Applied Microbiology and Biotechnology

, Volume 98, Issue 13, pp 5823–5837 | Cite as

Application of new metabolic engineering tools for Clostridium acetobutylicum

  • Tina Lütke-EverslohEmail author


The renewed interests in clostridial acetone-butanol-ethanol (ABE) fermentation as a next-generation biofuel source led to significantly intensified research in the past few years. This mini-review focuses on the current status of metabolic engineering techniques available for the model organism of ABE fermentation, Clostridium acetobutylicum. A comprehensive survey of various application examples covers two general issues related to both basic and applied research questions: (i) how to improve biofuel production and (ii) what information can be deduced from respective genotype/phenotype manipulations. Recently developed strategies to engineer C. acetobutylicum are summarized including the current portfolio of altered gene expression methodologies, as well as systematic (rational) and explorative (combinatorial) metabolic engineering approaches.


Biofuel Butanol Clostridia Knockout Mutagenesis Screening 


  1. Al-Hinai MA, Fast AG, Papoutsakis ET (2012) Novel system for efficient isolation of Clostridium double-crossover allelic exchange mutants enabling markerless chromosomal gene deletions and DNA integration. Appl Environ Microbiol 78(22):8112–8121PubMedCentralPubMedGoogle Scholar
  2. Al-Hinai MA, Jones MAA, Papoutsakis ET (2014) σK of Clostridium acetobutylicum is the first known sporulation-specific sigma factor with two developmentally separated roles, one early and one late in sporulation. J Bacteriol 196(2):287–299Google Scholar
  3. Alper H, Stephanopoulos G (2009) Engineering for biofuels: exploiting innate microbial capacity or importing biosynthetic potential? Nat Rev Microbiol 7(10):715–723PubMedGoogle Scholar
  4. Alsaker KV, Paredes C, Papoutsakis ET (2010) Metabolite stress and tolerance in the production of biofuels and chemicals: gene-expression-based systems analysis of butanol, butyrate, and acetate stresses in the anaerobe Clostridium acetobutylicum. Biotechnol Bioeng 105(6):1131–1147PubMedGoogle Scholar
  5. Bankar SB, Survase SA, Ojamo H, Granström T (2013) Biobutanol: the outlook of an academic and industrialist. RSC Adv 3(47):24734–24757Google Scholar
  6. Berezina OV, Zakharova NV, Yarotsky CV, Zverlov VV (2012) Microbial producers of butanol. Appl Biochem Microbiol 48(7):625–638Google Scholar
  7. Bi C, Jones SW, Hess DR, Tracy MBP, Papoutsakis ET (2011) SpoIIE is necessary for asymmetric division, sporulation, and expression of σF, σE, and σG but does not control solvent production in Clostridium acetobutylicum ATCC 824. J Bacteriol 193(19):5130–5137PubMedCentralPubMedGoogle Scholar
  8. Biot-Pelletier D, Martin VJ (2014) Evolutionary engineering by genome shuffling. Appl Microbiol Biotechnol. doi: 10.1007/s00253-014-5616-8 PubMedGoogle Scholar
  9. Bond-Watts BB, Bellerose RJ, Chang MCY (2011) Enzyme mechanism as a kinetic control element for designing synthetic biofuel pathways. Nat Chem Biol 7(4):222–227PubMedGoogle Scholar
  10. Borden JR, Papoutsakis ET (2007) Dynamics of genomic-library enrichment and identification of solvent tolerance genes for Clostridium acetobutylicum. Appl Environ Microbiol 73(9):3061–3068PubMedCentralPubMedGoogle Scholar
  11. Borden JR, Jones SW, Indurthi D, Chen Y, Terry Papoutsakis E (2010) A genomic-library based discovery of a novel, possibly synthetic, acid-tolerance mechanism in Clostridium acetobutylicum involving non-coding RNAs and ribosomal RNA processing. Metab Eng 12(3):268–281PubMedCentralPubMedGoogle Scholar
  12. Bowring SN, Morris JG (1985) Mutagenesis of Clostridium acetobutylicum. J Appl Microbiol 58(6):577–584Google Scholar
  13. Branduardi P, de Ferra F, Longo V, Porro D (2014) Microbial n-butanol production from Clostridia to non-Clostridial hosts. Eng Life Sci 14(1):16–26Google Scholar
  14. Caspeta L, Buijs NAA, Nielsen J (2013) The role of biofuels in the future energy supply. Energy Environ Sci 6(4):1077–1082Google Scholar
  15. Chu S, Majumdar A (2012) Opportunities and challenges for a sustainable energy future. Nature 488(7411):294–303PubMedGoogle Scholar
  16. Clark SW, Bennett GN, Rudolph FB (1989) Isolation and characterization of mutants of Clostridium acetobutylicum ATCC 824 deficient in acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase (EC and in other solvent pathway enzymes. Appl Environ Microbiol 55(4):970–976PubMedCentralPubMedGoogle Scholar
  17. Collas F, Kuit W, Clement B, Marchal R, Lopez-Contreras AM, Monot F (2012) Simultaneous production of isopropanol, butanol, ethanol and 2,3-butanediol by Clostridium acetobutylicum ATCC 824 engineered strains. AMB Express 2(1):45PubMedCentralPubMedGoogle Scholar
  18. Cooksley CM, Zhang Y, Wang H, Redl S, Winzer K, Minton NP (2012) Targeted mutagenesis of the Clostridium acetobutylicum Acetone-Butanol-Ethanol fermentation pathway. Metab Eng 14(6):630–641PubMedGoogle Scholar
  19. Dai Z, Dong H, Zhu Y, Zhang Y, Li Y, Ma Y (2012) Introducing a single secondary alcohol dehydrogenase into butanol-tolerant Clostridium acetobutylicum Rh8 switches ABE fermentation to high level IBE fermentation. Biotechnol Biofuels 5(1):44PubMedCentralPubMedGoogle Scholar
  20. Desai RP, Papoutsakis ET (1999) Antisense RNA strategies for metabolic engineering of Clostridium acetobutylicum. Appl Environ Microbiol 65(3):936–945PubMedCentralPubMedGoogle Scholar
  21. Desai RP, Harris LM, Welker NE, Papoutsakis ET (1999) Metabolic flux analysis elucidates the importance of the acid-formation pathways in regulating solvent production by Clostridium acetobutylicum. Metab Eng 1(3):206–213PubMedGoogle Scholar
  22. Dietrich JA, McKee AE, Keasling JD (2010) High-throughput metabolic engineering: advances in small-molecule screening and selection. Annu Rev Biochem 79:563–590PubMedGoogle Scholar
  23. Dietrich JA, Shis DL, Alikhani A, Keasling JD (2013) Transcription factor-based screens and synthetic selections for microbial small-molecule biosynthesis. ACS Synth Biol 2(1):47–58PubMedGoogle Scholar
  24. Dong H, Zhang Y, Dai Z, Li Y (2010) Engineering Clostridium strain to accept unmethylated DNA. PLoS One 5(2):e9038Google Scholar
  25. Dong H, Tao W, Dai Z, Yang L, Gong F, Zhang Y, Li Y (2012a) Biobutanol. Adv Biochem Eng Biotechnol 128:85–100PubMedGoogle Scholar
  26. Dong H, Tao W, Zhang Y, Li Y (2012b) Development of an anhydrotetracycline-inducible gene expression system for solvent-producing Clostridium acetobutylicum: a useful tool for strain engineering. Metab Eng 14(1):59–67PubMedGoogle Scholar
  27. Dong H, Tao W, Gong F, Li Y, Zhang Y (2014) A functional recT gene for recombineering of Clostridium. J Biotechnol 173:65–67Google Scholar
  28. Dürre P, Hollergschwandner C (2004) Initiation of endospore formation in Clostridium acetobutylicum. Anaerobe 10(2):69–74PubMedGoogle Scholar
  29. Dusséaux S, Croux C, Soucaille P, Meynial-Salles I (2013) Metabolic engineering of Clostridium acetobutylicum ATCC 824 for the high-yield production of a biofuel composed of an isopropanol/butanol/ethanol mixture. Metab Eng 18:1–8PubMedGoogle Scholar
  30. Ezeji T, Milne C, Price ND, Blaschek HP (2010) Achievements and perspectives to overcome the poor solvent resistance in acetone and butanol-producing microorganisms. Appl Microbiol Biotechnol 85(6):1697–1712PubMedGoogle Scholar
  31. Feustel L, Nakotte S, Dürre P (2004) Characterization and development of two reporter gene systems for Clostridium acetobutylicum. Appl Environ Microbiol 70(2):798–803PubMedCentralPubMedGoogle Scholar
  32. Fontaine L, Meynial-Salles I, Girbal L, Yang X, Croux C, Soucaille P (2002) Molecular characterization and transcriptional analysis of adhE2, the gene encoding the NADH-dependent aldehyde/alcohol dehydrogenase responsible for butanol production in alcohologenic cultures of Clostridium acetobutylicum ATCC 824. J Bacteriol 184(3):821–830Google Scholar
  33. Gao K, Li Y, Tian S, Yang X (2012a) Screening and characteristics of a butanol-tolerant strain and butanol production from enzymatic hydrolysate of NaOH-pretreated corn stover. World J Microbiol Biotechnol 28(10):2963–2971PubMedGoogle Scholar
  34. Gao X, Zhao H, Zhang G, He K, Jin Y (2012b) Genome shuffling of Clostridium acetobutylicum CICC 8012 for improved production of acetone-butanol-ethanol (ABE). Curr Microbiol 65(2):128–132PubMedGoogle Scholar
  35. Gheshlaghi R, Scharer JM, Moo-Young M, Chou CP (2009) Metabolic pathways of clostridia for producing butanol. Biotechnol Adv 27(6):764–781PubMedGoogle Scholar
  36. Girbal L, Mortier-Barriere I, Raynaud F, Rouanet C, Croux C, Soucaille P (2003) Development of a sensitive gene expression reporter system and an inducible promoter-repressor system for Clostridium acetobutylicum. Appl Environ Microbiol 69(8):4985–4988PubMedCentralPubMedGoogle Scholar
  37. Green EM, Bennett GN (1996) Inactivation of an aldehyde/alcohol dehydrogenase gene from Clostridium acetobutylicum ATCC 824. Appl Biochem Biotechnol 57–58:213–221PubMedGoogle Scholar
  38. Green EM, Boynton ZL, Harris LM, Rudolph FB, Papoutsakis ET, Bennett GN (1996) Genetic manipulation of acid formation pathways by gene inactivation in Clostridium acetobutylicum ATCC 824. Microbiology 142(8):2079–2086PubMedGoogle Scholar
  39. Gronenberg LS, Marcheschi RJ, Liao JC (2013) Next generation biofuel engineering in prokaryotes. Curr Opin Chem Biol 17(3):462–471PubMedGoogle Scholar
  40. Grupe H, Gottschalk G (1992) Physiological events in Clostridium acetobutylicum during the shift from acidogenesis to solventogenesis in continuous culture and presentation of a model for shift induction. Appl Environ Microbiol 58(12):3896–3902PubMedCentralPubMedGoogle Scholar
  41. Gu Y, Jiang Y, Wu H, Liu X, Li Z, Li J, Xiao H, Shen Z, Dong H, Yang Y, Li Y, Jiang W, Yang S (2011) Economical challenges to microbial producers of butanol: feedstock, butanol ratio and titer. Biotechnol J 6(11):1348–1357PubMedGoogle Scholar
  42. Harris L, Blank L, Desai RP, Welker NE, Papoutsakis ET (2001) Fermentation characterization and flux analysis of recombinant strains of Clostridium acetobutylicum with an inactivated solR gene. J Ind Microbiol Biotechnol 27(5):322–328Google Scholar
  43. Harris LM, Welker NE, Papoutsakis ET (2002) Northern, morphological, and fermentation analysis of spo0A inactivation and overexpression in Clostridium acetobutylicum ATCC 824. J Bacteriol 184(13):3586–3597Google Scholar
  44. 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 Meth 70(3):452–464Google Scholar
  45. Heap JT, Pennington OJ, Cartman ST, Minton NP (2009) A modular system for Clostridium shuttle plasmids. J Microbiol Meth 78(1):79–85Google Scholar
  46. 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 Meth 80(1):49–55Google Scholar
  47. 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(8):e59PubMedCentralPubMedGoogle Scholar
  48. Hönicke D, Janssen H, Grimmler C, Ehrenreich A, Lütke-Eversloh T (2012) Global transcriptional changes of Clostridium acetobutylicum cultures with increased butanol:acetone ratios. N Biotechnol 29(4):485–493PubMedGoogle Scholar
  49. Hou X, Peng W, Xiong L, Huang C, Chen X, Chen X, Zhang W (2013) Engineering Clostridium acetobutylicum for alcohol production. J Biotechnol 166(1–2):25–33PubMedGoogle Scholar
  50. Jang YS, Lee J, Malaviya A, Seung DY, Cho JH, Lee SY (2012a) Butanol production from renewable biomass: rediscovery of metabolic pathways and metabolic engineering. Biotechnol J 7(2):186–198PubMedGoogle Scholar
  51. Jang YS, Lee JY, Lee J, Park JH, Im JA, Eom MH, Lee SH, Song H, Cho JH, Seung do Y, Lee SY (2012b) Enhanced butanol production obtained by reinforcing the direct butanol-forming route in Clostridium acetobutylicum. MBio 3(5):e00314–12PubMedCentralPubMedGoogle Scholar
  52. Jang YS, Malaviya A, Cho C, Lee J, Lee SY (2012c) Butanol production from renewable biomass by clostridia. Bioresour Technol 123:653–663PubMedGoogle Scholar
  53. Jang YS, Park JM, Choi S, Choi YJ, Seung DY, Cho JH, Lee SY (2012d) Engineering of microorganisms for the production of biofuels and perspectives based on systems metabolic engineering approaches. Biotechnol Adv 30(5):989–1000PubMedGoogle Scholar
  54. Jang YS, Malaviya A, Lee J, Im JA, Lee SY, Eom MH, Cho JH, Seung DY (2013a) Metabolic engineering of Clostridium acetobutylicum for the enhanced production of isopropanol-butanol-ethanol fuel mixture. Biotechnol Prog 29(4):1083–1088PubMedGoogle Scholar
  55. Jang YS, Malaviya A, Lee SY (2013b) Acetone-butanol-ethanol production with high productivity using Clostridium acetobutylicum BKM19. Biotechnol Bioeng 110(6):1646–1653PubMedGoogle Scholar
  56. Jang YS, Woo HM, Im JA, Kim IH, Lee SY (2013c) Metabolic engineering of Clostridium acetobutylicum for enhanced production of butyric acid. Appl Microbiol Biotechnol 97(21):9355–9363PubMedGoogle Scholar
  57. Jang YS, Im JA, Choi SY, Lee JI, Lee SY (2014) Metabolic engineering of Clostridium acetobutylicum for butyric acid production with high butyric acid selectivity. Metab Eng. doi: 10.1016/j.ymben.2014.03.004i PubMedGoogle Scholar
  58. Janssen H, Grimmler C, Ehrenreich A, Bahl H, Fischer RJ (2012) A transcriptional study of acidogenic chemostat cells of Clostridium acetobutylicum—solvent stress caused by a transient n-butanol pulse. J Biotechnol 161(3):354–365PubMedGoogle Scholar
  59. Jia K, Zhang Y, Li Y (2010) Systematic engineering of microorganisms to improve alcohol tolerance. Eng Life Sci 10(5):422–429Google Scholar
  60. Jia K, Zhu Y, Zhang Y, Li Y (2011) Group II intron-anchored gene deletion in Clostridium. PLoS One 6(1):e16693PubMedCentralPubMedGoogle Scholar
  61. Jia K, Zhang Y, Li Y (2012) Identification and characterization of two functionally unknown genes involved in butanol tolerance of Clostridium acetobutylicum. PLoS One 7:e38815PubMedCentralPubMedGoogle Scholar
  62. Jiang Y, Xu C, Dong F, Yang Y, Jiang W, Yang S (2009) Disruption of the acetoacetate decarboxylase gene in solvent-producing Clostridium acetobutylicum increases the butanol ratio. Metab Eng 11(4–5):284–291PubMedGoogle Scholar
  63. Jin C, Yao M, Liu H, Lee CFF, Ji J (2011) Progress in the production and application of n-butanol as a biofuel. Renew Sust Energ Rev 15(8):4080–4106Google Scholar
  64. 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 173:7–9PubMedGoogle Scholar
  65. Jones DT, Woods DR (1986) Acetone-butanol fermentation revisited. Microbiol Rev 50(4):484–524PubMedCentralPubMedGoogle Scholar
  66. Jones SW, Tracy BP, Gaida SM, Papoutsakis ET (2011) Inactivation of σF in Clostridium acetobutylicum ATCC 824 blocks sporulation prior to asymmetric division and abolishes σE and σG protein expression but does not block solvent formation. J Bacteriol 193(10):2429–2440PubMedCentralPubMedGoogle Scholar
  67. Junelles AM, Janati-Idrissi R, Kanouni A, Petitdemange H, Gay R (1987) Acetone-butanol fermentation by mutants selected for resistance to acetate and butyrate halogen analogues. Biotechnol Lett 9(3):175–178Google Scholar
  68. Jurgens G, Survase S, Berezina O, Sklavounos E, Linnekoski J, Kurkijärvi A, Väkevä M, van Heiningen A, Granström T (2012) Butanol production from lignocellulosics. Biotechnol Lett 34(8):1415–1434PubMedGoogle Scholar
  69. 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 Biotechnol 19(12):1162–1167PubMedGoogle Scholar
  70. Kellermann SJ, Rentmeister A (2014) Current developments in cellulase engineering. ChemBioEng Rev 1(1):6–13Google Scholar
  71. Kim HJ, Turner TL, Jin Y-S (2013) Combinatorial genetic perturbation to refine metabolic circuits for producing biofuels and biochemicals. Biotechnol Adv 31(6):976–985PubMedGoogle Scholar
  72. Kovács K, Willson BJ, Schwarz K, Heap JT, Jackson A, Bolam DN, Winzer K, Minton NP (2013) Secretion and assembly of functional mini-cellulosomes from synthetic chromosomal operons in Clostridium acetobutylicum ATCC 824. Biotechnol Biofuels 6(1):117PubMedCentralPubMedGoogle Scholar
  73. Krutsakorn B, Honda K, Ye X, Imagawa T, Bei X, Okano K, Ohtake H (2013) In vitro production of n-butanol from glucose. Metab Eng 20:84–91PubMedGoogle Scholar
  74. Kuehne SA, Minton NP (2012) ClosTron-mediated engineering of Clostridium. Bioeng Bugs 3(4):247–254Google Scholar
  75. Kuit W, Minton NP, López-Contreras AM, Eggink G (2012) Disruption of the acetate kinase (ack) gene of Clostridium acetobutylicum results in delayed acetate production. Appl Microbiol Biotechnol 94(3):729–741PubMedCentralPubMedGoogle Scholar
  76. Kumar M, Gayen K (2011) Developments in biobutanol production: new insights. Appl Energy 88(6):1999–2012Google Scholar
  77. Lee SY, Bennett GN, Papoutsakis ET (1992a) Construction of Escherichia coli-Clostridium acetobutylicum shuttle vectors and transformation of Clostridium acetobutylicum strains. Biotechnol Lett 14(5):427–432Google Scholar
  78. Lee SY, Mermelstein LD, Bennett GN, Papoutsakis ET (1992b) Vector construction, transformation, and gene amplification in Clostridium acetobutylicum ATCC 824. Ann N Y Acad Sci 665:39–51PubMedGoogle Scholar
  79. Lee SY, Park JH, Jang SH, Nielsen LK, Kim J, Jung KS (2008) Fermentative butanol production by clostridia. Biotechnol Bioeng 101(2):209–228PubMedGoogle Scholar
  80. Lee J, Jang YS, Choi SJ, Im JA, Song H, Cho JH, Seung do Y, Papoutsakis ET, Bennett GN, Lee SY (2012) Metabolic engineering of Clostridium acetobutylicum ATCC 824 for isopropanol-butanol-ethanol fermentation. Appl Environ Microbiol 78(5):1416–1423PubMedCentralPubMedGoogle Scholar
  81. Lehmann D, Lütke-Eversloh T (2011) Switching Clostridium acetobutylicum to an ethanol producer by disruption of the butyrate/butanol fermentative pathway. Metab Eng 13(5):464–473PubMedGoogle Scholar
  82. Lehmann D, Hönicke D, Ehrenreich A, Schmidt M, Weuster-Botz D, Bahl H, Lütke-Eversloh T (2012a) Modifying the product pattern of Clostridium acetobutylicum: physiological effects of disrupting the acetate and acetone formation pathways. Appl Microbiol Biotechnol 94(3):743–754PubMedGoogle Scholar
  83. Lehmann D, Radomski N, Lütke-Eversloh T (2012b) New insights into the butyric acid metabolism of Clostridium acetobutylicum. Appl Microbiol Biotechnol 96(5):1325–1339PubMedGoogle Scholar
  84. Lemmel SA (1985) Mutagenesis in Clostridium acetobutylicum. Biotechnol Lett 7:711–716Google Scholar
  85. Li F, Hinderberger J, Seedorf H, Zhang J, Buckel W, Thauer RK (2008) Coupled ferredoxin and crotonyl coenzyme A (CoA) reduction with NADH catalyzed by the butyryl-CoA dehydrogenase/Etf complex from Clostridium kluyveri. J Bacteriol 190:843–850PubMedCentralPubMedGoogle Scholar
  86. Li Z, Xiao H, Jiang W, Jiang Y, Yang S (2013) Improvement of solvent production from xylose mother liquor by engineering the xylose metabolic pathway in Clostridium acetobutylicum EA 2018. Appl Biochem Biotechnol 171(3):555–568PubMedGoogle Scholar
  87. Li HG, Luo W, Wang Q, Yu XB (2014) Direct fermentation of gelatinized cassava starch to acetone, butanol, and ethanol using Clostridium acetobutylicum mutant obtained by atmospheric and room temperature plasma. Appl Biochem Biotechnol. doi: 10.1007/s12010-014-0765-x Google Scholar
  88. Lim JH, Seo SW, Kim SY, Jung GY (2013) Model-driven rebalancing of the intracellular redox state for optimization of a heterologous n-butanol pathway in Escherichia coli. Metab Eng 20:56–62PubMedGoogle Scholar
  89. Linhová M, Branská B, Patáková P, Lipovsky J, Fribert P, Rychtera M, Melzoch K (2012) Rapid flow cytometric method for viability determination of solventogenic clostridia. Folia Microbiol 57(4):307–311Google Scholar
  90. Liu S, Qureshi N (2010) How microbes tolerate ethanol and butanol. N Biotechnol 26(3–4):117–121Google Scholar
  91. Liu L, Zhang L, Tang W, Gu Y, Hua Q, Yang S, Jiang W, Yang C (2012a) Phosphoketolase pathway for xylose catabolism in Clostridium acetobutylicum revealed by 13C metabolic flux analysis. J Bacteriol 194(19):5413–5422PubMedCentralPubMedGoogle Scholar
  92. Liu XB, Gu QY, Yu XB, Luo W (2012b) Enhancement of butanol tolerance and butanol yield in Clostridium acetobutylicum mutant NT642 obtained by nitrogen ion beam implantation. J Microbiol 50(6):1024–1028PubMedGoogle Scholar
  93. Luan G, Cai Z, Gong F, Dong H, Lin Z, Zhang Y, Li Y (2013) Developing controllable hypermutable Clostridium cells through manipulating its methyl-directed mismatch repair system. Protein Cell 4(11):854–862PubMedGoogle Scholar
  94. Lütke-Eversloh T, Bahl H (2011) Metabolic engineering of Clostridium acetobutylicum: recent advances to improve butanol production. Curr Opin Biotechnol 22(5):634–647PubMedGoogle Scholar
  95. Lütke-Eversloh T, Stephanopoulos G (2005) Feedback inhibition of chorismate mutase/prephenate dehydrogenase (TyrA) of Escherichia coli: generation and characterization of tyrosine-insensitive mutants. Appl Environ Microbiol 71(11):7224–7228PubMedCentralPubMedGoogle Scholar
  96. Mann MS, Lütke-Eversloh T (2013) Thiolase engineering for enhanced butanol production in Clostridium acetobutylicum. Biotechnol Bioeng 110(3):887–897PubMedGoogle Scholar
  97. Mann MS, Dragovic Z, Schirrmacher G, Lütke-Eversloh T (2012) Over-expression of stress protein-encoding genes helps Clostridium acetobutylicum to rapidly adapt to butanol stress. Biotechnol Lett 34(9):1643–1649PubMedGoogle Scholar
  98. Mao S, Luo Y, Zhang T, Li J (2010) Proteome reference map and comparative proteomic analysis between a wild type Clostridium acetobutylicum DSM 1731 and its mutant with enhanced butanol tolerance and butanol yield. J Proteome Res 9(6):3046–3061PubMedGoogle Scholar
  99. Mao S, Luo Y, Bao G, Zhang Y, Li Y, Ma Y (2011) Comparative analysis on the membrane proteome of Clostridium acetobutylicum wild type strain and its butanol-tolerant mutant. Mol BioSyst 7(5):1660–1677PubMedGoogle Scholar
  100. Mariano AP, Filho RM (2012) Improvements in biobutanol fermentation and their impacts on distillation energy consumption and wastewater generation. Bioenergy Res 5(2):504–514Google Scholar
  101. Mascal M (2012) Chemicals from biobutanol: technologies and markets. Biofuels Bioprod Bioref 6(4):483–493Google Scholar
  102. Medkor N, Zerdani I, Sattar S (2010) Isolation of Clostridium acetobutylicum ATCC824 mutants using propionic and isovaleric acid halogen analogues as suicide substrates. Int J Microbiol Res 1(1):22–25Google Scholar
  103. Mermelstein LD, Papoutsakis ET (1993) In vivo methylation in Escherichia coli by the Bacillus subtilis phage ϕ3T I methyltransferase to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol 59(4):1077–1081PubMedCentralPubMedGoogle Scholar
  104. Mermelstein LD, Welker NE, Bennett GN, Papoutsakis ET (1992) Expression of cloned homologous fermentative genes in Clostridium acetobutylicum ATCC 824. Nat Biotechnol 10(2):190–195Google Scholar
  105. Mermelstein LD, Papoutsakis ET, Petersen DJ, Bennett GN (1993) Metabolic engineering of Clostridium acetobutylicum ATCC 824 for increased solvent production by enhancement of acetone formation enzyme activities using a synthetic acetone operon. Biotechnol Bioeng 42(9):1053–1060PubMedGoogle Scholar
  106. Moholkar VS, Ranjan A, Mayank R (2012) Economics of biobutanol: a review. Res J Pharm Biol Chem Sci 3(4):901–913Google Scholar
  107. 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):319–330Google Scholar
  108. Ni Y, Sun Z (2009) Recent progress on industrial fermentative production of acetone-butanol-ethanol by Clostridium acetobutylicum in China. Appl Microbiol Biotechnol 83(3):415–423PubMedGoogle Scholar
  109. Nicolaou SA, Gaida SM, Papoutsakis ET (2010) A comparative view of metabolite and substrate stress and tolerance in microbial bioprocessing: from biofuels and chemicals, to biocatalysis and bioremediation. Metab Eng 12(4):307–331PubMedGoogle Scholar
  110. Oultram JD, Loughlin M, Swinfield TJ, Brehm JK, Thompson DE, Minton NP (1988) Introduction of plasmids into whole cells of Clostridium acetobutylicum by electroporation. FEMS Microbiol Lett 56(1):83–88Google Scholar
  111. Papoutsakis ET (2008) Engineering solventogenic clostridia. Curr Opin Biotechnol 19(5):420–429PubMedGoogle Scholar
  112. Paredes CJ, Alsaker KV, Papoutsakis ET (2005) A comparative genomic view of clostridial sporulation and physiology. Nat Rev Microbiol 3(12):969–978PubMedGoogle Scholar
  113. Pasteur L (1862) Quelques résultats nouveaux relatifs aux fermentations acétique et butyrique. Bulletin de la Société Chimique de Paris:52-53Google Scholar
  114. Patáková P, Linhová M, Rychtera M, Paulova L, Melzoch K (2013) Novel and neglected issues of acetone-butanol-ethanol (ABE) fermentation by clostridia: Clostridium metabolic diversity, tools for process mapping and continuous fermentation systems. Biotechnol Adv 31(1):58–67PubMedGoogle Scholar
  115. Ranjan A, Moholkar VS (2012) Biobutanol: science, engineering, and economics. Int J Energy Res 36(3):277–323Google Scholar
  116. Rellos P, Ma J, Scopes RK (1997) Alteration of substrate specificity of Zymomonas mobilis alcohol dehydrogenase-2 using in vitro random mutagenesis. Protein Expr Purif 9(1):83–90PubMedGoogle Scholar
  117. Ren C, Gu Y, Hu S, Wu Y, Wang P, Yang Y, Yang C, Yang S, Jiang W (2010) Identification and inactivation of pleiotropic regulator CcpA to eliminate glucose repression of xylose utilization in Clostridium acetobutylicum. Metab Eng 12(5):446–454PubMedGoogle Scholar
  118. Ren C, Gu Y, Wu Y, Zhang W, Yang C, Yang S, Jiang W (2012) Pleiotropic functions of catabolite control protein CcpA in butanol-producing Clostridium acetobutylicum. BMC Genomics 13(1):349PubMedCentralPubMedGoogle Scholar
  119. Rothstein DM (1986) Clostridium thermosaccharolyticum strain deficient in acetate production. J Bacteriol 165:319–320PubMedCentralPubMedGoogle Scholar
  120. Sanchez S, Demain AL (2008) Metabolic regulation and overproduction of primary metabolites. Microb Biotechnol 1(4):283–319PubMedCentralPubMedGoogle Scholar
  121. Scheel M, Lütke-Eversloh T (2013) New options to engineer biofuel microbes: development and application of a high-throughput screening system. Metab Eng 17C:51–58Google Scholar
  122. Scotcher MC, Bennett GN (2005) SpoIIE regulates sporulation but does not directly affect solventogenesis in Clostridium acetobutylicum ATCC 824. J Bacteriol 187(6):1930–1936PubMedCentralPubMedGoogle Scholar
  123. Servinsky MD, Germane KL, Liu S, Kiel JT, Clark AM, Shankar J, Sund CJ (2012) Arabinose is metabolized via a phosphoketolase pathway in Clostridium acetobutylicum ATCC 824. J Ind Microbiol Biotechnol 39(12):1859–1867PubMedGoogle Scholar
  124. 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(11):963–965PubMedGoogle Scholar
  125. Shen CR, Lan EI, Dekishima Y, Baez A, Cho KM, Liao JC (2011) Driving forces enable high-titer anaerobic 1-butanol synthesis in Escherichia coli. Appl Environ Microbiol 77(9):2905–2915PubMedCentralPubMedGoogle Scholar
  126. Sillers R, Al-Hinai MA, Papoutsakis ET (2009) Aldehyde-alcohol dehydrogenase and/or thiolase overexpression coupled with CoA transferase downregulation lead to higher alcohol titers and selectivity in Clostridium acetobutylicum fermentations. Biotechnol Bioeng 102(1):38–49PubMedGoogle Scholar
  127. Soucaille P, Figge R, Croux C (2008) Process for chromosomal integration and DNA sequence replacement in clostridia. International Patent WO 2008/040387Google Scholar
  128. Steiner E, Dago AE, Young DI, Heap JT, Minton NP, Hoch JA, Young M (2011) Multiple orphan histidine kinases interact directly with Spo0A to control the initiation of endospore formation in Clostridium acetobutylicum. Mol Microbiol 80(3):641–654PubMedCentralPubMedGoogle Scholar
  129. Tangney M, Mitchell WJ (2007) Characterisation of a glucose phosphotransferase system in Clostridium acetobutylicum ATCC 824. Appl Microbiol Biotechnol 74(2):398–405PubMedGoogle Scholar
  130. Thomas L, Joseph A, Gottumukkala LD (2014) Xylanase and cellulase systems of Clostridium sp.: an insight on molecular approaches for strain improvement. Bioresour Technol. doi: 10.1016/j.biortech.2014.01.140 Google Scholar
  131. Thomason MK, Storz G (2010) Bacterial antisense RNAs: how many are there, and what are they doing? Annu Rev Genet 44:167–188PubMedCentralPubMedGoogle Scholar
  132. Thormann K, Dürre P (2001) Orf5/SolR: a transcriptional repressor of the sol operon of Clostridium acetobutylicum? J Ind Microbiol Biotechnol 27(5):307–313Google Scholar
  133. Thormann K, Feustel L, Lorenz K, Nakotte S, Dürre P (2002) Control of butanol formation in Clostridium acetobutylicum by transcriptional activation. J Bacteriol 184(7):1966–1973PubMedCentralPubMedGoogle Scholar
  134. 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):4951–4965Google Scholar
  135. Tomas CA, Beamish J, Papoutsakis ET (2004) Transcriptional analysis of butanol stress and tolerance in Clostridium acetobutylicum. J Bacteriol 186(7):2006–2018PubMedCentralPubMedGoogle Scholar
  136. Tracy BP, Gaida SM, Papoutsakis ET (2008) Development and application of flow-cytometric techniques for analyzing and sorting endospore-forming clostridia. Appl Environ Microbiol 74(24):7497–7506Google Scholar
  137. Tracy BP, Gaida SM, Papoutsakis ET (2010) Flow cytometry for bacteria: enabling metabolic engineering, synthetic biology and the elucidation of complex phenotypes. Curr Opin Biotechnol 21(1):85–99PubMedGoogle Scholar
  138. Tracy BP, Jones SW, Papoutsakis ET (2011) Inactivation of σE and σG in Clostridium acetobutylicum illuminates their roles in clostridial-cell-form biogenesis, granulose synthesis, solventogenesis, and spore morphogenesis. J Bacteriol 193(6):1414–1426PubMedCentralPubMedGoogle Scholar
  139. Tracy BP, Jones SW, Fast AG, Indurthi DC, Papoutsakis ET (2012) Clostridia: the importance of their exceptional substrate and metabolite diversity for biofuel and biorefinery applications. Curr Opin Biotechnol 23(3):364–381PubMedGoogle Scholar
  140. Truffaut N, Hubert J, Reysset G (1989) Construction of shuttle vectors useful for transforming Clostridium acetobutylicum. FEMS Microbiol Lett 58(1):15–20Google Scholar
  141. Tummala SB, Welker NE, Papoutsakis ET (1999) Development and characterization of a gene expression reporter system for Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol 65(9):3793–3799PubMedCentralPubMedGoogle Scholar
  142. Tummala SB, Junne SG, Papoutsakis ET (2003a) Antisense RNA downregulation of coenzyme A transferase combined with alcohol-aldehyde dehydrogenase overexpression leads to predominantly alcohologenic Clostridium acetobutylicum fermentations. J Bacteriol 185(12):3644–3653PubMedCentralPubMedGoogle Scholar
  143. Tummala SB, Welker NE, Papoutsakis ET (2003b) Design of antisense RNA constructs for downregulation of the acetone formation pathway of Clostridium acetobutylicum. J Bacteriol 185(6):1923–1934PubMedCentralPubMedGoogle Scholar
  144. Ventura JRS, 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):7505–7516PubMedGoogle Scholar
  145. Wang Y, Li X, Milne CB, Janssen H, Lin W, Phan G, Hu H, Jin YS, Price ND, Blaschek HP (2013) Development of a gene knockout system using mobile group II introns (Targetron) and genetic disruption of acid production pathways in Clostridium beijerinckii. Appl Environ Microbiol 79(19):5853–5863PubMedCentralPubMedGoogle Scholar
  146. Wiesenborn D, Rudolph F, Papoutsakis E (1988) Thiolase from Clostridium acetobutylicum ATCC 824 and its role in the synthesis of acids and solvents. Appl Environ Microbiol 54(11):2717–2722Google Scholar
  147. Wietzke M, Bahl H (2012) The redox-sensing protein Rex, a transcriptional regulator of solventogenesis in Clostridium acetobutylicum. Appl Microbiol Biotechnol 96(3):749–761PubMedGoogle Scholar
  148. Willims DR, Young DI, Young M (1990) Conjugative plasmid transfer from Escherichia coli to Clostridium acetobutylicum. J Gen Microbiol 136(5):819–826Google Scholar
  149. Woolston BM, Edgar S, Stephanopoulos G (2013) Metabolic engineering: past and future. Annu Rev Chem Biomol Eng 4:259–288PubMedGoogle Scholar
  150. Xiao H, Gu Y, Ning Y, Yang Y, Mitchell WJ, Jiang W, Yang S (2011) Confirmation and elimination of xylose-metabolic bottlenecks in glucose-PTS-deficient Clostridium acetobutylicum to realize simultaneous utilization of glucose, xylose and arabinose. Appl Environ Microbiol 77(22):7886–7895PubMedCentralPubMedGoogle Scholar
  151. Xue C, Zhao X-Q, Liu C-G, Chen L-J, Bai F-W (2013) Prospective and development of butanol as an advanced biofuel. Biotechnol Adv 31(8):1575–1584PubMedGoogle Scholar
  152. Xue C, Zhao J-B, Chen L-J, Bai F-W, Yang S-T, Sun J-X (2014) Integrated butanol recovery for an advanced biofuel: current state and prospects. Appl Microbiol Biotechnol. doi: 10.1007/s00253-014-5561-6 Google Scholar
  153. Yang H, Li J, Shin H-d DG, Liu L, Chen J (2014) Molecular engineering of industrial enzymes: recent advances and future prospects. Appl Microbiol Biotechnol 98(1):23–29PubMedGoogle Scholar
  154. Young M, Minton NP, Staudenbauer WL (1989) Recent advances in the genetics of the clostridia. FEMS Microbiol Rev 63(4):301–325Google Scholar
  155. Zhang YX, Perry K, Vinci VA, Powell K, Stemmer WPC, Del Cardayré SB (2002) Genome shuffling leads to rapid phenotypic improvement in bacteria. Nature 415(6872):644–646PubMedGoogle Scholar
  156. Zhao Y, Hindorff LA, Chuang A, Monroe-Augustus M, Lyristis M, Harrison ML, Rudolph FB, Bennett GN (2003) Expression of a cloned cyclopropane fatty acid synthase gene reduces solvent formation in Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol 69(5):2831–2841PubMedCentralPubMedGoogle Scholar
  157. Zhu L, Dong H, Zhang Y, Li Y (2011) Engineering the robustness of Clostridium acetobutylicum by introducing glutathione biosynthetic capability. Metab Eng 13(4):426–434PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  1. 1.Abteilung Mikrobiologie, Institut für BiowissenschaftenUniversität RostockRostockGermany

Personalised recommendations