title: Using synthetic biology to increase nitrogenase activity.
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.
  
  
  RESULTS
  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. oxytoca nifSU (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. oxytoca nifSU recovers 50.1 % of wild-type (Paenibacillus) activity. However, K. oxytoca nifWZM and nifQ can not increase activity.
  
  
  CONCLUSIONS
  The combined assembly of the potential Paenibacillus electron transporter genes (pfoABfldA) with K. oxytoca nifSU 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.
authors.affiliation: Key Laboratory for Agrobiotechnology and Key Laboratory of Soil Microbiology of Agriculture Ministry, China Agricultural University, Yuanmingyuan West Road No. 2, Haidian District, Beijing, 100193, People's Republic of China. lixinxin-2002@163.com.
authors.forename: Xin-Xin
authors.initials: XX
authors.lastname: Li
authors.affiliation: Key Laboratory for Agrobiotechnology and Key Laboratory of Soil Microbiology of Agriculture Ministry, China Agricultural University, Yuanmingyuan West Road No. 2, Haidian District, Beijing, 100193, People's Republic of China. 897043466@qq.com.
authors.forename: Qi
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Key Laboratory for Agrobiotechnology and Key Laboratory of Soil Microbiology of Agriculture Ministry, China Agricultural University, Yuanmingyuan West Road No. 2, Haidian District, Beijing, 100193, People's Republic of China. wsw_lxm2012@163.com.
authors.forename: Xiao-Meng
authors.initials: XM
authors.lastname: Liu
authors.affiliation: Key Laboratory for Agrobiotechnology and Key Laboratory of Soil Microbiology of Agriculture Ministry, China Agricultural University, Yuanmingyuan West Road No. 2, Haidian District, Beijing, 100193, People's Republic of China. shw_126@126.com.
authors.forename: Hao-Wen
authors.initials: HW
authors.lastname: Shi
authors.affiliation: Key Laboratory for Agrobiotechnology and Key Laboratory of Soil Microbiology of Agriculture Ministry, China Agricultural University, Yuanmingyuan West Road No. 2, Haidian District, Beijing, 100193, People's Republic of China. chensf@cau.edu.cn.
authors.forename: San-Feng
authors.initials: SF
authors.lastname: Chen
mesh_terms: D004579:Electron Transport; D004926:Escherichia coli / Q000378:metabolism; D005798:Genes, Bacterial; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D007709:Klebsiella / Q000201:enzymology / Q000235:genetics; D005810:Multigene Family; D009591:Nitrogenase / Q000378:metabolism*; D056507:Paenibacillus / Q000201:enzymology / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics; D058615:Synthetic Biology / Q000379:methods*
chemical_list: D007506:Iron-Sulfur Proteins; D009591:Nitrogenase