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Metabolic engineering of Corynebacterium glutamicum by synthetic small regulatory RNAs

  • Dehu Sun
  • Jiuzhou Chen
  • Yu Wang
  • Mingyue Li
  • Deming Rao
  • Yanmei Guo
  • Ning Chen
  • Ping ZhengEmail author
  • Jibin SunEmail author
  • Yanhe Ma
Biotechnology Methods - Short Communication

Abstract

Corynebacterium glutamicum is an important platform strain that is wildly used in industrial production of amino acids and various other biochemicals. However, due to good genomic stability, C. glutamicum is more difficult to engineer than genetically tractable hosts. Herein, a synthetic small regulatory RNA (sRNA)-based gene knockdown strategy was developed for C. glutamicum. The RNA chaperone Hfq from Escherichia coli and a rationally designed sRNA consisting of the E. coli MicC scaffold and a target binding site were proven to be indispensable for repressing green fluorescent protein expression in C. glutamicum. The synthetic sRNA system was applied to improve glutamate production through knockdown of pyk, ldhA, and odhA, resulting almost a threefold increase in glutamate titer and yield. Gene transcription and enzyme activity were down-regulated by up to 80%. The synthetic sRNA system developed holds promise to accelerate C. glutamicum metabolic engineering for producing valuable chemicals and fuels.

Keywords

Corynebacterium glutamicum Gene knockdown Glutamate Hfq Synthetic sRNA 

Notes

Acknowledgements

This work was funded by the National Natural Science Foundation of China (31700044 and 31870044), the Key Research Program of Chinese Academy of Sciences (ZDRW-ZS-2016-2), the International Partnership Program of Chinese Academy of Sciences (153D31KYSB20170121), the Tianjin Municipal Science and Technology Commission (15PTCYSY00020) and the first Special Support Plan for Talents Development and High-level Innovation and Entrepreneurship Team of the Tianjin Municipal City.

Data availability

All data generated or analysed during this study are included in this published article and its supplementary information files.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10295_2018_2128_MOESM1_ESM.pdf (415 kb)
Supplementary material 1 (PDF 415 kb)

References

  1. 1.
    Aiba H (2007) Mechanism of RNA silencing by Hfq-binding small RNAs. Curr Opin Microbiol 10:134–139.  https://doi.org/10.1016/j.mib.2007.03.010 CrossRefGoogle Scholar
  2. 2.
    Cao Y, Li X, Li F, Song H (2017) CRISPRi-sRNA: transcriptional-translational regulation of extracellular electron transfer in Shewanella oneidensis. ACS Synth Biol 6:1679–1690.  https://doi.org/10.1021/acssynbio.6b00374 CrossRefGoogle Scholar
  3. 3.
    Cho C, Lee SY (2017) Efficient gene knockdown in Clostridium acetobutylicum by synthetic small regulatory RNAs. Biotechnol Bioeng 114:374–383.  https://doi.org/10.1002/bit.26077 CrossRefGoogle Scholar
  4. 4.
    Cho JS, Choi KR, Prabowo CPS, Shin JH, Yang D, Jang J, Lee SY (2017) CRISPR/Cas9-coupled recombineering for metabolic engineering of Corynebacterium glutamicum. Metab Eng 42:157–167.  https://doi.org/10.1016/j.ymben.2017.06.010 CrossRefGoogle Scholar
  5. 5.
    Cleto S, Jensen JV, Wendisch VF, Lu TK (2016) Corynebacterium glutamicum metabolic engineering with CRISPR interference (CRISPRi). ACS Synth Biol 5:375–385.  https://doi.org/10.1021/acssynbio.5b00216 CrossRefGoogle Scholar
  6. 6.
    Dietrich C, Nato A, Bost B, Le Marechal P, Guyonvarch A (2009) Regulation of ldh expression during biotin-limited growth of Corynebacterium glutamicum. Microbiology 155:1360–1375.  https://doi.org/10.1099/mic.0.022004-0 CrossRefGoogle Scholar
  7. 7.
    Jiang Y, Qian F, Yang J, Liu Y, Dong F, Xu C, Sun B, Chen B, Xu X, Li Y, Wang R, Yang S (2017) CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum. Nat Commun 8:15179.  https://doi.org/10.1038/ncomms15179 CrossRefGoogle Scholar
  8. 8.
    Keilhauer C, Eggeling L, Sahm H (1993) Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon. J Bacteriol 175:5595–5603.  https://doi.org/10.1128/jb.175.17.5595-5603.1993 CrossRefGoogle Scholar
  9. 9.
    Kim B, Park H, Na D, Lee SY (2014) Metabolic engineering of Escherichia coli for the production of phenol from glucose. Biotechnol J 9:621–629.  https://doi.org/10.1002/biot.201300263 CrossRefGoogle Scholar
  10. 10.
    Kim J, Hirasawa T, Sato Y, Nagahisa K, Furusawa C, Shimizu H (2009) Effect of odhA overexpression and odhA antisense RNA expression on Tween-40-triggered glutamate production by Corynebacterium glutamicum. Appl Microbiol Biotechnol 81:1097–1106.  https://doi.org/10.1007/s00253-008-1743-4 CrossRefGoogle Scholar
  11. 11.
    Kovacs L, Csanadi A, Megyeri K, Kaberdin VR, Miczak A (2005) Mycobacterial RNase E-associated proteins. Microbiol Immunol 49:1003–1007.  https://doi.org/10.1111/j.1348-0421.2005.tb03697.x CrossRefGoogle Scholar
  12. 12.
    Lahir A, Stimple SD, Wood DW, Lease RA (2017) Retargeting a dual-acting sRNA for multiple mRNA transcript regulation. ACS Synth Biol 6:648–658.  https://doi.org/10.1021/acssynbio.6b00261 CrossRefGoogle Scholar
  13. 13.
    Liu J, Wang Y, Lu Y, Zheng P, Sun J, Ma Y (2017) Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum. Microb Cell Fact 16:205.  https://doi.org/10.1186/s12934-017-0815-5 CrossRefGoogle Scholar
  14. 14.
    Maeda T, Sakai T, Wachi M (2009) The Corynebacterium glutamicum NCgl2281 gene encoding an RNase E/G family endoribonuclease can complement the Escherichia coli rng:cat mutation but not the rne-1 mutation. Biosci Biotechnol Biochem 73:2281–2286.  https://doi.org/10.1271/bbb.90371 CrossRefGoogle Scholar
  15. 15.
    Na D, Yoo SM, Chung H, Park H, Park JH, Lee SY (2013) Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nat Biotechnol 31:170–174.  https://doi.org/10.1038/nbt.2461 CrossRefGoogle Scholar
  16. 16.
    Noh M, Yoo SM, Kim WJ, Lee SY (2017) Gene expression knockdown by modulating synthetic small RNA expression in Escherichia coli. Cell Syst 5:418–426.  https://doi.org/10.1016/j.cels.2017.08.016 CrossRefGoogle Scholar
  17. 17.
    Peng F, Wang X, Sun Y, Dong G, Yang Y, Liu X, Bai Z (2017) Efficient gene editing in Corynebacterium glutamicum using the CRISPR/Cas9 system. Microb Cell Fact 16:201.  https://doi.org/10.1186/s12934-017-0814-6 CrossRefGoogle Scholar
  18. 18.
    Ruan Y, Zhu L, Li Q (2015) Improving the electro-transformation efficiency of Corynebacterium glutamicum by weakening its cell wall and increasing the cytoplasmic membrane fluidity. Biotechnol Lett 37:2445–2452.  https://doi.org/10.1007/s10529-015-1934-x CrossRefGoogle Scholar
  19. 19.
    Schultz C, Niebisch A, Gebel L, Bott M (2007) Glutamate production by Corynebacterium glutamicum: dependence on the oxoglutarate dehydrogenase inhibitor protein OdhI and protein kinase PknG. Appl Microbiol Biotechnol 76:691–700.  https://doi.org/10.1007/s00253-007-0933-9 CrossRefGoogle Scholar
  20. 20.
    Song CW, Lee J, Lee SY (2015) Genome engineering and gene expression control for bacterial strain development. Biotechnol J 10:56–68.  https://doi.org/10.1002/biot.201400057 CrossRefGoogle Scholar
  21. 21.
    Wang Y, Cao G, Xu D, Fan L, Wu X, Ni X, Zhao S, Zheng P, Sun J, Ma Y (2018) A novel Corynebacterium glutamicum L-glutamate exporter. Appl Environ Microbiol 84:e02691-02617.  https://doi.org/10.1128/aem.02691-17 Google Scholar
  22. 22.
    Wang Y, Liu Y, Liu J, Guo Y, Fan L, Ni X, Zheng X, Wang M, Zheng P, Sun J, Ma Y (2018) MACBETH: multiplex automated Corynebacterium glutamicum base editing method. Metab Eng 47:200–210.  https://doi.org/10.1016/j.ymben.2018.02.016 CrossRefGoogle Scholar
  23. 23.
    Yoo SM, Na D, Lee SY (2013) Design and use of synthetic regulatory small RNAs to control gene expression in Escherichia coli. Nat Protoc 8:1694–1707.  https://doi.org/10.1038/nprot.2013.105 CrossRefGoogle Scholar
  24. 24.
    Zeller ME, Csanadi A, Miczak A, Rose T, Bizebard T, Kaberdin VR (2007) Quaternary structure and biochemical properties of mycobacterial RNase E/G. Biochem J 403:207–215.  https://doi.org/10.1042/bj20061530 CrossRefGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2019

Authors and Affiliations

  1. 1.College of BiotechnologyTianjin University of Science and TechnologyTianjinChina
  2. 2.Key Laboratory of Systems Microbial BiotechnologyChinese Academy of SciencesTianjinChina
  3. 3.Tianjin Institute of Industrial BiotechnologyChinese Academy of SciencesTianjinChina

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