KR20190001426A - Method of producing product using microorganism containing Daucus carota Hsp17.7 gene - Google Patents

Abstract

Provided is a method for producing a product by culturing a microorganism in the genus Escherichia comprising carrot Hsp17.7 genes. The present invention relates to a method for producing alcohol, comprising a step of culturing a microorganism including genetic mutation for improving a level of a polypeptide of an amino acid of SEQ ID NO: 1 in a culture medium, wherein the microorganism is a microorganism in the genus Escherichia.

Description

[0001] The present invention relates to a method for producing a product using microorganisms of the genus Escherichia including the carrot Hsp17.7 gene,

And a method for producing a product by culturing Escherichia genus microorganisms containing the carrot Hsp17.7 gene.

Heat shock protein (HSP) is a protein expressed under heat and inanimate stress. They are classified into five different families based on their molecular weight: HSP100, HSP90, HSP70, HSP60, and small HSP (sHSP) (12-42 kDa). sHSP is abundant and diverse in plants. Four species of sHSP were found in Drosophila melanogaster, 10 species in Homo sapiens, 16 species in Caenorhabditis elegans, and 19 species in Arabidopsis thaliana. These are believed to give increased tolerance to inanimate stress in plants.

However, there have been no disclosures regarding Escherichia genus microorganisms into which the carotene Hsp17.7 gene has been introduced, and whether the microorganisms have resistance to acetate and alcohol.

Zhang, Natl. Genet. 20: 123-128, 1998

One aspect provides a method of producing a product comprising culturing a microorganism in a medium comprising a genetic variation that increases the level of a polypeptide of the amino acid sequence of SEQ ID NO: 1.

One aspect is a method for producing a product comprising culturing a microorganism comprising a genetic mutation which increases the level of a polypeptide of the amino acid sequence of SEQ ID NO: 1 in a medium, wherein the microorganism is an Escherichia genus microorganism ≪ / RTI >

The polypeptide may be a heat shock protein Hsp17.7 derived from carrot (Daucus carota L.). The genetic variation includes any that increases the level of the polypeptide. The genetic variation may be, for example, increasing the number of copies of the gene encoding the polypeptide. Increasing the copy number may be by introducing the gene from the outside. Such introduction may include transformation, transduction, transfection, or electroporation. The gene may be one having the nucleotide sequence of SEQ ID NO: 2.

The genetic variation may be such that the gene encoding the polypeptide is introduced into the genome of the microorganism. The gene may be introduced by homologous recombination with the genome. The homologous recombination can be carried out, for example, when the strain is Escherichia coli, the sequence of the insertion site of the genome, that is, the lipoprotein (Lpp) gene promoter-Hsp17.7 gene-flanked in the yddE pseudogene Introducing a lipase recombinant target (Frt) cassette into E. coli, and selecting it.

The Escherichia genus microorganism may be E. coli.

The product may be an alcohol, an amino acid, or a protein. The alcohol may be a C1-C20 alcohol, for example, a C1-C15 alcohol, a C1-C10 alcohol, or a C1-C6 alcohol. The alcohol may be methanol, ethanol, propanol, butanol, 1,4-butanediol, pentanol, or hexanol. The amino acid may be lysine, threonine, arginine, or tryptophan. The protein may be an antibody, an enzyme, or a hormone.

The microorganism may further comprise a gene encoding an enzyme or a polypeptide constituting a product production pathway. For example, when the product is an alcohol, the gene may be one encoding an enzyme belonging to enzyme code (EC) 1.1.1.1. The gene may be a gene encoding an alcohol dehydrogenase.

In addition, the microorganism may be one in which a gene encoding an enzyme or protein constituting a pathway for degrading or decreasing the concentration of the product is inactivated or attenuated.

The step of culturing may be performed according to a conventional method known in the art. The medium to be used for the above-mentioned cultivation is a medium containing a sugar and a carbohydrate such as glucose, saccharose, lactose, fructose, maltose, starch and cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, Fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid may be included individually or as a mixture. The medium may be, for example, a nitrogen source such as peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate Or as a mixture. The medium may include, for example, potassium dihydrogenphosphate, dipotassium hydrogenphosphate or the corresponding sodium-containing salts as a source. The medium may include, for example, metal salts such as magnesium sulfate or iron sulfate necessary for growth. Essential growth materials such as amino acids and vitamins or suitable precursors may also be added to the culture in the culture. The material may be added to the culture in a suitable manner, for example, batchwise or continuously, during the course of the culture. The culture may be carried out under aerobic conditions.

The method may further comprise separating the desired product from the resulting culture. The separation may vary depending on the desired product. For example, when the target product is an alcohol, it may include performing distillation. For example, when the target product is a protein, centrifugation or filtration of the culture medium may be performed to separate the cells, and performing cell disruption, precipitation, dialysis, ion or affinity chromatography.

In the above method, the culture may be an aerobic culture, and the culture medium may include glucose as a carbon source. The aerobic culture may further include culturing in the state of being in contact with the atmosphere, or blowing an oxygen-containing gas such as air in a medium. The aerobic culture may include stirring the culture. In the aerobic culture, the oxygen concentration may be at least 50%, for example, 50% to 100%, 50% to 150%, or 100% to 200% of the saturated concentration in air. The glucose in the culture medium may be used in an amount of 1 to 20 w / v%, 1 to 15 w / v%, 1 to 10 w / v%, 5 to 20 w / v% / v)%, 5 to 10 (w / v)%, or 3 to 10 (w / v)%.

The culture may comprise from 0.5% to 30.0%, 0.5% to 20.0%, 0.5% to 15.0%, 0.5% to 10.0%, 0.5% to 5.0%, 1.0% to 30.0%, 1.0% to 20.0% From 3.0% to 30.0%, from 2.5% to 30.0%, from 3.0% to 30.0%, from 2.0% to 20.0%, from 2.5% to 15.0%, from 3.0% to 10.0% % ≪ / RTI > to 3.5% ethanol. The ethanol may be present in the pre-incubation period or a portion thereof. The portion may be at least 50% of the time of the pre-culture. The ethanol may be present at the end of the incubation.

The culture may be performed at a concentration of 10 mM to 300 mM, for example, 10 mM to 200 mM, 10 mM to 100 mM, 50 mM to 200 mM, 50 mM to 200 mM, 50 mM to 100 mM, And the like. The acetate may be present in the pre-incubation period or a portion thereof. The portion may be at least 50% of the time of the pre-culture. The ethanol may be present at the end of the incubation.

FIG. 1 is a diagram illustrating a process of fabricating a DNA construct.
Figures 2a-2f show growth when cultured in medium containing the indicated concentrations of 0 mM, 50 mM, 100 mM, 120 mM, 150 mM, and 200 mM sodium acetate.
Figures 3a-3f show growth when cultured in medium containing the indicated concentrations of 0%, 1%, 2%, 2.5%, 3%, and 3.5% (v / v) ethanol.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1 : Carrot to genome Hsp17 .7 Production of transgenic Escherichia coli transfected with the gene

Escherichia coli Hsp17.7 gene was introduced into Escherichia coli genome based on RecE / RecT recombination (Zhang, Natl. Genet. 20: 123-128, 1998) to produce transformed E. coli.

Specifically, a DNA construct containing the "Lpp gene promoter-Hsp17.7 gene-Frt cassette" in which the sequence of the insertion site and the yddE gene was juxtaposed was prepared. The Frt cassette contains a kanamycin selection marker.

The Lpp gene promoter and the Hsp17.7 gene contained the Lpp gene (NCBI accession no .: NC_000913.2) promoter specific (SEQ ID NOS: 3 and 4) and the Hsp17.7 gene (SEQ ID NO: 2) ) Were amplified from E. coli and carrot, respectively. Frt cassettes were also amplified from FRT cassettes included in Quick & Easy E. coli Gene Deletion Kit (Gene Bridges) by PCR using Frt cassette specific primers (SEQ ID NOS: 7 and 8). The amplified Lpp gene promoter and the Hsp17.7 gene were joined by two-step extension PCR without a primer. In addition, the amplified Lpp gene promoter, the Hsp17.7 gene, and the Frt cassette were ligated by two-step extension PCR without a primer. SEQ ID NO: 3 (Is-Lpp_F) and 8 (Frt-Is_R) primers contain the sequence of the insertion site, yddE gene.

FIG. 1 is a diagram illustrating a process of fabricating a DNA construct. 1, Is-Lpp_F, Lpp-Hsp17.7_R, Lpp-Hsp17.7_F, Hsp17.7_Frt_R, Hsp17.7_Frt_F, and Frt-Is_R represent primers of SEQ ID NOS: 3 to 8, respectively.

Next, according to the protocol of Quick and Easy E. coli Gene Deletion Kit (Gene Bridges GmbH), a Red / ET expression plasmid was introduced into E. coli BL21 (DE3) to promote homologous recombination. The E. coli and the plasmid were mixed, electroporation was performed, and then cultured in an ampicillin-containing LB medium to obtain E. coli into which the plasmid was introduced.

Next, electroporation in which the selected plasmid-introduced E. coli was mixed with the DNA construct was performed, followed by culturing in LB medium containing canaminic acid to select surviving cells.

Genomic DNA was extracted from the selected cells using the ExiPrep Bacterial Genomic DNA kit (Bioneer, Daejeon) and the Hsp17.7 gene was inserted into the genome by PCR using Hsp17.7 gene-specific primers (SEQ ID NOS: 5 and 6) Respectively. On the other hand, the Hsp17.7 gene was not identified in cells containing only empty plasmids.

In addition, it was confirmed whether Hsp17.7 protein accumulated in the E. coli cells selected above. The cell culture cultured in the LB medium containing kanamycin was centrifuged, and the obtained cell pellet was dissolved in the protein extraction buffer, and subjected to ultrasonic treatment. The supernatant obtained by centrifugation was subjected to SDS-PAGE gel electrophoresis to obtain Coomassie blue dye , Or transferred to a nitrocellulose membrane, followed by immunoblot analysis. Immunoblotting was performed using a polyclonal antibody against the Hsp17.7 protein using the ECL Plus system. As a result, Hsp17.7 was significantly expressed in the transformed E. coli cells as compared with the control.

Example  2: Identification of resistance to acetate and ethanol

The effect of the transformed E. coli obtained in the examples on the growth was confirmed by culturing in a medium containing acetate and ethanol, respectively.

The transformed E. coli, the control group i.e., the Red / ET expression plasmid only was introduced in E. coli, and inoculated with the wild-type E. coli as a negative control at the same concentration in Luria-Betani (LB) medium and every hour incubation for 9 hours water OD ₆₀₀ value And the cell growth was confirmed. At this time, the medium contained acetate, ethanol, or both at the indicated concentrations.

The results are shown in Figs. 2A to 2F and Figs. 3A to 3F.

Figures 2a-2f show growth when cultured in medium containing the indicated concentrations of 0 mM, 50 mM, 100 mM, 120 mM, 150 mM, and 200 mM sodium acetate.

Tables 1 and 2 also show cell concentrations for 9 hours in LB medium containing ethanol and sodium acetate at the indicated concentrations, respectively.

Strain Ethanol concentration and cell concentration (OD600) Nonstress One% 2% 2.5% 3% 3.5% Wild type 2.27 2.18 2.08 2.04 1.85 1.59 Control group 2.16 2.04 1.96 1.89 1.51 0.87 Transfected cells 2.36 2.22 2.15 2.14 1.98 1.96

Strain Sodium acetate concentration and cell concentration (OD600) Nonstress 50 mM 100 mM 120 mM 150 mM 200 mM Wild type 2.27 2.15 2.07 2.04 1.79 1.49 Control group 2.16 1.97 1.03 0.84 0.58 0.27 Transfected cells 2.36 2.25 2.27 2.18 2.14 2.02

Figures 3a-3f show growth when cultured in medium containing the indicated concentrations of 0%, 1%, 2%, 2.5%, 3%, and 3.5% (v / v) ethanol. As shown in Figs. 2A to 2F and Figs. 3A to 3F, the transformed E. coli maintained higher growth than the control.

Therefore, the transformed E. coli was resistant not only to acid such as acetate but also to alcohol such as ethanol. Therefore, the above-mentioned transformed E. coli can be effectively used for producing the product under acidic and / or alcohol-containing conditions. Thus, the transformed E. coli may further comprise a gene encoding a product, or a gene encoding a polypeptide used to produce an intermediate necessary for production of the product.

<110> Sangmyung University Industry-Academy Cooperation Foundation <120> Method of producing product using microorganism containing Daucus          carota Hsp17.7 gene <130> PN117973KR <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 157 <212> PRT <213> Daucus carota L. <400> 1 Met Ser Ile Ile Pro Ser Phe Phe Gly Gly Arg Arg Ser Asn Val Phe   1 5 10 15 Asp Pro Phe Ser Leu Asp Val Trp Asp Pro Phe Lys Asp Phe Pro Leu              20 25 30 Val Thr Ser Ser Ala Ser Glu Phe Gly Lys Glu Thr Ala Ala Phe Val          35 40 45 Asn Thr His Ile Asp Trp Lys Glu Thr Pro Gln Ala His Val Phe Lys      50 55 60 Ala Asp Leu Pro Gly Leu Lys Lys Glu Glu Val Lys Val Glu Leu Glu  65 70 75 80 Glu Gly Lys Val Leu Gln Ile Ser Gly Glu Arg Asn Lys Glu Lys Glu                  85 90 95 Glu Lys Asn Asp Lys Trp His Arg Val Glu Arg Ser Ser Gly Lys Phe             100 105 110 Leu Arg Arg Phe Arg Leu Pro Glu Asn Ala Lys Val Asp Glu Val Lys         115 120 125 Ala Ala Met Glu Asn Gly Val Leu Thr Val Thr Val Pro Lys Val Glu     130 135 140 Ile Lys Lys Pro Glu Val Lys Ala Ile Asp Ile Ser Gly 145 150 155 <210> 2 <211> 474 <212> DNA <213> Daucus carota <400> 2 atgtcgatca ttccaagctt ttttggaggc cggagaagca acgttttcga cccattctct 60 cttgatgtat gggatccttt caaagatttt cctcttgtta cctcctctgc ctctgagttc 120 ggaaaagaga ctgcagcgtt tgtcaatacg cacattgact ggaaggagac tccccaggct 180 catgtgttca aggccgatct tccagggctg aagaaagaag aggtgaaggt agagcttgaa 240 gaaggaaagg ttcttcagat cagcggagag aggaacaaag agaaggagga gaagaacgac 300 aagtggcacc gagtggaacg cagcagtggc aaatttctga ggaggttcag gcttccagag 360 aatgcaaagg tggatgaggt gaaagctgct atggagaatg gggtgttgac tgttactgtt 420 ccaaaggtgg agatcaagaa gcccgaggtc aaggccattg atatttctgg ttaa 474 <210> 3 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ccggatcttc cacaatacca atcgcaggcg agaacatgcg accctgtaat attgcttt 58 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tggaatgatc gacattatta ataccctcta 30 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 tagagggtat taataatgtc gatcattcca 30 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gagaatagga acttcttaac cagaaatatc 30 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gatatttctg gttaagaagt tcctattctc 30 <210> 8 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ggtacgccgg gctttgaact gccgctggag ggtgaagtac gcgcgaatac gactcactat 60 agggc 65

Claims (10)

1. A method for producing an alcohol comprising culturing a microorganism comprising a genetic mutation which increases the level of a polypeptide of the amino acid sequence of SEQ ID NO: 1 in a medium, wherein the microorganism is an Escherichia genus. The method according to claim 1, wherein the genetic variation increases the number of copies of the gene encoding the polypeptide. 3. The method of claim 2, wherein the gene has the nucleotide sequence of SEQ ID NO: 2. The method according to claim 1, wherein the genetic variation is that a gene encoding the polypeptide is introduced into the genome of the microorganism. The method according to claim 1, wherein the microorganism further comprises a gene encoding an enzyme constituting an alcohol production pathway. 5. The method according to claim 4, wherein the gene is alcohol dehydrogenase. The method according to claim 1, wherein the culture is performed in the presence of 0.5% to 5.0% ethanol. The method according to claim 1, wherein the culture is performed in the presence of 10 mM to 300 mM acetate. The method according to claim 1, wherein the alcohol is a C1-C6 alcohol. The method according to claim 1, wherein the alcohol is ethanol or 1,4-butanediol.

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