The presentation discuss about the present studies going on regarding biological nitrogen fixation in cereals. The material collected is recent one and includes all the potential studies and researches going around the world. the information given includes the pathways, the genetics, molecular mechanism, and results of various experiments and potential solutions. It describe all the possible ways and shows the future possibilities to achieve this dream.

1. A biotechnology dream: nitrogen fixing
cereal crops
Speaker: Sharma Deepak D.
Reg. no.: 1010119041
Major guide: Dr. V. P. Patel
Minor guide: Dr. V. B. Parekh
Course no: MBB-692
Date: 16/07/2021
Time: 3:00- 4:00 pm

2. Introduction
Biological Nitrogen Fixation
Mechanism of Symbiosis
Mechanism of Nitrogen fixation
Strategies to Transfer Symbiotic Nitrogen Fixation to Non-leguminous Crops
Case studies
Conclusion
2
Outline of the seminar
2

3. Introduction
The agriculture sector contributes 64% of world’s economic production.
Provides employment to 53% of the population of India (April, 2017).
Cereals like rice, wheat, maize, sorghum and millets are the crops with total
annual yields of 2000 million tons whereas two-third population consumes
only wheat worldwide
The world population today is 7.7 billion of whom 3.84 billion
people are fed by synthetic fertilizers.
World population is estimated to rise to 8.6 billion by 2030 and we
would need 185 mha more harvested area to feed this population.
The global nitrogen demand has been increasing by 1.3% p.a. and is
estimated to reach 132 mt by 2030 (101 mt in 2010).
3

4. • It represents about 2 % of the total plant
dry matter that enters the food chain.
• Nitrogen is an essential macronutrient
and is a component of proteins, nucleic
acids, and the energy currency of cells,
ATP.
• It is a component of the photosynthetic
pigment chlorophyll.
• It is a component of other important
alkaloids, hormones, etc.
Fig. 1. Nitrogen containing compounds.
(Buchanan et al., 2015)
4
Role of nitrogen in plants

5. Fig. 2. Relative contribution of the main crop categories to total N fertilizer
consumption (2018) in the main fertilizer markets. (Heffer, 2019)
Year 2018 2019 2020
Nitrogen (N) 115 376 117 116 118 763
Phosphate (P2O5 44 120 45 013 45 858
Potash (K2O) 34 894 35 978 37 042
Total (N+ P2O5 +K2O) 194 390 198 107 201 663
Table 1. World demand for fertilizer nutrient use, 2018-20 (000’ tonnes) (FAO, 2020)
5

6. Chemical Fertilizers
Urea (46%)
Ammonium Nitrate (33-
34%)
Di-ammonium Phosphate
(18%)
Organic Fertilizers
Bulky Organic Manure
1) FYM – 0.5%
2) Sheep and Goat Manure – 3%
3) Poultry Manure – 3.03%
Concentrated Organic Manure
1) Oilcakes – Mahua cake – 2.5%
Safflower cake – 7.9% N
2) Animal based Manures (meals)
Steamed bone meal – 1-2%
Horn and Hoof meal – 13%
Natural Sources
Fixation by lightening
Biological nitrogen
fixation
Sources of nitrogen in agriculture
6

7. Fig. 3. Contribution of various sources to the
fixed nitrogen in soil
(Buchanan et al. 2015) 7

8. 8
Limitations of synthetic nitrogen fertilizers
8

9. Biological Nitrogen Fixation is the fixation of elemental dinitrogen (N2), from
the atmosphere, by soil micro organisms through a reductive process into
ammonia.
Fig. 4. Primary nitrogen fixing genera
(Rogers and Oldroyd, 2014)
Biological Nitrogen Fixation
9

10. (Mus et al., 2016)
Fig. 5. Schematic representation of the different associations
between diazotrophs and plant hosts. 10

11. Fig. 6. Plant-microbe interaction in the soil
Flavone Chalcone Isoflavone
Fig. 7. Diverse structures of flavonoids
(Buchanan et al.,2015) 11
Mechanism of symbiosis
Rhizobial Symbiosis

12. Fig. 8. Perception of Nod Factors by plant membrane
receptors
Contd.
Cell membrane
Nuclear
membrane
(Rogers and Oldroyd, 2014) 12
• The rhizobium–legume symbiosis is
initiated by flavonoids released from the
plant roots that stimulate rhizobia to
produce the signalling molecule Nod
factor.
• The perception of Nod factor drives
developmental responses in the compatible
plant host that prepares the plant to
accommodate the endosymbiotic partner.
• Perception of Nod factor initiates two
coordinated genetic programmes: the
initiation of cell divisions in the root cortex
and the development of an infection thread
(or IT) in the root epidermis.

13. Fig. 9. Regulation by CCaMk
Contd.
(Buchanan et al.,2015) 13
• Nodulation signalling downstream of calcium spiking.
• Calcium spiking is perceived by the binding of calcium and calmodulin (CaM) to
CCaMK.

14. Fig. 10. Infection to nodule formation
Contd.
14
(Santi et al., 2013)

15. Fig. 11. Events that lead to nitrogen‐fixing cells.
Cont.
(Buchanan et al., 2015) 15

16. Fig. 12. Structures of MoFe and Fe proteins of nitrogenase, and electron flow
through the two enzymes. (Buchanan et al., 2015)
nifH
nifDK
16
Mechanism of Nitrogen Fixation

17. A) Components I and II are dissociated; II is
ready for reduction.
B) ATP binds to component II, which receives
electrons from an electron donor
(ferredoxin or flavodoxin); binding of ATP
induces an allosteric conformational change
which allows association of the two
proteins. Electrons flow from the [4Fe-4S]
cluster on II to the P cluster on I.
C) Electrons are further shuttled to the iron-
molybdenum cofactor (FeMoco), and ATP
is hydrolised to ADP. This step is repeated
several times before a molecule of N2 can
bind to FeMoco.
D) The protein complex dissociates, and
nitrogenase reduces dinitrogen to ammonia
and dihydrogen..
Fig. 13. A general catalytic mechanism scheme for nitrogenase
17

18. Effect of Oxygen
18
Fig. 15. Gene regulation based on free
oxygen concentration
Fig. 14. Mechanisms to maintain low free
oxygen.
2) Spatial or temporal separation of
photosynthesis and nitrogen fixation.
1)
(Buchanan et al. 2015) 18

19. Cyanobacterial symbiosis
Fig. 16. Symbiosis between Cyanobacteria and Gunnera
N2 starvation
19
(Santi et al., 2013)

20. Fig. 17. Nutrient exchange between plant cell and diazotrophic bacteria.
(Mus et al., 2013) 20
Asn, asparagine; Asp, aspartate;
KG, alpha ketoglutarate; AmtB,
ammonia transporter; Co,
cobalt; cyt bd, cytochrome bd;
DctA, dicarboxylate transporter;
Glu, glutamate; Gln, glutamine;
GOGAT, glutamate synthase;
GS, glutamine synthetase;
HCO3, bicarbonate; Mo,
molybdenum; NH3, ammonia;
N2ase, nitrogenase; Nod factors,
nodulation factors; NFR, Nod
factor receptor; OAA,
oxaloacetate; P, phosphorus; S,
sulfur

21. Fig. 18. Association of diazotrophs with plants as a potential gateway to sustainable
agriculture: strategies, tools, and challenges for engineering symbiotic nitrogen fixation.
Strategies to transfer SNF to Non-leguminous crops
Identify the gens in CSP
and use them to establish
RNS
Inoculate non-legumes
with endophytic
diazotrophs
Engineer nitrogen fixing
plants
Use endophytic bacteria as
a chassis for nitrogen
fixation
Create a biased rhizosphere
to encourage transkingdom
signalling
(Mus et al., 2016)
21

22. Table. 2. Major genomic loci detected for BNF in different legume species
22

23. Table 3. Functions of NOD, NIF, and FIX genes
Gene Function References
nod gene
nod M Nod factor synthesis Merrick (1992)
nod L Determine host range Gottfert (1993)
nod E Determine host range Gottfert (1993)
nod F Encodes a specific acyl carrier protein used to acylatethe nod factor specified by nod A Merrick (1992)
nod D Encodes a regulatory protein , Nod D that controlstranscription of other nod genes Merrick (1992)
nod A Direct synthesis of nod factor backbone Gottfert (1993)
nod B Direct synthesis of nod factor backbone Gottfert (1993)
nod C Direct synthesis of nod factor backbone Gottfert (1993)
nod I Membrane proteins that help in exporting nod factors Kondorosi et al. (1991)
nod J Membrane proteins that help in exporting nod factors Kondorosi et al. (1991)
fix gene
fix LJ Oxygen-responsive two-component regulatory system involved in positive control of
fixK and nifA
David et al. (1988)
fix K Positive regulator of fixNOQP, nifA; negative regulatorof nifA and fixK Batut et al. (1989)
fix NOQP Microaerobically induced, membrane-bound cyto-chrome oxidase Boistard et al. (1991)
fix GHIS Redox process-coupled cation pump Kahn et al. (1989)
fix ABCX Unknown function; required for nitrogenase activity; FixX shows similarity to
ferredoxins
Earl et al. (1987)
fix R Unknown function; not essential for nitrogen fixation Thony et al. (1987) 23

24. nif gene
nifD
α subunit of dinitrogenase. Forms an α2 ß2 tetramer with ß subunit interface. FeMo-co, the site substrate
reduction, is present buried within the α subunit of dinitrogenase
Dean and Jacobson (1992)
nifK ß subunits of dinitrogenase. ß clusters are present at ßsubunit interface
nifH
Fe protein subunit of dinitrogenase reductase. Obligate electron donor to dinitrogenase during
dinitrogenaseturnover. Also is required for FeMo-co biosynthesis
nifN Required for FeMo cofactor biosynthesis Allen et al. (1994)
nifV Encodes a homocitrate synthase. Homocitrate is anorganic component of FeMo cofactor Hoover et al. (1987)
nifB
Required for FeMo cofactor biosynthesis. Metabolicproduct. NifB-co is the specific Fe and S donor to
FeMo-co
Shahet al. (1999)
nifQ Incorporation of Mo into FeMo cofactor. Proposed tofunction in early MoO 2 processing Imperial et al. (1984)
nifE Forms ɑ2ß2 tetramer with nifN. Required for FeMocofactor biosynthesis Allen et al. (1994)
nifX Not essential for nitrogen fixation; required for FeMocofactor biosynthesis Shah et al. (1999)
nifU Involved in mobilization of Fe-S cluster synthesis andrepair Yuvaniyama et al. (2000)
nifS Involved in mobilization of Fe-S cluster synthesis andrepair Zheng et al. (1993)
nifY Associates with MoFe protein and dissociates uponFeMo cofactor insertion Homer et al. (1993)
nifM
Required for the maturation of nifH and Fe protein maturation. Putative peptidyl-prolyl cis/trans
isomerase
Dean and Jacobson (1992)
nifW Involved in stability of dinitrogenase. Proposed to pro-tect dinitrogenase from O inactivation Kim and Burgess (1996)
nifF Flavodoxin required for electron transfer to the Feprotein, Physiologic electron donor to nifH Thorneley et al. (1992)
nifJ
Pyruvate flavodoxin (ferredoxin) oxidoreductase involved in electron transport to nitrogenase; couples
pyruvate oxidation to reduction of the nifF product
Shah et al. (1988)
nifA Positive regulator of nif transcription Dixon (1998)
nifL Negative regulatory protein Dixon (1998) 24
Cont.

25. Case study

26. (Liu et al., 2018)
(Washington University, USA)
Case study I
Objective
• Engineer nitrogenase activity in the nondiazotrophic oxygenic photosynthetic cyanobacterium Synechocystis
sp. PCC 6803 through the transfer of 35 nitrogen fixation (nif) genes from the diazotrophic cyanobacterium
Cyanothece sp. ATCC 51142.
26

27. Materials and methods
Microorganisms, culture conditions, and media
• All cyanobacterial strains, including Cyanothece 51142,
Synechocystis 6803, and engineered strains were
cultured BG11 medium
• Yeast and E. coli strains yeast extract-peptone-dextrose
plus adenine (YPAD) medium and LB medium resp.
Construction of recombinant plasmids and
engineered strains
• Plasmids constructed by DNA assembler and Gibson
assembly.
• The sequences of all of the plasmids constructed in this
study were verified (Genewiz, NJ).
• pSyNif-1 and pSyNif-2 were introduced into the WT
strain through triparental conjugation and other
recombinant plasmids by natural transformation.
RT-PCR and q-PCR.
• RNA samples from culture (BG110) used for RT-PCR. cDNA
generated after reverse transcription was used as the template for PCR
to validate the transcription of genes.
• q-PCR was performed on RNA samples extracted from culture grown
in BG110 medium under light/dark conditions.
Measurement of nitrogen fixation activity
• Nitrogen fixation activity was measured by an Acetylene Reduction Assay
In vivo 15N2 incorporation assay
Isotope ratios were measured by elemental analyzer-
isotope ratio mass spectrometry and values are indicated as
Δ15N %, where the number represents a linear transform of
the 15N/14N isotope ratios.
Western blot analysis.
• PCR amplification of nifH gene and nifD gene from the genomic
DNA of Cyanothece 51142.
• The protein purified from E. coli BL21 (DE3) transformed with
plasmids pET28a-nifH and pET28a-nifD used as positive control.
• Proein extracted from Cyanobacterial cells cultured in N-free medium
and Immunodetection was performed using Western blotting Luminol
reagent.
27

28. Introduction of nif genes into Synechocystis 6803
(A) Phylogeny of cyanobacteria. (B) Genetic organization of the nif cluster and (C) the role of each gene product in Cyanothece 51142: Three structural
proteins (nifHDK; green), necessary cofactors (blue), accessory proteins (orange), ferredoxins (purple), and hypothetical proteins (brown). (D) Plasmid
pSyNif-1 containing the entire nif cluster. The backbone (gray) is from broad host plasmid pRSF1010, which can replicate in Synechocystis 6803. The yeast
helper fragment (black) contains CEN6 and ARS as an ori and ura3 as a selection marker. (E) Transcription of all 35 genes in engineered Synechocystis 6803.
Fig. 19. Introduction of nif genes into Synechocystis 6803.
Results
28

29. Scheme showing the top-down method to determine the
minimal nif cluster.
Fig. 20. The minimal nif cluster required for nitrogen fixation activity in Synechocystis 6803.
Nitrogen fixation activity in engineered strains.
The minimal required gene cluster for nitrogen fixation activity
29

30. • To increase the RNA expression levels of the
nitrogenase-related genes,
• Three small endogenous plasmids in Synechocystis
6803: pCA2.4, pCB2.4, and pCC5.2 maintained
higher transcriptional levels than those in a
pRSF1010-based plasmid, because of the higher
copy numbers of these three plasmids within
Synechocystis
• By replacing RSF1010 backbone of plasmid pSyNif-
2 by these endogenous episomes and then transferred
the plasmids to Synechocystis 6803,
• Generating three strains, TSyNif-9, TSyNif-10, and
TSyNif-11, with the chassis of pCA2.4, pCB2.4, and
pCC5.2, respectively.
• Genes nifH, nifD, and nifK in strain TSyNif-9
showed transcription levels that were several fold
higher than in TSyNif-2. In addition, nitrogen
fixation activity was increased by another 2- to 3-
fold,
Fig. 21. Enhancement of transcription levels of nif genes leads to higher
nitrogen fixation activity.
Improvement of nitrogen fixation activity.
30

31. Fig. 22. Expression of uptake hydrogenase improves O2 tolerance
of nitrogenase.
Improvement of O2 tolerance by introduction of
hydrogenase uptake
• In order to test the oxygen sensitivity of nitrogen fixation
activity in TSyNif-2, a measured amount of oxygen was
added to the headspace of cultures grown in BG110 media
to generate micro-oxic conditions of 0.5% and 1.0% of O2
in the sealed testing bottles. The activity dropped more than
10-fold and 60-fold resp.
• To enhance O2 tolerance under the same conditions, genes
coding for the uptake hydrogenase enzyme from
Cyanothece 51142 were introduced into the chromosome of
the TSyNif-2 strain.
• The structural genes for this hydrogenase, hupS and hupL,
present together in a single operon in Cyanothece 51142,
were transformed into TSyNif-2, generating strain TSyNif-
12
• HupW is required for the maturation of HupL protein.Thus,
the hupSLW genes organized in two operons were
transformed into TSynif-2 to generateTSyNif-13.
• The expression of hup genes in TSyNif-12 and TSyNif-13
was assessed by RT-PCR
31

32. (Geddes et al., 2019)
(University of Oxford, UK)
Case study II
Objective:
• To engineer barley plants that exude scyllo-inosamine (SIA) into the rhizosphere.
• To establish synthetic SIA-mediated transkingdom signalling between transgenic barley plants and R.
leguminosarum.
32

33. Methods
Bacterial strains and growth media
• Escherichia coli strains were grown in
Luria–Bertani (LB) medium
• Rhizobium leguminosarum and
Sinorhizobium meliloti strains were
grown in tryptone yeast (TY) medium
or universal minimal salts (UMS)
Plant materials and sterilization
• Medicago sativa, Pisium sativum,
Medicago truncatula ecotype
Jester and barley seeds were
sterilized with standard protocol.
Bacterial genetic manipulations and plasmid construction.
• All plasmids were verified by restriction digest and DNA
sequencing. Plasmids were transferred by conjugation into rhizobia
by tri-parental mating with pRK2013.
Generation of constructs for plant engineering.
• For transient expression, R. leguminosarum idhA and S. meliloti L5-
30 mosB were expressed under the control of CaMV 35s promoter
and Lotus japonicus Ubiquitin1 (LjUBI1) promoter, respectively.
• S. meliloti mosDEF and M. crystallinum IMT were expressed
transiently under the control of 35s and LjUBI1 promoters,
respectively.
• In barley transgenic lines, idhA and mosB were expressed under the
control of Zea mays ubiquitin1 promoter and Oryza sativa ubiquitin1
promoter, respectively.
• Constructs for plant engineering were assembled using golden gate
cloning. Recombinant clones were verified by PCR and DNA
sequence analysis.
33

34. Metabolite extraction and GC-MS analysis of root nodules.
• Ten germinated M. sativa or two germinated P. sativum seedlings
per pot were inoculated with 1 × 107 colony-forming units (CFUs)
of rhizobia in distilled water 3 days after planting.
• Plants were grown for 6 to 8 weeks before harvesting pink N2-
fixing nodules by hand.
• For metabolite extraction, 750 μl of CHCl3 and 1400 μl of dH2O
was added, samples were vortexed and then centrifuged for 15 min
at a relative centrifugal force (RCF) of 2200.
• GC-MS analysis was performed using the LJS_TMSI protocol at
the University of Oxford Department of Plant Sciences. GC-MS
was performed with the LJS Golm Stardard protocol.
Protein purification
• Purification of HIS-tagged proteins
from the lysate was performed
using a His-Spin Protein Miniprep
Kit (Zymo Research).
• Purity of purified proteins was
assessed by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis
analysis and proteins were
quantified using a Qubit 2.0
Fluorometer (Thermo Fisher)
according to the manufacturer’s
instructions.
Transient expression in Nicotiana benthamiana.
• Recombinant plasmids were mobilised into A. tumefaciens GV3101:pMP90 by electroporation.
• A transformed single colony was grown in LB. A. tumefaciens strains were mixed with an equal volume of a P19
suppressor strain and infiltrated into the underside of N. benthamiana leaves using a needleless 1 ml syringe.
• Leaf discs were harvested 3 days after infiltration, frozen in liquid nitrogen and the extracted metabolites were analysed
by GC-MS.
34

35. Hairy-root transformation in Medicago truncatula.
• A. rhizogenes AR1193 was transformed with
recombinant constructs by electroporation.
• Germinated seedlings of M. truncatula cultivar
Jester were transformed with A. rhizogenes
AR1193 using standard protocols.
Stable transformation of Hordeum vulgare.
• Recombinant binary vectors were transferred by
electroporation into A. tumefaciens AGL1 strain.
GC-MS analysis of engineered plant tissue.
• GC-MS analysis was performed using the
Eng_Plant protocol
• MassHunter qualitative analysis software
(Agilent) was used to determine the peak area of
rhizopine in EIC (m/z 245) chromatograms of the
standards and plant samples.
Bacterial luciferase biosensor screening assays
• Rhizopine lux biosensor was inoculated on the
engineered roots and plants were imaged each
day post-inoculation.
• Bioluminescence images were analysed for
quantification using imaging software IndiGO
(Berthold Technologies) and data were
expressed as the ratio of luminescence to
surface (cps mm−2).
Analysis of growth and bioreporter expression
in culture.
• Analysis of expression of bioreporters in free-
living culture was performed with either a
FLUOstar OMEGA (Lite) or CLARIOstar
plate reader (BMG Labtech).
• Data are expressed as relative luminescence
units calculated from total luminescence per
well/ culture density measured by OD595.
35

36. Results
Rhizopine exudation during rhizobial
symbiosis
a) Chemical structures of rhizopines and
related cyclitols.
b) NightOwl images of bioluminescence
of R. leguminosarum Rlv3841/pOPS0046
rhizopine lux biosensor on the surface of
M. sativa roots nodulated by S. meliloti
L5-30 wild-type (rhizopine+) and S.
meliloti L5-30 mosB:pK19 (rhizopine –,
Fig. 2a).
c) Specificity of induction of rhizopine
lux biosensor with rhizopines compared to
other chemically similar plant polyols
d) Induction curves showing the dynamic
range and sensitivity of the rhizopine lux
biosensor with chemically synthesised
SIA 1. Fig. 23. Detection of rhizopine exudation with a rhizopine biosensor 36

37. Fig. 24. Discovery of a natural and synthetic pathway for rhizopine synthesis.
37
a) Metabolites from nodules formed
by wild-type S. meliloti L5-30
compared to mosB:pK19 and
mosE:pK19 mutants.
b) Wild-type S. meliloti Rm1021
maintaining an empty vector
(EV) and expressing mosDEF.
c) GC-MS chromatograms (TIC) of
MosB in vitro assay in the
absence of protein or with MosB
added.
d) Proposed natural pathway of 3-O-
MSI biosynthesis in S. meliloti
L5-30
e) Linked in vitro assay of inositol
dehydrogenase IolG and MosB
f) Extracts prepared from tobacco
leaves agroinfiltrated with either
empty vector (control) or IdhA or
MosB or IdhA-MosB together
g) Proposed synthetic pathway of
SIA 1 synthesis

38. • a, b GC-MS TICs of extracts prepared
from M. truncatula transgenic roots (a)
and transgenic T0 barley seedlings (b)
transformed with empty vector (control)
or IdhA-MosB together.
• Highlighted peaks indicate scyllo-
inosamine 1 (orange), scyllo-inosose 7
(green) and myo-inositol 3 (dark blue).
• c, d NightOwl images showing
bioluminescence of Rlv3841/pOPS0046
rhizopine lux biosensor on the surface of
M. truncatula transgenic roots (c) and T0
barley seedlings (d) transformed with
empty vector (control) or IdhA-MosB
together (engineered).
Fig. 25. Rhizopine biosynthesis and signalling in M. truncatula and barley roots 38

39. a) Confocal fluorescence microscopy images showing
green fluorescent protein (GFP) fluorescence of
Rlv3841::mTn7-mCherry/pOPS0761 biosensor [with
constitutive mCherry fluorescence] on the surface of T1
barley seedlings transformed with empty vector
(control) or IdhA-MosB together (engineered).
Z-stack projections of green fluorescence channel (left,
GFP), red fluorescence channel (middle, mCherry) and
all channels merged (right, bright-field) are shown
(scale bars, 100 µm).
b) Three-dimensional images of GFP/mCherry mean
intensity ratios in Rlv3841::mTn7-mCherry/pOPS0761
biosensor on the surface of T1 barley seedlings
transformed with empty vector (left, control) or IdhA-
MosB together (middle, engineered).
Cool colours (purple) indicate low GFP/mCherry
intensity ratio, and warm colours (red) indicate a high
GFP/mCherry intensity ratio.
Fig. 26. Fluorescent microscopy of rhizopine-mediated
transkingdom signalling
39

40. (Bloch et al., 2020)
(Berkeley, USA)
Case study III
Objective
• Identification and isolation of diazotrophs that closely associate with key crops to improve nitrogen
contributions by BNF
• Gene editing to disrupt regulatory networks linking nitrogen sensing, fixation, and assimilation
40

41. Materials and methods
Isolation of Kosakonia sacchari PBC6.1
• Isolated bacterial Colonies from corn seedling
that emerged were tested for the presence of the
nifH gene by colony PCR
Fluorescence microscopy
• PBC6.1 was transformed with plasmid PB114-
RFP, a plasmid containing the pSC101 origin of
replication, chloramphenicol resistance cassette,
and a gene encoding red fluorescent protein (RFP)
under the control of a strong constitutive promoter.
• Transformed cell suspension was inoculated
directly on the seed at the time of seeding.
• Root sections were imaged on a 6D Widefield
Nike TI inverted fluorescence microscope
Initial field trial with isolated diazotrophs
• Corn seed was coated with a culture suspension of
6 strains selected from 49 strains isolated
• Root samples were processed and assayed for
colonization of the inoculant diazotrophs
Extraction of the root-associated microbiome
• Genomic DNA extraction was performed with the ZR-96 Quick-
gDNA kit, and RNA extraction was performed using the RNeasy kit.
Root colonization assay
• Quantification of root colonization was done by using quantitative
PCR. For each experiment, the colonization numbers were compared
with UTC seedling
Acetylene reduction assay (ARA)
• A modified version of the ARA (Temme et al., 2012) was used to
measure nitrogenase activity in pure culture conditions.
Ammonium excretion assay
• Supernatant from the reactor broth was assayed for free ammonium
using the Megazyme Ammonia Assay kit normalized to biomass at
each time point.
41

42. Gene editing
• The genome modifications described in Fig. 2 were
generated using genome editing methods described in
a recently published patent application on our guided
microbial remodeling platform (Bloch et al., 2019b).
• Genome-edited strains were cured of all plasmids used
to carry out genome editing by repeated subculturing
followed by sequence verification of the desired edits.
Greenhouse assays to measure nifH transcription
in PBC6.29, PBC6.99, PBC6.38, and PBC6.94
• Plants grown in nitrogen free background. Each seed was
inoculated with either sterile phosphate-buffered saline
(controls) or an equal volume of microbial suspension
using cells diluted to a set OD.
• At 2 or 4 weeks after planting, plants were harvested, and
nucleic acids were extracted from root tissue.
Colonization of each strain was measured.
• For microbial RNA quantification, microbial RNA was
extracted using the RNeasy Kit (Qiagen) The extracted
RNA was used for NanoString analysis of nifA, nifH, and
rpoB microbial transcript on an nCounter Sprint.
• Field trials to measure colonization and nifH
transcription were carried out at California, Puerto Rico
and Illinois
Re-isolation of edited strains from field corn
root samples
• Isolates were screened using PCR primers specific to
the mutations, and those with the correct band sizes
were further verified by Sanger sequencing of the 16S
rRNA region.
• Between two and 20 clones of each re-isolated strain
were then purified and assessed for ARA activity
relative to the original inoculant strain
42

43. Results
Fig. 27. Kosakonia sacchari PBC6.1 contains well-
characterized nitrogen regulatory pathways.
• PBC6.1 has a genome of at least 5.4 Mbp, and a nif gene cluster and a
nitrogen metabolic regulatory network.
• The nifLA operon directly regulates the rest of the nif cluster through
transcriptional activation by NifA and nitrogen- and oxygen-dependent
repression of NifA by NifL.
• Glutamine synthetase (GS) is responsible for rapid assimilation of newly
fixed nitrogen in nitrogen limiting conditions.
• GlnE- adenylylation- attenuate or deadenylylation- restore activity
• The nifLA operon and GS (encoded by the glnA gene) are regulated by
the PII protein regulatory cascade,
• No nitrogen- GlnD modifies PII proteins, leads to the up-regulation of
nitrogen fixation and assimilation pathways.
• Exogenous nitrogen- GlnD removes the covalent modification from the
PII proteins, leading to the repression of nitrogen fixation and
assimilation genes
43

44. Fig. 28. Mutated sequences of the key genes of the nitrogen fixation and
assimilation regulatory network of PBC6.1.
Remodel the regulatory networks of PBC6.1
1) Disruption of nifL can abolish inhibition of NifA and improve
nif expression in the presence of both oxygen and exogenous
fixed nitrogen, expressing nifA under the control of a
nitrogen-independent promoter could decouple nitrogenase
biosynthesis from the PII protein regulatory cascade (Fig. 2a)
2) Truncation of the GlnE protein to delete its adenylyl-removing
(AR) domain would lead to constitutively adenylylated GS,
limiting ammonium assimilation by the microbe and leading
to ammonium build up and release (Fig. 2b)
3) Abolishing expression of AmtB, the transporter responsible
for uptake of ammonium, could lead to greater extracellular
ammonium by preventing reuptake of excreted ammonium
(Fig. 2c)
4) Deletion of the GlnD protein would lead to a constant nitrogen
sufficiency signal by eliminating the ability of the cells to
covalently modify PII proteins (Fig. 2d).
5) Strain with the nifH gene deleted to serve as a negative control
for nitrogenase expression (Fig. 2e).
44

45. Table 4. List of isolated and remodeled K. sacchari strains used in this work
Strain Genotype
PBC6.1 WT
PBC6.15 ΔnifL::Prm5
PBC6.29 ΔnifL::Prm5 ΔglnEAR1
PBC6.99 ΔnifL::Prm5 ΔglnEAR1 ΔglnD
PBC6.90 ΔnifL::Prm5 ΔglnEAR1 ΔnifH
PBC6.14 ΔnifL::Prm1
PBC6.37 ΔnifL::Prm1 ΔglnE AR2
PBC6.38 ΔnifL::Prm1 ΔglnEAR1
PBC6.93 ΔnifL::Prm1 ΔglnE AR2 ΔamtB
PBC6.94 ΔnifL::Prm1 ΔglnE AR1 ΔamtB
45

46. Fig. 29. Edited strains of PBC6.1 fix nitrogen independently of nitrogen status.
• To assess the sensitivity of
PBC6.1 to exogenous nitrogen
and to predict activity in a
fertilized field, nitrogenase
activity in pure culture was
measured with the classical ARA
in the presence and absence of
fixed nitrogen.
• The wild-type strain exhibits
repression of nitrogenase activity
as glutamine concentrations
increase, while remodeled strains
show nitrogenase activity in the
presence of 5 mM glutamine
(Gln) or NH4
+.
• PBC6.90 was not tested in 5 mM
NH4
+; PBC6.99 was not tested in
5 mM glutamine.
46

47. Fig. 30. Edited strains of PBC6.1 excrete fixed nitrogen into their
environment.
• ∆nifL::Prm5 mutation alone (PBC6.15) was
insufficient to confer an ammonium excretion
phenotype, the ∆nifL::Prm1 mutation alone
(PBC6.14) led to significant excretion of ammonium.
• The ∆glnEAR mutations led to an increase in
ammonium excretion when stacked with the
∆nifL::Prm mutations, supporting our hypothesis that
down-regulation of GS activity would lead to
ammonium excretion
• The ∆glnD mutation led to an additional increase in
ammonium excretion, probably by causing a decrease
in glnA expression.
• The ammonium excretion phenotype conferred by the
∆glnEAR and ∆glnD mutations came with a
corresponding decrease in growth rates, similar to
what was observed in the acetylene reduction assay.
• The ∆amtB mutations had no apparent effect on
ammonium excretion or growth rate when stacked
with the ∆nifL::Prm and ∆glnEAR mutations.
• These results suggest that the edited strains may be
able to fix nitrogen and transfer it to the crop in
fertilized field conditions.
47

48. Fig. 31. Greenhouse experiments demonstrate rhizosphere nifH transcription in fertilized corn.
• To determine whether the
edited microbes were able
to express nitrogenase in
the rhizosphere of fertilized
plants, inoculated corn
plants in greenhouse assays
with PBC6.1 and a subset
of edited strains to measure
colonization of the
cornrhizosphere and nifH
transcription therein.
48
• black bars represent samples
analyzed using primers
targeting the nifH–por2
intergenic region, and hatched
bars represent samples
analyzed using primers
targeting the ∆nifL::Prm5
genotype

49. • Root samples were collected between 2
and 5 weeks after planting for nucleic
acid extraction to verify the presence of
the strain and expression of the nifH gene
• Root samples were collected, the root-
associated microbiome was extracted,
and nifH transcription (a, c, e, g) and
colonization (b, d, f, h) were quantified.
• At all locations, remodeled strains show
increased normalized nifH transcript
levels and similar colonization when
compared with PBC6.1.
• In (b, d, f, and h), black bars represent
samples analyzed using primers targeting
the nifH–por2 intergenic region; shaded
and hatched bars represent samples
analyzed using primers targeting
the ∆nifL::Prm1 and ∆nifL::Prm5
genotypes, respectively.
Fig 32. Remodeled strains colonize corn roots and express nitrogenase in diverse
locations, soil types, and nitrogen levels. 49

50. Fig 33. Corn inoculated with PBC6.1 and its derivatives exhibited
an increase in grain yield above UTC in the Puerto Rico field trial.
Fig. 34. Nitrogenase activity is observed in clones re-
isolated from field grown root samples.
50

51. • The creation of artificial symbioses or associations between diazotrophs and crops is a primary goal in
agriculture to reduce the demand for chemical nitrogen fertilizers
• Utilization of diazotrophs isolated from non-legumes in other non-leguminous crops has proved to be a
successful method to transfer nitrogen fixation
• Engineering nitrogen fixation activity in photosynthetic nondiazotrophic cyanobacteria paved the way to
resolve the problem of O2 sensitivity of nitrogenase enzyme.
• The minimal cluster for 24 nif genes for nitrogenase activity will provide a useful framework for refactoring
genes. But biosynthesis of fully functional nitrogenase is a complex process as genes with the same
designations in different species occasionally have alternative functions.
• The confirmation of the chemical structure of rhizopines, and their secretion into the rhizosphere, provides a
unique opportunity to use them as target molecules for engineering plant control of root bacteria (biased
rhizosphere)
• Rhizopine transkingdom signalling could control synthetic symbioses to deliver nitrogen to cereal crops.
• Through gene edited strains in which nitrogenase biosynthesis is decoupled from the regulatory networks that
sense and respond to cellular nitrogen status can fix and excrete significant quantities of nitrogen into their
environment at various levels of exogenous nitrogen is a first step toward developing strains that can replace
synthetic fertilizers in cereal crop production
Conclusion
51

52. Thank you