CN104136600B - Recombinant microorganism with improved putrescine production capacity and method for preparing putrescine - Google Patents

Abstract

The present invention relates to a recombinant microorganism having an improved ability to produce putrescine in high yield, wherein the activity of NCgl0101 in a Corynebacterium microorganism modified to produce putrescine is weakened; and also relates to a method for producing putrescine using the microorganism.

Description

Recombinant microorganism with improved putrescine production capacity and method for preparing putrescine

technical field

The present invention relates to a recombinant microorganism with improved ability to produce putrescine, and a method for producing putrescine using the microorganism.

Background technique

Putrescine (or 1,4-butanediamine) is a class of polyamines, like spermidine and spermine, found in Gram-negative bacteria and fungi. Since putrescine is present in a wide range of concentrations in various species, it is estimated to play an important role in microbial metabolism. Putrescine is usually chemically synthesized from succinonitrile and acrylonitrile from propylene. The chemical synthesis employs substances derived from petrochemicals as starting materials and also utilizes toxic chemical substances, therefore, the method is not environmentally friendly and has a problem of petroleum consumption.

In order to solve these problems, many studies have attempted to develop methods for the biosynthesis of putrescine using microorganisms, which is more environmentally friendly and reduces energy consumption. According to current knowledge, there are two microbial pathways for the biosynthesis of putrescine. In one pathway, glutamate produces ornithine, which decarboxylates to putrescine. Another pathway is that ornithine synthesizes arginine, arginine produces agmatine, and then synthesizes putrescine from agmatine. Furthermore, other methods of synthesizing putrescine utilize target microorganisms transformed with enzymes involved in known putrescine synthesis pathways. For example, WO09/125924 discloses a method for high-yield production of putrescine by inactivating the pathway involved in the decomposition and utilization of putrescine in Escherichia coli (E. Arginine pathway inactivation, and ornithine biosynthesis pathway enhancement. A document published in 2010 disclosed a method for producing high-concentration putrescine by introducing a protein that converts ornithine into putrescine into a non-putrescine-producing Corynebacterium strain and increasing its active. In addition, a method for producing putrescine from arginine by introducing arginine decarboxylase and agmatinase derived from Escherichia coli into the strain is also disclosed. In this regard, the ornithine pathway produces about 50 times more putrescine than the arginine pathway (Schneider et al., Appl. Microbiol. Biotechnol. 88:4, 859-868, 2010).

Contents of the invention

technical problem

Against this background, the present inventors found that putrescine can be produced at a high yield in microorganisms belonging to the genus Corynebacterium by attenuating or removing the activity of the NCgl0101 protein, thereby completing the present invention.

Technical solutions

One of the objects of the present invention is to provide a recombinant microorganism of the genus Corynebacterium capable of producing putrescine at a high yield, which has been modified to have NCgl0101 activity attenuated compared to the endogenous activity.

Another object of the present invention is to provide a method for producing putrescine using the microorganism.

Beneficial effect

When putrescine is produced using the microorganism of the genus Corynebacterium of the present invention having an improved putrescine-producing ability, it is modified such that the activity of NCg10101 is attenuated compared with endogenous activity, and thus it can produce putrescine at a high yield. Therefore, the microorganism can be widely used to produce putrescine more efficiently.

Description of drawings

Fig. 1 is a schematic diagram showing the relative position of genes encoding NCgl0100, NCgl0101, NCgl0102, NCgl0103 and NCgl0104 on the chromosome (chromosome) of wild-type Corynebacterium glutamicum ATCC13032 bacterial strain; and

Fig. 2 has shown the test result of doing growth comparison between the recombinant strains prepared by the present invention, wherein, 1, 2, 3, 4, 5 and 6 are pHC139T, pHC139T-P(CJ7)-NCgl0100, pHC139T-P(CJ7 )-tNCgl0100, pHC139T-P(CJ7)-NCgl0101, pHC139T-P(CJ7)-NCgl0102-NCgl0103 and pHC139T-P(CJ7)-NCgl0104 were prepared by introducing KCCM11138P respectively.

best way

To achieve the above object, in one aspect, the present invention provides a recombinant microorganism of the genus Corynebacterium with improved putrescine-producing ability, which is modified by weakening or removing the activity of NCg10101 protein, which has The amino acid sequence shown in SEQ ID NO.17 or SEQ ID NO.19.

The term "NCg10101" as used herein denotes a protein exhibiting metal-dependent enzymatic activity, expressed in Corynebacterium glutamicum, the function of which has not been fully understood. It includes the M20 family of peptidases or the metal binding domain or aminopropoxy of the aminobenzoyl-glutamate utilization protein (AbgB). AbgB of Escherichia coli constitutes an AbgB-containing aminobenzoyl-glutamate hydrolase, which hydrolyzes aminobenzoyl-glutamate to anthranilate and glutamate. Anthranilate is known to serve as a precursor for folate synthesis, but its relationship to putrescine production is unknown.

The NCgl0101 protein of the present invention may comprise the amino acid sequence shown in SEQ ID NO:17 or SEQ ID NO:19. However, it is not limited thereto because the amino acid sequence of the protein may vary depending on the kind or strain of microorganisms. In other words, it may be a mutant protein or an artificial variant whose amino acid sequence contains one or more of the amino acid sequences shown in SEQ ID NO: 17 or SEQ ID NO: 19. Substitution, deletion, insertion or addition of several amino acids, as long as it helps to improve putrescine-producing ability by weakening the activity of the protein. The "several" herein may vary according to the position or type of the three-dimensional structure of the amino acid residues in the protein, but especially means 2-20, especially 2-10, more especially 2-5. Furthermore, amino acid substitutions, deletions, insertions, additions or inversions include those due to artificial variants or natural mutations depending on the individual or species of microorganisms.

The polynucleotide encoding the amino acid sequence of the present invention may include a polynucleotide sequence encoding the following protein: a protein having an amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 19, or an amino acid sequence thereof that is identical to SEQ ID NO: 17 or A protein having 80% or higher, especially 90% or higher, more particularly 95% or higher, especially 97% or higher homology of the amino acid sequence shown in SEQ ID NO: 19, as long as the protein has a homology with NCg10101 protein-like activity. Most particularly the polynucleotide sequence shown in SEQ ID NO:16 or SEQ ID NO:18.

The term "homology" means the identity between two amino acid sequences, which can be detected by methods well known to those skilled in the art, using BLAST2.0 to calculate parameters such as score, identity and similarity.

In addition, the polynucleotide sequence encoding NCg10101 of the present invention can hybridize with the polynucleotide shown in SEQ ID NO: 16 or the probe prepared therefrom under "stringent conditions", and can also be a protein encoding NCg10101 that can function normally modified polynucleotides. "Stringent conditions" as used herein refers to conditions that allow specific hybridization between polynucleotides, as specifically described in, for example, "Molecular Cloning" (Laboratory Manual, edited by J. Sambrook et al., 2nd edition, Cold Spring Harbor Laboratory Publishing, Cold Spring Harbor, New York, 1989) or "Recent Methods in Molecular Biology" (FMAusubel et al., eds., John Wiley & Sons, New York). For example, perform hybridization at 65°C in hybridization buffer (3.5×ssc, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5mM NaH ₂ PO ₄ (pH 7), 0.5% SDS, 2mM EDTA ). SSC is 0.15M sodium chloride/0.15M sodium citrate at pH 7. After hybridization, the DNA-transferred membrane was washed with 2xSSC at room temperature, followed by 0.1-0.5xssc/0.1xSDS at 68°C.

The activity of the NCg10101 protein of the present invention can be weakened in the following ways: 1) partial or complete deletion of the polynucleotide encoding the protein, 2) modification of the expression control sequence to reduce the expression of the polynucleotide, 3) modification of the sequence on the chromosome or 4) Their combination.

In the above, by using a chromosomal gene-inserted vector, a polynucleotide encoding a protein can be partially or completely missing. The length of a "partial" deletion may vary depending on the type of polynucleotide, but is particularly 2bp-300bp, more particularly 2bp-100bp, more particularly 1bp-5bp.

The polynucleotide expression can also be reduced by modifying the expression control sequence in the following manner: through the deletion of nucleotide sequence, insertion, conservative or non-conservative substitution or their combination, mutations are induced in the expression control sequence to further weaken the expression control sequence. activity, or to replace the expression control sequence with a less active sequence. Expression control sequences include sequences encoding promoters, operator sequences, ribosomal binding sites, and sequences that control termination of transcription and translation.

In addition, the polynucleotide sequence on the chromosome can be modified to weaken the activity of the protein by inducing mutations in the sequence to further weaken the sequence by deletion, insertion, conservative or non-conservative substitution of the nucleotide sequence, or a combination thereof activity, or to replace the polynucleotide sequence with a modified sequence in order to obtain a weaker protein activity.

At the same time, the Corynebacterium microorganism of the present invention with enhanced putrescine-producing ability can be further modified, thereby weakening the activity of ornithine carbamoyltransferase (ArgF) involved in the synthesis of arginine from ornithine compared with the endogenous activity and the activity of a protein (NCg10101) involved in glutamate export. Furthermore, microorganisms of the genus Corynebacterium can be modified by additionally introducing ornithine decarboxylase (ODC) activity. Compared with the endogenous activity, Corynebacterium can be further modified to enhance the activity of acetylglutamic acid synthase to convert glutamic acid into acetylglutamic acid, and to enhance the activity of ornithine acetyltransferase (ArgJ) to convert acetyl ornithine amino acid into ornithine activity, enhance the activity of acetylglutamate kinase (ArgB) to convert acetylglutamate into acetylglutamyl phosphate, and enhance the activity of acetylglutamyl phosphate reductase (ArgC) to convert acetylglutamine acyl phosphate into acetylglutamate semialdehyde, and enhance the activity of acetylornithine aminotransferase (ArgD) to convert acetylglutamate semialdehyde into acetylornithine, thereby enhancing putrescine precursor Biosynthetic pathway of ornithine (Sakanyan V et al., Microbiology. 142:1, 99-108, 1996).

In this example, ArgF, NCg11221, ODC, ArgC, ArgJ, ArgB and ArgD may have (but not specifically limited to) the amino acid sequences shown in SEQ ID NO.: 20, 21, 22, 23, 24, 25, 26, respectively , or amino acid sequences having 80% or higher, especially 90% or higher, more particularly 95% or higher, especially 97% or higher homology to these sequences.

The term "ornithine decarboxylase (ODC)" as used herein refers to an enzyme that utilizes ornithine to produce putrescine, and ODC requires pyridoxal phosphate (pyridoxal 5'-phosphate, PLP) as a coenzyme. ODC is found in most Gram-negative bacteria, but also in some intestinal bacteria, such as Gram-positive bacteria-lactic acid bacteria. Escherichia coli has two genes encoding ODC, one is speC, which is continuously expressed at a specific concentration, and the other speF is under specific conditions (in the presence of ornithine above a certain concentration and at low pH) Express. Depending on the species, some species, such as E. coli, have two ODCs, while others have only one. Such as Escherichia sp., Shigella sp., Citrobacter sp., Salmonella sp. and Enterobacter sp. Species have two ODCs (speC, speF), while strains such as Yersinia sp., Klebsiella sp., Erwinia sp. have one ODC (speC). In lactic acid bacteria, ODC is expressed by a gene (speF) that can be induced under conditions of low pH or sufficient ornithine and histidine.

The activity of ODC can be introduced into the recombinant microorganism of the genus Corynebacterium of the present invention using genes encoding ODC derived from a plurality of different species. The polynucleotide encoding ODC may include (but not limited to) a polynucleotide encoding a protein consisting of the following amino acid sequence: the amino acid sequence shown in SEQ ID NO.: 22, or 70% or higher, particularly is an amino acid sequence of 80% or higher, more particularly 90% or higher homology.

In addition, ornithine decarboxylase (ODC) activity can be introduced into microorganisms by various methods well known in the art; for example, a method of inserting a polynucleotide comprising a nucleotide sequence encoding ODC into a chromosome, introducing a multinuclear A method for introducing nucleotides into microorganisms, a method for inserting a modified or enhanced promoter into the upstream of the ODC coding nucleotide sequence, and a method for inserting mutations into the ODC coding nucleotide sequence. More particularly, if a nucleotide sequence encoding ODC is introduced, the known CJ7 promoter can be used as a promoter to control its expression.

In addition, the enhancement of ArgC, ArgJ, ArgB and ArgD activities can be achieved by: 1) increasing the number of polynucleotides encoding the enzyme; 2) modifying the expression control sequence to increase the expression of the polynucleotide; 3) modifying the enzyme on the chromosome The coding polynucleotide sequence to enhance the activity of the enzyme; or 4) a combination of the above.

In method 1), the copy number increase of the polynucleotide encoding the enzyme can be achieved by operatively linking the polynucleotide to a vector, or inserting it into the chromosome of the host cell. More specifically, the polynucleotide copy number of the host cell can be increased by introducing a vector capable of independent replication and functioning, wherein the polynucleotide encoding the enzyme of the present invention is operably linked; or by introducing a vector capable of inserting the polynucleotide The vector of the host cell chromosome increases the copy number of the polynucleotide in the host cell, wherein the polynucleotides are operably linked.

The term "vector" as used herein refers to a DNA construct containing the nucleotide sequence of a polynucleotide encoding a target protein, which is operably linked to appropriate regulatory sequences to express the target protein in a suitable host. Regulatory sequences include a promoter to initiate transcription, any operator sequences to control transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control termination of transcription and translation. The vector can be transfected into a suitable host, then replicate and function independently of the host genome, and can be integrated into the chromosome itself.

In the present invention, any vector known in the art can be used without any particular limitation as long as it can replicate in a host. Examples of commonly used vectors are plasmids, cosmids, viruses, and bacteriophages, either in their native or recombinant state. For example, pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, while pBR system, pUC system, pBluescriptII system, pGEM system, pTZ system, pCL system and pET system can be used as plasmid vectors. The vectors that can be used in the present invention are not particularly limited, and known expression vectors can be used. Specifically, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors can be used. More specifically, pACYC177, pCL, pCC1BAC vectors can be used.

In addition, the vector that can insert the polynucleotide encoding the target protein into the chromosome of the host cell can be specifically, for example, the shuttle vector pECCG112 (Korean Patent Laid-Open No. 1992-0000933), which can be used in Escherichia coli and Coryneform bacteria. Self-replicating in , but not limited to.

In addition, the polynucleotide encoding the target protein in the chromosome can be replaced with a new polynucleotide using the vector inserted by the chromosomal gene. Chromosomal insertion of polynucleotides can be achieved by any method known in the art, for example, by homologous recombination. Since the vector of the present invention can be inserted into the chromosome by inducing homologous recombination, a selectable marker can be additionally included to confirm the successful insertion of the gene into the chromosome. The selectable marker is used to select cells transformed with the vector, in other words, to determine whether the polynucleotide of interest has been inserted. Markers can be used that can provide a selectable phenotype such as drug resistance, auxotrophy, tolerance to toxic substances or expression of surface proteins. In the context of treatment with a selective substance, only cells expressing the selectable marker will survive, or the cells will display a different phenotype, thus allowing the selection of successfully transformed cells by this method.

As used herein, the term "transformation" refers to introducing a vector containing a polynucleotide encoding a target protein into a host cell such that the protein can be expressed in the cell. A transforming polynucleotide includes all polynucleotides encoding a target protein that can be expressed in a host cell regardless of its location, whether inserted into the host cell's chromosome or located extrachromosomally. In addition, the polynucleotides include DNA and RNA encoding target proteins. The polynucleotide can be introduced in any form as long as it can be introduced into a host cell and expressed. For example, polynucleotides can be introduced into host cells in the form of expression cassettes (gene constructs), which contain all the necessary elements for self-expression. The expression cassette typically includes a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. In addition, a polynucleotide can be introduced into a host cell on its own and operably linked to sequences required for expression by the host cell.

The term "operably linked" as used herein refers to a functional linkage between a promoter sequence that initiates or mediates transcription of a polynucleotide encoding a target protein and said polynucleotide.

In addition, method 2) can be implemented as follows, modifying the expression control sequence to enhance the expression of the polynucleotide of the present invention: introducing a mutation in the sequence by deletion, insertion, conservative or non-conservative substitution of the nucleotide sequence, or a combination thereof, or Replaced with activity-enhancing nucleotide sequences. The expression control sequence includes a promoter, an operator sequence, a ribosome binding site coding sequence, and a sequence controlling transcription and translation termination.

A strong heterologous promoter can be linked upstream of a polynucleotide expression unit to replace the original promoter. Examples of strong promoters are pcj7 promoter, lysCP1 promoter, EF-Tu promoter, groEL promoter, aceA or aceB promoter etc., more specifically, operably linked to lysCP1 promoter or pcj7 promoter derived from coryneform bacteria To enhance expression of the polynucleotide encoding the enzyme. In this paper, the lysCP1 promoter is an enhanced promoter obtained by replacing the nucleotide sequence of the promoter region of the polynucleotide encoding aspartate kinase and aspartate semialdehyde dehydrogenase. Expression of a kinase gene that is strong enough to increase the activity of the corresponding enzyme 5-fold compared to wild type (International Application Publication No. 2009-096689). In addition, the pcj7 promoter was identified to express and have strong promoter activity in Corynebacterium ammoniagenes and Escherichia, and it was also highly expressed in Corynebacterium glutamicum (Korean Patent No. 0620092).

In addition, method 3) can be implemented as described below (but not particularly limited thereto) to modify the polynucleotide sequence on the chromosome: to induce sequence mutation through deletion, insertion, conservative or non-conservative substitution of nucleic acid sequence or their combination to The activity of said sequence is enhanced, or substituted by an activity-enhanced nucleotide sequence.

The microorganisms of the present invention are microorganisms with putrescine-producing ability, including prokaryotic microorganisms, which express a protein comprising the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 19, which may be, for example, Escherichia sp. .), Shigella sp., Citrobacter sp., Salmonella sp., Enterobacter sp., Yersinia sp. sp.), Klebsiella sp., Erwinia sp., Corynebacterium sp., Brevibacterium sp., Lactobacillus sp. ), Sllenomanas sp. and Vibrio sp.

The microorganism of the present invention is specifically a microorganism of the genus Corynebacterium, more specifically Corynebacterium glutamicum.

In one embodiment of the present invention, a Corynebacterium microorganism with deposit number KCCM11138P (Korean Patent Publication No. 2012-0064046) is modified to have the ability to produce putrescine at a high concentration by enhancing the biosynthetic pathway of putrescine . Specifically, the putrescine-producing strain KCCM11138P is a putrescine-overproducing strain in which strain ATCC13032 has deleted the gene encoding ornithine transcarbamylase (ArgF) in order to promote ornithine accumulation and encode the glutamate transporter ( glutamate exporter, NCgl1221) in order to increase intracellular glutamate, introduce the gene encoding ornithine decarboxylase (speC), and increase the expression level of ornithine biosynthesis gene (argCJBD).

In another embodiment of the present invention, the putrescine producing strain DAB12-a based on Corynebacterium glutamicum ATCC13869 was modified. This strain ATCC13869 is based on the same genotype as KCCM11138P, which is a putrescine-producing strain based on Corynebacterium glutamicum ATCC13032. Specifically, the putrescine-producing strain DAB12-a was obtained from the ATCC13869 strain obtained from the American Type Culture Collection (ATCC), in which the gene encoding ornithine carbamoylase (ArgF) and the protein encoding NCgl1221 were deleted. The gene encoding ornithine decarboxylase (ODC) from Escherichia coli (speC) was introduced, and the promoter of the ornithine biosynthesis gene operon (argCJBD) was replaced with an enhanced promoter.

According to one embodiment of the present invention, a microorganism of the genus Corynebacterium (KCCM11138P) has the ability to produce putrescine, and the microorganism is prepared by: deleting the gene encoding ornithine carbamoyltransferase (ArgF) and participating in extraglutamic acid The coding gene of this glutamate transporter (NCgl1221) replaced the promoter of the ArgCJBD gene cluster itself encoding the enzyme involved in the synthesis of ornithine from glutamic acid, and the coding gene of ornithine decarboxylase (ODC) (speC ) into the chromosome of wild-type Corynebacterium glutamicum ATCC13032. Based on KCCM11138P, a clone (A15) that grew well on a medium containing a high concentration of putrescine was selected, and it was confirmed that the selected A15 contained genes encoding NCgl0100, NCgl0101, NCgl0102, NCgl0103 and NCgl0104 (Example 1). In addition, due to the gene encoding NCg10101 among the 5 types of genes, the microorganisms grew in a medium containing a high concentration of putrescine (Example 2). In terms of the characteristics of the NCg10101-encoding gene, it was confirmed that putrescine production was decreased in a strain overexpressing the gene encoding NCg10101 (Example 3), and that putrescine production was increased in a strain in which the gene encoding NCg10101 was deleted (Example 4).

Therefore, the present inventors prepared a Corynebacterium glutamicum strain with improved putrescine-producing ability by removing the NCg10101 gene in the putrescine-producing strain KCCM11138P, named the strain Corynebacterium glutamicum CC01-0244, and deposited it in Korea. Microorganism Collection Center (hereinafter abbreviated as "KCCM"), the date of deposit was December 26, 2011, and the accession number was KCCM11241P.

To achieve the above object, in another aspect of the present invention, the present invention relates to the method for producing putrescine, comprising the following steps:

Cultivating a microorganism of the genus Corynebacterium with improved putrescine production ability, the microorganism is modified to have a weakened NCg10101 protein activity, and the NCg10101 protein has the amino acid sequence shown in SEQ ID NO.17 or SEQ ID NO.19; and

Putrescine was isolated from the culture solution obtained in the above steps.

The culture method of the present invention can be carried out under suitable medium and culture conditions known in the art. Those skilled in the art can easily adjust and adopt the culture method according to the selected strain. Examples of culture methods include, but are not limited to, batch, continuous, and fed-batch culture. The medium must suitably meet the requirements of the particular strain.

The medium must suitably meet the requirements of the particular strain. Media for various microorganisms are described, for example, in Manual of Methods for General Bacteriology, American Society for Bacteriology, (Washington, D.C., USA, 1981). The following can be utilized as carbon sources for the medium: sugars and carbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose), milk fat and fats (e.g., soybean oil, sunflower oil, peanut oil, and coconut oil), fatty acids (for example, palmitic acid, stearic acid, and linoleic acid), alcohols (for example, glycerol and ethanol), and organic acids (for example, acetic acid). These substances may be used alone or in combination. The following nitrogen sources can be utilized: nitrogen-containing organic compounds (e.g., peptone, yeast extract, beef extract, malt extract, corn steep liquor, soybean meal, and urea) or non-organic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate), and these substances can also be used alone or in combination. The following substances can be utilized as sources of phosphorus: potassium dihydrogen phosphate or dipotassium hydrogen phosphate or their corresponding sodium-containing salts. In addition, the medium can also include metal salts necessary for growth (for example, magnesium sulfate or iron sulfate), and finally, in addition to the above-mentioned substances, necessary growth-promoting substances such as amino acids and vitamins can be used. In addition to the culture medium, suitable precursors can also be added. The feed material can be provided in the culture medium at one time or in sufficient amount during the cultivation.

The pH of the culture broth can be adjusted by suitable basic compounds such as sodium hydroxide, potassium hydroxide or ammonia, or acidic compounds such as phosphoric or sulfuric acid. Foaming can be adjusted with anti-foaming agents such as fatty acid polyethylene glycol esters. Oxygen or an oxygen-containing gas mixture, such as air, may be introduced to maintain an aerobic environment for the culture. The culture temperature is usually 20-45°C, especially 25-40°C. Culture can be continued until putrescine production reaches the desired upper limit, usually within 10-160 hours. Putrescine can be released into the medium, or contained in the cells.

As for the method of collecting and recovering putrescine produced in the culture method of the present invention, it can be obtained from the culture medium by a suitable method known in the art according to the culture method (for example, batch, continuous or fed-batch culture). Recover the target substance.

Embodiments of the present invention

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are for illustrative purposes only, and the present invention should not be limited to these examples.

Example 1: Library preparation and clone screening for selection of effective genes for putrescine biosynthesis

To screen effective genes for putrescine biosynthesis from the chromosome of the wild-type coryneform bacterium strain, a chromosome library of the wild-type coryneform bacterium strain was prepared. In detail, the chromosome extracted from the wild-type Corynebacterium glutamicum ATCC13032 strain was randomly cut with the restriction enzyme Sau3AI, a fragment of 5-8 kb was selected therefrom, and then cloned into the E. coli shuttle vector pECCG122 (Korean Patent Publication No. 1992-0000933) to prepare a chromosome library.

To select effective putrescine biosynthesis genes from the thus prepared coryneform bacterium chromosomal library, colonies growing in a medium containing a high concentration of putrescine were obtained.

Simultaneously, these libraries were introduced into a microorganism of the genus Corynebacterium (KCCM11138P) having putrescine-producing ability to prepare respective transformants. Choose to contain 0.35M putrescine (10g/l glucose, 0.4g/l MgSO ₄ 7H ₂ O, 4g/l NH ₄ Cl, 1g/l KH ₂ PO ₄ , 1g/l K ₂ HPO ₄ , 2g/l l Urea, 10mg/l FeSO ₄ 7H ₂ O, 1mg/l MnSO ₄ 5H ₂ O, 5mg/l Niacinamide, 5mg/l Thiamine Hydrochloride, 0.1mg/l Biotin, 1mM Arginine, 25mg /l kanamycin, 0.35M putrescine, pH 7.0) transformants grown in minimal medium.

The strain KCCM11138P disclosed in the patent applied by the present inventor (Korean Patent Publication No. 2012-0064046) was prepared by deleting the ornithine carbamoylase encoded in the chromosome of the wild-type Corynebacterium glutamicum strain ATCC13032 (ArgF) and glutamate transporter (NCgl1221), the gene encoding ornithine decarboxylase (ODC) (speC) derived from Escherichia coli W3110 strain was introduced into the chromosome, and the replacement code was involved in the synthesis of ornithine from glutamate The promoter of the argCJBD gene cluster of acid enzyme was used to prepare each transformant. As a result, 275 colonies were selected, and colonies that grew well in a medium containing a high concentration of putrescine were subsequently identified. Each library clone was obtained and reintroduced into a putrescine producing strain. Subsequently, a colony growing well in a medium containing a high concentration of putrescine was identified, and thus a clone (A15) was finally selected. The selected clones were identified by sequencing. As a result, it was confirmed that the clone contained a total of 5 ORFs of NCgl0100, NCgl0101, NCgl0102, NCgl0103 and NCgl0104 from which 436 amino acids at the N-terminal were removed ( FIG. 1 ). Fig. 1 is a schematic diagram showing the relative positions of the genes encoding NCgl0100, NCgl0101, NCgl0102, NCgl0103 and NCgl0104 on the chromosome of wild-type C. glutamicum ATCC13032 strain.

Example 2: Identification of efficient genes for putrescine synthesis in A15 clone

Example 2-1: Cloning of 5 genes of A15 clone and preparation of transformants

The nucleotide sequence of the A15 clone obtained in Example 1 is already known. Based on the nucleotide sequence of the previously reported ATCC13032 strain, NCgl0100-F and NCgl0100-R shown in SEQ ID NO.1 and 2 were constructed as primers for amplifying the NCgl0100 gene, and NCgl0100-R shown in SEQ ID NO.2 and 3 R and tNCgl0100-F are used as primers for amplifying the tNCgl0100 gene (wherein the 436 amino acids at the N-terminal have been removed), NCgl0101-F and NCgl0101-R shown in SEQ ID NO.4 and 5 are used as primers for amplifying the NCgl0101 gene, NCgl0102-F and NCgl0103-R shown in SEQ ID NO.6 and 7 are used as primers for amplifying NCgl0102 and NCgl0103 genes, and NCgl0104-F and NCgl0104-R shown in SEQ ID NO.8 and 9 are used as primers for amplifying NCgl0104 genes primers. In addition, P(CJ7)-F and P(CJ7)-R shown in SEQ ID NO.10 and 11 were constructed as primers for amplifying the expression promoter P(CJ7) (or pcj7) (Korean Patent No. 10-0620092) (Table 1).

Subsequently, use the chromosome of the ATCC13032 strain as a template and each primer shown in SEQ ID NO.1-9 to carry out PCR (denaturation at 95°C for 30 seconds, annealing at 50°C for 30 seconds, extension at 72°C for 1 minute to 1 minute and 30 seconds , 25 rounds) to amplify 5 types of gene fragments. In addition, PCR was performed using the chromosome of Corynebacterium ammoniagenes as a template and the primers shown in SEQ ID NO.10 and 11 to amplify the promoter fragment.

The 5 kinds of genes cut by KpnI and XbaI and the CJ7 promoter cut by EcoRV and KpnI were ligated into the expression vector pHC139T cut by EcoRV and XbaI (Korean Patent No. 10-0860932), thereby preparing a total of 5 types of expression vectors, pHC139T-P (CJ7 )-NCgl0100, pHC139T-P(CJ7)-tNCgl0100, pHC139T-P(CJ7)-NCgl0101, pHC139T-P(CJ7)-NCgl0102-NCgl0103 and pHC139T-P(CJ7)-NCgl0104.

Table 1

Preparation of primers for strains expressing 5 genes contained in A15 clone

NCgl0100-F (SEQ ID NO.1) GCGCAT ATGAGCTCAACAACCTCAAAAACC NCgl0100-R (SEQ ID NO.2) GCGTCTAGA TTATCCTTCGAGGAAGATCGCAG tNCgt0100-F (SEQ ID NO.3) GCGCAT ATGTGGACGCTGATGGCTGC NCgl0101-F (SEQ ID NO.4) GCGCAT ATGAGTACTGACAATTTTTCTCCAC NCgl0101-R (SEQ ID NO.5) GCGTCTAGA CTAAGCCAAATAGTCCCCTAC NCgl0102-F (SEQ ID NO.6) GCGCAT ATGGATGAACGAAGCCGGTTTG NCgl0103-R (SEQ ID NO.7) GCGTCTAGATTAATCAATGAAGACGAATACAATTCC NCgl0104-F (SEQ ID NO.8) GCGCATATGGCGGGTGACAAATTGTGG NCgl0104-R (SEQ ID NO.9) GCGTCTAGATTAGGACAGTTCCGCTGGAGC P(CJ7)-F(SEQ ID NO.10) CAGATATCGCCGGCATAGCCTACCGATG P(CJ7)-R(SEQ ID NO.11) GCGTCTAGAGATATCAGTGTTTCCTTTCG

The Class 5 expression vectors thus prepared and the control pHC139T were introduced into the KCCM11138P strain of Example 1 by electroporation, and then inoculated on a BHIS plate containing 25 μg/ml kanamycin to select transformants.

Example 2-2: Find effective genes for putrescine

Transformants that grew well in a medium containing a high concentration of putrescine were selected from a total of 6 types of transformants obtained in Example 2-1 in the same manner as in Example 1 (Fig. 2). Fig. 2 is the test result of comparative growth situation between the prepared transformants of the present invention, wherein 1, 2, 3, 4, 5 and 6 represent the bacterial strains that introduce respectively 6 types of following expression vectors: pHC139T, pHC139T-P (CJ7) - NCgl0100, pHC139T-P(CJ7)-tNCgl0100, pHC139T-P(CJ7)-NCgl0101, pHC139T-P(CJ7)-NCgl0102-NCgl0103 and pHC139T-P(CJ7)-NCgl0104. As shown in Figure 2, only the transformant (No. 4) introduced with pHC139T-P(CJ7)-NCgl0101 showed good growth in the medium containing high concentration of putrescine, so NCg10101 was selected as an effective gene for putrescine biosynthesis .

Example 3: Evaluation of the ability of NCgl0101-overexpressing strains to produce putrescine

The putrescine-producing ability of the strain overexpressing the NCg10101 gene, which was identified as an effective gene in Example 2, was evaluated. A strain for evaluation was prepared by introducing pHC139T-P(CJ7)-NCgl0101 into the putrescine-producing strain KCCM11138P.

The pHC139T-P(CJ7)-NCgl0101 prepared in Example 2-1 and the pHC139T vector as a control group were introduced into the putrescine-producing strain KCCM11138P by electroporation, and then inoculated on a BHIS plate containing 25 μg/ml kanamycin to select transformation body. These transformants were named KCCM11138P/pHC139T and KCCM11138P/pHC139T-P(CJ7)-NCgl0101 respectively. At 30°C, the two transformants thus selected were mixed with 1 mM arginine (1% glucose, 1% polypeptone, 0.5% yeast extract, 0.5% beef extract, 0.25% NaCl, 0.2% urea, 100 μl 50% NaOH, 2% agar, pH6.8, each 1L) on CM plates for 24 hours, and then a loop of cells The culture was inoculated in 25 ml of the titration medium containing 25 μg/ml kanamycin shown in Table 2, and cultured with shaking at 200 rpm at 30° C. for 96 hours. During fermentation, all prepared strains were cultured in the medium with 1 mM arginine.

Table 2

composition Concentration (per 1L) glucose 8% Soy Protein 0.25% Corn Steep Steep Solids 0.5% (NH ₄ ) ₂ SO ₄ 4% Urea 0.15% KH ₂ PO ₄ 0.1% MgSO ₄ 7H ₂ O 0.05% Biotin 100μg Thiamine Hydrochloride 3000μg calcium-pantothenic acid 3000μg Nicotinamide 3000μg CaCO ₃ 5%

The results are shown in Table 3, when NCg10101 was overexpressed, the production of putrescine decreased.

table 3

strain type Putrescine (g/L) KCCM 11138P/pHC139T 9.5

KCCM 11138P/pHC139T-P(CJ7)-NCgl0101 5.1

Example 4: Evaluation of the ability of NCg10101-deleted strains to produce putrescine

Example 4-1: Preparation of NCg10101-deleted strains using ATCC13032-based putrescine-producing strains

According to Example 3, NCg10101 overexpression increases cell growth in media containing high concentrations of putrescine, but reduces putrescine production. Based on this result, the effect of NCg10101 deletion on putrescine production ability was examined.

In detail, based on the NCg10101 nucleotide sequence of the ATCC13032 strain, NCgl0101-del-F1_BamHI and NCgl0101-del-R1_SalI shown in SEQ ID NO.12 and 13 were constructed as primers to obtain homologous recombination fragments of the N-terminal region of NCg10101. NCgl0101-del-F2_SalI and NCgl0101-del-R2_XbaI shown in SEQ ID NO.14 and 15 were constructed as primers to obtain homologous recombination fragments of the C-terminal region of NCg10101 (Table 4). Using these two pairs of primers, fragments of the N-terminal and C-terminal regions of the NCg10101 gene were prepared by PCR. The PCR products were treated with BamHI&SalI and SalI&XbaI respectively, and cloned into the pDZ vector treated with BamHI&XbaI. The cloned plasmid was named pDZ-NCgl0101(K/O).

Table 4

Preparation of primers for NCgl0101-deletion strain

NCgl0101-del-F1_BamHI (SEQ ID NO.12) CGGGATCC CGGATTCCCTGCGATCATTG NCgl0101-del-R1_SalI (SEQ ID NO.13) ACGCGTCGAC CAGTCGACGGAACTTGTGGAG NCgl0101-del-F2_SalI (SEQ ID NO.14) ACGCGTCGAC GGCAACGACTCCGAAACCTTC NCgl0101-del-R2_XbaI (SEQ ID NO.15) CTAGTCTAGA CTGGATCCTCATGAATGCGC

The pDZ-NCgl0101(K/O) vector prepared for obtaining the KCCM11138PΔNCgl0101 strain was introduced into the KCCM11138P strain by electroporation, and then inoculated on a BHIS plate containing 25 μg/ml kanamycin. The successful insertion of the vector into the chromosome was confirmed by observing whether the colonies on the solid medium containing X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) were blue. The primary chromosomal insertion strain was cultured with shaking in nutrient medium (30°C, 8 hours), then diluted from 10-4 to 10-10, and inoculated on solid medium containing X-gal. Although most colonies appear as blue colonies, a small number of colonies appear as white colonies. Finally, the NCg10101 gene-deleted strain was screened out, and the double-crossover white colonies were identified by PCR using the primers shown in SEQ ID NO.12 and 15. The variant thus identified was designated KCCM11138PΔNCgl0101.

Example 4-2: Preparation of NCg10101-deleted strains using putrescine-producing strains based on ATCC13869

Utilize putrescine-producing strain DAB12-a (argF-deletion, NCgl1221-deletion, introduction of Escherichia coli speC and arg operon-argCJBD promoter-substitution strain) based on Corynebacterium glutamicum ATCC13869 to produce NCg10101 deletion strain, the former and based on The putrescine-producing strain KCCM11138P of Corynebacterium glutamicum ATCC13032 has the same genotype.

In detail, in order to identify the amino acid sequence of the gene encoding NCg10101 and its expressed protein derived from Corynebacterium glutamicum ATCC13869, the genomic DNA of Corynebacterium glutamicum ATCC13869 was used as a template and a pair of primers SEQ ID NO.12 and 15 (NCgl0101-del-F1_BamHI, NCgl0101-del-R2_XbaI) were subjected to PCR. In this embodiment, the PCR reaction was carried out for 30 rounds: 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. PCR products were separated by electrophoresis and their sequences were analyzed. After sequence analysis, it was identified that the gene encoding NCg10101 derived from Corynebacterium glutamicum ATCC13869 contained the nucleotide sequence shown in SEQ ID NO.18, and the encoded protein contained the amino acid sequence shown in SEQ ID NO.19. When the amino acid sequence of NCg10101 derived from Corynebacterium glutamicum ATCC13032 was compared with the amino acid sequence of NCg10101 derived from Corynebacterium glutamicum ATCC13869, they showed a sequence homology of 98%.

For deleting the coding gene derived from NCg10101 of Corynebacterium glutamicum ATCC13869, the genomic DNA of Corynebacterium glutamicum ATCC13869 was used as a template and two pairs of primers listed in Table 4, in the same manner as in Example 4-1, by PCR The N-terminal and C-terminal regions of the NCg10101 gene were amplified. Then, the PCR products were treated with BamHI&SalI and SalI&XbaI respectively, and cloned into the pDZ vector treated with BamHI&XbaI, thereby constructing plasmid pDZ-2'NCgl0101(K/O).

The plasmid pDZ-2'NCgl0101 (K/O) was transformed into Corynebacterium glutamicum DAB12-a in the same manner as in Example 4-1, and a strain in which the gene encoding NCg10101 was deleted was selected. The selected C. glutamicum variant was named DAB12-aΔNCgl0101.

Example 4-3: Evaluation of the ability of NCg10101-deleted strains to produce putrescine

In order to study the effect of NCg10101 deletion on the putrescine-producing ability of the putrescine-producing strain, the Corynebacterium glutamicum variants prepared in Example 4-1 and 4-2 were compared.

In detail, putrescine-producing abilities of two types of Corynebacterium glutamicum variants (KCCM11138PΔNCgl0101 and DAB12-aΔNCgl0101) were evaluated in the same manner as in Example 3. As shown in Table 5 below, deletion of NCg10101 was found to increase putrescine production.

table 5

strain type Putrescine (g/L) KCCM11138P 9.8 KCCM 11138PΔNCgl0101 11.3 DAB12-a 10.1 DAB12-aΔNCgl0101 11.0

Taken together, the results of Examples 3 and 4 show that in wild-type Corynebacterium glutamicum strains, overexpression of the gene encoding NCg10101 reduces putrescine production, while deletion of the gene increases putrescine production, indicating that NCg10101 directly affects putrescine Biosynthesis.

Therefore, in the above examples, the inventors prepared a Corynebacterium glutamicum strain with improved putrescine-producing ability by deleting the NCg10101 gene in the putrescine-producing strain KCCM11138P, and named the strain Corynebacterium glutamicum CC01- 0244, and deposited with the Korean Collection of Microorganisms (hereinafter abbreviated as "KCCM") as an international depositary institution under the Budapest Treaty on December 26, 2011, with accession number KCCM11241P.

Based on the above description, those skilled in the art should understand that the present invention can be implemented in other forms without changing the technical concept or essential technical features. In this regard, the above-described embodiments serve to illustrate aspects of the invention, but are not intended to limit the scope of the invention. It should be understood that the scope of the present invention includes any changes and modifications derived from the meaning, scope and equivalent concepts of the following claims, rather than the specific description above.

Claims (11)

1. A recombinant microorganism of the genus Corynebacterium that produces putrescine ability improves, wherein, in the microorganism of the genus Corynebacterium that has the ability to produce putrescine, the protein of the amino acid sequence shown in SEQ ID NO:17 or SEQ ID NO:19 An activity is modified to be attenuated or removed compared to its endogenous activity. 2. The microorganism according to claim 1, wherein the activity of ornithine decarboxylase is further introduced thereinto. 3. The microorganism according to claim 2, wherein the amino acid sequence of the ornithine decarboxylase is as shown in SEQ ID NO:22. 4. The microorganism of claim 1, wherein the activity of one or more substances selected from the group is further weakened compared to its endogenous activity: ornithine carbamoyltransferase and glutamic acid Transporter NCgl1221. 5. The microorganism according to claim 4, wherein the amino acid sequence of ornithine carbamoyltransferase is as shown in SEQ ID NO:20, and the amino acid sequence of glutamate transporter NCgl1221 is as shown in SEQ ID NO:21 shown. 6. The microorganism of claim 1, wherein the activity of one or more substances selected from the group consisting of acetyl gamma glutayl phosphate reductase, acetyl glutamate synthase or ornithine acetyl transferase, acetylglutamate kinase, and acetylornithine aminotransferase. 7. The microorganism according to claim 6, characterized in that, acetyl gamma glutamate phosphate reductase, acetyl glutamate synthase or ornithine acetyltransferase, acetyl glutamate kinase and acetyl ornithine aminotransferase The amino acid sequences of are shown in SEQ ID NO:23, 24, 25 and 26, respectively. 8. The microorganism according to claim 1, characterized in that, the weakening of the protein activity is through: 1) partial or complete deletion of the polynucleotide encoding the protein, 2) reduced expression of the polynucleotide, 3) Modifying the polynucleotide sequence of the chromosome to weaken the activity of the protein, or 4) their combination. 9. The microorganism according to claim 1, which is Corynebacterium glutamicum. 10. A method for producing putrescine, comprising cultivating a modified Corynebacterium microorganism whose ability to produce putrescine is improved, wherein the activity of the protein of the amino acid sequence shown in SEQ ID NO:17 or SEQ ID NO:19 is compared to its endogenous activity is attenuated or eliminated; and putrescine is isolated from the obtained cell culture. 11. The method for producing putrescine according to claim 10, wherein the microorganism belonging to the genus Corynebacterium is Corynebacterium glutamicum.