Analysis Of Genome Sequence And Nif Genes And Heterologous Expression Of Nitrogenase Genes From Nitrogen-fixing Paenibacillus

Paenibacillus is a genus of Gram-positive, endospore-forming bacterium. Nitrogen-fixing Paenibacillus species have the potential application in agriculture. So the study of Paenibacillus, including genomic information and resources has a great significance.In the genome of Paenibacillus sabinae T27,10 genes organized in a transcriptional unit are located within a 11 kb nif cluster. It is composed of niJB, niJH, nifD, nifK, nifE, nifN, nifX, hesA, orf1and nifV. In addition, it also contains three nifB genes, two nifH genes, one nifE gene, one nifN gene, two nifH-like genes and five pairs of nifDK-like genes. qRT-PCR analysis showed that, unlike nif genes, the transcription of nifHDK-like genes were not regulated by ammonium and oxygen, and nifH-like or nifD-like genes could not restore the nitrogenase activity of Klebsiella oxytoca nifH- and nifD- mutant strains, respectively, suggesting that nifHDK-like genes were not involved in nitrogen fixation.The nitrogenase activity in the recombinant Escherichia coli 78-7, carrying the nif cluster from Paenibacillus polymyxa WLY78, was only 10% of that observed in wild-type Paenibacillus. In order to increase the nitrogenase activity in the heterologous host,28 selected genes from Paenibacillus polymyxa WLY78 and Klebsiella oxytoca M5a1 were placed under control of Paenibacillus nif promoter in two different vectors, then they were separately or combinatorially transferred to E. coli 78-7. Results demonstrate that Paenibacillus sufCBSUD (Fe-S cluster assembly) and the potential electron transport genes pfoAB,fldA and fer could increase nitrogenase activity. Also, K. oxytoca nifSU (Fe-S cluster assembly) and nifFJ (electron transport specific for nitrogenase) could increase nitrogenase activity. Especially, combined assembly of the potential electron transporter genes (pfoABfldA) of Paenibacillus with K. oxytoca nifSU recovered 50.1% of wild-type(Paenibacillus) activity. However, K. oxytoca nifWZM and nifQ could not increase activity. pfoAB and fldA genes have been proposed to function in electron transfer to nitrogenase in P. polymyxa WLY78. In addition, we also did some pilot study of the heterogeneous expression of the nif cluster in the eukaryotic yeast Pichia pastoris GS115.