KR20120122995A - Method for Preparing Transformed E coli for Over-expression of Fatty Acid Biosynthesis Pathway and Transformed E coli Prepared by the Same - Google Patents

Abstract

PURPOSE: A method for preparing transformed E.coli for overexpression of fatty acid biosynthesis pathway is provided to enhance genetic stability of the transformed E.coli. CONSTITUTION: A method for preparing a co-transformed E.coli comprises: a step of inserting an aceE(pyruvate dehydrogenase, decarboxylase component E1) gene and accA(acetyl-CoA carboxylase, carboxytransferase) gene into an expression vector; and a step of electroporation of E.coli with the expression vector and transforming. A fatty acid biosynthesis comprises a step of culturing co-transformed E.coli and biosynthesizing fatty acid; and a step of collecting the synthesized fatty acid.

Description

Method for preparing transformed E. coli for overexpression of fatty acid biosynthetic pathway and transformed E coli prepared by the above method {Method for Preparing Transformed E coli for Over-expression of Fatty Acid Biosynthesis Pathway and Transformed E coli Prepared by the Same}

The present invention relates to a method for producing a transgenic E. coli for overexpression of a fatty acid biosynthetic pathway and to a transgenic E. coli prepared by the method.

The interest in bioenergies such as ethanol, butanol and biodiesel is increasing due to the limited amount of fossil fuel resources that show increasing energy use and sustained price increases in line with global economic growth. All of the aforementioned types of bioenergy can be used as fuel for power generation or transportation, but interest in compounds in the form of hydrocarbons, which is a new renewable energy resource, is particularly increasing due to some shortcomings of performance and production methods. Accordingly, interest in recombinant strains capable of producing long-chain fatty acids as metabolites is also increasing.

The synthesis of fatty acids in a microorganism in vivo begins with acetyl-CoA, and then carbon atoms are attached to the hydrocarbon chain two atoms at a time, and the chain becomes longer. Catabolic reaction proceeds in the opposite direction. That is, starting from the carboxyl group and ending with acetyl-CoA, fragments of the hydrocarbon chain and acetyl-CoA are E. coli ( Escherichia coli ) provides energy that is oxidized in the citric acid cycle (TCA cycle) and stored in the form of ATP. The first step in fatty acid biosynthesis is the transport of acetyl-CoA (an essential precursor for fatty acid synthesis) into the cytoplasm. Acetyl-CoA can be produced by β-oxidation of fatty acids or decarboxylation of pyruvic acid (decomposition of certain amino acids may produce acetyl-CoA). In the cytoplasm, acetyl-CoA is carboxylated to produce malonyl-CoA, an important intermediate in fatty acid biosynthesis. As shown in the equation below, this reaction is catalyzed by an acetyl-CoA carboxylase complex, which is composed of three enzymes and requires ^{Mn 2 + and biotin as well as ATP for enzymatic activity.} Acetyl-CoA carboxylase is composed of three proteins: biotin carboxylase, biotin transport protein, and carboxyl group transferase.

Fatty acid biosynthesis is achieved by successively adding two carbon units to the growth chain. Two of the three carbon atoms in the malonyl group of malonyl-CoA are added to the fatty acid chain at each stage of the biosynthetic reaction. This reaction, like malonyl-CoA formation, requires a multienzyme complex that is in the cytoplasm and is not bound to the membrane. This complex, made up of individual enzymes, refers to fatty acid synthase. The acyl carrier protein (ACP), which is a part of the fatty acid synthase complex, is involved in the binding of the acetyl group of acetyl-CoA to increase the carbon number of fatty acids.

Attempts and efforts corresponding to the purpose of overexpression of acetyl-CoA and malonyl-CoA, which aimed at the active metabolic flow of the fatty acid synthesis pathway using microorganisms, have been constantly made. Escherichia coli is an enzyme production system that has more metabolic information than other organisms, and has been widely used in the production of recombinant proteins because almost all information on genes is revealed.

Ethan S. James and John E. Cronan. Expression of Two Escherichia on the role of acetyl-CoA and malonyl Co-A in the metabolism of the introduction stage for fatty acid biosynthesis in Escherichia coli (Ethan S. James and John E. Cronan. coli Acetyl-CoA Carboxylase Subunits Is Autoregulated, THE JOURNAL OF BIOLOGICAL CHEMISTRY , 279(4):25202527(2004)). In addition, it is shown in references that malonyl-Co is converted to malonyl-ACP by ACP transacylase, and the metabolic network for fatty acid synthesis proceeds (Satyanrayana subrahmanyam and John E. Cronan, JR., Overproduction of a Functional Fatty Acid Biosynthetic Enzyme Blocks Fatty Acid Synthesis in Escherichia coli , JOURNAL OF BACTERIOLOGY , 180(17):4596-4602(1998)).

However, most studies have centered on the internal cycle of the fatty acid biosynthesis pathway, that is, the metabolic pathway after malonyl-ACP, and discuss the production and inhibition of fatty acids by the mechanism of the fab family of genes. Therefore, research from the carbon source for fatty acid biosynthesis to the introduction of the fatty acid synthesis pathway is insufficient.

Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of cited papers and patent documents are incorporated by reference in this specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

The present inventors have made intensive research efforts to develop a method for producing transformed E. coli that can stably and effectively overexpress the fatty acid biosynthetic pathway. As a result, the aceE gene and accA By inserting a gene into one expression vector and developing a method of transforming E. coli with the expression vector, the present invention was completed.

Accordingly, an object of the present invention is to provide a method for preparing a transgenic E. coli for overexpression of a fatty acid biosynthetic pathway.

Another object of the present invention is to provide a transgenic E. coli for overexpression of the fatty acid biosynthetic pathway prepared by the above method.

Another object of the present invention is to provide a method for biosynthesizing fatty acids using the transformed E. coli prepared above.

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

According to one aspect of the present invention, the present invention provides a method for preparing a transgenic E. coli for overexpression of a fatty acid biosynthetic pathway comprising the following steps:

(a) aceE (pyruvate dehydrogenase, decarboxylase component E1) Inserting the gene and accA (acetyl-CoA carboxylase, Carboxytransferase) gene into one expression vector; And

(b) the aceE Transforming E. coli with the gene and the expression vector into which the accA gene is inserted.

According to another aspect of the present invention, the present invention is aceE prepared by the method of the present invention described above. E. coli cotransformed with gene and accA gene is provided.

The present inventors have made intensive research efforts to develop a method for producing transformed E. coli that can stably and effectively overexpress the fatty acid biosynthetic pathway. As a result, the aceE gene and accA By inserting a gene into one expression vector and developing a method of transforming E. coli with the expression vector, the present invention was completed.

Recently, it has been reported that fatty acids can be used as bioenergy for transportation fuels. Accordingly, the present invention proposes a method for developing mutant strains for an improved fatty acid biosynthesis pathway as a general concern for renewable energy. Fatty acid refers to a chain-shaped saturated or unsaturated monocarboxylic acid chemically or biochemically, and this name is given because glycerol and fatty acids are separated when fat is hydrolyzed. The simplest fatty acid is butyric acid (4 carbons), but fatty acids that exist in nature have caprylic acid or more, all of which have an even number of carbons. Fatty acid is Escherichia coli It can be biosynthesized from a carbon source in vivo through a specific pathway, and the present invention provides a method of improving the metabolic flow of the fatty acid biosynthesis (FAS) pathway. Escherichia coli Fatty acid biosynthesis (FAS) pathway from glucose to fatty acid in vivo is schematically illustrated in FIG. 1.

In the present invention, the method of improving the metabolic flow of the fatty acid biosynthesis (FAS) pathway is to help the flow to FAS (fatty acid biosynthesis) by overexpressing acetyl-CoA and malonyl-CoA. To achieve this enzyme to convert pyruvate into acetyl -CoA (pyruvate dehydrogenase) expressing accA encoding the enzyme (acetyl-CoA carboxylase) to switch to the carbonyl -CoA the aceE gene and acetyl -CoA words, responsible for the production of Included in the vector and using the expression vector Escherichia ( Escherichia coli ) is transformed.

The aceE And the coding portion of the accA gene is preferable to sequence only the nucleotide sequence including the ribosomal binding site (RBS) and the part necessary for enzyme expression so as to have a minimum length while attempting the overexpression function of the enzyme. This is to reduce the metabolic burden of host cells.

In one embodiment, the aceE The gene is characterized by consisting of the nucleotide sequence of the first sequence in the sequence listing. More preferably, the aceE The gene has a ribosomal binding site (RBS) bound upstream of the nucleotide sequence of the first sequence in the sequence listing.

In another embodiment, the accA The gene is characterized by consisting of the nucleotide sequence of the second sequence in the sequence listing. More preferably, the aceA The gene is one in which the ribosomal binding site (RBS) of SEQ ID NO: 5 is bound upstream of the nucleotide sequence of SEQ ID NO: 2.

The aceE And the nucleic acid encoding the accA gene can be operably linked to an expression control sequence and inserted into an expression vector. In the above, "operably linked to" means that one nucleic acid fragment is associated with another nucleic acid fragment, and its function or expression is affected by the other nucleic acid fragment. In addition, the term'expression control sequence' refers to a DNA sequence that controls the expression of a nucleic acid sequence operably linked in a specific host cell. Such regulatory sequences include promoters to initiate transcription, any operator sequences to regulate transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences that regulate termination of transcription and translation.

* In the present invention, aceE And the promoter bound to the nucleic acid encoding the accA gene is used that has a strong expression ability, preferably a trc promoter can be used.

In one embodiment, the expression vector of the present invention comprises a promoter (trc promoter) sequence, aceE and accA gene sequences, and a bacterial-derived terminator sequence, wherein the sequences are linked in 5'-3' order It is desirable to be. This is aceE and accA The gene corresponds to the expression sequence according to the position in the chromosome of E. coli, and the biosynthetic pathway in which acetyl-CoA is first produced and consumed in the cell growth curve is applied to the production of malonyl-CoA.

In another embodiment, the expression vector further comprises a lac I gene. The lac I gene allows the host cell to regulate the trc promoter, whether or not it contains a gene encoding a Lac repressor. In this case above a ceE And accA In order to induce gene expression, Isopropyl-β-D-thiogalactopyranoside (IPTG) may be used.

In addition, the term'expression vector' in the present invention refers to a plasmid, viral vector, or other mediator known in the art capable of inserting a nucleic acid encoding a structural gene and expressing the nucleic acid in a host cell. It may be a plasmid vector.

As the plasmid, any carrier known in the art may be used as long as it is a carrier capable of replication and expression in E. coli. For example, although not limited thereto, it may be a pJE101, pJE102, and pJE103 expression vector (plasmid), and the expression vector may preferably be introduced into a backbone vector containing as the minimum sequence of the third sequence of the sequence listing. .

AceE of the present invention And the expression vector containing the accA gene is a method known in the art, for example, but is not limited thereto, _{after making a competent cell using a CaCl 2} buffer, and then introducing the expression vector into a host cell by a heat shock (42° C.) method. It can be introduced into the host cell by an injection method, a transformation method by electric shock, a transient transfection, microinjection, cell fusion, a calcium phosphate precipitation method, an electroporation method, or the like. In order to increase the stable production and efficiency of the transformant, it is preferable to use a transformation method by electric shock.

As the host cell to be transformed to overexpress the fatty acid biosynthetic pathway, a wild species that can be applied as an industrial microorganism and has excellent function is selected as E. Gram-negative bacteria ( Escherichia coli ) genus characteristics can be identified and compared to select the wild species with the most excellent function. The microorganism of the genus Escherichia is Escherichia , which is a gram-negative E. coli coli , and include, for example, MG1655, W3110, DH5α, XL1-Blue and BL21. Preferably it is E. coli K-12 MG1655.

The microorganism of the genus Escherichia for increasing the production of acetyl-CoA and malonyl-CoA of the present invention inherently has a fatty acid biosynthesis pathway, but a gene encoding an enzyme for producing fatty acid, i.e. aceE And accA It is a strain in which a gene is additionally introduced, and a nucleotide sequence encoding an enzyme for producing the fatty acid is amplified, that is, overexpressed. In this regard, the term'amplification' is used to increase the copy number of the gene(s), e.g., by using a strong promoter or by using a gene encoding a suitable enzyme with high activity and optionally using these means together. It describes increasing the intracellular activity of one or more enzymes encoded by the microbe.

According to another aspect of the present invention, the present invention comprises the steps of (a) biosynthesizing fatty acids by culturing the co-transformed E. coli of the present invention described above; And (b) obtaining the biosynthesized fatty acid.

The method of the present invention is aceE Gene and accA Since the transgenic E. coli of the present invention co-transformed with a gene is used, the descriptions in common between the two are omitted in order to avoid undue complexity in the present specification.

aceE Gene and accA The cultivation of the transformed E. coli co-transformed with the gene may be cultured by a conventional E. coli culture method known in the art. For example, co-transformed E. coli can be cultured at 37°C in LB medium.

Preferably, step (a) is carried out in the presence of an expression inducing agent, IPTG.

The step of separating the fatty acid biosynthesized in the co-transformed E. coli may be carried out according to a conventional separation or purification method known in the art (see B. Aurousseau et al., Journal). of the American 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 the case of culturing the co-transformed E. coli prepared using the above-described manufacturing method, the fatty acid biosynthetic pathway is overexpressed, and thus fatty acids that can be used as a beneficial energy source can be easily obtained.

Therefore, in the present invention, in order to develop a strain that efficiently produces a specific metabolite to increase intracellular production of substances at a higher level of the fatty acid biosynthesis pathway, understanding the genomic information and metabolic network of microorganisms. At the same time, the present invention was completed by developing a recombinant strain through the insertion of a target gene by applying it to wild-type E. coli.

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

(I) The present invention provides a method for preparing a transgenic E. coli for overexpression of a fatty acid biosynthetic pathway comprising the following steps:

(a) aceE Gene and accA Inserting the gene into one expression vector; And

(b) the aceE Transforming E. coli with the gene and the expression vector into which the accA gene is inserted.

(Ii) The method for producing transformed E. coli for overexpression of the fatty acid biosynthetic pathway of the present invention is aceE Gene and accA Since the gene is inserted into one expression vector, E. coli is transformed with the vector, the genetic stability of the transformed E. coli is high, and the effect of overexpressing the fatty acid biosynthetic pathway is very excellent.

1 is Escherichia coli It is a schematic showing the pathway from glucose to fatty acid biosynthesis (FAS) in vivo.
Figure 2 is Escherichia coli Such as AceE to pTrc99A, an expression vector for bacteria It is a schematic diagram of the structure in which the gene is inserted. It is named pJE101.
Figure 3 is Escherichia coli AccA to pTrc99A, an expression vector for bacteria such as It is a schematic diagram of the structure in which the gene is inserted. It is named pJE102.
Figure 4 is Escherichia coli This is a schematic diagram of a structure in which aceE and accA genes are inserted into pTrc99A, an expression vector for bacteria such as. It is named pJE103.
Figure 5 is a recombinant Escherichia coli growth curve of the wild species Escherichia coli This is a graph showing the result of comparison with.
Figure 6 is a recombinant Escherichia The amounts of acetic acid (Fig. 6a) and malonic acid (Fig. 6b) analyzed outside the cells of coli were determined by the wild species Escherichia. This is the result of comparison with the experimental result of coli.
Fig. 7 shows the amounts of acetic acid (Fig. 7a) and malonic acid (Fig. 7b) analyzed outside the cells of Escherichia coli containing pJE103, pJE101. Or it is the result of comparison with the experimental results of E. coli containing only pJE102.
Figure 8 shows the amounts of acetic acid (Figure 8a) and malonic acid (Figure 8b) analyzed outside the cells of Escherichia coli containing pJE103, pJE101. And This is the result of comparison with the results of the E. coli experiment including all pJE102.
9 is a recombinant Escherichia The amounts of acetic acid (Fig. 9a) and malonic acid (Fig. 9b) analyzed in the cells of coli were determined by the wild species Escherichia. This is the result of comparison with the experimental result of coli.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and that the scope of the present invention is not limited by these examples according to the gist of the present invention for those of ordinary skill in the art to which the present invention pertains. It will be self-evident.

Example

Example 1: Preparation of plasmid pJE101, pJE102, pJE103 and expressing the gene of the aceE accA

Polymerase chain reaction (PCR, DaKaRa Korea) of aceE and accA encoding four enzymes for the production of acetyl-CoA and malonyl-CoA using the chromosome of E. coli K-12 as a template. It was cloned through the method. The aceE and accA gene sequences and the names of enzymes encoded by each are summarized in Table 1 below, and primers and restriction enzymes used to amplify each gene are summarized in Table 2 below.

Gene sequence
(GenBank license number) gene Production enzyme Sequence Listing first sequence
(GeneID: 944834) aceE pyruvate dehydrogenase,
decarboxylase component E1 Sequence Listing 2
(GeneID: 944895) accA acetyl-CoA carboxylase,
carboxytransferase, alpha subunit

gene Primer sequence Restriction enzyme aceE F 5'-CCATGGATGAACGAAACCGTTGACGGCG-3' Nco I R 5'-GAGCTCTTACGCCAGACGCGGGTTAACT-3' Sac I accA F 5'-GGATCCATGAGTCTGAATTTCCTTGATTTT-3' BamH I R 5'-TCTAGATTACGCGTAACCGTAGCTCATC-3' Xba I

The genes in Table 1 were amplified using the primers listed in Table 2. The amplified product was introduced into the pTrc99A vector (SEQ ID NO: 3, Seon-Won Kim et al., Biotechnol . Prog . , 23:599-605 (2007)) using the restriction enzymes listed in Table 2, respectively, and aceE Gene , accA Genes and pJE101 , pJE102, and pJE103 containing aceE and accA genes were prepared.

Figures 2, 3 and 4 is a view showing a map of the pJE101 (including aceE gene), pJE102 (including accA gene), and pJE103 (including the aceE gene accA) La named vector. Each polymerase chain reaction was performed at 95° C./ under conventional 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)). After performing once at 5 minutes (denaturation), 60°C/1 minute (annealing), 72/1 minute (extension), 95°C/1 minute (denaturation), 60°C/30 seconds (annealing), 72/1 It was repeated 30 times with an extension. In the last step, for stable elongation, the reaction was performed at 95°C/1 minute (denaturation), 60°C/1 minute (annealing), and 72/5 minutes (extension). The DNA amplified after the polymerase chain reaction was confirmed on a 1% agarose gel, purified, and used for restriction enzyme reaction. Each of the DNA fragments treated with the restriction enzyme was inserted into the multicloning site of the pTrc99A vector as disclosed in FIGS. 2, 3 and 4. Transformed E. coli (E. coli AG1 competent cell, Stratagene) was extracted and treated with the restriction enzyme of the original insertion site to compare the size of the cut DNA by electrophoresis on a 1% agarose gel.

Example 2: E. coli transformant ( transformant ) Of the manufacture

In the case of E. coli, which is commonly used for cloning, a plasmid is put into the host cell by the heat shock (42°C) method after making competent cells using _{CaCl 2 buffer, but in the present invention, stable production and efficiency of transformants are improved.} For this, a transformation method by electric shock was used.

For the transformation method by electric shock, 200 µl of wild species E. coli culture medium cultured for 16 hours was added to 50 ml of LB medium (10 g/L Tripton, 10 g/L NaCl, 5 g/L) in a 250 ml flask. yeast extract), and when the absorbance of the culture solution reached 0.6 at a wavelength of 600 nm, 50 ml of the culture solution corresponding to the entire culture solution was centrifuged to separate the supernatant and cells. The collected cells were repeatedly washed 2-3 times with 5 ml of 10% glycerol, and then diluted with 100-200 µl of 10% glycerol. The diluted cells were dispensed with 50 µl and 1–3 µl of plasmid was added.

The dispensed 50 µl of the liquid containing the above plasmid was put in a cuvette for electroporation and electric shock (1800 V, 25 µF, 200Ω) was applied. After adding 1 ml of LB medium (10 g/L Tripton, 10 g/L NaCl, 5 g/L yeast extract) prepared in advance, it was incubated at 37°C for 1 hour. The cultured transformants were cultured in a selective medium (LB solid medium to which an ampicillin (50 µg/ml) was added) until a single cell was generated.

By the above method, three types of recombinant E. coli (transformants) into which the expression vector of the configuration shown in FIGS. 2, 3 and 4 were inserted were completed. In addition, since the recombinant E. coli having the plasmid of pJE101 of FIG. 2 and the plasmid of pJE102 of FIG. 3 at the same time uses the transformation method described above, the E. coli into which pJE101 is inserted was additionally confirmed according to the above method, and then the same method. PJE102 was inserted. Recombinant E. coli having both pJE101 and pJE102 was identified using PCR primers after extracting the plasmid inside, and the size of the inserted gene was also confirmed by electrophoresis.

Example 3: Culture of E. coli and production of proteins from recombinant strains

Each of the bacteria whose transformation accuracy was confirmed was incubated in LB medium for 12 hours, made into a bacterial solution for storage so that the glycerol concentration was 25%, and then stored at -80°C until the culture experiment. Incubation was carried out by incubating 1 ml of the storage bacteria solution in 3 ml of LB medium to which MPicillin (50 µg/ml) contained in a 10 ml bottom tube dp was added for 24 hours, and then again MP contained in a 500 ml flask. Inoculate 1/200 of the pre-culture solution in 200 ml of LB medium (10 g/L Tripton, 10 g/L NaCl, 5 g/L yeast extract) to which silin (50 µg/ml) was added to 37°C, 170 rpm Incubated for 24 hours in the state (FIG. 5). For protein expression, when the absorbance of the culture medium reached 0.6 at a wavelength of 600 nm, 1 mM of IPTG (isopropylthio-β-D-galactoside, sigma, USA) as an inducer was added to induce expression of the recombinant protein.

The growth curves of the four recombinant E. coli species and wild species cultured according to the above are shown in FIG. 5.

Example 4: Preparation of acetic acid from a recombinant E. coli intracellular (acetic acid ) and malonic acid

The concentration of acetic acid and malonic acid in the culture medium is determined by centrifugation (10 minutes, 4500 rpm) of the sample collected at a specific time during cultivation at 4°C. For extraction of, the following process was carried out.

The cells separated from the supernatant are changed into a fluid liquid by diluting the cells with 250 µl of methanol placed at -80°C below -80°C and checking after 5-10 minutes. This process was repeated 3 times and centrifuged at -9°C and the supernatant was stored (-80°C). The solution obtained by repeating the above process again with the remaining cells was combined to obtain a total of 500 µl of a sample. This is an intracellular substance, extracted from metabolites in cells.

The supernatant (extracellular material) separated from 50 ml of the culture medium and the material extracted from the cells (intracellular material) were purified by using a 0.2 μm filter, respectively, 500 μl, and then subjected to HPLC quantitative analysis under the conditions below. The results are shown in FIGS. 6 to 9.

HPLC conditions for acetic acid and malonic acid analysis item Condition HPLC model (Younglin Machine, Korea), UV730D (Younglin Machine, Korea) column Aminex HPX-87H (Biorad, USA) Flow rate 0.6 mi/min Injection volume 30 μl Mobile phase 0.005N sulfuric acid Oven temperature 50℃ Working time 30 minutes UV detection wavelength 210nm

As a result of the experiment, it was found that the recombinant E. coli showed a large difference in the production and secretion tendency of acetic acid and malonic acid analyzed in and outside the cells of wild E. coli (FIGS. 6 to 9).

Referring to Figure 7, in the case of Escherichia coli containing pJE103, pJE101 Alternatively, it can be seen that the amount of acetic acid and malonic acid analyzed extracellularly was significantly increased than that of Escherichia coli containing only pJE102.

In addition, referring to Figure 8, in the case of Escherichia coli containing pJE103 pJE101 And It can be seen that the amounts of acetic acid and malonic acid analyzed extracellularly were significantly increased compared to E. coli containing all of pJE102.

9, the production of acetic acid and malonic acid was not confirmed in wild species from 12 hours after the initiation of culture, but intracellular production of acetic acid and malonic acid was maintained by induction of expression by IPTG in 4 types of recombinant E. coli. can confirm.

Example 5: genetic stability of recombinant E. coli ( genetic stability ) compare

aceE And accA method for transformation of Escherichia coli with both genes are (i), and a method for inserting a single expression vector (pJE103) containing all aceE and accA gene as shown in Figure 3 in E. coli, (ii) and Figs. 2 and 3 As aceE There is a method of injecting two expression vectors into E. coli, one containing the gene (pJE101) and one containing the accA gene (pJE102).

E. coli transformed with pJE103 (including aceE and accA genes) vector according to the method (i) has high genetic stability to the vector for transformation, so descendant E. coli has almost the same ability to produce acetic acid and malonic acid as ancestor E. coli. Shown. On the other hand, pJE103 according to the process (including the aceE gene) pJE101 according to the method of (ii) vector and pJE102 (including accA gene) For transfection the E. coli by a vector of genetic stability of the vector for transfection (i) ( aceE and accA genes), compared to the vector-transformed E. coli, the descendant E. coli slightly decreased the acetic acid and malonic acid production capacity compared to the ancestor E. coli, but this was higher than the acetic acid and malonic acid produced by the wild E. coli. Have productivity. In order to confirm the genetic stability of E. coli transformed with pJE103 (including aceE and accA genes) vector according to the method (i), the recombinant was cultured in selective culture medium for 24 hours, and then the vector was maintained in the cell. when determine the number of objects of the strain, 89% was maintained, it was found to be 70% in the plasma one E. coli as pJE101 (including aceE gene) vector, and the pJE102 (including accA gene) vector according to the method of (ii).

As described above, a specific part of the present invention has been described in detail, and it is obvious that this specific technology is only a preferred embodiment for those of ordinary skill in the art, and the scope of the present invention is not limited thereto. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> Industry-University Cooperation Foundation Sogang University <120> Method for Preparing Transformed E coli for Over-expression of Fatty Acid Biosynthesis Pathway and Transformed E coli Prepared by the Same <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 2664 <212> DNA <213> Escherichia coli strain K-12, aceE <400> 1 atgtcagaac gtttcccaaa tgacgtggat ccgatcgaaa ctcgcgactg gctccaggcg 60 atcgaatcgg tcatccgtga agaaggtgtt gagcgtgctc agtatctgat cgaccaactg 120 cttgctgaag cccgcaaagg cggtgtaaac gtagccgcag gcacaggtat cagcaactac 180 atcaacacca tccccgttga agaacaaccg gagtatccgg gtaatctgga actggaacgc 240 cgtattcgtt cagctatccg ctggaacgcc atcatgacgg tgctgcgtgc gtcgaaaaaa 300 gacctcgaac tgggcggcca tatggcgtcc ttccagtctt ccgcaaccat ttatgatgtg 360 tgctttaacc acttcttccg tgcacgcaac gagcaggatg gcggcgacct ggtttacttc 420 cagggccaca tctccccggg cgtgtacgct cgtgctttcc tggaaggtcg tctgactcag 480 gagcagctgg ataacttccg tcaggaagtt cacggcaatg gcctctcttc ctatccgcac 540 ccgaaactga tgccggaatt ctggcagttc ccgaccgtat ctatgggtct gggtccgatt 600 ggtgctattt accaggctaa attcctgaaa tatctggaac accgtggcct gaaagatacc 660 tctaaacaaa ccgtttacgc gttcctcggt gacggtgaaa tggacgaacc ggaatccaaa 720 ggtgcgatca ccatcgctac ccgtgaaaaa ctggataacc tggtcttcgt tatcaactgt 780 aacctgcagc gtcttgacgg cccggtcacc ggtaacggca agatcatcaa cgaactggaa 840 ggcatcttcg aaggtgctgg ctggaacgtg atcaaagtga tgtggggtag ccgttgggat 900 gaactgctgc gtaaggatac cagcggtaaa ctgatccagc tgatgaacga aaccgttgac 960 ggcgactacc agaccttcaa atcgaaagat ggtgcgtacg ttcgtgaaca cttcttcggt 1020 aaatatcctg aaaccgcagc actggttgca gactggactg acgagcagat ctgggcactg 1080 aaccgtggtg gtcacgatcc gaagaaaatc tacgctgcat tcaagaaagc gcaggaaacc 1140 aaaggcaaag cgacagtaat ccttgctcat accattaaag gttacggcat gggcgacgcg 1200 gctgaaggta aaaacatcgc gcaccaggtt aagaaaatga acatggacgg tgtgcgtcat 1260 atccgcgacc gtttcaatgt gccggtgtct gatgcagata tcgaaaaact gccgtacatc 1320 accttcccgg aaggttctga agagcatacc tatctgcacg ctcagcgtca gaaactgcac 1380 ggttatctgc caagccgtca gccgaacttc accgagaagc ttgagctgcc gagcctgcaa 1440 gacttcggcg cgctgttgga agagcagagc aaagagatct ctaccactat cgctttcgtt 1500 cgtgctctga acgtgatgct gaagaacaag tcgatcaaag atcgtctggt accgatcatc 1560 gccgacgaag cgcgtacttt cggtatggaa ggtctgttcc gtcagattgg tatttacagc 1620 ccgaacggtc agcagtacac cccgcaggac cgcgagcagg ttgcttacta taaagaagac 1680 gagaaaggtc agattctgca ggaagggatc aacgagctgg gcgcaggttg ttcctggctg 1740 gcagcggcga cctcttacag caccaacaat ctgccgatga tcccgttcta catctattac 1800 tcgatgttcg gcttccagcg tattggcgat ctgtgctggg cggctggcga ccagcaagcg 1860 cgtggcttcc tgatcggcgg tacttccggt cgtaccaccc tgaacggcga aggtctgcag 1920 cacgaagatg gtcacagcca cattcagtcg ctgactatcc cgaactgtat ctcttacgac 1980 ccggcttacg cttacgaagt tgctgtcatc atgcatgacg gtctggagcg tatgtacggt 2040 gaaaaacaag agaacgttta ctactacatc actacgctga acgaaaacta ccacatgccg 2100 gcaatgccgg aaggtgctga ggaaggtatc cgtaaaggta tctacaaact cgaaactatt 2160 gaaggtagca aaggtaaagt tcagctgctc ggctccggtt ctatcctgcg tcacgtccgt 2220 gaagcagctg agatcctggc gaaagattac ggcgtaggtt ctgacgttta tagcgtgacc 2280 tccttcaccg agctggcgcg tgatggtcag gattgtgaac gctggaacat gctgcacccg 2340 ctggaaactc cgcgcgttcc gtatatcgct caggtgatga acgacgctcc ggcagtggca 2400 tctaccgact atatgaaact gttcgctgag caggtccgta cttacgtacc ggctgacgac 2460 taccgcgtac tgggtactga tggcttcggt cgttccgaca gccgtgagaa cctgcgtcac 2520 cacttcgaag ttgatgcttc ttatgtcgtg gttgcggcgc tgggcgaact ggctaaacgt 2580 ggcgaaatcg ataagaaagt ggttgctgac gcaatcgcca aattcaacat cgatgcagat 2640 aaagttaacc cgcgtctggc gtaa 2664 <210> 2 <211> 960 <212> DNA <213> Escherichia coli strain K-12, accA <400> 2 atgagtctga atttccttga ttttgaacag ccgattgcag agctggaagc gaaaatcgat 60 tctctgactg cggttagccg tcaggatgag aaactggata ttaacatcga tgaagaagtg 120 catcgtctgc gtgaaaaaag cgtagaactg acacgtaaaa tcttcgccga tctcggtgca 180 tggcagattg cgcaactggc acgccatcca cagcgtcctt ataccctgga ttacgttcgc 240 ctggcatttg atgaatttga cgaactggct ggcgaccgcg cgtatgcaga cgataaagct 300 atcgtcggtg gtatcgcccg tctcgatggt cgtccggtga tgatcattgg tcatcaaaaa 360 ggtcgtgaaa ccaaagaaaa aattcgccgt aactttggta tgccagcgcc agaaggttac 420 cgcaaagcac tgcgtctgat gcaaatggct gaacgcttta agatgcctat catcaccttt 480 atcgacaccc cgggggctta tcctggcgtg ggcgcagaag agcgtggtca gtctgaagcc 540 attgcacgca acctgcgtga aatgtctcgc ctcggcgtac cggtagtttg tacggttatc 600 ggtgaaggtg gttctggcgg tgcgctggcg attggcgtgg gcgataaagt gaatatgctg 660 caatacagca cctattccgt tatctcgccg gaaggttgtg cgtccattct gtggaagagc 720 gccgacaaag cgccgctggc ggctgaagcg atgggtatca ttgctccgcg tctgaaagaa 780 ctgaaactga tcgactccat catcccggaa ccactgggtg gtgctcaccg taacccggaa 840 gcgatggcgg catcgttgaa agcgcaactg ctggcggatc tggccgatct cgacgtgtta 900 agcactgaag atttaaaaaa tcgtcgttat cagcgcctga tgagctacgg ttacgcgtaa 960 960 <210> 3 <211> 4176 <212> DNA <213> Artificial Sequence <220> <223> Vector backbone pTrc99A <400> 3 gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc 60 ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc 120 gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc 180 tgaaatgagc tgttgacaat taatcatccg gctcgtataa tgtgtggaat tgtgagcgga 240 taacaatttc acacaggaaa cagaccatgg aattcgagct cggtacccgg ggatcctcta 300 gagtcgacct gcaggcatgc aagcttggct gttttggcgg atgagagaag attttcagcc 360 tgatacagat taaatcagaa cgcagaagcg gtctgataaa acagaatttg cctggcggca 420 gtagcgcggt ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg 480 atggtagtgt ggggtctccc catgcgagag tagggaactg ccaggcatca aataaaacga 540 aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc 600 ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg 660 tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg 720 acggatggcc tttttgcgtt tctacaaact ctttttgttt atttttctaa atacattcaa 780 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 840 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 900 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 960 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 1020 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 1080 tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 1140 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 1200 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 1260 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 1320 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 1380 cgatgcctac agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 1440 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 1500 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 1560 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 1620 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 1680 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 1740 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 1800 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 1860 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 1920 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 1980 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 2040 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 2100 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 2160 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 2220 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 2280 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 2340 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 2400 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 2460 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 2520 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 2580 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 2640 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 2700 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc 2760 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc 2820 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg 2880 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa 2940 ttcgcgcgcg aaggcgaagc ggcatgcatt tacgttgaca ccatcgaatg gtgcaaaacc 3000 tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa 3060 ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac cgtttcccgc 3120 gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg 3180 gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg 3240 ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg 3300 attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc 3360 ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg 3420 atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc ctgcactaat 3480 gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat tattttctcc 3540 catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca ccagcaaatc 3600 gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat 3660 aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga ctggagtgcc 3720 atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg 3780 ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg 3840 cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag ctcatgttat 3900 atcccgccgt caaccaccat caaacaggat tttcgcctgc tggggcaaac cagcgtggac 3960 cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt gcccgtctca 4020 ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg 4080 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg 4140 caacgcaatt aatgtgagtt agcgcgaatt gatctg 4176

Claims (4)

(a) aceE (pyruvate dehydrogenase, decarboxylase component E1) having the nucleotide sequence of SEQ ID NO: 1 Inserting an accA (acetyl-CoA carboxylase, Carboxytransferase) gene having the nucleotide sequence of the gene and the sequence listing second sequence into one expression vector including the sequence listing the third sequence as the minimum sequence; The expression vector comprises a trc promoter sequence, aceE gene sequence, accA gene sequence, rrnB terminator sequence and lacI gene; And (b) in the aceE gene and accA the gene is producing E. coli in the inserted expression vector transfected method of producing a transformed E. coli for the expression of fatty acid biosynthesis comprising the steps of converting by an electric shock aceE gene and accA gene E. coli co-transformed.
(a) culturing the co-transformed Escherichia coli of claim 1 to biosynthesize fatty acids; And (b) obtaining the biosynthesized fatty acid.
The Escherichia coli according to claim 1 or 2, wherein the fatty acid is acetic acid or malonic acid.
The Escherichia coli according to claim 1, wherein the co-transformed Escherichia coli has a growth potential of at least 85% with respect to wild type Escherichia coli.

Images