KR20120041826A - Recombinant microorganism having vitamin b-12 synthesis capability and preparation method of 3-hydroxypropionic acid therewith - Google Patents

Abstract

PURPOSE: A method for preparing 3-hydroxypropionic acid using a recombinant microorganism is provided to obtain 3-hydroxypropionic acid without vitamin B-12. CONSTITUTION: A recombinant microorganism with vitamin B-12 productivity is prepared by transforming a recombinant vector containing a gene encoding glycerol dehydratase and a gene encoding glycerol dehydratase reactivase. The recombinant vector additionally contains a gene encoding aldehyde dehydrogenase. Microorganisms with vitamin B-12 productivity include genus Pseudomonas, genus Klebsiella, genus Propionibacterium, genus Rhodopseudomonas, or genus Rhizobium. The gene encoding glycerol dehydratase is derived from the genome DNA of Klebsiella pneumoniae DSM 2026.

Description

Recombinant microorganism having vitamin B-12 synthesis capability and preparation method of 3-hydroxypropionic acid therewith}

The present invention relates to a recombinant microorganism having a vitamin B-12 producing ability and a method for preparing 3-hydroxypropionic acid using the same.

Recently, the production of bio-based fuels has been in the spotlight due to the sharp rise in oil prices and serious environmental problems. Biodiesel, one of the biobased fuels, is produced by transesterification of triglycerides from vegetable oils or animal fats.

Mass production of biodiesel has led to a surge in the production of by-product glycerol, producing about 100 million gallons of glycerol per billion gallons of biodiesel. As glycerol production surges, one billion tons of glycerol is produced worldwide. As a result, the price of glycerol has decreased by a factor of ten over the last two years. The market price for unrefined glycerol is $ 350 per tonne in 2004 and is now known at $ 200 per tonne.

In comparison, the price of glucose (glucose) is now known to be about $ 500 per ton and is expected to increase gradually. Given the current trend, we believe the continued decline in glycerol prices is inevitable.

Microorganisms can use glycerol as a carbon source, which can be used to produce a variety of fermentation products. 3-Hydroxypropionic acid (3-HP; C ₃ H ₆ O ₃ -MW 90.08) is a compound with various applications in chemical processes and can be produced from renewable resources such as glycerol.

3-HP is a weak acid with a pKa of 4.51 at 25 ° C., a non-chiral organic acid with structural three carbons, isomeric (2-hydroxypropionic acid) and isomers. It is also amorphous, pale yellow like syrup, specific gravity of 1.25 and refractive index of 1.45.

3-HP is an important synthetic intermediate used in many chemical processes: 1,3-propanediol (C ₃ H ₈ O ₂ -MW 76.09), acrylic acid (C ₃ H ₄ O ₂ -MW 72.06), methyl acrylate ( C ₄ H ₆ O ₂ -MW 86.09), acrylamide (C ₃ H ₅ NO-MW 71.08), ethyl 3-hydroxypropionic acid C ₅ H ₁₀ O ₃ -MW 118.13), malonic acid (C ₃ H ₄ O ₄ -MW 104.06), propionlactone (C ₃ H ₄ O ₂ -MW 72.06), acronitrile (C ₃ H ₄ N-MW 53.06), etc. are used as raw materials to produce. The global market for 3-HP-related products is 3.6 million tons and is expected to rise 5% annually from current demand and prices.

3-HP may be produced by a chemical process using ethylene cyanohydrin, beta-idopionic acid, beta-bromopropionic acid, beta-chloropropionic acid, beta-propiolactone, acrylic acid, or the like as intermediates. However, most of these chemicals are toxic and carcinogenic. In addition, it consumes a large amount of energy under the condition of high temperature and pressure, and has a disadvantage of emitting a large amount of pollutants.

In addition, biological 3-HP production is achieved by the chloroplexus Orantiacus , a photodependent nutritional microorganism. The microorganism grows autotrophically and photodependently, producing 3-HP as an intermediate. Other microorganisms that produce 3-HP from glycerol include Desulfovibrio carbinolicus , Desulfovibrio fructosovorans , Lactobacillus reuteri , and Pelopacter veneace . Pelobacter venetianus , Ilyobacter polytropus and the like are known.

However, since the metabolic pathway is complicated, it is difficult to efficiently control the process, and thus, a decrease in production yield and productivity is expected. Therefore, it is essential to design a synthetic route that does not exist in vivo.

As shown in Scheme 1, 3-HP can be produced from glycerol through a dehydration and oxidation process catalyzed by two enzymes biologically. The first enzyme, glycerol dehydratase, dehydrates glycerol to catalyze the intermediate 3-hydroxypropionaldehyde (3-HPA) production reaction. The second enzyme, aldehyde dehydrogenase, oxidizes the intermediate 3-HPA to produce the final product, 3-HP.

Scheme 1

Enzymes with high specificity for 3-HPA have not been identified to date. Only recently have Escherichia coli K-12 of AldH protein. (SM Raj et al, Process Biochemastry, 43:. 1440-1446,2008), azo Kgsadh RY-I protein in Bras rilrum chamber lens (Azospirillum brasilense) (Seiya Watanabe et al, The Journal of Biological chemistry Vol. 282, NO. 9, pp. 6685-6695, March 2, 2007) and Puuc protein of DSM 2026 from Klebsiella pneumoniae (Subramanian Mohan Raj et al., Biotechnology and Bioprocess Engineering, 15: 1- 1, 2010), etc. have been found to have high activity against 3-HPA, and DhaB and AldH or DhaB and Kgsadh-I derived from Klebsiella pneumoniae were expressed in E. coli at high concentrations of 38.7 g / L. 3-HP production was possible (Chelladurai Rathnasingh et al., Biotechnol. Bioeng ., 104 (4): 729, 2009).

On the other hand, the biggest problem when using E. coli to express such genes is the use of vitamin B-12, an expensive auxiliary substrate. The first enzyme, glycerol dehydratase, which dehydrates glycerol to produce 3-HPA, requires vitamin B-12.E. Coli does not have the ability to produce this vitamin B-12 on its own. Because.

The solution to the expensive problem of using vitamin B-12 is to use a strain that has the ability to produce vitamin B-12 itself in addition to Escherichia coli as a host, but through the genetic manipulation of these strains to produce 3-HP from glycerol Successful cases have not been reported.

Therefore, the present inventors have tried to produce 3-HP from glycerol without supplying vitamin B-12 from the outside through a microorganism capable of producing vitamin B-12 by itself, glycerol to a microorganism having the ability to produce vitamin B-12 A recombinant microorganism transduced with a recombinant vector consisting of a gene encoding dehydratase and a gene encoding a glycerol dehydratase reactivation factor, or a gene encoding an aldehyde dehydrogenase to the recombinant vector The recombinant microorganisms transduced with the included recombinant vector were prepared, thereby confirming that the recombinant microorganism produced 3-HP from glycerol without supplying vitamin B-12 and completing the present invention.

An object of the present invention consists of a gene encoding glycerol dehydratease and glycerol dehydrase reactivation enzyme, or additionally 3-hydroxypropionaldehyde dehydrogenase, to microorganisms capable of producing vitamin B-12 by themselves. It is to provide a recombinant microorganism transduced with a recombinant vector.

In addition, another object of the present invention to provide a method for producing 3-HP using the recombinant microorganism.

In order to achieve the above object, the present invention provides a gene encoding glycerol dehydratase and a gene encoding glycerol dehydratase reactivase in a microorganism having a vitamin B-12 producing ability. Provided is a recombinant microorganism characterized by transducing a recombinant vector consisting of.

More specifically, the present invention is transfected with a recombinant vector consisting of the dhaB gene encoding glycerol dehydratase and the gdrAB gene encoding glycerol dehydratase reactivase to a microorganism having a vitamin B-12 production ability It provides a recombinant microorganism characterized by.

In addition, the recombinant vector may further comprise a gene encoding an aldehyde dehydrogenase, that is, puuC gene.

More specifically, the present invention provides a dhaB gene encoding glycerol dehydratase , a gdrAB gene encoding glycerol dehydratase reactivase and a aldehyde dehydrogenase (aldehyde dehydrogenase) in a microorganism having vitamin B-12 production ability. Provided is a recombinant microorganism characterized by transducing a recombinant vector consisting of a puuC gene encoding a).

The microorganisms having the vitamin B-12 producing ability used in the present invention are Genus Pseudomonas , Genus Klebsiella , Genus Propionibacterium , Genus Rhodopseudomonas , and Genus Rhodopseudomonas . It may be any one microorganism selected from the group consisting of genus Rhizobium .

More specifically, the microorganisms having the ability to produce vitamin B-12 include Pseudomonas denitrificans , Klebsiella pneumoniae , Propionibacterium shermanii , and Rhododdomonas . Rhodopseudomonas protamicus and It may be any one microorganism selected from the group consisting of Rhizobium cobalaminogenum .

In particular, the microorganism having the vitamin B-12 production capacity is preferably Pseudomonas denitrificans (Accession No. KCCM 2026).

In addition, the gene encoding the glycerol dehydratase may be derived from genomic DNA of Klebsiella pneumoniae DSM 2026, the gene encoding the glycerol dehydratase reactivase is Klebsi Klebsiella pneumoniae Can be derived from the genomic DNA of the DSM 2026, a gene encoding the aldehyde-dihydro centipede rise is derived kgsadh from E. coli (Escherichia coli) the aldH gene, azo RY rilrum bra chamber lens (Azospirillum brasilense) derived from K-12 -I gene and puuC gene derived from genomic DNA of Klebsiella pneumoniae DSM 2026.

The recombinant microorganism according to the present invention is preferably Pseudomonas denitrificans pPBG1 (GSB0167, Accession No. KCTC 11672BP).

Hereinafter, the present invention will be described in more detail.

In the recombinant microorganism, glycerol dehydratase is an enzyme derived from Klebsiella pneumoniae , converts glycerol to 3-HPA, and is a protein that is encoded by the dhaB1 gene (KPN_03489). Large subunit or α subunit la called), a protein that is coded dhaB2 gene (KPN_03488) suspended gastric (Medium subunit or β subunit called called), d haB3 gene (KPN_03487) the protein subunits (small subunit or γ subunit encoding It is composed of three proteins.

In addition, glycerol dehydratase reactivation factor is a protein derived from Klebsiella pneumoniae , and serves to maintain the activity of the catalyst during the catalytic reaction of glycerol dehydratase , gdrA gene ( KPN_03486) is GdrA protein and gdrB gene (potein ID encoding; the AAA 74261) it consists of the protein encoding GdrB.

In addition, the aldehyde-dihydro centipede rise is Escherichia coli (Escherichia coli) to the aldH gene coding derived from K-12 ALDH protein, azo RY rilrum bra chamber lens (Azospirillum brasilense) the kgsadh-I KGSADH-I to the gene coding derived from Protein or Klebsiella pneumoniae As one of the PuuC proteins encoded by the puuC gene (KPN_01018) derived from DSM 2026, 3-HPA is converted to 3-HP.

The present invention also provides a method for producing 3-hydroxypropionic acid (3-HP), wherein the recombinant microorganism is cultured in a medium containing glucose and glycerol as a carbon source, and 3-hydroxypropionic acid is recovered from the culture solution. To provide.

In addition, the present invention comprises the steps of culturing the recombinant microorganism in a culture medium containing glucose and glycerol as a carbon source; And it provides a method for producing 3-HP comprising the step of producing 3-HP from the culture.

The culturing step is preferably carried out under aerobic conditions, vitamin B-12 may be added in the step of producing 3-hydroxypropionic acid from the glycerol.

In addition, the process of culturing the recombinant microorganism and 3-HP can be carried out using a culture method and separation and purification method commonly known in the conventional fermentation industry.

The recombinant microorganism according to the present invention can efficiently produce 3-HP from inexpensive glycerol without the addition of expensive vitamin B-12, thereby producing 3-HP while reducing the production price of the target metabolite such as 3-HP. It can be used very usefully as a strain.

1 is a cleavage map of the recombinant vector pPBG1,
2 is a cleavage map of the recombinant vector pPBGA1,
3 is a batch fermentation result of Pseudomonas dinitrippicans pPBG1.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited by these examples.

In particular, in the following examples, a method of producing 3-HP from glycerol without adding vitamin B-12 using Pseudomonas denitrificans as a host cell, but other microorganisms having the ability to produce vitamin B-12 Fields such as Klebsiella pneumoniae , Propionibacterium shermanii , Rhodopseudomonas protamicus , Rhizobium cobalaminogenum The same result can be obtained even when using strains such as the host cell will be apparent to those skilled in the art.

Example 1 Isolation of Genes That Convert Glycerol to 3-HP

1-1. from the pUCP19 vector bla  Amplification of promoter genes

Bla promoter gene from the pUCP19 vector (HP Schweizer, Elsevier Science Publishers, Gene, 97 (1991) 109-112) was subjected to PCR using a forward primer (SEQ ID NO: 1) and a reverse primer (SEQ ID NO: 2) to bla Promoter genes were amplified and digested with restriction enzymes HindIII and SacI.

1-2. Klebsiella New Monica from DSM 2026 dhaB  Gene amplification

Genomic DNA from the Klebsiella pneumoniae DSM 2026 strain was subjected to PCR using a forward primer (SEQ ID NO: 3) and a reverse primer (SEQ ID NO: 4) to dhaB1, dhaB2, dhaB3 and dhaB4 (gdrA). ) Genes were amplified and digested with restriction enzymes KpnI and AflII.

Internal primers for amplifying the dhaB gene include kp_dhaB_Int_FP1 (SEQ ID NO: 5); kp_dhaB_Int_FP2 (SEQ ID NO: 6); kp_dhaB_Int_FP3 (SEQ ID NO: 7); kp_dhaB_Int_FP4 (SEQ ID NO: 8); kp_dhaB_Int_FP5 (SEQ ID NO: 9); kp_dhaB_Int_FP6 (SEQ ID NO: 10); And kp_dhaB_Int_FP7 (SEQ ID NO: 11) were used.

1-3. Klebsiella New Monica from DSM 2026 gdrB  Gene amplification

Genomic DNA from Klebsiella pneumoniae DSM 2026 strain was subjected to PCR using a forward primer (SEQ ID NO: 12) and a reverse primer (SEQ ID NO: 13) to amplify the gdrB gene and cleaved with restriction enzymes AflII and SacI. .

1-4. Klebsiella New Monica from DSM 2026 puuC Gene amplification

PCR was performed twice to amplify the puuC gene including the bla promoter gene. Genomic DNA was amplified from the Klebsiella pneumoniae DSM 2026 strain using a forward primer (SEQ ID NO: 14) and a reverse primer (SEQ ID NO: 15).

Thereafter, the genes were amplified using the forward primer (SEQ ID NO: 16) and the reverse primer (SEQ ID NO: 15) from the amplified PCR product and digested with restriction enzymes Xba I and Sac I.

1-5. Amplification of the kanamycin resistance gene from the pKD4 plasmid

The kanamycin resistance gene from the pKD4 vector (Kirill et., Proceedings of the National Academy of Sciences, 97, (2000), 12) was subjected to PCR using a forward primer (SEQ ID NO: 17) and a reverse primer (SEQ ID NO: 18). The kanamycin resistance gene was amplified and digested with restriction enzyme NdeI.

Example 2. Construction of Gene Recombinant Vector for 3-HP Production

2-1. Construction of Recombinant Vector pPBG1

DhaB ( dhaB1, dhaB2, dhaB3, dhaB4 ( gdrA )) and gdrB PCR fragments amplified in Example 1 were respectively conjugated to the pGEM-T vector. And the prepared plasmids were introduced into E. coli XL1-blue, respectively.

pTBGA (pGEM-T / dhaB) plasmid was digested with restriction enzymes Kpn I and Afl Ⅱ, pTGB (pGEM-T / gdrB) plasmid was digested with restriction enzymes Afl Ⅱ and Sac I.

Plasmid, pTBGAB (pGEM-T / dhaB1234 and gdrB ), prepared by conjugating two DNA fragments, was cloned into pUCP19 vector, an expression vector, to obtain a recombinant plasmid pPBG1 (see FIG. 1).

2-2. Construction of Recombinant Vector pPBGA1

The pucC PCR fragment containing the bla promoter amplified in Example 1 was conjugated to the pGEM-T vector. And the produced plasmid was introduced into E. coli XL1-blue.

pTBPP (pGEMT / bla_promoter_pucC) plasmid was digested with Xba I and Sac I restriction enzymes and cloned into pPBG1, a recombinant expression vector, to obtain recombinant plasmid pPBBGA1 (see FIG. 2).

Example 3. Preparation of recombinant strain for 3-HP production

3-1. E.coli Construction of XL1-blue / pPBG1 Strains

E. coli XL1-blue was plated on LB solid medium and incubated at 37 ° C. for 36 hours, and then colonies were inoculated in 10 mL of LB liquid medium and incubated for 12 hours. Fully grown culture was inoculated 1% in 100 mL LB medium and incubated in a 200 rpm, 37 ℃ shaking incubator. After about 4 to 5 hours, when the OD600 was about 0.3 to 0.4, the cells were obtained by centrifugation at 4 ° C, 4,500 rpm, and 20 minutes, and the cells were resuspended with 200 mL of a 10% glycerol solution at 4 ° C. . And cells were obtained by centrifugation at 4 ℃, 5,500 rpm, 20 minutes.

The glycerol solution used for resuspension was cut in half and run two times in succession, and then the cell concentrate was resuspended so that the volume ratio of the cells and the glycerol solution was 1: 1. E. The cultured expression vector was cultured by mixing the obtained cell concentrate with pPBG1 prepared in Example 2-1, and then performing electroporation under the conditions of 2.5 kV, 25 μF, and 400 ohms . Coli XL1-blue was introduced.

After the electric shock, 1 mL of LB liquid medium was added and incubated for 1 hour in a 200 rpm, 37 ℃ shaking incubator. The culture was plated in LB solid medium containing the antibiotic ampicillin (final concentration 50 µg / L) and incubated at 37 ° C for at least 12 hours. To over 12 hours, the formed colonies were cultured in an LB liquid medium containing an antibiotic ampicillin, E. coli XL1-blue pPBG1 was prepared, which was then E. coli XL1-blue / It was named pPBG1 strain.

3-2. E.coli Construction of XL1-blue / pPBGA1 Strains

E. coli XL1-blue was transformed using pPBGA1 prepared in Example 2-2 in the same manner as described in Example 3-1, and was named E. coli XL1-blue / pPBGA1 strain.

3-3. Preparation of Pseudomonas Dinitripicans_pPBG1 Strain

Pseudomonas dinitrippicans colonies were inoculated in 5 mL of LB liquid medium and incubated at 30 ° C. for 12 hours. Fully grown cultures were inoculated 1% in 20 mL of LB broth and incubated at OD ₆₀₀ for 20 minutes at 42 ° C. and then incubated on 4 ° C. ice for 30 minutes to stop growth. Thereafter, 5 ml of the culture solution was centrifuged at 4 ° C., 2,500 rpm, and 5 minutes to obtain cells. The cells were washed twice with 2 ml of 300 mM sucrose at 4 ° C. and stored at 4 ° C. ice for 10 minutes.

The expression vector was cultured by Pseudomonas denite by mixing 1.5 μg of the recombinant vector pPBG1 of Example 2-1 in 70 μl of 300 mM sucrose and incubating on 4 ° C. ice for 2 minutes and performing electroporation. Introduced to Lipicans.

After the electric shock, 500 μl of SOC liquid medium was added thereto, and then incubated for 2 hours in a 200 rpm, 37 ° C. shaking incubator. The culture was plated in LB solid medium containing the antibiotic kanamycin (final concentration 30 μg / L) and incubated at 37 ° C. for at least 12 hours. Formed colonies were cultured in an LB liquid medium containing kanamycin antibiotic for at least 12 hours, and Pseudomonas dinitripicans pPBG1 was prepared and named Pseudomonas Dinitripicans / pPBG1 strain.

Example 4 Determination of Glycerol Dehydratase and Aldehyde Dehydrogenase Activity

The recombinant microorganism prepared in Example 3 was preinoculated in 5 mL of LB liquid medium and incubated at 37 ° C. for 12 hours. Fully grown culture was inoculated 1% in 50 mL of the same liquid medium and incubated in 200 rpm, 37 ℃ shaking incubator.

When the OD ₆₀₀ of the cultured cells reached 2.5, the cells precipitated by centrifugation were washed twice with potassium phosphate buffer solution (100 mM, pH 8.0), suspended in the same buffer, and then pressed with French press (FA-078A). , Thermo Electron Corp .; Waltham, MA, USA) and then crushed at a pressure of 1,250 psi, the cell debris was removed using a centrifuge and the cell extract supernatant was used to measure the enzyme activity.

4-1. Determination of Glycerol Dehydratase Activity

The activity of glycerol dehydratase in the prepared cell extract (crude cell) was measured by the following method. Appropriate amount of cell extract was added to potassium phosphate reaction solution containing 35 mM potassium chloride (35 mM, pH 8.0) and pre-incubated at 37 ° C. in 5 volumes, followed by 50 mM glycerol and 15 μM of vitamin B-12. Was added and the final volume was 0.5 ml, followed by reaction for 1 minute.

An equal volume of citric acid (100 mM) was added to terminate the reaction. The resulting 3-HPA was measured by HPLC (Agilent 1100 series, USA).

Enzyme active unit 1.0 U of glycerol dehydratase was defined as the amount of enzyme required to produce 1 μmol of 3-HPA for 1 minute.

Recombinant microorganism dhaB active unit
(μmol 3-HPA / mg protein / min) E. coli XL1-blue / pPBG 9.3 E. coli XL1-blue / pPBGA 5.9 Pseudomonas denitrificans / pPBG 2.8 Pseudomonas denitrificans (WT) 0.0

As a result, as shown in Table 1, in the recombinant microorganism Pseudomonas Dinitripicans DSM 2026 / pPBG according to the present invention, the enzyme activity of glycerol dehydratase was 2.8 U / mg, but Pseudomonas is the parent strain. Dinitripicans (WT) showed no activity at all.

E. coli XL1-blue / pPBG and E. coli XL1-blue / pPBGA had enzymatic activities of 9.3 U / mg and 5.9 U / mg of glycerol dehydratase, respectively.

There was a difference in activity of the recombinant microorganisms, and all of the activity was appropriate, and the transformation was well done.

4-2. Determination of the activity of aldehyde dehydrogenase

The activity of aldehyde dihydrogenase was measured by the following method. Appropriate amount of cell extract was added to potassium phosphate reaction solution (50 mM, pH 7.0) containing 1 mM 2-mercapto ethanol and pre-incubated for 5 minutes at 30 ° C., followed by 1 mM aldehyde and 2 mM NAD ( The reaction was initiated with P) ⁺ to a final volume of 0.5 ml.

The activity of the enzyme was examined by measuring the reduction of NAD (P) ⁺ to NAD (P) H at 340 nm. The amount of NAD (P) H produced was determined using a molar extinction coefficient (Δε ₃₄₀ ) 6.22 × 10 ³ M ⁻¹ cm ⁻¹ .

The active unit 1.0 U of aldehyde dehydrogenase was defined as the amount of enzyme required to reduce 1 μmol of NAD (P) ⁺ to NAD (P) H for 1 minute.

Recombinant microorganism Specific activity:
μmol NADH / mg protein / min (U / mg) E. coli XL1-blue / pPBG1 0 E. coli XL1-blue / pPBGA1 0.05 Pseudomonas denitrificans / pPBG1 0.02 Pseudomonas denitrificans (WT) 0.03

As a result, as shown in Table 2, the enzymatic activity of the aldehyde dehydrogenase of the E. coli XL1-blue / pPBGA1 strain was 0.05 U / mg, and the aldehyde dehydrozine of the E. coli XL1-blue / pPBG1 strain. Although the enzyme activity of izu was not shown, the enzyme activity of aldehyde dehydrogenase was 0.02 U / mg in P. denitrificans / pPBG1 strain. This indicates that P. denitrificans / pPBG1 can produce 3-HP without enzymatic expression of foreign aldehyde dehydrogenase.

Wild P. denitrificans (WT) showed enzymatic activity of aldehyde dehydrogenase of 0.03 U / mg. Therefore, it was found that wild P. denitrificans (WT) strain produced its own aldehyde dehydrogenase based on 3-hydroxy propionaldehyde.

Example 5. Transformed Pseudomonas denitrificans Of Production of 3-HP in pPBG1 Cultures

Recombinant strain P. denitrificans / pPBG1 prepared in Example 3-3 was preinoculated in 10 mL of M9 liquid medium containing 2 g / L yeast extract was incubated for 12 hours at 37 ℃. Fully grown cultures were cultured in M9 liquid medium (pH 7.0) with 20 mM glucose and 2 g / L yeast extract.

At this time, 100 mM potassium phosphate buffer solution was used to maintain the pH of the culture. Culture of the cells was incubated at a temperature of 37 ℃ 200 rpm using a 250mM Erlenmeyer flask containing 50mL of medium.

Then, when OD ₆₀₀ reached 1.0, 6.5 mM of glucose and 50 mM of glycerol were added.

As a result, as shown in FIG. 3, up to 15.85 mmol / L of 3-HP was obtained at 29 hours incubation time without adding any vitamin B-12. At this time, the concentration of the microorganism was 1.13 mg / l and the ratio of 3-HP produced relative to the glycerol used was about 0.33 (yield 33%). In addition, the entire culture was carried out under aerobic conditions. Meanwhile, wild P. denitrificans (WT) were cultured in the same manner. Regardless of incubation time, 3-HP was not produced at all.

Therefore, it could be confirmed that the 3-HP production capacity of the recombinant strain P. denitrificans / pPBG1 was shown in FIG. 3 due to the action of glycerol dehydratase and glycerol dehydratase reactivating enzyme introduced into the plasmid.

As described above in detail specific parts of the present invention, it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, thereby not limiting the scope of the present invention. will be. Therefore, the substantial scope of the present invention should be defined by the technical spirit of the claims.

Korea Research Institute of Bioscience and Biotechnology KCTC11672BP 20100326

<110> Institute for research and industry cooperation, PNU <120> Recombinant microorganism having vitamin B-12 synthesis          capability and preparation method of 3-hydroxypropionic acid          therewith <130> DP-2010-0140 <160> 18 <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 <210> 14 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for puuC gene <400> 14 gataaatgct tcaataatat tgaaaaagga agagtttatg atgaattttc agcacct 57 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for puuC gene <400> 15 agagagctct caagactcca gggcaat 27 <210> 16 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PCR product <400> 16 gcatctagat ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata 60 aatgcttcaa t 71 <210> 17 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for kanamycin resistant gene <400> 17 gataacatat gtcacgctgc cgcaagc 27 <210> 18 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for kanamycin resistant gene <400> 18 attccatatg ggggtgggcg aagaactcca gc 32

Claims (12)

A recombinant vector consisting of a gene encoding glycerol dehydratase and a gene encoding a glycerol dehydratase reactivase is transfected into a microorganism having a vitamin B-12 producing ability. A recombinant microorganism having a vitamin B-12 producing ability. The method of claim 1, wherein the recombinant vector is a recombinant microorganism having a vitamin B-12 production capacity, characterized in that further comprises a gene encoding aldehyde dehydrogenase (aldehyde dehydrogenase). The method according to claim 1 or 2, wherein the microorganisms having the ability to produce vitamin B-12 is Genus Pseudomonas , Genus Klebsiella , Genus Propionibacterium , Genus Rhodopseudomonas genus Rhodopseudomonas ) And Recombinant microorganism having a vitamin B-12 producing ability, characterized in that any one microorganism selected from the group consisting of genus Rhizobium . The method of claim 3, wherein the microorganisms having the ability to produce vitamin B-12 are Pseudomonas denitrificans , Klebsiella pneumoniae , Propionibacterium shermanii , Rhodo Rhodopseudomonas protamicus and Recombinant microorganisms having a vitamin B-12 producing ability , characterized in that any one of the microorganisms selected from the group consisting of Rhizobium cobalaminogenum . The recombinant microorganism having the vitamin B-12 producing ability according to claim 4, wherein the microorganism having the vitamin B-12 producing ability is Pseudomonas denitrificans (Accession No. KCCM 2026). The method of claim 1 or claim 2, wherein the gene encoding the glycerol dehydratase is recombinant, having the ability to produce vitamin B-12, characterized in that derived from genomic DNA of Klebsiella pneumoniae DSM 2026 microbe. The method of claim 1 or 2, wherein the gene encoding the glycerol dehydratase reactivase is Klebsiella pneumoniae ( Klebsiella pneumoniae ) A recombinant microorganism having vitamin B-12 producing ability, which is derived from genomic DNA of DSM 2026. The method according to claim 2, wherein the aldehyde D gene encoding a hydro centipede rise is Escherichia coli (Escherichia coli) K-12 The aldH gene, azo RY rilrum derived from Bra chamber lens (Azospirillum brasilense) when the kgsadh-I gene and keulrep derived from Elle pneumoniae ( Klebsiella pneumoniae ) Recombinant microorganisms having a vitamin B-12 production capacity , characterized in that selected from the group consisting of puuC gene derived from genomic DNA of DSM 2026. The recombinant microorganism of claim 1 or 2, wherein the recombinant microorganism is Pseudomonas denitrificans pPBG1 (GSB0167, Accession No. KCTC 11672BP). Culturing the recombinant microorganism of any one of claims 1 to 9 in a culture medium containing glucose and glycerol as a carbon source; And
Producing 3-hydroxypropionic acid from the culture solution
Method for producing 3-hydroxypropionic acid, characterized in that comprises a. The method for preparing 3-hydroxypropionic acid according to claim 10, wherein the culturing is performed under aerobic conditions. The method for producing 3-hydroxypropionic acid according to claim 10 or 11, wherein vitamin B-12 is added in the step of producing 3-hydroxypropionic acid from the glycerol.

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