KR20140122606A - Microorganism for biosynthesis of adipate and method for producing adipate using the same - Google Patents

Abstract

According to an embodiment of the present invention, a microorganism including polynucleotide coding succinyl-CoA:acetyl-Coa transferase or mutant thereof, polynucleotide coding 3-oxoadipil-CoA transferase or mutant thereof, polynucleotide coding ADH6 protein or mutant thereof, polynucleotide coding BDHA protein or mutant thereof, polynucleotide coding 4HD protein or mutant thereof and polynucleotide coding ALD5 protein or mutant thereof can produce adipate effectively by having a new metabolic pathway through introduction of exotic gene. This transformed microorganism can produce adipate efficiently as compared with chemical synthesis, and thus can be used industrially usefully.

Description

TECHNICAL FIELD The present invention relates to a microorganism for adipate biosynthesis and a method for producing adipate using the same.

To a microorganism having a novel target path capable of synthesizing adipate and a method for efficiently producing adipate using the same.

The adipate is one of the organic acids having the formula C ₆ H ₁₀ O ₄ . In nature, it is also contained in plants such as sugar beet. Nylon resin raw materials, plastic plasticizers, dyes, pharmaceuticals, and the like. In particular, it can be used to produce nylon which is a linear polyamide produced by condensing an adipate with hexamethylenediamine. It is used to make different kinds of fibers.

Other uses for adipates are for the production of plasticizers, unsaturated polyesters and polyester polyols. Other applications include polyurethane, its use in the production of lubricant components, and its use as a food ingredient, such as flavorants and gelling aids.

Adipates were prepared from various fats using oxidation. Some chemical synthesis processes for adipate synthesis use excessive amounts of strong nitric acid and may also be based on the oxidation of cyclohexanone, ketone, cyclohexanol or pure cyclohexanol.

However, with the increasing demand for adipate, a variety of methods have been developed which can produce adipates more efficiently than these chemical syntheses. Recently, a method of producing adipate using microorganisms has been studied. However, up to now, a method for efficiently producing adipate using microorganisms has not been studied much.

It has been confirmed that adipate can be synthesized using a new metabolic pathway while studying biosynthesis of adipate using microorganisms.

One aspect provides a microorganism comprising a polynucleotide encoding an ADH6 protein or variant thereof and a polynucleotide encoding an ALD5 protein or variant thereof.

Another aspect provides a method for culturing the microorganism to produce an adipate.

One aspect is a polynucleotide encoding succinyl-coA: acetylcoaitransferase or variant thereof, a polynucleotide encoding 3-oxoadipyl-coAtransferase or a variant thereof, an alcohol dehydrogenase 6 (ADH6) protein or A polynucleotide encoding a variant thereof, a polynucleotide encoding a D-beta-hydroxybutyrate dehydrogenase (BDHA) protein or a variant thereof, a polynucleotide encoding a 4HBD (NAD-dependent 4-hydroxybutyrate dehydrogenase) protein or a mutant thereof, and a polynucleotide encoding an aldehyde dehydrogenase 5) protein or a polynucleotide encoding a variant thereof.

At this time, succinyl-co A: acetylcoaitransferase is an enzyme having a function of converting succinyl-coe and acetyl coenzyme to 3-oxoadipyl-coenzyme. The succinyl-CoA: acetyl CoAtransferase may be one derived from Aspinobacterium, E. coli, Pseudomonas aeruginosa or Pseudomonas putida. The protein may have the amino acid sequence of SEQ ID NO: 1. In addition, the protein may be a succinyl-CoA: acetyl CoAtransferase variant in which one or more amino acids are substituted, added or deleted as long as the protein retains its activity. The polynucleotide encoding succinyl-CoA: acetyl koAt transferase may have the nucleotide sequence of SEQ ID NO: 2.

In addition, a polynucleotide encoding succinyl-CoA: acetyl CoAtransferase having the amino acid sequence of SEQ ID NO: 1 may be included in the vector. The usable vector is not particularly limited, and known expression vectors can be used. In addition, the vector may include a promoter.

In addition, 3-oxoadipyl-coAtransferase is an enzyme having a function of converting 3-oxoadipyl-coe and succinate into 3-oxo adipate. The 3-oxoadipyl-coAtransferase may be one derived from Asnithobacterium, Escherichia coli, Pseudomonas aeruginosa or Pseudomonas putida. The protein may have the amino acid sequence of SEQ ID NO: 3. In addition, the protein may be a succinyl-CoA: acetyl CoAtransferase variant in which one or more amino acids are substituted, added or deleted as long as the protein retains its activity. The polynucleotide encoding the 3-oxoadipyl-coaitransferase may have the nucleotide sequence of SEQ ID NO: 4.

In addition, the polynucleotide encoding the 3-oxoadipyl-coAtransferase having the amino acid sequence of SEQ ID NO: 3 may be included in the vector. The usable vector is not particularly limited, and known expression vectors can be used. In addition, the vector may include a promoter.

The ADH6 protein is also referred to as alcohol dehydrogenase 6, and is an enzyme having a function of converting alcohol into aldehyde or ketone, or converting aldehyde or ketone into alcohol. At this time, it functions by reducing the nicotinamide adenine dinucleotide. The ADH6 protein may be derived from Saccharomyces cerevisiae or Mycobacterium tuberculosis. The ADH6 protein may have the amino acid sequence of SEQ ID NO: 5. In addition, the protein may be an ADH6 variant in which one or more amino acids are substituted, added or deleted as long as the protein retains its activity. The polynucleotide encoding the ADH6 protein may have the nucleotide sequence of SEQ ID NO: 6.

In addition, a polynucleotide encoding ADH6 having the amino acid sequence of SEQ ID NO: 5 may be included in the vector. The usable vector is not particularly limited, and known expression vectors can be used. In addition, the vector may comprise a promoter.

The ALD5 protein is also called an aldehyde dehydrogenase 5, and is an enzyme having a function of oxidizing aldehyde. The ALD5 protein may be derived from Saccharomyces cerevisiae. The ALD5 protein may have the amino acid sequence of SEQ ID NO: 7. In addition, the protein may be an ALD5 variant in which one or more amino acids are substituted, added or deleted, so long as the protein retains its activity. The polynucleotide encoding the ALD5 protein may have the nucleotide sequence of SEQ ID NO: 8.

In addition, a polynucleotide encoding ALD5 having the amino acid sequence of SEQ ID NO: 7 may be included in the vector. The usable vector is not particularly limited, and known expression vectors can be used. In addition, the vector may include a promoter.

The BDHA protein is also referred to as D-beta-hydroxybutyrate dehydrogenase. It is an enzyme having a function of converting an alcohol to an aldehyde or a ketone, or converting an aldehyde or a ketone to an alcohol. At this time, it functions by reducing the nicotinamide adenine dinucleotide. The BDHA protein may be derived from (Ribium meliloti). The BDHA protein may have the amino acid sequence of SEQ ID NO: 9. In addition, the protein may be a BDHA mutant in which one or more amino acids are substituted, added or deleted, so long as the protein retains its activity. The polynucleotide encoding the BDHA protein may have the nucleotide sequence of SEQ ID NO.

In addition, a polynucleotide encoding ADH6 having the amino acid sequence of SEQ ID NO: 5 may be included in the vector. The usable vector is not particularly limited, and known expression vectors can be used. In addition, the vector may comprise a promoter.

The 4HBD protein is also called 4-hydroxybutyrate dehydrogenase, and is an enzyme having a function of converting an alcohol into an aldehyde or a ketone, or converting an aldehyde or a ketone into an alcohol. At this time, it functions by reducing the nicotinamide adenine dinucleotide. The 4HBD protein may be derived from Clostridium clubeieri. The 4HBD protein may have the amino acid sequence of SEQ ID NO: 11. In addition, the protein may be a 4HBD variant in which one or more amino acids are substituted, added or deleted, so long as the protein retains its activity. The polynucleotide encoding the 4HBD protein may have the nucleotide sequence of SEQ ID NO: 12.

Further, a polynucleotide encoding 4HBD having the amino acid sequence of SEQ ID NO: 11 may be included in the vector. The usable vector is not particularly limited, and known expression vectors can be used. In addition, the vector may comprise a promoter.

The promoter may be a lambda PL promoter, trp promoter, lac promoter, T7 promoter, pPro promoter, or gapA promoter.

In addition, the promoter is operably linked to a sequence encoding the gene. The term "operably linked" means a functional linkage between a nucleotide sequence (e.g., a promoter, a signal sequence, an array of transcription factor binding sites, a terminator or an enhancer) and other nucleotide sequences, Regulates transcription and / or translation of the nucleotide sequence encoding the gene.

At this time, the microorganism into which the polynucleotide is introduced may be general microorganisms that synthesize succinylcohols including Escherichia coli and acetylcohols

In addition, a vector containing the polynucleotide may be introduced into the microorganism to transform the microorganism.

As used herein, the term " transformation " means introducing a gene into a microorganism so that it can be expressed in a microorganism. The transformed gene may be either inserted into the chromosome of the microorganism or located outside the chromosome, so long as it can be expressed in the microorganism. In addition, the gene includes DNA and RNA as a polynucleotide capable of encoding a polypeptide. The gene may be introduced in any form as long as it can be introduced and expressed in microorganisms. For example, the gene may be introduced into the microorganism in the form of an expression cassette, which is a polynucleotide construct containing all the elements necessary for its expression. The expression cassette typically includes a promoter operably linked to the gene, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. In addition, the gene may be introduced into the host cell itself or in the form of a polynucleotide construct, and operably linked to the sequence necessary for expression in the microorganism.

In another aspect, there is provided a polynucleotide encoding succinyl-CoA: acetylcoautitransferase or a variant thereof, a polynucleotide encoding 3-oxoadipyl-coAtransferase or a variant thereof, an alcohol dehydrogenase 6 (ADH6) Polynucleotide encoding a D-beta-hydroxybutyrate dehydrogenase (BDHA) protein or variant thereof, a polynucleotide encoding a 4HBD (NAD-dependent 4-hydroxybutyrate dehydrogenase) protein or a variant thereof, and a polynucleotide encoding ALD5 aldehyde dehydrogenase 5) a polynucleotide encoding a protein or a variant thereof; And obtaining an adipate in the culture.

At this time, the microorganism may be E. coli. In addition, the microorganism can synthesize acetyl-CoA and succinyl-CoA.

The cultivation of the microorganism having the ability to produce a fermentation product can be carried out according to a suitable culture medium and culture conditions known in the art. Such a culturing process can be easily adjusted by those skilled in the art depending on the microorganism selected. Methods of culturing include, but are not limited to, batch, continuous, and fed-batch cultivation.

The medium used for the culture should suitably meet the requirements of the particular microorganism. The medium comprises various carbon sources, nitrogen sources and trace element components.

Carbon sources that can be used for the microorganism culture medium include glucose, sucrose, lactose, fructose, carbohydrates such as maltose, starch and cellulose, fats such as soybean oil, sunflower oil, castor oil and coconut oil, fatty acids such as palmitic acid, stearic acid and linoleic acid , Glycerol and alcohols such as ethanol, organic acids such as acetic acid, but are not limited thereto. The carbon sources may be used alone or in combination.

Nitrogen sources available for microbial culture media include organic nitrogen sources such as peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL) and soybean wheat, and urea, such as ammonium sulfate, ammonium phosphate, ammonium carbonate, ammonium nitrate But are not limited to, inorganic nitrogen sources. The nitrogen sources may be used alone or in combination.

The microbial culture medium may comprise potassium dihydrogenphosphate, dipotassium hydrogenphosphate and the corresponding sodium-containing salts as a source of phosphorus. It may also include metal salts such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins, and suitable precursors and the like may be included in the medium. The microorganism culture medium or the individual components may be added to the culture solution batchwise or continuously.

In addition, during culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the microorganism culture medium in an appropriate manner to adjust the pH of the culture medium. In addition, bubble formation can be suppressed by using a defoaming agent such as fatty acid polyglycol ester during the culture.

In order to maintain the aerobic state of the culture liquid, oxygen or an oxygen-containing gas (for example, air) is injected into the culture liquid. In addition, oxygen may be blocked or nitrogen may be injected to maintain the anaerobic state of the culture liquid. The temperature of the culture medium is usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C. The incubation period can be continued until the desired amount of quinolinic acid is obtained, preferably 10 to 160 hours.

Microorganisms according to one aspect did not produce adipate in a wild-type state. However, introduction of a foreign gene has a new metabolic pathway, and thus it is possible to effectively produce adipate. These transformed microorganisms can produce adipate efficiently as compared with chemical synthesis, and thus can be industrially useful.

Figure 1 shows a new pathway for biosynthesis of adipate.

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

EXAMPLES Example 1: Path discovery for biosynthesis of adipate

The reaction mechanism of the enzyme was analyzed and the starting material and the production material were summarized. In addition, species specificity of the enzyme was summarized. In addition, various reaction pathways for the production of adipate in the presence of Gibbs free energy in the action of the enzyme were discovered through computer simulation.

The pathway considered to be the most efficient in the synthesis of the double adipate is as follows, and the enzymes acting on the pathway are shown in Tables 1 and 2 below.

<Reaction Scheme 1>

3-Oxoadipate -> 3-Hydroxyhexanediolic acid -> Adipic acid

Adipate synthesis route 1: conversion of 3-oxo adipate to 3-hydroxyhexanediol acid Gene name Protein name GenBankID Organism Reference uniprotID E.C no. ADH6 NADP-dependent alcohol dehydrogenase 6 Z54141 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (Baker's yeast) 11742541 Q04894 1.1.1.2 bdhA D-beta-hydroxybutyrate dehydrogenase AL591985 Rhizobium meliloti 9922248 O86034 1.1.1.30 4hbD NAD-dependent 4-hydroxybutyrate dehydrogenase CP000673 Clostridium kluyveri 8550525 P38945 1.1.1.61

Adipate synthesis route 2: conversion of 3-hydroxyhexanediol acid to adipate Gene name Protein name GenBankID Organism Reference uniprotID E.C no. ALD5 Aldehyde dehydrogenase 5, mitochondrial U56605 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (Baker's yeast) 9473035 P40047 1.2.1.4

To confirm that the new adipate biosynthetic pathway actually works in microorganisms, microorganisms containing enzymes that operate in the novel adipate synthesis pathway were prepared.

Example 2. Preparation of a strain producing adipate

2.1. Cloning gene related to adipate production and production of recombinant vector for chromosome insertion

(Pseudomonas putida KT2440), 3-oxoadipyl-coAtransferase (Saccraomyces cerevisiae / Mycobacterium tuberculosis, adh6, Information on the gene information and the surrounding base sequence of bdhA, 4hbD (Table 1 microorganism) and ald5 (microorganism of Table 2) were obtained from NIH GeneBank of the US National Institutes of Health. Primers were prepared.

A polynucleotide encoding succinyl-co A: acetylcoaitransferase from Saccharomyces cerevisiae using a primer, a polynucleotide encoding 3-oxoadipyl-coaitransferase, a polynucleotide encoding an ADH6 protein Polynucleotides, polynucleotides encoding bdhA, polynucleotides encoding 4hbD, and polynucleotides encoding ALD5 protein were cloned.

The thus cloned polynucleotides were introduced into a vector to which the gapA promoter was coupled.

2.2. Introduction of adipate production-related genes

Vector comprising a polynucleotide encoding succinyl-CoA: acetyl coaitransferase prepared in Example 2.1), a vector comprising a polynucleotide encoding 3-oxoadipyl-coAtransferase, a vector comprising a polynucleotide encoding ADH6 protein , A vector comprising a polynucleotide encoding bdhA, a vector comprising a polynucleotide encoding hbD, and a vector comprising a polynucleotide encoding an ALD5 protein, into a Escherichia coli Respectively.

Example 3. Production of adipate using a strain producing adipate

The transformed Escherichia coli was inoculated into a 250 ml corn-baffle flask containing 25 ml of the complex liquid medium and inoculated with Escherichia coli that had not been transformed, Escherichia coli with only one of the genes introduced therein, and a strain into which both genes were introduced, 0.0 &gt; 200 &lt; / RTI &gt; rpm for 24 hours. Adipate concentrations were measured at constant time intervals. As a result, it was confirmed that adipate was produced in Escherichia coli in which adh6 and ald5 genes were introduced. Herein, the Escherichia coli is a strain including a vector comprising a polynucleotide encoding succinyl-CoA: acetyl CoAtransferase and a vector comprising a polynucleotide encoding 3-oxoadipyl-coAtransferase.

From these results, it was confirmed that adipate was produced in Escherichia coli containing a polynucleotide encoding a polynucleotide encoding an ADH6 protein and a polynucleotide encoding an ALD5 protein, and the predicted adipate pathway was found to work in Escherichia coli Respectively.

A microorganism including a polynucleotide encoding an ADH6 protein or a mutant thereof and a polynucleotide encoding an ALD5 protein or a mutant thereof has a novel metabolic pathway and is capable of efficiently producing adipate. In addition, such a metabolic pathway can be applied to various microorganisms, and microorganisms having such a metabolic pathway can efficiently produce adipate and thus can be industrially useful.

<110> Samsung Electronics Co. Ltd <120> Microorganism for biosynthesis of adipate and method for          producing adipate using the same <130> PN099033 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 213 <212> PRT <213> Pseudomonas putida <400> 1 Met Thr Ile Thr Lys Lys Leu Ser Arg Thr Glu Met Ala Gln Arg Val   1 5 10 15 Ala Ala Asp Ile Gly Aly Tyr Val Asn Aly Gly Ily Gly Ala              20 25 30 Pro Thr Leu Val Ala Asn Tyr Leu Gly Asp Lys Glu Val Phe Leu His          35 40 45 Ser Glu Asn Gly Leu Leu Gly Met Gly Pro Ser Pro Ala Pro Gly Glu      50 55 60 Glu Asp Asp Asp Leu Ile Asn Ala Gly Lys Gln His Val Thr Leu Leu  65 70 75 80 Thr Gly Gly Ala Phe Phe His His Ala Asp Ser Phe Ser Met Met Arg                  85 90 95 Gly Gly His Leu Asp Ile Ala Val Leu Gly Ala Phe Gln Val Ser Val             100 105 110 Lys Gly Asp Leu Ala Asn Trp His Thr Gly Ala Glu Gly Ser Ile Pro         115 120 125 Ala Val Gly Gly Ala Met Asp Leu Ala Thr Gly Ala Arg Gln Val Phe     130 135 140 Val Met Met Asp His Leu Thr Lys Thr Gly Glu Ser Lys Leu Val Pro 145 150 155 160 Glu Cys Thr Tyr Pro Leu Thr Gly Ile Ala Cys Val Ser Arg Ile Tyr                 165 170 175 Thr Asp Leu Ala Val Leu Glu Val Thr Pro Glu Gly Leu Lys Val Val             180 185 190 Glu Ile Cys Ala Asp Ile Asp Phe Asp Glu Leu Gln Lys Leu Ser Gly         195 200 205 Val Pro Leu Ile Lys     210 <210> 2 <211> 642 <212> DNA <213> Pseudomonas putida <400> 2 atgaccatca ccaaaaagct ctcccgcacc gagatggccc aacgcgtggc cgcagacatc 60 caggaaggcg cgtacgtaaa cctgggcatc ggcgcaccga ccctggtggc caactacctg 120 ggcgacaagg aagtgttcct gcacagcgag aacggcctgc tgggcatggg cccaagccct 180 gcgccgggcg aggaagacga tgacctgatc aacgccggca agcagcacgt caccctgctg 240 cggccgcgg gacatcgctg tactgggcgc cttccaggtg tcggtcaagg gcgacctggc caactggcac 360 acgggtgccg aaggctcgat cccggccgta ggcggtgcaa tggacctggc caccggcgcc 420 cgccaggtgt tcgtgatgat ggaccacctg accaagaccg gcgaaagcaa gctggtgccc 480 gagtgcacct acccgctgac cggtatcgct tgcgtcagcc gcatctacac cgacctggcc 540 gtactggaag tgacacctga agggctgaaa gtggtcgaaa tctgcgcgga catcgacttt 600 gacgagctgc agaaactcag tggcgtgccg ctgatcaagt ga 642 <210> 3 <211> 231 <212> PRT <213> Pseudomonas putida <400> 3 Met Ile Asn Lys Thr Tyr Glu Ser Ile Ala Ser Ala Val Glu Gly Ile   1 5 10 15 Thr Asp Gly Ser Thr Ile Met Val Gly Gly Phe Gly Thr Ala Gly Met              20 25 30 Pro Ser Glu Leu Ile Asp Gly Leu Ile Ala Thr Gly Ala Arg Asp Leu          35 40 45 Thr Ile Ile Ser Asn Aslan Aly Gly Asn Gly Glu Ile Gly Leu Ala Ala      50 55 60 Leu Leu Met Ala Gly Ser Val Arg Lys Val Val Cys Ser Phe Pro Arg  65 70 75 80 Gln Ser Asp Ser Tyr Val Phe Asp Glu Leu Tyr Arg Ala Gly Lys Ile                  85 90 95 Glu Leu Glu Val Val Pro Glu Gly Asn Leu Glu Arg Ile Arg Ala             100 105 110 Ala Gly Ser Gly Ile Gly Ala Phe Ser Ser Thr Gly Tyr Gly Thr         115 120 125 Leu Leu Ala Glu Gly Lys Glu Thr Arg Glu Ile Asp Gly Arg Met Tyr     130 135 140 Val Leu Glu Met Pro Leu His Ala Asp Phe Ala Leu Ile Lys Ala His 145 150 155 160 Lys Gly Asp Arg Trp Gly Asn Leu Thr Tyr Arg Lys Ala Ala Arg Asn                 165 170 175 Phe Gly Pro Ile Met Ala Met Ala Ala Lys Thr Ala Ile Ala Gln Val             180 185 190 Asp Gln Val Val Glu Leu Gly Glu Leu Asp Pro Glu His Ile Ile Thr         195 200 205 Pro Gly Ile Phe Val Gln Arg Val Val Ala Val Thr Gly Ala Ala Ala     210 215 220 Ser Ser Ile Ala Lys Ala Val 225 230 <210> 4 <211> 696 <212> DNA <213> Pseudomonas putida <400> 4 ttgatcaata aaacgtacga gtccatcgcc agcgcggtgg aagggattac cgacggttcg 60 accatcatgg tcggtggctt cggcacggct ggcatgccgt ccgagctgat cgatggcctc 120 attgccaccg gtgcccgcga cctgaccatc atcagcaaca acgccggcaa cggcgagatc 180 ggcctggccg ccctgctcat ggcaggcagc gtgcgcaagg tggtctgctc gttcccgcgc 240 cagtccgact cctacgtgtt cgacgaactg taccgcgccg gcaagatcga gctggaagtg 300 gtcccgcagg gcaacctggc cgagcgtatc cgcgccgcag gctccggcat tggtgcgttc 360 ttctcgccaa ccggctacgg caccctgctg gccgagggca aggaaacccg tgagatcgat 420 ggccgcatgt acgtgctgga aatgccgctg cacgccgact tcgcactgat caaggcgcac 480 aagggtgacc gttggggcaa cctgacctac cgcaaggccg cccgcaactt cggcccgatc 540 atggccatgg ctgccaagac cgccatcgcc caggtcgacc aggtcgtcga actcggtgaa 600 ctggacccgg aacacatcat caccccgggt atcttcgtcc agcgcgtggt cgccgtcacc 660 ggtgctgccg cttcttcgat tgccaaagct gtctga 696 <210> 5 <211> 360 <212> PRT <213> Saccharomyces cerevisiae ATCC 204508 <400> 5 Met Ser Tyr Pro Glu Lys Phe Glu Gly Ile Ala Ile Gln Ser His Glu   1 5 10 15 Asp Trp Lys Asn Pro Lys Lys Thr Lys Tyr Asp Pro Lys Pro Phe Tyr              20 25 30 Asp His Asp Ile Asp Ile Lys Ile Glu Ala Cys Gly Val Cys Gly Ser          35 40 45 Asp Ile His Cys Ala Ala Gly His Trp Gly Asn Met Lys Met Pro Leu      50 55 60 Val Val Gly His Glu Ile Val Gly Lys Val Val Lys Leu Gly Pro Lys  65 70 75 80 Ser Asn Ser Gly Leu Lys Val Gly Gln Arg Val Gly Val Gly Ala Gln                  85 90 95 Val Phe Ser Cys Leu Glu Cys Asp Arg Cys Lys Asn Asp Asn Glu Pro             100 105 110 Tyr Cys Thr Lys Phe Val Thr Thr Tyr Ser Gln Pro Tyr Glu Asp Gly         115 120 125 Tyr Val Ser Gln Gly Gly Tyr Ala Asn Tyr Val Val Val His Glu His     130 135 140 Phe Val Val Pro Ile Pro Glu Asn Ile Pro Ser His Leu Ala Ala Pro 145 150 155 160 Leu Leu Cys Gly Gly Leu Thr Val Tyr Ser Pro Leu Val Arg Asn Gly                 165 170 175 Cys Gly Pro Gly Lys Lys Val Gly Ile Val Gly Leu Gly Gly Ile Gly             180 185 190 Ser Met Gly Thr Leu Ile Ser Lys Ala Met Gly Ala Glu Thr Tyr Val         195 200 205 Ile Ser Arg Ser Ser Arg Lys Arg Glu Asp Ala Met Lys Met Gly Ala     210 215 220 Asp His Tyr Ile Ala Thr Leu Glu Glu Gly Asp Trp Gly Glu Lys Tyr 225 230 235 240 Phe Asp Thr Phe Asp Leu Ile Val Val Cys Ala Ser Ser Leu Thr Asp                 245 250 255 Ile Asp Phe Asn Ile Met Pro Lys Ala Met Lys Val Gly Gly Arg Ile             260 265 270 Val Ser Ile Ser Ile Pro Glu Gln His Glu Met Leu Ser Leu Lys Pro         275 280 285 Tyr Gly Leu Lys Ala Val Ser Ile Ser Tyr Ser Ala Leu Gly Ser Ile     290 295 300 Lys Glu Leu Asn Gln Leu Leu Lys Leu Val Ser Glu Lys Asp Ile Lys 305 310 315 320 Ile Trp Val Glu Thr Leu Pro Val Gly Glu Ala Gly Val His Glu Ala                 325 330 335 Phe Glu Arg Met Glu Lys Gly Asp Val Arg Tyr Arg Phe Thr Leu Val             340 345 350 Gly Tyr Asp Lys Glu Phe Ser Asp         355 360 <210> 6 <211> 1083 <212> DNA <213> Saccharomyces cerevisiae ATCC 204508 <400> 6 atgtcttatc ctgagaaatt tgaaggtatc gctattcaat cacacgaaga ttggaaaaac 60 ccaaagaaga caaagtatga cccaaaacca ttttacgatc atgacattga cattaagatc 120 gaagcatgtg gtgtctgcgg tagtgatatt cattgtgcag ctggtcattg gggcaatatg 180 aagatgccgc tagtcgttgg tcatgaaatc gttggtaaag ttgtcaagct agggcccaag 240 tcaaacagtg ggttgaaagt cggtcaacgt gttggtgtag gtgctcaagt cttttcatgc 300 ttggaatgtg accgttgtaa gaatgataat gaaccatact gcaccaagtt tgttaccaca 360 tacagtcagc cttatgaaga cggctatgtg tcgcagggtg gctatgcaaa ctacgtcaga 420 gttcatgaac attttgtggt gcctatccca gagaatattc catcacattt ggctgctcca 480 ctattatgtg gtggtttgac tgtgtactct ccattggttc gtaacggttg cggtccaggt 540 aaaaaagttg gtatagttgg tcttggtggt atcggcagta tgggtacatt gatttccaaa 600 gccatggggg cagagacgta tgttatttct cgttcttcga gaaaaagaga agatgcaatg 660 aagatgggcg ccgatcacta cattgctaca ttagaagaag gtgattgggg tgaaaagtac 720 tttgacacct tcgacctgat tgtagtctgt gcttcctccc ttaccgacat tgacttcaac 780 attatgccaa aggctatgaa ggttggtggt agaattgtct caatctctat accagaacaa 840 cacgaaatgt tatcgctaaa gccatatggc ttaaaggctg tctccatttc ttacagtgct 900 ttaggttcca tcaaagaatt gaaccaactc ttgaaattag tctctgaaaa agatatcaaa 960 atttgggtgg aaacattacc tgttggtgaa gccggcgtcc atgaagcctt cgaaaggatg 1020 gaaaagggtg acgttagata tagatttacc ttagtcggct acgacaaaga attttcagac 1080 tag 1083 <210> 7 <211> 520 <212> PRT <213> Saccharomyces cerevisiae ATCC 204508 <400> 7 Met Leu Ser Arg Thr Arg Ala Ala Ala Pro Asn Ser Arg Ile Phe Thr   1 5 10 15 Arg Ser Leu Leu Arg Leu Tyr Ser Gln Ala Pro Leu Arg Val Pro Ile              20 25 30 Thr Leu Pro Asn Gly Phe Thr Tyr Glu Gln Pro Thr Gly Leu Phe Ile          35 40 45 Asn Gly Glu Phe Val Ala Ser Lys Gln Lys Lys Thr Phe Asp Val Ile      50 55 60 Asn Pro Ser Asn Glu Glu Lys Ile Thr Thr Val Tyr Lys Ala Met Glu  65 70 75 80 Asp Asp Val Asp Glu Ala Val Ala Ala Lys Lys Ala Phe Glu Thr                  85 90 95 Lys Trp Ser Ile Val Glu Pro Glu Val Arg Ala Lys Ala Leu Phe Asn             100 105 110 Leu Ala Asp Leu Val Glu Lys His Gln Glu Thr Leu Ala Ala Ile Glu         115 120 125 Ser Met Asp Asn Gly Lys Ser Leu Phe Cys Ala Arg Gly Asp Val Ala     130 135 140 Leu Val Ser Lys Tyr Leu Arg Ser Cys Gly Gly Trp Ala Asp Lys Ile 145 150 155 160 Tyr Gly Asn Val Ile Asp Thr Gly Lys Asn His Phe Thr Tyr Ser Ile                 165 170 175 Lys Glu Pro Leu Gly Val Cys Gly Gln Ile Ile Pro Trp Asn Phe Pro             180 185 190 Leu Leu Met Trp Ser Trp Lys Ile Gly Pro Ala Leu Ala Thr Gly Asn         195 200 205 Thr Val Leu Lys Pro Ala Glu Thr Thr Pro Leu Ser Ala Leu Phe     210 215 220 Ala Ser Gln Leu Cys Gln Glu Ala Gly Ile Pro Ala Gly Val Val Asn 225 230 235 240 Ile Leu Pro Gly Ser Gly Arg Val Val Gly Glu Arg Leu Ser Ala His                 245 250 255 Pro Asp Val Lys Lys Ile Ala Phe Thr Gly Ser Thr Ala Thr Gly Arg             260 265 270 His Ile Met Lys Val Ala Ala Asp Thr Val Lys Lys Val Thr Leu Glu         275 280 285 Leu Gly Gly Lys Ser Pro Asn Ile Val Phe Ala Asp Ala Asp Leu Asp     290 295 300 Lys Ala Val Lys Asn Ile Ala Phe Gly Ile Phe Tyr Asn Ser Gly Glu 305 310 315 320 Val Cys Cys Ala Gly Ser Arg Ile Tyr Ile Gln Asp Thr Val Tyr Glu                 325 330 335 Glu Val Leu Gln Lys Leu Lys Asp Tyr Thr Glu Ser Leu Lys Val Gly             340 345 350 Asp Pro Phe Asp Glu Glu Val Phe Gln Gly Ala Gln Thr Ser Asp Lys         355 360 365 Gln Leu His Lys Ile Leu Asp Tyr Val Asp Val Ala Lys Ser Glu Gly     370 375 380 Ala Arg Leu Val Thr Gly Gly Ala Arg His Gly Ser Lys Gly Tyr Phe 385 390 395 400 Val Lys Pro Thr Val Phe Ala Asp Val Lys Glu Asp Met Arg Ile Val                 405 410 415 Lys Glu Glu Val Phe Gly Pro Ile Val Thr Val Ser Lys Phe Ser Thr             420 425 430 Val Asp Glu Val Ile Ala Met Ala Asn Asp Ser Gln Tyr Gly Leu Ala         435 440 445 Ala Gly Ile His Thr Asn Asp Ile Asn Lys Ala Val Asp Val Ser Lys     450 455 460 Arg Val Lys Ala Gly Thr Val Trp Ile Asn Thr Tyr Asn Asn Phe His 465 470 475 480 Gln Asn Val Pro Phe Gly Gly Phe Gly Gln Ser Gly Ile Gly Arg Glu                 485 490 495 Met Gly Glu Ala Ala Leu Ser Asn Tyr Thr Gln Thr Lys Ser Val Arg             500 505 510 Ile Ala Ile Asp Lys Pro Ile Arg         515 520 <210> 8 <211> 1563 <212> DNA <213> Saccharomyces cerevisiae ATCC 204508 <400> 8 atgctttctc gcacaagagc tgcagctccg aattccagaa tattcactag aagcttgtta 60 cgtctttatt ctcaagcacc attacgcgtt ccaattactc ttccaaatgg tttcacctac 120 gaacagccaa cagggttatt catcaatggt gaatttgttg cctcgaagca aaagaaaacg 180 tttgacgtga tcaatccatc taacgaagaa aagataacaa ctgtatacaa ggctatggaa 240 gatgatgttg atgaagccgt tgcagcggct aaaaaagctt ttgaaacgaa gtggtctatt 300 gtagagccgg aggttcgcgc taaagcttta ttcaatctcg ctgacttggt tgagaaacac 360 caagaaacac tggctgccat tgagtcaatg gataatggta agtcattgtt ttgtgcgcgc 420 ggtgacgtcg ctttagtatc taaatacttg cgttcttgcg gtggttgggc agataaaatc 480 tacggtaacg ttattgacac aggtaaaaac cattttacct actcaattaa ggaaccatta 540 ggcgtttgcg gccaaataat cccttggaac ttccctttat tgatgtggtc atggaaaatt 600 gggcctgctc tggctacagg taacaccgtc gtattgaaac ccgctgaaac aacaccttta 660 tctgcccttt tcgcttccca gttgtgtcag gaagcaggca tacccgctgg tgtagtcaat 720 atccttccgg gttccggtag agttgttgga gaaagattga gtgcacaccc agacgtgaag 780 aagattgctt ttacaggctc tactgccacc ggccgccata ttatgaaggt cgctgccgat 840 actgtcaaga aagtcacttt ggagctggga ggtaaatcac caaatattgt gtttgctgac 900 gctgatctag ataaagccgt caagaacatt gccttcggta ttttttacaa ctctggtgaa 960 gtttgctgcg ctggttccag aatatacatt caagatacag tatacgagga ggtgttgcaa 1020 aaactaaagg attacaccga gtcactaaag gtcggtgacc catttgatga ggaagttttc 1080 caaggtgctc aaacatctga caaacagctg cataaaattt tagactatgt cgatgtagca 1140 aaatcagagg gggctcgtct tgtgactgga ggggccagac atggcagtaa aggttatttt 1200 gtcaagccaa cagtgtttgc tgatgtcaaa gaagatatga gaattgttaa ggaggaagtg 1260 tttggtccca ttgtaactgt atccaagttt tctactgttg atgaagtgat tgctatggca 1320 aatgattctc aatatgggtt agccgcaggt attcacacta acgatattaa caaggctgtt 1380 gatgtgtcca aaagagtgaa agctggtact gtttggataa atacctataa caacttccac 1440 caaaatgttc ctttcggtgg cttcggccag tcaggtattg gccgtgaaat gggtgaggct 1500 gctttaagta actacactca aacaaaatct gtcagaattg ccattgacaa gccaattcgt 1560 tga 1563 <210> 9 <211> 258 <212> PRT <213> Rhizobium meliloti <400> 9 Met Thr Lys Thr Ala Val Ile Thr Gly Ser Thr Ser Gly Ile Gly Leu   1 5 10 15 Ala Ile Ala Arg Thr Leu Ala Lys Ala Gly Ala Asn Ile Val Leu Asn              20 25 30 Gly Phe Gly Ala Pro Asp Glu Ile Arg Thr Val Thr Asp Glu Val Ala          35 40 45 Gly Leu Ser Ser Gly Thr Val Leu His His Pro Ala Asp Met Thr Lys      50 55 60 Pro Ser Glu Ile Ala Asp Met Met Ala Met Val Ala Asp Arg Phe Gly  65 70 75 80 Gly Ala Asp Ile Leu Val Asn Asn Ala Gly Val Gln Phe Val Glu Lys                  85 90 95 Ile Glu Asp Phe Pro Val Glu Gln Trp Asp Arg Ile Ile Ala Val Asn             100 105 110 Leu Ser Ser Ser Phe His Thr Ile Arg Gly Ala Ile Pro Pro Met Lys         115 120 125 Lys Lys Gly Trp Gly Arg Ile Ile Asn Ile Ala Ser Ala His Gly Leu     130 135 140 Val Ala Ser Pro Phe Lys Ser Ala Tyr Val Ala Ala Lys His Gly Ile 145 150 155 160 Met Gly Leu Thr Lys Thr Val Ala Leu Glu Val Ala Glu Ser Gly Val                 165 170 175 Thr Val Asn Ser Ile Cys Pro Gly Tyr Val Leu Thr Pro Leu Val Glu             180 185 190 Lys Gln Ile Pro Asp Gln Ala Arg Thr Arg Gly Ile Thr Glu Glu Gln         195 200 205 Val Ile Asn Glu Val Met Leu Lys Gly Gln Pro Thr Lys Lys Phe Ile     210 215 220 Thr Val Glu Gln Val Ala Ser Leu Ala Leu Tyr Leu Ala Gly Asp Asp 225 230 235 240 Ala Ala Gln Ile Thr Gly Thr His Val Ser Met Asp Gly Gly Trp Thr                 245 250 255 Ala Gln         <210> 10 <211> 777 <212> DNA <213> rhizobium meliloti <400> 10 atgaccaaga ctgcggtgat aacgggttcc acgagcggca tcggattggc gatcgcccgg 60 accctggcga aggccggtgc caatatcgtc ctgaacggct tcggtgcgcc ggacgagatc 120 aggaccgtca cggatgaagt cgcaggcctg agctccggta cggtgcttca tcacccggcc 180 gacatgacca agccctccga aatcgccgac atgatggcga tggttgccga tcgcttcggc 240 ggcgccgata tcctcgtcaa caatgccggc gtgcagttcg ttgaaaagat cgaggatttt 300 ccggtcgagc aatgggaccg gatcatcgcc gtcaatctct cctcctcctt tcacaccatt 360 cgtggcgcca ttccgccgat gaagaagaag ggctggggcc ggatcatcaa tatcgcgtcc 420 gctcatggcc tcgtggcctc ccccttcaag tccgcctatg tcgccgccaa gcatggtatc 480 atggggttga cgaagactgt ggcgctggag gtggcggaga gcggtgtcac cgtgaactcg 540 atctgccccg gctacgttct gacgccgctc gtcgaaaagc agataccgga tcaggcgaga 600 acgcgcggca tcaccgagga acaggtgatc aacgaggtga tgctcaaggg acagccgacg 660 aaaaagttca tcaccgtcga acaggttgcc tccctggcgc tctatcttgc aggcgacgat 720 gccgcccaga tcaccgggac gcatgtttcg atggatggcg gctggacggc gcagtag 777 <210> 11 <211> 371 <212> PRT <213> Clostridium kluyveri <400> 11 Met Lys Leu Leu Lys Leu Ala Pro Asp Val Tyr Lys Phe Asp Thr Ala   1 5 10 15 Glu Glu Phe Met Lys Tyr Phe Lys Val Gly Lys Gly Asp Phe Ile Leu              20 25 30 Thr Asn Glu Phe Leu Tyr Lys Pro Phe Leu Glu Lys Phe Asn Asp Gly          35 40 45 Ala Asp Ala Val Phe Gln Glu Lys Tyr Gly Leu Gly Glu Pro Ser Asp      50 55 60 Glu Met Ile Asn Asn Ile Ile Lys Asp Ile Gly Asp Lys Gln Tyr Asn  65 70 75 80 Arg Ile Ile Ala Val Gly Gly Gly Ser Val Ile Asp Ile Ala Lys Ile                  85 90 95 Leu Ser Leu Lys Tyr Thr Asp Asp Ser Leu Asp Leu Phe Glu Gly Lys             100 105 110 Val Pro Leu Val Lys Asn Lys Glu Leu Ile Ile Val Pro Thr Thr Cys         115 120 125 Gly Thr Gly Ser Glu Val Thr Asn Val Ser Val Ala Glu Leu Lys Arg     130 135 140 Arg His Thr Lys Lys Gly Ile Ala Ser Asp Glu Leu Tyr Ala Thr Tyr 145 150 155 160 Ala Val Leu Val Pro Glu Phe Ile Lys Gly Leu Pro Tyr Lys Phe Phe                 165 170 175 Val Thr Ser Ser Val Asp Ala Leu Ile His Ala Thr Glu Ala Tyr Val             180 185 190 Ser Pro Asn Ala Asn Pro Tyr Thr Asp Met Phe Ser Val Lys Ala Met         195 200 205 Glu Leu Ile Leu Asn Gly Tyr Met Gln Met Val Glu Lys Gly Asn Asp     210 215 220 Tyr Arg Val Glu Ile Ile Glu Asp Phe Val Ile Gly Ser Asn Tyr Ala 225 230 235 240 Gly Ile Ala Phe Gly Asn Ala Gly                 245 250 255 Tyr Pro Ile Gly Gly Asn Tyr His Val Pro His Gly Glu Ala Asn Tyr             260 265 270 Leu Phe Phe Thr Glu Ile Phe Lys Thr Tyr Tyr Glu Lys Asn Pro Asn         275 280 285 Gly Lys Ile Lys Asp Val Asn Lys Leu Leu Ala Gly Ile Leu Lys Cys     290 295 300 Asp Glu Ser Glu Ala Tyr Asp Ser Leu Ser Gln Leu Leu Asp Lys Leu 305 310 315 320 Leu Ser Arg Lys Pro Leu Arg Glu Tyr Gly Met Lys Glu Glu Glu Ile                 325 330 335 Glu Thr Phe Ala Asp Ser Val Ile Glu Gly Gln Gln Arg Leu Leu Val             340 345 350 Asn Asn Tyr Glu Pro Phe Ser Arg Glu Asp Ile Val Asn Thr Tyr Lys         355 360 365 Lys Leu Tyr     370 <210> 12 <211> 1116 <212> DNA <213> Clostridium kluyveri <400> 12 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

Claims (22)

A polynucleotide encoding succinyl-CoA: acetyl-CoAtransferase or a variant thereof, a polynucleotide encoding 3-oxoadipyl-coAtransferase or a variant thereof, an alcohol dehydrogenase 6 (ADH6) protein or a variant thereof A polynucleotide encoding a D-beta-hydroxybutyrate dehydrogenase (BDHA) protein or a variant thereof, a polynucleotide encoding a 4HBD (NAD-dependent 4-hydroxybutyrate dehydrogenase) protein or a variant thereof, and an aldehyde dehydrogenase (ALD5) A microorganism comprising a polynucleotide encoding a protein or a variant thereof. The microorganism according to claim 1, wherein the succinyl-CoA: acetyl CoAtransferase is derived from Pseudomonas putida. 3. The microorganism according to claim 2, wherein the succinyl-CoA: acetyl CoAtransferase has the amino acid sequence of SEQ ID NO: 1. 2. The microorganism according to claim 1, wherein the polynucleotide encoding succinyl-CoA: acetylcoatetransferase has the nucleotide sequence of SEQ ID NO: 2. The microorganism according to claim 1, wherein the 3-oxoadipic-coAtransferase is derived from Pseudomonas putida. 6. The microorganism according to claim 5, wherein the 3-oxoadipyl-coAtransferase has the amino acid sequence of SEQ ID NO: 3. 3. The microorganism according to claim 1, wherein the polynucleotide encoding the 3-oxoadipyl-coeote transferase has the nucleotide sequence of SEQ ID NO: 4. The microorganism according to claim 1, wherein the ADH6 protein is derived from Saccharomyces cerevisiae. 9. The microorganism according to claim 8, wherein the ADH6 protein has the amino acid sequence of SEQ ID NO: 5. The microorganism according to claim 1, wherein the polynucleotide encoding the ADH6 protein has the nucleotide sequence of SEQ ID NO: 6. The microorganism according to claim 1, wherein the ALD5 protein is derived from Saccharomyces cerevisiae. 12. The method of claim 11, wherein said ALD5 protein has the amino acid sequence of SEQ ID NO: 7. The microorganism according to claim 1, wherein the polynucleotide encoding the ALD5 protein has the nucleotide sequence of SEQ ID NO: 8. The microorganism according to claim 1, wherein the BDHA protein is derived from Ribium meliloti. 15. The microorganism according to claim 14, wherein the BDHA protein has the amino acid sequence of SEQ ID NO: The microorganism according to claim 1, wherein the polynucleotide encoding the BDHA protein has the nucleotide sequence of SEQ ID NO: 10. The microorganism according to claim 1, wherein the 4HBD protein is Clostridium clumerii. [Claim 18] The 4HBD protein of claim 17, Wherein the microorganism has an amino acid sequence. The microorganism according to claim 1, wherein the polynucleotide encoding the 4HBD protein has the nucleotide sequence of SEQ ID NO: 12. The microorganism according to claim 1, wherein the microorganism synthesizes acetyl-CoA and succinyl-CoA. The microorganism according to claim 1, wherein the microorganism produces adipate. Culturing the microorganism according to any one of claims 1 to 19; And
A method for producing adipeptide comprising the step of obtaining an adipate in a culture.

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