Abstract
Saccharopolyspora spinosa produces tetra-cyclic macrolide spinosyns, a group of highly efficient pesticidal agents. However, this species lacks efficient vectors for genetic manipulation. In this study, the circular plasmid pCM32 was newly isolated from Saccharopolyspora endophytica YIM 61095. The complete nucleotide sequence of pCM32 consists of 14,611 bp and is predicted to encode 17 open reading frames (ORFs). Interestingly, a putative int gene in pCM32 was predicted by homologous alignment to encode an integrase belonging to the tyrosine family of integrases/recombinases. Plasmid pCM238 containing this int locus derived from pCM32 could be transferred by conjugation from Escherichia coli into Sa. spinosa at a high frequency. Integration of pCM238 in the host chromosome was demonstrated as site-specific recombination (at the tRNA Ser gene) via a 56-bp core sequence within the attP/attB sites. Plasmid pCM265, a shuttle vector containing the int and attP sequences of pCM32, was constructed to introduce foreign genes into Sa. spinosa. The production of spinosad approximately doubled in Sa. spinosa NRRL18395 after introducing pCM265-derived plasmids carrying the genes for phosphofructokinase (PFK) or anthranilate synthase. These results indicate that plasmid pCM32 is an actinomycete integrative and conjugative element (AICE) and that its derived integrative vectors are useful for efficiently introducing foreign DNA into Sa. spinosa.
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References
Alves AM, Euverink GJ, Bibb MJ, Dijkhuizen L (1997) Identification of ATP-dependent phosphofructokinase as a regulatory step in the glycolytic pathway of the actinomycete Streptomyces coelicolor A3(2). Appl Environ Microbiol 63:956–961
Bierman M, Logan R, O’Brien K, Seno ET, Rao RN, Schoner BE (1992) Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 116:43–49
Boeck LD, Chio H, Eaton TE, Godfrey OW, Michel KH, Nakatsukasa WM, Yao RC (1994) Process for producing A83543 compounds. US Patent 5362634
Borodina I, Krabben P, Nielsen J (2005) Genome-scale analysis of Streptomyces coelicolor A3(2) metabolism. Genome Res 15:820–829
Borodina I, Siebring J, Zhang J, Smith CP, van Keulen G, Dijkhuizen L, Nielsen J (2008) Antibiotic overproduction in Streptomyces coelicolor A3 (2) mediated by phosphofructokinase deletion. J Biol Chem 283:25186–25199
Esposito D, Scocca JJ (1997) The integrase family of tyrosine recombinases: evolution of a conserved active site domain. Nucleic Acids Res 25:3605–3614
Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95–98
Hosted TJ, Wang T, Horan AC (2005) Characterization of the Micromonospora rosaria pMR2 plasmid and development of a high G+C codon optimized integrase for site-specific integration. Plasmid 54:249–258
Kieser T, Bibb MJ, Buttler MJ, Chater KC, Hopwood DA (2000) Practical Streptomyces genetics. John Innes Foundation, Norwich
Kirst HA (2010) The spinosyn family of insecticides: realizing the potential of natural products research. J Antibiot (Tokyo) 63:101–111
Li J, Zhao GZ, Qin S, Huang HY, Zhu WY, Xu LH, Li WJ (2009) Saccharopolyspora tripterygii sp. nov., an endophytic actinomycete isolated from the stem of Tripterygium hypoglaucum. Int J Syst Evol Microbiol 59:3040–3044
Lin C, Paradkar AS, Vining LC (1998) Regulation of an anthranilate synthase gene in Streptomyces venezuelae by a trp attenuator. Microbiology 144:1971–1980
Luo Y, Ding X, Xia L, Huang F, Li W, Huang S, Tang Y, Sun Y (2011) Comparative proteomic analysis of Saccharopolyspora spinosa SP06081 and PR2 strains reveals the differentially expressed proteins correlated with the increase of spinosad yield. Proteome Sci 9:40
Matsushima P, Baltz RH (1994) Transformation of Saccharopolyspora spinosa protoplasts with plasmid DNA modified in vitro to avoid host restriction. Microbiology 140:139–143
Matsushima P, Broughton MC, Turner JR, Baltz RH (1994) Conjugal transfer of cosmid DNA from Escherichia coli to Saccharopolyspora spinosa: effects of chromosomal insertions on macrolide A83543 production. Gene 146:39–45
Mazodier P, Petter R, Thompson C (1989) Intergeneric conjugation between Escherichia coli and Streptomyces species. J Bacteriol 171:3583–3585
Mertz F, Yao R (1990) Saccharopolyspora spinosa sp. nov. isolated from soil collected in a sugar mill rum still. Int J System Bacteriol 40:34–39
Pan Y, Yang X, Li J, Zhang R, Hu Y, Zhou Y, Wang J, Zhu B (2011) Genome sequence of the spinosyns-producing bacterium Saccharopolyspora spinosa NRRL 18395. J Bacteriol 193:3150–3151
Qin Z, Peng K, Zhou X, Liang R, Zhou Q, Chen H, Hopwood DA, Kieser T, Deng Z (1994) Development of a gene cloning system for Streptomyces hygroscopicus subsp. yingchengensis, a producer of three useful antifungal compounds, by elimination of three barriers to DNA transfer. J Bacteriol 176:2090–2095
Qin S, Li J, Zhao GZ, Chen HH, Xu LH, Li WJ (2008) Saccharopolyspora endophytica sp. nov., an endophytic actinomycete isolated from the root of Maytenus austroyunnanensis. Syst Appl Microbiol 31:352–357
Qin S, Chen HH, Klenk HP, Kim CJ, Xu LH, Li WJ (2010) Saccharopolyspora gloriosae sp. nov., an endophytic actinomycete isolated from the stem of Gloriosa superba L. Int J Syst Evol Microbiol 60:1147–1151
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor
Sparks TC, Thompson GD, Kirst HA, Hertlein MB, Mynderse JS, Turner JR, Worden TV (1999) Fermentation-derived insect control agents. Meth Biotechnol 5:171–188
Strobel RJ, Nakatsukasa WM (1993) Response surface methods for optimizing Saccharopolyspora spinosa, a novel macrolide producer. J Ind Microbiol Biotechnol 11:121–127
Tang SK, Wang Y, Cai M, Zhi XY, Lou K, Xu LH, Jiang CL, Li WJ (2009) Saccharopolyspora halophila sp. nov., a novel halophilic actinomycete isolated from a saline lake in China. Int J Syst Evol Microbiol 59:555–558
te Poele EM, Bolhuis H, Dijkhuizen L (2008) Actinomycete integrative and conjugative elements. Anton Leeuw Int J G 94:127–143
Thompson GD, Dutton R, Sparks TC (2000) Spinosad—a case study: an example from a natural products discovery programme. Pest Manag Sci 56:696–702
Wang T, Chen Z, Cheng Q, Zhou M, Tian X, Xie P, Zhong L, Shen M, Qin Z (2012) Characterization of replication and conjugation of plasmid pWTY27 from a widely distributed Streptomyces species. BMC Microbiol 7:253
Wang X, Zhang C, Wang M, Lu W (2014) Genome-scale metabolic network reconstruction of Saccharopolyspora spinosa for spinosad production improvement. Microb Cell Fact 13:41
Xue C, Duan Y, Zhao F, Lu W (2013) Stepwise increase of spinosad production in Saccharopolyspora spinosa by metabolic engineering. Biochem Eng J 72:90–95
Yuan LJ, Zhang YQ, Guan Y, Wei YZ, Li QP, Yu LY, Li WJ, Zhang YQ (2008) Saccharopolyspora antimicrobica sp. nov., an actinomycete from soil. Int J Syst Evol Microbiol 58:1180–1185
Zhang X, Bao Y, Shi X, Ou X, Zhou P, Ding X (2012) Efficient transposition of IS204-derived plasmids in Streptomyces coelicolor. J Microbiol Methods 88:67–72
Acknowledgments
We are grateful to Dr. Adam Jones for reading and correcting the manuscript. This study was supported by grants from the National High Technology Research and Development Program of China (2012AA022107, 2012AA021703) and the National Basic Research Program of China (2011CBA00801).
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Chen, J., Xia, H., Dang, F. et al. Characterization of the chromosomal integration of Saccharopolyspora plasmid pCM32 and its application to improve production of spinosyn in Saccharopolyspora spinosa . Appl Microbiol Biotechnol 99, 10141–10149 (2015). https://doi.org/10.1007/s00253-015-6871-z
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DOI: https://doi.org/10.1007/s00253-015-6871-z