Metabolome Analysis Of Riboflavin–producing Bacillus Subtilis

According to comparison and optimization, a scheme of metabolomics analysis by LC MS was developed. The cold methanol (60% v/v) quenching protocol has been used in our laboratory for instant quenching of metabolic activity, however, cell leakage was found during the quenching procedure. Therefore, quick ?ltration method without quenching process was selected to separate cells from culture. In addition, we also compared three different extraction procedures, and finally found that combination with the acetonitrile: methanol: water and methanol: chloroform extraction method was more suitable for our research.The above metabolomics analysis method was applied in our research to distinguish various riboflavin–producing Bacillus subtilis by principal components analysis (PCA) and 16 metabolites were determined by qualification analysis based on m/z of metabolites. The outcome showed that: Compared with the B.subtilis RH33, Ru5P, DRL, RIB were significantly increased in B.subtilis RH33 SVZ, B.subtilis RH33 SVG and B.subtilisRH33 VGZ, whereas, PYR, CIT, SUC, MAL, OAA had great reduction. The result illustrated that amplification of the key genes zwf and gnd can effectively increase the PP pathway flux, lower TCA cycle flux, change the flux distribution of glycolytic pathway and pentose phosphate pathway metabolic, thus contribute to improve the yield of riboflavin.Meanwhile, due to the increase of PP flux, 5 phosphoribose and NADPH for synthesis of the purine nucleotides and amino acids was adequately supplied, and finally led to the increase of AIR, AMP, ADP, ATP, and Valine, Lysine, Phenylalanine, Glutamate.