JP2013013353A - Method for producing 1,3-propanediol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing 1,3-propanediol from a glycerol using gene recombination Escherichia coli to improve productivity of 1,3-propanediol.SOLUTION: In the method, bacterial cells containing at least glycerol dehydratase and 1,3-propanediol oxidoreductase are added to a medium containing a fermentation substrate as a carbon source with ethanolamine or 2-amino-1-propanol and cultivated to produce 1,3-propanediol.

Description

  The present invention relates to a method for producing 1,3-propanediol.

  1,3-propanediol is a useful compound with a wide range of uses, such as as a monomer used in the production of polyesters and polyurethanes, and as a starting material for the synthesis of cyclic compounds.

  The production method of 1,3-propanediol is roughly classified into a chemical synthesis method and a microbial fermentation method. Examples of the chemical synthesis method include a Degussa method using acrolein as a raw material and a Shell method using ethylene oxide as a raw material. It is done.

  On the other hand, examples of the microbial fermentation method include a method using anaerobic bacteria belonging to the genus Klebsiella and Clostridium, and a method using genetically modified Escherichia coli (for example, Patent Documents 1 and 2). See).

  In Non-Patent Document 1, Klebsiella pneumoniae belonging to the genus Klebsiella is used as a method of utilizing anaerobic bacteria, and glycerol dehydratase and glycerol dehydratase reactivation factor (hereinafter referred to as “GD / GDR”). Glycerol is dehydrated to give 3-hydroxypropionaldehyde, and 1,3-propanediol oxidoreductase (hereinafter also abbreviated as “PDH”) is added to add 3-hydroxypropionaldehyde. A method is described in which hydroxypropionaldehyde is converted to 1,3-propanediol to obtain 1,3-propanediol. Since the method described in Non-Patent Document 1 is a method based on general anaerobic fermentation from glycerol, it is necessary to strictly block oxygen and is not suitable for industrial production.

  Patent Document 1 discloses, as a method of using genetically modified Escherichia coli, a gene DAR1, encoding glycerol-3-phosphate (G3P) phosphatase, which is isolated from Saccharomyces cerevisiae using glucose as a raw material. The genes dhaB1, dhaB2, dhaB3 encoding GPP1, glycerol dehydratase isolated from Klebsiella pneumoniae, genes orfX, orfZ encoding glycerol dehydratase reactivation factor, and 1,3-propane Using E. coli strain RJ8 / pAH48 / pDT29 recombined with dhaT, a gene encoding diol oxidoreductase, using glucose as a raw material, A method is described for conversion to 3-propanediol to obtain 1,3-propanediol.

  In Patent Document 2, glycerol is dehydrated by using a cell and / or a cell-treated product containing diol dehydratase and / or glycerol dehydratase and diol dehydratase reactivation factor and / or glycerol dehydratase reactivation factor. A method for producing 1,3-propanediol by obtaining 3-hydroxypropionaldehyde and hydrogenating this 3-hydroxypropionaldehyde by a chemical synthesis method using no enzyme in the liquid phase is described. That is, the method described in Patent Document 2 includes a chemical synthesis method.

Special table 2006-512907 gazette JP 2005-102533 A

Enzyme Microb. Tech. 20, 82-86 (1997)

  The object of the present invention is to produce 1,3-propanediol, which improves the productivity of 1,3-propanediol using genetically modified E. coli when producing 1,3-propanediol from a fermentable substrate. Is to provide a method.

  As a result of intensive studies to solve the above problems, the present invention has been reached as follows.

  (1) A culture containing at least glycerol dehydratase and 1,3-propanediol oxidoreductase, and adding ethanolamine or 2-amino-1-propanol to a medium containing a fermentable substrate as a carbon source and culturing. To produce 1,3-propanediol.

  (2) Ethanolamine or 2-amino-1-propanol is added to a medium containing at least glycerol-dehydratase and 1,3-propanediol oxidoreductase as a carbon source from a fermentable substrate containing glycerol. In this method, 1,3-propanediol is produced by culturing.

  (3) Cellulose comprising glycerol dehydratase, glycerol dehydratase reactivating factor, 1,3-propanediol oxidoreductase, and adenosyltransferase, ethanolamine in a medium containing a fermentable substrate containing glycerol as a carbon source, Alternatively, it is a method for producing 1,3-propanediol by adding 2-amino-1-propanol and culturing.

  (4) The method for producing 1,3-propanediol according to any one of (1) to (3) above, wherein 1,3-propanediol is produced under microaerobic culture conditions.

  (5) A microbial cell comprising glycerol dehydratase, glycerol dehydratase reactivating factor, and adenosyltransferase is an E. coli strain, and E. coli is used as the host E. coli. E. coli W3110, E. coli. E. coli JM107, E. coli. coli JM109, E. coli. coli DH1, E. coli. coli DH5, E. coli. The method for producing 1,3-propanediol according to any one of (1) to (4) above, which is a single species selected from the group consisting of E. coli DH5α.

  The method for producing 1,3-propanediol according to the present invention provides a method for increasing the production amount of 1,3-propanediol further than the conventional method.

  A method for producing 1,3-propanediol in the embodiment of the present invention will be described below.

[Method for producing 1,3-propanediol]
In the method for producing 1,3-propanediol in the present embodiment, a cell comprising at least glycerol dehydratase and 1,3-propanediol oxidoreductase, ethanolamine in a medium containing glycerol as a single carbon source, or This is a method for producing 1,3-propanediol by adding 2-amino-1-propanol and culturing.

  In another embodiment of the present invention for producing 1,3-propanediol, glycerol dehydratase, glycerol dehydratase reactivation factor, 1,3-propanediol oxidoreductase, and a cell containing glycerol Is a method of producing 1,3-propanediol by adding ethanolamine or 2-amino-1-propanol to a medium with a single carbon source and culturing.

  In the present embodiment, glycerol dehydratase is not particularly limited, and may be derived from any source having / expressing this enzyme. Specifically, there are microorganisms belonging to the genus Klebsiella, Citrobacter genus, Clostridium genus, Lactobacillus genus, Enterobacter genus, Caloramator genus, Salmonella genus and Listeria genus. Citrobacter intermedium, Lactobacillus reuteri, Lactobacillus buchneri, Lactobacillus brevis, Enterobacter agglomerans, Clostridium butyricum, Caloramator viterbensis, Lactobacillus collinoides, Lactobacillus hilgardii, Salmonella typhimurium, Listeria cytoplasm These glycerol dehydratases may be used alone or as a mixture of two or more.

  The method for isolating glycerol dehydratase from the above sources is not particularly limited, and is similar to the above-described methods for separating and isolating enzymes such as extraction and column chromatography (for example, HPLC). It can be isolated from any microorganism.

In the present invention, the term “glycerol dehydratase reactivation factor” means a factor that catalyzes the reaction of glycerol → 3-hydroxypropionaldehyde + H ₂ O again to promote (reactivate) the activity of dehydratase that has been deactivated. . More specifically, coenzyme B12 is involved in the reaction of glycerol → 3-hydroxypropionaldehyde + H ₂ O catalyzed by dehydratase, but glycerol dehydratase is a reaction of glycerol → 3-hydroxypropionaldehyde + H ₂ O. When the enzyme is developed and catalyzed, the coenzyme B12 is crushed and the active center is deactivated. Here, the glycerol dehydratase is replaced by replacing the coenzyme B12 crushed by the glycerol dehydratase reactivation factor with a new coenzyme B12. The glycerol dehydratase can be reused in the dehydration reaction of glycerol. In this invention, what has the effect | action which reactivates glycerol dehydratase in this way is called "glycerol dehydratase reactivation factor." The glycerol dehydratase reactivation factor is not particularly limited as long as it has the above-described action, and a known glycerol dehydratase reactivation factor that activates inactivated glycerol dehydratase is used. .

  Here, “glycerol dehydratase” is sometimes abbreviated as “GD”, and “glycerol dehydratase reactivation factor” is abbreviated as “GDR”. Further, “glycerol dehydratase and glycerol dehydratase reactivating factor” may be abbreviated as “GD / GDR” hereinafter.

  The inventors of the present invention have introduced a recombinant E. coli incorporating glycerol dehydratase, glycerol dehydratase reactivating factor, 1,3-propanediol oxidoreductase, and adenosyltransferase into a medium containing glycerol as a single carbon source, ethanolamine, Alternatively, the inventors have found that the production amount of 1,3-propanediol is improved by adding 2-amino-1-propanol and culturing.

  In the method for producing 1,3-propanediol in the present embodiment, the cells are cultured under microaerobic culture conditions. Here, “microaerobic culture” in the present specification means culturing at a dissolved oxygen concentration / saturated oxygen concentration of 1/1000 to 1/10, and means an aerobic culture compared to anaerobic culture. The dissolved oxygen concentration / saturated oxygen concentration is preferably 1/500 to 1/30, more preferably 1/200 to 1/40. Here, the “microaerobic culture conditions” in the present specification may be maintained for at least a fixed time in the production process of 1,3-propanediol, and are not necessarily continuously in the production process of 1,3-propanediol. It is not limited to the case where it is maintained.

  In the present embodiment, E. coli that can be used is E. coli. E. coli W3110, E. coli. E. coli JM107, E. coli. coli JM109, E. coli. coli DH1, E. coli. coli DH5, E. coli. a single species selected from the group consisting of E. coli DH5α.

  In the present embodiment, “cells comprising glycerol dehydratase, glycerol dehydratase reactivator, 1,3-propanediol oxidoreductase, and adenosyltransferase” are, for example, plasmid pSPKGRT, strain E. coli. E. coli W3110 was transformed into E. coli. E. coli W3110 (pSPKGRT) can be used, and is a genetically modified E. coli strain into which the genes shown in Table 1 have been introduced.

  Here, “dhaB1”, “dhaB2” and “dhaB3” shown in Table 1 are genes encoding the functionally linked activity of glycerol dehydratase, and all are derived from Klebsiella pneumoniae. Further, “orfX” and “orfZ” shown in Table 1 are genes encoding a dehydratase reactivation gene, and “dhaT” is a gene encoding 1,3-propanediol oxidoreductase, It is derived from Klebsiella pneumoniae. In addition, “KoADT” shown in Table 1 is a gene encoding the functionally linked activity of adenosyltransferase and is derived from Klebsiella oxytoca.

In the present embodiment, one or two or more types of fermentable substrates are appropriately selected from the following general carbon sources in consideration of the assimilation of the host microorganism to be used.
Sugars:
glucose,
Fructose,
Maltose,
Galactose.
Natural carbohydrates:
starch,
Starch hydrolyzate,
Molasses,
Molasses,
wheat,
Corn, etc.
Alcohols:
Glycerol,
methanol,
Ethanol etc.
Fatty acids:
Acetic acid,
Gluconic acid,
Pyruvic acid,
Citric acid and so on.
Amino acids:
glycine,
glutamine,
Asparagine etc.
Hydrocarbons:
Normal paraffin etc.

  The ethanolamine or 2-aminopropan-1-one added to the medium is, for example, 0.1 to 5 g / L, and preferably 1.0 to 1.5 g / L.

  Here, as a culture medium composition in the present embodiment, a known culture composition is used, and those skilled in the art can appropriately select a culture medium composition for the above-described cells having the ability to produce 1,3-propanediol. As the medium of the present embodiment, for example, a common growth medium such as an LB medium, an SD medium, a YM medium, or the like is used as a preferable growth culture solution of the present invention.

  The concentration of glycerol, which is a carbon source and a raw material, added to the medium can be prevented from being excessive or insufficient by selecting the addition amount from the range of 30 to 80 g / L.

  In addition to glycerol, a nitrogen source is added to the medium. In the present embodiment, as described above, as the nitrogen source, ethanolamine or 2-amino-1-propanol is an essential component, and various nitrogen compounds that can be used for normal fermentation are used as appropriate. Preferred inorganic nitrogen sources are ammonium salts and nitrates. Preferred organic nitrogen sources are amino acids, yeast extract, peptones, meat extract, liver extract, digested serum powder and the like. More preferred inorganic nitrogen sources are ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium hydrogen phosphate, potassium nitrate and sodium nitrate. More preferred nitrogen sources are arginine, citrulline, ornithine, lysine, yeast extract, and peptones.

  Furthermore, in addition to the carbon source glycerol and the nitrogen source, other organic or inorganic substances suitable for culturing the cells used in the present embodiment or the treated cells can be added to the medium. For example, when the above-mentioned vitamin B12 (cyanocobalamin) is an essential component, and the growth and activity of obligate anaerobic microorganisms can be enhanced by adding inorganic compounds such as cofactors such as vitamins and various salts to the medium. There is also. For example, the following can be mentioned as microbial growth cofactors derived from inorganic compounds, vitamins and animals and plants.

  Examples of inorganic compounds include potassium dihydrogen phosphate, magnesium sulfate, manganese sulfate, sodium chloride, cobalt chloride, calcium chloride, zinc sulfate, copper sulfate, alum, sodium molybdate, potassium chloride, boric acid, nickel chloride, Examples include sodium tungstate, sodium selenate, and ferrous ammonium sulfate.

  In addition to the essential component vitamin B12, vitamins include, for example, biotin, folic acid, pyridoxine, thiamine, riboflavin, nicotinic acid, pantothenic acid, thiooctoic acid, and p-aminobenzoic acid.

  Methods for producing a culture solution by adding these inorganic compounds, vitamins, or growth cofactors are known. Therefore, a culture solution is prepared according to a manufacturing method as appropriate. The medium can be liquid, semi-solid, or solid. In the method for producing 1,3-propanediol in the present embodiment, a preferable medium form is a liquid medium.

  The cells in the present embodiment can be cultured according to a known microorganism culturing method, except under the above-described microaerobic culture conditions. For industrial production, it is also possible to use a continuous fermentation system that can continuously supply a medium and a substrate gas and that has a mechanism for recovering the culture.

  In the culture of bacterial cells in the present embodiment, a predetermined amount of oxygen is mixed into the continuous culture system to achieve microaerobic conditions. As the incubator, a commonly used culture tank can be used as it is. Culture tanks that can also be used for the culture of anaerobic microorganisms are commercially available. By substituting oxygen mixed in the culture tank with an inert gas such as nitrogen or a substrate gas, the above-mentioned range of fine tanks is available. You can create an aerobic atmosphere.

  For example, an anaerobic jar can be a bioreactor for culturing anaerobic microorganisms. The anaerobic culture jar is composed of an airtight container made of metal, glass, or synthetic resin, and can block the inside from oxygen in the atmosphere. Furthermore, the anaerobic culture jar can be equipped with a mechanism for removing molecular oxygen contained in the space inside the anaerobic culture jar or in the culture solution. For example, nitrogen, helium, hydrogen, carbon dioxide gas, or the like can be maintained in an anaerobic state by aeration and stirring from the bottom.

  In the present embodiment, an additional function can be given to the culture tank. For example, in addition to a commonly used stirring and mixing tank, a bubble column type or draft tube type culture tank can also be used. The cells or the treated cells are released and dispersed by the mixed gas blown into the liquid medium, and the cells or the treated cells can be brought into sufficient contact with the medium. In addition, cells or cells while dripping moisture onto a highly breathable slag such as a biotrickling filter, other ceramic-based inorganic fillers, or packed layers of organic synthetic materials such as polypropylene. It is also possible to incubate the processed material while inhaling gas. Furthermore, the used microbial cells or processed microbial cells can be used by immobilizing them on carrageenan gel, alginic acid gel, acrylamide gel, chitin, cellulose, agar, etc. by a conventional method.

  In the present embodiment, a person skilled in the art can use a known method in order to separate the cultured bacterial cells or the processed bacterial cells from the culture product solution. A preferable separation method is a method using a hollow fiber type ultrafiltration or microfiltration membrane having filtration performance and concentration performance. In addition, in order to obtain a filtration rate sufficient for separation of the bacterial cells or the treated bacterial cells and the product solution, a membrane with an appropriate fractional molecular weight may be selected according to the used bacterial cells or the treated bacterial cells. 1,3-propanediol can be recovered from the culture solution separated from the culture tank through the ultrafiltration membrane. A method for purifying the recovered 1,3-propanediol is known. For example, the culture is removed by centrifugation or the like, the supernatant is concentrated under reduced pressure, dried to dryness, and then ethyl acetate, butyl acetate, toluene , Extraction with an organic solvent such as xylene, hexane, heptane, methyl isobutyl ketone, methyl-t-butyl ether, or halogen. The extract can be purified by further operations such as silica gel chromatography and crystallization. Depending on the shape of the culture tank, a stirrer or the like can be used to sufficiently stir the medium. By agitating the culture in the culture tank, it is possible to increase the chance of contacting the medium components and necessary gas with the anaerobic microorganisms, and to optimize the production efficiency of 1,3-propanediol. A single or mixed gas such as hydrogen, nitrogen, carbon dioxide, or the like can also be supplied as micro or nano bubbles.

  The pH of the culture is preferably from 3.0 to 9.0, more preferably from 5.5 to 8.5, for sufficient growth of the cells or the treated cells. In order to increase the amount of 1,3-propanediol recovered, the temperature of the culture tank is not particularly limited, but 28 to 37 ° C is preferable, and 32 to 34 ° C is more preferable. Can do.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

  Hereinafter, in each Example, glycerol and 1,3-propanediol were quantified by the following method.

Determination of glycerol and 1,3-propanediol:
After sterilizing 1 mL of the culture solution, it was filtered through a 0.45 μm polytetrafluoroethylene (PTFE) membrane to obtain a sample for high performance liquid chromatography measurement.

[High-performance liquid chromatography conditions]
Column: ULTRON PS-80H (8.0 mm × 300 mm)
Mobile phase: 10 mM H ₂ SO ₄ aqueous solution Flow rate: 0.7 mL / min
Column temperature: 40 ° C
Detection: Refractive index (RI)
Retention time: 14.8 minutes for glycerol and 19.0 minutes for 1,3-propanediol.

Measurement of OD ₆₀₀ during culture:
After the culture solution was diluted 47 times with water, the turbidity at 600 nm was measured using SmartSpec 3000 manufactured by BioRad.

[Example 1] (Construction of a plasmid containing a 1,3-propanediol biosynthesis gene)
Using the genome extracted from Klebsiella pneumoniae ATCC25955 as a template, glycerol dehydratase gene (SEQ ID NO: 1), glycerol dehydratase reactivation factor gene (SEQ ID NO: 2), and 1,3-propanediol oxidoreductase gene (SEQ ID NO: 3), respectively PCR amplification was performed. Next, the adenosyltransferase gene (SEQ ID NO: 4) was PCR amplified using the genome extracted from Klebsiella oxytoca ATCC 8724 as a template. Each DNA fragment obtained was ligated using pSE420U (plasmid modified from the multicloning site of plasmid vector pSE420 manufactured by Invitrogen, WO 2006/132145) and Takara Ligation Kit to transform Escherichia coli JM109 strain. The obtained transformed strain was grown in an LB medium containing ampicillin, a plasmid was extracted from the grown transformant, and a plasmid in which it was confirmed that all genes were inserted was designated as pSPKGRT.

[Example 2] (Production of 1,3-propanediol by flask culture)
With pSPKGRT obtained in Example 1, W3110, JM107, JM109, DH1, DH5, and DH5α were transformed to produce production strains. Next, each strain was grown on 7 mL of LB medium to prepare a preculture, and 2.5 mL of the strain was inoculated into 1,3-propanediol production medium. The 1,3-propanediol production medium was charged with 50 mL in a 500 mL pleated Erlenmeyer flask.

The composition of the medium is 60 g / L glycerol, 6.0 g / L KH ₂ PO ₄ , 0.5 g / L yeast extract (Far East), 3.0 g / L (NH ₄ ) ₂ SO ₄ , 2.0 g / L MgSO _4. -Heptahydrate, 1.0 g / L citric acid, 1.0 g / L ethanolamine, mineral solution 10 mL / L, 2 mg / L cyanocobalamin, 0.02 mM IPTG, pH 7.5. The mineral solution was prepared by dissolving 2 g of nitrilotriacetic acid with 300 mL of ion-exchanged water and adjusting the pH to 6.0 with KOH, then 1 g of MnSO ₄ .1 hydrate, 0.8 g of Fe (SO ₄ ) _2. (NH ₄ ) ₂ · 6 hydrate, 0.2 g CoCl ₂ · 6 hydrate, 0.002 g ZnSO ₄ · 7 hydrate, 0.02 g CuSO ₄ · 2 hydrate, 0.02 g NiCl ₂ · 6 hydrate, 0.02 g Na ₂ MnO ₄ · 2 hydrate, 0.02 g Na ₂ SeO ₄ , 0.02 g Na ₂ WO ₄ were dissolved, and 1 L was added with ion-exchanged water. did.

After inoculating the preculture, the culture was started at a temperature of 33 ° C. and a rotation speed of 145 rpm. After 16 hours, after confirming that the OD ₆₀₀ was 2 to 4, the rotation speed was reduced to 70 rpm and the culture was continued. The production concentration of 1,3-propanediol during the culture is shown in Table 1. As a comparative example, the results of culturing at 145 rpm are shown in Table 2, and the results of culturing in the same manner as in Example 1 in a medium without ethanolamine are shown in Table 3. Production increased by culturing under microaerobic conditions, and further increased by adding ethanolamine.

[Comparative Example 1]
The experiment was performed in accordance with Example 2 except that the culture was performed at a rotation speed of 145 rpm throughout. The results are shown in Table 3.

[Comparative Example 2]
Performed according to Example 2 except that no ethanolamine was added. The results are shown in Table 4.

[Example 3] (Production of 1,3-propanediol by a minijar)
W3110 (pSPKGRT) and JM109 (pSPKGRT) prepared in Example 2 were grown in 50 mL of LB medium to prepare a preculture, and inoculated into 1,3-propanediol production medium. The inoculum was 14 mL and the production medium was 1.2 L. The components of the production medium are the same as in Example 2. After the inoculation, the culture was started while controlling the temperature at 33 ° C., the stirring speed at 600 rpm, the aeration rate at 0.5 vvm, and the pH at 7.2, and the dissolved oxygen concentration from the time when the OD ₆₀₀ became 2-3. Culturing was continued while controlling the stirring speed so that became 2% of saturated oxygen concentration. Table 5 shows the amount of 1,3-propanediol produced during the culture.

Claims (5)

  By culturing cells containing at least glycerol dehydratase and 1,3-propanediol oxidoreductase by adding ethanolamine or 2-amino-1-propanol to a medium containing a fermentable substrate as a carbon source, A process for producing 1,3-propanediol, which comprises producing 3-propanediol.   Culturing a cell comprising at least glycerol dehydratase and 1,3-propanediol oxidoreductase with ethanolamine or 2-amino-1-propanol added to a medium containing a fermentable substrate containing glycerol as a carbon source. A process for producing 1,3-propanediol, which comprises producing 1,3-propanediol.   In a cell containing glycerol dehydratase, glycerol dehydratase reactivating factor, 1,3-propanediol oxidoreductase, and adenosyltransferase, ethanolamine in a medium containing a fermentable substrate containing glycerol as a carbon source, or 2- A method for producing 1,3-propanediol, wherein 1,3-propanediol is produced by culturing with addition of amino-1-propanol.   The method for producing 1,3-propanediol according to any one of claims 1 to 3, wherein 1,3-propanediol is produced under microaerobic culture conditions.   A microbial cell comprising glycerol dehydratase, a glycerol dehydratase reactivating factor, and adenosyltransferase is an E. coli strain, and E. coli is used as the host E. coli. E. coli W3110, E. coli. E. coli JM107, E. coli. coli JM109, E. coli. coli DH1, E. coli. coli DH5, E. coli. The method for producing 1,3-propanediol according to any one of claims 1 to 4, wherein the method is a single species selected from the group consisting of E. coli DH5α.