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.     

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.     

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.     

Nutrient regulation of epothilone biosynthesis in heterologous and native production strains

Fermentation media with different initial concentrations of ammonium and phosphate salts were used to study the inhibitory effects of those ions on growth and production of epothilone in Sorangium cellulosum and Myxococcus xanthus. The native epothilone producer, S. cellulosum was more sensitive to ammonium and phosphate than the heterologous producer, M. xanthus. An ammonium concentration of 12 mM reduced epothilone titers by 90% in S. cellulosum but by only 40% in M. xanthus. When 5 mM phosphate was added to the medium, production in both strains was 60% lower. Higher phosphate concentrations had little additional effect on M. xanthus titers, but epothilone production with 17 mM extra-cellular phosphate in S. cellulosum was 95% lower than in the control condition. The effect of iron supplementation to the fermentation medium was also investigated. Both strains showed best production with 20 microM iron added to the medium.     

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.     

Soraphinol C, a new free-radical scavenger from Sorangium cellulosum

A new compound named soraphinol C (1) was isolated from myxobacteria Sorangium cellulosum KM1001 a soil isolate, together with a structurally related known compound, 4-hydroxysattabacin (2). These compounds were isolated by silica gel column chromatography and recycling preparative HPLC, consecutively. The structures of the compounds were determined on the basis of combined spectroscopic analyses. Compounds 1 and 2 exhibited antioxidant activity as a radical scavenger in the experiment using a hydrophilic free-radical initiator, 2,2'-azobis(2-amidinopropane)dihydrochloride with ORAC values of 0.956 and 0.617, respectively.     

Insights into the complex biosynthesis of the leupyrrins in Sorangium cellulosum So ce690

The anti-fungal leupyrrins are secondary metabolites produced by several strains of the myxobacterium Sorangium cellulosum. These intriguing compounds incorporate an atypically substituted γ-butyrolactone ring, as well as pyrrole and oxazolinone functionalities, which are located within an unusual asymmetrical macrodiolide. Previous feeding studies revealed that this novel structure arises from the homologation of four distinct structural units, nonribosomally-derived peptide, polyketide, isoprenoid and a dicarboxylic acid, coupled with modification of the various building blocks. Here we have attempted to reconcile the biosynthetic pathway proposed on the basis of the feeding studies with the underlying enzymatic machinery in the S. cellulosum strain So ce690. Gene products can be assigned to many of the suggested steps, but inspection of the gene set provokes the reconsideration of several key transformations. We support our analysis by the reconstitution in vitro of the biosynthesis of the pyrrole carboxylic starter unit along with gene inactivation. In addition, this study reveals that a significant proportion of the genes for leupyrrin biosynthesis are located outside the core cluster, a 'split' organization which is increasingly characteristic of the myxobacteria. Finally, we report the generation of four novel deshydroxy leupyrrin analogues by genetic engineering of the pathway.     

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。     

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).     

Assessment of fed-batch, semicontinuous, and continuous epothilone D production processes

Epothilone D is a member of a class of potent antineoplastic natural products produced by myxobacteria. Previously, we have described a fed-batch epothilone D production process in which an adsorber resin is incorporated into the bioreactor setup to capture and stabilize the product in situ, preventing its degradation within the bioreactor. The capture of epothilone D by these relatively large resin beads enables the development of continuous and semicontinuous culturing systems incorporating bead retention mechanisms to completely retain the product within the bioreactor, increasing the epothilone D product titer by almost 3-fold in both cases over a baseline fed-batch system. These product retention strategies, described here for production of the epothilones, are generally applicable to any system using adsorber resins as a method to capture product during a microbial cultivation.     

Diversity of epothilone producers among Sorangium strains in producer‐positive soil habitats

Large‐scale surveys show that the anti‐tumour compounds known as epothilones are produced by only a small proportion of Sorangium strains, thereby greatly hampering the research and development of these valuable compounds. In this study, to investigate the niche diversity of epothilone‐producing Sorangium strains, we re‐surveyed four soil samples where epothilone producers were previously found. Compared with the < 2.5% positive strains collected from different places, epothilone producers comprised 25.0–75.0% of the Sorangium isolates in these four positive soil samples. These sympatric epothilone producers differed not only in their 16S rRNA gene sequences and morphologies but also in their production of epothilones and biosynthesis genes. A further exploration of 14 soil samples collected from a larger area around a positive site showed a similar high positive ratio of epothilone producers among the Sorangium isolates. The present results suggest that, in an area containing epothilone producers, the long‐term genetic variations and refinements resulting from selective pressure form a large reservoir of epothilone‐producing Sorangium strains with diverse genetic compositions.     

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.     

纤维堆囊菌的供谢产物及其生物学活性分析

纤维堆囊菌不同菌株不但表现细胞和子实体形态的差异,而且供谢产物的组成和生物学活性也存在差异。纤维堆囊菌对革兰氏阴性细菌不表现任何抑制活性,部分菌株可抑制革兰氏阳性细菌;但所有菌株对真菌和肿瘤细胞有广泛和强烈的抑制作用。薄层层析显示,纤维堆囊菌的次级代谢产物组分较多,且大多数组分具有不同程度的抑制真菌和肿细胞的活性。在筛选中发现四株菌的代谢产物能够促进微管蛋白聚合,其中So33-1活性组分的薄层层析Rf值与已知的Epothilone A相似,而So81的则有较大的差异。研究结果表明纤维堆囊菌是很好的筛选抗真核生物活性的天然化合物资源。     

Production of an exopolysaccharide bioflocculant by Sorangium cellulosum

AIMS: To isolate a new exopolysaccharide bioflocculant produced by the myxobacterium Sorangium cellulosum NUST06, and to characterize its chemical composition and expolysaccharide production relative to carbon source. METHODS AND RESULTS: Exopolysaccharide levels and biomass production by S. cellulosum NUST06 were analysed relative to carbon source. Glucose in the medium at a level of 3 g l(-1) completely inhibited cell growth and exopolysaccharide production, but low concentrations of glucose (1-2 g l(-1)) could stimulate cell utilization of starch. The chemical composition and flocculating activity of the NUST06 exopolysaccharide was investigated. The flocculant comprised 38.3% proteins and 58.5% carbohydrates, of which glucose, mannose and glucuronic acid were present at 51.3%, 39.2% and 10.5%, respectively. The flocculating activity of the NUST06 flocculant depended strongly on cations. CONCLUSIONS: It is feasible to produce an exopolysaccharide bioflocculant by the strain NUST06 in a mineral salts medium using starch as a carbon source. SIGNIFICANCE AND IMPACT OF THE STUDY: This strain may be advantageous for commercial bioflocculant production and may enrich existing knowledge of myxobacteria.     

Synthesis and SAR of C12–C13-oxazoline derivatives of epothilone A

The SAR of a series of new epothilone A derivatives with a 2-substituted-1,3-oxazoline moiety trans-fused to the C12–C13 bond of the deoxy macrocycle have been investigated with regard to tubulin polymerization induction and cancer cell growth inhibition. Significant differences in antiproliferative activity were observed between different analogs, depending on the nature of the substituent at the 2-position of the oxazoline ring. The most potent compounds showed comparable activity with the natural product epothilone A. Modeling studies provide a preliminary rationale for the observed SAR.     

Myxobacteria isolated in Israel as potential source of new anti-infectives

AIMS: To evaluate the patterns of the production of antimicrobial compounds by Israeli myxobacteria newly isolated from soil samples and barks by a battery of isolation and purification methods. METHODS AND RESULTS: A total of 100 myxobacteria belonging to five of the 12 described genera, were isolated from 48 soil and 45 tree bark samples collected in different areas inside the State of Israel. Four isolation methods based on the peculiar metabolic and cell cycle aspects of myxobacteria, were combined with purification procedures and optimization of cultivation conditions. Ninety-seven strains were fermented and screened for antimicrobial activities. Production of antimicrobial activities was detected in 62 isolates. More than 50% of the collection (54 strains) was able to inhibit Escherichia coli growth. CONCLUSIONS: The results of this study support the idea that myxobacterial strains can be isolated from particular habitats and then cultivated and screened for their capacity to produce secondary metabolites endowed with antibacterial and antifungal activities. Myxovirescin, a typical poliketide myxobacterial antibiotic, has been identified in one Israeli isolate. Althiomycin, a thiazolyl peptide, which inhibits prokaryotic protein synthesis, usually produced by actinomycetes, was detected in three strains selected in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: The results confirm that myxobacteria are prolific producers of a variety of bioactive secondary metabolites including antibacterial and antifungal compounds, being their high frequency of anti-Gram-negative activities particularly appealing for the current anti-infective research. So far their screening has often been hampered because their isolation is time-consuming and are quite difficult to handle and cultivate. In this paper we demonstrate that a proper combination of isolation, purification and cultivation methods allow their pharmaceutical exploitation.     

Isolation and characterization of the epothilone biosynthetic gene cluster from Sorangium cellulosum

The epothilone biosynthetic gene cluster was isolated from Sorangium cellulosum strain SMP44. The gene cluster contains seven genes and spans approx. 56kb. The genes encoding the PKS, epoA, epoC, epoD, epoE, and epoF, are divided into nine modules. The EpoB protein is a non-ribosomal peptide synthetase (NRPS) that catalyzes formation of the thiazole found in the epothilones. EpoK is a P450 enzyme responsible for the epoxidation of epothilones C and D to epothilones A and B, respectively. EpoK was expressed in Escherichia coli, and the purified protein was shown to convert epothilone D to epothilone B in vitro.     

Epothilone D affects cell cycle and microtubular pattern in plant cells

Epothilones, macrocyclic lactones from culture filtrates of the myxobacterium Sorangium cellulosum, are known as taxol-like microtubular drugs in human medicine. To date, nothing is known about the effect of epothilones on microtubules (MTs) in plant cells and/or on the plant cell cycle. As shown in this report, the treatment of tomato cell suspension cultures with epothilone D produced a continuous increase in the mitotic index. Dose-response curves revealed that epothilone D alters the mitotic index at concentrations as low as 1.5 microM. Mitotic arrest was already visible after only 2 h of treatment, and 55% of the cells were arrested after 24 h. As shown by immunocytological methods, abnormal spindles are formed during metaphase, which leads to a random distribution of chromosomes in the whole cell and prevents the formation of a metaphase plate. The process of chromosome decondensation does not seem to be affected, because micronuclei form at the same place with the distributed chromosomes. This suggests that epothilone D influences the stability of plant MTs mainly during metaphase of the mitotic cycle. In metaphase, the effects of epothilone D seem to be irreversible, because cells with an abnormal spindle could not be recovered after removal of the drug.