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Genetic transformation system for Bacillus velezensis NSZ-YBGJ001 and curing of the endogenous plasmid pBV01

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Abstract

Objectives

To construct a genetic transformation system for Bacillus velezensis NSZ-YBGJ001 and identify the origin element in an endogenous plasmidpBV01 for curing pBV01 by plasmid incompatibility.

Results

A plasmid pUBC01 was constructed, and then an electrotransformation system for B. velezensis NSZ-YBGJ001 was developed, which reached ~ 1000 transformants per microgram of pUBC01 DNA. Additionally, a 7276-bp circular plasmid pBV01 with a G + C content of 37.5% was isolated from B. velezensis NSZ-YBGJ001 and analyzed via sequence analysis. To cure pBV01, an incompatible plasmid pBV02 harboring the replication element of pBV01 was developed and functionally replicated in both Bacillus subtilis WB600 and B. velezensis NSZ-YBGJ001. pBV01 was cured through introduction of pBV02 into B. velezensis NSZ-YBGJ001 after serial subculturing for approximately 40 generations. Finally, another plasmid, pBV03, was constructed based on pBV-ori, and exogenous genes in pBV03 could be efficiently expressed in B. subtilis.

Conclusions

The results of this study, including the genetic transformation system, plasmid-curing strategy, and exogenous gene expression, will support genetic manipulation of B. velezensis to promote its application in biocontrol and industry.

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Acknowledgements

This work was supported by the National Transgenic Major Program (2019ZX08010-004), the National Natural Science Foundation of China (31671802), the Central Public-interest Scientific Institution Basal Research Fund (Y2019XK19), the Xinjiang Key Research and Development Program of China (2018B01006-1).

Supplementary information

Supplementary Table 1—Strains and plasmids developed in this study.

Supplementary Table 2—Primers used for PCR in this study.

Supplementary Fig. 1—Schematic representation of an incompatible plasmid pBV02.

Author information

Author notes

  1. Hui Tian and Bo Liu have contributed equally to this work.

Authors and Affiliations

  1. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081, China

    Hui Tian, Bo Liu, Juan Yang, Xinxin Xu, Yuhong Zhang & Wei Zhang

  2. Beijing Plant Protection Station, No. 9 Beisanhuan Zhonglu, Beijing, 100026, China

    Chengfang Zhou & Zhijun Lu

Authors
  1. Hui Tian
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  2. Bo Liu
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  3. Juan Yang
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  4. Chengfang Zhou
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  5. Xinxin Xu
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  6. Yuhong Zhang
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  7. Zhijun Lu
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  8. Wei Zhang
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Correspondence to Zhijun Lu or Wei Zhang.

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Tian, H., Liu, B., Yang, J. et al. Genetic transformation system for Bacillus velezensis NSZ-YBGJ001 and curing of the endogenous plasmid pBV01. Biotechnol Lett 43, 1595–1605 (2021). https://doi.org/10.1007/s10529-021-03127-9

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  • DOI: https://doi.org/10.1007/s10529-021-03127-9

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