# Patent

## Title
Microorganisms for producing putrescine and method for producing putrescine using same

## Abstract

The present invention relates to a putrescine-producing microorganism and a method for producing putrescine using the same. To be more specific, the present invention is directed to a microorganism given the ability to produce putrescine which is generated by blocking a biosynthetic pathway from ornithine to arginine, increasing the intracellular level of glutamate, enhancing the biosynthetic pathway of ornithine from glutamate, and introducing extracellular ornithine decarboxylase; and a method for producing putrescine by 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/KR2011/009478, which was filed on Dec. 8, 2011, which claims priority to Korean Patent Application Nos. 10-2011-0130595, filed Dec. 7, 2011 and 10-2010-0124867, filed Dec. 8, 2010. 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_023_00US_ST25.txt. The text file is 50 KB, was created on, Sep. 9, 2013, and is being submitted electronically via EFS-Web.
TECHNICAL FIELD
The present invention relates to a putrescine-producing microorganism and a method for producing putrescine using the same.
BACKGROUND ART
Polyamine is a substance present in most of the living cells. Spermidine or spermine belonging to polyamine is found in various species such as bacteria, fungus, and animals. Putrescine or 1,4-butanediamine which is a precursor in spermidine and spermine metabolism is found in a gram-negative bacteria or fungus, and it is present in wide range of concentration in various species suggesting that it has an important role in metabolic pathway.
Putrescine is a building block in a synthesis of polyamine nylon-4, 6 which is produced by reacting putrescine with adipic acid. To be used in manufacture of a processed plastic as a raw material, putrescine is usually produced by chemical synthesis involving conversion of propylene to acrylonitrile and to succinonitrile. This chemical synthesis consists of three-step process comprising catalytic oxidation reaction which consumes a lot of energy, reaction using a toxic chemical such as cyanide, and hydrogenation reaction that uses high-pressure hydrogen. Production of putrescine by chemical synthesis is not environmentally friendly and also consumes a lot of energy leading to depletion of petroleum resource. Therefore, a more environmentally friendly and energy-effective method involving biomass utilization needs to be developed for putrescine production.
In microorganism, a biosynthetic pathway of putrescine is the same as L-arginine biosynthetic pathway from glutamate to ornithine synthesis step. Putrescine can be synthesized by two pathways such as ornithine decarboxylation or arginine decarboxylation. These two pathways produce the energy required for metabolism or allow the cell to have resistance to oxidative stress. A method for production of putrescine at a high concentration by transformation of E. coli and Corynebacterium has been reported. Production of putrescine in E. coli can be achieved by increasing expression level of ornithine decarboxylase and glutamate acetyltransferase. Also, putrescine can be produced at high concentration by removing spermidine and acetylputrescine synthetic pathways which degrade or utilize putrescine (Qian. Z D. et al., Biotechnol. Bioeng. 104:4, 651-662, 2009, International Patent Publication No. WO06/005603. International Patent Publication No. WO09/125924). Meanwhile, in Corynebacterium sp. strain which lacks putrescine synthetic pathway, putrescine may be produced from ornithine through insertion of ornithine decarboxylase gene derived from E. coli or putrescine may be produced from L-arginine by insertion of the gene of L-arginine decarboxylase and agamatinase derived from E. coli. Ornithine pathway actually can produce about 50 times higher amount of putrescine than L-arginine pathway (Schneider et al., Appl. Microbiol. Biotechnol. 88:4, 859-868, 2010).
Meanwhile, it was found that E. coli can grow normal in the presence of 44 g/L putrescine, while Corynebacterium glutamicum can grow normal in the presence of 66 g/L putrescine. Therefore, it seems more effective to use Corynebacterium sp. strain which can survive at higher concentration of putrescine than E. coli in development of microorganism for producing putrescine.
Corynebacterium sp. strains are commercially applicable microorganism that is widely used in production of amino acid, nucleic acids, enzymes, and antibiotic analogs. In Corynebacterium sp. strain, L-arginine is synthesized from glutamate by enzymes expressed from the gene of arginine operon composed of argCJBDFRGH. Arginine operon genes that take the most important role in biosynthesis of L-arginine uses intracellularly synthesized L-glutamate as a substrate for arginine synthesis. FIG. 2 shows a schematic diagram of a synthetic pathway of arginine from glutamate in Corynebacterium sp. strain. In arginine synthetic pathway, ArgJ converts glutamate to N-acetylglutamate, ArgB converts N-acetylglutamate to N-acetylglutamyl phosphate, ArgC converts N-acetylglutamyl phosphate to N-acetylglutamate semialdehyde, ArgD converts N-acetylglutamate semialdehyde to N-acetylornithine, ArgJ converts N-acetylornithine to ornithine, ArgF converts ornithinie to L-citrulline, ArgG converts L-citrulline to argininosuccinate, and ArgH converts argininosuccinate to arginine.
Previously known arginine-producing strains were developed by increasing the expression level of enzyme involved in arginine biosynthesis through introducing mutation to arginine operon or mutating promoter. Among the genes in arginine operon, argR which regulates and suppresses the expression of arginine operon gene and argB which is inhibited by arginine concentration have been targeted in many studies to increase arginine production level (Korea Patent Publication No. 2010-0060909).
Putrescine biosynthetic pathway is the same as arginine biosynthetic pathway from glutamate to ornithine synthesis step. Then putrescine is produced from the synthesized ornithine by ornithine decarboxylase (ODC). Therefore, in order to prepare a strain capable of producing high amount of putrescine, a sufficient amount of ornithine has to be made first. When glutamate was added to the argF- and argR-deleted strain of wild-type E. coli W3110, ornithine production level was increased by 20%. Also in addition to the pathway from glutamate to ornithine, when the pathway from glutamate to proline synthesis was blocked by knocking out proB gene which encodes γ-glutamylkinase involved in the first step thereof, the ornithine production level was increased as well. This suggests that when the intracellular level of glutamate is increased, it has positive effects on ornithine production in the cell. In a study of high yield production of glutamate which is a precursor of ornithine, Corynebacterium glutamicum has been studied for a long time. In this regard, it has been reported that the glutamate exporting activity of Corynebacterium glutamicum is enhanced when the cell lacks biotin or when the cell is treated with penicillin G or fatty acid ester surfactant. This result suggests that when the cell wall is damaged, glutamate can be exported better through cytoplasm.
NCgl1221 protein derived from Corynebacterium glutamicum wild-type strain (ATCC 13032) is known to promote the betain export and has a similar amino acid sequence as that of yggB which codes for a mechanosensitive channel protein (Korea Patent Publication No. 2010-0017581).
DISCLOSURE
Technical Problem
In effort of developing a strain capable of producing higher yield of putrescine, the present inventors have generated a strain producing high level of putrescine by blocking the biosynthetic pathway from ornithine to arginine, increasing the intracellular level of glutamate, enhancing the biosynthetic pathway from glutamate to ornithine, and by introducing an exogenous gene of ornithine decarboxylase which can synthesize putrescine from ornithine, thereby completing the present invention.
Technical Solution
One object of the present invention is to provide a microorganism given a ability to produce putrescine.
Another object of the present invention is to provide a method for producing putrescine by using the microorganism.
Advantageous Effect
A microorganism given the ability to produce putrescine of the present invention may be widely utilized for more effective putrescine production.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the biosynthetic pathway of putrescine and relevant genes in the transformed Corynebacterium glutamicum of the present invention.
FIG. 2 shows the biosynthetic pathway of arginine in the known Corynebacterium glutamicum. 
FIG. 3 shows the vector pDZ to be inserted into the chromosome of Corynebacterium sp. strain.



BEST MODE
As one aspect to achieve the object of the present invention, the present invention provides a putrescine-producing microorganism, wherein the microorganism is modified to have diminished activity of an ornithine carbamoyltransferase and a protein involved in glutamate export, i.e., NCgl1221 compared to the endogenous activity thereof, and an activity of ornithine decarboxylase (ODC) is introduced into the microorganism.
As used herein, “ornithine carbamoyltransferase (OCT)” refers to a catalytic enzyme that mediates the reaction between carbamoyl phosphate and ornithine to synthesize citrulline and phosphoric acid. OCT is present in a liver of urea-excreting animals as well as plant and microorganism, and in microorganism it is involved in arginine synthesis. The OCT enzyme comprises catalytic domain and regulatory domain, and when ornithine binds to the regulatory domain the enzyme activity is inhibited.
E. coli K12 strain has two types of OCT (ArgF and ArgI), and intestinal microorganism including E. coli B and W strains have OCT protein similar to ArgI. OCTs encoded by argF and argI have different amino acid sequences from each other, but they are considered as isoenzyme having the same function (EMBO J. (1982) 1:853-857). Corynebacterium sp. strain only has OCT encoded by argF gene. OCT only acts in the synthetic pathway from ornithine to arginine, and thus if the OCT activity is diminished, the intracellular ornithine production level can be increased.
The present invention provides a Corynebacterium sp. strain wherein the synthetic pathway from ornithine to arginine is blocked to inhibit the conversion of a putrescine precursor, i.e. ornithine, to arginine. For this purpose, a transformant strain was prepared by deletion of the gene coding for the ornithine carbamoyl transferase.
The ornithine carbamoyltransferase may be a protein having an amino acid sequence of SEQ ID No. 28 or an amino acid sequence having 70% or more homology thereto, and preferably 80% or more, more preferably 90% or more homology thereto, but is not limited thereto.
As used herein, “homology” refers to the similarity in nucleotide sequences of gene coding for a protein or amino acid sequences. When homology is sufficiently high, products of the corresponding gene may be the same or have a similar activity.
As used herein, “protein involved in glutamate export” refers to a type of mechanosensitive channels which function to export the intracellularly produced glutamate to extracellular environment.
The present invention provides a Corynebacterium sp. strain given the ability to produce putrescine. For this purpose, a transformant strain that can maintain high cellular level of glutamate was prepared by deleting the gene coding for a protein involved in export of glutamate which is a substrate for the putrescine precursor, i.e. ornithine.
By increasing the intracellular level of glutamate, i.e., a precursor of ornithine, an ornithine biosynthetic pathway can be stimulated. In the present invention, glutamate exporting can be reduced or inhibited by diminishing the NCgl1221 activity.
The removed protein involved in glutamate export may be a protein having an amino acid sequence of SEQ ID No. 30 or an amino acid sequence having 70% or more homology thereto, and more preferably having 80% or more homology, even more preferably having 90% or more homology thereto, but is not limited thereto.
The activity of the ornithine carbamoyltransferase and the protein involved in glutamate export can be diminished by a method selected from the group consisting of (1) a partial or full deletion of a gene coding for the protein, (2) modification of an expression regulatory sequence for suppressing the gene expression, (3) modification of the nucleotide sequence on chromosome for diminishing the protein activity, and 4) a combination thereof, but is not limited thereto.
A partial or full deletion of a polynucleotide coding for the protein can be done by introducing a vector for chromosomal insertion into a microorganism, thereby substituting the polynucleotide coding for an endogenous target protein on chromosome with a partially removed polynucleotide or a marker gene. The length “partial” may vary depending on the type of polynucleotide, but specifically it refers to a length of 1 to 300 nucleotides, preferably 1 to 100 nucleotides, and more preferably 1 to 50 nucleotides.
Also, modification of an expression regulatory sequence for reducing expression of the polynucleotide can be done by inducing a modification on the expression regulatory sequence through deletion, insertion, non-conservative or conservative substitution of nucleotide sequence, or a combination thereof in order to diminish the activity of expression regulatory sequence, or by replacing the expression regulatory sequence with a nucleotide sequence having weaker activity. The expression regulatory sequence includes a promoter, an operator sequence, a sequence coding for ribosome-binding site, and a sequence regulating the termination of transcription and translation.
Furthermore, modification of a polynucleotide sequence on chromosome, which codes for the enzyme of the present invention can be done by inducing a mutation on the sequence through deletion, insertion, non-conservative or conservative substitution of polynucleotide sequence, or a combination thereof in order to diminish the enzymatic activity, or by replacing the sequence with a polynucleotide sequence which is modified to have weaker activity.
A transformant microorganism in which a precursor of putrescine, ornithine, is accumulated in the cell is prepared by different method from former methods for generating ornithine-producing strain (Korea Patent Publication No. 2010-0060909), that is, a method for increasing the ornithine production by eliminating or diminishing the function of ArgR which is a transcription inhibitor of arginine biosynthetic pathway, and additionally by deleting ornithine carbamoyltransferase gene and introducing unregulated N-acetylglutamate synthase.
As used herein, “endogenous activity” refers to the activity of enzyme that a microorganism possesses in its native state. In the present invention, endogenous activity refers to the activity of ornithine carbamoyl transferase and a protein involved in glutamate export, i.e., NCgl1221 that a microorganism naturally possesses. Also, as used herein, “modified to have a weaker activity than an endogenous activity” refers to the state where an ornithine carbamoyl transferase and a protein involved in glutamate export, i.e., NCgl1221 do not function properly due to gene deletion or mutation and thus the activity of ornithine carbamoyl transferase and a protein involved in glutamate export, i.e., NCgl1221 that a microorganism naturally possesses is weakened.
As used herein, “ornithine decarboxylase (ODC)” refers to an enzyme that produces putrescine using ornithine. The ODC requires pyridoxal 5′-phosphate (PLP) as a coenzyme. ODC is present in most of gram-negative bacteria, but it may be present in some of intestinal bacteria such as lactobacillus among gram-positive bacteria. E. coli possesses two types of genes coding for ODC. One of them is speC which is constitutively expressed at a constant concentration, and the other gene is speF whose expression is induced only under certain conditions (level of ornithine higher than a certain concentration and low pH). Depending on the species, some species have two types of ODC enzymes like in E. coli, or have only one type of ODC. To be specific, species including Escherichia sp., Shigella sp., Citrobacter sp., Salmonella sp., and Enterobacter sp. have two types of ODC (i.e., speC and speF), while species including Yersinia sp., Klebsiella sp., and Erwinia sp. have only one type of ODC (speC). In lactobacillus, ODC is expressed from one type of gene (speF), which is induced by low pH or high concentration of ornithine and histidine.
The ODC may be a protein coded by the amino acid sequence of SEQ ID No. 41 or the amino acid sequence having 70% or morehomology, more preferably 80% or more homology, even more preferably 90% or more homology, but is not limited thereto. As described above, the activity of ornithine decarboxylase (ODC) can be introduced by using various methods known in the art, for example, an insertion of a polynucleotide comprising ODC-coding nucleotide sequence into the chromosome, insertion of the polynucleotide into a vector system to be introduced to a microorganism, insertion of a promoter with improved activity on upstream of ODC-coding nucleotide sequence or insertion of an ODC-coding gene with a modified promoter, and insertion of a variant of ODC-coding nucleotide sequence. More preferably, when inserting the ODC-coding nucleotide sequence, a CJ7 promoter of SEQ ID No. 42 may be used as a promoter to regulate the expression of ODC.
In the example of the present invention, a microorganism of Corynebacterium sp. strain given the ability to produce putrescine is provided. To prepare a transformant capable of producing putrescine, the microorganism was inserted with the nucleotide sequence coding for ornithine decarboxylase in the chromosome which can synthesize putrescine from ornithine.
Putrescine produced by a microorganism may be degraded, in E. coli by an intracellular degradation pathway, into spermidine, acetyl putrescine, and gamma-aminobutyric acid (GABA). It is known that ODC is present in most of gram-negative strains, but not in Corynebacterium species. Therefore, when Corynebacterium sp. strains are used to generate a putrescine-producing strain, introduction of exogenous ODC is required.
As used herein, “a microorganism given the ability to produce putrescine” or “putrescine-producing microorganism” refers to a microorganism generated by giving the putrescine-producing ability to a mother strain which did not have the ability to produce putrescine intrinsically. The microorganism given the ability to produce putrescine or the putrescine-producing microorganism may be, but is not limited to, the microorganism wherein the activities of acetylglutamate synthase which converts glutamate to N-acetylglutamate, ornithine acetyltransferase (ArgJ) that converts acetyl ornithine to ornithine, acetylglutamate kinase (ArgB) that converts acetyl glutamate to N-acetylglutamyl phosphate, N-acetyl-gamma-glutamyl-phosphate reductase (ArgC) that converts acetyl glutamyl phosphate to N-acetyl glutamate semialdehyde, and acetylornithine aminotransferase (ArgD) that converts acetyl glutamate semialdehyde to N-acetylornithine, are enhanced compared to an intrinsic activity thereof, in order to enhance the biosynthetic pathway from glutamate to ornithine synthesis, thereby enhancing the productivity of ornithine which is used as a starting material for putrescine synthesis, and the microorganism that is transformed to introduce the gene coding for ornithine decarboxylase (speC), thereby acquiring the ability to produce putrescine from ornithine.
Here, the N-acetyl-gamma-glutamyl-phosphate reductase (ArgC), acetylglutamate synthase or ornithine acetyltransferase (ArgJ), acetylglutamate kinase (ArgB), and acetylornithine aminotransferase (ArgD) may preferably have, but is not limited to, an amino acid sequence of SEQ ID Nos. 33, 35, 37, and 39 respectively, or an amino acid sequence having 70% or more homology, more preferably 80% or more homology, and even more preferably 90% or more homology thereto. The activity of ArgC, ArgJ, ArgB, and ArgD can be enhanced by a method selected from the group consisting of 1) increase of the copy number of a polynucleotide coding for the protein, 2) modification of an expression regulatory sequence for increasing the polynucleotide expression, 3) modification of the polynucleotide sequence on a chromosome for enhancing an activity of the enzyme, and 4) a combination thereof.
To be specific, various methods can be used to increase the enzymatic activity in a microorganism in general. For example, the expression level of a polynucleotide can be increased by increasing the copy number of the polynucleotide through transformation involving plasmid insertion, homologous recombination, conjugation, and translocation; modifying an expression regulatory sequence of the polynucleotide; amplifying a gene coding for a regulatory factor which stimulates the polynucleotide expression; or by deleting or inhibiting a gene coding for a regulatory factor which suppresses the polynucleotide expression. To be more specific, the expression level of a polynucleotide can be increased by operably linking a gene fragment comprising the polynucleotide to a multicopy vector which can be replicated in Corynebacterium sp. strains, by introducing single or multiple copies of the polynucleotide to the chromosome, or replacing an expression regulatory sequence comprising the promoter of polynucleotide by the sequence having an improved activity.
For instance, the argCJBD gene group may be transformed into a microorganism by using pHC139T vector to prepare a microorganism with a significantly improved ability to produce ornithine compared to a wild-type strain. Alternatively, a microorganism in which ornithine biosynthetic pathway is enhanced may be prepared by improving a promoter region regulating the expression of argCJBD gene in the chromosome of microorganism to enhance the same or by replacing a promoter region by a promoter with more improved activity. In particular, a method for improving promoter region may involve, for replacing a promoter within the chromosome, preparing a gene fragment comprising nucleotide sequences of both terminal sites adjacent to the target site on the chromosome and a promoter sequence to be inserted in the same form as in the original chromosome and following the same gene deletion method using a pDZ vector published by Korea Patent Publication No. 2009-0082702, but is not limited thereto. Here, the improved promoter may preferably be, but is not limited to, the pcj7 (or P(CJ7)) promoter having a nucleotide sequence of SEQ ID No. 42 (Korea Patent Registration No. 0620092). The pDZ vector may preferably be, but is not limited to, a vector represented by a cleavage map of FIG. 3.
As used herein, “vector” refers to a DNA construct comprising a nucleotide sequence of gene which is operably linked to an appropriate expression regulatory sequence to express a target gene in a suitable host cell. The expression regulatory sequence comprises a promoter that can initiate transcription, an optional operator sequence for regulating the transcription, a sequence coding for a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. Examples of conventional vectors include a natural or recombinant plasmid, cosmid, virus and bacteriophage. For instance, pWE15, M13, λEMBL3, λEMBL4, λFIXII, λDASHII, λZAPII, λgt10, λgt11, Charon4A, and Charon21A can be used as a phage vector or cosmid vector. As a plasmid vector, pDZ vector, pBR type, pUC type, pBluescriptII type, pGEM type, pTZ type, pCL type and pET type may be used. A usable vector is not particularly limited, and any known expression vector, preferably pDZ vector, can be used.
Meanwhile, the microorganism of the present invention may be, but is not limited to, a transformant of the microorganism belonging to Escherichia sp., Shigella sp., Citrobacter sp., Salmonella sp., Enterobacter sp. Yersinia sp., Klebsiella sp., Erwinia sp., Corynebacterium sp., Brevibacterium sp., Lactobacillus sp., Selenomanas sp., or Vibrio sp. which does not possess putrescine metabolic pathway, while having the activity of ornithine carbamoyl transferase and a protein involved in glutamate export, i.e., NCgl1221.
Preferably the microorganism of the present invention may be a transformant in which ornithine is accumulated by reducing or inactivating the ornithine carbamoyl transferase activity, the intracellular level of glutamate is increased by reducing or inactivating the activity of a protein involved in glutamate export, ornithine is overproduced by increasing the expression level of argCJBD gene group which is involved in arginine biosynthesis, and exogenous gene coding for ornithine decarboxylase (ODC) that converts ornithine to putrescine is introduced, thereby making the cell capable of producing putrescine.
Preferably, the putrescine-producing microorganism of the present invention may be Corynebacterium sp. strain, and more preferably Corynebacterium glutamicum. To be more specific, a wild-type strain Corynebacterium glutamicum ATCC 13032 or a glutamate-overproducing strain KCCM-10785P (Korea Patent Publication No. 2008-0034334) may be used, but is not limited thereto. The KCCM-10785P strain is a glutamate-overproducing strain generated by deleting cg2624 (NCBI LOCUS ID YP_226636) and cg2115 (NCBI LOCUS ID YP_226173) genes in a glutamate-producing strain (KFCC-11074) which was generated by using mutagen such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG).
Although glutamate overproduction by deletion of cg2624 and cg2115 have not been identified prior to the above publication, cg2624 is identified as pcaR, which is an IclR family regulatory protein, and cg2115 is identified as sugR, which is a transcriptional regulator of sugar metabolism.
As shown in the ornithine synthetic pathway of FIG. 2, in order to increase the production level of ornithine, it is required to increase the amount of a starting material, glutamate, block the pathway where the synthesized ornithine is converted into arginine, and increase the amount of enzyme involved in ornithine biosynthesis or enhance the activity thereof. Likewise, a transformed microorganism given the ability to produce putrescine can be prepared by increasing ornithine productivity, introducing a gene coding for ornithine decarboxylase (ODC) which can synthesize putrescine from ornithine to a microorganism lacking putrescine metabolic pathway, thereby inducing overproduction of ornithine.
According to the examples of the present invention, the following strains are prepared: argF-deleted Corynebacterium glutamicum strain (ATCC 13032 ΔargF and KCCM-10785P ΔargF) (Example 1), argF- and NCgl1221-deleted Corynebacterium glutamicum strain (ATCC 13032 ΔargF ΔNCgl1221 and KCCM-10785P ΔargF ΔNCgl1221) (Example 2), argF- and NCgl1221-deleted and argCJBD-inserted Corynebacterium glutamicum strain (ATCC 13032 ΔargF ΔNCgl1221/pHC139T-argCJBD(Cgl) and KCCM-10785P ΔargF ΔNCgl1221/pHC139T-argCJBD(Cgl)) (Example 3-1), argF- and NCgl1221-deleted Corynebacterium glutamicum strain with substitution of the promoter of argCJBD gene group in a chromosome (ATCC 13032 ΔargF ΔNCgl1221 P(CJ7)-argCJBD and KCCM-10785P ΔargF ΔNCgl1221 P(CJ7)-argCJBD) (Example 3-2), argF- and NCgl1221-deleted Corynebacterium glutamicum strain inserted with ODC-coding speC gene in the chromosome (ATCC 13032 ΔargF ΔNCgl1221 bioAD::P(CJ7)-speC(Ec) and KCCM-10785P ΔargF ΔNCgl1221 bioAD::P(CJ7)-speC(Ec)), argF- and NCgl1221-deleted Corynebacterium glutamicum strain inserted with ODC-coding speC gene and argCJBD gene group in the chromosome (ATCC 13032 ΔargF ΔNCgl1221 bioAD::P(CJ7)-speC(Ec)/pHC139T-argCJBD(Cgl) and KCCM-10785P ΔargF ΔNCgl1221 bioAD::P(CJ7)-speC(Ec)/pHC139T-argCJBD(Cgl)) and argF- and NCgl1221-deleted Corynebacterium glutamicum with substitution of the promoter of argCJBD gene group and insertion of speC gene in the chromosome (ATCC 13032 ΔargF ΔNCgl1221 P(CJ7)-argCJBD bioAD::P(CJ7)-speC(Ec) and KCCM-10785P ΔargF ΔNCgl1221 P(CJ7)-argCJBD bioAD::P(CJ7)-speC(Ec)). When putrescine productivity of these strains was compared, it was found that the argF- and NCgl1221-deleted Corynebacterium glutamicum strain with substitution of the promoter of argCJBD gene group and insertion of speC gene in the chromosome (ATCC 13032 ΔargF ΔNCgl1221 P(CJ7)-argCJBD bioAD::P(CJ7)-speC(Ec) and KCCM-10785P ΔargF ΔNCgl1221 P(CJ7)-argCJBD bioAD::P(CJ7)-speC(Ec)) showed excellent putrescine productivity (Tables 7 and 8).
As a result, the present inventors have named the putrescine-producing strain with the highest productivity as “CC01-0064 (ATCC 13032 ΔargF ΔNCgl1221 P(CJ7)-argCJBD bioAD::P(CJ7)-speC(Ec))”, and deposited a sample of the strain under Budapest treaty to Korean Culture Center of Microorganisms (KCCM) located in Hongje 1-dong, Seodaemun-gu, Seoul, Korea in Nov. 24, 2010 with Accession No. KCCM11138P.
As another aspect to achieve the object of the present invention, the present invention provides a method for producing putrescine, comprising the step of (i) obtaining a cell culture by culturing a putrescine-producing microorganism; and (ii) recovering putrescine from the cultured microorganism or cell culture.
In the method of producing putrescine, culturing the microorganism may preferably be done by batch culture, continuous culture, and fed-batch culture known in the art, but is not limited thereto. Furthermore, as for the culturing condition, an optimal pH of 5 to 9, preferably pH 6 to 8, and most preferably pH 6.8 can be maintained by using a basic chemical (for example: sodium hydroxide, potassium hydroxide or ammonia) or acidic chemical (for example: phosphoric acid or sulfuric acid). Also, an aerobic condition can be maintained by adding oxygen or oxygen-containing gas mixture to a cell culture. The culturing temperature may be maintained at 20° C. to 45° C., and preferably at 25° C. to 40° C. In addition, it is preferable to culture for about 10 to 160 hours. The putrescine produced by the above culturing may be excreted to a culture medium or remain inside the cell.
Furthermore, the medium for culturing may comprise sugar and carbohydrate (for example: glucose, sucrose, lactose, fructose, maltose, molasse, starch and cellulose), oil and fat (for example: soybean oil, sunflower seed oil, peanut oil and coconut oil), fatty acid (for example: palmitic acid, stearic acid and linoleic acid), alcohol (for example: glycerol and ethanol), and organic acid (for example: acetic acid) individually or in combination as a carbon source; nitrogen-containing organic compound (for example: peptone, yeast extract, meat juice, malt extract, corn solution, soybean meal powder and urea), or inorganic compound (for example: 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; other essential growth-stimulating substances including metal salts (for example: magnesium sulfate or iron sulfate), amino acids, and vitamins.
Mode for Invention
Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples.
Example 1: Preparation of argF-Deleted Corynebacterium glutamicum Strain
In this example, an argF-deleted strain was prepared from a wild-type Corynebacterium glutamicum strain ATCC 13032 and a glutamate-overproducing strain KCCM-10785P which was generated by deleting cg2624 and cg2115 genes in a glutamate-producing strain KFCC-11074 generated by using mutagen such as NTG (Korea Patent Publication No. 2008-0034334) in order to block a synthetic pathway of arginine from ornithine. The arginine biosynthetic genes of Corynebacterium glutamicum ATCC 13032 are organized in an operon having a form of argCJBDFRGH, and a deletion target argF gene (SEQ ID No. 27) is present adjacent to the genes coding for enzymes involved in ornithine synthetic pathway on the chromosome. Thus, a plasmid for deleting argF gene was prepared based on the nucleotide sequence of argD and argR which are located adjacent to the deletion target argF gene.
To be specific, based on the nucleotide sequence of argD and argR of the ATCC 13032 strain, a homologous recombination fragment adjacent to the N-terminal sequence of argF and a homologous recombination fragment adjacent to the C-terminal sequence of argF were constructed. For this, the fragment adjacent to the N-terminal sequence of argF was obtained by PCR using the genomic DNA from ATCC 13032 strain as a template, and primers (SEQ ID Nos. 1 and 2) (28 cycle of denaturation for 30 seconds at 94° C., annealing for 30 seconds at 55° C., and extension for 30 seconds at 72° C.). Likewise, the fragment adjacent to the C-terminal sequence of argF was obtained by PCR using the genomic DNA from ATCC 13032 strain as a template, and primers (SEQ ID Nos. 3 and 4) under same PCR condition (Table 1).







TABLE 1 







Primers for preparing argF-deleted strain (ΔargF)









Name
SEQ ID No.
Sequence(5′-3′)





argF-del-F1_BamHI
1
CGGGATCCTGGCCGTACCGGCGATTTCT




(SEQ ID NO: 1)


 


argF-del-R1_SalI
2
CGCGTCGACAAGTTTGAGTCCTTTATGCG




(SEQ ID NO: 2)


 


argF-del-F2_SalI
3
CGCGTCGACGACATGTCCCTTGGCTCAAC




(SEQ ID NO: 3)


 


argF-del-R2_XbaI
4
TGCTCTAGAAGTAATTCACCTAGTTCTTTACC




(SEQ ID NO: 4)









The above-prepared homologous recombination fragment adjacent to the N-terminal sequence of argF was restriction digested with BamHI and SalI, and the homologous recombination fragment adjacent to the C-terminal sequence of argF was restriction digested with SalI and XbaI. Then each of the cleaved fragments were inserted into the pDZ vector which was also restriction digested with BamHI and XbaI, thereby producing a plasmid pDZ-argF(K/O).
The above-prepared plasmid pDZ-argF(K/O) was transformed into the ATCC 13032 strain and KCCM-10785P strain. Then, the transformed strains were plated and cultured on BHIS plate (Braine heart infusion 37 g/l, sorbitol 91 g/l, agar 2%) which contains kanamycin (25 μg/ml) and X-gal(5-bromo-4-chloro-3-indolin-β-D-galactoside), while letting the colonies to grow on the plate. Among the colonies formed on the plate, colonies with blue colour was collected to select for the strain inserted with the plasmid pDZ-argF(K/O).
The above-selected strains were cultured with shaking in 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, pH 6.8) at 30° C. for 8 hours. Subsequently, each cell culture was serially diluted from 10−4 to 10−10. Then the diluted samples were plated and cultured on an X-gal-containing solid medium, letting the colonies to grow. Among the colonies formed on the plate, only the white colonies which appear at relatively low frequency were collected to select for the argF-deleted strains.
Successful insertion of the plasmid pDZargF(K/O) into the above-selected strains was confirmed by performing PCR using the chromosomal DNA from the above-selected strain as a template, and primers of SEQ ID Nos. 1 and 4. Through this PCR confirmation, it was confirmed that the above-selected strain is the argF-deleted strain (i.e., ATCC 13032 ΔargF and KCCM-10785P ΔargF).
Example 2: Preparation of argF- and NCgl1221-Deleted Corynebacterium glutamicum Strain
The NCgl1221 gene encoding a protein involved in glutamate export was further deleted in ATCC 13032 ΔargF strain and KCCM-10785P ΔargF strain obtained in Example 1 in order to increase the intracellular level of glutamate which is an ornithine precursor.
To be specific, based on the nucleotide sequence (SEQ ID No. 29) of NCgl1221 of the ATCC 13032 strain, a homologous recombination fragment adjacent to the N-terminal sequence of NCgl1221 and a homologous recombination fragment adjacent to the C-terminal sequence of NCgl1221 were constructed. For this, the fragment adjacent to the N-terminal adjacent sequence of NCgl1221 was generated by PCR using the genomic DNA from ATCC 13032 strain as a template and primers (SEQ ID Nos. 5 and 6), and the fragment adjacent to the C-terminal sequence of NCgl1221 was generated by PCR using the genomic DNA from ATCC 13032 strain as a template and primers (SEQ ID Nos. 7 and 8) under the same PCR condition as in Example 1 (Table 2).







TABLE 2 







Primers for preparing NCgl1221-deleted strain










SEQ ID



Name
No.
Sequence(5′-3′)





NCgl1221-del-F1_BamHI
5
CGGGATCCGTCCAAGCCAAGCCGATTTCAAC




(SEQ ID NO: 5)


 


NCgl1221-del-R1_SalI
6
ACGCGTCGACCCACTCGGCGCTTGATAATAC




(SEQ ID NO: 6)


 


NCgl1221-del-F2_SalI
7
ACGCGTCGACCTGGAACAAGAACTCTCCAGC




(SEQ ID NO: 7)


 


NCgl1221-del-R2_XbaI
8
CTAGTCTAGA GGTTGGTGCTTCCACTGCTG




(SEQ ID NO: 8)









The above-prepared homologous recombination fragment adjacent to the N-terminal sequence of NCgl1221 was restriction digested with BamHI and SalI. Likewise, the homologous recombination fragment adjacent to the C-terminal sequence of NCgl1221 was restriction digested with SalI and XbaI. Then each of the cleaved fragments was inserted into the pDZ vector that was cleaved with BamHI and XbaI, thereby producing a plasmid pDZ-NCgl1221(K/O).
The above-prepared plasmid pDZ-NCgl1221(K/O) was transformed into ATCC 13032 ΔargF strain and KCCM-10785P ΔargF strain. Then, the transformed strains were plated and cultured on BHIS plate (Braine heart infusion 37 g/l, sorbitol 91 g/l, agar 2%) which contains kanamycin (25 μg/ml) and X-gal(5-bromo-4-chloro-3-indolin-β-D-galactoside), while letting the colonies to grow on the plate. Among the colonies formed on the plate, colonies with blue colour was collected to select for the strain inserted with the plasmid pDZ-NCgl1221(K/O).
The above-selected strains were cultured with shaking in CM medium at 30° C. for 8 hours. Subsequently, each cell culture was serially diluted from 10−4 to 10−10. Then the diluted samples were plated and cultured on an X-gal-containing solid medium, letting the colonies to grow. Among the colonies formed on the plate, only the white colonies which appear at relatively low frequency were collected to select for the NCgl1221-deleted strains.
Successful insertion of the plasmid pDZ-NCgl1221(K/O) into the above-selected strains was confirmed by performing PCR using the chromosomal DNA from the above-selected strain as a template, and primers of SEQ ID Nos. 5 and 8. The selected NCgl1221-deleted strains were named as ATCC 13032 ΔargF ΔNCgl1221 or KCCM-10785P ΔargF ΔNCgl1221 accordingly.
Example 3: Preparation of argCJBD Gene-Inserted Corynebacterium glutamicum Strain
Example 3-1: Cloning of argCJBD Gene and Preparation of Transformant
In this example, a vector inserted with argC, argJ, argB, and argD genes (SEQ ID Nos. 32, 34, 36, and 38 respectively) was prepared and a transformant was prepared by introducing the same, in order to enhance the ornithine synthetic pathway by increasing the copy number of argCJBD operon (SEQ ID No. 31, comprising the promoter region) which codes for the enzymes involved in a synthetic pathway of ornithine from glutamate.
First, PCR was performed to obtain argCJBD gene by using the chromosome of ATCC 13032 strain as a template and primers (SEQ ID Nos. 9 and 10) (30 cycles of denaturation for 40 seconds at 95° C., annealing for 40 seconds at 55° C., and extension for 150 seconds at 72° C.), thereby obtaining a gene fragment having a size of 4,900 bp.







TABLE 3 







Primers to obtain argCJBD gene fragment of


ATCC 13032










SEQ ID



Name
No.
Sequence(5′-3′)





P_argC-5-
 9
CGGGGTACCCTCCTCCAGCAGCTCTAGCTC


KpnI

(SEQ ID NO: 9)


 


argD-3_XbaI
10
TGCTCTAGAAAGTTTGAGTCCTTTATGCG




(SEQ ID NO: 10)









The above-prepared gene fragment was run through gel electrophoresis on 0.8% agarose gel, and a band of the target size was cut and DNA sample was isolated therefrom. The isolated DNA was restriction digested with KpnI and XbaI to obtain a fragment, then the cleaved fragment was cloned into a pHC139T-gfp vector (Korea Patent Publication No. 2008-0074286), thereby producing an expression vector pHC139T-argCJBD(Cgl).
Subsequently, the expression vector pHC139T-argCJBD(Cgl) prepared for increasing the production level of ornithine in the cell was introduced into ATCC 13032 ΔargF ΔNCgl1221 strain and KCCM-10785P ΔargF ΔNCgl1221 strain through electroporation. Then, a successful transformant was selected by plating the transformed cells on BHIS plate containing 25 μg/ml kanamycin. Finally, each of the selected transformants was named as ATCC 13032 ΔargF ΔNCgl1221/pHC139T-argCJBD(Cgl) and KCCM-10785P ΔargF ΔNCgl1221/pHC139T-argCJBD(Cgl) accordingly.
Example 3-2: Substitution of the Promoter of argCJBD Gene in the Chromosome
In this example, a promoter of argCJBD was substituted with CJ7 promoter which was newly developed by the present applicant in the chromosome, in order to increase the expression level by removing the regulation of the argCJBD gene which codes for the enzymes involved in a synthetic pathway of ornithine from glutamate.
First, a homologous recombination fragment comprising a CJ7 promoter and a nucleotide sequence of both terminal sites of the promoter was prepared.
To be specific, the nucleotide sequence of 5′-terminal site of CJ7 promoter was obtained by performing PCR using the genomic DNA from ATCC 13032 strain as a template and primers (SEQ ID Nos. 11 and 12) (28 cycles of denaturation for 30 seconds at 94° C., annealing for 30 seconds at 55° C., and extension for 30 seconds at 72° C.). Likewise, the nucleotide sequence of CJ7 promoter region was obtained by PCR using primers (SEQ ID Nos. 13 and 14) under same PCR condition, and the nucleotide sequence of 3′-terminal site of CJ7 promoter was obtained by PCR using primers (SEQ ID Nos. 15 and 16) under same PCR condition.







TABLE 4 







Primers for substituting the promoter of


argCJBD gene










SEQ



Name
ID No.
Sequence (5′-3′)





argC-L-5-
11
CGGGATCCGCAACGCTTGCGGTGAGAGA


BamHI

(SEQ ID NO: 11)


 


argC-L-3-
12
CCGGAATTCCTGGAAGTGGTCGAAGAAGA


EcoRI

(SEQ ID NO: 12)


 


CJ7-5-EcoRI
13
CCGGAATTCGCCGGCATAGCCTACCGATG




(SEQ ID NO: 13)


 


CJ7-3-XbaI
14
TGCTCTAGAGATATCAGTGTTTCCTTTCG




(SEQ ID NO: 14)


 


argC-R-5-
15
TGCTCTAGAATGATAATGCATAACGTGTA


XbaI

(SEQ ID NO: 15)


 


argC-R-3-
16
ACGCGTCGACGCTTTCCGGAGGTGTTGTAC


SalI

(SEQ ID NO: 16)









The above-prepared 5′-terminal site fragment of promoter (argC-L) was restriction digested with BamHI and EcoRI, the CJ7 promoter region fragment was restriction digested with EcoRI and XbaI, and the 3′-terminal site fragment of promoter (argC-R) was restriction digested with XbaI and SalI. Then each of the cleaved PCR products was cloned into the pDZ vector which was also restriction digested with BamHI and SalI, thereby producing an expression vector pDZ-CJ7(arg) in which the promoter of argCJBD was substituted with CJ7 promoter.
The above-prepared expression vector pDZ-CJ7(arg) was transformed into ATCC 13032 ΔargF ΔNCgl1221 strain and KCCM-10785P ΔargF ΔNCgl1221 strain through electroporation. Then, the transformants were cultured with shaking in CM medium (30° C., 8 hours), and the cell culture was serially diluted from 10−4 to 10−10. Then, the diluted samples were placed and cultured on BHIS plate containing 25 μg/ml kanamycin and X-gal, letting the colonies to grow.
The white colonies which appear at low frequency were isolated from most of the blue colonies, thereby selecting only the strain where the arg promoter was successfully substituted with CJ7 promoter through double crossover. Successful substitution of argCJBD promoter in chromosome by the introduced expression vector pDZ-CJ7(arg) was confirmed by performing PCR using the genomic DNA from the above-selected strains as a template and primers (SEQ ID Nos. 13 and 16) (28 cycles of denaturation for 30 seconds at 94° C., annealing for 30 seconds at 55° C., and extension for 60 seconds at 72° C.). Finally, the confirmed strains were named as ATCC 13032 ΔargF ΔNCgl1221 P(CJ7)-argCJBD and KCCM-10785P ΔargF ΔNCgl1221 P(CJ7)-argCJBD accordingly.
Example 4: Corynebacterium glutamicum Strain Introduced with speC Gene
A speC gene coding for ODC of E. coli which synthesizes putrescine from ornithine was introduced within the inactivated biotin synthesis-related gene in the chromosome of Corynebacterium glutamicum strain.
Example 4-1: Preparation of Expression Vector Comprising a Gene Fragment of ODC
In order to introduce the speC gene derived from E. coli (SEQ ID No. 40) into Corynebacterium glutamicum strain, speC gene was cloned into the vector along with a CJ7 promoter having SEQ ID No. 42 such that speC is expressed from the CJ7 promoter.
First, a nucleotide sequence of CJ7 promoter region was obtained by performing PCR using p117-CJ7-gfp (Korea Patent Registration No. 10620092) as a template and primers (SEQ ID Nos. 17 and 18) (30 cycles of denaturation for 40 seconds at 94° C., annealing for 40 seconds at 55° C., and extension for 60 seconds at 72° C.), and a nucleotide sequence of peC-coding region was obtained by performing PCR using the chromosome of wild-type E. coli strain W3110 as a template and primers (SEQ ID Nos. 19 and 20) under the same PCR condition.







TABLE 5 







Primers to obtain the P(CJ7)-speC gene fragment









Name
SEQ ID No.
Sequence (5′-3′)





CJ7-5-KpnI
17
CGGGGTACCGCCGGCATAGCCTACCGATG




(SEQ ID NO: 17)


 


CJ7-3
18
p-GATATCAGTGTTTCCTTTCG




(SEQ ID NO: 18)


 


speC(Ec)-5
19
p-ATCATGAAATCAATGAATATTGCCG




(SEQ ID NO: 19)


 


speC(Ec)-3_XbaI
20
TGCTCTAGATTACTTCAACACATAACCGTACAAC




(SEQ ID NO: 20)









The nucleotide sequences of CJ7 promoter region and speC-gene coding region were restriction digested with KpnI and XbaI, and cloned into the pHC139T-gfp vector which was also treated with KpnI and XbaI, thereby generating an expression vector pHC139T-P(CJ7)-speC(Ec) which comprises the genes for CJ7 promoter and ODC-coding region on downstream thereof.
Example 4-2: Preparation of Transformant
Since biotin synthesis-related genes are partially deleted in Corynebacterium sp. strain, in this example, speC gene derived from E. coli was introduced in between the biotin synthetic genes bioA and bioD.
To be specific, to use both terminal sites of P(CJ7)-speC(Ec) gene fragment comprised in the expression vector pHC139T-P(CJ7)-speC(Ec) prepared in Example 4-1 as homologous recombination sites in the Corynebacterium sp. strain chromosome, each of the terminal sites of P(CJ7)-speC(Ec) was cloned to have the nucleotide sequence of bioA and bioD. For this, bioA gene fragment was obtained by performing PCR using the genome of ATCC 13032 strain as a template and primers (SEQ ID Nos. 21 and 22) (28 cycles of denaturation for 40 seconds at 94° C., annealing for 30 seconds at 55° C., and extension for 60 seconds at 72° C.) Likewise, bioD gene fragment was obtained by performing PCR under the same PCR condition using the same template but different primers (SEQ ID Nos. 25 and 26). Then the P(CJ7)-speC(Ec) gene fragment was obtained by PCR using the expression vector pHC139T-P(CJ7)-speC(Ec) as a template and primers (SEQ ID Nos. 23 and 24) under the same PCR condition.







TABLE 6 







Primers for insertion of P(CJ7)-speC gene fragment within


the chromosome (bioA, bioD)









Name
Seq ID No.
Sequence(5′-3′)





bioA-5-BamHI
21
CGGGATCCTGCGCGAGCTTGATCACCGA




(SEQ ID NO: 21)


 


bioA-3-ScaI
22
AAAAGTACTGCCTTGCCCACACACATGAT




(SEQ ID NO: 22)


 


P(CJ7)-5-ScaI
23
AAAAGTACTGCCGGCATAGCCTACCGATG




(SEQ ID NO: 23)


 


speC(Ec)-3-EcoRI
24
CCGGAATTCTTACTTCAACACATAACCGTACAAC




(SEQ ID NO: 24)


 


bioD-5-EcoRI
25
CCGGAATTCGCTGTTTTGGCGGATGAGAG




(SEQ ID NO: 25)


 


bioD-3-XbaI
26
TGCTCTAGACGCAAAAAGGCCATCCGTCA




(SEQ ID NO: 26)









The above-prepared bioA gene fragment was restriction digested with BamHI and ScaI, P(CJ7)-speC(Ec) gene fragment was restriction digested with ScaI and EcoRI, and bioD gene fragment was restriction digested with EcoRI and XbaI. Then these digested PCR products were cloned into the pDZ vector which was also treated with BamHI and XbaI, thereby generating the expression vector pDZ-bioAD-P(CJ7)-speC(Ec) for insertion of speC gene into the chromosome.
The above-prepared expression vector pDZ-bioAD-P(CJ7)-speC(Ec) was transformed into ATCC 13032 ΔargF ΔNCgl1221 strain, ATCC 13032 ΔargF ΔNCgl1221 P(CJ7)-argCJBD strain, KCCM-10785P ΔargF ΔNCgl1221 strain, and KCCM-10785P ΔargF ΔNCgl1221 P(CJ7)-argCJBD strain through electroporation, thereby generating the transformant of each strain.
Each of these transformants was cultured with shaking in CM medium (30° C., 8 hours), and the cell culture was serially diluted from 10−4 to 10−10. Then the diluted cell culture was plated and cultured on BHIS plate containing 25 μg/ml kanamycin and X-gal, letting the colonies form.
White colonies appear at relatively low frequency compared to majority of the colonies which have blue colour. By selecting the white colonies, only the strains where P(CJ7)-speC was inserted into the chromosome through double crossover could be selected. A successful insertion of P(CJ7)-speC gene in between bioA and bioD in the chromosome by the expression vector pDZ-bioAD-P(CJ7)-speC(Ec) was confirmed by performing PCR using the genomic DNA obtained from each of the selected strains as a template and primers (SEQ ID Nos. 21 and 26) (28 cycles of denaturation for 30 seconds at 94, annealing for 30 seconds at 55, and extension for 120 seconds at 72). Finally selected strains were named as ATCC 13032 ΔargF ΔNCgl1221 bioAD::P(CJ7)-speC(Ec), ATCC 13032 ΔargF ΔNCgl1221 P(CJ7)-argCJBD bioAD::P(CJ7)-speC(Ec), KCCM-10785P ΔargF ΔNCgl1221 bioAD::P(CJ7)-speC(Ec) or KCCM-10785P ΔargF ΔNCgl1221 P(CJ7)-argCJBD bioAD::P(CJ7)-speC(Ec) accordingly.
Furthermore, the pHC139T-argCJBD(Cgl) vector prepared in Example 3-1 was transformed into ATCC 13032 ΔargF ΔNCgl1221 bioAD::P(CJ7)-speC(Ec) strain and KCCM-10785P ΔargF ΔNCgl1221 bioAD::P(CJ7)-speC(Ec) strain. The prepared transformants were named as ATCC 13032 ΔargF ΔNCgl1221 bioAD::P(CJ7)-speC(Ec)/pHC139T-argCJBD(Cgl) and KCCM-10785P ΔargF ΔNCgl1221 bioAD::P(CJ7)-speC(Ec)/pHC139T-argCJBD(Cgl) accordingly.
Example 5: Effect of argF and NCgl1221 Deletion, Enhancement of argCJBD Expression Level, and Insertion of speC Gene in Improvement of Putrescine Productivity
Example 5-1: Analysis of Putrescine Productivity in ATCC 13032 Corynebacterium glutamicum 
In order to confirm the effect of argF deletion, NCgl1221 deletion, enhancement of argCJBD expression level, and insertion of speC gene in putrescine productivity of ATCC 13032 Corynebacterium glutamicum strain, the putrescine productivities of the strains which were generated in Examples 2 to 4 were compared.
To be specific, each of the strains generated in Examples 2 to 4 (ATCC 13032 ΔargF ΔNCgl1221 (Test Group 1), ATCC 13032 ΔargF ΔNCgl1221/pHC139T-argCJBD(Cgl) (Test Group 2), ATCC 13032 ΔargF ΔNCgl1221 P(CJ7)-argCJBD (Test Group 3), ATCC 13032 ΔargF ΔNCgl1221 bioAD::P(CJ7)-speC(Ec) (Test Group 4), ATCC 13032 ΔargF ΔNCgl1221 bioAD::P(CJ7)-speC(Ec)/HC139T-argCJBD(Cgl) (Test Group 5) and ATCC 13032 ΔargF ΔNCgl1221 P(CJ7)-argCJBD bioAD::P(CJ7)-speC(Ec) (Test Group 6)) was spreaded on Corn Meal Agar (CMA) plate containing 11.8% (w/v) agar and cultured at 37 for 24 hours. Then, each of the cultured strains was inoculated into 25 ml titer medium comprising 1 mM arginine (2% (w/v) glucose, 1% (w/v) polypeptone, 0.5% (w/v) yeast extract, 0.5% (w/v) (NH4)2SO4, 0.15% (w/v) urea, 0.4% (w/v) KH2PO4, 0.8% (w/v) K2HPO4, 0.05% (w/v) MgSO4, 100 μg/l biotin and 1 mg/l thiamine) and further cultured with shaking at 30 and 200 rpm for 72 hours. Then the concentration of ornithine and putrescine produced therefrom was measured and compared (Table 7). Also, strain ATCC 13032 with no genomic modification was used as a control group.







TABLE 7







Comparison of putrescine productivity in each of the mutant


strains derived from ATCC 13032 strain









Test group
Ornithine(g/L)
Putrescine(g/L)





Control
0.0
0.0


1
6.0
0.0


2
6.4
0.0


3
7.7
0.0


4
0.1
5.2


5
0.1
6.2


6
0.2
8.1









As shown in Table 7, when argF and NCgl1221 genes were deleted or when argF and NCgl1221 genes were deleted and argCJBD gene expression level was increased, only the production of ornithine was observed but putrescine was not produced in the cell. This might be caused by absence of speC gene which codes for ODC that synthesizes putrescine from ornithine in Corynebacterium glutamicum strain.
On the other hand, in three types of strains which were inserted with E. coli-derived speC gene as prepared in Example 4-2, ornithine was rarely present in the cell while production of putrescine was observed. This result suggests that through insertion of E. coli-derived speC gene which expresses ODC, putrescine could be synthesized from ornithine by ODC.
Also, when the production level of ornithine in the strains from Test Groups 1 to 3 was compared with the production level of putrescine in the strains from Test Groups 4 to 6 which were generated by inserting speC gene into the strains of Test Groups 1 to 3, it was evident that the production level of putrescine was comparable to the production level of ornithine. Furthermore, compared to when exogenous argCJBD gene was inserted, when the expression level of endogenous argCJBD gene was increased the production levels of ornithine and putrescine were improved.
Example 5-2: Examination of the Putrescine Productivity of Glutamate-Producing Corynebacterium glutamicum KCCM-10785P Strain
In order to confirm the effect of argF deletion, NCgl1221 deletion, enhancement of argCJBD expression level, and insertion of speC gene in putrescine productivity of glutamate-overproducing Corynebacterium glutamicum strain KCCM-10785P, putrescine productivity of each of the strains generated in Examples 2 to 4 was compared.
To be specific, each of the strains generated in Examples 2 to 4 (KCCM-10785P ΔargF ΔNCgl1221 (Test Group 1), KCCM-10785P ΔargF ΔNCgl1221/pHC139T-argCJBD(Cgl) (Test Group 2), KCCM-10785P ΔargF ΔNCgl1221 P(CJ7)-argCJBD (Test Group 3), KCCM-10785P ΔargF ΔNCgl1221 bioAD::P(CJ7)-speC(Ec) (Test Group 4), KCCM-10785P ΔargF ΔNCgl1221 bioAD::P(CJ7)-speC(Ec)/HC139T-argCJBD(Cgl) (Test Group 5), and KCCM-10785P ΔargF ΔNCgl1221 P(CJ7)-argCJBD bioAD::P(CJ7)-speC(Ec) (Test Group 6)) was inoculated in the same condition as described in Example 5-1 and cultured with shaking at 30 and 200 rpm for 48 hours. Then the concentration of ornithine produced in each cell culture was measured and compared (Table 6). Also, KCCM-10785P strain with no genomic modification was used as a control group.







TABLE 8







Comparison of putrescine productivity in each of the strains


derived from KCCM-10785P strain













Glutamate
Ornithine
Putrescine



Test group
(g/L)
(g/L)
(g/L)
















Control
15.5
0.0
0.0



1
5.2
7.6
0.0



2
4.8
7.9
0.0



3
2.0
9.0
0.0



4
1.4
1.7
5.9



5
0.1
1.3
6.8



6
0.0
0.1
9.5










As shown in Table 8, when argF and NCgl1221 genes were deleted even in a glutamate-overproducing strain or when argF and NCgl1221 genes were deleted and the expression level of argCJBD gene was increased, only the production of ornithine was observed, but no putrescine was produced in the cell.
Meanwhile, production of putrescine was observed only in three types of strains which were introduced with E. coli-derived speC gene as prepared in Example 4-2. This result suggests that through introduction of E. coli-derived speC gene, the ODC enzyme expressed therefrom could synthesize putrescine from ornithine.
When the production levels of glutamate and ornithine in the strains of Test Groups 1 to 3 were compared, it was observed that as the production amount of glutamate was reduced, the production amount of ornithine was increased comparatively. Also, when the production level of ornithine in the strains of Test Groups 1 to 3 was compared with the production level of putrescine in the strains of Test Groups 4 to 6 which were generated by inserting speC gene into the strains of Test Groups 1 to 3, the production level of putrescine was comparable with the production level of ornithine. In addition, compared to when the exogenous argCJBD gene was inserted, when the expression level of intrinsic argCJBD gene was increased the production level of ornithine and putrescine was increased. In conclusion, it was confirmed that as the production level of glutamate in the cell is increased, the amount of ornithine is also increased, which in turn enhances the production level of putrescine in the end.
Overall, the present inventors have named the strain prepared in Example 4-2, which was proved to have an excellent ability to produce putrescine, as “CC01-0064 (ATCC 13032 ΔargF ΔNCgl1221 P(CJ7)-argCJBD bioAD::P(CJ7)-speC(Ec))”, and deposited this strain under Budapest treaty to Korean Culture Center of Microorganisms (KCCM) located in Hongje 1-dong, Seodaemun-gu, Seoul, Korea in Nov. 24, 2010 with Accession No. KCCM11138P.
Based on the above description, it should be understood by those skilled in the art that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention without departing from the technical idea or essential features of the invention as defined in the following claims. 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.

## Claims

1. A putrescine-producing recombinant microorganism, wherein the microorganism has been modified to reduce endogenous ornithine carbamoyltransferase activity and glutamate exporter activity compared to the corresponding unmodified microorganism, wherein the microorganism has been modified to express an ornithine decarboxylase (ODC), wherein the microorganism is a Corynebacterium glutamicum, 
wherein the glutamate exporter prior to the modification has an amino acid sequence comprising SEQ ID NO: 30, wherein the ornithine carbamoyltransferase prior to the modification has an amino acid sequence comprising SEQ ID NO: 28, and
wherein the ornithine carbamoyltransferase activity and the glutamate exporter activity are reduced by a method selected from the group consisting of (i) a partial or full deletion of a gene coding for an endogenous ornithine carbamoyltransferase or an endogenous glutamate exporter, (ii) a modification of a regulatory region of a gene encoding an endogenous ornithine carbamoyltransferase or an endogenous glutamate exporter to reduce the gene expression, (iii) a modification of a gene coding for an endogenous ornithine carbamoyltransferase or coding for an endogenous glutamate exporter to reduce endogenous ornithine carbamoyltansferase activity or endogenous glutamate exporter activity, and (iv) any combination of (i), (ii), or (iii).

2. The putrescine-producing recombinant microorganism of claim 1, wherein the ornithine decarboxylase has an amino acid sequence comprising SEQ ID NO: 41.

3. The putrescine-producing recombinant microorganism of claim 1, wherein the microorganism is modified to express the ODC by a method selected from the group consisting of an insertion of a polynucleotide encoding the ODC into a chromosome, insertion of a polynucleotide encoding the ODC into a vector system to be introduced into the microorganism, insertion of a promoter with improved activity upstream of a polynucleotide encoding the ODC, and insertion of a gene encoding the ODC with a modified promoter.

4. The putrescine-producing recombinant microorganism of claim 1, wherein endogenous N-acetyl-gamma-glutamyl-phosphate reductase (ArgC) activity, endogenous N-acetylglutamate synthase or ornihthine acetyltransferase (ArgJ) activity, endogenous acetylglutamate kinase (ArgB) activity, and endogenous acetylornithine aminotransferase (ArgD) activity which are involved in biosynthesis of ornithine are further enhanced compared to the corresponding unmodified microorganism,
wherein the ArgC activity, ArgJ activity, ArgB activity, and ArgD activity are enhanced by a method selected from the group consisting of (i) increasing of the copy number of a gene encoding ArgC, ArgJ, ArgB, or ArgD, (ii) modifying a regulatory region of a gene encoding the ArgC, ArgJ, ArgB, or ArgD to increase expression, (iii) a modification of an endogenous gene coding for ArgC, ArgJ, ArgB, or ArgD to enhance ArgC, ArgJ, ArgB, or ArgD activity, and (iv) any combination of (i), (ii), or (iii).

5. The putrescine-producing recombinant microorganism of claim 4, wherein the ArgC, ArgJ, ArgB, and ArgD have an amino acid sequence comprising SEQ ID NOs: 33, 35, 37, and 39 respectively.

6. The putrescine-producing recombinant microorganism of claim 1, wherein the Corynebacterium glutamicum is Corynebacterium glutamicum KCCM11138P.

7. A method for producing putrescine, comprising the step of (i) obtaining a cell culture by culturing the putrescine-producing recombinant microorganism according to claim 1; and (ii) recovering putrescine from the cultured microorganism or cell culture.