Abstract
Background 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. ResultsIn 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.ConclusionThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0442-6) contains supplementary material, which is available to authorized users.
Highlights
Nitrogen fixation has been established in protokaryotic model Escherichia coli by transferring a minimal nif gene cluster composed of 9 genes from Paenibacillus sp
Our results demonstrate that Fe–S cluster assembly system and electron transport system from Paenibacillus or K. oxytoca can increase E. coli nitrogenase activity mediated by the minimal nif gene cluster composed of 9 genes
WLY78 could enable E. coli to fix nitrogen, suggesting that the assembly of Fe–S clusters for the nitrogenase was provided by E. coli iron-sulfur cluster assembly systems
Summary
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. The Fe protein is a γ2 homodimer bridged by the biochemical properties and structure of molybdenum nitrogenases are remarkably similar when purified from diverse bacteria and archaea, the organization and numbers of nif genes required for the synthesis and assembly of the enzyme varies greatly among these nitrogen-fixing species [6,7,8]. WLY78 possesses a minimal and compact nif gene cluster consisting of 9 genes (nifBnifHnifDnifKnifEnifNnifXhesAnifV) (Fig. 1) [9, 10]. This variability in nif genes content is undoubtedly determined by the environmental lifestyle of each diazotroph on one hand, the minimal nif gene sets are probably
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