Effect Of Expression Of RhtA And TolC On Heme Synthesis In Escherichia Coli

Heme is a porphyrin compound containing iron ions.It acts as a cofactor for various enzymes such as peroxidase and has many important biological functions.The aim of this study was to investigate the effect of the expression of heme precursor transporter protein in Escherichia coli(E.coli)on heme synthesis and metabolism.By constructing precursor transporter of gene rht A and tol C deleted strains,the changes of heme and its precursor content were analyzed.Based on transcriptome and q RT-PCR results,the regulation mechanism of heme synthesis in deleted strains was elaborated.The main results are as follows:(1)Regulation of heme precursor transporter expression.By Red homologous recombination,we constructed E.coli rhtA deletion,tol C deletion and tol C,rhtA double deletion strains WT-R,WT-T and WT-RT.The complementary strains p ET-R,p ET-T and p RT-TR with rht A,tol C and double deletion genes were constructed by one-step cloning.The plasmids p EA overexpressing hem A gene were transfected into WT,WTR,WT-T and WT-RT,respectively.The strains p EA,p EA-R,p EA-T and p EA-RT were obtained.The results of growth curve showed that the deletion of tol C and rht A had no significant effect on the growth of E.coli.(2)Effect of heme precursor transporter protein expression on heme synthesis.Compared with the original strain WT,extracellular 5-aminolevulinic acid(ALA)of WT-R decreased by 23%,bilinogen(PBG)increased by 15%,intracellular uroporphyrin III(UIII),coproporphyrin III(CIII)and protoporphyrin IX(PPIX)increased by 25%,15% and 18%,respectively.Heme increased by 12%;WT-T ALA decreased by 30%,PBG decreased by 39%.UIII,CIII and PPIX increased 4,14 and 6 times respectively,heme decreased 26%;WT-RT ALA decreased 30%,PBG decreased 12%,UIII,CIII and PPIX increased 19%,3 and 15 times respectively,Heme increased 4%.Compared with p EA,p EA-R decreased ALA by 16%,PBG by 14%,UIII,CIII and PPIX by 55%,64% and 470%,Heme by 24%,p EA-T ALA by 16%,PBG by 5%,CIII and PPIX by 17 and 1.8 times,Heme by 24%.The ALA,PBG,UIII,CIII and PIX of p EA-RT strains decreased by 24%,15%,40%,33 and 19 times,respectively,and Heme increased by 5%.(3)Analysis of the mechanism of heme synthesis in gene-deleted strains.The effect of heme precursor transporter protein expression on heme synthesis.The deletion of tol C and rht A results in the up-regulation or little change of hem H transcription,while the deletion of tol C results in the down-regulation of glutamyl-t RNA synthetase(Glutamylt RNA synthetase)gene glt X and hem A,which inhibits the synthesis of heme and ALA to some extent,resulting in the decrease of accumulation of ALA and heme,the decrease of extracellular PBG and the further flow of metabolites to heme synthesis.Unlike rht Adeficient strains,the decrease of extracellular ALA caused by tol C deletion may be due to the decrease of total ALA caused by tol C deletion.The accumulation of intracellular porphyrin in tol C-deficient strain WT-T and tol C-deficient hem A strain p EA-T was also over-expressed,but the heme production decreased compared with the control strain,which may be related to iron deficiency caused by tol C deficiency.The results of transcriptome analysis showed that transcription regulators arcA and ferrous transporter feoB were down-regulated and global transcription regulator fur was up-regulated,resulting in the difference of heme production.