Using synthetic biology to increase nitrogenase activity.
Using synthetic biology to increase nitrogenase activity.
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利用合成生物学提高固氮酶活性。
DOI：
10.1186/s12934-016-0442-6
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发表时间：
2016-02-20
影响因子：
6.4
通讯作者：
Chen SF
中科院分区：
文献类型：
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作者：
Li XX;Liu Q;Liu XM;Shi HW;Chen SF
将类芽孢杆菌WLY 78的nifB、nifH、nifD、nifK、nifE、nifN、nifX、hesA和nifV 9个基因组成的最小nif基因簇转入大肠杆菌，建立了固氮的原生态模型。而重组E.大肠杆菌78-7中的表达量仅为野生型类芽孢杆菌中观察到的表达量的10%。因此，有必要通过合成生物学来提高固氮酶的活性。 为了提高固氮酶在异源宿主中的活性，将来自类芽孢杆菌（Paenibacillussp.WLY78）和产酸克雷伯氏菌（Klebsiellaoxytoca）的共28个基因分别置于两种不同载体的类芽孢杆菌（Paenibacillusnif）启动子控制下，然后将它们分别或组合转移到重组E. coli78 -7。我们的研究结果表明，类芽孢杆菌suf操纵子（Fe-S簇组装）和潜在的电子传递基因pfoAB，fldA和fer可以提高固氮酶活性。所以，K。oxytocanifSU（Fe-S簇组装）和nifFJ（固氮酶特异性电子传递）可以提高固氮酶活性。特别是将潜在的类芽孢杆菌电子转运蛋白基因（pfoABfldA）与K. oxytocanifSU恢复了野生型（类芽孢杆菌属）活性的50.1%。然而，K. oxytocanifWZM和nifQ不能增加活性。将潜在的类芽孢杆菌电子转运蛋白基因（pfoABfldA）与K. oxytocanifSU在重组E. coli78 -7。本研究结果将为提高异源宿主固氮酶活性提供有价值的见解，并将为用最小nif基因工程改造禾谷类植物提供指导。本文的在线版本（doi：10.1186/s12934-016-0442-6）包含补充材料，可供授权用户使用。
Nitrogen fixation has been established in protokaryotic model Escherichia coli by transferring a minimal nif gene cluster composed of 9 genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV) from Paenibacillus sp. WLY78. However, the nitrogenase activity in the recombinant E. coli 78-7 is only 10 % of that observed in wild-type Paenibacillus. Thus, it is necessary to increase nitrogenase activity through synthetic biology. In order to increase nitrogenase activity in heterologous host, a total of 28 selected genes from Paenibacillus sp. WLY78 and Klebsiella oxytoca were placed under the control of Paenibacillus nif promoter in two different vectors and then they are separately or combinationally transferred to the recombinant E. coli 78-7. Our results demonstrate that Paenibacillus suf operon (Fe–S cluster assembly) and the potential electron transport genes pfoAB, fldA and fer can increase nitrogenase activity. Also, K. oxytocanifSU (Fe–S cluster assembly) and nifFJ (electron transport specific for nitrogenase) can increase nitrogenase activity. Especially, the combined assembly of the potential Paenibacillus electron transporter genes (pfoABfldA) with K. oxytocanifSU recovers 50.1 % of wild-type (Paenibacillus) activity. However, K. oxytocanifWZM and nifQ can not increase activity. The combined assembly of the potential Paenibacillus electron transporter genes (pfoABfldA) with K. oxytocanifSU recovers 50.1 % of wild-type (Paenibacillus) activity in the recombinant E. coli 78-7. Our results will provide valuable insights for the enhancement of nitrogenase activity in heterogeneous host and will provide guidance for engineering cereal plants with minimal nif genes. The online version of this article (doi:10.1186/s12934-016-0442-6) contains supplementary material, which is available to authorized users.