Abstract
A recombinant butanol pathway composed of Clostridium acetobutylicum ATCC 824 genes, thiL, hbd, crt, bcd-etfB-etfA, and adhe1 (or adhe) coding for acetyl-CoA acetyltransferase (THL), β-hydroxybutyryl-CoA dehydrogenase (HBD), 3-hydroxybutyryl-CoA dehydratase (CRT), butyryl-CoA dehydrogenase (BCD), butyraldehyde dehydrogenase (BYDH), and butanol dehydrogenase (BDH), under the tac promoter control was constructed and was introduced into Escherichia coli. The functional expression of these six enzymes was proved by demonstrating the corresponding enzyme activities using spectrophotometric, high performance liquid chromatography and gas chromatography analyses. The BCD activity, which was not detected in E. coli previously, was shown in the present study by performing the procedure from cell extract preparation to activity measurement under anaerobic condition. Moreover, the etfA and etfB co-expression was found to be essential for the BCD activity. In the case of BYDH activity, the adhe gene product was shown to have higher specificity towards butyryl-CoA compared to the adhe1 product. Butanol production from glucose was achieved by the highly concentrated cells of the butanologenic E. coli strains, BUT1 with adhe1 and BUT2 with adhe, under anaerobic condition, and the BUT1 and BUT2 strains were shown to produce 4 and 16-mM butanol with 6- and 1-mM butyrate as a byproduct, respectively. This study reports the novel butanol production by an aerobically pregrown microorganism possessing the genes of a strict anaerobe, Clostridium acetobutylicum.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Boynton ZL, Bennet 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
Brown DP, Ganova-Raeva L, Green BD, Wilkinson SR, Young M, Youngman P (1994) Characterization of spo0A homologues in diverse Bacillus and Clostridium species identifies a probable DNA-binding domain. Mol Microbiol 14:411–426
Cornillot E, Nair RV, Papoutsakis ET, Soucaille P (1997) The genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824 reside on a large plasmid whose loss leads to degeneration of the strain. J Bacteriol 179:5442–5447
Dürre P, Kuhn A, Gottwald M, Gottschalk G (1987) Enzymatic investigtations on butanol dehydrogenase and butyraldehyde dehydrogenase in extracts of Clostridium acetobutylicum. Appl Microbiol Biotechnol 26:268–272
Engel PC, Massey V (1971) Green butyryl-coenzyme A dehydrogenase. An enzyme-acyl-coenzyme A complex. Biochem J 125:889–902
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
George HA, Johnson JL, Moore WE, Holdeman LV, Chen JS (1983) Acetone, isopropanol, and butanol production by Clostridium beijerinckii (syn. Clostridium butylicum) and Clostridium aurantibutyricum. Appl Environ Microbiol 45:1160–1163
Gottwald M, Hippe H, Gottschalk G (1984) Formation of n-butanol from D-glucose by strains of the “Clostridium tetanomorphum” group. Appl Environ Microbiol 48:573–576
Hartmanis MG, 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
Jenkins LS, Nunn WD (1987) Regulation of the ato operon by the atoC gene in Escherichia coli. J Bacteriol 169:2096–2102
Jones DT, Woods DR (1986) Acetone-butanol fermentation revisited. Microbiol Rev 50:484–524
Keis S, Bennett CF, Ward VK, Jones DT (1995) Taxonomy and phylogeny of industrial solvent-producing clostridia. Int J Syst Bacteriol 45:693–705
Lehman TC, Hale DE, Bhala A, Thorpe C (1990) An acyl-coenzyme A dehydrogenase assay utilizing the ferricenium ion. Anal Biochem 186:280–284
Mitchell WJ (1998) Physiology of carbohydrate to solvent conversion by clostridia. Adv Microb Physiol 39:31–130
Nair RV, Bennett GN, Papoutsakis ET (1994) Molecular characterization of an aldehyde/alcohol dehydrogenase gene from Clostridium acetobutylicum ATCC 824. J Bacteriol 176:871–885
Nakata K, Inui M, Kos PB, Vertès AA, Yukawa H (2003) Vectors for the genetic engineering of corynebacteria. In: Saha BC (ed) Symposium Series No.855 Fermentation Biotechnology. American Chemical Society, Washington, DC, pp 175–191
Nölling J, Breton G, Omelchenko MV, Makarova KS, Zeng Q, Gibson R, Lee HM, Dubois J, Qiu D, Hitti J, 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 183:4823–4838
O’Neill HM, Butler G, Mayhew SG (1995) Cloning of electron-transferring flavoprotein from Megasphaera elsdenii. Biochem Soc Trans 23:379S
Pace CP, Stankovich MT (1987) Redox properties of electron-transferring flavoprotein from Megasphaera elsdenii. Biochim Biophys Acta 911:267–276
Petersen DJ, Welch RW, Rudolph FB, Bennett GN (1991) Molecular cloning of an alcohol (butanol) dehydrogenase gene cluster from Clostridium acetobutylicum ATCC 824. J Bacteriol 173:1831–1834
Rangarajan ES, Li Y, Ajamian E, Iannuzzi P, Kernaghan SD, Fraser ME, Cygler M, Matte A (2005) Crystallographic trapping of the glutamyl-CoA thioester intermediate of family I CoA transferases. J Biol Chem 280:42919–42928
Ravagnani A, Jennert KC, Steiner E, Grunberg R, Jefferies JR, Wilkinson SR, Young DI, Tidswell EC, Brown DP, Youngman P, Morris JG, Young M (2000) Spo0A directly controls the switch from acid to solvent production in solvent-forming clostridia. Mol Microbiol 37:1172–1185
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Van Berkel WJ, Van den Berg WA, Muller F (1988) Large-scale preparation and reconstitution of apo-flavoproteins with special reference to butyryl-CoA dehydrogenase from Megasphaera elsdenii. Hydrophobic-interaction chromatography. Eur J Biochem 178:197–207
Whitfield CD, Mayhew SG (1974) Purification and properties of electron-transferring flavoprotein from Peptostreptococcus elsdenii. J Biol Chem 249:2801–2810
Wiesenborn DP, Rudolph FB, Papoutsakis ET (1988) Thiolase from Clostridium acetobutylicum ATCC 824 and its role in the synthesis of acids and solvents. Appl Environ Microbiol 54:2717–2722
Acknowledgement
We thank Crispinus A. Omumasaba for critical reading of the manuscript. This work was financially supported in part by a grant from the Ministry of Economy, Trade and Industry (METI).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Inui, M., Suda, M., Kimura, S. et al. Expression of Clostridium acetobutylicum butanol synthetic genes in Escherichia coli . Appl Microbiol Biotechnol 77, 1305–1316 (2008). https://doi.org/10.1007/s00253-007-1257-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-007-1257-5