Abstract
Sixteen Tn916-induced mutants of Clostridium acetobutylicum were selected that were defective in the production of acetone and butanol. Formation of ethanol, however, was only partially affected. The strains differed with respect to the degree of solvent formation ability and could be assigned to three different groups. Type I mutants (2 strains) were completely defective in acetone and butanol production and contained one or three copies of Tn916 in the chromosome. Analysis of the mutants for enzymes responsible for solvent production revealed the presence of a formerly unknown, specific acetaldehyde dehydrogenase. The data obtained also strongly indicate that the NADP+-dependent alcohol dehydrogenase is in vivo reponsible for ethanol formation, whereas the NAD+-dependent alcohol dehydrogenase is probably involved in butanol production. No activity of this enzyme together with all other enzymes in the acetone and butanol pathway could be found in type I strains. All tetracycline-resistant mutants obtained did no longer sporulate.
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Abbreviations
- AADC:
-
acetoacetate decarboxylase
- AcaDH:
-
acetaldehyde dehydrogenase
- BuaDH:
-
butyraldehyde dehydrogenase
- CoA-TF:
-
acetoacetyl coenzyme A: acetate/butyrate: coenzyme A transferase
- NAD-ADH, NAD+ :
-
dependent alcohol dehydrogenase
- NADP-ADH, NADP+ :
-
dependent alcohol dehydrogenase
References
Andersch W, Bahl H, Gottschalk G (1983) Level of enzymes involved in acetate, butyrate, acetone and butanol formation by Clostridium acetobutylicum. Eur J Appl Biotechnol 18:327–332
Bahl H, Gottschalk G (1988) Microbial production of butanol/acetone. In: Rehm H-J, Reed G (eds) Biotechnology, vol 6b. VCH, Weinheim, pp 1–30
Bahl H, Andersch W, Gottschalk G (1982) Continuous production of acetone and butanol by Clostridium acetobutylicum in a two-stage phosphate-limited chemostat. Eur J Appl Microbiol Biotechnol 15:201–205
Bertram J, Dürre P (1989) Conjugal transfer and expression of streptococcal transposons in Clostridium acetobutylicum. Arch Microbiol 151:551–557
Clark SW, Bennett GN, Rudolph FB (1989) Isolation and characterization of mutants of Clostridium acetobutylicum ATCC 824 deficient in acetoacetyl-coenzyme A: acetate/butyrate: coenzyme A-transferase (EC 2.8.3.9) and in other solvent pathway enzymes. Appl Environ Microbiol 55:970–976
Costilow RN (1981) Biophysical factors in growth. In: Gerhardt P, Murray RGE, Costilow RN, Nester EW, Wood WA, Krieg NR, Phillips GB (eds) Manual of methods for general bacteriology. American Society for Microbiology, Washington, D.C., pp 68–78
Davies R (1943) Studies on the acetone-butanol fermentation. 4. Acetoacetic acid decarboxylase of Cl. acetobutylicum. Biochem J 37:230–238
Dürre P, Kuhn A, Gottschalk G (1986) Treatment with allyl alcohol selects specifically for mutants of Clostridium acetobutylicum defective in butanol synthesis. FEMS Microbiol Lett 36:77–81
Dürre P, Kuhn A, Gottwald M, Gottschalk G (1987) Enzymatic investigations in butanol dehydrogenase and butyraldehyde dehydrogenase in extracts of Clostridium acetobutylicum. Appl Microbiol Biotechnol 26:268–272
Gawron-Burke C, Clewell DB (1984) Regeneration of insertionally inactivated streptococcal DNA fragments after excision of transposon Tn916 in Escherichia coli: Strategy for targeting and cloning of genes from Gram-positive bacteria. J Bacteriol 159:214–221
Hirashima A, Childs G, Inouye M (1973) Differential inhibitory effects of antibiotics on the biosynthesis of envelope proteins of Escherichia coli. J Mol Biol 79:373–389
Janati-Idrissi R, Junelles AM, El Kanouni A, Petitdemange H, Gay R (1987) Sélection de mutants de Clostridium acetobutylicum défectifs dans la production d'acétone. Ann Inst Pasteur/Microbiol 138:313–323
Jones DT, van der Westhuizen A, Long S, Allcock ER, Reid SJ, Woods DR (1982) Solvent production and morphological changes in Clostridium acetobutylicum. Appl Environ Microbiol 43:1434–1439
Jones DT, Woods DR (1986) Acetone-butanol fermentation revisited. Microbiol Rev 50:484–524
Long S, Jones DT, Woods DR (1984) Initiation of solvent production, clostridial stage and endospore formation in Clostridium acetobutylicum P262. Appl Microbiol Biotechnol 20:256–261
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Meinecke B, Bahl H, Gottschalk G (1984) Selection of an asporogenous strain of Clostridium acetobutylicum in continuous culture under phosphate limitation. Appl Environ Microbiol 48:1064–1065
O'Brien RW, Morris JG (1971) Oxygen and the growth and metabolism of Clostridium acetobutylicum. J Gen Microbiol 68:307–318
Palosaari N, Rogers P (1988) Purification and properties of the inducible coenzyme A-linked butyraldehyde dehydrogenase from Clostridium acetobutylicum. J Bacteriol 170:2971–2976
Petitdemange H, Cherrier C, Begone JM, Gay R (1977) Étude des activités NADH et NADPH-ferrédoxine oxydoréductasiques chez Clostridium acetobutylicum. Can J Microbiol 23:152–160
Piovant M, Varenne S, Pagès JM, Lazdunski C (1978) Preferential sensitivity of syntheses of exported proteins to translation inhibitors of low polarity in Escherichia coli. Mol Gen Genet 164:265–274
Reysset G, Sebald M (1985) Conjugal transfer of plasmid antibiotic resistance from streptococci to Clostridium acetobutylicum. Ann Inst Pasteur/Microbiol 136B:275–282
Rogers P (1986) Genetics and biochemistry of Clostridium relevant to development of fermentation processes. Adv Appl Microbiol 31:1–60
Rogers P, Palosaari N (1987) Clostridium acetobutylicum mutants that produce butyraldehyde and altered quantities of solvents. Appl Environ Microbiol 53:2761–2766
Wiesenborn DP, Rudolph FB, Papoutsakis ET (1989) Coenzyme A transferase from Clostridium acetobutylicum ATCC 824 and its role in the uptake of acids. Appl Environ Microbiol 55:323–329
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Bertram, J., Kuhn, A. & Dürre, P. Tn916-induced mutants of Clostridium acetobutylicum defective in regulation of solvent formation. Arch. Microbiol. 153, 373–377 (1990). https://doi.org/10.1007/BF00249008
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DOI: https://doi.org/10.1007/BF00249008