Abstract
Objective
To develop an inducible CRISPR/Cas9–Recombinase A (RecA) system to manipulate genes in Nonomuraea gerenzanensis effectively.
Results
Compared with traditional homologous recombination, the inducible CRISPR/Cas9 system achieved 68.8% editing efficiency, whereas, with both the inducible Cas9 and the overexpressed RecA, the efficiency of the combined genome editing system reached 100%. The dbv23-deleted mutant obtained by the inducible CRISPR/Cas9–RecA system was confirmed to produce more A40926 with an approximate yield of 200 mg L−1 than that of around 150 mg L−1 produced by the wild-type strain.
Conclusions
This inducible CRISPR/Cas9–RecA system was successfully constructed and can be utilized as an efficient genome editing tool for Actinomyces able to shorten editing time simultaneously.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Baltz RH (2016) Genetic manipulation of secondary metabolite biosynthesis for improved production in Streptomyces and other actinomycetes. J Ind Microbiol Biotechnol 43:343–370. https://doi.org/10.1007/s10295-015-1682-x
Cai Y et al (2019) In vivo genome editing rescues photoreceptor degeneration via a Cas9/RecA-mediated homology-directed repair pathway. Sci Adv. https://doi.org/10.1126/sciadv.aav3335
Cao J et al (2016) An easy and efficient inducible CRISPR/Cas9 platform with improved specificity for multiple gene targeting. Nucleic Acids Res. https://doi.org/10.1093/nar/gkw660
Cho S, Shin J, Cho BK (2018) Applications of CRISPR/Cas system to bacterial metabolic engineering. Int J Mol Sci. https://doi.org/10.3390/ijms19041089
Cui L, Bikard D (2016) Consequences of Cas9 cleavage in the chromosome of Escherichia coli. Nucleic Acids Res 44:4243–4251. https://doi.org/10.1093/nar/gkw223
Gunnarsson N, Bruheim P, Nielsen J (2003) Production of the glycopeptide antibiotic A40926 by Nonomuraea sp. ATCC 39727: influence of medium composition in batch fermentation. J Ind Microbiol Biotechnol 30:150–156. https://doi.org/10.1007/s10295-003-0024-6
Huang H, Zheng G, Jiang W, Hu H, Lu Y (2015) One-step high-efficiency CRISPR/Cas9-mediated genome editing in Streptomyces. Acta Biochim Biophys Sin (Shanghai) 47:231–243. https://doi.org/10.1093/abbs/gmv007
Kim HM, Ahn BE, Lee JH, Roe JH (2015) Regulation of a nickel-cobalt efflux system and nickel homeostasis in a soil actinobacterium Streptomyces coelicolor. Metallomics 7:702–709. https://doi.org/10.1039/c4mt00318g
Marcone GL, Carrano L, Marinelli F, Beltrametti F (2010) Protoplast preparation and reversion to the normal filamentous growth in antibiotic-producing uncommon actinomycetes. J Antibiot (Tokyo) 63:83–88. https://doi.org/10.1038/ja.2009.127
Ojha D, Neelakanteshwar Patil K (2019) Molecular and functional characterization of the Listeria monocytogenes RecA protein: insights into the homologous recombination process. Int J Biochem Cell Biol. https://doi.org/10.1016/j.biocel.2019.105642
Rudolph MM, Vockenhuber MP, Suess B (2015) Conditional control of gene expression by synthetic riboswitches in Streptomyces coelicolor. Methods Enzymol 550:283–299. https://doi.org/10.1016/bs.mie.2014.10.036
Shapiro RS, Chavez A, Collins JJ (2018) CRISPR-based genomic tools for the manipulation of genetically intractable microorganisms. Nat Rev Microbiol 16:333–339. https://doi.org/10.1038/s41579-018-0002-7
Sosio M, Stinchi S, Beltrametti F, Lazzarini A, Donadio S (2003) The gene cluster for the biosynthesis of the glycopeptide antibiotic A40926 by Nonomuraea species. Chem Biol 10:541–549. https://doi.org/10.1016/S1074-5521(03)00120-0
Sosio M, Canavesi A, Stinchi S, Donadio S (2010) Improved production of A40926 by Nonomuraea sp. through deletion of a pathway-specific acetyltransferase. Appl Microbiol Biotechnol 87:1633–1638. https://doi.org/10.1007/s00253-010-2579-2
Acknowledgements
This work was supported by the Natural Science Foundation of Shandong Province (Grant No. ZR2015CL001).
Supporting information
Supplementary File 1
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Yue, X., Xia, T., Wang, S. et al. Highly efficient genome editing in N. gerenzanensis using an inducible CRISPR/Cas9–RecA system. Biotechnol Lett 42, 1699–1706 (2020). https://doi.org/10.1007/s10529-020-02893-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-020-02893-2