KR101493587B1 - Novel Pseudomonas SP2 and Preparation Method of 3-Hydroxypropionic acid Therewith - Google Patents

Abstract

The present invention relates to a novel Pseudomonas strain having the ability to produce vitamin B ₁₂ under aerobic conditions and a method for producing 3-hydroxypropionic acid using the same, and more particularly to a Pseudomonas SP2 strain of deposit number KACC91720P, Hydroxypropionic acid with a low-cost carbon source in a highly efficient and environmentally-friendly bioprocess

Description

A novel Pseudomonas SP2 strain and a method for producing 3-hydroxypropionic acid using the Pseudomonas sp. Strain,

The present invention relates to a novel Pseudomonas strain having the ability to produce vitamin B ₁₂ under aerobic conditions and a method for producing 3-hydroxypropionic acid using the same.

Vitamin B ₁₂ (vitamin B ₁₂ ) is one of the nonpolymer biological molecules found in liver extracts by Ninot and Murphy in 1926. Dimethylbenzylimidazole (DMB) nucleotides, aminopropanol functional groups, cobalt Orin ring, and the like. This cobalt-bonded cyanide group is called cyano-cobalamin. In vivo, the cyano group is replaced with adenosyl or methyl group to exist as adenosyl-cobalamin or methyl-cobalamin, It is an essential cofactor for metabolism.

Despite it discovered the chemical structure of vitamin B _12, and vitamin B ₁₂ because the commercial production of complex features of this structure has been carried out only by biological fermentation. Strain to be used for commercial production of vitamin B ₁₂ is known, so four kinds can be classified according to whether the oxygen demand of the vitamin B ₁₂ biosynthesis genes into the strain using a strain with an anaerobic route using aerobic path. Examples of strains capable of producing vitamin B ₁₂ from aerobic include Pseudomonas sp. denitrificans ), and the strains that can be produced under anaerobic conditions include Salmonella typhimurium ), Propionibacterium freudenreichii subsp. shermanii ( Propionibacterium freudenreichii subsp. shermanii ) And Bacillus megaterium megaterium ). These strains have been reported to require about 30 or more genes and a similar number of enzymatic steps for the synthesis of vitamin B ₁₂ .

Thus, strains capable of synthesizing vitamin B ₁₂ can be developed as host strains for the production of important metabolites, and have attracted the attention of researchers in related fields. Among them, Pseudomonas dinuclearpiana, which can produce vitamin B ₁₂ under aerobic conditions, has the advantage of effectively producing 3-hydroxypropionic acid (3-HP) from glycerol (Korean Patent Application No. 10-2010-0083654).

The production of 3-HP from glycerol is a step in which glycerol is converted to 3-hydroxypropionaldehyde (3-HPA) by glycerol dihydrate (DhaB), which requires vitamin B ₁₂ , and that 3-HPA is converted to NAD ⁺ Is oxidized to 3-HP by aldehyde dehydrogenase (AldH) as a coenzyme. The 3-HP thus produced can be used not only as a crosslinking agent for polymer coating agents, as a lubricant for metals, as an antistatic agent for fibers, but also as a synthetic agent for acrylic acid, 1,3-propanediol, acrylamide, malonic acid, propionactone and acronitrile It can be used as raw material.

Currently, the production of vitamin B ₁₂ itself is possible and the metabolic pathway to glycerol is best studied with Klebsiella pneumonia strains were used for 3-HP production. However, the production conditions of vitamin B ₁₂ requiring anaerobic conditions made it difficult to regenerate NAD ⁺ , thereby failing to improve the production of 3-HP over a certain level . Therefore, it is required to develop a new strain showing high productivity of vitamin B ₁₂ under aerobic condition and exhibiting rapid cell growth.

Accordingly, the present invention provides a novel Pseudomonas strain having vitamin B ₁₂ production ability under aerobic conditions and a method for producing 3-hydroxypropionic acid using the same.

In order to solve the above problems, the present invention provides a Pseudomonas SP2 strain of the deposit number KACC91720P, which produces vitamin B ₁₂ under aerobic conditions, and a method for producing vitamin B ₁₂ using the same.

The present invention also provides a recombinant strain in which the glycerol dehydratase gene (DhaB) and the glycerol dehydratase reactivase gene (GdrAB) are transfected into the above Pseudomonas sp. Strain, and 3 -Hydroxypropionic < / RTI > acid.

The Pseudomonas sp2 strain of Accession No. KACC91720P according to the present invention can produce vitamin B- ₁₂ at high efficiency under aerobic conditions, thus minimizing the NAD ⁺ regeneration problem as an enzyme coenzyme such as aldehyde dehydrogenase . Therefore, the strain can be used as an economical and useful host strain to be applied to the production of 3-hydroxypyropionic acid synthesized with an enzyme such as glycerol dihydrate and aldehyde dihydrogenase.

1 is a flow diagram of the SP2 strain of the present invention.
Figure 2 shows the expiration and microflora cultures of the strain SP2 of the present invention.
3 is a SEM photograph of the SP2 strain of the present invention.
FIG. 4 is a graph showing the yield of 3-HP after overexpressing the gene for regeneration of glycerol dihydrate and glycerol dihydrate in the SP2 strain of the present invention.

The present invention provides Pseudomonas sp2 strain of Accession No. KACC91720P which produces vitamin B ₁₂ under aerobic conditions.

The inventors of the present invention have been studying to isolate a strain producing vitamin B ₁₂ under aerobic conditions, and have obtained a new isolate of Pseudomonas sp. SP2 from the anaerobic digestion tank. The growth characteristics, production amounts of metabolites, fatty acid components, B ₁₂ production capacity, and confirmed that the new isolate, Pseudomonas sp. SP2, has potential as a host strain for the production of biomolecules requiring vitamin B ₁₂ as a coenzyme. It was confirmed by the National Academy of Agricultural Sciences Under accession number KACC91720P and completed the present invention.

In one embodiment of the present invention, the Pseudomonas sp. Strain is selected from the group consisting of glycerol, glucose, fructose, maltose, lactose, sucrose or galactose. It may be used as a carbon source. Vitamin B ₁₂ is synthesized when cultured under aerobic conditions in a medium containing the carbon source.

Accordingly, the present invention provides a method for producing Pseudomonas sp. Strain, comprising culturing the Pseudomonas sp. Strain in an aerobic condition in a medium containing a carbon source; And it provides a process for the production of vitamin B ₁₂ comprising the step of obtaining the vitamin B ₁₂ from the culture medium.

The Pseudomonas sp. Strain of the present invention can produce vitamin B- ₁₂ at high efficiency under aerobic conditions, and thus can minimize the NAD ⁺ regeneration problem used as a coenzyme for enzymes such as aldehyde dehydrogenase. Therefore, an enzyme such as glycerol dihydrate is introduced into the strain to produce a recombinant strain, which can be used as an economical and useful host strain by applying it to the production of 3-hydroxypyropionic acid.

Accordingly, the present invention provides a recombinant strain in which a glycerol dehydratase gene (DhaB) and a glycerol dehydratase reactivase gene (GdrAB) are transfected into Pseudomonas sp2 strain and a 3- Hydroxypropionic acid. ≪ / RTI >

In one embodiment of the present invention, the glycerol dehydratase gene (DhaB) and the glycerol dehydratase reactivase gene (GdrAB) are expressed by Klebsiella pneumoniae ) derived from the genomic DNA of DSM2026.

The glycerol dihydratator is an enzyme derived from Klebsiella pneumoniae that converts glycerol to 3-HPA and is a protein that is encoded by the dhaB1 gene (KPN_03489), a large subunit or an alpha subunit ), DhaB2 A protein encoded by the gene (KPN_03488) (called a medium subunit or a beta subunit), d haB3 (Small subunit or γ subunit), a protein encoded by the gene (KPN_03487).

In addition, the glycerol dihydratase reactivation factor is a protein derived from Klebsiella pneumoniae , which functions to maintain the activity of the catalyst during the catalytic reaction of glycerol dihydrate , and gdrA Consists GdrB protein which is coded; (AAA 74261 potein ID) gene (KPN_03486) coding GdrA gdrB protein and gene.

Transformation of the glycerol dehydratase gene (DhaB) and the glycerol dehydratase reactivase gene (GdrAB) can be carried out by using a recombinant vector containing the gene and an expression vector Lt; RTI ID = 0.0 > intracellular < / RTI >

The method for producing 3-hydroxypropionic acid is characterized in that glycerol dehydratase gene (DhaB) and glycerol dehydratase reactivase gene (GdrAB) are transduced into the Pseudomonas sp2 strain, Culturing the recombinant strain in a medium containing a carbon source; And obtaining 3-hydroxypropionic acid (3-HP) from the culture broth.

In one embodiment of the present invention, the culturing may be performed under aerobic conditions, and the carbon source may be glycerol and glucose, but the present invention is not limited thereto.

In the following examples of the present invention, the recombinant strain was cultured in a medium containing glycerol and glucose. As a result, it was confirmed that 3-HP was produced up to 16.24 mmol / L for 29 hours without addition of vitamin B ₁₂ . The ratio of produced 3-HP to glycerol used was about 0.32 (yield 32%).

Therefore, the Pseudomonas sp. SP2 strain of the present invention can produce vitamin B ₁₂ under aerobic conditions, and 3-hydroxypropionic acid (3-hydroxypropionic acid) using glycerol dihydrate (DhaB), which requires vitamin B ₁₂ as a coenzyme, HP), which is a strain having excellent properties.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Pseudomonas species SP2 Isolation and identification

1-1. Pseudomonas species SP2 Separation of

Samples were collected from anaerobic digestion tanks to isolate new strains. The collected samples were centrifuged at 8000 rpm for 10 minutes to remove heavy particles. The supernatant was continuously diluted with sterilized water, and then 5 g / L of glucose, 50 g / ml of ampicillin and cetrimide 0.3 g / L was added to the Pseudomonas sp. Plates were cultured at 37 ℃ for 24 hours. Pseudomonas sp. SP2 (SP2), which showed similar growth characteristics to Pseudomonas sp. The SP2 strain was aerobically cultured on LB agar medium and the colonies showed a light cream color. The SP2 strain was deposited at the National Institute of Agricultural Science and Technology, Center for Agricultural Genetic Resources on May 15, 2012 (Accession No .: KACC91720P).

1-2. 16S rDNA  Pseudomonas species by amplification SP2 Confirmation of

Genomic DNA from the newly isolated Pseudomonas sp. SP2 was subjected to PCR (polymerase chain reaction) using the primers shown in Table 1 below to obtain 1575 bp 16S ribosomal DNA. The PCR products were ligated to the pCDFduet-1 vector using the One-Step Isothermal DNA Assembly method. The PCR conditions for amplification (35 cycles) consisted of initial denaturation at 96 ° C for 10 min, denaturation at 96 ° C for 1 min, annealing at 65 (Insert), 47 ° C (vector) for 30 seconds, extension (polymerization) process at 68 ° C for 2 minutes (insert), 5 minutes (vector) and final extension at 72 ° C for 20 minutes.

Primer Sequence 16S rDNA  And vitamin  B ₁₂ gene Cloning primer pCDF-PD 16S INS IAFP TTA ACT TTA ATA AGG AGA TAT AAT TGA ACG CTG GCG GCA GGC C pCDF-PD 16S INS IARP GTA CAA TAC GAT TAC TTT CTG TTC GAA TCG TCT AAC CAG TGA ATC AAG pCDF-PD 16S VEC IARP TAT ATC TCC TTA TTA AAG TTAA pCDF-PD 16S VEC IAFP TCG AAC AGA AAG TAA TCG TAT TGT AC pCDF-PD CobB INS IAFP1 ATT TTG TTT AAC TTT AAT AAG GAG ATA TAA TGC CTT CAA GGC CGG GAT CAA GGC GCA GA pCDF-PD CobB VEC IARP2 TAT ATC TCC TTA TTA AAG TTA AAC AAA AT pCDF-PD CobB VEC IAFP2 AAT TTT GTT TAA CTT TAA TAA GGA GAT ATA pCDF-PD CobB INS IARP1 GGC CGT GTA CAA TAC GAT TAC TTT CTG TTC GAT CAT GGT CGA AAC AGG GCC ACC GCG GCG TCC G pCDF-PD CobQ INS IAFP TTA ACT TTA ATA AGG AGA TAT AAT GAC CTC GCC GAC GGG GCA GGC CGA pCDF-PD CobQ INS IARP TGT ACA ATA CGA TTA CTT TCT GTT CGA CTA CAG CCC GCA TAG TGC TTT CAA TCG Sequencing primer pCDF-PD 16S SEQ FP1 AGC GGA TAA CAA TTC CCC TGTA pCDF-PD 16S SEQ RP2 TAT ATC TCC TTC TTA TAC TTA ACT pCDF-CobB SEQ729FP CTG GCC GAC CTC GAC GCC CGC CTG pCDF-CobB SEQ783RP GCT GGC GGC CAG GGC ATC GGC GGC G pCDF-CobQ SEQ819FP CAC CTG GAA GCC GAG GAC GCC ATC pCDF-CobQ SEQ869RP GCC GCT CTT GGC GGT CTG GCG GAG

Cosmotech Co. Ltd. (Seoul, Korea), and the results of gene sequencing were compared using NCBI BLAST as a reference species of bacteria registered on the genomic database bank. As a result, it was confirmed that the sequence data of SP2 had 99% or more homology with the sequence of Pseudomonas nitroreducens . The SP2 isolate isolated from the above 16S rDNA analysis was classified as Pseudomonas sp. And the isolate was named as Pseudomonas sp. SP2, which was identified as a new strain producing vitamin B ₁₂ under aerobic conditions. The 16S rDNA gene sequence of SP2 was deposited with the NCBI nucleotide sequence database under accession number JX298094.

1-3. Vitamin B ₁₂ Pseudomonas spp. By synthetic gene amplification SP2 Confirmation of

Pseudomonas by dinayi pecan tree's (Pseudomonas denitrificans) are amplified with the cobB cobQ gene region known as genes required for synthesis of vitamin B ₁₂ in the aerobic and analyzed the sequences and identifying the SP2. The cobB and cobQ fragments were amplified from the genomic DNA of the isolates using the primers shown in Table 1 to obtain 1300 bp and 1500 bp gene fragments, respectively. The PCR products were ligated to the pCDFduet-1 vector using the One-Step Isothermal DNA Assembly method. The PCR conditions for amplification (35 cycles) were 96 ° C for 10 min, denaturation at 95 ° C for 1 min, annealing at 67 ° C (Insert), 62 ° C (vector) for 1 minute, extension (polymerization) for 68 ° C for 2 minutes (insert), 5 minutes (vector) and final extension for 72 ° C for 20 minutes.

Cosmotech Co. Ltd. (Seoul, Korea), and the results of gene sequencing were compared using NCBI BLAST as a reference species of bacteria registered on the genomic database bank. As a result, it was confirmed that the cob gene sequence data of SP2 has 83% or more homology with the cob gene sequence data of Azotobacter vinelandii . A novel isolated strain of SP2 and Pseudomonas dinayi pecan tree's (Pseudomonas denitrificans) vitamin B ₁₂ synthesis genes of the strain ATCC13867 and cobB cobQ gene, SP2 was classified as a new strain capable of synthesizing vitamin B ₁₂ under aerobic conditions. Based on the above results, a phylogenetic analysis was conducted to examine the relationship between the newly isolated bacteria and the previously described strains. As a result, as shown in FIG. 1, the new strain SP2 is an independent strain belonging to Pseudomonas species .

1-4. Pseudomonas SP2 Biochemical analysis of

Biochemical analysis of SP2 strains was performed using API 20E microtest kit (Himedia, Mumbai, India). The results are shown in Table 2.

As shown in Table 2, the strain SP2 is selected from the group consisting of mannitol, xylose, mannose, cellobiose, rahmnose, xylito, arabinose, Inositol, sorbitol and the like do not grow, but they do not grow in a medium such as glycerol, glucose, fructose, maltose, lactose, sucrose, galactose, galactose). It was also found to have no ability to produce hydrogen sulfide while having the ability to reduce nitrite.

Test SP2 Test SP2 Lactose + alpha -Methyl-D-Glucoside - Xylose - Rhamnose - Maltose + Cellobiose - Fructose + Melezitose - Dextrose + alpha -Methyl-D-Mannoside - Galactose + Xylitol - Raffinose - ONPG - Trehalose - Esculin hydrolysis - Melibiose - D-Arabinose - Sucrose + Citrate utilization + L-Arabinose - Malonate utilization - Mannose - Sorbose - Inulin - Indol - Sodium Gluconate - Methyl red - Glycerol + Voges Proskauer's - Salicin - Citrate utilization + Dulcitol - Lysine utilization - Inositol - Ornithine - Sorbitol - Urease - Mannitol - Phenylalanine Deaminase - Adonitol - Nitrate reduction + Arabitol - H ₂ S Production - Erythritol -

+: Positive (90% or more), -: Negative (90% or more)

From the above results, it was found that SP2 strain can be developed as a useful strain for synthesizing 3-HP from glycerol, based on the fact that the novel strain SP2 of the present invention can utilize glycerol as a carbon source.

< Example  2> SP2  Flask experiment using strain

The growth and metabolism of the new strain SP2 of the present invention was investigated under aerobic and micro conditions. The SP2 strain was cultured in a 250 mL Erlenmeyer flask containing LB medium at a temperature of 37 DEG C and a stirring speed of 200 rpm in a shaking incubator. For the aerobic condition and the microbial condition, the amount of oxygen in the upper space of the flask was adjusted by changing the amount of LB medium to 50 mL and 150 mL, respectively. Samples were taken periodically to analyze OD and metabolites, and the maximum specific growth rate (μ _max ) was measured at OD ₆₀₀ between 2 and 4 hours. The growth curve graph is shown in Fig. 2 (Symbol: ● - SP2 aerobic, ○ - SP2 aerobic).

2-1. Aerobic culture

The maximum specific growth rate (μ _max ) was 0.91 ± 0.5h ^{- 1} when the SP2 strain was cultured under aerobic conditions, and the OD ₆₀₀ was 3.23 after 8 hours. Pyruvate was produced in almost the same amount as the formate as a major metabolite. These results indicate that SP2 can be used as a commercial strain in various fields through the development of strains.

2-2. SP2 Wow Aerobic  culture

When the SP2 of culture in the conditions Miho group exhibited a 0.56 OD ₆₀₀ value of each of the eighth hour. It was observed that the growth rate was low until 8 hours and it was not growing anymore, and the metabolites were not produced either. These results indicate that the SP2 strain is an aerobic strain requiring oxygen for active growth, and that it is a strain that produces almost no vitamin B ₁₂ under microbial conditions.

< Example  3> SP2  Morphological observation of strain

Scanning electron microscopy (SEM) was used for morphological observation of SP2 strain. The strain was cultured in LB medium in aerobic condition, and the cell pellet was collected by centrifugation at 4 ° C for 10 minutes. The recovered cell pellet was resuspended in 0.1 M sodium crocodylate (pH 7.2) with a fixing solution containing glutaraldehyde (2.5%) and paraformaldehyde (2.0%), and then poly-L- Lt; RTI ID = 0.0 &gt; lysine. &Lt; / RTI &gt; Secondary fixation was carried out using a solution containing potassium ferricyanide (1.5%) and osmium oxide (1.0%) in 0.1M sodium cacodylate (pH 7.2). The primary and secondary fixation procedures were carried out at 4 ° C for 3 hours. After the secondary fixation, the ethanol content was changed to 50, 60, 70, 80, 90, and 95%, and then dried twice with hexamethyldisilazane. Finally, the dried cells were sputter-coated with gold at 30 mM for 150 seconds, and the coated cells were observed for microstructure at 12 kV using a scanning electron microscope (Quanata ^TM 250 FEG, FEI Company, Hillsboro, OR). The resulting photograph is shown in Fig. 3, the SP2 strain of the present invention was elliptical, and the surface of the tablets was slightly rough and elongated.

< Example  4> Assessment of susceptibility to antibiotics

Antibiotic testing was performed using the disk diffusion method proposed by Murray. The strains were cultured overnight at 37 ° C using Mueller-Hinton agar medium (Diffco) and HiMedia Icosa-GI-Minus designed for Gram negative microorganisms and the inhibition area for each antibiotic was sensitized according to the criteria set by NCCLS , intermediate, and resistant. The results are shown in Table 3 below.

Antibiotic group Antibiotics (μg) Suppression region diameter  ( mm ) SP2 Classification Aminoglycoside



Amikacin (30) 20 S Gentamicin (10) 22 S Kanamycin (30) 28 S Streptomycin (30) 20 R Tobramycin (10) 26 S Carboxypenicillin

Carbenicillin (50) 0 R Ampicillin (50) 0 R Augmentin (30) 25 NA β-lactamase inhibitors



Imipenem (10) 27 S Ticarcillin (75) 27 S Ciprofloxacillin (5) 35 S Gatifloxacin (5) 28 S Levofloxacin (5) 31 S Fluoroquinolone



Moxifloxacin (5) 26 NA Nalidixic acid (30) 18 I Norfloxacin (10) 29 S Ofloxacin (5) 30 S Sparfloxacin (5) 26 I Cephalosporin
Cefpodoxime (10) 0 R Cetriaxone (30) 32 S Lipopeptides Colistin (10) 7 R Sulphonamide Co-Trimoxazole (25) 21 NA

S: Sensitive, I: Intermediate, R: Resistant

The susceptibility of SP2 to antibiotics was evaluated by the above-mentioned method, and it was confirmed that it was resistant to most aminoglycoside antibiotics except streptomycin, imipenem, and cefpodoxime . Several strains of Pseudomonas strains have been reported to exhibit virulence by secreting exopolysaccharide alginate or forming alginate glycocalyx out of the cell. The two secretions are known to interfere with the infiltration of other aminoglycoside antibiotics except for streptomycin, imipenem, and cephodoxime as an ionic disorder substance against aminoglycoside antibiotics. Thus, the above results indicate that the SP2 strain has some pathogenicity. It was also found that SP2 was resistant to colistin and moderately susceptible to Nalidixic acid and Fluoroquinolone.

< Example  5> Fatty acid analysis

For fatty acid analysis, strain SP2 was inoculated into TSB (Tryptic Soy Broth) medium according to the MIDI system protocol and cultured at 37 ° C and 200 rpm for 24 hours. Next, heat was applied to the cell walls of the cultured cells for saponification of the lipids at 100 ° C for 30 minutes with NaOH-methanol, and then the extracted lipids were heated with HCl-methanol at 80 ° C for 10 hours Esterified. The fatty acid thus treated was analyzed by gas chromatography. The results of the component analysis are shown in Table 4 below.

SP2 Total lipid (% lipids ) 50% Fatty acid composition (% total Lipid weight ) C10: 0 3OH 2.23 C12: 0 3.53 C12: 0 2OH 5.33 C12: 0 3OH 4.05 C14: 0 3.44 C16: 1? 7c / 15 iso 2OH 9.03 C16: 0 39.18 C17: 0 cyclo 8.70 C18: 1? 7c /? 9t /? 12t 23.01 19: 0 cyclo? 8c 1.49

Referring to Table 4, the amount of lipid contained in the SP2 strain was 50%, and it was confirmed that 10 kinds of fatty acids were contained.

< Example  6> Vitamin B ₁₂ Extraction of

Vitamin B ₁₂ is available in Zhang et al . &lt; / RTI &gt; SP2 cells were cultured overnight in LB medium under aerobic conditions, and cell pellets were obtained by centrifugation. The obtained cell pellet was resuspended in 20 ml of 0.1% KCN in 100 mM MES solution (pH 6.0), sterilized at 121 ° C for 10 minutes, and then cooled at room temperature. Next, centrifugation was carried out at 10,000 rpm at 25 ° C for 10 minutes to remove the solids present in the sample, 1 g of Na ₂ SO ₄ was added to the separated supernatant, mixed well and centrifuged again. Next, as a full-scale step for extracting vitamin B ₁₂ , 10 mL of the supernatant formed after centrifugation was collected in a 50 mL tube, and 1 g Na ₂ SO ₄ and 10 mL benzyl alcohol were added to the supernatant. This procedure was repeated two more times with 5 mL benzyl alcohol, and then all of the benzyl alcohol-reacted extracts were collected and mixed thoroughly with 10 mL of chloroform and 2.0 mL of water. The solution was centrifuged at 10,000 rpm at 25 ° C for 10 minutes, and the absorbance of vitamin B ₁₂ on the aqueous solution was measured at 582 nm on the basis of water. In addition, commercially available vitamin B ₁₂ (Sigma-Aldrich, MO, USA) was dissolved with sterilized water in a concentration range of 1.0 to 100 μM, and then a standard curve was prepared and the concentration of vitamin B ₁₂ was calculated based thereon.

As a result, SP2 strain was observed to produce 11.1 ± 0.5 μg / mL of vitamin B ₁₂ . This level might be compared to the production of vitamin B ₁₂ from other Pseudomonas species reported to date in the production of vitamin B ₁₂ it shows that the strain SP2 can be a useful strain.

< Example  7> SP2 Vitamin B produced by ₁₂ Performance analysis as a coenzyme

Embodiment function as a vitamin B ₁₂ coenzyme extracted in Example 6 was evaluated by measuring the activity of the vitamin B ₁₂ DhaB requirement enzyme. DhaB activity was measured by Raj et al . &lt; / RTI &gt; DhaB overexpressing Klebsiella &lt; RTI ID = 0.0 &gt; pneumoniae recombinant strain was added to a solution containing 35 mM potassium phosphate solution (pH 8.0) and 50 mM potassium chloride and pre-incubated at 37 DEG C for 5 minutes. Then, 50 mM glycerol and 15 mu M vitamin B ₁₂ was added to the pre-incubated solution and reaction was carried out for 1 minute. Finally, the same amount (0.5 mL) of 100 mM citric acid was added to the total reaction volume, and the reaction was terminated after 1 minute. The unit of DhaB activity was defined as the amount of enzyme required to produce 1 μM of 3-HPA in glycerol per minute.

The activity of DhaB enzyme was measured using vitamin B ₁₂ extracted by the above method and showed 0.040 U / mg of enzyme activity. When vitamin B ₁₂ purchased from Sigma-Aldrich was used, it showed the same activity as vitamin B ₁₂ extracted from SP2 strain. This means that vitamin B ₁₂ produced in the newly isolated SP2 strain performs well as a coenzyme of DhaB. Therefore, the above results suggest that SP2 can be developed as a host strain for the production of platform chemicals such as 3-HP from glycerol using vitamin B ₁₂ requirement DhaB.

< Example  8> DhaB  And GdrAB  Enzyme Cloned  Recombination SP2 Lt; RTI ID = HP Production of

To evaluate the possibility of 3-HP production of the novel isolate SP2, recombinant SP2 was constructed in which each gene encoding glycerol dihydratase and glycerol dihydratase reactivation enzyme was cloned. The recombinant strains were also used to investigate 3-HP production on flask scale.

8-1. dhaB Wow gdrAB &Lt; / RTI &gt; over- SP2  Production of strain

(SEQ ID NO: 1: GACGAG CTT CTA AAT ACA TTC AAA TAT) reverse primer (SEQ ID NO: 2: GAC GAG CTC TCT AGA GGT ACC ACT CTT CCT TTT TCA ATA) to amplify the bla promoter gene from the pUCP19 vector The amplified PCR fragments were digested with restriction enzymes Hind III and Sac I.

The glycerol dihydratase (DhaB) gene is expressed by Klebsiella (SEQ ID NO. 3: AGT GGT ACC ATG AAA AGA TCA AAA CGA TTT GC) and reverse primer (SEQ ID NO: 4: AT CTT AAG CCT GAT TAA TTC GCC TGA CCG GCC AGT) from the genomic DNA of the strain S. pneumoniae DSM2026 PCR was performed. The amplified PCR fragments dhaB1 , dhaB2 , dhaB3 and dhaB4 (gdrA) were digested with restriction enzymes Kpn Ⅰ and Afl Ⅱ. The internal primers for amplifying the dhaB gene include kp_dhaB_Int_FP1 (SEQ ID NO: 5: CTG GCG CTG TGG GTC GGT TCG), kp_dhaB_Int_FP2 (SEQ ID NO: 6: TTT CTC CGG CTA CAG CGC GGT), kp_dhaB_Int_FP3 ATG AAC GCG), kp_dhaB_Int_FP4 (SEQ ID NO: 8: CGC ATT CTG GCT ATC TAT AAC), kp_dhaB_Int_FP5 (SEQ ID NO: 9: CTC TCG CAT CTA TCT TAA CG), kp_dhaB_Int_FP6 (SEQ ID NO: 10: TGG ATA CGT TTA TTC CGC GCA) And kp_dhaB_Int_FP7 (SEQ ID NO: 11: CGA TAA AAA AT ACC CGC TGG).

The amplification of the gdrB gene was also performed using a forward primer (SEQ ID NO: 12: GTG CAG CTT AAG AAA ATG TCG CTT TCA CCG CCA GGC GTA) and reverse primer (SEQ ID NO: 13: A TTA GAG CTC TCA GTT TCT CTC) from Klebsiella pneumoniae DSM2026 ACT TAA CGG). The amplified PCR fragment was digested with restriction enzymes Afl Ⅱ and Sac Ⅰ .

The amplified bla promoter and dhaB ( dhaB1 , dhaB2 , dhaB3 and dhaB4 ( gdrA )) and gdrB were cloned into the expression vector pUC19 vector to obtain a recombinant plasmid pPBG1. The prepared plasmid was introduced into a new isolate SP2 by electroporation, and the SP2 recombinant strain into which pPBG1 was introduced was named Pseudomonas sp2 / pPBG1.

8-2. Pseudomonas SP2 / pPBG1  The recombinant strains 3- HP production

The recombinant strain Pseudomonas sp2 / pPBG1 was preliminarily cultured in M9 liquid medium (pH 7.0) containing 2 g / L of yeast extract for 12 hours at 37 ° C., and then the grown medium was cultured in a medium containing 20 mM glucose and 2 g / L of yeast extract (PH 7.0) at a concentration of 0.1 OD ₆₀₀ and then cultured. At this time, 100 mM potassium phosphate buffer solution (pH 7.0) was used to maintain the pH of the culture solution. The cells were also cultured in a 250 mL Erlenmeyer flask containing 50 mL of M9 medium in a shaking incubator maintained at 37 DEG C, and the stirring speed was set to 200 rpm to be aerobic condition. Then, when the OD ₆₀₀ reached 1.0, 6.5 mM of glucose and 50 mM of glycerol were added. The resultant graph is shown in Fig. 4 (Symbol: ▲ - Glycerol, △ - Glucose, ● - 3 - HP, ○ - cell growth).

Referring to FIG. 4, it was observed that 3-HP up to 16.24 mmol / L could be produced for 29 hours without addition of vitamin B ₁₂ . At this time, the concentration of the microorganism was 1.3 mg / l, and the ratio of 3-HP produced relative to the glycerol used was about 0.32 (yield 32%). On the other hand, when Pseudomonas sp2 wild type (WT) was cultured in the same manner, production of 3-HP was not observed at all.

Therefore, the 3-HP production capacity of the recombinant strain Pseudomonas sp2 / pPBG1 using the newly isolated isolate SP2 was found to be due to the action of the glycerol dihydrate and the glycerol dihydratase re-activating enzyme introduced into the plasmid.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that such detail is solved by the person skilled in the art without departing from the scope of the invention. will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

National Academy of Agricultural Sciences KACC91720 20120716

<110> Institute for research and industry cooperation, PNU <120> Novel Pseudomonas SP2 and Preparation Method of 3-Hydroxypropionic acid Therewith <130> DP-2012-0598 <160> 13 <170> Kopatentin 1.71 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for bla promoter gene <400> 1 ggcgaagctt ctaaatacat tcaaatat 28 <210> 2 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for bla promoter gene <400> 2 gacgagctct ctagaggtac cactcttcct ttttcaata 39 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for dhaB gene <400> 3 agtggtacca tgaaaagatc aaaacgattt gc 32 <210> 4 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for dhaB gene <400> 4 atcttaagcc tgattaattc gcctgaccgg ccagt 35 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Internal primer for dhaB gene <400> 5 ctggcgctgt tggtcggttc g 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Internal primer for dhaB gene <400> 6 tttctccggc tacagcgcgg t 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Internal primer for dhaB gene <400> 7 tagcttctgc cgatgaacgc g 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Internal primer for dhaB gene <400> 8 cgcattctgg ctatctataa c 21 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Internal primer for dhaB gene <400> 9 ctctcgcatc tatcttaacg 20 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Internal primer for dhaB gene <400> 10 tggatacgtt tattccgcgc a 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Internal primer for dhaB gene <400> 11 cgataaaaaa atacccgctg g 21 <210> 12 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for gdrB gene <400> 12 gtgcagctta agaaaatgtc gctttcaccg ccaggcgta 39 <210> 13 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for gdrB gene <400> 13 attagagctc tcagtttctc tcacttaacg g 31

Claims (8)

Production of vitamin B ₁₂ under aerobic conditions using glycerol, glucose, fructose, maltose, lactose, sucrose or galactose as carbon source Pseudomonas sp2 strain of accession number KACC91720P. delete Culturing the Pseudomonas sp2 strain of claim 1 in an aerobic condition in a medium containing a carbon source; And
Process for producing a vitamin B _12, comprising the step of obtaining the vitamin B ₁₂ from the culture medium. A recombinant strain in which a glycerol dehydratase gene (DhaB) and a glycerol dehydratase reactivase gene (GdrAB) are transduced into the Pseudomonas sp2 strain according to claim 1. 5. The method of claim 4,
The glycerol dehydratase gene (DhaB) and the glycerol dehydratase reactivase gene (GdrAB) can be obtained from Klebsiella pneumoniae ) &lt; / RTI &gt; derived from the genomic DNA of DSM2026. Culturing the recombinant strain of claim 4 in a medium containing a carbon source; And
Hydroxypropionic acid from the culture. &Lt; RTI ID = 0.0 &gt; 3-Hydroxypropionic &lt; / RTI &gt; acid. The method according to claim 6,
Wherein the step of culturing is carried out under aerobic conditions. The method according to claim 6,
Lt; RTI ID = 0.0 &gt; 3-hydroxypropionic &lt; / RTI &gt; acid, wherein the carbon source is glycerol and glucose.

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