeks. Thus, biosynthesis of fumonisin B1 is regulated by a mechanism involving nitrogen metabolite repression, suggesting that control strategies that target the regulatory elements of nitrogen metabolism may be effective at reducing the risk of fumonisin contamination in food.     

NUTRITIONAL REQUIREMENTS FOR OVULE FORMATION IN EXCISED PISTILS OF NIGELLA

The nutritional requirements for ovule formation in Nigella saliva L. were investigated by growing excised pistils on defined media. Pistils grown on a medium containing the minerals of Murashige and Skoog produced significantly more ovules than on a medium containing the minerals of Bilderback. When the nitrogen, sulfate, and phosphate of the Bilderback medium were adjusted to levels comparable to those of the Murashige and Skoog medium, a similar number of ovules was formed. The effect of different forms and concentrations of nitrogen on ovule formation and pistil growth was investigated. High concentrations of nitrate (40 mil) favored pistil growth and ovule formation, but comparable levels of ammonium were toxic. When ammonium at concentrations above 10 mM was added to nitrate media, ovule formation was inhibited. A medium containing low concentrations of ammonium (10 mM) and nitrate (5 mM) supported more ovule formation and pistil growth in young pistils than a low-nitrate (5 mM) medium without ammonium. However, ovule formation on a medium containing 10 mM ammonium and 5 mM nitrate was significantly less than on a medium containing only 15 mM nitrate. Low concentrations of organic nitrogen in the form of α-alanine (1 mM) and γ-aminobutyric acid (5 mM) supported ovule formation and pistil growth similar to a high nitrate medium.     

Novel electrochemical-enzymatic model which quantifies the effect of the solution Eh on the kinetics of ferrous iron oxidation with Acidithiobacillus ferrooxidans

The heterologous production of epothilone D in Myxococcus xanthus was improved by 140-fold from an initial titer of 0.16 mg/L with the incorporation of an adsorber resin, the identification of a suitable carbon source, and the implementation of a fed-batch process. To reduce the degradation of epothilone D in the basal medium, XAD-16 (20 g/L) was added to stabilize the secreted product. This greatly facilitated its recovery and enhanced the yield by three-fold. The potential of using oils as a carbon source for cell growth and product formation was also evaluated. From a screen of various oils, methyl oleate was shown to have the greatest impact. At the optimal concentration of 7 mL/L in a batch process, the maximum cell density was increased from 0.4 g dry cell weight (DCW)/L to 2 g DCW/L. Product yield, however, depended on the presence of trace elements in the production medium. With an exogenous supplement of trace metals to the basal medium, the peak epothilone D titer was enhanced eight-fold. This finding demonstrates the significant role of metal ions in cell metabolism and in epothilone biosynthesis. To further increase the product yield, a continuous fed-batch process was used to promote a higher cell density and to maintain an extended production period. The optimized fed-batch cultures consistently yielded a cell density of 7 g DCW/L and an average production titer of 23 mg/L.     

林肯链霉菌合成林可霉素代谢调节的研究

在摇瓶条件下研究了葡萄糖、铵盐、磷酸盐对林可霉素产生菌林肯链霉菌的生长及林可霉素生物合成的影响。发酵过程中林可霉素的合成主要发生在菌体生长期,逐渐下降。使用6%的葡萄糖未发现通常所说的“葡萄糖效应”。0.2%铵盐有利于细胞生长,但0.8%NH+4对林可霉素的生物合成具有抑制作用。发酵48h后补加0.6% NH^＋_４,对林可霉素的生成没有显著影响。0.05%～0.1%磷酸盐对林可霉素合成具有较强的抑制作用。并就磷酸盐对菌体由初级代谢转向次级代谢的作用作了初步考察。     

Utilization of UF-permeate for production of beta-galactosidase by lactic acid bacteria

Four lactobacilli strains (Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacilus casei and Lactobacillus reuteri) were grown in MRS broth and three lactococci strains (Streptococcus thermophilus, Lactococcus lactis subsp. Lactis and Lactococcus lactis subsp. lactis biovar. diacetilactis) were grown in M17 broth. L. reuteri and S. thermophilus were chosen on the basis of the best mean beta-galactosidase activity of 10.44 and 10.01 U/ml respectively, for further studies on permeate-based medium. The maximum production of beta-galactosidase by L. reuteri was achieved at lactose concentration of 6%, initial pH 5.0-7.5, ammonium phosphate as nitrogen source at a concentration of 0.66 g N/L and incubation temperature at 30 degrees C/24 hrs to give 6.31 U/ml. While in case of S. thermophilus, maximum beta-galactosidase production was achieved at 10% lactose concentration of permeate medium, supplemented with phosphate buffer ratio of 0.5:0.5 (KH2PO4:K2HPO4, g/L), at initial pH 6.0-6.5, ammonium phosphate (0.66g N/L) as nitrogen source and incubation temperature 35 degrees C for 24 hrs to give 7.85 U/ml.     

Regulation of biosynthesis of bacilysin byBacillus subtilis

Summary Production of the dipeptide antibiotic bacilysin byBacillus subtilis 168 was growth associated and showed no evidence of repression by glucose or sucrose. Carbohydrates other than glucose and sucrose yielded lower specific titers of bacilysin. Bacilysin production in three such carbon sources (maltose, xylose, ribose) was delayed until growth slowed down. Ammonium salts were poor for bacilysin production when used as the sole nitrogen source. When added to the standard medium containing glutamate, they suppressed antibiotic production. Aspartate was slightly better than glutamate for antibiotic production as sole nitrogen source. No other nitrogen source tested, including inorganic, organic or complex, approached the activity of glutamate or aspartate. When added to glutamate, casamino acids, phenylalanine and alanine (a substrate of bacilysin synthetase) suppressed bacilysin production while stimulating growth. Phosphate provided for optimum growth and production at 7.5 mM and both processes were inhibited at higher concentrations. Ferric citrate stimulated growth and inhibited bacilysin production, the effects being due to both the iron and the citrate components. Elimination of ferric citrate stimulated production as did increasing the concentration of Mn to its optimum concentration of 6.6×10^–4M.     

Effects of phosphate, glucose, and ammonium on cell growth and lincomycin production by Streptomyces lincolnensis in chemically defined media

Cell growth and lincomycin production were measured in batch cultures of Streptomyces lincolnensis in chemically defined media. In these fermentations the specific rate of antibiotic production was maximal during growth and always declined at the end of the growth phase. It was found that both phosphate and ammonium salts, while required for cell growth, had negative effects on antibiotic production. By increasing the concentration of magnesium sulfate, it was possible to increase both the production rates and final titers of lincomycin. The mechanism for this effect was found to be the reduction of soluble phosphate in the medium through the precipitation of ammonium magnesium phosphate. Lincomycin production rates were not inhibited by glucose at concentrations of up to 30 g/L.     

The effect of nitrate and ammonium concentrations on growth and alkaloid accumulation of Atropa belladonna hairy roots

The growth, the alkaloid production, as well as the scopolamine/hyoscyamine ratio of two clones of belladonna hairy roots were studied. The effects of nitrate and ammonium concentrations on these cultures were investigated. A rise in ammonium concentration caused the decline of the hairy roots, while nitrate had a marked effect on the alkaloid content. The alkaloid production obtained with 15.8 mM of NO3- and 20.5 mM of NH4+ was 1.2-1.4 times higher than that obtained when the roots were grown in the standard Murashige and Skoog medium (MS medium, 39.5 mM of NO3- and 20.5 mM of NH4+). The nitrate and ammonium concentrations in the culture medium also had a strong influence on the scopolamine/hyoscyamine ratio. When nitrate or ammonium concentrations were raised, that ratio also was increased 2-3-fold. The hairy root clones originating from transformations with two distinct strains of Agrobacterium had similar responses.     

Complete genome sequence of the myxobacterium Sorangium cellulosum

The genus Sorangium synthesizes approximately half of the secondary metabolites isolated from myxobacteria, including the anti-cancer metabolite epothilone. We report the complete genome sequence of the model Sorangium strain S. cellulosum So ce56, which produces several natural products and has morphological and physiological properties typical of the genus. The circular genome, comprising 13,033,779 base pairs, is the largest bacterial genome sequenced to date. No global synteny with the genome of Myxococcus xanthus is apparent, revealing an unanticipated level of divergence between these myxobacteria. A large percentage of the genome is devoted to regulation, particularly post-translational phosphorylation, which probably supports the strain's complex, social lifestyle. This regulatory network includes the highest number of eukaryotic protein kinase-like kinases discovered in any organism. Seventeen secondary metabolite loci are encoded in the genome, as well as many enzymes with potential utility in industry.     

Improving conjugation efficacy of <Emphasis Type="Italic">Sorangium cellulosum</Emphasis> by the addition of dual selection antibiotics

The conjugation protocols in myxobacterium Sorangium cellulosum are often inapplicable due to the strain-specific sensitivity to the presence of Escherichia coli cells or the resistances to many antibiotics. Here we report that the conjugative transfer of the mobilizable plasmid pCVD442 from E. coli DH5alpha (lambda pir) to Sorangium strains could be greatly increased by the presence of low doses of dual selection antibiotics in the mating medium. The improvement was efficient in either E. coli-tolerant or sensitive Sorangium strains. For those phleomycin and hygromycin tolerant Sorangium strains, chloramphenicol-resistance gene was developed as a new selectable marker by driving the resistance gene with the aphII promoter. Using the improved protocol, the epothilone biosynthetic pathway was inactivated by an insertion mutation in the biosynthetic genes of the producing Sorangium strains.