WO2023181069A1 - Genetic modification of microbes for improved nitrate uptake for crops - Google Patents
Genetic modification of microbes for improved nitrate uptake for crops Download PDFInfo
- Publication number
- WO2023181069A1 WO2023181069A1 PCT/IN2023/050273 IN2023050273W WO2023181069A1 WO 2023181069 A1 WO2023181069 A1 WO 2023181069A1 IN 2023050273 W IN2023050273 W IN 2023050273W WO 2023181069 A1 WO2023181069 A1 WO 2023181069A1
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- WO
- WIPO (PCT)
- Prior art keywords
- nitrite
- composition
- ammonia
- nitrate
- bacteria
- Prior art date
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- 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
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- 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
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- C12Y107/02—Oxidoreductases acting on other nitrogenous compounds as donors (1.7) with a cytochrome as acceptor (1.7.2)
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- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Definitions
- the present invention relates to genetic modification of both Nitrogen fixing and non fixing microbes for improved Nitrate uptake for the crops.
- the present invention relates to up regulation of genes to increase the activity of enzymes selected from the group consisting of ammonia monooxygenase, Hydroxylamine oxidoreductase, Nitrite oxido reducatse for nitrate uptake by the crops (Ammonia oxidation to Nitrate formation).
- the Present invention further relates to down regulation/deletion of Nitrite reductase enzyme (Nitrite to ammonia conversion) and Nitric acid reductase (Nitric oxide (NO) to Nitrous oxide N 2 O) and Nitrous oxide reductase ( N 2 O to N2)to overcome the denitrification step.
- Nitrite reductase enzyme Nitrite to ammonia conversion
- Nitric acid reductase Nitric oxide (NO) to Nitrous oxide N 2 O
- Nitrous oxide reductase N 2 O to N2
- the Present Invention relates to the genetic modification of microbes converts ammonia to Nitrite, incorporation of the gene clusters responsible for Nitrite oxido reducatse enzyme to introduce the machinery for (Nitrite to Nitrate conversion in the soil).
- the Present Invention relates to the genetic modification of microbes converts Nitrite to Nitrate, incorporation of gene cluster responsible for Ammonia to Nitrite conversion. More precisely up regulation of ammonia monooxygenase and Hydroxylamine oxidoreductase.
- the present invention also relates to a composition comprising genetically modified microbes which is favourable for uptake of Nitrate in all crops
- the present invention relates to increase in Nitrification process in soil for the benefit of crops to assimilate Nitrogen.
- Nitrate is significant nitrogen source for microbes and plants. Nitrogen is an essential macronutrient for plants and is a key component of amino acids, proteins, nucleic acids, enzymes, and chlorophyll.
- Plants take up nitrogen in the form of nitrate (NO 3 -) and ammonium (NH 4 + ) ions. Atmospheric nitrogen is converted by microorganisms into ammonia, nitrite and nitrate that can be used by the plants. Nitrogen fixation is carried out in soil by microorganisms termed diazotrophs that include bacteria such as azotobacter and archaea or bacteria living symbiotically in nodules on the roots of legumes.
- Nitrification is a microbial process by which reduced nitrogen compounds, primarily ammonia are sequentially oxidized to nitrite and nitrate. Nitrification is the biological oxidation of ammonia to nitrite followed by the oxidation of the nitrite to nitrate occurring in separate organisms or direct ammonia oxidation to nitrate in comammox bacteria. The transformation of ammonia to nitrite is usually the rate limiting step of nitrification. Nitrification is an important step in the nitrogen cycle in soil. Nitrification is an aerobic process performed by small groups of autotrophic bacteria and archaea.
- Ammonia is converted into nitrite by ammonia oxidizing organisms: ammonia oxidizing archaea (AOA) and ammonia oxidizing bacteria (AOB) (Koops and PommereningRbser, 2001; Spang et al., 2010). Ammonia oxidation is an aerobic process and typically occurs in aphotic zones due to light inhibition (French et al., 2012).
- the first step of ammonia oxidation is catalyzed by ammonia monooxygenase (AMO), producing hydroxylamine as a result of the loss of two electrons. Hydroxylamine is then converted to nitrite by hydroxylamine oxidoreductase (HAO) and nitrite is oxidized to nitrate by nitrite oxidoreductase.
- AMO ammonia monooxygenase
- HEO hydroxylamine oxidoreductase
- AOB ammonia-oxidizing bacteria
- AOA ammonia- oxidizing archaea
- the second step - oxidation of nitrite into nitrate - is done by bacteria (nitrite- oxidizing bacteria, NOB) from the taxa Nitrospirae, Nitrospinae, Proteobacteria and Chloroflexi. They are present in soil, geothermal springs, freshwater and marine ecosystems.
- WO2017011602 Al discloses methods of increasing nitrogen fixation in a non- leguminous plant.
- Tire methods comprises exposing the plant to a plurality of bacteria having one or more genetic variations introduced into one or more genes or non- coding polynucleotides 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.
- WO2020245841 discloses genetic modification of microbes for improved nitrogen fixation and its delivery to crop-plants for assimilation. It discloses gene modifications of the nif gene cluster comprising up regulation of positive regulators, down regulation of negative regulators and over expression of structural genes to achieve enhanced nitrogen fixation.
- Nitrate is increased by the genticially modified microbes. Nitrate is readily taken by all the crops as the preferred Nitrogen source.
- Nitrite reductase enzyme Nitrite to ammonia conversion
- Nitric acid reductase Nitric oxide (NO) to Nitrous oxide N2O
- Nitrous oxide reducatse N2O to N2
- the present invention provides gene manipulation of microbes for the enhanced activity of enzymes selected from the group consisting of Ammonia monooxygenase, Hydroxylamine oxidoreductase and Nitrite oxido reducatse enzymes and Nitrogenase enzyme.
- the present invention provides the down regulation/deletion of Nitrite reductase enzyme (Nitrite to ammonia conversion) and Nitric acid reductase (Nitric oxide (NO) to Nitrous oxide N 2 O) and Nitrous oxide reducatse (N 2 O to N2) to overcome the denitrification step.
- Nitrite reductase enzyme Nitrite to ammonia conversion
- Nitric acid reductase Nitric oxide (NO) to Nitrous oxide N 2 O
- Nitrous oxide reducatse N 2 O to N2
- the present invention provides the genetic modification of micro-organisms for improving Nitrogen fixation and Nitrate uptake by the crops.
- the present invention provides genetic modification of microbes that result in increased expression of enzymes that convert ammonia to Nitrite and incorporation of the gene clusters that are responsible for the increased activity of Nitrite oxido reducatse enzyme that convert Nitrite to Nitrate in the soil.
- the present invention provides genetic modification of microbes that convert Nitrite to Nitrate and Ammonia to Nitrite by up regulation of enzymes Ammonia monooxygenase and Hydroxylamine oxidoreductase.
- the present invention provides involvement of CRISPR/Cas technology used preferentially for gene manipulation of microbes.
- Figure 1 Depicts impact of overexpression of Nitrification process on enhanced concentrations of Nitrite and Nitrate.
- Figure 2 Depicts impact of deletion of Denitrification process in addition to overexpression of Nitrification process, on enhanced concentrations of Nitrite and Nitrate.
- Ammonia is oxidized to Nitrite via Hydroxylamine.
- Two different enzymes are involved in the process - Ammonia monooxygenase and Hydroxylamine oxidoreductase.
- Ammonia monooxygenase catalyses the conversion of Ammonia to Hydroxylamine (NH2OH).
- Hydroxylamine oxidoreductase then further oxidizes Hydroxylamine to Nitrite.
- Nitrite is then converted into Nitrate by the Nitrite oxidoreductase. Therefore, the present inventors have genetically modified micro-organisms to enhance the sysnthesis of the aforesaid enzymes to increase nitrogen fixation and Nitrate uptake by the crops.
- the present invention provides gene manipulations in endophytic/ epiphytic/ rhizospheric microbes that results in increased expression of enzymes selected from the group comprising Ammonia monooxygenase, Hydroxylamine reducatse and Nitrate oxido reducatse enzymes and Nitrogenase enzyme.
- the present invention provides microbes selected from nitrogen fixing bacteria.
- the bacteria may include Proteobacteria (such as Methylobacter, Erwinia, Acinetobacter, Beijernickia, Sphingomona, Novosphingobium, Ochrobactrum, Gluconacetobacter etc.), Firmicutes (such as Clostridium sp. etc.), Cyanobacteria (such as cyanobacteria sp.), Actinobacteria (such as Frankia, Arthrobacter, Agromyces, Corynebacterium, Mycobacterium, Micromonospora, Propionibacteria etc.) and Bacteroidetes (such as Flavobacterium etc).
- Proteobacteria such as Methylobacter, Erwinia, Acinetobacter, Beijernickia, Sphingomona, Novosphingobium, Ochrobactrum, Gluconacetobacter etc.
- Firmicutes such as Clostridium sp. etc.
- the present invention provides a composition comprising genetically modified micro-organism(s) consisting of deletion/downregulation of Nitrite reductase enzyme (Nitrite to ammonia conversion) and Nitric acid reductase (Nitric oxide (NO) to Nitrous oxide N 2 O) and Nitrous oxide reducatse gene leading to an increase in nitrogenase activity, wherein the said micro- organism is an endophyte, an epiphyte and a rhizospheric microbe.
- composition of the presnt invention comprises genetically modified micro-organism(s) consisting of upregulation of Ammonia monooxygenase, Hydroxylamine oxidoreductase, Nitrite oxido reducatse genes wherein there is an improved nitrate production by these modified microbes compared to that of the wild type.
- the gene integration process consists of plasmid vector, suicidal/ integration vector.
- the Integration vector is carrying a DNA construct comprising a nucleotide sequence represented by SEQ ID NO 7.
- SEQ ID NO.7 consisting of 2816bp gene (SEQ ID NO 1) with promoter (SEQ ID NO 6), within Nael sites of deletion gene (SEQ ID No 4).
- the plasmid vector is expressed in miro-organism selected from the group comprising of endophytic bacteria, epiphytic bacteria, rhizospheric bacteria and fungi.
- the bacteria is selected from the group comprising ofcultivable bacteris (viz) Nitrococcus, Nitrospira, Pseudomonas.
- the bacterium is selected from the group comprising ofNitrobacter.
- the present invention provides a process for the preparation of genetically modified micro-organism(s) for improved Nitrate production and its delivery to crop-plants for assimilation,
- the present invention provides the genetic modification of micro-organisms for improving Nitrogen fixation and Nitrate uptake by the crops.
- the present invention provides the down regulation/deletion of Nitrite reductase enzyme (Nitrite to ammonia conversion) and Nitric acid reductase (Nitric oxide (NO) to Nitrous oxide N 2 O) and Nitrous oxide reducatse (N 2 O to N2) to overcome the denitrification step.
- Nitrite reductase enzyme Nitrite to ammonia conversion
- Nitric oxide (NO) Nitric oxide
- Nitrous oxide reducatse N 2 O to N2
- the Present Invention provides genetic modification of microbes converts ammonia to Nitrite, incorporation of the gene clusters responsible for Nitrite oxido reducatse enzyme to introduce the machinery for (Nitrite to Nitrate conversion in the soil).
- the Present Invention provides genetic modification of microbes converts Nitrite to Nitrate, incorporation of gene cluster responsible for Ammonia to Nitrite conversion. More precisely up regulation of ammonia monooxygenase and Hydroxylamine oxidoreductase.
- the present invention provides a process for gene modification of Nitrogen fixing microbes; wherein said microbes reduce glucose uptake and in turn increase the Nitrogen fixation to the plants.
- the organisms can be selected from but not limted to Methylobacterium, Methyloversatilis, Sphingomonas, Bosea, Altererythrobacter, Brevundimonas, Rubrivivax, Niveispirillum, Dinoroseobacter shibae.
- Other microbes includes Nitrosopumilus maritimus, Nitrospira inopinata, Rhodobacter sphaeroides, Cupriavidus necator, Nigrospora oryzae, Azospirillum lipoferum, Rhodopseudomonas palustris , Bradyrhizobium japonicum , Ralstonia eutropha , Cyanobacteria, Epichloe typhina, Rhodococcus, Xanthobacter species Nitrosomonas, Nitrosospira, Nitrosococcus, and Nitrosolobus.
- fungi contemplated by the present invention include, but are not limited to Aspergillus, Candida, Chlamydomonas, Chrysosporium, Cryotococcus, Fusarium, Kluyveromyces, Neotyphodium, Neurospora, Penicillium (e.g. P. chrysogenum), Pichia, Saccharomyces, Trichoderma and Xanthophyllomyces.
- Coli Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula, Zymomonas and Saccharomyces, e.g., Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Kluyveromyces lactis, Saccharomyces lactiss.
- the present invention provides endophytes selected from the group comprising Proteobacteria (such as Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea, Serratia, Rahnella, Azospirillum, Azorhizobium, Azotobacter, Duganella, Delftia, Bradyrhizobiun, Sinorhizobium and Halomonas), Firmicutes (such as Bacillus, Paenibacillus, Lactobacillus, Mycoplasma, and Acetabacterium) and Actinobacteria (such as Streptomyces, Rhodacoccus, Microbacterium, and Curtobacterium).
- Proteobacteria such as Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea, Serratia
- Bacteria that can be produced by the methods disclosed herein include Azotobacter sp., Bradyrhizobium sp., Klebsiella sp., and Sinorhizobium sp.
- the bacteria may be selected from the group consisting of Azotobacter vinelandii or Azotobacter chroococcum, Bradyrhizobium japonicum, Klebsiella pneumoniae, and Sinorhizobium meliloti.
- the bacteria may be of the genus Enterobacter and Rahnella.
- certain fungi comprising Saccharomyces cerevisiae and Trichoderma harzianum are selected for genetic modification for nitrogen fixation.
- nitrogen compounds like ammonia are sequentially oxidized to nitrite and nitrate.
- ammonia (NH 3 ) or ammonium (NH 4 + ) is oxidised to nitrite (NO 2 -) by ammonia-oxidizing bacteria (e.g. Nitrosomonas) and in the next step nitrite (NO 2 -) is oxidised to nitrate (NO 3 - ) by the nitrite -oxidizing bacteria (e.g. Nitrobacter).
- CRISPR/Cas9 genetic variations are brought about by CRISPR/Cas9, by subjecting DNA to mutagens and other options for specifically inducing cleavage at a target site are available, such as zinc finger nucleases, TALE nuclease (TALEN) systems, and meganuclease.
- TALEN TALE nuclease
- CRISPR/Cas9 Clustered regularly interspaced short palindromic repeatsj/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids.
- the Cas9 protein (or functional equivalent and/or variant thereof, i.e., Cas9-like protein) naturally contains DNA endonuclease activity that depends on association of the protein with two naturally occurring or synthetic RNA molecules called crRNA and tracrRNA (also called guide RNAs).
- the two molecules are covalently linked to form a single molecule (also called a single guide RNA (“sgRNA”).
- a single molecule also called a single guide RNA (“sgRNA”).
- the Cas9 or Cas9-like protein associates with a DNA-targeting RNA (which term encompasses both the two-molecule guide RNA configuration and the single-molecule guide RNA configuration), which activates the Cas9 or Cas9-like protein and guides the protein to a target nucleic acid sequence.
- Cas9 or Cas9-like protein retains its natural enzymatic function, it will cleave target DNA to create a double-strand break, which can lead to genome alteration (i.e., editing: deletion, insertion (when a donor polynucleotide is present), replacement, etc.), thereby altering gene expression.
- Some variants of Cas9 (which variants are encompassed by the term Cas9-like) have been altered such that they have a decreased DNA cleaving activity (in some cases, they cleave a single strand instead of both strands of the target DNA, while in other cases, they have severely reduced to no DNA cleavage activity).
- mutagens that create primarily point mutations and short deletions, insertions, transversions, and/or transitions, including chemical mutagens or radiation, may be used to create genetic variations.
- Mutagens include, but are not limited to, ethyl methanesulfonate, methylmethane sulfonate, N-ethyl-N- nitrosurea, triethylmelamine, N-methyl-N-nitrosourea, procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N'- nitro- Nitrosoguanidine, nitrosoguanidine, 2-aminopurine, 7,12 dimethyl- benz(a)anthracene, ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctan
- Example 1 Upregulation of genes to enhance Nitrification enzymes such as Ammonia monooxygenase, Hydroxylamine oxidoreductase, Nitrite oxido reducatse
- Plants take up nitrogen in the form of nitrate (NO ⁇ ) and ammonium (NHi + ) ions.
- the step of Ammonia oxidation to nitrate formation is catalysed by enzymes Ammonia monooxygenase and Hydroxylamine oxidoreductase s and Nitrite oxido reducatse.
- mutagenesis was performed in and around the active sites of the genes that code for enzymes Ammonia monooxygenase and Hydroxylamine oxidoreductases and Nitrite oxido reducatse.
- a library of mutants was constructed for each gene. These libraries were then screened for increased enzyme activity.
- Engineered strains were subjected to growth and ammonia consumption assay at controlled conditions. Wild type cells were also experimented in similar conditions, to test the increased Ammonia conversion finally to Nitrate.
- Nitrate, Nitrite and Ammonia were analysed by Abcam-make colorimetric sensors.
- Nitrate level increased by 1.5 fold in final engineered strain, as compared to unmodified wild type strain.
- Nitrate/Nitrite ratio got increased by 2 fold, from ratio of 1.5 in wild type to ratio 3 in Final engineered strain.
- Example 2 Impact of overexpression of Nitrification process on enhanced concentrations of Nitrite and Nitrate:
- Nitrospira sp. were manipulated for over expression of Nitrification enzymes Ammonia monooxygenase, Hydroxylamine oxidoreductase and Nitrite oxidoreductase sequentially, resulting in Engineered strains for Ammonia oxidation, as well as Nitrite oxidation to yield Nitrate finally. Analysis of Nitrite, Ammonia and Nitrate by sensor-based testing, showed Nitrate level increase by 2 fold between Ammonia oxidation and Nitrite oxidation, whereas, about 1.5 fold increase in Nitrate when compared to unmodified Wild type Nitrospira sp (Figure 1).
- Example 3 Down regulation /deletion of Denitrification enzymes such as Nitrite reductase.
- Nitrate level increased by 1.5 fold in final engineered strain, as compared to unmodified wild type strain, and 2 fold reduction in Nitric oxide (denitrification product). Nitrate/Nitrite ratio got increased by 2 fold, from ratio of 1.4 in wild type to ratio 5.6 in Final engineered strain.
- Example 4 Impact of deletion of Denitrification process in addition to overexpression of Nitrification process, on enhanced concentrations of Nitrite and Nitrate:
- Nitrite reductase and Nitrate reductase are manipulated by deletion of Denitrification enzymes, in addition to over expression of Nitrification enzymes, resulting in Engineered strains for further efficient Ammonia oxidation, as well as Nitrite oxidation to yield enhanced amounts of Nitrate finally.
- N2O reductase (nos gene) from Rhizhobium type of microbes was incorporated for additional N fixation.
Abstract
The present invention provides genetic modification of microbes for improved Nitrate uptake for the crops. More particularly, the present invention relates to gene modifications comprising up regulation of enzymes selected from the group consisting of Ammonia monooxygenase, Hydroxylamine oxidoreductase and Nitrite oxido reducatse and down regulation/deletion of enzymes selected from the group consisting of Nitrite reductase enzyme, Nitric acid reductase and Nitrous oxide reductase to achieve enhanced nitrogen fixation.
Description
“Genetic modification of microbes for Improved Nitrate uptake for Crops”
TECHNICAL FIELD OF THE INVENTION:
The present invention relates to genetic modification of both Nitrogen fixing and non fixing microbes for improved Nitrate uptake for the crops.
More particularly, the present invention relates to up regulation of genes to increase the activity of enzymes selected from the group consisting of ammonia monooxygenase, Hydroxylamine oxidoreductase, Nitrite oxido reducatse for nitrate uptake by the crops (Ammonia oxidation to Nitrate formation).
The Present invention further relates to down regulation/deletion of Nitrite reductase enzyme (Nitrite to ammonia conversion) and Nitric acid reductase (Nitric oxide (NO) to Nitrous oxide N2O) and Nitrous oxide reductase ( N2O to N2)to overcome the denitrification step.
The Present Invention relates to the genetic modification of microbes converts ammonia to Nitrite, incorporation of the gene clusters responsible for Nitrite oxido reducatse enzyme to introduce the machinery for (Nitrite to Nitrate conversion in the soil).
The Present Invention relates to the genetic modification of microbes converts Nitrite to Nitrate, incorporation of gene cluster responsible for Ammonia to Nitrite conversion. More precisely up regulation of ammonia monooxygenase and Hydroxylamine oxidoreductase.
The present invention also relates to a composition comprising genetically modified microbes which is favourable for uptake of Nitrate in all crops
The present invention relates to increase in Nitrification process in soil for the benefit of crops to assimilate Nitrogen.
BACKGROUND AND PRIOR ART OF THE INVENTION:
Nitrate is significant nitrogen source for microbes and plants. Nitrogen is an essential macronutrient for plants and is a key component of amino acids, proteins, nucleic acids, enzymes, and chlorophyll.
Plants take up nitrogen in the form of nitrate (NO3-) and ammonium (NH4 +) ions. Atmospheric nitrogen is converted by microorganisms into ammonia, nitrite and nitrate that can be used by the plants. Nitrogen fixation is carried out in soil by microorganisms termed diazotrophs that include bacteria such as azotobacter and archaea or bacteria living symbiotically in nodules on the roots of legumes.
Nitrification is a microbial process by which reduced nitrogen compounds, primarily ammonia are sequentially oxidized to nitrite and nitrate. Nitrification is the biological oxidation of ammonia to nitrite followed by the oxidation of the nitrite to nitrate occurring in separate organisms or direct ammonia oxidation to nitrate in comammox bacteria. The transformation of ammonia to nitrite is usually the rate limiting step of nitrification. Nitrification is an important step in the nitrogen cycle in soil. Nitrification is an aerobic process performed by small groups of autotrophic bacteria and archaea.
Ammonia is converted into nitrite by ammonia oxidizing organisms: ammonia oxidizing archaea (AOA) and ammonia oxidizing bacteria (AOB) (Koops and PommereningRbser, 2001; Spang et al., 2010). Ammonia oxidation is an aerobic process and typically occurs in aphotic zones due to light inhibition (French et al., 2012). The first step of ammonia oxidation is catalyzed by ammonia monooxygenase (AMO), producing hydroxylamine as a result of the loss of two
electrons. Hydroxylamine is then converted to nitrite by hydroxylamine oxidoreductase (HAO) and nitrite is oxidized to nitrate by nitrite oxidoreductase.
Ammonia oxidation
The oxidation of ammonia into nitrite is performed by two groups of organisms, ammonia-oxidizing bacteria (AOB) and ammonia- oxidizing archaea (AOA) AOB can be found among the Betaproteobacteria and Gammaproteobacteria. Since discovery of AOA in 2005, two isolates have been cultivated: Nitrosopumilus maritimus and Nitrososphaera viennensis. In soils the most studied AOB belong to the genera Nitrosomonas and Nitrosococcus. When comparing AOB and AOA, AOA dominate in both soils and marine environments, suggesting that Thaumarchaeota may be greater contributors to ammonia oxidation in these environments.
Nitrite oxidation
The second step - oxidation of nitrite into nitrate - is done by bacteria (nitrite- oxidizing bacteria, NOB) from the taxa Nitrospirae, Nitrospinae, Proteobacteria and Chloroflexi. They are present in soil, geothermal springs, freshwater and marine ecosystems.
Complete ammonia oxidation - comammox
Ammonia oxidation to nitrate in a single step within one organism was predicted in 2006 discovered in 2015 in species Nitrospira inopinata. Its pure culture was obtained in 2017 representing revolution in understanding of nitrification process. WO2017011602 Al discloses methods of increasing nitrogen fixation in a non- leguminous plant. Tire methods comprises exposing the plant to a plurality of bacteria having one or more genetic variations introduced into one or more genes or non- coding polynucleotides 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.
WO2020245841 discloses genetic modification of microbes for improved nitrogen fixation and its delivery to crop-plants for assimilation. It discloses gene modifications of the nif gene cluster comprising up regulation of positive regulators, down regulation of negative regulators and over expression of structural genes to achieve enhanced nitrogen fixation.
In the present invention availability of Nitrate is increased by the genticially modified microbes. Nitrate is readily taken by all the crops as the preferred Nitrogen source.
In recent years, the synthesis of nitrogen fixing systems has become increasingly common due to advances in synthetic biology. An important goal of nitrogen fixation research is the extension of this phenotype to non leguminous plants. Therefore, there is a need in the art to provide for genetic modification of microbes for improved Nitrogen fixation and Nitrate uptake by the crops.
OBJECT OF THE INVENTION:
It is an object of the present invention to provide for the gene manipulation of microbes for the enhanced activity of enzymes selected from the group consisting of ammonia monooxygenase, Hydroxylamine oxidoreducatse and Nitrite oxido reducatse enzymes and Nitrogenase enzyme.
It is another object of the present invention to provide for the down regulation/deletion of Nitrite reductase enzyme (Nitrite to ammonia conversion) and Nitric acid reductase (Nitric oxide (NO) to Nitrous oxide N2O) and Nitrous oxide reducatse (N2O to N2) to overcome the denitrification step .
It is another object of the present invention to provide a composition comprising genetically modified microbes which is favourable for fixing atmospheric nitrogen and uptake of Nitrate to crop-plant for assimilation in leguminous and non-leguminous plants.
It is another object of the present invention to provide for genetic modification of microbes that convert ammonia to Nitrite, incorporation of the gene clusters that are responsible for increased activity of Nitrite oxido reducatse enzyme that converts Nitrite to Nitrate in the soil.
It is another object of the present invention to provide genetic modification of microbes converts Nitrite to Nitrate, incorporation of gene cluster responsible for Ammonia to Nitrite conversion. More precisely up regulation of ammonia monooxygenase and Hydroxylamine oxidoreductase
It is an object of the present invention to provide a process for gene modification of Nitrogen fixing microbes; wherein said microbes reduce glucose uptake and in turn increase the Nitrogen fixation to the plants.
It is a further object of the present invention to provide involvement of CRISPR/Cas technology used preferentially for gene manipulation of microbes.
SUMMARY OF THE INVENTION:
In a main aspect, the present invention provides gene manipulation of microbes for the enhanced activity of enzymes selected from the group consisting of Ammonia monooxygenase, Hydroxylamine oxidoreductase and Nitrite oxido reducatse enzymes and Nitrogenase enzyme.
In another aspect, the present invention provides the down regulation/deletion of Nitrite reductase enzyme (Nitrite to ammonia conversion) and Nitric acid reductase (Nitric oxide (NO) to Nitrous oxide N2O) and Nitrous oxide reducatse (N2O to N2) to overcome the denitrification step.
In yet another aspect, the present invention provides the genetic modification of micro-organisms for improving Nitrogen fixation and Nitrate uptake by the crops.
In yet another aspect, the present invention provides genetic modification of microbes that result in increased expression of enzymes that convert ammonia to Nitrite and incorporation of the gene clusters that are responsible for the increased activity of Nitrite oxido reducatse enzyme that convert Nitrite to Nitrate in the soil.
In another aspect, the present invention provides genetic modification of microbes that convert Nitrite to Nitrate and Ammonia to Nitrite by up regulation of enzymes Ammonia monooxygenase and Hydroxylamine oxidoreductase.
In yet another aspect, the present invention provides involvement of CRISPR/Cas technology used preferentially for gene manipulation of microbes.
BRIEF DESCRIPTION OF THE DRAWINGS:
Figure 1: Depicts impact of overexpression of Nitrification process on enhanced concentrations of Nitrite and Nitrate.
Figure 2: Depicts impact of deletion of Denitrification process in addition to overexpression of Nitrification process, on enhanced concentrations of Nitrite and Nitrate.
DETAILED DESCRPITION OF THE INVENTION
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.
Ammonia is oxidized to Nitrite via Hydroxylamine. Two different enzymes are involved in the process - Ammonia monooxygenase and Hydroxylamine oxidoreductase. In the first step of this process ammonia monooxygenase catalyses the conversion of Ammonia to Hydroxylamine (NH2OH). Hydroxylamine oxidoreductase then further oxidizes Hydroxylamine to Nitrite.
Nitrite is then converted into Nitrate by the Nitrite oxidoreductase. Therefore, the present inventors have genetically modified micro-organisms to enhance the sysnthesis of the aforesaid enzymes to increase nitrogen fixation and Nitrate uptake by the crops.
In a preferred embodiment, the present invention provides gene manipulations in endophytic/ epiphytic/ rhizospheric microbes that results in increased expression of enzymes selected from the group comprising Ammonia monooxygenase, Hydroxylamine reducatse and Nitrate oxido reducatse enzymes and Nitrogenase enzyme.
Accordingly, the present invention provides microbes selected from nitrogen fixing bacteria.
The bacteria may include Proteobacteria (such as Methylobacter, Erwinia, Acinetobacter, Beijernickia, Sphingomona, Novosphingobium, Ochrobactrum, Gluconacetobacter etc.), Firmicutes (such as Clostridium sp. etc.), Cyanobacteria (such as cyanobacteria sp.), Actinobacteria (such as Frankia, Arthrobacter, Agromyces, Corynebacterium, Mycobacterium, Micromonospora, Propionibacteria etc.) and Bacteroidetes (such as Flavobacterium etc).
In an embodiment, the present invention provides a composition comprising genetically modified micro-organism(s) consisting of deletion/downregulation of Nitrite reductase enzyme (Nitrite to ammonia conversion) and Nitric acid reductase (Nitric oxide (NO) to Nitrous oxide N2O) and Nitrous oxide reducatse gene leading to an increase in nitrogenase activity, wherein the said micro- organism is an endophyte, an epiphyte and a rhizospheric microbe.
In another embodiment, the composition of the presnt invention comprises genetically modified micro-organism(s) consisting of upregulation of Ammonia monooxygenase, Hydroxylamine oxidoreductase, Nitrite oxido reducatse genes
wherein there is an improved nitrate production by these modified microbes compared to that of the wild type.
In another embodiment, the gene integration process consists of plasmid vector, suicidal/ integration vector.
In another embodiment, the Integration vector is carrying a DNA construct comprising a nucleotide sequence represented by SEQ ID NO 7.
In another embodiment, the SEQ ID NO.7 consisting of 2816bp gene (SEQ ID NO 1) with promoter (SEQ ID NO 6), within Nael sites of deletion gene (SEQ ID No 4).
In another embodiment, the plasmid vector is expressed in miro-organism selected from the group comprising of endophytic bacteria, epiphytic bacteria, rhizospheric bacteria and fungi.
In another embodiment, the bacteria is selected from the group comprising ofcultivable bacteris (viz) Nitrococcus, Nitrospira, Pseudomonas.
In yet another embodiment, the bacterium is selected from the group comprising ofNitrobacter.
In another embodiment, the present invention provides a process for the preparation of genetically modified micro-organism(s) for improved Nitrate production and its delivery to crop-plants for assimilation,
In another embodiment, the present invention provides the genetic modification of micro-organisms for improving Nitrogen fixation and Nitrate uptake by the crops. In yet another embodiment, the present invention provides the down regulation/deletion of Nitrite reductase enzyme (Nitrite to ammonia conversion) and Nitric acid reductase (Nitric oxide (NO) to Nitrous oxide N2O) and Nitrous oxide reducatse (N2O to N2) to overcome the denitrification step.
In a further embodiment, The Present Invention provides genetic modification of microbes converts ammonia to Nitrite, incorporation of the gene clusters responsible for Nitrite oxido reducatse enzyme to introduce the machinery for (Nitrite to Nitrate conversion in the soil).
In yet another embodiment, the Present Invention provides genetic modification of microbes converts Nitrite to Nitrate, incorporation of gene cluster responsible for Ammonia to Nitrite conversion. More precisely up regulation of ammonia monooxygenase and Hydroxylamine oxidoreductase.
In another embodiment, the present invention provides a process for gene modification of Nitrogen fixing microbes; wherein said microbes reduce glucose uptake and in turn increase the Nitrogen fixation to the plants.
The organisms can be selected from but not limted to Methylobacterium, Methyloversatilis, Sphingomonas, Bosea, Altererythrobacter, Brevundimonas, Rubrivivax, Niveispirillum, Dinoroseobacter shibae.
Other microbes includes Nitrosopumilus maritimus, Nitrospira inopinata, Rhodobacter sphaeroides, Cupriavidus necator, Nigrospora oryzae, Azospirillum lipoferum, Rhodopseudomonas palustris , Bradyrhizobium japonicum , Ralstonia eutropha , Cyanobacteria, Epichloe typhina, Rhodococcus, Xanthobacter species Nitrosomonas, Nitrosospira, Nitrosococcus, and Nitrosolobus.
Examples of fungi contemplated by the present invention include, but are not limited to Aspergillus, Candida, Chlamydomonas, Chrysosporium, Cryotococcus, Fusarium, Kluyveromyces, Neotyphodium, Neurospora, Penicillium (e.g. P. chrysogenum), Pichia, Saccharomyces, Trichoderma and Xanthophyllomyces.
Further, Clostridium acetobutylicum, C. Beijerinckii, C. accharoperbutylacetonicum, C. saccharobutylicum, C. aurantibutyricum, C.
tetanomorphum), Zymomonas, Escherichia (e.g., E. Coli), Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula, Zymomonas and Saccharomyces, e.g., Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Kluyveromyces lactis, Saccharomyces lactiss.
In a further embodiment, the present invention provides endophytes selected from the group comprising Proteobacteria (such as Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea, Serratia, Rahnella, Azospirillum, Azorhizobium, Azotobacter, Duganella, Delftia, Bradyrhizobiun, Sinorhizobium and Halomonas), Firmicutes (such as Bacillus, Paenibacillus, Lactobacillus, Mycoplasma, and Acetabacterium) and Actinobacteria (such as Streptomyces, Rhodacoccus, Microbacterium, and Curtobacterium).
Bacteria that can be produced by the methods disclosed herein include Azotobacter sp., Bradyrhizobium sp., Klebsiella sp., and Sinorhizobium sp.
The bacteria may be selected from the group consisting of Azotobacter vinelandii or Azotobacter chroococcum, Bradyrhizobium japonicum, Klebsiella pneumoniae, and Sinorhizobium meliloti. The bacteria may be of the genus Enterobacter and Rahnella.
In another embodiment, certain fungi comprising Saccharomyces cerevisiae and Trichoderma harzianum are selected for genetic modification for nitrogen fixation.
In the process of nitrification reduced nitrogen compounds like ammonia are sequentially oxidized to nitrite and nitrate. In the first step ammonia (NH3) or ammonium (NH4 +) is oxidised to nitrite (NO2-) by ammonia-oxidizing bacteria (e.g. Nitrosomonas) and in the next step nitrite (NO2-) is oxidised to nitrate (NO3- ) by the nitrite -oxidizing bacteria (e.g. Nitrobacter).
In the present invention, genetic variations are brought about by CRISPR/Cas9, by subjecting DNA to mutagens and other options for specifically inducing cleavage at a target site are available, such as zinc finger nucleases, TALE nuclease (TALEN) systems, and meganuclease.
Accoridngly, CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeatsj/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. The Cas9 protein (or functional equivalent and/or variant thereof, i.e., Cas9-like protein) naturally contains DNA endonuclease activity that depends on association of the protein with two naturally occurring or synthetic RNA molecules called crRNA and tracrRNA (also called guide RNAs). In some cases, the two molecules are covalently linked to form a single molecule (also called a single guide RNA (“sgRNA”). Thus, the Cas9 or Cas9-like protein associates with a DNA-targeting RNA (which term encompasses both the two-molecule guide RNA configuration and the single-molecule guide RNA configuration), which activates the Cas9 or Cas9-like protein and guides the protein to a target nucleic acid sequence. If the Cas9 or Cas9-like protein retains its natural enzymatic function, it will cleave target DNA to create a double-strand break, which can lead to genome alteration (i.e., editing: deletion, insertion (when a donor polynucleotide is present), replacement, etc.), thereby altering gene expression. Some variants of Cas9 (which variants are encompassed by the term Cas9-like) have been altered such that they have a decreased DNA cleaving activity (in some cases, they cleave a single strand instead of both strands of the target DNA, while in other cases, they have severely reduced to no DNA cleavage activity).
Further, mutagens that create primarily point mutations and short deletions, insertions, transversions, and/or transitions, including chemical mutagens or radiation, may be used to create genetic variations. Mutagens include, but are not
limited to, ethyl methanesulfonate, methylmethane sulfonate, N-ethyl-N- nitrosurea, triethylmelamine, N-methyl-N-nitrosourea, procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N'- nitro- Nitrosoguanidine, nitrosoguanidine, 2-aminopurine, 7,12 dimethyl- benz(a)anthracene, ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane, diepoxybutane, and the like), 2-methoxy-6- chloro-9 [3 -(ethyl -2 -chloro- ethyl)aminopropylamino] acridine dihydrochloride and formaldehyde.
Examples: Following examples are given by way of illustration therefore should not be construed to limit the scope of the invention.
Example 1: Upregulation of genes to enhance Nitrification enzymes such as Ammonia monooxygenase, Hydroxylamine oxidoreductase, Nitrite oxido reducatse
Plants take up nitrogen in the form of nitrate (NO<) and ammonium (NHi+) ions. The step of Ammonia oxidation to nitrate formation is catalysed by enzymes Ammonia monooxygenase and Hydroxylamine oxidoreductase s and Nitrite oxido reducatse.
Genetic modifications were performed on these genes in bacterial strain capable of Ammonia oxidation completely to nitrate. This resulted in increased enzyme activity of these enzymes and thereby improved Nitrogen fixation and nitrate uptake by the plant.
To identify such modifications, mutagenesis was performed in and around the active sites of the genes that code for enzymes Ammonia monooxygenase and Hydroxylamine oxidoreductases and Nitrite oxido reducatse. A library of mutants was constructed for each gene. These libraries were then screened for increased enzyme activity.
Engineered strains were subjected to growth and ammonia consumption assay at controlled conditions. Wild type cells were also experimented in similar conditions, to test the increased Ammonia conversion finally to Nitrate. Nitrate, Nitrite and Ammonia were analysed by Abcam-make colorimetric sensors. Nitrate level increased by 1.5 fold in final engineered strain, as compared to unmodified wild type strain. Nitrate/Nitrite ratio got increased by 2 fold, from ratio of 1.5 in wild type to ratio 3 in Final engineered strain.
Example 2: Impact of overexpression of Nitrification process on enhanced concentrations of Nitrite and Nitrate:
Nitrospira sp. were manipulated for over expression of Nitrification enzymes Ammonia monooxygenase, Hydroxylamine oxidoreductase and Nitrite oxidoreductase sequentially, resulting in Engineered strains for Ammonia oxidation, as well as Nitrite oxidation to yield Nitrate finally. Analysis of Nitrite, Ammonia and Nitrate by sensor-based testing, showed Nitrate level increase by 2 fold between Ammonia oxidation and Nitrite oxidation, whereas, about 1.5 fold increase in Nitrate when compared to unmodified Wild type Nitrospira sp (Figure 1).
Example 3: Down regulation /deletion of Denitrification enzymes such as Nitrite reductase.
Genetic modification was done in the microbes to reduce/inhibit the Nitrite to ammonia conversion. This resulted in reduction /inhibition of denitrification step, and hence reduction of denitrification compound such as Nitric oxide (NO).
Combination of denitrication process deletion and overexpression of nitrification process enzymes resulted in further increased amounts of Nitrite and Nitrate, and reduced levels of Nitric oxide.
Engineered strains were subjected to growth and ammonia consumption assay at controlled conditions. Wild type cells were also experimented in similar conditions, to test the increased Ammonia conversion finally to Nitrate. Nitrate,
Nitrite and Ammonia were analysed by Abcam-make colorimetric sensors. Nitrate level increased by 1.5 fold in final engineered strain, as compared to unmodified wild type strain, and 2 fold reduction in Nitric oxide (denitrification product). Nitrate/Nitrite ratio got increased by 2 fold, from ratio of 1.4 in wild type to ratio 5.6 in Final engineered strain.
Example 4: Impact of deletion of Denitrification process in addition to overexpression of Nitrification process, on enhanced concentrations of Nitrite and Nitrate:
Nitrospira sp. manipulated by deletion of Denitrification enzymes Nitrite reductase and Nitrate reductase, in addition to over expression of Nitrification enzymes, resulting in Engineered strains for further efficient Ammonia oxidation, as well as Nitrite oxidation to yield enhanced amounts of Nitrate finally. Analysis of Nitrite, Ammonia and Nitrate by sensor-based testing, showed Nitrate level increased by 1.5 fold in final engineered strain, as compared to unmodified wild type strain, and 2 fold reduction in Nitric oxide (denitrification product) (Figure 2)
Example 5: Incorporation of Nitrous oxide reduction process
Inorder to utilize the opportunity of itrous oxide reduction, as addition nitrogen foixation process, Genetic modification were focused on enabling Nitrous oxide (N2O) reduction to molecular Nitrogen (N2), by heterologous expression of N2O reductase. N2O reductase (nos gene) from Rhizhobium type of microbes was incorporated for additional N fixation.
Claims
1. A composition comprising genetically modified micro-organism(s) consisting of deletion/downregulation of Nitrite reductase enzyme (Nitrite to ammonia conversion) and Nitric acid reductase (Nitric oxide (NO) to Nitrous oxide N2O) and Nitrous oxide reducatse gene leading to an increase in nitrogenase activity, wherein the said micro-organism is an endophyte, an epiphyte and a rhizospheric microbe.
2. The composition as claimed in claim 1 wherein composition comprising genetically modified micro-organism(s) consisting of upregulation of Ammonia monooxygenase, Hydroxylamine oxidoreductase, Nitrite oxido reducatse genes wherein there is an improved nitrate production by these modified microbes compared to that of the wild type.
3. The composition as claimed in claim 1, wherein the gene integration process consists of plasmid vector, suicidal/ integration vector.
4. The composition as claimed in claim 2, wherein the Integration vector is carrying a DNA construct comprising a nucleotide sequence represented by SEQ ID NO 7.
5. The composition as claimed in claim 4, wherein SEQ ID NO.7 consisting of 2816bp gene (SEQ ID NO 1) with promoter (SEQ ID NO 6), within Nael sites of deletion gene (SEQ ID No 4).
6. The composition as claimed in claim 1, wherein the said plasmid vector is expressed in miro-organism selected from the group comprising of endophytic bacteria, epiphytic bacteria, rhizospheric bacteria and fungi.
7. The composition as claimed in claim 1, wherein the bacteria is selected from the group comprising ofcultivable bacteris (viz) Nitrococcus, Nitrospira, Pseudomonas.
8. The composition as claimed in claim 1, wherein the bacterium is selected from the group comprising of Nitrobacter.
9. A process for the preparation of genetically modified micro-organism(s) for improved Nitrate production and its delivery to crop-plants for assimilation,
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