Cited by 174Microbial Metabolic Engineering and BioproductionBacterial Genetics and BiotechnologyRNA and protein synthesis mechanisms

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Design of Antisense RNA Constructs for Downregulation of the Acetone Formation Pathway of Clostridium acetobutylicum

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Tummala, Seshu B., Welker, Neil E., Papoutsakis, Eleftherios T.·2003·Journal of Bacteriology

DOI 10.1128/jb.185.6.1923-1934.2003PMC PMC150123

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Abstract

We investigated the effect of antisense RNA (asRNA) structural properties on the downregulation efficacy of enzymes in the acetone-formation pathway (acetoacetate decarboxylase [AADC] and coenzyme A-transferase [CoAT]) of Clostridium acetobutylicum strain ATCC 824. First, we generated three strains, C. acetobutylicum ATCC 824 (pADC38AS), 824(pADC68AS), and 824(pADC100AS), which contain plasmids that produce asRNAs of various lengths against the AADC (adc) transcript. Western analysis showed that all three strains exhibit low levels of AADC compared to the plasmid control [ATCC 824(pSOS95del)]. By using computational algorithms, the three different asRNAs directed toward AADC, along with previously reported clostridial asRNAs, were examined for structural features (free nucleotides and components). When the normalized metrics of these structural features were plotted against percent downregulation, only the component/nucleotide ratio correlated well with in vivo asRNA effectiveness. Despite the significant downregulation of AADC in these strains, there were no concomitant effects on acetone formation. These findings suggest that AADC does not limit acetone formation and, thus, we targeted next the CoAT. Using the component/nucleotide ratio as a selection parameter, we developed three strains [ATCC 824 (pCTFA2AS), 824(pCTFB1AS), and 824(pCOAT11AS)] which express asRNAs to downregulate either or both of the CoAT subunits. Compared to the plasmid control strain, these strains produced substantially low levels of acetone and butanol and Western blot analyses showed significantly low levels of both CoAT subunits. These results show that CoAT is the rate-limiting enzyme in acetone formation and strengthen the hypothesis that the component/nucleotide ratio is a predictive indicator of asRNA effectiveness.

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Authors: Tummala, S. B., Welker, N. E., & Papoutsakis, E. T.
Affiliations: Northwestern University (US)
Department of Chemical Engineering
ORCID: Papoutsakis, Eleftherios T.
Venue: Journal of Bacteriology
Publisher: American Society for Microbiology
Published: 2003-03-15
Volume: 185
Issue: 6
Pages: 1923-1934
ISSN: 0021-9193 (ISSN-L)
1098-5530
DOI: 10.1128/jb.185.6.1923-1934.2003
PMC: PMC150123
OpenAlex: W2098508815
Cited by: 174
References: 33
Citations by year: 2026: 4 · 2025: 4 · 2024: 2 · 2023: 5 · 2022: 1 · 2021: 5 · 2020: 3 · 2019: 10 · 2018: 11 · 2017: 6 · 2016: 11 · 2015: 15 · 2014: 16 · 2013: 8 · 2012: 14
FWCI: 1.03
Type: journal article
Language: en
Update notices: correction (2020-12-20)
Open access: green
europepmc.org
Licence: journals.asm.org/non-commercial-tdm-license (tdm)
Locations: Journal of Bacteriology (publishedVersion)
Europe PMC (PubMed Central) (publishedVersion)
PubMed Central (publishedVersion)
PubMed (publishedVersion)
Funders: National Science Foundation
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 › RNA and protein synthesis mechanisms
Keywords: antisense RNA, RNA interference, Clostridium acetobutylicum, acetone production, acetoacetate decarboxylase, gene downregulation, rate-limiting enzyme
MeSH terms: Acetone, Carboxy-Lyases, Clostridium, Culture Media, Nucleic Acid Conformation, Coenzyme A-Transferases, Down-Regulation, RNA, Antisense, Bioreactors

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Design of Antisense RNA Constructs for Downregulation of the Acetone Formation Pathway of Clostridium acetobutylicum

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