KR20140001587A - Production of alcohol method using bio-ethanol production capacity of recombinant ralstonia eutropha - Google Patents

Abstract

The present invention relates to a method for producing ethanol using an ethanol producible recombinant strain and, more specifically, to a recombinant strain having improved bio-ethanol producibility by redesign of metabolism and a method for producing ethanol using the recombinant strain. In the method for producing ethanol according to the present invention, using a recombinant photosynthetic bacterium, an electrochemical reducing power is an energy source and carbon dioxide is the only carbon source and pyruvate is converted into acetaldehyde using sugar provided as a substrate, and the acetaldehyde can be converted into ethanol, thereby effectively producing the bio-ethanol.

Description

[0001] The present invention relates to a method for producing an alcohol using a recombinant strain for producing ethanol,

The present invention relates to a method for producing an alcohol using a recombinant strain for ethanol production, and more particularly, to a recombinant strain having improved bio-ethanol production ability using metabolic engineering and a method for producing bio-ethanol using the recombinant strain.

Concerns over skyrocketing oil prices and energy depletion have heightened interest in alternative energy production. Bioethanol is the most active source of alternative fuels for transportation. This is the initial stage of the development of the electric vehicle or the hydrogen vehicle, which is the final technical objective of the automobile, and it is still necessary to have an alternative energy to replace it.

At present, bioethanol can be mixed with gasoline, so it is a representative renewable resource energy in addition to biodiesel. Carbon dioxide that is generated when bioethanol is burned is an exception in the calculation of greenhouse gas defined by the Kyoto Protocol. In addition, unlike other cleaner fuels that require separate infrastructures (such as gas stations) to be deployed, it can be easily deployed in existing infrastructure, making early commercialization easy.

On the other hand, bioethanol production through biochemical transformation of biomass is very environmentally friendly, and a research base for maximizing its efficiency is essential. Ethanol production through the metabolic engineering re-design of microorganisms has led to the production of pyruvate decarboxylase and alcohol dehydrogenase II of Zymomonas mobilis in Escherichia coli ( Escherichia coli ) to produce E. coli strain KO11, which has been successfully produced.

Similarly, pyruvate decarboxylase and alcohol dehydrogenase II were introduced into Bacillus subtilis , but relatively little oxygen was used compared to the oxidation process Lactate, acetate, and 2,3-butanediol are produced under the fed fermentation conditions, and only a trace amount of ethanol is produced, resulting in low ethanol production efficiency .

KR 10-1073145 B1, Oct. 10, 2011 KR 10-0955945 B1, April 26, 2010

An object of the present invention is to provide a method for producing a transformant comprising a recombinant vector capable of biosynthesizing a sugar provided as a substrate of a photosynthetic-chemically synthesized strain with ethanol and producing ethanol using the transformant .

It is also an object of the present invention to provide a composition for producing alcohol containing the transformant.

In order to achieve the above object, the present invention is an electrochemical reduction force as an energy source, the recombinant Ralstonia eutropha ( Ralstonia) using carbon dioxide as the only carbon source The present invention also provides a method for producing an alcohol, which comprises culturing a strain of E. coli.

The present invention relates to a method for the treatment of Ralstonia < RTI ID = 0.0 > The present invention also provides a composition for producing an alcohol comprising the strain eutropha .

Alcohol production method according to the present invention by using a recombinant photosynthesis-chemical synthesis strain electrochemical reduction power as the energy source, carbon dioxide as the only carbon source, pyruvate (pyruvate) using a sugar provided as a substrate (acetaldehyde ( By converting to acetaldehyde), it can be converted to ethanol has the advantage of producing bio-ethanol efficiently.

1 shows the nucleotide sequence (SEQ ID NO: 1) of the PDC gene used in the production of the recombinant vector according to the present invention,
2 shows the nucleotide sequence of the ADH gene (SEQ ID NO: 2) used in the preparation of the recombinant vector according to the present invention,
FIG. 3 shows RT-PCR results of PDC gene and ADH gene according to the present invention,
FIG. 4 is a graph showing the distribution of the < RTI ID = 0.0 > Ralstonia < / RTI > (A) and a cross-sectional view (B) of an electrochemical reactor for the production of alcohol by using an eutropha strain,
FIG. 5 is a graph showing the effect of the Ralstonia < RTI ID = 0.0 > eutropha ) and the ethanol production.
(○: recombinant strain of Staphylococcus aureus strain cultured in a general reactor, ●: strain of recombinant Stoney utropha cultured in an electrochemical reactor, Δ: strain KCTC22469 cultured in a general reactor, ▲: Cultured strain KCTC22469)

Hereinafter, the present invention will be described in detail.

Ethanol production using microorganisms is performed by oxidizing glucose to pyruvate and then producing acetaldehyde by pyruvate decarboxylase (PDC) And the resulting acetaldehyde has a major metabolic pathway that is converted to ethanol by alcohol dehydrogenase (ADH).

The inventors of the present invention conducted a continuous research on ethanol production through a metabolic engineering redesign, and found that the metabolism pathway of photosynthetic-chemical synthesis strain having a physiological function capable of selectively controlling independent nutrition and heterotrophic metabolism in accordance with environmental conditions It is possible to efficiently produce a large amount of ethanol economically from the sugar provided as a substrate. Thus, the present invention has been completed.

In the present invention, "photosynthetic-chemical synthesis strain" is a typical heterotrophic bacterium that produces reductive power and energy required for growth depending on respiratory metabolism. However, in an environment rich in hydrogen and deficient in oxygen, Refers to a strain that synthesizes organic carbon through a pentose phosphate pathway.

In the present invention, an electrochemical reductive power is used as an energy source, carbon dioxide is used as a unique carbon source, and Ralstonia < RTI ID = 0.0 > The present invention also provides a method for producing an alcohol, which comprises culturing a strain of E. coli.

In the present invention, the culture is preferably performed using an electrochemical bioreactor, but is not limited thereto.

More specifically, in the bio-electrochemical reactor, electrical energy is converted to a biochemical reducing power through an electrochemical oxidation reaction. The bio-electrochemical reactor used in the present invention is designed to have a cathode and an anode separated by a membrane. An electrochemical reduction reaction can be produced from electrical energy using such an electrochemical reactor.

The electrochemical reactor for culturing the carbon dioxide-immobilized bacteria shown in FIG. 4 is an electrochemical reactor for concentrating and cultivating chemical-independent nutrients capable of growing using carbon dioxide and electrochemical reduction power as unique carbon sources and energy, respectively. The reducing power is transferred to the bacteria through the medium, the oxygen (oxidizing power) generated from the anode is discharged to the outside, and the bacteria can receive the electrochemical reduction power through the electron transfer medium included in the medium.

Specifically, when a DC power source is connected to a cathode and a cathode, the chemical-independent bacteria of the inorganic material medium provided in the anode reaction tank are supplied with reducing power, and in the anode reaction vessel, hydrogen and oxygen are generated as the water is electrolyzed. The hydrogen is separated into electrons and proton so that the electrons are transferred to the medium through the cathode by the electric driving force of the power supply, And is delivered to the medium. The electrons transferred to the medium reduce the electron transfer mediator and the reduced electron transfer mediates the bacterial cell metabolism and reduces NAD to NADH. In this process, hydrogen ions are introduced into the cell to generate ATP and react with NAD to generate NADH, which is a source of hydrogen ions necessary for NADH production. At this time, the direct current power between the cathode and anode of the electrochemical incubator is in the range of 1 to 10 volts.

Chemical independent autotrophic bacteria have the ability to synthesize organic compounds from carbon dioxide using specific chemical reactions or light energy. They are bacteria that grow and survive completely in the medium of inorganic compounds only. They are ammonium chloride, potassium dibasic potassium phosphate, sodium hydrogen carbonate Electrochemical reductive power is converted to biochemical reductive power to an inorganic medium containing trace elements such as graphite nonwoven fabric, electron transfer medium and trace elements such as Mg, Ca, Mo, Ni, Se, Fe, Mn, Cu, Since carbon dioxide can be grown as a unique carbon source, bacteria can be concentrated and cultured.

As described above, since the separated oxidation reaction and the reduction reaction are absolutely required growth environments for the growth of the chemically independent nutrient bacteria, the electrochemical reactor for the enrichment culture of the carbon dioxide- It is useful for cultivation of bacteria.

In the present invention, Ralstonia < RTI ID = 0.0 > eutropha ) strain is a pyruvate decarboxylase (PDC) gene; And an alcohol dehydrogenase (ADH) gene; Which is a recombinant strain of Streptomyces utrophilus.

More specifically, the PDC gene and the ADH gene can obtain a base sequence from Zymomonas mobilis, which is a typical ethanol-producing strain among bacteria, but the present invention is not limited thereto.

In the present invention, the PDC gene preferably has the nucleotide sequence as set forth in SEQ ID NO: 1, but preferably has 80% homology with the nucleotide sequence set forth in SEQ ID NO: 1 in consideration of degeneracy of the genetic code, , 85% homology, more preferably 90% homology, and most preferably 95% homology are included in the pyruvate dicarboxylase gene of the present invention.

 In the present invention, it is preferable that the ADH gene has the nucleotide sequence shown in SEQ ID NO: 2. However, considering the degeneracy of the genetic code, the ADH gene preferably has 80% homology with the nucleotide sequence shown in SEQ ID NO: 2, A gene having 85% homology, more preferably 90% homology, and most preferably 95% homology is included in the alcohol dihydrogenase gene of the present invention.

In the present invention, the recombinant vector may contain expression-suppressing fragments having various functions for inhibiting, amplifying, or inducing expression, a marker for selecting a transformant, a gene resistant to antibiotics, And a gene encoding a signal for secretion of the gene.

In the present invention, the above-mentioned Ralstonia < RTI ID = 0.0 > eutropha ) strain may be prepared by transforming KCTC22469, but is not limited thereto.

The recombinant Stoneyutropha strain of the present invention can be introduced by using, for example, an electroporation method, a polyethylene glycol method or the like.

In the present invention, cultivation of Ralstonia eutropha can be carried out by inoculating seed cells into a bioelectrochemical reactor after seed culture. At this time, the seed culture is carried out by adding 30 to 500 mM of glucose and 0.05 to 10 g / L of yeast extract to M9 minimal medium, injecting carbon dioxide for initial adaptation, , Preferably 50 to 350 mM of glucose and 1 to 5 g / L of yeast extract, and a temperature of 25 to 30 [deg.] C is further preferable, but not always limited thereto.

The composition of the M9 minimal medium for the seed culture is 6 to 24 g / L of disodium hydrogenphosphate (Na ₂ HPO ₄ ), 3 to 12 g / L of potassium dihydrogenphosphate (KH ₂ PO ₄ ), 0.05 to 5 g / 2 g / L, 1 to 3 g / L ammonium chloride (NH ₄ Cl), 0.05 to 2 mM magnesium sulfate (MgSO ₄ ) and 0.05 to 2 mM calcium chloride (CaCl ₂ ). The seed culture is preferably cultured for about one month.

The thus cultured Ralstonia < RTI ID = 0.0 > eutropha ) may be inoculated into an electrochemical reactor to start the reaction.

It is preferable that the medium for the main culture is an M9 minimal medium. It is also preferred that the culture is performed by adding 30 to 500 mM of glucose and 0.05 to 10 g / L of yeast extract to the medium initially. In the present culturing, 2.5 L of a medium having the following composition is put into a 3 L bio-electrochemical reactor, followed by culturing, but it is preferable to conduct stationary culture, but the present invention is not limited thereto.

The composition of the M9 minimal medium for the above cultivation is as follows: disodium hydrogenphosphate 6 to 24 g / L, potassium dihydrogenphosphate 3 to 12 g / L, sodium chloride 0.05 to 2 g / L, ammonium chloride 1 to 3 g / To 2 mM magnesium sulfate and 0.05 to 2 mM calcium chloride is preferably used but not limited thereto. Alternatively, it is preferable to use a medium containing 30 to 500 mM of glucose and 0.05 to 10 g / L of yeast extract in the medium, but is not limited thereto.

In the present invention, the culture is performed by supplying carbon dioxide at a rate of 0.1 to 1 ml / min. It is more preferable to supply the culture at a rate of 0.2 ml / min.

In addition, Ralstonia < RTI ID = 0.0 > eutropha ) is preferably 1 to 10 V and the incubation temperature is 15 to 35 ° C. When the voltage of the electrochemical reactor is 1 to 3 V and the incubation temperature is 25 To 30 < 0 > C is more preferable, but is not limited thereto.

In the present invention, the carbon dioxide used as the carbon source of the bio-electrochemical reactor can be carbon dioxide which is filled in the gas bag, and the gas is continuously circulated by using a peristaltic pump to allow the carbon dioxide to be uniformly dispersed in the medium . In the present invention, a 3 L electrochemical reactor was used for culture of Ralstonia eutropha strain, and it is possible to cultivate in a larger volume for the production of more ethanol.

The present invention relates to a method for the treatment of Ralstonia < RTI ID = 0.0 > The present invention also provides a composition for producing an alcohol comprising the strain eutropha .

The ethanol product compositions of the present invention include, for example, polypeptides, broths, cell lysates, purified or unpurified enzyme extracts or polypeptides suitable for culturing the transformant and producing ethanol using the saccharide as a substrate do.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the following Examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention.

Repeated descriptions of the same technical constitution and operation as those of the conventional art will be omitted.

[ Example  1] Production of recombinant vector

(One) pdc Wow adh  Gene sequencing secured

Zymomonas The DNA sequence of the pdc gene and the adh gene derived from the mobilis gene was obtained through the gene search program of the US National Bioinformatics Center.

The nucleotide sequence of the pdc gene (2.1 kb) and the nucleotide sequence of the adh gene (1.8 kb) are shown in FIGS. 1 and 2, respectively.

(2) pdc Wow adh  gene Cloning

In the step (1) A primer for cloning the nucleotide sequence of the pdc gene (2.1 kb) and the adh gene (1.8 kb) was prepared and shown in Table 1 below.

After amplification by PCR using the primers shown in Table 1, the amplified genes were ligated to TA vectors, respectively, and the nucleotide sequences of the corresponding genes were confirmed by sequencing.

The amplified genes were cloned into the pLOI308 vector and finally a recombinant pLOI308-10 vector (see APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1988, p.397-404) into which pdc and adh genes were inserted was prepared.

[ Example  2] Recombinant vector-introduced recombinant vector Ralstonia  Utropa ( Ralstonia  eutropha ) Preparation of Strains

For transformation, Ralstonia eutropha ) host cells were purchased from KCTC22469, a biotechnology center of the Korea Biotechnology Research Institute.

The recombinant pLOI308-10 vector prepared in Example 1 was transformed into KCTC22469 using an electroporation method to obtain a transformed host cell.

In order to confirm the insertion of the recombinant vector in the transformed host cell, denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, and incubation at 72 ° C for 30 seconds were carried out using the primers shown in Table 1 The presence of the gene ( pdc gene, adh gene) was confirmed by an enzymatic method under the condition of extracellular extention, and is shown in Fig.

[ Example  3] Transformed recombination at electrochemical reductive conditions Ralstonia  Utropa ( Ralstonia eutropha ) Ethanol production

The transformed recombinant strains of S. rutostylus tropheia identified in Example 2 were respectively added to 100 ml of M9 minimal medium in an amount of 270 mM glucose and 3 g / L of yeast extract, The cells were incubated at 28 ° C for about one month after carbon dioxide injection for initial adaptation.

The reaction was initiated by inoculating the cultured strain with the culture solution in the bioelectrochemical reactor.

The electrochemical bioreactor used was a 3 L bioelectrochemical reactor, and after the addition of 2.5 L of medium, the culture was started. Carbon dioxide (CO2) charged in a gas bag (carbon dioxide storage) was used as the carbon source, To continuously circulate the gas to uniformly disperse carbon dioxide in the medium. At this time, the supplied voltage was 2 V, and the incubation temperature was 28 to 32 ° C.

The medium was a M9 minimal medium, and yeast extract was added at an initial concentration of 0.1 g / L. The composition of the M9 minimal medium used consisted of disodium hydrogenphosphate 12 g / L, potassium dihydrogenphosphate 6 g / L, sodium chloride 1 g / L, ammonium chloride 2 g / L, 1 mM magnesium sulfate and 0.1 mM calcium chloride. This culture was subjected to stationary culture.

KCTC22469 strain in which the recombinant pLOI308-10 vector was not inserted into the control was cultured under the same conditions as above.

After 2 weeks of incubation, the concentration of ethanol produced was measured using an appropriate amount of cell culture mixture.

The concentration of ethanol produced was analyzed using a YSI biochemistry analyzer (Model 2700, Yellow Springs Instrument Co., Inc) equipped with a YSI 2786 membrane.

As a result, it was confirmed that 39 mM of ethanol was produced while 86 mM of glucose was consumed in the transformant recombinant strain of Streptomyces tropheia identified in Example 2, while the recombinant pLOI308-10 vector, which is a control, was inserted In the case of KCTC22469 strain, it was confirmed that no ethanol was produced at all.

 [ Comparative Example  1] in the absence of electrochemical reducing power Transformed  Recombination Ralstonia Utopia ( Ralstonia eutropha ) Ethanol production

In the ethanol production method of Example 3, the transformed recombinant strain Streptomyces utrophilus strain was cultured in the same manner except that the strain was cultured in the absence of electrochemical reductive power (general reactor).

KCTC22469 strain in which the recombinant pLOI308-10 vector was not inserted into the control was cultured under the same conditions as above.

The concentration of ethanol produced was analyzed using a YSI biochemistry analyzer (Model 2700, Yellow Springs Instrument Co., Inc) equipped with a YSI 2786 membrane.

As a result, it was confirmed that ethanol was produced at a concentration of 18 mM during the consumption of 70 mM of glucose, whereas the KCTC22469 strain without the recombinant pLOI308-10 vector, which was a control, was not produced at all.

The results of the above Examples and Comparative Examples show that some of the pyruvic acid produced during the fermentation of glucose was decarboxylated by acetaldehyde by PDC and acetaldehyde was reduced by ADH and converted to ethanol.

Also, the alcohol production method of the above-described embodiment introduces a gene capable of inducing the production of ethanol into a photosynthetic-chemically synthesized strain to induce pyruvic acid produced in the oxidation process of glucose to be converted into alcohol by the biochemically produced reducing power And that the productivity of ethanol can be improved to more than two times by culturing the bacteria in a bioelectrochemical reactor designed to convert electrochemical reduction power into metabolic energy and reducing power.

100: Negative electrode reaction tank 110: Medium
120: culture solution 130: sample port
200: anode reaction tank 210: water
300: membrane
400: cathode tank cover 410: external connection
420: cathode connection part 421: cathode line
430: anode connection part 431: oxygen discharge pipe
440: Carbon dioxide entry / exit 441: Carbon dioxide sparger
442: Carbon dioxide discharge pipe
500: electrochemical reactor
600: gas circulation device
700 carbon dioxide storage

<110> KOREA ATOMIC ENERGY RESEARCH INSTITUTE <120> Production of alcohol method using bio-ethanol production          capacity of recombinant Ralstonia eutropha <160> 6 <170> Kopatentin 1.71 <210> 1 <211> 2146 <212> DNA <213> Unknown <220> <223> PDC gene <400> 1 tgatcctgcc ctgtcttgtt tggaattgat gaggccgttc atgacaacag ccggaaaaat 60 tttaaaacag gcgtcttcgg ctgctttagg tctcggctac gtttctacat ctggttctga 120 ttcccggttt acctttttca aggtgtcccg ttcctttttc ccctttttgg aggttggtta 180 tgtcctataa tcacttaatc cagaagcggg cgtttagctt tgtccatcat ggttgtttat 240 cgctcatgat cgcggcatgt tctgatattt ttcctctaaa aaagataaaa agtcttttcg 300 cttcggcaga agaggttcat catgaacaaa aattaggcat ttttaaaaat gcctatagct 360 aaatcaggaa cgacacttta gaggtttctg ggtcatcctg attcagacat agtgttttga 420 atatatggag taagcaatga gttatactgt cggtacctat ttagcggagc ggcttgtcca 480 gattggtctc aagcatcact tcgcagtcgc gggcgactac aacctcgtcc ttcttgacaa 540 cctgcttttg aacaaaaaca tggagcaggt ttattgctgt aacgaactga actgcggttt 600 cagtgcagaa ggttatgctc gtgccaaagg cgcagcagca gccgtcgtta cctacagcgt 660 tggtgcgcat tccgcattcg atgctatcgg tggcgcctat gcagaaaacc ttccggttat 720 cctgatctcc ggtgctccga acaacaacga ccacgctgct ggtcatgtgt tgcatcatgc 780 tcttggcaaa accgactatc actatcagtt ggaaatggcc aagaacatca cggccgccgc 840 tgaagcgatt tacaccccgg aagaagctcc ggctaaaatc gatcacgtga ttaaaactgc 900 tctcgcgaag aagaagccgg tttatctcga aatcgcttgc aacattgctt ccatgccctg 960 cgccgctcct ggaccggcaa gtgcattgtt caatgacgaa gccagcgacg aagcatcctt 1020 gaatgcagcg gttgacgaaa ccctgaaatt catcgccaac cgcgacaaag ttgccgtcct 1080 cgtcggcagc aagctgcgcg ctgctggtgc tgaagaagct gctgttaaat tcaccgacgc 1140 tttgggcggt gcagtggcta ctatggctgc tgccaagagc ttcttcccag aagaaaatcc 1200 gcattacatt ggtacctcat ggggcgaagt cagctatccg ggcgttgaaa agacgatgaa 1260 agaagccgat gcggttatcg ctctggctcc tgtcttcaac gactactcca ccactggttg 1320 gacggatatc cctgatccta agaaactggt tctcgctgaa ccgcgttctg tcgttgtcag 1380 acgcattcgc ttccccagcg ttcatctgaa agactatctg acccgtttgg ctcagaaagt 1440 ttccaagaaa accggttctt tggacttctt caaatccctc aatgcaggtg aactgaagaa 1500 agccgctccg gctgatccga gtgctccgtt ggtcaacgca gaaatcgccc gtcaggtcga 1560 agctcttctg accccgaaca cgacggttat tgctgaaacc ggtgactctt ggttcaatgc 1620 tcagcgcatg aagctcccga acggtgctcg cgttgaatat gaaatgcagt ggggtcacat 1680 tggttggtcc gttcctgccg ccttcggtta tgccgtcggt gctccggaac gtcgcaacat 1740 cctcatggtt ggtgatggtt ccttccagct gacggctcag gaagttgctc agatggttcg 1800 cctgaaactg ccggttatca tcttcttgat caataactat ggttacacca tcgaagttat 1860 gatccatgat ggtccgtaca acaacatcaa gaactgggat tatgccggtc tgatggaagt 1920 gttcaacggt aacggtggtt atgacagcgg tgctgctaaa ggcctgaagg ctaaaaccgg 1980 tggcgaactg gcagaagcta tcaaggttgc tctggcaaac accgacggcc caaccctgat 2040 cgaatgcttc atcggtcgtg aagactgcac tgaagaattg gtcaaatggg gtaagcgcgt 2100 tgctgccgcc aacagccgta agcctgttaa caagctcctc tagttt 2146 <210> 2 <211> 1797 <212> DNA <213> Unknown <220> <223> ADH gene <400> 2 aagcttaatt taaaggcaaa atcggttacc acatctcaat tattaaacaa tacttcataa 60 taaaaagaca actttttcat aatttgcata agtcttgatg taaaaaatac atatttagaa 120 agaacaagca gccttgctca tcaccgttgt cgcgagtaga aaaatctcgg ctttcagaaa 180 aagaggccgc ttcgttaagc agactataaa tgtgctggaa taaagcgaac cccttgatct 240 gataaaactg atagacatat tgcttttgcg ctgcccgatt gctgaaaatg cgtaaaattg 300 gtgattttac tcgttttcag gaaaaacttt gagaaaacgt ctcgaaaacg ggatgaaaac 360 gcaaaaacaa tagaaagcga tttcgcgaaa atggttgttt tcgggttgtt gctttaaatt 420 agtatgtagg gtgaggttat agctatggct tcctcaactt tttatattcc tttcgtcaac 480 gaaatggggg aaggttcgct tgaaaaagca atcaaggatc ttaacggcag cggctttaaa 540 aatgcgctga tcgtttctga tgctttcatg aacaaatccg gtgttgtgaa gcaggttgct 600 gacctgttga aagcacaggg tattaattct gctgtttatg atggcgttat gccgaacccg 660 actgttaccg cagttctgga aggccttaag atcctgaagg ataacaattc agatttcgtc 720 atctccctcg gtggtggttc tccccatgac tgcgccaagg ccatcgctct ggtcgcaacc 780 aatggtggtg aagtcaaaga ctacgaaggt atcgacaaat ctaagaaacc tgccctgcct 840 ttgatgtcaa tcaacacgac ggctggtacg gcttctgaaa tgacgcgttt ctgcatcatc 900 actgatgaag tccgtcacgt taagatggcc attgttgacc gtcacgttac cccgatggtt 960 tccgtcaacg atcctctgtt gatggttggt atgccaaaag gcctgaccgc cgccaccggt 1020 atggatgctc tgacccacgc atttgaagcc tattcttcaa cggcagctac tccgatcacc 1080 gatgcttgcg ccttgaaggc tgcgtccatg atcgctaaga atctgaagac cgcttgcgac 1140 aacggtaagg atatgccagc tcgtgaagct atggcttatg cccaattcct cgctggtatg 1200 gccttcaaca acgcttcgct tggttatgtc catgctatgg ctcaccagtt gggcggctac 1260 tacaacctgc cgcatggtgt ctgcaacgct gttctgcttc cgcatgttct ggcttataac 1320 gcctctgtcg ttgctggtcg tctgaaagac gttggtgttg ctatgggtct cgatatcgcc 1380 aatctcggtg ataaagaagg cgcagaagcc accattcagg ctgttcgcga tctagctgct 1440 tccattggta ttccagcaaa tctgaccgag ctgggtgcta agaaagaaga tgtgccgctt 1500 cttgctgacc acgctctgaa agatgcttgt gctctgacca acccgcgtca gggtgatcag 1560 aaagaagttg aagaactctt cctgagcgct ttctaatttc aaaacaggaa aacggttttc 1620 cgtcctgtct tgattttcaa gcaaacaatg ccttcgattt ctaatcggag gcatttgttt 1680 ttgtttattg caaaaacaaa aaatattgtt acaaattttt acaggctatt aagcctaccg 1740 tcataaataa tatgccattt aaagcctatt atcaggattg tcgccccgat tggatcc 1797 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> P223 forward primer <400> 3 ctcacgaaga gcagttttaa tcacg 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> P223 reverse primer <400> 4 tccttcttga caaccggctt ttgaa 25 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ADH forward primer <400> 5 aagcaatcaa ggatcttaac ggca 24 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ADH reverse primer <400> 6 caacttcttt ctgatcaccc tg 22

Claims (17)

Recombinant Ralstonia with electrochemical reduction as energy source and carbon dioxide as the only carbon source eutropha ) Alcohol production method comprising the step of culturing the strain. The method of claim 1,
The culturing is an alcohol production method, characterized in that carried out using a biochemical bioreactor (electrochemical bioreactor). The method of claim 1,
The strains include pyruvate decarboxylase (PDC) genes; And an alcohol dehydrogenase (ADH) gene; Alcohol production method, characterized in that the introduced recombinant Ralstonia utropa strain. The method of claim 3, wherein
The PDC gene and ADH gene is an alcohol production method, characterized in that the gene derived from zymomonas mobilis . 5. The method of claim 4,
The PDC gene has a nucleotide sequence of SEQ ID NO: 1, the ADH gene has an nucleotide sequence of SEQ ID NO: 2, characterized in that the alcohol production method. The method of claim 3, wherein
The strain is an alcohol production method, characterized in that prepared by transforming KCTC22469. The method of claim 1,
The culturing is performed by supplying carbon dioxide at a rate of 0.1 to 1 ml / min alcohol production method The method of claim 1,
The culture was 6 to 24 g / L disodium hydrogen phosphate, 3 to 12 g / L potassium dihydrogen phosphate, 0.05 to 2 g / L sodium chloride, 1 to 3 g / L ammonium chloride, 0.05 to 2 mM magnesium sulfate, and 0.05 to Alcohol production method characterized in that performed using a medium containing 2 mM calcium chloride. The method of claim 8,
The medium is alcohol (glucose) and yeast extract (yeast extract) further comprising the alcohol production method. The method of claim 1,
The culturing is an alcohol production method, characterized in that carried out at a temperature of 15 to 35 ℃. The method of claim 10,
The culture is alcohol production method characterized in that the stationary culture. The method of claim 1,
The production method includes a step of seed culture before cultivation; And inoculating it into a biochemical reactor; Alcohol production method characterized in that it further comprises. 13. The method of claim 12,
The seed culture is alcohol production method, characterized in that carried out at a temperature of 15 to 35 ℃. Recombinant Ralstonia eutropha ) Alcohol-containing composition comprising a strain. The method of claim 14,
The strain is zymomonas mobilis pyruvate decarboxylase (PDC) gene from mobilis ); And an alcohol dehydrogenase (ADH) gene; Is a recombinant Ralstonia utropa strain introduced. 16. The method of claim 15,
Wherein the PDC gene has the nucleotide sequence set forth in SEQ ID NO: 1, and the ADH gene has the nucleotide sequence set forth in SEQ ID NO: 2. The method according to any one of claims 14 to 16,
The strain is characterized in that the alcohol producing ability.

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