Microbial Metabolic Flux And Pathway Analysis

Microbial metabolic process was studied in this paper. The E.coil central carbon metabolicnetwork is studied firstly. Based on Flux Balnace Analysis(FBA), E.coli1.1566and△sdhAB、△ackA-pta gene knockout mutants were selected to construct metabolic network. UsingCellNetAnalyzer software, with the greatest growth rate (μ) for optimization objectivefunction, normalization was finished with the rate of extracellular-carbohydrate entered to cellas standard. The metabolic flux of wild-type E. coli central carbon pathway was obtained.And the different between gene knockout mutants and wild-type E. coli metabolic flux wasanalyzed. Through metabolic flux analysis and study these single-gene knockout on themetabolism of E. coli, the main metabolic characteristics of the strains were understand.To objectively reflect the real situation of microbial cell metabolomics, a rapid microbialquenching method and standardization extraction method of intracellular metabolites wasestablished, and a suitable GC-MS condition was selected to carry out qualitative andquantitative analysis. Acetonitrile was regarded as the most efficient chemical for theirquenching and metabolite extracting ability. The following derivatization method was decidedby D-optimal design including temperature, derivatization agent, solvent and time parameters.Finally, the combination of temperature of75℃,60μL BSTFA+1%TMS as derivatizationagent,50μL pyridine as solvent and45min incubation time was thought to be the bestcondition which could gave the most peak number and peak area. Lay the foundation forfurther studying the metabolic pathway.Terephthalic acid is an important chemical raw material. A method for the determination ofterephthalic acid, p-tolualdehyde, p-methylbenzyl alcohol and p-toluic acid by HPLC wasestablished at the process of biocatalysis producing terephthalic acid. Hypersil SAX columnwas used. The mobile phase was2.5mol/L NH4H2PO4containing10%acetonitrile, pH was4.32. The flow rate was0.8mL/min, the temperature of column was30℃and detectedwavelength was254nm. In this work, each component was completely separated anddetermined in7minutes. The recovery accorded with detecting requirement. The method wasused to analyze the main metabolites in fermentation broth at different ferment times, which were produced at the process of biocatalysis producing terephthalic acid by stenotrophomonasmaltophilia and comamonas testosteroni. The metabolites in cell including organic acids,amino acids, fatty acids and sugars were detected by GC-MS. According to the analyticresults of HPLC and GC-MS, the pathway of producing terephthalic acid bystenotrophomonas maltophilia and comamonas testosteroni coordinated catalysis wasdiscussed.