Heme is a kind of porphyrin compounds containing iron, which widely exists in animals, plants and microorganisms. Ferrous ion is located in the center of heme and forms a stable conjugate structure with porphyrin ring. Heme has many key biological functions. It participates in a variety of biological processes such as energy metabolism, electron transfer, oxidative stress and signal transduction in cells. Besides, it is also an important biological iron supplement. Therefore, heme has a very wide range of applications in practical production and application. In Escherichia coli, heme is synthesized by C5 pathway through multi-step enzymatic catalytic reaction, which is highly conserved. At present, the research on the regulation of heme synthesis pathway mainly focuses on the upstream of the pathway, indicating that the addition of precursor 5-aminolevulinic acid (ALA) will significantly increase the yield of heme. However, excessive ALA will be toxic to cells, which is not conducive to cell survival and heme synthesis. In addition, the cost of exogenous ALA addition is relatively high. Therefore, we took the last step of heme synthesis pathway, that is, protoporphyrin IX combines with ferrous iron to form heme as the research object, regulated the expression of related genes that might affect this step, and studied the effect of downstream pathway modification on heme synthesis in E. coli. The ferrochelatase gene hemH, iron uptake regulator gene fur and a hemoprotein gene efeB participating in ion transportation were targeted. The heme contents in recombinants, the fur knockout mutant EcoΔfur, the EcoΔfur harboring the plasmid pET28a ligated with hemH (EcoΔfur/pEH), efeB (EcoΔfur/pEE) or both hemH and efeB (EcoΔfur/pEHE) were compared. The overexpression of hemH in EcoΔfur could inhibit heme synthesis. Previous studies have reported that the deletion of fur gene reduced the total iron content in E. coli and hemH overexpression could not promote heme synthesis. In this study the heme synthesis of the recombinant strain EcoΔfur/pEH was inhibited, indicating that there might be a more complicated regulation relation between fur and hemH. EfeB is a dye decolorizing peroxidase (DyP) using heme as cofactor and has specific iron transport function. Overexpression of efeB in fur deficient E. coli alleviated the decrease of heme content caused by fur deletion. Although the heme content of the recombinant strain EcoΔfur/pEE was lower than that of parent strain, the expression of efeB gene showed a positive effect on heme synthesis. Previous studies have shown that co-expression of hemH with hemoprotein gene helps the insertion of heme into the hemoprotein and improves emzyme’s activity. In this study, the co-overexpression of hemH and efeB also increased heme content significantly in EcoΔfur/pEHE, about 1.72 time that of the original strain E. coli BL21 (DE3). The results of this study provide some basic information of ion related hemH, fur and efeB genes on heme synthesis in E. coli, however further relationship among these three genes should be investigated in detail. The insertion of ion into protoporphyrin IX to form heme will not only be benefit for heme fermentation, but also be useful to produce the recombinant hemoproteins with high biological activities.
LIU Qi
,
LI Mengmeng
,
DING Liangliang
,
ZHANG Xin
,
WANG Yue
,
TANG Lei
. Effect of regulation of iron related genes in Escherichia coli on heme synthesis[J]. Food and Fermentation Industries, 2023
, 49(6)
: 98
-104
.
DOI: 10.13995/j.cnki.11-1802/ts.032077
[1] FRANKENBERG N, MOSER J, JAHN D.Bacterial heme biosynthesis and its biotechnological application[J].Applied Microbiology and Biotechnology, 2003, 63(2):115-127.
[2] SHIMIZU T, HUANG D Y, YAN F, et al.Gaseous O-2, NO, and CO in signal transduction:Structure and function relationships of heme-based gas sensors and heme-redox sensors[J].Chemical Reviews, 2015, 115(13):6 491-6 533.
[3] FIEGE K, QUEREBILLO C J, HILDEBRANDT P, et al.Improved method for the incorporation of heme cofactors into recombinant proteins using Escherichia coli nissle 1917[J].Biochemistry, 2018, 57(19):2 747-2 755.
[4] STOJILJKOVIC I, PERKINS-BALDING D.Processing of heme and heme-containing proteins by bacteria[J].DNA and Cell Biology, 2002, 21(4):281-295.
[5] RYTER S W.Significance of heme and heme degradation in the pathogenesis of acute lung and inflammatory disorders[J].International Journal of Molecular Sciences, 2021, 22(11):5509.
[6] QUINTERO-GUTIÉRREZ A G, GONZÁLEZ-ROSENDO G, SÁNCHEZ-MUÑOZ J, et al.Bioavailability of heme iron in biscuit filling using piglets as an animal model for humans[J].International Journal of Biological Sciences, 2008, 4(1):58-62.
[7] FILLAT M.F.The FUR (ferric uptake regulator) superfamily:Diversity and versatility of key transcriptional regulators[J].Archives of Biochemistry and Biophysics, 2014, 546:41-52.
[8] SEO S W, KIM D, LATIF H, et al.Deciphering Fur transcriptional regulatory network highlights its complex role beyond iron metabolism in Escherichia coli[J].Nature Communications, 2014, 5:4910.
[9] STURM A,SCHIERHORN A,LINDENSTRAUSS U, et al.YcdB from Escherichia coli reveals a novel class of Tat-dependently translocated hemoproteins[J].Journal of Biological Chemistry, 2006, 281(20):13 972-13 978.
[10] 丁亮亮,刘进生,顾鹏帅, 等.大肠杆菌过氧化物酶EfeB在细胞氧化应激中的作用[J].食品与发酵工业, 2020, 46(17):33-39.
DING L L, LIU J S, GU P S, et al.The role of peroxidase EfeB in Escherichia coli under cell oxidative stress[J] Food and Fermentation Industries, 2020, 46 (17):33-39.
[11] MIETHKE M, MONTEFERRANTE C G, MARAHIEL M A, et al.The Bacillus subtilis EfeUOB transporter is essential for high-affinity acquisition of ferrous and ferric iron[J].Biochimica et Biophysica Acta.Molecular Cell Research, 2013, 1 833(10):2 267-2 278.
[12] PRANAWIDJAJA S, CHOI S I, LAY B W, et al.Analysis of heme biosynthetic pathways in a recombinant Escherichia coli[J].Journal of Microbiology and Biotechnology, 2015, 25(6):880-886.
[13] SUDHAMSU J, KABIR M, AIROLA M V, et al.Co-expression of ferrochelatase allows for complete heme incorporation into recombinant proteins produced in E.coli[J].Protein Expression and Purification, 2010, 73(1):78-82.
[14] 陈丹园. 大肠杆菌血红素合成途径关键酶基因的表达与调控[D].无锡:江南大学,2018.
CHEN D Y.Expression and regulation of genes coding for the key enzyme in heme synthstic pathway in E.coli [D].Wuxi:Jiangnan University, 2018.
[15] 李蒙蒙. 铁摄取调节子与亚铁鳌合酶基因表达对大肠杆菌血红素合成的调控[D].无锡:江南大学,2019.
LI M M.Regulation of iron uptake regulator and ferrochelatase gene expression on heme synthesis of Escherichia coli [D].Wuxi:Jiangnan University, 2019.
[16] 陈丹园,沈云杰,杨燕, 等.关键酶基因的过表达与环境因素对大肠杆菌血红素合成的调控[J].食品与发酵工业, 2018, 44(11):7-14.
CHEN D Y, SHEN Y J, YANG Y, et al.Regulation of heme synthesis in Escherichia coli by overexpression of genes for the key enzymes and environmental factors[J]. Food and Fermentation Industries, 2018, 44 (11):7-14.
[17] GROSSE C, SCHERER J, KOCH D, et al.A new ferrous iron-uptake transporter, EfeU (YcdN), from Escherichia coli[J].Molecular Microbiology, 2006, 62(1):120-131.
[18] 马蓉,徐昊,丁锐, 等.大肠杆菌多基因共表达策略[J].中国生物工程杂志, 2012, 32(4):117-122.
MA R,XU H,DING R, et al.The strategy of gene coexpression in Escherichia coli[J].China Biotechnology, 2012, 32 (4):117-122.
[19] GRAVES P E, HENDERSON D P, HORSTMAN M J, et al.Enhancing stability and expression of recombinant human hemoglobin in E.coli:Progress in the development of a recombinant HBOC source[J].Biochimica Et Biophysica Acta-Proteins and Proteomics, 2008, 1 784(10):1 471-1 479.
[20] FENG C Q, PAN M, TANG L.5-Aminolevulinic acid level and dye-decolorizing peroxidase expression regulate heme synthesis in Escherichia coli[J].Biotechnology Letters, 2022, 44(2):271-277.
[21] DE ARMAS-RICARD M, LEVICÁN G, KATZ A, et al.Cellular levels of heme affect the activity of dimeric glutamyl-tRNA reductase[J].Biochemical and Biophysical Research Communications, 2011, 405(1):134-139.
[22] YU T H, TAN S I, YI Y C, et al.New insight into the codon usage and medium optimization toward stable and high-level 5-aminolevulinic acid production in Escherichia coli[J].Biochemical Engineering Journal, 2022, 177:108259.
[23] ZHANG J L, WENG H J, ZHOU Z X, et al.Engineering of multiple modular pathways for high-yield production of 5-aminolevulinic acid in Escherichia coli[J].Bioresource Technology, 2019, 274:353-360.
[24] 张良程. 大肠杆菌中5-氨基乙酰丙酸转运蛋白的研究[D].天津:天津科技大学,2016.
ZHANG L C.Study on 5-aminolevulinic acid transporters in Escherichia coli [D].Tianjin:Tianjin University of Science and Technology, 2016.
