Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 490120
Title Establishment of markerless gene deletion tools in thermophilic Bacillus smithii and construction of multiple mutant strains
Author(s) Bosma, E.F.; Weijer, A.H.P. van de; Vlist, L.; Vos, W.M. de; Oost, J. van der; Kranenburg, R. van
Source Microbial Cell Factories 14 (2015). - ISSN 1475-2859 - 12 p.
Department(s) Microbiology
Publication type Refereed Article in a scientific journal
Publication year 2015
Abstract BACKGROUND: Microbial 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. RESULTS: Clean 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 ¿ldhL ¿sigF mutant strain. Next, we demonstrated that the system can be used repetitively by creating B. smithii triple mutant strain ET 138 ¿ldhL ¿sigF ¿pdhA, from which also the gene encoding the a-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. CONCLUSIONS: In 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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