KR20160131341A

KR20160131341A - Novel genes involved in production of C5-C8 organic acid, vector comprising the genes, microorganism transformed with the vector, and method for producing C5-C8 organic acid using the microorganism

Info

Publication number: KR20160131341A
Application number: KR1020150063414A
Authority: KR
Inventors: 상병인; 이태호; 전병승
Original assignee: 한양대학교 산학협력단
Priority date: 2015-05-06
Filing date: 2015-05-06
Publication date: 2016-11-16
Also published as: KR101766955B1

Abstract

The present invention relates to a novel gene involved in C5-C8 organic acid production, a vector containing the gene, a microorganism transformed with the vector, and a method for producing C5-C8 organic acid using the same, , A gene coding for an enzyme involved in C5-C8 organic acid biosynthesis, and a method for producing C5-C8 organic acid using the genes.
According to the present invention, by providing a novel gene involved in C5-C8 organic acid production, it is possible to provide C5-C8 organic acid with improved production yield.

Description

A novel gene involved in the production of C5-C8 organic acid, a vector containing the gene, a microorganism transformed with the vector, and a method for producing C5-C8 organic acid using the same. the genes, microorganism transformed with the vector, and the method for producing C5-C8 organic acid using the microorganism}

The present invention relates to a novel gene involved in C5-C8 organic acid production, a vector containing the gene, a microorganism transformed with the vector, and a method for producing C5-C8 organic acid using the same.

80% of the energy used on the earth is produced from fossil fuels, and most of the raw materials such as plastic raw materials, synthetic rubber, solvents, paints and adhesives are produced from petrochemical processes. Therefore, it is necessary to continuously supply fossil fuels, but due to the limitation of the reserves, it is urgent to develop new alternative fuels.

Currently, available alternative energy sources are bio energy, terrestrial heat, wind power, intelligence, and tidal power, among which bio-energy is highly utilized as fuel for transportation and less than 30% , And to as much as 90%. Bio-energy is a sustainable source of energy made from biomass in the natural world. It is produced from plants, agriculture and environmental waste such as corn, sugarcane, and waste cellulose, There is an effect of reducing waste by using various organic wastes without increasing carbon dioxide (CO ₂ ). It does not contain heavy metals and other harmful substances, and liquid biofuels have the advantage of being mixed with existing automotive liquid fuels. As a biofuel derived from such renewable vegetable raw materials, a lot of research has already been carried out on ethanol as a C2 material and butanol as a C4 material as a transportation fuel, but aviation fuel requires a material having a higher carbon number It is necessary to develop new biofuels.

Thus, strains producing organic acids, which are precursors of the biofuel, have been developed. For example, Clostridium Tyrobutylicum S1 (KCTC 12103BP) has been disclosed as a strain producing butyric acid, which is a typical C4 material Patent Document 1), a strain producing hexanoic acid which is a C6 substance, Clostridium clubeieri and Megaspera eldenii have been disclosed (Non-Patent Document 1). However, since the production amount is insufficient, it is applied to the production process of biofuels This is insufficient.

Korean Patent Publication No. 10-2013-0077296

B. H. Kim and G. M. Gadd, Bacterial physiology and metabolism. Cambridge University Press, Cambridge (2008)

Disclosure of the Invention The present invention has been conceived to solve the problems described above, and it is an object of the present invention to provide a novel gene involved in C5-C8 organic acid production, a vector containing the gene, a microorganism transformed with the vector, and a method for producing C5- .

In order to solve the above problems,

A gene encoding an enzyme involved in C5-C8 organic acid biosynthesis, which comprises at least one base sequence selected from the group consisting of SEQ ID NOS: 1-8.

According to the present invention, the nucleotide sequence of SEQ ID NO: 1 encodes Acetyl-CoA acetyltransferase (THL), the nucleotide sequence of SEQ ID NO: 2 encodes 3-hydroxyacyl-CoA dehydrogenase (HBD) The nucleotide sequence of SEQ ID NO: 4 encodes Acyl-CoA dehydrogenase (ACDH), the nucleotide sequence of SEQ ID NO: 5 is Acetyl-CoA transferase (ACT) And the nucleotide sequence of SEQ ID NO: 6 encodes butyryl-CoA dehydrogenase (BCDH), the nucleotide sequence of SEQ ID NO: 7 encodes an electron transfer flavoprotein alpha subunit (ETF A) The sequence may be one encoding the electron transfer flavoprotein beta subunit (ETF B).

According to the present invention, the gene may be derived from Megasphaera hexanoica strain (KFCC11466P).

According to the present invention, the C5 organic acid is pentanoic acid, the C6 organic acid is hexanoic acid, the C7 organic acid is heptanoic acid, and the C8 organic acid may be octanoic acid.

The present invention also provides a vector comprising a gene encoding an enzyme involved in C5-C8 organic acid biosynthesis.

According to the present invention, said vector comprises a first vector comprising a gene represented by SEQ ID NO: 1 or SEQ ID NO: 10; And a second vector comprising the gene represented by SEQ ID NO: 4.

According to the present invention, the second vector may further comprise at least one gene selected from the genes represented by SEQ ID NOS: 2 to 3 and SEQ ID NOS: 5 to 9.

The present invention also provides a microorganism having the ability to produce a C5-C8 organic acid, which is transformed by said vector.

According to the present invention, the microorganism may be selected from the group consisting of bacteria, yeast and fungi.

According to the present invention, the microorganism may be a gene encoding an enzyme involved in lactate biosynthesis, a gene encoding an enzyme involved in acetate biosynthesis, a gene encoding an enzyme involved in ethanol biosynthesis, And a gene coding for an enzyme involved in the biosynthesis of succinate may be weakened or deleted in E. coli.

The present invention also provides a method for producing a C5-C8 organic acid by culturing the microorganism.

According to the present invention, by providing a novel gene involved in C5-C8 organic acid production, it is possible to provide C5-C8 organic acid with improved production yield.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing the entire gene map and gene information of megasperahexanoicus.
Fig. 2 is an analysis of RNA expression genes in the culture of megasperahexanoic acid strains without addition of acetic acid and butyric acid. Fig.
FIG. 3 is a graph showing RPKM (Read Per Kilobase per Millon mapped reads) values of RNA expression genes in the culture of megasperahexanoic acid by adding acetic acid and butyric acid.
FIG. 4 is a graph showing RPKM values of genes isolated from megasperahexanoicus strains.
FIG. 5 is a diagram showing a hexanoic acid biosynthetic pathway derived based on the RNA expression level of megasperahexanoicus.
6 is a diagram showing a hexanoic acid biosynthesis pathway of E. coli in which the lactate biosynthesis pathway, the acetate biosynthesis pathway, and the ethanol biosynthesis pathway are blocked, which is used in the present invention.
Fig. 7 is a diagram showing a vector containing a gene necessary for the hexanoic acid biosynthesis pathway, which was used in the production of the transformed microorganism according to Example 1. Fig.
8 is a diagram showing a vector containing a gene necessary for the hexanoic acid biosynthesis pathway used in the production of the microorganism transformed according to Example 2. Fig.
9 is a diagram showing a vector containing a gene necessary for a hexanoic acid biosynthetic pathway used in the production of a microorganism transformed according to Example 3. Fig.
10 is a diagram showing a vector containing a gene necessary for the hexanoic acid biosynthesis pathway used for the production of the transformed microorganism according to Example 4. Fig.
11 is a diagram showing a vector containing a gene necessary for the hexanoic acid biosynthesis pathway used for the production of the transformed microorganism according to Example 5. Fig.
12 is a diagram showing a vector containing a gene necessary for a hexanoic acid biosynthetic pathway, which was used in the production of a microorganism transformed according to Comparative Example 1. FIG.
13 is a diagram showing a vector containing a gene necessary for the hexanoic acid biosynthesis pathway used for the production of the transformed microorganism according to Comparative Example 2. Fig.
FIG. 14 is a graph showing the production yield of hexanoic acid of the transformed microorganisms according to Examples and Comparative Examples of the present invention. FIG.
15 is a graph showing the production of hexanoic acid of a transformed microorganism according to Example 1 of the present invention.

Hereinafter, the present invention will be described in more detail.

The term "deletion" in the present invention is a concept encompassing the mutation, substitution or deletion of a part or whole base of the gene, or introduction of a certain base so that the gene is not expressed or expressed, All of which block the biosynthetic pathway involved in the enzyme of the gene of interest.

In the present invention, "attenuated" 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.

In the present invention, "microorganism" includes algae, fungi, protozoa, filamentous fungi, yeast and viruses.

In the present invention, a "vector" may be any nucleic acid molecule having the purpose of transferring a specific gene into a host cell, and generally includes a self-replicating sequence, a genomic insertion sequence, a phage or nucleotide sequence, a linear or circular, Of DNA or RNA. In particular, it may be one having a foreign gene and having a factor that facilitates transformation of a specific host cell in addition to the foreign gene. Vectors generally include sequences that direct transcription and translation of appropriate genes, selectable markers, and sequences that allow autologous replication or chromosome insertion. Specific examples of the vector include, but are not limited to, a plasmid vector, a phage or a cosmid vector.

In the present invention, genes encoding enzymes involved in C5-C8 organic acid biosynthesis were successfully isolated from Megasphaera hexanoica strain (KFCC11466P).

Specifically, as described in the following examples, the entire gene of the megasperahexanoicus strain was decoded (Fig. 1), and genes with high levels of RNA expression among the genes involved in the production of organic acids were selected (Fig. 2 4).

Accordingly, the present invention provides genes encoding enzymes involved in C5-C8 organic acid biosynthesis, including at least one base sequence selected from the group consisting of SEQ ID NOS: 1-8.

The nucleotide sequence of SEQ ID NO: 1 encodes Acetyl-CoA acetyltransferase (THL), the nucleotide sequence of SEQ ID NO: 2 encodes 3-hydroxyacyl-CoA dehydrogenase (HBD) (ACT), the nucleotide sequence of SEQ ID NO: 4 encodes Acyl-CoA dehydrogenase (ACDH), the nucleotide sequence of SEQ ID NO: 5 encodes Acetyl-CoA transferase , The nucleotide sequence of SEQ ID NO: 6 encodes butyryl-CoA dehydrogenase (BCDH), the nucleotide sequence of SEQ ID NO: 7 encodes an electron transfer flavoprotein alpha subunit (ETF A), the nucleotide sequence of SEQ ID NO: transfer flavoprotein beta subunit (ETF B).

Here, the C5 organic acid is pentanoic acid, the C6 organic acid is hexanoic acid, the C7 organic acid is heptanoic acid, and the C8 organic acid is octanoic acid.

As can be seen from the results of the following examples, the vector according to the present invention is characterized in that the vector comprises a first vector comprising the gene represented by SEQ ID NO: 1 which codes for THL or a first vector comprising Beta keto thiolase (BKTB) A first vector comprising a gene represented by SEQ ID NO: 10; And a second vector coding for ACDH and comprising a gene represented by SEQ ID NO: 4.

The second vector comprises a gene of SEQ ID NO: 2 encoding HBD, a gene of SEQ ID NO: 3 encoding CRT, a gene of SEQ ID NO: 5 encoding ACT, a gene of SEQ ID NO: 6 encoding BCDH, More preferably at least one gene selected from the genes of SEQ ID NO: 7, SEQ ID NO: 8 encoding ETF B and SEQ ID NO: 9 encoding TER.

In order to improve the productivity of the C5-C8 organic acid, the microorganism may be a gene encoding an enzyme involved in lactate biosynthesis, a gene encoding an enzyme involved in acetate biosynthesis, It is preferable that one or more genes selected from the group consisting of a gene encoding an enzyme involved and a gene encoding an enzyme involved in succinate biosynthesis are weakened or deleted.

In the following examples, Escherichia coli MG1655 was used as a host microorganism. However, if the same gene to be deleted is deleted by using other Escherichia coli, bacteria, yeast and fungus, and an enzyme gene involved in C5-C8 organic acid biosynthesis is introduced, It will be said that the object of the invention can be achieved.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments and the like. It will be apparent to those skilled in the art, however, that these examples are provided for further illustrating the present invention and that the scope of the present invention is not limited thereto.

1. Strain isolation

To separate microorganisms producing excess hexanoic acid, a gastrointestinal tract containing gastric juice was purchased at a wastewater market located in Majang-dong, Seoul, Korea, and anaerobically treated. The anaerobically treated gastric juice was inoculated into Reinforced Clostridia medium (difco) containing 5 g / L hexanoic acid and incubated at 37 ° C for 3 days. This enrichment culture was subcultured several times in the same medium. The cultures were cultured in Reinforced Clostridia medium (difco) medium to form colonies. Each of the selected colonies was inoculated into a Reinforced Clostridia medium (difco) liquid medium and cultured at 37 ° C for 3 days. Then, hexanoic acid production was measured by gas chromatography (GC). As a result, microorganisms producing the greatest amount of hexanoic acid were isolated and named Megasphaera hexanoica. The strain was deposited on September 30, 2009 at the Korea Microorganism Conservation Center, a microorganism depository organization under the Budapest Treaty, and received the deposit number KFCC11466P.

2. Dielectric analysis

Genome analysis of megasperahexanoicas was performed by GSL FLX454 and Illumina High-Sic equipment, which was commissioned by Chunlab, a specialist in genome analysis, and the complete genetic map was analyzed using CL genomics, a bioinformatic program provided by ChunLab. Respectively.

FIG. 1 shows the entire gene map of megasperahexanoicas obtained by the above process. As a result of the dielectric analysis, a dielectric consisting of one contig was ensured, the dielectric size was 28778511 bp, the G + C ratio was 49%, 2821 cryptic sequences, 18 rRNAs and 53 tRNAs, It can be inferred that the point of change is the origin of replication.

3. Organic acid production-related genes RNA Through expression level analysis C5 - C8 Analysis of organic acid biosynthesis pathway

RNA transcriptomic analysis was performed to analyze genes involved in the production of C5-C8 organic acids and their biosynthetic pathway.

The RNA transcriptome analysis was performed by dividing the hexanoic acid into two conditions, that is, the condition of overproduction of hexanoic acid and the condition of little production. The RNA transcriptomic analysis was performed by dividing the time by 9 hours of initial logarithmic growth and 18 hours of saturation of production, Were compared. RNA extraction was performed using an RNA extraxction kit for cueogen bacteria, and the eluted samples were frozen below -70 ° C.

Samples were analyzed by Chun Lap and the results were analyzed using CLRNAseq, which is provided free from Chun Lap. Four samples were statistically normalized, and the expression levels were compared according to the condition and time, and genetic resources with high expression levels were obtained.

FIG. 2 is a graph showing RNA expression genes when Megasperra hexanoic acid was cultured without addition of acetic acid and butyric acid. FIG. 3 shows the results of analysis of RNA expression genes FIG. 4 is a graph showing RPKM values of genes isolated from megasperahexanoyika strains. FIG. 4 is a graph showing RPKM values. FIG.

Thus, it was confirmed that the genes represented by SEQ ID NOS: 1 to 8 among the genes derived from the megasperahexanoicus strain were the most influential factors for the production of C5-C8 organic acids.

In addition, a hexanoic acid biosynthetic pathway and related enzymes were derived based on the above-described RNA expression levels, and the results are shown in FIG.

4. Production of a vector containing the gene according to the present invention

(One). Preparation of first vector

(1-1) a first vector comprising a gene encoding THL

In order to insert the gene coding for THL into the pCOLA vector, primer sets of SEQ ID NOS: 11 and 12 below were used, NDEI was inserted in front of the primer set, and XHOI restriction enzyme region was inserted in the rear. The PCR fragment amplified by PCR was treated with NDE1 and XHOI, and the pCOLA vector was also treated with NDEI and XHOI, followed by ligation using a ligation kit, Mighty Mix. The ligated plasmid was inserted into DH5 alpha, and colony PCR was performed using the same primer to confirm that the gene was inserted into the vector.

[SEQ ID NO: 11] THL f: 5'-AAAAACATATGAAAAATGTGGTTATTGTGTC-3 '

[SEQ ID NO: 12] THL r: 5'-TTTTTGAGCTCAATCTTAAGTAGTGTGTAAAATTACCG-3 '

(1-2) a first vector comprising a gene encoding BKTB

In order to insert the gene encoding BKTB into the pCOLA vector, primer sets of SEQ ID NOS: 13 and 14 were used, NDEI was inserted in front of the primer set, and XHOI restriction enzyme site was inserted in the rear. The PCR fragments amplified by PCR were treated with NDE1 and XHOI. The pCOLA vector was also treated with NDE1 and XHOI, and then subjected to ligation using a ligation kit, Mighty Mix. The ligated plasmid was inserted into DH5 alpha, and colony PCR was performed using the same primer to confirm that the gene was inserted into the vector.

[SEQ ID NO: 13] BKTB f: 5'-AAAAACATATGACCCGTGAAGTTGTC-3 '

[SEQ ID NO: 14] BKTB r: 5'-TTTTTGAGCTCAATTTAAGCTACGCAAGCTTCTAC-3 '

(2) Production of the second vector

(2-1) Preparation of a vector containing a gene coding for HBD, CRT, ACDH, ACT

First, genes encoding HBD and CRT were attached and inserted into pCDF vector together. Primer sets of SEQ ID NOS: 15 and 16 below were used. ECOROI was inserted in the front of the primer and NOTI restriction enzyme region was inserted in the rear.

[SEQ ID NO: 15] HBD f: 5'-AAAAAGAATTCGATGTTCAAGAAAGTGATGGTCATT-3 '

[SEQ ID NO: 16] CRT r: 5'-TTTTTCGCCGGCGAATAAGCGGAAACTTCAGGCGAA-3 '

The primers set forth in SEQ ID NOS: 17 and 18 below were used to remove the histag in front of the HBD and remove the Ecori region in the constructed vector. At this time, the entire vector was amplified by PCR using Pfux Polymerase (Biopharmaceuticals) and ligated to the blunt end without restriction enzyme treatment.

[SEQ ID NO: 17] HBD2 f: 5'-ATGTTCAAGAAAGTGATGGTCATT-3 '

[SEQ ID NO: 18] PCOLA r: 5'-TACCGACGACGGGTACCATATA-3 '

Then, in order to insert the gene coding for ACT, primer sets of SEQ ID NOs: 19 and 20 shown below were used, and NDE1 and XHOI were used as restriction enzymes.

[SEQ ID NO: 19] ACT f: 5'-AAAAACATATGTATAAACTGTCGCAAATCGCT-3 '

[SEQ ID NO: 20] ACT r: 5'-TTTTTGAGCTCAATCATAAGGCAAAAACTCCAAAAG-3 '

Next, primers set forth in SEQ ID NOs: 21 and 22 were used as a template for the cDNA of Megasupera hexanoic acid, and ACDH was inserted followed by a ligated horse binding site (RBS) and an Ecori restriction enzyme region, and ACDH Were inserted.

[SEQ ID NO: 21] ACDH f: 5'-AAAAACATATGGGTTATATTCTTAACAAAGACCA-3 '

[SEQ ID NO: 22]

ACDH r: 5'-TTTTTGTATACGGAGGACTTAAGAATCACAAAGAAACATTAGACCGG-3 '

(2-2) Preparation of a vector comprising a gene coding for HBD, CRT, ACDH, BCDH, ETF A, ETF B, ACT

First, a gene coding for HBD and CRT was attached and inserted into a pCDF vector in the same manner as in (2-1) above.

Next, genes coding for BCDH, ETF A, ETF B and ACT were attached and inserted together, and the following primer sets of SEQ ID NO: 23 and SEQ ID NO: 20 were used.

[SEQ ID NO: 23] BCDH f: 5'-AAAAACATATGATGGATATCTCTAGAATGGACTTC-3 '

[SEQ ID NO: 20] ACT r: 5'-TTTTTGAGCTCAATCATAAGGCAAAAACTCCAAAAG-3 '

Next, primers set forth in SEQ ID NOS: 21 and 22 were used as a template for megasperahexanoic acid cDNA, ACDH was inserted into the region of the Ribuxial Binding Site (RBS) and Ecori restriction enzyme region, and ACDH The coding gene was inserted.

(2-3) Preparation of a vector containing a gene coding for HBD, CRT, ACDH, ETF A, ETF B, and ACT

After securing the plasmid of (2-2), the ECORI restriction enzyme region inserted at the back of the ACDH and the ECORI restriction enzyme region at the back of BCDH were used to insert the ACDH, followed by ligation with ECORI restriction enzyme.

(2-4) Preparation of a vector containing a gene encoding HBD, CRT, ACDH, TER, ACT

An ACDH fragment was inserted into a vector into which a gene encoding HBD, CRT, TER and ACT of (2-6) was inserted, using a restriction enzyme NDEI site and the primer set of SEQ ID NOS: 21 and 22, respectively.

(2-5) Preparation of a vector containing a gene coding for HBD, CRT, BCDH, ETF A, ETF B, and ACT

Using the same method as in (2-2) above, genes encoding HBD and CRT were attached and inserted into a pCDF vector, and genes coding for BCDH, ETF A, ETF B and ACT were attached and inserted together.

(2-6) Preparation of a vector containing a gene encoding HBD, CRT, TER, ACT

In order to insert the gene coding for TER into the pCDF vector, the primer set of SEQ ID NOs: 24 and 25 shown below was used, NDEI restriction enzyme was used for the front, HINDIII restriction enzyme was used for the rear, and the vector of (2-5) After digesting with NDEI and HINDIII restriction enzymes, the PCR amplified TER fragment was inserted.

[SEQ ID NO: 24] TER f: 5'-AAAAACATATGATTGTTAAACCGATGGTCC-3 '

[SEQ ID NO: 25] TER r: 5'-TTTTTTTCGAAAGTTTACGCTAGTTTCGCAAGGT-3 '

5. Transformed by the vector according to the invention C5 - C8 Production of microorganisms capable of producing organic acids

(1) deletion of the ldh, pta, adhe, and frda genes

In order to further delete the ldhA, pta, adhe and frda genes in Escherichia coli MG1655, the method using the pKDA 208 containing the recombinant pKM 208 plasmid containing the primers of SEQ ID Nos. 26 to 33 and the pKDA having the kanamycin resistance gene was used. ldhA (a gene encoding lactate dehydrogenase), pta (a gene encoding phosphotransacetylase), adhe (a gene encoding alcohol dehydrogenase), and the frda gene.

[SEQ ID NO: 26] ldhA1stup: 5'-AAATATTTTTAGTAGCTTAAATGTGATTCAACATCACTGGAG AAAGTCTTGTGTAGGCTGGAGCTGCTTC-3 '

[SEQ ID NO: 27] ldhA1stdo: 5'-ATTGGGGATTATCTGAATCAGCTCCCCTGGGTTGCAGGGGAG CGGCAAGATCCTCCTTAGTTCCTATTCC-3 '

[SEQ ID NO: 28] FRDA f: 5'-CTTACCCTGAAGTACGGGGCTGTGGGATAAAACAATCTGGAGGA ATGTCGTGTAGGCTGGAGCTGCTTC-3 '

[SEQ ID NO: 29] FRDA r: 5'-TATCGACTTCCGGGTTATAGCGCACCACCTCAATTTTCAGGTTTT TCATCTCCTCCTTAGTTCCTATTCC-3 '

[SEQ ID NO: 30] PTA f: 5'-GGTGCTGTTTTGTAACCCGCCAAATCGGCGGTAACGAAAGAGGAT AAACCGTGTAGGCTGGAGCTGCTTC-3 '

[SEQ ID NO: 31] PTA r: 5'-TTATTTCCGGTTCAGATATCCGCAGCGCAAAGCTGCGGATGATGA CGAGAATATCCTCCTTAGTTCCTATTCC-3 '

[SEQ ID NO: 32] ADHE f: 5'-ATTCGAGCAGATGATTTACTAAAAAAGTTTAACATTATCAGGAGA GCATTGTGTAGGCTGGAGCTGCTTC-3 '

[SEQ ID NO: 33] ADHE r: 5'-AAAAAACGGCCCCAGAAGGGGCCGTTTATATTGCCAGACAGCGCT ACTGATCCTCCTTAGTTCCTATTCC-3 '

(2) C5 - C8 Production of microorganisms capable of producing organic acids

(1-1) vector and (2-1) vector (Example 1), (1-1) vector and (2-1) vector in which the above-mentioned (1-1) vector and (2-1) vector are introduced into the deleted Escherichia coli MG1655 of the ldh, pta, -2) (Example 2), the microorganisms (Example 3) in which the vectors of (1-1) and (2-3) were introduced, the vectors (1-1) (Example 4), the microorganism (Example 5) into which the above (1-2) vector and (2-4) vector were introduced, the vector of (1-1) (Comparative Example 1), and the microorganism (Comparative Example 2) in which the vector of (1-1) and the vector of (2-6) were introduced.

6. Hexanoic acid Production comparison experiment

(1) The microorganism produced according to the above 5 (2) was cultured to carry out a comparative experiment on the production of hexanoic acid.

The medium was cultured at 30 ° C using LB medium containing 1.5 g / L of butyrate and 20 g / L of glucose and induced when the cell growth was between 0.6 and 0.8 with 1 mM IPTG.

As a result, it was confirmed that the production amount of hexanoic acid of the transformed microorganisms according to Examples 1 to 5 was much higher than that of Comparative Examples 1 to 2. [ Unlike Comparative Examples 1 and 2, in all of Examples 1 to 5, genes coding for ACDH were included. Thus, it was confirmed that genes coding for ACDH were the most important factors affecting production of hexanoic acid. In addition, it was confirmed that a gene coding for THL, HBD, CRT, ACT, BCDH, ETF A, ETF B and ACT is a factor contributing to the production of hexanoic acid (FIG. 14).

(2) As described above, it was confirmed that the gene coding for ACDH had the greatest influence on the production of hexanoic acid. As a result, the production of hexanoic acid of the transformed microorganism was confirmed according to Example 1.

The culture medium was TB medium supplemented with nitrogen source and 20 g / L of glucose and 5 g / L of butyrate were added and cultured at 30 ℃. Cell growth was induced with 1 mM IPTG when the cell growth was between 0.6 and 0.8. The pH was adjusted by adding W / V 5% CaCO3 with induction. Sampling was carried out at intervals of 12 hours using a sterilized syringe.

As a result, it was confirmed that the transformed microorganism according to Example 1 produced up to a maximum of XX g / L of hexanoic acid over time. As a result, the microorganism into which the genes according to the present invention were introduced had excellent organic acid production ability (Fig. 15).

Name of depository: Korea Microorganism Conservation Center (Domestic)

Accession number: KFCC11466P

Date of deposit: 20090930

<110> Industry-University Cooperation Foundation Hanyang University <120> Novel genes involved in the production of C5-C8 organic acid, vector comprising the genes, microorganism transformed with the vector, and method for producing C5-C8 organic acid using the 마이크로organism <130> JKP-0050 <160> 33 <170> KoPatentin 3.0 <210> 1 <211> 1194 <212> DNA <213> Unknown <220> <223> Megasphaera hexanoica <220> <221> gene <222> (1) (1194) <223> gene coding Acetyl-CoA acetyltransferase <400> 1 cgtactccta ttcaaagacg taacagctgt ccagctcggt atcgttgctg ttaaagctgc gatcgaacgt 120 gctaaagttc ctgtagatca gattgatgaa gttatcatgg gtcatgttct gacagctggc 180 tgcggcgaaa acacagcccg ccaggtagct ctccattccg gtattcctca ggaagttcct 240 gcatttacta tcaataaact gtgcgggtcc ggccttcgtg ctgtatccct cggcgctcag 300 cagattgaac tcggcgatgc tgactgcgta atcgtcggcg gtatggaatc catgtccaac 360 gctccgtatg ttatcgcaaa agcacgccgt ggctatcgta tgggcaatgg tgtcctcgaa 420 gatacgatgc tccgtgatgg cctggtttgc acagaaaatg gctaccacat gggcgtaacg 480 gcagaaaaca tcgcatcccg ctttggcgta acccgtcagc agcaggatga atgcgcttac 540 aattcccaga tgcgtgctgc caaagcccag gctgaaggca aattcgatgc tcagatcgct 600 cctgtaacga tccacaaccg caaaaaaggc gatatcgtca ttacgaagga tgaacatatc 660 cgtccggaaa ccacactgga aggcctcgct aaattaaaac cggctttcac aaaagacggc 720 acagttacag ctggtaacgc ttccggtatc aacgatgctg cttgcgcact ggttctcatg 780 agcaagaaaa aagccgaaga actcggtatc aaaccgattg ctgaaatcct cgactgggct 840 tcagcaggtg ttgaaccggc tatcatgggt actggtccta tcccggcctg ccataaactg 900 ttcaagaaaa ccggcctcaa aatggaagac ttcgaactgg ttgaattgaa cgaagctttc 960 gcagctcagg ctgtatattg ctgccagcag ctcggcgctg acatgagcaa aacaaacatc 1020 tatggttccg gtatctccct cggtcacccc gtaggctgct ccggtgcccg tatcctcacg 1080 actctgctct atgcattggc agaaccgggc cgcaatggtt cccgttatgg cctcgcttcc 1140 ctgtgcatcg gcggtggcca gggtacagcc gtagcaatta aaatgtgcga ctaa 1194 <210> 2 <211> 858 <212> DNA <213> Unknown <220> <223> Megasphaera hexanoica <220> <221> gene &Lt; 222 > (1) .. (858) <223> gene coding 3-hydroxyacyl-CoA dehydrogenase <400> 2 atgttcaaaa aagtcatggt tattggtgca ggtacgatgg gttccggtat tgctcaggta 60 tttgctgaac acggtgtaga tgttatcctg aacgatatca aacaggaatt catcgatggc 120 ggcatgaaaa aaatcgacaa acagctgacc agaaaagtta cgaaaggtaa aatgacggaa 180 gaccagaaag ctgaaatcat gggccgtctg accggtatcg tagaaatcac ggaagacaac 240 atgaaagatg ttgaactcgt tgtcgaagct gctatcgaag acgaaaagat caaatgcggc 300 atcttcaaga acctcgatga aaaatgcccg gctaatacaa tcctcgcttc caatacgtct 360 tccctgccta tcaccaagat tgctacggct acgaaacgtc cggaaaaagt tatcggcatg 420 cacttcttca atccggctcc ggtcatgaaa ctggtcgaaa tcatcaaagg tatcgcaact 480 gcgatgcta cgacgaaagc cgttgtagaa tgcgctgaag gccttggcaa atcccctgtt 540 gaagttgaag acttcccggg cttcgctgct aaccgtgtcg tcgttccgat gctgaatgaa 600 gcatgctatg ccctcatgga aggcgttgct tccaaagaag gtatcgacgc tgtctgcaaa 660 ctcggctaca accatccgat gggaccgctc gaactgtgcg acctgattgg taacgatgtc 720 gttctccacg ttatggaagt tctctatgaa ggctttggcg atccgaaata tcgtccgtgc 780 ccgctcctcc gtaaatacgt tgctgccggc tggctcggcc gcaagacggg caaaggcttc 840 tacgattaca gcaaataa 858 <210> 3 <211> 783 <212> DNA <213> Unknown <220> <223> Megasphaera hexanoica <220> <221> gene &Lt; 222 > (1) .. (783) <223> gene coding 3-hydroxyacyl-CoA dehydratase <400> 3 atggaatttg aaaacattct cttcaccgta gaagaaggta ttgctacaat tacgatcaac 60 cgtccgaaac aggctaacgc cctgaacgcc gctacggtcc gtgatattga aaaagccgtt 120 gactacattg ccggctcgaa agacgtacag gtagtcatca ttaccggtgc tggcgacaaa 180 ttcttcgttg ctggtgcaga tatcaaagaa atggctgatt acaacccgaa acaggctggt 240 gactggggca catatggtgc tggcgtcatc accaagattg aaagactgcc acagccggtt 300 atctgcgctg taaacggcta tgctctgggt ggcggctgcg aaatctccat ggcctgcgat 360 ttccgttatg cttcggataa cgccgtattc ggccagccgg aagtcggctt gggcatcatc 420 cccggcttcg gtggcacaca gcgtcttgca cgcctcgtag gtcctggcat ggcaaaagaa 480 atcatcctga gcaaccagaa tattgattcg gctgaagctc tccgcatcgg cctggtcaac 540 aaagtcgttc ctcaggctga attaatgccg gctgccatca agaccgctaa gaaaatcatg 600 aaacagggtc ctgtagcagt acagatcgct aagaaagcta tcaacaacgg cttacagtgc 660 gacatcatta caggtatcca gttcgaaagc aatgtcttcg gcctctgctt tgctacaaaa 720 gatcagaagg aaggcatgaa agcgttcatg gaaaaacgta aagccgattt caaaggcgaa 780 taa 783 <210> 4 <211> 1209 <212> DNA <213> Unknown <220> <223> Megasphaera hexanoica <220> <221> gene &Lt; 222 > (1) <223> gene coding Acyl-CoA dehydrogenase <400> 4 atgggttata ttcttaacaa agaccagatg gacatcgtta atttggcaaa agactatgca 60 gtaaagaaag ttatgccgct cgtaccggaa ctggacaacg ctccggaact gacaccggaa 120 acagaagaag gcaaaaaact tctcgacctc tatcagggcg ctgttgacct tggcctgacg 180 actctggaaa ttccggaaga atacggcggc cttggccagg actactacac cgtagccgct 240 gcatacgaag aattgtccaa agttgatgct ggctttgcta cagcagtagc cgcttcttcc 300 ctcggcctga aaccgatcct ccagcacggc aatgacgaac agaaaaaact ctatgccgat 360 ttcctcaacg gcgaaaagac gtctgaaaaa gctccggtac cgggcttctg cgcattctgc 420 ttaaccgaac cggatgctgg ttctgatgct tccaacagca agacgacagc taaaaaagtt 480 ggtgacgaat atattatcga cggcacgaaa tgcttcatca ccaacggcgg cgttgcttcc 540 gtttacaccg tattcgctgt aacggataag acgaagggcg taaaaggcat ctctgctttc 600 atcgttgaac gtgaccgcga aggcgtttcc attggcgctg aagaaaacaa gatgggcatc 660 cgcctctcca acacgacaga agttatcttc caggacgttc atatcccggc tgatcacctc 720 gtaggcgaag aaggcaaagg cttcatctat gctatgcaga cactggacct cgcccgtcct 780 atgattggtg ccctggcagt tggtattgct cagcgcggta tcgacgaagc tgttaaatat 840 gctcaggttc gtcagacatt tggcaagccg atcatcaaac acgaagctat cgctttcaag 900 ctggctgata tggatatcca gacagaagtt ggccgttcct ccatcatcaa cttcctcaac 960 aaatactatg acgaaagaaa gaatggccgt tacaactact cccgtgaagc tgctatctgc 1020 aaatgcttct gcggcgatat gtgcgtacag gtagctctcg atgctatcca gatcctcggc 1080 ggtttcggct acagccgtga atatccggtt gaaaaactcg ttcgtgatgc taaaatcatg 1140 cagatctatg aaggcaccaa cgaagtacag agaatcgtta tctccggcca gattacaaag 1200 aaacactaa 1209 <210> 5 <211> 1359 <212> DNA <213> Unknown <220> <223> Megasphaera hexanoica <220> <221> gene &Lt; 222 > (1) .. (1359) <223> gene coding Acetyl-CoA transferase <400> 5 atgtacaaac tttcacaaat tgcagaagag taccaaaaga aactcgtgac gccgcaggaa 60 gcggcagctg tcgtcaaaag cggagaccgt gtatcgtacg gccttggctg ttcggcaccg 120 tatgatacgg ataaagcgct ggccgaccat atcaataagg atggcttgaa agatgtggaa 180 atcatcgatg cgacgctgat tcaggatcat ccgttcttta cctatacgga aacggaatcc 240 aacgatcagg tccgtttcgt atcgggccat ttcaatggat tcgaccgcaa gatgaataaa 300 gccggccgct gctggttcat gccgctcctc tttaatgaac tgccgaaata ctggagccat 360 aaaaaagtgg atgtcgccat tttccaggta catccgatgg ataaatgggg caacttcaat 420 ctggggcctc aggtagccga tttaaggggc attctcaaat cggcggataa ggtcatcgta 480 gaagtcaatc agaaaatgcc gaaggcactg ggctatgaaa cggaattgaa tattgccgat 540 gtcgatttca tcgtcgaagg gtccaatccg gatatgccga ttgtcccgaa taaaccgtcg 600 accccggtcg atgataaaat tgccagcttc gtcgtaccga tgatcaaaga tggcagtacg 660 ctgcagctcg gtatcggcgg gattccgtcc gctattggcc ataagctggc agaatcggat 720 gtcaaagacc tgagcggtca tacggaaatg ctggtagatc cctatgtcga attgtacgaa 780 gccgggaaaa ttaccggtaa aaagaatcgc gacagaggta aaatcatgta taccttcgcc 840 ggtggcacac agcgtctgta tgattttatc gatgacaacc agatcgtgtt caatgcaccg 900 gtcaactatg tcaacaatat caatgtcgtt gccagcattg acaactttgt ttctatcaac 960 agctgcatca accttgacct ttatggtcag gtctgcgccg aatcggcagg ttaccgccat 1020 atcagtggta ctggcggtgc gctggacttc gcacagggag cctatctttc cgaaggtggc 1080 cagggcttca tctgcgtcca ttcgacgcgt aaactgaaag atggcagcct ggaatcgctg 1140 atccgcccaa ctctgactcc gggatctgtc gtcacgacac cgcgttcggc cgttcactac 1200 atcgttaccg aatacggcgt agccctcctc aaaggccagt ccacatggca gcgggcagaa 1260 gcgctgatta atattgctca tcctgatttc cgggaagaac tcattaaaga agcagaaaaa 1320 atgggcatct ggacgaagac gagcaagaca gaatactga 1359 <210> 6 <211> 1152 <212> DNA <213> Unknown <220> <223> Megasphaera hexanoica <220> <221> gene &Lt; 222 > (1) <223> gene coding Butyryl-CoA dehydrogenase <400> 6 atggatttta aattgaatga tctgcagaaa gacatcgtta aaaccgtcca tgattattgt 60 gaaaagaaaa taaaaccgga tatcggcgaa cgcgatcacg caggtgattt tggcgaagaa 120 catgtaaaag ctctgatgca ggacatgggt attgccggta tttactatcc tgaagaatac 180 ggtggaatgg gcaatgacgg cggcgatgta ctgacgtata tcctctgcgt ggaagaaatt 240 gccaaatatg atgccggtat ggcagcaacc gtttccgctt ccatttccct cggtaccaac 300 cccatctggc agtatggtac ggaagaacag aagaaaaaat atctcgaacc gctcgtaacg 360 ggtgaaaaac tcggtgcgtt ctgcctgacc gaaccgggtg ccggcaccga cgctgctatg 420 cagcagacga cggctgtcaa ggatggcgat cattacatcc tcaacggcaa caagattttc 480 atcaccaacg gcggtaaggc cgatacgtac atcgtatttg ccatgacgga taaatccaaa 540 ggcacgaaag gcatttccgc ctttatcgtt gaaaaaggct gggaaggctt cacctttggc 600 aagaaagaag acaaactggg catccatacg tcccagacta tggaactcat cttccaggat 660 gtcaaagttc cggcagaaaa cctgctcggt gaagaaggaa aaggcttcat catcgctatg 720 cagaccctcg acggcggccg tatcggcatc gctgcccagg ctctcggcat cgctgaatct 780 gcccttgacg acgctgtcgc ctattcgaaa gaacgtgtac agttcggtcg tccgctctgc 840 aaattccaga acgtttcctt caaactggct gatatgaaga tgaagatcga agcagcccgt 900 ctcctggttt acaaagcagc cgaagccaaa caggccggcg gccgtttctc tctggaagct 960 gccattgcca agagaatggc ttccgatatc gctatggaag ttacgacaga agctgtccag 1020 atctttggcg gctatggcta caccgaagat taccccgttg cccgtcatat gcgcgacgcc 1080 aagattacgc agatctatga aggcaccaac gaagtacagc tcatggttac atcgggcttc 1140 ctgctcaaat aa 1152 <210> 7 <211> 1008 <212> DNA <213> Unknown <220> <223> Megasphaera hexanoica <220> <221> gene &Lt; 222 > (1) .. (1008) <223> gene coding Electron transfer flavoprotein alpha subunit <400> 7 atggatttag cagaatacaa aggcatttat gtcattgctg aacaattcga aggcaaattg 60 cgcaacgtat ctttcgaact gttgggtcag gcccgcgttt tggctgacac catcggtgac 120 gaagttggcg ctatcttgat tggtaaagat gtaaaaccgc tggcacagga actgattgcc 180 cacggtgcac ataaagtata tgtatatgac gatccgaaac tggaaaacta caacacgacg 240 gcatatgcaa aagtcatttg cgatttcttc catgaagaaa aaccgaatgt attcctcgta 300 ggcgctacga acatcggccg tgacctcggc ccccgtattg ccaactccct ccagaccggc 360 cttacggctg actgcacggg cctggctgtt gacgacgacg gcaagaccat cgtttggact 420 cgtccggctc tcggcggcaa catcatggct gaaattattt gcccggacaa ccgcccgcag 480 atgggtaccg ttcgcccgaa cgtattcaaa aaaccggaag ctgatcctaa tgccaaaggt 540 gaagttatcg aaaagactgc taacctcagc gacgctgatt tcatgaccaa attcgttgaa 600 ctcatcaaaa tgggcggcga aggcatcaaa atcgaagaag ctgacgttat cgtatccggc 660 ggccgtggca tgaatggtcc tgaaccgttc actggcatgc tgaaagacct cgctgacgtt 720 ctcggcggtg ctgtaggcgc ttcccgtgct gctgttgacg ctggctggat cgatgctctc 780 catcaggtag gccagacagg taagacggta ggtccgaaaa tctacattgc ctgcggtatt 840 tccggcgcta tccagcatct cgctggcatg agtggttctg actgcgttat cgccatcaac 900 aaagatgaag atgctcctat cttcaaagtt tgcgattatg gtatcgtagg cgacgcattc 960 aagatcgttc cgctcctgac tgctgctatc aaaaaaatca aaggctaa 1008 <210> 8 <211> 816 <212> DNA <213> Unknown <220> <223> Megasphaera hexanoica <220> <221> gene <222> (1). (816) <223> gene coding Electron transfer flavoprotein beta subunit <400> 8 atggaaatat tggtatgtgt caaacaggtt ccggacacag cagaagtaaa aattgatccc 60 gtaaaacata ccgtcattcg tgccggtgta ccgaacatct tcaatccgtt cgaccagaac 120 gctttggaag ctgctcttca gttaaaagat aaccagggcg caaaggtaac tttgctctcc 180 atggggcccc cgcaggctaa agacgttctc cgtgaaggtc tggctatggg cgctgatgaa 240 gcatacctcc tcacagaccg caaagtcggc ggttccgata cgctggctac aggctattgc 300 ctggctcagg ctgttaaaaa agtagctgaa ctcaaaggca tcgaacagtt cgatatcatc 360 ctttgcggca aacaggctat tgatggtgat acggcacagg tcggcccgca gatcgcctgc 420 gaactcggca tcccgcagat tacctatgca gcagctatcg aagtaaacga agaagctaaa 480 tgcgtaaaag taaaacagca gaacgaagaa ggctacatca ttacggaagc taacttccct 540 gtcctgatta cggctgttaa agaattgaac gaaccgcgct tcccgaccat ccgtggcacg 600 atgaaagcaa aacgtcgcga aattcctgaa ctctccgctg atgacgttaa agctgacgaa 660 accaaaatcg gcctgagcgg ctccccgacg aaagttcgta agattttcac accgcctcag 720 agaacacagg gcctcatcat tcctgttgaa gatgacaacg accaggcagc tgttgatacc 780 ttgatggaaa aactgactgc tcagaaaatc atttaa 816 <210> 9 <211> 1194 <212> DNA <213> Unknown <220> <223> unknown <220> <221> gene <222> (1) (1194) <223> gene coding trans-2-enoyl-CoA reductase <400> 9 atgattgtta aaccgatggt ccgtaataat atctgtctga atgctcaccc gcaaggctgt 60 aagaaaggcg tggaagacca aattgaatat accaaaaaac gtattacggc agaagtgaaa 120 gccggcgcaa aagctccgaa aaacgtgctg gttctgggtt gcagcaatgg ctatggtctg 180 gcttctcgca ttaccgccgc gtttggctac ggtgcagcta cgatcggcgt tagtttcgaa 240 aaagcaggtt ccgaaaccaa atatggcacg ccgggttggt acaacaatct ggcttttgat 300 gaagcggcca aacgtgaagg cctgtatagt gtcaccattg atggtgacgc gttctccgat 360 gaaattaaag cacaggtgat cgaagaagcg aaaaagaaag gcattaaatt tgacctgatc 420 gtttacagcc tggcatctcc ggtccgtacc gatccggaca cgggtatcat gcataaatct 480 gtgctgaaac cgtttggcaa aaccttcacg ggtaaaaccg ttgatccgtt cacgggcgaa 540 ctgaaagaaa ttagcgcgga accggccaac gatgaagaag cagctgcgac cgtcaaagtg 600 atgggcggtg aagactggga acgttggatc aaacagctga gtaaagaagg cctgctggaa 660 gaaggttgca ttaccctggc gtattcctac atcggcccgg aagcaaccca agctctgtat 720 cgcaaaggca cgattggtaa agcgaaagaa catctggaag cgaccgccca ccgtctgaac 780 aaagaaaatc cgtcaatccg cgccttcgtt tcggtcaata aaggtctggt tacccgtgca 840 tcagctgtga ttccggttat cccgctgtac ctggcatcgc tgtttaaagt catgaaagaa 900 aaaggcaacc atgaaggttg tattgaacag atcacccgcc tgtatgccga acgtctgtac 960 cgcaaagatg gtacgattcc ggtggacgaa gaaaatcgta ttcgcatcga tgactgggaa 1020 ctggaagaag atgtccaaaa agccgtgagc gccctgatgg aaaaagttac cggcgaaaac 1080 gcggaatctc tgacggatct ggccggttat cgtcacgact ttctggcgag taatggtttt 1140 gatgttgaag gcattaacta cgaagcagaa gtggaacgct ttgatcgcat ttga 1194 <210> 10 <211> 1190 <212> DNA <213> Ralstonia eutropha <220> <221> gene &Lt; 222 > (1) .. (1190) <223> gene coding beta keto thiolase <400> 10 atgacccgtg aagttgtcgt tgtctcaggc gttcgcaccg caatcggcac ctttggcggc 60 tcgctgaagg atgtggcacc ggcagaactg ggcgcgctgg ttgtgcgtga agcactggct 120 cgcgcgcagg tgtctggcga tgacgttggt catgtcgtgt ttggcaacgt gattcaaacc 180 gaaccgcgtg atatgtatct gggtcgtgtt gcagcagtca acggtggtgt gaccatcaat 240 gccccggcac tgacggttaa tcgtctgtgc ggcagcggtc tgcaggcgat tgtgtctgca 300 gctcaaacca tcctgctggg cgatacggac gttgcgattg gcggtggcgc agaaagcatg 360 tctcgtgcac cgtacctggc accggcagca cgttggggtg cacgcatggg cgatgctggt 420 ctggtggaca tgatgctggg cgcgctgcat gatccgtttc atcgtattca catgggtgtc 480 accgctgaaa acgtggcgaa agaatatgat atctcacgcg cccagcaaga cgaagcagct 540 ctggaatcac accgtcgcgc atcggcggcc attaaagcgg gctactttaa ggatcagatc 600 gtgccggttg tctcgaaagg ccgtaagggt gatgttacct tcgatacgga cgaacatgtc 660 cgtcacgacg cgaccattga tgacatgacg aaactgcgcc cggtcttcgt gaaggaaaat 720 ggcaccgtta cggctggcaa cgcgagtggt ctgaatgatg cagctgcggc cgtggttatg 780 atggaacgtg ctgaagcgga acgtcgcggt ctgaaaccgc tggcacgtct ggtgtcctat 840 ggccacgcag gtgttgaccc gaaagcaatg ggcattggtc cggtgccggc caccaagatc 900 gcactggaac gtgctggtct gcaggtttct gatctggacg tcattgaagc caacgaagca 960 tttgcagctc aggcctgcgc agtcacgaaa gcgctgggcc tggatccggc caaggttaac 1020 ccgaatggca gtggtatttc cctgggtcat ccgatcggcg ctaccggtgc gctgatcacg 1080 gttaaagcac tgcacgaact gaatcgtgtc cagggtcgtt acgcactggt gaccatgtgt 1140 attggtggcg gtcaaggtat tgcggccatc ttcgaacgca tcgaatttaa 1190 <210> 11 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> THL forward primer <220> <221> primer_bind <222> (1) (31) <400> 11 aaaaacatat gaaaaatgtg gttattgtgt c 31 <210> 12 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> THL reverse primer <220> <221> primer_bind <222> (1) (38) <400> 12 tttttgagct caatcttaag tagtgtgtaa aattaccg 38 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> BKTB forward primer <220> <221> primer_bind <222> (1) (26) <400> 13 aaaaacatat gacccgtgaa gttgtc 26 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> BKTB reverse primer <220> <221> primer_bind <222> (1) <400> 14 tttttgagct caatttaagc tacgcaagct tctac 35 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> HBD forward primer <220> <221> primer_bind <222> (1) (36) <400> 15 aaaaagaatt cgatgttcaa gaaagtgatg gtcatt 36 <210> 16 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> CRT reverse primer <220> <221> primer_bind <222> (1) (36) <400> 16 tttttcgccg gcgaataagc ggaaacttca ggcgaa 36 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> HBD2 forward primer <220> <221> primer_bind <222> (1) <400> 17 atgttcaaga aagtgatggt catt 24 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> pCOLA reverse primer <220> <221> primer_bind <222> (1) (22) <400> 18 taccgacgac gggtaccata ta 22 <210> 19 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> ACT forward primer <220> <221> primer_bind <222> (1) (32) <400> 19 aaaaacatat gtataaactg tcgcaaatcg ct 32 <210> 20 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> ACT reverse primer <220> <221> primer_bind <222> (1) (36) <400> 20 tttttgagct caatcataag gcaaaaactc caaaag 36 <210> 21 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> ACDH forward primer <220> <221> primer_bind &Lt; 222 > (1) <400> 21 aaaaacatat gggttatatt cttaacaaag acca 34 <210> 22 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> ACDH reverse primer <220> <221> primer_bind <222> (1) (47) <400> 22 tttttgtata cggaggactt aagaatcaca aagaaacatt agaccgg 47 <210> 23 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> BCDH forward primer <220> <221> primer_bind <222> (1) <400> 23 aaaaacatat gatggatatc tctagaatgg acttc 35 <210> 24 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> TER forward primer <220> <221> primer_bind &Lt; 222 > (1) <400> 24 aaaaacatat gattgttaaa ccgatggtcc 30 <210> 25 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> TER reverse primer <220> <221> primer_bind &Lt; 222 > (1) <400> 25 tttttttcga aagtttacgc tagtttcgca aggt 34 <210> 26 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> ldhA forward primer <220> <221> primer_bind <222> (1) (70) <400> 26 aaatattttt agtagcttaa atgtgattca acatcactgg agaaagtctt gtgtaggctg 60 gagctgcttc 70 <210> 27 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> ldhA reverse primer <220> <221> primer_bind <222> (1) (70) <400> 27 attggggatt atctgaatca gctcccctgg gttgcagggg agcggcaaga tcctccttag 60 ttcctattcc 70 <210> 28 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> FRDA forward primer <220> <221> primer_bind <222> (1) (70) <400> 28 cttaccctga agtacggggc tgtgggataa aaacaatctg gaggaatgtc gtgtaggctg 60 gagctgcttc 70 <210> 29 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> FRDA reverse primer <220> <221> primer_bind <222> (1) (70) <400> 29 tatcgacttc cgggttatag cgcaccacct caattttcag gtttttcatc tcctccttag 60 ttcctattcc 70 <210> 30 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> PTA forward primer <220> <221> primer_bind <222> (1) (70) <400> 30 ggtgctgttt tgtaacccgc caaatcggcg gtaacgaaag aggataaacc gtgtaggctg 60 gagctgcttc 70 <210> 31 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> PTA reverse primer <220> <221> primer_bind <222> (1) (73) <400> 31 ttatttccgg ttcagatatc cgcagcgcaa agctgcggat gatgacgaga atatcctcct 60 tagttcctat tcc 73 <210> 32 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> ADHE forward primer <220> <221> primer_bind <222> (1) (70) <400> 32 attcgagcag atgatttact aaaaaagttt aacattatca ggagagcatt gtgtaggctg 60 gagctgcttc 70 <210> 33 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> ADHE reverse primer <220> <221> primer_bind <222> (1) (70) <400> 33 aaaaaacggc cccagaaggg gccgtttata ttgccagaca gcgctactga tcctccttag 60 ttcctattcc 70

Claims

A gene encoding an enzyme involved in C5-C8 organic acid biosynthesis, comprising at least one base sequence selected from the group consisting of SEQ ID NOS: 1 to 8;

The method according to claim 1,
Wherein the nucleotide sequence of SEQ ID NO: 1 encodes Acetyl-CoA acetyltransferase (THL), the nucleotide sequence of SEQ ID NO: 2 encodes 3-hydroxyacyl-CoA dehydrogenase (HBD) (ACT), the nucleotide sequence of SEQ ID NO: 4 encodes Acetyl-CoA dehydrogenase (ACDH), the nucleotide sequence of SEQ ID NO: 5 encodes Acetyl-CoA transferase The nucleotide sequence of SEQ ID NO: 6 encodes butyryl-CoA dehydrogenase (BCDH), the nucleotide sequence of SEQ ID NO: 7 encodes an electron transfer flavoprotein alpha subunit (ETF A), the nucleotide sequence of SEQ ID NO: 8 encodes an electron transfer flavoprotein beta subunit (ETF B). < RTI ID = 0.0 > 8. < / RTI >

The method according to claim 1,
Wherein the gene is derived from a strain of Megasphaera hexanoica (KFCC11466P). The gene encoding an enzyme involved in C5-C8 organic acid biosynthesis.

The method according to claim 1,
Wherein the C5 organic acid is pentanoic acid, the C6 organic acid is hexanoic acid, the C7 organic acid is heptanoic acid, and the C8 organic acid is octanoic acid.

A vector comprising the gene according to claim 1.

6. The method of claim 5,
Said vector comprising a first vector comprising a gene of SEQ ID NO: 1 or SEQ ID NO: 10; And a second vector comprising a gene represented by SEQ ID NO: 4.

The method according to claim 6,
Wherein the second vector further comprises at least one gene selected from the genes represented by SEQ ID NOS: 2 to 3 and SEQ ID NOS: 5 to 9.

A microorganism having the ability to produce C5-C8 organic acids transformed by the vector according to claim 5.

9. The method of claim 8,
Wherein the microorganism is selected from the group consisting of bacteria, yeast and mold.

10. The method of claim 9,
The microorganism includes a gene encoding an enzyme involved in lactate biosynthesis, a gene encoding an enzyme involved in acetate biosynthesis, a gene encoding an enzyme involved in ethanol biosynthesis, and a succinate ) A gene coding for an enzyme involved in biosynthesis is weakened or deleted in the microorganism.

A method for producing a C5-C8 organic acid by culturing the microorganism of any one of claims 8 to 10.