KR102028161B1 - Process for preparing 2,3-butanediol using transformant - Google Patents

Abstract

The present invention provides a recombinant vector for producing 2,3-butanediol, comprising a gene encoding a methanol dehydrogenase, a gene encoding an aldehyde lyase, and a gene encoding acetoin reductase, wherein each of the genes 2,3- is introduced. Transformant microorganism for producing butanediol, composition for producing 2,3-butanediol comprising the recombinant vector or transforming microorganism, kit for producing 2,3-butanediol comprising the composition and culturing the transforming microorganism It relates to a 2,3-butanediol production method. By using the transformed microorganism of the present invention, it is possible to produce 2,3-butanediol from ethanol without producing a by-product by a continuous enzymatic reaction, and thus widely used for economic production of 2,3-butanediol. Could be.

Description

Process for preparing 2,3-butanediol using transformant

The present invention relates to a method for producing 2,3-butanediol using a transformed microorganism, and more particularly, to a gene encoding a methanol dehydrogenase, a gene encoding an aldehydease, and a gene encoding acetoin reductase. Recombinant vector for producing 2,3-butanediol, comprising a transformed microorganism for producing 2,3-butanediol wherein each gene is introduced, the composition for producing 2,3-butanediol comprising the recombinant vector or a transforming microorganism, the composition It relates to a 2,3-butanediol production kit comprising a 2,3-butanediol production method comprising culturing the transforming microorganism.

There is a growing interest in global warming around the world, and the main cause is known as greenhouse effect such as carbon dioxide and methane. In order to solve this problem, researches on producing high value-added products from carbon dioxide by using microorganisms using carbon dioxide have been actively conducted. Recently, a synthetic biocatalyst designed by computer was designed to convert carbon dioxide and metabolic pathways were utilized. A rebuilding study was conducted.

The main sources of methane are biogas, which is the product of anaerobic digestion, and natural gas produced naturally. The main components of biogas are methane and carbon dioxide, 70-90% of natural gas is methane and the remainder consists of ethane and a small amount of propane. Recently, the production of shale gas, a natural gas produced in the shale layer, has rapidly increased, and there is a high interest in the use of methane and ethane. As a result, research on methane magnetization, a microorganism with the ability to utilize methane, is being actively conducted, and methane monooxygenase (MMO) possessed by methane bacteria can use a wide range of substrates. It also has the ability to oxidize longer alkanes such as ethane, and methanol dehydrogenase (MDH) is also known to be able to use a wider range of substrates, oxidizing multiple carbon alcohols. have.

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

Under these backgrounds, the present inventors have diligently expressed methanol dehydrogenase, aldehyde lyase, and acetoin reductase to develop a method for producing 2,3-butanediol more effectively using transformed microorganisms. When using a transformed microorganism transformed to make it possible to produce 2,3-butanediol from ethanol, the present invention was completed.

One object of the present invention is to provide a recombinant vector for producing 2,3-butanediol comprising a gene encoding a methanol dehydrogenase, a gene encoding an aldehyde lyase and a gene encoding acetoin reductase.

Another object of the present invention is to provide a transformed microorganism for producing 2,3-butanediol to which each gene is introduced.

Another object of the present invention to provide a composition for producing 2,3-butanediol comprising the recombinant vector or transformed microorganism.

Still another object of the present invention is to provide a kit for producing 2,3-butanediol containing the composition.

Still another object of the present invention is to provide a method for producing 2,3-butanediol comprising culturing the transformed microorganism.

The present inventors conducted various studies to develop a method for producing 2,3-butanediol more effectively using a transformed microorganism, and as a result, the conversion of ethanol to acetaldehyde and the conversion of acetaldehyde to acetoin Next, a method for producing 2,3-butanediol with converted acetoin was developed through a transgenic microorganism. That is, the methanol dehydrogenase expressed in the microorganism can convert ethanol to acetaldehyde in addition to the activity of converting methanol into formaldehyde, and the aldehyde lyase connects aldehyde compounds such as formaldehyde or acetaldehyde to form DHA and acetoin. When the acetoin reductase is able to convert the acetoin to 2,3-butanediol, and a transgenic microorganism capable of expressing all three enzymes, It was confirmed that 2,3-butanediol can be produced from ethanol. In particular, when the chain reaction of the enzyme is carried out, only the main reaction product such as acetaldehyde or acetoin is produced, and no additional by-products are generated, resulting in a high yield of the final product. . In addition, in the case of further expressing hydrogen methane oxide expressed in the normal methane magnetization bacteria, by converting the ethane in the air to ethanol, 2,3-butanediol can be produced from the ethane in the air, 2,3-butanediol It was confirmed that the productivity of can be significantly improved.

As such, a method for producing 2,3-butanediol from ethanol through a transforming microorganism capable of expressing methanol dehydrogenase, aldehyde lyase and acetoin reductase is not known at all, and was first developed by the present inventors. It became.

As one embodiment for achieving the above object, the present invention is a recombinant for producing 2,3-butanediol comprising a gene encoding a methanol dehydrogenase, a gene encoding an aldehyde lyase and a gene encoding acetoin reductase Provide a vector.

The term "methanol dehydrogenase (MDH)" of the present invention refers to an enzyme that catalyzes a reaction for converting methanol into formaldehyde, and it is known to use NAD ⁺ as energy for performing such an enzyme reaction. As the methanol dehydrogenase, all kinds of enzymes known in the art may be used. For example, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxybutyryl- CoA dehydrogenase (3-hydroxybutyryl-CoA dehydrogenase), alcohol dehydrogenase (Alcohol dehydrogenase), aldo-keto reductase (Aldo-keto reductase) and the like. For example, in the present invention, a transformed microorganism was prepared using the methanol dehydrogenase gene of SEQ ID NO: 1 optimized for expression in E. coli.

As used herein, the term "aldehyde lyase" refers to an enzyme that exhibits the activity of removing an aldehyde group from a compound. For example, three molecules of formaldehyde may be reacted to produce DHA, or two molecules of acetaldehyde may be reacted to produce acetoin. As the aldehyde lyase, all kinds of enzymes known in the art may be used. For example, formolase (FLS), aldolase, benzaldehyde lyase, Propioin synthase, and the like. For example, in the present invention, a transformed microorganism was prepared using the formolase gene of SEQ ID NO: 2 optimized for expression in E. coli.

As used herein, the term "acetoin reductase" refers to an enzyme that catalyzes the reaction of acetoin to 2,3-butanediol. The acetoin reductase may be, for example, alcohol dehydrogenase, and the like, and may be, for example, 2,3-butanediol dehydrogenase. For example, in the present invention, a transformed microorganism was prepared using the budABC gene encoding the 2,3-butanediol dehydrogenase.

In addition, each said enzyme is one or more in one or more positions of the amino acid sequence which comprises each enzyme as long as it shows each activity used by this invention (position or kind in the three-dimensional structure of the amino acid residue of a protein) Depending on the specific amino acid sequence, specifically 2 to 20, preferably 2 to 10, more preferably 2 to 5) amino acids may include amino acid sequences substituted, deleted, inserted, added or inverted. At least 80%, specifically at least 90%, more specifically at least 95%, and even more specifically at 99%, relative to the amino acid sequence of each enzyme as long as it can maintain or enhance the activity of each enzyme. If a protein having the above homology is an amino acid sequence having a biological activity substantially the same as or corresponding to that of each enzyme, some sequences may be deleted, modified, or replaced. Alternatively, if having an amino acid sequence which is added also, and included in the scope of the present invention, which is apparent to those skilled in the art.

As used herein, the term "homology" refers to the percent identity between two polynucleotides or polypeptide moieties. Homology between sequences from one moiety to another may be determined by known techniques. For example, homology can be determined by aligning sequence information and directly aligning sequence information between two polynucleotide molecules or two polypeptide molecules using readily available computer programs. The computer program may be BLAST (NCBI), CLC Main Workbench (CLC bio), MegAlign ™ (DNASTAR Inc), or the like. In addition, homology between polynucleotides can be determined by hybridization of polynucleotides under conditions of stable double-stranding between homologous regions, followed by digestion with single-strand-specific nucleases to determine the size of the digested fragments.

In another embodiment, the present invention Methanol dehydrogenase  Gene coding, Aldeha Id Rise  The gene that encodes and Acetoin  Provided is a transformed microorganism for producing 2,3-butanediol having a gene encoding a reductase.

The methanol dehydrogenase, aldehyde lyase and acetoin reductase are the same as described above.

The term "transforming microorganism" of the present invention, also referred to as a transformant, a cell or microorganism which is mutated to express the target protein after the polynucleotide encoding the target protein is introduced into the host using a vector. Means. In this case, the polynucleotide introduced into the host cell may be any form as long as it can be introduced into and expressed in the host cell.

The transformed microorganism provided by the present invention may be prepared by introducing an expression vector into which a polynucleotide sequence encoding each enzyme is introduced into a host cell. In this case, the host cell that can be used is not particularly limited as long as it is a host cell capable of expressing each enzyme by introducing a polynucleotide sequence encoding each enzyme of the present invention, but preferably suitable for application to a bioconversion process. Cultureable unicellular prokaryotic or eukaryotic cells can be used, more preferably E. coli, yeast, etc., and most preferably E. coli BL21 (DE3) cells.

The transformed microorganism of the present invention can produce 2,3-butanediol from ethanol contained in the medium by the continuous reaction of the enzyme expressed therefrom. That is, methanol dehydrogenase converts ethanol in the medium to acetaldehyde, the acetaldehyde is converted to acetoin by aldehyde lyase, and the acetoin is converted to 2,3-butanediol by acetoin reductase. As such, when the chain reaction of the enzyme is performed, only a main reaction product such as acetaldehyde or acetoin is produced, and no additional by-products are generated, resulting in a high yield of the final product. .

On the other hand, in order to improve the productivity of 2,3-butanediol produced by such a method, the transforming microorganism of the present invention may be an additional gene encoding a methanase.

The term "methane monooxygenase (MMO)" of the present invention means an enzyme capable of catalyzing the oxidation reaction of oxidizing methane to produce methanol. The methanase is not particularly limited as long as it exhibits an activity of catalyzing a reaction for producing methanol from methane. For example, Pseudomonas sp. And Rhodococcus sp. Brevibacterium sp., Comanonas sp., Acinetobacter sp., Arthrobacter sp., Brachymonas sp., Or It can be a methanase derived from microorganisms such as Methylosinus sp.

In the present invention, since the methanase can absorb ethane in the air and convert it into ethanol, it can be used to improve the productivity of 2,3-butanediol by using ethane cheaper than ethanol as a raw material.

In addition In another embodiment, the present invention provides a recombinant vector, a transformed microorganism or a Culture products  2,3-containing Butanediol  It provides a composition for production.

As described above, in the transformed microorganism into which the gene encoding the three enzymes is introduced, 2,3-butanediol may be produced from ethanol by a chain reaction of the three enzymes, and thus, the transformed microorganism or its culture. Water can be used to produce 2,3-butanediol from ethanol in high yields.

In addition, the recombinant vector including the gene encoding the three enzymes may also be used as an active ingredient of the composition for producing 2,3-butanediol.

In the present invention, the culture product of the transforming microorganism is not particularly limited as long as it can be used to produce 2,3-butanediol in high yield, for example, the culture of the transforming microorganism, culture supernatant, Lysates, fractions thereof, and the like, and as another example, a culture supernatant obtained by centrifuging a culture of a transforming microorganism, a lysate obtained by physically or sonicating a transforming microorganism, the culture, a culture supernatant , Fractions obtained by applying the crushed material to a method such as centrifugation, chromatography, and the like.

In another embodiment, the present invention provides the above 2,3- Butanediol  2,3- comprising compositions for production Butanediol  For production Kit  to provide.

The kit for producing 2,3-butanediol of the present invention may be used to produce 2,3-butanediol, including the composition for producing 2,3-butanediol, but is not particularly limited thereto, and one or more kinds thereof are suitable for the reaction. Other components of the composition, solution or device may be included, specific examples of the buffer used for the production of 2,3-butanediol, the reaction vessel for performing the production of 2,3-butanediol, the production of 2,3-butanediol It may include a shake incubator for performing, a timer for performing the 2,3-butanediol production reaction.

As another embodiment, the present invention comprises the step of culturing the transforming microorganism in a medium containing ethanol, 2,3- from ethanol Butanediol  Provide a way to produce.

In this case, the transformed microorganism is the same as described above.

In addition, the concentration of ethanol in the medium is not particularly limited, but may be, for example, 0.01 to 6% (v / v), and as another example, 0.1 to 2% (v / v). And as another example, it may be 1.7% (v / v).

In addition, it may further comprise the step of recovering 2,3-butanediol from the culture obtained through the culture.

As used herein, the term "culture" means a method of growing microorganisms under appropriately artificially controlled environmental conditions.

In the present invention, the method of culturing the transformed microorganism can be carried out using a method well known in the art. Specifically, the culture is not particularly limited as long as it can produce 2,3-butanediol from the transformed microorganism, but is continuously cultured in a batch process or in a fed batch or repeated fed batch process can do.

The medium used for culturing should meet the requirements of the particular strain in an appropriate manner while controlling the temperature, pH, etc. under aerobic conditions in a conventional medium containing a suitable carbon source, nitrogen source, amino acids, vitamins and the like. Carbon sources that can be used include sugars and carbohydrates such as glucose, xylose, sucrose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic Acids, stearic acid, fatty acids such as linoleic acid, alcohols such as glycerol, ethanol, organic acids such as acetic acid and the like can additionally be used, and these materials can 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 product, skim soy cake or its degradation product Can be. These nitrogen sources may be used alone or in combination. The medium may include, as personnel, monopotassium phosphate, dipotassium phosphate and corresponding sodium-containing salts. Personnel that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. In addition, as the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins can be used.

In addition, suitable precursors to the culture medium may be used. The raw materials described above may be added batchwise, fed-batch or continuous in a suitable manner to the culture in the culture process, but is 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, antifoaming agents such as fatty acid polyglycol esters can be used to inhibit bubble generation. Inject oxygen or an oxygen-containing gas (eg air) into the culture to maintain aerobic conditions. The temperature of the culture is usually 27 ° C to 37 ° C, preferably 30 ° C to 35 ° C. Incubation is continued until the maximum amount of production of the peptide is obtained. For this purpose it is usually achieved in 10 to 100 hours.

In addition, the step of recovering 2,3-butanediol from the reaction solution may be carried out by a method known in the art such as dialysis, centrifugation, filtration, solvent extraction, chromatography, crystallization. For example, the reaction solution may be centrifuged to remove transformed microorganisms, and the obtained supernatant may be subjected to a solvent extraction method to recover the desired 2,3-butanediol. Any method that can recover the 2,3-butanediol by combining known experimental methods according to the properties of butanediol can be used without particular limitation.

By using the transformed microorganism of the present invention, it is possible to produce 2,3-butanediol from ethanol without producing a by-product by a continuous enzymatic reaction, and thus widely used for economic production of 2,3-butanediol. Could be.

1 is a graph showing the results of analyzing the activity of formolae expressed in transformed Escherichia coli, (○) indicates the level of acetaldehyde as a substrate, and (●) indicates the level of acetoin as a reaction product.
Figure 2 is a graph showing the results of quantitative analysis of the level of acetoin produced from transforming Escherichia coli BL21-pMDHFLS2 in a medium condition containing ethanol and glucose alone or together.
3 is a culture of Recombinant strain producing acetoin expressing MDH and FLS cultured in a medium containing ethanol and Recombinant strain producing 2,3-butanediol expressing MDH, FLS and bdh1 It is a graph showing the results of analyzing the components contained in the product through HPLC.

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

Example  1: Preparation of Transgenic E. Coli

The E. coli gene of methanol dehydrogenase and the formolase (FLS) gene, which is a kind of aldehydease, were introduced to produce a transformant Escherichia coli capable of biosynthesizing 2,3-butanediol from ethanol.

Example  1-1: Obtaining Recombinant Vectors

First, the mdh gene encoding MAD ⁺ -dependent methanol dehydrogenase derived from Bacillus strain (Bacillus methanolicus MGA3) was synthesized by changing to an optimized sequence for expression in Escherichia coli (SEQ ID NO: 1). Mdh gene was cloned into pACYCDuet ™ -1 vector (Novagen) to obtain recombinant vector pMDH.

Second, formolase (FLS) gene (SEQ ID NO: 2), a kind of aldehyde lyase, was synthesized, and the synthesized FLS gene was cloned into a pACYCDuet ™ -1 vector (Novagen) to obtain a recombinant vector pFLS. .

Third, the synthesized FLS gene was cloned into pET21b (+) vector (Novagen) to obtain recombinant vector pFLS_2.

Fourth, the synthesized mdh gene and FLS gene were cloned into pACYCDuet ™ -1 vector (Novagen) to obtain a recombinant vector pMDHFLS.

Fifth, a budABC gene encoding 2,3-butanediol dehydrogenase (bdh1), a kind of acetoin reductase, was synthesized, and the synthesized budABC gene was cloned into a pAWP89 vector (Addgene). Recombinant vector pBUD was obtained.

Example  1-2: Production of Transgenic E. Coli

Four recombinant vectors synthesized in Example 1-1 were introduced into Escherichia coli (E. coli BL21 (DE3)), and each transformed Escherichia coli was prepared.

Specifically, recombinant vector pMDH was introduced into Escherichia coli to prepare transformed Escherichia coli BL21-pMDH; Recombinant vector pFLS was introduced into Escherichia coli to produce transformed Escherichia coli BL21-pFLS; Recombinant vector pMDHFLS was introduced into E. coli to produce transformed E. coli BL21-pMDHFLS; Recombinant vectors pMDH and pFLS_2 were introduced together into E. coli to produce transformed E. coli BL21-pMDHFLS2; Recombinant vectors pMDH, pFLS_2 and pBUD were introduced together into E. coli to produce transformed E. coli BL21-pMDHFLSBUD.

Each of the prepared E. coli was cultured overnight in 10mL LB medium, and the incubated culture solution was inoculated in LB medium and cultured to 0.5 to 0.6 based on OD600, and then 0.1 mM isopropyl β-D-1-. Thiogalactopyranoside (IPTG) was added to induce transcription of the gene, and protein was expressed for 4.5 hours at 37 ° C or 24 hours at 15 ° C after the addition of IPTG.

Example  2: 2,3- using transgenic E. coli Butanediol  production

Example  2-1: Formolase  Activity analysis

Formolase was produced in the transformed Escherichia coli BL21-pFLS prepared in Example 1-2, followed by centrifugation to obtain the cells, and the reaction buffer (0.082% Monosodium phosphate monohydrate, 1.32% Potassium phosphate and 100 mM NaPO _4). , pH 8.0).

Subsequently, PMSF and lysozyme were added to the suspension, followed by application to an ultrasonic crusher, and the cells were crushed. The crushed cells were centrifuged to obtain a supernatant containing formolase.

100 mM NaPO ₄ (pH 8.0), 2 mM MgSO ₄ , 134 mM acetaldehyde and 0.1 mM TPP were added to the obtained supernatant, and reacted at room temperature for 24 hours. Quantitative analysis confirmed the activity of the formolase (FIG. 1).

1 is a graph showing the results of analyzing the activity of formolae expressed in transformed Escherichia coli, (○) indicates the level of acetaldehyde as a substrate, and (●) indicates the level of acetoin as a reaction product. As shown in FIG. 1, it was confirmed that formolase expressed in the transformed E. coli of the present invention exhibited a constant level of enzyme activity from 2 hours.

Example  2-2: using transformed Escherichia coli Acetoin  production

BL21-pMDHFLS2 was cultured in the transformed Escherichia coli prepared in Example 1-2, two introduced genes were expressed, and then reacted in LB medium or M9 medium containing ethanol or glucose, and then as a reaction product. Quantitative analysis of the produced acetoin was performed (FIG. 2).

Figure 2 is a graph showing the results of quantitative analysis of the level of acetoin produced from transforming Escherichia coli BL21-pMDHFLS2 in a medium condition containing ethanol and glucose alone or together.

As shown in Figure 2, only when using the ethanol-containing LB medium, it was confirmed that a high yield of acetoin from BL21-pMDHFLS2.

Example  2-3: 2,3- using transformed E. coli Butanediol  production

Among the transformed Escherichia coli prepared in Example 1-2, transformed Escherichia coli BL21-pMDHFLS2 and transformed Escherichia coli BL21-pMDHFLS2 were added 1.7% (v / v) ethanol of transformed Escherichia coli BL21-pMDHFLSBUD. After culturing in the added LB culture, the reaction product in the culture was analyzed by HPLC (FIG. 3). At this time, wild type Escherichia coli was used as a control.

3 is a culture of Recombinant strain producing acetoin expressing MDH and FLS cultured in a medium containing ethanol and Recombinant strain producing 2,3-butanediol expressing MDH, FLS and bdh1 It is a graph showing the results of analyzing the components contained in the product through HPLC. As shown in Figure 3, transforming Escherichia coli expressing MDH and FLS was able to produce only acetoin using ethanol, transgenic Escherichia coli expressing FLS and bdh1, as well as acetoin using ethanol, 2,3- It was confirmed that butanediol can be produced.

As shown in the above results, 2,3-butanediol dehydrogenase, which is a kind of methanol dehydrogenase (MDH), formaldehyde (FLS), which is a kind of aldehyde lyase, and acetoin reductase, E. coli, which can express an enzyme (2,3-butanediol dehydrogenase, bdh1), was able to produce 2,3-butanediol from ethanol through a chain reaction of the expressed enzyme.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Process for preparing 2,3-butanediol using transformant <130> KPA161710-KR <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 1164 <212> DNA <213> Artificial Sequence <220> <223> recombinant methanol dehydrogenase <400> 1 atggaaatga cgaacactca gtcagccttc tttatgccta gcgtaaattt gttcggcgcg 60 ggtagcgtga atgaggtcgg tacccgcttg gcagatttag gtgtgaaaaa agcgctgctg 120 gttaccgatg cgggtctgca cggcttggga ctgtcggaaa aaattagtag cattatccgt 180 gccgccgggg tcgaagtgtc gatttttcct aaagcggagc cgaatcctac agataagaat 240 gtggccgaag gcttggaagc gtacaacgcg gaaaactgcg atagcattgt gactttgggc 300 ggcggtagca gccatgatgc cggcaaagca atcgcgttgg tggcagcgaa cggcggcaaa 360 attcacgact atgaaggcgt cgatgtgagt aaagaaccta tggtcccact tattgccatt 420 aataccaccg caggtacggg tagcgagctg accaagttca ctattatcac tgacaccgaa 480 cgtaaagtga agatggcaat cgtggataag catgtgactc cgaccctgag tattaatgac 540 ccagagttga tggtaggtat gccgcctagc ctgaccgcgg cgactgggct tgatgcgttg 600 actcatgcga tcgaagcata tgtctcaacc ggtgccaccc cgatcaccga tgcgcttgcg 660 attcaagcga tcaaaatcat cagcaaatat ctgccgcgcg cggtggccaa tggcaaagat 720 attgaagccc gtgaacaaat ggcgtttgca cagagcttag cgggtatggc ctttaataac 780 gcgggattag gctatgtgca tgccatcgca catcaactgg gcggctttta taactttcct 840 catggcgttt gtaatgccgt gctgttacct tacgtttgtc gttttaattt aattagcaaa 900 gtggaacgct acgccgaaat cgcggcgttc ttaggcgaaa atgtggatgg cctgagcacg 960 tatgacgcgg cggaaaaagc gattaaagcc attgaacgca tggcgaaaga cctgaacatt 1020 cctaaaggtt ttaaggagtt aggcgcgaaa gaagaggata tcgaaaccct ggcaaagaat 1080 gcgatgaaag acgcgtgcgc gttgactaac cctcgcaagc cgaagctgga ggaggtcatt 1140 caaatcatta aaaatgcgat gtaa 1164 <210> 2 <211> 1725 <212> DNA <213> Artificial Sequence <220> <223> recombinant formolase <400> 2 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 gcgcg 1725

Claims (18)

For producing 2,3-butanediol, which includes both a gene encoding methanol dehydrogenase (MDH), a gene encoding aldehyde lyase and a gene encoding acetoin reductase Recombinant vector.
The method of claim 1,
The methanol dehydrogenase is to convert ethanol to acetaldehyde, 2,3-butanediol production recombinant vector.
The method of claim 1,
The methanol dehydrogenase is expressed from the polynucleotide of SEQ ID NO: 1, 2,3-butanediol production recombinant vector.
The method of claim 1,
The aldehyde lyase is to convert acetaldehyde into acetoin, recombinant vector for producing 2,3-butanediol.
The method of claim 1,
The aldehyde lyase is formolase (formolase, FLS), 2,3-butanediol production recombinant vector.
The method of claim 5,
The formolase is expressed from the polynucleotide of SEQ ID NO: 2, 2,3-butanediol production recombinant vector.
The method of claim 1,
The acetoin reductase is to convert acetoin to 2,3-butanediol, recombinant vector for producing 2,3-butanediol.
The method of claim 1,
The acetoin reductase is 2,3-butanediol dehydrogenase (2,3-butanediol dehydrogenase, BDH1), 2,3-butanediol production recombinant vector.
The method of claim 1,
The recombinant vector further comprises a gene encoding a methane oxidase (methane monooxygenase, MMO), 2,3-butanediol production recombinant vector.
The method of claim 9,
The methanase is a recombinant vector for producing 2,3-butanediol which is to convert ethane in the ethanol in the atmosphere.
A trait for producing 2,3-butanediol, in which a gene encoding methanol dehydrogenase (MDH), a gene encoding aldehyde lyase and a gene encoding acetoin reductase were introduced. Transition microorganisms.
The method of claim 11,
The transforming microorganism is a gene that encodes methane oxidase (methane monooxygenase, MMO) is further introduced, the transformation microorganism for 2,3-butanediol production.
The recombinant vector of claim 1, wherein the recombinant microorganism of claim 11 or 12, or a culture product of the transformed microorganism, comprising 2,3-butanediol.
The method of claim 13,
The culture product is selected from the group consisting of the culture of the transformed microorganism, culture supernatant, lysate and fractions thereof, 2,3-butanediol production composition.
A kit for producing 2,3-butanediol, comprising the composition for producing 2,3-butanediol of claim 13.
A method for producing 2,3-butanediol from ethanol, comprising culturing the transformed microorganism of claim 11 or 12 in a medium comprising ethanol.
The method of claim 16,
The concentration of ethanol in the medium is 0.01 to 6% (v / v).
The method of claim 16,
It further comprises the step of recovering 2,3-butanediol from the culture obtained through the culture.

Images