KR100531671B1 - Method for the production of 1,3-propanediol from fermentable carbon sources using mixed cultures of two different organisms - Google Patents

Abstract

The invention (a) NAD ⁺ - dependent dehydrogenase having activity claim NAD ⁺ transformants in a gene encoding a polypeptide which converts NADH to the conversion of glycerol to produce a 1,3-propanediol producing organisms and glycerol production organism organism : Mixing at a cell ratio of the 1,3-propanediol producing organism = 1: 0.01 to 1: 1; (b) mixing the glycerol producing organism and the 1,3-propanediol producing organism under microaerobic conditions in the presence of at least one carbon source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides or single carbon substrates. Culturing; And (c) separating 1,3-propanediol from the culture, thereby producing 1,3-propanediol by co-culture of a glycerol producing organism and a 1,3-propanediol producing organism. to provide.

Description

Method for the production of 1,3-propanediol from fermentable carbon sources using mixed cultures of two different organisms}

The present invention relates to a method for producing 1,3-propanediol from fermentable carbon sources using two different organisms under the same culture conditions. More specifically 1,3-propanediol from a given carbon source through co-culture of an organism capable of producing glycerol from a fermentable carbon source and another organism capable of bioconverting the produced glycerol back to 1,3-propanediol It is about the production of.

1,3-propanediol is a very valuable monomer used in the production of polyesters, polyethers, polyurethanes and cyclic compounds used in fibers and carpets.

Various chemical routes to the production of 1,3-propanediol are well known in the art. For example, ethylene oxide can be converted to 1,3-propanediol by a catalyst in the presence of phosphine, water, carbon monoxide and hydrogen and acid. It is also possible to produce 1,3-propanediol from hydrocarbons, such as glycerol, in the presence of carbon monoxide and hydrogen by catalytic reduction in the solution phase of acrolein followed by reduction or by catalysts with group VIII elements of the periodic table. Although it is possible to produce 1,3-propanediol by these methods, it requires a high temperature or high pressure during the production process, which not only incurs a considerable production cost but also generates waste water containing various substances that cause environmental pollution. Due to the production and environmental problems.

Biological production of 1,3-propanediol from glycerol is also a long known method. Strains that produce 1,3-propanediol from glycerol in nature include Citrobacter, Clostridium, Klebsiella, and Lactobacillus. The conversion of glycerol to 1,3-propanediol using glycerol as the only carbon source in anaerobic conditions without other exogenous reducing receptors. The conversion of glycerol to 1,3-propanediol is divided into two stages through two different enzymatic reactions. In the first stage, dehydratase converts glycerol to 3-hydroxypropionaldehyde and 3-hydroxypropionaldehyde. In the second step, 3-hydroxypropionaldehyde is reduced to 1,3-propanediol by NADH-dependent oxidoreductase. Separately, 1,3-propanediol-producing strains provide the carbon and energy needed for bacterial growth through oxidative metabolic pathways of glycerol. Glycerol is first oxidized to dihydroxyacetone (DHA) by NAD ⁺ -dependent glycerol dehydrogenase, where NADH is produced. Subsequently it is converted to dihydroxyacetone phosphate (DHAP) by DHA kinase. The dihydroxyacetone phosphate produced is used for ATP production through glycolysis and the TCA cycle. Strains that produce 1,3-propanediol in nature, such as Citrobacter freundii and Klebsiella pneumoniae, include the four enzymes, glycerol dehydratase (dhaB). ), 1,3-propanediol oxidoreductase (dhaT), glycerol dehydrogenase (dhaD) and dihydroxyacetone kinase (dihydroxyacetone kinase (dhaK) It is related. Thus, the genes encoding these enzymes are organized in the form of dha regulons in which expression of genes can be coordinated. In addition, factors that help reactivate glycerol dihydratase (orfX, orfZ) are also included in the dha regulator. There have been many reports of producing 1,3-propanediol from glycerol by cloning dha regulators involved in the production of 1,3-propanediol from glycerol and expressing it in E. coli. For example, Tong et al. Introduced and expressed cosmids, including dha regulators of Klebsiella pneumoniae (dhaB, dhaT, dhaD, and dhaK), in Escherichia coli. , 3-propanediol was produced (Applied and Environmental Microbiololgy, 57: 3541 (1991)).

As with 1,3-propanediol, glycerol can be produced through chemical and biological pathways. The production of glycerol by the chemical route is through raw materials derived from petroleum such as acrolein, allyl chloride or propylene oxide, but as with 1,3-propanediol, significant production costs And disadvantages of generating environmental pollutants.

There is also a well known method of biological production of glycerol. Representative glycerol-producing strains include yeasts such as S. cerevisiae, C. magnoliae, P. farinose, and C. glycerinogenes. In addition, bacteria such as B. subtilis and algae such as D. tertiolecta are known as production strains. These strains can produce glycerol with glucose or other carbon sources via the fructose-1,6-bisphosphate pathway in glycolysis.

Representative glycerol biosynthetic pathways of known glycerol producing strains are as follows. In short, it is the pathway by which carbon sources such as glucose are bioconverted to glycerol through glycolysis. Glucose is converted to glucose-6-phosphate by hexokinase in the presence of ATP. Glucose-6-phosphate is converted to fructose-6-phosphate by glucose-phosphate isomerase followed by the action of 6-phosphofructokinase To fructose-1,6-bisphosphate, and back to dihydroxyacetone phosphate (DHAP) by aldolase. After this step, it was converted to glycerol-3-phosphate by NADH-dependent G3PDH, followed by glycerol-3-phosphate phosphatase. Dephosphorylation by means of glycerol (Agarwal et al., Adv. Biochem. Engrg. 41: 114 (1990)).

Conventionally, genes encoding glycerol-3-phosphate dehydrogenase that convert DHAP to glycerol-3-phosphate as a gene involved in the glycerol biosynthetic pathway are known. For example, Wang et al. Cloned GPD1 and DAR1 encoding glycerol-3-phosphate dehydrogenase from S. diastaticus (Wang et al., J. Bact. 176: 7091 (1994), Albertyn et al. Cloned GPD1 from S. cerevisiae (Albertyn et al., Mol. Cell Biol. 14; 4135 (1994)). Phosphatases (GPP1, GPP2) which dephosphorylate glycerol-3-phosphate to glycerol were also cloned from Saccharomyces cerevisiae by Norbeck et al. (Norbeck et al., J Biol. Chem. 271; 13875 (1996)). ). The GPD1 and GPP2 genes of Saccharomyces cerevisiae have been shown to be osmosensitivity by the above documents and biosynthesize glycerol as a means to protect themselves.

Recently, literature (International Patent Publications WO9635796, WO9821339, and WO0112833) have been reported that present a method for biologically producing 1,3-propanediol from glucose or other carbon sources using a single microorganism. These documents suggest a method of converting a carbon source such as glucose to glycerol and then back to 1,3-propanediol. For example, genes encoding NADH-dependent glycerol-3-phosphate dehydrogenase (NADH-dependent G3PDH) enzymes and genes encoding glycerol-3-phosphate phosphatase (EC3.1.3.21) may be produced by hosts such as E. coli. Cells are introduced and transformed to biosynthesize glycerol from glucose, which in turn encodes a gene encoding coenzyme B12-dependent dihydratase and a NADH-dependent 1,3-propanediol oxidoreductase in the transformed cells. The gene was added to make a recombinant organism and then to enable the production of 1,3-propanediol from glucose. However, this biological production method produces a 1,3-propanediol by using a single carbon source by a single microorganism, thereby transferring the fermentable carbon source to the 1,3-propanediol production pathway and maintaining a carbon source for cell growth. It is necessary to maintain the proper coordination of migration to the cell, while maintaining the proper coordination of the NADH supply as energy to maintain cell growth and the NADH supply of two molecules as the reducing equivalent receptor required for the production of 1,3-propanediol. There are difficulties in things.

A method of co-culturing a strain producing glycerol from glucose with a strain producing 1,3-propanediol from glycerol and finally producing 1,3-propanediol from glucose has been reported in the literature (US Pat. No. 5,599,689). . The production method is a strain that can produce glycerol from glucose in nature, for example, glycerol such as Saccharomyces cerevisiae, Pichia miso and Zygosaccharomyces rouxii Among E. coli introduced K. pneumoniae, C. freundii, or dha regulators, which can produce 1,3-propanediol from glycerol in one species and in nature. The 1,3-propanediol producing strain is co-cultured to produce glycerol from glucose from the former strain, and the latter absorbs the glycerol discharged into the medium to produce 1,3-propanediol. The production method presented in this document produces 1,3-propanediol from glucose under anaerobic culture conditions. The reason why the co-culture production method follows anaerobic culture conditions is that the activity of glycerol-3-phosphate dehydrogenase involved in glycerol production is NADH dependent in the case of glycerol producing strains such as Saccharomyces cerevisiae. . In other words, the Saccharomyces cerevisiae strain produces glycerol by reoxidizing NADH through the glycerol production pathway in anaerobic conditions without oxygen. In addition, in the case of 1,3-propanediol producing strains such as Klebsiella pneumoniae used in co-culture, the activity of the oxidoreductase enzyme that converts 3-hydroxypropionaldehyde into 1,3-propanediol NADH dependent, producing 1,3-propanediol in the absence of oxygen. These strains convert NADH into NAD ⁺ by glycerol-3-phosphate dehydrogenase enzyme and oxidoreductase enzyme in anaerobic conditions without oxygen, and use NAD ⁺ produced as an electron acceptor instead of oxygen. It produces the necessary energy. However, since the co-culture production method is carried out under anaerobic conditions, the NADH and fermentable carbon source generated in the strain are reduced by other genes induced by anaerobic expression, for example, lactate or lactate. It can be used to make ethanol, succinate, etc., so it can be competitive with the 1,3-propanediol production pathway. Therefore, when producing 1,3-propanediol from the fermentable carbon source according to the production method has a disadvantage in that the productivity is low. In addition, under anaerobic culture conditions, there is a problem that it is difficult to produce a large amount of 1,3-propanediol from glucose in a short time due to the deterioration of the strain.

It is therefore an object of the present invention to provide a method for producing 1,3-propanediol with high efficiency from a low cost carbon source such as glucose through coculture of different organisms.

The present invention provides a method for producing 1,3-propanediol by co-culture of a glycerol producing organism and a 1,3-propanediol producing organism, comprising the following steps:

(a) NAD ⁺ - dependent dehydrogenase claim having activity for the NAD ⁺ transformed with a gene encoding a polypeptide of 1,3-propanediol and glycerol producing organism to produce an organism which converts NADH glycerol producing organisms: the first , 3-propanediol producing organism = 1: mixing at a cell ratio of from 0.01 to 1: 1;

(b) mixing the glycerol producing organism and the 1,3-propanediol producing organism under microaerobic conditions in the presence of at least one carbon source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides or single carbon substrates. Culturing; And

(c) separating 1,3-propanediol from the culture.

In step (a) of the present invention, the glycerol producing organism may be selected from recombinant organisms transformed with Aspergillus, Saccharomyces, or genes involved in glycerol production. The recombinant organisms include Escherichia, Citroacter, Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus, Shizozosaccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Debaryomyces, Muco, Torulopsis, Methylobacter, Salmonella, Bacillus, Streptomyces, Pseudomonas and Corynebacterium It may be selected from the group of the genus composed. Preferably, E. coli of the genus Escherichia.

In addition, genes involved in glycerol production include genes encoding polypeptides having glycerol-3-phosphate dehydrogenase activity and genes encoding polypeptides having glycerol-3-phosphatase activity, but are not limited thereto. no. Examples of the gene encoding the polypeptide having the glycerol-3-phosphate dehydrogenase activity include one or more genes selected from the group consisting of GPD1, GPD2, GPD3, DAR1, gpsA, GUT2, glpD and glpABC. In addition, examples of genes encoding polypeptides having glycerol-3-phosphatase activity include, but are not limited to, one or more genes selected from the group consisting of GPP1 and GPP2.

The 1,3-propanediol producing organism is Citrobacter, Enterobacter, Clostridium, Klebsiella, Lactobacillus genus, or 1,3-propane It may be selected from recombinant organisms transformed with the genes involved in diol production. The recombinant organism is Escherichia, Aerobacter, Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida (Candida), Hansenula, Debaryomyces, Muco, Torulopsis, Methylobacter, Salmonella, Bacillus, Streptomyces Streptomyces, Pseudomonas and Corynebacterium may be selected from the group of the genus. Preferably, E. coli of the genus Escherichia.

In the method of the present invention, a gene involved in the production of 1,3-propanediol, comprising: (a) a gene encoding a polypeptide having at least one dihydratase activity; (b) a gene encoding at least one dihydratase reactivation factor; And (c) a gene encoding a polypeptide having at least one 1,3-propanediol oxidoreductase activity. The gene encoding the polypeptide having dihydratase activity may be selected from the group consisting of a gene encoding glycerol dihydratase and a gene encoding diol dihydratase. In addition, the dihydratase reactivation factor may be selected from the group consisting of orfX and orfZ derived from the dha regulator. However, genes involved in the production of 1,3-propanediol are not limited to the above examples, and any one known in the art can be used.

In the present invention, 1,3-propanediol producing organisms NAD ⁺ - is transformed organism by gene that encodes a polypeptide that can have dependent dehydrogenase activity convert NAD ⁺ to NADH. The NAD ⁺ -dependent dehydrogenase may be an endogenous gene derived from a host organism or may be an exogenous gene derived from other microorganisms such as other bacteria, yeasts or fungi. For example, NAD ⁺ -dependent malate dehydrogenase (sfcA) and NAD ⁺ -dependent alpha-ketoglutarate dehydrogenase and Saccharomyces cerevisiae derived from E. coli DH5α. NAD ⁺ -dependent isocitrate dehydrogenase (IDH) derived from Saccharomyces cerevisiae, NAD ⁺ -dependent formate dehydrogenase (FDH) derived from Staphylococcus aureus, etc. There is this. In the embodiment of the present invention, NAD ⁺ -dependent formate dehydrogenase (FDH) derived from Staphylococcus aureus was used as an example.

In the present invention, "transformation" includes those transformed by the gene itself and its transformation cassette. The "transformation cassette" refers to a vector having a foreign gene and having a factor that facilitates transformation of a specific host cell in addition to the foreign gene. Here, the vector is a self-replicating sequence, a genomic insertion sequence, a phage or nucleotide sequence, linear or circular, single or double stranded DNA or RNA. Generally, vectors include sequences that direct the transcription and translation of appropriate genes, selection markers, and sequences that allow self-replicating or chromosomal insertion. Specific examples of the vector include plasmid vectors (pSE, pBR, pUC, pBluscriptII, pGEM, pTZ, and pET) and phage or cosmid vectors (pWE15, M13, EMBL3, EMBL4, FIX II, DASH II). , ZAP II, gt11, Charon4A, Charon21A), and the like.

In step (a) of the present invention, the glycerol producing organism: the 1,3-propanediol producing organism is preferably mixed in a ratio of 1: 0.01 to 1: 1, but is not limited thereto.

In step (b) of the present invention, the carbon source used may vary depending on the type of glycerol producing organism and 1,3-propanediol producing organism being selected. Examples of the carbon source include glucose and fructose as monosaccharides, sucrose, lactose and maltose as oligosaccharides, starch as polysaccharides, CO ₂ and the like as monosaccharides, but are limited to these examples. It may be used as long as it is known in the art as a carbon source. The co-culture conditions of the present invention vary depending on the type of organism used, and can be cultured under anaerobic conditions or microaerobic conditions. Preferably it is cultured in microaerobic conditions. In the present invention, "microaerobic conditions" refer to conditions having an oxygen partial pressure in atmospheric pressure, that is, an oxygen partial pressure lower than about 0.2 atm. In the case of liquid culture of microorganisms, "microaerobic conditions" refers to an oxygen concentration of 20% or less of the dissolved oxygen tension (DOT) in pure water under the same temperature and pressure. Preferably the oxygen concentration means 20% DOT or less, preferably 10% DOT or less, more preferably 5% DOT, and most preferably 1% DOT or less. In the present invention, by using a silicone foam stopper instead of the face used as a microbial culture flask stopper for aerobic cultivation of microorganisms in general, the microbial culture is naturally fine by blocking the smooth oxygen supply in the culture flask. Aerobic culture conditions were maintained.

In step (c) of the present invention, 1,3-propanediol may be separated according to conventional separation methods known in the art. For example, after removing the biomass from the culture by filtration or centrifugation, the obtained supernatant may be separated by a method such as distillation or extraction.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example

Example 1 Preparation of Expression Cassette of Glycerol 3-Phosphate Dehydrogenase (GPD1)

Genomic DNA was extracted from Saccharomyces cerevisiae (KCTC 0119) strain, which was distributed by the Korea Institute of Bioscience and Biotechnology. Using the extracted DNA as a template, polymerase chain reaction (PCR) was carried out using synthetic primers (SEQ ID NOs: 1 and 2) containing the nucleotide sequences of the 5'-terminal NheI recognition site and the 3'-terminal SacI recognition site, and GPD1 ( NCBI X76859, SGD GPD1 / YDL022W) was amplified isolated. Amplified isolated GPD1 gene fragment was cloned into pCR2.1 vector using TOPO TA Cloning kit (Invitrogen, USA) to prepare pCGD1.

After the electrophoresis was performed by treating the prepared pCGD1 with NheI and SacI restriction enzymes, GPD1 gene fragments were recovered using a Qiagen gel recovery kit. In addition, the expression vector pSE380 (Invitrogen, USA) was treated with NheI and SacI restriction enzymes and then treated with AP (alkaline phosphatase) again to recover the pSE380 vector as described above. The expression cassette pSGD1 was constructed by cloning the NDL-SecI-generated GPD1 gene fragment into NheI / SacI / AP-treated pSE380 vectors into the same site.

Example 2 Preparation of Expression Cassette of Glycerol 3-Phosphatase (GPP2)

Synthetic primer (SEQ ID NO: 3) comprising a nucleotide sequence of 5 'terminal NheI recognition site and 3' terminal SacI recognition site using genomic DNA of Saccharomyces cerevisiae (KCTC 0119) as a template And 4) was used to perform polymerase chain reaction (PCR) and amplification isolation of GPP2 (SGD HOR2 / YER062C). Amplified isolated GPP2 gene fragment was cloned into pCR2.1 vector using TOPO TA Cloning kit (Invitrogen, USA) to prepare pCGP2.

The prepared pCGP2 was treated with NheI and SacI restriction enzymes as in Example 1 to recover the GPP2 gene fragment, which was cloned into NheI / SacI / AP (alkaline phosphatase) treated expression vector pSE380 into the same site to express the expression cassette pSGP2. Produced.

Example 3: co-expression cassette and transgenic E. coli of GPD1 and GPP2 genes

A method of inserting a GPP2 gene segment into a pSGD1 plasmid by allowing pSGD1 and pSGP2, whose expression is regulated by each trc promoter and transcription terminator, to be transcribed by one trc promoter into one expression vector Used.

First, in order for two genes to be transcribed by one promoter, a target gene must be located between the promoter and the terminator, and a ribosome-binding site (RBS) is included in front of the gene sequences. Must have a genetic structure (operon)

In this example, a new pCRGP2 containing a ribosome binding sequence and a GPP2 gene fragment was constructed. Synthetic primer (SEQ ID NO: 5) having a 5 'terminal SacI recognition site comprising a ribosomal binding sequence present in pSE380 using the pSGP2 plasmid cloned in Example 2 as a template and the 3' terminal SacI recognition used in Example 2 Polymerase chain reaction (PCR) was carried out using a synthetic primer having a site (SEQ ID NO: 4) and amplification and isolation of GPP2 including ribosomal binding sequence (RBS). Amplified isolated RBS / GPP2 gene segments were cloned into pCR2.1 vector using TOPO TA Cloning kit (Invitrogen, USA) to prepare pCRGP2.

The prepared pCRGP2 was treated with SacI restriction enzyme to recover the RBS / GPP2 gene fragment, and the pSGD1 prepared in Example 1 was cloned into the same site of the SacI / AP-treated expression vector to prepare an expression cassette named pSG1. . The expression cassette was introduced into E. coli host cells (DH 5α) to prepare E. coli DH5α (pCJP-005) which was finally transformed.

Example 4: Production of Glycerol from Transformed Escherichia Coli DH5α (pCJP-005)

In this example, the production of glycerol from glucose was confirmed using the transformed E. coli DH5α (pCJP-005) prepared in Example 3.

First, one colony (E. coli) grown with Escherichia coli DH5α (pCJP-005) was inoculated in 3 ml of LB medium containing 100 µg / ml of ampicillin and incubated at 37 ° C for 16 hours. 1 ml of the E. coli culture was added to a 250 ml shake flask containing 50 ml of the following glycerol production medium, the medium inlet was blocked with a silicone stopper, and then cultured at 50 rpm and 200 rpm for 48 hours at 37 ° C. .

The glycerol production medium is sterilized by adding 8 g of KH ₂ PO _4, 8 g of (NH ₄ ) ₂ HPO ₄ , 2.05 g of MgSO ₄ , 40 mg of CaCl _2, 40 mg of FeSO ₄ , and a trace element solution to 1 L of distilled water. . The glycerol production medium contains 10 ml of trace element solution. The trace element liquid composition was composed of 0.8 g of CoCl ₂ ㆍ 6H ₂ O, ZnSO ₄ ㆍ 7H ₂ O 0.4g, Na ₂ MoO ₄ ㆍ 2H ₂ O 0.4g, CuCl ₂ ㆍ 2H ₂ O 0.2g, H ₃ BO _{3 in} 1 L of distilled water. 0.1 g, HCl (37%) is included. In addition, separately sterilized glucose and yeast extract (20g / L and 5g / L, respectively) to the glycerol production medium, mixed with antibiotics (ampicillin 100㎍ / ㎖) and IPTG (1mM) Add before E. coli inoculation.

HPLC module type (Waters 510 pump, Waters 717 Autosampler, Waters 400 RI detector, SP 4290 Intergrator) was used to analyze the amount of glycerol produced and glucose consumed.

As a result of E. coli culture analysis, glycerol production was not found in the control group transformed with E. coli pSE380 vector, but in the experimental group using E. coli DH5α (pCJP-005), 1.562 g / L at 50 rpm and 3.980 g / L at 200 rpm Glycerol was produced. From the results of the analysis, the conversion of glucose to glycerol was relatively different from that of microaerobic culture conditions (50 rpm) with relatively low dissolved oxygen concentrations, and glycerol biosynthesis under microaerobic culture conditions (200 rpm) with slightly higher dissolved oxygen concentrations. This high was confirmed (see Table 1).

Table 1. Conversion of glucose to glycerol by transgenic E. coli DH5α (pSE380) and DH5α (pCJP-005) (unit: g / L)

Transformant Stirring speed Consumed Glucose Produced Glycerol DH5α (pSE380) control 50 rpm 10.000 0.000 200 rpm 10.000 0.000 DH5α (pCJP-005) experimental group 50 rpm 6.484 1.562 200 rpm 10.000 3.980

Example 5 Subcloning and Transgenic Escherichia Coli of Klebsiella pneumoniae

In this embodiment, the glycerol dihydratase gene (dhaB), dihydratase reactivation factors (orfX, orfZ) of Klebsiella pneumoniae, which has the activity of converting glycerol to 1,3-propanediol And prior to subcloning the dha regulatory (NCBI acession U30903) containing NADH-dependent 1,3-propanediol oxidoreductase (dhaT), the National Institute of Biological Information (US) (NCBI) (www.ncbi.nlm.nih.gov) gene search was used.

Genomic DNA was extracted from a Klebsiella pneumoniae (ATCC 25955) strain from the American Type Culture Collection (ATCC) using a Qiagen chromosome recovery kit. In this embodiment, the gene group size of the dha regulators to be subcloned is 9 kb or more, and thus, it is difficult to amplify the target gene group through a single polymerase chain reaction (PCR). Subcloning of the dha regulators was performed by dividing into group B), amplifying and then combining into one group.

First, two pairs of synthetic primers were prepared to amplify the two gene subgroups. One pair is a primer having a 5'-terminal HindIII recognition site (SEQ ID NO: 6) and a primer containing a 3'-terminal KpnI cleavage sequence of the dha regulatory base (SEQ ID NO: 7). The other pair uses a primer (SEQ ID NO: 8) and a 3 'terminal EcoRI recognition site containing a 5' terminal KpnI cleavage sequence of the dha regulator internal sequence to link with the front 3 'terminal KpnI of the dha regulatory constant. Primer (SEQ ID NO: 9).

Synthetic primer (SEQ ID NO: 6) comprising the base sequences of the 5'-terminal HindIII recognition site and the 3'-terminal KpnI recognition site using genomic DNA as a template for amplifying the dha regulation gene group A (SEQ ID NO: 6). And 7) was used to perform polymerase chain reaction (PCR) and genogroup A was amplified and isolated. Amplified isolated dha regulator gene group A fragment was cloned into pCR2.1 vector using TOPO TA Cloning kit (Invitrogen, USA) to prepare pCDA.

Synthetic primer (SEQ ID NO: 8) comprising the base sequences of the 5'-terminal KpnI recognition site and the 3'-terminal EcoRI recognition site, using genomic DNA as the template for amplifying dha regulation gene group B (SEQ ID NO: 8). And 9) were used to perform polymerase chain reaction (PCR) and genogroup B was amplified and isolated. Amplified isolated dha regulator gene group B fragment was cloned into pCR2.1 vector using TOPO TA Cloning kit (Invitrogen, USA) to prepare pCDB.

To combine the Dha regulatory gene groups A and B, first, a vector was created to combine the two gene groups into one. Since the size of the gene group to be inserted can be very large, cloning efficiency can be reduced, and ampicillin among antibiotic resistance markers in the vector is processed by processing DraI restriction enzyme to reduce the size of the existing pCR2.1 vector (3.9kb). ) -Resistance markers were removed. Therefore, this vector (pCR2.1 / Kan) has only resistance markers for kanamycin antibiotics. The prepared pCDA was treated with HindIII / KpnI to recover a gene group A fragment, and the expression vector, pCR2.1 / Kan, was cloned into the same site treated with HindIII / KpnI / AP to prepare pCR / Kan-A. In addition, pCDB was treated with KpnI / EcoRI to recover genogroup B fragments, which were cloned into the same region of the previously prepared pCR / Kan-A vector treated with KpnI / EcoRI / AP, and a group of dha regulatory genes named pCRRZ. An expression cassette was produced. In addition, pCRRZ was treated with HindIII / XbaI to recover the dha regulatory gene fragment, which was cloned into the same region of pCL1920 vector, a low copy number vector prepared by HindIII / XbaI / AP, and another dha named pCLRZ. The regulation gene expression cassette was produced. The promoters and terminators of the expression cassettes were designed to express genes related to 1,3-propanediol production from glycerol using those of Klebsiella pneumoniae dha regulator itself, which is not provided externally artificially. Subsequently, pCRRZ and pCLRZ were introduced into E. coli host cells (DH5α) to prepare E. coli DH5α (pCRRZ) and DH5α (pCLRZ) which were finally transformed.

Example 6: Production of 1,3-propanediol from transformed E. coli DH5α (pCLRZ)

In this example, the production of 1,3-propanediol from glycerol was confirmed using the transformed E. coli DH5α (pCLRZ) prepared in Example 5.

First, one colony (colon) grown with E. coli DH5α (pCLRZ) was inoculated in 3 ml of LB medium containing 100 µg / ml of spectinomycin and incubated at 37 ° C for 16 hours. 1 ml of the E. coli culture was added to a 250 ml shake flask containing 50 ml of 1,3-propanediol production medium, and the medium inlet was blocked with a silicone foam stopper, and then cultured at 50 rpm and 200 rpm for 48 hours at 37 ° C.

The 1,3-propanediol production medium was put in 1L of distilled water, Na ₂ HP ₄ 6g, KH ₂ PO ₄ 3g, NH ₄ Cl 2g, NaCl 0.5g, yeast extract 5g and MgSO ₄ 2mM, Sterilized by adjusting the pH to 6.8. In addition, the sterilized glycerol was mixed with the 1,3-propanediol production medium at 20 g / L and mixed therein, and inoculated with E. coli with spectinomycin (100 μg / ml) and coenzyme B12 (30 nM). Add before.

The HPLC module type (Waters 510 pump, Waters 717 Autosampler, Waters 400 RI detector, SP 4290 Intergrator) was used to analyze the amount of 1,3-propanediol and glycerol consumed.

As a result of E. coli culture analysis, 1,3-propanediol production was not found in the control group transformed with E. coli pCL1920 vector, but in the experimental group using DH5α (pCLRZ) 6.060g / L at 50rpm microaerobic culture conditions, the At slightly higher aerobic conditions (200 rpm), 1.994 g / L of 1,3-propanediol was produced. The results showed that the conversion of glycerol to 1,3-propanediol showed high 1,3-propanediol biosynthesis at 50 rpm microaerobic culture conditions with relatively low dissolved oxygen concentration in the culture medium (Table 2).

Table 2. Production of 1,3-propanediol from glycerol by transgenic E. coli DH5α (pCLRZ) (g / L)

Transformant Stirring speed Consumed Glycerol 1,3-propanediol production DH5α (pCL1920) control 50 rpm 8.895 0.000 200 rpm 9.870 0.000 DH5α (pCLRZ) experimental group 50 rpm 10.085 6.060 200 rpm 10.661 1.994

Example 7: Production of 1,3-propanediol from glucose through co-culture of glycerol-producing E. coli DH5α (pCJP-005) and 1,3-propanediol production E. coli DH5α (pCRRZ)

In this example, the transformed E. coli DH5α (pCJP-005) and E. coli DH5α (pCRRZ) produced in Example 3 and Example 5 were co-cultured to confirm the production of 1,3-propanediol from glucose.

First, 3 ml of LB medium containing 100 μg / ml of ampicillin and 25 μg / ml of kanamycin, each colony grown with E. coli DH5α (pCJP-005) and E. coli DH5α (pCRRZ). Were inoculated respectively and incubated at 37 ° C. for 16 hours. 1 ml of each E. coli culture was added to a 250 ml shake flask containing 50 ml of 1,3-propanediol production integrated medium, and the media inlet was closed with a silicone foam stopper and a face stopper, respectively, and then at 37 ° C. for 24 hours, Incubated at 200 rpm for 48 hours.

The 1,3-propanediol production integrated medium was placed in 1 L of distilled water, Kg ₂ PO ₄ 8g, (NH ₄ ) ₂ HPO ₄ 8g, MgSO ₄ 2.05g, CaCl ₂ 40mg, FeSO ₄ 40mg and a trace element solution, pH Sterilized to 6.7. The 1,3-propanediol production integrated medium contains 10 ml of trace element solution. The trace element liquid composition was composed of 0.8 g of CoCl ₂ ㆍ 6H ₂ O, ZnSO ₄ ㆍ 7H ₂ O 0.4g, Na ₂ MoO ₄ ㆍ 2H ₂ O 0.4g, CuCl ₂ ㆍ 2H ₂ O 0.2g, H ₃ BO _{3 in} 1 L of distilled water. 0.1 g, HCl (37%) is included. In addition, separately sterilized glucose and yeast extract (10 g / L and 5 g / L, respectively) to the 1,3-propanediol production integrated medium, and mixed with IPTG (1 mM) and coenzyme ( coenzyme) B12 (30 nM) is added before E. coli inoculation.

The HPLC module type (Waters 510 pump, Waters 717 Autosampler, Waters 400 RI detector, SP 4290 Intergrator) was used to analyze the amount of 1,3-propanediol, glycerol and glucose consumed.

As a result of E. coli culture analysis, when E. coli DH5α (pCJP-005) and E. coli DH5α (pCRRZ) were co-cultured in aerobic culture conditions using the presence cap, glycerol was 3.328 g / L and 3.112 for 24 hours and 48 hours, respectively. g / L was produced, but 1,3-propanediol was not produced, and under aerobic culture conditions (200 rpm) using a silicone foam stopper, glycerol and 1,3-propanediol were 3.367 g / L for 24 hours of incubation, respectively. It was confirmed that 1.533g / L and 0.790g / L were produced in L, 0.472g / L and 48 hours of incubation, respectively. The results showed that glycerol biosynthesis from glucose was similar to Example 4 under microaerobic culture conditions (200rpm) using silicone foam stopper, but it was confirmed that the conversion from glycerol to 1,3-propanediol was not excellent. . This is because, as in Example 6, the biosynthesis of 1,3-propanediol from glycerol is better at culture conditions with slightly lower dissolved oxygen concentrations. In addition, it was confirmed that the production of 1,3-propanediol from the glycerol at all in aerobic culture conditions with a smooth oxygen supply through the face stopper.

Table 3. Co-culture of transformed E. coli DH5α (pCJP-005) & DH5α (pCRRZ)

Production of 1,3-propanediol from glucose (g / L)

Co-culture transformants stopper Incubation time (hours) Glucose consumed (g) Produced Glycerol (g / l) 1,3-propanediol produced (g / l) DH5α (pCJP-005) & DH5α (pCRRZ) Silicone foam stopper 24 10.000 3.367 0.472 48 10.000 1.533 0.790 Face plug 24 10.000 3.328 0.000 48 10.000 3.112 0.000

Example 8 NAD ⁺ Dependence Subcloning and Transgenic Escherichia Coli of Dehydrogenase Gene

In this embodiment, NAD ⁺ - dependent dehydrogenase claim having activity as an exogenous gene of the gene, which encodes a polypeptide capable of converting NAD ⁺ to NADH Staphylococcus aureus (Staphylococcus aureus) of NAD ⁺ - dependent PO Mate dehydrogenase (FDH) (NCBI acession AP004822.1) was subcloned and introduced into E. coli DH5α (pCLRZ) containing the dha regulator of Klebsiella pneumoniae.

Genomic DNA was extracted from a Staphylococcus aureus (ATCC 6538) strain from the American Type Culture Collection (ATCC) using a Qiagen chromosome recovery kit. Using the extracted DNA as a template, polymerase chain reaction (PCR) was carried out using synthetic primers (SEQ ID NOs: 10 and 11) including the nucleotide sequences of the 5'-terminal NdeI recognition site and the 3'-terminal NsiI recognition site, and FDH Amplification was isolated. Amplified isolated FDH gene fragment was cloned into pCR2.1 vector using TOPO TA Cloning kit (Invitrogen, USA) to prepare pCFDH. Since the FDH gene has been cloned from the start codon without its own promoter, it may be normally operated by introducing a promoter from the outside. For the normal expression of FDH, the promoter of ptsG gene whose expression is induced by glucose in medium among the promoters present in the host cell and the promoter of sucA gene whose expression is increased in the presence of glycerol in medium were cloned and introduced into the FDH gene. Synthetic primer (ptsG promoter: SEQ ID NO: 12,13; sucA promoter: SEQ ID NO: 14,15) containing the nucleotide sequences of the 5 'terminal HindIII recognition site and the 3' terminal NdeI recognition site, respectively, using genomic DNA of E. coli DH5α as a template The polymerase chain reaction (PCR) was performed and the ptsG promoter and sucA promoter were amplified and isolated. Amplified isolated ptsG promoter and sucA promoter fragments were cloned into pCR2.1 vector using TOPO TA Cloning kit (Invitrogen, USA) to prepare pCPP and pCSP, respectively.

PCFDH was treated with NdeI and SpeI restriction enzymes, followed by electrophoresis, and the FDH gene fragment was recovered using a Qiagen gel recovery kit. In addition, pCPP and pCSP were treated with NdeI and SpeI restriction enzymes, and then AP (alkaline phosphatase) was retreated, and pCPP and pCSP were recovered as described above. An expression cassette pCPF and pCSF were prepared by cloning the FDH gene fragment cut with the NdeI and SpeI prepared above into the same site with the NdeI / SpeI / AP treated pCPP and pCSP vectors. Subsequently, pCPF and pCSF were introduced into E. coli DH5α (pCLRZ) prepared in Example 5 to prepare transformed Escherichia coli DH5α (pCLRZ / pCPF) and DH5α (pCLRZ / pCSF).

Example 9: Production of 1,3-propanediol from transformed E. coli DH5α (pCLRZ / pCPF) and DH5α (pCLRZ / pCSF)

In this example, the production of 1,3-propanediol from glycerol was confirmed using the transformed E. coli DH5α (pCLRZ / pCPF) and E. coli DH5α (pCLRZ / pCSF) prepared in Example 8.

First, one colony in which Escherichia coli DH5α (pCLRZ / pCPF) and Escherichia coli DH5α (pCLRZ / pCSF) was grown, each containing 100 µg / ml ampicillin and 100 µg / ml spectinomycin Inoculated in 3 ml of LB medium and incubated for 16 hours at 37 ℃. 1 ml of each E. coli culture was added to a 250 ml shake flask containing 50 ml of 1,3-propanediol production medium, the medium inlet was blocked with a silicone foam stopper, and then cultured at 200 rpm for 24 hours at 37 ° C.

The 1,3-propanediol production medium was put in 1L of distilled water, Na ₂ HP ₄ 6g, KH ₂ PO ₄ 3g, NH ₄ Cl 2g, NaCl 0.5g, yeast extract 5g and MgSO ₄ 2mM, Sterilized by adjusting the pH to 6.8. In addition, separately sterilized glycerol was mixed at 20 g / L to the 1,3-propanediol production medium, and antibiotics (ampicillin 100 ㎍ / ㎖, spectinomycin 100 ㎍ / ㎖) and coenzyme B12 (30 nM) ) Is added before E. coli inoculation.

The HPLC module type (Waters 510 pump, Waters 717 Autosampler, Waters 400 RI detector, SP 4290 Intergrator) was used to analyze the produced 1,3-propanediol and the amount of glycerol consumed.

As a result of E. coli culture analysis, it was confirmed that the control group transformed with E. coli pCLRZ produced 1.177 g / L of 1,3-propanediol in 200 rpm culture of microaerobic conditions. In contrast, the experimental groups of transformed E. coli DH5α (pCLRZ / pCPF) and transformed E. coli DH5α (pCLRZ / pCSF) produced 3.163 g / L and 6.120 g / L, respectively. These results suggest that the introduction of NAD ⁺ -dependent formate dehydrogenase (FDH) gene can increase the amount of NADH available in transgenic E. coli, thus activating the NADH-dependent oxidoreductase. Enzyme (active form) increased to induce the mass production of the anaerobic 1,3-propanediol even under microaerobic culture conditions (see Table 4).

Table 4. Transgenic E. coli DH5α (pCLRZ / pCPF) and DH5α (pCLRZ / pCSF)

1,3-propanediol production from glycerol (g / L)

Transformant Consumed Glycerol 1,3-propanediol produced DH5α (pCLRZ) 4.398 1.177 DH5α (pCLRZ / pCPF) 5.415 3.163 DH5α (pCLRZ / pCSF) 11.008 6.120

Example 10 NAD ⁺ Dependent Formate Subcloning of Dehydrogenase Gene into pCRRZ and Construction of Transgenic E. Coli

In this example, by subcloning the NAD ⁺ -dependent formate dehydrogenase gene (FDH) into the transformation cassette pCRRZ prepared in Example 5, the dha regulator genes and the formate dehydrogena by one cassette The first gene was allowed to be expressed simultaneously.

First, pCPF containing the FDH gene induced by glucose prepared in Example 8 was treated with NsiI restriction enzymes, followed by electrophoresis, and then ptsG promoter-FDH gene fragment using Qiagen gel recovery kit. Was recovered. In addition, pCRRZ prepared in Example 5 was treated with NsiI restriction enzyme, and then AP (alkaline phosphatase) was again treated and pCRRZ was recovered as described above. An expression cassette pCJP-023 was constructed by subcloning the ptsG promoter-FDH gene fragment cleaved with NsiI into the same region of the NsiI / AP treated pCRRZ vector. Thereafter, pCJP-023 was introduced into Escherichia coli DH5α to finally produce transformed Escherichia coli DH5α (pCJP-023).

Example 11: Production of 1,3-propanediol from glucose through co-culture of glycerol-producing E. coli DH5α (pCJP-005) and 1,3-propanediol production E. coli DH5α (pCJP-023)

In this example, co-cultivation of each of the transformed Escherichia coli DH5α (pCJP-005) and Escherichia coli DH5α (pCJP-023) prepared in Example 3 and Example 10 confirmed the production of 1,3-propanediol from glucose. It was.

First, 3 ml of colony (coony) from which Escherichia coli DH5α (pCJP-005) and Escherichia coli DH5α (pCJP-023) were grown, each containing 100 µg / ml of ampicillin and 25 µg / ml of kanamycin Each was inoculated in LB medium and incubated at 37 ° C. for 16 hours. 1 ml of each E. coli culture was added to a 250 ml shake flask containing 50 ml of 1,3-propanediol production integrated medium, the medium inlet was closed with a silicone foam stopper, and then 200 rpm for 24 hours at 37 ° C. for 48 hours. Incubated at.

The 1,3-propanediol production integrated medium is the same as in Example 7, and if necessary for the culture experiment, formate (formate; pH 6.8, 30 mM) is added before inoculation of E. coli.

HPLC module types (Waters 510 pump, Waters 717 Autosampler, Waters 400 RI detector, SP 4290 Intergrator) were used to analyze the amount of 1,3-propanediol, residual glycerol and glucose.

E. coli culture analysis revealed that formate dehydrogenase gene was co-cultured with E. coli DH5α (pCJP-005) and E. coli DH5α (pCJP-023) incorporating NAD ⁺ -dependent formate dehydrogenase gene (FDH). It can be seen that the productivity of 1,3-propanediol increased more than two times compared to the control without introducing. In addition, it was also confirmed that the productivity of 1,3-propanediol was increased when co-culture of transgenic E. coli was added by adding formate to the 1,3-propanediol production integrated medium. The cause of the increase in 1,3-propanediol productivity due to the addition of formate in the medium is the formate absorbed into E. coli by NAD ⁺ -dependent formate dehydrogenase (FDH) introduced into 1,3-propanediol producing E. coli. Is converted to NADH, which is required for 1,3-propanediol production (see Table 5).

Table 5. Production of 1,3-propanediol from glucose by co-culture of transgenic E. coli DH5α (pCJP-005) & DH5α (pCRRZ) or DH5α (pCJP-005) & DH5α (pCJP-023) (g / L)

Coculture transformants Incubation time Formate addition Consumed Glucose Produced Glycerol 1,3-propanediol produced DH5α (pCJP-005) & DH5α (pCRRZ) 24 hours radish 10.000 3.367 0.472 48 hours 10.000 1.533 0.790 DH5α (pCJP-005) & DH5α (pCJP-023) 24 hours 10.000 1.464 1.487 48 hours 10.000 0.000 1.632 24 hours U 10.000 0.870 1.719 48 hours 10.000 0.000 1.861

Example 12: Production of 1,3-propanediol from glucose according to the mixing ratio of E. coli DH5α (pCJP-005) and 1,3-propanediol production in co-culture of E. coli DH5α (pCJP-023)

In this example, 1,3-propane from glucose was co-cultured by varying the mixing ratio of each of the transformed E. coli DH5α (pCJP-005) and E. coli DH5α (pCJP-023) prepared in Example 3 and Example 10. Diol production difference was confirmed.

First, 3 ml of colony (coony) in which E. coli DH5α (pCJP-005) and E. coli DH5α (pCJP-023) were grown, each containing 100 μg / ml of ampicillin and 25 μg / ml of kanamycin Each was inoculated in LB medium and incubated at 37 ° C. for 16 hours. 0.01 ml to 1 ml of each E. coli culture was added to a 250 ml shake flask containing 50 ml of the same 1,3-propanediol production integrated medium as in Example 7 to which Formate (pH 6.8, 30 mM) was added. The medium inlet was blocked with a silicone foam stopper and then incubated at 37 rpm for 50 hours at 50 rpm and 200 rpm (see Table 6).

HPLC module types (Waters 510 pump, Waters 717 Autosampler, Waters 400 RI detector, SP 4290 Intergrator) were used to analyze the amount of 1,3-propanediol, glycerol and glucose consumed.

As a result of E. coli culture analysis, the production of 1,3-propanediol was higher when the initial mixing ratio of glycerol-producing E. coli (DH5α (pCJP-005)) was equal to or higher than that of 1,3-propanediol producing E. coli (DH5α (pCJP-023)). This increase was confirmed. In addition, it was confirmed that the productivity of 1,3-propanediol was excellent at 200 rpm culture conditions where the dissolved oxygen concentration was relatively higher than the micro-aerobic culture conditions of 50 rpm (see Table 6).

In the case of the literature suggesting a method for producing 1,3-propanediol from glucose through a coculture method similar to the present invention (US Pat. No. 5,599,689), the proportion of 1,3-propanediol-producing organisms is higher than that of glycerol-producing organisms. In this case, while the production of 1,3-propanediol is increased, in the present invention, when the ratio of glycerol producing organic gas is higher (e.g., glycerol producing organic gas: 1,3-propanediol producing organic gas = 1: 0.1, 1: 0.5) ), The production of 1,3-propanediol increased. In addition, in the present invention, it was confirmed that the yield of 1,3-propanediol was superior to that of the literature even under similar ratio conditions (1: 0.1 <ratio <1: 0.5).

Another difference between the document (US Pat. No. 5,599,689) and the present invention is that it produces 1,3-propanediol in microaerobic culture conditions in which a small amount of oxygen is provided, rather than anaerobic culture methods. In anaerobic culture conditions to organisms that use the NAD ⁺ generated in the oxygen rather than sparse by generating NAD ⁺ using NADH as an electron acceptor to produce energy for the cells. Therefore, anaerobic culturing is preferred because NADH is required to produce 1,3-propanediol from glucose. However, the present invention artificially increased the amount of NADH available in an organism by introducing and expressing a formate dehydrogenase gene capable of providing NADH to the organism. This enhances the anaerobic metabolic pathway for the use of NADH in microaerobic cultures, where small amounts of oxygen are provided to control the redox balance of NADH / NAD ^{+ in} the organism. Even in microaerobic culture conditions in which oxygen is provided, 1,3-propanediol can be mass produced from glucose.

Table 6. Comparison of 1,3-propanediol production difference from glucose according to the mixing ratio of co-culture of transgenic E. coli DH5α (pCJP-005) and DH5α (pCJP-023)

 Initial glucose concentration of 10 g / L units (g / L)

Coculture transformants Mixing ratio Stirring speed Consumed Glucose Produced Glycerol 1,3-propanediol produced DH5α (pCJP-005) & DH5α (pCJP-023) 1: 1 50 rpm 10.000 0.000 0.450 1: 0.01 200 rpm 10.000 3.140 0.100 1: 0.1 10.000 0.000 1.805 1: 0.5 10.000 0.000 1.680 1: 1 10.000 0.000 1.476 0.5: 1 10.000 0.000 0.974 0.1: 1 10.000 0.000 0.000 0.01: 1 10.000 0.000 0.000

* Mixing ratio = glycerol production E. coli: 1,3-propanediol production E. coli

* Mixing ratio example; 1: 1 = 1 ml: 1 ml, 1: 0.01 = 1 ml: 0.01 ml

Transgenic Escherichia coli DH5α (pCJP-005) and DH5α (pCJP-023) used in the present invention were deposited on the Korea Microorganism Conservation Center, an international depository, on October 17, 2003, respectively, Accession No. KCCM-10518 and KCCM-10519. Was granted.

According to the method for producing 1,3-propanediol of the present invention, an NADH produced in each organism through co-culture of a glycerol producing organism and a 1,3-propanediol producing organism under the same microaerobic conditions As a result, the production of 1,3-propanediol from glucose can be efficiently achieved by using it for energy production for growth or appropriately for glycerol production or 1,3-propanediol production.

According to the present invention, induction of a large amount of NADH in a recombinant organism through the introduction of a gene having a dehydrogenase activity capable of reducing NAD ⁺ to NADH into a 1,3-propanediol producing organism in large quantities in anaerobic culture conditions Not only enables the mass production of 1,3-propanediol produced under microaerobic culture conditions, but also increases the growth of organisms by culturing the organisms under anaerobic and microaerobic conditions. The productivity improvement of 3-propanediol can be achieved. In addition, anaerobic or aerobic culture conditions require the process of completely removing or supplying a large amount of oxygen due to the nature of the production process, and incurring considerable production costs in this process, while microaerobic culture conditions have lower production costs. In this respect, it can be an advanced production method of comparative advantage industrially.

<110> CJ Corporation <120> Method for the production of 1,3-propanediol from fermentable carbon sources using mixed cultures of two different organisms <160> 15 <170> KopatentIn 1.71 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gctagcgatg tctgctgctg ctga 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gagctcctaa tcttcatgta gatc 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gctagcaatg ggattgacta ctaa 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gagctcttac catttcaaca gatc 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gagctcaggt aattataacc cggg 24 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ggaagcttaa accatccata atatgcc 27 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ttagctgcag accgtacaga gattg 25 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 tgcttatcca gcggatggac 20 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ggaattccct gaaatagcac gttaaag 27 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gggtcatatg tcaaacggtg ccgttt 26 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 atgcatagcc gtgactatac ttagatg 27 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 aagcttgtgc aacttctcca atgatct 27 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 catatgtgaa gatgatcctg agtatgg 27 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 aagcttatcc ggcctacaag tcattac 27 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gttctgcata tggatccctt aagcatc 27

Claims (15)

(a) NAD ⁺ - dependent dehydrogenase claim having activity of NAD ⁺ transformed with a gene encoding a polypeptide which converts NADH into 1,3- propanediol producing organism, and glycerol production organism the glycerol producing organism: the 1,3-propanediol producing organism = 1: mixing at a cell ratio of from 0.01 to 1: 1; (b) mixing the glycerol producing organism and the 1,3-propanediol producing organism under microaerobic conditions in the presence of at least one carbon source selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides or single carbon substrates. Culturing; And (c) separating 1,3-propanediol from the culture, the method of producing 1,3-propanediol by co-culture of glycerol producing organisms and 1,3-propanediol producing organisms. The method of claim 1 wherein the glycerol producing organism is selected from Aspergillus, Saccharomyces, or recombinant organisms transformed with genes involved in glycerol production. The method of claim 2, wherein the recombinant organism is Escherichia, Citrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus, Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Devari Debaryomyces, Mucor, Torulopsis, Methylobacter, Salmonella, Bacillus, Streptomyces, Pseudomonas and Corynes The method is selected from the group consisting of Corynebacterium. The method of claim 1, wherein the 1,3-propanediol producing organism is Citrobacter (Enteritacter), Enterobacter (Clostridium), Klebsiella (Klebsiella), Lactobacillus (Lactobacillus) genus, Or a recombinant organism transformed with a gene involved in 1,3-propanediol production. The method of claim 4, wherein the recombinant organism is Escherichia, Aerobacter, Schizosaccharomyces, Zygosaccharomyces, Pichia, Cluj Veromai Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Salmonella, Bacillus ), Streptomyces, Pseudomonas and Corynebacterium. The method of claim 3, wherein the gene involved in glycerol production encodes a polypeptide having glycerol-3-phosphate dehydrogenase activity selected from the group consisting of GPD1, GPD2, GPD3, DAR1, gpsA, GUT2, glpD and glpABC. How to be a gene. The method of claim 3, wherein the gene involved in glycerol production is a gene encoding a polypeptide having glycerol-3-phosphatase activity selected from the group consisting of GPP1 and GPP2. The gene according to claim 5, which is involved in 1,3-propanediol production, (a) a gene encoding a polypeptide having at least one dihydratase activity; (b) a gene encoding at least one dihydratase reactivation factor; And (c) a gene comprising a gene encoding a polypeptide having at least one 1,3-propanediol oxidoreductase activity. The method of claim 8, wherein the gene encoding the polypeptide having dihydratase activity is selected from the group consisting of a gene encoding glycerol dihydratase and a gene encoding diol dihydratase. The method of claim 8, wherein said dihydratase reactivation factor is selected from the group consisting of orfX and orfZ derived from dha regulators. The method of claim 1, wherein the 1,3-propanediol producing organism comprises a gene encoding a polypeptide having at least one NAD ⁺ -dependent dehydrogenase activity and converting NAD ⁺ to NADH. The method of claim 11, wherein the gene is an endogenous gene derived from the host organism or an exogenous gene derived from another organism. The method of claim 1 wherein the NAD ⁺ - dependent dehydrogenase claim has an active gene encoding the polypeptide to convert NAD ⁺ to NADH is Staphylococcus aureus (Staphylococcus aureus) by NAD ⁺ derived from-dependent formate Dehydrogenase (FDH). The method of claim 1 wherein the NAD ⁺ - including a dependent formate dehydrogenase The (FDH), formate-dependent dehydrogenase claim having activity NAD ⁺ is a gene encoding a polypeptide which converts a NADH NAD ⁺ Method of culturing in the medium. The method of claim 1 wherein the carbon source is glucose.