WO2020245841A1

WO2020245841A1 - Genetic modification of endophytic/epiphytic/rhizospheric microbes for improved nitrogen fixation for crops

Info

Publication number: WO2020245841A1
Application number: PCT/IN2020/050494
Authority: WO
Inventors: Rahul Raju KANUMURU
Original assignee: Fertis India Pvt. Ltd.
Priority date: 2019-06-04
Filing date: 2020-06-03
Publication date: 2020-12-10

Abstract

The present invention relates to genetic modification of microbes for improved nitrogen fixation and its delivery to crop-plants for assimilation.More particularly, the present invention relates to gene modifications of the nifgene cluster comprising up regulation of positive regulators, down regulation of negativeregulators and over expression of structural genes to achieve enhanced nitrogenfixation.

Description

Genetic modification of endophytic/epiphytic/rhizospheric microbes for Improved

Nitrogen Fixation for Crops

TECHNICAL FIELD OF THE INVENTION:

Further, the present invention relates to genetic modification of associated genes such as amtB (gene for Ammonium transporter), glnA (gene for Glutamine synthetase), gls (gene forGlutaminase), such that enhanced nitrogen fixation is achieved leading to itsassimilation in crop plants. CRISPR/Cas9 technology is used preferentially for gene manipulation of microbes.

BACKGROUND AND PRIOR ART OF THE INVENTION:

Nitrogen is a critical limiting element for plant growth and production. It is a major component of chlorophyll, the most important pigment needed for photosynthesis, as well as amino acids, the key building blocks of proteins. It is also found in other important bio molecules, such as ATP and nucleic acids. Even though nitrogen is one of the most abundant elements, plants can only utilize reduced forms of this element. In order to utilize N₂, itmust be combined with hydrogen. The combining of hydrogen with N₂ is referred to as nitrogen fixation.

Plants acquire these forms of“combined” nitrogen by the addition of ammonia and/or nitrate fertilizer or manure to soil orthrough the release of these compounds during organic matter decomposition, or the conversion of atmospheric nitrogen into compounds by natural processes, such as lightning, and biological nitrogen fixation. Biological nitrogen fixation is a process whereby atmospheric N₂ is reduced to NH₃ and this process is catalyzed by microbial enzyme system comprising of nitrogenase. Since, plant cannot reduce atmospheric N₂ to NH₃; microorganisms having the capacity to fix atmospheric N₂ can be used as efficient biofertilizer.Nitrogen fixing endophytic bacteria has an edge over the rhizospheric counterpart because the endophyte lives inside the plant tissues and thereby faces less competition and makes available the fixed nitrogen directly to the plants.

According to the most recent estimate by the UN, there are 7.3 billion people and may reach 9.7 billion by 2050 resulting in an increase in food demand from 59% to 98% bv 2050.Therefore. farmers will need to increase crop production, either by increasing the amount of agricultural land to grow crops or by enhancing productivity on existing agricultural lands. In recent years, 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.

Considerable progress has been made in understanding the development of the nitrogen-fixing symbiosis between rhizobia and legumes, however, inducingand harbouring nitrogen- fixing nodules on non-leguminous crops has still not been achieved and used commercially.

PCT Publication No. W02017011602 discloses methods of increasing nitrogen fixation in non-leguminous plants comprising exposing the plant to a plurality of bacteria comprising one or more genetic variations introduced into one or more genesof 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. Further, the said method involves one or more genes or non-coding polynucleotides of the bacteria’s nitrogen fixation or assimilation genetic regulatory network are selected from the group consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifU, nifS, nifY, nifW, nifZ, nifM, nifF, nifB, and nifQ. However, it is important to note that the inventors of WO’602 have discussed gene modification mainly with respect to specific gene (nif) sequence, however, they have not demonstrated any such gene modification. Moreover, there are a plethora of uncharacterized prokaryotic and eukaryotic micro-organisms, which have not been used by the inventors of WO’602 for nitrogen fixation. In view of the aforementioned loopholes in the art, there is a need in the art to meet the challenge of providing sufficient supplemental sources of nitrogen, such as in fertilizer, to help to continue to increase with the growing need for increased food production.

OBJECT OF THE INVENTION:

It is an object of the present invention to provide a composition comprising genetically modified microbeswhich is favourable for fixing atmospheric nitrogen and its delivery to crop-plant for assimilation in leguminous and non-leguminous plants.

It is another object of the present invention to provide gene manipulation of microbeswhich may be endophytic, epiphytic or rhizospheric in nature.

It is a further object of the present invention to genetically modify themicrobes whi chare most common and prevalent in majority of crops and have been un-cultivable and uncharacterized till date.

SUMMARY OF THE INVENTION:

In a main aspect, the present invention provides for the genetic modification ofmicro- organisms selected from the group comprising of endophytic bacteria, an epiphytic bacteria, a rhizosphere bacteria, for improving nitrogen fixation abilities by CRISPR- Cas9 compared to wild type characteristic strains.

In an aspect, the present invention provides genetic modified microorganisms which are most common and prevalent in majority of crops and have been un-cultivable and uncharacterized. The present invention provides genetically modified microbes favourable for fixing atmospheric nitrogen and its delivery to crop-plant for assimilation.

The present invention provides a composition comprising genetically engineered micro- organism(s) consisting of deletion of nif L gene leading to an increase in the function of nifA product in nitrogenase activity, wherein the said micro-organism is uncharacterized and non-cultivated, wherein the said modiciation is carried out by CRISPR/Cas9 method.

In another aspect, the present invention providesgene manipulation in microbesresulting in deletion of NifL, increased expression of NifA or glutaminase; decreased expression of, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB, decreased adenylyl- removing activity of GlnEanddecreased uridylyl-removing activity of GlnD.

In one aspect, the present invention provides a process for enhancing nitrogen fixing abilities in leguminous and non-leguminonus plants.

In yet another aspect, the present invention provides a process for the preparation of genetically modified micro-organism(s) for improved nitrogen fixation and its delivery to crop-plants for assimilation by CRISPR/Cas9 technology.

(a) cloning a 20 bp protospacer sequence (SEQ ID NO.2) targeting the nifL gene in pAzCRISPR vector between the regions spanning nucleotides 3000 to 3900 bp of SEQ ID NO.7.

(b) transforming pCasAz vector having SEQ ID NO.6 containing the Cas9 and l-red recombination system into a host cell,

(c) mobilizing pAzCRISPR of step(a) and pCasAz of step (b) into a micro-organism wherein the said micro-organism is an endophyte, an epiphyte and a rhizospheric microbe, wherein the said micro-organism is uncharacterized and non-cultivated, wherein the said modiciation is carried out by CRISPR/Cas9 process.

A total of three cultivable and three non-cultivable microbes that were modified using the CRISPR/CAS9 method are given under table 1. The process of modification explained for Azoarcus sp. has been extended to the other five microbes. After confirmation, the nitrogen assimilation abilities of the six microbes were confined by Kjel-Dahl method (Fig. 11 and 12) using nitrogen-free growth medium. The overall experimental process to carry out the transformation and gene modification is simimar for the other remaining microbes in the present invention. In the current invention, genetically modified micro-organism(s) consisting of deletion/downregulation of nif L gene leading to an increase in nifA expression innitrogenase activity, wherein the said micro-organism is an endophyte, an epiphyte and a rhizospheric microbe. The mirobes in the current claim are not part of any other patent or claim involving endophyte work.where modification is not limited to the deletion/downregulation of nifL gene but also the upregulation of nifA gene wherein there is an improved nitrogen fixation by these modified microbes compared to that of the wild type, to be assimilated by the crop plants.

DETAILED DESCRIPTION OF DRAWINGS:

Figure 1 depicts an overview of NiFA/NifL/rnfOperon orientation (in blue arrows) in Azoarcus genome organization. The black arrows represent primers designed for PCR amplification and confirmation of the inserts into CRISPR plasmids.

Figure 2 depicts the amplification of nifA and rnfA gene from genomic DNA from Azoarcus sp. Lane 1, 1 kb DNA ladder; lane 2, Amplification of nifA gene; lane 3, amplification of rnfA gene.

Figure 3 depicts thePCR confirmation of Invitro fusion nifA and rnfA genes. Lane M- lkb DNA ladder; lanel-amplification of repair template.

Figure 4 depicts theGenetic organization of nifAL operon and rnf operon in wild type and post invitro fusion.

Figure 5 depicts complete sequence map of pAzCRISPR (in-house designed plasmid DNA designed for Azoarcus sp. improvement over the wild type). pAzCRISPR consists of 5678bp with selective restriction sites; selective antibiotic resistance markers; guide RNA (gRNA) scaffold; Ori sequence.

Figure 6 depicts thePCR confirmation of recombinant plasmid pAzCRISPR assembled with spacer and repair template. Lane M, 1 kb DNA ladder; lane 1-9, amplification of repair template and spacer cloned in pAzCRISPR plasmid. NifL spacer sequence was used as the forward primer and rnfA sequence was used as the reverse primer.

Figure 7 depicts the complete sequence map of pCasAz (in-house designed plasmid DNA designed for Azoarcus sp. improvement over the wild type). pCasAzconsists of 12924bp with selective restriction sites; selective antibiotic resistance markers; Cas9 sequence cassette; arabinose promoter; gene sequence; insertion site; Ori sequence. Figure 8 depicts PCR confirmation of cas9 gene. Lane M, 1 kb DNA ladder; lane 1 : negative control; lane 2: positive control; lanes 3-4: Azoarcus sp. with pCasAz sequence.

Figure 9 depicts BamHl restriction and release of oriT (680 bp) from pAzCRISPR assembled with spacer and repair template.

Figure 10 depicts Lane M: 1 kb DNA ladder; Lane 1 : negative control; lane 2: PCR amplification of spacer and repair template (2.5kbp) cloned in pCasAz vector.

Figure 11 depicts Nitrogen estimation using Kj el -Dahl method. Three cultivable microbes were grown in nitrogen-free medium for estimation. Methylobacterium extorquens; Beijerinckia indica; Azoarchus communis were used in the present invention.

Figure 12 depicts Nitrogen estimation by Kj el -Dahl method. Uncultivable microbes were grown in nitrogen-free medium for ammonia estimation. (P.S. = Pseudomonas sp.; B.S .= Bacillus sp.; SP: Sphingomonas sp. were found to exhibit highest nitrogen fixing ability are shown in the figure.)

Source of biological material: Cultivable microbes ( Methylobacterium extorquens; Beijerinckia indica; Azoarchus communis ) and un-cultivable and uncharacterised Pseudomonas sp.; Bacillus sp. and Sphingomonas sp (have been isolated by the process established in the present Applicants’s PCX Publication No. WOWO2019/058390) details are given below:

Table 1 : List of cultivable and uncultivable microbes used in the present study for genome modification for higher nitrogen fixing capability using CRISPR/Cas9 method.

DETAILED DESCRIPTION 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.

In a most preferred embodiment, the present invention provides a genetically modified micro-organism which is uncultivable and uncharacterized for improving nitrogen fixing abilities in leguminous and non-leguminous plants.

Accordingly, the present invention provides genetic modification of an endophytic bacteria, an epiphytic bacteria, a rhizosphere bacteria, and eukaryotic organisms for improving the nitrogen fixation abilitiesin leguminous and non-leguminous plants. These endophytes were improved from their wild type characteristic strains using CRISPR-Cas9 tools. The said endophytic bacterium is Azoarcus indigens ; an epiphytic bacterium, is Methylobacterium extorquens and a rhizosphere bacterium, Beijerinckia indica for improving the nitrogen fixation abilities.

The present invention employs CRISPR/Cas9 methodology. CRISPR is being widely used in plant science for improving genetic traits, develop better varieties, enhanced disease resistance etc. CRISPR-Cas9 based methodology is similar in procedural context among various sets of experiments in plant and animal models, but differs extensively with the sequences involving modifications/improvements based on the requirements. The CRISPR/Cas9 system has been harnessed in the present invention to provide a simple, RNA-programmable method to selectively edit genomes; create gene knockouts by insertion/deletion or knockings. Accordingly, in the present invention to give rise to a gene variation, a single guide RNA (sgRNA) is generated to direct the Cas9 nuclease to a specific genomic location. Cas9-induced double strand breaks are repaired via the NHEJ DNA repair pathway. The basic principle of CRISPR/Cas9- mediated gene editing involves a singleguide RNA (sgRNA), consisting of a crRNA sequence that is specific to the DNA target, and a tracrRNA sequence that interacts with the Cas9 protein, binds to a recombinant form of Cas9 protein that possess DNA endonuclease activity. The resulting complex will lead to a target-specific double- stranded DNA cleavage. The cleavage site will be repaired by a nonhomologous end joining (NHEJ) DNA repair pathway.

In one preferred embodiment, the present invention provides a pAzCRISPR vector construct represented by Seq Id No. 7 consisting of 5678bp with selective restriction sites; selective antibiotic resistance markers; guide RNA (gRNA) scaffold; Ori sequence (Fig 5).

The main role of pAzCRISPR plasmid in the present invention is to act as a repair construct that harbour the guide RNA (gRNA) which is the gene sequence specific compelentary to the target (host) genome sequence (here the region of NifL is the target for deletion where the sequence complementary to the N and C terminus coding regions are included in the gRNA). The region is regarded as the repair template that caries out the required modification in the host genome as a result of specific RNA-DNA hybrid identified by the Cas9 nuclease for excision and gene editing. The gRNA in pAzCRISPR is cloned between the regions spanning nucleotide 3000 to 3900 of Seq Id No.7. The repair template was cloned in Xbal and Xhol site of the in-house designed pAzCRISPR vector.

The present invention provides pCasAz vector containing the Cas9 and l-red recombination system was at first transformed into E. coli S17 l pir strain. E. coli S17 l pir strain serves as a donor strain for bi-parental mating which was the sole possinility to mobilize the pCasAz into Azoarcus sp. was used in the current study. pCasAz vector consists of 12924 bp having SEQ ID NO 6 including selective restriction sites; antibiotic resistance markers; Cas9 sequence cassette; arabinose promoter; gene sequence; insertion site and Ori sequence (Fig 7).

In the present invention, NIF-regulon was used for necessary modifications to generate enhanced nitrogen fixing ability microbe species viz. Azoarcus sp. The genomic organisation of Nil· (Nitrogen fixation) regulon.The organization of NiFA-NifL-rnfA operon and its orientation (shown in blue arrows) in Azoarcus genome organization is shown in Fig.1.

In a preferred embodiment, the present invention provides gene manipulation in endophytic/epiphytic/rhizospheric microbesthat results in increased expression of NifA or glutaminase; decreased expression of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB, decreased adenylyl-removing activity of GlnE, and decreased uridylyl- removing activity of GlnD.

Accordingly, the present invention provides microbes selected from nitrogen fixing bacteria subjected to mutation as follows:

N-fixing bacteria isolated according to methods of the disclosure can comprise a plurality of different bacterial taxa in combination. 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.

In a further embodiment, the present invention provides endophytes selected from the group comprising Proteobacteria (such as Azoarcus, Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea, Serratia, Rahnella, Azospirillum, Azorhizobium, Azotobacter, Duganella, Delflia, Bradyrhizobiun, Sphingomonas, Sinorhizobium and Halomonas), Firmicutes (such as Bacillus, Paenibacillus, Lactobacillus, Mycoplasma, and Acetabaclerium)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, an epiphytic bacterium (Methylobacterium extorquens) and a rhizosphere bacterium ( Beijerinckia indica) are selected for genetic modification for nitrogen fixation.

In another embodiment, certain fungi comprising Saccharomyces cerevisiae and Trichoderma harzianum are selected for genetic modification for nitrogen fixation.

In another embodiment, the present invention provides the horizontal transfer of nif gene cluster into endophytic strains of E. coli for the purpose of biological nitrogen fixation.

The microbes may be isolated from terrestrial environment, including its soils, plants, fungi, animals (including invertebrates) and other biota. Additionally, microbes can be isolated from insects, nematodes and algae.Further, uncultivable microbes especially endophytes were isolated from various crops and subsequently the common isolate present in all crops will be genetically modified for higher N-fixation and its transport to plant cell for assimilation.

In an embodiment, the present invention provides one or more genetic variations or combinations thereof selected from the group comprising a knock-out mutation; altering a regulatory sequence of a target gene; and inserting a heterologous regulatory sequence into the native sequence.

Accordingly, in one of the embodiment, the mutation in the microbe is performed in a gene selected from the group comprising ofnifA, nifL, ntrB, ntrC, glnA, glnB, glnK, draT, amtB, glnD, glnE, nifJ, nifH, nifD, nifK , nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, niffi, and nifQ.

The negative regulators is selected from the group comprising nifL (Gene for Nitrogen fixation regulatory protein, NifL), ntrB (Gene for FeMo cofactor biosynthesis protein, NifB, glnA (Gene for glutamine synthetase, GS), glnB (Gene for Nitrogen regulatory protein R-P 1), glnK (Gene for Nitrogen regulatory protein P-II 2), draT (Gene for Dinitrogenase reductase ADP-ribosyl transferase), amtB (Gene for ammonium transporter), glnE (Gene for GS adenylyl transferase) and glnD (Gene for Bifunctional uridylyltransferase).

The potentialmethod of genetic variation to control negative regulatorsis selected from a combination of the methods selected from the group comprising of Knock-out (null mutation), Point mutation through site directed mutagenesis (affecting the active site of protein) and deletion mutation (affecting the domain of protein). The positive regulator is selected from the group comprising nifA (Gene for Nif-specific regulatory protein) and GLS (Gene for Glutaminase).

The potential method of genetic variation to control positive regulators is selected from over expression of gene and increased expression through a strong promoter/weak start codon can be swapped out with an ATG for better translational efficiency/different RBS (ribosome binding site) with higher translational initiation efficiency.

Further, over expression of structural genes of nitrogenase (nifH, nifD and nifK) will be taken up for enhanced nitrogenase activity.

Elucidation for gene manipulation studies:

• NifA is the positive regulator of nif gene cluster

• NifL inhibits NifA activity by protein interaction

• NifL is regulated by products of glnD and glnK

• NtrB regulates NtrC which in turn regulates nifL and nifA

• AmtB (Ammonium transporter) activates NifL through GlnK

• GlnA (Glutamine synthetase, GS) and GLS (Glutaminase) are inversely related and GlnA down regulation and GLS up regulation in microbe will disrupt ammonia assimilation and enables its export from microbe to plant.

• GlnE (GS adenylyl transferase) and GlnB (Nitrogen regulatory protein P-II 1): Both are involved in regulation of glutamine synthetase

• DraT (Dinitrogenase reductase ADP-ribosyl transferase) down regulation will decrease the inactivation of nitrogenase in the microbe once the nitrogen fixing enzymes are produced at sufficient level (in high nitrogen condition in the cell) Further in some embodiments, the effect of above mentioned gene manipulations in microbes under study was determined by transcriptomics, proteomics. A global investigation of engineered nitrogen fixing strains will be carried out with respect to metaproteogenomics in order to understand the complexity of nif gene regulation and thereby enhanced nitrogen fixation and ultimately aiming at its delivery to and assimilation in plant cell.

GM microbes (Heterologous expression) with enhanced nitrogen fixation:

Genetically modified microbes for better nitrogen fixation will be achieved through:

• Null mutation/point mutation/deletion mutation of negative regulators of nif gene

• Heterologous expression of positive regulators of nif gene (either single copy or multi copy)

• Heterologous expression of nitrogenase structural genes (nifHDK gene cluster) that codes for proteins with higher specific activity

Non-GM microbes (homologous manipulation) with enhanced nitrogen fixation:

Non-genetically modified microbes will be used for organic farming and will be achieved through:

• Null mutation of negative regulators of nif gene

• Homologous over expression of positive regulators of nif gene

• Homologous over expression of nitrogenase structural genes (nifHDK gene cluster)

In another embodiment, genetically modified microbial consortia were designed based on different growth stages of crop. For instance, for soil application microbial consortia consisted of microbes which colonize root. Subsequently, consortia for foliar application consisted of GM microbes which colonize shoot and leaves.

In another preferred embodiment, the present invention provides a method of increasing nitrogen fixation ina non-leguminous plant, the said method comprising exposing the plant with improved nitrogen fixing microbes; themicrobes comprising one or more genetic variations introduced into one or more genes or non-coding polynucleotide of the microbe’s nitrogen fixation and/or assimilation genetic regulatory network, such that the genetically modified microbe is capable of fixing atmospheric nitrogen and delivering it to plant for its assimilation.

In some embodiments, the genetically modified bacteria or fungi of the present disclosure are present in the plant in an amount of at least 10³ cfu, 10⁴ cfu, 10⁵ cfu, 10⁶ cfu, 10⁷ cfu, 10⁸ cfu, 10⁹ cfu, 10¹⁰ cfu, 10¹¹ cfu, or 10¹² cfu per gram of fresh or dry weight of the plant.

Nitrogen assimilation in plants:

Nitrogen assimilation is the formation of organic nitrogen compounds such as amino acids from inorganic nitrogen compounds like nitrate or ammonia.

Plants absorb nitrogen from soil in the form of nitrate or ammonium.

• In aerobic soils it is nitrate which is predominant form of nitrogen that is absorbed.

• However, in flooded, anaerobic soils like rice fields, ammonia can predominate.

Ammonium ions are absorbed through ammonia transporters.

Further, nitrate is taken up by several nitrate transporters. Ultimately nitrate is converted to ammonia by reduction reaction in two steps. Nitrate is first reduced to nitrite in cytosol by nitrate reductase. Next, nitrite is reduced to ammonia in chloroplasts or plastids by nitrite reductase.

Importantly, diazotrophic microbes reduce atmospheric nitrogen and converts in to ammonia and supply to the plant. So, ultimately nitrogen reaches the chloroplast either in the form of nitrite or ammonia. Nitrite reduced to ammonia in chloroplast and thus available, total ammonia in chloroplast is converted to glutamine by Glutamine synthetase (GS). In the next step, Glutamate synthase (GOGAT) transfers amide group glutamine onto a 2-oxoglutarate molecule producing two glutamates. Further, transaminations are carried out to make other amino acids, most commonly asparagine from glutamine. The enzyme involved is asparaginase synthetase. So, in order to up regulate assimilation of ammonia into amino acids in plants through endophyte, enzymes such as GS and GOGATfrom endophyte to chloroplasts/plastids of plant cells are targeted. In yet another preferred emboidment, the present invention provides a process for the preparation of genetically modified micro-organism(s) for improved nitrogen fixation and its delivery to crop-plants for assimilation by CRISPR/Cas9 technology.

(a) cloning a 20 bp protospacer sequence having SEQ ID NO.2 targeting the nifL gene in pAzCRISPR vector between the region spanning nucleotide 3000 to 3900 bp of SEQ ID NO.7.

(c) mobilizing pAzCRISPR of step(a) and pCasAz of step (b) into a micro-organism, wherein the said micro-organism is an endophyte, an epiphyte and a rhizospheric microbe, wherein the said micro-organism is uncharacterized and non-cultivated, wherein the said modiciation is carried out by CRISPR/Cas9 process.

From the six microbes tested, three belonged to uncultivable microbes (1) Pseudomonas sp.; (2) Bacillus sp. (3) Sphingomonas sp. were used for strain improvement using CRISPR/Cas9 tools. The other three cultivable microbes which were improved in the present study using CRISP R/Cas9 tools were (1) Methyl oh acted um extorquens (2) Azoarcus sp. (3) Beijerinckia indica. Based on the results obtained from Kjeldahl method, highest amount of nitrogen assimilation was observed from an uncultivable Pseudomonas sp. (1368mg/L) (Fig. 11) followed by Methyl ob acterium extorquens (1128.75mg/L) (Fig.12).

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: Isolation of genomic DNA from Azoarcus sp.

Total genomic DNA was extracted from 5ml culture of Azoarcus sp. grown to late-log phase using Qiagen blood and tissue kit following the manufacturer’s instructions and confirmed in agarose gel electrophoresis. Example 2: Designing of spacer targeting nif-L gene and off-target prediction

The nifL gene was identified from the genomic DNA of Azoarcus sp. The 20 bp protospacer sequence specific for ntfL gene adjacent to the PAM site was designed which would be targeted by Cas9 protein and introduce a double-stranded break in the genome. Off-target was predicted using CasOT software (Xiao et al. 2014). The possible protospacer sequence in nifL gene along with PAM site was searched against the Azoarcus sp. genome to identify off-targets. Primers were designed that could confirm the protospacer sequences.

Example 3: Amplification and invitro fusion of nifA and rnfA gene

Primers were designed to amplify upstream sequence of nifA and downstream to rnfA genes to nifL using genomic DNA of Azoarcus sp. as template. nifA and rnfA genes were PCR amplified and confirmed yielding 1.5 kb and 950 bp products respectively (Fig. 2). The PCR products were restriction double digested with appropriate enzymes and ligated. The ligation product was purified using Qiagen gel extraction kit according to the manufacturer’s instructions. The purified product was used as the template to amplify invitro fused nifA-rnfA genes using primers nifA specific reverse and rnfA specific reverse (Fig. 3). This fused product severs as a repair template for homologous recombination. The genetic organization of nifA ; nifL and rnfA operons in wild type and post invitro fusion is shown in Fig. 4.

Example 4: Cloning of spacer and repair template into pAzCRISPR plasmid.

The repair template was cloned in Xbal and Xho\ site of the in-house designed pAzCRISPR vector. pAzCRISPR vector construct consists of 5678bp with selective restriction sites; selective antibiotic resistance markers; guide RNA (gRNA) scaffold; Ori sequence (Fig 5). 1 mg of plasmid and invitro fused repair template was digested with Xbal and Xhol for 3 hours and the linearized vector and PCR product were eluted using Qiagen gel extraction kit following the manufacturer’s protocol. The vector and insert (1 :3) were ligated using T4 DNA ligase overnight at 16°C. The ligation mixture was inactivated at 75° C for 10 mins and transformed into E. coli DH5a strain. The recombinant plasmids were confirmed by PCR using nifL spacer as the forward primer and rnfA reverse primer. 1 mg of the recombinant plasmid was cloned into E. coli DH5a by calcium-chloride method followed by heat-shock (quenched plasmid DNA sample was mixed with pre-chilled DH5a and incubated for 10 min) and subjected to 42°C heat shock treatment for 40s and immediately quenched on ice for 5min. The transformed E. coli DH5a cells were plated in LA-selective plates for screening transformed bacterial colonies. The recombinant plasmids were confirmed by PCR using nifL spacer region as the forward primer and rnfA spanning sequence as the reverse primer. PCR was pefromed in Eppendorf Thermal Cycler with 35 cycles with required dNTP mixture; DNA polymerase, buffers. The 20 bp protospacer sequence targeting nifL gene was cloned in Bsal site of pAzCRISPR vector by Golden gateway cloning (Fig 5).

Example 5: Mobilisation of pCasAz into Azoarcus sp.

For an efficient mobilization of the pCasAz vector (in-house designed) containing the Cas9 and l red recombination system into Azoarcus sp., was initially transformed to E. coli S17 l pir strain, which serves as donor strain for bi-parental mating was used in the current study. The pCasAz consists of 12924bp having SEQ ID NO.6 with selective restriction sites; selective antibiotic resistance markers; Cas9 sequence cassette; arabinose promoter; gene sequence; insertion site; Ori sequence (Fig 6). The pCasAz plasmid was extracted from E. coli DH5a strain and lmg of plasmid was transformed into E. coli S17 l pir using CaCI₂ mediated method. E. coli S17 l pir strain harboring pCasAz plasmid was confirmed by PCR amplification of the cas9 gene. 50 ml Azoarcus sp. was grown to late log phase in CV ethanol medium. Similarly, 50 ml E. coli S17 l pir harboring pCasAz was grown in minimal medium supplemented with 0.4% glucose, proline (40 mg/ml), threonine (40 mg/ml) and thiamine (2 mg/ml). Both cultures were mixed in 3 :1 ratio and spotted on CV ethanol medium supplemented with 0.4% glucose, proline (40 mg/ml), threonine (40 mg/ml) and thiamine (2 mg/ml). The plate was incubated overnight at 30° C. The cells were scraped from plate suspended in CV ethanol and plated on CV ethanol medium containing tetracycline (10 mg/ml). Azoarcus sp. harboring pCasAz was PCR confirmed for cas9 sequence (Fig. 7).

Example 6: Cloning of oriT in pAzCRISPR plasmid with spacer and repair template

To mobilize pAzCRISPR plasmid assembled with spacer and repair template, the origin of transfer ( oriT) gene was cloned in BamHl site of recombinant pAzCRISPR construct. The oriT gene was amplified from pCasAz vector and cloned in the pGEM T vector. Recombinant pAzCRISPR vector and recombinant pGEM_T vector was digested with BamHl restriction enzyme. The digested products were eluted using Qiagen gel extraction kit. The vector and insert were ligated using T4 DNA ligase. The ligation product was transformed into E. coli S17 l pir strain. The recombinant clones were confirmed by the release of oriT gene from pAzCRISPR vector using BamHl yielding a 680 bp product (Fig. 9).

Example 7: Mobilization of pAzCRISPR-oriT, spacer and repair template into Azoarcus sp. harboring pCasAz

The donor E. coli S17 l pir strain harboring pAzCRISPR construct assembled with spacer and repair template was mixed with Azoarcus sp. harboring pCasAz in 1 :3 ratio. The cells were spotted on LB medium and incubated at 30° C overnight. Post incubation the cells were scrapped and plated on LB medium containing tetracycline (10 mg/ml) and ampicillin (100 mg/ml). The transformants were confirmed by PCR amplification of cas9 gene-specific for pCasAz and ampicillin resistant cassette specific for pAzCRISPR plasmid.

Example 8: Cloning of spacer and repair template in pCasAz

As an alternate strategy, the guide RNA and repair template were cloned in pCasAz vector. The pCasAz plasmid was digested with Xhol enzyme and end filled using T4 DNA polymerase. The end filled vector was purified using Qiagen gel extraction kit. Using NifL F spacer and RnfA reverse primers encompassing spacer and repair template was amplified using Pfu polymerase which yielded a 2.5 kb product. The blunt-ended PCR product was ligated to pCasAz plasmid post end filling. The ligation mixture was transformed into E. coli S17 l pir strain. The transformants were confirmed by PCR amplification of cas9 gene and spacer and repair template.

Example 9: Mobilisation of pCasAz spacer and repair template into Azoarcus sp.

Azoarcus sp. was grown to late log phase in CV ethanol medium. Similarly, E. coli S17 l pir harboring pCasAz assembled with spacer and repair template was grown in minimal medium supplemented with 0.4% glucose, proline (40 mg/ml), threonine (40 mg/ml) and thiamine (2 mg/ml). Both donor and recipient cultures were mixed in 3 :1 ratio and spotted on CV ethanol medium supplemented with 0.4% glucose, proline (40 mg/ml), threonine (40 mg/ml) and thiamine (2 mg/ml). The plate was incubated overnight at 30°C. The cells were scraped from plate suspended in CV ethanol and plated on CV ethanol medium containing tetracycline (10 mg/ml). Azoarcus sp. harboring pCasAz with spacer and repair template was confirmed by PCR amplification of spacer and repair template (Fig. 10).

Example 10: Isolation of uncultivable endophytes using I-Chip from different plant sources capable of nitrogen fixation

The present invention using Ichip was useful in isolation of both cultivable and uncultivable endophytes followed by their identification via 16S rRNA/ITS gene sequencing (Reference: WO2019/058390 of the present applicant). In order to prepare inoculum for Ichip, 1-10 % of surface sterilized leaves/ stem/roQt/floiver/ seed and fruit (w/v) were crushed in sterile PBS and different dilutions (10^-2 to 10^-6) were plated on nutrient agar plate to enumerate the CFU from used plant sample. Same procedure was employed to process root, stem, seed, flower and fruit samples of the plant. Based on CFU count the same inoculum was diluted with 0.8% melted agar with minimum growth media, so as to achieve CFU of countable numbers. This was done so that cultivable endophyte can be obtained for later comparison with uncultivable endophytes after in-situ incubation via Ichip. After 4 week in-situ incubation, colonies were isolated from Ichip followed by DNA extraction and PCR using RAPD primers. Based on RAPD profile of endophytes isolated from different plant sources, and Ichip as in-situ incubation device, 3 endophytes were found to be different than group of cultivable endophytes with nitrogen fixation abilities.

Example 11: Determination of total nitrogen content assimilated by the microbes by Kjeldahl method

Nitrogen is one of the five major elements found in organic materials such as protein. The Kjeldahl method of nitrogen analysis is the worldwide standard for calculating the protein content in a wide variety of materials ranging from human and animal food, fertilizer, waste water and fossil fuels. In our study we followed the Kjeldahl procedure as described by Saha et al., 2012. Briefly, the Kjeldahl method consists of three steps, which have to be carefully carried out in sequence: (a) The sample is first digested in strong sulfuric acid in the presence of a catalyst, which helps in the conversion of the amine nitrogen to ammonium ions.

(b) The ammonium ions are then converted into ammonia gas, heated and distilled. The ammonia gas is led into a trapping solution where it dissolves and becomes an ammonium ion once again,

(c) Finally the amount of the ammonia that has been trapped is determined by titration with a standard solution, and a calculation made to determine the nitrogen content in mg/L.

Based on the experimental results obtained from Kjeldahl method, the nitrogen fixing ability of six microbes was determined. From the six microbes tested, three belonged to uncultivable microbes (1) Pseudomonas sp.; (2) Bacillus sp. (3) Sphingomonas sp. were modified using CRISPR/Cas9 tools. The other three cultivable microbes which were improved in the present study using CRISPR/Cas9 tools were (1) Methyl ob acterium extorquens (2) Azoarcus sp. (3) Beijerinckia indica. Based on the results obtained from Kjeldahl method, highest amount of nitrogen assimilation was observed from an uncultivable Pseudomonas sp. (1368mg/L) (Fig. 11) followed by Methylobacterium extorquens (1128.75mg/L) (Fig.12).

Advantages of the present invention:

• The present invention provides an in-house designed CRISPR based vector (pAzCRISPR) and a Cas9 based vector (pCasAz) such that it is possible to accommodate other genes of interest with respect to the nitrogenase complex or other operon from other bacterial strains. Therefore, the present pAzCRISPR and and pCasAz vector serve as a broad range vector for novel or unreported nitrogen fixing organisms.

• The present invention provides three un-cultivable endophytes exhibiting good nitrogen fixing properties which were improved upon from the wild type strain using the same research methodology as employed for Azoarcus strain. More number of nitrogen fixing microbes could be used as a combination or consortium to enhance the overall nitrogen fixation by the plant.

• In the present invention, multiple strategies have been implemented; nifL has been deleted from the nitrogenase complex along with improvement with respect to modification of the nifA promoter region to facilitate a much better nifA product generation and nitrogenase activity.

SEQUENCE LISTING

Claims

We claim,

1. A composition comprising genetically modified micro-organism(s) consisting of deletion/downregulation of nif L gene leading to an increase in nifA expression in nitrogenase activity, wherein the said micro-organism is an endophyte, an epiphyte and a rhizospheric microbe.

wherein the said micro-organism is uncharacterized and non-cultivated, wherein the said modiciation is carried out by CRISPR/Cas9 process.

2. The composition as claimed in claim 1 where modification is not limited to the deletion/downreguation of nifL gene but also the upregulation of nifA gene wherein there is an improved nitrogen fixation by these modified microbes compared to that of the wild type, to be assimilated by the crop plants.

3. The composition as claimed in claim 1 where modification is not limited to nifL and nifA gene; but selected from the group comprising NtrB/AmtB/GlnA/GlnE/DraT, wherein there is an improved nitrogen fixation by these modified microbes compared to that of the wild type, to be assimilated by the crop plants

4. The composition as claimed in claim 1, wherein the CRISPR/Cas9 process consists of plasmid vector.

5. The composition as claimed in claim 2, wherein the plasmid vector of pAzCRISPR is carrying a DNA construct comprising a nucleotide sequence represented by SEQ ED NO. 7.

6. The composition as claimed in claim 4, wherein SEQ ED NO.7 consisting of 5678bp with selective restriction sites; spacer, selective antibiotic resistance markers; guide RNA (gRNA) scaffold and Ori sequence.

7. The composition as claimed in claim 2, wherein the plasmid vector of pCasAz is carrying a DNA construct comprising a nucleotide sequence represented by SEQ ED NO. 6.

8. The composition as claimed in claim 6, wherein SEQ ID NO.6 is a DNA construct consisting of 12924bp with selective restriction sites; selective antibiotic resistance markers; Cas9 sequence cassette; arabinose promoter; gene sequence; insertion site and Ori sequence.

9. 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.

10. The composition as claimed in claim 1, wherein the bacteria is selected from the group comprising ofcultivable bacteris (viz) Methylobacterium extorquens; Beijerinckia indica; Azoarchus communis and uncultivable bacteria adapted to cultivabe using Ichip method (viz) Pseudomonas sp.; Bacillus sp. and Sphingomonas sp.

11. The composition as claimed in claim 1, wherein the fungi is selected from the group comprising of Saccharomyces cerevisiae and Trichoderma harzianum.

12. The composition as claimed in claim 1, wherein the bacterium is selected from the group comprising of E.coli.

13. A process for the preparation of genetically modified micro-organism(s) for improved nitrogen fixation and its delivery to crop-plants for assimilation by CRISPR/Cas9 technology.

(d) cloning a 20 bp protospacer sequence having SEQ ID NO.2 targeting the nifL gene in pAzCRISPR vector between the region spanning nucleotide 3000 to 3900 bp of SEQ ID NO 7.

(e) transforming pCasAz vector having SEQ ID NO.6 containing the Cas9 and l-red recombination system into a host cell,

(f) mobilizing pAzCRISPR of step(a) and pCasAz of step (b) into a micro organism. wherein the said micro-organism is an endophyte, an epiphyte and a rhizospheric microbe. wherein the said micro-organism is uncharacterized and non-cultivated, wherein the said modiciation is carried out by CRISPR/Cas9 process.