Engineering Nitrogen Fixation Activity in an Oxygenic Phototroph

ABSTRACT Biological 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...

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Autores principales:	Deng Liu, Michelle Liberton, Jingjie Yu, Himadri B. Pakrasi, Maitrayee Bhattacharyya-Pakrasi
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	cyanobacteria N2 fixation O2 tolerance photosynthesis synechocystis Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/f90c9c99f4da4a02a7eb9f38382e4297
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spelling	oai:doaj.org-article:f90c9c99f4da4a02a7eb9f38382e42972021-11-15T16:00:25ZEngineering Nitrogen Fixation Activity in an Oxygenic Phototroph10.1128/mBio.01029-182150-7511https://doaj.org/article/f90c9c99f4da4a02a7eb9f38382e42972018-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01029-18https://doaj.org/toc/2150-7511ABSTRACT Biological 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. 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 N2 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.Deng LiuMichelle LibertonJingjie YuHimadri B. PakrasiMaitrayee Bhattacharyya-PakrasiAmerican Society for MicrobiologyarticlecyanobacteriaN2 fixationO2 tolerancephotosynthesissynechocystisMicrobiologyQR1-502ENmBio, Vol 9, Iss 3 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	cyanobacteria N2 fixation O2 tolerance photosynthesis synechocystis Microbiology QR1-502
spellingShingle	cyanobacteria N2 fixation O2 tolerance photosynthesis synechocystis Microbiology QR1-502 Deng Liu Michelle Liberton Jingjie Yu Himadri B. Pakrasi Maitrayee Bhattacharyya-Pakrasi Engineering Nitrogen Fixation Activity in an Oxygenic Phototroph
description	ABSTRACT Biological 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. 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 N2 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.
format	article
author	Deng Liu Michelle Liberton Jingjie Yu Himadri B. Pakrasi Maitrayee Bhattacharyya-Pakrasi
author_facet	Deng Liu Michelle Liberton Jingjie Yu Himadri B. Pakrasi Maitrayee Bhattacharyya-Pakrasi
author_sort	Deng Liu
title	Engineering Nitrogen Fixation Activity in an Oxygenic Phototroph
title_short	Engineering Nitrogen Fixation Activity in an Oxygenic Phototroph
title_full	Engineering Nitrogen Fixation Activity in an Oxygenic Phototroph
title_fullStr	Engineering Nitrogen Fixation Activity in an Oxygenic Phototroph
title_full_unstemmed	Engineering Nitrogen Fixation Activity in an Oxygenic Phototroph
title_sort	engineering nitrogen fixation activity in an oxygenic phototroph
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/f90c9c99f4da4a02a7eb9f38382e4297
work_keys_str_mv	AT dengliu engineeringnitrogenfixationactivityinanoxygenicphototroph AT michelleliberton engineeringnitrogenfixationactivityinanoxygenicphototroph AT jingjieyu engineeringnitrogenfixationactivityinanoxygenicphototroph AT himadribpakrasi engineeringnitrogenfixationactivityinanoxygenicphototroph AT maitrayeebhattacharyyapakrasi engineeringnitrogenfixationactivityinanoxygenicphototroph
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Engineering Nitrogen Fixation Activity in an Oxygenic Phototroph

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