JP2024008564A - Method for producing amino acid and/or organic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing an amino acid and/or an organic acid.

SOLUTION: The present disclosure provides a method for producing an amino acid and/or an organic acid, wherein the method includes the step for culturing cyanobacteria with a reduced glycogen content. The present disclosure provides a method for producing an amino acid and/or an organic acid, using carbon dioxide as the raw material, wherein the method includes the steps of (a) culturing cyanobacteria with a reduced glycogen content, in the presence of carbon dioxide, and (b) extracting an amino acid and/or an organic acid from the culture medium in the step (a).

SELECTED DRAWING: None

COPYRIGHT: (C)2024,JPO&INPIT

Description

The present disclosure relates to methods of producing amino acids and/or organic acids. More specifically, the present disclosure relates to a method of producing an amino acid and/or an organic acid using cyanobacteria with reduced glycogen content, and a method of producing a chemical product using the amino acid/or organic acid as a raw material.

Currently, amino acids are produced industrially and are widely used in seasonings, feed, medicines, supplements, etc. In such industrial production of amino acids, fermentation production of amino acids is typically carried out using microorganisms such as Corynebacterium, but since it is necessary to supply sugars such as glucose as a carbon source, , its production cost is high.

The present inventors have discovered that by using cyanobacteria with reduced glycogen content, a large amount of various amino acids are released outside the cells. Based on this knowledge, the present disclosure provides a method for producing amino acids and/or organic acids using cyanobacteria with reduced glycogen content.

Accordingly, the present disclosure provides:
(Item 1)
A method for producing amino acids and/or organic acids, comprising:
A method comprising culturing cyanobacteria with reduced glycogen content.
(Item 2)
The method according to the above item, wherein the cyanobacteria is genetically modified to have a reduced glycogen content compared to wild-type cyanobacteria grown under the same conditions.
(Item 3)
The method according to any one of the above items, wherein the cyanobacteria include cyanobacteria in which enzyme activity contributing to glycogen synthesis is suppressed or deficient, and/or cyanobacteria in which enzyme activity contributing to glycogen decomposition is enhanced.
(Item 4)
The enzyme contributing to glycogen synthesis includes glucose-1-phosphate adenylyltransferase (GlgC) or a similar enzyme thereof, and glycogen synthase (GlgA) or a similar enzyme thereof, according to any one of the above items. Method.
(Item 5)
The method according to any one of the above items, wherein the enzyme contributing to glycogenolysis includes glycogen phosphorylase (GlgP) or an enzyme similar thereto.
(Item 6)
The method according to any one of the above items, wherein the blue-green algae is a blue-green algae that can inducibly express a gene encoding ppGpp synthase.
(Item 7)
The method according to any one of the above items, wherein the gene encoding the ppGpp synthase includes the yjbM gene derived from Bacillus subtilis.
(Item 8)
The amino acids and/or organic acids include alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, cysteine, A method according to any one of the preceding items, comprising pyruvic acid, 2-oxoglutaric acid, lactic acid, malic acid, succinic acid, and fumaric acid.
(Item 9)
The method according to any one of the above items, wherein the amino acid and/or organic acid comprises an aromatic amino acid.
(Item 10)
The method according to any one of the above items, wherein the cyanobacteria consume carbon dioxide as a carbon source.
(Item 11)
A method for producing p-coumaric acid, comprising the step of converting an amino acid produced by the method according to any one of the above items into p-coumaric acid.
(Item A1)
A method for producing amino acids and/or organic acids using carbon dioxide as a raw material, the method comprising:
(a) culturing cyanobacteria with reduced glycogen content in the presence of carbon dioxide;
(b) obtaining an amino acid and/or an organic acid from the culture solution of step (a).
(Item A2)
The method according to any one of the above items, wherein the cyanobacteria is genetically modified to have a reduced glycogen content compared to wild-type cyanobacteria grown under the same conditions.
(Item A3)
The method according to any one of the above items, wherein the cyanobacteria include cyanobacteria in which enzyme activity contributing to glycogen synthesis is suppressed or deficient, and/or cyanobacteria in which enzyme activity contributing to glycogen decomposition is enhanced.
(Item A4)
The enzyme contributing to glycogen synthesis includes glucose-1-phosphate adenylyltransferase (GlgC) or a similar enzyme thereof, and glycogen synthase (GlgA) or a similar enzyme thereof, according to any one of the above items. Method.
(Item A5)
The method according to any one of the above items, wherein the enzyme contributing to glycogenolysis includes glycogen phosphorylase (GlgP) or an enzyme similar thereto.
(Item A6)
The method according to any one of the above items, wherein the blue-green algae is a blue-green algae that can inducibly express a gene encoding ppGpp synthase.
(Item A7)
The method according to any one of the above items, wherein the gene encoding the ppGpp synthase includes the yjbM gene derived from Bacillus subtilis.
(Item A8)
The amino acids and/or organic acids include alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, cysteine, A method according to any one of the preceding items, comprising pyruvic acid, 2-oxoglutaric acid, lactic acid, malic acid, succinic acid, and fumaric acid.
(Item A9)
The method according to any one of the above items, wherein the amino acid and/or organic acid comprises an aromatic amino acid.
(Item A10)
The method according to any one of the above items, wherein the cyanobacteria consume carbon dioxide as a carbon source.
(Item A11)
A method for producing p-coumaric acid, comprising the step of converting an amino acid produced by the method according to any one of the above items into p-coumaric acid.
(Item B1)
A method for producing amino acid-containing or amino acid-based chemical products using carbon dioxide as a raw material, the method comprising:
(a) culturing cyanobacteria with reduced glycogen content in the presence of carbon dioxide;
(b) obtaining amino acids from the culture solution of step (a);
(c) a step of producing a chemical product using the amino acid as a raw material.
(Item B2)
The method according to any one of the above items, wherein the cyanobacteria is genetically modified to have a reduced glycogen content compared to wild-type cyanobacteria grown under the same conditions.
(Item B3)
The method according to any one of the above items, wherein the cyanobacteria include cyanobacteria in which enzyme activity contributing to glycogen synthesis is suppressed or deficient, and/or cyanobacteria in which enzyme activity contributing to glycogen decomposition is enhanced.
(Item B4)
The enzyme contributing to glycogen synthesis includes glucose-1-phosphate adenylyltransferase (GlgC) or a similar enzyme thereof, and glycogen synthase (GlgA) or a similar enzyme thereof, according to any one of the above items. Method.
(Item B5)
The method according to any one of the above items, wherein the enzyme contributing to glycogenolysis includes glycogen phosphorylase (GlgP) or an enzyme similar thereto.
(Item B6)
The method according to any one of the above items, wherein the blue-green algae is a blue-green algae that can inducibly express a gene encoding ppGpp synthase.
(Item B7)
The method according to any one of the above items, wherein the gene encoding the ppGpp synthase includes the yjbM gene derived from Bacillus subtilis.
(Item B8)
The amino acids and/or organic acids include alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, cysteine, A method according to any one of the preceding items, comprising pyruvic acid, 2-oxoglutaric acid, lactic acid, malic acid, succinic acid, and fumaric acid.
(Item B9)
The method according to any one of the above items, wherein the amino acid and/or organic acid comprises an aromatic amino acid.
(Item B10)
The method according to any one of the above items, wherein the cyanobacteria consume carbon dioxide as a carbon source.
(Item B11)
The method according to any one of the above items, wherein the chemical product comprises a bioplastic raw material, a food additive, a cosmetic additive, and/or a nutrient source for cell culture.

In the present disclosure, it is intended that one or more of the features described above may be provided in further combinations in addition to the combinations specified. Additionally, additional embodiments and advantages of the present disclosure will be apparent to those skilled in the art upon reading and understanding the following detailed description, as appropriate.

Note that features and significant actions and effects of the present disclosure other than those described above will become clear to those skilled in the art by referring to the following embodiments section and drawings.

According to the present disclosure, cyanobacteria produce amino acids through photosynthesis and release large amounts of various amino acids outside the cells, making it possible to construct an amino acid production system with significantly less environmental impact than conventional amino acid fermentation methods. can.

FIG. 1 is a schematic diagram showing the glycogen biosynthesis pathway in cyanobacteria. FIG. 2 is a schematic diagram showing an introduction construct for a cyanobacteria strain in which glycogen synthesis has been inactivated, according to an embodiment of the present disclosure. FIG. 3 is a schematic diagram showing a method for culturing cyanobacteria of the present disclosure in an embodiment of the present disclosure. FIG. 4 is a graph showing cell proliferation and glycogen accumulation of cyanobacteria in one embodiment of the present disclosure. FIG. 5 is a graph showing the amount of amino acids excreted from the blue-green algae of the present disclosure in an embodiment of the present disclosure. FIG. 6 is a graph showing the amount of amino acids excreted from the blue-green algae of the present disclosure in an embodiment of the present disclosure. FIG. 7 is a graph showing the amount of organic acid discharged from the blue-green algae of the present disclosure in an embodiment of the present disclosure.

Hereinafter, the present disclosure will be described while showing the best mode. Throughout this specification, references to the singular should be understood to include the plural unless specifically stated otherwise. Accordingly, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to also include the plural concept, unless specifically stated otherwise. Further, it should be understood that the terms used herein have the meanings commonly used in the art unless otherwise specified. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification (including definitions) will control.

Definitions of terms particularly used in this specification and/or basic technical contents will be explained below as appropriate.

As used herein, "about" means ±10% of the following numerical value.

As used herein, the term "amino acid" is broadly interpreted and refers to a compound having an amino group (-NH ₂ ) and a carboxy group (-COOH) in one molecule. As used herein, "amino acid" includes an organic compound having an amino group (NH ₂ ) and a carboxyl group (COOH) in the form of a salt, and an organic compound having an amino group (NH 2 ) and a carboxyl group (COOH); These include those that are used for bonds (for example, amide bonds, ester bonds) (for example, those in proteins), and those that contain imino groups instead of amino groups (for example, proline). In certain embodiments, an "amino acid" can be an alpha-amino acid, where the amino and carboxyl groups are attached to the same carbon atom. As used herein, "amino acid" refers to amino acids that mainly constitute proteins found in living organisms (alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine). Amino acids can be classified based on the type of side chain, including basic amino acids (lysine, arginine, histidine), acidic amino acids (aspartic acid, glutamic acid), neutral amino acids (asparagine, alanine, isoleucine, glycine, Glutamine, cysteine, threonine, serine, tyrosine, phenylalanine, proline, valine, methionine, leucine), aromatic amino acids (tyrosine, phenylalanine, tryptophan, histidine), hydroxyl amino acids (serine, threonine, tyrosine), and sulfur-containing amino acids (cysteine) , methionine), etc. Amino acids of the present disclosure include natural amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to natural amino acids. Amino acid analog refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, ie, hydrogen, carboxyl group, amino group, R group, etc. Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as the natural amino acid. Amino acid mimetics refer to chemical compounds that have a structure that differs from the general chemical structure of amino acids, but that function in a manner similar to natural amino acids.

As used herein, "aromatic amino acid" means a hydrophobic amino acid having an aromatic ring and an aromatic heterocycle. Aromatic amino acids include phenylalanine, tryptophan, tyrosine, and the like.

In this specification, "organic acid" is a general term for organic compounds exhibiting acidity. "Organic acids" include carboxylic acids having a carboxy group and sulfonic acids having a sulfo group. Carboxylic acids are further classified according to the number of carboxy groups in the molecule, including monocarboxylic acids (e.g., formic acid, acetic acid, propionic acid, quinic acid, etc.), dicarboxylic acids (e.g., oxalic acid, malonic acid, succinic acid, malic acid, etc.). , tartaric acid, etc.), tricarboxylic acids (for example, citric acid, aconitic acid, etc.), and the like. Furthermore, carboxylic acid also includes sugar acids (eg, ascorbic acid, gluconic acid, glucuronic acid, tartaric acid, etc.), which are compounds in which the oxygen group of a monosaccharide is substituted with carboxylic acid.

As used herein, "reduction" is interpreted broadly and means any negative change that results in a lesser amount of any molecule, substance, compound, composition, or activity thereof. For example, "reduced" includes a change such that it is less than its natural state or before any treatment or modification or the like. "Decrease" includes any decrease in a statistically significant amount, including a decrease in individual values, median values, or mean values. Thus, a decrease is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 % decrease.

As used herein, "enzyme activity" is broadly interpreted and refers to the catalytic effect exerted by an enzyme. In this specification, a change in "enzyme activity" includes a change in the catalytic effect of an enzyme due to the amount of transcription (mRNA amount) or translation amount (amount of protein) of a gene encoding an enzyme or protein.

As used herein, "suppression" or "deficiency" refers to gene expression (which may be at the transcription level or translation level) or the function of the gene product that is lower than its natural state or before modification. Suppression or deficiency of enzyme activity refers to a decrease in the expression level (either transcription level or translation level) of the gene encoding the enzyme or the function of the gene product.

As used herein, "enhancement" refers to an increase in gene expression (at the transcription level or translation level) or the function of the gene product compared to the natural state or before modification, and the enzyme activity Enhancement refers to an increase in the expression level (either transcription level or translation level) of the gene encoding the enzyme or the function of the gene product.

As used herein, "modification" refers to insertion of a base sequence into intracellular DNA, deletion of a portion of the base sequence of intracellular DNA, or a combination thereof.

As used herein, the term "modified organism" refers to an organism whose DNA base sequence has been modified relative to a reference organism. Reference organisms include, but are not limited to, naturally occurring organisms and organisms that are only recognized as such after being modified.

As used herein, "gene" refers to a factor that defines genetic traits, and "gene" may refer to "polynucleotide," "oligonucleotide," and "DNA." The gene may be endogenous or exogenous in the target organism. Furthermore, these known genes can be used as appropriate. As a gene, it can be used regardless of its origin. In other words, genes may be derived from organisms of other species or genera other than the target organism, or may be derived from organisms such as animals, plants, fungi (molds, etc.), and bacteria. It may be something. Information regarding such genes can be appropriately obtained by those skilled in the art by accessing websites such as NCBI (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov). These genes may be genes encoding proteins that have a certain relationship with sequence information disclosed in databases, etc., as long as they have each activity. In one such embodiment, a gene encoding a protein having the activity to be enhanced by the present invention is composed of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted, or added to the disclosed amino acid sequence. Can be mentioned. The amino acid mutations to the disclosed amino acid sequence, ie, deletions, substitutions, insertions, or additions, may be of any one type or a combination of two or more types. Further, the total number of these mutations is not particularly limited, but is preferably about 1 or more and 10 or less. More preferably, the number is 1 or more and 5 or less. Examples of amino acid substitutions are preferably conservative substitutions, and specifically include substitutions within the following groups. (glycine, alanine) (valine, isoleucine, leucine) (aspartic acid, glutamic acid) (asparagine, glutamine) (serine, threonine) (lysine, arginine) (phenylalanine, tyrosine).

As used herein, "introducing" a gene refers to introducing an exogenous or endogenous gene, preferably a functional gene, into, for example, a chromosomal genome using an appropriate introduction technique. Genes can be introduced using vectors such as phages and plasmids, and natural transformation methods, conjugation methods, protoplast-PEG methods, electroporation methods, and the like can also be used. Furthermore, by using targeted gene recombination methods known in the art, it is also possible to introduce an exogenous functional gene by replacing it with an endogenous functional gene. Note that the foreign functional gene is a gene that does not originally exist in the chromosomal genome of the organism, and may be a gene derived from another biological species or a synthetic gene produced by PCR or the like. Gene introduction also includes converting an existing genome into a desired gene by performing genome editing.

(Preferred embodiment)
Preferred embodiments of the present disclosure will be described below. The embodiments provided below are provided for a better understanding of the present disclosure, and the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can take the description in this specification into account and make appropriate modifications within the scope of the present disclosure. Further, the following embodiments of the present disclosure can be used alone or in combination.

In one aspect of the present disclosure, a method for producing amino acids and/or organic acids is provided, the method comprising culturing cyanobacteria with reduced glycogen content.

In one embodiment, in the methods of the present disclosure, culturing the cyanobacteria comprises culturing under phototrophic conditions or photoautotrophic conditions. Phototrophic conditions are used in a general sense and refer to conditions under which organisms grow and produce organic matter through photosynthesis using carbon dioxide as a carbon source. Photoautotrophic conditions are used in a general sense and refer to conditions under which organisms grow and produce organic matter through photosynthesis using only carbon dioxide as a carbon source. Therefore, in one embodiment of the present disclosure, the cyanobacteria used in the method of the present disclosure may consume carbon dioxide as a carbon source. The light irradiation conditions under phototrophic conditions may be either natural light or artificial light, and the intensity can be adjusted as appropriate depending on the density of algal bodies in the aqueous medium, the depth of the culture tank, etc. For example, natural or artificial light of 30-2000 μmol photons/m ² /s, preferably 30-1000 μmol photons/m ² /s, more preferably 50-600 μmol photons/m ² /s may be used. Within the above range, cyanobacteria can photosynthesize and multiply smoothly. Light irradiation may be continuous or periodic. For outdoor large-scale cultivation, only sunlight may be used as a light source, so that a day/night cycle exists, to minimize costs and avoid the additional expense of artificial lighting.

The medium may be any aqueous solution that can be used for seed culture and/or main culture, and is preferably an aqueous solution containing a nitrogen source, an inorganic salt, etc., as described below. Specifically, depending on the type of blue-green algae, artificial or natural seawater or fresh water (for example, distilled water) may be used as the aqueous medium. For example, use BG-11 medium (J Gen Microbiol 111: 1-61 (1979)); HSM medium and TAP medium (Cryogenic Science, 67:17-21 (2009)), Cramer-Myers medium (CM medium), etc. can do.

In one embodiment, the cyanobacteria used in the methods of the present disclosure can be cultured consuming only carbon dioxide as a carbon source, so there is no need to add organic materials such as lactose or glucose to the medium.

In one embodiment, the culture temperature of the cyanobacteria of the present disclosure is usually about 15 to about 40°C, preferably about 20 to about 37°C, more preferably about 25 to about 35°C. The pH of the aqueous medium is adjusted to any pH suitable for the growth of cyanobacteria, for example, about pH 5 to about 10, preferably about pH 6 to about 9, more preferably about pH 6 to about 8. Can be done. The pH can be adjusted as appropriate by adding carbon dioxide, hydrochloric acid, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, calcium hydroxide, magnesium hydroxide, and the like.

Regarding the method for producing amino acids and/or organic acids of the present disclosure, the cyanobacteria used in the method of the present disclosure can be precultured in advance. For example, culture can be performed through steps such as (1) pre-culture, (2) pre-culture, and (3) main culture. Aerated culture can be performed at any culture stage. For aerated culture, air or air mixed with carbon dioxide can be aerated into the aqueous medium. The culture period is (1) for pre-pre-culture, about 4 days to about 10 days, preferably about 4 days to about 7 days, more preferably about 5 days to about 6 days (OD750 is about 1.5 to about 2.0), (2) Preculture for about 3 days to about 10 days, preferably about 3 days to about 6 days, more preferably about 4 days to about 5 days (OD750 is about 1.5 to about 2 .0), (3) In the main culture, about 1 day to about 20 days, preferably about 4 days to about 16 days, more preferably about 8 days to about 14 days (OD750 is about 3 to about 10). be.

In one embodiment, as described below, when using cyanobacteria in which the expression of a gene encoding guanosine 4-phosphate (ppGpp) synthase is promoted, the timing to start inducing ppGpp synthesis is from the start of the main culture. After that, for example, about 48 hours (about 2 days) to about 120 hours (about 5 days), more preferably about 72 hours (about 3 days) to about 96 hours (about 4 days). Can be done.

By culturing as described above, amino acids and/or organic acids are released from the cyanobacteria used in the method of the present disclosure into the aqueous medium. In one embodiment, an enzyme having only the synthetic activity of ppGpp and hardly catalyzing its decomposition (for example, YjbM derived from Bacillus subtilis) is used as the ppGpp synthase, thereby reducing the intracellular ppGpp concentration. The amount of amino acids and/or organic acids released outside the cell can be increased through an increase in .

In one embodiment, various cyanobacteria can be used as the cyanobacteria. Blue-green algae (cyanobacteria) are typically single-celled prokaryotes that perform oxygenic photosynthesis, and can be classified into orders such as Chroococcalales, Nostocales, Uremales, Pleurocapsales, and Stigonematales. can. The Chloococcal orders include the genus Aphanocapsa, the genus Aphanotheke, the genus Camaesiphon, the genus Chloococcus, the genus Crocosphaera, the genus Cyanobacteria, the genus Cyanobium, the genus Cyanotheke, the genus Dactylococcopsis, the genus Gloeobacter, the genus Gloeocapsa, the genus Gloeotheke, and the genus Euhalotheke. The genus Haloteke, Johannes Baptistia, Melismopedia, Microcystis, Rhabdoderma, Synechococcus, Synechocystis, Thermosynechococcus, etc. The Nostocales include Coleodesmium, Fremiella, Microchaete, Lexia, Spirillestis, Tryposhrix, Anabaena, Anabaenopsis, Aphanizomenon, Aurosila, Cyanospira, Cylindrospermopsis, and Cylindro. The genus Spermum, Nodularia, Nostoc, Lychelia, Callothrix, Gloeotrychia, Skitonema, etc. are included. The order Arthrospira, Geitrelinema, Halomicronema, Halospirulina, Catagnimene, Leptolymphia, Limnothrix, Lymbya, Microcoleus, Uremo, Phormidium, Planctotricoides, and Planktos. The genus Rix, Plectonema, Limnothrix, Pseudoanabena, Schizothrix, Spirulina, Symproca, Trichodesmium, Ciconema, etc. are included. The Pleurocapsales include the genus Chloococcidiopsis, the genus Dermocarpa, the genus Dermocarpera, the genus Myxosarcina, the genus Pleurocapsa, the genus Stanieria, the genus Xenococcus, and the like. The Stygonema order includes Capsocilla, Chlorogloeopsis, Fischerella, Hapalosiphon, Mastigochladopsis, Mastigochladus, Nostochhopsis, Stigonema, Symphionema, Symphionemopsis, Umezakia, and Westi. Includes the genus Elopsis.

In one embodiment, the cyanobacteria used in the methods of the present disclosure can be genetically modified to have reduced glycogen content compared to wild-type cyanobacteria grown under the same conditions. . Examples of such cyanobacteria include cyanobacteria in which enzyme activities that contribute to glycogen synthesis, such as glycogen synthase, are suppressed or deficient, and/or cyanobacteria in which enzyme activities that contribute to glycogen decomposition, such as glycogen degrading enzymes, are enhanced. be able to.

As shown in the glycogen biosynthesis pathway of cyanobacteria in Figure 1, glycogen in cyanobacteria is produced by glucose-1-phosphate, which is synthesized from carbon dioxide via the Calvin cycle (CBB cycle), into GlgC, GlgA, and It is catalyzed and produced by various enzymes such as GlgB. Accordingly, in one embodiment of the present disclosure, genes contributing to glycogen synthesis, such as glgC encoding glucose-1-phosphate adenylyltransferase (GlgC) and glgA encoding glycogen synthase (GlgA), are deleted. Amino acids and/or organic acids can be produced by creating algae strains and culturing them under phototrophic conditions.

In one embodiment, enzymes that contribute to glycogen synthesis can include, in addition to GlgC and GlgA, similar enzymes that play an equivalent role.

As shown in the glycogen biosynthesis pathway of cyanobacteria in Figure 1, in cyanobacteria, glycogen is biosynthesized from glucose-1-phosphate derived from the Calvin cycle (CBB cycle) by glycogen phosphorylase (GlgP). Decomposed into 1-phosphoric acid. Therefore, in one embodiment, a blue-green algae strain that highly expresses a gene that contributes to glycogen degradation, such as glgP encoding glycogen phosphorylase (GlgP), is created and cultured under phototrophic conditions to produce amino acids and/or Organic acids can be produced.

In one embodiment, enzymes that contribute to glycogenolysis can include, in addition to GlgP, analogous enzymes that play a role equivalent to its enzymatic activity. The enzyme similar to GlgP may be any enzyme produced by a gene having the same function as GlgP.

In one embodiment, the GlgP of the present disclosure may be a protein comprising or consisting of an amino acid sequence having about 80% or more identity with the amino acid sequence of the enzyme, as long as its enzymatic activity is retained; It is preferably 85% or more, more preferably about 90% or more, even more preferably about 95% or more, and even more preferably about 99% or more.

Furthermore, GlgP of the present disclosure has 1 to about 20, preferably 1 to about 10, more preferably 1 to several (5, 4, 3) amino acids in the amino acid sequence of the enzyme, as long as its enzymatic activity is maintained. or 2) an amino acid sequence in which 1 to about 20 amino acids, preferably 1 to about 10, and more preferably 1 to several (5, 4, 3, or 2) amino acids are deleted from the amino acid sequence of the enzyme. an amino acid sequence in which 1 to about 20, preferably 1 to about 10, more preferably 1 to several (5, 4, 3, or 2) amino acids have been inserted into the amino acid sequence of the enzyme. Amino acid sequence, 1 to about 20, preferably 1 to about 10, more preferably 1 to several (5, 4, 3 or 2) amino acids in the amino acid sequence of the enzyme are substituted with other amino acids. The protein may include an amino acid sequence or a combination of these amino acid sequences, or may be a protein consisting of these amino acid sequences. Substitutions between amino acids with similar properties (e.g. glycine and alanine, valine, leucine and isoleucine, serine and threonine, aspartic acid and glutamic acid, asparagine and glutamine, lysine and arginine, cysteine and methionine, phenylalanine and tyrosine, etc.) For example, there may be a larger number of permutations.

In one embodiment, when producing cyanobacteria in which the enzyme activity that contributes to glycogen synthesis is suppressed or deleted and/or in which the enzyme activity that contributes to glycogen decomposition is enhanced, a gene encoding the target enzyme is generated. Examples include a method of introducing a mutation into a target enzyme, a method of replacing the base sequence encoding the target enzyme with another base sequence, and a method of newly introducing a base sequence derived from the same or different species. Examples of methods for introducing mutations into enzyme genes include site-specific mutation introduction methods. Specific methods for introducing site-specific mutations include methods using SOE-PCR reactions, ODA methods, Kunkel methods, and the like. In addition, Site-Directed Mutagenesis System Mutan-SuperExpress Km kit (Takara Bio Inc.), Transformer TM Site-Directed Mutagenesis kit (Clonetech Inc.), KOD-Pl Even if you use a commercially available kit such as us-Mutagenesis Kit (Toyobo), good. Furthermore, the target gene can also be obtained by imparting random genetic mutations and then performing enzyme activity evaluation and genetic analysis using appropriate methods.

In one embodiment, when producing cyanobacteria in which enzyme activity that contributes to glycogen synthesis is suppressed or deleted, a nucleotide sequence encoding an enzyme that contributes to glycogen synthesis, such as GlgC or GlgA, is inserted into a gene resistant to drugs such as gentamicin. You can also replace it with .

In general, the amino acid sequence encoding an enzyme protein does not necessarily exhibit enzyme activity unless the entire region of the sequence is conserved, and there are regions that do not affect enzyme activity even if the amino acid sequence changes. known to exist. In such regions that are not essential for enzyme activity, the enzyme's original activity can be maintained even if mutations such as amino acid deletions, substitutions, insertions, or additions are introduced. Also in the present disclosure, when producing blue-green algae with enhanced enzyme activity that contributes to glycogenolysis, it is possible to use a protein that maintains glycogenolysis ability and has a partially mutated amino acid sequence.

In one embodiment, when producing blue-green algae with enhanced enzyme activity that contributes to glycogenolysis, transformants can be produced using various known genetic engineering techniques, and the desired enzyme activity can be increased. There are no particular limitations on the method as long as it can be achieved, but for example, (1) introducing the glgP gene originally possessed by the host cyanobacterium to be genetically modified into a different genomic site and expressing it with a stronger promoter; (2) After introducing a mutation that strengthens the enzyme activity into the glgP gene originally possessed by the host cyanobacteria that is the target of genetic modification, the gene is introduced into a different genomic site and expressed using a stronger promoter. (3) introducing a glycogen phosphorylase gene with strong enzymatic activity derived from a foreign organism into the genome and expressing it with a strong promoter; You can also do it.

In other embodiments, methods for promoting the expression of the glgP gene or the gene encoding the ppGpp synthase described below include, for example, a method of introducing the above gene into a host cyanobacterium, a method of introducing the above gene into a host cyanobacterium, Examples include methods of modifying the expression control region (promoter, terminator, etc.) of the gene in algae. Furthermore, a plurality of the above genes may be introduced into the host cyanobacteria by this method, the expression control regions of the plurality of genes may be modified, or a combination thereof may be used.

In the present specification, cyanobacteria are defined as cyanobacteria in which the enzyme activity of the protein of interest is suppressed or deleted, cyanobacteria in which the enzyme activity of the protein of interest is increased, or cyanobacteria in which the expression of the gene encoding the protein of interest is promoted or deleted. Cells such as algae that are created through genetic modification are also called "transformants," the cells that have undergone genetic modification are called "hosts," and the cells that have the original genotype of that species are called "wild strains." ” is also called. In genetic modification, not only wild strains but also transformants can serve as hosts. Transformants of blue-green algae in which the enzyme activity of the target protein of the present disclosure is suppressed, deleted, or enhanced are preferably used in the method for producing amino acids and/or organic acids and the method for producing p-coumaric acid of the present disclosure. Can be done. Furthermore, the transformant of cyanobacteria in which the enzyme activity of the target protein of the present disclosure is suppressed, deleted, or enhanced may be a cyanobacteria in which the enzyme activity of one or more target proteins is enhanced, Furthermore, the enzyme activity of other target proteins may be suppressed or deleted.

In one embodiment, a transformant as described above can be obtained by introducing a gene of interest into a host by a known method. Specifically, we prepare recombinant vectors and gene expression cassettes that can express the target gene in host cells, genome editing constructs and RNAi constructs that target the target gene, and introduce these into host cells. They can be produced by transforming host cells. Blue-green algae in which the enzyme activity of the target protein of the present disclosure is suppressed or deleted, blue-green algae in which the enzyme activity of the target protein is increased, or blue-green algae in which the expression of the gene encoding the target protein is promoted or deleted, etc. The host can be appropriately selected from among the cyanobacteria mentioned above.

The plasmid vector for gene expression or the parent vector (plasmid) of the gene expression cassette is a vector that can introduce the gene encoding the protein of interest into the host and express the gene within the host cell. Good to have. For example, a vector having an expression control region such as a promoter or terminator depending on the type of host to be introduced, and a vector having a selection marker etc. can be used. Further, it may be a vector that has a replication origin such as a plasmid and autonomously replicates outside the chromosome, or a vector that is integrated into the chromosome.

Specific examples of expression vectors that can be used in the present disclosure include pBluescript (pBS) vectors (manufactured by Stratagene), pSTV vectors (manufactured by Takara Bio), and pUC vectors (manufactured by Takara Bio). , pET vector (manufactured by Merck), pGEX vector (manufactured by Cytiva), pCold vector (manufactured by Takara Bio), pHY300PLK (manufactured by Takara Bio), pUB110 (AmericanType Culture Collection), pBR322 (manufactured by Takara Bio) ), pRS403 (Stratagene), pMW218/219 (Nippon Gene), pRI vector (Takara Bio), pBI vector (Inplanta Innovations), and IN3 vector (Inplanta Innovations). Can be mentioned.

Furthermore, the type of promoter that regulates the expression of the gene encoding the protein of interest incorporated into the expression vector can be appropriately selected depending on the type of host used. Promoters that can be preferably used in the present disclosure include lac promoter, trp promoter, tac promoter, trc promoter, T7 promoter, SpoVG promoter, Rubisco operon (rbc) promoter, PSI reaction center protein (psaAB) promoter, and PSII D1 protein. (psbA) promoter, c-phycocyanin β subunit (cpcB) promoter, cauliflower mozile virus 35SRNA promoter, and housekeeping gene (eg, tubulin, actin, ubiquitin, etc.) promoters.

Furthermore, the type of selection marker for confirming that the desired trait has been obtained can be appropriately selected depending on the type of host used. Selectable markers that can be used in the present disclosure include, for example, gentamicin resistance gene, ampicillin resistance gene, chloramphenicol resistance gene, erythromycin resistance gene, neomycin resistance gene, kanamycin resistance gene, spectinomycin resistance gene, tetracycline resistance gene , blasticidin S resistance gene, bialaphos resistance gene, zeocin resistance gene, paromomycin resistance gene, and hygromycin resistance gene. Additionally, selectable markers associated with auxotrophy can also be used.

A gene encoding a protein of interest can be introduced into a vector by a conventional method such as ligation. Furthermore, it is preferable that the codons of the gene to be introduced be optimized according to the frequency of codon usage in the host microalgae. Information on codons used by various organisms is available from the Codon Usage Database (www.kazusa.or.jp/codon/).

The transformation method can be appropriately selected from various known methods depending on the type of host used. Examples include natural transformation methods, transformation methods using calcium ions, protoplast transformation methods, electroporation methods, lipofection methods, methods using Agrobacterium, particle gun methods, and conjugation methods.

The target gene introduced into the host cyanobacterium is preferably integrated into the genome of the cyanobacterium by homologous recombination or the like. The location on the genome where the target gene is integrated can be set as appropriate.

Selection of transformants into which a DNA fragment containing a gene of interest has been introduced can be performed using a selection marker or the like. For example, drug resistance acquired by a transformant as a result of introducing a drug resistance gene into a host cell together with a target DNA fragment during transformation can be used as an indicator. Furthermore, introduction of the target DNA fragment can also be confirmed by PCR or the like using the genome as a template.

Examples of methods for promoting the expression of a target gene by modifying the expression regulatory region of the gene in a host having the target gene on its genome include the following methods.

"Expression control region" refers to promoters, operators, terminators, etc., and these sequences are generally involved in regulating the expression level (transcription level, translation level) of adjacent genes. In a host having the above target gene on its genome, expression of genes such as GlgP and ppGpp synthase can be promoted by modifying the expression control region of the gene to promote expression of the gene.

Examples of methods for modifying the expression control region include promoter replacement. In a host having the various desired genes described above on its genome, expression of the various desired genes can be promoted by replacing the promoter of the gene with a promoter with higher transcriptional activity.

The above-mentioned promoter modification can be performed according to various known methods such as homologous recombination. Specifically, a linear DNA fragment containing the upstream and downstream regions of the target promoter and another promoter in place of the target promoter is constructed, and this is incorporated into the host cell, and the target promoter in the host genome is Two-fold homologous recombination occurs between the upstream and downstream sides of As a result, the target promoter on the genome is replaced with another promoter fragment, making it possible to modify the promoter. A method for modifying a target promoter by homologous recombination is described, for example, in Besher et al., Methods in molecular biology, 1995, vol. 47, p. This can be done by referring to documents such as 291-302.

In one embodiment, the amino acid and/or organic acid produced by the method of the present disclosure is not particularly limited as long as it is an amino acid and/or organic acid that can be biosynthesized by blue-green algae, and includes, for example, alanine, arginine, asparagine, Aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, cysteine, pyruvate, 2-oxoglutaric acid, lactic acid, malic acid, succinic acid , and fumaric acid. In one embodiment, the amino acids produced by the methods of the present disclosure can include aromatic amino acids such as phenylalanine, tryptophan, tyrosine, and the like.

Examples of amino acid metabolic pathways include the following.
For example, aromatic amino acids are synthesized via the shikimate pathway. In the shikimate pathway, chorismate is synthesized starting from a reaction in which phosphoenolpyruvate and erythrose-4-phosphate are condensed to synthesize 3-deoxy-D-arabino-heptulosone-7-phosphate. The aromatic amino acids phenylalanine, tyrosine, and tryptophan are then synthesized using chorismic acid as a substrate. Control of the shikimate pathway also controls all aromatic amino acids and secondary metabolites involved in this pathway. Enzymes of the aromatic amino acid synthesis pathway catalyze the biosynthesis of aromatic amino acids from erythrose 4-phosphate. The aromatic amino acid synthesis pathway includes a group of enzymes that biosynthesize aromatic amino acids such as tyrosine, phenylalanine, and tryptophan from erythrose 4-phosphate biosynthesized in the pentose phosphate pathway via chorismate. Furthermore, pyruvate carboxylase (eg, PYC1, PYC2, etc.) is an enzyme that biosynthesizes oxaloacetate from pyruvate biosynthesized in the glycolytic system. Pyruvate dehydrogenase (PDA1) is a complex enzyme that synthesizes acetyl-CoA from pyruvate biosynthesized through glycolysis. Generally, as aromatic amino acid synthesis pathway genes, there are genes that play the same role as ARO1, ARO2, ARO3, ARO4, ARO7, ARO8, ARO9, TYR1, PHA2, TRP2, TRP3, TRP4, TRP1, TRP3, TRP5, etc. in yeast. Among these, ARO1, ARO7, ARO8, ARO9, PHA2, and TRP5 are preferably targeted for enhancement.

Pathways related to the synthesis and metabolism of succinic acid and 2-oxoglutaric acid, as well as enzymes that catalyze them, are exemplified in Nature Catalysis 2, 18-33 (2019).

For other amino acid and/or organic acid synthesis and metabolic pathways, as well as the enzymes that catalyze them, see, for example, Eukaryot Cell. 2006 Feb; 5(2): 272-276., Current Opinion in Microbiology 32, 151-158 (2016), Annual Review of Plant Biology 67, 153-78 (2016), Pest Management Science 76(12), 3896-3904 (2020), etc., and in one embodiment, the present disclosure The cyanobacteria used in the method may be modified to be able to synthesize or metabolize these.

Furthermore, in the synthesis of glutamic acid from L-lactic acid in cyanobacteria, L-lactic acid is converted to pyruvate by NAD-independent L-lactate dehydrogenase, and pyruvate is converted to glutamic acid. Such a pathway is exemplified in, for example, PLoS One 9(7), e103380 (2014), and in one embodiment, the cyanobacteria used in the method of the present disclosure are those capable of synthesizing and metabolizing these. Modifications may be made.

In another aspect of the present disclosure, there is provided a method for producing p-coumaric acid, which includes a step of converting the amino acid produced by the method described above into p-coumaric acid.

Regarding the method for producing p-coumaric acid of the present disclosure, the method comprises transferring the amino acid, preferably aromatic amino acid, released into an aqueous medium by the above-mentioned method for producing an amino acid and/or organic acid of the present disclosure to the aqueous medium. Among them, it may be directly converted to p-coumaric acid. The conversion can be carried out using, for example, enzymes such as phenylalanine ammonia lyase, trans-cinnamic acid-4-monooxygenase, and tyrosine ammonia lyase, which are enzymes that catalyze the reaction of producing p-coumaric acid or its precursor using an aromatic amino acid as a substrate. This may be done by introducing into cyanobacteria a gene encoding p-coumaric acid, or enzymes involved in the conversion of p-coumaric acid from aromatic amino acids (e.g., phenylalanine ammonia-lyase, trans-cinnamic acid-4) into an aqueous medium. - Monooxygenase, tyrosine ammonia lyase, etc.) may be added. In general, phenolic compounds such as p-coumaric acid are known to inhibit the growth of cyanobacteria, and as described above, amino acids such as aromatic amino acids were directly converted to p-coumaric acid in an aqueous medium. In this case, as the conversion progresses, the conversion efficiency etc. normally decrease. In one embodiment, the blue-green algae used in the method of the present disclosure can be a blue-green algae in which expression of a gene encoding guanosine tetraphosphate (ppGpp) synthase is promoted. As a result, when adopting a method of accumulating ppGpp in cyanobacterial cells, it is possible to improve the resistance of cyanobacteria to p-coumaric acid, which in turn has the effect of avoiding a decrease in conversion efficiency. can.

In one embodiment, the amino acid and/or organic acid produced by the method for producing an amino acid and/or organic acid of the present disclosure is recovered, purified, etc., and, for example, the aromatic amino acid as described above is converted to p-coumaric acid. It can also be converted separately using enzymes involved in .

The ppGpp synthase is not particularly limited as long as it is an enzyme that synthesizes ppGpp (or pppGpp) using GDP (or GTP) as a substrate, but it is preferably an enzyme that has only ppGpp (or pppGpp) synthetic activity. By using an enzyme that only has ppGpp synthetic activity and hardly catalyzes its decomposition, amino acid release can be increased through increasing the intracellular ppGpp concentration. Specifically, such enzymes include ppGpp synthases such as YjbM derived from Bacillus subtilis.

In one embodiment, the ppGpp synthetase of the present disclosure may be a protein comprising or consisting of an amino acid sequence having about 80% or more identity with the amino acid sequence of the enzyme, as long as its enzymatic activity is retained. , preferably about 85% or more, more preferably about 90% or more, even more preferably about 95% or more, and even more preferably about 99% or more.

Furthermore, the ppGpp synthase of the present invention has 1 to about 20, preferably 1 to about 10, more preferably 1 to several (5, 4) amino acids in the amino acid sequence of the enzyme, as long as its enzymatic activity is maintained. , 3 or 2) amino acids deleted, the amino acid sequence of the enzyme has 1 to about 20, preferably 1 to about 10, more preferably 1 to several (5, 4, 3 or 2) amino acids deleted. ) amino acid sequence, 1 to about 20, preferably 1 to about 10, more preferably 1 to several (5, 4, 3, or 2) amino acids are inserted into the amino acid sequence of the enzyme. 1 to about 20, preferably 1 to about 10, more preferably 1 to several (5, 4, 3, or 2) amino acids in the amino acid sequence of the enzyme are substituted with other amino acids. or a combination of these amino acid sequences, or may be a protein consisting of these amino acid sequences. Substitutions between amino acids with similar properties (e.g. glycine and alanine, valine, leucine and isoleucine, serine and threonine, aspartic acid and glutamic acid, asparagine and glutamine, lysine and arginine, cysteine and methionine, phenylalanine and tyrosine, etc.) For example, there may be a larger number of permutations.

Phenylalanine ammonia-lyase (PAL) is not particularly limited as long as it is an enzyme that uses L-phenylalanine as a substrate and catalyzes a reaction that produces trans-cinnamic acid and ammonia, but specifically, for example, EC 4.3 .1.24, EC 4.3.1.25 enzymes, etc.

Trans-cinnamic acid-4-monooxygenase (C4H) is particularly limited if it is an enzyme that catalyzes a reaction that produces 4-hydroxycinnamic acid (p-coumaric acid) using trans-cinnamic acid as a substrate. However, specific examples include enzymes of EC 1.14.13.11.

Tyrosine ammonia-lyase (TAL) is not particularly limited as long as it is an enzyme that catalyzes the reaction of converting L-tyrosine to p-coumaric acid, but specifically, for example, EC 4.3.1.23, EC 4.3.1.25.

When using cyanobacteria in which the expression of genes encoding phenylalanine ammonia-lyase, trans-cinnamate-4-monooxygenase, tyrosine ammonia-lyase, etc. is promoted in the conversion to p-coumaric acid, these converts aromatic amino acids into p-coumaric acid in cyanobacterial cells by introducing a gene for cyanobacteria, and/or converts aromatic amino acids released into the aqueous medium into p-coumaric acid during culturing of cyanobacteria. It can also be converted to .

The amino acid, organic acid, or p-coumaric acid produced by the method of the present disclosure can be separated and purified from an aqueous medium by various known separation and purification methods, if necessary. Specifically, after separating cyanobacteria and their products by ultrafiltration membrane separation, centrifugation, concentration, etc., they are purified by known methods such as column methods and crystallization methods, and then dried to obtain crystals. Examples include a method of collecting as a sample.

In one embodiment of the present disclosure, p-coumaric acid produced by the production method of the present disclosure can be used as a raw material for aromatic polymers.

(General technology)
The molecular biological techniques, biochemical techniques, and microbiological techniques used herein are well known and commonly used in the art, and are described, for example, in Sambrook J. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F. M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F. M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Green Pub. Associates and Wiley-Interscience; Innis, M. A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F. M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Green Pub. Associates; Ausubel, F. M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Green Pub. Associates; Innis, M. A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F. M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annua l updates; Sninsky, J. J. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, Separate Volume Experimental Medicine "Gene Transfer & Expression Analysis Experimental Methods" Yodosha, 1997, etc., and these are relevant parts (possibly all) in this specification. is used as a reference.

Regarding DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, gene synthesis and fragment synthesis services such as GeneArt, GenScript, and Integrated DNA Technologies (IDT) can be used, as well as other services such as Gait. , M. J. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M. J. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, R. L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G.; M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G. T. (I996). Bioconjugate Techniques, Academic Press, etc., and relevant portions of these are incorporated herein by reference.

In this specification, "or" is used when "any one" of the items listed in the sentence can be adopted. The same goes for "or." In this specification, when "within a range" of "two values" is specified, the range includes the two values themselves.
All references, including scientific literature, patents, patent applications, etc., cited herein are herein incorporated by reference in their entirety to the same extent as if each were specifically indicated.

The present disclosure has been described above with reference to preferred embodiments for ease of understanding. The present disclosure will now be described based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided for the purpose of limiting the present disclosure. Therefore, the scope of the disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the claims.

(Example 1: Production of transformants)
The following transformants were produced using the cyanobacterium Synechococcus elongates PCC 7942. For a strain lacking the glgC (glucose-1-phosphate adenylyltransferase) gene, a gentamicin resistance cassette was used, as in the construct shown in FIG.

First, a plasmid carrying this construct was prepared, and E. coli into which this plasmid had been introduced was cultured with shaking (culture conditions: 50 mL LB/3 x 200 mL flasks, 37° C., 160 rpm, o/n). Plasmids were extracted from E. coli using the NucleoBond (registered trademark) Xtra Midi kit, and the DNA concentration was confirmed to be 1 μg/μL or higher using NanoDrop.

Subsequently, the cyanobacteria to be used were cultured with shaking in a Biotron incubator for 4 to 5 days until the OD ₇₅₀ = approximately 1.0 (Culture conditions: 70 mL BG-11/200 mL Erlenmeyer flask with rim, 50 to 70 μmol/m ² / s, 30° C., 100 rpm, initial OD ₇₅₀ =0.1). 100 μL of the cyanobacteria culture solution was dispensed into three 1.5 mL tubes, and 1 μg or more (for example, 3 μg, 5 μg) of plasmid was added to the tubes and mixed well by pipetting. The tube was wrapped in aluminum foil and incubated at room temperature for 24 hours with gentle stirring on a rotator. After removing the aluminum foil and allowing it to stand on a laboratory bench for 30 minutes, the entire cell suspension was seeded onto a membrane placed on a BG-11 agar medium.

After static culture for 3 days in a Biotron incubator (culture conditions: 50-70 μmol/m ² /s, 30° C.), the membrane was transferred to a BG-11 agar plate containing gentamicin. The cells were cultured statically in a Biotron incubator until colonies appeared (culture conditions: 50 to 70 μmol/m ² /s, 30°C), and the colonies were isolated and cultured with shaking in a Biotron incubator until growth was observed ( Culture conditions: 6 mL BG-11/6-well plate containing gentamicin, 50-70 μmol/m ² /s, 30° C., 100 rpm). Segregation check was performed by PCR.

(Example 2: Cultivation of blue-green algae)
A total of three strains of cyanobacteria were used: Synechococcus elongates PCC 7942, a glgC-deficient strain of Synechococcus elongates PCC 7942, and a glgC-deficient and yjbM expression-induced strain of Synechococcus elongates PCC 7942.

First, as a pre-culture, thaw a frozen stock of blue-green algae and culture it under the following conditions until OD750 = 1.5 to 2.0 (about 1 week), and then subculture until OD750 = 0.1. (N=1). Subsequently, as pre-culture, the cells were cultured under the following conditions until OD750 = 1.5 to 2.0 (about 4 days), and subcultured until OD750 = 0.1 (N = 3). Furthermore, as main culture, the cells were cultured for 10 days under the following conditions. In the main culture, a two-tiered flask shown in FIG. 3 was used. When inducing the expression of the yjbM gene, IPTG was added at a final concentration of 1 mM on the third day of culture.

Culture conditions [pre-culture]
Medium: 70mL BG-11/200mL Erlenmeyer flask _CO2 concentration: (not supplied)
Light conditions: ~50 μmol photons/m ² /s Daylight white fluorescent lamp Temperature: 30°C
Concussion: 100rpm

[Preculture]
Medium: 150mL BG-11/500mL Erlenmeyer flask _CO2 concentration: (not supplied)
Light conditions: ~50 μmol photons/m ² /s Daylight white fluorescent lamp Temperature: 30°C
Concussion: 100rpm

[Main culture]
Medium: 70mL BG-11/two-tiered flask _CO2 concentration: 1%
Light conditions: 110-120μmol photons/m ² /s daylight white fluorescent lamp temperature: 30℃
Concussion: 100rpm

(Example 3: Glycogen measurement)
The amount of glycogen in each cyanobacteria cultured in Example 2 was measured.

<Extraction>
5 mg of dried bacterial cells DW was weighed into an Eppendorf tube, 100 μL of 30% KOH (w/v) was added thereto, heated at 90° C. for 90 minutes, and then cooled on ice. After cooling for several minutes, 300 μL of ice-cold EtOH was added and stirred by vortexing. After standing still for 1 hour, it was centrifuged (4°C, 5000g, 5 minutes). The supernatant was discarded by decantation, 300 μL of ice-cold EtOH was added, and the mixture was centrifuged (4° C., 10,000 g, 5 minutes). Furthermore, the supernatant was carefully discarded using a tip, and 300 μL of ice-cold EtOH was added, followed by centrifugation (4° C., 14,000 g, 10 minutes). Further, the supernatant was carefully discarded using a tip, and the mixture was dried in a dry heat oven at 80°C with the lid opened for 30 minutes, and then stored at -30°C.

<Hydrolysis>
100 μL of MilliQ water was added to the dried glycogen extract, and the mixture was shaken with a maximizer at 1800 rpm and 25° C. for 10 minutes. This is called a glycogen extract. After shaking, centrifuge at 14,000 g and 25°C for 5 minutes, with the maximizer set at 50°C. The reagents listed in the table below were placed in a 2 mL Eppendorf tube with a lock (manufactured by Eppendorf) in order from the top, and pipetted gently to mix.

When the temperature of the maximizer reached 50° C., a sample was added thereto, and the sample was slowly shaken at 200 rpm for 2 hours to react. At this time, the heater was set at 95°C and turned on, and after the reaction was completed, the enzyme was deactivated by treatment at 95°C for 20 minutes. After the heat treatment, the samples were taken out and cooled to room temperature. Centrifugation was performed at 14,000g for 5 minutes at 4°C, and the supernatant was analyzed by HPLC. The results are shown in Figure 4. Glycogen accumulation was completely abolished in the ΔglgC strain.

(Example 4: Metabolome analysis)
Metabolome analysis was performed using the transformant produced in Example 1. 500 μL of ice cold CHCl ₃ was added to 500 μL of the supernatant of each cultured cyanobacterium, and the mixture was vortexed. Centrifugation was performed (4° C., 14000 g, 5 min), and 400 μL of the upper phase was transferred to an Amicon Ultra filter tube, followed by further centrifugation (4° C., 14000 g, 90 min). 248 μL of the solution that had passed through the filter was transferred to another Eppendorf tube, and 1 μL each of 100 mM-PIPES and 100 mM-Methionine sulfone were added thereto. Mix well by vortexing, store at -80°C, and analyze by CE-TOFMS. The results are shown in Figures 5-7. From this result, it was found that various amino acids including glutamic acid, tyrosine, glutamine, aspartic acid, asparagine, and threonine are excreted from the cells in large quantities. In addition to increases in pyruvic acid and 2-oxoglutaric acid, increases in extracellular concentrations of lactic acid, malic acid, succinic acid, and fumaric acid were also observed. Furthermore, among these, concentrations of substances other than glutamic acid, aspartic acid, and asparagine were further increased by inducing expression of the yjbM gene.

(Example 5: Example of GlgP activity enhancement)
A construct in which a highly active glycogen phosphorylase gene derived from another species (such as Corynebacterium callunae) is linked downstream of the trc promoter is prepared and introduced into the genome of the cyanobacterium Synechococcus elongates PCC 7942.
Transformation is performed in the same manner as in Example 1. Culture is performed under the same conditions as in Example 2 while inducing the expression of glycogen phosphorylase by adding IPTG. The culture supernatant is collected, and the amino acids released outside the cells are measured by the same metabolome analysis as in Example 4.
From the above, it can be confirmed that various amino acids and the like are increased even when the activity of glycogen phosphorylase is enhanced.

(Example 6: Example of suppressing GlgA activity)
A glgA-deficient strain is created by introducing a gentamicin resistance cassette onto the glgA gene (Synpcc7942_2518) of the cyanobacterium Synechococcus elongates PCC 7942.
Transformation is performed in the same manner as in Example 1. The glgA-deficient strain is cultured under the same conditions as in Example 2. The culture supernatant is collected, and the amino acids released outside the cells are measured by the same metabolome analysis as in Example 4.
From the above, it can be confirmed that various amino acids and the like increase even when the activity of enzymes contributing to glycogen synthesis is suppressed.

(Example 7: Example of recovery of amino acids and/or organic acids)
After removing cells from the culture medium by centrifugation, amino acids can be recovered using an appropriate ion exchange resin and/or precipitant. As a result, the amino acids and organic acids produced as in Examples 1 to 6 are recovered.

(Example 8: Production of p-coumaric acid)
By introducing a tyrosine ammonia-lyase gene into amino acid-producing cyanobacteria, we create p-coumaric acid-producing cyanobacteria that convert tyrosine to p-coumaric acid within their cells. Alternatively, yeast that displays tyrosine ammonia lyase on the cell surface is produced, and the yeast is added to the cyanobacterial culture supernatant and allowed to react, thereby converting tyrosine contained in the supernatant into p-coumaric acid.
As described above, p-coumaric acid is produced from the amino acid obtained by the method of the present disclosure.

(Example 9: Example of plastic production using p-coumaric acid)
Functional polymers are synthesized by copolymerizing p-coumaric acid with other organic acids (caffeic acid, ricinoleic acid, etc.). As a result, plastics can be produced using p-coumaric acid obtained as in Example 8.

(Note)
As described above, although the present disclosure has been illustrated using the preferred embodiments thereof, it is understood that the scope of the present disclosure should be interpreted only by the claims. The patents, patent applications, and other documents cited herein are hereby incorporated by reference to the same extent as if the contents themselves were specifically set forth herein. That is understood.

According to the present disclosure, various amino acids can be obtained at low production costs simply by culturing cyanobacteria. In addition, amino acids, which can be used as raw materials for bioplastics, food additives, and nutritional sources for cell culture, can be produced from carbon dioxide alone as a carbon source, so they are expected to be applied in industrial fields such as industrial products and the food industry. .

Claims (33)

A method for producing amino acids and/or organic acids, comprising:
A method comprising culturing cyanobacteria with reduced glycogen content. 2. The method of claim 1, wherein the cyanobacteria have been genetically modified to have reduced glycogen content compared to wild-type cyanobacteria grown under the same conditions. 3. The method according to claim 1, wherein the cyanobacteria include cyanobacteria in which enzyme activity contributing to glycogen synthesis is suppressed or deficient, and/or cyanobacteria in which enzyme activity contributing to glycogen decomposition is enhanced. 4. The method according to claim 3, wherein the enzymes contributing to glycogen synthesis include glucose-1-phosphate adenylyltransferase (GlgC) or a similar enzyme thereof, and glycogen synthase (GlgA) or a similar enzyme thereof. 5. The method according to claim 3, wherein the enzyme contributing to glycogenolysis comprises glycogen phosphorylase (GlgP) or an enzyme similar thereto. The method according to any one of claims 1 to 5, wherein the cyanobacteria are capable of inducibly expressing a gene encoding ppGpp synthase. 7. The method according to claim 6, wherein the gene encoding the ppGpp synthase includes the yjbM gene derived from Bacillus subtilis. The amino acids and/or organic acids include alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, cysteine, A method according to any one of claims 1 to 7, comprising pyruvic acid, 2-oxoglutaric acid, lactic acid, malic acid, succinic acid, and fumaric acid. The method according to any one of claims 1 to 8, wherein the amino acids and/or organic acids include aromatic amino acids. The method according to any one of claims 1 to 9, wherein the cyanobacteria consume carbon dioxide as a carbon source. A method for producing p-coumaric acid, comprising the step of converting the amino acid produced by the method according to claims 1 to 10 into p-coumaric acid. A method for producing amino acids and/or organic acids using carbon dioxide as a raw material, the method comprising:
(a) culturing cyanobacteria with reduced glycogen content in the presence of carbon dioxide;
(b) obtaining an amino acid and/or an organic acid from the culture solution of step (a). 13. The method of claim 12, wherein the cyanobacteria have been genetically modified to have reduced glycogen content compared to wild-type cyanobacteria grown under the same conditions. The method according to claim 12 or 13, wherein the cyanobacteria include cyanobacteria in which enzyme activity contributing to glycogen synthesis is suppressed or deficient, and/or cyanobacteria in which enzyme activity contributing to glycogen decomposition is enhanced. 15. The method according to claim 14, wherein the enzymes contributing to glycogen synthesis include glucose-1-phosphate adenylyltransferase (GlgC) or a similar enzyme thereof, and glycogen synthase (GlgA) or a similar enzyme thereof. 16. The method according to claim 14 or 15, wherein the enzyme contributing to glycogenolysis comprises glycogen phosphorylase (GlgP) or an enzyme similar thereto. The method according to any one of claims 12 to 16, wherein the cyanobacteria are capable of inducibly expressing a gene encoding ppGpp synthetase. 18. The method according to claim 17, wherein the gene encoding the ppGpp synthase includes the yjbM gene derived from Bacillus subtilis. The amino acids and/or organic acids include alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, cysteine, 19. A method according to any one of claims 12 to 18, comprising pyruvic acid, 2-oxoglutaric acid, lactic acid, malic acid, succinic acid, and fumaric acid. The method according to any one of claims 12 to 19, wherein the amino acids and/or organic acids include aromatic amino acids. The method according to any one of claims 12 to 20, wherein the cyanobacteria consume carbon dioxide as a carbon source. A method for producing p-coumaric acid, comprising the step of converting the amino acid produced by the method according to any one of claims 12 to 21 into p-coumaric acid. A method for producing amino acid-containing or amino acid-based chemical products using carbon dioxide as a raw material, the method comprising:
(a) culturing cyanobacteria with reduced glycogen content in the presence of carbon dioxide;
(b) obtaining amino acids from the culture solution of step (a);
(c) a step of producing a chemical product using the amino acid as a raw material. 24. The method of claim 23, wherein the cyanobacteria are genetically modified to have reduced glycogen content compared to wild-type cyanobacteria grown under the same conditions. 25. The method according to claim 23 or 24, wherein the cyanobacteria include cyanobacteria in which enzyme activity contributing to glycogen synthesis is suppressed or deficient, and/or cyanobacteria in which enzyme activity contributing to glycogen decomposition is enhanced. 26. The method according to claim 25, wherein the enzymes contributing to glycogen synthesis include glucose-1-phosphate adenylyltransferase (GlgC) or a similar enzyme thereof, and glycogen synthase (GlgA) or a similar enzyme thereof. 27. The method according to claim 25 or 26, wherein the enzyme contributing to glycogenolysis comprises glycogen phosphorylase (GlgP) or a similar enzyme thereof. The method according to any one of claims 23 to 27, wherein the cyanobacteria are capable of inducibly expressing a gene encoding ppGpp synthase. 29. The method according to claim 28, wherein the gene encoding the ppGpp synthase includes the yjbM gene derived from Bacillus subtilis. The amino acids and/or organic acids include alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, cysteine, 30. The method of any one of claims 23 to 29, comprising pyruvic acid, 2-oxoglutaric acid, lactic acid, malic acid, succinic acid, and fumaric acid. The method according to any one of claims 23 to 30, wherein the amino acids and/or organic acids include aromatic amino acids. A method according to any one of claims 23 to 31, wherein the cyanobacteria consume carbon dioxide as a carbon source. 33. The method according to any one of claims 23 to 32, wherein the chemical product comprises a bioplastic raw material, a food additive, a cosmetic additive, and/or a nutrient source for cell culture.