KR101686216B1 - Method for preparing fatty acid using recombinant Escherichia coli - Google Patents

Abstract

The present invention relates to recombinant Escherichia coli which is transformed to polynucleotide encoding thioesterase and polynucleotide encoding methylmalonyl-CoA carboxyltransferase, to a manufacturing method thereof, and a method for manufacturing fatty acid using the same. The recombinant Escherichia coli of the present invention shows increased malonyl-CoA productivity by introduction of methylmalonyl-CoA carboxyltransferase gene, and increased fatty acid productivity according to the same. Especially, the recombinant Escherichia coli of the present invention has excellent fatty acid productivity compared to Escherichia coli modified to overexpress ACC, thereby being used for producing oleochemicas.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a recombinant Escherichia coli,

The present invention relates to a method for producing a fatty acid using recombinant E. coli, and more particularly, to a method for producing a fatty acid by transforming wild-type Escherichia coli with a polynucleotide encoding a thiostere and a polynucleotide encoding a methylmalonyl-CoA carboxyltransferase And a method for producing a fatty acid by culturing the recombinant E. coli.

Oleochemicals are aliphatic compounds that are derived from fats and fatty acids. Containing chemicals cosmetics, shampoos, are widely used consumer goods from detergents to areas such as industrial applications such as paints, lubricants, pharmaceuticals, bio-plastics, bio-fuels (Brian et al., Metab. Eng. 29, 1-11 (2015)). Containing chemical products are generally produced using fatty acids obtained from vegetable oils or animal fats as precursors, but recently, a method of producing fatty acids in E. coli has been attracting attention due to a surge in demand and resource exhaustion.

In order to efficiently produce fatty acids in Escherichia coli, metabolism that over-expresses acetyl-CoA carboxylase (ACC) by over-expressing thioesterase or eliminating the fatty acid degradation pathway in Escherichia coli Engineering methods have been used.

As an example of a method for eliminating the fatty acid degradation pathway in E. coli, a method of inactivating fadD and fadE , which are important genes of fatty acid degradation pathway, prevents decomposition of produced fatty acid and thereby increases the production amount of fatty acid 3 to 10 times exhibited (Lu et al., Metab. Eng. 10, 333339 (2008), Lennen et al., Biotechnol. Bioeng. 106, 193-202 (2010), Yu et al., Proc. Natl. Acad. Sci . USA 108, 1864318648 (2011) ).

In the case of thioesterase, the enzyme that catalyzes the last step in the pathway of fatty acid synthesis in microorganisms, the leader peptide of thioesterase is removed to increase the accessibility of the substrate and the enzyme so that the fatty acid is 35 times (Steen et al. , Nature 463, 559562 (2010)).

Furthermore, the method of increasing ACC expression along with the overexpression of thioesterase showed a 30% increase in fatty acid synthesis (Lu et al. , Metab . Eng . 10, 333339 (2008), Lennen et al. , Biotechnol . Bioeng ., 106, 193202 (2010)).

The concentration of malonyl-CoA, a precursor of the fatty acid that is the starting material of various chemical products, remains very low (<5 uM) in the cells (Yoshichika et al. , Journal of General Microbiology 134, 2249-2253 (1988)). This small amount of substrate and complex regulator of ACC expression is a stumbling block in the production of small fatty acids.

Accordingly, the present inventors have made efforts to increase the fatty acid production ability by using a metabolic pathway derived from a microorganism other than the original metabolic pathway of wild-type E. coli, and by introducing the methylmalonyl-CoA carboxyltransferase gene into E. coli It is possible to increase the fatty acid production ability, and the present invention has been completed.

 Brian et al., Metab. Eng. 29, 1-11 (2015)  Lu et al., Metab. Eng. 10, 333-339 (2008)  Lennen et al., Biotechnol. Bioeng. 106, 193-202 (2010)  Yu et al., Proc. Natl. Acad. Sci. USA 108, 18643-18648 (2011)  Steen et al., Nature 463, 559-562 (2010)  Yoshichika et al., Journal of General Microbiology 134, 2249-2253 (1988)

Accordingly, an object of the present invention is to provide a method for producing a fatty acid using recombinant E. coli produced using a metabolic pathway other than ACC.

Another object of the present invention is to provide a recombinant Escherichia coli having an increased fatty acid producing ability, which is produced by using a metabolic pathway other than ACC.

In order to accomplish the above object, the present invention provides a method for producing (1) a wild-type E. coli comprising a polynucleotide encoding thiosterease and a polynucleotide encoding methylmalonyl-CoA carboxyltransferase ; &Lt; / RTI &gt; And (2) culturing the transformed Escherichia coli. The present invention also provides a method for producing a fatty acid from a recombinant Escherichia coli.

In order to achieve these and other objects, the present invention provides a recombinant E. coli transformed with a polynucleotide encoding thyroid protease and a polynucleotide encoding methylmalonyl-CoA carboxyltransferase.

The present invention shows that the introduction of the methylmalonyl-CoA carboxyltransferase gene into wild-type Escherichia coli can increase the fatty acid production ability, and the recombinant Escherichia coli according to the present invention is superior to Escherichia coli that has been engineered to overexpress ACC , The present invention can be usefully used in the production of containing chemicals.

Fig. 1 schematically shows the metabolic pathway of methylmalonyl-CoA carboxyltransferase introduced into E. coli.
Fig. 2 shows a cleavage map of the plasmid pBbB6c-tesA expressing thyroid protease.
Fig. 3 shows a cleavage map of the plasmid pBbA2k-accDABC expressing acetyl-CoA carboxylase.
Fig. 4 shows a cleavage map of plasmid pBbA2k-MMC1 expressing methylmalonyl-CoA carboxyltransferase derived from P. bacterium.
5 shows a cleavage map of the plasmid pBbA2k-MMC2 expressing methylmalonyl-CoA carboxyltransferase derived from Bailonella pavula.
6 is a graph comparing the fatty acid production capacities of the strains of Comparative Examples 1 and 2 and Examples 1 and 2.

(1) transforming a wild-type E. coli with a polynucleotide encoding thiosterease and a polynucleotide encoding methylmalonyl-CoA carboxyl transferase; And (2) culturing the transformed Escherichia coli. The present invention also provides a method for producing a fatty acid from a recombinant Escherichia coli.

The method for producing a fatty acid according to the present invention comprises preparing a recombinant Escherichia coli having an increased fatty acid producing ability from wild-type E. coli and culturing the same to obtain a fatty acid.

In step (1) of the method according to the present invention, the wild-type E. coli is transformed with a polynucleotide encoding thyroid protease and a polynucleotide encoding methyl malonyl-CoA carboxyl transferase .

Examples of the wild-type E. coli used in the step (1) include, but are not limited to, E. coli MG1655, W3110 and BL21 (DE3).

The polynucleotide encoding thyroidase used for producing the recombinant E. coli of the above step (1) may be derived from Escherichia coli, for example, Escherichia coli MG1655, preferably represented by SEQ ID NO: 34 . As shown in the fatty acid production pathway shown in FIG. 1, when fatty acids are produced from malonyl-CoA via an acyl-acyl carrier protein, the thiosterase produces fatty acids from acyl-ACP Lt; / RTI &gt; In the case of wild-type Escherichia coli, the amount of expression of thioesterase is low and the ability to produce fatty acid is not excellent. On the other hand, the recombinant E. coli transformed with the polynucleotide encoding the thyroid protease can exhibit excellent fatty acid production ability by overexpressing thyrosultase .

In addition, the polynucleotide encoding the methylmalonyl-CoA carboxyltransferase used to produce the recombinant E. coli of step (1) may be derived from Propionibacterium freudenreichii shermanii . The polynucleotide encoding the methylmalonyl-CoA carboxyltransferase derived from the above-mentioned P. bacterium, P. licheniformis is represented by m18870 having the nucleotide sequence of SEQ ID NO: 35, mmdA having the nucleotide sequence of SEQ ID NO: 36, A hypothetical protein having the nucleotide sequence of SEQ ID NO: 37 and BCCP having the nucleotide sequence of SEQ ID NO: 38.

Alternatively, the polynucleotide encoding the methylmalonyl-CoA carboxyltransferase used to produce the recombinant E. coli according to the present invention may be derived from veillonella parvula .

The polynucleotide encoding the methylmalonyl-CoA carboxyltransferase derived from bailronella fulbora comprises an amino acid sequence selected from the group consisting of mmdA having the nucleotide sequence of SEQ ID NO: 39, mmdB having the nucleotide sequence of SEQ ID NO: 40, nucleotide sequence of SEQ ID NO: MmdC having the nucleotide sequence of SEQ ID NO: 42, and mmdE having the nucleotide sequence of SEQ ID NO: 43.

The methylmalonyl-CoA carboxyltransferase catalyzes the production of malonyl-CoA and pyruvate from acetyl-CoA and oxaloacetate in the fatty acid production pathway shown in FIG. Therefore, the recombinant E. coli transformed with the polynucleotide encoding the methylmalonyl-CoA carboxyltransferase can produce malonyl-CoA and exhibit excellent fatty acid-producing ability.

In step (1) of the present invention, the wild-type E. coli is a plasmid containing a polynucleotide encoding thyroid protease, preferably a plasmid pBbB6c-tesA having a cleavage map as shown in Fig. 2, and methyl malonyl- A plasmid containing a polynucleotide encoding a transferase, preferably a plasmid pBbA2k-MMC1 having a cleavage map as shown in Fig. 4, or a plasmid pBbA2k-MMC2 having a cleavage map as shown in Fig.

The transformation can be carried out by sequential or simultaneous transformation of the wild-type E. coli with the two plasmids. The order of the plasmids used for sequential transformation of the wild type Escherichia coli into the two plasmids is not particularly limited. For example, wild-type Escherichia coli is transformed with a plasmid containing a polynucleotide encoding thyroid protease Can then be transformed with a polynucleotide encoding methylmalonyl-CoA carboxyltransferase, and vice versa.

The transformation can be carried out by conventional methods known in the art, and examples thereof include calcium phosphate method, liposome method, electroporation method, microinjection method and virus use method.

In step (2) of the method according to the invention, the recombinant E. coli produced in step (1) is cultured in a culture medium to obtain a fatty acid. Examples of the culture medium include, but are not limited to, LB medium, M9 medium, and the like.

0.01 to 1 mM of IPTG and 5 to 200 nM of tetracycline may be added to the culture medium.

The culture obtained through the culturing process may further be subjected to a conventional method known for obtaining a fatty acid, for example, a Sherlock Microbial Identification System method.

On the other hand, the present invention provides a recombinant E. coli transformed with a polynucleotide encoding thyroid protease and a polynucleotide encoding methylmalonyl-CoA carboxyl transferase.

The process for producing the recombinant E. coli according to the present invention is as described above.

The recombinant E. coli according to the present invention produces a high concentration of malonyl-CoA by expressing methylmalonyl-CoA carboxyltransferase which catalyzes the step of producing methylmalonyl-CoA from acetyl-CoA, and malonyl- CoA to produce a high concentration of fatty acids from malonyl-CoA by expressing a thiostere catalyzing one of the steps of producing the fatty acid from the CoA. In particular, the recombinant E. coli according to the present invention can be useful for the production of a chemical product containing a higher yield of fatty acid than recombinant E. coli which has been conventionally engineered to overexpress ACC.

EXAMPLES Hereinafter, the present invention will be described in detail with reference to Examples. However, the following Examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Reference example : Strain, plasmid and primer

The strains and plasmids used in the following Comparative Examples and Examples are shown in Table 1, and the primers are shown in Table 2.

Comparative Example  One: Cyoestraze  Production of recombinant Escherichia coli expressing

For comparison with the recombinant E. coli according to the present invention, recombinant E. coli expressing thiostarase was prepared as follows.

Comparative Example 1: Preparation of recombinant Escherichia coli expressing thioesterase

For comparison with the recombinant E. coli according to the present invention, recombinant E. coli expressing thiostarase was prepared as follows.

<1-1> Construction of expression plasmids

( TesA ) gene was amplified by PCR using Tesla FP (SEQ ID NO: 1) and TesA RP (SEQ ID NO: 2) using the MG1655 genome of thiostrease as a template and primers. The amplified tesA was digested with Eco RI and Xho I to obtain a fragment of SEQ ID NO: 34, followed by ligation to a pBbB6c-RFP plasmid (Joint BioEnergy Institute, USA) treated with the same restriction enzyme to express thyrotase To construct plasmid &quot; pBbB6c-tesA &quot;. The cleavage map of the plasmid pBbB6c-tesA is shown in Fig.

<1-2> Preparation of Recombinant Escherichia coli

Wild-type E. coli MG1655 (Blattner et al. Science 277, 5331, 1453-1474 (1997); ATCC 47076) was transformed with the expression plasmid pBbB6c-tesA prepared in the above Comparative Example 1-1 by electroporation. The transformant was cultured in an agar plate containing 30 μg / mL of chloramphenicol to prepare MG-pBbB6c-tesA, a recombinant Escherichia coli expressing thyroid protease

Comparative Example 2: Preparation of recombinant Escherichia coli expressing thioesterase and acetyl-CoA carboxylase (ACC)

For comparison with the recombinant E. coli according to the present invention, recombinant E. coli overexpressing thiostearase and acetyl-CoA carboxylase (ACC) was prepared as follows.

&Lt; 2-1 > Production of Expression Plasmid

Polynucleotide fragments encoding the four subunits accA, accB, accC and accD of acetyl-CoA carboxylase were obtained from the genome of wild-type E. coli MG1655 by PCR using the primers of SEQ ID NOS: 3 to 10.

Specifically, the primers of SEQ ID NOs: 3 and 4 were amplified to amplify accA, the primers of SEQ ID NOs: 5 and 6 were amplified to amplify accB, the primers of SEQ ID NOs: 7 and 8 were amplified to amplify accC, The primers of SEQ ID NOs: 9 and 10 were added to the prepared PCR mixture. The PCR mixture (5X Phusion buffer, 0.016 mM dNTP, 0.4 uM of each primer, 0.1 ng / ul genomic DNA, 0.016 U / ul phusion polymerase) was subjected to 30 cycles of denaturation for 30 seconds, denaturation for 30 seconds, annealing for 15 seconds, PCR was performed with a total of 30 cycles of 5 minutes to amplify each subunit.

The polynucleotide fragment encoding the accD thus obtained was digested with Bgl II and Xho I and then ligated to a pBbA2k-RFP plasmid (Joint BioEnergy Institute, USA) treated with the same restriction enzyme. Each polynucleotide fragment encoding accA, accB and accC was then digested with BamHI and XhoI and then sequentially ligated to the accD polynucleotide fragment in the pBbA2k-RFP plasmid, resulting in the expression of acetyl-CoA carboxylase The plasmid pBbA2k-accDABC was prepared. The cleavage map of the plasmid pBbA2k-accDABC prepared above is shown in Fig.

&Lt; 2-2 > Preparation of recombinant Escherichia coli

Recombinant E. coli MG-pBbB6c-tesA expressing thyroid hormone prepared in Comparative Example < 1-2 > was raised to an OD of 0.6, and the expression plasmid pBbA2k-accDABC prepared in the comparative example <2-1> And transformed by electroporation. Then, the transformant was plated on agar plates containing 30 ug / ml of chloramphenicol and 50 ug / ml of kanamycin to prepare recombinant Escherichia coli expressing thiostrease and acetyl-CoA carboxylase. The thus-produced strain was named 'MG-pBbB6c-tesA-pBbA2k-ACC'.

Example  One: Cyoestrase  And Methyl malonyl - CoA  Carboxyl transferase ( methylmalonyl - CoA  carboxyltransferase) (1) Preparation of recombinant Escherichia coli

<1-1> Methyl malonyl - CoA Of carboxyltransferase  secure

In order to obtain methyl malonyl-CoA carboxyltransferase, the gene sequence of 1) enzyme in methylmalonyl-CoA carboxyltransferase of the microorganism has been revealed, and 2) the intermediate metabolite of the enzyme reaction 3) the substrate specificity and the activity of the enzyme are confirmed in the cell, and the propionibacterium ( Propionibacterium &lt; RTI ID = 0.0 &gt; freudenreichii sharkiiii- derived methylmalonyl-CoA carboxyltransferase was selected. The methylmalonyl-CoA carboxyltransferase derived from the above-mentioned propionibacterium promoter lyschizmani is composed of m18870, mmdA, hypothetical protein and BCCP.

The genome was extracted from the strain using the plasmid kit (geneall, 100-102) after obtaining the Propionibacterium aviumiensis (KCTC5753) from the microorganism resource center (KCTC). In order to obtain m18870, mmdA, hypothetical protein and BCCP fragment constituting methylmalonyl-CoA carboxyltransferase from the above-mentioned genome, the primers of SEQ ID NOs: 11 and 12 for m18870 and the primers of SEQ ID NOs: 13 and 14 Primers of SEQ. ID. Nos. 15 and 16 for hypothetical proteins, primers of SEQ. ID. Nos. 17 and 18 for BCCP and PCR using the genomes of P. bacterium. To amplify each gene. PCR was performed using primers of SEQ ID NOS: 19 and 20 and pBbB6c (Joint BioEnergy Institute, USA) as a template in order to amplify a transcription termination terminator dbl terminator for termination of transcription.

Through the above PCR, m18870 having the nucleotide sequence of SEQ ID NO: 35, mmdA having the nucleotide sequence of SEQ ID NO: 36, hypothetical protein having the nucleotide sequence of SEQ ID NO: 37 and BCCP fragment having the nucleotide sequence of SEQ ID NO: 38 were obtained.

<1-2> Production of expression plasmid

A plasmid expressing methylmalonyl-CoA carboxyltransferase was prepared by combining m18870, mmdA, hypothetical protein and BCCP obtained in the above Example <1-1>.

Specifically, the plasmid pBbA2k-RFP (Joint BioEnergy Institute, USA) for E. coli was amplified by PCR using the primers of SEQ ID NOs: 31 and 32 to obtain a plasmid.

On the other hand, 5X ISO buffer (0.5M Tris-HCl (pH7.5) , 50mM MgCl 2, 4mM dNTP, 50mM DTT, 5mM NAD, 0.25g / mL PEG-8000) using an assembly master mixture Gibson (Gibson assembly master mixture ; 1X ISO buffer, 5.3 U / ul Taq ligase, 0.03 U / ul Phusion, 0.005 U / ul exonuclease). 150 μL of the above Gibson assembly master mixture was mixed with 100 ng (total 50 μL) of the fragment obtained in Example <1-1>, and then reacted at 50 ° C. for 1 hour. The competent E. coli was transformed with 5 μL of each of the above reaction solutions, and then plated on agar plates containing 50 μg / mL of kanamycin to obtain methylmalonyl-CoAcar from propionibacterium pre- To obtain plasmid ' pBbA2k-MMC1 ' which expresses a positive transferase. FIG. 4 shows cleavage maps of the plasmid pBbA2k-MMC1 prepared above.

&Lt; 1-3 > Preparation of recombinant Escherichia coli

Recombinant Escherichia coli MG-pBbB6c-tesA expressing thyroid protease prepared in Comparative Example < 1-2 > was transformed with the expression plasmid pBbA2k-MMC1 prepared in Example <1-2> And methylmalonyl-CoA carboxyltransferase were prepared. The thus prepared strain was designated as 'MG-pBbB6c-tesA-pBbA2k-MMC1'.

Example  2: Cyoestrase  And Methyl malonyl - CoA  Carboxyl transferase ( methylmalonyl - CoA  (2) Preparation of Recombinant Escherichia coli Expressing Carboxyl Transporase (2)

<2-1> Methyl malonyl - CoA Of carboxyltransferase  secure

In place of the propionibacterium flavonoid strain Kishi marjani strain used in the above Example <1-1>, vilonella pvulla (v eillonella parvula- derived methylmalonyl-CoA carboxyltransferase was selected. The methylmalonyl-CoA carboxyltransferase derived from bailronella favula is composed of mmdA, mmdB, mmdC, mmdD and mmdE.

Biolonella papulata strain (KCTC5019) was obtained from the microorganism resource center (KCTC), and the genome was extracted from the strain using a plasmid kit (geneall, 100-102). In order to obtain the mmdA, mmdB, mmdC, mmdD, and mmdE fragments constituting the methyl malonyl-CoA carboxyltransferase from the genome, primers of SEQ ID NOs: 21 and 22 for mmdA and SEQ ID NOs: 23 and 24 27 and 28 for mmdD and primers of SEQ ID Nos. 29 and 30 for mmdE were used as primers of SEQ ID Nos. 25 and 26 for mmdC and genomes of Vailronella pavula were used for primers of mmdC, PCR was performed as a template to amplify each gene.

Mmd having the nucleotide sequence of SEQ ID NO: 39, mmdB having the nucleotide sequence of SEQ ID NO: 40, mmdC having the nucleotide sequence of SEQ ID NO: 41, mmdD having the nucleotide sequence of SEQ ID NO: 42, and nucleotide sequence of SEQ ID NO: Lt; RTI ID = 0.0 &gt; mmdE &lt; / RTI &gt;

<2-2> Construction of Expression Plasmid

A plasmid expressing methylmalonyl-CoA carboxyltransferase was prepared by combining the mmdA, mmdB, mmdC, mmdD and mmdE obtained in Example <2-1>.

Specifically, the plasmid pBbA2k-RFP (Joint BioEnergy Institute, USA) for E. coli was amplified by PCR using the primers of SEQ ID NOs: 31 and 33 to obtain a plasmid.

On the other hand, 5X ISO buffer (0.5M Tris-HCl (pH 7.5 ), 50mM MgCl 2, 4mM dNTP, 50mM DTT, 5mM NAD, 0.25g / mL PEG-8000) using an assembly master mixture Gibson (Gibson assembly master mixture; 1X ISO buffer, 5.3 U / ul Taq ligase, 0.03 U / ul Phusion, 0.005 U / ul exonuclease). 150 μL of the Gibson assembly master mixture was mixed with 100 ng (total 50 μL) of the fragment obtained in Example <2-1>, and then reacted at 50 ° C. for 1 hour. The competent E. coli was transformed with 5 mu L of each reaction solution, and the transformant was plated on an agar plate containing 50 mu g / mL kanamycin to obtain methyl malonyl-CoA carboxyltransfer from Veilonella pavula Plasmid ' pBbA2k-MMC2 ' The cleavage map of the plasmid pBbA2k-MMC2 prepared above is shown in Fig.

&Lt; 2-3 > Preparation of recombinant Escherichia coli

Recombinant Escherichia coli MG-pBbB6c-tesA expressed in Comparative Example <1-2> expressing thyrothera was transformed with the expression plasmid pBbA2k-MMC2 prepared in Example <2-2> And methylmalonyl-CoA carboxyltransferase were prepared. The thus prepared strain was named 'MG-pBbB6c-tesA-pBbA2k-MMC2'.

Test Example  1: Fatty acid Generation  compare

PBbB2c-ACC, MG-pBbB6c-tesA-pBbA2k-MMC1 and MG-pBbB6c-tesA prepared in Comparative Examples 1 and 2 and Examples 1 and 2 were used as the recombinant Escherichia coli MG-pBbB6c-tesA, MG-pBbB6c-tesA- The fatty acid production ability of -pBbA2k-MMC2 was measured as follows.

Specifically, each strain was grown in LB medium at 37 占 폚 for 15 to 18 hours and then cultured at 37 占 폚 for 24 hours in M9-minimal medium containing glucose. When the optical density (600 nm) of the cells was 0.4 to 0.7 in each culture, 0.3 mM IPTG was added for expression of tesA and the expression of ACC and methylmalonyl-CoA carboxyltransferase 25 nM of tetracycline was added.

After the cultivation, the fatty acid concentration in the culture liquid was measured by gas chromatography. Specifically, 500 ul of the culture was immersed in 2 ml Eppendorf tube, and then 500 ul ethyl acetate, 50 ul HCl and 50 ul 1 g / L methylnonecanoate were added. Thereafter, the mixture was vortexed for 30 seconds to strongly mix the culture medium with ethyl acetate to transfer the fatty acid in the culture medium to the ethyl acetate layer. Then, centrifugation was performed at 12,000 rpm to separate the culture solution layer and the ethyl acetate layer. A separate 500 ul ethyl acetate layer was placed in a fresh 1.5 ml eppendorf tube. The above procedure was repeated by adding 500 ul of ethyl acetate to the remaining culture. Then, 500 ul of ethyl acetate layer was placed in 1.5 ml eppendorf tube to make a total of 1000 ul of ethyl acetate. Then, 500 ul of an extract (500 ul of 1000 ul of ethyl acetate) was added to a GC (gas chromatography) vial and 100 ul of methanol: HCl (9: 1) and 100 ul of TMS-diazomethane were added. (hood). During this process fatty acids become methylated through fatty acid methyl esters and can be easily measured via GC. Fatty acid concentration was measured using Agilent 7890A (GC).

The fatty acid measurement results are shown in Fig.

As shown in FIG. 6, the E. coli of Comparative Example 2 expressing thyroid protease and ACC increased the fatty acid production by about 31% as compared to the E. coli of Comparative Example 1 expressing thyroid protease alone. In contrast, the E. coli strains of Examples 1 and 2 expressing thiostrease and methylmalonyl-CoA carboxyltransferase were found to have increased fatty acid yields by about 61% and about 64%, respectively.

These results show that the introduction of thiostere and methylmalonyl-CoA carboxyltransferase can enhance the fatty acid production ability in E. coli.

<110> UNIST ACADEMY-INDUSTRY RESEARCH CORPORATION <120> Method for preparing fatty acid using recombinant Escherichia          coli <130> FPD201505-0127 <160> 43 <170> KoPatentin 3.0 <210> 1 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> TesA FP <400> 1 aaagaattca aaagatcttt taagaaggag atatacatat ggcggacacg ttattgat 58 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> TesA RP <400> 2 ttactcgagt tatgagtcat gatttacta 29 <210> 3 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> AccA FP <400> 3 tcaaaagatc ttttaagaag gagatataca tatgagtctg aatttccttg attttg 56 <210> 4 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> AccA RP <400> 4 tccttactcg agtttggatc cttacgcgta accgtagctc atcagg 46 <210> 5 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> AccB FP <400> 5 tcaaaagatc ttttaagaag gagatataca tatggatatt cgtaagatta aaaaac 56 <210> 6 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> AccB RP <400> 6 tccttactcg agtttggatc cttactcgat gacgaccagc ggctcg 46 <210> 7 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> AccC FP <400> 7 tcaaaagatc ttttaagaag gagatataca tatgctggat aaaattgtta ttgcca 56 <210> 8 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> AccC RP <400> 8 tccttactcg agtttggatc cttatttttc ctgaagaccg agtttt 46 <210> 9 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> AccD FP <400> 9 tcaaaagatc ttttaagaag gagatataca tatgagctgg attgaacgaa ttaaaa 56 <210> 10 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> AccD RP <400> 10 tccttactcg agtttggatc ctcaggcctc aggttcctga tccggt 46 <210> 11 <211> 61 <212> DNA <213> Artificial Sequence <220> <223> m18870 FP <400> 11 tcaaaagatc ttctgctgag gaggccacaa tttaaaatga gtccgcgaga aattgaggtt 60 t 61 <210> 12 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> m18870 RP <400> 12 ttagtgtacc cccgcctata ggtagtcacg cctgctgaac ggtgacttcg 50 <210> 13 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> mmde FP <400> 13 ctacctatag gcgggggtac actaaatggc tgaaaacaac aatttgaagc 50 <210> 14 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> MMDA RP <400> 14 cttaaatatt tccccccgca ttcaatcagc aggggaagtt tccatgcttc 50 <210> 15 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> hypothetical protein FP <400> 15 ttgaatgcgg ggggaaatat ttaagatggc tgatgaggaa gagaaggacc 50 <210> 16 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> hypothetical protein RP <400> 16 agcgaccctc ctacgacttg tgtgttcaac gaatggaatg gttctgcaga 50 <210> 17 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> BCCP FP <400> 17 acacacaagt cgtaggaggg tcgctatgaa actgaaggta acagtcaacg 50 <210> 18 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> BCCP RP <400> 18 agatccttac tcgagtttgg atcctcagcc gatcttgatg agaccctgac 50 <210> 19 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Dbl term FP <400> 19 ggatccaaac tcgagtaagg atctccaggc atcaaataaa acgaaaggc 49 <210> 20 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Dbl term RP <400> 20 atatccctag gtataaacgc agaaaggccc acccga 36 <210> 21 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> mmde FP <400> 21 gaaaagaatt caaaagatct taagcagaaa tataaggtgg tcgggatggc aacagtgcag 60 gaaaaaatcg 70 <210> 22 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> MMDA RP <400> 22 aaaaatcctc aacatcggga tgaacttata atggaatatt accatgtttc 50 <210> 23 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> mmdB FP <400> 23 gttcatcccg atgttgagga tttttatgga ggcttttgct gttgcgatac 50 <210> 24 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> mmdB RP <400> 24 agatccgtct cctaagtctg gccgcttagt ggttggacaa catagcaagc 50 <210> 25 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> mmdC FP <400> 25 gcggccagac ttaggagacg gatctatgaa aaaattcaac gttacagtaa 50 <210> 26 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> mmdC RP <400> 26 aatatgttcc ttttaatgtg ttagtttagc caagaacaac catgtcatcg 50 <210> 27 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> mmdD FP <400> 27 actaacacat taaaaggaac atattatgga aggacaagca gttactacca 50 <210> 28 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> mmdD RP <400> 28 ccttacctcc cacgcctgat ggtggttaac ctctaccgct taagcgacct 50 <210> 29 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> mmdE FP <400> 29 ccaccatcag gcgtgggagg taaggatgag caatgctaca acaactaacg 50 <210> 30 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> mmdE RP <400> 30 ccttactcga gtttggatcc ttatcgatta ccacgcaaac ggcct 45 <210> 31 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Vector FP (propionibactariem) <400> 31 ggatccaaac tcgagtaagg atct 24 <210> 32 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Vector RP (propionibactariem) <400> 32 tttaaattgt ggcctcctca gcagaagatc ttttgaattc ttttctctat 50 <210> 33 <211> 40 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Vector RP (veillonella) <400> 33 agatcttttg aattcttttc tctatcactg atagggagtg 40 <210> 34 <211> 552 <212> DNA <213> Escherichia coli <400> 34 atggcggaca cgttattgat tctgggtgat agcctgagcg ccgggtatcg aatgtctgcc 60 agcgcggcct ggcctgcctt gttgaatgat aagtggcaga gtaaaacgtc ggtagttaat 120 gccagcatca gcggcgacac ctcgcaacaa ggactggcgc gccttccggc tctgctgaaa 180 cagcatcagc cgcgttgggt gctggttgaa ctgggcggca atgacggttt gcgtggtttt 240 cagccacagc aaaccgagca aacgctgcgc cagattttgc aggatgtcaa agccgccaac 300 gctgaaccat tgttaatgca aatacgtctg cctgcaaact atggtcgccg ttataatgaa 360 gt; tttatggaag aggtctacct caagccacaa tggatgcagg atgacggtat tcatcccaac 480 cgcgacgccc agccgtttat tgccgactgg atggcgaagc agttgcagcc tttagtaaat 540 catgactcat aa 552 <210> 35 <211> 1518 <212> DNA <213> Propionibacterium freudenreichii shermanii <400> 35 atgagtccgc gagaaattga ggtttccgag ccgcgcgagg ttggtatcac cgagctcgtg 60 ctgcgcgatg cccatcagag cctgatggcc acacgaatgg caatggaaga catggtcggc 120 gcctgtgcag acattgatgc tgccgggtac tggtcagtgg agtgttgggg tggtgccacg 180 tatgactcgt gtatccgctt cctcaacgag gatccttggg agcgtctgcg cacgttccgc 240 aagctgatgc ccaacagccg tctccagatg ctgctgcgtg gccagaacct gctgggttac 300 cgccactaca acgacgaggt cgtcgatcgc ttcgtcgaca agtccgctga gaacggcatg 360 gacgtgttcc gtgtcttcga cgccatgaat gatccccgca acatggcgca cgccatggct 420 gccgtcaaga aggccggcaa gcacgcgcag ggcaccattt gctacacgat cagcccggtc 480 cacaccgttg agggctatgt caagcttgct ggtcagctgc tcgacatggg tgctgattcc 540 atcgccctga aggacatggc cgccctgctc aagccgcagc cggcctacga catcatcaag 600 gccatcaagg acacctacgg ccagaagacg cagatcaacc tgcactgcca ctccaccacg 660 ggtgtcaccg aggtctccct catgaaggcc atcgaggccg gcgtcgacgt cgtcgacacc 720 gccatctcgt ccatgtcgct cggcccgggc cacaacccca ccgagtcggt tgccgagatg 780 ctcgagggca ccgggtacac caccaacctt gactacgatc gcctgcacaa gatccgcgat 840 cacttcaagg ccatccgccc gaagtacaag aagttcgagt cgaagacgct tgtcgacacc 900 tcgatcttca agtcgcagat ccccggcggc atgctctcca acatggagtc gcagctgcgc 960 gcccagggcg ccgaggacaa gatggacgag gtcatggcag aggtgccgcg cgtccgcaag 1020 gccgccggct tcccgcccct ggtcaccccg tccagccaga tcgtcggcac gcaggccgtg 1080 ttcaacgtga tgatgggcga gtacaagagg atgaccggcg agttcgccga catcatgctc 1140 ggctactacg gcgccagccc ggccgatcgc gatccgaagg tggtcaagtt ggccgaggag 1200 cagtccggca agaagccgat cacccagcgc ccggccgatc tgctgccccc cgagtgggag 1260 gagcagtcca aggaggccgc ggccctcaag ggcttcaacg gcaccgacga ggacgtgctc 1320 acctatgcac tgttcccgca ggtcgctccg gtcttcttcg agcatcgcgc cgagggcccg 1380 c99cgtgg ctctcaccga tgcccagctg aaggccgagg ccgagggcga cgagaagtcg 1440 ctcgccgtgg ccggtcccgt cacctacaac gtgaacgtgg gcggaaccgt ccgcgaagtc 1500 accgttcagc aggcgtga 1518 <210> 36 <211> 1575 <212> DNA <213> Propionibacterium freudenreichii shermanii <400> 36 ggagcagctc gcagagcagc gccaggtgat cgaagccggt ggcggcgaac gtcgcgtcga gaagcaacat 120 tcccagggta agcagaccgc tcgtgagcgc ctgaacaacc tgctcgatcc ccattcgttc 180 gacgaggtcg gcgctttccg caagcaccgc accacgttgt tcggcatgga caaggccgtc 240 gtcccggcag atggcgtggt caccggccgt ggcaccatcc ttggtcgtcc cgtgcacgcc 300 gcgtcccagg acttcacggt catgggtggt tcggctggcg agacgcagtc cacgaaggtc 360 gtcgagacga tggaacaggc gctgctcacc ggcacgccct tcctgttctt ctacgattcg 420 ggcggcgccc ggatccagga gggcatcgac tcgctgagcg gttacggcaa gatgttcttc 480 gccaacgtga agctgtcggg cgtcgtgccg cagatcgcca tcattgccgg cccctgtgcc 540 ggtggcgcct cgtattcgcc ggcactgact gacttcatca tcatgaccaa gaaggcccat 600 atgttcatca cgggccccca ggtcatcaag tcggtcaccg gcgaggatgt caccgctgac 660 gaactcggtg gcgctgaggc ccatatggcc atctcgggca atatccactt cgtggccgag 720 gacgacgacg ccgcggagct cattgccaag aagctgctga gcttccttcc gcagaacaac 780 actgaggaag catccttcgt caacccgaac aatgacgtca gccccaatac cgagctgcgc 840 gacatcgttc cgattgacgg caagaagggc tatgacgtgc gcgatgtcat tgccaagatc 900 gtcgactggg gtgactacct cgaggtcaag gccggctatg ccaccaacct cgtgaccgcc 960 ttcgcccggg tcaatggtcg ttcggtgggc atcgtggcca atcagccgtc ggtgatgtcg 1020 ggttgcctcg acatcaacgc ctctgacaag gccgccgaat tcgtgaattt ctgcgattcg 1080 ttcaacatcc cgctggtgca gctggtcgac gtgccgggct tcctgcccgg cgtgcagcag 1140 gagtacggcg gcatcattcg ccatggcgcg aagatgctgt acgcctactc cgaggccacc 1200 gtgccgaaga tcaccgtggt gctccgcaag gcctacggcg gctcctacct ggccatgtgc 1260 aaccgtgacc ttggtgccga cgccgtgtac gcctggccca gcgccgagat tgcggtgatg 1320 gt; gacgccatgc gcgccgagaa gatcgaggag taccagaacg cgttcaacac gccgtacgtg 1440 gccgccgccc gcggtcaggt cgacgacgtg attgacccgg ctgatacccg tcgaaagatt 1500 gcttccgccc tggagatgta cgccaccaag cgtcagaccc gcccggcgaa gaagcatgga 1560 aacttcccct gctga 1575 <210> 37 <211> 249 <212> DNA <213> Propionibacterium freudenreichii shermanii <400> 37 atggctgatg aggaagagaa ggacctgatg atcgccacgc tcaacaagcg cgtcgcgtca 60 ttggagtctg agttgggttc actccagagc gatacccagg gtgtcaccga ggacgtactg 120 acggccattt cggccgccgt tgcggcctat ctcggcaacg atggatcggc tgaggtcgtc 180 catttcgccc cgagcccgaa ctgggtccgc gagggtcgtc gggctctgca gaaccattcc 240 attcgttga 249 <210> 38 <211> 372 <212> DNA <213> Propionibacterium freudenreichii shermanii <400> 38 atgaaactga aggtaacagt caacggcact gcgtatgacg ttgacgttga cgtcgacaag 60 tcacacgaaa acccgatggg caccatcctg ttcggcggcg gcaccggcgg cgcgccggca 120 ccgcgcgcag caggtggcgc aggcgccggt aaggccggag agggcgagat tcccgctccg 180 ctggccggca ccgtctccaa gatcctcgtg aaggagggtg acacggtcaa ggctggtcag 240 accgtgctcg ttctcgaggc catgaagatg gagaccgaga tcaacgctcc caccgacggc 300 aaggtcgaga aggtccttgt caaggagcgt gacgccgtgc agggcggtca gggtctcatc 360 aagatcggct ga 372 <210> 39 <211> 1530 <212> DNA <213> veillonella parvula <400> 39 atggcaacag tgcaggaaaa aatcgagtta ttgcacgaaa aactagctaa agttaaagct 60 ggtggcggtg aaaaacgcgt tgagaaacaa catgctcaag gtaaaatgac tgctcgcgaa 120 cgcttggcta aattgttcga tgataattcc ttcgttgaac ttgatcaatt tgttaaacat 180 cgttgtgtta acttcggtca agaaaagaaa gaattaccag gcgaaggtgt agtaacaggt 240 tatggtacta tcgatggtcg tttagtatat gcattcgcac aagatttcac tgtagaaggt 300 ggctctcttg gtgaaatgca tgctgctaaa atcgttaaag tacaacgttt agcaatgaaa 360 atgggtgctc ctattgttgg tatcaacgat tctggcggtg ctcgtattca agaagcagta 420 gatgcccttg ctggttacgg taaaattttc tttgaaaata caaatgcatc tggtgttatt 480 ccacaaattt ccgtaatcat gggtccatgt gcaggcggtg ctgtatattc tccagcattg 540 actgacttca tctacatggt taaaaatact tctcaaatgt tcgtcactgg tcctgcagta 600 atcaaatctg taactggtga agaagtaact gctgaagatc ttggtggtgc aatggctcac 660 aactccgtgt ctggtgttgc tcactttgca gctgaaaatg aagatgattg tatcgctcaa 720 attcgttact tattaggctt cttgccatcc aacaatatgg aagatgctcc attggtagat 780 actggtgacg atccaactcg tgaggatgaa agcttgaata gcttgttgcc agataacagt 840 aacatgcctt acgatatgaa agatgttatc gcagctactg tagataatgg cgaatactat 900 gaagtacaac cattctatgc tacaaatatc attacttgct tcgctcgttt tgatggtcaa 960 tccgttggta tcattgctaa ccaaccaaaa gtaatggctg gttgcttgga cattaacgct 1020 tctgacaaat cttcccgttt catccgtttc tgtgatgctt tcaatattcc aatcgttaac 1080 tttgttgacg ttcctggttt cttgcctggc acaaatcaag aatggggcgg tatcattcgt 1140 catggtgcta aaatgttata tgcttactct gaagctacag taccaaaaat tactgttatc 1200 actcgtaaag catatggcgg ttcctacctc gctatgtgtt cccaagattt aggcgcagac 1260 caagtatatg cttggcctac atccgaaatc gctgtaatgg gtcctgctgg tgcagctaat 1320 atcatcttca agaaagacga agataaagat gctaaaacag ctaaatatgt agaagagttt 1380 gcgactcctt acaaagctgc cgaacgtggc ttcgtagatg ttgtaatcga acctaaacaa 1440 actcgtccag cagttatcaa cgctttggca atgcttgcaa gtaaacgcga aaaccgtgct 1500 ccaaagaaac atggtaatat tccattataa 1530 <210> 40 <211> 1122 <212> DNA <213> veillonella parvula <400> 40 atggaggctt ttgctgttgc gatacaatct gttattaacg atagcgggtt cctcgcattt 60 acaacaggca atgctattat gattcttgta ggtttgatcc tattgtattt agcatttgct 120 cgtgagttcg aaccattatt gttgggtccg attgcgtttg gttgtttact tgccaatatt 180 cctcgtaacg gtttcgaaga aggcgttatg gcgcttatta gtgcaggtat ctcccaagaa 240 atcttcccac ctttaatttt ccttggtgta ggtgcaatga ctgactttgg tccactcatt 300 gccaatccta aaacattgct tttaggggct gcagctcaaa ttggtgtatt tgctgcctta 360 ggtggcgcaa tgatgcttgg ctttacagct caagaagcgg ctgctattgg tatcatcggc 420 ggtgctgatg gtcctacatc catttactta gctactaagc tagctcctca tttattaggt 480 gctatcgcgg ttgccgcata ttcctatatg tccttggtac cgttgattca accacctgta 540 atgaaactct tcactactca aaaagaacgt gaaattgtta tggaacaatt gcgcgaagta 600 acacgttttg aaaaaatcgt gttcccaatc gttgcaacga tcttcatttc cttattgctt 660 ccttccatta catccctttt aggtatgttg atgttaggta acttgttccg tgaatctggc 720 gtaactgatc gtttatccga tacttctcaa aacgcattga tcaatacagt tacaattttc 780 ttagcaactg gtactggctt gacaatgagt gcggaacact tcttaagctt agaaaccatc 840 aaaattattc ttttaggctt attcgcattt atttgcggta cagctggtgg cgtattgttc 900 ggtaaattga tgagcttagt agatggtggt aaaacaaatc cacttattgg ttctgccggt 960 gtatctgcgg ttccaatggc agctcgcgta tctcaagtag taggtgcgaa agctaaccca 1020 gctaacttct tgctcatgca tgctatggga cctaacgtag ctggcgttat cggtacagca 1080 gtagctgcag gtacaatgct tgctatgttg tccaaccact aa 1122 <210> 41 <211> 384 <212> DNA <213> veillonella parvula <400> 41 atgaaaaaat tcaacgttac agtaaatggt acagcatatg atgtagaagt taatgaagtg 60 aaagcagcgg ctcctgcagc agctcctaaa gcagctccag cagcagctcc agctcctaaa 120 gcagctcctg caccagctcc tgctgcagca gcagctccag ttccagcagg tgctgaaact 180 gtaaaagctc caatgcctgg taaaatctta tctgtagcag tatctgctgg tcaagcagtt 240 aaaaaaggcg aaactttgtt gattcttgaa gctatgaaaa tgcaaaatga aatcgcagct 300 ccacatgatg ctgtagtttc cgaagttcgc gtatctgcta accaaactgt atccactggc 360 gatgacatgg ttgttcttgg ctaa 384 <210> 42 <211> 348 <212> DNA <213> veillonella parvula <400> 42 atggaaggac aagcagttac taccaatcct tggttaatca tggcgattaa tatgacagtt 60 gtatttgctg tattgatagc tttaggtatt cttatggaaa tcgtacattt aatcgatcct 120 actaagaaga aaaaagaagc accagcagca actgctcctg ttgctactcc aacagctact 180 cctgtagcgc cagcaaatgc atctgctcaa aatgaggatg aagtagtagc agctatcgta 240 ggtgccattg tggcgatggg gtactcatct gaacaaattg catctattcg acctacagca 300 accagtgcta aatggcgctt ggaaggtcgc ttaagcggta gaggttaa 348 <210> 43 <211> 168 <212> DNA <213> veillonella parvula <400> 43 atgagcaatg ctacaacaac taacggtaaa actccatctc aagatgtagt agcagtaatc 60 gttggtgcat tagcggcaat gggttattcc gctgatcaaa tcgcgcatat ccgtccaatc 120 gtaagctaca attggaaaat ggaaggccgt ttgcgtggta atcgataa 168

Claims (8)

(1) transforming a wild-type E. coli with a polynucleotide encoding thiostosterase and a polynucleotide encoding methylmalonyl-CoA carboxyltransferase; And
(2) culturing the transformed Escherichia coli
Lt; / RTI &gt;
M18870 wherein the polynucleotide encoding said methylmalonyl-CoA carboxyltransferase is composed of the nucleotide sequence of SEQ ID NO: 35, mmdA consisting of the nucleotide sequence of SEQ ID NO: 36, the hypothetical protein consisting of the nucleotide sequence of SEQ ID NO: 37, 38. A method for producing a fatty acid from recombinant E. coli comprising BCCP consisting of the nucleotide sequence of SEQ ID NO: 38.
4. The method of claim 1, wherein the polynucleotide encoding the thiostere is represented by SEQ ID NO: 34.
delete delete The recombinant E. coli according to claim 1, wherein the recombinant E. coli is transformed with a plasmid comprising a polynucleotide encoding thiosterease and a plasmid comprising a polynucleotide encoding methylmalonyl-CoA carboxyltransferase Way.
6. The method according to claim 5, wherein the plasmid comprising the polynucleotide encoding the thyroid protease is a plasmid pBbB6c-tesA having the cleavage map shown in Fig.
6. The method according to claim 5, wherein the plasmid comprising the polynucleotide encoding the methylmalonyl-CoA carboxyltransferase is the plasmid pBbA2k-MMC1 having the cleavage map shown in Fig.
As a recombinant E. coli transformed with a polynucleotide encoding thyroid protease and a polynucleotide encoding methylmalonyl-CoA carboxyltransferase,
M18870 wherein the polynucleotide encoding said methylmalonyl-CoA carboxyltransferase is composed of the nucleotide sequence of SEQ ID NO: 35, mmdA consisting of the nucleotide sequence of SEQ ID NO: 36, the hypothetical protein consisting of the nucleotide sequence of SEQ ID NO: 37, A recombinant Escherichia coli comprising a BCCP consisting of the nucleotide sequence of SEQ ID NO: 38.

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