KR101170497B1 - Method for Preparing Ethanol or Organic Acid Using Recombinant Prokaryote Overexpressed fabA Gene - Google Patents

Abstract

The present invention relates to a method for producing ethanol or organic acid using an ethanol resistant strain, and more specifically, a recombinant prokaryotic nucleus overexpressed with β-hydroxydecanoyl thio ester dehydrase gene ( fabA). It relates to a method for producing ethanol or organic acid, characterized in that the use of microorganisms.

According to the present invention, even if ethanol produced by the microorganism during the fermentation of ethanol and organic acid accumulates at a high concentration in the medium, strain growth is not inhibited and high concentration of ethanol and organic acid can be produced.

Ethanol resistant, fabA, fermentation, ethanol production, organic acid production

Description

Method for Preparing Ethanol or Organic Acid Using Recombinant Prokaryote Microorganism Overexpressed Baea Gene {Method for Preparing Ethanol or Organic Acid Using Recombinant Prokaryote Overexpressed fabA Gene}

The present invention relates to a method for producing ethanol or organic acid using an ethanol resistant strain, and more specifically, a recombinant prokaryotic nucleus overexpressed with β-hydroxydecanoyl thio ester dehydrase gene ( fabA). It relates to a method for producing ethanol or organic acid, characterized in that the use of microorganisms.

Production of organic acids and alcohols through metabolic fermentation process of microorganisms is a generalized technology, and organic acids and alcohols produced during fermentation act as inhibitors in microbial fermentation and growth. In general, the ethanol resistance of microorganisms has been found to be closely related to the fatty acid composition of the cell membrane, and when the ethanol concentration in the medium increases, the saturated fatty acid (C16: 0) content of the cell membrane decreases and the unsaturated fatty acid (C16: 1, C18) decreases. (1) increases ( J. Bacteriol. 125: 670, 1976; Pharmacol. Biochem. Behav. 13 Suppl 1: 191-195, 1980,; Appl. Environ. Microbiol. 2003, 69: 1499-1503) .

Saccharomyces cerevisiae , a representative ethanol-producing strain, is resistant to ethanol when the unsaturated fatty acid generating gene is overexpressed or the fatty acid is added to the medium artificially to increase the amount of unsaturated fatty acid. It has been shown to improve ( Appl. Environ. Microbiol . 69: 1499-1503, 2003; Appl. Environ. Microbiol . 1996, 62: 4309, 1996).

For prokaryotes there is a need to produce recombinant strains that are rapidly degraded even at about 5% ethanol to be resistant at the same and higher alcohol concentrations.

β-Hydroxydecanoyl thio ester dehydrase (beta-hydroxyldecanoyl thio ester dehydrase, fabA) is a trans-beta-hydroxyl-Acid during intracellular fatty acid synthesis. In addition to its ability to convert 2-unsaturated acyl-ACEs, it has the ability to convert trans-2-unsaturated acyl-ACEs into cis-3-decenoyl-ACEs, an enzyme essential for the production of unsaturated fatty acids. Known as Interestingly, overexpression of the FabA enzyme alone has been reported to increase the saturated fatty acid content of the cell membrane ( Biochemistry . 22: 5897, 1983).

On the other hand, delta 5-unsaturase (Δs) derived from Bacillus subtilis (Δ5 desaturase, des) is known to have a function of introducing a double bond to the saturated fatty acids present in the cell membrane, and in the case of overexpressing the des gene in E. coli It has been reported to increase the contents of unsaturated fatty acids 5-cis-palmitoreic acid (16: 1 Δ5) and epedrenic acid (18: 2 Δ5 Δ11) in cell membranes ( J. Bacteriol ., 180: 2194). , 1998).

The present inventors pay attention to the fact that when the microbial strain is exposed to alcohols, the ratio of unsaturated fatty acids in the cell membrane is increased by the biological defense mechanism, thereby artificially modifying the ratio of saturated fatty acids and unsaturated fatty acids in these cell membranes to obtain resistance to ethanol. The study was conducted under the assumption that it could be.

Accordingly, the present inventors have made intensive efforts to develop prokaryotic microorganisms having high ethanol resistance. As a result, the present inventors confirmed that the recombinant E. coli overexpressing the β-hydroxydecanoyl thio ester dehydrase gene is resistant to high concentrations of ethanol and the present invention. To complete.

An object of the present invention is to provide a method for producing ethanol or organic acid using ethanol-resistant prokaryotic microorganisms that can grow in a high concentration of ethanol-containing medium.

In order to achieve the above object, the present invention comprises the steps of culturing a prokaryote microorganism overexpressing the β-hydroxydecanoyl thio ester dehydrase gene ( fabA) ; And it provides a method for producing ethanol or organic acid comprising the step of obtaining ethanol from the culture.

In addition, the present invention comprises the steps of culturing a prokaryote microorganism overexpressing the β-hydroxydecanoyl thio ester dehydrase gene ( fabA) ; And it provides a method for producing an organic acid comprising the step of obtaining an organic acid from the culture.

According to the present invention, even if ethanol produced by the microorganism during the fermentation of ethanol and organic acid accumulates at a high concentration in the medium, strain growth is not inhibited and high concentration of ethanol and organic acid can be produced.

From a consistent point of view, in another aspect, the present invention provides a method of culturing a prokaryotic microorganism in which the β-hydroxydecanoyl thio ester dehydrase gene ( fabA) is overexpressed; And it relates to a method for producing ethanol or organic acid comprising the step of recovering ethanol or organic acid from the culture.

In the present invention, prokaryotic microorganisms overexpressed the β-hydroxydecanoyl thio ester dehydrase gene ( fabA ) are β-hydroxydecanoyl thio ester dehydrase gene (β -hydroxydecanoyl thio ester dehydrase gene: fabA) is or transformed with the inserted vector, β- hydroxy decanoyl thioester di Hydra dehydratase gene (β-hydroxydecanoyl dehydrase gene thio ester: fabA) is inserted in a recombinant prokaryotic chromosome Microorganisms can be used.

Saccharomyces cerevisiae , a representative ethanol-producing strain, is resistant to ethanol when an unsaturated fatty acid gene is overexpressed or an unsaturated fatty acid is added to the medium to artificially increase the amount of unsaturated fatty acid. It has been shown to improve ( Appl. Environ. Microbiol . 69: 1499-1503, 2003; Appl. Environ. Microbiol . 1996, 62: 4309, 1996).

In one embodiment of the present invention, desaturase derived from Bacillus subtilis (degenerates C16: 1 by desaturating the Δ5 position of C16: 0) in J. Bacteriol., 180: 2194, 1998) Recombinant Escherichia coli was prepared by overexpressing the gene to increase the content of unsaturated fatty acid and decreased the content of saturated fatty acid. As a result, the ethanol resistance was decreased as compared to the control. .

In one embodiment of the present invention, E. coli overexpresses the β-hydroxydecanoyl thio ester dehydrase (FabA) gene to increase the content of saturated fatty acids and decrease the content of unsaturated fatty acids. Recombinant Escherichia coli ( E. coli / pBR-fabA) was prepared and examined for ethanol resistance, interestingly it was confirmed that the resistance to ethanol is improved compared to the control. Therefore, in the present invention, the prokaryotic microorganism may be characterized as E. coli.

On the other hand, the recombinant strains ( E. coli / pBR-des-fabA) overexpressing the desaturase gene and fabA gene at the same time canceled each other's influence and showed similar fatty acid composition and ethanol resistance as the control.

In the present invention, the method for recovering ethanol or organic acid from the culture medium of the variant may use a conventional separation technique, for example, distillation, electrodialysis, pervaporation, chromatography, solvent extraction, reaction extraction, etc. In order to separate substances of high purity, these may be used in combination.

In the present invention, the gene can be introduced into a variety of vectors, including plasmid vector, bacteriophage vector, cosmid vector, YAC (Yeast Artificial Chromosome) vector. For the purposes of the present invention, it is preferred to use plasmid vectors.

Typical plasmid vectors that can be used for such purposes include (a) a replication initiation point that allows for efficient replication to include hundreds of plasmid vectors per host cell, and (b) host cells transformed with the plasmid vector. It has a structure comprising an antibiotic resistance gene and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Although no appropriate restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods facilitates ligation of the vector and foreign DNA.

Various vectors, including the T-vector used in the present invention, contain a multicloning site (MCS), which is a collection of several restriction enzyme cleavage sites, which are located in the lacZ gene. Since MCS does not destroy the reading frame of the lacZ gene, lacZ is expressed in a suitable host cell to synthesize β-galaccosidase enzyme having biological activity. When the host cell is cultured in a medium containing X-gal, a colorless chemical, the enzyme breaks down X-gal and creates an insoluble blue substance. Therefore, host cell colonies transformed with plasmid vectors without insertion of foreign DNA into MCS are blue. Conversely, when foreign DNA is inserted into the MCS, the lacZ reading frame of the plasmid vector is broken and β-galactosidase is not produced. As a result, colorless host cell colonies are formed. In the method of manufacturing a DNA chip according to the present invention, the vector can be used more usefully because it confirms whether the DNA desired to be integrated on the slide is normally cloned into the vector.

After ligation, the vector should be transformed into the appropriate host cell. In the method for producing a DNA chip according to the present invention, the preferred host cell is a prokaryotic cell. Suitable prokaryotic host cells include E. coli strain DH5a, E. coli strain JM101, E. coli K12 strain 294, E. coli strain W3110, E. coli strain X1776, E. coli XL-1Blue (Stratagene) and E. coli B And the like. However, E. coli strains such as FMB101, NM522, NM538 and NM539 and other prokaryotic species and genera may also be used. In addition to the aforementioned E. coli , strains of the genus Agrobacterium, such as Agrobacterium A4. Bacillus subtilis (Bacillus subtilis) with the same Bashile (bacilli), S. typhimurium (Salmonella typhimurium) or Serratia dry sense another enteric bacteria, and various Pseudomonas (Pseudomonas) in the strain, such as (Serratia marcescens) as a host cell Can be used.

Prokaryotic transformation can be readily accomplished using the calcium chloride method described in section 1.82 of Sambrook et al., Supra. Optionally, electroporation ( EMBO J. , 1: 841,1982) can also be used to transform these cells.

As the method for inserting the gene on the chromosome of the host cell in the present invention can be used a commonly known genetic engineering method, for example retrovirus vector, adenovirus vector, adeno-associated virus vector, herpes simplex virus vector , Poxvirus vectors, lentiviral vectors or non-viral vectors.

As the vector used for overexpression of the gene according to the present invention, an expression vector known in the art may be used, and it is preferable to use a pET family vector (Novagen). When the cloning is performed using the pET family vector, histidine groups are bound to the ends of the expressed protein, and thus the protein can be effectively purified. In order to separate the expressed protein from the cloned gene, a general method known in the art may be used, and specifically, it may be separated using a chromatographic method using Ni-NTA His-binding resin (Novagen). .

The expression “expression control sequence” in the present invention means a DNA sequence essential for the expression of a coding sequence operably linked in a particular host organism. Such regulatory sequences include promoters for performing transcription, any operator sequence for regulating such transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation. For example, suitable control sequences for prokaryotes include promoters, optionally operator sequences, and ribosomal binding sites. Eukaryotic cells include promoters, polyadenylation signals, and enhancers. The factor that most influences the amount of gene expression in the plasmid is the promoter. As the promoter for high expression, an SRα promoter, a promoter derived from a cytomegalovirus, and the like are preferably used. To express the DNA sequences of the invention, any of a wide variety of expression control sequences can be used in the vector. Useful expression control sequences include, for example, early and late promoters of SV40 or adenovirus, lac system, trp system, TAC or TRC system, T3 and T7 promoters, major operator and promoter regions of phage lambda, fd code protein Regulatory region of, promoter for 3-phospho glycerate kinase or other glycolysis enzymes, promoters of the phosphatase such as Pho5, promoter of yeast alpha-crossing system and gene expression of prokaryotic or eukaryotic cells or viruses Other sequences known to modulate and various combinations thereof. The T7 promoter can be usefully used to express the proteins of the invention in E. coli.

Nucleic acids are "operably linked" when placed in a functional relationship with other nucleic acid sequences. This may be genes and regulatory sequence (s) linked in such a way as to allow gene expression when appropriate molecules (eg, transcriptional activating proteins) bind to regulatory sequence (s). For example, DNA for a pre-sequence or secretion leader is operably linked to DNA for a polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when positioned to facilitate translation. In general, "operably linked" means that the linked DNA sequence is in contact, and in the case of a secretory leader, is in contact and present within the reading frame. However, enhancers do not need to touch. Linking of these sequences is performed by ligation (linking) at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers according to conventional methods are used.

As used herein, the term “expression vector” generally refers to a fragment of DNA that is generally double stranded as a recombinant carrier into which fragments of heterologous DNA have been inserted. Here, heterologous DNA refers to heterologous DNA, which is DNA not naturally found in host cells. Once the expression vector is in the host cell, it can replicate independently of the host chromosomal DNA and several copies of the vector and its inserted (heterologous) DNA can be produced.

As is well known in the art, to raise the expression level of a transfected gene in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that function in the selected expression host. Preferably, the expression control sequence and the gene of interest are included in one expression vector including the bacterial selection marker and the replication origin. If the host cell is a eukaryotic cell, the expression vector must further comprise an expression marker useful in the eukaryotic expression host.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example 1: fabA  And des  Preparation of Gene Overexpression Strains

In order to obtain β-hydroxydecanoyl thio ester dehydrase gene ( fabA ) and Δ5 desaturase gene ( des ), E. coli strains and Bacillus subtilis were respectively cultured to obtain chromosomal DNA.

The chromosomal DNA of E. coli strain (W3110) was used as a template to amplify the beta-hydroxyacyl- ACE dehydratase gene ( fabA ) by PCR.

fabA gene fragment amplification primers

SEQ ID NO: 5- TCTAGA ATG GTAGATAAACGCG-3 (fabA- XbaI -F)

SEQ ID NO: 2: 5- GGATCC GCTAGC TCA GGCATTCTTCCGC- 3 (fabA- BamHI - NheI -R)

Using the chromosomal DNA of Bacillus subtilis as a template, the Δ5 desaturase gene ( des ) was amplified by PCR, and the following DNA fragments were used as primer sets.

primers for amplifying des gene fragments

SEQ ID NO: 5- TCTAGA ATG ACTGAACAAACCA-3 (des- XbaI -F)

SEQ ID NO: 4: 5- GGATCC CTCGAG TCA GGCATTCTTCCGC- 3 (des- BamHI -R)

The amplified fabA or des gene DNA fragments were cloned using pGEM T Easy vector (Promega, USA) to confirm the nucleotide sequence. To induce overexpression of the fabA or des genes, the following primers were used to insert the lacZ promoter (PlacZ), a potent promoter upstream of each gene (FIG. 1).

Primer for amplifying the lacZ promoter upstream of the fabA gene

SEQ ID NO: 5- AGATCT GTTAACGCGCAACGCAAT-3 (PlacZ-fabA-F)

SEQ ID NO: 6: 5- GGATCC TCTAGA GCTGTTTCCTGTGT-3 (PlacZ-fabA- BamHI - XbaI -R)

Primer for amplifying lacZ promoter upstream of des gene

SEQ ID NO: 5- GCTAGC GCGCAACGCAAT-3 (PlacZ-des- NheI -F)

SEQ ID NO: 8: 5- GGATCC TCTAGA GCTGTTTCCTGTGT-3 (PlacZ-des- BamHI - XbaI -R)

The pBR-fabA and pBR-des plasmids were prepared by recloning the fabA or des genes linked to the lacZ promoter with pBR322 vectors, respectively, and at the same time, the pBR-fabA-des plasmid was prepared to express the fabA and des genes simultaneously. The pBR-fabA, pBR-des and pBR-fabA-des plasmids were introduced into E. coli DH5α by thermal shock, respectively, to prepare a recombinant strain, and a control strain was prepared by introducing an expression vector pBR322.

Example 2 Analysis of Resistance of Ethanol to Recombinant Strains

In order to analyze the ethanol resistance of the recombinant strain transformed with the β-hydroxydecanoyl thio ester dehydrase gene ( fabA ) and the Δ5 desaturase gene ( des ) prepared in Example 1, the recombinant strains were 10 μg / Cell proliferation was confirmed by culturing in LB medium containing ml tetracycline, 0.5 mM IPTG and ethanol at concentrations of 0%, 1%, 2% and 3%, respectively. Culture conditions were culture volume, 50 ml / 250 ml, the culture temperature 37 ℃, suspension speed 120 rpm. Inoculation of 1 ml of the preculture strain grown at 600 nm to absorbance 2.0 to investigate the growth of the cells at 3 hour intervals.

As a result, as shown in FIG. 2, the cell proliferation of the control at 2% ethanol concentration was sharply reduced, whereas the recombinant strain (pBR-fabA) overexpressing the fabA gene showed ethanol resistance and showed continuous cell growth. Meanwhile, the recombinant strain (pBR-des) overexpressed the des gene was more sensitive to ethanol than the control. Recombinant strain (pBR-fabA-des) overexpressing both fabA and des genes showed similar ethanol resistance as control. At 3% ethanol concentration, the cell proliferation of all the recombinant strains was severely inhibited, but the ethanol resistance pattern was maintained as it is.

Example 3 Analysis of Cellular Fatty Acid Composition of Recombinant Strains

Changes in the cell membrane fatty acid composition of the recombinant strains prepared in Example 1 were analyzed. The culture medium of each recombinant strain was centrifuged at 10,000 rpm at 4 ℃ to recover the cells, washed with 20 ml of 1 M sorbitol and dried with a vacuum rotor. Next, a solution obtained by mixing sulfuric acid and methanol at 4:96 was added thereto, reacted at 90 ° C. for 1 hour to methylate, and the fatty acid was extracted with nucleic acid. The extracted fatty acids were analyzed by GC Agilent Technologies 6890N (HP-5 column).

As shown in Table 1, the recombinant Escherichia coli (pBR-fabA) overexpressed the fabA gene was found to have a higher content of saturated fatty acid (SFA) and lower content of unsaturated fatty acid (UFA) compared to the control (pBR322). In contrast, recombinant E. coli overexpressing the des gene showed the opposite result. On the other hand, overexpression of the fabA and des genes simultaneously counteracted the effect of each gene on fatty acid composition.

Figure 1 shows the manufacturing process of a recombinant vector containing the fabA gene and the des gene according to the present invention.

Figure 2 shows the growth curve of the ethanol concentration in the medium of the recombinant strain according to the present invention.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Method for Preparing Ethanol or Organic Acid Using Recombinant          Prokaryote Overexpressed fabA Gene <130> P09-B023 <160> 8 <170> Kopatentin 1.71 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 tctagaatgg tagataaacg cg 22 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggatccgcta gctcaggcat tcttccgc 28 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tctagaatga ctgaacaaac ca 22 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ggatccctcg agtcaggcat tcttccgc 28 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 agatctgtta acgcgcaacg caat 24 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ggatcctcta gagctgtttc ctgtgt 26 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gctagcgcgc aacgcaat 18 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ggatcctcta gagctgtttc ctgtgt 26  

Claims (8)

culturing prokaryotic microorganisms with increased ethanol resistance by overexpressing the β-hydroxydecanoyl thio ester dehydrase gene ( fabA) ; And recovering ethanol from the culture solution. The method of claim 1, wherein the recombinant prokaryotic microorganism is transformed with a vector into which the β-hydroxydecanoyl thio ester dehydrase gene ( fabA ) is inserted. . The method for producing ethanol according to claim 1, wherein the recombinant prokaryotic microorganism has a β-hydroxydecanoyl thio ester dehydrase gene ( fabA ) inserted into a chromosome. The method of claim 1, wherein the prokaryotic microorganism is E. coli. delete delete delete delete

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