# Patent

## Title
Recombinant microorganism having an enhanced ability to produce putrescine and a method for producing putrescine using the same

## Abstract

The present invention relates to a recombinant microorganism having enhanced ability to produce putrescine at high yield, wherein the activity of NCg10101 is weakened in a microorganism of genus Corynebacterium that has been modified to produce putrescine, and a method for producing putrescine using the same.

## Background
TECHNICAL FIELD
The present invention relates to recombinant microorganisms having an enhanced ability to produce putrescine and a method for producing putrescine using the same.
BACKGROUND ART
Putrescine (or 1,4-butanediamine) is a type of polyamine, such as spermidine and spermine, and is found in gram-negative bacteria and fungi. Since putrescine is present in a wide range of concentrations in various species, it is expected to play an important role in the metabolism of microorganisms. Putrescine is commonly produced by chemical synthesis through acrylonitrile and succinonitrile from propylene. The chemical synthesis uses the substances derived from petrochemicals as starting materials and uses toxic chemicals, and thus it is not environment-friendly and has a problem of oil depletion.
In order to resolve these problems, there has been much research on developing a method for biosynthesis of putrescine by using microorganisms, that is more environment-friendly and reduces energy consumption. According to current knowledge, putrescine can be biosynthesized through two pathways. In one pathway, ornithine is produced from glutamate and the ornithine is decarboxylated to synthesize putrescine. In the other pathway, arginine is synthesized from the ornithine, agmatine is produced from the arginine, and then putrescine is synthesized from the agmatine. In addition, there are other methods for synthesizing putrescine by using a target microorganism which is transformed with the enzymes involved in the known synthetic pathways of putrescine. For example, WO09/125924 discloses a method for producing putrescine at high yield by inactivating the pathway involved in the decomposition and utilization of putrescine in E. coli, by inactivating the pathway in which ornithine, a precursor of putrescine, is converted to arginine, and by enhancing the biosynthetic pathway of ornithine. An article published in 2010 discloses a method for producing putrescine at high concentration by introducing and enhancing the protein that converts ornithine to putrescine into Corynebacterium strains which are not capable of producing putrescine. In addition it discloses a method for producing putrescine from arginine by introducing E. coli-derived arginine decarboxylase and agmatinase into the strains. In this regard, the ornithine pathway produced about 50 times higher amount of putrescine than the arginine pathway (Schneider et al., Appl. Microbiol. Biotechnol. 88:4, 859-868, 2010).
DISCLOSURE
Technical Problems
In this background, the present inventors identified that putrescine can be produced at high yield in a microorganism of genus Corynebacterium by weakening or removing the activity of NCg10101 protein (SEQ ID NOS: 17 or 19), thereby completing the present invention.
Technical Solution
One objective of the present invention is to provide a recombinant microorganism of genus Corynebacterium capable of producing putrescine at high yield, which is modified to have the weakened NCg10101 (SEQ ID NOS: 17 or 19) activity, compared to the endogenous activity thereof.
Another objective of the present invention is to provide a method for producing putrescine using the microorganism.
Advantageous Effect
When the microorganism of genus Corynebacterium having an improved ability to produce putrescine of the present invention is used for the production of putrecine, it is modified to weaken NCg10101 (SEQ ID NOS: 17 or 19) activity compared to the endogenous activity thereof, and therefore, it can be produce putrescine at high yield. Accordingly, the microorganism can be widely used for the more effective production of putrescine.



DESCRIPTION OF FIGURES
FIG. 1 represents a schematic diagram showing the relative positions of genes encoding NCg10100 (SEQ ID NO: 27), NCg10101 (SEQ ID NOS: 17 or 19), NCg10102 (SEQ ID NO: 29), NCg10103 (SEQ ID NO: 30), and NCg10104 (SEQ ID NO: 31), which are on the chromosome of the wild type Corynebacterium glutamicum ATCC 13032 strain; and
FIG. 2 represents the test result of growth comparison between the recombinant strains prepared in the present invention, in which 1, 2, 3, 4, 5 and 6 are strains prepared by introducing pHC139T, pHC139T-P(CJ7)-NCg10100, pHC139T-P(CJ7)-tNCg10100, pHC139T-P(CJ7)-NCg10101, pHC139T-P(CJ7)-NCg10102-NCg10103, and pHC139T-P(CJ7)-NCg10104 into KCCM11138P, respectively.



BEST MODE
In one aspect to achieve the above objectives, the present invention provides a recombinant microorganism of genus Corynebacterium having an enhanced ability to produce putrescine, which is modified by weakening or removing the activity of NCg10101 protein having an amino acid sequence represented by SEQ ID NO. 17 or SEQ ID NO. 19, compared to the endogenous activity thereof.
As used herein, the term “NCg10101” means a protein showing the activity of a metal-dependent enzyme, which is expressed in Corynebacterium glutamicum (SEQ ID NOS: 17 or 19), and whose function is not yet fully known. It includes a metal binding domain of peptidase M20 family or aminobenzoyl-glutamate utilization protein (AbgB). The AbgB of E. coli constitutes aminobenzoyl-glutamate hydrolase with AbgA to hydrolyze aminobenzoyl-glutamate to aminobenzoate and glutamate. The aminobenzoate is known to be used as a precursor for folate synthesis, but its relationship with putrescine productivity has not been known.
NCg10101 protein of the present invention may comprise the amino acid sequence represented by SEQ ID NO: 17 or SEQ ID NO: 19. However, it is not limited thereto, because there may be the difference in the amino acid sequence of the protein depending on the microbial species or strains. In other words, it can be a mutant protein or artificial variant with an amino acid sequence comprising substitution, deletion, insertion, or addition of one or several amino acids at one or more locations of the amino acid sequence represented by SEQ ID NO: 17 or SEQ ID NO: 19, as long as it can help increase the ability to produce putrescine by weakening the activity of the protein. Herein, “several” may differ depending on the location or type in the three-dimensional structure of amino acid residues of the protein, but specifically means 2 to 20, specifically 2 to 10, and more specifically 2 to 5. In addition, the substitution, deletion, insertion, addition or inversion of the amino acid includes those caused by artificial variants or natural mutation, based on the difference in the individual or species of microorganism.
The polynucleotide encoding the amino acid sequence of the present invention may comprise the polynucleotide sequence encoding the protein having amino acid sequence represented by SEQ ID NO: 17 or SEQ ID NO: 19, or the amino acid sequence of 80% or more, specifically 90% or more, more specifically 95% or more, and particularly specifically 97% or more homology with the same, as long as it has similar activity as the NCg10101 protein. The most specifically, it may be the polynucleotide sequence represented by SEQ ID NO: 16 or SEQ ID NO: 18.
The term “homology” refers to the identity between two amino acid sequences and may be determined by the well known method to those skilled in the art, using BLAST 2.0 to compute the parameter such as score, identity and similarity.
In addition, the polynucleotide sequence encoding the polypeptide with the amino acid sequence of NCg10101 (SEQ ID NOS: 17 or 19) of the present invention can be hybridized with the polynucleotide of SEQ ID. NO: 16 or the probe prepared from the same under ‘stringent conditions’, and may be a modified polynucleotide sequence encoding the NCg10101 protein (SEQ ID NOS: 17 or 19) which normally functions. As used herein, “stringent conditions” refer to conditions which allow the specific hybridization between the polynucleotide, and are described specifically, for example, in Molecular Cloning (A Laboratory Manual, J. Sambrook et al., Editors, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, N. Y., 1989) or Current Protocols in Molecular Biology (F. M. Ausubel et al., Editors, John Wiley & Sons, Inc., New York). For example, the hybridization is carried out in the hybridization buffer of 65° C. (3.5×SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH2PO4 (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride/0.15 M sodium citrate of pH 7. After hybridization, the membrane to which DNA is transferred is rinsed with 2×SSC at room temperature and then rinsed again with 0.1 to 0.5×SSC/0.1×SDS at a temperature of 68° C.
The activity of NCg10101 protein (SEQ ID NOS: 17 or 19) in the present invention can be weakened by 1) a partial or whole deletion of a polynucleotide encoding the protein, 2) modifying an expression regulatory sequence to reduce the expression of the polynucleotide, 3) a modification of the polynucleotide sequence on chromosome or 4) a combination thereof.
In the above, a partial or whole deletion of a polynucleotide encoding the protein can be performed by substituting the polynucleotide encoding an endogenous target protein in the chromosome to a marker gene or a polynucleotide which partial nucleotide sequence was deleted, with a vector for chromosomal gene insertion. The length of the “partial” deletion depends on the type of polynucleotide, but is specifically 2 bp to 300 bp, more specifically 2 bp to 100 bp, and further more specifically 1 bp to 5 bp.
Also, to decrease the polynucleotide expression, an expression regulatory sequence may be modified by inducing mutations in the expression regulatory sequence through deletion, insertion, conservative or non-conservative substitution of nucleotide sequence or a combination thereof to further weaken the activity of the expression regulatory sequence, or by replacing the expression regulatory sequence with the sequence having weaker activity. The expression regulatory sequence may include a sequence encoding promoter, operator sequence, ribosomal binding site and the sequence controlling the termination of transcription and translation.
In addition, the polynucleotide sequence on chromosome to weaken the activity of the protein may be modified by inducing mutations in the sequence through deletion, insertion, conservative or non-conservative substitution of nucleotide sequence or a combination thereof to further weaken the activity of the sequence, or by replacing the polynucleotide sequence with the modified sequence to have weaker activity of the protein.
Meanwhile, a microorganism of genus Corynebacterium having enhanced ability to produce putrescine of the present invention may be further modified to weaken the activity of ornithine carbamoyltransferase (ArgF) involved in the synthesis of arginine from ornithine and the activity of protein (NCg11221; SEQ ID NO: 21) involved in exporting glutamate, compared to the endogenous activity thereof. In addition, the microorganism of Corynebacterium genus may be modified by additionally introducing the activity of ornithine decarboxylase (ODC). Also, the microorganism of genus Corynebacterium may be further modified to enhance the activity of acetyl glutamate synthase to convert glutamate to acetyl glutamate or ornithine acetyltransferase (ArgJ) to convert acetyl ornithine to ornithine, the activity of acetyl glutamate kinase (ArgB) to convert acetyl glutamate to acetyl glutamyl phosphate, the activity of acetyl gamma glutamyl phosphate reductase (ArgC) to convert acetyl glutamyl phosphate to acetyl glutamate semialdehyde, and the activity of acetyl ornithine amino transferase (ArgD) to convert acetyl glutamate semialdehyde to acetyl ornithine, compared to the endogenous activities thereof, thereby enhancing the biosynthetic pathway of ornithine, a putrescine precursor (Sakanyan V et al., Microbiology. 142:1, 99-108, 1996).
In this case, the ArgF, NCg11221, ODC, ArgC, ArgJ, ArgB and ArgD may have, but are not specifically limited to, the amino acid sequences represented by SEQ ID. NO: 20, 21, 22, 23, 24, 25, 26, respectively, or the amino acid sequences with 80% or more, specifically 90% or more, more specifically 95% or more, and most specifically 97% or more homology with the same.
As used herein, the term “ornithine decarboxylase (ODC)” refers to an enzyme that produces putrescine using ornithine, and the ODC requires pyridoxalphosphate (Pyridoxal 5′-phosphate, PLP) as a coenzyme. The ODC is found in most Gram-negative bacteria and may be found in some of the intestinal bacteria such as Lactobacillus of Gram-positive bacteria. E. coli has two types of genes encoding ODC, one of which, speC, is expressed continuously at the certain concentration and the other, speF, is expressed under specific conditions (the presence of ornithine at higher than certain concentrations and low pH). Depending on species, some species, like E. coli, have two kinds of ODC, and others have only one type. The species such as Escherichia sp., Shigella sp., Citrobacter sp., Salmonella sp., and Enterobacter sp. have two kinds of ODC (speC, speF), and the strains of Yersinia sp., Klebsiella sp., Erwinia sp., have one kind of ODC (spec). In case of lactobacillus, ODC is expressed in one type of gene (speF), and is known to be induced to be expressed under the conditions of low pH or abundant ornithine and histidine.
ODC activity may be introduced to the recombinant microorganism of genus Corynebacterium of the present invention using genes encoding ODC derived from the various species. The polynucleotide encoding the ODC may include, but is not limited to, the polynucleotide encoding the protein consisting of the amino acid sequence represented by SEQ ID NO: 22 and the amino acid sequence of 70% or more, specifically 80% or more, more specifically 90% or more homology with the same.
In addition, the introduction of ornithine decarboxylase (ODC) activity to the microorganisms may be performed by the various methods well known in the art; for example, the method to insert the polynucleotide including a nucleotide sequence encoding ODC to chromosome, the method to introduce the polynucleotide to the microorganisms by introducing to the vector system, the method to insert the promoter which is modified or has improved activity to the upper region of nucleotide sequence encoding ODC, and the method to insert mutation to the nucleotide sequence encoding ODC. More specifically, if the nucleotide sequence encoding ODC is introduced, known CJ7 promoter may be used as a promoter to control the expression of the same.
In addition, the enhancement of the activity of ArgC, ArgJ, ArgB and ArgD can be achieved by 1) an increase of the copy number of polynucleotide encoding the enzyme, 2) a modification of the expression regulatory sequence to increase the polynucleotide expression, 3) a modification of the polynucleotide sequence encoding the enzyme on chromosome to enhance the activity of the enzyme or 4) a combination thereof.
In method 1), the increase of the copy number of polynucleotide encoding the enzyme can be achieved by operably linking the polynucleotide to the vector or by inserting the same to the chromosome of the host cell. More specifically, the copy number of polynucleotide of the host cell can be increased by introducing a vector that is capable of replicating and functioning independently, wherein the polynucleotide encoding the enzyme of the present invention is operably linked, or by introducing the vector capable of inserting the polynucleotide into the chromosome of the host cell, wherein the polynucleotide is operably linked.
As used herein, the term “vector” refers to the DNA construct comprising the nucleotide sequence of the polynucleotide encoding the target protein operably linked to the proper regulatory sequence to express the target protein in the proper host. The regulatory sequence includes the promoter which can initiate transcription, any operator sequence to control the transcription, the sequence to encode the appropriate mRNA ribosome binding site, and the sequence to control the termination of transcription and translation. The vector may be transfected into a suitable host, and then may be replicated or function independently from the host genome, and may be integrated into the genome itself.
In the present invention, any vector which is known in the art may be used without any specific limitation as long as it can be replicated in the host. Examples of commonly used vectors are plasmid, cosmid, virus and bacteriophage in natural state or recombinant state. For example, pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A can be used as a phage vector or cosmid vector, and pBR system, pUC system, pBluescriptll system, pGEM system, pTZ system, pCL system and pET system can be used as a plasmid vector. The vector which can be used in the present invention is not particularly limited and the known expression vectors can be used. Specifically, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors can be used. Most specifically, pACYC177, pCL, pCC1BAC vectors can be used.
In addition, the vector which can insert the polynucleotide encoding the target protein into chromosome of a host cell may specifically be, for example, a shuttle vector, pECCG112 (Korean Patent Publication No. 1992-0000933) which is able to replicate by itself both in E. coli and Coryneform bacteria, but is not limited thereto.
In addition, the polynucleotide encoding the target protein in the chromosome may be replaced by a new polynucleotide by using a vector for chromosomal gene insertion. The insertion of the polynucleotide to the chromosome can be achieved by any method known in the art, for example, by homologous recombination. Since the vector of the present invention may be inserted into the chromosome by inducing a homologous recombination, the selection marker may be additionally included to confirm a successful gene insertion into the chromosome. A selection marker is for screening the cells which are transformed with the vector, in other words, for determining whether the target polynucleotide is inserted. The markers that provide selectable phenotypes such as drug resistance, auxotrophy, resistance to toxic agents or expression of surface proteins may be used. In an environment treated with a selective agent, only the cells expressing the selection marker can survive or cells show a different phenotype, and thus the successfully transformed cells can be selected through this method.
As used herein, the term “transformation” refers to the introduction of the vector comprising a polynucleotide encoding the target protein into the host cell so that the protein can be expressed in the cell. The transformed polynucleotide includes all polynucleotide which encode target proteins that can be expressed in the host cell regardless of the location, whether it is inserted into the chromosome of the host cell or located outside the chromosome. In addition, the polynucleotide includes DNA and RNA encoding the target protein. The polynucleotide may be introduced in any form as long as it can be introduced into the host cell and expressed. For example, the polynucleotide can be introduced into a host cell in a form of an expression cassette which is gene construct, comprising all the required elements for self-expression. The expression cassette typically includes a promoter operably linked to the polynucleotide, transcription termination signal, ribosomal binding site, and translation termination signal. The expression cassette may be the form of expression vector capable of self-replication. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to the sequences required for the expression of host cell.
As used herein, the term “operably linked” refers to the functional connection between the promoter sequence initiating or mediating the transcription of polynucleotide encoding the target protein and the polynucleotide.
In addition, the method 2) modification of the expression regulatory sequence to increase the expression of the polynucleotide in the present invention may be performed by inducing the mutation of the sequence through deletion, insertion, conservative or non-conservative substitution of nucleotide sequence or a combination thereof, or by substitution by the nucleotide sequence with enhanced activity. The expression regulatory sequence includes promoter, operator sequence, sequence encoding ribosomal binding sites, and sequence to control the termination of transcription and translation.
A strong heterologous promoter may be linked to the upper of expression unit of the polynucleotide instead of original promoters. An example of a strong promoter is pcj7 promoter, lysCP1 promoter, EF-Tu promoter, groEL promoter, aceA or aceB promoter, etc., and more specifically lysCP1 promoter or pcj7 promoter derived from Corynebacterium is operably linked to enhance the expression of polynucleotide encoding the enzyme. Herein, lysCP1 promoter, which is an improved promoter through substitution of the nucleotide sequence of the promoter region of polynucleotide encoding aspartate kinase and aspartate semialdehyde dehydrogenase, is strong enough to increase the activity of the corresponding enzyme by 5 times compared to the wild type through enhancement of expression of aspartate kinase gene (International Patent Publication No. 2009-096689). In addition, the pcj7 promoter was identified to be expressed in Corynebacterium ammoniagenes and Escherichia and to have a strong promoter activity, and can be expressed in Corynebacterium glutamicum as well in high intensity (Korean Patent No. 0620092).
In addition, the method 3) modification of the polynucleotide sequence on chromosome may be performed, but are not specifically limited to, by inducing the mutation of the sequence through deletion, insertion, conservative or non-conservative substitution of nucleotide sequence or a combination thereof to enhance the activity of the sequence, or by substitution by the nucleotide sequence having enhanced activity.
The microorganism in the present invention, which is a microorganism having the ability to produce putrescine, includes prokaryotic microorganism, wherein the protein comprising amino acid sequence represented by in SEQ ID NO: 17 or SEQ ID NO: 19 is expressed, and may be, for example, the microorganism of Escherichia sp., Shigella sp., Citrobacter sp., Salmonella sp., Enterobacter sp., Yersinia sp., Klebsiella sp., Erwinia sp., Corynebacterium sp., Brevibacterium sp., Lactobacillus sp., Sllenomanas sp., and Vibrio sp.
The microorganism in the present invention is specifically the microorganism of genus Corynebacterium and may more specifically be of Corynebacterium glutamicum. 
An embodiment of the present invention, the microorganism of genus Corynebacterium of accession number KCCM11138P (Korean Patent laid-open No. 2012-0064046), which has the ability to produce putrescine in a high concentration through enhanced putrescine-biosynthesis pathway, was modified. Specifically, the putrescine-producing strain KCCM11138P is the putrescine-overproducing strain, wherein the gene encoding ornithine carbamoyltransferase (ArgF) for accumulating ornithine and the gene encoding glutamate exporter (NCg11221; SEQ ID NO: 21) for increasing intracellular glutamate are deleted from ATCC13032 strains, the gene encoding ornithine decarboxylase (spec) is introduced, and the expression level of ornithine biosynthesis genes (argCJBD) is increased.
Another embodiment of the present invention, Corynebacterium glutamicum ATCC13869-based putrescine-producing strain DAB12-a was modified. The strain ATCC13869 was based on the same genotype as the KCCM11138P, which is putrescine-producing strain, based on Corynebacterium glutamicum ATCC13032. Specifically, putrescine-producing strain DAB12-a is from ATCC13869 strain obtained from American Type Culture Collection (ATCC), wherein the gene encoding ornithine carbamoyltransferase (ArgF) and the gene encoding the protein NCg11221 (SEQ ID NO: 21) to export glutamate are deleted, the gene (spec) encoding ornithine decarboxylase (ODC) derived from E. coli is introduced, and the promoter of ornithine biosynthesis gene operon (argCJBD) is replaced with the improved promoter.
According to one embodiment of the present invention, a microorganism of genus Corynebacterium (KCCM11138P) has an ability to produce putrescine, which is prepared by deletion of the gene encoding ornithine carbamoyl transferase (ArgF) and the gene encoding the glutamate exporter (NCg11221; SEQ ID NO: 21) involved in glutamate export, replacement of the own promoter of ArgCJBD gene cluster encoding an enzyme involved in the synthesis of ornithine from glutamate, and introduction of the gene (spec) encoding ornithine decarboxylase (ODC) into the chromosome in the wild-type Corynebacterium glutamicum ATCC13032. Based on KCCM11138P, a clone (A15) growing well in a medium containing high concentration of putrescine was selected, and it was confirmed that the selected A15 includes genes encoding NCg10100 (SEQ ID NO: 27), NCg10101 (SEQ ID NOS: 17 or 19), NCg10102 (SEQ ID NO: 29), NCg10103 (SEQ ID NO: 30) and NCg10104 (SEQ ID NO: 31) (Example 1). In addition, the microorganism grows in the medium containing high concentration of putrescine due to the gene encoding NCg10101 (SEQ ID NOS: 17 or 19) among the five types of genes (Example 2). As regards character of the gene encoding NCg10101 (SEQ ID NOS: 17 or 19), it was confirmed that putrescine production was reduced in a strain in which the gene encoding NCg10101 (SEQ ID NOS: 17 or 19) is overexpressed (Example 3), and putrescine production was increased in a strain in which the gene encoding NCg10101 (SEQ ID NOS: 17 or 19) is deleted (Example 4).
Accordingly, the present inventors named the Corynebacterium glutamicum strain having an enhanced ability to produce putrescine, which is prepared by removing the NCg10101 (SEQ ID NOS: 17 or 19) gene in the putrescine-producing strain KCCM 11138P, as Corynebacterium glutamicum CC01-0244, and deposited in the Korean Culture Center of Microorganisms (hereinafter, abbreviated to “KCCM”) on Dec. 26, 2011, with Accession No. KCCM11241P.
In another aspect of the present invention to achieve the above objectives, the present invention relates to a method for producing putrescine, comprising the steps of:
culturing the microorganism of genus Corynebacterium having an enhanced ability to produce putrescine, which is modified to have the weakened activity of NCg10101 protein (SEQ ID NOS: 17 or 19) having an amino acid sequence represented by SEQ ID NO. 17 or SEQ ID NO. 19; and

isolating putrescine from the culture broth obtained in the above step.

The culturing process in the present invention may be carried out in appropriate medium and under culturing conditions known in the art. Those skilled in the art can easily adjust and use the culturing process depending on selected strains. An example of the culturing process includes batch, continuous and fed-batch type cultures, but is not limited thereto. The culture medium may have to appropriately satisfy the requirements of a specific strain.
The culture medium may have to appropriately satisfy the requirements of specific strains. Culture media for various microorganisms are disclosed (for example, “Manual of Methods for General Bacteriology” from American Society for Bacteriology (Washington D.C., USA, 1981)). As a source of carbon in the medium, sugar and carbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose), butterfat 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), etc. may be used. These substances may be used individually or as a mixture. As a source of nitrogen, nitrogen-containing organic compound (e.g., peptone, yeast extract, beef extract, malt extract, corn steep liquor, soybean meal powder and urea) or inorganic compound (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate) may be used and these substances also may be used individually or as a mixture. As a source of phosphorus, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salt may be used. In addition, the culture medium may comprise metal salt (e.g., magnesium sulfate or iron sulfate) which is essential for the growth, and finally, essential growth-promoting substances such as amino acids and vitamins, may be used in addition to the above-mentioned substances. The appropriate precursor may be added in addition to the culture medium. The feed substance may be provided in the culture at once or adequately while culturing.
The pH of the culture may be adjusted by a proper basic compound (e.g., sodium hydroxide, potassium hydroxide or ammonia) or acidic compound (e.g., phosphoric acid or sulfuric acid). Foaming may be adjusted by an anti-foaming agent such as fatty acid polyglycolester. Aerobic condition of the culture may be maintained by introducing oxygen or oxygen-containing gas mixtures, for example, air. Culturing temperature may be typically 20 to 45° C., specifically 25 to 40° C. Culturing may be continued until the production of putrescine reaches the desired maximum, it may be usually achieved in 10 to 160 hours. Putrescine may be released into culture medium, or contained in the cell.
For the method for collecting and recovering the produced putrescine in the culturing process of the present invention, the target substance may be recovered from the culture medium using the appropriate known method in the art depending on the culture method, for example, batch, continuous or fed-batch type culture.
Mode for Invention
Hereinafter, the present invention will be described in more detail with the following Examples. However, these Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples.
Example 1
Library Preparation for Selection of Effective Genes for Putrescine Biosynthesis and Selection of Clones
In order to screen effective genes for putrescine biosynthesis from the chromosome of the wild-type Corynebacterium strain, a chromosome library of the wild-type Corynebacterium strain was prepared. In detail, the chromosome extracted from the wild-type Corynebacterium glutamicum ATCC 13032 strain was randomly cleaved with the restriction enzyme Sau3AI, and fragments of 5 to 8 kb were selected therefrom, and then cloned into an E. coli-Corynebacterium shuttle vector pECCG122 (Korean Patent laid-open No. 1992-0000933) to prepare a chromosome library.
In order to select effective genes for putrescine biosynthesis from the Corynebacterium chromosome library thus prepared, colonies growing in a medium containing high concentration of putrescine were obtained.
Meanwhile, the libraries were introduced into a microorganism of Corynebacterium genus (KCCM11138P) having an ability to produce putrescine, so as to prepare each of transformants. The transformants which were able to grow in a minimal medium containing 0.35 M putrescine (10 g/l of glucose, 0.4 g/l of MgSO4.7H2O, 4 g/l of NH4Cl, 1 g/l of KH2PO4, 1 g/l of K2HPO4, 2 g/l of urea, 10 mg/l of FeSO4.7H2O, 1 mg/l of MnSO4.5H2O, 5 mg/l of nicotinamide, 5 mg/l of thiamine hydrochloride, 0.1 mg/l of biotin, 1 mM arginine, 25 mg/l of kanamycin, 0.35 M putrescine, pH 7.0) were selected. The strain KCCM11138P is disclosed in a patent applied by the present inventors (Korean Patent laid-open No. 2012-0064046), which was prepared by deleting genes encoding ornithine carbamoyltransferase (argF) and glutamate exporter (NCg11221; SEQ ID NO: 21) in the chromosome of the wild type Corynebacterium glutamicum strain ATCC 13032, introducing a gene (spec) encoding ornithine decarboxylase (ODC) derived from the wild type E. coli W3110 strain into the chromosome, and replacing the promoter of argCJBD gene cluster encoding the enzyme involved in the synthesis of ornithine from glutamate, so as to prepare each of transformants. As a result, 275 colonies were selected, and colonies growing well in the medium containing high concentration of putrescine were secondarily identified. Each library clone was obtained and introduced into the putrescine strain again. Thereafter, colonies growing well in the medium containing high concentration of putrescine were identified and thus a clone (A15) was finally selected. This selected clone was identified by sequencing. As a result, it was confirmed that the clone comprises total 5 ORFs that encode NCg10100 (SEQ ID NO: 27), NCg10101 (SEQ ID NOS: 17 or 19), NCg10102 (SEQ ID NO: 29), NCg10103 (SEQ ID NO: 30) and NCg10104 (SEQ ID NO: 31), of which 436 amino acids at the N-terminus were removed (FIG. 1). FIG. 1 is a schematic diagram showing the relative positions of genes encoding NCg10100 (SEQ ID NO: 27), NCg10101 (SEQ ID NOS: 17 or 19), NCg10102 (SEQ ID NO: 29), NCg10103 (SEQ ID NO: 30) and NCg10104 (SEQ ID NO: 31), which are on the chromosome of the wild type Corynebacterium glutamicum ATCC 13032 strain.
Example 2
Identification of Effective Genes for Putrescine Synthesis in A15 Clone
Example 2-1
Cloning of 5 Genes in A15 Clone and Preparation of a Transformant
The nucleotide sequence of the A15 clone obtained in Example 1 was already known. Based on the nucleotide sequence of ATCC13032 strain previously reported, NCg10100-F and NCg10100-R represented by SEQ ID NOs. 1 and 2 as primers for amplification of the gene that encodes NCg10100 (SEQ ID NO: 27), NCg10100-R and tNCg10100-F represented by SEQ ID NOs. 2 and 3 as primers for amplification of the gene that encodes tNCg10100 (SEQ ID NO: 28) of which 436 amino acids at the N-terminus were removed, NCg10101-F and NCg10101-R represented by SEQ ID NOs. 4 and 5 as primers for amplification of gene that encodes NCg10101 (SEQ ID NOS: 17 and 19), NCg10102-F and NCg10103-R represented by SEQ ID NOs. 6 and 7 as primers for amplification of both the genes that encode NCg10102 (SEQ ID NO: 29) and NCg10103 (SEQ ID NO: 30), and NCg10104-F and NCg10104-R represented by SEQ ID NOs. 8 and 9 as primers for amplification of the gene that encodes NCg10104 (SEQ ID NO: 31) were constructed. In addition, P(CJ7)-F and P(CJ7)-R represented by SEQ ID NOs. 10 and 11 as primers for amplification of the expression promoter P(CJ7) (or pcj7) (Korean Patent No. 10-0620092) were constructed (Table 1).
Thereafter, PCR was carried out using the chromosome of ATCC 13032 strain as a template and each of the primer represented by SEQ ID NOs. 1 to 9 (denaturation at 95° C. for 30 seconds, annealing at 50° C. for 30 seconds, and extension at 72° C. for 1 minute ̃1 minute 30 seconds, 25 cycles), so as to amplify 5 types of gene fragments. In addition, PCR was carried out using the chromosome of Corynebacterium ammoniagenes as a template and primers represented by SEQ ID NOs. 10 and 11 so as to amplify the promoter fragment.
5 genes cleaved with KpnI and XbaI, and CJ7 promoter cleaved with EcoRV and KpnI were ligated into an expression vector pHC139T (Korean Patent No. 10-0860932) cleaved with EcoRV and XbaI, so as to prepare total 5 types of expression vectors, pHC139T-P(CJ7)-NCg10100, pHC139T-P(CJ7)-tNCg10100, pHC139T-P(CJ7)-NCg10101, pHC139T-P(CJ7)-NCg10102-NCg10103, and pHC139T-P(CJ7)-NCg10104.







TABLE 1





Primers for preparation of strains 


expressing 5 genes contained in A15 clone


















NCg10100-F (SEQ ID NO. 1)
GCGCAT ATGAGCTCAAC




AACCTCAAAAACC



 



NCg10100-R (SEQ ID NO. 2)
GCGTCTAGA TTATCCTT




CGAGGAAGATCGCAG



 



tNCgt0100-F (SEQ ID NO. 3)
GCGCAT ATGTGGACGCT




GATGGCTGC



 



NCg10101-F (SEQ ID NO. 4)
GCGCAT ATGAGTACTGA




CAATTTTTCTCCAC



 



NCg10101-R (SEQ ID NO. 5)
GCGTCTAGA CTAAGCCA




AATAGTCCCCTAC



 



NCg10102-F (SEQ ID NO. 6)
GCGCAT ATGGATGAACG




AAGCCGGTTTG



 



NCg10103-R (SEQ ID NO. 7)
GCGTCTAGATTAATCAAT




GAAGACGAATACAATTCC



 



NCg10104-F (SEQ ID NO. 8)
GCGCATATGGCGGGTGAC




AAATTGTGG



 



NCg10104-R (SEQ ID NO. 9)
GCGTCTAGATTAGGACAG




TTCCGCTGGAGC



 



P(CJ7)-F (SEQ ID NO. 10)
CAGATATCGCCGGCATAG




CCTACCGATG



 



P(CJ7)-R (SEQ ID NO. 11)
GCGTCTAGAGATATCAGT




GTTTCCTTTCG










5 types of the expression vectors thus prepared and a control group pHC139T were introduced into the KCCM11138P strain of Example 1 by electroporation, and then spread on BHIS plates containing 25 μg/ml kanamycin to select transformants.
Example 2-2
Search of Effective Genes for Putrescine
From the total 6 types of the transformants obtained in Example 2-1, transformants growing well in the medium containing high concentration of putrescine were selected in the same manner as in Example 1 (FIG. 2). FIG. 2 is the test result of comparing growth between the transformants prepared in the present invention, in which 1, 2, 3, 4, 5 and 6 represent strains introduced with the 6 types of expression vectors, pHC139T, pHC139T-P(CJ7)-NCg10100, pHC139T-P(CJ7)-tNCg10100, pHC139T-P(CJ7)-NCg10101, pHC139T-P(CJ7)-NCg10102-NCg10103 and pHC139T-P(CJ7)-NCg10104, respectively. As shown in FIG. 2, only the transformant (No. 4) introduced with pHC139T-P(CJ7)-NCg10101 showed excellent growth in the medium containing high concentration of putrescine, and thus NCg10101 (SEQ ID NOS: 17 or 19) was selected as the effective gene for putrescine biosynthesis.
Example 3
Evaluation of the Ability to Produce Putrescine in NCg10101-Overexpressing Strain
The ability to produce Putrescine of the strain overexpressing the NCg10101 (SEQ ID NOS: 17 or 19) gene which was identified as the effective gene in Example 2 was evaluated. A strain for evaluation was prepared by introducing pHC139T-P(CJ7)-NCg10101 into the putrescine-producing strain KCCM11138P.
pHC139T-P(CJ7)-NCg10101 prepared in Example 2-1 and pHC139T vector as a control group were introduced into the putrescine-producing strain KCCM 11138P by electroporation, and then spread on BHIS plates containing 25 μg/ml kanamycin to select transformants. The transformants were named as KCCM 11138P/pHC139T, and KCCM 11138P/pHC139T-P(CJ7)-NCg10101, respectively. These two transformants thus selected were cultured in CM plates containing 1 mM arginine (1% glucose, 1% polypeptone, 0.5% yeast extract, 0.5% beef extract, 0.25% NaCl, 0.2% urea, 100 μl of 50% NaOH, 2% agar, pH 6.8 per 1 L) at 30° C. for 24 hours, and then a loop of cell culture was inoculated in 25 ml of titer medium of Table 2 containing 25 μg/ml kanamycin, and cultured with shaking at 200 rpm at 30° C. for 96 hours. All of the prepared strains were cultured with addition of 1 mM arginine in the medium during fermentation.










TABLE 2







Composition
Concentration (per 1 L)









Glucose
  8%



Soybean protein
0.25%



Corn steep solids
 0.5%



(NH4)2SO4
  4%



Urea
0.15%



KH2PO4
 0.1%



MgSO47H2O
0.05%



Biotin
 100 μg



Thiamine Hydrochloride
3000 μg



Calcium-Panthotenic Acid
3000 μg



Nicotinamide
3000 μg



CaCO3
  5%










As a result, as shown in Table 3, when NCg10101 (SEQ ID NOS: 17 or 19) was overexpressed, putrescine production was reduced.










TABLE 3








Putrescine



Strain type
(g/L)









KCCM 11138P/pHC139T
9.5



KCCM 11138P/pHC139T-P(CJ7) -NCg10101
5.1










Example 4
Evaluation of the Ability to Produce Putrescine in NCg10101-Deleted Strain
Example 4-1
Preparation of NCg10101-Deleted Strain in ATCC 13032-Based Putrescine-Producing Strain
NCg10101 (SEQ ID NOS: 17 or 19) overexpression increased cell growth in the medium containing high concentration of putrescine, but decreased putrescine production according to Example 3. On the basis of this result, the effect of NCg10101 (SEQ ID NOS: 17 or 19) deletion on the ability to produce putrescine was examined.
In detail, based on the NCg10101 nucleotide sequence of ATCC 13032 strain (SEQ ID NOS: 16 or 18), NCg10101-del-F1_BamHI and NCg10101-del-R1_SalI represented by SEQ ID NOs. 12 and 13 as primers were constructed to obtain a homologous recombinant fragment of the N-terminal region of NCg10101 (SEQ ID NOS: 17 or 19). NCg10101-del-F2_SalI and NCg10101-del-R2_XbaI represented by SEQ ID NOs. 14 and 15 as primers were constructed to obtain a homologous recombinant fragment of the C-terminal region of NCg10101 (SEQ ID NOS: 17 or 19) (Table 4). The fragments of the N-terminal and C-terminal regions of NCg10101 gene (SEQ ID NOS: 16 or 18) were prepared by PCR using the two pairs of the primers. The PCR products were treated with BamHI & SalI and SalI & XbaI, respectively and cloned into a pDZ vector treated with BamHI & XbaI. The cloned plasmid was named as pDZ-NCg10101(K/O).







TABLE 4





Primers for preparation of 


NCg10101-deleted strains
















NCg10101-del-F1_BamHI 
CGGGATCC 


(SEQ ID NO. 12)
CGGATTCCCTGCGATCATTG


 


NCg10101-del-R1_SalI 
ACGCGTCGAC 


(SEQ ID NO. 13)
CAGTCGACGGAACTTGTGGAG


 


NCg10101-del-F2_SalI 
ACGCGTCGAC 


(SEQ ID NO. 14)
GGCAACGACTCCGAAACCTTC


 


NCg10101-del-R2_XbaI 
CTAGTCTAGA 


(SEQ ID NO. 15)
CTGGATCCTCATGAATGCGC









The pDZ-NCg10101(K/O) vector prepared for obtaining the KCCM 11138P ΔNCg10101 strain was introduced into KCCM 11138P strain by electroporation, and then spread on the BHIS plate containing 25 μg/ml kanamycin. The successful insertion of the vector in the chromosome was confirmed by observing whether the colony was blue on the solid medium containing X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). The primary chromosome inserted strain was shaking-cultured in a nutrient medium (30° C., 8 hours), was then diluted from 10−4 to 10−10, and spread on the solid medium containing X-gal. While a majority of colonies appeared as blue colony, a low proportion of colonies appeared as white colonies. The NCg10101 (SEQ ID NOS: 17 or 19) gene-deleted strains were finally selected by double crossover with the white colonies, and identified by PCR using the primers represented by SEQ ID NOs. 12 and 15. The variant thus identified was named as KCCM 11138P ΔNCg10101.
Example 4-2
Preparation of NCg10101-Deleted Strain in ATCC 13869-Based Putrescine-Producing Strain
Corynebacterium glutamicum ATCC13869-based putrescine-producing strain DAB12-a (argF-deleted, NCg11221 (SEQ ID NOS: or 19)-deleted, E. coli speC-introduced, and arg operon-argCJBD promoter-substituted strain), which has the same genotype as that of the putrescine-producing strain KCCM11138P based on Corynebacterium glutamicum ATCC13032, was used to prepare NCg10101 (SEQ ID NOS: 17 or 19)-deleted strains.
In detail, in order to identify the gene encoding NCg10101 (SEQ ID NOS: 17 or 19) derived from Corynebacterium glutamicum ATCC13869 and the amino acid sequence of the protein expressed therefrom, PCR was carried out using the genomic DNA of Corynebacterium glutamicum ATCC13869 as a template and a pair of primers, SEQ ID NOs. 12 and 15 (NCg10101-del-F1_BamHI, NCg10101-del-R2_XbaI). Here, PCR reaction was carried out with 30 cycles of denaturation at 95° C. for 30 seconds, annealing at 53° C. for 30 seconds, and extension at 72° C. for 2 minutes and 30 seconds. The PCR products were separated by electrophoresis and their sequences were analyzed. Through sequence analysis, it was identified that the gene encoding NCg10101 (SEQ ID NOS: 17 or 19) derived from Corynebacterium glutamicum ATCC13869 includes a nucleotide sequence represented by SEQ ID NO. 18 and the protein encoded thereby includes an amino acid sequence represented by SEQ ID NO. 19. When the amino acid sequences of NCg10101 (SEQ ID NOS: 17 or 19) derived from Corynebacterium glutamicum ATCC13032 and that of NCg10101 (SEQ ID NOS: 17 or 19) derived from Corynebacterium glutamicum ATCC13869 were compared, they showed 98% sequence homology.
In order to delete the gene encoding NCg10101 (SEQ ID NOS: 17 or 19) derived from Corynebacterium glutamicum ATCC13869, the region of N-terminal and C-terminal of NCg10101 gene (SEQ ID NOS: 16 or 18) were amplified by PCR using a genomic DNA of Corynebacterium glutamicum ATCC13869 as a template and two pairs of primers listed in Table 4 in the same manner as Example <4-1>. Then, the PCR products were treated with BamHI & SalI and SalI & XbaI, respectively and then cloned into the pDZ vector treated with BamHI & XbaI, thereby constructing a plasmid pDZ-2′NCg10101(K/O).
The plasmid pDZ-2′NCg10101(K/O) was transformed into Corynebacterium glutamicum DAB12-a in the same manner as in Example <4-1>, and the strain in which the gene encoding NCg10101 (SEQ ID NOS: 17 or 19) is deleted was selected. The selected Corynebacterium glutamicum variant was named as DAB12-a ΔNCg10101.
Example 4-3
Evaluation of the Ability to Produce Putrescine in NCg10101-Deleted Strain
In order to investigate the effect of NCg10101 (SEQ ID NOS: 17 or 19) deletion on the ability to produce putrescine in the putrescine-producing strain, the Corynebacterium glutamicum variants prepared in Examples <4-1> and <4-2> was compared.
In detail, the ability to putrescine in two types of Corynebacterium glutamicum variants (KCCM11138P ΔNCg10101 and DAB12-a ΔNCg10101) was evaluated in the same manner as in example 3. As shown in the following Table 5, putrescine production was found to be increased by NCg10101 (SEQ ID NOS: 17 or 19) deletion.










TABLE 5







Strain type
Putrescine (g/L)



















KCCM 11138P
9.8



KCCM 11138P ΔNCg10101
11.3



DAB12-a
10.1



DAB12-a ΔNCg10101
11.0










Taken together, the results of Examples 3 and 4 show that putrescine production was decreased by overexpression of the gene encoding NCg10101 (SEQ ID NOS: 17 or 19) and increased by deletion of the gene in the wild type Corynebacterium glutamicum strain, indicating that NCg10101 (SEQ ID NOS: 17 or 19) directly affects putrescine biosynthesis.
Accordingly, the present inventors named the Corynebacterium glutamicum strain having an improved ability to produce putrescine, which was prepared by deleting the NCg10101 gene (SEQ ID NOS: 16 or 18) in the putrescine-producing strain KCCM 11138P in the above Example, as Corynebacterium glutamicum CC01-0244, and deposited in Korean Culture Center of Microorganisms (hereinafter, abbreviated to as “KCCM”) which is international depositary authority under the Budapest Treaty on Dec. 26, 2011, with Accession No. KCCM11241P.
Based on the above descriptions, those skilled in the art will understand that the present invention may be conducted in other forms without changing the technical idea or essential technical features. In this regard, the Examples described above are to illustrate the invention in all respects, but not to limit the scope of the invention. It shall be understood that the scope of the present invention comprises any changes or modified forms derived from the meaning, scope and equivalent concept of the following claims rather than the detailed descriptions in the above.

## Claims

1. A recombinant Corynebacterium glutamicum having enhanced ability to produce putrescine, wherein the activity of a protein having an amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 19 is down regulated or removed, compared to the endogenous activity thereof.

2. The recombinant Corynebacterium glutamicum according to claim 1, wherein ornithine decarboxylase activity is introduced into the recombinant Corynebacterium glutamicum.

3. The recombinant Corynebacterium glutamicum according to claim 2, wherein the ornithine decarboxylase has the amino acid sequence of SEQ ID NO: 22.

4. The recombinant Corynebacterium glutamicum according to claim 1, wherein ornithine carbamoyltransferase (ArgF) activity and/or glutamate exporter activity are down regulated compared to the endogenous activity thereof.

5. The recombinant Corynebacterium glutamicum according to claim 4, wherein the ArgF has the amino acid sequence of SEQ ID NO: 20, and glutamate exporter has the amino acid sequence of SEQ ID NO: 21.

6. The recombinant Corynebacterium glutamicum according to claim 1, wherein one or more activities selected from the group consisting of acetyl gamma glutamyl phosphate reductase (ArgC) activity, acetyl glutamate synthase activity or ornithine acetyltransferase (ArgJ) activity, acetyl glutamate kinase (ArgB) activity, and acetyl ornithine amino transferase (ArgD) activity are further enhanced.

7. The recombinant Corynebacterium glutamicum according to claim 6, wherein ArgC, ArgJ, ArgB and ArgD have the amino acid sequences of SEQ ID NOs: 23, 24, 25, and 26, respectively.

8. The recombinant Corynebacterium glutamicum according to claim 1, wherein the activity of the protein is down regulated by 1) a partial or whole deletion of a polynucleotide encoding the protein, 2) a reduction of the polynucleotide expression, 3) a modification of the polynucleotide sequence on chromosome to down regulate the activity of the protein or 4) a combination thereof.

9. A method for producing putrescine, comprising culturing a recombinant Corynebacterium glutamicum having enhanced ability to produce putrescine in a cell culture broth, wherein the activity of a protein having an amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 19 is down regulated or removed in the recombinant Corynebacterium glutamicum compared to the endogenous activity thereof; obtaining the cell culture broth; and isolating putrescine from the obtained cell culture broth.