WO2023181065A1

WO2023181065A1 - Genetic modification of endophytic/ epiphytic/rhizospheric microbes for improved co2 fixation for crops

Info

Publication number: WO2023181065A1
Application number: PCT/IN2023/050269
Authority: WO
Inventors: Rahul Raju KANUMURU
Original assignee: Fertis India Pvt. Ltd.
Priority date: 2022-03-20
Filing date: 2023-03-20
Publication date: 2023-09-28

Abstract

The invention discloses 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, the present invention discloses the 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.

Description

“Genetic modification of endophytic/ epiphytic/rhizospheric microbes for improved CO₂ fixation for crops’’

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

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 tire soil carbon, which is depleting, and to enhance the soil productivity, and also to enhance the crop growth.

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).

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). 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 carboxy lase/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 non- leguminous 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 non- leguminous 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.

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 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 necessities ofprocess and pathways in Agri-beneficial organisms to assimilate CH₄ or N₂O and usage for production of biomass and production of agri- beneficial 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.

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. 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 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.

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 NOT 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, Alcaligene, Saccharomyces cerevisiae, Saccharomyces lactis, Brevibacterium, , Kluyveromyces lactis, Epichloe typhina, Enterococcus, Corynebacterium, Arthobacter, Pichia, Zymomonas, Saccharomyces carlsbergensis, Salmonella, Zymomonas, Rhodacoccus, Escherichia (e.g., E. Coli), Hansenula, Firmicutes, Rubrivivax, Dinoroseobacter shibae, Methylobacterium nodularis, 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, Epichloetyphina, 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 repeatsj/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 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-N- nitrosurea, 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 dimethyl- benz(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.

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 Ci 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. 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 contract (Sequence ID: 7) was transformed into Methylobacterium spp, using the standard electroporation procedure (1800 V, 25 pF, 200Q) 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 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 (Xbal) 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.

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<212> DNA

<213> FDH

<400> 2 atggccctca tcaaggaaat cgactacggc acgccgatcc gcgtcgccga gcagacggtg 60 tcgctgacca tcgacggcat ggccgtgacg gtgccggccg gcacctccgt gatggccgcg 120 gcgatgaccg cgggcacgca gatccccaag ctctgcgcca ccgactcgct ggagccctc 180 ggctcctgcc gcctctgcct cgtggagatc gagggacggc gcggcacgcc cgcctcctgc 240 accacgccgg ccgagaacgg catggtggtg cacacgcaga ccgacaagct cgcgcgcctg 300 cgcaagggcg tgatggagct ctacatctcc gatcacccgc tcgactgcct gacctgcgcg 360 gcgaacggcg attgcgagct gcagacgcag gcgggcgtcg tcggcctgcg cgacgtgcgc 420 tacggctacg agggcgacaa ccacgtccgc ccgagctccg agcgctacct gccgaaggac 480 gagtcgaacc cgtatttcac ctacgacccg tcgaagtgca tcgtctgcaa tcgctgcgtg 540 cgggcctgcg aggaggtgca gggcacctc gcgctgacca tcgccggccg cggcttcgac 600 agccgcgtcg ccgccggccc gacgaactc atggaatccg agtgcgtctc gtgcggcgcc 660 tgcgtgcagg cctgcccgac cgcgacgctc caggagaagt cgatccacga atacggccag 720 ccggagcacg ccgaggtcac gacctgcgcc tattgcggcg tcggctgctc cttcaaggcc 780 gagatgcagg gcgaccgcgt cgtgcgcatg gtgccctaca agggcggcaa ggcgaatgac 840 ggccatagct gcgtgaaggg ccgcttcgcc tacggctacg ccactcacaa ggaccgcatc 900 accaagccga tgatccggga gaagatcacg gatccgtggc gcgaggtcac ctgggaggag 960 gcgatcgacc gggcggcctc cgagttcaag cggatccagg ccacctacgg caaggatcg 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 agtcaccgg cctcgatccc 1560 gaacaggtgc gcggcgcggc gcggctctac gccacgggcg gcgcggccgg catctattac 1620 gggctgggcg tcaccgagca cagccagggc tcgaccatgg tgatgggcat ggccaacatc 1680 gccatggcca ccggcaacat cggcaagctc ggtgcgggcg taaacccctt gcgcggccag 1740 aacaacgtgc aaggatcctg cgacatgggc tcgtccccc acgagctcac cggctaccgc 1800 cacgtctcgg acgatgccac ccgcgagagc tcgaggcga 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 gatgggagg gcacggtgct gctctctaac gcgctgggct acccgatgaa ctacagccac 2220 ccatccgaga tcatggacga gatcgcggcc ctcaccccga gcttcaccgg ggtgtcctat 2280 gccaaactcg aggaactcgg ctcggtacag tggccctgca atgagaaggc gccgctcggt 2340 acgccgatga tgcacgtgga ccgcttcgtg cgcggcaagg gccggtcat 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 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 gtggccgacc agccggaccg ggcgactcg 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 tgtcgact 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 tcggctcga 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 atctctcca gaagctggtc 1260 cgcctcggct tcggctccaa caacgtcgac cattgcaccc ggctctgcca cgcctcctct 1320 gtggcggccc tgatggaggg gctgaactcg ggcgccgtga ccgcgccctt ctcggcggcg 1380 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 aggcgtctg gggccagtcc ctcgatccga agcgcgggct gaccgtggtc 2040 gagatcatgc gggcgatcca tgccggcgag atccgcggca tgtcatcga gggcgagaac 2100 ccggccatgt cggatcccga cctcaaccac gcccggcacg cgctggcgat gctcgaccat 2160 ctcgtcgtgc aggacctgt cctcaccgag acggccttcc acgccgacgt ggtgctgccg 2220 gcctccgcct tcgccgagaa ggcgggcagc ttcaccaaca cggaccggcg cgtccagatc 2280 gcccagcccg tcgtgccgcc cccgggcgac gcgcgccagg attggtggat catccaggaa 2340 ctcgcccggc ggatggggct cgactggagc tatgccggcc cggccgacgt gttcgccgag 2400 atggcgcagg tcatgccctc gctcgccaac atcacctggg agcgcctgga gcgcgagggc 2460 gccgtgacct acccggtcga cgcgcccgac aagccgggca acgagatcat cttctacgac 2520 ggctcccga 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 cgtccggac 2820 ggcatggtgt tcatgccctt ctgctacgcg gaggccgcgg ccaacctcct caccaccccc 2880 gccctcgatc cgctgggcct gatccccgag tcaagtct 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 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 gcacctgtc 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 tcatcgacaa gatcccgttc ggcgcgctga tgaacaaggg cctgacgatc cgcacgggcc 300 agacccacgt caatcgctgg agcgacgacc tgctgcggcg gategaggag ggteagateg 360 atccctcctt cgtgatcacc cataccgagc cgctggagcg cgggcccgag atgtacaaga 420 ccttccgcga caagcaggac ggetgeatea aggtegtget 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 eggetagett gcagtgggct tacatggcga tagctagact 60 gggcggtttt atggacagca agcgaaccgg aattgccagc tggggcgccc tetggtaagg 120 ttgggaagcc etgeaaagta aactggatgg ctttcttgcc gccaaggatc tgatggcgca 180 ggggatcaag atctgatcaa gagacaggat gaggategtt tcgcatgatt gaacaagatg 240 gattgeaege aggtteteeg gccgcttggg tggagagget atteggetat gactgggcac 300 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg 360 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc 420 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gtgtcactg 480 aagcgggaag ggactggctg ctatgggcg aagtgccggg gcaggatctc ctgtcatctc 540 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc 600 ttgatccggc tacctgccca tcgaccacc aagcgaaaca tcgcatcgag cgagcacgta 660 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg 720 cgccagccga actgtcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg 780 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat 840 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc 900 gtgatattgc tgaagagct ggcggcgaat gggctgaccg cttcctcgtg ctttacggta 960 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag 1020 tgctgcgcga gatgatctat gtatgccgac ccggcggcct gatctcgat cccggcgtct 1080 ac 1082

<210> 7 <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 catggccctc atcaaggaaa tcgactacgg cacgccgatc cgcgtcgccg agcagacggt 780 gtcgctgacc atcgacggca tggccgtgac ggtgccggcc ggcacctccg tgatggccgc 840 ggcgatgacc gcgggcacgc agatccccaa gctctgcgcc accgactcgc tggagccct 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 gccgctcgc ctacggctac gccactcaca aggaccgcat 1620 caccaagccg atgatccggg agaagatcac ggatccgtgg cgcgaggtca cctgggagga 1680 ggcgatcgac cgggcggcct ccgagttcaa gcggatccag gccacctacg gcaaggatc 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 ctcggctcg 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 gcatctata 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 caacgcgcca ggacttcgca tcaccaacat gctcgatgag gccgtcgatg gcagcttcaa 2640 gggcatgtac atccagggcg aggacatcgc gcagtccgat cccgacaccc atcacgtcac 2700 gtcaggcctc aaggcgatgg aatgcatcgt cgtgcaggac ctgttcctga acgagacggc 2760 aaaatacgcc cacgtctcc tgcccggagc ctcattcctg gagaaggacg gcacctcac 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 acccacaat cgcgctggca 3240 cgaggaggac gtgctggaga tccacccct 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 gcaggtccgg cgtaccaacc ggccctccga ctggcaggcg aagttctacg agggagattt 3540 ctccctgacc cggatcgccc aggccgcggc ggagtgagaa cccataaaat gtgatcgtcc 3600 gcggatcggt tgatcccggc ggcgacggcg cggccggtcc gccatgggtc atgtccggct 3660 ccggtcatc 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 tcgagctcg 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 agtgcaatct ctgcgtccgc gcctgccgcg aggtccaggt caacgacgtg atcggcatgg 4500 cctaccgctc ggccgggtcc aaggtggtgt tcgactcga cgacccgatg ggcggctcga 4560 cctgcgtcgc ctgcggcgag tgcgtgcagg cctgtccgac cggggcgctg atgccctcgg 4620 cctatctcga cgcgaacgag acccgggtcg tctatcccga ccgtgaggtc gcctcgctct 4680 gcccctatg cggtgtcggc tgccaggtct cctacaaggt caaggacgag cgcatcgtct 4740 atgccgaggg cctgaacggc ccggccaacc acaaccggct ctgcgtgaag ggccgcttcg 4800 gctcgacta 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 cgccctctc ggcggcgctc gatgccgagg tgatcatcgt catcggggcc aaccccaccg 5220 tgaaccaccc ggtcgcggcg accttcctca agaatgcggt gaagcagcgc ggcgccaagc 5280 tgatcgtcat ggatccgcgc cggcaggtgc tgtcccggca cgcctacagg cacctcgcct 5340 tcaagccggg ctcggacgtg gcgatgctga acgcgatgct gaacgtcatc atcgaggaga 5400 agctctacga cgagcagtac atcgccgggt acaccgagaa ctcgaggcg ctgcggcaga 5460 agatcgtcga ctcacgccc 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 tccgcctcg ccgagaaggc gggcagctc accaacacgg 6060 accggcgcgt ccagatcgcc cagcccgtcg tgccgccccc gggcgacgcg cgccaggat 6120 ggtggatcat ccaggaactc gcccggcgga tggggctcga ctggagctat gccggcccgg 6180 ccgacgtgt cgccgagatg gcgcaggtca tgccctcgct cgccaacatc acctgggagc 6240 gcctggagcg cgagggcgcc gtgacctacc cggtcgacgc gcccgacaag ccgggcaacg 6300 agatcatctt ctacgacggc ttcccgaccg agagcgggcg cgccaagatc gtgccggcgg 6360 cgatcgtgcc cccggacgag gtgcccgaca ccgagtccc 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 aagtctgcg 6720 cggcccgggt ctcgcccgtc cgggccgcgc cgccgatcgc cgccgagtga gagtgcgacc 6780 aatgcaagcg cggctagct gcagtgggct tacatggcga tagctagact gggcggtttt 6840 atggacagca agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc 6900 ctgcaaagta aactggatgg ctttcttgcc gccaaggatc tgatggcgca ggggatcaag 6960 atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gatgcacgc 7020 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat 7080 cggctgctct gatgccgccg tgtccggct gtcagcgcag gggcgcccgg ttctttttgt 7140 caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg 7200 gctggccacg acgggcgttc ctgcgcagc tgtgctcgac gtgtcactg aagcgggaag 7260 ggactggctg ctatgggcg 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 tgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg 7620 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc 7680 tgaagagctt ggcggcgaat gggctgaccg ctcctcgtg ctttacggta tcgccgctcc 7740 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag tgctgcgcga 7800 gatgatctat gtatgccgac ccggcggcct gatctcgatt cccggcgtct ac 7852

Claims

WE CLAIM,

1. 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. 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.