Expression of cloned homologous fermentative genes in Clostridium acetobutylicum ATCC 824

Mermelstein, Lee D.; Welker, Neil E.; Bennett, George N.; Papoutsakis, Eleftherios T.

doi:10.1038/nbt0292-190

Cited by 250Microbial Metabolic Engineering and BioproductionBacterial Genetics and BiotechnologyFungal and yeast genetics research

Expression of cloned homologous fermentative genes in Clostridium acetobutylicum ATCC 824

Mermelstein, Lee D., Welker, Neil E., Bennett, George N., Papoutsakis, Eleftherios T.·1992·Nature Biotechnology

DOI 10.1038/nbt0292-190

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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 in 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. coli 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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Some well-studied non-acetogenic clostridia, such as C. acetobutylicum, C. beijerinckii, C. difficile, and C. botulinum, have transformation protocols that were established as early as 1988.
“Some of the well-studied, non-acetogenic clostridia, such as the ABE model organisms C. acetobutylicum (Mermelstein et al., 1992) and C. beijerinckii (Oultram et al., 1988), and important pathogens, such as C. difficile (Ackermann et al., 2001) and C. botulinum (Zhou and Johnson, 1993), have transformation protocols that were established as early as 1988.”
From Gas Fermentation—A Flexible Platform for Commercial Scale Production of Low-Carbon-Fuels and Chemicals from Waste and Renewable Feedstocks (2016) · cites this paper as Mermelstein et al., 1992

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Authors: Mermelstein, L. D., Welker, N. E., Bennett, G. N., & Papoutsakis, E. T.
Affiliations: Northwestern University (US)
Rice University (US)
ORCID: Bennett, George N.
Papoutsakis, Eleftherios T.
Venue: Nature Biotechnology
Publisher: Springer Science and Business Media LLC
Published: 1992-02
Volume: 10
Issue: 2
Pages: 190-195
ISSN: 0733-222X (ISSN-L)
1087-0156
1546-1696
DOI: 10.1038/nbt0292-190
OpenAlex: W2009188183
Cited by: 249
References: 31
Citations by year: 2025: 4 · 2024: 5 · 2023: 4 · 2022: 6 · 2021: 7 · 2020: 6 · 2019: 12 · 2018: 10 · 2017: 4 · 2016: 17 · 2015: 8 · 2014: 18 · 2013: 14 · 2012: 12
FWCI: 2.91
Type: journal article
Language: en
Open access: closed
Licence: www.springer.com/tdm (unspecified)
Locations: Nature Biotechnology (publishedVersion)
PubMed (publishedVersion)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Fungal and yeast genetics research
Keywords: Clostridium acetobutylicum, acetoacetate decarboxylase, acetone production, butyrate production, heterologous expression, Bacillus subtilis, shuttle vector, electrotransformation, restriction enzymes, metabolic engineering
MeSH terms: Bacillus subtilis, Carboxy-Lyases, Cloning, Molecular, Clostridium, DNA, Bacterial, Escherichia coli, Fermentation, Genes, Bacterial, Genetic Vectors, Phosphate Acetyltransferase, Plasmids, Restriction Mapping

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Expression of cloned homologous fermentative genes in Clostridium acetobutylicum ATCC 824

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