Quantitative metabolomics of the thermophilic methylotroph Bacillus methanolicus

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.

Transaldolase in Bacillus methanolicus: biochemical characterization and biological role in ribulose monophosphate cycle

“…It has previously been shown that B. methanolicus uses the SBPase variant of the RuMP cycle [61] and three separate metabolomics studies have identified S7P in methanol grown MGA3 cells [13,34,43]. Our results presented here may indicate that B. methanolicus also possesses an active Ta variant of the RuMP cycle for regeneration of the formaldehyde acceptor Ru5P.…”

Methanol-Essential Growth of Corynebacterium glutamicum: Adaptive Laboratory Evolution Overcomes Limitation due to Methanethiol Assimilation Pathway

“…Since methanol is more reduced than glucose, it supports production of reduced compounds. Although omics data of native methylotrophs have been generated in the past decade [6,15,16,27], natural methanol utilization has not yet been fully understood, which makes the transfer of methylotrophy to non-methylotrophs a demanding challenge [5,19]. Here, we developed methanol-dependent C. glutamicum strains through rpe and rpi deletions that functioned as metabolic cut-offs to force methanol-utilization for biomass formation ( Figure 1A,B).…”

“…Although there have been recent advances in the development of tools for system engineering of B. methanolicus [15][16][17], the genetic toolbox for these methylotrophic bacteria is still limited and metabolic engineering to optimize carbon fluxes for increased product titers or yields remains a challenge to this day [18]. Thus, the demand to transfer methylotrophy to mesophilic bacteria with well-established engineering toolset has been high in recent years [19].…”

Microbial metabolomics: essential definitions and the importance of cultivation conditions for utilizing Bacillus species as bionematicides

“…; Carnicer et al . ; Patejko et al . ), can therefore be used to detect and qualitatively and quantitatively characterize discriminatory metabolite features with nematicidal potential.…”

“…In this context, the identification of a feature means that the feature was identified as a discriminatory feature explaining the statistical differences between metabolite profiles from different Bacillus species. Metabolomics, defined as the comprehensive analysis of all the metabolites present within an organism (Villas-Bôas et al 2007;Carnicer et al 2016;Patejko et al 2017), can therefore be used to detect and qualitatively and quantitatively characterize discriminatory metabolite features with nematicidal potential.…”