Page 49 - Application Notebook - Solution for Food Development

LAAN-A-LM-E102

Application Liquid Chromatography Mass Spectrometry

News Application of Metabolomics to Microbial Breeding

No.C131

Microorganisms have been used for many years to n LC/MS Analysis of Escherichia coli Extract
produce useful materials in a wide range of industrial We cultured E. coli in minimal media to which
fields, including food, biotechnology, and energy. In the thiosulfuric acid or sulfuric acid was added as a sulfur
food sector, microorganisms are used to produce foods source. To evaluate metabolic changes during culture,
by the fermentation process, such as alcoholic some E. coli were collected from the culture suspension
beverages and fermented foods, and in the were collected at 3, 4, 5, 6, 7, 8, and 9 hours of culture.
biotechnology sector microorganisms are used for the The optical density (OD) of the collected E. coli was
large scale production of amino acids and antibiotics. In measured before the media components and E. coli were
the energy sector, it is anticipated that microorganism quickly separated by filtration. E. coli extract was then
will be used for biofuel production, though lowering prepared by breaking down the isolated E. coli in
the cost presents an outstanding problem. Although methanol. After removing methanol by centrifugal
microorganisms are already used in the production of a concentration, the extract was adjusted to an
variety of useful materials, genetic modification and appropriate dilution with ultrapure water and used for
breeding is still performed with the aim of improving LC/MS analysis. Metabolites were analyzed
production efficiency. Metabolomics presents useful simultaneously using the analytical conditions of an ion
tools for the evaluation of metabolic changes during pairing method (LCMS-8040) and non-ion pairing
microorganism breeding, and for understanding method (LCMS-8050) obtained from an LCMS method
metabolic changes related to a target material and its package [primary metabolites]. Table 1 shows the
precursors and intermediates. By deepening our analytical conditions of each method. Fig. 1 also shows
understanding of the metabolic pathways involved in an MRM chromatogram (both at 6 hours into culture) for
material production, metabolomics is expected to result each analytical method obtained by analysis of E. coli
in more efficient materials production. In this article, we extract after culture in thiosulfate-containing medium.
discuss an example of LC/MS analysis of how the sulfur- The main peaks detected when the ion pairing method
containing metabolites vary during culture of a was used were amino acids, coenzymes, and nucleic
cysteine-producing Escherichia coli (E. coli) when either acid-related compounds, and the main peaks detected
thiosulfuric acid or sulfuric acid is added as the sulphur when the non-ion pairing method was used were amino
source during cysteine synthesis. acids, organic acids, and nucleic acid-related compounds.

Table 1 Analytical Conditions for Ion Pairing Method and Non-Ion Pairing Method
Ion Pairing Method (LC Analytical Conditions) Non-Ion Pairing Method (LC Analytical Conditions)
Column : RP column Column : RP column
Mobile Phase A : 15 mmol/L Acetate, 10 mmol/L Tributylamine - Water Mobile Phase A : 0.1 % Formic acid - Water
Mobile Phase B : Methanol Mobile Phase B : 0.1 % Formic acid - Acetonitrile
Flowrate : 0.3 mL/min Flowrate : 0.25 mL/min
Mode : Gradient elution Mode : Gradient elution

（×100,000） （×100,000）
4.5 Glu
4.0 Ion pairing method 5.0 Non-ion pairing method
3.5 NAD 4.0 Adenosine
3.0 Glu GSH
2.5 AMP 3.0 AMP
2.0 Gln Acetyl-CoA
1.5 2.0 Val Trp
1.0 Asp 1.0 Pantothenate Phe
0.5 G6P
0.0 0.0
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0
Fig. 1 MRM Chromatograms of Escherichia coli Extract After Culture in Thiosulfate-Containing Medium