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

  • Applied genetics and molecular biotechnology
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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.

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References

  • 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 18:327–332

    Article  CAS  Google Scholar 

  • Bergmeyer HU (1983) Methods in enzymatic analysis. Verlag Chemie Weinheim, Germany

    Google Scholar 

  • 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 178:3015–3024

    CAS  Google Scholar 

  • 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 72:248–254

    Article  CAS  Google Scholar 

  • Brekasis D, Paget MS (2003) A novel sensor of NADH/NAD+ redox poise in Streptomyces coelicolor A3(2). EMBO J 22:4856–4865

    Article  CAS  Google Scholar 

  • Datta R, Zeikus JG (1985) Modulation of acetone–butanol–ethanol fermentation by carbon monoxide and organic acids. Appl Environ Microbiol 49:522–529

    CAS  Google Scholar 

  • Desai RP, Papoutsakis ET (1999) Antisense RNA strategies for metabolic engineering of Clostridium acetobutylicum. Appl Environ Microbiol 65:936–945

    CAS  Google Scholar 

  • 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 1:206–213

    Article  CAS  Google Scholar 

  • Doremus MG, Linden JC, Moreira AR (1985) Agitation and pressure effects on acetone–butanol fermentation. Biotechnol Bioeng 27(6):852–860

    Article  CAS  Google Scholar 

  • Dürre P (2008) Fermentative butanol production: bulk chemical and biofuel. Ann NY Acad Sci 1125:353–362

    Article  Google Scholar 

  • 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 26:268–272

    Article  Google Scholar 

  • 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 175:6959–6969

    CAS  Google Scholar 

  • 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 188:5469–5478

    Article  CAS  Google Scholar 

  • 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 184:821–830

    Article  CAS  Google Scholar 

  • Gerischer U, Dürre P (1992) mRNA analysis of the adc gene region of Clostridium acetobutylicum during the shift to solventogenesis. J Bacteriol 174(2):426–433

    CAS  Google Scholar 

  • Girbal L, Soucaille P (1998) Regulation of solvent production in Clostridium acetobutylicum. Trends Biotechnol 16:11–16

    Article  CAS  Google Scholar 

  • 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 176:6146–6147

    CAS  Google Scholar 

  • Girbal L, Croux C, Vasconcelos I, Soucaille P (1995) Regulation of metabolic shifts in Clostridium acetobutylicum ATCC 824. FEMS Microbiol Rev 17:287–297

    Article  CAS  Google Scholar 

  • 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 71:2777–2781

    Article  CAS  Google Scholar 

  • Gorwa MF, Croux C, Soucaille P (1996) Molecular characterization and transcriptional analysis of the putative hydrogenase gene of Clostridium acetobutylicum ATCC 824. J Bacteriol 178:2668–2675

    CAS  Google Scholar 

  • Gottschal JC, Morris JG (1981) Non‐production of acetone and butanol by Clostridium acetobutylicum during glucose‐ and ammonium‐limitation in continuous culture. Biotech Lett 3:525–530

    Google Scholar 

  • 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 87:4645–4649

    Article  CAS  Google Scholar 

  • 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 20:1–15

    Article  CAS  Google Scholar 

  • 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 58:3896–3902

    CAS  Google Scholar 

  • 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 188:7062–7071

    Article  CAS  Google Scholar 

  • 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):322–328

    Article  CAS  Google Scholar 

  • Hartmanis MGN, Gatenbeck S (1984) Intermediary metabolism in Clostridium acetobutylicum: levels of enzymes involved in the formation of acetate and butyrate. Appl Environ Microbiol 47:1277–1283

    CAS  Google Scholar 

  • 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 70:452–464

    Article  CAS  Google Scholar 

  • 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 80:49–55

    Article  CAS  Google Scholar 

  • Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59

    Article  CAS  Google Scholar 

  • 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 29:485–493

    Google Scholar 

  • 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 30:585–595

    Article  Google Scholar 

  • 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 87:2209–2226

    Article  CAS  Google Scholar 

  • 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 11:284–291

    Article  CAS  Google Scholar 

  • 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):420–424

    Article  CAS  Google Scholar 

  • Jones DT, Woods DR (1986) Acetone–butanol fermentation revisited. Microbiol Rev 50:484–524

    CAS  Google Scholar 

  • 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:R114

    Article  Google Scholar 

  • 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 48:764–770

    CAS  Google Scholar 

  • 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 94:729–741

  • Lee SY, Park JH, Jang SH, Nielsen LK, Kim J, Jung KS (2008) Fermentative butanol production by clostridia. Biotechnol Bioeng 101:209–228

    Article  CAS  Google Scholar 

  • Lee JY, Jang YS, Lee J, Papoutsakis ET, Lee SY (2009) Metabolic engineering of Clostridium acetobutylicum M5 for highly selective butanol production. Biotechnol J 4:1432–1440

    Article  CAS  Google Scholar 

  • Lehmann D, Lütke-Eversloh T (2011) Switching Clostridium acetobutylicum to an ethanol producer by disruption of the butyrate/butanol fermentative pathway. Metab Eng 13:464–473

    Article  CAS  Google Scholar 

  • 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 94:743–754

    Article  CAS  Google Scholar 

  • Lütke-Eversloh T, Bahl H (2011) Metabolic engineering of Clostridium acetobutylicum: recent advances to improve butanol production. Curr Opin Biotechnol 22:634–647

    Article  Google Scholar 

  • 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 59:1077–1081

    CAS  Google Scholar 

  • 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 24:159–167

    CAS  Google Scholar 

  • Monot F, Martin JR, Petitdemange H, Gay R (1982) Acetone and butanol production by Clostridium acetobutylicum in a synthetic medium. Appl Environ Microbiol 44:1318–1324

    CAS  Google Scholar 

  • Münch R, Hiller K, Barg H, Heldt D, Linz S, Wingender E, Jahn D (2003) PRODORIC: prokaryotic database of gene regulation. Nucleic Acids Res 31:266–269

    Article  Google Scholar 

  • 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):5442–5447

    Google Scholar 

  • 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 76:1142–1161

    Article  CAS  Google Scholar 

  • Peguin S, Goma G, Delorme P, Soucaille P (1994) Metabolic flexibility of Clostridium acetobutylicum in response to methyl viologen addition. Appl Microbiol Biotechnol 42:611–616

    Article  CAS  Google Scholar 

  • 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 12:420–428

    Article  CAS  Google Scholar 

  • 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 13:186–193

    Article  CAS  Google Scholar 

  • 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):1145–1157

    Article  CAS  Google Scholar 

  • 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 27:681–694

    Article  CAS  Google Scholar 

  • Sambrock J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  • Sauer U, Santangelo JD, Treuner A, Buchholz M, Dürre P (1995) Sigma factor and sporulation genes in Clostridium. FEMS Microbiol Rev 17(3):331–340

    Article  CAS  Google Scholar 

  • 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 1:115–124

    Google Scholar 

  • 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 13:43–54

    Article  CAS  Google Scholar 

  • Soucaille P, Figge R, Croux C (2006) Process for chromosomal integration and DNA sequence replacement in Clostridia. Dépôt PCT n° PCT/EP2006/066997

  • Terracciano JS, Kashket ER (1986) Intracellular conditions required for initiation of solvent production by Clostridium acetobutylicum. Appl Environ Microbiol 52:86–91

    CAS  Google Scholar 

  • Thormann K, Dürre P (2001) Orf5/SolR: a transcriptional repressor of the sol operon of Clostridium acetobutylicum? J Ind Microbiol Biotechnol 27(5):307–313

    Article  CAS  Google Scholar 

  • 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):1966–1973

    Article  CAS  Google Scholar 

  • 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):1443–1450

    CAS  Google Scholar 

  • Walter KA, Mermelstein LD, Papoutsakis ET (1994) Studies of recombinant Clostridium acetobutylicum with increased dosages of butyrate formation genes. Ann NY Acad Sci 721:69–72

    Article  CAS  Google Scholar 

  • 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 69:466–478

    Article  CAS  Google Scholar 

  • 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 408:670–683

    Article  CAS  Google Scholar 

  • Wiesenborn DP, Rudolph FB, Papoutsakis ET (1989a) Phosphotransbutyrylase from Clostridium acetobutylicum ATCC 824 and its role in acidogenesis. Appl Environ Microbiol 55:317–322

    CAS  Google Scholar 

  • 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 55:323–329

    CAS  Google Scholar 

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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.

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  1. Abteilung Mikrobiologie, Institut für Biowissenschaften, Universität Rostock, Albert-Einstein-Str. 3, 18051, Rostock, Germany

    Mandy Wietzke & Hubert Bahl

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  1. Mandy Wietzke
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Correspondence to Hubert Bahl.

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Wietzke, M., Bahl, H. The redox-sensing protein Rex, a transcriptional regulator of solventogenesis in Clostridium acetobutylicum . Appl Microbiol Biotechnol 96, 749–761 (2012). https://doi.org/10.1007/s00253-012-4112-2

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