Regulation Of Iron Uptake Regulator And Ferrochelatase Gene Expression On Heme Synthesis Of Escherichia Coli

Heme is a kind of porphyrin compound with chelated iron ions in the middle,and participates in biological functions such as oxygen transport and electron transfer of cells.Increases its biosynthesis can promote enzyme activity of peroxidase with heme as a prosthetic group,and heme is used as a pigment additive and iron supplement in the food and pharmaceutical industry.Therefore,heme synthesis has certain application value in the food and medicine industry and production of recombinant protein with heme as a supplement,but the mechanism of heme metabolism regulation is still unclear.In this study,Escherichia coli was used as the research strain,and iron uptake regulator gene fur and ferrochelatase gene hemH were targeted.The effects of iron,and expression levels of fur and hemH on the regulation of heme biosynthesis were investigated by overexpression and deletion of fur as well as hemH overexpression.The main research contents and results are as follows:(1)The overexpression of the fur regulates the heme synthesis of Escherichia coli.By constructing the homologous recombinant strain Eco/pEF with fur overexpression,it was found that Fur inhibited the growth of the cells while the intracellular iron ions decreased,and the heme decreased by 3.55 times.The gene gltX,hemL and hemB were up-regulated by 4.93 times,2.55 times,and 4.61 times,respectively.However,due to the down-regulation of hemA,the ALA rate of the precursor substance of synthetic heme was decreased which led to the decrease of heme content in Eco/pEF.Under the condition of iron limit,fur has a certain promoting effect on heme synthesis,and the content of heme is 2.22 times higher than that of the original one.Fur has up-regulated the transcription levels of genes hemB,hemL hemH by 5.59 times,2.60 times,and 2.40 times,respectively,resulting in the promotion of heme biosynthesis under iron limitation.(2)The deletion of fur regulates the heme synthesis of Escherichia coli.By constructing the fur deletion strain Eco?fur,it was found that fur deletion did not affect the growth of the cells,but the heme content decreased by 3.83 times.The loss of fur reduced the expression of hemL,hemE and hemH by 6.37 times,6.43 times and 1.68 times,respectively.The precursor porphyrins did not accumulate significantly.The growth of the strain was inhibited under the condition of iron restriction.The synthesis of heme was further reduced after gene deletion.The expression levels of hemL,hemE and hemH were down-regulated by 11.06 times,5.41 times and 1.75 times,respectively.The protoporphyrin IX,a precursor of heme,was accumulated.(3)Overexpression of hemH regulates the heme synthesis of Escherichia coli.By examining the heme synthesis of the recombinant Eco/pEH.The heme content did not increase in Eco/pEH.The heme content under the iron-limited condition was increased by 3.83 times compared with the LB culture condition.Overexpression of hemH down-regulated hemB and hemF by 8.01 times and 3.13 times,respectively,and the transcription level of hemG was upregulated by 3.56 times under iron limitation.Through metabolomics analysis,it was found that changes in related differential metabolites and pathways led to the enhancement of the heme synthesis pathway,resulting in a relatively higer heme content of recombinant strain Eco/pEH under iron limitation.