Recombinant nitrogen fixing microorganism and uses thereof
A recombinant microorganism is provided herein, in particular, a recombinant microorganism of the Azotobacteraceae family. The recombinant Azotobacter microorganism is capable of fixing atmospheric nitrogen continuously in the presence of oxygen and externally fixed nitrogen sources. The present invention further provides a process for production of the recombinant microorganism and a composition comprising the recombinant microorganism for use as biofertilizers and/or for use in the preparation of a fertilizer composition. The recombinant microorganism produced by this invention is an environmental friendly, highly beneficial microorganism.
1. A recombinant microorganism comprising a disrupted chromosomal nifL gene and a chromosomal nifA gene operably linked to a heterologous constitutive promoter, wherein the microorganism is capable of fixing atmospheric nitrogen in presence of fixed nitrogen and oxygen and does not comprise antibiotic resistance marker gene.
2. The recombinant microorganism as claimed in claim 1 , wherein the microorganism is selected from a group consisting of Green sulfur bacteria, Firmibacteria, Thallobacteria, Heliobacteria, Cyanobacteria, Campylobacter , Proteobacteria, Archaeobacteria and Propionispira.
3. The recombinant microorganism as claimed in claim 1 , wherein the microorganism is Azotobacter spp.
4. The recombinant microorganism as claimed in claim 3 , wherein the Azotobacter is selected from a group consisting of Azotobacter chroococcum, Azotobacter vinelandii, Azotobacter armenaicus, Azotobacter beijerinckii, Azotobacter nigricans and Azotobacter paspali.
5. A recombinant Azotobacter having Accession number MTCC 5679, wherein the Azotobacter is capable of fixing atmospheric nitrogen in presence of fixed nitrogen and oxygen and does not comprise antibiotic resistance marker gene.
6. The recombinant microorganism as claimed in claim 1 , wherein the nifL gene is disrupted by insertional mutagenesis, deletion of genomic DNA, targeted gene disruption or introduction of a genomic or episomal vector.
7. A recombinant DNA molecule comprising a disrupted chromosomal nifL gene and a chromosomal nifA gene operably linked to a heterologous constitutive promoter.
8. A recombinant vector comprising the recombinant DNA molecule as claimed in claim 7 .
9. A composition comprising the recombinant microorganism as claimed in claim 1 .
10. The composition as claimed in claim 9 , wherein the composition optionally comprises a carrier.
11. The composition as claimed in claim 10 , wherein the carrier is an agriculturally acceptable carrier.
12. The composition as claimed in claim 9 , wherein the composition is a biofertilizer.
13. The composition as claimed in claim 9 , optionally comprising one or more constituents selected from a group comprising sources of potassium, phosphorus, carbon, nitrogen, iron, sulphur, calcium, magnesium, zinc, trace elements, growth factors, pesticides, anti-termites, bacteria, algae.
14. A process for producing a recombinant microorganism, as claimed in claim 1 , said process comprising:
(a) disrupting a chromosomal nifL gene comprising insertion of antibiotic resistance marker gene into said chromosomal nifL gene;
(b) inserting said disrupted nifL gene and a chromosomal nifA gene into a chromosome of a microorganism, wherein said disrupted nifL gene is upstream of said chromosomal nifA gene;
(c) replacing said antibiotic resistance marker gene with a heterologous constitutive promoter operably linked to said nifA gene.
15. The process as claimed in claim 14 , wherein said insertion of antibiotic resistance marker gene is carried out by interposon.
16. Method for enhancing fertility of a soil comprising: preparing a fertilizer composition comprising the recombinant microorganism as claimed in claim 1 ; and
providing said fertilizer composition to said soil.