MICROORGANISMS FOR THE PRODUCTION OF INORGANIC NITROGEN COMPOUNDS

Abstract

The invention proposes a method for developing a novel process for the sustainable production of inorganic nitrogen compounds in vitro / in vivo. The invention proposes the development of a novel microbial system for the efficient synthesis of ammonia and its oxidized derivatives. This novel microbial system technique can be implemented on a variety of different microbial hosts: modulating non-nitrogen-fixing microorganisms to fix nitrogen, or improving the nitrogen-fixing capacity of nitrogen-fixing microorganisms to synthesize ammonia or oxidize ammonia to produce ammonia and its oxidized derivatives, such as hydroxylamine, nitrate, nitrite, etc.

Description

[0001] GENETIC MODIFICATION OF MICROBES FOR PRODUCTION OF NITROGEN-CONTAINING, NON-CARBON COMPOUNDS

[0002] TECHNICAE FIELD OF THE INVENTION:

[0003] The present invention relates to a novel method for sustainable in vitro and in vivo production of non-carbon, nitrogen containing compounds including but not limited to Ammonia, Nitrate and Nitrite.

[0004] The present invention relates to a novel method that includes gene modifications in microbes, more specifically involving gene modifications resulting in Cl carbon fixation / increased C 1 carbon fixation with or without Hydrogen fixation with or without ammonia fixation and / or increased ammonia fixation with or without induced nitrogen fixation or increased nitrogen fixation with or without increased uptake / synthesis / regeneration of anyone or all compounds including ATP, NADH, NADPH, Pyruvate and Acetyl CoA with or without uptake / utilisation / reduction enhanced utilisation / reduction of Nitrate, Nitrite, N2O, N2O2 and / or carbon containing substrates.

[0005] The present invention additionally relates to gene modifications in microbes, more specifically involving gene modifications for enhancing transcription and / or translation of proteins involved in (i) uptake / fixation Carbon and / or (ii) uptake / fixation of ammonia and / or (iii) fixation of Nitrogen and / or (iv) uptake / fixation of Hydrogen and / or (v) uptake / synthesis / regeneration of anyone or all compounds including phosphate, ATP, NADH, NADPH, Pyruvate, CoA and Acetyl CoA and / or (vi) production of Nitrogen-containing, non-carbon compounds

[0006] The present invention also relates to gene modifications in microbes, more specifically involving gene modifications resulting in increased uptake / synthesis / regeneration of Phosphate and / or Hydrogen and / or CoenzymeA for (i) uptake / fixation Carbon and / or (ii) uptake / fixation of ammonia and / or (iii) fixation of Nitrogen and / or (iv) uptake / fixation of Hydrogen and / or (v) uptake / synthesis / regeneration of anyone or all compounds including phosphate, ATP, NADH, NADPH, Pyruvate, CoA and Acetyl CoA. and / or (vi) production of Nitrogen-containing, non-carbon compounds.

[0007] The present invention for production of Nitrogen-containing, non-carbon compounds, also relates to gene modifications in microbes, more specifically involving gene modifications resulting in increased uptake / synthesis / regeneration of Phosphate and / or Hydrogen and / or CoenzymeA for (i) enhanced carbon fixation and / or (ii) nitrogen fixation and / or (iii) ammonia fixation and / or (iv) uptake / synthesis / regeneration of anyone or all compounds including ATP, NADH, NADPH, Pyruvate and Acetyl CoA.

[0008] BACKGROUND AND PRIOR ART OF THE INVENTION:

[0009] US 20180297906A1 discloses methods including genetically modified bacterial strains for increasing nitrogen fixation in a non - leguminous plant. The modifications include either in genes or non - coding polynucleotides such as promoters of the bacteria's nitrogen fixation or assimilation genetic regulatory network, for enhancing the ability for fixing atmospheric nitrogen in the presence of exogenous nitrogen and produce 1 % or more of the fixed nitrogen in the plant. The patent focuses on only Nitrogen fixation process and related modifications, and there is no mention of the enhancement in carbon substrates, their assimilation and redox energy requirement.

[0010] WO2021222567A2 discloses methods and systems utilized for genetically modified bacterial strains comprising modifications in genes involved in regulation of nitrogen fixation. The modification in gene regulating nitrogen fixation results either in constitutive expression / activity of NifA in nitrogen limiting / non-nitrogen limiting conditions, decreased activity of GlnD and GlnE resulting in increased ammonium excretion.

[0011] The publication Tatemichi et al. AMB Expr (2020) 10: 150 describes the impact of sugar transport deletion on ammonia production, wherein combined effect of PtsG deletion and glnA deletion impacted on increased Ammonia release.

[0012] Few other references elaborate the ATP regeneration directly from AMP using enzyme combinations. The E. coli enzyme Poly phosphate kinase (PPK) catalyzes the kination of not only ADP but also other nucleoside diphosphates (NDP) using poly phosphate as a phosphate donor, resulting in nucleoside triphosphates (NTP). Polyphosphate-AMP-phosphotransferase (PAP), found in few microbes like Acinetobacter and Myxococcus, catalyzes the phospho-conversion of AMP to ADP using poly(P) as a phosphate donor. Combining PPK and PAP results in direct conversion of AMP to ATP regeneration

[0013] US20230107986A1 discloses genetically engineered microorganisms for production of carbon-based products of interest, such as sugars, alcohols, chemicals, amino acids, polymers, fatty acids and their derivatives, hydrocarbons, isoprenoids, and intermediates thereof, in engineered and / or evolved methylotrophs. The modifications in microorganisms occur in pathways and mechanisms which convert Cl compounds such as formate, formic acid, formaldehyde or methanol to organic carbon compounds.

[0014] US20170183665A1 provides disclosure for genetically modified microorganisms utilizing recombinant carbon fixation enzymes for CO2 fixation for production of a first essential biomass precursor. The genetic modifications in the microorganism occurs in carbon fixation pathways and enzymes associated with it particularly Calvin-Benson-Bassham cycle (C3 cycle) and Rubisco, Prk etc.

[0015] US8048661B2 discloses genetically modified microorganisms encoding modified pathways for enhancing carbon flux through acetyl-CoA. The methods of modification include altering the expression of enzymes in a reductive TCA or Wood-Ljungdahl pathway and increasing the availability of reducing equivalents in the presence of carbon monoxide or hydrogen.

[0016] US20200277636A1 discloses synthetic or genetically engineered microorganisms comprising methane, methanol utilizing pathways for conversion of Methane, methanol to organic compounds, industrial products, chemicals and intermediates. The patent provides methods for converting non-methanotropic, non- methylotropic microorganism into methanotropic, methylotropic microorganisms by incorporation of methane oxidizing and methanol-oxidizing metabolic pathways.

[0017] The technical limitation with the prior art for the production of nitrogencontaining, non-carbon containing compounds:

[0018] • Carbon, Nitrogen and Hydrogen substrate limitation: Less efficient / inefficient in uptake and utilisation of Carbon. Nitrogen and hydrogen substrates including sugars, organic acids and Cl compounds such as CO2, methane, methanol, Ammonia, Nitrate, Nitrite, N2O, H2, etc.

[0019] • Carbon, Nitrogen and Hydrogen loss: Carbon substrates used in various metabolic pathways and wasted in form of organic acids, CO2, CH4, alcohols, NH3, N2O, N2O2, etc.

[0020] • Redox energy inefficiency and imbalance: In the metabolic processes, the generated electrons and protons are not properly utilised due to inefficient synthesis / imbalance of ATP, NADH, NADPH, in in-vivo and in-vitro synthesis of nitrogen-containing, non-carbon compounds, and also for carbon and Nitrogen assimilation.

[0021] • Substrates and intermediates for carbon fixation, nitrogen fixation, Hydrogen fixation and regeneration of redox energy compounds.

[0022] • Energy molecules: In the metabolic processes, the generated Pyruvate and Acetyl-CoA are not utilised efficiently due to pyruvate loss in form of CO2 and Acetyl CoA diverted to non-specific, non-energy generation pathways.

[0023] OBJECT OF THE INVENTION:

[0024] The present invention relates to a novel method for sustainable in vitro and in vivo production of non-carbon, nitrogen containing compounds including but not limited to Ammonia, Nitrate and Nitrite.

[0025] It is an object of the present invention to provide microbes with gene modifications in nitrogen fixation for overproduction of ammonia, nitrate, nitrite, etc.

[0026] It is another object of the present invention to provide microbes with gene modifications in carbon fixation for overproduction / increased availability of redox energy compounds such as and not limited to ATP, NADH, NADPH, Hydrogen, Phosphate, Coenzyme-A, Pyruvate, required for enhanced production of Ammonia, nitrate, nitrite, etc.

[0027] It is another object of the present invention to provide above genetic modifications in epiphytes, endophytes, rhizosphere / free-living microbes.

[0028] SUMMARY OF THE INVENTION:

[0029] The present invention relates to a novel method for sustainable in vitro and in vivo production of non-carbon, nitrogen containing compounds including but not limited to Ammonia, Nitrate and Nitrite.

[0030] In one aspect, the present invention provides genetically modified microbes with modifications in both carbon and nitrogen fixation to enhance production of nitrogen-containing, non-carbon compounds such as Ammonia, Nitrate, Nitrite, etc. The present invention relates to a novel method that includes gene modifications in microbes, more specifically involving gene modifications resulting in carbon fixation / increased carbon fixation and / or nitrogen and / or ammonia fixation / increased nitrogen fixation and / or ammonia fixation with or without increased synthesis / regeneration of anyone or all compounds including ATP, NADH, NADPH, Phosphate, Hydrogen, Coenzyme-A, Pyruvate and Acetyl CoA with or without utilisation / reduction enhanced utilisation / reduction of Nitrate, Nitrite, N2O, N2O2 and / or carbon containing substrates

[0031] The present invention for production of Nitrogen-containing, non-carbon compounds, also relates to gene modifications in microbes, more specifically involving gene modifications resulting in increased synthesis / regeneration of Phosphate and / or Hydrogen and / or Coenzyme A for (i) enhanced carbon fixation and / or (ii) nitrogen fixation and / or (iii) ammonia fixation and / or (iv) synthesis / regeneration of anyone or all compounds including ATP, NADH, NADPH, Pyruvate and Acetyl CoA

[0032] BRIEF DESCRIPTION OF THE DRAWINGS:

[0033] Figure 1: Depicts impact of downregulation of ammonia uptake process in microbes, which leads to an increase in the release of ammonia into the environment.

[0034] Figure 2: Depicts impact of enhanced activity of Nitrogenase and related factors, on increased Nitrogen fixation and Ammonia release.

[0035] Figure 3: Depicts impact of different carbon metabolism of Cl compounds on increased carbon metabolism.

[0036] Figure 4: Depicts impact of over expression of Nitrification process, due to the overexpression of genes involved in conversion of Ammonia to Nitrite and Nitrate Figure 5: Depicts impact of deletion of de nitrification process, due to the deletion / downregulation of genes involved in reduction of Nitrite and Nitrite to Nitric oxide and Nitrous oxide

[0037] Figure 6: Depicts the pathway of CO2 fixation through Formate-Formaldehyde route, wherein overexpression of formate dehydrogenase under high active promoter enhanced the rate of CO2 fixation. Atmospheric carbon dioxide is converted into formic acid with the help of formate dehydrogenase and NADH. Formic acid is then converted into formaldehyde which further diverted into central carbon metabolism, serine pathway, and Ribulose monophosphate pathway.

[0038] Figure 7: Depicts the pathway of Methane fixation and assimilation due to heterologous expression of methane monooxygenase and further Methanol assimilation route towards carbon metabolism. Atmospheric Methane is absorbed within the cell by methane monooxygenase located in the membrane, converted into methanol which further reduced into formaldehyde by methanol dehydrogenase enzyme (MDH). This single-carbon aldehyde is then directed towards the central carbon metabolism for the synthesis of sugars, amino acids. The modified microbes express heterologous methane monooxygenase and methanol dehydrogenase to increase the carbon assimilation within the cell.

[0039] Figure 8: Depicts Formaldehyde assimilation route, by conversion of formaldehyde into D-arabino hexulose 6-phosphate with the help of hexulose 6- phosphate synthase (HPS) and ribulose monophosphate. Further, the synthesized Arabino sugar is then converted into intermediate fructose 6 phosphate by phosphohexose isomerase (PHI) and finally glyceraldehyde 3 -phosphate which further enters into the central carbon assimilation pathway. Our invention is related to the recombinant strain expressing heterologous Hexulose -6-phosphate synthase and isomerase (Hpsi2) which has both synthase and isomerase function to increase the carbon assimilation within the cell. Figure 9: Depicts the pathway of CO2 fixation through modified CBB pathway. Calvin-Benson-Bassham (CBB) pathway involves cyclic movement of carbon between the sugar molecule by fixing the atmospheric carbon dioxide with the help of phosphoribulo kinase (PRK) and Ribulose bisphosphate carboxylase and oxygenase (RuBisCO). The generated 3 carbon GA3P directed towards glycolysis and sugar phosphate recycling. Our invention related to the expression of heterologous Phosphoribulokinase (PRK) and compact RuBisCO for the CO2 assimilation.

[0040] Figure 10: Depicts the novel process of NADH regeneration with simultaneous carbon assimilations. Formate dehydrogenase reduces the carbon dioxide into formic acid resulting in the generation of NADH from NAD+. Similarly, glyceraldehyde 3 phosphate dehydrogenase uses the formed NADH to produce Phospho glyceric acid (PGA). Over expression of FDH and GAPDH under the control of GAPDH promoter generates NADH which can be used in central carbon assimilatory pathway.

[0041] Figure 11: Depicts the novel, inventive process of Nitrogenase metal specificity by promoter exchange. Modification and replacement of Anf Promoter with NifH promoter to increase electron delivery to Fe-nitrogenase, pathways activated by NifA. Incorporation of promoter such as nifH promoter (nifHDK operon) in place of anf promoter (of anfHDGK operon), shifts the specificity to Fe (Iron) irrespective of presence or absence of Molybdenum. Promoter exchange also impacts on enhanced Nitrogenase activity.

[0042] Figure 12: Depicts the pathway of Ammonia generation and fixation in amino acid metabolism. Nitrogen fixation in the recombinant strain over expressing native / heterologous nitrogenase and inactivating glnA. GOGAT: Glutamine oxoglutarate aminotransferase, Gin: glutamine, Glu: glutamate, OG: Oxoglutarate Figure 13: Depicts the pathway of Ammonia conversion to nitrate and Nitrate, and further N2O conversion and N2 production. Complete ammonium oxidation happens by enhanced nitrification process and deletion / downregulation of denitrification process. Further enablement of N2O reduction to N2 helps in Greenhouse gas remediation and utilisation of atmospheric pollutant N2O as an additional nitrogen source.

[0043] Figure 14: Depicts the Limitations in Ammonia, Nitrate, Nitrite production methods as per the prior art methods

[0044] Figure 15: Depicts the novel, Inventive solution with novel methods to overcome limitations in Ammonia, Nitrate, Nitrite production by gene manipulations of microbe for efficient carbon and nitrogen fixation, as well as for enhanced redox energy generation and regenerations.

[0045] Figure 16: Picture gives an overall description about the modifications performed to increase the Ammonia and related energy supply and regeneration of metabolites, with respect to carbon assimilation.

[0046] Figure 17: Picture depicts the Modified Nitrogenase gene construct for genome integration. Gene integration construct of Nitrogenase includes expression of Nitrogenase under NifH promoter, cloned with flanking sequences of Fdm gene partial sequences to facilitate integration with the Fdm gene in genome, resulting in simultaneous deletion of Fmn gene and integration of Nitrogenase under modified promoter.

[0047] DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention relates to a novel method for sustainable in vitro and in vivo production of non-carbon, nitrogen containing compounds including but not limited to Ammonia, Nitrate and Nitrite.

[0049] In one aspect, the present invention provides genetically modified microbes with modifications in both carbon and nitrogen fixation to enhance production of nitrogen-containing, non-carbon compounds such as Ammonia, Nitrate, Nitrite, etc.

[0050] The present invention relates to a novel method that includes gene modifications in microbes, more specifically involving gene modifications resulting in Cl carbon fixation / increased C 1 carbon fixation with or without Hydrogen fixation with or without ammonia fixation and / or increased ammonia fixation with or without induced nitrogen fixation or increased nitrogen fixation with or without increased uptake / synthesis / regeneration of anyone or all compounds including ATP, NADH, NADPH, Pyruvate and Acetyl CoA with or without uptake / utilisation / reduction enhanced utilisation / reduction of Nitrate, Nitrite, N2O, N2O2 and / or carbon containing substrates.

[0051] The present invention additionally relates to gene modifications in microbes, more specifically involving gene modifications for enhancing transcription and / or translation of proteins involved in (i) uptake / fixation Carbon and / or (ii) uptake / fixation of ammonia and / or (iii) fixation of Nitrogen and / or (iv) uptake / fixation of Hydrogen and / or (v) uptake / synthesis / regeneration of anyone or all compounds including phosphate, ATP, NADH, NADPH, Pyruvate, CoA and Acetyl CoA and / or (vi) for in-vitro / in-vivo / intra cellular / extra cellular production of non-Carbon Nitrogen-containing compounds (including but not limited to Ammonia, nitrate, nitrite etc. and their derivatives / salts).

[0052] The present invention also relates to gene modifications in microbes, more specifically involving gene modifications resulting in increased uptake / synthesis / regeneration of Phosphate and / or Hydrogen and / or CoenzymeA for (i) uptake / fixation Carbon and / or (ii) uptake / fixation of ammonia and / or (iii) fixation of Nitrogen and / or (iv) uptake / fixation of Hydrogen and / or (v) uptake / synthesis / regeneration of anyone or all compounds including phosphate, ATP, NADH, NADPH, Pyruvate, CoA and Acetyl CoA. and / or (vi) for in- vitro / in-vivo / intra cellular / extra cellular production of non-Carbon Nitrogencontaining compounds (including but not limited to Ammonia, nitrate, nitrite etc. and their derivatives / salts).

[0053] In yet another embodiment, the present invention provides methods of Ammonia uptake and further oxidation of Ammonia leading to Nitrification reactions, thereby enhancing nitrogen fixation in terms of Nitrate and Nitrite. One embodiment of the invention focus on process of enhanced Nitrogen fixation creating demand for enhanced availability of ATP, NADH and electrons, thereby enhancing the carbon fixation process. The current invention developed a novel process of integrating the carbon fixation and nitrogen fixation in an interdependent, mutually controlled system, thereby resulting in enhanced carbon and nitrogen fixation and impacting on product formation, which is explained in the following embodiments.

[0054] In one embodiment, the present invention provides process for microbes with genetic modification in nitrogen fixation for enhanced carbon fixation.

[0055] In another embodiment, the present invention provides process for microbes with genetic modification in carbon fixation for enhanced nitrogen fixation.

[0056] In a preferred embodiment, the present invention provides a process for modifying genes of microbes to decrease glucose uptake by them and enhance ammonia accumulation by the plants.

[0057] Nitrogen fixation and Ammonia production:

[0058] One of the embodiment of the present invention focus on enhanced Nitrogen fixation and ammonia production. The excess requirement of Redox energy compounds for Nitrogen fixation including ATP, NADH and electrons / hydrogen is supported by carbon assimilation / enhanced carbon assimilation.

[0059] Another embodiment of the current invention provides genetic modifications in microorganisms for enhancing carbon flux through increased uptake / assimilation of carbon compounds including but not limited to CO2, methane, methanol, formaldehyde, sugars like monosaccharides, disaccharides, or organic acids, which in turn increases the NADH, ATP and electron / hydrogen generation, which is sufficient for Nitrogen fixation. Therefore by carbon fixation / enhancing carbon fixation and / or assimilation, nitrogen fixation can also be increased. The current invention developed a novel process of integrating the carbon fixation and nitrogen fixation in an interdependent, mutually controlled system, thereby resulting in enhanced carbon and nitrogen fixation and impacting on product formation, which is explained in the following embodiments.

[0060] In one embodiment, the present invention provides a novel, inventive process for microbes with genetic modification in nitrogen fixation for enhanced carbon fixation.

[0061] In another embodiment, the present invention provides a novel, inventive process for microbes with genetic modification in carbon fixation for enhanced nitrogen fixation.

[0062] The objective of the present invention is to enhance the process of nitrogen fixation by modifying the Nitrogenase enzyme and related controlling genes for enhanced production of nitrogen-containing, non-carbon containing compounds.

[0063] In yet another embodiment, the present invention provides increased nitrogen fixation in microbe(s) for the production of Nitrogen-containing, non-carbon containing compounds by modification and / or overexpression of native Nitrogenase variants such as Iron-containing nitrogenase, molybdenum-based nitrogenase, vanadium-based nitrogenase, bimetallic nitrogenase, nitrogenase -like enzymes, and bacterial chlorophylls (BchL, BchM, BchB) to enhance Nitrogenase activity and improve Nitrogen fixation. Furthermore, the invention also involves manipulation of Nitrogenase related Regulatory genes, such as nifA, nifL, fix, mf genes, to enhance Nitrogenase activity and glutaminase, which hydrolyses glutamine to glutamate and ammonia, resulting in increased Nitrogen fixation.

[0064] In another embodiment, the Nitrogenase is expressed under the control of the specific promoter, enabling preference for Iron and / or Molybdenum and / or Vanadium and / or Bimetallic containing Nitrogenases. In yet another embodiment, the present invention provides inventive process of genetically modified microbe, wherein the said enhanced Nitrogen fixation is achieved by manipulation of Nitrogenase regulatory genes including but not limited to regulatory proteins, such as nifA and / or nifL, and / or fix genes and / or operons such as fixABCX, fixNOQP, fixH, fixJ, fixR, fixK, fixL, and / or rnf cluster genes such as rnfABCDEG.

[0065] Ammonia assimilation and Nitrate / Nitrite production

[0066] In one embodiment, the present invention provides a method for enhanced Ammonia production by deletion / downregulation of the activity of ammonia uptake enzymes such as Glutaminases, Glutamine synthase, and their isoforms, including GlnA, GlnD, and GlnE. Downregulating the activity of these enzymes in addition to Ammonia transorter downregulation reduces the assimilation of ammonia in amino acid synthesis, protein synthesis and thereby resulting in enhanced release of Ammonia.

[0067] In another embodiment, the present invention provides a method for enhanced uptake of Ammonia, by upregulating the activity of ammonia uptake enzymes such as Glutaminases, Glutamine synthase, and their isoforms, including GlnA, GlnD, and GlnE. These enzymes play a crucial role in the assimilation of ammonia in amino acid synthesis, protein synthesis and further metabolism.

[0068] In another embodiment, the present invention provides enhanced Ammonia uptake and oxidation of Ammonia to Nitrate and Nitrite, involving nitrification enzymes such as and not limited to Ammonia monooxygenase, Hydroxyl amine oxidase, Nitrate reductase, etc. the list of enzymes may also include deletion / downregulation of enzymes involved in denitrification process such as and not limited to nitrate reductase, Nitrite reductase for preventing the formation of Nitric oxide (NO) and further Nitrous oxide (N2O). In another embodiment, the toxicity caused by N2O is managed by fixation / reduction of Nitrous oxide-N2O (which is a greenhouse gas), by homologous / heterologous expression of genes for native / modified N2O reductase such as and not limited to nosR, nosZ, nosD, nosF, nosY, nosL, nosX, etc.

[0069] One embodiment describes the importance of Ammonia utilisation / fixation in metabolism for generation of amino acids, proteins etc. Enzyme glutamine synthetase (GS) coded by GlnA gene plays a crucial role in the assimilation of ammonia into organic nitrogen compounds and in the regulation of the nitrogen metabolism by controlling the intracellular levels of glutamine, which serves as a signal molecule for the regulation of several genes involved in nitrogen metabolism.

[0070] In yet another embodiment, the present invention provides genetically modified microbe, wherein said enhanced production of carbon-containing, non-Nitrogen containing natural or unnatural compounds is achieved by upregulation of Ammonia utilizing enzymes including Glutaminases, Glutamine synthase and its related enzymes like GDH, GlnA, GlnD, GlnE and Ammonia transporters like AmtA, AmtB, etc.

[0071] Accordingly, the present invention applies a novel combinatorial approach of manipulation of Glucose transporter gene, ptsG gene, glutamine synthase gene, glnA gene and Ammonia transporter gene, amtB gene resulting in reduced glucose uptake, and reduced ammonia uptake by microbes and finally resulting in enhanced ammonia release.

[0072] In some embodiments, the gene modification results in decreased expression of glnA, wherein the gene modification comprises replacing the native promoter of glnA with a promoter with lower activity.

[0073] In some embodiments, the genetically engineered bacterium comprising a modification in glnA which results in decreased expression or activity of glnA, finally leading to enhanced Ammonia concentration and release. Carbon fixation:

[0074] In one embodiment, the present invention details the methods of assimilation of various carbon compounds including but not limited to C 1 compounds like CO2, Methane, Methanol, formaldehyde, formate, etc., and organic acids, sugars, and other carbohydrates.

[0075] Another embodiment of the invention focuses on pathway engineering for carbon fixation including but not limited to, either individually or in combinations of pathways such as the Calvin Benson pathway (CBP) of CO 2 fixation to pentose sugars by Rubisco, Formate dehydrogenase mode of CO2 fixation, other pathways such as the reductive citric acid cycle (rTCA), the reductive acetyl-CoA pathway (Wood-Ljungdahl pathway), the 3-hydroxy propionate bicycle (3HP-bicycle), the 3-hydroxypropionate / 4-hydroxybutyrate cycle (3HP / 4HB cycle), and dicarboxylate / 4-hydroxybutyrate cycle (DC / HB), fixation of other CO2 or Cl compounds such as Methanol, Methane, Formaldehyde, etc.

[0076] Another embodiment provides a method of fixation of CO2 by Formateformaldehyde route, wherein Formate dehydrogenase (FDH) catalyses the conversion of carbon dioxide (CO2) into formic acid. Pyruvate formate-lyase is another enzyme that plays a crucial role in the conversion of Carbon dioxide to Formate. This enzyme is responsible for the synthesis of formate from pyruvate, and is an important part of the reductive acetyl-CoA pathway and Pentose phosphate pathway.

[0077] The present invention aims to generate a novel, inventive combinatorial genetic makeup of enzymes that are crucial for carbon fixation / CO2 fixation, as well as CH4, specifically enzymes involved in the Reductive pentose phosphate cycle, Reductive TCA cycle, gluconeogenesis and Calvin cycle, methane assimilation pathway, RuMP cycle etc. These enzymes include RuBisCO, PEP carboxylase, and Fructose bisphosphatase for CO2 assimilation, Methane assimilation pathways involving Methane Monooxygenase and Methanol Dehydrogenase (MDH) and / or Alcohol oxidase (AOX) enzymes, as well as enzymes for formaldehyde sequestration through genetic modifications in formaldehyde fixation system in RuMP, XuMP, Serine Cycle.

[0078] Another embodiment provides the details on modification of PEP carboxylase (phosphoenolpyruvate carboxylase) involvemnet in carbon fixation, specifically in the Reductive TCA cycle. PEP carboxylase catalyzes the conversion of phosphoenolpyruvate (PEP) to oxaloacetate, a key step in the Reductive TCA cycle. The present invention aims to genetically modify or over express PEP carboxylase to increase its activity and enhance the efficiency of the Reductive TCA cycle.

[0079] Another embodiment of the invention focuses on gene modifications for improved gluconeogenesis and the Calvin cycle. Fructose bisphosphatase is an enzyme involved in gluconeogenesis and the Calvin cycle. It catalyzes the conversion of fructose 1,6-bisphosphate to fructose 6-phosphate, an important step in the synthesis of glucose. In the Calvin cycle, fructose bisphosphatase plays a role in the regeneration of RuBP, which is necessary for the continued operation of the cycle.

[0080] Another major embodiment focuses on methane assimilation pathway, which happens through aerobic methane oxidation involving methane monooxygenase (MMO) enzymes - soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO), which catalyzes the oxidation of methane to methanol. The methanol formed from Methane can enter into central carbon metabolism through methanol oxidation to formaldehyde involving methanol dehydrogenase, or Alcohol oxidase (AOX) enzyme. The formaldehyde formed is then converted into different organic compounds through Serine cycle, RuMP and XuMP pathway. The present invention also focuses on CO2 sequestration by genetically modified microbes to increase the intracellular availability of CO2 through bicarbonate ion. The bicarbonate ion formed from CO2 is fixed to generate nitrogen containing compounds. Carbonic Anhydrase is responsible for CO2 sequestration by converting CO2 to Bicarbonate ion (HCO3'). The present invention focuses on increasing the expression or improving the catalytic activity of Carbonic Anhydrase.

[0081] In another embodiment, the formaldehyde sequestration is enhanced by the incorporation of homologous or heterologous genes for the Formaldehyde fixation system of the Ribulose mono phosphate (RuMP) pathway, involving and not limited to 3-hexulose-6-phosphate synthase (HPS), 6-phospho-3-hexuloisomerase (PHI) enzymes, and also by the incorporation of homologous and / or heterologous genes for the formaldehyde fixation system of Serine-threonine pathway involving, and not limited to FtfL, formate-THF ligase; Fch, methenyl-THF cyclohydrolase; MtdA, methylene -THF dehydrogenase.

[0082] In another embodiment, the methanol sequestration is enhanced by overexpression of enzyme such as and not limited to Methanol dehydrogenase and / or alcohol dehydrogenase.

[0083] In another embodiment, the CO2 sequestration via the said carbon fixation pathways is enhanced by the increased availability of CO2 / HCO3 (bicarbonate ions) through overexpression of enzymes such as and not limited to Carbonic anhydrase, Carbon concentrating mechanism (CCM), carboxysomes, etc.

[0084] In another embodiment, the present invention provides increase in the availability of CO2, by increasing the carbon assimilation pathway-related genes such as RuBisCO, CCMs, and carbonic anhydrase, which results in an increase in the energy supply required in the form of ATP and NADH in the nitrogen fixation for various nitrogenases in producing NH?1which can be utilized / diverted further in Nitrogen-containing, non-carbon compounds production.

[0085] In another embodiment, the present invention provides process for heterologous genetic modifications for carbon fixation in non-carbon fixing microorganisms wherein genes for carbon fixation such as Rubisco, Phosphohexoisomerase, Hexose phosphate synthase, Methane Monooxygenase etc. are taken from methanotrophs / methylotrophs but not limited to Galionellci spp., Methylomicrobium spp. etc.

[0086] In another embodiment, the present invention provides genetic control of Nitrogen / Carbon assimilation by homologous and / or heterologous modification of carbon uptake transporters such as PtsG, PtsN or PtsF or ManZ or LacY and carbonic anhydrase.

[0087] Regeneration of Hydrogen, Phosphate, ATP, NADH, NADPH:

[0088] In another embodiment, the present invention provides enhanced production of carbon & nitrogen-containing natural or unnatural compounds by enhanced uptake of hydrogen electrons, either extracellular or intracellular through hydrogenases.

[0089] In another embodiment, the present invention provides Hydrogen uptake / fixation for enhancing the Carbon fixation, Nitrogen fixation, synthesis / regeneration of ATP / ADP, NADH / NADPH by expression of Hydrogenase enzymes such as and not limited to uptake Hydrogenase, Hue Hydrogenase, CO / CO2 dependent hydrogenase (CODH), etc.

[0090] In another embodiment, the phosphate regeneration is for enhanced synthesis / regeneration / recycling of AMP / ADP / ATP and / or polyphosphates, redox carriers such as NADP+ / NADPH, and sugar phosphates involved in metabolic pathways such as and not limited to Glycolysis, TCA cycle, CBB pathway, pentose phosphate pathway, RuMP pathway, Serine pathway, etc. In another embodiment, the present invention provides methods for enhanced regeneration of Pyruvate, NADH, and ATP required for increased Nitrogen fixation. Pyruvate is a crucial metabolic intermediate that plays a vital role in several metabolic pathways such as gluconeogenesis, the TCA cycle, and amino acid biosynthesis. NADH and ATP, on the other hand, are essential cofactors involved in several biochemical reactions, including respiration, nitrogen fixation, and protein synthesis.

[0091] One embodiment of the present invention focuses on gene modifications for Malate -aspartate shuttle (MAS) involving the enzymes malate dehydrogenase in the mitochondrial matrix and intermembrane space, aspartate aminotransferase in the mitochondrial matrix and intermembrane space, malate-alpha-ketoglutarate antiporter in the inner membrane and the glutamate -aspartate antiporter in the inner membrane; and glycerol-3 -phosphate shuttle involving the enzymes Cytoplasmic glycerol-3 -phosphate dehydrogenase (cGPD) and mitochondrial glycerol-3 -phosphate dehydrogenase (mGPD). cGPD transfers an electron pair from NADH to dihydroxyacetone phosphate (DHAP), forming glycerol-3 - phosphate (G3P) and regenerating NAD+ needed to generate energy via glycolysis, and mGPD catalyzes the oxidation of G3P by FAD, regenerating DHAP in the cytosol and forming FADH2 in the mitochondrial matrix.

[0092] In another embodiment, the present invention provides enhanced production of Nitrogen-containing, non-carbon containing natural or unnatural compounds is achieved with enhanced regeneration / recycling of ATP / ADP / phosphate and / or NADH / NADPH, with or without enhanced hydrogen uptake / fixation, wherein the enzymes for NADH regeneration includes and not limited to all Dehydrogenases and oxidoreductases, Other enzymes for synthesis / regeneration of NADH includes but not limited to NADH oxidase, ADP-ribosyl cyclase, SARM-1, NAD hydrolases, mono (ADP-ribosyl)transferases, Nicotinic acid phosphor ribosyl transferase. NADH:NADPH transhydrogenase, poly(ADP- ribose) polymerases, nicotinamide N-methyl transferase (NNMT), sirtuins etc. Enzymes for ATP regeneration includes but not limited to ATP synthase, ATP cyclase, Malate - Aspartate shuttle and glycerol-3 -phosphate shuttle enzymes, Polyphosphate-AMP-phosphotransferase (PAP), Polyphosphate kinase, ATP synthesis / recycling via acid production pathway involving enzymes such as and not limited to Acetate kinase / propionate kinase / Butyrate kinase, CoA-acylating dehydrogenase, Phosphotrans acetylase / propionylase / Butyrylase, aldehyde dehydrogenase, lactate dehydrogenase, and the like .

[0093] In another embodiment, the present invention provides enhanced production of Nitrogen-containing, non-carbon containing natural or unnatural compounds is achieved with enhanced regeneration / recycling of Co enzyme A (CoA) and the enzymes for CoA (and Acetyl CoA / Propionyl CoA, etc.) regeneration / recycling includes such as and not limited to CoA transferases, CoA-acylating aldehyde dehydrogenase, CoA-dependent propionaldehyde dehydrogenase, phosphotransacetylase, Acetyl CoA-synthase (ACS), Acetyl coenzyme-A carboxylase (ACC), ACC-catalysed biotin carboxylase (BC), carboxyltransferase (CT), and the like.

[0094] In another embodiment, the present invention provides enhanced production of Nitrogen-containing, non-carbon containing natural or unnatural compounds is achieved by expression of Homologous / heterologous, native / modified enzymes responsible for increased pyruvate availability include but not limited to (1) enzymes involved in increasing pyruvate synthesis towards downstream product formation pathways and (2) enzymes involved in preventing pyruvate loss in form of CO2, wherein said enzymes for increasing pyruvate include and not limited to Pyruvate synthase, Pyruvate kinase, Pyruvate decarboxylase, carrier proteins like mitochondrial pyruvate carrier proteins etc., wherein such enzymes are overexpressed and / or deleted / downregulated.

[0095] In another embodiment, the present invention provides process to enhance Nitrogen / Carbon fixation by homologous / heterologous modification of genes involved in polysaccharides (starch, cellulose, hemicellulose and pectin) degradation and utilization. The enzymes include but not limited to cellulase, exo- glucanase, endo-glucanase, cellobiohydrolase, xylanase, Pectin esterase, lignin peroxidase, etc

[0096] In one embodiment, modification processes are developed to generate salts and derivatives of Ammonia, Nitrate and Nitrite such as and not limited to Ammonium nitrate, potassium nitrate, calcium nitrate, sodium nitrate, potassium nitrate, magnesium nitrate, sodium nitrite, Ammonium sulphate, Monoammonium phosphate (MAP), Diammonium phosphate (DAP), nitro glycerin etc. these salts and derivatives can be used in combination with our engineered microbes for additional nitrogen fixation.

[0097] In another embodiment, the present invention provides processes of combining enhanced microbes and their combinations, applied along with or without other microbes / inorganic / organic materials in any form such as and not limited to Ammonium sulphate, urea, super phosphate, ammonium phosphate, limestone, gypsum, muriate of potash, etc. these applications of combinations will result in further enhancement of Nitrogen compound release to crops and hence high yield of crops in good health.

[0098] In one embodiment, the invention provides process for utilisation of different carbohydrates, in particular various polysaccharide such as and not limited to starch, cellulose, hemicellulose, pectin by enzymes for breakdown / degradation to monomeric / dimeric sugar as substrates, wherein the enzymes for degradation includes but not limited to amylase, cellulase, exoglucanase, endo glucanase, cellbiohydrolase, xylanase, P-glucosidases, mannanase. Xyloglucanases, Pectin esterases (PEs), pectin methyl esterase, etc.

[0099] In another embodiment, the microbe is enginnered with ability to utilize or breakdown varieties of nutrients (carbon and / or nitrogen source), such as and not limited to organic and / or inorganic substrates in any form including gaseous / liquid / solid and their combinations, including but not limited to carbohydrates like mosaccharides / disaccharides / oligosaccharides / polysaccharides, etc., organic acids, proteins, lipids, pectin, inorganic & organic phosphates / phosphites (including phytic acid), nitrate / nitrite / Nitrous oxide (N2O), Trimethyl Amine (TMA), sulfate / sulfides (including H2S), synthetic / non-synthetic compounds including but not limited to plastics / fibres / rubber / resins / chemicals, etc. (like polyethylene terepthalate (PET), Ethylene glycol (EG), polyurethane), under in vivo and / or in vitro applications by homologous and / or heterologous expression of native or modified enzymes including but not limited to cellulase, exo- glucanase, endo-glucanase, cellobiohydrolase, xylanase, pectinases, pectin methyl esterase, pectin acetyl esterase, lignin peroxidase, proteases, peptidases, invertase, amylase, sugar isomerases, lipases, amidases, phytase, phosphatases, nitrate reductase, nitrite reductase, N2O reductase, sulfate adenylyltransferase (Sat), adenylyl sulfate reductase (AprBA), dissimilatory sulfite reductase (DsrAB), ATP sulfurylase (ATPS), Adenosine 5 '-phosphosulfate reductase (APSR), APS kinase (APSK), Terephthalate 1,2-dioxygenase, Terephthalate reductase, Dihydrodiol dehydrogenase, Glycolate oxidase, Glyoxylate carboligase, hydroxy pyruvate isomerase, protocatechuate decarboxylase, Feruloyl esterase, TMADH- trimethylamine dehydrogenase; DMADH-dimethylamine dehydrogenase; MMADH-monomethylamine dehydrogenase, Thioredoxin reductase, Thioredoxin, dihydro folate reductase, etc.

[0100] In another embodiment, the genetically modified microbe is enabled with enzymes for degradation of pesticides applied and thus reducing the toxicity caused by chemical pesticide / insecticide sprayed on the crops. This includes modification in microbes for enhancing production or inducing production of compounds for breakdown / denaturing / detoxifying / degrading toxic compounds such as and not limited to pesticides, aflatoxin, insecticides, etc. Due to the degrading enzymes, chemical pesticides / insecticides act as additional nutrient for the patented microbe. In another embodiment, the enhanced / production of Nitrate / Nitrite (including their derivatives) and their salts is achieved by upregulation and / or downregulation / deletion of homologous and / or heterologous enzymes such as and not limited Ammonium monooxygenase, Hydroxylamine oxidoreductase, Nitrate reductase, Nitrite reductase, etc, by co-fermentation / individual fermentation integrated with final conversion process.

[0101] In another embodiment, the present invention provides salts of nitrate / nitrite salts including but not limited to Ammonium nitrate, sodium nitrate, potassium nitrate, calcium nitrate, magnesium nitrate, sodium nitrite, Ammonium sulphate, Monoammonium phosphate (MAP), Diammonium phosphate (DAP), nitro glycerine etc.

[0102] In another embodiment, the enhanced microbes and the combinations thereof of the present invention may be applied along with or without other microbes / inorganic / organic materials in any form such as and not limited to Ammonium sulphate, urea, super phosphate, ammonium phosphate, limestone, gypsum, muriate of potash, and the like.

[0103] In another embodiment, the present invention provides homologous / heterologous enzyme / s which are native and / or modified enzyme, expressed under native and / or modified promoter, wherein modification includes but not limited to pointmutations, point-mutations, upregulation and / or downregulation / silencing / deletion, epigenetic mechanisms, and the like.

[0104] The microorganisms can be selected from but not limited to Methylobacterium, Methyloversatilis, Sphingomonas, Bosea, Altererythrobacter, Brevundimonas, Rubrivivax, Niveispirillum, Dinoroseobacter shibae.

[0105] Other microbes includes Nitro sopumilus maritimus, Nitrospira inopinata, Rhodobacter sphaeroides, Cupriavidus necator, Nigrospora oryzae, Azospirillum lipoferum, Rhodopseudomonas palustris, Bradyrhizobium japonicum, Ralstonia eutropha, Cyanobacteria, Epichloe typhina, Rhodococcus, Xanthobacter species Nitrosomonas, Nitrosospira, Nitrosococcus, and Nitrosolobus .

[0106] In a further embodiment, the present invention provides certain fungi comprising Aspergillus, Candida, Chlamydomonas, Chrysosporium, Cryotococcus, Fusarium, Kluyveromyces, Neotyphodium, Neurospora, Penicillium (e.g. P. chrysogenum), Pichia, Saccharomyces, Trichoderma and Xanthophyllomyces to be selected for genetic modification.

[0107] Further, Clostridium acetobutylicum, C. Beijerinckii, C. accharoperbutylacetonicum, C. saccharobutylicum, C. aurantibutyricum, C. tetanomorphum), Zymomonas, Escherichia (e.g., E. Coli), Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula, Zymomonas and Saccharomyces, e.g., Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Kluyveromyces lactis, Saccharomyces lactiss.

[0108] In a further embodiment, the present invention provides endophytes selected from the group comprising Proteobacteria (such as Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea, Se"atia, Rahnella, Azospirillum, Azorhizobium, Azotobacter, Duganella, Delftia, Bradyrhizobiun, Sinorhizobium and Halomonas)' , Firmicutes (such as Bacillus, Paenibacillus, Lactobacil / us, Mycoplasma, and Acetabacterium) and Actinobacteria (such as Streptomyces, Rhodacoccus, Microbacterium, and Curtobacterium).

[0109] Bacteria that can be produced by the methods disclosed herein include Azotobacter sp., Bradyrhizobium sp., Klebsiella sp., and Sinorhizobium sp. The bacteria may be selected from the group consisting of Azotobacter vinelandii or Azotobacter chroococcum, Bradyrhizobium japonicum, Klebsiella pneumoniae, and Sinorhizobium meliloti. The bacteria may be of the genus Enterobacter and Rahnella. In another embodiment, certain fungi comprising Saccharomyces cerevisiae and Trichoderma harzianum are selected for genetic modification for nitrogen fixation.

[0110] In another embodiment, the, Nitrogenase and Nitrogenase regulatory genes are manipulated for increased Nitrogen fixation in combination with or without carbon fixation in novel microbes such as Methylobacterium symbioticum, Frankici sp.

[0111] In another embodiment, the present invention provides microorganisms including but not limited to Proteobacteria such as Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea, Serratia, Rahnella, Azospirillum, Azorhizobium, Azotobacter, Beijerinckia, Duganella, Delftia, Bradyrhizobiun, Sinorhizobium and Halomonas'. Alpha-proteobacteria such as Methylobacterium spp., Methylorubrum, Methylomonas, Methylosarcina, Methylococcus, Methylobacterium symbioticum; Firmicutes such as Bacillus, Paenibacillus, Lactobacillus, Mycoplasma, and Acetabacterium and Actinobacteria such as Streptomyces, Rhodacoccus, Microbacterium, and Curtobacterium, Frankia sp., other microbes such as Rhodobacter sphaeroides, Pseudomonas stutzeri, Cupriavidus necator, Nigrospora oryzae, Azospirillum lipoferum, Rhodopseudomonas palustris, Rhodobacter spp., Bradyrhizobium japonicum, Ralstonia eutropha , Flavobacterium, Cyanobacteria, Epichloe typhina, Rhodococcus, Xanthobacter spp.; non-diazotrophs such as E.coli, Bacillus, Lactobacillus, yeasts like Saccharomyces, Pichia, Archaea bacteria etc.

[0112] In another embodiment, the present invention provides enzymes for increasing pyruvate include and not limited to Pyruvate synthase, Pyruvate kinase, Pyruvate decarboxylase, carrier proteins like mitochondrial pyruvate carrier proteins etc., wherein such enzymes are overexpressed and / or deleted / downregulated. In yet another embodiment, the present invention provides enzymes involved in Nitrogen fixation and / or carbon fixation, are overproduced by inclusion of Transcriptional and / or Translational enhancers such as and not limited to UNA1, UNA2, UNB, oligomers, and the like.

[0113] One of the strategies for increasing recombinant protein production is enhancement at transcription / translation level. Transcriptional and translational enhancers increase mRNA stability and translation efficiency. Addition of transcription enhancers such as oligomers in vector results in increased protein expression. Similarly, addition of transcription / translation enhancers UNA1, UNA2 and UNB results in increased protein expression.

[0114] Examples:.

[0115] Example 1:

[0116] Enhancing Ammonia release in modified microbe by Down-regulation / Deletion of genes involved in Ammonia uptake, including glnA and amtB genes

[0117] Nitrogen fixation in diazotrophs was improved by reducing glucose uptake and increasing ammonia accumulation.

[0118] Ammonia available for extracellular release was increased by blocking the pathways involved in Ammonia uptake, which includes gene such as Glutamine synthase (GlnA) and Ammonia transporter (AmtB) for ammonia intake towards the cell.

[0119] The reduction of glucose uptake and promotion of amino acid catabolism resulted in improved Nitrogen fixation and nitrate uptake by the plant. This is because the microbes, which have undergone genetic modifications, now have a decreased ability to consume glucose, which makes them more reliant on alternative nitrogen sources like ammonia and amino acids. This enhances the efficiency of Nitrogen fixation, which is an essential process for the growth of plants. The improved Nitrogen fixation and nitrate uptake have beneficial effects on plant growth and yield.

[0120] The use of a promoter with lower activity ensures that the expression or activity of these genes is reduced but not completely eliminated, which is important for the survival and growth of the microbial strains. The modifications made to these genes can be applied to different bacterial strains to enhance Nitrogen fixation and nitrate uptake in plants, making it a valuable technique in agricultural and horticultural industries.

[0121] In this patent, genetic modifications were made to the ptsG gene, glnA gene (Sequence ID: 3), and amtB gene (Sequence ID: 4) in bacterial strains. By disrupting the ptsG gene, glucose uptake was reduced, and by disrupting the glnA gene (Sequence ID: 3) and amtB gene (Sequence ID: 4), amino acid catabolism was promoted. The gene modification process involved replacing the native promoter of these genes with a promoter with lower activity, resulting in decreased expression or activity of these enzymes.

[0122] The strain with downregulated ammonia uptake was analysed for ammonia release in comparison to unmodified wild type strain, under same conditions. The final strain, with down-regulated activity of ammonia uptake, resulted in an almost 2-fold increase in ammonia release, which will have profound effect in field applications and be very much beneficial to plants (figure 1).

[0123] Example 2:

[0124] Enhancement of the Nitrogenase activity and regulator, for increased Nitrogen fixation in diazotrophs for enhanced Ammonia release to plants.

[0125] To enhance ammonia release to plants, gene modifications were performed to increase Nitrogenase activity and regulator in diazotrophs. The incorporation of Fe-Nitrogenase (Sequence ID: 1) from microbes such as Azotobacter, Rhodobacter sp., and Methylobacterium spp. was carried out to facilitate Molybdenum-independent Nitrogenase activity.

[0126] To increase nitrogenous activity, the protein with the highest nitrogenous activity was mined from the available database using the NCBI data server and bioinformatic approaches such as BLAST. These modifications result in an increase in Nitrogen fixation and ammonia release, which is beneficial for plant growth and yield (Figure 2).

[0127] Steps of gene modifications of Gene

[0128] • To overexpress Fe-Nitrogenase (Sequence ID: 1) copy of the heterologous gene was expressed under the control of high strength nifH (Sequence ID: 8), a constitutive promoter from Rhodobacter spp.,

[0129] • The gene sequence coding for Fe-Nitrogenase (Sequence ID: 1) from Rhodobacter sp. was cloned by the Gene synthesis method flanking sequence ID 6 and 7 as overlapping integration sites.

[0130] • The expression cassette was integrated into the Methylobacterium spp., genome using homologous recombination at formaldehyde dismutase (fdm) to delete the fdm sequence as well as simultaneous integration of Fe-nitrogenase gene cassette.

[0131] • The expression cassette was cloned inside Nael restriction digestion sequences available in fdm between to get the final integration cassette (Sequence ID: 9).

[0132] • The final integration cassette was taken in gene cloning vector pUC57 (Genscript plasmid), by gene synthesis, as depicted in Figure 17.

[0133] Gene transformation by homologous recombination:

[0134] Methylobacterium spp., electrocompetent cells were transformed with Integration cassette plasmid by standard methods. • Integration was targeted to be at fdm sites in the genome of Methylobacterium spp.

[0135] • Transformants were screened for integration by colony PCR. Confirmed strains were isolated and stored properly.

[0136] • Engineered strains were subjected to growth and Ammonia Production assay at controlled conditions. Wild-type cells were also experimented in similar conditions, to test the increased Ammonia production by using the Nessler reagent.

[0137] Manipulations also include overexpression / multi-copy expression of Nitrogenase controller proteins such as NifA. All these manipulations were incorporated on the strain with downregulated Gin activity, and finally resulted in an almost 4-fold increase in N2 fixation, in terms of Ammonia production

[0138] Example 3:

[0139] Enablement of Autotropism by the introduction of pathways related to the utilization of Cl compound.

[0140] The introduction of pathways related to the utilization of Cl compounds such as Methane, CO2, Formaldehyde, Methanol etc., as sources of carbon and energy, facilitates the microbe to be autotrophic and reduces dependency on sugar and organic acid substrates, either from an external supply or from the plants.

[0141] Gene modification includes incorporation / overexpression of enzymes involved in methane assimilation, such as Methane monooxygenase (Methylomicrobium alcaliphilum), alcohol oxidase / dehydrogenase, and CO2 fixation via Formate dehydrogenase (FDH), RuBisCO (Galionella sp), and entry into regular metabolic pathways, finally resulting in ATP and NADH generation.

[0142] Enhanced ATP and NADH regeneration and increased carbon metabolism positively impact increased N2 fixation, and hence increased ammonia release. These modifications result in a more sustainable and efficient process for nitrogen fixation, which is beneficial for plant growth and crop yield (Figure 3).

[0143] Example 4: Genetic engineering microbes such as denitrifying microbes for production of Nitrogen-containing compounds, Nitrate & Nitrite:

[0144] Part 1: Upregulation of genes to enhance Nitrification enzymes such as Ammonia monooxygenase, Hydroxylamine oxidoreductase, Nitrite oxido reductase as an example to produce the nitrogen containing compounds

[0145] Plants take up nitrogen in the form of nitrate (NO3') and ammonium (NH41) ions. The step of Ammonia oxidation to nitrate formation is catalysed by enzymes Ammonia monooxygenase and Hydroxylamine oxidoreductases and Nitrite oxido reductase.

[0146] Genetic modifications were performed on these genes in bacterial strain capable of Ammonia oxidation completely to nitrate. This resulted in increased enzyme activity of these enzymes and thereby improved Nitrogen fixation and nitrate uptake by the plant.

[0147] To identify such modifications, mutagenesis was performed in and around the active sites of the genes that code for enzymes Ammonia monooxygenase and Hydroxylamine oxidoreductases and Nitrite oxido reductase. A library of mutants was constructed for each gene. These libraries were then screened for increased enzyme activity.

[0148] Engineered strains were subjected to growth and ammonia consumption assay at controlled conditions. Wild type cells were also experimented in similar conditions, to test the increased Ammonia conversion finally to Nitrate. Nitrate, Nitrite and Ammonia were analysed by Abeam -make colorimetric sensors. Nitrate level increased by 1.5 -fold in final engineered strain, as compared to unmodified wild type strain. Nitrate / Nitrite ratio got increased by 2-fold, from ratio of 1.5 in wild type to ratio 3 in Final engineered strain. Part 2: Impact of overexpression of Nitrification process related genes on enhancing Nitrite and Nitrate concentrations as an example for the nitrogen containing compounds production:

[0149] Nitrospira sp. were manipulated for over expression of Nitrification enzymes Ammonia monooxygenase, Hydroxylamine oxidoreductase and Nitrite oxidoreductase sequentially (as depicted in Figure 13), resulting in Engineered strains for Ammonia oxidation, as well as Nitrite oxidation to yield Nitrate finally. Analysis of Nitrite, Ammonia and Nitrate by sensor-based testing, showed Nitrate level increase by 2-fold between Ammonia oxidation and Nitrite oxidation, whereas, about 1.5 -fold increase in Nitrate when compared to unmodified Wild type Nitrospira sp (Figure 4).

[0150] Part 3: Down regulation / deletion of Denitrification enzymes such as Nitrite reductase.

[0151] Genetic modification was done in the microbes to reduce / inhibit the Nitrite to ammonia conversion. This resulted in reduction / inhibition of denitrification step, and hence reduction of denitrification compound such as Nitric oxide (NO), as depicted in Figure 13.

[0152] Combination of dentification process deletion and overexpression of nitrification process enzymes resulted in further increased amounts of Nitrite and Nitrate, and reduced levels of Nitric oxide.

[0153] Engineered strains were subjected to growth and ammonia consumption assay at controlled conditions. Wild type cells were also experimented in similar conditions, to test the increased Ammonia conversion finally to Nitrate. Nitrate, Nitrite and Ammonia were analysed by Abeam -make colorimetric sensors. Nitrate level increased by 1.5 -fold in final engineered strain, as compared to unmodified wild type strain, and 2-fold reduction in Nitric oxide (denitrification product). Nitrate / Nitrite ratio got increased by 2-fold, from ratio of 1.4 in wild type to ratio 5.6 in Final engineered strain. Part 4: Impact of deletion of genes related to Denitrification process in addition to overexpression of genes related to Nitrification process, on enhanced concentrations of Nitrite and Nitrate:

[0154] Nitrospira sp. manipulated by deletion of Denitrification enzymes Nitrite reductase and Nitrate reductase, in addition to over expression of Nitrification enzymes, resulting in Engineered strains for further efficient Ammonia oxidation, as well as Nitrite oxidation to yield enhanced amounts of Nitrate finally. Analysis of Nitrite, Ammonia and Nitrate by sensor-based testing, showed Nitrate level increased by 1.5-fold in final engineered strain, as compared to unmodified wild type strain, and 2 fold reduction in Nitric oxide (denitrification product) (Figure 5)

[0155] Part 5: Gene manipulations for greenhouse gas -Nitrous oxide reduction process to improve the nitrogen containing compounds production:

[0156] In order to utilize the opportunity of nitrous oxide reduction, as addition nitrogen fixation process, Genetic modification were focused on enabling Nitrous oxide (N2O) reduction to molecular Nitrogen (N2), by heterologous expression of N2O reductase. N2O reductase (nos gene) from Rhizhobium type of microbes was incorporated for additional N fixation.

[0157] SEQUENCE LISTING

[0158] Sequence Listing Information:

[0159] DTD Version: Vl_3

[0160] File Name: Sequence listing_ 202241064230. xml

[0161] Software Name: WIPO Sequence

[0162] Software Version: 2.3.0

[0163] Production Date: 2023-12-08

[0164] General Information:

[0165] Current application / IP Office: IN

[0166] Current application / Application number: 202241064230

[0167] Current application / Filing date: 2022-11-10

[0168] Current application / Applicant file reference: 4230

[0169] Earliest priority application / IP Office: IN

[0170] Earliest priority application / Application number: 202241064230

[0171] Earliest priority application / Filing date: 2022-11-10

[0172] Applicant name: Fertis India Pvt. Ltd.

[0173] Applicant name / Language: en

[0174] Invention title: GENETIC MODIFICATION OF MICROBES FOR

[0175] PRODUCTION OF NITROGEN-CONTAINING, NON-CARBON

[0176] COMPOUNDS ( en )

[0177] Sequence Total Quantity: 16

[0178] Sequences:

[0179] Sequence Number (ID): 1

[0180] Length: 888

[0181] Molecule Type: DNA

[0182] Features Location / Qualifiers:

[0183] - source, 1..888

[0184] > mol_type, genomic DNA

[0185] > organism, Fe-Nitrogenase

[0186] Residues: atgggcaaac tccgtcagat cgccttctac ggcaaaggtg gtatcggcaa gtcgaccacc 60 tcgcagaaca ccctcgccgc gctggtcgag atgggtcaga agatcctcat cgtcggctgc 120 gaccccaagg ctgacagcac ccgtctgatc ctgaacacca agctgcagga caccgtgctg 180 cacctggccg ccgaggccgg ttcggtcgaa gatctggaag tcgaagacgt cgtgaaaatc 240 ggctacaagg gcatcaaatg caccgaagcc ggcggtccgg agccgggggt tggctgcgcc 300 ggccgtggcg tcatcaccgc gatcaactc ctgaagaaa acggcgccta tgacgatgtg 360 gactatgtgt cctatgacgt tctgggcgac gtggtctgcg gcggcttcgc catgccgatc 420 cgtgaaaaca aggcgcagga aatctacatc gtcatgtcgg gcgagatgat ggcgctttac 480 gccgccaaca acatcgccaa gggcatcctg aaatatgcga actcgggcgg cgtgcgtctg 540 ggcgggctga tctgcaacga acgcaagacc gaccgcgagc tggaactggc cgaagcgctg 600 gccgccaagc tgggctgcaa gatgatccac tcgtgccgc gcaacaacgt cgtgcaacat 660 gccgaactgc gccgcgaaac cgtgatccaa tacgatccga cctgcagcca ggcgcaggaa 720 taccgcgaac tggcccgcaa gatccacgag aactcgggca agggcgtcat cccgaccccg 780 atcacgatgg aagagctgga agagatgctg atggatttcg gcatcatgca atcggaagaa 840 gatcgcgaaa agcagatcgc cgagatggaa gccgcgatga aggcctga 888

[0187] Sequence Number (ID): 2 Length: 1605

[0188] Molecule Type: DNA

[0189] Features Location / Qualifiers:

[0190] - source, 1..1605

[0191] > mol_type, genomic DNA

[0192] > organism, NifA regulator

[0193] Residues: atggatggaa gcctcaaggg cgcgtcgagc acacactggc gtacgcagga gatgttcctc 60 gtccaggagg tcatgaacct gatcggcaag ggcctgcgcc tggatcaggt cgcgcgagag 120 atgcttcatc tcctgtccga gatcgtcgga ctgaaccggg gacggatcgt cctgaaagac 180 cccgacggca agggacaccg cattgcctac gcctatgggc tgacccgcga ggaggtcgct 240 cggggccgct acgcgccggg cgagggcatc accggacgcg tcatccagga cggtcacctc 300 atcatcgtgc aggacatcga caaggacccg accttcctcg ggcgggcggt ggcgcgggcg 360 cagcttcccc gcggcgccgt gtcgttcctg gcactgccga tccgcgttga tcacaggacg 420 gtcggcgcca ttgcctgcca ccgcatccgc caccgggagc gggcgctctc ggacgatctc 480 accatcctgc gcatcatcgc cacgatggtc agccagttgc tgaccctgaa cgagcgggtg 540 gagcagaaga cgcgggcgct cgaggaacac aacgacatgc tggcgcgcga gctgcgcatc 600 aagcgcgccc gttacggcat catcggaacc tcgcccgcgc tgctgcgcgc cctcgcccaa 660 gtggagaagg tcgcgagcgc gaccgcgagc gtgctgctgc tcggcgagtc cggaaccggc 720 aaggaactct tcgcccgggc gctccacctt gcgagtccgc gccgagaccg gccctcatc 780 aaggtcaatt gcgcggcgat ccccgacagc ctgtcgaat ccgagctgt cgggcacgag 840 cgcggcgcct tcaccggggc cgtcgatgcg cgggccggct ggtcgagca ggcgagcggc 900 ggcacgatct tcctcgacga gatcggcgag atgccggcgg tcctccagac caagctgctg 960 cgcacacttc aggaggggac ggtcgtacgc ctcggcggca agcgcgagat ccgggtcgat 1020 atgcgcctcg tcgccgcgac gaaccgggat ctcgccaccg aggtcgccca tggccgcttc 1080 cgagaggact tatctaccg cctcaacgtc gtgccgatca ccctgcctcc gctggcggag 1140 cggcgcagcg acatccccga tctggtgctg cactcctca cccaggcgaa ccagaacaac 1200 cagcgcaacg tcaacctgac ccagggcgcg gtcacgcatc tcgcccgcca gtcctggccg 1260 ggcaacatcc gccagctcgc caactcatc gagcggctcg tgctgctggc aagcgaaggt 1320 gtctggacg ccgaggacgt gcgtccgatg ctcgaaggtg ccgtcgtcac ggcgcccccg 1380 ctgctcgctc ccgcctcggc cccgttcttc tcgcccgaac gggaagcggc gttgcgcgcg 1440 atgggcaccg tgcgccccta tctgccggcc gactcgcacg gccacgaaca gttgcgcatg 1500 gccctggccc aggccggcgg caataaaacg cgggcggccc agcgcctcgg gctcaccgaa 1560 cgccaatttt cctatcgctg gcgcaagctc cagccggcac cctga 1605

[0194] Sequence Number (ID): 3

[0195] Length: 1410

[0196] Molecule Type: DNA

[0197] Features Location / Qualifiers:

[0198] - source, 1..1410

[0199] > mol_type, genomic DNA

[0200] > organism, Glutamine synthase (Gin)

[0201] Residues: atgaatacgg ccgccgatgt gctgaaggcc atcaaggaca atgacgtcaa gtacgtcgac 60 ttccgcttca ccgacccgcg cggcaagtgg cagcacgtga ccttcgacgt ctccctgatc 120 gacgaggaga tgttccagga cggcaccatg ttcgacggct cgtcgatcgc tggttggaag 180 gcgatcaacg aatccgacat gctgctgatg cccgatccgg tcacggcctg catggatccg 240 ttcttctcgg cctccaccat gtcgatcgtc tgcgacgtgc tcgagccctc gactggcgag 300 ccctatgccc gcgatccgcg ctcgaccgcc aagctcgccg aggcgtatct gcgctccacc 360 ggcatcggcg acacgatctt tgtcggcccc gaggccgagt tcttcgtgtt cgacgacgtg 420 aagtcggcg ccgaccccta ccataccggc tccagctcg actcgaccga gctgccgacc 480 aacggcttca ccgatacga gggcggcaac ctcggccacc gggtgcagac caagggcggc 540 tacttccccg tcccgccgca ggattcggct caggacatgc gcggcgagat gctggctgcg 600 atgcagtcca tgggcgtgaa ggtcgagaag caccaccacg aggtggcgtc ggcccagcac 660 gaactcggca tgaagtcga cacgctgacc ctgctcgccg accacatgca gatctataag 720 tactgcatcc acaacgtgc gcagagctac ggcaagtccg cgaccttcat gcccaagccc 780 gtctacggcg acaacggctc gggcatgcac gtgcaccagt cgatctggaa ggacggcaag 840 ccgctgtttg ccggcgacaa gtatgccgac ctcagccagg aatgcctgtg gtacatcggc 900 ggcatcatca agcacgccaa ggcgctgaac gccttcacca acccgtccac caactcctac 960 aagcgtctgg tgccaggcta cgaggccccc gtgctgctgg cctattcggc ccgcaaccgc 1020 tcggcctcct gccgtatccc gtggacgacg aacccgaagg ccaagcgcgt cgaggtccgc 1080 ttcccggatc cgatggccaa cccctacctc gcctctcgg cgctgctgat ggccggcctc 1140 gacggcatca tcaacaagat cgatccgggc ccggcgatgg acaaggatct ctacgacctg 1200 cccccgcgcg agctgaagaa gatcccgacc gtctgcggct cgcttcgtga ggcgctgcag 1260 aacctcgaca aggaccgcgc ctcctcaag gccggcggcg tgttctcgga cgaccagatc 1320 gactcgttca tcgagctgaa gatggccgag gtgctgcgct acgagatgac cccgcacccg 1380 atcgagttcg tgcagtacta ctcgctgtaa 1410

[0202] Sequence Number (ID): 4

[0203] Length: 1497

[0204] Molecule Type: DNA

[0205] Features Location / Qualifiers:

[0206] - source, 1..1497

[0207] > mol_type, genomic DNA

[0208] > organism, Ammonium transporter (Amt) Residues: atgaaacttc gtaatcttct cgcgctcgga ctgggaggag ccgcccttgg cctcctcttc 60 gtcgagccgt cgctcgcgca ggcgccggcg gtcgagcccg ccgtcaccgc ggccgcaccg 120 gtgcccaaca agggcgacac ggcgtggatg ctgctctcgg cgatcctcgt cctgctgatg 180 accgtacccg gcctcgccct gttctacggc ggcctcgtgc gcaccaagaa catgctctcg 240 gtgctgaccc agatcttcgc gatcgcctcg atcgtctgcc tgctgtgggt gacctacggc 300 tacagcctcg ccttcaccaa tggcggcggc ctcaatgact tcgtcggcgg cttctcgaag 360 gccttcctca agggcgtcga cgccaactcg gtggcggcca ccttctccaa cggcgtcgtg 420 atccccgaat acgtctacat ctgcttccag atgacgttcg cgatgatcac ccccggcctc 480 atcgtcggcg ccttcgccga gcggatgaag ttctcggcgc tggtcgtgtt cacgatcctc 540 tgggtcacgc tgatctactt cccgatggcc cacatggttt ggtactgggg cggtccggac 600 gtcttcgccg acgcggcccg caagctcgcc gccgccggtg gcgaggccaa tgccgcggcc 660 aaggccgagt acgacgcggt gctgggcgat gccggcatgc tcttcaagtg gggcgccctc 720 gacttcgccg gcggcaccgt cgtgcacatc aacgcaggca tcgccggcct cgtcggctgc 780 ctgatgctcg gcaagcgcat cggctacggc cgcgacctgc tggctccgca ctcgctgacc 840 atgaccacga tcggcgcctc gctgctctgg gtcggctggt tcggcttcaa cgccggctcg 900 aacctcgaag ccaacggcgc cgcgggtctg gcgatgatca acaccttcgt tgccaccgcc 960 gctgctgccg tctcctggct gttcgtggaa tgggccgcca agggtaagcc gtcgctcctc 1020 ggcatgctct cgggcgccat tgccggcctc gtcgccgtca ccccggccgc cggctttgcc 1080 ggcccgatgg gctcgatcgt tctgggtctg gccgccggcg cgatctgctt cgtgatgtgc 1140 tccaccgtga agaacgcgct gggctacgac gactccctcg acgtgttcgg cgtgcactgc 1200 atcggcggca tcctcggtgc catcgccacg ggtatcctgg tctcgcccga tctcggcggc 1260 gccggcatcc ccgactacac caccaagccc ggtgagctga ccgtcggtgc ctacgacatg 1320 gccgcccagg tcatcatcca ggcgaaggcg gtgggcttca ccatcctgtg gtccggcatc 1380 ggctcggcga tcctctacaa gctcgtcgat ctgacgatcg gcctgcgcgt gacgcaggaa 1440 gaagagcgcg agggcctcga catcgccgac cacggcgagc gcgcctacaa ctactga 1497

[0209] Sequence Number (ID): 5

[0210] Length: 2853

[0211] Molecule Type: DNA

[0212] Features Location / Qualifiers:

[0213] - source, 1..2853

[0214] > mol_type, genomic DNA organism, Formate dehydrogenase (fdh)

[0215] Residues: atgaccctca tcaaggaaat cgactacggc acgccgatcc ggctcaatga gcagacggtg 60 acgctgacca tcgacggcga gagcgtgacc gtgcccgcgg gcacctcggt catggccgcc 120 gcgatgcata tgggcaccaa gatcccgaag ctctgcgcta cggattcgct cgagccgttc 180 ggctcctgcc ggatgtgcct cgtggagatc gacggccgcc gcggcgcgcc cgcctcctgc 240 accaccccgg ccgagaatgg catggtcgtg cacacgcaaa ccgacaagct gcatcggctg 300 cgcaagggcg tgatggagct ctacatctcc gaccacccgc tcgactgcct gacctgcgcc 360 gccaacggcg attgcgagct gcaagatact gcaggtcagg tcggcctgcg cgaggtgcgc 420 tacggctatg acggcgacaa ccacgtcaag ccggcctccg accgctacct gcccaaggac 480 gagtcgaacc cctacttcac ctacgatccg tcgaagtgca tcgtctgcaa ccgctgcgtc 540 cgcgcctgcg aggagacgca gggcaccttc gcgctgacca tcgagggccg cggcttcgac 600 agccgcgtgg cggcgggccc gaccaacttc atgcagtccg aatgcgtgtc ctgcggcgcc 660 tgcgtgcagg cctgcccgac cgcgacgctg caggagaaga cgatccacca atacggccag 720 ccggaccatt ccgaggtcac gacctgcgcc tattgcggcg tcggctgcgc cttcaaggcc 780 gagatgcagg gcgacaaggt tgtccgcatg gtgccctaca agggcggcaa ggccaacgag 840 ggccatagct gcgtcaaggg ccgcttcgcc tacggctacg ccacccacaa ggaccgcatc 900 accaagccga tgatccgtga gaagatcacg gatccgtggc gcgaggtgtc gtgggaggag 960 gcgatcaacc acgccgcctc cgagttcaag cgcatccagg cgacgtatgg ccgcgactcg 1020 gtcggcggca tcacctcgtc gcgctgcacc aacgaggaag cctacctcgt ccagaagctg 1080 gtgcgcgccg ccttcggcaa caacaacgtc gatacctgcg cccgcgtctg ccactcgccg 1140 accggctacg gcctgatgtc cacgctcggc acctccgccg gcacgcagga cttcaagtcg 1200 gtcgaggaat ccgacgtgat cctcgtcatc ggcgccaacc cgaccgacgg tcaccccgtc 1260 ttcggctcgc ggatgaagaa gcggctgcgt gaaggcgccc gcctcatcgt cgccgacccg 1320 agaaaaatcg acctcgtgaa gtcgccccac atccgggccg accatcacct gccgctcaag 1380 cctggctcca acgtcgcctt catcaacgcc ttcgcccacg tcatcgtcac ggaagggctg 1440 atcgccgagg actacgtccg cgagcgctgc gatctggccg agttcgagtc ctgggcccgg 1500 ttcatcgccg aggagcgcaa ctcgccggag gccgcgcagg ccatcaccgg cgtcgatccg 1560 caggagatcc gcgccgcggc ccggctctac gccaccggcg gcaaggcggc gatctactac 1620 gggctcggcg tgaccgagca cagccagggc tcgaccatgg tgatgggcat ggccaacatc 1680 gccatggcca ccggcaatat cggcatggtg ggtgcgggcg tgaacccgct gcgcggccag 1740 aacaacgtgc agggctcctg cgacatgggc tcgtcccgc acgagctgcc gggctaccgc 1800 cacgtctcgg acgacgccac ccgcgagagc ttcgaggcga tctggggggc caagctcgac 1860 aacgcgccgg gcctgcgcat caccaacatg ctggacgagg ccgtcggcgg cagctcaag 1920 ggcatgtaca tccagggcga ggacatcgcg cagtccgacc ccgacaccca tcacgtcacc 1980 tccggcctca aggccatgga gtgcatcgtg atccaggacc tgtcctgaa cgagaccgcc 2040 aaatacgccc acgtctcct gccgggcgcc tcgttcctgg agaaggacgg caccttcacc 2100 aatgccgagc gccgcatctc ccgcgtgcgc aaagtgatgg ccccgatggg cggctacggc 2160 gattgggagg gcacggtgct gctcgccaac gcgctcggct acaagatgga gtacacccac 2220 ccgtccgaga tcatggacga gatcgcggcg ctcaccccga gctcgccgg cgtctcctac 2280 gacaagctgg aggagctggg ctcgatccag tggccgtgca acgagaaggc gccgctcggc 2340 acgccgatga tgcacgtcga ccggttcgtg cgcggcaagg gccgctcat gatcacggaa 2400 tacgtgccca ccgacgagcg gaccacgggc aagttcccgc tgatcctcac cacgggccgc 2460 atcctctcgc agtacaatgt cggcgcgcag acgcggcgga ccgagaactc ccgctggcac 2520 gaggaagacg tgctggagat ccacccctc gacgcggaga tgcgcggcat cgtcgatggc 2580 gacctcgtcg ccctggagag ccgctcgggc gacatcgcgc tgaaggccaa ggtgaccgag 2640 cggatgcagc cggggatcgt ctacacgacg ttccaccacg ccaagaccgg cgccaacgtc 2700 atcaccaccg actattcgga ctgggcgacc aactgccccg aatacaaggt gacggcggtg 2760 caggtgcggc gcacgaaccg gccctccgac tggcaggcga agtctacga ggaggacttc 2820 tcgctcaccc gcatcgccga agcggcggaa tag 2853

[0216] Sequence Number (ID): 6 Length: 300

[0217] Molecule Type: DNA Features Location / Qualifiers:

[0218] - source, 1..300

[0219] > mol_type, genomic DNA

[0220] > organism, Overlapping sequence (5') for Nitrogenase integration Residues: atgcgtgcac tggtgtggca cggaacccag gacgtccggt gcgactcggt tcctgatccg 60 gagatcgagc acgagcgcga cgccatcatc aaggtcacga gttgcgccat ctgcggctcg 120 gacctgcacc tgttcgacca tttcataccc acgatgaagt cgggcgacat cctcggccac 180 gagaccatgg gcgaggtggt cgaggtgggc tcggcggcca agtccaagct caaggtcggc 240 gaccgggtgg tgatcccctt cacgatcatc tgcggcgcat gcgaccagtg caagcgcggc 300

[0221] Sequence Number (ID): 7

[0222] Length: 300

[0223] Molecule Type: DNA

[0224] Features Location / Qualifiers:

[0225] - source, 1..300

[0226] > mol_type, genomic DNA

[0227] > organism, Overlapping sequence (3') for Nitrogenase integration Residues: gtcctgcgcg agatgatcta tgtctgccgg cccgccggca cgctctcggt gcccggcgtc 60 tatggcggcc tcatcgacaa gatcccgttc ggcgcgctga tgaacaaggg cctgacgatc 120 cgcacgggcc agacccacgt caatcgctgg agcgacgacc tgctgcggcg gategaggag 180 ggteagateg atccctcctt cgtgatcacc cataccgagc cgctggagcg cgggcccgag 240 atgtacaaga ccttccgcga caagcaggac ggetgeatea aggtegtget caagccctga 300

[0228] Sequence Number (ID): 8

[0229] Length: 200

[0230] Molecule Type: DNA

[0231] Features Location / Qualifiers:

[0232] - source, 1..200

[0233] > mol_type, genomic DNA

[0234] > organism, nifH promoter

[0235] Residues: ggcccgacaa aaccccgccc gacaccgcag gccctacaat ttgtcggcct tgttccattt 60 cgaacaaaac cttcaacacc atgatttege gtcattattt gegaaaatte cggttggcac 120 gatggetget gtagaagctg tgagcccggt taggaaccgt ctcgatattc gtgaagcacc 180 aacccccaag ggagccacac 200

[0236] Sequence Number (ID): 9 Length: 5119

[0237] Molecule Type: DNA

[0238] Features Location / Qualifiers:

[0239] - source, 1..5119

[0240] > mol_type, genomic DNA

[0241] > organism, Final integration cassette for Nitrogenase Residues: atgcgtgcac tggtgtggca cggaacccag gacgtccggt gcgactcggt tcctgatccg 60 gagatcgagc acgagcgcga cgccatcatc aaggtcacga gttgcgccat ctgcggctcg 120 gacctgcacc tgttcgacca tttcataccc acgatgaagt cgggcgacat cctcggccac 180 gagaccatgg gcgaggtggt cgaggtgggc tcggcggcca agtccaagct caaggtcggc 240 gaccgggtgg tgatcccctt cacgatcatc tgcggcgcat gcgaccagtg caagcgcggc 300 ggcccgacaa aaccccgccc gacaccgcag gccctacaat ttgtcggcct tgttccattt 360 cgaacaaaac cttcaacacc atgatttcgc gtcattattt gcgaaaattc cggttggcac 420 gatggctgct gtagaagctg tgagcccggt taggaaccgt ctcgatattc gtgaagcacc 480 aacccccaag ggagccacac atgacccgca agatcgccat ttacggcaaa ggtggtatcg 540 gcaaatcgac gaccacccag aacaccgccg cagcccttgc ctttttccac gaaaagaacg 600 tcttcatcca cggctgtgac ccgaaagccg acagcacccg gctgatcctg ggcggtctgc 660 cgcagcagac ggtgatggac acgctgcgca tcgagggcgc cgagcgcgtc accgtggaca 720 aggtcgtgaa gaccggcttc aaggacatcc gctgcgtgga atcgggcggg ccggagccgg 780 gcgtgggctg cgccggtcgc ggcgtcatca ccgccatcga cctgatggaa gaaaacgaag 840 cctacagcga agaccttgat ttcctgttct tcgacgtgct gggcgacgtg gtttgcggcg 900 gcttcgccat gcccattcgc gacggcaagg cggaggaagt ctatatcgtc gcctcgggcg 960 agatgatggc gatctatgcc gccaacaaca tctgcaaggg tctggcgaaa tacgcccggc 1020 aatcgggcgt gcgtctgggc gggatcatct gcaacagccg caatgtcgat ggcgaaaagg 1080 aattccttga ggaattcacc aaggccatcg gcaccaagat gatccacttc gtgccgcgcg 1140 acaacatcgt gcaaaaggcc gagttcaaca agcagaccgt gaccgaattc cagcccgagg 1200 ccaatcaggc gcaggaatac cgcgaactcg gccgcaagat catcgagaac gaggatttcg 1260 tgatcccgaa gccgctcgcc atggatgagc tggaagccat ggtcgtcaaa tacggcctga 1320 tggactgatc ctccggggcg ccgcgcccgg cgccctttcc ccgaatttcc atggagttag 1380 gccgatgccc taccatgagt ttgaagtctc gaaatgcatc cccgagcggc gcgagcacgc 1440 ggtgatgaag gcggcgggcg aggatctgac ctcttgcctg ccgaaaggct acctgaacac 1500 gatccccggc acgatttccg aacgcggctg cgcctatgc ggcgcaaaac acgtgatcgg 1560 cacgccgatg aaggacgtga tccacatctc ccacgggccc aatggctgca cctatgacac 1620 ctggcagacg aagcgctaca tctcggacaa tgacaacttc cagttgaaat acaccttgc 1680 gaccgacgtg aaggaaaagc acgtcgtctt cggcgccgag gggctgctga agaaatcgat 1740 gcacgaggcc tttgacgcct tcccgaacat caagcggatg acggtctatc agacctgcac 1800 gacggcgctg atcggcgatg acgtcgatgc catcgccaag gaagtgatgg aagaacgcgg 1860 cgatgtcgat gtgtcgtct gcaactcgcc cgggtcgcg gggcccagcc aatcgggcgg 1920 tcaccacaag atcaacatcg cctggctgaa ccagaaagtc ggcacggtcg agccggacta 1980 cctaggcgaa cacgtcatca actacgtggg cgaatacaac atccagggcg accaggaggt 2040 gatgatcgac tatttcaacc gcatgggcat tcaggtgctg tcgacctca ccggcaacgg 2100 cagctatgac agcctgcgga tgatgcaccg cgcgcatctg aacgtgctcg aatgcgcgcg 2160 gtctgccgaa tacatctgcg acgaactccg cgcccgctac ggcatcccc ggctggatat 2220 cgacggcttc ggcttcgagc cgctcgccaa ttcgctgcgc aaggtcgcgc tcttcttcgg 2280 catcgaggac aaggccgagg cgatcatcgc cgaggaatat gcgaagtgga agccgcagct 2340 ggactggtac aaggaacggc tgaagggcaa gaaggtctgc ctctggccgg gcggatcgaa 2400 gctctggcac tgggcccatg cgatcgaaga ggaaatgggc ctcaaggtcg tgtcggtcta 2460 taccaagttc ggccatcagg gcgacatgga aaagggcgtc tcgcgttgcg gcgagggggc 2520 ctggcgatc gacgacccga acgagctgga atcggtcgaa gccatcgaga tgctgaagcc 2580 cgacatcatc ttcaccggca aacgccccgg cgaatcgtc aagaaacacg gcgtccccta 2640 tctgaacgcc catgcctatc acaacgggcc ttacaagggc ttcgagggct gggtccgct 2700 cgctcgcgac atctacaacg cgatctatc gccgatgcgg cagctggcgg cgctggatat 2760 ttccgccccc gatgcggcca tcacctcggg ctccgcacc gccaagatga acgccgatct 2820 gaccgtttcg gatgaggtca agttcagcga ggtgctgcac gaatacaccg gcaaatacga 2880 ctcgattgcc gagatccgcg cccgcaatca ggcctatgcc gccgagcaga aagcgctccg 2940 cgacgccgt caacctgccg ccgaatgagg gacagatgac cgatatcagc gaaaaactcg 3000 atccgctcgt cgatacatc atgaagaact gcctgtggca gttcaactcg cgcggctggg 3060 accggctcaa gcagaatgcc gggatcctgt cgcagacctg cgagatcctg tgcggcgagg 3120 agccggtgca tgaaaccgcg atggaccgct gctactgggt cgatgcggtg atcctgtcgc 3180 gcgcctacaa ggcccgcttc ccctggctga tggccatgac caagcccgag atcaagagcc 3240 tgttcaaggc gtgcacgag aagatcgacc atctcaccgt tcacggctcg ctcaataccg 3300 agctgaccgt cccgcattat tgaaaggaag accccaatga ctgccaggt cacccagaag 3360 gcgcgggaag gcacgatcaa cccgatcttc acctgccagc ccgccggggc gcaatcgcc 3420 tcgatcggga tcaaggatg catcggcatc gttcacggcg gccagggctg cgtgatgtc 3480 gtgcgtctgc tgatctcgca gcacatgaag gaaagcttcg agatcgcctc gtcgtcggtc 3540 catgaagatg gcgcggtct cggcgcgctc gaccgtgtcg aaaccgcggt cgaggtgctt 3600 ttgacccgct accccgatgt gaaggtggtg ccgatcatca ccacctgctc gaccgagatc 3660 atcggcgacg acgtggacgg gctgctttcg aaactcgaag acgagtgct gccgaccaaa 3720 ttccccggcc gcgaagtgca tctgctcacc gtgcatgcc cgagctcgt tggctcgatg 3780 atcaccggct atgacaaggc ggtgcatgat ttcgtgaaga aattcgcgac gaaggacgag 3840 cccagcgaca agatcaacct gatcaccggc tgggtgaacc cgggcgatgt gaaggagctg 3900 aaacaccttc tggaggtgat ggaggtcaag gcgaacgtgc tctttgaagt cgaaagcttc 3960 gacagcccgc tgatgcccga tctggaacac cattcgcacg gctcgaccac gatcgaggat 4020 ctgcgcgaca cggccaatgc gaaaggcacc atcgcgctca atcgctatga aggcatgaag 4080 gccgccgat acctgaagaa gaagttcaag gttcccgcgg tgatcgggcc gaccccggtc 4140 ggcatccgca acaccgatgc ctcctgaaa gcggtctccg agatgaccgg ccagccgatc 4200 ccggcgcagc tggtcaagga acggggcctt gcgctcgatg ccatcgccga catcggccac 4260 atgtttctgg ccgacaaaag ggtggcgatc tatgcgaacc ccgatctggc catcggcctg 4320 accgagttct gcctgacct cgaaatgaag ccgaaactgc tgcttctggg cgatgacaac 4380 tcgggctatg tgaaagaccc ccgcgtgctg gcgctgcagg aaaacgcgcc ggatctggaa 4440 atcgtgacga acgccgattt ctgggatctg gaaagccgca tccagcaagg gctcgaactc 4500 gatctgatcc tcggccatc caagggccgg ttcatctcga tcgactacaa ggtgccgatg 4560 gtccgcgtgg gctcccgac ctacgaccgg gcagggatgt atcgccatcc ggtgctgggc 4620 tacggcgggg cgatgtcct tgccgaaacc atggccaaca cgcttttcgc cgacatggag 4680 gcgaagaaaa acaaggaatg gatcctcaac gtgtggtgac gtctgaagcc ggaactgcct 4740 gacggcagat gcgaaccgtc cctcccccgc tcacccgggg gagggacggg gccgagatgg 4800 cgcgggcacg aacggcctgg tcctgcgcga gatgatctat gtctgccggc ccgccggcac 4860 gctctcggtg cccggcgtct atggcggcct catcgacaag atcccgtcg gcgcgctgat 4920 gaacaagggc ctgacgatcc gcacgggcca gacccacgtc aatcgctgga gcgacgacct 4980 gctgcggcgg atcgaggagg gtcagatcga tccctcctc gtgatcaccc ataccgagcc 5040 gctggagcgc gggcccgaga tgtacaagac cttccgcgac aagcaggacg gctgcatcaa 5100 ggtcgtgctc aagccctga 5119 Sequence Number (ID): 10

[0242] Length: 2916

[0243] Molecule Type: DNA

[0244] Features Location / Qualifiers:

[0245] - source, 1..2916

[0246] > mol_type, genomic DNA

[0247] > organism, Ammonia monooxygenase (Amo) Residues: atggcggcag agcggggcta tgacatttcg cagtggtatg attcgcggcc gtggaagatt 60 gggtggtttg cgatgttggc gattgggatt ttttgggtat tgtatcagcg gacgtttggg 120 tattcgcacg ggttggactc gatgacgccg gagtttgacg cggtgtggat ggggttgtgg 180 cggtttaaca ttgtggccaa tgcgttattt tttgcgattg cggtggggtg gatttgggtg 240 acgcgggatc ggaatttggc gaatttggat ccgaagacgg agttgaagcg gtatttttac 300 tggatggggt ggttggcctg ctatgtgtgg ggggtctatt atgcgggcag ttacacattg 360 gagcaggatg cggcgtggca tcaggtgatc attcgggaca cgagttttac ggcgagtcac 420 attgtggcgt tttacgggac attcccgttg tacattacgt gcggggtgtc gagttatctg 480 tatgcgcaga cgcggttacc gttgtatagc caggcgacgt cgtttccgtt ggtggcggcg 540 gtggtggggc cgatgttcat cttaccgaac gtggggctca atgagtgggg ccatgcgttt 600 tggtttgtgg acgagttgtt tgcggcgccg ttgcattggg gctttgtgac gttgggctgg 660 tgcgggttgt ttggggcggc gggcggcgtg gcggcgcaga ttgtgagccg catgtcgaac 720 ttggcggatg tgatttggaa cggggcgccc aagagcatcc tggatccgtt ccccagccag 780 gtcaacccca acgccaaagc aggatactga atgtttagaa cggatgaaat aatcaaagcc 840 gccaagttgc ctccagaggg agtggcgatg tcgcggcaca ttgattacat ttactttatt 900 cctattttgt tcgtgaccat catcggaact tttcacatgc acacggcttt gttgtgcggt 960 gactgggatt tctggttgga ttggaaggat cggcagtggt ggccgattgt gactcccatc 1020 acaacaatta ccttctgcgc agcccttcaa tactataact gggtcaatta tcgtcagccg 1080 tttggggcaa cgataaccat tttagcgtta ggtgccggaa aatgggttgc ggtttacacc 1140 tcttggtggt ggtggtccaa ctatccgcca aatttcgtca tgccggccac gttgcttcct 1200 agcgccttgg ttcttgattt caccttgttg ctaactagaa actggacttt gaccgcagtg 1260 atcggggcct ggatgtacgc gattttgttc tatccgagca attggcctat ctttgcttac 1320 agccatactc cgctgtggt ggatgggacc ttgctttcat gggccgacta tatgggcttt 1380 atgtatgtgc ggaccggaac tcctgaatat atccgtatga ttgaagtgg gtcgctgcgg 1440 acgtcggtg ggcacagcac gatgatttcc tcgttcttg ctgcattcgc ctcttcattg 1500 atgtacatcc tgtggtggca gtttggaaag tttttctgca cgtcctattt ctacttcacg 1560 gatgacaaga agcgaacgac caaagtttac gatgtctttg cctatgcaac attggctcac 1620 gcggataagg ccaaactctc tggggggaaa gcatgaatga acgtcaaaca cgtcttcaag 1680 ctgtggatgc tgggattctg cggagtggcg acgttggcgt tcacgccggt gtttgatgct 1740 gctccagttc ttgctcacgg ggagcgtcg caagaaccgt ttctgcggat gcgcaccgtg 1800 aatggtatg acactgaatg ggtggggaaa agcactgcgg taaatgatgt tacatacatg 1860 aggggcaagt ttcatctgtc tgaagactgg cctcgtgcgg tagtgaaacc ccatcgaacg 1920 ttcgtcaatg tcggctctcc tagctccgtc tttgtgcggt taagcacgaa ggtggtggg 1980 gtgccgatgt ttgtgtctgg tcctatggaa atcgggcgtg attatgaata tgagatcacg 2040 ttgaaggcga gacttcctgg acatcatcac attcacccta tgttttctgt taaagaggct 2100 ggtcccatg ccggaccggg tgggtggatg gatatcacgg gccgatacgc tgattttaca 2160 aacccgatca agactctgac gggggaaaca tttgactcgg aaacagaggg tgggatgacc 2220 ggaattatgt ggcatatatt ctgggcatct gtgcctgt tctgggtggg ttggttcatg 2280 gttcgcccga tgtacttgat tcgggctcgt gtgcttgcgg cttatggtga tgaacttctg 2340 ttggatccgg ttgatcgcaa gctcgcaata ggtcttctcg tatttacggt ggcggttgtc 2400 actatcggtt atctcgctgc ggaggcgaag catcctatta ccgtgcccct gcaggctggt 2460 gaagcaaagg ttaaaccgct tcctataaaa ccgaatccat tggtggttga agtcacccac 2520 gccgaatatg acgtgccggg tcgtgctctt cgtatgacgg ttcacgccac taacaatggg 2580 actgagcctg tcagtatcgg tgaatcaca acggctggta tcgatttac aaataaagta 2640 ggagcagcga agctcgatcc gaactatcca caggagcta ttgctacagc cggactgacc 2700 atggataatg aggctccgat acagccgggt cagactgtg acattcacat agaatcaaag 2760 gatgtctat gggaggttca gcggctggt gacatctc acgatccgga tcagcggttt 2820 gctgggtgt tgatgtcatg gactgaatcg ggagaacgtc ttataaccc cgtgtgggct 2880 cctgtgcttc ctgtctttac acgattggga gcataa 2916

[0248] Sequence Number (ID): 11

[0249] Length: 1710

[0250] Molecule Type: DNA Features Location / Qualifiers:

[0251] - source, 1..1710

[0252] > mol_type, genomic DNA

[0253] > organism, Hydroxylamine oxido reductase (Hao) Residues: atgagcagca agtttgcgca gaagtttgtg atgctcgtct gcggcgtttt actggccgca 60 ccggcgcaag cggacttccc gacggtgccg aaggaaactt atgaggcact gaagatcgac 120 cgttcggctt ctccaaagga gctgtacgag gctctggtga aaaggtacct cgatcctgag 180 caaggggccg gaaagggaat ttacggtcag tactgggagc ccgtcacttt cagtaagtat 240 ttcgatccca cgacgttcta taagcccccg caggcagtca aggaagtcgc aagccgtgag 300 cagtgcgtca aatgtcatac ggatgaatcg ccggggtggg tggctgcgtg gaagcgcagc 360 acgcatgcca atctggataa gatccgtaaa ctgacgccca aggatgagac ctattacaag 420 aaggcgaagc ttgagcaaat cgaggaaaac cttcggtcga tgggcaagtt gggcaagggg 480 gagcagctaa aagaagttgg gtgcattgat tgtcactttg aaatcaatgc caaagggaag 540 gccgatcatc gcaaggacat taagctggcg acggcgaaca cctgcggtac gtgccatctt 600 caggaattcg ctgaacgaga atcggagcgt gacaccatca tatggccaaa agatcagtgg 660 cccaaggggc gtccttcgca tgccttggat tataaggcta acgtggaagt ggacgtctat 720 gccgggatgc ctcaaagaga aatcgccgat ggctgtaccg gttgtcacgt caatcagaac 780 aagtgcgata cttgtcactc gcgtcatgaa ttctcagtcg cggaatcgcg caagccggaa 840 aattgcgctc agtgtcacag tggggccgat cacaacaact gggaggccta ctccctctcc 900 aagcatggat tgaagtatca gagagacaaa gataagtgga atttcaatat tcccattaaa 960 gaagccattg ccaagggtgg attgagtgcc ccgacatgcc aattctgcca catggaatat 1020 caaggtaaga taacccacaa cgtggtgcgc aaggttcgat gggccaacta tccgttcgtt 1080 cctggcatcc gagagaatat ctcgaccgat tggtcggaaa agcggctcga tgcgtgggtt 1140 aaaacctgta cgaactgtca ctcggaaagt tacgcccgcg cctggatgga gttcatggat 1200 aaaggtacgt actccggtct tgataagtac gacgaggctc atcacgtagt cgaagagcag 1260 tacaaggccg ggttgttgac ggggcaaaaa accaaccggc ccgcaccgcc tcctccggtg 1320 aaggacggct ttgagcagtt cttccagatt tactggtcaa agggaaataa cccggcggcc 1380 aacgagctca agctgttcga aatggcggag gatcacctgg ttcaattgca cgtcagcttg 1440 gctcaccagt attggggata tacctatacg gttggttggg ctgcgatgaa tcgggcctat 1500 gtggaaatca tggacgatga cactcggctt aaggaaacac aaaagttgtt ggcccgtgtc 1560 gataagcttg agagcgagcg caagcactct ctgcttgatc tcgatggccc gggcgggaag 1620 ctgtcgatcg gggggttggg cggaggtatg atgctggccg gtacgtggc aatgctggc 1680 tggcgtcgac gagagaggaa agatcgttga 1710

[0254] Sequence Number (ID): 12

[0255] Length: 2448

[0256] Molecule Type: DNA

[0257] Features Location / Qualifiers:

[0258] - source, 1..2448

[0259] > mol_type, genomic DNA

[0260] > organism, Nitrate reductase (napA)

[0261] Residues: atggttccgc gacggctaca aaaagccgtc gcaggcttcg ccacggcgaa tcgtgtctcg 60 accatcctct ttgctcttga cttgaataga ccgatcaaat cggttattga acacctgtct 120 ctccgacact ggcgaacaag cacggtctca ccggagcaag gttgtctcat gatcgatggg 180 acggacgaac cgaccggctt catcagccgc cgtgatctgc tcaagctggc gggaacgtcg 240 gcctgtgcag cctgcctcct tcccctgacc ggttgtgaaa acctctggaa acgacagccg 300 gccatcaccg tcaatcgatg ggagaaaggc gtttgtcggt tttgtggaac aggctgcggc 360 gtcatgatcg gactgaaagg cgacaaggtc gtcgatgtca cgggagacga ggaagcccat 420 aaccgtggac gattatgcat taaagggctt gcgactcgcg acattcttta ctctcccggc 480 cgcctcctcc atccgatgat ccgaaaaaac ggcgaacttg agcgagccac gtggaatgag 540 gcgatggacc tcgtcgctca acgattcaag gacgccatcg aaacctttgg accggacagt 600 gtggccttct acggcagcgg acaactgctg acggaagaaa gttacacggc gaacaaactg 660 tttaaagctg ggatcggcac caacaacgtg gacggcaacc cacggctgtg catggcgtcc 720 gcggtcaccg gctacacctc gacatttgga aaagacgagc cgcccggttg ctatgaagat 780 atcgaccatg ccgactgttt tttcatcacc ggctccaatg ccctggaatg tcatccgatc 840 atttgggaac ggattcaaga ccgcaagcgg agccgacccg gtacgacgat catcatcgtc 900 gacccgcgca gaaccttcac ggcacagcac gcggatcttc accttcaaat cgttcccggc 960 acggacgtcg ccctgtataa cgccatgatg cacgtgttcg tggaacggaa cttcgtcgac 1020 cgtgacatgg ttgagcagta cctctctttt caagaaggcg accggcccag ctcattcacc 1080 gacttcaaaa cccatcttgc gcggtacagc ccggaacggg ttgcggacat ctgcggggtc 1140 agcgcgacgg ccatcgaga agcggcgttt ctgtttgcgt cgtccaaagc caccatgtct 1200 cttggacta tgggattgaa ccaacaggtc cagggcaccg cctccaaccg actggtctgc 1260 gccatgcatc tgttgaccgg tcatttcggt cggcccggcg ccactccct ttcgctcacg 1320 ggacagccga acgcgtgcgg aggcgtccgc gacaccggca gcctcgctca cgcattgccc 1380 aacggacgag tcgtcaccaa tccggcgcat cggcaagaaa tggaagatct gtggaatgtg 1440 ccgcgtggac gactcagcac caagccaggc ctccatgctg tagcactctt tgatgccatg 1500 gctcgtggat cagtgaaatg tgcctgatc atgtgcacga atccaggaca gacgcttcct 1560 gatatcaacg cctatcgccg cggcatggaa aacgttttct tggtcgtcgt tgatgccttt 1620 catcccaccg aaacgaccat attcgccgac gtggttcttc ccgccgctct ctgggtggaa 1680 aaggaagggg tcaaaggcaa tggagaacga cggtatcatc tctcgcccaa gctggttgat 1740 ccgccgggtg aggccaaaag tgatttagag attctggtcg agttagcgga ccgcctcggc 1800 tatggtcaac tcatcacggc caggacaccc aaagacgtgt gggacgaatg gcgcaagatt 1860 tcggcacact ccgagtataa cttcgagggt atcacgtatg aacggctcaa gcaggaacgg 1920 ggtctcttgt ggccctgtcc ctccgagacc catcctggca ccaaacggcg atacgtgccg 1980 ggagaagatc ccatcgccac aggaagcggg cggttcgact tttatggcaa accggacagg 2040 cgggccatca tctggctgga ccatcaagaa ggtctccctg accctgtac accggagttt 2100 cccttgctct tcaacacagg tcggattctg gaacatggc atacgatgac catcaccggc 2160 cgagtcccta ctctcaatt catcacccc gactatctg agatccaccc ctacgatgcc 2220 catgccttga ctatcaggga cggcgagccg gtcatcgtga caagccgccg cggagagatc 2280 gagctccgtg ctcgtgtgac ggatgccgtc cgtcccggga cggtgttgc caccatgcat 2340 tcggcaaggc atctcatcaa tcaggttacc caggcggcgc acgacccctt ctcgaagcag 2400 ccggcctaca aacggtgcgc cgtctcggtc aggcggaagc cggtgtaa 2448

[0262] Sequence Number (ID): 13 Length: 954

[0263] Molecule Type: DNA

[0264] Features Location / Qualifiers:

[0265] - source, 1..954

[0266] > mol_type, genomic DNA

[0267] > organism, Nitrite reductase (nirK)

[0268] Residues: atgcagaggt ttcgtgtgct cacataacg ctcgggcttg gcgtcctct cggagccagc 60 ggatgtatca cgatgccggc atctgccggg gcaaaggttc atgacgtcac gtcacggcg 120 accgaatcgg aaatcgtcat cgacggcaac gggacgaagt acaaggcctg gacgtttaac 180 ggtcaaatgc cgggcccggt tgttcgggtc acgcagggcg ataccgtcaa cttcaccttg 240 atcggccaca aggacaacgc ttttccgcat tcaatggact tccatgcggc cgagctcgac 300 ttcttgaaaa actaccacaa gacggtcgga ccgggcgaga cacataaat ctcctcgtg 360 gcgaagaaac ccggcgtgt tttctaccat tgcggcgcga gccccatgat ccagcacgtc 420 gcccgcggca tgttcggcgc catcatcgtc gaccccaaag attcgagcgt gtggcccaaa 480 gccgatcgag aatcgtgtt ggtgcagtcc gagctgtgga agaatcctga caacgtccag 540 gccatgtttg accggaagtg ggaccatacg gtctcaacg gcggcatctt taaataccat 600 ccgtctttc ccggcggaga accgctggaa gtcaaggtcg gcgagcgggt acgaatctac 660 tttgtgaacg ccggccccaa tgaattctcg gcgctccatc ccatcgcgga aatctgggac 720 gccgtctatg aaagcggcaa tcctcaaac aagtcatcg gagttcagac tatgtcgtc 780 gggccaggca gcgcggccac cttcgacatg atcgccgatg agccgggagc ctatcccgtc 840 gtgacccact ccttgaccgg ggccctccgg ggtgccatcg cggtggtggt cgccaacaag 900 aatcccaagg actataaagg tcagtgatg ccaaacaccc ctggaaccc gtaa 954

[0269] Sequence Number (ID): 14 Length: 1953

[0270] Molecule Type: DNA

[0271] Features Location / Qualifiers:

[0272] - source, 1..1953

[0273] > mol_type, genomic DNA

[0274] > organism, Nitrous oxide reductase (nos)

[0275] Residues: atgagcgaca gcgacaacat caagggcgtc agccggcgaa cgctgctggg gaccacggcg 60 gccgctgcgg gcgtgggact tgccggcggg gcggtggtaa caaaggacgg cgcgggcttc 120 gtctcgaccg ccgacgcgca gaccaaatct gccgcaccca aggcaccgcc ggcgcgtccg 180 gccgtgcaga agaccgaagt cgcgccgggc gagctcgacg aatattacgt cttcttttcg 240 agtggccaat ccggcgagat gcggatcatc gggctcccct cgatgcgcga actgatgcgc 300 gttccggtct tcaaccgctg cagcgcgacc ggctggggcc agaccaacga aagcctgaag 360 gttttgacag aagggatgca gccggcgacc cgcgaatcc tcaagaaccg tggcggcacg 420 ttcatgaacg gcgacctcca ccatccgcac gtctcctca cggacggcac ctacgacggc 480 cgctacgcct tcatgaacga caaggccaac agccgcgtag cgcgggtgcg gctcgacgtc 540 atgaagtgcg acaagattat cgaactgccg aatcagcaca ccgtccacgg ccttcggctg 600 cagaagtatc cacgaaccgg atatgtgttc tgcaacgggg aagacggcgt tccgctgccg 660 aacgacggca aggtctcga caacccgaag gaatatcact ccatctcac ggcgctcgac 720 ggcgatacca tgaaagtggc ctggcaggtg atggtagcg gcaatctcga caacgtcgat 780 tccgactacc aaggcaaata ctgctctcg acctgttaca acgccgaaga aggcgtcact 840 ctggccgaga tgaccgccaa cgagcaggac tgggtcgtca tcttcaacct caagcggatc 900 gaggaggcag tgaagaaggg tgatttcaag gaaatgaacg gcgtgcccgt gatcgacggt 960 cgcaagggct cgccctacac gcgctacgtt ccggtgtcga acaatccgca tggcatgaat 1020 accgcgcccg acggcatcca catcgtcgcg gcaggcaagc tgtcgccgac ggtgacggtg 1080 atggacgttc ggctgttcga ccaactgttc gacgacaaga tcaaaccgcg cgacgtcgtg 1140 gtcgccgagc ccgaactcgg cctcgggccg ctgcatacgg cctacgacgg caagggcaac 1200 gcctacacga cgttgtcct cgacagccag gtcgtcaaat ggaacatcga tctcgccaaa 1260 cgagccttta agggcgagaa ggtcgatccc atcatccaga agctcgacgt ccactatcag 1320 cctggccaca atcactcctc gatgggtcag accaaggagg ccgacggaaa atggctgatc 1380 tcgctgaaca agttttcgaa ggaccgcttt ctcaacgtcg gcccgctgaa gccggagaac 1440 gatcagttga tcgacatctc gggcgaccag atgaaactgg tccatgacgg tcctagcttc 1500 gccgagccgc atgacgccac catcgtgcac cgttccaaga tcaacccgat ctcgatctgg 1560 gaccgcgccg atccgatgtt tgccgatgcg gtcaagcaag ccaaggccga cgggatcaac 1620 ctggaggcgg atccaagat catccgcgac ggcaacaagg tgcgggtcta tatgacctcg 1680 accgctccgg cgtcgggct cgagcagtc caggtcaagc agggcgatca ggtcaccgtc 1740 tacatcacca acatcgatgc ggtcgaagac ctgacgcacg gcttctgcat cgtgaactac 1800 ggtatccaga tggaggtcgc gccgatggcg accgcgtcgg tgtcgttcag cgccgacaag 1860 gcgggcgtct actggtacta ctgctcctgg tctgccacg ccatgcacat ggagatgaag 1920 ggccgcatgt tcgtcgagcc gaaatcggtc tga 1953

[0276] Sequence Number (ID): 15

[0277] Length: 200

[0278] Molecule Type: DNA Features Location / Qualifiers:

[0279] - source, 1..200

[0280] > mol_type, genomic DNA

[0281] > organism, Gap promoter

[0282] Residues: ccaggatacg ggcgcaagtc atgccgggaa ccggctctga tatacacgta aattcccccg 60 gtttttcctc cgaaacgacc gtatcgacgc gggtttgccc ctttggtata acttgacaaa 120 atcgggacgc catcgatact atcgggccca ccaggccggt acttttcacc ggcctatttt 180 tcttgaagga ccacctcgct 200

[0283] Sequence Number (ID): 16

[0284] Length: 1225

[0285] Molecule Type: DNA

[0286] Features Location / Qualifiers:

[0287] - source, 1..1225

[0288] > mol_type, genomic DNA

[0289] > organism, Integration cassette (NirK deletion & Amo overexpression)

[0290] Residues: atgcagaggt ttcgtgtgct cacattaacg ctcgggcttg gcgtcctctt cggagccagc 60 ggatgtatca cgatgccggc atcaacgttt gcgccaagct tcctgctgaa catcaaaggc 120 aagaaaacat ctgttgtcaa agacagcatc cttgaacaag gacaattaac agttaacaaa 180 taaaaacgca aaagaaaatg ccgatatcct attggcattt tcttttattt cttatcaaca 240 taaaggtgaa tcccataggg caggagctaa ggaagctaaa atggagaaaa aaatcactgg 300 atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc 360 agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac 420 cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat 480 gaatgctcat ccggaattcc gtatggcaat gaaagacggt gagctggtga tatgggatag 540 tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat cgctctggag 600 tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg tggcgtgtta 660 cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt tcgtctcagc 720 caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg acaactctt 780 cgcccccgtt tcaccatgg gcaaatata tacgcaaggc gacaaggtgc tgatgccgct 840 ggcgatcag gttcatcatg ccgtttgtga tggcttccat gtcggcagaa tgcttaatga 900 attacaacag tactgcgatg agtggcaggg cggggcgtaa gccggcccca atgaattctc 960 ggcgctccat cccatcgcgg aaatctggga cgccgtctat gaaagcggca atcctcaaa 1020 caagttcatc ggagtcaga cttatgtcgt cgggccaggc agcgcggcca cctcgacat 1080 gatcgccgat gagccgggag cctatcccgt cgtgacccac tcctgaccg gggccctccg 1140 gggtgccatc gcggtggtgg tcgccaacaa gaatcccaag gactataaag gtcagtgat 1200 gccaaacacc cctggaacc cgtaa 1225

Claims

WE CLAIM,1. A method for genetically modifying microbes for in-vitro / in-vivo / intra cellular / extra cellular production of non-Carbon Nitrogen-containing compounds (including but not limited to Ammonia, nitrate, nitrite etc. and their derivatives / salts) wherein the modifications include enhanced / induced intracellular / extracellular Cl Carbon uptake / fixation / utilization (a) with or without enhanced / induced intracellular and / or extracellular Hydrogen uptake / utilization (b) with or without enhanced / induced nitrogen fixation (c) with or without enhanced uptake / synthesis / regeneration of ATP / NADH / NADPH; (d) with or without enhanced synthesis / concentration of pyruvate and acetyl-CoA; (e) with or without enhanced intracellular and / or extracellular Ammonia uptake / utilization or induced Ammonia uptake / utilization; (f) with or without enhanced intracellular and / or extracellular Nitrate / Nitrite / N2O / N2O2 uptake / utilization or induced Nitrate / Nitrite / N2O / N2O2 uptake / utilization; (g) with or without enhanced intracellular and / or extracellular utilization / uptake of carbon containing substrates like carbohydrate, organic acids, etc., (h) any combination of (a) to (h), wherein the said micro-organism can be a natural or induced diazotroph, epiphyte, endophyte, endosymbiont, rhizosphere, free-living, ruminant / non-ruminant gut microbe etc.

2. The method as claimed in claim 1, wherein the said modifications include homologous and / or heterologous expression(s) of genes for enhanced / induced Carbon and / or Nitrogen and / or Ammonia / Nitrate / Nitrite / N2O / N2O2 and / or Hydrogen fixation and / or uptake / synthesis / regeneration of ATP / NADH / NADPH and / or enhanced / induced Pyruvate and Acetyl CoA synthesis / increased concentration and / or enhanced intracellular and / or extracellular utilization / uptake of carbon containing substrates, their promoter / s and / orassociated regulatory gene / s, or a combination thereof wherein the said gene / s are native / modified / novel. The method as claimed in claim 1, wherein said genetically modified microbes belonging to endophyte, epiphyte or Rhizospheric / free living microbes including but not limited to, Enterobacter, Serratia, Azorhizobium, cyanobacteria, Gluconacetobacter, Acetobacter, Beijerinckia, Duganella, Delftia, Sinorhizobium, Bradyrhizobiun, Halomoncis , Methylobacterium spp., Methylobacterium symbioticum, Methylorubrum, Methylomonas, Bacillus, Paenibacillus, Lactobacillus, Mycoplasma, Acetabacterium, Streptomyces, Rhodococcus, Frankia sp, Microbacterium, Curtobacterium, Bradyrhizobium japonicum, Ralstonia eutropha, Epichloe typhina, Rhodococcus, Saccharomyces,Pichia, Methylobacterium spp., Methylobacterium symbioticum, Methylorubrum, Paenibacillus, Bacillus, Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Herbaspirillum, Pantoea, Serratia, Rahnella, Azospirillum, Azotobacter, Azorhizobium, Beijerinckia, Duganella, Bradyrhizobiun, Sinorhizobium, Methylomonas, Methylosarcina, Methylococcus, Nitrospira, Nitromonas, Nictrobacter, Bacillus, Paenibacillus,Streptomyces, Rhodobacter sphaeroides, Cupriavidus necator, Rhodobacter spp, Azospirillum lipoferum, Rhodopseudomonas palustris, Flavobacterium, Xanthobacter spp, Pichia, Saccharomyces, etc. The method of genetic modification of Diazotroph / induced diazotroph, wherein enhanced Carbon fixation is also achieved by enhancing Nitrogen fixation. The method of genetic modification of Diazotroph / induced diazotroph, wherein enhanced Nitrogen fixation is also achieved by enhancing Carbon fixation. The method as claimed in claims 1 and 4, Nitrogen fixation is increased through homologous and / or heterologous expression or modification of Nitrogenase enzyme / s including, but not limited to Iron-containing nitrogenase and / or molybdenum-based nitrogenase and / or vanadium-based nitrogenase, bimetallic nitrogenase, nitrogenase-like enzymes, bacterial chlorophylls (BchL, BchM, BchB).

7. The method as claimed in claims 1 and 6, where in the said Nitrogenase is expressed under control of specific promoter, enabling preference for Iron and / or Molybdenum and / or Vanadium and / or Bimetallic containing Nitrogenases8. The method as claimed in claim 1 and 4, the said enhanced Nitrogen fixation is achieved by manipulation of Nitrogenase regulatory genes including but not limited to regulatory proteins, such as nifA and / or nifL, and / or fix genes and / or operons such as fixABCX, fixNOQP, fixH, fixJ, fixR, fixK, fixL, and / or mf cluster genes such as mfABCDEG, etc.

9. The method as claimed in claim 6 and 8, wherein the said homologous / heterologous enzymes are native and / or modified, where enzyme modifications include but are not limited to point-mutations, upregulation and / or downregulation / silencing / deletion, epigenetic machanisms.

10. The method as claimed in claim 1, wherein enhanced ammonia production is achieved by reduced / arrested ammonia uptake / utilization, wherein ammonia uptake modification is achieved by deletion and / or downregulation of ammonia utilization enzymes including, but not limited to AmtA, AmtB, GlnA, GlnD, GlnE, GlnK etc.

11. The method as claimed in claim 1, wherein enhanced production of Ammonia, Nitrite, Nitrate, etc., natural or unnatural, is achieved with enhanced regeneration / recycling of ATP / ADP / phosphate and / or NADH / NADPH, with or without enhanced hydrogen uptake / fixation. The enzymes for NADH regeneration includes and not limited to all Dehydrogenases and oxidoreductases. Enzymes for ATP regeneration includes but not limited to ATP synthase, ATP cyclase, Malate - Aspartate shuttle and glycerol-3 -phosphate shuttle enzymes, Polyphosphate-AMP- phosphotransferase (PAP), Polyphosphate kinase, ATP synthesis / recycling via acid production pathway involving enzymes such as and not limited to Acetate kinase / propionate kinase / Butyrate kinase, CoA-acylating dehydrogenase, Phosphotrans acetylase / propionylase / Butyrylase, aldehyde dehydrogenase, lactate dehydrogenase, etc.

12. The method as claimed in claim 1, wherein enhanced production of carbon-containing, non-Nitrogen containing natural or unnatural compounds is achieved with enhanced regeneration / recycling of Co enzyme A (Co A) and the enzymes for Co A (and Acetyl Co A / Propionyl CoA, etc.) regeneration / recycling includes such as and not limited to CoA transferases, CoA-acylating aldehyde dehydrogenase, CoA- dependent propionaldehyde dehydrogenase, phosphotransacetylase, Acetyl CoA-synthase (ACS), Acetyl coenzyme-A carboxylase (ACC), ACC- catalyzed biotin carboxylase (BC), carboxyltransferase (CT), etc.

13. The method as claimed in claim 1, wherein Nitrogen / carbon assimilation is controlled by manipulation of homologous and / or heterologous carbon uptake transporters including but not limited to PtsG, PtsN or PtsF or ManZ or LacY14. The method as claimed in claim 1 and 5, wherein the said microbe is engineered for increased carbon / Nitrogen fixation through heterologous and / or Homologous expression of enzymes of carbon fixation pathway including but not limited to enzymes involved in Calvin cycle and / or 3- hydroxy propionate cycle and / or reductive acetyl-CoA pathway and / or reductive TCA cycle and / or dicarboxylate 4-hydroxybutyrate cycle and / or 3 -hydroxy propionate / 4-hydroxybutyrate cycle and / or RuMP pathway and / or serine pathway and / or Formate-Formaldehyde pathway, to utilize compounds including but not limited to Cl carbon compounds like CO2, Methane, Methanol, Formic acid / Formaldehyde, etc.

15. The method as claimed in claim 14, wherein the said homologous / heterologous enzymes are native and / or modified, where enzyme modifications include but are not limited to point-mutations, epigenetic mechanisms, etc. Wherein, the said enhanced Cl carbon fixation can also be used to facilitate partial autotrophism, reduction / dependency on carbohydrate substrates.

16. The method as claimed in claim 1 and 14, wherein manipulation of the Calvin pathway includes but not limited to overexpression of homologous or heterologous enzyme, Rubisco (ribulose bisphosphate carboxylase / oxygenase) with or without phosphoribulose kinase (PRK), as depicted in Figure 7.

17. The method as claimed in claim 1 and 14, wherein manipulation of Formate route includes but not limited to over-expression of homologous or heterologous enzymes such as Formate dehydrogenase (FDH), Formylmethanofuran Dehydrogenase, Formolase, etc., with or without point mutations for increased catalytic activity and thermostability.

18. The method of carbon fixation as claimed in claim 1 and 14, wherein methane (CH4) fixation and assimilation includes homologous and / or heterologous over-expression of Methane monooxygenase (MMO) enzymes, including and not limited to sMMO and / or pMMO operons, for soluble and particulate methane assimilation, respectively.

19. The method as claimed in claim 1 and 14, wherein formaldehyde sequestration is enhanced by the incorporation of homologous or heterologous genes for the Formaldehyde fixation system of the Ribulose mono phosphate (RuMP) pathway, involving and not limited to 3- hexulose-6-phosphate synthase (HPS), 6-phospho-3 -hexuloisomerase (PHI) enzymes, and also by the incorporation of homologous and / or heterologous genes for the formaldehyde fixation system of Serinethreonine pathway involving, and not limited to FtfL, formate -THF ligase; Fch, methenyl-THF cyclohydrolase; MtdA, methylene-THF dehydrogenase, Formolase, etc.

0. The method as claimed in claim 1 and 14, wherein methanol sequestration is enhanced by overexpression of enzyme such as and not limited to Methanol dehydrogenase and / or alcohol dehydrogenase.

1. The method as claimed in claim 1 and 14, wherein the CO2 sequestration via the said carbon fixation pathways is enhanced by increased availability of CO2 / HCO3 (bicarbonate ions) through overexpression of enzymes suchas and not limited to Carbonic anhydrase, carbon concentrating mechanism (CCM), carboxysome, etc.

22. The method as claimed in claim 1, are modified with hydrogenases for enhanced uptake of hydrogen electrons, either extracellular or intracellular, for further enhancing the Carbon and Nitrogen assimilation and generation / regeneration of ATP / ADP, NADH / NADPH, with or without enhanced phosphate regeneration, and for enhanced production of carbon & nitrogen-containing compounds. The hydrogen uptake is achieved by expression of Hydrogenase enzymes such as and not limited to uptake Hydrogenase, Hue Hydrogenase, CO / CO2 dependent hydrogenase (CODH), etc.

23. As claimed in claim 21, enhanced CO2 assimilation is also achieved with uptake of electrons in the form of Hydrogen for CO2 reduction to formic acid.

24. The method as claimed in claim 22, where in phosphate regeneration is for generation / regeneration / recycling of AMP / ADP / ATP and / or polyphosphates, redox carriers such as NADP+ / NADPH, and sugar phosphates involved in metabolic pathways such as and not limited to Glycolysis, TCA cycle, CBB pathway, pentose phosphate pathway, RuMP pathway, Serine pathway, etc.

25. The method as claimed in claim 1, the said microbe can be modified for breakdown / utilization / enhanced utilization of varied substrates such as and not limited to organic and / or inorganic substrates in any form including gaseous / liquid / solid and combination thereof, including but not limited to carbohydrates (including but not limited to monosaccharides / disaccharides / oligosaccharides / polysaccharides, etc.), organic acids, proteins, lipids, pectin, inorganic & organic phosphates / phosphites (including phytic acid), nitrate / nitrite / Nitrous oxide (N2O), Trimethyl Amine (TMA), sulfate / sulfides (including H2S), synthetic / nonsynthetic compounds including but not limited to plastics / fibers / rubber / resins / chemicals, etc. (like polyethylene terephthalate (PET), Ethyleneglycol (EG), polyurethane), under in vivo and / or in vitro applications by homologous and / or heterologous expression of native or modified enzymes including but not limited to cellulase, exo-glucanase, endo- glucanase, cellobiohydrolase, xylanase, pectinases, pectin methyl esterase, pectin acetyl esterase, lignin peroxidase, proteases, peptidases, Endopeptidase, exopeptidases, asparaginyl endopeptidases, butelase, dihydrofolate reductase, invertase, amylase, sugar isomerases, lipases, amidases, phytase, phosphatases, nitrate reductase, nitrite reductase, N2O reductase, sulfate adenylyl transferase (Sat), adenylyl sulfate reductase (AprBA), dissimilatory sulfite reductase (DsrAB), ATP sulfurylase (ATPS), Adenosine 5 '-phosphosulfate reductase (APSR), APS kinase (APSK), Terephthalate 1,2-dioxygenase, Terephthalate reductase, Dihydrodiol dehydrogenase, Glycolate oxidase, Glyoxylate carboligase, hydroxy pyruvate isomerase, protocatechuate decarboxylase, Feruloyl esterase, TMADH-trimethylamine dehydrogenase; DMADH- dimethylamine dehydrogenase; MMADH-monomethylamine dehydrogenase, H2O2 peroxidase, peroxygenase, natural and / or Artificial metalloenzymes, Flavoenzymes etc. The method as claimed in claim 1, wherein enhanced Carbon and / or Nitrogen fixation is achieved through the expression of Homologous / heterologous, native / modified enzymes responsible for increased pyruvate availability include but not limited to (1) enzymes involved in increasing pyruvate synthesis towards downstream product formation pathways and (2) enzymes involved in preventing pyruvate loss in form of CO2. The enzymes for increasing pyruvate include and not limited to Pyruvate synthase, Pyruvate kinase, Pyruvate decarboxylase, carrier proteins like mitochondrial pyruvate carrier proteins etc., wherein such enzymes are overexpressed and / or deleted / downregulated. The method a as claimed in claims 1, 35 and 36 , wherein enzymes involved in uptake / fixation of carbon and / or Nitrogen and / or Ammonia and / or Hydrogen and / or uptake / synthesis / regeneration of ATP, NADH,NADPH, Phosphate, Pyruvate, CoA, Acetyl-CoA and / or production of carbon and Nitrogen-containing compounds, are overexpressed by the inclusion of Transcriptional and / or Translational enhancers such as and not limited to UNA1, UNA2, UNB, oligomers, etc. The method as claimed in claim 1 and 5, wherein the fixation / reduction of nitrogen compound, Nitrous oxide-N2O (greenhouse gas), is achieved by homologous / heterologous expression of genes for native / modified N2O reductase such as and not limited to nosR, nosZ, nosD, nosF, nosY, nosL, nosX, etc. The method as claimed in claim 1, wherein the said modification in microbes is also for enhancing production or inducing production of compounds for breakdown / denaturing / detoxifying / degrading toxic compounds such as and not limited to pesticides, aflatoxin, insecticides, etc. The method as claimed in claims 4 and 5, wherein assimilation is enhanced by integrating the regeneration / recycling of the metabolic intermediates including but not limited to Hydrogen, Phosphate / ATP, NADH / NADPH, Pyruvate, Coenzyme A-CoA / Acetyl CoA resulting in stable / enhanced metabolic efficiency of the host microbe. The method as claimed inclaim 1, wherein the enhanced / production of Nitrate / Nitrite (including their derivatives) and their salts is achieved by upregulation and / or downregulation / deletion of homologous and / or heterologous enzymes such as and not limited Ammonium monooxygenase, Hydroxylamine oxidoreductase, Nitrate reductase, Nitrite reductase, etc, by co-fermentation / individual fermentation integrated with final conversion process. The method as claimed in claim 31, the said salts of ammonia / nitrate / nitrite include but not limited to Ammonium nitrate, sodium nitrate, potassium nitrate, calcium nitrate, magnesium nitrate, sodium nitrite, Ammonium sulphate, Monoammonium phosphate (MAP), Diammonium phosphate (DAP), nitro glycerine etc.

33. The combination described in claim 1, wherein the said enhanced microbes and the combinations thereof can be applied along with or without other microbes / inorganic / organic materials in any form such as and not limited to Ammonium sulphate, urea, super phosphate, ammonium phosphate, limestone, gypsum, muriate of potash, etc.

34. The method as claimed in claims 10 to 31, wherein the said homologous / heterologous enzyme / s are native and / or modified enzyme, expressed under native and / or modified promoter, where modification includes but not limited to point-mutations, point-mutations, upregulation and / or downregulation / silencing / deletion, epigenetic mechanisms, etc.

35. The present invention additionally relates to a method for gene modifications in microbes, more specifically involving gene modifications for enhancing transcription and / or translation of proteins involved in (i) uptake / fixation Carbon and / or (ii) uptake / fixation of ammonia and / or (iii) fixation of Nitrogen and / or (iv) uptake / fixation of Hydrogen and / or (v) uptake / synthesis / regeneration of anyone or all compounds including phosphate, ATP, NADH, NADPH, Pyruvate, CoA and Acetyl CoA and / or (vi) for in-vitro / in-vivo / intra cellular / extra cellular production of nonCarbon Nitrogen-containing compounds (including but not limited to Ammonia, nitrate, nitrite etc. and their derivatives / salts).

36. The present invention also relates to a method for gene modifications in microbes, more specifically involving gene modifications resulting in increased uptake / synthesis / regeneration of Phosphate and / or Hydrogen and / or CoenzymeA for (i) uptake / fixation Carbon and / or (ii) uptake / fixation of ammonia and / or (iii) fixation of Nitrogen and / or (iv) uptake / fixation of Hydrogen and / or (v) uptake / synthesis / regeneration of anyone or all compounds including phosphate, ATP, NADH, NADPH, Pyruvate, CoA and Acetyl CoA. and / or (vi) for in-vitro / in-vivo / intra cellular / extra cellular production of non-Carbon Nitrogen-containing compounds (including but not limited to Ammonia, nitrate, nitrite etc. and their derivatives / salts).

37. The method as claimed in claim 1, wherein the said modifications include homologous and / or heterologous expression(s) of respective genes, their promoter / s and / or associated regulatory gene / s, or a combination thereof wherein the said gene / s are native / modified / novel.

38. The composition as claimed in claim 1, wherein the microbial composition includes single microbe modified or a consortium of microbes modified for production of Nitrogen-containing, non-carbon compounds.