Expression And Regulation Of Genes Coding For The Key Enzyme In Heme Synthstic Pathway In E.coli

Heme is an important iron porphyrin compound,which is involved in biological functions including iron transport,antioxidation,signal regulation,and formation of most hemoproteins.In this study,Escherichia coli was targeted to explore the control of heme production by regulation of the key enzymes expression in the heme synthesis pathway.The mechanism of heme synthesis in the recombinants was analyzed by transcriptomics.And the specific activity of peroxidase was increased by co-expression of gene which promoted heme synthesis with ascorbate peroxidase gene?apx?.The main findings are as follows:?1?Overexpression of key enzyme genes and the regulation of heme synthesis by environmental factors in E.coli.HemA gene was found to be beneficial to the synthesis of porphyrins by constructing homologous recombinant Eco/pEX,Eco/pEA,Eco/pEB,Eco/pED,Eco/pEH,and Eco/pEAD containing the gltX,hemA,hemB,hemD,hemH,and hemAD genes.And the heme production in Eco/pEA was up to 26.53?mol·L^-1,which is 8.08 times higher than that of the control.While the overexpression of other genes and co-expression of hemA and hemD had no significant effect.The effect of temperature,dissolved oxygen,and exogenous additives on the production of porphyrin precursors 5-aminolevulinic acid?5-aminolevulinic acid?and final production heme were analyzed and it was found that Fe²⁺,Glu,or Glc all increased the content of 5-ALA and heme,but the influence of temperature and dissolved oxygen on 5-ALA and heme are inconsistent.With the condition of 0.2 mmol·L^-1 Glc being added at 37°C in shaking flask containing 200 m L liquid medium,heme production of Eco/pEA was highest,reaching 34.45?mol·L^-1,which was 10.79 times higher than the control.?2?Analysis of the mechanism of heme synthesis in recombinant bacteria.Eco/pEA can easily form inclusion bodies after induction.Although there are differences in heme production under different inducing conditions,they have all promoted heme production.RNA-seq analysis shows that:The transcriptional up-regulation of hemA affects the accumulation of heme in the bacteria and increased heme yield obviously.However,if the degree of hemA transcription is too high,transcription of hemD,hemF and arcA was down-regulated,and hemD and hemF expression was unfavorable for the accumulation of heme.At the same time,the up-regulation of hemA caused the inhibition glycolysis,pyruvate metabolism,fatty acid degradation and glutathione metabolism,while the Gly and succinyl-CoA synthesis related genes such as sucD,gcvP were up-regulated,indicating Gly and succinly-CoA may play an important role in heme synthesis.In addition,qRT-PCR analysis revealed that the overexpression of hemB up-regulated hemA and hemH while down-regulated hemD;the over-expression of hemH caused the up-regulation of hemA and hemB while down-regulated hemD;hemB and hemH may inhibit heme synthesis.Under induced conditions,the glutamate dehydrogenase gene gdhA and glutamate metabolic enzyme gene asnA in Eco/pEB and Eco/pEH are also up-regulated indicating gdhA and asnA may decrease the heme production.?3?Regulation of heme synthesis in heterologous expression of peroxidase gene.hemA and apx were co-expressed in strain Eco/pEA-28A.It was found that overexpression of hemA increased intracellular heme content.The APX specific activity increased significantly from the58.23 U·g^-1 to 495.20 U·g^-1.