WO2015064648A1 - Algue verte produisant un acide gras - Google Patents

Algue verte produisant un acide gras Download PDF

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WO2015064648A1
WO2015064648A1 PCT/JP2014/078791 JP2014078791W WO2015064648A1 WO 2015064648 A1 WO2015064648 A1 WO 2015064648A1 JP 2014078791 W JP2014078791 W JP 2014078791W WO 2015064648 A1 WO2015064648 A1 WO 2015064648A1
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medium
strain
treatment
culture
gene
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雄太 長坂
臼田　佳弘
陽子 桑原
鈴木　茂雄
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味の素株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C12N1/125—Unicellular algae isolates
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/89—Algae ; Processes using algae

Definitions

  • the present invention relates to green algae that produce fatty acids and use thereof.
  • Soybean seeds and oil palm (oil ⁇ palm) fruits which are oil plants generally used as a raw material for edible fats and oils, contain about 20% fat.
  • microalgae are known to produce fats and oils, and the yield of fats and oils per area of such microalgae is large for oil plants.
  • Exceed. In green algae, for example, it is known that SUHL0708 strain belonging to the genus Desmodesmus accumulates about 28% of fat and oil on an average dry weight of alga body during the culture period (Patent Document 1).
  • recovery of fats and oils from alga bodies requires steps such as alga body separation, dehydration, cell disruption, and oil purification, and is complicated and difficult.
  • fatty acids are produced by subjecting a culture of Chlorella kessleri to a medium temperature (Patent Document 2).
  • Patent Document 2 there is no known strain that produces fatty acids by a medium temperature treatment in green algae belonging to the genus Desmodemus.
  • An object of the present invention is to provide a green algae that generates fatty acids and a method for using the same.
  • the present invention can be exemplified as follows.
  • a green alga that belongs to the genus Desmodesmus and accumulates 25% (w / w) or more of fatty acid per dry weight of the alga when the alga is subjected to intermediate temperature treatment.
  • a green alga selected from the group consisting of AJ7846 strain (FERM BP-22252), AJ7847 strain (FERM BP-22253), and derivatives thereof.
  • the method wherein the bacterium has been modified so as to increase the ability to assimilate fatty acids.
  • the bacterium is a bacterium belonging to the family Enterobacteriaceae or a coryneform bacterium.
  • the method wherein the bacterium is Escherichia coli, Pantoea ananatis, or Corynebacterium glutamicum.
  • the organic solvent is methanol.
  • the method, wherein the medium-low temperature treatment is performed at a temperature of 5 ° C. to 60 ° C. and lower than the medium temperature treatment.
  • the algae of the present invention are green algae that accumulate (generate) fatty acids when the algal bodies are subjected to intermediate temperature treatment.
  • the algae of the present invention may be green algae that accumulate (generate) 25% (w / w) or more of fatty acids per dry weight of algal bodies when the algal bodies are subjected to intermediate temperature treatment.
  • the algae of the present invention may belong to the genus Desmodesmus.
  • Desmodesmus algatas (Desmodesmus armatus), Desmodesmus communis, Desmodesmus rkpirkollei, Desmodesmus ⁇ musstats Perforatus (Desmodesmus perforatus), Desmodesmus intermedius, Desmodesmus brasiliensis, Desmodesmus elegans, Desmodesmus desgans, Desmodesmus elegans ), Desmodesmus Pseudoserratus, Desmodes Maximus (Desmodes) mus maximus), and Desmodesmus bicellularis.
  • algae of the present invention include green algae selected from AJ7846 strain (FERM BP-22252), AJ7847 strain (FERM BP-22253), and derivatives thereof.
  • the AJ7846 and AJ7847 strains are considered to be related to the genus Desmodesmus such as Desmodesmus armatus and Desmodesmus communis.
  • the base sequence of 18S rDNA of AJ7846 strain is shown in SEQ ID NO: 4. According to BLAST analysis of 18S rDNA, AJ7846 strain shows 99.71% homology to Desmodesmus armatus var. Subalternans CCAP 276 / 4A strain and 99.24% homology to Desmodesmus communis CCAP 276 / 4B strain.
  • the base sequence of 18S rDNA of AJ7847 strain is shown in SEQ ID NO: 3. According to BLAST analysis of 18S rDNA, AJ7847 strain shows 99.77% homology to Desmodesmus armatus var. Subalternans CCAP 276 / 4A strain and 99.31% homology to Desmodesmus communis CCAP 276 / 4B strain.
  • the above-mentioned “derivative strain” means a strain constructed with the AJ7846 strain or AJ7847 strain as the parent strain (ancestor strain) and having the same or higher fatty acid producing ability as the parent strain (ancestral strain).
  • the derivative strain may be bred by artificial modification, for example.
  • Artificial alteration includes alteration by genetic engineering techniques and alteration by mutation treatment. Mutation treatments include X-ray irradiation, UV irradiation, and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ethylmethanesulfonate (EMS), and methylmethanesulfonate (MMS). ) And the like.
  • stock produced naturally for example at the time of use of a parent strain (ancestor strain) may be sufficient.
  • derivative strains include mutant strains that are naturally generated when the AJ7846 strain or AJ7847 strain is cultured.
  • a derivative strain may be constructed by one type of modification or may be constructed by two or more types of modification.
  • “Having a fatty acid production capacity equal to or higher than that of the parent strain” means that when the derived strain is cultured and the algal cells are subjected to intermediate temperature treatment, 70% or more, 80% or more of the amount of fatty acid produced under the same conditions in the parent strain , 90% or more, or 95% or more of fatty acids may be produced.
  • the algae of the present invention can be used for the production of, for example, fatty acids, fatty acid esters, sugar glycerol, or combinations thereof. Specifically, by culturing the algae of the present invention and appropriately treating the obtained algal bodies, fatty acids, fatty acid esters, sugar glycerol, or combinations thereof are produced.
  • fatty acids, fatty acid esters, and sugar glycerol are collectively referred to as “target substances”.
  • processes for generating target substances may be collectively referred to as “target substance generation processes”.
  • the method of the present invention includes culturing the algae of the present invention in a medium, subjecting the algal bodies obtained by the culture to a target substance generation treatment, and recovering the target substance from the treated product, Is a method for producing a target substance.
  • algae refers to algae cells obtained by culturing algae in a medium.
  • “treating / subjecting a target object (algae or a processed product thereof) under a specific condition may be read as“ incubating ”the target object under the specific condition.
  • the culture method is not particularly limited as long as the algae of the present invention can grow.
  • the culture conditions can be appropriately set by those skilled in the art.
  • the culture can be performed, for example, under normal conditions used for culturing microalgae.
  • algae such as Chlorella algae, Arthrospira algae (Spirulina), and Dunaliella salina are cultivated industrially on a large scale (Spolaore). , P. et al. 2006. J. Biosci. Bioeng. 101: 87-96).
  • the culture may be performed with reference to such knowledge, for example.
  • Culturing is autotrophic culture using photosynthesis without using organic compounds, heterotrophic culture using organic compounds without using photosynthesis, or mixed nutrition using both photosynthesis and organic compounds. It can be carried out by culture (mixotrophic culture). The culture may usually be performed by autotrophic culture.
  • Culture may be performed in an open system or a closed system.
  • culture can be performed in an open culture system called an open pond.
  • culture can be performed in a closed culture system called a closed photobioreactor.
  • the medium used for the culture is not particularly limited as long as the algae of the present invention can grow.
  • the culture medium may contain, for example, a nitrogen source and various inorganic salts.
  • the culture medium may contain other components, such as a carbon source, as needed.
  • a person skilled in the art can appropriately set the type and concentration of the medium components.
  • a normal medium used for culturing microalgae can be used.
  • As such a medium specifically, for example, 0.3 ⁇ HSM medium (Oyama, Y. et al. 2006. Planta-224: 646-654), 0.2 ⁇ Gambogue medium (Izumo, A. et al. 2007.
  • TAP medium Algae are also known to accumulate fats and oils in the algae when the nitrogen source is depleted (Thompson GA Jr. 1996. Biochim. Biophys. Acta 1302: 17-45).
  • a medium in which the concentration of the nitrogen source is limited may be used for culturing the algae of the present invention.
  • the culture can be performed using a liquid medium.
  • the culture temperature may be, for example, 20 to 40 ° C., preferably 25 to 35 ° C., more preferably around 30 ° C.
  • the initial pH of the medium may be, for example, near neutrality. Near neutral may be, for example, pH 7-9.
  • Near neutral may be, for example, pH 7-9.
  • a suitable inorganic or organic acidic or alkaline substance can be used for pH adjustment.
  • the culture may be performed with aeration.
  • the aeration amount may be, for example, 0.1 to 2 vvm (volume per volume per minute) as an aeration amount per minute per culture medium volume.
  • the culture medium may be further supplied to CO 2.
  • the supply amount of CO 2 may be, for example, 0.5 to 5% (v / v) with respect to the aeration amount.
  • CO 2 and air may be supplied separately to the culture solution, or mixed and supplied to the culture solution.
  • light is supplied to the culture system.
  • the light can be supplied using a suitable light source. Examples of the light source include a white fluorescent lamp, a white light emitting diode, a high pressure sodium lamp, and sunlight. These light sources may be used in appropriate combination.
  • the illuminance of light may be, for example, 1,000 to 10,000 lux.
  • the culture solution may be appropriately stirred or circulated.
  • Various operations such as light supply, air supply, CO 2 supply, agitation, and circulation may be performed continuously or intermittently.
  • the culture period may be, for example, 1 to 40 days.
  • the culture can be performed by batch culture, fed-batch culture, continuous culture, or a combination thereof.
  • cultivation may be performed by dividing into seed culture and main culture.
  • the main culture may be performed, for example, by inoculating 1-50% (v / v) of the seed culture solution in the main culture medium.
  • the culture conditions for the seed culture and the main culture may or may not be the same.
  • both seed culture and main culture may be performed by batch culture.
  • seed culture may be performed by batch culture, and main culture may be performed by fed-batch culture or continuous culture.
  • the algal bodies of the algae of the present invention are produced in the medium.
  • the algal bodies may be subjected to a target substance generation process while contained in the medium, or may be collected from the medium and then subjected to a target substance generation process.
  • the algal bodies may be subjected to a target substance generation treatment after being appropriately pretreated.
  • Examples of the pretreatment include dilution, concentration, freezing, thawing, and drying. These pretreatments may be appropriately combined.
  • the pretreatment can be appropriately selected according to various conditions such as the type of target substance generation treatment.
  • the method for recovering the algal cells from the medium is not particularly limited, and for example, a known method (Grima, E. M. et al. 2003. Biotechnol. Advances 20: 491-515) can be used.
  • algal bodies can be recovered from the culture medium by methods such as natural sedimentation, centrifugation, and filtration. At that time, a flocculant may be used.
  • the collected algal bodies can be appropriately washed using an appropriate medium.
  • the collected alga bodies can be appropriately resuspended using an appropriate medium.
  • Examples of the medium that can be used for washing and suspension include an aqueous medium (aqueous solvent) such as water and an aqueous buffer, an organic medium (organic solvent) such as methanol, and a mixture thereof.
  • aqueous medium such as water and an aqueous buffer
  • organic medium organic solvent
  • the medium can be appropriately selected according to various conditions such as the type of target substance generation treatment.
  • the algal bodies may be subjected to a target substance generation treatment after being diluted or concentrated to a desired degree, for example.
  • Algae body is diluted or concentrated so that the algal body concentration in the suspension is, for example, 25 g / L or more, or 250 g / L or more in terms of dry weight, and then the target substance generation treatment May be used.
  • the algal bodies can be diluted using an appropriate medium as described above. Concentration of algal bodies can be performed, for example, by precipitating algal bodies and removing the supernatant appropriately. Further, the algae can be concentrated by, for example, freeze-drying or evaporation.
  • the algal bodies may be frozen once and then subjected to a target substance generation treatment.
  • the freezing temperature may be, for example, 0 ° C. or lower, ⁇ 20 ° C. or lower, or ⁇ 50 ° C. or lower, and may be ⁇ 80 ° C. or higher.
  • the freezing time may be, for example, 1 hour or longer and 24 hours or shorter. Moreover, you may repeat freeze-thaw.
  • the pH of the reaction system may be adjusted to be weakly acidic or weakly alkaline.
  • the weak acidity may be, for example, pH 3.0 to 7.0, pH 4.0 to 6.0, or pH 4.5 to 6.0.
  • the weak alkalinity may be, for example, pH 7.5 to 12.0, pH 9.0 to 11.0, or pH 9.0 to 10.5.
  • the pH can be adjusted using, for example, an acidic substance such as hydrochloric acid, or an alkaline substance such as NaOH or KOH. By adjusting the pH, the algal bodies may or may not be hydrolyzed.
  • Fatty acid production by medium temperature treatment Fatty acids can be produced by subjecting algal bodies obtained by culture to medium temperature treatment. That is, one aspect of the method of the present invention includes culturing the algae of the present invention in a medium, subjecting the algal bodies obtained by the culture to a medium temperature treatment, and recovering fatty acids from the treated product, Is a method for producing a fatty acid. In the present invention, only one fatty acid may be produced, or two or more fatty acids may be produced.
  • “Medium temperature treatment” refers to treatment at an intermediate temperature.
  • the intermediate temperature treatment for example, the treatment at the intermediate temperature described in WO2011 / 013707 can be referred to.
  • the algal bodies can be subjected to intermediate temperature treatment in a state suspended in an appropriate medium as described above.
  • the “medium temperature” is not particularly limited as long as it is a temperature at which a fatty acid is generated.
  • the intermediate temperature can be appropriately set according to various conditions such as processing time.
  • the intermediate temperature may be, for example, 35 ° C. or higher, 40 ° C. or higher, 45 ° C. or higher, or 50 ° C. or higher. Further, the intermediate temperature may be, for example, 70 ° C. or lower, 65 ° C.
  • the time for the medium temperature treatment can be appropriately set according to various conditions such as the treatment temperature.
  • the time for the medium temperature treatment may be, for example, 1 hour or more, or 5 hours or more.
  • the intermediate temperature treatment time may be, for example, 48 hours or less, or 24 hours or less.
  • the pH of the medium temperature treatment is not particularly limited as long as the fatty acid is generated by the medium temperature treatment.
  • the pH of the medium temperature treatment may be, for example, pH 3.0 to 11.0.
  • the pH of the medium temperature treatment may be, for example, weakly acidic, near neutral, or weakly alkaline.
  • the weak acidity may be in the above weak acid range, for example, pH 4.5 to 6.0.
  • Near neutral may be, for example, pH 7.0-9.0.
  • the weak alkalinity may be in the weak alkalinity range as described above, for example, pH 9.0 to 10.5.
  • the pH of the medium temperature treatment is preferably pH 4.5 to 9.5, more preferably pH 4.5 to 6.5.
  • the pH of the medium temperature treatment is preferably pH 4.5 to 9.5, more preferably pH 5.5 to 7.5.
  • the medium temperature treatment may be performed by standing or may be performed while stirring or shaking.
  • the medium temperature treatment may be performed continuously or intermittently. Reaction conditions such as treatment temperature may or may not be constant throughout the medium temperature treatment. That is, for example, the algal cells may be continuously treated at the same temperature (hereinafter, also referred to as “continuous intermediate temperature treatment”), or may be treated by changing the temperature in the middle.
  • the continuous intermediate temperature treatment may be performed, for example, in the above-exemplified intermediate temperature range and the above-exemplified intermediate temperature treatment range.
  • the aspect which reduces temperature on the way is mentioned, for example.
  • the temperature of the first stage intermediate temperature treatment may be, for example, the intermediate temperature range exemplified above.
  • the temperature of the second stage intermediate temperature treatment may be, for example, 30 ° C or higher, 35 ° C or higher, or 40 ° C or higher.
  • the temperature of the second stage intermediate temperature treatment may be, for example, 55 ° C. or lower, 50 ° C. or lower, or 45 ° C. or lower.
  • the time of the first stage intermediate temperature treatment may be, for example, 1 minute or more, 5 minutes or more, 10 minutes or more, or 20 minutes or more.
  • the time of the first stage intermediate temperature treatment may be, for example, 120 minutes or less, or 60 minutes or less.
  • the second stage intermediate temperature treatment time may be, for example, 1 hour or more, 2 hours or more, or 4 hours or more.
  • the time of the second stage intermediate temperature treatment may be, for example, 20 hours or less, or 15 hours or less.
  • Fatty acids can be recovered from the processed product by the medium temperature treatment. Normally, a large amount of fatty acid can be contained in the algae in the treated product. Therefore, it is preferable to extract the fatty acid from the algal body after the intermediate temperature treatment and recover the fatty acid.
  • the method for extracting the fatty acid is not particularly limited, and for example, a known method can be used.
  • a technique for extracting fats and oils from general algae can be used. Examples of such methods include organic solvent treatment, ultrasonic treatment, bead crushing treatment, acid treatment, alkali treatment, enzyme treatment, hydrothermal treatment, supercritical treatment, microwave treatment, electromagnetic field treatment, and pressing treatment.
  • the treated product by the medium temperature treatment may be used for extraction of fatty acid as it is, or may be used for extraction of fatty acid after appropriate treatment such as concentration, dilution, drying and the like.
  • the treated product obtained by the intermediate temperature treatment may be separated into a precipitate (algae) and a supernatant by centrifugation or the like. In that case, fatty acids can be extracted from the precipitate.
  • Processed products by intermediate temperature treatment are diluted or concentrated so that the concentration of the precipitate is, for example, 250 ⁇ g / L or less, or 125 g / L or less in terms of dry weight, and then used for fatty acid extraction. Good.
  • the organic solvent treatment it is preferable to treat a treated product having a precipitate concentration of 125 g / L or less.
  • the organic solvent treatment it is preferable to separate the precipitate from the supernatant and treat it.
  • the organic solvent treatment may or may not be performed after drying the treated product by the intermediate temperature treatment.
  • the organic solvent used for the organic solvent treatment is not particularly limited as long as the fatty acid can be extracted from the treated product by the medium temperature treatment.
  • the organic solvent include alcohols such as methanol, ethanol, 2-propanol, butanol, pentanol, hexanol, heptanol, and octanol, ketones such as acetone, ethers such as dimethyl ether and diethyl ether, methyl acetate, and ethyl acetate. Such as esters, alkanes such as n-hexane, and chloroform.
  • the organic solvent one kind of organic solvent may be used, or two or more kinds of organic solvents may be used in combination.
  • the pH of the alkali treatment is not particularly limited as long as it is a pH at which a fatty acid can be extracted from a treated product by a medium temperature treatment.
  • the pH of the alkali treatment is usually pH 8.5 or higher, preferably pH 10.5 or higher, more preferably pH 11.5 or higher, and pH 14 or lower.
  • the temperature for the alkali treatment is usually 30 ° C. or higher, preferably 50 ° C. or higher, more preferably 70 ° C. or higher.
  • the temperature of the alkali treatment may be preferably 120 ° C. or lower.
  • the alkali treatment time may be usually 10 minutes or longer, preferably 30 minutes or longer, more preferably 50 minutes or longer.
  • the alkali treatment time may be preferably 150 minutes or less.
  • An alkaline substance such as NaOH or KOH can be used for the alkali treatment.
  • the recovery of the eluted fatty acid can be performed by a known method used for separation and purification of compounds. Examples of such a method include an ion exchange resin method and a membrane treatment method. These methods can be used in appropriate combination.
  • the collected fatty acid may contain components such as algal bodies, medium components, moisture, components used for various treatments, and metabolic byproducts of the algae of the present invention in addition to the fatty acids.
  • the fatty acid may be purified to the desired degree. Fatty acid purity is, for example, 30% (w / w) or higher, 50% (w / w) or higher, 70% (w / w) or higher, 90% (w / w) or higher, or 95% (w / w) ) Or more.
  • the fatty acid ester can be produced by subjecting the algal bodies obtained by the culture to a two-stage reaction of medium temperature treatment and medium to low temperature treatment in the presence of alcohol. That is, one embodiment of the method of the present invention includes culturing the algae of the present invention in a medium, subjecting the algae obtained by the culture to a medium temperature treatment, and treating the medium temperature treated product in the presence of alcohol. It is a method for producing a fatty acid ester, comprising subjecting to a low-temperature treatment and recovering the fatty acid ester from the treated product of the medium-low temperature treatment. In the present invention, only one fatty acid ester may be produced, or two or more fatty acid esters may be produced.
  • the two-stage reaction for example, the two-stage reaction described in WO2012 / 099172 can be referred to.
  • the two-stage reaction includes an intermediate temperature treatment which is a first stage treatment and a medium and low temperature treatment in the presence of alcohol which is a second stage treatment.
  • the second stage treatment is a treatment for producing a fatty acid ester.
  • the first stage treatment is a treatment for changing the state of the algal bodies of the algae of the present invention so as to promote the production of fatty acid esters in the second stage treatment.
  • “Medium temperature treatment” means treatment at medium temperature. “Medium and low temperature treatment” refers to treatment at medium and low temperatures. “Medium temperature” refers to a temperature lower than the medium temperature. “Medium temperature” and “medium low temperature” are not particularly limited as long as they are temperatures at which fatty acid esters are formed by a two-stage reaction. “Medium temperature” and “medium temperature” can be appropriately set according to various conditions such as processing time.
  • the temperature (medium temperature) of the first stage treatment may be, for example, 35 ° C. or higher, 40 ° C. or higher, 45 ° C. or higher, or 50 ° C. or higher.
  • the temperature (intermediate temperature) of the 1st process may be 70 degrees C or less, 65 degrees C or less, or 60 degrees C or less, for example.
  • the processing time for the first stage may be, for example, 1 minute or more, 5 minutes or more, 10 minutes or more, or 20 minutes or more.
  • the time for the first step may be, for example, 120 minutes or less, or 60 minutes or less.
  • the temperature of the second stage treatment may be, for example, 5 ° C or higher, 20 ° C or higher, or 30 ° C or higher. Further, the temperature of the second stage treatment (medium / low temperature) may be, for example, 60 ° C. or lower, 50 ° C. or lower, or 45 ° C. or lower.
  • the processing time for the second stage may be, for example, 10 minutes or longer, 30 minutes or longer, 1 hour or longer, or 2 hours or longer. Further, the time for the second stage may be, for example, 15 hours or less, 10 hours or less, or 5 hours or less.
  • the pH of the two-stage reaction is not particularly limited as long as the fatty acid ester is produced by the two-stage reaction.
  • the pH of the medium temperature treatment may be, for example, pH 3.0 to 11.0.
  • the pH of the two-stage reaction may be, for example, weakly acidic, near neutral, or weakly alkaline.
  • the first-stage treatment and the second-stage treatment may be performed by standing or may be performed while stirring or shaking.
  • Reaction conditions such as the treatment temperature may or may not be constant throughout the first stage of treatment.
  • Reaction conditions such as treatment temperature may or may not be constant throughout the second stage treatment.
  • the temperature of the reaction system is lowered and the second stage treatment is performed in the presence of alcohol.
  • the processed product of the first step may be used for the second step as it is, or may be used for the second step after appropriately performing treatments such as concentration and dilution.
  • the alcohol may be present in the reaction system so as to come into contact with the processed product of the first step.
  • an alcohol may be added to the processed product of the first stage, or a processed product of the first stage may be added to the alcohol.
  • the algal bodies may be separated from the processed product of the first stage treatment, and the separated algal bodies may be mixed with the reaction liquid for the second stage treatment containing alcohol.
  • the concentration of alcohol in the reaction system in the second stage treatment is usually 5% (v / v) or more, preferably 10% (v / v) or more, more preferably 20% (v / v) or more. It may be. Further, the alcohol concentration in the reaction system in the second stage treatment is usually 70% (v / v) or less, preferably 60% (v / v) or less, more preferably 50% (v / v). It may be the following.
  • the alcohol used for the second stage treatment is not particularly limited as long as the fatty acid ester is generated by the two-stage reaction.
  • lower alcohols having 5 or less carbon atoms such as methanol, ethanol, propanol, isopropanol, butanol, pentanol, ethylene glycol, hexanol, heptanol, octanol, nonanol, decanol, undecanol
  • Examples thereof include higher alcohols having 6 or more carbon atoms such as dodecanol, tridecanol, and tetradecanol.
  • the fatty acid ester can be recovered from the processed product of the two-stage reaction.
  • generated fatty acid ester may be contained abundantly in the algal body in a processed material. Therefore, it is preferable to extract the fatty acid ester from the algal body after the two-step reaction and recover the fatty acid ester.
  • ⁇ Fatty acid ester production by a two-step reaction does not require addition of a catalyst.
  • the reason is that the lipase originally possessed by the algae of the present invention is likely to act on lipids by the first treatment, and the lipase allows lipids such as fats and oils, ceramides, phospholipids, sugars. It is thought that this is because a transesterification reaction occurs between lipid (Glycolipid) and the like and alcohol added from outside.
  • the transesterification reaction by lipase is generally promoted in the presence of an organic solvent other than alcohols. Therefore, for example, an amount of an organic solvent effective to promote the transesterification reaction may be added to the reaction system during the second stage treatment.
  • organic solvent examples include hexane, heptane, isooctane, chloroform, ethyl acetate, and petroleum ether.
  • the collected fatty acid ester can be used as it is or in combination with pharmaceuticals, cosmetics, foods and drinks.
  • the blending amount of the fatty acid ester is not particularly limited as long as the function of the fatty acid ester is exhibited.
  • the blending amount of the fatty acid ester is not particularly limited, and may be, for example, 1 ppm (w / w) or more, 100 ppm (w / w) or more, or 1% (w / w) or more.
  • the amount of the fatty acid ester is not particularly limited, and may be, for example, 100% (w / w) or less, 10% (w / w) or less, or 1% (w / w) or less.
  • Sugar glycerol can be produced by subjecting the algal cells obtained by the culture to a medium temperature treatment and / or an organic solvent treatment. That is, one embodiment of the method of the present invention includes culturing the algae of the present invention in a medium, subjecting the algae obtained by the culture to a medium temperature treatment and / or organic solvent treatment, and from a treated product of the treatment. Recovering sugar glycerol, a method for producing sugar glycerol.
  • the medium temperature treatment and organic solvent treatment used for the production of sugar glycerol may be collectively referred to as a sugar glycerol production treatment.
  • “Sugar glycerol” refers to a compound having a structure in which a sugar is glycosidically bonded to a hydroxyl group of glycerol.
  • the sugar may be bound to any hydroxyl group of glycerol.
  • the sugar may be bonded to only one of the three hydroxyl groups of glycerol, or may be bonded to two or three hydroxyl groups. In the present invention, only one sugar glycerol may be produced, or two or more sugar glycerols may be produced.
  • the type of sugar is not particularly limited.
  • the sugar may be a monosaccharide, a polysaccharide, or a derivative thereof.
  • monosaccharides include glucose and galactose.
  • Polysaccharide refers to a saccharide composed of two or more monosaccharides. That is, the polysaccharide here includes disaccharides and oligosaccharides.
  • the polysaccharide may be linear or may have a branched chain.
  • the polysaccharide may be composed of one kind of monosaccharide or may be composed of two or more kinds of monosaccharides.
  • the degree of polymerization of the polysaccharide is not particularly limited, and may be, for example, 2 to 50, 2 to 10, or 2 to 5.
  • Examples of the polysaccharide include a polysaccharide containing galactose as a constituent sugar. Specific examples of the polysaccharide containing galactose as a constituent sugar include digalactose.
  • a “sugar derivative” refers to a sugar in which components such as atoms and functional groups have been introduced, substituted, or removed.
  • modification introduction, replacement, and removal of components are collectively referred to as “modification”.
  • the location to be modified is not particularly limited, and may be, for example, on a carbon atom, on an oxygen atom, or other location. There may be one place to be modified, or two or more places. There may be one type of modification, or two or more types.
  • the sugar derivative include deoxy sugar, amino sugar, sugar acid, and sugar alcohol.
  • the functional group to be introduced include an acetyl group, an amino group, an alkyl group, and a sulfonyl group (—SO 3 —R).
  • R in the sulfonyl group (—SO 3 —R) is not particularly limited, and may be, for example, a hydrogen atom (H) or an alkyl group.
  • sugar glycerol examples include galactosylglycerol, digalactosylglycerol, and sulfoquinovosylglycerol.
  • Galactosylglycerol is a sugar glycerol in which galactose is bonded to the hydroxyl group of any one carbon of glycerol.
  • Digalactosylglycerol is a sugar glycerol in which digalactose is bound to the hydroxyl group of any one carbon of glycerol.
  • Sulfoquinovosyl glycerol is a sugar glycerol in which sulfo quinose is bound to the hydroxyl group of any one carbon of glycerol. Unless otherwise specified, all of galactosylglycerol, digalactosylglycerol, and sulfoquinovosylglycerol have sugars bound to the hydroxyl group at the 1st carbon of glycerol, and sugars bound to the hydroxyl group at the 2nd carbon of glycerol. Or a saccharide bonded to the hydroxyl group at the 3-position carbon of glycerol, or a mixture thereof.
  • ⁇ Medium temperature treatment> For the medium temperature treatment, the description regarding the medium temperature treatment in the above ⁇ 2-2> can be applied.
  • Organic solvent treatment refers to treatment with an organic solvent.
  • the conditions for the organic solvent treatment are not particularly limited as long as sugar glycerol is produced by the organic solvent treatment.
  • the organic solvent treatment can be performed by bringing the algal bodies into contact with the organic solvent.
  • the collected alga bodies may be suspended in an organic solvent, or an organic solvent may be added to the alga body suspension.
  • organic solvent examples include alcohols such as methanol, ethanol, 2-propanol, butanol, pentanol, hexanol, heptanol and octanol, ketones such as acetone, ethers such as dimethyl ether and diethyl ether, methyl acetate and ethyl acetate. Esters, alkanes such as n-hexane, benzene, phenol, and chloroform.
  • organic solvent a water-soluble thing is preferable.
  • alcohol is preferable and methanol is more preferable.
  • the organic solvent one kind of organic solvent may be used, or two or more kinds of organic solvents may be used in combination.
  • the organic solvent may be a pure product or a mixture with other components.
  • examples of other components include an aqueous medium (aqueous solvent) such as water and an aqueous buffer solution. That is, for example, an aqueous solution of an organic solvent can be used for the organic solvent treatment.
  • the organic solvent concentration in the mixture may be, for example, 10% (v / v) or more, or 20% (v / v) or more, 90% (v / v) or less, 70% (v / v) or less Or 50% (v / v) or less.
  • the concentration of the organic solvent in the reaction solution for performing the organic solvent treatment may be, for example, 10% (v / v) or more, or 20% (v / v) or more, 90% (v / v) or less, It may be 70% (v / v) or less, or 50% (v / v) or less.
  • the organic solvent treatment time may be, for example, 10 minutes or longer, 30 minutes or longer, or 1 hour or longer. Further, the time of the organic solvent treatment may be, for example, 10 hours or less, 5 hours or less, or 3 hours or less.
  • the temperature of the organic solvent treatment may or may not be controlled. The temperature of the organic solvent treatment may be, for example, 10 to 70 ° C. or room temperature. The organic solvent treatment may be performed by standing or may be performed while stirring or shaking.
  • sugar glycerol is produced in the reaction supernatant and / or in the algal bodies.
  • sugar glycerol can be confirmed by a known method used for detection or identification of a compound. Examples of such a method include HPLC, LC / MS, GC / MS, and NMR. These methods can be used in appropriate combination.
  • the produced sugar glycerol can be collected by a known method used for separation and purification of compounds. Examples of such a method include an ion exchange resin method and a membrane treatment method. These methods can be used in appropriate combination.
  • sugar glycerol accumulates in the algal bodies, for example, if the algal bodies are crushed by means such as ultrasonic waves, and the sugar glycerol is recovered from the supernatant obtained by removing the algal bodies by means such as centrifugation. Good.
  • the collected sugar glycerol may contain components such as algal bodies, medium components, moisture, components used for various treatments, and metabolic byproducts of the algae of the present invention in addition to sugar glycerol.
  • the sugar glycerol may be purified to the desired degree.
  • the purity of sugar glycerol is, for example, 30% (w / w) or higher, 50% (w / w) or higher, 70% (w / w) or higher, 90% (w / w) or higher, or 95% (w / w) w) That's it.
  • the recovered sugar glycerol can be used as it is or in combination with pharmaceuticals, cosmetics, foods and drinks.
  • the amount of sugar glycerol blended is not particularly limited as long as the function of sugar glycerol is exhibited.
  • the blending amount of sugar glycerol is not particularly limited, and may be, for example, 1 ppm (w / w) or more, 100 ppm (w / w) or more, or 1% (w / w) or more.
  • the amount of sugar glycerol is not particularly limited, and may be, for example, 100% (w / w) or less, 10% (w / w) or less, or 1% (w / w) or less.
  • Glycerol glycerol may have functions such as prebiotics, ⁇ -amylase activation, moisturizing, or cell activation.
  • the treated product (medium-temperature treated product) by the intermediate temperature treatment described in ⁇ 2-2> above can be used, for example, as a carbon source for L-amino acid fermentation (WO2011) / 013707). That is, the present invention includes (A) culturing the algae of the present invention in a medium, (B) subjecting the algal cells obtained by the culture to a medium temperature treatment, and (C) a bacterium having L-amino acid-producing ability.
  • L-amino acid fermentation may be performed in the same manner as normal L-amino acid fermentation using bacteria, except that a medium containing a medium-temperature treated product is used.
  • the intermediate temperature processed product used for L-amino acid fermentation may be a processed product itself by intermediate temperature treatment. It may be subjected to a treatment such as drying, extraction, and centrifugation, or may be a component such as a fatty acid recovered from a treated product by an intermediate temperature treatment.
  • the processed product used for L-amino acid fermentation may be a fatty acid.
  • the fatty acid may or may not contain a component other than the fatty acid.
  • the fatty acid may be purified to the desired degree.
  • the fatty acid may be a free form or a salt thereof, or a mixture thereof.
  • the salt include alkali metal salts such as sodium salt and potassium salt.
  • Alkali metal salts of fatty acids are highly water-soluble, and are micellized and retained in water, so that they can be efficiently used by the bacteria of the present invention.
  • fatty acids it is preferable to increase the solubility of fatty acids by performing a treatment for promoting homogenization of fatty acids so that the bacterium of the present invention can use fatty acids more efficiently.
  • Examples of the treatment for promoting homogenization include emulsification.
  • Emulsification can be carried out, for example, by adding an emulsification accelerator or a surfactant.
  • Examples of the emulsification accelerator include phospholipids and sterols.
  • As the surfactant for example, a surfactant generally used in the field of biology can be used.
  • nonionic surfactants include, for example, polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan monooleate (Tween 80), alkyl glucosides such as n-octyl ⁇ -D-glucoside, Sucrose fatty acid esters such as sugar stearate, polyglycerin fatty acid esters such as polyglycerol stearate, Triton X-100 (TritonTriX-100), polyoxyethylene (20) cetyl ether (Brij-58), nonylphenol ethoxy Rate (Tergitol NP-40).
  • the surfactant include zwitterionic surfactants such as alkylbetaines such as N, N-dimethyl-N-dodecylglycine betaine.
  • examples of the treatment for promoting homogenization include homogenizer treatment, homomixer treatment, ultrasonic treatment, high pressure treatment, and high temperature treatment.
  • homogenizer treatment and / or ultrasonic treatment are preferable.
  • the treatment for promoting homogenization is preferably performed under alkaline conditions in which fatty acids can exist stably.
  • the alkaline condition is preferably pH 9 or more, more preferably pH 10 or more.
  • fats and oils may remain in the precipitate obtained by centrifuging the medium temperature treatment. Fats and oils produce fatty acids and glycerol by hydrolysis. The fatty acid and / or glycerol thus obtained may be used as a carbon source for L-amino acid fermentation as it is or after being appropriately purified.
  • the hydrolysis of fats and oils can be performed enzymatically using lipase, for example (WO2011 / 013707).
  • you may perform the hydrolysis of fats and oils chemically. Examples of the chemical hydrolysis method include a continuous high-temperature hydrolysis method in which oil and fat are in countercurrent contact with water under high temperature (250-260 ° C.) and high pressure (5-6 MPa).
  • the supernatant obtained by centrifuging the medium-temperature treated product may contain compounds such as glycerol, glucose, and starch fragments. These compounds may be used as carbon sources for L-amino acid fermentation as they are or after being appropriately purified.
  • the fragmented product of starch produces glucose by hydrolysis. The glucose thus obtained may be used as it is or after being appropriately purified as a carbon source for L-amino acid fermentation. For example, a supernatant with an increased glucose concentration thus obtained may be used. Hydrolysis of the starch fragment can be carried out enzymatically using, for example, amylase (WO2011 / 013707).
  • Bacteria used for L-amino acid fermentation The bacterium of the present invention is a bacterium having L-amino acid-producing ability.
  • “bacteria having L-amino acid-producing ability” means bacteria that have the ability to accumulate in the medium or in the cells so that the target L-amino acid can be produced and recovered when cultured in the medium.
  • the bacterium having L-amino acid-producing ability may be a bacterium capable of accumulating a larger amount of the target L-amino acid in the medium than the unmodified strain.
  • Non-modified strains include wild strains and parent strains.
  • the bacterium having L-amino acid-producing ability is a bacterium that can accumulate the target L-amino acid in an amount of 0.5 g / L or more, more preferably 1.0 g / L or more in the medium. May be.
  • L-amino acids include basic amino acids such as L-lysine, L-ornithine, L-arginine, L-histidine, L-citrulline, L-isoleucine, L-alanine, L-valine, L-leucine, glycine, etc.
  • Aliphatic amino acids amino acids which are hydroxymonoaminocarboxylic acids such as L-threonine and L-serine, cyclic amino acids such as L-proline, aromatic amino acids such as L-phenylalanine, L-tyrosine and L-tryptophan, L- Examples thereof include sulfur-containing amino acids such as cysteine, L-cystine and L-methionine, acidic amino acids such as L-glutamic acid and L-aspartic acid, and amino acids having an amide group in the side chain such as L-glutamine and L-asparagine.
  • the bacterium of the present invention may have only one L-amino acid producing ability or may have two or more L-amino acid producing ability.
  • the term “amino acid” may mean an L-amino acid unless otherwise specified.
  • the L-amino acid produced may be a free form, a salt thereof, or a mixture thereof. That is, in the present invention, the term “L-amino acid” may mean a free L-amino acid, a salt thereof, or a mixture thereof, unless otherwise specified. Examples of the salt will be described later.
  • bacteria belonging to the family Enterobacteriaceae and coryneform bacteria examples include bacteria belonging to the family Enterobacteriaceae and coryneform bacteria.
  • NCBI National Center for Biotechnology Information
  • the Escherichia bacterium is not particularly limited, but includes bacteria classified into the genus Escherichia by classification known to microbiologists.
  • Escherichia bacteria include, for example, Neidhardt et al. (Backmann, B. J. 1996. Derivations and Genotypes of some mutant derivatives of Escherichia coli K-12, p. 2460-2488. Table 1.
  • F. D. Nehard (ed.) “Escherichia, coli, and Salmonella, Cellular, and Molecular, Biology / Second Edition, American, Society, for Microbiology, Press, Washington, DC).
  • bacteria belonging to the genus Escherichia include Escherichia coli.
  • Specific examples of Escherichia coli include Escherichia coli W3110 (ATCC11027325) and Escherichia coli MG1655 (ATCC 47076) derived from the prototype wild-type strain K12.
  • the bacteria belonging to the genus Enterobacter are not particularly limited, but include bacteria classified into the genus Enterobacter by classification known to microbiologists.
  • Enterobacter bacteria include Enterobacter agglomerans and Enterobacter aerogenes.
  • Specific examples of Enterobacter agglomerans include the Enterobacter agglomerans ATCC12287 strain.
  • Specific examples of Enterobacter aerogenes include Enterobacter aerogenes ATCC13048, NBRC12010 (BiotechonolonBioeng.eng2007 Mar 27; 98 (2) 340-348), AJ110637 (FERM BP-10955) .
  • Enterobacter bacteria include those described in European Patent Application Publication No. EP0952221. Some Enterobacter agglomerans are classified as Pantoea agglomerans.
  • Pantoea bacterium is not particularly limited, and examples include bacteria classified into the Pantoea genus by classification known to microbiologists.
  • Examples of the genus Pantoea include Pantoea ⁇ ananatis, Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea.
  • Pantoea Ananatis LMG20103 strain AJ13355 strain (FERM ⁇ BP-6614), AJ13356 strain (FERM BP-6615), AJ13601 strain (FERM BP-7207), SC17 strain (FERM BP) -11091), and SC17 (0) strain (VKPM B-9246).
  • Pantoea bacterium also includes a bacterium reclassified as Pantoea in this way.
  • Examples of the genus Erwinia include Erwinia amylovora and Erwinia carotovora.
  • Examples of Klebsiella bacteria include Klebsiella planticola.
  • coryneform bacteria examples include bacteria belonging to genera such as Corynebacterium genus, Brevibacterium genus, and Microbacterium genus.
  • coryneform bacteria include the following species. Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium callunae Corynebacterium glutamicum Corynebacterium lilium Corynebacterium melassecola Corynebacterium thermoaminogenes (Corynebacterium efficiens) Corynebacterium herculis Brevibacterium divaricatum (Corynebacterium glutamicum) Brevibacterium flavum (Corynebacterium glutamicum) Brevibacterium immariophilum Brevibacterium lactofermentum (Corynebacterium glutamicum) Brevibacterium roseum Brevibacterium saccharolyticum Brevibacterium thiogenitalis Corynebacterium ammoniagenes (Corynebacterium stationis) Brevibacterium album Brevibacterium cerinum Microbacterium ammoniaphilum
  • coryneform bacteria include the following strains. Corynebacterium acetoacidophilum ATCC 13870 Corynebacterium acetoglutamicum ATCC 15806 Corynebacterium alkanolyticum ATCC 21511 Corynebacterium callunae ATCC 15991 Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, FERM BP-734 Corynebacterium lilium ATCC 15990 Corynebacterium melassecola ATCC 17965 Corynebacterium efficiens (Corynebacterium thermoaminogenes) AJ12340 (FERM BP-1539) Corynebacterium herculis ATCC 13868 Corynebacterium glutamicum (Brevibacterium divaricatum) ATCC 14020 Corynebacterium glutamicum (Brevibacterium flavum) ATCC 13826, ATCC 14067, AJ124
  • corynebacteria belonging to the genus Brevibacterium has been classified as a genus of corynebacteria, but bacteria integrated into the genus corynebacteria (Int. J. Syst. Bacteriol., 41, 255 (1991)) are also available. included.
  • Corynebacterium stationis which was previously classified as Corynebacterium ammoniagenes, includes bacteria that have been reclassified as Corynebacterium stationis by 16S rRNA sequencing (Int. J Syst. Evol. Microbiol., 60, 874-879 (2010)).
  • strains can be sold, for example, from the American Type Culture Collection (address 12301 Parklawn Drive, Rockville, Maryland 20852 P.O. Box 1549, Manassas, VA 20108, United States States of America). That is, a registration number corresponding to each strain is given, and it is possible to receive a sale using this registration number (see http://www.atcc.org/). The registration number corresponding to each strain is described in the catalog of American Type Culture Collection.
  • the bacterium of the present invention may inherently have L-amino acid-producing ability or may have been modified to have L-amino acid-producing ability.
  • a bacterium having L-amino acid-producing ability can be obtained, for example, by imparting L-amino acid-producing ability to the bacterium as described above, or by enhancing the L-amino acid-producing ability of the bacterium as described above. .
  • L-amino acid-producing ability can be imparted or enhanced by a method conventionally used for breeding amino acid-producing bacteria such as coryneform bacteria or Escherichia bacteria (Amino Acid Fermentation, Academic Publishing Center, Inc., 1986). (May 30, 1st edition issued, see pages 77-100). Examples of such methods include acquisition of auxotrophic mutants, acquisition of L-amino acid analog-resistant strains, acquisition of metabolic control mutants, and recombination with enhanced activity of L-amino acid biosynthetic enzymes. The creation of stocks. In the breeding of L-amino acid-producing bacteria, properties such as auxotrophy, analog resistance, and metabolic control mutation that are imparted may be single, or two or more.
  • L-amino acid biosynthetic enzymes whose activities are enhanced in breeding L-amino acid-producing bacteria may be used alone or in combination of two or more.
  • imparting properties such as auxotrophy, analog resistance, and metabolic control mutation may be combined with enhancing the activity of biosynthetic enzymes.
  • An auxotrophic mutant, an analog resistant strain, or a metabolically controlled mutant having L-amino acid production ability is subjected to normal mutation treatment of the parent strain or wild strain, and the auxotrophic, analog It can be obtained by selecting those exhibiting resistance or metabolic control mutations and having the ability to produce L-amino acids.
  • Normal mutation treatments include X-ray and ultraviolet irradiation, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methane sulfonate (EMS), methyl methane sulfonate (MMS), etc. Treatment with a mutagen is included.
  • the L-amino acid-producing ability can be imparted or enhanced by enhancing the activity of an enzyme involved in the target L-amino acid biosynthesis. Enhancing enzyme activity can be achieved, for example, by modifying bacteria so that expression of a gene encoding the enzyme is enhanced.
  • Methods for enhancing gene expression include increasing the gene copy number and increasing gene transcription and translation. Increasing the copy number of a gene can be achieved, for example, by introducing a vector carrying the gene into the host or by introducing the gene onto the chromosome of the host.
  • Increasing gene transcription and translation can include, for example, a promoter, an SD sequence (RBS), or a spacer region between the RBS and the start codon (eg, a sequence immediately upstream of the start codon (5′-UTR)), etc. This can be achieved by modifying the expression regulatory region.
  • RBS SD sequence
  • start codon eg, a sequence immediately upstream of the start codon (5′-UTR)
  • the L-amino acid-producing ability can be imparted or enhanced by reducing the activity of an enzyme that catalyzes a reaction that branches from the biosynthetic pathway of the target L-amino acid to produce a compound other than the target L-amino acid. It can be carried out.
  • an enzyme that catalyzes a reaction that produces a compound other than the target L-amino acid by branching from the biosynthetic pathway of the target L-amino acid includes enzymes involved in the degradation of the target amino acid. It is.
  • the reduction of the enzyme activity can be achieved, for example, by modifying the bacterium so that the expression of the gene encoding the enzyme is reduced or by destroying the gene.
  • Decreasing gene expression is, for example, regulating expression of a promoter, an SD sequence (RBS), or a spacer region between the RBS and the start codon (eg, a sequence immediately upstream of the start codon (5′-UTR)), etc. This can be achieved by modifying the region. Disrupting a gene can be achieved, for example, by deleting part or all of the gene.
  • L-amino acid-producing bacteria and methods for imparting or enhancing L-amino acid-producing ability are given below.
  • any of the modifications exemplified below for imparting or enhancing the properties of L-amino acid-producing bacteria and L-amino acid-producing ability may be used alone or in appropriate combination.
  • Examples of the method for imparting or enhancing L-glutamic acid-producing ability include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-glutamic acid biosynthetic enzymes is increased. .
  • Such enzymes include, but are not limited to, glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthase (gltBD), isocitrate dehydrogenase (icdA), aconite hydratase (acnA, acnB), citrate synthase (GltA), methyl citrate synthase (prpC), phosphoenolpyruvate carboxylase (ppc), pyruvate carboxylase (pyc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenol Pyruvate synthase (ppsA), enolase (eno), phosphoglyceromutase (pgmA, pgmI), phosphoglycerate kinase (pgk),
  • the parentheses are examples of abbreviations for genes encoding the enzymes (the same applies to the following description).
  • these enzymes it is preferable to enhance the activity of one or more enzymes selected from, for example, glutamate dehydrogenase, citrate synthase, phosphoenolpyruvate carboxylase, and methyl citrate synthase.
  • Strains belonging to the family Enterobacteriaceae that have been modified to increase expression of the citrate synthase gene, phosphoenolpyruvate carboxylase gene, and / or glutamate dehydrogenase gene include those disclosed in EP1078989A, EP955368A, and EP952221A Can be mentioned.
  • Examples of strains belonging to the family Enterobacteriaceae that have been modified to increase the expression of the Entner-Doudoroff pathway genes (edd, eda) include those disclosed in EP1352966B.
  • Examples of coryneform bacteria modified to increase the expression of the glutamate synthetase gene (gltBD) include those disclosed in WO99 / 07853.
  • the method for imparting or enhancing the ability to produce L-glutamic acid is, for example, selected from enzymes that catalyze a reaction that branches from the biosynthetic pathway of L-glutamic acid to produce a compound other than L-glutamic acid.
  • a method of modifying the bacterium so that the activity of the further enzyme is reduced can also be mentioned.
  • Examples of such enzymes include, but are not limited to, isocitrate lyase (aceA), ⁇ -ketoglutarate dehydrogenase (sucA, odhA), phosphotransacetylase (pta), acetate kinase (ack), acetohydroxyacid synthase (ilvG ), Acetolactate synthase (ilvI), formate acetyltransferase (pfl), lactate dehydrogenase (ldh), alcohol dehydrogenase (adh), glutamate decarboxylase (gadAB), succinate dehydrogenase (sdhABCD), 1-pyrroline-5-carboxylate
  • An example is dehydrogenase (putA).
  • aceA isocitrate lyase
  • sucA, odhA phosphotransacetylase
  • ack acetate kinase
  • ack acetohydroxy
  • Escherichia bacteria with reduced or deficient ⁇ -ketoglutarate dehydrogenase activity and methods for obtaining them are described in US Pat. Nos. 5,378,616 and 5,573,945.
  • a method for reducing or eliminating ⁇ -ketoglutarate dehydrogenase activity in enteric bacteria such as Pantoea bacteria, Enterobacter bacteria, Klebsiella bacteria, Erwinia bacteria, and the like are disclosed in U.S. Patent No. 6,197,559, U.S. Patent No. 6,682,912, This is disclosed in US Pat. No. 6,331,419, US Pat. No. 8,129,151, and WO2008 / 075483.
  • bacteria belonging to the genus Escherichia with reduced or deficient ⁇ -ketoglutarate dehydrogenase activity include the following strains.
  • E. coli W3110sucA Kmr
  • E. coli AJ12624 (FERM BP-3853)
  • E. coli AJ12628 (FERM BP-3854)
  • E. coli AJ12949 (FERM BP-4881)
  • E. coli W3110sucA is a strain obtained by disrupting the sucA gene encoding the ⁇ -ketoglutarate dehydrogenase of E. coli W3110. This strain is completely deficient in ⁇ -ketoglutarate dehydrogenase activity.
  • Coryneform bacteria with reduced or deficient ⁇ -ketoglutarate dehydrogenase activity and methods for obtaining them are described in WO2008 / 075483.
  • Specific examples of coryneform bacteria with reduced or deficient ⁇ -ketoglutarate dehydrogenase activity include the following strains.
  • L-glutamic acid-producing bacteria or parent strains for inducing them include Pantoea ananatis AJ13355 strain (FERM BP-6614), Pantoea ananatis SC17 strain (FERM BP-11091), Pantoea ananatis SC17 (0) strain (VKPM B) -9246) and the like.
  • the AJ13355 strain is a strain isolated as a strain capable of growing on a medium containing L-glutamic acid and a carbon source at low pH from soil in Iwata City, Shizuoka Prefecture.
  • the SC17 strain is a strain selected from the AJ13355 strain as a low mucus production mutant (US Pat. No. 6,596,517).
  • examples of L-glutamic acid-producing bacteria and parent strains for inducing them also include Pantoea bacteria with reduced or deficient ⁇ -ketoglutarate dehydrogenase activity.
  • Pantoea bacteria with reduced or deficient ⁇ -ketoglutarate dehydrogenase activity include the AJ13356 strain (US Pat. No. 6,331,419) which is the E1 subunit gene (sucA) deficient strain of the ⁇ -ketoglutarate dehydrogenase of the AJ13355 strain, and the SC17sucA strain which is the sucA gene deficient strain of the SC17 strain ( US Pat. No. 6,596,517).
  • the AJ13356 strain was founded on February 19, 1998 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute for Product Evaluation Technology, Patent Biological Depositary Center, Postal Code: 292-0818, Address: Chiba, Japan. Deposited at Kisarazu City Kazusa Kamashichi 2-5-8 120) under the accession number FERM P-16645 and transferred to the international deposit under the Budapest Treaty on 11 January 1999 and given the accession number FERM BP-6616 ing.
  • the SC17sucA strain was also assigned the private number AJ417.
  • the AJ13355 strain was identified as Enterobacter agglomerans at the time of its isolation, but has recently been reclassified as Pantoea anaananatis by 16S rRNA nucleotide sequence analysis and the like. Therefore, the AJ13355 strain and the AJ13356 strain are deposited as Enterobacter agglomerans in the above depository organization, but are described as Pantoea ananatis in this specification.
  • L-glutamic acid-producing bacteria or parent strains for inducing them include Pantoea bacteria such as Pantoea ananatis SC17sucA / RSFCPG + pSTVCB strain, Pantoea ananatis AJ13601 strain, Pantoea ananatis NP106 strain, and Pantoea ananatis NA1 strain .
  • Pantoea bacteria such as Pantoea ananatis SC17sucA / RSFCPG + pSTVCB strain, Pantoea ananatis AJ13601 strain, Pantoea ananatis NP106 strain, and Pantoea ananatis NA1 strain .
  • the SC17sucA / RSFCPG + pSTVCB strain is different from the SC17sucA strain in that the plasmid RSFCPG containing the citrate synthase gene (gltA), the phosphoenolpyruvate carboxylase gene (ppc), and the glutamate dehydrogenase gene (gdhA) derived from Escherichia coli, and Brevi
  • This is a strain obtained by introducing a plasmid pSTVCB containing a citrate synthase gene (gltA) derived from bacteria lactofermentum.
  • the AJ13601 strain was selected from the SC17sucA / RSFCPG + pSTVCB strain as a strain resistant to a high concentration of L-glutamic acid at low pH.
  • the NP106 strain is a strain obtained by removing the plasmid RSFCPG + pSTVCB from the AJ13601 strain.
  • AJ13601 shares were registered with the National Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute for Product Evaluation Technology, Biological Depositary Center, Postal Code: 292-0818, Address: Chiba, Japan. Deposited at Kisarazu City Kazusa Kamashika 2-5-8 120) under the accession number FERM P-17516, transferred to an international deposit based on the Budapest Treaty on July 6, 2000 and given the accession number FERM BP-7207 ing.
  • L-glutamic acid-producing bacteria or parent strains for inducing them include strains in which both ⁇ -ketoglutarate dehydrogenase (sucA) activity and succinate dehydrogenase (sdh) activity are reduced or deficient (JP 2010) -041920).
  • specific examples of such strains include, for example, a pantoea ananatis NA1 sucAsdhA double-deficient strain and a Corynebacterium glutamicum 140ATCC14067 odhAsdhA double-deficient strain (Corynebacterium glutamicum 8L3G ⁇ SDH strain) (Japanese Patent Laid-Open No. 2010-041920).
  • examples of L-glutamic acid-producing bacteria or parent strains for inducing them include auxotrophic mutants.
  • the auxotrophic mutant include E. coli VL334thrC + (VKPM B-8961) (EP 1172433).
  • E. coli VL334 (VKPM B-1641) is an L-isoleucine and L-threonine auxotroph having a mutation in the thrC gene and the ilvA gene (US Pat. No. 4,278,765).
  • E. coli VL334thrC + is an L-isoleucine-requiring L-glutamic acid-producing bacterium obtained by introducing a wild type allele of the thrC gene into VL334. The wild type allele of the thrC gene was introduced by a general transduction method using bacteriophage P1 grown on cells of wild type E. coli K12 strain (VKPM B-7).
  • examples of L-glutamic acid-producing bacteria or parent strains for inducing them also include strains resistant to aspartic acid analogs. These strains may be deficient in ⁇ -ketoglutarate dehydrogenase activity, for example.
  • Specific examples of strains resistant to aspartate analogs and lacking ⁇ -ketoglutarate dehydrogenase activity include, for example, E. coli AJ13199 (FERM BP-5807) (US Pat. No. 5,908,768), and L-glutamic acid.
  • E. coli FFRM P-12379 US Pat. No. 5,393,671
  • E. coli AJ13138 FERM BP-5565
  • a bacterium is modified so that the activity of D-xylulose-5-phosphate-phosphoketolase and / or fructose-6-phosphate phosphoketolase is increased.
  • There is also a method to do (Special Table 2008-509661). Either one or both of D-xylulose-5-phosphate-phosphoketolase activity and fructose-6-phosphate phosphoketolase activity may be enhanced.
  • D-xylulose-5-phosphate phosphoketolase and fructose-6-phosphate phosphoketolase may be collectively referred to as phosphoketolase.
  • D-xylulose-5-phosphate-phosphoketolase activity is the consumption of phosphoric acid to convert xylulose-5-phosphate into glyceraldehyde-3-phosphate and acetyl phosphate, and one molecule of H 2 O Means the activity of releasing. This activity is measured by the method described in Goldberg, M. et al. (Methods Enzymol., 9,515-520 (1966)) or L. Meile (J. Bacteriol. (2001) 183; 2929-2936). be able to.
  • fructose-6-phosphate phosphoketolase activity means that phosphoric acid is consumed, fructose 6-phosphate is converted into erythrose-4-phosphate and acetyl phosphate, and one molecule of H 2 O is released. Means activity. This activity is measured by the method described in Racker, E (Methods Enzymol., 5, 276-280 (1962)) or L. Meile (J. Bacteriol. (2001) 183; 2929-2936). be able to.
  • Examples of a method for imparting or enhancing L-glutamic acid producing ability include, for example, enhancing expression of yhfK gene (WO2005 / 085419) and ybjL gene (WO2008 / 133161) which are L-glutamic acid excretion genes. It is done.
  • Examples of methods for imparting or enhancing L-glutamic acid-producing ability for coryneform bacteria include methods for imparting resistance to organic acid analogs and respiratory inhibitors, and methods for imparting sensitivity to cell wall synthesis inhibitors. It is done. Specific examples of such a method include, for example, a method for imparting monofluoroacetic acid resistance (Japanese Patent Laid-Open No. 50-113209), a method for imparting adenine resistance or thymine resistance (Japanese Patent Laid-Open No. 57-065198), and urease.
  • JP 52-038088 Method of weakening (JP 52-038088), method of imparting malonic acid resistance (JP 52-038088), method of imparting resistance to benzopyrones or naphthoquinones (JP 56-1889), HOQNO Method for imparting resistance (Japanese Patent Laid-Open No. 56-140895), method for imparting resistance to ⁇ -ketomalonic acid (Japanese Patent Laid-Open No. 57-2689), method for imparting guanidine resistance (Japanese Patent Laid-Open No. 56-35981), sensitivity to penicillin And the like (JP-A-4-88994).
  • resistant or susceptible bacteria include the following strains: Corynebacterium glutamicum (Brevibacterium flavum) AJ3949 (FERM BP-2632; see JP 50-113209) Corynebacterium glutamicum AJ11628 (FERM P-5736; see JP-A-57-065198) Corynebacterium glutamicum (Brevibacterium flavum) AJ11355 (FERM P-5007; see JP 56-1889) Corynebacterium glutamicum AJ11368 (FERM P-5020; see JP-A-56-1889) Corynebacterium glutamicum (Brevibacterium flavum) AJ11217 (FERM P-4318; see JP-A-57-2689) Corynebacterium glutamicum AJ11218 (FERM P-4319; see JP 57-2689) Corynebacterium glutamicum (Brevibacterium flavum) AJ11564 (FERM P-
  • Examples of a method for imparting or enhancing L-glutamic acid producing ability for coryneform bacteria include a method for enhancing expression of the yggB gene and a method for introducing a mutant yggB gene having a mutation introduced into the coding region ( WO2006 / 070944).
  • the yggB gene encodes a mechanosensitive channel.
  • the yggB gene of Corynebacterium glutamicum ATCC13032 corresponds to a complementary sequence of the sequences 1,336,091 to 1,337,692 in the genome sequence registered in the NCBI database under GenBank Accession No. NC_003450, and is also called NCgl1221.
  • the YggB protein encoded by the yggB gene of Corynebacterium glutamicum ATCC13032 is registered as GenBank accession No. NP_600492.
  • Examples of the method for imparting or enhancing L-glutamine production ability include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-glutamine biosynthesis enzymes is increased.
  • Examples of such an enzyme include, but are not limited to, glutamate dehydrogenase (gdhA) and glutamine synthetase (glnA).
  • the activity of glutamine synthetase may be enhanced by disrupting the glutamine adenylyltransferase gene (glnE) or the PII regulatory protein gene (glnB) (EP1229121).
  • the method for imparting or enhancing L-glutamine production ability is, for example, selected from an enzyme that catalyzes a reaction that branches from the biosynthetic pathway of L-glutamine to produce a compound other than L-glutamine.
  • an enzyme that catalyzes a reaction that branches from the biosynthetic pathway of L-glutamine to produce a compound other than L-glutamine.
  • a method of modifying the bacterium so that the activity of the further enzyme is reduced can also be mentioned.
  • Such an enzyme is not particularly limited, and includes glutaminase.
  • L-glutamine-producing bacteria or parent strains for inducing them examples include coryneform bacteria (EP1229121, EP1424398) with enhanced activity of glutamate dehydrogenase (gdhA) and / or glutamine synthetase (glnA), and coryneforms with reduced glutaminase activity Type bacteria (Japanese Patent Laid-Open No. 2004-187684).
  • the L-glutamine-producing bacterium or the parent strain for inducing it is a strain belonging to the genus Escherichia having a mutant glutamine synthetase in which the tyrosine residue at position 397 of glutamine synthetase is substituted with another amino acid residue. (US Patent Application Publication No. 2003-0148474).
  • Corynebacterium glutamicum (Brevibacterium flavum) AJ11573 (FERM P-5492; JP 56-161495) Corynebacterium glutamicum (Brevibacterium flavum) AJ11576 (FERM BP-10381; JP 56-161495) Corynebacterium glutamicum (Brevibacterium flavum) AJ12212 (FERM P-8123; JP-A 61-202694)
  • Examples of the method for imparting or enhancing L-proline production ability include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-proline biosynthesis enzymes is increased.
  • Such enzymes include glutamate-5-kinase (proB), ⁇ -glutamyl-phosphate reductase, pyrroline-5-carboxylate reductase (putA).
  • proB glutamate-5-kinase
  • PDA pyrroline-5-carboxylate reductase
  • the proB gene German Patent No. 3127361
  • the proB gene German Patent No. 3127361 encoding glutamate-5-kinase in which feedback inhibition by L-proline is released can be suitably used.
  • a method for imparting or enhancing L-proline production ability for example, a method of modifying bacteria so that the activity of an enzyme involved in L-proline degradation is reduced.
  • an enzyme include proline dehydrogenase and ornithine aminotransferase.
  • L-proline-producing bacteria or parent strains for deriving them include, for example, E. coli NRRL B-12403 and NRRL B-12404 (British Patent No. 2075056), E. coli VKPM B-8012 ( Russian patent application 2000124295), E. coli plasmid variant described in German Patent 3127361, Bloom FR et al (The 15th Miami winter symposium, 1983, p.34), E. coli plasmid variant, 3, E. coli 702 strain (VKPMB-8011) resistant to 4-dehydroxyproline and azatidine-2-carboxylate, E. coli 702ilvA strain (VKPM B-8012) (EP 1172433) which is a 702 ilvA gene-deficient strain Is mentioned.
  • L-threonine producing bacteria examples include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-threonine biosynthetic enzymes is increased. .
  • enzymes include, but are not limited to, aspartokinase III (lysC), aspartate semialdehyde dehydrogenase (asd), aspartokinase I (thrA), homoserine kinase (thrB), threonine synthase ( threonine synthase) (thrC), aspartate aminotransferase (aspartate transaminase) (aspC).
  • aspartokinase III lysC
  • aspartate semialdehyde dehydrogenase aspartokinase I
  • thrB homoserine kinase
  • thrC threonine synthase
  • aspartate aminotransferase aspartate transaminase
  • the L-threonine biosynthesis gene may be introduced into a strain in which threonine degradation is suppressed.
  • strains in which threonine degradation is suppressed include E. coli TDH6 strain lacking threonine dehydrogenase activity (Japanese Patent Laid-Open No. 2001-346578).
  • the activity of the L-threonine biosynthetic enzyme is inhibited by the final product L-threonine. Therefore, in order to construct an L-threonine-producing bacterium, it is preferable to modify the L-threonine biosynthetic gene so that it is not subject to feedback inhibition by L-threonine.
  • the thrA, thrB, and thrC genes constitute a threonine operon, and the threonine operon forms an attenuator structure. Expression of the threonine operon is inhibited by isoleucine and threonine in the culture medium, and is suppressed by attenuation.
  • Enhanced expression of the threonine operon can be achieved by removing the leader sequence or attenuator in the attenuation region (Lynn, S. P., Burton, W. S., Donohue, T. J., Gould, R. M., Gumport, R. I., and Gardner, J. F. J. Mol. Biol. 194: 59-69 1987 (1987); WO02 / 26993; WO2005 / 049808; WO2003 / 097839).
  • the threonine operon may be constructed so that a gene involved in threonine biosynthesis is expressed under the control of a lambda phage repressor and promoter (see European Patent No. 0593792).
  • Bacteria modified so as not to be subjected to feedback inhibition by L-threonine can also be obtained by selecting a strain resistant to ⁇ -amino- ⁇ -hydroxyvaleric acid (AHV), which is an L-threonine analog.
  • HAV ⁇ -amino- ⁇ -hydroxyvaleric acid
  • the threonine operon modified so as not to be subjected to feedback inhibition by L-threonine is improved in the expression level in the host by increasing the copy number or being linked to a strong promoter.
  • An increase in copy number can be achieved by introducing a plasmid containing a threonine operon into the host.
  • An increase in copy number can also be achieved by transferring the threonine operon onto the host genome using a transposon, Mu phage, or the like.
  • examples of a method for imparting or enhancing L-threonine production ability include a method for imparting L-threonine resistance to a host and a method for imparting L-homoserine resistance.
  • the imparting of resistance can be achieved, for example, by enhancing the expression of a gene that imparts resistance to L-threonine or a gene that imparts resistance to L-homoserine.
  • genes that confer resistance include rhtA gene (Res. Microbiol. 154: 123-135 (2003)), rhtB gene (European Patent Application Publication No. 0994190), rhtC gene (European Patent Application Publication No.
  • L-threonine-producing bacteria or parent strains for deriving them include, for example, E. coli TDH-6 / pVIC40 (VKPM B-3996) (US Patent No. 5,175,107, US Patent No. 5,705,371), E. coli 472T23 / pYN7 (ATCC 98081) (U.S. Patent No. 5,631,157), E. coli NRRL-21593 (U.S. Patent No. 5,939,307), E. coli FERM BP-3756 (U.S. Patent No. 5,474,918), E. coli FERM BP-3519 and FERM BP-3520 (U.S. Patent No. 5,376,538), E.
  • E. coli MG442 (Gusyatiner et al., Genetika (in Russian), 14, 947-956 (1978)), E. coli VL643 and VL2055 ( EP 1149911 A), and E. coli VKPM B-5318 (EP 0593792 B).
  • VKPM B-3996 strain is a strain obtained by introducing plasmid pVIC40 into TDH-6 strain.
  • the TDH-6 strain is sucrose-assimilating, lacks the thrC gene, and has a leaky mutation in the ilvA gene.
  • the VKPM B-3996 strain has a mutation that imparts resistance to a high concentration of threonine or homoserine in the rhtA gene.
  • the plasmid pVIC40 is a plasmid in which a thrA * BC operon containing a mutant thrA gene encoding an aspartokinase homoserine dehydrogenase I resistant to feedback inhibition by threonine and a wild type thrBC gene is inserted into an RSF1010-derived vector (US Patent) No. 5,705,371).
  • This mutant thrA gene encodes aspartokinase homoserine dehydrogenase I substantially desensitized to feedback inhibition by threonine.
  • the strain VKPM B-5318 is non-isoleucine-requiring and retains the plasmid pPRT614 in which the control region of the threonine operon in the plasmid pVIC40 is replaced with a temperature-sensitive lambda phage C1 repressor and a PR promoter.
  • VKPM B-5318 was assigned to Lucian National Collection of Industrial Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia) on May 3, 1990 under the accession number VKPM B-5318 Has been deposited internationally.
  • the thrA gene encoding aspartokinase homoserine dehydrogenase I of E. coli has been revealed (nucleotide numbers 337-2799, GenBank accession NC_000913.2, gi: 49175990).
  • the thrA gene is located between the thrL gene and the thrB gene in the chromosome of E. coli K-12.
  • the thrB gene encoding homoserine kinase of Escherichia coli has been elucidated (nucleotide numbers 2801 to 3733, GenBank accession NC_000913.2, gi: 49175990).
  • the thrB gene is located between the thrA gene and the thrC gene in the chromosome of E. coli K-12.
  • the thrC gene encoding threonine synthase from E.coli has been elucidated (nucleotide numbers 3734 to 5020, GenBank accession NC_000913.2, gi: 49175990).
  • the thrC gene is located between the thrB gene and the yaaX open reading frame in the chromosome of E. coli K-12.
  • thrA * BC operon containing a mutant thrA gene encoding an aspartokinase homoserine dehydrogenase I resistant to feedback inhibition by threonine and a wild type thrBC gene is known in the threonine-producing strain E. coli VKPM B-3996. It can be obtained from plasmid pVIC40 (US Pat. No. 5,705,371).
  • the rhtA gene of E. coli is present at 18 minutes of the E. coli chromosome close to the glnHPQ operon, which encodes a glutamine transport system element.
  • the rhtA gene is the same as ORF1 (ybiF gene, nucleotide numbers 764 to 1651, GenBank accession number AAA218541, gi: 440181), and is located between the pexB gene and the ompX gene.
  • the unit that expresses the protein encoded by ORF1 is called rhtA gene (rht: resistant toosehomoserine andeonthreonine (resistant to homoserine and threonine)).
  • the asd gene of E. coli has already been clarified (nucleotide numbers 3572511 to 3571408, GenBank accession NC_000913.1, gi: 16131307), and can be obtained by PCR using primers prepared based on the nucleotide sequence of the gene ( White, TJ et al., Trends Genet., 5, 185 (1989)).
  • the asd gene of other microorganisms can be obtained similarly.
  • the aspC gene of E. ⁇ ⁇ coli has already been clarified (nucleotide numbers 983742 to 984932, GenBank accession NC_000913.1, gi: 16128895), and obtained by PCR using a primer prepared based on the nucleotide sequence of the gene be able to.
  • the aspC gene of other microorganisms can be obtained similarly.
  • coryneform bacteria having L-threonine-producing ability examples include Corynebacterium acetoacidophilum AJ12318123 (FERM BP-1172) (see US Patent No. 5,188,949).
  • Examples of a method for imparting or enhancing L-lysine production ability include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-lysine biosynthesis enzymes is increased.
  • Such enzymes include, but are not limited to, dihydrodipicolinate synthase (dapA), aspartokinase III (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate Deaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (US Pat. No.
  • phosphoenolpyruvate carboxylase ppc
  • aspartate semialdehyde dehydrogenase aspartate semialdehyde dehydrogenase
  • Asd aspartate aminotransferase (aspartate transaminase)
  • dapF diaminopimelate epi Diaminopimelate epimerase
  • dapD tetrahydrodipicolinate succinylase
  • dapE succinyl-diaminopimelate deacylase
  • aspartase aspartase (195) ).
  • dihydrodipicolinate reductase diaminopimelate decarboxylase, diaminopimelate dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, diaminopimelate epimerase, aspartate semialdehyde dehydrogenase, tetrahydrodipicolinate succinylase, and
  • the activity of one or more enzymes selected from succinyl diaminopimelate deacylase is enhanced.
  • a gene (cyo) (EP 1170376 A) involved in energy efficiency, a gene encoding nicotinamide nucleotide transhydrogenase (pntAB) ( US Pat. No. 5,830,716), ybjE gene (WO2005 / 073390), or combinations thereof may have increased expression levels.
  • Aspartokinase III (lysC) is subject to feedback inhibition by L-lysine.
  • a mutant lysC gene encoding aspartokinase III that has been desensitized to feedback inhibition by L-lysine is used. It may be used (US Pat. No.
  • a method for imparting or enhancing L-lysine producing ability for coryneform bacteria for example, a method of modifying the bacteria so that the activity of the lysine excretion system (lysE) is increased (WO97 / 23597). ).
  • the lysE gene of Corynebacterium glutamicum ATCC 13032 corresponds to the complementary sequence of the 1329712-1330413 sequence in the genome sequence registered in the NCBI database as GenBank accession NC_006958 (VERSION NC_006958.1 GI: 62388892).
  • the LysE protein of Corynebacterium glutamicum ATCC13032 is registered as GenBank accession YP_225551 (YP_225551.1 GI: 62390149).
  • the method for imparting or enhancing L-lysine production ability is, for example, selected from enzymes that catalyze the reaction of branching from the biosynthetic pathway of L-lysine to produce compounds other than L-lysine.
  • enzymes that catalyze the reaction of branching from the biosynthetic pathway of L-lysine to produce compounds other than L-lysine.
  • a method of modifying the bacterium so that the activity of the further enzyme is reduced can also be mentioned.
  • Such enzymes include, but are not limited to, homoserine dehydrogenase, lysine decarboxylase (US Pat. No. 5,827,698), and malic enzyme (WO2005 / 010175). .
  • L-lysine-producing bacteria or parent strains for inducing them include mutants having resistance to L-lysine analogs.
  • L-lysine analogs inhibit the growth of bacteria such as Enterobacteriaceae and coryneform bacteria, but this inhibition is completely or partially released when L-lysine is present in the medium.
  • the L-lysine analog is not particularly limited, and examples thereof include oxalysine, lysine hydroxamate, S- (2-aminoethyl) -L-cysteine (AEC), ⁇ -methyllysine, and ⁇ -chlorocaprolactam.
  • Mutant strains having resistance to these lysine analogs can be obtained by subjecting bacteria to normal artificial mutation treatment.
  • L-lysine-producing bacteria or parent strains for deriving them include, for example, E. coli AJ11442 (FERM BP-1543, NRRL B-12185; see U.S. Pat. No. 4,346,170) and E. coli VL611. Can be mentioned. In these strains, feedback inhibition of aspartokinase by L-lysine is released.
  • L-lysine-producing bacteria or parent strains for inducing them include E. coli WC196 strain.
  • the WC196 strain was bred by conferring AEC resistance to the W3110 strain derived from E. coli K-12 (US Pat. No. 5,827,698).
  • the WC196 strain was named E.
  • Preferred L-lysine producing bacteria include E.coli WC196 ⁇ cadA ⁇ ldc and E.coli WC196 ⁇ cadA ⁇ ldc / pCABD2 (WO2010 / 061890).
  • WC196 ⁇ cadA ⁇ ldc is a strain constructed by disrupting the cadA and ldcC genes encoding lysine decarboxylase from the WC196 strain.
  • WC196 ⁇ cadA ⁇ ldc / pCABD2 is a strain constructed by introducing plasmid pCABD2 (US Pat. No. 6,040,160) containing a lysine biosynthesis gene into WC196 ⁇ cadA ⁇ ldc.
  • WC196 ⁇ cadA ⁇ ldc was named AJ110692, and on October 7, 2008, National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (currently, National Institute of Technology and Evaluation, Patent Biological Depositary Center, ZIP Code: 292-0818, Address: 2-5-8 120, Kazusa Kamashitsu, Kisarazu City, Chiba Prefecture, Japan) was deposited under the accession number FERM BP-11027.
  • pCABD2 is a mutant dapA gene encoding dihydrodipicolinate synthase (DDPS) derived from Escherichia coli having a mutation that is desensitized to feedback inhibition by L-lysine, and a mutation that is desensitized to feedback inhibition by L-lysine.
  • a mutant lysC gene encoding aspartokinase III derived from Escherichia coli, dapB gene encoding dihydrodipicolinate reductase derived from Escherichia coli, and ddh encoding a diaminopimelate dehydrogenase derived from Brevibacterium lactofermentum Contains genes.
  • a preferable L-lysine-producing bacterium includes E.coli AJIK01 strain (NITE BP-01520).
  • the AJIK01 strain was named E. coli AJ111046.
  • Patent Microorganisms Deposit Center Postal Code: 292-0818, Address: Kazusa Kama, Kisarazu City, Chiba Prefecture, Japan
  • No. 2-5-8 120
  • coryneform bacteria having L-lysine-producing ability examples include, for example, AEC-resistant mutant strains (Corynebacterium glutamicum (Brevibacterium lactofermentum) AJ11082 (NRRL 470 B-11470) strain, etc .; Japanese Patent Publication No. 56-1914, Japanese Patent Publication No. 56- No. 1915, No. 57-14157, No. 57-14158, No. 57-30474, No. 58-10075, No. 59-4993, No. 61-35840 No.
  • L-homoserine for its growth Mutants that require amino acids such as (see Japanese Patent Publication Nos. 48-28078 and 56-6499); exhibit resistance to AEC, and further L-leucine, L-homoserine, L-proline, L-serine Mutants requiring amino acids such as L-arginine, L-alanine and L-valine (US Pat. No.
  • Examples of the method for imparting or enhancing L-arginine-producing ability include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-arginine biosynthesis enzymes is increased. .
  • Examples of such enzymes include, but are not limited to, N-acetylglutamate synthase (argA), N-acetylglutamylphosphate reductase (argC), ornithine acetyltransferase (argJ), N-acetylglutamate kinase (argB), acetylornithine Examples include transaminase (argD), acetylornithine deacetylase (argE) ornithine carbamoyltransferase (argF), argininosuccinate synthase (argG), argininosuccinate lyase (argH), and carbamoyl phosphate synthase (carAB).
  • argA N-acetylglutamate synthase
  • argC N-acetylglutamylphosphate reductase
  • argJ ornithine acetyltransferase
  • N-acetylglutamate synthase (argA) gene examples include mutant N-acetylglutamate synthase in which amino acid residues corresponding to the 15th to 19th positions of the wild type are substituted and feedback inhibition by L-arginine is released. It is preferable to use a gene to be encoded (European Application Publication No. 1170361).
  • L-arginine-producing bacteria or parent strains for inducing the same include, for example, E. coli 237 strain (VKPM B-7925) (US Patent Application Publication 2002/058315 A1), mutant N-acetylglutamic acid Its derivative strain ⁇ ⁇ ( Russian patent application No. 2001112869, EP1170361A1) introduced with the argA gene encoding synthase, E.237coli 382 strain (VKPM B-7926) 237 (VKPM B-7926) EP1170358A1) and E. coli 382ilvA + strain, which is a strain in which the wild-type ilvA gene derived from E. coli K-12 strain is introduced into 382 strain.
  • E. coli 237 strain VKPM B-7925
  • US Patent Application Publication 2002/058315 A1 mutant N-acetylglutamic acid Its derivative strain ⁇ ⁇
  • E.237coli 382 strain VKPM B-7926
  • L-arginine-producing bacteria or parent strains for inducing them include strains having resistance to amino acid analogs and the like.
  • Such strains include, for example, ⁇ -methylmethionine, p-fluorophenylalanine, D-arginine, arginine hydroxamic acid, S- (2-aminoethyl) -cysteine, ⁇ -methylserine, ⁇ -2-thienylalanine, or Examples include Escherichia coli mutants having resistance to sulfaguanidine (see JP-A-56-106598).
  • L-arginine-producing bacteria or parent strains for inducing them include strains lacking ArgR, an arginine repressor (US Patent Application Publication No. 2002-0045223), and strains that have increased intracellular glutamine synthetase activity. Examples thereof include coryneform bacteria such as (US Patent Application Publication No. 2005-0014236).
  • L-arginine-producing bacteria or parent strains for inducing them include mutants of coryneform bacteria having resistance to amino acid analogs and the like.
  • examples of such a strain include, in addition to 2-thiazolealanine resistance, a strain having L-histidine, L-proline, L-threonine, L-isoleucine, L-methionine, or L-tryptophan auxotrophic No. 54-44096); strains resistant to ketomalonic acid, fluoromalonic acid, or monofluoroacetic acid (JP 57-18989); strains resistant to argininol (Japanese Patent Publication No.
  • a strain resistant to X-guanidine (X is a fatty chain or a derivative thereof) (Japanese Patent Laid-Open No. 2-186995); a strain resistant to arginine hydroxamate and 6-azauracil (Japanese Patent Laid-Open No. 57-150381) ).
  • Specific examples of coryneform bacteria having the ability to produce L-arginine include the following strains.
  • L-citrulline and L-ornithine-producing bacteria share a biosynthetic pathway with L-arginine.
  • N-acetylglutamate synthase argA
  • N-acetylglutamylphosphate reductase argC
  • ornithine acetyltransferase argJ
  • N-acetylglutamate kinase argB
  • acetylornithine transaminase argD
  • WO 2006-35831 By increasing the enzyme activity of deacetylase (argE), the ability to produce L-citrulline and / or L-ornithine can be imparted or enhanced (WO 2006-35831).
  • Examples of the method for imparting or enhancing L-histidine production ability include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-histidine biosynthesis enzymes is increased.
  • Examples of such an enzyme include, but are not limited to, ATP phosphoribosyltransferase (hisG), phosphoribosyl-AMP cyclohydrolase (hisI), phosphoribosyl-ATP pyrophosphohydrolase (hisI), phosphoribosylformimino-5-aminoimidazole carboxamide ribonucleoside.
  • tide isomerase (hisA), amide transferase (hisH), histidinol phosphate aminotransferase (hisC), histidinol phosphatase (hisB), and histidinol dehydrogenase (hisD).
  • hisA tide isomerase
  • hisH amide transferase
  • hisC histidinol phosphate aminotransferase
  • hisB histidinol phosphatase
  • hisD histidinol dehydrogenase
  • L-histidine biosynthetic enzymes encoded by hisG and hisBHAFI are known to be inhibited by L-histidine. Therefore, the ability to produce L-histidine can be imparted or enhanced, for example, by introducing a mutation that confers resistance to feedback inhibition in the ATP phosphoribosyltransferase gene (hisG) ( Russian Patent No. 2003677 and No. 2). 2119536).
  • L-histidine-producing bacteria or parent strains for inducing them include, for example, E. coli 24 strain (VKPM B-5945, RU2003677), E. coli NRRL B-12116-B-12121 (US Patent) No. 4,388,405), E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (U.S. Patent No. 6,344,347), E. coli H-9341 (FERM BP-6674) (EP1085087) E. coli AI80 / pFM201 (US Pat. No. 6,258,554), E.
  • E. coli FERM P-5038 and 5048 into which a vector carrying DNA encoding an L-histidine biosynthetic enzyme was introduced Japanese Patent Laid-Open No. 56-005099
  • E. coli strain EP1016710A
  • E. coli 80 strain VKPM B
  • Sulfaguanidine DL-1,2,4-triazole-3-alanine
  • streptomycin -7270 Russian Patent No. 2119536
  • Examples of the method for imparting or enhancing L-cysteine production ability include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-cysteine biosynthesis enzymes is increased.
  • Examples of such an enzyme include, but are not limited to, serine acetyltransferase (cysE) and 3-phosphoglycerate dehydrogenase (serA).
  • Serine acetyltransferase activity can be enhanced, for example, by introducing a mutant cysE gene encoding a mutant serine acetyltransferase resistant to feedback inhibition by cysteine into bacteria.
  • Mutant serine acetyltransferases are disclosed, for example, in JP-A-11-155571 and US Patent Publication No. 20050112731. Further, the 3-phosphoglycerate dehydrogenase activity can be enhanced by introducing, for example, a mutant serA gene encoding a mutant 3-phosphoglycerate dehydrogenase resistant to feedback inhibition by serine into a bacterium. Mutant 3-phosphoglycerate dehydrogenase is disclosed, for example, in US Pat. No. 6,180,373.
  • the method for imparting or enhancing L-cysteine production ability is selected from, for example, an enzyme that catalyzes a reaction that branches from the biosynthesis pathway of L-cysteine to produce a compound other than L-cysteine.
  • an enzyme that catalyzes a reaction that branches from the biosynthesis pathway of L-cysteine to produce a compound other than L-cysteine Alternatively, a method of modifying the bacterium so that the activity of the further enzyme is reduced can also be mentioned.
  • examples of such enzymes include enzymes involved in the degradation of L-cysteine.
  • the enzyme involved in the degradation of L-cysteine is not particularly limited, but cystathionine- ⁇ -lyase (metC) (Japanese Patent Laid-Open No. 11-155571, Chandra et.
  • examples of methods for imparting or enhancing L-cysteine production ability include enhancing the L-cysteine excretion system and enhancing the sulfate / thiosulfate transport system.
  • proteins of the L-cysteine excretion system include proteins encoded by the ydeD gene (JP 2002-233384), proteins encoded by the yfiK gene (JP 2004-49237), emrAB, emrKY, yojIH, acrEF, bcr, And each protein encoded by each gene of cusA (Japanese Patent Laid-Open No.
  • sulfate / thiosulfate transport system protein examples include proteins encoded by the cysPTWAM gene cluster.
  • L-cysteine-producing bacteria or parent strains for deriving them include, for example, E. coli JM15 (US Patent) transformed with various cysE alleles encoding mutant serine acetyltransferase resistant to feedback inhibition. No. 6,218,168, Russian Patent Application No. 2003121601), E. coli W3110 (US Pat.No. 5,972,663), cysteine desulfhydrase, which has an overexpressed gene encoding a protein suitable for excretion of substances toxic to cells Examples include E. coli strain (JP11155571A2) with reduced activity and E. coli W3110 (WO01 / 27307A1) with increased activity of the transcriptional control factor of the positive cysteine regulon encoded by the cysB gene.
  • coryneform bacteria having L-cysteine-producing ability examples include coryneform bacteria in which intracellular serine acetyltransferase activity is increased by retaining serine acetyltransferase with reduced feedback inhibition by L-cysteine (for example, JP-A-2002-233384).
  • Examples of a method for imparting or enhancing L-serine production ability include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-serine biosynthetic enzymes is increased.
  • Examples of such enzymes include, but are not limited to, 3-phosphoglycerate dehydrogenase (serA), phosphoserine transaminase (serC), and phosphoserine phosphatase (serB) (Japanese Patent Laid-Open No. 11-253187).
  • the 3-phosphoglycerate dehydrogenase activity can be enhanced, for example, by introducing a mutant serA gene encoding a mutant 3-phosphoglycerate dehydrogenase resistant to feedback inhibition by serine into bacteria. Mutant 3-phosphoglycerate dehydrogenase is disclosed, for example, in US Pat. No. 6,180,373.
  • L-serine-producing bacteria or parent strains for inducing them examples include coryneform bacteria that are resistant to azaserine or ⁇ - (2-thienyl) -DL-alanine and lack L-serine resolution. (JP-A-10-248588).
  • coryneform bacteria include, for example, Corynebacterium glutamicum (Brevibacterium flavum) AJ13324 (FERM P-16128) ⁇ ⁇ , which is resistant to azaserine and lacks L-serine resolution, and ⁇ - (2- Corynebacterium glutamicum (Brevibacterium flavum) AJ13325 (FERM ⁇ ⁇ P-16129) ⁇ ⁇ that is resistant to thienyl) -DL-alanine and lacks the resolution of L-serine (Japanese Patent Laid-Open No. 10-248588).
  • L-methionine producing bacteria examples include L-threonine-requiring strains and mutants having resistance to norleucine (Japanese Patent Laid-Open No. 2000-139471).
  • examples of L-methionine-producing bacteria or parent strains for deriving them also include strains that retain mutant homoserine transsuccinylase that is resistant to feedback inhibition by L-methionine (Japanese Patent Laid-Open No. 2000-139471). , US20090029424).
  • L-methionine is biosynthesized with L-cysteine as an intermediate, L-methionine production ability can be improved by improving L-cysteine production ability (Japanese Patent Laid-Open No. 2000-139471, US20080311632).
  • L-methionine-producing bacteria or parent strains for inducing them include, for example, E. coli AJ11539 (NRRL B-12399), E. coli AJ11540 (NRRL B-12400), E. coli AJ11541 (NRRL B-12401), E. coli AJ11542 (NRRL B-12402) (British Patent No. 2075055), E. coli 218 strain (VKPM B-8125) having resistance to norleucine, an analog of L-methionine (Russian Patent No. 2209248) No.), 73 shares (VKPM B-8126) (Russian Patent No. 2215782), E.
  • coli AJ13425 (FERM P-16808) (Japanese Patent Laid-Open No. 2000-139471).
  • the AJ13425 strain lacks a methionine repressor, weakens intracellular S-adenosylmethionine synthetase activity, and produces intracellular homoserine transsuccinylase activity, cystathionine ⁇ -synthase activity, and aspartokinase-homoserine dehydrogenase II.
  • L-threonine-requiring strain derived from E. coli W3110 with enhanced activity.
  • Examples of the method for imparting or enhancing the ability to produce L-leucine include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-leucine biosynthesis enzymes is increased. .
  • Examples of such an enzyme include, but are not limited to, an enzyme encoded by a gene of leuABCD operon.
  • a mutant leuA gene US Pat. No. 6,403,342
  • encoding isopropyl malate synthase from which feedback inhibition by L-leucine has been released can be suitably used.
  • L-leucine-producing bacteria or parent strains for inducing the same include, for example, leucine-resistant E. coli strains (eg, 57 strains (VKPM B-7386, US Pat. No. 6,124,121)), ⁇ - E. coli strains resistant to leucine analogs such as 2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoroleucine (JP-B-62-34397 and JP-A-8-70879), WO96 And strains belonging to the genus Escherichia such as E. coli strain and E. coli H-9068 (JP-A-8-70879) obtained by the genetic engineering method described in / 06926.
  • leucine-resistant E. coli strains eg, 57 strains (VKPM B-7386, US Pat. No. 6,124,121)
  • ⁇ - E. coli strains resistant to leucine analogs such as 2-thienylalan
  • Coryneform bacteria having L-leucine-producing ability include, for example, Corynebacterium amicglutamicum (Brevibacterium lactofermentum) AJ3718 (FERM P-2516), which is resistant to 2-thiazolealanine and ⁇ -hydroxyleucine, and is auxotrophic for isoleucine and methionine. Is mentioned.
  • Examples of the method for imparting or enhancing L-isoleucine producing ability include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-isoleucine biosynthesis enzymes is increased.
  • Examples of such an enzyme include, but are not limited to, threonine deaminase and acetohydroxy acid synthase (JP-A-2-458, FR 0356739, and US Pat. No. 5,998,178).
  • L-isoleucine-producing bacteria or parent strains for inducing them include mutants having resistance to 6-dimethylaminopurine (Japanese Patent Laid-Open No. 5-304969), thiisoleucine, isoleucine hydroxamate, etc.
  • Escherichia bacteria such as mutant strains resistant to isoleucine analogs and mutant strains resistant to DL-ethionine and / or arginine hydroxamate in addition to isoleucine analogs (JP-A-5-130882).
  • coryneform bacteria having the ability to produce L-isoleucine examples include, for example, coryneform bacteria (JP 2001-169788) in which a brnE gene encoding a branched-chain amino acid excretion protein is amplified, and protoplast fusion with L-lysine-producing bacteria.
  • coryneform bacteria imparted with isoleucine-producing ability Japanese Patent Laid-Open No. Sho 62-74293
  • coryneform bacteria enhanced with homoserine dehydrogenase Japanese Patent Laid-Open No. Sho 62-91193
  • threonine hydroxamate resistant strain Japanese Patent Laid-Open No.
  • Examples of a method for imparting or enhancing L-valine production ability include a method of modifying a bacterium so that the activity of one or more enzymes selected from L-valine biosynthetic enzymes is increased.
  • Examples of such enzymes include, but are not limited to, enzymes encoded by genes of ilvGMEDA operon and ilvBNC operon. ilvBN encodes acetohydroxy acid synthase, and ilvC encodes isomeroreductase (WO 00/50624).
  • the ilvGMEDA operon and the ilvBNC operon are subject to expression suppression (attenuation) by L-valine, L-isoleucine, and / or L-leucine. Therefore, in order to enhance the enzyme activity, it is preferable to remove or modify the region necessary for attenuation and to cancel the expression suppression by the produced L-valine.
  • the threonine deaminase encoded by the ilvA gene is an enzyme that catalyzes the deamination reaction from L-threonine to 2-ketobutyric acid, which is the rate-limiting step of the L-isoleucine biosynthesis system. Therefore, for L-valine production, it is preferable that the ilvA gene is disrupted and the threonine deaminase activity is reduced.
  • the method for imparting or enhancing L-valine-producing ability is, for example, selected from enzymes that catalyze a reaction that branches from the biosynthetic pathway of L-valine to produce a compound other than L-valine.
  • enzymes that catalyze a reaction that branches from the biosynthetic pathway of L-valine to produce a compound other than L-valine.
  • a method of modifying the bacterium so that the activity of the further enzyme is reduced can also be mentioned.
  • examples of such enzymes include, but are not limited to, threonine dehydratase involved in L-leucine synthesis and enzymes involved in D-pantothenic acid synthesis (International Publication No. 00/50624).
  • L-valine-producing bacterium or the parent strain for deriving the same include, for example, the E. coli strain (US Pat. No. 5,998,178) that has been modified to overexpress the ilvGMEDA operon.
  • examples of L-valine-producing bacteria or parent strains for deriving them also include strains having mutations in aminoacyl t-RNA synthetases (US Pat. No. 5,658,766).
  • examples of such a strain include E. coli VL1970 having a mutation in the ileS gene encoding isoleucine tRNA synthetase.
  • E. coli VL1970 was accepted on June 24, 1988 at Lucian National Collection of Industrial Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia) under the accession number VKPM B-4411. It has been deposited.
  • examples of L-valine-producing bacteria or parent strains for deriving the same also include mutant strains (WO96 / 06926) that require lipoic acid for growth and / or lack H + -ATPase. .
  • L-valine-producing bacteria or parent strains for inducing them include strains having resistance to amino acid analogs and the like.
  • Such strains include, for example, L-isoleucine and L-methionine requirement, coryneform bacterial strains resistant to D-ribose, purine ribonucleoside, or pyrimidine ribonucleoside and capable of producing L-valine.
  • L-alanine producing bacteria examples include coryneform bacteria lacking H + -ATPase (Appl Microbiol Biotechnol. 2001 Nov; 57 (4): 534-40) and aspartic acid ⁇ -Coryneform bacteria with enhanced decarboxylase activity (JP 07-163383 A).
  • L-tryptophan producing bacteria L-phenylalanine producing bacteria, L-tyrosine producing bacteria>
  • methods for imparting or enhancing L-tryptophan production ability, L-phenylalanine production ability, and / or L-tyrosine production ability include biosynthesis of L-tryptophan, L-phenylalanine, and / or L-tyrosine.
  • Biosynthetic enzymes common to these aromatic amino acids are not particularly limited, but 3-deoxy-D-arabinohepturonic acid-7-phosphate synthase (aroG), 3-dehydroquinate synthase (aroB) Shikimate dehydrogenase (aroE), shikimate kinase (aroL), 5-enolic acid pyruvylshikimate 3-phosphate synthase (aroA), chorismate synthase (aroC) (European Patent No. 763127). Expression of genes encoding these enzymes is controlled by a tyrosine repressor (tyrR), and the activity of these enzymes may be enhanced by deleting the tyrR gene (European Patent No. 763127).
  • tyrR tyrosine repressor
  • L-tryptophan biosynthesis enzyme examples include, but are not limited to, anthranilate synthase (trpE), tryptophan synthase (trpAB), and phosphoglycerate dehydrogenase (serA).
  • trpE anthranilate synthase
  • trpAB tryptophan synthase
  • serA phosphoglycerate dehydrogenase
  • L-tryptophan production ability can be imparted or enhanced by introducing DNA containing a tryptophan operon.
  • Tryptophan synthase consists of ⁇ and ⁇ subunits encoded by trpA and trpB genes, respectively.
  • anthranilate synthase is subject to feedback inhibition by L-tryptophan
  • a gene encoding the enzyme into which a mutation that releases feedback inhibition is introduced may be used.
  • phosphoglycerate dehydrogenase is feedback-inhibited by L-serine
  • a gene encoding the enzyme into which a mutation that releases feedback inhibition is introduced may be used to enhance the activity of the enzyme.
  • L-tryptophan-producing ability is imparted or enhanced by increasing the expression of an operon consisting of malate synthase (aceB), isocitrate lyase (aceA), and isocitrate dehydrogenase kinase / phosphatase (aceK). (WO2005 / 103275).
  • the L-phenylalanine biosynthetic enzyme is not particularly limited, and examples thereof include chorismate mutase and prefenate dehydratase. Chorismate mutase and prefenate dehydratase are encoded by the pheA gene as a bifunctional enzyme. Since chorismate mutase-prefenate dehydratase is feedback-inhibited by L-phenylalanine, in order to enhance the activity of the enzyme, a gene encoding the enzyme into which a mutation that releases feedback inhibition is introduced may be used.
  • the L-tyrosine biosynthetic enzyme is not particularly limited, and examples thereof include chorismate mutase and prephenate dehydrogenase. Chorismate mutase and prefenate dehydrogenase are encoded by the tyrA gene as a bifunctional enzyme. Since chorismate mutase-prefenate dehydrogenase is feedback-inhibited by L-tyrosine, to enhance the activity of the enzyme, a gene encoding the enzyme into which a mutation that releases feedback inhibition is introduced may be used.
  • the L-tryptophan, L-phenylalanine, and / or L-tyrosine producing bacterium may be modified so that biosynthesis of aromatic amino acids other than the target aromatic amino acid is lowered.
  • L-tryptophan, L-phenylalanine, and / or L-tyrosine-producing bacteria may be modified so that the by-product uptake system is enhanced.
  • By-products include aromatic amino acids other than the desired aromatic amino acid. Examples of genes encoding uptake systems of by-products include, for example, uptake systems of tnaB and mtr, which are L-tryptophan uptake systems, and pheP, L-tyrosine, which are genes encoding uptake systems of L-phenylalanine. TyrP, which is a gene coding for (EP1484410).
  • E. coli JP4735 / pMU3028 carrying a mutant trpS gene encoding a partially inactivated tryptophanyl-tRNA synthetase. DSM10122) and JP6015 / pMU91 (DSM10123) (U.S. Patent No. 5,756,345)
  • E. coli SV164 with trpE allele encoding anthranilate synthase not subject to feedback inhibition by tryptophan
  • phosphoglycerate dehydrogenase not subject to feedback inhibition by serine E. coli SV164 pGH5
  • coryneform bacteria having L-tryptophan-producing ability include, for example, Corynebacterium glutamicum 118AJ12118 (FERM BP-478 patent 01681002) resistant to sulfaguanidine, a strain into which tryptophan operon has been introduced (JP 63240794), coryneform And a strain into which a gene encoding shikimate kinase derived from a type bacterium has been introduced (Japanese Patent Laid-Open No. 01994749).
  • E. coli AJ12739 (tyrA :: Tn10, tyrR) (VKPM) lacking chorismate mutase-prefenate dehydrogenase and tyrosine repressor B-8197) (WO03 / 044191)
  • E. coli HW1089 (ATCC 55371) (U.S. Patent No. 5,354,672), carrying a mutant pheA34 gene encoding chorismate mutase-prefenate dehydratase with released feedback inhibition
  • E.Coli MWEC 101-b KR8903681
  • E.coli NRRL B-12141 NRRL B-12145
  • NRRL B-12146 NRRL B-12147
  • US Pat. No. 4,407,952 E.coli NRRL B-12141
  • NRRL B-12145 NRRL B-12146
  • NRRL B-12147 US Pat. No. 4,407,952
  • E. coli K-12 that retains a gene encoding chorismate mutase-prefenate dehydratase in which feedback inhibition is released.
  • ⁇ W3110 (tyrA) / pPHAB> (FERM BP-3566)
  • L-phenylalanine-producing bacteria or parent strains for inducing them include, for example, strains belonging to the genus Escherichia in which the activity of the protein encoded by the yedA gene or the yddG gene is increased (US2003 / 0148473, US2003 / 0157667, WO03 / 044192).
  • coryneform bacteria having the ability to produce L-phenylalanine include, for example, Corynebacterium amicglutamicum BPS-13 strain FER (FERM BP-1777), Corynebacterium glutamicum K77 (FERM BP-2062) having reduced phosphoenolpyruvate carboxylase or pyruvate kinase activity Corynebacterium glutamicum K78 (FERM BP-2063) (European Patent Publication No. 331145, Japanese Patent Laid-Open No. 02-303495) and tyrosine-requiring strain (Japanese Patent Laid-Open No. 05-049489).
  • coryneform bacteria having the ability to produce L-tyrosine include Corynebacterium glutamicum AJ11655 (FERM P-5836) (Japanese Patent Publication No. 2-6517), Corynebacterium glutamicum (Brevibacterium lactofermentum) AJ12081 (FERM P-7249) -70093).
  • examples of a method for imparting or enhancing L-amino acid-producing ability include a method of modifying a bacterium so that the activity of discharging L-amino acid from the bacterium cell is increased.
  • the activity to excrete L-amino acids can be increased, for example, by increasing the expression of a gene encoding a protein that excretes L-amino acids.
  • genes encoding proteins that excrete various amino acids include b2682 gene (ygaZ), b2683 gene (ygaH), b1242 gene (ychE), and b3434 gene (yhgN) (Japanese Patent Laid-Open No. 2002-300874) .
  • examples of a method for imparting or enhancing L-amino acid producing ability include a method for modifying bacteria so that the activity of a protein involved in sugar metabolism or a protein involved in energy metabolism is increased.
  • Proteins involved in sugar metabolism include proteins involved in sugar uptake and glycolytic enzymes.
  • genes encoding proteins involved in sugar metabolism include glucose 6-phosphate isomerase gene (pgi; WO 01/02542 pamphlet), phosphoenolpyruvate synthase gene (pps; EP 877090 specification) , Phosphoenolpyruvate carboxylase gene (ppc; WO 95/06114 pamphlet), pyruvate carboxylase gene (pyc; WO 99/18228 pamphlet, European application 1092776), phosphoglucomutase gene (Pgm; WO 03/04598 pamphlet), fructose diphosphate aldolase gene (pfkB, fbp; WO 03/04664 pamphlet), pyruvate kinase gene (pykF; WO 03/008609 pamphlet), transaldolase Gene (talB; WO03 / 008611 pamphlet), fumarase residue Examples include
  • genes encoding proteins involved in energy metabolism include a transhydrogenase gene (pntAB; US Pat. No. 5,830,716), a cytochrome bo type oxidase (cyoB; European Patent Application Publication No. 1070376) Is mentioned.
  • the bacterium of the present invention may be modified, for example, so as to enhance the fatty acid assimilation ability.
  • modifications may include reducing the expression of the fadR gene, enhancing the expression of one or more genes selected from the group consisting of the fadL, fadE, fadD, fadB, and fadA genes, and the cyoABCDE operon. Examples thereof include enhancing expression and combinations thereof (Japanese Patent Laid-Open No. 2011-167071).
  • the fadR gene encodes a negative transcription factor for the fad regulon (DiRusso, C. C. et al. 1992. J. Biol. Chem. 267: 8685-8691; DiRusso, C. C. et al. 1993. Mol Microbiol. 7: 311-322).
  • the fad regulon includes the fadL, fadE, fadD, fadB, and fadA genes, which encode proteins involved in fatty acid metabolism.
  • the fadR gene and fad regulon are found, for example, in bacteria belonging to the family Enterobacteriaceae.
  • the fadR gene of Escherichia coli K12 MG1655 strain corresponds to the sequence at positions 124161-1234880 in the genome sequence of the same strain (GenBank accession No. NC_000913).
  • the FadR protein of Escherichia coli K12 MG1655 strain is registered under GenBank accession No. NP_415705.
  • the fadL gene encodes an outer membrane transporter capable of taking up long-chain fatty acids (Kumar, G. B. and Black, P. N. 1993. J. Biol. Chem. 268: 15469-15476; Stenberg, F. et al. 2005. J. Biol. Chem. 280: 34409-34419).
  • the fadL gene of Escherichia coli K12 MG1655 strain corresponds to the sequence from 2459328 to 2460668 in the genome sequence of the same strain (GenBank accession No. NC_000913).
  • the FadL protein of Escherichia coli K12 MG1655 strain is registered under GenBank accession No. NP_416846.
  • the fadD gene catalyzes the reaction to produce fatty acyl-CoA (fatty-acyl-CoA) from long-chain fatty acids (fatty-acyl-CoA-synthetase activity) and encodes a protein incorporated through the inner membrane ( Dirusso, C. C. and Black, P. N. 2004. J. Biol. Chem. 279: 49563-49566; Schmelter, T. et al. 2004. J. Biol. Chem. 279: 24163-24170).
  • the fadD gene of Escherichia coli K12 MG1655 strain corresponds to a complementary sequence of the sequences 160885 to 1887770 in the genome sequence (GenBank ⁇ accession No. NC_000913) of the same strain.
  • the FadD protein of Escherichia coli K12 MG1655 strain is registered under GenBank accession No. NP_416319.
  • the fadE gene encodes a protein having an acyl-CoA dehydrogenase activity that catalyzes a reaction to oxidize fatty acyl-CoA (O'Brien, W. J. and Frerman, F. E. 1977. J. Bacteriol. 132: 532-540; Campbell, J. W. and Cronan, J. E. 2002. J. Bacteriol. 184: 3759-3764).
  • the fadE gene of Escherichia coli K12 MG1655 strain corresponds to a complementary sequence of the sequences from 240859 to 243303 in the genome sequence of the same strain (GenBank accession No. NC_000913).
  • the FadE protein of Escherichia coli K12 MG1655 strain is registered under GenBank accession No. NP_414756.
  • the fadB gene encodes the ⁇ subunit of the fatty acid oxidation complex.
  • the ⁇ subunit includes enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyacyl-CoA epimerase, ⁇ 3-cis- ⁇ 2 -Has four activities of trans-enoyl CoA isomerase ( ⁇ 3-cis- ⁇ 2-trans-enoyl-CoA isomerase) (Pramanik, A. et al. 1979. J. Bacteriol. 137: 469-473; Yang, S. Y. and Schulz, H. 1983. J. Biol. Chem. 258: 9780-9785).
  • the fadB gene of Escherichia coli K12 MG1655 strain corresponds to the complementary sequence of the 4026805-4028994 position in the genome sequence of the same strain (GenBank accession No. NC_000913).
  • the FadB protein of Escherichia coli K12 MG1655 strain is registered under GenBank accession No. NP_418288.
  • the fadA gene encodes the ⁇ subunit of the fatty acid oxidation complex.
  • the ⁇ subunit has 3-ketoacyl-CoA thiolase activity (Pramanik, A. et al. 1979. J. Bacteriol. 137: 469-473).
  • the fadA gene of Escherichia coli K12 MG1655 strain corresponds to a complementary sequence of the 4025632 to 4026795 positions in the genome sequence (GenBank accession No. NC_000913) of the same strain.
  • the FadA protein of Escherichia coli K12 MG1655 strain is registered under GenBank accession No. YP_026272.
  • the fadA and fadB genes form the fadBA operon (Yang, S. Y. et al. 1990. J. Biol. Chem. 265: 10424-10429).
  • the expression of the entire fadBA operon may be enhanced.
  • the cyoABCDE operon encodes a cytochrome bo-terminal oxidase complex, which is one of the terminal oxidases.
  • cyoB gene has subunit I
  • cyoA gene has subunit II
  • cyoC gene has subunit III
  • cyoC gene has subunit IV
  • cyoE gene has heme O synthase activity.
  • the cyo operon is found, for example, in bacteria belonging to the family Enterobacteriaceae.
  • the cyoABCDE gene of Escherichia coli K12 MG1655 strain is complementary to the sequences of 449887 to 450834, 447874 to 449865, 447270 to 448884, 446941 to 447270, and 446039 to 446929 in the genome sequence of the same strain (GenBank accession No.
  • CyoABCDE protein of Escherichia coli K12 MG1655 strain is registered under GenBank accession No. NP_414966, NP_414965, NP_414964, NP_414963, and NP_414962, respectively.
  • the gene used for bacterial breeding is not limited to the above-exemplified genes or genes having a known base sequence, as long as it encodes a protein having the original function maintained, and may be a variant thereof.
  • the variant may be, for example, a homologue or artificially modified gene of the above-exemplified gene or a gene having a known base sequence.
  • the gene used encodes a protein in which the original function is maintained, one or several amino acids at one or several positions are substituted, deleted, or deleted in the amino acid sequence of a known protein. It may be a gene encoding a protein having an inserted or added amino acid sequence.
  • the function of the protein is usually 70% or more, preferably 80% or more, more preferably 90% or more with respect to the protein before one or several amino acids are substituted, deleted, inserted or added. Can be maintained.
  • the “one or several” is different depending on the position of the amino acid residue in the three-dimensional structure of the protein and the type of amino acid residue, but specifically 1 to 50, 1 to 40, 1 to 30 It means 1 to 20, preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5, and particularly preferably 1 to 3.
  • substitution, deletion, insertion, or addition of one or several amino acids described above is a conservative mutation that maintains the protein function normally.
  • a typical conservative mutation is a conservative substitution.
  • Conservative substitution is a polar amino acid between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid. In this case, between Gln and Asn, when it is a basic amino acid, between Lys, Arg, and His, when it is an acidic amino acid, between Asp and Glu, when it is an amino acid having a hydroxyl group Is a mutation that substitutes between Ser and Thr.
  • substitutions considered as conservative substitutions include substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys, substitution from Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp Substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe substitution, Leu to Ile, Met, Val or Phe substitution, Substitution from Lys to Asn, Glu, Gln, His or Arg, substitution from Met to Ile, Leu, Val or Phe, substitution from Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala Substitution, substitution from Trp to Phe or Tyr, substitution
  • the gene having a conservative mutation as described above is 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, particularly with respect to the entire amino acid sequence of a known protein.
  • it may be a gene encoding a protein having 99% or more homology and maintaining the original function.
  • “homology” may refer to “identity”.
  • the gene used was hybridized under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a complementary sequence to all or part of the known gene sequence, and the original function was maintained. It may be DNA encoding a protein. “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, highly homologous DNAs, for example, 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more between DNAs having homology.
  • the probe used for the hybridization may be a part of a gene complementary sequence.
  • a probe can be prepared by PCR using an oligonucleotide prepared on the basis of a known gene sequence as a primer and a DNA fragment containing these base sequences as a template.
  • hybridization washing conditions include 50 ° C., 2 ⁇ SSC, and 0.1% SDS.
  • the gene to be used may be one in which an arbitrary codon is replaced with an equivalent codon as long as it encodes a protein whose original function is maintained.
  • the gene used may be modified so as to have an optimal codon according to the codon usage frequency of the host to be used.
  • the medium used for L-amino acid fermentation is not particularly limited as long as it contains a medium-temperature-treated product and the bacterium of the present invention can grow and L-amino acid is produced.
  • a normal medium used for culturing microorganisms such as bacteria can be used.
  • the medium may contain a component selected from a carbon source, a nitrogen source, a phosphate source, a sulfur source, and other various organic components and inorganic components, if necessary, in addition to the medium-temperature treated product.
  • the type and concentration of the medium component may be appropriately set according to various conditions such as the type of bacteria used and the type of L-amino acid to be produced.
  • the medium-temperature treated product may or may not be used as the sole carbon source. That is, in L-amino acid fermentation, other carbon sources may be used in combination with the medium-temperature processed product.
  • Other carbon sources are not particularly limited as long as they can be assimilated by the bacterium of the present invention to produce L-amino acids. Specific examples of other carbon sources include glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, molasses, starch hydrolyzate, biomass hydrolyzate, and other sugars, acetic acid, fumaric acid, citric acid, etc.
  • the ratio of the carbon source derived from the medium-temperature processed product in the total carbon source is, for example, 5% by weight or more, 10% by weight or more, 20% by weight or more, preferably 30% by weight or more. More preferably, it may be 50% by weight or more.
  • one type of carbon source may be used, or two or more types of carbon sources may be used in combination.
  • the nitrogen source include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen sources such as peptone, yeast extract, meat extract, and soybean protein degradation product, ammonia, and urea.
  • Ammonia gas or ammonia water used for pH adjustment may be used as a nitrogen source.
  • the nitrogen source one kind of nitrogen source may be used, or two or more kinds of nitrogen sources may be used in combination.
  • the phosphoric acid source examples include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphoric acid.
  • phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate
  • phosphate polymers such as pyrophosphoric acid.
  • the phosphoric acid source one type of phosphoric acid source may be used, or two or more types of phosphoric acid sources may be used in combination.
  • the sulfur source include inorganic sulfur compounds such as sulfate, thiosulfate, and sulfite, and sulfur-containing amino acids such as cysteine, cystine, and glutathione.
  • the sulfur source one kind of sulfur source may be used, or two or more kinds of sulfur sources may be used in combination.
  • organic and inorganic components include, for example, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium and calcium; vitamin B1, vitamin B2, vitamin B6 and nicotine Examples include vitamins such as acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components such as peptone, casamino acid, yeast extract, and soybean protein degradation products containing these.
  • inorganic salts such as sodium chloride and potassium chloride
  • trace metals such as iron, manganese, magnesium and calcium
  • vitamin B1, vitamin B2, vitamin B6 and nicotine include vitamins such as acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components such as peptone, casamino acid, yeast extract, and soybean protein degradation products containing these.
  • vitamins such as acid, nicotinamide, and vitamin B12
  • amino acids amino acids
  • nucleic acids amino acids
  • organic components such as peptone, casamino acid, yeast extract, and soybean
  • L-lysine producing bacteria often have an enhanced L-lysine biosynthetic pathway and weakened L-lysine resolution. Therefore, when culturing such L-lysine-producing bacteria, for example, one or more amino acids selected from L-threonine, L-homoserine, L-isoleucine, and L-methionine are supplemented to the medium. Is preferred.
  • L-glutamic acid when L-glutamic acid is produced by coryneform bacteria, it is preferable to limit the amount of biotin in the medium, or to add a surfactant or penicillin to the medium. In order to suppress foaming during culture, it is preferable to add an appropriate amount of a commercially available antifoaming agent to the medium.
  • Culture conditions are not particularly limited as long as the bacterium of the present invention can grow and L-amino acids are produced.
  • the culture can be performed, for example, under normal conditions used for culture of microorganisms such as bacteria.
  • the culture conditions may be appropriately set according to various conditions such as the type of bacteria used and the type of L-amino acid to be produced.
  • Cultivation can be performed using a liquid medium.
  • the culture medium of the bacterium of the present invention cultured in a solid medium such as an agar medium may be directly inoculated into a liquid medium, or the bacterium of the present invention seeded in a liquid medium is used as a liquid for main culture.
  • the medium may be inoculated. That is, the culture may be performed separately for seed culture and main culture. In that case, the culture conditions of the seed culture and the main culture may or may not be the same.
  • the amount of the bacterium of the present invention 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.
  • Culture can be performed 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.
  • each medium component may be contained in the initial medium, the fed-batch medium, or both.
  • the type of component contained in the initial culture medium may or may not be the same as the type of component contained in the fed-batch medium.
  • concentration of each component contained in a starting culture medium may be the same as the density
  • the concentration of the medium-temperature treated product in the medium is not particularly limited as long as the bacterium of the present invention can use the medium-temperature treated product as a carbon source.
  • the medium temperature treatment product may be contained in the medium such that the fatty acid concentration in the medium is 10 w / v% or less, preferably 5 w / v% or less, more preferably 2 w / v% or less. Further, the medium temperature treatment product is, for example, such that the fatty acid concentration in the medium is 0.2 w / v% or more, preferably 0.5 w / v% or more, more preferably 1.0 w / v% or more. May be contained.
  • the intermediate temperature treatment product may be contained in the initial culture medium, the fed-batch medium, or both in the concentration range exemplified above.
  • the medium-temperature treated product When the medium-temperature treated product is contained in the fed-batch medium, the medium-temperature treated product has, for example, a fatty acid concentration in the medium after fed-batch of 5 w / v% or less, preferably 2 w / v% or less, more preferably You may contain in a feeding medium so that it may become 1 w / v% or less.
  • the medium-temperature treated product has, for example, a fatty acid concentration in the medium after fed-batch of 0.01 w / v% or more, preferably 0.02 w / v% or more. More preferably, it may be contained in the fed-batch medium so as to be 0.05 w / v% or more.
  • the medium-temperature treated product may be contained in the concentration range exemplified above when it is used only as a carbon source. Further, the intermediate-temperature treated product may be contained in the concentration range exemplified above when another carbon source is used in combination. In addition, when the intermediate temperature treated product is used in combination with another carbon source, for example, the concentration range obtained by appropriately correcting the concentration range exemplified above according to the ratio of the carbon source derived from the middle temperature treated product in the total carbon source, etc. It may be contained. In addition, you may apply mutatis mutandis when the description regarding the density
  • the medium-temperature treated product may or may not be contained in the medium in a certain concentration range during the entire culture period.
  • the medium-temperature processed product may be insufficient for some period. “Insufficient” means that the required amount is not satisfied.
  • the concentration in the medium may be zero.
  • Partial period refers to, for example, a period of 1% or less, a period of 5% or less, a period of 10% or less, a period of 20% or less, a period of 30% or less, or a period of the whole culture period, or It may be a period of 50% or less.
  • cultivation may mean the whole period of main culture, when culture
  • the culture can be performed aerobically, for example.
  • the culture can be performed by aeration culture or shaking culture.
  • the oxygen concentration may be controlled to, for example, 5 to 50%, preferably about 10% of the saturated oxygen concentration.
  • 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, magnesium hydroxide, etc. can do.
  • the culture temperature may be, for example, 20 to 45 ° C, preferably 25 ° C to 37 ° C.
  • the culture period may be, for example, 1 hour or more, 4 hours or more, 10 hours or more, or 15 hours or more, and may be 168 hours or less, 120 hours or less, 90 hours, or 72 hours or less. Specifically, 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 bacterium of the present invention is lost. By culturing the bacterium of the present invention under such conditions, L-amino acids accumulate in the cells and / or in the medium.
  • fed-batch culture or continuous culture fed-batch may be continued throughout the entire culture period or only during a part of the culture period.
  • multiple feedings may be performed intermittently.
  • the duration of one feeding is, for example, 30% or less, preferably 20% or less, more preferably 10% of the total time of the plurality of feedings.
  • the start and stop of fed batch may be repeated so that:
  • the second and subsequent feedings are controlled so that they are started when the carbon source in the fermentation medium is depleted in the immediately preceding feeding stop phase.
  • Carbon source depletion can be detected, for example, by increasing pH or increasing dissolved oxygen concentration.
  • extraction of the culture solution may be continued throughout the entire culture period, or may be continued only during a part of the culture period. Further, in continuous culture, a plurality of culture solutions may be extracted intermittently. Extraction and feeding of the culture solution may or may not be performed simultaneously. For example, the feeding may be performed after the culture solution is extracted, or the culture solution may be extracted after the feeding.
  • the amount of the culture solution to be withdrawn is preferably the same as the amount of the medium to be fed.
  • the “same amount” may be, for example, an amount of 93 to 107% with respect to the amount of medium to be fed.
  • the withdrawal may be started within 5 hours, preferably within 3 hours, more preferably within 1 hour after the start of fed-batch.
  • the bacterial cells can be reused by recovering L-amino acid from the extracted culture medium and recirculating the filtration residue containing the bacterial cells in the fermenter (French Patent No. 2669935). ).
  • L-glutamic acid when producing L-glutamic acid, it is also possible to carry out the culture while precipitating L-glutamic acid in the medium using a liquid medium adjusted to conditions under which L-glutamic acid is precipitated.
  • the conditions under which L-glutamic acid precipitates are, for example, pH 5.0 to 3.0, preferably pH 4.9 to 3.5, more preferably pH 4.9 to 4.0, and particularly preferably around pH 4.7. (European Patent Application Publication No. 1078989).
  • cultivation may be performed at the said pH in the whole period, and may be performed at the said pH only for a part of period.
  • 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.
  • a method of fermenting basic amino acid using bicarbonate ion and / or carbonate ion as a main counter ion of basic amino acid may be used.
  • basic amino acids can be produced while reducing the amount of sulfate ions and / or chloride ions that have been conventionally used as counter ions for basic amino acids.
  • L-amino acid can be confirmed by a known method used for detection or identification of a compound. Examples of such a method include HPLC, LC / MS, GC / MS, and NMR. These methods can be used in appropriate combination.
  • the produced L-amino acid can be recovered by a known method used for separation and purification of compounds. Examples of such a method include an ion exchange resin method, a membrane treatment method, a precipitation method, and a crystallization method. These methods can be used in appropriate combination. In the case where L-amino acid accumulates in the microbial cells, for example, the microbial cells are crushed with ultrasonic waves, and the microbial cells are removed by centrifugation from the supernatant obtained by ion exchange resin method or the like. Amino acids can be recovered. The recovered L-amino acid may be a free form, a salt thereof, or a mixture thereof.
  • Examples of the salt include sulfate, hydrochloride, carbonate, ammonium salt, sodium salt, and potassium salt.
  • L-lysine may be free L-lysine, L-lysine sulfate, L-lysine hydrochloride, L-lysine carbonate, or a mixture thereof.
  • L-glutamic acid may be free L-glutamic acid, sodium L-glutamate (MSG), ammonium L-glutamate (monoammonium L-glutamate), or a mixture thereof. .
  • ammonium L-glutamate in the fermentation broth is crystallized by adding an acid, and equimolar sodium hydroxide is added to the crystals to obtain sodium L-glutamate (MSG).
  • MSG sodium L-glutamate
  • you may decolorize by adding activated carbon before and after the crystallization see Industrial crystallization of sodium glutamate, Journal of the Seawater Society of Japan, Vol. 56, No. 5, Tetsuya Kawakita).
  • L-amino acid is precipitated in the medium, it can be recovered by centrifugation or filtration.
  • the L-amino acid precipitated in the medium may be isolated together after crystallization of the L-amino acid dissolved in the medium.
  • the recovered L-amino acid may contain components other than the L-amino acid, such as bacterial cells, medium components, water, and bacterial metabolic byproducts.
  • the purity of the recovered L-amino acid is, for example, 30% (w / w) or higher, 50% (w / w) or higher, 70% (w / w) or higher, 80% (w / w) or higher, 90% (W / w) or more, or 95% (w / w) or more.
  • Example 1 Acquisition of a high-accumulation strain of fatty acids of microalgae (1) Cultivation of water or soil samples Water or soil samples were collected from ponds, rivers, paddy fields, etc. in various parts of Japan.
  • the culture solution in which the growth of green algae was confirmed as described above was applied to a plate medium of 0.2 ⁇ Gamborg's B5 medium, and cultured for 2 weeks under the same conditions as described above except that it was not shaken. At that time, when preferential growth of contaminating bacteria was observed on the plate medium, the culture solution was sterilized by hypochlorous acid treatment. Specifically, after diluting a sodium hypochlorite solution having an effective chlorine concentration of 8.5 to 17.5% 100 times with sterilized water, the diluted solution is mixed with the culture solution so that the effective chlorine concentration is about 10 ppm, It was allowed to stand at room temperature for 10 minutes.
  • a sodium thiosulfate solution adjusted to 10,000 ppm was added to the medium so as to be about 10 times the amount of added effective chlorine.
  • the treated culture solution was applied to a plate medium of 0.2 ⁇ Gamborg's'B5 medium and cultured for 2 weeks.
  • a single colony was scraped with a platinum loop from a plate medium in which good growth of green algae was confirmed, applied to a plate medium of 0.2 ⁇ Gamborg's B5 medium, and further cultured for 2 weeks to obtain an algal isolate.
  • the two strains thus obtained were designated as AJ7847 strain (FERM BP-22253) and AJ7846 strain (FERM BP-22252).
  • 18S rDNA region amplification universal primers (primer set 1: SEQ ID NOs: 1 and 2) for green algae were used. Molecular phylogenetic analysis was performed using rDNA as an index. The sequenced 18S rDNA region sequences are shown in SEQ ID NO: 3 (AJ7847 strain) and SEQ ID NO: 4 (AJ7846 strain). Using these sequences, BLAST search from NCBI database (http://www.ncbi.nlm.nih.gov/Blast.cgi) was used to obtain highly homologous green alga-derived 18S rDNA sequence data. I made a tree.
  • ClustalX2 was used for creating multiple alignments
  • Sea View was used for editing
  • NJplot was used for displaying and editing phylogenetic trees.
  • the phylogenetic tree was created based on the ClustalX2 neighborhood join method, assuming that the bootstrap random number was 111 and the bootstrap number was 1000.
  • the obtained phylogenetic tree is shown in FIG. From these results, it was revealed that the AJ7847 and AJ7846 strains are related to the genus Desmodesmus. It was confirmed that the AJ7847 strain and the AJ7846 strain showed a high homology of more than 99% with respect to the known strains Desmodesmus armatus var. Subalternans CCAP 276 / 4A and Desmodesmus communis CCAP 276 / 4B.
  • Example 2 Cultivation evaluation of green algae strain (1) Cultivation of green algae strain A colony on a plate medium of an isolated green algae strain scraped with a platinum loop was added 50 mL of 0.2 ⁇ Gamborg's B5 medium. The cells were inoculated into mL Erlenmeyer flasks and cultured for one week. These cultures were seeded in a flask to which 10 mL of fresh 0.2 ⁇ Gamborg's B5 medium was added so that the turbidity at a wavelength of 750 nm immediately after the addition was 0.25.
  • the algal bodies used for the measurement of the amount of fatty acids were prepared as follows. 1 mL of the culture solution cultured by the method described in (1) above was dispensed into a 1.5 mL tube, frozen (-80 ° C, 30 minutes), and then incubated at 50 ° C for 20 hours. Next, centrifugation (12,000 rpm, 5 minutes) was performed to precipitate algal bodies. In addition, 1 mL of the culture solution was dispensed into a 1.5 mL tube, and the alga body precipitated by centrifugation (12,000 rpm, 5 minutes) was defined as an untreated alga body.
  • the obtained dried product was dissolved and diluted to an appropriate concentration using 2-propanol, and the fatty acid content was measured using a fatty acid colorimetric kit (Wako Pure Chemicals, LabAssay TM NEFA). The measurement was performed by measuring the absorbance using a 96-well microplate and an absorbance plate reader according to the protocol attached to the kit.
  • the isolated green algae strains, AJ7847 strain and AJ7846 strain both showed higher values of fatty acid production and fatty acid content per algal body than the related strains (Table 1). It should be noted that fatty acids were not detected in any strain under conditions using medium temperature treated untreated algal cells.
  • the present invention provides green algae that produce fatty acids.
  • the green algae can be used for the production of fatty acids, fatty acid esters, sugar glycerol, or combinations thereof.

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Abstract

　La présente invention concerne une algue verte qui produit un acide gras; et un procédé d'utilisation correspondant. Une algue verte telle que la souche AJ7846 (FERM BP -22252) ou la souche AJ7847 (FERM BP -22253), ou des souches dérivées de celles-ci, est mise en culture et au moyen d'un traitement approprié des phycobionts ainsi obtenus, un acide gras, un ester d'acide gras, un glycérol de sucre, ou une combinaison de ce qui précède, est produit.
PCT/JP2014/078791 2013-11-01 2014-10-29 Algue verte produisant un acide gras WO2015064648A1 (fr)

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