KR20120041827A - Recombinant microorganism transformed with puuc gene encoding 3-hydroxypropionaldehyde dehydrogenase and method of preparing 3-hydroxypropionic acid or co-preparing 1,3-propanediol and 3-hydroxypropionic acid therewith - Google Patents

Abstract

PURPOSE: A recombinant microorganism is provided to produce 1,3-pripandiol and 3-hydroxypropionic acid without vitamin B-12. CONSTITUTION: A recombinant microorganism produces 3-hydroxypropionic acid or 3-hydroxypropionic acid and 1,3-propandiol simultaneously by transducing a recombinant vector containing puuC gene encoding aldehyde dehydrogenase. The recombinant vector additionally contains dhaB gene encoding glycerol dehydratase and gdrAB gene encoding glycerol dehydratase reactivase. The puuC gene encoding aldehyde dehydrogenase is derived from the genomic DNA Klebsiella pneumoniae DSM 2026.

Description

Recombinant microorganism transformed with puuC gene coding 3-hydroxypropionaldehyde dehydrogenase and method of preparing 3-hydroxypropionic acid or co-preparing 1,3-propanediol and 3-hydroxypropionic acid therewith}

The present invention relates to a recombinant microorganism transduced with the puuC gene encoding aldehyde dehydrogenase and a method for preparing 3-hydroxypropionic acid alone or 1,3-propanediol and 3-hydroxypropionic acid using the same. It is about.

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

Glycerol dehydratase, the first enzyme required for the process of Scheme 1, is well known in Klebsiella pneumoniae, which has an excellent ability to produce 1,3-propanediol (1,3-PDO) from glycerol (Mu Y et al., Biotechnol.Letters.28 : 1755-1759 (2006) This enzyme requires vitamin B-12, a coenzyme for the reaction.

However, enzymes with high specificity for 3-HPA have not been found so far. 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).

But Klebsiella Newmonia As a result, aldehyde dehydrogenase having high activity against 3-HPA has not been reported yet. If Klebsiella Newmonia Klebsiella pneumoniae with high DhaB activity, if appropriate aldehyde dehydrogenase derived from By simply overexpressing this enzyme in the cell, 3-HP production is possible.

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, Klebsiella pneumoniae dehydratase derived from Klebsiella pneumoniae that dehydrates glycerol to produce 3-HPA, necessarily requires vitamin B-12, and E. coli has the ability to produce this vitamin B-12. This is because they do not have their own.

The solution to the expensive vitamin B-12 problem is to use a strain that has the ability to produce vitamin B-12 on its own in addition to E. coli. To date, microorganisms known to produce vitamin B-12 by themselves are Pseudomonas dinitripicans , Klebsiella pneumoniae . However, Pseudomonas Dinitripicans has no ability to metabolize glycerol and genetic engineering techniques are not well developed. Klebsiella pneumoniae, on the other hand, has the ability to produce vitamin B-12 on its own and has a high activity of glycerol dehydratase, but dehydrogenase that produces 3-HP by specifically acting on 3-HPA. Is not yet known.

Therefore, the present inventors have tried to produce 3-HP from glycerol without supplying vitamin B-12 from the outside through Klebsiella pneumoniae, a microorganism having excellent glycerol metabolism ability and capable of producing vitamin B-12 by itself. , Klebsiella New Monica Find and characterize puuC , a gene that encodes an enzyme that has excellent ability to convert 3-HPA into 3-HP in high efficiency, and is characterized by Klebsiella pneumoniae Was overexpressed within. In addition, by deleting the DhaT gene, which reduces the intermediate product 3-HPA to 1,3-PDO, in Klebsiella pneumoniae, it is possible to produce 3-HPA alone or 3-HPA and 1,3-PDO simultaneously from glycerol. It was possible to produce a strain that can be. Furthermore, the present invention was completed by confirming that the puuC gene can be produced in high efficiency by expressing the puuC gene instead of aldH or kgsadh -I in recombinant E. coli having DhaB and GdrAB activities.

An object of the present invention is the gene necessary for transformation of various bacteria to produce 1,3-PDO from glycerol alone or simultaneously with 3-HP, namely 3-hydroxypropion aldehyde dehydrogenase It is to provide a recombinant microorganism transformed with a recombinant vector containing a puuC gene encoding.

 Further, another object of the present invention is to include puuC gene encoding 3-hydroxypropion aldehyde dehydrogenase, and additionally, a gene encoding glycerol dehydratase and a gene encoding glycerol dehydratase reactivation enzyme. It is to provide a recombinant microorganism transformed with a recombinant vector consisting of.

In addition, another object of the present invention is to use a recombinant microorganism to produce 3-HP alone or 3-HP and 1,3-PDO simultaneously from glycerol without the use of an expensive substrate, vitamin B-12. To provide.

In order to achieve the above object, the present invention is a 3-hydroxypropionic acid alone, or 3-hydroxypropionic acid and 1, from glycerol by transducing a recombinant vector consisting of puuC gene encoding aldehyde dehydrogenase (aldehyde dehydrogenase) It provides a recombinant microorganism characterized in that it simultaneously produces 3-propanediol.

The recombinant vector may further include a dhaB gene encoding glycerol dehydratase and a gdrAB gene encoding glycerol dehydratase reactivase.

The microorganism used as a host in the present invention can be used as any microorganism with or without vitamin B-12 production ability, for example, genus Escherichia , genus Klebsiella , Pseudomonas genus ( genus Pseudomonas ), genus Propionibacterium , genus Rhodopseudomonas , and It may be any one microorganism selected from the group consisting of genus Rhizobium .

More specifically, the microorganism is Escherichia coli BL21, Klebsiella pneumoniae , Klebsiella pneumoniae △ dhaT (Klebsiella pneumoniae △ dhaT ), Pseudomonas dinitripicans ( Pseudomonas denitrificans ), Propionibacterium shermanii , Rhodopseudomonas protamicus and It may be any one microorganism selected from the group consisting of Rhizobium cobalaminogenum .

The puuC gene encoding the aldehyde dehydrogenase, the dhaB gene encoding the glycerol dehydratase and the gdrAB gene encoding the glycerol dehydratase reactivase are respectively Klebsiella pneumoniae Can be derived from the genomic DNA of DSM 2026.

In addition, the recombinant microorganism is Escherichia coli / pCDFDuet_dhaB_gdrAB_pQE80L_puuC; GSB0181 (KCTC 11674BP), Klebsiella pneumoniae / pUC19_puuC; GSB0098 (KCTC 11671BP) and Klebsiella pneumoniae ΔdhaT / pUC19_puuC; GSB0163 (KCTC 11673BP) can be any one selected from the group consisting of.

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) that consists of protein and GdrA gdrB gdrB protein gene coding for coding.

Aldehyde dehydrogenase is also an enzyme derived from Klebsiella pneumoniae , which converts 3-HPA to 3-HP and is encoded by the PuuC gene (KPN_01018). It is composed of protein.

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

In addition, the present invention comprises the steps of culturing the recombinant microorganism in a culture medium containing glycerol as a carbon source; And it provides a method for the simultaneous production of 3-HP and 1,3-PDO comprising the step of producing 3-HP and 1,3-PDO from the culture at the same time.

Whether the recombinant microorganism according to the present invention is used as a host and glycerol is used as a carbon source to produce 3-HP alone or simultaneously to produce 3-HP and 1,3-PDO depends on the oxygen supply in the culture step. Can be adjusted.

That is, when the culture step is carried out under aerobic conditions, 3-HP can be produced alone, and 3-HP can be produced alone by deleting the dhaT gene which reduces 3-HPA to 1,3-PDO, whereas the culture If the step is carried out under anaerobic or partial aerobic conditions, it is possible to produce 3-HP and 1,3-PDO simultaneously. For example, oxygen concentration can be maintained at 20% or higher for 3-HP production alone, and oxygen concentration at 20% or lower for simultaneous production of 3-HP and 1,3-PDO.

Vitamin B-12 may be added from the glycerol alone in the 3-HP production step or in the simultaneous production of 3-HP and 1,3-PDO.

In addition, the process of culturing the recombinant microorganism and 3-HP and 1,3-PDO can be carried out using a culture method and a separate purification method known in the conventional fermentation industry.

Recombinant microorganism according to the present invention can efficiently produce 3-HP or 1,3-PDO from low-cost glycerol without the addition of expensive vitamin B-12, the desired metabolite such as 3-HP or 1,3-PDO It can be very useful as a strain producing 3-HP or 1,3-PDO while reducing the production cost of.

1 is a cleavage map of the recombinant vector pUC19_puuC.
2 is a batch fermentation result carried out under aerobic conditions of Escherichia coli BL21 / pCDFDuet_dhaB_gdrAB_pQE80L_puuC.
3A to 3C are batch fermentation results of Klebsiella pneumoniae / pUC19_puuC (a: anaerobic conditions, b: partial aerobic conditions, c: aerobic conditions).
4A to 4D show the results of batch fermentation (a, b, c) and fed-batch fermentation (d) of Klebsiella pneumoniae ΔdhaT / pUC19_puuC (a: anaerobic conditions, b: partial aerobic conditions, c: aerobic conditions, d: Incubated under aerobic conditions and then fermented under partial aerobic conditions).

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, the following examples disclose a method of producing 3-HP or 1,3-PDO from glycerol using Klebsiella pneumoniae and Escherichia coli as host cells. Other microorganisms with the ability to produce vitamin B-12, such as Pseudomonas denitrificans , 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. 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: 1) and a reverse primer (SEQ ID NO: 2) to dhaB1, dhaB2, dhaB3 and dhaB4 (gdrA). ) Genes were amplified and digested with restriction enzymes EcoRI and HindIII.

1-2. 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: 3) and a reverse primer (SEQ ID NO: 4) to amplify the gdrB gene and digested with restriction enzymes HindIII and AfIII. .

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

Genomic DNA from the Klebsiella pneumoniae DSM 2026 strain was subjected to PCR using a forward primer (SEQ ID NO: 5) and a reverse primer (SEQ ID NO: 6) to amplify the puuC gene and to carry out the restriction enzyme BamHI. Cleavage with HindIII.

1-4. Klebsiella New Monica from DSM 2026 puuC Gene amplification and lac promoter gene amplification from the pUC19 vector

Overlapping PCR was performed to amplify the puuC gene, including the lac promoter gene.

The lac promoter gene was amplified by PCR using a forward primer (SEQ ID NO: 7) and a reverse primer (SEQ ID NO: 8) containing a part of the puuC gene from the pUC19 vector (New england biolabs # 3041L).

The genomic DNA was then PCR from the Klebsiella pneumoniae DSM 2026 strain using a forward primer (SEQ ID NO: 9) and a reverse primer containing the HindIII site (SEQ ID NO: 10) containing the 3 ′ prime portion of the lac promoter gene. PuuC gene was amplified.

PCR was performed using the amplified two PCR product lac promoter gene and puuC gene using a forward primer (SEQ ID NO: 7) and a reverse primer (SEQ ID NO: 10) including a HindIII site.

Thereafter, phospho nucleokinase was treated to add a phosphate group at the 5 ′ prime end of the PCR product.

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

PCR of the kanamycin resistance gene from the pKD4 vector (Kirill et., Proceedings of the National Academy of Sciences, 97, (2000), 12) using a forward primer (SEQ ID NO: 11) and a reverse primer (SEQ ID NO: 12) The kanamycin resistance gene was amplified and digested with restriction enzymes HindIII and NdeI.

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

2-1. Recombinant vector pLB0024 (pCDFDuet_ dhaB_gdrAB ) Production

DhaB ( dhaB1, dhaB2, dhaB3, dhaB4 ( gdrA )) and gdrB PCR fragments amplified in Examples 1-1 and 1-2 were respectively conjugated to the pGEM-T vector. And the prepared plasmids were introduced into E. coli XL1-blue, respectively.

pLB0006 (pGEM- T_dhaB ) plasmid was digested with restriction enzymes EcoRI and HindIII, and pLB0023 (pGEM-T_ gdrB ) plasmid was digested with restriction enzymes HindIII and AfIII.

The plasmid prepared by conjugating two DNA fragments was cloned into pCDFDuet vector, an expression vector, to obtain recombinant plasmid pLB0024.

2-2. Construction of Recombinant Vector pLB0037 (pQE80L_puuC)

The puuC gene PCR fragment amplified in Example 1-3 was conjugated to the pGEM-T vector. And the produced plasmid was introduced into E. coli XL1-blue.

pLB0107 (pGEM-T_puuC) plasmid was digested with BamHI and HindIII restriction enzymes and cloned into pQE80L vector, a recombinant expression vector, to obtain recombinant plasmid pLB0037.

2-3. Construction of Recombinant Vector pLB0066 (pUC19_puuC)

The pucC PCR fragment containing the lac promoter amplified in Examples 1-4 was conjugated to the pUC19 vector. And the produced plasmid was introduced into E. coli DH5α.

The plasmid was digested with HindIII and NdeI restriction enzymes and conjugated with the kanamycin cassette amplified in Example 1-5.

Thereafter, the produced plasmid was cloned into pUC19 vector, which is a recombinant expression vector, to obtain recombinant plasmid pLB0066 (see FIG. 1).

Example 3. Klebsiella pneumoniae DdhaT  Mutantism

      Overlapping PCR was performed to knock out the dhaT gene from Klebsiella pneumoniae DSM 2026 strain genomic DNA.

      PCR was performed using a forward primer (SEQ ID NO: 13) containing a BamHI site from the dhaT gene upstream and a reverse primer (SEQ ID NO: 14) containing a 5 ′ prime portion of the dhaT gene downstream from genomic DNA.

Next, PCR was performed using a forward primer (SEQ ID NO: 15) containing a 3 ′ prime portion of dhaT Upstream and a reverse primer (SEQ ID NO: 16) containing a NotI site from the dhaT gene Downstream.

The two fragments amplified above were subjected to PCR using a forward primer (SEQ ID NO: 13) and a reverse primer (SEQ ID NO: 16) to obtain gene fragments in the upstream and downstream directions of the dhaT gene except for the dhaT gene.

Fragments of both fragments of the dhaT gene were cleaved with BamHI and NotI restriction enzymes and re-conjugated to the pGEMT-vector, followed by electroporation at 2.5 kV, 25 μF, and 400 ohms to introduce into E. coli _DH5α. It was then plated on X-gal / IPTG empicillin solid medium to confirm conjugation and then conjugated to pKOV vector (AJ, Phillips et., J. Bacteriology (1997) 179: 6228-6237) useful for knock-out.

Then, the conjugated vector was introduced into the Klebsiella pneumoniae DSM 2026 strain by electroporation, and 1 mL of LB liquid medium was added thereto, followed by incubation for 1 hour in a 200 rpm, 30 ° C. shaking incubator.

During incubation, fragments of pKOV vectors complementary to both genes, including the dhaT of Klebsiella pneumoniae DSM 2026 strain genomic DNA, were replaced. Thereafter, chloramphenicol (25 μg / ml) LB solid medium was incubated at 42 ° C. for 24 hours. This is because the pKOV vector is not replicated by the pSC101 gene at 42 ° C.

Colonies of the cultured solid medium were inoculated in 200 uL LB liquid medium and immediately stepped into chloramphenicol (25 μg / ml) LB solid medium and 5% sucrose LB solid medium, respectively.

Thereafter, 16 hours of incubation at 42 ° C. resulted in colonies cultured only in chloramphenicol (25 μg / ml) LB solid medium without incubation in 5% sucrose LB solid medium. PCR was performed using SEQ ID NO: 18).

Selected colonies equal in length to both fragments of the dhaT gene conjugated to the pKOV vector were selected for chloramphenicol (25 μg / ml) LB solid medium and 5% sucrose LB solid medium, respectively, and then chloramphenicol (25 μg / ml) LB solid medium. Klebsiella pneumoniae DdhaT was prepared by selecting colonies which were cultured in E. coli but not in 5% sucrose LB solid medium, and were named as GSB0162 strains.

Example 4. Preparation of recombinant strain for the production of 3-hydroxypropionic acid

4-1. GSB0181 ( Escherichia coli Preparation of strain BL21 / pLB0011_pLB0037) E.Coli BL21 / pCDFDuet_dhaB_gdrAB_pQE80L_puuC)

E. coli BL21 was plated on LB solid medium and incubated at 37 ° C. for 14 hours, and colonies were incubated in 10 mL of LB liquid medium for 12 hours. Fully grown culture was inoculated 1% in 100 mL LB liquid medium and incubated in a 200 rpm, 37 ℃ shake incubator. After 4 to 5 hours, when the OD600 was about 0.3 to 0.4, 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 the cells were obtained by centrifugation at 4 ℃, 4,500 rpm, 20 minutes. The glycerol solution used for resuspension was cut in half and run twice in succession, and then the cell concentrate was resuspended so that the volume ratio of the cells and the glycerol solution was 1: 1. By mixing the obtained cell concentrate and pLB0024 and pLB0037 prepared in Examples 2-1 and 2-2, and then performing the electroporation under the conditions of 2.5 kV, 25 μF, 400 ohms, the expression The vector was introduced into cultured E. coli BL21.

After the electric shock, 1 mL of LB liquid medium was added and incubated for 1 hour in a 200 rpm, 37 ℃ shake incubator. The culture solution was plated in LB solid medium containing the antibiotic ampicillin (final concentration 100 µg / L) and striptomycin (final concentration 50 µg / L) and incubated for 12 hours at 37 ° C. The colonies formed were incubated for more than 12 hours in LB liquid medium containing ampicillin and strimycin antibiotics, and E. coli BL21 / pLB0024_pLB0037 was prepared and named as GSB0181 strains. The deposit was made on March 26 with accession number KCTC 11674BP.

4-2. GSB0098 ( Klebsiella pneumoniae / pLB0066) Construction of Strains ( K.pneumoniae / pUC19_lac_promoter_puuC_HindⅢ_Kana_NdeI)

Klebsiella pneumoniae was plated in LB solid medium and incubated for 14 hours at 37 ℃, colonies were incubated with 10 mL of LB liquid medium containing 0.7 mM EDTA and incubated for 12 hours. The fully grown culture was inoculated 1% in 100 mL of LB liquid medium containing 0.7 mM EDTA and incubated in a 200 rpm, 37 ℃ shake incubator. After 4 to 5 hours, when the OD600 was about 0.3 to 0.4, 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 the cells were obtained by centrifugation at 4 ℃, 4,500 rpm, 20 minutes. The glycerol solution used for resuspension was cut in half and run twice in succession, and then the cell concentrate was resuspended so that the volume ratio of the cells and the glycerol solution was 1: 1. After mixing the obtained cell concentrate and the pLB0066 vector prepared in Example 2-3, the expression vector was cultured by performing electroporation at 2.5 kV, 25 μF, 400 ohms. Introduced in Ciella Pneumoniae.

After the electric shock, 1 mL of LB liquid medium was added and incubated for 1 hour in a 200 rpm, 37 ℃ shake incubator. The culture solution was plated in LB solid medium containing the antibiotic ampicillin (final concentration 100 µg / L) and kanamycin (final concentration 50 µg / L) and incubated at 37 ° C for at least 12 hours. The colonies formed were incubated in LB liquid medium containing ampicillin and kanamycin antibiotics for 12 hours or longer to prepare Klebsiella pneumoniae / pLB0066. Deposited with KCTC 11671BP.

4-3. GSB0163 ( Klebsiella pneumoniae _ △ dhaT / pLB0066) Preparation of strain K.pneumoniae △ dhaT / puuC_HindⅢ_Kana_NdeI)

Example 4-2 by transfection methods and pLB0066 recombinant vector of Example 2-3 in the same manner as described in the Klebsiella pneumoniae dhaT _ △ strain were prepared the Klebsiella pneumoniae dhaT _ △ / pLB0066, this strain, named GSB0163 It was deposited with KCTC 11673BP on March 26, 2010 to the Korea Institute of Bioscience and Biotechnology, Gene Resource Bank.

Example 5 Determination of Glycerol Dehydratase and Aldehyde Dehydrogenase Activity

The recombinant strain prepared in Example 4 was inoculated in LB liquid medium and cultured at 37 ° C. When OD ₆₀₀ = 2.5 of the cultured cells, the precipitated cells were washed twice with potassium phosphate buffer solution (50 mM, pH 7.0) after centrifugation, and then suspended in the same buffer to serve as 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.

5-1. Determination of Glycerol Dehydratase Activity

The activity of glycerol dehydratase in cell extracts was measured by HPLC (HPLC, Agilent 1100 series; CA, USA). Appropriate amount of cell extract was added to potassium phosphate reaction solution (50 mM, pH 8.0) containing 35 mM potassium chloride, preincubated at 37 ° C. for 5 minutes, and then 50 mM glycerol and 15 μM of vitamin B-12 were added. The reaction was added for 1 minute with the final volume of 0.5 ml.

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 BL21 / pCDFDuet_dhaB_gdrAB_pQE80L_puuC 1.07 ± 0.3 Klebsiella pneumoniae (Wild type) 6.79 ± 0.5 Klebsiella pneumoniae / pUC19_puuC 20.8 ± 1.2 Klebsiella pneumoniae △ dhaT 8.7 ± 0.6 Klebsiella pneumoniae △ dhaT / pUC19_puuC 10.2 ± 0.9

As a result, as shown in Table 1, the glycerol dehydratase activity of the recombinant strain overexpressed by introducing pCDFDuet_dhaB_gdrAB_pQE80L_puuC into E. coli BL21 was 1.07 ± 0.3 μmol / mg, and 6.79 ± 0.5 for Klebsiella pneumoniae (Wild type). μmol / mg is indicated.

Recombinant strains incorporating pUC19_puuC into Klebsiella pneumoniae (wild type) had 20.8 ± 1.2 μmol / mg and strains from which dhaT gene was removed from Klebsiella pneumoniae (wild type) had 8.7 ± 0.6 μmol / mg activity in Klebsiella pneumoniae (wild type). Recombinant strain in which pUC19_puuC was introduced into the removed strain exhibited glycerol dehydratase activity of 10.2 ± 0.9 μmol / mg.

5-2. 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 solution (50 mM, pH 7.0 or 8.0), pre-incubated at 45 ° C. for 5 minutes, and then 1.8 mM aldehyde and 0.76 mM NAD (P) ⁺ were added to a final volume of 0.5. The reaction was initiated to ml.

The activity of the enzyme was determined by measuring the rate at which NAD (P) ⁺ is reduced 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.

Enzyme source ^a Total active
(μmolNADH / ml / min) Protein (mg / ml) Specificity
(U / mg) refine
(fold) Cell extract 19.374 ± 1.55 6.19 ± 0.49 3.13 ± 0.25 1.00 Tablet PuuC 264.26 ± 21.14 9.53 ± 0.76 27.73 ± 2.21 8.86

^a No activity on E. coli BL21 host cells.

Substrate specificity of the aldehyde dehydrogenase was measured at 45 ° C., pH 8.0 using various types of aldehydes in the presence of coenzyme NAD ⁺ or NADP ⁺ . The aldehydes used in the present invention are as follows: formaldehyde (HCHO, MW 30.03), acetaldehyde (CH ₃ CHO, MW 44.05), 3-hydroxypropionaldehyde (CH ₂ (OH) CH ₂ CHO, MW 74.2) , Propionaldehyde (CH ₃ CH ₂ CHO, MW 58.09), butylaldehyde (CH ₃ (CH ₂ ) ₂ CHO, MW 72.11), aldehyde (CH ₃ (CH ₂ ) ₃ CHO, MW 86.13), isoaldehyde ( (CH ₃ ) ₂ CHCH ₂ CHO, MW 86.13), 2-puraldehyde (C ₄ H ₃ OCHO, MW 96.1), benzaldehyde (C ₆ H ₅ CHO, MW 106.13).

Substrate specificity of the aldehyde dehydrogenase according to the present invention, as shown in Table 3, most of the aldehydes showed higher specificity when using NAD ⁺ than coenzyme NADP ⁺ and aldehydes, butyl aldehyde, isovalaldehyde, 3 High specificity was obtained when coenzyme NAD ⁺ was used in the order of -hydroxypropion aldehyde. In particular, 3-hydroxypropion aldehyde dropped about 90% in activity when using NADP ⁺ . It may be said that Klapsiella pneumoniae PuuC has high specificity for NAD ⁺ .

Quality Specificity (U / mg) NAD ⁺ NADP ⁺ 3-hydroxypropionaldehyde 22.75 ± 1.34 2.37 ± 0.19 Formaldehyde (C1) 0.55 ± 0.02 1.75 ± 0.14 Acetaldehyde (C2) 16.84 ± 1.16 1.97 ± 0.17 Propionaldehyde (C3) 18.53 ± 1.59 1.89 ± 0.15 Butylaldehyde (C4) 31.92 ± 2.31 2.02 ± 0.16 Valaldehyde (C5) 33.58 ± 2.66 1.99 ± 0.16 Isobalaldehyde 27.32 ± 1.74 1.81 ± 0.12 2-puraldehyde 5.19 ± 0.97 1.45 ± 0.11 Benzaldehyde 7.24 ± 1.05 1.61 ± 0.14

Kinetic properties of the purified aldehyde dihydrogenase were investigated for oxidation of 3-hydroxypropion aldehyde as shown in Table 4.

It was 34.08 Umg ^-1 protein is the maximum reaction rate of the oxidation-butyl aldehyde (V _max), the maximum reaction rate (V _max) of 3-hydroxy-propionaldehyde oxidation was 22.25 Umg ^-1 protein.

The catalytic efficiency (k _cat / K _m ) of 3-hydroxypropionaldehyde and butylaldehyde oxidation for NAD ⁺ was 41.44 X 10 ³ M ^-1 s ^-1 and 117.15 X 10 ³ M ^-1 s ^-1 .

Popular vagina ^a Coenzyme V _max
(Umg ^-1 protein) K _m
(mM) k _cat
(s ^-1 ) k _cat / K _m
× 10 ³ (M ^-1 s ^-1 ) 3-hydroxypropionaldehyde NAD ⁺ 22.25 ± 1.78 0.48 ± 0.04 19.81 ± 1.58 41.44 ± 3.32 Butylaldehyde NAD ⁺ 34.08 ± 2.73 0.26 ± 0.02 30.34 ± 2.43 117.15 ± 9.37 Valaldehyde NAD ⁺ 33.26 ± 2.66 0.14 ± 0.01 29.61 ± 2.37 207.07 ± 16.6 Isobal
Aldehyde NAD ⁺ 28.61 ± 2.29 0.21 ± 0.02 25.47 ± 2.04 121.87 ± 9.75 3-hydroxypropionaldehyde ^b NAD ⁺ 25.46 ± 2.04 0.09 ± 0.01 22.67 ± 1.81 254.69 ± 20.40

^a substrate varies between 0.1 mM and 2 mM

^b 3-HPA varies between 1.8 mM and NAD ⁺ varies between 0.025 mM and 0.8 mM

Example 6. Production of 3-hydroxypropionic acid in transformed GSB0181 culture

The recombinant strain E. coli BL21 / pCDFDuet_dhaB_gdrAB_pQE80L_puuC prepared in Example 4-1 was preinoculated in 10 mL of M9 liquid medium and preincubated at 37 ° C for 12 hours. Fully grown cultures were cultured in M9 liquid medium (pH 7.0) to which glycerol 100 mM and yeast extract 0.2 g / L were added.

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 200 rpm at 37 ℃ using a 250 mM Erlenmeyer flask containing 50 mL of medium.

Thereafter, when OD ₆₀₀ reached 0.6, 100 uM of IPTG was added, and 4 μM of vitamin B-12 was added over 5 times at 2 hour intervals and incubated for 30 hours.

As a result, a maximum of 52.77 mmol / L of 3-HP was obtained in a 30 hour incubation as shown in FIG. 2. In addition, the ratio of 3-HP produced relative to the glycerol used was about 0.62 (yield 62%) and the entire culture was under aerobic conditions.

These results show that PuuC has high aldehyde dehydrogenase activity against 3-HPA, and its activity is well expressed in the produced GSB0181 recombinant strain.

Example 7 Simultaneous Production of 3-Hydroxypropionic Acid or 3-Hydroxypropionic Acid and 1,3-propanediol in Transformed GSB0098 and GSB0163 Cultures

The recombinant strains prepared in Examples 4-2 and 4-3 were preinoculated in 10 mL of LB liquid medium and preincubated at 37 ° C. for 12 hours. Fully grown cultures were cultured in M9 liquid medium (pH 7.0) to which glycerol 100 mM and yeast extract 0.2 g / L were added.

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 200 rpm (aerobic), 30 rpm (partly aerobic), 100 rpm (anaerobic) at a temperature of 37 ° C. using a 250 mM Erlenmeyer flask containing 50 mL of medium. In the case of aerobic and partly aerobic, the outside air is continuously transferred into the flask during the incubation period using a sponge stopper. In contrast, in the case of anaerobic use, the stopper is used to completely insulate the alien air from the flask using a screw stopper. It was.

Thereafter, when OD ₆₀₀ reached 0.6, 1 mM of IPTG was added and the total incubation time was 33 hours.

7-1. GSB0098 ( Klebsiella pneumoniae / pUC19_puuC) alone to produce 3-HP or simultaneous production of 3-HP and 1,3-PDO

Figure 3a is a batch fermentation of anaerobic Klebsiella pneumoniae / pUC19_puuC, 1,3-PDO was able to obtain a maximum of 29.07 ± 3.1 mmol / L, 3-HP was 1.86 ± 0.2 mmol / L. Figure 3b is a batch fermentation of Klebsiella pneumoniae / pUC19_puuC cultured in aerobic partially, 1,3-PDO was obtained 21.61 ± 2.5 mmol / L, 3-HP was 3.38 ± 0.4 mmol / L. That is, in both cases a large amount of 1,3-PDO was obtained and the production of 3-HP was insignificant. In anaerobic or partially aerobic state, the concentration of intracellular NADH is very high, indicating that most of 3-HPA produced by DhaB is converted to 1,3-PDO rather than 3-HP. Therefore, in this condition, the presence of PuuC in the recombinant strain Klebsiella pneumoniae / pUC19_puuC has a very limited effect on the total glycerol metabolism.

FIG. 3c shows a batch fermentation of aerobic cultured Klebsiella pneumoniae / pUC19_puuC, yielding 5.1 ± 0.2 mmol / L of 1,3-PDO and 4.5 ± 0.3 mmol / L of 3-HP. Under the same conditions, Klebsiella pneumoniae (WT) without PuuC cloning produced 5.4 ± 0.4 mmol / L of 1,3-PDO and 2.4 ± 0.2 mmol / L of 3-HP. Compared with anaerobic or partially aerobic, 1,3-PDO production was reduced compared to 3-HP production. However, the yield of 3-HP is very low compared to the glycerol used, which means that under aerobic conditions, most of the glycerol is used for cell growth via the glycerol kanase pathway rather than the glycerol dehydratase (DhaB) pathway ( Appl. Microbiol. Biotechnol. 50: 24-29, 1998).

The recombinant strain Klebsiella pneumoniae / pUC19_puuC overexpressing puuC in Klebsiella pneumoniae was able to produce 1,3-PDO and 3-HP simultaneously under all conditions. In addition, the ratio or amount changed depending on the amount of oxygen supplied. Although yields were not satisfactory, especially in the yield of 3-HP, it was demonstrated that Klebsiella pneumoniae / pUC19_puuC could simultaneously produce 1,3-PDO and 3-HP without the addition of vitamin B-12. This was judged by the action of the aldehyde dehydrogenase enzyme encoded by PuuC introduced into the plasmid.

7-2. GSB0163 ( Klebsiella pneumoniae 3-HP alone production with ΔDhaT / pUC19_puuC) or simultaneous production of 3-HP and 1,3-PDO

Klebsiella pneumoniae GSB0098, ie, Klebsiella pneumoniae / pUC19_puuC, produced less than 3-HP when compared to 1,3-PDO, due to excessively high activity of glyceraldehyde oxidoreductase (DhaT). It was estimated.

Therefore, GSB0163, ie, Klebsiella pneumoniae ΔDhaT / pUC19_puuC, was prepared to remove dhaT and lower its activity.

4A shows the results of batch fermentation of anaerobic Klebsiella pneumoniae ΔDhaT / pUC19_puuC, where 3-HP was 27.19 ± 2.9 mmol / L and 1,3-PDO was 23.72 ± 2.1 mmol / L.

On the other hand, Klebsiella pneumoniae ΔDhaT without overexpressing PuuC was able to obtain 1.9 ± 0.2 mmol / L of 3-HP and 17.61 ± 0.8 mmol / L of 1,3-PDO.

It was confirmed that 3-HP increased about 15 times and 1,3-PDO increased about 1.3 times due to the action of the aldehyde dehydrogenase enzyme introduced into the plasmid without adding any vitamin B-12.

FIG. 4B shows the results of batch fermentation of Klebsiella pneumoniae ΔDhaT / pUC19_puuC cultured in aerobic partially to obtain 17.62 ± 1.5 mmol / L of 3-HP and 18.44 ± 1.8 mmol / L of 1,3-PDO. In comparison, 3-HP production of Klebsiella pneumoniae ΔDhaT under the same conditions was 2.58 ± 0.4 mmol / L, and 1,3-PDO showed 22.83 ± 2.0 mmol / L.

4C shows the results of batch fermentation of aerobic cultured Klebsiella pneumoniae ΔDhaT / pUC19_puuC, yielding 7.17 ± 0.9 mmol / L of 3-HP and 4.12 ± 0.7 mmol / L of 1,3-PDO.

Figure 4d shows the result of fed-batch fermentation of Klebsiella pneumoniae ΔDhaT / pUC19_puuC fermented partially aerobic after incubation to 1.0 O.D600 in aerobic, 177.6 mM 3-HP and 220.8 mM 1,3-PDO through 24 hours fermentation Could get

At this time, a 5 L bioreactor was used, the working volume was 1 L, the initial glycerol concentration was 100 mM and the pH was maintained at 7.0. The air inflow rate was 1.0 VVM for cell growth under initial aerobic conditions and 0.1 VVM for partial aerobic maintenance after 1 mM IPTG induction. Product production yield (sum of 3-HP and 1,3-PDO) relative to glycerol consumed for 24 hours was 51%.

Thus, the recombinant strains prepared and cultured above produced up to 177.6 mM 3-HP and 220.8 mM 1,3-PDO in fed-batch fermentation without addition of vitamin B-12.

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 KCTC11671BP, KCTC11673BP, KCTC11674BP 20100326

<110> Institute for research and industry cooperation, PNU <120> Recombinant microorganism transformed with puuC gene encoding          3-hydroxypropionaldehyde dehydrogenase and method of preparing          3-hydroxypropionic acid or co-preparing 1,3-propanediol and          3-hydroxypropionic acid therewith <130> DP-2010-0205 <160> 18 <170> KopatentIn 1.71 <210> 1 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for dhaB gene <400> 1 gaattcatga aaagatcaaa acgatttgca gtact 35 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for dhaB gene <400> 2 aagcttgatc tcccactgac caaagctgg 29 <210> 3 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for gdrB gene <400> 3 aagcttgagg ggaccgtcat gtcgctttca ccgccag 37 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for gdrB gene <400> 4 cttaagtcag tttctctcac ttaacggc 28 <210> 5 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for puuC gene <400> 5 aagcttgagg ggaccgtcat gtcgctttca ccgccag 37 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for puuC gene <400> 6 cttaagtcag tttctctcac ttaacggc 28 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for lac promoter gene <400> 7 ctggcacgac aggtttcccg actg 24 <210> 8 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for lac promoter gene <400> 8 ggtgctgaaa attcatagct gtttcctgtg tg 32 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for puuC gene <400> 9 cacacaggaa acagctatga attttcagca cc 32 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for puuC gene <400> 10 aagctttcaa gactccaggg caatccagat 30 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for kanamycin resistant gene <400> 11 gataaaagct ttcacgctgc cgcaagc 27 <210> 12 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for kanamycin resistant gene <400> 12 attccatatg ggggtgggcg aagaactcca gc 32 <210> 13 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for dhaT gene knock-out <400> 13 attggatccg gtgacaaaat aacgctgac 29 <210> 14 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for dhaT gene knock-out <400> 14 cgttgccttt acgcgggttt aagttaacgt ttggcacc 38 <210> 15 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for dhaT gene knock-out <400> 15 ggtgccaaac gttaacttaa acccgcgtaa aggcaacg 38 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for dhaT gene knock-out <400> 16 aattgcggcc gcgtagcaga cgcccagttg 30 <210> 17 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 17 gcacacgaaa tctaccgaca gtttcactat gaa 33 <210> 18 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 18 acgaactgtt ttaacgggcc gctctcggcc atattg 36

Claims (14)

Characterization of the production of 3-hydroxypropionic acid alone or 3-hydroxypropionic acid and 1,3-propanediol simultaneously from glycerol by transducing a recombinant vector consisting of the puuC gene encoding aldehyde dehydrogenase. Recombinant microorganisms. The method of claim 1, wherein the recombinant vector further comprises a dhaB gene encoding glycerol dehydratase and a gdrAB gene encoding glycerol dehydratase reactivase. Recombinant microorganisms. The method of claim 1 or 2, wherein the microorganism is genus Escherichia , genus Klebsiella , genus Pseudomonas , genus Propionibacterium , genus Propionibacterium , genus Rhodocdomonas . genus Rhodopseudomonas ) and Recombinant microorganism, characterized in that any one microorganism selected from the group consisting of genus Rhizobium . The method of claim 3, wherein the microorganism is Escherichia coli BL21, Klebsiella pneumoniae , Klebsiella pneumoniae △ dhaT (Klebsiella pneumoniae Δ dhaT ), Pseudomonas Dinitripicans ( Pseudomonas denitrificans ), Propionibacterium shermanii , Rhodopseudomonas protamicus and Recombination microorganism, characterized in that selected from the group consisting of Rhizobium cobalaminogenum ( Rhizobium cobalaminogenum ). The recombinant microorganism according to claim 1 or 2, wherein the puuC gene encoding the aldehyde dehydrogenase is derived from genomic DNA of Klebsiella pneumoniae DSM 2026. The recombinant microorganism of claim 2, wherein the dhaB gene encoding glycerol dehydratase is derived from genomic DNA of Klebsiella pneumoniae DSM 2026. The method of claim 2, wherein the gdrAB gene encoding the glycerol dehydratase reactivase is Klebsiella pneumoniae A recombinant microorganism, which is derived from genomic DNA of DSM 2026. The method according to claim 1 or 2, wherein the recombinant microorganism is Escherichia coli / pCDFDuet_dhaB_gdrAB_pQE80L_puuC; GSB0181 (KCTC 11674BP), Klebsiella pneumoniae / pUC19_puuC; GSB0098 (KCTC 11671BP) and Klebsiella pneumoniae ΔdhaT / pUC19_puuC; Recombinant microorganism, characterized in that selected from the group consisting of GSB0163 (KCTC 11673BP). Culturing the recombinant microorganism of any one of claims 1 to 8 in a culture medium containing 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 of claim 9, wherein the culturing is performed in aerobic conditions. The method for producing 3-hydroxypropionic acid according to claim 9 or 10, wherein vitamin B-12 is added in the step of producing 3-hydroxypropionic acid from the glycerol. Culturing the recombinant microorganism of any one of claims 1 to 8 in a culture medium containing glycerol as a carbon source; And
Simultaneously producing 3-hydroxypropionic acid and 1,3-propanediol from the culture solution
Simultaneous preparation method of 3-hydroxypropionic acid and 1,3-propanediol comprising a. The method of claim 12, wherein the culturing is performed under anaerobic or partial aerobic conditions. 13. The method of claim 12 or 13, wherein 3-hydroxypropionic acid and 1,3-, characterized in that the addition of vitamin B-12 in the step of simultaneously producing 3-hydroxypropionic acid and 1,3-propanediol from the glycerol Simultaneous preparation of propanediol.

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