KR101765219B1 - Recombinant E.coli producing unsaturated fatty acid, and method for producing unsaturated fatty acid using the same - Google Patents

Abstract

The present invention relates to a recombinant Escherichia coli producing cis-vaccenic acid in an unsaturated fatty acid, and a process for producing an unsaturated fatty acid using the same. The recombinant Escherichia coli producing the unsaturated fatty acid according to the present invention uses three types of genes fabB , fabF and fabA in the fatty acid synthesis elongation stage as a promoter of cspA having the genetic information of the cold shock protein, The recombinant plasmid was prepared by cloning into a plasmid (pCOLD1) which was characterized by increasing the expression efficiency, and the recombinant plasmid was introduced into E. coli BL21 (DE3), which is a host strain, The expression efficiency of the three genes is increased. In addition, when the recombinant Escherichia coli is cultured at a low temperature to produce a fatty acid, the proportion and amount of vaccenic acid in the unsaturated fatty acid is greatly increased, and the unsaturated fatty acid can be effectively produced.

Description

TECHNICAL FIELD The present invention relates to a recombinant E. coli producing an unsaturated fatty acid and a method for producing the unsaturated fatty acid using the same,

The invention of the unsaturated fatty acid cis - relates to a recombinant Escherichia coli, and a method for producing unsaturated fatty acids using the same to produce a foil sensan (cis -vaccenic acid).

At present, the rapid growth of the global market is based on increased use of energy. It is urgent to develop energy to replace oil as energy prices are rising due to the depletion of oil energy. For this purpose, interest in development of natural energy such as solar power or wind power is gaining attention. However, there are disadvantages such as low energy density and intermittent use, and thus there are many restrictions on the supply. In addition, oil prices in the domestic energy market, which relies heavily on imports of fuels, are expected to rise by 36% YoY in 2011 (see KNOC's press release). Respectively. In particular, since international oil prices directly affect the price of ordinary people's lives, it is essential to produce new alternative energy with low dependence on oil.

One of the most popular alternative energy sources is biofuel, a kind of renewable energy that uses biomass to produce not only organisms but also by-products from metabolism. Typical examples of biofuels are bioethanol, biodiesel, methanol, biogas, and solid fuels, which are currently used as transportation fuels. As shown in FIG.

Biodiesel is a single alkyl ester mixture with a chemically long fatty acid ring represented by the structure: Because biodiesel is very similar to light oil, it has the advantage of being able to be used without replacing the current internal combustion engine. It can be transported and sold through existing facilities and is considered as a representative alternative energy for transportation fuel. The quality of biodiesel is determined according to the raw material. In the case of containing a large amount of saturated fatty acid, the oxidation stability of the biodiesel is excellent, but the low temperature fluidity is poor in winter. On the other hand, when there are many unsaturated fatty acids, the low temperature fluidity is good, but when the number of double bonds is large, the oxidation stability is low. Accordingly, the most stable raw materials are oleic acid: is known as biodiesel content is more than 60% of the (oleic acid, octadecenoic acid, C18 1Δ 9).

The fatty acid composition ratios of the plant biodiesel and the physical properties of the biodiesel produced from the plant fat are shown in Tables 1 and 2, respectively.

Biodiesel composition Raw material C16: 0 C16: 1 C18: 0 C18: 1 C18: 2 C18: 3 palm oil 42.6 0.3 4.4 40.5 10.1 0.2 Soybean oil 13.9 0.3 2.1 23.2 56.2 4.3 Rapeseed oil 3.5 0 0.9 64.1 22.3 8.2

Kinematic viscosity
(40, mm ² / s) Cetanean Low calorific value Cloud point
(° C) Low temperature filter
Clogging point (℃) flash point
(° C) Oxidation stability (h), 110 ° C palm oil 5.7 62 33.5 13 12 164 4.0 Soybean oil 4.0 45 33.5 One -4 178 2.1 Rapeseed oil 4.4 54.4 - -3.3 -13 - 7.6

On the other hand, Escherichia coli uses a different type of fatty acid synthesis (Type 2) than that of mammals, and research has been carried out through this. The fatty acid synthesis type 2 system has basic enzymes in the initiation step and the elongation step of the acyl group, and the step of synthesizing the fatty acid of such enzyme is shown in Fig. The fatty acids required for E. coli are produced by this repetitive synthesis, and the saturated fatty acids produced mainly include palmitic acid (hexadecanoic acid) having 16 carbon atoms and the cis-unsaturated fatty acid having a double bond at the carbon number of 18, there are Toledo and palmitoleic acid (palmitoleic acid, 9- cis -hexadecenoic acid ): sensan foil (1 △ 11 cis -vaccenic acid, cis -11-octadecanoic acid, C18).

Fab (fatty acid biosynthesis) genes present in Escherichia coli are enzyme-producing genes involved in fatty acid synthesis. The fatty acid biosynthetic pathway consists largely of an initiation step, an elongation step, a termination step, and a beta-oxidation step. The fatty acid synthesis involved in the kidney stage The genes are fabA, fabB, and fabF . fabB with the genetic information of the β-ACP dehydrase Hydroxydecanoyl / isomerase fabA and KASI with the genetic information of the (β-ketoacyl-ACP (acyl -carrier-protein) synthaseI) gene is essential for the synthesis of unsaturated fatty acids. fabF that with fabB carry the genetic information for the KASII (β-ketoacyl-ACP synthaseII ) is located in the fatty acid elongation steps, by bonding the carbonyl -ACP words, the fatty acid -ACP increased two by two carbon atoms of the fatty acid of extending the length of the fatty acid It plays a role. These three enzymes are involved in the direct pathway to biosynthesis of palmitoic acid, palmitoleic acid, and cis- valencenic acid (C18: 1Δ9), which are the main constituents of E. coli cell membranes, - KASII, an enzyme that plays a key role in the production of Park Sung San, is characterized by its ability to be expressed at low temperatures. Enzyme and gene information of fatty acid biosynthesis of E. coli are shown in Table 3.

Many studies have been carried out to produce a target protein by transforming and expressing a desired gene in E. coli using a plasmid or producing a metabolite by a protein acting as an enzyme. In the case of recombinant E. coli transformed with the recombinant plasmid, the culture is temporarily stopped when cultured at a low temperature, and the recombinant plasmid protein expression is decreased in most of the recombinant E. coli. The cold shock protein encoded by the plasmid pCOLD1 is specifically induced to induce expression of the target gene recombined in pCOLD1 at a low temperature. The gene having the genetic information of the cold shock protein is cspA . The plasmid (pCOLD1, Takara) is a protein expression vector using the promoter of the gene cspA . Using this promoter, the target protein expression is up to 60% of the bacterial protein and can express a protein that is not expressed in the conventional expression system, and can be used in various E. coli.

gene Expression protein Change of phenotype in case of mutation aas Acyl-ACP synthetase lysophosphatidyl
ethanolamine accumulation accA Acetyl-CoA carboxylase carboxyltransferase α subunit accB Acetyl-CoA carboxylase BCCP subunit accC Acetyl-CoA carboxylase biotin carboxylase subunit accD Acetyl-CoA carboxylase carboxyltransferase β subunit acpP ACP ACP structural gene acpS ACP synthetase apo-ACP accumulation cfa Cyclopropane fatty acid synthase cells lack cyclopropane fatty acids fabA β-Hydroxydecanoyl ACP
dehydrase / isomerase Unsaturated fatty acid auxotroph fabAup - Saturated fatty acid and production fabB β-Ketoacyl-ACP synthase I Unsaturated fatty acid auxotroph fabD Malonyl-CoA: ACP transacylase Saturated fatty acids and auxotrophs of unsaturated fatty acids are required depending on temperature fabE Acetyl-CoA carboxylase - fabF β-Ketoacyl-ACP synthase II Controlled by temperature fabG β-Ketoacyl-ACP synthase - fabH β-Ketoacyl-ACP synthase Ⅲ - fabi Enoyl-ACP reductase - fabZ 3-hydroxyacyl-ACP dehydratase - fadD Long-chain acyl-CoA synthetase - fadR Transcriptional regulator of fabA - fatA Use of trans unsaturated fatty acids lpxA UDP-Glc-NAc acyltransferase Lipid A accumulation orf-17 Putative dehydrase - plsB sn -Glycerol-3-phosphate acyltransferase glycerol-3-phosphate self-nutrition plsC 1-Acylglycerol phosphaste acyltransferase - plsX Unknown PlsB ^- required for phenotype tesA Thioesterase I Enzymatic deficiency tesB Thioesterase II Enzymatic deficiency

As described above, interest in fatty acids that can be converted into biodiesel as alternative energy for fossil fuels is increasing, and interest in E. coli biosynthesizing fatty acids is also increasing. The current biodiesel is made using vegetable oil. In the case of biodiesel containing a large amount of unsaturated fatty acids, it has the advantage of not freezing at low temperatures. For example, in the case of biodiesel using rapeseed oil, it has an oleic acid content of more than 64%, which is lower than other biodiesel. However, in the case of vegetable oil, energy production using microorganisms has been attracting attention due to problems such as a rise in grain prices, a shortage of agricultural land, and an increase in land price. In the case of microorganisms, the fatty acid is biosynthesized as a fat form constituting the cell membrane. Accordingly, interest in E. coli that can be genetically manipulated has been increased, and studies on this have been made.

Therefore, there is an urgent need to develop a strain capable of increasing the composition of unsaturated fatty acid, which is advantageous in physical properties when converted to biodiesel as part of the development of new and renewable energy.

In accordance with this demand, the present inventors have sought to develop a novel strain that improves the productivity of unsaturated fatty acids.

The present inventors have previously developed a novel recombinant Escherichia coli which produces cis- vicenic acid among unsaturated fatty acids.

As a background of the present invention, the present inventors have found that recombinant Escherichia coli producing cis- valencenic acid among unsaturated fatty acids and fatty acids using the same in Korean Patent No. 10-1402108 (May 26, 2014) And Korean Patent No. 10-1275090 (Jun. 10, 2013) discloses a recombinant Escherichia coli producing a fatty acid and a method for producing a fatty acid using the recombinant Escherichia coli.

Numerous cited documents and patent documents are referred to and cited throughout this specification. The disclosures of the cited documents and patents are incorporated herein by reference in their entirety to more clearly describe the state of the art to which the present invention pertains and the content of the present invention.

Korean Patent No. 10-1402108 (May 26, 2014) Korean Patent No. 10-1275090 (June 10, 2013)

The present inventors have made efforts to develop a strain capable of increasing the composition of unsaturated fatty acids. As a result, two genes, FabB (KASI and KASII), which have information on the enzymes (KASI, KASII) acting at the last stage of carbon chain elongation and fatty acid production in the fatty acid synthesis elongation step using cspA promoter having genetic information of cold shock protein , recombinant plasmids containing FabA and KASI as well as the gene fabA having the information of the enzyme (β-Hydroxydecanoyl ACP dehydrase / isomerase) participating in the unsaturated fatty acid production step were prepared and the transformed recombinant E. coli was prepared. By culturing the recombinant E. coli at a low temperature and extracting fatty acids therefrom, it was confirmed that the ratio of cis- valencenoic acid in unsaturated fatty acids was greatly increased, thereby completing the present invention.

It is therefore an object of the present invention to provide a recombinant plasmid vector.

Another object of the present invention is of an unsaturated fatty acid cis - to provide a recombinant mutant microorganism to produce foil sensan (cis -vaccenic acid).

It is still another object of the present invention to provide a method for producing unsaturated fatty acids.

Hereinafter, the present invention will be described in detail.

According to one aspect of the present invention, the present invention provides a recombinant vector comprising: (i) a promoter operable in prokaryotic cells; And (ii) a nucleotide ( fabF , SEQ ID NO: 1) encoding a beta-keto acyl-acyl carrier protein synthase II of E. coli K-12 MG1655; A nucleotide ( FabB , SEQ ID NO: 2) coding for beta-ketoacyl-acyl transferase protein synthase I (KASI, β-keto acyl-acyl carrier protein synthase I) of E. coli K-12 MG1655; And a nucleotide ( fabA , SEQ ID NO: 3) coding for beta -hydroxydecanoyl thioester dehydrase / isomerase of E. coli K-12 MG1655; Lt; RTI ID = 0.0 > plasmid < / RTI > vector.

In a preferred embodiment of the present invention, the recombinant plasmid is pCOLD1 :: fabF, fabB, fabA .

The term "vector" as used herein refers to a DNA construct containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in an appropriate host, and specifically includes a promoter sequence, a terminator sequence, Genes, and other suitable sequences. Such vectors may be plasmids, phages, cosmids, and the like (Molecular Cloning: Laboratory Manual: Second Edition, Cold Spring Harbor Laboratory Press (1989), Sambrook et al. The preparation of these vectors, mutagenesis, sequencing, introduction of DNA into cells, and gene expression and protein analysis are described in detail in Current Protocols in Molecular Biology, edited by Ausubel et al., John Wiley & Sons (1992). Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. Plasmids are the most commonly used form of the current vector, and in this specification 'plasmid' and 'vector' are sometimes used interchangeably. For the purposes of the present invention, the vector is a vector suitable for expression in prokaryotic cells and a plasmid vector expressing the desired protein.

As used herein, the term "operably linked" refers to an expression control sequence that is linked to regulate transcription and translation of a polynucleotide sequence encoding a protein of interest, and wherein the nucleotide sequence under the control of an expression control sequence (including a promoter) And maintaining the correct decoding frame so that a polypeptide of the desired protein is expressed and encoded by the nucleotide sequence.

As used herein, the term "promoter" means a minimal sequence sufficient to direct transcription.

In the present invention, promoters which can be used for expressing a target protein in prokaryotes include, for example, cspA, T7, tac, trc, lac, lpp, phoA, recA, araBAD, proU, cst-1, tetA, cadA, -lac, starvation promoters, T7-lac operator, T3-lac operator, T5-lac operator, T4 gene 32, nprM-lac operator.

Preferably, cspA, T7, tac, lac, T7-lac operator, T3-lac operator, T5-lac operator, T4 gene 32, more preferably cspA, T7, tac, T7-lac operator can be used.

In a preferred embodiment of the present invention, the promoter is a cspA promoter.

The cspA promoter possesses genetic information of a cold shock protein that enhances gene expression efficiency at low temperatures and exists in pCOLD1.

Therefore, the expression of the target protein can be more stably induced at low temperature using the cspA promoter (SEQ ID NO: 4).

According to another aspect of the present invention, the present invention provides a recombinant Escherichia coli transformed by said vector.

In a preferred embodiment of the present invention, the recombinant E. coli produces an unsaturated fatty acid.

In a preferred embodiment of the present invention, the unsaturated fatty acid is cis-vaccenic acid.

In a preferred embodiment of the present invention, the recombinant E. coli is E. coli BL21 (DE3) :: pCOLD1 :: fabF, fabB, fabA (accession number: KCTC18324P).

The recombinant Escherichia coli producing fatty acid according to the present invention is a recombinant Escherichia coli producing Escherichia coli K-12 (pCOLD1), which is characterized by increasing the expression efficiency of genes at low temperatures by using a promoter of cspA having genetic information of a cold shock protein Nucleotides ( fabF , SEQ ID NO: 1) carrying the genetic information of beta-ketoacyl-acyl carrier protein synthase II (KASII, β-keto acyl-acyl carrier protein synthase II) of MG1655 ; Nucleotide ( FabB , SEQ ID NO: 2) carrying the genetic information of beta-ketoacyl-acyl carrier protein synthase I (KASI, β-keto acyl-acyl carrier protein synthase I) of E. coli K-12 MG1655; And a nucleotide ( fabA , SEQ ID NO: 3) having genetic information of β-hydroxydecanoyl thioester dehydrase / isomerase of E. coli K-12 MG1655 . ≪ / RTI >

The expressions "having genetic information" and "having genetic information" used in referring to a gene herein refer to coding a target gene and are used interchangeably herein.

The gene information used in the present invention is based on the genetic information of E. coli K-12 MG1655, and information of the enzymes (KASI, KASII) acting at the last stage of carbon chain elongation and fatty acid production in the fatty acid elongation step utilizes a fabA gene with the genetic information of the two genes fabB, enzymes (β-Hydroxydecanoyl ACP dehydrase / isomerase ) involved in the step of generating an unsaturated fatty acid with fabF and KASI with.

Specifically, Escherichia coli (E.coli K-12 MG1655) in the gene of interest (fabB, fabF, fabA) a recombinant plasmid pEcoli-Nterm 6xHN :: fabF, fabB , fabA ( Republic of Korea Patent No. 10-1402108 prepared using (Polymerase chain reaction) is carried out using a template as a template to obtain a target gene.

In particular, a recombinant plasmid is prepared by performing restriction enzyme reaction and ligation reaction on the obtained nucleotide ( fabB , fabF , fabA ) and a plasmid (pCOLD1) for protein expression [pCOLD1 :: fabF , fabB, fabA ].

In the case of the replication branch of the plasmid for protein expression, pCOLD1 has a cspA promoter which contains ColE1 and possesses an ampicillin resistance gene and genetic information of a cold shock protein which enhances gene expression efficiency at low temperature, It has a lac operator for the purpose of regulating expression.

E. coli XL-1 blue :: pCOLD1 :: fabF , fabB, fabA ] was prepared by the heat shock transformation method using the recombinant plasmid prepared above. The above prepared recombinant E. coli was confirmed that grows from a circular colony form in a solid LB medium containing antibiotics, a gene of the plasmid treated colonies with restriction enzymes, extracts of the culture and the plasmid in a liquid LB medium supplemented with ampicillin containing (fabB , fabF , fabA ) and E. coli K-12 MG1655 were found to be free of errors.

The prepared recombinant E. coli BL21 (DE3) :: pCOLD1 :: fabF , fabB, fabA ) was deposited on September 17, 2014 with KCTC of the Korea Research Institute of Bioscience and Biotechnology, KCTC18324P I got a security number.

According to another aspect of the present invention, the present invention provides a process for preparing an unsaturated fatty acid comprising the steps of:

(a) primary culturing the recombinant E. coli;

(b) secondary culturing the first-cultured recombinant E. coli of step (a) at a low temperature of 10 캜 to 20 캜;

(c) separating the culture medium and the culture medium by centrifuging the culture solution obtained in the step (b);

(d) adding a mixed solution of sulfuric acid and methanol to the cells separated in the step (c), filling the mixture with nitrogen, and performing an esterification reaction; And

(e) adding hexane and distilled water to the reaction solution of step (d) to separate the layers.

Preferably, the secondary culture of step (b) is at a low temperature of 15 캜 to 18 캜, most preferably 17 캜.

In a preferred embodiment of the present invention, the method further comprises analyzing the unsaturated fatty acid composition of the layer separated in step (e).

Any of the methods known in the art can be used as long as the above analysis can analyze the composition of the fatty acid.

In a preferred embodiment of the present invention, the unsaturated fatty acid is cis-vaccenic acid.

For example, the prepared recombinant E. coli BL21 (DE3) :: pCOLADuet ^TM -1 :: accA, accB, accC, accD + pEcoliNterm 6xHN :: fabF, fabB, fabA + pCDF-1b :: `tesA a) primary culture in 37 ℃ to grow until mid-exponential group to move to the low temperature of 17 ℃ after induction with IPTG 2 culture drive, and centrifuged After separating the microbial cells and the medium, a mixed solution of sulfuric acid and methanol was added to the separated microbial cells, and the microbial cells were filled with nitrogen. Then, the esterification reaction was carried out, and hexane and distilled water were added to the reaction mixture. The layer is subjected to gas chromatography to determine the composition of the unsaturated fatty acid.

According to the method of the present invention, the ratio and amount of the unsaturated fatty acid vaccenic acid has increased remarkably.

The method of the present invention produces an unsaturated fatty acid using the above-mentioned recombinant E. coli, and redundant description thereof is omitted in order to avoid the excessive complexity of the present specification.

As described above, the recombinant E. coli according to the present invention can be effectively used as a biofuel by effectively producing an unsaturated fatty acid.

The recombinant E. coli producing the fatty acid according to the present invention can be produced by using three kinds of genes fabB , fabF and fabA in the fatty acid synthesis elongation stage as a promoter of cspA having the genetic information of the cold shock protein, The recombinant plasmid is prepared by cloning into a plasmid (pCOLD1) which is characterized by increasing the efficiency, and the recombinant plasmid is introduced into E. coli BL21 (DE3), which is a host strain, There is an effect of increasing the expression efficiency of three genes. In addition, when the fatty acid was prepared using the recombinant E. coli, the ratio of the unsaturated fatty acid was not genetically modified with the recombinant E. coli BL21 (DE3) :: pEcoli-Nterm 6xHN :: fabF, fabB, fabA ) The ratio of the unsaturated fatty acids in E. coli BL21 (DE3) is greatly increased, and unsaturated fatty acids can be effectively produced.

1 shows a fatty acid synthesis route and an unsaturated fatty acid synthesis route.
Fig. 2 is a diagram showing information of a plasmid (pCOLD1) used for increasing protein expression efficiency in E. coli at low temperatures.
Fig. 3 is a diagram showing the sizes of recombinant plasmids (pCOLD1 :: fabF , fabB, fabA ) and fabF + fabB + fabA .
FIG. 4 is a diagram showing the structure and size change of a recombinant plasmid in which nucleotides including gene information of plasmid pCOLD1 and fabB, fabF and fabA for expression of the protein in E. coli are combined.
FIG. 5 is a diagram showing a structure and size change of a recombinant plasmid in which a nucleotide including gene information of the genes pEcoli-Nterm 6xHN and genes fabF, fabB, and fabA is combined for use as a template for the genetic information of fabB, fabF and fabA .
FIG. 6 is a diagram showing the nucleotide sequence analysis results of the fabF, fabB, and fabA genes bound to the plasmid pCOLD1 for expression of the protein in E. coli.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example  1: Preparation of recombinant plasmid containing nucleotide having information of target gene

One. PCR Gene amplification using

The gene information used in the present invention is based on the genetic information of E. coli K-12 MG1655, and it is known that the enzymes (KASI, KASII) acting at the last stage of carbon chain elongation and fatty acid production in the fatty acid synthesis elongation step two genes with information fabB (SEQ ID NO: 1), fabF (SEQ ID NO: 2) and the KASI gene with the information on the enzyme (β-Hydroxydecanoyl ACP dehydrase / isomerase ) involved in the unsaturated fatty acid generation step fabA (SEQ ID NO: 3 with ) Was used. The nucleotide sequence information was referenced from KEGG (Kyoto Encyclopedia of Genes and Genomes, www.genome.jp/kegg/ ), and PCR was carried out using the prepared primers to obtain the desired gene Respectively. Primer preparation and restriction enzymes for amplification of the target gene are shown in Table 4.

Target gene Expression enzyme Plasmid primer Restriction enzyme fabB KASI
(β-keto acyl-acyl carrier protein synthase I) pEcoli-Nterm 6xHN 5'-GGA AGA TCT GAA GGA GAT ATA CCA TGA AAC GTG CAG TGA TTA CTG GCC-3 ' BglII,
NotI 5'-ATA AGA ATG CGG CCG CTT AAT CTT TCA GCT TGC GCA TTA CCA-3 ' fabF KASII
(β-keto acyl-acyl carrier protein synthaseII) pEcoli-Nterm 6xHN 5'-AAA ACT GCA GGT GTG TCT AAG CGT CGT GTA GTT GTGA-3 ' PstI,
SalI 5'-ACG CGT CGA CTT AGA TCT TTT TAA AGA TCA AAG AAC C-3 ' fabA β-Hydroxydecanoyl ACP dehydrase / isomerase pEcoli-Nterm 6xHN 5'-CCT TAA TTA AGG AAG GAG GGA TGG TAG ATA AAC GCG AAT CC-3 ' PacI,
XbaI 5'-GCT CTA GAG CTC AGA AGG CAG ACG TAT CCT G-3 ' fabF, fabB, fabA pCOLD1  5'-AAA ACT GCA GGT AGT GTC TAA GCG TCG TGT AGT TGT GA-3 ' PstI, XbaI  5'-GCT CTA GAT CAG AAG GCA GAC GTA TCC TGG AAC AG C-3 '

For the genes fabF, fabB and fabA corresponding to the recombinant plasmids (pEcoli-Nterm 6xHN :: fabF, fabB, fabA ) used as the template gene for the polymerase chain reaction, the template gene of E. coli was obtained by the following method. First, after E. coli K12 MG1655 was cultured, the E. coli culture was centrifuged and the supernatant was taken. 5 ml of lysozyme solution was added to the supernatant, mixed and mixed until a clear viscous appeared. When viscosity appeared, 2 ml of 10% SDS (sodium dodecyl sulfate) solution was added and reacted for 30 minutes. The mixture was dispensed in 0.7 ml portions into microtube, and PCI solution (phenol, chloroform, isoamyl alcohol) was added to the mixture until the layer separation did not occur. The supernatant was collected, treated with PCI solution in the same manner, and centrifuged. DNA was separated by addition of 3M acetate and twice the volume of ethanol, adjusted to pH 6 by 1/10 of the collected supernatant volume. The isolated DNA was used as a template and amplified with polymerase chain reaction together with primers of respective genes ( fabB , fabF , fabA ). The polymerase chain reaction was carried out under denaturation temperature of 94 ° C., annealing temperature of 65 ° C. and extension temperature of 72 ° C. under the usual conditions, and the reaction was performed 30 times in total. The polymerase used in the reaction was NEB (New England Biolabs Inc.). The amplified genetic material was subjected to purification and confirmed in 0.7% agarose gel.

2. Preparation of recombinant plasmids containing nucleotides with information of the gene of interest

The obtained gene nucleotide and protein expression vector were cleaved using a common restriction enzyme that did not cleave other regions except the terminal region and confirmed by electrophoresis. The band of the confirmed agarose gel was cut and dissolved at 50 ° C, followed by purification and ligation at 4 ° C to form a circular form in which pCOLD1 of linear type was combined with fabF, fabB, and fabA Plasmids (pCOLD1 :: fabF , fabB, fabA ) were prepared. The sizes of the recombinant plasmids (pCOLD1 :: fabF , fabB, fabA ) and fabF + fabB + fabA were confirmed in FIG. The structure and size changes of the recombinant plasmids in which the nucleotides including the plasmid pCOLD1 and the gene information of fabB, fabF and fabA are combined are shown in FIG. And are shown in fabB, fabF, the use as a template for the genetic information of fabA plasmid pEcoli-Nterm 6xHN gene fabF, fabB, Figure 5 structure, and size change of a recombinant plasmid that combines an oligonucleotide containing the genetic information of fabA.

Example 2: Preparation of recombinant Escherichia coli transformed with recombinant plasmid

1. Confirmation of synthesis error of recombinant plasmid

Before putting the plasmid (pCOLD1 :: fabF , fabB, fabA ) for the purpose of carrying the target gene into the host cell , it is necessary to confirm the synthesis error of the recombinant plasmid, and to improve the efficiency of transformation of the vector into E. coli E Competent cells were prepared using E. coli XL1-Blue ( E. coli XL1-B) to perform primary transformation ( E. coli XL-1 blue :: pCOLD1 :: fabF , fabB, fabA ).

The competent cells were prepared by the following procedure. E. coli XL1-Blue cultured at 37 ° C was subcultured, collected at the exponential period of the growth curve, and stored at 0 ° C for 30 minutes. The supernatant was removed by centrifugation at 5,000 rpm, and the MgCl ₂ solution was added to the cell wall for 30 minutes at 0 ° C to facilitate the plasmid transfer. The solution was centrifuged again to remove the supernatant, and CaCl ₂ solution was added and the mixture was subjected to secondary treatment at 0 ° C for 60 minutes. Recombinant plasmids (pCOLD1 :: fabF , fabB, fabA ) containing an external gene were added thereto and reacted at 0 ° C for 30 minutes, heat-treated in a hot water bath at 42 ° C for 30 seconds, (Heat-shock method). The sample subjected to the thermal shock transformation method was cultured at 37 ° C for 1 hour to help normal growth of the transformed cells. The cells were cultured in a solid LB medium (LB agar plate, tryptone 10 g / L, NaCl 5 g / L, yeast extract 5 g / L, agar 15 g / L) containing an antibiotic (ampicillin) To a certain extent.

The transformed sample was found to grow in a small round colony form in solid LB medium containing antibiotics. The colonies were cultured in 5 ml of liquid LB medium containing antibiotics and plasmids were extracted using a general plasmid extraction kit. The extracted plasmid was digested with the restriction enzyme treated for ligation after the PCR, and the size of the plasmid and the nucleotide having the information of the target gene were cut and compared with the gene of E. coli K-12 MG1655. In addition, it was confirmed that there is no synthetic error occurring during the mutation of the transformation process or the polymerase chain reaction by confirming that the genomic sequence analysis result of the above plasmid and the genetic information disclosed in KEGG are identical. This is shown in FIG.

Although recombinant plasmid pCOLD1 :: fabF, fabB, sequencing and sequence analysis of the sequence one nucleotide at a result of fabB gene of E. coli K-12 MG1655 a fabB fabA gene of the E. coli K-12 MG1655 and different from each other, It was confirmed that the amino acid sequence in the case of translation into amino acid and the protein having the steric structure as the function of the enzyme were consistent with each other.

2. Preparation of recombinant E. coli E. coli BL21 (DE3) :: pCOLD1 :: fabF , fabB, fabA )

In order to insert the recombinant plasmid (pCOLD1 :: fabF , fabB, fabA ) in which the synthetic error was checked in 1 above, E. coli BL21 (DE3) and a recombinant plasmid ( E. coli BL21 (DE3) :: pCOLD1 :: fabF , fabB, fabA ) were prepared by repeating the heat shock transformation method.

Therefore, the recombinant E. coli BL21 (DE3) :: pCOLD1 :: fabF , fabB, fabA ) prepared above was deposited with the KCTC of the BRC Center of the Korea Biotechnology Research Institute on September 17, KCTC18324P.

The prepared recombinant E. coli, the target gene and the recombinant plasmid are shown in Table 5.

Recombinant E. coli Target gene The recombinant plasmid E. coli BL21 (DE3) :: pCOLD1 :: fabF , fabB, fabA fabF, fabB, fabA pCOLD1 :: fabF , fabB, fabA

Example 3 Production of Fatty Acids Using Recombinant Escherichia coli

Recombinant E. coli BL21 (DE3) :: pCOLD1 :: fabF , fabB, fabA ) prepared in Example 2 was placed in 200 ml of liquid LB medium containing ampicillin as an antibiotic and cultured for a total of 27 hours. First, the cells were cultured in a shaking incubator at 37 DEG C while being rotated at a speed of 200 rpm. Considering that the expression of KAS II is efficient at low temperature after the addition of 1.0 mM IPTG (Isopropyl-β-D-thiogalactopyranoside) for the purpose of gene expression when the OD of the culture reached 0.6 ~ 0.8, The cells were cultured at low temperature, centrifuged to separate the cells and the medium, and lyophilized to remove moisture from the cells. To prepare a sample for analysis, a mixed solution of sulfuric acid and methanol (volume ratio of 5: 100) was added to a test tube containing cells, and the mixture was filled with nitrogen. The test tube was sealed, subjected to an esterification reaction at 90 DEG C for 30 minutes, and cooled at room temperature. Then, hexane and distilled water were added and mixed on a stirrer. The layers were separated by centrifugation, and the hexane layer of the upper layer in which FAMEs (fatty acid methyl esters) were dissolved was analyzed. The instrument used for the analysis was gas chromatography (Agilent 6890N), and the column used was an Agilent 19091N-133 capillary column.

As shown in Table 6, the recombinant Escherichia coli of the present invention was analyzed for the unsaturated fatty acid composition. As a result, it was found that the recombinant strain of the present invention contained not only a wild-type strain ( E. coli BL21 (DE3)) but also a positive control E. coli BL21 (DE3) :: pEcoli-Nterm 6xHN :: fabF, fabB, fabA transformed with the recombinant plasmids (pEcoli-Nterm 6xHN :: fabF, fabB, fabA ) disclosed in EP-10-1402108 , And it was confirmed that the ratio and amount of cis-vaccenic acid was improved remarkably.

Korea Biotechnology Research Institute KCTC18324P 20140917

<110> INHA-INDUSTRY PARTNERSHIP INSTITUTE <120> Recombinant E. coli producing unsaturated fatty acid, and method <130> INHA1.146P <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 1242 <212> DNA <213> Escherichia coli <400> 1 gtgtctaagc gtcgtgtagt tgtgaccgga ctgggcatgt tgtctcctgt cggcaatacc 60 gtagagtcta cctggaaagc tctgcttgcc ggtcagagtg gcatcagcct aatcgaccat 120 ttcgatacta gcgcctatgc aacgaaattt gctggcttag taaaggattt taactgtgag 180 gacattatct cgcgcaaaga acagcgcaag atggatgcct tcattcaata tggaattgtc 240 gctggcgttc aggccatgca ggattctggc cttgaaataa cggaagagaa cgcaacccgc 300 attggtgccg caattggctc cgggattggc ggcctcggac tgatcgaaga aaaccacaca 360 tctctgatga acggtggtcc acgtaagatc agcccattct tcgttccgtc aacgattgtg 420 aacatggtgg caggtcatct gactatcatg tatggcctgc gtggcccgag catctctatc 480 gcgactgcct gtacttccgg cgtgcacaac attggccatg ctgcgcgtat tatcgcgtat 540 ggcgatgctg acgtgatggt tgcaggtggc gcagagaaag ccagtacgcc gctgggcgtt 600 ggtggttttg gcgcggcacg tgcattatct acccgcaatg ataacccgca agcggcgagc 660 cgcccgtggg ataaagagcg tgatggtttc gtactgggcg atggtgccgg tatgctggta 720 cttgaagagt acgaacacgc gaaaaaacgc ggtgcgaaaa tttacgctga actcgtcggc 780 tttggtatga gcagcgatgc ttatcatatg acgtcaccgc cagaaaatgg cgcaggcgca 840 gctctggcga tggcaaatgc tctgcgtgat gcaggcattg aagcgagtca gattggctac 900 gttaacgcgc acggtacttc tacgccggct ggcgataaag ctgaagcgca ggcggtgaaa 960 accatcttcg gtgaagctgc aagccgtgtg ttggtaagct ccacgaaatc tatgaccggt 1020 cacctgttag gtgcggcggg tgcagtagaa tctatctact ccatcctggc gctgcgcgat 1080 caggctgttc cgccaaccat caacctggat aacccggatg aaggttgcga tctggatttc 1140 gtaccgcacg aagcgcgtca ggttagcgga atggaataca ctctgtgtaa ctccttcggc 1200 ttcggtggca ctaatggttc tttgatcttt aaaaagatct aa 1242 <210> 2 <211> 1217 <212> DNA <213> Escherichia coli <400> 2 aaacgtgcag tgattactgg cctgggcatt gtttccagca tcggtaataa ccagcaggaa 60 gtcctggcat ctctgcgtga aggacgttca gggatcactt tctctcagga gctgaaggat 120 tccggcatgc gtagccacgt ctggggcaac gtaaaactgg ataccactgg cctcattgac 180 cgcaaagttg tgcgctttat gagcgacgca tccatttatg cattcctttc tatggagcag 240 gcaatcgctg atgcgggcct ctctccggaa gcttaccaga ataacccgcg cgttggcctg 300 attgcaggtt ccggcggcgg ctccccgcgt ttccaggtgt tcggcgctga cgcgatgcgc 360 ggcccgcgcg gcctgaaagc ggttggcccg tatgtggtca ccaaagcgat ggcatccggc 420 gtttctgcct gcctcgccac cccgtttaaa attcatggcg ttaactactc catcagtccg 480 cgtgtgcgac ttccgcacac tgtatcggta acgcagtaga gcagatccaa ctgggcaaac 540 aggacatcgt gtttgctggc ggcggcgaag agctgtgctg ggaaatggct tgcgaattcg 600 acgcaatggg tgcgctgtct actaaataca acgacacccc ggaaaaagcc tcccgtactt 660 acgacgctca ccgtgacggt ttcgttatcg ctggcggcgg cggtatggta gtggttgaag 720 agctggaaca cgcgctggcg cgtggtgctc acatctatgc tgaaatcgtt ggctacggcg 780 caacctctga tggtgcagac atggttgctc cgtctggcga aggcgcagta cgctgcatga 840 agatggcgat gcatggcgtt gataccccaa tcgattacct gaactcccac ggtacttcga 900 ctccggttgg cgacgtgaaa gagctggcag ctatccgtga agtgttcggc gataagagcc 960 cggcgatttc tgcaaccaaa gccatgaccg gtcactctct gggcgctgct ggcgtacagg 1020 aagctatcta ctctctgctg atgctggaac acggctttat cgccccgagc atcaacattg 1080 aagagctgga cgagcaggct gcgggtctga acatcgtgac cgaaacgacc gatcgcgaac 1140 tgaccaccgt tatgtctaac agcttcggct tcggcggcac caacgccacg ctggtaatgc 1200 gcaagctgaa agattaa 1217 <210> 3 <211> 518 <212> DNA <213> Escherichia coli <400> 3 atggtagata aacgcgaatc ctatacaaaa gaagaccttc ttgcctctgg tcgcggtgac 60 tgtttggcgc taaaggcccg caattgccag caccgaacat gctgatgatg gaccgtgtgg 120 tcaaaatgac cgaaacgggt ggtaacttcg acaaagggta tgttgaagca gaactggata 180 tcaatccgga tctgtggttc ttcggatgcc actttattgg cgatccggtt atgccgggat 240 gcctgggcct ggacgcaatg tggcagctgg tagggttcta cctcggctgg ctgggcggcg 300 aaggtaaagg ccgcgcgctg ggcgttggcg aagtgaaatt cactggtcag gtactgccga 360 cagcgaaaaa agtgacctac cgtattcact ttaaacgcat tgttaaccgt cgtctgatta 420 tgggcctggc ggatggcgaa gtgctggttg atggtcgtct gatctatacc gccagcgacc 480 tgaaagtcgg tctgttccag gatacgtctg ccttctga 518 <210> 4 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> cspA promoter <400> 4 ccgattaatc ataaatatga aaaataattg ttgcatcacc cgccaatgcg tggcttaatg 60 cacatca 67 <210> 5 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> forward primer for fabB gene <400> 5 ggaagatctg aaggagatat accatgaaac gtgcagtgat tactggcc 48 <210> 6 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for fabB gene <400> 6 ataagaatgc ggccgcttaa tctttcagct tgcgcattac ca 42 <210> 7 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> forward primer for fabF gene <400> 7 gt; <210> 8 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for fabF gene <400> 8 acgcgtcgac ttagatcttt ttaaagatca aagaacc 37 <210> 9 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> forward primer for fabA gene <400> 9 ccttaattaa ggaaggaggg atggtagata aacgcgaatc c 41 <210> 10 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for fabA gene <400> 10 gctctagagc tcagaaggca gacgtatcct g 31 <210> 11 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> forward primer for fabF, fabB and fabA gene <400> 11 aaaactgcag gtagtgtcta agcgtcgtgt agttgtga 38 <210> 12 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for fabF, fabB and fabA gene <400> 12 gctctagatc agaaggcaga cgtatcctgg aacagac 37

Claims (10)

Operatively linked in the 5 'to 3' direction,
(i) a cspA promoter operable in prokaryotic cells; And
(ii) a nucleotide ( fabF , SEQ ID NO: 1) encoding a beta-ketoacyl-acyl transferase protein synthetase II of E. coli K-12 MG1655 (KASII, β-keto acyl-acyl carrier protein synthaseII); A nucleotide ( FabB , SEQ ID NO: 2) coding for beta-ketoacyl-acyl transferase protein synthase I (KASI, β-keto acyl-acyl carrier protein synthase I) of E. coli K-12 MG1655; And a nucleotide ( fabA , SEQ ID NO: 3) encoding beta-hydroxydecanoyl thioester dehydrase / isomerase of E. coli K-12 MG1655 A recombinant Escherichia coli producing cis-vaccenic acid transformed with a recombinant plasmid vector, wherein the recombinant Escherichia coli is E. coli BL21 (DE3) :: pCOLD1 :: fabF, fabB, fabA, Wherein the recombinant Escherichia coli is a recombinant Escherichia coli deposited with KCTC18324P. delete 2. The recombinant E. coli according to claim 1, wherein the recombinant plasmid is pCOLD1 :: fabF, fabB, fabA . delete delete delete delete A process for the preparation of cis-vaccenic acid comprising the steps of:
(a) primary culturing the recombinant E. coli of claim 1;
(b) secondary culturing the first-cultured recombinant E. coli of step (a) at a low temperature of 10 캜 to 20 캜;
(c) separating the culture medium and the culture medium by centrifuging the culture solution obtained in the step (b);
(d) adding a mixed solution of sulfuric acid and methanol to the cells separated in the step (c), filling the mixture with nitrogen, and performing an esterification reaction; And
(e) adding hexane and distilled water to the reaction solution of step (d) to separate the layers. delete delete

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