RECOMBINANT MICROORGANISM INCLUDING GENETIC MODIFICATION THAT INCREASES ACTIVITY OF NITRIC OXIDE REDUCTASE AND METHOD OF REDUCING CONCENTRATION OF NITRIC OXIDE IN SAMPLE BY USING THE SAME

Abstract

A recombinant microorganism including a genetic modification that increases activity of nitric oxide reductase in the recombinant microorganism, a composition for reducing a concentration of nitric oxide in a sample, the composition including the recombinant microorganism, and a method of reducing a concentration of nitric oxide in a sample, are disclosed.

Claims

1. A recombinant microorganism comprising a genetic modification that increases activity of nitric oxide reductase in the recombinant microorganism.

2. The recombinant microorganism of claim 1, wherein the genetic modification increases the copy number of a gene encoding the nitric oxide reductase.

3. The recombinant microorganism of claim 1, wherein the nitric oxide reductase is a nitric oxide reductase derived from the genus Escherichia, a nitric oxide reductase derived from the genus Paracoccus, or a combination thereof.

4. The recombinant microorganism of claim 1, wherein the nitric oxide reductase is a nitric oxide reductase derived from Escherichia coli, a nitric oxide reductase derived from Paracoccus versutus, or a combination thereof.

5. The recombinant microorganism of claim 1, wherein the nitric oxide reductase is a flavodiiron protein or cytochrome c-dependent nitric oxide reductase having identification number EC 1.7.2.5.

6. The recombinant microorganism of claim 1, wherein the nitric oxide reductase comprises a polypeptide having 75% or greater sequence identity to the amino acid sequence of SEQ ID NOS: 1, 2, 3, or 4.

7. The recombinant microorganism of claim 1, wherein the recombinant microorganism belongs to the genus Escherichia or the genus Paracoccus.

8. A composition for reducing a concentration of nitric oxide in a sample, the composition comprising a recombinant microorganism comprising a genetic modification that increases activity of nitric oxide reductase in the recombinant microorganism.

9. The composition of claim 8, wherein the genetic modification increases the copy number of a gene encoding the nitric oxide reductase.

10. The composition of claim 8, wherein the nitric oxide reductase is a flavodiiron protein or a cytochrome c-dependent nitric oxide reductase5.

11. The composition of claim 8, wherein the nitric oxide (NO) is in a form of Fe(II)(L)—NO, and is a complex formed by chelating a chelating agent L with Fe.sup.2+ and NO.

12. The composition of claim 8, wherein the sample is in a liquid or a gas state.

13. The composition of claim 8, wherein the recombinant microorganism belongs to the genus Escherichia or the genus Paracoccus.

14. A method of reducing a concentration of nitric oxide in a sample, the method comprising contacting a recombinant microorganism comprising a genetic modification that increases activity of nitric oxide reductase in the recombinant microorganism with the nitric oxide-containing sample and reducing the concentration of nitric oxide in the sample.

15. The method of claim 14, wherein the genetic modification increases the copy number of a gene encoding the nitric oxide reductase.

16. The method of claim 14, wherein the nitric oxide reductase is a flavodiiron protein (FDP) or a cytochrome c-dependent nitric oxide reductase.

17. The method of claim 14, wherein the nitric oxide (NO) is in a form of Fe(II)(L)—NO, and is a complex formed by chelating a chelating agent L with Fe.sup.2+ and NO.

18. The method of claim 14, wherein the contacting is performed in a sealed container.

19. The method of claim 14, wherein the contacting comprises culturing or incubating the recombinant microorganism in the presence of the nitric oxide-containing sample.

20. The method of claim 14, wherein the contacting comprises culturing the recombinant microorganism in a sealed container and under conditions in which the recombinant microorganism grows.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The above and other aspects, features, and advantages of certain embodiments of the disclosure will be more apparent from the following description taken in conjunction with the accompanying drawings, in which:

[0053] FIG. 1 is a graph which shows the conversion of Fe(II)EDTA-.sup.15NO to .sup.15N.sub.2O over time using recombinant Escherichia coli (E. coli) in which a nitric oxide reductase (NOR) pathway is enhanced;

[0054] FIG. 2 is a graph which shows the conversion of Fe(II)EDTA-.sup.15NO to .sup.15N.sub.2 using recombinant Paracoccus versutus (P. versutus) in which the NOR pathway is enhanced; and

[0055] FIG. 3 is a graph which shows the conversion of Fe(II)EDTA-.sup.15NO to .sup.15N.sub.2 using recombinant Paracoccus versutus in which the NOR pathway is enhanced.

DETAILED DESCRIPTION

[0056] Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects.

[0057] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. “At least one” is not to be construed as limiting “a” or “an.” As used herein, “a,” “an,” “the,” and “at least one” do not denote a limitation of quantity, and are intended to cover both the singular and plural, unless the context clearly indicates otherwise. For example, “an element” has the same meaning as “at least one element,” unless the context clearly indicates otherwise. “Or” means “and/or.” As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. It will be further understood that the terms “comprises” and/or “comprising,” or “includes” and/or “including” when used in this specification, specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof.

[0058] “About” or “approximately” as used herein is inclusive of the stated value and means within an acceptable range of deviation for the particular value as determined by one of ordinary skill in the art, considering the measurement in question and the error associated with measurement of the particular quantity (i.e., the limitations of the measurement system). For example, “about” can mean within one or more standard deviations, or within ±30%, 20%, 10% or 5% of the stated value.

[0059] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0060] Hereinafter, the present disclosure will be described in more detail with reference to exemplary embodiments. However, these exemplary embodiments are only for illustrating the present disclosure, and the scope of the present disclosure is not limited to these exemplary embodiments.

EXAMPLE 1

Examination and Improvement of Ability of E. coli to Convert Fe(II)EDTA-NO to N.SUB.2.O Through Genetic Engineering of E. coli Nitric Oxide (NOR)

1.1. Preparation of Mutant and Recombinant E. Coli

[0061] norV and norW genes were deleted from E. coli W3110 strain using a one-step inactivation method (K A Datsenko and B L Wanner, Proc. Natl. Acad. Sci. USA., 2000 Jun. 6; 97(12):6640-5).

[0062] To delete the norVW gene, PCR was performed using a pKD3 vector as a template and the oligonucleotides of SEQ ID NOs: 9 and 10 as primers. The obtained DNA fragment was electroporated into competent cells of the E. coli W3110 strain expressing λ-red recombinase to prepare a norVW gene-deleted mutant strain. To examine the deletion of the norVW gene, colony PCR was performed using primers of SEQ ID NOs: 11 and 12. As a result, the norVW gene-deleted W3110, designated delta norVW (ΔnorVW) strain was obtained.

[0063] Next, a recombinant strain was prepared, in which E. coli-derived norVW gene was overexpressed in E. coli W3110. In detail, E. coli W3110 was cultured in a medium, E. coli genomic DNA (gDNA) was extracted from the culture, and PCR was performed using the gDNA as a template and oligonucleotides of SEQ ID NOs: 13 and 14 as a primer set, to amplify the E. coli norVW gene including the nucleotide sequences of SEQ ID NOs: 5 and 6. PCR amplification was performed using a vector pIND4 (AC Ind et al. Appl Environ Microbiol. 2009 Oct; 75(20): 6613-5) as a template and oligonucleotides of SEQ ID NOs: 15 and 16 as a primer set to obtain a vector fragment. The norVW gene was ligated to the vector pIND4 using a method according to an InFusion Cloning Kit (Clontech Laboratories, Inc.) to prepare a norVW-overexpressing vector pIND4-norVW. At this time, expression of the norVW gene was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG).

[0064] The norVW-overexpressing vector was introduced into E. coli W3110 cells by an electroporation method (Sambrook, J & Russell, D. W., New York: Cold Spring Harbor Laboratory Press, 2001) to prepare a strain in which the ability to reduce Fe(II)EDTA-NO was improved. The transformed strain was obtained by selection on an LB plate containing kanamycin (50 μg/ml).

1.2. Examination and Improvement of Ability to Convert Fe(II)EDTA-NO to N.SUB.2.O

[0065] The norVW gene-deleted E. coli W3110 (ΔnorVW) was cultured in an LB medium at 30° C. with shaking at 230 rotations per minute (rpm). The norVW gene-overexpressing E. coli W3110 strain (W3110/pIND4-norVW) was cultured in an LB medium at 30° C. with shaking at 230 rpm to induce norVW gene expression by adding 0.5 mM IPTG. Next, the norVW-deleted E. coli and the norVW gene-overexpressing E. coli cells were isolated. The isolated cells were added to an M9 medium containing 5 grams per liter (g/L) glucose and 5 millimolar (mM) Fe(II)EDTA-.sup.15NO at pH 7.0 to obtain and OD.sub.600 of 1, and as a result, a reaction mixture was obtained.

[0066] 30 milliliters (mL) of the reaction mixture was added to a 60-mL serum bottle, and cultured at 30° C. with shaking at 140 rpm for 5 hours. The serum bottle was maintained in an anaerobic chamber and under anaerobic conditions. A control group was the same as above, except that a control strain, i.e., E. coli including an empty vector, was used.

[0067] Next, the gas in the headspace of the reaction serum bottle was sampled and the production amount of .sup.15N.sub.2O was analyzed by gas chromatography-mass spectrometry (GC-MS).

[0068] The results are shown in FIG. 1. FIG. 1 is a graph of .sup.15N.sub.2O amount (%) versus time (hr), which shows the results of converting Fe(II)EDTA-.sup.15NO to .sup.15N.sub.2O using the recombinant E. coli in which the NOR pathway was enhanced.

[0069] As shown in FIG. 1, the norVW gene-deleted E. coli W3110 (ΔnorVW strain) lost 100% of its ability to convert Fe(II)EDTA-.sup.15NO to .sup.15N.sub.2O. Further, the norVW-overexpressing E. coli strain showed remarkably improved ability to reduce Fe(II)EDTA-.sup.15NO to .sup.15N.sub.2O, as compared with the control group.

EXAMPLE 2

Improvement of Ability to Reduce Fe(II)EDTA-NO to N.SUB.2 .Using Recombinant Paracoccus Versutus Strain including E. Coli Nitric Oxide (NOR) Genes (norV, norW)

2.1. Preparation of Recombinant Paracoccus Versutus Strain

[0070] Into a natural denitrifying bacterium, Paracoccus versutus strain DSM 582 (hereinafter, referred to as ‘Pv’.), the recombinant vector including the E. coli-derived norVW gene obtained in Example 1 was introduced. The E. coli-derived norVW-overexpressing vector was introduced into Paracoccus versutus DSM 582 cells by electroporation (Sambrook, J & Russell, D. W., New York: Cold Spring Harbor Laboratory Press, 2001) to prepare a strain in which the ability to reduce Fe(II)EDTA-NO to N.sub.2 was improved. The transformed strain (Pv/pIND4-Ec norVW) was obtained by selection on an LB plate containing kanamycin (50 μg/ml). The recombinant vector was pIND4-Ec norVW, which was prepared in the same manner as the method described in Example 1. As a result, a Pv/pIND4-Ec norVW strain was finally obtained.

2.2. Improvement of Ability to Reduce Fe(II)EDTA-NO to N.SUB.2

[0071] The recombinant Paracoccus versutus strain (Pv/pIND4-Ec norVW), in which E. coli-derived norVW gene was introduced into a Paracoccus versutus strain, was cultured in an LB medium at 30° C. with shaking at 230 rpm to induce expression of the norVW gene by adding 0.5 mM IPTG. Next, the norVW-overexpressing Paracoccus versutus cells were isolated.

[0072] The isolated cells were added to an M9 medium containing 5 g/L glucose and 5 mM Fe(II)EDTA-.sup.15NO at pH 7.0 to obtain an OD.sub.600 of 1, and as a result, a reaction mixture was obtained.

[0073] 30 mL of the reaction mixture was added to a 60-mL serum bottle, and cultured at 30° C. with shaking at 140 rpm for 5 hours. The serum bottle was maintained in an anaerobic chamber and under anaerobic conditions. A control group was the same as above, except that a Paracoccus versutus control strain, i.e., Paracoccus versutus including an empty vector, was used.

[0074] Next, the gas in the headspace of the reaction serum bottle was sampled and the production amount of .sup.15N.sub.2O was analyzed by GC-MS.

[0075] The results are shown in FIG. 2. FIG. 2 is a graph of .sup.15N.sub.2O amount (%) versus test sample, which shows the results of converting Fe(II)EDTA-.sup.15NO to .sup.15N.sub.2 using the recombinant Paracoccus versutus in which the NOR pathway was enhanced.

[0076] As shown in FIG. 2, Pv/pIND4-Ec norVW demonstrated a remarkably improved ability to reduce Fe(II)EDTA-.sup.15NO to .sup.15N.sub.2, as compared with the control group.

EXAMPLE 3

Improvement of Ability to Reduce Fe(II)EDTA-NO to N.SUB.2 .Using Recombinant Paracoccus Versutus Strain Overexpressing Paracoccus Versutus-Derived Nitric Oxide Genes (norC, norB)

3.1. Preparation of Recombinant Paracoccus Versutus Strain

[0077] A recombinant Pv strain overexpressing NorCB gene which is derived from a natural denitrifying bacterium, Paracoccus versutus DSM 582 (Pv) strain, was prepared. In detail, Paracoccus versutus DSM 582 was cultured in an LB medium, and Paracoccus versutus DSM 582 gDNA was extracted from the culture, and PCR was performed using this gDNA as a template and the oligonucleotides of SEQ ID NOs: 17 and 18 as a primer set to amplify the Paracoccus versutus DSM 582 norCB gene including nucleotide sequences of SEQ ID NOs: 7 and 8. PCR amplification was performed using a vector pIND4 (AC Ind et al., Appl Environ Microbiol., 2009 Oct; 75(20), pp. 6613-5) as a template and oligonucleotides of SEQ ID NOs: 19 and 20 as a primer set to obtain a vector fragment. The norCB gene (SEQ ID NO:21) was ligated to the vector pIND4 using a method described in an InFusion Cloning Kit (Clontech Laboratories, Inc.) to prepare a norCB-overexpressing vector pIND4-Pv norCB. At this time, expression of the norCB gene was induced by IPTG.

[0078] The norCB-overexpressing vector was introduced into Paracoccus versutus DSM 582 cells by an electroporation method (Sambrook, J & Russell, D. W., New York: Cold Spring Harbor Laboratory Press, 2001) to prepare a strain, in which ability to reduce Fe(II)EDTA-NO was improved. The transformed strain (Pv/pIND4-Pv NorCB) was obtained by selection of an LB plate containing kanamycin (50 μg/ml).

3.2. Improvement of Ability to Reduce Fe(II)EDTA-NO to N.SUB.2

[0079] The Pv/pIND4-Pv NorCB strain was cultured in an LB medium at 30° C. with shaking at 230 rpm, and NorCB gene expression was induced by adding 0.5 mM IPTG. Next, the NorCB gene-overexpressing Pv/pIND4-Pv NorCB cells were isolated. The isolated cells were added to an M9 medium containing 5 g/L glucose and 5 mM Fe(II)EDTA-.sup.15NO at pH 7.0 to obtain an OD.sub.600 of 1, and as a result, a reaction mixture was obtained.

[0080] 30 mL of the reaction mixture was added to a 60-mL serum bottle, and cultured at 30° C. with shaking at 140 rpm for 5 hours. The serum bottle was maintained in an anaerobic chamber and under anaerobic conditions. A control group was the same as above, except that a control strain, i.e., E. coli including an empty vector was used.

[0081] Next, the gas in the headspace of the reaction serum bottle was sampled and the production amount of .sup.15N.sub.2 was analyzed by GC-MS.

[0082] The results are shown in FIG. 3. FIG. 3 is a graph of .sup.15N.sub.2O concentration (%) versus test sample, which shows results of converting Fe(II)EDTA-.sup.15NO to .sup.15N.sub.2 using the recombinant Paracoccus versutus in which the NOR pathway was enhanced. As shown in FIG. 3, NorCB-overexpressing Paracoccus versutus strain showed remarkably improved ability to reduce Fe(II)EDTA-.sup.15NO to .sup.15N.sub.2, as compared with the control group.

[0083] It should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should be considered as available for other similar features or aspects in other embodiments. While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope as defined by the following claims.