WO2017115855A1 - ガス攪拌式発酵装置 - Google Patents
ガス攪拌式発酵装置 Download PDFInfo
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- WO2017115855A1 WO2017115855A1 PCT/JP2016/089141 JP2016089141W WO2017115855A1 WO 2017115855 A1 WO2017115855 A1 WO 2017115855A1 JP 2016089141 W JP2016089141 W JP 2016089141W WO 2017115855 A1 WO2017115855 A1 WO 2017115855A1
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- metal film
- sintered metal
- culture tank
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- UAEPNZWRGJTJPN-UHFFFAOYSA-N CC1CCCCC1 Chemical compound CC1CCCCC1 UAEPNZWRGJTJPN-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
- C12M29/08—Air lift
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/02—Stirrer or mobile mixing elements
- C12M27/04—Stirrer or mobile mixing elements with introduction of gas through the stirrer or mixing element
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/04—Apparatus for enzymology or microbiology with gas introduction means
- C12M1/08—Apparatus for enzymology or microbiology with gas introduction means with draft tube
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/18—Flow directing inserts
- C12M27/24—Draft tube
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
- C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
- C12P5/026—Unsaturated compounds, i.e. alkenes, alkynes or allenes
Definitions
- the present invention relates to a gas stirring type fermentation apparatus. Furthermore, it is related with the manufacturing method of a chemical substance.
- Patent Document 1 discloses a method for producing isoprene by culturing a microorganism having an isoprene-producing ability represented by Bacillus subtilis.
- flammable substances such as isoprene are also included.
- combustible substances When combustible substances are to be manufactured, the specification of a fermentation apparatus that takes into account the risk of fire and explosion due to continued combustion is required. There are three factors that cause combustion and explosion: a combustible substance, oxygen, and an ignition source. To prevent combustion and explosion, it is necessary to remove at least one of these three components.
- a combustible material such as isoprene is produced by a fermentation method, there are three elements: “combustible material to be produced”, “oxygen”, and “electric energy of a stirrer that is an ignition source”. There is concern about the possibility of combustion and explosion (hereinafter also referred to as “explosion”).
- Non-Patent Document 1 discloses an airlift reactor capable of releasing gas from a nozzle provided at the bottom of a culture tank and stirring the culture solution in the culture tank.
- Patent Document 2 discloses a technique for performing aerobic culture by dispersing and supplying air, oxygen, or a mixed gas thereof to a culture tank using an air diffuser provided with a sintered metal film.
- Non-Patent Document 1 the gas released from the nozzle forms bubbles in the culture tank.
- the culture solution in the culture tank is agitated by the movement of the bubbles.
- the mixing performance of the culture solution is reduced compared to conventional fermentation equipment that uses a mechanical stirrer, so that chemical substances cannot be produced sufficiently. Found that there is a case.
- aerobic culture is performed using the technique described in Non-Patent Document 1, it is necessary to release an oxygen-containing gas from a nozzle to stir the culture solution and supply oxygen to the culture solution. It has also been found that, compared with a conventional fermentation apparatus that stirs a culture solution with a stirrer, the shearing and dispersing action of bubbles is reduced, and the oxygen supply performance to the culture solution may be significantly reduced.
- Patent Document 2 does not exclude the use of a mechanical stirrer, and there is a concern about the possibility of an explosion or the like when producing a flammable substance.
- An object of the present invention is to provide a gas-stirring fermentation apparatus excellent in culture medium mixing performance, and particularly to provide a gas-stirring fermentation apparatus that can be applied to aerobic culture with a high oxygen demand.
- the present invention includes the following contents.
- a culture tank a draft tube arranged inside the culture tank;
- the gas supply pipe includes a gas discharge portion made of a sintered metal film,
- a gas stirring type fermentation apparatus characterized in that a culture solution in a culture tank can be stirred by a gas released from a gas releasing unit.
- the discharge gas linear velocity (gas release amount [m 3 / s] / sintered metal film surface area [m 2 ]) of the gas release portion made of the sintered metal film is 0.04 m / s or less.
- the device according to [1] or [2]. [4] When the inner diameter of the culture tank is D 1 and the inner diameter of the draft tube is D 2 , D 1 and D 2 satisfy the relationship 0.7 ⁇ D 2 / D 1 [1] to [3] The apparatus in any one of. [5] A gas containing a microorganism capable of producing a chemical substance is discharged from a gas discharge part made of a sintered metal film, and the medium containing the released gas is allowed to pass through the inside of the draft tube. A method for producing a chemical substance, comprising stirring a culture solution and culturing a microorganism to produce a chemical substance. [6] The method according to [5], wherein the chemical substance includes a combustible substance.
- a gas-stirring fermentation apparatus excellent in culture medium mixing performance particularly a gas-stirring fermentation apparatus that can be applied to aerobic culture with a high oxygen demand.
- FIG. 1 is a schematic view showing a gas stirring type fermentation apparatus in one embodiment of the present invention.
- FIG. 2 is a schematic diagram for explaining the circulation flow of the culture solution in the gas stirring type fermentation apparatus in one embodiment of the present invention.
- Drawing 3 is a mimetic diagram for explaining the suitable size and arrangement of the gas stirring type fermentation device of the present invention.
- FIG. 2 is a view showing a pAH162-Para-mvaES plasmid carrying the faecalis-derived mvaES operon.
- FIG. 5 is a diagram showing a map of pAH162-mvaES.
- FIG. 1 is a schematic view showing a gas stirring type fermentation apparatus in one embodiment of the present invention.
- FIG. 2 is a schematic diagram for explaining the circulation flow of the culture solution in the gas stirring type fermentation apparatus in one embodiment of the present invention.
- Drawing 3 is a mimetic diagram for explaining
- FIG. 6 shows a plasmid for pAH162-MCS-mvaES chromosome fixation.
- FIG. 7 is a diagram showing a set of chromosome-fixing plasmids that retain the mvaES gene under the transcriptional control of (A) P lldD , (B) P phoC , or (C) P pstS .
- FIG. 8 is a diagram showing an outline of the construction of the pAH162- ⁇ attL-KmR- ⁇ attR vector.
- FIG. 9 shows an expression vector for pAH162-Ptac chromosome fixation.
- FIG. 10 is a diagram showing codon optimization in the KDyI operon obtained by chemical synthesis.
- FIG. 11 shows (A) pAH162-Tc-Ptac-KDyI and (B) pAH162-Km-Ptac-KDyI chromosome-fixing plasmids that retain the codon-optimized KDyI operon.
- FIG. It is a figure which shows the chromosome fixed plasmid holding the mevalonate kinase gene derived from palidicola.
- FIG. 13 shows maps of genomic variants of (A) ⁇ ampC :: attB phi80 , (B) ⁇ ampH :: attB phi80 , and (C) ⁇ crt :: attB phi80 .
- FIG. 14 shows maps of genomic variants of A) ⁇ crt :: pAH162-P tac -mvk (X) and (B) ⁇ crt :: P tac -mvk (X).
- FIG. 15 shows (A) ⁇ ampH :: pAH162-Km-P tac -KDyI, (B) ⁇ ampC :: pAH162-Km-P tac -KDyI, and (C) ⁇ ampC :: P tac -KDyI. It is a figure which shows a map.
- FIG. 15 shows (A) ⁇ ampH :: pAH162-Km-P tac -KDyI, (B) ⁇ ampC :: pAH162-Km-P tac -KDyI, and (C) ⁇ ampC :: P tac -KDyI. It is a figure which shows a map.
- FIG. 15 shows (A) ⁇ ampH :
- FIG. 16 shows maps of chromosome modifications of (A) ⁇ ampH :: pAH162-Px-mvaES and (B) ⁇ ampC :: pAH162-Px-mvaES.
- FIG. 17 is a diagram showing (A) growth and (B) isoprene production amount (mg / Batch) by culturing the isoprene-producing microorganism SWITCH-PphoC ⁇ gcd / IspSM strain.
- FIG. 18 is a graph showing dissolved oxygen (DO) concentration in the culture of the isoprene-producing microorganism SWITCH-PphoC ⁇ gcd / IspSM strain.
- DO dissolved oxygen
- the gas stirring type fermentation apparatus of the present invention is: A culture tank; A draft tube arranged inside the culture tank; A gas supply pipe for supplying gas to the inside of the culture tank; Including The gas supply pipe includes a gas discharge portion made of a sintered metal film, The culture solution in the culture tank can be stirred by the gas released from the gas release part.
- the gas-stirring fermenter is a fermenter that stirs a culture solution only by a gas flow action, unlike a conventional fermenter equipped with a mechanical stirrer.
- the gas agitating fermentation apparatus when gas is released from a gas discharge portion installed in the culture tank, bubbles composed of the released gas are generated.
- the gas discharge part is installed in the lower part of a culture tank, and the produced
- bubbles rise in the culture tank the bubbles tend to collect at the center of the culture tank due to the wall effect of the culture tank. As a result, there is a difference in the apparent density of the culture solution between the central portion in the culture tank and the outside (wall surface side) portion.
- the culture solution rises with bubbles at the center of the culture tank and falls at the outer side (wall surface side). Thereby, the circulation flow of a culture solution generate
- the above apparent density difference can be further increased, and stirring of the culture solution can be promoted.
- the gas release part and the draft tube are aligned so that a culture solution containing a large amount of bubbles and having a low apparent density passes through the inside of the draft tube.
- the apparent density difference of the culture solution can be increased between the inside and the outside of the draft tube, and the stirring of the culture solution can be promoted.
- an upward flow of the culture solution is generated inside the draft tube (hereinafter, the action of expressing the upward flow of the culture solution is also referred to as “gas lift action”), and a downward flow of the culture solution is generated outside the draft tube. .
- the present invention achieves the conventional gas stirring type fermenter by using a gas releasing part made of a sintered metal film as the gas releasing part in the gas stirring type fermenting apparatus in which the draft tube is arranged inside the culture tank. It achieves an excellent culture medium mixing performance. Furthermore, in the present invention, when an oxygen-containing gas is used as the gas released from the gas release part, excellent oxygen supply performance can be realized to the extent that it can be applied to aerobic culture with a high oxygen demand. .
- the gas stirring type fermentation apparatus of the present invention is suitable for producing various chemical substances, particularly hydrophobic substances.
- Hydrophobic substances have low solubility in the culture medium, and can be easily separated and recovered.
- the hydrophobic substance may be recovered from the lower part of the culture tank.
- the hydrophobic substance may be recovered from the upper part of the culture tank.
- the hydrophobic substance refers to a substance exhibiting low solubility in the culture solution to such an extent that it can be easily separated from the culture solution to be used.
- the solubility (normal temperature) of the hydrophobic substance in the culture solution is preferably 10 g / kg or less, more preferably 5 g / kg or less, 1 g / kg or less, or 0.1 g / kg or less because separation and recovery are easy. It is.
- the hydrophobic substance to be produced is not particularly limited as long as it can be produced by fermentation.
- hydrogen saturated hydrocarbon such as methane
- unsaturated carbon such as ethylene, propylene, butadiene, isobutene, and isoprene.
- the gas stirred fermenter of the present invention is also suitable for producing a hydrophilic or water-miscible substance.
- a hydrophilic or water-miscible substance means a high solubility in a culture solution to such an extent that a purification treatment such as distillation treatment or membrane separation treatment is required to separate the substance from the culture solution used. Or a substance exhibiting miscibility.
- the solubility (normal temperature) of the hydrophilic or water-miscible substance in the culture solution exceeds 10 g / kg, and is usually 20 g / kg or more, or 30 g / kg or more.
- Such a hydrophilic or water-miscible substance is not particularly limited as long as it can be produced by a fermentation method, and examples thereof include lower (C 1 -C 6 ) alcohol compounds such as ethanol and propanol.
- Isoprenoid compounds can be synthesized via two different metabolic pathways that converge to IPP and its isomer, DMAPP.
- an isoprenoid compound consists of one or more isoprene units having the molecular formula (C 5 H 8 ) n .
- the precursor of the isoprene unit is isopentenyl pyrophosphate or dimethylallyl pyrophosphate.
- 30,000 isoprenoid compounds have been identified and new compounds have been identified.
- Isoprenoids are also known as terpenoids. The difference between terpenes and terpenoids is that terpenoids are hydrocarbons, whereas terpenoids contain additional functional groups.
- Terpenes are classified according to the number of isoprene units in the molecule [for example, hemiterpene (C5), monoterpene (C10), sesquiterpene (C15), diterpene (C20), sesterterpene (C25), triterpene (C30), Sescal terpenes (C35), tetraterpenes (C40), polyterpenes, norisoprenoids].
- monoterpenes include pinene, nerol, citral, camphor, menthol, limonene, carvone, and linalool.
- sesquiterpenes include nerolidol and farnesol.
- diterpenes examples include phytol and vitamin A1.
- Squalene is an example of a triterpene and carotene (provitamin A1) is a tetraterpene (Nature Chemical Biology 2,674 to 681 (2006); Nature Chemical Biology 5,283 to 291 (2009); Nature Reviews 9 Microbiols 37 947 (2005); Adv Biochem Eng Biotechmol (DOI: 10.1007 / 10 — 2014 — 288)).
- the isoprenoid compound is isoprene (monomer).
- a microorganism having an ability to produce an isoprenoid compound has a dimethylallyl diphosphate supply route.
- Dimethylallyl diphosphate (DMAPP, dimethylallyl diphosphate) is a precursor of peptidoglycan and electron acceptors (menaquinone and the like) and is known to be essential for the growth of microorganisms (Fujisaki et al., J. Biochem). , 1986; 99: 1137-1146).
- Examples of the dimethylallyl diphosphate supply route include a methyl erythritol phosphate (MEP) route and a mevalonic acid (MVA) route.
- DMAPP which is a material of an isoprenoid compound (for example, a substrate for isoprene synthesis)
- the isoprenoid compound-producing microorganism is an aerobic microorganism.
- the isoprenoid compound-producing microorganism is preferably cultured under aerobic conditions.
- the concentration of dissolved oxygen in the medium may be a concentration sufficient for the growth of microorganisms.
- the concentration of dissolved oxygen in the medium that is sufficient for the growth of aerobic microorganisms is not particularly limited as long as it is a concentration that can promote the growth of aerobic microorganisms, for example, 1 ppm or more, 3 ppm or more, 5 ppm or more, 7 ppm or more, or 7 .22 ppm or more may be used.
- the concentration of dissolved oxygen for the growth of aerobic microorganisms may also be, for example, 0.3 ppm or less, 0.15 ppm or less, or 0.05 ppm or less.
- the gas stirring fermentation apparatus of the present invention can be widely applied to the production of chemical substances by fermentation.
- the chemical substance to be manufactured contains a flammable substance
- the specification of the fermenter considering the danger of fire and explosion due to continued combustion is required.
- an auxiliary agent such as an organic solvent used for production contains a flammable substance
- the specification of the fermentation apparatus considering the risk of fire or explosion due to continued combustion is required.
- the chemical substance itself to be produced is flammable, ignitable or explosive
- an auxiliary agent such as an organic solvent used for production is flammable, ignitable or explosive.
- the explosibility of substances and auxiliaries can be investigated by general test methods that measure the explosive limits of combustible gases and vapors.
- a mixed gas is put into an explosion test container, an ignition source is activated, and the presence or absence of an explosion is detected by a temperature sensor and a pressure sensor. The same operation is repeated by changing the concentration of the mixed gas, and an explosion occurs.
- the range can be determined (Standard Practice for Determining Limits of Flammability of Chemicals at Elevated Temperature and Pressure, ASTM E918-83 (2011)).
- JIS standard JIS K 2265 is known.
- the chemical substance to be manufactured contains a flammable substance and the flammable substance is manufactured by aerobic culture, there is a concern about the possibility of an explosion or the like.
- the flammable substance contains a flammable gas
- the flammable gas and oxygen are likely to coexist in the upper part of the culture tank.
- the “electric energy of the stirrer as an ignition source” is excluded from factors that cause an explosion or the like. Is possible. Thereby, in the gas stirring type fermentation apparatus of the present invention, it is possible to reduce the possibility of explosion or the like.
- the oxygen utilization efficiency is further extremely high, and the concentration of oxygen discharged to the upper part of the culture tank can be kept low. Therefore, it is possible to adjust the atmosphere in the upper part of the culture tank so as not to enter the combustion range of the combustible gas, and the possibility of explosion or the like can be further reduced. Therefore, the gas stirring type fermentation apparatus of the present invention is particularly suitable for producing a combustible substance.
- the flammable substance is a substance that is liquid at 1 atm. 20 ° C. and has a flash point of 70 ° C. or less, or a lower limit of the combustion limit of the substance is 10% or less, or a lower limit of the limit oxygen concentration. A substance whose value is 15% or less.
- fermentation using a two-phase extraction fermentation in which a hydrophobic substance is cultured together with an organic substance may be performed, and the organic layer containing the hydrophobic substance may be recovered as a gas.
- the organic substance used in the two-phase extraction fermentation is preferably an organic solvent that is immiscible with water, but is not limited thereto.
- the organic substance used is preferably an organic solvent that is harmless to microorganisms used for fermentation production.
- organic solvents examples include corn oil, dodecane, hexadecane, oleyl alcohol, butyl oleate, butyl phthalate, dodecanol, bis (2-ethylhexyl) phthalate, farnesene, isopropyl myristate, butanol, cyclohexane, n-tetradecane, and the like.
- Bennan TC et al., Alleviating monoterpene toxicity using a two-phase extractive biofermentation for the bioproduction mechanism.) 3-2522, 2012 is not intended to be limited to this.
- microorganisms that are resistant to organic solvents harmful to microorganisms such as toluene and benzene can be used (Kosuke Honda et al. “Manufacturing in the non-aqueous world using organic solvent-resistant microorganisms” environmental biotechnology Academic Journal, Vol. 6, No. 2, p. 109-114, 2006).
- the produced hydrophobic substance has moved to the organic layer and can be removed from the fermentation medium by dispensing operation.
- the hydrophobic substance may be removed from the fermentation medium in combination with other types of organic extractants.
- Gas stripping may be performed by passing a gas such as air, nitrogen, or carbon dioxide through the fermentation medium, thereby forming a hydrophobic substance-containing gas phase.
- Hydrophobic product can be obtained using methods known in the art, for example, using a cold water trap to concentrate the hydrophobic material, or by scrubbing the gas phase with a solvent.
- it may be recovered from the hydrophobic substance-containing gas phase by combining unit operations such as cooling, absorption, adsorption, and membrane separation.
- the culture tank is not particularly limited as long as it can be provided with a gas supply pipe and a draft tube provided with a gas release portion made of a sintered metal film to be described later.
- Properties specific gravity, flammability, etc.), production scale, culture method (batch culture method, fed-batch culture method, continuous culture method, etc.), culture conditions (aerobic conditions, anaerobic conditions, etc.) ) Etc., an appropriate culture tank may be selected. The culture solution will be described later.
- the draft tube is not particularly limited as long as the above-described gas lift action can be achieved, and a known draft tube (also referred to as “gas lift pipe”) may be used.
- the draft tube is generally arranged inside the culture tank so that the tube axis direction is perpendicular to the horizontal plane.
- the gas superficial velocity in the draft tube may be determined to achieve a desired mixing performance, but is preferably 0.001 m / s or more, more preferably 0.005 m / s or more, and still more preferably 0.01 m / s. That's it.
- the upper limit of the gas superficial velocity is preferably high from the viewpoint of the mixing performance of the culture solution, but can usually be 0.2 m / s or less, 0.1 m / s or less, 0.05 m / s or less, or the like.
- an oxygen-containing gas is released from the gas discharge section, and the culture solution is stirred and oxygen is supplied to the culture solution.
- the gas stirring type fermentation apparatus of the present invention as long as the discharge gas linear velocity of the gas discharge portion made of a sintered metal film, which will be described later, is below a certain value, the gas superficial velocity in the draft tube is simply over a wide range.
- the inventors of the present invention have found that remarkably superior oxygen supply performance can be realized as compared with a conventional apparatus using a hole nozzle or the like.
- the gas stirring type fermentation apparatus of the present invention covers a wide range of gas superficial velocities in the draft tube as long as the linear gas velocity of the gas releasing portion made of the sintered metal film is 0.04 m / s or less.
- the oxygen utilization efficiency based on the sodium sulfite method also called oxygen dissolution efficiency and oxygen transfer efficiency
- the oxygen utilization is 10 times higher than that of the conventional apparatus using a single hole nozzle.
- Efficiency can be realized. Therefore, the concentration of oxygen discharged to the upper part of the culture tank can be kept low, and the atmosphere of the upper part of the culture tank can be adjusted so that it does not enter the combustion range of combustible gas. It is possible to further reduce the possibility.
- the gas supply pipe has a function of supplying gas to the inside of the culture tank.
- the gas supply pipe is provided with a gas discharge portion made of a sintered metal film. The details of the gas supplied to the inside of the culture tank will be described later.
- the gas that has passed through the gas supply pipe is discharged from the gas discharge portion made of the sintered metal film into the culture tank.
- the sintered metal film has a large number of pores, and the gas is released into the culture vessel as fine bubbles according to the diameter of the pores.
- an oxygen transfer capacity coefficient K L a (where K L is a liquid boundary film mass transfer coefficient, and a is a unit volume) serving as an index of oxygen supply performance.
- K L is a liquid boundary film mass transfer coefficient
- a is a unit volume
- the average pore diameter of the sintered metal film is preferably 20 ⁇ m or less, more preferably. Is 10 ⁇ m or less, more preferably 8 ⁇ m or less, 6 ⁇ m or less, or 5 ⁇ m or less.
- the lower limit of the average pore diameter of the sintered metal film is not particularly limited, but can usually be 0.1 ⁇ m or more, 0.5 ⁇ m or more, 1 ⁇ m or more.
- the sintered metal film can be manufactured by pressing and sintering a metal powder having a uniform particle size distribution. It is possible to adjust the average pore diameter of the obtained sintered metal film by changing the average particle diameter, sintering temperature, etc. of the metal powder.
- the material of the sintered metal film include nickel, stainless steel, inconel, titanium, and the like. From the viewpoint of mechanical strength, chemical resistance, thermal shock resistance, etc., a sintered metal film made of stainless steel is preferable.
- the gas discharge part made of the sintered metal film is aligned with the draft tube so that the culture solution containing the gas released from the gas discharge part passes through the inside of the draft tube.
- the gas discharge part which consists of a sintered metal film is arrange
- the mixing performance of the culture solution may vary. Specifically, in the range where the released gas linear velocity is 0.04 m / s or less, if the gas superficial velocity in the draft tube is the same, even if the released gas linear velocity is changed, the mixing performance of the culture solution is almost unchanged. There is no effect. That is, when the linear velocity of the released gas is 0.04 m / s or less, the influence of the gas superficial velocity is dominant in the mixing performance of the culture solution.
- the discharge gas linear velocity of the gas discharge portion made of the sintered metal film is 0.04 m / s or less.
- the lower limit of the released gas linear velocity is preferably 0.005 m / s or more, more preferably 0.01 m / s or more, 0.02 m / s or more, or 0 from the viewpoint of easily achieving the desired gas superficial velocity. 0.03 m / s or more.
- the linear velocity of the released gas of the gas releasing portion made of the sintered metal film is 0.04 m / s or less.
- the discharge gas linear velocity is 0.04 m / s or less, it is possible to achieve oxygen utilization efficiency exceeding 70% over a wide range of gas superficial velocity in the draft tube.
- the present inventors have found that, in the range where the released gas linear velocity exceeds 0.04 m / s, the oxygen utilization efficiency decreases as the released gas linear velocity increases.
- the discharge gas linear velocity of the gas release part made of a sintered metal film is , Preferably 0.04 m / s or less, more preferably in the range of 0.005 to 0.04 m / s, still more preferably in the range of 0.01 to 0.04 m / s, and 0.02 to 0.04 m / s.
- the range is 0.03 to 0.04 m / s.
- the shape and dimensions of the gas discharge part made of the sintered metal film are not particularly limited as long as the above-mentioned preferable discharge gas linear velocity can be achieved.
- a plurality of gas discharge portions made of a sintered metal film may be provided.
- the gas stirring type fermentation apparatus of the present invention may include other elements necessary for producing a chemical substance by a fermentation method.
- Such other elements include, for example, a culture solution supply pipe for supplying a culture solution to the inside of the culture tank, a base compound supply pipe for supplying a basic compound for pH adjustment to the inside of the culture tank, and a manufactured chemical substance.
- Examples include a collection tube to be collected, a gas discharge tube for taking out the gas from the upper part of the culture tank, and a temperature controller for adjusting the temperature of the culture tank. These may use known elements commonly used in fermenters.
- FIG. 1 shows a schematic diagram of a gas stirring type fermentation apparatus according to an embodiment of the present invention.
- a gas agitating fermentation apparatus 10 includes a culture tank 1, a draft tube 2 disposed inside the culture tank, and a gas supply pipe 3 that supplies gas to the inside of the culture tank.
- a gas release part 4 made of a sintered metal film.
- the gas stirring type fermentation apparatus 10 can stir the culture solution 5 in the culture tank with the gas released from the gas release unit.
- FIG. 1 shows an embodiment provided with one gas discharge part made of a sintered metal film. In order to achieve a suitable discharge gas linear velocity and to achieve a desired gas superficial velocity, sintering is performed. A plurality of gas discharge portions made of a metal film may be provided.
- FIG. 2 is a schematic diagram for explaining the circulation flow of the culture solution in the gas stirring type fermentation apparatus in one embodiment of the present invention.
- the meaning of each symbol is the same as in FIG.
- the gas that has passed through the gas supply pipe 3 is released from the gas release portion 4 made of a sintered metal film into the culture tank 1.
- the sintered metal film has a large number of pores, and the gas is released into the culture vessel as fine bubbles according to the diameter of the pores.
- a culture solution containing many bubbles and having a low apparent density rises inside the draft tube 2. Outside of the draft tube, the culture solution with a low bubble content and high apparent density falls.
- an upward flow of the culture solution is generated inside the draft tube, and a downward flow of the culture solution is generated outside the draft tube. Therefore, a circulating flow of the culture solution is generated inside the culture tank, and the culture solution is stirred.
- FIG. 3 shows a schematic diagram for explaining suitable dimensions and arrangement of the gas stirring type fermentation apparatus of the present invention. The meaning of each symbol is the same as in FIG.
- the gas-stirring fermentation apparatus of the present invention can be used from the viewpoint of further enhancing the mixing performance of the culture solution. It is preferable to satisfy the relationship of 0.7 ⁇ D 2 / D 1 .
- the D 2 / D 1 ratio is more preferably 0.75 or more, further preferably 0.8 or more, 0.82 or more, 0.84 or more, 0.86 or more, or 0 from the viewpoint of the mixing performance of the culture solution. .88 or more.
- the upper limit of the D 2 / D 1 ratio is preferably 0.98 or less, more preferably 0.96 or less, still more preferably 0.94 or less, or 0.92 or less from the viewpoint of the mixing performance of the culture solution.
- the draft tube 2 is preferably arranged coaxially with the culture tank 1.
- the gas-stirring fermentation apparatus of the present invention has the following conditions (i), ( It is preferable to satisfy at least one of ii). (I): 1 ⁇ (H L ⁇ H 2max ) / D 1 (Ii): H 2min / D 2 ⁇ 2
- the (H L -H 2max ) / D 1 ratio is more preferably 1.5 or more, and further preferably 2 or more.
- the upper limit of the (H L -H 2max ) / D 1 ratio is not particularly limited as long as a smooth circulating flow of the culture solution can be achieved, but it can usually be 5 or less, 4 or less, and the like.
- the H 2min / D 2 ratio is more preferably 1.5 or less, still more preferably 1 or less, 0.9 or less, 0.8 or less, 0.8. 7 or less, or 0.6 or less.
- the lower limit of the H 2min / D 2 ratio is not particularly limited as long as a smooth circulating flow of the culture solution can be achieved, but is usually 0.1 or more, 0.2 or more, 0.3 or more, 0.4 or more, etc. obtain.
- the gas stirring type fermentation apparatus of the present invention can realize stirring of a culture solution only by a gas flow action, and a mechanical stirrer can be substantially eliminated.
- the oxygen utilization efficiency is extremely high, and the concentration of oxygen discharged to the upper part of the culture tank can be kept low. Therefore, it is possible to adjust the atmosphere in the upper part of the culture tank so as not to enter the combustion range, and even if a mechanical stirrer is used, the possibility of explosion or the like can be reduced.
- the method for producing a chemical substance of the present invention comprises: Gas is released from a gas discharge part made of a sintered metal film into a culture solution containing microorganisms capable of producing chemical substances, and the culture solution containing the released gas is allowed to pass through the inside of the draft tube. Stirring, and culturing microorganisms to produce chemicals.
- the chemical substance to be manufactured, the gas discharge part made of a sintered metal film, and the draft tube are as described in the above [Gas Stirring Fermenter].
- the method for producing a chemical substance of the present invention is characterized in that the culture solution is agitated by the gas lift action of the gas released from the gas releasing part made of a sintered metal film, and can be achieved by the conventional gas stirring type fermentation method. Achieves excellent mixing performance of culture broth. Furthermore, when an oxygen-containing gas is used as the gas released from the gas release part, excellent oxygen supply performance can be realized to the extent that it can be applied to aerobic culture with a high oxygen demand.
- the advantageous effects described for the gas stirring type fermentation apparatus of the present invention are similarly applied to the chemical substance production method of the present invention.
- microorganisms capable of producing chemical substances include 1) microorganisms that inherently have the ability to produce chemical substances, and 2) inherently have no ability to produce chemical substances, or substantially It includes both microorganisms that do not have, but have been introduced with chemical production genes by genetic recombination and have acquired chemical production ability.
- microorganisms having the ability to produce chemical substances various microorganisms are known depending on the type of chemical substance, and these known microorganisms may be widely used in the present invention.
- the present invention can be widely applied to microorganisms to be developed in the future.
- Gram-positive bacteria examples include Bacillus genus bacteria, Listeria genus bacteria, Staphylococcus genus bacteria, Streptococcus genus bacteria, Enterococcus genus bacteria, Clostridium (Clostridium genus)
- Examples include bacteria, bacteria belonging to the genus Corynebacterium, and bacteria belonging to the genus Streptomyces, and bacteria belonging to the genus Bacillus and bacteria belonging to the genus Corynebacterium are preferred.
- Bacillus genus bacteria include Bacillus subtilis, Bacillus anthracis, Bacillus cereus, and the like, and Bacillus subtilis is more preferable.
- bacteria belonging to the genus Corynebacterium include Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium cornebacteria and the like. -Glutamicum is more preferred.
- Examples of the gram-negative bacteria include Escherichia bacteria, Pantoea bacteria, Salmonella bacteria, Vibrio bacteria, Serratia bacteria, Enterobacter bacteria Escherichia bacteria, Pantoea bacteria, Enterobacter bacteria are preferable.
- Escherichia bacteria As the bacterium belonging to the genus Escherichia, Escherichia coli is preferable.
- Examples of the bacteria belonging to the genus Pantoea include Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans, Pantoea citrea, Pantoea pantoea, and Pantoea pantoea. Ananatis (Panthea ananatis) and Pantoea citrea are preferred.
- Pantoea bacterium strains exemplified in European Patent Application Publication No. 09522121 may be used.
- Representative strains of the genus Pantoea include, for example, Pantoea ananatis AJ13355 strain (FERM BP-6614) and Pantoea ananatis AJ13356 strain (FERM BP-6615) disclosed in European Patent Application No. 0952212.
- Examples of Enterobacter genus bacteria include Enterobacter agglomerans, Enterobacter aerogenes, and the like, with Enterobacter aerogenes being preferred.
- a strain exemplified in European Patent Application Publication No. 09522121 may be used as the Enterobacter bacterium.
- Enterobacter bacteria include, for example, Enterobacter agglomerans ATCC 12287 strain, Enterobacter aerogenes ATCC 13048 strain, Enterobacter aerogenes NBRC 12010 strain (Biotechnol Bioeng. 2007 Mar 27; 98 (2): 340-348. ), Enterobacter aerogenes AJ11037 (FERM BP-10955) strain, and the like.
- Enterobacter aerogenes AJ110737 strain was deposited on August 22, 2007 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1-6 Higashi 1-chome, 1-chome, Tsukuba, Ibaraki 305-8565, Japan) Deposited as FERM P-21348, transferred to an international deposit under the Budapest Treaty on March 13, 2008, and given the receipt number of FERM BP-10955.
- Examples of the fungi include, for example, the genus Saccharomyces, the genus Schizosaccharomyces, the genus Yarrowia, the genus Trichoderma, the genus Aspergillus, Examples include microorganisms, and microorganisms belonging to the genus Saccharomyces, Schizosaccharomyces, Yarrowia, or Trichoderma are preferred.
- Saccharomyces Saccharomyces (Saccharomyces) genus, for example, Saccharomyces carlsbergensis (Saccharomyces carlsbergensis), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomyces Deer statics (Saccharomyces diastaticus), Saccharomyces Dougurashi (Saccharomyces douglasii), Saccharomyces Kuruibera (Saccharomyces kluyveri), Saccharomyces norbensis, Saccharomyces obiformis, and Saccharomyces olviformis. S. cerevisiae is preferred.
- Schizosaccharomyces As a microorganism belonging to the genus Schizosaccharomyces, Schizosaccharomyces pombe is preferable. As a microorganism belonging to the genus Yarrowia, Yarrowia lipolytica is preferable.
- Trichoderma microorganisms of the genus, for example, Trichoderma Harujianumu (Ttichoderma harzianum), Trichoderma Koningi (Trichoderma koningii), Trichoderma Rongifurakiamu (Trichoderma longibrachiatum), Trichoderma reesei (Trichoderma reesei), Trichoderma viride (Trichoderma viride And Trichoderma reesei is preferred.
- Microorganisms that have essentially no or substantially no ability to produce isoprenoid compounds introduce a gene encoding an isoprenoid compound synthase that is an enzyme of the dimethylallyl diphosphate supply pathway using an expression vector, or on a chromosome. It is possible to confer the ability to produce isoprenoid compounds by introducing it into the gene by genetic recombination.
- examples of the dimethylallyl diphosphate supply route include a methyl erythritol phosphate (MEP) route and a mevalonic acid (MVA) route.
- MEP methyl erythritol phosphate
- MVA mevalonic acid
- the methylerythritol phosphate (MEP) pathway is a biosynthetic pathway of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (Nat. Prod. Rep. 16 (5): 565-574 1999. ). Since Methylerythritol phosphate (MEP) and 1-deoxy-D-xylulose-5-phosphate (DXP or DOXP) are biosynthesized as metabolic intermediates, the MEP pathway, DXP pathway, DOXP pathway, and MEP / DOXP pathway be called.
- IPP isopentenyl diphosphate
- DMAPP dimethylallyl diphosphate
- Examples of enzymes involved in the methylerythritol phosphate (MEP) pathway include 1-deoxy-D-xylulose-5-phosphate synthase (EC: 2.2.1.7, Example 1, Dxs, ACCESSION ID NP_414954; Example 2, AT3G21500, ACCESSION ID NP_566686; Example 3, AT4G15560, ACCESSION ID NP_193291; Example 4, AT5G11380, ACCESSION ID NP_001078570), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (EC: 1.1.
- Example 1 Dxr, ACCESSION ID NP_414715; Example 2, AT5G62790, ACCESSION ID NP_001190600), 4 Diphosphocytidyl-2-C-methyl-D-erythritol synthase (EC: 2.7.7.60; Example 1, IspD, ACCESSION ID NP_417227; Example 2, AT2G02500, ACCESSION ID NP_565286), 4-diphosphocytidyl-2-C- Methyl-D-erythritol kinase (EC: 2.7.1.148; Example 1, IspE, ACCESSION ID NP_415726; Example 2, AT2G26930, ACCESSION ID NP_180261), 2-C-methyl-D-erythritol-2,4- Cycloniphosphate synthase (EC: 4.6.1.12; Example 1, IspF, ACCESSION ID NP_417226;
- the mevalonic acid (MVA) pathway is a biosynthetic pathway for synthesizing isopentenyl diphosphate and dimethylallyl diphosphate, which are starting synthetic substances of isoprenoids, from acetyl CoA.
- Examples of enzymes involved in the mevalonate (MVA) pathway include, for example, mevalonate kinase (EC: 2.7.1.36; Example 1, Erg12p, ACCESSION ID NP — 013935; Example 2, AT5G27450, ACCESSION ID NP — 001190411), phosphomevalonic acid Kinase (EC: 2.7.4.2; Example 1, Erg8p, ACCESSION ID NP_013947; Example 2, AT1G31910, ACCESSION ID NP_001185124), diphosphomevalonate decarboxylase (EC: 4.1.1.33; Example 1, Mvd1p , ACCESSION ID NP_014441; Example 2, AT2G38700, ACCESS
- Example 1 Hmg2p, ACCESSION ID NP_013555; Example 2, Hmg1p, ACCESSION ID NP_013636; Example 3, AT1G76490, ACCESSION ID NP_177775; Example 4, AT2G17370, ACCESSION NP. 5, MvaA, ACCESSION ID P13702), acetyl-CoA-C-acetyltransferase / hydroxymethylglutaryl-CoA reductase (EC: 2.3.1.9/1.1.1.34, eg, MvaE, ACCESSION ID) AAG02439).
- one or more enzymes involved in the mevalonate (MVA) pathway eg, phosphomevalonate kinase, diphosphomevalonate decarboxylase, acetyl-CoA-C-acetyltransferase / hydroxymethylglutaryl-CoA reductase, hydroxymethylglutaryl
- MVA mevalonate
- the gene encoding (-CoA synthase) may be placed under the control of a growth promoter reverse-dependent promoter.
- the isoprenoid compound-producing microorganism may further have an enhanced pathway for synthesizing dimethylallyl diphosphate (DMAPP), which is a material of an isoprenoid compound (eg, a substrate for isoprene synthase).
- DMAPP dimethylallyl diphosphate
- IPP isopentenyl diphosphate
- DMAPP dimethylallyl diphosphate
- an expression vector for one or more enzymes involved in the mevalonate pathway and / or methylerythritol phosphate pathway associated with the production of IPP and / or DMAPP may be introduced into the isoprenoid compound-producing microorganism.
- Such an enzyme expression vector may be an integrative vector or a non-integrative vector.
- Such enzyme expression vectors further express together or individually a plurality of enzymes (eg, 1, 2, 3 or 4 or more) involved in the mevalonate pathway and / or the methylerythritol phosphate pathway.
- it may be an expression vector for polycistronic mRNA.
- the origin of one or more enzymes involved in the mevalonate pathway and / or the methylerythritol phosphate pathway may be homologous or heterologous to the host.
- the origin of the enzyme involved in the mevalonate pathway and / or methylerythritol phosphate pathway is heterologous to the host, for example, the host is a bacterium as described above (eg, E. coli), and the mevalonate pathway
- the enzyme involved may be derived from a fungus (eg, Saccharomyces cerevisiae).
- the expression vector introduced into the host may express an enzyme involved in the mevalonate pathway.
- the isoprenoid compound synthase expression vector may be an integral vector or a non-integration vector.
- the culture solution preferably contains a carbon source.
- the carbon source include carbohydrates such as monosaccharides, disaccharides, oligosaccharides, and polysaccharides; invert sugar obtained by hydrolyzing sucrose; glycerol; carbon atoms such as methanol, formaldehyde, formate, carbon monoxide, and carbon dioxide Compound of number 1; oil such as corn oil, palm oil, soybean oil; acetate; animal oil; animal oil; fatty acid such as saturated fatty acid and unsaturated fatty acid; lipid; phospholipid; glycerolipid; monoglyceride, diglyceride, triglyceride, etc.
- the culture solution preferably contains a hydrogen-containing material among the materials exemplified as the carbon source.
- the culture solution preferably further contains a nitrogen source, inorganic ions and other organic trace components as required.
- a nitrogen source, inorganic ions, and other organic trace components any conventionally known components may be used.
- the method of the present invention can be performed in a system having a liquid phase and a gas phase.
- a closed system for example, a reactor such as a fermenter or a fermentation tank can be used in order to avoid disappearance due to diffusion of the produced isoprene.
- a medium containing an isoprene-producing microorganism can be used as the liquid phase.
- the gas phase is the space above the liquid phase in the system, also called head space, and contains fermentation gas. Isoprene has a boiling point of 34 ° C.
- isoprene produced in the liquid phase can be easily transferred into the gas phase, the isoprene-producing reaction by the isoprene-producing microorganism in the liquid phase (enzymatic reaction by isoprene synthase) is always inclined toward the isoprene producing side. You can also.
- Isoprene has an explosive limit of 1.0 to 9.7% (w / w) (eg, Brandes et al., Physikalish Technology Bureaualtalt (PTB), 2008) and has an explosive property, and isoprene is a gas phase. This is because the explosion range fluctuates depending on the mixing ratio with oxygen (see U.S. Pat. No. 8,420,360B2, FIG. 24), so that it is necessary to control the oxygen concentration in the gas phase from the viewpoint of avoiding the explosion.
- the oxygen concentration in the gas phase can be controlled by supplying a gas with a controlled oxygen concentration into the system.
- the gas supplied into the system may contain gas components other than oxygen, such as nitrogen, carbon dioxide, and argon. More specifically, the oxygen concentration in the gas phase can be controlled by adding an inert gas so that the oxygen concentration is equal to or lower than the critical oxygen concentration of the gas having the explosion range. An inert gas is desirable as a gas component other than oxygen.
- the oxygen concentration-controlled gas is supplied into the liquid phase, whereby the oxygen concentration in the gas phase is indirectly controlled. This is because the oxygen concentration in the gas phase can be controlled by adjusting the dissolved oxygen concentration in the liquid phase as follows.
- Oxygen in the gas supplied into the liquid phase dissolves in the liquid phase and eventually reaches a saturated concentration.
- dissolved oxygen in the liquid phase is consumed by the metabolic activity of the microorganism to be cultured, and as a result, the dissolved oxygen concentration falls below the saturation concentration.
- oxygen in the gas phase or oxygen in the newly supplied gas can move to the liquid phase due to gas-liquid equilibrium. That is, the oxygen concentration in the gas phase decreases depending on the oxygen consumption rate of the microorganism. It is also possible to control the oxygen concentration in the gas phase by controlling the oxygen consumption rate of the microorganism to be cultured.
- the oxygen consumption rate is increased, and the oxygen concentration in the gas phase is 9% (v / v) or less (eg, 5% (v / v) or less, 0.8 % (V / v) or less, 0.6% (v / v) or less, 0.5% (v / v) or less, 0.4% (v / v) or less, 0.3% (v / v)
- it can be set to 0.2% (v / v) or less, or 0.1% (v / v) or less) or substantially 0% (v / v).
- the oxygen concentration in the initially supplied gas can be set low. Therefore, the oxygen concentration in the gas phase can be set in consideration of the oxygen consumption rate by the microorganisms in the liquid phase and the oxygen concentration in the gas to be supplied.
- Cultivation can be performed using a liquid medium.
- a microorganism cultured in a solid medium such as an agar medium may be directly inoculated into a liquid medium, or a microorganism cultured with a seed in a liquid medium may be inoculated into a liquid medium for main culture.
- the culture may be performed separately for seed culture and main culture.
- the culture conditions of seed culture and main culture may be the same or different.
- the amount of microorganisms contained in the medium at the start of culture is not particularly limited.
- a seed culture solution having an OD660 of 4 to 8 may be added at 0.1 to 30% by mass, preferably 1 to 10% by mass with respect to the medium for main culture at the start of culture.
- Culturing can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof.
- the culture medium at the start of the culture is also referred to as “initial culture medium”.
- a medium supplied to a culture system (fermentor) in fed-batch culture or continuous culture is also referred to as “fed-batch medium”.
- supplying a feeding medium to a culture system in fed-batch culture or continuous culture is also referred to as “fed-batch”.
- cultivation is performed by dividing into seed culture and main culture, for example, both seed culture and main culture may be performed by batch culture. Further, for example, seed culture may be performed by batch culture, and main culture may be performed by fed-batch culture or continuous culture.
- Cultivation may be performed under aerobic conditions, may be performed under slight aerobic conditions, or may be performed under anaerobic conditions.
- the culture is preferably performed under microaerobic conditions or anaerobic conditions.
- the aerobic condition means that the dissolved oxygen concentration in the liquid medium is 0.33 ppm or more, preferably 1.5 ppm or more, which is a detection limit by the oxygen membrane electrode.
- the microaerobic condition means that oxygen is supplied to the culture system, but the dissolved oxygen concentration in the liquid medium is less than 0.33 ppm.
- Anaerobic conditions refer to conditions where oxygen is not supplied to the culture system.
- the culture may be performed under the conditions selected above during the entire period, or may be performed under the conditions selected above only during a part of the period.
- “culturing under aerobic conditions” means that culture is performed under aerobic conditions during at least a part of the whole period of culture.
- “culturing under microaerobic conditions” means that the culture is performed under microaerobic conditions during at least a part of the entire culture period.
- “culturing under anaerobic conditions” means that culturing is performed under anaerobic conditions in at least a part of the entire period of culture.
- the “partial period” may be, for example, a period of 50% or more, 70 or more, 80% or more, 90% or more, 95% or more, or 99% or more of the entire culture period.
- cultivation may mean the whole period of main culture, when culture
- the dissolved oxygen concentration in the liquid medium can be reduced by means such as reducing the aeration volume and stirring speed, culturing without sealing the container and aerated with an inert gas containing carbon dioxide gas. Achieving anaerobic or anaerobic conditions.
- the pH of the medium may be, for example, pH 3 to 10, preferably pH 4.0 to 9.5. During the culture, the pH of the medium can be adjusted as necessary. The pH of the medium is adjusted using various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, calcium hydroxide, and magnesium hydroxide. can do.
- various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, calcium hydroxide, and magnesium hydroxide. can do.
- the medium may contain carbonate ions, bicarbonate ions, carbon dioxide gas, or a combination thereof.
- These components may be supplied, for example, by microbial metabolism or may be supplied from carbonates and / or bicarbonates used for pH adjustment. Moreover, these components can also be supplied by separately adding carbonic acid, bicarbonate, a salt thereof, or carbon dioxide as necessary.
- Specific examples of the carbonate or bicarbonate salt include, for example, calcium carbonate, magnesium carbonate, ammonium carbonate, sodium carbonate, potassium carbonate, ammonium bicarbonate, sodium bicarbonate, and potassium bicarbonate.
- Carbonate ions and / or bicarbonate ions may be added at a concentration of, for example, 0.001 to 5M, preferably 0.1 to 3M, more preferably 1 to 2M.
- carbon dioxide is contained, for example, 50 mg to 25 g, preferably 100 mg to 15 g, more preferably 150 mg to 10 g of carbon dioxide may be contained per liter of the solution.
- the culture temperature may be, for example, 20 ° C. to 45 ° C., preferably 25 ° C. to 37 ° C.
- the culture period may be, for example, 10 hours to 120 hours.
- the culture may be continued, for example, until the carbon source in the medium is consumed or until the activity of the microorganism is lost.
- gas is released from a gas discharge portion made of a sintered metal film, and the culture solution containing the released gas is passed through the inside of the draft tube to stir the culture solution.
- the culture medium containing gas has a low apparent density and usually rises perpendicular to the horizontal plane. Therefore, in the method of the present invention, a draft tube is provided so that a tube axis direction thereof is perpendicular to a horizontal plane so that a gas-containing culture medium having a low apparent density passes through the inside of the draft tube. It is preferable to release gas from a gas discharge portion provided in the vicinity of the lower opening.
- the culture medium stirring mechanism by the gas lift action is as described above.
- the gas released from the gas discharge part may be determined according to the culture conditions (aerobic conditions, anaerobic conditions, etc.) of the microorganisms.
- the gas released from the gas release part is preferably an oxygen-containing gas.
- the oxygen-containing gas is not particularly limited as long as it has an oxygen concentration sufficient for culturing microorganisms under aerobic conditions. For example, air, oxygen-enriched air, pure oxygen, and an inert gas (nitrogen) Etc.).
- the gas released from the gas releasing part is not particularly limited as long as it does not substantially contain oxygen, such as nitrogen, carbon dioxide, hydrogen, methane, carbon monoxide, and These mixed gases are mentioned.
- the preferable range of the gas discharge linear velocity of the gas discharge portion made of the sintered metal film and the gas superficial velocity of the draft tube is as described in the above [Gas Stirring Fermenter].
- the linear velocity of the released gas of the gas releasing portion made of a sintered metal film is preferably 0.04 m / s or less.
- the method of the present invention may further include recovering the chemical substance.
- a chemical substance has low solubility in a culture solution and can be easily separated and collected.
- the chemical substance may be recovered as a liquid, or the chemical substance may be recovered as a gas.
- purification and isolation treatment may be performed by a known method.
- Preparation Example 1 Microaerobic inducible isoprenoid compound-producing microorganism (SWITCH-Plld / IspSM), phosphate deficiency-inducing isoprenoid compound-producing microorganism (SWITCH-PphoC / IspSM, SWITCH-PpstS / IspSM), arabinose-induced isoprenoid compound production Construction of microorganisms (SWITCH-Para / IspSM) 1-1) Construction of pMW-Para-mvaES-Trp 1-1-1) Chemical synthesis of movaE gene derived from Enterococcus faecalis Acetyl-CoA acyltransferase coding and hydroxymethyl-encoding enzyme The base sequence of mvaE and the amino acid sequence are already (Base sequence ACCESSION numbers: AF29009.1, (1479..3890), amino acid sequence ACCESSION numbers: AAG02439) (J.
- the amino acid sequence of Enterococcus faecalis-derived mvaE protein and the nucleotide sequence of the gene are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
- the mvaE gene was transformed into E. coli. E. coli for efficient expression in E. coli.
- An mvaE gene optimized for E. coli codon usage was designed and named EFmvaE. This base sequence is shown in SEQ ID NO: 7.
- the mvaE gene was chemically synthesized and then cloned into pUC57 (GenScript), and the resulting plasmid was named pUC57-EFmvaE.
- coli An mvaS gene optimized for E. coli codon usage was designed and named EFmvaS. This base sequence is shown in SEQ ID NO: 10. The mvaS gene was chemically synthesized and then cloned into pUC57 (GenScript), and the resulting plasmid was named pUC57-EFmvaS.
- arabinose-inducible mevalonate pathway upstream gene expression vector was constructed by the following procedure. E. coli by PCR using the plasmid pKD46 as a template and the synthetic oligonucleotides shown in SEQ ID NO: 11 and SEQ ID NO: 12 as primers. A PCR fragment containing Para comprising the araC and araBAD promoter sequences from E. coli was obtained. A PCR fragment containing the EFmvaE gene was obtained by PCR using the plasmid pUC57-EFmvaE as a template and the synthetic oligonucleotides shown in SEQ ID NO: 13 and SEQ ID NO: 14 as primers.
- a PCR fragment containing the EFmvaS gene was obtained by PCR using the plasmid pUC57-EFmvaS as a template and the synthetic oligonucleotides shown in SEQ ID NO: 15 and SEQ ID NO: 16 as primers.
- a PCR fragment containing the Ttrp sequence was obtained by PCR using the plasmid pSTV-Ptac-Ttrp as a template and the synthetic oligonucleotides shown in SEQ ID NO: 17 and SEQ ID NO: 18 as primers.
- Prime Star polymerase (manufactured by Takara Bio Inc.) was used for PCR to obtain these four PCR fragments.
- the reaction solution was prepared according to the composition attached to the kit, and 30 cycles of reaction at 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 1 minute / kb were performed.
- Synthetic oligonucleotides shown in SEQ ID NO: 11 and SEQ ID NO: 14 using a PCR product containing purified Para and a PCR product containing EFmvaE gene as a template, and a PCR product containing purified EFmvaS gene and a PCR product containing Ttrp as a template.
- PCR was performed using 15 and the synthetic oligonucleotide shown in SEQ ID NO: 18 as primers.
- Plasmid pMW219 (manufactured by Nippon Gene Co., Ltd.) was digested with SmaI according to a conventional method. A PCR product containing pMW219 and purified Para and EFmvaE genes, and a PCR product containing EFmvaS gene and Ttrp after SmaI digestion were ligated using In-Fusion HD Cloning Kit (Clontech). The resulting plasmid was named pMW-Para-mvaES-TTrp.
- the KpnI-SalI fragment of pMW-Para-mvaES-Ttrp was cloned into the SphI-SalI recognition site of pAH162- ⁇ attL-TcR- ⁇ attR.
- E.I. E. coli under the control of the Para Para promoter and repressor gene araC.
- the pAH162-Para-mvaES plasmid carrying the faecalis-derived mvaES operon was constructed (FIG. 4).
- a set of plasmids for immobilizing chromosomes holding mvaES genes under the control of different promoters was constructed.
- a polylinker containing I-SceI, XhoI, PstI and SphI recognition sites was inserted into the only HindIII recognition site located upstream of the mvaES gene.
- annealing was performed using primers 1 and 2 (Table 5) and polynucleotide kinase.
- the obtained double-stranded DNA fragment was 5 'phosphorylated with polynucleotide kinase, and the obtained phosphorylated fragment was inserted into pAH162-mvaES plasmid cleaved with HindIII by ligation reaction.
- the resulting pAH162-MCS-mvaES plasmid (FIG. 6) is convenient for cloning the promoter while maintaining the desired orientation in front of the mvaES gene.
- DNA fragments carrying the regulatory regions of the lldD, phoC and pstS genes were obtained from P. cerevisiae.
- the P tac promoter was inserted into the HindIII-SphI recognition site of the pAH162- ⁇ attL-Tc R - ⁇ attR vector (Minaeva NI et al., BMC Biotechnol., 2008; 8:63). As a result, an expression vector pAH162-P tac for chromosome fixation was constructed. The sequence of the cloned promoter fragment was determined and confirmed to be the sequence as designed. A map of pAH162-P tac is shown in FIG.
- FIG. 11 A DNA fragment (FIG. 10) carrying the cerevisiae-derived PMK, MVD and yIDI genes was subcloned into the SphI-KpnI restriction enzyme recognition site of the chromosome fixing vector pAH162-Ptac.
- the DNA sequence containing the chemically synthesized KDyI operon is shown in SEQ ID NO: 43.
- the resulting plasmid pAH162-Tc-Ptac-KDyI carrying the Ptac-KDyI expression cassette is shown in FIG. 11 (A).
- SC17 (0) P. ananatis AJ13355 is a ⁇ Red resistant derivative (Katashkina JI et al., BMC Mol Biol., 2009; 10:34); The annotated complete genome sequence of ananatis AJ13355 is available as PRJDA 162073 or GenBank accession numbers AP012032.1 and AP012033.1. pMWattphi plasmid [Minaeva NI et al. , BMC Biotechnol.
- the DNA fragment used to replace the crt operon with attL phi80 -kan-attR phi80 was amplified in a reaction using primers 19 and 20 (Table 1).
- the pMWattphi plasmid (Minaeva NI et al., BMC Biotechnol., 2008; 8:63) was used as a template in this reaction.
- the resulting integrant was named SC17 (0) ⁇ crt :: attL phi80 -kan-attR phi80 .
- Primers 21 and 22 (Table 1) were combined with SC17 (0) ⁇ crt :: attL phi80 -kan-attR phi80 . It was used for PCR verification of the chromosomal structure.
- the pAH162-Ptac-mvk (M. palidicola) plasmid was prepared according to a previously reported protocol (Andrewa IG et al., FEMS Microbiol Lett., 2011; 318 (1): 55-60) SC17 (0) ⁇ crt :: attB It was integrated into the attB phi80 site of phi80 . Plasmid integration was confirmed by polymerase chain reaction using primers 21 and 23 and primers 22 and 24 (Table 1). As a result, an SC17 (0) ⁇ crt :: pAH162-P tac -mvk (M. palidicola) strain was obtained.
- FIG. 14 (A) A map of the ⁇ crt :: pAH162-P tac -mvk (M. palidicola) modification is shown in FIG. 14 (A).
- the genetic traits of SC17 (0) ⁇ crt :: pAH162-P tac -mvk (M. palladicola) via a genomic DNA electroporation technique (Katashkina JI et al., BMC Mol Biol., 2009; 10:34).
- the obtained strain uses the tetracycline resistance gene tetRA as a marker.
- SWITCH strain set The pAH162-Km-Ptac-KDyI plasmid was prepared according to the previously reported protocol (Andrewa IG et al. FEMS Microbiol Lett. 2011; 318 (1): 55-60), SC17 (0 ) ⁇ ampH :: attB ⁇ 80 ⁇ ampC :: attB ⁇ 80 ⁇ crt :: P tac -mvk (M.palladicola) / pAH123-cat was integrated into the chromosome of the strain. Cells were seeded on LB agar containing 50 mg / L kanamycin.
- Proliferated Km R clones were tested in a PCR reaction using primers 11 and 15 and primers 11 and 17 (Table 1). Strains carrying the pAH162-Km-Ptac-KDyI plasmid integrated into ⁇ ampH :: attB ⁇ 80 or ⁇ ampC :: attB ⁇ 80 m were selected. Maps of ⁇ ampH :: pAH162-Km-Ptac-KDyI and ⁇ ampC :: pAH162-Km-Ptac-KDyI chromosome variants are shown in FIGS. 15 (A) and (B).
- pAH162-Px-mvaES (where, Px is the one of the following regulatory region: araC-P ara (E.coli) , P lldD, P phoC, P pstS) a previously reported protocol [Andreeva IG et al. , FEMS Microbiol Lett. , 2011; 318 (1): 55-60] using the pAH123-cat helper plasmid, SC17 (0) ⁇ ampC :: pAH162-Km-P tac -KDyI ⁇ ampH pH :: attB phi80 ⁇ crt :: P tac -mvk ( M.
- Preparation Example 2 Construction of SC17 (0) ⁇ gcd and SWITCH-PphoC ⁇ gcd and introduction of isoprene synthase
- the Ananatis gcd gene encodes glucose dehydrogenase; Ananatis is known to accumulate gluconic acid during aerobic growth (Andreeva IG et al., FEMS Microbiol Lett. 2011 May; 318 (1): 55-60).
- ⁇ Red-dependent integration of DNA fragments obtained by PCR using primers gcd-attL and gcd-attR (Table 2) and pMW118-attL-kan-attR plasmid as template Minaeva NI et al., BMC Biotechnol.
- Primers gcd-t1 and gcd-t2 are used for PCR analysis of the resulting integrants.
- the kanamycin resistance marker gene was obtained by standard ⁇ Ing / Xis mediated procedures [Katashkina JI et al., BMC Mol Biol. 2009; 10:34].
- the resulting strain is named SWITCH-PphoC ⁇ gcd strain.
- a competent cell of SWITCH-PphoC ⁇ gcd strain was prepared according to a standard method, and pSTV28-Ptac-IspSM (WO2013 / 179722), a vector for expressing isoprene synthase derived from Mucuna, was introduced by electroporation.
- the resulting isoprenoid compound-producing microorganism was named SWITCH-PphoC ⁇ gcd / IspSM.
- the gas stirring type fermentation apparatus includes a draft tube disposed inside and a gas supply pipe for supplying gas to the inside of the culture tank, and the gas supply pipe is made of a sintered metal film having an average pore diameter of 5.0 ⁇ m. A discharge part is provided.
- the culture conditions are pH 6.8 (controlled with ammonia gas), 34 ° C., and 2.3 L / min (2.3 L / min) so that the linear velocity of the released gas of the gas releasing portion made of the sintered metal film is 0.03 m / sec.
- the isoprene concentration in the fermentation exhaust gas is measured using a multi-gas analyzer (F10 manufactured by GASERA).
- the oxygen and carbon dioxide concentrations in the fermentation exhaust gas are measured using a gas analyzer (DEX-1562A manufactured by Able Co., Ltd.).
- Example 2 Culture evaluation of SWITCH-PphoC ⁇ gcd / IspSM A culture test of the isoprene-producing microorganism SWITCH-PphoC ⁇ gcd / IspSM was performed using a gas-stirring fermentation apparatus.
- a 3 L volume gas stirring type fermenter (BMA-02PI type manufactured by Able Co., Ltd.) was used for the culture.
- the gas-stirring fermentation apparatus includes a culture tank, a draft tube disposed inside the culture tank, and a gas supply pipe that supplies gas to the inside of the culture tank, and the gas supply pipe has an average pore diameter of 5.
- the gas discharge part which consists of a 0-micrometer sintered metal film was provided.
- the inner diameter D 1 of the culture vessel is 7.6 cm
- the inner diameter D 2 of the draft tube is 5.5cm
- the top of the height H 2max draft tube from the bottom of the culture vessel 33.1Cm draft from the bottom of the culture tank
- the lowermost height H 2min of the tube was 9.8 cm.
- the glycerol stock of SWITCH-PphoC ⁇ gcd / IspSM strain was thawed, and 50 ⁇ L of the cell suspension was evenly applied to 6 LB plates containing 60 mg / L chloramphenicol, and precultured at 34 ° C. for 16 Incubate for hours. Subsequently, 2.0 L of fermentation medium was poured into the culture tank of the gas stirring type fermentation apparatus.
- the fermentation medium performs heat sterilization at 120 ° C. for 20 minutes, and after cooling, mixes A section and B section 1: 1.
- the culture temperature is 34 ° C.
- aseptic air is 2.3 L / min (oxygen concentration: 21% (v / v)) so that the linear velocity of the released gas of the gas releasing part made of a sintered metal film is 0.03 m / sec. was fed into the fermentation medium.
- the pH of the fermentation medium was controlled to 6.8 using ammonia gas, and the culture was performed for 24 hours while measuring the DO concentration in the fermentation medium using a galvanic DO sensor (SDOU-10L160-125 manufactured by Able Co., Ltd.). Went.
- the DO concentration at the start of cultivation before inoculation was 21%, and the DO concentration in the saturated sodium sulfite solution was 0%.
- Ammonia gas for pH adjustment was supplied from the upper part of the fermentation apparatus by connecting a gas supply pipe dedicated to supplying ammonia gas to the fermentation medium, and the culture temperature was controlled using a silicon rubber heater and cooling water.
- a glucose medium containing 0.07 mL / L of a deformed GD-113K adjusted to 700 g / L is continuously added so that the glucose concentration in the fermentation medium is in the range of 20 g / L to 40 g / L. did.
- Sampling is performed as appropriate after the start of culture. Analysis of D. value and glucose concentration was performed.
- concentration of isoprene in the fermentation exhaust gas is measured using a multi-gas analyzer (F10 manufactured by GASERA), and the oxygen and carbon dioxide concentrations in the fermentation exhaust gas are measured using a gas analyzer (DEX-1562A manufactured by Able Co., Ltd.). It was measured.
- the OD value was measured at 600 nm after diluting the fermentation broth 101 times with a spectrophotometer (U-2900, manufactured by Hitachi High-Tech Science Co., Ltd.).
- the gas release section includes a culture layer, a draft tube arranged inside the culture tank, and a gas supply pipe for supplying gas to the inside of the culture tank, and the gas supply pipe is made of a sintered metal film. It was demonstrated that a chemical substance (isoprene) can be produced by using a gas stirring fermenter equipped with
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Abstract
Description
[1] 培養槽と、
培養槽の内部に配置されたドラフトチューブと、
培養槽の内部へとガスを供給するガス供給管と、
を含み、
ガス供給管が、焼結金属膜からなるガス放出部を備え、
ガス放出部から放出されるガスにより培養槽内の培養液を攪拌可能としたことを特徴とする、ガス攪拌式発酵装置。
[2] 焼結金属膜の平均細孔径が20μm以下である、[1]に記載の装置。
[3] 焼結金属膜からなるガス放出部の放出ガス線速(ガス放出量[m3/s]/焼結金属膜の表面積[m2])が、0.04m/s以下である、[1]又は[2]に記載の装置。
[4] 培養槽の内径をD1、ドラフトチューブの内径をD2としたとき、D1とD2が、0.7<D2/D1の関係を満たす、[1]~[3]のいずれかに記載の装置。
[5] 化学物質の生産能を有する微生物を含む培養液に、焼結金属膜からなるガス放出部からガスを放出し、放出されたガスを含有する培養液をドラフトチューブの内部を通過させて培養液を攪拌すること、及び
微生物を培養して化学物質を製造すること
を含む、化学物質の製造方法。
[6] 化学物質が、可燃性物質を含む、[5]に記載の方法。
[7] 化学物質が、疎水性物質である、[5]又は[6]に記載の方法。
[8] 化学物質が、イソプレノイド化合物である、[5]~[7]のいずれかに記載の方法。
[9] 焼結金属膜の平均細孔径が20μm以下である、[5]~[8]のいずれかに記載の方法。
[10] 焼結金属膜からなるガス放出部の放出ガス線速(ガス放出量[m3/s]/焼結金属膜の表面積[m2])が、0.04m/s以下である、[5]~[9]のいずれかに記載の方法。
本発明のガス攪拌式発酵装置は、
培養槽と、
培養槽の内部に配置されたドラフトチューブと、
培養槽の内部へとガスを供給するガス供給管と、
を含み、
ガス供給管が、焼結金属膜からなるガス放出部を備え、
ガス放出部から放出されるガスにより培養槽内の培養液を攪拌可能としたことを特徴とする。
培養槽は、後述する焼結金属膜からなるガス放出部を備えたガス供給管とドラフトチューブとを設けることが可能である限りにおいて特に限定されない。製造対象である化学物質の性質(比重、可燃性の有無等)や製造規模、培養方法(バッチ培養法、流加培養法、連続培養法等)、培養条件(好気性条件、嫌気性条件等)などに応じて、適切な培養槽を選択してよい。なお、培養液については後述することとする。
ドラフトチューブは、上記のガスリフト作用を達成し得る限りにおいて特に限定されず、公知のドラフトチューブ(「ガスリフト管」とも呼ばれる。)を使用してよい。
ガス供給管は、培養槽の内部へとガスを供給する機能を有する。本発明のガス攪拌式発酵装置において、ガス供給管は、焼結金属膜からなるガス放出部を備えることを特徴とする。なお、培養槽の内部へと供給されるガスの詳細は後述することとする。
(i):1≦(HL-H2max)/D1
(ii):H2min/D2≦2
本発明の化学物質の製造方法は、
化学物質の生産能を有する微生物を含む培養液に、焼結金属膜からなるガス放出部からガスを放出し、放出されたガスを含有する培養液をドラフトチューブの内部を通過させて培養液を攪拌すること、及び
微生物を培養して化学物質を製造すること
を含む。
バシラス(Bacillus)属細菌としては、例えば、枯草菌(Bacillus subtilis)、炭疽菌(Bacillus anthracis)、セレウス菌(Bacillus cereus)等が挙げられ、枯草菌(Bacillus subtilis)がより好ましい。
コリネバクテリウム(Corynebacterium)属細菌としては、例えば、コリネバクテリウム・グルタミカム(Corynebacterium glutamicum)、コリネバクテリウム・エフィシエンス(Corynebacterium efficiens)、コリネバクテリウム・カルナエ(Corynebacterium callunae)等が挙げられ、コリネバクテリウム・グルタミカムがより好ましい。
エシェリヒア(Escherichia)属細菌としては、エシェリヒア・コリ(Escherichia coli)が好ましい。
パントエア(Pantoea)属細菌としては、例えば、パントエア・アナナティス(Pantoea ananatis)、パントエア・スチューアルティ(Pantoea stewartii)、パントエア・アグロメランス(Pantoea agglomerans)、パントエア・シトレア(Pantoea citrea)等が挙げられ、パントエア・アナナティス(Pantoea ananatis)、パントエア・シトレア(Pantoea citrea)が好ましい。また、パントエア属細菌としては、欧州特許出願公開第0952221号に例示された株を使用してもよい。パントエア属細菌の代表的な株としては、例えば、欧州特許出願公開第0952221号に開示されるパントエア・アナナティスAJ13355株(FERM BP-6614)およびパントエア・アナナティスAJ13356株(FERM BP-6615)が挙げられる。
エンテロバクター(Enterobacter)属細菌としては、例えば、エンテロバクター・アグロメランス(Enterobacter agglomerans)、エンテロバクター・アエロゲネス(Enterobacter aerogenes)等が挙げられ、エンテロバクター・アエロゲネス(Engerobacter aerogenes)が好ましい。また、エンテロバクター属細菌としては、欧州特許出願公開第0952221号に例示された菌株を使用してもよい。エンテロバクター属細菌の代表的な株としては、例えば、エンテロバクター・アグロメランスATCC12287株、エンテロバクター・アエロゲネスATCC13048株、エンテロバクター・アエロゲネスNBRC12010株(Biotechnol Bioeng. 2007 Mar 27;98(2):340-348)、エンテロバクター・アエロゲネスAJ110637(FERM BP-10955)株等が挙げられる。エンテロバクター・アエロゲネスAJ110637株は、2007年8月22日付で独立行政法人 産業技術総合研究所 特許生物寄託センター(〒305-8566 日本国茨城県つくば市東1丁目1番地1 中央第6)に受託番号FERM P-21348として寄託され、2008年3月13日にブダペスト条約に基づく国際寄託に移管され、FERM BP-10955の受領番号が付与されている。
サッカロミセス(Saccharomyces)属の微生物としては、例えば、サッカロミセス・カールスベルゲンシス(Saccharomyces carlsbergensis)、サッカロミセス・セレビシエー(Saccharomyces cerevisiae)、サッカロミセス・ディアスタティクス(Saccharomyces diastaticus)、サッカロミセス・ドウグラシー(Saccharomyces douglasii)、サッカロミセス・クルイベラ(Saccharomyces kluyveri)、サッカロミセス・ノルベンシス(Saccharomyces norbensis)、サッカロミセス・オビフォルミス(Saccharomyces oviformis)が挙げられ、サッカロミセス・セレビシエー(Saccharomyces cerevisiae)が好ましい。
シゾサッカロミセス(Schizosaccharomyces)属の微生物としては、シゾサッカロミセス・ポンベ(Schizosaccharomyces pombe)が好ましい。
ヤロウイア(Yarrowia)属の微生物としては、ヤロウィア・リポリティカ(Yarrowia lipolytica)が好ましい。
トリコデルマ(Trichoderma)属の微生物としては、例えば、トリコデルマ・ハルジアヌム(Ttichoderma harzianum)、トリコデルマ・コニンギー(Trichoderma koningii)、トリコデルマ・ロンギフラキアム(Trichoderma longibrachiatum)、トリコデルマ・リーゼイ(Trichoderma reesei)、トリコデルマ・ビリデ(Trichoderma viride)が挙げられ、トリコデルマ・リーゼイ(Trichoderma reesei)が好ましい。
1-1)pMW-Para-mvaES-Ttrpの構築
1-1-1)Enterococcus faecalis由来mvaE遺伝子の化学合成
acetyl-CoA acetyltransferaseとhydroxymethlglutaryl-CoAreductaseをコードするEnterococcus faecalis由来mvaEの塩基配列、及びアミノ酸配列はすでに知られている(塩基配列のACCESSION番号:AF290092.1、(1479..3890)、アミノ酸配列のACCESSION番号:AAG02439)(J.Bacteriol.182(15),4319-4327(2000))。Enterococcus faecalis由来mvaEタンパク質のアミノ酸配列、及び遺伝子の塩基配列を配列番号5、及び配列番号6にそれぞれ示す。mvaE遺伝子をE.coliで効率的に発現させるためにE.coliのコドン使用頻度に最適化したmvaE遺伝子を設計し、これをEFmvaEと名付けた。この塩基配列を配列番号7に示す。mvaE遺伝子は化学合成された後、pUC57(GenScript社製)にクローニングされ、得られたプラスミドをpUC57-EFmvaEと名付けた。
hydroxymethylglutaryl-CoA synthaseをコードするEnterococcus faecalis由来mvaSの塩基配列、及びアミノ酸配列はすでに知られている(塩基配列のACCESSION番号:AF290092.1、complement(142..1293)、アミノ酸配列のACCESSION番号:AAG02438)(J.Bacteriol.182(15),4319-4327(2000))。Enterococcus faecalis由来mvaSタンパク質のアミノ酸配列、及び遺伝子の塩基配列を配列番号8、及び配列番号9にそれぞれ示す。mvaS遺伝子をE.coliで効率的に発現させるためにE.coliのコドン使用頻度に最適化したmvaS遺伝子を設計し、これをEFmvaSと名付けた。この塩基配列を配列番号10に示す。mvaS遺伝子は化学合成された後、pUC57(GenScript社製)にクローニングされ、得られたプラスミドをpUC57-EFmvaSと名付けた。
アラビノース誘導型メバロン酸経路上流遺伝子発現ベクターは次の手順で構築した。プラスミドpKD46を鋳型として配列番号11と配列番号12に示す合成オリゴヌクレオチドをプライマーとしたPCRによりE.coli由来araCとaraBADプロモーター配列からなるParaを含むPCR断片を得た。プラスミドpUC57-EFmvaEを鋳型として配列番号13と配列番号14に示す合成オリゴヌクレオチドをプライマーとしたPCRによりEFmvaE遺伝子を含むPCR断片を得た。プラスミドpUC57-EFmvaSを鋳型として配列番号15と配列番号16に示す合成オリゴヌクレオチドをプライマーとしたPCRによりEFmvaS遺伝子を含むPCR断片を得た。プラスミドpSTV-Ptac-Ttrpを鋳型として配列番号17と配列番号18に示す合成オリゴヌクレオチドをプライマーとしたPCRによりTtrp配列を含むPCR断片を取得した。これら4つのPCR断片を得るためのPCRにはPrime Starポリメラーゼ(タカラバイオ(株)製)を用いた。反応溶液はキットに添付された組成に従って調整し、98℃にて10秒、55℃にて5秒、72℃にて1分/kbの反応を30サイクル行った。精製したParaを含むPCR産物とEFmvaE遺伝子を含むPCR産物を鋳型として配列番号11と配列番号14に示す合成オリゴヌクレオチドを、精製したEFmvaS遺伝子を含むPCR産物とTtrpを含むPCR産物を鋳型として配列番号15と配列番号18に示す合成オリゴヌクレオチドをプライマーとしてPCRを行った。その結果、ParaとEFmvaE遺伝子、EFmvaSとTtrp含むPCR産物を取得した。プラスミドpMW219((株)ニッポンジーン製)は常法に従ってSmaI消化した。SmaI消化後pMW219と精製したParaとEFmvaE遺伝子を含むPCR産物、EFmvaS遺伝子とTtrpを含むPCR産物はIn-Fusion HD Cloning Kit(Clontech社製)を用いて連結した。得られたプラスミドは、pMW-Para-mvaES-Ttrpと命名した。
メバロン酸経路の上流および下流遺伝子を保有する組込み型プラスミドを構築するため、pAH162-λattL-TcR-λattR vector(Minaeva NI et al.,BMC Biotechnol.2008;8:63)を用いた。
tetAR遺伝子を含むpAH162-λattL-TcR-λattR(Minaeva NI et al.,BMC Biotechnol.,2008;8:63)のAatII-ApaIフラグメントを、プライマー9および10(表1)、ならびにpUC4Kプラスミド(Taylor LAおよびRose RE.,Nucleic Acids Res.,16,358,1988)を鋳型として用いたPCRで得られたDNAフラグメントと置換した。その結果、pAH162-λattL-KmR-λattRが得られた(図8)。
attLphi80およびattRphi80に隣接したkan遺伝子、ならびに標的染色体部位に相同な40bp配列を含むPCR増幅DNAフラグメントのλRed依存的な組込み(Katashkina JI et al.,BMC Mol Biol.,2009;10:34)、続いて、カナマイシン耐性マーカーのファージphi80 Int/Xis依存的な除去(Andreeva IG et al.,FEMS Microbiol Lett.,2011;318(1):55-60)を含む2段階の手法を用いて、ΔampH::attBphi80およびΔampC::attBphi80染色体改変を、P.ananatis SC17(0)株に段階的に導入した。SC17(0)は、P.ananatis AJ13355のλRed耐性誘導体である(Katashkina JI et al.,BMC Mol Biol.,2009;10:34);P.ananatis AJ13355の注釈付完全ゲノム配列は、PRJDA162073またはGenBankアクセッション番号AP012032.1およびAP012033.1として利用可能である。pMWattphiプラスミド[Minaeva NI et al.,BMC Biotechnol.,2008;8:63]を鋳型として用いて、プライマー11および12、ならびにプライマー13および14(表1)をプライマーとして用いて、それぞれampHおよびampC遺伝子領域への組込みに使用されるDNAフラグメントを生成した。プライマー15および16、ならびにプライマー17および18(表1)を、得られた染色体改変物のPCR検証に用いた。
その後、ゲノムDNAエレクトロポレーション手法(Katashkina JI et al.,BMC Mol Biol.,2009;10:34)を介してSC17(0)Δcrt::pAH162-Ptac-mvk(M.paludicola)の遺伝形質をSC17(0) ΔampC::attBphi80 ΔampH::attBphi80へ移行させた。得られた株はテトラサイクリン耐性遺伝子tetRAをマーカーとして利用している。tetRAマーカー遺伝子を含むpAH162-Ptac-mvk(M.paludicola)組込み型プラスミドのベクター部分を、既報のpMW-intxis-catヘルパープラスミド[Katashkina JI et al.,BMC Mol Biol.,2009;10:34]を用いて除去した。その結果、マーカー遺伝子欠損株SC17(0) ΔampH::attBφ80 ΔampC::attBφ80 Δcrt::Ptac-mvk(M.paludicola)を得た。Δcrt::Ptac-mvk(M.paludicola)ゲノム改変物のマップを、図14(B)に示す。
pAH162-Km-Ptac-KDyIプラスミドを、既報のプロトコル(Andreeva IG et al. FEMS Microbiol Lett. 2011;318(1):55-60)にしたがい、SC17(0)ΔampH::attBφ80 ΔampC::attBφ80 Δcrt::Ptac-mvk(M.paludicola)/pAH123-cat株の染色体に組み込んだ。50mg/Lカナマイシンを含むLBアガー上に細胞を撒いた。増殖したKmRクローンを、プライマー11および15、ならびにプライマー11および17(表1)を用いたPCR反応で試験した。ΔampH::attBφ80またはΔampC::attBφ80mに組み込まれたpAH162-Km-Ptac-KDyIプラスミドを保持する株を選択した。ΔampH::pAH162-Km-Ptac-KDyIおよびΔampC::pAH162-Km-Ptac-KDyI染色体改変物のマップを、図15(A)および(B)に示す。
pAH162-Px-mvaES(ここで、Pxは、以下の調節領域のうちの一つである:araC-Para(E.coli)、PlldD、PphoC、PpstS)を、既報のプロトコル[Andreeva IG et al.,FEMS Microbiol Lett.,2011;318(1):55-60]にしたがってpAH123-catヘルパープラスミドを用いてSC17(0) ΔampC::pAH162-Km-Ptac-KDyI ΔampH::attBphi80 Δcrt::Ptac-mvk(M.paludicola)およびSC17(0) ΔampC::attBphi80 ΔampH::pAH162-Km-Ptac-KDyI Δcrt::Ptac-mvk(M.paludicola)レシピエント株のattBphi80部位に挿入した。その結果、SWITCH-Px-1およびSWITCH-Px-2とそれぞれ名付けられた2セットの株を得た。ΔampH::pAH162-Px-mvaESおよびΔampC::pAH162-Px-mvaES染色体改変物のマップを図16に示す。
常法に従いSWITCH菌のエレクトロコンピテントセルを作成し、エレクトロポレーションによりムクナ由来イソプレンシンターゼの発現プラスミドであるpSTV28-Ptac-IspSM(WO2013/179722)を導入した。得られたイソプレノイド化合物生成微生物をそれぞれ、SWITCH-Para/IspSM、SWITCH-Plld/IspSM、SWITCH-PpstS/IspSM、SWITCH-PphoC/IspSMと命名した。
P.アナナティスのgcd遺伝子は、グルコースデヒドロゲナーゼをコードしており、P.アナナティスは好気性増殖中にグルコン酸を蓄積することが知られている(Andreeva IGら,FEMS Microbiol Lett.2011 May;318(1):55-60)。
プライマーgcd-attLおよびgcd-attR(表2)及び鋳型としてpMW118-attL-kan-attRプラスミドを用いるPCRで得られたDNAフラグメントのλRed依存的な組込み(Minaeva NIら,BMC Biotechnol.2008;8:63)により、gcd遺伝子が破壊されたSC17(0)Δgcd株を構築する。組込み体を確認するため、プライマーgcd-t1およびgcd-t2(表2)を用いる。
SC17(0)Δgcd株のゲノムDNAを、Wizard Genomic DNA Purification Kit(プロメガ)を用いて単離し、既報の方法〔Katashkina JIら,BMC Mol Biol.2009;10:34〕にしたがってSWITCH-PphoC株のマーカーを含まない誘導体中に電気形質転換する。その結果、SWITCH-PphoC-Δgcd(KmR)株を得る。プライマーgcd-t1およびgcd-t2(表2)を、得られた組込み体のPCR解析に用いる。カナマイシン耐性マーカー遺伝子を、標準的なλIng/Xis媒介手法〔Katashkina JIら,BMC Mol Biol.2009;10:34〕にしたがって得る。得られた株を、SWITCH-PphoC Δgcd株と命名する。
SWITCH-PphoC Δgcd株のコンピテントセルを標準的な方法にしたがって調製し、ムクナ由来のイソプレンシンターゼ発現用ベクターであるpSTV28-Ptac-IspSM(WO2013/179722)をエレクトロポレーションにより導入した。得られたイソプレノイド化合物生成微生物を、SWITCH-PphoC Δgcd/IspSMと命名した。
1)イソプレン生成微生物SWITCH―PphoCΔgcd/IspSM株のジャー培養条件
イソプレン生成微生物SWITCH―PphoCΔgcd/IspSM株の菌体増殖のため、ジャー培養を行う。ジャー培養には3L容積のガス攪拌式発酵装置(エイブル(株)製)を使用する。グルコース培地は表3に示す組成になるように調整する。前培養としてクロラムフェニコール(60mg/L)を含むLBプレートにイソプレン生成微生物SWITCH―PphoCΔgcd/IspSM株を塗布し、34℃にて16時間培養を実施する。2Lのグルコース培地を3L容積のガス攪拌式発酵装置に投入後、充分に増殖したプレート7枚分の菌体を接種し、培養を開始する。ガス攪拌式発酵装置は内部に配置されたドラフトチューブと、培養槽の内部へとガスを供給するガス供給管とを含み、ガス供給管が平均細孔径5.0μmの焼結金属膜からなるガス放出部を備える。培養条件は、pH6.8(アンモニアガスにて制御)、34℃であり、焼結金属膜からなるガス放出部の放出ガス線速が0.03m/secとなるように2.3L/min(酸素濃度:20%(v/v)の空気を培地中に供給する。ガルバニ式DOセンサー(エイブル(株)製)を用いて培養液中の溶存酸素(Dissolved Oxigen,DO)濃度を測定し、48時間培養を行う。培養中は、培地中のグルコース濃度が15g/L以上になるよう500g/Lに調整したグルコースを連続的に添加する。
発酵排気ガス中のイソプレン濃度はマルチガスアナライザー(GASERA社製 F10)を用いて測定する。発酵排気ガス中の酸素、炭酸ガス濃度はガス分析装置(エイブル(株)製 DEX-1562A)を用いて測定する。
ガス攪拌式発酵装置を用いて、イソプレン生成微生物SWITCH-PphoC Δgcd/IspSM株の培養試験を実施した。培養には3L容積のガス攪拌式発酵装置(エイブル(株)製 BMA-02PI型)を使用した。該ガス攪拌式発酵装置は、培養槽と、培養槽の内部に配置されたドラフトチューブと、培養槽の内部へとガスを供給するガス供給管とを含み、ガス供給管が平均細孔径5.0μmの焼結金属膜からなるガス放出部を備えた。培養槽の内径D1は7.6cm、ドラフトチューブの内径D2は5.5cm、培養槽の底部からのドラフトチューブの最上部の高さH2maxは33.1cm、培養槽の底部からのドラフトチューブの最下部の高さH2minは9.8cmであった。
SWITCH-PphoC Δgcd/IspSM株のグリセロールストックを融解し、菌体懸濁液50μLを6枚の60mg/Lのクロラムフェニコールを含むLBプレートに均一に塗布し、前培養として34℃にて16時間培養した。
次いで、発酵培地2.0Lをガス攪拌式発酵装置の培養槽に注入した。ここで、発酵培地は、表4記載のA区とB区をそれぞれ1L調製した後、120℃で20分加熱滅菌を行い、放冷後、A区とB区を1:1で混合し、クロラムフェニコール(60mg/L)を添加して調製した。得られた前培養プレート6枚分の菌体の全量を発酵培地に植菌し、培養を開始した。培養槽の底部からの培養液の液面高さHLは34.8cmであった。
培養温度は34℃とし、焼結金属膜からなるガス放出部の放出ガス線速0.03m/secとなるように2.3L/min(酸素濃度:21%(v/v))の無菌空気を発酵培地中に供給した。発酵培地のpHはアンモニアガスを用いて6.8に制御し、ガルバニ式DOセンサー(エイブル(株)製 SDOU-10L160-125)を用いて発酵培地中のDO濃度を測定しながら、24時間培養を行った。植菌前の培養開始時のDO濃度を21%とし、飽和亜硫酸ナトリウム溶液中のDO濃度を0%とした。pH調整用のアンモニアガスは、発酵装置上部からアンモニアガス供給専用のガス供給管を発酵培地に繋いで供給し、培養温度はシリコンラバーヒーターと冷却水を用いて制御した。培養中は、発酵培地中のグルコース濃度が20g/Lから40g/Lの範囲になるように、700g/Lに調整したディスホームGD-113K 0.07mL/Lを含むグルコース培地を連続的に添加した。
2 ドラフトチューブ
3 ガス供給管
4 焼結金属膜からなるガス放出部
5 培養液
10 ガス攪拌式発酵装置
Claims (10)
- 培養槽と、
培養槽の内部に配置されたドラフトチューブと、
培養槽の内部へとガスを供給するガス供給管と、
を含み、
ガス供給管が、焼結金属膜からなるガス放出部を備え、
ガス放出部から放出されるガスにより培養槽内の培養液を攪拌可能としたことを特徴とする、ガス攪拌式発酵装置。 - 焼結金属膜の平均細孔径が20μm以下である、請求項1に記載の装置。
- 焼結金属膜からなるガス放出部の放出ガス線速(ガス放出量[m3/s]/焼結金属膜の表面積[m2])が、0.04m/s以下である、請求項1又は2に記載の装置。
- 培養槽の内径をD1、ドラフトチューブの内径をD2としたとき、D1とD2が、0.7<D2/D1の関係を満たす、請求項1~3のいずれか1項に記載の装置。
- 化学物質の生産能を有する微生物を含む培養液に、焼結金属膜からなるガス放出部からガスを放出し、放出されたガスを含有する培養液をドラフトチューブの内部を通過させて培養液を攪拌すること、及び
微生物を培養して化学物質を製造すること
を含む、化学物質の製造方法。 - 化学物質が、可燃性物質を含む、請求項5に記載の方法。
- 化学物質が、疎水性物質である、請求項5又は6に記載の方法。
- 化学物質が、イソプレノイド化合物である、請求項5~7のいずれか1項に記載の方法。
- 焼結金属膜の平均細孔径が20μm以下である、請求項5~8のいずれか1項に記載の方法。
- 焼結金属膜からなるガス放出部の放出ガス線速(ガス放出量[m3/s]/焼結金属膜の表面積[m2])が、0.04m/s以下である、請求項5~9のいずれか1項に記載の方法。
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CN109415672A (zh) | 2019-03-01 |
US20180327705A1 (en) | 2018-11-15 |
JPWO2017115855A1 (ja) | 2018-10-18 |
EP3399015A4 (en) | 2019-08-21 |
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