title: Establishment of markerless gene deletion tools in thermophilic Bacillus smithii and construction of multiple mutant strains.
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 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.
  
  
  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.
authors.affiliation: Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB, Wageningen, The Netherlands. elleke.bosma@wur.nl.
authors.forename: Elleke F
authors.initials: EF
authors.lastname: Bosma
authors.affiliation: Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB, Wageningen, The Netherlands. tom.vandeweijer@wur.nl.
authors.forename: Antonius H P
authors.initials: AH
authors.lastname: van de Weijer
authors.affiliation: Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB, Wageningen, The Netherlands. laurensvdvlist@gmail.com.
authors.forename: Laurens
authors.initials: L
authors.lastname: van der Vlist
authors.affiliation: Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB, Wageningen, The Netherlands. willem.devos@wur.nl.
authors.forename: Willem M
authors.initials: WM
authors.lastname: de Vos
authors.affiliation: Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB, Wageningen, The Netherlands. john.vanderoost@wur.nl.
authors.forename: John
authors.initials: J
authors.lastname: van der Oost
authors.affiliation: Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB, Wageningen, The Netherlands. r.van.kranenburg@corbion.com.|Corbion, Arkelsedijk 46, 4206 AC, Gorinchem, The Netherlands. r.van.kranenburg@corbion.com.
authors.forename: Richard
authors.initials: R
authors.lastname: van Kranenburg
mesh_terms: D001407:Bacillus / Q000378:metabolism*; D005285:Fermentation; D017353:Gene Deletion; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D016296:Mutagenesis
chemical_list: D007658:Ketone Oxidoreductases; C053164:pyruvate dehydrogenase (NADP+)