title: Quantitative metabolomics of the thermophilic methylotroph Bacillus methanolicus.
abstract: BACKGROUND
  The gram-positive bacterium Bacillus methanolicus MGA3 is a promising candidate for methanol-based biotechnologies. Accurate determination of intracellular metabolites is crucial for engineering this bacteria into an efficient microbial cell factory. Due to the diversity of chemical and cell properties, an experimental protocol validated on B. methanolicus is needed. Here a systematic evaluation of different techniques for establishing a reliable basis for metabolome investigations is presented.
  
  
  RESULTS
  Metabolome analysis was focused on metabolites closely linked with B. methanolicus central methanol metabolism. As an alternative to cold solvent based procedures, a solvent-free quenching strategy using stainless steel beads cooled to -20 °C was assessed. The precision, the consistency of the measurements, and the extent of metabolite leakage from quenched cells were evaluated in procedures with and without cell separation. The most accurate and reliable performance was provided by the method without cell separation, as significant metabolite leakage occurred in the procedures based on fast filtration. As a biological test case, the best protocol was used to assess the metabolome of B. methanolicus grown in chemostat on methanol at two different growth rates and its validity was demonstrated.
  
  
  CONCLUSIONS
  The presented protocol is a first and helpful step towards developing reliable metabolomics data for thermophilic methylotroph B. methanolicus. This will definitely help for designing an efficient methylotrophic cell factory.
authors.affiliation: Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, 31077, Toulouse, France.|INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, 31400, Toulouse, France.|CNRS, UMR5504, 31400, Toulouse, France.
authors.forename: Marc
authors.initials: M
authors.lastname: Carnicer
authors.affiliation: Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, 31077, Toulouse, France.|INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, 31400, Toulouse, France.|CNRS, UMR5504, 31400, Toulouse, France.
authors.forename: Gilles
authors.initials: G
authors.lastname: Vieira
authors.affiliation: Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway.
authors.forename: Trygve
authors.initials: T
authors.lastname: Brautaset
authors.affiliation: Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, 31077, Toulouse, France.|INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, 31400, Toulouse, France.|CNRS, UMR5504, 31400, Toulouse, France.
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Portais
authors.affiliation: Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, 31077, Toulouse, France. stephanie.heux@insa-toulouse.fr.|INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, 31400, Toulouse, France. stephanie.heux@insa-toulouse.fr.|CNRS, UMR5504, 31400, Toulouse, France. stephanie.heux@insa-toulouse.fr.|LISBP-INSA de Toulouse, 135 Avenue de Rangueil, 31077, Toulouse Cedex 04, France. stephanie.heux@insa-toulouse.fr.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Heux
mesh_terms: D001407:Bacillus / Q000254:growth & development / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D055442:Metabolome / Q000187:drug effects*; D055432:Metabolomics; D000432:Methanol / Q000494:pharmacology*; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D000432:Methanol
keywords: Bacillus methanolicus; Methanol; Quantitative metabolomics; Quenching