为了阐明铁摄取调节子基因fur对大肠杆菌血红素合成的影响,对fur基因进行了同源过表达,考察了菌体生长、铁离子浓度及血红素合成变化,并分析了血红素合成途径关键基因的表达水平。结果表明,原始菌株在限铁条件下的血红素量比在非限铁条件下的血红素量下降2.54倍;fur过表达对菌体的生长有抑制作用,胞内的铁离子减少;非限铁条件下,过表达菌株Eco/pEF的血红素量比原始菌株下降了3.55倍;而过表达菌株Eco/pEF在限铁条件下的血红素量比在非限铁条件下的血红素量增加3.10倍。fur过表达引起hemA下调,而gltX、hemB、hemC分别上调4.93倍、4.61倍和9.39倍;菌株Eco/pEF在限铁条件下hemB与hemG分别上调了5.59倍和4.49倍。虽然fur过表达使血红素合成途径基因表达水平上调,但并未导致血红素大量合成。该结果为通过铁离子摄取调控胞内血红素的水平提供了新的理论支持。
In order to elucidate the effects of iron uptake regulator gene fur on heme synthesis in Escherichia coli, fur was homogenously overexpressed. The growth, the concentration of iron ions and the changes in heme synthesis and the expression level of key genes in heme synthesis pathway were analyzed. The results showed that the heme content in the original strain under iron limited condition was 2.54 times lower than that under non-iron limited condition. Overexpression of fur inhibited the growth and decreased the concentration of intracellular iron ions. Under non-iron limited condition, the heme content in fur overexpressed strain Eco/pEF decreased by 3.55 times compared with that of the original strain and the heme content in Eco/pEF under iron limited condition increased by 3.10 times compared with that under non-iron limited condition. Overexpression of fur down-regulated hemA, while gltX, hemB and hemC were up-regulated by 4.93, 4.61 and 9.39 times, respectively. Under iron limited condition, hemB and hemG in Eco/pEF were up-regulated by 5.59 and 4.49 times, respectively. Although fur overexpression up-regulated the expression levels of the genes involved in heme synthesis pathway, heme content did not increase significantly. These results provide theoretical support for regulating intracellular heme levels by iron ion uptake.
[1] HEINEMANN I U, JAHN M, JAHN D. The biochemistry of heme biosynthesis[J]. Archives of Biochemistry & Biophysics, 2008, 474(2): 238-251.
[2] FRANKENBERG N, MOSER J, JAHN D. Bacterial heme biosynthesis and its biotechnological application[J]. Applied Microbiology and Biotechnology, 2003, 63(2): 115-127.
[3] 黄加保.芥蓝抗坏血酸过氧化物酶在大肠杆菌中的基因表达及酶学性质研究[D].无锡:江南大学,2013:33-38.
[4] RAMZI A B, HYEON J E, HAN S O. Improved catalytic activities of a dye-decolorizing peroxidase (DyP) by overexpression of ALA and heme biosynthesis genes in Escherichia coli[J]. Process Biochemistry,2015,50(8):1 272-1 276.
[5] 卫乐红,时亚文,陈石良,等.血红素铁的制备及应用研究进展[J].食品与药品,2013,15(5):357-360.
[6] DAILEY H A, DAILEY T A, GERDES S, et al. Prokaryotic heme biosynthesis: Multiple pathways to a common essential product[J]. Microbiology & Molecular Biology Reviews, 2017, 81(1): e00 048-16.
[7] FILLAT M F. The FUR (ferric uptake regulator) superfamily: Diversity and versatility of key transcriptional regulators[J]. Archives of Biochemistry & Biophysics, 2014, 546(6):41-52.
[8] MCHUGH J P, RODRIGUEZQUIÑONES F, ABDUL-TEHRANI H, et al. Global iron-dependent gene regulation in Escherichia coli a new mechanism for iron homeostasis[J]. Journal of Biological Chemistry, 2003, 278(32): 29 478-29 486.
[9] ANDREWS S C, ROBINSON A K, RODRI′GUEZQUIÑONES F. Bacterial iron homeostasis[J]. FEMS Microbiology Reviews,2003,27(2-3):215-237.
[10] LEE M J, CHUN S J, KIM H J, et al. Porphyrin derivatives from a recombinant Escherichia coli grown on chemically defined medium[J]. Journal of Microbiology & Biotechnology,2012,22(12):1 653-1 658.
[11] 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.
[12] 陆超. 固氮施氏假单胞菌铁吸收调节蛋白Fur参与铁转运和固氮调控的研究[D].北京:中国农业科学院,2016:37-38.
[13] MICHENER J K, NIELSEN J, SMOLKE C D. Identification and treatment of heme depletion attributed to overexpression of a lineage of evolved P450 monooxygenases [J]. Proceedings of the National Academy of Sciences of the United States of America,2012,109(47): 19 504-19 509.
[14] ZHANG J, KANG Z, CHEN J, et al. Optimization of the heme biosynthesis pathway for the production of 5-aminolevulinic acid in Escherichia coli [J]. Scientific Reports,2015,5:8 584-8 591.
[15] CORNELIS P, WEI Q, ANDREWS S C, et al. Iron homeostasis and management of oxidative stress response in bacteria[J]. Metallomics, 2011, 3(6):540-549
[16] LAU K, KREWULAK K D, VOGEL H J. Bacterial ferrous iron transport: The Feo system [J]. FEMS Microbiology Reviews, 2015, 40(2): 273-298.
[17] CARTRON M L, MADDOCKS S, GILLINGHAM P, et al. Feo-transport of ferrous iron into bacteria [J]. Biometals, 2006,19(2):143-157.
[18] LIU S, ZHANG G, LI X, et al. Microbial production and applications of 5-aminolevulinic acid [J]. Applied Microbiology and Biotechnology, 2014, 98(17):7 349-7 357.
[19] SCHAECHTER M. Encyclopedia of Microbiology(Third Edition)[M]. Salt Lake City:Academic Press,2009: 194-209.
[20] PRANAWIDJAJA S, CHOI S I, LAY B W, et al. Analysis of heme biosynthetic pathways in a recombinant Escherichia coli [J]. Journal of Microbiology Biotechnology, 2014, 25(6):880-886.
[21] KAMMLER M, SCHÕN C, HANTKE K. Characterization of the ferrous iron uptake system of Escherichia coli [J]. Journal of Bacteriology, 1993,175(19):6 212-6 219.
[22] NANDAL A, HUGGINS C C O, WOODHALL M R, et al. Induction of the ferritin gene (ftnA) of Escherichia coli by Fe2+-Fur is mediated by reversal of H-NS silencing and is RyhB independent[J]. Molecular Microbiology,2010,75(3):637-657.
[23] LI F, WANG Y, GONG K, et al. Constitutive expression of RyhB regulates the heme biosynthesis pathway and increases the 5-aminolevulinic acid accumulation in Escherichia coli [J]. FEMS Microbiology Letters,2014,350(2):209-215.
[24] HERNÁNDEZ J A, LÓPEZ-GOMOLLÓV S, BES M T, et al. Three fur homologues from Anabaena sp. PCC7120: Exploring reciprocal protein-promoter recognition[J]. FEMS Microbiology Letters,2004,236(2):275-282.
[25] GONZÁLEZ A, BES M T, VALLADARES A, et al. FurA is the master regulator of iron homeostasis and modulates the expression of tetrapyrrole biosynthesis genes in Anabaena sp. PCC 7120 [J]. Environmental Microbiology, 2012, 14(12): 3 175-3 187.
[26] FU H H, LIU L, DONG Z, et al. Dissociation between iron and heme biosynthesis is largely accountable for respiration defects of Shewanella oneidensis fur mutants[J]. Applied and Environmental Microbiology,2018,84(8):18.
[27] COLPA D I, FRAAIJE M W. High overexpression of dye decolorizing peroxidase TfuDyP leads to the incorporation of heme precursor protoporphyrin IX [J]. Journal of Molecular Catalysis B: Enzymatic,2016,134(PtB):372-377.
[28] NAKAHIGASHI K, NISHIMURA K, MIYAMOTO K, et al. Photosensitivity of a protoporphyrin-accumulating, light-sensitive mutant (visA) of Escherichia coli K-12 [J]. Proceedings of the National Academy of Sciences, 1991, 88(23): 10 520-10 524.
