KR101334973B1 - Method for Preparing Transformed Escherichia coli for Over-expression of Fatty Acid - Google Patents

Abstract

The present invention relates to a method for producing a transformed Escherichia coli for fatty acid overexpression, and more particularly, (a) fabB (3- oxoacyl- [acyl-transport-protein] synthase I), fabG (3- oxoacyl- Nucleotide sequence encoding [acyl-transfer-protein] reductase), fabZ (3R-hydroxymyristol acyl transfer protein dehydratase ) or fabI ( innoyl- [acyl-transport-protein] reductase) Inserting at least one nucleotide sequence selected from the group consisting of into an expression vector; And (b) transforming Escherichia coli with an expression vector into which nucleotides encoding the fabB , fabG , fabZ, and fabI are inserted. According to the present invention, the present invention can provide a method for producing a transformant Escherichia coli for overexpression of a fatty acid biosynthetic pathway, and provides a recombinant Escherichia coli of a fatty acid elongation step of converting glucose to hydrocarbon. The present invention can also increase the production of fatty acid biosynthetic metabolites of microorganisms according to one or more gene combinations.

Description

Method for Preparing Transformed Escherichia coli for Over-expression of Fatty Acid}

The present invention relates to a method for producing a transforming Escherichia coli for fatty acid overexpression.

Recently, countries around the world have been devoting their efforts to developing eco-friendly bioenergy to prepare for the era of high oil prices and reducing greenhouse gas.In Korea, the need for production of fuel bioenergy is increasing. For industrialization, research for new bioenergy production technology is important.

There is increasing interest in bioenergy, such as ethanol, butanol and biodiesel, due to the increasing use of energy as a result of global economic growth and the limited availability of fossil fuel resources. Although it can be used as a transport fuel, due to some drawbacks of performance and production methods, there is a growing interest in compounds in the form of hydrocarbons, which are new renewable energy resources. Accordingly, there is a growing interest in recombinant strains capable of producing long chain fatty acids as metabolites.

The present invention provides a method for improving the metabolic flow of the fatty acid biosynthesis (FAS) pathway as a study to provide an improved method for biologically producing hydrocarbons from fermentable carbon sources in a single microorganism. In one aspect of the invention, a fatty acid elongation step recombination method to regulate the flow of metabolism to convert glucose to hydrocarbons is to facilitate the flow of the renal loop by overexpressing enzymes of the fatty acid biosynthetic pathway. To realize this, the enzyme converts malonyl-acp (acyl-carrying-protein) to 3-oxohexadecanoyl- [acp] and 3-oxostyroyl- [acp] (3-oxoacyl- [acp] Syntase ) encoding the fabB gene, 3- oxohexadecanoyl- [acp] and 3- oxostyroyl- [acp], is selected from (R) -3-hydroxy-palmitoyl- [acp] and (R ) -3-hydroxy-octa decanoyl - acp - enzyme (3-oxo acyl transition to [acyl-transport-protein] reductase kinase) a transformant using a fabG encoding the E. coli (Escherichia coli ), and in addition, (R) -3-hydroxy-palmitoyl- [acp] and (R) -3-hydroxy-octadecanoyl- [acp] are trans-hexatec-2-ino FabZ encoding (3R) -hydroxymyristol- [acp] dehydratase, an enzyme that converts to mono- [acp] and trans-octadec-2- inoyl- [acp], is also introduced into the strain, Finally, ino converts trans-hexadec-2-inoyl- [acp] and trans-octadec-2-inoyl- [acp] to hexa-dicanoyl- [acp] and octadecanoyl- [acp]. Development of a fatty acid-producing recombinant strain is achieved by inserting the fabI gene for overproduction of one- [acp] reductase. This transgenic Escherichia coli was used to identify the difference between the production of the wild type Escherichia coli and malonic acid and fatty acid.

Fatty acid refers to carboxylic acid chemically or biochemically. It refers to a chain-shaped saturated or unsaturated monocarboxylic acid, which is named because hydrolysis of fat results in the separation of glycerol and fatty acids. The simplest fatty acid is butyric acid (four carbon), but the natural fatty acids have more than eight carbons (caprylic acid) and all have even carbon numbers.

Fatty acid synthesis in microorganisms starts with acetyl-CoA, which then leads to longer carbon chains by attaching two atoms of carbon atoms to the hydrocarbon chain at a time. Catabolism proceeds in the opposite direction. In other words, starting with the carboxyl group and ending with acetyl-CoA, the fragment of the hydrocarbon chain and acetyl-CoA are Escherichia coli ) provides energy that is oxidized in the TCA cycle and stored in the form of ATP.

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

ACP is a special form of coenzyme A (CoA) that is specifically used for fatty acid biosynthesis. Acetyl transacylase synthesizes acetyl-ACP and carries other acyl groups other than acetyl and malonyl transacylase synthesizes malonyl-ACP. Acyl-malonyl-ACP condensing enzyme (β-ketoacyl-ACP synthase) is condensed with acetyl-ACP and malonyl-ACP to produce acetoacetyl-ACP. As a result, CO ₂ added to malonyl-CoA is eliminated in this process. This reaction corresponds to a decarboxylation reaction which enables a thermodynamically unfavorable reaction.

β-ketoacyl-ACP reductase reduces acetoacetyl-ACP to make D-β-hydroxybutyryl-ACP. The use of NADPH as a reducing coenzyme and the type D, not L, are different from the oxidation process. 3-hydroxyacyl-ACP dehydratase is synthesized by crotonyl-ACP through a dehydration reaction. Innoyl-ACP reductase synthesizes butyryl-ACP using NADPH as the reducing agent.

The synthesized butyryl-ACP returns to the previous stage and continues to react with the β-ketoacyl-ACP synthetase and merge with malonyl-ACP. This cycle continues until 16 carbon chains are present. Since β-ketoacyl-ACP synthase cannot accept larger substrates, the reaction ends with palmitoyl-ACP. Palmitoyl-ACP is hydrolyzed and converted to palmitated and ACP. As a result, 7 molecules of maloyl-CoA and 1 molecule of acetyl-CoA are used to synthesize 1 molecule of palmitate. Since one molecule of ATP is required for the synthesis of maloyl-CoA and two molecules of NADPH are used for each circuit, seven molecules of ATP and 14 molecules of NADPH are required.

Attempts and efforts have been made consistently with the purpose of overexpressing enzymes that have actively metabolized the FAS pathway using microorganisms. E. coli is an enzyme production system that is known for a lot of metabolic information compared to other organisms, almost all of the information on the gene is widely used in recombinant protein production.

For information on the reaction mechanisms of genes and metabolic pathways that are key to fatty acid biosynthesis, see H. Marrakchi, Y.-M. nWhang and CO Rock, Mechanistic diversity and regulation of Type II fatty acid synthesis, Biochemical Society Transactions, Vol. 30 (6), 1050-1055, 2002. The role of individual genes in the renal cycle in the metabolism of fatty acid biosynthesis in Escherichia coli is mentioned in the Mechanistic diversity and regulation of Type II fatty acid synthesis, Biochemical Society Transactions, Vol. 30 (6), 1050-1055, 2002 have. In addition, it was shown in the literature that fatty acid synthesis metabolic networks by various enzymes in fatty acid biosynthesis proceed (Current understanding of fatty acid biosynthesis and the acyl carrier protein , Biochem. J, 430, 19, 2010).

Therefore, in the present invention, to set up the target gene and metabolites for the intracellular production of the specific step substances in the renal cycle of the various genes of the fatty acid biosynthetic metabolic pathway, and to develop a strain that efficiently produces a specific metabolite The present invention has been completed by developing a recombinant strain through insertion of a target gene while understanding the genomic information and metabolic network of the microorganism and applying it to the wild type E. coli.

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

The present inventors were stable and effectively working to develop a method for producing a transformed E. coli capable of over-expressing the fatty acid biosynthesis pathway as a result fabB (3- oxo-acyl - [acyl-transfer-protein] synthase Ⅰ), fabG (3- Coding for oxoacyl- [acyl-transport-protein] reductase), fabZ (3R-hydroxymyristol acyl transport protein dehydratase ) or fabI ( innoyl- [acyl-transport-protein] reductase) The present invention has been completed by developing a method of inserting one or more nucleotide sequences selected from the group consisting of nucleotide sequences into one expression vector and transforming E. coli with the expression vector.

It is therefore an object of the present invention to provide a method for producing a transformant Escherichia coli for fatty acid overexpression.

Another object of the present invention is to provide a transformant Escherichia coli for fatty acid overexpression prepared by the above method.

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

According to one aspect of the invention, the present invention provides a method for producing a transforming Escherichia coli for fatty acid overexpression comprising:

(a) fabB (3- oxoacyl- [acyl-transport-protein] synthase I), fabG (3- oxoacyl- [acyl-transport-protein] reductase), fabZ (3R-hydroxymyristol acyl Inserting into the expression vector one or more nucleotide sequences selected from the group consisting of nucleotide sequences encoding transport protein dehydratase ) or fabI ( inoyl- [acyl-transport-protein] reductase); And

(b) transforming Escherichia coli with an expression vector into which the nucleotides encoding the fabB , fabG , fabZ and fabI are inserted.

The present inventors earnestly researched for the development of strains that stably and effectively produce fatty acid biosynthetic metabolites through genetic engineering, and as a result fabB , fabG , fabZ and fabI When E. coli is transformed with a gene, it is confirmed that the fatty acid biosynthetic pathway is overexpressed very effectively since the base material is induced into the fatty acid circuit through two pathways, thus completing the present invention.

3-Oxoacyl- [acyl-transfer-protein] synthase I (3-Oxoacyl- [acyl-carrier-protein] synthase I) of the present invention is malonyl-acp (acyl-carrier-protein) 3-oxohexadecanoyl- [acp ] And the catalyst involved in the process of conversion to 3-oxostearoyl- [acp], the fabB gene is the gene encoding it.

3-oxoacyl- [acyl-transfer-protein] synthase I expressed in the expression vector of the present invention is Escherichia Preference is given to using those derived from coli ). More preferably, it is possible to express 3-oxoacyl- [acyl-transport-protein] synthase I of E. coli K-12 MG1655, and the 3-oxoacyl- [acyl-transport-protein] synthase I The nucleotide sequence encoding the nucleotide sequence may be more preferably represented by SEQ ID NO: 1.

In addition, the 3-oxoacyl- [acyl-transport-protein] reductase (3-Oxoacyl- [acp] reductase) of the present invention is 3-oxohexadecanoyl- [acp] and 3-oxostearoyl- [acp] (R It is a catalyst involved in the conversion to) -3-hydroxy-palmitoyl- [acp] and (R) -3-hydroxy-octadecanoyl- [acp], and the fabG gene encodes it.

The 3-oxoacyl- [acyl-transfer-protein] reductase expressed in the expression vector of the present invention is Escherichia coli. Preference is given to using those derived from coli ). More preferably it is possible to express 3-oxoacyl- [acyl-transport-protein] reductase of E. coli K-12 MG1655, said 3-oxoacyl- [acyl-transport-protein] reductase The nucleotide sequence encoding the may be more preferably represented by the sequence listing second sequence.

The 3R-hydroxymyristol acyl carrier protein dehydratase of the present invention is (R) -3-hydroxy-palmitoyl- [acp] and (R) -3-hydroxy-octadecanoyl It is a catalyst involved in the conversion of-[acp] to trans-hexadec-2-enoyl- [acp] and trans-octadec-2-enoyl- [acp], and the fabZ gene encodes it.

The 3R-hydroxy myristol acyl transfer protein dehydratase expressed in the expression vector of the present invention is Escherichia Preference is given to using those derived from coli ). More preferably, the nucleotide encoding the 3R-hydroxy myristol acyl transfer protein dehydratase of E. coli K-12 MG1655, and the nucleotide encoding the 3R-hydroxy myristol acyl transfer protein dehydratase. The sequence may be more preferably represented by the sequence listing third sequence.

Innoyl- [acyl-transfer-protein] reductases of the invention are trans-hexadec-2-enoyl- [acp] and trans-octadec-2-enoyl- [acp] hexa. It is a catalyst involved in the process of conversion to -decanoyl- [acp] and octadecanoyl- [acp], and the fabI gene is the gene encoding it.

The innoyl- [acyl-transfer-protein] reductase expressed in the expression vector of the present invention is Escherichia Preference is given to using those derived from coli ). More preferably, the nucleotide encoding the innoyl- [acyl-transport-protein] reductase of E. coli K-12 MG1655, and the nucleotide encoding the inoyl- [acyl-transport-protein] reductase The sequence may be more preferably represented by SEQ ID NO: 4 sequence.

As described above, each of the nucleotide sequences encoding the enzymes involved in the fatty acid biosynthesis process of (a) to (b) may be operably linked to an expression control sequence and inserted into the expression vector. 'Operably linked to' as used herein means that one nucleic acid fragment is combined with another nucleic acid fragment so that its function or expression is affected by another nucleic acid fragment. Also, an " expression control sequence " means a DNA sequence that regulates the expression of a nucleic acid sequence operably linked to a particular host cell. Such regulatory sequences include promoters for initiating transcription, any operator sequences for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the termination of transcription and translation.

The expression vector prepared in the present invention is constructed so as to express a desired gene in a host cell. Generally, an expression vector comprises a construct for the expression of a promoter-gene-transcription termination sequence.

In the present invention, the promoter contained in the expression vector is preferably used having a strong expression ability.

Preferably, the expression vector of the present invention comprises a nucleotide sequence of the promoter sequence and the gene of interest (structural gene) for bacterial use, and the sequences are preferably linked in 5'-3 'order.

The expression vector further includes a lacZ gene. The lacZ gene encodes β-galactosidase, and in this case, Isopropyl-β-D-thiogalactopyranoside (IPTG) can be used to induce the expression of a nucleotide encoding a structural gene.

According to a preferred embodiment of the present invention, the expression vector may include the entirety of one or more nucleotide sequences selected from the group consisting of fabB , fabG , fabZ and fabI . However, in the expression vector of the present invention, the genes are inserted into a sequence including only a base sequence including a ribosomal binding site (RBS) and an essential part of the enzyme expression so that the genes have a minimum length including an overexpression function of the enzyme. It is desirable in terms of reducing the metabolic burden of the cells. In the embodiment of the present invention, the expression vector pSTV28 including the fifth sequence as the minimum sequence was used.

The expression vector used in the present invention means a plasmid, viral vector or other medium known in the art capable of inserting a nucleic acid encoding a structural gene and capable of expressing the nucleic acid in a host cell, preferably a plasmid It may be a vector.

As the plasmid, any carrier known in the art may be used as long as it is a carrier capable of replication and expression in E. coli.

More preferably, the expression vector is a promoter sequence and fabB Contains the sequence of the gene, named ' E. coli JES1020'.

More preferably, the expression vector is a promoter sequence and fabG Contains the sequence of the gene, named ' E. coli JES1021'.

More preferably. The expression vector is a promoter sequence and fabZ Contains the sequence of the gene, named ' E. coli JES1022'.

More preferably, the expression vector is a promoter sequence and fabI Contains the sequence of the gene, named ' E. coli JES1023'.

More preferably, the expression vector is a promoter sequence, fabB Sequence and fabI of gene Contains the sequence of the gene, named ' E. coli JES1024'.

More preferably, the expression vector is a promoter sequence, fabB It contains the sequence of the gene and the sequence of the fabG gene, named ' E. coli JES1025'.

More preferably. The expression vector is a promoter sequence, fabZ Sequence and fabI of gene Contains the sequence of the gene, named ' E. coli JES1026'.

More preferably, the expression vector is a promoter sequence, fabB Gene sequence, fabZ It contains the sequence of the gene and the sequence of the fabG gene, named ' E. coli JES1027'.

More preferably, the expression vector is a promoter sequence, fabB Gene sequence, fabZ It contains the sequence of the gene and the sequence of the fabI gene, named ' E. coli JES1028'.

More preferably, the expression vector is a promoter sequence, fabB Gene sequence, fabZ Sequence of gene, sequence of fabG gene and fabI Contains the sequence of the gene, named ' E. coli JES1029'.

Preferably, the promoter of the expression vector prepared in the present invention is a lac promoter.

FabB , fabZ , fabG of the present invention And fabI The expression vector containing the gene is a method known in the art, such as, but not limited to, making a competent cell using CaCl ₂ buffer, and then inserting the expression vector into the host cell by thermal shock (42 ° C.). , Can be introduced into the host cell by a transformation method by electric shock, transient transfection, microinjection, cell fusion, calcium phosphate precipitation, electroporation, and the like. In order to stably manufacture and improve the efficiency of the transformant, it is preferable to use a transformation method by electric shock.

Host cell Escherichia coli to be transformed to overexpress the fatty acid biosynthetic pathway is selected as a wild species that is applicable to industrial microorganisms and excellent in function. Escherichia coli negative for bacteria gram coli ) can identify and compare the characteristics of the genus and select the most powerful wild species. The microorganism of the genus Escherichia is Escherichia , a bacterium Gram-negative Escherichia coli. coli , for example MG1655, W3110, DH5α, XL1-Blue and BL21. Preferably E. coli K-12 MG1655.

Microorganisms of the genus Escherichia for increasing the production of hexadecanoic acid and octadecanoic acid of the present invention inherently have a fatty acid biosynthesis pathway, but enzymes for producing fatty acids The gene that encodes fabB , fabZ , fabG And / or fabI A gene is additionally introduced and a nucleotide sequence encoding an enzyme for producing the fatty acid is amplified, i.e. overexpressed. In this regard, the term 'amplification' refers to a suitable DNA by, for example, using a strong promoter or using a gene encoding a suitable enzyme with high activity and optionally using these means together to increase the copy number of the gene (s). Increasing the intracellular activity of one or more enzymes encoded by in a microorganism is described.

According to another aspect of the present invention, the present invention provides a transformant Escherichia coli prepared by the method of the present invention described above.

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

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

(a) The present invention can provide a method for producing transformed Escherichia coli for overexpression of a fatty acid biosynthetic pathway.

(b) The present invention provides recombinant E. coli at the fatty acid elongation stage, which converts glucose into hydrocarbons.

(c) The present invention can increase the production of fatty acid biosynthetic metabolites of microorganisms according to one or more gene combinations.

1 is Escherichia coli This diagram shows the fatty acid biosynthetic pathway from glucose in vivo.
2 is Escherichia Schematic representation of the structure with the fabB gene inserted into pSTV28, a bacterial expression vector such as coli . Called pJE120.
3 Escherichia coli FabG to pSTV28, an expression vector for bacteria Schematic representation of the structure in which the gene is inserted. Named pJE121.
4 is Escherichia coli FabZ in pSTV28, an expression vector for bacteria Schematic representation of the structure in which the gene is inserted. Named pJE122.
5 Escherichia coli This is a schematic diagram of a structure in which the fabI gene is inserted into pSTV28, which is an expression vector for bacteria. Named pJE123.
FIG. 6 is a cross- coli This is a schematic diagram of a structure in which genes of fabB and fabI are inserted into pSTV28, which is a bacterial expression vector. Named pJE124.
7 Escherichia coli This is a schematic diagram of a structure in which the genes of fabB and fabG are inserted into pSTV28, which is a bacterial expression vector. Called pJE125.
8 is Escherichia coli This is a schematic diagram of the structure in which the genes of fabZ and fabI are inserted into pSTV28, which is a bacterial expression vector. Named pJE126.
9 Escherichia coli This is a schematic diagram of the structure in which the genes of fabB , fabG and fabZ are inserted into pSTV28, a bacterial expression vector. Named pJE127.
10 Escherichia coli This is a schematic diagram of the structure in which genes of fabB , fabZ and fabI are inserted into pSTV28, which is a bacterial expression vector. Named pJE128.
11 is Escherichia coli PSTV28 , an expression vector for bacteria such as fabB and fabG Schematic representation of the structure in which the genes of fabZ and fabI are inserted. Named pJE129.
12 Recombinant Escherichia coli growth curves in wild species Escherichia coli This graph shows the result of comparing with. Legend indicates that wtE.coliMG1655 means wild type E. coli MG1655, E. coli.B is E. coli JES1020, E.coli.G is E. coli JES1021, E.coli.Z is E. coli JES1022, E. coli.I is E. coli JES1023, E.coli.BI is E. coli JES1024, E.coli.BG is E. coli JES1025, E.coli.ZI is E. coli JES1026, E.coli.BGZ E. coli JES1027, E. coli.BZI means E. coli JES1028, E. coli.BGZI means E. coli JES1029.
Figure 13 shows the difference in the production of hexadecanoic acid (C16), octadecanoic acid (C18) in the cells of wild species E. coli and recombinant E. coli analyzed by GC-MS.

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

Example

Preparation of plasmids pJE120, pJE121, pJE122, pJE123, pJE124, pJE125, pJE126, pJE127, pJE128 and pJE129 for the expression of each gene combination

With the chromosome of Escherichia coli K-12 as a template, 3-oxohexadecanoyl- [acp], 3-oxostyroyl- [acp], (R) -3-hydroxy-palmitoyl- [acp], (R ) -3-hydroxy-octadecanoyl- [acp], trans-hexadec-2-inoyl- [acp], trans-octadec-2-inoyl- [acp], hexa-decanoyl- [acp ] And nucleotide genes ( fabbank , NCBI) of fabB, fabG, fabZ, and fabI, which encode enzymes for the production of octadecanoyl- [acp] , were cloned by polymerase chain reaction (PCR, DaKaRa Korea). 1-2).

Gene sequence
(genebank authorization number) gene Production enzyme Sequence Listing Second Sequence
(GeneID: 946799) fabB 3-oxoacyl- [acyl-carrying-protein] synthase I Sequence Listing Third Sequence
(Gene ID: 945645) fabG 3-oxoacyl- [acyl-carrying-protein] reductase SEQ ID NO: 4 Sequence
(Gene ID: 944888) fabZ (3R) -hydroxymyristol acyl transporter protein dehydratase SEQ ID NO: 5 Sequence
(Gene ID: 945870) fabi Innoyl- [acyl-carrying-protein] reductase

gene Primer sequence Restriction enzyme fabB Forward 5'-GAGCTCCAGAGGTATTGAATGAAACGTGC-3 ' Sac i Reverse 5'-GGTACCTTAATCTTTCAGCTTGCGCA-3 ' Kpn I fabG Forward 5'-CCCGGGAAGAGGAAAATCATGAATTTTGAAG-3 ' Sma I Reverse 5'-GGATCCCAAATCGCGGTCAGACCATGTA-3 ' BamH I fabZ Forward 5'-GGATCCAGGAAGAGTATCTTGACTACTAACACTCATAC-3 ' BamH I Reverse 5'-GTCGACTCAGGCCTCCCGGCTA-3 ' Sal I fabi Forward 5'-GTCGACAGGATTAAAGCTATGGGTTTTCTTT-3 ' Sal I Reverse 5'-GCATGCTTATTTCAGTTCGAGTTCGTTCA-3 ' Sph i

The genes of Table 1 were amplified using the primers listed in Table 2. The amplified product was introduced into the pSTV28 vector (SEQ ID NO: 9, Seon-Won Kim et al., Biotechnol . Prog. 2007, 23, 599-605) using the restriction enzymes listed in Table 2 to pJE120, pJE121, pJE122, pJE123, pJE124, pJE125, pJE126, pJE127, pJE128 and pJE129 were prepared.

2 to 11 show maps of the vectors named pJE120, pJE121, pJE122, pJE123, pJE124, pJE125, pJE126, pJE127, pJE128 and pJE129. Each polymerase chain reaction was carried out under normal reaction conditions (10 mM Tris-HCl (pH 9.0), 50 mM KCI, 0.1% Triton X-100, 2 mM MgSO ₄ , Taq DNA Polymerase (DaKaRa)). 1 time at 5 ° C./5 min (modification), 60 ° C./1 min (annealing), 72/1 min (amplification), then 95 ° C./1 min (modification), 60 ° C./30 sec (annealing), 72 Thirty replicates were performed at / 1 min (amplification). In the final step, 95 ° C / 1 min (modification), 60 ° C / 1 min (annealing), and 72/5 min (amplification) reactions were performed for stable amplification. The DNA amplified after the polymerase chain reaction was identified and purified on 1% agarose gel and used for restriction enzyme reaction. Each restriction enzyme treated DNA manipulation was inserted into the multicloning site of the pSTV28 vector as described in FIGS. 2, 3, 4 and 5. E. coli transformed to confirm the insertion of the inserted gene at each step Plasmids were extracted from DH5α competent cells (HIT-DH5α, Korea) and treated with restriction enzymes at the original insertion sites to compare the size of the cut DNA by electrophoresis on 1% agarose gel.

E. coli transformants ( transformant )

In general, Escherichia coli, which is widely used for cloning, prepares competent cells using CaCl ₂ buffer and inserts plasmid into host cells by a thermal shock (42 ° C.) method. For the transformation method by electroporation was used.

For the electroshock transformation method, 50 μl of LB (10 g / L tryptone, 10 g / L NaCl, 5 g / L) in 200 μl of wild E. coli culture cultured for 16 hours in a 250 mL flask When the absorbance of the broth was inoculated into the yeast extract) medium and reached 0.6 at 600 nm wavelength, 50 ml of the broth corresponding to the entire broth was centrifuged to separate the supernatant from the cells. The obtained cells were repeatedly washed 2-3 times with 5 ml of 10% glycerol, and then diluted with 100-200 µl of 10% glycerol. Dispense 50 μl of diluted cells and add 1-3 μl of plasmid.

An aliquot of 50 μl of liquid containing the plasmid is placed in an electroporation cuvette and subjected to electric shock (1800 v, 25 uF, 200 Ω). 1 ml LB (10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract) prepared in advance is added and incubated at 37 ° C. for 1 hour. The cultured transformants are incubated in the selective medium chloramphenicol (30 μg / ml) with the added LB solid medium until a single cell is produced.

 FIG. 2 Navi The construct of FIG. 11 was prepared, and 10 recombinant E. coli strains into which an expression vector was inserted were developed.

Development strain Genotype E. coli JES1020 Escherichia coli K12 MG1655 :: pSTV28 :: fabB E. coli JES1021 Escherichia coli K12 MG1655 :: pSTV28 :: fabG E. coli JES1022 Escherichia coli K12 MG1655 :: pSTV28 :: fabZ E. coli JES1023 Escherichia coli K12 MG1655 :: pSTV28 :: fabI E. coli JES1024 Escherichia coli K12 MG1655 :: pSTV28 :: fabB :: fabI E. coli JES1025 Escherichia coli K12 MG1655 :: pSTV28 :: fabB :: fabG E. coli JES1026 Escherichia coli K12 MG1655 :: pSTV28 :: fabZ :: fabI E. coli JES1027 Escherichia coli K12 MG1655 :: pSTV28 :: fabB :: fabG :: fabZ E. coli JES1028 Escherichia coli K12 MG1655 :: pSTV28 :: fabB :: fabZ :: fabI E. coli JES1029 Escherichia coli K12 MG1655 :: pSTV28 :: fabB :: fabG :: fabZ :: fabI

Culture of Escherichia Coli and Production of Proteins from Recombinant Strains

Each bacterium whose transformation accuracy was confirmed was incubated in LB medium for about 12 hours, and made into a storage solution so that the glycerol concentration was 25%, and stored at -80 ° C until the culture experiment. The culture was incubated for 12 hours in 1 ml of the electrolytic storage solution in 3 ml of LB to which chloramphenicol (50 µg / ml) in a 10 ml tube was added, and then again to chloramphenicol (50 µg / ml in 500 ml flask). 200 ml of LB (10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract) medium to which ml) was added was inoculated with 1/200 electric culture solution at 37 ° C. and 170 rpm for 24 hours. Incubated (Figure 12). When protein absorbance reached 0.6 at 600 nm wavelength, 1 mM IPTG (isopropylthio-β-D-galactoside, sigma, USA), an inducer, was added to induce expression of recombinant proteins.

Of recombinant E. coli From within the cell  Fatty acids ( fatty acid ) Measure

In the examples herein, the culture medium cultured under the conditions mentioned in 'Cultivation of E. coli and Production of Proteins from Recombinant Strains' contained in cells when the amount of E. coli was sufficient for 4 hours and 24 hours after addition of IPTG. Fatty acid extraction was performed in the following manner to determine fatty acids:

The extraction process was divided into 5 stages. As the first process culture stage, 5 ml of the culture solution was centrifuged (4500 rpm, 10 minutes), and E. coli was stored at -80 ° C. In the second process, the recombinant E. coli stored in the saponification step was vortexed for 5-10 seconds with 1 ml of Solution 1 (NaOH 45 g, MeOH 150 ml, distilled water 150 ml). After reacting at 100 ° C. for 5 minutes, the solution was vortexed again for 5-10 seconds, then reacted again at 100 ° C. for 25 minutes, and then cooled. The third process is a methylation step. 2 ml of solution 2 (6 N HCl 325 ml, MeOH 275 ml) was added and vortexed for 5-10 seconds, followed by reaction at 80 ° C. for 10 minutes. After the reaction was over, it quickly cooled down. The fourth step is an extraction step, in which 1.25 ml of solution 3 (hexane: methyl tet-butyl ester = 1: 1) is added, shaken up and down for 10 minutes, and the bottom layer is extracted and discarded. The fifth step is a washing step to facilitate the analysis by GC. 3 ml of solution 4 (NaOH 10.8 g, distilled water 900 ml) is added to the remaining supernatant of the previous step, shaken up and down for 5 minutes, and the supernatant is extracted. Was analyzed by GC / MS.

The CG / MS used in the present invention was 5975 series MSD (Hewlett-Packard (HP), Germany) and Agilent 7890A (Hewlett-Packard (HP), Germany) and used an HP-5 column (30m × 0.32 mm, film thickness 0.25). The GC temperature conditions for analysis were 40 ° C./5 min, 3 ° C./min up to 220 ° C., 3 ° C./min up to 250 ° C. and 250 ° C./5 min, and the MS interface temperature was 160 ° C.

Hexadecanoic acid (C16) and octadecanoic acid (C18) were extracted and analyzed by this extraction method. As a result, the fatty acid, hexadecanoic acid (C16) and octadecanoic acid (C18) of recombinant E. coli overexpressed the kidney cycle genes involved in fatty acid synthesis than wild species were produced in excess (FIG. 13). In particular, considering the growth rate of E. coli, fabB , fabG , fabZ , compared with wild species, are not particularly inhibited by the inserted gene. or consisting of fabI genes E. coli , which is a strain transformed by inserting one or more genes selected from the group into an expression vector. JES1020- E.coli JES1029) can be seen that the fatty acid productivity is significantly improved compared to the wild species (Figs. 12 and 13). The results shown in Figure 13 shows wild E. coli Compared to MG1655, the peak of the product fatty acid was found to be significantly higher. Through this invention , fabB , fabG and fabZ are located in the elongation stage inside fatty acid biosynthesis. And The fabI gene proved to play an important role in enhancing fatty acid production.

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

Attach an electronic file to a sequence list

Claims (19)

Method for producing a transforming Escherichia coli for fatty acid overexpression comprising:
(a) a fab sequence encoding fabB (3- oxoacyl- [acyl-transport-protein] synthase I) comprising the nucleotide sequence of SEQ ID NO: 1, fabG comprising the nucleotide sequence of SEQ ID NO: 2 FabZ (3R-hydroxymyristol acyl transfer protein dehydratase ) comprising a nucleotide sequence encoding (3- oxoacyl- [acyl-transport-protein] reductase) and the nucleotide sequence of SEQ ID NO: 3 Inserting a nucleotide sequence encoding A into the expression vector; And
(b) transforming Escherichia coli with an expression vector into which the nucleotide sequences encoding the fabB , fabG and fabZ are inserted.
delete delete delete delete delete delete delete delete delete delete delete The method of claim 1, wherein the expression vector is a promoter sequence, fabB Sequence of gene, sequence of fabZ gene and fabG Method for producing a transformant E. coli, characterized in that comprising the sequence of the gene.
delete delete The method of claim 13, wherein the promoter is a lac promoter.
The method of claim 1, wherein the expression vector is introduced into a backbone vector comprising the minimum sequence of SEQ ID NO 5 sequence.
According to claim 1, wherein the step of transforming E. coli (b) is a method for producing transformed E. coli, characterized in that the electroshock method.
The transformed E. coli produced by the method of any one of claims 1, 13 and 16 to 18.

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