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Characteristics and activity analysis of epothilone operon promoters from Sorangium cellulosum strains in Escherichia coli

  • Applied genetics and molecular biotechnology
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Abstract

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.

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References

  • Ayers DG, Auble DT, deHaseth PL (1989) Promoter recognition by Escherichia coli RNA polymerase. Role of the spacer DNA in functional complex formation. J Mol Biol 207(4):749–756. doi:10.1016/0022-2836(89)90241-6

    Article  PubMed  CAS  Google Scholar 

  • Bollag DM, McQueney PA, Zhu J, Hensens O, Koupal L, Liesch J, Goetz M, Lazarides E, Woods CM (1995) Epothilones, a new class of microtubule-stabilizing agents with a taxol-like mechanism of action. Cancer Res 55(11):2325–2333

    PubMed  CAS  Google Scholar 

  • Brodskii LI, Ivanov VV, Kalaidzidis YL, Leontovich AM, Nikolaev VK, Feranchuk SI, Drachev VA (1995) GeneBee-NET: internet based server for biopolymer structure analysis. Biochem Moscow 60(8):923–928

    Google Scholar 

  • Brosius J (1984) Plasmid vectors for the selection of promoters. Gene 27(2):151–160. doi:10.1016/0378-1119(84):90136-90137

    Article  PubMed  CAS  Google Scholar 

  • Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD (2003) Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res 31(13):3497–3500. doi:10.1093/nar/gkg500

    Article  PubMed  CAS  Google Scholar 

  • Davidson RC, Blankenship JR, Kraus PR, de Jesus BM, Hull CM, D'Souza C, Wang P, Heitman J (2002) A PCR-based strategy to generate integrative targeting alleles with large regions of homology. Microbiology 148(Pt 8):2607–2615

    PubMed  CAS  Google Scholar 

  • Dong H, Li YZ, Hu W (2004) Analysis of purified tubulin in high concentration of glutamate for application in high throughput screening for microtubule-stabilizing agents. Assay Drug Dev Technol 2(6):621–628. doi:10.1089/adt.2004.2.621

    Article  PubMed  CAS  Google Scholar 

  • Fu J, Wenzel S, Perlova O, Wang J, Gross F, Tang Z, Yin Y, Stewart A, Müller R, Zhang Y (2008) Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition. Nucleic Acids Res 36(17):e113. doi:10.1093/nar/gkn499

    Article  PubMed  Google Scholar 

  • Gerth K, Bedorf N, Höfle G, Irschik H, Reichenbach H (1996) Epothilons A and B: antifungal and cytotoxic compounds from Sorangium cellulosum (Myxobacteria). Production, physico-chemical and biological properties. J Antibiot (Tokyo) 49(6):560–563

    Article  CAS  Google Scholar 

  • Gong GL, Sun X, Liu XL, Hu W, Cao WR, Liu H, Liu WF, Li YZ (2007) Mutation and a high-throughput screening method for improving the production of Epothilones of Sorangium. J Ind Microbiol Biotechnol 34(9):615–623. doi:10.1007/s10295-007-0236-2

    Article  PubMed  CAS  Google Scholar 

  • Harley CB, Reynolds RP (1987) Analysis of E. coli promoter sequences. Nucleic Acids Res 15(5):2343–2361

    Article  PubMed  CAS  Google Scholar 

  • Hawley DK, McClure WR (1983) Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res 11(8):2237–2255. doi:10.1093/nar/11.8.2237

    Article  PubMed  CAS  Google Scholar 

  • Hu W, Dong H, Li YZ, Hu XT, Han GJ, Qu YB (2004) A high-throughput model for screening anti-tumor agents capable of promoting polymerization of tubulin in vitro. Acta Pharmacol Sin 25(6):775–782

    PubMed  CAS  Google Scholar 

  • Julien B, Shah S (2002) Heterologous expression of epothilone biosynthetic genes in Myxococcus xanthus. Antimicrob Agents Chemother 46(9):2772–2778. doi:10.1128/AAC.46.9.2772-2778.2002

    Article  PubMed  CAS  Google Scholar 

  • Julien B, Shah S, Ziermann R, Goldman R, Katz L, Khosla C (2000) Isolation and characterization of the epothilone biosynthetic gene cluster from Sorangium cellulosum. Gene 249(1–2):153–160. doi:10.1016/S0378-1119(00)00149-9

    Article  PubMed  CAS  Google Scholar 

  • Kowalski RJ, Giannakakou P, Hamel E (1997) Activities of the microtubule-stabilizing agents epothilones A and B with purified tubulin and in cells resistant to paclitaxel (Taxol®). J Biol Chem 272(4):2534–2541. doi:10.1074/jbc.272.4.2534

    Article  PubMed  CAS  Google Scholar 

  • Lau J, Frykman S, Regentin R, Ou S, Tsuruta H, Licari P (2002) Optimizing the heterologous production of epothilone D in Myxococcus xanthus. Biotechnol Bioeng 78(3):280–288. doi:10.1002/bit.10202

    Article  PubMed  CAS  Google Scholar 

  • Lesnik EA, Sampath R, Levene HB, Hendersor TJ, McNeil JA, Ecker DJ (2001) Prediction of rho-independent transcriptional terminators in Escherichia coli. Nucleic Acids Res 29(17):3583–3594. doi:10.1093/nar/29.17.3583

    Article  PubMed  CAS  Google Scholar 

  • Li ZF, Zhao JY, Xia ZJ, Shi J, Liu H, Wu ZH, Hu W, Liu WF, Li YZ (2007) Evolutionary diversity of ketoacyl synthases in cellulolytic myxobacterium Sorangium. Syst Appl Microbiol 30(3):189–196. doi:10.1016/j.syapm.2006.06.002

    Article  PubMed  Google Scholar 

  • Molnár I, Schupp T, Ono M, Zirkle R, Milnamow M, Nowak-Thompson B, Engel N, Toupet C, Stratmann A, Cyr DD, Gorlach J, Mayo JM, Hu A, Goff S, Schmid J, Ligon JM (2000) The biosynthetic gene cluster for the microtubule-stabilizing agents epothilones A and B from Sorangium cellulosum So ce90. Chem Biol 7(2):97–109. doi:10.1016/S1074-5521(00)00075-2

    Article  PubMed  Google Scholar 

  • Müller R (2009) Biosynthesis and heterologous production of epothilones. Fortschr Chem Org Naturst 90:29–53. doi:10.1007/978-3-211-78207-1_2

    Article  PubMed  Google Scholar 

  • Mulligan ME, Brosius J, McClure WR (1985) Characterization in vitro of the effect of spacer length on the activity of Escherichia coli RNA polymerase at the TAC promoter. J Biol Chem 260(6):3529–3538

    PubMed  CAS  Google Scholar 

  • Mutka SC, Carney JR, Liu Y, Kennedy J (2006) Heterologous production of epothilone C and D in Escherichia coli. Biochemistry 45(4):1321–1330. doi:10.1021/bi052075r

    Article  PubMed  CAS  Google Scholar 

  • Ni X, Westpheling J (1997) Direct repeat sequences in the Streptomyces chitinase-63 promoter direct both glucose repression and chitin induction. Proc Natl Acad Sci U S A 94(24):13116–13121. doi:10.1073/pnas.94.24.13116

    Article  PubMed  CAS  Google Scholar 

  • Ormen O, Regue MQ, Tomas JM, Granum PE (2003) Studies of aerolysin promoters from different Aeromonas spp. Microb Pathog 35(5):189–196. doi:10.1016/S0882-4010(03)00124-4

    Article  PubMed  CAS  Google Scholar 

  • Park SR, Park JW, Jung WS, Han AR, Ban YH, Kim EJ, Sohng JK, Sim SJ, Yoon YJ (2008) Heterologous production of epothilones B and D in Streptomyces venezuelae. Appl Microbiol Biotechnol 81(1):109–117. doi:10.1007/s00253-008-1674-0

    Article  PubMed  CAS  Google Scholar 

  • Reese MG (2001) Application of a time-delay neural network to promoter annotation in the Drosophila melanogaster genome. Comput Chem 26(1):51–56. doi:10.1016/S0097-8485(01)00099-7

    Article  PubMed  CAS  Google Scholar 

  • Reichenbach H, Dworkin M (1992) The myxobacteria. In: Balows A, Trüper HG, Dworkin M, Harder W, Schleifer KH (eds) The prokaryotes. 2nd edn. Springer, Berlin Heidelberg New York, pp 3416–3487

    Google Scholar 

  • Stefano JE, Gralla JD (1982) Spacer mutations in the lac ps promoter. Proc Natl Acad Sci USA 79(4):1069–1072. doi:10.1073/pnas.79.4.1069

    Article  PubMed  CAS  Google Scholar 

  • Su D-S, Balog A, Meng D, Bertinato P, Danishefsky SJ, Zheng Y-H, Chou T-C, He L, Horwitz SB (1997) Structure–activity relationship of the epothilones and the first in vivo comparison with paclitaxel. Angw Chem 36(19):2093–2096. doi:10.1002/anie.199720931

    Article  CAS  Google Scholar 

  • Tang L, Shah S, Chung L, Carney J, Katz L, Khosla C, Julien B (2000) Cloning and heterologous expression of the epothilone gene cluster. Science 287(5453):640–642. doi:10.1126/science.287.5453.640

    Article  PubMed  CAS  Google Scholar 

  • Tu Y, Chen GP, Wang YL (2007) Autonomously replicating plasmid transforms Sorangium cellulosum So ce90 and induces expression of green fluorescent protein. J Biosci Bioeng 104(5):385–390. doi:10.1263/jbb.104.385

    Article  PubMed  CAS  Google Scholar 

  • Warne SE, deHaseth PL (1993) Promoter recognition by Escherichia coli RNA polymerase: Effects of single base pair deletions and insertions in the spacer DNA separating the −10 and −35 regions are dependent on spacer DNA sequence. Biochemistry 32(24):6134–6140. doi:10.1021/bi9813431

    Article  PubMed  CAS  Google Scholar 

  • Xia ZJ, Wang J, Hu W, Liu H, Gao XZ, Wu ZH, Zhang PY, Li YZ (2008) Improving conjugation efficacy of Sorangium cellulosum by the addition of dual selection antibiotics. J Ind Microbiol Biotechnol 35(10):1157–1163. doi:10.1007/s10295-008-0395-9

    Article  PubMed  CAS  Google Scholar 

  • Yan ZC, Wang B, Li YZ, Gong X, Zhang HQ, Gao PJ (2003) Morphologies and phylogenetic classification of cellulolytic myxobacteria. Syst Appl Microbiol 26(1):104–109. doi:10.1078/072320203322337380

    Article  PubMed  Google Scholar 

  • Zor T, Selinger Z (1996) Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Anal Biochem 236(2):302–308. doi:10.1006/abio.1996.0171

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was financially supported by the National Natural Science Foundation (grant nos. 30900027 and 81128016) of China and China Postdoctoral Science Foundation (grant no. 2012M521323). The funding agencies had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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  1. State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, 250100, China

    Li-Ping Zhu, Zhi-Feng Li, Xin Sun, Shu-Guang Li & Yue-Zhong Li

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  1. Li-Ping Zhu
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  2. Zhi-Feng Li
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Correspondence to Zhi-Feng Li.

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Zhu, LP., Li, ZF., Sun, X. et al. Characteristics and activity analysis of epothilone operon promoters from Sorangium cellulosum strains in Escherichia coli . Appl Microbiol Biotechnol 97, 6857–6866 (2013). https://doi.org/10.1007/s00253-013-4830-0

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