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Expression of Shewanella frigidimarina fatty acid metabolic genes in E. coli by CRISPR/cas9-coupled lambda Red recombineering

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Abstract

Objective

To construct a clustered, regularly interspaced, short palindromic repeats (CRISPR)/cas9 system and use this system to obtain a recombinant Escherichia coli strain possessing the fatty acid metabolism genes from a lipid-rich marine bacterium.

Results

The fatty acid regulatory transcription factor (fadR), delta9 (Δ9 desaturase) and acetyl-CoA carboxylase (acc) genes were cloned from Shewanella frigidimarina. The fatty acid regulatory transcription factor (fadD) and phosphoenolpyruvate carboxylase inactivated strains were used to construct the fadR/delta9 and acc knock-in strains, which are both markerless and “scar”-less, and identified the change in fatty acid composition in the recombinant strains. There was no change in fatty acid composition between the wild-type strain and recombinant strains. All strains had 11:0, 12:0, 13:0, 14:0, 15:0, 16:0, 17:1, 17:0 and 18:0 fatty acids, with 16:0 and 18:0 fatty acids being dominant. The total lipid content of each recombinant strain was higher than the wild-type strain, with a maximum of 13.1 %, nearly 5.3 % higher than wild-type strain.

Conclusion

The CRISPR/cas9 system, in conjunction with λ-Red recombinases, can rapidly and efficiently edit the E. coli genome. The CRISPR/cas9 recombineering machinery can be modified to select biotechnologically-relevant bacteria other than E. coli.

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Acknowledgments

This work was supported by the Basic Scientific Fund for National Public Research Institutes of China (Grant No. 2015T05), Marine Renewable Energy Funds Project (Grant No. 201305022 GHME2001SW02) and Taishan Scholar award. We sincerely thank Sheng Yang from Shanghai Institutes for Biological Sciences for kindly providing pTargetF and pCas.

Supporting information

Supplementary protocols—Total lipid extraction and FAME preparation.

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

Supplementary Table 2—Primers used in this study.

Supplementary Fig. 1—The TIC of FAME Mix and sample using GC–MS.

Author information

Authors and Affiliations

  1. College of Life Sciences, Shihezi University, Shihezi, 832000, People’s Republic of China

    Jun Xia & Jian-bo Zhu

  2. College of Chemical Engineering, Qingdao University, Qingdao, 266071, People’s Republic of China

    Ling Wang, Ying-hua Lou & Lei Shi

  3. The First Institute of Oceanography, State Oceanic Administration of China, Qingdao, 266061, People’s Republic of China

    Cheng-jun Sun, Ming-gang Zheng & Li Zheng

Authors
  1. Jun Xia
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  2. Ling Wang
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  3. Jian-bo Zhu
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  4. Cheng-jun Sun
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  5. Ming-gang Zheng
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  6. Li Zheng
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  7. Ying-hua Lou
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  8. Lei Shi
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Corresponding author

Correspondence to Ming-gang Zheng.

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Xia, J., Wang, L., Zhu, Jb. et al. Expression of Shewanella frigidimarina fatty acid metabolic genes in E. coli by CRISPR/cas9-coupled lambda Red recombineering. Biotechnol Lett 38, 117–122 (2016). https://doi.org/10.1007/s10529-015-1956-4

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  • DOI: https://doi.org/10.1007/s10529-015-1956-4

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