A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation

Abstract

Biological nitrogen fixation (BNF) is the reduction of N2 into NH3 in a group of prokaryotes by an extremely O2-sensitive protein complex called nitrogenase. Transfer of the BNF pathway directly into plants, rather than by association with microorganisms, could generate crops that are less dependent on synthetic nitrogen fertilizers and increase agricultural productivity and sustainability. In the laboratory, nitrogenase activity is commonly determined by measuring ethylene produced from the nitrogenase-dependent reduction of acetylene (ARA) using a gas chromatograph. The ARA is not well suited for analysis of large sample sets nor easily adapted to automated robotic determination of nitrogenase activities. Here, we show that a reduced sulfonated viologen derivative (S2Vred) assay can replace the ARA for simultaneous analysis of isolated nitrogenase proteins using a microplate reader. We used the S2Vred to screen a library of NifH nitrogenase components targeted to mitochondria in yeast. Two NifH proteins presented properties of great interest for engineering of nitrogen fixation in plants, namely NifM independency, to reduce the number of genes to be transferred to the eukaryotic host; and O2 resistance, to expand the half-life of NifH iron-sulfur cluster in a eukaryotic cell. This study established that NifH from Dehalococcoides ethenogenes did not require NifM for solubility, [Fe-S] cluster occupancy or functionality, and that NifH from Geobacter sulfurreducens was more resistant to O2 exposure than the other NifH proteins tested. It demonstrates that nitrogenase components with specific biochemical properties such as a wider range of O2 tolerance exist in Nature, and that their identification should be an area of focus for the engineering of nitrogen-fixing crops.

Functional expression of the nitrogenase Fe protein in transgenic rice

“…3c). This indicated that OsNifM Av expression was sufficient in all three rice lines, which was consistent with recent results obtained in S. cerevisiae 22 .…”

“…The nifH Ht gene was placed under a stronger promoter than nifM Av because the former encodes the most abundant Nif protein and it is part of the nitrogenase complex. Although there is no study proving that NifM is essential for NifH in plant mitochondria, NifM has been shown to be required for NifH solubility in heterologous expression systems including E. coli , yeast mitochondria, and plant chloroplasts 14 , 16 , 22 . Although we were unable to obtain clear western blot signals for Os NifM Av in the plant extracts, the fact that Os NifH Ht was soluble indicated that Os NifM Av was expressed at sufficient levels to facilitate NifH polypeptide maturation, as recently shown for NifH Ht in S. cerevisiae 22 .…”

“…Although there is no study proving that NifM is essential for NifH in plant mitochondria, NifM has been shown to be required for NifH solubility in heterologous expression systems including E. coli , yeast mitochondria, and plant chloroplasts 14 , 16 , 22 . Although we were unable to obtain clear western blot signals for Os NifM Av in the plant extracts, the fact that Os NifH Ht was soluble indicated that Os NifM Av was expressed at sufficient levels to facilitate NifH polypeptide maturation, as recently shown for NifH Ht in S. cerevisiae 22 . In callus, both Os NifH Ht and Os NifM Av gave rise to bands of the expected size (~33 kDa for both proteins) when analyzed by SDS-PAGE and western blot, indicating that the proteins were correctly processed and stable in rice mitochondria (Fig.…”

Genome-Wide Characterization of Nitrogenase Reductase (nifH) Genes in the Sweet Potato [Ipomoea batatas (L.) Lam] and Its Wild Ancestors

“…nifH is a marker gene, and researchers have been able to characterize aspects of the diversity and ecology of nitrogen-fixing bacteria and archaea. The biological nitrogen fixation of plants, rather than by association with microorganisms, can generate crops that are less dependent on synthetic nitrogen fertilizers and can increase agricultural productivity and sustainability [ 50 ]. The sweet potato, the seventh largest food crop in the world [ 2 ], grows well in nitrogen-poor infertile soils, and it is believed that sweet potato endo- and epiphytic microorganisms play important roles in acting upon this growth ability in this plant [ 21 , 22 , 23 , 24 ].…”

The Sea's best kept secret: the use of seaweed as a source of biohydrogen for clean and renewable energy

“…24 Recently, a new spectrophotometric method to measure nitrogenase activity has been developed. 25 Similarly, quantification of hydrogenase can be done spectrophotometrically via the reduction product of methyl viologen, which is oxidized by the hydrogenase enzyme. 26 These biological catalysts are responsible for the metabolic mechanism associated with the formation and production of biohydrogen, with biological engineering required to further optimize the interaction of the enzymes with the substrate, in this case, organic materials.…”