Applied Microbiology and Biotechnology

, Volume 98, Issue 1, pp 313–323 | Cite as

Improvement of ClosTron for successive gene disruption in Clostridium cellulolyticum using a pyrF-based screening system

  • Gu-Zhen Cui
  • Jie Zhang
  • Wei Hong
  • Chenggang Xu
  • Yingang Feng
  • Qiu Cui
  • Ya-Jun Liu
Applied genetics and molecular biotechnology


Clostridium includes a number of species, such as thermophilic Clostridium thermocellum and mesophilic Clostridium cellulolyticum, producing biofuels and chemicals from lignocellulose, while genetic engineering is necessary to improve wild-type strains to fulfill the requirement of industrialization. ClosTron system is widely used in the gene targeting of Clostridium because of its high efficiency and operability. However, the targetron plasmid present in cell hinders the successive gene disruption. To solve this problem, a pyrF-based screening system was developed and implemented in C. cellulolyticum strain H10 in this study for efficient targetron plasmid curing. The screening system was composed of a pyrF-deleted cell chassis (H10ΔpyrF) constructed via homologous recombination and a PyrF expression cassette located in a targetron plasmid containing an erythromycin resistance gene. With the screening system, the gene targeting could be achieved following a two-step procedure, including the first step of gene disruption through targetron transformation and erythromycin selection and the second step of plasmid curing by screening with 5-fluoroorotic acid. To test the developed screening system, successive inactivation of the major cellulosomal exocellulase Cel48F and the scaffoldin protein CipC was achieved in C. cellulolyticum, and the efficient plasmid curing was confirmed. With the assistance of the pyrF-based screening system, the targetron plasmid-cured colonies can be rapidly selected by one-plate screening instead of traditional days' unguaranteed screening, and the successive gene disruption becomes accomplishable with ClosTron system with improved stability and efficiency, which may promote the metabolic engineering of Clostridium species aiming at enhanced production of biofuels and chemicals.


Clostridium cellulolyticum ClosTron Targetron Plasmid curing PyrF 



We thank Dr. Weihong Jiang and Dr. Sheng Yang from the Institute of Plant Physiology and Ecology, Shanghai, People's Republic of China for providing plasmid pSY6. We thank Dr. Yin Li and Dr. Hongjun Dong from the Institute of Microbiology, Chinese Academy of Sciences, Beijing, People's Republic of China for the helpful discussion. This work was supported by the National Basic Research Program of China (973 Program, grant 2011CB707404), the Key Technologies R&D Program from the Ministry of Science and Technology of China (grant 2011BAD22B02), and the Instrument Developing Project of the Chinese Academy of Sciences (grant no. YZ201138).

Supplementary material

253_2013_5330_MOESM1_ESM.pdf (234 kb)
ESM 1 (PDF 233 kb)


  1. Abdou L, Boileau C, de Philip P, Pages S, Fierobe HP, Tardif C (2008) Transcriptional regulation of the Clostridium cellulolyticum cip-cel operon: a complex mechanism involving a catabolite-responsive element. J Bacteriol 190:1499–1506PubMedCentralPubMedCrossRefGoogle Scholar
  2. Berzin V, Kiriukhin M, Tyurin M (2013) “Curing” of plasmid DNA in acetogen using microwave or applying an electric pulse improves cell growth and metabolite production as compared to the plasmid-harboring strain. Arch Microbiol 195:181–188PubMedCrossRefGoogle Scholar
  3. Boeke JD, LaCroute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197:345–346PubMedCrossRefGoogle Scholar
  4. Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  5. Brana H, Benada O, Navratil O, Cejka K, Hubacek J (1983) Stability of the hybrid plasmid pIM138 and its curing by some eliminating agents. Folia Microbiol (Praha) 28:441–445CrossRefGoogle Scholar
  6. Chen Y, McClane BA, Fisher DJ, Rood JI, Gupta P (2005) Construction of an alpha toxin gene knockout mutant of Clostridium perfringens type A by use of a mobile group II intron. Appl Environ Microbiol 71:7542–7547PubMedCentralPubMedCrossRefGoogle Scholar
  7. Chung D, Farkas J, Westpheling J (2013) Overcoming restriction as a barrier to DNA transformation in Caldicellulosiruptor species results in efficient marker replacement. Biotechnol Biofuels 6:82PubMedCentralPubMedCrossRefGoogle Scholar
  8. 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 89:201–208PubMedCrossRefGoogle Scholar
  9. Desvaux M (2005) Clostridium cellulolyticum: model organism of mesophilic cellulolytic clostridia. FEMS Microbiol Rev 29:741–764PubMedCrossRefGoogle Scholar
  10. Desvaux M, Guedon E, Petitdemange H (2000) Cellulose metabolism by Clostridium cellulolyticum growing in batch culture on defined medium. Appl Environ Microbiol 66:2461–2470PubMedCentralPubMedCrossRefGoogle Scholar
  11. Dong H, Zhang Y, Dai Z, Li Y (2010) Engineering Clostridium strain to accept unmethylated DNA. PLoS ONE 5:e9038PubMedCentralPubMedCrossRefGoogle Scholar
  12. Enyeart PJ, Chirieleison SM, Dao MN, Perutka J, Quandt EM, Yao J, Whitt JT, Keatinge-Clay AT, Lambowitz AM, Ellington AD (2013) Generalized bacterial genome editing using mobile group II introns and Cre-lox. Mol Syst Biol 9:685PubMedCentralPubMedCrossRefGoogle Scholar
  13. Frazier CL, San Filippo J, Lambowitz AM, Mills DA (2003) Genetic manipulation of Lactococcus lactis by using targeted group II introns: generation of stable insertions without selection. Appl Environ Microbiol 69:1121–1128PubMedCentralPubMedCrossRefGoogle Scholar
  14. Guedon E, Desvaux M, Petitdemange H (2002) Improvement of cellulolytic properties of Clostridium cellulolyticum by metabolic engineering. Appl Environ Microb 68:53–58CrossRefGoogle Scholar
  15. Heap JT, Ehsaan M, Cooksley CM, Ng YK, Cartman ST, Winzer K, Minton NP (2012) Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker. Nucleic Acids Res 40:e59PubMedCentralPubMedCrossRefGoogle Scholar
  16. Heap JT, Kuehne SA, Ehsaan M, Cartman ST, Cooksley CM, Scott JC, Minton NP (2010) The ClosTron: mutagenesis in Clostridium refined and streamlined. J Microbiol Methods 80:49–55PubMedCrossRefGoogle Scholar
  17. Heap JT, Pennington OJ, Cartman ST, Carter GP, Minton NP (2007) The ClosTron: a universal gene knock-out system for the genus Clostridium. J Microbiol Methods 70:452–464PubMedCrossRefGoogle Scholar
  18. Higashide W, Li Y, Yang Y, Liao JC (2011) Metabolic engineering of Clostridium cellulolyticum for isobutanol production from cellulose. Appl Environ Microbiol 77:2727–2733PubMedCentralPubMedCrossRefGoogle Scholar
  19. Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68PubMedCrossRefGoogle Scholar
  20. Hovi M, Sukupolvi S, Edwards MF, Rhen M (1988) Plasmid-associated virulence of Salmonella enteritidis. Microb Pathog 4:385–391PubMedCrossRefGoogle Scholar
  21. Jennert KC, Tardif C, Young DI, Young M (2000) Gene transfer to Clostridium cellulolyticum ATCC 35319. Microbiology 146:3071–3080PubMedGoogle Scholar
  22. Jia K, Zhu Y, Zhang Y, Li Y (2011) Group II intron-anchored gene deletion in Clostridium. PLoS ONE 6(1):e16693PubMedCentralPubMedCrossRefGoogle Scholar
  23. Karberg M, Guo H, Zhong J, Coon R, Perutka J, Lambowitz AM (2001) Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria. Nat Biotech 19:1162–1167CrossRefGoogle Scholar
  24. Kuehne SA, Heap JT, Cooksley CM, Cartman ST, Minton NP (2011) ClosTron-mediated engineering of Clostridium. Methods Mol Biol 765:389–407PubMedCrossRefGoogle Scholar
  25. Lambowitz AM, Zimmerly S (2011) Group II introns: mobile ribozymes that invade DNA. Cold Spring Harb Perspect Biol 3(8):a003616Google Scholar
  26. Li Y, Tschaplinski TJ, Engle NL, Hamilton CY, Rodriguez M Jr, Liao JC, Schadt CW, Guss AM, Yang Y, Graham DE (2012) Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations. Biotechnol Biofuels 5:2PubMedCentralPubMedCrossRefGoogle Scholar
  27. Lin L, Song H, Ji Y, He Z, Pu Y, Zhou J, Xu J (2010) Ultrasound-mediated DNA transformation in thermophilic gram-positive anaerobes. PLoS ONE 5:e12582PubMedCentralPubMedCrossRefGoogle Scholar
  28. Liu H, Han J, Liu X, Zhou J, Xiang H (2011) Development of pyrF-based gene knockout systems for genome-wide manipulation of the archaea Haloferax mediterranei and Haloarcula hispanica. J Genet Genomics 38:261–269PubMedCrossRefGoogle Scholar
  29. Lynd LR, van Zyl WH, McBride JE, Laser M (2005) Consolidated bioprocessing of cellulosic biomass: an update. Curr Opin Biotechnol 16:577–583PubMedCrossRefGoogle Scholar
  30. Maamar H, Abdou L, Boileau C, Valette O, Tardif C (2006) Transcriptional analysis of the cip-cel gene cluster from Clostridium cellulolyticum. J Bacteriol 188:2614–2624PubMedCentralPubMedCrossRefGoogle Scholar
  31. Maamar H, Valette O, Fierobe HP, Belaich A, Belaich JP, Tardif C (2004) Cellulolysis is severely affected in Clostridium cellulolyticum strain cipCMut1. Mol Microbiol 51:589–598PubMedCrossRefGoogle Scholar
  32. Olson DG, Tripathi SA, Giannone RJ, Lo J, Caiazza NC, Hogsett DA, Hettich RL, Guss AM, Dubrovsky G, Lynd LR (2010) Deletion of the Cel48S cellulase from Clostridium thermocellum. Proc Natl Acad Sci U S A 107:17727–17732PubMedCentralPubMedCrossRefGoogle Scholar
  33. Pavlostathis SG, Miller TL, Wolin MJ (1988) Fermentation of insoluble cellulose by continuous cultures of Ruminococcus albus. Appl Environ Microb 54:2655–2659Google Scholar
  34. Perret S, Maamar H, Belaich JP, Tardif C (2004) Use of antisense RNA to modify the composition of cellulosomes produced by Clostridium cellulolyticum. Mol Microbiol 51:599–607PubMedCrossRefGoogle Scholar
  35. Rattanachaikunsopon P, Phumkhachorn P (2009) Glass bead transformation method for Gram-positive bacteria. Braz J Microbiol 40:923–926PubMedCrossRefGoogle Scholar
  36. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  37. Sato T, Fukui T, Atomi H, Imanaka T (2005) Improved and versatile transformation system allowing multiple genetic manipulations of the hyperthermophilic archaeon Thermococcus kodakaraensis. Appl Environ Microbiol 71:3889–3899PubMedCentralPubMedCrossRefGoogle Scholar
  38. Shao L, Hu S, Yang Y, Gu Y, Chen J, Jiang W, Yang S (2007) Targeted gene disruption by use of a group II intron (targetron) vector in Clostridium acetobutylicum. Cell Res 17:963–965PubMedCrossRefGoogle Scholar
  39. Spengler G, Molnar A, Schelz Z, Amaral L, Sharples D, Molnar J (2006) The mechanism of plasmid curing in bacteria. Curr Drug Targets 7:823–841PubMedCrossRefGoogle Scholar
  40. Suzuki H, Murakami A, Yoshida K (2012) Counterselection system for Geobacillus kaustophilus HTA426 through disruption of pyrF and pyrR. Appl Environ Microbiol 78:7376–7383PubMedCentralPubMedCrossRefGoogle Scholar
  41. Svetlitchnyi VA, Kensch O, Falkenhan DA, Korseska SG, Lippert N, Prinz M, Sassi J, Schickor A, Curvers S (2013) Single-step ethanol production from lignocellulose using novel extremely thermophilic bacteria. Biotechnol Biofuels 6:31PubMedCentralPubMedCrossRefGoogle Scholar
  42. Tardif C, Maamar H, Balfin M, Belaich JP (2001) Electrotransformation studies in Clostridium cellulolyticum. J Ind Microbiol Biot 27:271–274CrossRefGoogle Scholar
  43. Tripathi SA, Olson DG, Argyros DA, Miller BB, Barrett TF, Murphy DM, McCool JD, Warner AK, Rajgarhia VB, Lynd LR, Hogsett DA, Caiazza NC (2010) Development of pyrF-based genetic system for targeted gene deletion in Clostridium thermocellum and creation of a pta mutant. Appl Environ Microbiol 76:6591–6599PubMedCentralPubMedCrossRefGoogle Scholar
  44. Uhlin BE, Nordstrom K (1975) Plasmid incompatibility and control of replication: copy mutants of the R-factor R1 in Escherichia coli K-12. J Bacteriol 124:641–649PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  1. 1.Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of BioEnergy and Bioprocess TechnologyChinese Academy of SciencesQingdaoPeople’s Republic of China
  2. 2.Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess TechnologyChinese Academy of SciencesQingdaoPeople’s Republic of China
  3. 3.University of Chinese Academy of SciencesBeijingPeople’s Republic of China

Personalised recommendations