WO2024121870A1 - Modification génétique de microbes pour la production de composés non azotés contenant du carbone - Google Patents

Modification génétique de microbes pour la production de composés non azotés contenant du carbone Download PDF

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WO2024121870A1
WO2024121870A1 PCT/IN2023/051163 IN2023051163W WO2024121870A1 WO 2024121870 A1 WO2024121870 A1 WO 2024121870A1 IN 2023051163 W IN2023051163 W IN 2023051163W WO 2024121870 A1 WO2024121870 A1 WO 2024121870A1
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acid
carbon
enhanced
limited
fixation
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Rahul Raju KANUMURU
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Fertis India Pvt. Ltd.
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Definitions

  • 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 cl carbon fixation/increased cl 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
  • 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.
  • prk phospho- ribulo-kinase
  • RuBisCo Ribulose-Bisphosphate Carboxylase
  • 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 Cl compounds such as formate, formic acid, formaldehyde or methanol to organic carbon 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 Cl 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.
  • 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 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.
  • 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
  • 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).
  • MDH methanol dehydrogenase enzyme
  • 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.
  • PHI phosphohexose isomerase
  • 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.
  • Hpsi2 heterologous Hexulose -6-phosphate synthase and isomerase
  • FIG. 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).
  • PRK phosphor-ribulo kinase
  • RuBisCO Ribulose bisphosphate carboxylase and oxygenase
  • 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.
  • PRK phosphor-ribulo kinase
  • RuBisCO Ribulose bisphosphate carboxylase and oxygenase
  • 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.
  • PGA Phospho-glyceric acid
  • Figure 11 Depicts the novel, inventive process of Nitrogenase metal specificity by promoter exchange. Modification and replacement of Anf Promoter with NifH promoter to increase electron delivery to Fe-nitrogenase, pathways activated by NifA. Incorporation of promoter such as nifH promoter (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.
  • 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 cl carbon fixation/increased cl 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.
  • 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.
  • 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.
  • the present invention details the methods of assimilation of various carbon compounds including but not limited to Cl 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 dicarboxy late/4-hydroxybutyrate cycle (DC/HB), fixation of other CO2 or Cl compounds such as Methanol, Methane, Formaldehyde, etc.
  • CBP Calvin Benson pathway
  • rTCA reductive citric acid cycle
  • Wood-Ljungdahl pathway the reductive acetyl-CoA pathway
  • HP-bicycle 3-hydroxy propionate bicycle
  • 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 Formateformaldehyde route, where in Formate dehydrogenase (FDH) catalyses the conversion of carbon dioxide (CO2) into formic acid.
  • FDH Formate dehydrogenase
  • 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.
  • PEP carboxylase phosphoenolpyruvate carboxylase
  • PEP carboxylase catalyzes the conversion of phosphoenolpyruvate (PEP) to oxaloacetate, a key step in the Reductive TCA cycle.
  • PEP carboxylase aims to genetically modify or over express PEP carboxylase to increase its activity and enhance the efficiency of the Reductive TCA 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.
  • fructose bisphosphatase plays a role in the regeneration of RuBP, which is necessary for the continued operation of the cycle.
  • 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.
  • MMO methane monooxygenase
  • sMMO methane monooxygenase
  • pMMO particulate methane monooxygenase
  • the methanol formed from Methane can enter into central carbon metabolism through methanol oxidation to formaldehyde involving methanol dehydrogenase, or Alcohol oxidase (AOX) enzyme.
  • AOX Alcohol oxidase
  • 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.
  • CBB Calvin- Benson-Bassham
  • RuMP formaldehyde is condensed with ribulose monophosphate to produce fructose-6- phosphate.
  • XuMP pathway formaldehyde is condensed with xylulose monophosphate to produce fructose-6-phosphate.
  • 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.
  • the microorganisms may be genetically engineered to improve their ability to utilize carbon from different sources.
  • 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.
  • RuMP Ribulose mono phosphate
  • HPS 3-hexulose-6-phosphate synthase
  • PHI 6-phospho-3 -hexuloisomerase
  • the methanol sequestration is enhanced by overexpression of enzyme such as and not limited to Methanol dehydrogenase and/ or alcohol dehydrogenase.
  • the CO2 sequestration via the said carbon fixation pathways is enhanced by the increased availability of CO2/ HCOs (bicarbonate ions) through overexpression of enzymes such as and not limited to Carbonic anhydrase, Carbon concentrating mechanism (CCM), carboxysomes, etc.
  • CO2/ HCOs bicarbonate ions
  • 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 (HCOs')-
  • the present invention focuses on increasing the expression or improving the catalytic activity of Carbonic Anhydrase.
  • 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.
  • 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.
  • 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.
  • the present invention provides a novel, inventive process for microbes with genetic modification in nitrogen fixation for enhanced carbon fixation.
  • the present invention provides a novel, inventive process for microbes with genetic modification in carbon fixation for enhanced nitrogen fixation.
  • 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.
  • 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.
  • 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.
  • 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.
  • the invention also involves manipulation of Nitrogenase related Regulatory genes, such as nifA, nifL, fix, mf genes, to enhance Nitrogenase activity and glutaminase, which hydrolyses glutamine to glutamate and ammonia, resulting in increased Nitrogen fixation.
  • Nitrogenase related Regulatory genes such as nifA, nifL, fix, mf genes, to enhance Nitrogenase activity and glutaminase, which hydrolyses glutamine to glutamate and ammonia, resulting in increased Nitrogen fixation.
  • 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.
  • 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 mf cluster genes such as mfABCDEG
  • Nitrogenase regulatory genes including but not limited to regulatory proteins, such as nifA and/ or nifL, and/ or fix genes and/ or operons such as fixABCX, fixNOQP, fixH, fixJ, fixR, fixK, fixL, and/ or mf cluster genes such as mfABCDEG
  • 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.
  • ammonia uptake enzymes such as Glutaminases, Glutamine synthase, and their isoforms, including GlnA, GlnD, and GlnE.
  • GlnA enzyme glutamine synthetase
  • 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.
  • Ammonia utilizing enzymes including Glutaminases, Glutamine synthase and its related enzymes like GDH, GlnA, GlnD, GlnE and Ammonia transporters like AmtA, AmtB, etc.
  • 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.
  • the present invention provides Hydrogen uptake/ fixation for enhancing the Carbon fixation, Nitrogen fixation, synthesis/ regeneration of ATP/ ADP, NADH/ NADPH by expression of Hydrogenase enzymes such as and not limited to uptake Hydrogenase, Hue Hydrogenase, CO/CO2 dependent hydrogenase (CODH), etc.
  • the phosphate regeneration is for enhanced synthesis/ regeneration/ recycling of AMP/ ADP/ ATP and/ or polyphosphates, redox carriers such as NADPVNADPH, 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.
  • 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 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).
  • MAS Malate-aspartate shuttle
  • 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.
  • DHAP dihydroxyacetone phosphate
  • G3P glycerol-3 - phosphate
  • NAD+ glycerol-3 - phosphate
  • 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.
  • the enzymes for NADH regeneration includes and not limited to all Dehydrogenases and oxidoreductases
  • Enzymes for ATP regeneration includes but not limited to ATP synthase, ATP cyclase, Malate - Aspartate shuttle and glycerol-3 -phosphate shuttle enzymes, Polyphosphate-AMP-phosphotransferase (PAP), Polyphosphate kinase, ATP synthesis/ recycling via acid production pathway involving enzymes such as and not limited to Acetate kinase/ propionate kinase/ Butyrate kinase, CoA-acylating dehydrogenase, Phosphotrans acetylase/ propionylase/ Butyrylase, aldehyde dehydrogenase, lactate dehydrogenase, and the like .
  • 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.
  • CoA transferases CoA-acylating aldehyde dehydrogenase, CoA-dependent propionaldehyde dehydrogenase, phosphotransacetylase, Acetyl CoA-synthase (ACS
  • 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.
  • the present invention provides genetically modified microbe, wherein said modifications support production/ enhanced production of carbon-containing, non-Nitrogen natural or unnatural compounds.
  • 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.
  • 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.
  • 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, D
  • 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.
  • 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, glycerogly colip ids, 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.
  • 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
  • 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.
  • 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, Camosic 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 Kl-phylloquinone, vitamin K2 -mena)
  • 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.
  • MUFA mono unsaturated fatty acids
  • 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.
  • the present genetically engineered microbe with enhanced nitrogen and carbon fixation can be used as production host for sugar production from Cl compound such as and not limited to CO2, CH4, Formaldehyde, etc.
  • 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.
  • 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.
  • SEC serotonin
  • 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.
  • 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.
  • non- nitrogenous organic compounds such as organic acids, solvents, terpenoids, PHAs, PHBs, ply lactate, carotenoids, biodiesel and so on.
  • 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.
  • 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.
  • 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.
  • 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.
  • enzymes including but not limited to sucrose-phosphate synthase, sucrose-phosphate phosphatase, Fructokinase, glucose-6-phosphate isomerase, phosphoglucomutase, fructose-
  • 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.
  • 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.
  • carbohydrates including but not limited to monosaccharides/ disaccharides/ oligosaccharides/ polysaccharides, etc.
  • organic acids including but not limited to monosaccharides/ disaccharides/ oligosacchari
  • 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 such as butelase 1 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.
  • 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.
  • 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.
  • 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.
  • transcription/translation level 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.
  • 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.
  • assimilation is enhanced by integrating the regeneration/ recycling of the metabolic intermediates including but not limited to Hydrogen, Phosphate/ ATP, NADH/ NADPH, Pyruvate, Coenzyme A-CoA/ Acetyl CoA resulting in stable/ enhanced metabolic efficiency of the host microbe.
  • the metabolic intermediates including but not limited to Hydrogen, Phosphate/ ATP, NADH/ NADPH, Pyruvate, Coenzyme A-CoA/ Acetyl CoA resulting in stable/ enhanced metabolic efficiency of the host microbe.
  • the 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, Alterery
  • 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, Rey ranella mas siliens is, Alcaligene, Saccharomyces cerevisiae, Saccharomyces lactis, Brevibacterium, , Kluyveromyces lactis, Epichloe 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.
  • 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,
  • Proteobacteria such as Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea, Serratia, Rahnella
  • Example 1 Novel method for Overproduction of carbon-containing, nonnitrogen compound by genetically enhanced microbe.
  • Methylobacterium spp was genetically modified for enhanced production of formic acid by:
  • Nitrogenase and related regulator genes including
  • FDH Formic acid dehydrogenase
  • 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 Co A.
  • PPK Polyphosphate kinase
  • PAP Polyphosphate AMP-Phospho transferase
  • Example 2 Enhanced CO2 fixation by overexpression of CBB pathway enzymes Rubisco and PRK-Phospho ribulo kinase:-
  • 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.
  • GDFD Glutathione dependent formaldehyde dehydrogenase
  • Carbon compounds including Cl 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).
  • GHG green house gas
  • 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.
  • 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).
  • 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 Co A.
  • PPK Polyphosphate kinase
  • PAP Polyphosphate AMP-Phospho transferase
  • 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.

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Abstract

La présente invention concerne un nouveau procédé de production durable in vitro et in vivo de composés non azotés, contenant du carbone. Ledit nouveau procédé implique des modifications génétiques de diazotrophes et/ou de diazotrophes induits, avec l'une ou l'autre ou des combinaisons des éléments suivants : (i) une fixation accrue de l'azote, (ii) avec ou sans fixation du carbone, (iii) une stabilité métabolique accrue par une régénération accrue de l'un ou de tous les composés, y compris l'ATP, le NADH, le NADPH, le phosphate, l'hydrogène, le Coenzyme-A et le pyruvate. La présente invention concerne des modifications génétiques de microbes pour une fixation accrue de l'azote, avec ou sans fixation du carbone, avec un meilleur recyclage de l'ATP et du NADH, conduisant à la production in-vitro et in-vivo de composés non azotés contenant du carbone, y compris, mais sans s'y limiter, des acides organiques, des acides gras, des solvants et des sucres, des substances phénoliques, des gibbérellines, des proto-alcaloïdes, des stéroïdes, des structures polymériques comme les terpènes et les terpénoïdes, les caroténoïdes, les acides gras, les lipides, les polyhydroxy-alcénoates (PHA, PHB), le poly-lactate, le biodiesel, etc.
PCT/IN2023/051163 2022-12-09 2023-12-11 Modification génétique de microbes pour la production de composés non azotés contenant du carbone WO2024121870A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170183665A1 (en) * 2014-05-22 2017-06-29 Yeda Research And Development Co. Ltd. Recombinant microorganisms capable of carbon fixation
US20200095620A1 (en) * 2017-11-15 2020-03-26 Unist(Ulsan National Institute Of Science And Technology) Recombinant microorganism and method for production of formic acid by using same
WO2020245841A1 (fr) * 2019-06-04 2020-12-10 Fertis India Pvt. Ltd. Modification génétique de microbes endophytes/épiphytes/rhizosphériques pour une fixation améliorée de l'azote pour des cultures
WO2021084526A1 (fr) * 2019-10-31 2021-05-06 Yeda Research And Development Co. Ltd. Bactéries autotrophes génétiquement modifiées pour la conversion de co2 en matériaux organiques

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170183665A1 (en) * 2014-05-22 2017-06-29 Yeda Research And Development Co. Ltd. Recombinant microorganisms capable of carbon fixation
US20200095620A1 (en) * 2017-11-15 2020-03-26 Unist(Ulsan National Institute Of Science And Technology) Recombinant microorganism and method for production of formic acid by using same
WO2020245841A1 (fr) * 2019-06-04 2020-12-10 Fertis India Pvt. Ltd. Modification génétique de microbes endophytes/épiphytes/rhizosphériques pour une fixation améliorée de l'azote pour des cultures
WO2021084526A1 (fr) * 2019-10-31 2021-05-06 Yeda Research And Development Co. Ltd. Bactéries autotrophes génétiquement modifiées pour la conversion de co2 en matériaux organiques

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