Applied Microbiology and Biotechnology

, Volume 99, Issue 2, pp 1011–1022 | Cite as

Engineering Clostridium acetobutylicum with a histidine kinase knockout for enhanced n-butanol tolerance and production

  • Mengmeng Xu
  • Jingbo Zhao
  • Le Yu
  • I-Ching Tang
  • Chuang Xue
  • Shang-Tian YangEmail author
Bioenergy and biofuels


Clostridium acetobutylicum JB200, a mutant strain of C. acetobutylicum ATCC 55025 obtained through strain evolution in a fibrous bed bioreactor, had high butanol tolerance and produced up to ~21 g/L butanol from glucose in batch fermentation, an improvement of ~67 % over the parental strain (~12.6 g/L). Comparative genomic analysis revealed a single-base deletion in the cac3319 gene leading to C-terminal truncation in its encoding histidine kinase (HK) in JB200. To study the effects of cac3319 mutation on cell growth and fermentation, the cac3319 gene in ATCC 55025 was disrupted using the ClosTron group II intron-based gene inactivation system. Compared to ATCC 55025, the cac3319 HK knockout mutant, HKKO, produced 44.4 % more butanol (18.2 ± 1.3 vs. 12.6 ± 0.2 g/L) with a 90 % higher productivity (0.38 ± 0.03 vs. 0.20 ± 0.02 g/L h) due to increased butanol tolerance, confirming, for the first time, that cac3319 plays an important role in regulating solvent production and tolerance in C. acetobutylicum. This work also provides a novel metabolic engineering strategy for generating high-butanol-tolerant and high-butanol-producing strains for industrial applications.


ABE fermentation Butanol tolerance C. acetobutylicum Histidine kinase Gene knockout Metabolic engineering 



This work was supported in part by the National Science Foundation STTR program (Grant No. IIP-1026648). We would like to thank Prof. N.P. Minton and Dr. J.T. Heap (University of Nottingham, UK) for providing the plasmid pAN2 and Hopen Yang for copyediting the manuscript.

Supplementary material

253_2014_6249_MOESM1_ESM.pdf (53 kb)
ESM 1 (PDF 53 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Mengmeng Xu
    • 1
  • Jingbo Zhao
    • 1
  • Le Yu
    • 1
  • I-Ching Tang
    • 2
  • Chuang Xue
    • 3
  • Shang-Tian Yang
    • 1
    Email author
  1. 1.William G. Lowrie Department of Chemical and Biomolecular EngineeringThe Ohio State UniversityColumbusUSA
  2. 2.Bioprocessing Innovative CompanyDublinUSA
  3. 3.Department of Life Science and BiotechnologyDalian University of TechnologyDalianChina

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