Design of Antisense RNA Constructs for Downregulation of the Acetone Formation Pathway of Clostridium acetobutylicum
Open accessAbstract
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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By applying antisense RNA, up to 90% reduction in gene expression together with high level of specificity were reported for clostridia.
“By applying asRNA, up to 90% reduction in gene expression together with high level of specificity were reported for clostridia (Desai and Papoutsakis, 1999; Tummala et al., 2003; Scotcher et al., 2005).”
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 Tummala et al., 2003
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- Authors
- Tummala, S. B., Welker, N. E., & Papoutsakis, E. T.
- Affiliations
- Northwestern University (US)
- Department of Chemical Engineering
- 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
- 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
- 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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