KR101145405B1 - Mutant Blocked in Glycerol Oxidaion Pathway for Producing 1,3-Propanediol - Google Patents

Abstract

The present invention provides a microbial variant in which a gene encoding a transcriptional activator or a gene encoding a dihydroxyacetone kinase is deleted or inactivated in a microorganism having the ability to produce 1,3-propanediol using glycerol as a carbon source and the above variant. It relates to a method for producing 1,3-propanediol using.

Using the variant according to the present invention, when producing 1,3-propanediol from glycerol using a conventional strain, it is possible to produce only 1,3-propanediol with almost no inevitably produced by-products, the cost in the purification process Can reduce the cost.

Description

Mutant Blocked in Glycerol Oxidaion Pathway for Producing 1,3-Propanediol

The present invention provides a microbial variant in which a gene encoding a transcriptional activator or a gene encoding a dihydroxyacetone kinase is deleted or inactivated in a microorganism having the ability to produce 1,3-propanediol using glycerol as a carbon source and the above variant. It relates to a method for producing 1,3-propanediol using.

1,3-propanediol is a material that can be used as a synthetic raw material such as polyester, polyether, or polyurethanes. Fiber and plastic films such as high-performance clothing, carpets, and automobile fabrics It is used for various purposes such as. In particular, polytrimethylene terephtalate (PTT) produced by the polymerization reaction of 1,3-propanediol and terephtalic acid has excellent physical properties and has a lower point than polyethylene terephthalate (PET) at 228 ° C. It is attracting attention as a next-generation textile material that can replace PET in the future due to its substantial utility. In addition, plastics and polymers made of 1,3-propanediol as monomers have better optical stability than products made of butanediol and ethylene glycol. In addition, since 1,3-propanediol can be used as a polyglycol-type lubricant and solvent, its commercial value is higher than that of glycerol.

1,3-propanediol can be produced by chemical synthesis and microbial fermentation. Chemical production methods include converting ethylene oxide to 1,3-propanediol by hydroformylation (US Pat. No. 3,687,981) and converting acrolein to 1,3-propanediol by hydration. (US Pat. No. 5,015,789). However, since these chemical processes require high temperature or high pressure during the production of 1,3-propanediol, the production cost is high and there is a problem of generating waste oil containing environmental pollutants.

Bakteo (Citrobacter) in a flexible anaerobic strains sheet by biological methods, Clostridium (Clostridium), Enterobacter bakteo (Enterobacter), one Rio bakteo (Ilyobacter), keurep when Ella (Klebsiella), Lactobacillus bacteria (Lactobacillus), Tupelo bakteo ( Pelobacter ) and the like to produce 1,3-propanediol from glycerol using microorganisms (U.S. Patent No. 5,254,467), the genetic engineering method of the leap to amplify the glycerol dehydrogenase gene in Lactobacillus or 1, 3-propanediol may be produced using the knocked out variant, but the ability to produce 1,3-propanediol may be improved (US 2007/0148749), but using genetically engineered variants, There is no prior art on how to reduce by-products that accumulate large amounts in the production of 1,3-propanediol.

In the metabolic process of converting glycerol to 1,3-propanediol using the microorganism, various kinds of oxidative metabolites are produced in large quantities. In particular, 2,3-butanediol is a glycerol oxidative metabolite and has a similar boiling point to 1,3-propanediol, which acts as a big constraint in the purification process.

Accordingly, the present inventors have made efforts to develop microorganisms that produce only 1,3-propanediol without generating metabolic by-products including 2,3-butanediol in glycerol metabolism by metabolic methods. To block oxidative metabolic pathways that produce by-products in glycerol metabolic pathways, and to produce variants having only reduced metabolic pathways that produce 1,3-propanediol, and to produce 1,3-propanediol using the variants. When it was confirmed that no metabolism by-products such as 2,3-butanediol were produced, the present invention was completed.

It is an object of the present invention to provide a variant in which the glycerol oxidation pathway is blocked in a microorganism having the ability to produce 1,3-propanediol based on glycerol in order to produce 1,3-propanediol from glycerol without the production of by-products. It is.

Another object of the present invention is to provide a method for producing 1,3-propanediol, wherein the variant is cultured in a medium containing glycerol.

In order to achieve the above object, the present invention, in a microorganism having the ability to produce 1,3-propanediol using glycerol as a carbon source, a gene encoding a transcriptional activator or a gene encoding a dihydroxyacetone kinase is deleted or It provides an inactivated microbial variant.

The present invention also comprises the steps of culturing the microbial variant in a medium containing glycerol to produce 1,3-propanediol; And it provides a method for producing 1,3-propanediol comprising the step of recovering the produced 1,3-propanediol.

The invention also relates to the glycerol dehydrogenase gene (DhaD), transcriptional activator gene (DhaR), 1,3-propanediol oxidoreductase gene (DhaT) and glycerol dehydrolase reactivator II gene ( A vector containing a gene encoding a 1,3-propanediol oxidoreductase is introduced into a Krebssiella pneumoniae variant (AK strain) from which DhaBA2) is deleted, or a 1,3-propanediol oxydori It provides a variant having a 1,3-propanediol generating ability is inserted into the chromosome of the variant (AK strain) ductase gene.

The invention also relates to the glycerol dehydrogenase gene (DhaD), transcriptional activator gene (DhaR), 1,3-propanediol oxidoreductase gene (DhaT) and glycerol dehydrolase reactivator II gene ( Vector containing a gene encoding a 1,3-propanediol oxidoreductase and a gene encoding a glycerol dehydratase reactivator in a Krebsiella pneumoniae variant (AK strain) that has deleted DhaBA2) 1,3 wherein a gene encoding 1,3-propanediol oxidoreductase and a gene encoding a glycerol dehydratase reactivator are inserted into the chromosome of the variant (AK strain) It provides a variant having a propanediol generating ability.

The present invention also relates to Krebselanus, in which the transcriptional activator gene (DhaR), the 1,3-propanediol oxidoreductase gene (DhaT) and the glycerol dehydratase reactivator II gene (DhaBA2) are deleted. A vector containing a gene encoding 1,3-propanediol oxidoreductase is introduced into the monadic variant (AR strain), or a gene encoding a 1,3-propanediol oxidoreductase is introduced into the variant (AR strain). It provides a variant having the ability to produce 1,3-propanediol inserted in the chromosome of the strain).

The present invention also relates to Krebselanus, in which the transcriptional activator gene (DhaR), the 1,3-propanediol oxidoreductase gene (DhaT) and the glycerol dehydratase reactivator II gene (DhaBA2) are deleted. Monia variants (AR strains) have introduced a vector containing a gene encoding a 1,3-propanediol oxidase reductase and a gene encoding a glycerol dehydratase reactivator, or 1,3- A gene encoding propanediol oxidoreductase and a gene encoding a glycerol dehydratase reactivator provide a variant having the ability to produce 1,3-propanediol inserted into a chromosome of the variant (AR strain). .

The present invention also comprises the steps of culturing the microbial variant in a medium containing glycerol to produce 1,3-propanediol; And it provides a method for producing 1,3-propanediol comprising the step of recovering the produced 1,3-propanediol.

Using the variant according to the present invention, when producing 1,3-propanediol from glycerol using a conventional strain, it is possible to produce only 1,3-propanediol with almost no inevitably produced by-products, the cost in the purification process Can reduce the cost.

In one aspect, the present invention is a microorganism having a ability to produce 1,3-propanediol using glycerol as a carbon source, a microorganism in which a gene encoding a transcriptional activator or a gene encoding a dihydroxyacetone kinase is deleted or incapable It is about a variant.

In the present invention, the gene encoding glycerol dehydrogenase may be additionally deleted or disabled.

Glycerol metabolism is metabolized through two oxidative and reductive metabolic pathways (FIG. 1). First, in oxidative metabolism, glycerol is oxidized to dehydroxyacetone (DHA) by the action of NAD + dependent glycerol dehydrogenase to produce NADH, and by the action of DHA kinase It is converted into dehydroxyacetone phosphate (DHAP) and then metabolized through glycolysis and used as carbon and energy source for growth. In the oxidative metabolism, 2,3-butanediol, acetic acid, ethanol, lactic acid And by-products such as succinic acid are produced.

On the other hand, in reductive metabolism, glycerol is converted to 3-hydroxypropionaldehyde by the action of dehyratase and then 1,3- by NADH dependent oxidoreductase. NAD + is formed while reducing to propanediol.

The oxidation and reduction metabolic pathways of glycerol are closely related to each other in order to maintain intracellular NAD + -NADH balance, and the genes encoding the four enzymes-glycerol dehyratase ( dhaB ), 1 , 3-propanediol oxidoreductase (1,3-propanediol reducatse, dhaT ), glycerol dehydrogenase ( dhaD ), dehydroxyacetone kinase ( dhaK ) -are usually clusters on chromosomes ( clusters) and regulated to the same regulon by co-existing transcriptional regulator DhaR.

Variants of the present invention by deleting or disabling the gene encoding the protein responsible for the oxidation pathway during the glycerol metabolism process, without generating by-products such as 2,3-butanediol, ethanol, lactic acid, succinic acid, through a reduction pathway It is a strain that produces only 1, 3-propanediol.

In the present invention, the protein responsible for the glycerol oxidation pathway of the variant may be characterized in that the glycerol dehydrogenase, transcriptional activator and dihydroxyacetone kinase.

In the present invention, the mutant sheet to bakteo (Citrobacter), Clostridium (Clostridium), Enterobacter bakteo (Enterobacter), one Rio bakteo (Ilyobacter), keurep when Ella (Klebsiella), Lactobacillus (Lactobacillus) and fellow bakteo It may be characterized in that it is selected from the group consisting of ( Pelobacter ).

In the present invention, the variant is Krebsiella pneumoniae, the gene encoding the enzyme responsible for the glycerol oxidation pathway is the glycerol dehydrogenase gene (DhaD), transcription activator gene (DhaR) and dihydroxyacetone kinase It may be characterized as a gene (DhaK, DhaL, DhaM and DhaK ').

In one aspect, the present invention provides a glycerol dehydrogenase gene (DhaD), a transcriptional activator gene (DhaR), a 1,3-propanediol oxidoreductase gene (DhaT), and a chromosome of a Krebsiella pneumoniae strain. Deletion of the glycerol dehydratase reactivation factor II gene (DhaBA2) to produce a Krebssiella pneumoniae variant (AK strain), 1,3-propane which is a gene responsible for the glycerol reduction pathway of the deleted gene Transformation with a recombinant vector comprising a diol oxidoreductase gene (DhaT) and a glycerol dehydratase reactivator II gene (DhaBA2) to restore the reduction pathway and consequently the glycerol dehydrogenase gene (DhaD) and the transcript activator gene (DhaR) was the only deletion variants (FIG. 2).

Thus, the variant of the present invention may be characterized in that the glycerol dehydrogenase gene (DhaD) and the transcriptional activator gene (DhaR) are deleted or disabled.

In this process, the lacZ promoter (P _lacZ ) is inserted upstream of the gene so that the reduction pathway genes are no longer controlled by the DhaR regulator, so that the expression of the gene can be artificially controlled using an inducer.

In another aspect, the invention provides a transcriptional activator gene (DhaR), a 1,3-propanediol oxidoreductase gene (DhaT) and a glycerol dehydratase reactivator II in the chromosome of a Krebsiella pneumoniae strain. A Krebsiella pneumoniae variant (AR strain), in which the gene (DhaBA2) was deleted, was produced, and the 1,3-propanediol oxidoreductase gene (DhaT), which is a gene responsible for the glycerol reduction pathway among the deleted genes, and A recombinant vector containing a glycerol dehydratase reactivator II gene (DhaBA2) was transformed to restore the reduction pathway, resulting in a variant in which only the transcriptional activator gene (DhaR) was deleted (FIG. 2). ).

Therefore, the variant of the present invention may be characterized in that the transcriptional activator gene (DhaR) is deleted or disabled.

In another aspect, the present invention is a microorganism having a ability to produce 1,3-propanediol using glycerol as a carbon source, a microorganism in which a gene encoding a transcriptional activator or a gene encoding a dihydroxyacetone kinase is deleted or incapable The present invention relates to a method for producing 1,3-propanediol, wherein the variant is cultured in a medium containing glycerol.

In one aspect, the present invention provides a Klebsiella pneumoniae strain in which a glycerol dehydrogenase gene (DhaD) and a transcriptional activator gene (DhaR) are deleted and a transcriptional activator gene (DhaR). The strain was confirmed to produce 1,3-propanediol without producing other byproducts of the oxidation pathway except for a small amount of acetic acid in a medium containing glycerol.

In another aspect, the invention provides a glycerol dehydrogenase gene (DhaD), a transcriptional activator gene (DhaR), a 1,3-propanediol oxidoreductase gene (DhaT) and a glycerol dehydratase reactivator. A vector containing a gene encoding 1,3-propanediol oxidoreductase was introduced into the Krebs. Erythropneumoniae variant (AK strain), in which the II gene (DhaBA2) was deleted, or 1,3-propanediol The present invention relates to a variant having 1,3-propanediol generating ability in which a gene encoding oxidoreductase is inserted into a chromosome of the variant (AK strain).

In order to prepare the above variant, DhaB enzyme of dha regulation is carried out by homologous recombination method using homologous recombination of Krebsiella pneumoniae MGH78578 strain (hereinafter referred to as "Cu") with plasmid DNA cured. Reactivation factor, DhaT gene, DhaR regulator and DhaD gene was replaced with apramycin resistance gene to prepare a recombinant strain lacking both oxidation and reduction pathways (hereinafter referred to as "AK" strain).

In order to prepare the above variant, DhaB enzyme of dha regulation is carried out by homologous recombination method using homologous recombination of Krebsiella pneumoniae MGH78578 strain (hereinafter referred to as "Cu") with plasmid DNA cured. A recombinant strain was prepared in which the reactivation factor, the DhaT gene, and the DhaR regulator were substituted with the apramycin resistance gene (named AR). Thus, a recombinant strain was prepared in which the oxidation and reduction pathways were deleted (hereinafter referred to as the "AR" strain). ).

In addition, we analyzed the proliferation and glycerol metabolism of the Klebsiella pneumoniae recombinant strains AK and AR, which were removed from the glycerol anaerobic society. AK and AR strains were cultured in the medium to which glycerol was added to investigate the growth of the cells, and at the same time, the residual amount of glycerol and the production of metabolites including 1,3-propanediol were analyzed by chromatography. As a result, oxidative metabolites such as 2,3-butanediol except for acetic acid were not produced at all, which is consistent with the genetic background in which the glycerol anaerobic metabolism was closed in AK and AR strains.

In the present invention, in order to recover the glycerol reduction pathway of the AK strain and the AR strain in which the oxidation and reduction pathways of glycerol were blocked, a glycerol reduction pathway recovery plasmid was prepared. The plasmid DNA is a gene having a DhaB reactivation enzyme gene ( orfW ) -orfX DNA fragment and 1,3-propanediol oxidoreductase activity { dhaT , yqhD (from E. coli ), or yqhD homologous gene ( Krepsi Ela pneumoniae)} was amplified and inserted downstream of the lacZ promoter of the pGEM TEasy vector.

In order to analyze the characteristics of the AK and AR strains transformed with the glycerol reduction pathway repair plasmid DNA, the metabolites in the culture solution were analyzed while culturing the recombinant strains in a medium to which glycerol, tetracycline, and IPTG were added. Recombinant strains derived from Klebsiella pneumoniae Cu consumed all of the added glycerol after a certain time after incubation, whereas recombinant strains derived from AK and AR showed a somewhat slower rate of glycerol consumption than recombinant strains derived from Cu. It was confirmed that the production capacity of, 3-propanediol was restored (FIG. 8). Both yqhD genes from E. coli or Klebsiella pneumoniae have been shown to restore the glycerol reduction pathway as well as the dhaT gene. On the other hand, AK, AR-derived recombinant strains that block the glycerol oxidation pathway was still found to produce no other oxidation pathway metabolites except for a small amount of acetic acid (FIG. 8).

In another aspect, the glycerol dehydrogenase gene (DhaD), transcriptional activator gene (DhaR), 1,3-propanediol oxidoreductase gene (DhaT) and glycerol dehydrolase reactivator II gene ( Vector containing a gene encoding a 1,3-propanediol oxidoreductase and a gene encoding a glycerol dehydratase reactivator in a Krebsiella pneumoniae variant (AK strain) that has deleted DhaBA2) 1,3 wherein a gene encoding 1,3-propanediol oxidoreductase and a gene encoding a glycerol dehydratase reactivator are inserted into the chromosome of the variant (AK strain) It relates to a variant having a propanediol generating ability.

In another aspect, the present invention relates to a Kreb, in which the transcriptional activator gene (DhaR), the 1,3-propanediol oxidoreductase gene (DhaT), and the glycerol dehydratase reactivator II gene (DhaBA2) are deleted. A vector containing a gene encoding a 1,3-propanediol oxidoreductase is introduced into a siela pneumoniae variant (AR strain), or a gene encoding a 1,3-propanediol oxidoreductase is The present invention relates to a variant having 1,3-propanediol generating ability inserted into a chromosome of a variant (AR strain).

In another aspect, the present invention relates to a Kreb, in which the transcriptional activator gene (DhaR), the 1,3-propanediol oxidoreductase gene (DhaT), and the glycerol dehydratase reactivator II gene (DhaBA2) are deleted. A vector containing a gene encoding 1,3-propanediol oxidoreductase and a gene encoding a glycerol dehydratase reactivator is introduced into the siela pneumoniae variant (AR strain), or 1, Genes encoding 3-propanediol oxidoreductase and genes encoding glycerol dehydratase reactivator are present in the variant having the ability to produce 1,3-propanediol inserted into the chromosome of the above-mentioned variant (AR strain). It is about.

In the present invention, "deletion" of a gene means a state in which the gene is deleted on a chromosome or a plasmid to be unable to produce a protein encoded by the gene, and "disabling" of the gene means that the gene is inserted, translocated, By some deletion, it means a state in which the gene cannot be encoded.

Cultivation of the variant according to the present invention can be carried out according to well-known methods, conditions such as the culture temperature and time, the pH of the medium can be appropriately adjusted. Recovery of the 1,3-propanediol from the culture medium of the variant may use a conventional separation technique, such as distillation, electrodialysis, pervaporation, chromatography, solvent extraction, reaction extraction, etc. These may be used in combination to separate high materials.

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 to these examples.

Hereinafter, in the Examples, the variant was prepared by blocking all of the glycerol redox pathway of the crab ciella pneumoniae strain, and having the 1,3-propanediol production capacity without producing by-products by restoring the glycerol reduction pathway again. Variants were prepared, but by blocking only the glycerol oxidation pathway of microorganisms having 1,3-propanediol-producing ability from glycerol, it is possible to prepare variants having 1,3-propanediol-producing ability without producing by-products. Will be self-evident.

Example 1 Preparation of Recombinant Strain Blocked with Glycerol Redox Metabolic Pathway

For the redesign of the glycerol metabolic cycle, a recombinant strain was prepared as a base strain, which completely blocked the metabolic circuit from glycerol in Klebsiella pneumoniae MGH 78578 (ATCC 700721). Antimicrobial resistance characteristics of the Klebsiella pneumoniae MGH78578 strain were analyzed to obtain a selection marker that could be used during the recombination process. Apramycin (50 ㎍ / ml) could be used as an antibiotic marker for selection of recombinant strains. Confirmed. In addition, the plasmids present in the original strain were removed by a curing method, and tetracycline was added as an available selection marker. Plasmid DNA is cured the keurep when Ella pneumoniae MGH78578 strain (named Cu) to the to the parent strain dha a homologous recombination method LES gyulron a DhaB enzyme reactivation factor (Fig. 2), DhaT gene, DhaR regulator and By replacing the DhaD gene with apramycin resistance gene, a recombinant strain AK lacking both oxidation and reduction pathways was prepared. At the same time, a recombinant strain AR was prepared in which the DhaB enzyme reactivation factor, DhaT gene and DhaR regulator were substituted with apramycin resistance gene. At this time, the DhaR-dependent self-promoter upstream of the DhaB gene was replaced with an artificially controlable lacZ promoter.

(1) Cure of Plasmid DNA from Klebsiella pneumoniae

Inoculated with Krebssiella pneumoniae MGH78578 in liquid medium (LB) containing 1% trypton, 0.5% yeast extract, 0.5% NaCl, incubated at 180 rpm at 37 ° C for 12 hours, and then 1% trypton, 0.5% yeast Passage was inoculated in a liquid medium containing extract, 0.5% NaCl, 2 mM Tris-Cl buffer, 0.1% EtBr and incubated at 37 rpm for 180 hours at 37 rpm. After three repetitions in the same manner, the cultures were plated in solid LB medium without antibiotics and incubated at 37 ° C. for 12 hours to isolate single colonies. The isolated colonies were inoculated in medium with or without tetracycline to select colonies that could no longer grow in medium with tetracyline. Plasmid DNA was isolated from the selected colonies and confirmed by agarose electrophoresis to finally select strains that lost plasmid DNA. The selected strain was named Klebsiella pneumoniae MGH78578 Cu, and after confirming that the cell proliferation and glycerol metabolism characteristics are not significantly different from the MGH78578 strain, it was used as a parent strain for the preparation of recombinant strains.

(2) Preparation of Recombinant Strain Blocked with Glycerol Metabolic Circuits

To isolate the chromosomal DNA of the Krebssiella pneumoniae MGH78578 strain, the strain was incubated for 12 hours in LB liquid medium (50 ml), and the cells were recovered by centrifugation at 14,000 ㅧ g for 10 minutes at 10 ° C. The recovered cells were washed with 50 mM Tris-Cl (pH 8.0), resuspended in 3 ml of 25% sucrose, 175 µl of TES buffer, 20 µl SDS, and 100 µl of 0.5 mM EDTA, and left at 37 ° C for 30 minutes. . 40 μl of RNase (10 mg / ml in TE buffer) was added and left at 37 ° C. for 20 minutes, and 250 μl of Proteinase K (10 mg / ml in TE buffer) was added to remove protein for 1 hour at 50 ° C. Treated. 900 μl of 5 M NaCl was added and centrifuged at 14,000 ㅧ g for 10 minutes to recover the supernatant, followed by induction of chromosomal DNA by adding 2 volumes of anhydrous alcohol, and centrifugation at 14,000 ㅧ g at 4 ° C. for 15 minutes. DNA was precipitated, washed with 70% ethanol and dried. The separated chromosomal DNA was confirmed by electrophoresis on 0.7% (U / V) agarose gel.

DNA fragments for preparing plasmid for homologous recombination using the extracted chromosomal DNA as a template were amplified by PCR using the following primer sets (FIG. 2). PCR reaction conditions were 2 μl template DNA, 2 μl each primer DNA, 0.2 mM dNTP mix, 2 mM MgCl ₂ , 5 μl 10 μg buffer (excluding Mg ²⁺ ), 0.05 U / μl Taq polymerase and 30.5 μl tertiary distilled water. 50 µl of the final reaction solution of 1 cycle was denatured at 94 ° C for 30 seconds, annealed at 52 ° C for 30 seconds, and amplified at 72 ° C for 1 minute.

dhaBI gene fragment amplification primers

SEQ ID NO: 5'-TCTAGAATGAAAAGATCAAAACGATTT-3 '(dhaBI XbaI-480 bpF)

SEQ ID NO: 5'-GGATCCGTCAGCGGCAATCTGCAC-3 '(dhaBI BamHI-480 bpR)

dhaK gene fragment amplification primers

SEQ ID NO: 5'-AAGCTTCATGCTCTCCGGCGCCTGTC-3 '(dhaK HindIII-200-700 bpF)

SEQ ID NO: 5'-AGATCTATTTGGTCCAGCGAGCTGAAGC-3 '(dhaK BglII-200-700 bpR)

dhaR gene fragment amplification primers

SEQ ID NO: 5'-AGATCTCCTGGGATTTCGCGACGGCA-3 '(dhaR bglII-200-700 bpF)

SEQ ID NO: 5'-AAGCTTTCGACAATCGGTTTTAAGGTG-3 '(dhaR HindIII-200-700 bpR)

Primer for amplifying Apr gene fragment

SEQ ID NO: 5'-GTTAACCTGACGCCGTTGGATACACC-3 'Apr HpaI F

SEQ ID NO: 8'-AGATCTAAAAGCTTATGAGCTCAGCCAATCGA-3 'Apr HindIII-BglIIR

The amplified DNA fragment was cloned using pGEM TEasy vector to confirm the nucleotide sequence, and then plasmid DNA was prepared as shown in FIG. 3.

In the method shown in Fig. 3A, the plasmid DNA to which the amino terminus (dhaB ')-LacZ promoter (P _lacZ ) -Apramycin resistance gene-DhaK gene amino acid (dhaK') of the DhaB gene for preparing an AK strain was connected The amino terminal (dhaB ')-LacZ promoter (P _lacZ ) -Apramycin resistance gene-DhaR gene of the DhaR gene for preparing an AR strain was linked by the method shown in FIG. 3B. Plasmid DNA was prepared.

DNA fragments obtained by treatment of the plasmid with Bam HI- Bgl II were introduced into the Klebsiella pneumoniae Cu strain by electroshock method, and recombinant strains forming colonies were isolated in apramycin-added medium. The obtained colony was inferred that there is an insertion of a Bam HI- Bgl II DNA fragment containing the apramycin resistance gene in the chromosome took place, carried out by the Southern block port homologous recomination This was exactly the final check have occurred in the dha LES gyunron, as a result , dha LES gyulron of DhaB enzyme reactivation factor, DhaT gene, DhaR regulator and DhaD gene is removed, and the lacZ promoter and the apramycin was a resistance gene to obtain a recombinant strain AK strain insertion, DhaB enzyme reactivation factor, DhaT gene and DhaR The recombinant strain AR was removed and a regulator was inserted, and the lacZ promoter and apramycin resistance gene were inserted.

Example 2 Characterization of Klebsiella pneumoniae AK, AR Strains

The proliferation and glycerol metabolism of the Klebsiella pneumoniae recombinant strains AK and AR, which were removed from the glycerol anaerobic community, were analyzed. AK and AR strains were inoculated in a medium containing glycerol, and then cultured at 180 rpm at 37 ° C. for 12 hours, and then examined for the degree of growth of the cells, and at the same time, the residual amount of glycerol and 1,3-propanediol in the culture supernatant were included. The production of metabolites was analyzed by chromatography {(model name: Agilent 1200 (refactive index detector, RID), column: Aminex HPX-87H (Bio-Rad) 300 mm x 78 mm, solvent: 65:35 deionized water-acetonitrile (0.005 MH ₂ SO ₄ ), flow rate 0.5 ml / min} .AK and AR strains showed twice as slow cell growth rate as compared to the parent strain Cu, which was used as a control. Propandiol production was found to be 40% and 20%, respectively (Fig. 5), oxidative metabolites such as 2,3-butanediol except for acetic acid were not produced at all, which is AK, AR recombination Glycerol anaerobic metabolism in lung strain The results are consistent with a genetic background.

Example 3: Preparation of Glycerol Reduction Pathway Recovery Strains

(1) Preparation of Plasmid DNA for Glycerol Reduction Pathway Repair

DhaB reactivating enzyme gene ( orfW ) -orfX DNA fragment and gene with 1,3-propanediol oxidoreductase activity { dhaT , yqhD (from E. coli ), or yqhD homology gene, using the following primer sequences (From Klebsiella pneumoniae)} was amplified and cloned into a pGEM TEasy vector to confirm the nucleotide sequence, thereby preparing plasmid DNA as shown in FIG. 5.

SEQ ID NO: 5'-AGATCTATGAGCTATCGTATGTTTGA-3 '(dhaT-BglII F)

SEQ ID NO: 10'5-CTCGAGAAGCTTCAGAATGCCTGGCGGAAAAT-3 '(dhaT-HindIII / XhoI R)

SEQ ID NO: 5'-AGATCTATGAACAACTTTAATCTGCAC-3 '(yqhD-BglII F)

SEQ ID NO: 12'5-AGATCTATGAATAATTTCGACCTGCA-3 '(yqhD-HindIII / XhoI R)

SEQ ID NO: 13: 5'-AGATCTATGAATAATTTCGACCTGCA-3 '(yqhD Kle BglII F)

SEQ ID NO: 14'5-CTCGAGAAGCTTAGCGTGCAGCCTCGTAAAT-3 '(yqhD Kle HindIII, XhoI R)

5 will illustrating a process of manufacturing a DhaT gene and DhaB reactivate gene plasmid DNA plasmid or DhaT including gene only contain the keurep when Ella pneumoniae downstream lacZ promoter, Figure 6 is on the downstream lacZ promoter 1,3-propanediol oxidoreductase-derived E. coli-derived plasmid DNA containing the YqhD (E) gene and DhaB reactivation enzyme gene or plasmid DNA containing only the YqhD (E) gene is shown. FIG. 7 shows only a plasmid or YqhD (K) gene containing a 1,3-propanediol oxidoreductase active YqhD (K) gene and dhaB reactivating enzyme gene derived from Krebsiella pneumoniae downstream of the lacZ promoter. It shows the process of constructing the included plasmid DNA.

(2) Preparation of Recombinant Strain with Restored Glycerol Reduction Path

Plasmid DNA containing the gene encoding the six kinds of 1,3-propanediol oxidoreductase activating enzyme prepared above and plasmid DNA containing pBR322 and DhaB reactivase gene as a control, glycerol anaerobic Recombinant strains with restored glycerol reduction pathways were prepared by introducing into AK and AR strains with closed metabolic circuits (Table 1). Recombinant strains in which each plasmid was introduced into the parent strain Cu strain were used as controls. The synthetic strain prepared in this example was deposited in the Genetic Resource Center of the Korea Research Institute of Bioscience and Biotechnology (Table 2).

Recombinant strain and plasmid DNA used or produced in the present invention Significant Genotype Strains E. coli DH5a Cloning Host K. pneumoniae Cu Tet ^R contained Plasmid DNA curing K. pneumoniae MGH 78578 K. pneumoniae AK ( orfY-dhaT-orfW-orfX-dhaR-dhaD ) :: P _LacZ -Apr ^R K. pneumoniae AR ( orfY-dhaT-orfW-orfX-dhaR ) :: P _LacZ -Apr ^R Plasmids pV pBR322 pVO pBR322-P _LacZ orfW-orfX pVOT pBR322-P _LacZ orfW-orfX- P _LacZ dhaT pVT pBR322-P _LacZ dhaT pVOQ pBR322-P _LacZ orfW-orfX- P _LacZ yqhD ( E. coli ) pVQ pBR322-P _LacZ yqhD ( E. coli ) pVOK pBR322-P _LacZ orfW-orfX -P _LacZ yqhD ( K. pneumoniae ) pVK pBR322-P _LacZ yqhD ( K. pneumoniae )

Accession number of the recombinant strain according to the present invention Strain name Deposit number Klebsiella pneumoniae AK KCTC11419BP Klebsiella pneumoniae AR KCTC11420BP Klebsiella pneumoniae AK-VOT KCTC11421BP Klebsiella pneumoniae AK-VOK KCTC11422BP Klebsiella pneumoniae AR-VOT KCTC11423BP Klebsiella pneumoniae AR-VOK KCTC11424BP

Example 4 Characterization of Recombinant Strains with Restored Glycerol Reduction Pathway

The recombinant strains prepared in Example 3 were inoculated in a medium (50 ml) to which glycerol (2%), tetracycline (10 μg / ml) and IPTG (0.5 mM) were added, and then cultured at 37 ° C. and 120 rpm. Metabolites were analyzed. Recombinant strains derived from Klebsiella pneumoniae Cu consumed all the added glycerol after 14 hours after incubation, whereas AK and AR-derived recombinant strains showed a slower rate of glycerol consumption than the recombinant strains derived from Cu. The production capacity of 1,3-propanediol was confirmed to have recovered (FIG. 8). Both YqhD genes from E. coli or Klebsiella pneumoniae have been shown to restore the glycerol reduction pathway as well as the DhaT gene. On the other hand, AK, AR-derived recombinant strains that block the glycerol oxidation pathway was still found to produce no other oxidation pathway metabolites except for a small amount of acetic acid (FIG. 8).

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 is a schematic diagram showing a reduction pathway for producing 1,3-propanediol and an oxidation pathway for producing byproducts during glycerol metabolism.

Figure 2 illustrates a method for producing a variant according to the invention using the structure of the dha regulator.

Figure 3 shows a method for producing a plasmid DNA for producing a variant according to the present invention, A is the amino terminus (dhaB ') -LacZ promoter (P _lacZ ) -Apramycin resistance gene of the DhaB gene for preparing the AK strain- A method for preparing plasmid DNA linked to the amino terminus (dhaK ') of the DhaK gene is shown. B is the amino terminus (dhaB')-LacZ promoter (P _lacZ ) -Apramycin resistance gene-DhaR of the DhaB gene to prepare an AR strain. It shows a method for producing plasmid DNA linked to the amino terminal (dhaR ') of the gene.

Figure 4 is a graph analyzing the glycerol metabolite of Krebsiella pneumoniae AK, AR strain.

Figure 5 shows a method for producing plasmid DNA containing only the DhaT gene or DhaT gene of the DhaT gene and DhaB reactivation enzyme gene of the Krebsiella pneumoniae downstream of the lacZ promoter.

6 shows only the plasmid or YqhD (E) gene containing the 1,3-propanediol oxidoreductase active YqhD (E) gene ( E. coli ) and the DhaB reactivating enzyme gene derived from E. coli downstream of the lacZ promoter. A schematic diagram illustrating the process of constructing the plasmid DNA.

7 is a plasmid or YqhD (K) containing a 1,3-propanediol oxidoreductase-activated YqhD (K) ( K. pneumoniae ) gene and a DhaB reactivating enzyme gene derived from Krebsiella pneumoniae downstream of the lacZ promoter. ) Shows a method for producing plasmid DNA containing only genes.

Figure 8 is a graph analyzing the glycerol metabolism of the Krebssiella pneumoniae Cu, AK and AR-derived recombinant strains according to the present invention.

<110> Korea Research Institute of Bioscience and Biotechnology <120> MUTANT BLOCKED IN GLYCEROL OXIDAION PATHWAY FOR PRODUCING          1,3-PROPANEDIOL <130> P08-B173 <160> 14 <170> KopatentIn 1.71 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 tctagaatga aaagatcaaa acgattt 27 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggatccgtca gcggcaatct gcac 24 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 aagcttcatg ctctccggcg cctgtc 26 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 agatctattt ggtccagcga gctgaagc 28 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 agatctcctg ggatttcgcg acggca 26 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 aagctttcga caatcggttt taaggtg 27 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gttaacctga cgccgttgga tacacc 26 <210> 8 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 agatctaaaa gcttatgagc tcagccaatc ga 32 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 agatctatga gctatcgtat gtttga 26 <210> 10 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ctcgagaagc ttcagaatgc ctggcggaaa at 32 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 agatctatga acaactttaa tctgcac 27 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 agatctatga ataatttcga cctgca 26 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 agatctatga ataatttcga cctgca 26 <210> 14 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 ctcgagaagc ttagcgtgca gcctcgtaaa t 31  

Claims (12)

Citrobacter, Clostridium, Enterobacter, which have the ability to produce 1,3-propanediol using glycerol as a carbon source and are involved in dihydroxyacetone kinase in the glycerol oxidation pathway In a microorganism selected from the group consisting of Ilyobacter, Klebsiella, Lactobacillus and Pelobacter, a gene encoding a transcriptional activator or a dihydroxyacetone kinase is encoded. Microbial variant, characterized in that the gene is deleted or disabled. The microbial variant according to claim 1, wherein the gene encoding glycerol dehydrogenase is additionally deleted or disabled. delete The microorganism of claim 1, wherein the microorganism is Krebsiella pneumoniae, the transcriptional activator gene is DhaR, and the dihydroxyacetone kinase gene is selected from the group consisting of DhaK, DhaL, DhaM, and DhaK '. Variants. 5. The microbial variant according to claim 4, wherein the glycerol dehydrogenase gene (DhaD) and the transcriptional activator gene (DhaR) are deleted or disabled. The microorganism variant according to claim 4, wherein the transcriptional activator gene (DhaR) is deleted or disabled. Culturing the microbial variant of any one of claims 1, 2 and 4 to 6 in a medium containing glycerol to produce 1,3-propanediol; And recovering the generated 1,3-propanediol. Glycerol dehydrogenase gene (DhaD), transcriptional activator gene (DhaR), 1,3-propanediol oxidoreductase gene (DhaT) and glycerol dehydrolase (DhaB) enzyme reactivator II gene (dhaBA2 A vector containing a gene encoding a 1,3-propanediol oxidoreductase is introduced into a Krebs. Neumoniae variant having a deletion of) or a gene encoding a 1,3-propanediol oxidoreductase. Is a Klebsiella pneumoniae variant having a 1,3-propanediol generating ability is inserted into the chromosome of the variant. Glycerol dehydrogenase gene (DhaD), transcriptional activator gene (DhaR), 1,3-propanediol oxidoreductase gene (DhaT) and glycerol dehydrolase (DhaB) enzyme reactivator II gene (dhaBA2 A vector containing a gene encoding 1,3-propanediol oxidoreductase and a gene encoding a glycerol dehydratase (DhaB) enzyme reactivation factor II in a Krebs. 1, wherein the gene encoding 1,3-propanediol oxidoreductase and the gene encoding glycerol dehydratase (DhaB) enzyme reactivation factor II are inserted into the chromosome of the variant, Krebsciella pneumoniae variants having the ability to produce 3-propanediol. Krebsiella pneumoniae that lacks the transcriptional activator gene (DhaR), 1,3-propanediol oxidoreductase gene (DhaT), and glycerol dehydratase (DhaB) enzyme reactivator II gene (dhaBA2) A vector containing a gene encoding a 1,3-propanediol oxidoreductase is introduced into a mutant, or a gene encoding a 1,3-propanediol oxidoreductase is inserted into a chromosome of the variant. Krebsciella pneumoniae variants having the ability to produce 3-propanediol. Krebsiella pneumoniae that lacks the transcriptional activator gene (DhaR), 1,3-propanediol oxidoreductase gene (DhaT), and glycerol dehydratase (DhaB) enzyme reactivator II gene (dhaBA2) Subtypes have introduced a vector containing a gene encoding 1,3-propanediol oxidoreductase and a gene encoding glycerol dehydratase (DhaB) enzyme reactivation factor II, or 1,3-propane A gene encoding the diol oxidoreductase and a gene encoding the glycerol dehydratase (DhaB) enzyme reactivation factor II are Krebsiella pneumoniae having the ability to produce 1,3-propanediol inserted into the chromosome of the variant. Monia variant. Culturing the microbial variant of any one of claims 8-11 in a medium containing glycerol to produce 1,3-propanediol; And recovering the generated 1,3-propanediol.

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