Advertisement

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

Abstract

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.

Keywords

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

Abbreviations

sgRNA

Single-guide RNA

HDarm

Homolog-directed repair arm

GFP

Green fluorescent protein

scFv

Single-chain variable fragment

SUMO

Small ubiquitin-related modifier

NT-proBNP

N-terminal pro-brain natriuretic peptide

Notes

Acknowledgements

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)

References

  1. 1.
    Becker J, Zelder O, Hafner S, Schroder H, Wittmann C (2011) From zero to hero-design-based systems metabolic engineering of Corynebacterium glutamicum for l-lysine production. Metab Eng 13:159–168.  https://doi.org/10.1016/j.ymben.2011.01.003 CrossRefGoogle Scholar
  2. 2.
    Woo HM, Park JB (2014) Recent progress in development of synthetic biology platforms and metabolic engineering of Corynebacterium glutamicum. J Biotechnol 180:43–51.  https://doi.org/10.1016/j.jbiotec.2014.03.003 CrossRefGoogle Scholar
  3. 3.
    Becker J, Wittmann C (2012) Bio-based production of chemicals, materials and fuels -Corynebacterium glutamicum as versatile cell factory. Curr Opin Biotechnol 23:631–640.  https://doi.org/10.1016/j.copbio.2011.11.012 CrossRefGoogle Scholar
  4. 4.
    Park SH, Kim HU, Kim TY, Park JS, Kim SS, Lee SY (2014) Metabolic engineering of Corynebacterium glutamicum for l-arginine production. Nat Commun 5:4618.  https://doi.org/10.1038/ncomms5618 CrossRefGoogle Scholar
  5. 5.
    Liu X, Yang Y, Zhang W, Sun Y, Peng F, Jeffrey L, Harvey L, McNeil B, Bai Z (2016) Expression of recombinant protein using Corynebacterium glutamicum: progress, challenges and applications. Crit Rev Biotechnol 36:652–664Google Scholar
  6. 6.
    Heider SA, Wendisch VF (2015) Engineering microbial cell factories: metabolic engineering of Corynebacterium glutamicum with a focus on non-natural products. Biotechnol J 10:1170–1184CrossRefGoogle Scholar
  7. 7.
    Jacobi AM, Rettig GR, Turk R, Collingwood MA, Zeiner SA, Quadros RM, Harms DW, Bonthuis PJ, Gregg C, Ohtsuka M, Gurumurthy CB, Behlke MA (2017) Simplified CRISPR tools for efficient genome editing and streamlined protocols for their delivery into mammalian cells and mouse zygotes. Methods 121–122:16–28.  https://doi.org/10.1016/j.ymeth.2017.03.021 CrossRefGoogle Scholar
  8. 8.
    Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S (2015) Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 81:2506–2514.  https://doi.org/10.1128/AEM.04023-14 CrossRefGoogle Scholar
  9. 9.
    Jakočiūnas T, Jensen MK, Keasling JD (2016) CRISPR/Cas9 advances engineering of microbial cell factories. Metab Eng 34:44–59CrossRefGoogle Scholar
  10. 10.
    DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res 41:4336–4343.  https://doi.org/10.1093/nar/gkt135 CrossRefGoogle Scholar
  11. 11.
    Wang T, Wei JJ, Sabatini DM, Lander ES (2014) Genetic screens in human cells using the CRISPR-Cas9 system. Science 343:80–84CrossRefGoogle Scholar
  12. 12.
    Cleto S, Jensen JV, Wendisch VF, Lu TK (2016) Corynebacterium glutamicum metabolic engineering with CRISPR interference (CRISPRi). ACS Synth Biol 5:375–385CrossRefGoogle Scholar
  13. 13.
    Park J, Shin H, Lee S-M, Um Y, Woo HM (2018) RNA-guided single/double gene repressions in Corynebacterium glutamicum using an efficient CRISPR interference and its application to industrial strain. Microb Cell Fact 17:4CrossRefGoogle Scholar
  14. 14.
    Jiang Y, Qian F, Yang J, Liu Y, Dong F, Xu C, Sun B, Chen B, Xu X, Li Y (2017) CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum. Nat Commun 8:15179CrossRefGoogle Scholar
  15. 15.
    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 42:157–167CrossRefGoogle Scholar
  16. 16.
    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
  17. 17.
    Ahmad M, Hirz M, Pichler H, Schwab H (2014) Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol 98:5301–5317CrossRefGoogle Scholar
  18. 18.
    Zhang J, Ye F, Lan L, Jiang H, Luo C, Yang C-G (2011) Structural switching of Staphylococcus aureus Clp protease a key to understanding protease dynamics. J Biol Chem 286:37590–37601CrossRefGoogle Scholar
  19. 19.
    Trentini DB, Suskiewicz MJ, Heuck A, Kurzbauer R, Deszcz L, Mechtler K, Clausen T (2016) Arginine phosphorylation marks proteins for degradation by a Clp protease. Nature 539:48CrossRefGoogle Scholar
  20. 20.
    Wang F, Mei Z, Qi Y, Yan C, Hu Q, Wang J, Shi Y (2011) Structure and mechanism of the hexameric MecA-ClpC molecular machine. Nature 471:331–335.  https://doi.org/10.1038/nature09780 CrossRefGoogle Scholar
  21. 21.
    Engels S, Schweitzer JE, Ludwig C, Bott M, Schaffer S (2004) clpC and clpP1P2 gene expression in Corynebacterium glutamicum is controlled by a regulatory network involving the transcriptional regulators ClgR and HspR as well as the ECF sigma factor σH. Mol Microbiol 52:285–302CrossRefGoogle Scholar
  22. 22.
    Ziegler K, Benz R, Schulz GE (2008) A putative α-helical porin from Corynebacterium glutamicum. J Mol Biol 379:482–491CrossRefGoogle Scholar
  23. 23.
    Bott M, Brocker M (2012) Two-component signal transduction in Corynebacterium glutamicum and other corynebacteria: on the way towards stimuli and targets. Appl Microbiol Biotechnol 94:1131–1150CrossRefGoogle Scholar
  24. 24.
    Rodseth RN, Biccard BM, Le Manach Y, Sessler DI, Buse GAL, Thabane L, Schutt RC, Bolliger D, Cagini L, Cardinale D (2014) The prognostic value of pre-operative and post-operative B-type natriuretic peptides in patients undergoing noncardiac surgery: B-type natriuretic peptide and N-terminal fragment of pro-B-type natriuretic peptide: a systematic review and individual patient data meta-analysis. J Am Coll Cardiol 63:170–180CrossRefGoogle Scholar
  25. 25.
    Mueller T, Gegenhuber A, Poelz W, Haltmayer M (2005) Diagnostic accuracy of B type natriuretic peptide and amino terminal proBNP in the emergency diagnosis of heart failure. Heart 91:606–612.  https://doi.org/10.1136/hrt.2004.037762 CrossRefGoogle Scholar
  26. 26.
    Maisel AS, Krishnaswamy P, Nowak RM, McCord J, Hollander JE, Duc P, Omland T, Storrow AB, Abraham WT, Wu AH (2002) Rapid measurement of B-type natriuretic peptide in the emergency diagnosis of heart failure. N Engl J Med 347:161–167CrossRefGoogle Scholar
  27. 27.
    Emdin M, Passino C, Prontera C, Fontana M, Poletti R, Gabutti A, Mammini C, Giannoni A, Zyw L, Zucchelli G, Clerico A (2007) Comparison of brain natriuretic peptide (BNP) and amino-terminal ProBNP for early diagnosis of heart failure. Clin Chem 53:1289–1297.  https://doi.org/10.1373/clinchem.2006.080234 CrossRefGoogle Scholar
  28. 28.
    Blin K, Pedersen LE, Weber T, Lee SY (2016) CRISPy-web: an online resource to design sgRNAs for CRISPR applications. Synth Syst Biotechnol 1:118–121CrossRefGoogle Scholar
  29. 29.
    Yan M-Y, Yan H-Q, Ren G-X, Zhao J-P, Guo X-P, Sun Y-C (2017) CRISPR-Cas12a-assisted recombineering in bacteria. Appl Environ Microbiol 83:e00947-00917Google Scholar
  30. 30.
    Maffitt M, Auldridge M, Sen S, Floyd S, Krerowicz A, Uphoff M, Thompson J, Mead D, Steinmetz E (2015) Rapid screening for protein solubility and expression. Nat Methods 12(6):586.  https://doi.org/10.1038/nmeth.f.382
  31. 31.
    Zhao Z, Liu X, Zhang W, Yang Y, Dai X, Bai Z (2016) Construction of genetic parts from the Corynebacterium glutamicum genome with high expression activities. Biotechnol Lett 38:2119–2126CrossRefGoogle Scholar
  32. 32.
    Seferian KR, Tamm NN, Semenov AG, Tolstaya AA, Koshkina EV, Krasnoselsky MI, Postnikov AB, Serebryanaya DV, Apple FS, Murakami MM (2008) Immunodetection of glycosylated NT-proBNP circulating in human blood. Clin Chem 54:866–873CrossRefGoogle Scholar
  33. 33.
    Choi JW, Yim SS, Lee SH, Kang TJ, Park SJ, Jeong KJ (2015) Enhanced production of gamma-aminobutyrate (GABA) in recombinant Corynebacterium glutamicum by expressing glutamate decarboxylase active in expanded pH range. Microb Cell Fact 14:21CrossRefGoogle Scholar
  34. 34.
    Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, Heckl D, Ebert BL, Root DE, Doench JG (2014) Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343:84–87CrossRefGoogle Scholar
  35. 35.
    Liu X, Yang S, Wang F, Dai X, Yang Y, Bai Z (2017) Comparative analysis of the Corynebacterium glutamicum transcriptome in response to changes in dissolved oxygen levels. J Ind Microbiol Biotechnol 44:181–195.  https://doi.org/10.1007/s10295-016-1854-3 CrossRefGoogle Scholar
  36. 36.
    Rozkov A (2001) Control of proteolysis of recombinant proteins in Escherichia coli. BioteknologiGoogle Scholar
  37. 37.
    Selas Castiñeiras T, Williams SG, Hitchcock AG, Smith DC (2018) E. coli strain engineering for the production of advanced biopharmaceutical products. FEMS Microbiol Lett 365:fny162CrossRefGoogle Scholar
  38. 38.
    Costa-Riu N, Maier E, Burkovski A, Krämer R, Lottspeich F, Benz R (2003) Identification of an anion-specific channel in the cell wall of the Gram-positive bacterium Corynebacterium glutamicum. Mol Microbiol 50:1295–1308CrossRefGoogle Scholar
  39. 39.
    Matsuda Y, Itaya H, Kitahara Y, Theresia NM, Kutukova EA, Yomantas YAV, Date M, Kikuchi Y, Wachi M (2014) Double mutation of cell wall proteins CspB and PBP1a increases secretion of the antibody Fab fragment from Corynebacterium glutamicum. Microb Cell Fact 13:56CrossRefGoogle Scholar
  40. 40.
    Lee Y, Nasution O, Choi E, Choi I-G, Kim W, Choi W (2015) Transcriptome analysis of acetic-acid-treated yeast cells identifies a large set of genes whose overexpression or deletion enhances acetic acid tolerance. Appl Microbiol Biotechnol 99:6391–6403CrossRefGoogle Scholar

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

Personalised recommendations