| Escherichia coli was selected as a laboratory model strain for the research on molecular biology,bioengineering,and metabolomics because of its easy cultivation and genetic background.Metabolomics is the basis for qualitative and quantitative research on intracellular and extracellular small molecule metabolites.In this research,the growth of E.coli JM109 at different nutritional levels was studied,focusing on the absorption and utilization of amino acids under different circumstances.The purpose is to analyze the utilization and transformation of amino acids,to understand the amino acid metabolism characteristics of E.coli,and to prepare for the improvement of the culture medium to promote the growth and the fermentation performance of bacteria and for the design of transformation pathways.In the research,the level of metabolic control phenomena was also studied by regulating the enzyme production and enzyme activity of GABA synthetic as a model.A set of integral test methods of amino acid metabolism was established.A group of applicable FMOC-Cl derived amino acid parameters was optimized as follows: sample amount 40 ?L,0.2 mol/L amino acid derivative buffer 40 ?L,FMOC-Cl solution 20 ?L,acetonitrile 80 ?L,deionized water 90 ?L,set at room temperature for 10 min.A set of suitable methods was established for quenching the strains and permeating the cell as a basis for the subsequent detection and analysis of intracellular substances.The method for detecting metabolites using HPLC-MS and the standard curve of amino acid were established for the accurately qualitative and quantitative analysis of amino acids.The results showed that 14 amino acids were identified in LB including Arg 0.249 g/L,Glu 0.031 g/L,Gly 0.218 g/L,Ala 0.035 g/L,Pro 0.040 g/L,Met 0.007 g/L,Ser 0.042 g/L,Val 0.043 g/L,Trp 0.014 g/L,Leu/Ile 0.144 g/L,Phe 0.094 g/L,Lys 0.080 g/L and Thr 0.024 g/L.The consumption of amino acid after the growth of E.coli JM109(p UC19)in LB medium under standard conditions were: Arg 100%,Gly 47%,Ala 100%,Met 100%,Leu/Ile 31%,Lys 52%,Pro 100%,Thr 100%,Ser 100%,Trp 100% and Glu 100%.the production of amino acid were: Val 12%,Phe 12%.accumulation of five amino acids in the cell were: Glu,Ala,Val and Leu/Ile.comparison between intercellular and extracellular amino acid showed that E.coli has a strong ability for the active transmission of Glu and Ala into the cells.The results of E.coli cultured with a single amino acid as the mainly nitrogen source showed that: Glu and Asp were better utilized by the strains,and the strains grew well;higher concentrations of new substances were produced by the cultivation using Cys,Tyr,and Val,and the strains grew poorly;the strains cultured with Trp and Pro changed the mediums to acid and the strains grew in general,with the remaining amount was very high;the remaining amount of amino acids was high after the cultivation using His,Thr and Gly,but biomass was very high,especially the cultivation using His,its growth amount was 50% higher than that of using other amino acids,though the remaining amount was over 40%,which indicated that there might be a special physiological mechanism.In the future design of E.coli metabolic pathways,according to the remaining cell situation,Glu,Phe,Pro,His,Thr and Gly may be more suitable as reactants for biotransformation in simple medium.under eutrophic conditions,Glu,Ala,Val,Leu,Ileu and Gly,Phe and Lys may be more suitable as biotransformation reactants.Under standard fermentation condition in LB medium with different p H:the amino acids Ser,Thr,Pro and Trp were all exhausted;Glu was completely depleted in extracellular at different p H,and the high concentration was retained in the cell;the highest amount of internal accumulation and the lowest amount of extracellular accumulation of Leu/Ile was obtained at the p H 5.52;Arg absorbtion was blocked at the p H 4.58.Val and Phe were amino acids produced.Under standard fermentation condition in LB medium with different temperature: the extracellular amino acids Ser,Thr,Pro,and Trp were exhausted;Glu was completely depleted at different temperatures and a higher intercellular concentration was retained;Val and Phe were amino acids produced,and the extracellular production was higher with temperature changed;the absorption of Gly was blocked at 35 ?.The isocaudarner Sau 3A1 and Bam HI were used to construct a random library,and the strains were screened for significantly changed the metabolic pathway.Finally,strain 17 that produced a new substance was found.The gene sequencing confirmed that it contained aspartic acid decarboxylase.The product identified by mass spectrometry should be ?-Hydroxypropionic acid(3-AP).It was interesting that strain 17 produced 3-AP only in the condition of LB medium,but not in Asp transforming medium.There may be some kind of regulation mechanism,which need further analysis and research.In the study of GABA production using from Glu transformation by Escherichia coli,as a model,it was found that(1)the yield of GABA transformation was significantly affected by p H,and the yield in p H 2.6 was 40 times higher than that of p H 7.2;(2)a large amount of glutamate decarboxylase was produced in LB medium.The amount of production in GI medium reduced by more than 93%;(3)the activity of glutamate decarboxylase in the glucose-containing conversion solution was 15 times higher than that in LB(p H 2.6);(4)with 5 g/L glucose added in LB medium,the amount of glutamate decarboxylase produced decreased by 76%;(5)E.coli cultured in acidic and neutral LB fermentation medium for 9 h,a large and equal amount of glutamate decarboxylase was produced in both of the two mediums.Based on these phenomena,it was possible to gain insight into the complex regulatory mechanism of GABA production using Glu transformation by E.coli,which cannot be fully explained by existing theories.These research results provided a valuable window or the further research area on the metabolic regulation mechanism of E.coli. |
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