KR20070096347A - Mutants having a producing ability of 4-hydroxybutyrate and method for preparing 4hb using the same - Google Patents

Abstract

Mutants having highly producing ability of 4-hydroxybutyrate(4HB) and a method for producing 4-hydroxybutyrate by using the same mutants are provided to mass produce 4-hydroxybutyrate from highly succinate-producing microorganisms by inserting a 4-hydroxybutyrate-producing gene and deleting a succinate-producing gene. Mutants having highly producing ability of 4-hydroxybutyrate is characterized in that a gene encoding an enzyme converting succinate into 4-hydroxybutyrate is introduced or amplified and gene associated with conversion of succinate semialdehyde into succinate is deleted. The mutant is bacterium, yeast and fungus, wherein the bacterium is selected from Rumen bacterium, Corynebacterium sp., Brevibacterium sp. and Escherichia coli; the enzyme gene converting succinate into 4-hydroxybutyrate is selected from Cat1(succinyl-CoA transferase) gene of SEQ ID NO:1, SucD(succinate semialdehyde dehydrogenase) gene of SEQ ID NO:2, 4hbD(4-hydroxybutyrate dehydrogenase) gene of SEQ ID NO:3 and GHB(4-hydroxybutyrate dehydrogenase) gene of SEQ ID NO:4; and the gene associated with conversion of succinate semialdehyde into succinate is GabD(succinic semialdehyde dehydrogenase) gene of SEQ ID NO:5.

Description

Mutants having a Producing Ability of 4-Hydroxybutyrate and Method for Preparing 4HB Using the Same}

1 is a schematic diagram showing a route for producing 4HB from succinic acid.

The present invention relates to a variant having 4HB (4-hydroxybutyrate) generating ability and a method for producing 4HB using the same, and more specifically, to convert succinic acid (succinate) to 4HB (4-hydroxybutyrate) to microorganisms having high succinic acid performance The 4HB high-performance variant in which the gene of the enzyme was introduced and the gene involved in converting succinate semialdehyde to succinic acid and the variant was deleted, and the variant was cultured in a medium containing carbohydrate, and then 4HB (from the culture medium). It relates to a method for producing 4HB, characterized in that 4-hydroxybutyrate) is obtained.

Biodegradable polymer materials have been suggested as an alternative to synthetic polymer materials, which constitute a major axis of the pollution problem. Accordingly, various biodegradable polymer materials have been developed. One of them, poly-β-hydroxybutyrate, is a biodegradable polymer that accumulates in various microorganisms under nutritional imbalance, and has excellent properties such as biodegradability, moisture resistance, piezoelectricity, and biocompatibility. have. Among them, 4HB (4-hydroxybutyrate) is a representative example of polyhydroxyalkanoate (PHA), and has similar characteristics to polyester, and exhibits a wide range of physical properties ranging from crystalline plastics to high elastic rubber. Much research is underway as degradable plastics.

In addition, 4HB can be easily converted to various 4 carbon atoms such as 1,4-butanediol, gammabutyrolactone (GBL) and THF. The various chemicals are used throughout the chemical industry as polymers, solvents, fine chemical intermediates, and the like. Currently, most of the four carbon-carbon chemicals are synthesized from 1,4-butanediol, maleic anhydride, etc., but as the oil price rises, the production cost is increasing, and development of a process to supplement and replace the chemical production process is required. There is a situation. Thus, biological processes have been proposed as an alternative to chemical production.

On the other hand, succinic acid is a dicarboxylic acid having 4 carbon atoms and is a kind of organic acid produced when microorganisms are cultured under anaerobic conditions. Currently, various microorganisms are used as succinic acid producing strains, and production prices are decreasing due to the development of efficient fermentation and separation and purification processes. In addition, 4HB can be generated from succinic acid, and once produced, 4HB can be derived from various organic acids having 4 carbon atoms.

Representative patent applications related to the method for increasing the production efficiency of succinic acid include the applicant's international patent application publication number WO 2005/052135, in which the lumen producing high concentration of succinic acid with little other organic acid produced. Disclosed is a bacterial strain and a method for producing succinic acid using the same. In addition, Korean Patent Application No. 10-2004-60149 discloses a method for producing E. coli mutants capable of producing succinic acid at high concentration, and Korean Patent Application Nos. 10-2005-0076301, 10-2005-0076317, and 10-2005-0076348 discloses a method for producing succinic acid using a novel gene.

Thus, in the art using a microorganism capable of producing a high concentration of succinic acid disclosed in the patent, the variant having the ability to produce 4HB (4-hydroxybutyrate), which is a precursor of four carbon atoms chemically used throughout the chemical industry and this There is an urgent need for a method for biological preparation of 4HB.

Therefore, the present inventors have made efforts to prepare 4-hydroxybutyrate (4HB) using microorganisms capable of producing succinic acid at high concentrations, and as a result, 4HB biosynthesis related genes derived from Clostridium strain can be produced in large quantities. It was introduced into the microorganisms to obtain 4HB producing microbial variants, which confirmed that the microbial variants produced 4HB efficiently and completed the present invention.

As a result, the main object of the present invention is that the gene of the enzyme converting succinate to 4HB (4-hydroxybutyrate) is introduced or amplified, and the gene involved in converting succinate semialdehyde to succinic acid is deleted. It is to provide a variant having 4HB high performance characterized in that.

Another object of the present invention is to provide a method for producing 4HB, characterized in that after culturing the variant in a medium containing a carbohydrate, 4HG (4-hydroxybutyrate) from the culture solution.

In order to achieve the above object, the present invention is a microorganism having a high performance of succinic acid, the gene of the enzyme for converting succinate (succinate) to 4HB (4-hydroxybutyrate) is introduced or amplified, succinate semialdehyde (succinate semialdehyde) Provided is a variant having 4HB high performance, characterized in that the gene involved in the conversion to succinic acid is deleted.

In the present invention, the variant having high succinic acid performance may be selected from the group consisting of bacteria, yeast and fungi, wherein the bacteria are lumen bacteria, Corynebacterium genus, Brevibacterium ( Brevibacterium ) genus and E. coli may be selected from the group consisting of.

In the present invention, the lumen bacteria, a gene encoding lactic acid dehydrogenase ( ldhA ) and a gene encoding pyruvate-formic acid degrading enzyme ( pfl ) is deleted, and other organic acids are hardly produced under anaerobic conditions. It may be characterized by having a characteristic of producing succinic acid at a high concentration.

In the present invention, the lumen bacterium, a gene encoding lactic acid dehydrogenase ( ldhA ), a gene encoding pyruvate-formic acid degrading enzyme ( pfl ), a gene encoding phosphotransacetylase ( pta ) and acetic acid The gene encoding the kinase ( ackA ) is deleted, and in anaerobic conditions, it can be characterized in that it has a characteristic of generating succinic acid at high concentration with little generation of other organic acids.

In the present invention, the lumen bacteria, the gene encoding lactic acid dehydrogenase ( ldhA ), the gene encoding pyruvate-formic acid degrading enzyme ( pfl ) and the gene encoding phosphopyruvic acid carboxylase ( ppc ) is deleted In the anaerobic condition, the organic acid may be characterized by having a high concentration of succinic acid with little generation of other organic acids.

In the present invention, the lumen bacteria is genus Mannheimia sp .) , Actinobacillus sp. and Anaerobiospirillum sp. may be selected from the group consisting of, but is not limited thereto. In the case of the lumen bacteria, it may be characterized by the genus Mannheimia sp. , And preferably, the succinsis producers MBEL55E (KCTC 0769BP) and the genus LPK (KCTC 10558BP) , LPK4 and LPK7 (KCTC 10626BP) may be selected from the group consisting of.

In the present invention, the E. coli is deleted both the gene encoding the glucose phosphatase ( ptsG ) and the gene encoding pyruvate kinase ( pykA and pykF ), while generating little other organic acid under anaerobic conditions It may be characterized by having a characteristic of producing a high concentration of succinic acid, preferably, the E. coli variant may be characterized as W3110GFA.

In the present invention, the gene of the enzyme for converting the succinic acid to 4HB and the gene involved in converting the succinate semialdehyde to succinic acid may be characterized in that it is derived from Clostridium kluyveri . The gene of the enzyme for converting succinic acid into 4HB is a gene encoding Cat1 (succinyl-CoA transferase), a gene encoding sucD (succinate semialdehyde dehydrogenase), a gene encoding 4hbD (4-hydroxybutyrate dehydrogenase), and GHB (4 -hydroxybutyrate dehydrogenase) may be selected from the group consisting of genes.

In the present invention, the gene encoding Cat1 is SEQ ID NO: 1, the gene encoding SucD is SEQ ID NO: 2, the gene encoding 4hbD is SEQ ID NO: 3 and the gene encoding GHB has the nucleotide sequence of SEQ ID NO: 4 The gene involved in converting succinate semialdehyde into succinic acid may be a gene encoding Succinic semialdehyde dehydrogenase (GabD), and the gene encoding GabD is a sequence. It may be characterized by having the nucleotide sequence of the number 5.

In the present invention, the gene encoding the DctA (C4-dicarboxylate transport protein) involved in the transport of succinic acid may be further introduced or amplified, wherein the gene encoding the DctA is a base of SEQ ID NO: 6 It may be characterized by having a sequence.

In the present invention, a gene encoding GabD is deleted from a microorganism having high succinic acid performance, and a gene encoding Cat1, a gene encoding SucD, a gene encoding 4hbD, and a gene encoding GHB are all introduced or amplified. It provides a 4HB microbial variant having a high performance, characterized in that.

In the present invention, the gene encoding the DctA (C4-dicarboxylate transport protein) involved in the transport of succinic acid may be further introduced or amplified.

The present invention also provides a method for producing 4HB, wherein the variant is cultured in a medium containing carbohydrate, and 4HG (4-hydroxybutyrate) is obtained from the culture solution.

The present invention also provides a method for producing 4HB, wherein the variant is cultured in a medium containing carbohydrate, and 4HG (4-hydroxybutyrate) is obtained from the culture solution.

Hereinafter, the present invention will be described in detail.

Lumen bacteria among the succinic acid producing microorganisms of the present invention were prepared according to the method disclosed in the applicant's patent application WO 2005/052135. In other words, in the lumen bacterium Mannheimia succiniciproducens 55E, the lactic acid dehydrogenase gene ( ldhA ) and pyruvate- formatease gene ( pfl ) were deleted, resulting in mutant strain Mannheimia sp. LPK (KCTC 10558BP) was produced, and mutant strains ( Mannheimia ) were deleted from the phosphotransacetylacetylase gene ( pta ), acetic acid kinase gene ( ackA ) and phosphopyruvic acid carboxylase gene ( ppc ), respectively, in the LPK strain. sp. LPK7 and LPK4) were produced, and then cultured under anaerobic conditions, it was confirmed that succinic acid was produced in high yield.

In addition, among the succinic acid producing microorganism of the present invention, E. coli was prepared according to the method disclosed in the applicant's applicant's domestic patent publication No. 10-2006-0011345. That is, the gene coding for glucose phophotransferase (ptsG) and pyruvate kinase in the W3110 strain transformed with the recombinant expression vector pTrcEBG expressing bacteriophage red operon (exo-beta-gam) Two genes (pykA, pykF) encoding (pyruvate kinase) were deleted to obtain Escherichia coli mutant strain W3110GFA, which was cultured under anaerobic conditions, and W3110GFA was used rather than using the parent strain W3110 strain. When it was confirmed that the productivity is greatly improved.

In the present invention, enzymes for converting succinic acid to 4HB are enzymes used for 4HB (4-hydroxybutyate) biosynthesis derived from Clostridium kluyveri. Of course, Clostridium clui berry does not produce 4HB, but the enzymes cloned in the strains were found to play an important role in 4HB production.

In the present invention, the enzyme for converting succinic acid to 4HB is SucD (succinate) for converting succinate to succinyl-CoA and Cat1 (succinyl-CoA transferase) and succinyl-CoA to succinate semialdehyde, as shown in FIG. 4-hydroxybutyrate dehydrogenase (GHB) and 4-hydroxybutyrate dehydrogenase (GHB), which convert semialdehyde dehydrogenase) and succinate semialdehyde into 4-hydroxybutyrate.

In addition, in order to achieve the object of the present invention, it is very important to efficiently use succinic acid. To meet this, succinic acid-producing microorganisms are depleted by succinic semialdehyde dehydrogenase (gabD) which is involved in converting Succinic semialdehyde into Succininate. It is characterized by. In addition, it is possible to amplify the DctA enzyme involved in the transport of succinic acid for efficient in vivo delivery of succinic acid released into the body of the microorganism.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

In particular, the examples in the lumen of the bacteria, which produce a high concentration acid deletion mutant genetic characters from Mannheimia sp Mannheimia sp. Using only LPK (KCTC 10558BP), LPK7 and LPK4 and E. coli mutant strain W3110GFA, only 4HB was produced. It will be obvious to those of ordinary skill in the industry.

In addition, the following examples exemplify specific media and culture methods, but as reported in the literature (Lee et al. , Bioprocess Biosyst. Eng. , 26:63, 2003; Lee et al. , Appl. Microbiol. Biotechnol. , 58: 663, 2002; Lee et al, Biotechnol Lett, 25:......... 111, 2003; Lee et al, Appl Microbiol Biotechnol, 54:23, 2000; Lee et al, Biotechnol Bioeng. 72:41, 2001), or other mediums such as whey and corn steep liguor, or other methods such as fed-batch culture and continuous culture. It will be obvious to those of ordinary skill in the industry.

Example  1: Manufacturing method of microorganism having high succinic acid performance

1-1 Preparation of Lumen Bacteria with High Succinic Acid Performance

Lumen bacteria, the succinic acid producing microorganisms of the present invention, were prepared according to the method disclosed in the applicant's patent application WO 2005/052135. In other words, in the lumen bacterium Mannheimia succiniciproducens 55E, the lactic acid dehydrogenase gene ( ldhA ) and pyruvate- formatease gene ( pfl ) were deleted, resulting in mutant strain Mannheimia sp. LPK (KCTC 10558BP) was produced, and mutant strains ( Mannheimia ) were deleted from the phosphotransacetylacetylase gene ( pta ), acetic acid kinase gene ( ackA ) and phosphopyruvic acid carboxylase gene ( ppc ), respectively, in the LPK strain. sp. LPK7 and LPK4).

Preparation of Escherichia Coli with High Performance of 1-2 Succinic Acid

E. coli, a succinic acid-producing microorganism of the present invention, was prepared according to the method disclosed in Korean Patent Publication No. 10-2006-0011345 of the applicant. In other words, in the W3110 strain transformed with the recombinant expression vector pTrcEBG expressing the bacteriophage red operon (exo-beta-gam), the gene (ptsG) and pyruvate kinase encoding glucose phophotransferase Two genes (pykA, pykF) encoding (pyruvate kinase) were deleted to obtain E. coli mutant strain W3110GFA.

Example  2: 4 HB  Conversion enzyme Cloning

2-1 Cloning of Genes Encoding 4HB Converting Enzymes (Cat1, SucD, 4hbD, GHB)

The inventors have performed DNA polymerase chain reaction (PCR) using oligonucleotide primers synthesized based on a known gene sequence (L21902) for cloning the gene cat1 encoding Cat1 from Clostridium cluiberry DSM 555. cat1 gene was amplified.

Primers used in the PCR amplification experiment are as follows.

[SEQ ID NO: 7] cat1f-BspHI aaaaatcaat gagtaaaggg ataaagaatt cac

[SEQ ID NO: 8] cat1b-XbaI gctctagatt atttcatact accagttttt ataaa

PTrc99Cat1 was prepared by inserting the amplified cat1 gene into pTrc99A (Amersham Pharmacia Biotech) expression vector digested with Nco I / Xba I to make an expression vector.

In addition, the DNA polymerase chain reaction (PCR) was performed using oligonucleotide primers synthesized based on a known gene sequence (L21902) to clone the SucD and 4hbD operon genes. sucD4hbD gene was amplified.

Primers used in the PCR amplification experiment are as follows.

[SEQ ID NO: 9] SucDf-BspHI aaaaatcaat gagtaatgaa gtatctataa aag

[SEQ ID NO: 10] 4hbDb-XabI gctctagatt agataaaaaa gaggacattt cacaatatgg

PTrc99SucD4hbD was prepared by inserting the amplified sucD4hbD gene into pTrc99A (Amersham Pharmacia Biotech) expression vector digested with Nco I / Xba I to make an expression vector.

In addition, using an oligonucleotide primer synthesized based on a known gene sequence (L36817) to clone the GHB gene of SEQ ID NO: 4, a DNA polymerase chain reaction (PCR) was performed. The ghb gene was amplified.

Primers used in the PCR amplification experiment are as follows.

[SEQ ID NO: 11] H16 GHBf-BspHI aaaaatcaat ggcgtttatc tactatctg

[SEQ ID NO: 12] H16 GHBb-XbaI gctctagatt acatggactg ctcaagcata c

2-2 Cloning of the Gene Encoding DctA Involved in the Transport of Succinic Acid

For cloning the gene encoding DctA involved in the transport of succinic acid in Escherichia coli, an oligonucleotide primer synthesized based on a known gene sequence (NC_000913) was used to clone the gene dctA encoding the DctA of SEQ ID NO: 6 from Escherichia coli W3110. DNA polymerase chain reaction (PCR) dctA gene was amplified.

Primers used in the PCR amplification experiment are as follows.

[SEQ ID NO: 13] DctAf-EcoRI ggaattcatg aaaacctctc tgtttaaaag c

SEQ ID NO: 14 DctAb-XbaI gctctagatt aagaggataa ttcgtgcgtt ttgcc

The amplified dctA to make an expression vector The gene p10499A (Park et al ., FEMS Microbiol. Lett 214: 217, 2002) p10499DctA was prepared by cutting and inserting into an Eco RI / Xba I expression vector.

Example  3: 4 HB  With high performance Variant  making

3-1 Deletion of GabD-coding Genes in Succinic High-Performance Microorganisms

E. coli, a succinic acid-producing microorganism of the present invention, was prepared according to the method disclosed in Korean Patent Publication No. 10-2006-0011345 of the applicant. That is, the E. coli mutant strain W3110GFA-1, which lost the GabD gene of SEQ ID NO: 5 in the W3110GFA strain transformed with the recombinant expression vector pTrcEBG expressing bacteriophage red operon (exo-beta-gam), was obtained.

3-2 Introduction of Genes Prepared in Example 2

The 4HB converting enzyme clone cloned in Example 2 was introduced into W3110GFA-1 from which GabD was prepared in 3-1 was removed.

Example  4: 4 HB  purchase

W3110GFA-1 transformed with a recombinant plasmid expressing cat1 , sucD , 4 hbD , and dctA genes prepared in Example 3 was cultured as set forth in the applicant's patent 10-2006-0011345. That is, the Escherichia coli mutant strain (W3110GFA-1) prepared in Example 3 was cultured aerobically under medium conditions containing CaCO ₃ excess, and the product produced therefrom was analyzed.

First, 10 ml of LB medium was prepared, and inoculated with E. coli, followed by preculture at 37 ° C. for 12 hours. Then, 30 g glucose, 2 g KH ₂ PO ₄ , 10 g (NH ₄ ) ₂ SO ₄ · 7H ₂ O, 10 mg MnCl ₂ · 4H ₂ O, 10 mg FeSO ₄ · 4H ₂ O, 20 g CaCO ₃ , 50 mg FeSO ₄ per liter of distilled water 7H ₂ O, 10mg CaCl ₂ , 11mg ZnSO ₄ 7H ₂ O, 2.5mg MnSO ₄ 5H ₂ O, 5mg CuSO ₄ 5H ₂ O, 0.5mg (NH ₄ ) Mo ₇ O ₂₄ 4H ₂ 0, 0.1 5 ml of E. coli W3110GFA mutant strains were inoculated into a 250 ml flask containing 100 mg of medium having a pH of 7.0 NaB ₄ O ₇ 10H ₂ O and adjusted to pH 7.0 with KOH, and incubated at 30 ° C. for 120 hours at 120 rpm. Then, the produced 4HB was extracted with chloroform or ethyl acetate after lowering the pH of the culture solution to 2 with sulfuric acid.

As described in detail above, according to the present invention, there is provided a variant having 4HB (4-hydroxybutyrate) generating ability, which is a precursor of four carbon atoms, which is importantly used throughout the chemical industry, and a method of biologically manufacturing 4HB using the same. It works. In addition, 4HB prepared by the present invention can be easily converted to various 4 carbon atoms such as 1,4-BDO (1,4-butanediol), GBL (gammabutyrolactone) and THF (tetrahydrofuran).

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> LG Chem, Ltd. <120> Mutants Having a Producing Ability of 4-Hydroxybutyrate and          Method for Preparing 4HB Using the Same <130> P06-B024 <160> 14 <170> KopatentIn 1.71 <210> 1 <211> 1617 <212> DNA <213> Clostridium kluyveri <400> 1 atgagtaaag ggataaagaa ttcacaattg aaaaaaaaga atgtaaaggc tagtaatgtg 60 gcagaaaaga ttgaagagaa agttgaaaaa acagataagg ttgttgaaaa ggcagctgag 120 gttactgaaa aacgaattag aaacttgaag cttcaggaaa aagttgtaac agcagatgtg 180 gcagctgata tgatagaaaa cggtatgatt gttgcaatta gcggatttac tccttccggg 240 tatcctaaag aagtacctaa agcattgact aaaaaagtta atgccttaga ggaagaattc 300 aaggtaacac tttatacagg ttcatctaca ggagccgata tagacggaga atgggcaaaa 360 gcaggaataa tagaaagaag aattccatat cagacaaatt ctgatatgag gaaaaaaata 420 aatgatggtt ctattaagta tgctgatatg catttaagcc atatggctca atatattaat 480 tattctgtaa ttcctaaagt agatatagct ataatagagg cagtagctat tacagaagaa 540 ggggatatta ttccttcaac aggaattgga aatacagcta cttttgtgga aaatgcagat 600 aaggtaatag tggaaattaa tgaggctcaa ccgcttgaat tggaaggtat ggcagatata 660 tatacattaa aaaaccctcc aagaagagag cccataccta tagttaatgc aggcaatagg 720 atagggacca catatgtgac ctgtggttct gaaaaaatat gcgctatagt gatgacaaat 780 acccaggata aaacaagacc tcttacagaa gtgtctcctg tatctcaggc tatatccgat 840 aatcttatag gatttttaaa taaagaggtt gaagagggaa aattacctaa gaacctgctt 900 cctatacagt caggagttgg aagtgtagca aatgcagttt tggccggact ttgtgaatca 960 aattttaaaa atttgagttg ttatacagaa gttatacagg attctatgct gaagcttata 1020 aaatgtggta aagcagatgt ggtgtcaggc acttccataa gtccttcacc ggagatgttg 1080 cctgagttca taaaggacat aaatttcttt agagaaaaga tagtattaag accacaggaa 1140 ataagtaata atccagagat agcaagaaga ataggagtta tatccataaa cactgctttg 1200 gaagtagata tatatggtaa tgtaaactcc actcatgtta tgggaagcaa aatgatgaat 1260 ggtataggcg gttctggaga ctttgccaga aatgcatatt tgactatatt cactacagag 1320 tctatcgcca aaaaaggaga tatatcatct atagttccta tggtatccca tgtggatcat 1380 acagaacatg atgtaatggt aattgttaca gaacagggag tagcagattt aagaggtctt 1440 tctcctaggg aaaaggccgt ggctataata gaaaattgtg ttcatcctga ttacaaggat 1500 atgcttatgg aatattttga agaggcttgt aagtcatcag gtggaaatac accacataat 1560 cttgaaaaag ctctttcctg gcatacaaaa tttataaaaa ctggtagtat gaaataa 1617 <210> 2 <211> 1419 <212> DNA <213> Clostridium kluyveri <400> 2 atgagtaatg aagtatctat aaaagaatta attgaaaagg caaaggcggc acaaaaaaaa 60 ttggaagcct atagtcaaga acaagttgat gtactagtaa aagcactagg aaaagtggtt 120 tatgataatg cagaaatgtt tgcaaaagaa gcagttgaag aaacagaaat gggtgtttat 180 gaagataaag tagctaaatg tcatttgaaa tcaggagcta tttggaatca tataaaagac 240 aagaaaactg taggcataat aaaagaagaa cctgaaaggg cacttgttta tgttgctaag 300 ccaaagggag ttgtggcagc tactacgcct ataactaatc cagtggtaac tcctatgtgt 360 aatgcaatgg ctgctataaa gggcagaaat acaataatag tagcaccaca tcctaaagca 420 aagaaagttt cagctcatac tgtagaactt atgaatgctg agcttaaaaa attgggagca 480 ccagaaaata tcatacagat agtagaagca ccatcaagag aagctgctaa ggaacttatg 540 gaaagtgctg atgtagttat tgctacaggc ggtgctggaa gagttaaagc tgcttactcc 600 agtggaagac cagcttatgg cgttggacct ggaaattcac aggtaatagt tgataaggga 660 tacgattata acaaagctgc acaggatata ataacaggaa gaaaatatga caatggaatt 720 atatgttctt cagagcaatc agttatagct cctgctgaag attatgataa ggtaatagca 780 gcttttgtag aaaatggggc attctatgta gaagatgagg aaacagtaga aaagtttaga 840 tcaactttat ttaaagatgg aaaaataaac agcaagatta taggtaaatc cgtccaaatt 900 attgcggatc ttgcaggagt aaaagtacca gaaggtacta aggttatagt acttaagggt 960 aaaggtgcag gagaaaaaga tgtactttgt aaagaaaaaa tgtgtccagt tttagtagca 1020 ttgaaatatg atacttttga agaagcagtt gaaatagcta tggctaatta tatgtatgaa 1080 ggagctggtc atacagcagg catacattct gacaatgacg agaacataag atatgcaaga 1140 actgtattac ctataagcag attagttgta aatcagcctg caactactgc tggaggaact 1200 gtattaccta taagcagatt agttgtaaat cagcctgcaa ctactgctgg aggaagtttc 1260 aataatggat ttaaccctac tactacacta ggctgcggat catggggcag aaacagtatt 1320 tcagaaaatc ttacttacga gcatcttata aatgtttcaa gaatagggta tttcaataaa 1380 gaagcaaaag ttcctagcta tgaggaaata tggggataa 1419 <210> 3 <211> 1116 <212> DNA <213> Clostridium kluyveri <400> 3 atgaagttat taaaattggc acctgatgtt tataaatttg atactgcaga ggagtttatg 60 aaatacttta aggttggaaa aggtgacttt atacttacta atgaattttt atataaacct 120 ttccttgaga aattcaatga tggtgcagat gctgtatttc aggagaaata tggactcggt 180 gaaccttctg atgaaatgat aaacaatata attaaggata ttggagataa acaatataat 240 agaattattg ctgtaggggg aggatctgta atagatatag ccaaaatcct cagtcttaag 300 tatactgatg attcattgga tttgtttgag ggaaaagtac ctcttgtaaa aaacaaagaa 360 ttaattatag ttccaactac atgtggaaca ggttcagaag ttacaaatgt atcagttgca 420 gaattaaaga gaagacatac taaaaaagga attgcttcag acgaattata tgcaacttat 480 gcagtacttg taccagaatt tataaaagga cttccatata agttttttgt aaccagctcc 540 gtagatgcct taatacatgc aacagaagct tatgtatctc caaatgcaaa tccttatact 600 gatatgttta gtgtaaaagc tatggagtta attttaaatg gatacatgca aatggtagag 660 aaaggaaatg attacagagt tgaaataatt gaggattttg ttataggcag caattatgca 720 ggtatagctt ttggaaatgc aggagtggga gcggttcacg cactctcata tccaataggc 780 ggaaattatc atgtgcctca tggagaagca aattatctgt tttttacaga aatatttaaa 840 acttattatg agaaaaatcc aaatggcaag attaaagatg taaataaact attagcaggc 900 atactaaaat gtgatgaaag tgaagcttat gacagtttat cacaactttt agataaatta 960 ttgtcaagaa aaccattaag agaatatgga atgaaagagg aagaaattga aacttttgct 1020 gattcagtaa tagaaggaca gcagagactg ttggtaaaca attatgaacc tttttcaaga 1080 gaagacatag taaacacata taaaaagtta tattaa 1116 <210> 4 <211> 1149 <212> DNA <213> Clostridium kluyveri <400> 4 atggcgttta tctactatct gacccacatc cacctggatt tcggcgcggt aagcctgctc 60 aagtccgaat gcgagcgcat cggcatccgc cgcccgttgc tggtgaccga caagggcgtg 120 gtcgccgcgg gagtggcgca gcgtgccatc gatgcaatgc agggcctgca ggttgcggta 180 ttcgatgaaa ccccgtcgaa cccgaccgag gccatggtgc gcaaggccgc cgcacaatac 240 cgcgaggccg gctgcgacgg gctggtggca gtgggcggcg gctcgtcgat cgacctcgcc 300 aagggcatcg ccatcctggc cacgcatgag ggcgagctga ccacctatgc caccatcgaa 360 ggcggcagcg ccaggatcac cgacaaggcg gcgccgctga tcgcggtgcc caccacctcg 420 ggcaccggca gcgaggtggc gcgcggcgcc atcatcatcc tggacgacgg ccgcaagctg 480 ggcttccatt cctggcattt gctgcccaag tccgccgtct gcgacccgga actgacgctg 540 gggctgccgg ccgggctgac cgcggccacc ggcatggatg cgatcgcgca ctgcatcgag 600 accttcctgg cccccgcctt caacccgccc gcggacggca ttgcgctgga cgggctggag 660 cgcggctggg gccatatcga acgcgccacc cgcgacggtc aggaccgcga cgcacgcctg 720 aacatgatga gcgcgtcgat gcagggcgca atggcgttcc agaaggggct gggctgcgtg 780 cattcgctgt cgcacccgct gggcgggctg aagatcgacg gccgcaccgg cctgcaccac 840 ggcacgctca acgcggtggt gatgccggcg gtgctgcgct tcaacgccga tgcgcccacg 900 gtggtgcgcg acgaccgcta cgcacgcctg cgccgcgcca tgcacctgcc cgacggcgcc 960 gatatcgcgc aggccgtgca cgacatgacc gtgcgcctgg gcctgcccac cgggctgcgt 1020 cagatgggtg tcaccgagga catgttcgac aaggtgattg ccggtgcgct ggtcgaccat 1080 tgccacaaga ccaacccgaa agaagccagc gccgcggatt atcggcgtat gcttgagcag 1140 tccatgtag 1149 <210> 5 <211> 1449 <212> DNA <213> Escherichia coli <400> 5 atgaaactta acgacagtaa cttattccgc cagcaggcgt tgattaacgg ggaatggctg 60 gacgccaaca atggtgaagc catcgacgtc accaatccgg cgaacggcga caagctgggt 120 agcgtgccga aaatgggcgc ggatgaaacc cgcgccgcta tcgacgccgc caaccgcgcc 180 ctgcccgcct ggcgcgcgct caccgccaaa gaacgcgcca ccattctgcg caactggttc 240 aatttgatga tggagcatca ggacgattta gcgcgcctga tgaccctcga acagggtaaa 300 ccactggccg aagcgaaagg cgaaatcagc tacgccgcct cctttattga gtggtttgcc 360 gaagaaggca aacgcattta tggcgacacc attcctggtc atcaggccga taaacgcctg 420 attgttatca agcagccgat tggcgtcacc gcggctatca cgccgtggaa cttcccggcg 480 gcgatgatta cccgcaaagc cggtccggcg ctggcagcag gctgcaccat ggtgctgaag 540 cccgccagtc agacgccgtt ctctgcgctg gcgctggcgg agctggcgat ccgcgcgggc 600 gttccggctg gggtatttaa cgtggtcacc ggttcggcgg gcgcggtcgg taacgaactg 660 accagtaacc cgctggtgcg caaactgtcg tttaccggtt cgaccgaaat tggccgccag 720 ttaatggaac agtgcgcgaa agacatcaag aaagtgtcgc tggagctggg cggtaacgcg 780 ccgtttatcg tctttgacga tgccgacctc gacaaagccg tggaaggcgc gctggcctcg 840 aaattccgca acgccgggca aacctgcgtc tgcgccaacc gcctgtatgt gcaggacggc 900 gtgtatgacc gttttgccga aaaattgcag caggcagtga gcaaactgca catcggcgac 960 gggctggata acggcgtcac catcgggccg ctgatcgatg aaaaagcggt agcaaaagtg 1020 gaagagcata ttgccgatgc gctggagaaa ggcgcgcgcg tggtttgcgg cggtaaagcg 1080 cacgaacgcg gcggcaactt cttccagccg accattctgg tggacgttcc ggccaacgcc 1140 aaagtgtcga aagaagagac gttcggcccc ctcgccccgc tgttccgctt taaagatgaa 1200 gctgatgtga ttgcgcaagc caatgacacc gagtttggcc ttgccgccta tttctacgcc 1260 cgtgatttaa gccgcgtctt ccgcgtgggc gaagcgctgg agtacggcat cgtcggcatc 1320 aataccggca ttatttccaa tgaagtggcc ccgttcggcg gcatcaaagc ctcgggtctg 1380 ggtcgtgaag gttcgaagta tggcatcgaa gattacttag aaatcaaata tatgtgcatc 1440 ggtctttaa 1449 <210> 6 <211> 1287 <212> DNA <213> Escherichia coli <400> 6 atgaaaacct ctctgtttaa aagcctttac tttcaggtcc tgacagcgat agccattggt 60 attctccttg gccatttcta tcctgaaata ggcgagcaaa tgaaaccgct tggcgacggc 120 ttcgttaagc tcattaagat gatcatcgct cctgtcatct tttgtaccgt cgtaacgggc 180 attgcgggca tggaaagcat gaaggcggtc ggtcgtaccg gcgcagtcgc actgctttac 240 tttgaaattg tcagtaccat cgcgctgatt attggtctta tcatcgttaa cgtcgtgcag 300 cctggtgccg gaatgaacgt cgatccggca acgcttgatg cgaaagcggt agcggtttac 360 gccgatcagg cgaaagacca gggcattgtc gccttcatta tggatgtcat cccggcgagc 420 gtcattggcg catttgccag cggtaacatt ctgcaggtgc tgctgtttgc cgtactgttt 480 ggttttgcgc tccaccgtct gggcagcaaa ggccaactga tttttaacgt catcgaaagt 540 ttctcgcagg tcatcttcgg catcatcaat atgatcatgc gtctggcacc tattggtgcg 600 ttcggggcaa tggcgtttac catcggtaaa tacggcgtcg gcacactggt gcaactgggg 660 cagctgatta tctgtttcta cattacctgt atcctgtttg tggtgctggt attgggttca 720 atcgctaaag cgactggttt cagtatcttc aaatttatcc gctacatccg tgaagaactg 780 ctgattgtac tggggacttc atcttccgag tcggcgctgc cgcgtatgct cgacaagatg 840 gagaaactcg gctgccgtaa atcggtggtg gggctggtca tcccgacagg ctactcgttt 900 aaccttgatg gcacatcgat atacctgaca atggcggcgg tgtttatcgc ccaggccact 960 aacagtcaga tggatatcgt ccaccaaatc acgctgttaa tcgtgttgct gctttcttct 1020 aaaggggcgg caggggtaac gggtagtggc tttatcgtgc tggcggcgac gctctctgcg 1080 gtgggccatt tgccggtagc gggtctggcg ctgatcctcg gtatcgaccg ctttatgtca 1140 gaagctcgtg cgctgactaa cctggtcggt aacggcgtag cgaccattgt cgttgctaag 1200 tgggtgaaag aactggacca caaaaaactg gacgatgtgc tgaataatcg tgcgccggat 1260 ggcaaaacgc acgaattatc ctcttaa 1287 <210> 7 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 aaaaaatcaat gagtaaaggg ataaagaatt cac 33 <210> 8 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gctctagatt atttcatact accagttttt ataaa 35 <210> 9 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 aaaaaatcaat gagtaatgaa gtatctataa aag 33 <210> 10 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gctctagatt agataaaaaa gaggacattt cacaatatgg 40 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 aaaaatcaat ggcgtttatc tactatctg 29 <210> 12 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gctctagatt acatggactg ctcaagcata c 31 <210> 13 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 ggaattcatg aaaacctctc tgtttaaaag c 31 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 gctctagatt aagaggataa ttcgtgcgtt ttgcc 35

Claims (22)

In a microorganism having high succinic acid performance, a gene of an enzyme converting succinate to 4HB (4-hydroxybutyrate) is introduced or amplified, and a gene involved in converting succinate semialdehyde into succinic acid is deleted. Variants having 4HB high performance, characterized in that there is. The variant according to claim 1, wherein the variant having high succinic acid performance is selected from the group consisting of bacteria, yeast and mold. The variant according to claim 2, wherein the bacterium is selected from the group consisting of lumen bacteria, Corynebacterium genus, Brevibacterium genus and Escherichia coli. The method of claim 3, wherein the lumen bacteria, the gene encoding lactic acid dehydrogenase ( ldhA ) and the gene encoding pyruvate-formic acid degrading enzyme ( pfl ) is deleted, almost no other organic acid produced under anaerobic conditions Variant characterized in that it has the property of producing a high concentration of succinic acid without. The method of claim 3, wherein the lumen bacterium is a gene encoding lactic acid dehydrogenase ( ldhA ), a gene encoding pyruvate-formic acid degrading enzyme ( pfl ), a gene encoding phosphotransacetylacetylase ( pta ) and A variant, characterized in that the gene encoding acetic acid kinase ( ackA ) is deleted and that succinic acid is produced at high concentration with little generation of other organic acids under anaerobic conditions. The method of claim 3, wherein the lumen bacterium is a gene encoding lactic acid dehydrogenase ( ldhA ), a gene encoding pyruvate-formic acid degrading enzyme ( pfl ) and a gene encoding phosphopyruvic acid carboxylase ( ppc ). A variant, characterized in that it has been deleted and has a characteristic of producing high concentrations of succinic acid in the anaerobic conditions with little generation of other organic acids. 7. The lumen bacterium of claim 4, wherein the lumen bacterium is of the genus Mannheimia. sp .) , Actinobacillus sp .) and Unerobiospirium sp. ( Anaerobiospirillum sp.) variants. 8. The variant according to claim 7, wherein the lumen bacterium is of the genus Mannheimia sp . The method of claim 8, wherein the lumen bacteria are selected from the group consisting of S. nichia succinsis producers MBEL55E (KCTC 0769BP), the genus LPK (KCTC 10558BP), LPK4 and LPK7 (KCTC 10626BP) Variants. The method of claim 3, wherein the E. coli is deleted both the gene encoding the glucose phosphatase ( ptsG ) and the gene encoding pyruvate kinase ( pykA and pykF ), and rarely other organic acids are produced under anaerobic conditions A variant that has the characteristic of producing succinic acid at a high concentration. The variant of claim 10, wherein the E. coli variant is W3110GFA. According to claim 1, wherein the gene of the enzyme for converting the succinic acid to 4HB and the gene involved in the conversion of the succinate semialdehyde (succinate semialdehyde) to succinic acid variant, characterized in that derived from Clostridium kluyveri . According to claim 1, wherein the gene of the enzyme for converting succinic acid to 4HB is a gene encoding Cat1 (succinyl-CoA transferase), a gene encoding a succinate semialdehyde dehydrogenase (SucD), 4hbD (4-hydroxybutyrate dehydrogenase) And a gene selected from the group consisting of genes encoding 4-hydroxybutyrate dehydrogenase (GHB). The method of claim 13, wherein the gene encoding Cat1 is SEQ ID NO: 1, the gene encoding SucD is SEQ ID NO: 2, 4hbD gene is a gene encoding SEQ ID NO: 3 and GHB is the nucleotide sequence of SEQ ID NO: 4 Variants characterized by having. The variant according to claim 1, wherein the gene involved in converting succinate semialdehyde into succinic acid is a gene encoding Succinic semialdehyde dehydrogenase (GabD). The variant according to claim 15, wherein the gene encoding GabD has a nucleotide sequence of SEQ ID NO: 5. 17. The variant according to any one of claims 1 to 16, wherein the gene encoding DctA (C4-dicarboxylate transport protein) involved in the transport of succinic acid is further introduced or amplified. The variant according to claim 17, wherein the gene encoding the DctA has a nucleotide sequence of SEQ ID NO: 6. In the microorganism having high succinic acid performance, the gene encoding GabD is deleted, and the gene encoding Cat1, the gene encoding SucD, the gene encoding 4hbD and the gene encoding GHB are all introduced or amplified. Microbial variant having 4HB high performance. 20. The variant according to claim 19, wherein the gene encoding DctA (C4-dicarboxylate transport protein) involved in the transport of succinic acid is further introduced or amplified. 17. The preparation of 4HB, wherein the variant of any one of claims 1 to 6 and 10 to 16 is cultured in a medium containing carbohydrate, and then 4HG (4-hydroxybutyrate) is obtained from the culture solution. Way. The method of claim 4, wherein the mutant of claim 19 or 20 is cultured in a medium containing carbohydrate, and then 4HG (4-hydroxybutyrate) is obtained from the culture solution.

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