A Minimal Nitrogen Fixation Gene Cluster from Paenibacillus sp WLY78 Enables Expression of Active Nitrogenase in Escherichia coli (Retracted article. See vol. 11, 2016)
A Minimal Nitrogen Fixation Gene Cluster from Paenibacillus sp WLY78 Enables Expression of Active Nitrogenase in Escherichia coli (Retracted article. See vol. 11, 2016)
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DOI：
10.1371/journal.pgen.1003865
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发表时间：
2013-10-01
期刊：
影响因子：
4.5
通讯作者：
Li, Jilun
中科院分区：
文献类型：
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作者：
Wang, Liying;Zhang, Lihong;Li, Jilun
大多数生物固氮是由钼依赖性固氮酶催化的，这是一种由两种蛋白质组成的酶复合物，其中含有三种不同的金属簇。固氮生物含有固氮 nif 基因的共同核心，该基因编码酶的结构亚基和合成金属簇所需的成分。然而，固氮细菌和古细菌之间固氮生长所需的 nif 基因的补充存在显着差异。在这项研究中，我们在类芽孢杆菌基因组中鉴定了一个由九个 nif 基因组成的最小 nif 基因簇。 WLY78，一种从竹子根际分离的革兰氏阳性兼性厌氧菌。我们证明该生物体中的 nif 基因被组织为一个操纵子，包括 nifB、nifH、nifD、nifK、nifE、nifN、nifX、hesA 和 nifV，并且 nif 簇受位于 nifB 上游的 sigma(70) (sigma(A)) 依赖性启动子的控制。为了研究固氮营养的遗传要求，我们将类芽孢杆菌 nif 簇转移到大肠杆菌中。当从天然 nifB 启动子或 T7 启动子表达时，最小的 nif 基因簇能够在该宿主中合成具有催化活性的固氮酶。缺失分析表明，除了核心 nif 基因外，hesA 在固氮中也发挥着重要作用，并且对钼的可用性有反应。类芽孢杆菌中的 nif 转录是根据氮可用性和外部氧浓度来调节的，而来自 nifB 启动子的转录在大肠杆菌中是组成型的，表明 nif 转录的负调节在异源宿主中被绕过。这项研究证明了在具有至少九个 nif 基因的非固氮生物体中进行固氮工程的潜力。
Most biological nitrogen fixation is catalyzed by molybdenum-dependent nitrogenase, an enzyme complex comprising two component proteins that contains three different metalloclusters. Diazotrophs contain a common core of nitrogen fixation nif genes that encode the structural subunits of the enzyme and components required to synthesize the metalloclusters. However, the complement of nif genes required to enable diazotrophic growth varies significantly amongst nitrogen fixing bacteria and archaea. In this study, we identified a minimal nif gene cluster consisting of nine nif genes in the genome of Paenibacillus sp. WLY78, a gram-positive, facultative anaerobe isolated from the rhizosphere of bamboo. We demonstrate that the nif genes in this organism are organized as an operon comprising nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV and that the nif cluster is under the control of a sigma(70) (sigma(A))-dependent promoter located upstream of nifB. To investigate genetic requirements for diazotrophy, we transferred the Paenibacillus nif cluster to Escherichia coli. The minimal nif gene cluster enables synthesis of catalytically active nitrogenase in this host, when expressed either from the native nifB promoter or from the T7 promoter. Deletion analysis indicates that in addition to the core nif genes, hesA plays an important role in nitrogen fixation and is responsive to the availability of molybdenum. Whereas nif transcription in Paenibacillus is regulated in response to nitrogen availability and by the external oxygen concentration, transcription from the nifB promoter is constitutive in E. coli, indicating that negative regulation of nif transcription is bypassed in the heterologous host. This study demonstrates the potential for engineering nitrogen fixation in a non-nitrogen fixing organism with a minimum set of nine nif genes.