# Patent

## Title
Microorganisms for producing putrescine and process for producing putrescine using them

## Abstract

The present invention relates to a recombinant microorganism capable of producing putrescine at high yield due to inactivated activity of a protein having an amino acid sequence represented by SEQ ID NO: 2 in the microorganism, and a method of producing putrescine using the microorganism.

## Background
CROSS-REFERENCES TO RELATED APPLICATIONS
This application is a U.S. national phase application of International PCT Patent Application No. PCT/KR2015/004087, which was filed on Apr. 24, 2015, which claims priority to Korean Patent Application Nos. 10-2014-0049766, filed Apr. 25, 2014. These applications are incorporated herein by reference in their entireties.
STATEMENT REGARDING SEQUENCE LISTING
The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is HANO_049_00US_ST25.txt. The text file is 35 KB, was created on Oct. 25, 2016, and is being submitted electronically via EFS-Web.
TECHNICAL FIELD
The present invention relates to a recombinant microorganism for producing putrescine and a method of producing putrescine using the same.
BACKGROUND ART
Biogenic amines (BAs) are nitrogen compounds mainly produced by decarboxylation of amino acids, amination of aldehyde and ketone, and transamination. Such biogenic amines are low molecular weight compounds, which are synthesized during metabolism of microorganisms, plants, and animals, and thus are known as components easily found in cells thereof. Especially, polyamine such as spermidine, spermine, putrescine (or 1,4-butanediamine), cadaverine, etc., are substances present in most living cells.
Among them, putrescine is an important raw material which synthesizes polyamine nylon-4,6 by reacting with adipic acid, and is produced mainly by chemical synthesis through acrylonitrile and succinonitrile from propylene.
In addition, a method for producing putrescine at high concentration by transformation of E. coli and genus Corynebacterium has been disclosed (International Publication No. WO06/005603; International Publication No. WO09/125924; Qian Z D et al., Biotechnol. Bioeng. 104(4): 651-662, 2009; Schneider et al., Appl. Microbiol. Biotechnol. 88(4): 859-868, 2010; and Schneider et al., Appl. Microbiol. Biotechnol. 95: 169-178, 2012). Further, research on putrescine transporter regarding E. coli, yeast, plant and animal cells has been actively conducted (K Igarashi, Plant Physiol. Biochem. 48: 506-512, 2010).
Meanwhile, the present inventors demonstrated that membrane proteins encoding NCgl2522 function as a putrescine exporter in a microorganism of genus Corynebacterium, which includes a putrescine synthesis pathway, and confirmed that putrescine may be produced at a high yield by enhancing NCgl2522 activity from to the endogenous activity thereof (KR Patent Application No. 10-2013-0030020)
In addition, NCgl2523 gene is a multidrug-resistance-related transcription factor which belongs to TetR family, and is known to act as an NCgl2522 expression inhibitor (Hirochi et. al. J Biol. Chem. 280:46, 38711-38719. 2005).
In this regard, the present inventors have continuously conducted research and confirmed enhanced putrescine production by depletion of NCgl2523 which constitutes NCgl2522 operon, in the manner similar to the effects of enhancing NCgl2522 activity, thereby completing the present invention.
DISCLOSURE
Technical Problem
An objective of the present invention is to provide a recombinant microorganism capable of producing putrescine at high yield.
Another objective of the present invention is to provide a method of producing putrescine at high yield using the microorganism.
Technical Solution
In one aspect to achieve the above objectives, the present invention provides a microorganism of genus Corynebacterium having putrescine productivity, in which a protein having an amino acid sequence represented by SEQ ID NO: 2 is inactivated
In one exemplary embodiment, the present invention provides a microorganism of genus Corynebacterium having putrescine productivity, wherein an activity of ornithine decarboxylase (ODC) is further introduced into the microorganism.
In another exemplary embodiment, the present invention provides a microorganism of genus Corynebacterium having putrescine productivity, in which the ODC has an amino acid sequence represented by SEQ ID NO: 10.
In still another exemplary embodiment, the present invention provides a microorganism of genus Corynebacterium having putrescine productivity, wherein acetyltransferase activity is further inactivated in the microorganism.
In still another embodiment, the present invention provides a microorganism of genus Corynebacterium having putrescine productivity, wherein the acetyltransferase comprises an amino acid represented by SEQ ID NO: 15 or 16.
In still another embodiment, the present invention provides a microorganism of genus Corynebacterium having putrescine productivity, in which the microorganism is Corynebacterium glutamicum. 
In another aspect, the present invention provides a method of producing putrescine including:
i) culturing a microorganism of genus Corynebacterium having putrescine productivity wherein a protein having an amino acid sequence represented by SEQ ID NO: 2 is inactivated in a culture medium; and
ii) separating putrescine from a cultured microorganism or the culture medium obtained from the above step.
In an exemplary embodiment, the present invention provides a method of producing putrescine, in which the microorganism of genus Corynebacterium is Corynebacterium glutamicum. 
Hereinafter, the present invention will be described in detail.
In one aspect, the present invention relates to a microorganism of genus Corynebacterium having putrescine productivity, wherein a protein having an amino acid sequence represented by SEQ ID NO: 2, thus NCgl2523, is inactivated
As used herein, the term “NCgl2523” refers to a multidrug-resistance-related transcription factor which belongs to TetR family, and is known to act as an Ncgl2522 expression inhibitor (Hirochi et. al., J Biol. Chem. 280(46): 38711-38719, 2005).
In the present invention, NCgl2523 is a protein having an amino acid of SEQ ID NO: 2 or an amino acid sequence having 70% or more, more specifically 80% or more, even more specifically 90% or more, much more specifically 98% or more, and most specifically 99% or more homology to the sequence, and is not limited thereto as long as it is a protein having the activity of an NCgl2522 expression inhibitor.
Further, because amino acid sequences of proteins showing the activity may differ depending on species or strain of microorganisms, it is not limited thereto. It is obvious that a protein having an amino acid sequence in which the sequence is partially deleted, modified, substituted, or inserted is included in the scope of the present invention, as long as a sequence having homology to the sequence shows biological activity practically equivalent or corresponding to a protein of SEQ ID NO: 2.
As used herein, the term “homology” refers to similarity between given amino acid sequences or nucleotide sequences and may be represented in percentage. Herein, the homology sequence which have identical or similar activity with a given amino acid sequence or nucleotide sequence is indicated by “% homology”. For example, homology may be examined by using conventional software calculating mediated parameters such as score, identity, similarity, etc., and more specifically using BLAST 2.0, or by comparing sequences via Southern hybridization under defined stringent conditions. Appropriate hybridization conditions may be defined by the scope of the art, and may be determined by one of ordinary skill in the art using known methods (i.e., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, N.Y., 1989; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).
As long as it has an activity similar to those of NCgl2523 proteins, a polynucleotide encoding NCgl2523 of the present invention may include an amino acid sequence of SEQ ID NO: 2, or a polynucleotide encoding a protein having 70% or more, specifically 80% or more, more specifically 90% or more, much more specifically 95% or more homology thereto, much more specifically 98% or more, and most specifically 99% or more homology to the sequence, and may especially include a nucleotide sequence of SEQ ID NO: 1 or 3.
Further, a polynucleotide encoding NCgl2523 of the present invention may be hybridized in stringent conditions with a probe of a nucleotide sequence represented by SEQ ID NO: 1 or 3, or a probe derived from the nucleotide sequence, and may be a variant encoding functionally normal NCgl2523. As used herein, the term “stringent conditions” refers to conditions which enable a specific hybridization between polynucleotides. For example, such stringent conditions are described in detail in literature (i.e., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, N.Y., 1989; F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).
In the present invention, it was confirmed that when NCgl2523, which constitutes an NCgl2522 operon, was deleted in a microorganism of genus Corynebacterium having putrescine productivity, putrescine production increased, similarly to when NCgl2522 activity was enhanced. In this regard, the present invention may provide a recombinant microorganism showing putrescine production at a high yield by inhibiting NCgl2522 expression resulting from inactivating NCgl2523 activity. Therefore, as disclosed in the embodiment of the present invention, by inactivating the corresponding NCgl2523 and genes encoding amino acids similar thereto, putrescine productivity may be enhanced in a microorganism having amino acids having sequences similar to that of NCgl2523, in which such amino acids consist of NCgl2522 and an operon, or a microorganism in which NCgl2523 acts similarly as an NCgl2522 expression modulator.
As used herein, the term “inactivation” refers to not expressing a gene encoding a corresponding polypeptide, showing a certain reduction in gene expression, not producing a corresponding functional polypeptide, even when expressed.
In addition, inactivation refers not only to completely inactivating a gene encoding a corresponding polypeptide, but also to a weakened or significantly reduced expression compared to the wild-type, thereby practically not expressing the gene. Therefore, gene inactivation may be complete (knockout) or partial (for example, a hypomorph which shows gene expression below the normal level, or a product of a mutant gene which shows partial reduction in activity in effect of a hymorph).
In particular, in the present invention, inactivation of NCgl2523 may be induced by:
1) a partial or complete deletion of a polynucleotide encoding the protein;
2) modification of a regulatory sequence to decrease an expression of the polynucleotide;
3) modification of the polynucleotide sequence on chromosome in order to weaken the protein activity; and
4) a combination thereof,
without being particularly limited thereto.
1) a partial or complete deletion of a polynucleotide encoding the protein may be performed by replacing a polynucleotide encoding endogenous target proteins or chromosomes with a partially removed polynucleotide or a marker gene using a vector for chromosomal insertion into a microorganism. The “partial” may vary depending on the type of polynucleotides, but specifically refers to 1 to 300, more specifically to 1 to 100, and even more specifically 1 to 50.
As used herein, the term “vector” refers to a DNA construct including a nucleotide sequence encoding a desired protein, which is operably linked to an appropriate expression regulatory sequence to express the desired protein in a suitable host cell. The regulatory sequence includes a promoter that can initiate transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosome-binding site, and a sequence regulating the termination of transcription and translation. After the vector is transformed into a suitable host cell, it can replicate or function independently of the host genome, and can be integrated into the genome itself.
The vector used in the present invention is not particularly limited, as long as it is able to replicate in host cells, and any vector known in the art may be used. Examples of conventional vectors may include a natural or recombinant plasmid, cosmid, virus, and bacteriophage. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc., may be used as a phage vector or cosmid vector, and pBR types, pUC types, pBluescriptII types, pGEM types, pTZ types, pCL types, pET types, etc., may be used as a plasmid vector. A vector usable in the present invention is not particularly limited, and any known expression vector may be used. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, or pCC1BAC vector may be used.
Further, the polynucleotide encoding a desired protein in chromosomes may be replaced by a mutated polynucleotide using a vector for chromosomal insertion. The insertion of the polynucleotide into the chromosome may be performed by any method known in the art such as homologous recombination, without being particularly limited thereto.
As used herein, the term “transformation” refers to introduction of vectors including a polynucleotide encoding target proteins into host cells so that proteins encoded by the polynucleotide are expressed in host cells. As long as the transformed polynucleotide can be expressed in a host cell, it includes all whether it is integrated into chromosomes of host cells or exists extrachromosomally. Further, the polynucleotide includes DNA and RNA encoding target proteins. The polynucleotide may be introduced in any form, as long as it can be introduced into host cells and expressed therein. For example, the polynucleotide may be introduced into host cells in the form of an expression cassette which is a gene construct including all elements required for its autonomous expression. Typically, the expression cassette includes a promoter operably linked to the polynucleotide, transcriptional termination signals, ribosome-binding sites, or translation termination signals. The expression cassette may be in the form of a self-replicable expression vector. Also, the polynucleotide may be introduced into host cells as it is and operably linked to sequences required for expression in host cells, without being limited thereto.
Further, the term “operably linked” refers to a functional linkage between a polynucleotide sequence encoding desired proteins and a promoter sequence which initiates and mediates transcription of the polynucleotide sequence.
Next, 2) modification of a regulatory sequence to decrease an expression of the polynucleotide may be performed by inducing modification in a regulatory sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof in a nucleotide sequence or replacing with a nucleotide with a weaker activity, in order to weaken the activity of the regulatory sequence, without being particularly limited thereto. The regulatory sequence may include a promoter, an operator sequence, a sequence encoding ribosome-binding site, and a sequence regulating termination of transcription and translation, without being limited thereto.
Further, 3) modification of the polynucleotide sequence on chromosome may be performed by inducing modification in a regulatory sequence deletion, insertion, non-conservative or conservative substitution, or a combination thereof in a nucleotide sequence, or replacing with a nucleotide with a weaker activity, in order to weaken the protein activity, without being limited thereto,
As used herein, the term “a microorganism having putrescine productivity” or “a microorganism producing putrescine” refers to a microorganism naturally having putrescine productivity or a microorganism, in which putrescine productivity is incorporated into a parent strain having no putrescine productivity.
The microorganism producing putrescine may be, without being particularly limited thereto, a microorganism having improved productivity of ornithine to be used as a raw material for putrescine biosynthesis, in which the microorganism is modified to have higher activities of acetylglutamate synthase converting glutamate to acetylglutamate (N-acetylglutamate) or ornithine acetyltransferase (ArgJ) converting acetyl ornithine to ornithine, acetylglutamate kinase (ArgB) converting acetyl glutamate to acetylglutamyl phosphate (N-acetylglutamyl phosphate), acetyl-gamma-glutamyl phosphate reductase (ArgC) converting acetyl glutamyl phosphate to acetyl glutamate semialdehyde (N-acetyl glutamate semialdehyde), or acetylornithine aminotransferase (ArgD) converting acetyl glutamate semialdehyde to acetylornithine (N-acetylornithine), compared to the endogenous activity, in order to enhance the biosynthesis pathway from glutamate to ornithine glutamate.
Further, the microorganism is modified to inactivate endogenous activity of ornithine carbamoyltransfrase (ArgF) involved in arginine synthesis from ornithine, glutamate exporter (NCgl1221), and/or acetyltransferase which acetylizes putrescine and/or is modified to introduce activity of ornithine decarboxylase (ODC).
Here, the ornithine carbamoyltransfrase (ArgF), glutamate exporter (NCgl1221), ornithine decarboxylase (ODC), acetyl-gamma-glutamyl-phosphate reductase (ArgC), acetyl glutamate synthase or ornithineacetyltransferase (ArgJ), acetylglutamate kinase (ArgB), and acetylornithine aminotransferase (ArgD), may include specifically an amino acid sequence each represented by SEQ ID NO: 8, 9, 10, 11, 12, 13, and 14 or an amino acid sequence having 70% or more, more specifically 80% or more, even more specifically 90% or more homology to the sequence, without being particularly limited thereto.
Further, an acetyltransferase, which acetylizes putrescine, may include specifically an amino acid sequence represented by SEQ ID NO: 15 or 16 or an amino acid sequence having 70% or more, more specifically 80% or more, even more specifically 90% or more homology to the sequence, without being particularly limited thereto.
In particular, an increase in activity in the present invention may be carried out by:
1) an increase in the copy number of polynucleotides encoding the enzyme;
2) modification of a regulatory sequence to increase the polynucleotide expression;
3) modification of a polynucleotide sequence on chromosome to enhance activity of the enzyme; or
4) modification to enhance by a combination thereof,
without being limited thereto.
1) an increase in the copy number of polynucleotides may be performed in the form operably liked to a vector, or by chromosomal insertion into host cells, without being particularly limited thereto. In particular, it may be performed by introducing a vector, to which a polynucleotide encoding an enzyme of the present invention enzyme operably linked and which may be copied and function independently of host cells into host cells, or by introducing a vector to which the polynucleotide is operably linked and which is able to insert the polynucleotide into choromosomes in host cells into host cells, thereby increasing the copy number of polynucleotides in chromosomes of the host cells.
Next, 2) modification of a regulatory sequence to increase the polynucleotide expression may be performed by inducing modification in a sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof, or replacing with a nucleotide sequence with enhanced activity, in order to enhance the activity the regulatory sequence, without being particularly limited thereto. The regulatory sequence may include a promoter, an operator sequence, a sequence encoding a ribosome-binding site, a sequence regulating termination of transcription and translation, etc., without being particularly limited thereto.
In the upstream of the polynucleotide expression unit, a strong heterologous promoter may be linked instead of the original promoter. Examples of the strong promoter are CJ7 promoter, lysCP1 promoter, EF-Tu promoter, groEL promoter, aceA or aceB promoter, etc., and more specifically, may be operably linked to a Corynebacterium-originated promoter, lysCP1 promoter (WO2009/096689), or CJ7 promoter (KR Patent No. 0620092 and WO2006/065095) and increase an expression of a polynucleotide encoding the enzyme, but are not limited thereto.
Furthermore, 3) modification of a polynucleotide sequence on chromosome may be performed by inducing modification in a regulatory sequence by deletion, insertion, non-conservative or conservative substitution or a combination there of in a nucleotide sequence, or by replacing with a polynucleotide sequence which is modified to have enhanced activity, in order to enhance activity of the polynucleotide sequence, without being particularly limited thereto.
Further, inactivation of ornithine carbamoyltransfrase (ArgF), glutamate exporter, and acetyl transferase may be performed by methods of inactivating NCgl2523 as previously mentioned:
1) a partial or complete deletion of a polynucleotide encoding the protein;
2) modification of a regulatory sequence to decrease an expression of the polynucleotide;
3) modification of the polynucleotide sequence on chromosome in order to weaken the protein activity; and
4) a combination thereof,
without being particularly limited thereto.
Meanwhile, a microorganism of the present invention is a microorganism having putrescine productivity and includes prokaryotic microorganisms expressing proteins including an amino acid represented by SEQ ID NO: 2. Examples of such are Escherichia sp., Shigella sp., Citrobacter sp., Salmonella sp., Enterobacter sp., Yersinia sp., Klebsiella sp., Erwinia sp., Corynebacterium sp., Brevibacterium sp., Lactobacillus sp., Selenomanas sp., Vibrio sp., Pseudomonas sp., Streptomyces sp., Arcanobacterium sp., Alcaligenes sp. microorganisms, etc. In particular, a microorganism of the present invention may be a microorganism of genus Corynebacterium, and more specifically Corynebacterium glutamicum, but is not limited thereto.
In one exemplary embodiment, the present invention uses strains having high-concentration putrescine productivity due to an enhanced putrescine biosynthesis pathway from glutamate, which are microorganisms of genus Corynebacterium deposited under Accession Nos. KCCM11138P (KR Patent No. 2012-0064046) and KCCM11240P (KR Patent No. 2012-0003634).
In another exemplary embodiment, the present invention uses KCCM11138P and KCCM11240P, which are putrescine-producing strains derived from Corynebacterium glutamicum ATCC13032, and DAB12-a (KR Patent No. 2013-0082478) and DAB12-b (KR No. 2013-0082478; DAB12-a ΔNCgl1469), which are putrescine-producing strains derived from Corynebacterium glutamicum ATCC13869, each having identical genotypes. ATCC13869 strain may be obtained from American Type Culture Collection (ATCC).
In particular, the present inventors named a microorganism of genus Corynebacterium having enhanced putrescine productivity due to inactivation of NCgl2523 activity in Corynebacterium glutamicum KCCM11240P, which is a putrescine-producing strain, as Corynebacterium glutamicum CC01-0844, and deposited it to Korean Culture Center of Microorganisms (KCCM) under Budapest Treaty as Accession No. KCCM11520P on Feb. 25, 2014.
In another aspect, the present invention provides a method of producing putrescine including:
i) culturing a microorganism of genus Corynebacterium having enhanced putrescine productivity wherein a protein having an amino acid sequence represented by SEQ ID NO: 2 is inactivated in a culture medium; and
ii) separating putrescine from the cultured microorganism or the culture medium obtained from the above step.
In the method, culturing the microorganism may be performed by known batch culturing methods, continuous culturing methods, fed-batch culturing methods, etc., without being particularly limited thereto. Here, culture conditions may be maintained at optimal pH (e.g., pH 5 to 9, specifically pH 6 to 8, and most specifically pH 6.8) using basic compounds (e.g., sodium hydroxide, potassium hydroxide, or ammonia) or acidic compounds (e.g., phosphoric acid or sulfuric acid), and at an aerobic condition by oxygen or oxygen-containing gas mixture to a cell culture, without being particularly limited thereto. The culture temperature may be maintained at 20° C. to 45° C. and specifically at 25° C. to 40° C., and cultured for about 10 to 160 hours. Putrescine produced by the cultivation may be exported to the culture medium or remain in cells.
Further, the used culture medium may include sugar and carbohydrate (e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose), oil and fat (e.g., soybean oil, sunflower seed oil, peanut oil, and coconut oil), fatty acid (e.g., palmitic acid, stearic acid, and linoleic acid), alcohol (e.g., glycerol and ethanol), and organic acid (e.g., acetic acid) individually or in combination as a carbon source; nitrogen-containing organic compounds (e.g., peptone, yeast extract, meat juice, malt extract, corn solution, soybean meal powder, and urea), or inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate) individually or in combination as a nitrogen source; potassium dihydrogen phosphate, dipotassium phosphate, or sodium-containing salt corresponding thereto individually or in combination as a phosphorus source; and other essential growth-stimulating substances including metal salts (e.g., magnesium sulfate or iron sulfate), amino acids, and vitamins, without being limited thereto.
Separating putrescine produced in the culturing step of the present invention may be performed using a suitable method known in the art depending on culturing methods such as batch, continuous or fed-batch culturing methods, etc., thereby collecting desired amino acids from the culture.
Advantageous Effects
A microorganism of genus Corynebacterium having enhanced putrescine productivity of the present invention is modified to inactivate activity of a protein including an amino acid sequence of SEQ ID NO: 2, thereby inducing enhanced activity of a protein which is expected to be a putrescine exporter, specifically NCgl2522, and increasing putrescine export to the outside of cells, and thus may effectively produce putrescine.



DESCRIPTION OF DRAWINGS
FIG. 1 is a schematic diagram showing combined structures of NCgl2523 and the adjacent. In particular, FIG. 1 is a schematic diagram showing a combined structure of NCgl2522-NCgl2523-NCgl2524 which is a combined structure of adjacent genes of a microorganism including NCgl2523 (Type 1); a structure of combined NCgl2522-NCgl2523 and individual NCgl2524 (Type 2); or a combined structure of NCgl2522-NCgl2523 (Type 3).



MODE FOR INVENTION
Hereinafter, the present invention will be described in more detail with reference to Examples. However, the Examples are for illustrative purposes only, and thus the scope of the present invention is not intended to be limited by the Examples.
Example 1
Preparation of NCgl2523-Deleted Strains and Verification of its Putrescine Productivity
<1-1> Preparation of NCgl2523-Deleted Strains in ATCC13032-Derived Putrescine-Producing Strains
In order to verify whether deletion of NCgl2523 derived from Corynebacterium glutamicum ATCC13032 has an effect in putrescine productivity, vectors for deletion of a gene encoding NCgl2523 were prepared.
In particular, based on the nucleotide sequence of a gene encoding NCgl2523 represented by SEQ ID NO: 1, a pair of primers of SEQ ID NOs: 4 and 5 for obtaining homologous recombination fragments at the N-terminal region of NCgl2523, and a pair of primers of SEQ ID NOs: 6 and 7 for obtaining homologous recombination fragments at the C-terminal region of NCgl2523 were constructed as shown in Table 1.








TABLE 1







NCgl2523-del-F1_BamHI
CGGGATCCATGACTACCTCGCAGC


(SEQ ID NO: 4)
GTTTC


NCgl2523-del-R1_SalI
ACGCGTCGACCTAGTGCGCATTAT


(SEQ ID NO: 5)
TGGCTCC


NCgl2523-del-F2 SalI
ACGCGTCGACAGCCATGCTGGAAA


(SEQ ID NO: 6)
CAATTCTCG


NCgl2523-del-R2 XbaI
CTAGTCTAGAGAGAGCTGCGCATA


(SEQ ID NO: 7)
GTACTG









PCR was performed using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and two pairs of primers, thereby amplifying PCR fragments at the N-terminal and C-terminal regions of NCgl2523 gene. Desired fragments were obtained via electrophoresis of the PCR fragments. Here, PCR reaction was repeated for 30 cycles including 30 seconds of denaturation at 95° C., 30 seconds of annealing at 55° C., and 30 seconds of extension at 72° C. The thus obtained fragments of the N-terminal and C-terminal regions were treated with restriction enzymes BamHI and SalI, and restriction enzymes SalI and XbaI, respectively. The treated fragments were cloned into pDZ vectors treated with restriction enzymes BamHI and XbaI, thereby constructing pDZ-1'NCgl2523(K/O) plasmids.
The pDZ-1'NCgl2523 (K/O) plasmids were each transformed into KCCM11138P (KR Patent No. 10-1348461) and KCCM11240P (KR Patent No. 2013-0082478) by electroporation to obtain transformants. For colony formation, the transformants were plated and cultured on BHIS plate media (Braine heart infusion (37 g/L), sorbitol (91 g/L), and agar (2%)) containing kanamycin (25 μg/mL) and X-gal (5-bromo-4-chloro-3-indolin--D-galactoside). From the formed colonies, blue colonies were selected as strains introduced with pDZ-1'NCgl2523(K/O) plasmids.
The selected strains is cultured in a CM medium (glucose 10 g/L, polypeptone (10 g/L), yeast extract (5 g/L), beef extract (5 g/L), NaCl (2.5 g/L), urea (2 g/L), and pH 6.8) at 30° C. for 8 hours. After serial dilution of each cell culture from 10−4 to 10−10, the diluted samples were plated and cultured in an X-gal-containing solid medium to form colonies. From the formed colonies, white colonies, which appeared at a relatively low frequency, were finally selected as strains with deletion of a gene encoding NCgl2523 by a secondary crossover.
PCR was performed using a pair of primers of SEQ ID NOs: 4 and 7 to confirm deletion of a gene encoding NCgl2523 in the finally selected strains. The Corynebacterium glutamicum mutants were each named as KCCM11138P ΔNCgl2523 and KCCM11240P ΔNCgl2523.
<1-2> Preparation of NCgl2523-Deleted Strains in ATCC13869-Derived Putrescine-Producing Strains
NCgl2523-deleted strains were constructed from DAB12-a (KR Patent No. 2013-0082478) and DAB12-b (KR Patent No. 2013-0082478; DAB12-a ΔNCgl1469), which are putrescine-producing strains derived from Corynebacterium glutamicum ATCC13869.
In particular, in order to verify a sequence of NCgl2523 gene and the expressed protein therefrom derived from Corynebacterium glutamicum ATCC13869, PCR was performed using genomic DNA of Corynebacterium glutamicum ATCC13869 as a template and a pair of primers of SEQ ID NOs: 4 and 7. Here, PCR reaction was repeated for 30 cycles including 30 seconds of denaturation at 95° C., 30 seconds of annealing at 55° C., and 1 minute 30 seconds of extension at 72° C.
By separating the thus obtained PCR products via electrophoresis and analyzing by sequencing, a gene encoding NCgl2523 derived from Corynebacterium glutamicum ATCC13869 was found to include a nucleotide sequence represented by SEQ ID NO: 3. Further, an amino acid sequence of proteins encoded by the gene was compared to an amino acid sequence of NCgl2523 (SEQ ID NO: 2) derived from Corynebacterium glutamicum ATCC13032, and the result showed 100% homology.
In order to delete a gene encoding NCgl2523 derived from Corynebacterium glutamicum ATCC13869, PCR was performed using genomic DNA of Corynebacterium glutamicum ATCC13869 as a template and two pairs of primers described in Table 1 as in Example <1-1>, and PCR fragments of the N-terminal C-terminal regions of NCgl2523 gene were amplified, thereby obtaining desired fragments via electrophoresis. Here, PCR reaction was repeated for 30 cycles including 30 seconds of denaturation at 95° C., 30 seconds of annealing at 55° C., and 30 seconds of extension at 72° C. The obtained fragments of the N-terminal and C-terminal regions were treated with restriction enzymes BamHI and SalI, and restriction enzymes SalI and XbaI, respectively. The treated fragments were cloned into pDZ vectors treated with restriction enzymes BamHI and XbaI, thereby constructing pDZ-2'NCgl2523(K/O) plasmids.
By transforming pDZ-2'NCgl2523 (K/O) into each of Corynebacterium glutamicum DAB12-a and DAB12-b in the same manner as described in Example <1-1>, strains with deletion of a gene encoding NCgl2523 were selected. The selected Corynebacterium glutamicum mutants were named as DAB12-a ΔNCgl2523 and DAB12-b ΔNCgl2523.
<1-3> Evaluation of Putrescine Productivity of NCgl2523-Deleted Strains
In order to verify the effects of NCgl2523 deletion on putrescine production in putrescine-producing strains, Corynebacterium glutamicum mutants constructed in Example <1-1> and <1-2> were compared for putrescine productivity.
In particular, 4 different types of Corynebacterium glutamicum mutants (KCCM11138P ΔNCgl2523, KCCM11240P ΔNCgl2523, DAB12-a ΔNCgl2523, and DAB12-b ΔNCgl2523) and 4 different types of parent strains (KCCM11138P, KCCM11240P, DAB12-a, and DAB12-b) were each plated on 1 mM arginine-containing CM plate media (1% glucose, 1% polypeptone, 0.5% yeast extract, 0.5% beef extract, 0.25% NaCl, 0.2% urea, 100 μL of 50% NaOH, and 2% agar, at pH 6.8, base on 1 L) and cultured at 30° C. for 24 hours. After inoculating each of the cultured stains using a platinum loop in 25 mL of titer media (8% Glucose, 0.25% soybean proteins, 0.50% corn steep solids, 4% (NH4)2SO4, 0.1% KH2PO4, 0.05% MgSO4.7H2O, 0.15% urea, 100 g of biotin, 3 mg of thiamine hydrochloride, 3 mg of calcium-pantothenic acid, 3 mg of nicotinamide, and 5% of CaCO3, based on 1 L), shake culturing was carried out at 30° C. and 200 rpm for 98 hours. 1 mM arginine was added to the media for culturing all strains. The concentration of putrescine produced from each culture was measured, and results are shown in Table 2.










TABLE 2







Strain
Putrescine (g/L)









KCCM11138P 
 9.8



KCCM11138P ΔNCgl2523
11.8



KCCM11240P (−)
12.4



KCCM11240P ΔNCgl2523
14.9



DAB12-a
10.2



DAB12-a ΔNCgl2523
12.2



DAB12-b
13.1



DAB12-b ΔNCgl2523
15.2










As shown in Table 2, putrescine production was increased in 4 kinds of NCgl2523-deleted Corynebacterium glutamicum mutants.
Example 2
Measurement of Intercellular Putrescine Concentrations in NCgl2523-Deleted Strains
To examine changes in intercellular putrescine concentration in Corynebacterium glutamicum mutants having inactivated NCgl2523 activity, intercellular putrescine concentrations were measured in Corynebacterium glutamicum mutant KCCM11240P ΔNCgl2523 and parent strain KCCM11240P by extraction using an organic solvent. Intracellular metabolite analysis was carried out in accordance with a method described in literature (Nakamura J et al., Appl. Environ. Microbiol., 73(14): 4491-4498, 2007).
First, after inoculating each of Corynebacterium glutamicum mutant KCCM11240P ΔNCgl2523 and parent strain KCCM11240P in 25 ml of 1 mM arginine-containing CM liquid media (1% glucose, 1% polypeptone, 0.5% yeast extract, 0.5% beef extract, 0.25% NaCl, 0.2% urea, and 100 μL of 50% NaOH, at pH 6.8, based on 1 L), shake culturing was carried out at 30° C. and 200 rpm. When cell growth reached the exponential phase during cultivation, cells were isolated from the culture media by rapid vacuum filtration (Durapore HV, 0.45 m; Millipore, Billerica, Mass.). The cell-adsorbed filter was washed twice with 10 ml of cooled water and emerged in methanol-containing 5 M morpholine ethanesulfonic acid and 5 M methionine sulfone for 10 minutes. The extraction liquid obtained therefrom was mixed well with an equal volume of chloroform and 0.4-fold volume of water. Only the aqueous phase was applied to a spin column to remove protein contaminants. The filtered extraction liquid was analyzed by capillary electrophoresis mass spectrometry, and the results are shown in Table 3.










TABLE 3







Strain
Putrescine (mM)









KCCM11240P
7



KCCM11240P ΔNCgl2523
1










As shown in Table 3, the intercellular putrescine concentration was significantly reduced in Corynebacterium glutamicum mutants having inactivated NCgl2523 activity compared to that of parent strain KCCM11240P.
It is suggested that when NCgl2523 is deleted in Corynebacterium glutamicum mutant KCCM11240P, inhibition of NCgl2522 expression is withdrawn and NCgl2522 activity is enhanced, thereby enhancing putrescine-exporting ability and subsequently exporting putrescine produced inside the cell to the outside of cells efficiently.
Example 3
Ortholog Search of NCgl2523 Gene and Gene Context Analysis
Ortholog search was conducted for NCgl2523, which is derived from Corynebacterium glutamicum ATCC13032, using KEGG, MetaCyc Database and NCBI blastP. Via gene cluster, a combined structure of NCgl2523, and NCgl2522 and NCgl2524, which constitute an operon in the genome of each microorganism was examined.
The gene name of NCgl2522 is cgmA, which is a major facilitator superfamily permease belonging to DHA2 family, and the gene name of NCgl2523 is cgmR, which is TetR-family transcriptional regulator. NCgl2524 has not been attributed with a function, but is a major facilitator superfamily permease belonging to UMF1 family.
According to the analysis results, while NCgl2522 and NCgl2523 mostly constitute the same operon, NCgl2524 may be present in the same operons (Type 1), present in a different position in the genome (Type 2), or not present in the genome (Type 3), depending on microorganisms.
In this regard, each of analyzed microorganisms was classified into 3 types according to a combined structure of genes adjacent to NCgl2523. In particular, microorganisms which are expected to have NCgl2523 were classified into a combined structure of NCgl2522-NCgl2523-NCgl2524 (Type 1); a structure of combined NCgl2522-NCgl2523 and individual NCgl2524 (Type 2); or a combined structure of NCgl2522-NCgl2523 (Type 3) (FIG. 1). The classification results are shown in Table 4.









TABLE 4






Combined



Type
structure
Corresponding microorganisms








Microorganism
Acidovorax avenae, Actinobaculum sp., Actinomyces sp.,



having
Actinomyces vaccimaxillae, Actinoplanes missouriensis,



NCgl2523
Actinosynnema mirum, Agrobacterium tumefaciens, alpha




proteobacterium, Amycolatopsis mediterranei, Amycolatopsis




orientalis, Arcanobacterium haemolyticum, Arthrobacter




aurescens, Arthrobacter sp., Bdellovibrio bacteriovorus,




Beutenbergia cavernae, Bordetella bronchiseptica, Bordetella




pertussis, Bordetella parapertussis, Brachybacterium




paraconglomeratum, Clavibacter michiganensis subsp.,




Corynebacterium atypicum, Corynebacterium callunae,




Corynebacterium casei, Corynebacterium diphtheriae,




Corynebacterium glutamicum, Corynebacterium glutamicum




AR1, Corynebacterium glutamicum ATCC 13032,




Corynebacterium glutamicum ATCC 13869, Corynebacterium




glutamicum ATCC 14067, Corynebacterium glutamicum ATCC




21831, Corynebacterium glutamicum K051, Corynebacterium




glutamicum MB001, Corynebacterium glutamicum R,




Corynebacterium glutamicum SCgG1, Corynebacterium




glutamicum SCgG2, Corynebacterium glycinophilum,




Corynebacterium maris, Corynebacterium pseudotuberculosis,




Corynebacterium sp. ATCC 6931, Corynebacterium




terpenotabidum, Corynebacterium ulcerans, Corynebacterium




urealyticum, Corynebacterium variabile, Corynebacterium




vitaeruminis, Dermabacter sp. HFH0086, Enterobacter cloacae




EcWSU1, Enterobacter sp. R4-368, Gammaproteobacteria,




Granulicoccus phenolivorans, Hafnia alvei, Ilumatobacter




coccineus, Isoptericola variabilis, Janthinobacterium




agaricidamnosum, Ketogulonicigenium vulgare, Microbacterium




sp., Microbacterium testaceum, Micrococcus luteus,




Micromonospora aurantiaca, Micromonospora sp.,




Mycobacterium gilvum, Nakamurella multipartita, Nesterenkonia




alba, Nesterenkonia sp., Nocardia brasiliensis, Nocardia




cyriacigeorgica, Nocardia farcinica, Nocardiopsis dassonvillei,




Nocardiopsi, kunsanensis, Nocardiopsis sp., Nocardiopsis




valliformis, Nocardiopsis xinjiangensis, Ochrobactrum anthropi,




Ochrobactrum intermedium, Paracoccus aminophilus,




Paracoccus sp. 5503, Pectobacterium carotovorum subsp.




carotovorum PCC21, Promicromonospora sukumoe,




Propionibacterium acidipropionici, Propionibacterium




freudenreichii, Proteobacteria, Providencia stuartii ATCC 33672,




Pseudomonas aeruginosa, Pseudomonas cremoricolorata,




Pseudomonas geniculata, Pseudomonas stutzeri, pseudonocardia




dioxanivorans, Renibacterium salmoninarum, Rhodococcus equi,




Rhodococcus erythropolis, Rhodococcus jostii, Rhodococcus




opacus B4, Rhodococcus opacus PD630, Rhodococcus




pyridinivorans, Saccharomonospora viridis, Saccharopolyspora




erythraea, Salinispora, Salinispora arenicola, Salinispora




pacifica, Salinispora tropica, Sanguibacter keddieii, Serratia




liquefaciens, Serratia marcescens, Serratia plymuthica, Serratia




proteamaculans, Serratia sp., Sodalis sp. HS1, Sphingobium




chinhatense, Stenotrophomonas maltophilia, Stenotrophomonas




sp., Streptococcus anginosus, Streptomyces, Streptomyces




alboviridis, Streptomyces albus, Streptomyces atroolivaceus,




Streptomyces baarnensis, Streptomyces cyaneofuscatus,




Streptomyces fulvissimus, Streptomyces globisporus,




Streptomyces griseus, Streptomyces mediolani, Streptomyces




scopuliridis, Streptomyces sp., Xanthomonas citri pv. citri,




Xenorhabdus nematophila, Yaniella halotolerans, Yersinia




enterocolitica


1
Combined
Corynebacterium callunae, Corynebacterium casei,



structure of
Corynebacterium glutamicum AR1, Corynebacterium glutamicum



NCgl2522-NCgl
ATCC 13032, Corynebacterium glutamicum ATCC 13869,



2523-NCgl2524
Corynebacterium glutamicum ATCC 14067, Corynebacterium




glutamicum ATCC 21831, Corynebacterium glutamicum K051,




Corynebacterium glutamicum MB001, Corynebacterium




glutamicum R, Corynebacterium glutamicum SCgG1,




Corynebacterium glutamicum SCgG2, Corynebacterium




vitaeruminis


2
Structure of 
Actinoplanes missouriensis, Actinosynnema mirum,



combined
Amycolatopsis mediterranei, Amycolatopsis mediterranei,



NCgl2522-NCgl
Amycolatopsis orientalis, Arcanobacterium haemolyticum,



2523; individual
Arthrobacter aurescens, Arthrobacter sp., Bordetella



NCgl2524
bronchiseptica, Bordetella parapertussis, Bordetella pertussis,




Clavibacter michiganensis, Corynebacterium glycinophilum,




Corynebacterium maris, Corynebacterium terpenotabidum,




Corynebacterium variabile, Isoptericola variabilis,




Microbacterium testaceum, Micromonospora aurantiaca,




Micromonospora sp. L5, Mycobacterium gilvum, Nakamurella




multipartita, Nocardia brasiliensis, Nocardia cyriacigeorgica,




Nocardia cyriacigeorgica, Nocardia farcinica, Nocardiopsis




dassonvillei, Nocardiopsis dassonvillei, Ochrobactrum anthropi,




Paracoccus aminophilus, Propionibacterium acidipropionici,




Pseudonocardia dioxanivorans, Renibacterium salmoninarum,




Rhodococcus equi, Rhodococcus pyridinivorans,




Saccharomonospora viridis, Saccharopolyspora erythraea,




Salinispora tropica, Streptomyces albus, Streptomyces




fulvissimus, Streptomyces griseus


3
Combined
Acidovorax avenae, Bdellovibrio bacteriovorus, Beutenbergia



structure of 
cavernae, Corynebacterium atypicum, Corynebacterium



NCgl2522-NCgl
diphtheriae, Corynebacterium pseudotuberculosis,



2523
Corynebacterium ulcerans, Corynebacterium urealyticum,




Enterobacter cloacae, Enterobacter sp., Hafnia alvei,




Ilumatobacter coccineus, Janthinobacterium agaricidamnosum,




Ketogulonicigenium vulgare, Pectobacterium carotovorum subsp.




carotovorum PCC21, Providencia stuartii, Pseudomonas




aeruginosa, Pseudomonas cremoricolorata, Pseudomonas




stutzeri, Sanguibacter keddieii, Serratia liquefaciens, Serratia




marcescens, Serratia plymuthica, Serratia proteamaculans,




Serratia sp., Sodalis sp. HS1, Stenotrophomonas maltophilia,




Xenorhabdus nematophila, Yersinia enterocolitica









From these results, it is expected that putrescine productivity may be enhanced as in the present invention by inactivating genes encoding NCgl2523 and an amino acid sequence similar thereto, for microorganisms having an amino acid including a sequence similar to NCgl2523 and consisting of an operon with a protein expected to be a putrescine exporter, such as NCgl2522, as in the classification.
Based on the above description, it should be understood by one of ordinary skill in the art that other specific embodiments may be employed in practicing the invention without departing from the technical idea or essential features of the present invention. In this regard, the above-described examples are for illustrative purposes only, and the invention is not intended to be limited by these examples. The scope of the present invention should be understood to include all of the modifications or modified form derived from the meaning and scope of the following claims or its equivalent concepts, rather than the above detailed description.
DEPOSIT DESIGNATION
Depository Authority: Korea Culture Center of Microorganisms
Accession No.: KCCM11520P
Date of Deposit: 20140225

## Claims

1. An isolated microorganism of genus Corynebacterium having putrescine productivity, wherein a protein having an amino acid sequence represented by SEQ ID NO: 2 is inactivated.

2. The microorganism of genus Corynebacterium having putrescine productivity according to claim 1, wherein an activity of ornithine decarboxylase (ODC) is further introduced into the microorganism.

3. The microorganism of genus Corynebacterium having putrescine productivity according to claim 2, wherein the ODC has an amino acid sequence represented by SEQ ID NO: 10.

4. The microorganism of genus Corynebacterium having putrescine productivity according to claim 1, wherein acetyltransferase activity is further inactivated in the microorganism.

5. The microorganism of genus Corynebacterium having putrescine productivity according to claim 4, wherein the acetyltransferase comprises an amino acid represented by SEQ ID NO: 15 or 16.

6. The microorganism of genus Corynebacterium having putrescine productivity according to claim 1, wherein the microorganism is Corynebacterium glutamicum.

7. A method of producing putrescine, comprising:
culturing a microorganism of genus Corynebacterium having putrescine productivity wherein a protein having an amino acid sequence represented by SEQ ID NO: 2 is inactivated, in a culture medium; and
separating putrescine from the cultured microorganism or the culture medium obtained from the above step.

8. The method of producing putrescine according to claim 7, wherein the microorganism of genus Corynebacterium is Corynebacterium glutamicum.