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

Abstract

A recombinant microorganism of the genus Escherichia, comprises a genetic modification that increases expression of a nosZ gene encoding NosZ, which is a nitrous oxide reductase, in the recombinant microorganism, wherein the recombinant microorganism comprises a nosR gene encoding NosR, a nosD gene encoding NosD, a nosF gene encoding NosF, a nosY gene encoding NosY, and an apbE gene encoding ApbE, and wherein the nosR gene, the nosD gene, the nosF gene, the nosY gene and the apbE gene are derived from a microorganism of the genus Pseudomonas, the genus Paracoccus, or a combination thereof.

Claims

1. A recombinant microorganism of the genus Escherichia, comprising a genetic modification that increases expression of a nosZ gene encoding NosZ, which is a nitrous oxide reductase, in the recombinant microorganism, wherein the recombinant microorganism comprises the nosZ gene, a nosR gene encoding NosR, a nosD gene encoding NosD, a nosF gene encoding NosF, a nosY gene encoding NosY, and an apbE gene encoding ApbE, and wherein the nosZ gene, the nosR gene, the nosD gene, the nosF gene, the nosY gene and the apbE gene are derived from a microorganism of the genus Pseudomonas, the genus Paracoccus, or a combination thereof.

2. The recombinant microorganism of claim 1, wherein the genetic modification comprises an increase in a copy number of the nosZ gene, a copy number of the nosR gene, a copy number of the nosD gene, a copy number of the nosF gene, a copy number of the nosY gene, a copy number of the apbE gene, or a combination thereof.

3. The recombinant microorganism of claim 1, wherein the NosZ is a polypeptide having 75% or greater sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7, the NosR is a polypeptide having 75% or greater sequence identity to the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 13, or SEQ ID NO: 16, the NosD is a polypeptide having 75% or greater sequence identity to the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 22, or SEQ ID NO: 25, the NosF is a polypeptide having 75% or greater sequence identity to the amino acid sequence of SEQ ID NO: 28, SEQ ID NO: 31, or SEQ ID NO: 34, the NosY is a polypeptide having 75% or greater sequence identity to the amino acid sequence of SEQ ID NO: 37, SEQ ID NO: 40, or SEQ ID NO: 43, and the ApbE is a polypeptide having 75% or greater sequence identity to the amino acid sequence of SEQ ID NO: 55, SEQ ID NO: 58, or SEQ ID NO: 61.

4. The recombinant microorganism of claim 1, wherein the nosZ gene and the nosR gene are comprised in a first vector, and the nosD gene, the nosF gene, the nosY gene, and the apbE gene are comprised in a second vector that is different from the first vector comprising the nosZ gene and the nosR gene.

5. The recombinant microorganism of claim 1, wherein the nosZ gene and the nosR gene are comprised in a first operon, and the nosD gene, the nosF gene, the nosY gene, and the apbE gene are comprised in a second operon that is different from the first operon comprising the nosZ gene and the nosR gene.

6. The recombinant microorganism of claim 1, wherein the microorganism does not comprise an exogenous nosL gene encoding a heterologous NosL.

7. A composition for reducing a concentration of nitrous oxide in a sample, the composition comprising: a recombinant microorganism of the genus Escherichia comprising a genetic modification that increases expression of a nosZ gene encoding NosZ, which is a nitrous oxide reductase, in the recombinant microorganism, wherein the recombinant microorganism comprises the nosZ gene, a nosR gene encoding NosR, a nosD gene encoding NosD, a nosF gene encoding NosF, a nosY gene encoding NosY, and an apbE gene encoding ApbE, and wherein the nosZ gene, the nosR gene, the nosD gene, the nosF gene, the nosY gene and the apbE gene are derived from a microorganism of the genus Pseudomonas, the genus Paracoccus, or a combination thereof.

8. The composition of claim 7, wherein the genetic modification comprises an increase in a copy number of the nosZ gene, a copy number of the nosR gene, a copy number of the nosD gene, a copy number of the nosF gene, a copy number of the nosY gene, a copy number of the apbE gene, or a combination thereof.

9. The composition of claim 7, wherein the NosZ is a polypeptide having 75% or greater sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7, the NosR is a polypeptide having 75% or greater sequence identity to the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 13, or SEQ ID NO: 16, the NosD is a polypeptide having 75% or greater sequence identity to the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 22, or SEQ ID NO: 25, the NosF is a polypeptide having 75% or greater sequence identity to the amino acid sequence of SEQ ID NO: 28, SEQ ID NO: 31, or SEQ ID NO: 34, the NosY is a polypeptide having 75% or greater sequence identity to the amino acid sequence of SEQ ID NO: 37, SEQ ID NO: 40, or SEQ ID NO: 43 and the ApbE is a polypeptide having 75% or greater sequence identity to the amino acid sequence of SEQ ID NO: 55, SEQ ID NO: 58, or SEQ ID NO: 61.

10. The composition of claim 7, wherein the nosZ gene and the nosR gene are comprised in a first vector, and the nosD gene, the nosF gene, the nosY gene, and the apbE gene are comprised in a second vector that is different from the first vector comprising the nosZ gene and the nosR gene.

11. The composition of claim 7, wherein the nosZ gene and the nosR gene are comprised in a first operon, and the nosD gene, the nosF gene, the nosY gene, and the apbE gene are comprised in a second operon that is different from the first operon comprising the nosZ gene and the nosR gene.

12. The composition of claim 7, wherein the recombinant microorganism does not comprise an exogenous nosL gene encoding a heterologous NosL.

13. A method of reducing a concentration of nitrous oxide in a sample, the method comprising contacting a recombinant microorganism with the nitrous oxide-containing sample to reduce the concentration of nitrous oxide in the sample, wherein the recombinant microorganism is of the genus Escherichia and comprises a genetic modification that increases expression of a nosZ gene encoding NosZ, which is a nitrous oxide reductase, in the recombinant microorganism, wherein the recombinant microorganism comprises the nosZ gene, a nosR gene encoding NosR, a nosD gene encoding NosD, a nosF gene encoding NosF, a nosY gene encoding NosY, and an apbE gene encoding ApbE, wherein the nosZ gene, the nosR gene, the nosD gene, the nosF gene, the nosY gene, and the apbE gene are derived from a microorganism of the genus Pseudomonas, the genus Paracoccus, or a combination thereof.

14. The method of claim 13, wherein the genetic modification comprises an increase in a copy number of the nosZ gene, a copy number of the nosR gene, a copy number of the nosD gene, a copy number of the nosF gene, a copy number of the nosY gene, a copy number of the apbE gene, or a combination thereof.

15. The method of claim 13, wherein the NosZ is a polypeptide having 75% or greater sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7, the NosR is a polypeptide having 75% or greater sequence identity to the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 13, or SEQ ID NO: 16, the NosD is a polypeptide having 75% or greater sequence identity to the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 22, or SEQ ID NO: 25, the NosF is a polypeptide having 75% or greater sequence identity to the amino acid sequence of SEQ ID NO: 28, SEQ ID NO: 31, or SEQ ID NO: 34, the NosY is a polypeptide having 75% or greater sequence identity to the amino acid sequence of SEQ ID NO: 37, SEQ ID NO: 40, or SEQ ID NO: 43 and the ApbE is a polypeptide having 75% or greater sequence identity to the amino acid sequence of SEQ ID NO: 55, SEQ ID NO: 58, or SEQ ID NO: 61.

16. The method of claim 13, wherein the nosZ gene and the nosR gene are comprised in a first vector, and the nosD gene, the nosF gene, the nosY gene, and the apbE gene are comprised in a second vector that is different from the first vector comprising the nosZ gene and the nosR gene.

17. The method of claim 13, wherein the nosZ gene and the nosR gene are comprised in a first operon, and the nosD gene, the nosF gene, the nosY gene, and the apbE gene are comprised in a second operon that is different from the first operon comprising the nosZ gene and the nosR gene.

18. The method of claim 13, wherein the recombinant microorganism does not comprise an exogenous nosL gene encoding a heterologous NosL.

19. The method of claim 13, wherein the contacting is performed under anaerobic conditions in a sealed container.

20. The method of claim 13, wherein the contacting comprises culturing or incubating the recombinant microorganism in the presence of the nitrous oxide-containing sample.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] 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:

[0052] FIG. 1A is an illustration of a norVW gene in the chromosome of E. coli;

[0053] FIG. 1B is a map of a pPs2/Pa2/Pv2 vector;

[0054] FIG. 1C is a map of a pPs4/Pa4/Pv4 vector;

[0055] FIG. 2 illustrates the genetic maps of the nitrous oxide reductase pathways of P. stutzeri, P. aeruginosa, and P. versutus;

[0056] FIG. 3 shows results of converting N.sub.2O to N.sub.2 using a recombinant E. coli including a pPs2/Pa2/Pv2 vector including a nosZ gene, a nosR gene, and a nosL gene, and a pPs4/Pa4/Pv4 vector including a nosD gene, a nosY gene, a nosF gene, a tatABC gene, and an apbE gene, in which nitrous oxide pathways are enhanced;

[0057] FIG. 4 is a graph of .sup.15N.sub.2 concentration (mM) versus test sample, which shows the results of converting N.sub.2O to N.sub.2 using recombinant E. coli in which the nitrous oxide pathway is enhanced;

[0058] FIG. 5A is an illustration of a norVW site in the chromosome of E. coli;

[0059] FIG. 5B is a map of a pPs1/Pa1/Pv1 vector;

[0060] FIG. 5C is a map of a pPs3/Pa3/Pv3 vector; and

[0061] FIG. 6 is a graph of .sup.15N.sub.2 concentration (mM) versus test sample, which shows the results of converting N.sub.2O to N.sub.2 using a recombinant E. coli including a pPs1/Pa1/Pv1 vector including a nosZ gene, a nosR gene, a nosL gene, a tatA, a tatB, and a tatC gene, and a pPs3/Pa3/pv3 vector including a nosD gene, a nosF gene, a nosY gene, an apbE gene, and in which nitrous oxide pathways are enhanced.

DETAILED DESCRIPTION

[0062] 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.

[0063] 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. 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.

[0064] “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.

[0065] 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,

[0066] 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: Development of Recombinant Microorganism of Genus Escherichia Having N.SUB.2.-Producing Ability

[0067] In this exemplary embodiment, a nosZ gene encoding a key enzyme nitrous oxide reductase (NosZ) and the accessory genes essential for activity of the NosZ enzyme, i.e., nosR, nosL, nosD, nosF, nosY, apbE, and tat genes, were extracted from nos operons or gene clusters and genomes of three kinds of natural denitrifying bacteria: Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus versutus. Each of the genes was codon-optimized for E. coli, and then introduced into E. coli. As a result, a recombinant microorganism of the genus Escherichia having N.sub.2-producing ability by converting N.sub.2O to N.sub.2, i.e., a recombinant E. coli, was prepared. To examine whether the recombinant E. coli had the N.sub.2-producing ability, the recombinant E. coli was cultured in the presence of a substrate containing a radioactive isotope .sup.15N, i.e., .sup.15N.sub.2O and FeEDTA-.sup.15NO, and then the amount of .sup.15N.sub.2 in the culture or in the upper air layer in the culture, was measured.

1. Identification of Genes Essential for Conversion of In Vivo N.sub.2O to N.sub.2 in E. coli

[0068] In detail, 8 different genes, i.e., nosZ, nosR, nosL, nosD, nosF, nosY, apbE, and tat genes, which exist in nitrous oxide reductase clusters of natural denitrifying bacteria of the genus Pseudomonas and the genus Paracoccus, e.g., Pseudomonas stutzeri (Ps), Pseudomonas aeruginosa (Pa), and Paracoccus versutus (Pv), were introduced in combination into E. coli to obtain recombinant E. coli. Next, the 6 different genes essential for the ability of the recombinant E. coli to convert in vivo N.sub.2O to N.sub.2, e.g., nosZ, nosR, nosD, nosF, nosY, and apbE genes, were determined by examining the ability of the obtained recombinant E. coli to convert in vivo N.sub.2O to N.sub.2.

[0069] The functions of the respective gene products are considered to be as follows. NosZ is a protein encoded by the nosZ gene, and is an enzyme that catalyzes a conversion reaction of N.sub.2O to N.sub.2, i.e., nitrous oxide reductase. NosZ is a 130 kDa, homodimeric metalloprotein including two copper centers, i.e., Cu.sub.A and Cu.sub.Z, in each monomer. NosR is a protein encoded by the nosR gene, and may be a polytopic membrane protein that serves as an electron donor for N.sub.2O reduction. NosD is a protein encoded by the nosD gene, and is essential for the formation of the [4Cu:2S] site Cu.sub.Z of the NosZ protein. NosD may provide sulfur (S) for NosZ. NosF and NosY are proteins encoded by the nosF gene and the nosY gene, respectively, and NosF and NosY together form a complex, e.g., a tetramer, to serve as an ABC transporter. ApbE is a protein encoded by the apbE gene, and may be a flavinyltransferase that transfers flavin to NosR.

2. Construction of Vector and Preparation of Recombinant E. coli Transformed with this Vector

(1) Construction of Vector

[0070] Expression vectors used in this exemplary embodiment were pET28a, pETDuet™-1, and pACYCDuet™-1 vectors. pET28a, in which a lac operator is operably linked to a T7 promoter, includes a kanamycin resistance Kan.sup.R gene as a selection marker. pETDuet™-1 vector, in which a lac operator is operably linked to a T7 promoter, includes an ampicillin resistance Amp.sup.R gene as a selection marker. pACYCDuet™1 vector, in which a lac operator is operably linked to a T7 promoter, includes a chloramphenicol resistance Cm.sup.R gene as a selection marker.

[0071] The NosZ has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7. The NosR has the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 13, or SEQ ID NO: 16. The NosD has the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 22, or SEQ ID NO: 25. The NosF has the amino acid sequence of SEQ ID NO: 28, SEQ ID NO: 31, or SEQ ID NO: 34. The NosY has the amino acid sequence of SEQ ID NO: 37, SEQ ID NO: 40, or SEQ ID NO: 43. The ApbE has the amino acid sequence of SEQ ID NO: 55, SEQ ID NO: 58, or SEQ ID NO: 61.

[0072] Further, the nosZ gene has the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 9. The nosR gene has the nucleotide sequence of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, 15, SEQ ID NO: 17, or SEQ ID NO: 18. The nosD gene has the nucleotide sequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 27. The nosF gene has the nucleotide sequence of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 36. The nosY gene has the nucleotide sequence of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 44, or SEQ ID NO: 45. The ApbE gene has the nucleotide sequence of SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 62, or SEQ ID NO: 63. Further, the NosL has the amino acid sequence of SEQ ID NO: 46, SEQ ID NO: 49, or SEQ ID NO: 52. The nosL gene has the nucleotide sequence of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, or SEQ ID NO: 54.

[0073] Further, the tatA, tatB, or tatC has the amino acid sequence of SEQ ID NO:64, SEQ ID NO: 67, SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 82, SEQ ID NO: 85, or SEQ ID NO: 88. The tatA, tatB, or tatC gene has the nucleotide sequence of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 84, 86, SEQ ID NO: 87, SEQ ID NO: 89, or SEQ ID NO: 90.

[0074] The origin and characteristics of the above-mentioned proteins and nucleotides encoding the same are listed in the sequence list. Among the above genes, genes used in the expression vector of E. coli in this exemplary embodiment were those optimized by considering the codon frequency used in E. coli for the nucleotide sequence of a natural gene, and information thereof is described in the sequence list.

[0075] FIG. 1A shows a norVW site in the chromosome of E. coli and maps of the pPs2/Pa2/Pv2 vector and the pPs4/Pa4/Pv4 vector introduced into E. coli. In FIG. 1A, norVW indicates nitrous oxide reductase of E. coli. In FIG. 1B pPs2/Pa2/Pv2 vector, nosZ (Z), nosR (R) and nosL (L) genes, derived from P. stutzeri, P. aeruginosa, and P. versutus, were commonly operably linked to a T7 promoter in pET28a vector, and a ribosome binding site (RBS) indicates an AAGGAG sequence, which is an E. coli RBS sequence. In this regard, the nosZ gene includes a sequence encoding his-tag.

[0076] The nosZ, nosR, and nosL genes were amplified from three different strains of microorganisms by PCR using primer sets and using DNA synthesized by codon optimization as a template, and the resulting products were introduced into the vector at an NcoI enzyme restriction site. For the nosZ, nosR, and nosL genes of P. stutzeri, primer sets of SEQ ID NOs: 91 and 92; SEQ ID NOs: 93 and 94; and SEQ ID NOs: 95 and 96 were used, respectively. For the nosZ, nosR and nosL genes of P. aeruginosa, primer sets of SEQ ID NOs: 97 and 98; SEQ ID NOs: 99 and 100; and SEQ ID NOs: 101 and 102 were used, respectively. For the nosZ, nosR and nosL genes of P. versutus, primer sets of SEQ ID NOs: 103 and 104; SEQ ID NOs: 105 and 106; and SEQ ID NOs: 107 and 108 were used, respectively.

[0077] In FIG. 1C, the pPs4/Pa4/Pv4 vector, nosD and nosY genes, derived from P. stutzeri, P. aeruginosa, or P. versutus, were commonly operably linked to a T7 promoter in a pACYC-duet vector, tatABC, apbE, and nosF genes are commonly operably linked to another T7 promoter, and RBS of each gene indicates an E. coli RBS AAGGAG sequence. The nosD, nosY, tatABC, apbE, and nosF genes from each of three strains of microorganisms were amplified by PCR using the following primer sets and DNA synthesized by codon optimization as a template. The resulting PCT products of the nosD and nosY genes were introduced into an NcoI enzyme restriction site, and the resulting products of the tatABC, apbE, and nosF genes were introduced into an NdeI enzyme restriction site. For the nosD, nosY, tatABC, apbE, and nosF genes of P. stutzeri, primer sets of SEQ ID NOs: 109 and 110; SEQ ID NOs: 111 and 112; SEQ ID NOs: 113 and 114; SEQ ID NOs: 115 and 116; and SEQ ID NOs: 117 and 118 were used, respectively. For the nosD, nosY, tatABC, apbE, and nosF genes of P. aeruginosa, primer sets of SEQ ID NOs: 119 and 120; SEQ ID NOs: 121 and 122; SEQ ID NOs: 123 and 124; SEQ ID NOs: 125 and 126; and SEQ ID NOs: 127 and 128 were used, respectively. For the nosD, nosY, tatABC, apbE, and nosF genes of P. versutus, primer sets of SEQ ID NOs: 129 and 130; SEQ ID NOs: 131 and 132; SEQ ID NOs: 133 and 134; SEQ ID NOs: 135 and 136; and SEQ ID NOs: 137 and 138 were used, respectively. The tatABC of P. stutzeri, P. aeruginosa, and P. versutus is a codon-optimized sequence including all of the tatA, tatB, and tatC genes, and has the nucleotide sequence of SEQ ID NO: 139, 140, or 141.

[0078] Further, in FIG. 1, each vector excluding the nosL gene or the nosR gene from pPs2/Pa2/Pv2, and each vector excluding the nosY gene, the tatABC gene, and the apbE gene; the tatABC gene, the apbE gene, and the nosF gene; the tatABC gene and the apbE gene; or the tatABC gene from pPs4/Pa4/Pv4, were prepared. One vector was selected from the pPs2/Pa2/Pv2-based vectors, and one vector was selected from the pPs4/Pa4/Pv4-based vectors, and these two vectors were introduced into E. coli such that gene combinations selected from the eight genes were expressed in the recombinant E. coli. The recombinant E. coli including these gene combinations was cultured in the presence of N.sub.2O or Fe(II)EDTA-NO, and the N.sub.2-producing ability thereof was examined to identify gene combinations essential for N.sub.2 production in E. coli. Here, with regard to apbE, one kind of P. stutzeri (Ps), i.e., Ps_apbE, one kind of P. aeruginosa (Pa), i.e., Pa_apbE, and one kind of P. versutus (Pv), i.e., Pv_apbE were introduced.

[0079] FIG. 2 shows the genetic maps of the nitrous oxide reductase pathways of P. stutzeri, P. aeruginosa, and P. versutus.

(2) Preparation of Recombinant E. coli Having N.sub.2-Producing Ability and Examination of Activity Thereof

[0080] Two vectors consisting of one vector of the pPs2/Pa2/Pv2-based vectors and one vector of pPs4/Pa4/Pv4-based vectors prepared in (1) were introduced into E. coli strain C43 (DE3) by transformation to prepare recombinant E. coli. The transformation was performed by electroporation. In this regard, each gene introduced into E. coli was a gene derived from the same strain.

(2.1) Culture for NosZ Maturation Stage

[0081] The recombinant E. coli was cultured at 37° C. in a 2×YT medium containing 50 micrograms per milliliter (μg/mL) riboflavin and 0.25 mM CuCl.sub.2 in an Erlenmeyer flask until the OD.sub.600 of the culture reached 0.6, and then 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added and the bacteria were cultured at 30° C. and with shaking at a speed of 140 rotations per minute (rpm) overnight to induce gene expression. Next, the cells were harvested and used for the subsequent N.sub.2 production reaction.

(2.2) Culture for Production of N.sub.2 from .sup.15N.sub.2O

[0082] The recombinant E. coli cells were added to an M9 medium (pH 7.0) containing 5 grams per liter (g/L) of glucose and 1.25 mM .sup.15N.sub.2O (g) in a serum bottle and cultured to a density of OD.sub.600=1 to prepare 30 mL of a culture mixture, which was then added to a 60-ml serum bottle and cultured at 30° C. and 140 rpm under stirring. The .sup.15N.sub.2O (g) concentration represents a concentration with respect to a volume of the culture upper layer. At this time, the bottle was sealed with a stopper to prepare anaerobic conditions. A control group was the same as above, except that E. coli including an empty vector was used.

[0083] 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.

[0084] The results are shown in FIG. 3 and in Table 1 below. FIG. 3 shows the results of converting N.sub.2O to N.sub.2 using the recombinant E. coli including the pPs2/Pa2/Pv2 vector including the nosZ gene, the nosR gene, and the nosL gene, and the pPs4/Pa4/Pv4 vector including the nosD gene, the nosY gene, the nosF gene, the tatABC gene, and the apbE gene, and in which the nitrous oxide pathways are enhanced.

[0085] As shown in FIG. 3, when the recombinant E. coli pPa2/pPa4 and pPs2/pPs4 were used, N.sub.2 production was remarkably increased 5 hours and 24 hours later, as compared with E. coli BL31(C43) including the empty vector. In particular, the largest N.sub.2 production was observed in the recombinant E. coli pPs2/pPs4. These results indicate that, even though the same nosZ, nosR, nosL, nosD, nosY, tatABC, apbE, and nosF genes are included, their expression levels in E. coli cells vary depending on their origin. In FIG. 3, pPa2/pPa4, pPs2/pPs4, and pPv2/pPv4 represent the recombinant E. coli including the pPs2 vector and the pPs4 vector, the recombinant E. coli including the pPs2 vector and the pPs4 vector, and the recombinant E. coli including the pPv2 vector and the pPv4 vector, respectively.

[0086] In addition, the production amount of N.sub.2 by recombinant E. coli including each gene combination selected from the eight genes is shown in Tables 1, 2 and 3 below. Table 1 shows the production amount of N.sub.2 (mM), when the recombinant E. coli including a combination of nos genes derived from P. stutzeri was cultured in the presence of N.sub.2O for 24 hours. Table 2 shows the production amount of N.sub.2 (mM), when the recombinant E. coli including a combination of nos genes derived from P. aeruginosa was cultured in the presence of N.sub.2O for 24 hours. Table 3 shows the production amount of N.sub.2 (mM), when the recombinant E. coli including a combination of nos genes derived from P. versutus was cultured in the presence of N.sub.2O for 24 hours.

TABLE-US-00001 TABLE 1 Genes in pACYC_DEUT-1 vector D, F, Y, apbE, D, F D, Y D, F, Y D, F, Y, apbE tatA Genes in Z — — — — — pET28a ZR — — — 0.22 0.23 vector ZRL — — — 0.31 0.32

TABLE-US-00002 TABLE 2 Genes in pACYC_DEUT-1 vector D, F, Y, apbE, D, F D, Y D, F, Y D, F, Y, apbE tatA Genes in Z — — — — — pET28a ZR — — — 0.07 0.07 vector ZRL — — — 0.08 0.08

TABLE-US-00003 TABLE 3 Genes in pACYC_DEUT-1 vector D, F, Y, apbE, D, F D, Y D, F, Y D, F, Y, apbE tatA Genes in Z — — — — — pET28a ZR — — — 0.06 0.06 vector ZRL — — — 0.07 0.07

[0087] As shown in Tables, 1, 2 and 3, it was confirmed that, among the eight genes of the nitrous oxide reductase pathway, six genes, i.e., nosZ, nosR, nosD, nosF, nosY, and apbE genes, are essential genes.

(2.3) Culture for Production of N.sub.2 from Fe(II)EDTA-.sup.15NO

[0088] The recombinant E. coli cells obtained in (2.1) were added to an M9 medium (pH 7.0) containing 5 g/L glucose and 1.25 mM Fe(II)EDTA-.sup.15NO at a cell density of OD.sub.600=1 to prepare a reaction mixture.

[0089] 30 mL of the reaction mixture was added to a 60-ml serum bottle and then cultured at 30° C. and 140 rpm under stirring. The serum bottle was maintained in an anaerobic chamber and allowed to be under anaerobic conditions. A control group was the same as above, except that E. coli including an empty vector was used.

[0090] 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.

[0091] The results are shown in FIG. 4. FIG. 4 shows the results of converting N.sub.2O to N.sub.2 using the recombinant E. coli in which nitrous oxide pathways are enhanced.

[0092] As shown in FIG. 4, when the recombinant E. coli pPa2/pPa4 and pPs2/pPs4 were used, N.sub.2 production was remarkably increased 5 hours and 24 hours later, as compared with E. coli BL31(C43) including the empty vector. In particular, the largest N.sub.2 production was observed in the recombinant E. coli pPs2/pPs4.

[0093] These results indicate that, even though the same nosZ, nosR, nosL, nosD, nosY, tatABC, apbE, and nosF genes are included, their expression levels in E. coli cells vary depend upon their origin. In FIG. 4, pPa2/pPa4, pPs2/pPs4, and pPv2/pPv4 represent the recombinant E. coli including the pPa2 vector and the pPa4 vector, the recombinant E. coli including the pPs2 vector and the pPs4 vector, and the recombinant E. coli including the pPv2 vector and the pPv4 vector, respectively.

Example 2: Evaluation of In Vitro Activity of Recombinant Nitrous Oxide Reductase NosZ

[0094] In this exemplary embodiment, a nosZ gene encoding the enzyme nitrous oxide reductase (NosZ) and accessory genes essential for activity of the NosZ enzyme, i.e., nosR, nosL, nosD, nosF, nosY, apbE, and tat genes, were extracted from nos operons or gene clusters and genomes of three different strains of natural denitrifying bacteria (Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus versutus), codon-optimized for E. coli, and then introduced into E. coli. As a result, a recombinant microorganism of the genus Escherichia having N.sub.2-producing ability by converting N.sub.2O to N.sub.2, i.e., a recombinant E. coli, was prepared. A cell lysate of the recombinant E. coli was obtained. To examine whether the cell lysate had the N.sub.2-producing ability, the recombinant E. coli was cultured in the presence of a substrate containing a radioactive isotope .sup.15N, i.e., .sup.15N.sub.2O and FeEDTA-.sup.15NO, and then the amount of .sup.15N.sub.2 in the culture thereof or the upper air layer in the culture was measured.

1. Construction of Vector and Preparation of Recombinant E. coli Transformed with this Vector

[0095] (1) Construction of Vector

[0096] Expression vectors used in this exemplary embodiment were pETDuet™-1 and pACYCDuet™1 vectors (Novagen). pETDuet™-1 vector, in which a lac operator is operably linked to a T7 promoter, includes an ampicillin resistance Amp.sup.R gene as a selection marker. pACYCDuet™1 vector, in which a lac operator is operably linked to a T7 promoter, includes a chloramphenicol resistance Cm.sup.R gene as a selection marker.

[0097] FIG. 5 shows a norVW site in the chromosome of E. coli and maps of the pPs1/Pa1/Pv1 vector and the pPs3/Pa3/Pv3 vector introduced into E. coli. In FIG. 5A, norVW refers to the nitrous oxide reductase of E. coli; in FIG. 5B the pPs2/Pa2/Pv2 vector, the nosZ, nosR and nosL genes, derived from P. stutzeri, P. aeruginosa, or P. versutus, were commonly operably linked to a T7 promoter in pET_deut vector, and a ribosome binding site (RBS) having an E. coli RBS AAGGAG sequence; and in FIG. 5C, the pPs4/Pa4/Pv4 vector the nosD, nosY, tatA, tatB, and tatC genes, derived from P. stutzeri, P. aeruginosa, and P. versutus, were commonly operably linked to a T7 promoter, and a ribosome binding site (RBS) has an E. coli RBS AAGGAG sequence. In this regard, the nosZ gene includes a sequence encoding his-tag.

[0098] The nosZ, nosR, and nosL genes of three kinds of microorganisms were amplified by PCR using primer sets described in Example 1, DNA was synthesized by codon optimization as a template, and the resulting products were introduced into NcoI enzyme restriction site. Further, the tatA, tatB and tatC genes of three kinds of microorganisms were amplified by PCR using primer sets described in Example 1 and DNA was synthesized by codon optimization as a template, and the resulting products were introduced into NdeI enzyme restriction site.

[0099] In the pPs3/Pa3/Pv3 vector, the nosD, nosF, and nosY genes derived from P. stutzeri, P. aeruginosa, or P. versutus, were commonly operably linked to a T7 promoter in pACYC-duet vector, apbE gene was commonly operably linked to a T7 promoter, and RBS of each gene has an E. coli RBS AAGGAG sequence. The nosD, nosF, nosY, and apbE genes of three kinds of microorganisms were amplified by PCR using primer sets described in Example 1 and DNA synthesized by codon optimization as a template, and the resulting products of the nosD, nosF, and nosY genes were introduced into NcoI enzyme restriction site, and the resulting product of the apbE gene was introduced into NdeI enzyme restriction site.

[0100] (2) Preparation of Recombinant E. coli Having N.sub.2-Producing Ability and Examination of Activity Thereof

[0101] Two vectors consisting of the pPs1/Pa1/Pv1 vector and the pPs4/Pa4/Pv4 vector prepared in (1) were introduced into E. coli C43 (DE3) by transformation to prepare recombinant E. coli. The transformation was performed by electroporation. In this regard, each gene introduced into E. coli was a gene derived from the same strain.

[0102] (2.1) Culture for NosZ Maturation Stage

[0103] The recombinant E. coli was cultured at 37° C. in a 2×YT medium containing 50 μg/mL riboflavin and 0.25 mM CuCl.sub.2 in an Erlenmeyer flask until the OD.sub.600 reached 0.6, and then 1 mM IPTG was added and the bacteria were cultured at 30° C. with shaking at 140 rpm overnight to induce gene expression. Next, the recombinant E. coli cells were sonicated in a lysis buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 10 mM imidazole, pH 8.0). As a result, a cell lysate was obtained, and NosZ was purified from the cell lysate by a general method using Ni-NTA affinity column and the following two buffers: Ni-NTA washing buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 20 mM imidazole, pH 8.0) and Ni-NTA elution Buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 250 mM imidazole, pH 8.0).

[0104] Subsequently, the purified NosZ was brought into contact with N.sub.2O and used in the N.sub.2 production reaction.

[0105] (2.2) Production of N.sub.2 from .sup.15N.sub.2O by Purified NosZ

[0106] 0.2 mg/ml of purified NosZ obtained in (2.1), 2.0 mM benzyl viologen, 1.0 mM sodium dithionite, and 1.25 mM .sup.15N.sub.2O(g) were added to water (pH 7.0) to prepare an aqueous reaction solution. 30 mL of the aqueous reaction solution was added to a 60 ml serum bottle, and cultured under stirring at 30° C. and 140 rpm. At this time, the bottle was sealed with a stopper to prepare anaerobic conditions. A control group was the same as above, except that an aqueous solution containing bovine serum albumin (BSA) was used.

[0107] 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.

[0108] The results are shown in FIG. 6. FIG. 6 shows results of converting N.sub.2O to N.sub.2 using NosZ derived from the recombinant E. coli including the pPs1/Pa1/Pv1 vector including the nosZ gene, the nosR gene, the nosL gene, the tatA, the tatB, and the tatC gene, and the pPs3/Pa3/pv3 vector including the nosD gene, the nosF gene, the nosY gene, and the apbE gene, in which nitrous oxide pathways are enhanced.

[0109] As shown in FIG. 6, when the recombinant E. coli pPa1/pPa3, pPs1/pPs3, and pPv1/pPv3-derived NosZ was used, N.sub.2 production was remarkably increased 5 hours and 24 hours later, as compared with the control group containing BSA. In particular, the largest N.sub.2 production was observed in the recombinant E. coli pPv1/pPv3.

[0110] These results indicate that, even though the same nosZ, nosR, nosL, nosD, nosY, tatABC, apbE, and nosF genes are included, their expression levels in E. coli cells vary depending on their origin, and they may not have activity in vivo, despite having in vitro activity.

[0111] In FIG. 6, pPa1/pPa3, pPs1/pPs3, and pPv1/pPv3 represent the recombinant E. coli including the pPa1 vector and the pPs3 vector, the recombinant E. coli including the pPs1 vector and the pPs3 vector, and the recombinant E. coli including the pPv1 vector and the pPv3 vector.

[0112] 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.