Construction of a restriction-less, marker-less mutant useful for functional genomic and metabolic engineering of the biofuel producer Clostridium acetobutylicum
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BACKGROUND: Clostridium acetobutylicum is a gram-positive, spore-forming, anaerobic bacterium capable of converting various sugars and polysaccharides into solvents (acetone, butanol, and ethanol). The sequencing of its genome has prompted new approaches to genetic analysis, functional genomics, and metabolic engineering to develop industrial strains for the production of biofuels and bulk chemicals. RESULTS: The method used in this paper to knock-out or knock-in genes in C. acetobutylicum combines the use of an antibiotic-resistance gene for the deletion or replacement of the target gene, the subsequent elimination of the antibiotic-resistance gene with the flippase recombinase system from Saccharomyces cerevisiae, and a C. acetobutylicum strain that lacks upp, which encodes uracil phosphoribosyl-transferase, for subsequent use as a counter-selectable marker. A replicative vector containing (1) a pIMP13 origin of replication from Bacillus subtilis that is functional in Clostridia, (2) a replacement cassette consisting of an antibiotic resistance gene (MLS (R) ) flanked by two FRT sequences, and (3) two sequences homologous to selected regions around target DNA sequence was first constructed. This vector was successfully used to consecutively delete the Cac824I restriction endonuclease encoding gene (CA_C1502) and the upp gene (CA_C2879) in the C. acetobutylicum ATCC824 chromosome. The resulting C. acetobutylicum Δcac1502Δupp strain is marker-less, readily transformable without any previous plasmid methylation and can serve as the host for the "marker-less" genetic exchange system. The third gene, CA_C3535, shown in this study to encode for a type II restriction enzyme (Cac824II) that recognizes the CTGAAG sequence, was deleted using an upp/5-FU counter-selection strategy to improve the efficiency of the method. The restriction-less marker-less strain and the method was successfully used to delete two genes (ctfAB) on the pSOL1 megaplasmid and one gene (ldhA) on the chromosome to get strains no longer producing acetone or l-lactate. CONCLUSIONS: The restriction-less, marker-less strain described in this study, as well as the maker-less genetic exchange coupled with positive selection, will be useful for functional genomic studies and for the development of industrial strains for the production of biofuels and bulk chemicals.
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Several counter-selectable markers to allow efficient screening for the rare second recombination event have been developed for clostridia, including upp, mazF, codA, and pyrE or pyrF.
“To facilitate this method, several counter-selectable markers to allow efficient screening for the rare second recombination event have been developed for clostridia including upp (encoding a uracil-phosphoribosyl-transferase) (Soucaille et al., 2008; Croux et al., 2016), mazF (E. coli toxin) (Al-Hinai et al., 2012a), codA (cytosine deaminase) (Cartman et al., 2012), and pyrE or pyrF (orotate”
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 Croux et al., 2016
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- Authors
- Croux, C., Nguyen, N.-P.-T., Lee, J., Raynaud, C., Saint-Prix, F., Gonzalez-Pajuelo, M., Meynial-Salles, I., & Soucaille, P.
- Affiliations
- Institut National des Sciences Appliquées de Toulouse (FR)
- Korea University (KR)
- METabolic Explorer (France) (FR)
- Corresponding author
- Soucaille, Philippe
- Venue
- Biotechnology for Biofuels
- Published
- 2016-02-02
- Volume
- 9
- Issue
- 1
- Pages
- 23-23
- ISSN
- 1754-6834 (ISSN-L)
- PMC
- PMC4736252
- OpenAlex
- W2261829810
- Cited by
- 41
- References
- 33
- Citations by year
- 2025: 1 · 2024: 2 · 2023: 2 · 2022: 3 · 2021: 6 · 2020: 1 · 2019: 3 · 2018: 10 · 2017: 8 · 2016: 5
- FWCI
- 4.51
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- cc-by
- Locations
- Biotechnology for Biofuels (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- HAL (submittedVersion, other-oa)
- Funders
- Seventh Framework Programme (PEOPLE-ITN-2008-237942)
- 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 › CRISPR and Genetic Engineering
- Keywords
- Clostridium acetobutylicum, gene deletion, gene replacement, uracil phosphoribosyltransferase, antibiotic resistance genes, type II restriction enzymes, counter-selection, acetone production, L-lactate production, functional genomics, metabolic engineering
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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
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