微生物学通报  2021, Vol. 48 Issue (3): 701−709

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潘梅, 刘爽欣, 唐蕾
PAN Mei, LIU Shuangxin, TANG Lei
谷氨酸代谢调控及hemA过表达促进大肠杆菌血红素合成
Enhancing heme synthesis in Escherichia coli by regulating glutamic acid metabolism and over-expressing hemA
微生物学通报, 2021, 48(3): 701-709
Microbiology China, 2021, 48(3): 701-709
DOI: 10.13344/j.microbiol.china.200434

文章历史

收稿日期: 2020-05-04
接受日期: 2020-07-04
网络首发日期: 2020-07-23
谷氨酸代谢调控及hemA过表达促进大肠杆菌血红素合成
潘梅1 , 刘爽欣2 , 唐蕾1,2     
1. 江南大学工业生物技术教育部重点实验室    江苏  无锡    214122;
2. 江南大学生物工程学院    江苏  无锡    214122
摘要: 【背景】 大肠杆菌(Escherichia coli)以谷氨酸为前体经C5途径合成有限的血红素。【目的】 探究胞内谷氨酸代谢及谷氨酰-tRNA还原酶基因(hemA)过表达对5-氨基乙酰丙酸(5-Aminolevulinic Acid,ALA)和血红素合成的影响。【方法】 通过Red同源重组敲除与谷氨酸代谢有关的mscSaroG,构建hemA表达载体并导入基因缺失菌株中。【结果】 mscS单敲除或mscSaroG双敲除对菌体生长无显著影响。与出发菌株相比,单敲除与双敲除菌株的谷氨酸含量均有所增加,ALA含量略微下降,血红素含量分别增加了11.6%和35.7%。在双敲除菌株中进一步过表达hemA后,胞内血红素含量增至47.603 μmol/L。【结论】 通过调控谷氨酸代谢流量与过表达hemA可促进血红素的合成,该结果为增强C5途径的血红素合成提供了新的思路。
关键词: 大肠杆菌    谷氨酰-tRNA还原酶    Red同源重组    谷氨酸    血红素    
Enhancing heme synthesis in Escherichia coli by regulating glutamic acid metabolism and over-expressing hemA
PAN Mei1 , LIU Shuangxin2 , TANG Lei1,2     
1. Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, Jiangsu 214122, China;
2. School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, China
Abstract: [Background] Escherichia coli uses glutamic acid as a precursor to synthesize limited heme through the C5 pathway. [Objective] The purpose of this study was to investigate the effects of the regulation of intracellular glutamic acid metabolism and over-expression of glutamyl-tRNA reductase gene (hemA) on 5-aminolevulinic acid (ALA) and heme synthesis. [Methods] Through Red homologous recombination, mscS and aroG which are related to glutamate metabolism were knocked out, and the hemA expression vector was constructed and introduced into the gene-knockout strains. [Results] The single and double gene-knockout had no significant effect on cell growth, but the content of glutamic acid increased, whereas that of ALA decreased slightly. The heme content in single and double gene-knockout strains increased by 11.6% and 35.7% compared with that in the parental strain respectively. [Conclusion] The over-expression of hemA in the double gene-knockout strain caused the increase of heme to 47.603 μmol/L. Heme synthesis can be promoted by regulation of intracellular glutamate metabolic flow and over-expression of hemA which provides a new approach for heme enhancement through C5 pathway.
Keywords: Escherichia coli    glutamyl-tRNA reductase    Red homologous recombination    glutamic acid    heme    

血红素是一类重要的含铁卟啉类化合物,参与电子转移、活性氧分解、底物氧化、基因表达调控等生物体内生化反应[1]。在实际应用中,血红素及其卟啉衍生物可用于食品增色、贫血病和卟啉症的治疗[2-4]。此外,以血红素为辅基的蛋白,如染料脱色过氧化物酶(Dye-Decolorizing Peroxidase,DyP)可用于废弃染料的降解[5]。目前以酶法和从生物材料中提取血红素存在产量低、环境不友好等缺点。可以利用微生物发酵法生产血红素和重组血红素蛋白,但是由于血红素的胞内合成受到严格调控,工业化困难。因此,通过代谢工程改造提高微生物细胞的血红素合成能力至关重要。

大肠杆菌(Escherichia coli)作为重要的基因工程宿主菌,主要以谷氨酸为前体经C5途径合成血红素[6] (图 1),其中hemA编码的谷氨酰-tRNA还原酶被认为是C5途径的关键酶[7]。以往的研究报道多以提高5-氨基乙酰丙酸(5-Aminolevulinic Acid,ALA)的胞内合成量以及增强ALA下游代谢流的方式提升胞内血红素的合成[8-10]。由于ALA合成的增强将拉动上游代谢,因此可通过上游的代谢工程改造,使胞内的碳代谢流更多地流向前体谷氨酸,结合过表达hemA,以提高血红素的含量。

图 1 大肠杆菌血红素合成的C5途径 Figure 1 The C5 pathway of heme synthesis in E. coli 注:DAHP:3-脱氧-D-阿拉伯庚酮糖-7磷酸;aroG:DAHP合成酶;gdhA:谷氨酸脱氢酶;mscS:机械敏感通道蛋白;gltX:谷氨酰-tRNA合成酶;hemA:谷氨酰-tRNA还原酶;hemL:ALA合成酶 Note: DAHP: 3-Deoxy-D-Arobino-heptulosonate-7-Phosphate; aroG: DAHP synthase; gdhA: Glutamate dehydrogenase; mscS: Mechanosensitive channel of small conductance protein; gltX: Glutamyl-tRNA synthetase; hemA: Glutamyl-tRNA reductase; hemL: ALA synthase

在调控谷氨酸代谢流方面,3-脱氧-D-阿拉伯庚酮糖-7-磷酸(3-Deoxy-D-Arobino-Heptulosonate- 7-Phosphate,DAHP)合成酶(aroG)作为DAHP合成酶的主要同工酶,与磷酸烯醇式丙酮酸羧化酶竞争底物磷酸烯醇式丙酮酸(Phosphoenolpyruvate,PEP)[11],催化芳香族氨基酸前体DAHP的合成,从而分流了PEP向草酰乙酸的代谢通量,进而影响α-酮戊二酸(α-Ketoglutarate,α-KG)和谷氨酸的生成[12]。小电导类机械敏感型离子通道(Mechanosensitive Channel of Small Conductance,MscS)在感知膜张力变化、保护细胞免受低渗透压的冲击中发挥作用[13],其在Ca2+调节、细胞分裂和氨基酸转运中具有生理功能[14]。最近有研究表明,在谷氨酸棒状杆菌中谷氨酸的转运是由MscCG (也称为NCgl1221)介导的。MscS类蛋白的N端结构域参与L-谷氨酸外泌[15],在敲除该基因后,胞内谷氨酸的积累增加[15-16]。大肠杆菌的MscS蛋白与谷氨酸棒状杆菌的MscCG同源,且N端结构域相似[16-17]。综上,本研究通过敲除aroGmscS,降低糖代谢中心途径中PEP向芳香族氨基酸的代谢流[11-12, 18-19]、阻塞机械敏感型离子通道,减少谷氨酸的外泌[15-17],提高因过表达hemA后可能导致的谷氨酸胞内供给不足。本文报道通过调控谷氨酸代谢及hemA过表达促进大肠杆菌血红素合成的研究结果。

1 材料与方法 1.1 材料

1.1.1 质粒、菌株与引物

实验所用的菌株和质粒见表 1,所用引物(表 2)由天霖生物科技有限公司(上海)合成。

表 1 菌株与质粒 Table 1 Strains and plasmids
菌株/质粒
Strains/Plasmids
特性
Features
来源
Sources
Strains
  E. coli BL21(DE3) Expression strain Our laboratory
  WTΔmscS E. coli BL21(DE3) deleted mscS This study
  WTΔmscSΔaroG E. coli BL21(DE3) deleted mscS and aroG This study
  WT/pET E. coli BL21(DE3) transformed with plasmid pET-28a Our laboratory
  WT/pEA E. coli BL21(DE3) transformed with plasmid pEA Our laboratory
  WTΔmscS/pEA WTΔmscS transformed with plasmid pEA This study
  WTΔmscSΔaroG/pEA WTΔmscSΔaroG transformed with plasmid pEA This study
Plasmids
  pET-28a, pET-22b Expression plasmids Our laboratory
  pKD46, pKD4, pCP20 Red homologous recombination deletion plasmids Our laboratory
  pBUKD Plasmid containing UmscS, KanM and DmscS This study
  pEA pET-28a inserted with hemA Our laboratory

表 2 本研究中所用引物 Table 2 Sequence of primers used in this study
引物名称
Primers name
引物序列
Primers sequence (5′→3′)
UmscSF ggataacaattccccTCTAGAGCGAAGATCAGCCGAACA (Xba I)
UmscSR acGTGCCGCCACGATGTTTA
DmscSF tcatatggaccTAAGCGGGTGAAAGAAGACA
DmscSR ttgtcgacggagctcGAATTCAGGCTGCCCAGTACAACAAA (EcoR I)
KmscSF taaacatcgtggcggcacGTGTAGGCTGGAGCTGCTTC
KmscSR acccgcttaGGTCCATATGAATATCCTCCTTAG
rmscSF GCGAAGATCAGCCGAACA
rmscSR AGGCTGCCCAGTACAACAAA
QmscSF GAACGAAGCTTATCTGCAGG
QmscSR GGTTCCACATCAAGTTGCC
UaroGF ATGAATTATCAGAACGACGATTTACGCATCAAAGAA ATCAAAGAGTTACTGTGTAGGCTGGAGCTGCTTC
DaroGR TTACCCGCGACGCGCTTTTACTGCATTCGCCAGTTGA CGTAACAGAGCATCATGGGAATTAGCCATGGTC
QaroGF CCCGTTTACACATTCTGACG
QaroGR TGATTCATCGGATACGCCAC
注:下划线为酶切位点
Note: The sequences of restriction sites are underlined

1.1.2 主要试剂和仪器

限制性内切酶、基因组提取、片段纯化和质粒提取试剂盒,TaKaRa公司;ClonExpress® MultiS One Step Cloning Kit,南京诺唯赞生物科技有限公司;抗生素(Kan、Amp和Cmr)、L-阿拉伯糖,生工生物工程(上海)股份有限公司;其他试剂均为国产或进口分析纯。

电转仪、凝胶成像仪,Bio-Rad公司;安捷伦高效液相色谱仪,Sykam公司;EnSpire多标记检测系统(酶标仪),PerkinElmer公司。

1.1.3 培养基

LB培养基(g/L):NaCl 10.0,胰蛋白胨10.0,酵母提取物5.0,琼脂粉20 (固体培养基加入)。pH 7.0,1×105 Pa灭菌20 min。需要添加抗生素的菌株,Kan、Amp和Cmr添加的终浓度分别为100、50和15 mg/L。

1.2 方法

1.2.1 基因敲除菌株的构建

基因打靶片段扩增方法参考文献[20]。基因敲除方法参考文献[21]。mscS敲除菌命名为WTΔmscSmscSaroG双缺失菌命名为WTΔmscSΔaroG

1.2.2 生长曲线测定

将各菌株于37 ℃、200 r/min活化过夜,次日以1%的比例转接于50 mL的LB培养基中。每间隔2 h取样,测定菌液在600 nm下的吸光度值,共测定12 h。

1.2.3 谷氨酸含量测定

使用氨基酸专用高效液相色谱仪测定谷氨酸含量,具体操作如下:活化菌体过夜,次日转接培养7 h。取培养后的菌体于4 ℃、8 000 r/min离心15 min,磷酸缓冲液洗涤菌体后重悬菌体,在低温条件下对细胞进行超声破碎,于4 ℃、10 000 r/min离心30 min,取4 mL上清液,加入4 mL浓盐酸,充氮气,放入120 ℃烘箱水解22 h。将水解样品加入10 mol/L NaOH中和,定容至25 mL。双层滤纸过滤,取1 mL澄清液于室温下12 000 r/min离心30 min。取400 μL上清液置于液相瓶中进行液相分析检测。

1.2.4 血红素及5-氨基乙酰丙酸浓度测定

参照文献[10]测定血红素浓度,参照文献[22]测定ALA含量。采用GraphPad Prism软件分析数据结果。

2 结果与分析 2.1 构建mscS基因缺失菌

以pBUKD质粒为模板、rmscSF和rmscSR为引物扩增出2 639 bp的打靶片段,导入含pKD46质粒的E. coli BL21(DE3)制备的电转感受态细胞中(图 2A),以QmscSF/QmscSR为鉴定引物对阳性单菌落进行PCR扩增验证。理论上,经PCR扩增后,同源重组成功的菌株应得到长度为2 704 bp的片段,而对照菌WT扩增后的片段长度应为1 950 bp。电泳结果显示:阳性菌PCR扩增后的条带大小与理论计算值相一致(图 2B),表明已成功进行同源重组。将pCP20质粒转入重组菌,通过QmscSF/QmscSR引物对阳性转化菌进行PCR扩增验证,理论上mscS缺失后PCR扩增可获得长度为1 311 bp的扩增片段,而对照菌为1 950 bp。电泳结果显示:菌落PCR扩增后的条带大小与理论计算值相一致(图 2C)。将PCR产物进行测序分析,得到的序列结果正确,表明mscS已成功缺失,将缺失菌株命名为WTΔmscS

图 2 mscS敲除电泳验证 Figure 2 Electrophoretic verification of mscS knockout 注:M:DL10000 DNA Marker。A:扩增打靶片段;1:打靶片段;B:同源重组验证;1-3:重组成功菌;4:WT对照;C:抗性消除验证;1:mscS缺失菌;2:WT对照 Note: M: DL10000 DNA Marker. A: Amplification of target fragment; 1: Target fragment; B: Verification of homologous recombination; 1-3: Strain with homologous recombinant; 4: WT control. C: Verification after elimination of resistance; 1: Strain with mscS deletion; 2: WT control
2.2 构建mscS/aroG双敲除菌

以pKD4质粒为模板、UaroGF/DaroGR为引物扩增出1 607 bp的打靶片段导入WTΔmscS中(图 3A),以QaroGF/QaroGR为鉴定引物对阳性单菌落进行扩增验证。理论上,经PCR扩增后,同源重组成功的菌株应得到长度为1 724 bp的扩增片段,而对照菌(WT)扩增后的片段长度应为1 180 bp。电泳结果显示:挑取的阳性菌落扩增后条带大小与理论值一致(图 3B),表明已成功进行同源重组。将pCP20质粒转入重组菌,通过QaroGF/QaroGR引物对阳性转化菌进行PCR扩增验证。理论上aroG缺失后可获得长度为331 bp的PCR扩增片段,而对照菌为1 180 bp。电泳结果显示:扩增得到的条带大小与理论值相一致(图 3C);对PCR产物进行测序分析,所得序列结果正确,表明aroG也已成功缺失,将缺失菌株命名为WTΔmscSΔaroG

图 3 mscS/aroG敲除电泳验证 Figure 3 Electrophoretic verification of mscS/aroG knockout 注:M:DL2000 DNA Marker。A:扩增打靶片段;1:打靶片段;B:同源重组验证;1:WT对照;2-4:重组成功菌;C:抗性消除验证;1:WT;2-3:mscS/aroG缺失菌 Note: M: DL2000 DNA Marker. A: Amplification of target fragment; 1: Target fragment; B: Verification of homologous recombination; 1: WT control; 2-4: Strains with homologous recombinant; C: Verification after elimination of resistance; 1: WT control; 2-3: Strains with mscS/aroG deletion
2.3 基因缺失对于大肠杆菌生长状态的影响

在LB培养基中培养对照菌(WT)和敲除菌株,测定不同培养时间下的菌体浓度绘制生长曲线(图 4)。结果表明,在不同的培养时间内,WTΔmscS与WT的菌体浓度几乎一致,无显著差异;在2-10 h内,WTΔmscSΔaroG的菌体浓度略低于WT;在10-12 h内又恢复到与WT一致的水平。

图 4 各菌株生长曲线 Figure 4 Growth curve of each strain

以上结果表明敲除mscSaroG对于大肠杆菌的生长没有显著影响,分析原因可能是由于:(1) 作为氨基酸转运的机械敏感型离子通道,MscS的缺失只会降低氨基酸的外泌,不会导致菌体生长所需氨基酸的缺乏,因此不会对细胞生长产生不利影响;(2) aroG仅是3个DAHP合成酶(aroGaroHaroF)之一[18],即使aroG缺失,aroFaroH仍然可以催化芳香族氨基酸的合成,满足细胞生长的需求。

2.4 基因缺失对于大肠杆菌谷氨酸积累的影响

对WT、WTΔmscS和WTΔmscSΔaroG的谷氨酸含量进行分析的结果表明,WTΔmscS和WTΔmscSΔaroG的谷氨酸含量分别为0.248 g/g-DCW和0.260 g/g-DCW,与WT相比,分别提升了2.0%和7.0%。mscSaroG的缺失并未造成胞内谷氨酸的大量积累,增加的谷氨酸是否用于血红素的合成需要进一步的分析。

2.5 基因缺失对于大肠杆菌ALA积累和血红素合成的影响

对血红素及ALA含量的分析表明,WTΔmscS与WTΔmscSΔaroG的胞内血红素含量分别为2.357 μmol/L与2.866 μmol/L,与WT相比,分别增加了11.6%和35.7% (图 5A);胞外ALA含量分别为8.822 mg/L和8.354 mg/L,与WT相比,分别下降了3.5%和8.6% (图 5B),推测ALA进一步转化成为了血红素。

图 5 各菌株的血红素(A)与ALA (B)含量比较 Figure 5 Contents of heme (A) and ALA (B) in each strain Note: *: P≤0.05; **: P < 0.01
2.6 过表达hemA对缺失菌ALA积累和血红素合成的影响

C5途径中hemA是关键基因,编码谷氨酰-tRNA还原酶,该酶参与ALA合成,是血红素合成途径中的限速步骤[23-24]。将hemA导入缺失菌中的结果表明,WT/pEA的血红素含量为34.117 μmol/L,WTΔmscS/pEA和WTΔmscSΔaroG/pEA的血红素含量比WT/pEA的血红素含量增加了34.4%和39.5%;其中,WTΔmscSΔaroG/pEA的血红素含量达到47.603 μmol/L,约为未过表达菌株WT的22倍(图 6A)。WT/pEA的ALA含量为10.827 mg/L,较未过表达菌株有较大提升,有利于血红素的合成(图 6B)。因此,在缺失菌株中过表达hemA可显著增强血红素的合成。

图 6 各菌株血红素(A)与ALA (B)含量 Figure 6 Contents of heme (A) and ALA (B) in each strain Note: *: P≤0.05
3 讨论与结论

血红素应用广泛,是大多数原核和真核生物细胞呼吸的重要辅助因子,也是优质的铁源[25],以血红素为辅基的蛋白对各种生物学过程至关重要。在以血红素为辅基的重组酶合成中,血红素不足是导致酶活性不高的重要因素[5, 26]。最新研究表明,血红素辅因子的充足供应对于可溶性和功能性血红蛋白的生产至关重要[26-27],为了提高血红蛋白的溶解度和稳定性,目前主要是通过添加血红素及其前体ALA,从而使新合成的蛋白质和血红素有效结合[1],因此提高血红素的含量有利于提高重组酶活性。在以往关于E. coli血红素合成代谢途径的研究中,侧重于ALA合成及之后的下游相关基因之间的调控和相互作用对血红素合成的影响。例如,Kwon等[28]将血红素合成途径中的hemAhemB/C/DhemEhemH共表达,在LB培养基中培养细胞获得3.300 μmol/L胞内血红素;Pranawidjaja等[29]通过共表达异源hemA和编码内源泛酸激酶coaA,并在培养基中添加琥珀酸、甘氨酸和FeCl3,胞内血红素含量达到9.100 μmol/g-细胞;翁焕娇[30]通过对血红素合成途径的模块化组装优化和补料分批发酵,胞内血红素产量达到1.399 mg/L。

与上述报道不同,本文主要关注了过表达内源hemA后上游前体物的供给问题,通过芳香氨基酸合成基因的缺失和谷氨酸外泌的阻塞,调控谷氨酸代谢流。结果表明,在基因缺失菌株中过表达hemA,血红素含量增至47.603 μmol/L,约为出发菌株的22倍。

本研究仅过表达了hemA,虽然过程简单且血红素量含量有明显提升,但未能结合ALA下游基因的表达调控。今后可根据已有的文献报道[10, 30]进行ALA下游基因的共表达或模块化设计,进一步拉动C5代谢流流向血红素。

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