摘要
血红素(heme)是一种金属卟啉化合物,在细胞中具有重要的生理功能,实际生产中应用广泛,目前对大肠杆菌(Escherichia coli)血红素合成途径调控的研究主要集中在上游,而下游部分较少。为了研究下游途径的改造对大肠杆菌血红素合成的影响,对合成途径最后一步的相关基因进行了不同的表达调控。研究结果表明,在铁摄取调节子(ferric uptake regulator,Fur)基因fur缺失菌中过表达亚铁鳌合酶(ferrochelatase)基因hemH会抑制血红素的合成;而过表达染料脱色过氧化物酶(dye-decolorizing peroxidase)基因efeB可以缓解因fur缺失而造成的血红素含量下降;efeB和hemH基因的共表达则可以有效提高血红素的含量,达到出发菌株E.coli BL21的1.72倍。研究结果为提升大肠杆菌血红素含量的研究提供了一定的策略和理论依据。
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.
作者
刘琪
李蒙蒙
丁亮亮
张鑫
王悦
唐蕾
LIU Qi;LI Mengmeng;DING Liangliang;ZHANG Xin;WANG Yue;TANG Lei(School of Biotechnology,Jiangnan University,Wuxi 214122,China;The Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2023年第6期98-104,共7页
Food and Fermentation Industries
基金
111引智计划(111-2-06)
国家轻工技术与工程一流学科自主课题资助(LITE2018-27)。