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Applied Microbiology and Biotechnology

, Volume 96, Issue 3, pp 749–761 | Cite as

The redox-sensing protein Rex, a transcriptional regulator of solventogenesis in Clostridium acetobutylicum

  • Mandy Wietzke
  • Hubert BahlEmail author
Applied genetics and molecular biotechnology

Abstract

Solventogenic clostridia are characterised by their biphasic fermentative metabolism, and the main final product n-butanol is of particular industrial interest because it can be used as a superior biofuel. During exponential growth, Clostridium acetobutylicum synthesises acetic and butyric acids which are accompanied by the formation of molecular hydrogen and carbon dioxide. During the stationary phase, the solvents acetone, butanol and ethanol are produced. However, the molecular mechanisms of this metabolic switch are largely unknown so far. In this study, in silico, in vitro and in vivo analyses were performed to elucidate the function of the CAC2713-encoded redox-sensing transcriptional repressor Rex and its role in the solventogenic shift of C. acetobutylicum ATCC 824. Electrophoretic mobility shift assays showed that Rex controls the expression of butanol biosynthetic genes as a response to the cellular NADH/NAD+ ratio. Interestingly, the Rex-negative mutant C. acetobutylicum rex::int(95) produced high amounts of ethanol and butanol, while hydrogen and acetone production were significantly reduced. Both ethanol and butanol (but not acetone) formation started clearly earlier than in the wild type. In addition, the rex mutant showed a de-repression of the bifunctional aldehyde/alcohol dehydrogenase 2 encoded by the adhE2 gene (CAP0035) as demonstrated by increased adhE2 expression as well as high NADH-dependent alcohol dehydrogenase activities. The results presented here clearly indicated that Rex is involved in the redox-dependent solventogenic shift of C. acetobutylicum.

Keywords

Biofuel Butanol ABE fermentation Redox regulation ClosTron 

Notes

Acknowledgments

The authors thank Tina Lütke-Eversloh, Department of Microbiology, University Rostock for many fruitful discussions and useful comments on the manuscript. We are also grateful to Nigel P. Minton and John T. Heap, University of Nottingham for kindly providing the ClosTron plasmids and to Philippe Soucaille, INSA, Toulouse for kindly providing pCons::upp.

Supplementary material

253_2012_4112_MOESM1_ESM.pdf (1.1 mb)
ESM 1 (PDF 1140 kb)

References

  1. Andersch W, Bahl H, Gottschalk G (1983) Levels of enzymes involved in acetate, butyrate, acetone and butanol formation by Clostridium acetobutylicum. Eur J Appl Microbiol Biotechnol 18:327–332CrossRefGoogle Scholar
  2. Bergmeyer HU (1983) Methods in enzymatic analysis. Verlag Chemie Weinheim, GermanyGoogle Scholar
  3. Boynton ZL, Bennett GN, Rudolph FB (1996) Cloning, sequencing, and expression of clustered genes encoding beta-hydroxybutyryl-coenzyme A (CoA) dehydrogenase, crotonase, and butyryl-CoA dehydrogenase from Clostridium acetobutylicum ATCC 824. J Bacteriol 178:3015–3024Google Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  5. Brekasis D, Paget MS (2003) A novel sensor of NADH/NAD+ redox poise in Streptomyces coelicolor A3(2). EMBO J 22:4856–4865CrossRefGoogle Scholar
  6. Datta R, Zeikus JG (1985) Modulation of acetone–butanol–ethanol fermentation by carbon monoxide and organic acids. Appl Environ Microbiol 49:522–529Google Scholar
  7. Desai RP, Papoutsakis ET (1999) Antisense RNA strategies for metabolic engineering of Clostridium acetobutylicum. Appl Environ Microbiol 65:936–945Google Scholar
  8. 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 1:206–213CrossRefGoogle Scholar
  9. Doremus MG, Linden JC, Moreira AR (1985) Agitation and pressure effects on acetone–butanol fermentation. Biotechnol Bioeng 27(6):852–860CrossRefGoogle Scholar
  10. Dürre P (2008) Fermentative butanol production: bulk chemical and biofuel. Ann NY Acad Sci 1125:353–362CrossRefGoogle Scholar
  11. Dürre P, Kuhn A, Gottwald M, Gottschalk G (1987) Enzymatic investigations on butanol dehydrogenase and butyraldehyde dehydrogenase in extracts of Clostridium acetobutylicum. Appl Microbiol Biotechnol 26:268–272CrossRefGoogle Scholar
  12. Fischer RJ, Helms J, Dürre P (1993) Cloning, sequencing, and molecular analysis of the sol operon of Clostridium acetobutylicum, a chromosomal locus involved in solventogenesis. J Bacteriol 175:6959–6969Google Scholar
  13. Fischer RJ, Oehmcke S, Meyer U, Mix M, Schwarz K, Fiedler T, Bahl H (2006) Transcription of the pst operon of Clostridium acetobutylicum is dependent on phosphate concentration and pH. J Bacteriol 188:5469–5478CrossRefGoogle Scholar
  14. 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 184:821–830CrossRefGoogle Scholar
  15. Gerischer U, Dürre P (1992) mRNA analysis of the adc gene region of Clostridium acetobutylicum during the shift to solventogenesis. J Bacteriol 174(2):426–433Google Scholar
  16. Girbal L, Soucaille P (1998) Regulation of solvent production in Clostridium acetobutylicum. Trends Biotechnol 16:11–16CrossRefGoogle Scholar
  17. Girbal L, Vasconcelos I, Soucaille P (1994) Transmembrane pH of Clostridium acetobutylicum is inverted (more acidic inside) when the in vivo activity of hydrogenase is decreased. J Bacteriol 176:6146–6147Google Scholar
  18. Girbal L, Croux C, Vasconcelos I, Soucaille P (1995) Regulation of metabolic shifts in Clostridium acetobutylicum ATCC 824. FEMS Microbiol Rev 17:287–297CrossRefGoogle Scholar
  19. Girbal L, Von Abendroth G, Winkler M, Benton PM, Meynial-Salles I, Croux I, Peters JW, Happe T, Soucaille P (2005) Homologous and heterologous overexpression in Clostridium acetobutylicum and characterization of purified clostridial and algal Fe-only hydrogenases with high specific activities. Appl Environ Microbiol 71:2777–2781CrossRefGoogle Scholar
  20. Gorwa MF, Croux C, Soucaille P (1996) Molecular characterization and transcriptional analysis of the putative hydrogenase gene of Clostridium acetobutylicum ATCC 824. J Bacteriol 178:2668–2675Google Scholar
  21. Gottschal JC, Morris JG (1981) Non‐production of acetone and butanol by Clostridium acetobutylicum during glucose‐ and ammonium‐limitation in continuous culture. Biotech Lett 3:525–530Google Scholar
  22. Grant SG, Jessee J, Bloom FR, Hanahan D (1990) Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci USA 87:4645–4649CrossRefGoogle Scholar
  23. Grimmler C, Janssen H, Krausse D, Fischer RJ, Bahl H, Dürre P, Liebl W, Ehrenreich A (2011) Genome-wide gene expression analysis of the switch between acidogenesis and solventogenesis in continuous cultures of Clostridium acetobutylicum. J Mol Microbiol Biotechnol 20:1–15CrossRefGoogle Scholar
  24. 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 58:3896–3902Google Scholar
  25. Gyan S, Shiohira Y, Sato I, Takeuchi M, Sato T (2006) Regulatory loop between redox sensing of the NADH/NAD(+) ratio by Rex (YdiH) and oxidation of NADH by NADH dehydrogenase Ndh in Bacillus subtilis. J Bacteriol 188:7062–7071CrossRefGoogle Scholar
  26. Harris LM, 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):322–328CrossRefGoogle Scholar
  27. Hartmanis MGN, Gatenbeck S (1984) Intermediary metabolism in Clostridium acetobutylicum: levels of enzymes involved in the formation of acetate and butyrate. Appl Environ Microbiol 47:1277–1283Google Scholar
  28. 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 70:452–464CrossRefGoogle Scholar
  29. 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 80:49–55CrossRefGoogle Scholar
  30. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59CrossRefGoogle Scholar
  31. 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 29:485–493Google Scholar
  32. Hüsemann MHW, Papoutsakis ET (1989) Comparison between in vivo and in vitro enzyme activities in continuous and batch fermentations of Clostridium acetobutylicum. Appl Microbiol Biotechnol 30:585–595CrossRefGoogle Scholar
  33. Janssen H, Döring C, Ehrenreich A, Voigt B, Hecker M, Bahl H, Fischer RJ (2010) A proteomic and transcriptional view of acidogenic and solventogenic steady-state cells of Clostridium acetobutylicum in a chemostat culture. Appl Microbiol Biotechnol 87:2209–2226CrossRefGoogle Scholar
  34. 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 11:284–291CrossRefGoogle Scholar
  35. Johnson JL, Toth J, Santiwatanakul S, Chen JS (1997) Cultures of “Clostridium acetobutylicum” from various collections comprise Clostridium acetobutylicum, Clostridium beijerinckii, and two other distinct types based on DNA-DNA reassociation. Int J Syst Bacteriol 47(2):420–424CrossRefGoogle Scholar
  36. Jones DT, Woods DR (1986) Acetone–butanol fermentation revisited. Microbiol Rev 50:484–524Google Scholar
  37. Jones SW, Paredes CJ, Tracy B, Cheng N, Sillers R, Senger RS, Papoutsakis ET (2008) The transcriptional program underlying the physiology of clostridial sporulation. Genome Biol 9:R114CrossRefGoogle Scholar
  38. Kim BH, Bellows P, Datta R, Zeikus JG (1984) Control of carbon and electron flow in Clostridium acetobutylicum fermentations: utilization of carbon monoxide to inhibit hydrogen production and to enhance butanol yields. Appl Environ Microbiol 48:764–770Google Scholar
  39. Kuit W, Minton NP, Lopez-Contreras AM, Eggink G (2012) Disruption of the acetate kinase (ack) gene of Clostridium acetobutylicum results in delayed acetate production. Appl Microbiol Biotechnol 94:729–741Google Scholar
  40. Lee SY, Park JH, Jang SH, Nielsen LK, Kim J, Jung KS (2008) Fermentative butanol production by clostridia. Biotechnol Bioeng 101:209–228CrossRefGoogle Scholar
  41. Lee JY, Jang YS, Lee J, Papoutsakis ET, Lee SY (2009) Metabolic engineering of Clostridium acetobutylicum M5 for highly selective butanol production. Biotechnol J 4:1432–1440CrossRefGoogle Scholar
  42. Lehmann D, Lütke-Eversloh T (2011) Switching Clostridium acetobutylicum to an ethanol producer by disruption of the butyrate/butanol fermentative pathway. Metab Eng 13:464–473CrossRefGoogle Scholar
  43. Lehmann D, Hönicke D, Ehrenreich A, Schmidt M, Weuster-Botz D, Bahl H, Lütke-Eversloh T (2012) Modifying the product pattern of Clostridium acetobutylicum: physiological effects of disrupting the acetate and acetone formation pathways. Appl Microbiol Biotechnol 94:743–754CrossRefGoogle Scholar
  44. Lütke-Eversloh T, Bahl H (2011) Metabolic engineering of Clostridium acetobutylicum: recent advances to improve butanol production. Curr Opin Biotechnol 22:634–647CrossRefGoogle Scholar
  45. Mermelstein LD, Papoutsakis ET (1993) In vivo methylation in Escherichia coli by the Bacillus subtilis phage phi3T I methyltransferase to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol 59:1077–1081Google Scholar
  46. Meyer CL, Roos JW, Papoutsakis ET (1986) Carbon monoxide gasing leads to alcohol production and butyrate uptake without acetone formation in continuous cultures of Clostridium acetobutylicum. Appl Microbiol Biotechnol 24:159–167Google Scholar
  47. Monot F, Martin JR, Petitdemange H, Gay R (1982) Acetone and butanol production by Clostridium acetobutylicum in a synthetic medium. Appl Environ Microbiol 44:1318–1324Google Scholar
  48. Münch R, Hiller K, Barg H, Heldt D, Linz S, Wingender E, Jahn D (2003) PRODORIC: prokaryotic database of gene regulation. Nucleic Acids Res 31:266–269CrossRefGoogle Scholar
  49. 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 179(17):5442–5447Google Scholar
  50. Pagels M, Fuchs S, Pané-Farré J, Kohler C, Menschner L, Hecker M, McNamarra PJ, Bauer MC, Von Wachenfeldt C, Liebeke M, Lalk M, Sander G, Von Eiff C, Proctor RA, Engelmann S (2010) Redox sensing by a Rex-family repressor is involved in the regulation of anaerobic gene expression in Staphylococcus aureus. Mol Microbiol 76:1142–1161CrossRefGoogle Scholar
  51. Peguin S, Goma G, Delorme P, Soucaille P (1994) Metabolic flexibility of Clostridium acetobutylicum in response to methyl viologen addition. Appl Microbiol Biotechnol 42:611–616CrossRefGoogle Scholar
  52. Pei J, Zhou Q, Jiang Y, Le Y, Li H, Shao W, Wiegel J (2010) Thermoanaerobacter spp. control ethanol pathway via transcriptional regulation and versatility of key enzymes. Metab Eng 12:420–428CrossRefGoogle Scholar
  53. Pei J, Zhou Q, Jing Q, Li L, Dai C, Li H, Wiegel J, Shao W (2011) The mechanism for regulating ethanol fermentation by redox levels in Thermoanaerobacter ethanolicus. Metab Eng 13:186–193CrossRefGoogle Scholar
  54. Ravcheev DA, Li X, Latif H, Zengler K, Leyn SA, Korostelev YD, Kazakov AE, Novichkov PS, Osterman AL, Rodionov DA (2012) Transcriptional regulation of central carbon and energy metabolism in bacteria by redox-responsive repressor rex. J Bacteriol 194(5):1145–1157CrossRefGoogle Scholar
  55. Roos JW, McLaughlin JK, Papoutsakis ET (1985) The effect of pH on nitrogen supply, cell lysis, and solvent production in fermentations of Clostridium acetobutylicum. Biotechnol Bioeng 27:681–694CrossRefGoogle Scholar
  56. Sambrock J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar
  57. Sauer U, Santangelo JD, Treuner A, Buchholz M, Dürre P (1995) Sigma factor and sporulation genes in Clostridium. FEMS Microbiol Rev 17(3):331–340CrossRefGoogle Scholar
  58. Shaheen R, Shirley M, Jones DT (2000) Comparative fermentation studies of industrial strains belonging to four species of solvent-producing clostridia. J Mol Microbiol Biotechnol 1:115–124Google Scholar
  59. Sickmier EA, Brekasis D, Paranawithana S, Bonanno JB, Paget MS, Burley SK, Kielkopf CL (2005) X-ray structure of a Rex-family repressor/NADH complex insights into the mechanism of redox sensing. Structure 13:43–54CrossRefGoogle Scholar
  60. Soucaille P, Figge R, Croux C (2006) Process for chromosomal integration and DNA sequence replacement in Clostridia. Dépôt PCT n° PCT/EP2006/066997Google Scholar
  61. Terracciano JS, Kashket ER (1986) Intracellular conditions required for initiation of solvent production by Clostridium acetobutylicum. Appl Environ Microbiol 52:86–91Google Scholar
  62. Thormann K, Dürre P (2001) Orf5/SolR: a transcriptional repressor of the sol operon of Clostridium acetobutylicum? J Ind Microbiol Biotechnol 27(5):307–313CrossRefGoogle Scholar
  63. 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):1966–1973CrossRefGoogle Scholar
  64. Vasconcelos I, Girbal L, Soucaille P (1994) Regulation of carbon and electron flow in Clostridium acetobutylicum grown in chemostat culture at neutral pH on mixtures of glucose and glycerol. J Bacteriol 176(5):1443–1450Google Scholar
  65. Walter KA, Mermelstein LD, Papoutsakis ET (1994) Studies of recombinant Clostridium acetobutylicum with increased dosages of butyrate formation genes. Ann NY Acad Sci 721:69–72CrossRefGoogle Scholar
  66. Wang E, Bauer MC, Rogstam A, Linse S, Logan DT, Von Wachenfeldt C (2008) Structure and functional properties of the Bacillus subtilis transcriptional repressor Rex. Mol Microbiol 69:466–478CrossRefGoogle Scholar
  67. Wang E, Ikonen TP, Knaapila M, Svergun D, Logan DT, Von Wachenfeldt C (2011) Small-angle X-ray scattering study of a rex family repressor: conformational response to NADH and NAD+ binding in solution. J Mol Biol 408:670–683CrossRefGoogle Scholar
  68. Wiesenborn DP, Rudolph FB, Papoutsakis ET (1989a) Phosphotransbutyrylase from Clostridium acetobutylicum ATCC 824 and its role in acidogenesis. Appl Environ Microbiol 55:317–322Google Scholar
  69. Wiesenborn DP, Rudolph FB, Papoutsakis ET (1989b) Coenzyme a transferase from Clostridium acetobutylicum ATCC 824 and its role in the uptake of acids. Appl Environ Microbiol 55:323–329Google Scholar

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© Springer-Verlag 2012

Authors and Affiliations

  1. 1.Abteilung Mikrobiologie, Institut für BiowissenschaftenUniversität RostockRostockGermany

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