KR101697368B1 - Enhanced Butanol Producing Recombinant Microorganisms and Method for Preparing Butanol Using the Same - Google Patents

Abstract

The present invention relates to a recombinant microorganism having improved butanol production ability, and a method for producing butanol using the recombinant microorganism. More particularly, the present invention relates to a recombinant microorganism having improved butanol production ability by using 2-ketoisovalerate as a precursor of L-valine as an intermediate, And a method for producing butanol using the microorganism. The recombinant microorganism according to the present invention is characterized in that a microorganism having the ability to produce 2-ketoisovalerate as a precursor of L-valine is added to a microorganism having the ability to produce 2-ketoisovalerate as a microorganism which encodes a VorABC enzyme which is an enzyme for converting 2-ketoisovalerate into isobutyryl- A gene encoding an enzyme that converts isobutyryl-CoA into butyryl-CoA, a gene that codes an enzyme that converts butyryl-CoA into butylaldehyde, and a gene that encodes an enzyme that converts butylaldehyde to butanol Is introduced.
Since the recombinant microorganism according to the present invention is easy to grow unlike the conventional Clostridium acetobutylicum and has a high possibility of improving the strain due to the manipulation of the additional metabolic flow, the recombinant microorganism according to the present invention is industrially produced microorganism of butanol, , It is useful as an industrially produced microorganism of butanol and has an effect of providing a recombinant microorganism having improved butanol production ability by simplifying the process of converting 2-ketoisovalerate to isobutyryl-CoA have.

Description

TECHNICAL FIELD The present invention relates to a recombinant microorganism having improved butanol productivity and a method for producing butanol using the recombinant microorganism,

The present invention relates to a recombinant microorganism having improved butanol productivity and a method for producing butanol using the same. More particularly, the present invention relates to a recombinant microorganism having improved butanol production ability by introducing a VorABC gene with an intermediate of 2-ketoisovalerate as a precursor of L- Which is capable of converting 2-ketoisovalerate into isobutyryl-CoA in one-step, and a process for producing butanol using the recombinant microorganism.

Recently, due to high oil prices and environmental problems, biofuel production using microorganisms has attracted great interest. Recently, bio-butanol has emerged as an alternative fuel for petrol, and the market size is increasing rapidly. Currently, 10 to 12 billion pounds of butanol are produced annually around the world, with a market size of 7 to 8.4 billion dollars (Lee, SY et al ., Biotechnology and Bioengineering 101: 209, 2008). In particular, biobutanol has the characteristics of being suitable as a fuel, such as energy density, volatility control, sufficient octane number, low impurity, energy efficiency higher than ethanol, better mixing with gasoline, . Therefore, mass production of the most suitable butanol as a substitute fuel using microorganisms can bring about the effect of substituting crude oil imports and environmental effects due to the reduction of greenhouse gas emissions.

Butanol can be produced by biological methods by anaerobic ABE (Acetone-Butanol-Ethanol) fermentation of Clostridial strain (Jones, DT and Woods, DR, Microbiol . Rev. , 50: 484, 1986; Rogers, P., Adv . Appl . Microbiol ., 31: 1, 1986; Lesnik, EA et al ., Necleic Acids Research , 29: 3583, 2001), which was a major source of butanol and acetone for more than 40 years until the 1950s. Clostridial strains are difficult to grow because of poor growth conditions, complete molecular biology tools and omics technology.

Therefore, production of butanol from Escherichia coli having excellent growth and various omics technology is required. In particular, since there is no development of strains using metabolic engineering or omics technology, there is unlimited development potential.

The butanol production pathway in Clostridium acetobutylicum is shown in FIG. 1 (Lee, SY et al., Biotechnology and Bioengineering 101: 209, 2008). However, some enzymes (crotonase, β-hydroxybutyryl-coenzyme A dehydrogenase, and butyryl-CoA dehydrogenase) have very low or very little activity (Boynton, ZL et al. J. Biotechnol ., 178: 3015-3024, 1996) it is difficult to catalyze the reaction effectively as in Clostridium acetoobutylicum.

Therefore, a butanol biosynthesis pathway was introduced to produce a recombinant bacterial having a butanol-producing ability, and butanol production was attempted using the recombinant bacteria, but the amount of the produced butanol was insignificant (WO 2007/041269 A2).

In addition, attempts have been made to produce various chain alcohols such as butanol and isobutanol from 2-ketoacid, a precursor of valine (Shota Atsumi et al ., Nature, 451, 2008) (US2007 / 0092957A1), the yield of butanol and isobutanol produced was low, although Shota Atsumi et al ., Nature , 451, 2008).

In addition, a metabolic circuit for converting 2-ketoisovalerate into butanol was newly constructed, and a microorganism having the ability to produce 2-ketoisovalerate as a precursor of L-valine was added with 2-ketoisovalerate as an isobutyl A recombinant microorganism having a butanol -producing ability was prepared by introducing bkdAB-lpdV , which is a gene coding for an enzyme that converts to l- CoA. However, bkdA1, bkdB, and bkdAB in the process of converting 2-ketoisovalerate to isobutyryl- The lpdV gene has been involved in complex interactions, making it difficult to control the expression levels of the three genes.

Accordingly, the present inventors have made intensive efforts to increase the efficiency of converting 2-ketoisovalerate to butanol by simplifying the process of converting 2-ketoisovalerate into isobutyryl-CoA. As a result, VorA, VorB and VorC genes were involved in the conversion of 2-ketoisovalerate into isobutyryl-CoA, and that recombinant microorganisms were introduced into the recombinant microorganism, and that the recombinant microorganism had higher butanol productivity And the present invention has been completed.

It is an object of the present invention to provide a recombinant microorganism having improved butanol production ability and a method for producing the recombinant microorganism.

It is another object of the present invention to provide a process for producing butanol using the recombinant microorganism.

It is still another object of the present invention to provide a recombinant microorganism having the ability to produce isobutyryl-CoA in a 2-ketoisovalerate having an introduced VorABC gene and to produce isobutyryl-CoA from 2-ketoisovalerate using the same Method.

In order to accomplish the above object, the present invention provides a microorganism having the ability to produce 2-ketoisovalerate, which is a precursor of L-valine, to a microorganism having the ability to produce 2-ketoisovalerate as an enzyme for converting 2-ketoisovalerate into isobutyryl- Ketoisovalerate ferredoxin reductase, alpha subunit), VorB (2-oxoisovalerate ferredoxin oxydorodicta (< RTI ID = 0.0 > oxidoreductase) and VorC (ketoisovalerate ferredoxin reductase, gamma subunit), a gene coding for the VorABC enzyme, isobutyryl-CoA is converted to butyryl-CoA A gene encoding an enzyme that converts butyryl-CoA to butylaldehyde, and a gene encoding an enzyme that converts butylaldehyde to butanol. Provides a method for producing a recombinant microorganism.

The present invention also relates to a microorganism having the ability to produce 2-ketoisovalerate which is a precursor of L-valine, which is produced by the above-mentioned method, and which has an ability to convert 2-ketoisovalerate into isobutyryl- (2-ketoisovalerate ferredoxin reductase, alpha subunit), VorB (2-oxoisovalerate ferredoxin oxydoryldexta) and VorC (2-ketoisovalerate ferredoxin reductase, Unit), a gene encoding an enzyme that converts isobutyryl-CoA to butyryl-CoA, a gene that codes for an enzyme that converts butyryl-CoA to butylaldehyde, and a gene encoding an enzyme that converts butylaldehyde A recombinant microorganism having a butanol-producing ability is provided, wherein a gene coding for an enzyme for converting the enzyme into butanol is introduced.

The present invention also relates to a microorganism having the ability to produce 2-ketoisovalerate as a precursor of L-valine, an enzyme which converts 2-ketoisovalerate into isobutyryl-CoA, VorA VorABC, which consists of VorB (2-oxoisovalerate peredoxin oxydoryldexta) and VorC (2-ketoisovalerate peredoxin reductase, gamma subunit) A gene encoding an enzyme that converts iso-butyryl-CoA into butyryl-CoA, a gene that codes an enzyme that converts butyryl-CoA into butylaldehyde, and an enzyme that converts butylaldehyde into butanol Encoding a gene coding for an enzyme involved in L-isoleucine biosynthesis, a gene encoding an enzyme involved in L-leucine biosynthesis, and an enzyme involved in D-pantothenic acid biosynthesis It provides a recombinant microorganism having high butanol producing ability, characterized in that L- and gene with a gene coding for the enzyme involved in valine biosynthesis is inactivated or deleted.

In the present invention, the gene coding for the enzyme involved in L-isoleucine biosynthesis is ilvA (a gene encoding threonine dehydratase), and the enzyme involved in L-leucine biosynthesis The coding gene is leuA (a gene encoding isopropylmalate synthase), and the gene coding for the enzyme involved in D-pantothenic acid biosynthesis is panB (3-methyl-2-oxo Is a gene coding for oxobutanoate hydroxymethyltransferase), and the gene coding for the enzyme involved in L-valine biosynthesis is ilvE (chain amino acid aminotransferase) Encoding gene).

In the present invention, the microorganism having the ability to produce 2-ketoisovalerate can be obtained by (a) deletion of a lacI gene (gene encoding a lac orerone repressor) gene; (b) ilvH (acetohydroxy acid synthase isozyme III) Elimination of feedback inhibition of genes; (c) replacing the native promoter comprising the attenuated region of ilvGMEDA (acetohydroxy acid synthase isozyme I) and ilvBN (acetohydroxy acid synthase isozyme II) operon with a strong promoter; And (d) introduction of an expression vector comprising a strong promoter.

The present invention also provides a method for producing butanol, wherein the produced recombinant microorganism is cultured to produce butanol, and then recovering butanol from the culture.

The present invention also relates to a microorganism having the ability to produce 2-ketoisovalerate as a precursor of L-valine, an enzyme which converts 2-ketoisovalerate into isobutyryl-CoA, VorA VorABC, which consists of VorB (2-oxoisovalerate peredoxin oxydoryldexta) and VorC (2-ketoisovalerate peredoxin reductase, gamma subunit) A recombinant microorganism having an ability to produce isobutyryl-CoA and a recombinant microorganism in the 2-ketoisovalerate obtained by introducing a gene coding for an enzyme ( VorABC ) are produced, and isobutyryl-CoA is produced , And recovering isobutyryl-CoA from the culture solution. The present invention also provides a method for producing isobutyryl-CoA from a 2-ketoisovaleric acid salt.

In addition, the present invention relates to an enzyme for converting 2-ketoisovalerate into isobutyryl-CoA, VorA (2-ketoisovalerate ferredoxin reductase, alpha subunit), VorB (2-oxoisovalerate Ketoisobaric acid salt using a VorABC enzyme composed of Vor AB (ferredoxin oxidoreductase) and VorC (2-ketoisovalerate ferredoxin reductase, gamma subunit), or a recombinant microorganism expressing the enzyme, Butyryl-CoA. ≪ / RTI >

The present invention has an effect of providing a recombinant microorganism improved in butanol production ability having a butanol biosynthesis ability from 2-ketoisovalerate as a precursor of L-valine. The recombinant microorganism according to the present invention is useful as industrially produced microorganisms of butanol because it is easy to grow unlike the existing Clostridium acetoobutylicum and has a high possibility of strain improvement due to the manipulation of additional metabolic flow, There is an effect of providing a recombinant microorganism having butanol solubility ability by simplifying the process of converting acid salt into isobutyryl-CoA.

1 shows the butanol production pathway of C. acetobutiliumum ATCC 824 strain.
2 shows a metabolic circuit in which butanol is biosynthesized from 2-ketoisovalerate in a microorganism having the ability to produce 2-ketoisovalerate as a precursor of L-valine.
Figure 3 shows the production process of the sacB homologous recombination pSacHR06 vector.
Fig. 4 shows the production process of the pKBRilvBNCD-ptac-adhEbdhAB vector.
Figure 5 shows the process by which 2-ketoisovalerate is converted to isobutyryl-CoA.
Figure 6 shows the pTac15k_VorABC_ptac_icmAB vector.

In the present invention, by introducing VorABC , a gene encoding an enzyme that converts 2-ketoisovalerate into isobutyryl-CoA, into a microorganism having the ability to produce 2-ketoisovalerate as a precursor of L-valine, 2- A recombinant microorganism having improved butanol production ability was constructed by constructing a metabolism circuit for converting ketoisovaleric acid to butanol to confirm whether the recombinant microorganism produced could synthesize butanol.

That is, a microorganism having the ability to produce 2-ketoisovalerate as a precursor of L-valine, a VorABC gene encoding an enzyme that converts 2-ketoisovalerate into isobutyryl-CoA, an isobutyryl-CoA When a gene encoding an enzyme that converts butyryl-CoA into butyryl-CoA, a gene that encodes an enzyme that converts butyryl-CoA into butylaldehyde, and a gene that encodes an enzyme that converts butylaldehyde into butanol are introduced, It is predicted that butanol will be produced from toisovalerate , especially by the VorABC gene The conversion of 2-ketoisovalerate into 1-step isobutyryl-CoA is expected to increase the butanol productivity.

The microorganisms having the ability to produce 2-ketoisovalerate can be bacteria, yeast, fungi and the like. Preferably, Corynebacterium genus, Brevibacterium genus and Escherichia coli are used. But there is no limitation as long as it has the ability to produce 2-ketoisovalerate.

Since the 2-ketoisovalerate is a precursor of valine, the microorganism having the ability to produce 2-ketoisovalerate can be obtained by using a mutant microorganism (Val) (Korean Patent No. 832,740) having L- In order to further increase the accumulation rate of 2-ketoisovalerate, a mutant microorganism (L-valine) having mutant microorganisms (Val) that weaken or delete the gene encoding an enzyme involved in L-valine biosynthesis .

In the present invention, "attenuation" is a concept encompassing the mutation, substitution, deletion, or deletion of some bases of the gene or reduction of the activity of the enzyme expressed by the gene by introducing a part of the base. And blocking all or a substantial portion of the biosynthetic pathway.

The term "amplification" in the present invention refers to amplification, substitution, or deletion of a part of the gene, introduction of a certain base, or introduction of a gene derived from another microorganism encoding the same enzyme to increase the activity of the corresponding enzyme .

2 shows a metabolic circuit in which butanol is biosynthesized from 2-ketoisovalerate in a microorganism having the ability to produce 2-ketoisovalerate as a precursor of L-valine.

In order to prepare butanol from 2-ketoisovalerate using a microorganism having the ability to produce 2-ketoisovalerate, a metabolic circuit for converting 2-ketoisovalerate to butanol was newly constructed, A gene encoding each of the enzymes that constitute it was introduced. That is, to a microorganism having the ability to produce 2-ketoisovalerate as a precursor of L-valine, an enzyme that converts 2-ketoisovalerate into isobutyryl-CoA, VorA (2-ketoisovalerate, Vor ABC enzyme consisting of VorB (2-oxoisovalerate ferredoxin oxydoryldexta) and VorC (2-ketoisovalerate ferredoxin reductase, gamma subunit) was coded A gene encoding an enzyme that converts isobutyryl-CoA into butyryl-CoA, a gene that codes an enzyme that converts butyryl-CoA to butylaldehyde, and a gene that codes an enzyme that converts butylaldehyde to butanol Lt; / RTI >

In the present invention, the enzyme that converts the isobutyryl-CoA to butyryl-CoA is an isobutyryl-CoA mutase, and the enzyme that converts the butyryl-CoA to butylaldehyde is butylaldehyde dihydro And the enzyme that converts the butyraldehyde to butanol is butanol dehydrogenase.

In the present invention, the gene encoding the enzyme for converting the 2-ketoisovalerate into isobutyryl-CoA may be characterized by being VorABC (SEQ ID NO: 46) derived from methanosarcina or acetiburance , That is, a gene encoding 2- ketoisovalerate peresoxin reductase is VorB derived from methanosarcine acetylborance , and a gene encoding 2- oxoisovalerate peresoxin oxidoreductase is Methanose And VorA and VorC derived from Leucina acetylborance . The gene encoding the enzyme for converting the isobutyryl-CoA into butyryl-CoA may be icmAB derived from Streptomyces arbormitilis . However, even if it is derived from another microorganism, There is no restriction as long as it is expressed and exhibits the same enzyme activity.

The gene coding for the enzyme that converts butyryl-CoA to butylaldehyde is adhE derived from Clostridium acetobutylicum . The butylaldehyde can be converted into butanol The gene coding for the enzyme may be bdhAB derived from Clostridium acetobutylicum. However, even if it is derived from another microorganism, there is no limitation as long as it is expressed in the introduced host cell and exhibits the same enzyme activity.

On the other hand, the microorganism having the ability to produce 2-ketoisovalerate as a precursor of L-valine of the present invention is preferably a microorganism that has been mutated to improve the accumulation rate of 2-ketoisovalerate. Specifically, the gene encoding the enzyme involved in L-isoleucine biosynthesis is mutated so as to increase the expression of the gene encoding the enzyme involved in 2-ketoisovalerate biosynthesis, and a gene encoding an enzyme involved in L-isoleucine biosynthesis and an enzyme involved in L- It is preferable to use a microorganism in which a gene coding for an enzyme involved in D-pantothenic acid biosynthesis and a gene coding for an enzyme involved in L-valine biosynthesis are weakened or deleted.

In the present invention, the gene coding for the enzyme involved in L-isoleucine biosynthesis is ilvA ( gene encoding threonine dehydratase), and the gene encoding the enzyme involved in L-leucine biosynthesis is leuA 2-isopropyl maleate synthase), and the gene coding for the enzyme involved in D-pantothenic acid biosynthesis is panB (a gene encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase Gene), and the gene coding for the enzyme involved in the L-valine biosynthesis is ilvE (a gene encoding amino acid transaminase of branched chain amino acid).

Further, in order to increase expression of a gene encoding an enzyme involved in 2-ketoisovalerate biosynthesis, (a) a gene encoding lacI (a gene encoding a lac operon inhibitory factor) is deleted, (b) ilvH Acetohydroxy acid synthetase isozyme III) Eliminate feedback inhibition of the gene; (c) replacing the original promoter comprising the attenuated region of ilvGMEDA (acetohydroxy acid synthase isozyme I) and ilvBN (acetohydroxy acid synthase isozyme II) operon with a strong promoter, and (d) Lt; / RTI > can be introduced.

In the present invention, the strong promoter may be selected from the group consisting of a trc promoter, a tac promoter, a T7 promoter, a lac promoter, and a trp promoter, wherein the expression vector comprising the strong promoter is selected from the group consisting of ilvB, ilvN, ilvC, and a vector containing the ilvD gene.

In one embodiment of the present invention, in order to prepare butanol from 2-ketoisovalerate, a mutant microorganism having an excellent accumulation rate of 2-ketoisovalerate is prepared, and then, a 2-ketoisovalerate A recombinant microorganism having a butanol production ability was prepared by introducing a gene encoding an enzyme involved in the production of butanol.

First, lacI gene was removed, feedback inhibition of ilvH was removed, and Escherichia coli W3110 competent cells, in which the original promoter including the attenuation region of ilvGMEDA and ilvBN operon were replaced by a tac promoter having strong transcription activity, Deletion of the lvA , panB, leuA and ilvE genes was induced to produce a mutant microorganism having an excellent accumulation rate of 2-ketoisovalerate.

Next, the branched chain ketoacid dehydrogenase derived from Pseudomonas putida KT2440 (E1 subunit: the dehydrogenase-decarboxylase, E2 subunit: the transacylase, E3: lipoamide dehydrogenase complex respectively encoded by bkdA , bkdB and lpdV genes) and Bacillus subtilis The process by which the branched chain ketoacid dehydrogenase (a complex of subunits encoded by the bkdA1 , bkdA2, bkdB and lpdV genes) is involved in valine catabolism and converts 2-ketoisovalerate into isobutyryl-CoA (Sokatch, JR et al, J. Bacteriol, 148 : 647-652, 1981; Sykes PJ et al, J. Bacteriol, 169:..... 1619-1625, 1987; Namba, Y., et al, J. Biol. Chem ., 244: 4437-4447) In view of the fact that the bkdA1 , bkdB and lpdV genes were made through complex interactions, it was difficult to control the expression levels of the three genes, and 2-ketoisovalerate was converted into iso Butyryl-CoA to be converted into one-step VorA (2-ketoisovalerate ferredoxin reductase, alpha subunit, SEQ ID NO: 43), VorB (2-oxoisovalerate ferredoxin oxydoryldexazate, SEQ ID NO: 44) derived from methanosarcina acetuborans, ) And VorC (2-ketoisovalerate ferredoxin reductase, gamma subunit, SEQ ID NO: 45) were cloned.

And the fact that the isobutyryl-CoA mutase derived from Streptomyces arbormitilis (a complex of the protein encoded by icma , icmB gene) converts isobutyryl-CoA to butyryl-CoA (Zerbe-Burkhardt , K., et al ., J. Biol. Chem ., 273: 6508-6517, 1998), to prepare butyryl-CoA from 2-ketoisovalerate, (Butyraldehyde dehydrogenase-encoding gene) derived from Acetobutylicum and bdhAB (a gene coding for butanol dehydrogenase of Clostridium acetobutylicum ) were further introduced to finally synthesize butanol from butyryl-CoA.

The process of converting 2-ketoisovalerate into isobutyryl-CoA by the bkdA , bkdB , and lpdV genes is a process in which the enzyme encoded by the bkdA1 gene converts 2-ketoisovalerate into 2-methyl-1-hydroxypropyl (2-methyl propanoyl) -dihydrilpoamide-E was converted to isobutyryl CoA by an enzyme encoded by the bkdB gene, which was then converted to 2- (2-methylpropanoyl) -dihydrilpoamide- And the enzyme encoded by the lpdV gene participates in the above process and is carried out through complex interactions as a whole, whereas when the VorABC gene is introduced, 2-ketoisovalerate is directly converted to isobutyryl-CoA (Fig. 5).

That is, the genes encoding ilvB (acetohydroxy acid synthase I large subunit), ilvN (genes encoding acetohydroxy acid synthase I small subunit) and ilvC (encoding acetohydroxy acid isomeroreductase), which are essential genes for 2-ketoisovalerate accumulation Gene encoding an enzyme that converts 2-ketoisovalerate into isobutyryl-CoA to a pKKilvBNCD vector in which a gene encoding the gene (e.g., gene) and ilvD (gene encoding dihydroxy-acid dehydratase) are sequentially cloned, isobutyryl- A vector encoding a gene encoding an enzyme that converts CoA to butyryl-CoA, a gene encoding an enzyme that converts butyryl-CoA to butylaldehyde, and a gene encoding an enzyme that converts butylaldehyde to butanol , Which is then introduced into a microorganism having the ability to produce 2-ketoisovalerate, Microorganisms were produced.

When the recombinant microorganism was cultured in a glucose-containing medium, it was confirmed that butanol was produced (Table 1).

Finally, in the present invention, a gene ( VorABC ) encoding an enzyme that converts 2-ketoisovalerate into isobutyryl-CoA is added to a microorganism having the ability to produce 2-ketoisovalerate as a precursor of L- , ( IcmAB ) encoding an enzyme that converts isobutyryl-CoA to butyryl-CoA, a gene ( adhE ) encoding an enzyme that converts butyryl-CoA to butylaldehyde , and an enzyme that converts butylaldehyde to butanol ( BdhAB ) was introduced into the recombinant microorganism to produce a recombinant microorganism, which confirmed that the butanol production ability of the recombinant microorganism was remarkably increased.

In addition, according to the present invention, since it is converted from 2-ketoisovalerate to isobutyryl-CoA by the VorABC enzyme, 2-ketoisovalerate is added to the microorganism having the ability to produce 2-ketoisovalerate, ( VorABC ) coding for an enzyme for conversion to butyryl-CoA is introduced to produce a recombinant microorganism, and the recombinant microorganism is cultured to produce isobutyryl-CoA. When the recombinant microorganism is used, It is possible to convert 2-ketoisovalerate into higher efficiency with isobutyryl-CoA.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

In particular, in the following examples, Escherichia coli W3110 was used as a host microorganism. However, it was found that an enzyme involved in biosynthesis of butanol from 2-ketoisovalerate was improved by using other Escherichia coli, bacteria, yeast and mold to improve L- And then using the same to produce butanol will be apparent to those skilled in the art.

In the following examples, only those genes derived from a specific strain are exemplified as genes to be introduced, but it will be apparent to those skilled in the art that there is no restriction as long as they are expressed in the introduced host cell and exhibit the same activity.

In the following example, it was confirmed that the keto group of 2-ketoacid was removed by the reaction of VorABC enzyme and the 2-ketoisovalerate was converted into isobutyryl-CoA by newly binding CoA group. However, this function of VorABC enzyme It would be obvious to one of ordinary skill in the art that the VorABC enzyme does not only cause this reaction to 2-ketoisovalerate, but also the function of the felodoxine oxydoroductase against several other 2-ketoacids .

In the following examples, only a specific medium and a culture method are exemplified. However, as described in the literature, when a medium different from a saccharified liquid such as whey or CSL (corn steep liquor) is used or fed- batch culture, continuous culture, etc. (Lee et al . , Bioprocess Biosyst . Eng . , 26: 63, 2003; Lee et al. , Appl. Microbiol . Biotechnol . , 58: 663, 2002; Lee et al . , Biotechnol . Lett . , 25: 111, 2003; Lee et al . , Appl . Microbiol . Biotechnol . , 54: 23,2000; Lee et al. , Biotechnol . Bioeng . , 72: 41, 2001) will be apparent to those skilled in the art.

Production of recombinant microorganism having butanol-producing ability using microorganisms having the ability to produce 2-ketoisovalerate and measurement of production ability of butanol

The present inventors' Korean Patents (a mutant microorganism improved in branched chain amino acid production ability and a method for producing branched chain amino acid using the same, registration number: 10-0832740, registered on May 21, 2008) A recombinant microorganism having a butanol production ability using a microorganism capable of producing valerate was prepared .

1-1: Production of microorganisms having the ability to produce 2-ketoisovalerate

1-1-1: Preparation of pSacHR06

PSacHR06 vector was constructed for the purpose of using sacB homologous recombination technique (Wohlleben et al., J. Bacteriol. , 174: 5462, 1992) derived from Bacillus subtilis for the substitution of specific bases or bases of chromosomal DNA ). Fig. 3 shows the production process of sacB homologous recombination pSacHR06.

First, the 1522 bp fragment obtained by digesting the pUC19 vector with Nde I and Ahd I was inserted into the pACYC177 vector (SEQ ID NO: 2) to replace the ampicillin resistance property of the pUC19 (New England Biolab) vector with the resistance property to kanamycin New England Biolabs) was digested with StuI and subjected to DNA condensation reaction with a 1340 bp fragment to obtain a pUC19KM vector.

Next, the 2.5kb fragment and the pBlu2KSP pUC19KM vector obtained by cutting with Pvu II (full name: pBluescriptIIKS ( +)) cutting the vector with Pvu II to obtain a vector pUC19KKS the 400bp fragment obtained by the condensation reaction. PCR was performed using the primers of SEQ ID NOS: 1 and 2 and the pUC19 vector template in order to facilitate the easy removal of the DNA replication origin in the vector. As a result, A DNA fragment having a replication origin was obtained. Cutting the fragment with Sal I and Dra III, and by cutting the vector with Sal I and Dra III pUC19KKS obtain a vector pUC19K reacted 1.5kb fragment and DNA condensation obtained. To introduce the sacB gene of Bacillus subtilis into the pUC19K vector, PCR was performed using the genomic DNA template of Bacillus subtilis and the primers of SEQ ID NOS: 3 and 4 to synthesize a DNA fragment having the sacB gene. The synthesized DNA fragment and pUC19K vector were digested with XbaI, SpeI and EcoRV enzymes and condensed to construct a new vector having the sacB gene, which was named pSacHR06.

The pSacHR06 vector has a sacB gene derived from Bacillus subtilis and can be used for sacB positive selection because it can easily remove the DNA replication origin and perform the recondensation reaction using a restriction enzyme.

[SEQ ID NO: 1] pucoriup: 5'-agccgtcgacgctagcgcatgcacgcgtgtgcacccatggga cgtcctcactgactcgctgcgctc-3 '

[SEQ ID NO: 2] pucorido: 5'-ggctcacaacgtggctagcgacgtcgtgcacccatgggttcc actgagcgtcagacc-3 '

[SEQ ID NO: 3] sacBf: 5'-actctctagacgcgggtttgttactgataa-3 '

[SEQ ID NO: 4] sacBr: 5'-gctagatatcaggatatcggcattttcttt-3 '

1-1-2: deletion of lacI gene

(Warner et al. , PNAS, 6: 97 (12): 6640-7) in E. coli W3110 (ATTC 39936), a microorganism having the ability to produce 2-ketoisovalerate, 6645, 2000), which is a gene encoding the repressor of the lac operon, deleted the lacI gene, which has the function of inhibiting transcription of the lac operon responsible for lactose degradation, and eliminated the antibiotic resistance.

[SEQ ID NO: 5] lacI_1stup: 5'-gtgaaaccagtaacgttatacgatgtcgcagagtatgccgg tgtctcttagattgcagcattacacgtcttg-3 '

[SEQ ID NO: 6] lacI_1stdo: 5'-tcactgcccgctttccagtcgggaaacctgtcgtgccagctg cattaatgcacttaacggctgacatggg-3 '

1-1-3: elimination of feedback inhibition of ilvH

(G) and the 50th base (C) of ilvH , a gene encoding acetohydroxy acid synthetase isozyme III, are replaced with A and T, respectively, by referring to a patent (US Pat. No. 6,737,255 B2) filed by Ajinomoto , And the chromosomal DNA of E. coli W3110 (ATTC 39936) was isolated and purified by a known method (Sambrook, et al ., Molecular cloning, 2nd ed., Cold Spring Harbor Laboratory Press, NY, 1989).

Namely, PCR was carried out using the primers of SEQ ID NOS: 7 and 8, 9 and 10, and Escherichia coli W3110 genomic DNA template, and the obtained two PCR fragments were mixed at the same concentration to obtain a template, Were used as primers to perform overlapping PCR. To the PCR fragment of 1280bp obtained in the same manner as described above was cut with Pst I and Sal I enzymes, and also inserted into a vector which pSacHR06 for the sacB homologous recombination digested with Pst I and Sal I enzymes, and then, sequencing, 41 th ilvH It was confirmed that the base (G) and the 50th base (C) were substituted with A and T, respectively.

The obtained vector was digested with NheI enzyme to delete the replication origin and then self-ligation was performed. Then, E. coli W3110 competent cells lacking the lacI gene prepared in the above 1-1-2 were added with electroporation- Respectively. Next, strains in which feedback inhibition was removed by sacB positive selection (Wohlleben et al ., J. Bacteriol ., 174: 5462, 1992) were obtained.

[SEQ ID NO: 7] ilvH1: 5'-gactctgcagggtgatcgagactctttggcggttgac-3 '

[SEQ ID NO: 8] ilvH2: 5'-ggaaaaaaggccaatcacgcggaataacgcgtctgattcattttcga gtaag-3 '

[SEQ ID NO: 9] ilvH3: 5'-cttactcgaaaatgaatcagacgcgttattccgcgtgattggccttt tttcc-3 '

[SEQ ID NO: 10] ilvH4: 5'-gctccgtcgaccagtttcacaattgccccttgcgtaaa-3 '

1-1-4: Replacement of the attenuation region of ilvGMEDA and ilvBN operons with the tac promoter

In order to induce the constitutive expression of acetohydroxoic acid synthase isozyme I and II in Escherichia coli W3110 in which the feedback inhibition of the lacI gene and ilvH produced in 1-1-3 was removed, acetohydroxoic acid synthase isozyme I And ilvBN and ilvGMEDA operons, respectively, encoding the transcriptional regulatory mechanism of the operon were replaced by a strong promoter, the tac promoter.

First, for the substitution of the ilvGMEDA operon attenuation region, PCR was carried out using a pair of primers and Escherichia coli W3110 genomic DNA template of SEQ ID NOS: 11 and 12, 13 and 14, respectively, and then PCR fragments ilvGatt1 and ilvGatt2 Respectively. By cutting the two PCR fragments thus obtained by each of Sal I / Bam HI and Eco RI / Pst I enzyme by cloning the appropriate enzyme cleavage site of the pKK223-3 vector (Pharmacia Biotech), and then, after confirming the nucleotide sequence in sequencing, Sal I and Pst I enzymes were cloned into the corresponding enzyme cleavage sites of the pSacHR06 vector and the original promoter containing the attenuated region was replaced with the tac promoter in the same manner as the above feedback inhibition removal.

for attenuating regions removal of ilvBN operon, SEQ ID NO: 15 and 16 and 17 and the one using a pair of the 18 each primer PCR and cloned in the same manner as in removing the ilvGMEDA attenuation zone, and then, the ilvGMEDA attenuation region removed from the Escherichia coli And electroporated into W3110 competent cells. As a result, the original promoter containing the ilvBN attenuation region could be replaced with the tac promoter.

[SEQ ID NO: 11] ilvGatt1f: 5'-gactgtcgacctaacttattggctgtaagctgttctgaggcc-3 '

[SEQ ID NO: 12] ilvGatt1r: 5'-gctcggatccgaatgttgttcccttcctcgtagttcatcc-3 '

[SEQ ID NO: 13] ilvGatt2f: 5'-gactgaattcatgaatggcgcacagtgggtggtacatgcg- 3 '

[SEQ ID NO: 14] ilvGatt2r: 5'-gctcctgcagtcaccgctggctaactggatatcttttggg-3 '

[SEQ ID NO: 15] ilvBatt1f: 5'-gactcgtcgacagagatggtagggcggataa-3 '

[SEQ ID NO: 16] ilvBatt1r: 5'-gctcggatcccacactgtattatgtcaaca-3 '

[SEQ ID NO: 17] ilvBatt2f: 5'-gactgaattcatggcaagttcgggcacaac-3 '

[SEQ ID NO: 18] ilvBatt2r: 5'-gctcactgcagtgcgtcacgaatgctttctt-3 '

1-1-5: deletion of ilvA , panB, ilvE and leuA genes

To eliminate the feedback inhibition of the lacI gene and ilvH obtained in 1-1-4 and to delete the ilvA , panB, ilvE and leuA genes in W3110 substituted with the tac promoter of the ilvGMEDA and ilvBN operon attenuation regions , 26 primer, ilvA (a gene encoding threonine dehydratase, the first enzyme of isoleucine biosynthesis pathway), panB (an enzyme necessary for pantothenate biosynthesis, 3-methyl- (A gene encoding 2-oxobutanoate hydroxymethyltransferase), ilvE (a gene encoding a branched chain amino acid aminotransferase among enzymes required for valine biosynthesis), and leuA (an enzyme required for leucine biosynthesis) A gene encoding isopropyl maleate synthase) and the like were deleted.

That is, the lacI gene produced in 1-1-4 was removed, the feedback inhibition of ilvH was removed, and the original promoter including the attenuation region of ilvGMEDA and ilvBN operon was replaced with a tac promoter having strong transcription activity. The deletion of the four genes was induced in tent cells.

[SEQ ID NO: 19] ilvA1stup: 5'-atggctgactcgcaacccctgtccggtgctccggaaggtgc cgaatatttgattgcagcattacacgtcttg-3 '

[SEQ ID NO: 20] ilvA1stdo: 5'-ctaacccgccaaaagaacctgaacgccgggttattggttt cgtcgtggccacttaacggctgacatggg-3 '

[SEQ ID NO: 21] panB1stup: 5'-atgaaaccgaccaccatctccttactgcagaagtacaaac aggaaaaaaagattgcagcattacacgtcttg-3 '

[SEQ ID NO: 22] panB1stdo: 5'-ttaatggaaactgtgttcttcgcccggataaacgccggact ccacttcagcacttaacggctgacatggg-3 '

[SEQ ID NO: 23] leuA1stup: 5'-atgagccagcaagtcattattttcgataccacattgcgcga cggtgaacagattgcagcattacacgtcttg-3 '

[SEQ ID NO: 24] leuA1stdo: 5'-ttcagaacgtgcaccatggctttggcagatgactcgacaat atcggtagcacttaacggctgacatggg-3 '

[SEQ ID NO: 25] ilvE1stup: 5'-atcttcgtcggtgatgttggcatgggagtaaacccgccag cgggatactctaggtgacactatagaacgcg-3 '

[SEQ ID NO: 26] ilvE1stdo: 5'-caggttttcgcctgcgccttcagagatataaccgttcacatc cagcgcgatagtggatctgatgggtacc-3 '

1-1-6: Construction of pKBRilvBNCD vector

The genes coding for ilvB (acetohydroxy acid synthase I large subunit), ilvN (encoding acetohydroxy acid synthase I small subunit), and genes coding for < RTI ID = ), ilvC (a gene encoding acetohydroxy acid isomeroreductase), and ilvD (a gene encoding dihydroxy-acid dehydratase) are successively cloned into a pKK223-3 expression vector (Pharmacia Biotech) to obtain a pKKilvBNCD vector, The sequence was confirmed. The pKKilvBNCD vector was obtained by ligation of the 8.0 kb fragment obtained by digesting the pKKilvBNCD vector with the Pci I and Sph I enzymes and the 1.9 kb fragment obtained by digesting the pBR322 vector with the Pci I and Sph I enzymes (see FIG. 4).

 [SEQ ID NO: 27] ilvBNf: 5'-actcgaattcatggcaagttcgggcacaacat-3 '

[SEQ ID NO: 28] ilvBNr: 5'-actcgaattcatggcaagttcgggcacaacat-3 '

[SEQ ID NO: 29] ilvCf: 5'-agtgctgcagacgaggaatcaccatggctaac-3 '

[SEQ ID NO: 30] ilvCrSX: 5'-gctcctgcagtctagagctagcgagctcttaacccgc-3 '

[SEQ ID NO: 31] ilvDf: 5'-actcgagctcgagacagacactgggagtaa-3 '

[SEQ ID NO: 32] ilvDr: 5'-tacgtctagattaaccccccagtttcgatt-3 '

1-1-7: Construction of pKBRilvBNCD-ptac-adhE-bdhAB vector

PCR was carried out using the pTac15K vector as a template and the primers of SEQ ID NO: 33 and SEQ ID NO: 34. The tac promoter thus obtained was digested with Xba I and inserted into the same restriction enzyme cleavage site of the pKBRilvBNCD vector prepared in Example 1-1-6 to obtain a pKBRilvBNCD-ptac vector.

The PCR reaction was carried out using the C. acetobutylicum ATCC 824 genomic DNA as a template and the primers of SEQ ID NO: 35 and SEQ ID NO: 36. The adhE gene thus obtained was digested with NheI and StuI and inserted into the same restriction enzyme cleavage site of the pKBRilvBNCD-ptac vector to obtain a pKBRilvBNCD-ptac-adhE vector.

Next, the PCR reaction was carried out using primers of SEQ ID NO: 37 and SEQ ID NO: 38 using C. acetobutylicum ATCC 824 genomic DNA as a template. The bdhAB gene thus obtained was digested with StuI and inserted into the same restriction enzyme cleavage site of the pKBRilvBNCD-ptac-adhE vector to prepare a pKBRilvBNCD-ptac-adhE-bdhAB vector (see FIG. 4).

[SEQ ID NO: 33] tacf: 5'-atcttctagaatcggaagctgtggtatggc-3 '

[SEQ ID NO: 34] tacrsn: 5'-gtgctctagaaggcctgatcagctagctgtttcctgtgtga-3 '

[SEQ ID NO: 35] adhEfn: 5'-acgcgctagcatgaaagttacaaatcaaaa-3 '

[SEQ ID NO: 36] adhErs: 5'-acgtaggcctatagtctatgtgcttcatga-3 '

[SEQ ID NO: 37] bdhfst: 5'-atgcaggcctcaacgaaaaattcaccccct-3 '

[SEQ ID NO: 38] bdhrst: 5'-acgtaggcctccggtaggtttacacagatt-3 '

1-1-8: Construction of pTac15K_VorABC vector

pTac15K_VorABC vector was prepared by cloning vorA , vorB , and vorC operon of Methanosarcina acetivorans with the primers of SEQ ID NOS: 39 and 40 in the pTac15K vector (p15A origin, low copies, KmR; KAISTMBEL stock).

[SEQ ID NO: 39] VorABCF-2: 5'-TATAGAGCTCATGGCAAAAAATGATGAAAA-3 '

[SEQ ID NO: 40] VorABCR-2: 5'-TATTTCTAGATCATACCTTATACTCTCTTGCCC-3 '

1-1-9: Construction of pTac15K_VorABC_ptac_icmAB vector

The pTac15K_VorABC_ptac_icmAB vector was prepared by cloning the icma and icmB genes of Streptomyces avermitilis into the pTac15K_VorABC vector using the primers of SEQ ID NOS: 41 and 42.

[SEQ ID NO: 41] tacicmABF-1: 5'-CGCGGCATGCGAAATGAGCTGTTGACAATT-3 '

[SEQ ID NO: 42] tacicmABR-1: 5'-TATAGCATGCCTACGCCCCGGCAGGCTGCC-3 '

1-2: Production of butanol-producing bacteria

The original promoter including the attenuated region of ilvGMEDA and ilvBN operon was substituted with the tac promoter, the feedback inhibition of ilvH was removed, and lacI , ilvA , panB , The microorganism producing the butanol was prepared by introducing the vector prepared in 1-1-6 to 1-1-9 into Escherichia coli W3110 strain {Val (Δ ilvE )} in which the leuA and ilvE genes were deleted.

1-3: Measurement of butanol production ability

The butanol-producing microorganisms prepared in 1-2 above were selected on LB plate medium supplemented with ampicillin and chloramphenicol at 50 占 퐂 / ml and 30 占 퐂 / ml, respectively. The transformant and the butanol-producing microorganism described in Korean Patent Application No. 10-2009-0059560 were inoculated in 10 ml of LB medium and pre-cultured at 37 ° C for 12 hours. Then, glucose (10 g / L) was added to a 250 ml flask containing 100 ml LB taken out at 80 ° C or higher after sterilization, and 1 ml of the preculture was inoculated and cultured at 31 ° C for 12 hours. As a result, as shown in Table 1, no butanol was produced in wild-type E. coli W3110, and recombinant microorganism bkdAB-lpdV was introduced as a gene encoding an enzyme that converts 2-ketoisovalerate to isobutyryl- (Korean Patent Application No. 10-2009-0059560), the recombinant mutant microorganism according to the present invention produces 32 mg / L butanol, while the recombinant microorganism into which bkdAB-lpdV is introduced produces 10 mg / L butanol Respectively. It was confirmed that the recombinant microorganism according to the present invention produced a larger amount of butanol, and a larger amount of butanol was expected to be produced in a large scale culture. These results indicate that butanol was successfully produced from 2-ketoisovalerate by overexpression of the VorABC , adhE, icmAB and bdhAB genes in the present invention and was also introduced into microorganisms having the ability to produce 2-ketoisovalerate It was confirmed that 2-ketoisovalerate was accumulated by the genetic manipulation and acted as an important precursor for the production of butanol.

Strain Butanol (mg / L) W3110 ND ¹ Val (△ ilvE) + pKBRilvBNCD- ptac-adhEbdhAB + pTac15KlpdVbkdA1-ptac-bkdA2BicmAB 10 Val (△ ilvE) + pKBRilvBNCD- ptac-adhEbdhAB + pTac15K_VorABC_icmAB 32

¹ Not detected.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Korea Advanced Institute of Science and Technology <120> Enhanced Butanol Producing Recombinant Microorganisms and Method          Preparing Butanol Using the Same <130> P10-B186 <160> 46 <170> Kopatentin 1.71 <210> 1 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 agccgtcgac gctagcgcat gcacgcgtgt gcacccatgg gacgtcctca ctgactcgct 60 gcgctc 66 <210> 2 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggctcacaac gtggctagcg acgtcgtgca cccatgggtt ccactgagcg tcagacc 57 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 actctctaga cgcgggtttg ttactgataa 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gctagatatc aggatatcgg cattttcttt 30 <210> 5 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta gattgcagca 60 ttacacgtct tg 72 <210> 6 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg cacttaacgg 60 ctgacatggg 70 <210> 7 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gactctgcag ggtgatcgag actctttggc ggttgac 37 <210> 8 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ggaaaaaagg ccaatcacgc ggaataacgc gtctgattca ttttcgagta ag 52 <210> 9 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 cttactcgaa aatgaatcag acgcgttatt ccgcgtgatt ggcctttttt cc 52 <210> 10 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gctccgtcga ccagtttcac aattgcccct tgcgtaaa 38 <210> 11 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gactgtcgac ctaacttatt ggctgtaagc tgttctgagg cc 42 <210> 12 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gctcggatcc gaatgttgtt cccttcctcg tagttcatcc 40 <210> 13 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gactgaattc atgaatggcg cacagtgggt ggtacatgcg 40 <210> 14 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 gctcctgcag tcaccgctgg ctaactggat atcttttggg 40 <210> 15 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gactcgtcga cagagatggt agggcggata a 31 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 gctcggatcc cacactgtat tatgtcaaca 30 <210> 17 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 gactgaattc atggcaagtt cgggcacaac 30 <210> 18 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 gctcactgca gtgcgtcacg aatgctttct t 31 <210> 19 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 atggctgact cgcaacccct gtccggtgct ccggaaggtg ccgaatattt gattgcagca 60 ttacacgtct tg 72 <210> 20 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 ctaacccgcc aaaaagaacc tgaacgccgg gttattggtt tcgtcgtggc cacttaacgg 60 ctgacatggg 70 <210> 21 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 atgaaaccga ccaccatctc cttactgcag aagtacaaac aggaaaaaaa gattgcagca 60 ttacacgtct tg 72 <210> 22 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 ttaatggaaa ctgtgttctt cgcccggata aacgccggac tccacttcag cacttaacgg 60 ctgacatggg 70 <210> 23 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 atgagccagc aagtcattat tttcgatacc acattgcgcg acggtgaaca gattgcagca 60 ttacacgtct tg 72 <210> 24 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 ttcagaacgt gcaccatggc tttggcagat gactcgacaa tatcggtagc acttaacggc 60 tgacatggg 69 <210> 25 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 atcttcgtcg gtgatgttgg catgggagta aacccgccag cgggatactc taggtgacac 60 tatagaacgc g 71 <210> 26 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 caggttttcg cctgcgcctt cagagatata accgttcaca tccagcgcga tagtggatct 60 gatgggtacc 70 <210> 27 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 actcgaattc atggcaagtt cgggcacaac at 32 <210> 28 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 actcgaattc atggcaagtt cgggcacaac at 32 <210> 29 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 agtgctgcag acgaggaatc accatggcta ac 32 <210> 30 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 gctcctgcag tctagagcta gcgagctctt aacccgc 37 <210> 31 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 actcgagctc gagacagaca ctgggagtaa 30 <210> 32 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 tacgtctaga ttaacccccc agtttcgatt 30 <210> 33 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 33 atcttctaga atcggaagct gtggtatggc 30 <210> 34 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 gtgctctaga aggcctgatc agctagctgt ttcctgtgtg a 41 <210> 35 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 acgcgctagc atgaaagtta caaatcaaaa 30 <210> 36 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 36 acgtaggcct atagtctatg tgcttcatga 30 <210> 37 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 37 atgcaggcct caacgaaaaa ttcaccccct 30 <210> 38 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 38 acgtaggcct ccggtaggtt tacacagatt 30 <210> 39 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 39 tatagagctc atggcaaaaa atgatgaaaa 30 <210> 40 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 40 tatttctaga tcatacctta tactctcttg ccc 33 <210> 41 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 41 cgcggcatgc gaaatgagct gttgacaatt 30 <210> 42 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 42 tatagcatgc ctacgccccg gcaggctgcc 30 <210> 43 <211> 482 <212> PRT <213> Methanosarcina acetivorans <400> 43 Met Lys Met Ala Ala Glu Ile Ile Asp Gly Glu Lys Val Ile Lys Lys   1 5 10 15 Pro Lys Ala Leu Tyr Ser Glu Tyr Pro Arg Lys Gly Gly Ala Ala Pro              20 25 30 Thr Ala Thr His Tyr Cys Pro Gly Cys Gly His Gly Val Leu His Lys          35 40 45 Leu Ile Ala Glu Ala Ile Asp Asp Leu Gly Ile Gln Asp Arg Thr Val      50 55 60 Met Ile Ser Pro Val Gly Cys Ala Val Phe Ala Tyr Tyr Tyr Phe Asp  65 70 75 80 Thr Gly Asn Ile Gln Val Ala His Gly Arg Ala Pro Ala Val Gly Thr                  85 90 95 Gly Val Ser Ser Ala Glu Glu Asn Ala Val Val Ile Ser Tyr Gln Gly             100 105 110 Asp Gly Asp Leu Ala Ser Ile Gly Leu Asn Glu Thr Leu Gln Ala Ala         115 120 125 Asn Arg Gly Glu Lys Leu Ala Val Phe Phe Val Asn Asn Thr Val Tyr     130 135 140 Gly Met Thr Gly Gly Gln Met Ala Pro Thr Thr Leu Ile Gly Glu Lys 145 150 155 160 Thr Thr Thr Pro Glu Gly Arg Asp Pro Arg Phe Ala Gly Tyr Pro                 165 170 175 Leu His Met Cys Glu Leu Leu Asp Asn Leu Lys Ala Pro Ile Phe Ile             180 185 190 Glu Arg Val Ser Val Ser Asp Ile Ser His Ile Arg Lys Ala Arg Lys         195 200 205 Ala Val Arg Lys Ala Leu Glu Val Gln Arg Asp Gly Lys Gly Tyr Ala     210 215 220 Phe Val Glu Val Leu Ala Ala Cys Pro Thr Asn Leu Arg Met Asp Ala 225 230 235 240 Glu Gln Ala Ile Gln Phe Ile Asn Glu Gln Met Glu Lys Glu Phe Pro                 245 250 255 Leu Arg Asn Phe Arg Asp Asn Phe Glu Ser Ala Glu Pro Leu His Arg             260 265 270 Gly Ile Ser Asp Phe Thr Thr Glu Ser Leu Glu Lys Leu Tyr Gly Ile         275 280 285 Glu Ala Glu Thr Gly Glu Lys Pro Ser Arg Thr Asp Phe Ala Pro Ile     290 295 300 Gln Thr Lys Ile Ala Gly Phe Gly Gly Gln Gly Val Leu Ser Met Gly 305 310 315 320 Leu Ile Leu Ala Gln Ala Gly Val Lys Ala Asn Leu Asn Ala Ser Trp                 325 330 335 Phe Pro Ser Tyr Gly Pro Glu Gln Arg Gly Gly Thr Ser Asn Cys Ser             340 345 350 Val Val Ile Ser Gly Gln Ala Ile Gly Ser Pro Thr Val Tyr Thr Pro         355 360 365 Asp Ile Leu Ile Ala Met Asn Arg Pro Ser Leu Glu Arg Phe Glu Gly     370 375 380 Ala Val Lys Glu Gly Gly Phe Ile Leu Tyr Asp Ser Thr Ile Gly Glu 385 390 395 400 Ala Glu Thr Pro Ala Gly Val Asn Ala Ile Ala Val Ala Thr Glu                 405 410 415 Lys Ala Lys Glu Ala Gly Asp Glu Arg Ala Asn Ser Phe Met Leu             420 425 430 Gly Val Leu Leu Gly Leu Asn Ala Thr Gly Leu Glu Glu Glu Ala Phe         435 440 445 Lys Glu Ala Leu Ala Glu Asn Phe Thr Gly Lys Pro Lys Val Ile Glu     450 455 460 Phe Asn Gln Gln Met Leu Glu Ala Gly Ala Ala Trp Ala Arg Glu Tyr 465 470 475 480 Lys Val         <210> 44 <211> 351 <212> PRT <213> Methanosarcina acetivorans <400> 44 Met Ala Thr Gln Leu Val Lys Gly Asn Ser Ala Ile Val Val Gly Ala   1 5 10 15 Leu Tyr Ala Gly Cys Asp Cys Phe Phe Gly Tyr Pro Ile Thr Pro Ala              20 25 30 Ser Glu Ile Leu His Asp Ala Ser Lys Tyr Phe Pro Met Ile Gly Arg          35 40 45 Lys Phe Val Gln Ala Glu Ser Glu Glu Ala Ala Ile Asn Met Val Phe      50 55 60 Gly Gly Ala Ser Ala Gly His Arg Val Met Thr Ser Ser Ser Gly Pro  65 70 75 80 Gly Ile Ser Leu Met Gln Glu Gly Ile Ser Tyr Leu Ala Gly Ala Glu                  85 90 95 Leu Pro Cys Val Leu Val Asp Ile Met Arg Ala Gly Pro Gly Leu Gly             100 105 110 Asn Ile Gly Pro Glu Gln Gly Asp Tyr Asn Gln Val Val Lys Gly Gly         115 120 125 Gly His Gly Asn Tyr Lys Asn Ile Val Leu Ala Pro Asn Ser Val Gln     130 135 140 Glu Met Cys Asp Phe Thr Met Lys Ala Phe Glu Leu Ala Phe Lys Tyr 145 150 155 160 Arg Asn Pro Ala Ile Val Leu Ala Asp Gly Val Leu Gly Gln Met Ile                 165 170 175 Glu Ser Leu Glu Phe Pro Lys Lys Ala Leu Thr Pro Glu Ile Asp Ala             180 185 190 Ser Trp Ala Val Asn Gly Thr Ala Glu Thr Arg Pro Asn Leu Ile Thr         195 200 205 Ser Ile Phe Leu Asp Phe Asn Glu Leu Gly Gln Phe Asn Glu Lys Leu     210 215 220 Gln Ala Lys Tyr Glu Leu Ile Lys Gln Asn Glu Val Asp Tyr Glu Glu 225 230 235 240 Tyr Met Thr Asp Asp Ser Ser Ile Val Ser Val Ser Ser Gyr Ile Ser                 245 250 255 Ser Arg Ile Cys Arg Ser Ala Val Asp Leu Ala Arg Lys Glu Gly Ile             260 265 270 Lys Val Gly Leu Phe Arg Pro Lys Thr Leu Phe Pro Phe Pro Glu Ala         275 280 285 Gln Leu Lys Ala Leu Ala Asp Lys Glu Ala Ser Phe Ile Ser Val Glu     290 295 300 Met Ser Asn Gly Gln Met Ile Asp Asp Ile Arg Leu Ala Ile Asp Cys 305 310 315 320 Ser Gln Pro Val Glu Leu Val Asn Arg Met Gly Gly Asn Leu Ile Thr                 325 330 335 Leu Asp Gln Ile Met Asp Lys Ile Arg Lys Val Ala Gly Glu Ala             340 345 350 <210> 45 <211> 82 <212> PRT <213> Methanosarcina acetivorans <400> 45 Met Ala Lys Asn Asp Glu Lys Glu Pro Tyr Pro Val Ile Asn Ile Leu   1 5 10 15 Glu Cys Lys Ala Cys Gly Arg Cys Leu Leu Ala Cys Pro Lys Asp Val              20 25 30 Leu Phe Met Ser Asp Asn Leu Asn Ala Arg Gly Tyr His Tyr Val Glu          35 40 45 Tyr Lys Gly Glu Gly Cys Ser Gly Cys Ala Ser Cys Tyr Tyr Thr Cys      50 55 60 Pro Glu Pro Leu Ala Leu Glu Ile His Ile Pro Leu Lys Lys Glu Glu  65 70 75 80 Ala Asp         <210> 46 <211> 2772 <212> DNA <213> Methanosarcina acetivorans <400> 46 atggcaaaaa atgatgaaaa agaaccgtac cctgttatca acattctgga gtgcaaggct 60 tgtgggcgct gccttcttgc ctgcccgaag gatgtgcttt tcatgagtga taacctgaac 120 gccaggggtt accactacgt agagtataaa ggagaaggct gttcaggctg tgcgagctgc 180 tactatacct gtcctgaacc ccttgcactg gaaatccata ttcccctgaa aaaggaggaa 240 gccgactaag gcaggataag gagggaaaaa gatggcaaca caactcgtaa aaggcaactc 300 cgcaatagtc gttggcgcac tttatgccgg atgtgactgt ttctttggat atcccattac 360 tccggcaagc gaaattctgc atgatgcatc caaatacttt cccatgatag gaaggaaatt 420 cgtgcaggcc gagtcagagg aggctgcaat taacatggtc ttcggggggg catcagccgg 480 gcacagggta atgacgtcct cctcaggccc aggaattagc ctgatgcagg aagggatctc 540 ttatcttgca ggcgcagagc ttccttgtgt gcttgtcgat ataatgaggg caggccctgg 600 actagggaac atcggacctg aacagggtga ttataaccag gtagtaaaag gtggagggca 660 cgggaattat aaaaatatcg tacttgcccc caactccgtg caggagatgt gtgactttac 720 catgaaggct tttgagctgg ccttcaagta caggaaccct gcaatcgtgc ttgctgacgg 780 agtgctcggg cagatgattg agtcccttga gtttccgaaa aaagccctta ctcctgaaat 840 cgatgcaagc tgggcagtca acggtacagc cgaaaccagg cctaacctga taacctctat 900 cttccttgac ttcaacgaac tcgggcagtt caacgagaaa ctgcaggcaa aatacgagct 960 gataaagcaa aatgaggttg actacgaaga atacatgacc gacgatgcct caatcgtgct 1020 tgtctcttac ggcataagca gcaggatctg caggtcagct gtagaccttg ccagaaagga 1080 aggcataaag gtggggctct tcaggccaaa gactcttttc ccattcccgg aggcgcagtt 1140 gaaagccctt gcagataagg aagcttcttt catctccgtg gagatgagca acgggcagat 1200 gatagatgac atcaggcttg caatcgactg ctcacagccc gtggaactgg taaaccgcat 1260 gggaggcaac ctgatcaccc ttgaccagat catggacaaa atcagaaagg tagcagggga 1320 ggcatgaaaa tggcagcaga aattattgac ggagaaaagg ttatcaaaaa gccaaaagcc 1380 ctgtactcgg aatacccgcg caaaggaggt gcagctccaa cagccaccca ctattgtcct 1440 ggctgcggac atggggtttt gcacaagctc attgccgagg caattgacga cctcggaatc 1500 caggacagga cggttatgat cagccctgtg ggatgtgcag tttttgctta ttattacttt 1560 gacacaggca atatccaggt tgcccacggc cgggctcctg ctgttggaac aggtgtctca 1620 agggctgaag aaaacgctgt agttatctcc taccagggcg acggagacct tgcttcgatc 1680 ggcctgaacg agacgctgca ggcggcaaac agaggagaaa agcttgcagt ttttttcgta 1740 aataacaccg tatacggcat gaccggcgga cagatggctc caacaaccct tatcggggaa 1800 aagacaacca caacccctga aggtcgcgac ccccgctttg caggctaccc gctccacatg 1860 tgtgagcttc tggacaacct gaaggctccg atttttatcg aaagagtctc ggtttccgat 1920 atctctcata tccgaaaagc ccgaaaagcc gttcgaaagg cgctcgaagt ccagcgtgat 1980 ggcaagggct atgcttttgt ggaagtcctt gcagcctgcc cgaccaacct taggatggat 2040 gcagagcagg ctatccagtt cattaacgag cagatggaaa aggaattccc gctcaggaac 2100 ttcagggaca atttcgaatc tgccgaaccc cttcaccgcg ggatcagtga ctttacaact 2160 gagtcccttg aaaagcttta cggaattgaa gcggaaaccg gagaaaaacc ctcaaggact 2220 gactttgctc cgatccagac caagattgca ggtttcggag gacagggtgt cctgagcatg 2280 ggccttatcc ttgcccaggc aggagtcaaa gcaaacctca atgcttcctg gttcccgtct 2340 tacggcccgg aacagcgcgg cggaacctca aactgctcgg tcgtgatttc aggtcaggct 2400 ataggctcgc ctacggtcta caccccagat atccttatag ccatgaaccg tccctccctc 2460 gagagattcg aaggggcagt aaaagaagga ggcttcatcc tctacgactc tacaatcggc 2520 gaagccgaga cccctgcagg cgtaaacgca attgcagtcc ctgcaaccga aaaagcaaaa 2580 gaagcaggtg atgagagagc cgcaaactcc ttcatgctcg gagtccttct gggcctgaac 2640 gcaaccggcc ttgaagaaga agccttcaaa gaagcccttg ccgaaaactt cacaggcaaa 2700 cccaaagtta ttgagtttaa ccagcagatg ctcgaagccg gggcagcatg ggcaagagag 2760 tataaggtat ga 2772

Claims (32)

A microorganism having the ability to produce 2-ketoisovalerate, which is a precursor of L-valine, is added to an enzyme that converts 2-ketoisovalerate into isobutyryl-CoA, VorA (2-keto (Alpha-subunit), VorB (2-oxoisovalerate ferredoxin oxydoryldexta), and VorC (2-ketoisovalerate ferredoxin reductase, gamma subunit) A gene encoding an enzyme that converts isobutyryl-CoA to butyryl-CoA, a gene that codes for an enzyme that converts butyryl-CoA to butylaldehyde (butyraldehyde), and a gene encoding butylaldehyde To a recombinant microorganism having a butanol-producing ability.
The process for producing a recombinant microorganism having butanol-producing ability according to claim 1, wherein the microorganism having the ability to produce 2-ketoisovalerate as a precursor of L-valine is selected from the group consisting of bacteria, yeast and fungi .
The method according to claim 1, wherein the microorganism having the ability to produce 2-ketoisovalerate as a precursor of L-valine is selected from the group consisting of Corynebacterium genus, Brevibacterium genus, and E. coli Wherein the recombinant microorganism has a butanol-producing ability.
2. The method according to claim 1, wherein the microorganism having the ability to produce 2-ketoisovalerate as a precursor of L-valine is selected from the group consisting of enzymes involved in L-isoleucine biosynthesis A gene encoding an enzyme involved in L-leucine biosynthesis, a gene encoding an enzyme involved in D-pantothenic acid biosynthesis, and a gene encoding an enzyme involved in L-valine biosynthesis are weakened or deleted Wherein the recombinant microorganism has a butanol-producing ability.
5. The method according to claim 4, wherein the gene coding for the enzyme involved in L-isoleucine biosynthesis is ilvA ( gene encoding threonine dehydratase), and the gene encoding the enzyme involved in L-leucine biosynthesis is leuA (A gene encoding 2-isopropyl maleate synthase), and the gene coding for the enzyme involved in D-pantothenic acid biosynthesis is panB (3-methyl-2-oxobutanoate hydroxymethyl transperase encoded Wherein the gene coding for the enzyme involved in L-valine biosynthesis is ilvE (gene encoding amino acid transaminase of branched chain amino acid).
5. The method for producing a recombinant microorganism having a butanol-producing ability according to claim 4, wherein the microorganism having the ability to produce 2-ketoisovalerate is further modified by a method selected from the group consisting of a) deletion of lacI (gene encoding the lac operon inhibitor) gene; (b) ilvH (acetohydroxy acid synthase isozyme III) Elimination of feedback inhibition of genes; (c) replacing the original promoter comprising the attenuator of ilvGMEDA (acetohydroxy acid synthase isozyme I) and ilvBN (acetohydroxy acid synthase isozyme II) operon with a strong promoter; And (d) introduction of an expression vector comprising a strong promoter.
[Claim 7] The method according to claim 6, wherein the strong promoter is selected from the group consisting of trc promoter, tac promoter, T7 promoter, lac promoter and trp promoter.
[Claim 7] The method according to claim 6, wherein the expression vector containing the strong promoter is a vector containing ilvB, ilvN , ilvC, and ilvD genes.
2. The method according to claim 1, wherein the enzyme that converts the isobutyryl-CoA to butyryl-CoA is isobutyryl-CoA mutase and the enzyme that converts the butyryl-CoA to butylaldehyde is butylaldehyde dehydrogenase , And the enzyme for converting the butylaldehyde to butanol is butanol dehydrogenase. The method for producing a recombinant microorganism having a butanol-producing ability according to claim 1,
2. The method according to claim 1, wherein the enzyme is selected from the group consisting of VorA (2-ketoisovalerate ferredoxin reductase, alpha subunit), VorB (2-oxoisobarbital), which is an enzyme that converts 2-ketoisovalerate into isobutyryl- acid ferredoxin oxy Torii virtue hydratase) and VorC (2- Kane toy small valeric acid ferredoxin reductase, a gene encoding the enzyme VorABC consisting of gamma subunit) is meta or industrial reusi (Methanosarcina) acetoxy TiVo lance (acetivorans) method of producing a recombinant microorganism having high butanol producing ability, characterized in that the derived VorABC.
The method of claim 1, wherein the gene coding for the enzyme to convert the isobutyryl -CoA as butyryl -CoA is a butanol productivity according to claim Streptomyces (Streptomyces) Arbor US subtilis (avermitilis) derived from the icmAB &Lt; / RTI &gt;
2. The method for producing a recombinant microorganism having a butanol-producing ability according to claim 1, wherein the gene coding for the enzyme for converting the butyryl-CoA to butylaldehyde is adhE derived from Clostridium acetoobutylicum .
2. The method for producing a recombinant microorganism having a butanol-producing ability according to claim 1, wherein the gene coding for the enzyme for converting butylaldehyde to butanol is bdhAB derived from Clostridium acetoobutylicum .
A microorganism having the ability to produce 2-ketoisovalerate as a precursor of L-valine was added to an enzyme that converts 2-ketoisovalerate into isobutyryl-CoA, VorA (2-ketoisovalerate, A gene coding for the VorABC enzyme consisting of VorB (2-oxoisovalerate peredoxin oxydoryldexta) and VorC (2-ketoisovalerate ferredoxin reductase, gamma subunit) , A gene encoding an enzyme that converts isobutyryl-CoA into butyryl-CoA, a gene encoding an enzyme that converts butyryl-CoA to butylaldehyde, and a gene encoding an enzyme that converts butylaldehyde to butanol Wherein the recombinant microorganism has a butanol-producing ability.
15. The recombinant microorganism having ability to produce butanol according to claim 14, wherein the microorganism having the ability to produce 2-ketoisovalerate as a precursor of L-valine is selected from the group consisting of bacteria, yeast and fungi.
15. The method according to claim 14, wherein the microorganism having the ability to produce 2-ketoisovalerate as a precursor of L-valine is selected from the group consisting of Corynebacterium genus, Brevibacterium genus and E. coli Wherein the recombinant microorganism has a butanol-producing ability.
15. The method according to claim 14, wherein the enzyme that converts isobutyryl-CoA to butyryl-CoA is isobutyryl-CoA mutase and the enzyme that converts the butyryl-CoA to butylaldehyde is butylaldehyde dehydrogenase , And the enzyme that converts the butyraldehyde to butanol is butanol dehydrogenase.
15. The method of claim 14, wherein the enzyme that converts the 2-ketoisovalerate into isobutyryl-CoA, VorA (2-ketoisovalerate ferredoxin reductase, alpha subunit), VorB The gene coding for the VorABC enzyme consisting of Vor AB (valereate ferredoxin oxydoryldactase) and VorC (2-ketoisovalerate ferredoxin reductase, gamma subunit) is VorABC derived from methanosarcina acetylborance The recombinant microorganism having the ability to produce butanol.
15. The recombinant microorganism having a butanol- producing ability according to claim 14, wherein the gene encoding the enzyme for converting the isobutyryl-CoA to butyryl-CoA is icmAB derived from Streptomyces arbormitilis .
15. The recombinant microorganism having butanol-producing ability according to claim 14, wherein the gene coding for the enzyme for converting the butyryl-CoA to butylaldehyde is adhE derived from Clostridium acetoobutylicum .
15. The recombinant microorganism having a butanol-producing ability according to claim 14, wherein the gene coding for the enzyme for converting butylaldehyde to butanol is bdhAB derived from Clostridium acetobutylicum.
A microorganism having the ability to produce 2-ketoisovalerate as a precursor of L-valine was added to an enzyme that converts 2-ketoisovalerate into isobutyryl-CoA, VorA (2-ketoisovalerate, A gene coding for the VorABC enzyme consisting of VorB (2-oxoisovalerate peredoxin oxydoryldexta) and VorC (2-ketoisovalerate ferredoxin reductase, gamma subunit) , A gene encoding an enzyme that converts isobutyryl-CoA into butyryl-CoA, a gene encoding an enzyme that converts butyryl-CoA to butylaldehyde, and a gene encoding an enzyme that converts butylaldehyde to butanol A gene encoding an enzyme involved in L-isoleucine biosynthesis, a gene encoding an enzyme involved in L-leucine biosynthesis, a gene encoding an enzyme involved in D-pantothenic acid biosynthesis, and a gene encoding L- Recombinant microorganism having high butanol producing ability, characterized in that in the gene coding for the enzyme involved in the biosynthesis is inactivated or deleted.
23. The method according to claim 22, wherein the gene coding for the enzyme involved in L-isoleucine biosynthesis is ilvA ( gene encoding threonine dehydratase), and the gene encoding the enzyme involved in L-leucine biosynthesis is leuA (A gene encoding 2-isopropyl maleate synthase), and the gene coding for the enzyme involved in D-pantothenic acid biosynthesis is panB (3-methyl-2-oxobutanoate hydroxymethyl transperase encoded Wherein the gene coding for the enzyme involved in L-valine biosynthesis is ilvE (gene encoding amino acid transaminase of branched chain amino acid).
The recombinant microorganism having ability to produce 2-ketoisovalerate according to claim 22, wherein the microorganism having the ability to produce 2-ketoisovalerate is further mutated by a method selected from the group consisting of: (a) lacI (gene encoding the lac operon inhibitor) deletion of the gene; (b) ilvH (acetohydroxy acid synthase isozyme III) Elimination of feedback inhibition of genes; (c) replacing the original promoter comprising the attenuation region of ilvGMEDA (acetohydroxy acid synthase isozyme I) and ilvBN (acetohydroxy acid synthase isozyme II) operon with a strong promoter; And (d) introduction of an expression vector comprising a strong promoter.
25. The recombinant microorganism having butanol-producing ability according to claim 24, wherein the expression vector comprising the strong promoter is a vector containing ilvB, ilvN , ilvC, and ilvD genes.
14. The method for producing butanol according to claim 14, wherein the recombinant microorganism is cultured to produce butanol, and then the butanol is recovered from the culture.
The method for producing butanol according to claim 22 or 24, wherein the recombinant microorganism is cultured to produce butanol, and then the butanol is recovered from the culture.
A microorganism having the ability to produce 2-ketoisovalerate as a precursor of L-valine was added to an enzyme that converts 2-ketoisovalerate into isobutyryl-CoA, VorA (2-ketoisovalerate, A gene coding for the VorABC enzyme consisting of VorB (2-oxoisovalerate peredoxin oxydoryldexta) and VorC (2-ketoisovalerate ferredoxin reductase, gamma subunit) ( VorABC ) is introduced into the recombinant microorganism. The recombinant microorganism having the ability to produce isobutyryl-CoA in the 2-ketoisovalerate.
28. The method according to claim 28, wherein the VorA is represented by SEQ ID NO: 43, VorB is represented by SEQ ID NO: 44, and VorC is represented by amino acid sequence of SEQ ID NO: 45. Recombinant microorganism.
A method for producing isobutyryl-CoA, which comprises culturing the recombinant microorganism of claim 28 or 29 to produce isobutyryl-CoA, and then recovering isobutyryl-CoA from the culture broth. How to produce.
VorA (2-ketoisovalerate ferredoxin reductase, alpha subunit), an enzyme that converts 2-ketoisovalerate into isobutyryl-CoA, VorB (2-oxoisovalerate ferredoxin oxydorudate VorABC enzyme consisting of Vor AB (Vor ABC) and VorC (2-Ketoisovalerate ferredoxin reductase, gamma subunit), or recombinant microorganism expressing the enzyme, was used to convert 2-ketoisovalerate into isobutyryl-CoA .
32. The method according to claim 31, wherein the VorA is represented by SEQ ID NO: 43, VorB is represented by SEQ ID NO: 44, and VorC is represented by amino acid sequence of SEQ ID NO: 45. Way.

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