DESC:TECHNICAL FIELD OF THE INVENTION:
The present invention relates to a novel method for sustainable in vitro and in vivo production of non-nitrogen, carbon containing compounds.
The present invention relates to a novel method that includes gene modifications in microbes, more specifically involving gene modifications resulting in uptake/fixation/induced fixation of nitrogen and/or hydrogen with or without induced ammonia uptake/fixation increased ammonia uptake/fixation with or without induced c1 carbon fixation/increased c1 carbon uptake/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.

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) nitrogen fixation and/or (ii) uptake/fixation of ammonia and/or (iii) uptake/fixation of carbon 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 non-Nitrogen and Carbon containing compounds

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) enhanced nitrogen fixation and/or (ii) uptake/fixation of ammonia and/or (iii) uptake/fixation of carbon and/ or (iv) uptake/synthesis/regeneration of anyone or all compounds including phosphate, ATP, NADH, NADPH, Pyruvate, CoA and Acetyl CoA and/or (v) production of non-Nitrogen and Carbon containing compounds
BACKGROUND AND PRIOR ART OF THE INVENTION:

PCT Publication No. W02017011602 discloses methods of increasing nitrogen fixation in non-leguminous plants comprising exposing the plant to a plurality of bacteria comprising one or more genetic variations introduced into one or more genes of the bacteria's nitrogen fixation or assimilation genetic regulatory network, such that the bacteria are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.

US20190211342 discloses genetic modification of non-autotrophic microorganisms to enhance the expression of enzymes recombinant phospho- ribulo-kinase (prk) and Ribulose-Bisphosphate Carboxylase (RuBisCo) to improve carbon fixation.
US 20180297906A1 discloses methods including genetically modified bacterial strains for increasing nitrogen fixation in a non - leguminous plant. The modifications include either within the genes or non-coding polynucleotides such as promoters of the bacteria's nitrogen fixation or assimilation genetic regulatory network. The genetically engineered bacterial strains are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen and produce 1% or more of the fixed nitrogen in the plant.
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.
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 C1 compounds such as formate, formic acid, formaldehyde or methanol to organic carbon compounds.

The technical limitation with the prior art for the production of carbon-containing, non-nitrogen organic compounds:

• Carbon, Nitrogen and Hydrogen substrate limitation: Less efficient/ inefficient in uptake and utilisation of Carbon. Nitrogen and hydrogen substrates including sugars, organic acids and C1 compounds such as CO2, methane, methanol, Ammonia, Nitrate, Nitrite, N2O, H2, etc.
• 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.
• 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 carbon-containing, non-nitrogen compounds, and also for carbon and Nitrogen assimilation.
• Substrates and intermediates for carbon fixation, nitrogen fixation, Hydrogen fixation and regeneration of redox energy compounds.
• 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.

OBJECT OF THE INVENTION:
The present invention relates to a novel method for sustainable in vitro and in vivo production of non-nitrogen, carbon-containing compounds.

It is an object of the present invention to overcome the technical limitations with the prior art for enhanced carbon fixation, nitrogen fixation, hydrogen fixation for production of non-nitrogen, carbon-containing compounds.

It is another object of the present invention to overcome Carbon, Nitrogen and Hydrogen substrate limitations, reducing their loss in metabolism, and reducing Redox imbalance.

It is another object of the present invention to provide methods for increased synthesis/ regeneration of ATP, NADH, NADPH, pyruvate, Coenzyme-A, Acetyl-CoA, Hydrogen, and phosphate required for enhanced nitrogen, carbon, Hydrogen assimilation, and for production of nitrogen and carbon-containing compounds.

The present invention additionally relates to the combination of increased nitrogen with and without CO2 fixation and with enhanced regeneration / recycling of energy & central metabolites such as Phosphate / ATP / NADH / Coenzyme A / Pyruvate to increase the production of non-Nitrogen, carbon-containing compounds including but not limited to organic acids such as lactic acid, acetic acid, formic acid, citric acid, oxalic acid, malic acid etc. and plant growth hormones such as ethylene, gibberellins, polymeric compounds like terpenoids, carotenoids, etc.

SUMMARY OF THE INVENTION:
The present invention relates to a novel method for sustainable in vitro and in vivo production of non-nitrogen, carbon containing compounds.

The present invention relates to gene modifications in microbes, more specifically involving gene modifications resulting in nitrogen and/or ammonia fixation with or without carbon fixation with/ 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.

The present invention for production of non-Nitrogen, carbon containing 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 CoenzymeA for (i) enhanced nitrogen fixation and/or (ii) ammonia fixation and/or (iii) carbon fixation and/or (iv) synthesis/regeneration of anyone or all compounds including ATP, NADH, NADPH, Pyruvate and Acetyl CoA

Description of Figures:
Figure 1: Depicts CO2 fixation to Formate production as a product of carbon-containing, non-nitrogen compounds
Figure 2: Depicts CO2 consumption profile of engineered microbe, expressing formate dehydrogenase for CO2 conversion to formic acid.
Figure 3: Depicts methane consumption profile of engineered microbe, expressing methane consumption genes for conversion of methane to methanol and further conversion to formaldehyde.
Figure 4: Pictorial representation of integrated process of carbon and Nitrogen uptake and redox energy regeneration/ recycling. Integrated generation/ regeneration and reuse of Carbon substrates, Hydrogen, Phosphate and Redox energy compounds ATP, NADH, NADPH results in enhanced rate of carbon assimilation as well as enhanced generation ATP, NADH and enhanced metabolism, finally resulting increased rate of product formation.
Figure 5: Depicts sugar production profile of free-living microbe
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.
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.
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 phosphor-ribulo 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 Phospho-ribulo kinase (PRK) and compact RuBisCO for the CO2 assimilation.
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.
Figure 11: Depicts the novel, inventive process of Nitrogenase metal specificity by promoter exchange. Modification and replacement of Anf Promoter with Nif H promoter to increase electron delivery to Fe-nitrogenase, pathways activated by NifA. Incorporation of promoter such as nif H promoter (nif HDK operon) in place of anf promoter (of anf HDGK operon), shifts the specificity to Fe (Iron) irrespective of presence or absence of Molybdenum. Promoter exchange also impacts on enhanced Nitrogenase activity.
Figure 12: Depicts the Limitations in production methods of Carbon-containing, non-nitrogen compounds, as per the prior art methods
Figure 13: Depicts the novel, Inventive solution with novel methods to overcome limitations in production methods of Carbon-containing, non-nitrogen compounds by gene manipulations of microbe for efficient carbon and nitrogen fixation, as well as for enhanced redox energy generation and regenerations.
Figure 14: Picture depicts the Rubisco-PRK gene construct for genome integration. Gene integration construct includes expression of Rubisco-PRK under Mxa promoter, cloned with flanking sequences of GDFD gene partial sequences to facilitate integration with the GDFD gene in genome, resulting in simultaneous deletion of GDFD gene and integration of Rubisco-PRK under modified promoter.

DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a novel method for sustainable in vitro and in vivo production of non-nitrogen, carbon containing compounds.

The present invention relates to methods for enhanced carbon fixation, nitrogen fixation, hydrogen fixation and efficient redox energy supply for production of carbon-containing, non-nitrogen compounds.
The present invention relates to a novel method that includes gene modifications in microbes, more specifically involving gene modifications resulting in uptake/fixation/induced fixation of nitrogen and/or hydrogen with or without induced ammonia uptake/fixation increased ammonia uptake/fixation with or without induced c1 carbon fixation/increased c1 carbon uptake/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.

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) nitrogen fixation and/or (ii) uptake/fixation of ammonia and/or (iii) uptake/fixation of carbon 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 natural or unnatural compounds, non-Nitrogen and Carbon containing compounds

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) enhanced nitrogen fixation and/or (ii) uptake/fixation of ammonia and/or (iii) uptake/fixation of carbon and/ or (iv) uptake/synthesis/regeneration of anyone or all compounds including phosphate, ATP, NADH, NADPH, Pyruvate, CoA and Acetyl CoA and/or (v) for in-vitro/in-vivo/intra cellular/extra cellular production of natural or unnatural compounds, non-Nitrogen and Carbon containing compounds.

In an embodiment, the present invention provides genetic modifications of microbes for increased nitrogen fixation, with and without carbon fixation, leading to production of non-nitrogen containing primary & secondary metabolites such as organic acids like succinic acid, formate, lactic acid, acetic acid, and solvents like methanol, ethanol, isopropanol, fatty acids, glycerol and lipids, phenolics, polyphenols, etc.
Carbon fixation:
In one embodiment, the present invention details the methods of assimilation of various carbon compounds including but not limited to C1 compounds like CO2, Methane, Methanol, formaldehyde, formate, etc., and organic acids, sugars, and other carbohydrates.
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 CO2 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 C1 compounds such as Methanol, Methane, Formaldehyde, etc.
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.
Another embodiment provides a method of fixation of CO2 by Formate-formaldehyde route, where in 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.

Another embodiment provides the details on modification of PEP carboxylase (phosphoenolpyruvate carboxylase) involvement 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.
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.
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.
Another embodiment elaborates the serine cycle, also known as the Calvin-Benson-Bassham (CBB) cycle, involves the condensation of formaldehyde with glycine to produce serine. Serine is then further metabolized to generate central carbon metabolites, such as 3-phosphoglycerate. In the RuMP pathway, formaldehyde is condensed with ribulose monophosphate to produce fructose-6-phosphate. In the XuMP pathway, formaldehyde is condensed with xylulose monophosphate to produce fructose-6-phosphate.
In another embodiment, the present invention involves optimizing the growth conditions of the microorganisms. This may include providing a suitable temperature, pH range, and nutrient composition to enhance carbon uptake. In addition, the microorganisms may be genetically engineered to improve their ability to utilize carbon from different sources.

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.

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

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.

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.
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 which can be utilized/diverted further in production of carbon-containing, non-nitrogen compounds.
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 Galionella spp., Methylomicrobium spp. etc.

Nitrogen fixation and Ammonia fixation:
One 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 the driving force for enhanced carbon assimilation.
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.

In one embodiment, the present invention provides a novel, inventive process for microbes with genetic modification in nitrogen fixation for enhanced carbon fixation.
In another embodiment, the present invention provides a novel, inventive process for microbes with genetic modification in carbon fixation for enhanced nitrogen fixation.
In another major embodiment, the present invention provides inventive process of combination of gene manipulations with respect to enhanced Nitrogenase the associated regulatory genes. Nitrogenases from any of the classes such as and not limited to Mo-Fe Nitrogenase or Fe-Nitrogenase or alternate Nitrogenases are manipulated. In addition to Nitrogenase gene manipulations, manipulations are targeted on associated genes such as AmtB, GlnA, GlnD, GlnE, NifA and NifL genes are manipulated for increased Nitrogen fixation and flux towards metabolism.
In yet another embodiment, the present invention provides increased nitrogen fixation in microbe(s) for the production of primary and secondary nitrogen metabolites 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, rnf genes, to enhance Nitrogenase activity and glutaminase, which hydrolyses glutamine to glutamate and ammonia, resulting in increased Nitrogen fixation.

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
In yet another embodiment, the present invention provides a method to upregulate 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 amno acid synthesis, protein synthesis and further metabolism.
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.
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.

Regeneration of Hydrogen, Phosphate, ATP, NADH, NADPH, Pyruvate, Coenzyme A:
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.

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, Huc Hydrogenase, CO/CO2 dependent hydrogenase (CODH), etc.

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.
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.

In another embodiment, the present invention provides enhanced production of Carbon-containing, non-nitrogen compounds, 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 .

In another embodiment, the present invention provides enhanced production of carbon-containing, non-Nitrogen 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.

In another embodiment, the present invention provides enhanced production of non-Nitrogen and Carbon-containing 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.
Carbon-containing, non-nitrogen compounds:
In yet another embodiment, the present invention provides genetically modified microbe, wherein said modifications support production/ enhanced production of carbon-containing, non-Nitrogen natural or unnatural compounds.
In yet another embodiment, the present invention provides in vitro production of organic acids, solvents like methanol, ethanol, or sugars, proto alkaloids, terpenes etc., useful as direct product or as raw material for different production arenas, wherein the production is in controlled conditions in laboratory culturing vessel or fermenters or Bioreactors.

In yet another embodiment, the present invention provides genetically modified microbe, wherein said carbon-containing, non-nitrogen containing natural or unnatural compounds includes such as and not limited to: carbohydrates, organic acids, vitamins, lipids, alcohols, phenolic compounds, aromatic compounds, aliphatic compounds, terpenes and terpenoids, biopolymers, other primary and/ or secondary metabolites, and the like.

In yet another embodiment, the present invention provides application of genetically modified microbe, for production of carbohydrates and their isomers/ epimers and/ or derivatives, include but not limited to natural or unnatural Monosaccharides (like Glucose, Fructose, Galactose, Allulose, Xylose, Xylulose, Arabinose, Ribose, Ribulose, Rhamnose, Sorbose, Tagatose, Idose, Glulose, Fucose), Disaccharides (like Sucrose, Maltose, Lactose, Cellobiose, Isomaltose, Isomaltulose, Trehalose, Trehalulose, Leucrose, Turanose), oligosaccharides (like malto oligosaccharides, iso-malto oligosaccharides, fructo oligosaccharides, milk oligosaccharides), polysaccharides (like Maltodextrin, Glucan, Dextran, xylan, pullulan, Glycan like capsular polysaccharides), sugar phosphates (like Glucose 1-phoshate, Glucose 6-phosphate, Fructose 6-phosphate, Fructose 1,6-bis phosphate, Phosphoenol pyruvate, Glyceraldehyde 3-phosphate, Dihydroxy acetone phosphate), etc.

In yet another embodiment, said organic acids include such as and not limited to natural or unnatural Acetic acid, Butyric acid, Formic acid, Lactic acid, Citric acid, Oxalic acid, Glutaric acid, Succinic acid, Malic acid, Fumaric acid, Pyruvic acid, Tartaric acid, Oxalo-acetic acid, Phosphoglyceric acid, Aconitic acid, Itaconic acid, Ascorbic acid (Vitamin C), etc.

In yet another embodiment, the present invention provides genetically modified microbe, wherein said lipids include but not limited to saturated/ unsaturated/ poly unsaturated fatty acids (like short, medium and long-chain fatty acids) Mono, di and tri-acyl glycerides, designer/ structured lipids, microbial oil, Fatty acid and their esters such as methyl esters, ethyl esters, structured oil, glycolipids, glyceroglycolipids, galactolipids, rhamnolipids, and the like. Mono unsaturated fatty acids include important fatty acids such as and not limited to Oleic acid (omega-9 fatty acid), trans-Vaccenic acid (omega-7 fatty acid), etc.

In yet another embodiment, the said alcohols include but not limited to natural or unnatural Methanol, Ethanol, Propanol, Iso-propanol, Butanol, Iso-butanol, Tertiary butanol, Hexanol, octanol, cyclo hexanol, sugar alcohols/ polyols (Glycerol, Sorbitol, Mannitol, Erythritol, Xylitol, Glucitol, Galactitol, Ribitol, Iditol, Gulitol, Fucitol), etc.

In yet another embodiment, the said phenolic and aromatic compounds and their derivatives include but not limited to natural or unnatural catechol, resorcinol, hydroxy quinone, phenolic acid like ferulic acid, Caffeic acid, cinnamic acid, coumaric acid, syringic acid, vanillic acid, salicylic acid, hydroxy benzoic acid, tannic acid, epigallocatechin gallate (EGCG), and gallic acid (GA), Ellagic acid, Rosmarinic acid, Trimethoxy Cinnamic Acid, Caffeate esters, Chicoric acid, Gentisic Acid, Dihydroxyhydrocinnamic acid, Carnosic acid, Caftaric acid, Hydroxy cinnamic acid, Diferulic acids, Vanillic acid, Vanillin, Acetosyringine, Quinones and derivatives such as and not limited to Semiquinone, menaquinone, Vitamin K (vitamin K1-phylloquinone, vitamin K2-menaquinone), steroids, sterols, Vitamin D (calciferol), Azulene, Guaiazulene, Vetivazulene, phyllodulcin, curcumin, poly phenols like Flavanoids, Nobiletin, Capsaicinoids, and the like.

In another embodiment, the genetically engineered microbe of the present invention will be useful for production of non-nitrogen primary and secondary metabolites. Primary metabolites include but not limited to Glycerol, Fatty acids including short-chain, medium-chain, long-chain fatty acids, either saturated or unsaturated, mono unsaturated fatty acids (MUFA), poly unsaturated fatty acids (PUFA), Docosahexaenoic acid (DHA), EPA etc., fat-soluble Vitamins such as Vitamin A (carotenoid, retinol), vitamin C (Ascorbic acid), vitamin D, Vitamin K; organic acids such as Lactic acid, acetic acid, succinic acid, fumaric acid, malic acid, citric acid, alcohols such as ethanol, propanol, iso -propanol, Butanol, other solvents like Acetone, isoamyl alcohol, and sugars like glucose, fructose, fibre sugars, disaccharides etc.

In another embodiment, the present invention provides increase in various secondary metabolites production in Nitrogen and CO2 fixing microorganisms. The secondary metabolites include but not limited to the purpose of stress responses, defense mechanisms, metal carrying, and signalling secondary metabolites. Examples of secondary metabolites include to carotenoids, terpenes and terpenoids, Gibberellin, phenolic acids, fumaric, coumaric acid, cinnamic acid, proto alkaloids, steroids, polymeric biomaterials such as poly hydroxy aldehydes (PHAs) and Poly hydroxy butyrates (PHBs), poly lactate, Alginate, biosurfactants such as Rhamno lipids, Glucan, Dextran polymers, poly phenolic compounds such as phenolic acids, Phyllodulcin, flavonoids, polyphenolic amides, polyphenols, oil and fats, biodiesel, etc.

In yet another embodiment, , the present genetically engineered microbe with enhanced nitrogen and carbon fixation, can be used as production host for sugar production from C1 compound such as and not limited to CO2, CH4, Formaldehyde, etc.

In yet another embodiment, the present invention is useful for the production of unique novel, secondary metabolites like decay-resistant biopolymers such as and not limited to Spropollenin, Suberin, etc. sporopollenin is carbon-hydrogen-oxygen containing, lipid- and phenolic-based biopolymer present in the outer exine layer of pollen walls. Similarly, suberin is a lipid- and phenolic-based polymer present in the cell walls of various external and internal tissue layers. Sporopollenin is the most robust organic compound in nature and is known as the diamond of plant kingdom. Sporopollenin exine capsules (SEC) are abundant in nature. They have good biocompatibility and no immunity. The rich carboxyl, hydroxyl and phenolic groups make SEC easy to be functionalized or complexed with nanomaterials. The plentiful nanochannels on SEC increases their specific surface area, supporting capture of cancer cell and biomolecules. The unique properties of SEC lead to their wide applications in drug delivery carriers, oral vaccine carriers, medical imaging, biosensing, cell growth scaffold, microreactor, micro robot, etc.

In yet another embodiment, the present invention provides incorporation of pathways or missing genes or rate-limiting genes necessary for production of non-nitrogenous organic compounds, such as organic acids, solvents, terpenoids, PHAs, PHBs, ply lactate, carotenoids, biodiesel and so on.

In yet another embodiment, the said aliphatic compounds include and not limited to natural or unnatural alkanes such as methane, ethane, long chain alkanes, alkanols and alkanoic acid; alkenes/ olefins such as Ethylene, long chain and/ or poly unsaturated alkenes, and the like.

In yet another embodiment, the terpenes/ terpenoids and their derivatives includes but not limited to natural or unnatural terpenes and terpenoids, isoprenes, isoprene pyrophosphate, monoterpenes, diterpenes, diterpenoids like Gibberellic acid, triterpenes, tetraterpenes like saponin, sesquiterpenes (like Abscisic acid), sesquiterpene lactones, and derivatives like carotenes, Vitamin A, carotenoid, retinol, retinal, retinoic acid, xanthophyll, menaquinone, lycopene and the like.

In yet another embodiment, the biopolymers include but not limited to PHA, PHB, Polylactate, Alginate, Agarose, cellulose acetate, methyl/ ethyl cellulose, hydroxy propyl cellulose, hydroxy propyl methyl cellulose, Sporopollenin, suberin, lignin and the like.

In yet another embodiment, said carbohydrates production including monosaccharides and disaccharides is achieved by homologous/ heterologous modification of enzymes including but not limited to sucrose-phosphate synthase, sucrose-phosphate phosphatase, Fructokinase, glucose-6-phosphate isomerase, phosphoglucomutase, fructose-1,6-bisphosphatase/ sedoheptulose-1,7-bisphosphatase, UTPglucose-1-phosphate uridylyl transferase; Rubisco, glucokinase, invertase, sugar isomerase, sugar epimerase, oxidoreductase, Formolase, Fructose 6-phosphate Aldolase, and the like.

In yet another embodiment, said 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, and the like.

In yet another embodiment, microbe can be modified for breakdown/ utilization/ enhanced utilization of varied substrates 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/ non-synthetic compounds including but not limited to plastics/ fibres/ rubber/ resins/ chemicals, etc.(like polyethylene terepthalate (PET), Ethylene glycol (EG), polyurethane, polybutylene succinate-PBS, PBS-adipate), 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, Glycosyl transferases, PBSase, proteases, peptidases, invertase, amylase, sugar isomerases, lipases, amidases, phytase, phosphatases, nitrate reductase, nitrite reductase, N2O reductase, sulphate adenylyl transferase (Sat), adenylyl sulphate reductase (AprBA), dissimilatory sulfite reductase (DsrAB), ATP sulfurylase (ATPS), Adenosine 5'-phosphosulfate reductase (APSR), APS kinase (APSK), Terephthalate 1,2-dioxygenase, Terephthalate reductase, Di-hydrodiol dehydrogenase, Glycolate oxidase, Glyoxylate carbo-ligase, hydroxy pyruvate isomerase, protocatechuate decarboxylase, Feruloyl esterase, TMADH-trimethylamine dehydrogenase; DMADH-dimethylamine dehydrogenase; MMADH-monomethylamine dehydrogenase, Thioredoxin reductase, Thioredoxin, dihydro folate reductase, and the like.

Proteases also include other enzymes such as Asparaginyl endopeptidases, Butelase, dihydro folate reductase, etc., which assist in improving thermal stability of the protein. Peptide backbone cyclization is commonly observed in nature and is increasingly applied to proteins and peptides to improve thermal and chemical stability and resistance to proteolytic enzymes and enhance biological activity. Ligase type asparaginyl endopeptidases (AEPs) such as butelase1 and AEP1 have been employed to facilitate peptide ligation and head-to-tail cyclization reactions invitro which removes cleavable N- and C termini, and thereby improves peptide stability against exopeptidases.

In yet another embodiment, the 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.

Gene modifications:
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 and/ or downregulation/ silencing/ deletion of either homologous or heterologous genes and related regulatory genes.

In yet another embodiment, the present invention provides genetically modified microbe, wherein said enzymes involved in Nitrogen fixation and/ or carbon fixation and/ or Ammonia fixation and/ or production of carbon-containing non-Nitrogen containing natural or unnatural compounds, are overexpressed by the inclusion of Transcriptional and/ or Translational enhancers such as and not limited to UNA1, UNA2, UNB, oligomers, and the like.
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.
In yet another embodiment, 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, epigenetic mechanisms, and the like.

In yet another embodiment, 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.

In another embodiment, the present invention provides Nitrogen fixing organisms including but not limted to Azospirillum lipoferum, Clostridium acetobutylicum, C. Beijerinckii, Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea, Serratia, Rahnella, Azospirillum, Azorhizobium, Duganella, Azotobacter sp, Delftia, Bradyrhizobium sp, Sinorhizobium sp, Halomonas, Xanthobacter, Klebsiella sp, Azotobacter vinelandii or Azotobacter chroococcum, Bacillus, Paenibacillus, Lactobacillus, Mycoplasma, Acetobacter, Rhodococcus, Cyanobacteria, Pseudomonas, Klebsiella, Altererythrobacter, Streptomyces, Microbacterium, Curtobacterium, Brevundimonas, C. accharoperbutylacetonicum, C. saccharobutylicum, C. aurantibutyricum, C. tetanomorphum, Niveispirillum, Azospirillum amazonense, Azoarcus, Bacillus amyloliquefaciens and Klebsiella.

In another embodiment, the present invention provides both Nitrogen and CO2 fixing organisms and not limited to Bradyrhizobium japonicum, Nitrospira inopinata, Rhodopseudomonas palustris, Azotobacter chrococcum, Azotobacter vinelandii, Sphingomonas, Nitrosopumilus maritimus, Methylobacterium, Rhodobacter sphaeroides, Reyranella massiliensis, Alcaligene, Saccharomyces cerevisiae, Saccharomyces lactis, Brevibacterium, , Kluyveromyces lactis, Epichloë typhinaEnterococcus, Corynebacterium, Arthobacter, Pichia, Zymomonas, Saccharomyces carlsbergensis, Salmonella, Zymomonas, Rhodacoccus, Escherichia (e.g., E. Coli), Hansenula, Firmicutes, Rubrivivax, Dinoroseobacter shibae, Methylobacterium nodulans, Methylobacterium radiotoleran, Methyloversatilis sp, Methylobacterium oryzae, Beijerinckia indica, Frankia spp., Synechocystis, Synechoccus sp., etc.

In yet another embodiment, the present invention provides genetically modified microbe, wherein said microbes are selected from the group consisting of 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., Methylobacterium symbioticum, Methylorubrum, Methylomonas, Methylosarcina, Methylococcus; Beta-proteobacteria such as Nitrospira, Nitromonas, Nitrobacter, etc; Firmicutes such as Bacillus, Paenibacillus, Lactobacillus, Mycoplasma, and Acetabacterium and Actinobacteria such as Streptomyces, Rhodacoccus, Frankia sp., Microbacterium, and Curtobacterium, other microbes such as Rhodobacter sphaeroides, Cupriavidus necator, Nigrospora oryzae, Azospirillum lipoferum, Rhodopseudomonas palustris, Rhodobacter spp., Bradyrhizobium japonicum, Ralstonia eutropha, Flavobacterium, Cyanobacteria, Epichloë typhina, Rhodococcus, Xanthobacter spp.; Algae/ Micro algae cyanobacteria such as Synechococcus spp., Synechocystis spp., Scenedesmus spp., Chlorella spp., Chlamydomonas, etc.; non-diazotrophs such as E.coli, Bacillus, Lactobacillus, yeasts like Saccharomyces and Pichia, Archaea bacteria and the like.

EXAMPLES
The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.

Example 1: Novel method for Overproduction of carbon-containing, non-nitrogen compound by genetically enhanced microbe.
Methylobacterium spp was genetically modified for enhanced production of formic acid by:
Enhanced Nitrogen fixation:
Enhanced Nitrogen fixation was achieved by modification of Nitrogenase and related regulator genes, including
1. Promoter exchange for the Nitrogenase – Expression of Nitrogenase under NifH promoter for Iron-specific Nitrogenase expression.
2. multi copy expression of positive regulator NifA,
3. Upregulation of Ammonia uptake genes Gln, GDH, for enhanced metabolism
Enhanced carbon fixation:
Carbon fixation in terms of CO2 uptake was enhanced by overexpression of Formic acid dehydrogenase (FDH)
Gene coding for FDH was amplified using Methylobacterium genomic DNA as template.
Enhanced ATP/ NADH regeneration
NADH regeneration by incorporation of FDH gene (utilizing NADH) and glyceraldehyde 3-phosphate dehydrogenase (generating NADH), in tandem under control of same promoter to enhance the rate of NADH recycling/ regeneration as well as simultaneous CO2 conversion to formic acid
ATP generation was enhanced by overexpression of ATP synthase as well as phosphate regeneration by combining PPK (Polyphosphate kinase) and PAP (Polyphosphate AMP-Phospho transferase) enzymes, resulting in conversion of AMP to ATP and simultaneous increment in Acetyl CoA.
Result:
The step-wise addition of gene constructs for carbon fixation, Nitrogen fixation and ATP/NADH/Phosphate regeneration resulted in 5 fold increased conversion of CO2 to Formic acid, as compared to 2 to 3 fold increase after FDH overexpression.
The conversion of CO2 into formic acid (Formate) is catalysed by the NADH dependent enzyme Formate dehydrogenase – FDH, which play play an essential role in energy generation during growth on C1 compounds. Diazotroph such as Methylobacterium species was manipulated for formate dehydrogenase (Sequence id: 1) multi copy expression, which resulted in enhanced CO2 fixation and conversion to formic acid, by expression under constitutive promoter of Glyceraldehyde 3-phosphate dehydrogenase- GAP promoter (Sequence id: 2).
The homologous multi-copy expression of FDH expressing cells showed maximum formate productivity which was 2 -3 times greater than that of wild type strain, as evident from Figure 1.

Example 2: Enhanced CO2 fixation by overexpression of CBB pathway enzymes Rubisco and PRK-Phospho ribulo kinase:-
Key enzymes in the carbon fixation CBB pathway include major rate-limiting enzymes such as Rubisco enzyme (Sequence id: 3), PRK-Phospho ribulo kinase (Sequence id: 4), by which CO2 enters the metabolism via glycolytic steps. Gene Modification in terms of upregulation of any one or two of these genes of enzymes in a strain resulted in increase of carbon fixation and carbon fluxing towards glycolytic pathway in the cells, and enhance microbe’s central metabolism.
Genetic modifications were performed on the above genes in bacterial cells such that the modifications result in increased carbon fixation. Modification, mutagenesis was performed in and around the active sites of the genes that code for Rubisco enzyme, PRK-Phospho ribulo kinase. A library of mutants was constructed for each gene. These libraries were then screened for increased carbon fixation. These libraries were created from wild-type cells, or from cells that have been modified to display increased carbon fixation.

Example 3: Impact of CO2 fixation pathway incorporation on CO2 absorption by the modified microbes:
Microbe modified for enhanced carbon fixation, by the way of pathway engineering, had the capacity of increased rate of carbon assimilation. Due to the increased expression of CO2 fixation pathway enzymes such as Rubisco enzyme (Sequence id: 3), PRK-Phospho ribulo kinase (Sequence id:4), carbon flux increased resulting in increased carbon uptake and absorption, as evident from Figure 2. Both the RuBisCO and PRK genes were integrated into Glutathione dependent formaldehyde dehydrogenase (GDFD) (Sequence ID: 6) in located in the genome of the Methylobacterium.

Example 4:
Establishment of methane assimilation pathway in microbes for utilizing methane as carbon source similar to other C1 carbon compounds like CO2. Methanol, etc.:-
Carbon compounds including C1 compounds like CO2, methane, methanol, formaldehyde are readily available sources for microbes. Enabling microbes to take up all these to utilize in metabolism will help in green house gas (GHG) remediation (CO2 and Methane are GHG).
Inorder to enable the microbes to take up methane, there was necessity to incorporate heterologous expression of Methane monooxygenase (sequence id: 5) under GAP promoter control. Methane monooxygenase catalyses the conversion of methane to methanol, which is further assimilated in metabolism as formaldehyde and enters the carbon assimilation process. Experimentally this resulted in increased methane assimilation which was originally not capable by the wild type strain, as evident from the Figure 3.

Example 5:
Novel method for Overproduction of carbon-containing, non-nitrogen compound (sugar) by genetically enhanced free living microbe (cyanobacteria)

Free living microbe such as cyanobacteria of Synechococcus spp. was modified for over production of sugars by novel methods of gene modification including enhanced nitrogen fixation, enhanced CO2 fixation and enhance energy metabolites (ATP/NADH/ Phosphate).

Enhanced Nitrogen fixation:
Enhanced Nitrogen fixation was achieved by incorporation of heterologous Nitrogenase and multi copy expression of homologous regulatory genes, including
1. Heterologous expression of Nitrogenase under NifH promoter for Iron-specific Nitrogenase expression.
2. multi copy expression of positive regulator NifA,
3. Upregulation of Ammonia uptake genes Gln, GDH, for enhanced metabolism
Enhanced carbon fixation:
Carbon fixation in terms of CO2 uptake by incorporation of genes including
1. Overexpression of native Rubisco
2. Overexpression of native Phospho ribulo kinase- PRK
Enhanced ATP/ NADH regeneration
NADH regeneration by incorporation of FDH gene (utilizing NADH) and glyceraldehyde 3-phosphate dehydrogenase (generating NADH), in tandem under control of same promoter to enhance the rate of NADH recycling/ regeneration as well as simultaneous CO2 conversion to formic acid
ATP generation was enhanced by overexpression of ATP synthase as well as phosphate regeneration by combining PPK (Polyphosphate kinase) and PAP (Polyphosphate AMP-Phospho transferase) enzymes, resulting in conversion of AMP to ATP and simultaneous increment in Acetyl CoA.
Enhanced sugar production
sugar production (such as sucrose) form cyanobacteria were enhanced by downregulation of invertase (to prevent sugar breakdown) and upregulation of sugar transporters.
Result:
The step-wise addition of gene constructs for carbon fixation, Nitrogen fixation and ATP/NADH/Phosphate regeneration resulted in 3 fold increased sugar production as compared to the unmodified strain, as depicted in figure 5.

SEQUENCE LISTING
Sequence Listing Information:
DTD Version: V1_3
File Name: Sequence listing_4226.xml
Software Name: WIPO Sequence
Software Version: 2.3.0
Production Date: 2023-12-08
General Information:
Current application / IP Office: IN
Current application / Application number: 202241064226
Current application / Filing date: 2022-11-10
Current application / Applicant file reference: 4226
Earliest priority application / IP Office: IN
Earliest priority application / Application number: 202241064226
Earliest priority application / Filing date: 2022-11-10
Applicant name: Fertis India Pvt. Ltd.
Applicant name / Language: en
Invention title: GENETIC MODIFICATION OF MICROBES FOR PRODUCTION OF CARBON-CONTAINING NON-NITROGEN COMPOUNDS ( en )
Sequence Total Quantity: 6
Sequences:
Sequence Number (ID): 1
Length: 2856
Molecule Type: DNA
Features Location/Qualifiers:
- source, 1..2856
> mol_type, genomic DNA
> organism, FDH
Residues:
atggccctca tcaaggaaat cgactacggc acgccgatcc gcgtcgccga gcagacggtg 60
tcgctgacca tcgacggcat ggccgtgacg gtgccggccg gcacctccgt gatggccgcg 120
gcgatgaccg cgggcacgca gatccccaag ctctgcgcca ccgactcgct ggagcccttc 180
ggctcctgcc gcctctgcct cgtggagatc gagggacggc gcggcacgcc cgcctcctgc 240
accacgccgg ccgagaacgg catggtggtg cacacgcaga ccgacaagct cgcgcgcctg 300
cgcaagggcg tgatggagct ctacatctcc gatcacccgc tcgactgcct gacctgcgcg 360
gcgaacggcg attgcgagct gcagacgcag gcgggcgtcg tcggcctgcg cgacgtgcgc 420
tacggctacg agggcgacaa ccacgtccgc ccgagctccg agcgctacct gccgaaggac 480
gagtcgaacc cgtatttcac ctacgacccg tcgaagtgca tcgtctgcaa tcgctgcgtg 540
cgggcctgcg aggaggtgca gggcaccttc gcgctgacca tcgccggccg cggcttcgac 600
agccgcgtcg ccgccggccc gacgaacttc atggaatccg agtgcgtctc gtgcggcgcc 660
tgcgtgcagg cctgcccgac cgcgacgctc caggagaagt cgatccacga atacggccag 720
ccggagcacg ccgaggtcac gacctgcgcc tattgcggcg tcggctgctc cttcaaggcc 780
gagatgcagg gcgaccgcgt cgtgcgcatg gtgccctaca agggcggcaa ggcgaatgac 840
ggccatagct gcgtgaaggg ccgcttcgcc tacggctacg ccactcacaa ggaccgcatc 900
accaagccga tgatccggga gaagatcacg gatccgtggc gcgaggtcac ctgggaggag 960
gcgatcgacc gggcggcctc cgagttcaag cggatccagg ccacctacgg caaggattcg 1020
gtcggcggca tcacctcgtc ccgctgcacc aacgaggagg cctacctcgt ccagaagctg 1080
gtgcgcgcgg ccttcggcaa caacaacgtc gatacctgcg cccgcgtctg ccactcgccg 1140
accggctacg gcctgatgtc gacgctcggc acctcggccg gcacccagga cttcgcctcg 1200
gtggcgcatt ccgacgtgat cctcgtcatc ggcgccaacc cgacggacgg ccatccggtc 1260
ttcggctcgc gcatgaagaa gcgcctgcgc gagggggcga agctcatcgt cgccgatccg 1320
cgcaagatcg acctcgtgaa gtcgccccac atcaaggcgg acttccacct gcccctgaag 1380
cccggctcca acgtcgcctt catcaactcg atcgcgcacg tcatcgtcac ggaagggctg 1440
atcgacgagg cctatatccg cgcgcgctgc gacctcggcg agttcgagtc ctgggcccgc 1500
ttcatcgcgg aggagcgcca ctcccccgag aaccagcagc agttcaccgg cctcgatccc 1560
gaacaggtgc gcggcgcggc gcggctctac gccacgggcg gcgcggccgg catctattac 1620
gggctgggcg tcaccgagca cagccagggc tcgaccatgg tgatgggcat ggccaacatc 1680
gccatggcca ccggcaacat cggcaagctc ggtgcgggcg taaacccctt gcgcggccag 1740
aacaacgtgc aaggatcctg cgacatgggc tcgttccccc acgagctcac cggctaccgc 1800
cacgtctcgg acgatgccac ccgcgagagc ttcgaggcga tctggggtgc caagctcgac 1860
aacgcgccag gacttcgcat caccaacatg ctcgatgagg ccgtcgatgg cagcttcaag 1920
ggcatgtaca tccagggcga ggacatcgcg cagtccgatc ccgacaccca tcacgtcacg 1980
tcaggcctca aggcgatgga atgcatcgtc gtgcaggacc tgttcctgaa cgagacggca 2040
aaatacgccc acgtcttcct gcccggagcc tcattcctgg agaaggacgg caccttcacc 2100
aatgccgagc gccgcatcag ccgcgtgcgc aaggtcatgc ccccgatggg cggctacggc 2160
gattgggagg gcacggtgct gctctctaac gcgctgggct acccgatgaa ctacagccac 2220
ccatccgaga tcatggacga gatcgcggcc ctcaccccga gcttcaccgg ggtgtcctat 2280
gccaaactcg aggaactcgg ctcggtacag tggccctgca atgagaaggc gccgctcggt 2340
acgccgatga tgcacgtgga ccgcttcgtg cgcggcaagg gccggttcat gatcaccgag 2400
ttcgtggcga ctgaggagcg cacgggggcg aagttcccgc tcatcctcac cacgggtcgg 2460
atcctctccc agtacaacgt cggcgctcag acccggcgca cccacaattc gcgctggcac 2520
gaggaggacg tgctggagat ccaccccttc gacgcggagc tgcgcggtat catggacggc 2580
gacctcgtcg ccctggagag ccgctcgggc gacatcgctc tgaaggccaa gatttcggag 2640
cgcatgcagc caggcgtggt ctacaccacc ttccaccacg ctaagaccgg cgccaacgtc 2700
atcaccaccg actattcgga ctgggccacg aactgccccg agtacaaagt gacggcggtg 2760
caggtccggc gtaccaaccg gccctccgac tggcaggcga agttctacga gggagatttc 2820
tccctgaccc ggatcgccca ggccgcggcg gagtga 2856

Sequence Number (ID): 2
Length: 200
Molecule Type: DNA
Features Location/Qualifiers:
- source, 1..200
> mol_type, other DNA
> organism, Gap Promoter
Residues:
tccgcggatc ggttgatccc ggcggcgacg gcgcggccgg tccgccatgg gtcatgtccg 60
gctccggttc atcgccggtt cagcgccggc agccacagag caatccgcat cgcggaggtg 120
ccgtcgggcc cccgccgcgc accgctcgcc gcctcggacg cccgctgcgt ggcgcccctt 180
aagcaggaag gaaacacgcc 200

Sequence Number (ID): 3
Length: 1413
Molecule Type: DNA
Features Location/Qualifiers:
- source, 1..1413
> mol_type, other DNA
> organism, RubisCO
Residues:
atggatcaat ctaatcgtta cgcgaatctg aatctcaaag aagaagattt gattaaaaat 60
ggcaagcacc tgttggttgc ttataagctg attccggcca aaggccacgg atttctggaa 120
gtcgctgctc acgttgcagc tgaatcttct accggcacca atgtggaagt gtccaccacc 180
gacgacttta ctcgtggtgt agatgctctg gtgtatgaaa tagacgaaac tgcatttggt 240
gacgaccctg ttaagggtgg cggcttgttt aaggtcgctt atccagttga attgttcgac 300
ccgaacctga ctgatggcac ttataacatc tctcatatgt ggtctctgat cctgggcaac 360
aatcagggta tgggcgacca tcaaggtctg cgtatgctgg acttcttggt tcccgagatg 420
atggtcagga aattcgatgg cccgtccgcc aatatcagca acctgtggaa ggtgctgggt 480
cgttcggaaa cagacggtgg ttacatcgcc ggtaccatca tcaagcctaa gttgggtctg 540
cgtcctgagc cattcgctaa agcctgctac gacttctggt tgggtggtga cttcatcaag 600
aacgatgaac cgcaagctaa tcaaccattc tgtccgatgg aagtggttat gccgaaagtg 660
gctgaagcca tggatcgcgc tcaacaggca accggtcagg ctaagttgtt ctcagctaac 720
atcacagccg attactacaa ggaaatgatc caccgcggtg atttcgtgct ggaaacattt 780
gccaagtaca attccgctag ccatgtggcc ttcctcgttg acggctttgt gaccggcccg 840
gctggcgtaa ccaccgcacg tcgcgagttc ccggacacct tcttgcactt ccaccgcgca 900
ggccacggtg ccgttacttc ctacaagtca ccaatgggta tggatccatt gtgctacatg 960
aagctggtgc gtttgatggg tgcgtccggt atgcataccg gtactatggg ttacggcaag 1020
atggaaggtc acggcaagga aactgtgctg gcctacatgc tggaacgcga tgagtgccaa 1080
ggcccttatt tctaccagaa gtggtacggt atgaaggcga ctacgcctat catctccggc 1140
ggtatgaacg cattgcgtct gccaggcttc ttccagaacc tcggccacgg caacgtgatc 1200
aatacctgcg gcggcggtgc cttcggtcac atcgacagcc cggcagcggg tggtatctca 1260
ctcggtcagg catacgattg ctggaaatca ggttccgatc ctatcgaata tgcgaagact 1320
cacaaagaat tcgcccgtgc gttcgaatct ttcccgaaag atggcgataa gctgttcgct 1380
ggctggcgcg aaaagttggg cgtgcacaag taa 1413

Sequence Number (ID): 4
Length: 861
Molecule Type: DNA
Features Location/Qualifiers:
- source, 1..861
> mol_type, genomic DNA
> organism, Phosphoribulose kinase
Residues:
atgtcggcgc gtcaccccat catctcggtc accggctcct cgggggccgg gacgacctcg 60
gtccgcaaca ccttcgagca gatcttccgc cgggaggacg tcagcgcggt ctatatcgag 120
ggcgacggct tccacgcctt cgaccgcgag accatgcgcg cgatgatggc ccgcgagccg 180
acgctgagcc acttcgcgcc gcgggccaac ctcctgccgg agctggagga ggtgttccgc 240
agctacagcg agtccggcac cggccgcacg cggcactacg cgcacgacgc gcatgatgcg 300
gcccgctacg gcaccccgga gggcaccttc tcggcctggg aggagttccc gccgggctcg 360
gacctgctgt tctacgaggg cctgcacggc tgcgtggtcg attcgagcgt ggacatcgcc 420
cgctatcccg atctcaagat cggcgtggtg ccggtgatca acctcgaatg gatccagaag 480
ctgcaccgcg accgctcgtt ccgcggctac tcgaccgagg ccgtcaccga cgtgatcctg 540
cggcggatgc ccgactacgt gcagacgatc tgcccgcaat tctcctggac ggacatcaac 600
ttccagcgcg tgccgacggt ggacacctcg aacccgttca tcgcccgctg gatccccacg 660
gccgacgagt cgatggtggt gatccggttc aagaacccga aggggatcga cttctcctat 720
ctggtctcga tgatccacga cagcttcatg agccgggcga actccatcgt gatcccgggc 780
ggcaagctcg acctcgcgat gcagctgatc ctgacgccga tcatcatgca gctcgtggag 840
cgcaggcggc ggctggcttg a 861

Sequence Number (ID): 5
Length: 4820
Molecule Type: DNA
Features Location/Qualifiers:
- source, 1..4820
> mol_type, genomic DNA
> organism, Methane mono-oxygenase
Residues:
atggctgcta caactgagtc agttaaagct gatgctgcag aagcaccact tttgaacaaa 60
aaaaatatca ttgccggcgc atctttgtat ctggttttct atgcgtgggt tcgttggtat 120
gaaggtgttt atggttggtc cgcaggtctg gattcattcg cacctgagtt cgaaacatac 180
tggatgaact tcctgtatat cgaaatggta ttagaagtat taacagcttc tgtattatgg 240
ggctacatct ggaaatctcg tgatcgtaaa gtaatgtcaa tcactccacg tgaagagttg 300
agaagacatt ttactcactg gacatggtta atgatgtacg gtatcgcgat ctacttcggt 360
gctagctact tcactgagca agatggtact tggcatcaaa ctatcgttcg tgatactgac 420
ttcactccaa gtcacatcat cgagttctac ttgagctacc caatctacat catcactggt 480
ggtgcttctt tcttgtatgc taaaacaaga cttccaactt accaacaagg tttgtctctg 540
caatacctgg ttgttgttgt aggtccgttc atgatcctgc ctaacgttgg tttgaacgag 600
tggggtcata ctttctggtt tatggaagaa ttgtttgttg ctcctctgca ctatggcttc 660
gtcttcttcg gatggtctgc cttgggtgta ttgggtgtaa tcaacatcga gctgggcgcg 720
ttgtccaagc tgctgaaaaa agacctggct taatcgctta gtctgaaaaa aactatctcc 780
tggtcgcttc ttcttaaccg aagaagcgat tcagaaaata gtaaaataaa taataatttt 840
taaatccttt aggaggtaag ctaatgagcg catctcaatc agctgttcgt tcacgcgcag 900
aagctgtcaa agtttctcgt acgttcgact acatgattct ctttacggtg ttttttgttg 960
ttctcggtgg ttatcatatc cactatatgt tgaccggcgg tgactgggat ttctggactg 1020
actggaaaga tagacgtcta tgggtaactg tagcaccaat cgtttcaatc actttccctg 1080
ctgctgttca agctgtactt tggtggcgct atcgcatcgc ttggggtgca actttgtgtg 1140
tcttaggtct gttactcggt gagtggatca acagatactt caacttctgg ggatggacat 1200
acttcccagt taactttgta ttcccttcaa acctgatgcc aggcgcaatc gtattagacg 1260
tcatcctgat gctttctaac agcatgactc tgactgcggt tgttggtggt ttggcttggg 1320
gcttactgtt ctatccaggt aactggccaa tcattgctcc attacatgtt cctgttgaat 1380
acaacggcat gatgatgact ctggctgacc ttcaaggtta ccactatgtt agaactggta 1440
ctcctgagta cattcgtatg gttgaaaaag gtacattgag aacatttggt aaagacgttg 1500
ctccagtatc agcgttcttc tctggtttcg taagtatctt gatttacttc ttgtggcact 1560
tcttcggaag ctggttcgga agtgaaaaat ttgtacaagc tgcttgatac aaacttaata 1620
atcaaaaacc ttggtatatt catttagaat ctaccgaatg cttacaaaac tattaattct 1680
ctgtaataga ggaggatata tgaaaataat aaaagacaaa gttgctaaat tgtcctttgt 1740
cgcactgctg gtaactgtaa cagcagcgat gttttacact ccgacagcat ctgctcatgg 1800
tgagaaatca caggcggcat tcatgcgtat gcggactatt cactggttcg acttgaactg 1860
gtctaaagac caagtttctg tcaatgaaac tatgtcaatt tcgggtaaat tccacgtttt 1920
cgcgggatgg cctgaaactg tcgataaacc agaagttgct tttttgaaca tcggtattcc 1980
tggtcctgta tttattcgtg caggttcttg gatcggtgga caattagttc ctcgttcagt 2040
atctttagag ctgggcgaaa cttatgagtt taaagttttg ttgaaagcac gtcgtccagg 2100
cgactggcac gttcatacaa tgatgaacgt tcaaggcggt ggtccaatca ttggtccagg 2160
taaatgggta actattactg gttctatggg tgatttcaaa aatccaatca ctacattgac 2220
tggtgaaacc attgatctgg aaacttatgc attagacggt gtctatggtt ggcatttgtt 2280
ctggtacctt ttgggtgtag cttggatggt atactggtgt cgtaaacctg tattcattcc 2340
acgcagaata gctgttgacg ctggcaaagc cgattctttg atcactccaa ctgacaaaaa 2400
agttggtatg gctttcgctg ccggtacttt ggcaatcgtt gctgtctcaa tgggtcaagc 2460
caacgagaaa tatcctgtta cgactccatt gcaagcaggt ttgatgcgcg gtattaaatc 2520
tttggaactt ccacaaccta cagtttctgt aaaagttgtt gatgcttctt accgtgtacc 2580
aggtcgtgca atgcaaatga ctttggaaat cactaacaac ggtgattctg cagttcgttt 2640
ggctgagttc aacacagctt ctgttcgttt cttggatgct gatgtatatg aagacgacac 2700
taactatcca gatgacttgt tagctgaaga aggtttgtct gttagcgata acagcccact 2760
tgctccaggc gaaacaagaa ctgttgacgt tactgcttct gacgcagcat gggaagttta 2820
ccgtttagct gacttgatct acgatccaga cagccgtttc gcgggtctgt tgttcttcat 2880
cgacgaagac ggcaaccgtc aaatgacaat ggtagatgct ccattgatcc caacattcat 2940
ctaacgtctg aagccggaac tgcctgacgg cagatgcgaa ccgtccctcc cccgctcacc 3000
cgggggaggg acggggccga gatggcgcgg gcacgaacgg cctgtgccgc ttgcaggccg 3060
agaaagcgcg ccatgcgacg cttgtacggt tgcgccttgc acgtccatgt gatctgacac 3120
cgcgatcgga aagccctggc ccgacggccg gccttccgcg gggtcggccg cgtcagaaag 3180
cccacgtcga tgggtcgcgg cgcggtcaga ataacagtct ccaaggggag caggcgagag 3240
acacatggca aaaccattga ttcaattggc actagattct ttggatttcg acaaaaccct 3300
ggaacttgct tcgcaagcgg cgccttatgt cgatatcatt gaaatcggta caccctgtat 3360
caagtttaac gggcttgaat tggtcaagga aattaaacgc agatttcctg ataaattgct 3420
gctggtcgat cttaaaacga tggatgccgg tgaatacgaa gccagcccat tctacgaagc 3480
cggtgccgat atctgcacgg tacttggaac atccggaatt tcaactatca aaggcgttat 3540
caatgcggct aaaaaatata atgccgaagt tcaagtcgat ttgattaacg tcgaggataa 3600
agcgagttgc gctgaggaag ctgtggcggc cggcgcacaa attatcggca ttcataccgg 3660
catcgacgcc caggcggccg gacagacccc ctttgcggat ttacaagatt tattgcgctt 3720
ggggttaaac actcgagtat cagttgccgg cggtatcaag cccgaaactg tacgcgatgt 3780
tgttgaggcc ggagttgaca taattgtcgt aggcggcgcg attaccggcg cgccatctcc 3840
agtcaatgcc gctcgaaaaa ttcaacgctt ggtcaatagc aagggcacgc atcgtgaatt 3900
tgtcgtcgat aagatttcgg atgtcttata tgcgaccgat gactcttatg atagaaaact 3960
gacgaacatg ctcgatcaag ctcgccgaat ttttgtctca ggcgcgggac ggtcgggctt 4020
gattggccgc tttttcgcga tgcgattgat gcatagcggt tacgatacca gtgtagtagg 4080
tgaaattgtt acacccagca tcaaacaagg cgatttgctc attataattt ccggctccgg 4140
cgaaaccgag cagttggtcg cattcactaa acgtgctcgc gagattggcg caaaaatcgt 4200
gctgatttca gcgaaaagcg agtcgacaat cggcgacatg gccgatgccg tgtttagagt 4260
cggcagcccc gaacagtatg gcaaggttgt cggtatgccg atgggtacgg ttttcgagct 4320
ttccacattg gcatttttgg aagcgaccat ctctcatgtc atccacgaaa aaggcattcc 4380
ggaggaagaa atgagatcga gacatgcgaa cctggaataa cgtctgaagc cggaactgcc 4440
tgacggcaga tgcgaaccgt ccctcccccg ctcacccggg ggagggacgg ggccgagatg 4500
gcgcgggcac gaacggcctg gtgctgcgcg agatgatcta tgtatgccga cccggcggcc 4560
tgatctcgat tcccggcgtc tacggcggct tggccgacaa gatcccgatg ggcgccgcaa 4620
tgaataaggg cctcacgttc cgcatgggac agacccacgt ccagcgctgg accgccgacc 4680
ttctgcgtcg gatcgaggag gagcagatcg atccctcctt cgtcatcacg cacgaggtct 4740
ccctcgacaa gggacccgag atgtatcgga tgttccggga caagcaggac agttgcatca 4800
aggtcgttct caaaccctga 4820

Sequence Number (ID): 6
Length: 1170
Molecule Type: DNA
Features Location/Qualifiers:
- source, 1..1170
> mol_type, genomic DNA
> organism, Glutathione dependent formaldehyde dehydrogenase (GDFD)
Residues:
atgcgcgcgc tctactggaa cggcgtcaac gacctgcgtg tcggaaccgt caaggatccc 60
gagatcgtca accctcgcga cgccatcctg aaggtcaggc tatccaccac ctgcggctcg 120
gacctgcact tcatcgacgg ctacatcccg accatgaagg ccggcgacgt gatcggccac 180
gagttcatgg gcgaggtcgt cgaggtcggg ccggcggtga aacagatcaa gaagggggat 240
cgggtcgtcg tcccctcgtt catcgcctgc ggtgaatgct ggtactgcca ggagaagctc 300
ttctcgctct gcgactgcac gcacccgaag ccggaactgc aggagccgct gttcggctat 360
ccgaccgccg gcatctacgg gtacacgcat gccttcgggg gttatgcggg ctcgcacgcc 420
gaatacatcc gcgtgccgca tgccgacgtg gactgcttca aggtgcccga cggcgtcggg 480
gacgaaacgg ccctgttcct ctcggacgcc gcacccacgg gctacatggg cgccgatttc 540
tgcggcatcc ggccgggcga caccgtcgcg gtctgggggt gcggcggcgt cggcctgatg 600
gcgcagcaga gcgcgcggct gatgggcgcc gagcgggtga tcgccatcga ccgcttcccc 660
gagcgcctcc ggatggcgcg cgagcacgcc ggctccgaga cgatcgacta cacgaaagtg 720
cacagcgtgc tggaggcgct gaaggagatg acgggcgggc gcggaccgga tgcctgcatc 780
gacgccgtcg ggatggaagc gcacggcacc ggaccgcaat acgcctacga ccgcgtgaag 840
caggtgttgc gcctcgagac ggatcgtgcc caggcgctgc gcgaggccgt catggcctgc 900
cgcaagggcg gtacgctgtc ggtgctcggc gtctacggga tgatcgacaa gttcccgatg 960
ggcgccatca tgaacaaggg catgacggtc cgcaccgccc agcaacacgg gcaggcctac 1020
atggaccggc tgctcgccca tgccgagaag ggcgagctca acccggccta tctcgccacc 1080
caccgcttct cgctcgagga cggaccgcgc ggctacgaca tgttcaagca caagacggac 1140
ggctgcgtgc gcgcggtgtt cgcgccgtag 1170

,CLAIMS:
1. A method for genetically enhancing microbes for in-vitro/in-vivo/intra cellular/extra cellular production of carbon containing, non-nitrogen containing natural or unnatural compounds wherein the modifications include enhanced/ induced nitrogen fixation and/ or enhanced/induced intracellular and/ or extracellular Hydrogen fixation/uptake/utilization (a) with or without enhanced/induced intracellular and/ or extracellular Ammonia uptake/utilization; (b) with or without enhanced/induced intracellular and/or extracellular C1 Carbon uptake/fixation/utilization; (c) with or without enhanced/induced synthesis/ regeneration/uptake 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 Nitrate/Nitrite/N2O/N2O2 uptake/utilization or induced Nitrate/Nitrite/N2O/N2O2 uptake/ utilization; (f) with or without enhanced intracellular and/ or extracellular utilization/ uptake of carbon containing substrates like carbohydrate, organic acids, etc.; (g) any combination of (a) to (f). Wherein the said microbe 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 said modifications comprise homologous and/ or heterologous expression(s) of enzymes for enhanced/induced Nitrogen and/ or Carbon 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/ or associated regulatory gene/s, or a combination thereof wherein the said gene/s are native/ modified/ novel.
3. 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, Halomonas, Methylobacterium spp., Methylobacterium symbioticum, Methylorubrum, Methylomonas, Bacillus, Paenibacillus, Lactobacillus, Mycoplasma, Acetabacterium, Streptomyces, Rhodococcus, Frankia sp, Microbacterium, Curtobacterium, Bradyrhizobium japonicum, Ralstonia eutropha, Epichloë 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.
4. The method as claimed in claim 1, wherein said carbon-containing, non-nitrogen containing natural or unnatural compounds includes such as and not limited to: carbohydrates, organic acids, vitamins, lipids, alcohols, phenolic compounds, aromatic compounds, aliphatic compounds, terpenes and terpenoids, biopolymers, other primary and/ or secondary metabolites, etc.
5. The method as claimed in claim 1, wherein enhanced production of carbon-containing, non-Nitrogen containing natural or unnatural compounds, is achieved through enhanced Nitrogen fixation by either homologous or heterologous Nitrogenase and related controlling genes, such as Iron-containing nitrogenase and/ or molybdenum-based nitrogenase and/ or vanadium-based nitrogenase, bimetallic nitrogenase, or nitrogenase-like enzymes, bacterial chlorophylls (BchL, BchM, BchB).
6. The method as claimed in claim 1, 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, etc.
7. The method as claimed in claim 1, wherein 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,AmtA, AmtB, etc.
8. The method as claimed in claim 1, wherein enhanced production of carbon-containing, non-Nitrogen containing natural or unnatural compounds is achieved by upregulation and/ or downregulation/ silencing/ deletion of either homologous or heterologous genes and related regulatory genes.
9. The method as claimed in claim 1, wherein enhanced production of carbon-containing, non-Nitrogen containing natural or unnatural compounds is achieved with enhanced synthesis/ regeneration 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. The 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.
10. 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 (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-catalyzed biotin carboxylase (BC), carboxyltransferase (CT), and the like.
11. The method as claimed in claim 1, wherein said enhanced production of carbon-containing, non-Nitrogen 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. The said enzymes for increasing pyruvate include and not limited to Pyruvate synthase, Pyruvate kinase, Pyruvate decarboxylase, carrier proteins like mitochondrial pyruvate carrier proteins and the like, wherein such enzymes are overexpressed and/ or deleted/ downregulated.
12. The method as claimed in claim 1, wherein the Carbon fixation process occurs through the expression of genes, including but not limited to genes 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 Pentose phosphate pathway and/or RuMP pathway and/or serine pathway and/ or Formate-Formaldehyde pathway and/ or formyl-CoA elongation pathway, to utilize compounds including but not limited to C1 carbon compounds like CO2, Methane, Methanol, Formic acid/ Formaldehyde, and the like.
13. The method as claimed in claim 12, wherein said enzymes are homologous and/ or heterologous enzymes, native and/ or modified, and the enzyme modifications include but are not limited to point mutations, epigenetic mechanisms, etc.
14. The method as claimed in claim 1 and 12, wherein said 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)
15. The method as claimed in claim 1 and 12, wherein said 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.
16. The method as claimed in claim 1 and 12, wherein said 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.
17. The method as claimed in claim 1 and 12, said 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.
18. The method as claimed in claim 1 and 12, wherein said methanol sequestration is enhanced by overexpression of enzyme such as and not limited to Methanol dehydrogenase and/ or alcohol dehydrogenase.
19. The method as claimed in claim 1 and 12, wherein 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, and the like.
20. The method as claimed in claim 1, wherein said enhanced production of carbon-containing, non-Nitrogen compounds is through enhanced uptake of hydrogen electrons, either extracellular or intracellular through hydrogenases, for further enhancing the Carbon and/or Nitrogen assimilation and/or generation/ regeneration of ATP/ ADP, NADH/ NADPH, with or without enhanced phosphate regeneration. The hydrogen uptake is achieved by expression of Hydrogenase enzymes such as and not limited to uptake Hydrogenase, Huc Hydrogenase, CO/CO2 dependent hydrogenase (CODH), and the like.
21. The method as claimed in claims 12 and 19, wherein the enhanced CO2 assimilation is also achieved with uptake of electrons in the form of Hydrogen for CO2 reduction to formic acid (carbon & hydrogen containing compound).
22. The method as claimed in claims 1, 9 and 20, wherein said phosphate regeneration is for enhanced synthesis/ regeneration/ recycling of AMP/ ADP/ ATP and/ or polyphosphates, redox carriers such as NADP+/NADPH, and sugar phophates involved in metabolic pathways such as and not limited to Glycolysis, TCA cycle, CBB pathway, pentose phosphate pathway, RuMP pathway, Serine pathway, and the like.
23. The method as claimed in claims 14 to 22, wherein said homologous/ heterologous enzyme/s are native and/ or modified enzymes, expressed under native and/ or modified promoter, where modification includes but not limited to point-mutations, epigenetic mechanisms, and the like.
24. The method as claimed in claim1, 39 and 40 wherein said enzymes involved in Nitrogen fixation and/ or carbon fixation and/ or Ammonia fixation and/ or production of carbon-containing non-Nitrogen containing natural or unnatural compounds, are overexpressed by the inclusion of Transcriptional and/ or Translational enhancers such as and not limited to UNA1, UNA2, UNB, oligomer, and the like.
25. The method as claimed in claim 4, wherein said carbohydrates and their isomers/ epimers and/ or derivatives, include but not limited to natural or unnatural Monosaccharides (like Glucose, Fructose, Galactose, Allulose, Xylose, Xylulose, Arabinose, Ribose, Ribulose, Rhamnose, Sorbose, Tagatose, Idose, Glulose, Fucose), Disaccharides (like Sucrose, Maltose, Lactose, Cellobiose, Isomaltose, Isomaltulose, Trehalose, Trehalulose, Leucrose, Turanose), oligosaccharides (like malto oligosaccharides, iso-malto oligosaccharides, fructo oligosaccharides, milk oligosaccharides), polysaccharides (like Maltodextrin, Glucan, Dextran, xylan, pullulan, Glycan like capsular polysaccharides), sugar phosphates (like Glucose 1-phoshate, Glucose 6-phosphate, Fructose 6-phosphate, Fructose 1,6-bis phosphate, Phosphoenol pyruvate, Glyceraldehyde 3-phosphate, Dihydroxy acetone phosphate), and the like.
26. The method as claimed in claim 4, wherein the said organic acids include such as and not limited to natural or unnatural Acetic acid, Butyric acid, Formic acid, Lactic acid, Citric acid, Oxalic acid, Glutaric acid, Succinic acid, Malic acid, Fumaric acid, Pyruvic acid, Tartaric acid, Oxalo-acetic acid, Phosphoglyceric acid, Aconitic acid, Itaconic acid, Ascorbic acid (Vitamin C), and the like.
27. The method as claimed in claim 4, wherein the said lipids include such as and not limited to saturated/ unsaturated/ poly unsaturated fatty acids (like short, medium and long-chain fatty acids) Mono, di and tri-acyl glycerides, designer/ structured lipids, microbial oil, Fatty acid and their esters such as methyl esters, ethyl esters, structured oil, glycolipids, glyceroglycolipids, galactolipids, rhamnolipids, and the like.
28. The method as claimed in claim 4, wherein the said alcohols such as and not limited to natural or unnatural Methanol, Ethanol, Propanol, Iso-propanol, Butanol, Iso-butanol, Tertiary butanol, Hexanol, octanol, cyclo hexanol, sugar alcohols/ polyols (Glycerol, Sorbitol, Mannitol, Erythritol, Xylitol, Glucitol, Galactitol, Ribitol, Iditol, Gulitol, Fucitol), and the like.
29. The method as claimed in claim 4, wherein the said phenolic and aromatic compounds and their derivatives include but not limited to natural or unnatural catechol, resorcinol, hydroxy quinone, phenolic acid like ferulic acid, Caffeic acid, cinnamic acid, coumaric acid, syringic acid, vanillic acid, salicylic acid, hydroxy benzoic acid, tannic acid, Ellagic acid, Rosmarinic acid, Trimethoxy Cinnamic Acid, Caffeate esters, Chicoric acid, Gentisic Acid, Dihydroxyhydrocinnamic acid, Carnosic acid, Caftaric acid, Hydroxy cinnamic acid, Diferulic acids, Vanillic acid, Vanillin, Acetosyringine, Quinones and derivatives such as and not limited to Semiquinone, menaquinone, Vitamin K (vitamin K1-phylloquinone, vitamin K2-menaquinone), steroids, sterols, Vitamin D (calciferol), Azulene, Guaiazulene, Vetivazulene, phyllodulcin, curcumin, poly phenols like Flavanoids, Nobiletin, Capsaicinoids, and the like.
30. The method as claimed in claim 4, wherein said aliphatic compounds include and not limited to natural or unnatural alkanes such as methane, ethane, long chain alkanes, alkanols and alkanoic acid; alkenes/ olefins such as Ethylene, long chain and/ or poly unsaturated alkenes, and the like.
31. The method as claimed in claim 4, wherein said terpenes/ terpenoides and their derivatives includes but not limited to natural or unnatural terpenes and terpenoides, isoprenes, isoprene pyrophosphate, monoterpenes, diterpenes, diterpenoids like Gibberellic acid, triterpenes, tetraterpenes like saponin, sesquiterpenes (like Abscisic acid), sesquiterpene lactones, and derivatives like carotenes, Vitamin A, carotenoid, retinol, retinal, retinoic acid, xanthophyll, menaquinone, lycopene and the like.
32. The method as claimed in claim 4. wherein said biopolymers includes but not limited to PHA, PHB, Polylactate, Alginate, Agarose, cellulose acetate, methyl/ ethyl cellulose, hydroxy propyl cellulose, hydroxy propyl methyl cellulose, Sporopollenin, suberin, lignin, and the like.
33. The method as claimed in claim 25, wherein the said carbohydrates production including monosaccharides and disaccharides is achieved by homologous/ heterologous modification of enzymes including but not limited to sucrose-phosphate synthase, sucrose-phosphate phosphatase, Fructokinase, glucose-6-phosphate isomerase, phosphoglucomutase, fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase, UTPglucose-1-phosphate uridylyl transferase; Rubisco, glucokinase, invertase, sugar isomerase, sugar epimerase, oxidoreductase, Formolase, Fructose 6-phosphate Aldolase, and the like.
34. 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, and the like.
35. 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/ non-synthetic compounds including but not limited to plastics/ fibres/ rubber/ resins/ chemicals, etc.(like polyethylene terepthalate (PET), Ethylene glycol (EG), polyurethane, polybutylene succinate-PBS, PBS-adipate), 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, Glycosyl transferases, PBSase, 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, and the like.
36. 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.
37. The method as claimed in claim 25 to 36, 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, epigenetic mechanisms, etc.
38. The method as claimed in claims 5 and 12, wherein carbon and Nitrogen assimilation is enhanced by integrating the synthesis/ regeneration 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.
39. The present invention additionally relates to a method of gene modifications in microbes, more specifically involving gene modifications for enhancing transcription and/or translation of proteins involved in (i) nitrogen fixation and/or (ii) uptake/fixation of ammonia and/or (iii) uptake/fixation of carbon 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-Nitrogen & Carbon containing natural or unnatural compounds.
40. The present invention also relates to a method of 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 nitrogen fixation and/or (ii) uptake/fixation of ammonia and/or (iii) uptake/fixation of carbon and/ or (iv) uptake/synthesis/regeneration of anyone or all compounds including phosphate, ATP, NADH, NADPH, Pyruvate, CoA and Acetyl CoA and/or (v) for in-vitro/in-vivo/intra cellular/extra cellular production of non-Nitrogen & Carbon containing natural or unnatural compounds.
41. The method as claimed in claim 39 and 40, wherein said modifications comprise homologous and/ or heterologous expression(s) of the respective enzymes, their promoter/s and/ or associated regulatory gene/s, or a combination thereof wherein the said gene/s are native/ modified/ novel.
42. The composition as claimed in claim 1, wherein the microbial composition includes single microbe modified or a consortium of microbes modified for production of non-Nitrogen, carbon -containing compounds.