Abstract
Streptomyces and other closely-related actinobacteria are important sources of bioactive molecules. Streptomyces synthetic biology and genetics empower therapeutic and agrichemical development through strain improvement and biosynthetic understanding. Such efforts rely on the availability of developed molecular toolsets. Among these tools, vectors that enable combinatorial chromosomal manipulations are particularly desirable. Towards developing tools for facile multiplex engineering, we herein describe the development of new integrating vectors derived from BD1 subgroup actinophage OzzyJ (ϕOZJ). By demonstrating the transformation of several Streptomyces spp. using ϕOZJ-derived vectors, we reveal their potential for strain engineering. We further report the development of new ϕC31 and ϕBT1-based vectors having orthogonal resistance, replication and integration features for concomitant transformation with our ϕOZJ-derived vectors. Importantly, the resulting compatible vector panel enabled us to demonstrate the transfer of up to three plasmids each into Streptomyces venezuelae, Streptomyces roseosporus and Streptomyces pristinaespiralis during a single conjugation experiment. To our knowledge this is the first documentation of conjugation-mediated multiplex plasmid transformation, a useful approach for rapid combinatorial strain development.
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Baltz RH (2012) Streptomyces temperate bacteriophage integration systems for stable genetic engineering of actinomycetes (and other organisms). J Ind Microbiol Biotechnol 39:661–672. https://doi.org/10.1007/s10295-011-1069-6
Baltz RH (2014) 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. https://doi.org/10.1021/sb3000673
Barka EA, Vatsa P, Sanchez L, Gaveau-Vaillant N, Jacquard C, Meier-Kolthoff JP, Klenk HP, Clement C, Ouhdouch Y, van Wezel GP (2016) Taxonomy, physiology, and natural products of actinobacteria. Microbiol Mol Biol Rev 80:1–43. https://doi.org/10.1128/MMBR.00019-15
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
Blaesing F, Mühlenweg A, Vierling S, Ziegelin G, Pelzer S, Lanka E (2005) Introduction of DNA into actinomycetes by bacterial conjugation from E. coli–an evaluation of various transfer systems. J Biotechnol 120:146–161. https://doi.org/10.1016/j.jbiotec.2005.06.023
Blodgett JAV, Oh D-C, Cao S, Currie CR, Kolter R, Clardy J (2010) Common biosynthetic origins for polycyclic tetramate macrolactams from phylogenetically diverse bacteria. Proc Natl Acad Sci USA 107:11692–11697. https://doi.org/10.1073/pnas.1001513107
Dayan FE, Cantrell CL, Duke SO (2009) Natural products in crop protection. Bioorg Med Chem 17:4022–4034
El-Tarabily KA, Sivasithamparam K (2006) Non-streptomycete actinomycetes as biocontrol agents of soil-borne fungal plant pathogens and as plant growth promoters. Soil Biol Biochem 38:1505–1520
Fayed B, Younger E, Taylor G, Smith MC (2014) A novel Streptomyces spp. integration vector derived from the S. venezuelae phage, SV1. BMC Biotechnol 14:51
Flett F, Mersinias V, Smith CP (1997) High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes. FEMS Microbiol Lett 155:223–229
Fogg PC, Colloms S, Rosser S, Stark M, Smith MC (2014) New applications for phage integrases. J Mol Bio 426:2703–2716
Fogg PC, Haley JA, Stark WM, Smith MC (2017) Genome integration and excision by a new Streptomyces bacteriophage, ϕJoe. Appl Environ Microbiol 83:e02767–e02816
Hirsch C, Ensign J (1976) Heat activation of Streptomyces viridochromogenes spores. J Bacteriol 126:24–30
Keiser T, Bibb M, Buttner M, Chater K, Hopwood D (2000) Practical Streptomyces genetics. The John Innes Foundation, Norwich
Lewin GR, Carlos C, Chevrette MG, Horn HA, McDonald BR, Stankey RJ, Fox BG, Currie CR (2016) Evolution and ecology of actinobacteria and their bioenergy applications. Ann Rev Microbiol 70:235–254
Mast Y, Weber T, Golz M, Ort-Winklbauer R, Gondran A, Wohlleben W, Schinko E (2011) Characterization of the ‘pristinamycin supercluster’ of Streptomyces pristinaespiralis. Microb Biotechnol 4:192–206. https://doi.org/10.1111/j.1751-7915.2010.00213.x
Mazodier P, Petter R, Thompson C (1989) Intergeneric conjugation between Escherichia coli and Streptomyces species. J Bacteriol 171:3583–3585
Medema MH, Breitling R, Takano E (2011) Synthetic biology in Streptomyces bacteria. Methods Enzymol 497:485–502. https://doi.org/10.1016/B978-0-12-385075-1.00021-4
Merrick CA, Zhao J, Rosser SJ (2018) Serine integrases: advancing synthetic biology. ACS Synth Biol 7:299–310. https://doi.org/10.1021/acssynbio.7b00308
Miao V, Coëffet-Le Gal M-F, Nguyen K, Brian P, Penn J, Whiting A, Steele J, Kau D, Martin S, Ford R, Gibson T, Bouchard M, Wrigley SK, Baltz RH (2006) Genetic engineering in Streptomyces roseosporus to produce hybrid lipopeptide antibiotics. Chem Biol 13:269–276. https://doi.org/10.1016/j.chembiol.2005.12.012
Miura T, Hosaka Y, Yan-Zhuo Y, Nishizawa T, Asayama M, Takahashi H, Shirai M (2011) In vivo and in vitro characterization of site-specific recombination of actinophage R4 integrase. J Gen Appl Microbiol 57:45–57
Muroi T, Kokuzawa T, Kihara Y, Kobayashi R, Hirano N, Takahashi H, Haruki M (2013) TG1 integrase-based system for site-specific gene integration into bacterial genomes. Appl Microbiol Biotechnol 97:4039–4048. https://doi.org/10.1007/s00253-012-4491-4
Myronovskyi M, Luzhetskyy A (2016) Native and engineered promoters in natural product discovery. Nat Prod Rep 33:1006–1019. https://doi.org/10.1039/c6np00002a
Newitt JT, Prudence SM, Hutchings MI, Worsley SF (2019) Biocontrol of cereal crop diseases using streptomycetes. Pathogens 8:78
Phelan RM, Sachs D, Petkiewicz SJ, Barajas JF, Blake-Hedges JM, Thompson MG, Reider Apel A, Rasor BJ, Katz L, Keasling JD (2017) Development of next generation synthetic biology tools for use in Streptomyces venezuelae. ACS Synth Biol 6:159–166. https://doi.org/10.1021/acssynbio.6b00202
Prakash D, Nawani N, Prakash M, Bodas M, Mandal A, Khetmalas M, Kapadnis B (2013) Actinomycetes: a repertory of green catalysts with a potential revenue resource. Biomed Res Int 2013:264020
Sambrook J, Fritsch E, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Press, New York
Stach JEM, Maldonado LA, Ward AC, Goodfellow M, Bull AT (2003) New primers for the class Actinobacteria: application to marine and terrestrial environments. Environ Microbiol 5:828–841
Van Dessel W, Van Mellaert L, Geukens N, Anné J (2003) Improved PCR-based method for the direct screening of Streptomyces transformants. J Microbiol Methods 53:401–403
Watve M, Tickoo R, Jog M, Bhole B (2001) How many antibiotics are produced by the genus Streptomyces? Arch Microbiol 176:386–390
Yuan WM, Crawford DL (1995) Characterization of Streptomyces lydicus WYEC108 as a potential biocontrol agent against fungal root and seed rots. Appl Environ Microbiol 61:3119–3128
Yuzawa S, Mirsiaghi M, Jocic R, Fujii T, Masson F, Benites VT, Baidoo EEK, Sundstrom E, Tanjore D, Pray TR, George A, Davis RW, Gladden JM, Simmons BA, Katz L, Keasling JD (2018) Short-chain ketone production by engineered polyketide synthases in Streptomyces albus. Nat Commun 9:4569. https://doi.org/10.1038/s41467-018-07040-0
Zotchev S, Caffrey P (2009) Genetic analysis of nystatin and amphotericin biosynthesis. Methods Enzymol 459:243–258. https://doi.org/10.1016/S0076-6879(09)04611-4
Acknowledgements
We are indebted to WUSTL faculty C. Schafer and K. Hafer for generously providing ϕWTV and ϕOZJ lysates and assembled genome sequences prior to public release. We are grateful to W. Metcalf (U-Illinois, Urbana) for pBTC034 and R. Baltz (Cognogen Bioconsulting) for Streptomyces griseofuscus. We thank former WUSTL Biol3493 students K. Lou, J. Alex and B. Makhdoom for designing the multiplex screening primers for ϕBT1-dependent plasmid integration and assisting in the isolation of the environmental strains used in this work. This material is based upon work supported by the National Science Foundation under NSF-CAREER 1846005 to J Blodgett.
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Ko, B., D’Alessandro, J., Douangkeomany, L. et al. Construction of a new integrating vector from actinophage ϕOZJ and its use in multiplex Streptomyces transformation. J Ind Microbiol Biotechnol 47, 73–81 (2020). https://doi.org/10.1007/s10295-019-02246-7
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DOI: https://doi.org/10.1007/s10295-019-02246-7