KR102257223B1 - Process for preparing acetoin using methanotrophs or transformant thereof - Google Patents

Abstract

The present invention comprises a wild-type methanogen or a gene encoding ack (acetate kinase), a gene encoding pta (phosphoate acetyltransferase), a gene encoding acs (acetyl-coenzyme A synthetase) and/or aor (aldehyde ferredoxin oxidoreductase). Acetoin production method comprising the step of culturing the transgenic methanogens in which the encoding gene is inactivated in an atmospheric condition containing ethane or in a medium containing ethane, transformation for production of acetoin in which the gene is inactivated It relates to a methanogen, a composition for producing acetoin comprising the transformed methanogen, and a kit for producing acetoin comprising the composition. If the method of producing acetoin from ethane using the transformed methanogens provided in the present invention is used, the productivity of acetoin can be improved while reducing the production amount of acetic acid produced from the methanogens. In addition, since acetoin can be produced from raw materials such as natural gas and sail gas including ethane, it will be widely used for economical production of acetoin.

Description

[Process for preparing acetoin using methanotrophs or transformant thereof]

The present invention relates to a method for producing acetoin using a methanogen or a transformant thereof, and more specifically, the present invention encodes a wild-type methanogen or a gene encoding ack (acetate kinase), pta (phosphoate acetyltransferase) Transgenic methanogens in which the gene encoding the gene, acs (acetyl-coenzyme A synthetase) and/or the gene encoding aor (aldehyde ferredoxin oxidoreductase) are inactivated, atmospheric conditions containing ethane, or containing ethane. Acetoin production method comprising the step of culturing in a medium, a transgenic methanogen for production of acetoin in which the gene is inactivated, a composition for producing acetoin containing the transgenic methanogen, and aceto comprising the composition It relates to a kit for the production of phosphorus.

The interest in global warming is increasing worldwide, and the main causes are known as the greenhouse effect such as carbon dioxide and methane. To solve this problem, research on producing high value-added products from carbon dioxide using microorganisms that use carbon dioxide is being actively conducted. Recently, to convert carbon dioxide, a synthetic biocatalyst designed by computer calculation was created, and the metabolic pathway using this was developed. A reconstruction study was conducted.

The main sources of methane are biogas, a product of anaerobic digestion, and naturally generated natural gas. The main components of biogas are methane and carbon dioxide, 70-90% of natural gas is methane, and the rest is composed of 5-15% ethane and a small amount of propane. Recently, the production of shale gas, which is a natural gas produced in the shale layer, has rapidly increased, and interest in the use of methane and ethane is high. As a result, research on methane-sugar bacteria, a microorganism with the ability to utilize methane, is actively being conducted, and methane monooxygenase (MMO) possessed by methane-sugar bacteria can use a wide range of substrates, so not only methane is used. In addition, it has the ability to oxidize longer alkanes such as ethane, and methanol dehydrogenase (MDH) is also known to be able to oxidize multi-carbon alcohols as it can use a wider range of substrates. have.

On the other hand, acetoin is widely used industrially, and it is converted into 2,3-butanediol and is used in various manufacturing industries such as printing ink, cosmetics, and solvents for synthesizing synthetic resins. In addition, biologically produced 2,3-butanediol has the advantage that it can be converted into useful substances such as methyl ethyl ketone (solvent) and 1,3-butadiene (used in the production of synthetic rubber) through a dehydration process. Such acetoin is produced using glucose as a raw material from microorganisms such as Aerobacter aerogenes, Klebsiella pneumonia, Klebsiella oxytoca, and Bacillus polymyxa. Has become. However, when the microorganisms are used, not only acetoin, but also ethanol, lactate, acetate, etc. are produced as by-products, and there is a disadvantage in that the production yield of acetoin is low. In order to overcome these shortcomings, various studies have been conducted. For example, Korean Patent No. 1562866 discloses a method of reducing the content of the same by-product and increasing the yield of acetoin using a recombinant Krebsiella oxytoca M1 strain.

Under this background, in order to develop a method for producing acetoin more effectively using methanogens, the present inventors, as a result of intensive research efforts, showed that a gene encoding a wild-type methanogen or ack (acetate kinase), phosphorate acetyltransferase (pta). ) Encoding gene, acs (acetyl-coenzyme A synthetase) encoding gene, aor (aldehyde ferredoxin oxidoreductase) encoding gene, etc. It was confirmed that acetoin can be produced more effectively from raw materials such as gas and shale gas, and the present invention was completed.

One object of the present invention is to provide a method for producing acetoin using wild-type methanogens or transgenic methanogens in which the ack gene, pta gene, acs gene and/or aor gene are inactivated.

Another object of the present invention is to provide a transgenic methanogen production for the production of acetoin in which the ack gene, the pta gene, the acs gene, the aor gene, and the like are deleted.

Another object of the present invention is to provide a composition for producing acetoin comprising the transformed methanogens.

Another object of the present invention is to provide a kit for producing acetoin comprising the composition.

One embodiment of the present invention for achieving the above object is a wild-type methanogen or A group consisting of a gene encoding ack (acetate kinase), a gene encoding pta (phosphoate acetyltransferase), a gene encoding acetyl-coenzyme A synthetase (acs), a gene encoding aldehyde ferredoxin oxidoreductase (aor), and combinations thereof It provides a method for producing acetoin comprising the step of culturing the transformed methanogens, in which the gene selected from is inactivated, in an atmospheric condition containing ethane or in a medium containing ethane.

The term "methanotrophs" of the present invention is also referred to as methane-oxidizing bacteria, and refers to prokaryotes that grow using methane as the sole carbon source. Most of the methanogens use methane in the atmosphere as a carbon source, but after converting methane into the form of pyruvic acid, they are used for metabolism in vivo.

In the present invention, the methanogens can be interpreted as meaning a methanogens capable of producing acetoin from ethane, and may be a wild-type methanogens, or acetoin production in the genome of the wild-type methanogens. A variety of genes related to is introduced, or various genes related to acetoin production present in the genome of the wild-type methanogens may be inactivated transgenic methanogens.

For example, methane magnetization bacteria to be used in the present invention, but particularly one that can improve the productivity of the acetonide of ethane are not limited to, as an example, Pseudomonas (Methylomonas) in, in bakteo (Methylobacter) methyl methyl , methyl Rhodococcus (Methylococcus) in methyl micro byum (Methylomicrobium) in methyl Los Blow (Methylosphaera) in methyl local bushes (Methylocaldum) in, Sar with a bus (Methyloglobus), a methylglutaryl methyl or when (Methylosarcina ) Genus, Methyloprofundus genus, Methylothermus genus, Methylohalobius genus, Methylogaea genus, Methylomarinum genus, methyl a beolreom Marino penalized by (Methylovulum) in methyl (Methylomarinovum), a Love column (Methylorubrum) genus Paracoccus (Methyloparacoccus) in, in city seutiseu (Methylocystis) as a Sinners (Methylosinus) in methyl methyl methyl methyl , it is a cellar (Methylocella) genus, methyl kaepsa (Methylocapsa) in methyl hydroperoxide Lula (Methylofurula) in methyl O CD pilreom (Methylacidiphilum) in the strain belonging like methyl O CD micro byum (Methylacidimicrobium) in methyl and , As another example, it may be a strain of the genus Methylomicrobium.

The transformed methanogens provided by the present invention can be interpreted as being a transformant in which various genes related to acetoin production present in the genome of the wild-type methanogens are inactivated. In the genome of the fungus, the gene coding for ack (acetate kinase), the gene coding for phosphoate acetyltransferase (pta), the gene coding for acetyl-coenzyme A synthetase (acs), and the gene coding for aldehyde ferredoxin oxidoreductase (aor) are each individually. It can be interpreted as being a transformed strain in an inactivated form or in combination.

The inactivated gene prevents the expression of each protein encoded by the gene, or is one or more at one or more positions in the amino acid sequence constituting each protein (position in the three-dimensional structure of amino acid residues of the protein). B, depending on the type, but specifically 2 to 20 amino acids, preferably 2 to 10, more preferably 2 to 5) amino acids in the form of substitution, deletion, insertion, addition or inversion of a protein in a mutated form By expressing the protein, even if the protein is expressed, it may be prevented from performing its normal function.

Since the transgenic methanogens as well as the methanogens can absorb ethane in the atmosphere and convert them into ethanol, not only when the ethane is included in the medium, but also the medium does not contain ethane and contains ethane. Even when atmospheric conditions are used, acetoin can be produced.

In addition, in order to improve the productivity of acetoin, the methanogens or transformed methanogens may have a form in which a gene encoding FLS (formolase) is additionally introduced. Since the FLS (formolase) is known to be capable of converting acetaldehyde to acetoin, the transformed methanogens into which a gene encoding the FLS (formolase) is additionally introduced can further improve the productivity of acetoin. .

At this time, the concentration of ethane included in the atmospheric condition is not particularly limited thereto, but as an example, it may be 0.01 to 6% (v/v), and as another example, it may be 0.1 to 2% (v/v). And, as another example, it may be 1.7% (v/v). In addition, when the medium contains ethane, the concentration of ethane contained in the medium is not particularly limited thereto, but may be 0.01 to 90% (v/v) as an example, and as another example, 0.1 to 50% ( v/v), and as another example, it could be 22% (v/v).

The term "culture" of the present invention means a method of growing the wild-type methanogens or transformed methanogens under appropriately artificially controlled environmental conditions.

In the present invention, the method of culturing the wild-type methanogens or transformed methanogens may be performed using a method widely known in the art. Specifically, the culture is not particularly limited as long as acetoin can be produced from ethane using the wild-type methanogens or transformed methanogens, but a batch process or injection batch or repeated injection batch process (fed batch or repeated fed) batch process) can be cultured continuously.

The medium used for cultivation must meet the requirements of a specific strain in an appropriate manner while controlling temperature, pH, etc. under aerobic conditions in a conventional medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin, and the like. Carbon sources that can be used include sugars and carbohydrates such as methane, glucose, xylose, sucrose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, etc. Fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, organic acids such as acetic acid, and the like may additionally be used, and these substances may be used individually or as a mixture. Nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation products, skim soybean cake or its degradation products, etc. I can. These nitrogen sources may be used alone or in combination. The medium may contain first potassium phosphate, second potassium phosphate, and a corresponding sodium-containing salt as personnel. Personnel that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salt. In addition, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used as the inorganic compound. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins can be used.

In addition, precursors suitable for the culture medium may be used. The above-described raw materials may be added in a batch, fed-batch, or continuous manner to the culture during the cultivation process, but are not particularly limited thereto. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid can be used in an appropriate manner to adjust the pH of the culture.

In addition, foaming can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. Oxygen or an oxygen-containing gas (eg air) is injected into the culture to maintain an aerobic condition. The temperature of the culture is usually 27°C to 37°C, preferably 30°C to 35°C. The cultivation is continued until the maximum amount of the peptide is obtained. For this purpose it is usually achieved in 10 to 100 hours.

In addition, it may further include the step of recovering acetoin from the culture obtained through the culture, the step of recovering acetoin from the culture is dialysis, centrifugation, filtration, solvent extraction, chromatography, crystallization It can be carried out by methods known in the art, such as. For example, the culture is centrifuged to obtain a supernatant from which wild-type methanogens or transformed methanogens are removed, and the obtained supernatant is applied to a solvent extraction method to recover the desired acetoin. In addition, any method capable of recovering the acetoin by combining known experimental methods according to the properties of the desired acetoin may be used without particular limitation.

Another embodiment of the present invention is a gene encoding ack (acetate kinase), a gene encoding pta (phosphoate acetyltransferase), a gene encoding acs (acetyl-coenzyme A synthetase), a gene encoding aor (aldehyde ferredoxin oxidoreductase) And a gene selected from the group consisting of a combination thereof is inactivated acetoin production transgenic methanogens, acetoin production composition comprising the transgenic methanogens, and acetoin production kit comprising the composition Provides.

As described above, since the transformed methanogens in which the three genes are individually or complexly inactivated can effectively produce acetoin from ethane, the use of the transformed methanogens or a culture thereof may result in ethane. In addition, acetoin can be produced in high yield from raw materials such as natural gas and sail gas including ethane.

In the present invention, as long as the culture product of the transformed methanase bacteria can be used to produce acetoin from ethane in a high yield, it is not particularly limited thereto, but as an example, the culture product of the transformed methanase bacteria, It may be a culture supernatant, a lysate, a fraction thereof, and the like, and as another example, a culture supernatant obtained by centrifuging the culture of the transformed methanogens, a lysate obtained by physically or ultrasonically treating a transformed microorganism It may be a fraction obtained by applying the culture, culture supernatant, lysate, etc. to a method such as centrifugation or chromatography.

On the other hand, the kit for producing acetoin of the present invention may be used to produce acetoin, including the composition for producing acetoin, but is not particularly limited thereto, but one kind or more suitable for the reaction Or an apparatus may be included, and specific examples include a buffer solution used for the production of acetoin, a reaction vessel for performing the acetoin production, a shaking incubator for performing the acetoin production, and a timer for performing the acetoin production reaction And the like.

If the method of producing acetoin from ethane using the transformed methanogens provided in the present invention is used, the productivity of acetoin can be improved while reducing the production amount of acetic acid produced from the methanogens. In addition, since acetoin can be produced from raw materials such as natural gas and sail gas including ethane, it will be widely used for economical production of acetoin.

1 shows three different wild-type methanogens (Methylomonas sp. DH-1 strain (A); Methylomicrobium alcaliphilum 20Z strain (B)); Methylosinus trichosporium OB3b strain expressing methane oxidase, methanol dehydrogenase, and aldehyde lyase. (C)) is a graph showing the result of comparing the concentrations of acetoin and other components produced from the culture when methane (left) or ethane (right) is provided as a substrate and cultured.
2 is a comparison of the concentrations of acetoin and other components produced from the culture of the FLS gene and the BudC gene introduced transgenic methanogen M. alcaliphilum strain 20Z, when cultured by providing ethane as a substrate. As a graph showing the results, (A) represents a culture of wild-type methanogens M. alcaliphilum strain 20Z as a control, and (B) represents a culture of transformed methanogens M. alcaliphilum strain 20Z.
Figure 3 is each culture obtained by culturing while supplying ethane to each of the methane-sugar bacteria in which acetate kinase (ack), phosphate acetyltransferase (pta), acetyl-coenzyme A synthetase (acs) or aldehyde ferredoxin oxidoreductase (aor) is deleted This is a graph showing the results of comparing the concentrations of acetoin and acetic acid contained in.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Production of acetoin using wild-type methanogens

Example 1-1: Cultivation of wild-type methanogens

Three wild-type methanogens (Methylomonas sp. DH-1 strain; Methylomicrobium alcaliphilum 20Z strain; and Methylosinus trichosporium OB3b strain) were each inoculated into the NMS medium, and methane was injected into the atmosphere in the flask to cultivate the methanogens. .

Example 1-2: Measurement of acetoin concentration in culture broth cultured using methane

After culturing while supplying methane to the three types of methanogens cultured in Example 1-1, changes in the cell concentration and acetoin concentration in the culture were measured (FIG. 1).

Example 1-3: Measurement of acetoin concentration after injecting ethane into a culture medium cultured using methane

After culturing while supplying ethane to the three types of methanogens cultured in Example 1-1, changes in the concentration of cells present in the culture, acetaldehyde, acetoin, and acetic acid as the cultivation time elapses Was measured (Fig. 1).

1 shows three different wild-type methanogens (Methylomonas sp. DH-1 strain (A); Methylomicrobium alcaliphilum 20Z strain (B)); Methylosinus trichosporium OB3b strain expressing methane oxidase, methanol dehydrogenase, and aldehyde lyase. (C)) is a graph showing the result of comparing the concentrations of acetoin and other components produced from the culture when methane (left) or ethane (right) is provided as a substrate and cultured.

As shown in FIG. 1, it was confirmed that each wild-type methanogen expressing methane oxidase, methanol dehydrogenase, and aldehyde lyase can produce acetoin from ethane through a chain reaction of the expressed enzyme.

Example 2: Production of acetoin using a gene-introduced methanogen

Acetoin was produced by using the formolase (FLS) gene and the acetoin reductase (BudC) gene.

Example 2-1: Construction of transformation vector

First, a gene encoding formolase (FLS), an enzyme that converts acetaldehyde to acetoin, was used as a template, and PCR was performed using the following primers to obtain a FLS gene amplification product.

FLS_F: 5'-GAAACAGCTATGGCTATGATTACTGGTGG-3' (SEQ ID NO: 1)

FLS_R: 5'-tgtgatgacTCACGCGCCGGATTGGAAAT-3' (SEQ ID NO: 2)

In addition, a gene encoding acetoin reductase (BudC), an enzyme that converts acetoin to 2,3-butanediol (2,3-BDO), was used as a template, and PCR was performed using the following primers to obtain a BudC gene amplification product. I did.

BudC_F: 5'-GGCGCGTGAgtcatcacaataaggaaaga-3' (SEQ ID NO: 3)

BudC_R: 5'-CGACAACTATTAGTTAAACACCATGCCGC-3' (SEQ ID NO: 4)

Finally, using the pAWP89 vector as a template, PCR was performed using the following primers to obtain an amplification product of the pAWP89 vector.

pAWP89_F: 5'- GGCGCGTGATTGTCGGGAAGATGCGTGAT-3' (SEQ ID NO: 5)

pAWP89_R: 5'- TTTAACTAATAGTTGTCGGGAAGATGCGT-3' (SEQ ID NO: 6)

The obtained FLS gene amplification product and the BudC gene amplification product (SEQ ID NO: 7) were introduced into the pAWP89 vector to prepare a methanogen transformation vector.

Example 2-2: Production of acetoin using transformed methanogens

The transformation vector prepared in Example 2-1 was introduced into the wild-type methanogen M. alcaliphilum strain 20Z by electroporation.

Approximately, after culturing M. alcaliphilum strain 20Z, after washing three times with water, 1 μg of the expression vector designed in the expression vector production experiment was transformed using the Gene Pulser XcellTM Electropotation system (Bio-rad). After electroporation, the cells were recovered, transferred to 10 ml NMS medium, stabilized by supplying methane substrate, plated on solid NMS medium supplemented with kanamycin, and cultured to obtain transformed methanogens.

2,3-butanediol (2,3-BDO), acetaldehyde, acetoin, and acetic acid present in the culture after culturing while supplying ethane to the obtained transformed methanogens. The change in concentration of was measured (Fig. 2). At this time, as a control, a culture of untransformed wild-type methanogen M. alcaliphilum strain 20Z was used.

2 is a comparison of the concentrations of acetoin and other components produced from the culture of the FLS gene and the BudC gene introduced transgenic methanogen M. alcaliphilum strain 20Z, when cultured by providing ethane as a substrate. As a graph showing the results, (A) represents a culture of wild-type methanogens M. alcaliphilum strain 20Z as a control, and (B) represents a culture of transformed methanogens M. alcaliphilum strain 20Z.

As shown in FIG. 2, the concentration of acetaldehyde and acetic acid in the culture did not show any difference between the wild-type strain and the transformed strain, but the concentration of 2,3-butanediol (2,3-BDO) and acetoin was a wild-type strain. Compared to, it was confirmed that it was significantly increased in the transformed strain. In the wild-type strain, 2,3-butanediol (2,3-BDO) was not produced, and acetoin was produced at a maximum of 15 mg/L, but in the transformed strain, 2,3-butanediol (2,3-BDO) was produced at a maximum of 1.1. It was confirmed that it was produced at mg/L, and acetoin was produced at a maximum of 30 mg/L.

Example 3: Production of acetoin using a gene-deleted methanogen

Acetoin was produced by using a methane magnetizing bacteria in which acetate kinase (ack), phosphate acetyltransferase (pta), acetyl-coenzyme A synthetase (acs) or aldehyde ferredoxin oxidoreductase (aor) was deleted.

Example 3-1: Preparation of transgenic methanogens in which the ack gene was deleted

First, the upstream 750bp of the gene (SEQ ID NO: 8) encoding acetate kinase (ack) present in the genome of the M. alcaliphilum strain 20Z strain was used as a template, and PCR was performed using the following primers to obtain an amplification product. I did.

ack_up_F: 5'-ttggtctgacagttaccaTGCATGTGCGGGGCTATAAA-3' (SEQ ID NO: 9)

ack_up_R: 5'-cccgatttcTTGATTGAGGAACTGCCGCT-3' (SEQ ID NO: 10)

Next, the downstream 750 bp of the gene encoding acetate kinase (ack) present in the genome of the M. alcaliphilum strain 20Z strain was used as a template, and PCR was performed using the following primers to obtain an amplification product.

ack_down_F: 5'-ctcaatcaaGAAATCGGGCTTGCCGAAAG-3' (SEQ ID NO: 11)

ack_down_R: 5'-caccgacaacctgcacatCGACCCGCCTTATTACACGA-3' (SEQ ID NO: 12)

Finally, using the pCM433kanT vector as a template, PCR was performed using the following primers to obtain an amplification product of the pCM433kanT vector.

pCM433kanT_F: 5'-ATGTGCAGGTTGTCGGTGTC-3' (SEQ ID NO: 13)

pCM433kanT_R: 5'-TGGTAACTGTCAGACCAAGTTTACTC-3' (SEQ ID NO: 14)

The obtained upstream amplification product and downstream amplification product were introduced into the pCM433kanT vector to obtain a recombinant vector, and then PCR was performed using the recombinant vector as a template and primers of SEQ ID NOs: 9 and 12 to obtain a gene fragment of 1500 bp. I did.

The 1500bp gene fragment obtained above was introduced into the pCM433kanT vector to prepare a transformation vector for ack deletion.

The prepared transformation vector for ack deletion was introduced into the wild-type methanogen M. alcaliphilum strain 20Z by electroporation.

Approximately, M. alcaliphilum strain 20Z was cultured, washed three times with water, and then 1 μg of a transformation vector for ack deletion was transformed using a Gene Pulser XcellTM Electropotation system (Bio-rad). After the electroporation, the cells were recovered, transferred to 10 ml NMS medium, stabilized by supplying methane substrate, plated on solid NMS medium to which kanamycin was added, and cultured to obtain a single colony. The obtained single colony was re-sprayed on a solid NMS medium to which sucrose was added to obtain cells. Using the obtained genome of the cells as a template, PCR was performed using primers of SEQ ID NOs: 9 and 12 to finally select transformants in which the 1500 bp gene fragment was not amplified.

Example 3-2: Preparation of transgenic methanogens in which the pta gene was deleted

First, 750bp upstream of the gene (SEQ ID NO: 15) encoding phosphate acetyltransferase (pta) present in the genome of M. alcaliphilum strain 20Z as a template, and PCR using the following primers to obtain an amplification product I did.

pta_up_F: 5'-ttggtctgacagttaccatcaacgaccgattgaaa-3' (SEQ ID NO: 16)

pta_up_R: 5'-gtaagtcggcggcgatacaggctcaa-3' (SEQ ID NO: 17)

Next, 750 bp downstream of the gene encoding phosphate acetyltransferase (pta) present in the genome of the M. alcaliphilum strain 20Z strain was used as a template, and PCR was performed using the following primers to obtain an amplification product.

pta_down_F: 5'-gtatcgccgccgacttaccgctgccc-3' (SEQ ID NO: 18)

pta_down_R: 5'-CACCGACAACCTGCACATacctttttcgtgcaaaa-3' (SEQ ID NO: 19)

The obtained upstream amplification product and downstream amplification product were introduced into the pCM433kanT vector to obtain a recombinant vector, and then PCR was performed using the recombinant vector as a template and primers of SEQ ID NOs: 16 and 19 to obtain a gene fragment of 1500 bp. I did.

The 1500bp gene fragment obtained above was introduced into the pCM433kanT vector to prepare a transformation vector for pta deletion.

The prepared transformation vector for pta deletion was introduced into the wild-type methanogen M. alcaliphilum strain 20Z by electroporation.

Approximately, M. alcaliphilum strain 20Z was cultured, washed three times with water, and then 1 μg of a transformation vector for ack deletion was transformed using a Gene Pulser XcellTM Electropotation system (Bio-rad). After the electroporation, the cells were recovered, transferred to 10 ml NMS medium, stabilized by supplying methane substrate, plated on solid NMS medium to which kanamycin was added, and cultured to obtain a single colony. The obtained single colony was re-sprayed on a solid NMS medium to which sucrose was added to obtain cells. Using the genome of the obtained cells as a template, PCR was performed using primers of SEQ ID NOs: 16 and 19 to finally select transformants in which the 1500 bp gene fragment was not amplified.

Example 3-3: Preparation of transgenic methanogens in which the acs gene was deleted

First, 750bp upstream of the gene (SEQ ID NO: 20) encoding acetyl-coenzyme A synthetase (acs) present in the genome of M. alcaliphilum strain 20Z as a template, and amplified by performing PCR using the following primers The product was obtained.

acsA_up_F: 5'-TTGGTCTGACAGTTACCAtaattaccgactcgcacg-3' (SEQ ID NO: 21)

acsA_up_R: 5'-gaggcgactttccggtgtaatgtgagc-3' (SEQ ID NO: 22)

Next, the downstream 750 bp of the gene encoding acetyl-coenzyme A synthetase (acs) present in the genome of the M. alcaliphilum strain 20Z strain was used as a template, and PCR was performed using the following primers to obtain an amplified product. .

acsA_down_F: 5'-acaccggaaagtcgcctcgaacgaaat-3' (SEQ ID NO: 23)

acsA_down_R: 5'-CACCGACAACCTGCACATggatttttcttcgtcgcg-3' (SEQ ID NO: 24)

The obtained upstream amplification product and the downstream amplification product were introduced into the pCM433kanT vector to obtain a recombinant vector, and then PCR was performed using the recombinant vector as a template and primers of SEQ ID NOs: 21 and 24 to obtain a gene fragment of 1500 bp. I did.

The 1500bp gene fragment obtained above was introduced into the pCM433kanT vector to prepare a transformation vector for acs deletion.

The prepared transformation vector for acs deletion was introduced into the wild-type methanogen M. alcaliphilum strain 20Z by electroporation.

Approximately, M. alcaliphilum strain 20Z was cultured, washed three times with water, and then 1 μg of a transformation vector for ack deletion was transformed using a Gene Pulser XcellTM Electropotation system (Bio-rad). After the electroporation, the cells were recovered, transferred to 10 ml NMS medium, stabilized by supplying methane substrate, plated on solid NMS medium to which kanamycin was added, and cultured to obtain a single colony. The obtained single colony was re-sprayed on a solid NMS medium to which sucrose was added to obtain cells. Using the obtained genome of the cells as a template, PCR was performed using primers of SEQ ID NOs: 21 and 24 to finally select transformants in which the 1500 bp gene fragment was not amplified.

Example 3-4: Preparation of transgenic methanogens in which the aor gene was deleted

First, the upstream 750bp of the gene (SEQ ID NO: 25) encoding the aldehyde ferredoxin oxidoreductase (aor) present in the genome of the M. alcaliphilum strain 20Z strain was used as a template, and PCR was performed using the following primers to obtain the amplified product. Obtained.

aor_up_F: 5'-TTGGTCTGACAGTTACCActaacgcaaaccgatga-3' (SEQ ID NO: 26)

aor_up_R: 5'-tagtgcctcctatagcgatttaatcga-3' (SEQ ID NO: 27)

Next, 750 bp downstream of the gene encoding aldehyde ferredoxin oxidoreductase (aor) present in the genome of the M. alcaliphilum strain 20Z strain was used as a template, and PCR was performed using the following primers to obtain an amplified product.

aor_down_F: 5'-tcgctataggaggcactatgcattatg-3' (SEQ ID NO: 28)

aor_down_R: 5'-ACCGACAACCTGCACATcgcttccttccaaaaaat-3' (SEQ ID NO: 29)

The obtained upstream amplification product and the downstream amplification product were introduced into the pCM433kanT vector to obtain a recombinant vector, and then PCR was performed using the recombinant vector as a template and primers of SEQ ID NOs: 26 and 29 to obtain a gene fragment of 1500 bp. I did.

The 1500bp gene fragment obtained above was introduced into the pCM433kanT vector to prepare a transformation vector for aor deletion.

The prepared transformation vector for aor deletion was introduced into the wild-type methanogen M. alcaliphilum strain 20Z by electroporation.

Approximately, M. alcaliphilum strain 20Z was cultured, washed three times with water, and then 1 μg of a transformation vector for ack deletion was transformed using a Gene Pulser XcellTM Electropotation system (Bio-rad). After the electroporation, the cells were recovered, transferred to 10 ml NMS medium, stabilized by supplying methane substrate, plated on solid NMS medium to which kanamycin was added, and cultured to obtain a single colony. The obtained single colony was re-sprayed on a solid NMS medium to which sucrose was added to obtain cells. Using the obtained genome of the cells as a template, PCR was performed using primers of SEQ ID NOs: 26 and 29 to finally select transformants in which the 1500 bp gene fragment was not amplified.

Example 3-5: Production of acetoin using transgenic methanogens with deletion of genes

After culturing while supplying ethane to each of the transformed methanogens prepared in Examples 3-1 to 3-4, changes in the concentration of acetoin and acetic acid present in the culture were measured as the cultivation time elapsed. Was done (Fig. 3). At this time, as a control, a culture of untransformed wild-type methanogen M. alcaliphilum strain 20Z was used.

Figure 3 is each culture obtained by culturing while supplying ethane to each of the methane-sugar bacteria in which acetate kinase (ack), phosphate acetyltransferase (pta), acetyl-coenzyme A synthetase (acs) or aldehyde ferredoxin oxidoreductase (aor) is deleted. This is a graph showing the result of comparing the concentrations of acetoin and acetic acid contained in.

As shown in FIG. 3, in the case of the wild-type strain, acetic acid was produced at the highest concentration, while the concentration of acetoin was relatively the lowest. In contrast, each of the transgenic methanogens in which the gene was deleted showed a relatively high concentration of acetoin and a relatively low concentration of acetic acid compared to the wild type. In particular, it was confirmed that acetoin was produced in a higher concentration than acetic acid in the transgenic methanogens in which aor was deleted.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, the embodiments described above are illustrative in all respects and should be understood as non-limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description and equivalent concepts are included in the scope of the present invention.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Process for preparing acetoin using transformed methanotrophs <130> KPA180803-KR-P1 <150> KR 10-2018-0080668 <151> 2018-07-11 <160> 29 <170> KopatentIn 2.0 <210> 1 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gaaacagcta tggctatgat tactggtgg 29 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tgtgatgact cacgcgccgg attggaaat 29 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ggcgcgtgag tcatcacaat aaggaaaga 29 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 cgacaactat tagttaaaca ccatgccgc 29 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ggcgcgtgat tgtcgggaag atgcgtgat 29 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 tttaactaat agttgtcggg aagatgcgt 29 <210> 7 <211> 2522 <212> DNA <213> Artificial Sequence <220> <223> recombinant DNA <400> 7 atggctatga ttactggtgg tgaactggtt gttcgtaccc tgattaaagc tggcgtagaa 60 catctgtttg gcctgcatgg cattcatatt gacaccattt ttcaggcttg cctggaccac 120 gacgtcccaa tcattgatac tcgccacgaa gcggcggcag gccacgctgc ggaaggttat 180 gcccgcgcgg gcgctaaact gggtgttgcc ctggtgaccg ctggcggtgg ctttaccaat 240 gccgttacgc cgatcgcgaa cgctcggacc gatcgcactc cggttctgtt cctgaccggt 300 tctggtgctc ttcgtgatga cgaaaccaac accctgcagg ccggtattga tcaggtggcc 360 atggcggccc cgatcacgaa atgggctcat cgtgttatgg caactgaaca catcccgcgt 420 ctggttatgc aggccattcg tgccgctctg agcgccccac gtggcccggt gctgctggat 480 ctgccatggg acatcctgat gaaccaaatc gatgaagatt ccgttatcat cccagacctg 540 gtgctgtctg ctcacggtgc ccatccagac ccggctgacc tggaccaggc tctggcactg 600 ctgcgtaaag ccgaacgccc agttatcgta ctgggctccg aggcgtcccg caccgcacgc 660 aagaccgcac tgagcgcatt cgtagcggcg accggtgtac cggttttcgc tgactatgaa 720 ggcctgtcca tgctgagcgg cctgccggac gctatgcgtg gcggcctggt gcagaacctg 780 tactcctttg caaaagctga tgcagctccg gacctggtac tgatgctggg tgctcgtttc 840 ggtctgaaca ccggtcatgg ttccggtcaa ctgatcccgc attctgctca ggtgatccag 900 gtggatccag acgcgtgtga actgggtcgc ctgcaaggca tcgcgctggg tatcgtggct 960 gatgtaggtg gcaccattga agcgctggct caggcgaccg cacaggacgc cgcgtggccg 1020 gaccgcggcg actggtgcgc caaggtaact gacctggccc aggagcgtta cgcttccatc 1080 gcggctaaat ccagctctga acatgcgctg cacccgttcc acgcttctca ggttatcgcg 1140 aaacacgtgg acgcaggcgt gaccgtcgtt gcggatggtg gcctgactta tctgtggctg 1200 tccgaagtta tgtctcgtgt caaaccaggc ggcttcctgt gccacggcta tctgaacagc 1260 atgggtgtag gcttcggtac tgccctgggt gcgcaggttg cggatctgga ggcaggtcgt 1320 cgtaccatcc tggtgaccgg cgacggctct gttggttatt ccattggcga attcgacacc 1380 ctggtacgca aacagctgcc gctgattgta attatcatga acaaccagtc ttggggctgg 1440 accctgcact ttcagcagct ggccgttggt cctaaccgtg tcaccggcac ccgcctggaa 1500 aatggttcct atcacggcgt tgctgcggca ttcggtgctg atggttacca cgtcgactct 1560 gtcgagagct tcagcgccgc tctggctcag gcactggcac acaaccgccc ggcatgcatc 1620 aacgttgctg tggccctgga cccgatcccg ccggaggaac tgatcctgat tggcatggac 1680 ccgtttgcgg gctccacgga gaatctgtat ttccaatccg gcgcgtgagt catcacaata 1740 aggaaagaaa aatgaaaaaa gtcgcacttg ttaccggcgc cggccagggg attggtaaag 1800 ctatcgccct tcgtctggtg aaggatggat ttgccgtggc cattgccgat tataacgacg 1860 ccaccgccaa agcggtcgcc tccgaaatca accaggccgg cggccgcgcc atggcggtga 1920 aagtggatgt ttctgaccgc gaccaggtat ttgccgccgt cgaacaggcg cgcaaaacgc 1980 tgggcggctt cgacgtcatc gtcaacaacg ccggcgtggc gccatccacg ccgatcgagt 2040 ccattacccc ggagattgtc gacaaagtct acaacatcaa cgtcaaaggg gtgatctggg 2100 gcatccaggc agcggtcgag gcctttaaga aagagggtca cggcgggaaa atcatcaacg 2160 cctgttccca ggccggccac gtcggcaacc cggagctggc ggtatatagc tcgagtaaat 2220 tcgcggtacg cggcttaacc cagaccgccg ctcgcgacct cgcgccgctg ggcatcacgg 2280 tcaacggcta ctgcccgggg attgtcaaaa cgccgatgtg ggccgaaatt gaccgccagg 2340 tgtccgaagc cgccggtaaa ccgctgggct acggtaccgc cgagttcgcc aaacgcatca 2400 ccctcggccg cctgtccgag ccggaagatg tcgccgcctg cgtctcctat cttgccagcc 2460 cggattctga ttatatgacc ggtcagtcat tgctgatcga cggcggcatg gtgtttaact 2520 aa 2522 <210> 8 <211> 1206 <212> DNA <213> Artificial Sequence <220> <223> recombinant DNA <400> 8 atgacaatac caaacggcaa cattctcgtg atcaacagcg gcagttcctc aatcaagtat 60 cgattgattg ctctgccgca agagcaggta ctggcagacg gcttgctgga acgcatcgga 120 gaacaggaaa gcaggatcat tcatagagcc gacgattcgg gtcgtttaaa tgaaatcaag 180 cagtcggtca tcgctgccga tcatcaccaa gccttcaagg cagtcttcga gattttgggc 240 gaaaattgct cggtcgatgc aataggccac cgcgtcgtgc atggcggcga tcggttctcc 300 ggccctgcct tggtcgatga cgatacgata gcgtcgatgc gcgcactctg ccgaatagcg 360 ccgctgcata atccggttaa cttgcttggc atcgagagtt gcttggctca tttcccgggc 420 gtaccacagg tggcggtatt cgatacggca tttcaccaaa cgatgccgcc ccacgcctat 480 cgttatgcga ttccggaaac ttggtatagc gattacggca tacgccgatt cggttttcac 540 ggcacctctc atcattatgt ggcgagacgg gccgccgaat ttatcggtaa acctttcgat 600 cgcagccatc tgattacttt gcatttgggc aatggcgcga gcgcaacagc gattgcaaac 660 ggccgctccg tcgatacgtc gatggggttt acgccgctgg aaggtttggt aatgggcacg 720 cgtagcggcg atttggaccc ggcaataccg ctatttgtcg aacaaaccga aaataccgac 780 acggacgcaa tcgaccgggc attgaaccgc gaatccggat taaaaggctt atgtggtacc 840 aacgacttaa gaaccgtgct cgaacaaaca aatgcaggcg atgaacgagc ccgcttggct 900 ctcgatctgt attgctatcg aatcaagaaa tatatcggcg cttactacgc ggtactcggc 960 gaagtcgacg ctctggtttt taccggcggc gtcggcgaaa acgcggccga agtgcgccgt 1020 ttagcctgcg aaggcctgtc gcgtctcggc atcgccattg atgaagcggc caatagcgac 1080 gtgaccggag ctatcgccga aatcgggctt gccgaaagtc gaacccgtat tctagtcatt 1140 aaaactgacg aagaattgca aattgcccgg gaagccatgg ctgtgcttga taaagatcac 1200 gcatga 1206 <210> 9 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ttggtctgac agttaccatg catgtgcggg gctataaa 38 <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 cccgatttct tgattgagga actgccgct 29 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ctcaatcaag aaatcgggct tgccgaaag 29 <210> 12 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 caccgacaac ctgcacatcg acccgcctta ttacacga 38 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 atgtgcaggt tgtcggtgtc 20 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 tggtaactgt cagaccaagt ttactc 26 <210> 15 <211> 2115 <212> DNA <213> Artificial Sequence <220> <223> recombinant DNA <400> 15 ctaagcagcc ggttttcgac cggtttcttg agcctgtatc gccgtgatca caacggtatt 60 gacgatatcg tttacggtac agcctcggct taaatcgttg accggtttat tcaagccttg 120 caaaataggc ccgatggcaa tcgctccgga cgagcgctga actgctttat aagtattgtt 180 gccggtattt agatcgggaa aaataaatac gttcgcttgc cctgccacct cgctgtccgg 240 cattttggtc ttggcaacac ggctatcgat cgccgcatcg tattgaatcg gcccttccag 300 tttcaattcc ggtttcaatc gtttagccaa ttcgactgcc gacctaacct tatcgaccgc 360 ctcgcctttt ccggactcgc cggttgaata agaaagcatc gcaacgcgtg gcgtgatatt 420 gaacatactc gcggtttgcg cggaactaat cgcaatatcg gctaattgtc tggaattagg 480 attcggattg accgcgcaat ccccataaac cagcacccgg tcttccaagc acatgaaaaa 540 aacgctagag acgatcgatg ttcccggttt ggttttgatg atttcaaagg ccggcctgat 600 cgtatgctgg gtcgtatgta ttgacccgga aaccattccg tcggcaagat tatggtgtac 660 catcatcgtc ccgaaaaaac tgacatctgt cataagatca aacgccaatt cgtaatgaac 720 gcccttatgc ttgcgtaact cgaaataagt ttcggcaaat ttctgacgca gttcggaagt 780 catcggatcg ataatctcga tgtcttctaa ttgcaaagcc atcgcagcga ttttttgctt 840 gatcgtttct tcattaccta gcagcgtcaa cttgacggct cccctaagga aaaggatttc 900 ggctgccctt aaaatgcgct cttcctcgcc ttcaggcaaa acaatatgct gcggattggc 960 tttggctcgc tgcaaaattt catattcgaa catgatcggc gtcacgcgcg gcgttttacg 1020 cgtaaacagg cgctgctgca gctcctgcat gttgatatta tcttcgacca aactcaaggc 1080 cgtggcgatc ttgcgttcat tattggaatt taaacgcgca tgtactcgag taacgttcat 1140 cgccgtcgtg aaggtgtcgg tggcgacacc taaaacggca aacggcaaag cccctaatcc 1200 ctcgattaag tattgcacct gcggcgcggg ctcttgctct ccagtcaaca gtaaaccggc 1260 aatttgcgga taacttttcg agtgataagc catcaaacaa gccagaatga tatccgaacg 1320 atccccgggc gtaatgatca aatcgccttt ctcaatgtag tcgagaaaat ccgaaactaa 1380 catcgccgca accttgtagt tataaacttc aaggttgagc gtttcgaaat catgcgataa 1440 aaactttgca ttcaacgacc tggcaatatc gcccatgctg ggcttttcca gtgacggttc 1500 ggagggtacg acataaacag gaaagttgga ttgcggatcg tgtccgaatt gatctctaag 1560 ttcgccaacc tgagccgccg gaacactgtt gatcaccgtt gccaacagga cagcattttt 1620 ttcctgaata tcatgtcgga gcccggccaa agcatcgatt atttgttcat tggatcgatt 1680 gcctccttga ataattggca tcaacagaca atctaaattg ttcgcgacat cggcattaaa 1740 atcgaactca aaaatcgact cgccgctttt ataatcggaa ccgacacaaa tcacatgatc 1800 cgattgaatt tttaagcttc ggtatttagc gagaatcagt tttattaaat cgtcataacg 1860 atcatgagcc aataggcgcc gagcttcctc atcggtgcat ccgtacatcg attcgtacgg 1920 ccactcgagc tgataacgat aggttactag ctcaatcaag tcgtcttttt cctctccggc 1980 atgaataata gggcgaaaaa aaccgatctt ttgggcaaaa ccggaaaaca tttccatcat 2040 agctagcatg atgaccgact taccgctgcc ctgctcggca ccggttatat aaatattttt 2100 agagacttgg gacat 2115 <210> 16 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 ttggtctgac agttaccatc aacgaccgat tgaaa 35 <210> 17 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 gtaagtcggc ggcgatacag gctcaa 26 <210> 18 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 gtatcgccgc cgacttaccg ctgccc 26 <210> 19 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 caccgacaac ctgcacatac ctttttcgtg caaaa 35 <210> 20 <211> 1938 <212> DNA <213> Artificial Sequence <220> <223> recombinant DNA <400> 20 atgactgaag tgaaaaccta tcctgtcgat cccgaactgt cagcccgagc tcacattaca 60 ccggaacgct ataaaacact ttatcgacaa tcgatcgatg aaccggaatc cttttgggcc 120 gaacaagccg aacaatttct cgactggttc aagccttggg ataaagtcat ggattacgac 180 tattcgaaag gctacatccg ctggttcgaa ggcggcgaac tcaatgtcag ctacaattgc 240 ttggaccggc atttagccac acgaggcgac caaactgcga ttatctggga aagcgacgat 300 cccgctcaag acaaaaaagt cagttaccgt gaattgcacc gagaagtttg caaattcgcc 360 aatgttctaa aaagccaagg cgtcaaaaaa ggcgaccgag tctgtattta cctgccgatg 420 atcgtcgaag cggccgtcgc caccctggcc tgtgcccgaa tcggcgcagt ccactccatc 480 gtgttcggcg gtttttcggc cgactcttta cgcgaccgca tacaagacgc cgactgccac 540 actgttatct gcgccgatga agggcttcgc ggcggcaaaa agataccgct caaaaccagt 600 gtcgaccagg cgatttcaca ctgcccgaat gtcaaaactg tcatcgtcat caaaaacacc 660 ggaaatgata ttccgtggac gccgaaacgc gatatttggt atcacgaagc gatgaaaacc 720 gcatcggaca actgcccgcc cgagacccta tccgccgaag acccgttatt cattctttac 780 acatcgggtt ctaccggtaa accgaagggc ctgctgcaca ccacgggcgg atatttatta 840 ttcgccgcga tgacgcataa atatgtattc gattatcacg acggcgatat ttattggtgc 900 accgccgata tcggctgggt caccggccat tcttacatca tttacggacc gctctgcaac 960 ggcgcaacga ctttgatgtt cgaaggtatt ccgacctacc ccgagcccga ccgattttgg 1020 cgcatcgtcg ataaacacca agtcaatatt ttttataccg cgccaactgc aattcgcgcc 1080 ttgatggcgc aaggcaacga ctttgtcaat cgcaccgacc gcagcagttt aagaatcctc 1140 ggcacggtcg gcgaaccgat caaccccgaa gcgtgggaat ggtattatca catcgtcggc 1200 aacgaacgct gccctatcat ggatacttgg tggcaaaccg aaaccggggg cattttgatc 1260 acgccgctgc ccggcgccat agcattgaaa ccgggctcgg cgacactgcc gttcttcggc 1320 gttaaaccgg aaattctcga caatgccggt aatgtactcg aaggcgaagc tgagggggtt 1380 ctagtattga gccgcccgtg gccgggtcag gccagaagta tttacggcga tcacaaccgt 1440 tttatcgaca cctattttaa aaattttccg ggctattatt ttgccggcga cggcgctcgc 1500 cgcgacaggg acggttatta ctggatcacc ggccgcgtcg atgatgtaat caatgtttcg 1560 ggccatcgca tggggaccgc agaagtcgaa agtgcgctgg tcttgcatga agacgtcgcc 1620 gaagccgccg tggtgggcta cccgcatcct atcaagggcc agggcattta tgcttatgtc 1680 acgctcaatt ccggcgttca cgcaagcgaa gctctcaagc aggaattggt cgatctggtt 1740 agaaaagaaa tcggagccat cgcccacccc gatatcattc aatgggcacc gggccttccg 1800 aaaacgcgct caggaaaaat catgcggcgt attttaagaa aagtcgcctc gaacgaaatc 1860 gacagcctag gcgatacctc cacgctggca gacccatcgg ttgtagatga aatcatcgat 1920 catagagcca ataaataa 1938 <210> 21 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 ttggtctgac agttaccata attaccgact cgcacg 36 <210> 22 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 gaggcgactt tccggtgtaa tgtgagc 27 <210> 23 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 acaccggaaa gtcgcctcga acgaaat 27 <210> 24 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 caccgacaac ctgcacatgg atttttcttc gtcgcg 36 <210> 25 <211> 1851 <212> DNA <213> Artificial Sequence <220> <223> recombinant DNA <400> 25 atggcctgga ccggaaacgt actacgcgtc aatttgaccg aacgtacctg cagcaaagaa 60 ccgcttaaca aagactgggc gctgagctat ttaggccagc gaggcctggc gactaaatat 120 ctaaccgaag aaatcgaccc caaaatcgat gctctatcgc cggacaataa attgattatg 180 gcaacagggc ctctgaccgg cacgccggca tcgaccgccg ggcgctattc ggttatcacc 240 aaaagcccgt tgaccggtac ggtcgcctgc tcgaattccg gcggttttat cggcatggaa 300 ttcaaaaacg ccggctggga tatgattatc ttcgaaggca aggcttcttc tccggtttat 360 ttgtatttgg aaaacgaaaa ggctcaattg ctgccggctg acgaaatctg gggtcagtcg 420 gtttggcagg ccgacgaatg gctgcataat cgtcatcaag atcccttatt gcggatcgcg 480 gcggttggtc gttccgccga acaaggttgt ttgttttcag ggatcatcaa tgatttacac 540 cgagctgccg ggcgttcagg cgtcggcacc gtgatggcag cgaaaaatct caaggccgtc 600 gctgttcgcg gcacgcgagg cgtcggcaat atcaaagacc cggttgcctt tatgcaagca 660 gtcaatgccg gtaaaaaagt actggctgag gggcctgtga ctgccgaagg cttgccgact 720 tacggtacgc aagtcttgat gaatgtcatc aacgaagtcg gcgcgatgcc tacccgaaat 780 ttcaaggacg tgcaattcga aggcgccgcg aagatttccg ccgaagcgat gcacgatccg 840 cgcccttcgg acggcaaacc gaatctggtc acgaatgccg cctgcttcgg ctgcacgatc 900 gcgtgcggac gcatttcgac gatagccggc ggccatttta cgatcacaaa caaaccgcaa 960 taccggggcg catcgggcgg tttggaatac gaggcggcct gggcattggg gtcggatacc 1020 ggcgtcgacg atctcgatgc gctgacctat gtcaatttct tatgcaacga agacggcatg 1080 gatccgattt cgctcggttc gacgatcgca gcggcgatgg agttatacga atccggagcg 1140 atcacgattg aacagaccgg tggcatcgaa ttgaaattcg gttcggccga agcgctggtt 1200 aaattgaccg agttgactgc taaaggcgaa ggcttcggac gcgaaatcgg tctcggctcg 1260 aaacgcttat gcgaaaaata cgggcgtccc gaattgtcga tgacggtcaa gggccaggag 1320 tttccggcat acgacccgcg cggcattcaa ggcatggggc tggcctatgc gacctcgaat 1380 cgcggcgcct gtcatttaag aggttatacg gtttcatcgg aaatcatggg tttgccggaa 1440 aagaccgacc ccttgattac cgacggcaaa gccgccttgg tgaaagcctt tcaggatgcg 1500 accgcagcgg tggactcagc cggattgtgc gtgttttcga ccttctcatg gacgatggcc 1560 gatatcgcgc cacagatcgc cgcagcctgc gacggcgact ggaccgtcga tagcttgctc 1620 gaaaccggcg agcgcatctg gaatttggag cgaaaattta atttggatgc gggcctcacc 1680 ggcgccgacg acaatttgcc cgagcgtttg ttaaaagaag ccgccaaaac ggggccggct 1740 aaaggcaagg tcaacgactt ggccaaaatg ctccccgaat attaccaatt gcgcggctgg 1800 acggcagacg gcataccgaa agccgaaacc ttgtcgcggc tgtcggtgta g 1851 <210> 26 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 ttggtctgac agttaccact aacgcaaacc gatga 35 <210> 27 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 tagtgcctcc tatagcgatt taatcga 27 <210> 28 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 tcgctatagg aggcactatg cattatg 27 <210> 29 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 accgacaacc tgcacatcgc ttccttccaa aaaat 35

Claims (10)

Any selected from the group consisting of a gene encoding ack (acetate kinase), a gene encoding pta (phosphoate acetyltransferase), a gene encoding acetyl-coenzyme A synthetase (acs), and a gene encoding aldehyde ferredoxin oxidoreductase (aor). A method for producing acetoin comprising the step of culturing the transformed methanogens in which one gene is inactivated in an atmospheric condition containing ethane or a medium containing ethane.
The method of claim 1,
The methane magnetization bacteria methyl Pseudomonas (Methylomonas) genus, methyl bakteo (Methylobacter) in methyl in Rhodococcus (Methylococcus) in micro byum (Methylomicrobium) in methyl Los Blow (Methylosphaera) in methyl local extra methyl ( Methylocaldum ) genus, Methyloglobus genus, Methylosarcina genus, Methyloprofundus genus, Methylothermus genus, Methylohalobius Genus, Methylogaea genus, Methylomarinum genus, Methylovulum genus, Methylomarinovum genus, Methylorubrum genus, Methyloparacoccus Methyloparacoccus ) genus, Methylosinus genus, Methylocystis genus, Methylocella genus, Methylocapsa genus, Methylofurula genus, Methylacidiphilum ( Methylacidiphilum ) genus or methyl acidimicrobium (Methylacidimicrobium) genus strain that is, acetoin production method.
The method of claim 1,
The gene encoding ack (acetate kinase) consists of the nucleotide sequence of SEQ ID NO: 8, the gene encoding the pta (phosphoate acetyltransferase) consists of the nucleotide sequence of SEQ ID NO: 15, and the acs (acetyl-coenzyme A synthetase) ) The gene encoding is composed of the nucleotide sequence of SEQ ID NO: 20, and the gene encoding the aor (aldehyde ferredoxin oxidoreductase) is composed of the nucleotide sequence of SEQ ID NO: 25, acetoin production method.
The method of claim 1,
The concentration of ethane in the medium is 0.01% to 90% (v / v) that, acetoin production method.
The method of claim 1,
The transgenic methanogen production method is that the gene encoding FLS (formolase) is additionally introduced.
The method of claim 1,
The method of producing acetoin further comprising the step of culturing the transformed methanogens and then recovering acetoin from the culture.
Any selected from the group consisting of a gene encoding ack (acetate kinase), a gene encoding pta (phosphoate acetyltransferase), a gene encoding acetyl-coenzyme A synthetase (acs), and a gene encoding aldehyde ferredoxin oxidoreductase (aor). Transgenic methanogens for the production of acetoin in which one gene is inactivated.
A composition for producing acetoin, comprising the transformed methanogens of claim 7 or a culture product thereof.
The method of claim 8,
The culture product is selected from the group consisting of a culture product of the transformed methanogens, a culture supernatant, a lysate, a fraction thereof, and a combination thereof, a composition for producing acetoin.
A kit for producing acetoin, comprising the composition for producing acetoin of claim 8.

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