pBluescript II SK(+) vector

Nitrogen fixation was established in the prokaryotic model Escherichia coli by transferring a minimal nif gene cluster consisting of 9 genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV) from Paenibacillus sp. WLY78. However, the nitrogenase activity in recombinant E. coli 78-7 was only 10% of that observed in wild-type Paenibacillus. To enhance heterologous host nitrogenase activity, a total of 28 genes were selected from  Paenibacillus sp. WLY78 and Klebsiella oxytoca were placed under the control of the Paenibacillus nif promoter in two different vectors and then transferred into recombinant E. coli 78-7 individually or in combination. Results indicate that the Paenibacillus suf operon (Fe-S cluster assembly) and the potential electron transport genes pfoAB, fldA, and fer can enhance nitrogenase activity. Furthermore, K. oxytoca nifSU (Fe-S cluster assembly) and nifFJ (nitrogenase-specific electron transport) can increase nitrogenase activity. In particular, combinatorial assembly of a latent Paenibacillus electron transporter gene (pfoABfldA) with K. oxytoc nifSU restored 50.1% of wild-type ( Paenibacillus ) activity. However, K. oxytoca nifWZM and nifQ were unable to increase activity.

Figure 1. Assembly and functional analysis of the K. oxytoca Fe-S cluster assembly system (nifUS) in E. coli 78-7. a Linear view of the Fe-S cluster assembly gene region in the pBluescript II SK (+) derived plasmid. b Relative nitrogenase activity of wild-type Paenibacillus sp. WLY78, E. coli 78-7 [pBluescriptII SK (+)] and E. coli 78-7 (nifUS). E. coli 78-7 [pBluescriptII SK (+)] was used as a control. (Li, Xin-Xin, et al. 2016)