WO2024121871A1 - Genetic modification of microbes for production of nitrogen and carbon-containing compounds - Google Patents

Genetic modification of microbes for production of nitrogen and carbon-containing compounds Download PDF

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WO2024121871A1
WO2024121871A1 PCT/IN2023/051164 IN2023051164W WO2024121871A1 WO 2024121871 A1 WO2024121871 A1 WO 2024121871A1 IN 2023051164 W IN2023051164 W IN 2023051164W WO 2024121871 A1 WO2024121871 A1 WO 2024121871A1
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nitrogen
fixation
carbon
enzymes
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Rahul Raju KANUMURU
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Fertis India Pvt. Ltd.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present invention relates to a novel method for sustainable in vitro and in vivo production of nitrogen and 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 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 Nitrogen and Carbon containing compounds
  • W02017011602 discloses methods of increasing nitrogen fixation in non- leguminous plants comprising exposing the plant to a plurality of bacteria (microbiome) 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.
  • microbiome 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 phosphoribulokinase (prk) and Ribulose-Bisphosphate Carboxylase (RuBisCo) to improve carbon fixation. It also discloses methods that include down-regulating genes in microorganisms using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas associated protein arrays.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • US 20210163374A1 discloses genetically engineered bacterial strain which is plant growth promoting bacterial strain and fixes atmospheric nitrogen in agricultural system and comprises the gene modifications in one or more genes from the group consisting of besll , beslll , yjbE , fhaB , pehA , glga , otsB , Dun , and cysZ for increased nitrogen fixation and colonization of plant.
  • 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.
  • US20170183665A1 provides disclosure for genetically modified microorganisms utilizing recombinant carbon fixation enzymes for CO2 fixation for production of a first essential biomass precursor.
  • the genetic modifications in the microorganism occurs in carbon fixation pathways and enzymes associated with it particularly Calvin-Benson-Bassham cycle (C3 cycle) and RuBisCO, Prk etc.
  • US10801045B2 discloses genetically engineered microorganisms wherein carbon fixation pathways are modified to make microorganism chemoautotrophic and efficiently utilizes inorganic carbon compounds such as formate, formic acid, methane, carbon monoxide, carbonyl sulfide, carbon disulfide, hydrogen sulfide, bisulfide anion, thiosulfate, elemental sulfur, molecular hydrogen, ferrous iron, ammonia, cyanide ion, and/or hydrocyanic acid and produce organic carbon products such as sugars, alcohols, chemicals, amino acids, polymers, fatty acids and their derivatives, hydrocarbons, isoprenoids, and intermediates etc.
  • inorganic carbon compounds such as formate, formic acid, methane, carbon monoxide, carbonyl sulfide, carbon disulfide, hydrogen sulfide, bisulfide anion, thiosulfate, elemental sulfur, molecular hydrogen, ferrous iron, ammoni
  • US8048661B2 discloses genetically modified microorganisms encoding modified pathways for enhancing carbon flux through acetyl-CoA.
  • the methods of modification include altering the expression of enzymes in a reductive TCA or
  • US9150888B2 discloses genetically engineered photoautotrophic microorganism for conversion of carbon dioxide and light into carbon-based products of interest such as ethanol, ethylene, chemicals, polymers, n-alkanes, isoprenoids, pharmaceutical products.
  • US20200277636A1 discloses synthetic or genetically engineered microorganisms comprising methane, methanol utilizing pathways for conversion of Methane, methanol to organic compounds, industrial products, chemicals and intermediates.
  • the patent provides methods for converting non-methanotrophic, non- methylotrophic microorganism into methanotrophic, methylotropic microorganisms by incorporation of methane oxidizing and methanol -oxidizing metabolic pathways.
  • 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 and nitrogen containing 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 nitrogen and carbon containing compounds.
  • the present invention relates to a novel method for sustainable in vitro and in vivo production of nitrogen and carbon containing compounds.
  • the present invention relates to a novel method that includes gene modifications in microbes, more specifically involving gene modifications resulting in nitrogen fixation and/or ammonia fixation or increased nitrogen fixation and/or ammonia fixation or carbon fixation/increased carbon fixation or Hydrogen fixation/increased Hydrogen fixation with increased synthesis/ regeneration of anyone or all compounds including ATP, NADH, NADPH, 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 Nitrogen and 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) Hydrogen fixation and/ or (v) synthesis/regeneration of anyone or all compounds including ATP, NADH, NADPH, Pyruvate and Acetyl CoA
  • Figure 1 Depicts CO2 consumption profile of the engineered microbe, modified with enhanced formate dehydrogenase expression.
  • Figure 2 Depicts Urea production by engineered microbes, modified with establishment of urea synthesis genes for expression of rate-limiting enzymes such as Ornithine trans-carbamylase, carbamyl phosphate synthase and Arginase.
  • rate-limiting enzymes such as Ornithine trans-carbamylase, carbamyl phosphate synthase and Arginase.
  • Urea production is achieved by diverting Nitrogen flux from amino acid synthesis pathway.
  • Figure 3 Depicts enhanced Protein production by engineered microbe, modified for Nitrogen flux towards amino acids over production as well as increased carbon and redox flux.
  • Figure 4 Depicts impact of different carbon metabolism of Cl compounds on increased carbon metabolism.
  • Figure 5 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 6 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.
  • MDH methanol dehydrogenase enzyme
  • the modified microbes express heterologous methane monooxygenase and methanol dehydrogenase to increase the carbon assimilation within the cell.
  • Figure 7 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. 8 Depicts the pathway of CO2 fixation through modified CBB pathway.
  • Calvin-Benson-Bassham (CBB) pathway involves cyclic movement of carbon between the sugar molecule by fixing the atmospheric carbon dioxide with the help of phosphoribulo kinase (PRK) and Ribulose bisphosphate carboxylase and oxygenase (RuBisCO).
  • PRK phosphoribulo 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 Phosphoribulokinase (PRK) and compact RuBisCO for the CO2 assimilation.
  • PRK phosphoribulo kinase
  • RuBisCO Ribulose bisphosphate carboxylase and oxygenase
  • Figure 9 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 10 Depicts the novel, inventive process of Nitrogenase metal specificity by promoter exchange. Modification and replacement of Anf Promoter with NifH promoter to increase electron delivery to Fe-nitrogenase, pathways activated by NifA. Incorporation of promoter such as nifH promoter (nifHDK operon) in place of anf promoter (of anfHDGK operon), shifts the specificity to Fe (Iron) irrespective of presence or absence of Molybdenum. Promoter exchange also impacts on enhanced Nitrogenase activity.
  • nifH promoter nifHDK operon
  • anf promoter of anfHDGK operon
  • Figure 11 Depicts the pathway of Ammonia generation and fixation in amino acid metabolism. Nitrogen fixation in the recombinant strain over expressing native/ heterologous nitrogenase and inactivating glnA.
  • GOGAT Glutamine oxoglutarate aminotransferase
  • Gin glutamine
  • Glu glutamate
  • OG Oxoglutarate
  • Figure 12 Depicts the Limitations in Nitrogen & carbon-containing compounds production methods as per the prior art methods
  • Figure 13 Depicts the novel, Inventive solution with novel methods to overcome limitations in Nitrogen & carbon-containing compounds production by gene manipulations of microbe for efficient carbon and nitrogen fixation, as well as for enhanced redox energy generation and regenerations.
  • Figure 14 Depicts gives an overall description about the modifications performed to increase the Ammonia and related energy supply and regeneration of metabolites, with respect to carbon assimilation.
  • Figure 15 Depicts the Modified Nitrogenase gene construct for genome integration.
  • Gene integration construct of Nitrogenase includes expression of Nitrogenase under NifH promoter, cloned with flanking sequences of Fdm gene partial sequences to facilitate integration with the Fdm gene in genome, resulting in simultaneous deletion of Fmn gene and integration of Nitrogenase under modified promoter.
  • the present invention relates to a novel method for sustainable in vitro and in vivo production of nitrogen and 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 and nitrogen 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) for in-vitro/in-vivo/intra cellular/extra cellular production of Nitrogen & Carbon containing natural or unnatural 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 Nitrogen & Carbon containing natural or unnatural compounds.
  • the present invention is focused on achieving specific objectives by utilizing genetic modifications of microbes to enhance their ability to fix nitrogen.
  • This process results in the production of a range of nitrogen-containing primary metabolites, including amino acids, proteins, enzymes, nucleotides, vitamins, nitrogen-containing organic acids, and other similar compounds.
  • These primary metabolites play a crucial role in supporting various cellular processes such as growth, repair, and maintenance.
  • One of the embodiment of the present invention focus on enhanced Nitrogen fixation and ammonia production.
  • the excess requirement of Redox energy compounds for Nitrogen fixation including ATP, NADH and electrons/hydrogen is supported by carbon assimilation/ enhanced carbon assimilation.
  • Another embodiment of the current invention provides genetic modifications in microorganisms for enhancing carbon flux through increased uptake/ assimilation of carbon compounds including but not limited to CO2, methane, methanol, formaldehyde, sugars like monosaccharides, disaccharides, or organic acids, which in turn increases the NADH, ATP and electron/hydrogen generation, which is sufficient for Nitrogen fixation. Therefore by carbon fixation/ enhancing carbon fixation and/ or assimilation, nitrogen fixation can also be increased.
  • the current invention developed a novel process of integrating the carbon fixation and nitrogen fixation in an interdependent, mutually controlled system, thereby resulting in enhanced carbon and nitrogen fixation and impacting on product formation, which is explained in the following embodiments.
  • 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 objective of the present invention is to enhance the process of nitrogen fixation by modifying the Nitrogenase enzyme and related controlling genes for enhanced production of nitrogen and carbon-containing compounds.
  • 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 details the methods of assimilation of various carbon compounds including but not limited to C 1 compounds like CO2, Methane, Methanol, formaldehyde, formate, etc., and organic acids, sugars, and other carbohydrates.
  • 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 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
  • 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.
  • 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.
  • 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 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 (HCOf).
  • the present invention focuses on increasing the expression or improving the catalytic activity of Carbonic Anhydrase.
  • 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/ HCO3 (bicarbonate ions) through overexpression of enzymes such as and not limited to Carbonic anhydrase, Carbon concentrating mechanism (CCM), carboxysomes, etc.
  • CO2/ HCO3 bicarbonate ions
  • enzymes such as and not limited to Carbonic anhydrase, Carbon concentrating mechanism (CCM), carboxysomes, etc.
  • the present invention provides increase in the availability of CO2, by increasing the carbon assimilation pathway-related genes such as RuBisCO, CCMs, and carbonic anhydrase, which results in an increase in the energy supply required in the form of ATP and NADH in the nitrogen fixation for various nitrogenases in producing NH 3 + which can be utilized/diverted further in Nitrogen and carbon-containing compounds production.
  • the present invention provides process for heterologous genetic modifications for carbon fixation in non-carbon fixing microorganisms wherein genes for carbon fixation such as Rubisco, Phosphohexoisomerase, Hexose phosphate synthase, Methane Monooxygenase etc. are taken from methanotrophs/methylotrophs but not limited to Galionellci spp., Methylomicrobium spp. etc.
  • the present invention provides genetic control of Nitrogen/ Carbon assimilation by homologous and/ or heterologous modification of carbon uptake transporters such as PtsG, PtsN or PtsF or ManZ or LacY and carbonic anhydrase.
  • 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 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.
  • 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 Nitrogen and Carbon containing natural or unnatural compounds is achieved with enhanced regeneration/ recycling of ATP/ ADP/ phosphate and/ or NADH/ NADPH, with or without enhanced hydrogen uptake/ fixation, wherein the enzymes for NADH regeneration includes and not limited to all Dehydrogenases and oxidoreductases, Other enzymes for synthesis/ regeneration of NADH includes but not limited to NADH oxidase, ADP-ribosyl cyclase, SARM-1, NAD hydrolases, mono (ADP-ribosyl)transferases, Nicotinic acid phosphor ribosyl transferase.
  • NADHNADPH 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 .
  • 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 Nitrogen and Carbon containing natural or unnatural compounds is achieved by expression of Homologous/ heterologous, native/ modified enzymes responsible for increased pyruvate availability include but not limited to (1) enzymes involved in increasing pyruvate synthesis towards downstream product formation pathways and (2) enzymes involved in preventing pyruvate loss in form of CO2, wherein said enzymes for increasing pyruvate include and not limited to Pyruvate synthase, Pyruvate kinase, Pyruvate decarboxylase, carrier proteins like mitochondrial pyruvate carrier proteins etc., wherein such enzymes are overexpressed and/ or deleted/ downregulated.
  • the present invention pertains to an embodiment for production of nitrogen and carbon containing compounds including but not limited to, amino acids, proteins, enzymes, nucleotides, nitrogen-containing vitamins like Vitamin-B and derivatives, amines, amides, etc. (whose functions include but not limited to metal-carrying metabolites, signalling metabolites, plant growth stimulants, bioherbicides, bioinsecticides, antimicrobial agents, antiparasitic agents, enzyme inhibitors, etc.)
  • the present invention provides increasing the production of nitrogen-containing compounds such as proteins in microorganisms that can fix nitrogen and carbon dioxide.
  • proteins are essential molecules that perform a wide range of functions in living organisms, including acting as enzymes, structural components, and signaling molecules.
  • the present invention provides methods for microbe modification for production of enzymes including pectinases, cellulases, phytases, lipases, and other types of enzymes such as hydrolases, oxidoreductases, lyases, transferases, ligases, and isomerases.
  • Pectinases are enzymes that break down pectin, a component of plant cell walls, and are used in various industries such as food, textiles, and paper production.
  • Cellulases are enzymes that break down cellulose, the main component of plant cell walls, and are used in the production of biofuels and animal feed.
  • Phytases are enzymes that break down phytic acid, a component of plant seeds that can reduce the bioavailability of certain minerals, and are used in animal feed to improve mineral absorption.
  • Lipases are enzymes that break down fats and are used in the production of various products such as detergents, cosmetics, and pharmaceuticals.
  • the present invention provides methods for increasing the production of nitrogen-containing compounds such as peptides in microorganisms that can fix nitrogen and carbon dioxide.
  • Peptides are short chains of amino acids that can perform various functions in living organisms, including acting as hormones, enzymes, and signalling molecules.
  • the present invention provides methods for increase in nitrogen-containing secondary metabolites such as non-ribosomal peptides.
  • Non-ribosomal peptides are a type of peptide that are not synthesized by ribosomes, but rather by multi-enzyme complexes known as non-ribosomal peptide synthetases (NRPS) which includes but not limited to Antibiotics, Siderophores, Toxins, Nitrogen storage polymers.
  • NRPS non-ribosomal peptide synthetases
  • flavonoids such as flavonoids and its derivatives
  • alkaloids like isoquinoline alkaloids, indole alkaloids, steroidal alkaloids, proto alkaloids, pseudo alkaloids, including but not limited to alkaloids like streptomycin, penicillins, palmatine, dragmacidin, Neofiscalin, etc., for use in antimicrobial purposes, signal transduction purposes
  • Amides like urea, guanidine, formamide, benzamide, etc. Amines like glucosamine, NAG (n-acetyl glucosamine), hydroxyl amine, etc.; N-
  • microbe modification for nutrient acquisition is targeted.
  • Microbes can be enabled to assimilate varied nutrients under in vivo and/ or in vitro applications including but not limited involved in food processing, nutrient dense food preparation, functional food additives such as proteases, pectinases, invertase, amylase, etc.; enzymes to extract bio-active components, probiotics, probiotics; enzymes involved in catabolic degradation/ utilization such as and not limited to cellulase, exo-glucanase, endo-glucanase, cellobiohydrolase, xylanase, Pectin esterase, lignin peroxidase, protease; enzymes for anabolism and metabolite conversion such as and not limited to sugar isomerases, phytase, invertase; enzymes such as Nitrate reductase, Nitrite reductase, sulfate adenylyl transferase (
  • the present invention provides modifications of microbes with ribozymes involved in gene regulatory circuits of the nitrogen fixation and or carbon fixation pathways for the controlled expression for the production of nitrogen and carbon-containing compounds.
  • the Nitrogen & Carbon containing natural or unnatural compounds include but not limited to amino acids, proteins, enzymes, cofactors, nucleotides, vitamins, flavonoids, alkaloids, peptides, proteins, amides, amines, Urea, Xanthines, N-glycosides, glucosinolates, non-protein amino acids, single cell protein, Nitrogen-containing organic acids, and the like.
  • the Nitrogen & Carbon containing natural or unnatural compounds are produced in vivo and in vitro.
  • the present invention provides methods of gene modifications in carbon fixation to enhance nitrogen fixation in microbes including epiphyte or endophyte or rhizosphere or free-living or ruminant/ non-ruminant gut microbe for in-vitro and in-vivo production of Nitrogen & Carbon containing primary and secondary metabolites.
  • the present invention provides process for gene modifications of microbes including epiphyte or endophyte or rhizosphere or free- living or ruminant/ non-ruminant gut microbe genetically modified for nitrogen fixation with/without carbon fixation for in-vitro and in-vivo production of Nitrogen & Carbon containing primary and secondary metabolites.
  • the present invention provides genetically modify non- diazotrophs with nitrogen fixation genes with/without carbon fixation for in-vitro and in-vivo production of Nitrogen & Carbon containing primary and secondary metabolites.
  • the present invention provides in addition to primary metabolites, the modified microbes also produce a variety of secondary metabolites. These metabolites serve multiple purposes such as stress responses, defense mechanisms, metal carrying, and signalling metabolites, plant growth stimulants, bioherbicides bio insecticides, antimicrobial agents, antiparasitic agents, and enzyme inhibitors, and many others.
  • the present invention provides a process of Nitrogen fixation that involves the conversion of nitrogen gas into essential amino acids such as ammonia, glutamate and glutamine. Once glutamate is produced, biosynthesis of amino acids such as leucine, phenylalanine and valine biosynthesis can begin. The process continues as transaminases catalyze the transfer of the amine from glutamate to synthesize other amino acids such as serine, aspartate, alanine, isoleucine, and tyrosine.
  • the ammonia oxidising bacteria particularly belongs to genus Nitrospira, and capable of oxidising Ammonia to Nitrogen products such as and not limited to Nitrate, Hydroxylamine and Nitrite.
  • the Ammonia oxidation is achieved using enzymes including but not limited to Ammonia monooxygenase, Hydroxylamine oxidoreductase, Nitrite oxidoreductase, and the like.
  • the amino acids include but not limited to proteinogenic and non-proteinogenic amino acids / their isomers/ their derivatives (including unnatural amino acids, amino acid based and amino conjugated compounds), such as Phenylalanine, Valine, Tryptophan, Threonine, Isoleucine, Methionine, Histidine, Leucine, Serine, Proline, Glutamine, Glutamic acid, para-nitro-L- phenylalanine (pN-Phe), hydroxyproline (Hyp), beta-alanine, citrulline (Cit), ornithine (Om), norleucine (Nle), 3-nitrotyrosine, nitroarginine, pyroglutamic acid (Pyr), Amino Benzoic acid and derivatives, a-hydroxy/ a-thio aminoacids, N- formyl -L-a-aminoacids like N-formyl Methionine, N-formyl Phenylalaine, Pipe
  • the present invention provides modified or unmodified proteins which can be recombinant (including synthetic proteins) and/ or native proteins which are derived and/ or fibrous and/ or globular proteins, such as and not limited Glyco/lipoproteins (like toxin protein), nutritive proteins, therapeutic proteins (like Interferons, Interleukins, modnilin, etc.), glomalin related soil protein (GRSP), lectin, antibody, anti-freeze proteins, Phospho proteins, methyl proteins, and the like.
  • Glyco/lipoproteins like toxin protein
  • nutritive proteins like Interferons, Interleukins, modnilin, etc.
  • therapeutic proteins like Interferons, Interleukins, modnilin, etc.
  • GRSP glomalin related soil protein
  • lectin lectin
  • antibody anti-freeze proteins
  • Phospho proteins methyl proteins, and the like.
  • the present invention provides nutritive proteins belongs to plant, animal, mammal, avian, insect proteins, such as and not limited to milk proteins, whey protein, casein, egg protein like ovalbumin, egg lipo proteins like phosvitin, livetin, porcine, soy protein, leghemoglobin, and the like.
  • the present invention provides Phospho proteins including but not limited to Immunoglobin Fc receptors, ovo-vitelline, calcineurines, uro-cortines, and the like.
  • the present invention provides methyl proteins including but not limited to CUL1 -ubiquitin protein ligase complex, G3BPl-stress granule protein, Enolase, and the like.
  • the present invention provides proteins which are also enzymes including hydrolases, oxidoreductases, lyases, transferases, ligases and isomerases class of enzymes.
  • the present invention provides protein and non-protein enzymes (like Ribozymes) are involved in the synthesis of primary and secondary metabolites such as nucleotide, nucleic acids, amino acids, proteins, enzymes, lipids, carbohydrates, cofactors, antibiotics, steroids, carotenoids, terpenoids, polymers, (like Poly(amino acids) including but not limited to poly glutamic acid, poly lysine, polyarginine, etc.
  • protein and non-protein enzymes like Ribozymes
  • primary and secondary metabolites such as nucleotide, nucleic acids, amino acids, proteins, enzymes, lipids, carbohydrates, cofactors, antibiotics, steroids, carotenoids, terpenoids, polymers, (like Poly(amino acids) including but not limited to poly glutamic acid, poly lysine, polyarginine, etc.
  • poly aspartate polyamides, polyurethanes, polyacrylamide, etc.
  • vitamins alkaloids, phenolics, organic acids, flavonoids, amines, peptides, urea, guanidine, glycosides, glucosinolates, non-protein amino acids, single cell protein and the like.
  • the present invention provides vitamins including enzyme cofactors/ coenzymes, such as and not limited to vitamin B and its isoforms/ derivatives like thiamine, thiamine pyrophosphate, riboflavin, niacin, pantothenic acid, biotin, folate, vitamin B6 (pyridoxine), vitamin B12 (cobalamine), P5P, Methylcobalamin, Hydroxycobalamine, THF, Coenzyme.
  • enzyme cofactors/ coenzymes such as and not limited to vitamin B and its isoforms/ derivatives like thiamine, thiamine pyrophosphate, riboflavin, niacin, pantothenic acid, biotin, folate, vitamin B6 (pyridoxine), vitamin B12 (cobalamine), P5P, Methylcobalamin, Hydroxycobalamine, THF, Coenzyme.
  • the present invention provides cofactors including but not limited to NADH/NAD, FADH/FAD, Flavin mono nucleotide (FMN), etc.
  • the present invention provides nucleotides including and not limited to Adenine, Guanine, Cytosine, Thymine, ATP, GTP, CTP, TTP, and the like.
  • the present invention provides alkaloids and its derivatives including but not limited to isoquinoline alkaloids, indole alkaloids, steroidal alkaloids, proto alkaloids, pseudo alkaloids, sesquiterpene alkaloids including but not limited to alkaloids like streptomycin, penicillins, palmatine, dragmacidin, Neofiscalin, Huperzine A and the like.
  • the present invention provides flavonoids and its derivatives include but not limited to chaicones, flavones, isoflavones, flavanones, flavonols, anthocyanidins, anthocyanins, auronidins, and the like.
  • the present invention provides amides include but not limited to urea, guanidine, carbamides, formamide, benzamide, etc., and the production is achieved by the homologous/ heterologous expression of enzymes such as and not limited to Carbamoyl phosphate synthase, Ornithine transcarbamylase, Argino succinate synthetase, Argino succinate lyase, Arginase, and the like.
  • enzymes such as and not limited to Carbamoyl phosphate synthase, Ornithine transcarbamylase, Argino succinate synthetase, Argino succinate lyase, Arginase, and the like.
  • the present invention provides amines and its derivatives include but not limited to hydroxyl amine, glucosamine, NAG (n-acetyl glucosamine) and their polymers such as and not limited to Chitosan, chito oligosaccharides, lipo chito oligosaccharides, and the like.
  • N-glycosides include but not limited to Amygdalin, prunasin, linamarin, lotaustralin, dhurrin, and the like.
  • the present invention provides peptides include but not limited to di/tri/ tetra/ oligo peptides, linear peptide, for the purposes as enzyme inhibitor peptides, signal peptides, neurotransmitter inhibitor peptides, carrier peptides such as oxytocin, angiotensin, natriuretic peptide, and the like.
  • the present invention provides Nitrogen-containing organic acids including but not limited to uric acid, muramic acid, theacrine, tetramethyl uric acid, sialic acid, and the like.
  • the present invention provides Xanthines and its derivatives including but not limited to paraxanthine, caffeine, Methylliberine, theobromine, theophylline, and the like.
  • the present invention provides nitrate/ nitrite and its derivatives include but not limited to alkyl nitrite, amyl nitrite and the like.
  • the present invention provides Nitrogen and carbon containing compounds also includes natural or unnatural chromophores/ fluorophores such as and not limited to Luciferin, Luminol, Fluorescent proteins (such as and not limited to Green/ Red Fluorescent protein GFP/ RFP), 4', 6- diamidino-2-phenylindole (DAPI), Fluorescein isothiocyanate (FITC), hemicyanine, boron-dipyrromethene, dicyanomethylene -4H-pyran, and rhodamine derivatives, Pacific blue fluorophores, 7-hydroxy-coumarin, Fluorinated Azido-Coumarin, etc.
  • natural or unnatural chromophores/ fluorophores such as and not limited to Luciferin, Luminol, Fluorescent proteins (such as and not limited to Green/ Red Fluorescent protein GFP/ RFP), 4', 6- diamidino-2-phenylindole (DAPI), Fluorescein isothiocyanate (
  • the microbe of the present invention 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 H 2 S), synthetic/ nonsynthetic 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/
  • methods are provided for enzyme expression in microbes for enhancing production or inducing production of natural or unnatural compounds for breakdown/ denaturing/ detoxifying/ degrading toxic compounds such as and not limited to pesticides, aflatoxin, insecticides, etc.
  • the present invention provides 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.
  • the present invention provides enzymes in microbes which is also useful for enhancing production or inducing production of natural or unnatural compounds for breakdown/ denaturing/ detoxifying/ degrading toxic compounds such as and not limited to pesticides, aflatoxin, insecticides, and the like.
  • the present invention provides application of in vitro and/ or in vivo production of nitrogen and carbon containing natural or unnatural compounds are including but not limited to use in agriculture/ Agro-industries
  • non-agriculture purposes including but not limited to pharma, nutrition, environment remediation, fuel, chemicals, materials, etc.
  • the present invention provides modifications of microbes with transcriptional/translational enhancers for increasing expression of Nitrogen/Carbon fixation genes for nitrogen compound production.
  • the present invention provides overexpression of enzymes involved in Nitrogen and/or Carbon fixation by inclusion of Transcriptional and/or Translational enhancers (UNA1, UNA2, UNB, oligomers, etc.)
  • 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.
  • the present invention incorporates gene manipulation of microbes for increasing nitrogen compound production by gene editing of Nitrogen/Carbon fixation genes through CRISPR/Cas technology
  • the present invention provides homologous/ heterologous enzymes are native and/ or modified, where enzyme modifications include but are not limited to point-mutations, epigenetic mechanisms, and the like.
  • the present invention provides homologous/ heterologous enzyme/s are native and/ or modified enzymes, expressed under native and/ or modified promoter, where modification includes but not limited to pointmutations.
  • the enzymes involved in Nitrogen fixation and/ or carbon fixation and/ or Ammonia fixation and/ or production of carbon and Nitrogencontaining 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.
  • the present invention provides Nitrogen fixing organisms 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
  • 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, Epichloe typhinaEnterococcus, Corynebacterium, Arthobacter, Pichia, Zymomonas, Saccharomyces carlsbergensis, Salmonella, Zymomonas,
  • Rhodacoccus Escherichia (e.g., E. Coli), Hansenula, Firmicutes, Rubrivivax, Dinoroseobacter shibae, Methylobacterium nodularis, Methylobacterium radiotoleran, Methyloversatilis sp, Methylobacterium oryzae, Beijerinckia indica, Frankia spp., Synechocystis, Synechoccus sp., etc
  • the microbes manipulated includes 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'.
  • 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, Nictrobacter, 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, Ralst
  • ammonia utilisation microbe also include Ammonia oxidising microbes such as and not limited to Nitrosomonas spp., Nitrosococcus spp., Nitrospira spp., Nitrospina, Nitrocystis, Nitrosomonas, Nitrosolobus, Nitrobacter, Pseudomonas and the like.
  • Ammonia oxidising microbes such as and not limited to Nitrosomonas spp., Nitrosococcus spp., Nitrospira spp., Nitrospina, Nitrocystis, Nitrosomonas, Nitrosolobus, Nitrobacter, Pseudomonas and the like.
  • the present invention provides amino acids, peptides, proteins, and other nitrogen-containing compounds production which can be done in a fermenter, or within the plant as an endophyte.
  • the invention seeks to optimize the process of Nitrogen fixation by facilitating the production of essential amino acids that are required for various cellular processes.
  • the biosynthesis of amino acids from glutamate and glutamine enables the production of several amino acids that are necessary for protein synthesis and overall cellular function. This process has enormous potential in various fields such as agriculture, biotechnology, and pharmaceuticals, where these amino acids and proteins can be utilized for various purposes.
  • the present invention provides increase protein production through genetic modifications of microbes.
  • protein production can be achieved through the use of a fermenter, a container in which microorganisms are grown under controlled conditions.
  • a fermenter By genetically modifying the microbes to produce more proteins, the fermenter can be used to produce large quantities of protein for commercial use.
  • the genetic modifications of the microbes aim to increase protein production through various methods, including fermenters, soil, and endophytes. This has the potential to increase the availability of protein for various applications, including food, animal feed, and industrial uses.
  • the present invention provides a process for making the genetically modified microbe, comprising : a. Enhanced Nitrogen fixation b. Enhanced Carbon fixation c. Enhanced synthesis/ regeneration of redox energy compounds
  • the present invention provides genetically modified microbe for production of Nitrogen and Carbon containing natural or unnatural compounds, comprising modification in Nitrogenase and related genes, Formate dehydrogenase, NifA, Ammonia assimilation genes.>
  • the present invention provides composition comprising the genetically modified microbe including a single microbe or a consortium of microbes modified for production of Nitrogen-containing, non-carbon compounds.
  • Example 1 Genetic engineering of the diazotroph bacteria for increase Nitrogen fixation and flux diversion for production of Nitrogen-containing product as Protein with insecticidal function: (Cry proteins)
  • the present invention includes genetic modification of nitrogen-fixing microorganisms for increased nitrogen fixation which leads to increased production of nitrogen compounds such as proteins for example: bi ope sticide s/toxin
  • Genes that were expressed includes CrylAb, CrylF, CrylAc, Cryl 1A etc, derived from Bacillus thurigenesis.
  • the strain was Methylobacterium spp .
  • Cry gene(s) to be expressed shall be redesigned using Methylobacterium preferred codon for optimal expression.
  • High-level expression of chromosomally integrated genes in Methylobacterium spp. were achieved under the control of different promoters - constutive promoter such as CoxB (gene id 4) derived from Methylobacterium spp. and inducible promoter such as strong methanol dehydrogenase promoter (PmxaF) (gene id 3) derived from Methylobacterium spp. using the homologous recombination system. Multiple copies of Cry genes were used for increased expression of Cry protein. Constructs were cloned in vector pUC57 with overlapping gene sequence for genome integration.
  • promoters - constutive promoter such as CoxB (gene id 4) derived from Methylobacterium spp. and inducible promoter such as strong methanol dehydrogenase promoter (PmxaF) (gene id 3) derived from Methylo
  • proteases enzymes will be secreted to destroy the recombinant cry protein, so that the protein will not be there in the environment.
  • Example 2 Impact of C02 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, PEP carboxylase, Fructose bisphosphatase, carbon flux increased resulting in increased carbon uptake and absorption, as evident from Figure 1.
  • CO2 fixation pathway enzymes such as Rubisco enzyme, PEP carboxylase, Fructose bisphosphatase
  • Example 3 Enhancing CO2 fixation by Altering Rubisco enzyme, PEP carboxylase, Fructose bisphosphatase enzymes, and impact on Nitrogen compound production: -
  • the CO2 fixation pathway can refer to the metabolic pathway in bacteria which enables the uptake of Cl carbon compound CO2 into the cell.
  • Key enzymes in the carbon fixation pathway include Rubisco enzyme, PEP carboxylase, Fructose bisphosphatase, by which the carbon enters the metabolism via glycolytic steps.
  • Gene Modification in terms of upregulation of any one, two, or three 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.
  • Example 4 Microbial Urea production as an example of Nitrogen product from the modified microbe -
  • Enhancing Nitrogen fixation and carbon fixation results in increased metabolism which can be diverted to production of general production of metabolic products or targeted production of Nitrogen compounds.
  • Urea H 2 N-CO-NH 2
  • C&N compound such as Urea (H 2 N-CO-NH 2 ).
  • Urea is generally produced as nitrogenous waste compound of purine-pyrimidine breakdown and amino acid breakdown.
  • the rate limiting enzymes of the Urea synthesis cycle such as Carbamoyl phosphate synthase (CPS), Ornithine transcarbamoylase (OTC) and Arginase (ARG) were overexpressed combine CO 2 and Ammonia to result in Urea finally.
  • CPS Carbamoyl phosphate synthase
  • OTC Ornithine transcarbamoylase
  • ARG Arginase
  • Example 5 Milk protein production as an example of Nitrogen compound production from the modified microbe -
  • Enhancing Nitrogen fixation and carbon fixation results in increased metabolism which can be diverted to production of general production of metabolic products or targeted production of Nitrogen compounds.
  • the increased metabolic flux out of increase carbon and nitrogen fixation was diverted for the increased production of C&N compound such as protein like Milk protein.
  • C&N compound such as protein like Milk protein.
  • Heterologous expression of gene for milk protein such as Lactalbumin was expressed under highly expressive promoter such as Alcohol oxidase or alcohol dehydrogenase promoter.
  • Milk protein was expressed as heterologous, secretory protein from the modified organism enabled with enhanced carbon and Nitrogen fixation.
  • host was modified to have enhanced ATP, NADH regeneration to facilitate further enhanced expression, inclusion of translational and transcriptional enhancers, further more increased the level expression, as evident from Figure 3.
  • the step-wise modifications increased the protein expression level of original single copy expression from 5 g/L upto 50 g/L.
  • Example 6 Enhanced carbon fixation CO2 fixation by overexpression of formaldehyde dehydrogenase
  • Example 7 Genetic engineering of microbe Methylobacterium for enhanced greenhouse gas remediation:
  • Genetic modification of the microbe particularly Methylobacterium spp, for greenhouse gas remediation but not limited to CO2 has been done by over expression of the FDH using the strong constitutive promoter (PGAP)-
  • the gene sequences (sequence ID: 6 and 7) along with promoter and integration flanking sites (sequence ID: 8 and 9) were synthesized by gene synthesis or PCR based overlap extension method and cloned in pUC57 vector using overlap extension PCR or restriction enzyme-based method.
  • Example 7 Novel process of gene integration followed for the integration of heterologous or homologous genes:
  • sequence ID: 6 and 7 sequence ID: 8 and 9 (Glutathione dependent formate dehydrogenase) has been chosen as the site of integration.
  • Example 8 Novel process of marker-free gene integration methodology: Antibiotic markers are necessary for initial deletion construct development and transformant screening.
  • antibiotic marker but not limited to Kanamycin, tetracycline or chloramphenicol was used as a selection marker.
  • the selection marker kanamycin along with promoter was amplified from pUC57 (genscript) and deletion construct was synthesized by placing the sequence ID 8 and 9 flanking the kanamycin marker (Sequence ID: 11). The complete construct was synthesized by overlap extension PCR or gene synthesis and clone in pUC57 vector with ampicillin selection marker.
  • the overall complete construct was linearized with restriction enzyme (Xbal) and transformed into Methylobacterium spp, using electroporation method and positive transformant were selected on kanamycin containing medium and confirmation along with selection marker was performed by either colony PCR or PCR with purified genomic DNA.
  • Xbal restriction enzyme
  • Another integration plasmid which is similar to the integration plasmid, where in the marker gene is replaced with gene cassettes for expression.
  • the expression cassette plasmid gets integrated at the same gene integration site and removes the marker gene out.
  • the strain is devoid of Antibiotic marker gene in genome.
  • the marker gene which was there in initial step was replaced with gene expression cassette.
  • mxaF promoter (PmxaF) Residues: caagcctcccc gcttggtcgg gccgcttcgc gagggcccgt tgacgacaac ggtgcgatgg 60 gtcccggccc cggtcaagac gatgccaata cgtgcgaca ctacgccttg gcacttttag 120 aatgccta tcgtcctgat aagaaatgtc cgaccagcta aagacatcgc gtccaatcaa 180 agcctagaaa atataggcga agggacgcta ataagtctt cataagaccg cgcaaatcta 240 aaaatatcct tagatcacg atgcggcact tcggatgact t

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Abstract

The present invention relates to a novel method for sustainable in vitro and in vivo production of nitrogen and carbon containing compounds such as and not limited to Proteins, Enzymes, peptides amino acids, nucleotides, vitamins, etc. The present invention relates to a novel method that includes gene modifications in microbes, more specifically involving gene modifications resulting in nitrogen fixation and/or ammonia fixation or increased nitrogen fixation and/or ammonia fixation or carbon fixation/increased carbon fixation or Hydrogen fixation/increased Hydrogen fixation with increased synthesis/ regeneration of anyone or all compounds including ATP, NADH, NADPH, Pyruvate and Acetyl CoA with or without utilisation/reduction enhanced utilisation/reduction of Nitrate, Nitrite, N2O, N2O2 and/or carbon containing substrates

Description

GENETIC MODIFICATION OF MICROBES FOR PRODUCTION OF NITROGEN AND CARBON-CONTAINING COMPOUNDS
TECHNICAE FIELD OF THE INVENTION:
The present invention relates to a novel method for sustainable in vitro and in vivo production of nitrogen and 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 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
Figure imgf000003_0001
and Acetyl CoA and/or (v) production of Nitrogen and Carbon containing compounds
BACKGROUND AND PRIOR ART OF THE INVENTION:
W02017011602 discloses methods of increasing nitrogen fixation in non- leguminous plants comprising exposing the plant to a plurality of bacteria (microbiome) 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 phosphoribulokinase (prk) and Ribulose-Bisphosphate Carboxylase (RuBisCo) to improve carbon fixation. It also discloses methods that include down-regulating genes in microorganisms using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas associated protein arrays.
US 20210163374A1 discloses genetically engineered bacterial strain which is plant growth promoting bacterial strain and fixes atmospheric nitrogen in agricultural system and comprises the gene modifications in one or more genes from the group consisting of besll , beslll , yjbE , fhaB , pehA , glga , otsB , trez , and cysZ for increased nitrogen fixation and colonization of plant.
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.
Figure imgf000004_0001
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.
US20170183665A1 provides disclosure for genetically modified microorganisms utilizing recombinant carbon fixation enzymes for CO2 fixation for production of a first essential biomass precursor. The genetic modifications in the microorganism occurs in carbon fixation pathways and enzymes associated with it particularly Calvin-Benson-Bassham cycle (C3 cycle) and RuBisCO, Prk etc.
US10801045B2 discloses genetically engineered microorganisms wherein carbon fixation pathways are modified to make microorganism chemoautotrophic and efficiently utilizes inorganic carbon compounds such as formate, formic acid, methane, carbon monoxide, carbonyl sulfide, carbon disulfide, hydrogen sulfide, bisulfide anion, thiosulfate, elemental sulfur, molecular hydrogen, ferrous iron, ammonia, cyanide ion, and/or hydrocyanic acid and produce organic carbon products such as sugars, alcohols, chemicals, amino acids, polymers, fatty acids and their derivatives, hydrocarbons, isoprenoids, and intermediates etc.
US8048661B2 discloses genetically modified microorganisms encoding modified pathways for enhancing carbon flux through acetyl-CoA. The methods of modification include altering the expression of enzymes in a reductive TCA or
Figure imgf000005_0001
Wood-Ljungdahl pathway and increasing the availability of reducing equivalents in the presence of carbon monoxide or hydrogen.
US9150888B2 discloses genetically engineered photoautotrophic microorganism for conversion of carbon dioxide and light into carbon-based products of interest such as ethanol, ethylene, chemicals, polymers, n-alkanes, isoprenoids, pharmaceutical products.
US20200277636A1 discloses synthetic or genetically engineered microorganisms comprising methane, methanol utilizing pathways for conversion of Methane, methanol to organic compounds, industrial products, chemicals and intermediates. The patent provides methods for converting non-methanotrophic, non- methylotrophic microorganism into methanotrophic, methylotropic microorganisms by incorporation of methane oxidizing and methanol -oxidizing metabolic pathways.
The technical limitation with the prior art for the production of nitrogen and carbon containing 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 and nitrogen containing compounds, and also for carbon and Nitrogen assimilation.
• Substrates and intermediates for carbon fixation, nitrogen fixation, Hydrogen fixation and regeneration of redox energy compounds.
Figure imgf000006_0001
• 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 nitrogen and 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 carbon and nitrogen 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.
It is an object of the present invention to provide genetic modifications of microbes for in-vivo and in-vitro production of nitrogen and carbon containing primary and secondary metabolites including but not limited to, amino acids, proteins, enzymes, nucleotides, nitrogen-containing vitamins like Vitamin-B and derivatives, Urea, Xanthines, N-glycosides, glucosinolates, non-protein amino acids, single cell protein, Nitrogen-containing organic acids, etc. (whose functions include but not limited to metal-carrying metabolites, signalling metabolites, plant
Figure imgf000007_0001
growth stimulants, bioherbicides, bioinsecticides, antimicrobial agents, antiparasitic agents, enzyme inhibitors, etc.)
SUMMARY OF THE INVENTION:
The present invention relates to a novel method for sustainable in vitro and in vivo production of nitrogen and carbon containing compounds.
The present invention relates to a novel method that includes gene modifications in microbes, more specifically involving gene modifications resulting in nitrogen fixation and/or ammonia fixation or increased nitrogen fixation and/or ammonia fixation or carbon fixation/increased carbon fixation or Hydrogen fixation/increased Hydrogen fixation with increased synthesis/ regeneration of anyone or all compounds including ATP, NADH, NADPH, 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 Nitrogen and 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) Hydrogen fixation and/ or (v) synthesis/regeneration of anyone or all compounds including ATP, NADH, NADPH, Pyruvate and Acetyl CoA
Description of Figures:
Figure 1: Depicts CO2 consumption profile of the engineered microbe, modified with enhanced formate dehydrogenase expression.
Figure 2: Depicts Urea production by engineered microbes, modified with establishment of urea synthesis genes for expression of rate-limiting enzymes such as Ornithine trans-carbamylase, carbamyl phosphate synthase and Arginase.
Figure imgf000008_0001
Urea production is achieved by diverting Nitrogen flux from amino acid synthesis pathway.
Figure 3: Depicts enhanced Protein production by engineered microbe, modified for Nitrogen flux towards amino acids over production as well as increased carbon and redox flux.
Figure 4: Depicts impact of different carbon metabolism of Cl compounds on increased carbon metabolism.
Figure 5: 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 6: 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 7: 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
Figure imgf000009_0001
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 8: Depicts the pathway of CO2 fixation through modified CBB pathway. Calvin-Benson-Bassham (CBB) pathway involves cyclic movement of carbon between the sugar molecule by fixing the atmospheric carbon dioxide with the help of phosphoribulo kinase (PRK) and Ribulose bisphosphate carboxylase and oxygenase (RuBisCO). The generated 3 carbon GA3P directed towards glycolysis and sugar phosphate recycling. Our invention related to the expression of heterologous Phosphoribulokinase (PRK) and compact RuBisCO for the CO2 assimilation.
Figure 9: 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 10: Depicts the novel, inventive process of Nitrogenase metal specificity by promoter exchange. Modification and replacement of Anf Promoter with NifH promoter to increase electron delivery to Fe-nitrogenase, pathways activated by NifA. Incorporation of promoter such as nifH promoter (nifHDK operon) in place of anf promoter (of anfHDGK operon), shifts the specificity to Fe (Iron) irrespective of presence or absence of Molybdenum. Promoter exchange also impacts on enhanced Nitrogenase activity.
Figure 11: Depicts the pathway of Ammonia generation and fixation in amino acid metabolism. Nitrogen fixation in the recombinant strain over expressing native/ heterologous nitrogenase and inactivating glnA. GOGAT: Glutamine oxoglutarate aminotransferase, Gin: glutamine, Glu: glutamate, OG: Oxoglutarate
Figure imgf000010_0001
Figure 12: Depicts the Limitations in Nitrogen & carbon-containing compounds production methods as per the prior art methods
Figure 13: Depicts the novel, Inventive solution with novel methods to overcome limitations in Nitrogen & carbon-containing compounds production by gene manipulations of microbe for efficient carbon and nitrogen fixation, as well as for enhanced redox energy generation and regenerations.
Figure 14: Depicts gives an overall description about the modifications performed to increase the Ammonia and related energy supply and regeneration of metabolites, with respect to carbon assimilation.
Figure 15: Depicts the Modified Nitrogenase gene construct for genome integration. Gene integration construct of Nitrogenase includes expression of Nitrogenase under NifH promoter, cloned with flanking sequences of Fdm gene partial sequences to facilitate integration with the Fdm gene in genome, resulting in simultaneous deletion of Fmn gene and integration of Nitrogenase under modified promoter.
DETAILED DESCRPITION OF THE INVENTION
The present invention relates to a novel method for sustainable in vitro and in vivo production of nitrogen and 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 and nitrogen 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
Figure imgf000011_0001
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 Nitrogen & Carbon containing natural or unnatural 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 Nitrogen & Carbon containing natural or unnatural compounds.
The present invention is focused on achieving specific objectives by utilizing genetic modifications of microbes to enhance their ability to fix nitrogen. This process results in the production of a range of nitrogen-containing primary metabolites, including amino acids, proteins, enzymes, nucleotides, vitamins, nitrogen-containing organic acids, and other similar compounds. These primary metabolites play a crucial role in supporting various cellular processes such as growth, repair, and maintenance.
Nitrogen fixation and energy requirements:
Figure imgf000012_0001
One of the embodiment of the present invention focus on enhanced Nitrogen fixation and ammonia production. The excess requirement of Redox energy compounds for Nitrogen fixation including ATP, NADH and electrons/hydrogen is supported by carbon assimilation/ enhanced carbon assimilation.
Another embodiment of the current invention provides genetic modifications in microorganisms for enhancing carbon flux through increased uptake/ assimilation of carbon compounds including but not limited to CO2, methane, methanol, formaldehyde, sugars like monosaccharides, disaccharides, or organic acids, which in turn increases the NADH, ATP and electron/hydrogen generation, which is sufficient for Nitrogen fixation. Therefore by carbon fixation/ enhancing carbon fixation and/ or assimilation, nitrogen fixation can also be increased.
The current invention developed a novel process of integrating the carbon fixation and nitrogen fixation in an interdependent, mutually controlled system, thereby resulting in enhanced carbon and nitrogen fixation and impacting on product formation, which is explained in the following embodiments.
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.
Nitrogenase and related genes:
The objective of the present invention is to enhance the process of nitrogen fixation by modifying the Nitrogenase enzyme and related controlling genes for enhanced production of nitrogen and carbon-containing compounds.
In yet another embodiment, the present invention provides increased nitrogen fixation in microbe(s) for the production of primary and secondary nitrogen
Figure imgf000013_0001
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, mf 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 mf cluster genes such as mfABCDEG
Ammonia fixation/ utilisation:
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
Figure imgf000014_0001
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.
Carbon fixation:
In one embodiment, the present invention details the methods of assimilation of various carbon compounds including but not limited to C 1 compounds like CO2, Methane, Methanol, formaldehyde, formate, etc., and organic acids, sugars, and other carbohydrates.
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 Cl compounds such as Methanol, Methane, Formaldehyde, etc.
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. 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,
Figure imgf000015_0001
and is an important part of the reductive acetyl-CoA pathway and Pentose phosphate pathway.
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 the details on modification of PEP carboxylase (phosphoenolpyruvate carboxylase) involvemnet in carbon fixation, specifically in the Reductive TCA cycle. PEP carboxylase catalyzes the conversion of phosphoenolpyruvate (PEP) to oxaloacetate, a key step in the Reductive TCA cycle. The present invention aims to genetically modify or over express PEP carboxylase to increase its activity and enhance the efficiency of the Reductive TCA cycle.
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.
Figure imgf000016_0001
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.
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 (HCOf). The present invention focuses on increasing the expression or improving the catalytic activity of Carbonic Anhydrase.
Figure imgf000017_0001
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.
In another embodiment, the present invention provides increase in the availability of CO2, by increasing the carbon assimilation pathway-related genes such as RuBisCO, CCMs, and carbonic anhydrase, which results in an increase in the energy supply required in the form of ATP and NADH in the nitrogen fixation for various nitrogenases in producing NH3 + which can be utilized/diverted further in Nitrogen and carbon-containing compounds production.
In another embodiment, the present invention provides process for heterologous genetic modifications for carbon fixation in non-carbon fixing microorganisms wherein genes for carbon fixation such as Rubisco, Phosphohexoisomerase, Hexose phosphate synthase, Methane Monooxygenase etc. are taken from methanotrophs/methylotrophs but not limited to Galionellci spp., Methylomicrobium spp. etc.
Figure imgf000018_0001
In another embodiment, the present invention provides genetic control of Nitrogen/ Carbon assimilation by homologous and/ or heterologous modification of carbon uptake transporters such as PtsG, PtsN or PtsF or ManZ or LacY and carbonic anhydrase.
Regeneration of Hydrogen, Phosphate, ATP, NADH, NADPH:
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, Hue 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.
Figure imgf000019_0001
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 Nitrogen and Carbon containing natural or unnatural compounds is achieved with enhanced regeneration/ recycling of ATP/ ADP/ phosphate and/ or NADH/ NADPH, with or without enhanced hydrogen uptake/ fixation, wherein the enzymes for NADH regeneration includes and not limited to all Dehydrogenases and oxidoreductases, Other enzymes for synthesis/ regeneration of NADH includes but not limited to NADH oxidase, ADP-ribosyl cyclase, SARM-1, NAD hydrolases, mono (ADP-ribosyl)transferases, Nicotinic acid phosphor ribosyl transferase. NADHNADPH 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 .
Figure imgf000020_0001
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 Nitrogen and Carbon containing natural or unnatural compounds is achieved by expression of Homologous/ heterologous, native/ modified enzymes responsible for increased pyruvate availability include but not limited to (1) enzymes involved in increasing pyruvate synthesis towards downstream product formation pathways and (2) enzymes involved in preventing pyruvate loss in form of CO2, wherein said enzymes for increasing pyruvate include and not limited to Pyruvate synthase, Pyruvate kinase, Pyruvate decarboxylase, carrier proteins like mitochondrial pyruvate carrier proteins etc., wherein such enzymes are overexpressed and/ or deleted/ downregulated.
Nitrogen and carbon containing compounds
The present invention pertains to an embodiment for production of nitrogen and carbon containing compounds including but not limited to, amino acids, proteins, enzymes, nucleotides, nitrogen-containing vitamins like Vitamin-B and derivatives, amines, amides, etc. (whose functions include but not limited to metal-carrying metabolites, signalling metabolites, plant growth stimulants, bioherbicides, bioinsecticides, antimicrobial agents, antiparasitic agents, enzyme inhibitors, etc.)
Figure imgf000021_0001
In another embodiment, the present invention provides increasing the production of nitrogen-containing compounds such as proteins in microorganisms that can fix nitrogen and carbon dioxide. Proteins are essential molecules that perform a wide range of functions in living organisms, including acting as enzymes, structural components, and signaling molecules.
The present invention provides methods for microbe modification for production of enzymes including pectinases, cellulases, phytases, lipases, and other types of enzymes such as hydrolases, oxidoreductases, lyases, transferases, ligases, and isomerases. Pectinases are enzymes that break down pectin, a component of plant cell walls, and are used in various industries such as food, textiles, and paper production. Cellulases are enzymes that break down cellulose, the main component of plant cell walls, and are used in the production of biofuels and animal feed. Phytases are enzymes that break down phytic acid, a component of plant seeds that can reduce the bioavailability of certain minerals, and are used in animal feed to improve mineral absorption. Lipases are enzymes that break down fats and are used in the production of various products such as detergents, cosmetics, and pharmaceuticals.
In another embodiment, the present invention provides methods for increasing the production of nitrogen-containing compounds such as peptides in microorganisms that can fix nitrogen and carbon dioxide. Peptides are short chains of amino acids that can perform various functions in living organisms, including acting as hormones, enzymes, and signalling molecules.
In another embodiment, the present invention provides methods for increase in nitrogen-containing secondary metabolites such as non-ribosomal peptides. Non- ribosomal peptides are a type of peptide that are not synthesized by ribosomes, but rather by multi-enzyme complexes known as non-ribosomal peptide synthetases (NRPS) which includes but not limited to Antibiotics, Siderophores, Toxins, Nitrogen storage polymers.
Figure imgf000022_0001
One major embodiment of the invention, details the list of Nitrogen and carbon containing secondary metabolites for varied purposes, such as and not limited to flavonoids, such as flavonoids and its derivatives include but not limited to chaicones, flavones, isoflavones, flavanones, flavanols, anthocyanidins, anthocyanins, auronidins, etc., for use in flavoring agents, cosmetics etc.; alkaloids like isoquinoline alkaloids, indole alkaloids, steroidal alkaloids, proto alkaloids, pseudo alkaloids, including but not limited to alkaloids like streptomycin, penicillins, palmatine, dragmacidin, Neofiscalin, etc., for use in antimicrobial purposes, signal transduction purposes; Amides like urea, guanidine, formamide, benzamide, etc., Amines like glucosamine, NAG (n-acetyl glucosamine), hydroxyl amine, etc.; N-gly coside like Amygdalin, prunasin, linamarin, lotaustralin, dhurrin, which are used as drug or precursors; peptides like di/tri/ tetra/ oligo peptides, linear peptide, to be used as enzyme inhibitor peptides, signal peptides, neurotransmitter inhibitor peptides, carrier peptides such as oxytocin, angiotensin, kutznerides, natriuretic peptide, etc.; nitrogen containing organic acids like uric acid, muramic acid, theacrine, tetramethyl uric acid, sialic acid, etc; xanthines and derivatives like to paraxanthine, caffeine, Methyl xanthine, theobromine, theophylline, etc, which are used in beverages.
In another embodiment, microbe modification for nutrient acquisition is targeted. Microbes can be enabled to assimilate varied nutrients under in vivo and/ or in vitro applications including but not limited involved in food processing, nutrient dense food preparation, functional food additives such as proteases, pectinases, invertase, amylase, etc.; enzymes to extract bio-active components, probiotics, probiotics; enzymes involved in catabolic degradation/ utilization such as and not limited to cellulase, exo-glucanase, endo-glucanase, cellobiohydrolase, xylanase, Pectin esterase, lignin peroxidase, protease; enzymes for anabolism and metabolite conversion such as and not limited to sugar isomerases, phytase, invertase; enzymes such as Nitrate reductase, Nitrite reductase, sulfate adenylyl transferase (Sat), adenylyl sulfate reductase (AprBA), and dissimilatory sulfite reductase (DsrAB), ATP sulfurylase (ATPS), Adenosine 5 '-phosphosulfate
Figure imgf000023_0001
reductase (APSR), APS kinase (APSK) involved in metabolic assimilation of toxic NOx/SOx gases such as and not limited to H2S, NO2/ NO3, N2O,
In another embodiment, the present invention provides modifications of microbes with ribozymes involved in gene regulatory circuits of the nitrogen fixation and or carbon fixation pathways for the controlled expression for the production of nitrogen and carbon-containing compounds.
In another embodiment, the Nitrogen & Carbon containing natural or unnatural compounds include but not limited to amino acids, proteins, enzymes, cofactors, nucleotides, vitamins, flavonoids, alkaloids, peptides, proteins, amides, amines, Urea, Xanthines, N-glycosides, glucosinolates, non-protein amino acids, single cell protein, Nitrogen-containing organic acids, and the like.
In another embodiment, the Nitrogen & Carbon containing natural or unnatural compounds are produced in vivo and in vitro.
In an embodiment, the present invention provides methods of gene modifications in carbon fixation to enhance nitrogen fixation in microbes including epiphyte or endophyte or rhizosphere or free-living or ruminant/ non-ruminant gut microbe for in-vitro and in-vivo production of Nitrogen & Carbon containing primary and secondary metabolites.
In another embodiment, the present invention provides process for gene modifications of microbes including epiphyte or endophyte or rhizosphere or free- living or ruminant/ non-ruminant gut microbe genetically modified for nitrogen fixation with/without carbon fixation for in-vitro and in-vivo production of Nitrogen & Carbon containing primary and secondary metabolites.
In another embodiment, the present invention provides genetically modify non- diazotrophs with nitrogen fixation genes with/without carbon fixation for in-vitro and in-vivo production of Nitrogen & Carbon containing primary and secondary metabolites.
Figure imgf000024_0001
In yet another embodiment, the present invention provides in addition to primary metabolites, the modified microbes also produce a variety of secondary metabolites. These metabolites serve multiple purposes such as stress responses, defense mechanisms, metal carrying, and signalling metabolites, plant growth stimulants, bioherbicides bio insecticides, antimicrobial agents, antiparasitic agents, and enzyme inhibitors, and many others.
In another embodiment, the present invention provides a process of Nitrogen fixation that involves the conversion of nitrogen gas into essential amino acids such as ammonia, glutamate and glutamine. Once glutamate is produced, biosynthesis of amino acids such as leucine, phenylalanine and valine biosynthesis can begin. The process continues as transaminases catalyze the transfer of the amine from glutamate to synthesize other amino acids such as serine, aspartate, alanine, isoleucine, and tyrosine.
In another embodiment, the ammonia oxidising bacteria particularly belongs to genus Nitrospira, and capable of oxidising Ammonia to Nitrogen products such as and not limited to Nitrate, Hydroxylamine and Nitrite.
In another embodiment, the Ammonia oxidation is achieved using enzymes including but not limited to Ammonia monooxygenase, Hydroxylamine oxidoreductase, Nitrite oxidoreductase, and the like.
In another embodiment, the amino acids include but not limited to proteinogenic and non-proteinogenic amino acids / their isomers/ their derivatives (including unnatural amino acids, amino acid based and amino conjugated compounds), such as Phenylalanine, Valine, Tryptophan, Threonine, Isoleucine, Methionine, Histidine, Leucine, Serine, Proline, Glutamine, Glutamic acid, para-nitro-L- phenylalanine (pN-Phe), hydroxyproline (Hyp), beta-alanine, citrulline (Cit), ornithine (Om), norleucine (Nle), 3-nitrotyrosine, nitroarginine, pyroglutamic acid (Pyr), Amino Benzoic acid and derivatives, a-hydroxy/ a-thio aminoacids, N-
Figure imgf000025_0001
formyl -L-a-aminoacids like N-formyl Methionine, N-formyl Phenylalaine, Piperazinic acid (a-hydrazino acid), serotonin, psilocybin, amino acid-piperine conjugates, piperoyl-amino acid conjugates lik piperoyl-L-valine, piperoyl-L- methionine, piperoyl-L-tryptophan, poly-R-(L-glutamic acid) conjugates of camptothecin, Z-L-lysine-quinine, Z-L-aspartic acid-quinine, Z-L-cysteine- quinine, quercetin-alanine, quercetin-valine, quercetin-lysine, icaritin-L-histidine, icaritin-L-arginine, icaritin-L-lysine, curcumin-amino acid conjugates, Tetrahydrocurcumin-L-glycine, tetrahydrocurcumin-L-valine and the like.
In another embodiment, the present invention provides modified or unmodified proteins which can be recombinant (including synthetic proteins) and/ or native proteins which are derived and/ or fibrous and/ or globular proteins, such as and not limited Glyco/lipoproteins (like toxin protein), nutritive proteins, therapeutic proteins (like Interferons, Interleukins, modnilin, etc.), glomalin related soil protein (GRSP), lectin, antibody, anti-freeze proteins, Phospho proteins, methyl proteins, and the like.
In another embodiment, the present invention provides nutritive proteins belongs to plant, animal, mammal, avian, insect proteins, such as and not limited to milk proteins, whey protein, casein, egg protein like ovalbumin, egg lipo proteins like phosvitin, livetin, porcine, soy protein, leghemoglobin, and the like.
In another embodiment, the present invention provides Phospho proteins including but not limited to Immunoglobin Fc receptors, ovo-vitelline, calcineurines, uro-cortines, and the like.
In another embodiment, the present invention provides methyl proteins including but not limited to CUL1 -ubiquitin protein ligase complex, G3BPl-stress granule protein, Enolase, and the like.
Figure imgf000026_0001
In another embodiment, the present invention provides proteins which are also enzymes including hydrolases, oxidoreductases, lyases, transferases, ligases and isomerases class of enzymes.
In another embodiment, the present invention provides protein and non-protein enzymes (like Ribozymes) are involved in the synthesis of primary and secondary metabolites such as nucleotide, nucleic acids, amino acids, proteins, enzymes, lipids, carbohydrates, cofactors, antibiotics, steroids, carotenoids, terpenoids, polymers, (like Poly(amino acids) including but not limited to poly glutamic acid, poly lysine, polyarginine, etc. poly aspartate, polyamides, polyurethanes, polyacrylamide, etc.) vitamins, alkaloids, phenolics, organic acids, flavonoids, amines, peptides, urea, guanidine, glycosides, glucosinolates, non-protein amino acids, single cell protein and the like.
In another embodiment, the present invention provides vitamins including enzyme cofactors/ coenzymes, such as and not limited to vitamin B and its isoforms/ derivatives like thiamine, thiamine pyrophosphate, riboflavin, niacin, pantothenic acid, biotin, folate, vitamin B6 (pyridoxine), vitamin B12 (cobalamine), P5P, Methylcobalamin, Hydroxycobalamine, THF, Coenzyme.
In another embodiment, the present invention provides cofactors including but not limited to NADH/NAD, FADH/FAD, Flavin mono nucleotide (FMN), etc.
In another embodiment, the present invention provides nucleotides including and not limited to Adenine, Guanine, Cytosine, Thymine, ATP, GTP, CTP, TTP, and the like.
In another embodiment, the present invention provides alkaloids and its derivatives including but not limited to isoquinoline alkaloids, indole alkaloids, steroidal alkaloids, proto alkaloids, pseudo alkaloids, sesquiterpene alkaloids including but not limited to alkaloids like
Figure imgf000027_0001
streptomycin, penicillins, palmatine, dragmacidin, Neofiscalin, Huperzine A and the like.
In another embodiment, the present invention provides flavonoids and its derivatives include but not limited to chaicones, flavones, isoflavones, flavanones, flavonols, anthocyanidins, anthocyanins, auronidins, and the like.
In another embodiment, the present invention provides amides include but not limited to urea, guanidine, carbamides, formamide, benzamide, etc., and the production is achieved by the homologous/ heterologous expression of enzymes such as and not limited to Carbamoyl phosphate synthase, Ornithine transcarbamylase, Argino succinate synthetase, Argino succinate lyase, Arginase, and the like.
In another embodiment, the present invention provides amines and its derivatives include but not limited to hydroxyl amine, glucosamine, NAG (n-acetyl glucosamine) and their polymers such as and not limited to Chitosan, chito oligosaccharides, lipo chito oligosaccharides, and the like.
In another embodiment, the present invention provides N-glycosides include but not limited to Amygdalin, prunasin, linamarin, lotaustralin, dhurrin, and the like.
In another embodiment, the present invention provides peptides include but not limited to di/tri/ tetra/ oligo peptides, linear peptide, for the purposes as enzyme inhibitor peptides, signal peptides, neurotransmitter inhibitor peptides, carrier peptides such as oxytocin, angiotensin, natriuretic peptide, and the like.
In another embodiment, the present invention provides Nitrogen-containing organic acids including but not limited to uric acid, muramic acid, theacrine, tetramethyl uric acid, sialic acid, and the like.
Figure imgf000028_0001
In another embodiment, the present invention provides Xanthines and its derivatives including but not limited to paraxanthine, caffeine, Methylliberine, theobromine, theophylline, and the like.
In another embodiment, the present invention provides nitrate/ nitrite and its derivatives include but not limited to alkyl nitrite, amyl nitrite and the like.
In another embodiment, the present invention provides Nitrogen and carbon containing compounds also includes natural or unnatural chromophores/ fluorophores such as and not limited to Luciferin, Luminol, Fluorescent proteins (such as and not limited to Green/ Red Fluorescent protein GFP/ RFP), 4', 6- diamidino-2-phenylindole (DAPI), Fluorescein isothiocyanate (FITC), hemicyanine, boron-dipyrromethene, dicyanomethylene -4H-pyran, and rhodamine derivatives, Pacific blue fluorophores, 7-hydroxy-coumarin, Fluorinated Azido-Coumarin, etc.
In another embodiment, the microbe of the present invention can be modified for breakdown/ utilization/ enhanced utilization of varied substrates such as and not limited to organic and/ or inorganic substrates in any form including gaseous/ liquid/ solid and combination thereof, including but not limited to carbohydrates (including but not limited to monosaccharides/ disaccharides/ oligosaccharides/ polysaccharides, etc.), organic acids, proteins, lipids, pectin, inorganic & organic phosphates/ phosphites (including phytic acid), nitrate/ nitrite/ Nitrous oxide (N2O), Trimethyl Amine (TMA), sulfate/ sulfides (including H2S), synthetic/ nonsynthetic compounds including but not limited to plastics/ fibres/ rubber/ resins/ chemicals, etc. (like polyethylene terepthalate (PET), Ethylene glycol (EG), polyurethane), under in vivo and/ or in vitro applications by homologous and/ or heterologous expression of native or modified enzymes including but not limited to cellulase, exo-glucanase, endo-glucanase, cellobiohydrolase, xylanase, pectinases, pectin methyl esterase, pectin acetyl esterase, lignin peroxidase, proteases, peptidases, Endopeptidase, exopeptidases, asparaginyl endopeptidases,
Figure imgf000029_0001
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 sulfiirylase (ATPS), Adenosine 5'- phosphosulfate reductase (APSR), APS kinase (APSK), Terephthalate 1,2- dioxygenase, Terephthalate reductase, Dihydrodiol dehydrogenase, Glycolate oxidase, Glyoxylate carboligase, hydroxy pyruvate isomerase, protocatechuate decarboxylase, Feruloyl esterase, TMADH-trimethylamine dehydrogenase; DMADH-dimethylamine dehydrogenase; MMADH-monomethylamine dehydrogenase, Thioredoxin reductase, Thioredoxin, dihydro folate reductase and the like.
In another embodiment, methods are provided for enzyme expression in microbes for enhancing production or inducing production of natural or unnatural compounds for breakdown/ denaturing/ detoxifying/ degrading toxic compounds such as and not limited to pesticides, aflatoxin, insecticides, etc.
In another embodiment, the present invention provides 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 another embodiment, the present invention provides enzymes in microbes which is also useful for enhancing production or inducing production of natural or unnatural compounds for breakdown/ denaturing/ detoxifying/ degrading toxic compounds such as and not limited to pesticides, aflatoxin, insecticides, and the like.
In another embodiment, the present invention provides application of in vitro and/ or in vivo production of nitrogen and carbon containing natural or unnatural compounds are including but not limited to use in agriculture/ Agro-industries
Figure imgf000030_0001
(including but not limited to animal husbandry, aquaculture, sericulture, apiculture, etc.) and non-agriculture purposes (including but not limited to pharma, nutrition, environment remediation, fuel, chemicals, materials, etc).
Gene editing/modifications
In another embodiment, the present invention provides modifications of microbes with transcriptional/translational enhancers for increasing expression of Nitrogen/Carbon fixation genes for nitrogen compound production.
In another embodiment, the present invention provides overexpression of enzymes involved in Nitrogen and/or Carbon fixation by inclusion of Transcriptional and/or Translational enhancers (UNA1, UNA2, UNB, oligomers, etc.)
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 a further embodiment, the present invention incorporates gene manipulation of microbes for increasing nitrogen compound production by gene editing of Nitrogen/Carbon fixation genes through CRISPR/Cas technology
In another embodiment, the present invention provides homologous/ heterologous enzymes are native and/ or modified, where enzyme modifications include but are not limited to point-mutations, epigenetic mechanisms, and the like.
In another embodiment, the present invention provides homologous/ heterologous enzyme/s are native and/ or modified enzymes, expressed under native and/ or
Figure imgf000031_0001
modified promoter, where modification includes but not limited to pointmutations.
In another embodiment, the enzymes involved in Nitrogen fixation and/ or carbon fixation and/ or Ammonia fixation and/ or production of carbon and Nitrogencontaining 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.
In another embodiment, the present invention provides Nitrogen fixing organisms 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, Epichloe typhinaEnterococcus, Corynebacterium, Arthobacter, Pichia, Zymomonas, Saccharomyces carlsbergensis, Salmonella, Zymomonas,
Figure imgf000032_0001
Rhodacoccus, Escherichia (e.g., E. Coli), Hansenula, Firmicutes, Rubrivivax, Dinoroseobacter shibae, Methylobacterium nodularis, Methylobacterium radiotoleran, Methyloversatilis sp, Methylobacterium oryzae, Beijerinckia indica, Frankia spp., Synechocystis, Synechoccus sp., etc
In another embodiment, the microbes manipulated includes 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, Nictrobacter, 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, Epichloe typhina, Rhodococcus, Xanthobacter spp.; non-diazotrophs such as E.coli, Bacillus, Lactobacillus, yeasts like Saccharomyces and Pichia, Archaea bacteria and the like.
In another embodiment, the ammonia utilisation microbe also include Ammonia oxidising microbes such as and not limited to Nitrosomonas spp., Nitrosococcus spp., Nitrospira spp., Nitrospina, Nitrocystis, Nitrosomonas, Nitrosolobus, Nitrobacter, Pseudomonas and the like.
In yet another embodiment, the present invention provides amino acids, peptides, proteins, and other nitrogen-containing compounds production which can be done in a fermenter, or within the plant as an endophyte.
Figure imgf000033_0001
The invention seeks to optimize the process of Nitrogen fixation by facilitating the production of essential amino acids that are required for various cellular processes. The biosynthesis of amino acids from glutamate and glutamine enables the production of several amino acids that are necessary for protein synthesis and overall cellular function. This process has enormous potential in various fields such as agriculture, biotechnology, and pharmaceuticals, where these amino acids and proteins can be utilized for various purposes.
In yet another embodiment, the present invention provides increase protein production through genetic modifications of microbes.
In another embodiment, protein production can be achieved through the use of a fermenter, a container in which microorganisms are grown under controlled conditions. By genetically modifying the microbes to produce more proteins, the fermenter can be used to produce large quantities of protein for commercial use.
The genetic modifications of the microbes aim to increase protein production through various methods, including fermenters, soil, and endophytes. This has the potential to increase the availability of protein for various applications, including food, animal feed, and industrial uses.
In another embodiment, the present invention provides a process for making the genetically modified microbe, comprising : a. Enhanced Nitrogen fixation b. Enhanced Carbon fixation c. Enhanced synthesis/ regeneration of redox energy compounds
In yet another embodiment, the present invention provides genetically modified microbe for production of Nitrogen and Carbon containing natural or unnatural compounds, comprising modification in Nitrogenase and related genes, Formate dehydrogenase, NifA, Ammonia assimilation genes.>
Figure imgf000034_0001
In another embodiment, the present invention provides composition comprising the genetically modified microbe including a single microbe or a consortium of microbes modified for production of Nitrogen-containing, non-carbon compounds.
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 : Genetic engineering of the diazotroph bacteria for increase Nitrogen fixation and flux diversion for production of Nitrogen-containing product as Protein with insecticidal function: (Cry proteins)
The present invention includes genetic modification of nitrogen-fixing microorganisms for increased nitrogen fixation which leads to increased production of nitrogen compounds such as proteins for example: bi ope sticide s/toxin
Genes that were expressed includes CrylAb, CrylF, CrylAc, Cryl 1A etc, derived from Bacillus thurigenesis. The gene sequence coding for the activated Cry toxin (around 65 kDa) The strain was Methylobacterium spp . Cry gene(s) to be expressed shall be redesigned using Methylobacterium preferred codon for optimal expression.
High-level expression of chromosomally integrated genes in Methylobacterium spp. were achieved under the control of different promoters - constutive promoter such as CoxB (gene id 4) derived from Methylobacterium spp. and inducible promoter such as strong methanol dehydrogenase promoter (PmxaF) (gene id 3)
Figure imgf000035_0001
derived from Methylobacterium spp. using the homologous recombination system. Multiple copies of Cry genes were used for increased expression of Cry protein. Constructs were cloned in vector pUC57 with overlapping gene sequence for genome integration.
Example: Crylab (gene id 2) with coxB promoter (SEQ ID NO4) with overlapping integration gene sequence i.e. left overlap region - 490 nucleotide sequence from UreA ( SEQ ID NO 5) and right overlap region -500 nucleotide sequence from UreC gene ( SEQ ID NO 6) was synthesized using gene synthesis approach and cloned in pUC57 vector ( from Genescript) using Ndel and Hindlll sites. The final construct consisted of overlapping right and left arm with gene cassette ( SEQ ID NO 7). BamHI and Bglll restriction sites were included at start and end of gene cassette to allow multiple copy cloning of gene cassette for multi-fold expression of Cry protein production.
1. In order to increase the nitrogen fixation in Methylobacterium, aminoacid assimilation pathway is blocked, so the ammonia will be getting accumulated and made avaialable to crops. Accumulation of ammonia in the cytoplasm does not allow the nitrogen content of the Methylobacterium to be used for protein synthesis and leads to excretion or removal of ammonia outside the cell. However, if the ammonia accumulation in the cytoplasm is blocked then the ammonia/nitrogen content can be channelized for protein production. The ammonia/nitrogen will be used as ingredients for amino acid production which will be used as building blocks for the heterologous protein expression as well as synthesis of housekeeping enzymes and other enzymes/structural proteins needed for cell survival.
2. At the end of the life cycle of the microbes, proteases enzymes will be secreted to destroy the recombinant cry protein, so that the protein will not be there in the environment.
Figure imgf000036_0001
Example 2: Impact of C02 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, PEP carboxylase, Fructose bisphosphatase, carbon flux increased resulting in increased carbon uptake and absorption, as evident from Figure 1.
Example 3: Enhancing CO2 fixation by Altering Rubisco enzyme, PEP carboxylase, Fructose bisphosphatase enzymes, and impact on Nitrogen compound production: -
The CO2 fixation pathway can refer to the metabolic pathway in bacteria which enables the uptake of Cl carbon compound CO2 into the cell. Key enzymes in the carbon fixation pathway include Rubisco enzyme, PEP carboxylase, Fructose bisphosphatase, by which the carbon enters the metabolism via glycolytic steps. Gene Modification in terms of upregulation of any one, two, or three 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, PEP carboxylase, or Fructose bisphosphatase. 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.
Genetic modifications were performed in these cells such that one or more genes in the carbon fixation pathway upregulated. A library of variants was constructed such that promoter activity for one or more genes in the carbon fixation pathway is increased. Due to the increase in carbon fixation ability, positive impact was
Figure imgf000037_0001
observed on increased production of Nitrogen compound, such as Urea in this example, as evident from figure 2.
Example 4: Microbial Urea production as an example of Nitrogen product from the modified microbe -
Enhancing Nitrogen fixation and carbon fixation results in increased metabolism which can be diverted to production of general production of metabolic products or targeted production of Nitrogen compounds.
In current invention, the increased metabolic flux out of increase carbon and nitrogen fixation was diverted for the increased production of C&N compound such as Urea (H2N-CO-NH2). Urea is generally produced as nitrogenous waste compound of purine-pyrimidine breakdown and amino acid breakdown. In current invention, we focused on urea synthesis by incorporating the genes and completing the Urea cycle pathway, starting from Carbamoyl phosphate synthase. The rate limiting enzymes of the Urea synthesis cycle such as Carbamoyl phosphate synthase (CPS), Ornithine transcarbamoylase (OTC) and Arginase (ARG) were overexpressed combine CO2 and Ammonia to result in Urea finally.
Example 5: Milk protein production as an example of Nitrogen compound production from the modified microbe -
Enhancing Nitrogen fixation and carbon fixation results in increased metabolism which can be diverted to production of general production of metabolic products or targeted production of Nitrogen compounds.
In current invention, the increased metabolic flux out of increase carbon and nitrogen fixation was diverted for the increased production of C&N compound such as protein like Milk protein. Heterologous expression of gene for milk protein such as Lactalbumin was expressed under highly expressive promoter such as Alcohol oxidase or alcohol dehydrogenase promoter. Milk protein was expressed as heterologous, secretory protein from the modified organism enabled with enhanced carbon and Nitrogen fixation. In addition, host was modified to have enhanced ATP, NADH regeneration to facilitate further enhanced
Figure imgf000038_0001
expression, inclusion of translational and transcriptional enhancers, further more increased the level expression, as evident from Figure 3. Overall, the step-wise modifications increased the protein expression level of original single copy expression from 5 g/L upto 50 g/L.
Example 6: Enhanced carbon fixation CO2 fixation by overexpression of formaldehyde dehydrogenase
The homologous expression or over expression of FDH (gene id: 6 and 7) in Methylobacterium spp. under the control of glyceraldehyde 3-phosphate dehydrogenase promoter (PGAP, sequence id: 5). Naturally, this promoter drives the expression of glyceraldehyde-3-phosphate dehydrogenase gene constitutively. The product of this gene catalyzes an important energy-yielding step in carbohydrate metabolism, the reversible oxidative phosphorylation of glyceraldehyde-3-phosphate in the presence of inorganic phosphate and nicotinamide adenine dinucleotide (NAD).
Example 7: Genetic engineering of microbe Methylobacterium for enhanced greenhouse gas remediation:
Genetic modification of the microbe particularly Methylobacterium spp, for greenhouse gas remediation but not limited to CO2 has been done by over expression of the FDH using the strong constitutive promoter (PGAP)- The gene sequences (sequence ID: 6 and 7) along with promoter and integration flanking sites (sequence ID: 8 and 9) were synthesized by gene synthesis or PCR based overlap extension method and cloned in pUC57 vector using overlap extension PCR or restriction enzyme-based method. Final integration construct (Sequence ID: 10) was transformed into Methylobacterium spp, using the standard electroporation procedure (1800 V, 25 pF, 200Q) and screened for presence of integrated construct in the chromosome by PCR based confirmation.
Example 7: Novel process of gene integration followed for the integration of heterologous or homologous genes:
Figure imgf000039_0001
Integration of homologous and heterologous genes require certain gene or genes to be deleted, for generation of space for integration. For the integration of FDH (Sequence ID: 6 and 7), sequence ID: 8 and 9 (Glutathione dependent formate dehydrogenase) has been chosen as the site of integration.
Example 8: Novel process of marker-free gene integration methodology: Antibiotic markers are necessary for initial deletion construct development and transformant screening. In this present invention, antibiotic marker but not limited to Kanamycin, tetracycline or chloramphenicol was used as a selection marker. The selection marker kanamycin along with promoter was amplified from pUC57 (genscript) and deletion construct was synthesized by placing the sequence ID 8 and 9 flanking the kanamycin marker (Sequence ID: 11). The complete construct was synthesized by overlap extension PCR or gene synthesis and clone in pUC57 vector with ampicillin selection marker. The overall complete construct was linearized with restriction enzyme (Xbal) and transformed into Methylobacterium spp, using electroporation method and positive transformant were selected on kanamycin containing medium and confirmation along with selection marker was performed by either colony PCR or PCR with purified genomic DNA. In the gene deletion site with the marker gene, another integration plasmid which is similar to the integration plasmid, where in the marker gene is replaced with gene cassettes for expression. Upon linearization and transformation of this plasmid, the expression cassette plasmid gets integrated at the same gene integration site and removes the marker gene out. Finally, the strain is devoid of Antibiotic marker gene in genome. The marker gene which was there in initial step was replaced with gene expression cassette.
Figure imgf000040_0001
SEQUENCE LISTING
Sequence Listing Information:
DTD Version: Vl_3
File Name: Sequence listing_4228.xml
Software Name: WIPO Sequence
Software Version: 2.3.0
Production Date: 2023-11-07
General Information:
Current application / IP Office: IN
Current application / Application number: 202241064228
Current application / Filing date: 2022-11-10
Current application / Applicant file reference: 4228
Earliest priority application / IP Office: IN
Earliest priority application / Application number: 202241064228
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 NITROGEN AND CARBON-CONTAINING
COMPOUNDS ( en )
Sequence Total Quantity: 11
Sequences:
Sequence Number (ID): 1
Length: 888
Molecule Type: DNA
Features Location/Qualifiers:
- source, 1..888
> mol_type, genomic DNA
> organism, Nitrogenase
Residues:
Figure imgf000041_0001
atgggcaaac tccgtcagat cgccttctac ggcaaaggtg gtatcggcaa gtcgaccacc 60 tcgcagaaca ccctcgccgc gctggtcgag atgggtcaga agatcctcat cgtcggctgc 120 gaccccaagg ctgacagcac ccgtctgatc ctgaacacca agctgcagga caccgtgctg 180 cacctggccg ccgaggccgg ttcggtcgaa gatctggaag tcgaagacgt cgtgaaaatc 240 ggctacaagg gcatcaaatg caccgaagcc ggcggtccgg agccgggggt tggctgcgcc 300 ggccgtggcg tcatcaccgc gatcaactc ctgaagaaa acggcgccta tgacgatgtg 360 gactatgtgt cctatgacgt tctgggcgac gtggtctgcg gcggcttcgc catgccgatc 420 cgtgaaaaca aggcgcagga aatctacatc gtcatgtcgg gcgagatgat ggcgctttac 480 gccgccaaca acatcgccaa gggcatcctg aaatatgcga actcgggcgg cgtgcgtctg 540 ggcgggctga tctgcaacga acgcaagacc gaccgcgagc tggaactggc cgaagcgctg 600 gccgccaagc tgggctgcaa gatgatccac tcgtgccgc gcaacaacgt cgtgcaacat 660 gccgaactgc gccgcgaaac cgtgatccaa tacgatccga cctgcagcca ggcgcaggaa 720 taccgcgaac tggcccgcaa gatccacgag aactcgggca agggcgtcat cccgaccccg 780 atcacgatgg aagagctgga agagatgctg atggatttcg gcatcatgca atcggaagaa 840 gatcgcgaaa agcagatcgc cgagatggaa gccgcgatga aggcctga 888
Sequence Number (ID): 2 Length: 3468
Molecule Type: DNA
Features Location/Qualifiers:
- source, 1..3468
> mol_type, genomic DNA
> organism, Insecticidal protein Cry 1 Ab codon optimized for Methylobacterium spp.
Residues: atggacaaca acccgaacat caacgaatgc atcccctaca actgcctgtc caacccggaa 60 gtggaagtcc tggggggcga acgcatcgaa accggctata ccccgatcga catctcgctg 120 tccctgacgc agttcctgct gagcgaattc gtccccggcg ccggcttcgt cctgggcctg 180 gtcgatatca tctggggcat cttcggcccc tcgcagtggg atgccttcct ggtgcagatc 240 gagcagctga tcaaccagcg catcgaggag ttcgcccgga accaggccat ctcgcgcctc 300 gagggcctct ccaacctgta ccagatttac gcggagtcgt tccgcgagtg ggaggccgat 360
Figure imgf000042_0001
cccaccaacc ccgcgctgcg cgaagagatg cgcatccagt tcaacgacat gaatcggcc 420 ctcaccaccg ccatccccct cttcgccgtg cagaactacc aggtgcccct gctctcggtc 480 tacgtccagg ccgccaacct ccatctgccg gtcctccggg acgtctcggt gtcggccag 540 cgctggggct tcgacgcggc caccatcaac tcgcgctaca acgacctgac ccggctcatc 600 ggcaactaca ccgatcacgc cgtccggtgg tacaacaccg gcctggagcg cgtgtggggc 660 ccggactcgc gggactggat tcgctataac cagtccgcc gcgagctcac cctcaccgtc 720 ctggatatcg tgagcctctt cccgaactac gattcccgca cgtacccgat ccgcaccgtc 780 tcgcagctca cccgggagat tatacgaac ccggtcctcg agaacttcga tggctcgtc 840 cggggctcgg cgcagggcat cgaggggagc atccgctcgc cgcacctcat ggatatcctc 900 aactccatca ccatctacac cgacgcgcat cggggcgagt actactggtc gggccaccag 960 atcatggcgt cgccggtggg gtcagcggc cccgaattca cgttcccgct gtacggcacc 1020 atgggcaacg cggccccgca gcagcgcatc gtcgcccagc tgggccaggg cgtctaccgc 1080 accctcagca gcaccctcta ccgccggccc ttcaacatcg gcatcaacaa ccagcagctc 1140 tcggtgctcg atggcaccga attcgcgtac gggaccagct cgaacctgcc cagcgccgtg 1200 tatcgcaagt cggggaccgt ggactcgctc gatgagatcc cgccccagaa caacaacgtg 1260 ccgccccggc agggcttctc ccaccgcctg tcccacgtct cgatgtccg ctcgggcttc 1320 tccaatcgt ccgtcagcat catccgcgcc ccgatgtca gctggattca ccggtccgcg 1380 gaatcaaca acatcatccc gtccagccag atcacccaga tccccctgac gaagtcgacc 1440 aacctcggct cgggcacgtc cgtcgtcaag ggcccgggct tcacgggcgg cgacatcctc 1500 cgccgcacct cccccggcca gattcgacc ctgcgcgtga atatcaccgc gcccctctcg 1560 cagcgctacc gcgtccgcat ccggtacgcg agcaccacca acctgcagtt ccacacctcg 1620 atcgacggcc gccccatcaa ccagggcaac ttcagcgcca ccatgtcctc gggctccaac 1680 ctccagagcg gctcgtccg caccgtcggc ttcaccacgc ccttcaact ctccaacggc 1740 tcctcggtgt tcaccctctc ggcccacgtc ttcaactcgg gcaacgaggt gtacatcgac 1800 cggatcgagt tcgtcccggc cgaggtgacg ttcgaggccg agtacgatct cgagcgcgcg 1860 cagaaggccg tgaatgagct gtcaccagc tcgaaccaga tcggcctgaa gacggacgtg 1920 accgactacc acatcgacca ggtctccaac ctggtcgagt gcctctcgga cgagttctgc 1980 ctcgacgaga agaaggaact cagcgagaag gtgaagcacg ccaagcgcct ctcggacgaa 2040 cgcaacctgc tgcaggaccc caacttccgc ggcatcaatc gccagctgga ccgcggctgg 2100 cgcggctcga cggacatcac gatccagggc ggggacgacg tcttcaagga gaactacgtg 2160 accctgctgg gcaccttcga cgagtgctac ccgacctacc tctaccagaa gatcgacgag 2220
Figure imgf000043_0001
tcgaagctca aggcgtacac gcgctaccag ctccgcggct acatcgagga cagccaggac 2280 atcgagattt acctgatccg ctacaacgcg aagcatgaga ccgtgaacgt gccgggcacc 2340 gggagcctgt ggcccctgtc cgccccctcg cccatcggca agtgcgcgca ccatcccac 2400 catttctcgc tcgacatcga cgtcggctgc acggatctca acgaggacct cggcgtgtgg 2460 gtgatcttca agatcaagac gcaggacggc cacgcccggc tggggaacct cgagttcctg 2520 gaggagaagc ccctcgtggg cgaggcgctg gcgcgcgtga agcgcgccga gaagaagtgg 2580 cgcggcaagc gcgagaagct cgagtgggag accaacatcg tctacaagga ggcgaaggag 2640 tcggtcgacg cgctctcgt gaactcgcag tacgatcgcc tccaggccga caccaacatc 2700 gccatgatcc acgcggcgga caagcgcgtc catccatcc gcgaggcgta tctgccggag 2760 ctgtcggtca tcccgggcgt caacgcggcg atcttcgagg agctcgaggg ccggatcttc 2820 accgccttct ccctgtacga cgcgcgcaac gtcatcaaga acggggactt caacaacggc 2880 ctcagctgct ggaacgtcaa gggccacgtc gacgtggagg agcagaacaa ccaccggtcc 2940 gtcctggtgg tgcccgaatg ggaggccgaa gtctcgcagg aggtgcgcgt gtgcccgggc 3000 cggggctaca tcctccgcgt gacggcgtac aaggagggct acggcgaggg ctgcgtgacc 3060 atccatgaga tcgagaacaa cacggatgag ctgaagtca gcaactgcgt cgaggaggaa 3120 gtgtacccga ataacacggt gacgtgcaac gactacaccg ccacgcagga ggagtacgag 3180 ggcacctaca cctcgcgcaa ccggggctat gacggcgcgt acgagtcgaa cagctcggtg 3240 ccggcggatt acgcgtcggc gtatgaggag aaggcctaca cggacgggcg ccgcgataac 3300 ccgtgcgaaa gcaaccgggg ctacggcgac tacacgccgc tcccggccgg ctacgtcacc 3360 aaggagctcg agtactccc ggagaccgac aaggtgtgga tcgagatcgg cgagaccgaa 3420 ggcaccttca tcgtggacag cgtggagctg ctgctgatgg aggagtga 3468
Sequence Number (ID): 3
Length: 400
Molecule Type: DNA
Features Location/Qualifiers:
- source, 1..400
> mol_type, genomic DNA
> organism, mxaF promoter (PmxaF) Residues: caagcctccc gcttggtcgg gccgcttcgc gagggcccgt tgacgacaac ggtgcgatgg 60
Figure imgf000044_0001
gtcccggccc cggtcaagac gatgccaata cgtgcgaca ctacgccttg gcacttttag 120 aatgccta tcgtcctgat aagaaatgtc cgaccagcta aagacatcgc gtccaatcaa 180 agcctagaaa atataggcga agggacgcta ataagtctt cataagaccg cgcaaatcta 240 aaaatatcct tagatcacg atgcggcact tcggatgact tccgagcgag cctggaacct 300 cagaaaaacg tctgagagat accgcgaggc cgaaaggcga ggcggttcag cgaggagacg 360 caggatgagc aggtttgtga catcagtctc ggccttggcg 400
Sequence Number (ID): 4 Length: 250
Molecule Type: DNA
Features Location/Qualifiers:
- source, 1..250
> mol_type, genomic DNA
> organism, coxB promoter (PcoxB)
Residues: cgatcacgcc cgcggggccg cgacgttgcg ccgccggacc gtcgctttcc tgccgcttgc 60 aggccgagaa agcgcgccat gcgacgcttg tacggttgcg ccttgcacgt ccatgtgatc 120 tgacaccgcg atcggaaagc cctggcccga cggccggcct tccgcggggt cggccgcgtc 180 agaaagccca cgtcgatggg tcgcggcgcg gtcagaataa cagtctccaa ggggagcagg 240 cgagagacac 250
Sequence Number (ID): 5 Length: 203
Molecule Type: DNA
Features Location/Qualifiers:
- source, 1..203
> mol_type, genomic DNA
> organism, glyceraldehyde 3 -phosphate dehydrogenase promoter (Pgap)
Residues: tccgcggatc ggttgatccc ggcggcgacg gcgcggccgg tccgccatgg gtcatgtccg 60
Figure imgf000045_0001
gctccggttc atcgccggtt cagcgccggc agccacagag caatccgcat cgcggaggtg 120 ccgtcgggcc cccgccgcgc accgctcgcc gcctcggacg cccgctgcgt ggcgcccctt 180 aagcaggaag gaaacacgcc atg 203
Sequence Number (ID): 6
Length: 2856
Molecule Type: DNA
Features Location/Qualifiers:
- source, 1..2856
> mol_type, genomic DNA
> organism, Formare Dehydrogenase 1
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
Figure imgf000046_0001
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 agtcgagtc ctgggcccgc 1500 ttcatcgcgg aggagcgcca ctcccccgag aaccagcagc agttcaccgg cctcgatccc 1560 gaacaggtgc gcggcgcggc gcggctctac gccacgggcg gcgcggccgg catctatac 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 tgtcctgaa cgagacggca 2040 aaatacgccc acgtctcct gcccggagcc tcattcctgg agaaggacgg cacctcacc 2100 aatgccgagc gccgcatcag ccgcgtgcgc aaggtcatgc ccccgatggg cggctacggc 2160 gattgggagg gcacggtgct gctctctaac gcgctgggct acccgatgaa ctacagccac 2220 ccatccgaga tcatggacga gatcgcggcc ctcaccccga gctcaccgg 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 cccacaatc gcgctggcac 2520 gaggaggacg tgctggagat ccacccctc 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): 7
Figure imgf000047_0001
Length: 2973
Molecule Type: DNA
Features Location/Qualifiers:
- source, 1..2973
> mol_type, genomic DNA
> organism, Formate dehydrogenase II
Residues: atgaacgacg gccccgatct ccacggcaag gcgacggacc ggaccgaggt ccgggcgcgg 60 acgcgccagg atgcgggcgg cgccgctccg gaggggcggc cgggcgcggg cggcccctat 120 tcgcagggcg ccaaggccgg tggccaggcc tcgcccgagc cgagcgggct tgtcggcctg 180 acggagcggc ccgcagcgcc gccgagcatc gcgttcgagc tcgacggcga gacggtcgag 240 gcgcggccgg gcgagaccat ctgggcggtc gccaagcgcc tcggcaccca catcccgcat 300 ctctgccaca agccggagcc cggctaccgg ccggacggca attgccgcgc ctgcatggtc 360 gagatcgagg gcgagcgcgt gctcgcggcc tcctgcaagc gcacgcccgc catcggcatg 420 aaggtgaaga ccgccaccga gcgcgcggag aaggcccgcg ccatggtgat ggaattgctg 480 gtggccgacc agccggaccg ggcgacttcg cacgatccga cctcgcattt ctgggcgcag 540 gccgatttcg tggacatcgc cgcgagccgc tttcccgcgg ccgagcgctg gcaggccgac 600 gcgagccatc cggccatgcg ggtgaacctc gatgcctgca tccagtgcaa tctctgcgtc 660 cgcgcctgcc gcgaggtcca ggtcaacgac gtgatcggca tggcctaccg ctcggccggg 720 tccaaggtgg tgttcgactt cgacgacccg atgggcggct cgacctgcgt cgcctgcggc 780 gagtgcgtgc aggcctgtcc gaccggggcg ctgatgccct cggcctatct cgacgcgaac 840 gagacccggg tcgtctatcc cgaccgtgag gtcgcctcgc tctgccccta ttgcggtgtc 900 ggctgccagg tctcctacaa ggtcaaggac gagcgcatcg tctatgccga gggcctgaac 960 ggcccggcca accacaaccg gctctgcgtg aagggccgct tcggcttcga ctacgtgcac 1020 catccccacc ggctgaccaa gcccctgatc cggctcgaca acgccccgaa ggacgcgaac 1080 gaccaggtcg atcccgccaa cccctggacg catttccgcg aggccacctg ggaggaggcc 1140 ctcgaccgcg ccgcggccgg gctgcggacg gtccgcgaca gccacggccc caaggcgctc 1200 gccggcttcg gctcggccaa gggctcgaac gaggaggcct atctcttcca gaagctggtc 1260 cgcctcggct tcggctccaa caacgtcgac cattgcaccc ggctctgcca cgcctcctct 1320 gtggcggccc tgatggaggg gctgaactcg ggcgccgtga ccgcgccctt ctcggcggcg 1380 ctcgatgccg aggtgatcat cgtcatcggg gccaacccca ccgtgaacca cccggtcgcg 1440
Figure imgf000048_0001
gcgacctcc tcaagaatgc ggtgaagcag cgcggcgcca agctgatcgt catggatccg 1500 cgccggcagg tgctgtcccg gcacgcctac aggcacctcg cctcaagcc gggctcggac 1560 gtggcgatgc tgaacgcgat gctgaacgtc atcatcgagg agaagctcta cgacgagcag 1620 tacatcgccg ggtacaccga gaacttcgag gcgctgcggc agaagatcgt cgacttcacg 1680 cccgagaaga tggaggccgt ctgcggcatc gaggccgcga ccctgcgcga ggtcgcgcgc 1740 ctctacgccc ggtcgaaggc ctcgatcatc ttctggggca tgggtatcag ccagcacgtc 1800 cacggcaccg acaactcgcg ctgcctgatc gccctggccc tcgtcaccgg ccagatcgga 1860 cggccgggca cggggctgca ccccctgcgc ggccagaaca acgtgcaggg cgcctccgat 1920 gcgggcctga tcccgatggt ctatccggac taccagtccg tcgagaaggc ggcggtgcgc 1980 gagctgttcg aggcgttctg gggccagtcc ctcgatccga agcgcgggct gaccgtggtc 2040 gagatcatgc gggcgatcca tgccggcgag atccgcggca tgtcatcga gggcgagaac 2100 ccggccatgt cggatcccga cctcaaccac gcccggcacg cgctggcgat gctcgaccat 2160 ctcgtcgtgc aggacctgtt cctcaccgag acggccttcc acgccgacgt ggtgctgccg 2220 gcctccgcct tcgccgagaa ggcgggcagc ttcaccaaca cggaccggcg cgtccagatc 2280 gcccagcccg tcgtgccgcc cccgggcgac gcgcgccagg atggtggat catccaggaa 2340 ctcgcccggc ggatggggct cgactggagc tatgccggcc cggccgacgt gttcgccgag 2400 atggcgcagg tcatgccctc gctcgccaac atcacctggg agcgcctgga gcgcgagggc 2460 gccgtgacct acccggtcga cgcgcccgac aagccgggca acgagatcat ctctacgac 2520 ggctcccga ccgagagcgg gcgcgccaag atcgtgccgg cggcgatcgt gcccccggac 2580 gaggtgcccg acaccgagtt cccgatggtg ctctcgaccg gccgggtgct ggagcattgg 2640 catacgggct cgatgacccg gcgcgccggc gtgctcgacg cgctggagcc cgaggcggtg 2700 gcctcctgg ccccgcgcga gctctaccgc ctcggcctcg agcccggcat gacgatgcgg 2760 ctcgagacgc ggcgcggcgc cgtcgaggtg aaggtccggt ccgaccgcga cgttccggac 2820 ggcatggtgt tcatgccctt ctgctacgcg gaggccgcgg ccaacctcct caccaccccc 2880 gccctcgatc cgctgggcct gatccccgag ttcaagtct gcgcggcccg ggtctcgccc 2940 gtccgggccg cgccgccgat cgccgccgag tga 2973
Sequence Number (ID): 8
Length: 521
Molecule Type: DNA
Features Location/Qualifiers:
Figure imgf000049_0001
- source, 1..521
> mol_type, genomic DNA
> organism, Glutathione dependent fomate dehydrogenase upstream flanking region
Residues: cacgggctcg aggtcacggc ccccttcggc gccgcgctcg ccgccgccct ggtggccgag 60 cgcggactca gcgaggtcgc ggtcaccgcg atcggccatc gccgcggcga gggcgtgctg 120 cgcgtcctgc cggcctagga gcagcggcgg gcgggcccga ccgccctccc ggacggcgtc 180 gagaccagag agtttctccg aactcttact ctgaagaccg gttctcccct cgagcgccgg 240 accccgcacg gggtccgcgc cccttcagtc ccgaggagag cgccgatgcg tgcactggtg 300 tggcacggaa cccaggacgt ccggtgcgac tcggttcctg atccggagat cgagcacgag 360 cgcgacgcca tcatcaaggt cacgagttgc gccatctgcg gctcggacct gcacctgttc 420 gaccatttca tacccacgat gaagtcgggc gacatcctcg gccacgagac catgggcgag 480 gtggtcgagg tgggctcggc ggccaagtcc aagctcaagg t 521
Sequence Number (ID): 9
Length: 616
Molecule Type: DNA
Features Location/Qualifiers:
- source, 1..616
> mol_type, genomic DNA
> organism, Glutathione dependent formate dehydrogenase downstream flanking region
Residues: tcaacttcga gaccgacagc gtgatcgagc gcctgaacgc gatgaccgcg ggcaagggcc 60 ccgagaaatg catcgacgcg gtcgggctcg aggctcacgc cgccggcacc gtcgatgcga 120 tgtacgaccg cgccaagcag gcgatgatgc tggagaccga ccggccgcat gtcctgcgcg 180 agatgatcta tgtctgccgg cccgccggca cgctctcggt gcccggcgtc tatggcggcc 240 tcatcgacaa gatcccgttc ggcgcgctga tgaacaaggg cctgacgatc cgcacgggcc 300 agacccacgt caatcgctgg agcgacgacc tgctgcggcg gategaggag ggteagateg 360 atccctcctt cgtgatcacc cataccgagc cgctggagcg cgggcccgag atgtacaaga 420
Figure imgf000050_0001
cctccgcga caagcaggac ggctgcatca aggtcgtgct caagccctga ctccacccgt 480 tcccctctc agaggaggtg ccgtcatggg ccagcacaat cccaggaacg tcctgccgcg 540 gaccgcgctg cgcgggcgct cgcaatccgt cgccgaccgc gtcgcgcagg ggctcgggct 600 ctctcgatc ggcctc 616
Sequence Number (ID): 10
Length: 7852
Molecule Type: DNA
Features Location/Qualifiers:
- source, 1..7852
> mol_type, genomic DNA
> organism, Complete integration construct
Residues: cacgggctcg aggtcacggc ccccttcggc gccgcgctcg ccgccgccct ggtggccgag 60 cgcggactca gcgaggtcgc ggtcaccgcg atcggccatc gccgcggcga gggcgtgctg 120 cgcgtcctgc cggcctagga gcagcggcgg gcgggcccga ccgccctccc ggacggcgtc 180 gagaccagag agtttctccg aactcttact ctgaagaccg gttctcccct cgagcgccgg 240 accccgcacg gggtccgcgc cccttcagtc ccgaggagag cgccgatgcg tgcactggtg 300 tggcacggaa cccaggacgt ccggtgcgac tcggttcctg atccggagat cgagcacgag 360 cgcgacgcca tcatcaaggt cacgagttgc gccatctgcg gctcggacct gcacctgttc 420 gaccatttca tacccacgat gaagtcgggc gacatcctcg gccacgagac catgggcgag 480 gtggtcgagg tgggctcggc ggccaagtcc aagctcaagg ttccgcggat cggttgatcc 540 cggcggcgac ggcgcggccg gtccgccatg ggtcatgtcc ggctccggtt catcgccggt 600 tcagcgccgg cagccacaga gcaatccgca tcgcggaggt gccgtcgggc ccccgccgcg 660 caccgctcgc cgcctcggac gcccgctgcg tggcgcccct taagcaggaa ggaaacacgc 720 catggccctc atcaaggaaa tcgactacgg cacgccgatc cgcgtcgccg agcagacggt 780 gtcgctgacc atcgacggca tggccgtgac ggtgccggcc ggcacctccg tgatggccgc 840 ggcgatgacc gcgggcacgc agatccccaa gctctgcgcc accgactcgc tggagccctt 900 cggctcctgc cgcctctgcc tcgtggagat cgagggacgg cgcggcacgc ccgcctcctg 960 caccacgccg gccgagaacg gcatggtggt gcacacgcag accgacaagc tcgcgcgcct 1020 gcgcaagggc gtgatggagc tctacatctc cgatcacccg ctcgactgcc tgacctgcgc 1080
Figure imgf000051_0001
ggcgaacggc gattgcgagc tgcagacgca ggcgggcgtc gtcggcctgc gcgacgtgcg 1140 ctacggctac gagggcgaca accacgtccg cccgagctcc gagcgctacc tgccgaagga 1200 cgagtcgaac ccgtatttca cctacgaccc gtcgaagtgc atcgtctgca atcgctgcgt 1260 gcgggcctgc gaggaggtgc agggcacctt cgcgctgacc atcgccggcc gcggctcga 1320 cagccgcgtc gccgccggcc cgacgaact catggaatcc gagtgcgtct cgtgcggcgc 1380 ctgcgtgcag gcctgcccga ccgcgacgct ccaggagaag tcgatccacg aatacggcca 1440 gccggagcac gccgaggtca cgacctgcgc ctattgcggc gtcggctgct cctcaaggc 1500 cgagatgcag ggcgaccgcg tcgtgcgcat ggtgccctac aagggcggca aggcgaatga 1560 cggccatagc tgcgtgaagg gccgcttcgc ctacggctac gccactcaca aggaccgcat 1620 caccaagccg atgatccggg agaagatcac ggatccgtgg cgcgaggtca cctgggagga 1680 ggcgatcgac cgggcggcct ccgagttcaa gcggatccag gccacctacg gcaaggattc 1740 ggtcggcggc atcacctcgt cccgctgcac caacgaggag gcctacctcg tccagaagct 1800 ggtgcgcgcg gccttcggca acaacaacgt cgatacctgc gcccgcgtct gccactcgcc 1860 gaccggctac ggcctgatgt cgacgctcgg cacctcggcc ggcacccagg acttcgcctc 1920 ggtggcgcat tccgacgtga tcctcgtcat cggcgccaac ccgacggacg gccatccggt 1980 cttcggctcg cgcatgaaga agcgcctgcg cgagggggcg aagctcatcg tcgccgatcc 2040 gcgcaagatc gacctcgtga agtcgcccca catcaaggcg gacttccacc tgcccctgaa 2100 gcccggctcc aacgtcgcct tcatcaactc gatcgcgcac gtcatcgtca cggaagggct 2160 gatcgacgag gcctatatcc gcgcgcgctg cgacctcggc gagttcgagt cctgggcccg 2220 cttcatcgcg gaggagcgcc actcccccga gaaccagcag cagttcaccg gcctcgatcc 2280 cgaacaggtg cgcggcgcgg cgcggctcta cgccacgggc ggcgcggccg gcatctatta 2340 cgggctgggc gtcaccgagc acagccaggg ctcgaccatg gtgatgggca tggccaacat 2400 cgccatggcc accggcaaca tcggcaagct cggtgcgggc gtaaacccct tgcgcggcca 2460 gaacaacgtg caaggatcct gcgacatggg ctcgttcccc cacgagctca ccggctaccg 2520 ccacgtctcg gacgatgcca cccgcgagag cttcgaggcg atctggggtg ccaagctcga 2580 caacgcgcca ggacttcgca tcaccaacat gctcgatgag gccgtcgatg gcagcttcaa 2640 gggcatgtac atccagggcg aggacatcgc gcagtccgat cccgacaccc atcacgtcac 2700 gtcaggcctc aaggcgatgg aatgcatcgt cgtgcaggac ctgttcctga acgagacggc 2760 aaaatacgcc cacgtcttcc tgcccggagc ctcattcctg gagaaggacg gcaccttcac 2820 caatgccgag cgccgcatca gccgcgtgcg caaggtcatg cccccgatgg gcggctacgg 2880 cgattgggag ggcacggtgc tgctctctaa cgcgctgggc tacccgatga actacagcca 2940
Figure imgf000052_0001
cccatccgag atcatggacg agatcgcggc cctcaccccg agcttcaccg gggtgtccta 3000 tgccaaactc gaggaactcg gctcggtaca gtggccctgc aatgagaagg cgccgctcgg 3060 tacgccgatg atgcacgtgg accgctcgt gcgcggcaag ggccggtca tgatcaccga 3120 gttcgtggcg actgaggagc gcacgggggc gaagtcccg ctcatcctca ccacgggtcg 3180 gatcctctcc cagtacaacg tcggcgctca gacccggcgc acccacaatt cgcgctggca 3240 cgaggaggac gtgctggaga tccacccctt cgacgcggag ctgcgcggta tcatggacgg 3300 cgacctcgtc gccctggaga gccgctcggg cgacatcgct ctgaaggcca agatttcgga 3360 gcgcatgcag ccaggcgtgg tctacaccac cttccaccac gctaagaccg gcgccaacgt 3420 catcaccacc gactatcgg actgggccac gaactgcccc gagtacaaag tgacggcggt 3480 gcaggtccgg cgtaccaacc ggccctccga ctggcaggcg aagtctacg agggagattt 3540 ctccctgacc cggatcgccc aggccgcggc ggagtgagaa cccataaaat gtgatcgtcc 3600 gcggatcggt tgatcccggc ggcgacggcg cggccggtcc gccatgggtc atgtccggct 3660 ccggtcatc gccggttcag cgccggcagc cacagagcaa tccgcatcgc ggaggtgccg 3720 tcgggccccc gccgcgcacc gctcgccgcc tcggacgccc gctgcgtggc gccccttaag 3780 caggaaggaa acacgccatg aacgacggcc ccgatctcca cggcaaggcg acggaccgga 3840 ccgaggtccg ggcgcggacg cgccaggatg cgggcggcgc cgctccggag gggcggccgg 3900 gcgcgggcgg cccctatcg cagggcgcca aggccggtgg ccaggcctcg cccgagccga 3960 gcgggcttgt cggcctgacg gagcggcccg cagcgccgcc gagcatcgcg ttcgagctcg 4020 acggcgagac ggtcgaggcg cggccgggcg agaccatctg ggcggtcgcc aagcgcctcg 4080 gcacccacat cccgcatctc tgccacaagc cggagcccgg ctaccggccg gacggcaatt 4140 gccgcgcctg catggtcgag atcgagggcg agcgcgtgct cgcggcctcc tgcaagcgca 4200 cgcccgccat cggcatgaag gtgaagaccg ccaccgagcg cgcggagaag gcccgcgcca 4260 tggtgatgga attgctggtg gccgaccagc cggaccgggc gactcgcac gatccgacct 4320 cgcatttctg ggcgcaggcc gatttcgtgg acatcgccgc gagccgcttt cccgcggccg 4380 agcgctggca ggccgacgcg agccatccgg ccatgcgggt gaacctcgat gcctgcatcc 4440 agtgcaatct ctgcgtccgc gcctgccgcg aggtccaggt caacgacgtg atcggcatgg 4500 cctaccgctc ggccgggtcc aaggtggtgt tcgactcga cgacccgatg ggcggctcga 4560 cctgcgtcgc ctgcggcgag tgcgtgcagg cctgtccgac cggggcgctg atgccctcgg 4620 cctatctcga cgcgaacgag acccgggtcg tctatcccga ccgtgaggtc gcctcgctct 4680 gcccctattg cggtgtcggc tgccaggtct cctacaaggt caaggacgag cgcatcgtct 4740 atgccgaggg cctgaacggc ccggccaacc acaaccggct ctgcgtgaag ggccgcttcg 4800
Figure imgf000053_0001
gcttcgacta cgtgcaccat ccccaccggc tgaccaagcc cctgatccgg ctcgacaacg 4860 ccccgaagga cgcgaacgac caggtcgatc ccgccaaccc ctggacgcat tccgcgagg 4920 ccacctggga ggaggccctc gaccgcgccg cggccgggct gcggacggtc cgcgacagcc 4980 acggccccaa ggcgctcgcc ggcttcggct cggccaaggg ctcgaacgag gaggcctatc 5040 tcttccagaa gctggtccgc ctcggcttcg gctccaacaa cgtcgaccat tgcacccggc 5100 tctgccacgc ctcctctgtg gcggccctga tggaggggct gaactcgggc gccgtgaccg 5160 cgcccttctc ggcggcgctc gatgccgagg tgatcatcgt catcggggcc aaccccaccg 5220 tgaaccaccc ggtcgcggcg accttcctca agaatgcggt gaagcagcgc ggcgccaagc 5280 tgatcgtcat ggatccgcgc cggcaggtgc tgtcccggca cgcctacagg cacctcgcct 5340 tcaagccggg ctcggacgtg gcgatgctga acgcgatgct gaacgtcatc atcgaggaga 5400 agctctacga cgagcagtac atcgccgggt acaccgagaa cttcgaggcg ctgcggcaga 5460 agatcgtcga ctcacgccc gagaagatgg aggccgtctg cggcatcgag gccgcgaccc 5520 tgcgcgaggt cgcgcgcctc tacgcccggt cgaaggcctc gatcatctc tggggcatgg 5580 gtatcagcca gcacgtccac ggcaccgaca actcgcgctg cctgatcgcc ctggccctcg 5640 tcaccggcca gatcggacgg ccgggcacgg ggctgcaccc cctgcgcggc cagaacaacg 5700 tgcagggcgc ctccgatgcg ggcctgatcc cgatggtcta tccggactac cagtccgtcg 5760 agaaggcggc ggtgcgcgag ctgttcgagg cgtctgggg ccagtccctc gatccgaagc 5820 gcgggctgac cgtggtcgag atcatgcggg cgatccatgc cggcgagatc cgcggcatgt 5880 tcatcgaggg cgagaacccg gccatgtcgg atcccgacct caaccacgcc cggcacgcgc 5940 tggcgatgct cgaccatctc gtcgtgcagg acctgttcct caccgagacg gccttccacg 6000 ccgacgtggt gctgccggcc tccgcctcg ccgagaaggc gggcagcttc accaacacgg 6060 accggcgcgt ccagatcgcc cagcccgtcg tgccgccccc gggcgacgcg cgccaggatt 6120 ggtggatcat ccaggaactc gcccggcgga tggggctcga ctggagctat gccggcccgg 6180 ccgacgtgtt cgccgagatg gcgcaggtca tgccctcgct cgccaacatc acctgggagc 6240 gcctggagcg cgagggcgcc gtgacctacc cggtcgacgc gcccgacaag ccgggcaacg 6300 agatcatct ctacgacggc tcccgaccg agagcgggcg cgccaagatc gtgccggcgg 6360 cgatcgtgcc cccggacgag gtgcccgaca ccgagtccc gatggtgctc tcgaccggcc 6420 gggtgctgga gcattggcat acgggctcga tgacccggcg cgccggcgtg ctcgacgcgc 6480 tggagcccga ggcggtggcc tcctggccc cgcgcgagct ctaccgcctc ggcctcgagc 6540 ccggcatgac gatgcggctc gagacgcggc gcggcgccgt cgaggtgaag gtccggtccg 6600 accgcgacgt tccggacggc atggtgtca tgcccttctg ctacgcggag gccgcggcca 6660
Figure imgf000054_0001
acctcctcac cacccccgcc ctcgatccgc tgggcctgat ccccgagtc aagttctgcg 6720 cggcccgggt ctcgcccgtc cgggccgcgc cgccgatcgc cgccgagtga gagtgcgacc 6780 aatgcaagcg cggctagctt gcagtgggct tacatggcga tagctagact gggcggtttt 6840 atggacagca agcgaaccgg aattgccagc tggggcgccc tctggtaagg tgggaagcc 6900 ctgcaaagta aactggatgg ctttctgcc gccaaggatc tgatggcgca ggggatcaag 6960 atctgatcaa gagacaggat gaggatcgt tcgcatgatt gaacaagatg gatgcacgc 7020 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat 7080 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt 7140 caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg 7200 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag 7260 ggactggctg ctatgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc 7320 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc tgatccggc 7380 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga 7440 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga 7500 actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg 7560 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg 7620 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatatgc 7680 tgaagagctt ggcggcgaat gggctgaccg ctcctcgtg cttacggta tcgccgctcc 7740 cgatcgcag cgcatcgcct tctatcgcct tctgacgag tcttctgag tgctgcgcga 7800 gatgatctat gtatgccgac ccggcggcct gatctcgatt cccggcgtct ac 7852
Sequence Number (ID): 11 Length: 1082
Molecule Type: DNA
Features Location/Qualifiers:
- source, 1..1082
> mol_type, genomic DNA
> organism, Kanamycin marker sequence Residues: gagtgcgacc aatgcaagcg cggctagctt gcagtgggct tacatggcga tagctagact 60 gggcggtttt atggacagca agcgaaccgg aattgccagc tggggcgccc tctggtaagg 120
Figure imgf000055_0001
ttgggaagcc ctgcaaagta aactggatgg ctttctgcc gccaaggatc tgatggcgca 180 ggggatcaag atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg 240 gattgcacgc aggtctccg gccgcttggg tggagaggct attcggctat gactgggcac 300 aacagacaat cggctgctct gatgccgccg tgtccggct gtcagcgcag gggcgcccgg 360 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc 420 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg 480 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc 540 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc 600 ttgatccggc tacctgccca tcgaccacc aagcgaaaca tcgcatcgag cgagcacgta 660 ctcggatgga agccggtct gtcgatcagg atgatctgga cgaagagcat caggggctcg 720 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg 780 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat 840 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg tggctaccc 900 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg ctcctcgtg ctttacggta 960 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tctgacgag ttcttctgag 1020 tgctgcgcga gatgatctat gtatgccgac ccggcggcct gatctcgatt cccggcgtct 1080 ac 1082

Claims

WE CLAIM,
1. A method for genetically modifying microbes for in-vitro/in-vivo/intra cellular/extra cellular production of Nitrogen & Carbon 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 Cl 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 phosphate/ ATP/ NADH/ NADPH and/or enhanced/induced Pyruvate and Actyl 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.
Figure imgf000057_0001
The method as claimed in claim 1, wherein said genetically modified microbes belonging to endophyte, epiphyte or Rhizospheric/free living microbes including but not limited to, Enterobacter, Serratia, Azorhizobium, cyanobacteria, Gluconacetobacter, Acetobacter, Beijerinckia, Duganella, Delftia, Sinorhizobium, Bradyrhizobiun, Halomoncis , Methylobacterium spp., Methylobacterium symbioticum, Methylorubrum, Methylomonas, Bacillus, Paenibacillus, Lactobacillus, Mycoplasma, Acetabacterium, Streptomyces, Rhodococcus, Frankia sp, Microbacterium, Curtobacterium, Bradyrhizobium japonicum, Ralstonia eutropha, Epichloe typhina, Rhodococcus, Saccharomyces,Pichia, Methylobacterium spp., Methylobacterium symbioticum, Methylorubrum, Paenibacillus, Bacillus, Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Herbaspirillum, Pantoea, Serratia, Rahnella, Azospirillum, Azotobacter, Azorhizobium, Beijerinckia, Duganella, Bradyrhizobiun, Sinorhizobium, Methylomonas, Methylosarcina, Methylococcus, Nitrospira, Nitromonas, Nictrobacter, Bacillus, Paenibacillus,
Streptomyces, Rhodobacter sphaeroides, Cupriavidus necator, Rhodobacter spp, Azospirillum lipoferum, Rhodopseudomonas palustris, Flavobacterium, Xanthobacter spp, Pichia, Saccharomyces, etc. The method as claimed in claim 1, wherein the said Nitrogen & Carbon containing natural or unnatural compounds include but not limited to amino acids, proteins, enzymes, cofactors, nucleotides, vitamins, flavonoids, alkaloids, peptides, proteins, amides, amines, Urea, Xanthines, N-glycosides, glucosinolates, non-protein amino acids, single cell protein, Nitrogen-containing organic acids, etc. The method as claimed in claim 4, the said Nitrogen & Carbon containing natural or unnatural compounds are produced in vivo and in vitro The method as claimed in claim 5, the application of in vitro and/ or in vivo production of nitrogen and carbon containing natural or unnatural compounds are including but not limited to use in agriculture/ Agroindustries (including but not limited to animal husbandry, aquaculture,
Figure imgf000058_0001
sericulture, apiculture, etc.) and non-agriculture purposes (including but not limited to pharma, nutrition, environment remediation, fuel, chemicals, materials, etc). The method as claimed in claim 1, wherein enhanced production of Nitrogen & Carbon 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). 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 mf cluster genes such as mfABCDEG, etc. The method as claimed in claims 7 and 8, wherein the said Nitrogenase/s and regulatory proteins are native enzyme and/ or modified, where enzyme modifications include but are not limited to point-mutations, epigenetic mechanisms, etc. The method as claimed in claim 1, wherein the said 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 method as claimed in claim 1, wherein enhanced production of Primary and secondary Nitrogen and Carbon containing metabolites is achieved by upregulation of Ammonia utilizing enzymes including but not limited to Glutaminases, Glutamine synthase, Glutamate dehydrogenase, Ammonia transporter, and related enzymes like GDH, GlnA, GlnD, GlnE, AmtA, AmtB, etc. The method as claimed in claim 1, wherein enhanced production of Nitrogen and Carbon containing natural or unnatural compounds is
Figure imgf000059_0001
achieved with enhanced regeneration/ recycling of ATP/ ADP/ phosphate and/ or NADH/ NADPH, with or without enhanced hydrogen uptake/ fixation. The enzymes for NADH regeneration includes and not limited to all Dehydrogenases and oxidoreductases. Enzymes for ATP regeneration includes but not limited to ATP synthase, ATP cyclase, Malate - Aspartate shuttle and glycerol-3 -phosphate shuttle enzymes, Polyphosphate-AMP- phosphotransferase (PAP), Polyphosphate kinase, ATP synthesis/ recycling via acid production pathway involving enzymes such as and not limited to Acetate kinase/ propionate kinase/ Butyrate kinase, CoA- acylating dehydrogenase, Phosphotrans acetylase/ propionylase/ Butyrylase, aldehyde dehydrogenase, lactate dehydrogenase, etc. 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 Co enzyme A (Co A) and the enzymes for Co A (and Acetyl Co A/ Propionyl CoA, etc.) regeneration/ recycling includes such as and not limited to CoA transferases, CoA-acylating aldehyde dehydrogenase, CoA- dependent propionaldehyde dehydrogenase, phosphotransacetylase, Acetyl CoA-synthase (ACS), Acetyl coenzyme-A carboxylase (ACC), ACC- catalyzed biotin carboxylase (BC), carboxyltransferase (CT), etc. The method as claimed in claim 1 and 2, wherein enhanced production of Nitrogen and Carbon containing natural or unnatural compounds is achieved by expression of Homologous/ heterologous, native/ modified enzymes responsible for increased pyruvate availability include but not limited to (1) enzymes involved in increasing pyruvate synthesis towards downstream product formation pathways and (2) enzymes involved in preventing pyruvate loss in form of CO2. The enzymes for increasing pyruvate include and not limited to Pyruvate synthase, Pyruvate kinase, Pyruvate decarboxylase, carrier proteins like mitochondrial pyruvate carrier proteins etc., wherein such enzymes are overexpressed and/ or deleted/ downregulated.
Figure imgf000060_0001
The method as claimed in claim 1, wherein production of nitrogen and carbon-containing natural or unnatural compounds is enhanced with or without Carbon fixation/enhanced carbon fixation. The method as claimed in claim 15, 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 Cl carbon compounds like CO2, Methane, Methanol, Formic acid/ Formaldehyde, etc. The method as claimed in claim 16, wherein the said homologous/ heterologous enzymes are native and/ or modified, where enzyme modifications include but are not limited to point-mutations, epigenetic mechanisms, etc. The method as claimed in claim 1 and 16, wherein manipulation of the Calvin pathway includes but not limited to overexpression of homologous or heterologous enzyme, Rubisco (ribulose bisphosphate carboxylase/ oxygenase) with or without phosphoribulose kinase (PRK), and regulatory genes such as and not limited to Rubisco activators like Rea, Hpyl, etc. The method as claimed in claim 1 and 16, wherein manipulation of formate route includes but not limited to over-expression of homologous or heterologous Formate dehydrogenase (FDH), Formyhnethanofuran Dehydrogenase, Formolase, etc., with or without point mutations for increased catalytic activity and thermostability. The method as claimed in claim 1 and 16, wherein methane (CH4) fixation and assimilation include homologous and/ or heterologous over-expression of Methane monooxygenase (MMO) enzymes, including and not limited
Figure imgf000061_0001
to sMMO and/ or pMMO operons, for soluble and particulate methane assimilation, respectively. The method as claimed in claim 1 and 16, wherein formaldehyde sequestration is enhanced by the incorporation of homologous or heterologous genes for the Formaldehyde fixation system of the Ribulose mono phosphate (RuMP) pathway, involving and not limited to 3- hexulose-6-phosphate synthase (HPS), 6-phospho-3 -hexuloisomerase (PHI) enzymes, and also by the incorporation of homologous and/or heterologous genes for the formaldehyde fixation system of Serinethreonine pathway involving, and not limited to FtfL, formate -THF ligase; Fch, methenyl-THF cyclohydrolase; MtdA, methylene-THF dehydrogenase. The method as claimed in claim 1 and 16, wherein methanol sequestration is enhanced by overexpression of enzyme such as and not limited to Methanol dehydrogenase and/ or alcohol dehydrogenase. The method as claimed in claims 1 and 16, 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, etc. The method as claimed in claim 1, the said enhanced production of carbon & nitrogen-containing natural or unnatural 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 synthesis/ 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, Hue Hydrogenase, CO/CO2 dependent hydrogenase (CODH), etc.
Figure imgf000062_0001
The method as claimed in claim 16 and 23, 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). The method as claimed in claim 1, 12 and 24, where in 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. The method as claimed in claims 18 to 24, wherein the 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. The method as claimed in claims 1, 57 and 58 , wherein enzymes involved in uptake/ fixation of Nitrogen and/ or carbon and/ or Ammonia and/ or Hydrogen and/ or uptake/ synthesis/ regeneration of ATP, NADH, NADPH, Phosphate, Pyruvate, CoA, Acetyl-CoA and/or production of carbon and Nitrogen-containing compounds, are overexpressed by the inclusion of Transcriptional and/ or Translational enhancers such as and not limited to UNA1, UNA2, UNB, oligomers, etc. The method as claimed in claim 1, wherein the said ammonia utilisation microbe also include Ammonia oxidising microbes such as and not limited to Nitrosomonas spp., Nitrosococcus spp., Nitrospira spp., Nitrospina, Nitrocystis, Nitrosomonas, Nitrosolobus, Nitrobacter, Pseudomonas etc. The method as claimed in claim 29, wherein the ammonia oxidising bacteria particularly belongs to genus Nitrospira, and capable of oxidising Ammonia to Nitrogen products such as and not limited to Nitrate, Hydroxylamine and Nitrite. The method as claimed in claim 29, wherein Ammonia oxidation is achieved using enzymes including but not limited to Ammonia
Figure imgf000063_0001
monooxygenase, Hydroxylamine oxidoreductase, Nitrite oxidoreductase, etc. The method as claimed in claim 4, wherein the said amino acids include but not limited to proteinogenic and non-proteinogenic amino acids / their isomers/ their derivatives (including unnatural amino acids, amino acid based and amino conjugated compounds), such as Phenylalanine, Valine, Tryptophan, Threonine, Isoleucine, Methionine, Histidine, Leucine, Serine, Proline, Glutamine, Glutamic acid, para-nitro-L-phenylalanine (pN-Phe), hydroxyproline (Hyp), beta-alanine, citrulline (Cit), ornithine (Om), norleucine (Nle), 3-nitrotyrosine, nitroarginine, pyroglutamic acid (Pyr), Amino Benzoic acid and derivatives, a-hydroxy/ a-thio aminoacids, N-formyl -L-a-aminoacids like N-formyl Methionine, N-formyl Phenylalaine, Piperazic acid (a-hydrazino acid), serotonin, psilocybin, amino acid-piperine conjugates, icaritin-L-lysine, curcumin-amino acid conjugates, Tetrahydrocurcumin-L-glycine, tetrahydrocurcumin-L-valine etc. The method as claimed in claim 4, wherein the said modified or unmodified proteins can be recombinant (including synthetic proteins) and/ or native proteins which are derived and/ or fibrous and/ or globular proteins, such as and not limited Glyco/lipoproteins (like toxin protein), nutritive proteins, therapeutic proteins (like Interferons, Interleukins, modnilin, etc.), glomalin related soil protein (GRSP), lectin, antibody, anti-freeze proteins, Phospho proteins, methyl proteins, etc. The method as claimed in claim 33, wherein the said nutritive proteins belongs to plant, animal, mammal, avian, insect proteins, such as and not limited to milk proteins, whey protein, casein, egg protein like ovalbumin, egg lipo proteins like phosvitin, livetin, porcine, soy protein, leghemoglobin, etc. The method as claimed in claim 33, wherein the said Phospho proteins includes but not limited to Immunoglobin Fc receptors, ovo-vitelline, calcineurines, urocortines, etc.
Figure imgf000064_0001
The method as claimed in claim 33. Wherein the said methyl proteins include but not limited to CUL1 -ubiquitin protein ligase complex, G3BP1- stress granule protein, Enolase, etc. The method as claimed in claim 33, wherein the said proteins are also enzymes including hydrolases, oxidoreductases, lyases, transferases, ligases and isomerases class of enzymes. The method as claimed in claims 4 and 33, the said protein and nonprotein enzymes (like Ribozymes) are involved in the synthesis of primary and secondary metabolites such as nucleotide, nucleic acids, amino acids, proteins, enzymes, lipids, carbohydrates, cofactors, antibiotics, steroids, carotenoids, terpenoides, polymers, (like Poly(amino acids) including but not limited to poly glutamicacid, poly lysine, polyarginine, etc,; poly aspartate, polyamides, polyurethanes, polyacrylamide, etc.) vitamins, alkaloids, phenolics, organic acids, flavonoids, amines, peptides, urea, guanidine, glycosides, glucosinolates, non-protein amino acids, single cell protein etc. The method as claimed in claim 4, wherein the said vitamins include enzyme cofactors/ coenzymes, such as and not limited to vitamin B and its isoforms/ derivatives like thiamine, thiamine pyrophosphate, riboflavin, niacin, pantothenic acid, biotin, folate, vitamin B6 (pyridoxine), vitamin B12 (cobalamine), P5P, Methylcobalamin, Hydroxycobalamine, THF, Coenzyme The method as claimed in claim 4, wherein the said cofactors include but not limited to NADH/NAD, FADH/FAD, Flavin mono nucleotide (FMN), etc. The method as claimed in claim 4, where in the said nucleotides include and not limited to Adenine, Guanine, Cytosine, Thymine, ATP, GTP, CTP, TTP, etc. The method as claimed in claim 4, wherein the said alkaloids and its derivatives include but not limited to isoquinoline alkaloids, indole alkaloids, steroidal alkaloids, proto alkaloids, pseudo
Figure imgf000065_0001
alkaloids, sesquiterpene alkaloids including but not limited to alkaloids like streptomycin, penicillins, palmatine, dragmacidin, Neofiscalin, Huperzine A etc. The method as claimed in claim 4, wherein the said flavonoids and its derivatives include but not limited to chaicones, flavones, isoflavones, flavanones, flavonols, anthocyanidins, anthocyanins, auronidins, etc. The method as claimed in claim 4, wherein the amides include but not limited to urea, guanidine, carbamides, formamide, benzamide, etc., and the production is achieved by the homologous/ heterologous expression of enzymes such as and not limited to Carbamoyl phosphate synthase, Ornithine trans-carbamylase, Argino succinate synthetase, Argino succinate lyase, Arginase, etc. The method as claimed in claim 4, wherein the said amines and its derivatives include but not limited to hydroxyl amine, glucosamine, NAG (n-acetyl glucosamine) and their polymers such as and not limited to Chitosan, chito oligosaccharides, lipo chito oligosaccharides, etc. The method as claimed in claim 4, where in the said N-glycosides include but not limited to Amygdalin, prunasin, linamarin, lotaustralin, dhurrin, etc. The method as claimed in claim 4, wherein the said peptides include but not limited to di/tri/ tetra/ oligo peptides, linear peptide, for the purposes as enzyme inhibitor peptides, signal peptides, neurotransmitter inhibitor peptides, carrier peptides such as oxytacin, angiotensin, natriuretic peptide, etc. The method as claimed in claim 4, wherein the Nitrogen-containing organic acids include but not limited to uric acid, muramic acid, theacrine, tetramethyl uric acid, sialic acid, etc. The method as claimed in claim 4, wherein the said Xanthines and its derivatives include but not limited to paraxanthine, caffeine, Methylliberine, theobromine, theophylline, etc.
Figure imgf000066_0001
The method as claimed in claim 4, wherein the said nitrate/ nitrite and its derivatives include but not limited to alkyl nitrite, amyl nitrite etc. The method as claimed in claim 4, the Nitrogen and carbon containing compounds also includes natural or unnatural chromophores/ fluorophores such as and not limited to Luciferin, Luminol, Fluorescent proteins (such as and not limited to Green/ Red Fluorescent protein GFP/ RFP), 4', 6- diamidino-2-phenylindole (DAPI), Fluorescein isothiocyanate (FITC), hemicyanine, boron-dipyrromethene, dicyanomethylene -4H-pyran, rhodamine derivatives, Pacific blue fluorophores, 7-hydroxy-coumarin, Fluorinated Azido-Coumarin, etc. The said chromophores/ fluorophores can also be used as biosensors. 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), Hydrogen peroxide (H2O2), synthetic/ non-synthetic compounds including but not limited to plastics/ fibres/ rubber/ resins/ chemicals, etc. (like polyethylene terepthalate (PET), Ethylene glycol (EG), polyurethane), under in vivo and/ or in vitro applications by homologous and/ or heterologous expression of native or modified enzymes including but not limited to cellulase, exo-glucanase, endo-glucanase, cellobiohydrolase, xylanase, pectinases, pectin methyl esterase, pectin acetyl esterase, lignin peroxidase, proteases, peptidases, endo-glucanase, cellobiohydrolase, xylanase, pectinases, pectin methyl esterase, pectin acetyl esterase, lignin peroxidase, proteases, peptidases, Endopeptidase, exopeptidases, asparaginyl endopeptidases, butelase, invertase, amylase, sugar isomerases,
Figure imgf000067_0001
lipases, amidases, phytase, phosphatases, nitrate reductase, nitrite reductase, N2O reductase, sulfate adenylyltransferase (Sat), adenylyl sulfate reductase (AprBA), dissimilatory sulfite reductase (DsrAB), ATP sulfurylase (ATPS), Adenosine 5 '-phosphosulfate reductase (APSR), APS kinase (APSK), Terephthalate 1,2-dioxygenase, Terephthalate reductase, Dihydrodiol dehydrogenase, Glycolate oxidase, Glyoxylate carboligase, hydroxy pyruvate isomerase, protocatechuate decarboxylase, Feruloyl esterase, TMADH-trimethylamine dehydrogenase; DMADH- dimethylamine dehydrogenase; MMADH-monomethylamine dehydrogenase, H2O2 peroxidase, peroxygenase, natural and/ or Artificial metalloenzymes, Flavoenzymes etc. The method as claimed in claim 1 and 5, wherein the fixation/ reduction of nitrogen compound, Nitrous oxide-N2O (greenhouse gas), is achieved by homologous/ heterologous expression of genes for native/ modified N2O reductase such as and not limited to nosR, nosZ, nosD, nosF, nosY, nosL, nosX, etc. The method as claimed in claim 37, wherein the said enzymes in microbes is also for enhancing production or inducing production of natural or unnatural compounds for breakdown/ denaturing/ detoxifying/ degrading toxic compounds such as and not limited to pesticides, aflatoxin, insecticides, etc. The method as claimed in claims 31 to 54, wherein the 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, upregulation and/ or downregulation/ silencing/ deletion, epigenetic mechanisms, etc. The method as claimed in claims 8 and 16, wherein assimilation is enhanced by integrating the regeneration/ recycling of the metabolic intermediates including but not limited to Hydrogen, Phosphate/ ATP, NADH/NADPH, Pyruvate, Coenzyme A-CoA/ Acetyl CoA resulting in stable/ enhanced metabolic efficiency of the host microbe.
Figure imgf000068_0001
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 Nitrogen & Carbon containing natural or unnatural compounds. 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 Nitrogen & Carbon containing natural or unnatural compounds. The method as claimed in claim 57 and 58, 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. A composition comprising the genetically modified microbe as claimed in claim 1, including a single microbe modified or a consortium of microbes modified for production of Nitrogen and carbon -containing compounds.
PCT/IN2023/051164 2022-12-09 2023-12-11 Genetic modification of microbes for production of nitrogen and carbon-containing compounds WO2024121871A1 (en)

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Citations (6)

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