Modification Of Heme Metabolic Pathway In Escherichia Coli And Heterologous Expression Of DyP Enzyme

Dye-decolorizing peroxidases（Dy Ps）are newly discovered peroxidases with heme as cofactor,which can be used for the degradation of lignin and a variety of dyes.At present,heterologous expression of Dy Ps has been achieved in engineering bacteria such as Escherichia coli,but the lack of cofactor results in low enzyme activity.Therefore,it is crucial to increase the yield of heme while efficiently expressing Dy P enzymes.In this study,Escherichia coli BL21（DE3）was used as the starting strain and the high-yielding heme strain was obtained by modifying the heme synthesis pathway,reducing the secretion of precursors,and regulating the expression of genes involved in the heme synthesis pathway.The modified heme high-yielding strains are used as the host strains for expressing the Dy P enzyme,and the enzyme activity of Tfu Dy P is greatly improved.The main research contents and results are as follows:（1）Enhancement of heme C5 pathway in E.coli.5-Aminolevulinic acid（ALA）,a key precursor for heme synthesis,was synthesized via C4 and C5 pathways,while ALA was synthesized via C5 pathway in E.coli.Firstly,the the hem A gene（encoding glutamyl-t RNA reductase）of C5 pathway was overexpressed in tol C-deleted E.coli WTΔT to increase the level of ALA in cells,which lead to the accumulation of precursor porphyrin;subsequently,the hem H gene（encoding Ferrochelatase）was co-expressed and the accumulated porphyrin was converted into heme,and the yield of heme was increased to 34.69μmol·L^-1,which was3.44 fold of that in WT.The yield of heme was further improved by adding ferrous ions,and finally the yield of heme was 40.18μmol·L^-1at the concentration of ferrous ions of 80μmol·L^-1,which was 3.98 fold of that in WT.（2）Construction of C4 pathway in E.coli.The hem L gene of C5 pathway was knocked out by Red homologous recombination,and the E.coli with C5 pathway-deleted was obtained.And then the ALA synthase（Rsp Hem A,hem A^R）from Rhodobacter sphaeroides was heterologously expressed,so that the WT△L/PCA^Rwith C4 pathway synthesis of heme was obtained.It was found that the heme yield of WT△L/PCA^Rreached 43.51μmol·L^-1,which was much higher than that of WT,C5 pathway overexpressed strains and strains of two pathway co-expression.Subsequently,the downstream genes hem B（encoding ALA dehydrataseand）and hem H of heme synthesis pathway was overexpressed,which further increased the heme yield to 59.46μmol·L^-1,which was 5.89 fold of that in WT.（3）Heterologous expression and enzyme activity analysis of heme peroxidase.The Tfu Dy P stored in our laboratory from Thermobifida fusca was introduced into the heme-producing strain obtained in this study to detect the enzyme activity.The results showed that the enzyme activity of WTΔL/p CA^R-p ED and WTΔT/p CA^R-p ED containing medium copy plasmid p ET28a expressing Tfu Dy P gene and low copy plasmid p CDFDuet1 expressing hem A^Rwas significantly improved to 30.25 U·mg^-1and 32.75 U·mg^-1,which were 5.98 and6.47 fold of that in the strain WT/p ED containing p ET28a expressing Tfu Dy P in WT.After Tfu Dy P was purified,the enzyme activity of the recombinant strain WTΔT/p CA^R-p ED was further increased to 110.63 U·mg^-1.The above results indicate that the regulation of heme metabolic pathway can improve the activity of recombinant Tfu Dy P with heme as cofactor.