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WO2015005451A1 - Procédé de production de 2-désoxy-scyllo-inosose - Google Patents

Procédé de production de 2-désoxy-scyllo-inosose Download PDF

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Publication number
WO2015005451A1
WO2015005451A1 PCT/JP2014/068497 JP2014068497W WO2015005451A1 WO 2015005451 A1 WO2015005451 A1 WO 2015005451A1 JP 2014068497 W JP2014068497 W JP 2014068497W WO 2015005451 A1 WO2015005451 A1 WO 2015005451A1
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WIPO (PCT)
Prior art keywords
medium
doi
phytic acid
deoxy
inosose
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Ceased
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PCT/JP2014/068497
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English (en)
Japanese (ja)
Inventor
大輔 宮澤
松本　和也
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Mitsui Chemicals Inc
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Mitsui Chemicals Inc
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Priority to JP2015526412A priority Critical patent/JP6321645B2/ja
Publication of WO2015005451A1 publication Critical patent/WO2015005451A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)

Definitions

  • the present invention relates to a method for producing 2-deoxy-siro-inosose.
  • 2-Deoxy-siro-inosose (hereinafter also referred to as “DOI”) is a useful substance used as a raw material for pharmaceuticals, chemical industrial resources, and the like.
  • DOI 2-Deoxy-siro-inosose
  • G-6- glucose-6-phosphate
  • P recombinant DOI synthase obtained using E. coli.
  • WO 2006/109479 a method for producing DOI from D-glucose by fermentation using E. coli expressing DOI synthase has also been developed. Thereby, it became possible to produce DOI from D-glucose obtained from plant-derived resources.
  • Fermentation includes the cultivation of microorganisms and the production of substances using the microorganisms grown by the cultivation as a catalyst.
  • Nutrient sources are required for the cultivation and growth of microorganisms.
  • Natural microorganism components such as yeast extract and corn steep liquor (hereinafter also referred to as “CSL”) are often used for ordinary microorganism culture media.
  • CSL is a by-product obtained by immersing corn grains in a sulfite solution in the process of producing corn starch. Soluble proteins, peptides, amino acids, sugars, and vitamins eluted from corn grains are contained in CSL.
  • CSL includes a unique component produced by lactic acid fermentation that occurs during the immersion of corn grains in a sulfite solution, and is therefore a highly nutritious natural medium component.
  • CSL is widely used as an important medium component for microbial culture in the production of antibiotics, vitamins, amino acids, enzymes, and the like because it is less expensive than yeast extract and the like. Even in DOI fermentation production, good productivity can be obtained by adding 30 g / L of CSL (3 w / v%) to the medium.
  • natural medium components also contain many organic substances that are not necessary for the growth of Escherichia coli and the production of substances.
  • DOI-producing Escherichia coli produces DOI
  • if such organic matter is contained in a large amount in the culture solution the purification load of the culture solution increases, and the purification and recovery process for obtaining DOI becomes complicated. Therefore, it has become clear that it is necessary to limit the amount of natural medium components in the whole medium to, for example, 1 w / v% or less.
  • the amount of the natural medium component used is reduced in order to reduce the purification load of the culture solution, the nutrient source is reduced, and it can be expected that the growth of microorganisms is limited and the productivity deteriorates.
  • DOI-producing Escherichia coli can be cultured in a medium containing a natural medium component of 1 w / v% or less based on the total medium, and the DOI accumulated in the medium is recovered.
  • DOI production method capable of suppressing the number of steps and maintaining the same DOI productivity as when DOI-producing Escherichia coli was cultured in a medium containing about 3 w / v% of a natural medium component with respect to the whole medium, and the production method Is to provide the DOI produced by.
  • a report showing that the DOI-producing E. coli of DOI-producing E.
  • DOI productivity improves by adding a specific amount of pentoses separately from a growth carbon source.
  • the present inventors have a group consisting of a specific amount of phytic acid component and a specific amount of pentose and / or gluconic acid even if the amount of CSL used is reduced.
  • the present inventors have found a method capable of maintaining DOI productivity equivalent to the case where the amount of CSL used is not reduced by using a medium containing at least one selected from the above.
  • Phytic acid component selected from the group consisting of a molar amount of phytic acid hydrolyzate and 0.05 w / v% or more and 0.27 w / v% or less of phytic acid and an equimolar amount of phytic acid salt based on the total medium. ; (C) Equimolar amounts of 0.01 w / v% to 0.45 w / v% pentose with respect to the whole medium and 0.01 w / v% to 0.45 w / v% with respect to the whole medium A component selected from the group consisting of gluconic acid. [2] The production method according to [1], wherein the medium has a pH of 5 to 6.
  • DOI-producing Escherichia coli can be cultured in a medium containing a natural medium component of 1.0 w / v% or less based on the total medium, and the number of steps when collecting DOI accumulated in the medium.
  • DOI production that can maintain the same DOI productivity as when DOI-producing Escherichia coli is cultured in a medium containing about 3 w / v% of a natural medium component with respect to the total medium. DOI can be provided.
  • the method for producing 2-deoxy-siro-inosose (DOI) of the present invention comprises a natural medium component (a) of 1.0 w / v% or less based on the total medium, At least one selected from the group consisting of the following component (b) and component (c); 2-deoxy-siro-inosose-producing Escherichia coli is cultured in a medium containing 2-deoxy-siro-inosose to produce 2-deoxy-siro-inosose (hereinafter also referred to as “DOI production step”), and the 2-deoxy-shiro produced -Purifying and recovering inosource from the medium (hereinafter also referred to as "DOI purification and recovery step").
  • DOI production step a medium containing 2-deoxy-siro-inosose to produce 2-deoxy-siro-inosose
  • DOI purification and recovery step 2-deoxy-shiro produced
  • Phytic acid component selected from the group consisting of a molar amount of phytic acid hydrolyzate and 0.05 w / v% or more and 0.27 w / v% or less of phytic acid and an equimolar amount of phytic acid salt based on the total medium. .
  • DOI-producing Escherichia coli can be cultured in a medium containing a natural medium component of 1.0 w / v% or less based on the total medium, and the DOI accumulated in the medium is purified.
  • the number of steps at the time of recovery can be suppressed, and the productivity of DOI equivalent to that obtained when DOI-producing Escherichia coli is cultured in a medium containing about 3 w / v% of a natural medium component with respect to the entire medium can be maintained.
  • the term “process” is not limited to an independent process, and is included in the term if the intended purpose of the process is achieved even when it cannot be clearly distinguished from other processes. .
  • a numerical range indicated using “to” indicates a range including the numerical values described before and after “to” as the minimum value and the maximum value, respectively.
  • the amount of each component in the composition when there are a plurality of substances corresponding to each component in the composition, the plurality of substances present in the composition unless otherwise specified. Means the total amount.
  • total medium refers to a medium placed in a culture container in the case of batch culture, in the case of fed-batch culture, refers to a combination of the initial medium and fed-batch medium, and in the case of continuous culture. It refers to the medium in the culture tank.
  • inactivation means an enzyme measured by any existing measurement system (unless otherwise specified, “enzyme” in this specification includes “factor” which does not exhibit enzyme activity alone).
  • enzyme in this specification includes “factor” which does not exhibit enzyme activity alone.
  • the activity in DOI-producing Escherichia coli before inactivation is defined as 100, the activity is 1/10 or less.
  • the “reduction” of the enzyme activity in the present invention refers to a state in which the activity of the enzyme is significantly reduced as compared with the state before the treatment by the gene recombination technique of the gene encoding the enzyme.
  • the term “enhancement” of “activity” in the present invention broadly means that various enzyme activities in DOI-producing Escherichia coli before the enhancement increase after the enhancement. There are no particular limitations on the method of strengthening as long as the activity of various enzymes possessed by DOI-producing Escherichia coli is enhanced.
  • the enhancement by an enzyme gene introduced from outside the cell is carried out by introducing a gene encoding an enzyme having a higher activity than the host-derived enzyme into the cell from outside the host microorganism by gene recombination technology. Adding the enzyme activity of the introduced enzyme gene, or replacing this enzyme activity with the enzyme activity inherent in the host, and further increasing the number of host-derived enzyme genes or enzyme genes from outside the cell to 2 or more Increasing and combinations thereof may be mentioned.
  • the enhancement by enhancing the expression of the enzyme gene in the microorganism includes specifically introducing a base sequence that enhances the expression of the enzyme gene into the microorganism from outside the microorganism of the host microorganism, and the enzyme possessed by the host microorganism on the genome. Enhancing the expression of an enzyme gene by substituting the promoter of the gene with another promoter, and combinations thereof can be mentioned.
  • “Providing” “activity” in the present invention broadly means that an enzyme gene is introduced from the outside to give a target enzyme activity to a microorganism that cannot find the target enzyme gene.
  • the method of impartation is not particularly limited as long as the activity of the target enzyme is given to the microorganism, and includes transformation with a plasmid carrying the enzyme gene, introduction of the enzyme gene into the genome, and combinations thereof. it can.
  • the promoter used for “enhancement” or “giving” of “activity” is not particularly limited as long as it can express a gene, but a constitutive promoter or an inducible promoter can be used.
  • KEGG Knowhouse of Genes and Genomes; http://www.genome.jp/kegg/) or NCBI (Kizo Encyclopedia) National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/gene/), etc.
  • NCBI Know Encyclopedia National Center for Biotechnology Information
  • only the gene information of each strain registered in KEGG or NCBI is used.
  • the enzyme activity can be imparted, for example, by introducing a gene encoding the enzyme into the cell from the outside of the host bacterium by gene recombination technology.
  • the introduced enzyme gene may be derived from any of the same or different cells from the host cell.
  • the phrase “by gene recombination technology” or the like means that a change in the nucleotide sequence is caused by the insertion of another DNA into the nucleotide sequence of the native gene, or the substitution, deletion or combination of a part of the gene. Any occurrences may be included, for example, may be obtained as a result of mutation.
  • a microorganism in which the activity of a factor or enzyme is inactivated refers to a microorganism whose native activity has been impaired by some method from outside the cell to inside the cell.
  • These microorganisms can be produced, for example, by destroying a gene encoding the protein or enzyme (gene disruption).
  • the gene disruption includes a mutation in the base sequence of the gene, insertion of another DNA, and deletion of a certain part of the gene so that the function of the gene cannot be exhibited. Can be mentioned.
  • the gene cannot be transcribed into mRNA, and the structural gene is not translated. Or, since the transcribed mRNA is incomplete, the amino acid sequence of the translated structural protein is mutated or deleted, and the original function cannot be performed.
  • the gene-disrupted strain can be produced by any method as long as a disrupted strain that does not express the enzyme or protein is obtained.
  • Various methods of gene disruption have been reported (natural breeding, addition of mutagen, ultraviolet irradiation, radiation irradiation, random mutation, transposon, site-specific gene disruption, etc.), but only certain specific genes can be disrupted.
  • gene disruption by homologous recombination is preferable. Homologous recombination techniques are described in J. Bacteriol. 161, 1219-1221 (1985) and J. MoI. Bacteriol. , 177, 1511-1519 (1995), Proc. Natl. Acad. Sci. U. S. A, 97, 6640-6645 (2000), etc., and these methods and their applications can be easily implemented by engineers in the same industry.
  • the present invention will be described in more detail.
  • the DOI production method of the present invention comprises a natural medium component (a) of 1.0 w / v% or less based on the total medium, and at least one selected from the group consisting of the following component (b) and component (c): Culturing DOI-producing Escherichia coli in a medium containing, and having a DOI production step of producing DOI: (B) 0.05 w / v% to 0.27 w / v% phytic acid, 0.05% / 0.27 w / v% phytic acid, etc. to the whole medium, etc.
  • Phytic acid component selected from the group consisting of a molar amount of phytic acid hydrolyzate and 0.05 w / v% or more and 0.27 w / v% or less of phytic acid and an equimolar amount of phytic acid salt based on the total medium. ; (C) Equimolar amounts of 0.01 w / v% to 0.45 w / v% pentose with respect to the whole medium and 0.01 w / v% to 0.45 w / v% with respect to the whole medium A component selected from the group consisting of gluconic acid.
  • the method for culturing DOI-producing Escherichia coli is not particularly limited as long as it can produce DOI satisfactorily, and examples thereof include palindromic culture, continuous culture, and fed-batch culture.
  • Palindrical culture refers to a culture method in which a medium is inoculated into a medium and the medium is not added until the end of the culture.
  • Continuous culture refers to a culture method in which the medium is extracted from the culture container while continuously or intermittently feeding the medium to the culture container being cultured.
  • the fed-batch culture refers to a culture method in which a medium is fed continuously or intermittently to a culture container being cultured, and the medium is not extracted from the culture container until the end of the culture.
  • fed-batch culture or continuous culture reduces osmotic stress on DOI-producing Escherichia coli by lowering the sugar concentration in the culture solution, and continues to provide a fresh medium. This is preferable from the viewpoint of improving production efficiency.
  • the culture conditions can be appropriately set depending on the type, amount, culture apparatus, etc. of DOI-producing E. coli to be cultured.
  • the culture temperature is preferably 20 ° C. or higher and 40 ° C. or lower, and more preferably 25 ° C. or higher and 30 ° C. or lower.
  • the pressure in the culture tank during the culture is preferably normal pressure (0.1 MPa) to 0.5 MPa, more preferably normal pressure (0.1 MPa) to 0.2 MPa.
  • the pH of the medium during culture can be adjusted to pH 4 to 7, for example. By adjusting the pH of the medium within this range, the DOI production efficiency of DOI-producing E. coli can be improved. It is more preferable to adjust the pH of the medium so that the pH is 5-6.
  • the measurement of pH is not particularly limited, but can be performed by, for example, a pH meter (model number: HM-30V, manufactured by Toa DKK Corporation).
  • a culture tank In culturing, it is common to use a culture tank that can control temperature, pH, aeration conditions, stirring speed, etc., but in the DOI production method of the present invention, a culture tank is used for culturing. It is not limited. When culturing using a culture tank, if necessary, seed culture may be performed in advance as a preculture, and this may be inoculated into a medium in a culture tank prepared in advance.
  • Ventilation does not necessarily require air to pass through the culture medium, and depending on the shape of the culture tank, it also includes upper surface ventilation that allows the air layer above the culture liquid to be ventilated while appropriately stirring the culture liquid. Any gas may be used as long as oxygen-containing gas can flow into it.
  • the dissolved oxygen concentration changes depending on the combination of internal pressure, stirring blade position, stirring blade shape, stirring speed, etc., so the following is an index based on the productivity of DOI and the amount of organic acids other than DOI.
  • the optimum condition can be obtained.
  • stirring speed Desirable results can be obtained under aeration conditions that can be achieved at 50 rpm to 2000 rpm, more preferably 0.05 L / min to 1 L / min at normal pressure and a stirring speed of 100 rpm to 1000 rpm.
  • the ventilation conditions are not limited to this. Further, it is not necessary to keep the above-described aeration conditions constant from the beginning of culture to the end, and aeration may be performed as part of the culture process.
  • a fed-batch medium may be added to the initial medium.
  • the initial culture medium refers to a medium that is present in the culture vessel when inoculating bacteria and starting culture.
  • a fed-batch medium refers to a medium fed to a culture vessel or the like during culture. Conditions such as the timing, amount, flow acceleration and the like of adding the fed-batch medium are not particularly limited.
  • a fed-batch medium of 1 w / w% to 300 w / w% relative to the weight of the initial medium is added to the initial medium weight every hour. Of 0.1 w / w% to 10 w / w%.
  • the medium is 1.0% w / v or less natural medium component (a) with respect to the total medium; At least one selected from the group consisting of the following component (b) and component (c); Containing.
  • Phytic acid component selected from the group consisting of a molar amount of phytic acid hydrolyzate and 0.05 w / v% or more and 0.27 w / v% or less of phytic acid and an equimolar amount of phytic acid salt based on the total medium.
  • C Equimolar amounts of 0.01 w / v% to 0.45 w / v% pentose with respect to the whole medium and 0.01 w / v% to 0.45 w / v% with respect to the whole medium
  • the medium may contain an aqueous medium such as water necessary for growth of DOI-producing Escherichia coli, a carbon source, a nitrogen source, inorganic salts, and the like.
  • the medium is preferably sterilized and used for culture.
  • the method for sterilizing the medium is not limited as long as the medium can be sterilized without microorganisms having growth ability.
  • Examples of the sterilization method include sterilization methods such as autoclaving (autoclave; for example, heat sterilization at 121 ° C. for 20 minutes) or filtration sterilization (for example, filtration with a filter having a pore diameter of 0.45 ⁇ m or 0.2 ⁇ m).
  • autoclaving autoclave; for example, heat sterilization at 121 ° C. for 20 minutes
  • filtration sterilization for example, filtration with a filter having a pore diameter of 0.45 ⁇ m or 0.2 ⁇ m.
  • one or more medium components may be sterilized separately from the other medium components, and each may be mixed after sterilization.
  • the medium in the present invention contains 1.0 w / v% or less of the natural medium component (a) with respect to the total medium.
  • the natural medium component (a) refers to a component used when preparing a natural medium. Specifically, peptone (derived from milk, animal meat, fish meat, soy protein, etc.), yeast extract, meat extract, malt extract, corn steep liquor, tomato juice, oatmeal, fruit juice, vegetable juice, soy milk, animal milk, skim milk, cereal Ingredients derived from natural products such as enzyme digests of potatoes, beans, seaweed or mushrooms, and molasses. Above all, by using corn steep liquor as a natural medium component, growth of E. coli and DOI productivity culture can be performed efficiently.
  • the natural medium component (a) contained in the medium is 1.0 w / v% or less with respect to the total medium, because the number of steps in the DOI purification and recovery process can be suppressed. Moreover, it is more preferable that the natural medium component is 0.6 w / v% or less based on the total medium. Moreover, it is preferable from a viewpoint of favorable DOI productivity that the natural culture medium component contained in a culture medium is 0.1 w / v% or more with respect to the whole culture medium. In addition, the natural medium component is more preferably 0.3 w / v% or more based on the total medium. About the range of the content rate of the natural culture medium component contained in a culture medium, it can set suitably combining an upper limit and a lower limit.
  • the amount of the natural medium component (a) contained in the medium (at the start of main culture) is preferably 0 to 2.2 w / v%, more preferably 0 to 1.8 w / v%. 0 to 1.0 w / v% is more preferable.
  • the amount of the natural medium component (a) contained in the medium (at the start of main culture) should be 0.5 w / v% to 1.8 w / v%. Is more preferable.
  • the amount of the natural medium component (a) in the whole medium is preferably 22 g / L or less because a culture solution with a reduced purification load can be obtained. In continuous culture or fed-batch culture, the natural medium component (a) can be added to one or both of the initial medium and the fed-batch medium.
  • the culture medium in this invention contains at least 1 sort (s) chosen from the group which consists of the following phytic acid component (b) and component (c) other than a specific amount of natural culture medium component (a).
  • Phytic acid component (b) 0.05 w / v% or more and 0.27 w / v% or less of phytic acid relative to the whole medium, 0.05 w / v% or more and 0.27 w / v% or less of the whole medium Selected from the group consisting of phytic acid equimolar amounts of phytic acid hydrolyzate and 0.05 w / v% or more and 0.27 w / v% or less of phytic acid to equimolar amounts of phytic acid salt Phytic acid component.
  • the medium in the present invention is 0.05 w / v% or more and 0.27 w / v% or less of phytic acid relative to the whole medium, and 0.05 w / v% or more and 0.27 w / v% or less of phytin relative to the entire medium It is selected from the group consisting of an equimolar amount of phytic acid hydrolyzate in acid and 0.05 w / v% or more and 0.27 w / v% or less of phytic acid in an equimolar amount of phytic acid salt based on the total medium.
  • a phytic acid component (b) can be contained.
  • phytic acid component examples include phytic acid, a hydrolyzate of phytic acid, and a salt of phytic acid.
  • Phytic acid is a hexaphosphate ester of myo-inositol and is also called inositol hexakisphosphate.
  • the mixed salt of magnesium and calcium phytate is extremely insoluble in water and is called phytin. It is abundant in plant seeds and seedlings such as cereals and is known to be the main storage form of phosphate. ing.
  • Phytic acid is hydrolyzed by a kind of phosphatase, phytase, to produce myo-inositol and inorganic phosphate through a six-step reaction in which six phosphates are released one by one.
  • the hydrolyzate of phytic acid is not particularly limited as long as it is a hydrolyzate of phytic acid, but myo-inositol produced by hydrolyzing phytic acid and free phosphoric acid from the viewpoint of improving the productivity of DOI. And a mixture thereof. Further, it may be a partial hydrolyzate of phytic acid to which 1 to 5 phosphoric acids are bonded. In addition, if the phosphoric acid ester of myo-inositol in which 1 to 5 phosphoric acids are bound to myo-inositol, these are not actually obtained by hydrolysis, but are chemically synthesized or used in cereals, beans, fruits. It may be obtained from an extract from meat, fish or the like.
  • the hydrolyzate of phytic acid in an equimolar amount to phytic acid means that the number of moles of myo-inositol or its phosphate ester in the hydrolyzate of phytic acid is equal to the number of moles of phytic acid.
  • the salt of phytic acid refers to phytic acid bound with calcium, magnesium, potassium, sodium, or the like, and a mixed salt of two or more thereof.
  • a salt of phytic acid with a partial hydrolyzate is also included in the phytic acid salt in the present invention.
  • phytic acid component (b) phytic acid, a hydrolyzate of phytic acid, or a salt of phytic acid may be used alone, or two or more thereof may be used in combination.
  • the phytic acid component (b) is preferably one kind selected from the group consisting of phytic acid, a hydrolyzate of phytic acid, and a salt of phytic acid.
  • the total content of the phytic acid component (b) is 0.05 w / v% or more and 0.27 w / v% or less or 0.05 w with respect to the whole medium. It is preferable that the molar amount of phytic acid is not less than / v% and not more than 0.27 w / v%.
  • the content of phytic acid is preferably 0.02 w / v% or more and 0.30 w / v% or less, and 0.03 w / v% or more and 0.20 w / v% or less with respect to the whole medium. Is more preferably 0.05 w / v% or more and 0.10 w / v% or less.
  • the content of the hydrolyzate of phytic acid is preferably equimolar to 0.02 w / v% or more and 0.30 w / v% or less of phytic acid, and is 0.03 w / v% with respect to the whole medium.
  • it is equimolar to 0.20 w / v% or less of phytic acid, and even more preferably equimolar to 0.05 w / v% or more of 0.10 w / v% or less of phytic acid.
  • the content of phytic acid salt is preferably equimolar to 0.02 w / v% or more and 0.30 w / v% or less of phytic acid, and is 0.03 w / v% or more and 0 to 0%.
  • it is equimolar to 20 w / v% or less of phytic acid, more preferably equimolar to 0.05 w / v% or more and 0.10 w / v% or less of phytic acid.
  • the content of the phytic acid component is preferably 0.4 mmol / L or more and 5.0 mmol / L or less, more preferably 0.5 mmol / L or more and 3.0 mmol / L or less, and 0.74 mmol / L. More preferably, it is 1.5 mmol / L or less.
  • the content of the phytic acid component means the total amount of phytic acid, phytic acid hydrolyzate and phytic acid salt contained in the whole medium. If the content of the phytic acid component is 0.74 mmol / L or more with respect to the whole medium, it is preferable because there is an effect of improving DOI productivity, and if it is 1.5 mmol / L or less, the production cost becomes too high.
  • the content of the phytic acid component of 0.74 mmol / L or more and 1.5 mmol / L or less with respect to the whole medium is 0.05 w / v% or more and 0 with respect to the whole medium when converted to the phytic acid content. It falls under 27w / v%.
  • a phytic acid component 0.05 w / v% or more and 0.27 w / v% or less of phytic acid with respect to the whole medium, or 0.05 w / v% or more and 0.27 w / v% or less with respect to the whole medium.
  • Either an equimolar amount of phytic acid hydrolyzate in phytic acid, or 0.05 w / v% to 0.27 w / v% of phytic acid in an equimolar amount of phytic acid It is more preferable to contain 1 type.
  • a phytic acid component may be added to one or both of the initial medium and the fed-batch medium.
  • phytic acid may be added to one or both of the initial culture medium and the feed culture medium, but it is more preferable to add to the initial culture medium.
  • phytic acid is known to be a fermentation promoter.
  • Yeast utilizes myo-inositol produced by hydrolyzing phytic acid with phytase.
  • myo-inositol is an indispensable substance that constitutes a major phospholipid as phosphatidylinositol.
  • J. et al. Biosci. Bioeng. Vol. 98, no. 2,107-113, 2004 shows that the accumulation of myo-inositol in cells increases the phosphatidylinositol content of the cell membrane and makes it ethanol resistant.
  • the membrane of E. coli does not contain phosphatidylinositol.
  • the medium in the present invention is a pentose of 0.01 w / v% or more and 0.45 w / v% or less of the whole medium and a pentose of 0.01 w / v% or more and 0.45 w / v% or less of the whole medium.
  • a component (c) selected from the group consisting of equimolar amounts of gluconic acid can be contained.
  • the pentose that can be used in the DOI production method of the present invention is not particularly limited as long as it can be metabolized by the pentose phosphate pathway of Escherichia coli. Specific examples include D-xylose, L-arabinose, D-ribose and the like.
  • the content of pentose in the whole medium is preferably 0.01 w / v% or more and 0.45 w / v% or less, and 0.02 w / v% or more and 0.10 w / v% or less with respect to the whole medium. More preferably, it is 0.02 w / v% or more and 0.05 w / v% or less.
  • the content of pentose means the total amount of D-xylose, L-arabinose, D-ribose and the like contained in the whole medium.
  • the content of gluconic acid in the entire medium is preferably equimolar to the pentose of 0.01 w / v% or more and 0.45 w / v% or less, and 0.02 w / v% or more and 0 More preferably, it is equimolar to a pentose of 10 w / v% or less, and more preferably equimolar to a pentose of 0.02 w / v% or more and 0.05 w / v% or less.
  • “gluconic acid” includes gluconate such as sodium gluconate, potassium gluconate and calcium gluconate in addition to gluconic acid.
  • the content rate of gluconic acid means the total amount of gluconic acid, sodium gluconate, potassium gluconate, calcium gluconate, etc. which are contained in the whole culture medium. Any one type of pentose and / or gluconic acid may be used alone, or two or more types may be used in combination.
  • the total content of pentose and gluconic acid is preferably 0.01 w / v% or more and 0.45 w / v% or less with respect to the entire medium, and 0.02 w / v % Or more and 0.10 w / v% or less is more preferable, and 0.02 w / v% or more and 0.05 w / v% or less is more preferable.
  • pentose and / or gluconic acid may be added to one or both of the initial medium and the fed-batch medium.
  • pentose and / or gluconic acid may be added to one or both of the initial medium and the fed-batch medium, but more preferably added to the fed-batch medium.
  • those metabolized by the pentose phosphate pathway of E. coli such as D-xylose and L-arabinose can be used.
  • the concentration of pentose and / or gluconic acid in the fed-batch medium is preferably 0.02 w / v% or more, more preferably in the range of 0.03 w / v% to 1.00 w / v%, in terms of pentose concentration.
  • a range of 0.04 w / v% to 0.10 w / v% is more preferable.
  • DOI-producing Escherichia coli has reduced or inactivated activity of Zwf and cannot send glucose to the pentose phosphate pathway, but Metabolic Engineering Vol. 6, pp164-174, 2004 reveals that zwf-disrupted E. coli can grow in the same manner as the original E. coli even in a minimal medium by activating the tricarboxylic acid cycle.
  • DOI productivity is greatly increased by adding D-xylose, L-arabinose or gluconic acid to the feed medium at a low concentration of 0.05 w / v%. Improved.
  • the medium in the present invention comprises a specific amount of a natural medium component (a), 0.05 w / v% or more and 0.27 w / v% or less of phytic acid relative to the whole medium, 0.05 w / v relative to the whole medium.
  • the content ratio of the phytic acid component and the pentose in the whole medium should be 0.9: 1 to 6.8: 1 on a mass basis. Is preferable from the viewpoint of DOI productivity.
  • the content ratio of phytic acid component and pentose in the whole medium is more preferably 1: 1 to 4: 1 on a mass basis, and 1.2: 1 to 3.5. More preferably, the ratio is 1.
  • the content ratio of the phytic acid component and pentose and / or gluconic acid in the whole medium is based on a substance amount. From the viewpoint of DOI productivity, 0.2: 1 to 1.8: 1 is preferable.
  • the content ratio of phytic acid component and pentose and / or gluconic acid in the whole medium is 0.3: 1 to 1.6: 1 based on the amount of substance. More preferably, it is more preferably 0.4: 1 to 0.9: 1.
  • a carbon source other than pentose or gluconic acid (hereinafter also referred to as “other carbon source”) is required for DOI raw materials, growth of E. coli, and the like.
  • the medium used in the production method of the present invention can contain other carbon sources in addition to the above-mentioned natural medium component (a) and phytic acid component (b) and / or component (c).
  • Other carbon sources include sucrose, starch, D-glucose, D-fructose and the like.
  • glucose is preferable as the other carbon source from the viewpoint of sugar as a raw material for DOI.
  • sucrose can also be preferably used.
  • the medium in the present invention is an aqueous medium such as water necessary for the growth of DOI-producing Escherichia coli in addition to the above-mentioned natural medium component (a) and phytic acid component (b) and / or pentose or gluconic acid (component (c)).
  • You may contain an antifoamer etc. for preventing foaming of the culture medium during culture, such as a medium, a nitrogen source, inorganic salts, and vitamins.
  • Inorganic salts, vitamins, and the like may be included as necessary as long as they are suitable for the growth of E. coli.
  • Examples of the medium containing inorganic salts and vitamins include, but are not limited to, a composition such as a known M9 medium.
  • the sugar as a carbon source may be added to the initial medium or may be added to the feed medium.
  • sugar it is preferable to add it at a concentration that does not cause osmotic stress on E. coli.
  • sugar is added to the fed-batch medium to a concentration of 25 w / v% to 50 w / v% of the fed-batch medium, and fed-batch is started simultaneously with the start of the main culture. It is preferable to do.
  • Nitrogen sources include nitrogen atom-containing components, ammonium chloride, ammonium sulfate, ammonium acetate, phosphoric acid derived from natural products (microorganisms, plants, animal milk, animal meat, etc.) that are 1.0 w / v% or less based on the total medium.
  • Inorganic acids such as ammonium or ammonium salts of organic acids, ammonia, other amino acids, nitrogen compounds and the like can be mentioned.
  • a person skilled in the art can appropriately set the type and amount of the nitrogen source.
  • the inorganic salts phosphates, magnesium salts, calcium salts, iron salts, manganese salts, sulfates and the like are used.
  • examples include calcium.
  • These inorganic salts may be used alone or in a combination of two or more.
  • the concentration of inorganic salts in the medium is usually about 0.001 to 1.0% (wt), although it varies depending on the inorganic salt used.
  • vitamins can also be included as needed. Examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, nicotinic acid and the like.
  • the person skilled in the art can also set the types and amounts of other components added to the medium as appropriate. In addition, it is preferable to add a small amount of a few components because the purification load at the time of DOI recovery is expected to be small.
  • examples of the DOI-producing Escherichia coli include Escherichia coli provided with the ability to fermentatively produce DOI from a carbon source.
  • the carbon source is preferably sugar.
  • a recombinant E. coli having improved DOI production activity by enhancing, inactivating, reducing or combining these enzyme activities involved in the DOI production activity of E. coli by genetic recombination is used. It is preferable.
  • Examples of the recombinant E. coli having improved DOI production activity include the recombinant E. coli described in International Publication No. 2010/053052.
  • the DOI-producing E. coli is preferably a recombinant E. coli comprising the following gene recombination (i) and one or more gene recombination selected from the following (ii) to (iii).
  • recombinant E. coli further comprising the following gene recombination (iv) is more preferable because DOI can be produced from sucrose.
  • recombinant Escherichia coli further comprising the gene recombination of (v) below is more preferable because it can promote glucose uptake and improve DOI productivity.
  • BtrC in (i) means a general term for enzymes that catalyze the reaction of producing DOI from glucose-6-phosphate.
  • BtrC is preferably derived from a genus Bacillus, a bacterium belonging to the genus Streptomyces, or derived from Paenibacillus, from the viewpoint of high enzyme activity.
  • Pii of (ii) is classified into enzyme number 5.3.1.9 according to the report of the International Biochemical Union (I.U.B) Enzyme Committee, and glucose-6-phosphate to fructose-6- A generic term for enzymes that reversibly catalyze isomerization to phosphoric acid.
  • (Iii) Zwf is classified into enzyme number 1.1.1.149 according to the report of the International Biochemical Union (I.U.B) enzyme committee, and glucose-6- with NADP + as a coenzyme.
  • CscA is classified into enzyme number 3.2.1.26 according to the report of the International Biochemical Union (I.U.B) Enzyme Committee, and produces D-glucose and D-fructose from sucrose. It is a generic term for enzymes that catalyze hydrolysis reactions.
  • Glf in (v) refers to a glucose transport protein derived from Zymomonas bacteria that transports extracellular D-glucose into the cell.
  • the gene recombination imparting or enhancing DOI synthase (BtrC) activity may be any gene recombination that introduces a gene encoding 2-deoxy-siro-inosose synthase (BtrC) into E. coli. Thereby, the DOI production capability of Escherichia coli is imparted or enhanced.
  • the gene recombination imparted with sucrose hydrolase (CscA) activity may be a gene recombination that specifically introduces a gene encoding CscA into Escherichia coli.
  • the genetic recombination imparted with the glucose transport promoting protein (Glf) activity may be any genetic recombination that introduces a gene encoding Glf into E. coli. This enhances the DOI production capacity of E. coli.
  • DOI synthase is an enzyme that produces DOI using glucose-6-phosphate as a substrate. Therefore, DOI-producing Escherichia coli uses DOI-producing Escherichia coli that has been genetically modified to inactivate or reduce the activities of phosphoglucose isomerase (Pgi) and glucose-6-phosphate dehydrogenase (Zwf). This is preferable because DOI can be efficiently produced from glucose. In this case, the DOI-producing E. coli cannot use D-glucose as an energy source. Therefore, DOI can be satisfactorily produced by adding sugar as an energy source to the medium in addition to D-glucose.
  • Pgi phosphoglucose isomerase
  • Zwf glucose-6-phosphate dehydrogenase
  • Examples of sugars that serve as energy sources other than D-glucose include D-fructose and D-mannose.
  • D-fructose examples include D-fructose and D-mannose.
  • sucrose hydrolase CscA
  • DOI-producing Escherichia coli having a gene encoding sucrose hydrolase (CscA) may convert D-glucose, which is a hydrolysis product of sucrose, into DOI and use D-fructose as an energy source. This is preferable because it becomes possible.
  • Sucrose is a sugar obtained from sugarcane, sugar beet, etc.
  • crude sugar or waste molasses containing sucrose is an important biomass material such as a raw material for bioethanol.
  • Escherichia coli K12-derived strains, B-derived strains, etc. cannot originally hydrolyze sucrose, but genetically modified Escherichia coli introduced with a gene encoding sucrose hydrolase (CscA) produces DOI using raw sugar or molasses as a raw material. This is preferable because it becomes possible.
  • the DOI-producing Escherichia coli according to the present invention has an inherent phosphoglucose isomerase (Pgi) from the viewpoint of improving DOI productivity and improving DOI productivity that enables production from raw materials containing sucrose such as crude sugar and molasses. ) And glucose-6-phosphate dehydrogenase (Zwf) are inactivated or reduced, and a DOI-producing E. coli having a gene encoding sucrose hydrolase (CscA) is more preferable.
  • Pgi phosphoglucose isomerase
  • Zwf glucose-6-phosphate dehydrogenase
  • CscA sucrose hydrolase
  • the inoculation amount, the inoculation method, and the like of the DOI-producing E. coli medium are not particularly limited.
  • the DOI production method of the present invention has a DOI purification / recovery step in which the produced 2-deoxy-siro-inosose is purified from the medium and recovered.
  • the purification and recovery step recovers the DOI obtained in the DOI production step, and is generally performed by purifying and recovering the DOI accumulated in the culture from the culture.
  • the culture in the present invention is a product obtained by culture, and specifically includes a culture medium, cultured DOI-producing E. coli, DOI produced by DOI-producing E. coli, and the like. Moreover, a culture solution etc. are mentioned as an example of a culture.
  • a conventionally known method can be used from a culture solution. For example, after microbial cells are removed by centrifugation or the like, the culture supernatant is treated with activated carbon to remove proteins, polymer components, etc., and the treatment solution is added to an ion exchange resin and eluted with distilled water. Further, fractions not containing impurities can be collected while measuring refractive index, pH, conductivity, etc., and the aqueous solution can be removed to recover DOI. According to the present invention, the amount of activated carbon or the amount of ion exchange resin used in the DOI purification and recovery step can be reduced by reducing the purification load.
  • the DOI of the present invention comprises 1.0% w / v or less natural medium component (a) relative to the whole medium, 0.05% w / v to 0.27w / v% phytic acid relative to the total medium, Equal molar amount of phytic acid hydrolyzate to 0.05 w / v% or more and 0.27 w / v% or less of phytic acid and 0.05 w / v% to 0.27 w / A phytic acid component (b) selected from the group consisting of equimolar amounts of a phytic acid salt to v% or less of phytic acid, and 0.01 w / v% or more and 0.45 w / v% or less of the whole medium At least one selected from the group consisting of component (c) selected from the group consisting of pentose of 0.01 w / v% or more and 0.45 w / v% or less of pen
  • a medium containing 2-deoxy-shiro-inosose It is obtained by a method for producing 2-deoxy-siro-inosose comprising culturing production E. coli to produce 2-deoxy-siro-inosose and recovering the produced 2-deoxy-siro-inosose from the medium. .
  • culturing production E. coli to produce 2-deoxy-siro-inosose and recovering the produced 2-deoxy-siro-inosose from the medium.
  • % means w / v%.
  • CAGGAATTCG CTATATCTGGG CTCTGCACG SEQ ID NO: 1
  • CAGTCTAGAG CAATACTCTT CTGATTGCGA SEQ ID NO: 2
  • CAGTCTAGCATCGTGTC Four types of oligonucleotide primers having the base sequence of 4) were synthesized.
  • the primer of SEQ ID NO: 1 has an EcoRI recognition site on the 5 ′ end side
  • the primers of SEQ ID NOS: 2 and 3 have an XbaI recognition site on the 5 ′ end side
  • the primer of SEQ ID NO: 4 has a PstI recognition site on the 5 ′ end side.
  • Escherichia coli B strain genomic DNA was prepared by the method described in Current Protocols in Molecular Biology (JohnWiley & Sons). Using the obtained genomic DNA as a template, a combination of a primer having the base sequence of SEQ ID NO: 1 and a primer having the base sequence of SEQ ID NO: 2, and a primer having the base sequence of SEQ ID NO: 3 and the base sequence of SEQ ID NO: 4 A DNA fragment of about 1.0 kb (hereinafter sometimes referred to as a pgi-L fragment and a pgi-R fragment) was amplified by performing PCR under normal conditions in combination with the primers having them. These DNA fragments were separated and collected by agarose electrophoresis.
  • the pgi-L fragment was digested with EcoRI and XbaI, and the pgi-R fragment was digested with XbaI and PstI, respectively.
  • the two digested fragments were mixed with EcoRI and PstI digests of the temperature sensitive plasmid pTH18cs1 (GenBank accession number AB019610), reacted with T4 DNA ligase, and transformed into Escherichia coli DH5 ⁇ competent cell (manufactured by Takara Bio Inc.). After conversion, a plasmid containing two fragments, the 5 ′ upstream vicinity fragment and the 3 ′ downstream vicinity fragment of pgi, was obtained. The resulting plasmid was named pTH ⁇ pgi.
  • This LB agar plate was cultured at 42 ° C. where no temperature-sensitive plasmid could be maintained, and the grown transformant was obtained as a strain in which the entire length of the plasmid was integrated into the Escherichia coli chromosome by extrachromosomal-chromosomal homologous recombination.
  • Genomic DNA was obtained from this strain, PCR was performed using this as a template, and the presence of the chloramphenicol resistance gene of pTH18cs1 on the chromosome, as well as the 5 ′ upstream vicinity region of pgi, and 3 ′ downstream The presence of a region homologous to each of the neighboring regions on the chromosome confirmed that the entire plasmid was a strain integrated into the Escherichia coli chromosome.
  • a strain in which the entire length of the plasmid was incorporated into the Escherichia coli chromosome was planted in a 100 ml baffled flask containing 20 ml of LB liquid medium not containing chloramphenicol, and this was cultured with shaking at 30 ° C. for 4 hours.
  • This culture solution was diluted with an LB liquid medium not containing chloramphenicol and spread on an LB agar medium not containing chloramphenicol.
  • 96 colonies grown at 42 ° C. were randomly selected and grown on LB agar medium containing no chloramphenicol or LB agar medium containing chloramphenicol, Chloramphenicol sensitive strains were selected.
  • genomic DNA was obtained from the selected strain, PCR was performed using this as a template, a strain lacking pgi was selected, and this was designated as a B ⁇ pgi strain.
  • CAGGAATTCA TGCGTTGCAG CACGATATC SEQ ID NO: 5
  • CAGTCTAGAT AACCCCGGTAC TTAAGCCAG SEQ ID NO: 6
  • the primer of SEQ ID NO: 5 has an EcoRI recognition site on the 5 ′ end side
  • the primers of SEQ ID NOS: 6 and 7 have an XbaI recognition site on the 5 ′ end side
  • the primer of SEQ ID NO: 8 has a PstI recognition site on the 5 ′ end side.
  • a DNA fragment of about 0.85 kb hereinafter sometimes referred to as zwf-L fragment
  • zwf-R 1.0 kb
  • the zwf-L fragment was digested with EcoRI and XbaI and the zwf-R fragment was digested with XbaI and PstI, respectively.
  • the two digested fragments were mixed with EcoRI and PstI digests of the temperature sensitive plasmid pTH18cs1, reacted with T4 DNA ligase, transformed into Escherichia coli DH5 ⁇ competent cell (manufactured by Takara Bio Inc.), and zwf
  • the resulting plasmid was named pTH ⁇ zwf.
  • Genomic DNA was obtained from this strain, PCR was performed using this as a template, and the chloramphenicol resistance gene of pTH18cs1 was present on the chromosome, and the region near the 5 ′ upstream of zwf and 3 ′ downstream The presence of a region homologous to each of the neighboring regions on the genome confirmed that the entire plasmid length was integrated into the Escherichia coli chromosome.
  • a strain in which the entire length of the plasmid was incorporated into the Escherichia coli chromosome was planted in a 100 ml baffled flask containing 20 ml of LB liquid medium not containing chloramphenicol, and this was cultured with shaking at 30 ° C. for 4 hours.
  • This culture solution was diluted with an LB liquid medium not containing chloramphenicol and spread on an LB agar medium not containing chloramphenicol.
  • 96 colonies grown at 42 ° C. were randomly selected and grown on LB agar medium containing no chloramphenicol or LB agar medium containing chloramphenicol, Chloramphenicol sensitive strains were selected.
  • genomic DNA was obtained from the selected strain, PCR was performed using this as a template, a strain lacking zwf was selected, and this was designated as B ⁇ pgi ⁇ zwf strain.
  • the primer of SEQ ID NO: 9 has an NdeI recognition site on the 5 ′ end side
  • the primer of SEQ ID NO: 10 has a HindIII, XhoI, SalI, BamHI, and SacI recognition site in this order from the 5 ′ end side.
  • the above DNA fragment was mixed with the Escherichia coli DH5 ⁇ competent cell (manufactured by Takara Bio Inc.) after mixing with the above-mentioned DNA fragment and the cloning vector pBR322 (GenBank accession number J01749) digested with NdeI and HindIII and using ligase.
  • pBR322 GenBank accession number J01749
  • the obtained colony was cultured overnight at 30 ° C. in an LB liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid was recovered from the obtained cells.
  • This plasmid was named pGAP.
  • the base sequence of the DOI synthase gene (btrC) possessed by Bacillus circulans has already been reported (GenBank accession number AB097196).
  • oligonucleotide primers having the base sequences of CACTGGAGCT CCGTGGGTGGA ATATAGACG ACTAAAACAA TTTG (SEQ ID NO: 11) and CAGGATCCTT ACAGCCCTTC (SEQ ID NO: 12) were respectively synthesized.
  • the primer of SEQ ID NO: 11 has a SacI recognition site and a 13-base GAPDH gene ribosome binding sequence in this order from the 5 ′ end.
  • the primer of SEQ ID NO: 12 has a BamHI recognition site on the 5 ′ end side.
  • a PCR method was performed under normal conditions using the Bacillus circulans genomic DNA as a template together with the above two types of primers, and the obtained DNA fragment was digested with restriction enzymes SacI and BamHI to obtain an about 1.1 kbp DOI synthase gene (btrC). ) Fragment was obtained. This DNA fragment was mixed with a fragment obtained by digesting plasmid pGAP with restriction enzymes SacI and BamHI, ligated with ligase, and then transformed into Escherichia coli DH5 ⁇ competent cell (manufactured by Takara Bio Inc.).
  • a transformant that grew on an LB agar plate containing 50 ⁇ g / mL of ampicillin was obtained.
  • the obtained colony was cultured overnight at 30 ° C. in an LB liquid medium containing 50 ⁇ g / mL of ampicillin, and plasmid pGAP-btrC was recovered from the obtained bacterial cells to construct a DOI synthase gene (btrC) expression vector. .
  • sucrose hydrolase gene (cscA) ⁇ Construction of DOI synthase gene (btrC) and sucrose hydrolase gene (cscA) expression vectors under the control of the GAPDH promoter>
  • the base sequence of the sucrose hydrolase gene (cscA) possessed by Escherichia coli O-157 has already been reported. That is, it is described in the Escherichia coli O-157 strain genomic sequence 3274383-3275816 described in GenBank accession number AE005174.
  • oligonucleotide primers having the base sequences of GCGGATCGCC TGGTGGAATA TATGACGCAA TCTCGATTGC (SEQ ID NO: 13) and GACGCGTCGA CTTAACCCAG TTGCCAGAGT GC (SEQ ID NO: 14) were synthesized.
  • the primer of SEQ ID NO: 13 has a BamHI recognition site and a 13-base GAPDH gene ribosome binding sequence in this order from the 5 ′ end.
  • the primer of SEQ ID NO: 14 has a SalI recognition site on the 5 ′ end side.
  • PCR is performed under normal conditions using the above two primers together with genomic DNA of Escherichia coli O-157 strain (SIGMA-ALDRICH: IRMM449) as a template, and the resulting DNA fragment is digested with restriction enzymes BamHI and SalI.
  • the sucrose hydrolase gene (cscA) fragment of about 1.4 kbp was obtained.
  • This DNA fragment was mixed with a fragment obtained by digesting plasmid pGAP-btrC with restriction enzymes BamHI and SalI, ligated with ligase, and transformed into Escherichia coli DH5 ⁇ competent cell (manufactured by Takara Bio Inc.).
  • a transformant that grew on an LB agar plate containing 50 ⁇ g / mL of ampicillin was obtained.
  • the obtained colonies were cultured overnight in an LB liquid medium containing 50 ⁇ g / mL of ampicillin at 30 ° C., and the plasmid pGAP-btrC-cscA was recovered from the obtained bacterial cells to obtain DOI synthase gene (btrC) and sucrose hydrolysate.
  • a degrading enzyme gene (cscA) expression vector was constructed.
  • oligonucleotide primers having the base sequences of CCTGTCGACG CTGGTGGAAT ATATGAGTTC TGAAAGTAGT CAGG (SEQ ID NO: 15) and CTACTCGAGC TACTTCTGGG (AGCGCCCACA (SEQ ID NO: 16)) were synthesized.
  • the primer of SEQ ID NO: 15 has a SalI recognition site and a 13-base GAPDH gene ribosome binding sequence in this order from the 5 ′ end.
  • the primer of SEQ ID NO: 16 has an XhoI recognition site on the 5 ′ end side.
  • a PCR method was performed under normal conditions using Zymomonas mobilis genomic DNA as a template together with the above two types of primers, and the obtained DNA fragment was digested with restriction enzymes SalI and XhoI to give a glucose transport-promoting protein gene of about 1.4 kbp ( glf) fragment was obtained.
  • This DNA fragment and the fragment obtained by digesting plasmid pGAP-btrC-cscA with restriction enzymes SalI and XhoI were mixed, ligated using ligase, and then transferred to Escherichia coli DH5 ⁇ competent cell (manufactured by Takara Bio Inc.).
  • the transformant was obtained by transforming and growing on an LB agar plate containing 50 ⁇ g / mL of ampicillin. The obtained colony was cultured overnight at 30 ° C. in an LB liquid medium containing 50 ⁇ g / mL of ampicillin, and the plasmid pGAP-btrC-cscA-glf was recovered from the obtained bacterial cells to obtain a DOI synthase gene (btrC), Sucrose hydrolase gene (cscA) and glucose transport promoting protein gene (glf) expression vectors were constructed.
  • btrC DOI synthase gene
  • cscA Sucrose hydrolase gene
  • glf glucose transport promoting protein gene
  • a 1 L culture tank (culture device BMJ-01 manufactured by ABLE) containing 350 g of the initial medium (CSL reduction medium) having the composition shown in Table 1 is sterilized, and 20 mL of the preculture solution is transferred to the culture tank (main culture). Started. With the start of culture, a fed-batch medium having the composition shown in Table 2 was fed at a rate of 0.1 g / min.
  • the fed-batch medium was prepared by separately sterilizing a glucose solution, a fructose solution, a solution containing potassium dihydrogen phosphate, ammonium sulfate, and sodium chloride, a magnesium sulfate solution, a calcium chloride solution, and a thiamine hydrochloride solution, respectively.
  • Example 1 DOI production by adding phytic acid to the initial medium Comparative Example except that phytic acid was added to the initial medium having the composition shown in Table 1 so that the concentration was 0.14 w / v% (2.11 mmol / L). The culture and the DOI accumulation amount were measured in the same manner as in 1. The accumulated DOI concentration at 48 hours was 66 g / L.
  • Example 2 D-xylose added to fed-batch medium to produce DOI As in Comparative Example 1, except that D-xylose was added to the fed-batch medium having the composition shown in Table 2 so that the concentration was 0.05 w / v%. Culture and DOI accumulation were measured. The DOI accumulation concentration at 48 hours was 58 g / L.
  • Example 3 Production of DOI by adding phytic acid to the initial medium and D-xylose to the fed-batch medium.
  • Phytic acid was added to the initial medium having the composition shown in Table 1 so that the concentration was 0.14 w / v%.
  • Culture and measurement of DOI accumulation were performed in the same manner as in Comparative Example 1 except that D-xylose was added to the feed medium having the composition of 2 so that the concentration was 0.05 w / v%.
  • the accumulated DOI concentration at 48 hours was 72 g / L.
  • Comparative Example 1 The results of Comparative Example 1, Examples 1 to 3 and Reference Example 1 are shown in Table 5.
  • DOI productivity decreased, but phytic acid was fed into the initial medium or D-xylose was fed. It was confirmed that by adding to the medium, productivity equivalent to that produced when the CSL concentration of the initial medium and the fed-batch medium was 3 w / v% was obtained.
  • Example 4 Production of DOI by adding phytic acid to fed-batch medium Cultured in the same manner as Comparative Example 1 except that phytic acid was added to the fed-batch medium having the composition shown in Table 2 so that the concentration was 0.14 w / v%. And the DOI accumulation amount was measured. The DOI accumulation concentration at 48 hours was 55 g / L. It was confirmed that phytic acid can obtain a greater effect when added to the initial culture medium than when added to the feed medium.
  • Example 5 Phytic acid and D-xylose are added to the initial medium to produce DOI.
  • the initial medium having the composition shown in Table 1 has phytic acid and 0.05 w / v% so as to be 0.14 w / v%.
  • culture and measurement of DOI accumulation were performed in the same manner as in Comparative Example 1 except that D-xylose was added.
  • the accumulated DOI concentration at 48 hours was 68 g / L. It was confirmed that D-xylose had a greater effect when added to the feed medium.
  • Example 6 DOI production by adding L-arabinose to fed-batch medium The same as Comparative Example 1 except that L-arabinose was added to the fed-batch medium having the composition shown in Table 2 at 0.05 w / v%. The culture and the amount of accumulated DOI were measured. The DOI accumulation concentration at 48 hours was 50 g / L. Similar to D-xylose, L-arabinose was also confirmed to improve productivity. From this result, it is considered that the same effect can be obtained even when pentose metabolized by the pentose phosphate pathway such as D-ribose and D-ribulose is added.
  • Example 7 Phytic acid was added to the initial medium, D-xylose was added to the feed medium, and DOI production. D-xylose was added to the feed medium having the composition shown in Table 2 to 0.025 w / v%. Except for the addition, the culture and the DOI accumulation amount were measured in the same manner as in Example 1. The accumulated DOI concentration at 48 hours was 66 g / L. In order to obtain higher productivity, it was confirmed that the concentration of D-xylose in the feed medium is preferably higher than 0.025 w / v%.
  • Example 8 Phytic acid was added to the initial medium, and D-xylose was added to the fed-batch medium to produce 0.09 w / v% (1.35 mmol / L) in the initial medium having the composition shown in Table 1.
  • culture and measurement of DOI accumulation amount were performed in the same manner as in Example 2 except that phytic acid was added.
  • the accumulated DOI concentration at 48 hours was 65 g / L.
  • the concentration of phytic acid in the initial medium is preferably higher than 0.09 w / v%.
  • Example 9 Myo-inositol and phosphate were added to the initial medium to produce DOI.
  • Phytine added to the initial medium having the composition shown in Table 1 at 0.14 w / v% (2.11 mmol / L).
  • the DOI accumulation concentrations at 48 hours were 47 g / L, 54 g / L, and 68 g / L, respectively. It was confirmed that when both myo-inositol and potassium dihydrogen phosphate were added, the same effects as when phytic acid was added were obtained.
  • Example 10 Calcium phytate was added to the initial medium, D-xylose was added to the feed medium, and DOI production was added to the initial medium having the composition shown in Table 1 at an equimolar concentration with 0.14 w / v% phytic acid. Except that calcium phytate was added, culture and measurement of DOI accumulation amount were performed in the same manner as in Example 2. The DO accumulation concentration at 48 hours was 70 g / L. It was confirmed that calcium phytate can achieve the same effect as phytic acid.
  • Example 11 Calcium phytate was added to the initial medium to produce DOI.
  • the DO accumulation concentrations at 48 hours were 62 g / L and 57 g / L, respectively.
  • Example 12 D-xylose was added to a fed-batch medium to produce DOI.
  • D-- was added to a fed-batch medium having the composition shown in Table 2 so that the concentration was 0.5 w / v% or 1.0 w / v%.
  • Culture and measurement of DOI accumulation were performed in the same manner as in Comparative Example 1 except that xylose was added.
  • the DO accumulation concentrations at 48 hours were 66 g / L and 58 g / L, respectively.
  • Example 13 DOI production by adding gluconic acid to fed-batch medium Except for adding gluconic acid to the fed-batch medium shown in Table 2 so as to have an equimolar concentration with 0.05 w / v% D-xylose.
  • the culture and the DOI accumulation amount were measured.
  • the DOI accumulation concentration at 48 hours was 63 g / L.
  • D-xylose and L-arabinose the effect of improving productivity was confirmed when gluconic acid was used.
  • Example 14 Phytic acid was added to the initial medium, D-xylose was added to the feed medium, and DOI was produced from sucrose. A feed medium having the composition shown in Table 6 was used, and 0.05 w / v% was used as the feed medium. In the same manner as in Example 1 except that D-xylose was added so that the pH of the culture was 5.5, and the amount of accumulated DOI was measured. The accumulated DOI concentration at 48 hours was 52 g / L. Even when sucrose was used as a raw material, it was confirmed that DOI could be produced satisfactorily in a CSL-reducing medium supplemented with phytic acid and D-xylose.
  • Example 15 Crude purification of DOI
  • sulfuric acid was added to the culture solution obtained in Example 3 or the culture solution obtained in Reference Example 1 to adjust the pH to 3.0. Centrifugation was performed at 8000 ⁇ g for 20 minutes in order to remove the cells.
  • 0.5 w / w% activated carbon of the centrifugation supernatant was added and stirred. In order to remove the activated carbon, it was filtered using a filter paper and a 0.45 ⁇ m membrane filter.
  • the transmittance at a wavelength of 400 nm of pure water in the spectrophotometer is 100%
  • the transmittance at a wavelength of 400 nm of the filtrate after removal of activated carbon is 40% in the filtrate of the culture solution obtained in Example 3.
  • the filtrate of the culture solution obtained in Reference Example 1 was 8%. It was confirmed that the culture solution obtained by the DOI production method according to the present invention has a small purification load.

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Abstract

　La présente invention concerne un procédé de production de 2-désoxy-scyllo-inosose consistant à mettre en culture des Escherichia coli productrices de 2-désoxy-scyllo-inosose dans un milieu contenant au moins un constituant choisi parmi (a) un constituant de milieu de culture naturel en quantité spécifique par rapport au milieu de culture complet, (b) un constituant tel que l'acide phytique contenu en quantité spécifique par rapport au milieu de culture complet et (c) un constituant tel que le pentose contenu en quantité spécifique par rapport au milieu de culture complet, afin de produire le 2-désoxy-scyllo-inosose ; et à récupérer le 2-désoxy-scyllo-inosose ainsi produit à partir du milieu.
PCT/JP2014/068497 2013-07-12 2014-07-10 Procédé de production de 2-désoxy-scyllo-inosose Ceased WO2015005451A1 (fr)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
WO2020085435A1 (fr) 2018-10-25 2020-04-30 株式会社Ihi Procédé de production de trihydroxybenzène
WO2020085436A1 (fr) * 2018-10-25 2020-04-30 株式会社Ihi Système de production de trihydroxybenzène

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WO2006112000A1 (fr) * 2005-03-30 2006-10-26 The Niigata Institute Of Science And Technology Procede permettant de synthetiser un 2-deoxy-scyllo-inosose au moyen d'une souche e. coli modifiee, procede de purification correspondant et 2-deoxy-scyllo-inosose ainsi obtenu
WO2010053052A1 (fr) * 2008-11-05 2010-05-14 三井化学株式会社 Bactérie apte à produire du 2-désoxy-scyllo-inosose (doi) et procédé pour la production de 2-désoxy-scyllo-inosose (doi) l'utilisant
WO2010109916A1 (fr) * 2009-03-26 2010-09-30 旭化成ケミカルズ株式会社 Nouvelle 2-désoxy-scyllo-inosose synthase

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WO2006112000A1 (fr) * 2005-03-30 2006-10-26 The Niigata Institute Of Science And Technology Procede permettant de synthetiser un 2-deoxy-scyllo-inosose au moyen d'une souche e. coli modifiee, procede de purification correspondant et 2-deoxy-scyllo-inosose ainsi obtenu
WO2010053052A1 (fr) * 2008-11-05 2010-05-14 三井化学株式会社 Bactérie apte à produire du 2-désoxy-scyllo-inosose (doi) et procédé pour la production de 2-désoxy-scyllo-inosose (doi) l'utilisant
WO2010109916A1 (fr) * 2009-03-26 2010-09-30 旭化成ケミカルズ株式会社 Nouvelle 2-désoxy-scyllo-inosose synthase

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YAMAUCHI N. ET AL.: "Biochemical studies on 2- deoxy-scyllo-inosose, an early intermediate in the biosynthesis of 2-deoxystreptamine", J. ANTIBIOT., vol. 5, no. 5, pages 756 - 766 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020085435A1 (fr) 2018-10-25 2020-04-30 株式会社Ihi Procédé de production de trihydroxybenzène
WO2020085436A1 (fr) * 2018-10-25 2020-04-30 株式会社Ihi Système de production de trihydroxybenzène
JP2020065494A (ja) * 2018-10-25 2020-04-30 株式会社Ｉｈｉプラントエンジニアリング トリヒドロキシベンゼンを製造するためのシステム
JP7260872B2 (ja) 2018-10-25 2023-04-19 株式会社Ihi トリヒドロキシベンゼンを製造するためのシステム
US11685704B2 (en) 2018-10-25 2023-06-27 Mitsui Chemicals, Inc. Trihydroxybenzene production method

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