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The cloning of microbial natural product biosynthetic gene clusters and their heterologous expression in a suitable host have proven to be a feasible approach to improve the yield of valuable natural products and to begin mining cryptic natural products in microorganisms. Myxobacteria are a prolific source of novel bioactive natural products with only limited choices of heterologous hosts that have been exploited. Here, we describe the use of Burkholderiales strain DSM 7029 as a potential heterologous host for the functional expression of myxobacterial secondary metabolites. Using a newly established electroporation procedure, the 56 kb epothilone biosynthetic gene cluster from the myxobacterium Sorangium cellulosum was introduced into the chromosome of strain DSM 7029 by transposition. Production of epothilones A, B, C, and D was detected despite their yields being low. Optimization of the medium, introduction of the exogenous methylmalonyl-CoA biosynthetic pathway, and overexpression of rare tRNA genes resulted in an approximately 75-fold increase in the total yields of epothilones to 307 μg L

Genome sequencing of cellulolytic myxobacterium Sorangium cellulosum reveals many open-reading frames (ORFs) encoding various degradation enzymes with low sequence similarity to those reported, but none of them has been characterized. In this paper, a predicted lipase gene (lipA) was cloned from S. cellulosum strain So0157-2 and characterized. lipA is 981-bp in size, encoding a polypeptide of 326 amino acids that contains the pentapeptide (GHSMG) and catalytic triad residues (Ser114, Asp250 and His284). Searching in the GenBank database shows that the LipA protein has only the 30% maximal identity to a human monoglyceride lipase. The novel lipA gene was expressed in Escherichia coli BL21 and the recombinant protein (r-LipA) was purified using Ni-NTA affinity chromatography. The enzyme hydrolyzed the p-nitrophenyl (pNP) esters of short or medium chain fatty acids (≤C(10)), and the maximal activity was on pNP acetate. The r- LipA is a cold-adapted lipase, with high enzymatic activity in a wide range of temperature and pH values. At 4 °C and 30 °C, the K(m) values of r-LipA on pNP acetate are 0.037 ± 0.001 and 0.174 ± 0.006 mM, respectively. Higher pH and temperature conditions promoted hydrolytic activity toward the pNP esters with longer chain fatty acids. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents. The results suggest that the r-LipA protein has some new characteristics potentially promising for industrial applications and S. cellulosum is an intriguing resource for lipase screening.

The anticancer compound epothilone B is biosynthesized by the myxobacteriumSorangium cellulosum; however, the fermentation characteristics for epothilone production in Sorangium cellulosum have not yet been reported. In this study, medium components for the production of epothilone B in the So0157-2 strain of S. cellulosum were statistically screened and optimized. First, the nutrients in the fermentation medium were optimized in one-factor-at-a-time, Plackett–Burman design, and Box–Behnken design experiments. Afterwards, three nutritional parameters were selected for the optimization of epothilone B production in shaking flask cultures using a central composite response surface methodology design; a polynomial equation model that related the medium components and epothilone B yield was established. The data were further analyzed using response surface plots and canonical mathematical model analyses with the SAS 8.0 software. After optimized, the yield of epothilone B increased to 82.0 ± 3 mg/l, 7.2-fold higher than the initial yield (11.3 ± 0.4 mg/l). Key words: Myxobacterium, Sorangium cellulosum, epothilone B, statistical optimization, response surface methodology.

To investigate the characters ofSorangiumstrains and the approaches for improving the production of epothilone, correlation of epothilone production and morphogenesis was studied in strains So0157-2, So0007-3andSo0003-3. On lean medium, So0157-2 could produce epothilones in a wide pH range (5.0-12.0), but formed fruiting bodies at high pH values (9.0-14.0). Formation of fruiting body did not affect the yield of epothilones in So0157-2. In So0007-3 and So0003-3, producing of epothilone was strongly coupled with the process of morphogenesis. It suggests that reduction of the association between morphogenesis and epothilone producing would prominently improve the epothilone production.

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 microg/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.

Aim To study the chemical constituents of Sorangium cellulosum So0157-2. Methods The chemical constituents were isolated and purified by column chromatography, and their structures were identified by spectroscopic analyses including 1D-, 2D-NMR data and MS analyses. Results Two epothilones were purified and identified as seco-epothilone A (1) and 1-methyl-seco-epothilone A (2). Conclusion 1 and 2 were obtained from this strain for the first time, and 1 was a new natural product.

Complex environmental conditions can significantly affect bacterial genome size by unknown mechanisms. The So0157-2 strain of Sorangium cellulosum is an alkaline-adaptive epothilone producer that grows across a wide pH range. Here, we show that the genome of this strain is 14,782,125 base pairs, 1.75-megabases larger than the largest bacterial genome from S. cellulosum reported previously. The total 11,599 coding sequences (CDSs) include massive duplications and horizontally transferred genes, regulated by lots of protein kinases, sigma factors and related transcriptional regulation co-factors, providing the So0157-2 strain abundant resources and flexibility for ecological adaptation. The comparative transcriptomics approach, which detected 90.7% of the total CDSs, not only demonstrates complex expression patterns under varying environmental conditions but also suggests an alkaline-improved pathway of the insertion and duplication, which has been genetically testified, in this strain. These results provide insights into and a paradigm for how environmental conditions can affect bacterial genome expansion.

Sorangium cellulosum, a cellulolytic myxobacterium, is capable of producing a variety of bioactive secondary metabolites. Epothilones are anti-eukaryotic secondary metabolites produced by some S. cellulosum strains. In this study, we analyzed interactions between 12 strains of S. cellulosum consisting of epothilone-producers and non-epothilone producers isolated from two distinct soil habitats. Co-cultivation on filter papers showed that different Sorangium strains inhibited one another's growth, whereas epothilone production by the producing strains changed markedly for most (73%) pairwise mixtures. Using a quantitative polymerase chain reaction, we demonstrated that the expression of epothilone biosynthetic genes in the epothilone producers typically changed significantly when these bacteria were mixed with non-producing strains. The results indicated that intraspecies interactions between different S. cellulosum strains not only inhibited the growth of partners, but also could change epothilone production.

The epothilones, compounds with anticancer mechanisms similar to that of paclitaxel (Taxol), are produced by strains of the myxobacterium Sorangium cellulosum, and the gene cluster responsible for epothilone biosynthesis is organised as a large operon. In this work, we showed that the 440-bp promoter regions of the operons from eight S. cellulosum strains have 94.27 % DNA sequence identity and 50 % variability in promoter activity in Escherichia coli. A primer extension analysis revealed two transcriptional start sites (TSSs) at 246 (TSS1) and 193 bp (TSS2) upstream of the translation start site (TLS), respectively. Promoter truncation determined that the basal promoter from the So0157-2 strain is located within a 264-bp region containing weak promoter activity; whereas in the 38-bp region upstream, the 264-bp promoter was required for the strong promoter activity, which was dramatically increased by 11-fold in average. There was a conserved stem-loop structure between TSS2 and the TLS, which was identified in E. coli as a negative regulatory element. In addition, the upstream non-conserved 357-bp non-coding region contributes to the promoter activity, increasing it by 1.5-fold. In conclusion, the expression of the epothilone operon non-coding region in E. coli is regulated by a double promoter (with -35 and -10 regions and two distinct TSSs), a stem-loop structure, and a distal non-coding region.

The cellulolytic myxobacterium Sorangium cellulosum is able to efficiently degrade many kinds of polysaccharides, but none of the enzymes involved have been characterized. In this paper, a xylanase gene (xynA) was cloned from S. cellulosum So9733-1 using thermal asymmetric interlaced PCR. The gene is composed of 1,209 bp and has only 52.27% G + C content, which is much lower than that of most myxobacterial DNA reported (67-72%). Gene xynA encodes a 402 amino acid protein that contains a single catalytic domain belonging to the glycoside hydrolase family 10. The novel xylanase gene, xynA, was expressed in Escherichia coli BL21 (DE3) and the recombinant protein (r-XynA) was purified by Ni-affinity chromatography. The r-XynA had the optimum temperature of 30-35°C and exhibited 33.3% activity at 5°C and 13.7% activity at 0°C. Approximately 80% activity was lost after 20-min pre-incubation at 50°C. These results indicate that r-XynA is a cold-active xylanase with low thermostability. At 30°C, the K (m) values of r-XynA on beechwood xylan, birchwood xylan, and oat spelt xylan were 25.77 ± 4.16, 26.52 ± 4.78, and 38.13 ± 5.35 mg/mL, respectively. The purified r-XynA displayed optimum activity at pH 7.0. The activity of r-XynA was enhanced by the presence of Ca(2+). The r-XynA hydrolyzed beechwood xylan, birchwood xylan, and xylooligosaccharides (xylotriose, xylotetraose, and xylopentose) to produce primarily xylose and xylobiose. To our knowledge, this is the first report on the characterization of a xylanase from S. cellulosum.

3-O-alpha-D-ribofuranosyl epothilone A (epothiloneoside A) is a major component of glycosylated epothilones in Sorangium cellulosum strain So0157-2. The production and glycosylation ratios of epothiloneoside A in both solid and liquid culture conditions with various pH values and carbon sources were studied. The results showed that glycosylation occurs whenever epothilones are produced, regardless of changes in pH values, production time curves, and different carbon sources. We suggest that glycosylation is a stable process, paralleling the biosynthesis of epothilones in the So0157-2 strain.

The taxonomy of myxobacteria is based mainly on their morphological characteristics. The genus Sorangium belongs to the myxobacterial suborder Sorangiineae. Strains in the genus were classified either as one species, Sorangium cellulosum, by ignoring divergent morphological characteristics, or into several species; however, the latter classification is based on some dubious morphological characteristics and is inconsistent with the phylogeny constructed from 16S rRNA gene sequences. In this study, two HSP60 (groEL1 and groEL2) genes were amplified and sequenced from 22 Sorangium strains. The groEL1 and groEL2 gene sequences were highly conserved in Sorangium strains, suggesting that these two paralogous genes both play important roles in the life cycle. The phylogeny constructed by the groEL genes was rather consistent with the morphological characteristics of sporangioles. Including information from the phylogenetic analysis and morphological characteristics, it is suggested that the genus Sorangium includes two species.

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.

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).