ORNITHINE DECARBOXYLASE VARIANT AND METHOD FOR PRODUCING PUTRESCINE BY USING SAME
20230287382 · 2023-09-14
Inventors
- Jaehun LEE (Seoul, KR)
- Hee-See MOON (Seoul, KR)
- Ae Ji JEON (Seoul, KR)
- Young Lyeol Yang (Seoul, KR)
- Byung-Gee KIM (Seoul, KR)
- Eun Young HONG (Seoul, KR)
Cpc classification
C12P13/00
CHEMISTRY; METALLURGY
International classification
Abstract
The present application relates to a variant of ornithine decarboxylase or protein, a polynucleotide encoding the same, a microorganism containing the same, and a method for producing putrescine using the same.
The present invention achieves effects of increasing putrescine productivity, production efficiency or production selectivity, suppressing side reactions, and saving the cost involved in purifying putrescine.
Claims
1. An ornithine decarboxylase variant, comprising an amino acid substitution at a position corresponding to position a) 713, b) 698, or c) 713 and 698 of SEQ ID NO: 1, and having at least 80% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1.
2. The ornithine decarboxylase variant according to claim 1, wherein the substitution of the amino acid at the 713th position is substitution with a hydrophobic amino acid, a basic amino acid, an acidic amino acid, a neutral amino acid, or an aromatic amino acid, except alanine.
3. The ornithine decarboxylase variant according to claim 1, wherein the substitution of the amino acid at the 713th position is A713L, A713I, A713V, A713R, A713D, A713W, or A713Q.
4. The ornithine decarboxylase variant according to claim 1, wherein the substitution of the amino acid at the 698th position is E698D.
5. The ornithine decarboxylase variant according to claim 1, wherein the substitution of the amino acid at the 713th position is A713L, A713I, A713V, A713R, A713D, A713W, or A713Q, and the substitution of the amino acid at the 698th position is E698D.
6. The ornithine decarboxylase variant according to claim 1, wherein the variant comprising a polypeptide selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NOS. 19 to 23.
7. A polynucleotide encoding the ornithine decarboxylase variant according to claim 1.
8. A microorganism containing ornithine decarboxylase, wherein the ornithine decarboxylase comprises a polypeptide of SEQ ID NO: 1 or a polypeptide comprising an amino acid substitution at a position corresponding to position a) 713, b) 698, or c) 713 and 698 of SEQ ID NO: 1 and having at least 80% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1.
9. The microorganism according to claim 8, wherein the microorganism is Escherichia sp. or Corynebacterium sp.
10. A method for producing putrescine, comprising culturing a microorganism, wherein the microorganism comprises an ornithine decarboxylase, wherein the ornithine decarboxylase comprises a polypeptide of SEQ ID NO: 1, or a polypeptide comprising an amino acid substitution at a position corresponding to position a) 713, b) 698, or c) 713 and 698 of SEQ ID NO: 1 and having at least 80% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1.
11. The method for producing putrescine according to claim 10, comprising accumulating the putrescine in a medium.
12. The method for producing putrescine according to claim 10, comprising recovering the putrescine from the cultured microorganism or the medium.
13. A method for increasing a purity of putrescine, comprising culturing a microorganism, wherein the microorganism comprises an ornithine decarboxylase, wherein the ornithine decarboxylase comprises a polypeptide of SEQ ID NO: 1, or a polypeptide comprising an amino acid substitution at a position corresponding to position a) 713, b) 698, or c) 713 and 698 of SEQ ID NO: 1 and having at least 80% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1.
14. A method for increasing a ratio of putrescine to cadaverine (Put/Cad), comprising culturing a microorganism, wherein the microorganism comprises an ornithine decarboxylase, wherein the ornithine decarboxylase comprises a polypeptide of SEQ ID NO: 1, or a polypeptide comprising an amino acid substitution at a position corresponding to position a) 713, b) 698, or c) 713 and 698 of SEQ ID NO: 1 and having at least 80% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1.
15. A method for producing polyamide with putrescine, wherein the putrescine is produced by culturing a microorganism comprising an ornithine decarboxylase, wherein the ornithine decarboxylase comprises a polypeptide of SEQ ID NO: 1, or a polypeptide comprising an amino acid substitution at a position corresponding to position a) 713, b) 698, or c) 713 and 698 of SEQ ID NO: 1 and having at least 80% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1.
16. A composition for use in producing polyamide, comprising a microorganism comprising an ornithine decarboxylase, wherein the ornithine decarboxylase comprises a polypeptide of SEQ ID NO: 1, or a polypeptide comprising an amino acid substitution at position corresponding to a position a) 713, b) 698, or c) 713 and 698 of SEQ ID NO: 1 and having at least 80% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0105]
[0106]
[0107]
[0108]
[0109]
[0110]
THE BEST FORM FOR CARRYING OUT THE INVENTION
[0111] Hereinafter, the present application will be described in more detail by Examples. However, since these Examples are intended to illustrate the present application by way of example, the scope of the present application is not limited by these Examples, and it will be apparent to those skilled in the technical field to which this application belongs.
Example 1. Comparison of Activities of Variously Derived Ornithine Decarboxylases
[0112] Substrate reactivities of ornithine decarboxylases derived from four microorganisms were compared. They are wild type ornithine decarboxylases derived from Lactobacillus saerimneri (inducible), Saccharomyces cerevisiae (inducible), E. coli (constitutive), E. coli (inducible), which were denoted as ODC_Lb, ODC_Sc, ODC_Ec, and ODC_Ef, respectively. After genes corresponding to the enzymes were inserted into a pET24ma vector, the proteins were expressed under the condition of 0.1 mM IPTG and 18° C., using E. coli BL21 (DE3). Then, the initial reaction rates were compared at 45° C. using 10% cell extract. The case of using ornithine of 4 mM and the case of using lysine of 4 mM as the substrates were compared, respectively.
[0113]
Example 2. Selection of Variation Site of Ornithine Decarboxylase
[0114] Since a crystal structure of Lactobacillus ornithine decarboxylase has been known, it is possible to predict a tunnel through which the substrate enters and exits the enzyme by analysis of the crystal structure. In order to select functional residues that perform saturation variation among the predicted tunnel sites, multiple sequence alignment was performed using sequence information of bioinformatics, and the positions of A696, V702, A713, and E698 were selected as variation positions from the N-terminal of the amino acid sequence of the ornithine decarboxylase used in the present invention.
[0115] Since residues conserving the amino acid residues at a specific position within the protein structure play a very important role in the structure and function of the protein and are particularly likely to play a direct role in the catalytic process, they are excluded as the variation residues.
Example 3 Performing Saturation Variation and Researching Variants on Functional Residues of Ornithine Decarboxylases
[0116] Saturation variation refers to the introduction of changes in various base arrangements at a designated position in a gene. The saturation variation also means inserting a variation through PCR by inserting a NNK codon, instead of the sequence that is intended to be mutated, on a primer of a complementary sequence that binds to a template strand. In this case, in the NNK codon, N means A, T, G, C of a nucleotide, and K means T, G of the nucleotide.
[0117] The saturation variation was performed using the NNK codon on the selected functional residues, followed by screening against the variant libraries. All the libraries were subjected to primary and secondary screenings through an entire cell reaction. The entire cell reaction refers to a reaction which uses cell contents by crushing the cells containing a specific enzyme or uses entire intact cells without separating and purifying the enzyme. The first screening was conducted through the entire cell reaction of ornithine, and variants showing the activity similar to or faster than the wild type upon comparison were selected as change in absorbance. The secondary screening was conducted through the entire cell response of lysine, and among the variants selected from the primary screening, variants that the reactivity to lysine is lower than that of the wild type were selected.
[0118] A specific activity when ornithine or lysine was used as a substrate was measured for the selected variant enzymes. The specific activity refers to the activity per unit amount of pure protein from which impurities and other proteins are removed through enzymatic purification, and is usually expressed as the number of unit per 1 mg, wherein an amount of enzyme that catalyzes a substrate change of 1 μmol per minute is 1 unit. Specifically, the Lactobacillus-derived wild type and variant ornithine decarboxylases were transformed into E. coli BL21 (DE3) and expressed using IPTG as an inducer in a culture volume of 50 mL, and then only pure proteins were purified from them using a Ni-NTA column. First, after expression of the proteins, the cells were crushed with a sonic crusher, and centrifuged to obtain a cell extract. The cell extract was put in a column equilibrated with 50 mM phosphate buffer solution (pH8.0) to which 300 mM sodium chloride was added, and then bound with a nickel resin at 0° C. for 1 hour. Subsequently, a protein that failed to bind to the resin were shed, and other proteins that were non-specifically bound were removed with a Tris buffer solution containing 50 mM imidazole. Finally, only the desired protein was eluted with a Tris buffer solution containing 250 mM imidazole. In order to remove the imidazole from the eluted protein, a desalting process using a filtration column was performed to finally obtain only the active protein. Thereafter, an amount of the protein was measured using a Bradford protein quantification kit, and the specific activity was measured using the same amount of protein.
[0119] When ornithine or lysine was used as a substrate, the specific activity of Lactobacillus-derived wild type and variant ornithine decarboxylases was measured by HPLC (High-Performance Liquid Chromatography) analysis. The reaction was performed at 50° C. for 30 minutes to 300 minutes to obtain an average value from the experiments three times. The initial reaction rate was measured when a conversion yield of 10% to 25% was shown. A cation exchange column was used and the moving phase consisted of 0.6 g/L citric acid, 4 g/L tartaric acid, 1.4 g/L ethyldiamine, 5% methanol and 95% water. The buffer solution of pH used was a citric-sodium citrate buffer in case of pH 5.0. The specific activity of the wild type and variant ornithine decarboxylases was shown in
[0120] As shown in
[0121] Specifically, in case ornithine was used as the substrate, the specific activities of the variants (A696E, V702G, A713L, A696E/A713L, V702G/A713L, A696E/V702G/A713L, E698D, E698D/A713L) were confirmed to be 19.9%, 4.3%, 89.4%, 12.8%, 4.9%, 0.1%, 75.6%, and 74.4%, respectively, compared to the specific activities of the wild types (
[0122] In case lysine was used as the substrate, the specific activities of the variant enzymes (A696E, V702G, A713L, A696E/A713L, V702G/A713L, A696E/V702G/A713L, E698D, E698D/A713L) were confirmed to be 16.9%, 0.6%, 42.4%, 4.4%, 0.9%, 0.7%, 50.8%, and 29.2%, respectively, compared to the specific activities of the wild type enzymes, which confirm suppression of the side reaction (
Example 4. Confirmation of Kinetic Parameters for Functional Residues of Ornithine Decarboxylases
[0123] Among the variants of ornithine decarboxylases used in Example 3, the properties of A713L, E698D, and E698D/A713L, which are the variants having the specific activity of 70% or more, were intended to be more closely identified. Lysine having various concentration conditions was used to compare kinetic parameters of the variants and the wild types. The kinetic parameter represents a substrate affinity and a substrate conversion capacity value of enzymes using substrate solutions with different concentrations.
[0124] Specifically, lysine concentration of 0.45 mM to 140 mM was used to confirm the kinetic parameter for lysine of the wild type and variant ornithine decarboxylases after protein purification. A buffer solution of pH was a citric-sodium citrate buffer of pH 5.0, and a reaction volume was performed at 200 μl. The analysis was conducted through the HPLC analysis method specified above, and was obtained as an average value of the experiments three times. The kinetic parameters of the wild type and variant lysine decarboxylases were shown in
[0125] As shown in
Example 5. Characteristic Analysis of Variant Ornithine Decarboxylase
[0126] This Example was intended to investigate, among the variants, the effect of the variant (A713L) of ornithine decarboxylase having the highest the specific activity of ornithine on the production of putrescine or cadaverine. A case where ornithine having a high concentration of 51.5 g/L (0.39 M) was used as a substrate and a case where lysine having a concentration of 2.57 g/L (17.6 mM) was used as the substrate were performed, respectively. In order to obtain suitable reaction conditions for the two substrates, a buffer concentration for titrating the pH under the two conditions was 0.1 M or 0.37 M.
[0127] Specifically, 0.1 mg of the wild type enzyme and the variant enzyme after purification of the proteins was finished were used for the reaction. 0.39 M ornithine or 17.6 mM lysine was used as the substrate, and 0.1 or 0.37 M citric-sodium citrate buffer (pH 5.0) was used as a buffer. 0.1 mM PLP coenzyme was used, and the reaction proceeded in the reaction volume of 2 mL at 50° C. The results thereon were shown in
[0128]
[0129]
Example 6. Comparing and Measuring Expression of Recombinant ODC Genes in Corvine Strain
[0130] A method of producing a recombinant gene for expressing ODC_Lb, ODC_Sc, ODC_Ec, and ODC_Ef, which are the ornithine decarboxylases derived from the four microorganisms mentioned in Example 1, is as follows.
[0131] Specifically, using Lactobacillus saerimneri (ACCESSION No. P43099), Saccharomyces cerevisiae (ACCESSION No. J02777.1), and Escherichia coli str. K-12 (ACCESSION no. BAA35349) genomic information, the ornithine decarboxylase genes were amplified in the gene coding region by PCR with the gene sequence listed in Table 1 below, and then the PCR products were treated with restriction enzymes and inserted into plasmids.
TABLE-US-00001 TABLE 1 Primer Primer sequence odc_Lb_F 5′ -ATATCATATGAGTTCTTCTCTTAAAATTGCT-3′ (SEQ ID NO. 32) odc_Lb_R 5′ - ATATCTCGAGGTTGTTGTAACGATCATCGTT-3′ (SEQ ID NO. 33) odc_Sc_F 5′ -TAAACCATGGGCATGTCTAGTACTCAAGTAGGA- (SEQ ID 3′ NO. 34) odc_Sc_R 5′ -ATATCTCGAGATCGAGTTCAGAGTCTATGTA-3′ (SEQ ID NO. 35) Odc_Ec_F 5′ - ATATCATATGAAATCAATGAATATTGCCGCC-3′ (SEQ ID NO. 36) Odc_Ec_R 5′ -ATATCTCGAGCTTCAACACATAACCGTACAACCG- (SEQ ID 3′ NO. 37) Odc_Ef_F 5′ -ATATCATATGTCAAAATTAAAAATTGCGGTT-3′ (SEQ ID NO. 38) Odc_Ef_R 5′ -ATATCTCGAGTAATTTTTCCCCTTTCAACAG-3′ (SEQ ID NO. 39)
[0132] The recombined genes were designed to allow additional translation of His-tag into protein C-terminal. E. coli DH5 alpha was used as a host strain for DNA manipulation, and E. coli BL21 (DE3) was used as the host strain for expression of C-terminal His6-tagged ODC gene. The recombinant E. coli BL21 was grown at 37° C. in a LB medium of 50 mL containing kanamycin of 50 mg/mL. When the culture solution reached 0.8 under OD600 condition, IPTG of 0.2 mM was added to the culture solution. After inducing expression of the protein at 18 to 30° C., cells were harvested. The cells were resuspended in a lysis buffer and sonicated to crush the cells. The obtained recombinant ODCs were purified at 4° C. with a Ni-NTA agarose resin from Quiagen in Hilden, Germany. Recombinant proteins were obtained using Centriplus YM-30 (Millipore, Bedford, Mass.) with a molecular mass cut off of 100 kDa. Expression results were shown in
[0133] When analyzing the results under the 30° C. expression induction condition on a SDS-PAGE gel, it can be confirmed that the expression levels of the recombinant ODC_Lb and ODC_Ec were higher than those of the ODC_Sc and ODC_Ef. However, in case of the ODC_Ec, it was confirmed that an amount of a soluble protein was significantly decreased when it was expressed under the medium temperature condition. Additionally, when the expression at 37° C. was performed, the amount of the soluble protein of ODC_Ec was further reduced.
[0134] It was intended to compare and evaluate an amount expressed as the soluble protein by expressing the ODC_Lb and ODC_Ec genes in Coryne strains. A CJ7 promoter (KCCM10617, Korean Patent Registration No. 10-0620092) was introduced in front of initiation codons of the ODC_Lb gene and the ODC_Ec gene. First, in order to obtain a gene containing the CJ7 promoter sequence, PCR was performed with the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, using the primer pair listed in Table 2 below. The PCR reaction was carried out by repeating, 30 times, processes consisting of denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds and elongation at 72° C. for 30 seconds.
TABLE-US-00002 TABLE 2 Primer Primer sequence Co-CJ7_5 5′ -GAAGGATCCATAGCCTACCGATGTAGAT-3′ (SEQ ID NO. 40) Co-CJ7_3 5′ -AATTCTAGAAGTGTTTCCTTTCGTTGGGTAC-3′ (SEQ ID NO. 41)
[0135] After electrophoresis was performed using 1.5% agarose gel, a product of the PCR nucleic acid having a size of 400 basepair (bp) was obtained. The obtained PCR product was purified using a PCR prep kit (GeneAll, Seoul). BamHI and XbaI were added to the purified PCR product and a pSCEC vector solution sample, and reacted with a restriction enzyme at 37° C. for 4 hours, and then subjected to the electrophoresis using 1.5% agarose gel. Thereafter, a band of the PCR nucleic acid product having a size of 400 bp and a band of the vector size were cut to obtain nucleic acid fragments using the Gel prep kit (GeneAll, Seoul). The purified CJ7 promoter fragments and vector of each 1 mg was ligated using a T4 ligase, and then electrophoresed to E. coli DH5 alpha strains at 2500 V. The recovered strains were plated on a LB plate medium containing 50 μg/L spectinomycin, and cultured at 37° C. for 1 day to select strains showing resistance. After selecting 18 strains and performing colony PCR with SEQ ID NOS. 9 and 10, PCR products having a size of 400 bp were identified. The production of pSCEC_cj7 having the CJ7 promoter was confirmed from the results of the colony PCR.
[0136] Based on the obtained pSCEC_cj7 vector, two genes of ODC_Lb and ODC_Ec, were amplified through the PCR using the primers listed in Table 3 below.
TABLE-US-00003 TABLE 3 Primer Primer sequence Co-ODC_Lb_5 5′ -TTATATTCTAGAAGTTCTTCTCTTAAAATTGCT- (SEQ ID 3′ NO. 42) Co-ODC_Lb_3 5′ -TTATATGTCGACGTTGTTGTAACGATCATCGTT- (SEQ ID 3′ NO. 43) Co-ODC_Ec_5 5′ -TTATATTCTAGAAAATCAATGAATATTGCCGCC- (SEQ ID 3′ NO. 44) Co-ODC_Ec_3 5′ -TTATATGTCGACCTTCAACACATAACCGTACAAC (SEQ ID CG-3′ NO. 45)
[0137] The obtained PCR product and the pSCEC_cj7 vector were treated with the restriction enzyme XbaI and SalI. The restriction enzyme-treated nucleic acids were gel prep to ligate ODC_Lb, ODC_Ec and pSCEC_cj7 nucleic acid fragments and inserted into E. coli DH5 alpha strain. PSCEC_cj7_ODC_Lb and pSCEC_cj7_ODC_Ec were obtained from the selected strains that the insertion was identified, respectively, and the electrophoresis was performed at 2500 V for microorganisms KCCM11240P, which are Corynebacterium sp. producing putrescine.
[0138] Colonies were formed by plating and culturing the strains on a BHIS plate medium (Braine heart infusion 37 g/l, sorbitol 91 g/l, agar 2%) containing 50 μg/L spectinomycin. It was confirmed that the selected strains can be cultured by shaking them in a CM medium (glucose 10 g/L, polypeptone 10 g/L, yeast extract 5 g/L, beef extract 5 g/L, NaCl 2.5 g/L, Urea 2 g/L, pH 6.8) containing 50 μg/L spectinomycin. 3 mL of the produced two Corynebacterium glutamicum variants were cultured, and then centrifugated to secure fungus bodies. The obtained fungus bodies were centrifuged after crushing the cells by an ultrasonic treatment method to obtain a solution containing soluble proteins.
[0139] The ODC_Lb and ODC_Ec proteins containing His-tag were purified, respectively, using a Ni-NTA Spin Column (Hilden, Germany). A concentration of the obtained proteins was measured using a nano drop. The concentration of the recombinant proteins calculated based on the measured values were 1.282 g/L ODC_Lb and 0.039 g/L ODC_Ec, respectively, which confirms that ODC_Lb secured the soluble proteins of 30 times or more compared to ODC_Ec in the Coryne strain.
[0140] When ODC_Lb was expressed using E. coli and Coryne strain as hosts under a medium temperature condition, it could be confirmed that high soluble proteins were produced with high expression level and normal protein folding.
Example 7. Preparation of Ornithine Decarboxylase Variant-Incorporated Putrescine Production Coryne Strain and Measurement of Putrescine Production Capacity
[0141] In order to investigate the effect of the ornithine decarboxylase variant of the present application on putrescine production, a strain incorporating the variant of ornithine decarboxylase into a microorganism of Corynebacterium sp. having improved putrescin production capacity was prepared.
[0142] Specifically, as the microorganism of Corynebacterium sp. having improved putrescin production capacity, the microorganism of Corynebacterium sp. (KCCM11240P) having putrescine production capacity disclosed in Korean Patent Laid-open Publication No. 2013-0082478 was used. The microorganism of Corynebacterium (KCCM11240P) is a microorganism in which NCg11469 is defective in a microorganism prepared from Corynebacterium glutamicum ATCC13032 (ATCC 13032 ΔargF ΔNCg11221 P (CJ7)-argCJBD bioAD::P (CJ7)-speC (Ec): KCCM11138P (Korean Patent Laid-open Publication No. 2012-0064046)).
[0143] A vector was prepared to substitute the ornithine decarboxylase in the putrescine-producing microorganism with a variant of ornithine decarboxylase derived from Lactobacillus. More specifically, DNAs of the ornithine decarboxylase variant derived from Lactobacillus prepared in Examples 1 and 3 were amplified using ODC_Lb_start (EcoRV)_5 and ODC_Lb_stop (MfeI)_3 primers disclosed in Table 4 below. Specifically, wild type and variant (E698D, A713L) of Lactobacillus ornithine decarboxylases were inserted into the prepared pET24ma vector to make each of them as a template, and PCR was performed using two primers of L-odc_start (EcoRV)_5 and L-odc_stop (MfeI)_3 shown in Table 4 below.
TABLE-US-00004 TABLE 4 Primer Primer sequence ODC_Lb_start 5′ -gcgatatcatgaaatcaatgaatattgccg-3′ (EcoRV)_5 (SEQ ID NO. 46) ODC_Lb_stop 5′ -gccaattggttgttgtaacgatcatc-3′ (MfeI)_3 (SEQ ID NO. 47)
[0144] The gene fragments obtained through PCR amplification were treated with EcoRV and MfeI restriction enzymes (37° C., 3 hours), and the gene fragments of wild type and variant (E698D, A713L) of ornithine decarboxylases derived from Lactobacillus were inserted into pDZ-bioAD-P (CJ7) vector produced by the method disclosed in Korean Patent Laid-open Publication No. 2012-0064046. EcoRV and MfeI restriction enzymes were used in the above method. The recombinant vectors (pDZ-ODC_Lb, pDZ-ODC_Lb_E698D, pDZ-ODC_Lb_A713L) for chromosomal insertion prepared by the above method were identified by sequence analysis.
[0145] To obtain Coryne strains in which the wild type and variant ornithine decarboxylases derived from Lactobacillus were inserted into the chromosome, each of the pDZ-ODC_Lb, pDZ-ODC_Lb_E698D, pDZ-ODC_Lb_A713L recombinant vectors prepared above was transfected to the KCCM11240P strain using electrophoresis, and then plated on a BHIS plate medium (37 g/l of brainheart infusion, 91 g/l of sorbitol, 2% of agar, 25 μg/ml of 1 L basis+kanamycin).
[0146] A successful insertion of the vector into the chromosome was determined by presence of a blue color in a solid medium containing X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). After the primary chromosome-inserted strains were cultured by shaking in a nutrient medium (30° C., 8 hours), serially diluted, respectively, and plated on the solid medium containing X-gal. While most colonies indicated the blue color, they were able to select white colonies at a low rate, and the selected colonies were secondly crossed over to be able to obtain strains that the final Lactobacillus ornithine decarboxylase variants were introduced into the chromosome. Finally, the strains was identified by sequence analysis of the variants. The identified strains were named as KCCM11240P::ODC_Lb, KCCM11240P::ODC_Lb_E698D, KCCM11240P::ODC_Lb_A713L.
[0147] In order to confirm the effect of putrescine production capacity of the putrescine-producing strains due to introduction of the wild type and variant ornithine decarboxylases derived from Lactobacillus, the putrescine production capacity was evaluated.
[0148] Specifically, after the strains produced above were cultured in a CM plate medium (1% glucose, 1% polypeptone, 0.5% yeast extract, 0.5% beef extract, 0.25% NaCl, 0.2% urea, 100% NaOH 100 μl, 2% agar, pH 6.8, 1 L basis) containing 1 mM arginine at 30° C. for 16 hours, the strains were inoculated with about one Inoculation loop (platinum) in a 25 ml titer medium having the composition of Table 5 below, and then cultured by shaking with 200 rpm at 30° C. for 24 hours. Arginine of 1 mM was added to all the produced strains in the medium and cultured for fermentation.
TABLE-US-00005 TABLE 5 Composition ingredient Concentration/Content (1 L basis) Glucose 8% Soy protein 0.25% Corn steep solid 0.50% (NH4) 2SO4 4% Urea 0.15% KH2PO4 0.10% MgSO4—7H2O 0.05% Biotin 100 ug Thiamine hydrochloride 3000 ug Calcium-pantothenic acid 3000 ug Nicotinamide 3000 ug CaCO.sub.3 5%
[0149] As a result, as shown in Table 6 below, after 12 hours of the culture, an amount of putrescine production from the strains into which the variant (A713L) of ornithine decarboxylase derived from Lactobacillus was introduced was increased by about 115% P compared to the strains having E. coli ornithine decarboxylase (KCCM11240P). In addition, the strains into which the variant (A713L) of ornithine decarboxylase was introduce showed increase of about 40% P compared to the strains having a wild type ornithine decarboxylase derived from Lactobacillus (KCCM11240P::ODC_Lb).
[0150] Further, an amount of cadaverine production from the strains (KCCM11240P::ODC_Lb_A713L) in which the variant (A713L) of ornithine decarboxylase was introduced was decreased by about 48% P compared to KCCM11240P due to the side reactions upon production of putrescine. Judging from the concentration of the residual glucose in the culture solution, it can be seen that the consumption of sugar increases for the same time, thereby increasing productivity.
TABLE-US-00006 TABLE 6 12 hrs Residual Put fold Cad fold glucose Strain (g/L) (%) (ppm) (%) (g/L) KCCM11240P 1.3 100 39.427 100 41.7 KCCM11240P::ODC_Lb 2 154 26.196 66 37.3 KCCM11240P::ODC_Lb_A713L 2.8 215 20.367 52 29 KCCM11240P::ODC_Lb_E698D 2.7 208 23.243 59 29.8
[0151] The above result indicates that, in virtue of the introduction of the variant ornithine decarboxylase derived from Lactobacillus, it is possible to not only produce putrescine of a higher concentration than before, judging from the sugar consumptions in the putrescine producing strains, but also has the effect of reducing cadaverine and improving productivity.
Example 8. Prediction of the Effect of Saturation Variation on the 713th Amino Acid Residue of Lactobacillus Ornithine Decarboxylase
[0152] Among the selected variants, the functional residue (A713L) in which the 713th alanine of Lactobacillus ornithine decarboxylase was substituted with leucine was substituted with other amino acids except alanine and leucine, and then introduced into putrescine-producing strain KCCM11240P. This Example was intended to investigate the effect of the functional residue (A713L) on putrescine production.
[0153] Specifically, a mutant strain substituted by a variant in which the 713th amino acid from the N-terminal in the amino acid sequence of SEQ ID NO. 1 is substituted with other amino acid, including a hydrophobic amino acid, was prepared. The variant was substituted instead of a wild type ornithine decarboxylase derived from E. coli in a chromosome of the microorganism of Corynebacterium sp. (KCCM11240P) having an enhanced putrescine production capacity. More specifically, in order to prepare vectors substituted with valine, one of the hydrophobic amino acids, arginine, one of the basic amino acids, aspartic acid, one of the acidic amino acids, glutamine, one of the neutral amino acids, and tryptophan, one of the aromatic amino acids, respectively, PCR was carried out using the pDZ-ODC_Lb vector prepared in Example 7 as a template and using the primers disclosed in Table 4 above and Table 7 below. Firstly, the PCR was performed on the front portion (5′) and the rear portion (3′), respectively, in the center of the site where the variation was to be caused, and then, secondly, the PCR was performed to combine the two PCR fragments. For example, in case of a variant (A713V) in which the 713th amino acid is replaced with valine from alanine, the front portion was amplified by PCR using ODC_Lb_start (EcoRV)_5 and ODC_Lb_A713V_3 primers, and the rear portion was amplified by PCR using ODC_Lb_A713V_5 and ODC_Lb_stop (MfeI)_3 primers. The two PCR fragments obtained through the primary PCR were used as a template for the secondary PCR, and the PCR was performed using ODC_Lb_start (EcoRV)_5 and ODC_Lb_stop (MfeI)_3 primers. The finally obtained variant A713V gene fragment of Lactobacillus ornithine decarboxylase was inserted into pDZ-bioAD-P (CJ7) vector in the same manner as in Example 7. The remaining variants A713R, A713D, A713W, and A713Q were also subjected to PCR in the same manner as above using the primers described in Table 7 below and inserted into the pDZ-bioAD-P (CJ7) vector. The prepared recombinant vectors for chromosome insertion (pDZ-ODC_Lb_A713V, pDZ-ODC_Lb_A713R, pDZ-ODC_Lb_A713D, pDZ-ODC_Lb_A713W, pDZ-ODC_Lb_A713Q) were identified by sequence analysis.
TABLE-US-00007 TABLE 7 Primer Primer sequence ODC_Lb_A713V_3 5′-aagaaggcgacaaggttgttgtgtacggtgaagtttacgatg-3′ (SEQ ID NO. 48) ODC_Lb_A713V_5 5′-catcgtaaacttcaccgtacacaacaaccttgtcgccttctt-3′ (SEQ ID NO. 49) ODC_Lb_A713R_9 5′-aagaaggcgacaaggttgttaggtacggtgaagtttacgatg-3′ (SEQ ID NO. 50) ODC_Lb_A713R_5 5′-catcgtaaacttcaccgtacctaacaaccttgtcgccttctt-3′ (SEQ ID NO. 51) ODC_Lb_A713D_3 5′-aagaaggcgacaaggttgttgactacggtgaagtttacgatg-3′ (SEQ ID NO. 52) ODC_Lb_A713D_5 5′-catcgtaaacttcaccgtagtcaacaaccttgtcgccttctt-3′ (SEQ ID NO. 53) ODC_Lb_A713Q_3 5′-aagaaggcgacaaggttgttcaatacggtgaagtttacgatg-3′ (SEQ ID NO. 54) ODC_Lb_A713Q_5 5′-catcgtaaacttcaccgtattgaacaaccttgtcgccttctt-3′ (SEQ ID NO. 55) ODC_Lb_A713W_3 5′-aagaaggcgacaaggttgtttggtacggtgaagtttacgatg-3′ (SEQ ID NO. 56) ODC_Lb_A713W_5 5′-catcgtaaacttcaccgtaccaaacaaccttgtcgccttctt-3′ (SEQ ID NO. 57)
[0154] In order to obtain strains in which variants in which the 713th alanine of Lactobacillus ornithine decarboxylase was substituted with other amino acid including a hydrophobic amino acid were inserted into the chromosome, each of pDZ-ODC_Lb_A713V, pDZ-ODC_Lb_A713R, pDZ-ODC_Lb_A713D, pDZ-ODC_Lb_A713W, and pDZ-ODC_Lb_A713Q recombinant vectors prepared above was transfected into KCCM11240P strain and selected in the same method as in Example 7 to obtain strains in which final variants of Lactobacillus ornithine decarboxylase were introduced into the chromosome. Finally, the strains were identified by sequence analysis of the variants. The identified strains were named as KCCM11240P::ODC_Lb_A713V, KCCM11240P::ODC_Lb_A713R, KCCM11240P::ODC_Lb_A713D, KCCM11240P::ODC_Lb_A713Q, KCCM11240P::ODC_Lb_A713W.
[0155] In order to confirm the effect of putrescine production capacity of the putrescine producing strain due to the introduction of the variant in which the 713th alanine of Lactobacillus ornithine decarboxylase is substituted with other amino acid including a hydrophobic amino acid, the putrescine production capacity was evaluated in the same method as in Example 7.
TABLE-US-00008 TABLE 8 12 hrs Residual Put fold Cad fold glucose Strain (g/L) (%) (ppm) (%) (g/L) KCCM11240P 1.3 100 39.024 100 40.9 KCCM11240P::ODC_Lb 2 154 27.283 70 37.6 KCCM11240P::ODC_Lb_A713L 2.8 215 20.069 51 29.8 KCCM11240P::ODC_Lb_A713V 2.2 169 24.123 62 31.6 KCCM11240P::ODC_Lb_A713R 2.5 192 22.961 59 25.9 KCCM11240P::ODC_Lb_A713D 2.3 177 23.615 61 33.4 KCCM11240P::ODC_Lb_A713Q 2.6 200 21.845 56 27.8 KCCM11240P::ODC_Lb_A713W 2.1 162 26.074 67 30
[0156] As a result, as shown in Table 8 above, the strains, into which the variant of Lactobacillus ornithine decarboxylase substituted with other amino acid including hydrophobic amino acid was introduced, indicated an increase in putrescine production of about 86% P in average after the culture of 12 hours, compared to the strain (KCCM11240P) having the wild type ornithine decarboxylase derived from E. coli. In addition, they showed an increase in putrescine production of about 21% P in average compared to the strain having the wild type ornithine decarboxylase derived from Lactobacillus.
[0157] Further, it can be seen that the cadaverine production caused by the side reactions upon producing the putrescine was decreased by about 41% P, and that the consumption of sugar was increased for the same time, thereby further increasing productivity, judging from a concentration of the residual glucose in the culture medium.
[0158] The above result indicates that, due to the variants substituted with valine, one of the hydrophobic amino acids, arginine, one of the basic amino acids, aspartic acid, one of the acidic amino acids, glutamine, one of the neutral amino acids, and tryptophan, one of the aromatic amino acids, in addition to the variant in which the 713th alanine of Lactobacillus ornithine decarboxylase is substituted with leucine, it is possible to not only produce putrescine of a higher concentration than before, judging from the sugar consumptions in the putrescine producing strains, but also has the effect of reducing cadaverine and improving productivity.
[0159] From the above descriptions, those skilled in the art to which the present application pertains will understand that the present application may be implemented in other specific form without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that Examples described above are illustrative in all respects and not restrictive. The scope of the present application should be construed to include the meaning and scope of the claims described below rather than the detailed description of the specification, and any modifications or modified forms derived from equivalent concepts thereof.