Applied Microbiology and Biotechnology

, Volume 102, Issue 18, pp 8107–8119 | Cite as

Improving the fermentation performance of Clostridium acetobutylicum ATCC 824 by strengthening the VB1 biosynthesis pathway

  • Zhengping Liao
  • Yukai Suo
  • Chuang Xue
  • Hongxin FuEmail author
  • Jufang WangEmail author
Bioenergy and biofuels


Vitamin B1 (VB1) is an essential coenzyme for carbohydrate metabolism and involved in energy generation in most organisms. In this study, we found that insufficient biosynthesis of VB1 in Clostridium acetobutylicum ATCC 824 is a major limiting factor for efficient acetone-butanol-ethanol (ABE) fermentation. In order to improve the fermentation performance of C. acetobutylicum ATCC 824, the VB1 biosynthesis pathway was strengthened by overexpressing the thiC, thiG, and thiE genes. The engineered strain 824(thiCGE) showed enhanced VB1 and energy synthesis, resulting in better growth, faster sugar consumption, higher solvents production, and lower acids formation than the wild-type strain in both VB1 free and normal P2 medium (1 mg/L). Compared with the wild-type strain, 824(thiCGE) produced 13.0 ± 0.1% or 12.7 ± 1.2% more butanol in VB1 free P2 medium when glucose or xylose was used as the substrate, respectively. When mixed sugar (glucose:xylose = 2:1) was used as the substrate in VB1 free P2 medium, the xylose consumption rate and butanol titer of 824(thiCGE) were 45.8 ± 1.9% and 20.4 ± 0.3% higher than those of the wild-type strain. All these results demonstrated that this metabolic engineering strategy could provide a new and effective way to improve the cellular performance of solventogenic clostridia. In addition, it may have some potential application value in ABE fermentation using simple medium and/or lignocellulosic biomass.


Clostridium acetobutylicum Vitamin B1 biosynthesis Carbohydrate metabolism Acetone-butanol-ethanol fermentation 



This work was funded by the National Natural Science Foundation of China (21676098), the Fundamental Research Funds for the Central Universities (2017PY013, 2017BQ084), and the China Postdoctoral Science Foundation Funded Project (2017M612667).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. Aristidou A, Penttilä M (2000) Metabolic engineering applications to renewable resource utilization. Curr Opin Biotechnol 11(2):187–198. CrossRefPubMedGoogle Scholar
  2. Backstrom AD, McMordie RAS, Begley TP (1995) Biosynthesis of thiamin I: the function of the thiE gene product. J Am Chem Soc 117(8):2351–2352. CrossRefGoogle Scholar
  3. Baer SH, Blaschek HP, Smith TL (1987) Effect of butanol challenge and temperature on lipid composition and membrane fluidity of butanol-tolerant Clostridium acetobutylicum. Appl Environ Microbiol 53(12):2854–2861. PubMedPubMedCentralGoogle Scholar
  4. Bazurto JV, Heitman NJ, Downs DM (2015) Aminoimidazole carboxamide ribotide exerts opposing effects on thiamine synthesis in Salmonella enterica. J Bacteriol 197(17):2821–2830. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Begley TP, Downs D, Ealick SE, McLafferty FW, Van Loon AP, Taylor S, Campobasso N, Chiu H-J, Kinsland C, Reddick JJ, Xi J (1999) Thiamin biosynthesis in prokaryotes. Arch Microbiol 171:293–300. CrossRefPubMedGoogle Scholar
  6. Dong W, Stockwell VO, Goyer A (2015) Enhancement of thiamin content in Arabidopsis thaliana by metabolic engineering. Plant Cell Physiol 56(12):2285–2296. CrossRefPubMedGoogle Scholar
  7. Ezeji TC, Qureshi N, Blaschek HP (2004a) Acetone butanol ethanol (ABE) production from concentrated substrate: reduction in substrate inhibition by fed-batch technique and product inhibition by gas stripping. Appl Microbiol Biotechnol 63(6):653–658. CrossRefPubMedGoogle Scholar
  8. Ezeji TC, Qureshi N, Blaschek HP (2004b) Butanol fermentation research: upstream and downstream manipulations. Chem Rec 4(5):305–314. CrossRefPubMedGoogle Scholar
  9. Formanek J, Mackie R, Blaschek HP (1997) Enhanced butanol production by Clostridium beijerinckii BA101 grown in semidefined P2 medium containing 6 percent maltodextrin or glucose. Appl Environ Microbiol 63(6):2306–2310. PubMedPubMedCentralGoogle Scholar
  10. Frank RA, Leeper FJ, Luisi BF (2007) Structure, mechanism and catalytic duality of thiamine-dependent enzymes. Cell Mol Life Sci 64(7–8):892–905. CrossRefPubMedGoogle Scholar
  11. Groot' WJ, van den Qever CE, Kossen NWF (1984) Pervaporation for simultaneous product recovery in the butanol/isopropanol batch fermentation. Biotechnol Lett 6(11):709–714. CrossRefGoogle Scholar
  12. Gu Y, Li J, Zhang L, Chen J, Niu L, Yang Y, Yang S, Jiang W (2009) Improvement of xylose utilization in Clostridium acetobutylicum via expression of the talA gene encoding transaldolase from Escherichia coli. J Biotechnol 143(4):284–287. CrossRefPubMedGoogle Scholar
  13. Guo T, Tang Y, Xi YL, He AY, Sun BJ, Wu H, Liang DF, Jiang M, Ouyang PK (2011) Clostridium beijerinckii mutant obtained by atmospheric pressure glow discharge producing high proportions of butanol and solvent yields. Biotechnol Lett 33(12):2379–2383. CrossRefPubMedGoogle Scholar
  14. Harris LM, DR P, WN E, PE T (2000) Characterization of recombinant strains of the Clostridium acetobutylicum butyrate kinase inactivation mutant: need for new phenomenological models for solventogenesis and butanol inhibition? Biotechnol Bioeng 67(1):1–11. CrossRefPubMedGoogle Scholar
  15. Harris L, Blank L, Desai R, Welker N, Papoutsakis E (2001) Fermentation characterization and flux analysis of recombinant strains of Clostridium acetobutylicum with an inactivated solR gene. J Ind Microbiol Biotechnol 27(5):322–328. CrossRefPubMedGoogle Scholar
  16. 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(3):452–464. CrossRefPubMedGoogle Scholar
  17. Heap JT, Pennington OJ, Cartman ST, Minton NP (2009) A modular system for Clostridium shuttle plasmids. J Microbiol Methods 78(1):79–85. CrossRefPubMedGoogle Scholar
  18. Isar J, Rangaswamy V (2012) Improved n-butanol production by solvent tolerant Clostridium beijerinckii. Biomass Bioenergy 37:9–15. CrossRefGoogle Scholar
  19. Jang Y-S, Lee JY, Lee J, Park JH, Im JA, Eom M-H, Lee J, Lee S-H, Song H, Cho J-H, Seung DY, Lee SY (2012) Enhanced butanol production obtained by reinforcing the direct butanol-forming route in Clostridium acetobutylicum. mBio 3(5):e00314–e00312. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Jin L, Zhang H, Chen L, Yang C, Yang S, Jiang W, Gu Y (2014) Combined overexpression of genes involved in pentose phosphate pathway enables enhanced D-xylose utilization by Clostridium acetobutylicum. J Biotechnol 173:7–9. CrossRefPubMedGoogle Scholar
  21. Kelleher NL, Taylor SV, Grannis D, Kinsland C, Chiu HJ, Begley TP, McLafferty FW (1998) Efficient sequence analysis of the six gene products (7-74 kDa) from the Escherichia coli thiamin biosynthetic operon by tandem high-resolution mass spectrometry. Protein Sci 7(8):1796–1801. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Lee SY, Park JH, Jang SH, Nielsen LK, Kim J, Jung KS (2008) Fermentative butanol production by clostridia. Biotechnol Bioeng 101(2):209–228. CrossRefPubMedGoogle Scholar
  23. Liao Z, Zhang Y, Luo S, Suo Y, Zhang S, Wang J (2017) Improving cellular robustness and butanol titers of Clostridium acetobutylicum ATCC824 by introducing heat shock proteins from an extremophilic bacterium. J Biotechnol 252:1–10. CrossRefPubMedGoogle Scholar
  24. Lütke-Eversloh T, Bahl H (2011) Metabolic engineering of Clostridium acetobutylicum: recent advances to improve butanol production. Curr Opin Biotechnol 22(5):634–647. CrossRefPubMedGoogle Scholar
  25. Manzetti S, Zhang J, van der Spoel D (2014) Thiamin function, metabolism, uptake, and transport. Biochemistry 53(5):821–835. CrossRefPubMedGoogle Scholar
  26. Medina-Silva R, Barros MP, Galhardo RS, Netto LE, Colepicolo P, Menck CF (2006) Heat stress promotes mitochondrial instability and oxidative responses in yeast deficient in thiazole biosynthesis. Res Microbiol 157(3):275–281. CrossRefPubMedGoogle Scholar
  27. Nair RV, Green EM, Watson DE, Bennett GN, Papoutsakis ET (1999) Regulation of the sol locus genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824 by a putative transcriptional repressor. J Bacteriol 181(1):319–330. PubMedPubMedCentralGoogle Scholar
  28. Nolling J, Breton G, Omelchenko MV, Makarova KS, Zeng Q, Gibson R, Lee HM, Dubois J, Qiu D, Hitti J, Production GTCSC, Finishing BT, Wolf YI, Tatusov RL, Sabathe F, Doucette-Stamm L, Soucaille P, Daly MJ, Bennett GN, Koonin EV, Smith DR (2001) Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. J Bacteriol 183(16):4823–4838. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Rapala-Kozik M, Kowalska E, Ostrowska K (2008) Modulation of thiamine metabolism in Zea mays seedlings under conditions of abiotic stress. J Exp Bot 59(15):4133–4143. CrossRefPubMedGoogle Scholar
  30. Settembre E, Begley TP, Ealick SE (2003) Structural biology of enzymes of the thiamin biosynthesis pathway. Curr Opin Struct Biol 13(6):739–747. CrossRefPubMedGoogle Scholar
  31. Shi A, Zhu X, Lu J, Zhang X, Ma Y (2013) Activating transhydrogenase and NAD kinase in combination for improving isobutanol production. Metab Eng 16:1–10. CrossRefPubMedGoogle Scholar
  32. Tittmann K (2009) Reaction mechanisms of thiamin diphosphate enzymes: redox reactions. FEBS J 276(9):2454–2468. CrossRefPubMedGoogle Scholar
  33. Tomas CA, Welker NE, Papoutsakis ET (2003) Overexpression of groESL in Clostridium acetobutylicum results in increased solvent production and tolerance, prolonged metabolism, and changes in the cell’s transcriptional program. Appl Environ Microbiol 69(8):4951–4965. CrossRefPubMedPubMedCentralGoogle Scholar
  34. Tseng GC, Oh M-K, Rohlin L, Liao JC, Wong WH (2001) Issues in cDNA microarray analysis: quality filtering, channel normalization, models of variations and assessment of gene effects. Nucleic Acids Res 29(12):2549–2557. CrossRefPubMedPubMedCentralGoogle Scholar
  35. Tunc-Ozdemir M, Miller G, Song L, Kim J, Sodek A, Koussevitzky S, Misra AN, Mittler R, Shintani D (2009) Thiamin confers enhanced tolerance to oxidative stress in Arabidopsis. Plant Physiol 151(1):421–432. CrossRefPubMedPubMedCentralGoogle Scholar
  36. van der Graaff E, Hooykaas P, Lein W, Lerchl J, Kunze G, Sonnewald U, Boldt R (2004) Molecular analysis of “de novo” purine biosynthesis in solanaceous species and in Arabidopsis thaliana. Front Biosci 9:1803–1816. CrossRefPubMedGoogle Scholar
  37. Ventura J-RS, Hu H, Jahng D (2013) Enhanced butanol production in Clostridium acetobutylicum ATCC 824 by double overexpression of 6-phosphofructokinase and pyruvate kinase genes. Appl Microbiol Biotechnol 97(16):7505–7516. CrossRefPubMedGoogle Scholar
  38. Wang L, Xia M, Zhang L, Chen H (2014) Promotion of the Clostridium acetobutylicum ATCC 824 growth and acetone–butanol–ethanol fermentation by flavonoids. World J Microbiol Biotechnol 30(7):1969–1976. CrossRefPubMedGoogle Scholar
  39. Webb E, Febres F, Downs DM (1996) Thiamine pyrophosphate (TPP) negatively regulates transcription of some thi genes of Salmonella typhimurium. J Bacteriol 178(9):2533–2538. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Wolak N, Tomasi M, Kozik A, Rapala-Kozik M (2015) Characterization of thiamine uptake and utilization in Candida spp. subjected to oxidative stress. Acta Biochim Pol 62(3):445–455. CrossRefPubMedGoogle Scholar
  41. Xu G-Q, Chu J, Zhuang Y-P, Wang Y-H, Zhang S-L (2008) Effects of vitamins on the lactic acid biosynthesis of Lactobacillus paracasei NERCB 0401. Biochem Eng J 38(2):189–197. CrossRefGoogle Scholar
  42. Xue C, Zhao J, Lu C, Yang S-T, Bai F, Tang IC (2012) High-titer n-butanol production by Clostridium acetobutylicum JB200 in fed-batch fermentation with intermittent gas stripping. Biotechnol Bioeng 109:2746–2756. CrossRefPubMedGoogle Scholar
  43. Yang ST, Zhao J (2013) Adaptive engineering of Clostridium for increased butanol production. US Patent 8450093Google Scholar
  44. Yang Y, Lang N, Yang G, Yang S, Jiang W, Gu Y (2016) Improving the performance of solventogenic clostridia by reinforcing the biotin synthetic pathway. Metab Eng 35:121–128. CrossRefPubMedGoogle Scholar
  45. Yu X, Liang X, Liu K, Dong W, Wang J, Zhou M-g (2015) The thiG Gene Is Required for Full Virulence of Xanthomonas oryzae pv. oryzae by Preventing Cell Aggregation. PLoS ONE 10(7):e0134237.
  46. Zeng WY, Tang YQ, Gou M, Sun ZY, Xia ZY, Kida K (2017) Comparative transcriptomes reveal novel evolutionary strategies adopted by Saccharomyces cerevisiae with improved xylose utilization capability. Appl Microbiol Biotechnol 101(4):1753–1767. CrossRefPubMedGoogle Scholar
  47. Zhang YI, Taylor S, Chiu H-J, P Begley T (1997) Characterization of the Bacillus subtilis thiC operon involved in thiamine biosynthesis. J Bacteriol 179:3030–3035. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Zhu L, Dong H, Zhang Y, Li Y (2011) Engineering the robustness of Clostridium acetobutylicum by introducing glutathione biosynthetic capability. Metab Eng 13(4):426–434. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.School of Biology and Biological EngineeringSouth China University of TechnologyGuangzhouChina
  2. 2.School of Life Science and BiotechnologyDalian University of TechnologyDalianChina
  3. 3.State Key Laboratory of Pulp and Paper EngineeringSouth China University of TechnologyGuangzhouChina

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