Distribution of Sorangium cellulosum

The myxobacterium Sorangium cellulosum exists and plays an important role in soil ecology by its ability to degrade cellulosic materials; it was found to be widely distributed in North American soils.     

纤维堆囊菌的多细胞形态发生过程

粘细菌是具有复杂多细胞行为的革蓝氏阴性细菌,纤维堆囊菌是粘细菌中唯一能够降解纤维素的粘细菌种属。本文通过扫描电子显微镜和相差光学显微镜,分析了纤维堆囊菌在纤维素基质上的生长和子实体形态发生的多细胞行为特征,给出了生长和子实体发育的模式图谱。     

纤维堆囊菌生长及限制因素的研究

纤维堆囊菌对可溶性淀粉、木聚糖、纤维素等复杂碳源的利用能力较强,简单碳源中只利用葡萄糖;KNO3、蛋白栋是较好的氮源,大多数氮源都能支持生长,表明该菌营养要求简单,能高效的得用处是里丰富的纤维素资源。堆囊菌的盐耐受能力较低,盐浓度高于1％的培养基中菌体几乎不能生长,Mg^2是生长必须的元素,Ca^2 对生长没有明显的作用,但是子实体的形态发生所必需的。培养基的起始pH为7.0时细胞生长较好,大于8.0或小于5.0不生长。纤维堆囊菌生长具有细胞密度依赖性,低密度的细胞不能生长。固定在滤纸上的细胞淋洗液（纤维素酶降解产物）对细胞生长具有明显促进作用,并能降低细胞生长的密度依赖性。     

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.     

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.     

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.     

产Epothilone B纤维堆囊菌的选育与发酵优化

目的:获得高产Epothilone B菌株和最佳营养条件及发酵条件。方法:采用紫外线照射的方法对实验室保存的1株纤纤堆囊菌AHB125进行诱变处理,并采用单因素和正交实验优化培养基中的碳源、氮源和发酵条件。结果:经诱变后获得1株遗传性能稳定变异菌株(SC4-56),Epothilone B产量为14.6mg/L,比初始菌株高195%;该菌株最佳碳源和氮源为6%玉米淀粉和3%蛋白胨,产量分别为20.5和21.8 mg/L;最佳发酵条件为:转速160r/min,温度32℃,接种量8%(V/V),装液量90 mL/500mL,优化后经验证实验,Epothilone B达28.24 mg/L。结论:获得了1株高产量变异菌株,并确定了最佳生产条件。     

Cloning and characterization of an rRNA methyltransferase from Sorangium cellulosum

A locus (kmr) responsible for aminoglycosides-resistance of Sorangium cellulosum was cloned and characterized in Myxococcus xanthus. The gene kmr encodes a putative rRNA methyltransferase. Expression of the complete ORF endowed the Myxococcus transformants with the resistance to aminoglycosidic antibiotics of kanamycin, apramycin, gentamycin, neomycin, and tobramycin at an extraordinary high-level (MIC, higher than 500 μg/ml). However, the gene did not function in Escherichia coli cells. In Sorangium genome, the gene kmr was followed by a putative integrase gene, and was highly homologous in different Sorangium strains. The Sorangium rRNA methyltransferase sequence was in low similarity to the reported 16S rRNA methyltransferases, and their resistance spectrums were also different. The results indicate that the rRNA methyltransferase (Kmr) in Sorangium strains is a new member of the rRNA methyltransferases family.     

Development of a mariner-based transposon for use in Sorangium cellulosum

In order to generate marked insertions in the myxobacterium Sorangium cellulosum, a transposon based on the eukaryotic mariner transposon was developed. The transposition frequency was increased with the use of a mutated tnp gene. The transposon randomly inserts into the chromosome, as demonstrated by targeted mutagenesis of the epoK gene.     

Development of a mariner-Based Transposon for Use in Sorangium cellulosum

In order to generate marked insertions in the myxobacterium Sorangium cellulosum, a transposon based on the eukaryotic mariner transposon was developed. The transposition frequency was increased with the use of a mutated tnp gene. The transposon randomly inserts into the chromosome, as demonstrated by targeted mutagenesis of the epoK gene.     

纤维堆囊菌生理生化及药敏特征

对纤维堆囊菌模式菌DSM14627的生理生化和药敏性进行了系统的考察,研究显示该茵能利用葡萄糖及纤维素,不能分解利用麦芽糖、蔗糖等12种碳源;磷酸酶、β-葡萄糖苷酶及明胶液化实验为阳性,过氧化氢酶、β-木糖苷酶等13项实验均阴性;对丙氟哌酸、万古霉素、多粘霉素B等15种抗生素敏感,对氨苄西林及苯唑青霉素等10种抗生素有极强抗性.     

Repeated batch production of epothilone B by immobilized Sorangium cellulosum

Production of extracellular epothilone B, one of the potent anticancer agents, by free and immobilized Sorangium cellulosum was studied using the repeated batch culture process. The concentration of alginate used in immobilization was directly related to the mass transfer rate of nutrients, mechanical stability, and the epothilone B production yield. With the optimized 3% (w/v) calcium alginate carrier, a prolonged repeated batch culture was investigated for the 5 repeated batches for 24 days. The maximum productivity of epothilone B obtained from the alginate-immobilized cells was 5.03 mg/l/day, which is 3 times higher than that of free cells (1.68 mg/l/day).     

Mutation in the rel gene of Sorangium cellulosum affects morphological and physiological differentiation

Interruption of the (p)ppGpp synthetase gene ( rel ) of Sorangium cellulosum So ce56 resulted in loss of ppGpp accumulation after norvaline treatment during exponential growth phase. The rel mutant failed to produce wild-type levels of the polyketides chivosazol and etnangien in production media. In wild-type cells expression of the chivosazol biosynthetic operon can be significantly increased by norvaline or α-methylglucoside. This induction does not occur in the rel mutant. The rel mutant also lost the capability to form multicellular fruiting bodies under nutrient starvation.     

Structural features and hypoglycaemic activity of an exopolysaccharide produced by Sorangium cellulosum

AIMS: To investigate the structural features and hypoglycaemic activity of an exopolysaccharide (EPS) produced by Sorangium cellulosum NUST06. METHODS AND RESULTS: The chemical structure of the EPS from S. cellulosum NUST06 was determined by gas-liquid chromatography, gas chromatography (GC), GC-mass spectrometry and nuclear magnetic resonance. The EPS was composed of a beta-D-(1-->4)-glucose backbone with alpha-D-(1-->6)-mannose side chains. The molecular weight of the EPS was approx. 2x10(5) Da. Healthy and alloxan-induced diabetic mice were used in the study. Blood glucose levels of the experimental animals during the trial period were analysed by a glucose test kit based on the glucose oxidase method. When 100 and 200 mg kg(-1) day(-1) of purified EPS was orally administered for 7 days, the serum glucose in alloxan-induced diabetic mice was reduced by 35.9 and 41.4% (P<0.01), and the serum glucose in healthy mice was reduced by 27.3 and 30.1% (P<0.05), respectively. CONCLUSIONS: The EPS produced by S. cellulosum NUST06 decreased blood glucose levels distinctly in both healthy and alloxan-induced diabetic mice. SIGNIFICANCE AND IMPACT OF THE STUDY: To elucidated the chemical structure of the EPS from S. cellulosum NUST06 and exploited the anti-diabetic potential of the EPS.     

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.     

Transfer of mobilizable plasmids to Sorangium cellulosum and evidence for their integration into the chromosome.

Recombinant vectors derived from the broad-host-range mobilizable plasmid pSUP2021 were constructed and transferred by IncP-mediated conjugation from Escherichia coli to Sorangium cellulosum, where they were integrated into the chromosome by homologous recombination and maintained stably. This appears to be the first system of gene transfer to S. cellulosum.     

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。     

Identification of an l-dopa decarboxylase gene from Sorangium cellulosum So ce90

During a screening program intended to identify genes encoding enzymes typical for secondary metabolism in Sorangium cellulosum So ce90, an aromatic amino acid decarboxylase gene (ddc) was detected. Expression of ddc in Escherichia coli and subsequent enzyme assays with cell-free extracts confirmed the proposed function derived from amino acid sequence comparisons. In contrast to other aromatic amino acid decarboxylases of eukaryotic origin, the S. cellulosum Ddc converted only L-dihydroxy phenylalanine. This is the first report of a gene encoding an L-dihydroxy phenylalanine decarboxylase in bacteria.     

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.