KR20160044695A - 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 recombinant E. coli producing, among unsaturated fatty acids, cis-vaccenic acid; and to a method for producing unsaturated fatty acids using the same. The recombinant E. coli producing unsaturated fatty acids according to the present invention constructs a system co-expressing 5 genes, i.e., accA, accB, accC, accD, fabD, and fabD located in one plasmid; 3 genes, i.e., fabB, fabF, and fabA, located in one plasmid; and tesA located in one plasmid. Also, the four recombinant plasmids are introduced into the recombinant E. coli (BL21(DE3) ΔfadD), which is a host strain having alleviated fatty acid reduction by removing fadD in a beta-oxidation step of fatty acid synthesis, and causes simultaneous transformation leading to a co-expression of 13 genes. In addition, when fatty acids are prepared by culturing the recombinant E. coli at a low temperature, the ratio and quantity of vaccenic acid, among unsaturated fatty acids, are greatly increased, so the unsaturated fatty acids 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. Acid-CoA is a precursor of fatty acids , and accA, accB, accC, and accD (acetyl-CoA carboxylase) play a role in reacting acetyl-CoA with malonyl-CoA. This reaction occurs when accA, accB, accC, accD are gathered together, resulting in slower fatty acid synthesis and rate-limiting. Malonyl -CoA is converted to maldonyl -ACP via fabD and used for the elongation step where the carbon number is increased. In particular, the fabB, fabF, and fabA genes are involved in the elongation stage and contain information on the genes fabA and KASI (β-ketoacyl-ACP (acyl-carrier-protein) synthaseI) that contain information on β-Hydroxydecanoyl ACP dehydrase / isomerase The gene fabB is an essential gene for the synthesis of unsaturated fatty acids. fabF gene that has information of the KASII (β-ketoacyl-ACP synthaseII ) with fabB is increased two by two the number of carbon atoms of the fatty acid by placing the fatty acid elongation steps, a carbonyl bond -ACP words, the fatty acid -ACP 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 pepsic acid, which are the major constituents of E. coli cell membrane. Especially, KASII is an enzyme that plays a key role in the production of cis- And the expression is good at low temperature.

In addition, there are the genes fabG, fabH, fabI, and fabZ which have information on enzymes (KAS, KASIII, enoyl-acyl carrier protein reductase, 3-hydroxy acyl-acyl carrier protein dehydratase)

In addition, at the end of fatty acid synthesis, tesA gene makes thioesterase I enzyme and converts fatty acyl-ACP to free fatty acid. However, in the case of tesA , it is present in the periplasm of E. coli and there is a limit to the rate of conversion of acyl-ACP to free fatty acid.

In addition, there is a gene fadD having a genetic information of an enzyme (long-chain acyl-Coenzyme A synthetase) involved in fatty acid reduction by converting a fatty acid into long chain acyl-ACP in a fatty acid synthesis beta-oxidation step .

Table 3 shows the enzymes involved in the fatty acid biosynthesis of Escherichia coli and gene information.

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.

In addition, the present invention uses the vector disclosed in Korean Patent No. 10-1390327 (Apr. 23, 2014) and Korean Patent Publication No. 10-2014-0010475 (Jan. 27, 2014).

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) Korean Patent No. 10-1390327 (Apr. 23, 2014) Korean Patent Publication No. 10-2014-0010475 (Jan. 27, 2014)

The present inventors have made efforts to develop a strain capable of increasing the composition of unsaturated fatty acids. As a result, accA, accB, accC, accD , malonyl-CoA having the information of an enzyme (ACC, Acetyl-CoA carboxylase) which is located at the beginning of fatty acid synthesis and reacted with malonyl- FabD with information of enzyme (FabD) synthesized by ACP and two genes fabB , fabF and KASI with information of enzymes (KASI, KASII) acting at the last stage of carbon chain elongation and fatty acid production in fatty acid elongation step The enzymes involved in the production of unsaturated fatty acids (β-Hydroxydecanoyl ACP dehydrase / isomerase), fabA , and other enzymes involved in fatty acid synthesis (KAS, KASIII, enoyl-acyl carrier protein reductase, 3-hydroxy acyl gene with the information of -acyl carrier protein dehydratase) fabG, fabH , fabI, fabZ thio S. atmospheres in the fatty acid synthesis terminating stage having a first I (thioesteraseI) information to remove the leader sequence and not the periplasmic (periplasm) Gene was present on the modified pojil prepare a recombinant plasmid containing the nucleotide sequence of `tesA. In addition, a host E. coli having the gene fadD having the information of the enzyme (FadD) involved in fatty acid reduction in the fatty acid synthesis beta-oxidation step is prepared, and the recombinant plasmid is introduced into the host E. coli, To prepare recombinant E. coli. The recombinant Escherichia coli was cultured at a low temperature and fatty acid extraction was performed to confirm that the ratio of cis- valencenic acid in unsaturated fatty acids was greatly increased, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a recombinant Escherichia coli which produces cis- vulcanic acid in unsaturated fatty acids

Another object of the present invention is to provide a process for producing an unsaturated fatty acid.

Hereinafter, the present invention will be described in detail.

According to one aspect of the present invention, the present invention provides a nucleotide ( accA , SEQ ID NO: 1) carrying the genetic information of a carboxyl-transferase alpha-unit (CT-alpha) of E. coli K-12 MG1655; A nucleotide ( accB , SEQ ID NO: 2) carrying the genetic information of the biotin carboxyl carrier protein (BCCP) of E. coli K-12 MG1655; A nucleotide ( accC , SEQ ID NO: 3) carrying the genetic information of biotin carboxylase of E. coli K-12 MG1655; A nucleotide ( accD , SEQ ID NO: 4) carrying the genetic information of a carboxyl-transferase beta-unit (CT-beta) of E. coli K-12 MG1655; Of E. coli K-12 MG1655 Nucleotides ( fabD , SEQ ID NO: 5) carrying the genetic information of malonyl-coenzyme A: malonyl-Coenzyme A (acyl carrier protein transacylase); Nucleotides ( fabF , SEQ ID NO: 6) carrying the genetic information of beta-ketoacyl-acyl carrier protein synthase II (KASII, β-keto acyl-acyl carrier protein synthase II) of E. coli K-12 MG1655; Nucleotides ( fabB , SEQ ID NO: 7) 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; Nucleotides ( fabA , SEQ ID NO: 8) carrying the genetic information of β-hydroxy decanoyl thioester dehydrase / isomerase of β-hydroxydecanoyl thioesterase / isomerase of E. coli K-12 MG1655; Nucleotides ( fabG , SEQ ID NO: 9) carrying the genetic information of the beta-keto acyl-acyl carrier protein synthase (KAS) of E. coli K-12 MG1655; Nucleotides ( fabH , SEQ ID NO: 10) carrying the genetic information of beta-ketoacyl-acyl transferase protein synthetase III (KASIII, β-keto acyl-acyl carrier protein synthase III) of E. coli K-12 MG1655; A nucleotide having a genetic information of an enoyl-acyl carrier protein reductase of E. coli K-12 MG1655 ( fabI , SEQ ID NO: 11); Nucleotides ( fabZ , SEQ ID NO: 12) carrying the genetic information of 3-hydroxyacyl-acyl carrier protein dehydratase of E. coli K-12 MG1655; And a nucleotide ( 'tesA, SEQ ID NO: 13) with genetic information modified to exist in the cytoplasm rather than the periplasm by removing the leader sequence from the thioesterase I of E. coli K-12 MG1655; , Which is a transformant obtained by transforming a host E. coli with a recombinant plasmid comprising at least one nucleotide selected from the group consisting of:

In a preferred embodiment of the present invention, the host E. coli is a nucleotide ( fadD ) having genetic information of a long-chain acyl-Coenzyme A synthetase of E. coli BL21 (DE3) No. 14) is deleted. E. coli BL21 (DE3)? FadD .

In a preferred embodiment, the recombinant plasmid is ^{pCOLADuet TM -1 :: accA, accB,} accC, accD, fabD + pACYCDuet TM -1 :: fabF, fabB, fabA + pTrc99A :: fabG, fabH, fabI, fabZ + pCDF-1b :: ` tesA .

In a preferred embodiment of the present invention, the recombinant E. coli is E. coli BL21 (DE3) ΔfadD :: pCOLADuet ^™ -1 :: accA, accB, accC, accD, fabD + pACYCDuet ^™ -1 :: fabF, fabA + pTrc99A :: fabG , fabH , fabI , fabZ + pCDF-1b :: ` tesA (accession number: KCTC18325P).

The recombinant Escherichia coli producing the fatty acid according to the present invention comprises a nucleotide ( fadD , SEQ ID NO: 14) having genetic information of a long-chain acyl-Coenzyme A synthetase of Escherichia coli BL21 (DE3) was being removed (BL21 (DE3) △ fadD) , E. coli K-12 of the MG1655 carboxyl transmitter buffer raise the alpha-unit (CT-α, carboxyltransferase α-unit) nucleotides have a genetic information of (accA, SEQ ID NO: 1), ( AccB , SEQ ID NO: 2) carrying the genetic information of biotin carboxyl carrier protein (BCCP) of E. coli K-12 MG1655, genetic information of biotin carboxylase (BC) of E. coli K-12 MG1655 have the nucleotide (accC, SEQ ID NO: 3), E. coli K-12 MG1655 of carboxyl transferase raised beta-unit (CT-β, β-carboxyltransferase unit) nucleotides have a genetic information of (accD, SEQ ID NO: 4), for Jang-Kyun K-12 of MG1655 Nucleotides ( fabD , SEQ ID NO: 5) carrying the genomic information of Malonyl-Coenzyme A: acyl carrier protein transacylase, beta- ketoacyl -acyl transferase of E. coli K-12 MG1655 Nucleotides ( fabF , SEQ ID NO: 6) carrying the genetic information of the protein synthetase II (KASII, β-keto acyl-acyl carrier protein synthaseII), beta- ketoacyl- acyl transfer protein synthetase I (KASI ( FabB , SEQ ID NO: 7) carrying the genetic information of β-keto acyl-acyl carrier protein synthase I, β- hydroxydecanoyl thioester hydrolase / isomerase (β- (KAS, β-keto acyl-acyl carrier protein (KAS)) of E. coli K-12 MG1655, a nucleotide ( FabA , SEQ ID NO: ( FabG , SEQ ID NO: 9), which contains the genetic information of the β-ketoacyl-acyl carrier protein synthase III (KASIII), and the beta-ketoacyl-acyl carrier protein synthase III of E. coli K-12 MG1655 ( FabH , SEQ ID NO: 10) carrying the genetic information of the enoyl-acyl carrier protein reductase of E. coli K-12 MG1655 ( fabI , SEQ ID NO: 11). Nucleotides ( fabZ , SEQ ID NO: 12) carrying the genetic information of 3-hydroxyacyl-acyl carrier protein dehydratase of E. coli K-12 MG1655, thioses of E. coli K-12 MG1655 (&Quot; tesA, SEQ ID NO: 13) " having genetic information modified to exist in the cytoplasm, not in the periplasm, by removing the leader sequence from the thioesterase I Lt; RTI ID = 0.0 > plasmid. ≪ / RTI >

The gene information used in the present invention is based on the genetic information of E. coli K-12 MG1655, and is located at the initiation stage of fatty acid synthesis, and genetic information of an enzyme (ACC) which reacts acetyl- CoA with malonyl- with accA, accB, accC, fabD the accD and malonyl -CoA with the genetic information of the enzyme (FadD) making a carbonyl -ACP sseuyige words that the stretching step, In addition to the two genes fabB , fabF and KASI, which have information on enzymes (KASI, KASII) acting at the last stage of carbon chain elongation and fatty acid elongation at the fatty acid elongation stage, enzymes involved in the unsaturated fatty acid production step (β-Hydroxydecanoyl ACP dehydrase / isomerase) gene with the genetic information of fabA, that in addition to a gene having information of an enzyme (KAS, KASIII, enoyl-acyl carrier protein reductase, 3-hydroxy acyl-acyl carrier protein dehydratase) which is involved in fatty acid synthesis elongation fabG, fabH, fabI, fabZ and fatty acyl -ACP (fatty acyl-ACP) to free fatty acid (free fatty acid) to a conversion enzyme, thio esterase I (thioesteraseI) information with a gene having a leader sequence that is removed information ' The base sequence of tesA is used.

Since tesA is present in the periplasm of E. coli, there is a limit to the rate at which acyl-ACP converts to free fatty acid. Thus, in the present invention, it is possible to increase the conversion speed by eliminating the lead sequence of tesA and making it exist in the cytoplasm.

The gene information used in the present invention is based on the genetic information of Escherichia coli BL21 (DE3), which converts fatty acids to long-chain acyl-ACP in the fatty acid synthesis beta-oxidation step, It is possible to remove the fadD gene having the genetic information of the enzyme involved in the reduction (long-chain acyl-Coenzyme A synthetase) from the host E. coli to alleviate the decrease of the fatty acid.

Specifically, E. coli K-12 MG1655 is cultured to obtain a template gene of E. coli by polymerase chain reaction (PCR), and the DNA is isolated by centrifugation.

The polymerase chain reaction was performed using primers of respective genes ( accA, accB, accC, accD, fabD, fabB , fabF , fabA , fabG, fabH , fabI, fabZ , and tesA ) Securing the gene. In particular, accB and accC are genes that are consecutively present on E. coli K-12 MG1655, resulting in nucleotides at the same time and retaining the acquired nucleotides ( accA, accB, accC, accD, fabD, fabB , fabF , fabA , fabG, fabH , fabI , fabZ, tesA) and performs a plasmid ^{(pCOLADuet TM -1, pACYCDuet TM -1} , pTrc99A, pCDF-1b) of a restriction enzyme reaction and ligation (ligation) reaction for protein expression to produce a recombinant plasmid [pCOLADuet ^{TM pCOLADuet TM -1 :: accA, accB,} accC, accD, fabD, pACYCDuet TM -1 :: fabF, fabB, fabA, pTrc99A :: fabG, fabH, fabI, fabZ, pCDF-1b :: tesA].

In order to remove the leader sequence, a primer was constructed in which the 75th nucleotide sequence was changed from C to T, and a recombinant plasmid was prepared by amplifying the PCR product using a site-directed mutagenesis method [pCDF-1b :: ` tesA ].

For the replication fork of a plasmid for protein expression, pCOLADuet ^TM -1 is ColA, pACYCDuet ^TM -1 is pACYC, pTrc99A are pBR322, pCDF-1b comprises the CDF, and the kanamycin resistance gene For the pCOLADuet ^TM -1, pACYCDuet ^TM - 1, the ampicillin-resistant gene for pTrc99A, the pCF-1b gene for streptomycin resistance, the T7 promoter and the trc promoter for base sequence analysis, and the lac operator Lt; / RTI >

E. coli XL-1 blue :: pCOLADuet ^TM -1 :: accA, accB, accC, accD, E. coli XL-1 blue :: pCOLADuet ^TM -1 :: accA, accB, accC, accD were prepared by heat shock transformation using the recombinant plasmid :: pACYCDuet ^TM -1 fabF , fabB , fabA, E. coli XL-1 blue :: pTrc99A :: fabG , fabH, fabI , fabZ, E. coli XL-1 blue :: pCDF-1b :: ` tesA ]. The recombinant E. coli thus produced was found to grow in the form of a circular colony in a solid LB medium containing an antibiotic. The colonies were cultured in a liquid LB medium containing kanamycin, chloramphenicol, ampicillin, and streptomycin, The gene sequences and errors of the plasmid genes ( accA, accB, accC, accD, fabD, fabB , fabF , fabA , fabG, fabH , fabI, fabZ and tesA ) and E. coli K-12 MG1655 Respectively.

The prepared recombinant Escherichia coli (E. coli BL21 (DE3) △ fadD :: pCOLADuet TM -1 :: accA, accB, accC, accD, fabD + pACYCDuet TM -1 :: fabF, fabB, fabA + pTrc99A :: fabG, fabH, fabI , fabZ + pCF -1b :: ` tesA ) was donated to KCTC (Korea Research Institute of Bioscience and Biotechnology) on September 17, 2014, and received the accession number of KCTC18325P.

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);

(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.

In a preferred embodiment of the present invention, the secondary culture of step (b) is cultured at a low temperature of 10 캜 to 20 캜, and most preferably at a low temperature of 15 캜 to 18 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, the unsaturated fatty acid is cis-a foil sensan (cis -vaccenic acid).

For example, the prepared recombinant E. coli BL21 (DE3) :: pCOLADuet ^™ -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 Escherichia coli producing the fatty acid according to the present invention comprises (1) one recombinant plasmid in which the five kinds of genes accA, accB, accC, accD and fabD in the starting stage of fatty acid synthesis are simultaneously expressed; (2) a recombinant plasmid in which three kinds of genes fabB , fabF and fabA in the fatty acid synthesis elongation stage are expressed simultaneously; (3) a recombinant plasmid in which four kinds of genes fabG, fabH , fabI and fabZ in the synthetic elongation stage of fatty acid are simultaneously expressed; And (4) a recombinant plasmid expressing `tesA 'in the termination step of fatty acid synthesis was constructed at the same time, and recombinant E. coli, which is a host strain in which fatty acid reduction is alleviated by eliminating fadD in the fatty acid synthesis beta- oxidation step ( E. The four recombinant plasmids are introduced into E. coli BL21 (DE3) [Delta] fadD and co-transformed to thereby express the three genes at the same time. In addition, when a fatty acid was prepared using the above recombinant E. coli, the ratio of cis- vicenic acid, one of the unsaturated fatty acids, was higher than that of Escherichia coli that did not undergo genetic manipulation, And the unsaturated fatty acid can be effectively produced.

1 is a view showing a process of fatty acid biosynthesis of E. coli.
Fig. 2 shows the structure and cloning site of the plasmid pCOLADuet ^TM- 1 for expression of the protein in E. coli.
3 shows the structure and cloning site of the plasmid pACYCDuet ^TM- 1 for expression of the protein in E. coli.
4 is a diagram showing the structure and cloning site of the plasmid pTrc99A for expression of the protein in E. coli.
5 is a diagram showing the structure and cloning site of plasmid pCDF-1b for protein expression in E. coli.
FIG. 6 is a diagram showing the structure and size of a plasmid pCOLADuet ^TM -1 for expression of the protein in E. coli , and a recombinant plasmid in which nucleotides including accA, accB, accC, accD, and fabD are combined.
7 is a diagram showing the structure and size change of a recombinant plasmid in which a nucleotide including the gene information of the plasmid pACYCDuet ^TM- 1 and the fabB, fabF, and fabA for the protein expression in E. coli is combined.
FIG. 8 is a diagram showing the structure and size change of a recombinant plasmid in which a nucleotide including the gene information of the plasmid pTrc99A and the fabG, fabH, fabI and fabZ for the expression of the protein in E. coli is combined.
9 is a graph showing the structure and size change of a plasmid pCDF-1b for protein expression in plasmid E. coli for expression of a protein in E. coli and a recombinant plasmid (pCDF-1b :: ` tesA ) in which nucleotides including tesA gene information are combined to be.
10 is a diagram showing a structure and size change of a plasmid pEcoli-Nterm 6xHN for use as a template for the genetic information of fabB, fabF and fabA , and a recombinant plasmid in which nucleotides including gene information of genes fabF, fabB and fabA are combined .
11 is a diagram showing the upstream sequence and the downstream sequence of the fadD gene in E. coli :: BL21 (DE3).
12 is a diagram showing a process for producing a recombinant plasmid (pKOV :: fadD upstream, fadD downstream; fadD fusion-pKOV) used for the removal of fadD gene in the host E. coli :: BL21 (DE3).
13 is shown whether or not the fadD gene from E. coli E. coli :: BL21 (DE3) fadD gene removed after the final selection process, there are two types of E. coli BL21 (DE3) genome (genomic DNA), which in the course of the .
14 is a diagram showing the nucleotide sequence analysis result of the accA gene bound to the plasmid pCOLADuet ^TM -1 for expression of the protein in E. coli.
15 is a diagram showing the nucleotide sequence analysis results of the accB and accC genes bound to the plasmid pCOLADuet ^TM -1 for expression of the protein in E. coli.
16 is a diagram showing the nucleotide sequence analysis result of the accD gene bound to the plasmid pCOLADuet ^TM -1 for expression of the protein in E. coli.
17 is a diagram showing the nucleotide sequence analysis result of the fab D gene bound to the plasmid pCOLADuet ^TM -1 for expression of the protein in E. coli.
18 is a diagram showing the nucleotide sequence analysis results of the fabF, fabB and fabA genes bound to the plasmid pACYCDuet ^TM -1 for expression of the protein in E. coli.
19 is a diagram showing the result of base sequence analysis of the fabG gene bound to the plasmid pTrc99A for expression of the protein in E. coli.
20 is a diagram showing the nucleotide sequence analysis result of the fabH gene bound to the plasmid pTrc99A for expression of the protein in E. coli.
FIG. 21 is a diagram showing a nucleotide sequence analysis result of a fabI gene bound to a plasmid pTrc99A for expression of a protein in E. coli.
22 is a diagram showing the nucleotide sequence analysis result of the fabZ gene bound to the plasmid pTrc99A for expression of the protein in E. coli.
23 is a diagram showing a nucleotide sequence analysis result of the 'tesA gene bound to the plasmid pCDF-1b 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 is located at the beginning of fatty acid synthesis, and information of an enzyme (ACC) that reacts acetyl- CoA with malonyl- accA (SEQ ID NO: 1), accB (SEQ ID NO: 2), accC (SEQ ID NO: 3), accD (SEQ ID NO: 4), making the words enzyme carbonyl -CoA as carbonyl -ACP sseuyige words that the stretching step (FadD) with of fabD with genetic information (SEQ ID NO: 5), and a fatty acid of two genes in the stretching step having information of an enzyme (KASI, KASII) acting on the kidney and the fatty acid generated final step in the carbon chain, fabF (SEQ ID NO: 6), fabB (SEQ ID NO: 7), a gene FabA having the genetic information of the enzyme (β-Hydroxydecanoyl ACP dehydrase / isomerase) participating in the unsaturated fatty acid production step together with KASI, and enzymes involved in the fatty acid synthesis elongation step KAS, KASIII, enoyl-acyl carrier protein reductase, 3-hydroxy gene with the information of the acyl-acyl carrier protein dehydratase) fabG ( SEQ ID NO: 9), fabH (SEQ ID NO: 10), fabI (SEQ ID NO: 11), fabZ (SEQ ID NO: 12) And the gene `tesA (SEQ ID NO: 13) which has information of thioesterase I, which is an enzyme that converts fatty acyl-ACP to free fatty acid, ) 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 accA carboxyltransferase α subunit pCOLADuet ^TM -1 5'-CGG ATC CGA TGA ATT TCC TTG ATT TTG AAC AGC-3 ' BamHI, SalI 5'-GCG TCG ACG TCT TAC GCG TAA CCG TAG-3 ' accB, accC BCCP unit, biotin carboxylase subunit pCOLADuet ^TM -1 5'-GAA GAT CTT CGG ATG GAT ATT CGT AAG ATT AAA AAA CTG ATC G-3 ' BglII, PacI 5'-CCT TAA TTA AGG TTA TTT TTC CTG AAG ACC GAG TTT CTC-3 ' accD carboxyltransferase β unit pCOLADuet ^TM -1 5'-GCG TCG ACG TCA AGG AGA TAT ACA TGA GCT GGA TTG AAC-3 ' SalI, NotI 5'-ATA AGA ATG CGG CCG CTC AGG CCT CAG GTT C-3 ' fabD malonyl-CoA: ACP transacylase pCOLADuet ^TM -1 5'-GGA ATT CCA TAT GGA ATT CCA TGA CGC AAT TTG CAT T -3 ' NdeI 5'-GAA GAT CTT CCT CCT TTT AAA GCT CGA GCG CC-3 ' BglII 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 pACYCDuet ^TM -1 5'-AAA AGG ATC CGG TGT CTA AGC GTC GTG TAG-3 ' BamHI, PstI 5'-AAA ACT GCA GTC AGA AGG CAG ACG TAT CCT G-3 ' fabG KAS
(β-keto acyl-acyl carrier protein synthase) pTrc99A 5'-GAA TTC GGA AAA TCA TGA ATT TTG AAG GAA -3 ' EcoRI, SacI 5'-GAG CTC TTA DGD GTA ACC GTA GCT CAT C -3 ' fabH KASIII
(β-keto acyl-acyl carrier protein synthaseIII) pTrc99A 5'-GGA TCC CCG AAA AGT GAC TGA GCG TACE -3 ' BamHI, XbaI 5'-TCT AGA CTA GAA ACG AAC CAG CGC GGA G -3 ' fabi enoyl-acyl carrier protein reductase pTrc99A 5'-TCT AGA AAG GAT TAA AGC TAT GGG TTT TCT -3 ' XbaI, SalI 5'-TCT AGA TTA TTT CAG TTC GAG TTC GTT CAT -3 ' fabZ 3-hydroxy acyl-acyl carrier protein dehydratase pTrc99A 5'-GAG CTC GGA AGA GTA TCT TGA CTA CTA AC -3 ' SacI, BamHI 5'-GGA TCC TCA GGC CTC CCG GCT ACG AGC -3 ' tesA thioesterase A pCDF-1b 5'-CGG GAT CCC ATG ATG AAC TTC AAC AAT GTT TTC CG-3 ' BamHI, PstI 5'-GGA AGA TCT GAA GGA GAT ATA CCA TGA AAC GTG CAG TGA TTA CTG GCC-3 ' `tesA modified thioesterase A pCDF-1b 5'-GTT AAC CTT CCG TGC CGC TGC AGC GGA CAC GTT ATT G-3 ' 5'-CAA TAA CGT GTC CGC TGC AGC GGC ACG GAA GGT TAA C-3 '

The template gene of E. coli for the polymerase chain reaction 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 thereto and mixed until no layer separation occurred. Then, the supernatant was collected and centrifuged in the same manner. 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 used for polymerase chain reaction with the primers of the respective genes ( accA, accB, accC, accD, fabD, fabF, fabB, fabA, fabG, fabH, The gene was amplified. 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 purified and identified on 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 캜 and subjected to purification and ligation at 4 캜 to obtain accA and accD which were bound to MCS1 of linear pCOLADuet ^TM -1 A circular plasmid (pACYCduet ^TM- 1 :: accA, accB, accC, accD, fabD ) in which accB , accC and fabD were combined with MCS2 and pACYCDuet ^TM -1 with fabF, ^{TM -1 :: fabF, fabB, fabA} ), pTrc99A the fabG, fabH, fabI, in which circular plasmid combining fabZ (pTrc99A :: fabG, fabH, fabI, fabZ), 1 that combines a linear tesA the pCDF-1b Circular plasmid (pCDF-1b :: tesA ) was prepared. In the case of tesA , a sequence-altered primer was prepared to change the 75th base of the sequence C to T, and a plasmid containing the mutated 'tesA' was prepared by PCR using the recombinant plasmid (pCDF-1b :: tesA ) . In this process, Stratagene product pfu Ultra HF DNA polymerase was used and pCDF-1b :: tesA was removed with DpnI to select site-directed mutated pCDF-1b :: `tesA .

The structures and cloning sites of the plasmids pCOLADuet ^TM- 1, pACYCDuet ^TM- 1, pCDF-1b and pTrc99A for expression of the protein in E. coli were shown in FIGS. 2, 3, 4 and 5, respectively. Plasmid pCOLADuet ^TM -1 The structural and size changes of the recombinant plasmids in which the nucleotides including the gene information of accA, accB, accC, accD and fabD are combined are shown in FIG. 6, and the nucleotides including the gene information of pACYCDuet ^TM -1 and fabB, fabF and fabA The structure and size of the recombinant plasmid were shown in FIG. 7, and the structure and size of the recombinant plasmid in which nucleotides including pTrc99A and fabG, fabH, fabI, and fabZ were incorporated are shown in FIG. 8, and the plasmid pCDF- structure, and size change of 1b and `tesA recombinant plasmid combining a nucleotide, including genetic information (the 75th base is tes a gene replaced by T in C) It is shown in Fig 9. And fabB, are shown in the structure and size variation is 10 of fabF, the use as a template for the genetic information of fabA plasmid pEcoli-Nterm 6xHN gene fabF, fabB, recombinant plasmid combining a nucleotide including a gene information of fabA.

Example 2: Genetic deletion of host E. coli used for the production of recombinant E. coli

1. Gene removal using pKOV vector

In the present invention, Escherichia coli ( E. coli BL21 (DE3)) for protein expression was used as E. coli used for fatty acid production. (Long-chain acyl-Coenzyme A synthetase), which converts fatty acids to long-chain acyl-ACP at the fatty acid synthesis beta-oxidation stage to alleviate fatty acid degradation The gene fadD (SEQ ID NO: 14) was removed from the host E. coli. In order to delete fadD , pKOV , a gene replacement vector, was used in the present invention. The method is shown below.

The pKOV vector contains the chloramphenicol antibiotic resistance gene and has a temperature-sensitive pSC101 origin of replication. Thus, the host can grow with the vector stably at 30 ° C, and the vector is lost at 42 ° C. And the pKOV vector has a sacB gene, which does not grow on a medium containing sucrose. Using this property, gene removal can be performed. First, the gene sequence of upstream and downstream of the gene to be deleted is cloned into MCS. The nucleotide sequence information was referenced from KEGG (Kyoto Encyclopedia of Genes and Genomes, www.genome.jp/kegg/ ). The resulting recombinant vector (pKOV :: fadD upstream, fadD downstream, fadD fusion-pKOV) is transformed into E. coli BL21 (DE3) and the temperature is changed from 30 to 42 ° C while culturing in an antibiotic medium. At this time, the recombinant vector is integrated with the genome of fadD of E. coli BL21 (DE3). When the temperature is changed to 30 ° C in a medium without antibiotics, the recombinant vector is cytoplasm again. At this time, culturing in a medium with sucrose at 42 ° C finally results in genetic exchange and loss of the recombinant vector, resulting in the removal of the gene and the removal of the gene. E. coli BL21 (DE3) [Delta] fadD ) in which the fadD gene was deleted was prepared by confirming the presence of the gene through PCR.

The sizes of the primers and the upstream and downstream sequences used in the fadD removal using the recombinant vector (pKOV :: fadD upstream, fadD downstream) are shown in Table 5. The upstream sequence and the downstream sequence of the fadD gene in E. coli :: BL21 (DE3) are shown in Fig. In addition, the production process of fadD fusion-pKOV is shown in Fig. 13 as to whether the fadD gene is present in two types of E. coli BL21 (DE3) genomes (genomic DNA) existing after the final screening process.

- primer Upstream
(1217bp) F 5'-GTCCGGCTTCGGAAGATTT-3 ' R 5'-CGATATCTGTGTTAAGTCAGTCGTCAGAC-3 ' Downstream
(1523 bp) F 5'-CGGATATCTTCTTCACCTCTAAAATGCGTG-3 ' R 5'-CGGAACTTCCCTGACTGATATT-3 '

Example 3: Preparation of recombinant Escherichia coli transformed with a recombinant plasmid

1. Confirmation of synthesis error of recombinant plasmid

Each of the plasmids for the purpose of carrying the gene of interest ^{(pCOLADuet TM -1 :: accA, accB} , accC, accD, fabD, pACYCDuet TM -1 :: fabF, fabB, fabA, pTrc99A :: fabG, fabH, fabI, fabZ , pCDF-1b :: loading a `tesA) into the host cell before, E. coli transformants for improved efficiency when switching to a recombinant plasmid and a synthetic error check of vector stability and host strain of E. coli XL1-Blue (E. coli XL1 -B ) were used to prepare competent cells for primary transformation. (E. coli XL-1 blue :: pCOLADuet TM -1 :: accA, accB, accC, accD, E. coli XL-1 blue :: pACYCDuet TM -1 fabF, fabB, fabA, E. coli XL-1 blue :: pTrc99A :: fabG , fabH , fabI , fabZ, E. coli XL-1 blue :: pCDF-lb :: ` tesA )

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. Wherein each recombinant plasmid containing the gene for the outer ^{(pCOLADuet TM -1 :: accA, accB} , accC, accD, fabD, pACYCDuet TM -1 :: fabF, fabB, fabA, pTrc99A :: fabG, fabH, fabI, fabZ, pCDF -1b :: ` tesA ), reacted at 0 ° C for 30 minutes, heat-treated in a hot water bath at 42 ° C for 30 seconds, and placed in ice at 0 ° C for 5 minutes sequentially (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 solid LB medium (LB agar plate, 10 g / L of tryptone, 5 g / L of NaCl, 5 g of yeast extract) containing antibiotics (kanamycin, chlorampinin, ampicillin, streptomycin) g / L, and agar 15 g / L).

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 Figs. 14, 15, 16, 17, 18, 19, 20, 21, 22 and 23.

During this recombinant plasmid pACYCDuet ^TM -1 :: 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, but it was confirmed that the amino acid sequence when the protein was translated into amino acid and the protein having the steric structure as the enzyme function coincide with each other.

2. Preparation of recombinant E. coli E. coli BL21 (DE3) fadD :: pCOLADuet ^TM -One:: accA, accB, accC, accD, fabD + pACYCDuet ^TM -One:: fabF, fabB, fabA + pTrc99A :: fabG , fabH , fabi , fabZ  + pCDF-1b :: ` tesA )

The recombinant plasmid check the synthesis error in the ^{1 (pCOLADuet TM -1 :: accA,} accB, accC, accD, fabD, pACYCDuet TM -1 :: fabF, fabB, fabA, pTrc99A :: fabG, fabH, fabI, fabZ , pCDF-1b :: ` tesA ), the host E. coli strain ( E. coli BL21 (DE3) ΔfadD ) prepared in Example 2 was treated in the same manner as in the above-mentioned method for producing a competent cell 1 and recombinant plasmids Was selected to produce a recombinant strain, and the above-mentioned method 1 was sequentially and repeatedly applied to the other recombinant plasmids with the recombinant strain to obtain a final recombinant E. coli BL21 (DE3 <sup>) △ fadD :: pCOLADuet TM -1 ::</sup> accA, accB, accC, accD, fabD + pACYCDuet TM -1 :: fabF, fabB, fabA + pTrc99A :: fabG, fabH, fabI, fabZ + pCDF-1b :: ` tesA ).

Thus, the recombinant E. coli BL21 (DE3) ΔfadD :: pCOLADuet ^™ -1 :: accA, accB, accC, accD, fabD + pACYCDuet ^™ -1 :: fabF, fabB, fabA + pTrc99A :: fabG , fabH , fabI , fabZ + pCF -1b :: ` tesA ) was donated to KCTC (Korea Research Institute of Bioscience and Biotechnology) on September 17, 2014, and received the accession number of KCTC18325P.

The prepared recombinant E. coli, the target gene and the recombinant plasmid are shown in Table 6.

Recombinant E. coli Target gene The recombinant plasmid E. coli BL21 (DE3) △ fadD :: pCOLADuet TM -1 :: accA, accB, accC, accD, fabD + pACYCDuet TM -1 :: fabF, fabB, fabA + pTrc99A :: fabG, fabH, fabI, fabZ + pCDF-1b :: `tesA accA, accB, accC, accD, fabD, fabF, fabB, fabA, fabG, fabH, fabI, fabZ, ` tesA pCOLADuet ^TM -1 :: accA, accB, accC, accD, fabD + pACYCDuet ^TM -1 :: fabF, fabB, fabA + pTrc99A :: fabG, fabH, fabI, fabZ + pCDF-1b :: `tesA

Example 4 Production of Fatty Acids Using Recombinant Escherichia coli

The recombinant E. coli BL21 (DE3) ΔfadD :: pCOLADuet ^™ -1 :: accA, accB, accC, accD, fabD + pACYCDuet ^™ -1 :: fabF, fabB, fabA + pTrc99A :: fabG , fabH , fabI , fabZ + pCDF-1b :: ` tesA ) was added to 200 ml of liquid LB medium containing antibiotics kanamycin, chloramphenicol, ampicillin, and streptomycin 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 7, when the unsaturated fatty acid composition of the recombinant E. coli of the present invention was analyzed, 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 No. 10-1402108 disclosed in E. coli BL21 (DE3) :: pCOLADuet -1 :: accA, accB, accC, accD + pEcoliNterm 6xHN :: fabF, fabB, fabA + pCDF-1b :: 'tesA, accession No. KCTC 12216BP). In particular, it was confirmed that the ratio and amount of cis-vaccenic acid was remarkably improved.

Korea Biotechnology Research Institute KCTC18325P 20140917

<110> INHA-INDUSTRY PARTNERSHIP INSTITUTE <120> Recombinant E. coli producing unsaturated fatty acid, and method          for producing unsaturated fatty acid using the same <130> INHA1.145P <160> 42 <170> Kopatentin 2.0 <210> 1 <211> 960 <212> DNA <213> Escherichia coli <400> 1 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> 2 <211> 1831 <212> DNA <213> Escherichia coli <400> 2 atggatattc gtaagattaa aaaactgatc gagctggttg aagaatcagg catctccgaa 60 ctggaaattt ctgaaggcga agagtcagta cgcattagcc gtgcagctcc tgccgcaagt 120 ttccctgtga tgcaacaagc ttacgctgca ccaatgatgc agcagccagc tcaatctaac 180 gcagccgctc cggcgaccgt tccttccatg gaagcgccag cagcagcgga aatcagtggt 240 cacatcgtac gttccccgat ggttggtact ttctaccgca ccccaagccc ggacgcaaaa 300 gcgttcatcg aagtgggtca gaaagtcaac gtgggcgata ccctgtgcat cgttgaagcc 360 atgaaaatga tgaaccagat cgaagcggac aaatccggta ccgtgaaagc aattctggtc 420 gaaagtggac aaccggtaga atttgacgag ccgctggtcg tcatcgagta acgaggcgaa 480 catgctggat aaaattgtta ttgccaaccg cggcgagatt gcattgcgta ttcttcgtgc 540 ctgtaaagaa ctgggcatca agactgtcgc tgtgcactcc agcgcggatc gcgatctaaa 600 acacgtatta ctggcagatg aaacggtctg tattggccct gctccgtcag taaaaagtta 660 tctgaacatc ccggcaatca tcagcgccgc tgaaatcacc ggcgcagtag caatccatcc 720 gggttacggc ttcctctccg agaacgccaa ctttgccgag caggttgaac gctccggctt 780 tatcttcatt ggcccgaaag cagaaaccat tcgcctgatg ggcgacaaag tatccgcaat 840 cgcggcgatg aaaaaagcgg gcgtcccttg cgtaccgggt tctgacggcc cgctgggcga 900 cgatatggat aaaaaccgtg ccattgctaa acgcattggt tatccggtga ttatcaaagc 960 ctccggcggc ggcggcggtc gcggtatgcg cgtagtgcgc ggcgacgctg aactggcaca 1020 atccatctcc atgacccgtg cggaagcgaa agctgctttc agcaacgata tggtttacat 1080 ggagaaatac ctggaaaatc ctcgccacgt cgagattcag gtactggctg acggtcaggg 1140 caacgctatc tatctggcgg aacgtgactg ctccatgcaa cgccgccacc agaaagtggt 1200 cgaagaagcg ccagcaccgg gcattacccc ggaactgcgt cgctacatcg gcgaacgttg 1260 cgctaaagcg tgtgttgata tcggctatcg cggtgcaggt actttcgagt tcctgttcga 1320 aaacggcgag ttctatttca tcgaaatgaa cacccgtatt caggtagaac acccggttac 1380 agaaatgatc accggcgttg acctgatcaa agaacagctg cgtatcgctg ccggtcaacc 1440 gctgtcgatc aagcaagaag aagttcacgt tcgcggccat gcggtggaat gtcgtatcaa 1500 cgccgaagat ccgaacacct tcctgccaag tccgggcaaa atcacccgtt tccacgcacc 1560 tggcggtttt ggcgtacgtt gggagtctca tatctacgcg ggctacaccg taccgccgta 1620 ctatgactca atgatcggta agctgatttg ctacggtgaa aaccgtgacg tggcgattgc 1680 ccgcatgaag aatgcgctgc aggagctgat catcgacggt atcaaaacca acgttgatct 1740 gcagatccgc atcatgaatg acgagaactt ccagcatggt ggcactaaca tccactatct 1800 ggagaaaaaa ctcggtcttc aggaaaaata a 1831 <210> 3 <211> 1831 <212> DNA <213> Escherichia coli <400> 3 atggatattc gtaagattaa aaaactgatc gagctggttg aagaatcagg catctccgaa 60 ctggaaattt ctgaaggcga agagtcagta cgcattagcc gtgcagctcc tgccgcaagt 120 ttccctgtga tgcaacaagc ttacgctgca ccaatgatgc agcagccagc tcaatctaac 180 gcagccgctc cggcgaccgt tccttccatg gaagcgccag cagcagcgga aatcagtggt 240 cacatcgtac gttccccgat ggttggtact ttctaccgca ccccaagccc ggacgcaaaa 300 gcgttcatcg aagtgggtca gaaagtcaac gtgggcgata ccctgtgcat cgttgaagcc 360 atgaaaatga tgaaccagat cgaagcggac aaatccggta ccgtgaaagc aattctggtc 420 gaaagtggac aaccggtaga atttgacgag ccgctggtcg tcatcgagta acgaggcgaa 480 catgctggat aaaattgtta ttgccaaccg cggcgagatt gcattgcgta ttcttcgtgc 540 ctgtaaagaa ctgggcatca agactgtcgc tgtgcactcc agcgcggatc gcgatctaaa 600 acacgtatta ctggcagatg aaacggtctg tattggccct gctccgtcag taaaaagtta 660 tctgaacatc ccggcaatca tcagcgccgc tgaaatcacc ggcgcagtag caatccatcc 720 gggttacggc ttcctctccg agaacgccaa ctttgccgag caggttgaac gctccggctt 780 tatcttcatt ggcccgaaag cagaaaccat tcgcctgatg ggcgacaaag tatccgcaat 840 cgcggcgatg aaaaaagcgg gcgtcccttg cgtaccgggt tctgacggcc cgctgggcga 900 cgatatggat aaaaaccgtg ccattgctaa acgcattggt tatccggtga ttatcaaagc 960 ctccggcggc ggcggcggtc gcggtatgcg cgtagtgcgc ggcgacgctg aactggcaca 1020 atccatctcc atgacccgtg cggaagcgaa agctgctttc agcaacgata tggtttacat 1080 ggagaaatac ctggaaaatc ctcgccacgt cgagattcag gtactggctg acggtcaggg 1140 caacgctatc tatctggcgg aacgtgactg ctccatgcaa cgccgccacc agaaagtggt 1200 cgaagaagcg ccagcaccgg gcattacccc ggaactgcgt cgctacatcg gcgaacgttg 1260 cgctaaagcg tgtgttgata tcggctatcg cggtgcaggt actttcgagt tcctgttcga 1320 aaacggcgag ttctatttca tcgaaatgaa cacccgtatt caggtagaac acccggttac 1380 agaaatgatc accggcgttg acctgatcaa agaacagctg cgtatcgctg ccggtcaacc 1440 gctgtcgatc aagcaagaag aagttcacgt tcgcggccat gcggtggaat gtcgtatcaa 1500 cgccgaagat ccgaacacct tcctgccaag tccgggcaaa atcacccgtt tccacgcacc 1560 tggcggtttt ggcgtacgtt gggagtctca tatctacgcg ggctacaccg taccgccgta 1620 ctatgactca atgatcggta agctgatttg ctacggtgaa aaccgtgacg tggcgattgc 1680 ccgcatgaag aatgcgctgc aggagctgat catcgacggt atcaaaacca acgttgatct 1740 gcagatccgc atcatgaatg acgagaactt ccagcatggt ggcactaaca tccactatct 1800 ggagaaaaaa ctcggtcttc aggaaaaata a 1831 <210> 4 <211> 915 <212> DNA <213> Escherichia coli <400> 4 atgagctgga ttgaacgaat taaaagcaac attactccca cccgcaaggc gagcattcct 60 gaaggggtgt ggactaagtg tgatagctgc ggtcaggttt tataccgcgc tgagctggaa 120 cgtaatcttg aggtctgtcc gaagtgtgac catcacatgc gtatgacagc gcgtaatcgc 180 ctgcatagcc tgttagatga aggaagcctt gtggagctgg gtagcgagct tgagccgaaa 240 gatgtgctga agtttcgtga ctccaagaag tataaagacc gtctggcatc tgcgcagaaa 300 gaaaccggcg aaaaagatgc gctggtggtg atgaaaggca ctctgtatgg aatgccggtt 360 gtcgctgcgg cattcgagtt cgcctttatg ggcggttcaa tggggtctgt tgtgggtgca 420 cgtttcgtgc gtgccgttga gcaggcgctg gaagataact gcccgctgat ctgcttctcc 480 gcctctggtg gcgcacgtat gcaggaagca ctgatgtcgc tgatgcagat ggcgaaaacc 540 tctgcggcac tggcaaaaat gcaggagcgc ggcttgccgt acatctccgt gctgaccgac 600 ccgacgatgg gcggtgtttc tgcaagtttc gccatgctgg gcgatctcaa catcgctgaa 660 ccgaaagcgt taatcggctt tgccggtccg cgtgttatcg aacagaccgt tcgcgaaaaa 720 ctgccgcctg gattccagcg cagtgaattc ctgatcgaga aaggcgcgat cgacatgatc 780 gtccgtcgtc cggaaatgcg cctgaaactg gcgagcattc tggcgaagtt gatgaatctg 840 ccagcgccga atcctgaagc gccgcgtgaa ggcgtagtgg tacccccggt accggatcag 900 gaacctgagg cctga 915 <210> 5 <211> 930 <212> DNA <213> Escherichia coli <400> 5 atgacgcaat ttgcatttgt gttccctgga cagggttctc aaaccgttgg aatgctggct 60 gatatggcgg cgagctatcc aattgtcgaa gaaacgtttg ctgaagcttc tgcggcgctg 120 ggctacgacc tgtgggcgct gacccagcag gggccagctg aagaactgaa taaaacctgg 180 caaactcagc ctgcgctgtt gactgcatct gttgcgctgt atcgcgtatg gcagcagcag 240 ggcggtaaag caccggcaat gatggccggt cacagcctgg gggaatactc cgcgctggtt 300 tgcgctggtg tgattgattt cgctgatgcg gtgcgtctgg ttgagatgcg cggcaagttc 360 atgcaagaag ccgtaccgga aggcacgggc gctatggcgg caatcatcgg tctggatgat 420 gcgtctattg cgaaagcgtg tgaagaagct gcagaaggtc aggtcgtttc tccggtaaac 480 tttaactctc cgggacaggt ggttattgcc ggtcataaag aagcggttga gcgtgctggc 540 gctgcctgta aagcggcggg cgcaaaacgc gcgctgccgt taccagtgag cgtaccgtct 600 cactgtgcgc tgatgaaacc agcagccgac aaactggcag tagaattagc gaaaatcacc 660 tttaacgcac caacagttcc tgttgtgaat aacgttgatg tgaaatgcga aaccaatggt 720 gatgccatcc gtgacgcact ggtacgtcag ttgtataacc cggttcagtg gacgaagtct 780 gttgagtaca tggcagcgca aggcgtagaa catctctatg aagtcggccc gggcaaagtg 840 cttactggcc tgacgaaacg cattgtcgac accctgaccg cctcggcgct gaacgaacct 900 tcagcgatgg cagcggcgct cgagctttaa 930 <210> 6 <211> 1242 <212> DNA <213> Escherichia coli <400> 6 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> 7 <211> 1217 <212> DNA <213> Escherichia coli <400> 7 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> 8 <211> 518 <212> DNA <213> Escherichia coli <400> 8 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> 9 <211> 735 <212> DNA <213> Escherichia coli <400> 9 atgaattttg aaggaaaaat cgcactggta accggtgcaa gccgcggaat tggccgcgca 60 attgctgaaa cgctcgcagc ccgtggcgcg aaagttattg gcactgcgac cagtgaaaat 120 ggcgctcagg cgatcagtga ttatttaggt gccaacggca aaggtctgat gttgaatgtg 180 accgacccgg catctatcga atctgttctg gaaaaaattc gcgcagaatt tggtgaagtg 240 gatatcctgg tcaataatgc cggtatcact cgtgataacc tgttaatgcg aatgaaagat 300 gaagagtgga acgatattat cgaaaccaac ctttcatctg ttttccgtct gtcaaaagcg 360 gtaatgcgcg ctatgatgaa aaagcgtcat ggtcgtatta tcactatcgg ttctgtggtt 420 ggtaccatgg gaaatggcgg tcaggccaac tacgctgcgg cgaaagcggg cttgatcggc 480 ttcagtaaat cactggcgcg cgaagttgcg tcacgcggta ttactgtaaa cgttgttgct 540 ccgggcttta ttgaaacgga catgacacgt gcgctgagcg atgaccagcg tgcgggtatc 600 ctggcgcagg ttcctgcggg tcgcctcggc ggcgcacagg aaatcgccaa cgcggttgca 660 ttcctggcat ccgacgaagc agcttacatc acgggtgaaa ctttgcatgt gaacggcggg 720 atgtacatgg tctga 735 <210> 10 <211> 954 <212> DNA <213> Escherichia coli <400> 10 atgtatacga agattattgg tactggcagc tatctgcccg aacaagtgcg gacaaacgcc 60 gatttggaaa aaatggtgga cacctctgac gagtggattg tcactcgtac cggtatccgc 120 gaacgccaca ttgccgcgcc aaacgaaacc gtttcaacca tgggctttga agcggcgaca 180 cgcgcaattg agatggcggg cattgagaaa gaccagattg gcctgatcgt tgtggcaacg 240 acttctgcta cgcacgcttt cccgagcgca gcttgtcaga ttcaaagcat gttgggcatt 300 aaaggttgcc cggcatttga cgttgcagca gcctgcgcag gtttcaccta tgcattaagc 360 gtagccgatc aatacgtgaa atctggggcg gtgaagtatg ctctggtcgt cggttccgat 420 gtactggcgc gcacctgcga tccaaccgat cgtgggacta ttattatttt tggcgatggc 480 gcgggcgctg cggtgctggc tgcctctgaa gagccgggaa tcatttccac ccatctgcat 540 gccgacggta gttatggtga attgctgacg ctgccaaacg ccgaccgcgt gaatccagag 600 aattcaattc atctgacgat ggcgggcaac gaagtcttca aggttgcggt aacggaactg 660 gcgcacatcg ttgatgagac gctggcggcg aataatcttg accgttctca actggactgg 720 ctggttccgc atcaggctaa cctgcgtatt atcagtgcaa cggcgaaaaa actcggtatg 780 tctatggata atgtcgtggt gacgctggat cgccacggta atacctctgc ggcctctgtc 840 ccgtgcgcgc tggatgaagc tgtacgcgac gggcgcatta agccggggca gttggttctg 900 cttgaagcct ttggcggtgg attcacctgg ggctccgcgc tggttcgttt ctag 954 <210> 11 <211> 789 <212> DNA <213> Escherichia coli <400> 11 atgggttttc tttccggtaa gcgcattctg gtaaccggtg ttgccagcaa actatccatc 60 gcctacggta tcgctcaggc gatgcaccgc gaaggagctg aactggcatt cacctaccag 120 aacgacaaac tgaaaggccg cgtagaagaa tttgccgctc aattgggttc tgacatcgtt 180 ctgcagtgcg atgttgcaga agatgccagc atcgacacca tgttcgctga actggggaaa 240 gtttggccga aatttgacgg tttcgtacac tctattggtt ttgcacctgg cgatcagctg 300 gatggtgact atgttaacgc cgttacccgt gaaggcttca aaattgccca cgacatcagc 360 tcctacagct tcgttgcaat ggcaaaagct tgccgctcca tgctgaatcc gggttctgcc 420 ctgctgaccc tttcctacct tggcgctgag cgcgctatcc cgaactacaa cgttatgggt 480 ctggcaaaag cgtctctgga agcgaacgtg cgctatatgg cgaacgcgat gggtccggaa 540 ggtgtgcgtg ttaacgccat ctctgctggt ccgatccgta ctctggcggc ctccggtatc 600 aaagacttcc gcaaaatgct ggctcattgc gaagccgtta ccccgattcg ccgtaccgtt 660 actattgaag atgtgggtaa ctctgcggca ttcctgtgct ccgatctctc tgccggtatc 720 tccggtgaag tggtccacgt tgacggcggt ttcagcattg ctgcaatgaa cgaactcgaa 780 ctgaaataa 789 <210> 12 <211> 456 <212> DNA <213> Escherichia coli <400> 12 ttgactacta acactcatac tctgcagatt gaagagattt tagaacttct gccgcaccgt 60 ttcccgttct tactggtgga tcgcgtgctg gattttgaag aaggtcgttt tctgcgcgca 120 gtaaaaaatg tctctgtcaa tgagccattc ttccagggcc atttccctgg aaaaccgatt 180 ttcccgggtg tgctgattct ggaagcaatg gcacaggcaa caggtattct ggcgtttaaa 240 agcgtaggaa aactggaacc gggtgagctg tactacttcg ctggtattga cgaagcgcgc 300 ttcaagcgcc cggtcgtgcc tggcgatcaa atgatcatgg aagtcacttt cgaaaaaacg 360 cgccgcggcc tgacccgttt taaaggggtt gctctggtcg atggtaaagt agtttgcgaa 420 gcaacgatga tgtgtgctcg tagccgggag gcctga 456 <210> 13 <211> 627 <212> DNA <213> Escherichia coli <400> 13 atgatgaact tcaacaatgt tttccgctgg catttgccct tcctgttcct ggtcctgtta 60 accttccgtg ccgctgcagc ggacacgtta ttgattctgg gtgatagcct gagcgccggg 120 tatcgaatgt ctgccagcgc ggcctggcct gccttgttga atgataagtg gcagagtaaa 180 acgtcggtag ttaatgccag catcagcggc gacacctcgc aacaaggact ggcgcgcctt 240 ccggctctgc tgaaacagca tcagccgcgt tgggtgctgg ttgaactggg cggcaatgac 300 ggtttgcgtg gttttcagcc acagcaaacc gagcaaacgc tgcgccagat tttgcaggat 360 gtcaaagccg ccaacgctga accattgtta atgcaaatac gtctgcctgc aaactatggt 420 cgccgttata atgaagcctt tagcgccatt taccccaaac tcgccaaaga gtttgatgtt 480 ccgctgctgc ccttttttat ggaagaggtc tacctcaagc cacaatggat gcaggatgac 540 ggtattcatc ccaaccgcga cgcccagccg tttattgccg actggatggc gaagcagttg 600 cagcctttag taaatcatga ctcataa 627 <210> 14 <211> 1686 <212> DNA <213> Escherichia coli <400> 14 ttgaagaagg tttggcttaa ccgttatccc gcggacgttc cgacggagat caaccctgac 60 cgttatcaat ctctggtaga tatgtttgag cagtcggtcg cgcgctacgc cgatcaacct 120 gcgtttgtga atatggggga ggtaatgacc ttccgcaagc tggaagaacg cagtcgcgcg 180 tttgccgctt atttgcaaca agggttgggg ctgaagaaag gcgatcgcgt tgcgttgatg 240 atgcctaatt tattgcaata tccggtggcg ctgtttggca ttttgcgtgc cgggatgatc 300 gtcgtaaacg ttaacccgtt gtataccccg cgtgagcttg agcatcagct taacgatagc 360 ggcgcatcgg cgattgttat cgtgtctaac tttgctcaca cactggaaaa agtggttgat 420 aaaaccgccg ttcagcacgt aattctgacc cgtatgggcg atcagctatc tacggcaaaa 480 ggcacggtag tcaatttcgt tgttaaatac atcaagcgtt tggtgccgaa ataccatctg 540 ccagatgcca tttcatttcg tagcgcactg cataacggct accggatgca gtacgtcaaa 600 cccgaactgg tgccggaaga tttagctttt ctgcaataca ccggcggcac cactggtgtg 660 gcgaaaggcg cgatgctgac tcaccgcaat atgctggcga acctggaaca ggttaacgcg 720 acctatggtc cgctgttgca tccgggcaaa gagctggtgg tgacggcgct gccgctgtat 780 cacatttttg ccctgaccat taactgcctg ctgtttatcg aactgggtgg gcagaacctg 840 cttatcacta acccgcgcga tattccaggg ttggtaaaag agttagcgaa atatccgttt 900 accgctatca cgggcgttaa caccttgttc aatgcgttgc tgaacaataa agagttccag 960 cagctggatt tctccagtct gcatctttcc gcaggcggag ggatgccagt gcagcaagtg 1020 gtggcagagc gttgggtgaa actgacagga cagtatctgc tggaaggcta tggccttacc 1080 ggtgtgcgc cgctggtcag cgttaaccca tatgatattg attatcatag tggtagcatc 1140 ggtttgccgg tgccgtcgac ggaagccaaa ctggtggatg atgatgataa tgaagtacca 1200 ccgggtcaac cgggtgagct ttgtgtcaaa ggaccgcagg tgatgctggg ttactggcag 1260 cgtccggatg ctacagatga gatcatcaaa aatggctggt tacacaccgg cgacatcgcg 1320 gtgatggatg aagaagggtt cctgcgcatt gtcgatcgta aaaaagacat gattctggtt 1380 tccggtttta acgtctatcc caacgagatt gaagatgtcg tcatgcagca tcctggcgta 1440 caggaagtcg cggctgttgg cgtaccttcc ggctccagtg gtgaagcggt gaaaatcttc 1500 gtagtgaaaa aagatccatc gcttaccgaa gagtcactgg tgaccttttg ccgccgtcag 1560 ctcacgggct acaaagtacc gaagctggtg gagtttcgtg atgagttacc gaaatctaac 1620 gtcggaaaaa ttttgcgacg agaattacgt gacgaagcgc gcggcaaagt ggacaataaa 1680 gcctga 1686 <210> 15 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for accA gene <400> 15 cggatccgat gaatttcctt gattttgaac agc 33 <210> 16 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for accA gene <400> 16 gcgtcgacgt cttacgcgta accgtag 27 <210> 17 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> forward primer for accB and accC gene <400> 17 gaagatcttc ggatggatat tcgtaagatt aaaaaactga tcg 43 <210> 18 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for accB and accC gene <400> 18 gaagatcttc ggatggatat tcgtaagatt aaaaaactga tcg 43 <210> 19 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> forward primer for accD gene <400> 19 gcgtcgacgt caaggagata tacatgagct ggattgaac 39 <210> 20 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for accD gene <400> 20 ataagaatgc ggccgctcag gcctcaggtt 30 <210> 21 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> forward primer for fabD gene <400> 21 ggaattccat atggaattcc atgacgcaat ttgcatt 37 <210> 22 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for fabD gene <400> 22 gaagatcttc ctccttttaa agctcgagcg cc 32 <210> 23 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> forward primer for fabB gene <400> 23 ggaagatctg aaggagatat accatgaaac gtgcagtgat tactggcc 48 <210> 24 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for fabB gene <400> 24 ataagaatgc ggccgcttaa tctttcagct tgcgcattac ca 42 <210> 25 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> forward primer for fabF gene <400> 25 gt; <210> 26 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for fabF gene <400> 26 acgcgtcgac ttagatcttt ttaaagatca aagaacc 37 <210> 27 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> forward primer for fabA gene <400> 27 ccttaattaa ggaaggaggg atggtagata aacgcgaatc c 41 <210> 28 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for fabA gene <400> 28 gctctagagc tcagaaggca gacgtatcct g 31 <210> 29 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer for fabF, fabB and fabA gene <400> 29 aaaaggatcc ggtgtctaag cgtcgtgtag 30 <210> 30 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for fabF, fabB and fabA gene <400> 30 aaaactgcag tcagaaggca gacgtatcct g 31 <210> 31 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer for fabG gene <400> 31 gaattcggaa aatcatgaat tttgaaggaa 30 <210> 32 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for fabG gene <400> 32 gagctcttad gdgtaaccgt agctcatc 28 <210> 33 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> forward primer for fabH gene <400> 33 ggatccccga aaagtgactg agcgtaca 28 <210> 34 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for fabH gene <400> 34 tctagactag aaacgaacca gcgcggag 28 <210> 35 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer for fabI gene <400> 35 tctagaaagg attaaagcta tgggttttct 30 <210> 36 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for fabI gene <400> 36 tctagattat ttcagttcga gttcgttcat 30 <210> 37 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer for fabZ gene <400> 37 gagctcggaa gagtatcttg actactaac 29 <210> 38 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for fabZ gene <400> 38 ggatcctcag gcctcccggc tacgagc 27 <210> 39 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> forward primer for tesA gene <400> 39 cgggatccca tgatgaactt caacaatgtt ttccg 35 <210> 40 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for tesA gene <400> 40 ggaagatctg aaggagatat accatgaaac gtgcagtgat tactggcc 48 <210> 41 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> forward primer for 'tesA gene <400> 41 gttaaccttc cgtgccgctg cagcggacac gttattg 37 <210> 42 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for 'tesA gene <400> 42 caataacgtg tccgctgcag cggcacggaa ggttaac 37

Claims (10)

A nucleotide ( accA , SEQ ID NO: 1) carrying the genetic information of a carboxyl-transferase alpha-unit (CT-alpha) of E. coli K-12 MG1655; A nucleotide ( accB , SEQ ID NO: 2) carrying the genetic information of the biotin carboxyl carrier protein (BCCP) of E. coli K-12 MG1655; A nucleotide ( accC , SEQ ID NO: 3) carrying the genetic information of biotin carboxylase of E. coli K-12 MG1655; A nucleotide ( accD , SEQ ID NO: 4) carrying the genetic information of a carboxyl-transferase beta-unit (CT-beta) of E. coli K-12 MG1655; Of E. coli K-12 MG1655 Nucleotides ( fabD , SEQ ID NO: 5) carrying the genetic information of malonyl-coenzyme A: malonyl-Coenzyme A (acyl carrier protein transacylase); Nucleotides ( fabF , SEQ ID NO: 6) carrying the genetic information of beta-ketoacyl-acyl carrier protein synthase II (KASII, β-keto acyl-acyl carrier protein synthase II) of E. coli K-12 MG1655; Nucleotides ( fabB , SEQ ID NO: 7) 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; Nucleotides ( fabA , SEQ ID NO: 8) carrying the genetic information of β-hydroxy decanoyl thioester dehydrase / isomerase of β-hydroxydecanoyl thioesterase / isomerase of E. coli K-12 MG1655; Nucleotides ( fabG , SEQ ID NO: 9) carrying the genetic information of the beta-keto acyl-acyl carrier protein synthase (KAS) of E. coli K-12 MG1655; Nucleotides ( fabH , SEQ ID NO: 10) carrying the genetic information of beta-ketoacyl-acyl transferase protein synthetase III (KASIII, β-keto acyl-acyl carrier protein synthase III) of E. coli K-12 MG1655; A nucleotide having a genetic information of an enoyl-acyl carrier protein reductase of E. coli K-12 MG1655 ( fabI , SEQ ID NO: 11); Nucleotides ( fabZ , SEQ ID NO: 12) carrying the genetic information of 3-hydroxyacyl-acyl carrier protein dehydratase of E. coli K-12 MG1655; And a nucleotide (` tesA, SEQ ID NO: 13) with genetic information modified to exist in the cytoplasm rather than the periplasm by removing the leader sequence from the thioesterase I of E. coli K-12 MG1655; Wherein the recombinant E. coli is a transformant obtained by transforming a host E. coli with a recombinant plasmid containing at least one nucleotide selected from the group consisting of: The host E. coli according to claim 1, wherein the host E. coli is a nucleotide ( fadD , SEQ ID NO: 14) having genetic information of a long-chain acyl-Coenzyme A synthetase of E. coli BL21 (DE3) E. coli BL21 (DE3)? FadD from which the E. coli strain has been removed. The method of claim 1, wherein the recombinant plasmid is <sup>pCOLADuet TM -1 :: accA, accB,</sup> accC, accD, fabD + pACYCDuet TM -1 :: fabF, fabB, fabA + pTrc99A :: fabG, fabH, fabI, fabZ + pCDF-1b :: ` tesA . 2. The recombinant E. coli according to claim 1, wherein the recombinant E. coli is E. coli BL21 (DE3) ΔfadD :: pCOLADuet ^™ -1 :: accA, accB, accC, accD, fabD + pACYCDuet ^™ -1 :: fabF, fabB, fabA + pTrc99A :: fabG , fabH , fabI , fabZ + pCF -1b :: ` tesA (accession number: KCTC18325P). The method of claim 1, wherein the unsaturated fatty acid is cis-recombinant E. coli, characterized in that the foil sensan (cis -vaccenic acid). A nucleotide ( accA , SEQ ID NO: 1) carrying the genetic information of a carboxyl-transferase alpha-unit (CT-alpha) of E. coli K-12 MG1655; A nucleotide ( accB , SEQ ID NO: 2) carrying the genetic information of the biotin carboxyl carrier protein (BCCP) of E. coli K-12 MG1655; A nucleotide ( accC , SEQ ID NO: 3) carrying the genetic information of biotin carboxylase of E. coli K-12 MG1655; A nucleotide ( accD , SEQ ID NO: 4) carrying the genetic information of a carboxyl-transferase beta-unit (CT-beta) of E. coli K-12 MG1655; Of E. coli K-12 MG1655 Nucleotides ( fabD , SEQ ID NO: 5) carrying the genetic information of malonyl-coenzyme A: malonyl-Coenzyme A (acyl carrier protein transacylase); Nucleotides ( fabF , SEQ ID NO: 6) carrying the genetic information of beta-ketoacyl-acyl carrier protein synthase II (KASII, β-keto acyl-acyl carrier protein synthase II) of E. coli K-12 MG1655; Nucleotides ( fabB , SEQ ID NO: 7) 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; Nucleotides ( fabA , SEQ ID NO: 8) carrying the genetic information of β-hydroxy decanoyl thioester dehydrase / isomerase of β-hydroxydecanoyl thioesterase / isomerase of E. coli K-12 MG1655; Nucleotides ( fabG , SEQ ID NO: 9) carrying the genetic information of the beta-keto acyl-acyl carrier protein synthase (KAS) of E. coli K-12 MG1655; Nucleotides ( fabH , SEQ ID NO: 10) carrying the genetic information of beta-ketoacyl-acyl transferase protein synthetase III (KASIII, β-keto acyl-acyl carrier protein synthase III) of E. coli K-12 MG1655; A nucleotide having a genetic information of an enoyl-acyl carrier protein reductase of E. coli K-12 MG1655 ( fabI , SEQ ID NO: 11); Nucleotides ( fabZ , SEQ ID NO: 12) carrying the genetic information of 3-hydroxyacyl-acyl carrier protein dehydratase of E. coli K-12 MG1655; And a nucleotide (` tesA, SEQ ID NO: 13) with genetic information modified to exist in the cytoplasm rather than the periplasm by removing the leader sequence from the thioesterase I of E. coli K-12 MG1655; Recombinant plasmids containing the oligonucleotide consisting of ^{(pCOLADuet TM -1 :: accA, accB} , accC, accD, fabD + pACYCDuet TM -1 :: fabF, fabB, fabA + pTrc99A :: fabG, fabH, fabI, fabZ + recombinant Escherichia coli producing cis- vaccenic acid and transformed with pCDF-1b :: ` tesA ). A process for preparing an unsaturated fatty acid comprising the steps of:
(a) primary culturing the recombinant Escherichia coli of any one of claims 1 to 6;
(b) secondary culturing the first-cultured recombinant E. coli of step (a);
(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. 8. The method according to claim 7, wherein the secondary culture of step (b) is cultured at a low temperature of 10 to 20 占 폚. The method of claim 7, further comprising analyzing the unsaturated fatty acid composition of the layer separated in step (e). The method of claim 9 wherein the unsaturated fatty acid is cis-night sensan (cis -vaccenic acid) method of producing a polyunsaturated fatty acids, characterized in that.

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