Science China Life Sciences

, Volume 60, Issue 9, pp 968–979 | Cite as

Reconstruction of a hybrid nucleoside antibiotic gene cluster based on scarless modification of large DNA fragments

  • Jiming Zhuo
  • Binbin Ma
  • Jingjing Xu
  • Weihong Hu
  • Jihui Zhang
  • Huarong TanEmail author
  • Yuqing TianEmail author
Research Paper


Genetic modification of large DNA fragments (gene clusters) is of great importance in synthetic biology and combinatorial biosynthesis as it facilitates rational design and modification of natural products to increase their value and productivity. In this study, we developed a method for scarless and precise modification of large gene clusters by using RecET/RED-mediated polymerase chain reaction (PCR) targeting combined with Gibson assembly. In this strategy, the biosynthetic genes for peptidyl moieties (HPHT) in the nikkomycin biosynthetic gene cluster were replaced with those for carbamoylpolyoxamic acid (CPOAA) from the polyoxin biosynthetic gene cluster to generate a ~40 kb hybrid gene cluster in Escherichia coli with a reusable targeting cassette. The reconstructed cluster was introduced into Streptomyces lividans TK23 for heterologous expression and the expected hybrid antibiotic, polynik A, was obtained and verified. This study provides an efficient strategy for gene cluster reconstruction and modification that could be applied in synthetic biology and combinatory biosynthesis to synthesize novel bioactive metabolites or to improve antibiotic production.


large DNA fragment PCR targeting Gibson assembly gene cluster hybrid antibiotic 


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This work was supported by grants from the Ministry of Science and Technology of China (2013CB734001 and 2015CB150600) and the National Natural Science Foundation of China (31370097 and 31571281).


  1. Baltz, R.H. (2009). Biosynthesis and genetic engineering of lipopeptides in Streptomyces roseosporus. Methods Enzymol 458, 511-531.CrossRefPubMedGoogle Scholar
  2. Baltz, R.H. (2013). Combinatorial biosynthesis of cyclic lipopeptide antibiotics: a model for synthetic biology to accelerate the evolution of secondary metabolite biosynthetic pathways. ACS Synth Biol 3, 748-758.CrossRefGoogle Scholar
  3. Bierman, M., Logan, R., O’Brien, K., Seno, E.T., Nagaraja Rao, R., and Schoner, B.E. (1992). Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp.. Gene 116, 43–49.CrossRefPubMedGoogle Scholar
  4. Bormann, C., Huhn, W., Zahner, H., Rathmann, R., Hahn, H., and Konig, W.A. (1985). Metabolic products of microorganisms.228.New nikkomycins produced by mutants of Streptomyces tendae. J Antibiot 38, 9–16.CrossRefPubMedGoogle Scholar
  5. Bormann, C., Mattern, S., Schrempf, H., Fiedler, H.P., and Zähner, H. (1989). Isolation of Streptomyces tendae mutants with an altered nikkomycin spectrum. J Antibiot 42, 913–918.CrossRefPubMedGoogle Scholar
  6. Bruntner, C., Lauer, B., Schwarz, W., Mohrle, V., and Bormann, C. (1999). Molecular characterization of co-transcribed genes from Streptomyces tendae Tu901 involved in the biosynthesis of the peptidyl moiety of the peptidyl nucleoside antibiotic nikkomycin. Mol Gen Genet 262, 102–114.PubMedGoogle Scholar
  7. Chao, R., Yuan, Y.B., and Zhao, H.M. (2015). Building biological foundries for next-generation synthetic biology. Sci China Life Sci 58, 658–665.CrossRefPubMedGoogle Scholar
  8. Chen, H., Hubbard, B.K., O’Connor, S.E., and Walsh, C.T. (2002). Formation of ß-hydroxy histidine in the biosynthesis of nikkomycin antibiotics. Chem Biol 9, 103–112.CrossRefPubMedGoogle Scholar
  9. Chen, W., Zeng, H., and Tan, H. (2000). Cloning, sequencing, and function of sanF: a gene involved in nikkomycin biosynthesis of Streptomyces ansochromogenes. Curr Microbiol 41, 312–316.CrossRefPubMedGoogle Scholar
  10. Datsenko, K.A., and Wanner, B.L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97, 6640–6645.CrossRefPubMedPubMedCentralGoogle Scholar
  11. Decker, H., Zähner, H., Heitsch, H., König, W.A., and Fiedler, H.P. (1991). Structure-activity relationships of the nikkomycins. J General Microbiol 137, 1805–1813.CrossRefGoogle Scholar
  12. Delzer, J., Fiedler, H.P., MÜLler, H., Zähner, H., Rathmann, R., Ernst, K., and König, W.A. (1984). New nikkomycins by mutasynthesis and directed fermentation. J Antibiot 37, 80–82.CrossRefPubMedGoogle Scholar
  13. Du, D., Wang, L., Tian, Y., Liu, H., Tan, H., and Niu, G. (2015). Genome engineering and direct cloning of antibiotic gene clusters via phage BT1 integrase-mediated site-specific recombination in Streptomyces. Sci Rep 5, 8740.CrossRefPubMedPubMedCentralGoogle Scholar
  14. Fiedler, H.P. (1984). Screening for new microbial products by high-performance liquid chromatography using a photodiode array detector. J Chromatography A 316, 487–494.CrossRefGoogle Scholar
  15. Fu, J., Bian, X., Hu, S., Wang, H., Huang, F., Seibert, P.M., Plaza, A., Xia, L., Müller, R., Stewart, A.F., and Zhang, Y. (2012). Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting. Nat Biotechnol 30, 440–446.CrossRefPubMedGoogle Scholar
  16. Funayama, S., and Isono, K. (1977). Biosynthesis of the polyoxins, nucleoside peptide antibiotics: biosynthetic pathway for 5-O-carbamoyl-2- amino-2-deoxy-L-xylonic acid (carbamoylpolyoxamic acid). Biochemistry 16, 3121–3127.CrossRefPubMedGoogle Scholar
  17. Gibson, D.G., Young, L., Chuang, R.Y., Venter, J.C., Hutchison, C.A., and Smith, H.O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Meth 6, 343–345.CrossRefGoogle Scholar
  18. Gust, B., Challis, G.L., Fowler, K., Kieser, T., and Chater, K.F. (2003). PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc Natl Acad Sci USA 100, 1541–1546.CrossRefPubMedPubMedCentralGoogle Scholar
  19. Isono, K. (1988). Nucleoside antibiotics: Structure, biological activity, and biosynthesis.. J Antibiot 41, 1711–1739.CrossRefPubMedGoogle Scholar
  20. Isono, K., Asahi, K., and Suzuki, S. (1969). Studies on polyoxins, antifungal antibiotics. 13. The structure of polyoxins. J Am Chem Soc 91, 7490–7505.CrossRefPubMedGoogle Scholar
  21. Isono, K., and Suzuki, S. (1968). The structures of polyoxins A and B. Tetrahedron Lett 9, 1133–1137.CrossRefPubMedGoogle Scholar
  22. Jiang, W., Zhao, X., Gabrieli, T., Lou, C., Ebenstein, Y., and Zhu, T.F. (2015). Cas9-assisted targeting of chromosome segments CATCH enables onestep targeted cloning of large gene clusters. Nat Commun 6, 8101.CrossRefPubMedPubMedCentralGoogle Scholar
  23. Kao, C.M., Katz, L., and Khosla, C. (1994). Engineered biosynthesis of a complete macrolactone in a heterologous host. Science 265, 509–512.CrossRefPubMedGoogle Scholar
  24. Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F., and Hopwood, D.A. (2000). Practical Streptomyces Genetics. (Norwich, UK: John Innes Foundation).Google Scholar
  25. Li, J., Li, L., Tian, Y., Niu, G., and Tan, H. (2011). Hybrid antibiotics with the nikkomycin nucleoside and polyoxin peptidyl moieties. Metab Eng 13, 336–344.CrossRefPubMedGoogle Scholar
  26. Li, R., Xie, Z., Tian, Y., Yang, H., Chen, W., You, D., Liu, G., Deng, Z., and Tan, H. (2009). polR, a pathway-specific transcriptional regulatory gene, positively controls polyoxin biosynthesis in Streptomyces cacaoi subsp. asoensis. Microbiology 155, 1819–1831.CrossRefPubMedGoogle Scholar
  27. Liao, G., Li, J., Li, L., Yang, H., Tian, Y., and Tan, H. (2009). Selectively improving nikkomycin Z production by blocking the imidazolone biosynthetic pathway of nikkomycin X and uracil feeding in Streptomyces ansochromogenes. Microb Cell Fact 8, 61.CrossRefPubMedPubMedCentralGoogle Scholar
  28. Liao, G., Li, J., Li, L., Yang, H., Tian, Y., and Tan, H. (2010). Cloning, reassembling and integration of the entire nikkomycin biosynthetic gene cluster into Streptomyces ansochromogenes lead to an improved nikkomycin production. Microb Cell Fact 9, 6.CrossRefPubMedPubMedCentralGoogle Scholar
  29. Liu, G., Tian, Y., Yang, H., and Tan, H. (2005). A pathway-specific transcriptional regulatory gene for nikkomycin biosynthesis inStreptomyces ansochromogenes that also influences colony development. Mol Microbiol 55, 1855–1866.CrossRefPubMedGoogle Scholar
  30. MacNeil, D.J., Gewain, K.M., Ruby, C.L., Dezeny, G., Gibbons, P.H., and MacNeil, T. (1992). Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene 111, 61–68.CrossRefPubMedGoogle Scholar
  31. Miao, V., Coëffet-Legal, M.F., Brian, P., Brost, R., Penn, J., Whiting, A., Martin, S., Ford, R., Parr, I., Bouchard, M., Silva, C.J., Wrigley, S.K., and Baltz, R.H. (2005). Daptomycin biosynthesis in Streptomyces roseosporus: cloning and analysis of the gene cluster and revision of peptide stereochemistry. Microbiology 151, 1507–1523.CrossRefPubMedGoogle Scholar
  32. Murakami, T., Burian, J., Yanai, K., Bibb, M.J., and Thompson, C.J. (2011). A system for the targeted amplification of bacterial gene clusters multiplies antibiotic yield in Streptomyces coelicolor. Proc Natl Acad Sci USA 108, 16020–16025.CrossRefPubMedPubMedCentralGoogle Scholar
  33. Niu, G., and Tan, H. (2015). Nucleoside antibiotics: biosynthesis, regulation, and biotechnology. Trends Microbiol 23, 110–119.CrossRefPubMedGoogle Scholar
  34. Nguyen, K.T., Ritz, D., Gu, J.Q., Alexander, D., Chu, M., Miao, V., Brian, P., and Baltz, R.H. (2006). Combinatorial biosynthesis of novel antibiotics related to daptomycin. Proc Natl Acad Sci USA 103, 17462–17467.CrossRefPubMedPubMedCentralGoogle Scholar
  35. Paget, M.S., Chamberlin, L., Atrih, A., Foster, S.J., and Buttner, M.J. (1999). Evidence that the extracytoplasmic function sigma factor sigmaE is required for normal cell wall structure in Streptomyces coelicolor A3(2). J Bacteriol 181, 204–211.PubMedPubMedCentralGoogle Scholar
  36. Pullan, S.T., Chandra, G., Bibb, M.J., and Merrick, M. (2011). Genome-wide analysis of the role of GlnR in Streptomyces venezuelae provides new insights into global nitrogen regulation in actinomycetes. BMC Genomics 12, 175.CrossRefPubMedPubMedCentralGoogle Scholar
  37. Sambrook, J., and Russell, D.W. (2001). Molecular Cloning: A Laboratory Manual, 3rd ed. (NY: Cold Spring Harbor Laboratory Press).Google Scholar
  38. Shao, Z., Zhao, H., and Zhao, H. (2009). DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Res 37, e16–e16.CrossRefGoogle Scholar
  39. Suzuki, S., Isono, K., Nagatsu, J., Mizutani, T., Kawashima, Y., and Mizuno, T. (1965). A new antibiotic, polyoxin A. J Antibiot 18, 131.PubMedGoogle Scholar
  40. Tsvetanova, B., Peng, L., Liang, X., Li, K., Yang, J., Ho, T., Shirley, J., Xu, L., Potter, J., Kudlicki, W., Peterson, T., and Katzen, F. (2011). Genetic assembly tools for synthetic biology. Methods Enzymol 498, 327–348.CrossRefPubMedGoogle Scholar
  41. Zhang, L., Wang, L., Wang, J., Ou, X., Zhao, G., and Ding, X. (2010). DNA cleavage is independent of synapsis during Streptomyces phage φBT1 integrase-mediated site-specific recombination. J Mol Cell Biol 2, 264–275.CrossRefPubMedGoogle Scholar

Copyright information

© Science China Press and Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.State Key Laboratory of Microbial Resources, Institute of MicrobiologyChinese Academy of SciencesBeijingChina
  2. 2.University of Chinese Academy of SciencesBeijingChina
  3. 3.School of Life SciencesYunnan UniversityKunmingChina

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