Metabolic Characteristics About E. Coli Based On Chromatography-mass Spectrometry

Microbial metabolomics can be divided into metabolic footprint (extracellular metabolites) and metabolic fingerprint (intracellular metabolites) analysis with the spatial distribution of the study. The main step of the latter can be summarized as metabolic quenching, metabolite extraction and metabolite detection. With the high resolution and high-sensitivity detection on the separation of complex biological systems, chromatography-mass spectrometry has a very wide range of applications in the field of metabolomics.E.coli AS1.1566was selected as the target subjected, gas chromatography-mass spectrometry (GC-MS) was used in the analysis. Firstly, the method was established by using-80℃physical saline as quenching agent. The blockage of enzymatic activities was satisfied as the calculated energy charge was about0.89which was consistent with the theoretical value. At the same time metabolite leakage was alleviated. The damage rate of the cell membrane quenched by physiological saline was less than6%, whereas, the traditional cold methanol method was over60%. After the D-optimal experimental design, when the proportion of methanol to water was21:79, we realized the maximization of the metabolites extraction efficiency (the desirability was0.81).E.coli AS1.1566and an unknown strain were selected to be analyzed in view of metabolic fingerprint analysis using the above method. In order to cover a wider range of compounds in the cell extract, liquid chromatography-mass spectrometry (LC-MS) metabolic fingerprint analysis was also performed. The acquired data was firstly processed through SAM analysis. The differential compounds were screened and identified and then subjected to metabolic pathway enrichment analysis (PEA) and topological analysis (PTA). The results indicated that the major difference between them was in the oxygen dependent energy metabolism and this conclusion was finally demonstrated by oxygen dependent culture experiment. It was showed that the unknown srrain was an absolute anaerobic bacterium.For the purpose of unknown gene function analysis, a high throughput screening method was developed based on96-well cell culture plate. Firstly, the retention time of48compounds was confirmed by GC-MS, and the retention indices were locked by the saturated alkane. Then48compounds with known concentrations were added to the96-well cell culture plate. Finally, E.colil.1566, yfcC gene knockout mutant and yfcC overexpression mutant were cultured pair-wise in96-well plates. Culture broth of the same strain was mixed and analyzed by GC-MS based metabolic footprint analysis. The results showed that the function of yfcC gene was related to4amino acids, sugars and organic acids metabolism.As previously mentioned, nucleosides has received extensive attention as one of the most important compounds metabolized in the microbial growth process. A quick and simple method was developed to quantify19nucleosides by using triple quadurupole liquid chromatography-tandem mass spectrometry (LC-MS/MS). The linearity of the detected nucleosides was excellent with R2>0.99and the limits of qualification and detection were ranged from0.05to20μg/L and0.02to10μg/L. seven nuclesides could be readily quantified in E. coli at different phases. Additionally, distinct decreased of the guanosine during the growth phase was discovered by using SAM analysis.