Lactose-inducible system for metabolic engineering of Clostridium ljungdahlii
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The development of tools for genetic manipulation of Clostridium ljungdahlii has increased its attractiveness as a chassis for autotrophic production of organic commodities and biofuels from syngas and microbial electrosynthesis and established it as a model organism for the study of the basic physiology of acetogenesis. In an attempt to expand the genetic toolbox for C. ljungdahlii, the possibility of adapting a lactose-inducible system for gene expression, previously reported for Clostridium perfringens, was investigated. The plasmid pAH2, originally developed for C. perfringens with a gusA reporter gene, functioned as an effective lactose-inducible system in C. ljungdahlii. Lactose induction of C. ljungdahlii containing pB1, in which the gene for the aldehyde/alcohol dehydrogenase AdhE1 was downstream of the lactose-inducible promoter, increased expression of adhE1 30-fold over the wild-type level, increasing ethanol production 1.5-fold, with a corresponding decrease in acetate production. Lactose-inducible expression of adhE1 in a strain in which adhE1 and the adhE1 homolog adhE2 had been deleted from the chromosome restored ethanol production to levels comparable to those in the wild-type strain. Inducing expression of adhE2 similarly failed to restore ethanol production, suggesting that adhE1 is the homolog responsible for ethanol production. Lactose-inducible expression of the four heterologous genes necessary to convert acetyl coenzyme A (acetyl-CoA) to acetone diverted ca. 60% of carbon flow to acetone production during growth on fructose, and 25% of carbon flow went to acetone when carbon monoxide was the electron donor. These studies demonstrate that the lactose-inducible system described here will be useful for redirecting carbon and electron flow for the biosynthesis of products more valuable than acetate. Furthermore, this tool should aid in optimizing microbial electrosynthesis and for basic studies on the physiology of acetogenesis.
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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 Banerjee et al., 2014
A lactose-inducible system (bgaR-PbgaL) from Clostridium perfringens was successfully adapted for use in C. ljungdahlii.
“A lactose-inducible system (bgaR-PbgaL) from Clostridium perfringens was successfully adapted for use in C. ljungdahlii (Banerjee et al., 2014) and a tetracycline inducible system (Tet3no) has been demonstrated in C. autoethanogenum (Walker and Köpke, 2015).”
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 Banerjee et al., 2014
C. ljungdahlii with plasmid expression of C. acetobutylicum acetone biosynthetic genes under a lactose inducible promoter produced up to 871 mg/L acetone from CO in bottles.
“| C. ljungdahlii | Plasmid expression of C. acetobutylicum acetone biosynthetic genes (thlA-ctfAB-adc) under lactose inducible promoter | Production of up to 871 mg/L acetone from CO in bottles | Banerjee et al., 2014 |”
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 Banerjee et al., 2014
Acetone production increased over 500x from H2/CO2 in C. ljungdahlii using the same acetone genes under control of an inducible system.
“Acetone production increased over 500x from H2/CO2 in C. ljungdahlii using the same genes but under control of an inducible system (Banerjee et al., 2014) and in A. woodii by combinatorial testing of different promoter and plasmid combinations (Hoffmeister et al., 2016).”
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 Banerjee et al., 2014
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- Authors
- Banerjee, A., Leang, C., Ueki, T., Nevin, K. P., & Lovley, D. R.
- Affiliations
- University of Massachusetts Amherst (US)
- Venue
- Applied and Environmental Microbiology
- Publisher
- American Society for Microbiology
- Published
- 2014-04-15
- Volume
- 80
- Issue
- 8
- Pages
- 2410-2416
- ISSN
- 0099-2240 (ISSN-L)
- 1098-5336
- PMC
- PMC3993169
- OpenAlex
- W2120580364
- Cited by
- 126
- References
- 34
- Citations by year
- 2026: 7 · 2025: 9 · 2024: 7 · 2023: 5 · 2022: 16 · 2021: 15 · 2020: 7 · 2019: 13 · 2018: 7 · 2017: 12 · 2016: 10 · 2015: 14 · 2014: 4
- FWCI
- 5.47
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Applied and Environmental Microbiology (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- U.S. Department of Energy (DEAR0000159)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Physical Sciences › Engineering › Biomedical Engineering › Biofuel production and bioconversion
- Physical Sciences › Environmental Science › Environmental Engineering › Microbial Fuel Cells and Bioremediation
- Keywords
- Clostridium ljungdahlii, aldehyde/alcohol dehydrogenase, ethanol production, acetate production, acetone production, acetogenesis, syngas fermentation, microbial electrosynthesis
- MeSH terms
- Acetone, Acetyl Coenzyme A, Alcohol Dehydrogenase, Ethanol, Carbon, Clostridium, Fructose, Lactose, Transcriptional Activation, Gene Expression, Gene Expression Regulation, Bacterial, Acetic Acid, Metabolic Engineering, Metabolic Flux Analysis
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