Advertisement

Applied Microbiology and Biotechnology

, Volume 103, Issue 13, pp 5391–5400 | Cite as

Development of a shuttle plasmid without host restriction sites for efficient transformation and heterologous gene expression in Clostridium cellulovorans

  • Teng Bao
  • Jingbo Zhao
  • Qianxia Zhang
  • Shang-Tian YangEmail author
Methods and protocols
  • 143 Downloads

Abstract

Clostridium cellulovorans capable of producing large amounts of acetate and butyrate from cellulose is a promising candidate for biofuels and biochemicals production from lignocellulosic biomass. However, the restriction modification (RM) systems of C. cellulovorans hindered the application of existing shuttle plasmids for metabolic engineering of this organism. To overcome the hurdle of plasmid digestion by host, a new shuttle plasmid (pYL001) was developed to remove all restriction sites of two major RM systems of C. cellulovorans, Cce743I and Cce743II. The pYL001 plasmid remained intact after challenge by C. cellulovorans cell extract. Post-electroporation treatments and culturing conditions were also modified to improve cell growth and colony formation on agar plates. With the improvements, the pYL001 plasmid, without in vivo methylation, was readily transformed into C. cellulovorans with colonies of recombinant cells formed on agar plates within 24 h. Three pYL001-derived recombinant plasmids free of Cce743I/Cce743II restriction sites, after synonymous mutation of the heterologous genes, were constructed and transformed into C. cellulovorans. Functional expression of these genes was confirmed with butanol and ethanol production from glucose in batch fermentations by the transformants. The pYL001 plasmid and improved transformation method can facilitate further metabolic engineering of C. cellulovorans for cellulosic butanol production.

Keywords

Restriction modification system Clostridium cellulovorans Methylation Plasmid Transformation 

Notes

Acknowledgements

This material is based upon work supported by the Department of Energy’s Office of Energy Efficiency and Renewable Energy (EERE) under the Bioenergy Technologies Office, Award Number DE-EE0007005.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

253_2019_9882_MOESM1_ESM.pdf (485 kb)
ESM 1 (PDF 484 kb)

References

  1. Bao T, Zhao J, Li J, Liu X, Yang ST (2019) n-Butanol and ethanol production by Clostridium cellulovorans overexpressing aldehyde/alcohol dehydrogenases from Clostridium acetobutylicum. Bioresour Technol 285:121316CrossRefGoogle Scholar
  2. Bayliss CD, Callaghan MJ, Moxon ER (2006) High allelic diversity in the methyltransferase gene of a phase variable type III restriction-modification system has implications for the fitness of Haemophilus influenzae. Nucleic Acids Res 34:4046–4059CrossRefGoogle Scholar
  3. Bruder MR, Pyne ME, Moo-Young M, Chung DA, Chou CP (2016) Extending CRISPR-Cas9 technology from genome editing to transcriptional engineering in the genus Clostridium. Appl Environ Microbiol 82:6109–6119CrossRefGoogle Scholar
  4. Cui GZ, Hong W, Zhang J, Li WL, Feng Y, Liu YJ, Cui Q (2012) Targeted gene engineering in Clostridium cellulolyticum H10 without methylation. J Microbiol Methods 89(3):201–208CrossRefGoogle Scholar
  5. Croux C, Lee J, Raynaud C, Saint-Prix F, Gonzalez-Pajuelo M, Meynial-Salles I, Soucaille P (2016) Construction of a restriction-less, marker-less mutant useful for functional genomic and metabolic engineering of the biofuel producer Clostridium acetobutylicum. Biotechnol Biofuels 9(1):23CrossRefGoogle Scholar
  6. Diner BA, Fan J, Scotcher MC, Wells DH, Whited GM (2018) Synthesis of heterologous mevalonic acid pathway enzymes in Clostridium ljungdahlii for the conversion of fructose and of syngas to mevalonate and isoprene. Appl Environ Microbiol 84(1):e01723–e01717Google Scholar
  7. Dong H, Zhang Y, Dai Z, Li Y (2010) Engineering clostridium strain to accept unmethylated DNA. PLoS One 5(2):e9038CrossRefGoogle Scholar
  8. Elhai J, Vepritskiy A, Muro-Pastor AM, Flores E, Wolk CP (1997) Reduction of conjugal transfer efficiency by three restriction activities of Anabaena sp. strain PCC 7120. J Bacteriol 179:1998–2005CrossRefGoogle Scholar
  9. Gaida SM, Liedtke A, Jentges AH, Engels B, Jennewein S (2016) Metabolic engineering of Clostridium cellulolyticum for the production of n-butanol from crystalline cellulose. Microb Cell Factories 15(6)Google Scholar
  10. Guss AM, Olson DG, Caiazza NC, Lynd LR (2012) Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellum. Biotechnol Biofuels 5(1):30CrossRefGoogle Scholar
  11. Heap JT, Pennington OJ, Cartman ST, Minton NP (2009) A modular system for Clostridium shuttle plasmids. J Microbiol Methods 78(1):79–85CrossRefGoogle Scholar
  12. Higashide W, Li Y, Yang Y, Liao JC (2011) Metabolic engineering of Clostridium cellulolyticum for production of isobutanol from cellulose. Appl Environ Microbiol 77:2727–2733CrossRefGoogle Scholar
  13. Huang CN, Liebl W, Ehrenreich A (2018) Restriction-deficient mutants and marker-less genomic modification for metabolic engineering of the solvent producer Clostridium saccharobutylicum. Biotechnol Biofuels 11(1):264CrossRefGoogle Scholar
  14. Jennert KC, Tardif C, Young DI, Young M (2000) Gene transfer to Clostridium cellulolyticum ATCC 35319. Microbiology 146:3071–3080CrossRefGoogle Scholar
  15. Jiang WY, Zhao JB, Wang ZQ, Yang ST (2014) Stable high-titer n-butanol production from sucrose and sugarcane juice by Clostridium acetobutylicum JB200 in repeated batch fermentations. Bioresour Technol 163:172–179CrossRefGoogle Scholar
  16. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science:1225829Google Scholar
  17. Joseph RC, Kim NM, Sandoval NR (2018) Recent developments of the synthetic biology toolkit for Clostridium. Front Microbiol 9:154CrossRefGoogle Scholar
  18. Klapatch TR, Demain AL, Lynd LR (1996) Restriction endonuclease activity in Clostridium thermocellum and Clostridium thermosaccharolyticum. Appl Microbiol Biotechnol 45:127–131CrossRefGoogle Scholar
  19. Kolek J, Sedlar K, Provaznik I, Patakova P (2016) Dam and Dcm methylations prevent gene transfer into Clostridium pasteurianum NRRL B-598: development of methods for electrotransformation, conjugation, and sonoporation. Biotechnol Biofuels 9:14CrossRefGoogle Scholar
  20. Lee SY, Mermelstein LD, Bennett GN, Papoutsakis ET (1992) Vector construction, transformation, and gene amplification in Clostridium acetobutylicum ATCC 824. Ann N Y Acad Sci 665:39–51CrossRefGoogle Scholar
  21. Lesiak JM, Liebl W, Ehrenreich A (2014) Development of an in vivo methylation system for the solventogen Clostridium saccharobutylicum NCP 262 and analysis of two endonuclease mutants. J Biotechnol 188:97–99CrossRefGoogle Scholar
  22. Lin PP, Mi L, Moriok AH, Yoshino MM, Konishi S, Xu SC, Papanek BA, Riley LA, Guss AM, Liao JC (2015) Consolidated bioprocessing of cellulose to isobutanol using Clostridium thermocellum. Metab Eng 31:44–52CrossRefGoogle Scholar
  23. Lin YL, Blaschek HP (1984) Transformation of heat-treated Clostridium acetobutylicum protoplasts with pUB110 plasmid DNA. Appl Environ Microbiol 48:737–742Google Scholar
  24. Mermelstein LD, Welker NE, Bennett GN, Papoutsakis ET (1992) Expression of cloned homologous fermentative genes in Clostridium acetobutylicum ATCC 824. BioTechnology 10:190–195Google Scholar
  25. Molitor B, Kirchner K, Henrich AW, Schmitz S, Rosenbaum MA (2016) Expanding the molecular toolkit for the homoacetogen Clostridium ljungdahlii. Sci Report 6:31518CrossRefGoogle Scholar
  26. Oh YH, Eom GT, Kang KH, Choi JW, Song BK, Lee SH, Park SJ (2015) Optimized transformation of newly constructed Escherichia coli-Clostridia shuttle vectors into Clostridium beijerinckii. Appl Biochem Biotechnol 177:226–236CrossRefGoogle Scholar
  27. Ou JF, Xu NN, Ernst P, Ma C, Bush M, Goh K, Zhao JB, Zhou LF, Yang ST, Liu XG (2017) Process engineering of cellulosic n-butanol production from corn-based biomass using Clostridium cellulovorans. Process Biochem 62:144–150CrossRefGoogle Scholar
  28. Papanek B, Biswas R, Rydzak T, Guss AM (2015) Elimination of metabolic pathways to all traditional fermentation products increases ethanol yields in Clostridium thermocellum. Metab Eng 32:49–54CrossRefGoogle Scholar
  29. Purdy D, O'Keeffe TA, Elmore M, Herbert M, McLeod A, Bokori-Brown M, Ostrowski A, Minton NP (2002) Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier. Mol Microbiol 46:439–452CrossRefGoogle Scholar
  30. Pyne ME, Bruder M, Moo-Young M, Chung DA, Chou CP (2014) Technical guide for genetic advancement of underdeveloped and intractable Clostridium. Biotechnol Adv 32:623–641CrossRefGoogle Scholar
  31. Pyne ME, Moo-Young M, Chung DA, Chou CP (2013) Development of an electrotransformation protocol for genetic manipulation of Clostridium pasteurianum. Biotechnol Biofuels 6(1):50CrossRefGoogle Scholar
  32. Richards DF, Linnett PE, Oultram JD, Young M (1988) Restriction endonucleases in Clostridium pasteurianum ATCC 6013 and C. thermohydrosulfuricum DSM 568. J Gen Microbiol 134:3151–3157Google Scholar
  33. Roberts RJ, Vincze T, Posfai J, Macelis D (2015) REBASE-a database for DNA restriction and modification: enzymes, genes and genomes. Nucleic Acids Res 43:D298–D299CrossRefGoogle Scholar
  34. Rose RE (1988) The nucleotide sequence of pACYC184. Nucleic Acids Res 16:355CrossRefGoogle Scholar
  35. Tamaru Y, Miyake H, Kuroda K, Nakanishi A, Kawade Y, Yamamoto K, Uemura M, Fujita Y, Doi RH, Ueda M (2010) Genome dequence of the cellulosome-producing mesophilic organism Clostridium cellulovorans 743B. J Bacteriol 192(3):901–902CrossRefGoogle Scholar
  36. Tian L, Papanek B, Olson DG, Rydzak T, Holwerda EK, Zheng T, Zhou J, Maloney M, Jiang N, Giannone RJ, Hettich RL, Guss AM, Lynd LR (2016) Simultaneous achievement of high ethanol yield and titer in Clostridium thermocellum. Biotechnol Biofuels 9:116CrossRefGoogle Scholar
  37. Wang J, Yang X, Chen C-C, Yang ST (2014) Engineering clostridia for butanol production from biorenewable resources: from cells to process integration. Curr Opin Chem Eng 6:43–54CrossRefGoogle Scholar
  38. Wang SH, Dong S, Wang PX, Tao Y, Wang Y (2017) Genome editing in Clostridium saccharoperbutylacetonicum N1-4 with the CRISPR-Cas9 system. Appl Environ Microbiol 83(10):e00233–e00217CrossRefGoogle Scholar
  39. Xu T, Li Y, Shi Z, Hemme CL, Li Y, Zhu Y, Van Nostrand JD, He Z, Zhou J (2015) Efficient genome editing in Clostridium cellulolyticum via CRISPR-Cas9 nickase. Appl Environ Microbiol AEM:00873Google Scholar
  40. Yang X, Xu M, Yang ST (2015) Metabolic and process engineering of Clostridium cellulovorans for biofuel production from cellulose. Metab Eng 32:39–48CrossRefGoogle Scholar
  41. Yang XR, Xu MM, Yang ST (2016) Restriction modification system analysis and development of in vivo methylation for the transformation of Clostridium cellulovorans. Appl Microbiol Biotechnol 100:2289–2299CrossRefGoogle Scholar
  42. Yu M, Zhang Y, Tang IC, Yang ST (2011) Metabolic engineering of Clostridium tyrobutyricum for n-butanol production. Metab Eng 13:373–382CrossRefGoogle Scholar
  43. Yu M, Du Y, Jiang W, Chang W-L, Yang ST, Tang IC (2012) Effects of different replicons in conjugative plasmids on transformation efficiency, plasmid stability, gene expression and n-butanol biosynthesis in Clostridium tyrobutyricum. Appl Microbiol Biotechnol 93:881–889CrossRefGoogle Scholar
  44. Yu L, Xu M, Tang I-C, Yang ST (2015) Metabolic engineering of Clostridium tyrobutyricum for n-butanol production through co-utilization of glucose and xylose. Biotechnol Bioeng 112:2134–2141CrossRefGoogle Scholar
  45. Zhang J, Zong WM, Hong W, Zhang ZT, Wang Y (2018) Exploiting endogenous CRISPR-Cas system for multiplex genome editing in Clostridium tyrobutyricum and engineer the strain for high-level butanol production. Metab Eng 47:49–59CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.William G. Lowrie Department of Chemical and Biomolecular EngineeringThe Ohio State UniversityColumbusUSA

Personalised recommendations