TY - JOUR
T1 - Engineering nitrogen fixation activity in an oxygenic phototroph
AU - Liu, Deng
AU - Liberton, Michelle
AU - Yu, Jingjie
AU - Pakrasi, Himadri B.
AU - Bhattacharyya-Pakrasi, Maitrayee
N1 - Funding Information:
We thank Huimin Zhao and his research group (University of Illinois) for introducing us to the use of the DNA Assembler method; Lingxia Zhao, Xiujun Duan, and Alicia Lohman for expert technical assistance; and members of the research groups of H.B.P., Costas Maranas, Tae Seok Moon, and Fuzhong Zhang for critical scientific discussions. D.L., M.L., J.Y., H.B.P., and M.B.-P. designed the experiments; D.L., M.L., J.Y., and M.B.-P. performed the experiments; D.L., M.L., H.B.P., and M.B.-P. wrote the paper. This study was supported by the National Science Foundation (MCB-1331194).
Publisher Copyright:
© 2018 Liu et al.
PY - 2018/5/1
Y1 - 2018/5/1
N2 - Biological nitrogen fixation is catalyzed by nitrogenase, a complex metallo-enzyme found only in prokaryotes. N 2 fixation is energetically highly expensive, and an energy-generating process such as photosynthesis can meet the energy demand of N 2 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 N 2 . 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 O 2 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 N 2 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. IMPORTANCE Application of chemically synthesized nitrogen fertilizers has revolutionized agriculture. However, the energetic costs of such production processes and the widespread application of fertilizers have raised serious environmental issues. A sustainable alternative is to endow to crop plants the ability to fix atmospheric N 2 in situ. One long-term approach is to transfer all nif genes from a prokaryote to plant cells and to express nitrogenase in an energy-producing organelle, chloroplast, or mitochondrion. In this context, 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.
AB - Biological nitrogen fixation is catalyzed by nitrogenase, a complex metallo-enzyme found only in prokaryotes. N 2 fixation is energetically highly expensive, and an energy-generating process such as photosynthesis can meet the energy demand of N 2 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 N 2 . 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 O 2 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 N 2 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. IMPORTANCE Application of chemically synthesized nitrogen fertilizers has revolutionized agriculture. However, the energetic costs of such production processes and the widespread application of fertilizers have raised serious environmental issues. A sustainable alternative is to endow to crop plants the ability to fix atmospheric N 2 in situ. One long-term approach is to transfer all nif genes from a prokaryote to plant cells and to express nitrogenase in an energy-producing organelle, chloroplast, or mitochondrion. In this context, 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.
KW - Cyanobacteria
KW - N2 fixation
KW - O2 tolerance
KW - Photosynthesis
KW - Synechocystis
UR - https://www.scopus.com/pages/publications/85048285044
U2 - 10.1128/mBio.01029-18
DO - 10.1128/mBio.01029-18
M3 - Article
C2 - 29871920
AN - SCOPUS:85048285044
SN - 2161-2129
VL - 9
JO - mBio
JF - mBio
IS - 3
M1 - e01029-18
ER -