Development of an anhydrotetracycline-inducible gene expression system for solvent-producing Clostridium acetobutylicum: a useful tool for strain engineering
Abstract
Clostridium acetobutylicum is an important solvent (acetone-butanol-ethanol) producing bacterium. However, a stringent, effective, and convenient-to-use inducible gene expression system that can be used for regulating the gene expression strength in C. acetobutylicum is currently not available. Here, we report an anhydrotetracycline-inducible gene expression system for solvent-producing bacterium C. acetobutylicum. This system consists of a functional chloramphenicol acetyltransferase gene promoter containing tet operators (tetO), Pthl promoter (thiolase gene promoter from C. acetobutylicum) controlling TetR repressor expression cassette, and the chemical inducer anhydrotetracycline (aTc). The optimized system, designated as pGusA2-2tetO1, allows gene regulation in an inducer aTc concentration-dependent way, with an inducibility of over two orders of magnitude. The stringency of TetR repression supports the introduction of the genes encoding counterselective marker into C. acetobutylicum, which can be used to increase the mutant screening efficiency. This aTc-inducible gene expression system will thus increase the genetic manipulation capability for engineering C. acetobutylicum.
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Gene expression reporter systems that function under anaerobic conditions have emerged, including the β-glucuronidase gene, chloramphenicol acetyltransferase gene, flavin-based fluorescent proteins, luxA-luxB reporter system, and lacZ gene from Thermoanaerobacterium thermosulfurogenes EM1.
“Gene expression reporter systems that function under anaerobic conditions have emerged, including the β-glucuronidase gene (gusA) (Girbal et al., 2003; Dong et al., 2012; Banerjee et al., 2014), chloramphenicol acetyltransferase gene (catP) (Heap et al., 2007; Zhang et al., 2015b), flavin-based fluorescent proteins (Drepper et al., 2007, 2010; Mukherjee and Schroeder, 2015), luxA-luxB reporter”
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 Dong et al., 2012
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Self-citationHassan Mahmoud, M. A.-R. (2014) · OPen Access Repositorium der Universität Ulm (OPARU) (Ulm University) · doi:10.18725/oparu-3312
Metabolism and cell-to-cell interactions of anaerobic syntrophic Clostridia co-cultures
Streett, H. (2025) · Library, Museums and Press - UDSpace (University of Delaware) · doi:10.58088/pe3w-rj36
Prospect of Metabolic Engineering for Biochemical Production
Ruhal, R., & Kataria, R. (2022) · Energy, environment, and sustainability · doi:10.1007/978-981-16-8682-5_20
9 Natural and Synthetic Gas for Productions of Liquid Fuels and Their Additives
Shah, Y. T. (2017) · CRC Press eBooks · doi:10.1201/9781315302355-10
Munir, S., Wan, S., Gao, X., Lai, M., Mu, Z., Wang, H., Liu, Z., Li, F., Xia, L., & Tan, Y. (2026) · Applied Microbiology and Biotechnology · doi:10.1007/s00253-026-13714-3
Lopez-Garcia, O. K., Hejtmancik, T., Pizarro‐Guajardo, M., Brehm, L., Brito-Silva, C., & Paredes‐Sabja, D. (2026) · ACS Synthetic Biology · doi:10.1021/acssynbio.5c00961
- Authors
- Dong, H., Tao, W., Zhang, Y., & Li, Y.
- Affiliations
- Chinese Academy of Sciences (CN)
- Corresponding author
- Li, Yin
- Venue
- Metabolic Engineering
- Publisher
- Elsevier BV
- Published
- 2012-01
- Volume
- 14
- Issue
- 1
- Pages
- 59-67
- ISSN
- 1096-7176 (ISSN-L)
- OpenAlex
- W2065708815
- Cited by
- 65
- References
- 47
- Citations by year
- 2026: 4 · 2025: 3 · 2024: 1 · 2023: 4 · 2022: 7 · 2021: 5 · 2020: 2 · 2019: 5 · 2018: 3 · 2017: 4 · 2016: 2 · 2015: 9 · 2014: 9 · 2013: 4 · 2012: 3
- FWCI
- 2.08
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Metabolic Engineering (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 › Gene Regulatory Network Analysis
- Keywords
- Clostridium acetobutylicum, anhydrotetracycline, inducible gene expression, TetR repressor, tet operator, acetone-butanol-ethanol production, strain engineering, mutant screening
- MeSH terms
- Cyanobacteria, Carbon Dioxide, 1-Butanol, Metabolic Engineering
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