WO2017002978A1 - Procédé de production de sucre rare - Google Patents

Procédé de production de sucre rare Download PDF

Info

Publication number
WO2017002978A1
WO2017002978A1 PCT/JP2016/069724 JP2016069724W WO2017002978A1 WO 2017002978 A1 WO2017002978 A1 WO 2017002978A1 JP 2016069724 W JP2016069724 W JP 2016069724W WO 2017002978 A1 WO2017002978 A1 WO 2017002978A1
Authority
WO
WIPO (PCT)
Prior art keywords
microorganism
phosphate
protein
psicose
activity
Prior art date
Application number
PCT/JP2016/069724
Other languages
English (en)
Japanese (ja)
Inventor
祐子 島並
田畑　和彦
Original Assignee
協和発酵バイオ株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 協和発酵バイオ株式会社 filed Critical 協和発酵バイオ株式会社
Priority to JP2017526463A priority Critical patent/JP6993227B2/ja
Publication of WO2017002978A1 publication Critical patent/WO2017002978A1/fr
Priority to JP2021197204A priority patent/JP7244613B2/ja

Links

Images

Classifications

    • 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
    • C12P19/02—Monosaccharides
    • 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
    • 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

Definitions

  • the present invention relates to an efficient production method of a rare sugar using a microorganism, a culture of the microorganism, or a processed product of the culture, and more particularly to a production method of D-psicose and a production method of D-alloheptulose.
  • rare sugars are defined as “sugars that rarely exist in nature” and are simple sugars with a small abundance in nature. In general, rare sugars are often low in yield in a synthetic reaction in an organic chemical synthesis method.
  • D-psicose a kind of rare sugar, has about 70% of the sweetness of sugar but has almost no calories, so it can be used as a low calorie sweetener.
  • an inhibitory action on blood pressure increase and an inhibitory action on visceral fat accumulation have been recognized.
  • Patent Document 1 discloses a method for producing psicose using a chemical isomerization reaction.
  • Patent Documents 2 and 3 and Non-Patent Documents 1 to 4 disclose enzymes related to biosynthesis of D-psicose.
  • Non-Patent Document 5 discloses a method of increasing reaction efficiency using boron.
  • D-alloheptulose a kind of rare sugar, is a kind of 7-carbon sugar, and it has been confirmed that it is contained in trace amounts in the roots of yellow-flowered nine cherry trees (Non-patent Document 6). There have been few reports.
  • a method for producing D-alloheptulose an enzyme method using an enzyme derived from Escherichia coli and a fermentation method using Corynebacterium glutamicum are disclosed (Non-patent Document 7).
  • the method for producing D-psicose using a chemical isomerization reaction cannot specifically produce D-psicose, so that a by-product sugar other than D-psicose is produced in a large amount, and the reaction efficiency is low. Many isomerized sugars remain as substrates.
  • the D-psicose production method using an enzyme related to the biosynthesis of D-psicose has a reaction efficiency of about 33%. Furthermore, even when the reaction efficiency to D-psicose is increased using boron, the conversion rate is about 60%.
  • an object of the present invention is to provide an efficient method for producing a rare sugar using a microorganism, a culture of the microorganism, or a processed product of the culture.
  • the present inventors have found that by using a microorganism that produces D-psicose-6-phosphate from a sugar having a D-psicose-6-phosphate dephosphorylation activity enhanced compared to the parent strain. It was found that D-psicose can be produced efficiently. Further, it was found that D-alloheptulose can be efficiently produced by using a microorganism having enhanced allulose-6-phosphate 3-epimerase activity as compared with the parent strain, and the present invention was completed based on these findings.
  • D-psicose-6-phosphate is produced from a sugar having an enhanced activity of the protein according to any one of the following [1] to [3] as compared to a parent strain A microorganism culture or a treated product of the culture, and (ii) a sugar as a substrate present in an aqueous medium to produce and accumulate D-psicose in the aqueous medium, and from that aqueous medium D-psicose
  • a method for producing D-psicose characterized in that [1] Protein consisting of the amino acid sequence represented by SEQ ID NO: 1 or 2 [2] Amino acid sequence wherein 1 to 10 amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 or 2
  • a mutant protein having D-psicose-6-phosphate dephosphorylation activity [3] comprising an amino acid sequence having 95% or more identity with the amino acid sequence
  • DNA encoding the protein according to any one of [1] to [3] of ⁇ 1> or ⁇ 2> [5] DNA consisting of the base sequence represented by SEQ ID NO: 50 or 51
  • DNA consisting of the base sequence represented by SEQ ID NO: 50 or 51
  • DNA encoding [7] DNA encoding a homologous protein comprising a base sequence having 95% or more identity with the base sequence represented by SEQ ID NO: 50 or 51 and having D-psicose-6-phosphate dephosphorylation activity ⁇ 4>
  • the microorganism according to any one of ⁇ 1> to ⁇ 3>, wherein the microorganism that produces D-psicose-6-phosphate from sugar is a microorganism in which allulose-6-phosphate 3-epime
  • a microorganism that produces D-psicose-6-phosphate from sugar further comprises D-allose-6-phosphate isomerase, RpiR, D-allose transport protein, and D-allose kinase, compared to the parent strain.
  • ⁇ 6> Any one of ⁇ 1> to ⁇ 5>, wherein the microorganism that produces D-psicose-6-phosphate from sugar is a microorganism in which 6-phosphofructokinase activity is further reduced or lost compared to the parent strain
  • ⁇ 7> (i) a microorganism culture having enhanced allulose-6-phosphate 3-epimerase activity as compared with the parent strain or a treated product of the culture as an enzyme source; and (ii) a sugar as a substrate in an aqueous medium.
  • D-alloheptulose in the aqueous medium, allowing D-alloheptulose to be collected from the aqueous medium, and producing D-alloheptulose.
  • a microorganism having enhanced allulose-6-phosphate 3-epimerase activity as compared to the parent strain is cultured in a medium, D-alloheptulose is produced and accumulated in the culture, and D-alloheptulose is collected from the culture.
  • a process for producing D-alloheptulose characterized in that ⁇ 9> The production method according to ⁇ 7> or ⁇ 8>, wherein the microorganism is a microorganism that produces sedheptulose-7-phosphate from sugar.
  • ⁇ 10> The production method according to any one of ⁇ 7> to ⁇ 9>, wherein the microorganism produces D-alloheptulose from D-alloheptulose-7-phosphate.
  • a microorganism having enhanced allulose-6-phosphate 3-epimerase activity as compared to the parent strain is a microorganism having enhanced activity of the protein according to any one of the following [8] to [10]: ⁇ 7
  • a protein comprising the amino acid sequence represented by SEQ ID NO: 52
  • the amino acid sequence represented by SEQ ID NO: 52 comprising an amino acid sequence in which 1 to 10 amino acids are deleted, substituted or added, and
  • a mutant protein having allulose-6-phosphate 3-epimerase activity [10] consisting of an amino acid sequence having 95% or more identity with the amino acid sequence represented by SEQ ID NO: 1 or 2, and allulose-6-phosphate 3 -Homologous protein having epimerase activity ⁇ 12>
  • the protein according to any one of the following [11] to [13], wherein a microorganism that produces D-alloheptulose from D-alloheptulose-7-phosphate is compared to the parent strain ⁇ 10> or ⁇ 11>, the production method according to ⁇ 10>, wherein the microorganism has enhanced activity.
  • a protein comprising the amino acid sequence represented by SEQ ID NO: 1 [12] consisting of an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1, and A mutant protein having D-psicose-6-phosphate dephosphorylation activity [13] consisting of an amino acid sequence having 95% or more identity with the amino acid sequence represented by SEQ ID NO: 1, and D-psicose-6-phosphorus
  • a microorganism that produces cedoheptulose-7-phosphate from a homologous protein ⁇ 13> sugar having acid dephosphorylation activity is a microorganism in which 6-phosphofructokinase activity is further reduced or lost compared to the parent strain ⁇ 9
  • the production method according to any one of> to ⁇ 12>.
  • ⁇ 14> The production method according to any one of ⁇ 1> to ⁇ 13>, wherein the sugar is at least one sugar selected from the group consisting of glucose, fructose, and sucrose.
  • the parent strain is a microorganism belonging to the genus Escherichia.
  • the present invention provides an efficient method for producing a rare sugar using a microorganism, a culture of the microorganism, or a processed product of the culture.
  • a microorganism that produces D-psicose-6-phosphate from a sugar having enhanced D-psicose-6-phosphate dephosphorylation activity as compared with the parent strain.
  • the D-allose metabolic system possessed by the microorganism can be used efficiently, production of by-product sugars and the remaining of the substrate can be suppressed, and D-psicose can be produced with high efficiency.
  • D-alloheptulose of the present invention by using a microorganism having enhanced activity of allulose-6-phosphate 3-epimerase as compared to the parent strain, sedheptulose-7-phosphate possessed by the microorganism can be converted to D- The activity of isomerizing to alloheptulose-7-phosphate can be used efficiently, and D-alloheptulose can be produced with high efficiency.
  • FIG. 1 (a) shows a schematic diagram of the metabolic system of D-allose possessed by microorganisms belonging to the genus Escherichia.
  • FIG. 1 (b) shows a schematic diagram of a specific example of D-psicose production by microorganisms used in the method for producing D-psicose of the present invention.
  • FIG. 2 shows a schematic diagram of an allose-utilizing operon of a microorganism belonging to the genus Escherichia.
  • microorganisms used in the method for producing D-psicose of the present invention and methods for producing the microorganisms include the microorganisms described in (1) to (4) below. .
  • a mutant protein consisting of a deleted, substituted or added amino acid sequence and having D-psicose-6-phosphate dephosphorylation activity [3] 95% or more, preferably 95% or more of the amino acid sequence represented by SEQ ID NO: 1 or 2 Homologous protein comprising an amino acid sequence having 97% or more, more preferably 98% or more, most preferably 99% or more identity, and having D-psicose-6-phosphate dephosphorylation activity (2) ) With the parent strain as a parent strain, and the microorganism with enhanced allulose-6-phosphate 3-epimerase activity compared to the parent strain. (3) The microorganism according to (1) or (2) is used as a parent strain and compared with the parent strain.
  • a microorganism in which the activity of at least one protein selected from the group consisting of -6-phosphate isomerase, RpiR, D-allose transport protein and D-allose kinase is reduced or lost (4) Any of (1) to (3) A microorganism in which 6-phosphofructokinase activity is reduced or lost as compared with the parental strain is described below as (1) to (4).
  • a mutant protein consisting of a deleted, substituted or added amino acid sequence and having D-psicose-6-phosphate dephosphorylation activity [3] 95% or more, preferably 95% or more of the amino acid sequence represented by SEQ ID NO: 1 or 2 Homologous protein comprising an amino acid sequence having 97% or more, more preferably 98% or more, most preferably 99% or more identity, and having D-psicose-6-phosphate dephosphorylation activity
  • D-psicose-6-phosphate dephosphorylation activity refers to an activity of dephosphorylation using D-psicose-6-phosphate as a substrate.
  • the “microorganism that produces D-psicose-6-phosphate from sugar” means that when the microorganism is cultured in a medium, D-psicose-6-phosphate is used as a starting substrate in the cells of the microorganism. This refers to microorganisms that produce and accumulate.
  • a mutated protein refers to a protein obtained by artificially deleting or substituting amino acid residues in the original protein, or inserting or adding amino acid residues into the protein.
  • Homologous proteins are proteins that are considered to have the same structure and function as the original protein, so that the gene encoding the protein has the same evolutionary origin as the gene encoding the original protein. It refers to the protein of living organisms.
  • an amino acid is deleted, substituted, inserted or added means that 1 to 10 amino acids are deleted, substituted, inserted or added at any position in the same sequence. Also good.
  • the number of amino acids to be deleted, substituted, inserted or added is 1 to 10, preferably 1 to 8, and most preferably 1 to 5.
  • the amino acid to be deleted, substituted, inserted or added may be a natural type or a non-natural type.
  • Natural amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L -Methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and the like.
  • amino acids included in the same group can be substituted for each other.
  • Group A leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine
  • B group aspartic acid, glutamic acid, isoaspartic acid, Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid group
  • C asparagine, glutamine group
  • D lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid group
  • E proline, 3 -Hydroxyproline, 4-hydroxyproline
  • F group serine, threonine, homoserine
  • G group phenyl, threonine
  • the above-mentioned mutant protein or homologous protein has D-psicose-6-phosphate dephosphorylation activity.
  • a recombinant DNA containing DNA encoding the protein is prepared by the method described later, A microorganism that is unable to confirm D-psicose-6-phosphate dephosphorylation activity with recombinant DNA, such as a microorganism obtained by transforming Escherichia coli W3110 strain, is cultured, and the protein is obtained from the resulting culture.
  • a cell extract containing sucrose is prepared, the fraction is contacted with D-psicose-6-phosphate as a substrate, and the resulting D-psicose is detected by high-speed chromatography or gas chromatography. be able to.
  • the parent strain refers to the original strain that is the subject of genetic modification and transformation.
  • the original strain to be transformed by gene transfer is also called a host strain.
  • the parent strain may be a wild strain as long as it is a microorganism that produces D-psicose-6-phosphate from sugar, and the wild strain does not have the ability to produce D-psicose-6-phosphate from sugar. In some cases, it may be a breeding strain that has been artificially imparted with the ability to produce D-psicose-6-phosphate from sugar.
  • a mechanism for controlling a biosynthetic pathway for producing D-psicose-6-phosphate from sugar (B) a method for enhancing expression of at least one enzyme involved in a biosynthetic pathway for producing D-psicose-6-phosphate from a sugar, (c) a method for relaxing or releasing at least one of A method for increasing the copy number of at least one enzyme gene involved in a biosynthetic pathway that produces psicose-6-phosphate, (d) a biosynthetic pathway that produces D-psicose-6-phosphate from a sugar A method of weakening or blocking at least one metabolic pathway branching to a metabolite other than the target substance, and (e) a cell line having a higher resistance to an analog of D-psicose-6-phosphate than a wild-type strain How to-option, and
  • Such a parent strain is preferably a prokaryote or yeast strain, more preferably belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc.
  • Prokaryotic organisms or yeast strains belonging to the genus Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Siwaniomyces, Pichia, Candida, etc. are most preferably Escherichia coli MG1655, Escherichia coli XL1-Blue.
  • Escherichia coli XL2-Blue Escherichia coli DH1, Escherichia coli MC1000, Escherichi coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.
  • the microorganism is a microorganism capable of producing D-psicose-6-phosphate from a sugar.
  • the microorganism is a recombinant DNA having a DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 or 2. Can be confirmed by culturing the transformed microorganism in a medium and detecting D-psicose accumulated in the culture using HPLC described later.
  • microorganisms (a) and (b) can be mentioned as microorganisms having enhanced activity of the protein according to any one of [1] to [3] as compared to the parental microorganism.
  • A obtained by modifying the gene encoding the protein according to any one of [1] to [3] on the chromosomal DNA of the parent strain, i) the ratio of the protein compared to the microorganism of the parent strain A microorganism having enhanced activity and ii) a microorganism having an increased transcription amount of the gene or a production amount of the protein as compared to the microorganism of the parent strain, and (b) a microorganism of the parent strain with a recombinant DNA containing a DNA encoding the protein.
  • microorganism having enhanced activity is 1 to 10 amino acids, preferably 1 to 5 amino acids, most preferably the amino acid sequence of the protein according to any one of the above [1] to [3] of the parent strain. Since it has a protein having an amino acid sequence in which 1 to 5 amino acids are substituted, there can be mentioned a microorganism having a mutant protein whose specific activity is enhanced as compared with the parent strain.
  • the microorganism having enhanced activity can be obtained by using the specific activity of the protein described in any one of the above [1] to [3] of the parent strain by a usual mutation treatment method, a gene replacement method using a recombinant DNA technique, or the like. It can be obtained by strengthening.
  • NTG N-methyl-N′-nitro-N-nitrosoguanidine
  • the gene replacement method using the recombinant DNA technique is used for mutation treatment using a mutation agent in vitro or error-prone PCR on the DNA encoding the protein according to any one of [1] to [3]. And a method of substituting the gene encoding the protein according to any one of [1] to [3] present on the chromosomal DNA of the parent strain by homologous recombination after the mutation is introduced. be able to.
  • the DNA encoding the protein according to any one of [1] to [3] is, for example, a probe DNA that can be designed based on the base sequence represented by SEQ ID NO: 50 or 51, In 1), it can be obtained by a method using PCR, which will be described later.
  • Examples of the homologous recombination method include a method using a plasmid for homologous recombination that can be prepared by ligating with a plasmid DNA having a drug resistance gene that cannot autonomously replicate in the host cell to be introduced.
  • a method using homologous recombination frequently used in Escherichia coli a method of introducing recombinant DNA using a homologous recombination system of lambda phage [Proc. Natl. Acad. Sci. USA, 97, 6641-6645 (2000)].
  • a selection method using the fact that Escherichia coli becomes sucrose-sensitive by Bacillus subtilis levanshuclase integrated on the chromosome together with the recombinant DNA, or the wild-type rpsL gene is incorporated into Escherichia coli having a mutant rpsL gene resistant to streptomycin.
  • the selection method utilizing the sensitivity of Escherichia coli to streptomycin [Mol. Ol Microbiol., 55, 137 (2005), Biosci. Biotechnol. Biochem., 71, 2905 (2007)], etc. Microorganisms in which the above target region has been replaced with recombinant DNA can be obtained.
  • the microorganism having enhanced specific activity of the protein according to any one of the above [1] to [3] compared to the parent strain means that, for example, a microorganism having a parent strain and a mutant protein is cultured, After treatment with sonic waves, etc., D-psicose-6-phosphate and other substrates are added to the treated product, and the resulting D-psicose is detected by high-speed chromatography or gas chromatography and confirmed. can do.
  • the microorganism having an increased amount or production amount of the protein includes a transcriptional regulatory region or a promoter region of the gene encoding the protein according to any one of [1] to [3] present on the chromosomal DNA of the parent strain.
  • the amount of the microorganism or the amount of the protein produced is increased by, for example, changing the transcription amount of the gene of the protein according to any one of the above [1] to [3] or the amount of the protein produced by a normal mutation. It can be obtained by enhancement using a treatment method, a gene replacement method using recombinant DNA technology, or the like.
  • Examples of the mutation treatment method include the methods described above.
  • the gene replacement method using the recombinant DNA technique comprises a transcription regulatory region and a promoter region of a gene encoding the protein according to any one of [1] to [3] possessed by the parent strain, for example, upstream of the initiation codon of the protein.
  • the DNA having a base sequence of 200 bp, preferably 100 bp, is subjected to mutation treatment in a test tube or error-prone PCR, etc., and then introduced into the DNA, and then present on the chromosomal DNA of the parent strain [1]-[ 3], a method for substitution with the gene encoding the protein according to any one of the above 3 using the homologous recombination method described above.
  • the above [1] to [3] of the parent strain by replacing the promoter region of the gene encoding the protein according to any one of the above [1] to [3] of the parent strain with a known strong promoter sequence, the above [1] to [3 ] The microorganism which the production amount of the protein as described in any one of [1] improved can also be acquired.
  • Such promoters in the case of using a microorganism belonging to the genus Escherichia as the parent strain, for example, trp promoter functional in Escherichia coli (Ptrp), lac promoter (Plac), P L promoter, P R promoter, P SE promoter and the like And promoters derived from Escherichia coli, phages, and the like. Further, for example, artificially constructed promoters such as a promoter in which two Ptrps are connected in series, a tac promoter, a lacT7 promoter and a letI promoter can also be mentioned.
  • examples thereof include an SPO1 promoter, an SPO2 promoter, a penP promoter that function in Bacillus subtilis.
  • P54-6 promoter [Appl. Microbiol. Biotechnol., 53, 674-6792000 (2000)] can be exemplified.
  • yeast strain When a yeast strain is used as the parent strain, examples thereof include PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock polypeptide promoter, MF ⁇ 1 promoter, CUP1 promoter and the like.
  • the microorganism obtained by the above method is a microorganism in which the transcription amount of the gene encoding the protein according to any one of [1] to [3] or the production amount of the protein is increased as compared with the parent strain.
  • the transcription amount of the gene of the microorganism can be confirmed by Northern blotting or the production amount of the protein of the microorganism by comparison with the parent strain by Western blotting.
  • the number of copies of the gene over the parent strain obtained by transforming the parent strain with a recombinant DNA containing the DNA encoding the protein according to any one of [1] to [3]
  • the microorganism having an increased number of microorganisms can be obtained by transforming a parental microorganism with a recombinant DNA containing a DNA encoding the protein according to any one of [1] to [3] above, and And microorganisms in which the gene is retained in addition to chromosomal DNA.
  • the DNA encoding the protein according to any one of [1] to [3] may be a DNA encoding a protein having the activity of the protein according to any one of [1] to [3]. Any of these may be used, but specific examples include one DNA selected from the group consisting of the following [4] to [7].
  • DNA encoding the protein according to any one of [1] to [3] [5] DNA consisting of the base sequence represented by SEQ ID NO: 50 or 51 [6] Homologous protein that hybridizes under stringent conditions with DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 50 or 51 and has D-psicose-6-phosphate dephosphorylation activity
  • DNA encoding [7] A nucleotide sequence having 95% or more, preferably 97% or more, more preferably 98% or more, most preferably 99% or more identity with the base sequence represented by SEQ ID NO: 50 or 51, and DNA encoding a homologous protein having D-psicose-6-phosphate dephosphorylation activity
  • the recombinant DNA containing the DNA of any one of [4] to [7] is capable of autonomously replicating in the parent strain or integrating into the chromosome and transcribing the DNA.
  • hybridize means that the DNA hybridizes to DNA having a specific base sequence or a part of the DNA. Therefore, the DNA having the specific base sequence or a part thereof can be used as a probe for Northern or Southern blot analysis, or can be used as an oligonucleotide primer for PCR analysis.
  • DNA used as a probe examples include DNA of at least 100 bases, preferably 200 bases or more, more preferably 500 bases or more.
  • DNA used as a primer DNA of at least 10 bases or more, preferably 15 bases or more can be raised.
  • DNA hybridization experiment methods are well known. For example, those skilled in the art can determine hybridization conditions according to the present specification. The hybridization conditions are described in Molecular Cloning 2nd Edition, 3rd Edition (2001), Methods General and Molecular act Bacteriology, ASM Press (1994), Immunology methods manual, Academic press (1996). Can be done according to other standard textbooks.
  • DNA that hybridizes under stringent conditions can be obtained by following the instructions attached to the commercially available hybridization kit.
  • commercially available hybridization kits include a random primed DNA labeling kit (manufactured by Roche Diagnostics) that prepares probes by a random prime method and performs hybridization under stringent conditions.
  • the above stringent conditions include a DNA-immobilized filter and probe DNA, 50% formamide, 5 ⁇ SSC (750 mmol / l sodium chloride, 75 mmol / l sodium citrate), 50 mmol / l phosphoric acid. After incubation overnight at 42 ° C. in a solution containing sodium (pH 7.6), 5 ⁇ Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g / l denatured salmon sperm DNA, for example about 65 ° C. The conditions which wash
  • the DNA capable of hybridizing under the above stringent conditions includes, for example, a DNA comprising the base sequence represented by SEQ ID NO: 50 or 51 when calculated based on the above-described parameters using BLAST, FASTA, etc. And DNA having at least 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more.
  • a recombinant DNA is prepared by inserting the DNA fragment downstream of the promoter of an appropriate expression vector.
  • the recombinant DNA is composed of a promoter, a ribosome binding sequence, the DNA described in any one of [4] to [7] above, and a transcription termination sequence. Recombinant DNA is preferred. A gene that controls the promoter may also be included.
  • a transcription termination sequence is not necessarily required for the expression of the DNA, but a transcription termination sequence is preferably arranged immediately below the structural gene.
  • D-psicose-6-phosphorus by replacing the base sequence of the portion encoding the protein having D-psicose-6-phosphate dephosphorylation activity so as to be an optimal codon for host expression, D-psicose-6-phosphorus
  • the expression level of the protein having acid dephosphorylation activity can be improved.
  • Information on codon usage in the parent strain used in the production method of the present invention can be obtained through a public database.
  • expression vectors include pColdI (manufactured by Takara Bio Inc.), pCDF-1b, pRSF-1b (all manufactured by Novagen), and pMAL-c2x (New England Biolabs).
  • PGEX-4T-1 (manufactured by GE Healthcare Bioscience), pTrcHis (manufactured by Invitrogen), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-30 (manufactured by Qiagen) PET-3 (manufactured by NOVAGEN), pKYP10 (Japanese Patent Laid-Open Publication No. 58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl.
  • any promoter can be used as long as it functions in cells of microorganisms belonging to the genus Escherichia, for example, trp promoter (P trp ), lac promoter (P lac ), P L promoter, P R promoter and P SE promoter, may be used promoters derived from Escherichia coli, phage and the like.
  • An artificially designed and modified promoter such as a promoter in which two Ptrps are connected in series, a tac promoter, a lacT7 promoter, and a letI promoter can also be used.
  • examples of expression vectors include pCG1 (Japanese Unexamined Patent Publication No. 57-134500), pCG2 (Japanese Unexamined Patent Publication No. 58-35197), and pCG4 (Japanese Special No. 57-183799), pCG11 (Japanese Unexamined Patent Publication No. 57-134500), pCG116, pCE54, pCB101 (all Japanese Unexamined Patent Publication No. 58-105999), pCE51, pCE52, pCE53 [any] In addition, Molecularoleand General Genetics, 196, 175 (1984)] can be mentioned.
  • any promoter in the case of using the above expression vector, any promoter can be used as long as it functions in cells of coryneform bacteria.
  • P54-6 promoter [Appl. Microbiol. Biotechnol., 53, 674-679 ( 2000)] can be used.
  • examples of the expression vector include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15 and the like.
  • any promoter in the case of using the above expression vector, any promoter can be used as long as it functions in yeast strain cells.
  • PHO5 promoter PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat Examples include promoters such as shock polypeptide promoter, MF ⁇ 1 promoter, CUP1 promoter and the like.
  • the introduction of the recombinant DNA as a plasmid capable of autonomous replication in the parent strain or the integration into the chromosome of the parent strain amplifies the gene originally possessed by the microorganism on the chromosomal DNA.
  • the gene introduced by transformation can be confirmed by a method of confirming an amplification product by PCR using an amplifiable primer set.
  • the increase in the transcription amount of the DNA or the production amount of the protein encoded by the DNA means that the transcription amount of the gene of the microorganism is Northern blotting or the production amount of the protein of the microorganism is Western blotting. Thus, it can be confirmed by a method of comparing with that of the parent strain.
  • the DNA encoding the protein of [1] above and the DNA of [5] above have, for example, a nucleotide sequence represented by SEQ ID NO: 50 (yniC gene) or a nucleotide sequence represented by SEQ ID NO: 51 (ybiV gene).
  • a microorganism preferably a microorganism belonging to the genus Escherichia coli, more preferably a chromosomal DNA library of Escherichia coli MG1655 strain using a probe DNA that can be designed based on the base DNA, or based on the base sequence It can be obtained by PCR [PCR Protocols, Academic Press (1990)] using a chromosomal DNA of the microorganism as a template, using a primer DNA that can be designed.
  • Escherichia coli MG1655 strain can be obtained from the National Institute of Technology and Evaluation Biotechnology Center (NITE Biological Resource Center).
  • the DNA encoding the mutant protein of [2] above can be obtained, for example, by subjecting it to error-prone PCR or the like using a DNA consisting of the base sequence represented by SEQ ID NO: 50 or 51 as a template.
  • PCR is performed using the DNA as a template to amplify a fragment A (a mutation is introduced at the 3 ′ end) from the 5 ′ end to the mutation introduction site of the DNA.
  • the DNA encoding the homologous protein of [3] above and the DNA of [6] and [7] above are, for example, 95% or more of the nucleotide sequence represented by SEQ ID NO: 50 or 51 with respect to various gene sequence databases
  • a nucleotide sequence having an identity of 97% or more, more preferably 98% or more, most preferably 99% or more is searched, or represented by SEQ ID NO: 2 or 4 with respect to various protein sequence databases.
  • amino acid sequence having 95% or more, preferably 97% or more, more preferably 98% or more, most preferably 99% or more identity with an amino acid sequence is searched, and based on the base sequence or amino acid sequence obtained by the search Using the probe DNA or primer DNA that can be designed in this manner and a microorganism having the DNA, It can be obtained by a method similar to the method of acquiring.
  • the obtained DNA described in any one of [4] to [7] above is directly or cleaved with an appropriate restriction enzyme and incorporated into a vector by a conventional method, and the obtained recombinant DNA is transformed into a host cell.
  • a base sequence analysis method usually used, for example, dideoxy method [Proc. Natl. Acad. Sci., USA, 74, 5463 (1977)] or 3700 DNA analyzer (Applied Biosystems) etc. By analyzing using an analyzer, the base sequence of the DNA can be determined.
  • Escherichia coli XL1-Blue Escherichia coli XL2-Blue
  • Escherichia coli DH1 Escherichia coli MC1000
  • Escherichia coli KY3276 Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101
  • Escherichia coli No. 49 Escherichia coli W3110, Escherichia coli NY49, Escherichia coli MP347, and Escherichia coli NM522.
  • Examples of the vector include pBluescript IIKS (+) (manufactured by Stratagene), pDIRECT [Nucleic Acids Res., 18, 6069 (1990)], pCR-Script Amp SK (+) (manufactured by Stratagene), pT7Blue (Manufactured by Novagen), pCR II (manufactured by Invitrogen) and pCR-TRAP (manufactured by Gene Hunter).
  • any method for introducing DNA into a host cell can be used.
  • a method using calcium ion [Proc. Natl. Acad. Sci., USA, 69, 2110]. (1972)], protoplast method (Japanese Patent Laid-Open No. 63-248394), electroporation method [Nucleic Acids Res., 16, 6127 (1988)] and the like.
  • the full-length DNA can be obtained by Southern hybridization or the like for a chromosomal DNA library using the partial length DNA as a probe.
  • the target DNA can also be prepared by chemical synthesis using an 8905 type DNA synthesizer manufactured by Perceptive Biosystems.
  • the recombinant DNA used in the production method of the present invention can be prepared by inserting the DNA fragment downstream of the promoter of an appropriate expression vector.
  • Examples of such recombinant DNA include Ptrp-yniC and Ptrp-ybiV described later in Examples.
  • Methods for introducing recombinant DNA as a plasmid capable of autonomous replication in the parent strain include, for example, a method using calcium ions [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)], protoplast method (Japan) And a method of electroporation [Nucleic Acids Res., ⁇ 16, 61271988 (1988)].
  • the microorganism produced by the above method is a microorganism in which the activity of the protein according to any one of the above [1] to [3] is enhanced as compared with the parent strain. 7] Confirmed by comparing the transcription amount of the DNA according to any one of [7], the production amount of the protein according to any one of [1] to [3] above, or the specific activity of the protein with that of the parent strain can do.
  • microorganisms examples include MGC and MGV described later in Examples.
  • Allulose-6-phosphate 3-epimerase activity is defined as fructose-6-phosphate. Is the activity of isomerizing to D-psicose-6-phosphate.
  • microorganism having enhanced allulose-6-phosphate 3-epimerase activity as compared with the parent strain include the following microorganisms (a) and (b).
  • B A microorganism obtained by transforming a parental microorganism with a recombinant DNA containing a DNA encoding the protein and having an increased copy number of the gene compared to the parental strain
  • the microorganism 1-10 amino acids, preferably 1-8 amino acids, most preferably 1-5 amino acids in the amino acid sequence of the protein having allulose-6-phosphate 3-epimerase activity of the parent strain
  • a microorganism having a mutant protein whose specific activity is enhanced as compared with the parent strain are examples of the protein having allulose-6-phosphate 3-epimerase activity of the parent strain.
  • the microorganism produced by the method described in 1 (1) above is used as a parent strain, and the specific activity of a protein having allulose-6-phosphate 3-epimerase activity of the parent strain is treated with a normal mutation treatment.
  • Examples of the mutation treatment method include the method described in 1 (1) above.
  • the gene replacement method using recombinant DNA technology is a method of mutating DNA encoding a protein having allulose-6-phosphate 3-epimerase activity by subjecting it to mutation treatment using a mutation agent in vitro or error-prone PCR.
  • the DNA encoding a protein having allulose-6-phosphate 3-epimerase activity is described in 1 (1) above using, for example, a probe DNA that can be designed based on the base sequence represented by SEQ ID NO: 42 It can be obtained by a method using PCR.
  • a microorganism having enhanced specific activity of a protein having allulose-6-phosphate 3-epimerase activity compared to the parent strain means that, for example, a microorganism having a parent strain and a mutant protein is cultured, and the culture is sonicated or the like. After the treatment, fructose-6-phosphate and other substrates are added to the treated product, and the amount of synthesized D-psicose-6-phosphate can be measured and compared by HPLC.
  • Microorganisms with increased protein production include at least one base in the base sequence of the transcriptional regulatory region or promoter region of a gene encoding a protein having an allulose-6-phosphate 3-epimerase activity present on the chromosomal DNA of the parent strain.
  • the promoter region of the gene present on the chromosomal DNA of the parent strain and a known strong promoter Obtained by replacing the over arrangement, and a microorganism expression level of the gene is increased.
  • Microorganisms with increased protein production include, for example, transcription of a gene of a protein having allulose-6-phosphate 3-epimerase activity of the parent strain, which is a microorganism constructed by the method described in 1 (1) above.
  • the amount of the protein or the amount of the protein produced can be obtained by enhancing using a usual mutation treatment method, a gene replacement method using a recombinant DNA technique, or the like.
  • Examples of the mutation treatment method include the method described in 1 (1) above.
  • the gene replacement method using the recombinant DNA technique is a transcriptional regulatory region and a promoter region of a gene encoding a protein having an allulose-6-phosphate 3-epimerase activity possessed by the parent strain, for example, 200 bp upstream of the start codon of the protein, preferably Is introduced into the DNA by subjecting the DNA having a base sequence of 100 bp to a mutation treatment in vitro or error-prone PCR, and then allulose-6-phosphate 3-epimerase present on the chromosomal DNA of the parent strain
  • Examples of the method include substitution using a gene encoding a protein having activity and the homologous recombination method described in 1 (1) above.
  • allulose-6-phosphate 3-epimerase activity from the parent strain can also be obtained by substituting the promoter region of the gene encoding the protein having parental allulose-6-phosphate 3-epimerase activity with a known strong promoter sequence. It is also possible to obtain a microorganism with improved production of protein having
  • Examples of such a promoter include the promoter described in 1 (1) above.
  • the microorganism obtained by the above method is a microorganism in which the transcription amount of a gene encoding a protein having allulose-6-phosphate 3-epimerase activity or the production amount of the protein is increased as compared with the parent strain, for example, The transcription amount of the gene of the microorganism can be confirmed by comparing with that of the parent strain by Northern blotting, or the production amount of the protein of the microorganism by Western blotting.
  • (B) Allulose-6-phosphate The number of copies of the gene increased compared to the parent strain obtained by transforming the parent strain with a recombinant DNA containing a DNA encoding a protein having 3-epimerase activity.
  • a microorganism the number of copies of the gene increased on the chromosomal DNA by transforming the parental microorganism with a recombinant DNA containing DNA encoding a protein having allulose-6-phosphate 3-epimerase activity.
  • Microorganisms and microorganisms having the above genes in addition to chromosomal DNA can be mentioned as plasmid DNA.
  • the protein having allulose-6-phosphate 3-epimerase activity may be any protein as long as it has the activity, and specifically, one protein selected from the group consisting of the following [1] to [3] Examples include proteins.
  • the amino acid sequence represented by SEQ ID NO: 52 consists of an amino acid sequence in which 1 to 10, preferably 1 to 8, and most preferably 1 to 5 amino acids are deleted, substituted or added, and Mutant protein having allulose-6-phosphate 3-epimerase activity, [3] An amino acid sequence having 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the amino acid sequence represented by SEQ ID NO: 52, and allulose-6 -Homologous protein with phosphate 3-epimerase activity
  • mutant protein having activity can be obtained by introducing a site-specific mutation into DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 52, for example, using the site-specific mutagenesis method described below. it can.
  • the number of amino acid residues to be deleted, substituted or added is not particularly limited, but is a number that can be deleted, substituted or added by a known method such as the above-mentioned site-specific mutation method, and is 1 to 10, The number is preferably 1 to 8, most preferably 1 to 5.
  • amino acid sequence represented by SEQ ID NO: 52 1 to 10, preferably 1 to 8, and most preferably 1 to 5 or more amino acids are deleted, substituted or added is any position in the same sequence. In which one or more amino acid residues may be deleted, substituted or added.
  • a mutant protein comprising an amino acid sequence in which 1 to 10, preferably 1 to 8, most preferably 1 to 5 amino acid residues are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 52,
  • Allulose-6-phosphate is a protein having 3-epimerase activity.
  • a transformant expressing a protein whose activity is to be confirmed using a DNA recombination method is prepared and cultured in a medium. It can be confirmed by measuring D-psicose-6-phosphate in the medium.
  • the DNA encoding a protein having allulose-6-phosphate 3-epimerase activity may be any DNA that encodes a protein having the activity. Specifically, the following [4] to [7] 1 DNA selected from the group consisting of [4] DNA encoding the protein according to any one of [1] to [3] above, [5] DNA having the base sequence represented by SEQ ID NO: 42, [6] DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 42 and encodes a homologous protein having allulose-6-phosphate 3-epimerase activity , [7] A base sequence having at least 95%, preferably 97%, more preferably 98%, most preferably 99% or more identity with the base sequence represented by SEQ ID NO: 42, and allulose- DNA encoding a homologous protein having 6-phosphate 3-epimerase activity
  • the description of “hybridize”, the method of DNA hybridization experiment, and the stringent conditions described above are the same as described in 1 (1) above.
  • the DNA capable of hybridizing under the above stringent conditions includes, for example, a DNA comprising the base sequence represented by SEQ ID NO: 42 when calculated based on the above-mentioned parameters using the above-mentioned BLAST, FASTA, etc. And DNA having at least 95%, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more.
  • a DNA fragment of an appropriate length containing a portion encoding the protein is obtained.
  • a transformant with an improved production rate can be obtained by substituting the base in the base sequence of the protein-encoding portion so as to be an optimal codon for expression in a host cell.
  • a recombinant DNA is prepared by inserting the DNA fragment downstream of the promoter of an appropriate expression vector.
  • a microorganism constructed by the method described in 1 (1) above By transforming a microorganism constructed by the method described in 1 (1) above with the recombinant DNA, a microorganism having an increased copy number of the gene encoding the protein than the parent strain can be obtained. .
  • a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the start codon is adjusted to an appropriate distance (eg, 6 to 18 bases).
  • a transcription termination sequence is not necessarily required, but a transcription termination sequence is placed directly under the structural gene. It is preferable to arrange.
  • the expression vector contains a promoter at a position where it can autonomously replicate in the above host cell or can be integrated into a chromosome, and can transcribe DNA encoding a protein having allulose-6-phosphate 3-epimerase activity. What is used is used.
  • the recombinant DNA containing the DNA is capable of autonomous replication in prokaryotes, and at the same time, a recombinant consisting of a promoter, a ribosome binding sequence, the DNA, and a transcription termination sequence. It is preferably body DNA. A gene that controls the promoter may also be included.
  • Examples of the expression vector and the promoter in the case of using the expression vector include the same expression vectors and promoters as described in 1 (1) above.
  • Examples of the recombinant DNA obtained by binding a DNA encoding a protein having an allulose-6-phosphate 3-epimerase activity to an expression vector include pUC19_alsE described later in the Examples.
  • DNA encoding a protein having the activity of allulose-6-phosphate 3-epimerase obtained by the above method can be obtained at any position of chromosomal DNA by using the homologous recombination method described in 1 (1) above. It is also possible to obtain a microorganism in which the copy number of the gene encoding the protein is increased as compared with the parent strain.
  • microorganism obtained by the above method has an increased number of copies of a gene encoding a protein having an allulose-6-phosphate 3-epimerase activity as compared to the parent strain. Can be used to confirm.
  • microorganisms examples include MGC / pUC19_alsE, MGV / pUC19_alsE, MGC ⁇ als ⁇ pfkA / pUC19_alsE and MGV ⁇ als ⁇ pfkA / pUC19_alsE described later in the examples.
  • the microorganism of (1) or (2) is selected from the group consisting of D-allose-6-phosphate isomerase, RpiR, D-allose transport protein, and D-allose kinase, compared to the parent strain.
  • D-allose-6-phosphate isomerase is a protein having an activity to isomerize D-allose-6-phosphate to D-psicose-6-phosphate. is there.
  • RpiR is a protein having an activity of suppressing gene expression by binding to an operator part of an allose-utilizing operon as a repressor and blocking transcription of the operon linked thereto.
  • the D-allose transport protein is a protein having an activity of transporting D-allose from outside the cell into the cell membrane.
  • D-allose kinase is a protein having the activity of converting D-allose into D-allose-6-phosphate.
  • the specific activity of the protein is higher than that of the parent strain 80% or less, preferably 50% or less, more preferably 30% or less, even more preferably 20% or less, particularly preferably 10% or less, most preferably reduced to 0%, and (b) compared to the parent strain,
  • the gene transcription amount or the protein production amount is 80% or less, preferably 50% or less, more preferably 30% or less, and still more preferably 20%.
  • the gene encoding D-allose-6-phosphate isomerase may be any gene encoding a protein having D-allose-6-phosphate isomerase activity. Specifically, the following [1] And one gene selected from the group consisting of [4]. [1] a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 53, [2] An amino acid sequence having 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the amino acid sequence represented by SEQ ID NO: 53, and D-allose A gene encoding a homologous protein having -6-phosphate isomerase activity, [3] a gene consisting of the base sequence represented by SEQ ID NO: 43, and [4] A gene that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 43 and encodes a homologous protein having D-allose-6-phosphate isomerase activity
  • the gene encoding RpiR may be any gene as long as it encodes a protein having an activity of blocking transcription of an allose-utilizing operon as a repressor. Specifically, from the following [1] to [4] One gene selected from the group consisting of: [1] a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 54, [2] An amino acid sequence having 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the amino acid sequence represented by SEQ ID NO: 54, and as a repressor A gene encoding a homologous protein having an activity to block transcription of an allose-utilizing operon, [3] a gene consisting of the base sequence represented by SEQ ID NO: 44, and [4] A homologous protein that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 44 and has the activity of blocking transcription of an allose-utilizing oper
  • the gene encoding the D-allose transport protein may be any gene as long as it encodes a protein having an activity of transporting D-allose from outside the cell into the cell membrane. Specifically, the following [1] to [1] One gene selected from the group consisting of [4] can be mentioned.
  • the gene encoding D-allose kinase may be any gene that encodes a protein having D-allose kinase activity, and is specifically selected from the group consisting of [1] to [4] below.
  • One gene is mentioned.
  • [1] a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 58, [2] An amino acid sequence having 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the amino acid sequence represented by SEQ ID NO: 58, and D-allose
  • hybridize the method of DNA hybridization experiment, and the stringent conditions described above are the same as described in 1 (1) above.
  • the DNA that can hybridize under the stringent conditions described above is represented by any one of SEQ ID NOs: 43 to 48, for example, when calculated based on the above-described parameters using BLAST or FASTA. Mention may be made of DNA having at least 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with a DNA comprising a base sequence.
  • the microorganism in which the activity of at least one protein selected from the group consisting of D-allose-6-phosphate isomerase, RpiR, D-allose transport protein, and D-allose kinase is reduced or lost as compared with the parent strain is, for example, 1
  • a microorganism obtainable by the method of (1) or (2) is a parent strain, and at least selected from the group consisting of D-allose-6-phosphate isomerase, RpiR, D-allose transport protein, and D-allose kinase of the parent strain
  • the activity of one protein can be obtained by reducing or losing the activity using a usual mutation treatment method, a gene replacement method using a recombinant DNA technique, or the like.
  • Examples of the mutation treatment method include the method described in 1 (1) above.
  • a gene replacement method using recombinant DNA technology a gene encoding at least one protein selected from the group consisting of D-allose-6-phosphate isomerase, RpiR, D-allose transport protein, and D-allose kinase in vitro.
  • One or more base substitutions, deletions or additions can be introduced into the above, and substitution can be performed using the homologous recombination method of 1 (1) above.
  • the genes encoding D-allose-6-phosphate isomerase, RpiR, D-allose transport protein, and D-allose kinase can be designed based on the base sequences represented by SEQ ID NOs: 43 to 48, respectively. Using DNA, it can be obtained by the method using PCR described in 1 (1) above.
  • Substitution or deletion of one or more bases in a gene encoding at least one protein selected from the group consisting of D-allose-6-phosphate isomerase, RpiR, D-allose transport protein, and D-allose kinase in vitro The method of introducing an addition and the method of replacing the gene prepared by the above method with the target region on the chromosomal DNA of the parent strain by homologous recombination etc. are the same as the method of 1 (1) above. .
  • a microorganism in which the activity of D-allose-6-phosphate isomerase is reduced or lost compared to the parent strain is, for example, that the parent strain and the microorganism are cultured in a medium containing D-allose-6-phosphate, and the culture solution This can be confirmed by comparing the ratio of D-psicose-6-phosphate in the microbial cells.
  • the fact that the activity of the D-allose transport protein is reduced or lost compared to the parent strain means that, for example, the parent strain and the microorganism are cultured in a medium containing D-allose, and the D-allose ratio in the culture solution is compared. This can be confirmed.
  • a microorganism in which the activity of D-allose kinase is reduced or lost compared to the parent strain is, for example, that the parent strain and the microorganism are cultured in a medium containing D-allose, and the D-allose- This can be confirmed by comparing the 6-phosphate ratio.
  • the transcription amount of the gene encoding at least one protein selected from the group consisting of D-allose-6-phosphate isomerase, RpiR, D-allose transport protein and D-allose kinase, or the production amount of the protein was reduced or lost.
  • microorganisms include those lacking the allose-utilizing operon. Specifically, for example, MG1655 ⁇ als described later in the examples can be given.
  • Microorganisms that have reduced or lost 6-phosphofructokinase activity compared to the parent strain have a base sequence of the gene encoding wild-type 6-phosphofructokinase that does not contain the mutation present on the chromosomal DNA. Obtained by introducing a deletion, substitution or addition, (a) the specific activity of the protein is 80% or less, preferably 50% or less, more preferably 30% or less, even more preferably 20% or less, compared to the parent strain Particularly preferably 10% or less, most preferably reduced to 0%, and (b) the transcription amount of the gene or the production amount of the protein is 80% or less, preferably 50% or less, more preferably compared to the parent strain. Can be mentioned microorganisms that have been reduced to 30% or less, more preferably 20% or less, particularly preferably 10% or less, and most preferably 0%. More preferred examples include microorganisms that are partially or completely deficient in the gene.
  • the gene encoding 6-phosphofructokinase may be any gene that encodes a protein having 6-phosphofructokinase activity, and specifically comprises the following [1] to [4].
  • One gene selected from the group can be mentioned.
  • a gene encoding a homologous protein having fructokinase activity [3] a gene consisting of the base sequence represented by SEQ ID NO: 49, and [4] A gene that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 49 and encodes a homologous protein having 6-phosphofructokinase activity
  • hybridize the method of DNA hybridization experiment, and the stringent conditions described above are the same as described in 1 (1) above.
  • the DNA that can hybridize under the above-mentioned stringent conditions includes, for example, at least a DNA comprising the base sequence represented by SEQ ID NO: 49 when calculated based on the above-described parameters using BLAST, FASTA, etc.
  • a DNA having 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more can be mentioned.
  • a microorganism in which the activity of 6-phosphofructokinase is reduced or lost compared to the parent strain is, for example, a microorganism that can be obtained by any one of the methods 1 (1) to (3) described above as a parent strain, and the 6-phosphofructokinase activity of the parent strain
  • the fructokinase activity can be obtained by reducing or losing it using a usual mutation treatment method, a gene replacement method using recombinant DNA technology, or the like.
  • Examples of the mutation treatment method include the method described in 1 (1) above.
  • one or more base substitutions, deletions or additions are introduced into a gene encoding a protein having 6-phosphofructokinase activity in vitro, and the above 1 (1) And a method of substitution using the homologous recombination method.
  • a gene encoding a protein having 6-phosphofructokinase activity is obtained by performing PCR described in 1 (1) above using a probe DNA that can be designed based on the base sequence represented by SEQ ID NO: 49, for example. It can be obtained by the method used.
  • the method of substituting the target region on the chromosomal DNA of the parent strain is the same as the method described in 1 (1) above.
  • 6-phosphofructokinase activity is reduced or lost compared to the parent strain is, for example, that the parent strain and the microorganism are cultured in a medium containing fructose-6-phosphate, and are cultured in the culture solution and in the microorganism cell. This can be confirmed by comparing the fructose 1,6-diphosphate or fructose 2,6-diphosphate ratio.
  • the amount of transcription of a gene encoding a protein having 6-phosphofructokinase activity or the production of the protein is reduced or lost.
  • the amount of transcription of the gene of the microorganism is expressed as follows: It can be confirmed by comparing the production amount of the protein of the microorganism with that of the parent strain by Northern blotting or by Western blotting.
  • microorganisms in which the transcription amount of a gene encoding a protein having 6-phosphofructokinase activity or the production amount of the protein is reduced or lost include MG1655 ⁇ als ⁇ pfkA described later in Examples.
  • D-psicose of the present invention 2.1. Method for producing D-psicose using sugar as substrate
  • the activity of the protein according to any one of [1] to [3] above as an enzyme source A culture of a microorganism that produces D-psicose-6-phosphate from sugar (hereinafter referred to as 2.1 microorganism) or a treated product of the culture, and (ii) a sugar as a substrate,
  • a method for producing D-psicose, characterized in that it is present in a medium, D-psicose is produced and accumulated in the aqueous medium, and D-psicose is collected from the aqueous medium, will be described below.
  • the method for culturing microorganisms can be performed according to a conventional method used for culturing microorganisms.
  • a medium for culturing the microorganism any of a natural medium and a synthetic medium can be used as long as it contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the microorganism, and can efficiently culture the microorganism. May be.
  • Any carbon source may be used as long as the microorganism can assimilate, for example, glucose, fructose, sucrose, molasses containing them, carbohydrates such as starch and starch hydrolysate, organic acids such as acetic acid and propionic acid, and the like. And alcohols such as ethanol and propanol.
  • nitrogen source examples include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented bacterial cells, digests thereof, and the like can be used.
  • inorganic salt examples include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.
  • the culture is preferably performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the culture temperature is preferably 15 to 40 ° C., and the culture time is usually 5 hours to 7 days.
  • the pH during the culture is preferably maintained at 3.0 to 9.0.
  • the pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.
  • antibiotics such as ampicillin and tetracycline may be added to the medium as needed during the culture.
  • an inducer may be added to the medium as necessary.
  • isopropyl- ⁇ -D-thiogalactopyranoside is used when cultivating a microorganism transformed with an expression vector using the lac promoter
  • indole acrylic is used when culturing a microorganism transformed with an expression vector using the trp promoter.
  • An acid or the like may be added to the medium.
  • the culture of microorganisms obtained by the above culture or the processed product of the culture is used as an enzyme source, the enzyme source and sugar are present in an aqueous medium, and D-psicose is generated and accumulated in the aqueous medium. By collecting D-psicose from the medium, D-psicose can be produced.
  • the processed product of the culture includes the concentrate of the culture, the dried product of the culture, the cells obtained by centrifuging or filtering the culture, and the drying of the cells.
  • a concentrate of the culture, a dried product of the culture, a microbial cell obtained by centrifuging or filtering the culture, a dried product of the microbial cell Functions similar to the culture as an enzyme source such as a lyophilized product, a surfactant-treated product of the microbial cell, a solvent-treated product of the microbial cell, an enzyme-treated product of the microbial cell, and an immobilized product of the microbial cell Examples include those containing viable cells to be retained, as well as sonicated products of the cells and mechanically ground products of the cells, most preferably the concentrate of the culture and the culture.
  • a dried product of the cell a cell obtained by centrifuging or filtering the culture, a dried product of the cell, a freeze-dried product of the cell, a surfactant-treated product of the cell, As a source of enzyme such as a solvent-treated product, an enzyme-treated product of the bacterial cell, and an immobilized product of the bacterial cell, Include those which Nde.
  • the amount of the enzyme source is preferably 1 to 500 g / l, more preferably 1 to 300 g / l for each enzyme source used.
  • the sugar concentration is preferably 0.1 mM to 10 M, more preferably 1 mM to 1 M.
  • sugar include glucose, fructose and sucrose.
  • aqueous medium examples include buffers such as water, phosphate, carbonate, acetate, borate, citrate and tris, alcohols such as methanol and ethanol, esters such as ethyl acetate, acetone and the like. Examples include ketones and amides such as acetamide.
  • the culture solution of the microorganism used as an enzyme source can be used as an aqueous medium.
  • a chelating agent such as phytic acid, a surfactant or an organic solvent may be added as necessary.
  • the surfactant include nonionic surfactants such as polyoxyethylene / octadecylamine (for example, Nimine S-215, manufactured by NOF Corporation), cetyltrimethylammonium / bromide and alkyldimethyl / benzylammonium chloride (for example, cation F2-40E).
  • Cationic surfactants such as Nippon Oil & Fats Co., Ltd., anionic surfactants such as lauroyl and sarcosinate, tertiary amines such as alkyldimethylamine (eg, tertiary amine FB, manufactured by Nippon Oil & Fats Co., Ltd.), etc. Any one may be used as long as it promotes the generation of psicose, and one kind or a mixture of several kinds may be used.
  • the surfactant is usually used at a concentration of 0.1 to 50 g / l.
  • Examples of the organic solvent include xylene, toluene, aliphatic alcohol, acetone, ethyl acetate and the like, and they are usually used at a concentration of 0.1 to 50 ml / l.
  • the reaction for producing D-psicose is carried out in an aqueous medium at pH 5 to 10, preferably pH 6 to 8, and 20 to 50 ° C. for 1 to 96 hours.
  • adenine, adenosine-5'-monophosphate (AMP), adenosine-5'-triphosphate (ATP), magnesium sulfate, magnesium chloride and the like can be added.
  • Adenine, AMP and ATP are usually used at a concentration of 0.01 to 100 mmol / l.
  • D-psicose produced in an aqueous medium can be quantified using a sugar analyzer manufactured by Dionex [Anal. Biochem., 189, 151 (1990)].
  • the D-psicose produced in the reaction solution can be collected by a usual method using activated carbon, ion exchange resin, or the like.
  • D-psicose is obtained from a sugar having enhanced activity of the protein according to any one of [1] to [3] above.
  • -Production of D-psicose characterized by culturing a microorganism producing 6-phosphate in a medium, producing and accumulating D-psicose in the culture, and collecting D-psicose from the culture The method will be described below.
  • the method for culturing the microorganism can be performed according to a usual method used for culturing microorganisms.
  • a medium for culturing the microorganism a medium containing a receptor carbohydrate in the medium described in 2.1 above can be used.
  • D-psicose can be produced by producing and accumulating D-psicose in the culture by the above culture and collecting D-psicose from the culture.
  • a method for quantifying D-psicose the method described in 2.1 above can be used.
  • D-psicose can be collected from the culture by combining an ion exchange resin method, a precipitation method and other known methods.
  • an ion exchange resin method for example, the microbial cells are crushed by ultrasonic waves, and the microbial cells are removed by centrifugation. Can be collected.
  • FIG. 1 (a) shows a schematic diagram of the metabolic system of D-allose possessed by microorganisms belonging to the genus Escherichia.
  • FIG. 1 (b) shows a schematic diagram of a specific example of D-psicose production by microorganisms used in the method for producing D-psicose of the present invention.
  • [1] the allose-utilizing operon is destroyed, and the expression of the gene (alsE) encoding allulose-6-phosphate 3-epimerase is enhanced compared to the parent strain, and [2 ] A gene encoding phosphofructokinase (pfkA) is disrupted, and [3] D-psicose is obtained by fermentation using a microorganism having enhanced D-psicose-6-phosphate dephosphorylation activity than the parent strain. The case of manufacturing will be described.
  • Fig. 2 shows a schematic diagram of the allose-utilizing operon of microorganisms belonging to the genus Escherichia (J. Bacteriol. 181, 7126-7130, 1999).
  • the allose-utilizing operon of microorganisms belonging to the genus Escherichia includes a gene encoding D-allose-6-phosphate isomerase (rpiB), a gene encoding repressor (rpiR), and D-allose transport.
  • rpiB D-allose-6-phosphate isomerase
  • rpiR gene encoding repressor
  • a gene (alsB) encoding a protein (binding protein), a gene (alsA) encoding a D-allose transport protein (ATPase), a gene (alsC) encoding a D-allose transport protein (membrane protein), and allulose-6 -Phosphorus is composed of a gene (alsE) and yjct (alsK) encoding 3-epimerase activity.
  • fructose-6-phosphate is metabolized to fructose-6-phosphate by disrupting the gene (pfkA) encoding 6-phosphofructokinase. By acting, it can be suppressed to become fructose 1,6-diphosphate or fructose 2,6-diphosphate, and fructose-6-phosphate as a substrate can be accumulated in the microorganism.
  • D-psicose-6-phosphate dephosphorylation activity is enhanced as compared with the parent strain, so that D-psicose-6-phosphate is dephosphorylated.
  • D-psicose can be produced with high efficiency.
  • Microorganisms used in the method for producing D-alloheptulose of the present invention and methods for producing the microorganisms examples include the microorganisms described in (2A) to (2C) below. .
  • the parent strain described in 1 (1) above can be used as the parent strain of the microorganism having enhanced allulose-6-phosphate 3-epimerase activity compared to the parent strain.
  • Examples of microorganisms having enhanced allulose-6-phosphate 3-epimerase activity as compared to the parent strain include the following microorganisms (a) and (b) similar to those described in 1 (2) above.
  • the fact that the specific activity of the protein having allulose-6-phosphate 3-epimerase activity is enhanced as compared with the parent strain is that, for example, the parent strain and the microorganism are cultured, and the culture is sonicated. This can be confirmed by adding sedoheptulose-7-phosphate and other substrates to the treated product, and measuring and comparing the amount of synthesized D-alloheptulose-7-phosphate by HPLC.
  • microorganism having enhanced allulose-6-phosphate 3-epimerase activity as compared with the parent strain include a microorganism having enhanced activity of the protein according to any one of [8] to [10] below than the parent strain. It is done.
  • a protein comprising the amino acid sequence represented by SEQ ID NO: 52
  • the amino acid sequence represented by SEQ ID NO: 52 comprising an amino acid sequence in which 1 to 10 amino acids are deleted, substituted or added
  • a mutant protein having allulose-6-phosphate 3-epimerase activity [10] consisting of an amino acid sequence having 95% or more identity with the amino acid sequence represented by SEQ ID NO: 1 or 2, and allulose-6-phosphate 3 -Homologous proteins with epimerase activity
  • a microorganism in which the activity of the protein described in any one of [8] to [10] is enhanced as compared with the parent strain can be obtained by the same method as described in 1 (1) above. It can be confirmed by a method similar to the method described in 1 (1) above.
  • the microorganism having an enhanced activity of the protein according to any one of [8] to [10] has an activity of isomerizing sedheptulose-7-phosphate to D-alloheptulose-7-phosphate. This activity of the protein has been revealed for the first time in the present invention.
  • microorganisms examples include MG 1655 / pUC19_alsE and MGC / pUC19_alsE described later in the examples.
  • (2B) A microorganism that produces sedheptulose-7-phosphate from a sugar having enhanced allulose-6-phosphate 3-epimerase activity as compared to the parent strain “to produce sedheptulose-7-phosphate from a sugar” When cultured in a medium, it means that sedoheptulose-7-phosphate is produced and accumulated in cells of the microorganism using sugar as a starting substrate.
  • the parent strain may be a wild strain as long as it is a microorganism that produces sedheptulose-7-phosphate from sugar, or an artificial strain if the wild strain does not have the ability to produce sedheptulose-7-phosphate from sugar.
  • it may be a breeding strain imparted with the ability to produce cedoheptulose-7-phosphate from sugar.
  • Microorganisms that produce D-alloheptulose-7-phosphate from sugars that have enhanced allulose-6-phosphate 3-epimerase activity than the parent strain can be obtained by the same method as described in 1 (2) above. it can.
  • microorganisms that produce D-alloheptulose-7-phosphate from sugar include microorganisms that have reduced or lost 6-phosphofructokinase activity compared to the parent strain.
  • a microorganism having a reduced or lost 6-phosphofructokinase activity compared to the parent strain can be obtained by the same method as described in 1 (4) above. It can confirm by the method similar to the method of description.
  • Allulose-6-phosphate 3-epimerase activity is enhanced compared to the parent strain, and further, 6-phosphofructokinase activity is reduced or lost, reducing the flow of carbon to the central metabolism and entering the pentose phosphate pathway. It is considered that the productivity of D-alloheptulose is improved by increasing the amount of inflow.
  • microorganisms examples include MG1655 ⁇ als ⁇ pfkA / pUC19_alsE and MGC ⁇ als ⁇ pfkA / pUC19_alsE described later in the Examples.
  • (2C) A microorganism that produces sedoheptulose-7-phosphate from sugar and D-alloheptulose from D-alloheptulose-7-phosphate, which has enhanced allulose-6-phosphate 3-epimerase activity compared to the parent strain.
  • “Microorganism producing D-alloheptulose from D-alloheptulose-7-phosphate” refers to D-alloheptulose-7-phosphate as a starting substrate when D-alloheptulose-7-phosphate is used as a starting substrate. It means generating and accumulating.
  • “generate sedoheptulose-7-phosphate from sugar and D-alloheptulose from D-alloheptulose-7-phosphate” means that sedoheptulose-7-phosphate is generated from sugar and sedoheptulose-7 -Production of D-alloheptulose using D-alloheptulose-7-phosphate obtained by isomerizing phosphoric acid as a substrate.
  • the parent strain may be a wild strain as long as it is a microorganism that produces sedheptulose-7-phosphate from sugar and D-alloheptulose-7-phosphate from D-alloheptulose.
  • microorganisms that produce D-alloheptulose from D-alloheptulose-7-phosphate include microorganisms in which the activity of the protein according to any one of the following [11] to [13] is enhanced as compared with the parent strain: .
  • a protein comprising the amino acid sequence represented by SEQ ID NO: 1 [12] consisting of an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1, and A mutant protein having D-psicose-6-phosphate dephosphorylation activity [13] consisting of an amino acid sequence having 95% or more identity with the amino acid sequence represented by SEQ ID NO: 1, and D-psicose-6-phosphorus Homologous protein with acid dephosphorylation activity
  • a microorganism in which the activity of the protein according to any one of [11] to [13] is enhanced as compared with the parent strain can be obtained by a method similar to the method described in 1 (1) above. It can be confirmed by a method similar to the method described in 1 (1) above.
  • microorganisms examples include MGC / pUC19_alsE and MGC ⁇ als ⁇ pfkA / pUC19_alsE described later in Examples.
  • Production method of D-alloheptulose of the present invention 4.1. Method for producing D-alloheptulose using sugar as a substrate Among the methods for producing D-alloheptulose of the present invention, (i) the above-mentioned 3.
  • a culture of the microorganisms (2A) to (2C) hereinafter referred to as 4.1 microorganisms described in 1.
  • a method for producing D-alloheptulose characterized in that it is present in a medium, D-alloheptulose is produced and accumulated in the aqueous medium, and D-alloheptulose is collected from the aqueous medium, will be described below.
  • the method described in 2.1 above can be used as the method for culturing the microorganism of 4.1.
  • 4.1 Microorganism culture obtained by culturing microorganisms or treated product thereof, and a dephosphorylation enzyme as an enzyme source, the enzyme source and sugar are present in an aqueous medium, and the aqueous medium D-alloheptulose can be produced by producing and accumulating D-alloheptulose therein and collecting D-alloheptulose from the medium.
  • the concentrate of the culture, the dried product of the culture, the culture is centrifuged, filtered, or the like Cells, dried product of the cells, freeze-dried product of the cells, surfactant-treated product of the cells, solvent-treated product of the cells, enzyme-treated product of the cells, and the What contains a living microbial cell that retains the same function as the culture as an enzyme source such as an immobilized product of the microbial cell, as well as a sonicated product of the microbial cell, a mechanically ground processed product of the microbial cell, Examples include crude enzyme extracts obtained from the treated cells and purified enzymes obtained from the treated cells.
  • any dephosphorylating enzyme may be used as long as it has an activity to dephosphorylate D-alloheptulose-7-phosphate.
  • the parent strain described in 1 (1) above originally has The phosphatase is preferably a phosphatase encoded by the yniC gene.
  • a chelating agent such as phytic acid, a surfactant, or an organic solvent may be added as necessary.
  • a surfactant and the organic solvent those described in 2.1 can be used.
  • D-alloheptulose produced in an aqueous medium can be quantified using a sugar analyzer manufactured by Dionex [Anal. Biochem., 189, 151 (1990)]. D-alloheptulose produced in the reaction solution can be collected by a usual method using activated carbon, ion exchange resin or the like.
  • D-alloheptulose-7-phosphate is produced from sugar, which has enhanced allulose-6-phosphate 3-epimerase activity compared to the parent strain.
  • a method for producing D-alloheptulose which comprises culturing a microorganism to be cultured in a medium, producing and accumulating D-alloheptulose in the culture, and collecting D-alloheptulose from the culture, is described below.
  • the method for culturing the microorganism can be performed according to a usual method used for culturing microorganisms.
  • a medium for culturing the microorganism a medium containing a receptor carbohydrate in the medium described in 2.1 above can be used.
  • D-alloheptulose can be produced by producing and accumulating D-alloheptulose in the culture by the above culture and collecting D-alloheptulose from the culture.
  • a method for quantifying D-alloheptulose the method described in 4.1 above can be used.
  • the collection of D-alloheptulose from the culture can be usually carried out by a combination of ion exchange resin method, precipitation method and other known methods.
  • D-alloheptulose accumulates in the microbial cells, for example, the microbial cells are crushed by ultrasonic waves, and the microbial cells are removed by centrifugation. Can be collected.
  • Example 1 Construction of microorganisms used in the production of D-psicose (1) Acquisition of DNA fragments used as markers in gene deletion and gene replacement Table 1 shows the DNA comprising the nucleotide sequence represented by “Primer set” in Table 1 as a primer set. PCR was carried out using the DNA described in the “template” as a template to amplify each DNA fragment.
  • Bacillus subtilis 168 strain genomic DNA was prepared by a conventional method.
  • the cat of the amplified DNA fragment contains about 200 bp upstream to about 100 bp downstream of the cat gene.
  • the amplified DNA fragment sacB contains about 300 bp upstream to about 100 bp downstream of the sacB gene.
  • a SalI recognition site is given to DNA consisting of the base sequences represented by SEQ ID NOs: 38 and 40.
  • the cat and sacB of the amplified DNA fragment were cut with the restriction enzyme SalI, and DNA ligation Kit Ver. 2 (manufactured by Takara Bio Inc.). PCR is performed using the ligation reaction solution as a template and the DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 39 and 41 as a primer set, and a DNA fragment containing the cat gene and sacB gene (hereinafter referred to as cat-sacB) fragment.
  • cat-sacB a DNA fragment containing the cat gene and sacB gene
  • AlsK upstream 1 and alsK upstream 2 contain about 1000 bp upstream from the start codon of the alsK gene.
  • rpiB downstream 1 and rpiB downstream 2 contain approximately 1000 bp downstream from the stop codon of the rpiB gene.
  • PCR was performed using as a template a mixture of alsK upstream 1, rpiB downstream 1, and cat-sacB fragment at equimolar ratios, and using a DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 3 and 6 as a primer set, A DNA fragment (hereinafter referred to as “als :: cat-sacB”) containing an allose-utilizing operon into which a cat-sacB fragment was inserted was obtained.
  • PCR is carried out using a mixture of alsK upstream 2 and rpiB downstream 2 in equimolar ratio as a template, and using DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 3 and 6 as a primer set, and rpiB, ripR, alsA, A DNA (hereinafter referred to as ⁇ als) fragment containing the region around the allose-utilizing operon lacking alsB, alsC, alsE, and alsK was obtained.
  • the als :: cat-sacB fragment was transformed into plasmid pKD46 [Datsenko, KA, Warner, BL, Proceedings of the National Academy of Science of the United States of America, Vol. 97. 6640-6645 ( 2000)] is transferred to the Escherichia coli MG1655 strain by electroporation, and a chloramphenicol resistant and sucrose sensitive transformant (allose-utilizing operon is transformed into als :: cat-sacB). A substituted transformant) was obtained.
  • the ⁇ als fragment was introduced into the transformant by electroporation to obtain a transformant exhibiting chloramphenicol sensitivity and sucrose resistance (als :: cat-sacB replaced with ⁇ als). . Furthermore, a transformant exhibiting ampicillin sensitivity (transformant from which pKD46 had been removed) was obtained.
  • the microorganism was named MG1655 ⁇ als.
  • PfkA upstream 1 and pfkA upstream 2 contain about 700 bp upstream from the start codon of the pfkA gene.
  • pfkA downstream 1 and pfkA downstream 2 contain about 800 bp downstream from the stop codon of the pfkA gene.
  • PCR was carried out using DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 9 and 12 as a primer set, using as a template a mixture of pfkA upstream 1, pfkA downstream 1, and cat-sacB fragment at an equimolar ratio, A DNA fragment (hereinafter referred to as pfkA :: cat-sacB) containing the pfkA peripheral region into which the cat-sacB fragment was inserted was obtained.
  • pfkA a DNA fragment
  • PCR was performed using a mixture of pfkA upstream 2 and pfkA downstream 2 in equimolar ratio as a template, using the DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 9 and 12 as a primer set, and pfkA gene lacking pfkA gene A DNA fragment (hereinafter referred to as ⁇ pfkA) containing the peripheral region was obtained.
  • the ⁇ pfkA fragment was introduced into the transformant by electroporation to obtain a transformant showing chloramphenicol sensitivity and sucrose resistance (transformant in which pfkA :: cat-sacB was replaced with ⁇ pfkA). It was. Furthermore, a transformant from which pKD46 was eliminated was obtained.
  • the microorganism was named MG1655 ⁇ als ⁇ pfkA.
  • telomere sequence For the trp promoter, use pTK31 (APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2007, Vol. 73, No. 20, p. 6378-6385) as a template, and for other amplified DNA fragments, use the genomic DNA of Escherichia coli MG1655 strain as a template. PCR was performed using DNA consisting of the base sequence represented by “Primer set” in Table 4 as a primer set, and each DNA fragment was amplified.
  • YniC upstream 1 and yniC upstream 2 contain about 1000 bp upstream from the start codon of the yniC gene.
  • the yniC downstream 1 contains about 1000 bp downstream from the stop codon of the yniC gene.
  • the yniC downstream 2 contains about 1000 bp downstream from the start codon of the yniC gene to the stop codon.
  • PCR was carried out using as a template a mixture of yniC upstream 1, yniC downstream 1, and cat-sacB fragment at equimolar ratios, and using a DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 17 and 20 as a primer set, A DNA fragment (hereinafter referred to as yniC :: cat-sacB) fragment containing the region surrounding yniC into which the cat-sacB fragment was inserted was obtained.
  • a Ptrp-yniC fragment was introduced into the transformant by electroporation, and a transformant exhibiting chloramphenicol sensitivity and sucrose resistance (yniC :: cat-sacB was replaced with Ptrp-yniC) Body). Furthermore, a transformant from which pKD46 was eliminated was obtained.
  • the microorganism was named MGC.
  • PCR was performed using pTK31 as a template, and other fragments using Escherichia coli MG1655 genomic DNA as a template and DNA consisting of the nucleotide sequence represented by “Primer set” in Table 5 as a primer set. Each DNA fragment was amplified.
  • the ybiV upstream 1 and the ybiV upstream 2 of the amplified DNA fragment contain about 1000 bp upstream from the start codon of the ybiV gene.
  • ybiV downstream 1 contains approximately 1000 bp downstream from the stop codon of the ybiV gene.
  • YbiV downstream 2 contains about 1000 bp downstream from the start codon of the ybiV gene to the stop codon.
  • PCR is carried out using a mixture of equimolar ratios of ybiV upstream 1, ybiV downstream, and cat-sacB fragment as a template, using the DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 29 and 32 as a primer set, and cat A DNA fragment (hereinafter referred to as ybiV :: cat-sacB) containing the region surrounding ybiV in which the sacB fragment was inserted was obtained.
  • a transformant in which the ybiV :: cat-sacB fragment was introduced into Escherichia coli MG1655 strain carrying pKD46 by electroporation and showed chloramphenicol resistance and sucrose sensitivity (ybiV gene was ybiV :: a transformant substituted with cat-sacB).
  • a Ptrp-ybiV fragment was introduced into the transformant by electroporation, and a transformant showing chloramphenicol sensitivity and sucrose resistance (ybiV :: cat-sacB was replaced with Ptrp-ybiV) Body). Furthermore, a transformant from which pKD46 was eliminated was obtained.
  • the microorganism was named MGV.
  • MGV ⁇ als ⁇ pfkA in which the trp promoter was inserted upstream of the ybiV gene of MG1655 ⁇ als ⁇ pfkA was constructed in the same manner.
  • MUC, MGV, MGC ⁇ als ⁇ pfkA and MGV ⁇ als ⁇ pfkA were transformed with pUC19_alsE to obtain MGC / pUC19_alsE, MGV / pUC19_alsE, MGC ⁇ als ⁇ pfkA / pUC19pAlsEp19As
  • Escherichia coli MG1655 and MG1655 ⁇ als ⁇ pfkA were transformed with pUC19_alsE to obtain MG1655 / pUC19_alsE and MG1655 ⁇ als ⁇ pfkA / pUC19_alsE, respectively.
  • Example 2 Manufacture of D-psicose The cells were cultured overnight, inoculated into a large test tube containing 5 mL of LB medium, and cultured with shaking at 30 ° C. for 12 hours. 100 mg / L ampicillin was added as needed.
  • Test tube production medium [glucose 20 g / L, magnesium sulfate heptahydrate 2 g / L, casamino acid 5 g / L, ammonium sulfate 2 g / L, citric acid 1 g / L, potassium dihydrogen phosphate 14 g / L, hydrogen hydrogen phosphate Potassium 16 g / L, thiamine hydrochloride 10 mg / L, ferrous sulfate heptahydrate 50 mg / L, manganese sulfate pentahydrate 10 mg / L, adjusted to pH 7.2 with sodium hydroxide solution, glucose and magnesium sulfate The heptahydrate aqueous solution is separately autoclaved, cooled, and mixed.
  • the culture solution was appropriately diluted and centrifuged, and the psicose concentration in the supernatant was quantified with a sugar analyzer ICS-5000 (manufactured by Thermo Fisher Scientific). The results are shown in Table 6.
  • the yniC gene and the ybiV gene have D-psicose-6-phosphate dephosphorylation activity, and D-psicose-6-phosphate dephosphorylation obtained by transforming the gene. It was found that D-psicose can be efficiently produced by using a microorganism that has enhanced phosphorylation activity and produces D-psicose-6-phosphate from sugar.
  • Example 3 Production of D-psicose using a treated product of the culture of microorganisms MG1655 / pUC19_alsE, MG1655 ⁇ als ⁇ pfkA / pUC19_alsE, MGC / pUC19_alsFs19, MGV / pUC19_als19, MGV / pUC19_als19
  • a large test tube containing 5 ml of LB medium [Bactotryptone (Difco) 10 g / l, Yeast extract (Difco) 5 g / l, Sodium chloride 5 g / l] containing 50 mg / ml of ampicillin. Incubate at 30 ° C. for 16 hours.
  • 10% of the culture solution is inoculated into an Erlenmeyer flask with baffle containing 50 ml of LB medium containing 50 mg / ml of ampicillin, and cultured at 30 ° C. and 220 rpm for 8 hours. Centrifuge 40 ml of the culture solution to obtain wet cells.
  • reaction buffer 0.1 M bicine buffer (pH 8.0), 10 mM manganese chloride
  • reaction buffer 0.1 M bicine buffer (pH 8.0), 10 mM manganese chloride
  • the mixture is centrifuged at 18,000 ⁇ g for 5 minutes to precipitate the membrane fraction, and then the supernatant is collected.
  • the supernatant is quantified for protein using the Bradford method and used for the reaction.
  • the reaction is performed by adding the above-described supernatant protein solution to a reaction buffer containing 12.5 mM glucose to a final concentration of 0.25 mg / ml and shaking at 30 ° C. and 330 rpm.
  • the reaction is terminated by adding a 1/4 amount of 6.7M phosphoric acid.
  • reaction solution After completion of the reaction, the reaction solution is appropriately diluted and centrifuged, and the psicose concentration of the supernatant is quantified with a sugar analyzer ICS-5000 (manufactured by Thermo Fisher Scientific).
  • Example 4 Production of D-alloheptulose MG1655 / pUC19_alsE, MG1655 ⁇ als ⁇ pfkA / pUC19_alsE, MGC / pUC19_alsE, MGV / pUC19_alsE, MGV / pUC19_alsE, MGC ⁇ alp ⁇ 19
  • the cells were cultured overnight at 30 ° C., inoculated into a large test tube containing 5 mL of LB medium, and cultured with shaking at 30 ° C. for 12 hours.
  • Test tube production medium [glucose 20 g / L, magnesium sulfate heptahydrate 2 g / L, casamino acid 5 g / L, ammonium sulfate 2 g / L, citric acid 1 g / L, potassium dihydrogen phosphate 14 g / L, dihydrogen phosphate Potassium 16 g / L, thiamine hydrochloride 10 mg / L, ferrous sulfate heptahydrate 50 mg / L, manganese sulfate pentahydrate 10 mg / L, adjusted to pH 7.2 with sodium hydroxide solution, glucose and magnesium sulfate The heptahydrate aqueous solution is separately autoclaved, cooled, and mixed.
  • Example 5 Production of D-alloheptulose using a processed product of a microorganism culture MG1655 / pUC19_alsE, MG1655 ⁇ als ⁇ pfkA / pUC19_alsE, MGC / pUC19_als19, MGf / pUC19_als19, MGC ⁇ p1919 was inoculated into a large test tube containing 5 ml of LB medium [Bactotryptone (Difco) 10 g / l, Yeast extract (Difco) 5 g / l, Sodium chloride 5 g / l] containing 50 mg / ml of ampicillin. Incubate at 30 ° C. for 16 hours.
  • 10% of the culture solution is inoculated into an Erlenmeyer flask with baffle containing 50 ml of LB medium containing 50 mg / ml of ampicillin, and cultured at 30 ° C. and 220 rpm for 8 hours. Centrifuge 40 ml of the culture solution to obtain wet cells.
  • reaction buffer 0.1 M bicine buffer (pH 8.0), 10 mM manganese chloride
  • reaction buffer 0.1 M bicine buffer (pH 8.0), 10 mM manganese chloride
  • the mixture is centrifuged at 18,000 ⁇ g for 5 minutes to precipitate the membrane fraction, and then the supernatant is collected.
  • the supernatant is quantified for protein using the Bradford method and used for the reaction.
  • the reaction is performed by adding the above-described supernatant protein solution to a reaction buffer containing 12.5 mM glucose to a final concentration of 0.25 mg / ml and shaking at 30 ° C. and 330 rpm.
  • the reaction is terminated by adding a 1/4 amount of 6.7M phosphoric acid.
  • reaction solution After completion of the reaction, the reaction solution is appropriately diluted, centrifuged, and the alloheptulose concentration in the supernatant is quantified with a sugar analyzer ICS-5000 (manufactured by Thermo Fisher Scientific).
  • the method for producing D-psicose of the present invention by using a microorganism that produces D-psicose-6-phosphate from a sugar having enhanced D-psicose-6-phosphate dephosphorylation activity as compared with the parent strain. Therefore, the metabolic system of D-allose possessed by microorganisms can be used efficiently, the production of by-product sugar and the remaining of the substrate can be suppressed, and D-psicose can be produced with high efficiency.
  • D-alloheptulose of the present invention by using a microorganism that produces D-alloheptulose-7-phosphate from a sugar having an allulose-6-phosphate 3-epimerase activity enhanced compared to the parent strain. D-alloheptulose can be produced with high efficiency.

Landscapes

  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

La présente invention vise à procurer un procédé efficace de production d'un sucre rare à l'aide d'un micro-organisme, une culture de micro-organismes ou le produit de la culture transformé. La présente invention concerne les éléments suivants : un procédé de production de D-psicose, au moyen du système métabolique D-allose d'un micro-organisme, au moyen d'un micro-organisme qui produit du D-psicose-6-phosphate à partir d'un sucre, dans lequel l'activité enzymatique de déphosphorylation du D-psicose-6-phosphate est améliorée ; et un procédé de production de D-alloheptulose au moyen d'un micro-organisme dans lequel l'activité allose-6-phosphate 3-épimérase est améliorée.
PCT/JP2016/069724 2015-07-02 2016-07-01 Procédé de production de sucre rare WO2017002978A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP2017526463A JP6993227B2 (ja) 2015-07-02 2016-07-01 希少糖の製造法
JP2021197204A JP7244613B2 (ja) 2015-07-02 2021-12-03 希少糖の製造法

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2015133709 2015-07-02
JP2015-133709 2015-07-02
JP2016-062331 2016-03-25
JP2016062331 2016-03-25

Publications (1)

Publication Number Publication Date
WO2017002978A1 true WO2017002978A1 (fr) 2017-01-05

Family

ID=57608595

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2016/069724 WO2017002978A1 (fr) 2015-07-02 2016-07-01 Procédé de production de sucre rare

Country Status (2)

Country Link
JP (2) JP6993227B2 (fr)
WO (1) WO2017002978A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018129275A1 (fr) * 2017-01-06 2018-07-12 Greenlight Biosciences, Inc. Production acellulaire de sucres
WO2018169957A1 (fr) * 2017-03-13 2018-09-20 Bonumose Llc Production enzymatique d'hexoses
KR20190013558A (ko) * 2017-07-31 2019-02-11 씨제이제일제당 (주) 신규한 싸이코스-6-인산 탈인산효소, 상기 효소를 포함하는 사이코스 생산용 조성물, 상기 효소를 이용하여 사이코스를 제조하는 방법
KR20190094199A (ko) * 2016-12-14 2019-08-12 보너모스 엘엘씨 D-알룰로스의 효소적 생산
US10421953B2 (en) 2013-08-05 2019-09-24 Greenlight Biosciences, Inc. Engineered proteins with a protease cleavage site
WO2020122504A1 (fr) * 2018-12-11 2020-06-18 씨제이제일제당 (주) Enzyme de déphosphorylation d'un nouvel acide psicose-6-phosphorique, composition de production de psicose comprenant celle-ci, et procédé de production de psicose faisant appel à celle-ci
CN111712570A (zh) * 2018-01-25 2020-09-25 中国科学院天津工业生物技术研究所 一种生产阿洛酮糖及其衍生物的工程菌株及其构建方法和应用
RU2776637C2 (ru) * 2017-01-06 2022-07-22 Гринлайт Байосайенсис, Инк. Бесклеточное получение сахаров
CN115074376A (zh) * 2022-04-28 2022-09-20 福州大学 一种利用重组大肠杆菌发酵高效合成d-阿洛酮糖的方法
JP2023500269A (ja) * 2019-10-28 2023-01-05 サムヤン コーポレイション フルクトース－６－リン酸３－エピマー化酵素およびその用途

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LI, ZIJIE ET AL.: "Synthesis of D-Sorbose and D- Psicose by Recombinant Escherichia coli", J. CARBOHYDR. CHEM., vol. 34, 29 July 2015 (2015-07-29), pages 349 - 357, XP055345959, ISSN: 0732-8303 *
WEI, MOHUI ET AL.: "Transforming Flask Reaction into Cell -Based Synthesis: Production of Polyhydroxylated Molecules via Engineered Escherichia coli", ACS CATALYSIS, vol. 5, 29 May 2015 (2015-05-29), pages 4060 - 4065, XP055345956, ISSN: 2155-5435 *
YANG. JIANGANG ET AL.: "Biosynthesis of Rare Ketoses Through Constructing a Recombination Pathway in an Engineered Corynebacterium glutamicum", BIOTECHNOL. BIOENG., vol. 112, pages 168 - 180, XP055345951, ISSN: 1097-0290 *
YUKO SHIMANAMI ET AL.: "Daichokin ni yoru D- Psicose no Kojundo Hakko Seisanho no Kaihatsu", PROCEEDINGS OF THE ANNUAL MEETING OF JAPAN SOCIETY FOR BIOSCIENCE, BIOTECHNOLOGY, AND AGROCHEMISTRY, 5 March 2016 (2016-03-05), pages 4F146, ISSN: 2186-7976 *

Cited By (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10421953B2 (en) 2013-08-05 2019-09-24 Greenlight Biosciences, Inc. Engineered proteins with a protease cleavage site
KR20190094199A (ko) * 2016-12-14 2019-08-12 보너모스 엘엘씨 D-알룰로스의 효소적 생산
JP2020513247A (ja) * 2016-12-14 2020-05-14 ボヌモーズ エルエルシー Ｄ−アルロースの酵素的生産
KR102581107B1 (ko) 2016-12-14 2023-09-21 보너모스, 인코포레이티드 D-알룰로스의 효소적 생산
JP2020503059A (ja) * 2017-01-06 2020-01-30 グリーンライト バイオサイエンシーズ インコーポレーテッドＧｒｅｅｎｌｉｇｈｔ Ｂｉｏｓｃｉｅｎｃｅｓ，Ｉｎｃ． 糖の無細胞的生産
RU2776637C2 (ru) * 2017-01-06 2022-07-22 Гринлайт Байосайенсис, Инк. Бесклеточное получение сахаров
WO2018129275A1 (fr) * 2017-01-06 2018-07-12 Greenlight Biosciences, Inc. Production acellulaire de sucres
CN110300800A (zh) * 2017-01-06 2019-10-01 绿光生物科技股份有限公司 糖的无细胞生产
US10704067B2 (en) 2017-01-06 2020-07-07 Greenlight Biosciences, Inc. Cell-free production of sugars
US10577635B2 (en) 2017-01-06 2020-03-03 Greenlight Biosciences, Inc. Cell-free production of sugars
JP7186167B2 (ja) 2017-01-06 2022-12-08 グリーンライト バイオサイエンシーズ インコーポレーテッド 糖の無細胞的生産
US10316342B2 (en) 2017-01-06 2019-06-11 Greenlight Biosciences, Inc. Cell-free production of sugars
WO2018169957A1 (fr) * 2017-03-13 2018-09-20 Bonumose Llc Production enzymatique d'hexoses
US11236320B2 (en) 2017-03-13 2022-02-01 Bonumose, Inc. Enzymatic production of hexoses
US10745683B2 (en) 2017-03-13 2020-08-18 Bonumose Llc Enzymatic production of hexoses
US11345909B2 (en) 2017-03-13 2022-05-31 Bonumose, Inc. Enzymatic production of hexoses
US11993796B2 (en) 2017-03-13 2024-05-28 Bonumose, Inc. Enzymatic production of hexoses
RU2766710C2 (ru) * 2017-03-13 2022-03-15 Бонамоуз, Инк. Ферментативное получение гексоз
JP2020529205A (ja) * 2017-07-31 2020-10-08 シージェイ チェイルジェダン コーポレーションＣｊ Ｃｈｅｉｌｊｅｄａｎｇ Ｃｏｒｐｏｒａｔｉｏｎ 新規のプシコース−６−リン酸脱リン酸酵素、前記酵素を含むプシコース生産用組成物、前記酵素を利用してプシコースを製造する方法
WO2019027173A3 (fr) * 2017-07-31 2019-04-04 씨제이제일제당(주) Nouvelle psicose-6-phosphate phosphatase, composition pour la production de psicose comprenant ladite enzyme, procédé pour la production de psicose au moyen de ladite enzyme
AU2018310072B2 (en) * 2017-07-31 2022-03-10 Cj Cheiljedang Corporation Novel psicose-6-phosphate phosphatase, composition for producing psicose including said enzyme, method for producing psicose using said enzyme
CN111819278A (zh) * 2017-07-31 2020-10-23 Cj第一制糖株式会社 新的阿洛酮糖-6-磷酸磷酸酶、包括所述酶的用于产生阿洛酮糖的组合物、利用所述酶产生阿洛酮糖的方法
CN111819278B (zh) * 2017-07-31 2024-06-18 Cj第一制糖株式会社 新的阿洛酮糖-6-磷酸磷酸酶及其用途
KR20190013558A (ko) * 2017-07-31 2019-02-11 씨제이제일제당 (주) 신규한 싸이코스-6-인산 탈인산효소, 상기 효소를 포함하는 사이코스 생산용 조성물, 상기 효소를 이용하여 사이코스를 제조하는 방법
TWI729305B (zh) * 2017-07-31 2021-06-01 南韓商Ｃｊ第一製糖股份有限公司 阿洛酮糖-6-磷酸磷酸酶、對所述酶進行編碼的核酸、用於生產d-阿洛酮糖的包括所述酶的組成物以及使用所述酶生產d-阿洛酮糖的方法
KR102086494B1 (ko) 2017-07-31 2020-03-10 씨제이제일제당 (주) 신규한 싸이코스-6-인산 탈인산효소, 상기 효소를 포함하는 사이코스 생산용 조성물, 상기 효소를 이용하여 사이코스를 제조하는 방법
CN111712570B (zh) * 2018-01-25 2023-07-28 中国科学院天津工业生物技术研究所 一种生产阿洛酮糖及其衍生物的工程菌株及其构建方法和应用
CN111712570A (zh) * 2018-01-25 2020-09-25 中国科学院天津工业生物技术研究所 一种生产阿洛酮糖及其衍生物的工程菌株及其构建方法和应用
WO2020122504A1 (fr) * 2018-12-11 2020-06-18 씨제이제일제당 (주) Enzyme de déphosphorylation d'un nouvel acide psicose-6-phosphorique, composition de production de psicose comprenant celle-ci, et procédé de production de psicose faisant appel à celle-ci
JP2023500269A (ja) * 2019-10-28 2023-01-05 サムヤン コーポレイション フルクトース－６－リン酸３－エピマー化酵素およびその用途
CN115074376A (zh) * 2022-04-28 2022-09-20 福州大学 一种利用重组大肠杆菌发酵高效合成d-阿洛酮糖的方法
CN115074376B (zh) * 2022-04-28 2023-11-14 福州大学 一种利用重组大肠杆菌发酵高效合成d-阿洛酮糖的方法

Also Published As

Publication number Publication date
JPWO2017002978A1 (ja) 2018-04-12
JP6993227B2 (ja) 2022-01-13
JP2022025157A (ja) 2022-02-09
JP7244613B2 (ja) 2023-03-22

Similar Documents

Publication Publication Date Title
JP7244613B2 (ja) 希少糖の製造法
JP7035024B2 (ja) テアニンの製造方法
JP2017523787A (ja) フィードバック抵抗性アセトヒドロキシ酸シンターゼ変異体及びそれを用いたｌ−バリンの生産方法
JP3946423B2 (ja) Ｎ−アセチルノイラミン酸の製造法
WO2022168992A1 (fr) Protéine ayant une activité de 1,3-fucosyltransférase, et procédé de production de sucre contenant du fucose
JP4318549B2 (ja) Ｎ−アセチルノイラミン酸の製造法
WO2023238843A1 (fr) Micro-organisme présentant une meilleure productivité de 3'-sialyllactose et procédé de production de 3'-sialyllactose
JP2013081404A (ja) 新規ジペプチド合成酵素およびそれを用いたジペプチドの製造法
JP6441806B2 (ja) Ｎ−アセチルノイラミン酸及びｎ−アセチルノイラミン酸含有糖質の製造法
CN116848248A (zh) 具有含有岩藻糖的糖质的转运活性的蛋白和含有岩藻糖的糖质的制造方法
JP5079272B2 (ja) シチジン‐５´‐一リン酸‐ｎ‐アセチルノイラミン酸およびｎ‐アセチルノイラミン酸含有糖質の製造法
WO2023038128A1 (fr) Micro-organisme recombiné utilisé pour produire de la cdp-choline, et procédé de production de cdp-choline utilisant ledit micro-organisme recombiné
WO2023153461A1 (fr) Procédé de production d'oligosaccharide ayant un squelette de lewis x
WO2023182528A1 (fr) Protéine ayant une activité alpha 1,2-fucosyltransférase et procédé de production de lacto-n-fucopentaose i (lnfpi)
WO2021261564A1 (fr) Procédé de production d'un dipeptide
WO2023182527A1 (fr) Procédé de production de lactodifucotétraose (ldft)
JP2013081406A (ja) 新規ジペプチド合成酵素およびそれを用いたジペプチドの製造法
WO2022176994A1 (fr) PROTEINE MODIFIÉE AYANT UNE ACTIVITÉ α1, 2-FUCOSYLTRANSFÉRASE ET PROCÉDÉ DE PRODUCTION D'HYDRATE DE CARBONE CONTENANT DU FUCOSE
WO2023210244A1 (fr) Protéine présentant une activité nampt et procédé de production de nmn
WO2021125245A1 (fr) Micro-organisme ayant une perméase de lactose modifiée, et procédé de production d'un oligosaccharide contenant du lactose
JP2022001556A (ja) 蛋白質及び３−ヒドロキシイソ吉草酸の製造方法
JP2021191241A (ja) β−ポリリンゴ酸の製造法
JP2022045001A (ja) バリオールアミン及びボグリボースの製造方法
JP2021019518A (ja) ビオラセイン又はデオキシビオラセインの製造法
JP2003274948A (ja) Ｕｄｐ−グルクロン酸の製造法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16818086

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2017526463

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16818086

Country of ref document: EP

Kind code of ref document: A1