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. 1996 Jun;178(11):3015–3024. doi: 10.1128/jb.178.11.3015-3024.1996

Cloning, sequencing, and expression of clustered genes encoding beta-hydroxybutyryl-coenzyme A (CoA) dehydrogenase, crotonase, and butyryl-CoA dehydrogenase from Clostridium acetobutylicum ATCC 824.

Z L Boynton 1, G N Bennet 1, F B Rudolph 1
PMCID: PMC178046  PMID: 8655474

Abstract

The enzymes beta-hydroxybutyryl-coenzyme A (CoA) dehydrogenase (BHBD), crotonase, and butyryl-CoA dehydrogenase (BCD) from Clostridium acetobutylicum are responsible for the formation of butyryl-CoA from acetoacetyl-CoA. These enzymes are essential to both acid formation and solvent formation by clostridia. Clustered genes encoding BHBD, crotonase, BCD, and putative electron transfer flavoprotein alpha and beta subunits have been cloned and sequenced. The nucleotide sequence of the crt gene indicates that it encodes crotonase, a protein with 261 amino acid residues and a calculated molecular mass of 28.2 kDa; the hbd gene encodes BHBD, with 282 residues and a molecular mass of 30.5 kDa. Three open reading frames (bcd, etfB, and etfA) are located between crt and hbd. The nucleotide sequence of bcd indicates that it encodes BCD, which consists of 379 amino acid residues and has high levels of homology with various acyl-CoA dehydrogenases. Open reading frames etfB and etfA, located downstream of bcd, encode 27.2- and 36.1-kDa proteins, respectively, and show homology with the fixAB genes and the alpha and beta subunits of the electron transfer flavoprotein. These findings suggest that BCD in clostridia might interact with the electron transfer flavoprotein in its redox function. Primer extension analysis identified a promoter consensus sequence upstream of the crt gene, suggesting that the clustered genes are transcribed as a transcriptional unit and form a BCS (butyryl-CoA synthesis) operon. A DNA fragment containing the entire BCS operon was subcloned into an Escherichia coli-C. acetobutylicum shuttle vector. Enzyme activity assays showed that crotonase and BHBD were highly overproduced in cell extracts from E. coli harboring the subclone. In C. acetobutylicum harboring the subclone, the activities of the enzymes crotonase, BHBD, and BCD were elevated.

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Selected References

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  1. Arigoni F., Kaminski P. A., Hennecke H., Elmerich C. Nucleotide sequence of the fixABC region of Azorhizobium caulinodans ORS571: similarity of the fixB product with eukaryotic flavoproteins, characterization of fixX, and identification of nifW. Mol Gen Genet. 1991 Mar;225(3):514–520. doi: 10.1007/BF00261695. [DOI] [PubMed] [Google Scholar]
  2. Becker D. F., Fuchs J. A., Banfield D. K., Funk W. D., MacGillivray R. T., Stankovich M. T. Characterization of wild-type and an active-site mutant in Escherichia coli of short-chain acyl-CoA dehydrogenase from Megasphaera elsdenii. Biochemistry. 1993 Oct 12;32(40):10736–10742. doi: 10.1021/bi00091a026. [DOI] [PubMed] [Google Scholar]
  3. Bedzyk L. A., Escudero K. W., Gill R. E., Griffin K. J., Frerman F. E. Cloning, sequencing, and expression of the genes encoding subunits of Paracoccus denitrificans electron transfer flavoprotein. J Biol Chem. 1993 Sep 25;268(27):20211–20217. [PubMed] [Google Scholar]
  4. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  5. Birktoft J. J., Holden H. M., Hamlin R., Xuong N. H., Banaszak L. J. Structure of L-3-hydroxyacyl-coenzyme A dehydrogenase: preliminary chain tracing at 2.8-A resolution. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8262–8266. doi: 10.1073/pnas.84.23.8262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bitar K. G., Perez-Aranda A., Bradshaw R. A. Amino acid sequence of L-3-hydroxyacyl CoA dehydrogenase from pig heart muscle. FEBS Lett. 1980 Jul 28;116(2):196–198. doi: 10.1016/0014-5793(80)80642-9. [DOI] [PubMed] [Google Scholar]
  7. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  8. Bross P., Engst S., Strauss A. W., Kelly D. P., Rasched I., Ghisla S. Characterization of wild-type and an active site mutant of human medium chain acyl-CoA dehydrogenase after expression in Escherichia coli. J Biol Chem. 1990 May 5;265(13):7116–7119. [PubMed] [Google Scholar]
  9. Cary J. W., Petersen D. J., Papoutsakis E. T., Bennett G. N. Cloning and expression of Clostridium acetobutylicum ATCC 824 acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase in Escherichia coli. Appl Environ Microbiol. 1990 Jun;56(6):1576–1583. doi: 10.1128/aem.56.6.1576-1583.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cary J. W., Petersen D. J., Papoutsakis E. T., Bennett G. N. Cloning and expression of Clostridium acetobutylicum phosphotransbutyrylase and butyrate kinase genes in Escherichia coli. J Bacteriol. 1988 Oct;170(10):4613–4618. doi: 10.1128/jb.170.10.4613-4618.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chung C. T., Miller R. H. A rapid and convenient method for the preparation and storage of competent bacterial cells. Nucleic Acids Res. 1988 Apr 25;16(8):3580–3580. doi: 10.1093/nar/16.8.3580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dietrichs D., Meyer M., Schmidt B., Andreesen J. R. Purification of NADPH-dependent electron-transferring flavoproteins and N-terminal protein sequence data of dihydrolipoamide dehydrogenases from anaerobic, glycine-utilizing bacteria. J Bacteriol. 1990 Apr;172(4):2088–2095. doi: 10.1128/jb.172.4.2088-2095.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Djordjevic S., Pace C. P., Stankovich M. T., Kim J. J. Three-dimensional structure of butyryl-CoA dehydrogenase from Megasphaera elsdenii. Biochemistry. 1995 Feb 21;34(7):2163–2171. doi: 10.1021/bi00007a009. [DOI] [PubMed] [Google Scholar]
  14. Earl C. D., Ronson C. W., Ausubel F. M. Genetic and structural analysis of the Rhizobium meliloti fixA, fixB, fixC, and fixX genes. J Bacteriol. 1987 Mar;169(3):1127–1136. doi: 10.1128/jb.169.3.1127-1136.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Engel P. C., Massey V. Green butyryl-coenzyme A dehydrogenase. An enzyme-acyl-coenzyme A complex. Biochem J. 1971 Dec;125(3):889–902. doi: 10.1042/bj1250889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Engel P. C., Massey V. The purification and properties of butyryl-coenzyme A dehydrogenase from Peptostreptococcus elsdenii. Biochem J. 1971 Dec;125(3):879–887. doi: 10.1042/bj1250879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fendrich G., Abeles R. H. Mechanism of action of butyryl-CoA dehydrogenase: reactions with acetylenic, olefinic, and fluorinated substrate analogues. Biochemistry. 1982 Dec 21;21(26):6685–6695. doi: 10.1021/bi00269a011. [DOI] [PubMed] [Google Scholar]
  18. Finocchiaro G., Colombo I., Garavaglia B., Gellera C., Valdameri G., Garbuglio N., Didonato S. cDNA cloning and mitochondrial import of the beta-subunit of the human electron-transfer flavoprotein. Eur J Biochem. 1993 May 1;213(3):1003–1008. doi: 10.1111/j.1432-1033.1993.tb17847.x. [DOI] [PubMed] [Google Scholar]
  19. Finocchiaro G., Ito M., Ikeda Y., Tanaka K. Molecular cloning and nucleotide sequence of cDNAs encoding the alpha-subunit of human electron transfer flavoprotein. J Biol Chem. 1988 Oct 25;263(30):15773–15780. [PubMed] [Google Scholar]
  20. Frischauf A. M., Lehrach H., Poustka A., Murray N. Lambda replacement vectors carrying polylinker sequences. J Mol Biol. 1983 Nov 15;170(4):827–842. doi: 10.1016/s0022-2836(83)80190-9. [DOI] [PubMed] [Google Scholar]
  21. GREEN D. E., MII S., MAHLER H. R., BOCK R. M. Studies on the fatty acid oxidizing system of animal tissues. III. Butyryl coenzyme A dehydrogenase. J Biol Chem. 1954 Jan;206(1):1–12. [PubMed] [Google Scholar]
  22. Hartmanis M. G., Gatenbeck S. Intermediary Metabolism in Clostridium acetobutylicum: Levels of Enzymes Involved in the Formation of Acetate and Butyrate. Appl Environ Microbiol. 1984 Jun;47(6):1277–1283. doi: 10.1128/aem.47.6.1277-1283.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Husain M., Steenkamp D. J. Electron transfer flavoprotein from pig liver mitochondria. A simple purification and re-evaluation of some of the molecular properties. Biochem J. 1983 Feb 1;209(2):541–545. doi: 10.1042/bj2090541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Husain M., Steenkamp D. J. Partial purification and characterization of glutaryl-coenzyme A dehydrogenase, electron transfer flavoprotein, and electron transfer flavoprotein-Q oxidoreductase from Paracoccus denitrificans. J Bacteriol. 1985 Aug;163(2):709–715. doi: 10.1128/jb.163.2.709-715.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jones D. T., Woods D. R. Acetone-butanol fermentation revisited. Microbiol Rev. 1986 Dec;50(4):484–524. doi: 10.1128/mr.50.4.484-524.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kelly D. P., Kim J. J., Billadello J. J., Hainline B. E., Chu T. W., Strauss A. W. Nucleotide sequence of medium-chain acyl-CoA dehydrogenase mRNA and its expression in enzyme-deficient human tissue. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4068–4072. doi: 10.1073/pnas.84.12.4068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kim J. J., Wang M., Paschke R. Crystal structures of medium-chain acyl-CoA dehydrogenase from pig liver mitochondria with and without substrate. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7523–7527. doi: 10.1073/pnas.90.16.7523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kim J. J., Wu J. Structure of the medium-chain acyl-CoA dehydrogenase from pig liver mitochondria at 3-A resolution. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6677–6681. doi: 10.1073/pnas.85.18.6677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Komuniecki R., McCrury J., Thissen J., Rubin N. Electron-transfer flavoprotein from anaerobic Ascaris suum mitochondria and its role in NADH-dependent 2-methyl branched-chain enoyl-CoA reduction. Biochim Biophys Acta. 1989 Jun 23;975(1):127–131. doi: 10.1016/s0005-2728(89)80210-5. [DOI] [PubMed] [Google Scholar]
  30. Lee S. Y., Mermelstein L. D., Bennett G. N., Papoutsakis E. T. Vector construction, transformation, and gene amplification in Clostridium acetobutylicum ATCC 824. Ann N Y Acad Sci. 1992 Oct 13;665:39–51. doi: 10.1111/j.1749-6632.1992.tb42572.x. [DOI] [PubMed] [Google Scholar]
  31. Lehman T. C., Hale D. E., Bhala A., Thorpe C. An acyl-coenzyme A dehydrogenase assay utilizing the ferricenium ion. Anal Biochem. 1990 May 1;186(2):280–284. doi: 10.1016/0003-2697(90)90080-s. [DOI] [PubMed] [Google Scholar]
  32. Lundberg N. N., Thorpe C. Inactivation of short-chain acyl-coenzyme A dehydrogenase from pig liver by 2-pentynoyl-coenzyme A. Arch Biochem Biophys. 1993 Sep;305(2):454–459. doi: 10.1006/abbi.1993.1446. [DOI] [PubMed] [Google Scholar]
  33. Matsubara Y., Indo Y., Naito E., Ozasa H., Glassberg R., Vockley J., Ikeda Y., Kraus J., Tanaka K. Molecular cloning and nucleotide sequence of cDNAs encoding the precursors of rat long chain acyl-coenzyme A, short chain acyl-coenzyme A, and isovaleryl-coenzyme A dehydrogenases. Sequence homology of four enzymes of the acyl-CoA dehydrogenase family. J Biol Chem. 1989 Sep 25;264(27):16321–16331. [PubMed] [Google Scholar]
  34. Matsubara Y., Kraus J. P., Ozasa H., Glassberg R., Finocchiaro G., Ikeda Y., Mole J., Rosenberg L. E., Tanaka K. Molecular cloning and nucleotide sequence of cDNA encoding the entire precursor of rat liver medium chain acyl coenzyme A dehydrogenase. J Biol Chem. 1987 Jul 25;262(21):10104–10108. [PubMed] [Google Scholar]
  35. Mermelstein L. D., Papoutsakis E. T. In vivo methylation in Escherichia coli by the Bacillus subtilis phage phi 3T I methyltransferase to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol. 1993 Apr;59(4):1077–1081. doi: 10.1128/aem.59.4.1077-1081.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mermelstein L. D., Welker N. E., Bennett G. N., Papoutsakis E. T. Expression of cloned homologous fermentative genes in Clostridium acetobutylicum ATCC 824. Biotechnology (N Y) 1992 Feb;10(2):190–195. doi: 10.1038/nbt0292-190. [DOI] [PubMed] [Google Scholar]
  37. Mermelstein L. D., Welker N. E., Petersen D. J., Bennett G. N., Papoutsakis E. T. Genetic and metabolic engineering of Clostridium acetobutylicum ATCC 824. Ann N Y Acad Sci. 1994 May 2;721:54–68. doi: 10.1111/j.1749-6632.1994.tb47376.x. [DOI] [PubMed] [Google Scholar]
  38. Minami-Ishii N., Taketani S., Osumi T., Hashimoto T. Molecular cloning and sequence analysis of the cDNA for rat mitochondrial enoyl-CoA hydratase. Structural and evolutionary relationships linked to the bifunctional enzyme of the peroxisomal beta-oxidation system. Eur J Biochem. 1989 Oct 20;185(1):73–78. doi: 10.1111/j.1432-1033.1989.tb15083.x. [DOI] [PubMed] [Google Scholar]
  39. Mullany P., Clayton C. L., Pallen M. J., Slone R., al-Saleh A., Tabaqchali S. Genes encoding homologues of three consecutive enzymes in the butyrate/butanol-producing pathway of Clostridium acetobutylicum are clustered on the Clostridium difficile chromosome. FEMS Microbiol Lett. 1994 Nov 15;124(1):61–67. doi: 10.1111/j.1574-6968.1994.tb07262.x. [DOI] [PubMed] [Google Scholar]
  40. Naito E., Ozasa H., Ikeda Y., Tanaka K. Molecular cloning and nucleotide sequence of complementary DNAs encoding human short chain acyl-coenzyme A dehydrogenase and the study of the molecular basis of human short chain acyl-coenzyme A dehydrogenase deficiency. J Clin Invest. 1989 May;83(5):1605–1613. doi: 10.1172/JCI114058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Osumi T., Ishii N., Hijikata M., Kamijo K., Ozasa H., Furuta S., Miyazawa S., Kondo K., Inoue K., Kagamiyama H. Molecular cloning and nucleotide sequence of the cDNA for rat peroxisomal enoyl-CoA: hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme. J Biol Chem. 1985 Jul 25;260(15):8905–8910. [PubMed] [Google Scholar]
  42. Pace C. P., Stankovich M. T. Redox properties of electron-transferring flavoprotein from Megasphaera elsdenii. Biochim Biophys Acta. 1987 Feb 25;911(3):267–276. doi: 10.1016/0167-4838(87)90067-7. [DOI] [PubMed] [Google Scholar]
  43. Powell P. J., Thorpe C. 2-octynoyl coenzyme A is a mechanism-based inhibitor of pig kidney medium-chain acyl coenzyme A dehydrogenase: isolation of the target peptide. Biochemistry. 1988 Oct 18;27(21):8022–8028. doi: 10.1021/bi00421a008. [DOI] [PubMed] [Google Scholar]
  44. Pramanik A., Pawar S., Antonian E., Schulz H. Five different enzymatic activities are associated with the multienzyme complex of fatty acid oxidation from Escherichia coli. J Bacteriol. 1979 Jan;137(1):469–473. doi: 10.1128/jb.137.1.469-473.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Shinzawa K., Inagaki T., Ohishi N., Ichihara C., Tsukagoshi N., Udaka S., Yagi K. Molecular cloning of a cDNA for alpha-subunit of rat liver electron transfer flavoprotein. Biochem Biophys Res Commun. 1988 Aug 30;155(1):300–304. doi: 10.1016/s0006-291x(88)81084-2. [DOI] [PubMed] [Google Scholar]
  47. Spratt S. K., Black P. N., Ragozzino M. M., Nunn W. D. Cloning, mapping, and expression of genes involved in the fatty acid-degradative multienzyme complex of Escherichia coli. J Bacteriol. 1984 May;158(2):535–542. doi: 10.1128/jb.158.2.535-542.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Van Berkel W. J., Van den Berg W. A., Müller F. Large-scale preparation and reconstitution of apo-flavoproteins with special reference to butyryl-CoA dehydrogenase from Megasphaera elsdenii. Hydrophobic-interaction chromatography. Eur J Biochem. 1988 Dec 1;178(1):197–207. doi: 10.1111/j.1432-1033.1988.tb14444.x. [DOI] [PubMed] [Google Scholar]
  49. Walter K. A., Bennett G. N., Papoutsakis E. T. Molecular characterization of two Clostridium acetobutylicum ATCC 824 butanol dehydrogenase isozyme genes. J Bacteriol. 1992 Nov;174(22):7149–7158. doi: 10.1128/jb.174.22.7149-7158.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Waterson R. M., Castellino F. J., Hass G. M., Hill R. L. Purification and characterization of crotonase from Clostridium acetobutylicum. J Biol Chem. 1972 Aug 25;247(16):5266–5271. [PubMed] [Google Scholar]
  51. Waterson R. M., Hill R. L. Enoyl coenzyme A hydratase (crotonase). Catalytic properties of crotonase and its possible regulatory role in fatty acid oxidation. J Biol Chem. 1972 Aug 25;247(16):5258–5265. [PubMed] [Google Scholar]
  52. Watmough N. J., Kiss J., Frerman F. E. Structural and redox relationships between Paracoccus denitrificans, porcine and human electron-transferring flavoproteins. Eur J Biochem. 1992 May 1;205(3):1089–1097. doi: 10.1111/j.1432-1033.1992.tb16877.x. [DOI] [PubMed] [Google Scholar]
  53. Whitfield C. D., Mayhew S. G. Purification and properties of electron-transferring flavoprotein from Peptostreptococcus elsdenii. J Biol Chem. 1974 May 10;249(9):2801–2810. [PubMed] [Google Scholar]
  54. Wierenga R. K., Terpstra P., Hol W. G. Prediction of the occurrence of the ADP-binding beta alpha beta-fold in proteins, using an amino acid sequence fingerprint. J Mol Biol. 1986 Jan 5;187(1):101–107. doi: 10.1016/0022-2836(86)90409-2. [DOI] [PubMed] [Google Scholar]
  55. Williamson G., Engel P. C. Butyryl-CoA dehydrogenase from Megasphaera elsdenii. Specificity of the catalytic reaction. Biochem J. 1984 Mar 1;218(2):521–529. doi: 10.1042/bj2180521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wilson R., Ainscough R., Anderson K., Baynes C., Berks M., Bonfield J., Burton J., Connell M., Copsey T., Cooper J. 2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans. Nature. 1994 Mar 3;368(6466):32–38. doi: 10.1038/368032a0. [DOI] [PubMed] [Google Scholar]
  57. Yang S. Y., Elzinga M. Association of both enoyl coenzyme A hydratase and 3-hydroxyacyl coenzyme A epimerase with an active site in the amino-terminal domain of the multifunctional fatty acid oxidation protein from Escherichia coli. J Biol Chem. 1993 Mar 25;268(9):6588–6592. [PubMed] [Google Scholar]
  58. Yang X. Y., Schulz H., Elzinga M., Yang S. Y. Nucleotide sequence of the promoter and fadB gene of the fadBA operon and primary structure of the multifunctional fatty acid oxidation protein from Escherichia coli. Biochemistry. 1991 Jul 9;30(27):6788–6795. doi: 10.1021/bi00241a023. [DOI] [PubMed] [Google Scholar]
  59. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  60. Youngleson J. S., Jones D. T., Woods D. R. Homology between hydroxybutyryl and hydroxyacyl coenzyme A dehydrogenase enzymes from Clostridium acetobutylicum fermentation and vertebrate fatty acid beta-oxidation pathways. J Bacteriol. 1989 Dec;171(12):6800–6807. doi: 10.1128/jb.171.12.6800-6807.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Youngleson J. S., Lin F. P., Reid S. J., Woods D. R. Structure and transcription of genes within the beta-hbd-adh1 region of Clostridium acetobutylicum P262. FEMS Microbiol Lett. 1995 Jan 15;125(2-3):185–191. doi: 10.1111/j.1574-6968.1995.tb07356.x. [DOI] [PubMed] [Google Scholar]

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