Abstract

ABSTRACTBiological nitrogen fixation is catalyzed by nitrogenase, a complex metalloenzyme found only in prokaryotes. N2 fixation is energetically highly expensive, and an energy-generating process such as photosynthesis can meet the energy demand of N2 fixation. However, synthesis and expression of nitrogenase are exquisitely sensitive to the presence of oxygen. Thus, engineering nitrogen fixation activity in photosynthetic organisms that produce oxygen is challenging. Cyanobacteria are oxygenic photosynthetic prokaryotes, and some of them also fix N2. Here, we demonstrate a feasible way to engineer nitrogenase activity in the nondiazotrophic cyanobacterium Synechocystis sp. PCC 6803 through the transfer of 35 nitrogen fixation (nif) genes from the diazotrophic cyanobacterium Cyanothece sp. ATCC 51142. In addition, we have identified the minimal nif cluster required for such activity in Synechocystis 6803. Moreover, nitrogenase activity was significantly improved by increasing the expression levels of nif genes. Importantly, the O2 tolerance of nitrogenase was enhanced by introduction of uptake hydrogenase genes, showing this to be a functional way to improve nitrogenase enzyme activity under micro-oxic conditions. To date, our efforts have resulted in engineered Synechocystis 6803 strains that, remarkably, have more than 30% of the N2 fixation activity of Cyanothece 51142, the highest such activity established in any nondiazotrophic oxygenic photosynthetic organism. This report establishes a baseline for the ultimate goal of engineering nitrogen fixation ability in crop plants.

Highlights

  • Biological nitrogen fixation is catalyzed by nitrogenase, a complex metalloenzyme found only in prokaryotes

  • Synechocystis 6803, the nondiazotrophic cyanobacterium utilized in this study, provides a model chassis for rapid investigation of the necessary requirements to establish diazotrophy in an oxygenic phototroph

  • Containing the entire nif cluster with 35 genes (Fig. 1D), was constructed using the DNA assembler method [32]. The chassis of this vector was based on pRSF1010 [33], and this self-replicating pSyNif-1 plasmid was transferred into Synechocystis 6803 by conjugation, generating the engineered strain TSyNif-1

Read more

Summary

Introduction

Biological nitrogen fixation is catalyzed by nitrogenase, a complex metalloenzyme found only in prokaryotes. The heterotrophic bacterium Escherichia coli has been successfully engineered for nitrogen fixation activity through transfer of nif genes from various diazotrophic species [13,14,15,16,17]. We successfully transferred and expressed this large nif gene cluster in nondiazotrophic Synechocystis 6803 with resultant N2 fixation activity.

Results
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call