Coenzyme A-transferase-independent butyrate re-assimilation in Clostridium acetobutylicum—evidence from a mathematical model
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The hetero-dimeric CoA-transferase CtfA/B is believed to be crucial for the metabolic transition from acidogenesis to solventogenesis in Clostridium acetobutylicum as part of the industrial-relevant acetone-butanol-ethanol (ABE) fermentation. Here, the enzyme is assumed to mediate re-assimilation of acetate and butyrate during a pH-induced metabolic shift and to faciliate the first step of acetone formation from acetoacetyl-CoA. However, recent investigations using phosphate-limited continuous cultures have questioned this common dogma. To address the emerging experimental discrepancies, we investigated the mutant strain Cac-ctfA398s::CT using chemostat cultures. As a consequence of this mutation, the cells are unable to express functional ctfA and are thus lacking CoA-transferase activity. A mathematical model of the pH-induced metabolic shift, which was recently developed for the wild type, is used to analyse the observed behaviour of the mutant strain with a focus on re-assimilation activities for the two produced acids. Our theoretical analysis reveals that the ctfA mutant still re-assimilates butyrate, but not acetate. Based upon this finding, we conclude that C. acetobutylicum possesses a CoA-tranferase-independent butyrate uptake mechanism that is activated by decreasing pH levels. Furthermore, we observe that butanol formation is not inhibited under our experimental conditions, as suggested by previous batch culture experiments. In concordance with recent batch experiments, acetone formation is abolished in chemostat cultures using the ctfa mutant.
KeywordsClostridium acetobutylicum ctfA mutant Acid re-assimilation pH-induced metabolic shift Mathematical modelling
The authors acknowledge support by the German Federal Ministry for Education and Research (BMBF FKZ 0315782D) and by the Biotechnology and Biological Sciences Research Council (UK) (BBSRC No. BB/I004475/1), as part of the European Transnational Network ‘Systems Biology of Microorganisms’ (SysMo) within the COSMIC consortium. N.P.M. also acknowledges funding received from the Biotechnology and Biological Sciences Research Council, as part of the BBSRC Sustainable Bioenergy Centre (BSBEC) initiative, for the programme ‘Second Generation, Sustainable Biofuels’ (BBSRC No. BB/G016224/1). We thank Ulf W. Liebal and Carola Berger for their critical comments and fruitful discussions. The responsibility for the content of this manuscript lies with the authors.
Conflict of interests
The authors declare that they have no conflict of interest.
- Al-Hinai M, Fast A, Papoutsakis E (2012) A novel system for efficient isolation of double-crossover allelic exchange mutants in Clostridium enabling markerless chromosomal gene deletions and DNA integration. Appl Environ Microbiol 78:8112–8121. doi: 10.1128/AEM.02214-12 PubMedCrossRefPubMedCentralGoogle Scholar
- Caspi R, Altman T, Dreher K, Fulcher C, Subhraveti P, Keseler I, Kothari A, Krummenacker M, Latendresse M, Mueller L, Ong Q, Paley S, Pujar A, Shearer A, Travers M, Weerasinghe D, Zhang P, Karp P (2012) The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases. Nucleic Acids Res 40:D742–D753. doi: 10.1093/nar/gkr1014 PubMedCrossRefPubMedCentralGoogle Scholar
- Dürre P, Fischer RJ, Kuhn A, Lorenz K, Schreiber W, Stürzenhofecker B, Ullmann S, Winzer K, Sauer U (1995) Solventogenic enzymes of Clostridium acetobutylicum: catalytic properties, genetic organization, and transcriptional regulation. FEMS Microbiol Rev 17:251–262. doi: 10.1111/j.1574-6976.1995.tb00209.x PubMedCrossRefGoogle 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. doi: 10.1128/JB.184.3.821-830.2002 PubMedCrossRefPubMedCentralGoogle 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. doi: 10.1159/000320973 PubMedCrossRefGoogle Scholar
- Haus S, Jabbari S, Millat T, Janssen H, Fischer RJ, Bahl H, King J, Wolkenhauer O (2011) A systems biology approach to investigate the effect of pH-induced gene regulation on solvent production by Clostridium acetobutylicum in continuous culture. BMC Syst Biol 5:10. doi: 10.1186/1752-0509-5-10 PubMedCrossRefPubMedCentralGoogle Scholar
- Janssen H, Döring C, Ehrenreich A, Voigt B, Hecker M, Bahl H, Fischer RJ (2010) A proteomic and transcriptional view of acidogenesis and solventogenesis in Clostridium acetobutylicum in a chemostat culture. Appl Microbiol Biotechnol 87:2209–2226. doi: 10.1007/s00253-010-2741-x PubMedCrossRefPubMedCentralGoogle Scholar
- Karp P, Paley S, Krummenacker M, Latendresse M, Dale J, Lee T, Kaipa P, Gilham F, Spaulding A, Popescu L, Altman T, Paulsen I, Keseler I, Caspi R (2010) Pathway Tools version 13.0: integrated software for pathway/genome informatics and systems biology. Brief Bioinform 11:40–79. doi: 10.1093/bib/bbp043 PubMedCrossRefPubMedCentralGoogle Scholar
- Kuehne S, Minton N (2012) ClosTron-mediated engineering of Clostridium. Bioengineered 3:247–254. doi: 10.4161/bioe.21004
- Madigan M, Martinko J, Dunlap P, Clark D, Brock T (2009) Brock Biology of Microorganisms, 12th edn. Pearson/Benjamin Cummings, San FransiscoGoogle Scholar
- Mao S, Luo Y, Zhang T, Li J, Bao G, Zhu Y, Chen Z, Zhang Y, Li Y, Ma Y (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 9:3046–3061. doi: 10.1021/pr9012078 PubMedCrossRefGoogle Scholar
- Millat T, Janssen H, Bahl H, Fischer RJ, Wolkenhauer O (2013a) Integrative modelling of pH-dependent enzyme activity and transcriptomic regulation of the acetone-butanol-ethanol fermentation of Clostridium acetobutylicum in continuous culture. Microbial Biotechnol 6:526–539. doi: 10.1111/1751-7915.12033 CrossRefGoogle Scholar
- Milne C, Eddy J, Raju R, Ardekani S, Kim PJ, Senger R, Jin YS, Blaschek H, Price N (2011) Metabolic network reconstruction and genome-scale model of butanol-producing strain Clostridium beijerinckii NCIMB 8052. BMC Syst Biol 5:130. doi: 10.1186/1752-0509-5-130 PubMedCrossRefPubMedCentralGoogle Scholar
- Nölling J, Breton G, Omelchenko M, Makarova K, Zeng Q, Gibson R, Lee H, Dubois J, Qiu D, Hitti J, GSC Production, Teams B Finishing, Wolf Y, Tatusov R, Sabathe F, Doucette-Stamm L, Soucaille P, Daly M, Bennett G, Koonin E, Smith D (2001) Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. J Bacteriol 183:4823–4838. doi: 10.1128/JB.183.16.4823-4838.2001 PubMedCrossRefPubMedCentralGoogle Scholar
- Rogers P, Gottschalk G (1993) Biochemistry and regulation of acid and solvent formation in clostridia. In: Woods DR (ed) The clostridia and biotechnology. Butterworth-Heinemann, London, pp 25–50Google Scholar
- Schaffer S, Isci N, Zickner B, Dürre P (2002) Changes in protein synthesis and identification of proteins specifically induced during solventogenesis in Clostridium acetobutylicum. Electrophoresis 23:110–121. doi: 10.1002/1522-2683(200201)23:1<110::AID-ELPS110>3.0.CO;2-G PubMedCrossRefGoogle Scholar
- Tummala S, Junne S, Papoutsakis E (2003a) Antisense RNA downregulation of coenzyme A transferase combined with alcohol-aldehyde dehydrogenase overexpression leads to predominantly alcohologenic Clostridium acetobutylicum fermentations. J Bacteriol 185:3644–3653. doi: 10.1128/JB.185.12.3644-3653.2003 PubMedCrossRefPubMedCentralGoogle Scholar