KR20140026208A - Recombinant micro-organisms for producing organic aicd and the method for producing organic acid by using thereof - Google Patents

Abstract

The present invention relates to recombinant micro-organisms for producing organic acid in which activities of acetyl-CoA acetyltransferase, β-Ketothiolase II, crotonaseand, 3-hydroxybutyryl-CoA dehydrogenase, and trans-enoyl-CoA reductase are enhanced, and to a method for producing organic acid using the same. In addition, the present invention relates to a method for producing the organic acid which includes the steps of: culturing the microorganisms; and collecting the organic acid from the microorganisms or the culture, and to the organic acid manufactured through the same. The conventional method for producing hexanoic acid has low productivity even after long term fermentation and biosynthesis of the hexanoic acid becomes difficult so that the present invention provides the microorganisms which are drastically developed in fermentation time and production amount by introducing genes provided in the present invention. By using the microorganisms, the present invention can contribute to biosynthesis of the hexanoic acid and growth of bioplastic industries caused by the same and be helpful for a novel method for producing the bioplastic which is simple and requires low costs. The present invention can drastically contribute to growth of the bioplastic industry.

Description

[0001] The present invention relates to a recombinant microorganism for producing an organic acid and a method for producing the same,

The present invention relates to a process for the production of acetyl-CoA acetyltransferase, beta-ketothiolase II, crotonase, 3-hydroxybutyryl-CoA dehydrogenase, -CoA dehydrogenase), and a trans-enoyl-CoA reductase, and a method for producing the microorganism. The present invention also relates to a method for culturing a microorganism, which comprises culturing the microorganism; And recovering the organic acid from the culture or microorganism, and an organic acid produced thereby.

With the depletion of fossil energy sources that are the long-term energy sources of mankind, we have been able to overcome the limitations of crude oil price rises and limitations, to prevent carbon dioxide emissions and environmental pollution from the production and use of petrochemical products. The move to the bio-based economy to utilize biomass is progressing actively.

In addition, as CO2 emissions from the use of petroleum-based products continue to increase, many countries are seeking policies and technology development to reduce carbon dioxide emissions as a result of the Kyoto Protocol, which aims to reduce CO2 emissions. Development and commercialization of biochemical products using biochemicals are underway as a solution.

Biomass is a renewable organic material that includes all plants, animals and microorganisms, including renewable organic matter extracted from crops and trees, agricultural and feed crops, agriculture and forest wastes, algae, . Unlike existing petroleum-derived materials, biomass is independent of fossil fuels, includes a variety of sources, and is environmentally friendly.

Bio-fuels and bio-based chemicals are being researched and developed using these biomass materials as biomaterials. In bio-fuels, bio-fuels such as bio-ethanol, biodiesel and bio-butanol A variety of bioplastics and biochemicals using biodegradable materials such as PLA, 1,3-PDO, C2-C4 building blocks such as bio-ethylene and butadiene have been researched and developed.

In particular, bioplastics generally refers to plastic monomers such as C3, C4, C5, and C6, which are obtained by using as a metabolic-fermentation process, and polymeric materials for the human and chemical industries produced by chemical or biological methods therefrom. The bioplastics industry, which is derived from plants or biodegradable, is showing rapid growth as market competitiveness has improved due to increased consumer preference for eco-friendly products, increased use regulation of refractory plastics, and rising prices of petroleum-based products. Accordingly, global demand for bioplastics in 2013 is expected to reach 900,000 tons and prices will reach US $ 2.6 billion. In 2015, the market will account for 1.5-4.8% of the entire plastic market, with a market size of 4 million to 12.5 million tons. It is expected to reach.

Currently, bioplastics production technologies based on C3, C4, C5, and C6 monomers are highly technically mature, but C5 and C6 monomer production technologies are currently technologically low. In particular, bioplastics using C3 and C4 monomers are already in the process of industrialization, but bioplastics using C5 and C6 monomers have been suffering from industrialization due to insufficient production technology of monomers. Therefore, the technology for producing C5 and C6 monomers and the production of bioplastics using the same are attracting attention as a mainstream development field in the bioplastics industry, and efficient techniques for producing C5 and C6 monomers are needed.

Under these circumstances, the present inventors have made extensive efforts to develop a strain capable of biosynthesizing hexanoic acid, which is a C6 monomer used as a precursor of bioplastics, at a higher yield. As a result, it has been found that acetyl- CoA acetyltransferase CoA acetyltransferase, beta-ketothiolase II, crotonase, 3-hydroxybutyryl-CoA dehydrogenase, and trans-inoyl- It was confirmed that hexanoic acid was obtained in a high yield in a strain into which a gene expressing a trans-enoyl-CoA reductase was introduced. In addition, it was confirmed that thioesterase II (II) or beta-ketothiol It was confirmed that the final product hexanoic acid was obtained in a high yield in the strain into which the gene expressing the Ⅰ (ß-Ketothiolase I) was introduced and that the C4 organic acid was produced as a byproduct, Completed.

One object of the present invention is to provide an acetyl-CoA acetyltransferase, beta-ketothiolase II, crotonase, 3-hydroxybutyryl-CoA dehydrogenase (3-hydroxybutyryl-CoA dehydrogenase), and a trans-enoyl-CoA reductase.

Another object of the present invention is to provide a method for producing a microorganism, which comprises culturing a vector expressing an acetyl-CoA acetyltransferase, beta-ketotiolase II, crotonose, 3-hydroxybutyryl-CoA dehydrogenase, and trans- And a step of introducing the microorganism into the microorganism.

Yet another object of the present invention is to provide a method for producing microorganisms, And recovering the organic acid from the culture or microorganism.

Another object of the present invention is to provide an organic acid produced by the above method.

In one aspect of the present invention, there is provided an acetyl-CoA acetyltransferase, a β-ketothiolase Ⅱ, a crotonase, (3-hydroxybutyryl-CoA dehydrogenase), and a trans-enoyl-CoA reductase (hereinafter referred to as " trans-enoyl-CoA reductase ").

The term "acetyl-CoA acetyltransferase (AtoB) " of the present invention refers to an enzyme that generates acetoacetyl-CoA from two acetyl-CoA by transferring an acetyl group to acetyl-Coenzyme A . Specifically, it acts on the formation and degradation of ketone bodies in mitochondria, and is an enzyme necessary for proper metabolism of isoleucine (NCBI Accession No. NC_012971.2).

The term "Beta-ketothiolase II, BktB" of the present invention refers to an enzyme that polymerizes a CoA compound having a carbon number of C2 to produce a compound such as C4 or C6 . In the present invention, β-ketothiolase II (bktB) refers to an enzyme that produces β-ketohexanoyl-CoA (C6) by adding two acetyl-CoA to butyryl-CoA, an intermediate metabolite of metabolism (NCBI Accession No. AF026544).

The term "Crotonase" in the present invention refers to an enzyme that binds a water molecule to an enoyl-CoA compound with an enoyl-CoA hydratase as another expression, Is an enzyme that mediates the action of decomposing water molecules to produce enoyl-coA compounds. In the present invention, it acts on 3-hydroxybutyryl-CoA to generate crotonyl-CoA (NCBI Accession No. U17110.1).

The term "3-hydroxybutyryl-CoA dehydrogenase" of the present invention mediates the action of releasing hydrogen from 3-hydroxybutyryl-CoA, which is interpreted as a kind of oxidation reaction . However, this reaction becomes a reversible reaction and can also mediate a kind of reducing action that forms 3-hydroxybutyryl-CoA by attaching hydrogen to Acetoacetyl-CoA. In the present invention, the 3-hydroxybutyryl-CoA dehydrogenase is used in the direction of producing 3-hydroxybutyryl-CoA by binding hydrogen to acetoacetyl-CoA (NCBI Accession No. P52041).

The term "trans-enoyl-CoA reductase" of the present invention means an enzyme that attaches hydrogen to trans-inoyl-CoA to reduce it. In the present invention, two hydrogen atoms are attached to crotonyl-CoA to produce butyryl-CoA, which is irreversible (NCBI Accession No. Q73Q47).

The sequence of the enzymes can be obtained from known databases and the like, but is not limited thereto. The amino acid coding for BktB, phbA, AtoB, TesB, Crt, Hbd, Ter or MCT1 is at least 70%, preferably at least 80%, more preferably at least 80% As long as the protein has an activity of each of the genes described above substantially as an amino acid sequence showing 90% or more, more preferably 95% or more, even more preferably 98% or more, and most preferably 99% , And it is also obvious that a protein variant having an amino acid sequence in which some of the sequences are deleted, modified, substituted, or added is also included within the scope of the present invention, as long as it is an amino acid sequence having substantially the same or corresponding biological activity as the sequence having such similarity .

The term "active" of the present invention means a function possessed by an enzyme, and the present invention includes all functions inherent in enzymes in vivo or functions expressed in vitro. Accordingly, in the present invention, the term "enhanced activity" means that the activities of the enzymes are improved more than the conventional ones, and the activity of the enzymes is beneficially changed in any aspect such as reaction rate, chemical equilibrium, it means.

The method of enhancing (or increasing) the activity in the present invention can be applied to various methods well known in the art. Examples of such methods include, but are not limited to, methods in which a polynucleotide comprising a nucleotide sequence encoding a specific enzyme is further inserted into a chromosome, or a method of introducing the polynucleotide into a vector system, A method of increasing the number of copies of a base sequence, a method of replacing with a strong promoter, a method of introducing a mutation into a promoter, and a method of mutating a gene. In one specific embodiment, a method of increasing the number of copies of the gene by introducing a gene encoding the enzyme into the vector and transforming the microorganism to enhance the activity of the enzymes in the genus Kluyveromyces.

The term "organic acid" of the present invention is a generic term of an organic compound having an acidity, and is an organic compound containing a carboxy group and a sulfone group. The phenomenon that the carbon compounds, mainly carbohydrates, are incompletely oxidized by the action of microorganisms, and various organic acids are produced and accumulated often occurs and is called organic acid fermentation. The kinds of organic acids to be produced are industrially produced by lactic acid, acetic acid, citric acid, gluconic acid and the like by fermentation method. The mechanism by which an organic acid is produced is often a metabolite intermediate when a carbon compound undergoes biodegradation, but it is also a simple oxidation product that does not undergo a change in its carbon skeleton.

In the present invention, the organic acid is preferably hexanoic acid, which is an organic acid having 6 carbon atoms, or butyryl acid, which is an organic acid of C4, and most preferably hexanoic acid. Hexanoic acid can be used as a precursor in the production of bioplastics. That is, hexanoic acid produced by the microorganism of the present invention is used as a precursor of bioplastics, and is a basis for mass production of bioplastics by a biochemical method.

The term "microorganism" of the present invention includes algae, bacteria, protozoa, fungi, yeast, and viruses called marginal creatures. Belongs. Minimal metabolism is carried out to maintain life according to the minimum living unit, and it can be adjusted to the desired direction by promoting or inhibiting the specific metabolic process by using genetic engineering method in such metabolic process.

In the present invention, the microorganism may be E. coli . Escherichia coli is a enteric bacterium that is a parasitic source of mammals, including humans. It is a tuberous anaerobic gram-negative bacterium that produces acid by decomposing glucose. In particular, E. coli K12 strain ( E. coli K-12) has been widely used as a host cell in research materials and biosynthesis industries in molecular biology and biotechnology.

In the present invention, the microorganism may be yeast. Yeast (yeast) is a microbe used for various fermentations and is the simplest form of eukaryotes. Preferably, the yeast may be a member of the genus Saccharomyces, Candida, Kluyveromyces, or Torulaspora, and most preferably, It may be cilius (Klluyveromyces marxianus).

In the present invention, Cluyveromyces membrane cyanosis is characterized by its ability to use various carbon sources, growth ability at high temperature, fast growth rate, and low tendency to produce ethanol (kryptt negative) when exposed to excessive sugar Lt; / RTI > Unlike common yeast strains, many strains have been reported for Cluyveromyces membrane cyanosis.

In the present invention, the microorganism may further be a microorganism whose activity of thioesterase II is enhanced.

The term "Thioesterase II, TesB" of the present invention is an enzyme which decomposes thioester bonds. In the present invention, the hexanoyl-CoA is cleaved to remove the CoA moiety so that the alcohol group OH is present at the terminal (Hexanoic acid) (NCBI Accession No. P0AGG2).

In order to produce the microorganism for producing an organic acid according to the present invention, it is most preferable to enhance the activity of the combination of AtoB, BktB, TesB, Crt, Hbd and Ter when based on E. coli.

In the present invention, the microorganism is further characterized in that the activity of alcohol dehydrogenase, lactate dehydrogenase, fumarate reductase, and phosphotransacetylase is weaker than the intrinsic activity Or the like.

In the present invention, an alcohol dehydrogenase is an enzyme that mediates the action of releasing hydrogen from alcohol and can be interpreted as a kind of oxidation reaction. However, this reaction by the present enzyme is a reversible reaction. Lactate dehydrogenase "in the present invention mediates the action of releasing hydrogen from lactate, which can be interpreted as an oxidation reaction. The term" fumarate dehydrogenase " reductase "is an enzyme that converts fumarate to succinic acid and plays an important role in anaerobic respiration metabolism of microorganisms. The term" phosphotransacetylase "of the present invention refers to an enzyme that converts acetyl-CoA and CoA As the enzyme that mediates the conversion reaction to acetyl phosphate, the reaction is a reversible reaction.

In the present invention, the term "intrinsic activity" means an active state of an enzyme having a microorganism in its natural state.

In the present invention, " intrinsic activity weakening " means deletion of all or part of the nucleotide sequence on the chromosome of a microorganism containing the nucleotide sequence encoding the endogenous protein, substitution of a part of the nucleotide sequence or insertion of one or more base pairs into the nucleotide sequence ≪ RTI ID = 0.0 > and / or < / RTI > In addition, the intrinsic activity may be weakened by mutation of the entire or a part of the expression regulatory sequence of the base sequence encoding the endogenous protein, or partial substitution or insertion, resulting in degradation or destruction of the expression inducing activity. The expression regulatory sequence may be located on the chromosomal nucleotide sequence above or below the nucleotide sequence, and is preferably, but not limited to, a promoter, an enhancer, and the like. Mutation of the gene into the chromosome can be accomplished by any method known in the art, for example, homologous recombination. In other words, the enzymes do not normally function due to gene deletion or mutation, which means that the enzymes that the microorganisms have in their natural state are weakened.

In the present invention, the activity-enhancing activity of thioesterase II, or the activity-weakened microorganism of alcohol dehydrogenase, lactate dehydrogenase, fumarate reductase, and transphosphorylated acetylase may be E. coli.

One embodiment, atoB and bktB gene in Escherichia coli of the present invention is pET a duet-1 vector, crt and hbd gene in pCOLA duet-1 vector, ter and tesB gene was introduced respectively into pACYC duet-1 vector (Fig. 2 ). In one embodiment of the present invention, the alcohol dehydrogenase, lactate dehydrogenase, fumarate reductase, and phosphate transacetase, which are inherently present in E. coli, were identified as "one-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. "Specifically, the homologous recombination system of genomic DNA was promoted by using red recombinase, and the kanamycin resistance system was used to replace the target gene region with the kanamycin cassette And only the strains in which the target gene was removed were selected. Finally, the kanamycin cassette was removed to obtain a strain in which the genes were disrupted.

In the present invention, the microorganism may be a microorganism enhanced in the activity of beta-ketothiolase I.

The term " Beta-ketothiolase I, PhdA "of the present invention means that a CoA compound having a carbon number of C2 is polymerized with Beta-Ketotiola II described above, C6, and so on. In the present invention, beta-keto-tiolase I (phdA) means an enzyme that produces β-ketohexanoyl-CoA (C6) by adding two acetyl-CoA to butyryl-CoA which is an intermediate metabolite of metabolism (NCBI Accession No. AF026544).

In the present invention, the microorganism having enhanced beta-ketothiola I activity may be yeast. It is most preferable to enhance the activity of the combination of AtoB, PhdA, BktB, Crt, Hbd, and Ter genes when yeast-based microorganisms are produced.

In the present invention, the microorganism may be a microorganism having the accession number KACC 93153B.

In one embodiment of the present invention there is provided a method for the treatment and / or prophylaxis of Clube Veromyces membrane cyanosis beta-ketotyolase I and II, acetyl-CoA acetyltransferase, crotonase, 3-hydroxybutyryl- CoA dehydrogenase and trans- -COA < / RTI > reductase was introduced via the pJSKM316GPD vector expressed in the Cluyeberomyces membrane cyanobacteria strain (Fig. 3).

In the present invention, the origins of the organic acid-producing enzymes are derived from Beta-ketotiolase I and Beta-ketotyolase II from Ralstonia eutropha; The acetyl CoA acetyltransferase and thioesterase are derived from Escherichia coli; The crotonate and 3-hydroxybutyryl-CoA dehydrogenase are derived from Clostridium acetobutylicum; The trans-inyl-CoA reductase may be derived from Treponema denticola.

In another embodiment, the present invention provides a method of producing an antibody that expresses an acetyl CoA acetyltransferase, a beta-ketotiolase II, a crotonate, a 3-hydroxybutyryl-CoA dehydrogenase, and a trans-inoyl-CoA reductase And introducing the vector into the microorganism.

The term "vector" of the present invention 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, Double stranded 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 plasmid vectors (pSE system, pBR system, pUC system, pBluscriptII system, pGEM system, pTZ system, pET system, pJSKM316GPD system, pCOLA system and pACYC system) and phage or cosmid vectors (pWE15, M13, EMBL3, EMBL4, FIX II, DASH II, ZAP II, gt11, Charon4A, Charon21A). In particular, the pET, pCOLA, or pACYC vector, which is an E. coli expression vector, can have a duet expression structure capable of simultaneously introducing and expressing two target proteins into a microorganism.

In one embodiment of the invention, atoB and bktB gene pET a duet-1 vector, crt and hbd gene in pCOLA duet-1 vector, ter and tesB gene was introduced respectively into pACYC duet-1 vector (Fig. 2) .

In the present invention, the vector may be a yeast expression vector capable of expressing a target protein in yeast. Preferably, the yeast expression vector may be a vector that integrates the desired protein on the genome of the yeast. On the other hand, the yeast expression vector may be preferably a pJSKM316GPD vector expressed in a strain of C. tuberosum cyanosis.

In one embodiment of the present invention, the enzyme is selected from the group consisting of Beta-ketothiolase I and II, acetyl-CoA acetyltransferase, crotonase, 3-hydroxybutyryl-CoA dehydrogenase and trans- The inoyl-CoA reductase was introduced through the pJSKM316GPD vector expressed in the Cluyeberomyces membrane cyanobacteria strain (Fig. 3).

The term "introduction" of the present invention encompasses any method whereby the exogenous gene is included in a vector, a host cell, or the like. The method of introducing the gene into a vector or introducing a vector or the like into a host cell or the like can be carried out by selecting a suitable standard technique as known in the art. In particular, in the present invention, the vector may be introduced into the microorganism by any method known in the art. For example, microinjection, electroporation, particle spraying a particle bombardment method, a direct muscle injection method, an insulator method, and a method using a transposon.

According to the embodiments of the present invention, 6 genes in addition to the E. coli are atoB and bktB gene in pET duet-1 vector, hbd and crt genes in pCOLA duet-1 vector, and ter tesB gene pACYC duet- 1 vector, respectively (Fig. 2). This was introduced into Escherichia coli. In addition, whether the hexane acid expression-related genes were normally expressed in E. coli was confirmed by SDS-PAGE and coomassie blue staining (Fig. 6).

According to a specific embodiment of the present invention, the six genes added to the yeast were inserted into a pJSKM316GPD vector capable of expression in Cluyveromyces membrane cyanosis (Fig. 3). This was introduced into Cl. Veromyces membriana. In addition, the hexane acid expression-related genes were implanted in the genome through random integration on the genome of the host microorganism (FIG. 4). It was confirmed that it was introduced into the genome and expressed normally through colony PCR confirmation and mRNA analysis by RT-PCR (FIG. 5).

In another embodiment, the present invention provides a method for culturing a microorganism, comprising culturing the microorganism; And recovering the organic acid from the culture or microorganism.

The cultivation of the recombinant yeast strain in the present invention can be carried out according to a well-known method, and it is preferable to culture in an anaerobic state in which no oxygen is introduced. In addition, the conditions such as the culture temperature, the culture time and the pH of the culture medium can be appropriately adjusted. Examples of suitable culture methods include fed-batch culture, batch culture, and cintinuous culture, and are preferably, but not exclusively, fed-batch culture.

The culture medium used should suitably meet the requirements of a particular strain. Culture media for various microorganisms are known (see, for example, " Manual of Methods for General Bacteriology ", from the American Society for Bacteriology (Washington D.C., USA, 1981)). The carbon source in the medium can be selected from the group consisting of sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats such as soybean oil, sunflower seed oil, peanut oil and coconut oil ), Fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture. Nitrogen sources include nitrogen-containing organic compounds such as peptone, yeast extract, juice, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, Ammonium), and these materials may also be used individually or as a mixture. As the phosphorus source, potassium dihydrogenphosphate, dipotassium hydrogenphosphate or the corresponding sodium-containing salts may be used. In addition, the culture medium may contain metal salts essential for growth (for example, magnesium sulfate or ferrous sulfate), and finally essential growth-promoting substances such as amino acids and vitamins may be used in addition to the above-mentioned substances. Additional suitable precursors may be added to the culture medium. The feed material may be added all at once to the culture, or may be supplied appropriately during the culture.

The pH of the culture can be adjusted by the appropriate use of a basic compound (eg sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (eg phosphoric acid or sulfuric acid). Foaming can be controlled using a defoaming agent such as a fatty acid polyglycol ester. The incubation temperature is usually 20 to 45 캜, preferably 25 to 40 캜, most preferably 30 캜. The culture can be continued until the desired amount of the target substance is maximally obtained, and the target substance may be released into the culture medium or contained in the cells.

Thus, the term "culture" of the present invention includes microorganisms and all substances present in an incubator in which the microorganisms are cultivated. In general, microorganisms themselves and substances containing microorganisms, enzymes, metabolites, etc., which are excreted in the medium.

The term "recovery" of the present invention means the process of separating a particular substance from a mixture. In particular, "a step of recovering an organic acid" means a process of separating an organic acid mixed with other substances as a metabolite to the culture and microorganism, and it is a method that is widely known in the art, Can be separated. The number of times of hexanol recovery may be appropriately determined according to the physical and chemical properties of the organic acid. For example, filtration, anion exchange chromatography, crystallization and HPLC may be used, but the present invention is not limited thereto.

In the present invention, the organic acid is preferably a metabolic normal butyric acid or hexanoic acid.

According to a specific embodiment of the present invention, hexanoic acid produced by culturing the microorganism was analyzed by Gas Chromatography of YL 6100 series model. HP-FFAP (30m x 0.25mm ID, 0.25μm) was used as the column and helium gas was used as the carrier. The oven was initially maintained at 100 ° C for 5 minutes, increased from 100 ° C to 240 ° C by 10 ° C per minute, and maintained at 240 ° C for 12 minutes. The injector was set to 1:50 split mode and the detector was set to 300 ° C. Butyric acid, valeric acid and hexanoic acid showed retention times of 10 min, 11 min and 12 min, respectively. However, the gas chromatography used in this example was used for the purpose of detecting how much experimental amount of hexanoic acid could be produced, not for commercial use. In this regard, the step of separating out the organic acid is suitable for separating the organic acid, especially hexanoic acid, in high purity and in large quantities for commercial use.

In another aspect, the present invention provides an organic acid prepared by the above-described method for producing an organic acid.

In the present invention, the organic acid is preferably a metabolic normal butyric acid or hexanoic acid. The organic acid prepared in the present invention can be used as a precursor of bioplastics.

According to the present invention, the production method of hexanoic acid has been hindered in the biosynthesis industry of hexanoic acid due to a low production amount despite the long fermentation. However, by introducing the gene proposed in the present invention, It provides microorganisms that have greatly improved in terms of time and yield. Therefore, the use of the microorganism of the present invention will greatly contribute to the hexanoic acid biosynthesis and the growth of the bioplastics industry thereby contributing to a new simple and inexpensive method of producing bioplastics.

Figure 1 is a schematic diagram of biosynthetic pathways for the production of organic acids, especially hexanoic acid.
Fig. 2 is a diagram showing a construction expression vector of a gene necessary for an organic acid in E. coli , in particular, a 1-hexanol hexanoic acid biosynthetic pathway.
Fig. 3 is a diagram showing a construction expression vector of a gene necessary for the biosynthesis pathway of an organic acid, particularly hexanoic acid 1-hexanol, in K. marxianus .
Fig. 4 is a schematic diagram for explaining the mechanism of random integration of a gene necessary for the biosynthesis pathway of an organic acid, especially hexanoic acid 1-hexanol, into a microorganism genome.
Fig. 5 is a diagram for identifying a biosynthetic gene transformed into K. marxianus .
Fig. 6 is a figure showing the biosynthetic gene transformed into E. coli . Fig.
FIG. 7 is a schematic diagram showing a method of gene disruption of genomic DNA of E. coli . FIG.
Fig. 8 is a diagram showing gene fragmentation of genomic DNA of E. coli .
9 is a diagram for analyzing the fermentation conditions of E. coli and the amount of hexanoic acid (C6) produced by each strain.
FIG. 10A is a view for analyzing the fermentation conditions of K. marxianus and the production amount of hexanoic acid (C6) for each strain.
FIG. 10B is a view for analyzing the fermentation conditions of K. marxianus and the production amount of hexanoic acid (C6) for each strain.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

Example  1. Production of hexanoic acid biosynthetic gene introduction strain

Escherichia coli coli) and Cluj Vero My process film cyano Taunus (Kluyveromyces Since marxianus can not produce hexanoic acid by itself, a strain that biosynthesizes hexanoic acid by introducing or disrupting the gene has been produced.

Example  1-1. Hexanoic acid biosynthetic pathway design

Although β-ketothiolase I and II (phbA, bktB) derived from Ralstonia eutropha, which is an enzyme producing C6 (hexanoic acid), were finally introduced into the native Escherichia coli, the recombinant strain did not produce C6 carboxylic acid as a fermentation product . This was because β-ketothiolase, which converts acetyl-CoA to butyryl-CoA, was not active, and a separate pathway was designed as a metabolic pathway for producing hexanoic acid, rather than using the existing β-ketothiolase pathway.

In order to solve the above problem, an intermediate product, Butyryl-CoA, was synthesized using crotonase (Crt) and trans-enoyl-CoA reductase (Ter) Mass production was induced and beta-ketothiolanes I and II (phbA, bktB) introduced in the above functioned to finally produce C6 (hexanoic acid) in a mass production.

The metabolic pathways involved in this process are as follows. Acetyl-CoA was produced in glucose and two acetyl-CoA molecules were further bound to acetoacetyl-CoA by acetyl-CoA acetyltransferase (AtoB). Acetoacetyl-CoA was reduced to 3-hydroxybutyl-CoA by NADH reduction by 3-hydroxybutyl-CoA dehydrogenase (Hbd). 3-hydroxybutyl-CoA was converted to crotonyl-CoA as a butyric acid intermediate by Crt. Crotonyl-CoA is converted to butyryl-CoA by Ter, which acts as an oxidoreductase in CH-CH bonds with NADP ⁺ and NAD ⁺ as acceptors. Butyryl-CoA is converted to Hexanol-CoA by the addition of acetyl-CoA by bktB, resulting in the production of 3-ketohexanoyl-CoA and Hbd, Crt and Ter genes. And thioesterase II (tesB) was acted upon to finally produce hexanoic acid (C6).

In addition, in order to solve the problem that the atoB gene converts acetyl-CoA to acetyl-CoA through β-oxidation reaction instead of acetoacetyl-CoA reaction in acetyl-CoA, acetyl-CoA directly converts acetoacetyl- But not by the pathway that produced malonyl-CoA, but by bypassing the pathway to acetoacetyl-CoA. The gene involved in this reaction is the malonyl-CoA carrier protein (MCT1) and Saccharomyces lt; / RTI > from S. cerevisiae .

Example  1-2. Identification of hexanoic acid biosynthetic pathway genes

The genes introduced to introduce the designed hexanoic acid biosynthetic pathway into the strain are shown in Table 1 below.

Introduction gene size and size gene origin SEQ ID NO: size BktB
(β-ketothiolase Ⅱ) Ralstonia eutropha One 394 aa (1182 bp) phbA
(β-ketothiolase I) Ralstonia eutropha 2 393 aa (1179 bp) AtoB
(acetyl-CoA acetyltransferase) Escherichia coli 3 394 aa (1185 bp) Tesb
(Thioesterase II) Escherichia coli 4 287 a.a (861 bp) Crt
(Crotonase) Clostridium  acetobutylicum 5 261 aa (861 bp) Hbd
(3-hydroxybutyryl-CoA dehydrogenase) Clostridium  acetobutylicum 6 282 aa (848 bp) Ter
(trans-enoyl-CoA reductase) Treponema denticola 7 398 aa (1194 bp) MCT1
(malonyl-CoA carrier protein) Saccharomyces  cerevisiae 8 361 aa (1083 bp)

However, K. marxianus There are plenty TesB endogenous activity of genes, TesB gene was not introduced separately.

Example  1-3. E. coli  Hexanoic acid ( C6 Construction of biosynthetic gene expression system

Six genes for biosynthesis of hexanoic acid in E. coli were introduced into three expression vectors. atoB and bktB gene in pET duet-1 vector, hbd and crt genes in pCOLA duet-1 vector, and ter tesB gene was introduced into each duet pACYC-1 vector. The primers used were as shown in Table 2 below.

Primers for introducing E. coli gene Restriction enzyme Primer sequence SEQ ID NO: atoB 5 'BamHI CGGGATCCGAAAAATTGTGTCATCGTCAGTGCG 9 3'HindⅢ CCCAAGCTTTTAATTCAACCGTTCAATCACCATC 10 bktB 5 'Nd GGAATTCCATATGACGCGTGAAGTGGTAGTGG 11 3'Xho I CCGGCTCGAGTCAGATACGCTCGAAGATGG 12 crt 5 'Nco I CATGCCATGGGCGAACTAAACAATGTCATCCTTGAAAAG 13 3'HindⅢ CCCAAGCTTCTATCTATTTTTGAAGCCTTCAATTTTTCTT 14 hbd 5 'Nd GGAATTCCATATGAAAAAGGTATGTGTTATAGGTGCAGG 15 3'Xho I CCGCTCGAGTTATTTTGAATAATCGTAGAAACCTTTTCCT 16 sweat 5 'Nco I CATGCCATGGGCATCGTCAAGCCAATGGTGCG 17 3'HindⅢ CCCAAGCTTTTAAATACGATCGAAACGTTCAACT 18 tesB 5 'Nd CATATGTTAATTGTGATTACGCATCA 19 3'Xho I CTCGAGATGAGTCAGGCGCTAAAAAATTTAC 20

The expression of the protein was confirmed by SDS-PAGE in order to confirm whether the hexanoic acid biosynthesis genes were normally transformed into E. coli . The strain was cultured in a 100 mL flask at 37 ° C and 250 rpm. To induce protein expression, protein expression was induced by the addition of OD ₆₀₀ at 0.5 to 0.1 mM IPTG. After IPTG addition, Time. The E. coli culture was recovered and the proteins were isolated by SDS-PAGE. The protein expression was confirmed by coommassie blue staining. AtoB and BktB introduced when overexpressed AtoB and BktB Proteins were identified at about 40 kDa, and Hbd and Crt The overexpressed Hbd and Crt proteins were detected at approximately 30 kDa and 28 kDa, respectively. In addition, Ter introduced 43 kDa and TesB 30 kDa, respectively (Fig. 6).

Example  1-4. K. marxianus  Hexanoic acid ( C6 Construction of biosynthetic gene expression system

Hexanoic acid (C6) biosynthetic genes were constructed by introducing the gene identified in Example 1-2 into the pJSKM316GPD vector, which is an expression vector for transformation in the existing K. marxianus . The primers used were as shown in Table 3 below.

K. marxianus introduction primer gene Restriction enzyme Primer sequence SEQ ID NO: atoB 5'Xba I CGGGATCCGAAAAATTGTGTCATCGTCAGTGCG 21 3 'BamHI CCCAAGCTTTTAATTCAACCGTTCAATCACCATC 22 phbA 5 'BamHI GGAATTCCATATGACGCGTGAAGTGGTAGTGG 23 3 'Xma I CCGGCTCGAGTCAGATACGCTCGAAGATGG 24 bktB 5'BamH CATGCCATGGGCGAACTAAACAATGTCATCCTTGAAAAG 25 3 'Xma I CCCAAGCTTCTATCTATTTTTGAAGCCTTCAATTTTTCTT 26 crt 5'BamH GGAATTCCATATGAAAAAGGTATGTGTTATAGGTGCAGG 27 3'EcoR I CCGCTCGAGTTATTTTGAATAATCGTAGAAACCTTTTCCT 28 hbd 5'BamH CATGCCATGGGCATCGTCAAGCCAATGGTGCG 29 3'EcoR I CCCAAGCTTTTAAATACGATCGAAACGTTCAACT 30 sweat 5'BamH CATATGTTAATTGTGATTACGCATCA 31 3'EcoR I CTCGAGATGAGTCAGGCGCTAAAAAATTTAC 32

In order to transform the hexanoic acid biosynthesis genes into K. marxianus , the transgenes were introduced into the genome by a random integration method. Specifically, a plasmid in which a hexanic acid biosynthetic gene constructed in expression vector pJSKM316GPD for transformation into K. marxianus was inserted was amplified by PCR from URA3 auxotroph to CYC terminator, and then the same concentration (50-100 ng / ≪ / RTI > ul). The introduced hexanoic acid biosynthesis genes were confirmed to be normally expressed in the genome through colony PCR confirmation on the plate and mRNA analysis by RT-PCR (FIG. 5).

Example  2. Related gene disruption E. coli  Strain production

Hexanoic acid production Alcohol dehydrogenase ( Adh ), Lactate dehydrogenase ( Ldh ), Lactate dehydrogenase ( Ldh ), and Lactate dehydrogenase ( Ldh ), which produce ethanol, lactic acid, succinic acid and acetic acid, Fumarate reductase, Frd ), phosphotransacetylase, Pta ) was crushed. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. "Specifically, a homologous recombination system of genomic DNA was promoted using a red recombinase, and a kanamycin resistance system was used to replace the target gene region with a kanamycin cassette The kanamycin cassette was finally removed to obtain a strain in which the target gene was disrupted (FIG. 7).

The kanamycin cassette replacement and gene disruption were confirmed through the confirmation primer by referring to the nucleotide sequence around the gene as a target of the obtained strain (Fig. 8).

Example  3. Fermentation conditions and hexanoic acid production of the produced strains

Example  3-1. E. coli  Analysis of strain fermentation conditions and hexanoic acid production

The recombinant E. coli strain expressing the hexanoic acid (C6) biosynthetic gene constructed in Example 1-3 was fermented in the following steps.

First, the strain was pre-cultured overnight in 5 ml of LB (10 g NaCl, 10 g tryptone, 5 g yeast extract / L) medium. The cells were cultured in 100 ml of LB (1% yeast extract, 2% peptone) medium and 1 L of TB (12 g tryptone, 24 g yeast extract, 2.31 g KH ₂ PO ₄ , 12.54 _{g K 2 HPO 4, 4 ml} glycerol / L) in the initial medium was inoculated with an OD ₆₀₀ was set at 0.1. When OD ₆₀₀ was increased to 0.5, induction was performed with IPTG 0.1 mM and fermentation was carried out for 7 hours under aerobic condition (1 vvm of oxygen, 600 rpm). Subsequent fermentation was carried out at 37 ° C, pH 6.8, under anaerobic conditions (1 vvm nitrogen, 250 rpm). Samples were taken every hour to confirm changes in the product.

The resulting hexanoic acid was analyzed by Gas Chromatography of YL 6100 series model. HP-FFAP (30m x 0.25mm ID, 0.25μm) was used as the column and helium gas was used as the carrier. The oven was initially maintained at 100 ° C for 5 minutes, increased from 100 ° C to 240 ° C by 10 ° C per minute, and maintained at 240 ° C for 12 minutes. The injector was set to 1:50 split mode and the detector was set to 300 ° C. Butyric acid, valeric acid and hexanoic acid showed retention times of 10 min, 11 min and 12 min, respectively. As a result of analyzing the samples collected every hour, it was confirmed that about 38 mg / L of hexanoic acid (C6) was detected and 200 mg / L of butyric acid (C4) was detected in the sample at 190 hours.

Example  3-2. K. marxianus  Analysis of strain fermentation conditions and hexanoic acid production

The recombinant K. marxianus strain expressing the hexanoic acid (C6) biosynthetic gene constructed in Example 1-4 proceeded to fermentation in the following steps.

First, the strain was pre-cultured overnight in 5 ml of YPD (1% yeast extract, 2% peptone, 2% glucose) medium and adjusted to pH 4.5 and pH 7.0 Were fermented at 37 ° C and 100 rpm under micro-aerobic and oxygen limited conditions in 50 ml of YP (1% yeast extract, 2% peptone, 20 g / L of glucose or 20 g / L of glucose). Samples were taken every hour to check the amount of fatty acid change. The resulting hexanoic acid was analyzed by Gas Chromatography. The amount of glucose and galactosis consumed in the medium was measured by DNS, a reducing sugar method. The analysis of samples taken every hour showed that hexanoic acid (C6) was detected at about 354 mg / L at 24 hours in micro-aerobic condition at pH 4.5 and 149 mg / L, 191 mg / L. When 20 g / L galactose was added to pH 7.0, hexane acid (C6) was detected at 150 mg / L and 200 mg / L at 16 and 24 hours under micro-aerobic and oxygen limited conditions, respectively. Thereafter, the amount of produced samples was reduced or not detected at the detection time.

According to the above results, it can be seen that the strains produced in Example 1 have an ability to produce an excessive amount of hexanoic acid under a certain condition. In particular, the conventional technology only produces about 20 mM of hexanoic acid through fermentation over 200 hours. However, the present invention provides a fermentation product containing about 354 mg / L of hexane Acid and can produce hexane acid in a thermophilic strain and thus have normal advantages. Therefore, it has been confirmed that the method of producing hexanoic acid using the strain has a superior effect in terms of time, yield, and manufacturing process as compared with the conventional technology.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Agricultural Biotechnology Research Institute KACC93153B 20120823

<110> Research & Business Foundation SUNGKYUNKWAN UNIVERSITY <120> Recombinant micro-organisms for producing organic aicd and the          method for producing organic acids by using thereof <130> PA120770KR <160> 32 <170> Kopatentin 2.0 <210> 1 <211> 1185 <212> DNA <213> Ralstonia eutropha beta-KETOTHIOLASE II <400> 1 atgacgcgtg aagtggtagt ggtaagcggt gtccgtaccg cgatcgggac ctttggcggc 60 agcctgaagg atgtggcacc ggcggagctg ggcgcactgg tggtgcgcga ggcgctggcg 120 cgcgcgcagg tgtcgggcga cgatgtcggc cacgtggtat tcggcaacgt gatccagacc 180 gagccgcgcg acatgtatct gggccgcgtc gcggccgtca acggcggggt gacgatcaac 240 gcccccgcgc tgaccgtgaa ccgcctgtgc ggctcgggcc tgcaggccat tgtcagcgcc 300 gcgcagacca tcctgctggg cgataccgac gtcgccatcg gcggcggcgc ggaaagcatg 360 agccgcgcac cgtacctggc gccggcagcg cgctggggcg cacgcatggg cgacgccggc 420 ctggtcgaca tgatgctggg tgcgctgcac gatcccttcc atcgcatcca catgggcgtg 480 accgccgaga atgtcgccaa ggaatacgac atctcgcgcg cgcagcagga cgaggccgcg 540 ctggaatcgc accgccgcgc ttcggcagcg atcaaggccg gctacttcaa ggaccagatc 600 gtcccggtgg tgagcaaggg ccgcaagggc gacgtgacct tcgacaccga cgagcacgtg 660 cgccatgacg ccaccatcga cgacatgacc aagctcaggc cggtcttcgt caaggaaaac 720 ggcacggtca cggccggcaa tgcctcgggc ctgaacgacg ccgccgccgc ggtggtgatg 780 atggagcgcg ccgaagccga gcgccgcggc ctgaagccgc tggcccgcct ggtgtcgtac 840 ggccatgccg gcgtggaccc gaaggccatg ggcatcggcc cggtgccggc gacgaagatc 900 gcgctggagc gcgccggcct gcaggtgtcg gacctggacg tgatcgaagc caacgaagcc 960 tttgccgcac aggcgtgcgc cgtgaccaag gcgctcggtc tggacccggc caaggttaac 1020 ccgaacggct cgggcatctc gctgggccac ccgatcggcg ccaccggtgc cctgatcacg 1080 gtgaaggcgc tgcatgagct gaaccgcgtg cagggccgct acgcgctggt gacgatgtgc 1140 atcggcggcg ggcagggcat tgccgccatc ttcgagcgta tctga 1185 <210> 2 <211> 1182 <212> DNA <213> Ralstonia eutropha beta-ketothiolase I <400> 2 atgaccgatg tagtgatcgt atcggcggtc cgtaccgccg tgggcaagtt tggcggttcg 60 ctggcgaaaa tccccgcgcc ggagctgggt gcggccgtga tccgcgaagc gctgtcgcgc 120 gccaaggtgg cgccggatca ggtcagcgaa gtcatcatgg gccaggtgct gaccgcgggt 180 tcgggccaga acccggcgcg ccaggcgttg atcaaggccg gcctgcccga catggtgccg 240 ggcatgacca tcaacaaggt gtgcggctcg ggcctgaagg ccgtgatgct ggccgccaac 300 gccatcgtcg ccggggatgc cgacatcgtc gtggccggcg ggcaggagaa catgtccgcc 360 gcgccgcacg tgctgcccgg ctcgcgcgac ggtttccgca tgggcgacac caagctcatc 420 gactcgatga tcgtggatgg gctgtgggac gtctacaacc agtaccacat gggcatcacc 480 gccgagaatg tcgccaagca gtacggcatc acgcgcgagg cccaggacgc attcgccgtg 540 gcttcgcaga acaaggcgga agccgcgcag aagtccggtc gcttcaatga cgagatcgtt 600 cccatcctga ttccgcagcg caagggcgac ccgatcgcct tcgcgcagga cgagttcgtc 660 cgccatggcg ccacgctgga atcgatgacg ggcctgaagc cggcattcga caaagccggc 720 acggtgacgg ccgccaatgc ctcgggcctc aacgacggcg gcgccgccgt ggtggtcatg 780 tcggccgccc gcgccaagga actgggtctg accccgctgg ccaccatccg cgcctacgcc 840 aatgccggcg tggacccgaa ggtgatgggc atgggcccgg tgccggcttc caagcgctgc 900 ctgtcgcgcg ccggctggtc ggtgggcgac ctggacctga tggagatcaa cgaggcgttt 960 gccgcccagg cgctggccgt gcaccagcag atgggctggg ataccgccaa ggtcaacgtc 1020 aacggcggcg cgattgccat cggtcacccc atcggcgcgt cgggctgccg catcctggtg 1080 acgctgctgc acgagatgca gaagcgcgac gcgaagaagg gcctggcctc gctgtgcatc 1140 ggcggcggca tgggcgtggc gctggcggtc gagcgcccgt ga 1182 <210> 3 <211> 1185 <212> DNA <213> Escherichia coli acetyl-CoA acetyltransferase <400> 3 atgaaaaatt gtgtcatcgt cagtgcggta cgtactgcta tcggtagttt taacggttca 60 ctcgcttcca ccagcgccat cgacctgggg gcgacagtaa ttaaagccgc cattgaacgt 120 gcaaaaatcg attcacaaca cgttgatgaa gtgattatgg gtaacgtgtt acaagccggg 180 ctggggcaaa atccggcgcg tcaggcactg ttaaaaagcg ggctggcaga aacggtgtgc 240 ggattcacgg tcaataaagt atgtggttcg ggtcttaaaa gtgtggcgct tgccgcccag 300 gccattcagg caggtcaggc gcagagcatt gtggcggggg gtatggaaaa tatgagttta 360 gccccctact tactcgatgc aaaagcacgc tctggttatc gtcttggaga cggacaggtt 420 tatgacgtaa tcctgcgcga tggcctgatg tgcgccaccc atggttatca tatggggatt 480 accgccgaaa acgtggctaa agagtacgga attacccgtg aaatgcagga tgaactggcg 540 ctacattcac agcgtaaagc ggcagccgca attgagtccg gtgcttttac agccgaaatc 600 gtcccggtaa atgttgtcac tcgaaagaaa accttcgtct tcagtcaaga cgaattcccg 660 aaagcgaatt caacggctga agcgttaggt gcattgcgcc cggccttcga taaagcagga 720 acagtcaccg ctgggaacgc gtctggtatt aacgacggtg ctgccgctct ggtgattatg 780 gaagaatctg cggcgctggc agcaggcctt acccccctgg ctcgcattaa aagttatgcc 840 agcggtggcg tgccccccgc attgatgggt atggggccag tacctgccac gcaaaaagcg 900 ttacaactgg cggggctgca actggcggat attgatctca ttgaggctaa tgaagcattt 960 gctgcacagt tccttgccgt tgggaaaaac ctgggctttg attctgagaa agtgaatgtc 1020 aacggcgggg ccatcgcgct cgggcatcct atcggtgcca gtggtgctcg tattctggtc 1080 acactattac atgccatgca ggcacgcgat aaaacgctgg ggctggcaac actgtgcatt 1140 ggcggcggtc agggaattgc gatggtgatt gaacggttga attaa 1185 <210> 4 <211> 861 <212> DNA <213> Escherichia coli Thioesterase II <400> 4 ttaattgtga ttacgcatca ccccttcctg aacggtcgag gcaaccagta cgccgtcttg 60 tgccgcgca atacagcagc cattcattca aattaaacgg gcgatggaac cacatggaat ggtcaatggt 180 ggcaatctga atccccggtt cgagaaaacc gatgccgtgc ggctgtagag ctaccggcag 240 gaagttaaga tcagaagcgt aaccgagcag atactgatga acgcgcaggt catccggcac 300 gctaccattt gcgcggatcc acacctgacg atgtggttct gcgacgtgac ctttcagtgg 360 gttatgaaac tccaccggac ggacttccag cggacgatcg cagatgaatt tatctttcag 420 cactggcggc agcaggtgcg ccagcgattg ggcgatttgc gtttccgaag ggaggccatc 480 aggcgctggc gcggacggca ttgttttttg atgttcgaaa cccgcttctg gtgcctggaa 540 agaggcagtc atataaaaaa tcggtttgcc gttttgaata gcagcaaccc ggcgggcgct 600 gaagctgtta ccgtcacgca gcgtttcgac atcataaata atcggcttct tactatcgcc 660 agggcgaaga aagtagctgt gaaacgaatg taccagccgc tcttcaggga cggtctcttt 720 tgcagcatac aaggcctgac ccacgacctg gccgccaaac acctggcgta aacctaaatc 780 ttcactctgg ccgcgaaaga gtccttcctc aattttttcc agatttaaca atgtcagtaa 840 attttttagc gcctgactc t 861 <210> 5 <211> 861 <212> DNA <213> Clostridium acetobutylicum Crotonase <400> 5 ggatccttga cggctagctc agtcctaggt acagtgctag ctcattctaa aaaaggagca 60 tctgtgatgg aactaaacaa tgtcatcctt gaaaaggaag gtaaagttgc tgtagttacc 120 attaacagac ctaaagcatt aaatgcgtta aatagtgata cactaaaaga aatggattat 180 gttataggtg aaattgaaaa tgatagcgaa gtacttgcag taattttaac tggagcagga 240 gaaaaatcat ttgtagcagg agcagatatt tctgagatga aggaaatgaa taccattgaa 300 ggtagaaaat tcgggatact tggaaataaa gtgtttagaa gattagaact tcttgaaaag 360 cctgtaatag cagctgttaa tggttttgct ttaggaggcg gatgcgaaat agctatgtct 420 tgtgatataa gaatagcttc aagcaacgca agatttggtc aaccagaagt aggtctcgga 480 ataacacctg gttttggtgg tacacaaaga ctttcaagat tagttggaat gggcatggca 540 aagcagctta tatttactgc acaaaatata aaggcagatg aagcattaag aatcggactt 600 gtaaataagg tagtagaacc tagtgaatta atgaatacag caaaagaaat tgcaaacaaa 660 attgtgagca atgctccagt agctgttaag ttaagcaaac aggctattaa tagaggaatg 720 cagtgtgata ttgatactgc tttagcattt gaatcagaag catttggaga atgcttttca 780 acagaggatc aaaaggatgc aatgacagct ttcatagaga aaagaaaaat tgaaggcttc 840 aaaaatagat agtgagtcga c 861 <210> 6 <211> 921 <212> DNA <213> Clostridium acetobutylicum 3-hydroxybutyryl-CoA dehydrogenase <400> 6 gtcgacttga cggctagctc agtcctaggt acagtgctag ctggcaagtc taaaggagca 60 tcacgaatga aaaaggtatg tgttataggt gcaggtacta tgggttcagg aattgctcag 120 gcatttgcag ctaaaggatt tgaagtagta ttaagagata ttaaagatga atttgttgat 180 agaggattag attttatcaa taaaaatctt tctaaattag ttaaaaaagg aaagatagaa 240 gaagctacta aagttgaaat cttaactaga atttccggaa cagttgacct taatatggca 300 gctgattgcg atttagttat agaagcagct gttgaaagaa tggatattaa aaagcagatt 360 tttgctgact tagacaatat atgcaagcca gaaacaattc ttgcatcaaa tacatcatca 420 ctttcaataa cagaagtggc atcagcaact aaaagacctg ataaggttat aggtatgcat 480 ttctttaatc cagctcctgt tatgaagctt gtagaggtaa taagaggaat agctacatca 540 caagaaactt ttgatgcagt taaagagaca tctatagcaa taggaaaaga tcctgtagaa 600 gtagcagaag caccaggatt tgttgtaaat agaatattaa taccaatgat taatgaagca 660 gttggatatat tagcagaagg aatagcttca gtagaagaca tagataaagc tatgaaactt 720 ggagctaatc acccaatggg accattagaa ttaggtgatt ttataggtct tgatatatgt 780 cttgctataa tggatgtttt atactcagaa actggagatt ctaagtatag accacataca 840 ttacttaaga agtatgtaag agcaggatgg cttggaagaa aatcaggaaa aggtttctac 900 gattattcaa aataactcga g 921 <210> 7 <211> 1286 <212> DNA <213> Treponema denticola trans-enoyl-CoA reductase <400> 7 ctcgagttga cggctagctc agtcctaggt acagtgctag ctacattaag gaggaagccg 60 atgatcgtca agccaatggt gcgcaataat atctgtctga acgctcaccc gcagggttgt 120 aaaaagggtg tagaagacca gattgaatac actaagaaac gcatcaccgc agaagttaaa 180 gcaggtgcca aagcaccgaa aaacgtcctg gtgctgggct gcagcaacgg ctacggtctg 240 gcaagccgca ttacggctgc attcggttac ggcgctgcta ctattggtgt tagcttcgaa 300 aaggcgggtt ctgaaaccaa atacggcact ccaggctggt acaacaacct ggcattcgac 360 gaagcagcga agcgtgaggg tctgtactct gttaccatcg acggtgacgc gttctctgac 420 gagatcaaag ctcaggttat cgaggaagct aaaaagaaag gtatcaaatt cgacctgatt 480 gtgtactccc tggcctctcc ggttcgtacc gacccggata ccggcatcat gcacaaaagc 540 gtactgaagc cgtttggcaa aaccttcact ggtaaaaccg ttgatccttt caccggcgag 600 ctgaaggaaa tctccgccga gccagctaac gatgaggagg ctgctgcgac cgttaaagtg 660 atgggtggcg aagactggga acgttggatc aaacaactgt ccaaggaagg tctgctggag 720 gagggctgta ttactctggc atattcttac atcggcccgg aggcgactca ggcactgtat 780 cgtaagggca ccatcggtaa agcgaaagaa catctggagg ccaccgctca ccgtctgaac 840 aaggaaaacc cgagcatccg tgctttcgtg tccgttaaca agggcctggt tacgcgcgct 900 tccgcagtaa ttccggtcat tccgctgtac ctggcttccc tgtttaaagt catgaaagaa 960 aaaggcaacc acgaaggttg tatcgaacaa attactcgcc tgtatgcgga gcgcctgtac 1020 cgtaaggatg gcactatccc ggttgatgaa gagaaccgca tccgcattga cgattgggaa 1080 ctggaagagg atgtacagaa agcggtttcc gcgctgatgg aaaaagtgac gggcgaaaac 1140 gcggaatccc tgacggatct ggcaggttac cgtcacgact ttctggcgtc taatggtttc 1200 gacgttgagg gtattaacta cgaggcagaa gttgaacgtt tcgatcgtat ttaatctaga 1260 acgccagggc atgagctctt aattaa 1286 <210> 8 <211> 1083 <212> DNA <213> Saccharomyces cerevisiae malonyl-CoA carrier protein <400> 8 atgaagctac taaccttccc aggtcaaggg acctccatct ccatttcgat attaaaagcg 60 ataataagaa acaaatcaag agaattccaa acaatactga gtcagaacgg caaggaatca 120 aatgatctat tgcagtacat cttccagaac ccttccagcc ccggaagcat tgcagtctgc 180 tccaaccttt tctatcaatt gtaccagata ctctcgaatc cttctgatcc tcaagatcaa 240 gcaccaaaaa atatgactaa gatcgattcc cccgacaaga aagacaatga acaatgttac 300 cttttgggtc actcgctagg cgagttaaca tgtctgagtg ttaattcact gttttcgtta 360 aaggatcttt ttgatattgc taattttaga aataagttaa tggtaacatc tactgaaaag 420 tacttagtag cccacaatat caacagatcc aacaaatttg aaatgtgggc actctcttct 480 ccgagggcca cagatttacc gcaagaagtg caaaaactac taaattcccc taatttatta 540 tcatcttcac aaaataccat ttctgtagca aatgcaaatt cagtaaagca atgtgtagtc 600 accggtctgg ttgatgattt agagtcctta agaacagaat tgaacttaag gttcccgcgt 660 ttaagaatta cagaattaac taacccatac aacatcccct tccataatag cactgtgttg 720 aggcccgttc aggaaccact ctatgactac atttgggata tattaaagaa aaacggaact 780 cacacgttga tggagttgaa ccatccaata atagctaact tagatggtaa tatatcttac 840 tatattcatc atgccctaga tagattcgtt aagtgttcaa gcaggactgt gcaattcacc 900 atgtgttatg ataccataaa ctctggaacc ccagtggaaa ttgataagag tatttgcttt 960 ggcccgggca atgtgattta taaccttatt cggagaaatt gtccccaagt ggacactata 1020 gaatacacct ctttagcaac tatagacgct tatcacaagg cggcagagga gaacaaagat 1080 tga 1083 <210> 9 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> E. col AtoB 5'BamHI <400> 9 cgggatccga aaaattgtgt catcgtcagt gcg 33 <210> 10 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> E. col AtoB 3'HindIII <400> 10 cccaagcttt taattcaacc gttcaatcac catc 34 <210> 11 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> E. col bktB 5'NdeI <400> 11 ggaattccat atgacgcgtg aagtggtagt gg 32 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> E. col bktB 3'XhoI <400> 12 ccggctcgag tcagatacgc tcgaagatgg 30 <210> 13 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> E. col crt 5'NcoI <400> 13 catgccatgg gcgaactaaa caatgtcatc cttgaaaag 39 <210> 14 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> E. col crt 3 'HindIII <400> 14 cccaagcttc tatctatttt tgaagccttc aatttttctt 40 <210> 15 <211> 39 <212> DNA <213> Artificial Sequence <220> E. col hbd 5'NdeI <400> 15 ggaattccat atgaaaaagg tatgtgttat aggtgcagg 39 <210> 16 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> E. col hbd 3'XhoI <400> 16 ccgctcgagt tattttgaat aatcgtagaa accttttcct 40 <210> 17 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> E. col ter 5'NcoI <400> 17 catgccatgg gcatcgtcaa gccaatggtg cg 32 <210> 18 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> E. col ter 3'HindIII <400> 18 cccaagcttt taaatacgat cgaaacgttc aact 34 <210> 19 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> E. col tesB 5'NdeI <400> 19 catatgttaa ttgtgattac gcatca 26 <210> 20 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> E. col tesB 3 'XhoI <400> 20 ctcgagatga gtcaggcgct aaaaaattta c 31 <210> 21 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> K. marxianus atoB 5'XbaI <400> 21 cgggatccga aaaattgtgt catcgtcagt gcg 33 <210> 22 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> K. marxianus atoB 3 'BamHI <400> 22 cccaagcttt taattcaacc gttcaatcac catc 34 <210> 23 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> K. marxianus phbA 5'BamHI <400> 23 ggaattccat atgacgcgtg aagtggtagt gg 32 <210> 24 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> K. marxianus phbA 53'XmaI <400> 24 ccggctcgag tcagatacgc tcgaagatgg 30 <210> 25 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> K. marxianus bktB 5'BamHI <400> 25 catgccatgg gcgaactaaa caatgtcatc cttgaaaag 39 <210> 26 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> K. marxianus bktB 3'XmaI <400> 26 cccaagcttc tatctatttt tgaagccttc aatttttctt 40 <210> 27 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> K. marxianus crt 5'BamHI <400> 27 ggaattccat atgaaaaagg tatgtgttat aggtgcagg 39 <210> 28 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> K.marxianus crt 3'EcoRI <400> 28 ccgctcgagt tattttgaat aatcgtagaa accttttcct 40 <210> 29 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> K. marxianus hbd 5'BamHI <400> 29 catgccatgg gcatcgtcaa gccaatggtg cg 32 <210> 30 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> K. marxianus hbd 3'EcoRI <400> 30 cccaagcttt taaatacgat cgaaacgttc aact 34 <210> 31 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> K. marxianus ter 5'BamHI <400> 31 catatgttaa ttgtgattac gcatca 26 <210> 32 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> K.marxianus ter 3'EcoRI <400> 32 ctcgagatga gtcaggcgct aaaaaattta c 31

Claims (19)

Acetyl-CoA acetyltransferase, beta-ketothiolase II, crotonase, 3-hydroxybutyryl-CoA dehydrogenase, ), And a trans-enoyl-CoA reductase activity of the microorganism.
The microorganism according to claim 1, wherein the activity of thioesterase II is further enhanced.
The method according to claim 2, wherein the activity of an alcohol dehydrogenase, a lactate dehydrogenase, a fumarate reductase, and a phosphotransacetylase is weaker than an intrinsic activity Microorganisms that have become.
4. The method according to claim 3, wherein the activity of the enzyme is attenuated by 1) deleting a part or the whole of the gene encoding the enzyme, 2) modification of the expression control sequence so that expression of the gene is decreased, and 3) A modification of said gene sequence on a chromosome, and 4) a combination thereof.
The microorganism according to claim 1, wherein the activity of β-ketothiolase I is further enhanced.
6. The microorganism according to claim 5, wherein the microorganism is the accession number KACC 93153B.
3. The microorganism according to claim 2, wherein the microorganism is Escherichia coli.
6. The microorganism according to claim 5, wherein the microorganism is yeast.
9. The microorganism according to claim 8, wherein the yeast is Saccharomyces sp., Candida sp., Kluyveromyces sp., Or Torulaspora sp.
10. The microorganism according to claim 9, wherein the yeast of the genus Kluyveromyces is Kluyveromyces marxianus.
The method according to any one of claims 1, 2 or 5, wherein the beta-ketotyolase I and beta-ketotyolase II are derived from Ralstonia eutropha; Acetyl CoA acetyltransferase and thioesterase II are from Escherichia coli; The crotonate and 3-hydroxybutyryl-CoA dehydrogenase are derived from Clostridium acetobutylicum; Wherein the trans-inyl-CoA reductase is derived from Treponema denticola.
A step of introducing into a microorganism a vector expressing an acetyl CoA acetyltransferase, beta-ketotiolase II, crotonase, 3-hydroxybutyryl-CoA dehydrogenase, and trans-inoyl-CoA reductase Wherein the microorganism is cultured in the presence of a microorganism.
13. The method of claim 12, further comprising introducing into a cell using a vector expressing Thioesterase II.
14. The method according to claim 13, further comprising the step of deleting some or all of the genes expressing an alcohol dehydrogenase, a lactate dehydrogenase, a fumarate reductase, and a transphosphorylated acetylase that are inherently present in the microorganism .
13. The method of claim 12, further comprising introducing a vector expressing beta-ketothiolase I into the microorganism.
Culturing the microorganism of any one of claims 1 to 10; And recovering the organic acid from the culture or microorganism.
17. The method of claim 16, wherein the organic acid is a hexanoic acid having 6 carbon atoms.
16. An organic acid prepared by the method of claim 16.
19. The organic acid according to claim 18, wherein the organic acid is a C 4 organic acid (butyric acid) or a C 6 organic acid (hexanoic acid).

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