Regulation Of Heme Synthesis And Heterologous Expression Of Heme Peroxidase In Escherichia Coli

Heme,a class of iron-containing porphyrin compounds,plays an important role in the metabolic activities of living organisms and serves as a cofactor for heme peroxidase,which is crucial to the enzyme activity.Heme peroxidase has a wide range of applications in biochemical assays,wastewater treatment,dye decolorization,etc.When it is heterologously expressed in bacteria,the enzymatic activity is often low,mainly due to the tight regulation of intracellular heme synthesis and its inefficient binding to the decofactor protein.Therefore,the regulatory mechanism of heme synthesis and the binding of heme to proteases need to be investigated.5-Aminolevulinic acid（5-ALA）is a key precursor substance for heme synthesis.In this study,Escherichia coli BL21（DE3）was used as the experimental object to investigate the regulatory mechanism of heme synthesis by regulating the level of 5-ALA exogenously;three different sources of peroxidase were also heterologously expressed to analyze the binding ability between this class of proteins and heme.The main research contents and results are as follows:（1）The effect of exogenous addition of 5-ALA on heme synthesis.Exogenous addition of 1000μmol·L^-1 5-ALA to LB medium can cause feedback inhibition of intracellular heme synthesis.The accumulation of key precursors and gene transcription levels in the heme synthesis pathway were detected.The conversation of 5-ALA and PBG was not affected by negative feedback,and the transcription levels of hem E,hem F,and hem G in the downstream of heme synthesis were down-regulated to various degrees,and the down-regulation of hem F was the most obvious.（2）The effect of endogenous regulation of 5-ALA on heme synthesis.As an amino acid secondary transporter on the cell membrane,Eam A is involved in the export of 5-ALA.Studies have confirmed that knockout of eam A is beneficial to the accumulation of intracellular 5-ALA and heme.The WTΔE/hem A strain obtained by overexpressing the key gene hem A on the basis of knockout of eam A decreased the intracellular heme content from12.3μmol·L^-1 to 9.5μmol·L^-1 compared with the WT/hem A strain overexpressing hem A alone,a decrease of 29.52%.The accumulation of precursors and gene transcription levels in the heme synthesis pathway were detected.The results showed that the production of intracellular 5-ALA and PBG decreased;the upstream genes glt X,hem A and hem L of 5-ALA synthesis were down-regulated to varying degrees,among which the downregulation of hem L is the most obvious,and the synthesis of 5-ALA is affected by the negative feedback of endogenous regulation.（3）The effect of heterologous expression of heme peroxidase on heme synthesis.Heterologous expression of dye decolorizing peroxidase（Tfu Dy P）from Thermobifida fusca was performed to bind free heme to Tfu Dy P in WTΔE/hem A strain and WT/hem A strain.Enzyme activity assays and spectroscopic analysis showed that the enzyme activity and R_Zvalues of Tfu Dy P derived from WTΔE/hem A-Dy P were higher than those of WT/hem A-Dy P.Examination of heme production and transcript levels of key genes revealed that expression of Dy P significantly increased intracellular heme content and transcript levels of hem A and hem L.These results suggested that the binding of heme to Tfu Dy P removed the feedback effect of free heme on 5-ALA synthesis.（4）Interaction between heme and heme peroxidase.To further analyse the binding of heme to heme peroxidase,heterologous expression of Ba APX from Brassica alboglabra L.,Rh Dyp B from Rhodococcus jostii RHA1 and Tfu Dy P in E.coli BL21（DE3）were investigated for their ability to bind heme.Enzymes were saturated with heme by supplement of hemin in the crude enzyme solution,purified and then subjected to spectroscopic analysis and molecular simulations.The results showed that Ba APX had the highest heme saturation and a more favourable structure for heme binding.