KR101990212B1 - A method for selectively producing C3-C8 alcohol using acyl CoA transferase from Megasphaera hexanoica - Google Patents

Abstract

The present invention relates to a novel gene encoding an Acyl CoA transferase involved in C3-C8 alcohol production, a vector comprising the gene and a gene encoding an alcohol dehydrogenase, a microorganism transformed with the vector, and C3-C8 alcohol And a method for selectively producing the same.
According to the present invention, by providing a novel gene encoding an Acyl CoA transferase involved in the production of C3-C8 alcohol, it is possible to produce C3-C8 alcohol with improved production yield and selectively.

Description

A method for selectively producing C3-C8 alcohol using Acyl CoA transferase derived from megasparrahexanoic acid {A method for selectively producing C3-C8 alcohol using acyl CoA transferase from Megasphaera hexanoica}

The present invention relates to a novel gene encoding an Acyl CoA transferase involved in C3-C8 alcohol production, a vector comprising the gene and a gene encoding an alcohol dehydrogenase, a microorganism transformed with the vector, and C3-C8 alcohol And a method for selectively producing 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, bioenergy, solar energy, wind power, intelligence, and tidal power generation are emerging as alternative energy sources. Among them, bioenergy is highly utilized as transportation fuel and less than 30% Due to its ability to reduce to as much as 90%, it is attracting worldwide attention. 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 biofuels such as Clostridium sp. Have been reported. However, these strains have limitations in producing high-carbon alcohol and can not selectively produce alcohols having various carbon numbers .

Disclosure of the Invention The present invention has been conceived in order to solve the above problems. In the present invention, a novel gene encoding Acyl CoA transferase involved in C3-C8 alcohol production, a vector containing the gene and a gene encoding Alcohol Dehydrogenase, And a method for selectively producing a C3-C8 alcohol using the microorganism.

In order to solve the above problems,

A gene encoding an enzyme involved in biosynthesis of a C3-C8 alcohol including any one of the nucleotide sequences selected from the group consisting of SEQ ID NOS: 1 to 8 is provided.

According to the present invention, the nucleotide sequence of SEQ ID NOS: 1 to 8 may be one encoding an Acyl CoA transferase (ACT) enzyme.

According to the present invention, the gene may be derived from Megasphaera hexanoica strain (KCCM11835P).

According to the present invention, the C3 alcohol is propanol, the C4 alcohol is butanol, the C5 alcohol is pentanol, the C6 alcohol is hexanol, the C7 alcohol is heptanol heptanol), and the C8 alcohol may be octanol.

The present invention also relates to a gene encoding an enzyme involved in biosynthesis of a C3-C8 alcohol comprising any one of the nucleotide sequences selected from the group consisting of SEQ ID NOS: 1 to 8; And a gene encoding an alcohol dehydrogenase (ADH) enzyme.

According to the present invention, the gene encoding the Alcohol Dehydrogenase (ADH) enzyme may include any one of the nucleotide sequences selected from the group consisting of SEQ ID NOS: 9 to 11.

According to the present invention, the gene represented by the nucleotide sequence of SEQ ID NO: 9 encodes Aldehyde-alcohol dehydrogenase (AdhE), the gene represented by the nucleotide sequence of SEQ ID NO: 10 encodes Aldehyde / alcohol dehydrogenase (AdhE2) , And the gene represented by the nucleotide sequence of SEQ ID NO: 11 may be one coding for Propionaldehyde dehydrogenase-Alcohol dehydrogenase (PduP-AdhA).

In addition, the present invention provides a microorganism transformed by the above-mentioned vector and capable of producing C3-C8 alcohol.

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

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 C3-C8 alcohol by culturing the microorganism.

According to the present invention, at least one of C3 to C8 organic acids may be added during the microbial culture.

According to the present invention, by providing a novel gene encoding an Acyl CoA transferase involved in the production of C3-C8 alcohol, it is possible to produce C3-C8 alcohol with improved production yield and selectively.

1 is a diagram showing a vector including a gene encoding Acyl CoA transferase (ACT) and a gene encoding Alcohol Dehydrogenase (ADH), which are used in the production of the transformed microorganism according to the embodiment of the present invention in FIG.
2 is a view showing a biosynthetic pathway of a C3-C8 alcohol of a transformed microorganism according to an embodiment of the present invention.
Fig. 3 is a diagram showing the entire gene map and gene information of the Megasperra hexanoika strain.
FIG. 4 is a graph showing production yields of C3-C8 alcohol produced by culturing the microorganism transformed according to Example 1-8 and Comparative Example 1 of the present invention with 48 hours of C3-C8 organic acid added thereto.
5 is a graph showing C5-C8 alcohol conversion, organic acid consumption, and alcohol conversion rate when cultured for 48 hours with C5-C8 organic acid added to the transformed microorganism according to Example 1-8 and Comparative Example 1 of the present invention .
FIG. 6 is a graph showing production yields of C3-C8 alcohol produced by culturing the transformed microorganism according to Example 1-8 and Comparative Example 1 of the present invention after culturing for 72 hours with C3-C8 organic acid added thereto. FIG.
FIG. 7 is a graph showing the conversion of C3-C8 alcohol produced by culturing the transformed microorganism according to Example 1-8 and Comparative Example 1 of the present invention for 72 hours by adding C3-C8 organic acid thereto.
8 is a graph showing the results of cell growth measurement when cultured for 72 hours with C3-C8 organic acid added to the transformed microorganism according to Example 1-8 and Comparative Example 1 of the present invention.
9 is a graph showing the conversion of C3-C8 alcohol produced by culturing for 72 hours with C3-C8 organic acid added to the transformed microorganism according to Example 1-8 and Comparative Example 1 of the present invention, -C8 alcohol. ≪ / RTI >
10 is a graph showing the amount of C4 alcohol produced when C4 organic acid is added to a microorganism using AdhE as an alcohol dehydrogenase (ADH) enzyme according to Example 9 of the present invention.
FIG. 11 is a graph showing the amount of C4 alcohol produced when a C4 organic acid is added to a microorganism using AdhE2 as an alcohol dehydrogenase (ADH) enzyme according to Example 1 of the present invention.
FIG. 12 is a graph showing the amount of C4 alcohol produced when a C4 organic acid was added to a microorganism using PduP-AdhA as an alcohol dehydrogenase (ADH) enzyme according to Example 10 of the present invention.
FIG. 13 is a graph showing the conversion and yield of C3-C8 alcohol in microorganisms according to Examples 1 and 9-10 of the present invention. FIG.
FIG. 14 is a graph showing the results of measurement of tolerance to butyric acid and hexanoic acid of a transformed microorganism according to Example 1 of the present invention. FIG.
15 is a graph showing C4 and C6 organic acid consumption and C4 and C6 alcohol production amounts of microorganisms transformed according to Example 1 of the present invention over time.
16 is a graph showing C4 and C6 organic acid consumption and C4 and C6 alcohol production amounts of microorganisms transformed according to Example 2 of the present invention over time.
17 is a graph showing C4 and C6 organic acid consumption amounts and C4 and C6 alcohol production amounts of microorganisms transformed according to Example 3 of the present invention over time.
18 is a graph showing C4 and C6 organic acid consumption amounts and C4 and C6 alcohol production amounts of microorganisms transformed according to Example 4 of the present invention over time.
19 is a graph showing C4 and C6 organic acid consumption amounts and C4 and C6 alcohol production amounts of microorganisms transformed according to Example 5 of the present invention over time.
Fig. 20 is a graph showing C4 and C6 organic acid consumption and C4 and C6 alcohol production amounts of microorganisms transformed according to Example 6 of the present invention over time. Fig.
FIG. 21 is a graph showing C4 and C6 organic acid consumption and C4 and C6 alcohol production amounts of the transformed microorganisms over time according to Example 7 of the present invention. FIG.
22 is a graph showing C4 and C6 organic acid consumption amounts and C4 and C6 alcohol production amounts of microorganisms transformed according to Example 8 of the present invention over time.
23 is a graph showing the amounts of C4 and C6 organic acids consumed and the yields of C4 and C6 alcohols of the microorganisms transformed according to Comparative Example 1 of the present invention over time.

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, " 8 Acyl CoAtransferases " capable of converting the C3-C8 organic acid outside the cell into acyl-CoA were extracted from megasparrahexanoic acid, and this gene was amplified using a gene encoding an alcohol dehydrogenase (ADH) And then simultaneously expressing C3-C8 alcohol from the external organic acid, thereby selectively producing C3-C8 alcohol.

For this purpose, genes encoding Acyl CoA transferase (ACT) enzymes involved in C3-C8 alcohol biosynthesis have been successfully isolated from Megasphaera hexanoica strain (KCCM11835P) in the present invention.

Next, as described in the following examples, the entire gene of the megasperahexanoicus strain was decoded (Fig. 3), and a gene encoding Acyl CoAtransferase capable of converting an organic acid of the C3-C8 into acyl-CoA Genes with high levels of RNA expression were selected.

Accordingly, the present invention provides a gene encoding an enzyme involved in biosynthesis of a C3-C8 alcohol, which comprises any one of the nucleotide sequences selected from the group consisting of SEQ ID NOS: 1-8.

In this case, the nucleotide sequences of SEQ ID NOS: 1 to 8 are characterized by encoding an Acyl CoA transferase (ACT) enzyme.

The gene is also characterized in that it is derived from Megasphaera hexanoica strain (KCCM11835P).

The C3 organic acid is propionic acid, the C3 alcohol is propanol, the C4 organic acid is butyric acid, the C4 alcohol is butanol, the C5 organic acid is pentanoic acid, the C5 alcohol is pentanol, C6 C8 organic acid is hexanoic acid, C6 alcohol is hexanol, C7 organic acid is heptanoic acid, C7 alcohol is heptanol, C8 organic acid is octanoic acid, C8 alcohol is octanoic acid, Octanol.

The present invention also relates to a gene encoding an enzyme involved in biosynthesis of a C3-C8 alcohol comprising any one of the nucleotide sequences selected from the group consisting of SEQ ID NOS: 1 to 8; And a gene encoding an alcohol dehydrogenase (ADH) enzyme.

Specifically, a gene encoding an Acyl CoA transferase (ACT) enzyme involved in the C3-C8 alcohol biosynthesis involving any one of the nucleotide sequences selected from the group consisting of SEQ ID NOS: 1 to 8 is inserted into the MCS I region of the pCDF duet vector And the gene encoding the alcohol dehydrogenase (ADH) enzyme was inserted into the MCS II region of the pCDF duet vector.

Herein, the gene coding for the alcohol dehydrogenase (ADH) enzyme may include any one of the nucleotide sequences selected from the group consisting of SEQ ID NOS: 9 to 11. Specifically, the gene represented by the nucleotide sequence of SEQ ID NO: (AdhE), the gene represented by the nucleotide sequence of SEQ ID NO: 10 encodes Aldehyde / alcohol dehydrogenase (AdhE2), and the gene represented by the nucleotide sequence of SEQ ID NO: 11 encodes Aldehyde-alcohol dehydrogenase dehydrogenase (PduP-AdhA).

At this time, the AdhE is Escherichia coli AdhE2 is derived from Clostridium acetobutylicum , PduP in PduP-AdhA is derived from Salmonella typhimurium , and AdhA is derived from Lactococcus lactis .

In addition, the present invention provides a microorganism transformed by the above-mentioned vector and capable of producing C3-C8 alcohol.

In order to improve the yield of the C3-C8 alcohol, the microorganism may be selected from a gene encoding an enzyme involved in lactate biosynthesis, a gene encoding an enzyme involved in acetate biosynthesis, And at least one gene selected from the group consisting of a gene encoding an enzyme involved in succinate biosynthesis and an enzyme involved in biosynthesis of succinate is weakened or deleted.

In the following examples, Escherichia coli MG1655 was used as a host microorganism. However, genes coding for Acyl CoA transferase (ACT) enzymes involved in the biosynthesis of C3-C8 alcohol using other Escherichia coli, bacteria, yeast and fungi and Alcohol Dehydrogenase (ADH) < / RTI > enzyme is introduced, it is said that the object of the present invention can be achieved.

The present invention also provides a method for producing a C3-C8 alcohol by culturing the microorganism.

At this time, at least one of C3 to C8 organic acids may be added during the culture of the microorganism. Specifically, when a C3 organic acid is added to the microorganism culture, a C4 alcohol is added to the C4 organic acid, a C5 alcohol is added to the C5 organic acid, a C6 alcohol is added to the C6 organic acid, a C7 alcohol is added to the C7 organic acid, When C8 organic acid is added, C8 alcohol is selectively produced. When a plurality of organic acids having a predetermined carbon number is added, a plurality of alcohols having a predetermined carbon number can be produced.

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

In the wuxi market in Majang-dong, Seoul, Korea, we purchased small intestine containing gastric juice and processed it anaerobically. 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 April 28, 2016 with the deposit number KCCM11835P to the Korea Microorganism Conservation Center, a microorganism depository organization under the Budapest Treaty.

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. 3 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.

(SEQ ID NO: 1: ACT 5510, SEQ ID NO: 2: ACT 3270, SEQ ID NO: 3: ACT 3280, SEQ ID NO: 4) encoding the Acyl CoA transferase (ACT) among genes derived from the above- (Denoted as ACT 23750, SEQ ID NO: 5: ACT 19420, SEQ ID NO: 6: ACT 7470 SEQ ID NO: 7 ACT 5060, SEQ ID NO: 8 ACT 24660) and SEQ ID NO: 12 (denoted ACT 27250).

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

The genes coding for the Acyl CoA transferase (ACT) enzymes comprising the nucleotide sequences of SEQ ID NOS: 1 to 8 and SEQ ID NO: 12 according to the present invention were inserted into the MCS I region of the pCDF duet-1 vector, A vector according to the present invention was prepared by inserting a gene encoding an alcohol dehydrogenase (ADH) enzyme comprising the nucleotide sequence shown in any one of SEQ ID NOS: 9 to 11 into each inserted vector into the MCS II region (FIG. 1) At this time, the expression level of the ACT gene was approximately 700,000 and the expression level of the ADH gene was adjusted to approximately 10000000-3000000. The specific manufacturing method is as follows.

(1) Preparation of a vector comprising SEQ ID NO: 1 and SEQ ID NO: 10

In order to insert the gene coding for ACT 5510 according to SEQ ID NO: 1 into the MCSI region of the pCDF duet-1 vector, primers set forth in SEQ ID NOs: 13 and 14 below, in which BAMHI was inserted in front of the primer set and HIND3 restriction enzyme region was inserted in the rear Were used. The PCR fragment amplified by PCR was treated with BAMHI and HIND3. The pCDF duet-1 vector was also treated with BAMHI and HIND3 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]: 5'-TATATGGATCCCATGTACAAACTTTCACAAATTGCAG-3 '

[SEQ ID NO: 14]: 5'-TATATAAGCTTTTAGTATTCTGTCTTGCTCGTCT-3 '

Next, in order to insert the gene coding for AdhE2 according to SEQ ID NO: 10 into the MCS II region of the pCDF duet-1 vector, NDEI was inserted in front of the primer set and SEQ ID NOS: 33 and 34 in which the XHOI restriction enzyme region was inserted at the rear Primer set was used. The PCR fragments amplified by PCR were treated with NDEI and XHOI. The pCDF duet-1 vector was also treated with NDEI 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: 33]: 5'-TATATCATATGATGAAAGTTACAAATCAAAAAGAACTAAAA-3 '

[SEQ ID NO: 34]: 5'-TATATGAGCTCTTAAAATGATTTTATATAGATATCCTTAAGTT-3 '

(2) Preparation of a vector comprising SEQ ID NO: 2 and SEQ ID NO: 10

In order to insert the gene encoding ACT 3270 according to SEQ ID NO: 2 into the MCSI region of the pCDF duet-1 vector, primers set forth in SEQ ID NOS: 15 and 16 below, in which BAMHI was inserted in front of the primer set and HIND3 restriction enzyme region was inserted in the rear Was used to prepare a vector. ≪ tb > < TABLE >

[SEQ ID NO: 15]: 5'-TATATGATCCCATGTCAGAATGGACGGATATGTA-3 '

[SEQ ID NO: 16]: 5'-TATATAAGCTTTTACCGTTTATTGCTCTTGCGC-3 '

(3) Preparation of a vector comprising SEQ ID NO: 3 and SEQ ID NO: 10

In order to insert the gene coding for ACT 3280 according to SEQ ID NO: 3 into the MCSI region of the pCDF duet-1 vector, primers set forth in SEQ ID NOs: 17 and 18 below, in which BAMHI and HIND3 restriction enzyme regions were inserted in front of the primer set, Was used to prepare a vector. ≪ tb > < TABLE >

[SEQ ID NO: 17]: 5'-TATATGAGCTCCATGGATGTAATGCAAGAATATGCC-3 '

[SEQ ID NO: 18]: 5'-TATATGTCGACTTAGATCAGGATATGCATTTTTTTAGC-3 '

(4) Preparation of a vector comprising SEQ ID NO: 4 and SEQ ID NO: 10

In order to insert the gene coding for ACT 23750 according to SEQ ID NO: 4 into the MCSI region of the pCDF duet-1 vector, primers set forth in SEQ ID NOS: 19 and 20 below, in which BAMHI was inserted in front of the primer set and HIND3 restriction enzyme region was inserted in the rear Was used to prepare a vector. ≪ tb > < TABLE >

[SEQ ID NO: 19]: 5'-TATATGAGCTCCATGACACAATATGAAACAATGTATGAA-3 '

[SEQ ID NO: 20]: 5'-TATATGTCGACTTACCAAATCATGTATTTGTCAACAG-3 '

(5) Preparation of a vector comprising SEQ ID NO: 5 and SEQ ID NO: 10

In order to insert the gene coding for ACT 19420 according to SEQ ID NO: 5 into the MCSI region of the pCDF duet-1 vector, primers set forth in SEQ ID NOS: 21 and 22 below, in which BAMHI was inserted in front of the primer set and HIND3 restriction enzyme region was inserted in the rear Was used to prepare a vector. ≪ tb > < TABLE >

[SEQ ID NO: 21]: 5'-TATATGGATCCCATGATGAATCAATGGCAGCGCAT-3 '

[SEQ ID NO: 22]: 5'-TATATAAGCTTTTACACAATAATATGCATCTTTCTGG-3 '

(6) Preparation of a vector comprising SEQ ID NO: 6 and SEQ ID NO: 10

In order to insert the gene coding for ACT 7470 according to SEQ ID NO: 6 into the MCSI region of the pCDF duet-1 vector, primers set forth in SEQ ID NOS: 23 and 24 below, in which BAMHI was inserted in front of the primer set and HIND3 restriction enzyme region was inserted in the back, Was used to prepare a vector. ≪ tb > < TABLE >

[SEQ ID NO: 23]: 5'-TATATGGATCCCATGGATTACCAGAGTGAATACATGA-3 '

[SEQ ID NO: 24]: 5'-TATATAAGCTTTTATCGTTTATGAGAAGCGCGC-3 '

(7) Preparation of a vector comprising SEQ ID NO: 7 and SEQ ID NO: 10

In order to insert the gene coding for ACT 5060 according to SEQ ID NO: 7 into the MCSI region of the pCDF duet-1 vector, primers set forth in SEQ ID NOs: 25 and 26 below, in which BAMHI and HIND3 restriction enzyme regions were inserted in front of the primer set, Was used to prepare a vector. ≪ tb > < TABLE >

[SEQ ID NO: 25]: 5'-TATATGGATCCCATGAATCCATTCGAAATATATCAGG-3 '

[SEQ ID NO: 26]: 5'-TATATAAGCTTTTATTTCTTGTTACTGTTCCTCCAG-3 '

(8) Preparation of a vector comprising SEQ ID NO: 8 and SEQ ID NO: 10

In order to insert the gene coding for ACT 24660 according to SEQ ID NO: 8 into the MCSI region of the pCDF duet-1 vector, primers set forth in SEQ ID NOS: 27 and 28 below, in which BAMHI was inserted in front of the primer set and HIND3 restriction enzyme region was inserted in the rear, Was used to prepare a vector. ≪ tb > < TABLE >

[SEQ ID NO: 27]: 5'-TATATGAGCTCCATGATTGACATTTCAGATCGCATA-3 '

[SEQ ID NO: 28]: 5'-TATATGTCGACTTACTTCATACTCCCTGTTTCCA-3 '

(9) Preparation of a vector comprising SEQ ID NO: 1 and SEQ ID NO: 9

In order to insert the gene coding for AdhE according to SEQ ID NO: 9 into the MCS II region of the pCDF duet-1 vector, a primer set of SEQ ID NOs: 31 and 32 shown below inserted with NDEI in front of the primer set and XHOI restriction enzyme region in the back A vector was prepared in the same manner as in (1) above, except that it was used.

[SEQ ID NO: 31]: 5'-GCGCCATATGGCTGTTACTAATGTCG-3 '

[SEQ ID NO: 32]: 5'-GCGCCTCGAGTTAAGCGGATTTTTTC-3 '

(10) Preparation of a vector comprising SEQ ID NO: 1 and SEQ ID NO: 11

In order to insert the gene coding for AdhE according to SEQ ID NO: 11 into the MCS II region of the pCDF duet-1 vector, a primer set of SEQ ID NOS: 35 and 36, in which NDEI was inserted in front of the primer set and an XHOI restriction enzyme region was inserted in the rear, A vector was prepared in the same manner as in (1) above, except that it was used.

[SEQ ID NO: 35]: 5'-GCGCCATATGAACACCAGCGAACTGGAAA-3 '

[SEQ ID NO: 36]: 5'-GCGCCTCGAGTTATTTGGTGAAGTCG-3 '

(11) Preparation of a vector comprising SEQ ID NO: 12 and SEQ ID NO: 10

In order to insert the gene encoding ACT 27250 according to SEQ ID NO: 12 into the MCSI region of the pCDF duet-1 vector, primers set forth in SEQ ID NOS: 29 and 30, in which BAMHI was inserted in front of the primer set and HIND3 restriction enzyme region was inserted in the rear, Was used to prepare a vector. ≪ tb > < TABLE >

[SEQ ID NO: 29]: 5'-TATATGGATCCCATGACTGGTATTGATGCACATATTT-3 '

[SEQ ID NO: 30]: 5'-TATATAAGCTTTTATCGTCTCGTCATCTCCTTAT-3 '

4. Production of microorganisms having C3-C8 alcohol production ability transformed by the vector according to the present invention

(1) deletion of ldhA, pta, adhe, and frda genes

In order to further delete the ldhA, pta, adhe and frda genes from Escherichia coli MG1655, pKM 208 plasmid carrying the recombinase-bearing pKM 208 plasmids using the primers of the following SEQ ID NOS: 37-44 and pKDA using the kanamycin resistance gene were 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: 37] ldhA1stup: 5'-AAATATTTTTAGTAGCTTAAATGTGATTCAACATCACTGGAG AAAGTCTTGTGTAGGCTGGAGCTGCTTC-3 '

[SEQ ID NO: 38] ldhA1stdo: 5'-ATTGGGGATTATCTGAATCAGCTCCCCTGGGTTGCAGGGGAG CGGCAAGATCCTCCTTAGTTCCTATTCC-3 '

[SEQ ID NO: 39] FRDA f: 5'-CTTACCCTGAAGTACGGGGCTGTGGGATAAAACAATCTGGAGGA ATGTCGTGTAGGCTGGAGCTGCTTC-3 '

[SEQ ID NO: 40] FRDA r: 5'-TATCGACTTCCGGGTTATAGCGCACCACCTCAATTTTCAGGTTTT TCATCTCCTCCTTAGTTCCTATTCC-3 '

[SEQ ID NO: 41] PTA f: 5'-GGTGCTGTTTTGTAACCCGCCAAATCGGCGGTAACGAAAGAGGAT AAACCGTGTAGGCTGGAGCTGCTTC-3 '

[SEQ ID NO: 42] PTA r: 5'-TTATTTCCGGTTCAGATATCCGCAGCGCAAAGCTGCGGATGATGA CGAGAATATCCTCCTTAGTTCCTATTCC-3 '

[SEQ ID NO: 43] ADHE f: 5'-ATTCGAGCAGATGATTTACTAAAAAAGTTTAACATTATCAGGAGA GCATTGTGTAGGCTGGAGCTGCTTC-3 '

[SEQ ID NO: 44] ADHE r: 5'-AAAAAACGGCCCCAGAAGGGGCCGTTTATATTGCCAGACAGCGCT ACTGATCCTCCTTAGTTCCTATTCC-3 '

(2) Production of microorganisms having C3-C8 alcohol production ability

In order to produce a microorganism having an alcohol biosynthesis pathway according to FIG. 2, a microorganism into which the vector according to the above-mentioned 3- (1) was introduced (Example 1: ACT_5510 ), The microorganism (Example 2: ACT_3270) into which the vector according to the above 3- (2) was introduced (Example 2: ACT_3270), the microorganism into which the vector according to the above 3- (3) (Example 4: ACT_23750), the microorganism into which the vector according to the above 3- (5) was introduced (Example 5: ACT_19420), the microorganism into which the vector according to the above 3- (6) Example 6: ACT_7460), microorganisms into which the vector according to the above 3- (7) (Example 7: ACT_5060), vector according to the above 3- (8) (Example 8: ACT_24660) (Example 9: using AdhE as ADH), the microorganism into which the vector according to (3- (10) above was introduced (Example 10: using PduP-AdhA as ADH) 3- (11) The introduction of micro-organisms: (Comparative Example 1 ACT_27250) was prepared.

5. Measurement of C3-C8 alcohol production capacity

Experiments related to the C3-C8 alcohol production ability of the transformed microorganisms according to Examples 1-10 and Comparative Example 1 were conducted.

FIG. 4 is a graph showing production yields of C3-C8 alcohol produced by culturing the microorganism transformed according to Example 1-8 and Comparative Example 1 of the present invention with 48 hours of C3-C8 organic acid added thereto. At this time, 1 g / L of C3-C8 organic acid was added to each of the microorganisms and cultured, and the amount of alcohol produced corresponding to the number of carbon atoms of the corresponding organic acid was measured. As a result of the measurement, it was confirmed that C3-C8 alcohol was selectively converted when C3-C8 organic acid was added to the transformed microorganism according to the present invention. In addition, all of the microorganisms according to Examples 1-8 showed excellent alcohol-selective productivity. Specifically, the microorganism according to Example 1 showed excellent conversion ability to all organic acids as a whole. In addition, the microorganisms according to Examples 4 and 6 showed the highest alcohol conversion rates with respect to the C4 organic acids. In the case of the microorganisms according to Examples 5 and 8, the higher the carbon number of the organic acid, the lower the conversion rate While the microorganism according to Example 7 showed an increase in conversion amount with respect to C4, C6 organic acid.

5 is a graph showing C5-C8 alcohol conversion, organic acid consumption, and alcohol conversion rate when cultured for 48 hours with C5-C8 organic acid added to the transformed microorganism according to Example 1-8 and Comparative Example 1 of the present invention . Thus, it was confirmed that the microorganisms according to Examples 1-8 of the present invention all had excellent alcohol conversion production capacity even for a high carbon number organic acid.

FIG. 6 shows the yield of C3-C8 alcohol produced by culturing the transformed microorganism with C3-C8 organic acid for 72 hours according to Example 1-8 and Comparative Example 1 of the present invention, and FIG. FIG. 8 is a graph showing the results of cell growth measurement, and FIG. 9 is a graph showing the conversion rates of C3-C8 alcohol produced by separating C3-C5 alcohol and C5-C8 alcohol. Thus, it was confirmed that the microorganisms according to Example 1-8 exhibited excellent alcohol-selective productivity and high conversion even when incubated for 72 hours. Especially, the microorganisms according to Example 1-3 had very high selectivity for all organic acids, And that the microorganisms according to Examples 4-8 also have various properties in converting organic acids into alcohols.

Next, the C3-C8 alcohol conversion amount and the production amount of the microorganism according to Examples 1, 9-10 of the present invention were measured, and the results are shown in Fig. As a result, it was confirmed that AdhE, AdhE2, and Pdup-AdhA, which are used as ADH enzymes, show high selectivity and alcohol conversion for C3-C8 organic acids. In particular, Pdup-AdhA has the highest alcohol conversion rate for C5-C7 organic acids and AdhE has the highest alcohol conversion for C8 organic acids.

Next, C4g organic acid was added to the microorganism according to Example 1, Example 9, and Example 10 of the present invention at 1 g / L, and the C4 alcohol conversion and production amount were measured during 48 hours of culture. As shown in Fig. At this time, in order to evaluate the efficiency of the enzyme itself, the cell was concentrated and the O.D value was adjusted to 10, and then the activity of the enzyme was measured by adding organic acid (Table 1).

The results showed that alcohol productivity was in the order of Pdup - AdhA, AdhE and AdhE2. AdhE showed higher productivity than AdhE2, but alcohol conversion of organic acid was somewhat lower.

Next, the microorganisms transformed according to Example 1 of the present invention were injected with butyric acid and hexanoic acid, and cultured, and the tolerance to these organic acids was measured. The results are shown in Fig. As a result of the measurement, it was confirmed that the optimum concentration of butyric acid was 30 mM and the optimum concentration of hexanoic acid was 15 mM.

Next, the amounts of C4 and C6 organic acids consumed and the yields of C4 and C6 alcohols of the transformed microorganisms were measured according to Examples 1-8 and Comparative Example 1, and the results are shown in Figs. 15 to 23 and Table 2 shows the results. At this time, the added C4 organic acid and C6 organic acid were added at the optimum concentrations derived from FIG.

As a result of the measurement, it was confirmed that all of the microorganisms exhibited the highest production efficiency in 24 hours of incubation. Also, it was confirmed that the microorganisms according to Example 1 showed the highest amounts of C4 and C6 organic acids consumed and C4 and C6 alcohols, respectively. The microorganisms according to Examples 1 to 8 had organic acid consumption rate, alcohol production rate and alcohol conversion rate , It was confirmed that the production rate of alcohol can be selectively controlled by using these characteristics.

Korea Microorganism Conservation Center (overseas) KCCM11835P 20160428

<110> Industry-University Cooperation Foundation Hanyang University <120> A method for activating C3-C8 alcohol using acyl CoA          transferase from Megasphaera hexanoica <130> JKP-0728 <150> KR 10-2016-0158433 <151> 2016-11-25 <160> 44 <170> KoPatentin 3.0 <210> 1 <211> 1359 <212> DNA <213> Unknown <220> <223> Megasphaera hexanoica <220> <221> gene &Lt; 222 > (1) .. (1359) <223> gene coding Acyl CoA transferase (ACT 5510) <400> 1 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> 2 <211> 1344 <212> DNA <213> Unknown <220> <223> Megasphaera hexanoica <220> <221> gene &Lt; 222 > (1) .. (1344) <223> gene coding Acyl CoA transferase (ACT 3270) <400> 2 atgtcagaat ggacggatat gtataaacaa aaactgacga ctccggaaca agcagttacc 60 cttgtaaaag acggcgactg ggtcgattat ggcatgacga cctcccagcc ggtactgctc 120 gataaagcac tggcgggccg caaagatgag ttacatgata ttaaagtacg ggaaaccatg 180 tccctgtttc cccgccaggt atgcgaatgc gaccccgatt gcgatacttt taccgtcatg 240 aactggcatc tcagcggata tgaccgcaag ctggcagcac agtcacgcat gttttttaat 300 cctatgtgtt tccgtaatga accgtcgatt taccgcaata tcattgatgt cgatgtggcg 360 atgattactg tagcgcctat ggataaacac ggatatttta actttggcct ttgtgcggcc 420 gtgaccgagg ctattacgaa aaaggctaaa aaagtcatcg tcgaagtcaa tccgcatatg 480 ccgcgctgtc ttggcggccg tggggaagcc atccatattt ctgacgtcga tgccatcgtc 540 gaagcgcccg atgtgccagt gccgactatt cctttcgtcc ttggaaacga tatcgatcag 600 aaaatagctt cttatatcgt cgaagccatt cccgatgggg caacattgca gctgggcatt 660 ggcgggctgc ctaacagtgt aggcgctatg attgccaaat cggatttgaa agatctgggt 720 gtccatacag aaatgctcgt cgacgcattc taccttatgg ctaaggaagg cagactgacg 780 aataaagtca aaaacctgaa ccgtgataaa ggggtctttg cctttgccct tggttctcag 840 gatttatatg actgggtaga tgacaatcct gctctggcaa cctttcccat cgattatgta 900 aatgatccgg ctgttatcgg acagattgat aattttattt ccatcaacag ttgcatcgaa 960 gtagacctct atggccaggt atcggccgaa tccatgggga cccaccagat cagtggcagc 1020 ggcggccagc tggacttcac cgacggtgcc taccggtcca gaggcggtaa gtccatcatc 1080 gccatgcacg cgacccatac ggataaaaag accggtacgg ttacgtccaa catcagcccc 1140 atcctgaaag ccggtacgac tgtcaccgat ccccgttccc agacgcactg gattgcgaca 1200 gaatacggca tggtcaacct catgggtaaa tcgacctggc agcgcgcaga agcccttatc 1260 ggcctggcac accccgattt ccgggacgaa ctcatcaaag aagcggacaa gatgcatatc 1320 tggcgcaaga gcaataaacg gtaa 1344 <210> 3 <211> 1302 <212> DNA <213> Unknown <220> <223> Megasphaera hexanoica <220> <221> gene <222> (1) 1302 <223> gene coding Acyl CoA transferase (ACT 3280) <400> 3 atggatgtaa tgcaagaata tgccgataag aaaaaaacgc cggaacagat tgccgcctgt 60 gtggaatcgg gctggtcctg ttgtgctgat atttcaggag ccatcccgcc ggtcctggcc 120 gatgctctgg gaaaacgggc cgctgccggg gatatacagg atgtgacctg tcatactctg 180 ctcgatgtga cgccgctggg gaccttatct tccgaagcct atccgaatat tacgcctgtt 240 acctggtttt ccggtggcgg cctgcgtaaa gcggccaacg aaggacgctg cgacatcatg 300 ccctgctatt accacgatgc gccgggactg tttgaacgat atatcgacat tgatgcgttc 360 tttgcccagg tatctcctat ggataagcac gggtatttca gcaccagcct gagcggttcc 420 tgcagtgccg ccatgatccg gaaagcccgc catatttatc tggaagtcaa tgatcagctg 480 ccgcgggtac tcacggcgcc gcaaatccat atttcacagg tcgatggact gtgtgaaacg 540 tcccatgccc tgcctatcgt gccgccggta cagattgatg aaatcagccg tactatcggc 600 ggttatattg ccgaagaagt gcctgacgga gcgacactgc agcttggtat cggcgctgta 660 ccagaagccg tgggtctggc cctcaaggat aagcatgatc tgggaatcca tacagaactg 720 tttacagaca gcatggtaga actcattgaa tgcggggctg tcaccaatac tagaaaaccg 780 attcaccggg gaaagactgt ggcaaccctg acctttggtt ctcagcgtat ttacgatttt 840 attgatgaca atccggcatt tctcatgtta cctgtcgatt atgtcaatga cccggcagtc 900 atcgcccgcc atccggactt tatttctgtc aatgcggcta ttgaagttga tttctttggt 960 caggcctgct ctgaatcgac aggaacgcgg catatatctg gtacaggtgg ccaggctgat 1020 tacgtgcgcg gtgccatctg ctcaccgggc ggaaaaagct ttattgcttt cccctctacg 1080 gcaaaaggag gcactatcag caaaatcgtg ccgacactga cgccgggagc catcgtttcg 1140 acgagtaaaa atgatattga ttacgtcgtt acagaatacg gcatcgccaa attacggggg 1200 aaaacccttt ctcagcggac aaaagccctg atttccattg cccatccgaa attccgggaa 1260 gagctgacct ttgctgctaa aaaaatgcat atcctgatct aa 1302 <210> 4 <211> 1335 <212> DNA <213> Unknown <220> <223> Megasphaera hexanoica <220> <221> gene <222> (1). (1335) <223> gene coding Acyl CoA transferase (ACT 23750) <400> 4 atgacacaat atgaaacaat gtatgaacag aaaaaaatga cagcagaaca ggcactccaa 60 ttgattcaaa gccgtgacta catgtttagt gcccaggctt caggggaacc ggcagctatt 120 ctcgataaac ttcagtactt aaaaaaaacg ggagttagag atacgattat taatacctgt 180 cttcccttaa aggattatcc atggctccat gatcaggaaa tgaaaggaat catgttccat 240 aatggctggt ttttctgcgg ccccctgcgt caatcccaga aggaaaaatt gacgagtgcc 300 gtgccacagc ggtctacgac tattttacgc cataccttag accggatccg atacgaagga 360 cgacgccctg ttgttctggc aaccgtatct cctatggata agaatggtta tatgacactt 420 tctgtcagcg ccatttatga acgcgatctg attaatgcgg gagcgctgac tattgtagaa 480 gtcaatccta atttcccaag aacgtttggt gatacactgg tccatgtttc tgaggttgca 540 gctgttgtag aatcagaccg gccgattccc tgtgccaaac tggcgccata tactgaaaca 600 gatgcggcta ttggcaggta tattgcagat ctcatagaag atggatccac aatccagctg 660 ggtattggca atattcccaa tgcagtagcc aatgaactga aagcaaagaa acatttgggg 720 attcatacgg aaatgtttac ggaaacgatg gttgatttga ttgaatgcgg cgctgtagac 780 aattcgcaaa aagggtttat gaatggatat tctgtctgct catttacgat gggaagccag 840 cggttatatg attttattga caataaccct tcggtccttt ttaagtcgag caccttcagt 900 aatgatccct atacgattag ccggaataac aagtttgttt ccattaatgc cagcctggaa 960 atcgacctga ccggtcagtg cgcgtcggaa accgtgggaa atctgcagtg gtccggaacg 1020 ggtggtcagt ctgaaactgt tcagggggca caaatgtcac caggaggcaa gtccattatt 1080 gccatgcatt ctacgtacac gacaaaagat gccgatggca agcccattct ccattctaaa 1140 attgttcctt tcctggctcc aggggcggcg gttacgacat cgcgcaatga taccgattac 1200 gttgttacgg aatatggtgt ggcctggctg cgcggtttaa atattaagca gcgtgccgaa 1260 tccttgatcc gcatagcaca tcctgatttt cgcgatgaac tgcgcaaagc tgttgacaaa 1320 tacatgattt ggtag 1335 <210> 5 <211> 1305 <212> DNA <213> Unknown <220> <223> Megasphaera hexanoica <220> <221> gene &Lt; 222 > (1) 1305 <223> gene coding Acyl CoA transferase (ACT 19420) <400> 5 atgatgaatc aatggcagcg catgtacgaa gaaaagaaga tgacggctga atccttggcc 60 aggcagttca aaaacgggga tgtctgtgtc agcacagggc aggtggcgga accgacggga 120 attctgcagg tactggcctc gtatgctccc gagctggacc tggaacatat ccgccattgt 180 gtactccttc ccctccggca gcaggagtac atgaaagccg gtatggagaa atatatttat 240 cacatttccc actttgtcag tgcttatgac aggaacatga tatgggaagg ccgcggcgat 300 tatatgccgg cccattacag tcaggtacca caggtgtgga ccagtgtcct gcctgagccg 360 gatgtgtttt acgctgccgt atcccctatg gatgagcacg ggtattttag tttcggtacg 420 gcggccgatt tgagtgaagt ccgtcatcat gcagaaaaaa tactggtaga agtaaatccg 480 actatgcccc ggtcgtttgg ttcctttatc catatttcag aagtagacgg tatatgggaa 540 aatgatgctc ccattacagt agtcgatccg ccgccggtta ccaatacgga ccttgccatc 600 gggcagatga ttgctgacga gatacccaat ggagccacgc tgcagctggg aattggcggc 660 attcccaatg cggtggcaca gagcctgctc gataagcgga atctgggcgt acatagtgaa 720 atgttttgtg acagcatggt tgatctgaca ctggccggag tcattactaa cggctgcaaa 780 cggattcatc gcggaacctc tgttgcgacc tttacctttg cttcccgcag gacctatgat 840 tttattgaca ataatcccgg tgtcgccttc ctgcccgtat cgtatgtcaa tgatccccgc 900 gtcattgcta tgaatgatca ggtcatttcc atcaattcgt gcatcgaaat cgacttgttc 960 ggccaggtat gctcggaaac gatgggaaca aagaactata gcggtgtagg cgggcagatt 1020 gatttcatcc gtggggccgc tgcttccaaa ggaggaaaat cgttcctcgc ttttacggca 1080 acggcaaaaa aagggaaagt gtccaagatt aaaccggtcc tcacagaagg agcctgtgtc 1140 agtacgacga gaaacgatgt cgattatgta gttactgagt ttggaatggc acggctgaaa 1200 gggctgacct gttcccagcg tgccaaagcg ctgattcgca ttgcagctcc tgctttccgg 1260 gaagaattaa cggaggcagc cagaaagatg catattattg tgtga 1305 <210> 6 <211> 1338 <212> DNA <213> Unknown <220> <223> Megasphaera hexanoica <220> <221> gene &Lt; 222 > (1) .. (1338) <223> gene coding Acyl CoA transferase (ACT 7470) <400> 6 atggattacc agagtgaata catgagcaaa cgctgtacac cgcagcaggc agtgcagctg 60 atccgcgacg gggactgggt cgattacacc cagggtatta cctttcccca gcttctcgat 120 gaggccctgg cagaacggac aggggaattg gccgatgtga agatccgctg catgttctgc 180 ctgaaacggc cgcagatatt ggaaaaagat ccccatagcg tatcttttac gtatcatacc 240 tggcatagca gccagataga ccgcagggat gtcaatgaag gccgggctta tttcgccccg 300 attcagtaca gtcatctggc agaatattac cgccggggaa tggcaccggt cgatgttgtc 360 atgatgaccg tttctcctat ggaccgtcat gggaacttca gttttgcctg caatccgtcg 420 gccactcagg gcgctctcga tgcagcagac cgcatcatcg tcgaagttaa ccccaacctg 480 ccggaagcgt acggcctggg aggagatttt attcatattt ccgatattga tgctatcgtg 540 gaaagtgatg taccggtgct tatggcaccc aatccgcccg tacaggaaat cgataagaaa 600 attgcggcct ggctgctccc ctatctgaaa gacggtatga cgctgcagat cggtgtcggc 660 ggtattccca atgccctggg gatgctcatt gccgattcgg atctgaaaga tctgggcatg 720 catacggaat atctcagtga tggctgcctg aaactgtatg aggcagggaa gataacgaat 780 cgcagaaaag aactcctgcc gggcaaaggc gtgtatggta cttgtgccgg cagcacggaa 840 ctgtacgatt tcgtcgatca caaccgggca ttactatcag cccctattga atatgtcaat 900 cacgtggatc agatccgtca gctgaaccag ttcgtttcta tcaatggctg cctggccgtc 960 gatttatatg gacaggtttg ctcggaatcg gctggcctct ggcatatcag cggctcgggg 1020 ggacaggtcg attttattag cggtgccttc tggtctcctc atgggcaggc ctttctgact 1080 atgccttcga cgtatacaga ccggcagggt atcgtacatt cacgcataca gccgtttttc 1140 tcacatggcg atatcgttac gacggtacgg gccatagctc cgtgtatcgt aacggaatac 1200 gggatagcag aactggaagg caggacgacg tggcagcggg cagaagcgct cattgccatt 1260 gcccatcccg atttccgcga atctctgatc cgtgcggcgg aagcccagca catatggcgc 1320 gcttctcata aacgataa 1338 <210> 7 <211> 1338 <212> DNA <213> Unknown <220> <223> Megasphaera hexanoica <220> <221> gene &Lt; 222 > (1) .. (1338) <223> gene coding Acyl CoA transferase (ACT 5060) <400> 7 ttgaatccat tcgaaatata tcaggaaaaa ctgcacactc ccgaagaggc agtgcatctc 60 gtccagtccg gcgactgggt cgattatagc cagacctgtt cctttccggc ggctctcgat 120 gcggctctgg cggcacggcg ggatgagctc actgacgtga aggtgcgcca cgccatatcg 180 atgcgccctg tccagatagt agaacaggac ccgcagcggc aggcctttac atacaacctc 240 tggcactgct cgggactgga tcgtaagtat atcgatacgg gacgggcttt cttctcgcct 300 atgatgttcc gcttctgcgg ctcctattac agccgcggcc aggcgccggt caacgtggcc 360 atggtgaccg tatcgcccat ggaccgctat ggcaatttca gctatggcct gactaactgc 420 tgtatgcagg aaatgctcga tgcggctgac cggattatcc tggaagtcaa tccccatatg 480 ccttttatat acggtatggc agatgatcac atcaatatcc gcgacgttga tgctgtcgtt 540 gaaaatgatc agcctctgag cgaagcccca agccggggag ccagtgaact ggataagcag 600 attgccgccc agatttttcc cttcatccac gatggagaca cactgcaact cggcatcggc 660 ggtatgccca atgccctggg gtcgctcatt gccgggtcag acctcaggga cctgggcatg 720 catcataac tcatgagcga tggctacctc gacctgtata aggccggcaa gatcaccaat 780 aagcgtaaga ccctgcagaa gggaaagggc gtattttcca tctgcagcgg atcgaaggaa 840 ctctatgagt tcctcgacca caatattgac attctgtcgg cgcccatgca ttatgtcaac 900 gcccggaaa cgattcgtca gctggaccat ttcgtttcca tcaatggctg cattgcctgc 960 gcctctacg gccaggtcag ctccgaatcg gcggggacgc ggcagatcag tgggaccggc 1020 ggccagcttg atttcgtcac cggtgcctat acggcagaac acggccggac cttcctggcc 1080 atggcgtcga gccgggtgga caagaaaggc gtccgccatt ccaatatcgt accatgcttt 1140 accggcggtg acatcatcac gacgccgcgg gcacagacca tgtacatcgt caccgaatac 1200 ggggcggtca atctggccgg acttacgacg tggcaacggg cagaaaaact gatcggtatc 1260 gctcatcccg atttccggga cgaactcatc aaagccgcag aacaacagaa aatctggagg 1320 aacagtaaca agaaatga 1338 <210> 8 <211> 1500 <212> DNA <213> Unknown <220> <223> Megasphaera hexanoica <220> <221> gene &Lt; 222 > (1) .. (1500) <223> gene coding Acyl CoA transferase (ACT 24660) <400> 8 atgattgaca tttcagatcg catacgaaat aaagcataca tgtccagagt tacgacggca 60 gaagaagcgg cgaagctgat tcatcccgat gatattgtgg ctgtatcggg gtttacaccg 120 gccgggtatc ccaaagccgt accgctggct ctggcgaagc ggatagaacg ggaacatttt 180 cagattacgc tgtatgctgg ggcatccatc ggtgatgaaa ttgatggggc cctggcacgg 240 gtacacggta tttcccgccg gttttcctat catacgaata aagatctgcg ccgcgaaatc 300 aatgatggca gtgtggctta tgccgattat cacgtcagcg tatttgccca gatgctaagg 360 gaagggttca tgaagcgccc cgatatcgtc gtggtggaag cggcagctat cacaaaagaa 420 gggaatctca tccctactac gtctgtagga acgacgccgg ccatgattga cgtgtgtcag 480 aaagttgtcg tcgaaatcaa tgtgacccag ccgctcagtc tggaagggat gcatgacgta 540 tacgatattc ccaatccgcc attccgtgta cccattccca tcgtccatac aggggaccgt 600 attgggaagc cgtatatcac ctgcggttgg gataaaatcg ttgctattgt tccctgtgat 660 attcccgatg ctccccggtc gtttaaacct gttgatgatg cgggccggaa aatgggaaat 720 ctcatcgtgc agttttttaa ggatgaagta gctaaaggcc ggctgccgga gcatctcctg 780 ccactccagt ccggtgtcgg atccgtagcc aatgcggtca ttcaggggct ggccaaaagt 840 gatttcgaac atttgtctat ctttacggaa gtgctccagg acggcatgtt taccctcatc 900 gatgccggga aagtcgatgc cgtatcgacg gcttctattt cagcatcacc ggaagggttg 960 aagcatttct atgaacacat cgatgagtac cggaaaaaaa tcgtcatacg accccaggaa 1020 atttctaata atcccgaagt gatccgccgc atcggagcta ttgccatgaa tacggccatt 1080 gagtttgata tctatggtca ggtcaattcg acacatatct gtggcagtcg tctcatgaat 1140 ggaattggcg gttccgggga ctttgcccgg gcgggatatc tgacgatttt ctttaccagt 1200 tcgacggcaa aaaacggtgc catcagttcc gtagttccca tgtgctccca cgtagatcac 1260 acggagcacg atgtggatgt cctctgtacg gaacagggca ttgcagacct gcgcggtctt 1320 tctcctgtcg aacgggcaag gactatcata gctaactgtg cccacccgga ttacagagac 1380 cagctcacag attatctgaa ccgggctatt gctgcaacgg gtagccagca tgaaccacag 1440 ctcctgaaag aggccctgtc atggcagcag cgctacctgg aaacagggag tatgaagtga 1500                                                                         1500 <210> 9 <211> 2676 <212> DNA <213> Unknown <220> <223> Escherichia coli MG1655 <220> <221> gene &Lt; 222 > (1) .. (2676) <223> gene coding AdhE <400> 9 atggctgtta ctaatgtcgc tgaacttaac gcactcgtag agcgtgtaaa aaaagcccag 60 cgtgaatatg ccagtttcac tcaagagcaa gtagacaaaa tcttccgcgc cgccgctctg 120 gctgctgcag atgctcgaat cccactcgcg aaaatggccg ttgccgaatc cggcatgggt 180 atcgtcgaag ataaagtgat caaaaaccac tttgcttctg aatatatcta caacgcctat 240 aaagatgaaa aaacctgtgg tgttctgtct gaagacgaca cttttggtac catcactatc 300 gctgaaccaa tcggtattat ttgcggtatc gttccgacca ctaacccgac ttcaactgct 360 atcttcaaat cgctgatcag tctgaagacc cgtaacgcca ttatcttctc cccgcacccg 420 cgtgcaaaag atgccaccaa caaagcggct gatatcgttc tgcaggctgc tatcgctgcc 480 ggtgctccga aagatctgat cggctggatc gatcaacctt ctgttgaact gtctaacgca 540 ctgatgcacc acccagacat caacctgatc ctcgcgactg gtggtccggg catggttaaa 600 gccgcataca gctccggtaa accagctatc ggtgtaggcg cgggcaacac tccagttgtt 660 atcgatgaaa ctgctgatat caaacgtgca gttgcatctg tactgatgtc caaaaccttc 720 gacaacggcg taatctgtgc ttctgaacag tctgttgttg ttgttgactc tgtttatgac 780 gctgtacgtg aacgttttgc aacccacggc ggctatctgt tgcagggtaa agagctgaaa 840 gctgttcagg atgttatcct gaaaaacggt gcgctgaacg cggctatcgt tggtcagcca 900 gcctataaaa ttgctgaact ggcaggcttc tctgtaccag aaaacaccaa gattctgatc 960 ggtgaagtga ccgttgttga tgaaagcgaa ccgttcgcac atgaaaaact gtccccgact 1020 ctggcaatgt accgcgctaa agatttcgaa gacgcggtag aaaaagcaga gaaactggtt 1080 gt; cgcgtttctt acttcggtca gaaaatgaaa acggcgcgta tcctgattaa caccccagcg 1200 tctcagggtg gtatcggtga cctgtataac ttcaaactcg caccttccct gactctgggt 1260 tgtggttctt ggggtggtaa ctccatctct gaaaacgttg gtccgaaaca cctgatcaac 1320 aagaaaaccg ttgctaagcg agctgaaaac atgttgtggc acaaacttcc gaaatctatc 1380 tacttccgcc gtggctccct gccaatcgcg ctggatgaag tgattactga tggccacaaa 1440 cgtgcgctca tcgtgactga ccgcttcctg ttcaacaatg gttatgctga tcagatcact 1500 tccgtactga aagcagcagg cgttgaaact gaagtcttct tcgaagtaga agcggacccg 1560 accctgagca tcgttcgtaa aggtgcagaa ctggcaaact ccttcaaacc agacgtgatt 1620 atcgcgctgg gtggtggttc cccgatggac gccgcgaaga tcatgtgggt tatgtacgaa 1680 catccggaaa ctcacttcga agagctggcg ctgcgcttta tggatatccg taaacgtatc 1740 tacaagttcc cgaaaatggg cgtgaaagcg aaaatgatcg ctgtcaccac cacttctggt 1800 acaggttctg aagtcactcc gtttgcggtt gtaactgacg acgctactgg tcagaaatat 1860 ccgctggcag actatgcgct gactccggat atggcgattg tcgacgccaa cctggttatg 1920 gacatgccga agtccctgtg tgctttcggt ggtctggacg cagtaactca cgccatggaa 1980 gcttatgttt ctgtactggc atctgagttc tctgatggtc aggctctgca ggcactgaaa 2040 ctgctgaaag aatatctgcc agcgtcctac cacgaagggt ctaaaaatcc ggtagcgcgt 2100 gaacgtgttc acagtgcagc gactatcgcg ggtatcgcgt ttgcgaacgc cttcctgggt 2160 gtatgtcact caatggcgca caaactgggt tcccagttcc atattccgca cggtctggca 2220 aacgccctgc tgatttgtaa cgttattcgc tacaatgcga acgacaaccc gaccaagcag 2280 actgcattca gccagtatga ccgtccgcag gctcgccgtc gttatgctga aattgccgac 2340 cacttgggtc tgagcgcacc gggcgaccgt actgctgcta agatcgagaa actgctggca 2400 tggctggaaa cgctgaaagc tgaactgggt attccgaaat ctatccgtga agctggcgtt 2460 caggaagcag acttcctggc gaacgtggat aaactgtctg aagatgcatt cgatgaccag 2520 tgcaccggcg ctaacccgcg ttacccgctg atctccgagc tgaaacagat tctgctggat 2580 acctactacg gtcgtgatta tgtagaaggt gaaactgcag cgaagaaaga agctgctccg 2640 gctaaagctg agaaaaaagc gaaaaaatcc gcttaa 2676 <210> 10 <211> 3180 <212> DNA <213> Unknown <220> <223> Clostridium acetobutylicum <220> <221> gene &Lt; 222 > (1) .. (3180) <223> gene coding AdhE2 <400> 10 atgaaagtta caaatcaaaa agaactaaaa caaaagctaa atgaattgag agaagcgcaa 60 aagaagtttg caacctatac tcaagagcaa gttgataaaa tttttaaaca atgtgccata 120 gccgcagcta aagaaagaat aaacttagct aaattagcag tagaagaaac aggaataggt 180 cttgtagaag ataaaattat aaaaaatcat tttgcagcag aatatatata caataaatat 240 aaaaatgaaa aaacttgtgg cataatagac catgacgatt ctttaggcat aacaaaggtt 300 gctgaaccaa ttggaattgt tgcagccata gttcctacta ctaatccaac ttccacagca 360 attttcaaat cattaatttc tttaaaaaca agaaacgcaa tattcttttc accacatcca 420 cgtgcaaaaa aatctacaat tgctgcagca aaattaattt tagatgcagc tgttaaagca 480 ggagcaccta aaaatataat aggctggata gatgagccat caatagaact ttctcaagat 540 ttgatgagtg aagctgatat aatattagca acaggaggtc cttcaatggt taaagcggcc 600 tattcatctg gaaaacctgc aattggtgtt ggagcaggaa atacaccagc aataatagat 660 gagagtgcag atatagatat ggcagtaagc tccataattt tatcaaagac ttatgacaat 720 ggagtaatat gcgcttctga acaatcaata ttagttatga attcaatata cgaaaaagtt 780 aaagaggaat ttgtaaaacg aggatcatat atactcaatc aaaatgaaat agctaaaata 840 aaagaaacta tgtttaaaaa tggagctatt aatgctgaca tagttggaaa atctgcttat 900 ataattgcta aaatggcagg aattgaagtt cctcaaacta caaagatact tataggcgaa 960 gtacaatctg ttgaaaaaag cgagctgttc tcacatgaaa aactatcacc agtacttgca 1020 atgtataaag ttaaggattt tgatgaagct ctaaaaaagg cacaaaggct aatagaatta 1080 ggtggaagtg gacacacgtc atctttatat atagattcac aaaacaataa ggataaagtt 1140 aaagaatttg gattagcaat gaaaacttca aggacattta ttaacatgcc ttcttcacag 1200 ggagcaagcg gagatttata caattttgcg atagcaccat catttactct tggatgcggc 1260 acttggggag gaaactctgt atcgcaaaat gtagagccta aacatttatt aaatattaaa 1320 agtgttgctg aaagaaggga aaatatgctt tggtttaaag tgccacaaaa aatatatttt 1380 aaatatggat gtcttagatt tgcattaaaa gaattaaaag atatgaataa gaaaagagcc 1440 tttatagtaa cagataaaga tttttttaaa cttggatatg ttaataaaat aacaaaggta 1500 ctagatgaga tagatattaa atacagtata tttacagata ttaaatctga tccaactatt 1560 gattcagtaa aaaaaggtgc taaagaaatg cttaactttg aacctgatac tataatctct 1620 attggtggtg gatcgccaat ggatgcagca aaggttatgc acttgttata tgaatatcca 1680 gaagcagaaa ttgaaaatct agctataaac tttatggata taagaaagag aatatgcaat 1740 ttccctaaat taggtacaaa ggcgatttca gtagctattc ctacaactgc tggtaccggt 1800 tcagaggcaa caccttttgc agttataact aatgatgaaa caggaatgaa atacccttta 1860 acttcttatg aattaccccc aaacatggca ataatagata ctgaattaat gttaaatatg 1920 cctagaaaat taacagcagc aactggaata gatgcattag ttcatgctat agaagcatat 1980 gtttcggtta tggctacgga ttatactgat gaattagcct taagagcaat aaaaatgata 2040 tttaaatatt tgcctagagc ctataaaaat gggactaacg acattgaagc aagagaaaaa 2100 atggcacatg cctctaatat tgcggggatg gcatttgcaa atgctttctt aggtgtatgc 2160 cattcaatgg ctcataaact tggggcaatg catcacgttc cacatggaat tgcttgtgct 2220 gtattaatag aagaagttat taaatataac gctacagact gtccaacaaa gcaaacagca 2280 ttccctcaat ataaatctcc taatgctaag agaaaatatg ctgaaattgc agagtatttg 2340 aatttaaagg gtactagcga taccgaaaag gtaacagcct taatagaagc tatttcaaag 2400 ttaaagatag atttgagtat tccacaaaat ataagtgccg ctggaataaa taaaaaagat 2460 ttttataata cgctagataa aatgtcagag cttgcttttg atgaccaatg tacaacagct 2520 aatcctaggt atccacttat aagtgaactt aaggatatct atataaaatc attttaagcc 2580 tatgtttcgg ttatggctac ggattatact gatgaattag ccttaagagc aataaaaatg 2640 atatttaaat atttgcctag agcctataaa aatgggacta acgacattga agcaagagaa 2700 aaaatggcac atgcctctaa tattgcgggg atggcatttg caaatgcttt cttaggtgta 2760 tgccattcaa tggctcataa acttggggca atgcatcacg ttccacatgg aattgcttgt 2820 gctgtattaa tagaagaagt tattaaatat aacgctacag actgtccaac aaagcaaaca 2880 gcattccctc aatataaatc tcctaatgct aagagaaaat atgctgaaat tgcagagtat 2940 ttgaatttaa agggtactag cgataccgaa aaggtaacag ccttaataga agctatttca 3000 aagttaaaga tagatttgag tattccacaa aatataagtg ccgctggaat aaataaaaaa 3060 gatttttata atacgctaga taaaatgtca gagcttgctt ttgatgacca atgtacaaca 3120 gctaatccta ggtatccact tataagtgaa cttaaggata tctatataaa atcattttaa 3180                                                                         3180 <210> 11 <211> 2556 <212> DNA <213> Unknown <220> <223> Salmonella typhimurium, Lactococcus lactis <220> <221> gene &Lt; 222 > (1) .. (2556) <223> gene coding PduP-AdhA <400> 11 catatgttga cggctagctc agtcctaggt acagtgctag cgattatgga taaggaggag 60 cctgcgatga acaccagcga actggaaacc ctgatccgta ccattctgag cgaacagtta 120 accaccccgg cacaaacccc ggttcaaccg caaggtaaag gcattttcca gagcgtttcc 180 gaagcgattg atgcagcaca tcaggcgttt ctgcgttatc aacagtgtcc gctgaaaacc 240 cgtagcgcta ttatctctgc gatgcgtcag gaactgaccc cgttattagc accgttagcc 300 gaagaaagcg ctaacgaaac cggtatgggc aacaaagagg acaaattcct gaaaaacaaa 360 gcggcgctgg ataacacccc gggcgttgaa gatttaacca ccaccgcatt aaccggcgac 420 ggcggtatgg ttctgtttga atacagcccg ttcggcgtta ttggctctgt tgcaccgagc 480 accaatccga ccgaaaccat catcaacaac tccatcagca tgctggcggc aggtaacagc 540 atttatttta gcccgcatcc gggcgcgaaa aaagttagcc tgaaactgat cagcctgatc 600 gaagaaattg cgtttcgttg ttgcggcatt cgtaacctgg ttgttaccgt tgcagaaccg 660 acctttgaag ctacccagca aatgatggcg catccgcgta ttgcggtttt agcaattacg 720 gt; gcaggtaatc cgccgtgtat tgttgacgaa accgcggatc tggtcaaagc ggcggaagat 840 attattaacg gcgcgagctt tgattataac ctgccgtgca tcgcggaaaa aagcctgatt 900 gtcgtcgaaa gcgtagcgga acgtttagtt cagcagatgc agacctttgg cgcattactg 960 ctgtctccgg cagataccga taaattacgc gcggtttgct taccggaagg ccaggcgaac 1020 aaaaaactgg ttggtaaatc cccgtctgcc atgttagaag cagcaggtat tgcagttccg 1080 gcaaaagcac cgcgtttact gattgcactg gttaacgctg acgatccgtg ggttacctct 1140 gaacaactga tgccgatgct gccggttgta aaagttagcg attttgatag cgcgctggca 1200 ttagcactga aagttgaaga gggcctgcat cataccgcaa ttatgcatag ccagaacgtc 1260 agccgtctga atttagcagc acgtaccctg caaaccagca tcttcgtcaa aaacggcccg 1320 agttacgcag gtattggcgt tggcggcgaa ggttttacca cctttaccat tgctaccccg 1380 accggtgaag gcaccacctc tgcacgtacc tttgcacgtt ctcgtcgttg cgttttaacg 1440 aacggcttta gcattcgcta agacgtcttg acggctagct cagtcctagg tacagtgcta 1500 gcaggaatgt aatcggagga ggaggaaatg aaagcggcgg tagttcgtca taatccggac 1560 ggttacgcgg atctggtcga aaaagaactg cgcgcaatta aaccgaacga agctctgctg 1620 gacatggaat actgcggcgt ttgtcatacc gatctgcacg tagctgctgg cgattatggt 1680 aacaaagcag gcaccgtgct gggtcatgaa ggtatcggca tcgtcaaaga aatcggcacc 1740 gatgttagta gcctgcaagt aggcgatcgt gttagcgttg cttggttttt cgaaggctgc 1800 ggccattgcg aatattgcgt ttctggtaac gagacctttt gccgcgaggt caaaaacgcg 1860 ggttattctg ttgacggcgg tatggctgaa gaagcgattg ttgtcgcgga ttacgcagtt 1920 aaagttccgg acggtctgga cccgattgaa gcaagtagca ttacctgcgc aggcgttacc 1980 acctacaaag cgatcaaagt tagcggcgtc aaaccgggcg attggcaggt aattttcggc 2040 gcaggtggtc tgggtaatct ggcaattcag tacgcgaaaa acgtcttcgg cgcaaaagtc 2100 atcgcggtcg atattaacca ggacaaactg aacctggcga aaaagattgg cgcagatgtg 2160 attattaaca gcggcgacgt taacccggtg gatgaaatca aaaagattac cggcggcctg 2220 ggtgcacaat ctgcgattgt gtgcgctgtt gcgcgtattg cgtttgaaca ggcagttgct 2280 tccctgaaac cgatgggtaa aatggtcgca gttgcactgc cgaataccga aatgaccctg 2340 tctgttccga cggttgtttt tgacggtgtt gaagttgcag gtagtctggt tggcacccgc 2400 ctggatctgg cagaagcatt tcagttcggc gcggaaggta aagttaaacc gattgtcgcg 2460 acccgtaaac tggaagaaat caacgacatc atcgacgaga tgaaagcggg caaaatcgag 2520 ggtcgtatgg tcatcgactt caccaaataa ctcgag 2556 <210> 12 <211> 444 <212> DNA <213> Unknown <220> <223> Megasphaera hexanoica <220> <221> gene &Lt; 222 > (1) <223> gene coding Acyl CoA transferase (ACT 27250) <400> 12 atgactggta ttgatgcaca tatttcatcg gcccacatgg tcctttcccg ggatctcaat 60 ccccacgata cgctcttcgc cgggcagggc acgtcctata tgatcgagtg cgccttcctg 120 gcggtacaga gttttctccg tacgccccat attgtctgtc tggggctgga cggcctgcgc 180 ttccttcatc ccgtccataa gggggataca atccgcgtag atagttccat cgtccatgcc 240 gggacgagcg gcatcggcgt gtatattact ctttcgctcc tgccggacgg accggtggcg 300 gcgtcgtgtt tcgtgtcgtt cgtccacatc gacgaagcga cggggcgggc tgtaccccac 360 ggtgtgacgc tggcggaact tccaccggag atggcccgcc ggcagaagca gtatctggca 420 tataaggaga tgacgagacg atga 444 <210> 13 <211> 37 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (37) <223> forward primer <400> 13 tatatggatc ccatgtacaa actttcacaa attgcag 37 <210> 14 <211> 34 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind &Lt; 222 > (1) <223> reverse primer <400> 14 tatataagct tttagtattc tgtcttgctc gtct 34 <210> 15 <211> 34 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind &Lt; 222 > (1) <223> forward primer <400> 15 tatatgatcc catgtcagaa tggacggata tgta 34 <210> 16 <211> 33 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (33) <223> reverse primer <400> 16 tatataagct tttaccgttt attgctcttg cgc 33 <210> 17 <211> 36 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (36) <223> forward primer <400> 17 tatatgagct ccatggatgt aatgcaagaa tatgcc 36 <210> 18 <211> 36 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (36) <223> reverse primer <400> 18 tatatgagct ccatggatgt aatgcaagaa tatgcc 36 <210> 19 <211> 39 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (39) <223> forward primer <400> 19 tatatgagct ccatgacaca atatgaaaca atgatatgaa 39 <210> 20 <211> 37 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (37) <223> reverse primer <400> 20 tatatgtcga cttaccaaat catgtatttg tcaacag 37 <210> 21 <211> 35 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) <223> forward primer <400> 21 tatatggatc ccatgatgaa tcaatggcag cgcat 35 <210> 22 <211> 37 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (37) <223> reverse primer <400> 22 tatataagct tttacacaat aatatgcatc tttctgg 37 <210> 23 <211> 37 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (37) <223> forward primer <400> 23 tatatggatc ccatggatta ccagagtgaa tacatga 37 <210> 24 <211> 33 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (33) <223> reverse primer <400> 24 tatataagct tttatcgttt atgagaagcg cgc 33 <210> 25 <211> 37 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (37) <223> forward primer <400> 25 tatatggatc ccatgaatcc attcgaaata tatcagg 37 <210> 26 <211> 36 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (36) <223> reverse primer <400> 26 tatataagct tttatttctt gttactgttc ctccag 36 <210> 27 <211> 36 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (36) <223> forward primer <400> 27 tatatgagct ccatgattga catttcagat cgcata 36 <210> 28 <211> 34 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind &Lt; 222 > (1) <223> reverse primer <400> 28 tatatgtcga cttacttcat actccctgtt tcca 34 <210> 29 <211> 37 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (37) <223> forward primer <400> 29 tatatggatc ccatgactgg tattgatgca catattt 37 <210> 30 <211> 34 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind &Lt; 222 > (1) <223> reverse primer <400> 30 tatataagct tttatcgtct cgtcatctcc ttat 34 <210> 31 <211> 26 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (26) <223> forward primer <400> 31 gcgccatatg gctgttacta atgtcg 26 <210> 32 <211> 26 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (26) <223> reverse primer <400> 32 gcgcctcgag ttaagcggat tttttc 26 <210> 33 <211> 41 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (41) <223> forward primer <400> 33 tatatcatat gatgaaagtt acaaatcaaa aagaactaaa a 41 <210> 34 <211> 43 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (43) <223> reverse primer <400> 34 tatatgagct cttaaaatga ttttatatag atatccttaa gtt 43 <210> 35 <211> 29 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (29) <223> forward primer <400> 35 gcgccatatg aacaccagcg aactggaaa 29 <210> 36 <211> 26 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (26) <223> reverse primer <400> 36 gcgcctcgag ttatttggtg aagtcg 26 <210> 37 <211> 70 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (70) <223> forward primer <400> 37 aaatattttt agtagcttaa atgtgattca acatcactgg agaaagtctt gtgtaggctg 60 gagctgcttc 70 <210> 38 <211> 70 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (70) <223> reverse primer <400> 38 attggggatt atctgaatca gctcccctgg gttgcagggg agcggcaaga tcctccttag 60 ttcctattcc 70 <210> 39 <211> 70 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (70) <223> forward primer <400> 39 cttaccctga agtacggggc tgtgggataa aaacaatctg gaggaatgtc gtgtaggctg 60 gagctgcttc 70 <210> 40 <211> 70 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (70) <223> reverse primer <400> 40 tatcgacttc cgggttatag cgcaccacct caattttcag gtttttcatc tcctccttag 60 ttcctattcc 70 <210> 41 <211> 70 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (70) <223> forward primer <400> 41 ggtgctgttt tgtaacccgc caaatcggcg gtaacgaaag aggataaacc gtgtaggctg 60 gagctgcttc 70 <210> 42 <211> 73 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (73) <223> reverse primer <400> 42 ttatttccgg ttcagatatc cgcagcgcaa agctgcggat gatgacgaga atatcctcct 60 tagttcctat tcc 73 <210> 43 <211> 70 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (43) <223> forward primer <400> 43 attcgagcag atgatttact aaaaaagttt aacattatca ggagagcatt gtgtaggctg 60 gagctgcttc 70 <210> 44 <211> 70 <212> DNA <213> Unknown <220> <223> Artificial Sequence <220> <221> primer_bind <222> (1) (70) <223> reverse primer <400> 44 aaaaaacggc cccagaaggg gccgtttata ttgccagaca gcgctactga tcctccttag 60 ttcctattcc 70

Claims (12)

A gene represented by the nucleotide sequence of SEQ ID NO: 1, a gene represented by the nucleotide sequence of SEQ ID NO: 3, a gene represented by the nucleotide sequence of SEQ ID NO: 4, a nucleotide sequence of SEQ ID NO: , A gene represented by the nucleotide sequence of SEQ ID NO: 6, a gene represented by the nucleotide sequence of SEQ ID NO: 7, and a gene represented by the nucleotide sequence of SEQ ID NO: 8, to the biosynthesis of the C3-C8 alcohol selected from the group consisting of Genes that encode enzymes involved. The method according to claim 1,
A gene coding for an enzyme involved in biosynthesis of a C3-C8 alcohol characterized in that each gene represented by the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 8 encodes an Acyl CoA transferase (ACT) enzyme. The method according to claim 1,
Each of the genes represented by the nucleotide sequences of SEQ ID NOS: 1 to 8 is involved in the biosynthesis of C3-C8 alcohol, which is derived from Megasphaera hexanoica strain deposited with Accession No. KCCM11835P Gene encoding the enzyme. The method according to claim 1,
Wherein the C3 alcohol is propanol, the C4 alcohol is butanol, the C5 alcohol is pentanol, the C6 alcohol is hexanol, the C7 alcohol is heptanol, the C8 alcohol is hexanol, A gene encoding an enzyme involved in biosynthesis of a C3-C8 alcohol characterized in that the alcohol is octanol. A gene encoding an enzyme involved in biosynthesis of the C3-C8 alcohol according to claim 1; And
A gene encoding an alcohol dehydrogenase (ADH) enzyme. 6. The method of claim 5,
Wherein the gene encoding the Alcohol Dehydrogenase (ADH) enzyme is represented by any one of the nucleotide sequences of SEQ ID NOS: 9 to 11. The method according to claim 6,
Wherein the gene represented by the nucleotide sequence of SEQ ID NO: 9 encodes Aldehyde-alcohol dehydrogenase (AdhE), the gene represented by the nucleotide sequence of SEQ ID NO: 10 encodes Aldehyde / alcohol dehydrogenase (AdhE2) Wherein the gene represented by the nucleotide sequence of SEQ ID NO: 1 encodes Propionaldehyde dehydrogenase-Alcohol dehydrogenase (PduP-AdhA). A microorganism having the ability to produce C3-C8 alcohols, transformed by the vector according to claim 5. 9. The method of claim 8,
Wherein the microorganism is selected from the group consisting of E. coli, bacteria, yeast and fungi. 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 C3-C8 alcohol by culturing the microorganism according to claim 8. 12. The method of claim 11,
Wherein at least one of C3 to C8 organic acids is added during the culture of the microorganism.

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