WO2010064744A1

WO2010064744A1 - Mutant blocked in glycerol oxidaion pathway for producing 1,3-propanediol

Info

Publication number: WO2010064744A1
Application number: PCT/KR2008/007145
Authority: WO
Inventors: Chul Ho Kim; Jeong-Woo Seo; Sun-Yeon Heo; Mi Young Seo; Baek Rock Oh; Jin-Oh Baek; Pil-Soo Seo; Min Ho Choi
Original assignee: Korea Research Institute of Bioscience and Biotechnology KRIBB
Current assignee: Korea Research Institute of Bioscience and Biotechnology KRIBB
Priority date: 2008-12-03
Filing date: 2008-12-03
Publication date: 2010-06-10
Anticipated expiration: 2011-06-03
Also published as: CN102395671B; CN102395671A

Abstract

The present invention relates to a microbial mutant in which the gene encoding transcription activator or the gene encoding dihydroxyacetone kinase in a microorganism having the ability to produce 1,3 -propanediol using glycerol as a carbon source is deleted or inactivated and to a method for producing 1,3- propanediol using the mutant. When the mutant according to the present invention is used, the production of byproducts which are inevitably obtained when producing 1,3 -propanediol from glycerol using prior strains can be minimized, and thus the cost of purification of 1,3-propandiol can be reduced.

Description

MUTANT BLOCKED IN GLYCEROL OXIDAION PATHWAY FOR PRODUCING 1,3-PROPANEDIOL

TECHNICAL FIELD

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

BACKGROUND ART

1,3-propanediol can be used as a raw material for synthesizing polyester, poly ether, polyurethanes or the like and is used in various applications, including fibers such as highly functional clothing, carpets, or automobile fabrics, and plastic films. Particularly, polytrimethylene terephtalate (PTT) which is produced by polymerization of 1,3-propanediol with terephtalic acid has excellent physical properties and a melting point of 228 ^°C which is lower than that of polyethylene terephthalate (PET). Thus, polytrimethylene terephtalate (PTT) has a higher utility and is receiving attention as a next-generation fiber material which can substitute for PET. Also, plastics and polymers produced from 1,3-propanediol as a monomer show excellent optical stability compared to that of products produced from butanediol or ethylene glycol. In addition, 1,3-propanediol can be used as a polyglycol-type lubricant and a solvent, and thus its commercial value is evaluated to be higher than that of glycerol.

1,3-propanediol can be produced by chemical synthesis or microbial fermentation. Chemical processes for producing 1,3-propanediol include a process of converting i ethylene oxide to 1 ,3 -propanediol by hydroformylation (US Patent No. 3,687,981) and a process of converting acrolein to 1,3 -propanediol by hydration (US Patent No. 5,015,789). However, such chemical processes have problems in that they require a high-temperature or high-pressure process during the production of 1,3- propanediol, leading to high production costs, and generate waste oil containing environmental pollutants.

Biological processes include a process of producing 1,3 -propanediol from glycerol using microorganisms such as Citrobacter, Clostridium, Enterobacter, Ilyobacter, Klebsiella, Lactobacillus, Pelobacter or the like, which are facultative anaerobic strains (US Patent No. 5,254,467). Also, genetic engineering techniques using such microbial strains include a case in which a mutant obtained by amplifying or knocking out the glycerol dehydrogenase gene from Lactobacillus is used to produce 1,3 -propanediol with increased productivity (US 2007/0148749). However, there is no prior art relating to a method for reducing byproducts which are accumulated in large amounts when producing 1,3 -propanediol using genetically engineered mutants. In a metabolic process of converting glycerol to 1,3 -propanediol using the above microorganisms, various kinds of oxidation metabolites are produced in large amounts. Particularly, 2,3-butanediol which is an oxidative metabolite of glycerol has a boiling point similar to that of 1,3- propanediol acts as a great hindrance in a process of purifying 1,3-propanediol.

Accordingly, the present inventors have made many efforts to develop a microorganism producing only 1,3-propanediol in glycerol metabolism by a metabolic engineering method without producing oxidative metabolic byproducts including 2,3-butanediol. As a result, the present inventors have constructed a mutant having only a reductive metabolic pathway producing 1,3-propanediol by using a genetic recombination technique to block an oxidative metabolic pathway producing byproducts in the glycerol metabolic pathway and have found that, when the mutant is used to produce 1,3-propanediol, oxidative metabolic byproducts including 2,3-butanediol are not produced, thereby completing the present invention.

SUMMARY OF INVENTION

It is, therefore, an object of the present invention to provide a mutant in which the oxidative pathway of glycerol is blocked in a microorganism having the ability to produce 1,3 -propanediol using glycerol as a carbon source in order to produce 1,3- propanediol from glycerol without producing byproducts.

Another object of the present invention is to provide a method for producing 1,3- propanediol, which comprises culturing the mutant in a medium containing glycerol.

To achieve the above objects, the present invention provides a microbial mutant in which a gene encoding a transcription activator or a gene encoding dihydroxyacetone kinase is deleted or inactivated in a microorganism having the ability to produce 1,3 -propanediol using glycerol as a carbon source.

The present invention also provides a method for preparing 1,3 -propanediol, which comprises producing 1,3 -propanediol by culturing said microbial mutant in a glycerol-containing medium; and recovering said 1,3 -propanediol produced.

The present invention also provides a mutant having the ability to produce 1,3- propanediol, in which a vector containing a gene encoding 1,3 -propanediol oxidoreductase is introduced in a Klebsiella pneumoniae strain (an AK strain) with a deletion of a glycerol dehydrogenase gene (DhaD), a transcription activator gene (DhaR), a 1,3 -propanediol oxidoreductase gene (DhaT) and a glycerol dehydratase reactivation factor II gene (DhaBA2) or in which the gene encoding 1,3- propanediol oxidoreductase is inserted into the chromosome of the mutant (the AK strain).

The present invention also provides a mutant having the ability to produce 1,3- propanediol, in which a vector containing a gene encoding 1,3 -propanediol oxidoreductase and a gene encoding glycerol dehydratase reactivation factor is introduced in a Klebsiella pneumoniae strain (an AK strain) with a deletion of a glycerol dehydrogenase gene (DhaD), a transcription activator gene (DhaR), a 1,3- propanediol oxidoreductase gene (DhaT) and a glycerol dehydratase reactivation factor II gene (DhaBA2) or in which the gene encoding 1,3 -propanediol oxidoreductase and the gene encoding glycerol dehydratase reactivation gene are inserted into the chromosome of the mutant (the AK strain).

The present invention also provides a mutant having the ability to produce 1,3- propanediol, in which a vector containing a gene encoding 1,3 -propanediol oxidoreductase is introduced in a Klebsiella pneumoniae strain (an AR strain) with a deletion of a transcription activator gene (DhaR), a 1,3-propanediol oxidoreductase gene (DhaT) and a glycerol dehydratase reactivation factor II gene (DhaBA2) or in which the gene encoding 1,3 -propanediol oxidoreductase is inserted into the chromosome of the mutant (the AR strain).

The present invention also provides a mutant having the ability to produce 1 ,3- propanediol, in which a vector containing a gene encoding 1,3-propanediol oxidoreductase and a gene encoding glycerol dehydratase reactivation factor is introduced in a Klebsiella pneumoniae strain (an AR strain) with a deletion of a transcription activator gene (DhaR), a 1,3-propanediol oxidoreductase gene (DhaT) and a glycerol dehydratase reactivation factor II gene (DhaBA2) or in which the gene encoding 1,3-propanediol oxidoreductase and the gene encoding glycerol dehydratase reactivation gene are inserted into the chromosome of the mutant (the AR strain). The present invention also provides a method for preparing 1,3 -propanediol, which comprises producing 1,3 -propanediol by culturing said mutant in a glycerol- containing medium; and recovering said 1,3 -propanediol produced.

Other features and aspects of the present invention will be more apparent from the following detailed description and the appended claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing a reductive pathway producing 1,3- propanediol and an oxidative pathway producing byproducts in a glycerol metabolic process.

FIG. 2 shows a method for preparing a mutant according to the present invention, shown using the structure of the dha regulon.

FIG. 3 shows methods for plasmid DNAs for preparing mutants according to the present invention. FIG. 3A shows a method for constructing a plasmid DNA, which is used to prepare an AK strain and comprises a linkage of DhaB gene amino terminus (dhaB')-LacZ promoter (PlacZ)-apramycin resistant gene-DhaK gene amino terminus (dhaK¹), and FIG. 3 B shows a method for constructing a plasmid DNA, which is used to prepare an AR strain and comprises a linkage of DhaB gene amino terminus (dhaB')-LacZ promoter (PlacZ)-apramycin resistant gene-DhaR gene amino terminus (dhaR¹).

FIG. 4 is a graphic diagram showing analysis results for glycerol metabolites of Klebsiella pneumoniae AK and AR strains.

FIG. 5 shows a method for constructing a plasmid DNA containing the DhaT gene and DhaB reactivation enzyme gene of Klebsiella pneumoniae downstream of the lacZ promoter or for constructing a plasmid DNA containing only the DhaT gene downstream of the lacZ promoter.

FIG. 6 is a schematic diagram explaining a process for constructing a plasmid DNA containing the 1,3 -propanediol oxidoreductase activity YqhD(E) gene (derived from E. colϊ) and the DhaB reactivation enzyme gene downstream of the lacZ promoter or for constructing a plasmid DNA containing only the YqhD(E) gene downstream of the lacZ promoter.

FIG. 7 shows a method for constructing the 1,3 -propanediol oxidoreductase activity YqhD(K) gene (derived from K. pneumoniae) and the DhaB reactivation enzyme gene downstream of the lacZ promoter or for constructing a plasmid DNA containing only the YqhD(K) gene downstream of the lacZ promoter.

FIG. 8 is a graphic diagram showing analysis results for glycerol metabolites of recombinant strains derived from Klebsiella pneumoniae Cu, AK and AR according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION, AND PREFERRED EMBODIMENTS

In one aspect, the present invention relates to a microbial mutant in which a gene encoding a transcription activator or a gene encoding dihydroxyacetone kinase is deleted or inactivated in a microorganism having the ability to produce 1,3- propanediol using glycerol as a carbon source.

In the mutant according to the present invention, preferably, the gene encoding glycerol dehydrogenase is additionally deleted or inactivated.

The glycerol metabolic pathway consists of two metabolic pathways, that is, oxidative and reductive metabolic pathways (FIG. 1). In the oxidative metabolic process, glycerol is oxidized to dehydroxyacetone (DHA) by NAD+ dependent glycerol dehydrogenase while producing NADH, which is then converted to dehydroxyacetone phosphate (DHAP) by DHA kinase. The dehydroxyacetone phosphate (DHAP) is metabolized through various pathways while being used as the carbon and energy sources required for growth. In the oxidative metabolic process, byproducts including 2,3-butanediol, acetic acid, ethanol, lactic acid and succinic acid are produced.

Meanwhile, in the reductive metabolic process, glycerol is converted to 3- hydroxypropionaldehyde by the action of dehyratase, and then reduced to 1,3- propanediol by the action of NADH dependent oxidoreductase while forming NAD+.

The oxidative and reductive pathways of glycerol are closely connected with each other in order to maintain the NAD+-NADH balance in cells, and the genes encoding the four enzymes, that is, glycerol dehyratase (dhaB), 1,3-propanediol reducatse (dhaT), glycerol dehydrogenase (dhaD) and dehydroxyacetone kinase (dhaK), are arranged as clusters on the chromosome and regulated in the same regulon by the coexisting transcription factor DhaR.

The mutant of the present invention is a microbial strain in which genes encoding proteins involved in the oxidative pathway of the glycerol metabolic process are deleted or inactivated and which produces only 1,3-propanediol through the reductive pathway without producing byproducts including 2,3-butanediol, ethanol, lactic acid and succinic acid.

In the present invention, the proteins involved in the glycerol oxidative pathway of the mutant are preferably glycerol dehydrogenase, a transcription activator and dihydroxyacetone kinase. In the present invention, the microorganism is preferably selected from the group consisting of Citrobacter, Clostridium, Enterobacter, Ilyobacter, Klebsiella, Lactobacillus and Pelobacter.

In the present invention, the microorganism is preferably Klebsiella pneumoniae, and the genes encoding enzymes involved in the glycerol oxidative pathway are a glycerol dehydrogenase gene (DhaD), a transcription activator gene (DhaR) and dihydroxyacetone kinase genes (DhaK, DhaL, DhaM and DhaK¹).

In one embodiment of the present invention, a Klebsiella pneumoniae mutant (an AK strain) was constructed by deleting the glycerol dehydrogenase gene (DhaD), the transcription activator gene (DhaR), the 1,3 -propanediol oxidoreductase gene (DhaT) and the glycerol dehydratase reactivation factor II gene (DhaBA2) from the chromosome of the Klebsiella pneumoniae strain. The mutant was transformed with a recombinant vector comprising the 1,3 -propanediol oxidoreductase gene (DhaT) and the glycerol dehydratase reactivation factor II gene (DhaBA2), which are genes involved in the reductive pathway of glycerol, thus restoring the reductive pathway. As a result, a mutant with a deletion of only the glycerol dehydrogenase gene (DhaD) and the transcription activator gene (DhaR) was constructed (FIG. 2).

Accordingly, the mutant of the present invention is characterized in that the glycerol dehydrogenase gene (DhaD) and the transcription activator gene (DhaR) are deleted or inactivated.

In this process, the lacZ promoter (PlacZ) was inserted upstream of the genes involved in the reductive pathway, such that the genes were no longer regulated by the DhaR regulator, whereby the expression of the genes could be artificially controlled using an inducer. In another embodiment of the present invention, a Klebsiella pneumoniae mutant (an AR strain) was constructed by deleting the transcription activator gene (DhaR), the 1,3 -propanediol oxidoreductase gene (DhaT) and the glycerol dehydratase reactivation factor II gene (DhaBA2) from the chromosome of the Klebsiella pneumoniae strain. The mutant was transformed with a recombinant vector comprising the 1,3 -propanediol oxidoreductase gene (DhaT) and the glycerol dehydratase reactivation factor II gene (DhaBA2), which are genes involved in the reductive pathway of glycerol, thus restoring the reductive pathway. As a result, a mutant with a deletion of only the transcription activator gene (DhaR) was constructed (FIG. 2).

Accordingly, the mutant of the present invention is characterized in that the transcription activator gene (DhaR) is deleted or inactivated.

In another aspect, the present invention relates to a method for producing 1,3- propanediol, which comprises culturing in a glycerol-containing medium a microbial mutant in which a gene encoding transcription activator or a gene encoding dihydroxyacetone kinase in a microorganism having the ability to produce 1,3 -propanediol using glycerol as a carbon source is deleted or inactivated.

In one embodiment of the present invention, it was confirmed that the Klebsiella pneumoniae strain with a deletion of the glycerol dehydrogenase gene (DhaD) and the transcription activator gene (DhaR) and the Klebsiella pneumoniae strain with a deletion of the transcription activator gene (DhaR) produced 1,3 -propanediol in a medium containing glycerol without producing byproducts of the oxidative pathway other than a small amount of acetic acid.

In still another aspect, the present invention relates to a mutant having the ability to produce 1,3 -propanediol, in which a vector containing a gene encoding 1,3- propanediol oxidoreductase is introduced in a Klebsiella pneumoniae strain (an AK strain) with a deletion of a glycerol dehydrogenase gene (DhaD), a transcription activator gene (DhaR), a 1,3-propanediol oxidoreductase gene (DhaT) and a glycerol dehydratase reactivation factor II gene (DhaBA2) or in which the gene encoding 1,3-propanediol oxidoreductase is inserted into the chromosome of the mutant (the AK strain).

To prepare the above-described mutant, the DhaB enzyme reactivation factor, DhaT gene, DhaR regulator and DhaD gene of the dha regulon were substituted with the apramycin-resistant gene by a homologous recombination method using a plasmid DNA-cured Klebsiella pneumoniae MGH78578 strain (hereinafter referred to as "Cu") as a parent strain, thus preparing a recombinant strain with a deletion of both oxidative and reductive pathways (hereinafter referred to as an "AK" strain).

To prepare the above-described mutant, the DhaB enzyme reactivation factor, DhaT gene and DhaR regulator of the dha regulon were substituted with the apramycin-resistant gene by a homologous recombination method using a plasmid DNA-cured Klebsiella pneumoniae MGH78578 strain (hereinafter referred to as "Cu") as a parent strain, thus preparing a recombinant strain with a deletion of both oxidative and reductive pathways (hereinafter referred to as an "AR" strain).

Also, the proliferation and glycerol metabolic characteristics of the Klebsiella pneumoniae recombinant strains (AK and AR strains) with a deletion of anaerobic metabolic pathways of glycerol were analyzed. For this purpose, the AK and AR strains were cultured in a medium supplemented with glycerol, and the degree of proliferation of the microbial cells was examined. In addition, the amount of glycerol present in the culture broth and the production of metabolites including

1,3-propanediol were analyzed by chromatography. As a result, it was shown that oxidation metabolites including 2,3-butanediol other than acetic acid were not produced, and this result was consistent with the genetic background in which the anaerobic metabolic pathways of glycerol in the AK and AR strains were blocked.

In order to restore the glycerol reductive pathway of each of the AK and AR strains in which the oxidative and reductive pathways have been blocked, a plasmid for restoring the reductive pathway of glycerol was prepared. The plasmid DNA was prepared by amplifying a DhaB reactivation enzyme gene {prfW)-orpC DNA fragment, the dhaT, yqhD (derived from E. colt) or yqhD homologous gene

(derived from Klebsiella pneumoniae) having 1,3 -propanediol oxidoreductase activity and inserting the amplified products downstream of the lacZ promoter of a pGEM TEasy vector.

In order to analyze the characteristics of the AK and AR strains transformed with the plasmid DNA for restoring the reductive pathway of glycerol, each of the recombinant strains was cultured in a medium supplemented with glycerol, tetracycline and IPTG, while metabolites in the culture broth were analyzed. The recombinant strains derived from Klebsiella pneumoniae Cu completely consumed the added glycerol after a given time of culture, whereas the recombinant strains derived from AK or AR showed glycerol consumption rates slightly slower than those of the Cu-derived recombinant strains, but their ability to produce 1,3- propanediol was found to be restored (FIG. 8). The two yqhD genes derived from E. coli or Klebsiella pneumoniae were shown to restore the reductive pathway of glycerol in the same manner as the dhaT gene. Meanwhile, it was found that the AK- or AR-derived recombinant strains in which the oxidative pathway of glycerol has been blocked still did not produce metabolites of the oxidative pathway other than a small amount of acetic acid (FIG. 8).

In still another aspect, the present invention relates to a mutant having the ability to produce 1,3 -propanediol, in which a vector containing a gene encoding 1,3- propanediol oxidoreductase and a gene encoding glycerol dehydratase reactivation factor is introduced in a Klebsiella pneumoniae strain (an AK strain) with a deletion of a glycerol dehydrogenase gene (DhaD), a transcription activator gene (DhaR), a 1,3 -propanediol oxidoreductase gene (DhaT) and a glycerol dehydratase reactivation factor II gene (DhaBA2) or in which the gene encoding 1,3- propanediol oxidoreductase and the gene encoding glycerol dehydratase reactivation gene are inserted into the chromosome of the mutant (the AK strain).

In yet still another aspect, the present invention relates to a mutant having the ability to produce 1,3-propanediol, in which a vector containing a gene encoding 1,3 -propanediol oxidoreductase is introduced in a, Klebsiella pneumoniae strain (an AR strain) with a deletion of a transcription activator gene (DhaR), a 1,3- propanediol oxidoreductase gene (DhaT) and a glycerol dehydratase reactivation factor II gene (DhaBA2) or in which the gene encoding 1,3-propanediol oxidoreductase is inserted into the chromosome of the mutant (the AR strain).

In yet still another aspect, the present invention relates to a mutant having the ability to produce 1,3-propanediol, in which a vector containing a gene encoding 1,3-propanediol oxidoreductase and a gene encoding glycerol dehydratase reactivation factor is introduced in a Klebsiella pneumoniae strain (an AR strain) with a deletion of a transcription activator gene (DhaR), a 1,3-propanediol oxidoreductase gene (DhaT) and a glycerol dehydratase reactivation factor II gene (DhaBA2) or in which the gene encoding 1,3-propanediol oxidoreductase and the gene encoding glycerol dehydratase reactivation gene are inserted into the chromosome of the mutant (the AR strain).

As used herein, the term "deletion" of genes refers to the state in which the genes were deleted from a chromosome or a plasmid, such that proteins encoded by the genes could not be produced. The term "inactivation" of genes refers to the state in which the genes were inserted, translocated or partially deleted, such that proteins encoded by the genes could not be produced. The culture of the mutant according to the present invention can be carried out according to a widely known method, and conditions including the culture temperature and time, the pH of medium, and the like can be suitably controlled. Collection of 1,3 -propanediol from the culture broth of the mutant can be carried out using conventional isolation techniques, for example, distillation, electrodialysis, evaporation, chromatography, solvent extraction, and reaction extraction, and these techniques may generally be used in combination to isolate highly pure substances.

Examples

Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are for illustrative purposes only and are not to be construed to limit the scope of the present invention.

In the following examples, a mutant in which the oxidative and reductive pathways of glycerol in the Klebsiella pneumoniae strain were all blocked, and the reductive pathway of glycerol was restored again, thus preparing a mutant having the ability to produce 1,3 -propanediol without producing byproducts. However, it will be obvious to those skilled in the art that a mutant having the ability to produce 1,3- propanediol without producing byproducts can be prepared by blocking only the oxidative pathway of glycerol in a microorganism having the ability to produce 1,3- propanediol from glycerol.

Example 1 : Preparation of recombinant strains in which oxidative-reductive metabolic pathways of glycerol were blocked

For a redesign of the glycerol metabolic pathway, a recombinant strain in which the glycerol metabolic pathway in Klebsiella pneumoniae MGH 78578 (ATCC 700721) was completely blocked was prepared as a base strain. In order to secure a selection marker which can be used in a genetic recombination process, the antibiotic resistance characteristics of the Klebsiella pneumoniae MGH 78578 strain were analyzed. As a result, it was found that apramycin (50 /zg/ml) could be used as an antibiotic marker for the selection of recombinant strains. Also, plasmids originally present in the strain were removed by a curing method, and tetracycline was added as a usable selection marker. Using a plasmid DNA-cured Klebsiella pneumoniae MGH 78578 strain (named "Cu") as a parent strain, the DhaB enzyme reactivation factor, DhaT gene, DhaR regulator and DhaD gene of the dha regulon (FIG. 2) were substituted with apramycin-resistant genes by a homologous recombination method, thus preparing a recombinant strain AK with a deletion of both oxidative and reductive pathways. At the same time, a recombinant strain AR was prepared by substituting the DhaB enzyme reactivation factor, the DhaT gene and the DhaR regulator with apramycin-resistant gene. Herein, the DhaR dependent promoter upstream of the DhaB gene was replaced with an artificially controllable lacZ promoter.

(1) Curing of plasmid DNA from Klebsiella pneumoniae

Klebsiella pneumoniae MGH78578 was inoculated into a liquid medium (LB) containing 1% tryptone, 0.5% yeast extract and 0.5% NaCl and was cultured at 37 ^°C at 180 rpm for 12 hours. Then, the cultured strain was subinoculated into a liquid medium containing 1% tryptone, 0.5% yeast extract, 0.5% NaCl, 2 mM Tris- Cl buffer and 0.1% EtBr and was cultured at 37 ^°C at 180 rpm for 12 hours. The culture was repeated three times in the same manner as described above, and then the culture broth was plated onto a solid LB medium containing no antibiotic and was cultured at 37 "C for 12 hours, and single colonies were isolated from the culture broth. The isolated colonies were inoculated into a medium supplemented with or without tetracycline, and colonies which no longer proliferated in the medium supplemented with tetracycline were selected. Plasmid DNA was isolated from the selected colonies and analyzed by agarose gel electrophoresis, and a strain lacking the plasmid DNA was finally selected. The selected strain was named "Klebsiella pneumoniae MGH78578 Cu" and used as a parent strain for preparing recombinant strains, after it was confirmed that the cellular proliferation and glycerol metabolic characteristics thereof did not significantly differ from those of the MGH78578 strain.

(2) Preparation of recombinant strains in which glycerol metabolic pathway was blocked

To isolate the chromosomal DNA of the Klebsiella pneumoniae MGH78578 strain, the strain was cultured in LB liquid medium (50 ml) for 12 hours, and then the cells were collected by centrifugation at 10 ^°C at 14,000^χg for 10 minutes. The collected cells were washed with 50 mM Tris-Cl (pH 8.0), resuspended in a solution containing 3 mi of 25% sucrose, 175 μi of TES buffer, 20% SDS and 100 μi of 0.5 mM EDTA and allowed to stand at 37 ^°C for 30 minutes. 40 ≠ of RNase (10 mg/mt in TE buffer) was added to the cells which were then allowed at 37 ^°C for 20 minutes. To remove proteins, 250 μi of proteinase K (10 mg/mt in TE buffer) was added to the cells which were then incubated at 50 "C for 1 hour. 900 μi of 5 M NaCl was added to the cells which were then centrifuged at 14,000 xg for 10 minutes to collect the supernatant. A two-fold volume of anhydrous alcohol was added to the collected supernatant to induce the precipitation of the chromosomal DNA. Then, the cell solution was centrifuged at 4⁰C at 14,000 xg for 15 minutes to precipitate the chromosomal DNA, and the precipitated DNA was washed with 70% ethanol, and then dried. The isolated chromosomal DNA was analyzed by electrophoresis on 0.7% (W/V) agarose gel.

DNA fragments for preparing a plasmid for homologous recombination were amplified by PCR using the extracted chromosomal DNA as a template and the following primer sets (FIG. 2). The PCR reaction performed using 50 μi of a final reaction solution containing 2 μi of template DNA, 2 μi of each primer DNA, 0.2 mM dNTP mix, 2 mM MgC12, 5 μi of 1Ox buffer (Mg2+ free), 0.05 \MlA of Taq polymerase and 30.5 μi of triple-distilled water for one cycle of denaturation at 94⁰C for 30 sec, annealing at 52 ^°C for 30 sec and extension at 72 ^°C for 1 min.

Primer for amplifying dhaBI gene fragment

SEQ ID NO: 1 : 5'-TCTAGAATGAAAAGATCAAAACGATTT-3'(dhaBI XbaI-480bpF) SEQ ID NO: 2: S'-GGATCCGTCAGCGGCAATCTGCAC-S'CdhaBI BamHI-480bpR)

Primer for amplifying dhaK gene fragment

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

SEQ ID NO: 4: 5'-AGATCTATTTGGTCCAGCGAGCTGAAGC-3'(dhaK BglII-200-700bpR)

Primer for amplifying dhaR gene fragment

SEQ ID NO: 5: 5'-AGATCTCCTGGGATTTCGCGACGGCA-3'(dhaR bglII-200-700bpF) SEQ ID NO: 6: 5'-AAGCTTTCGACA ATCGGTTTT AAGGTG-3 '(dhaR HindIII-200-700bpR)

Primer for amplifying Apr gene fragment SEQ ID NO: 7: 5'-GTTAACCTGACGCCGTTGGATACACC^' Apr Hpal F

SEQ ID NO: 8: 5'-AGATCTAAAAGCTTATGAGCTCAGCCAATCGA-S' Apr Hindlll-BglHR

The amplified DNA fragments were cloned using a pGEM TEasy vector, and the base sequence thereof was analyzed. Then, as shown in FIG. 3, plasmid DNAs were constructed.

In the method shown in FIG. 3A, a plasmid DNA for preparing an AK strain, which was comprised of a linkage of DhaB gene amino terminus (dhaB')-LacZ promoter (PlacZ)-apramycin resistant gene-DhaK gene amino terminus (dhaK'), was constructed. In the method shown in FIG. 3B, a plasmid DNA for preparing an AR strain, which was comprised of a linkage of DhaB gene amino terminus (dhaB')-LacZ promoter (PlacZ)-apramycin resistant gene-DhaR gene amino end (dhaR'), was constructed.

Each of the plasmids was treated with BamHI-BglU, and the collected DNA fragment was introduced into the Klebsiella pneumoniae Cu strain by electroporation. Then, recombinant strains forming colonies in a medium supplemented with apramycin were isolated. It was inferred that, in the obtained colonies, the insertion of the BamHl-BgHl DNA fragment containing the apramycin resistant gene into the chromosome occurred. It was finally confirmed by Southern blotting that homologous recombination occurred accurately in the dha regulon. As a result, a recombinant strain AK with a deletion of the DhaB enzyme reactivation factor, DhaT gene, DhaR regulator and DhaD gene of the dha regulon and an insertion of the lacZ promoter and the apramycin resistant gene was obtained, and a recombinant strain AR with a deletion of the DhaB enzyme reactivation factor, the DhaT gene and the DhaR regulator and an insertion of the lacZ promoter and the apramycin resistant gene was obtained.

Example 2: Analysis of characteristics of Klebsiella pneumoniae strains AK and AR

The proliferation and glycerol metabolic characteristics of the Klebsiella pneumoniae recombinant strains AK and AR in which the anaerobic metabolic pathway of glycerol was removed were analyzed. Each of the AK and AR strains was inoculated into a medium supplemented with glycerol and was cultured at 37 ^°C at 180 rpm for 12 hours. Then, the degree of proliferation of the cells was examined, and the amount of glycerol present in the culture supernatant and the production of metabolites including 1,3 -propanediol were analyzed by chromatography (device used: Agilent 1200 (refractive index detector, RID); column used: Aminex HPX-87H (Bio-Rad) 300 mm x 78 mm; solvent used: 65:35 deionized water-acetonitrile (0.005 M H₂SO₄); and flow rate: 0.5 ml/min). The AK and AR strains showed cell proliferation rates which were about two-fold slower than that of the parent strain Cu used as a control, and the glycerol consumption and 1,3 -propanediol production thereof were shown to be about 40% and 20%, respectively (FIG. 5). It was shown that oxidation metabolites including 2,3-butanediol other than acetic acid were not produced, and this result was consistent with the genetic background in which the anaerobic metabolic pathway of glycerol in each of the AK and AR recombinant strains was blocked.

Example 3: Preparation of strains in which reductive pathway of glycerol was restored

(1) Preparation of plasmid DNA for restoring reductive pathway of glycerol

A DhaB reactivation enzyme gene (orβV)-orβCDNA fragment and the dhaT, yqhD (derived from E. colϊ) or yqhD homologous gene (derived from Klebsiella pneumoniae) having 1,3 -propanediol oxidoreductase activity were amplified using the following primer sequences. The amplified genes were cloned into a pGEM TEasy vector, and the base sequences thereof were analyzed. Then, as shown in FIG. 5, plasmid DNAs were prepared.

SEQ ID NO: 9: S'-AGATCTATGAGCTATCGTATGTTTGA-S'fdhaT-Bglll F) SEQ ID NO: 10: S'-CTCGAGAAGCTTCAGAATGCCTGGCGGAAAAT-3'fdhaT-HindIII/XhoI R) SEO ID NO: 1 1 : 5'-AGATCTATGAACAACTTTAATCTGCAC-3'(yqhD-BglH F) SEQ ID NO; 12: 5'-AGATCTATGAATAATTTCGACCTGCA-3'(vqhD-HindIII/XhoI R) SEQ ID NO: 13: 5'-AGATCTATGAATAATTTCGACCTGCA-3'(vqhD KIe BgIH F) SEQ ID NO: 14: 5'-CTCGAGAAGCTTAGCGTGCAGCCTCGTAAAT-3'(yqhD KIe Hindlll, Xhol R)

FIG. 5 shows a process for constructing a plasmid DNA containing the DhaT gene and DhaB reactivation enzyme gene of Klebsiella pneumoniae downstream of the lacZ promoter or for constructing a plasmid DNA containing only the DhaT gene downstream of the lacZ promoter, and FIG. 6 shows a process for constructing a plasmid DNA containing the 1,3 -propanediol oxidoreductase activity YqhD(E) gene (derived from E. coli) and the DhaB reactivation enzyme gene downstream of the lacZ promoter or for constructing a plasmid DNA containing only the YqhD(E) gene downstream of the lacZ promoter. FIG. 7 shows a process for constructing a plasmid DNA containing the 1,3 -propanediol oxidoreductase activity YqhD(K) gene (derived from Klebsiella pneumoniae) and the DhaB reactivation enzyme gene downstream of the lacZ promoter or for constructing a plasmid DNA containing only the YqhD(K) gene downstream of the lacZ promoter.

(2) Preparation of recombinant strains in which reductive pathway of glycerol was restored

Each of the above-constructed six kinds of plasmid DNAs containing the gene encoding 1,3 -propanediol oxireductase activity enzyme, and control plasmid DNAs containing pBR322 and the DhaB reactivation enzyme gene was introduced by electroporation into each of the AK and AR strains in which the anaerobic metabolic pathway of glycerol was blocked, thus preparing recombinant strains in which the reductive pathway of glycerol was restored (Table 1). The recombinant strain in which each of the plasmids was introduced into the parent strain Cu was used as a control. The recombinant strains constructed in this Example were deposited at the Biological Resource Center in the Korea Research Institute of Bioscience and Biotechnology (Table 2). Table 1 : Recombinant strains and plasmid DNAs used or constructed in the present invention

Table 2: Deposit numbers of recombinant strains according to the present invention

Example 4: Analysis of characteristics of recombinant strains in which reductive pathway of glycerol was restored

Each of the recombinant strains prepared in Example 3 was inoculated into a medium (50 ml) supplemented with glycerol (2%), tetracycline (10 μg/ml) and

IPTG (0.5 mM) and was cultured at 37 ^°C at 120 rpm, while metabolites in the culture broth were analyzed. The recombinant strains derived from Klebsiella pneumoniae Cu completely consumed the added glycerol after 14 hours of culture, whereas the recombinant strains derived from AK or AR showed glycerol consumption rates slightly slower that those of the Cu-derived recombinant strains, but their ability to produce 1,3 -propanediol was found to be restored (FIG. 8). It was shown that the two YqhD genes derived from E. coli or Klebsiella pneumoniae restored the reductive pathway of glycerol in the same manner as the DhaT gene. Meanwhile, it was found that the AK- or AR-derived recombinant strains in which the oxidation pathway of glycerol was blocked still did not produce metabolites of the oxidative pathway other than a small amount of acetic acid (FIG. 8).

INDUSTRIAL APPLICABILITY

As described above in detail, when the mutant according to the present invention is used, the production of byproducts which are inevitably obtained when producing 1,3 -propanediol from glycerol using prior strains can be minimized, and thus the cost of purification of 1,3-propandiol can be reduced.

Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims

THE CLAIMS

What is Claimed is:

L A microbial mutant in which a gene encoding a transcription activator or a gene encoding dihydroxyacetone kinase is deleted or inactivated in a microorganism having the ability to produce 1,3 -propanediol using glycerol as a carbon source.

2. The microbial mutant according to claim 1, in which a gene encoding glycerol dehydrogenase is additionally deleted or inactivated.

3. The microbial mutant according to claim 1, wherein the microorganism is selected from the group consisting of Citrobacter, Clostridium, Enterobacter, Ilyobacter, Klebsiella, Lactobacillus and Pelobacter.

4. The microbial mutant according to claim 3, wherein said microorganism is Klebsiella pneumoniae, and the gene encoding a transcription activator is DhaR, and the gene encoding dihydroxyacetone kinase is selected from the group consisting of DhaK, DhaL, DhaM and DhaK'.

5. The microbial mutant according to claim 4, in which the glycerol dehydrogenase gene (DhaD) and the transcription activator gene (DhaR) are deleted or inactivated.

6. The microbial mutant according to claim 4, in which the transcription activator gene (DhaR) is deleted or inactivated.

7. A method for preparing 1,3-propanediol, the method comprises producing 1,3- propanediol by culturing the microbial mutant of any one claim among claims 1 -6 in a glycerol-containing medium; and recovering said 1,3-propanediol produced.

8. A mutant having the ability to produce 1,3-propanediol, in which a vector containing a gene encoding 1,3-propanediol oxidoreductase is introduced in a Klebsiella pneumoniae strain (an AK strain) with a deletion of a glycerol dehydrogenase gene (DhaD), a transcription activator gene (DhaR), a 1,3- propanediol oxidoreductase gene (DhaT) and a glycerol dehydratase reactivation factor II gene (DhaBA2) or in which the gene encoding 1,3-propanediol oxidoreductase is inserted into the chromosome of the mutant (the AK strain).

9. A mutant having the ability to produce 1,3-propanediol, in which a vector containing a gene encoding 1,3-propanediol oxidoreductase and a gene encoding glycerol dehydratase reactivation factor is introduced in a Klebsiella pneumoniae strain (an AK strain) with a deletion of a glycerol dehydrogenase gene (DhaD), a transcription activator gene (DhaR), a 1,3-propanediol oxidoreductase gene (DhaT) and a glycerol dehydratase reactivation factor II gene (DhaBA2) or in which the gene encoding 1,3-propanediol oxidoreductase and the gene encoding glycerol dehydratase reactivation gene are inserted into the chromosome of the mutant (the AK strain).

10. A mutant having the ability to produce 1,3-propanediol, in which a vector containing a gene encoding 1,3-propanediol oxidoreductase is introduced in a Klebsiella pneumoniae strain (an AR strain) with a deletion of a transcription activator gene (DhaR), a 1,3-propanediol oxidoreductase gene (DhaT) and a glycerol dehydratase reactivation factor II gene (DhaBA2) or in which the gene encoding 1,3-propanediol oxidoreductase is inserted into the chromosome of the mutant (the AR strain).

1 1. A mutant having the ability to produce 1,3 -propanediol, in which a vector containing a gene encoding 1,3 -propanediol oxidoreductase and a gene encoding glycerol dehydratase reactivation factor is introduced in a Klebsiella pneumoniae strain (an AR strain) with a deletion of a transcription activator gene (DhaR), a 1,3- propanediol oxidoreductase gene (DhaT) and a glycerol dehydratase reactivation factor II gene (DhaBA2) or in which the gene encoding 1,3 -propanediol oxidoreductase and the gene encoding glycerol dehydratase reactivation gene are inserted into the chromosome of the mutant (the AR strain).

12. A method for preparing 1,3 -propanediol, the method comprises producing 1,3- propanediol by culturing the mutant of any one claim among claims 8-11 in a glycerol-containing medium; and recovering said 1,3 -propanediol produced.