Method for using algae to produce d-glucose

Abstract

The present disclosure provides a circulation-type system that minimizes waste and environmental impact for the production of a substance by culturing cells or a microorganism. The present disclosure also provides a microorganism in which the Calvin (CBB) cycle occurs and into which a mechanism for converting glucose-1-phosphate (G1P) to glucose has been introduced or enhanced. The present disclosure also provides a combination of glucose-dependent cells or a glucose-dependent microorganism and cells or a microorganism that can produce and excrete glucose and a system and / or a kit that includes the combination.

Description

Method for producing D-glucose using algae

[0001] The present disclosure relates to a resource recycling method using cells or microorganisms, and more particularly to a method for producing D-glucose using algae and the microorganisms used therein.

[0002] Current food production, which relies on parts of plants and animals, such as grains and livestock, results in a great deal of waste. In recent years, cultured meat, which is made by culturing cells or microorganisms from edible parts, has attracted global attention and is being actively developed. However, problems remain, as the components of the culture medium used to culture cells or microorganisms are derived from grains and livestock, and are extremely expensive. Furthermore, measures will be needed to deal with the large amount of culture waste that will be generated as cultured meat becomes more widespread in the future.

[0003] D-glucose is an energy and carbon source for living organisms and is the most important nutrient source in recent biorefineries. Currently, D-glucose is supplied from terrestrial plants, but there is a need to develop a cheap and stable production method using algae (microalgae and cyanobacteria).

[0004] A known method for producing glucose using algae is to suppress the decomposition of glucose produced within the cells or microorganisms by disrupting the glucokine gene of the cyanobacterium Synechococcus elongatus PCC7942, thereby causing glucose to be secreted outside the cells or microorganisms (Non-Patent Document 1).

[0005] Zhang, S. , Sun, J. , Feng, D. et al. Unlocking the potentials of cyanobacterial photosynthesis for directly converting carbon dioxide into glucose. Nat Commun 14, 3425 (2023). https: / / doi.org / 10.1038 / s41467-023-39222-w

[0006] This disclosure focuses on carbohydrates commonly produced by algae (starch and glycogen) and invents a method for producing and secreting D-glucose in algae through carbohydrate degradation and conversion of intermediate metabolites in carbohydrate synthesis. Specifically, and without intending to be limiting, efficient D-glucose production is possible by introducing genes related to the production of sucrose, such as the glucose-1-phosphatase gene or the glucoamylase gene, regardless of the metabolic processes of sucrose production and its degradation by invertase. Furthermore, the D-glucose secretion and production ability of algae is improved by introducing genes related to release, such as the glucose-facilitated diffusion protein gene.

[0007] The present disclosure provides the following. (Conversion of G1P to glucose) [Item 1] A cell or microorganism having a Calvin Bacterium (CBB) cycle, wherein a mechanism for converting glucose-1-phosphate (G1P) to glucose has been introduced or enhanced. [Item 2] The cell or microorganism according to any one of the above items, wherein the cell or microorganism comprises a cyanobacterium. [Item 3] The cell or microorganism according to any one of the above items, wherein the conversion of glucose-1-phosphate (G1P) to glucose is achieved by an enzyme. [Item 4] The cell or microorganism according to any one of the above items, wherein the enzyme that converts glucose-1-phosphate (G1P) to glucose comprises glucose-1-phosphatase. [Item 5] The cell or microorganism according to any one of the above items, wherein the enzyme that converts glucose-1-phosphate (G1P) to glucose comprises Escherichia coli glucose-1-phosphatase (EcAGP, EcYihX, etc.). [Item 6] The cell or microorganism according to any one of the above items, wherein the conversion of G1P to glucose is achieved without blocking the conversion of G1P to glycogen. [Item 7] The cell or microorganism according to any one of the above items, further configured to attenuate or inhibit the conversion of glucose to glucose-6-phosphate (G6P). [Item 8] The cell or microorganism according to any one of the above items, wherein the conversion of glucose to glucose-6-phosphate (G6P) is achieved by an enzyme (e.g., Glk). [Item 9] The cell or microorganism according to any one of the above items, wherein a mechanism for excreting glucose outside the cell or microorganism has been introduced or enhanced. [Item 10] The cell or microorganism according to any one of the above items, wherein the mechanism for excreting glucose outside the cell or microorganism is achieved by an efflux protein. [Item 11] The cell or microorganism according to any one of the above items, wherein the efflux protein is a glucose diffusion facilitating protein (GLF) or a glucose transporter (GLUT).[Item 12] The cell or microorganism according to any one of the above items, wherein the efflux protein is achieved by Zymomonas mobilis glucose diffusion facilitating protein (ZmGLF) or a glucose transporter (GLUT). (Conversion of glycogen to glucose) [Item 13] A cell or microorganism having a Calvin (CBB) cycle, such as a cyanobacterium, into which a mechanism for converting glycogen to glucose has been introduced or enhanced. [Item 14] The cell or microorganism according to item 13, wherein the mechanism for converting glycogen to glucose is achieved by an enzyme that converts glycogen to glucose. [Item 15] The cell or microorganism according to any one of the above items, wherein the enzyme that converts glycogen to glucose includes glucoamylase. [Item 16] The cell or microorganism according to any one of the above items, wherein the enzyme that converts glycogen to glucose includes Rhizopus delemar glucoamylase (RdGA). [Item 17] The cell or microorganism according to any one of the above items, further configured to inhibit conversion of glucose to glucose-6-phosphate (G6P). [Item 18] The cell or microorganism according to any one of the above items, wherein the enzyme that converts glucose to glucose-6-phosphate (G6P) comprises glucokinase. [Item 19] The cell or microorganism according to any one of the above items, wherein the glucokinase comprises at least one selected from the group consisting of hexokinase, polyphosphate glucokinase, and polyphosphate glucokinase. [Item 20] The cell or microorganism according to any one of the above items, wherein a mechanism for exporting glucose to the outside of the cell or microorganism has been introduced or enhanced. [Item 21] The cell or microorganism according to any one of the above items, wherein the export is achieved by Zymomonas mobilis glucose diffusion facilitating protein (ZmGLF) or a glucose transporter (GLUT).(Conversion of glucose to glucose-6-phosphate) [Item 22] A cell or microorganism that is a microorganism having the Calvin Bacterium (CBB) cycle, such as cyanobacteria, and is configured to inhibit the conversion of glucose to glucose-6-phosphate (G6P). [Item 22A1] The cell or microorganism according to any one of the above items, wherein the inhibition is achieved by inhibiting glucokinase activity. [Item 22A2] The cell or microorganism according to any one of the above items, wherein the inhibition is achieved by disruption of a glucokinase activity gene. [Item 22A3] The cell or microorganism according to any one of the above items, wherein the mechanism for exporting glucose to the cell or microorganism is absent or weakened. [Item 23] The cell or microorganism according to any one of the above items, wherein the conversion of glucose to glucose-6-phosphate (G6P) is achieved by an enzyme (e.g., Glk). [Item 24] The cell or microorganism according to any one of the above items, further comprising an enzyme that converts glucose-1-phosphate (G1P) to glucose, and / or an enzyme that converts glycogen to glucose, which has been introduced or enhanced. [Item 25] The cell or microorganism according to any one of the above items, wherein the enzyme that converts glucose-1-phosphate (G1P) to glucose comprises Escherichia coli glucose-1-phosphatase (EcAGP, EcYihX, etc.). [Item 26] The cell or microorganism according to any one of the above items, wherein a mechanism for excreting glucose outside the cell or microorganism has been introduced or enhanced. [Item 27] ​​The cell or microorganism according to any one of the above items, wherein the mechanism for excreting glucose outside the cell or microorganism is achieved by an efflux protein. [Item 28] The cell or microorganism according to any one of the above items, wherein the efflux protein comprises Zymomonas mobilis glucose diffusion facilitating protein (ZmGLF) or a glucose transporter (GLUT). [Item 29] The cell or microorganism according to any one of the preceding items, wherein the cell or microorganism does not contain invertase. [Item 30] The cell or microorganism according to any one of the preceding items, wherein the cell or microorganism comprises a cyanobacterium.(Circular Economy) [Item 31] A combination of a glucose-requiring cell or microorganism and a cell or microorganism capable of producing and exporting glucose, or a system and / or kit comprising the same. [Item 32] The combination according to any one of the above items, a system and / or kit comprising the same, wherein the glucose-requiring cell or microorganism comprises an animal cell or microorganism. [Item 33] A method of producing cultured meat using the combination, system and / or kit according to any one of the above items, wherein the animal cell or microorganism is a cell or microorganism used to produce cultured meat. [Item 34] The combination according to any one of the above items, a system and / or kit comprising the same, or the method according to any one of the above items, wherein the cell or microorganism capable of producing and exporting glucose comprises a microorganism according to any one of the above items. [Item 35] The combination according to any one of the above items, a system and / or kit comprising the same, wherein the glucose-requiring cell or microorganism comprises yeast. [Item 36] The combination according to any one of the above items, a system and / or kit comprising the same, wherein the glucose-requiring cell or microorganism comprises Escherichia coli. [Item 37] A method for producing a fermented food using the combination, system, and / or kit described in any one of the above items, wherein the yeast or E. coli is a cell or microorganism used to produce a fermented food (including, but not limited to, alcohol). [Item 38] A combination described in any one of the above items, a system and / or kit comprising them, or a method described in any one of the above items, wherein the cell or microorganism capable of producing and exporting glucose comprises the cell or microorganism described in any one of the above items. [Item 39] A combination, system, and / or kit described in any one of the above items, or a method described in any one of the above items, wherein the cell or microorganism produces muconic acid. [Item 40] A method for producing a medium to which glucose is refed, comprising adding a cell or microorganism capable of producing and exporting glucose to a medium from which glucose has been consumed (e.g., a post-animal culture medium).[Item 41] A combination comprising cells or microorganisms capable of producing and exporting glucose, and cells or microorganisms capable of removing ammonia and / or cells or microorganisms capable of removing lactate, as well as a system and / or kit comprising them. [Item 42] The combination according to any one of the above items, wherein the cells or microorganisms capable of producing and exporting glucose, the cells or microorganisms capable of removing ammonia, and the cells or microorganisms capable of removing lactate are independently the same cells or microorganisms and / or different cells or microorganisms. [Item 43] The combination according to any one of the above items, comprising cells or microorganisms capable of producing and exporting glucose, and cells or microorganisms capable of removing lactate, as well as a system and / or kit comprising them. [Item 44] The combination according to any one of the above items, wherein the cells or microorganisms capable of producing and exporting glucose and / or the cells or microorganisms capable of removing lactate have the ability to remove ammonia, as well as a system and / or kit comprising them. [Item 45] The combination according to any one of the above items, further comprising cells or microorganisms capable of removing ammonia, as well as a system and / or kit comprising them. [Item 46] The combination according to any one of the above items, wherein the glucose production and export ability, the ammonia removal ability, and the lactate removal ability are each independently abilities that the cells or microorganisms originally have or abilities that have been introduced by modification, and a system and / or kit comprising the same. [Item 47] A method for producing substance X, comprising: 1) culturing a first cell or microorganism capable of producing substance X, 2) culturing a second cell or microorganism having glucose production and export ability using the medium obtained after the culture of 1), 3) culturing the first cell or microorganism in the medium obtained after the culture of 2) together with a third cell or microorganism having ammonia removal ability, and optionally together with a fourth cell or microorganism having lactate removal ability, 4) culturing the first cell or microorganism in the medium obtained after the culture of 2) or 3), and 5) repeating steps 2) and / or 3) as necessary.[Item 48] A method for culturing the first cells or microorganisms, comprising: 1) culturing a first cell or microorganism; 2) culturing a second cell or microorganism capable of producing and exporting glucose using the culture medium obtained after the culture of step 1); 3) culturing the second cell or microorganism in the culture medium obtained after the culture of step 2) together with a third cell or microorganism capable of removing ammonia, and optionally together with a fourth cell or microorganism capable of removing lactate; 4) culturing the first cell or microorganism in the culture medium obtained after the culture of step 2) or 3); and 5) repeating steps 2) and / or 3) as necessary. (c) in step (b), culturing the first cells or microorganisms together with a third cell or microorganism having the ability to remove ammonia, and, if necessary, a fourth cell or microorganism having the ability to remove lactate; (d) culturing the first cells or microorganisms in the culture medium after the culture of step (c); and (e) repeating steps (b) and / or (c) as necessary. [Item 48A1] A method for culturing the first cells or microorganisms, comprising the steps of: (a) culturing a first cell or microorganism; (b) culturing a second cell or microorganism having the ability to produce and export glucose using the culture medium obtained after the culture of step (a); (c) culturing the second cell or microorganism together with a third cell or microorganism having the ability to remove ammonia, and, if necessary, a fourth cell or microorganism having the ability to remove lactate; (d) culturing the first cells or microorganisms in the culture medium after the culture of step (c); and (e) repeating steps (b) and / or (c) as necessary. [Item 48A2] The culture method according to any one of the above items, wherein the second, third, and / or fourth cell or microorganism is a cyanobacterium. [Item 48A3] The method according to any one of the above items, wherein the cyanobacterium is a Synechococcus bacterium. [Item 48A4] The method according to any one of the above items, wherein the first cell or microorganism is an animal cell. [Item 48A5] The method according to any one of the above items, wherein the second, third, and / or fourth cell or microorganism is genetically modified to exhibit at least two, preferably all, of the following abilities: glucose production and excretion, ammonia removal, and lactate removal.[Item 48A6] A method for culturing the first cells or microorganisms, comprising: 1) culturing a first cell or microorganism; 2) culturing a second cell or microorganism capable of producing and exporting glucose using the culture medium obtained after the culture of step 1); 3) culturing the second cell or microorganism together with a third cell or microorganism capable of removing ammonia and lactate in the culture medium obtained after the culture of step 2); 4) culturing the first cell or microorganism in the culture medium obtained after the culture of step 2) or 3); and 5) repeating steps 2) and / or 3) as necessary. [Item 48B1] A method for culturing animal cells, comprising: (a) culturing first cells or microorganisms in a first culture tank; (b) supplying the culture solution after step (a) to a second culture tank containing second cells or microorganisms having the ability to remove ammonia and, if necessary, the ability to remove lactic acid, and bringing it into contact with the second cells or microorganisms; (c) supplying the culture solution after step (b) to a third culture tank containing third cells or microorganisms having the ability to produce and export glucose, and bringing it into contact with the third cells or microorganisms; (d) returning the culture solution after step (c) to the first culture tank and continuing the culturing of the first cells or microorganisms; and (e) continuously or intermittently repeating steps (b) to (d), wherein the second cells or microorganisms and the third cells or microorganisms are different from each other. [Item 48B2] The culture method according to any one of the above items, wherein the first culture vessel, the second culture vessel, and the third culture vessel are continuously connected via tubing. [Item 48B3] The method according to any one of the above items, wherein the second cell or microorganism is a cyanobacterium or Escherichia coli. [Item 48B4] The method according to any one of the above items, wherein the third cell or microorganism is a cyanobacterium. [Item 48B5] The culture method according to any one of the above items, wherein the first cell or microorganism is an animal cell. [Item 48B6] The method according to any one of the above items, wherein the second cell or microorganism and / or the third cell or microorganism are microorganisms genetically modified to exhibit their respective functions.[Item 48C1] A method for culturing a first cell or microorganism, comprising: (a) culturing a first cell or microorganism and obtaining a culture medium after the culture; (b) culturing a second cell or microorganism having the ability to remove lactate using the culture medium obtained in (a) and obtaining a first treated supernatant from the culture; (c) culturing a third cell or microorganism having the ability to produce and export glucose using the culture medium obtained in (a) and obtaining a second treated supernatant from the culture; (d) mixing the first treated supernatant obtained in (b) and the second treated supernatant obtained in (c) to prepare a regeneration medium; and (e) culturing the first cell or microorganism in the regeneration medium prepared in (d), wherein the second cell or microorganism and the third cell or microorganism are different microorganisms. [Item 48C2] The method according to any one of the above items, characterized in that in step (d), the first treated supernatant and the second treated supernatant are mixed at a predetermined volume ratio. [Item 48C3] The culture method according to any one of the above items, wherein the second cell or microorganism is a cyanobacterium or Escherichia coli. [Item 48C4] The method according to any one of the above items, wherein the third cell or microorganism is a cyanobacterium. [Item 48C5] The method according to any one of the above items, wherein the first cell or microorganism is an animal cell. [Item 48C6] The method according to any one of the above items, wherein the second cell or microorganism and / or the third cell or microorganism are microorganisms genetically modified to perform their respective functions. [Item 48C7] The method according to any one of the above items, comprising a step of changing the mixing ratio of the second cell or microorganism and the third cell or microorganism depending on the amount of each product. [Item 49] The method according to any one of the above items, wherein the cell or microorganism capable of producing and exporting glucose and the cell or microorganism capable of removing ammonia are the same cell or microorganism, or are different cells or microorganisms. [Item 50] The method according to any one of the above items, wherein the cell is an animal cell.[Item 51] The method according to any one of the above items, wherein the cells or microorganisms capable of producing and exporting glucose, the cells or microorganisms capable of removing ammonia, and the cells or microorganisms capable of removing lactate are each independently the cells or microorganisms described in any one of the above items. [Item 52] A combination comprising an animal cell or microorganism capable of producing and exporting substance X, a cell or microorganism capable of producing and exporting glucose, and a cell or microorganism capable of removing ammonia and / or a cell or microorganism capable of removing lactate, or a system and / or kit comprising the same. [Item 53] The combination according to any one of the above items, wherein substance X is an amino acid, an aromatic compound, a pigment, a protein, cultured meat, terpenoid, oil or fat, a fuel (including, but not limited to, ethanol, gas, petroleum, etc.), a pharmaceutical, an antibiotic, a polymer, etc., or a system and / or kit comprising the same.

[0008] It is contemplated that the present disclosure may provide one or more of the above-described features in combinations other than those explicitly stated. Further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

[0009] Therefore, the present disclosure provides a method for utilizing the capabilities of cells or microorganisms to establish a recycling-type cell or microbial culture and a more recycling-friendly production technology. ２ The present disclosure is applicable to all carbohydrate-producing algae and can be used in conjunction with existing invertase-mediated production methods. This will enable a variety of sustainable production methods to be achieved in the circular economy.

[0010] Figure 1 shows the results of cell proliferation tests for strains introduced with glucose-1-phosphatase, glucoamylase, and glucose efflux protein. A graph showing cell proliferation for each test strain (Figure 1, left) and the amount of glucose secreted at each time point (Figure 1, right) are shown. Figure 1A shows the results of cell proliferation tests for a wild-type strain without gene introduction and a strain introduced with glucose-1-phosphatase and glucose efflux protein. This figure shows the results of cell proliferation tests for strains introduced with glucose-1-phosphatase, glucoamylase, and glucose efflux protein. A graph showing cell proliferation for each test strain (Figure 1B, left) and the amount of glucose secreted at each time point (Figure 1B, right) are shown. This figure shows the results of cell proliferation tests for strains introduced with glucose-1-phosphatase, glucoamylase, and glucose efflux protein. A graph showing cell proliferation for each test strain (Figure 1C, left) and the amount of glucose secreted at each time point (Figure 1C, right) are shown. Figure 2 shows graphs showing glucose secretion for strains introduced with glucose-1-phosphatase and glucose efflux protein. Figure 3 is a graph showing that when muconic acid-producing E. coli was suspended in the culture supernatant of a strain into which glucose-1-phosphatase and a glucose export protein had been introduced, glucose in the culture supernatant was consumed. Figure 4 is a graph showing that when muconic acid-producing E. coli was suspended in the culture supernatant of a strain into which glucose-1-phosphatase and a glucose export protein had been introduced, protocatechuic acid, an intermediate in muconic acid production, was produced. Figure 5 is a graph showing that when muconic acid-producing E. coli was suspended in the culture supernatant of a strain into which glucose-1-phosphatase and a glucose export protein had been introduced, almost no muconic acid, the final product, was produced. Figure 6 shows an example of a gene insertion construct for D-glucose production. Figure 7 shows a non-limiting example of a D-glucose production method of the present disclosure. Figure 8 is a schematic diagram of an aromatic production test using E. coli using glucose produced by a glk-deficient AGP-GLF-introduced strain. FIG. 9 shows the changes in the amounts of glucose and pyruvic acid in the culture supernatant during circulating culture in which animal cells (C2C12 cells) and algae (KC0154 strain) to which lactate utilization ability and glucose excretion ability had been imparted were alternately used.FIG. 10 shows the changes in the amount of lactic acid and ammonia in the culture supernatant during circulating culture in which animal cells (C2C12 cells) and algae (KC0154 strain) conferred with lactate assimilation and glucose excretion capabilities were alternately used. FIG. 11 shows the change in viable cell count when C2C12 cells were cultured again (second culture cycle) using culture wastewater from animal cells (C2C12 cells). When the C2C12 cells were cultured for the second cycle after culturing without culturing the KC0154 strain conferred with lactate assimilation and glucose excretion capabilities, a decrease in viable cell count was observed over two days of culture. On the other hand, when the KC0154 strain was cultured with C2C12 cell culture wastewater and then the C2C12 cells were cultured for the second cycle, an increase in viable cell count and proliferation of the C2C12 cells were confirmed.

[0011] The present disclosure will now be described with reference to the best mode. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, it should be understood that terms used in this specification are used in the sense commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In the event of conflict, the present specification (including definitions) will prevail.

[0012] The following provides definitions of terms particularly used in this specification and / or explains basic technical content as appropriate.

[0013] As used herein, "about" may mean ±10% of the numerical value that follows, or, when referring to significant figures, may mean a range of numerical values ​​expressed as significant figures.

[0014] As used herein, the terms "assimilability" and "utilization" refer to the ability of an organism to take up a substance into cells or microorganisms and metabolize or consume it, and can be used interchangeably. As used herein, the term "removal ability" refers to the ability to remove a target substance XX (e.g., L-lactic acid) from a certain location, and the mechanism for this removal is via assimilation, uptake, metabolism, consumption, etc.

[0015] In this specification, "conferring or enhancing" some ability (also referred to as YY ability, where YY is a term that represents some ability, such as, but not limited to, assimilation ability) means that when an organism does not naturally have that ability, the organism is given that ability by, for example, genetically modifying the organism, or when an organism naturally has that ability, the organism is genetically modified to improve that ability.

[0016] As used herein, the terms "XX assimilation ability" and "XX assimilation" refer to the ability to take up a substance XX (e.g., L-lactic acid, glucose, etc.) into a cell or microorganism and metabolize or consume it, and can be used interchangeably. "XX removal ability" refers to the ability to remove XX (e.g., L-lactic acid, glucose, etc.) from a certain location (in this case, if XX is L-lactic acid, it is referred to as "(L-)lactic acid removal ability"), and the mechanism of this is via assimilation, uptake, metabolism, consumption, etc.

[0017] As used herein, "glucose derivatives or derivatives thereof" refers to compounds produced by conversion of glucose in organisms, cells, or microorganisms, or products produced by modifying, processing, etc. these compounds. As used herein, "lactic acid derivatives or derivatives thereof" refers to compounds produced by conversion of lactic acid in organisms, cells, or microorganisms, or products produced by modifying, processing, etc. these compounds.

[0018] As used herein, "modification" refers to changing the properties of a cell or microorganism, and can be achieved, for example, by inserting a foreign base sequence into the genomic sequence of a cell or microorganism, or by deleting a portion of the genomic sequence of a cell or microorganism, or a combination thereof.

[0019] As used herein, the term "modified organism" or "modified organism" refers to an organism whose properties have been altered relative to a reference organism, typically an organism in which the base sequence of the genome sequence has been altered. Examples of reference organisms include, but are not limited to, naturally occurring organisms, organisms that were unknown before modification, and organisms that did not exist before modification.

[0020] As used herein, the term "gene" refers to a factor that determines a genetic trait, and "gene" may refer to "polynucleotide," "oligonucleotide," and "DNA." Genes encoding such proteins may be endogenous or exogenous to the target organism. Furthermore, known genes may be used as appropriate. Genes may be used regardless of their origin. That is, genes may be derived from organisms of other species or genus than the target organism, or may be derived from organisms such as animals, plants, fungi (molds, etc.), and bacteria. Those skilled in the art can appropriately obtain information on such genes by accessing websites such as the NCBI (National Center for Biotechnology Information; http: / / www.ncbi.nlm.nih.gov). These genes may be genes encoding proteins that have a certain relationship to sequence information disclosed in databases, etc., as long as they have the respective activities. One such embodiment is a gene encoding a protein having an activity to be enhanced in the present invention, which comprises an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted, or added from the disclosed amino acid sequence. The amino acid mutations in the disclosed amino acid sequence, i.e., deletion, substitution, insertion, or addition, may be any one type, or a combination of two or more types. The total number of these mutations is not particularly limited, but is preferably between one and ten, and more preferably between one and five. Preferred examples of amino acid substitutions include conservative substitutions, specifically substitutions within the following groups: (glycine, alanine), (valine, isoleucine, leucine), (aspartic acid, glutamic acid), (asparagine, glutamine), (serine, threonine), (lysine, arginine), and (phenylalanine, tyrosine).

[0021] As used herein, "introduction" of a gene refers to the introduction of an exogenous or endogenous gene, preferably a functional gene, into, for example, a chromosomal genome, using an appropriate introduction technique. Gene introduction can be performed using vectors such as phages and plasmids, and natural transformation, conjugation, protoplast-PEG, and electroporation can also be used. Furthermore, by utilizing targeted gene recombination methods known in the art, an exogenous functional gene can be introduced by replacing an endogenous functional gene. An exogenous functional gene is a gene that is not originally present in the chromosomal genome of the organism, and can be a gene derived from another biological species or a synthetic gene created by PCR or the like. Gene introduction also includes converting an existing genome into a desired gene by genome editing.

[0022] As used herein, a "gene that synthesizes" a substance such as glucose refers to a gene that encodes an enzyme or the like involved in promoting the synthesis of a substance such as glucose in a living organism, cell, or microorganism, or a gene that promotes the expression of an enzyme or the like involved in promoting the synthesis of a substance such as glucose. For example, when pyruvate is a substrate, the enzymes of the Calvin cycle and enzymes responsible for converting fructose-6-phosphate to glucose via glucose-6-phosphate (e.g., glucose-6-phosphate isomerase) are part of the genes (products) that perform this synthesis.

[0023] As used herein, "attenuation" of a gene refers to reducing gene expression (which may be at the transcription level or translation level) or the function of a gene product compared to its natural state or before modification, and "deletion" of a gene refers to eliminating gene expression (which may be at the transcription level or translation level) or the function of a gene product, also referred to as "inhibition." As used herein, "attenuation" and "deletion" of a "gene" can be described as "attenuation" and "deletion" of the nucleic acid encoding the gene and its product, etc.

[0024] As used herein, "introduction" refers to externally introducing a target mechanism (e.g., an enzyme, etc.) into a living organism (e.g., a cell or a microorganism) that does not normally have the target mechanism present in the cell or microorganism, and "enhancement" refers to strengthening (quantitatively and / or qualitatively) the target mechanism in a living organism that already has the target mechanism present.

[0025] As used herein, the term "enzyme" refers to a biological substance, often a protein, that acts as a catalyst to promote chemical reactions in living organisms. Enzymes bind to specific substrates and accelerate the chemical reactions. After the reaction, the enzyme itself remains unchanged and can repeat the same reaction. Enzymes are involved in many important processes in the life activities of living organisms, such as metabolism, DNA replication, repair, and energy production. The specificity of an enzyme is determined by the structure of its active site, which allows it to recognize and act only on specific substrates. Enzymes exert their activity depending on conditions such as pH, temperature, and substrate concentration, and their activity can fluctuate with changes in these conditions. Therefore, the enzymes referred to herein are those that promote specific reactions based on their properties in the biological or chemical process of interest.

[0026] As used herein, the term "efflux protein" refers to a protein that is responsible for the process of transporting a biomolecule (e.g., a protein) synthesized within a cell or microorganism to the outside of the cell or microorganism or to the outside of the cell or microorganism membrane. An efflux protein has a sequence responsible for specific excretion. Targets for excretion include hormones, enzymes, antibodies, and components of the extracellular or microbial matrix, among other functions. Secretion by efflux proteins is important for the normal function of cells or microorganisms and communication between tissues, and abnormalities in this process can cause disease. Furthermore, efflux proteins affect the extracellular or extracellular environment and are involved in a wide range of biological processes, including signal transduction between cells or microorganisms, immune responses, and tissue repair and regeneration. Based on these properties, efflux proteins as used herein are responsible for secretion outside the cell or microorganism to fulfill a specific function. Examples of efflux proteins include diffusion-facilitating proteins.

[0027] As used herein, the term "diffusion-facilitating protein" refers to a protein that facilitates the transport of specific molecules through a cellular or microbial membrane. These proteins support passive diffusion, typically transporting molecules along a concentration gradient inside or outside a cell or microorganism. Diffusion-facilitating proteins function as channel proteins or carrier proteins, helping molecules such as glucose, amino acids, and ions pass through a cellular or microbial membrane. This allows cells or microorganisms to efficiently uptake necessary substances, maintaining their normal function. The activity of these proteins depends on their ability to selectively bind to specific molecules, resulting in smooth movement of substances through a cellular or microbial membrane. Furthermore, because diffusion-facilitating proteins do not require energy, efficient transport is possible while minimizing energy consumption. The diffusion-facilitating proteins referred to herein possess these properties and perform an important function supporting the transport of substances inside and outside a cell or microorganism.

[0028] As used herein, the term "enzyme that converts glycogen to glucose" refers to an enzyme that catalyzes the reaction of decomposing glycogen to produce glucose or a glucose derivative. This enzyme primarily includes an enzyme called glycogen phosphorylase, which plays a role in converting glycogen to glucose-1-phosphate (G1P). Debranching enzymes (glycogen debranching enzymes) also participate in this process, processing the branched structure of glycogen and promoting its degradation. G1P is then converted by phosphoglucomutase to glucose-6-phosphate (G6P), which can be used as glucose in cells or microorganisms as needed. This enzyme, as used herein, is essential for the efficient production of glucose from glycogen and plays an important role in energy supply and maintaining blood glucose levels.

[0029] As used herein, examples of "enzymes that convert glycogen to glucose" include the following enzymes. A specific, non-limiting example is Rhizopus delemar glucoamylase (RdGA), an enzyme that efficiently degrades glycogen and starch to produce glucose. Glycogen phosphorylase is a key enzyme that converts glycogen to glucose-1-phosphate (G1P). α-Amylase also hydrolyzes the α-1,4-bonds in glycogen and starch to produce oligosaccharides and dextrins. In addition, debranching enzymes eliminate the branched structure of glycogen and promote the action of glycogen phosphorylase. Another example is glucoamylase, an enzyme that degrades starch and glycogen to glucose, particularly hydrolyzing the terminal α-1,4-bonds. Isoamylase is an enzyme that hydrolyzes the α-1,6-bonds in glycogen and starch, cleaving the branched portions. Maltase also breaks down maltose (malt sugar) into glucose and is involved in the utilization of maltose, a by-product of glycogenolysis. Acid maltase is an enzyme that hydrolyzes glycogen in lysosomes to produce glucose. α-Glycosylase also plays a role in the breakdown of glycogen and starch to produce glucose. Finally, glycosyltransferase is an enzyme that transfers the short sugar chains remaining from glycogenolysis to other sugar molecules, enabling further degradation by glycogen phosphorylase. These enzymes play an important role in the process of glycogen degradation to produce glucose.

[0030] As used herein, the term "conversion of glucose to glucose-6-phosphate (G6P)" refers to the initial phosphorylation process of glucose molecules after their uptake into cells or microorganisms. This conversion is primarily catalyzed by enzymes called hexokinase or glucokinase. This enzymatic reaction adds a phosphate group to the 6-carbon atom of glucose to produce glucose-6-phosphate. This conversion is a critical step that allows glucose to remain within cells or microorganisms and be further utilized in metabolic pathways such as glycolysis, glycogen synthesis, and the pentose phosphate pathway. Conversion to G6P plays an essential role in energy supply, metabolic regulation, and maintaining the normal function of cells or microorganisms.

[0031] As used herein, "glucose-1-phosphate (G1P)" is a key intermediate in carbohydrate metabolism. G1P is particularly involved in the processes of glycogenolysis and glycogen synthesis. During glycogen degradation, glucose units are released as G1P by the enzyme glycogen phosphorylase. G1P is then converted to glucose-6-phosphate (G6P) by the enzyme phosphoglucomutase, which is further utilized in glycolysis and gluconeogenesis. G1P also functions as a precursor in glycogen synthesis. Specifically, G1P is converted to uridine diphosphate glucose (UDP-glucose), which is added to the glycogen chain by glycogen synthase. As a result, G1P plays a central role in energy storage and utilization. Enzymes that convert G1P to glycogen include glgCs such as glgC1. GlgC is a representative example of an enzyme that refers to glucose-1-phosphate adenylyl transferase. This enzyme plays an important role in the process of glycogen biosynthesis, catalyzing the reaction that produces ADP-glucose using glucose-1-phosphate (G1P) and ATP as substrates. The produced ADP-glucose is incorporated into glycogen chains by glycogen synthase, and glycogen synthesis proceeds. GlgC is an enzyme essential for glycogen synthesis to store energy, particularly in organisms such as plants and bacteria.

[0032] As used herein, "glucose-6-phosphate (G6P)" refers to the phosphorylated form of glucose molecules and is a central intermediate in glucose metabolism. G6P is involved in multiple metabolic pathways, including glycolysis, gluconeogenesis, and the pentose phosphate pathway. When glucose is taken up by cells or microorganisms, it is phosphorylated by hexokinase or glucokinase to produce G6P. This allows glucose to remain within the cell or microorganism and be used for metabolism. G6P is converted to pyruvate through glycolysis or used to produce NADPH and ribose-5-phosphate in the pentose phosphate pathway. It also functions as a precursor for glycogen synthesis. G6P plays an important role in energy production and the synthesis of biomolecules.

[0033] As used herein, the term "conversion of glucose to glucose-6-phosphate (G6P)" refers to any mechanism for converting glucose to glucose-6-phosphate (G6P), which is typically achieved by phosphorylation of glucose, and is usually achieved by an enzyme (e.g., glucokinase).

[0034] As used herein, "glucokinase activity" refers to one of the fundamental reactions in cellular carbohydrate metabolism, an enzyme activity that phosphorylates glucose to produce glucose-6-phosphate (G6P). Specifically, it catalyzes the reaction of glucose and adenosine triphosphate (ATP) as substrates, transferring a phosphate group derived from ATP to the 6-carbon position of glucose. The G6P produced by this reaction is supplied to glycolysis, the pentose phosphate pathway, and pathways for synthesizing polysaccharides such as glycogen and trehalose, and is used for energy acquisition and metabolite synthesis in cells. Glucokinase activity is present in a variety of organisms, including microorganisms, plant cells, and animal cells, and its expression level and activity are regulated according to the respective physiological conditions. In particular, it plays a central role in regulating glucose utilization efficiency and metabolic flux in cells, affecting growth, metabolic control, and even the production of secondary metabolites. Furthermore, "glucokinase activity" as used herein encompasses, but is not limited to, naturally occurring enzyme activity as well as the activity of mutants and artificial enzymes improved by molecular modification, genetic engineering, or evolutionary engineering techniques.

[0035] As used herein, "inhibition of glucokinase activity" refers to the effect of reducing or completely suppressing glucokinase activity in cells. This inhibition is achieved by direct or indirect interference with the glucose phosphorylation reaction. Specifically, this includes direct inhibition using small molecules, inhibitory peptides, antibodies, or other bioactive molecules that prevent binding to the catalytic site of glucokinase, as well as indirect inhibition via suppression of glucokinase gene expression, changes in post-translational modifications, or control of its intracellular localization. Such inhibitory effects reduce the conversion of glucose to glucose-6-phosphate (G6P), thereby regulating downstream metabolic fluxes such as glycolysis, the pentose phosphate pathway, and the polysaccharide synthesis pathway. As used herein, "inhibition of glucokinase activity" encompasses inhibitory effects in a variety of cellular systems, including microorganisms, plant cells, and animal cells. Furthermore, as used herein, "inhibition of glucokinase activity" includes, but is not limited to, not only naturally occurring inhibitors but also artificial inhibitors obtained by molecular modification or chemical synthesis, as well as means of suppressing expression using genetic engineering techniques.

[0036] As used herein, "disruption of a glucokinase activity gene" refers to a procedure that eliminates the function of a gene responsible for glucokinase activity in a cell, thereby eliminating or significantly reducing the enzymatic activity derived from the gene. This disruption can be achieved by introducing mutations, deletions, insertions, or frameshifts into the gene sequence, as well as by blocking transcription itself by modifying the promoter region or regulatory sequence. Specific techniques include gene knockout using homologous recombination, transposon insertion, and the introduction of loss-of-function mutations using the cleavage and repair process with targeted nucleases (e.g., CRISPR / Cas, TALEN, ZFN, etc.). This results in the cell losing its ability to convert glucose to glucose-6-phosphate (G6P), thereby controlling carbon flux to glycolysis and related metabolic pathways. As used herein, "disruption of a glucokinase activity gene" includes techniques applicable to any organism, including microorganisms, plant cells, and animal cells. This includes, but is not limited to, not only complete gene deletion but also the introduction of point mutations or partial deletions that eliminate the catalytic function of the enzyme.

[0037] As used herein, the phrase "having no or weakened mechanisms for excretion outside the cell or microorganism" refers to a state in which a cell or microorganism lacks or has impaired function of an excretion pathway that would normally transport substances to the medium or external environment. The term "excretion mechanism" as used herein includes membrane transporters, efflux pumps, secretion systems (e.g., types I to VI secretion systems), transporters, channels, or other active or passive transport mechanisms. Cells lacking or weakened such mechanisms are more likely to retain specific metabolites or exogenous substances within the cell, enabling control of metabolic pathways, substance accumulation, or increased intracellular concentrations. Specific examples include cases in which the function of a transport protein is lost through gene disruption, gene expression suppression, point mutation introduction, or structural modification, or when the expression level is reduced to weaken the transport ability. As used herein, the phrase "having no or weakened mechanisms for excretion outside the cell or microorganism" includes, but is not limited to, strains that naturally lack the excretion mechanism, strains in which the function has been eliminated through experimental modification, and strains that retain partial transport ability but with significantly reduced transport ability compared to conventional strains.

[0038] In this specification, the "Calvin (CBB) cycle" refers to a typical carbon dioxide fixation reaction in photosynthesis. Almost all green plants and photosynthetic bacteria have this cycle. More specifically, it is a series of chemical reactions known as the dark reactions of photosynthesis, which take place in the chloroplasts of plants and algae. In this cycle, carbon dioxide (CO ２ ) is fixed by the enzymes ribulose-1,5-bisphosphate (RuBP) and Rubisco (ribulose-1,5-bisphosphate carboxylase / oxygenase) to produce 3-phosphoglycerate. This is then reduced by glyceraldehyde-3-phosphate dehydrogenase to produce glyceraldehyde-3-phosphate. Finally, ribulose-5-phosphate is produced, which is then regenerated into ribulose-1,5-bisphosphate. This process uses ATP and NADPH, ultimately producing carbohydrates such as glucose.

[0039] As used herein, "organisms having the Calvin cycle" refer to any organism that has the Calvin cycle. This includes almost all green plants and photosynthetic bacteria, including, for example, cyanobacteria and hydrogen-oxidizing bacteria. Other examples of organisms that have the Calvin cycle (Calvin-Benson-Bassham cycle) include mainly photosynthetic plants, algae, cyanobacteria, purple sulfur bacteria, and purple non-sulfur bacteria, and specific examples include the following organisms: First, examples of plants include Oryza sativa, Triticum aestivum, Zea mays, Glycine max, Arabidopsis thaliana, Saccharum officinarum, Solanum tuberosum, Solanum lycopersicum, Helianthus annuus, Cucurbita pepo, etc. Also, Prunus spp., Fagus sylvatica, Acer spp., Pinus spp. Plants such as Olea europaea, Coffea arabica, Brassica oleracea, Spinacia oleracea, Perilla frutescens, and Rosa spp. also have the Calvin cycle. Examples of algae include Chlorella spp., Arthrospira spp., Chlamydomonas reinhardtii, Ulva spp., Laminaria spp., Undaria pinnatifida, Sargassum fusiforme, Spirogyra spp., and Diatoma spp. , Tetraselmis spp. Cyanobacteria include Anabaena spp., Nostoc spp., Prochloron spp., Synechocystis spp., Chlorococcum spp., Prochlorococcus spp., Chlorobium spp., Oscillatoria spp., Rhizobium spp., Synechococcus spp., and the like.Other photosynthetic organisms include Fucus spp., Zostera spp., Acromyrmex spp. (symbiotic bacteria with the Calvin cycle), Gloeobacter violaceus, Prochlorothrix hollandica, Thermodesulfovibrio spp., Chlorarachnion spp., Prochlorotrinia spp., and Chloronema spp. Hydrogen-oxidizing bacteria include Hydrogenophilus spp. and Cupriavidus spp., and purple non-sulfur bacteria include Rhodopseudomonas spp. All of these organisms have the Calvin cycle and the ability to fix carbon dioxide and synthesize organic matter.

[0040] As used herein, "algae" refers to organisms that produce oxygen through photosynthesis, excluding mosses, ferns, and spermatophytes, which are primarily terrestrial. Algae can produce their own oxygen and nutrients (e.g., glucose and amino acids) and grow when the environment necessary for photosynthesis is provided. Preferably, cyanobacteria are used in the present disclosure. The algae to which the present invention can be applied may be naturally occurring algae or algae grown by known culture methods.

[0041] As used herein, the term "conversion of glucose-1-phosphate (G1P) to glycogen" refers to any phenomenon in which glucose-1-phosphate (G1P) is converted into glycogen, and can usually be achieved by an enzyme (e.g., glucose-1-phosphate adenylyltransferase (glgC)) or the like.

[0042] As used herein, the term "product" refers to a substance produced, generated, or otherwise produced by an organism, cultured cells, or microorganism, and includes, for example, amino acids, proteins, sugars, and the like.

[0043] In one embodiment, the algae to which the present invention can be applied may be "microalgae" (also referred to as "unicellular algae"). As used herein, "microalgae" refers to algae in which each individual is composed of a single cell, and includes microalgae in which multiple microalgae gather to form colonies, and includes freshwater or marine, stenohaline or euryhaline microalgae. Examples of microalgae include blue-green algae (cyanobacteria), which are prokaryotic photosynthetic organisms characterized by having chlorophyll a and phycobilin pigments; green algae in which chlorophyll a and b are the primary pigments in the chloroplasts; and unicellular red algae in which chlorophyll a is the primary pigment.To give more detailed examples, in the green algae, there are Chlamydomonas reinhardtii (Japanese name: Chlamydomonas) (freshwater) of the Chlorophyceae order Chlamydomonadales, Dunaliella salina (Japanese name: Dunaliella) (marine) of the Dunaliella order, Volvox carteri (Japanese name: Volvox) (freshwater) of the Volvoxales, Chlorococcum littorale (marine) of the Chlorococcales, and Hydrodictyon reticulatum (Hydrodictyon nigricans) of the Chlorophyceae order. reticulatum (Japanese name: Amimidoro) (freshwater), Pediastrum duplex (Japanese name: Kunshoumo) (freshwater), Scenedesmus dimorphus (Japanese name: Ikadamo) (freshwater), Chlorella sp. (Japanese name: Chlorella) of the Trebouxiophyceae family, Chlorellales (e.g., Chlorella vulgaris (freshwater), Chlorella pyrenoidosa (freshwater), Chlorella erupsoidea (freshwater), Examples of such species include Chlorella ellipsoidea (freshwater), Chlorella regularis (freshwater), Euglena gracilis and Euglena proxima (Japanese name: Midorimushi) (freshwater) of the Euglenophyta, Euglenophyceae, Euglenales. Examples of unicellular cyanobacteria include Acaryochloris marina (marine), Spirulina subsalsa (freshwater), Arthrospira platensis (freshwater), and Synechococcus sp. (marine or freshwater) of the Cyanobacteria phylum.Examples of unicellular red algae include Cyanidium caldarium (Japanese name: Ideyukogome) (freshwater) of the Class Aquilegiae, Class Aquilegiae, Order Aquilegiae, and Galdieria partita (freshwater). Examples of unicellular charophytes include the genus Stichococcus (freshwater) of the Class Klebsormidiales, Class Aquilegiae, Class Aquilegiae, and Class Bquilegiae, and also include the unicellular algae Filamentous-ulvophyte, which belongs to the Class Ulva. In addition to the algae listed above, the algae to which the present invention can be applied may be genetically engineered versions of these algae, or algae other than those listed above. For example, the algae to which the present invention can be applied may be freshwater or marine, and / or stenohaline or euryhaline microalgae, but are preferably marine and / or euryhaline microalgae, such as, but not limited to, Chlorococcum littorale and algae of the genus Synechococcus.

[0044] As used herein, the term "component derived from" refers to an organism, cultured cell, or microorganism itself, a part of any of them, or a component produced by any of them.

[0045] As used herein, the terms "nutritional component" and "nutrient source" are used interchangeably and refer to components that are consumed or metabolized by organisms, cultured cells, or microorganisms as they grow or multiply, including, for example, sugars, amino acids, and the like, or complexes thereof.

[0046] As used herein, the terms "excreted component" and "excreted component" refer to a component that is released from inside a cell or microorganism to the outside during the process of culturing the cell or microorganism. Excretion can take any form, such as secretion, simple elimination of waste products, evaporation, or diffusion. This component also includes components that are toxic to the cell or microorganism (for example, L-lactic acid to certain animal cells or microorganisms).

[0047] As used herein, the terms "recycling," "circular," and "recycle" refer to the reuse of a substance or an organism composed of substances in a system, preferably all substances present in a system or an organism composed of substances, within the system without receiving supplies from the outside, thereby reproducing the target substance or organism composed of the substance. For example, the concepts of recycling, recycling, or circularity apply to the use of components excreted by a first organism, cultured cells, or microorganism as a nutrient source by a second organism, cultured cells, or microorganism, and the use of components excreted by the second organism, cultured cells, or microorganism as a nutrient source by the first organism, cultured cells, or microorganism.

[0048] As used herein, the term "mechanism" (which in English is a term intended to mean "configuration" or simply "element") is a term intended to mean an entity for achieving some purpose, and refers to a configuration, process, or means for achieving some purpose. Biochemically, mechanisms are often composed of some kind of protein or complex protein, and are often enzymes or combinations of enzymes, but are not limited to this, and can also include, for example, receptors and efflux proteins.

[0049] As used herein, the term "mechanism for converting glucose-1-phosphate (G1P) to glucose" refers to any means or substance (including single or multiple enzymes) that promotes the process of converting glucose-1-phosphate (G1P) to glucose. G1P is converted to glucose-6-phosphate (G6P), for example, by the action of an enzyme called phosphoglucomutase. In this reaction, the phosphate group of G1P moves to another position. This G6P is dephosphorylated by an enzyme called glucose-6-phosphatase in tissues such as the liver, becoming free glucose. These enzymes are examples of mechanisms.

[0050] As used herein, "blocking the conversion of X (e.g., G1P) to Y (e.g., glycogen)" refers to stopping the conversion of X to Y by some means. X and Y each independently refer to a substance or a complex thereof within a cell, and typically refer to different substances or complexes thereof. For example, when the conversion of X to Y is achieved by an enzyme, the enzyme is rendered non-functional (e.g., by modifying or knocking out the enzyme).

[0051] As used herein, "glucose production and export capability" refers to the ability of a cell or microorganism to produce glucose via a metabolic pathway and export the produced glucose outside the cell. Here, "production" includes the action of producing glucose intracellularly via a carbohydrate metabolic pathway (e.g., a gluconeogenesis pathway, a degradation pathway for polysaccharides such as starch and cellulose, or an artificial pathway introduced by metabolic engineering). "Excretion" includes the action of exporting such intracellularly produced glucose to the extracellular environment via a membrane transporter, transporter, channel, diffusion, or other mechanism. As used herein, "glucose production and export capability" includes not only organisms that naturally have the ability to produce and export glucose, but also cells or microorganisms whose capabilities have been introduced or enhanced by genetic engineering or metabolic engineering. It also includes, but is not limited to, systems that partially have either production or export capabilities but can supply glucose to the external environment as a result of combining both.

[0052] As used herein, "lactic acid removal ability" refers to the ability of cells or microorganisms to reduce the concentration of lactic acid present within the cell or culture environment by decomposing, converting, or transporting it. "Removal" here includes the action of incorporating lactic acid into a metabolic pathway and converting it into other metabolites (e.g., pyruvate, carbon dioxide, water, etc.), the action of taking up lactic acid from outside the cell and metabolizing it, or the action of binding or immobilizing lactic acid to make it unavailable. Specifically, typical mechanisms responsible for "lactic acid removal ability" include the oxidation reaction of lactic acid to pyruvate by lactate dehydrogenase (LDH), the degradation reaction by lactate oxidase, and fermentation pathways that utilize lactic acid. Furthermore, it also includes cases where lactic acid in the external environment is taken up into cells via membrane transporters or channels and processed by metabolism. As used herein, "lactic acid removal ability" includes, but is not limited to, microorganisms that naturally have the ability to assimilate lactic acid, as well as cells or microorganisms that have been conferred or enhanced with the ability to utilize lactic acid through genetic modification or metabolic engineering.

[0053] As used herein, "ammonia removal ability" refers to the ability of cells or microorganisms to reduce the concentration of ammonia present within the cells or in the culture environment by immobilizing, metabolically converting, or converting it to other compounds. "Removal" here includes the action of assimilating ammonia and using it as a nitrogen source, the action of incorporating it into a metabolic pathway and converting it into biomolecules such as amino acids, nucleotides, and proteins, or the action of fixing it to a stable nitrogen compound via glutamine synthetase or the like. Specifically, typical mechanisms responsible for "ammonia removal ability" include the assimilation of ammonia into glutamine by glutamine synthetase (GS), the incorporation into glutamate by glutamate dehydrogenase (GDH), or processing via a metabolic pathway similar to the urea cycle. Furthermore, in some microorganisms, nitrification, in which ammonia is taken up and oxidized to nitrate, nitrite, etc., or the action of converting it into other nitrogen compounds, also falls under this category. In this specification, the term "ammonia removal ability" includes, but is not limited to, cells and microorganisms that naturally have the ability to assimilate and utilize ammonia, as well as cells and microorganisms that have been given or enhanced ammonia processing ability through genetic modification, metabolic engineering, or introduction of an artificial metabolic pathway.

[0054] As used herein, "continuous" refers to a state in which a certain action, treatment, or step is carried out continuously without interruption. "Continuous" here includes cases in which a process or reaction is carried out continuously without interruption, cases in which steps or reactions are connected sequentially as a constant flow, and cases in which, even if the process or reaction is periodic, it is essentially continuous without interruption from an external perspective. For example, in a culture operation, "continuous" refers to a state in which the supply of medium and the removal of products are carried out without interruption, including so-called continuous culture processes. Furthermore, in chemical and enzymatic reactions, "continuous" includes a state in which the supply of substrates and the recovery of products are carried out continuously, and the reaction system proceeds while maintaining a stable state. As used herein, "continuous" includes not only completely uninterrupted continuity but also, but is not limited to, cases in which the process as a whole is recognized as being carried out integrally, rather than discontinuously, even if there are technically negligible short-term transitions or switches between steps.

[0055] As used herein, the term "intermittent" refers to a state in which a certain action, treatment, or process is not performed continuously but is repeatedly performed at regular time intervals. "Intermittent" here includes cases in which clearly defined cycles of on-off are repeated, cases in which external manipulation or stimulation is intermittently applied, or cases in which a process or reaction is performed with periodic or irregular interruptions. For example, in a culture operation, "intermittent" refers to batch culture or fed-batch culture in which medium is supplied or products are recovered at regular intervals. In a chemical or enzymatic reaction, "intermittent" also includes a mode in which reactants are supplied or products are removed at regular intervals rather than continuously. As used herein, "intermittent" encompasses a mode in which the entire process is not continuously operated, but is performed repeatedly to obtain a desired effect or product. Therefore, this term includes, but is not limited to, a method with periodic interruptions or a method operated intermittently as needed.

[0056] As used herein, "continuously connected" refers to a configuration in which multiple culture devices, reaction vessels, or processing units are seamlessly connected to one another, allowing for uninterrupted transfer of culture fluid, cell suspension, medium, or product. "Continuously connected" here includes cases where the devices are physically connected directly by piping or flow channels, or where uninterrupted flow is maintained via pumps, valves, etc. For example, a configuration in which a cell suspension cultured in a first culture vessel is constantly pumped to a second culture vessel by a pump, or a configuration in which the cells are sequentially transferred between culture vessels via a continuous flow channel, falls under "continuously connected." Furthermore, "continuously connected" does not simply mean a mechanical connection, but also includes the maintenance of an uninterrupted flow of culture fluid even during operation. As used herein, "continuously connected" includes not only a system in which multiple devices are arranged in complete series, but also includes, but is not limited to, a system in which multiple devices are constantly connected and operated while a portion of the culture fluid is branched and merged.

[0057] As used herein, "continuously connected via tubing" refers to a configuration in which multiple culture devices, reaction vessels, or processing units are connected by tubing, allowing for the continuous transfer of culture fluid, cell suspension, culture medium, or product through the tubing. The term "tubing" as used herein includes flexible tubing, rigid tubing, silicone tubing, resin tubing, sterile tubing, and other tubing materials that enable fluid transport. The term "continuously connected via tubing" refers not only to a simple mechanical connection by tubing, but also to maintaining an uninterrupted flow of culture fluid or the like through the tubing even during operation. For example, this includes a configuration in which a first fermentation tank and a second fermentation tank are directly connected by a sterile tubing, and liquid is constantly transferred by a pump, or a configuration in which a continuous flow path is formed via tubing and sequential processing is performed. As used herein, "continuously connected via tubing" includes, but is not limited to, a serial connection using a single tubing, as well as a configuration in which multiple devices are constantly connected by branching or merging multiple tubing.

[0058] As used herein, the term "treated supernatant" refers to the supernatant portion obtained after removing cells, microorganisms, cell debris, or solid components from a liquid culture medium by centrifugation, filtration, sedimentation, or other separation means, and refers to a liquid fraction that has undergone a specific treatment process. The term "treatment" as used herein includes sterilization, bacteria removal, concentration, dialysis, protein removal, chemical treatment, physical treatment, or a combination thereof. For example, if the supernatant obtained by centrifugation of a cell culture medium is subjected to sterile filtration using a 0.2 μm filter or the like, the resulting liquid portion falls under the category of "treated supernatant." This definition also includes liquid fractions in which specific components have been adjusted by dialysis or concentration after recovery of the supernatant from the culture medium. As used herein, "treated supernatant" is not limited to those that have undergone simple physical separation, but also includes, but is not limited to, liquids prepared by additional chemical or biological manipulation of the supernatant.

[0059] As used herein, a "system" (including multiple types of cells or microorganisms (e.g., A cells or microorganisms and B cells or microorganisms)) refers to a biological system in which A cells or microorganisms and B cells or microorganisms, each with a specific role, cooperate with each other to perform a unified physiological function as a whole. Because it has a biological meaning, it is sometimes called a biological system to distinguish it from other fields. In this system, A cells or microorganisms and B cells or microorganisms each have their own unique functions, but they depend on each other and work cooperatively to contribute to the homeostasis and maintenance of the body's functions. The roles of A cells or microorganisms and B cells or microorganisms are configured to complement each other depending on the purpose of the system. For example, A cells or microorganisms have a specific function and perform that function by cooperating with other cells or microorganisms, particularly B cells or microorganisms. For example, A cells or microorganisms can secrete signaling molecules and send instructions to B cells or microorganisms through them. Meanwhile, B cells or microorganisms receive signals from A cells or microorganisms and perform a specific response based on that information. Furthermore, when A cells or microorganisms and B cells or microorganisms cooperate in providing nutrients (e.g., glucose production and utilization), A cells or microorganisms produce glucose, and B cells or microorganisms can use the glucose to achieve another function (e.g., fermentation, meat production, etc.).

[0060] As used herein, the term "kit" refers to a unit in which the components to be provided (e.g., cells, reagents, raw materials, structural unit components, instructions, etc.) are provided, typically separated into two or more compartments. This kit format is preferred when the objective is to provide a composition that should not be provided in a mixed state for reasons of stability, but is preferably mixed immediately before use, or to provide a procedure for the recipient to synthesize or manufacture on-site or later. Such kits advantageously include instructions or manuals describing how to use the provided components or how to handle the reagents. When the kit is used herein as a reagent kit, the kit typically includes instructions describing how to use the various reagents, etc.

[0061] (Preferred Embodiments) 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 make appropriate modifications within the scope of the present disclosure in light of the description herein. In addition, the following embodiments of the present disclosure can be used alone or in combination.

[0062] (Modified Organism) In one aspect of the present disclosure, a modified organism, cultured cell, or microorganism is provided, wherein the modification includes imparting or enhancing the ability to produce D-glucose to the organism, cultured cell, or microorganism. D-glucose is one of the nutritional components of various organisms, and in one embodiment of the present disclosure, providing such a modified organism, cultured cell, or microorganism enables the modified organism, cultured cell, or microorganism to grow by utilizing D-glucose, and further enables components derived from the modified organism, cultured cell, or microorganism to serve as a nutrient source for the organism.

[0063] (Modification of Glucose-1-phosphate (G1P) Conversion) In one specific aspect of the present disclosure, the present disclosure provides a cell or microorganism having a Calvin (CBB) cycle, into which a mechanism for converting glucose-1-phosphate (G1P) to glucose has been introduced or enhanced. When the mechanism for converting glucose-1-phosphate (G1P) to glucose exists in nature, the naturally occurring enzyme may be enhanced (quantitatively or qualitatively, or both), or, when it exists in nature or is not present, it may be introduced from outside. Without wishing to be bound by theory, advantages of glucose production by introducing G1P include a simple glucose production pathway and low energy consumption. Furthermore, the overall balance of the invertase-mediated glucose production pathway (sucrose phosphate synthase sps, sucrose phosphate phosphatase spp, invertase inv, fructokinase fp) is such that two molecules of ATP are consumed to produce one molecule of glucose from one molecule of glucose-6-phosphate, whereas the G1P-mediated pathway can produce one molecule of glucose from one molecule of glucose-6-phosphate without consuming ATP, which can be said to lead to improved energy efficiency.

[0064] In one embodiment, the cells or microorganisms used in the present disclosure include cyanobacteria.

[0065] Various types of cyanobacteria can be used. Cyanobacteria are typically unicellular or microbial prokaryotes that perform oxygenic photosynthesis and can be classified into the Chroococcales, Nostocales, Oscillatoryes, Pleurocapsales, Stigonemales, etc. The Chroococcales include the genera Aphanocapsa, Aphanotheche, Chamaesiphon, Chroococcus, Crocosphera, Cyanobacteria, Cyanobium, Cyanotheche, Dactylococcopsis, Gloeobacter, Gloeocapsa, Gloeotheche, Euhalotheche, Halotheche, Johannesbaptistia, Melismopedia, Microcystis, Rhabdoderma, Synechococcus, Synechocystis, and Thermosynechococcus. The Nostocales order includes the genera Coreodesmium, Fremiella, Microchaete, Rexia, Spirirestis, Tryposthrix, Anabaena, Anabaenopsis, Aphanizomenon, Aurocira, Cyanospira, Cylindrospermopsis, Cylindrospermum, Nodularia, Nostoc, Rickeria, Kalothrix, Gloeotrichia, and Scytonema. The Oscillatory order includes the genera Arthrospira, Gaitrelinema, Halomicronema, Halospirulina, Catagnimene, Leptorynbya, Limnothrix, Lymbya, Microcoleus, Oscillatory order, Phormidium, Planctotrichoides, Planktothrix, Plectonema, Limnothrix, Pseudoanavena, Schizothrix, Spirulina, Symploca, Trichodesmium, and Chiconema. The Pleurocapsales includes the genera Chroococchidiopsis, Dermocarpa, Dermocarpella, Myxosarcina, Pleurocapsa, Stanieria, and Xenococcus. The Stigonemales include the genera Capsocilla, Chlorogloeopsis, Fischerella, Hapalosiphon, Mastigocladopsis, Mastigocladus, Nostochopsis, Stigonema, Symphionema, Symphionemopsis, Umezakia, and Westielopsis.

[0066] In one embodiment, the conversion of glucose-1-phosphate (G1P) to glucose used in the present disclosure is achieved by an enzyme. Specifically, the enzyme that converts glucose-1-phosphate (G1P) to glucose is typically glucose-1-phosphatase, and examples thereof include, but are not limited to, Escherichia coli glucose-1-phosphatase (EcAGP, EcYihX, etc.) and the ybbC gene encoding glucose-1-phosphatase of lactic acid bacteria.

[0067] In one embodiment of the present disclosure, the conversion to glucose is achieved without blocking the conversion of G1P to glycogen. Not blocking the conversion of G1P to glycogen is achieved by not modifying or knocking out the naturally occurring enzyme that converts G1P to glycogen.

[0068] In a more preferred embodiment of the present disclosure, the microorganism of the present disclosure is further configured to attenuate or inhibit the conversion of glucose to glucose-6-phosphate (G6P).

[0069] In one embodiment, such conversion of glucose to glucose-6-phosphate (G6P) is accomplished by an enzyme (eg, Glk).

[0070] In one specific embodiment, it may be advantageous to introduce or enhance a mechanism for excreting glucose outside the cell or microorganism of the present disclosure. By enhancing or introducing glucose excretion, glucose can be utilized in other cells or microorganisms or the cell or microorganism itself without the need for separate extraction, and can be advantageously used in a circular reaction.

[0071] In one embodiment, glucose is exported from cells or microorganisms via an efflux protein, such as, but not limited to, Zymomonas mobilis glucose diffusion facilitating protein (ZmGLF) and glucose transporter (GLUT).

[0072] (Modification of Glycogen to Glucose Conversion) In another aspect, the present disclosure provides a cell or microorganism having the Calvin Bacteria (CBB) cycle, such as a cyanobacterium, into which a mechanism for converting glycogen to glucose has been introduced or enhanced. The mechanism for converting glycogen to glucose may be enhanced (quantitatively or qualitatively, or both) by a naturally occurring enzyme if it exists in nature, or may be introduced from an external source if it does not exist in nature. Preferably, the mechanism for converting glycogen to glucose is achieved by an enzyme that converts glycogen to glucose.

[0073] In certain embodiments, enzymes that convert glycogen to glucose, such as Rhizopus delemar glucoamylase (RdGA), other types of glucoamylase, isoamylase, acid maltase, α-glycosylase, glycosyltransferase, etc., may also be evaluated for this function.

[0074] In a preferred embodiment, a microorganism having the glycogen-to-glucose conversion mechanism of the present disclosure introduced or enhanced therein is further configured to inhibit the conversion of glucose to glucose-6-phosphate (G6P). Without wishing to be bound by theory, it has been found that inhibition of this conversion results in an unexpected increase in glucose production or excretion, providing an advantageous aspect.

[0075] In one embodiment, conversion of glycogen to glucose is accomplished by glucoamylase. Alternatively, glycogen can be converted to glucose 1-phosphate by glycogen phosphorylase, and glucose 1-phosphate can be converted to glucose by glucose-1-phosphatase; such multiple enzyme reactions are within the scope of the present disclosure.

[0076] In a further preferred embodiment, it is advantageous that a mechanism for excreting glucose outside the cell or microorganism has been introduced or enhanced in the cell or microorganism of the present disclosure. Thus, the cell or microorganism may be one into which a mechanism for converting glycogen to glucose has been introduced or enhanced and a mechanism for excreting glucose outside the cell or microorganism has been introduced or enhanced, or one into which a mechanism for converting glycogen to glucose has been introduced or enhanced, a mechanism for converting glycogen to glucose has been introduced or enhanced, and a mechanism for excreting glucose outside the cell or microorganism has been introduced or enhanced.

[0077] In one embodiment, efflux in the present disclosure is achieved by proteins such as Zymomonas mobilis glucose diffusion facilitating protein (ZmGLF), a glucose transporter (GLUT).

[0078] In one embodiment, the cells or microorganisms used in the present disclosure include cyanobacteria.

[0079] Various types of cyanobacteria can be used. Cyanobacteria are typically unicellular or microbial prokaryotes that perform oxygenic photosynthesis and can be classified into the Chroococcales, Nostocales, Oscillatoryes, Pleurocapsales, Stigonemales, etc. The Chroococcales include the genera Aphanocapsa, Aphanotheche, Chamaesiphon, Chroococcus, Crocosphera, Cyanobacteria, Cyanobium, Cyanotheche, Dactylococcopsis, Gloeobacter, Gloeocapsa, Gloeotheche, Euhalotheche, Halotheche, Johannesbaptistia, Melismopedia, Microcystis, Rhabdoderma, Synechococcus, Synechocystis, and Thermosynechococcus. The Nostocales order includes the genera Coreodesmium, Fremiella, Microchaete, Rexia, Spirirestis, Tryposthrix, Anabaena, Anabaenopsis, Aphanizomenon, Aurocira, Cyanospira, Cylindrospermopsis, Cylindrospermum, Nodularia, Nostoc, Rickeria, Kalothrix, Gloeotrichia, and Scytonema. The Oscillatory order includes the genera Arthrospira, Gaitrelinema, Halomicronema, Halospirulina, Catagnimene, Leptorynbya, Limnothrix, Lymbya, Microcoleus, Oscillatory order, Phormidium, Planctotrichoides, Planktothrix, Plectonema, Limnothrix, Pseudoanavena, Schizothrix, Spirulina, Symploca, Trichodesmium, and Chiconema. The Pleurocapsales includes the genera Chroococchidiopsis, Dermocarpa, Dermocarpella, Myxosarcina, Pleurocapsa, Stanieria, and Xenococcus. The Stigonemales include the genera Capsocilla, Chlorogloeopsis, Fischerella, Hapalosiphon, Mastigocladopsis, Mastigocladus, Nostochopsis, Stigonema, Symphionema, Symphionemopsis, Umezakia, and Westielopsis.

[0080] (Modification of Glucose to G6P Conversion) In another aspect, a cell or microorganism is provided that is a microorganism having the Calvin Bacterium (CBB) cycle, such as a cyanobacterium, and is configured to inhibit the conversion of glucose to glucose-6-phosphate (G6P). Inhibition of the conversion of glucose to glucose-6-phosphate (G6P) can be achieved by inhibiting (including knocking out) or suppressing (quantitatively or qualitatively, or both) the naturally occurring enzyme, if it exists in nature, or by not introducing it from an exogenous source, if it does not exist in nature. Preferably, the conversion of glucose to glucose-6-phosphate (G6P) is achieved by knocking out an enzyme (e.g., Glk).

[0081] In a preferred embodiment, the inhibition of glucose conversion to glucose-6-phosphate (G6P) of the present disclosure is achieved by inhibiting glucokinase activity. Alternatively, the inhibition of glucose conversion to glucose-6-phosphate (G6P) of the present disclosure may be achieved by disrupting the glucokinase activity gene. This configuration enables more effective inhibition of glucose conversion to glucose-6-phosphate (G6P).

[0082] In one embodiment, the present disclosure does not include or weakens a mechanism for excreting glucose outside the cell or microorganism. By reducing the energy cost allocated to the mechanism for excreting glucose outside the cell or microorganism, highly efficient glucose synthesis is possible in the entire synthesis system.

[0083] In one embodiment, the present disclosure further comprises the introduction or enhancement of an enzyme that converts glucose-1-phosphate (G1P) to glucose, and / or the introduction or enhancement of an enzyme that converts glycogen to glucose.

[0084] In certain embodiments, the enzyme that converts glucose-1-phosphate (G1P) to glucose may be, for example, glucose-1-phosphatase (EC 3.1.3.10), and specifically may be any enzyme belonging to EC 3.1.3.10, such as Escherichia coli glucose-1-phosphatase (EcAGP and EcYihX, etc.), agp and yihX genes of Salmonella, Enterobacter, and Cronobacter, or the ybbC gene encoding glucose-1-phosphatase of Lactobacillus.

[0085] In a preferred embodiment, a mechanism for excreting glucose outside the cell or microorganism has been introduced or enhanced in a cell or microorganism configured to inhibit the conversion of glucose to glucose-6-phosphate (G6P) of the present disclosure. In a preferred embodiment, a cell or microorganism configured to inhibit the conversion of glucose to glucose-6-phosphate (G6P) of the present disclosure and into which an enzyme that converts glucose-1-phosphate (G1P) to glucose has been introduced or enhanced and / or an enzyme that converts glycogen to glucose has been introduced or enhanced has been introduced, a mechanism for excreting glucose outside the cell or microorganism has been introduced or enhanced.

[0086] In one embodiment, the mechanism by which glucose is exported from the cell or microorganism of the present disclosure is achieved by an export protein.

[0087] In specific embodiments, the efflux proteins of the present disclosure can be Zymomonas mobilis glucose diffusion facilitating protein (ZmGLF), glucose transporters (GLUTs), and the like.

[0088] The cells or microorganisms of the present disclosure may be cells or microorganisms that do not contain invertase, or cells or microorganisms that contain invertase. Preferably, it may be advantageous for the cells or microorganisms of the present disclosure not to contain invertase. Without wishing to be bound by theory, it may be advantageous that they are salt-tolerant, can be cultivated in seawater, and grow quickly. Since energy consumption in glucose production via the invertase pathway can lead to reduced cell or microbial growth and production, the absence of invertase may be an advantage in itself, or it may increase the possibility of utilizing fructose to convert to glucose. When deleting invertase, it is preferable to introduce G1P and a glucose efflux protein in addition to deleting glucokinase. This is because efficient exocrine secretion of glucose can be achieved from the cell or microorganism.

[0089] In one embodiment, the cells or microorganisms used in the present disclosure include cyanobacteria.

[0090] Various types of cyanobacteria can be used. Cyanobacteria are typically unicellular or microbial prokaryotes that perform oxygenic photosynthesis and can be classified into the Chroococcales, Nostocales, Oscillatoryes, Pleurocapsales, Stigonemales, etc. The Chroococcales include the genera Aphanocapsa, Aphanotheche, Chamaesiphon, Chroococcus, Crocosphera, Cyanobacteria, Cyanobium, Cyanotheche, Dactylococcopsis, Gloeobacter, Gloeocapsa, Gloeotheche, Euhalotheche, Halotheche, Johannesbaptistia, Melismopedia, Microcystis, Rhabdoderma, Synechococcus, Synechocystis, and Thermosynechococcus. The Nostocales order includes the genera Coreodesmium, Fremiella, Microchaete, Rexia, Spirirestis, Tryposthrix, Anabaena, Anabaenopsis, Aphanizomenon, Aurocira, Cyanospira, Cylindrospermopsis, Cylindrospermum, Nodularia, Nostoc, Rickeria, Kalothrix, Gloeotrichia, and Scytonema. The Oscillatory order includes the genera Arthrospira, Gaitrelinema, Halomicronema, Halospirulina, Catagnimene, Leptorynbya, Limnothrix, Lymbya, Microcoleus, Oscillatory order, Phormidium, Planctotrichoides, Planktothrix, Plectonema, Limnothrix, Pseudoanavena, Schizothrix, Spirulina, Symploca, Trichodesmium, and Chiconema. The Pleurocapsales includes the genera Chroococchidiopsis, Dermocarpa, Dermocarpella, Myxosarcina, Pleurocapsa, Stanieria, and Xenococcus. The Stigonemales include the genera Capsocilla, Chlorogloeopsis, Fischerella, Hapalosiphon, Mastigocladopsis, Mastigocladus, Nostochopsis, Stigonema, Symphionema, Symphionemopsis, Umezakia, and Westielopsis.

[0091] The cells or microorganisms of the present disclosure can be realized by introducing a construct such as that shown in FIG. 6 into a cell or microorganism, as a non-limiting example of a configuration example, and a conceptual non-limiting example of the production method is illustrated in FIG. 7. For example, the cells or microorganisms produce sucrose from fructose-6-phosphate and glucose-6-phosphate produced in the Calvin (CBB) cycle, and are relatively superior to microorganisms that produce glucose and fructose by decomposing sucrose with invertase. While not wishing to be bound by theory, contemplated advantages include, but are not limited to, advantageous conversion due to substantially complete conversion of glucose, as opposed to only half of the carbon sources of fructose-6-phosphate and glucose-6-phosphate produced in the Calvin (CBB) cycle being converted to glucose, as well as fewer steps leading to glucose production, less energy consumption, and / or better energy efficiency for glucose production.

[0092] In one embodiment, the presence or absence of a deletion of glucokinase, such as the glk gene, may be an important characteristic of some of the microorganisms disclosed herein. While not wishing to be bound by theory, it is generally believed that the deletion of glk is a major factor in increasing glucose production in many strains (e.g., Non-Patent Document 1), but the present disclosure unexpectedly enabled an increase in glucose production without deleting glk. For example, and not intended to be limiting, it has been found that in certain advantageous embodiments of the present disclosure, the combination of glucose-1-phosphatase and / or GLUT1 expression can more advantageously increase glucose production without deleting glk (e.g., production on the order of g / L can be achieved, making it possible to apply the production to industrial applications).

[0093] In another aspect, without wishing to be bound by theory, some embodiments of the present disclosure enable glucose to be secreted from the cell or outside the microorganism more efficiently than a microorganism in which a glycogen synthase gene (e.g., glgC, which is ADP-glucose pyrophosphorylase) is deleted, without deleting the glycogen synthase gene, and this is an effect that could not be predicted from the prior art.

[0094] In one embodiment, it may be advantageous for the microorganism of the present disclosure to use the AGP gene derived from Escherichia coli as G1P, because, without wishing to be bound by theory, it is believed that glucose can be more efficiently secreted outside the cell or the microorganism.

[0095] In one embodiment, while deficiency of enzymes such as glgA1 and glgA2 can result in poor growth, the present disclosure demonstrates that additional deletions (e.g., glucokinase (glk gene)) do not exacerbate the poor growth and instead increase glucose production.

[0096] (Circular Economy)

[0097] (Combination with other glucose-requiring cells or microorganisms (animal, fungus (yeast), bacterium (Escherichia coli, etc.)) In one aspect, the present disclosure provides a combination of a glucose-requiring cell or microorganism with a cell or microorganism having the ability to produce and export glucose. Any cells or microorganisms can be used as the glucose-requiring cell or microorganism and the cell or microorganism having the ability to produce and export glucose, as long as they have the ability, and such cells may be the same cell or microorganism, or different cells or microorganisms. Preferably, they are different cells or microorganisms. Although not intended to be limiting, in a certain embodiment, when these cells or microorganisms are combined in the same medium, it is advantageous that the medium conditions (components and pH) are suitable for both bacterial cells. When only the culture supernatant of a cell or microorganism having the ability to produce and export glucose is collected and used, the medium can be modified to be suitable for glucose-requiring cells by adding other additives.

[0098] In one aspect, the present disclosure provides a system comprising a glucose-requiring cell or microorganism and a cell or microorganism having the ability to produce and export glucose, wherein the glucose-requiring cell or microorganism and the cell or microorganism having the ability to produce and export glucose can be configured in such a manner that the glucose produced by the cell or microorganism having the ability to produce and export glucose can be utilized by the glucose-requiring cell or microorganism.

[0099] In one aspect, the present disclosure provides a kit comprising a glucose-requiring cell or microorganism and a cell or microorganism having the ability to produce and export glucose. In this kit, the glucose-requiring cell or microorganism and the cell or microorganism having the ability to produce and export glucose are configured in such a manner that glucose produced by the cell or microorganism having the ability to produce and export glucose can be utilized by the glucose-requiring cell or microorganism, thereby constituting a system, and instructions and the like can be included as necessary.

[0100] The instructions may include points to note when providing the cells. For example, and not intended to be limiting, when combining the cells in the same medium, the medium conditions (components and pH) must be suitable for both bacterial cells. On the other hand, when using only the culture supernatant of cells capable of producing and secreting glucose, the medium may be modified to a medium suitable for glucose-requiring cells by adding other additives. Therefore, when using a kit to configure the system of the present disclosure, an appropriate embodiment can be adopted, such as configuring the system in the same medium or configuring the system using the supernatant.

[0101] In one embodiment, the glucose-requiring cells or microorganisms include animal cells or microorganisms. In the case of animal cells or microorganisms, the culture medium must maintain an optimal pH. In a specific embodiment, examples of animal cells or microorganisms include, but are not limited to, Chinese hamster ovary (CHO) cells or microorganisms, rat liver-derived cells or microorganisms (RL-34), mouse myoblasts or microorganisms (C2C12), avian myoblasts or microorganisms (QM-7), chicken fibroblasts or microorganisms (DF-1), and insect cells or microorganisms.

[0102] In another embodiment, the animal cell or microorganism is a cell or microorganism used to produce cultured meat. In this embodiment, the disclosure provides a method of producing cultured meat using the methods of the disclosure.

[0103] In another embodiment, the present disclosure provides a combination, a system and / or a kit comprising the combination, or a method, wherein the cell or microorganism capable of producing and exporting glucose used in the present disclosure comprises a microorganism of the present disclosure (which may be any cell or microorganism described herein).

[0104] In one embodiment, the glucose-requiring cells or microorganisms of the present disclosure may include eukaryotes such as yeast, bacteria such as E. coli, and the like.

[0105] In one embodiment, the yeast used in the present disclosure is a cell or microorganism used to produce fermented foods, including but not limited to sake alcohol, miso, soy sauce, etc.

[0106] In one embodiment, the E. coli used in the present disclosure is a cell or microorganism used to produce fermented foods or useful substances (e.g., muconic acid).

[0107] In one embodiment, the cells or microorganisms capable of producing and emitting glucose used in the present disclosure may be a combination, a system and / or kit including the microorganism, or a method including the microorganism of the present disclosure. For example, laboratory-grown meat can be produced from animal cells or microorganisms cultured in a medium derived from grains or algae, and this can be achieved sustainably. Furthermore, an advantage of using an algae-derived medium is that it contains vitamins and algae secrete small amounts of amino acids into the medium, making it suitable for culturing glucose-requiring cells that require vitamins and amino acids.

[0108] (Upcycling of Spent Culture Medium) In one aspect, the glucose-producing cyanobacteria of the present disclosure can also be applied to upcycling of spent culture medium.

[0109] In one embodiment, the present disclosure provides a method for producing a medium to which glucose is replenished, comprising adding cells or microorganisms capable of producing and secreting glucose to a medium in which glucose has been consumed (such as, for example, a medium after animal culture, as a non-limiting example). Specific examples include, but are not limited to, the following: The medium in which glucose has been consumed is recovered by centrifugation, membrane exchange, or the like, and cells or microorganisms capable of producing and secreting glucose are added thereto and cultured for a certain period of time, thereby secreting glucose into the culture medium. The medium in which glucose has been secreted is then recovered, and glucose-requiring cells or microorganisms are added thereto, allowing the added cells or microorganisms to grow using glucose as a carbon source.

[0110] (Comprehensive Circular Methods) In one aspect, the present disclosure provides sustainable and circular methods of material production that enable a circular economy.

[0111] In relation to the present disclosure, the applicant provides a means to solve the problem that conventional culture media used in the culture of animal cells or microorganisms must be discarded because harmful substances (mainly ammonia and lactic acid) accumulate before the amino acids and inorganic substances are completely consumed. For example, ammonia can be removed by culturing microalgae in the used culture media (also referred to as "waste culture media" for convenience in this specification, but this does not mean that the used culture media is actually discarded). Furthermore, lactic acid can also be removed by introducing an L-lactate dehydrogenase gene into microalgae (WO 2022 / 250165 and WO 2023 / 106300). These findings can be combined with the present disclosure.

[0112] The discarded medium after culturing animal cells that can be applied to the present disclosure is a medium that can be normally used for cell culture, and it is possible to use the medium after culturing animal cells. The medium for animal cells before culturing that can be applied to the present invention is not limited as long as it is a known medium, but may be, for example, Eagle's medium, Dulbecco's modified Eagle's medium (DMEM), DMEM:F12 medium, Glasgow's minimum essential medium, Grace's insect medium, Ham's medium, Iscove's modified Eagle's medium, RPMI-1640 medium, L-15 medium, McCoy's 5A medium, M199 medium, etc., and any medium that can culture animal cells can be used.

[0113] As used herein, the term "animal cells" is used to include cells of vertebrates (mammals (also referred to as "mammals"), birds, reptiles, amphibians, or fish) or cells of invertebrates (e.g., sea squirts, arthropods (crustaceans (e.g., shrimp, crabs, etc.), insects, etc.), echinoderms (sea urchins, sea cucumbers, starfish, etc.), mollusks (e.g., shellfish, squid, octopus, etc.)). In one embodiment, the animal cell is derived from a vertebrate (e.g., a mammal such as a human, monkey, cow, whale, bear, deer, horse, pig, wild boar, sheep, rabbit, rat, mouse, hamster, goat, dog, or cat; a bird such as a chicken, duck, goose, quail, goat, or pheasant; a reptile such as a crocodile, lizard, snake, turtle, or soft-shelled turtle; an amphibian such as a frog, salamander, or newt; a tuna, salmon, trout, carp, shark, eel, pufferfish, or zebrafish). The cells may be cells of animals such as sea squirts, arthropods (crustaceans (e.g., shrimp, crabs, etc.), insects, etc.), echinoderms (sea urchins, sea cucumbers, starfish, etc.), mollusks (e.g., shellfish (including bivalves), squid, octopus, etc.)), or primary cells, established cell lines, pluripotent stem cells (e.g., ES cells, ntES cells, Muse cells, iPS cells), or tissue stem cells (e.g., mesenchymal stem cells), or cells induced to differentiate from any of these. Furthermore, the origin of animal cells in biological tissues may be, for example, muscle system (e.g., myoblasts), skin system (e.g., fibroblasts, keratinocytes), liver system (e.g., hepatic parenchymal cells), kidney system (e.g., kidney cells (e.g., HEK293)), cardiac system (e.g., cardiac muscle cells), digestive system (e.g., oral mucosal cells, intestinal epithelial cells, parietal cells), hematopoietic system (e.g., hematopoietic stem cells), reproductive tissue system (e.g., ovarian cells (e.g., CHO cells), sperm cells, uterine epithelial cells), mucosal system (e.g., epithelial cells), bone tissue system (e.g., osteoblasts, chondrocytes), or tissues other than these.

[0114] The culture method for animal cells that provides a medium applicable to the present disclosure can be a known culture method (e.g., 37°C, saturated water vapor, 5% CO ２ The method is not particularly limited, and may be in accordance with the following conditions:

[0115] In one embodiment, the composition containing the medium used for culturing algae after culturing animal cells may be adjusted to an osmotic pressure equivalent to or within a range of ±10% (preferably ±5%, more preferably ±3%) of the osmotic pressure of a standard medium typically used for culturing the target algae, such as a medium for freshwater algae (e.g., AF6 medium, C medium, URO medium, VT medium, etc.) or a medium for marine algae (e.g., ESM medium, f / 2 medium, IMR medium, MNK medium, C medium + 10% seawater, a mixture of Daigo IMK and Daigo Artificial Seawater SP, etc.). The osmotic pressure may be adjusted by measuring the osmotic pressure using a known method before culturing the algae, and adjusting the osmotic pressure as needed, for example, if the osmotic pressure is outside the range of ±10% (preferably ±5%, more preferably ±3%) of the osmotic pressure of the standard medium typically used for culturing the target algae. The osmotic pressure may be monitored during algal cultivation and adjusted as necessary, for example, if it is outside the range of ±10% (preferably ±5%, more preferably ±3%) of the osmotic pressure of a standard medium typically used for culturing the target algae. This can further enhance the algal growth effect. The osmotic pressure may be adjusted according to known methods. For example, if the osmotic pressure is high, the medium may be diluted with a hypotonic medium such as water (e.g., distilled water, ion-exchanged water, sterilized water). Alternatively, if the osmotic pressure is low, the medium may be adjusted by adding sodium salts (e.g., sodium chloride, sodium hydroxide, sodium carbonate, etc.), potassium salts (e.g., potassium chloride, potassium carbonate, potassium bicarbonate, potassium phosphate, etc.), magnesium salts (e.g., magnesium chloride, magnesium oxide, etc.), and / or calcium salts (e.g., calcium chloride, calcium carbonate, calcium hydroxide, etc.). For example, when the algae to be cultured is Chlorococcum littorale, the osmotic pressure of the resulting composition may be adjusted by adding sodium chloride, magnesium chloride, and potassium chloride to the waste animal cell culture medium.

[0116] In one embodiment, the composition provided by the present invention is a composition for culturing microalgae, for example, freshwater or marine, or stenohaline or euryhaline microalgae. In one aspect, the microalgae to which the present invention can be applied are preferably marine and / or euryhaline microalgae, such as Chlorococcum littorale and Synechococcus sp.

[0117] The conditions for culturing algae depend on the type of algae being cultured. The culture is carried out at a temperature of 5 to 40°C, preferably 10 to 35°C, and more preferably 10 to 30°C, for a period of usually 1 to 10 days, preferably 3 to 7 days, and may be aerated or anaerobic agitation culture, shaking culture, or static culture.

[0118] In one embodiment, in a specific embodiment of the present disclosure, when algae capable of nitrogen fixation are used, the addition of a nitrogen source is not necessary. However, in another embodiment, when algae lack or have insufficient nitrogen fixation ability, the addition of an additional nitrogen source may be necessary for algal growth. Urea, ammonia, and the like can be used as nitrogen sources, but are not limited thereto. The ammonia concentration of the composition used for algae cultivation is 0.5 mM or higher, which enables efficient cultivation of algae using ammonia as a nitrogen source. The upper limit of the ammonia concentration of the composition used for algae cultivation is not particularly limited, but since the composition contains a medium used after animal cell culture, it can be provided at a concentration that allows animal cells to be cultured. Therefore, although not limited thereto, the ammonia concentration of the composition used for algae cultivation can be provided at, for example, 0.5 mM to 10 mM, 0.5 mM to 5 mM, or 0.5 mM to 3 mM. Because ammonia can serve as a nitrogen source in algae cultivation, if ammonia is consumed by the cultivation, further ammonia may be added as a nitrogen source, or alternatively or additionally, nitrate may be added.

[0119] In one embodiment, the processes of the present disclosure may be carried out in animal cells or algae by carrying out some or all of the steps described in, for example, WO 2022 / 250165, the disclosure of which is incorporated herein by reference in its entirety.

[0120] In one embodiment, the process of the present disclosure may be carried out under pressure. As used herein, "under pressure" refers to atmospheric pressure, i.e., pressure conditions higher than 1 atmosphere, and may be carried out at, for example, 1.1 atmospheres or higher, 1.5 atmospheres or higher, 1.8 atmospheres or higher, or 2 atmospheres or higher. For example, the pressure may be 1.1 to 300 atmospheres, 1.5 to 200 atmospheres, 1.8 to 100 atmospheres, 2 to 50 atmospheres, for example, 2 to 20 atmospheres. The pressurized condition may be achieved by any device or method, and for example, the pressurized condition can be achieved by using an autoclave.

[0121] In some aspects of the present disclosure, it may be advantageous for organisms such as microalgae and cyanobacteria to utilize L-lactic acid, a major component excreted by cultured animal cells. Many microalgae and cyanobacteria may lack genes encoding L-lactic acid conversion enzymes. The following is a list of microalgae and cyanobacteria that have been confirmed to have NAD-independent L-lactate dehydrogenase (L-iLDH) and lactate 2-monooxygenase (LMO). In the table, species marked with a black circle are species that have L-iLDH or LMO, and can be used in the examples of the present disclosure, as an example.

[0122]

[0123]

[0124]

[0125] In one embodiment of the present disclosure, an L-lactate dehydrogenase gene and / or a lactate permease gene can be introduced into an organism such as cyanobacteria to confer L-lactate assimilation or lactate removal ability. Representative organisms to which such lactate removal ability can be imparted include cyanobacteria, microalgae, yeast, Escherichia coli, Bacillus subtilis, Corynebacterium, actinomycetes, filamentous fungi, lactic acid bacteria, or cultured cells of animals or plants. In one embodiment of the present disclosure, the modified organisms or cultured cells can be those that can be used in the resource-recycling food production system described below. In this case, the modified cells or microorganisms can be advantageous because they can supply glucose as a nutrient source to animal cells.

[0126] There are no particular limitations as long as the L-lactic acid removal ability is imparted or enhanced, and this includes gene modification, introduction, and / or deletion. For example, in one embodiment, a gene that imparts or enhances L-lactic acid assimilation or membrane permeability to the organisms or cultured cells described above can be introduced. In another embodiment, a gene responsible for synthesizing L-lactic acid or D-lactic acid can be attenuated or deleted.

[0127] Examples of genes that perform L-lactic acid assimilation include genes encoding NAD-independent L-lactate dehydrogenase (EC number 1.1.2.3) such as lldD from Escherichia coli, genes encoding lactate-2-monooxygenase (EC number 1.13.12.4) from Mycolicibacterium smegmatis, and genes encoding lactate oxidase (EC number 1.1.3.2) from Aerococcus viridans. In addition to lldD in E. coli, NAD-independent L-lactate dehydrogenase (EC number 1.1.2.3) has been reported to contribute to L-lactate utilization (e.g., Corynebacterium glutamicum (lldD gene) (Appl Environ Microbiol. 2005 Oct; 71(10): 5920-5928), Pseudomonas aeruginosa (lldA and lldD genes) (Environ Microbiol Rep. 2018 Oct; 10(5): 569-575), and Pseudomonas stutzeri (lldABC genes) (J Bacteriol. 2015 Jul;197(13):2239-2247.), Clostridium acetobutylicum (Archives of Microbiology volume 164, pages 36-42 (1995)), etc. In the present disclosure, L-lactic acid assimilation ability was imparted to cyanobacteria by introducing E. coli lldD, so L-lactic acid assimilation ability can be similarly imparted using enzymes of the same type, not just those derived from E. coli. Furthermore, many literature reports that various foreign genes, including those derived from E. coli, function in cyanobacteria.For example, as an example of successful production of compounds that are not naturally produced by cyanobacteria by introducing foreign genes, ACS Synth Biol. 2019 Dec 20; 8(12): 2701-2709 reported that astaxanthin was successfully produced by introducing the β-carotene ketolase gene and β-carotene hydroxylase gene derived from Brevundimonas sp. into the cyanobacterium Synechococcus sp. PCC 7002, and Front Bioeng Biotechnol. 2014 Jun 19; 2: 21 reported that astaxanthin was successfully produced by introducing the β-carotene ketolase gene and β-carotene hydroxylase gene derived from Brevundimonas sp. into the cyanobacterium Synechococcus sp. By introducing the L-limonene synthase gene derived from Mentha spicata and the (E)-α-bisabolene synthase gene derived from Abies grandis into PCC 7002, limonene and bisabolene were successfully produced, respectively. In Front Bioeng Biotechnol. 2015 Apr 24;3:48, it was reported that the cyanobacterium Synechococcus sp. In PCC 7002, the production of lauric acid was successfully achieved by introducing the lauroyl-acyl carrier protein thioesterase gene derived from Umbellularia californicay, and in Biosci Biotechnol Biochem. 2013; 77(5):966-70, the production of L-lactic acid was successfully achieved by introducing L-lactate dehydrogenase derived from Lactococcus lactis, Lactobacillus plantarum, and Lactobacillus rhamnosus into the cyanobacterium Synechocystis sp. PCC 6803.In Metab Eng. 2020 Jan; 57: 129-139, the production of aromatic amino acids was enhanced in the cyanobacterium Synechocystis sp. PCC 6803 by introducing the aroGfbr and tyrAfbr genes derived from Escherichia coli, and further by introducing the tyrosine / phenylalanine ammonia lyase gene derived from Trichosporon cutaneum and the 4-hydroxyphenlacetate 3-hydroxylase complex gene derived from Escherichia coli, the production of aromatic compounds that PCC 6803 does not originally produce was successfully achieved. Also, Microb Cell Fact. 2017 Feb 23;16(1):34, they successfully produced ethylene by introducing an ethylene-forming enzyme gene derived from Pseudomonas syringae into the cyanobacterium Synechocystis sp. PCC 6803. Therefore, in the present disclosure, it is believed that foreign genes other than those from E. coli can also be used to impart L-lactic acid assimilation ability to cyanobacteria.

[0128] Examples of genes that confer or enhance membrane permeability for L-lactic acid include genes encoding lactate permeases such as lldP ​​of E. coli, and genes encoding monocarboxylic acid transporters.

[0129] Examples of genes involved in the synthesis of L-lactic acid or D-lactic acid include genes encoding NAD-dependent L-lactate dehydrogenase (EC 1.1.1.27), genes encoding lactaldehyde dehydrogenase (EC 1.2.1.22), genes encoding malolactic enzymes (EC 4.1.1.101), and genes encoding D-lactate dehydrogenase (EC 1.1.1.28) such as ldhA from cyanobacteria.

[0130] In one embodiment of the present disclosure, the modified microorganism or cell can synthesize useful amino acids using components excreted by cultured animal cells, and therefore can also serve as a source of amino acids. Thus, in one embodiment of the present disclosure, the microorganism or cell of the present disclosure has enzymes and / or amino acid synthesis systems for, for example, amino acids such as glutamine, glutamic acid, alanine, valine, and leucine, and / or enzymes and / or enzymes for D-lactic acid, 3-hydroxypropionic acid, acrylic acid, formic acid, acetic acid, ethanol, 2,3-butanediol, butanol, acetone, isobutyric acid, isobutanol, isobutylene, butyric acid, 1-butanol, propanediol, and the like. The enzymes and / or synthesis systems for the synthesis of 1,4-butanediol, caproic acid, pentane, citric acid, succinic acid, fumaric acid, malic acid, itaconic acid, γ-aminobutyric acid, γ-butyrolactam, ornithine, putrescine, 6-aminocaproic acid, ε-caprolactam, ethylene, γ-butyrolactone, 1,4-butanediol, levulinic acid, adipic acid, cadaverine, 5-aminovaleric acid, δ-valerolactam, 1-propanol, or butane may be present. Examples of these enzymes and synthesis systems are described in Nature Catalysis 2, 18-33 (2019), for example.

[0131] In one aspect of the present disclosure, there is provided a method for culturing at least two types of cells, cell X and cell Y, in a circulatory manner, comprising the steps of: (a) providing cell Y with a component excreted by cell X; (b) providing cell X with a component derived from cell Y; (c) culturing cell X and cell Y; and (d) repeating steps (a) to (c) as necessary, wherein at least one of the components excreted by cell X is a component assimilable by cell Y, and at least one of the components derived from cell Y is a nutrient for cell X. In one embodiment of the present disclosure, this method amplifies only cells of edible tissues of animals and plants, thereby simultaneously reducing the supply of nutrients and the problem of culture waste, thereby providing a waste-free, environmentally friendly, and healthy circulatory culture food production system. Here, in this specification, the excreted component may be glucose, or may be a specific cell or microorganism of the present disclosure.

[0132] In one embodiment, a method for producing a desired product can be provided by using at least one of cells X and Y in the above-described method, which produces the desired product. In one embodiment of the present disclosure, cell X can be an animal cell, and for example, a cell that can be used for meat production through culture is preferred. In another embodiment, components derived from cell Y can also be preferably used directly as a nutrient source for cell X.

[0133] In another embodiment, the components excreted by cell X are not particularly limited as long as they can be assimilated by cell Y, and can include, for example, L-lactic acid, carbon dioxide, ammonia, urea, or phosphate. In another embodiment, the components derived from cell Y are not particularly limited as long as they can serve as nutrients for cell X, and can include, for example, glucose, glutamic acid, glutamine, alanine, arginine, asparagine, aspartic acid, cysteine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, pyruvic acid, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, folic acid, choline, starch, glycogen, and cellulose. In one embodiment of the present disclosure, the components excreted by cell X and the components derived from cell Y do not need to be essential nutrients for the other cell, as long as they can be assimilated. For example, L-lactic acid, which is excreted by animal cells, is not an essential nutrient for cyanobacteria, but can be assimilated by modified cyanobacteria.Similarly, ammonia, which is excreted by animal cells, is a compound that can be assimilated by cyanobacteria and is essential for cyanobacteria when there are no other nitrogen source compounds or when cyanobacteria with nitrogen fixing ability are not used.

[0134] In one embodiment, cells X and Y can be cultured and components excreted by or derived from one cell can be used as a nutrient source for the other cell, thereby forming a circulatory culture method. In this case, cells X and Y can be cultured co-cultured or ectopic. As used herein, "ectopic" culture means culturing in a spatially or physically separated location.

[0135] In another aspect of the present disclosure, there is provided a method for culturing animal cells by recycling components discharged from animal cell culture using an organism, the method comprising: (a) a step of causing an organism to consume the discharged components, the organism having the ability to assimilate the components; (b) a step of recovering a nutrient source for the animal cells from a culture medium of the organism; (c) a step of culturing the animal cells using the nutrient source; and (d) a step of repeating steps (a) to (c) as necessary.

[0136] In one embodiment of the present disclosure, a highly efficient, low-environmental-impact recycling cell culture system can be provided that cultivates algae capable of synthesizing organic compounds from inorganic substances through photosynthesis, and recycles carbon, nitrogen, and other materials. A three-dimensional organization system that produces only edible tissues by amplifying animal cells can also be provided. In such a recycling system, cell culture engineering, catalytic chemistry, and genetic engineering can be used to amplify animal cells using algae-derived nutrients and recycle culture wastewater. Furthermore, in one embodiment of the present disclosure, the disclosed method can reduce waste from conventional cultured food production techniques. Tissue engineering in the field of regenerative medicine can also be applied to the three-dimensional organization system from cells, and integrating the two systems can realize a bioeconomical cultured food production system that uses cells as food.

[0137] In one embodiment of the present disclosure, the nutrient source for animal cells can be a component excreted by an organism after assimilation by the organism itself, or a component constituting the organism itself, which can be used directly or indirectly after catalytic treatment. For example, in addition to the glucose provided herein, other nutrient sources for animal cells include glutamic acid, glutamine, alanine, arginine, asparagine, aspartic acid, cysteine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, pyruvic acid, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, folic acid, and choline. In another embodiment, carbohydrates (starch and glycogen) accumulated within the cells of microalgae and cyanobacteria, or cellulose in the cell walls, can be hydrolyzed, and the resulting glucose can be used as a nutrient source for animal cells.

[0138] In one embodiment of the present disclosure, the components discharged from the animal cell culture may be any components that can be assimilated and consumed by the organism and used to produce nutrients to be supplied to the animal cells, such as L-lactic acid, carbon dioxide, ammonia, urea, phosphate, etc. In one embodiment, the components discharged from the animal cell culture may be toxic to the animal cells, such as L-lactic acid, ammonia, urea, etc.

[0139] In one embodiment of the present disclosure, a system can be provided that enables the cultivation of algae using light energy as a power source, the supply of nutrients to animal and plant cells through algae decomposition, the cultivation of animal and plant cells that can be used as food, and the recycling of culture wastewater.Furthermore, by providing a three-dimensional organization system for forming amplified cells into three-dimensional tissues suitable for consumption, a recycling-based cultured food production system using cells as food can be provided.

[0140] In one embodiment, the method of the present disclosure requires efficient recycling of the substances contained in the culture wastewater, since the large amount of culture wastewater generated during cell culture places a burden on the environment. Therefore, in one embodiment, the method of the present disclosure can recycle the culture wastewater by comprehensively analyzing the components of the culture wastewater and selecting algae that can utilize these substances, in addition to utilizing glucose. The culture wastewater can also be recycled by using useful components, either directly or separated and selected by various techniques, as a nutrient source for the algae. In one embodiment, some of the wastewater-derived components can also be utilized in cell culture of animals and plants.

[0141] In one embodiment, the method of the present disclosure requires that the above processes be linked to operate stably and efficiently. Therefore, in one embodiment, the method of the present disclosure can provide a stable and efficient system that selects important parameters in each culture process and incorporates monitoring and control techniques for those parameters.

[0142] In one embodiment, the method of the present disclosure is required to achieve an organization from single cells that is equivalent to current foods in terms of nutritional value and texture. Therefore, in one embodiment, the method of the present disclosure can provide a production system for constructing three-dimensional tissues from cells by maximizing the self-organizing ability of animal cells and utilizing tissue engineering techniques developed in regenerative medicine research, thereby producing, for example, cultured sashimi or cultured chicken fillet. In one embodiment, since plant cells inherently have the ability to self-organize from single cells (callus cells), establishing control techniques for callus culture using nutrients derived from algae or animal cells can also provide a production system for producing cultured rice, cultured root vegetables, and cultured fruit pulp.

[0143] In one embodiment, the disclosed method is required to produce healthy and delicious food stably, safely, and without strain or waste. Therefore, in one embodiment, the disclosed method can provide a bioeconomical cultivated food production system by aseptically integrating a series of systems, monitoring and controlling the production process, and optimizing quality and efficiency.

[0144] In one embodiment, the method of the present disclosure can use organisms or cultured cells that produce, in addition to improving glucose utilization, glutamic acid, glutamine, alanine, arginine, asparagine, aspartic acid, cysteine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, pyruvate, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, folic acid, choline, starch, glycogen, or cellulose. Such organisms or cultured cells may contain these compounds or enzymes for their synthesis or metabolism, or may be modified to be able to synthesize or metabolize them. Pathways related to amino acid synthesis and metabolism, and the enzymes that catalyze them, are described, for example, in 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. In one embodiment, the organism or cultured cell may be modified so as to be capable of synthesizing or metabolizing these.

[0145] In one embodiment, the cells or microorganisms having modified glucose-producing ability of the present disclosure can be combined with a recycle culture of animal cells or microorganisms using spent medium from which ammonia and lactic acid have been removed. Because this technology removes harmful substances, combining the glucose-producing organisms of the present disclosure not only removes harmful substances from the spent medium, but also enables the efficient addition of nutrients that become depleted by the recycle culture (see WO2022 / 250165 and WO2023 / 106300 and the Examples herein).

[0146] In another aspect, the present disclosure provides a combination, a system, and / or a kit comprising cells or microorganisms capable of producing and exporting glucose and cells or microorganisms capable of removing ammonia and / or cells or microorganisms capable of removing lactate. With regard to ammonia removal, in addition to the description herein, reference may be made to WO2022 / 250165 and WO2023 / 106300, etc.

[0147] In another aspect, the present disclosure provides a method for producing a substance X, comprising: 1) culturing a first cell or microorganism capable of producing substance X; 2) culturing a second cell or microorganism capable of producing and exporting glucose using the culture medium obtained after the culture of 1); 3) if necessary, culturing a third cell or microorganism capable of removing ammonia and / or a fourth cell or microorganism capable of removing lactate in the culture medium obtained after the culture of 2); 4) culturing the first cell or microorganism in the culture medium obtained after the culture of 2) or 3); and 5) if necessary, repeating steps 2) and / or 3).

[0148] Substance X can include amino acids, aromatic compounds, pigments, proteins, cultured meat, terpenoids, oils and fats, fuels (including but not limited to ethanol, gas, petroleum, etc.), pharmaceuticals, antibiotics, polymers, etc.

[0149] The present disclosure provides a culture method comprising the steps of: 1) culturing a first cell or microorganism; 2) culturing a second cell or microorganism capable of producing and excluding glucose using the culture medium obtained from the culture of step 1); 3) optionally culturing a third cell or microorganism capable of removing ammonia and / or a fourth cell or microorganism capable of removing lactate in the culture medium obtained from step 2); 4) culturing the first cell or microorganism in the culture medium obtained from step 2) or 3); and 5) optionally repeating steps 2) and / or 3). Regarding ammonia removal, in addition to the description herein, reference may be made to WO 2022 / 250165 and WO 2023 / 106300. Examples of cells include, but are not limited to, animal cells. Furthermore, in a preferred embodiment, step 3) may be culturing the first cell or microorganism in the culture medium obtained from step 2) together with a cell or microorganism capable of removing ammonia and, if necessary, with a cell or microorganism capable of removing lactate.

[0150] In one embodiment, the present disclosure provides a method for culturing a first cell or microorganism, comprising: (a) culturing a first cell or microorganism; (b) culturing a second cell or microorganism capable of producing and exporting glucose using the culture medium obtained after the culture of (a); (c) culturing the first cell or microorganism together with a third cell or microorganism capable of removing ammonia and, optionally, a fourth cell or microorganism capable of removing lactate in the culture medium obtained after the culture of (b); (d) culturing the first cell or microorganism in the culture medium obtained after the culture of (c); and (e) repeating steps (b) and / or (c) as necessary, wherein at least two, preferably all, of the second cell or microorganism, the third cell or microorganism, and the fourth cell or microorganism are the same cell or microorganism that combines at least two, preferably all, of the abilities of producing and exporting glucose, removing ammonia, and removing lactate. In a preferred embodiment, the second, third, and / or fourth cell or microorganism may be a cyanobacterium. In a further preferred embodiment, the cyanobacteria may be a bacterium of the genus Synechococcus. In another preferred embodiment, the second, third, and / or fourth cells or microorganisms may be genetically modified to exhibit at least two, and preferably all, of the abilities of glucose production and export, ammonia removal, and lactate removal. According to this configuration, culturing using one or more cells or microorganisms having the abilities of glucose production and export, ammonia removal, and lactate removal, separate from the first cells or microorganisms, reduces the energy costs allocated to the abilities of glucose production and export, ammonia removal, and lactate removal in the first cells or microorganisms, and improves the culture efficiency of the first cells or microorganisms as a whole culture system.

[0151] In a preferred embodiment, the first cell or microorganism may be an animal cell.

[0152] In one embodiment, the present disclosure provides a method for culturing animal cells, comprising: (a) culturing first cells or microorganisms in a first culture tank; (b) supplying the culture solution obtained after step (a) to a second culture tank containing second cells or microorganisms having the ability to remove ammonia and, if necessary, the ability to remove lactate, and contacting the second cells or microorganisms with the second cells or microorganisms; (c) supplying the culture solution obtained after step (b) to a third culture tank containing third cells or microorganisms having the ability to produce and export glucose, and contacting the third cells or microorganisms with the third cells or microorganisms; (d) refluxing the culture solution obtained after step (c) back into the first culture tank and continuing to culture the first cells or microorganisms; and (e) continuously or intermittently repeating steps (b) to (d), wherein the second cells or microorganisms and the third cells or microorganisms are different from each other. According to this configuration, the efficiency of culturing the first cells or microorganisms is improved by circulating the cultivation of the first cells or microorganisms, the removal of ammonia and / or lactic acid, and the production and discharge of glucose.

[0153] In a preferred embodiment, the first culture tank, the second culture tank, and the third culture tank may be continuously connected via a tube. This configuration allows steps (a) to (e) to be performed more simply and precisely, and improves the culture efficiency of the first cells or microorganisms in the entire system.

[0154] In a more preferred embodiment, the second cell or microorganism may be a cyanobacterium or Escherichia coli. Furthermore, the third cell or microorganism may be a cyanobacterium. According to this configuration, by employing Escherichia coli and cyanobacteria, which are versatile model organisms, it is easy to design culture conditions according to the culture situation, and it becomes easy to improve the efficiency of culturing the first cell or microorganism as an entire system. Furthermore, the second cell or microorganism and / or the third cell or microorganism may be microorganisms that have been genetically modified to exhibit their respective functions. By employing this configuration, it becomes easy to design culture conditions according to the culture situation, and it becomes easy to improve the efficiency of culturing the first cell or microorganism as an entire system.

[0155] In a preferred embodiment, the first cell or microorganism may be an animal cell.

[0156] In one embodiment, the present disclosure provides a method for culturing a first cell or microorganism, comprising: (a) culturing a first cell or microorganism and obtaining a culture medium after the culture; (b) culturing a second cell or microorganism having the ability to remove lactate using the culture medium obtained in (a) and obtaining a first treated supernatant from the culture; (c) culturing a third cell or microorganism having the ability to produce and export glucose using the culture medium obtained in (a) and obtaining a second treated supernatant from the culture; (d) mixing the first treated supernatant obtained in (b) and the second treated supernatant obtained in (c) to prepare a regeneration medium; and (e) culturing the first cell or microorganism in the regeneration medium prepared in (d), wherein the second cell or microorganism and the third cell or microorganism are different microorganisms. According to this configuration, the cultivation efficiency of the first cell or microorganism is improved by circulating the cultivation of the first cell or microorganism, ammonia removal and / or lactic acid removal, and glucose production and discharge via the culture medium supernatant.

[0157] In a preferred embodiment, in step (d), the first treated supernatant and the second treated supernatant may be mixed at a predetermined volume ratio. According to this configuration, the first treated supernatant and the second treated supernatant are mixed at a predetermined volume ratio to form a medium suitable for culturing the first cell or microorganism, thereby improving the culture efficiency of the first cell or microorganism. The predetermined volume ratio may be set to an appropriate value each time depending on the concentrations of lactic acid, glucose, cell or microbial products, and other components in the first treated supernatant and / or the second treated supernatant. In other words, the method may include a step of changing the mixing ratio of the second cell or microorganism and the third cell or microorganism depending on the amount of their respective products.

[0158] In a more preferred embodiment, the second cell or microorganism may be a cyanobacterium or Escherichia coli. Furthermore, the third cell or microorganism may be a cyanobacterium. According to this configuration, by employing Escherichia coli and cyanobacteria, which are versatile model organisms, it is easy to design culture conditions according to the culture situation, and it becomes easy to improve the efficiency of culturing the first cell or microorganism as an entire system. Furthermore, the second cell or microorganism and / or the third cell or microorganism may be microorganisms that have been genetically modified to exhibit their respective functions. By employing this configuration, it becomes easy to design culture conditions according to the culture situation, and it becomes easy to improve the efficiency of culturing the first cell or microorganism as an entire system.

[0159] In a preferred embodiment, the first cell or microorganism may be an animal cell.

[0160] The genetically modified cyanobacteria that secrete and produce D-glucose (hereinafter referred to as glucose-producing cyanobacteria) of the present disclosure can also be applied to bioeconomic food production systems.

[0161] Examples of applications of the present disclosure include food production technologies such as "cultured meat," which is produced by culturing animal cells or microorganisms to form tissues, and "precision fermentation," which produces target food components (proteins, lipids, functional ingredients, etc.) by fermenting microorganisms into which specific genes have been introduced. These technologies require the mass cultivation of animal cells or microorganisms, and further require the supply of glucose as an energy source to the culture medium for culturing these cells or microorganisms; the technology of the present disclosure can solve these problems.

[0162] The present disclosure provides a method for supplying glucose with minimal environmental impact. For example, glucose production using microalgae is 10 times more energy efficient than production using plants. Furthermore, the present disclosure offers advantages such as eliminating the need for an extraction process to add glucose produced by microalgae to a culture medium as a nutrient. Specifically, the glucose-producing cyanobacteria of the present disclosure can be cultured in a medium for animal cells or microorganisms, and can secrete glucose directly into the medium. Furthermore, by using the medium after culturing the glucose-producing cyanobacteria, it is possible to culture animal cells or microorganisms using the glucose secreted by the cyanobacteria as a nutrient (see Examples). Because the present disclosure also eliminates the need for an extraction process, animal cells or microorganisms can be cultured efficiently with even less environmental impact, achieving bioeconomical production of cultured meat. In other words, by applying the glucose-producing cyanobacteria of the present disclosure to microbial culture, as in the case of animal cell or microorganism culture, effective precision fermentation can be achieved.

[0163] Furthermore, applications of the glucose-producing cyanobacteria disclosed herein are not limited to simply secreting glucose into the culture medium to achieve efficient cultivation of animal cells or microorganisms used in cultured meat or microorganisms performing precision fermentation; the glucose-producing cyanobacteria disclosed herein can also be applied to upcycling used culture medium.

[0164] The technology disclosed herein can also be applied to upcycling spent culture media. This disclosure avoids the need to discard culture media after animal cell or microbial culture because harmful substances (mainly ammonia and lactic acid) accumulate before amino acids and inorganic substances are completely consumed. Specifically, ammonia can be removed by culturing microalgae in spent culture media, and lactic acid can also be removed by introducing an L-lactate dehydrogenase gene into the microalgae ( WO 2022 / 250165 and WO 2023 / 106300 ). It is possible to recycle animal cells or microorganisms using spent culture media from which ammonia and lactic acid have been removed. Combining this with the glucose-producing cyanobacteria disclosed herein not only removes harmful substances from spent culture media, but also efficiently adds nutrients that become depleted during the recycle culture.

[0165] In one embodiment, the production of cultured meat can include the following steps: harvesting muscle cells from livestock muscle tissue, expanding the muscle cells in large quantities in vitro, using tissue engineering techniques to create a three-dimensional tissue, and then maturing the resulting three-dimensional tissue through tissue culture. Methods for three-dimensional organization of amplified cells include using a cell scaffold material and seeding cells onto the scaffold (EMBO reports 2019; 20: e47395, NPJ Sci Food 2021; 5: 6, ACS Appl Mater Interfaces 2021; 13: 32193-32204), or using spheroid technology (Biofabrication 2021; 13. doi: 10.1088 / 1758-5090 / ac23e3) and cell sheet technology (Circ Res 2002; 90: e40-48) without using a scaffold material.

[0166] In one embodiment, a method for producing cultured meat by culturing animal cells by recycling components discharged from animal cell culture in an organism is provided, the method comprising: (a) a step of causing an organism to consume the discharged components, the organism having the ability to assimilate the components, (b) a step of recovering a nutrient source for the animal cells from the organism, (c) a step of culturing the animal cells using the nutrient source, (d) a step of repeating steps (a) to (c) as necessary, and (e) a step of three-dimensionally organizing the cultured animal cells into cultured meat. In one embodiment, the method for producing cultured meat can also be used by appropriately selecting from the embodiments described elsewhere in this specification.

[0167] In one embodiment, a method for evaluating a modified strain is provided, the method comprising the steps of providing a microorganism or cell as described elsewhere herein and confirming the presence of the modification in the microorganism or cell. In one embodiment, the method for evaluating a modified strain can also be selected from the embodiments described elsewhere herein, as appropriate.

[0168] (General Techniques) The molecular biological, biochemical and microbiological techniques used herein are well known and commonly used in the art, and are described, for example, in Sambrook J. 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 annual updates; Sninsky, J. J. et al. (1999). These are described in PCR Applications: Protocols for Functional Genomics, Academic Press, a special edition of Experimental Medicine, "Gene Introduction & Expression Analysis Experimental Methods," Yodosha, 1997, and other publications, the relevant portions of which (possibly in their entirety) are incorporated herein by reference.

[0169] Regarding DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, gene synthesis and fragment synthesis services such as GeneArt, GenScript, Integrated DNA Technologies (IDT) and the like can be used. Other examples include 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). These are described in "Nucleic Acids in Chemistry and Biology," Oxford University Press; and "Bioconjugate Techniques," Academic Press, Hermanson, G. T. (1996), the relevant portions of which are incorporated herein by reference.

[0170] In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." In this specification, when it is specified that "within a range" of "two values," the range includes the two values ​​themselves.

[0171] All references cited herein, including scientific literature, patents, patent applications, and the like, are incorporated by reference in their entirety to the same extent as if each were specifically set forth.

[0172] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the scope of the claims.

[0173] Examples are described below. (Example 1: Creation of cyanobacteria with modified glucose production ability) Glucose is produced by dephosphorylation of glucose-1-phosphate or glucose-6-phosphate, but in many microalgae and cyanobacteria, genes encoding glucose-1-phosphatase (EC 3.1.3.10) or glucose-6-phosphatase (EC 3.1.3.9) have not been identified, and other phosphatases carry out catalytic reactions using glucose-1-phosphate or glucose-6-phosphate as non-specific substrates.

[0174] We focused on the yihX and agp genes of Escherichia coli as genes encoding glucose-1-phosphatase. We also focused on the glf gene derived from Zymomonas mobilis, which encodes glucose-facilitated diffusion protein for secreting glucose outside the cell or microorganism. We also focused on glucoamylase (EC 3.2.1.3), which hydrolyzes glycogen, a carbon storage substance within the cell or microorganism, as a gene contributing to glucose production.

[0175] Plasmid construction: Plasmids were constructed to introduce the yihX / agp gene from Escherichia coli, the glf gene from Zymomonas mobilis, and the ga gene from Rhizopus delemar into the glk gene site of the cyanobacterium Picosynechococcus (Synechococcus) sp. PCC 7002. (KCP0155, KCP0156, KCP0160, KCP0166) A plasmid (KCP0150) was constructed to insert an ampicillin resistance gene into the glk gene site in the cyanobacterium Picosynechococcus sp. PCC 7002 to delete the glk gene. A kanamycin resistance gene was inserted into the glgC gene site of PCC 7002 to construct a plasmid (KCP0167) for deleting the glgC gene involved in glycogen synthesis.

[0176] The list of constructed plasmids is shown in Table 4. A schematic diagram is shown in Figure 6.

[0177]

[0178] KCP0155 was constructed by inserting the psbA2 promoter, a codon-optimized agp gene, a ribosome binding site, and a codon-optimized glf gene downstream of the ampicillin resistance cassette of KCP0150.

[0179] KCP0156 was constructed by inserting the psbA2 promoter, a codon-optimized yihX gene, a ribosome binding site, and a codon-optimized glf gene downstream of the ampicillin resistance cassette of KCP0150.

[0180] KCP0160 was constructed by inserting the psbA2 promoter and a codon-optimized ga gene downstream of the ampicillin resistance cassette of KCP0150.

[0181] KCP0166 was constructed by inserting the lacI gene, the cLac143 promoter, and a codon-optimized ga gene downstream of the ampicillin resistance cassette of KCP0150.

[0182] A kanamycin resistance gene was inserted into the glgC gene site of the cyanobacterium Picosynechococcus sp. PCC 7002 to construct a plasmid, KCP0167, for disrupting the glgC gene.

[0183] Information on Escherichia coli agp and yihX (glucose-1-phosphatase, EC 3.1.3.10), Zymomonas mobilis glf (glucose facilitated diffusion protein), and Rhizopus delemar ga (glucoamylase, EC 3.2.1.3) was obtained from NCBI.

[0184] The amino acid sequences used are as follows: E. coli AGP (NP_415522.1): SEQ ID NO: 1 E. coli YihX (NP_418321.4): SEQ ID NO: 2 Z. mobilis GLF (GenBank: AVZ41684.1): SEQ ID NO: 3 Rhizopus delemar glucoamylase (GenBank: EIE75378.1): SEQ ID NO: 4

[0185] Based on these amino acid sequences, a codon-optimized base sequence was artificially synthesized using the cyanobacterium Synechococcus elongatus as a host (commissioned to GenScript).

[0186] The nucleotide sequences of each are as follows: E. coli agp (codon optimized): SEQ ID NO: 5 E. coli yihX (codon optimized): SEQ ID NO: 6 Z. mobilis glf (codon optimized): SEQ ID NO: 7 Rhizopus delemar ga (codon optimized): SEQ ID NO: 8

[0187] Transformation of cyanobacteria: The constructed plasmid was used to transform the cyanobacteria Picosynechococcus sp. PCC 7002, and the agp / yihX / glf / ga genes were introduced into the glk gene site. Transformation of a strain (KC0151) in which the agp gene and glf gene were introduced into the glk gene site and a strain (KC0152) in which the yihX gene and glf gene were introduced into the glk gene site was carried out using KCP0167 to disrupt the glgC gene site. The L-lactic acid-assimilating cyanobacterium strain KC0092, prepared as described in WO 2023 / 106300, was transformed, and the agp / glf genes were introduced into the glk gene site.

[0188] The cyanobacteria Picosynechococcus sp. PCC 7002 and KC0092 were grown in MA2 medium at 50 μmol photons / m ２ / s (daylight fluorescent lamp), 30℃, 100 rpm ７５０ The culture was cultured until the chromatogram showed a value of about 1. 100 μL of the culture medium was transferred to a 1.5 mL microtube, and 1 μg of the constructed plasmid was added. The culture was then gently stirred at room temperature for 24 hours in the dark, and the entire cell or microbial suspension was then inoculated onto MA2 agar medium with a filter placed over it. 50 μmol photons / m ２ The filter was transferred to MA2 agar medium containing 4 μg / mL carbenicillin. ２ The colony cells or microorganisms were cultured under conditions of 4 μg / mL carbenicillin-containing MA2 medium, 50 μmol photons / m ２ The cells were cultured under a neutral white fluorescent lamp (Natural White Fluorescent Lamp) at 30°C and 100 rpm. PCR was performed using the genomic DNA of the cultured cells or microorganisms as a template, and it was confirmed that the agp / yihX / glf / ga genes had been introduced into the glk gene site.

[0189] The cyanobacterial strains KC0151 and KC0152 prepared by the above procedure were cultured in MA2 medium at 50 μmol photons / m ２ / s (daylight fluorescent lamp), 30℃, 100 rpm ７５０The culture was cultured until the chromatogram showed a value of about 1. 100 μL of the culture medium was transferred to a 1.5 mL microtube, and 1 μg of the constructed plasmid was added. The culture was then gently stirred at room temperature for 24 hours in the dark, and the entire cell or microbial suspension was then inoculated onto MA2 agar medium with a filter placed over it. 50 μmol photons / m ２ The filter was transferred to MA2 agar medium containing 100 μg / mL kanamycin. ２ The colony cells or microorganisms were cultured under conditions of 100 μg / mL kanamycin-containing MA2 medium, 50 μmol photons / m ２ The cells were cultured under a neutral white fluorescent lamp (daylight) at 30° C. and 100 rpm. PCR was performed using the genomic DNA of the cultured cells or microorganisms as a template, and it was confirmed that the glgC gene site had been deleted.

[0190] A list of cyanobacterial strains is shown in Table 5. A schematic diagram of some of them is shown in Figure 6.

[0191]

[0192] Pre-culture for evaluation of glucose production amount of glucose-producing gene-transfected strains KC151, KC152, KC161, or KC166 was cultured in a two-stage flask in MA2 medium containing 4 μg / mL carbenicillin, 100 μmol photons / m ２ / s (daylight fluorescent lamp), 2% CO ２ The mixture was cultured at 30°C and 100 rpm for 3 days.

[0193] KC169 or KC170 was cultured in a two-tier flask in MA2 medium containing 4 μg / mL carbenicillin and 100 μg / mL kanamycin, 100 μmol photons / m ２ / s (daylight fluorescent lamp), 2% CO ２ The mixture was cultured at 30°C and 100 rpm for 3 days.

[0194] OD from the pre-culture medium ７５０The cells or microorganisms were subcultured so that the concentration of the medium reached 0.1, and then cultured in a two-stage flask in MA2 medium containing 100 μmol photons / m ２ / s (daylight fluorescent lamp), 2% CO ２ The strain was cultured at 30°C and 100 rpm for 14 days. For KC0166, samples were prepared with and without isopropyl β-d-thiogalactopyranoside (IPTG) added to a final concentration of 1 mM on the second day of the main culture.

[0195] During the main culture period, the growth of cells or microorganisms was monitored by measuring the optical density at 750 nm (OD ７５０ ) and the nitrate and glucose concentrations in the culture supernatant were measured by the following methods.

[0196] The only nitrogen source contained in MA2 medium is sodium nitrate. Nitrate absorbs light with a wavelength of 220 nm well, so the optical density (OD) at a wavelength of 220 nm was used to measure the nitrate concentration in the culture supernatant. ２２０ The cells or microorganisms were removed from the culture medium by centrifugation (8000×g, 5 min), and the OD ２２０ was measured.

[0197] To measure the glucose concentration in the culture supernatant, cells or microorganisms and cell or microbial debris were removed from the culture by centrifugation (8000 × g, 5 min) and filtration, and the filtrate was analyzed using an HPLC system (Shimadzu Corporation) (column: Bio-Rad Aminex HPX-87H column, mobile phase: 5 mM sulfuric acid, temperature: 50°C, flow rate: 0.6 mL / min, analysis time: 25 min). A similar analysis was performed on glucose standards of known concentrations to create a calibration curve, and the glucose concentration in the culture supernatant was calculated based on this curve.

[0198] Results: Compared to the wild-type strain PCC7002, the introduction of the agp / yihX / glf / ga gene into the glk gene site resulted in an OD ７５０ The glgC gene deletion strain had no effect on cell or microbial growth as an indicator, but cell or microbial growth was reduced in the glgC gene deletion strain (Figure 1).

[0199] When the glucose concentration in the culture supernatant was measured, glucose was not secreted in PCC7002, whereas glucose was secreted into the culture supernatant in the strain in which the agp / yihX / glf / ga genes were introduced into the glk gene site. The glf gene was introduced to secrete glucose outside the cell or microorganism, but glucose secretion was also confirmed in KC0161 and KC0166, which did not have the glf gene introduced, indicating that introduction of the glf gene is not necessarily essential for glucose production.

[0200] KC0151 (a strain into which the agp and glf genes had been introduced) achieved glucose production of 0.5 g / L on the 14th day of culture.

[0201] (Example 1A: Glucose secretion production using cyanobacteria) Glucose is produced by dephosphorylation of glucose-1-phosphate and glucose-6-phosphate, but even if glucose is produced by introducing glucose-1-phosphatase (EC 3.1.3.10) or glucose-6-phosphatase (EC 3.1.3.9), it may be phosphorylated by glucokinase (EC 2.7.1.2), and as a result, it may not contribute to an increase in glucose production. Therefore, the inventors considered it important to delete glucokinase in order to prevent glucose phosphorylation.

[0202] Plasmid construction: A plasmid (KCP0209) was constructed to introduce the glf gene from Zymomonas mobilis into the glk gene site of the cyanobacterium Picosynechococcus (Synechococcus) sp. PCC 7002. Plasmid KAP0019 was constructed to disrupt the glk gene (alr2973) of the cyanobacterium Nostoc (Anabaena) sp. PCC 7120.

[0203] A list of the constructed plasmids is shown in Table 6. KCP0209 was constructed by inserting the psbA2 promoter and a codon-optimized glf gene downstream of the ampicillin resistance cassette of KCP0150. KAP0019 was constructed by inserting the sacB gene, a spacer sequence for disrupting the glk gene, and 1000 bp upstream and downstream sequences of the glk gene into pSL2680.

[0204]

[0205] The glf (glucose facilitated diffusion protein) information for Zymomonas mobilis was obtained from NCBI. Z. mobilis GLF (GenBank: AVZ41684.1) MSSESSQGLVTRLALIAAIGGLLFGYDSAVIAAIGTPVDIHFIAPRHLSATAAASLSGMVVVAVLVGCVTGSLLSGWIGIRFGRRGGLLMSSICFVAAGFGAALTEKLFGTGGSALQIFCFFRFLAGLGIGVVSTLTPTYIAEIAPPDKRGQMVSGQQMAIVTGALTGYIFTWLLAHFGSIDWVNASGWCWSPASEGLIGIAFLLLLLTAPDTPHWLVMKGRHSEASKILARLEPQ ADPNLTIQKIKAGFDKAMDKSSAGLFAFGITVVFAGVSVAAFQQLVGINAVLYYAPQMFQNLGFGADTALLQTISIGVVNFIFTMIASRVVDRFGRKPLLIWGALGMAAMMAVLGCCFWFKVGGVLPLASVLLYIAVFGMSWGPVCWVVLSEMFPSSIKGAAMPIAVTGQWLANILVNFLFKVADGSPALNQTFNHGFSYLVFAALSILGGLIVARFVPETKGRSLDEIEEMWRSQK (SEQ ID NO: 25)

[0206]

[0207] Transformation of cyanobacteria: The constructed plasmid was used to transform the cyanobacteria Picosynechococcus sp. PCC 7002, and the Amp Ｒ The / glf gene was introduced.

[0208] The cyanobacterium Picosynechococcus sp. PCC 7002 was grown in MA2 medium at 50 μmol photons / m ２ / s (daylight fluorescent lamp), 30℃, 100 rpm ７５０ The cells were cultured until the chromatin density reached approximately 1.0 μg. 100 μL of the culture medium was transferred to a 1.5 mL microtube, and 1 μg of the constructed plasmid was added. The mixture was gently stirred at room temperature for 24 hours in the dark, and the entire cell suspension was seeded onto MA2 agar medium covered with a filter (Immobilon-NC membrane, pore size: 0.45 μm, Merck Millipore, Massachusetts, USA). The chromatin density was 50 μmol photons / m ２ The filter was transferred to MA2 agar medium containing 4 μg / mL carbenicillin. ２ The colony cells were cultured under conditions of 50 μmol photons / m (daylight fluorescent light) at 30°C until colonies were formed. ２ The cells were cultured under a neutral white fluorescent lamp (daylight) at 30°C and 100 rpm. PCR was performed using the genomic DNA of the cultured cells as a template to clone Amp into the glk gene site. Ｒ It was confirmed that the / glf gene had been introduced.

[0209] The constructed plasmid was used to transform the cyanobacterium Nostoc (Anabaena) sp. PCC 7120 to disrupt the glk gene (alr2973).

[0210] The cyanobacterium strain PCC 7120 was cultured in BG-11 medium at 50 μmol photons / m ２ / s (daylight fluorescent lamp), 30℃, 100 rpm ７５０The culture was cultured until the pH reached approximately 3. The culture solution was subjected to an ultrasonic cleaner for 10 minutes, centrifuged at 1500×g for 10 minutes, and the supernatant was discarded. The bacterial cells were suspended in BG-11 medium, centrifuged at 1500×g for 10 minutes, and the supernatant was discarded. This procedure was repeated twice to wash the bacterial cells. The bacterial cells were then resuspended in BG-11 medium and centrifuged at 50 μmol photons / m ２ The cells were cultured overnight under conditions of 1 / s (daylight fluorescent light), 30°C, and 100 rpm. The culture solution was centrifuged at 1500 x g for 10 minutes, suspended in 1 mL of BG-11 medium, and the number of filaments in a 1 / 1000 dilution was counted using a hemocytometer. ７ The culture solution for the filaments was mixed with E. coli carrying the three plasmids pRL443, pRL623, and KAP0019, and the entire mixture was inoculated onto a 5% LB medium-containing BG-11 agar medium covered with a filter (Immobilon-NC membrane, pore size: 0.45 μm). ２ The culture was left to stand for 1 day under conditions of 50 μmol photons / m (daylight fluorescent light) and 30°C, and then the filter was transferred to a BG-11 agar medium containing 100 μg / mL neomycin. ２ The colony cells were cultured under conditions of 50 μmol photons / m (daylight fluorescent light) at 30°C until colonies were formed. ２ The cells were cultured under a neutral white fluorescent lamp at 30°C and 100 rpm. PCR was performed using the genomic DNA of the cultured cells as a template to confirm that the glk gene site had been deleted. After that, the cells were cultured in BG-11 medium containing 5% sucrose, 50 μmol photons / m ２ The cells were cultured under a neutral white fluorescent lamp (Natural White Fluorescent Lamp) at 30°C and 100 rpm. PCR was performed using the genomic DNA of the cultured cells as a template, and it was confirmed that the sacB gene site, i.e., KAP0019, had been lost.

[0211] A list of cyanobacterial strains is shown in Table 7.

[0212]

[0213] Pre-culture of glucose production by transgenic strains (Picosynechococcus) KC160 or KC230 was cultured in a two-stage flask in MA2 medium containing 4 μg / mL carbenicillin, 100 μmol photons / mL ２ / s (daylight fluorescent lamp), 2% CO ２ The mixture was cultured at 30°C and 100 rpm for 3 days.

[0214] OD from the pre-culture medium ７５０ The cells were subcultured so that the cell density was 0.1, and then cultured in a two-stage flask containing MA2 medium, 100 μmol photons / m ２ / s (daylight fluorescent lamp), 2% CO ２ The mixture was cultured at 30°C and 100 rpm for 14 days.

[0215] During the main culture period, cell growth was monitored by optical density (OD) at 750 nm. ７５０ The nitrate and glucose concentrations in the culture supernatant were measured by the following method. The only nitrogen source contained in MA2 medium is sodium nitrate. Since nitrate absorbs light with a wavelength of 220 nm well, the nitrate concentration in the culture supernatant was measured by the optical density (OD) at a wavelength of 220 nm. ２２０ The cells were removed from the culture medium by centrifugation (8000×g, 5 min), and the OD ２２０ was measured.

[0216] To measure the glucose concentration in the culture supernatant, cells and cell debris were removed from the culture by centrifugation (8000 × g, 5 min) and filtration (pore size: 0.22 μm, Shimadzu GLC, Tokyo, Japan), and the filtrate was analyzed using an HPLC system (Shimadzu Corporation) (column: Bio-Rad Aminex HPX-87H column, mobile phase: 5 mM sulfuric acid, temperature: 50°C, flow rate: 0.6 mL / min, analysis time: 25 min). A similar analysis was performed on glucose standards of known concentrations to create a calibration curve, and the glucose concentration in the culture supernatant was calculated based on this curve.

[0217] Measurement of the glucose concentration in the culture supernatant revealed that PCC 7002 did not secrete glucose, whereas the glk gene-disrupted strain and the strain in which the glf gene was introduced into the glk gene site secreted glucose into the culture supernatant. The glf gene was introduced to secrete glucose outside the cells, but glucose secretion was also confirmed in KC0160, which does not have the glf gene introduced. This indicates that disruption of the glk gene leads to glucose secretion, and introduction of the glf gene increases glucose production (Figure 1B).

[0218] KC0230 (a strain into which the glf gene has been introduced) achieved glucose production of 0.3 g / L on the 14th day of culture.

[0219] Evaluation of glucose production by the glucose-producing gene-transfected strain (Nostoc) Pre-culture PCC 7120 or KA0015 was cultured in a two-stage flask in nitrate-free BG-11 medium, 100 μmol photons / m ２ / s (daylight fluorescent lamp), 5% CO ２ The mixture was cultured at 30°C and 100 rpm for about 7 to 10 days.

[0220] OD from the pre-culture medium ７５０ The cells were subcultured so that the cell density was 0.1, and then cultured in a two-stage flask containing nitrate-free BG-11 medium, 100 μmol photons / m ２ / s (daylight fluorescent lamp), 5% CO ２ The mixture was cultured at 30°C and 100 rpm for 49 days.

[0221] During the main culture period, cell growth was evaluated based on the dry weight of the cells (mg / L), and the glucose concentration in the culture supernatant was measured by the following method.

[0222] Cells and cell debris were removed from the culture medium by centrifugation (8000 × g, 5 min) and filtration (pore size: 0.22 μm, Shimadzu GLC), and the filtrate was analyzed using an HPLC system (Shimadzu Corporation) (column: Bio-Rad Aminex HPX-87H column, mobile phase: 5 mM sulfuric acid, temperature: 50°C, flow rate: 0.6 mL / min, analysis time: 25 min). A similar analysis was performed on glucose standards of known concentrations to create a calibration curve, and the glucose concentration in the culture medium supernatant was calculated based on this curve.

[0223] Results Compared to the wild-type strain PCC 7120, disruption of the glk gene affected cell growth, as measured by cell dry weight, resulting in a decrease in cell growth (Fig. 1C).

[0224] When the glucose concentration in the culture supernatant was measured, it was found that PCC 7120 did not secrete glucose, whereas the glk gene disruptant secreted glucose into the culture supernatant.

[0225] KA0015 (glk gene disruptant strain) achieved glucose production of 7.9 g / L on the 49th day of culture.

[0226] Example 2: Yeast In this example, the cyanobacteria of Example 1 are combined with yeast.

[0227] Glucose-producing cyanobacteria are cultured to produce glucose in the supernatant, which is then filtered and collected. Yeast is added to the supernatant and fermented under the desired conditions. Any nutrients that are lacking during yeast culture and pH adjustment are added as needed.

[0228] The yeasts and procedures that can be used are as follows: Yeasts include Saccharomyces cerevisiae, Saccharomyces bayanus, Candida utilis, Schizosaccharomyces pombe, Kluyveromyces lactis, Pichia pastoris, Hansenula polymorpha, Issatchenkia orientalis.

[0229] In this example, the pH of the glucose-producing cyanobacterial supernatant is checked and adjusted to the optimal pH range for yeast fermentation. A typical pH range is between 4.5 and 6.0. The yeast is grown in a separate nutrient medium before being added to the cyanobacterial supernatant. During the fermentation process, the temperature, pH, glucose concentration, and product (e.g., ethanol) concentration are regularly monitored. This allows for process control to maximize fermentation efficiency and achieve optimal yield. After fermentation is complete, the yeast is precipitated, and the supernatant is harvested to recover the product.

[0230] (Example 3: Aromatic production test by Escherichia coli using glucose produced by the glk-deficient AGP-GLF-introduced strain) Glucose obtained by culturing the glk-deficient AGP-GLF-introduced strain prepared in Example 1 was fed to Escherichia coli to produce aromatic compounds (details are shown in FIG. 8).

[0231] The glk-deficient AGP-GLF-introduced strain produced in Example 1 and the PCC7002 wild-type strain as a negative control were cultured in a two-tier flask for 7 days under the conditions of 4 mg / L carbonicillin-containing MAD2 medium, 100 μmol photons / m / s (neutral white fluorescent light), 2% CO, 30°C, and 100 rpm (preculture). ７５０ The cells were subcultured so that the OD = 1, and cultured in a two-stage flask in MAD2 medium at 100 μmol photons / m2 / s (daylight fluorescent light), 2% CO2, 30°C, and 100 rpm for 28 days (main culture). During the main culture period, cell growth was monitored by measuring the optical density at 750 nm (OD ７５０ The glucose concentration was measured by the following method: On the 28th day of the main culture, the entire culture medium was collected and centrifuged at 14,000×g for 30 minutes, and the supernatant was stored frozen (−30° C.).

[0232] The resulting supernatant was used to test aromatic production by E. coli. The muconic acid-producing E. coli strain was inoculated into 3 mL of LB medium (100 μg / mL ampicillin, 50 μg / mL spectinomycin, 50 μg / mL kanamycin) and cultured overnight at 37°C with a shaking speed of 200 rpm. The initial OD ６００The resulting cells were inoculated into 20 mL of LB medium (100 μg / mL ampicillin, 50 μg / mL spectinomycin, 50 μg / mL kanamycin) so that the OD = 0.01, and cultured overnight at 37°C with a shaking speed of 200 rpm. The resulting cells were then transferred to 3 mL of the supernatant of the glk-deficient AGP-GLF-introduced strain, the supernatant of the PCC7002 wild-type strain, and MAD2 medium (containing 1 g / L glucose) for OD analysis. ６００ = 1, and a production test was carried out at a shaking speed of 200 rpm and 37°C. After 1, 6, and 24 hours, 500 μL of the culture medium was sampled and centrifuged to obtain the supernatant, and the concentrations of the produced aromatic compounds (protocatechuic acid, catechol, muconic acid) and glucose were measured by HPLC.

[0233] To measure the glucose concentration in the culture supernatant, cells and cell debris were removed from the culture by centrifugation (8,000 × g, 5 min) and filtration, and the filtrate was analyzed by HPLC (Shimadzu Corporation) (column: Bio-Rad Aminex HPX-87H column, mobile phase: 5 mM sulfuric acid, temperature: 50°C, flow rate: 0.6 mL / min, analysis time: 25 min).

[0234] Aromatic acids (protocatechuic acid, catechol, and muconic acid) in the culture supernatant were measured using an HPLC system (Shimadzu Corporation) (column: InertSustain AQ-C18 (5 μm, 4.6 × 250 mm), mobile phase: A: 50 mM NaH ２ P.O. ４ in H2O (pH2.1, H ３ P.O. ４ ) B: Gradient analysis of 100% acetonitrile, temperature: 40°C, flow rate: 1 mL / min, analysis time: 30 min).

[0235] (Results) No significant difference in growth was observed between KC0151 and wild-type strain PCC7002 (Figure 1). On the other hand, glucose was not detected in the culture supernatant of wild-type strain PCC7002, but 1 g / L or more of glucose was obtained in the culture supernatant of KC0151 by day 26 (Figure 2).

[0236] The muconic acid-producing E. coli strain used possesses a plasmid expressing aroZ, aroY, and catA under the control of the trc promoter, which encode three reactions (dehydroshikimate to protocatechuate, protocatechuate to catechol, and catechol to muconate) that are intermediates in the shikimate pathway. These reactions are all derived from invasive species and are not present in wild-type E. coli strains. Furthermore, to increase muconic acid production, this muconic acid-producing E. coli strain lacks aroE, which encodes the enzyme that converts dehydroshikimate to shikimate. The strain also possesses two plasmids: a plasmid expressing aroG-tktA-ppsA under the control of the trc promoter, and a plasmid expressing aroB-aroD under the control of the trc promoter. Muconic acid-producing E. coli strains were suspended in the resulting culture supernatant, and consumption of the glucose produced by KC0151 was confirmed (Figure 3). Glucose consumption was also confirmed in MAD2 medium containing 1 g / L of glucose as a positive control. Measurement of aromatic compounds in the E. coli culture supernatant revealed that muconic acid (ccMA), the final product, was produced in the MAD2 medium containing glucose, but its production was barely observed in the KC0151 culture supernatant (Figure 5). Meanwhile, production of 0.1 g / L of protocatechuic acid was confirmed in the KC0151 supernatant (Figure 4). Production of these compounds was not confirmed in the culture supernatant of the wild-type strain PCC 7002. These results confirmed that fermentative production using E. coli is possible using glucose derived from cyanobacteria.

[0237] The gene sequences used are as follows: Bacillus thuringiensis aroZ (codon optimized): SEQ ID NO: 9 Klebsiella pneumoniae aroY (codon optimized): SEQ ID NO: 10 Pseudomonas putida catA (codon optimized): SEQ ID NO: 11 E. coli aroG (D146N): SEQ ID NO: 12 E. coli tktA: SEQ ID NO: 13 E. coli ppsA: SEQ ID NO: 14 E. coli aroB: SEQ ID NO: 15 E. coli aroD: SEQ ID NO: 16 The amino acid sequences are as follows: Bacillus thuringiensis aroZ (codon optimized): SEQ ID NO: 17 Klebsiella pneumoniae aroY (codon optimized): SEQ ID NO: 18 Pseudomonas putida catA (codon optimized): SEQ ID NO: 19 E. coli aroG (D146N): SEQ ID NO: 20 E. coli tktA: SEQ ID NO: 21 E. coli ppsA: SEQ ID NO: 22 E. coli aroB: SEQ ID NO: 23 E. coli aroD: SEQ ID NO: 24 See also Figure 8 for details of these.

[0238] (Example 4: Animal cell culture) We demonstrate the culture of animal cells using nutrients excreted by the cyanobacteria of the present disclosure, demonstrating that the present technology can be widely applied to cell culture.

[0239] 1. Materials and Methods 1-1. Cyanobacterial Culture In this example, the glucose-producing recombinant Synechococcus produced in Example 1 was maintained and cultured in an artificial climate chamber (Nippon Medical Chemical Machinery Manufacturing Co., Ltd.) using MA2 medium in an Erlenmeyer flask capped with an air-permeable silicone stopper (e.g., C-40) (culture conditions: temperature: 25°C; CO ２ Concentration: 1%; Stirring speed: 60 rpm; Photosynthetic photon flux density (PPFD): approximately 80 μmol / m ２ / s). DMEM (glucose- and pyruvate-free) is used as the medium for animal cells. This is used to demonstrate whether glucose can be replenished by culturing glucose-producing Synechococcus, and whether animal cells can be cultured in a carbohydrate-free medium. Under the same conditions as above, glucose-producing Synechococcus is cultured in a medium changed to DMEM (glucose- and pyruvate-free). The culture supernatant is then collected over time and used for the animal cells described below. The glucose content of the culture supernatant is measured using the hexokinase method.

[0240] (1) Composition of DMEM (glucose and pyruvic acid free) (i) Carbohydrates: Glucose: 0 mg / L, Pyruvate: 0 mg / L (ii) Amino Acids: L-Arginine: 84 mg / L, L-Cystine: 48 mg / L, L-Glutamine: 584 mg / L, Glycine: 30 mg / L, L-Histidine: 42 mg / L, L-Isoleucine: 105 mg / L, L-Leucine: 105 mg / L, L-Lysine: 146 mg / L, L-Methionine: 30 mg / L, L-Phenylalanine: 66 mg / L, L-Serine: 42 mg / L, L-Threonine: 95 mg / L, L-Tryptophan: 16 mg / L, L-Tyrosine: 71 mg / L, L-Valine: 94 mg / L (iii) Vitamins: Pantothenic acid: 4 mg / L, choline chloride: 4 mg / L, folic acid: 4 mg / L, i-inositol: 7.2 mg / L, niacinamide: 4 mg / L, pyridoxine: 4 mg / L, riboflavin: 0.4 mg / L, thiamine: 4 mg / L. (iv) Minerals: CaCl2: 200 mg / L, KCl: 400 mg / L, Fe(NO3)3.9H2O: 0.10 mg / L, MgSO4: 98 mg / L, NaCl: 6400 mg / L, NaHCO3: 3700 mg / L, NaH2PO4: 109 mg / L, phenol red: 15 mg / L.

[0241] 1-2. Cell culture and cell viability test Animal cells were C2C12 cells (ATCC (registered trademark) CRL-1772 ＴＭ ) and QM7 cells (ATCC® CRL-1962 ＴＭAnimal cells are cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, Missouri, USA) supplemented with 10% fetal bovine serum (FBS, Thermo-Fischer Scientific, Massachusetts, USA) and 1% penicillin-streptomycin (P / S, Invitrogen, Carlsbad, California, USA) at 37°C in 5% CO. ２ The cells were cultured in a humidified atmosphere containing HCl. Viable cell counts were assessed by XTT assay (Biological Industries, Connecticut, USA) or trypan blue exclusion test. Animal cells were seeded at a density of 10,000–100,000 cells / well in 48- or 96-well plates (AGC Technoglass Co., Shizuoka, Japan) and cultured overnight in DMEM supplemented with 10% FBS and 1% P / S. After overnight culture, the medium was discarded and the cells were washed twice with phosphate-buffered saline (PBS, Sigma-Aldrich). The cells were then cultured for 1–3 days in glucose-containing DMEM, glucose-free DMEM, or glucose-free DMEM containing glucose-producing Synechococcus. Viable cell counts were then quantitatively analyzed by XTT assay or trypan blue exclusion test.

[0242] The composition of glucose-containing DMEM is as follows: (i) Carbohydrates: Glucose: 1000 mg / L or 4500 mg / L, Pyruvate: 110 mg / L (ii) Amino Acids: L-Arginine: 84 mg / L, L-Cystine: 48 mg / L, L-Glutamine: 584 mg / L, Glycine: 30 mg / L, L-Histidine: 42 mg / L, L-Isoleucine: 105 mg / L, L-Leucine: 105 mg / L, L-Lysine: 146 mg / L, L-Methionine: 30 mg / L, L-Phenylalanine: 66 mg / L, L-Serine: 42 mg / L, L-Threonine: 95 mg / L, L-Tryptophan: 16 mg / L, L-Tyrosine: 71 mg / L, L-Valine: 94 mg / L (iii) Vitamins: Pantothenic acid: 4 mg / L, Choline chloride: 4 mg / L, Folic acid: 4 mg / L, i-inositol: 7.2 mg / L, Niacinamide: 4 mg / L, Pyridoxine: 4 mg / L, Riboflavin: 0.4 mg / L, Thiamine: 4 mg / L (iv) Minerals: CaCl ２ :200mg / L ・KCl: 400mg / L ・Fe(NO ３ ) ３ ・9H ２ O: 0.10mg / L ・MgSO ４ :98mg / L ・NaCl: 6400mg / L ・NaHCO ３ :3700mg / L ・NaH ２ P.O. ４ : 109 mg / L Phenol red: 15 mg / L

[0243] 2. Results 2-1. Cell culture using glucose-producing Synechococcus cultured in glucose-free DMEM We investigated whether the culture supernatant of glucose-producing Synechococcus can be used as a carbohydrate source for animal cell culture. When animal cells are cultured in glucose-free DMEM, a significant decrease in cell viability is observed compared to when cultured in nutrient-containing medium. However, when cultured in glucose-free DMEM with glucose-producing Synechococcus, no decrease in cell viability is observed. These results indicate that when glucose-producing Synechococcus is supplied to a carbohydrate-free medium, animal cells can utilize the glucose and become cultured.

[0244] Example 5: Recirculating Culture This example demonstrates recirculating culture using animal cells and cyanobacteria produced by the present invention and prior art. This is a technology for improving the efficiency of cultivated meat production and biopharmaceutical production using animal cells, with reduced environmental impact and lower costs.

[0245] 1. Materials and Methods 1-1. Animal Cell Culture (1st Cycle) and Culture Waste Collection. Animal muscle cells (C2C12 cells or QM7 cells), the cell source for cultured meat production, are cultured for 2-3 days in the fetal bovine serum (FBS) and glucose-containing DMEM described above (or serum-free medium using culture supernatant from rat hepatocyte RL34 cells (JCRB0247), which produce growth factors for animal muscle cells) (1st cycle of circulation culture). The culture waste is collected. The amounts of glucose, pyruvate (nutrients for animal cells), ammonia, and lactate (waste products of animal cells) in the culture waste or the animal cell preculture medium are quantitatively analyzed. The amounts of glucose, pyruvate, ammonia, and lactate are measured using the hexokinase UV method, the pyruvate oxidase enzymatic method, the modified Fujii-Okuda method, and the lactate oxidase enzymatic method (SRL), respectively.

[0246] 1-2. Cyanobacterial Cultivation Using Culture Waste (First Cycle of Cyanobacterial Cultivation) Using the animal cell culture waste recovered in 1-1, the glucose-producing genetically modified Synechococcus produced in Example 1 is cultured in the same manner as above. At this time, a lactate-utilizing Synechococcus (a genetically modified cyanobacterium that converts lactic acid, a waste product of animal cells, into pyruvate, a nutrient for animal cells) from a prior patented technology is cultured simultaneously or under the same conditions as above using the culture waste after the above culture. The culture supernatant is then collected over time and used for animal cell culture (second cycle). The amounts of glucose, pyruvate, ammonia, and lactate in the culture supernatant and in the animal cell culture waste before cyanobacterial cultivation are measured.

[0247] 1-3. Animal cell culture (second cycle) and collection of culture waste fluid Animal cells (C2C12 cells, QM7 cells, etc.) are cultured for 2-3 days using the cyanobacterial culture waste fluid collected in 1-2 (second cycle of circulating culture). At this time, animal cells are also cultured using the animal cell culture waste fluid before cyanobacterial culture. The cell count after culture is measured using a trypan blue exclusion test. The glucose, pyruvate, ammonia, and lactate amounts in the culture supernatant and the culture waste fluid before animal cell culture are also measured. The collected culture waste fluid is used to culture the genetically modified Synechococcus produced using the present invention and prior patent technology in the same manner as described in 1-2 (second cycle of cyanobacterial culture).

[0248] 2. Results 2-1. Animal cell culture (first cycle) Compared to the medium before animal cell culture, the animal cell culture resulted in a significant decrease in glucose and pyruvic acid, and a significant increase in lactate and pyruvic acid.

[0249] 2-2. Cyanobacterial cultivation using culture wastewater (first cycle of cyanobacterial cultivation) Cultivation using the cyanobacteria produced in this invention increases the amount of glucose in the culture wastewater and reduces the amount of ammonia. Furthermore, culturing the lactate-utilizing Synechococcus produced using the prior patented technology significantly reduces the amounts of lactic acid and ammonia in the culture wastewater, and greatly increases the amount of pyruvic acid.

[0250] 2-3. Animal Cell Culture (Second Cycle) When animal cells such as C2C12 cells or QM7 cells are cultured using culture wastewater without culturing cyanobacteria, a significant decrease in cell viability is observed. This is thought to be due to a lack of nutrients such as glucose and pyruvate, and the accumulation of waste products such as lactate and ammonia. However, when culture wastewater from the cultivation of glucose-producing Synechococcus or lactate-utilizing Synechococcus is used, no decrease in cell viability is observed, and cell proliferation is observed. This result is thought to be due to the supply of glucose and pyruvate to the culture wastewater, which clears ammonia and lactate.

[0251] (Example 6: Circular economy application (1)) In this example, as an example of a circular economy application, a cyanobacteria having the ability to utilize lactate and excrete glucose is cultured in a medium for cyanobacteria for a certain period of time, and then inoculated into the culture solution after animal cell culture.

[0252] After culturing for a certain period in the culture medium after animal cell culture, glucose was secreted into the medium, and lactic acid and ammonia were consumed. The cyanobacteria were cultured under the following conditions: temperature: 30°C; CO ２ Concentration: 2%; Stirring speed: 100 rpm; Light intensity: approximately 100 μmol / m ２ / s.

[0253] Briefly, the KC0154 strain, which has been imparted with the ability to utilize lactate and excrete glucose, is cultured in MA2 medium for about a week and then inoculated into the culture medium after culturing animal cells for 2-3 days. KC0154 is then cultured in the medium after animal cell culture for about a week, and the amounts of glucose, pyruvate, ammonia, and lactate in the medium before and after culturing KC0154 are measured. It can be confirmed that the amounts of glucose and pyruvate increase and the amounts of ammonia and lactate decrease after culturing KC0154. More details are as follows.

[0254] 1. Materials and Methods 1-1. Cyanobacterial Culture In this study, we used a genetically modified Synechococcus (KC0154) with the ability to utilize lactate and produce and excrete glucose. This strain was created by combining prior patented technology with the present invention. It was maintained and cultured in an artificial climate chamber (Nippon Medical and Chemical Machinery Manufacturing Co., Ltd.) using MA2 medium in an Erlenmeyer flask capped with an air-permeable silicone stopper (e.g., C-40) (culture conditions: temperature: 25°C; CO ２ Concentration: 1%; Stirring speed: 60 rpm; Photosynthetic photon flux density (PPFD): approximately 80 μmol / m ２ / s). DMEM (glucose- and pyruvate-free) is used as the medium for animal cells. This is used to demonstrate whether glucose can be replenished by culturing glucose-producing Synechococcus, and whether animal cells can be cultured in a carbohydrate-free medium. Under the same conditions as above, genetically modified Synechococcus with lactate utilization and glucose production and excretion capabilities is cultured in a medium changed to DMEM (glucose- and pyruvate-free). The culture supernatant is then collected over time and used for the animal cells described below. The glucose content of the culture supernatant is measured using the hexokinase method.

[0255] (1) Composition of DMEM (glucose and pyruvic acid free) (i) Carbohydrates: Glucose: 0 mg / L, Pyruvate: 0 mg / L (ii) Amino Acids: L-Arginine: 84 mg / L, L-Cystine: 48 mg / L, L-Glutamine: 584 mg / L, Glycine: 30 mg / L, L-Histidine: 42 mg / L, L-Isoleucine: 105 mg / L, L-Leucine: 105 mg / L, L-Lysine: 146 mg / L, L-Methionine: 30 mg / L, L-Phenylalanine: 66 mg / L, L-Serine: 42 mg / L, L-Threonine: 95 mg / L, L-Tryptophan: 16 mg / L, L-Tyrosine: 71 mg / L, L-Valine: 94 mg / L (iii) Vitamins: Pantothenic acid: 4 mg / L, choline chloride: 4 mg / L, folic acid: 4 mg / L, i-inositol: 7.2 mg / L, niacinamide: 4 mg / L, pyridoxine: 4 mg / L, riboflavin: 0.4 mg / L, thiamine: 4 mg / L. (iv) Minerals: CaCl2: 200 mg / L, KCl: 400 mg / L, Fe(NO3)3.9H2O: 0.10 mg / L, MgSO4: 98 mg / L, NaCl: 6400 mg / L, NaHCO3: 3700 mg / L, NaH2PO4: 109 mg / L, phenol red: 15 mg / L.

[0256] 1-2. Cell culture and cell viability test Animal cells were C2C12 cells (ATCC (registered trademark) CRL-1772 ＴＭ ) and QM7 cells (ATCC® CRL-1962 ＴＭ Animal cells are cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, Missouri, USA) supplemented with 10% fetal bovine serum (FBS, Thermo-Fischer Scientific, Massachusetts, USA) and 1% penicillin-streptomycin (P / S, Invitrogen, Carlsbad, California, USA) at 37°C in 5% CO. ２The cells were cultured in a humidified atmosphere containing HCl. Viable cell counts were assessed by XTT assay (Biological Industries, Connecticut, USA) or trypan blue exclusion test. Animal cells were seeded at a density of 10,000–100,000 cells / well in 48- or 96-well plates (AGC Technoglass Co., Shizuoka, Japan) and cultured overnight in DMEM supplemented with 10% FBS and 1% P / S. After overnight culture, the medium was discarded and the cells were washed twice with phosphate-buffered saline (PBS, Sigma-Aldrich). The cells were then cultured for 1–3 days in glucose-containing DMEM, glucose-free DMEM, or glucose-free DMEM containing glucose-producing Synechococcus. Viable cell counts were then quantitatively analyzed by XTT assay or trypan blue exclusion test.

[0257] The composition of glucose-containing DMEM is as follows: (i) Carbohydrates: Glucose: 1000 mg / L or 4500 mg / L, Pyruvate: 110 mg / L (ii) Amino Acids: L-Arginine: 84 mg / L, L-Cystine: 48 mg / L, L-Glutamine: 584 mg / L, Glycine: 30 mg / L, L-Histidine: 42 mg / L, L-Isoleucine: 105 mg / L, L-Leucine: 105 mg / L, L-Lysine: 146 mg / L, L-Methionine: 30 mg / L, L-Phenylalanine: 66 mg / L, L-Serine: 42 mg / L, L-Threonine: 95 mg / L, L-Tryptophan: 16 mg / L, L-Tyrosine: 71 mg / L, L-Valine: 94 mg / L (iii) Vitamins: Pantothenic acid: 4 mg / L, Choline chloride: 4 mg / L, Folic acid: 4 mg / L, i-inositol: 7.2 mg / L, Niacinamide: 4 mg / L, Pyridoxine: 4 mg / L, Riboflavin: 0.4 mg / L, Thiamine: 4 mg / L (iv) Minerals: CaCl ２ :200mg / L ・KCl: 400mg / L ・Fe(NO ３ ) ３ ・9H ２ O: 0.10mg / L ・MgSO ４:98mg / L ・NaCl: 6400mg / L ・NaHCO ３ :3700mg / L ・NaH ２ P.O. ４ : 109 mg / L Phenol red: 15 mg / L

[0258] 2. Results 2-1. Cell culture using glucose-free DMEM cultured with recombinant Synechococcus capable of lactate utilization and glucose production and export. This study investigated whether the culture supernatant of recombinant Synechococcus capable of lactate utilization and glucose production and export can be used as a carbohydrate source for animal cell culture. When animal cells are cultured in glucose-free DMEM, a significant decrease in cell viability is observed compared to when cultured in nutrient-containing medium. However, when cultured in glucose-free DMEM cultured with recombinant Synechococcus capable of lactate utilization and glucose production and export, no decrease in cell viability is observed. These results indicate that when recombinant Synechococcus capable of lactate utilization and glucose production and export is cultured in a carbohydrate-free medium, glucose can be supplied to animal cells, allowing them to utilize and cultivate.

[0259] In this example, we demonstrate circulating culture using animal cells and cyanobacteria produced by the present invention and prior art. This is a technology for improving the efficiency of cultivated meat production and biopharmaceutical production using animal cells, with reduced environmental impact and cost.

[0260] 1. Materials and Methods 1-1. Animal Cell Culture (1st Cycle) and Culture Waste Collection. Animal muscle cells (C2C12 cells or QM7 cells), the cell source for cultured meat production, are cultured for 2-3 days in the fetal bovine serum (FBS) and glucose-containing DMEM described above (or serum-free medium using culture supernatant from rat hepatocyte RL34 cells (JCRB0247), which produce growth factors for animal muscle cells) (1st cycle of circulation culture). The culture waste is collected. The amounts of glucose, pyruvate (nutrients for animal cells), ammonia, and lactate (waste products of animal cells) in the culture waste or the animal cell preculture medium are quantitatively analyzed. The amounts of glucose, pyruvate, ammonia, and lactate are measured using the hexokinase UV method, the pyruvate oxidase enzymatic method, the modified Fujii-Okuda method, and the lactate oxidase enzymatic method (SRL), respectively.

[0261] 1-2. Cyanobacterial culture using culture wastewater (first cycle of cyanobacterial culture) Using the animal cell culture wastewater recovered in 1-1, a genetically modified Synechococcus (KC0154) capable of lactic acid utilization and glucose production and excretion is cultured in the same manner as above. The culture supernatant is then collected over time and used for animal cell culture (second cycle). The amounts of glucose, pyruvate, ammonia, and lactic acid in the culture supernatant and in the animal cell culture wastewater before cyanobacterial culture are measured.

[0262] 1-3. Animal cell culture (second cycle) and collection of culture waste fluid Animal cells (e.g., C2C12 cells or QM7 cells) are cultured for 2-3 days using the cyanobacterial culture waste fluid collected in 1-2 (second cycle of circulating culture). At this time, animal cells are also cultured using the animal cell culture waste fluid before cyanobacterial culture. The cell count after culture is measured using a trypan blue exclusion test. The glucose, pyruvate, ammonia, and lactate amounts in the culture supernatant and the culture waste fluid before animal cell culture are also measured. The collected culture waste fluid is used to culture a genetically modified Synechococcus capable of lactic acid utilization and glucose production and excretion (second cycle of cyanobacterial culture).

[0263] 2. Results 2-1. Animal cell culture (first cycle) Compared to the medium before animal cell culture, the animal cell culture resulted in a significant decrease in glucose and pyruvic acid, and a significant increase in lactate and ammonia.

[0264] 2-2. Cyanobacteria cultivation using culture wastewater (first cycle of cyanobacteria cultivation) By culturing using the cyanobacteria produced by this invention, the amount of glucose in the culture wastewater increases due to the effects of this invention, and the amount of pyruvic acid increases due to the effects of the prior patented technology. In addition, the amount of ammonia decreases due to the cultivation of cyanobacteria, and the amount of lactic acid decreases significantly due to the effects of the prior patented technology.

[0265] 2-3. Animal Cell Culture (Second Cycle) When animal cells such as C2C12 and QM7 cells are cultured using culture wastewater without culturing cyanobacteria, a significant decrease in cell viability is observed. This is thought to be due to a lack of nutrients such as glucose and pyruvate, and the accumulation of waste products such as lactate and ammonia. However, when culture wastewater from culture of genetically modified Synechococcus strains capable of lactate utilization and glucose production and excretion is used, no decrease in cell viability is observed and cell proliferation is observed. This result is thought to be due to the supply of glucose and pyruvate to the culture wastewater, which clears ammonia and lactate.

[0266] Using these examples as a reference, animal cells such as C2C12 cells and QM7 cells can be used as cell sources for cultured meat production, and therefore can also be applied to cultured meat.

[0267] (Example 7: Circular economy application (2)) In this example, as an example of a circular economy application, circulatory culture using cyanobacteria capable of lactic acid assimilation and glucose excretion was demonstrated.

[0268] 1. Materials and Methods 1-1. Animal Cell Culture (1st Cycle) and Recovery of Culture Wastewater The composition of glucose-containing DMEM is as follows: (i) Carbohydrates: Glucose: 1000 mg / L, Pyruvate: 110 mg / L (ii) Amino Acids: L-Arginine: 84 mg / L, L-Cystine: 48 mg / L, L-Glutamine: 584 mg / L, Glycine: 30 mg / L, L-Histidine: 42 mg / L, L-Isoleucine: 105 mg / L, L-Leucine: 105 mg / L, L-Lysine: 146 mg / L, L-Methionine: 30 mg / L, L-Phenylalanine: 66 mg / L, L-Serine: 42 mg / L, L-Threonine: 95 mg / L, L-Tryptophan: 16 mg / L, L-Tyrosine: 71 mg / L, L-Valine: 94 mg / L (iii) Vitamins: Pantothenic acid: 4 mg / L Choline chloride: 4 mg / L, Folic acid: 4 mg / L, i-inositol: 7.2 mg / L, Niacinamide: 4 mg / L, Pyridoxine: 4 mg / L, Riboflavin: 0.4 mg / L, Thiamine: 4 mg / L (iv) Minerals: CaCl2: 200 mg / L, KCl: 400 mg / L, Fe(NO3)3.9H2O: 0.10 mg / L, MgSO4: 98 mg / L, NaCl: 6400 mg / L, NaHCO3: 3700 mg / L, NaH2PO4: 109 mg / L, Phenol red: 15 mg / L Animal muscle cells (C2C12 cells), the cell source for cultivated meat production, were cultured for three days (first cycle of circulating culture) in the above-described DMEM medium supplemented with 10% fetal bovine serum (FBS), and the culture effluent was collected. The amounts of glucose and pyruvate (nutrients for animal cells), ammonia, and lactate (waste products of animal cells) in the culture medium before and after animal cell culture were quantitatively analyzed. The amounts of glucose, pyruvate, ammonia, and lactate were measured using the hexokinase UV method, the pyruvate oxidase enzymatic method, the modified Fujii-Okuda method, and the lactate oxidase enzymatic method, respectively. These biochemical analyses were performed by SRL Corporation (Tokyo, Japan).

[0269] 1-2. Cyanobacterial culture using culture wastewater (first cycle of cyanobacterial culture) The culture wastewater of the animal cells collected in 1-1 was used to induce freeze stock of a genetically modified Synechococcus (KC0154) capable of lactic acid utilization and glucose excretion, and the OD ７５０ The culture was continued until the OD = 4. KC0154 was collected by centrifugation (8000 × g, 5 min, 25 ° C.). ７５０ The cells were inoculated so that the OD = 0.5 and cultured in a two-tier flask for about 5 days (preculture). The preculture solution was centrifuged to recover the cells, and the OD ７５０ The culture waste liquid was inoculated so that the β-glucan concentration became 5, and the culture was carried out in a two-tier flask for about 6 days (main culture, CO concentration: 5%, temperature: 30°C). The light intensity was adjusted to 150 μmol photons / m 24 hours after the start of the main culture. ２ / s to 360 μmol photons / m ２ The algae culture solution was then collected and centrifuged (8000 × g, 5 min, 25°C) to collect the supernatant, which was then used for animal cell culture (second cycle). The amounts of glucose, pyruvate, ammonia, and lactic acid in the animal cell culture wastewater after cyanobacterial culture were measured.

[0270] 1-3. Animal Cell Cultivation (Second Cycle) and Collection of Culture Wastewater Animal cells (C2C12 cells) were cultured for two days using the cyanobacterial culture wastewater collected in 1-2 (second cycle of circulating culture). At the same time, as a comparative experiment, animal cell culture was also performed using animal cell culture wastewater without cyanobacterial culture. The cell count of the animal cells after culture was measured using a trypan blue exclusion test. Furthermore, the amounts of glucose, pyruvate, ammonia, and lactate in the cyanobacterial culture wastewater after the second cycle of animal cell culture were measured. It is also possible to use the collected second cycle of animal cell culture wastewater to culture a genetically modified Synechococcus (KC0154) capable of lactate utilization and glucose excretion, as described in 1-2 (second cycle of cyanobacterial culture).

[0271] 2. Results 2-1. Animal cell culture (first cycle) Compared to the medium before animal cell culture, the culture of animal cells resulted in a significant decrease in glucose and pyruvate (Figure 9), and an increase in lactate and ammonia (Figure 10).

[0272] 2-2. Cyanobacterial culture using animal cell culture wastewater (first cycle of cyanobacterial culture) By culturing genetically modified cyanobacteria capable of lactate assimilation and glucose excretion, the amount of glucose in the culture wastewater increased (Figure 9), and the amounts of lactate and ammonia decreased (Figure 10). Furthermore, the amount of pyruvic acid increased significantly (Figure 9). This is thought to be due to pyruvic acid synthesized from lactate by KC0154 being excreted outside the cells.

[0273] 2-3. Animal Cell Culture (Second Cycle) When animal cell (C2C12) culture wastewater was used without culturing cyanobacteria (KC0154) for the second cycle of C2C12 cell culture (comparison experiment), a significant decrease in viable cell count was observed (Figure 11). This is thought to be due to a lack of nutrients such as glucose and pyruvate and the accumulation of waste products such as lactate and ammonia. However, when a genetically modified Synechococcus (KC0154) capable of lactate utilization and glucose excretion was cultured using the animal cell (C2C12) culture wastewater and then the C2C12 cells were cultured for the second cycle, no decrease in cell viability was observed and C2C12 cells proliferated (Figure 11). This result is thought to be due to the supply of nutrients such as glucose and pyruvate to the animal cell culture wastewater by culturing KC0154, thereby clearing the waste products ammonia and lactate. In fact, the consumption of glucose and pyruvate increased with the second cycle of animal cell culture, as observed with KC0154 culture (Figure 9). On the other hand, the consumption of lactate and ammonia, which decreased with KC0154 culture, increased with the second cycle of animal cell culture (Figure 10).

[0274] (Example 8) Circular Economy Application (3) In this example, as an example of a circular economy application, a continuous circulation culture system is demonstrated in which an animal cell culture tank and two types of microbial culture tanks, each capable of lactate assimilation and glucose excretion, are connected by tubes.

[0275] 1. Materials and Methods

[0276] 1-1. Microorganisms and animal cells used・Animal cells: Animal muscle cells (C2C12 cells) are used as the cell source for cultured meat production.・Lactic acid-utilizing microorganisms: A genetically modified cyanobacterium (hereinafter referred to as "Strain LA") with an enhanced ability to utilize lactic acid and produce pyruvic acid is used.・Glucose-excreting microorganisms: A genetically modified cyanobacterium (hereinafter referred to as "Strain GL") with the ability to excrete glucose produced by photosynthesis outside the cell is used.

[0277] 1-2. Basal Medium For the initial culture of animal cells, a medium containing glucose-containing DMEM supplemented with 10% fetal bovine serum (FBS) is used, which has the following composition: (i) Carbohydrates: Glucose: 1000 mg / L or 4500 mg / L; Pyruvate: 110 mg / L; (ii) Amino acids (same as in Example 7, omitted); (iii) Vitamins (same as in Example 7, omitted); (iv) Minerals (same as in Example 7, omitted).

[0278] 1-3. Construction of a continuous circulation culture system Three independent culture tanks are prepared: an animal cell culture tank (reactor A), a lactic acid-assimilating microorganism culture tank (reactor B), and a glucose-excreting microorganism culture tank (reactor C). The culture medium of animal cells cultured in reactor A is continuously sent to reactor B via a pump. The culture medium in reactor B, in which lactic acid has been assimilated, is then sent to reactor C. The culture medium to which glucose has been supplied in reactor C is passed through a microorganism removal filter (pore size: 0.2 μm, Thermo-Fischer Scientific) and then returned to reactor A. A continuous culture medium circulation system is constructed by connecting these flow paths with tubing.

[0279] 1-4. Culture Conditions and Measurements: Reactor A (animal cells): C2C12 cells were cultured at a temperature of 37°C and a CO2 concentration of 5%. Reactor B (Strain LA): Strain LA was cultured at a temperature of 30°C and a CO2 concentration of 5% with 150 μmol photons / m2 / s of light. Reactor C (Strain GL): Strain GL was cultured at a temperature of 30°C and a CO2 concentration of 5% with 150 μmol photons / m2 / s of light. During system operation, the culture medium was periodically sampled from the inlet and outlet of each reactor. The concentrations of glucose, pyruvate, ammonia, and lactate were measured using the hexokinase UV method, the pyruvate oxidase enzymatic method, the modified Fujii-Okuda method, and the lactate oxidase enzymatic method, respectively. The number of animal cells in reactor A is measured by a trypan blue exclusion test. As a comparative experiment, a system is also operated in which the culture medium of reactor A is circulated directly without passing through reactors B and C.

[0280] 2. Results 2-1. Removal of lactic acid and ammonia in reactor B The culture medium sent from reactor A contains high concentrations of lactic acid and ammonia produced by the metabolism of animal cells. When this culture medium passes through reactor B, the action of strain LA significantly reduces the lactic acid concentration. Ammonia is also assimilated as a nitrogen source, and its concentration decreases. Furthermore, because strain LA produces pyruvic acid from lactic acid, an increase in the pyruvic acid concentration is observed at the outlet of reactor B.

[0281] 2-2. Supply of glucose in reactor C When the culture medium treated in reactor B flows into reactor C, Strain GL produces glucose through photosynthesis and excretes it into the culture medium. This resupplies the glucose consumed by the animal cells, increasing the glucose concentration in the culture medium.

[0282] 2-3. Maintenance of growth of animal cells under continuous circulation culture In a comparative experiment, when the culture medium was circulated without microbial treatment, the growth of animal cells quickly stopped due to the depletion of nutrients and the accumulation of waste products, and a significant decrease in the number of viable cells was observed.

[0283] On the other hand, in the continuous circulation system of this example, the culture medium is regenerated by the action of reactors B and C. That is, the waste products lactic acid and ammonia are removed, and the nutrients glucose and pyruvic acid are supplied. By returning this regenerated culture medium to reactor A, the animal cells maintain a high survival rate over a long period of time and show stable proliferation. This result demonstrates that this system is an effective means for enabling the long-term stable culture of animal cells.

[0284] (Example 9) Circular Economy Application (4) In this example, as an example of a circular economy application, we demonstrate that a regeneration medium prepared by separately culturing a microorganism capable of utilizing lactate and a microorganism capable of excreting glucose and mixing the culture supernatants from these microorganisms is effective for culturing animal cells. This method has the advantage that the mixing ratio can be adjusted according to the processing capacity of each microorganism, and the composition of the regeneration medium can be optimized.

[0285] 1. Materials and Methods 1-1. Microorganisms and Animal Cells Used - Animal cells: Animal muscle cells (C2C12 cells) are used. - Lactic acid-utilizing microorganisms: Genetically modified cyanobacteria (hereinafter referred to as "Strain LA") that have the ability to efficiently utilize lactic acid are used. - Glucose-excreting microorganisms: Genetically modified cyanobacteria (hereinafter referred to as "Strain GL") that have the ability to excrete glucose produced by photosynthesis outside the cells are used.

[0286] 1-2. Animal cell culture (first cycle) and preparation of culture wastewater: C2C12 cells were cultured for 3 days (first cycle of circulation culture) using a medium consisting of DMEM with the same composition as in Example 7 supplemented with 10% fetal bovine serum (FBS). After culture, the culture medium was collected and cellular components were removed by centrifugation (1,000 × g, 5 minutes, 4°C) and sterilization with a filter (pore size: 0.2 μm, Thermo-Fischer Scientific). This was used in subsequent experiments as "animal cell culture wastewater."

[0287] 1-3. Individual Cultivation of Microorganisms and Recovery of Culture Supernatant The animal cell culture wastewater prepared in 1-2 is divided into two equal parts, and each part is used for the following microbial culture. (a) Treatment with Strain LA: Strain LA is inoculated into the animal cell culture wastewater and cultured for three days under conditions of light irradiation (150 μmol photons / m2 / s), 5% CO2 concentration, and 30°C. After culture, the bacterial cells are removed by centrifugation (8,000 × g, 10 minutes, 4°C), and the supernatant is collected. This is referred to as "treated supernatant A (lactic acid utilization)." (b) Treatment with Strain GL: Strain GL is inoculated into the animal cell culture wastewater and cultured for three days under conditions of light irradiation (150 μmol photons / m2 / s), 5% CO2 concentration, and 30°C. After the cultivation, the cells are removed by centrifugation (8,000×g, 10 minutes, 4° C.), and the supernatant is collected. This is designated as "treated supernatant B (glucose supply)."

[0288] 1-4. Preparation of regeneration medium The "treated supernatant A" and "treated supernatant B" obtained in 1-3 are mixed at a volume ratio of 1:1. After mixing, the mixture is sterilized using a filter (pore size: 0.2 μm, Thermo-Fischer Scientific) and used as the "regeneration medium."

[0289] 1-5. Animal Cell Culture (Second Cycle) C2C12 cells were cultured for two days using the following three types of media: (1) This Example: "Regeneration Medium" prepared in 1-4; (2) Comparative Example 1: Untreated "Animal Cell Culture Wastewater" obtained in 1-2; (3) Comparative Example 2: Freshly prepared DMEM medium. After culture, the number of viable cells in each well was measured using a trypan blue exclusion test. The concentrations of glucose, pyruvate, ammonia, and lactate in the medium before and after culture were also measured.

[0290] 2. Results 2-1. Changes in culture fluid components due to treatment with each microorganism When "animal cell culture wastewater" was treated with each microorganism, characteristic changes in the components were observed. In "treated supernatant A," the action of Strain LA significantly reduced the lactic acid concentration and increased the pyruvic acid concentration. On the other hand, in "treated supernatant B," the action of Strain GL significantly increased the glucose concentration and reduced the ammonia concentration as a nitrogen source.

[0291] 2-2. Animal cell culture using regenerated medium (second cycle) C2C12 cells cultured in Comparative Example 1 (untreated culture wastewater) showed a significant decrease in viable cell count due to nutrient depletion and waste accumulation. In contrast, when cultured in the "regenerated medium" of this example, cell viability remained high and significant cell proliferation was observed, although not equivalent to that of Comparative Example 2 (fresh medium).

[0292] These results indicate that by mixing "treated supernatant A" and "treated supernatant B" in an appropriate ratio, lactic acid, an inhibitor of animal cell growth, was removed while the essential nutrients glucose and pyruvate were supplied in a balanced manner.

[0293] This method suggests the possibility of precisely controlling the quality of the regenerated medium by flexibly changing the mixing ratio depending on the processing capacity of each microorganism and the desired culture conditions, and can be said to be an extremely practical approach.

[0294] (Note) As described above, the present disclosure has been illustrated using preferred embodiments thereof, but it is understood that the scope of the present disclosure should be interpreted solely by the claims. It is understood that the patents, patent applications, and other documents cited in this specification are incorporated by reference into this specification in their entirety as if the contents themselves were specifically set forth herein. This application claims priority to Japanese Patent Application No. 2024-152689, filed with the Japan Patent Office on September 4, 2024, the specification of which is incorporated by reference in its entirety into this application.

[0295] According to the present disclosure, it is possible to construct a recycling system that minimizes waste and environmental impact in the anticipated production of substances through cell or microbial culture. This makes it possible to develop a highly efficient, low-environmental-impact recycling cell or microbial culture system, which is expected to be applied in industrial fields such as cell or microbial agriculture, cultured food production, and recycling systems.

[0296]

Claims

1. Cells or microorganisms having a Calvin (CBB) cycle, wherein a mechanism for converting glucose-1-phosphate (G1P) to glucose has been introduced or enhanced.

2. The cell or microorganism according to claim 1, wherein the conversion from G1P to glucose is achieved without blocking the conversion of G1P to glycogen.

3. The cell or microorganism according to claim 1 or 2, further configured to attenuate or inhibit the conversion of glucose to glucose-6-phosphate (G6P).

4. The cell or microorganism according to any one of claims 1 to 3, wherein a mechanism for the release of glucose outside the cell or microorganism has been introduced or enhanced.

5. Cells or microorganisms having a Calvin (CBB) cycle, such as cyanobacteria, in which a mechanism for converting glycogen to glucose has been introduced or enhanced.

6. The cell or microorganism according to claim 5, further configured to inhibit the conversion of glucose to glucose-6-phosphate (G6P).

7. The cell or microorganism according to any one of claims 5 or 6, wherein a mechanism for the release of glucose outside the cell or microorganism has been introduced or enhanced.

8. Microorganisms having a Calvin (CBB) cycle, such as cyanobacteria, whose cells or microorganisms are configured to inhibit the conversion of glucose to glucose-6-phosphate (G6P).

9. The cell or microorganism according to claim 8, wherein the inhibition is achieved by inhibition of glucokinase activity.

10. The cell or microorganism according to claim 8, wherein the inhibition is achieved by disruption of the glucokinase active gene.

11. The cell or microorganism according to claim 9 or 10, wherein it does not have or has a weakened mechanism for the glucose to be released outside the cell or microorganism.

12. The cell or microorganism according to any one of claims 8 to 11, further comprising an enzyme introduced or enhanced to convert glucose-1-phosphate (G1P) to glucose, and / or an enzyme introduced or enhanced to convert glycogen to glucose.

13. The cell or microorganism according to any one of claims 8 to 12, wherein a mechanism for the release of glucose outside the cell or microorganism has been introduced or enhanced.

14. The cell or microorganism according to any one of claims 1 to 13, wherein the cell or microorganism does not contain invertase.

15. The cell or microorganism according to any one of claims 1 to 14, wherein the cell or microorganism includes cyanobacteria.

16. Combinations of glucose-requiring cells or microorganisms with cells or microorganisms capable of glucose production or efflux, systems and / or kits containing them.

17. The combination according to claim 16, a system and / or kit comprising the same, wherein the glucose-requiring cells or microorganisms include animal cells or microorganisms.

18. A method for producing cultured meat using the combination, system and / or kit according to claim 16 or 17, wherein the animal cells or microorganisms are cells or microorganisms used to produce cultured meat.

19. The cells or microorganisms having glucose production and excretion capacity include the cells or microorganisms described in any one of claims 1 to 15, the combination described in claim 16 or 17, a system and / or kit containing them, or the method described in claim 18.

20. The combination according to claim 16, a system and / or kit comprising the glucose-requiring cells or microorganisms, wherein the glucose-requiring cells or microorganisms include yeast.

21. The combination according to claim 16, a system and / or kit comprising the glucose-requiring cell or microorganism, wherein the glucose-requiring cell or microorganism includes Escherichia coli.

22. A method for producing a fermented food using the combination, system and / or kit according to claim 20 or 21, wherein the yeast or Escherichia coli is a cell or microorganism used to produce a fermented food.

23. The cells or microorganisms having glucose production and excretion capacity include the cells or microorganisms described in any one of claims 1 to 15, the combination described in claim 20 or 21, a system and / or kit containing them, or the method according to claim 22.

24. A method for producing a glucose-replenished culture medium, comprising adding a culture medium from which glucose has been consumed, and cells or microorganisms capable of producing or purging glucose.

25. Combinations, systems, and / or kits comprising cells or microorganisms having glucose production and excretion capabilities, cells or microorganisms having ammonia removal capabilities, and / or cells or microorganisms having lactate removal capabilities.

26. The combination according to claim 25, a system and / or kit comprising the same, wherein the cell or microorganism having glucose production and excretion ability, the cell or microorganism having ammonia removal ability, and the cell or microorganism having lactic acid removal ability are each independently the same cell or microorganism and / or different cells or microorganisms.

27. The combination according to claim 25 or 26, comprising cells or microorganisms capable of producing and exporting glucose and cells or microorganisms capable of removing lactate, and a system and / or kit comprising the combination.

28. A combination, a system and / or a kit comprising the combination described in any one of claims 25 to 27, wherein the cells or microorganisms capable of producing and exporting glucose and / or the cells or microorganisms capable of removing lactate have the ability to remove ammonia.

29. A method for producing substance X, comprising: 1) culturing a first cell or microorganism capable of producing substance X; 2) culturing a second cell or microorganism capable of glucose production and excretion using the culture medium after culturing in 1); 3) culturing together with a third cell or microorganism capable of ammonia removal in the culture medium of 2), and optionally together with a fourth cell or microorganism capable of lactic acid removal; 4) culturing the first cell or microorganism in the culture medium after culturing in 2) or 3); and 5) repeating 2) and / or 3) as necessary.

30. A method for culturing a first cell or microorganism, comprising: 1) a step of culturing a first cell or microorganism; 2) a step of culturing a second cell or microorganism having glucose production and excretion ability using the culture medium after culturing in 1); 3) a step of culturing together with a third cell or microorganism having ammonia removal ability in the culture medium of 2), and optionally together with a fourth cell or microorganism having lactic acid removal ability; 4) a step of culturing the first cell or microorganism in the culture medium after culturing in 2) or 3); and 5) a step of repeating 2) and / or 3) as necessary.

31. A method for culturing a first cell or microorganism, comprising: (a) culturing a first cell or microorganism; (b) culturing a second cell or microorganism having glucose production and efflux ability using the culture medium after culturing in (a); (c) culturing in step (b) together with a third cell or microorganism having ammonia removal ability and, if necessary, a fourth cell or microorganism having lactic acid removal ability; (d) culturing the first cell or microorganism in the culture medium after culturing in (c); and (e) repeating steps (b) and / or (c) as necessary, wherein at least two, preferably all, of the second cell or microorganism, the third cell or microorganism, and the fourth cell or microorganism are the same cell or microorganism possessing at least two, preferably all, of glucose production and efflux ability, ammonia removal ability, and lactic acid removal ability.

32. The culture method according to claim 31, wherein the second, third and / or fourth cell or microorganism is cyanobacteria.

33. The method of claim 32, wherein the cyanobacteria is a Synechococcus bacterium.

34. The method according to any one of claims 31 to 33, wherein the first cell or microorganism is an animal cell.

35. The method according to any one of claims 31 to 34, wherein the second, third and / or fourth cell or microorganism is genetically modified to exhibit at least two, preferably all, of the following: glucose production and excretion ability, ammonia removal ability, and lactic acid removal ability.

36. A method for culturing a first cell or microorganism, comprising: 1) a step of culturing a first cell or microorganism; 2) a step of culturing a second cell or microorganism having glucose production and excretion ability using the culture medium after culturing in 1); 3) a step of culturing together with a third cell or microorganism having ammonia removal ability and lactic acid removal ability in the culture medium of 2); 4) a step of culturing the first cell or microorganism in the culture medium after culturing in 2) or 3); and 5) a step of repeating 2) and / or 3) as necessary.

37. A method for culturing animal cells, comprising: (a) culturing first cells or microorganisms in a first culture tank; (b) supplying the culture medium after step (a) to a second culture tank containing second cells or microorganisms having ammonia removal ability and, if necessary, lactic acid removal ability, and bringing the second cells or microorganisms into contact with the first cells or microorganisms; (c) supplying the culture medium after step (b) to a third culture tank containing third cells or microorganisms having glucose production and excretion ability, and bringing the third cells or microorganisms into contact with the first cells or microorganisms; (d) refluxing the culture medium after step (c) to the first culture tank and continuing the cultivation of the first cells or microorganisms; and (e) continuously or intermittently repeating steps (b) to (d), characterized in that the second cells or microorganisms and the third cells or microorganisms are different cells or microorganisms from each other.

38. The culture method according to claim 37, characterized in that the first culture tank, the second culture tank, and the third culture tank are continuously connected via a tube.

39. The method of claim 37 or 38, wherein the second cell or microorganism is a cyanobacterium or Escherichia coli.

40. The method of any one of claims 37 to 39, wherein the third cell or microorganism is a cyanobacterium.

41. The culture method according to any one of claims 37 to 40, wherein the first cell or microorganism is an animal cell.

42. The method of any one of claims 37 to 41, wherein the second cell or microorganism and / or the third cell or microorganism are microorganisms genetically modified to perform their respective functions.

43. A method for culturing a first cell or microorganism, comprising the steps of: (a) culturing a first cell or microorganism and obtaining a culture medium thereafter; (b) culturing a second cell or microorganism having lactic acid removal ability using the culture medium obtained in (a), and obtaining a first treated supernatant from the culture; (c) culturing a third cell or microorganism having glucose production and excretion ability using the culture medium obtained in (a), and obtaining a second treated supernatant from the culture; (d) mixing the first treated supernatant obtained in (b) and the second treated supernatant obtained in (c) to prepare a regenerated culture medium; and (e) culturing the first cell or microorganism in the regenerated culture medium prepared in (d), characterized in that the second cell or microorganism and the third cell or microorganism are different cells or microorganisms from each other.

44. The method according to claim 43, characterized in that, in step (d) above, the first treated supernatant and the second treated supernatant are mixed in a predetermined volume ratio.

45. The culture method according to claim 43 or 44, wherein the second cell or microorganism is cyanobacteria or Escherichia coli.

46. ​​The method according to any one of claims 43 to 45, wherein the third cell or microorganism is a cyanobacterium.

47. The method according to any one of claims 43 to 46, wherein the first cell or microorganism is an animal cell.

48. The method according to any one of claims 43 to 47, wherein the second cell or microorganism and / or the third cell or microorganism are genetically modified microorganisms that perform their respective functions.

49. The method according to any one of claims 43 to 48, comprising the step of changing the mixing ratio of the second cell or microorganism and the third cell or microorganism according to the amount of each product.

50. The method according to claim 29 or 30, wherein the cell or microorganism having glucose production and excretion ability and the cell or microorganism having ammonia removal ability are the same cell or microorganism, or are different cells or microorganisms.

51. Combinations, systems, and / or kits comprising cells or microorganisms capable of producing substances, cells or microorganisms capable of glucose production and excretion, cells or microorganisms capable of ammonia removal and / or cells or microorganisms capable of lactic acid removal.

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