ArticleApplied and environmental microbiology2014
Development of a markerless knockout method for Actinobacillus succinogenes.
Article in Applied and environmental microbiology, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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Who cites it
13 citing papers in PubMed, 28 citations in OpenAlex.
- Formate-driven in situ COBiotechnology for biofuels and bioproducts · 2026Article
- Construction and Characterization of Novel Shuttle Expression Vectors for Actinobacillus succinogenes.Biotechnology and applied biochemistry · 2025Article
- Production of Succinic Acid by Metabolically EngineeredJournal of microbiology and biotechnology · 2024Article
- Development of highly efficient and specific base editors in Actinobacillus succinogenes for enhancing succinic acid production.Biotechnology for biofuels and bioproducts · 2023Article
- Comparative Transcriptome Analysis Reveals the Molecular Mechanisms of Acetic Acid Reduction by Adding NaHSOPolish journal of microbiology · 2023Article
- Effects of down-regulation of ackA expression by CRISPR-dCpf1 on succinic acid production in Actinobacillus succinogenes.AMB Express · 2023Article
- One step forward, two steps back: Transcriptional advancements and fermentation phenomena in Actinobacillus succinogenes 130Z.PloS one · 2021Article
- Metabolic Regulation of Organic Acid Biosynthesis inFrontiers in bioengineering and biotechnology · 2019Article
- Opportunities, challenges, and future perspectives of succinic acid production by Actinobacillus succinogenes.Applied microbiology and biotechnology · 2018Review
- Prediction of reaction knockouts to maximize succinate production by Actinobacillus succinogenes.PloS one · 2018Article
- Metabolic Engineering of Actinobacillus succinogenes Provides Insights into Succinic Acid Biosynthesis.Applied and environmental microbiology · 2017Article
- Respiratory glycerol metabolism of Actinobacillus succinogenes 130Z for succinate production.Journal of industrial microbiology & biotechnology · 2014Article
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5 authors at 1 institution in 1 country.
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No grant is acknowledged in the PubMed record.
Abstract
Actinobacillus succinogenes is one of the best natural succinate-producing organisms, but it still needs engineering to further increase succinate yield and productivity. In this study, we developed a markerless knockout method for A. succinogenes using natural transformation or electroporation. The Escherichia coli isocitrate dehydrogenase gene with flanking flippase recognition target sites was used as the positive selection marker, making use of A. succinogenes's auxotrophy for glutamate to select for growth on isocitrate. The Saccharomyces cerevisiae flippase recombinase (Flp) was used to remove the selection marker, allowing its reuse. Finally, the plasmid expressing flp was cured using acridine orange. We demonstrate that at least two consecutive deletions can be introduced into the same strain using this approach, that no more than a total of 1 kb of DNA is needed on each side of the selection cassette to protect from exonuclease activity during transformation, and that no more than 200 bp of homologous DNA is needed on each side for efficient recombination. We also demonstrate that electroporation can be used as an alternative transformation method to obtain knockout mutants and that an enriched defined medium can be used for direct selection of knockout mutants on agar plates with high efficiency. Single-knockout mutants of the fumarate reductase and of the pyruvate formate lyase-encoding genes were obtained using this knockout strategy. Double-knockout mutants were also obtained by deleting the citrate lyase-, β-galactosidase-, and aconitase-encoding genes in the pyruvate formate lyase knockout mutant strain.
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