Effect Of Genetic Recombination Of Key Enzymes In Heme Synthetic Pathway On Heterologous Expression Of TfuDyP In Escherichia Coli

Dye-decolorizing peroxidases（DyPs）are a group of heme-containing proteins that can efficiently degrade substrates such as anthraquinone and azo dyes.As a result,they have great potential for applications in textile wastewater treatment and other fields.The direct extraction of DyPs from the original strain has the problems of complicated separation steps and high cost.Heterologous expression of DyPs in Escherichia coli is an important method to solve the above problems,but the lack of intracellular heme content limits the improvement of the activity of heterologously expressed DyPs,and the effect of gene expression of key enzymes regulating heme synthesis pathway on the heterologous expression of DyPs is not clear.In this study,the DyP from Thermobifida fusca（TfuDyP）was used as the research object,and the mutant strain WT-RT of Escherichia coli BL21（DE3）,which weakened the secretion of heme precursors was used as the host.By co-expressing the downstream key gene of the heme synthetic pathway in E.coli and iron transport genes to promote the transformation of porphyrin to heme,the intracellular heme content was increased.The expression level of TfuDyP in the host cell and fermentation conditions were optimized to improve the enzyme activity,and to investigate the effect of gene recombination on heme synthetic pathway on the heterologous expression of TfuDyP.The main research contents and results are as follows:（1）Transformation of porphyrin to heme in WT-RT strain.5-aminolevulinic acid（5-ALA）and protoporphyrinⅨ（PPⅨ）are important precursors in the heme synthetic pathway of E.coli,and rhtA and tol C encode the transporters of 5-ALA and PPⅨ,respectively.Knocking out rhtA and tol C had no significant effect on bacterial growth.Compared to the wild-type strain WT,the porphyrin and heme contents of knockout strain WT-RT were significantly increased.Exogenous addition of Fe²⁺increased the heme content of the WT-RT strain to a maximum of 29.44μmol·g^-1 DCW,while adding Fe³⁺increased the heme content to 38.22μmol·g^-1 DCW,1.78 times that of the WT strain.Efe B,encoding transferrin was overexpressed in WT-RT and strain RT-pEE was obtained.The heme content decreased significantly in RT-pEE,while co-expression of hemH and efeB significantly increased the heme content compared to the RT-pEE strain.After exogenous addition of Fe²⁺in the co-expression strain,the heme content reached a maximum of 30.85μmol·g^-1 DCW.（2）The effects of expression vectors and co-expression of key heme synthetic genes on TfuDyP enzyme activity and intracellular heme content.TfuDyP was expressed using the pRSFDuet-1 plasmid in the WT-RT strain,and the enzyme activity reached 3.93 U·mg^-1,with a heme content of 25.39μmol·g^-1 DCW.Co-expression of hemH and TfuDyP leds to a significant decrease in TfuDyP activity,and only the strain expressing TfuDyP using the pET-28a plasmid and hemH using the pCDFDuet-1 plasmid showed a slight increase in heme content compared to the strain expressing TfuDyP using the pET-28a plasmid alone.Hem F,encoding coproporphyrinogenⅢoxidase was co-expressed with TfuDyP using pRSFDuet-1plasmid and strain RT-pRSF-DF was obtained.The heme concentration of strain RT-pRSF-DF was 38.17μmol·g^-1 DCW,but the TfuDyP enzyme activity decreased to 1.24 U·mg^-1.Rea-time fluorescence quantitative PCR analysis showed that,with the overexpression of hemF,the expressions of glt X,hemA,hemL,hemD,hemE and hemG in the heme synthetic pathway were up-regulated at different levels,while hemB,hemC and hemH were all down-regulated,among which hemH was the most significant.The transcription level of TfuDyP was down-regulated 1.73 times,resulting in a decrease in TfuDyP enzyme activity.（3）The effect of fermentation conditions on the activity of recombinant TfuDyP.Optimize the fermentation conditions of RT-pRSF-DF,included adding different concentrations of Fe²⁺,Fe³⁺and 5-ALA,changing LB medium to TB medium.Exogenous addition of different concentrations of Fe²⁺,Fe³⁺and 5-ALA promoted the heme content and enzyme activity of TfuDyP.The optimal fermentation conditions were as follows:TB medium was used,induction temperature was 30℃for 18 h.As a result,heme content was 51.99μmol·g^-1 DCW,and enzyme activity reached 6.21 U·mg^-1.