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Expression of Cloned Homologous Fermentative Genes in Clostridium Acetobutylicum ATCC 824

Bio/Technology volume 10, pages 190–195 (1992)Cite this article

Abstract

We have previously cloned the acetone-formation pathway gene, encoding acetoacetate decarboxylase (adc), and butyrate-formation pathway gene, encoding phosphotransbutyrylase (ptb), of Clostridium acetobutylicum ATCC 824 via Escherichia coli. Here we report their subcloning in Bacillus subtilis and transfer to strain ATCC 824 via electrotransformation, where the corresponding enzyme activities were expressed at elevated levels, using pFNK1, a new B. subtilis/C. acetobutylicum shuttle vector. Plasmid pFNK1 was used because shuttle vectors that function in E. coil were unable to electrotransform ATCC 824 unless they became deleted in the E. coli-plasmid regions. The difficulties with shuttle vectors that function in E. coli are probably due to the presence of a restriction endonuclease in ATCC 824. This endonuclease recognizes the sequence 5′-GCNGC-3′, which is prevalent in E. coli plasmids but occurs infrequently in pFNK1 and C. acetobutylicum genes. Cloning of genes in C. acetobutylicum is critical for redirecting the cellular metabolism (metabolic engineering) as well as for genetic studies of this industrial organism.

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Author notes

  1. Eleftherios T. Papoutsakis: Corresponding author.

Authors and Affiliations

  1. Northwestern University, Dept. of Chemical Engineering, Evanston, IL, 60208

    Lee D. Mermelstein & Eleftherios T. Papoutsakis

  2. Dept. of Biochemistry, Molecular Biology and Cell Biology, Evanston, IL, 60208

    Neil E. Welker

  3. Rice University, Dept. of Biochemistry and Cell Biology, P.O. Box 1892, Houston, TX, 77251

    George N. Bennett

Authors
  1. Lee D. Mermelstein
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  2. Neil E. Welker
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  3. George N. Bennett
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  4. Eleftherios T. Papoutsakis
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Mermelstein, L., Welker, N., Bennett, G. et al. Expression of Cloned Homologous Fermentative Genes in Clostridium Acetobutylicum ATCC 824. Nat Biotechnol 10, 190–195 (1992). https://doi.org/10.1038/nbt0292-190

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