KR101686214B1 - Method for producing dicarboxylic acid by omega oxidation in mutant escherichia coli - Google Patents

Abstract

The present invention relates to a mutant Escherichia coli capable of producing dicarboxylic acid through omega oxidation, a producing method thereof, and a method for producing dicarboxylic acid using the mutant E. coli. According to the method of the present invention, in the E. coli, the dicarboxylic acid can be produced from a fatty acid such as vegetable oil with an excellent yield.

Description

METHOD FOR PRODUCING DICARBOXYLIC ACID BY OMEGA OXIDATION IN MUTANT ESCHERICHIA COLI BACKGROUND OF THE INVENTION [0001]

The present invention relates to a method for producing dicarboxylic acid through oxidation of omega in E. coli, and to a mutant E. coli for producing dicarboxylic acid.

C ₁₂ Α, ω-dicarboxylic acid (LDCA), which is a long-chain fatty acid, is widely used as a raw material in the production of perfume, pressure-sensitive adhesive, polyamide, polyester and lubricant. LDCA has been mostly produced from petroleum through chemical conversion processes, which have the risk of securing resources due to depletion of petroleum resources. It also emits greenhouse gases such as carbon dioxide, which are the main cause of global warming. Accordingly, there is a need to develop a method for producing a compound such as LCDA using a fatty acid derived from a reproducible feed-stock.

Omega-oxidation is an essential metabolic pathway for the production of dicarboxylic acids in microorganisms. Unlike beta-oxidation in fatty acid oxidation, eukaryotic cells only have eukaryotic cells (Angela Cintolesi et al ., Syst Biol Med ., 5, 575-585 (2013)).

The first step in omega-oxidation is the oxidation of omega-hydroxylated fatty acids (ω-hydroxy), which is mediated by the cytochrome P450 monooxygenase (CYP450) at the fatty acid chain termini and the NADPH cytochrome P450 reductase fatty acid. Then, the omega-hydroxylated fatty acid may undergo the dicarboxylic acid-producing step mediated by the aldehyde dehydrogenase through the production of oxo-fatty acid (oxofatty acid) mediated by the alcohol dehydrogenase.

Among the CYP450s, CYP153A was identified as Marinobacter as derived from aquaeolei), the C _{10 -20} saturated fatty acid and C16: 1-18: 1 indicates the range of the active fatty acid, such as an unsaturated fatty acid, the substrate conversion rate with high omega-hydroxide acids of 63-93% of (Honda Malca S et al ., Chem Commun (Camb) , 48, 5115-7 (2012)).

Also, according to Scheps D et al ., Microb Biotechnol., 6, 694-707 (2013), the NCP is derived from Bacillus megaterium . When NCP is combined with CYP153A, in C ₁₂ fatty acids, the chain end position selectivity of the Omega fatty acids of C ₁₂ hydroxide is more than 95%.

The present inventors have succeeded in transforming Escherichia coli into a recombinant vector containing heterologous genes related to omega oxidation so as to produce dicarboxylic acid (DCA) directly from a fatty acid to produce α, ω-dicarboxylic acid, particularly C ₁₂ and C ₁₄ dicarboxylic acid. The present invention has been completed based on this finding.

Angela Cintolesi et al., Syst Biol Med., 5, 575-585 (2013) Honda Malca S et al., Chem Commun (Camb), 48, 5115-7 (2012) Scheps D et al., Microb Biotechnol., 6, 694-707 (2013)

Accordingly, an object of the present invention is to provide a method for producing dicarboxylic acid from omega-oxidation from mutant E. coli.

Another object of the present invention is to provide the mutant Escherichia coli.

In order to achieve the above object,

(1) knock out fadE and fadR genes of wild-type Escherichia coli and then bind the cytochrome P450 monooxygenase and cytochrome 450 reductase binding genes ( CYP - NCP ), ADH1 , ALD5 Transforming the E. coli with an expression vector comprising an AlkL gene to produce a mutant E. coli; And

Is selected from the group consisting of _1: 2, the mutant culture of E. coli, and the harvesting of the tissue phase cell _{and, C 10, C 12, C} 14, C 16, C 16 to the _{medium: 1,} C ₁₈ and C ₁₈ A method for producing dicarboxylic acid through omega oxidation in E. coli, comprising the step of adding a saturated fatty acid or an unsaturated fatty acid.

In order to accomplish the above-mentioned other objects, the present invention provides a method for screening a wild-type Escherichia coli fadE and fadR genes knocked out and detecting a CYP-NCP binding gene, ADH1 , ALD5 And an Escherichia coli mutant which expresses the AlkL gene and produces dicarboxylic acid through omega oxidation.

By using the method of the present invention, omega oxidation, which is a characteristic of only eukaryotic cells, is possible in E. coli, which is a prokaryotic cell, so that .alpha.,. Omega.-dicarboxylic acid can be produced from various vegetable oils composed of fatty acids having different lengths in E. coli.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing a process for producing a dicarboxylic acid from a fatty acid through omega oxidation in a mutant E. coli of the present invention. FIG.
2 shows a cleavage map of a recombinant plasmid for producing the mutant E. coli according to the present invention.
Fig. 3 is an SDS-PAGE gel photograph showing the presence or absence of protein expression of the mutant E. coli of the present invention.
Figs. 4 and 5 show the amounts of dicarboxylic acid produced according to the presence or absence of the auxiliary substrate in the biotransformation of the mutant E. coli of the present invention.
Fig. 6 shows the amount of dicarboxylic acid produced according to the temperature during biotransformation of the mutant E. coli of the present invention.
FIGS. 7 and 8 show the presence or absence of thiourea in the biotransformation of the mutant E. coli of the present invention and the amount of dicarboxylic acid produced according to the presence or absence of ALA, respectively.
Fig. 9 shows the production amounts of dicarboxylic acid according to the presence or absence of thiourea addition in the biotransformation of the mutant E. coli of the present invention.
10 shows the results of gas chromatography (GC) of the dicarboxylic acid produced in the mutant E. coli of the present invention.

(1) Knock out fadE and fadR genes of wild-type Escherichia coli and then bind the cytochrome P450 monooxygenase and cytochrome 450 reductase binding genes ( CYP-NCP ), ADH1 , ALD5 and AlkL genes Transforming the Escherichia coli with an expression vector containing Escherichia coli to prepare a mutant Escherichia coli; Is selected from the group consisting of _1: and (2) the mutant culture of E. coli, and harvesting the tissue phase cell _{and, C 10, C 12, C} 14, C 16, C 16 to the _{medium: 1,} C ₁₈ and C ₁₈ A method for producing dicarboxylic acid through omega oxidation in E. coli, comprising the step of adding a saturated fatty acid or an unsaturated fatty acid.

In the method of the present invention, step 1 is a step of knocking out the fadE and fadR genes of wild-type E. coli and then transforming the E. coli with an expression vector containing CYP-NCP , ADH1 , ALD5 and AlkL genes to prepare mutant E. coli to be.

In the present invention, the host cell may be a prokaryotic cell, preferably a wild-type Escherichia coli such as MG1655 (Blattner et al. Science 277, 5331, 1453-1474 (1997)).

In the present invention, in order to inactivate the beta-oxidation process of the host cell, that is, E. coli, fadE , which is a gene encoding the acyl CoA dehydrogenase in the E. coli, is isolated and removed. Further, when a large amount of fat present in the acyl CoA inside the cell to inhibit the function of the fadR gene which acts as inhibition of a gene related to the beta oxidation, the fadR The gene is isolated and knocked out. Thus, a preferred E. coli that can be used in the present invention may be wild-type E. coli MG1655 knocked out fadE and fadR genes.

In the present invention, "knockout" means that the activity of a specific enzyme is inactivated or removed so that the enzyme does not exhibit the activity of the enzyme.

In this step, knockout of the fadE and fadR genes in wild-type E. coli can be performed using a gene knockout method known in the art.

Also, in the above step, CYP - NCP , ADH1 , ALD5 And an expression vector containing the AlkL gene. The cleavage map of the expression vector is shown in Fig.

The vector system of the present invention can be constructed through various methods known in the art, and specific methods for this are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001) , Which is incorporated herein by reference.

The CYP - NCP gene encodes a fusion protein of cytochrome P450 monooxygenase and cytochrome 450 reductase. Specifically, the CYP - NCP is a gene coding for a fusion protein prepared through domain binding of CYP153A, a monooxygenase derived from Marley Bacteria aquaeye, and NCP derived from Bacillus magaterium. In the case of Escherichia coli, a fatty acid is converted to an omega-hydroxylated fatty acid .

Figure 1 shows the process of producing dicarboxylic acid from fatty acids through omega oxidation.

The CYP - NCP gene is described in Scheps D et al., Microb. Biotechnol., 6, 694-707 (2013), and specifically can be represented by the nucleotide sequence of SEQ ID NO: 16.

The ADH1 gene is expressed on Saccharomyces cerevisiae cerevisiae) a gene encoding an alcohol dehydrogenase derived from, for example, be represented by the base sequence of SEQ ID NO: 19. By expressing the ADH1 gene in E. coli, the omega hydroxylated fatty acid converted by the CYP - NCP gene can be converted into oxy fatty acid.

The ALD5 gene is Candida Tropical faecalis (Candida tropicalis) as the gene encoding the aldehyde dehydrogenase derived from, for example, it is represented by the base sequence of SEQ ID NO: 18. By expressing the ALD5 gene in E. coli, the oxy fatty acid converted by the ADH1 gene can be converted to a dicarboxylic acid.

The AlkL genes Pseudomonas footage is (Pseudomonas putida) a gene encoding a transporter of an alkane derived from, for example, be represented by the base sequence of SEQ ID NO: 17. By expressing the AlkL gene in E. coli, the converted dicarboxylic acid can be transported to the outside of the cell.

The expression vector used in the present invention may contain a strong promoter capable of promoting transcription (such as a tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL? Promoter, pR? Promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter), a ribosome binding site for initiation of translation, and a transcription / translation termination sequence.

The vectors that can be used in the present invention include plasmids such as Bglbrick vector, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, (e.g., pLAFR1, pHV14, pGEX series, pET series, and pUCP19), phage (e.g., lambda-Charon, lambda Delta z1 and M13 or the like) or viruses, but preferably a specific gene to be used for dicarboxylic acid biosynthesis A Bglbrick vector capable of effectively controlling the expression of the inserted gene from the outside can be used.

On the other hand, the vector of the present invention includes, as a selection marker, an antibiotic resistance gene commonly used in the art, for example, ampicillin, gentamicin, carbinicillin, chloramphenicol, streptomycin, kanamycin, And resistance genes for tetracycline.

The expression vector of the present invention includes a promoter sequence, a nucleotide sequence of the gene to be expressed (structural gene), and a terminator sequence, and the sequences are preferably linked in the order of 5 '→ 3'.

It is preferable that the expression vector of the present invention insert only a nucleotide sequence containing a ribosomal binding site (RBS) and a part essential for expression of the enzyme, in terms of reducing the metabolic burden of E. coli.

The CYP - NCP , ADH1 , ALD5 And an AlkL gene can be introduced into E. coli knocked out with fadE and fadR genes to produce transformed E. coli transformed.

How to carry the vector of the invention into the E. coli is known in the art, for example, CaCl ₂ method (Cohen, SN et al, Proc Natl Acac Sci USA, 9:..... 2110-2114 (1973)), one method (Cohen, SN et al, Proc Natl Acac Sci USA, 9: 2110-2114 (1973); and Hanahan, D., J. Mol Biol, 166:....... 557-580 (1983 ) And an electroporation method (Dower, WJ et al ., Nucleic Acids Res., 16: 6127-6145 (1988)). However, in the present invention, in order to stably produce a transformant and improve its efficiency It is preferable to use a transformation method by electroporation.

In the process of the invention, step 2, after culturing the mutant of E. coli, the harvesting of the tissue phase cells, and the medium _{C 10, C 12, C 14} , C 16, C 16: 1, C 18 and C _{18: 1} or a saturated or unsaturated fatty acid selected from the group consisting of

As the culture medium for culturing the above-mentioned mutant E. coli, Escherichia coli culture medium known in the art may be used. For example, Luria-Bertani (LB) medium can be used. The culture medium may be a medium supplemented with a suitable antibiotic such as kanamycin, ampicillin or chloramphenicol.

The cultivation of the mutant E. coli can be carried out in the presence of an expression inducing agent, isopropyl beta-D-thiogalactopyranoside (IPTG), which is added between 0.5 and 0.6 at OD 600.

In the step 2, it is preferable to add a substance selected from the group consisting of 5-aminolevulinic acid (ALA), thiourea, phosphate buffer, antibiotic, and combinations thereof when the mutant E. coli is cultured. Preferably, 5-aminolevulinic acid can be added. By adding the 5-aminolevulinic acid, it acts as a precursor of heme to increase the monoxygenase activity, thereby improving the production yield of dicarboxylic acid Can be improved. The 5-aminolevulinic acid may be added in an amount of 0.25 mM based on the total volume of the medium. In addition, by adding thiourea, it acts to purify by oxidizing hydrogen peroxide which is toxic to cells that may occur in monooxygenase.

In the step 2, at the time of harvesting the cells in the resting phase, the above culture broth was centrifuged at 12,000 g for 15 minutes at 4 ° C. After collecting only the pellets, the cells were washed twice with 0.1M, pH 7.4 potassium phosphate buffer, .

In the step 2, in suspending cells of a discontinuous discontinuous period for use in biotransformation of a fatty acid into a dicarboxylic acid, the biotransformation medium may be selected from the group consisting of potassium phosphate buffer, antibiotic, tween 80, trace element solution, It is preferable to add a substance selected from the group consisting of combinations. After resuspending cells of 50 g _cww / L in a potassium phosphate buffer, preferably 0.1 M, pH 7.4, 0.5% Tween 80, a detergent that can effectively dissolve the fatty acid, was added and the fatty acid (C ₁₂ , C _14, etc.) was added in an amount of 1 g / L, and then filter sterilized trace element solution (g / L: 2.4 g FeCl ₃ _6H ₂ O, 0.3 g CoCl ₂ _6H ₂ O, 0.15 g CuCl ₂ _2H ₂ O, 0.3 g ZnCl ₂ , 0.3 g Na ₂ MO ₄ _ ₂ H ₂ O, 0.075 g H ₃ BO ₃ , and 0.495 g MnCl ₂ _ ₄ H ₂ O).

The fatty acid may be selected from the group consisting of C ₁₀ , C ₁₂ , C ₁₄ , C ₁₆ and C ₁₈ saturated fatty acids and C _{16 : 1} and C _{18 : 1} unsaturated fatty acids as long chain fatty acids. The fatty acids may be converted to dicarboxylic acids such as hexadecanedioic acid, tetradecanedioic acid and the like via the process of the present invention.

The temperature at the time of biotransformation of the fatty acid to the dicarboxylic acid is 25 to 37 ° C, and stirring at 200 rpm, preferably 30 ° C and 200 rpm, can improve the production yield of dicarboxylic acid.

Further, according to the present invention, the conversion is preferably performed in the absence of a co-substrate selected from the group consisting of glycerol, glucose, and a combination thereof, from the viewpoint of the yield of production of the dicarboxylic acid.

The step of isolating the biosynthesized dicarboxylic acid in the mutant E. coli can be carried out according to a conventional separation or purification method known in the art (B. Aurousseau et al., Journal of the American Society of Oil Chemists' Society , 57 (3): 1558-9331 (1980); Frank C. Magne et al., Journal of the American Oil Chemists Society, 34 (3): 127-129 (1957)).

In addition, the present invention is that of the wild-type E. coli fadE and fadR gene knocked out, CYP - NCP Binding genes, ADH1 , ALD5 And an Escherichia coli mutant which expresses the AlkL gene and produces dicarboxylic acid through omega oxidation.

The Escherichia coli and CYP - NCP Binding genes, ADH1 , ALD5 And the AlkL gene are as described above in connection with the above production method.

According to the present invention, it is possible to produce a dicarboxylic acid by introducing an omega oxidation system, which is a metabolic process not found in wild type prokaryotes, for example, E. coli, into E. coli and converting the fatty acid to dicarboxylic acid in E. coli. Therefore, according to the present invention, it is also possible to produce dicarboxylic acids from fatty acids such as various vegetable oils and the like with excellent yield even in Escherichia coli.

[Example]

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

<Materials and supplies>

Escherichia coli (DH10B) strain was used for cloning, using wild-type E. coli MG1655 as the parent strain (Blattner et al., Science 277, 5331, 1453-1474 (1997)).

The Bglbrick plasmid (pBbA6C and pBbE6K; A = p15A replicon; E = colE1 replicon; 6 = PLlacO promoter; C = chloramphenicol resistance and K = kanamycin resistance) was extracted from the lab stock and the expression construct (Lee et al ., J Biol. Eng., 5:12 (2011)).

Other plasmids, such as the pSIM5 plasmid for λ-Red recombinant expression plasmids (Datsenko and Wanner et al., PNAS, 97, 6640-6645 (2000)); a pCP20 plasmid for the yeast FLP recombinant gene controlled by cI repressor and temperature sensitive replication (Cherepanov and Wackernagel, Gene, 158 (1): 9-14 (1995)); And pKD13 (Datsenko and Wanner et al., PNAS, 97, 6640-6645 (2000)) as a template plasmid for gene disruption, and resistant genes flanked by the FRT region were obtained from each lap cell stock.

All materials including fatty acids and DCA were obtained from Sigma-Aldrich, and restriction enzymes and DNA ligase (New England Biolabs Inc.) and Phusion high-Fidelity DNA polymerase (Thermo scientific) were used for cloning and plasmid construction . Oligonucleotides were synthesized with reference to Korea and E. coli were grown at 37 ° C in LB medium supplemented with appropriate antibiotics such as 50 μg / ml kanamycin, 100 μg / ml ampicillin or 30 μg / ml chloramphenicol.

In addition, the strains and plasmids used in the following examples are shown in Table 1, and the sequences of primers and used genes used in the production of expression vectors are shown in Tables 2 and 3, respectively.

Example  1: Manufacture of Omega-Oxidized Escherichia coli

A mutant E. coli capable of performing omega oxidation in the wild type Escherichia coli was prepared according to the following method.

(1-1) fade  And / or fadR  Gene knockout

(Lambda-Red) and FLP-mediated (Lambda-Red) in accordance with the method described in Datsenko and Wanner et al., PNAS, 97, 6640-6645 (2000) FadE , a gene encoding an acyl CoA dehydrogenase, was knocked out using the primers of SEQ ID NOS: 3 and 4 shown in Table 2 using a FLP-mediated site-specific recombination system.

Also, when a large amount of fatty acyl CoA is present in the cell, the fadR gene, which is a fatty acid metabolic regulator acting as an inhibitor of the gene related to beta oxidation, is knocked out using the primers of SEQ ID NOS: 1 and 2.

Specifically, wild-type MG1655 strain was transformed with pSim5 plasmid capable of expressing lambda-red recombinase, and then recovered at 30 DEG C for 1 hour. Colonies cultured overnight at 30 &lt; 0 &gt; C in chloramphenicol agar medium were then selected. On the other hand, kanamycin cassettes were amplified by PCR from pKD13 plasmids using the primers of SEQ ID NOS: 1, 2 and 3, 4, and the selected cells were transformed with the amplified kanamycin cassette. Then, the transformed cells were restored for about 2 hours to 3 hours, and colonies cultured at 37 占 폚 were selected for curing in kanamycin agar medium. The selected cells were transformed with a PCP20 plasmid capable of expressing the flipperase and then restored for 1 hour and 30 minutes. The cells were cultured overnight at 30 ° C in an ampicillin agar medium, and the cultured colonies were finally cultured in a normal LB agar medium after stirring in a general LB medium at 42 ° C.

The E. coli MG1655 was refers to the E. coli MG1655 and the fadE fadR gene has been removed through the process as "MG-Δ fadEΔ fadR", also, only remove fadE gene was referred to as "MG-Δ fadE".

(1-2) Construction of an expression vector containing an omega-oxidized gene

(CYP153A) derived from Marinobacterium aquaeyrae (DSM 11845), Bacillus margatta (Bacillus spp.) According to the method of Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (ADCC) derived from NADPH: cytochrome P450 reductase (NCP), saccharomyces cerevisiae (KCTC 7296), and aldehyde dehydrogenase (ADCC) derived from Candida tropicalis (ATCC 20336) (ALD5) DNA was amplified using Phusion high-Fidelity DNA polymerase (Thermo scientific). The Pseudomonas putida-alkane transporter (AlkL) gene cloned in pGEM-B1 (Biona, Korea) was synthesized in Bioneer (Korea).

Specifically, the CYP - NCP binding gene was prepared by binding CYP153A and the NCP domain with reference to the document (Scheps D et al ., Microb Biotechnol 6, 694-707 (2013)). Maq_F / Maq_R primers of SEQ ID NOs: 5 and 6 for CYP153A and NCP_F / NCP_R primers of SEQ ID NOs: 7 and 8 for NCP , respectively. The two genes were amplified by 98 At 30 ° C for 20 seconds, at 60 ° C for 30 seconds, and at 72 ° C for 60 seconds for 30 cycles, then at 72 ° C for 5 minutes using overlap extension PCR. At this time, a 3XGGS tandem region was inserted between the CYP153A and the NCP as a linker.

The fusion gene CYP - NCP obtained above was transformed into pBbA6C plasmid (Lee et al ., J Biol Eng, 5:12 (2011), A = p15A replicon, E = colE1 replicon, 6 = PLlacO promoter, C = Clms Page number 16 &gt; plasmid was cloned into the Bgl II and Xho I sites of the plasmid pBbA6C-FP (Kramfenicol resistance and K = kanamycin resistance).

AlkL was amplified using the AlkL_F / AlkL_R primer of SEQ ID NOS: 9 and 10, followed by 2 cycles at 98 ° C for 2 minutes, at 98 ° C for 2 minutes, at 98 ° C for 20 seconds, at 55 ° C for 30 seconds and at 72 ° C for 15 seconds , And 72 &lt; 0 &gt; C for 5 minutes. Next, the plasmid pBbA6C-FP prepared above was digested with restriction enzymes Pvu I and Xho I, and the amplified AlkL was ligated with DNA ligase (New England Biolabs Inc.) to obtain pBbA6C-FP / AlkL A plasmid was prepared.

On the other hand, the ADH1 gene encoding the alcohol dehydrogenase was amplified by PCR using the ADH1_F / ADH1_R primer of SEQ ID NOS: 11 and 12 at 98 ° C for 2 minutes, at 98 ° C for 20 seconds, at 57 ° C for 30 seconds, After the cycle, the amplified product was amplified by PCR under the condition of 72 ° C for 5 minutes. The ALD5 gene coding for the aldehyde dehydrogenase was amplified by PCR using the ALD5_F / ALD5_R primer of SEQ ID NOS: 13 and 14 at 98 ° C for 2 minutes, 20 sec., 58 sec. For 30 sec. And 72 sec. For 30 sec. 30 cycles, and 72 sec. For 5 min. The two genes were ligated by overlap extension PCR under conditions of 2 minutes at 98 占 폚, 30 cycles at 98 占 폚 for 20 seconds, 60 占 폚 for 30 seconds and 72 占 폚 for 50 seconds, and 72 占 폚 for 5 minutes , the Nde I / Xho I site of the pBbE6K plasmid (Lee et al ., 2011, A = p15A replicon, E = colE1 replicon, 6 = PLlacO promoter, C = chloramphenicol resistance and K = kanamycin resistance) . ADH-ALD was amplified from pBbE6K using the ALD_F / ADH1_R primers of SEQ ID NOS: 15 and 12, and then digested with XhoI .

Finally, the pBbA6C-FP / AlkL plasmid prepared above was digested with Xho I, and the ALD-ADH product was ligated with DNA ligase (New England Biolabs Inc.) to obtain "pBbA6C-ω "Plasmids were prepared. The cleavage map of the plasmid is shown in Fig.

(1-3) Production of mutant E. coli for omega-oxidation

The MG1655 (MG-fadE Δ) strain, or fadE fadR gene and the MG1655 (MG-Δ fadEΔfadR) strain removal manufacturing fadE gene is removed from the 1-1, the ω-pBbA6C plasmid prepared in 1-2 , And the transformed strains were referred to as Eω and REω, respectively.

Experimental Example  1: Confirmation of protein expression in the prepared strain

Whether or not the protein expression was properly performed in the mutant E. coli REω prepared in the above example was analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gels) electrophoresis as follows.

First, the E. coli E. coli REω was grown at an optical density of 600 to 0.5, and the expression was induced using 0.1 mM IPTG. Then, the cells were cultured at 25 DEG C for 16 hours, and the cells were collected, and the loading buffer was added, and then the mixture was heated at 95 DEG C for 20 minutes. Whole-cell lysates were electrophoresed with 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gels) using the Laemmli method. Protein bands were stained with Coomassie brilliant blue R-250 to confirm protein expression. At this time, the molecular weight of each gene was analyzed using the online ExPasy tool. A photograph of the SDS-PAGE gel is shown in Fig.

In Figure 3 M will represent the total fraction of the marker, lane 1 and 2 are each mutant E. coli and wild type E. coli (control), a is a CYP - NCP fusion protein (120 kD) is, b is an aldehyde dehydrogenase (ALD1, 54 kD), c is an alcohol dehydrogenase ( ADH5 , 37 kD), and d is an alkane transporter ( AlkL , 25 kD), indicating that protein expression is normal in mutant E. coli REω.

Experimental Example  2: Conversion of fatty acid to dicarboxylic acid

(2-1) Preparation of Escherichia coli in the resting cell state

Single colonies of the mutant Escherichia coli (Eω and RE ω) prepared in the above example were cultured overnight in 5 ml of LB medium at 37 ° C. and 200 rpm. 500 L of mutant strain cultured overnight was inoculated into 50 ml of LB medium and cultured at 37 ° C and 200 rpm. Then, the culture was inoculated into 1 L of LB medium at a ratio of 1: 100, 600 to 0.5-0.6, 0.1 mM IPTG was added to induce expression.

Then, 0.25 mM of 5-aminolevulinic acid, a precursor of reduced hematin, was added and cultured at 25 ° C and 200 rpm for 20 hours. The cells were then recovered by centrifugation at 25 DEG C and 12,000 g for 15 minutes and washed twice with potassium phosphate buffer (0.1 M, pH 7.4).

(2-2) Mutation In E. coli  Measurement of dicarboxylic acid production using omega oxidation

E. coli (50 g _cww / L) in the resting state obtained in 2-1 above was resuspended in 0.1 M potassium phosphate buffer (pH 7.4), and then 1 g / L of C ₁₂ Fatty acids and C ₁₄ Fatty acids, respectively. Then, a 1X concentration filter-filtered aqueous trace element solution (g / L: 2.4 g FeCl ₃ _ ₆ H ₂ O, 0.3 g CoCl ₂ _ ₆ H ₂ O, 0.15 g CuCl ₂ _ ₂ H ₂ O, 0.3 g ZnCl ₂ , 0.3 g Na ₂ MO _{4 _2H 2 O, 0.075gH 3 BO 3} , and _{0.495g MnCl 2 _4H 2 O) (} Zhou L et al., Curr. Microbiol., 62, 981-989 (2011) was added), and 30 μg / mL chloramphenicol . 0.5% of TWEEN80 was added to dissolve the fatty acid.

The mixed solution was stirred and cultured in a shaking incubator under the conditions of 30 ° C. and 200 rpm, and the amount of dicarboxylic acid produced was measured using a gas chromatography / flame ionization detector (GC / FID) Respectively.

Fatty acid 1 g / L generated from a result of measuring the amount of dicarboxylic acid, mutant E. coli REω of the present invention is from about 300 mg / L (307.69 mg / L) after about 16 hours when given away to put the C ₁₄ fatty acid C ₁₄ dicarboxylic acids was confirmed generated, it was confirmed that C ₁₂ dicarboxylic acid is generated in the case given away into the C ₁₂ fatty acid, 150 mg / L (149.92 mg / L) after about 16 hours.

Experimental Example  3: Identification of Factors Affecting Dicarboxylic Acid Production

(3-1) Effect of auxiliary substrate, temperature and cell concentration on dicarboxylic acid production

To determine whether the amount of dicarboxylic acid produced varies with the presence or absence of the auxiliary substrate, the temperature and the cell concentration in the conversion of Experimental Example 2-2. Conversion was performed in a shaking incubator (30 ° C and 200 rpm), samples were collected and quantified using GC / FID.

First, in order to confirm the effect of the auxiliary substrate, the case of adding 1% (w / v) glycerol and 0.4% (w / v) D-glucose as an auxiliary substrate in the conversion in Experimental Example 2-2 and the case The content of dicarboxylic acid was measured every 4 hours and the results are shown in Figs. 4 and 5, respectively.

As shown in FIGS. 4 and 5, when the auxiliary substrate was not added, the yield was improved more than two times as compared with the case where the auxiliary substrate was added. In addition, it was confirmed that E. coli transformed for 24 hours without addition of an auxiliary substrate exhibited about 2-fold improvement in production yield as compared with the yield of dicarboxylic acid production of mutant Escherichia coli REω transformed with the auxiliary substrate for 48 hours I could.

The content of C ₁₂ dicarboxylic acid measured in the most favorable conversion conditions (i.e., when the conversion is carried out for 12 hours) is 158 mg / L, and C ₁₄ The content of dicarboxylic acid was 410 mg / L.

Further, in order to confirm the influence of the temperature, the strain of RE? Was transformed at 25 占 폚, 30 占 폚 and 37 占 폚 during the transformation in Experimental Example 2. As a result, as shown in FIG. 6, it was found that conversion at 30 ° C was preferable in terms of yield of dicarboxylic acid production. The data presented in Figure 6 are expressed as the mean of three experiments, and the error bars represent standard deviations.

(3- 2 dicarboxylic acid  The 5- Aminolevulinic acid  And Thiourea  effect

To confirm the effect of 5-aminolefic acid (ALA), a haeme pro-cursor, on the yield of dicarboxylic acid production yield of mutant E. coli REω, 0.25 mM ALA was added together with 0.1 mM IPTG to produce mutant E. coli REω Induction of heterologous gene transduction, and subsequent transformation. The results of dicarboxylic acid production with or without addition of ALA in the presence of thiourea are shown in FIG. 7, and the results of production of dicarboxylic acid with or without addition of ALA under the absence of thiourea are shown in FIG. The data presented in Figures 7 and 8 are expressed as the mean of three experiments, and the error bars represent the standard deviation.

As a result, the addition of ALA significantly affected the yield of dicarboxylic acid production ( P <0.01) compared to the case without ALA at the time of conversion of E. coli E. coli. It can be seen that hemifrocursor containing a monooxygenase enzyme such as CYP153A exhibits high production yield regardless of the presence or absence of thiourea through ALA supplementation.

Further, thiourea was added to a biotransformation medium (Doi et al ., Appl Microbiol Biotechnol, 98 (2), 629-639 (2014)) to a final concentration of 1 mM, followed by biotransformation to obtain dicarboxylic acid Production was measured. The results are shown in Fig. The data presented in Figure 9 are presented as the mean of three experiments, and the error bars represent the standard deviation.

As shown in FIG. 9, when thiourea was added, the yield of C ₁₄ dicarboxylic acid in RE ω was greatly increased ( P <0.01), but C ₁₂ The yield of dicarboxylic acid was not significantly affected ( P > 0.05).

(3-3) GC / FID Product Analysis by

In order to quantify the amount of omega dicarboxylic acid (dodecanedioic acid and tetradecanoic acid) of mutant E. coli in the above methods, the following procedure was performed.

First, the cell-free supernatant obtained using centrifugation was acidified with HCl and extracted with an equal volume of 1 g / LC ₁₉ -ethylacetate as an internal standard. The organic layer was then collected and vortexed twice and derivatized using (trimethylsilyl) diazomethane solution (TMS). The silylated derivative obtained above was analyzed and quantified with GC (Agilent 7890A, USA) equipped with FID and capillary column (Supelco-Nukol, 15 m x 0.53 mm ID, 0.50 m film).

The temperature program was set at 120 ° C for 2 minutes, and the linear temperature gradient was from 10 ° C / min to 220 ° C. The concentration of the biotransformation product was measured based on calibration curves (R ² > 0.99). Mass spectral detector (Agilent, 5975C, USA) was obtained at 70 eV by electron impact ionization.

The gas chromatographic analysis results of the dicarboxylic acid produced in RE ω are shown in FIG.

As a result, the shaking in the culture test, and from Eω REω both C ₁₂ and C ₁₄ fatty acids, respectively dodecanedioic acid (C ₁₂ Dicarboxylic acid; C ₁₂ -DCA) and tetradecanedione (C ₁₄ dicarboxylic acid; C ₁₄ -DCA).

The maximum amounts of C ₁₂ -DCA and C ₁₄ -DCA produced after 48 hours of transformation in RE ω-mutant E. coli were 75.53 mg / L and 157.24 mg / L, respectively. In particular, the production of C ₁₂ -DCA and C ₁₄ -DCA produced after transformation for 48 hours was 142.08% and 204.37% higher than that of E. coli, respectively, in RE ω-mutant E. coli. This is because wild type MG1655 Escherichia coli fadR It is thought that the deletion of the gene promoted the transport of fatty acids in the cells by increasing the expression of the fadL / D gene, which plays a role of fatty acid transport.

In the case of C ₁₂ -DCA, the conversion efficiency was found to be 14.76% in E. coli E. coli. On the other hand, the production efficiency of C ₁₄ -DCA was 18.04%. A linear relationship between production yield and time was clearly observed. RE ω-mutant E. coli began to transform faster than E ω and produced C ₁₄ -DCA more than C ₁₂ -DCA.

On the other hand, omega-hydroxylated fatty acids can be synthesized by converting fatty acids in mutant E. coli transformed with a vector containing CYP153A / AlkL gene using ancillary substrates (glycerol and glucose). However, in the present invention, an auxiliary substrate is not added to synthesize dicarboxylic acid by Baeyer bilayer monooxygenase / esterase. In addition, the maximum product formation rate and maximum product concentration were higher when glucose was not added during the conversion. This means that glucose used as a carbon source and energy source does not help to convert oleic acid to 10-hydroxystearic acid (Jeon et al., Process Biochemistry, 47 (6 ), 941-947 (2012)).

Glycerin and glucose induce alkane degradation and inhibit the introduction of dicarboxylic acid producing enzymes in C. tropicallis and Y. lipolytica strains (Schindler et al ., 1990, Seghezzi et al ., 1992, Nazarko, et al ., 2004). When glucose was added alone to the medium, not all strains produced C ₁₂ -DCA and C ₁₄ -DCA. Glucose strongly inhibits the expression of regulon genes both in the presence and absence of oleate. Therefore, in the present invention, it is preferable that the fatty acid conversion of the mutant E. coli is carried out without any auxiliary substrate.

<110> UNIST ACADEMY-INDUSTRY RESEARCH CORPORATION <120> METHOD FOR PRODUCING DICARBOXYLIC ACID BY OMEGA OXIDATION IN          MUTANT ESCHERICHIA COLI <130> FPD201505-0128 <160> 19 <170> KoPatentin 3.0 <210> 1 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> FadR_del_F <400> 1 tctggtatga tgagtccaac tttgttttgc tgtgttatgg aaatctcact gtgtaggctg 60 gagctgcttc 70 <210> 2 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> FadR_del_R <400> 2 aacaacaaaa aacccctcgt ttgaggggtt tgctctttaa acggaaggga attccgggga 60 tccgtcgacc 70 <210> 3 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> FadE_del_F <400> 3 ccatatcatc acaagtggtc agacctccta caagtaaggg gcttttcgtt gtgtaggctg 60 gagctgcttc 70 <210> 4 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> FadE_del_R <400> 4 ttacgcggct tcaactttcc gcactttctc cggcaacttt accggcttcg attccgggga 60 tccgtcgacc 70 <210> 5 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Maq_F <400> 5 atagatcttt taagaaggag atatacatat gccaacactg cccagaac 48 <210> 6 <211> 85 <212> DNA <213> Artificial Sequence <220> <223> Maq_R <400> 6 ctgttcagtg ctaggtgaag gaatgctgcc gccgctgccg ccgctgccgc cactgttcgg 60 tgtcagtttg accatcaacc tggaa 85 <210> 7 <211> 83 <212> DNA <213> Artificial Sequence <220> <223> NCP_F <400> 7 gcggcagcat tccttcacct agcactgaac agtctgctaa aaaagtacgc aaaaaggcag 60 aaaacgctat aatacgccgc tgc 83 <210> 8 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> NCP_R <400> 8 attctcgagc gatatcgatc gttattaccc agcccacacg tcttttg 47 <210> 9 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> AlkL_F <400> 9 ggtcgatcgt ttaagaagga gatatacata tgagtttttc taattataaa gtaatcgcga 60 tgccg 65 <210> 10 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> AlkL_R <400> 10 atctcgagtt attagaaaac atatgacgca ccaa 34 <210> 11 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> ADH1_F <400> 11 tttaagaagg agatatacac atgtctatcc cagaaactca 40 <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> ADH1_R <400> 12 cttactcgag ttatttagaa gtgtcaacaa cgt 33 <210> 13 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> ALD5_F <400> 13 atacatatgt ctttgccagt cgtcaccaa 29 <210> 14 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> ALD5_R <400> 14 gtgtatatct ccttcttaaa agatccttaa gtgagcttaa ttctaacag 49 <210> 15 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> ALD_F <400> 15 aggcctcgag aattgtgagc ggataacaat tgac 34 <210> 16 <211> 3223 <212> DNA <213> Artificial Sequence <220> <223> CYP-NCP <400> 16 atgccaacac tgcccagaac atttgacgac attcagtccc gactgattaa cgccacctcc 60 agggtggtgc cgatgcagag gcaaattcag ggactgaaat tccttaatgag cgccaagagg 120 aagaccttcg gcccacgccg accgatgccc gaattcgttg aaacacccat cccggacgtt 180 aacacgctgg cccttgagga catcgatgtc agcaatccgt ttttataccg gcagggtcag 240 tggcgcgcct atttcaaacg gttgcgtgat gaggcgccgg tccattacca gaagaacagc 300 cctttcggcc ccttctggtc ggtaactcgg tttgaagaca tcctgttcgt ggataagagt 360 cacgacctgt tttccgccga gccgcaaatc attctcggtg accctccgga ggggctgtcg 420 gtggaaatgt tcatagcgat ggatccgccg aaacacgatg tgcagcgcag ctcggtgcag 480 ggagtagtgg caccgaaaaa cctgaaggag atggaggggc tgatccgatc acgcaccggc 540 gatgtgcttg acagcctgcc tacagacaaa ccctttaact gggtacctgc tgtttccaag 600 gaactcacag gccgcatgct ggcgacgctt ctggattttc cttacgagga acgccacaag 660 ctggttgagt ggtcggacag aatggcaggt gcagcatcgg ccaccggcgg ggagtttgcc 720 gatgaaaatg ccatgtttga cgacgcggca gacatggccc ggtctttctc caggctttgg 780 cgggacaagg aggcgcgccg cgcagcaggc gaggagcccg gtttcgattt gatcagcctg 840 ttgcagagca acaaagaaac gaaagacctg atcaatcggc cgatggagtt tatcggtaat 900 ttgacgctgc tcatagtcgg cggcaacgat acgacgcgca actcgatgag tggtggcctg 960 gtggccatga acgaattccc cagggaattt gaaaaattga aggcaaaacc ggagttgatt 1020 ccgaacatgg tgtcggaaat catccgctgg caaacgccgc tggcctatat gcgccgaatc 1080 gccaagcagg atgtcgaact gggcggccag accatcaaga agggtgatcg agttgtcatg 1140 tggtacgcgt cgggtaaccg ggacgagcgc aaatttgaca accccgatca gttcatcatt 1200 gatcgcaagg acgcacgaaa ccacatgtcg ttcggctatg gggttcaccg ttgcatgggc 1260 aaccgtctgg ctgaactgca actgcgcatc ctctgggaag aaatactcaa gcgttttgac 1320 aacatcgaag tcgtcgaaga gcccgagcgg gtgcagtcca acttcgtgcg gggctattcc 1380 aggttgatgg tcaaactgac accgaacagt ggcggcagcg gcggcagcgg cggcagcatt 1440 ccttcaccta gcactgaaca gtctgctaaa aaagtacgca aaaaggcaga aaaaggcaga 1500 aaacgctcat aatacgccgc tgcttgtgct atacggttca aatatgggaa cagctgaagg 1560 aacggcgcgt gatttagcag atattgcaat gagcaaagga tttgcaccgc aggtcgcaac 1620 gcttgattca cacgccggaa atcttccgcg cgaaggagct gtattaattg taacggcgtc 1680 ttataacggt catccgcctg ataacgcaaa gcaatttgtc gactggttag accaagcgtc 1740 tgctgatgaa gtaaaaggcg ttcgctactc cgtatttgga tgcggcgata aaaactgggc 1800 tactacgtat caaaaagtgc ctgcttttat cgatgaaacg cttgccgcta aaggggcaga 1860 aaacatcgct gaccgcggtg aagcagatgc aagcgacgac tttgaaggca catatgaaga 1920 atggcgtgaa catatgtgga gtgacgtagc agcctacttt aacctcgaca ttgaaaacag 1980 tgaagataat aaatctactc tttcacttca atttgtcgac agcgccgcgg atatgccgct 2040 tgcgaaaatg cacggtgcgt tttcaacgaa cgtcgtagca agcaaagaac ttcaacagcc 2100 aggcagtgca cgaagcacgc gacatcttga aattgaactt ccaaaagaag cttcttatca 2160 agaaggagat catttaggtg ttattcctcg caactatgaa ggaatagtaa accgtgtaac 2220 agcaaggttc ggcctagatg catcacagca aatccgtctg gaagcagaag aagaaaaatt 2280 agctcatttg ccactcgcta aaacagtatc cgtagaagag cttctgcaat acgtggagct 2340 tcaagatcct gttacgcgca cgcagcttcg cgcaatggct gctaaaacgg tctgcccgcc 2400 gcataaagta gagcttgaag ccttgcttga aaagcaagcc tacaaagaac aagtgctggc 2460 aaaacgttta acaatgcttg aactgcttga aaaatacccg gcgtgtgaaa tgaaattcag 2520 cgaatttatc gcccttctgc caagcatacg cccgcgctat tactcgattt cttcatcacc 2580 tcgtgtcgat gaaaaacaag caagcatcac ggtcagcgtt gtctcaggag aagcgtggag 2640 cggatatgga gaatataaag gaattgcgtc gaactatctt gccgagctgc aagaaggaga 2700 tacgattacg tgctttattt ccacaccgca gtcagaattt acgctgccaa aagaccctga 2760 aacgccgctt atcatggtcg gaccgggaac aggcgtcgcg ccgtttagag gctttgtgca 2820 ggcgcgcaaa cagctaaaag aacaaggaca gtcacttgga gaagcacatt tatacttcgg 2880 ctgccgttca cctcatgaag actatctgta tcaagaagag cttgaaaacg cccaaagcga 2940 aggcatcatt acgcttcata ccgctttttc tcgcatgcca aatcagccga aaacatacgt 3000 tcagcacgta atggaacaag acggcaagaa attgattgaa cttcttgatc aaggagcgca 3060 cttctatatt tgcggagacg gaagccaaat ggcacctgcc gttgaagcaa cgcttatgaa 3120 aagctatgct gacgttcacc aagtgagtga agcagacgct cgcttatggc tgcagcagct 3180 agaagaaaaa ggccgatacg caaaagacgt gtgggctggg taa 3223 <210> 17 <211> 690 <212> DNA <213> Pseudomonas putida <400> 17 atgagttttt ctaattataa agtaatcgcg atgccggtgt tggttgctaa ttttgttttg 60 ggggcggcca ctgcatgggc gaatgaaaat tatccggcga aatctgctgg ctataatcag 120 ggtgactggg tcgctagctt caatttttct aaggtctatg tgggtgagga gcttggcgat 180 ctaaatgttg gagggggggc tttgccaaat gctgatgtaa gtattggtaa tgatacaaca 240 cttacgtttg atatcgccta ttttgttagc tcaaatatag cggtggattt ttttgttggg 300 gtgccagcta gggctaaatt tcaaggtgag aaatcaatct cctcgctggg aagagtcagt 360 gaagttgatt acggccctgc aattctttcg cttcaatatc attacgatag ctttgagcga 420 ctttatccat atgttggggt tggtgttggt cgggtgctat tttttgataa aaccgacggt 480 gctttgagtt cgtttgatat taaggataaa tgggcgcctg cttttcaggt tggccttaga 540 tatgaccttg gtaactcatg gatgctaaat tcagatgtgc gttatattcc tttcaaaacg 600 gacgtcacag gtactcttgg cccggttcct gtttctacta aaattgaggt tgatcctttc 660 attctcagtc ttggtgcgtc atatgttttc 690 <210> 18 <211> 1479 <212> DNA <213> Candida tropicalis <400> 18 atgtctttgc cagtcgtcac caaactcact actcctaagg gtctctccta caaccaacca 60 ttaggtttgt tcatcaacaa cgagttcgtt gttccaaaat ccaagcaaac cttcgaagtc 120 ttctcccctt ccaccgaaga gaagatcacc gatgtctacg aagctttagc cgaagatgtc 180 gacgttgctg ccgaagcagc ttacgccgcc taccacaacg actgggccct tggtgctcca 240 gaacaaagag ccaagatctt gctcaagttg gccgacttgg tcgaagaaca cgccgagacc 300 ttggcccaga tcgaaacctg ggacaacggt aagtccttgc agaacgccag aggcgatatc 360 ggattcactg ccgcttactt tagatcctgt ggtggatggg ccgacaagaa caccggtgac 420 aacatcaaca ccggtggcac ccaccttact tacacccaga gagtcccatt ggtgtgtggt 480 caaatcatcc cttggaacgc aagtaccttg atggccagtt ggaagcttgg tcccgttatc 540 gctaccggtg gtaccactgt gcttaaatca gctgaagcta ccccattagc tgtcttgtac 600 ctcgcccaat tgttagttga agccggtctt ccaaagggtg tcgttaacat tgtttccggt 660 ttcggtacca ctgccggttc cgctatcgct agccatccaa agatcgacaa ggtcgccttt 720 actggttcca ccaacaccgg taagatcatc atgaagttgg ctgcggagtc caacttgaag 780 aaggtcactt tggaattggg tggtaagtcc ccacacattg ttttcaacga cgctgacttg 840 gaccgcgccg tcagctactt ggttgctgcc attttcagta actccggcga gacctgtgct 900 gccggatccc gtgtcttggt gcaatccggt gtctacgacg aagttgttgc taagttcaag 960 aagggcgccg aggccgttaa agttggtgac ccattcgacg aagaaacctt catgggttcc 1020 caagtcaacg aagtccaatt gtctagaatc ttgcaataca tcgagctggg taaggaacaa 1080 ggtgccactg ttgtcaccgg tggtggtaga gccggggaca agggttactt cgtcaagcca 1140 actattttcg ccgacgttca caaggacatg actatcgtca aggaagaaat ctttggtcct 1200 gttgtctccg tcgtcaaatt cgataccatt gaagaagcta tcgctttggc taacgactcc 1260 gaatacggtt tggccgctgg tatccacacc actaacatca gcaccggtgt caccgtcgct 1320 aacagaatca agtccggtac tgtctgggtc aacacttaca atgacttgca ccccatggtt 1380 ccattcggtg gtttcggcgc ttctggtatc ggcagagaaa tgggtgcaga agtcatgaag 1440 gaatacaccg aagttaaggc tgttagaatt aagctcact 1479 <210> 19 <211> 1044 <212> DNA <213> Saccharomyces cerevisiae <400> 19 atgtctatcc cagaaactca aaaaggtgtt atcttctacg aatcccacgg taaattggaa 60 cacaaggata ttccagttcc aaagccaaag gccaacgaat tgttgatcaa cgttaagtac 120 tctggtgtct gtcacaccga cttgcacgct tggcacggtg actggccatt gccagttaag 180 ctaccattag tcggtggtca cgaaggtgcc ggtgtcgttg tcggcatggg tgaaaacgtt 240 aagggctgga agatcggtga ctacgccggt atcaaatggt tgaacggttc ttgtatggcc 300 tgtgaatact gtgaattggg taacgaatcc aactgtcctc acgctgactt gtctggttac 360 acccacgacg gttctttcca acaatacgct accgctgacg ctgttcaagc cgctcacatt 420 cctcaaggta ccgacttggc ccaagtcgcc cccatcttgt gtgctggtat caccgtctac 480 aaggctttga agtctgctaa cttgatggcc ggtcattggg ttgccatttc cggtgctgcc 540 ggtggtctag gttctttggc tgttcaatac gccaaggcta tgggttacag agtcttgggt 600 attgacggtg gtgaaggtaa ggaagaatta ttcagatcca tcggtggtga agtcttcatt 660 gacttcacta aggaaaagga cattgtcggt gctgttctaa aggccactga cggtggtgct 720 cacggtgtca tcaacgtttc cgtttccgaa gccgctattg aagcttctac cagatacgtt 780 agagctaacg gtaccaccgt tttggtcggt atgccagctg gtgccaagtg ttgttctgat 840 gtcttcaacc aagtcgtcaa gtccatctct attgttggtt cttacgtcgg taacagagcc 900 gacaccagag aagctttgga cttcttcgcc agaggtttgg tcaagtctcc aatcaaggtt 960 gtcggcttgt ctaccttgcc agaaatttac gaaaagatgg aaaagggtca aatcgttggt 1020 agatacgttg ttgacacttc taaa 1044

Claims (8)

(1) knock out fadE and fadR genes of wild-type Escherichia coli, and then express the expression of the cytochrome P450 monooxygenase and cytochrome 450 reductase ( CYP-NCP ), ADH1 , ALD5 and AlkL gene Transforming the Escherichia coli with a vector to produce a mutant Escherichia coli; And
Is selected from the group consisting of _1: 2, the mutant culture of E. coli, and the harvesting of the tissue phase cell _{and, C 10, C 12, C} 14, C 16, C 16 to the _{medium: 1,} C ₁₈ and C ₁₈ Adding a saturated fatty acid or an unsaturated fatty acid,
Wherein the CYP-NCP binding gene, ADH1 , ALD5 and AlkL genes are composed of the nucleotide sequences shown in SEQ ID NOs: 16, 19, 18 and 17, respectively.
delete The method according to claim 1,
Wherein the expression vector has a cleavage map as shown in Fig.
The method according to claim 1,
The step (2) further comprises the step of adding a substance selected from the group consisting of 5-aminolevulinic acid (ALA), thiourea, phosphate buffer, antibiotic, tween 80, trace element solution and combinations thereof . &Lt; / RTI &gt;
The method according to claim 1,
Wherein step (2) is carried out at a temperature of from 25 to &lt; RTI ID = 0.0 &gt; 37 C. &lt; / RTI &gt;
The method according to claim 1,
Wherein step (2) is performed in the absence of a co-substrate selected from the group consisting of glycerol, glucose, and combinations thereof.
A mutant E. coli producing knockout of fadE and fadR genes of wild-type E. coli and producing dicarboxylic acid through omega oxidation by expressing CYP-NCP binding gene, ADH1 , ALD5 and AlkL gene,
Wherein the CYP-NCP binding gene, ADH1 , ALD5, and AlkL genes are composed of the nucleotide sequences of SEQ ID NOS: 16, 19, 18 and 17, respectively. delete

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