KR101482680B1 - Method for Preparing Fatty acid using Transformed E.coli introducing Trans-2-enoyl-CoA reductase - Google Patents

Abstract

The present invention relates to a method for producing a fatty acid using a transformed Escherichia coli. The method comprises the steps of (a) preparing a nucleotide encoding a trans-2-enoyl-CoA reductase of Pseudomonas sp . Inserting the sequence into an expression vector to prepare a recombinant vector; (b) transforming the recombinant vector into E. coli to produce a transformed E. coli; (c) culturing the transformed E. coli to prepare a fatty acid; And (d) obtaining the fatty acid. The present invention also provides a method for producing a fatty acid using the transformed E. coli. According to the present invention, the transformed E. coli of the present invention exhibits a 2-4-fold increased fatty acid production ability as compared with wild-type E. coli. The fatty acid production method of the present invention can produce harmful waste and energy consumption by producing a fatty acid through an environmentally friendly biological process.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for preparing a fatty acid using trans-2-enoyl-CoA reductase,

The present invention relates to a method for producing fatty acids using transformed E. coli with trans-2-enoyl-CoA reductase.

There is a need to develop environmentally friendly new bioprocesses that can replace the chemical industry produced from fossil fuels, that is, use biomass as a raw material to produce waste that is harmful to humans and minimize energy consumption. Although interest in bioethanol, biodiesel, biogas and butanol represented by bioenergy is increasing, all of the types of bioenergy mentioned can be used as a fuel for power generation or transport, but some disadvantages of practical application and production methods , There is a growing interest in hydrocarbon-based compounds as a new renewable energy resource. Accordingly, there is a growing interest in recombinant strains capable of producing fatty acids as metabolites.

Attempts and efforts have been constantly being made for the purpose of overexpression of fatty acid production in the fatty acid pathway metabolism of microorganisms. In particular, Escherichia coli is known for its metabolic information in comparison with other organisms in the enzyme production system, and information about genes is widely known and widely used for the production of recombinant proteins. Escherichia coli is a manageable microorganism with good growth potential and easy path control.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made efforts to produce hydrocardon-type compounds as renewable energy resources that can replace the chemical industry. As a result, a nucleotide sequence encoding a trans-2-enoyl-CoA reductase of Pseudomonas species was inserted into an expression vector and introduced into Escherichia coli to produce a fatty acid The present invention has been completed.

Accordingly, an object of the present invention is to provide a process for producing fatty acids.

Another object of the present invention is to provide a fatty acid.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for producing a fatty acid using a transformed E. coli comprising the steps of:

(a) preparing a recombinant vector by inserting a nucleotide sequence encoding a trans-2-enoyl-CoA reductase of Pseudomonas species into an expression vector;

(b) transforming the recombinant vector into E. coli to produce a transformed E. coli;

(c) culturing the transformed E. coli to prepare a fatty acid; And

(d) obtaining the fatty acid.

The present inventors have made efforts to produce hydrocardon-type compounds as renewable energy resources that can replace the chemical industry. As a result, a nucleotide sequence encoding a trans-2-enoyl-CoA reductase of Pseudomonas species was inserted into an expression vector and introduced into Escherichia coli to produce a fatty acid Method.

The fatty acid production method of the present invention will be described in detail as follows:

Step (a): Preparation of recombinant vector

As a first step for preparing a fatty acid of the present invention, a nucleotide sequence encoding a trans-2-enoyl-CoA reductase of Pseudomonas sp . And inserted into an expression vector to prepare a recombinant vector.

In the present invention, the microorganism used for preparing the fatty acid is Pseudomonas sp ., Preferably Pseudomonas sp. putida ), Pseudomonas crmolorolorata cremoricolorata , Pseudomonas sp. fulva , Pseudomonas monteilii , Pseudomonas sp. oryzihabitans ), Pseudomonas parafulba ( Pseudomonas a parafulva) or Pseudomonas replicon Cedar City kogeulro (Pseudomonas plecoglossicida).

According to a preferred embodiment of the present invention, a nucleotide encoding trans-2-enoyl-CoA reductase is amplified using the chromosome of Pseudomonas putida F1 as a template for producing fatty acids in the present invention.

Preferably, the nucleotide encoding the trans-2-enoyl-CoA reductase is SEQ ID NO: 3.

In the present invention, the enzyme to be introduced into Escherichia coli is trans-2-enoyl-CoA reductase. The trans-2-enoyl-CoA reductase has the activity of catalyzing the following reaction:

[Reaction Scheme 1]

[Reaction Scheme 2]

The nucleotide sequence of trans-2-enoyl-CoA reductase used in the present invention is interpreted to include a nucleotide sequence showing substantial identity to the nucleotide sequence in addition to the above-mentioned sequence. The above substantial identity is determined by aligning the nucleotide sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using algorithms commonly used in the art to obtain a minimum of 80% Homology, more preferably at least 90% homology, and most preferably at least 95% homology. Alignment methods for sequence comparison are well known in the art. Various methods and algorithms for alignment are described by Smith and Waterman, Adv . Appl . Math . 2: 482 (1981) ; Needleman and Wunsch, J. Mol . Bio . 48: 443 (1970); Pearson and Lipman, Methods in Mol . Biol . 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988); Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc . Acids Res. 16: 10881-90 (1988); Huang et al., Comp . Appl . BioSci . 8: 155-65 (1992) and Pearson et al., Meth. Mol . Biol . 24: 307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol . Biol . 215: 403-10 (1990)) is accessible from National Center for Biological Information (NBCI) It can be used in conjunction with sequence analysis programs such as blastx, tblastn and tblastx. BLSAT is available at http://www.ncbi.nlm.nih.gov/BLAST/. A method for comparing sequence homology using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

The genes are introduced into the expression vector and expressed in E. coli. As used herein, the term "expression vector" is a linear or circular DNA molecule consisting of a fragment encoding a polypeptide of interest operably linked to an additional fragment provided in the transcription of the expression vector. Such additional fragments include promoter and termination coding sequences. The expression vector also includes at least one replication origin, at least one selection marker, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or contain both elements.

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) This document is incorporated herein by reference.

Each nucleotide sequence encoding enzymes involved in the fatty acid production of the present invention can be inserted into an expression vector operably linked to an expression control sequence. The term 'operably linked to' as used herein means that one nucleic acid fragment is combined with another nucleic acid fragment so that its function or expression is affected by other nucleic acid fragments. Also, an " expression control sequence " means a DNA sequence that regulates the expression of a nucleic acid sequence operably linked to a particular host cell. Such regulatory sequences include promoters for initiating transcription, any operator sequences for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the termination of transcription and translation.

The vector of the present invention can typically be constructed as a vector for expression. When the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (e.g., lac promoter, tac promoter, lacUV5 promoter, lpp promoter, pL ^貫 promoter, pR ^貫 promoter, rac5 Promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter), a ribosome binding site for initiation of detoxification, and a transcription / translation termination sequence. Promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol., 158: 1018-1024 (1984)) when E. coli (e.g. HB101, BL21, ) And the left promoter of phage lambda (pL ^lambda promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14: 399-445 (1980)). Preferably, the promoter used in the vector of the present invention is the lac promoter.

The vector injected into the host cell may be expressed in host cells, in which case a large amount of acetylenic reductase is obtained. For example, when the expression vector comprises a lac promoter, the host cell may be treated with IPTG (Isopropylthio-β-D-Galactoside) to induce gene expression.

Meanwhile, vectors that can be used in the present invention include plasmids such as pUCP19, pUC18, pTrc99A, pSTV28, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9 , such as pHC79, pIJ61, pLAFR1, pHV14, pET22b, pGEX series and pET series, etc.), phage (such as λgt4λB, λ-Charon, λΔz1 and M13) However, pUCP19 is preferably used.

The expression vector of the present invention includes the promoter sequence and the nucleotide sequence of the gene to be expressed (structural gene), and it is preferable that the sequences are linked in the order of 5'-3 '.

Preferably, the expression vector comprises a lac promoter sequence and the lac promoter sequence is operatively linked to a nucleotide sequence encoding the trans-2-enoyl-CoA reductase.

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

According to a preferred embodiment of the present invention, the selectable marker used in the present invention is ampicillin.

The vector of the present invention is characterized in that the trans-2-enoyl-CoA reductase gene contains only a nucleotide sequence including a RBS (ribosomal binding site) and an essential part for enzyme expression so as to have an enzyme over- Is preferable in terms of reducing the metabolic burden of host cells.

Step (b): Preparation of transformed E. coli

Next, the recombinant vector of step (a) is transformed into E. coli to prepare a transformed E. coli.

Host cells capable of continuously cloning and expressing the vector of the present invention in a stable manner can be any host cell known in the art, such as E. coli MG1655, E. coli JM109, E. coli Such as Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, and Salmonella typhimurium, such as BL21 (DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, And Staphylococcus aureus and various Pseudomonas species.

According to a preferred embodiment, the host cell used for the production of fatty acids in the present invention is a Shogun (E. coli), and more preferably E. coli MG1655.

Methods for delivering the vector of the present invention into a host cell can be carried out using a CaCl ₂ method (Cohen, SN et al., Proc . Natl . Acac. Sci . USA , 9: 2110-2114 SN et al, Proc Natl Acac Sci USA, 9: 2110-2114 (1973); and Hanahan, D., J. Mol Biol, 166:....... 557-580 (1983)) and electroporation method (Dower, WJ et al., Nucleic. Acids Res . , 16: 6127-6145 (1988)).

Preferably, electroporation is used to introduce the recombinant vector of the invention into the host cell.

Step (c): Preparation of fatty acid

Then, the transformed E. coli of step (b) is cultured to prepare a fatty acid.

According to a preferred embodiment of the present invention, a growth similar to that of wild-type Escherichia coli is shown in a culture medium (for example, LB medium or minimal medium) containing a carbon source (for example, glucose) (FIG.

Step (d): Acquisition of fatty acid

Finally, the fatty acid of step (c) is obtained.

Preferably, the transformed E. coli of the present invention exhibits a 2-4-fold increased fatty acid production ability as compared to wild-type E. coli (FIG. 6).

The transformed Escherichia coli of the present invention has growth similar to that of wild-type Escherichia coli and exhibits excellent fatty acid production ability.

According to another aspect of the present invention, there is provided a fatty acid prepared according to the fatty acid producing method of the present invention.

The fatty acid of the present invention is a hydrocarbon-type compound produced by a biological process and can be used as energy such as petroleum, natural gas and coal.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a method for producing fatty acids using the transformed E. coli.

(b) The transformed E. coli of the present invention exhibits a 2-4-fold increased fatty acid production ability as compared with wild-type E. coli.

(c) The fatty acid production method of the present invention can produce harmful wastes and energy consumption by producing fatty acids through environmentally friendly biological processes.

1, It is a scheme showing the metabolism of butanoate from glucose in E. coli in vivo.
Fig. 2 is a graph coli) - Pseudomonas (Pseudomonas) encoding the shuttle vector in trans-2-2- enoic room -CoA reductase kinase (trans-2-enoyl-CoA reductase, EC 1.3.1.44 in pUCP9) is a schematic view showing the plasmid is the gene has been inserted to be.
FIG. 3 is a schematic representation of a plasmid in which atoDA (acetyl-CoA: acetoacetyl-CoA transferase, alpha subunit and beta subunit encoding gene) is inserted into pUCP9 .
FIG. 4 shows a gene coding for trans 2-enoyl-CoA reductase (EC 1.3.1.44) in pUCP9 and a gene encoding atoDA (acetyl-CoA: acetoacetyl-CoA transferase, alpha subunit And a beta subunit) is inserted into the plasmid.
5 shows the growth curves of the transformed E. coli and wild-type E. coli according to the present invention by media. (A) is the minimal medium, (B) is the minimal medium containing 10 g / L of glucose, (C) is the LB medium, and (D) is the LB medium containing 10 g / L of glucose.
Fig. 6 shows the results obtained by adding the transformed E. coli and wild-type E. coli of the present invention to the LB medium containing 10 g / L of glucose (B) and 10 g / L of glucose (D) It was extracted from 24 o'clock and quantified.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Preparation of recombinant plasmid

Coli chromosome in the mold (E. coli MG1655) and acetyl -CoA: acetoacetyl -CoA transferase kinase alpha subunit (acetyl-CoA: acetoacetyl-CoA transferase, alpha subunit) and acetyl -CoA: acetoacetyl -CoA Using primers for the gene sequences of atoD and atoA (Genbank, NCBI) coding for the enzyme acetyl-CoA (acetoacetyl-CoA transferase, beta subunit) And cloned by a chain reaction (PCR, DaKaRa Korea) method.

2-enoyl-CoA reductase, which is not involved in the fatty acid synthesis of Pseudomonas aeruginosa by using the chromosome of Pseudomonas putida F1 as a template, CoA reductase, EC 1.3.1.44) was prepared and cloned (Table 1 and Table 2).

gene Production enzyme atoD Acetyl-CoA: acetoacetyl-CoA transferase (alpha subunit) atoA Acetyl-CoA: acetoacetyl-CoA transferase (beta subunit) 1.3.1.44 Trans-2-enoyl-CoA reductase (trans-2-enoyl-CoA reductase)

gene Primer sequence Restriction enzyme ATODA Forward 5'-CTGCAGTAAAAAATGAAAACAAAATTGATGAC-3 ' PstI Reverse 5'-AAGCTTTCATAAATCACCCCGTTGCGTAT-3 ' HindIII 1.3.1.44 Forward 5'-CCCGGGACGAGGTACAGACATTGGCCAT-3 ' Sma I Reverse 5'-GGATCCTTACAGCTCGACGCAGTCGAAC-3 ' BamHI

The primers of Table 2, which were designed to specifically amplify the genes of Table 1, were amplified. ATODA And And amplified with each RBS (ribosome binding site) of the 1.3.1.44 gene.

Each of the polymerase chain reactions was performed under the usual reaction conditions (10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 2 mM MgSO ₄ , Taq DNA polymerase (DaKaRa) 1 minute (denaturation), 62 [deg.] C / 30 sec (annealing) and 72 [deg.] C / 1 minute (elongation). As a final step, the cells were reacted at 95 ° C / 1 min (denaturation), 60 ° C / 1 min (annealing) and 72 ° C / 5 min (elongation) for stable elongation. After PCR, the amplified DNA was purified on 0.8% agarose gel, purified and used for T-vector cloning. ligation was performed on the pGEM-T easy vector (DaKaRa) to obtain a recombinant plasmid pGEM :: atoDA And pGEM :: 1.3.1.44. The recombinant plasmids were transformed in E. coli to produce transformed E. coli.

Example  2: Identification of recombinant plasmids

The recombinant plasmids pGEM-T :: 1.3.1.44 of Example 1 and pGEM-T: ato AD and PUCP19 (Schweizer HP, Escherichia - Pseudomonas shuttle vectors derived from pUC18 / 19, Gene , (1991) 97: 109-121) which is a shuttle vector of Escherichia coli and Pseudomonas was transferred to a 37 ° C water bath ) For about 2 hours. Each of the DNA fragments was cut and ligated to a multicloning site of the pUCP19 vector using a T4 ligase (Dakara, Japan) as described in Figures 2 and 3 at 16 ° C to obtain a recombinant plasmid . In order to confirm the insertion of the inserted gene in each step, E. coli was transformed, and the recombinant plasmid was extracted and treated with the restriction enzyme at the original insertion site. The size of the cloned DNA was electrophoresed on 0.8% agarose gel A comparison method was used.

As a result, PCR products were introduced into pUCP19 vector to prepare pJS31, pJS32 and pJS33. Figures 2, 3 and 4 show a map of the vector named pJS31 (including genes encoding 1.3.1.44), pJS32 (including atoDA genes) and pJS33 (including genes encoding atoDA and 1.3.1.44) Fig.

Example  3: Preparation of transformed E. coli

In the present invention, a transformation method by electroporation was used in order to stably produce the transformed E. coli and increase the efficiency thereof.

30 μl (0.1%) of wild-type E. coli MG1655 culture medium cultured for 16 hrs was inoculated into 3 ml of LB (10 g / L tryptone, 10 g / L NaCl and 5 g / L yeast extract), and when the absorbance of the culture reached 0.6 at 600 nm wavelength, 3 ml of the culture solution corresponding to the whole culture was centrifuged (12000 rpm, 1 minute ) To separate the supernatant and the cells. The harvested cells were washed once with 1 ml of 10% glycerol, and then centrifuged again (12,000 rpm, 1 minute) to separate the supernatant and cells. The supernatant was removed and the cells were suspended in 80 [mu] l of 10% glycerol. 1 [mu] l of recombinant plasmid (pJS31, pJS32 or pJS33) was added to the suspended cells.

80 [mu] l of the liquid containing the above plasmid was transferred to a Gene pulser Xcell (BIO-RAD, USA) packed in an electroporation cuvette (BIO-RAD, USA) Electric shock (2500 v, 25 μF, 200 Ω) was applied. 1 ml of LB prepared beforehand (10 g / L of tryptone, 10 g / L of NaCl and 5 g / L of yeast extract) was added, followed by shaking culture at 200 rpm and 37 ° C for 1 hour. The cultured E. coli was cultured in medium supplemented with ampicillin (50 占 퐂 / ml) until single cells were produced. The recombinant plasmids pJS31, pJS32 or pJS33 were transformed into E. coli SGJS31, E. coli SGJS32 and E. coli transformed into E. coli MG1655 SGJS33. In addition, when the same vector was used to transform the empty vector into wild-type Escherichia coli, the fatty acid production was different (Table 3).

E. coli transformed Characteristic E. coli SGJS30 Escherichia coli :: pUCP19 E. coli SGJS31 Escherichia coli :: pUCP19 :: 1.3.1.44 E. coli SGJS32 Escherichia coli :: pUCP19 :: ATOD E. coli SGJS33 Escherichia coli :: pUCP19 :: 1.3.1.44 :: atoDA

Example  4: Characterization of transformed E. coli

The transformed Escherichia coli transformed into Escherichia coli was pre-cultured in LB (containing ampicillin 50 μg / ml) for about 16 hours and the culture was inoculated again into LB (containing ampicillin 50 μg / ml) ㎚, and stored at -80 ℃ for the culture experiment.

The transformant Escherichia coli developed above was pre-cultured in 3 ml of LB supplemented with ampicillin (50 μg / ml) for 16 hours, followed by addition of ampicillin 50 (10 g / L of tryptone, 10 g / L of NaCl and 5 g / L of yeast extract) supplemented with 200 mL of the culture medium containing 0.5% Lt; / RTI > The wild type Escherichia coli to be compared with the transformed Escherichia coli was tested under the same conditions as the Escherichia coli transformed in LB containing no ampicillin. For protein expression, when the absorbance of the culture reached 0.6 at 600 nm wavelength, IPTG (isopropylthio-β-D-galactoside, Sigma, USA) was added (0.5 mM / Lt; / RTI > The growth curves of the transformed E. coli and the wild type Escherichia coli developed for the purpose of the present invention and the species for comparing the production increase of the fatty acid content by the expression of the initial derivative according to the above example are shown in FIG.

In the present invention, the ability of transforming E. coli to grow in minimal medium (MM) and LB medium was confirmed. Medium composition components of the minimal medium are shown in Table 4.

Minimal medium (MM) ingredient g / L Na ₂ SO ₄ 4 (NH _{4) 2} SO ₄ 5.4 NH ₄ Cl 1.0 K ₂ HPO ₄ 7.3 NaH ₂ PO ₄ H ₂ O 1.8 (NH _{4) 2} -H- citrate 12 MgSO ₄ 0.25 Thiamine 0.02

In the minimal medium containing no sugar, all of the transformed E. coli did not grow well. In the medium containing glucose (glucose 10 g / L) In the LB medium, the initial culture was very fast in the medium containing the saccharide, but it did not grow much more than when it was not contained after consumption.

The reason that the transfected E. coli was not much different from the control (the transformed E. coli transformed with the public vector) is considered to be not so large as toxicity or inhibition by gene insertion or overexpression. This is because the coding part of the gene includes the overexpression function of the enzyme so that the ribosomal binding site (RBS) and the nucleotide sequence including the essential part for expression of the enzyme are set to have the minimum length and the metabolism of the host cell It is expected to reduce the burden (metabolic burden) and to develop the sequence of the two genes to be connected in order according to the production route.

Example  5: Fatty acids ( fatty acid ) Measure

When the amount of the cells was sufficient for 24 hours after addition of IPTG in the culture medium cultured under the condition of Example 4, fatty acid extraction was carried out in the following manner in order to measure the fatty acid in the transformed E. coli. The extraction process was divided into 5 stages. In the first step culturing, 5 ml of the culture was centrifuged (4500 rpm, 10 minutes), and the transformed E. coli was stored at -80 ° C. In the second step, 1 ml of the first solution (45 g of NaOH, 150 ml of MeOH, 150 ml of desalted distilled water) was added to the transformed Escherichia coli stored in the saponification step and vortexed for 5-10 seconds. After reacting at 100 ° C for 5 minutes, vortex for 5 to 10 seconds and then reacted again at 100 ° C for 25 minutes. The third step is a methylation step. 2 ml of the second solution (325 ml of 6N HCl, 275 ml of MeOH) is added, vortexed for 5-10 seconds, and reacted at 80 ° C for 10 minutes. After the reaction was completed, the heat was turned off quickly. The fourth step is an extration step in which 1.25 ml of a third solution (Haxane / Methyl tert-Butyl Ester = 1/1) is added and shaken up and down for 10 minutes After the layers were separated, the lower layer was extracted and removed. In the fifth step, 3 ml of the fourth solution (NaOH 10.8 g, desalted distilled water 900 ml) was added to the remaining supernatant to facilitate the analysis by GC, and the supernatant was shaken up and down for 5 minutes, Were extracted and analyzed by GC / MS.

The CG / MS used in the present invention was a 5975 series MSD and Agilent 7890A and HP-5 column (30 m × 0.32 mm, film thickness 0.25 μ). GC analysis was performed at 100 DEG C for 5 minutes, 220 DEG C at 5 DEG C / min, and 250 DEG C for 5 minutes.

FIG. 6 shows results of fatty acid extraction analysis of the transformed E. coli and wild-type E. coli for enhancing the fatty acid content according to the above example. It was confirmed that the transformed Escherichia coli SGJS31 transfected with the trans-2-enoyl-CoA reductase gene, which is a foreign gene of Pseudomonas putida, increased up to 2.6 times more than wild-type Escherichia coli in the LB medium Could. As shown in FIG. 1, it was confirmed that the introduction of the gene also affected the fatty acid biosynthetic pathway, thereby enhancing the fatty acid. On the other hand, E. coli transformed with the atoDA gene and the trans-2-enoyl-CoA reductase gene had a decreased ability to produce fatty acid than Escherichia coli transformed with trans-2-enoyl-CoA reductase alone. These results indicate that trans-2-enoyl-CoA reductase gene overexpression of the endogenous gene, atoDA gene, in trans-2-enoyl-CoA reductase gene Fatty acid production capacity, and these results provide important clues to the control step in fatty acid mass production using the transformed E. coli.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Industry-University Cooperation Foundation Sogang University <120> Method for Preparing Fatty acid using Transformed E. coli          introducing Trans-2-enoyl-CoA reductase <130> PN120326 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 663 <212> DNA <213> AToD <400> 1 atgaaaacaa aattgatgac attacaagac gccaccggct tctttcgtga cggcatgacc 60 atcatggtgg gcggatttat ggggattggc actccatccc gcctggttga agcattactg 120 gaatctggtg ttcgcgacct gacattgata gccaatgata ccgcgtttgt tgataccggc 180 atcggtccgc tcatcgtcaa tggtcgagtc cgcaaagtga ttgcttcaca tatcggcacc 240 aacccggaaa caggtcggcg catgatatct ggtgagatgg acgtcgttct ggtgccgcaa 300 ggtacgctaa tcgagcaaat tcgctgtggt ggagctggac ttggtggttt tctcacccca 360 acgggtgtcg gcaccgtcgt agaggaaggc aaacagacac tgacactcga cggtaaaacc 420 tggctgctcg aacgcccact gcgcgccgac ctggcgctaa ttcgcgctca tcgttgcgac 480 accttggca acctgaccta tcaacttagc gcccgcaact ttaaccccct gatagccctt 540 gcggctgata tcacgctggt agagccagat gaactggtcg aaaccggcga gctgcaacct 600 gaccatattg tcacccctgg tgccgttatc gaccacatca tcgtttcaca ggagagcaaa 660 taa 663 <210> 2 <211> 650 <212> DNA <213> atoA <400> 2 tggatgcgaa acaacgtatt gcgcgccgtg tggcgcaaga gcttcgtgat ggtgacatcg 60 ttaacttagg gatcggttta cccacaatgg tcgccaatta tttaccggag ggtattcata 120 tcactctgca atcggaaaac ggcttcctcg gtttaggccc ggtcacgaca gcgcatccag 180 atctggtgaa cgctggcggg caaccgtgcg gtgttttacc cggtgcagcc atgtttgata 240 gcgccatgtc atttgcgcta atccgtggcg gtcatattga tgcctgcgtg ctcggcggtt 300 tgcaagtaga cgaagaagca aacctcgcga actgggtagt gcctgggaaa atggtgcccg 360 gtatgggtgg cgcgatggat ctggtgaccg ggtcgcgcaa agtgatcatc gccatggaac 420 attgcgccaa agatggttca gcaaaaattt tgcgccgctg caccatgcca ctcactgcgc 480 aacatgcggt gcatatgctg gttactgaac tggctgtctt tcgttttatt gacggcaaaa 540 tgtggctcac cgaaattgcc gacgggtgtg atttagccac cgtgcgtgcc aaaacagaag 600 ctcggtttga agtcgccgcc gatctgaata cgcaacgggg tgatttatga 650 <210> 3 <211> 1212 <212> DNA <213> 1.3.1.44 <400> 3 ttggccatca ttcatcctaa ggttcgcggt ttcatctgca ccacgactca cccgaagggc 60 tgcgagctca acgtccgtga ccagatcgaa gccacccgca agctgggcgt acgcgaggat 120 ggcccgaaga aggtcctggt catcggtgct tccagcggct acggcctggc tgcccgcatc 180 acggcggcct tcggcttcaa ggctgacacc ctgggcgtgt tcttcgaaaa accaggcacc 240 gagaccaagg ccggcactgc tggctggtac aacgctgccg ccttcgacaa gttcgccaag 300 gccgagggcc tgtacagcaa gtcgatcaac ggtgacgcct tctctgacga agcccgcgcc 360 aaggtcatcg agctgatcaa gaacgaaatg ggtggcaagg tcgacctggt catctactcg 420 ctggcttcgc ccgtgcgcaa gctgccgcag accggtgaag tcattcgctc ggcactcaag 480 ccgatcggcc agccatacaa gtcgaccgcc atcgacacca acaaggacac catcatcgag 540 gccagcatcg agccggccac cgagcaggaa attgccgata ccgtcaccgt catgggtggc 600 caggactggc agctgtggat cgatgcgctg gctggcgcca atgtactggc cgaaggcgct 660 cgcaccgtgg ccttcagcta tatcggcagc gatatcacct ggccgatcta ctggcacggt 720 gcactgggcc aggccaagca ggacctggac gaaactgccc tgcgtctgaa ccagaagctg 780 gccggtgaag tgaaaggcgg cgccaatgtg gcggtgctca agtcggtggt tacccaggcc 840 agctcggcca tcccggtgat gccactgtac ctgtcgatgg tcttcaagat catgcaggag 900 aaaggtgttc acgaaggcac ccaggaccag ctcgaccgca tgtaccgcga ccgtatgtac 960 cgcaccgacg gtgcgccggc agaggtcgac gagaaggggc gcctgcgcct ggacgactgg 1020 gaactgcgtg acgacgtgca aaacgcctgc aaggccctgt ggccacaggt gaccaccgag 1080 aacctgttcg agctgaccga ctacgccggt tacaagaagc agttcctcaa cctgttcggc 1140 ttcgagcgcg ctgacgtcaa ctacgacgaa gacgtggcca ccgatgtgaa gttcgactgc 1200 gtcgagctgt aa 1212

Claims (7)

A method for producing a fatty acid using transformed E. coli comprising the steps of:
(a) preparing a recombinant vector by inserting a nucleotide sequence encoding trans-2-enoyl-CoA reductase of Pseudomonas putida into an expression vector;
(b) transforming the recombinant vector into E. coli to produce a transformed E. coli;
(c) culturing the transformed E. coli to prepare a fatty acid; And
(d) obtaining the fatty acid.
delete 2. The method of claim 1, wherein the nucleotide sequence encoding the trans-2-enoyl-CoA reductase is SEQ ID NO: 3.
The recombinant vector of claim 1, wherein the recombinant vector comprises a lac promoter sequence and the lac promoter sequence is operatively linked to a nucleotide sequence encoding the trans-2-enyl-CoA reductase. How to.
2. The method of claim 1, wherein the transformation employs electroporation.
The method according to claim 1, wherein the transformed E. coli exhibits a 2-4-fold increase in fatty acid production ability as compared to that of wild-type E. coli.
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