DESC:TECHNICAL FIELD OF THE INVENTION:
The invention relates to the development of genetically modified microbes, either
autotrophic or non-autotrophic endophytes, epiphytes and Rhizopsphere microbes,
for efiiecient utilization of CO2 as raw material source for diversed product
formation.
In particular, engineered microbes developed in this invention are meant to
efficiently perform GHG sequestration specifically CO2 and resulting in
sustainable production of Agri-related compounds not limited toAmmonia,
Nitrate, Nitraite, Urea, Nitrate-compounds, and value-added chemicals not limited
to aminoacids, peptides ,proteins, enzymes, nucleotides, vitamins, nitrogen
containing secondary metabolites, produced for the purpose of plant growth,
increase of yield, yield quality stress responses, defense mechanisms, metal
carrying, and signalling metabolites, plant growth stimulants, bioherbicides
bioinsecticides, antimicrobial agents, antiparasitic agents, peptides, enzyme
inhibitors, as well as for production of non-Agri compunds not limited to
heterologous protein and enzyme production, peptides, biosimilars production.
Major focus of the current invention is towards manipulation of microbes for
efficient uptake of GHG gases not limited to CO2 and utilization of them for
product formation.
Inventive part of the current invention lies in the incorporation of metabolic
pathways or missing genes of the pathway or combination of genes, necessary for
CO2 uptake and assimilation in metabolic pathways, leading to biomass synthesis
as well as increased product formation.
One embodiment of the invention relates to the Incorporation of pathways for CO2
uptake and assimilation not limited to the following pathways, genes and enzymes
1. Completion of CBB (Calvin-Bentham-Bassham pathway) by way of
introduction of Rubisco genes,
2. Reductive citric acid cycle, leading to formation of Pyruvate and
oxaloactetae
3. Reductive acetyl-CoA route, leading to Formate and acetyl-CoA formation
3
4. 3-hydroxypropionate cycle, resulting in methylmalanoyl CoA and
Glyoxylate production
5. 3-hydroxypropionate/4-hydroxybutyrate cycle, resulting in formation of
succinyl-CoA and pyruvate
6. Dicarboxylate/4-hydroxybutyrate cycle, resulting in pyruvate, succinyl
CoA and acetyl-CoA
Incorporation of the entire genes of above said pathwys, or few of the missing
genes or facilitating over expression of rate-limiting genes, divert the Carbon flux,
in terms of CO2/HCO3- towards either increase pyruvate formation or Acetyl-CoA
or intermediates of TCA cycle, amino acid synthesis pathway or fatty acid
synthesis.
Carbon capture, sequestration & utilization (CCSU) have been widely recognized
as an efficient option for reducing the atmospheric CO2 concentration. Generally,
CO2 capture can be regarded as the process of capturing waste CO2 from specific
sources, such as fossil fuel power plants; CO2 sequestration can be regarded as the
process of transporting/depositing enriched CO2 to a storage site (mineralization
or landfill); and CO2 utilization can be regarded as the process of directly using
CO2 as a reaction medium or transforming renewable CO2 into useful chemicals,
materials or fuels.
The present invention also relates to genetic modification of microbes with
improved CO2 fixation, more particularly, the present invention relates to genetic
modification of microbes to increase the activity of the enzyme selected from the
group consisting of Formate dehydrogenase (FDH) preferably oxygen-tolerant
FDH and pyruvate formate-lyase for Carbon dioxide to reduced to Formate.
Formate enter in to reductive acetyl coA pathway or Pentose phosphate pathway
to make pyruvate and build the biomass for microbes
It is a further object of the present invention to increase soil organic carbon
content using microbes. The purpose of CO2 fixation is to enhance the soil
carbon, which is depleting, and to enhance the soil productivity, and also to
enhance the crop growth.
4
BACKGROUND AND PRIOR ART OF THE INVENTION:
Soil microorganisms play a key role in the fractionation of C and N compounds
which enter the soil environment. Indeed, many of the biogeochemical processes
in soils are microbially mediated and a wide range of autotrophic and
heterotrophic microorganisms are involved in below-ground soil processes that
include mineralization, oxidation and assimilation of C and N into forms available
to the plants.
Soil microbes are capable of CO2 from the soil and atmosphere. Apart from
surface photosynthetic CO2 fixation and chemoautotrophic fixation, dark
anaplerotic (i.e.non-photosynthetic) fixation of CO2 is especially important for
provision of C-skeletons for amino acid synthesis.
Heterotrophic organisms are also known to require CO2 for growth, although CO2
provides only a certain percentage of the biomass C of these organisms.
Furthermore, it was recently found that, during growth on certain oxo-compounds,
not only anaerobic bacteria, but also aerobic Rhodococcus and Xanthobacter
species perform carboxylation reactions of the substrate, which contribute
substantially to biomass formation from CO2. Thiobacillus sp. was also shown to
incorporate more than 10% of the cell C from CO2 during both mixotrohic and
heterotrophic growth.
Agri-beneficial microbes are capable of incorporating CO2 into biomass via six
natural carbon fixation pathways (Bar-Even et al., 2012). Since the discovery of
the Calvin-Benson-Bassham (CBB) cycle in the 1940s and 1950s, another five
CO2 assimilation mechanisms have been elucidated namely, the reductive citric
acid cycle (rTCA), the reductive acetyl-CoA pathway (Wood-Ljungdahl
pathway), the 3-hydroxypropionate bicycle (3HP bicycle), the 3-
hydroxypropionate/4-hydroxybutyrate cycle (3HP/4HB cycle), and
dicarboxylate/4-hydroxybutyrate cycle (DC/HB).
5
For common microorganisms, the CBB cycle (Reductive pentose phosphate
cycle) is the most important mechanism of CO2 fixation. The entire cycle is
composed of 13 steps and three stages, consisting of carboxylation, reduction and
regeneration. Fructose bisphosphatase or sedohrptulose 1, 7 bisphosphatase play a
role in regeneration of RuBP. RuBP stands for ribulose bisphosphate and is a 5
carbon compound involved in the Calvin cycle; Atmospheric carbon dioxide is
combined with RuBP to form a 6 carbon compound, with the help of an enzyme
called RuBisCO. RuBisCO is the first enzyme utilized in the process of carbon
fixation and its enzymatic activity is highly regulated. It is found in the mesophyll
cells.
Microbes generate energy via the oxidation of acetate derived from carbohydrates,
fats, and proteins into carbon dioxide through citric acid cycle (TCA) or Krebs
cycle. Microbes also undergo reverse TCA or reverse Krebs cycles to produce
various carbon compounds from carbon dioxide and water with the help of
various enzymes including PEP carboxylase.
Gluconeogenesis involves generation of glucose from non-sugar carbon substrates
such as pyruvate, (S)-lactate, glycerol, and glucogenic amino acids. The process is
the reversal of the glycolysis pathway. In order to enable the pathway to flow in
the direction of glucose production, the reactions are catalyzed by glycolytic
enzymes fructose-bisphosphatase and water dikinase in the opposite direction.
Low levels of growth and CO2 fixation efficiency have largely limited industrial
applications of microbes including cyanobacteria (Blankenship et al.,
2011; Kushwaha et al., 2018). The CO2 capturing rate of ribulose-1,5-
bisphosphate carboxylase/oxygenase (RuBisCO) in the CBB cycle is an order of
magnitude lower than the average of central metabolic enzymes, and efforts to
improve RuBisCO kinetic properties to improve CO2 fixation efficiency have
attained only limited success so far (Antonovsky et al., 2017; Liang et al., 2018).
6
US20190211342 discloses genetic modification of non-autotrophic
microorganisms to enhance the expression of enzymes recombinant
phosphoribulokinase (prk) and Ribulose-Bisphosphate Carboxylase (RuBisCo) to
improve carbon fixation. It also discloses methods that include down-regulating
genes in microorganisms using CRISPR arrays.
WO2019185861 discloses genetic modification of microbes to enhance the
activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) capable
of using carbon dioxide as the only source of carbon. It also discloses methods to
genetically modify microorganisms through site-directed mutagenesis in the
native gene coding for the NusG / SPT5 protein using CRISPR technology.
In recent years, the synthesis of carbon fixing systems has become increasingly
common due to advances in synthetic biology. There is also a need for art to
improve the microbial CO2 fixation efficiency and extend this phenotype to nonleguminous
plants. Accordingly, the present invention provides a composition
comprising genetically modified microbes with improved carbon fixation ability
capable of delivering to crop-plant for assimilation in leguminous and nonleguminous
plants.
OBJECT OF THE INVENTION:
The invention relates to a composition comprising genetically modified microbes
not limited to endophytes, ephiphytes and free living Rhizoshperic microbes
favourable for fixing CO2. These engineered artificial autotrophs efficiently do the
CO2 fixation and sustainable production of value-added chemicals not limited to
aminoacids,peptides,proteins, enzymes, nucleotides, vitamins, nitrogen containing
secondary metabolites, produced for the purpose of plant growth, increase of
yield, yield quality stress responses, defense mechanisms, metal carrying, and
signalling metabolites, plant growth stimulants, bioherbicides
bioinsecticides,antimicrobial agents, antiparasitic agents, peptides, enzyme
inhibitors, etc.
7
It is another object of the present invention to provide gene manipulation of
microbes selected from the group comprising of endophytic bacteria, an epiphytic
bacteria, or a rhizosphere bacteria.
It is an object of the present invention relates to increased activity of organic acid
such as alpha keto gluconic acid and others such as citric acid, malic acid, fumaric
acid lactic acid, acetic acid, and oxalic acid secretion to solubilize the soil bound
phosphate and other soil bound nutrients such as silica, zinc, calcium, magnesium
and others .Also prevent these nutrient being locked in the soil to make available
to crops. The microbial biomass consists mostly of bacteria and fungi, which
decompose crop residues and organic matter in soil. This process releases
nutrients, such as nitrogen (N), into the soil that are available for plant uptake.
It is an object of the present invention relates to providing carbon source to the
microbes present in the soil. In soil the microbial biomass is usually ‘starved’
because soil is too dry or doesn’t have enough organic carbon.By the action of
photosynthetic and other microbe’s action atmospheric CO2 fixed and provide
carbon source to the microbes present in the soil.
It is an object of the present invention relates to provide soluble and insoluble
carbon produced by non pathogenic fungi and other microbes to contribute to the
increase in soil organic carbon.
It is an object of the present invention relates to the soil microbial community is
also an important factor influencing reduction of soil pH. Soil microbe’s role in
maintaining soil productivity through biochemical processes such as litter
decomposition and nutrient recycling.Soil microbial community also takes care of
plant disease resistance
8
More particularly, the present invention relates to genetic modification of
enzymes selected from a group consisting of Rubisco enzyme of Reductive
pentose phosphate cycle, PEP carboxylase of Reductive TCA cycle, and Fructose
bisphosphatase that convert fructose 1,6-bisphosphate to fructose6
phosphate in gluconeogenesis and in Calvin cycle, for the improved carbon
fixation/CO2 fixation.
It is further object of the present Invention relates to genetic modification of
microbes with improved CO2 fixation, more particularly, the present invention
relates to genetic modification of microbes to increase the activity of the enzyme
selected from the group consisting of Formate dehydrogenase (FDH) preferably,
oxygen-tolerant FDH and pyruvate formate-lyase for Carbon dioxide to reduced
to Formate. Formate enters in to reductive acetyl COA pathway or Pentose
phosphate pathway to make pyruvate and build the biomass for microbes.
It is a further object of the present invention to facilitate methane assimilation and
N2O assimilation, which are also major contributors for rise in green ghouse
effect. Methane emission from animal wastes and rice field is major problem to
overcome. Extensive application of Chemical fertilizers leads to leaching out and
wastage of of Nitrogen compounds such as Urea, by action non-Agri beneficial
organisms, which denitrify and convert to N2). N2O generation in huge volumes
from Agri fields is a major cause of environmental pollution. Hence, there are
neccesities ofprocess and pathways in Agri-beneficial organisms to assimilate
CH4 or N2O and usage for production of biomass and production of agribeneficial
products, proteins, nitrogen compounds, which help in plant health and
yield improvement, as well as soil fertility and environmental remediation.
Overall, the major focus of the current patent is on environmental remediation by
assimilation of CO2for production agr- reletaed and nn-agri related compounds,
finally resulting in reduction of environmental pollution, improvement in plant
yield and soil fertility, and production of varied products from Agri-microbial
culture practices.
9
SUMMARY OF THE INVENTION:
In a main aspect of the present invention relates to a composition comprising
genetically modified microbes, favourable for fixing green house gases such as
CO2, CH4 and N2O, and deliver to crop for assimilation, also improves the soil
fertility.
Accordingly, the present invention relates to genetic modification of enzymes
selected from a group consisting of Rubisco enzyme of Reductive pentose
phosphate cycle, PEP carboxylase of Reductive TCA cycle, and Fructose
bisphosphatase that converts fructose-1,6-bisphosphate to fructose 6-
phosphate in gluconeogenesis and the Calvin cycle.
It is yet another aspect of the present invention relates to genetic modification of
microbes with improved CO2 fixation, more particularly, the present invention
relates to genetic modification of microbes to increase the activity of the enzyme
selected from the group consisting of Formate dehydrogenase (FDH) preferably,
oxygen-tolerant FDH and pyruvate formate-lyase for Carbon dioxide to reduced
to Formate . Formate enters in to reductive acetyl COA pathway or Pentose
phosphate pathway to make pyruvate and build the biomass for microbes.
In another aspect, the present invention provides for the genetic modification of
micro-organisms selected from a group comprising of endophytic bacteria, an
epiphytic bacteria, a rhizosphere bacteria, for improving carbon fixation.
It is a further object of the present invention to increase soil organic carbon
content using microbes. The purpose of carbon fixation is to enhance the soil
carbon, which is depleting, and to enhance the soil productivity, and also to
enhance the crop growth.
10
It is a further object of the present invention to increase specific chemicals which
enhance photosynthesis and other processes in the host resulting in enhanced
vegetative growth.
Further, the present invention relates to assimilation of CH4 and N2O by microbes
for conversion to usedful compounds and finally resulting in improvemtnt in plant
health, soil fertility and CO2 gas remediation.
Other objective of the present invention is to enable CO2 assimilation pathways in
autotrophs as well as non-autotrophs, for agri as well as non-agri applications.
Highlight objective of the invention is enhancement of CO2 assimilation, by the
way of enhancing downstream assimilation activities and product formation. By
enhancing the product formation activities, Nitronase functions, protein and
biomass synthesis creates demand for more energy and ATP supply, which will
make increased assimilation of GHG gases maily CO2.
BRIEF DESCRIPTION OF THE DRAWINGS:
Figure 1: CO2 fixation and conversion to Formic acid by catalytic activity of
Formate dehydrogenase: Gas chromatography (GC) analysis of CO2 fixation to
form formate, resulted in almost 75% conversion of CO2 to formate by
Methylobacterium spp FDH enzyme, in controlled conditions.
Figure 2: Incorporation of Heterologous Rubisco in Methylobacterium spp., and
generation of Autotrophism, enabled CO2 fixation in CBB pathway, leading to
formation of 6 carbon sugar from 5 carbon sugar and further entry of compounds
into general sugar metabolic pathways.
Figure 3: CO2 consumption profile of engineered microbes: Engineering the
microbes for CO2 consumption, by incorporation of Formate dehydrogenase and
11
overexpression, resulted in more than 50% consumption of supplied CO2 as the
sole carbon source, without additional sugar.
DETAILED DESCRPITION OF THE INVENTION:
The invention will now be described in detail in connection with certain preferred
and optional embodiments, so that various aspects thereof may be more fully
understood and appreciated.
To accomplish the objectives of the invention, the present invention discloses a
composition comprising genetically modified microbes, favourable for fixing
green house gases such as CO2, CH4 and N2O, and its deliver to all plants/crops
and soil for assimilation.
In an embodiment, the present invention relates to genetic modification of
microbial enzymes selected from a group consisting of Rubisco enzyme of
Reductive pentose phosphate cycle, PEP carboxylase of Reductive TCA cycle,
and Fructose bisphosphatase that converts fructose-1, 6-bisphosphate to fructose
6-phosphate in gluconeogenesis and the Calvin cycle.
In another embodiment, the present invention relates to genetic modification of
micro-organisms selected from a group comprising of an endophytic bacteria, an
epiphytic bacteria, and a rhizosphere bacteria, for improving CO2 fixation.
In an embodiment, the present invention provides a composition comprising
genetically modified micro-organism(s) consisting of modification of _ Rubisco
enzyme of Reductive pentose phosphate cycle, PEP carboxylase of Reductive
TCA cycle, and Fructose bisphosphatase that converts fructose-1,6-bisphosphate
to fructose 6-phosphate, Formate dehydrogenase wherein the said micro-organism
is an endophyte, an epiphyte and a rhizospheric microbe.
In an embodiment, the present invention provides a process carried for gene
modification consists of sucicidal venctor or plasmid.
12
In an embodiment, the present invention provides a the plasmid vector of carrying
a DNA construct comprising a nucleotide sequence represented by SEQ ID NO. 7.
In an embodiment, the present invention provides a SEQ ID NO: 7 consisting of
7852 bp with selective restriction sites along with required modified gene
sequences.
In an embodiment, the plasmid vector carrying a DNA construct comprising a
nucleotide sequence represented by SEQ ID NO. 2 and 3.
In an embodiment, the plasmid vector is expressed in miro-organism selected
from the group comprising of endophytic bacteria, epiphytic bacteria, rhizospheric
bacteria and fungi.
In an embodiment, the bacteria is selected from the group comprising of
cultivable bacteris (viz) Methylobacterium extorquens; Beijerinckia indica;
Azoarchus communis and uncultivable bacteria adapted to cultivabe using Ichip
method (viz) Pseudomonas sp.; Bacillus sp. And Sphingomonas sp.
In an embodiment, the present invention provides a process for the preparation of
genetically modified micro-organism(s) for improved nitrogen fixation and its
delivery to crop-plants for assimilation by homologous recombinations.
1. Cloning a of a sequence having SEQ ID NO 2 and 3. targeting the
Glutathione dependent formate dehydrogenase gene in integration vector
or sucidal vector flanking the region SEQ ID NO.4 and 5.
2. Transforming pUC57 vector harboring SEQ ID NO.7 containing the FDH
into a host cell, wherein the said micro-organism is an endophyte, an
epiphyte and a rhizospheric microbe. wherein the said micro-organism is
uncharacterized and non-cultivated.
In another embodiment, the present invention provides the microbes includes, and
not limited to, Bradyrhizobium japonicum, Nitrospira inopinata,
Rhodopseudomonas palustris, Sphingomonas, Nitrosopumilus maritimus,
Methylobacterium, Rhodobacter sphaeroides, Reyranella massiliensis,
13
Alcaligene, Saccharomyces cerevisiae, Saccharomyces lactis, Brevibacterium, ,
Kluyveromyces lactis, Epichloë typhina, Enterococcus, Corynebacterium,
Arthobacter, Pichia, Zymomonas, Saccharomyces carlsbergensis, Salmonella,
Zymomonas, Rhodacoccus, Escherichia (e.g., E. Coli), Hansenula, Firmicutes,
Rubrivivax, Dinoroseobacter shibae, Methylobacterium nodulans,
Methylobacterium radiotoleran, Methyloversatilis sp, Methylobacterium oryzae,
Beijerinckiaindica. Ralstonia eutropha/ Cupriavidus necator, Methyloversatilis,
Nigrospora oryzae, Candida, Reyranella massiliensis,
Novosphingobiumaromaticivorans, Microbacterium, Acidovorax, Bordetella.
Phomopsis liquidambaris , Nitrosopumilus maritimus, Ralstonia eutrophus/
Cupriavidus necator, Nitrospira inopinata , Nigrospora oryzae ,
Rhodopseudomonas palustris ,Periconia spp, Paenibacillus
beijingensis,Thiobacillus sp,Sinorhizobium meliloti ,Methyloversatilissp ,
Novosphingobiumaromaticivorans, Acidovorax, Reyranella massiliensis,
Microbacterium, Bordetella, Methyloversatilissp, Cyanobacteria,
Rhodobactercapsulatus, Epichloëtyphina, Metallosphaera, Sulfolobus ,
Archaeoglobus, Cenarchaeum spp, Clostridium autoethanogenum, Xanthobacter
flavus, Oligotropha carboxidovorans, Acidithiobacillus thiooxidans,
Desulfobacter hydrogenophilus, Thiomicrospira denitrificans, Candida,
Reyranella massiliensis.
In a further embodiment, the present invention relates to involvement of
CRISPR/Cas technology used preferentially for gene manipulation of microbes.
In the present invention, genetic variations are brought about by CRISPR/Cas9,
by subjecting DNA to mutagens and other options for specifically inducing
cleavage at a target site are available, such as zinc finger nucleases, TALE
nuclease (TALEN) systems, and meganuclease.
Accoridngly, CRISPR/Cas9 (Clustered regularly interspaced short palindromic
repeats)/CRISPR-associated (Cas) systems provide bacteria and archaea with
adaptive immunity against viruses and plasmids by using CRISPR RNAs
(crRNAs) to guide the silencing of invading nucleic acids. The Cas9 protein (or
14
functional equivalent and/or variant thereof, i.e., Cas9-like protein) naturally
contains DNA endonuclease activity that depends on association of the protein
with two naturally occurring or synthetic RNA molecules called crRNA and
tracrRNA (also called guide RNAs). In some cases, the two molecules are
covalently linked to form a single molecule (also called a single guide RNA
(“sgRNA”). Thus, the Cas9 or Cas9-like protein associates with a DNA-targeting
RNA (which term encompasses both the two-molecule guide RNA configuration
and the single-molecule guide RNA configuration), which activates the Cas9 or
Cas9-like protein and guides the protein to a target nucleic acid sequence. If the
Cas9 or Cas9-like protein retains its natural enzymatic function, it will cleave
target DNA to create a double-strand break, which can lead to genome alteration
(i.e., editing: deletion, insertion (when a donor polynucleotide is present),
replacement, etc.), thereby altering gene expression. Some variants of Cas9
(which variants are encompassed by the term Cas9-like) have been altered such
that they have a decreased DNA cleaving activity (in some cases, they cleave a
single strand instead of both strands of the target DNA, while in other cases; they
have severely reduced to no DNA cleavage activity).
Further, mutagens that create primarily point mutations and short deletions,
insertions, transversions, and/or transitions, including chemical mutagens or
radiation, may be used to create genetic variations. Mutagens include, but are not
limited to, ethyl methanesulfonate, methylmethanesulfonate, N-ethyl-Nnitrosurea,
triethylmelamine, N-methyl-N-nitrosourea, procarbazine,
chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer,
melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N'-
nitro- Nitrosoguanidine, nitrosoguanidine, 2-aminopurine, 7,12 dimethylbenz(
a)anthracene, ethylene oxide, hexamethylphosphoramide, bisulfan,
diepoxyalkanes (diepoxyoctane, diepoxybutane, and the like), 2-methoxy-6-
chloro-9[3-(ethyl-2-chloro- ethyl)aminopropylamino] acridinedihydrochloride and
formaldehyde.
15
EXAMPLES
The following examples, which include preferred embodiments, will serve to
illustrate the practice of this invention, it being understood that the particulars
shown are by way of example and for purpose of illustrative discussion of
preferred embodiments of the invention.
Example 1: Homologous expression of FDH in Methylobacterium spp.,
Formate dehydrogenase play an essential role in energy generation during growth
on C1 compounds. The conversion of CO2 into formate offers key advantages for
carbon recycling, and formate dehydrogenase (FDH) enzymes are at the centre of
intense research in this patent, due to the “green” advantages the bioconversion
can offer, namely substrate and product selectivity and specificity, in reactions run
at ambient temperature and pressure and neutral pH.
Methylobacterium spp, is reported to show remarkable activity converting carbon
dioxide into formate. Formate dehydrogenase from M. spp, was verifed as the key
responsible enzyme for the conversion of carbon dioxide to formate. The
homologus expression of FDH expressing cells showed maximum formate
productivity which was 2 -3 times greater than that of wild type. M. spp, FDH
was successfully engineered to elevate the production of formate from CO2 after
elucidating key responsible enzyme for the conversion of CO2 to formate.
Example 2: Altering Rubisco enzyme, PEP carboxylase, Fructose
bisphosphatase:-
The CO2 fixation pathway can refer to metabolic pathway in bacteria which
enables the uptake of carbon into the cell. Key enzymes in the carbon fixation
pathway can include Rubisco enzyme, PEP carboxylase, Fructose bisphosphatase.
Modification or upregulation of any one, two, or three of these genes of enzymes
in a bacterial strain may increase carbon fixation pathway in the cells, and may
enhance the soil carbon, soil productivity, and also enhance the crop growth.
16
Example 3: Impact of CO2 fixation pathway incorporation on CO2 absorption by
microbes
Example 4: The homologous expression or over expression of FDH (SEQ ID
NO: 2 and SEQ ID NO: 3) in Methylobacterium spp. under the control of
glyceraldehyde 3-phosphate dehydrogenase promoter (PGAP, SEQ ID NO: 1).
Naturally, this promoter drives the expression of glyceraldehyde-3-phosphate
dehydrogenase gene constitutively. The product of this gene catalyzes an
important energy-yielding step in carbohydrate metabolism, the reversible
oxidative phosphorylation of glyceraldehyde-3-phosphate in the presence of
inorganic phosphate and nicotinamide adenine dinucleotide (NAD).
Example 5: Genetic modification of the microbe particularly Methylobacterium
spp, for green house gas remediation but not limited to CO2 has been done by over
expression of the FDH (Sequence ID: 2 and 3) using the strong constitutive
promoter (PGAP). The gene sequences (sequence ID: 2 and 3) along with promoter
and integration flanking sites (sequence ID: 4 and 5) were synthesized by gene
synthesis or PCR based overlap extension method and cloned in pUC57 vector
using over lap extension PCR or restriction enzyme-based method. Final
integration contruct (Sequence ID: 7) was transformed into Methylobacterium
spp, using the standard electroporation procedure (1800 V, 25 µF, 200O) and
screened for presence of integrated construct in the chromosome by PCR based
confirmation.
Example 6: Integration of heterologous or homologous genes require certain gene
or genes to be deleted. For the integration of FDH (Sequence ID: 2 and 3),
sequence ID: 4 and 5 has been chosen as the site of integration.
Example 7: Antiobiotic markers are necessary for initial deletion construct
development and transformant screening. In this present invention, antibiotic
17
marker but not limited to Kanamycin, tetracycline or chloramphenicol was used as
a selection marker. The selection marker kanamycin along with promoter was
amplified from pUC57 (genscript) and deletion construct was synthesized by
placing the sequence ID 4 and 5 flanking the kanamycin marker (Sequence ID: 6).
The complete construct was synthesized by over lap extension PCR or gene
synthesis and clone in pUC57 vector with ampicillin selection marker. The overall
complete construct was linearized with restriction enzyme (Xba1) and
transformed into Methylobacterium spp, using electroporation method and
positive transformant were selected on kanamycin containg medium and
confirmation of sequence ID 4 and 5 along with selection marker was performed
by either colony PCR or PCR with purified genomic DNA.
18
SEQUENCE LISTING
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<120> Genetic modification of endophytic/ epiphytic/rhizospheric
microbes for improved CO2 fixation for crops
<130> 004
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<141> 2021-09-20
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ggccatagct gcgtgaaggg ccgcttcgcc tacggctacg ccactcacaa ggaccgcatc 900
accaagccga tgatccggga gaagatcacg gatccgtggc gcgaggtcac ctgggaggag 960
gcgatcgacc gggcggcctc cgagttcaag cggatccagg ccacctacgg caaggattcg 1020
gtcggcggca tcacctcgtc ccgctgcacc aacgaggagg cctacctcgt ccagaagctg 1080
gtgcgcgcgg ccttcggcaa caacaacgtc gatacctgcg cccgcgtctg ccactcgccg 1140
accggctacg gcctgatgtc gacgctcggc acctcggccg gcacccagga cttcgcctcg 1200
gtggcgcatt ccgacgtgat cctcgtcatc ggcgccaacc cgacggacgg ccatccggtc 1260
ttcggctcgc gcatgaagaa gcgcctgcgc gagggggcga agctcatcgt cgccgatccg 1320
cgcaagatcg acctcgtgaa gtcgccccac atcaaggcgg acttccacct gcccctgaag 1380
cccggctcca acgtcgcctt catcaactcg atcgcgcacg tcatcgtcac ggaagggctg 1440
atcgacgagg cctatatccg cgcgcgctgc gacctcggcg agttcgagtc ctgggcccgc 1500
ttcatcgcgg aggagcgcca ctcccccgag aaccagcagc agttcaccgg cctcgatccc 1560
gaacaggtgc gcggcgcggc gcggctctac gccacgggcg gcgcggccgg catctattac 1620
gggctgggcg tcaccgagca cagccagggc tcgaccatgg tgatgggcat ggccaacatc 1680
21
gccatggcca ccggcaacat cggcaagctc ggtgcgggcg taaacccctt gcgcggccag 1740
aacaacgtgc aaggatcctg cgacatgggc tcgttccccc acgagctcac cggctaccgc 1800
cacgtctcgg acgatgccac ccgcgagagc ttcgaggcga tctggggtgc caagctcgac 1860
aacgcgccag gacttcgcat caccaacatg ctcgatgagg ccgtcgatgg cagcttcaag 1920
ggcatgtaca tccagggcga ggacatcgcg cagtccgatc ccgacaccca tcacgtcacg 1980
tcaggcctca aggcgatgga atgcatcgtc gtgcaggacc tgttcctgaa cgagacggca 2040
aaatacgccc acgtcttcct gcccggagcc tcattcctgg agaaggacgg caccttcacc 2100
aatgccgagc gccgcatcag ccgcgtgcgc aaggtcatgc ccccgatggg cggctacggc 2160
gattgggagg gcacggtgct gctctctaac gcgctgggct acccgatgaa ctacagccac 2220
ccatccgaga tcatggacga gatcgcggcc ctcaccccga gcttcaccgg ggtgtcctat 2280
gccaaactcg aggaactcgg ctcggtacag tggccctgca atgagaaggc gccgctcggt 2340
acgccgatga tgcacgtgga ccgcttcgtg cgcggcaagg gccggttcat gatcaccgag 2400
ttcgtggcga ctgaggagcg cacgggggcg aagttcccgc tcatcctcac cacgggtcgg 2460
atcctctccc agtacaacgt cggcgctcag acccggcgca cccacaattc gcgctggcac 2520
gaggaggacg tgctggagat ccaccccttc gacgcggagc tgcgcggtat catggacggc 2580
gacctcgtcg ccctggagag ccgctcgggc gacatcgctc tgaaggccaa gatttcggag 2640
22
cgcatgcagc caggcgtggt ctacaccacc ttccaccacg ctaagaccgg cgccaacgtc 2700
atcaccaccg actattcgga ctgggccacg aactgccccg agtacaaagt gacggcggtg 2760
caggtccggc gtaccaaccg gccctccgac tggcaggcga agttctacga gggagatttc 2820
tccctgaccc ggatcgccca ggccgcggcg gagtga 2856
<210> 3
<211> 2973
<212> DNA
<213> FDH
<400> 3
atgaacgacg gccccgatct ccacggcaag gcgacggacc ggaccgaggt ccgggcgcgg 60
acgcgccagg atgcgggcgg cgccgctccg gaggggcggc cgggcgcggg cggcccctat 120
tcgcagggcg ccaaggccgg tggccaggcc tcgcccgagc cgagcgggct tgtcggcctg 180
acggagcggc ccgcagcgcc gccgagcatc gcgttcgagc tcgacggcga gacggtcgag 240
gcgcggccgg gcgagaccat ctgggcggtc gccaagcgcc tcggcaccca catcccgcat 300
ctctgccaca agccggagcc cggctaccgg ccggacggca attgccgcgc ctgcatggtc 360
gagatcgagg gcgagcgcgt gctcgcggcc tcctgcaagc gcacgcccgc catcggcatg 420
aaggtgaaga ccgccaccga gcgcgcggag aaggcccgcg ccatggtgat ggaattgctg 480
23
gtggccgacc agccggaccg ggcgacttcg cacgatccga cctcgcattt ctgggcgcag 540
gccgatttcg tggacatcgc cgcgagccgc tttcccgcgg ccgagcgctg gcaggccgac 600
gcgagccatc cggccatgcg ggtgaacctc gatgcctgca tccagtgcaa tctctgcgtc 660
cgcgcctgcc gcgaggtcca ggtcaacgac gtgatcggca tggcctaccg ctcggccggg 720
tccaaggtgg tgttcgactt cgacgacccg atgggcggct cgacctgcgt cgcctgcggc 780
gagtgcgtgc aggcctgtcc gaccggggcg ctgatgccct cggcctatct cgacgcgaac 840
gagacccggg tcgtctatcc cgaccgtgag gtcgcctcgc tctgccccta ttgcggtgtc 900
ggctgccagg tctcctacaa ggtcaaggac gagcgcatcg tctatgccga gggcctgaac 960
ggcccggcca accacaaccg gctctgcgtg aagggccgct tcggcttcga ctacgtgcac 1020
catccccacc ggctgaccaa gcccctgatc cggctcgaca acgccccgaa ggacgcgaac 1080
gaccaggtcg atcccgccaa cccctggacg catttccgcg aggccacctg ggaggaggcc 1140
ctcgaccgcg ccgcggccgg gctgcggacg gtccgcgaca gccacggccc caaggcgctc 1200
gccggcttcg gctcggccaa gggctcgaac gaggaggcct atctcttcca gaagctggtc 1260
cgcctcggct tcggctccaa caacgtcgac cattgcaccc ggctctgcca cgcctcctct 1320
gtggcggccc tgatggaggg gctgaactcg ggcgccgtga ccgcgccctt ctcggcggcg 1380
24
ctcgatgccg aggtgatcat cgtcatcggg gccaacccca ccgtgaacca cccggtcgcg 1440
gcgaccttcc tcaagaatgc ggtgaagcag cgcggcgcca agctgatcgt catggatccg 1500
cgccggcagg tgctgtcccg gcacgcctac aggcacctcg ccttcaagcc gggctcggac 1560
gtggcgatgc tgaacgcgat gctgaacgtc atcatcgagg agaagctcta cgacgagcag 1620
tacatcgccg ggtacaccga gaacttcgag gcgctgcggc agaagatcgt cgacttcacg 1680
cccgagaaga tggaggccgt ctgcggcatc gaggccgcga ccctgcgcga ggtcgcgcgc 1740
ctctacgccc ggtcgaaggc ctcgatcatc ttctggggca tgggtatcag ccagcacgtc 1800
cacggcaccg acaactcgcg ctgcctgatc gccctggccc tcgtcaccgg ccagatcgga 1860
cggccgggca cggggctgca ccccctgcgc ggccagaaca acgtgcaggg cgcctccgat 1920
gcgggcctga tcccgatggt ctatccggac taccagtccg tcgagaaggc ggcggtgcgc 1980
gagctgttcg aggcgttctg gggccagtcc ctcgatccga agcgcgggct gaccgtggtc 2040
gagatcatgc gggcgatcca tgccggcgag atccgcggca tgttcatcga gggcgagaac 2100
ccggccatgt cggatcccga cctcaaccac gcccggcacg cgctggcgat gctcgaccat 2160
ctcgtcgtgc aggacctgtt cctcaccgag acggccttcc acgccgacgt ggtgctgccg 2220
gcctccgcct tcgccgagaa ggcgggcagc ttcaccaaca cggaccggcg cgtccagatc 2280
gcccagcccg tcgtgccgcc cccgggcgac gcgcgccagg attggtggat catccaggaa 2340
25
ctcgcccggc ggatggggct cgactggagc tatgccggcc cggccgacgt gttcgccgag 2400
atggcgcagg tcatgccctc gctcgccaac atcacctggg agcgcctgga gcgcgagggc 2460
gccgtgacct acccggtcga cgcgcccgac aagccgggca acgagatcat cttctacgac 2520
ggcttcccga ccgagagcgg gcgcgccaag atcgtgccgg cggcgatcgt gcccccggac 2580
gaggtgcccg acaccgagtt cccgatggtg ctctcgaccg gccgggtgct ggagcattgg 2640
catacgggct cgatgacccg gcgcgccggc gtgctcgacg cgctggagcc cgaggcggtg 2700
gccttcctgg ccccgcgcga gctctaccgc ctcggcctcg agcccggcat gacgatgcgg 2760
ctcgagacgc ggcgcggcgc cgtcgaggtg aaggtccggt ccgaccgcga cgttccggac 2820
ggcatggtgt tcatgccctt ctgctacgcg gaggccgcgg ccaacctcct caccaccccc 2880
gccctcgatc cgctgggcct gatccccgag ttcaagttct gcgcggcccg ggtctcgccc 2940
gtccgggccg cgccgccgat cgccgccgag tga 2973
<210> 4
<211> 521
<212> DNA
<213> site of integration of FDH
<400> 4
cacgggctcg aggtcacggc ccccttcggc gccgcgctcg ccgccgccct ggtggccgag 60
26
cgcggactca gcgaggtcgc ggtcaccgcg atcggccatc gccgcggcga gggcgtgctg 120
cgcgtcctgc cggcctagga gcagcggcgg gcgggcccga ccgccctccc ggacggcgtc 180
gagaccagag agtttctccg aactcttact ctgaagaccg gttctcccct cgagcgccgg 240
accccgcacg gggtccgcgc cccttcagtc ccgaggagag cgccgatgcg tgcactggtg 300
tggcacggaa cccaggacgt ccggtgcgac tcggttcctg atccggagat cgagcacgag 360
cgcgacgcca tcatcaaggt cacgagttgc gccatctgcg gctcggacct gcacctgttc 420
gaccatttca tacccacgat gaagtcgggc gacatcctcg gccacgagac catgggcgag 480
gtggtcgagg tgggctcggc ggccaagtcc aagctcaagg t 521
<210> 5
<211> 616
<212> DNA
<213> site of integration of FDH
<400> 5
tcaacttcga gaccgacagc gtgatcgagc gcctgaacgc gatgaccgcg ggcaagggcc 60
ccgagaaatg catcgacgcg gtcgggctcg aggctcacgc cgccggcacc gtcgatgcga 120
tgtacgaccg cgccaagcag gcgatgatgc tggagaccga ccggccgcat gtcctgcgcg 180
agatgatcta tgtctgccgg cccgccggca cgctctcggt gcccggcgtc tatggcggcc 240
27
tcatcgacaa gatcccgttc ggcgcgctga tgaacaaggg cctgacgatc cgcacgggcc 300
agacccacgt caatcgctgg agcgacgacc tgctgcggcg gatcgaggag ggtcagatcg 360
atccctcctt cgtgatcacc cataccgagc cgctggagcg cgggcccgag atgtacaaga 420
ccttccgcga caagcaggac ggctgcatca aggtcgtgct caagccctga ctccacccgt 480
tccccttctc agaggaggtg ccgtcatggg ccagcacaat cccaggaacg tcctgccgcg 540
gaccgcgctg cgcgggcgct cgcaatccgt cgccgaccgc gtcgcgcagg ggctcgggct 600
cttctcgatc ggcctc 616
<210> 6
<211> 1082
<212> DNA
<213> kanamycin marker
<400> 6
gagtgcgacc aatgcaagcg cggctagctt gcagtgggct tacatggcga tagctagact 60
gggcggtttt atggacagca agcgaaccgg aattgccagc tggggcgccc tctggtaagg 120
ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc gccaaggatc tgatggcgca 180
ggggatcaag atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg 240
gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac 300
28
aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg 360
ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc 420
ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg 480
aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc 540
accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc 600
ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta 660
ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg 720
cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg 780
tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat 840
tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc 900
gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta 960
tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag 1020
tgctgcgcga gatgatctat gtatgccgac ccggcggcct gatctcgatt cccggcgtct 1080
ac 1082
<210> 7
29
<211> 7852
<212> DNA
<213> artificial sequence
<220>
<223> artificial sequence
<400> 7
cacgggctcg aggtcacggc ccccttcggc gccgcgctcg ccgccgccct ggtggccgag 60
cgcggactca gcgaggtcgc ggtcaccgcg atcggccatc gccgcggcga gggcgtgctg 120
cgcgtcctgc cggcctagga gcagcggcgg gcgggcccga ccgccctccc ggacggcgtc 180
gagaccagag agtttctccg aactcttact ctgaagaccg gttctcccct cgagcgccgg 240
accccgcacg gggtccgcgc cccttcagtc ccgaggagag cgccgatgcg tgcactggtg 300
tggcacggaa cccaggacgt ccggtgcgac tcggttcctg atccggagat cgagcacgag 360
cgcgacgcca tcatcaaggt cacgagttgc gccatctgcg gctcggacct gcacctgttc 420
gaccatttca tacccacgat gaagtcgggc gacatcctcg gccacgagac catgggcgag 480
gtggtcgagg tgggctcggc ggccaagtcc aagctcaagg ttccgcggat cggttgatcc 540
cggcggcgac ggcgcggccg gtccgccatg ggtcatgtcc ggctccggtt catcgccggt 600
tcagcgccgg cagccacaga gcaatccgca tcgcggaggt gccgtcgggc ccccgccgcg 660
caccgctcgc cgcctcggac gcccgctgcg tggcgcccct taagcaggaa ggaaacacgc 720
30
catggccctc atcaaggaaa tcgactacgg cacgccgatc cgcgtcgccg agcagacggt 780
gtcgctgacc atcgacggca tggccgtgac ggtgccggcc ggcacctccg tgatggccgc 840
ggcgatgacc gcgggcacgc agatccccaa gctctgcgcc accgactcgc tggagccctt 900
cggctcctgc cgcctctgcc tcgtggagat cgagggacgg cgcggcacgc ccgcctcctg 960
caccacgccg gccgagaacg gcatggtggt gcacacgcag accgacaagc tcgcgcgcct 1020
gcgcaagggc gtgatggagc tctacatctc cgatcacccg ctcgactgcc tgacctgcgc 1080
ggcgaacggc gattgcgagc tgcagacgca ggcgggcgtc gtcggcctgc gcgacgtgcg 1140
ctacggctac gagggcgaca accacgtccg cccgagctcc gagcgctacc tgccgaagga 1200
cgagtcgaac ccgtatttca cctacgaccc gtcgaagtgc atcgtctgca atcgctgcgt 1260
gcgggcctgc gaggaggtgc agggcacctt cgcgctgacc atcgccggcc gcggcttcga 1320
cagccgcgtc gccgccggcc cgacgaactt catggaatcc gagtgcgtct cgtgcggcgc 1380
ctgcgtgcag gcctgcccga ccgcgacgct ccaggagaag tcgatccacg aatacggcca 1440
gccggagcac gccgaggtca cgacctgcgc ctattgcggc gtcggctgct ccttcaaggc 1500
cgagatgcag ggcgaccgcg tcgtgcgcat ggtgccctac aagggcggca aggcgaatga 1560
cggccatagc tgcgtgaagg gccgcttcgc ctacggctac gccactcaca aggaccgcat 1620
31
caccaagccg atgatccggg agaagatcac ggatccgtgg cgcgaggtca cctgggagga 1680
ggcgatcgac cgggcggcct ccgagttcaa gcggatccag gccacctacg gcaaggattc 1740
ggtcggcggc atcacctcgt cccgctgcac caacgaggag gcctacctcg tccagaagct 1800
ggtgcgcgcg gccttcggca acaacaacgt cgatacctgc gcccgcgtct gccactcgcc 1860
gaccggctac ggcctgatgt cgacgctcgg cacctcggcc ggcacccagg acttcgcctc 1920
ggtggcgcat tccgacgtga tcctcgtcat cggcgccaac ccgacggacg gccatccggt 1980
cttcggctcg cgcatgaaga agcgcctgcg cgagggggcg aagctcatcg tcgccgatcc 2040
gcgcaagatc gacctcgtga agtcgcccca catcaaggcg gacttccacc tgcccctgaa 2100
gcccggctcc aacgtcgcct tcatcaactc gatcgcgcac gtcatcgtca cggaagggct 2160
gatcgacgag gcctatatcc gcgcgcgctg cgacctcggc gagttcgagt cctgggcccg 2220
cttcatcgcg gaggagcgcc actcccccga gaaccagcag cagttcaccg gcctcgatcc 2280
cgaacaggtg cgcggcgcgg cgcggctcta cgccacgggc ggcgcggccg gcatctatta 2340
cgggctgggc gtcaccgagc acagccaggg ctcgaccatg gtgatgggca tggccaacat 2400
cgccatggcc accggcaaca tcggcaagct cggtgcgggc gtaaacccct tgcgcggcca 2460
gaacaacgtg caaggatcct gcgacatggg ctcgttcccc cacgagctca ccggctaccg 2520
ccacgtctcg gacgatgcca cccgcgagag cttcgaggcg atctggggtg ccaagctcga 2580
32
caacgcgcca ggacttcgca tcaccaacat gctcgatgag gccgtcgatg gcagcttcaa 2640
gggcatgtac atccagggcg aggacatcgc gcagtccgat cccgacaccc atcacgtcac 2700
gtcaggcctc aaggcgatgg aatgcatcgt cgtgcaggac ctgttcctga acgagacggc 2760
aaaatacgcc cacgtcttcc tgcccggagc ctcattcctg gagaaggacg gcaccttcac 2820
caatgccgag cgccgcatca gccgcgtgcg caaggtcatg cccccgatgg gcggctacgg 2880
cgattgggag ggcacggtgc tgctctctaa cgcgctgggc tacccgatga actacagcca 2940
cccatccgag atcatggacg agatcgcggc cctcaccccg agcttcaccg gggtgtccta 3000
tgccaaactc gaggaactcg gctcggtaca gtggccctgc aatgagaagg cgccgctcgg 3060
tacgccgatg atgcacgtgg accgcttcgt gcgcggcaag ggccggttca tgatcaccga 3120
gttcgtggcg actgaggagc gcacgggggc gaagttcccg ctcatcctca ccacgggtcg 3180
gatcctctcc cagtacaacg tcggcgctca gacccggcgc acccacaatt cgcgctggca 3240
cgaggaggac gtgctggaga tccacccctt cgacgcggag ctgcgcggta tcatggacgg 3300
cgacctcgtc gccctggaga gccgctcggg cgacatcgct ctgaaggcca agatttcgga 3360
gcgcatgcag ccaggcgtgg tctacaccac cttccaccac gctaagaccg gcgccaacgt 3420
catcaccacc gactattcgg actgggccac gaactgcccc gagtacaaag tgacggcggt 3480
33
gcaggtccgg cgtaccaacc ggccctccga ctggcaggcg aagttctacg agggagattt 3540
ctccctgacc cggatcgccc aggccgcggc ggagtgagaa cccataaaat gtgatcgtcc 3600
gcggatcggt tgatcccggc ggcgacggcg cggccggtcc gccatgggtc atgtccggct 3660
ccggttcatc gccggttcag cgccggcagc cacagagcaa tccgcatcgc ggaggtgccg 3720
tcgggccccc gccgcgcacc gctcgccgcc tcggacgccc gctgcgtggc gccccttaag 3780
caggaaggaa acacgccatg aacgacggcc ccgatctcca cggcaaggcg acggaccgga 3840
ccgaggtccg ggcgcggacg cgccaggatg cgggcggcgc cgctccggag gggcggccgg 3900
gcgcgggcgg cccctattcg cagggcgcca aggccggtgg ccaggcctcg cccgagccga 3960
gcgggcttgt cggcctgacg gagcggcccg cagcgccgcc gagcatcgcg ttcgagctcg 4020
acggcgagac ggtcgaggcg cggccgggcg agaccatctg ggcggtcgcc aagcgcctcg 4080
gcacccacat cccgcatctc tgccacaagc cggagcccgg ctaccggccg gacggcaatt 4140
gccgcgcctg catggtcgag atcgagggcg agcgcgtgct cgcggcctcc tgcaagcgca 4200
cgcccgccat cggcatgaag gtgaagaccg ccaccgagcg cgcggagaag gcccgcgcca 4260
tggtgatgga attgctggtg gccgaccagc cggaccgggc gacttcgcac gatccgacct 4320
cgcatttctg ggcgcaggcc gatttcgtgg acatcgccgc gagccgcttt cccgcggccg 4380
agcgctggca ggccgacgcg agccatccgg ccatgcgggt gaacctcgat gcctgcatcc 4440
34
agtgcaatct ctgcgtccgc gcctgccgcg aggtccaggt caacgacgtg atcggcatgg 4500
cctaccgctc ggccgggtcc aaggtggtgt tcgacttcga cgacccgatg ggcggctcga 4560
cctgcgtcgc ctgcggcgag tgcgtgcagg cctgtccgac cggggcgctg atgccctcgg 4620
cctatctcga cgcgaacgag acccgggtcg tctatcccga ccgtgaggtc gcctcgctct 4680
gcccctattg cggtgtcggc tgccaggtct cctacaaggt caaggacgag cgcatcgtct 4740
atgccgaggg cctgaacggc ccggccaacc acaaccggct ctgcgtgaag ggccgcttcg 4800
gcttcgacta cgtgcaccat ccccaccggc tgaccaagcc cctgatccgg ctcgacaacg 4860
ccccgaagga cgcgaacgac caggtcgatc ccgccaaccc ctggacgcat ttccgcgagg 4920
ccacctggga ggaggccctc gaccgcgccg cggccgggct gcggacggtc cgcgacagcc 4980
acggccccaa ggcgctcgcc ggcttcggct cggccaaggg ctcgaacgag gaggcctatc 5040
tcttccagaa gctggtccgc ctcggcttcg gctccaacaa cgtcgaccat tgcacccggc 5100
tctgccacgc ctcctctgtg gcggccctga tggaggggct gaactcgggc gccgtgaccg 5160
cgcccttctc ggcggcgctc gatgccgagg tgatcatcgt catcggggcc aaccccaccg 5220
tgaaccaccc ggtcgcggcg accttcctca agaatgcggt gaagcagcgc ggcgccaagc 5280
tgatcgtcat ggatccgcgc cggcaggtgc tgtcccggca cgcctacagg cacctcgcct 5340
35
tcaagccggg ctcggacgtg gcgatgctga acgcgatgct gaacgtcatc atcgaggaga 5400
agctctacga cgagcagtac atcgccgggt acaccgagaa cttcgaggcg ctgcggcaga 5460
agatcgtcga cttcacgccc gagaagatgg aggccgtctg cggcatcgag gccgcgaccc 5520
tgcgcgaggt cgcgcgcctc tacgcccggt cgaaggcctc gatcatcttc tggggcatgg 5580
gtatcagcca gcacgtccac ggcaccgaca actcgcgctg cctgatcgcc ctggccctcg 5640
tcaccggcca gatcggacgg ccgggcacgg ggctgcaccc cctgcgcggc cagaacaacg 5700
tgcagggcgc ctccgatgcg ggcctgatcc cgatggtcta tccggactac cagtccgtcg 5760
agaaggcggc ggtgcgcgag ctgttcgagg cgttctgggg ccagtccctc gatccgaagc 5820
gcgggctgac cgtggtcgag atcatgcggg cgatccatgc cggcgagatc cgcggcatgt 5880
tcatcgaggg cgagaacccg gccatgtcgg atcccgacct caaccacgcc cggcacgcgc 5940
tggcgatgct cgaccatctc gtcgtgcagg acctgttcct caccgagacg gccttccacg 6000
ccgacgtggt gctgccggcc tccgccttcg ccgagaaggc gggcagcttc accaacacgg 6060
accggcgcgt ccagatcgcc cagcccgtcg tgccgccccc gggcgacgcg cgccaggatt 6120
ggtggatcat ccaggaactc gcccggcgga tggggctcga ctggagctat gccggcccgg 6180
ccgacgtgtt cgccgagatg gcgcaggtca tgccctcgct cgccaacatc acctgggagc 6240
gcctggagcg cgagggcgcc gtgacctacc cggtcgacgc gcccgacaag ccgggcaacg 6300
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agatcatctt ctacgacggc ttcccgaccg agagcgggcg cgccaagatc gtgccggcgg 6360
cgatcgtgcc cccggacgag gtgcccgaca ccgagttccc gatggtgctc tcgaccggcc 6420
gggtgctgga gcattggcat acgggctcga tgacccggcg cgccggcgtg ctcgacgcgc 6480
tggagcccga ggcggtggcc ttcctggccc cgcgcgagct ctaccgcctc ggcctcgagc 6540
ccggcatgac gatgcggctc gagacgcggc gcggcgccgt cgaggtgaag gtccggtccg 6600
accgcgacgt tccggacggc atggtgttca tgcccttctg ctacgcggag gccgcggcca 6660
acctcctcac cacccccgcc ctcgatccgc tgggcctgat ccccgagttc aagttctgcg 6720
cggcccgggt ctcgcccgtc cgggccgcgc cgccgatcgc cgccgagtga gagtgcgacc 6780
aatgcaagcg cggctagctt gcagtgggct tacatggcga tagctagact gggcggtttt 6840
atggacagca agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc 6900
ctgcaaagta aactggatgg ctttcttgcc gccaaggatc tgatggcgca ggggatcaag 6960
atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc 7020
aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat 7080
cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt 7140
caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg 7200
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gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag 7260
ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc 7320
tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc 7380
tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga 7440
agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga 7500
actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg 7560
cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg 7620
tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc 7680
tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc 7740
cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag tgctgcgcga 7800
gatgatctat gtatgccgac ccggcggcct gatctcgatt cccggcgtct ac 7852 ,CLAIMS:A composition comprising genetically modified micro-organism(s)
consisting of modification of _ Rubisco enzyme of Reductive pentose
phosphate cycle, PEP carboxylase of Reductive TCA cycle, and Fructose
bisphosphatase that converts fructose-1,6-bisphosphate to fructose 6-
phosphate, Formate dehydrogenase wherein the said micro-organism is an
endophyte, an epiphyte and a rhizospheric microbe.
2. The composition as claimed in claim 1, wherein the process carried for
gene modification consists of sucicidal venctor or plasmid.
3. The composition as claimed in claim 2, wherein the plasmid vector of
carrying a DNA construct comprising a nucleotide sequence represented
by SEQ ID NO. 7.
4. The composition as claimed in claim 4, wherein SEQ ID NO: 7 consisting
of 7852 bp with selective restriction sites along with required modified
gene sequences.
5. The composition as claimed in claim 2, wherein the plasmid vector
carrying a DNA construct comprising a nucleotide sequence represented
by SEQ ID NO. 2 and 3.
6. The composition as claimed in claim 1, wherein the said plasmid vector is
expressed in miro-organism selected from the group comprising of
endophytic bacteria, epiphytic bacteria, rhizospheric bacteria and fungi.
7. The composition as claimed in claim 1, wherein the bacteria is selected
from the group comprising of cultivable bacteris (viz) Methylobacterium
extorquens; Beijerinckia indica; Azoarchus communis and uncultivable
bacteria adapted to cultivabe using Ichip method (viz) Pseudomonas sp.;
Bacillus sp. And Sphingomonas sp.
8. A process for the preparation of genetically modified micro-organism(s)
for improved nitrogen fixation and its delivery to crop-plants for
assimilation by homologous recombinations.
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a. Cloning a of a sequence having SEQ ID NO 2 and 3. targeting the
Glutathione dependent formate dehydrogenase gene in integration
vector or sucidal vector flanking the region SEQ ID NO.4 and 5.
b. Transforming pUC57 vector harboring SEQ ID NO.7 containing
the FDH into a host cell, wherein the said micro-organism is an
endophyte, an epiphyte and a rhizospheric microbe. wherein the
said micro-organism is uncharacterized and non-cultivated.