Art

[1]

The present invention relates to novel mutant ornithine dicarboxylate raised relates to a protein and use thereof.

[2]

BACKGROUND

[3]

Putrescine (or 1,4-butane-diamine) is an important raw material for the production of polyamide-4,6, including nylon 4,6. Was produced in a manner by the hydrogenation of succinonitrile (succinonitrile) produced by nitrile (acrylonitrile) acryloyl by hydrogen cyanide was added (hydrogen cyanide) in an industrial scale. The route of synthesis of these chemicals requires as a raw material a non-reproduction of the petrochemical product, a relatively high temperature with respect to a multi-phase (multi step), multi-reactor design (muti-reactor design), as well as an expensive catalyst systems and the pressure is required. In addition, because toxic and highly flammable reactants in conventional chemical synthesis routes is not good from an environmental aspect. Thus, from a biomass-derived carbon sources it can be reproduced as an alternative to the chemical production process and require a production of putrescine, recently bio-chemical process for producing microorganisms through the environmentally friendly is getting a lot of attention. Putrescine god is a type of polyamines that are found in the full range of organisms ranging from plants and animals from bacteria. The concentration of putrescine in E. coli has been very high as about 2.8g / l. In addition, microorganisms gajyeoseo potentially good resistance to high concentrations of polyamines, may grow or survive under the high concentration of the polyamine exist. For example, Corynebacterium glutamicum ( Corynebacterium glutamicum ) have been reported that can grow under more cadaverine (cadaverine) present 30 g / L. Therefore, there is research into using microorganisms is continuously made ​​as to produce an industrially available high concentration of the polyamine used. However, research to produce polyamines through microorganism is still not reach the state to a level that can industrially apply to the Then polyamine strain development of high yield should be conducted in the future continue (Qian ZG, et al, Biotechnol Bioeng, 104.: 651-662, 2009; Schneider J, et al, Appl Microbiol Biotechnol, 88:. 859-868, 2010).

[4]

[5]

On the other hand, dicarboxylic ornithine decarboxylase (Ornithine decarboxylase, ODC) is an enzyme that converted by enzymes present in most of the microorganisms, the ornithine to putrescine. ODC from the E. coli is typically forms an homodimer form, and is formed in the active site of the dimer interface. The reaction mechanism of the ODC is needed the PLP (pyridoxal phosphate) as a cofactor and, PLP that has formed a Schiff base (Schiff base) to a lysine residue of the active site of the enzyme substrate (substrate) of ornithine, lysine residue and replace after that, decarboxylation (decarboxylation) is a reaction occurs. When God created putrescine ODC is again formed as a PLP Schiff base.

[6]

[7]

Putrescine existing production, introduced in strains of Corynebacterium spp ODC activity has been reported as very low in protein-coding genes in E. coli speC. Therefore, in order to develop the putrescine-producing strain of high yield, improvement of the enzyme ODC is the last step in the production of putrescine path can be very important. However, the present structure and the reaction mechanism involved in the study increased as the active came experiment is in progress, such as introducing a mutation to the case for the ODC protein has not been reported.

[8]

Detailed Description of the Invention

SUMMARY

[9]

The present inventors have examples effort results in order to improve the ODC protein shown to putrescine to produce shoes one less play an important role in active, that with a new variation by discovering the location enhanced by introducing a mutation in that position putrescine production capacity variations ODC protein was produced, it was confirmed that existing putrescine by ODC protein god introducing mutated microorganisms to produce putrescine in God and can be produced in a yield, and completed the present invention.

[10]

Technical Solution

[11]

One object of the present invention is to provide a novel mutant ornithine dicarboxylate raised (ornithine decarboxylase, ODC) protein.

[12]

Another object of the invention is to provide a vector and a transformant transformed with the vector comprising the nucleic acid molecule, the nucleic acid molecule encoding a protein with the mutant ODC.

[13]

It is another object of the L- ornithine (L-ornithine), ODC protein was added to the above variation in the L- ornithine L- ornithine or a mixture containing the fermentation broth comprising the step of reacting, putrescine production of the present invention to provide a process.

[14]

It is another object of the present invention having the ability Corey putrescine production in Corynebacterium ( Corynebacterium sp. To provide an improved ability to introduce a recombinant microorganism the mutant ODC protein, putrescine production in microorganisms).

[15]

Another object of the present invention is the genus Corynebacterium (having improved putrescine-producing ability by introducing the above variations ODC protein Corynebacterium sp. Culturing a) microorganisms: putrescine and from the culture obtained in the above step water comprising the step of separating the gods putrescine to provide a new production method.

[16]

Effects of the Invention

[17]

Ornithine digital camera decarboxylase protein mutated in accordance with the present invention not only it increases the putrescine switching activity up to 21-fold compared to wild-type, increasing significantly the existing putrescine putrescine during the introduction of the production strains gods production activity bar, may be widely utilized in a more efficient mass production of putrescine an alternative to traditional chemical synthetic routes.

[18]

Brief Description of the Drawings

[19]

1 is a view comparing measured putrescine conversion activity of the protein, or the introduction of the E165A mutant I163A ODC protein in E. coli and the native protein. Specifically, according to the conversion reaction occurs there is an increase in pH to determine the pH increased to phenol red was confirmed an increase in the absorbance. ODC as compared to wild-type protein was confirmed that the conversion activities of putrescine I163A or E165A, or ODC protein was introduced two variations is superior.

[20]

Best Mode for Carrying out the Invention

[21]

In some embodiments for achieving the above object, the present invention has an amino acid sequence set forth in SEQ ID NO: 1 ornithine dicarboxylate raised (ornithine decarboxylase, ODC) beonjjae isoleucine 163 (isoleucine), and 165 amino acid residues from the N- terminus of beonjjae the at least one selected from the group consisting of glutamic acid (glutamic acid) amino acid residues is mutated, provides for a novel mutation variant ODC protein.

[22]

[23]

As used herein, the term is "ornithine dicarboxylic acid Reyes (ornithine decarboxylase, ODC)" is an enzyme that catalyzes the first step and got to the final stage of the path to synthesize the production of putrescine from ornithine, polyamine Scheme. To produce an L- ornithine to putrescine as a substrate, it acts as pyridoxal phosphate (Pyridoxal phosphate, PLP) This co-factor.

[24]

[Reaction]

[25]

L- ornithine putrescine <=> CO + 2

[26]

In the present invention raise ornithine dicarboxylate (ornithine decarboxylase, ODC) is specifically may be derived from E. coli ODC, more specifically Escherichia coli ( Escherichia coli may be an ODC having the amino acid sequence set forth in) derived from SEQ ID NO: 1.

[27]

[28]

In the present invention, the method for securing the ODC (ornithine decarboxylase) is capable of a variety of methods well known in the art applies. An example of the method is screening of a useful enzyme resources by generally containing the codon optimized to obtain the enzyme in E. coli with high efficiency used DNA synthesis techniques and the bulk dielectric information chemical method based on the organisms of the microorganism to enzyme expression It can be obtained through the method, and are not limited thereto.

[29]

[30]

In the present invention, "Mutated ODC protein" means the amino acid sequence one or more amino acids of the protein refers to the ODC The ODC protein added, removed, or replaced. In the present invention, specifically refers to an effective increase in the activity compared to the wild type protein to the ODC protein by the mutation. In the present invention, variations may be used without limitation generally known methods are known in the art as a way to improve the enzyme, whereby there is a strategy such as rational design (rational design) and induce evolution (directed evolution). For example, rational design strategy has the amino acid position of a specific location, such as a specified mutation introduction (site-directed mutagenesis), and the induction strategy has evolved and a method that causes the random variations (random mutagenesis). Moreover, without an external operation by a natural mutation, 163 the second and / or amino acid residue 165 of SEQ ID NO: 1 can be mutated. As used herein, the term "an ODC protein mutant", "ODC variant" and "variant speC" may be used interchangeably.

[31]

The invention of the variation of ODC protein Specifically, E. coli (Escherichia coli) derived from SEQ ID NO: 1 as set forth the amino acid sequence of having ornithine dicarboxylate raised (ornithine decarboxylase, ODC) of the N-terminus from the 163 beonjjae isoleucine (isoleucine) amino and residues, and / or 165 beonjjae glutamic acid (glutamic acid) amino acid residues may be mutated. Examples is above 165 beonjjae glutamic acid is substituted with alanine (alanine), glycine (glycine), serine (serine) or Val (valine) or, wherein the 163 beonjjae isoleucine may be substituted with alanine (alanine), glycine (glycine), serine (serine) or Val (valine). In addition, the 163 beonjjae isoleucine and 165 beonjjae glutamic acid as ODC protein mutated at the same time, 163 beonjjae isoleucine and 165 beonjjae glutamic acid may be substituted by each selected from the group consisting of alanine, valine, serine and glycine, and specific examples 163 beonjjae isoleucine and glutamic acid, respectively 165 beonjjae alanine-alanine, alanine-valine, serine-valine or valine - may be substituted with valine.

[32]

In the embodiment of the present invention, if the introduction of mutations in the 163, 165 amino acids of the wild-type ODC in various combinations, to confirm that the increased ability putrescine production, the position of the most to produce an enhanced ODC variants capability putrescine production It was identified as an important location. In particular, via a mutation for replacing an amino acid present in the critical transition location to a small residue (small residue), amino acids (alanine, serine, valine, or glycine), it was confirmed that the increase in production ability putrescine.

[33]

Also, ODC protein mutated in this invention may be composed of the amino acid sequence described by any of SEQ ID NO: 34 to SEQ ID NO: 57. Specifically, it may be constituted by SEQ ID NO: 34 to SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 49 and SEQ ID NO: amino acid sequence of any one of the substrate 57. This 163 beonjjae isoleucine, or the amino acid sequence for the ODC protein mutated at 165 beonjjae glutamic acid is a small residue, the containing the mutation and at least having excellent putrescine conversion activity than the wild-type 50% with the sequence from the N-terminus, 60% , 70%, and may include without 75%, 80%, 85%, 90%, 95%, limited to a polypeptide having a homology of more than 97% or 99%.

[34]

[35]

As used herein, the term "homology" refers to the percent of identity between two polynucleotide or polypeptide Mo ET. Between sequences from one parent to the other one of the parent ET ET homology can be determined by the known art. For example, it is possible to directly align the sequence information between two polypeptide molecules or two polynucleotide molecules is determined homology between two Mo ET by using computer programs available for sequence alignment information, and readily available. In addition, after a polynucleotide homology between a polynucleotide hybridizing under conditions of forming a stable duplexes between homologous regions, single-stranded-specific nuclease by decomposing agent can be determined by determining the amount of degradation fragments.

[36]

The term in the invention, "homologous" includes a protein homologous in all grammatical forms and spelling variations form a superfamily, and proteins derived from other species origin, refers to the relationship between the protein having a "common evolutionary origin". Such proteins (and their encoding genes) have sequence homology, which is reflected by the high degree of sequence similarity. However, if generally used as "homology" in the present invention are to be qualified by the jerk-type, such as "high" will have a sequence similarity speaking not meant to be a common evolutionary origin.

[37]

In the present invention, the term "sequence similarity" means the degree of identity or correspondence between the nucleotide sequences or amino acid sequences of proteins that may or may not share a common evolutionary origin. In one embodiment, two amino acid sequences are at least 21% of the polypeptide match for a given length of the amino acid sequences (specifically at least about 50%, about 75%, more specifically, 90%, 95%, 96%, 97 when% or 99%), "substantially homologous" or is "substantially similar". Substantially homologous sequences can be confirmed by using a standard software which is used in the data bank, or, for example, comparing the sequences by Southern hybridization (southern hybridization) experiments under stringent conditions as defined for the particular system. Appropriate hybridization conditions is within the technical scope defined (eg. Sambrook et al., 1989, see infra).

[38]

[39]

In the specific embodiment of the invention, the structural analysis of the ODC protein derived from E. coli, induced mutations through rational design strategies based on the structure information. Mutation to widen the opening portion (entrance region) of the passage for the substrate access to the active site (V156, D160, I163, E165, Q691) and mutant (N153, D309 for stabilizing the co-factor of PLP coupled to the active site ) was designed and manufactured (examples 1 and 2). Specifically, the activity of ODC protein when through the mutation to replace a residue bulky inlet region of the passage with the alanine small residues (small residue) substituted with alanine the 163 amino acid of isoleucine and 165 amino acid of glutamic acid from the N-terminal this was found to significantly increase (example 3). Meanwhile, ODC protein by introducing mutations into mutant V156A, D160A, Q691A, N153D, N153E, D309E mutation in the remaining six species, including the PLP for stabilizing the activity is greatly reduced compared to the wild type or the results were almost gone. Accordingly, it sikineunde 163 beonjjae isoleucine and glutamic acid 165 beonjjae of ODC protein derived from E. coli (SEQ ID NO: 1) increases the activity of the protein was identified as an important residue. The introduction of the mutation is located using the primer and the PCR described in Table 2 were made through the specified mutations.

[40]

Further, in the embodiments of the present invention, additionally the 163 beonjjae isoleucine and 165 beonjjae glutamic acid by the which are in addition to substitution with alanine another small residue serine (serine), Val (valine) or even replaced with a glycine (glycine), the It was to optimize the variation of the residue (example 4 and Table 4). After a 163 and 165 amino acid residue to introduce a single mutation in each of glycine (G, glycine), serine (S, serine), valine (V, valine), when the 163 amino acid moiety, with serine and 165 amino acid residues in the case when one is substituted with valine kcat / K M was confirmed that each representing a 4.4-fold, 6.9-fold increase in activity compared to the wild-type value (Table 5). Based on these results, the introduction of the double mutant was confirmed for both the active moiety. If you have a single transition introduce all I163S and E165V union activity is highest when introducing compared to showing the 8-fold increase in activity compared to wild-type, 163 and 165 amino acid if both are replaced by valine residues 21.3-fold increase in activity compared to wild-type showing the highest activity was found with a (example 4 and Table 5).

[41]

Overall, the increase in ODC activity enzyme variants K M , rather than reduce the value of the kcat due to the increase in value of kcat / K M was an increase in value. This suggests that the structure of the ODC enzyme is changed in a direction to increase the speed to be converted into a product of putrescine than increasing polarity binding affinity for an enzyme of the ornithine substrate due to mutations introduced.

[42]

[43]

ODC enzyme activity measurement method of the present invention to convert the tin ornithine to putrescine is used in the reaction for the ODC enzyme mediated. Specifically, ODC enzyme ornithine molecules of water consumed dog when you switch to tin molecule putrescine and putrescine God with carbon dioxide molecules and OH - are the molecular ion produced. Accordingly, since the overall pH is increased, measuring the increase in pH at 559 nm as one of the phenol red (phenol red) of the pH indicator, the reaction proceeds thereby in proportion to the pH is increased amount increases the value of the absorbance. Indirectly measure the amount putrescine using such characteristics reactions.

[44]

[45]

As used herein, the term to "ornithine" is a basic amino acid that plays an important role in the ornithine circuit, in particular L- ornithine is widely found in plants, animals, microorganisms. Typically, ornithine and the relation factor production in vivo with a tin circuits play an important role for the spirit. In addition, in vivo and it may be arginine, glutamic acid, proline and converted each other to the a- ketone acid, oxalic acid and the glycidyl group transmission. Through this, as a substrate to produce the amine (putrescine) by ornithine decarboxylase it is synthesized from a dicarboxylic polyamine. According to the present invention may be L- ornithine butynyl can be used in particular as a substrate for ornithine decarboxylase digital camera.

[46]

In the present invention, the term, "putrescine" is widely distributed in the normal components in a living body present in a material produced by the hydrolysis reaction of dicarboxylic acids or of ornithine Agde Martin, putrefaction. Configure the ribosome as a kind of a polyamine, and has a function to facilitate or promote the growth of the cells RNA synthesis. In particular, industrially and is available for the relevant raw material for the production of polyamide-4,6, including nylon 4,6, a material that is still a need for studies for the mass production.

[47]

[48]

As another aspect, the invention provides a nucleic acid molecule encoding an ODC mutated protein of the invention.

[49]

As used herein, the term "nucleic acid molecule" is a DNA and has a meaning that includes the RNA molecule comprehensively, basic structural unit of the nucleotide is a natural nucleotide, as well as sugar or base region modified analogs in the nucleic acid molecule (analogue) includes do.

[50]

[51]

As another aspect, the present invention provides a vector comprising a nucleic acid molecule encoding a mutated ODC protein of the present invention.

[52]

In the present invention, the term "vector" refers to any vehicle for the cloning and / or transformation of the base into a host cell. The vector may be a replicon (replicon) that may lead to replication of fragments coupled to combine the different DNA fragments. Which functions as the self-unit of the "replicon" is DNA replication in vivo, i.e., possible replication by modulation of self, any genetic unit (e. G., Plasmid, phage, cosmid, chromosome, virus) refers to . The term "vector" includes viral and non-viral vehicle for introducing a nucleotide into a host cell in vitro, ex vivo or in vivo. The term "vector" may also include a mini spherical DNA. For example, the vector may be a plasmid that does not have a bacterial DNA sequence. Removal of bacterial DNA sequences rich in CpG region may reduce the transgene expression silencing was done to get a more sustained expression from plasmid DNA vectors. The term "vector" may also include a transposon, or an artificial chromosome.

[53]

In the present invention, vectors are vectors comprising a nucleic acid molecule encoding an ODC protein mutated according to the present invention is not specifically limited to, mammalian cells (for example, man, monkey, rabbit, rat, hamster, mouse cells, etc.), plant cells, yeast cells, insect cells or bacterial cells and in eukaryotic or prokaryotic cells comprising (e.g., E. coli, etc.) can be a vector that can replicate and / or express the nucleic acid molecule. Specifically connected operably to an appropriate promoter so that the expression of the polynucleotide in a host cell, can be a vector that includes at least one selectable marker, more specifically, a phage, plasmid, cosmid, mini- chromosome, virus, retrovirus vectors or the like may be introduced form the said polynucleotide.

[54]

pET system (novagen) using the T7 promoter which is conventionally used in the art are well known, and can take advantage of a variety of expression systems known in the art are not limited. Specifically, the vector comprising a nucleic acid molecule encoding an ODC protein mutated in the present invention may be in the pET28a vector.

[55]

[56]

In one specific embodiment of the present invention, position via the PCR - a nucleic acid molecule encoding a mutant designated ODC protein was inserted into the pET28a vector. Through this, the variation in ODC (speC) expression vector, pET28a-speC_I163A, pET28a-speC_I163G, pET28a-speC_I163S, pET28a-speC_I163V, pET28a-speC_E165A, pET28a-speC_E165S, pET28a-speC_E165G, pET28a-speC_E165V, pET28a-speC_I163A_E165A, pET28a -speC_I163S_E165V, pET28a-speC_I163A_E165V, was prepared in the pET28a-speC_I163V_E165V, whether introduced mutations was confirmed by sequencing.

[57]

[58]

As another aspect, the present invention provides a transformant transformed with the vector.

[59]

Transformants in the present invention as long as it can express a variant of the invention the ODC vector is introduced is not particularly limited, transformed Escherichia coli ( E. coli ), genus Corynebacterium, Streptomyces MRS, Salmonella typhimurium bacterial cells, such as Mu Solarium; Yeast cells; Fungal cells, such as blood teeth Paz Laboratories; Draw Joe Pilar, insect cells such as Spodoptera Sf9 cells; CHO (Chinese hamster ovary cells, chinese hamster ovary cells), SP2 / 0 (mouse myeloma), human lymph subregion (human lymphoblastoid), COS, NSO (mouse myeloma), 293T, bow melanoma cells, HT-1080, BHK ( baby hamster kidney cells, animal cells, such as baby hamster kidney cells), HEK (human embryonic kidney cells, human embryonic kidney cells), PERC.6 (human retina cells); Or it may be a plant cell.

[60]

[61]

In yet one aspect, the present invention is L- ornithine (L-ornithine), including the step of reacting ODC protein was added to the above variation in the L- ornithine L- ornithine or a mixture containing fermentation broth, putrescine It provides a new production method.

[62]

L- ornithine, putrescine and mutated protein such as ODC is as described above.

[63]

In the present invention, a substance for putrescine mutated ODC protein and the reaction for the preparation may be a L- ornithine, L- ornithine L- ornithine or a mixture containing fermentation broth. L- ornithine and the mixture containing the tin mean L- ornithine and the mixture is mixed with other ingredients that existed separately, L- ornithine is fermented through a process of fermentation by L- ornithine is generated or the content is rising It can mean a fermentation broth containing L- ornithine sufficient for the reaction, but is not limited thereto.

[64]

For example, through a process of fermentation with respect to a method and the resulting fermentation broth to produce a L- ornithine, conventional bars are disclosed in US Patent Application No. No. 3,668,072, it may be consulted to herein ( E.coli , ATCC 21104) .

[65]

[66]

In the present invention, the mutant protein may be a fermentation broth of ODC which contains the purified ODC protein mutation or mutant microorganisms ODC protein. Specifically, the microorganism used to prepare the microbial fermentation broth may be a microorganism expressing the ODC protein mutated according to the present invention, more specifically, with a vector comprising a nucleic acid molecule encoding an ODC protein mutated in this invention transformants can be converted transformant microorganisms.

[67]

[68]

As another aspect, the present invention produces putrescine Corey has a function in Corynebacterium ( Corynebacterium sp. Provided enhanced microbial capability) has been introduced ODC protein, the mutant microorganisms, putrescine production.

[69]

The term in the invention, "microorganism" is the wild-type microorganism or a natural or artificially includes both happened microbial genetic modification, the foreign gene inserted, or due to a reason such as the activity of the intrinsic gene is enhanced or weakened weakening the specific mechanism or microorganism may be enhanced.

[70]

As used herein, the term "putrescine produced Corridor has the ability Corynebacterium genus ( Corynebacterium sp. Refers to the genus Corynebacterium microorganisms with putrescine production capacity) microorganism" through the natural type or mutated. The Leeum of Corynebacterium microorganisms in culture with the putrescine was already known. However, the putrescine production capability was significantly lower status or genetic mechanism acting on the principle of the production mechanism unknown. Thus, by the Corridor Four of the putrescine production capacity in the invention it tumefaciens in microorganisms is a natural type isolates itself or an external putrescine production mechanisms and genes are inserted with, or to enhance the activity of the intrinsic genes or weaken improved putrescine refers to the genus Corynebacterium microorganism have the producing ability.

[71]

In the present invention, "the genus Corynebacterium microorganism" specifically Corynebacterium glutamicum, Corynebacterium ammonia to Ness, Brevibacterium lactose flops momentum ( Brevibacterium lactofermentum ), Brevibacterium Playa Pan ( Brevibacterium flavu m), Corynebacterium thermoaminogenes amino to Ness ( Corynebacterium thermoaminogenes ), Corynebacterium epi syeonseu ( Corynebacterium efficiens or the like), but is not limited thereto. More specifically, in the present invention, the genus Corynebacterium microorganism is a high concentration of putrescine even when exposed to a new four Corey are more immune to cell growth and survival tumefaciens glutamicum ( Corynebacterium glutamicum may be). For example, weaken the activity of the protein than the endogenous activity NCgl1469 putrescine may be a new improved production capability Corynebacterium glutamicum KCCM11240P (KCCM11138P △ NCgl1469) strains, but is not limited thereto. As putrescine and the production strain was a deletion of the gene coding for NCgl1469 KCCM11240P strains in order to block the path to the synthesis of putrescine N- acetyl God putrescine in the cell, International Patent Publication WO2013 / 105827 strains disclosed in to be.

[72]

[73]

In the specific embodiment of the present invention, the weakens the activity of the NCgl1469 protein than the endogenous activity putrescine production capability enhanced genus Corynebacterium microorganism (KCCM11240P (KCCM11138P △ NCgl1469)) on the basis of the putrescine conversion activity the increased ODC I163S / E165V (speC) variants were produced mutant was introduced into the chromosome in wild-type speC (example 6). The mutant Corynebacterium glutamicum named CC01-0578 and 10 June 2013 under the Budapest Treaty on the International Culture Center of Microorganisms Depositary agency Korea (Korea Culture Center of Microorganisms, KCCM ) was deposited with the accession number KCCM11425P.

[74]

[75]

As another aspect, the invention is the genus Corynebacterium (having improved putrescine-producing ability by introducing a mutated ODC protein of the present invention Corynebacterium sp. Culturing a) microbes; Putrescine and provides a production method including the step of separating putrescine from the culture obtained in the above step.

[76]

Specifically, the genus Corynebacterium microorganism may be Corynebacterium glutamicum Corey kumil Tommy, more specifically, Corynebacterium glutamicum may be CC01-0578 (accession No. KCCM11425P) strain.

[77]

[78]

As used herein, the term "culture" means that the growth conditions in a controlled environment with a microorganism appropriately artificially. Method in the present invention using a microorganism of the genus Corynebacterium is cultured putrescine can be carried out using methods well known in the art. Specifically, the culture is a batch process, injection process batch or repeated injection arrangement (fed batch or repeated fed batch process) in a continuous culture may be, but is not limited to that.

[79]

The medium used to culture should meet the requirements of the particular strains in a suitable manner. Corynebacterium sp for the culture medium has been known (e.g., Manual of Methods for General Bacteriology. American Society for Bacteriology. Washington DC, USA, 1981). In that can be used glycogen is glucose, saccharose, lactose, paroxetine lactose, maltose, starches, sugars and carbohydrates such as cellulose, soybean oil, sunflower oil, castor oil, such as coconut oil and fat, palmitic acid, stearic acid, , fatty acids such as linoleic acid, glycerol, alcohols such as ethanol, may be included the organic acids such as acetic acid. These materials may be used individually or as a mixture. With which can be used nitrogen source include peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean, for wheat, and the element or the inorganic compound, for example, may include ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. Nitrogen sources can also be used separately or as a mixture. By personnel that may be used are phosphoric acid or the corresponding sodium, potassium hydrogen phosphate or potassium susoyi-containing salts can be included. In addition, the culture medium may contain metal salts such as magnesium sulfate or iron sulfate needed for growth. Finally, in addition to the above materials can be used is essential growth substances such as amino acids and vitamins. In addition, suitable precursors may be used in the culture medium. The raw materials can be added to a batch or continuously by any suitable way to the culture in the culture process.

[80]

Using a base or an acid compound or phosphoric acid compound such as sulfuric acid, such as sodium hydroxide, potassium hydroxide, and ammonia in a suitable manner it is possible to adjust the pH of the culture. In addition, by using an antifoaming agent such as fatty acid polyglycol ester can suppress foam generation. It can be injected containing gas (eg, air), oxygen or oxygen into the culture in order to maintain aerobic conditions. A culture temperature of water is usually 20 ℃ to 45 ℃, specifically may be a 25 ℃ to 40 ℃. Culture is continued until the maximum obtainable amount of God putrescine desired. For this purpose usually can be achieved in 10 to 160 hours. Putrescine or God can be released into the culture medium, contained in the cells.

[81]

[82]

How to produce putrescine according to the present invention comprises the step of recovering putrescine from the cell or the culture. The method for recovering putrescine from the cells or the culture is known in the art, for example, centrifugation, filtration, anion exchange chromatography, crystallization and HPLC, but may be used such as, but is not limited to these examples.

[83]

Mode for the Invention

[84]

It will now be described in more detail to the present invention the following examples. They are, however, but the scope of the invention intended for the purpose of illustrating the invention by way of example to be limited to these Examples.

[85]

[86]

Example 1. ODC (ornithine decarboxylase) structural analysis and design variants

[87]

[88]

In general, it is known that E. coli has two types of ODC. One is inducible ODC (speF) that when the surrounding pH is acidic condition expression induction, the other is a constitute ODC (speC) which is involved in producing a diamine, such as putrescine gods (Applebaum DM, et al, Biochemistry, 16.: 1590-1581, 1977). Among them it was chosen constitute ODC speC involved in the putrescine produced in a gene of interest.

[89]

[90]

In up to now been found that the structure of the bacteria there ODC of parahaemolyticus and Lactobacillus bacteria, ODC (speC) in E. coli of which is predicted to have a similar structure to the Lactobacillus 30a ODC. Therefore, Lactobacillus ODC 3D structure based on the E. coli ODC (speC) in the amino acid sequence alignment comparing the GeneDoc program by using the (align) was (Momany C, et al., J Mol Biol, 4: 849-854, 1995) . Comparison of the amino acid sequence of Escherichia coli and Lactobacillus bacteria speC 30a of the ODC 53% sequence identity, 65% similarity to the two enzymes was found to be very similar proteins. Therefore, RCSB Protein Data Bank at me with Lactobacillus 30a ODC (PDB ID: 1ORD) structures based on homology modeling the structure of the E. coli speC (homology modeling) was proceeding. As a result, the overall framework of the protein was almost similar, PLP (pyridoxal phosphate) and amino acid sequence of the active site (active site) was also almost the same involved in binding.

[91]

For both enzymes structure analysis ODC of the there is present in a dimeric form of cells, the active sites formed in the boundary surface (interface) of the dimer, the inlet portion (entrance region) of the passage narrowed to the substrate access to the active site It was analyzed by one. Therefore, coming substrate is example by up to the active site to give widen the entrance area to be converted the product quickly, by targeting a bulky one residue in the inlet region were designed mutations to introduce a mutation for substituting a small residues (V156 , D160, I163, E165, Q691).

[92]

Additional mutations were also designed with respect to the active site residues to stabilize around a co-factor of PLP to which is bonded to the active site (N153, D309).

[93]

[94]

Example 2. E. coli ODC (speC) gene cloning and expression

[95]

[96]

Was typically used to pET28a (Novagen) vector system is used for enzyme expression In order to express the E. coli gene speC. Preferentially speC gene was amplified by PCR using primers disclosed in Table 1 to the chromosome of the E. coli wild-type strain W3110 as a template. Gene fragment obtained by PCR amplification with vector pET28a was inserted into the pET28a vector speC gene fragment by using restriction enzymes XhoI and NdeI after the treatment (37 ° C, 3 hours), the conventional ligation techniques.

[97]

[98]

Table 1 [Table 1]
primer Primer sequences
speC_start (NdeI) _5 (SEQ ID NO: 2) Cagccatatgaaatcaatga-5'-3 '
speC_stop (XhoI) _3 (SEQ ID NO: 3) Ggtgctcgagttacttcaac 5'-3 '

[99]

[100]

Whether the introduction of mutations speC expression vector (pET28a-speC) produced in the above was confirmed by sequence analysis.

[101]

[102]

Was introduced a mutation to alanine substitution in the embodiment (alanine) residue, respectively for a small residue on the target 1, to stabilize the PLP according to the position to combine with each other PLP was introduced mutations.

[103]

By the vector pET28a-speC as prepared in the above as a template, PCR was performed using primers disclosed in Table 1 and Table 2 below. 1, a car proceeds each PCR with respect to the front part (5 ') and the rear part (3') around the area you want to cause a mutation to introduce a mutation in the speC gene, and then combine the two PCR fragments as Secondary It was carried out the PCR. For example, speC V156A cases, the front part speC_start (NdeI) _5 (SEQ ID NO: 2) and speC_V156A_3 (SEQ ID NO 5) Primers for use in PCR amplification and, the back portion is speC_V156A_5 (SEQ ID NO: 4) and speC_stop (XhoI ) _3 (SEQ ID NO: 3) was amplified by PCR using primers. Primary using PCR Two PCR fragment obtained through a template of the second PCR, and using the speC_start (NdeI) _5 (SEQ ID NO: 2) and speC_stop (XhoI) _3 (SEQ ID NO: 3) primers was performed by PCR. V156A speC gene obtained finally was inserted into the pET28a vector was made in the same manner as speC gene fragment. Other variations remainder was also inserted into the pET28a vector by using primers described in Table 2, the PCR proceeds in the same manner as described above.

[104]

speC mutant expression vectors prepared in the above (pET28a-speC_V156A, pET28a-speC_D160A, pET28a-speC_I163A, pET28a-speC_E165A, pET28a-speC_Q691A, pET28a-speC_N153D, pET28a-speC_N153E, pET28a-speC_D309E) through the mutation whether the introduction of the sequence analysis confirmed.

[105]

[106]

Table 2 [Table 2]
Entrance area variations
speC_V156A_5 (SEQ ID NO: 4) Gctgacgcaaaattgggcgatctgctta 5'-3 '
speC_V156A_3 (SEQ ID NO: 5) 5'-ccaattttgcgtcagcgttacacatatc-3 '
speC_D160A_5 (SEQ ID NO: 6) Attgggcgctctgcttattcatgaagga 5'-3 '
speC_D160A_3 (SEQ ID NO: 7) 5'-aagcagagcgcccaattttacgtcagcg-3 '
speC_I163A_5 (SEQ ID NO: 8) 5'-ctgcttgctcatgaaggatcggcgaaag 3 '
speC_I163A_3 (SEQ ID NO: 9) Ttcatgagcaagcagatcgcccaatttt 5'-3 '
speC_E165A_5 (SEQ ID NO: 10) Attcatgcaggatcggcgaaagatgcgc 5'-3 '
speC_E165A_3 (SEQ ID NO: 11) Cgatcctgcatgaataagcagatcgccc-5'-3 '
speC_Q691A_5 (SEQ ID NO: 12) 5'-gagctggcaggtgtttatagcgaaaccg-3 '
speC_Q691A_3 (SEQ ID NO: 13) 5'-aacacctgccagctccggcgaaaatccc-3 '
PLP stabilizing mutations
speC_N153D_5 (SEQ ID NO: 14) 5'-tatgtgtgacgctgacgtaaaattgggc 3 '
speC_N153D_3 (SEQ ID NO: 15) 5'-gtcagcgtcacacatatcggcgcgaaag 3 '
speC_N153E_5 (SEQ ID NO: 16) 5'-tatgtgtgaagctgacgtaaaattgggc 3 '
speC_N153E_3 (SEQ ID NO: 17) Gtcagcttcacacatatcggcgcgaaag-5'-3 '
speC_D309E_5 (SEQ ID NO: 18) Ctgtttgaatccgcgtgggtcggttatgaa 5'-3 '
speC_D309E_3 (SEQ ID NO: 19) Cgcggattcaaacagaatgtaatcacaca-5'-3 '

[107]

[108]

Example 3. putrescine synthesis activity measurement of ODC (speC) mutant enzyme

[109]

[110]

3-1. ODC mutant enzymes obtained

[111]

By introducing into the E. coli with a genotype of pET28a-DE3 speC mutant vector prepared in Example 2 it was produced in a strain that can be obtained for the enzyme.

[112]

Expression of the mutant vector pET28a-speC was reference to the pET System Manual (Novagen). Specifically, the cells were cultured 16 hours 37 ° C, 200 rpm conditions by screening a single colony of each of the strains inoculated into 3 ml LB broth (kanamycin + 50 ug / ml) in LB medium plate. This again new 15 ml LB medium (+ 50 ug kanamycin / ml) in the re-inoculated to OD 600 0.6 in the condition that the same culture conditions in the raised immediately, so that the final concentration of 0.5mM IPTG was added to 18 ° C, 180rpm , enzyme expression was induced by culturing for 20 hours.

[113]

After induction enzyme expression were isolated by ultrasonic crushing the obtained cells were used after centrifugation the supernatant was obtained therefrom for the primary activity evaluation. Were conducted to evaluate the second active after refining in order to identify the characteristics of the added enzyme, the enzyme using a his-tag expression by binding to the enzyme through a pET vector were isolated with Ni-NTA column. The tablets were used Chelating Excellose spin kit (Bioprogen). The process as ODC (SpeC wild-type and mutant) enzymes are obtained as expressed by 8% SDS PAGE through a soluble form was confirmed that each can be obtained from the supernatant.

[114]

[115]

3-2. Putrescine of ODC (speC) new synthetic variant enzyme activity measurements

[116]

Using as a substrate for ornithine putrescine synthetic whether degree obtained in the examples carried out to evaluate a 3-1 ODC (SpeC wild type and variants) of enzyme activity was measured by ODC. Putrescine activity evaluation conditions of the ODC activity was to identify the synthesis proceeds with reference to the literature report (Vienozinskiene J, et al, Anal Biochem, 146: 180-183, 1985).

[117]

That is, when ornithine one molecule be putrescine god made ​​by ODC enzyme water molecules dogs consumed and putrescine God and carbon dioxide molecules and OH with - so to be the ion-molecule generated increases the overall pH (Scheme 1) . Increased pH is the change of absorbance at 559nm by one of phenol red (phenol red) of the pH indicator, it is in proportion to the increase of the pH value of the amount of increase in the absorbance. The amount of putrescine produced by indirectly using the characteristic of being able to measure the amount of putrescine produced by this reaction characteristic was measured.

[118]

[Scheme 1]

[119]

L-ornithine + H2O -> putrescine + CO2 + OH-

[120]

[121]

For the primary evaluation of ODC enzyme activity, the supernatant before purification gave the same according to the concentration of the protein quantification. The reaction conditions are enzymes 30ug supernatant, 10mM ornithine, 1.25uM made after the reaction solution so that the PLP, the pH change was monitored with 40uM phenol red.

[122]

The measurement result is an increase in ODC mutant enzyme than the wild-type putrescine was the formation rate of the I163A and E165A. The remaining 6 jong V156A variant, the introduction of mutations in ODC D160A, Q691A, N153D, N153E, D309E showed the aspect of the change in absorbance at 559nm with few (see FIG. 1).

[123]

The first was his-tag purified quantified in order to narrow down the characteristics of ODC and E165A mutant I163A two enzymes screened by the screening by measuring the concentration of ornithine putrescine synthesis rate. ODC enzyme concentration used was 10ug, ornithine concentration was measured by a pH change such as the phenol red in the range 0.15 ~ 10mM.

[124]

[125]

Table 3 [Table 3]
ODC 효소 K M (mM) kcat(sec-1) kcat/KM(sec-1M-1) fold(kcat/KM)
WT (wild type) 1.5 1.6 1.1 x 103 1.0
I163A mutant 0.7 1.8 2.6 x 103 2.4
E165A mutant 1.1 2.4 2.2 x 103 2.0

[126]

[127]

From the results above, the design through the ODC structure analysis I163A, E165A mutant enzyme ODC K compared to the wild-type M , the value 53%, 27%. Reduced, it was found that also (binding affinity) of the substrate is increased binding affinity of ornithine. Also, kcat values ​​I163A mutant compared to the WT was 12.5%, E165A mutant has been found that also increase the ability to convert the tin ornithine according to show the value increased by 50% to putrescine. Finally, the representing characteristics of the enzyme activity kcat / K M was calculated values, I163A mutant compared to the WT was confirmed that 2.4 times, E165A mutant was 2 times higher (Table 3).

[128]

[129]

Example 4. ODC (speC) mutations optimization

[130]

[131]

Been identified as in Example 3 was introduced into a mutant of the amino acid of the various kinds of small size with respect to the relevant amino acid residues 163 (isoleucine), and 165 amino acid (glutamic acid) on ODC activity. Mutation introduction was carried out according to the same method as described in Example 1. The primers were as follows in Table 4 used for it. After further introducing a single mutation, respectively 163, 165 in position, mutation combinations increased ODC activity was evaluated in each position making the introduction of the double mutant (double mutant).

[132]

[133]

Table 4 Table 4
primer Primer sequences
speC_I163G_5 (SEQ ID NO: 20) Ctgcttggtcatgaaggatcggcgaaagat-5'-3 '
speC_I163G_3 (SEQ ID NO: 21) 5'-ttcatgaccaagcagatcgcccaatttt-3 '
speC_I163S_5 (SEQ ID NO: 22) Ctgctttctcatgaaggatcggcgaaagat-5'-3 '
speC_I163S_3 (SEQ ID NO: 23) Ttcatgagaaagcagatcgcccaatttt-5'-3 '
speC_I163V_5 (SEQ ID NO: 24) Ctgcttgttcatgaaggatcggcgaaagat-5'-3 '
speC_I163V_3 (SEQ ID NO: 25) Ttcatgaacaagcagatcgcccaatttt 5'-3 '
speC_E165G_5 (SEQ ID NO: 26) Attcatggaggatcggcgaaagatgcgc 5'-3 '
speC_E165G_3 (SEQ ID NO: 27) Cgatcctccatgaataagcagatcgccc-5'-3 '
speC_E165S_5 (SEQ ID NO: 28) Attcattcaggatcggcgaaagatgcgc 5'-3 '
speC_E165S_3 (SEQ ID NO: 29) Cgatcctgaatgaataagcagatcgccc-5'-3 '
speC_E165V_5 (SEQ ID NO: 30) Attcatgtaggatcggcgaaagatgcgc 5'-3 '
speC_E165V_3 (SEQ ID NO: 31) Cgatcctacatgaataagcagatcgccc-5'-3 '

[134]

[135]

The ODC mutants introduced into the primer of Table 4 are then purified according to the methods disclosed in Examples 2 and 3 were measured putrescine synthesis rate. Putrescine synthesis rate measurement results are shown in Table 5 for the manufacture of ODC variants.

[136]

[137]

Table 5 Table 5
ODC 효소 K M (mM) kcat (sec-1) kcat/KM(sec-1M-1) fold(kcat/KM)
WT (wild type) 1.5 1.6 1.1 x 103 1.0
I163G mutant 1.7 4.2 2.5 x 103 2.3
I163S variants 1.5 7.4 4.8 x 103 4.4
I163V variant 1.3 4.4 3.5 x 103 3.2
E165G variants 3.0 5.6 1.9 x 103 1.7
E165S mutant 1.9 10.1 5.2 x 103 4.7
E165V mutant 1.4 10.9 7.6 x 103 6.9
I163A E165A mutant 1.5 6.4 4.1 x 103 3.7
I163S E165V mutant 1.2 10.5 8.8 x 103 8.0
I163A E165V mutant 0.9 6.3 6.8 x 103 6.2
I163V E165V mutant 1.1 25.7 2.3 x 103 21.3

[138]

[139]

After introducing a single mutation 163, 165 amino acid residues as shown in Table 5, each of glycine (G, glycine), serine (S, serine), valine (V, valine), the case of 163 residues has a serine, in the case of the 165 residues if replaced by valine kcat / K M showed a 4.4-fold, 6.9-fold increase in activity compared to wild-type value. Based on these results, the introduction of the double mutant was confirmed for both the active moiety. Surprisingly, a single mutation was introduced in the indicated highest I163S and double mutations E165V If you change if you gave more than 163, 165 replaced by valine residues in both the highest increase of 21.3 times compared to wild-type activity of the combination.

[140]

Overall, the increase in ODC activity enzyme variants K M , rather than reduce the value of the kcat due to the increase in value of kcat was the increase in / KM values. This suggests that the structure of the ODC enzyme is changed in a direction to increase the speed to be converted into a product of putrescine than increasing polarity binding affinity for an enzyme of the ornithine substrate due to mutations introduced.

[141]

[142]

Example 5. ODC ornithine Tre strains expressing mutant enzyme that manufactures and Pew Latin temperament new conversion measurement

[143]

[144]

The mutation optimized ODC enzyme variants in Example 4 to evaluate whether the effects seen in changing teen ornithine to putrescine within the actual microbes.

[145]

Specifically, with the strain produced by introducing a mutant vector pET28a-speC in E. coli DE3 having genotype it was the experiment. In LB medium plate was screened by a single colony of each strain was inoculated in 3 ml LB (+ kanamycin 50 ug / ml) liquid culture medium 16 ° C 37 hours, 200 rpm condition. This new re-inoculation 25 ml LB (+ kanamycin 50 ug / ml and 0.2% glucose) material in a liquid medium to OD 600 was cultured so that the value is 0.5 to 0.6. Since 0.5 mM IPTG was added to induce the expression of ODC (speC), by centrifugation after 20 h of incubation in 18 ° C, 200rpm condition by removing the supernatant to obtain a cell. Aggregates (pellet) form obtained in the cell re-1X M9 minimal medium (3.37mM Na 2 HPO 4, 2.2mM KH 2 PO 4, 0.86mM NaCl, 0.94mM NH 4 Cl) in the resuspended to OD 600 value in the adjusted to 20, followed by a final reaction volume to be 10 ml by further addition of the substrate and co-factor in 10mM ornithine 0.5 uM to PLP. The reaction conditions are 25 ° C, while shaking at 200 rpm conditions were measured putrescine quantitative measurement of putrescine concentration switch to using TNBS to sampling by the hour (Ngo TT, et al, Anal Biochem, 160:. 290 -293, 1987).

[146]

TNBS method was performed an analysis by diluting 50 times the supernatant after centrifugation of the culture medium in the sampling. To give a sample to be analyzed was diluted in 0.5 ml mix well were added 1 ml 4N NaOH, into the 1-pentanol to 2 ml gave here and mix well again. After centrifugation for 5 min at 2000 rpm and the supernatant ml 0.1 M Na 1 2 B 4 O 7 (pH 8.0) to put in a new tube containing 1 ml was mixed well. Re-mixing after the addition of 10mM TNBS 1 ml, 2 ml DMSO into the mix after, Absorbance was measured at 426nm taking the supernatant after centrifuging.

[147]

[148]

Table 6. Table 6
0 hr 2 hr 4 hr
put (mM) Conversion (%) put (mM) Conversion (%) put (mM) Conversion (%)
WT wild-type 1.6 16 5.7 57 8.6 86
Mutant I163V 1.5 15 7.5 75 9.8 98
E165V mutant 1.6 16 7.9 79 10.1 100
I163V E165V 변이체 1.6 16 7.7 77 10.0 100

[149]

[150]

For the wild-type and ODC variants three kinds of results progress putrescine conversion measurement experiment, ODC variants are relative increase in ornithine speed to convert Teen with putrescine compared to wild type in the 2 hours of reaction sample of about 32-39% degree the results are shown. Switching speed difference between ODC variants almost no did not have greater activity differences seen in the purified liver ODC variants seen in in vitro experiments, in the case of the wild-type showed the result that the reaction was completely finished even after four hours the variant to react at 4 hours this pattern showed almost complete. As the result, in the in vivo experiments the enzyme conversion strain was confirmed in an increased effectiveness of ODC activity variant.

[151]

[152]

Example 6. ODC variants introduced putrescine and putrescine-producing strain manufacturing production capacity measure

[153]

[154]

The prepared putrescine conversion activity increased by introducing a mutant ODC in real putrescine-producing strain affects the putrescine productivity was measured putrescine production capacity.

[155]

[156]

6-1. ODC variants introduced putrescine-producing strain produced

[157]

Weaken the activity of NCgl1469 protein than the endogenous activity by putrescine on the basis of a new production capability enhancements genus Corynebacterium microorganisms (KCCM11240P), the putrescine switching activity increased ODC (speC) mutant chromosomes in my wild speC mutant strains were produced which introduced.

[158]

The putrescine production capability improved Corynebacterium glutamicum (KCCM-11240P) strain is a strain disclosed in International Patent Publication WO2013 / 105827 Lake, disclosed in the parent strain patents filed (International Patent Publication WO2012 / 077995 No.) Pugh Trail Corridor with new production capabilities were made on the basis of the Corynebacterium genus microorganisms (KCCM11138P). A more specific manufacturing method microorganism strain Corynebacterium genus having Corey NCgl1469 the N- terminal portion and a C- terminal portion pDZ vector putrescine-producing ability by cloning based on the nucleotide sequence of the NCgl1469 gene of the strain ATCC13032 (KCCM11138P) after introduction of the strain using the electroporation method, kanamycine (25 ㎍ / ㎖) it was then plated on selective medium is contained. Successful insertion of the vector chromosomes was screened a colony showing a blue color in the medium containing X-gal (5- bromo-4-chloro-3-indolyl -β-D- galactoside). Primary dilution after chromosomal insertion strains cultured in a nutrient medium to the cross end NCgl1469 gene deletion strain by (cross over) by selecting white kolronieul appears at a lower rate and plated on medium without antibiotics containing X-gal It was selected. Finally, the KCCM11138P △ NCgl1469 strain by deletion of the gene coding for the protein NCgl1469 involved in the putrescine degradation pathway, one of putrescine N- acetyl God putrescine degradation pathway by which new cells in all of the strains produced KCCM11138P ability than the strain producing putrescine and putrescine-producing strain is enhanced.

[159]

Specifically, the Example 2, and was amplified using primers _3 speC_start (BamHI) _5, speC_stop (XbaI) from the start to the ODC (speC) DNA of the mutant produced in 4 Table 7. Specifically, by using two primers of the produced pET28a-speC variant (I163S, I163V, I163S E165V) speC_start (BamHI) which the vector as a template, disclosed in Table 7 _5, speC_stop (XbaI) _3 PCR It was performed.

[160]

[161]

Table 7 [Table 7]
primer Primer sequences
speC_start (BamHI) _5 (SEQ ID NO: 32) Cgcggatccatgaaatcaatgaatattgc-5'-3 '
speC_stop (XbaI) _3 (SEQ ID NO: 33) 5'-gctctacattacttcaacacataaccgt-3'

[162]

[163]

After a gene fragment and vector pDZ obtained through PCR amplification by BamHI and XbaI restriction enzyme treated with (37 ° C, 3 hours), using conventional ligation (ligation) technique a gene fragment of speC variants in pDZ vector It was inserted. For the production of recombinant vector inserted into the chromosome (pDZ-speC_I163S, pDZ-speC_I163V, pDZ-speC_I163S E165V) it was confirmed by sequence analysis.

[164]

[165]

speC variants are to obtain the strain inserted into a chromosome, the pDZ-speC_I163S prepared in the above, pDZ-speC_I163V, pDZ-speC_I163S E165V After recombination transformant injection using the vector electroporation each to KCCM11240P strain BHIS plate medium (brain heart infusion 37 g / l, sorbitol 91 g / l, agar 2%, were plated on 25 ug kanamycin 1L reference + / ml).

[166]

Successful insertion of the vector is a chromosomal blue color in a solid medium containing X-gal (5- bromo-4-chloro-3-indolyl -β-D- galactoside) was determined by whether or not the natanaeneunga. After the shaking culture primary chromosome inserted strain in a nutrient medium (30 ° C, 8 hours), to each serial dilution, it was plated on solid medium containing X-gal. Most were colonies can be screened for white colonies of a lower rate, on the other hand, bluish, screened colonies were obtained the strain introduced in the final speC variants by a secondary cross (cross over) are chromosomes. Finally strain was confirmed by sequence analysis of mutants. It was named the strain identified as KCCM11240P :: speC_I163S, KCCM11240P :: speC_I163V, KCCM11240P :: speC_I163S E165V, naming KCCM11240P :: speC_I163S E165V of them as Corynebacterium glutamicum CC01-0578, and the Budapest Treaty on 10 June 2013 under the accession to the international agency, Korea Culture Center of microorganisms (Korea Culture Center of microorganisms, KCCM) was deposited as accession number KCCM11425P.

[167]

[168]

6-2. ODC variants introduced putrescine-producing strain of putrescine production capacity measure

[169]

In order to confirm the effect of putrescine-producing strain of putrescine production capacity due to the ODC (speC) variants introduced to evaluate the putrescine-producing ability to target a strain produced in Example 6-1.

[170]

[171]

Specifically, the in production strains of 1mM arginine is included CM plate medium (glucose 1%, polypeptone 1%, yeast extract 0.5%, beef extract 0.5%, NaCl 0.25%, urea 0.2%, 50% NaOH 100ul, agar 2%, pH 6.8, by a platinum degree inoculate 1L basis) 16 time, and then titer medium 25 ml that has to Table 8 composition incubated at 30 ° C in the following, for 24 hours it at 200 rpm at 30 ° C shaken and incubated. It was cultured by the addition of 1mM arginine in the fermentation medium during the production for all the strains.

[172]

[173]

Table 8 Table 8
Composition Concentration / content (1L basis)
glucose 8 %
Soy Protein 0.25 %
Corn steep solid 0.5 %
(NH4)2SO4 4 %
Element 0.15 %
KH 2 PO 4 0.1 %
MgSO 4 -7 H 2 O 0.05 %
Biotin 100 and
Thiamine hydrochloride 3000 and
Calcium pantothenate 3000 and
Nicotinamide 3000 and
CaCO3 5 %

[174]

[175]

As a result, as shown in Table 9, in the introduction of the ODC (speC) mutant strain with increased activity, it showed a putrescine production is 37 ~ 105% increase in the aspect of the 12 hours sample.

[176]

Such results show that the results ODC variants are introduced because doemeuro consumption compared to putrescine-producing strain can produce a higher concentration of putrescine per existing in.

[177]

[178]

Table 9. [Table 9]
Strain 12 hr
Put (g/L) fold (%)
KCCM11240P 1.3 100
KCCM11240P I163S mutant 2.7 205
KCCM11240P E165V mutant 2.7 193
KCCM11240P I163S E165V mutant 1.8 137

[179]

[180]

From the above description, those skilled in the art will appreciate that the present invention without changing the technical spirit or essential features may be embodied in other specific forms. In this regard, the embodiments described above are illustrative and will in every way should be understood as not limiting it. The scope of the invention should be construed that all changes or variations derived from the meaning and scope of the claims and the equivalent concepts to be described later, rather than the description above within the scope of the invention.

[181]

[182]

Claims

[Claim 1]

Ornithine dicarboxylate raised (ornithine decarboxylase, ODC) beonjjae isoleucine 163 (isoleucine), and 165 amino acid residues glutamic acid beonjjae (Glutamic acid) at least one selected from the group consisting of amino acid residues from the N- terminus of an amino acid sequence set forth in SEQ ID NO: 1 this will be a mutated, the mutant protein ODC.

[Claim 2]

According to claim 1, wherein the glutamic acid 165 beonjjae alanine (alanine), Glycine (Glycine), serine (Serine) or the valine that is, variation in ODC protein substituted with (Valine).

[Claim 3]

According to claim 1, wherein the isoleucine 163 beonjjae alanine (alanine), Glycine (Glycine), serine (Serine) or the valine that is, variation in ODC protein substituted with (Valine).

[[4]

According to claim 1, wherein the 163 beonjjae isoleucine and 165 beonjjae glutamic acid as ODC protein mutated at the same time, 163 beonjjae isoleucine and 165 beonjjae glutamic each alanine-alanine, alanine-one substituted by valine-valine, serine-valine or valine which, ODC mutated protein.

[Claim 5]

According to claim 1, SEQ ID NO: 34 to SEQ ID NO, mutated ODC protein consisting of the amino acid sequence described by any one of 57.

[6.]

Claim 1 to a nucleic acid molecule encoding a protein of any one of ODC of 5 mutations.

[7.]

It claims 1 to 5, a vector comprising a nucleic acid molecule encoding a protein of any one of variations of the ODC wherein.

[8.]

The transformed with the transgenic vector 7 protest.

[9.]

L- ornithine (L-ornithine), including the step of L- ornithine L- ornithine or the mixture was added to the fermentation broth first to Claim 5, wherein variation of any one of the ODC protein in the reaction includes, Pugh Tre new manufacturing methods.

[10.]

According to claim 9, wherein the mutated protein is the ODC is contained in the purified ODC protein mutation or mutations ODC protein microorganism fermentation broth, putrescine production method.

[11.]

Putrescine produced in corridor with the ability Corynebacterium ( Corynebacterium sp. ) Of claims 1 to 5, wherein any one of ODC mutated protein was introduced, putrescine production capability improved recombinant microbial organisms.

[12.]

According to claim 11, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum ( Corynebacterium glutamicum ) would in the recombinant microorganism.

[13.]

(a) the genus Corynebacterium (having 1 to claim 5, wherein any one of Mutated ODC protein is enhanced putrescine-producing ability introduction of Corynebacterium sp. culturing a) microorganism: and (b) the (a) to Pew from the culture obtained in step includes the step of separating putrescine Trail new production methods.

[14.]

14. The method of claim 13 wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum ( Corynebacterium glutamicum would in), putrescine production method.