Quantitative metabolomics of the thermophilic methylotroph Bacillus methanolicus.

Marc Carnicer,Stephanie Heux,Trygve Brautaset,Jean-Charles Portais,Gilles Vieira

doi:10.1186/s12934-016-0483-x

Abstract

BackgroundThe 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.ResultsMetabolome 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.ConclusionThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0483-x) contains supplementary material, which is available to authorized users.

Highlights

The gram-positive bacterium Bacillus methanolicus MGA3 is a promising candidate for methanol-based biotechnologies
Precision of the protocol without cell separation In previous sections, we demonstrated that when B. methanolicus cells are separated from the medium prior extraction, most of the metabolites are lost in the washing solution
We demonstrated that, like in many prokaryotic cells, significant leakage of metabolites occurs in B. methanolicus, might hampering the use of protocols which include cell separation for metabolomics

Summary

Introduction

The gram-positive bacterium Bacillus methanolicus MGA3 is a promising candidate for methanol-based biotechnologies. Industrial biotechnology mainly uses sugars and molasses as carbon sources. These raw materials come from plants and require cultivable land which is increasingly needed to produce food for human populations. Among the different microorganisms able to use C1 sources, the gram-positive facultative methylotroph Bacillus methanolicus is a possible cell factory for the industrial production of l-lysine, l-glutamate and cadaverine from methanol at elevated temperatures [2,3,4]. B. methanolicus MGA3 (ATCC 53907) growths at 50 °C and assimilates methanol by using the ribulose monophosphate (RuMP) pathway [5]. Genes involved in this metabolic pathway are located in the pBM19 plasmid and are upregulated upon growth on methanol

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Results

Conclusion