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By comparing optimization, established the use of LC MS analysis means producing riboflavin Bacillus subtilis metabolomics research methods . Comparison, cold methanol ( 60% ) inactivation of bacteria used in the experiments , a certain degree of leakage of intracellular metabolites phenomenon and collected by rapid filtration cells overcomes this shortcoming . Additionally, this article compares three different extraction methods, by comparing the test results to determine the acetonitrile : methanol: water extraction method , and methanol: chloroform extraction method used in combination is more suitable for the present study . Finalized by rapid filtration cells were harvested , acetonitrile : methanol: water and methanol : chloroform extraction method used in combination intracellular metabolites, LC-MS detected metabolites genomics research methods. Be established metabolomics research methods used in four different producing riboflavin engineered bacteria , intracellular metabolites of its positive and negative ion ESI MS data analysis PCA , initially identified 16 kinds of major differences between metabolites and metabolite profiling based on comparative study of different genotypes of strains metabolic reasons for the differences . Produced by four different intracellular riboflavin engineered bacteria metabolites detected : Compared with B.subtilis RH33 , B.subtilis RH33 SVZ, B.subtilis RH33 SVG and B.subtilis RH33 VGZ in 5 - phosphate nuclear ketose , 6,7 dimethyl 2,4- dioxo- tetrahydro- 8 ribose pteridine alcohol , have different levels of riboflavin significantly increased , and pyruvic acid, citric acid, succinic acid , malic acid , oxaloacetic acid so there are different degrees of reduction . Illustrates the amplification of the pentose phosphate pathway by the key and the gnd gene zwf effectively increase its throughput, decrease the citric acid cycle flow , changing the glycolytic pathway and the pentose phosphate pathway metabolism in the distribution of traffic and thus help riboflavin production increase . Meanwhile , since the pentose phosphate pathway flux increases , purines , nucleotides, amino acids and other metabolites of the required 5 ribose synthesis and reducing power NADPH, that nucleotides 5 amino imidazole , AMP, ADP, ATP and valine , lysine , phenylalanine and glutamic acid increased in varying degrees .
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