| Heme is a kind of iron-containing porphyrin compounds.It participates in important physiological functions such as cell electron transporting respiratory chain,active oxygen decomposition and catalytic oxidation of substrates in vivo.It can also be used as a cofactor combined with peroxidase to play a catalytic oxidation role,and its content will affect the enzyme activity.In this study,Escherichia coli BL21(DE3)was taken as the starting strain,and the C5 pathway of heme synthesis was taken as the research object.Through molecular modification,the upstream glutamate metabolic flow and the transporter expression of intermediate 5-aminolevulinic acid(ALA)of the heme synthesis pathway were regulated.And the changes of heme contents and transcription level of heme synthetic genes were investigated.Further,the recombinant E.coli strains were used as hosts to express heme peroxidase DyP and the culture conditions were optimized to explore the effects on enzyme activity.The main research contents and results are as follows:(1)The regulation of glutamate metabolism flux and overexpressing of glutamyl-tRNA reductase(hemA)can promote heme synthesis.Mechanosensitive channel of small conductance(MscS)and 3-deoxy-?-arabinoheptulosonate-7 phosphate synthase(AroG)were used for regulation.MscS has physiological functions in osmotic pressure regulation,cell division and amino acid transport.AroG requires PEP to synthesize aromatic amino acids,which diverts the metabolic flux to TCA,which in turn affects the production of glutamate by ?-KG.The reaction catalyzed by HemA is the rate-limiting step of the C5 pathway,and its overexpression can increase the heme content.The Red homologous recombination was used to construct mscS and aroG double-deletion E.coli strain(WT?MG),the complementary strain was constructed by one-step cloning(WT?MG/pRMG).The growth curve showed that the double-deletion of mscs and aroG had no significant effect on the growth of E.coli.Compared with the starting strain(WT),the glutamic acid content of WT?MG increased by 6.3%,extracellular ALA decreased by 8.9%,and intracellular heme increased by 31.1%.After overexpression of hem A gene,the intracellular heme content was further increased,indicating that regulating the metabolic flux of upstream of the heme synthetic pathway can promote heme synthesis to some extent.(2)The regulation of ALA transportation and overexpression of hemA can promote heme synthesis.EamA is a kind of amino acid secondary transport membrane protein,which has been confirmed to mediate ALA transport in E.coli.The Red homologous recombination was used to knock out eamA to construct a single deletion strain WT?E,and the complementary strain was constructed(WT?E/pBE).The growth curve of WT?E was almost the same as that of WT,and there was no significant difference in bacterial growth density.Compared with WT,extracellular ALA of WT?E decreased by 21.9% and intracellular heme increased by 42.9%,indicating that EamA is indeed involved in ALA transport.After overexpression of hemA,the heme content also increased significantly,13 times that of the deletion bacteria,indicating that regulating the transport of intermediate products of C5 pathway has a positive effect on the final product heme synthesis.(3)Heterogeneous expression of heme peroxidase in gene-deficient bacteria.DyP has been heteroexpressed of in E.coli in our laboratory,but the enzyme activity is low due to the lack of heme cofactor.Therefore,the DyP gene was transferred into the recombinant bacteria WT?MG and WT?E,which were modified by the above heme pathway to detect enzyme activity.The results showed that the specific enzyme activity was improved,especially in the eamA-deficient E.coli expressing DyP,with 51.9% increase compared with the control group.The enzyme activity of recombinant E.coli was further increased by optimizing the culture conditions.The results show that through the regulation of heme pathway can increase the activity of heterologous peroxidase with heme as prosthetic group. |
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