KR20140026207A - Recombinant micro-organisms for producing hexanol and the method for producing hexanol by using thereof - Google Patents

Abstract

The present invention relates to microorganisms for producing hexanol which activation of cytochrome P450 monooxygenase is enhanced and a microorganism producing method. Also, the present invention relates to a hexanol production method comprising a step of culturing the microorganisms; and a step of collecting hexanol from the cultures or microorganisms and hexanol produced therefrom. According to the present invention, an existing 1-hexanol production method has low production in spite of long-time fermentation thereby blocking an biosynthesis industry of 1-hexanol, however, as genes presented in the present invention are introduced, microorganisms which have been significantly advanced in fermentation time and production volume are provided. Accordingly, when using microorganisms, it is believed to significantly contribute to 1-hexanol bio synthesis and the growth of a bio-plastic industry and is helpful for a simple and low-cost bio-plastic production method.

Description

Recombinant micro-organisms for producing hexanol and the method for producing hexanol by using approximately}

The present invention relates to a microorganism for producing hexanol with enhanced activity of cytochrome p450 monooxygenase and a method for producing the microorganism. In addition, the present invention comprises the steps of culturing the microorganism; And it relates to a hexanol production method and hexanol prepared through the step of recovering hexanol from the culture or microorganism.

As fossil energy sources, the long-time energy sources of mankind, have been depleted, existing petrochemical-based industries have been used to overcome the limits of continuous price rises and saturation of crude oil, and to prevent carbon dioxide emissions and environmental pollution from petrochemical production and use. There is an active shift towards bio-based economies that want to use biomass.

In addition, with the entry into force of the Kyoto Protocol, which aims to reduce carbon dioxide emissions from the use of petroleum-derived products, many countries are seeking to develop policies and technologies to reduce carbon dioxide emissions. Biochemical products using this are recognized as a solution and are being developed and commercialized.

Among them, biomass is a renewable organic substance, which includes plants, animals, and microorganisms, which are renewable organic substances extracted from crops and trees, agricultural products and feed crops, agricultural and forest waste, algae, and municipal waste. This includes. Unlike conventional petroleum-derived materials, biomass has the advantage of being independent of fossil fuels, containing a variety of sources, and being environmentally friendly.

As a raw material, biotechnology and bio-based chemicals are being researched and developed using biotechnology as a raw material. In the biofuel field, biofuels such as bioethanol, biodiesel, and biobutanol for transport fuel replacement In the biochemical field, various bioplastics and biochemicals using PLA, 1,3-PDO, C2 ~ C4 building blocks such as bioethylene and butadiene have been researched, developed and produced.

In particular, bioplastics collectively refers to plastic monomers such as C3, C4, C5, C6 obtained by using metabolic-fermentation processes, and polymers for human body and chemical industry, which are produced by chemical or biological methods therefrom. The plant-derived or biodegradable bioplastics industry is growing at a rapid pace as market competitiveness improves with increasing consumer preference for environmentally friendly products, increased regulations on the use of hardly degradable plastics, and rising prices for petroleum-based products. Accordingly, the global demand for bioplastics in 2013 is expected to reach 900,000 tons, with a price of $ 2.6 billion.In 2015, it accounts for 1.5-4.8% of the entire plastic market, and the market size ranges from 4 million to 12.5 million tons. It is expected to reach.

Currently, bioplastics production technology based on C3, C4, C5, and C6 monomers has a very high level of technical maturity, but C5 and C6 monomers are currently at a technically low level. In particular, bioplastics using C3 or C4 monomers are already being industrialized, but bioplastics using C5 and C6 monomers are having difficulty in industrialization due to the lack of monomer production technology. Therefore, C5 and C6 monomer production technology and bioplastics production technology using the same have attracted attention as the main development field in the bioplastics industry in the future, for this purpose, efficient C5 and C6 monomer production technology is required.

Under these backgrounds, the present inventors have diligently researched to develop a strain capable of biosynthesizing a higher yield of hexanol, a C6 monomer used as a precursor of bioplastics, and thus, cytochrome P450 monooxygenase. It was confirmed that hexanol was obtained in a high yield in the strain in which the gene was expressed. In addition, acetyl-CoA acetyltransferase, beta-ketothiolase Ⅱ (ß-Ketothiolase II), Crotonase, 3-hydroxybutyryl-CoA dehydrogenase, alcohol dehydrogenase and trans-enoyl-CoA reductase It was confirmed that the final product hexanol was obtained in high yield in a strain that increased the production of beta-ketohexanoyl-CoA by introducing a gene expressing W, thereby completing the present invention.

One object of the present invention is to provide a microorganism for producing hexanol with enhanced activity of cytochrome P450 monooxygenase.

Another object of the present invention is to provide a method for preparing the microorganism, comprising introducing a vector expressing a cytochrome p450 monooxidant into the microorganism.

Another object of the present invention is to culture the microorganism; And it provides a method for producing hexanol, comprising the step of recovering hexanol from the culture or microorganism.

Still another object of the present invention is to provide a hexanol prepared by the hexanol production method.

As one embodiment for achieving the above object, the present invention provides a microorganism for hexanol (hexanol) production, characterized in that the activity of cytochrome P450 monooxygenase is enhanced.

As used herein, the term "cytochrome P450 monooxygenase (CYP)" is essential for an organism that performs oxidative metabolism to various exogenous substances such as most drugs or environmental substances or endogenous substances such as steroids or lipids. It is a catalytic enzyme. The enzyme has a heme (prosthetic group) as a prosthetic group (iron ions can be complexed enzyme).

Cytochrome p450 monooxidant in the context of the present invention functions to produce hexanol through the process of oxidizing beta-ketohexanoyl-CoA (β-ketohexanoyl-CoA). The sequence thereof may be obtained from a known database, for example, but may be, but is not limited to, GenBank Accession No.AY505118 of NCBI. The amino acid encoding the CYP is at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, most preferably An amino acid sequence showing at least 99% similarity includes any protein having an activity of substantially oxidizing beta-ketohexanoyl-CoA (beta-ketohexanoyl-CoA), without limitation, and as a sequence having such similarity, substantially cytochrome p450 single If it is an amino acid sequence having the same or corresponding biological activity as the oxidizing agent, it is obvious that protein variants having amino acid sequences in which some sequences are deleted, modified, substituted, or added are also included within the scope of the present invention.

In one embodiment of the present invention, hexanol production is obtained by culturing a recombinant strain in which cytochrome p450 monooxidant (CYP) having a nucleotide sequence of SEQ ID NO: 9 is introduced into a Kluyveromyces marxianus ( K. marxianus ) strain. It was confirmed (FIGS. 5A, 5B and 5C). In particular, as shown in Figure 5a, it was confirmed that in the strain of the CYP gene produced 190 mg / L hexanol in 4 hours, compared to the hexanol is not produced in the natural type K. marxianus strain .

As used herein, the term "activity" means a function of an enzyme, and the present invention includes all functions that are inherent in enzymes in vivo or exhibited in vitro. Accordingly, in the present invention, "enhancing activity" means that the activity of the enzymes is improved than before, which means that the activity of the enzymes is changed beneficially in any one aspect, such as reaction rate, chemical equilibrium, and reaction energy. it means.

In the present invention, a method of enhancing (or increasing) the activity may be applied to various methods well known in the art. Examples of the method include, but are not limited to, encoding a specific enzyme by a method of additionally inserting a polynucleotide comprising a nucleotide sequence encoding a specific enzyme into a chromosome or introducing the polynucleotide into a vector system. There are a method of increasing the number of copies of the nucleotide sequence, a method of replacing with a strong promoter, a method of introducing a mutation into a promoter, and a method by genetic variation. In one specific embodiment, a method of increasing the copy number of a gene by introducing a gene encoding the enzyme into a vector and transforming the microorganism in order to enhance the activity of the enzymes in an Escherichia or Cl. Can be used.

As used herein, the term "hexanol" refers to an aliphatic saturated alcohol having 6 carbon atoms, having a formula of C ₆ H ₁₃ OH, and containing 17 isomers. N of n-hexanol is determined by the position of alcohol group (OH). In the present invention, preferably, the hexanol may be 1-hexanol. 1-hexanol is an alcohol having a structure of CH ₃ (CH ₂ ) ₅ OH, which can be used as a precursor in the manufacture of bioplastics. That is, hexanol prepared by the microorganism of the present invention is used as a precursor of bioplastics, and becomes a basis for producing bioplastics in large quantities by bio and chemical methods.

As used herein, the term "microorganism" includes algae, bacteria, protozoa, fungi, yeast and marginal organisms. Belong. As it is a minimum living unit, it performs minimal metabolic processes to maintain life, and it can be adjusted in a desired direction by promoting or inhibiting specific metabolic processes by using genetic engineering methods for these metabolic processes.

In the present invention, the microorganism may be yeast. Yeast is a microorganism used in various fermentations and is the simplest form of eukaryotes. Preferably, the yeast may be of the genus Saccharomyces, Candida, Kluyveromyces, or Torulaspora, and most preferably the genus Cluiveromyces membrane. May be Klluyveromyces marxianus.

In the present invention, Kluyveromyces maximanus is characterized by its ability to use various carbon sources, its ability to grow at high temperatures, its rapid growth rate, and its low tendency to produce ethanol when exposed to excess sugar (creptree negative type). Having yeast strain. Unlike common yeast strains, many strains have been reported.

In the present invention, the microorganism may be a microorganism having the accession number KACC 93152B.

The microorganisms for hexanol production of the present invention are acetyl CoA acetyltransferase (AtoB), beta-ketothiolase I (ß-Ketothiolase I, phbA), beta-ketothiolase II (ß-Ketothiolase II, BktB), Crotonase (Crt), 3-hydroxybutyryl-CoA dehydrogenase (3-hydroxybutyryl-CoA dehydrogenase (Hbd), and trans-inoyl-CoA reductase (trans) -enoyl-CoA reductase, Ter) activity may be further enhanced.

As used herein, the term "acetyl-CoA acetyltransferase" refers to an enzyme that generates acetoacetyl-CoA from two acetyl-CoAs by transferring an acetyl group to acetyl CoA (acetyl-coenzyme A). Specifically, it exists in the mitochondria and acts in the formation and degradation of ketone bodies and is an enzyme necessary for proper metabolism of isoleucine (NCBI Accession No. NC_012971.2)

As used herein, the terms "beta-ketothiolase I" and "beta-ketothiolase II" are condensation of a CoA compound having C2 carbon atoms. It means an enzyme produced by a compound such as C4 or C6. In the present invention, β-ketothiolase I (phbA) and II (bktB) refer to an enzyme that produces β-ketohexanoyl-CoA (C6) by adding two acetyl-CoAs to butyryl-CoA, an intermediate metabolite of metabolism ( NCBI Accession No. AF026544).

As used herein, the term "Crotonase" refers to an enzyme that binds a water molecule to an enoyl-CoA compound by, in other words, an enoyl-CoA hydratase, and reversibly a hydroxy group. It is an enzyme that also mediates the action of decomposing water molecules in CoA compounds that produce enoyl-coA compounds. In the present invention, it is used in the direction of producing crotonyl-CoA by acting on 3-hydroxybutyryl-CoA (NCBI Accession No. U17110.1).

As used herein, the term "3-hydroxybutyryl-CoA dehydrogenase" mediates the release of hydrogen from 3-hydroxybutyryl-CoA, which is interpreted as a kind of oxidation reaction. Can be. However, this reaction can also be a reversible reaction, which can also mediate a kind of reductive action to produce 3-hydroxybutyryl-CoA by attaching hydrogen to Acetoacetyl-CoA. In the present invention, 3-hydroxybutyryl-CoA dehydrogenase is used in the direction of generating 3-hydroxybutyryl-CoA by binding hydrogen to acetoacetyl-CoA (NCBI Accession No. P52041).

As used herein, the term “trans-enoyl-CoA reductase” refers to an enzyme that reduces hydrogen by attaching hydrogen to trans-inoyl-CoA. In the present invention, two hydrogens are attached to crotonyl-CoA to produce butyryl-CoA, which is an irreversible reaction (NCBI Accession No. Q73Q47).

The microorganism for producing hexanol of the present invention may be one in which the activity of alcohol dehydrogenase (AdhE2) is further enhanced.

The term "alcohol dehydrogenase" in the present invention mediates the action of hydrogen to desorb from alcohol, which can be interpreted as a kind of oxidation reaction. However, the reaction by this enzyme is a reversible reaction. In the present invention, the alcohol dehydrogenase is used to generate butyraldehyde by desorbing hydrogen from 1-Butanol and to generate butyryl-CoA by desorbing hydrogen from butyraldehyde (NCBI Accession No.).

In the present invention, the microorganism may be a microorganism that is preferably Accession Number KACC 93154B.

In the present invention, the enzymes are preferably beta-ketothiolase I and beta-ketothiolase II derived from Ralstonia eutropha; Acetyl CoA acetyltransferase and thioesterase are derived from Escherichia coli; Alcohol dehydrogenase, crotonase and 3-hydroxybutyryl-CoA dehydrogenase are derived from Clostridium acetobutylicum; Trans-inoyl-CoA reductase is from Treponema denticola; Cytochrome p450 monooxidants may be derived from Rhodococus ruber, but include without limitation any protein having the activity of the enzyme, and some if the amino acid sequence having substantially the same or corresponding biological activity as the enzyme It is obvious that protein variants whose sequences have deleted, modified, substituted, or added amino acid sequences are also included within the scope of the present invention.

As another aspect, the present invention provides a method for producing the microorganism, comprising introducing into the microorganism a vector expressing a cytochrome p450 monooxidant.

As used herein, the term "vector" may be any nucleic acid molecule for the purpose of delivering a specific gene into a host cell, and is generally self-replicating sequence, genome insertion sequence, phage or nucleotide sequence, linear or circular, single or Double stranded DNA or RNA. In particular, it may have a foreign gene and may have a factor that facilitates transformation of a specific host cell in addition to the foreign gene. Generally, vectors include sequences that direct the transcription and translation of appropriate genes, selection markers, and sequences that allow self-replicating or chromosomal insertion. Specific examples of the vector include plasmid vectors (pSE, pBR, pUC, pBluscriptII, pGEM, pTZ, pET, pJSKM316GPD) and phage or cosmid vectors (pWE15, M13, EMBL3, EMBL4, FIX II). , DASH II, ZAP II, gt11, Charon4A, Charon21A), but are not limited thereto.

In the present invention, the vector may be a yeast expression vector capable of expressing the protein of interest in yeast. Preferably, the yeast expression vector may be a vector which integrates a target protein on the genome of the yeast. On the other hand, the yeast expression vector may be a pJSKM316GPD vector preferably expressed in Kluyveromyces maximans strain.

As used herein, the term "introduction" includes any method for allowing an external gene to be included in a vector or a host cell. The method of introducing the gene into a vector or introducing a vector or the like into a host cell may be performed by selecting a suitable standard technique as known in the art. In particular, the method of introducing a vector into the microorganism in the present invention can be used by selecting any method known in the art, but is not limited to this, microijection (microijection), electroporation (electroporation), particle injection method (particle bombardment), direct muscle injection method, an insulator (insulator) and a method using a transposon can be appropriately selected and applied.

According to a specific embodiment of the present invention, the eight additional genes were inserted into a pJSKM316GPD vector expressable in Kluyveromyces macxanus (FIG. 2). It was introduced into Cluj Veromaises Maxianus. In addition, the eight genes related to 1-hexanol expression were inherently included in the genome through a process of random integration on the genome of the host microorganism (FIG. 3). The colony PCR and RT-PCR mRNA analysis confirmed that it was normally expressed and introduced into the genome (FIG. 4).

As another aspect, the present invention comprises the steps of culturing the microorganism; And it provides a method for producing hexanol, comprising the step of recovering hexanol from the culture or microorganism.

Cultivation of the recombinant yeast strain in the present invention can be carried out according to well-known methods, it is preferable to culture in an anaerobic state in which oxygen is not introduced. In addition, conditions such as the incubation temperature, the incubation time and the pH of the medium may be appropriately adjusted. Suitable culture methods include fed-batch culture, batch culture and continuous culture (cintinuous culture), and the like, but preferably fed-batch culture, but is not limited thereto.

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)). Carbon sources in the medium include sugars and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), fats and fats (e.g. 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, organic acids such as acetic acid, and the like. 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. Potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium containing salts can be used as the phosphorus source. 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 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 antifoams such as fatty acid polyglycol esters. The culture temperature is usually 20 to 45 ° C, preferably 25 to 40 ° C, most preferably 30 ° C. Cultivation can be continued until the desired amount of desired material is obtained, and the desired material can be discharged into the culture medium or contained in the cells.

Thus, the "culture" of the present invention includes microorganisms and all materials present in the incubator in which the microorganisms are cultured. In general, it means a substance containing microorganisms themselves and various enzymes, metabolites, etc. excreted in the medium.

As used herein, the term "recovery" refers to the process of separating a particular substance from a mixture. In particular, the step of recovering hexanol refers to a process of separating hexanol mixed with other substances as a metabolite such as the culture and microorganism, which is a cell or culture by a method well known in the art. It can be separated from the medium. Hexanol recovery may use a known method suitable according to the physical and chemical properties of the hexanol, for example, filtration, anion exchange chromatography, crystallization and HPLC may be used, but is not limited thereto.

In the present invention, the hexanol is preferably 1-hexanol in the metabolic process of the present invention.

According to a specific embodiment of the present invention, 1-Hexanol produced by culturing the microorganism was analyzed by gas chromatography. The equipment used YL 6100 series machine and the column was DB-FFAP (30m x 0.25mm ID, 0.25μm) and helium gas was used as carrier gas. The oven was initially kept at 100 ° C. for 5 minutes, increased by 10 ° C. per minute from 100 ° C. to 250 ° C., and held at 250 ° C. for 12 minutes. The injector was set at 1:50 split mode and the detector at 300 ° C. 1-hexanol had a retention time of 4.14 minutes. However, the gas chromatography used in this example was used for the purpose of detecting how much can be produced experimentally, not for the commercial use of 1-hexanol. In this regard, the step of separating the hexanol is a method capable of separating the high-purity and large quantities of 1-hexanol for commercial use.

As another aspect, the present invention provides a hexanol prepared by the hexanol production method.

In the present invention, the hexanol is preferably 1-hexanol in the metabolic process of the present invention. Hexanol prepared in the present invention can be used as a precursor of bioplastics.

According to the present invention, the production method of the 1-hexanol in the past despite a long time fermentation is low production, which has been an obstacle to the biosynthesis industry of 1-hexanol, the fermentation time and the introduction of the gene proposed in the present invention It provides microorganisms that are greatly developed in terms of yield. For this reason, the use of the microorganism of the present invention is expected to contribute greatly to the growth of the 1-hexanol biosynthesis and thereby the bioplastics industry, and will help in the production of a new simple and low-cost bioplastics.

1 is a schematic diagram of the biosynthetic pathway for 1-hexanol production.
FIG. 2 is a diagram showing construct expression vectors of genes required for the 1-hexanol biosynthesis pathway. FIG.
Figure 3 is a schematic diagram illustrating a mechanism for random integration transformation of the gene required for the 1-hexanol biosynthetic pathway into the microbial genome.
4 is a diagram confirming the biosynthetic gene transformed in K. marxianus .
FIG. 5a is a diagram of supplying glucose as a carbon source and analyzing 1-hexanol production amount of each strain under micro-aerobic fermentation conditions. FIG.
FIG. 5b is a diagram of supplying galactose as a carbon source and analyzing 1-hexanol production of each strain under micro-aerobic fermentation conditions. FIG.
FIG. 5c is a diagram of supplying galactose as a carbon source and analyzing 1-hexanol production of each strain under oxygen limited fermentation conditions. FIG.

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. 1- Hexanol  Production of biosynthetic gene introduction strain

Since Kluyveromyces marxianus cannot produce 1-Hexanol on its own, it was intended to produce a strain that biosynthesizes 1-Hexanol.

Example  1-1. Hexanol  Biosynthetic Metabolic Pathway Design

Ralstonia , an enzyme that produces C6 (hexanoic acid), a precursor of 1-hexanol, to native E. coli Although the beta-ketothiolase I (phbA) and II (bktB) derived from eutropha were introduced, the recombinant strain did not produce C6 carboxylic acid as a fermentation product. This is because the beta-ketothiolase, which converts acetyl-CoA to butyryl-CoA, is inactive.Therefore, a metabolic pathway that produces hexanol is used instead of the existing beta-ketothiolase. Design was made (FIG. 1).

In order to solve this problem, Butyryl-CoA, an intermediate product, was synthesized using Crotonase (CRT) and Trans-enoyl-CoA reductase (Ter) in different biosynthetic pathways. Induced mass production, and thus beta-ketothiolase I and II (phbA, bktB) were introduced to function to finally mass-produce 1-hexanol.

The metabolic pathways introducing the process are as follows. Acetyl-CoA is produced in glucose, and one more molecule of acetyl-CoA is bound to two acetoacetyl-CoA by acetyl-CoA acetyltransferase (AtoB). Acetoacetyl-CoA was converted to 3-hydroxybutyl-CoA by NADH reduction by 3-hydroxybutyl-CoA dehydrogenase (Hbd). 3-hydroxybutyl-CoA was converted to crotonyl-CoA, a butyric acid intermediate by Crt. Crotonyl-CoA is converted to butyryl-CoA by Ter, which acts as an oxidoreductase in CH-CH bonds using NADP ⁺ and NAD ⁺ as acceptors. Butyryl-CoA produced hexanoic acid (C6) by adding two acetyl-CoAs by phbA and bktB. Finally, hexanoic acid was converted to n-hexanol by monooxygenation with P450 monooxygenase substrate.

In addition, the atoB gene directly resolves acetoacetyl-CoA from acetyl-CoA to acetyl-CoA through a β-oxidation reaction rather than acetoacetyl-CoA to acetyl-CoA. Bypass route was established to acetoacetyl-CoA via malonyl-CoA rather than to produce. The gene involved in this reaction is the malonyl-CoA carrier protein (MCT1) and Saccharomyces Introduced separately from cerevisiae .

Example  1-2. Gene Identification of 1-Hexanol Biosynthesis Metabolic Pathway

Genes introduced to introduce the designed 1-Hexanol biosynthetic metabolic pathway into the strain are shown in Table 1 below.

Gene Derivation and Size gene origin SEQ ID NO: size BktB
(β-ketothiolase II) Ralstonia eutropha One 394 a.a (1182 bp) phbA
(β-ketothiolase I) Ralstonia eutropha 2 393 a.a (1179 bp) AtoB
(acetyl-CoA acetyltransferase) Escherichia coli 3 394 a.a (1185 bp) Crt
(Crotonase) Clostridium  acetobutylicum 4 261 a.a (861 bp) Hbd
(3-hydroxybutyryl-CoA dehydrogenase) Clostridium  acetobutylicum 5 282 a.a (848 bp) Ter
(trans-enoyl-CoA reductase) Treponema denticola 6 398 a.a (1194 bp) CYP
(cytochrome P450 monooxygenase) Rhodococus ruber 7 818 a.a (2454 bp) AdhE2
(alcohol dehydrogenase) Clostridium acetobutylicum 8 858 a.a (2574 bp) MCT1
(malonyl-CoA carrier protein) Saccharomyces cerevisiae 9 361 a.a (1083 bp)

Example  1-3. K. marxianus  My 1- Hexanol  Biosynthetic Gene Expression System

1-Hexanol biosynthetic genes were constructed by introducing the genes identified in Examples 1-2 into the pJSKM316GPD vector, which is an expression vector for transformation in K. marxianus . Primers used for this are shown in Table 3 below.

K. marxianus introduction primer gene Restriction enzyme Primer sequence SEQ ID NO: atoB 5'XbaⅠ CTAGTCTAGAATGAAAAATTGTGTCATCGT 10 3'BamHⅠ GATCGGATCCTTAATTCAACCGTTCAATCA 11 phbA 5'BamHⅠ GATCGGATCCGCACTGACGTTGTCATCGTATC 12 3'XmaⅠ CCGGCCCGGGTTATTTGCGCTCGACTGCC 13 bktB 5'BamHⅠ GATCGGATCCACGCGTGAAGTGGTAGTGG 14 3'XmaⅠ CCGGCCCGGGTCAGATACGCTCGAAGATGG 15 crt 5'BamHⅠ GATCGGATCCATGGAACTAAACAATGTCAT 16 3'EcoRⅠ AATTGAATTCCTATCTATTTTTGAAGCCTT 17 hbd 5'BamHⅠ GATCGGATCCATGAAAAAGGTATGTGTTAT 18 3'EcoRⅠ AATTGAATTCTTATTTTGAATAATCGTAGA 19 ter 5'BamHⅠ GATCGGATCCATGATCGTCAAGCCAATGGTGCG 20 3'EcoRⅠ AATTGAATTCTTAAATACGATCGAAACGTTCAACT 21 CYP 5'XbaⅠ GATCGGATCCATGATCGTCAAGCCAATGGTGCG 22 3'SpeⅠ AATTGAATTCTTAAATACGATCGAAACGTTCAACT 23

In order to transform K. marxianus into the 1-Hexanol biosynthetic genes, the transgenes were introduced on the genome by a random integration method. Specifically, the plasmid in which the 1-Hexanol biosynthetic genes constructed in the expression vector pJSKM316GPD for transforming K. marxianus was inserted was amplified by PCR (polymerase chain reaction) from URA3 auxotroph to CYC terminator and then the same concentration (one plasmid). Transformation at 50-100 ng / μl) (FIG. 2 and FIG. 3). The introduced 1-hexanol biosynthesis genes were confirmed to be normally expressed and introduced into the genome through colony PCR and RT-PCR mRNA analysis on the plate (Fig. 4).

Example  2. K. marxianus  Fermentation Conditions and 1- of Strains Hexanol  Generation analysis

1-Hexanol biosynthetic gene expression recombinant K.marxianus strain constructed in Example 1-3 was fermented 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 50 ml of YP (1) was adjusted to pH 7.0. % yeast extract, 2% peptone, Galactose 20g / L or Glucose 20g / L) were fermented at 37 ℃ and 100rpm under micro-aerobic and oxygen limited conditions, respectively. Samples were taken every hour to determine the amount of change in fatty acids. The produced 1-Hexanol was analyzed by Gas Chromatography. The equipment used YL 6100 series machine and the column was DB-FFAP (30m x 0.25mm ID, 0.25μm) and helium gas was used as carrier gas. The oven was initially kept at 100 ° C. for 5 minutes, increased by 10 ° C. per minute from 100 ° C. to 250 ° C., and held at 250 ° C. for 12 minutes. The injector was set at 1:50 split mode and the detector at 300 ° C. 1-hexanol had a retention time of 4.14 minutes. The amount of glucose and galactose consumed was measured using DNS, the quantitative method of reducing sugar. The amount of glucose and galactos consumed in the medium was measured using DNS, a quantitative reducing sugar method.

The analysis of the samples collected each time, only P450 CYP153A K. marxianus integration in the transformant strain CYP, AtoB, Crt, Hbd, Ter , bktB, phbA, CYP153A P450 reductase is the transformed random integration in K. marxianus Strain H6C, AtoB, Crt, Hbd, Ter, AdhE2, bktB, phbA, CYP153A P450 reductase randomly integrated into K. marxianus were fermented using glucose under micro-aerobic conditions at pH 7.0 . The CYP strain was found to be about 190 mg / L in the sample at 4 hours, the H6C strain was detected at about 159 mg / L in the sample at 4 hours, and the H7C strain was detected at about 164 mg / L 1-hexanol in the sample at 8 hours. It was. In addition, the fermentation was performed using galactose at pH 7.0 under micro-aerobic and oxygen limited conditions. Under micro-aerobic conditions, CYP strains were about 162 mg / L in the 4 hour sample, H6C strains were about 140 mg / L in the 4 hour sample, and H7C strains were about 150 mg / L 1-hexanol in the 12 hour sample. It confirmed that this was detected. Finally, under oxygen limited conditions, CYP strains were about 157 mg / L in the 4 hour sample, H6C strains were about 130 mg / L in the 8 hour sample, and H7C strains were about 140 mg / L in the 4 hour sample. It was confirmed that hexanol was detected. Thereafter, the amount produced was reduced or not detected compared to the detection time.

According to the above results, it can be seen that the strains prepared by Example 1 have the ability to produce excessive 1-Hexanol under certain conditions. In particular, existing techniques only produce 1-Hexanol less than about 50 mg / L through fermentation over 50 hours (Dekishima Y et al; J Am). Chem Soc . 2011 Aug 3; 133 (30): 11399-401), the present invention produced about 130-190 mg / L of 1-Hexanol with only 4-12 hours of fermentation, corresponding to 1 / 12.5-1 / 4 thereof. Thus, it was confirmed that the 1-Hexanol production method using the strain had superior efficiency of 2.6 times to 4 times the efficiency and 4 to 12 times the efficiency in terms of production compared to the existing 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 KACC93152B 20120823 Agricultural Biotechnology Research Institute KACC93154B 20120823

<110> Research & Business Foundation SUNGKYUNKWAN UNIVERSITY <120> Recombinant micro-organisms for producing hexanol and the method          for producing hexanol by using <130> PA120771KR <160> 23 <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 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> Clostridium acetobutylicum Crotonase <400> 4 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> 5 <211> 921 <212> DNA <213> Clostridium acetobutylicum 3-hydroxybutyryl-CoA dehydrogenase <400> 5 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 gttggtatat 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> 6 <211> 1286 <212> DNA <213> Treponema denticola trans-enoyl-CoA reductase <400> 6 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> 7 <211> 2403 <212> DNA <213> Rhodococus ruber cytochrome P450 monooxygenase <400> 7 atgtcaacga gttcaagtac aagtaatgac atccaggcaa aaataattaa cgccacatcc 60 aaagtcgtgc caatgcatct acagatcaag gcactaaaaa acttgatgaa ggtgaagcgg 120 aagaccattg gcacttcccg ccctcaggtg cactttgttg aaaccgattt gcctgacgtc 180 aatgatttgg cgatagaaga tatcgatacg agtaaccctt ttttataccg acaaggtaag 240 gcgaatgcgt actttaagcg gttgcgtgat gaagcgccgg tgcactacca gaagaacagt 300 gctttcgggc cgttctggtc ggtaacacgc tacgaagata ttgtcttcgt ggacaagagc 360 catgatttgt tttccgccga accccaaatt atcttgggtg atcctccgga aggcctgtcg 420 gttgaaatgt tcatcgctat ggatcctccc aagcacgacg tacagcgtcg ggcagtccag 480 ggtgttgttg cgcccaagaa cctgaaagaa atggaaggac tgatccgcaa gcgcaccggg 540 gacgtactcg atagcctgcc gttggacact ccgttcaact gggtgccggt ggtgtcgaaa 600 gagctgaccg ggcgcatgct cgcctcactg ttagatttcc cgtatgacga acgcgaaaaa 660 ctggttggct ggtcggatcg attgtccggc gcgtcctcgg caaccggcgg cgagtttacg 720 aatgaagatg tgttttttga tgatgctgca gatatggcgt gggctttctc caagctttgg 780 cgtgataaag aagcccgtca aaaagcaggt gaagagccgg gtttcgattt gatcagcatg 840 cttcagtcca atgaagacac aaaagatctg atcaatcgtc ctttggaatt cattggtaat 900 ctcgcgttgt tgattgttgg cggtaatgac accacgcgta actcaatgag cgggggggtg 960 ctggctttaa atcagttccc agagcaattc gagaagctaa aggcgaaccc aaagcttatc 1020 cccaattggt ctctgaaata ttcgctggca acgccgcttg cgtatatgcg ccgggttgcc 1080 aagcaggatg tggagctgaa cggacagacc atcaagaagg gtgatcgcgt gctgatgtgg 1140 tatgcgtcgg gcaaccagga tgagagaaaa tttgagaatc ctgagcaatt catcatcgac 1200 cgcaaagata cgcgtaacca tgtgtcgttt ggttatgggg ttcaccgttg tatgggcaac 1260 cgccttgccg aactgcagct gcgtattctg tgggaagagc ttctccctcg ctttgaaaac 1320 atcgaagtga tcggtgagcc ggagcgcgtg caatcgaact ttgtgcgggg ctattccaag 1380 atgatggtta agttgacggc taaaaaacaa ttcgtgctgc accgccatca accggtcacc 1440 atcggagaac ccgccgcccg ggcggtgtcc cgcaccgtca ccgtcgagcg cctggaccgg 1500 atcgccgacg acgtgctgcg cctcgtcctg cgcgacgccg gcggaaagac attacccacg 1560 tggactcccg gcgcccatat cgacctcgac ctcggcgcgc tgtcgcgcca gtactccctg 1620 tgcggcgcgc ccgatgcgcc gagctacgag attgccgtgc acctggatcc cgagagccgc 1680 ggcggttcgc gctacatcca cgaacagctc gaggtgggaa gcccgctccg gatgcgcggc 1740 cctcggaacc atttcgcgct cgaccccggc gccgagcact acgtgttcgt cgccggcggc 1800 atcggcatca ccccagtcct ggccatggcc gaccacgccc gcgcccgggg gtggagctac 1860 gaactgcact actgcggccg aaaccgttcc ggcatggcct atctcgagcg tgtcgccggg 1920 cacggtgacc gggccgccct gcacgtgtcc gaggaaggca cccggatcga cctcgccgcc 1980 ctcctcgccg agcccgcccc cggcgtccag atctacgcgt gcgggcccgg gcggctgctc 2040 gccggactcg aggacgcgag ccggaactgg cccgacgggg cgctgcacgt cgagcacttc 2100 acctcgtccc tcgcggcgct cgatccggac gtcgagcacg ccttcgacct cgaactgcgt 2160 gactcggggc tgaccgtgcg ggtcgaaccc acccagaccg tcctcgacgc gttgcgcgcc 2220 aacaacatcg acgtgcccag cgactgcgag gaaggcctct gcggctcgtg cgaggtcgcc 2280 gtcctcgacg gcgaggtcga ccatcgcgac acggtgctga ccaaggccga gcgggcggcg 2340 aaccggcaga tgatgacctg ctgctcgcgt gcctgtggcg accgcctggc cctgcgcctc 2400 tga 2403 <210> 8 <211> 2589 <212> DNA <213> Clostridium acetobutylicum alcohol dehydrogenase <400> 8 tctagaatga aggttacaaa tcagaaggag ttgaagcaaa agttaaatga attgagagaa 60 gcacagaaga aattcgccac ttacactcag gaacaagtcg ataagatatt caagcaatgt 120 gcaatcgccg cagccaagga gcgtattaat cttgctaaac tagctgtcga agaaactggt 180 attggacttg tcgaagacaa gatcatcaag aaccatttcg ccgccgagta catctacaac 240 aagtataaga atgaaaagac ttgtggtata attgatcacg atgattcctt aggcatcact 300 aaggttgctg agcctattgg tattgttgct gccattgttc caaccaccaa cccaacatcc 360 actgctatct tcaagtcttt gatctccttg aaaactagaa acgcaatttt cttttctcca 420 catccaagag ctaagaagtc caccatcgca gctgcaaagt taattttgga tgctgccgtt 480 aaggctggtg cccctaagaa catcattggt tggattgatg agccatctat tgaattgtct 540 caagatctaa tgtccgaagc cgatattatc ttggcaactg gtggcccatc aatggtcaag 600 gctgcatata gtagtggtaa accagctatt ggtgttggtg ctggcaacac accagctatc 660 attgatgaat ctgctgatat cgacatggcc gtgtcatcta ttatattaag taagacatac 720 gacaacggcg ttatctgcgc ttccgaacaa tccatcctag ttatgaactc catctacgag 780 aaggtgaagg aagagttcgt gaagaggggt tcctacattt taaaccaaaa cgaaatagcc 840 aagatcaagg aaaccatgtt taaaaacggt gccatcaacg cagacatcgt gggtaaaagt 900 gcctacatca ttgccaaaat ggctggtatt gaagttccac agaccacaaa gatcttaatc 960 ggtgaggttc aatctgttga aaaatctgaa ttgttctcac atgaaaagtt gtcaccagtg 1020 cttgccatgt acaaagtcaa ggactttgat gaagccttga agaaggctca acgtttgata 1080 gagcttggtg gtagtggcca tacatcctca ttatacatcg actcacaaaa caacaaggat 1140 aaggtgaaag agttcggttt ggctatgaaa acctccagaa cattcattaa tatgccatcc 1200 tcacaaggtg cttctggtga tttgtacaat tttgccattg ccccttcttt cactctagga 1260 tgtggcacct ggggtggtaa ttctgtctct caaaacgtgg aaccaaagca cctattgaac 1320 atcaagtccg ttgctgaaag aagagaaaac atgctatggt tcaaggttcc tcaaaagatc 1380 tatttcaagt atggttgctt gagattcgct ttgaaggaat tgaaggacat gaacaaaaag 1440 agagcattta tcgtcaccga caaggacttg ttcaagttgg gatacgtcaa caaaataact 1500 aaggtgttgg acgagatcga catcaagtac tccatcttca ctgacatcaa atcagatcct 1560 actattgata gtgttaagaa gggtgctaaa gagatgttga actttgagcc agataccatt 1620 atttcaattg gtggtggttc tccaatggac gctgccaagg ttatgcactt attgtacgaa 1680 tacccagaag cagaaattga aaacttggcc atcaatttca tggatatccg taagagaatc 1740 tgtaattttc ctaagttggg aaccaaggca atatctgtgg ccatcccaac caccgctggt 1800 acaggttctg aagcaactcc atttgctgtt ataaccaacg acgagactgg tatgaagtac 1860 ccattgacct cttacgaatt gaccccaaac atggctatta tcgatactga attaatgctt 1920 aacatgccaa ggaagcttac cgctgctacc ggcattgatg ctttggtcca tgctatcgaa 1980 gcttatgtct ccgtcatggc tactgactat accgatgaat tggcccttag agctatcaag 2040 atgattttca agtacctacc tagagcttac aaaaacggta ctaatgatat tgaagctaga 2100 gaaaagatgg cacacgcctc taacatcgct ggaatggctt ttgctaacgc atttttgggt 2160 gtttgtcact ctatggcaca caagttgggt gctatgcacc atgtcccaca cggtattgca 2220 tgtgctgtct tgattgaaga agttatcaaa tacaacgcta cagactgtcc aaccaaacag 2280 actgctttcc cacagtataa gtcccctaat gccaagagaa aatacgctga gatcgccgaa 2340 tacttgaacc taaagggaac cagtgatacc gaaaaggtca ccgcccttat cgaggcaatt 2400 tccaagttga agattgactt gtccatccca caaaatattt ctgctgcagg tatcaacaag 2460 aaggacttct acaacacttt agacaaaatg tctgaactag cattcgacga ccaatgtaca 2520 accgctaatc ctcgttatcc attaatctct gaattgaaag acatttacat caagagtttc 2580 taactcgag 2589 <210> 9 <211> 1083 <212> DNA <213> Saccharomyces cerevisiae malonyl-CoA carrier protein <400> 9 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> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> K.marxianus AtoB 5'XbaI <400> 10 ctagtctaga atgaaaaatt gtgtcatcgt 30 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> K.marxianus AtoB 3'BamHI <400> 11 gatcggatcc ttaattcaac cgttcaatca 30 <210> 12 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> K.marxianus phbA 5'BamHI <400> 12 gatcggatcc gcactgacgt tgtcatcgta tc 32 <210> 13 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> K.marxianus phbA 3'XmaI <400> 13 ccggcccggg ttatttgcgc tcgactgcc 29 <210> 14 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> K.marxianus bktB 5'BamHI <400> 14 gatcggatcc acgcgtgaag tggtagtgg 29 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> K.marxianus bktB 3'XmaI <400> 15 ccggcccggg tcagatacgc tcgaagatgg 30 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> K.marxianus crt 5'BamHI <400> 16 gatcggatcc atggaactaa acaatgtcat 30 <210> 17 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> K.marxianus crt 3 'EcoCo <400> 17 aattgaattc ctatctattt ttgaagcctt 30 <210> 18 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> K.marxianus hbd 5'BamHI <400> 18 gatcggatcc atgaaaaagg tatgtgttat 30 <210> 19 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> K.marxianus hbd 3'EcoRI <400> 19 aattgaattc ttattttgaa taatcgtaga 30 <210> 20 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> K.marxianus ter 5'BamHI <400> 20 gatcggatcc atgatcgtca agccaatggt gcg 33 <210> 21 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> K.marxianus ter 3 'EcoRI <400> 21 aattgaattc ttaaatacga tcgaaacgtt caact 35 <210> 22 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> K.marxianus CYP 5'XbaI <400> 22 gatcggatcc atgatcgtca agccaatggt gcg 33 <210> 23 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> K.marxianus CYP 3'SpeI <400> 23 aattgaattc ttaaatacga tcgaaacgtt caact 35

Claims (18)

A microorganism for producing hexanol, in which the activity of cytochrome P450 monooxygenase is enhanced.
The microorganism of claim 1, wherein the microorganism is Accession Number KACC 93152B.
The method of claim 1, further comprising acetyl-CoA acetyltransferase, beta-ketothiolase I, beta-ketothiolase II, croto Microorganisms having enhanced activity of Crotonase, 3-hydroxybutyryl-CoA dehydrogenase, and trans-enoyl-CoA reductase.
The microorganism according to claim 3, wherein the activity of alcohol dehydrogenase is further enhanced.
The microorganism according to claim 4, wherein the microorganism is Accession Number KACC 93154B.
The method according to claim 1, 3 or 4, wherein the enhancing activity of the enzyme comprises 1) an increase in the number of copies of the polynucleotide encoding the enzyme, 2) a modification of the expression control sequence to increase the expression of the polynucleotide, 3) a microorganism carried out by a method selected from the group consisting of a modification of said polynucleotide sequence on a chromosome and a combination thereof 4) to enhance the activity of said enzyme.
The microorganism of claim 6, wherein the copy number increase of the polynucleotide encoding the enzyme is performed by introducing a vector expressing the enzyme into the microorganism.
The microorganism of claim 1, wherein the microorganism is yeast.
The microorganism of claim 8, wherein the yeast is Saccharomyces, Candida, Kluyveromyces, or Torulaspora.
10. The microorganism according to claim 9, wherein the genus yeast of Kluyveromyces is Klluyveromyces marxianus.
The method according to claim 1, 3 or 4, wherein beta-ketothiolase I and beta-ketothiolase II are derived from Ralstonia eutropha; Acetyl CoA Acetyltransferase is derived from Escherichia coli; Alcohol dehydrogenase, crotonase and 3-hydroxybutyryl-CoA dehydrogenase are derived from Clostridium acetobutylicum; Trans-inoyl-CoA reductase is from Treponema denticola; Cytochrome p450 monooxidant is a microorganism derived from Rhodococus ruber (Rhodococus ruber).
A method for producing a microorganism according to claim 1, comprising introducing into a microorganism a vector expressing a cytochrome p450 monooxidant.
13. Acetyl CoA acetyltransferase, beta-ketothiolase I, beta-ketothiolase II, crotonase, 3-hydroxybutyryl-CoA dehydrogenase, and trans-inoyl-CoA Further comprising introducing into the microorganism a vector expressing a reductase.
The method of claim 13, further comprising introducing the vector expressing an alcohol dehydrogenase into the microorganism.
Culturing the microorganism of any one of claims 1 to 5 or 7 to 10; And recovering hexanol from the culture or microorganism.
The method of claim 15, wherein the hexanol is 1-hexanol (1-Hexanol) having 6 carbon atoms.
Hexanol prepared by the method of claim 15.
The hexanol of claim 17, wherein the hexanol is 1-hexanol having 1 to 6 carbon atoms.

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