KR20200040981A - Transformed methanotrophs for producing putrescine and uses thereof - Google Patents

Abstract

The present invention relates to transformed methanotrophs for producing putrescine, in which the activity of spermidine synthase is weakened or inactivated compared to its endogenous activity, and the activities of constitutive ornithine decarboxylase and putrescine transporters are enhanced compared to their endogenous activities; a production method of putrescine using the transformed methanotrophs; and a composition for putrescine production comprising the transformed methanotrophs. The transformed methanotrophs of the present invention exhibit high tolerance to putrescine due to adaptive evolution, and thus are not inhibited in growth by endogenously produced putrescine. Moreover, the transformed methanotrophs can produce putrescine in high yield from C1 carbon sources via enhanced putrescine production pathways, and thus may be usefully used in an eco-friendly and economical high-throughput production of putrescine.

Description

Transformed methanotrophs for producing putrescine and uses thereof}

In the present invention, the activity of spermidine synthase is weakened or inactivated compared to the intrinsic activity, and the activity of ornithine decarboxylase constitutive and putrescine transporter is intrinsic. A transformed methane magnetizing bacterium for putrescine production enhanced compared to activity; Method for producing putrescine using the transformed methane magnetization bacteria; And it relates to a composition for producing putrescine comprising the transformed methane magnetization.

Methane gas is the main component of natural gas and shale gas, and is known to cause greenhouse effect. Unlike liquid hydrocarbons represented by petroleum, methane is a gas in itself, making it difficult to convert to other substances of high value and difficult to use as fuel.

Meanwhile, methanotrophic bacteria are microorganisms that can grow methane as the only energy source, and were first isolated by Sohngen in 1906. Methane magnetization bacteria have enzymes called methane monooxygenases (MMO), which can easily convert methane to methanol under normal temperature and pressure. In methane magnetization bacteria, biosynthesis of carbon compounds from methane is carried out by a ribulose monophosphate cycle (RuMP cycle) and a serine cycle, and generally type I methane magnetization bacteria are ribulose monophosphate The circuit, type II methane magnetization bacteria are known to synthesize biomass using the serine circuit (J Microbiol Biotechnol. Hwang IY et al., 2014 Dec 28; 24 (12): 1597-605). Efforts have been made to utilize physiological properties of methane magnetizing bacteria to convert methane to high value-added organic compounds, while at the same time reducing harmful methane gas and utilizing it as an energy source.

Putrescine (putrescine, 1,4-diaminobutane) is an important platform chemical in various industries, with a demand of about 10,000 tonnes per year (Scott et al., 2007). Putrescine is known to help in skin care, sperm formation, and nerve protection in the pharmaceutical field, and is known to have an effect on tumor formation and tissue hypertrophy. In addition, putrescine can be used as a substitute for pesticides when cultivating plants, and is known to increase the resistance to cold, contamination and salt, and to prevent wilting. Meanwhile, putrescine is an important raw material for synthesizing polyamine nylon-4,6 by reacting with adipic acid, and is mainly produced by chemical synthesis from propylene through acrylonitrile and succinonitrile. However, such a chemical synthesis method has a high energy consumption, is not environmentally friendly, and furthermore has a problem of exhaustion of petroleum resources.

Against this background, the present inventors conducted a thorough research on a method for producing putrescine, using a biomass that is more environmentally friendly and can reduce energy consumption, and as a result, transformed methane that can use methane as a carbon source. The present invention was completed by confirming a method capable of producing putrescine in a more eco-friendly, economical and high yield through magnetizing bacteria.

One object of the present invention is that the activity of spermidine synthase is weakened or inactivated compared to the intrinsic activity, ornithine decarboxylase constitutive and putrescine transporter. It is to provide a transformed methane magnetizing bacterium for putrescine production, whose activity is enhanced compared to intrinsic activity.

Another object of the present invention is to provide a method for preparing putrescine using the transformed methane magnetizing bacteria.

Another object of the present invention is to provide a composition for the production of putrescine containing the transformed methane magnetizing bacteria.

Specifically, it is as follows. Meanwhile, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. In addition, the scope of the present invention is not limited by the specific descriptions described below.

One aspect of the present invention for achieving the above object is the activity of the spermidine synthase (spermidine synthase) is weakened or inactivated compared to the intrinsic activity, ornithine decarboxylase constitutive and putre It provides a transformed methane magnetizing bacterium for putrescine production, wherein the activity of the putrescine transporter is enhanced compared to the intrinsic activity.

In addition, another aspect of the present invention, in the transformed methane magnetizing bacteria, lactate dehydrogenase (lactate dehydrogenase) and acetic acid phosphatase (acetate kinase) activity is weakened or inactivated compared to the intrinsic activity, transformation Provide methane magnetization bacteria.

The inventors of the present invention, as a more environmentally friendly and economical method for producing putrescine, as a result of earnestly researching methods using biomass such as methane, surprisingly, when using the transformed methane magnetizing bacteria of the present invention with high yield It was confirmed that the production of putrescine is possible. The production of high yield futresine through methane magnetization bacteria using methane as a carbon source is unknown at all, and it can be said that the significance is very large in that it was first developed through the present invention.

In the present invention, the term "Futresin" is a compound having a colorless crystal having a molecular weight of 88 having a chemical formula of C ₄ H ₁₂ N ₂ , by decarboxylation of ornithine or hydrolysis of agmatine. It means the material that is produced. Specifically, putrescine is ornithine decarboxylase constitutive ( speC ) or inducible ornithine decarboxylase ( speF ) from ornithine in vivo , or arginine ) Can be synthesized by arginine decarboxylase and agmatinase. Although putrescine is present in decay, it is widely distributed as a normal component in living organisms and is included in ribosomes as a type of polyamine. Meanwhile, putrescine is known to promote cell growth and RNA synthesis in vivo.

The term “methanotroph” in the present invention means a bacterium that uses methane as a main carbon source or energy source. The methane magnetization bacteria may mean a host strain that is the target of transformation in the present invention, and also for the purposes of the present invention, methane is used as a carbon source, and pyruvate is used as a starting material, and finally, putrescine is passed through ornithine and arginine. Any strain that can be produced can be included without limitation. In addition, it is apparent to those skilled in the art that a strain capable of using methane among methylotrophs using a C1 compound such as methane, methanol, and methylamine as an energy source may also be included in the methane magnetization bacteria of the present invention.

The methane magnetization bacterium is not particularly limited due to that can be a C1 compounds such as methane as an energy source, methyl Pseudomonas genus (Methylomonas), methyl bakteo in (Methylobacter), methyl Rhodococcus genus (Methylococcus), methyl Ross The genus Ferra ( Methylosphaera ), Methylocaldum , Methyloglobus , Methylosarcina , Methyloprofundus , Methylothermusmus ), Methylohalobius ( Methylohalobius ), Methylogaea ( Methylogaea ), Methylomarinum ( Methylomarinum ), Methylovulum ( Methylovulum ), Methylomarinovum ( Methylomarinovum ), Methyllorum in (Methylorubrum), methyl Paracoccus genus (Methyloparacoccus), methyl Sinners in (Methylosinus), methyl seutiseu in (Methylocystis), cellar in (Methylocella), kaepsa in (Methylocapsa) methyl methyl upon by a methyl hydroperoxide rule It may be a strain of the genus La (Methylofurula), Methylacidiphilum , Methylacidimicrobium , or Methylomicrobium , specifically Methylomicrobium alcaliphilum . It may be 20Z. The bio-conversion process using methane magnetization bacteria is economically advantageous because it can use relatively inexpensive methane as a carbon source, and also has an advantage in environmental aspects such as prevention of greenhouse gas emission in that it can use methane in the atmosphere. In addition, there is no loss of oxygen compared to the process using glucose, and the yield of the final production material is also high.

In the present invention, the term "transformation methane magnetizing bacteria" refers to a strain that has been transformed by removing or introducing a gene of the methane magnetizing bacteria. Specifically, in the transformed methane magnetizing bacteria of the present invention, the activity of spermidine synthase in the methane magnetizing bacteria is weakened or inactivated compared to the intrinsic activity, and ornithine decarboxylase constitutive ) And putrescine transporter (putrescine transporter) activity may mean enhanced methane magnetization compared to the intrinsic activity, but is not limited thereto. In the present invention, the transformed methane magnetizing bacteria may have a significantly improved putrescine production capacity compared to the wild type.

In addition, in the present invention, the transformed methane magnetization bacteria may have improved putrescine resistance by adaptive evolution.

In the present invention, the term “adaptive evolution” refers to a type of evolution that involves evolutionary changes in which an organism can adapt to a specific environment, and for the purpose of the present invention, the strain M. alcaliphilum 20Z having low resistance to putrescine is low. It may be to increase the resistance to putrescine by exposure to putrescine dihydrochloride, but is not particularly limited thereto.

In one embodiment of the present invention, in order to increase resistance to putrescine of M. alcaliphilum 20Z, exposure to 100 mM putrescine dihydrochloride was repeated while culturing while stirring at 30 ° C and 230 rpm. , A strain having high resistance to putrescine was obtained, and the strain was confirmed to be able to grow normally even after 72 hours of exposure to putrescine (FIG. 3).

In the present invention, the term “spermidine synthase” refers to an enzyme that catalyzes the reaction of producing spermidine and methylthioadenosine from putrescine. The gene encoding spermidine synthetase ( speE ) may be composed of the nucleotide sequence of SEQ ID NO: 1, and 70% or more of the sequence of SEQ ID NO: 1, specifically 80% or more, more specifically 90% or more , More specifically 95% or more, most specifically 99% or more as a nucleotide sequence exhibiting homology, if substantially capable of expressing a protein having a spermidine synthase activity, may be included without limitation.

In one embodiment of the present invention, whether or not putrescine can accumulate in the transformed M. alcaliphilum 20Z strain that deleted the gene of spermidine synthase ( speE ) that catalyzes the conversion of putrescine to spermidine As a result, as a result, it was confirmed that the WT ΔspeE strain in which the gene was deleted can accumulate 0.35 mg / l putrescine after 96 hours of culture (FIG. 5).

In the present invention, the term "ornithine decarboxylase constitutive" refers to an enzyme that catalyzes a reaction to produce putrescine by removing the carboxyl group of L-ornithine. The gene ( speC ) encoding the ornithine decarboxylase may be composed of the nucleotide sequence of SEQ ID NO: 2, 70% or more with the sequence of SEQ ID NO: 2, specifically 80% or more, and more specifically 90 % Or more, more specifically 95% or more, and most specifically 99% or more, as a base sequence showing homology, which may substantially express ornithine decarboxylase protein, and may be included without limitation.

In the present invention, the term "putrescine transporter (putrescine transporter)" is a protein that catalyzes the absorption and release of putrescine, and the gene encoding the putrescine transporter ( potE ) may be composed of the nucleotide sequence of SEQ ID NO: 3 And a sequence of 70% or more, specifically 80% or more, more specifically 90% or more, more specifically 95% or more, and most specifically 99% or more of the sequence of SEQ ID NO: 3 If it is possible to express a protein having substantially putrescine transporter activity, it can be included without limitation.

In one embodiment of the present invention, to further increase the production of putrescine in M. alcaliphilum 20Z, the ornithine decarboxylase gene ( speC ) and the putrescine transporter gene ( potE ) are inserted under the control of the tac promoter. M. alcaliphilum 20Z was transformed with one pAWP89-speCE plasmid and cultured in NMS medium containing methanol or methane. As a result, it was confirmed that the transformed methanogenic WT speCE can accumulate 2.27 mg / l putrescine after 96 hours of culture. Furthermore, in the case of the strain (WT ΔspeE speCE) in which pAWP89-speCE was additionally introduced into the WT ΔspeE strain in which the spermidine synthase activity was inactivated, it was confirmed that 2.72 mg / L of putrescine was produced after 96-hour culture. (Fig. 5).

In the present invention, the term “lactate dehydrogenase” is an enzyme that catalyzes the reaction of converting pyruvic acid to lactic acid, and the gene encoding the lactic acid dehydrogenase ( ldh ) consists of the nucleotide sequence of SEQ ID NO: 4 It may be, and the sequence of SEQ ID NO: 4 and 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% or more, and most specifically, a base showing at least 99% homology When it is possible to express a protein having substantially lactic acid dehydrogenase activity as a sequence, it can be included without limitation.

The term “acetate kinase” in the present invention is an enzyme that catalyzes the reaction of converting acetyl-CoA (Acetyl-CoA) produced from pyruvic acid to acetate, a gene encoding the acetic acid phosphatase ( ack ) may be composed of the nucleotide sequence of SEQ ID NO: 5, the sequence of SEQ ID NO: 5 and 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% or more, Most specifically, as a base sequence showing at least 99% homology, a protein having substantially acetic acid phosphatase activity can be included without limitation.

In one embodiment of the present invention, a WT Δldh Δack ΔspeE having ldh and ack genes deleted for the WT ΔspeE strain and a WT Δldh Δack ΔspeE speCE strain introduced with pAWP89-speCE added thereto are prepared, and the yield of putrescine is evaluated. As a result, it was confirmed that the WT Δldh Δack ΔspeE strain produced about 0.88 mg / L and the highest 3.75 mg / L putrescine in the case of the WT Δldh Δack ΔspeE speCE strain.

Each of the enzymes or proteins described above is an amino acid in which one or more amino acids at one or more positions of the amino acid sequence constituting each enzyme or protein are substituted, deleted, inserted, added, or reversed, as long as each activity used in the present invention is exhibited. It may contain a sequence, as long as the activity of each enzyme or protein, 80% or more, specifically 90% or more, more specifically 95% or more, more specifically, with respect to the amino acid sequence of each enzyme or protein It is obvious to those skilled in the art that amino acids having substantially the same or corresponding activity as each enzyme or protein are included in the scope of the present invention.

In the present invention, the term "weakened or inactivated" means that the activity of the enzyme or protein is reduced or not expressed at all, compared to the wild type or its intrinsic activity, or even when expressed, the activity is absent or reduced. The weakening or inactivation of the enzyme or protein may be performed by any method known in the art. Specifically, deletion of a part or all of the polynucleotide encoding the enzyme, modification of the expression control sequence to decrease the expression of the nucleotide, modification of the nucleotide sequence on the chromosome to weaken the activity of the protein, or combinations thereof It may be performed in a selected method, but is not particularly limited thereto. The method of deleting part or all of the polynucleotide encoding the protein is performed by replacing a polynucleotide encoding an intrinsic target protein in a chromosome with a polynucleotide or a marker gene in which some nucleic acid sequences are deleted through a vector for chromosomal insertion in bacteria. Can be.

The term "enhancing" in the present invention means that the activity of the enzyme or protein is increased compared to the wild type or its intrinsic activity. Enhancing the activity of the enzyme or protein can be performed by any method known in the art.

Another aspect of the present invention for achieving the above object is the activity of the spermidine synthase (spermidine synthase) is weakened or inactivated compared to the intrinsic activity, ornithine decarboxylase constitutive and It is to provide a method for producing putrescine, comprising culturing a transformed methane magnetizing bacterium for putrescine production, wherein the activity of a putrescine transporter is enhanced compared to an intrinsic activity. The spermidine synthetase, ornithine decarboxylase, putrescine transporter, weakened or inactivated, enhanced, putrescine, transformed methane magnetizing bacteria are as described above.

In the present invention, the term "cultivation" refers to growing under environmental conditions artificially controlled cells or tissues of interest. The environmental conditions typically include nutrients, temperature, osmotic pressure, pH, gas composition, light, etc., but it is the medium that directly affects the medium, and the medium can be largely divided into a liquid medium and a solid medium. Cultivation of the transformed methane magnetizing bacteria of the present invention can be performed using methods well known in the art.

Specifically, the culture is not particularly limited as long as it can produce putrescine from the transformed methanogenic bacteria, but is continuously cultured in a batch process or in a fed batch or repeated fed batch process. can do. As the medium used for culture, NMS (nitrate mineral salts) medium known to be used for cultivation of methane magnetization bacteria may be used, and a medium in which the components contained in the medium or the contents thereof are properly adjusted according to the methane magnetization bacteria may be used. However, this is not particularly limited. In the present invention, the culture temperature of the transformed methane magnetization bacteria may be 15 ° C to 45 ° C, specifically 20 ° C to 40 ° C, more specifically 25 ° C to 35 ° C, for smooth contact between methane and the strain, 150rpm to 300rpm , Specifically, may be stirred at 180 rpm to 270 rpm, more specifically 200 rpm to 250 rpm, but is not limited thereto.

In the present invention, the method for preparing the putrescine may be culturing the transformed methane magnetizing bacteria in a culture medium containing a C1 carbon source. In addition, in the present invention, the method for preparing putrescine may be to increase the production amount of putrescine by adding NH ₄ Cl to the culture medium. Specifically, when the transformed methane magnetizing bacteria (ΔldhΔackΔspeE1ΔspeE2) was added and cultured with NO ₃ ^2- and additional NH ₄ Cl in NMS medium, it was confirmed that the production of putrescine was significantly increased (Table 2).

In the present invention, the manufacturing method may further include the step of recovering putrescine from the culture medium. The step of recovering the putrescine may be performed by a suitable method known in the art according to the culture method. Specifically, the known methods for recovering putrescine are not particularly limited, but centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (for example, ammonium sulfate precipitation), chromatography Methods such as HPLC, ion exchange, affinity, hydrophobicity and size exclusion can be used.

Another aspect of the present invention for achieving the above object is the activity of the spermidine synthase (spermidine synthase) is weakened or inactivated compared to the intrinsic activity, ornithine decarboxylase constitutive (ornithine decarboxylase constitutive) and Provided is a composition for the production of putrescine comprising a transformed methanogen for producing putrescine, wherein the activity of the putrescine transporter is enhanced compared to the intrinsic activity.

The spermidine synthetase, ornithine decarboxylase, putrescine transporter, attenuated or inactivated, enhanced, putrescine, transformed methane magnetizing bacteria are as described above.

The transformed methane magnetizing bacterium of the present invention shows high resistance to putrescine by adaptive evolution, so growth is not inhibited by putrescine produced by itself, and putrescine is removed from the C1 carbon source by strengthening the putrescine production pathway. Since it can be produced with high yield, it can be used for mass production of eco-friendly and economical putrescine.

1 shows the metabolic engineering pathway for the production of putrescine in M. alcaliphilum 20Z.
Figure 2 shows the putresin resistance of the M. alcaliphilum 20Z strain, grown in NMS medium at 30 ° C in an exponential growth phase (OD ₆₀₀ 0.4), and various concentrations of putrescine dihydrochloride (putrescine) dihydrochloride) to measure absorbance and evaluate the strain growth.
Figure 3 shows the increase in putrescine resistance of the M. alcaliphilum 20Z strain according to adaptive evolution. Empty circles represent the M. alcaliphilum 20Z strain (control) in NMS medium, and filled circles represent the M. alcaliphilum 20Z strain exposed to 100 mM putrescine dihydrochloride.
Figure 4 shows the growth curve in the NMS medium of the wild-type and transformed M. alcaliphilum 20Z strain.
Figure 5 shows a comparison of the production level of putrescine in the wild-type and transformed M. alcaliphilum 20Z strains.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Experimental Example 1: Materials and Methods

The strains ( M. alcaliphilum 20Z, 20Z speE and 20Z speCE, etc.) and plasmids (pCM433-speE1 and pAWP89-speCE, etc.) used in the present invention are shown in Table 1 below. Putrescine is a substance exhibiting toxicity to cells. In the present invention, the speC , speE , and potE genes optimized for the codon preference of M. alcaliphilum 20Z and the strain having increased adaptability (tolerance) to putrescine through adaptive evolution were used.

[Table 1]

Methylomicrobium alcaliphilum 20Z strain is 1.0 g / L MgSO ₄ .7H ₂ O, 0.02 g / L CaCl ₂ .6H ₂ O, 1.0 g / L KNO ₃ , 15 g / L NaCl, 2 ml / L trace element solution (Na ₂ -EDTA 0.5 g / l, FeSO ₄ .7H ₂ O 1 g / l, F2-EDTA 0.75 g / l, ZnSO ₄ .7H ₂ O 0.8 g / l, MnCl ₂ .4H ₂ O 0.005 g / l, H ₃ BO ₃ 0.03 g / l, 6H ₂ O with CoCl ₂ 0.05 g / l, Cu-EDTA 0.4 g / l, CuCl ₂ .2H ₂ O 0.6 g / l, NiCl ₂ . 6H ₂ O 0.002 g / l, Na ₂ MoO ₄ .2H ₂ O 0.05 g / l), phosphate buffer (KH ₂ PO ₄ 54.4 g / l, Na ₂ HPO ₄ .12H ₂ O 143.4 g / l) 2 ml / L, cultured in NMS (nitrate mineral salts medium) medium containing 4.5 ml / L of a 1 M solution of NaHCO ₃ , 0.5 ml / L of a 1 M solution of Na ₂ CO ₃ was sterilized by a filter before use (Ojala et al., 2011). The liquid culture was grown in a 500 ml baffle flask sealed with a screw cap in an atmosphere of 30% methane or 0.2% methanol at 30 ° C.

Specifically, as can be seen in Table 2 below, in the process, NO ₃ ^2- and additional NH ₄ Cl were added to the NMS medium and cultured, and the result was cultured from M. alcaliphilum 20Z-derived transformed methane magnetizing bacteria (ΔldhΔackΔspeE1ΔspeE2). It was confirmed that the production of tressin increased more.

[Table 2]

Experimental Example 2: Plasmid production

All plasmids used in the present invention were produced using Gibson Assembly (NEB, England). Primers of the invention designed using Primer 3 software and made with Macrogen (Seoul, South Korea) are listed in Table 3 below. The homology sequence for Gibson Assembly is shown in lower case, and the ribosome binding site is shown in bold.

[Table 3]

Genomic DNA of E. coli K12 strains W3110 and M. alcaliphilum 20Z were isolated using a Wizard® Genomic DNA Purification Kit (Promega, USA). PCR was performed using Lamp Pfu polymerase (BioFACT, Korea). Plasmids were prepared using Genome compiler software. Plasmid pAWP89-speCE amplifies ornithine decarboxylase constitutive (speC) and putrescine transporter (potE) from genomic DNA of E. coli K12 strain W3110 with the primers shown in Table 3 above. , Gibson assembly method. The sacB counter selection plasmid pCM433-speE1 is a spermidine synthase ( speE) through exchange of unlabeled alleles by amplification and binding of two adjacent homologous regions upstream and downstream of the target gene. ).

Experimental Example 3: Methylrobium Alkali Pilum 20Z ( M. alcaliphilum  20Z) strain transformation

M. alcaliphilum 20Z competent cells were prepared as follows: The culture of M. alcaliphilum 20Z was grown to an OD ₆₀₀ of 0.6 to 0.8, and the cells were obtained and centrifuged at 5000 g, 4 ° C. for 10 minutes. Washed twice with cold sterile water. After the resulting cell pellet was resuspended in cold sterile water, 50 μl of the cell suspension was used for electroporation. After gently mixing the competent cells with 500 ng of DNA plasmid, the mixture was transferred to a cold 1 mm cuvette (Bio-Rad). Electroporation was performed using a Gene Pulser Xcell ™ electroporation system (Bio-Rad) set at 1.3 kV, 25 kV and 200 Ω (Yan X. et al., 2016). After electroporation, 1 ml of room temperature NMS medium was added and resuspended, and then transferred to 20 ml medium in a 250 ml serum bottle containing 0.2% methanol. After incubating the cells at 30 ° C. for 24 hours, cells were obtained and plated on a selective plate.

Experimental Example 4: Analysis method

Cell growth was observed by measuring optical density at 600 nm with a Beckman spectrophotometer using a 1.5 ml cuvette with a 1 cm path length. The putrescine concentration was analyzed by high performance liquid chromatography (HPLC, Jasco Corporation, Japan) using a Symmetry C18 column (5 μm, Waters corporation, Massachusetts, USA) with a UV spectrophotometer detector at 360 nm. Putrescine was derivatized with o-phthaldialdehyde (OPA, Sigma, St. Louis, MO) using previously described methods (2007, Yildirim et al, 2007). The OPA reagent was prepared as follows: 0.2 g of OPA was dissolved in 9.0 ml of methanol, and 0.4 ml (pH 9.0) of borate buffer 1.0 ml and finally 160 ml of 2-mercaptoethanol were added. After adding 50 μl of sample and 450 μl of water to 400 μl of methanol, the mixture was derivatized using 100 μl of OPA reagent. The resulting mixture was immediately filtered through 0.2 μm PVDF syringe filter (Whatman, Maidstone, UK) and injected into HPLC. The composition of mobile phase A was pH 7.2, 55% methanol in 0.1M sodium acetate, and mobile phase B was methanol. The column temperature was maintained at 25 ° C. For separation, a mobile phase of 0.8 ml / min was used in the following gradient: 1-6 min, 100% A; 6-10 min, linear gradient change from 0% to 30% of solvent B; 10-15 min, linear change from 30% to 50% of solvent B; 15-19 min, linear gradient change from 50% to 100% of solvent B; 19-23 min, solvent B maintained at 100%; 23-25 min, linear gradient change from 100% to 30% of solvent B; 25-28 min, linear gradient change from 30% to 0% of solvent B. Standard putrescine was prepared in water and treated as described above.

Example 1: Adaptive evolution Methylrobium Alkali Pilum 20Z ( M. alcaliphilum  20Z) increased putrescine resistance

1-1: M. alcaliphilum  Confirm 20Z putrescine tolerance

M. alcaliphilum 20Z was incubated exponentially and exposed to putrescine dihydrochloride at different concentrations (20, 50, 100, 200 and 400 mM), respectively. As a result, putrescine, especially at putrescine concentrations above 50 mM,M. alcaliphilum 20ZThe growth rate of was significantly reduced. Specifically, optical density (OD₆₀₀) Slowly increased during the first 12 hours, but rapidly decreased after 12 hours of exposure, and it was confirmed that the added putrescine dihydro chloride caused cell lysis (FIG. 2). In other words, thisM. alcaliphilum 20ZLow putrescine toleranceM. alcaliphilum 20ZIt means that it can be a major cause of inhibiting the production of putrescine using metabolism.

1-2: by adaptive evolution M. alcaliphilum  Increased putrescine tolerance of 20Z

As confirmed in Example 1-1, as the concentration of putrescine increases, the growth rate of M. alcaliphilum 20Z decreases remarkably, for 40 days to increase the resistance of M. alcaliphilum 20Z to putrescine. Adaptive evolution was performed. Specifically, M. alcaliphilum 20Z was grown in NMS medium to OD ₆₀₀ 0.2, exposed to 100 mM putrescine dihydrochloride, stirred at 30 ° C. at 230 rpm and incubated for 5 days. The cells were then transferred to fresh NMS to restore and repeat the cycle. As a result, after 5 cycles, a strain having high resistance even at a concentration of 100 mM putrescine was obtained (FIG. 3). That is, from this, the putrescine-resistant strain has a slightly decreased growth rate after 72 hours of exposure to putrescine, but can grow normally, and thus can be used for the production of putrescine using the metabolism of the methanogenic M. alcaliphilum 20Z Was confirmed.

Example 2: Preparation of transformed methane magnetization for production of putrescine and confirmation of increase in putrescine production

Due to low resistance to putrescine and activation of the putrescine utilization pathway, the accumulation of putrescine could not be confirmed in wild-type strain M. alcaliphilum 20Z (FIG. 5). First, spermidine synthase It was confirmed whether putrescine could accumulate in the transformed M. alcaliphilum 20Z strain that deleted the gene ( speE ). Specifically, the vector pCM433-speE1 was composed of two adjacent homologous regions upstream and downstream of the spermidine synthase gene ( speE , 282 amino acids), and knocked out the gene through the exchange of unlabeled alleles. . As a result, it was confirmed that the WT Δ speE strain in which the spermidine synthase activity was inactivated was able to accumulate 0.35 mg / l of putrescine after 96 hours of culture (FIG. 5).

To increase the production of putrescine in M. alcaliphilum 20Z, overexpression of the ornithine decarboxylase constitutive ( speC ) and additionally putrescine transporter ( potE ) under the control of the tac promoter It was confirmed whether or not putrescine production. Specifically, the two genes were amplified and inserted into the pAWP89 vector to prepare a pAWP89-speCE plasmid. Then, M. alcaliphilum 20Z was transformed with pAWP89-speCE plasmid, and cultured in NMS medium containing 0.2% methanol or 30% methane in the atmosphere. As a result, it was confirmed that the WT speCE strain having enhanced ornithine decarboxylase and putrescine transporter activity can accumulate 2.27 mg / l putrescine after 96 hours of culture (FIG. 5). Furthermore, in the case of a strain (WT Δ speE speCE) in which pAWP89-speCE was additionally introduced to the WT Δ speE strain in which the spermidine synthase activity was inactivated, 2.72 mg / L of putrescine was produced after incubation for 96 hours. Was confirmed.

In addition, in order to further increase the production of putrescine, the gene encoding the lactic acid dehydrogenase ( ldh ) and the gene encoding the acetic acid phosphatase ( ack ) are further deleted to prevent the production of major bypass products, lactic acid and acetic acid, , Reducing the flow from pyruvate to the TCA circuit. Specifically, in the same manner as described above for the WT Δ speE strain, the WT Δ ldh Δ ack Δ speE deleted with the ldh and ack genes and the WT Δ ldh Δ ack Δ speE speCE strain in which pAWP89-speCE was additionally introduced therein As a result of evaluating the production amount of putrescine, WT Δ ldh Δ ack Δ speE strain produces about 0.88 mg / L, and WT Δ ldh Δ ack Δ speE speCE strain produces the highest 3.75 mg / L putrescine. Was confirmed.

That is, the methane magnetizing bacteria of the present invention, which have undergone the process of adaptive evolution to have resistance to putrescine, are not inhibited by growth of putrescine produced by themselves, and the spermidine synthase activity is weakened or inactive, ornithine The decarboxylase and putrescine transporter activities are enhanced, and the lactic acid dehydrogenase and acetic acid phosphatase activities are weakened or inactivated, and thus can be usefully used to produce putrescine in large quantities. In addition, the method for producing putrescine using the transformed methane magnetization bacteria is meaningful in that it can produce putrescine more environmentally and economically than the conventional chemical synthesis method.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without changing its technical spirit or essential characteristics. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the following claims rather than the above detailed description and equivalent concepts thereof.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Transformed methanotrophs for producing putrescine and uses          thereof <130> KPA180340-KR <160> 15 <170> KopatentIn 2.0 <210> 1 <211> 849 <212> DNA <213> Methylomicrobium alcaliphilum 20Z <400> 1 atgaatccga ccgaatggtt taccgaacaa gccccgggcg ccggctcggc cttttcgttg 60 aaaatcaaac gcaaattgca tgaagaacaa tcggattttc aatttttgga aatctatgaa 120 accgaagatt ttggcaattt gatggtcatc gatggctgca ccatggtctc gacccgcgat 180 aatttttttt atcatgaaat gatgtcgcat ccggtcttgt ttacccatcc gaatccgaaa 240 cgcgtctgga tcatcggcgg cggcgattgc ggcaccttga aagaagtctt gaaacatccg 300 ggcgtcgaac aagtcgtcca aatcgatatc gatgaacgcg tcacccgctt ggccgaaatc 360 tattttccgg aattgtgcga atcgaatggc gatccgcgcg ccgaattgaa atttatcgat 420 ggcatcaaat gggtcaaaga tgccgccccg ggctcggtcg atatcatcat cgtcgattcg 480 accgatccgg tcggcccggc cgaaggcttg tttggcgaag ccttttatcg cgattgcttg 540 aattgcttgt cggaaaatgg catggtcatc caacaatcgg aatcggcctt gtatcatttg 600 aatttgatgc aagccatgcg caatgccatg tcggccgccg gctttgatca tttgcaaacc 660 ttgttttttc cgcaatgcat ctatccgtcg ggctggtggt cggccaccat cgccggcaaa 720 aaatcgttgg gctcgtttcg ccaagaagat tcggccaata aaccgtttac caccgaatat 780 tataatgtcg atatccatcg cgccgccttg gcccaaccgg aatttgtcaa acgcgccttt 840 ggcggctaa 849 <210> 2 <211> 2136 <212> DNA <213> Escherichia coli K-12 W3110 <400> 2 atgaaatcga tgaatatcgc cgcctcgtcg gaattggtct cgcgcttgtc gtcgcatcgc 60 cgcgtcgtcg ccttgggcga taccgatttt accgatgtcg ccgccgtcgt catcaccgcc 120 gccgattcgc gctcgggcat cttggccttg ttgaaacgca ccggctttca tttgccggtc 180 tttttgtatt cggaacatgc cgtcgaattg ccggccggcg tcaccgccgt catcaatggc 240 aatgaacaac aatggttgga attggaatcg gccgcctgcc aatatgaaga aaatttgttg 300 ccgccgtttt atgatacctt gacccaatat gtcgaaatgg gcaattcgac ctttgcctgc 360 ccgggccatc aacatggcgc cttttttaaa aaacatccgg ccggccgcca tttttatgat 420 ttttttggcg aaaatgtctt tcgcgccgat atgtgcaatg ccgatgtcaa attgggcgat 480 ttgttgatcc atgaaggctc ggccaaagat gcccaaaaat ttgccgccaa agtctttcat 540 gccgataaaa cctattttgt cttgaatggc acctcggccg ccaataaagt cgtcaccaat 600 gccttgttga cccgcggcga tttggtcttg tttgatcgca ataatcataa atcgaatcat 660 catggcgcct tgatccaagc cggcgccacc ccggtctatt tggaagcctc gcgcaatccg 720 tttggcttta tcggcggcat cgatgcccat tgctttaatg aagaatattt gcgccaacaa 780 atccgcgatg tcgccccgga aaaagccgat ttgccgcgcc cgtatcgctt ggccatcatc 840 caattgggca cctatgatgg caccgtctat aatgcccgcc aagtcatcga taccgtcggc 900 catttgtgcg attatatctt gtttgattcg gcctgggtcg gctatgaaca atttatcccg 960 atgatggccg attcgtcgcc gttgttgttg gaattgaatg aaaatgatcc gggcatcttt 1020 gtcacccaat cggtccataa acaacaagcc ggcttttcgc aaacctcgca aatccataaa 1080 aaagataatc atatccgcgg ccaagcccgc ttttgcccgc ataaacgctt gaataatgcc 1140 tttatgttgc atgcctcgac ctcgccgttt tatccgttgt ttgccgcctt ggatgtcaat 1200 gccaaaatcc atgaaggcga atcgggccgc cgcttgtggg ccgaatgcgt cgaaatcggc 1260 atcgaagccc gcaaagccat cttggcccgc tgcaaattgt ttcgcccgtt tatcccgccg 1320 gtcgtcgatg gcaaattgtg gcaagattat ccgacctcgg tcttggcctc ggatcgccgc 1380 tttttttcgt ttgaaccggg cgccaaatgg catggctttg aaggctatgc cgccgatcaa 1440 tattttgtcg atccgtgcaa attgttgttg accaccccgg gcatcgatgc cgaaaccggc 1500 gaatattcgg attttggcgt cccggccacc atcttggccc attatttgcg cgaaaatggc 1560 atcgtcccgg aaaaatgcga tttgaattcg atcttgtttt tgttgacccc ggccgaatcg 1620 catgaaaaat tggcccaatt ggtcgccatg ttggcccaat ttgaacaaca tatcgaagat 1680 gattcgccgt tggtcgaagt cttgccgtcg gtctataata aatatccggt ccgctatcgc 1740 gattatacct tgcgccaatt gtgccaagaa atgcatgatt tgtatgtctc gtttgatgtc 1800 aaagatttgc aaaaagccat gtttcgccaa caatcgtttc cgtcggtcgt catgaatccg 1860 caagatgccc attcggccta tatccgcggc gatgtcgaat tggtccgcat ccgcgatgcc 1920 gaaggccgca tcgccgccga aggcgccttg ccgtatccgc cgggcgtctt gtgcgtcgtc 1980 ccgggcgaag tctggggcgg cgccgtccaa cgctattttt tggccttgga agaaggcgtc 2040 aatttgttgc cgggcttttc gccggaattg caaggcgtct attcggaaac cgatgccgat 2100 ggcgtcaaac gcttgtatgg ctatgtcttg aaataa 2136 <210> 3 <211> 1320 <212> DNA <213> Escherichia coli K-12 W3110 <400> 3 atgtcgcaag ccaaatcgaa taaaatgggc gtcgtccaat tgaccatctt gaccatggtc 60 aatatgatgg gctcgggcat catcatgttg ccgaccaaat tggccgaagt cggcaccatc 120 tcgatcatct cgtggttggt caccgccgtc ggctcgatgg ccttggcctg ggcctttgcc 180 aaatgcggca tgttttcgcg caaatcgggc ggcatgggcg gctatgccga atatgccttt 240 ggcaaatcgg gcaattttat ggccaattat acctatggcg tctcgttgtt gatcgccaat 300 gtcgccatcg ccatctcggc cgtcggctat ggcaccgaat tgttgggcgc ctcgttgtcg 360 ccggtccaaa tcggcttggc caccatcggc gtcttgtgga tctgcaccgt cgccaatttt 420 ggcggcgccc gcatcaccgg ccaaatctcg tcgatcaccg tctggggcgt catcatcccg 480 gtcgtcggct tgtgcatcat cggctggttt tggttttcgc cgaccttgta tgtcgattcg 540 tggaatccgc atcatgcccc gtttttttcg gccgtcggct cgtcgatcgc catgaccttg 600 tgggcctttt tgggcttgga atcggcctgc gccaataccg atgtcgtcga aaatccggaa 660 cgcaatgtcc cgatcgccgt cttgggcggc accttgggcg ccgccgtcat ctatatcgtc 720 tcgaccaatg tcatcgccgg catcgtcccg aatatggaat tggccaattc gaccgccccg 780 tttggcttgg cctttgccca aatgtttacc ccggaagtcg gcaaagtcat catggccttg 840 atggtcatgt cgtgctgcgg ctcgttgttg ggctggcaat ttaccatcgc ccaagtcttt 900 aaatcgtcgt cggatgaagg ctattttccg aaaatctttt cgcgcgtcac caaagtcgat 960 gccccggtcc aaggcatgtt gaccatcgtc atcatccaat cgggcttggc cttgatgacc 1020 atctcgccgt cgttgaattc gcaatttaat gtcttggtca atttggccgt cgtcaccaat 1080 atcatcccgt atatcttgtc gatggccgcc ttggtcatca tccaaaaagt cgccaatgtc 1140 ccgccgtcga aagccaaagt cgccaatttt gtcgcctttg tcggcgccat gtattcgttt 1200 tatgccttgt attcgtcggg cgaagaagcc atgttgtatg gctcgatcgt cacctttttg 1260 ggctggacct tgtatggctt ggtctcgccg cgctttgaat tgaaaaataa acatggctaa 1320                                                                         1320 <210> 4 <211> 924 <212> DNA <213> Methylomicrobium alcaliphilum 20Z <400> 4 atgaaaatag ccataatcgg agccggccat gtcggttctt cgttagccta cgcattggtg 60 ttgaagggct tgggtaatca tttgttgtta gtcaatcgcg atgcgaccaa agctttgggc 120 aatgccttgg atttacagca taccctggct ttttgcgaac ggccgatgca aatcgagggc 180 ggctcgcttg aagaggcggt gggctgcgat atcgtcgcga tcaccgcttc ggagccgatg 240 acaaaaggta tgacttctcg tatggaatta ggacaagcca atgtcgagtt gttcaagtcg 300 ttgattccaa tgctggccga aaataatcct gaagctgttt ttattatcat cacgaacccg 360 gtcgatgtga tgacttgttg ggctacgcgg gtttcggagt tgcccccttc aagaatcgtc 420 ggcatcggca cgctcgtcga ttcggcacgt ttcagaaccc tattatccaa ggccgaacag 480 attcatcccg atgatttaag agcctatatt cttggcgaac acggaccgaa tcagtttccg 540 gtatttagcc atgcttcggc cggcggcgaa ccgataaccg atagtccaag gcatagagag 600 atttttgaag aggtcaacaa ggccggtttc gaggtttatc gattgaaagg ctataccaat 660 ttcgcgattg cttcagccgc ttgcgaagtc attagaacca tcgttcacga cgaacaccga 720 acgatgccgc tcagtacttg cttcgatgat tggcagggaa tcaaagacaa ttgcttcagc 780 attccggtcg tactgggtcg ttccggtatt atccgatatt tacagcccga cctgaacagt 840 ctggagcgcg aagctctggt gcatacggcc aaaattgttc gagctaatat cgatagttta 900 ttgtttggag taggtgaaag gtga 924 <210> 5 <211> 1206 <212> DNA <213> Methylomicrobium alcaliphilum 20Z <400> 5 atgacaatac caaacggcaa cattctcgtg atcaacagcg gcagttcctc aatcaagtat 60 cgattgattg ctctgccgca agagcaggta ctggcagacg gcttgctgga acgcatcgga 120 gaacaggaaa gcaggatcat tcatagagcc gacgattcgg gtcgtttaaa tgaaatcaag 180 cagtcggtca tcgctgccga tcatcaccaa gccttcaagg cagtcttcga gattttgggc 240 gaaaattgct cggtcgatgc aataggccac cgcgtcgtgc atggcggcga tcggttctcc 300 ggccctgcct tggtcgatga cgatacgata gcgtcgatgc gcgcactctg ccgaatagcg 360 ccgctgcata atccggttaa cttgcttggc atcgagagtt gcttggctca tttcccgggc 420 gtaccacagg tggcggtatt cgatacggca tttcaccaaa cgatgccgcc ccacgcctat 480 cgttatgcga ttccggaaac ttggtatagc gattacggca tacgccgatt cggttttcac 540 ggcacctctc atcattatgt ggcgagacgg gccgccgaat ttatcggtaa acctttcgat 600 cgcagccatc tgattacttt gcatttgggc aatggcgcga gcgcaacagc gattgcaaac 660 ggccgctccg tcgatacgtc gatggggttt acgccgctgg aaggtttggt aatgggcacg 720 cgtagcggcg atttggaccc ggcaataccg ctatttgtcg aacaaaccga aaataccgac 780 acggacgcaa tcgaccgggc attgaaccgc gaatccggat taaaaggctt atgtggtacc 840 aacgacttaa gaaccgtgct cgaacaaaca aatgcaggcg atgaacgagc ccgcttggct 900 ctcgatctgt attgctatcg aatcaagaaa tatatcggcg cttactacgc ggtactcggc 960 gaagtcgacg ctctggtttt taccggcggc gtcggcgaaa acgcggccga agtgcgccgt 1020 ttagcctgcg aaggcctgtc gcgtctcggc atcgccattg atgaagcggc caatagcgac 1080 gtgaccggag ctatcgccga aatcgggctt gccgaaagtc gaacccgtat tctagtcatt 1140 aaaactgacg aagaattgca aattgcccgg gaagccatgg ctgtgcttga taaagatcac 1200 gcatga 1206 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pAWP89-ptac-For <400> 6 tagttgtcgg gaagatgcgt 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> pAWP89-ptac-Rev <400> 7 agctgtttcc tgtgtgaata 20 <210> 8 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> FWD_primer_speC <400> 8 cacacaggaa acagctatga aatcaatgaa tattgcc 37 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> REV_primer_speC <400> 9 gtgaatacct ttacttcaac acataaccgt 30 <210> 10 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> FWD_primer_potE <400> 10 tgttgaagta aaggtattca cacaggaaac agctatgagt caggctaaat cga 53 <210> 11 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> REV_primer_potE <400> 11 gcatcttccc gacaactatt aaccgtgttt atttttcagt 40 <210> 12 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> FWD_primer_speE F1 <400> 12 tggtctgaca gttaccagcc ggaaatgatg aagtcca 37 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> REV_primer_speE F1 <400> 13 gggatttctt gcgcattagt tatcgaggag 30 <210> 14 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> FWD_primer_speE F2 <400> 14 taatgcgcaa gaaatccctg cagtgttga 29 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> REV_primer_speE F2 <400> 15 ccgacaacct gcacatttcg tttaaggtct gtgcga 36

Claims (15)

The activity of spermidine synthase is weakened or inactivated compared to the intrinsic activity, and the activity of ornithine decarboxylase constitutive and putrescine transporter is compared to the intrinsic activity Enhanced, transformed methane magnetizing bacteria for putrescine production.
According to claim 1, wherein the lactic acid dehydrogenase (lactate dehydrogenase) and acetic acid phosphatase (acetate kinase) activity is weakened or inactivated compared to the intrinsic activity, the transformed methanogenic bacteria.
According to claim 1, The gene encoding the spermidine synthetase ( speE ) is composed of the nucleotide sequence of SEQ ID NO: 1, transformed methane magnetizing bacteria.
According to claim 1, wherein the gene encoding the ornithine decarboxylase ( speC ) is composed of the nucleotide sequence of SEQ ID NO: 2, the transformed methane magnetizing bacteria.
According to claim 1, The gene encoding the putrescine transporter ( potE ) is composed of the nucleotide sequence of SEQ ID NO: 3, the transformed methanogenic bacteria.
The method of claim 2, wherein the gene encoding the lactic acid dehydrogenase ( ldh ) is composed of the nucleotide sequence of SEQ ID NO: 4, the transformed methanogenic bacteria.
According to claim 2, wherein the gene encoding the acetic acid phosphatase ( ack ) is composed of the nucleotide sequence of SEQ ID NO: 5, the transformed methanogenic bacteria.
The method of claim 1 wherein the methane magnetization bacteria methyl Pseudomonas genus (Methylomonas), methyl bakteo in (Methylobacter), methyl Rhodococcus genus (Methylococcus), methyl Los Blow in (Methylosphaera), in methyl local bushes (Methylocaldum) in , Methyloglobus , Methylosarcina , Methyloprofundus , Methylothermusmus , Methylohalobius , Methylohalobius The genus Logea ( Methylogaea ), Methylomarinum , Methylovulum , Methylomarinovum , Methylorubrum , Methyloparacoccus , Methylosinus , Methylocystis , Methylocella , Methylocapsa , Methylofurula, Methylacidiphilum , Methyl acid microphone Transgenic methane magnetizing bacteria, which are strains of the genus Lobium ( Methylacidimicrobium ) or Methylomicrobium .
According to claim 1, wherein the methane magnetization bacteria are methylomicrobium alkalphilum ( Methylomicrobium alcaliphilum ) 20Z, transformed methane magnetization bacteria.
The method of claim 1, wherein the transformed methane magnetization bacteria, the enhanced ability to produce putrescine compared to the wild type, transformed methane magnetization bacteria.
According to claim 1, wherein the transformed methane magnetization bacteria, the adaptive evolution (adaptive evolution) will improve the putrescine resistance, the transformed methane magnetization bacteria.
A method for producing putrescine, comprising culturing the transformed methane magnetizing bacteria of any one of claims 1 to 11 in a culture medium containing a C1 carbon source.
The method of claim 12, further comprising recovering putrescine from the culture medium.
The method of claim 12, wherein NH ₄ Cl is additionally added to the culture medium to increase the amount of putrescine produced.
A composition for the production of putrescine, comprising the transformed methane magnetizing bacteria of any one of claims 1 to 11.

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