ArticleMicrobial cell factories2015
Establishment of markerless gene deletion tools in thermophilic Bacillus smithii and construction of multiple mutant strains.
Article in Microbial cell factories, 2015. 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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13 citing papers in PubMed, 25 citations in OpenAlex.
- Development of a markerless tool for targeted chromosome modification in the thermophilic and methylotrophic bacterium Bacillus methanolicus.Microbial cell factories · 2025Article
- Efficient markerless genetic manipulation ofApplied and environmental microbiology · 2024Article
- Development of a Cas12a-Based Genome Editing Tool for Moderate Thermophiles.The CRISPR journal · 2021Article
- Genome Editing of the Anaerobic Thermophile Thermoanaerobacter ethanolicus Using Thermostable Cas9.Applied and environmental microbiology · 2020Article
- Genetic Tools and Techniques for Recombinant Expression in Thermophilic Bacillaceae.Microorganisms · 2018Review
- Isolation of levoglucosan-utilizing thermophilic bacteria.Scientific reports · 2018Article
- In vivo selection of sfGFP variants with improved and reliable functionality in industrially important thermophilic bacteria.Biotechnology for biofuels · 2018Article
- Characterizing a thermostable Cas9 for bacterial genome editing and silencing.Nature communications · 2017Article
- Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9.ACS synthetic biology · 2017Article
- Development of an efficient technique for gene deletion and allelic exchange in Geobacillus spp.Microbial cell factories · 2017Article
- Development and implementation of rapid metabolic engineering tools for chemical and fuel production inBiotechnology for biofuels · 2017Article
- Contributory roles of two l-lactate dehydrogenases for l-lactic acid production in thermotolerant Bacillus coagulans.Scientific reports · 2016Article
- Complete genome sequence of thermophilic Bacillus smithii type strain DSM 4216(T).Standards in genomic sciences · 2016Article
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Authors and funding
6 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundMicrobial conversion of biomass to fuels or chemicals is an attractive alternative for fossil-based fuels and chemicals. Thermophilic microorganisms have several operational advantages as a production host over mesophilic organisms, such as low cooling costs, reduced contamination risks and a process temperature matching that of commercial hydrolytic enzymes, enabling simultaneous saccharification and fermentation at higher efficiencies and with less enzymes. However, genetic tools for biotechnologically relevant thermophiles are still in their infancy. In this study we developed a markerless gene deletion method for the thermophile Bacillus smithii and we report the first metabolic engineering of this species as a potential platform organism.
resultsClean deletions of the ldhL gene were made in two B. smithii strains (DSM 4216(T) and compost isolate ET 138) by homologous recombination. Whereas both wild-type strains produced mainly L-lactate, deletion of the ldhL gene blocked L-lactate production and caused impaired anaerobic growth and acid production. To facilitate the mutagenesis process, we established a counter-selection system for efficient plasmid removal based on lacZ-mediated X-gal toxicity. This counter-selection system was applied to construct a sporulation-deficient B. smithii ΔldhL ΔsigF mutant strain. Next, we demonstrated that the system can be used repetitively by creating B. smithii triple mutant strain ET 138 ΔldhL ΔsigF ΔpdhA, from which also the gene encoding the α-subunit of the E1 component of the pyruvate dehydrogenase complex is deleted. This triple mutant strain produced no acetate and is auxotrophic for acetate, indicating that pyruvate dehydrogenase is the major route from pyruvate to acetyl-CoA.
conclusionsIn this study, we developed a markerless gene deletion method including a counter-selection system for thermophilic B. smithii, constituting the first report of metabolic engineering in this species. The described markerless gene deletion system paves the way for more extensive metabolic engineering of B. smithii. This enables the development of this species into a platform organism and provides tools for studying its metabolism, which appears to be different from its close relatives such as B. coagulans and other bacilli.
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