Triple deletion of clpC, porB, and mepA enhances production of small ubiquitin-like modifier-N-terminal pro-brain natriuretic peptide in Corynebacterium glutamicum

  • Feng Peng
  • Xiuxia Liu
  • Xinyue Wang
  • Jing Chen
  • Meng Liu
  • Yankun Yang
  • Zhonghu BaiEmail author
Metabolic Engineering and Synthetic Biology - Original Paper


In our previous work, a two-plasmid CRISPR/Cas9 system was constructed for genome editing in Corynebacterium glutamicum. To increase the transformation efficiency and simplify the plasmid curing steps, an all-in-one CRISPR/Cas9 system was constructed for efficient genome editing. In addition, to research proteolysis during the production of recombinant proteins and generate a host for enhanced expression of recombinant proteins, the system was used to delete three genes, clpC, porB, and mepA in C. glutamicum CGMCC1.15647, which encoded the Clp protease subunit ClpC, anion selective channel protein B, and metallopeptidase A, respectively. After the evaluation of different plasmids and hosts, small ubiquitin-like modifier-N-terminal pro-brain natriuretic peptide (SUMO-NT-proBNP), an important protein used for the diagnosis of mild heart failure was successfully expressed in the triple mutant ΔclpCΔporBΔmepA, which exhibit threefold higher levels of protein expression compared with the wild-type. In conclusion, we created a simplified CRISPR tool for genome editing in C. glutamicum, provided a method to generate a host for enhanced expression of recombinant proteins and successfully expressed SUMO-NT-proBNP in C. glutamicum. This tool and method will greatly facilitate genetic engineering and metabolic optimization of this important platform organism.


Corynebacterium glutamicum CRISPR/Cas9 Genome editing Protease SUMO-NT-proBNP 



Single-guide RNA


Homolog-directed repair arm


Green fluorescent protein


Single-chain variable fragment


Small ubiquitin-related modifier


N-terminal pro-brain natriuretic peptide



This work was supported by the National Natural Science Foundation of China (21808082), the Natural Science Foundation of Jiangsu Province (BK20150148), the 111 Project (111-2-06) and national first-class discipline program of Light Industry Technology and Engineering (LITE2018-24).

Compliance with ethical standards

Conflict of interests

The authors declare that they have no conflict of interest.

Supplementary material

10295_2018_2091_MOESM1_ESM.docx (717 kb)
Supplementary material 1 (DOCX 716 kb)


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Copyright information

© Society for Industrial Microbiology and Biotechnology 2018

Authors and Affiliations

  • Feng Peng
    • 1
    • 2
    • 3
    • 4
  • Xiuxia Liu
    • 1
    • 2
    • 3
    • 4
  • Xinyue Wang
    • 1
    • 2
    • 3
    • 4
  • Jing Chen
    • 1
    • 2
    • 3
    • 4
  • Meng Liu
    • 2
  • Yankun Yang
    • 1
    • 2
    • 3
    • 4
  • Zhonghu Bai
    • 1
    • 2
    • 3
    • 4
    Email author
  1. 1.The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of BiotechnologyJiangnan UniversityWuxiChina
  2. 2.National Engineering Laboratory for Cereal Fermentation TechnologyJiangnan UniversityWuxiChina
  3. 3.The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of BiotechnologyJiangnan UniversityWuxiChina
  4. 4.Jiangsu Provincial Research Center for Bioactive Product Processing TechnologyJiangnan UniversityWuxiChina

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