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.     

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.     

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).     

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.     

Improvement of epothilone B production by in situ removal of ammonium using cation exchange resin in Sorangium cellulosum culture

A cation exchange resin was added at different stages in the growth of Sorangium cellulosum cultures to remove excess ammonium, which has a negative influence on the production of the anticancer agent, epothilone. When 8 g L⁻¹ of cation exchange resin were added at the mid-death phase, the ammonium removal reached 83% and the maximum production of epothilone was 14.3 mg L⁻¹, which was 2.6 times greater than that of the control culture.     

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.     

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。     

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.     

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.     

Origins of Replication in Sorangium cellulosum and Microcystis aeruginosa.

The genome of Sorangium cellulosum has recently been completely sequenced, and it is the largest bacterial genome sequenced so far. In their report, Schneiker et al. (in Complete genome sequence of the myxobacterium Sorangium cellulosum, Nat. Biotechnol., 2007, 25, 1281-1289) concluded that 'In the absence of the GC-skew inversion typically seen at the replication origin of bacterial chromosomes, it was not possible to discern the location of oriC'. In addition, the complete genome of Microcystis aeruginosa NIES-843 has also been recently sequenced, and in this report, Kaneko et al. (in Complete genomic structure of the bloom-forming toxic cyanobacterium Microcystis aeruginosa NIES-843, DNA Res., 2007, 14, 247-256) concluded that 'there was no characteristic pattern, according to GC skew analysis'. Therefore, oriC locations of the above genomes remain unsolved. Using Ori-Finder, a recently developed computer program, in both genomes, we have identified candidate oriC regions that have almost all sequence hallmarks of bacterial oriCs, such as asymmetrical nucleotide distributions, being adjacent to the dnaN gene, and containing DnaA boxes and repeat elements.     

Heterologous production of epothilone C and D in Escherichia coli

The epothilones are a family of polyketide natural products that show a high potential as anticancer drugs. They are synthesized by the action of a hybrid nonribosomal peptide synthetase/polyketide synthase in the myxobacterium Sorangium cellulosum. In this work, the genes encoding the entire cluster,epoA, epoB, epoC, epoD, epoE, and epoF, were redesigned and synthesized to allow for expression in Escherichia coli. The expression of the largest of the proteins, EpoD, also required the protein be separated into two polypeptides with compatible module linkers. Using a combination of lowered temperature, chaperone coexpression, and alternative promoters, we succeeded in producing a soluble protein from all genes in the epothilone cluster. The entire synthetic epothilone cluster was then expressed in a strain of E. coli modified to enable polyketide biosynthesis, resulting in the production of epothilones C and D. Furthermore, feeding a thioester of the normal substrate for EpoD to cells expressing the epoD, epoE, and epoF genes also led to the production of epothilones C and D. The design of the synthetic epothilone genes together with E. coli expression provides the ideal platform for both the biochemical investigation of the epothilone PKS and the generation of novel biosynthetic epothilone analogues.     

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.     

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。