(MTP)-Nif fusion proteins (B, S, H, Y) were effectively co-expressed, thus
establishing the viability of reconstituting the complete complex of nitrogenase in
plant cells. Yang et al. (2017) reported that few of the plant electron transport chains
(ETCs) can be recruited to provide the reducing equivalents crucial for N 2 fixation.
They used E. coli as a chassis to find out about the compatibility of Mo and Fe-only
nitrogenases with ETC modules from a range of organelle of the target plant. They
replaced bacterial ETC module with genes encoding ferredoxin-NADPH
oxidoreductases (FNRs) and their cognate ferredoxin counterparts from plant
organelles and observed compatibility of FNR-ferredoxin module from chloroplasts
and root plastids with both types of nitrogenase. However, an analogous mitochondrial ETC module could not transfer electrons to nitrogenase. According to the
authors, this incompatibility of mitochondrial ETC could be overcome with hybrid
modules consisting of mitochondrial NADPH-dependent adrenodoxin oxidoreductase and the Anabaena ferredoxins FdxH or FdxB. Thus, Burén and Rubio (2018)
pointed out that while engineering nitrogenase it is significant to consider the
integration of nif gene components from different origins. Beside confirming expression and organelle targeting, it is also important to analyse whether Nif proteins
accrue in soluble forms within the eukaryotic cell.
Simplification of the N 2 fixation challenge is decisively significant because it
reduces the number of modules that have to be transferred into plant organelles.
Additionally, it will assist in finding lost or limiting steps in the course of enabling
plant cells with N 2 fixing ability. Ten proteins are mandatory to sustain N 2 fixation in
E. coli. Plant host can provide proteins necessary for coupling cellular metabolism to
N 2 fixation (NifJ and NifF) and proteins mandatory for organizing Fe and sulphur for
metal co-factor assembly (NifU and NifS). NifV, already produced by a few
eukaryotes, catalyzes the formation of homocitrate making the active site of nitrogenase accessible (extensively reviewed by Vicente and Dean 2017). Thus, expression
of the rest of the five microbial proteins is essential to assemble a first-generation
N 2 -fixing non-leguminous plant, for example, nifB, an S-adenosylmethionine–
dependent enzyme associated with nitrogenase catalytic site (Vicente and Dean
2017).
15.3.3 Transfer of N 2 Fixing Traits to Microorganisms Closely
Associated With Non-Leguminous Crops
To achieve host control of a microbe, cereals have to manage N 2 fixation by
epiphytic bacteria (growing on the root surface), endophytic bacteria (growing inside
roots) or by bacteria that inhabit engineered nodules (Mueller et al. 2012). One of the
approaches is to enhance the competitiveness of a particular microbe in the rhizosphere is to provide specialized carbon source that the broad-spectrum microbiota
cannot catabolise (Savka et al. 2002). Oger et al. (1997) and Savka and Farrand
(1997) confirmed that expression of opine synthetic genes in tobacco along with the
introduction of opine catabolism genes in Pseudomonas provided competitive gain
for colonization to opine catabolising strain over the wild-type strains. Plant
448
P. Singla
establishing the viability of reconstituting the complete complex of nitrogenase in
plant cells. Yang et al. (2017) reported that few of the plant electron transport chains
(ETCs) can be recruited to provide the reducing equivalents crucial for N 2 fixation.
They used E. coli as a chassis to find out about the compatibility of Mo and Fe-only
nitrogenases with ETC modules from a range of organelle of the target plant. They
replaced bacterial ETC module with genes encoding ferredoxin-NADPH
oxidoreductases (FNRs) and their cognate ferredoxin counterparts from plant
organelles and observed compatibility of FNR-ferredoxin module from chloroplasts
and root plastids with both types of nitrogenase. However, an analogous mitochondrial ETC module could not transfer electrons to nitrogenase. According to the
authors, this incompatibility of mitochondrial ETC could be overcome with hybrid
modules consisting of mitochondrial NADPH-dependent adrenodoxin oxidoreductase and the Anabaena ferredoxins FdxH or FdxB. Thus, Burén and Rubio (2018)
pointed out that while engineering nitrogenase it is significant to consider the
integration of nif gene components from different origins. Beside confirming expression and organelle targeting, it is also important to analyse whether Nif proteins
accrue in soluble forms within the eukaryotic cell.
Simplification of the N 2 fixation challenge is decisively significant because it
reduces the number of modules that have to be transferred into plant organelles.
Additionally, it will assist in finding lost or limiting steps in the course of enabling
plant cells with N 2 fixing ability. Ten proteins are mandatory to sustain N 2 fixation in
E. coli. Plant host can provide proteins necessary for coupling cellular metabolism to
N 2 fixation (NifJ and NifF) and proteins mandatory for organizing Fe and sulphur for
metal co-factor assembly (NifU and NifS). NifV, already produced by a few
eukaryotes, catalyzes the formation of homocitrate making the active site of nitrogenase accessible (extensively reviewed by Vicente and Dean 2017). Thus, expression
of the rest of the five microbial proteins is essential to assemble a first-generation
N 2 -fixing non-leguminous plant, for example, nifB, an S-adenosylmethionine–
dependent enzyme associated with nitrogenase catalytic site (Vicente and Dean
2017).
15.3.3 Transfer of N 2 Fixing Traits to Microorganisms Closely
Associated With Non-Leguminous Crops
To achieve host control of a microbe, cereals have to manage N 2 fixation by
epiphytic bacteria (growing on the root surface), endophytic bacteria (growing inside
roots) or by bacteria that inhabit engineered nodules (Mueller et al. 2012). One of the
approaches is to enhance the competitiveness of a particular microbe in the rhizosphere is to provide specialized carbon source that the broad-spectrum microbiota
cannot catabolise (Savka et al. 2002). Oger et al. (1997) and Savka and Farrand
(1997) confirmed that expression of opine synthetic genes in tobacco along with the
introduction of opine catabolism genes in Pseudomonas provided competitive gain
for colonization to opine catabolising strain over the wild-type strains. Plant
448
P. Singla