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Abstract

Some major producers of useful bioactive natural products belong to the genus or related actinobacteria. Genetic engineering of these bacteria and the pathways that synthesize their valuable products often relies on serine integrases. To further improve the flexibility and efficiency of genome engineering via serine integrases, we explored whether multiple integrating vectors encoding orthogonally active serine integrases can be introduced simultaneously into recipients via conjugal transfer and integration. Pairwise combinations of donors containing vectors encoding orthogonal serine integrases were used in each conjugation. Using donors containing plasmids (of various sizes) encoding either the φBT1 or the φC31 integration systems, we observed reproducible simultaneous plasmid integration into and at moderate frequencies after conjugation. This work demonstrated how site-specific recombination based on orthogonal serine integrases can save researchers time in genome engineering experiments in .

Funding
This study was supported by the:
  • Biotechnology and Biological Sciences Research Council (Award BB/K003356/1)
    • Principle Award Recipient: MargaretC. M. Smith
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2021-12-08
2022-01-29
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References

  1. Rutherford K, Van Duyne GD. The ins and outs of serine integrase site-specific recombination. Curr Opin Struct Biol 2014; 24:125–131 [View Article] [PubMed]
    [Google Scholar]
  2. Thorpe HM, Smith MC. In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Proc Natl Acad Sci U S A 1998; 95:5505–5510 [View Article] [PubMed]
    [Google Scholar]
  3. Gregory MA, Till R, Smith MCM. Integration site for Streptomyces phage phiBT1 and development of site-specific integrating vectors. J Bacteriol 2003; 185:5320–5323 [View Article] [PubMed]
    [Google Scholar]
  4. Kim AI, Ghosh P, Aaron MA, Bibb LA, Jain S et al. Mycobacteriophage Bxb1 integrates into the Mycobacterium smegmatis groEL1 gene. Mol Microbiol 2003; 50:463–473 [View Article] [PubMed]
    [Google Scholar]
  5. Hong Y, Hondalus MK. Site-specific integration of Streptomyces PhiC31 integrase-based vectors in the chromosome of Rhodococcus equi. FEMS Microbiol Lett 2008; 287:63–68 [View Article] [PubMed]
    [Google Scholar]
  6. Saha S, Zhang W, Zhang G, Zhu Y, Chen Y et al. Activation and characterization of a cryptic gene cluster reveals a cyclization cascade for polycyclic tetramate macrolactams. Chem Sci 2017; 8:1607–1612 [View Article] [PubMed]
    [Google Scholar]
  7. Sosio M, Giusino F, Cappellano C, Bossi E, Puglia AM et al. Artificial chromosomes for antibiotic-producing actinomycetes. Nat Biotechnol 2000; 18:343–345 [View Article] [PubMed]
    [Google Scholar]
  8. Fogg PCM, Colloms S, Rosser S, Stark M, Smith MCM. New applications for phage integrases. J Mol Biol 2014; 426:2703–2716 [View Article] [PubMed]
    [Google Scholar]
  9. Xu Z, Thomas L, Davies B, Chalmers R, Smith M et al. Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome. BMC Biotechnol 2013; 13:87 [View Article] [PubMed]
    [Google Scholar]
  10. Gomide MS, Sales TT, Barros LRC, Limia CG, de Oliveira MA et al. Genetic switches designed for eukaryotic cells and controlled by serine integrases. Commun Biol 2020; 3:255 [View Article] [PubMed]
    [Google Scholar]
  11. Zhao J, Pokhilko A, Ebenhöh O, Rosser SJ, Colloms SD. A single-input binary counting module based on serine integrase site-specific recombination. Nucleic Acids Res 2019; 47:4896–4909 [View Article] [PubMed]
    [Google Scholar]
  12. Yang L, Nielsen AAK, Fernandez-Rodriguez J, McClune CJ, Laub MT et al. Permanent genetic memory with >1-byte capacity. Nat Methods 2014; 11:1261–1266 [View Article] [PubMed]
    [Google Scholar]
  13. Colloms SD, Merrick CA, Olorunniji FJ, Stark WM, Smith MCM et al. Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination. Nucleic Acids Res 2014; 42:e23. [View Article] [PubMed]
    [Google Scholar]
  14. Smith MC. Phage‐encoded serine integrases and other large serine recombinases. Mobile DNA III 2015253–272
    [Google Scholar]
  15. Stark WM. Making serine integrases work for us. Curr Opin Microbiol 2017; 38:130–136 [View Article] [PubMed]
    [Google Scholar]
  16. Haginaka K, Asamizu S, Ozaki T, Igarashi Y, Furumai T et al. Genetic approaches to generate hyper-producing strains of goadsporin: the relationships between productivity and gene duplication in secondary metabolite biosynthesis. Biosci Biotechnol Biochem 2014; 78:394–399 [View Article] [PubMed]
    [Google Scholar]
  17. Li L, Zheng G, Chen J, Ge M, Jiang W et al. Multiplexed site-specific genome engineering for overproducing bioactive secondary metabolites in actinomycetes. Metab Eng 2017; 40:80–92 [View Article] [PubMed]
    [Google Scholar]
  18. Elmore JR, Dexter GN, Francis R, Riley L, Huenemann J et al. The SAGE genetic toolkit enables highly efficient, iterative site-specific genome engineering in bacteria. bioRxiv 2020
    [Google Scholar]
  19. Fayed B, Ashford DA, Hashem AM, Amin MA, El Gazayerly ON et al. Multiplexed integrating plasmids for engineering of the erythromycin gene cluster for expression in Streptomyces spp. and combinatorial biosynthesis. Appl Environ Microbiol 2015; 81:8402–8413 [View Article] [PubMed]
    [Google Scholar]
  20. Gomez-Escribano JP, Bibb MJ. Engineering Streptomyces coelicolor for heterologous expression of secondary metabolite gene clusters. Microb Biotechnol 2011; 4:207–215 [View Article] [PubMed]
    [Google Scholar]
  21. Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA. Practical Streptomyces Genetics John Innes Foundation Norwich; 2000
    [Google Scholar]
  22. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. Cold spring harbor laboratory press 1989
    [Google Scholar]
  23. Gao H, Murugesan B, Hoßbach J, Evans SK, Stark WM et al. Integrating vectors for genetic studies in the rare Actinomycete Amycolatopsis marina. BMC Biotechnol 2019; 19:1–10 [View Article]
    [Google Scholar]
  24. Rowe CJ, Cortés J, Gaisser S, Staunton J, Leadlay PF. Construction of new vectors for high-level expression in actinomycetes. Gene 1998; 216:215–223 [View Article] [PubMed]
    [Google Scholar]
  25. Gao H, Taylor G, Evans SK, Fogg PCM, Smith MCM. Application of serine integrases for secondary metabolite pathway assembly in Streptomyces. Synth Syst Biotechnol 2020; 5:111–119 [View Article] [PubMed]
    [Google Scholar]
  26. Ko B, D’Alessandro J, Douangkeomany L, Stumpf S, deButts A et al. Construction of a new integrating vector from actinophage ϕOZJ and its use in multiplex Streptomyces transformation. J Ind Microbiol Biotechnol 2020; 47:73–81 [View Article] [PubMed]
    [Google Scholar]
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