JP2021106614A - Method for producing organic acid - Google Patents

Abstract

To provide an environmentally friendly and effective method for producing organic acid that does not use a fossil fuel resource, and can effectively produce organic acid from synechocystis.SOLUTION: Culturing synechocystis in aqueous medium containing carbonate ion and/or bicarbonate ion, and recovering organic acid being intracellular metabolite. Furthermore, culturing synechocystis in which one or a plurality of kinds of enzymatic function selected from phosphoenolpyruvic acid carboxylase (PEP carboxylase), pyruvate ferredoxin oxidoreductase and phosphoglucomutase is enhanced in aqueous medium, and recovering organic acid being intracellular metabolite.SELECTED DRAWING: Figure 7

Description

The present invention relates to a method for producing an organic acid using microalgae.

This application claims the priority of Japanese application Japanese Patent Application Nos. 2015-78889 and 2015-228518, which are incorporated herein by reference.

Succinic acid belongs to organic acids and is used as a raw material for polymers such as polyester and polyamide. In addition, organic acids such as lactic acid and succinic acid are widely used as synthetic raw materials for foods, pharmaceuticals, and other chemical products. These organic acids are currently produced industrially from raw materials derived from fossil fuel resources, but _{the global environment is concerned about the depletion of fossil fuel resources in recent years and the increase in carbon dioxide (CO 2} ) in the atmosphere. From the background of the problem, production from sugar-based biomass such as starch and cellulose is expected as one of the renewable energies.

Microalgae are aquatic organisms that can use light to produce sugar energy from _{CO 2.} Since it is aquatic, it can avoid competition with food and land use, and is attracting attention as a promising biological system for bioenergy production. However, a common challenge in the production of bio-based chemicals is to enable mass production and reduce prices. Therefore, improvement in productivity efficiency is required, but one of the major problems when using microalgae is low cell density. A photosynthetic evaluation system for microalgae has been developed by the present inventors (Non-Patent Documents 1 and 2), and a method for increasing cell density by identifying and enhancing factors that determine the growth of microalgae has been sought. Various studies are underway.

Regarding the growth of microalgae, when each of the flavodiiron proteins Flv1 and Flv3 is deleted in Synechocystis sp. PCC6803, which is one of the cyanobacteria, under fluctuating light. It has been reported that cell proliferation and photosynthesis of Synechocystis speratus were inhibited (Non-Patent Document 3).

There is a disclosure of a method for producing succinic acid, which comprises a step of converting an organic raw material into succinic acid in the presence of a microorganism or a processed product thereof in an aqueous medium (Patent Document 1). Here, it is disclosed that the production rate of succinic acid is increased by setting the concentration of the alkali metal succinate in the aqueous medium within a specific range and then adding ammonia and / or ammonium salt. However, a method using microalgae has not been studied.

It is desired to develop a method for producing organic acids more efficiently by an environmentally friendly method without using fossil fuel resources. For example, little is known about extracellular production of microalgae and organic acids including succinic acid, and metabolites of microalgae have not been fully elucidated.

Hasunuma T et al., 2010, J Exp Bot 61: 1041-1051 Hasunuma T et al., 2013, J Exp Bot 64: 2943-2954 Allahverdiyeva Y et al., 2013, PNAS 110: 4111-4116

International Publication WO2013 / 69786

An object of the present invention is to provide an environment-friendly and effective method for producing an organic acid without using fossil fuel resources.

As a result of diligent studies to solve the above problems, the present inventors _{effectively recycle CO 2} and light energy from microalgae directly to recover organic acids, which are intracellular metabolites. We have found that it is possible to produce an organic acid, and as a result, it is possible to provide an environment-friendly and effective method for producing an organic acid, and completed the present invention. Specifically, Synechocystis is cultured in an aqueous medium containing carbonate ion and / or bicarbonate ion to recover an organic acid which is an intracellular metabolite. Further , phosphoenolpyruvate carboxylase (PEP carboxylase), pyruvate ferredoxin oxidoreductase, and one or more enzyme-enhanced cinecocystis selected from phosphoglucomutase are cultured in an aqueous medium, and cells are cultured. By recovering organic acids, which are internal metabolites.

That is, the present invention comprises the following.
1. 1. A method for producing an organic acid from Synechocystis , which comprises the following steps (A) and (B):
(A) Phosphoenol pyruvate carboxylase (PEP carboxylase), pyruvate ferredoxin oxidoreductase, and pyruvate ferredoxin oxidoreductase in an aqueous medium containing 0 to 500 mM carbonate ion and / or bicarbonate ion under dark and anaerobic culture conditions. A step of culturing one or more enzyme-enhanced cinecocystis selected from phosphoglucomase;
(B) A step of recovering the organic acid produced from Synechocystis of (A).
2. An aqueous medium with a carbonate ion and / or bicarbonate ion content of 0-500 mM is (1) filled with carbon dioxide and / or (2) sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, carbonate. The production method according to item 1 above, which is an aqueous medium by adding any one or more carbonates selected from magnesium.
3 . The production method according to item 2 above, wherein the filling of carbon dioxide is carried out until it becomes saturated in an aqueous medium.
4 . The method for producing an organic acid according to any one of the above items 1 to 3 , wherein the organic acid is an aliphatic dicarboxylic acid.
5 . The method for producing an organic acid according to any one of the above items 1 to 3 , wherein the organic acid is any one or more selected from succinic acid, lactic acid and acetic acid.
6 . A method for producing any one or more organic acids selected from succinic acid, acetic acid and lactic acid from Synechocystis , which comprises the following steps (a) and (b):
(A) A step of culturing Synechocystis with enhanced PEP carboxylase function in an aqueous medium containing 100 to 500 mM carbonate ion and / or bicarbonate ion in a dark place and under anaerobic culture conditions;
(B) A step of recovering any one or more organic acids selected from succinic acid, acetic acid and lactic acid produced from Synechocystis of the above (a).
7 . A method for producing any one or more organic acids selected from succinic acid, lactic acid and acetic acid from Synechocystis , which comprises the following steps (c) and (d):
(C) A step of culturing Synechocystis with enhanced pyruvate ferredoxin oxidoreductase function in an aqueous medium containing 100 to 500 mM carbonate ion and / or bicarbonate ion in a dark place and under anaerobic culture conditions;
(D) A step of recovering any one or more organic acids selected from succinic acid, lactic acid and acetic acid produced from Synechocystis of the above (c).
8 . A method for producing any one or more organic acids selected from succinic acid, lactic acid and acetic acid from Synechocystis , which comprises the following steps (e) and (f):
(E) A step of culturing Synechocystis with enhanced phosphoglucomutase function in an aqueous medium under dark and anaerobic culture conditions;
(F) A step of recovering any one or more organic acids selected from succinic acid, lactic acid and acetic acid produced from Synechocystis of the above (e).

The citric acid cycle in the microalgae can be activated by culturing the microalgae in an aqueous medium containing carbonate ion and / or bicarbonate ion. According to the method for producing an organic acid of the present invention, glycogen synthesized by photosynthesis of microalgae and a carbon source taken from an aqueous medium are effectively utilized to effectively produce an organic acid which is an intracellular metabolite. Can be done. Furthermore, by culturing microalgae with enhanced NADPH-O ₂ oxidoreductase function and / or rate-determining enzyme function in glycolysis from glycogen to citric acid cycle in an aqueous medium, it is more effectively organic. Can produce acid. As a result, it is possible to produce organic acids in an environmentally friendly and effective manner without using fossil fuel resources.

It is a figure which shows the production result of the organic acid by microalgae (Synechocystis spp.). (Example 1) It is a figure which shows the production result of the organic acid by microalgae (Synechocystis spp.). (Example 1) It is a figure which shows the production result of the organic acid by a microalga (Euglena). (Example 2) It is a figure which shows the production result of the organic acid by the microalgae (Chlamydomonas). (Example 3) It is a figure which shows the production result of the organic acid by the microalgae (Synechocystis spp.) Under the CO _{2 aeration condition.} (Example 4) It is a figure which shows the production pathway of succinic acid by microalgae. (Example 5) It is a figure which shows the production result of the organic acid by the microalga (Synechocystis) which overexpressed PEP carboxylase. (Example 5) It is a figure which shows the production method of PCC6803 (PGMox) which overexpressed PGM. (Example 6) It is a figure which shows the production result of the organic acid by PCC6803 (PGMox). (Example 6) It is a figure which shows the manufacturing method of PCC6803 (PFORox) which overexpressed PFO. (Example 7) It is a figure which shows the production result of the organic acid by PCC6803 (PFORox). (Example 7) It is a result figure which confirmed the production of lactic acid and succinic acid by culture of Flv3 overexpressing microalgae. (Example 8) It is a result figure which confirmed the intracellular accumulation amount of glycogen and ATP by culture of Flv3 overexpressing microalgae. (Example 9) It is a figure which shows the time-dependent change ^{of the 13} C labeling rate of a microalga (Synechocystis) intracellular metabolite. (Reference example 1) It is a figure which shows the intracellular metabolic pathway of microalgae (Synechocystis spp.). (Reference example 1) It is a result figure which measured the cell density and the intracellular accumulation amount of glycogen by the culture of microalgae with time. (Reference example 2) It is a result figure which measured the intracellular accumulation amount of acetic acid, lactic acid and succinic acid by culture of microalgae with time. (Reference example 3) It is a figure which shows the result of having investigated the intracellular metabolism profile by culture of microalgae. It is a figure which shows the result of having measured the intracellular accumulation amount of phosphoenolpyruvate, pyruvic acid, and lactic acid in the citric acid cycle with time. (Reference example 4). It is a figure which shows the result of having investigated the intracellular metabolism profile by culture of microalgae. It is a figure which shows the result of having measured the intracellular accumulation amount of phosphoenolpyruvate, malic acid, fumaric acid and succinic acid in the citric acid cycle with time. (Reference example 4) It is a figure which confirmed the use of ATP accompanying the production of organic acid by culture of microalgae. That is, it is a figure which shows the result of confirming the use of ATP by measuring the time course change of ATP, ADP and AMP. (Reference example 5)

The present invention relates to a method for producing an organic acid, and more particularly to a method for producing an organic acid from microalgae, which comprises the following steps.
(A) A step of culturing microalgae in an aqueous medium containing 20 to 2000 mM carbonate ion and / or bicarbonate ion;
(B) A step of recovering the organic acid produced from the microalgae of the above (A).

1. 1. Microalgae As used herein, the term "microalgae" refers to microorganisms that have chlorophyll and perform photosynthesis. _{Microalgae can immobilize CO 2} in the atmosphere by photosynthesis to synthesize sugars (eg, glycogen), while generating oxygen (O _{2 ) from water (H 2} O) (“oxygen-evolving photosynthesis”). Also called). The microalgae may have a unicellular morphology or may have a colony morphology (eg, filaments, sheets or balls). The microalgae may propagate in either the ocean or freshwater.

The microalgae of the present invention are either prokaryotic cyanobacteria (orchid algae) or eukaryotic organisms (eg, green algae, diatomaceae, whirlpool algae, red algae, placeno algae, Euglena algae, true eye spot algae, etc.). There may be. Examples of cyanobacteria (Cyanobacteria) include Synechocystis, Arthrospira, Spirulina, Anabaena, Synechococcus, Thermosynechococcus. Examples include the genus Stock (Nostoc), the genus Prochlorococcu, the genus Microcystis, and the genus Gloeobacter. Eukaryotic organisms include, for example, green algae such as Chlamydomonas, Chlorella, Dunaliella, Hematococcus, Volvox, Botryococcus; Genus (Rhizosolenia), Ketoceros (Chaetoceros), Cyclotella, Cylindrotheca, Navicula, Phaeodactylum, Thalassiosira, Fistulifera, etc. Kind; whirlpool algae such as Amphidinium, Symbiodinium; red algae such as Cyanidioschyzon, Phorphyridium; Ostreococcus, etc. Placeno algae; Euglena algae such as the genus Euglena; genuine eye spot algae such as the genus Nannochloropsis. For example, the microbial species of microalgae include Synechocystis sp. PCC6803, Synechococcus sp. PCC7002, and Arthrospira platensis (also referred to as "Spirulina"). Spirulina maxima, Spirulina subsalsa, Anabaena sp. PCC7120, Chlamydomonas reinhardtii, Chlamydomonas reinhardtii, Chlamydomonas chlamydomonas sp.・ Chlorella pyrenoidosa, Dunaliella salina, Dunaliella sp., Hematococcus pluvialis, Volvox carteri, Botriococcus braunii, Botry Cyclotella cryptica, Cylindrotheca fusiformis, Navicula saprophila, Phaeodactylum tricornutum, Phaeodactylum tricornutum, Thalassiosila pseudonana sp. Amphidinium sp., Symbiodinium microadriaticum, Synichocystis merolae, Porphyridium sp., Ostreococcus tauri , Euglena gracilis, Nanno chloropsis oculata) and the like.

The microalgae that can be used in the method of the present invention may be wild-type or may be microalgae modified to effectively produce organic acids. As the modification method, a method known per se or any method developed in the future can be applied, and the modification can be performed by, for example, a method such as gene recombination. Examples of such microalgae include transgenic microalgae capable of overexpressing an enzyme capable of enhancing the production of organic acids. For example, _{microalgae with enhanced NADPH-O 2} oxidoreductase function and / or rate-determining enzyme function in glycolysis from glycogen to the citric acid cycle can be utilized.

As used herein, the term "NADPH-O ₂ oxidoreductase" refers to the conversion of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to oxidized nicotinamide adenine dinucleotide phosphate (NADP ⁺ _{) and from O 2} to H. An enzyme that catalyzes the production of _{2 O.} In the photosynthetic photosystem I possessed by microalgae, the enzyme can convert NADPH generated by conversion from ^{NADP + by electron transfer from reduced ferredoxin to NADP +} again and generate water from oxygen.

Examples of the NADPH-O ₂ oxidoreductase of the present invention include flavodiiron protein (hereinafter, also referred to as “Flv”). Examples of flavoproteins include Flv3, Flv1, and the like, and Flv3 is particularly preferable. Examples of flavoproteins include those derived from the above-mentioned microalgae. Flv3 and Flv1 are involved in the _{photoreduction of O 2 in photosynthetic photosystem I.} For example, as Flv3, those derived from the above-mentioned microalgae can be mentioned. For example, the flv3 gene (sll0550: nucleotide sequence and amino acid sequence of the coding region shown in SEQ ID NOs: 1 and 2) derived from Synechocystis sp. PCC6803 can be used for transformation of microalgae. In the present specification, for example, Flv3 or Flv1 is specified by the amino acid sequence disclosed in the database for Flv3 or Flv1, and one or several amino acids in the disclosed amino acid sequence are deleted, substituted or added. It is also possible to specify an amino acid sequence having the above-mentioned amino acid sequence and having the enzyme activity required in the present invention. Alternatively, an amino acid sequence having 70% or more sequence identity with the amino acid sequence disclosed in the database for Flv3 or Flv1 and having the enzymatic activity required in the present invention can also be specified.

_{The gene related to NADPH-O 2} oxidoreductase used in the present invention refers to a gene capable of expressing the above-mentioned flavodi iron protein, specifically, a gene capable of expressing Flv3 or Flv1, and particularly preferably Flv3. A gene that can be expressed. Specifically, it may be a DNA having a base sequence disclosed in a database, or a gene that hybridizes with a DNA having a base sequence complementary to the DNA under stringent conditions. Stringent conditions are those disclosed, for example, on pages 1.101 to 1.104 of Molecular Cloning, 2nd. Ed., Cold Spring Harbor Laboratory 1989, New York. Such functional oligonucleotides are also included in the gene for _{NADPH-O 2} oxidoreductase of the present invention as long as they can hybridize to the DNA, resulting in sequence-specific binding.

Such genes are modeled on, for example, nucleic acids derived from DNA extracted from various organisms, various cDNA libraries, genomic DNA libraries, etc. using primers designed based on disclosed or known base sequences. For example, it can be obtained as a nucleic acid fragment by performing PCR amplification. Further, it can be obtained as a nucleic acid fragment by hybridization using a nucleic acid derived from the above library or the like as a template and a DNA fragment which is a part of a gene encoding the enzyme to be expressed or expressed in the present invention as a probe. Can be done. Alternatively, the gene may be synthesized as a nucleic acid fragment by various nucleic acid sequence synthesis methods known in the art such as a chemical synthesis method. The gene may be codon-optimized to optimize expression in the host microorganism. Codon optimization can be performed using any means or device commonly used by those skilled in the art.

As used herein, the term "microalgae with enhanced _{NADPH-O 2} oxidoreductase function" refers to "microalgae transformed to express or enhance the expression of _{NADPH-O 2 oxidoreductase".} As used herein, the term "expressing or enhancing the expression of _{NADPH-O 2} oxidoreductase" means that the expression of the gene related to _{NADPH-O 2 oxidoreductase is enhanced.} In the present specification, the form in which _{the expression of the gene related to NADPH-O 2 oxidoreductase is enhanced is the NADPH-O 2} oxide, as compared with the form before the modification for enhancing the expression of these genes is performed in the microalgae of the present invention. It is not particularly limited as long as it is in a form in which an increase in the production amount or activity of reductase is confirmed. Modifications that enhance gene expression may be made by methods known per se or by any method developed in the future. In an embodiment in which gene expression is enhanced, for example, any endogenous gene is linked under the control of a stronger promoter (which may be either a constitutive promoter or an inducible promoter). Examples include embodiments. In addition, an embodiment in which either an endogenous gene and / or an exogenous gene is additionally introduced can be mentioned. Any additionally introduced gene is preferably retained operably by a strong promoter, such as a constitutive promoter. The enhancement of expression is also referred to herein as "overexpression".

As the promoter, any promoter that functions in microalgae can be used. For example, if the microalgae are cyanobacteria (cyanobacteria), promoters such as sbDII, psbA3, psbA2, nirA, petE, nrsRS, nrsABCD, ndhF3, rbcL, rbcX, glnA, atp1, atp2, petF1 that can be derived from cyanobacteria. Can be mentioned.

An example of a plasmid vector for introducing the above gene into microalgae is the pTCP2031V vector. pTCP2031V vectors include the psbA2 (slr1311) promoter, as well as part of the coding region of slr2030 and slr2031 (as a platform for homologous recombination), and recombinant plasmids containing chloramphenicol-resistant cassettes (Satoh S et al.,, 2001, J. Biol. Chem. 276, 4293-4297; Horiuchi M et al., 2010, Biochem. J. 431, 135-140).

A recombinant construct, for example, an expression vector or a chromosome-integrated vector prepared as described above can be introduced into a host microalgae to prepare a transformed microalgae.

In many cases, a gene homologous recombination method can be used for transformation of transformed microalgae (particularly cyanobacteria). For gene homologous recombination methods, for example, the pTCP2031V vector can be preferably used. For the introduction of an expression vector or a replicable plasmid for transformation, a method known per se or any method developed in the future can be applied. For example, the electroporation method, the protoplast-PEG method, the microinjection method, the particle gun method, the calcium phosphate method, the lipofection method, the calcium ion method and the like can be mentioned.

The transformant is selected by using a selectable marker contained in the expression vector used for gene transfer or the chromosome-integrated vector. Antibiotics or drugs according to the selectable marker can be added to the medium suitable for each host microorganism. As such a selection medium, any medium suitable for the growth of microalgae can be used. For example, BG-11 agar medium (eg, described in Rippka R et al., 1979, J Gen Microbiol 111: 1-61: can be used for cyanobacteria); HSM agar medium and TAP agar medium (these are eg, eg. Fukuzawa et al., 2009, Agar Science, 67: 17-21: Can be used for eukaryotic organisms such as cyanobacteria). First, the transformant is selected based on this selectable marker, and then the transformant is selected by analyzing the expression of _{the target gene (that is, the NADPH-O 2 oxidoreductase gene) or its product.} Can be done. The NADPH-O ₂ oxidoreductase expression product can be confirmed by, for example, Western blotting.

Examples of rate-determining enzymes in glycolysis herein include phosphoenolpyruvate carboxylase (PEP carboxylase: PEPC), phosphoglucomutase (PGM: phosphoglucomutase), and pyruvate ferredoxin oxidoreductase (PFO). (See FIG. 6).

As used herein, PEP carboxylase refers to an enzyme that synthesizes oxaloacetate from phosphoenolpyruvate and CO _{2 in the C 4 pathway of the carbon dioxide fixation pathway.} As used herein, phosphoglucomutase (PGM) is an enzyme that mutually converts glucose-1-phosphate (G1P) and glucose-6-phosphate (G6P) in the metabolism of glycogen. In the present specification, pyruvate ferredoxin oxidoreductase (PFO) is classified as an oxidoreductase and is an enzyme that mutually converts pyruvate and acetyl-CoA in glycolysis.

2. Aqueous medium In the above, the "aqueous medium" refers to an aqueous solution used for seed culture and / or main culture. As will be described later, it is preferably an aqueous solution containing a nitrogen source, an inorganic salt and the like. Specifically, artificial or natural seawater or fresh water (for example, distilled water) may be used depending on the microalgae. For example, BG-11 medium (J Gen Microbiol 111: 1-61 (1979)); HSM medium and TAP medium (low temperature science, 67: 17-21 (2009)), Cramer-Myers medium (CM medium), etc. are used. can do. Specifically, a medium having the composition shown in Table 1 below may be used.

In order to efficiently carry out the production reaction of the organic acid in the culture, an organic raw material may be added to the medium as a carbon source. The organic raw material used for the main culture is not particularly limited as long as the microalcohol can be assimilated and proliferated, but usually, carbohydrates such as galactose, lactose, glucose, fructose, sucrose, saccharose, starch and cellulose; glycerol, Fructose sugars such as polyalcohols such as mannitol, xylitol, and ribitol are used and can be selected according to the target organic acid, and can be selected from general organic raw materials. For example, glucose, sucrose, or fructose is preferable, and glucose or sucrose is particularly preferable. Further, a starch saccharified solution containing the fermentable sugar, molasses and the like are also used, and the fermentable sugar may be a sugar solution extracted from plants such as sugar cane, sugar beet and sugar maple. These organic raw materials can be used alone or in combination. The aqueous medium can contain carbonate ion, bicarbonate ion or CO ₂ .

3. 3. Culturing conditions The culturing temperature of microalgae is usually 15 to 40 ° C, preferably 20 to 37 ° C, and more preferably 25 ° C to 35 ° C. The pH of the aqueous medium can be adjusted to any pH suitable for the growth of microalgae, such as pH 5-10, preferably pH 6-9, more preferably pH 6-8. The pH can be appropriately adjusted by adding sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, calcium hydroxide, magnesium hydroxide and the like.

Regarding the method for producing an organic acid of the present invention, microalgae can be pre-cultured in advance. For example, it can be cultured through steps such as (1) pre-culture, (2) pre-culture, (3) main culture (photoautotrophic), and (4) main culture (anaerobic, dark place). In the cultures (1) to (3), aeration culture can be performed, and the culture period is preferably 3 to 14 days, more preferably 3 to 5 days, respectively. For aerated culture, air or _{air mixed with CO 2} can be aerated through an aqueous medium.

In (3) main culture (photoautotroph) of the present specification, "photoautotroph" is used in a general sense, and microalgae _{produce sugar from CO 2} and water by photosynthesis, and this is used as an energy source. A mechanism for growth. The light irradiation conditions during photoautotrophic nutrition may be either natural light or artificial light, and the intensity thereof can be appropriately adjusted depending on the density of algae in the aqueous medium, the depth of the culture tank, and the like. For example, 30~2000μmol photons m ^-2 s ^-1, preferably, 30~1000μmol photons m ^-2 s ^-1, more preferably, 50~600μmol photons m ^-2 s natural light or artificial light of ^-1 can be used .. Within the above range, microalgae can photosynthesize and proliferate smoothly. The light irradiation may be continuous or periodic. For large outdoor cultures, light / dark cycles may be provided to minimize costs and avoid additional costs of artificial lighting.

In (4) main culture (anaerobic, dark place) of the present specification, "anaerobic culture" means culturing with the dissolved oxygen concentration in the solution kept low. In order to make the condition anaerobic, for example, the container is sealed and reacted without airflow, _{an inert gas such as nitrogen gas (N 2} ) is supplied for reaction, or an inert gas containing _{CO 2 is aerated.} Method can be used. In the process of anaerobic and dark conditions, the organic acid is discharged into the aqueous medium.

It is important that (4) main culture (anaerobic, dark place) of the present specification contains carbonate ion, bicarbonate ion and / or CO _2. The carbonate ion and bicarbonate ion preferably contain 20 to 2000 mM, preferably 20 to 500 mM, and more preferably 20 to 150 mM. The introduction of carbonate and / or bicarbonate into an aqueous medium is selected from (1) CO ₂ filling and / or (2) sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate. It can be done by adding any one or more carbonates. When _{filling with CO 2} , it can be filled until saturated. When CO ₂ is saturated, the carbonate ion concentration becomes 20 to 2000 mM.

4. Recovery of Organic Acids Produced from Microalgae Organic acids produced by the above methods are separated and purified from an aqueous medium by a method known per se or any separation or purification method developed in the future, if necessary. can do. Specifically, it is separated from microalgae and its products by ultrafiltration membrane separation, centrifugation, concentration, etc., then purified by a known method such as a column method or a crystallization method, and dried to form crystals. Examples include a method of collecting. In the present invention, the organic acid to be produced is not particularly limited, but is an intracellularly metabolized organic acid produced in the citric acid cycle, and specifically, an organic carboxylic acid. Examples of the organic carboxylic acid include succinic acid, lactic acid, acetic acid, fumaric acid, 2-ketoglutaric acid, malic acid, citric acid and the like. Among the organic acids, aliphatic dicarboxylic acids such as succinic acid, fumaric acid, 2-ketoglutaric acid and malic acid are preferable, and succinic acid is particularly preferable.

5. Production of Organic Acids By recovering organic acids produced from microalgae by the above method, it is possible to produce organic acids in an environmentally friendly and effective manner without using fossil fuel resources. That is, the photosynthesis of microalgae and the carbon source incorporated into the microalgae make it possible to produce organic acids from biomass, and it is possible to produce organic acids in an environmentally friendly and effective manner. The supply of carbonate ion and / or bicarbonate ion to the aqueous medium can be effectively utilized by utilizing, for example, _{CO 2 in} the atmosphere industrially emitted in the manufacturing process of electricity, steel and the like. It has an excellent effect on the natural environment in that it can effectively utilize _{CO 2} in the atmosphere. Further, according to the microalgae of the present invention, the aqueous medium used as biomass can utilize not only fresh water but also seawater, and can be stably and effectively utilized regardless of the depletion of water resources and the limit of cultivated land. be able to.

6. Citric Acid Cycle The present invention provides a method for activating the citric acid cycle in microalgae, which comprises culturing microalgae in an aqueous medium having a carbonate ion and / or bicarbonate ion content of 20 to 2000 mM. Also extends. According to the method of the present invention, the citric acid cycle is activated, and as a result, the production of organic acid, which is an intracellular metabolite, is enhanced.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

(Example 1) Production of organic acid by microalgae (Synechocystis sp. PCC6803) In this example, Synechocystis sp. PCC6803 (Synechocystis sp. PCC6803) glucose tolerance (GT) (Williams JGK, 1988, Methods Enzymol 167:) 766-778) (hereinafter referred to as "PCC6803 (GT)" in this example and each example) was used.

(1) Culture before before
PCC6803 (GT) colonies grown on BG-11 agar medium (BG-11 containing 1.5% Agar) were taken with a loop loop, added to an aqueous medium (70 mL), and 50 μmol under aeration conditions at pH 7.8. The cells ^{were cultured in photons m -2} s- ¹ at 30 ° C. for 4 to 5 days. _{Here, as an aqueous medium, a culture medium containing 17.6 mM NaNO 3} , 20 mM Hepes-KOH in a BG-11 liquid medium (Rippka R et al., 1J Gen Microbiol 111: 1-61 (1979)) was used. The algae density was measured by _{OD 750} using a Shimadzu UV mini spectrophotometer (ultraviolet-visible spectrophotometer: manufactured by Shimadzu Corporation). Post-culture OD ₇₅₀ ranged from 1 to 1.5. Ventilation means ventilation by air unless otherwise specified. The same applies to the following examples.

(2) pre-culture above the (1) PCC6803 (GT) was pre-preculture in, OD ₇₅₀ is added to the aqueous medium (0.99 mL) such that 0.1, aeration at pH 7.8, 50 [mu] mol photons m ^{- The} cells were cultured at 30 ° C. for 4 to 5 days at 2 s- ^1. Here, as an aqueous medium, a culture medium containing 17.6 mM NaNO ₃ , 20 mM Hepes-KOH in a BG-11 liquid medium was used. Post-culture OD ₇₅₀ ranged from 1 to 1.5.

(3) Main culture (light independent use)
PCC6803 (GT) pre-cultured in (2) above was added to an aqueous medium (70 mL) so that _{OD 750} became 0.4, and 120 μmol photons m ^-2 s- ¹ _{at pH 7.8 under CO 2 aeration conditions.} Incubated at 30 ° C. for 3 days. Here, as an aqueous medium, a culture medium containing 5 mM NH ₄ Cl and 50 mM Hepes-KOH in a BG-11 liquid medium was used. Post-culture OD ₇₅₀ was 6-7. After culturing, PCC6803 (GT) was recovered by filtration through a filter (polytetrafluoroethylene membrane, pore size 1 μm).

(4) Main culture (anaerobic / dark place)
The PCC6803 (GT) recovered in (3) above was added to an aqueous medium (10 mL) so _{that the OD 750 became 20.} Here, 100 mM Hepes-KOH (pH 7.8) was used as the aqueous medium. 0-500 mM sodium hydrogen carbonate (LVDS ₃ ) was added to the aqueous medium, and after 10 minutes of N ₂ aeration, culture was started under anaerobic conditions, and 72 hours later, the aqueous medium containing PCC6803 (GT) was recovered. ..

The aqueous medium recovered in (4) above was centrifuged at 14000 g and 4 ° C for 5 minutes, respectively, and the supernatant was recovered and filtered using a 0.45 μm pore size Mini-UniPrep (manufactured by GE Healthcare Japan Co., Ltd.). did. For the filtrate, the amount of succinic acid, lactic acid, acetic acid and glycogen produced was measured by high performance liquid chromatography (HPLC) column (Aminex HPX-87H; manufactured by Bio-Rad) and refractive index detector (RID-10A; Shimadzu Corporation). Measured by HPLC equipped with (manufactured by Mfg. Co., Ltd.). The production of fumalic acid, 2-ketoglutaric acid, malic acid, and citrate is described in Non-Patent Document 2 by extracting intracellular metabolites and capillary electrophoresis / mass spectrometry (CE / MS) system (CE: Liquid Chromatography-Triple Quadrupole Mass Spectrometry (LC / QqQ-MS) Controlled by Agilent G7100; MS, Agilent G6224AA LC / MSD TOF; Agilent Technologies) and Mass Hunter Workstation Data Acquisition Software (Agilent Technologies) It was analyzed and measured using a system (LC: Agilent 1200 series; MS, Agilent 6460 with Jet Stream Technology; manufactured by Agilent Technologies). As a result, the amount of organic acids (succinic acid, lactic acid, acetic acid, fumaric acid, 2-ketoglutaric acid, malic acid, citric acid) produced tended to increase as _{the LVDS 3 concentration increased (Fig. 1, Fig. 1,).} 2). It was confirmed that succinic acid, lactic acid and acetic acid can be effectively produced by PCC6803 (GT) by adjusting the LVDS _{3 concentration.}

(Example 2) Production of organic acid by microalgae (Euglena) In this example, Euglena (Euglena gracilis (NIES-48)) strain was used as a microbial species of microalgae. Hereinafter referred to as "NIES-48".

(1) Culture before before
Colonies of NIES-48 grown on Cramer-Myers agar medium (CM medium containing 1.5% Agar) were taken with a loop loop, added to an aqueous medium (70 mL), and 50 μmol photons m under aeration conditions at pH 7.8. Incubate at 30 ° C for 4-5 days at ^-2 s- ^1. Here, as the aqueous medium, a culture medium containing 7.5 mM (NH ₄ ) ₂ HPO ₄ in the CM medium having the composition shown in Table 2 was used. Post-culture OD ₇₅₀ ranged from 1 to 1.5.

(2) Pre-culture Add NIES-48 pre-cultured in (1) above _{to an aqueous medium (150 mL) so that the OD 750} becomes 0.1, and under aeration conditions at pH 3.5, 50 μmol photons m- ^2. The cells ^{were cultured in s-1} at 30 ° C. for 4 to 5 days. Here, as an aqueous medium, a culture medium containing 7.5 mM (NH ₄ ) ₂ HPO ₄ in a CM medium was used. Post-culture OD ₇₅₀ ranged from 1 to 1.5.

(3) Main culture (light independent use)
Add NIES-48 pre-cultured in (2) above to an aqueous medium (70 mL) so that _{OD 750 becomes 0.4, and under CO 2} aeration conditions at pH 3.5, at 120 μmol photons m ^-2 s- ¹ . The cells were cultured at 30 ° C. for 5 days. Here, as an aqueous medium, a culture medium containing 3 mM (NH ₄ ) ₂ HPO ₄ was used as a CM medium. Post-culture OD ₇₅₀ was 6-7. After culturing, NIES-48 was recovered by centrifugation (3000 g, 5 minutes, 4 ° C).

(4) Main culture (anaerobic / dark place)
The NIES-48 recovered in (3) above was added to an aqueous medium (10 mL) so _{that the OD 750 became 20.} Here, 100 mM Hepes-KOH (pH 7.8) was used as the aqueous medium. _{Then, LVDS 3} was added to the aqueous medium so as to be 0, 100, and 200 mM, and _{after N 2} aeration for 10 minutes, the culture was started at 30 ° C. with stirring at 170 rpm under anaerobic conditions, and the cells were cultured for 72 hours. .. The aqueous medium containing NIES-48 was recovered while maintaining the sealed state.

The aqueous medium containing NIES-48 cultured in (4) above was centrifuged at 14000 g and 4 ° C. for 5 minutes, respectively, and the supernatant was collected and filtered by the same method as in Example 1. Further, the production amounts of succinic acid, lactic acid, acetic acid and citric acid were measured by the same method as in Example 1. As a result, _{the production amounts of succinic acid, lactic acid and acetic acid increased at 100 mM of LVDS 3} concentration, and the production amounts of lactic acid and acetic acid tended to decrease at 200 mM (Fig. 3). On the other hand, the amount of citric acid produced tended to increase as the concentration of _{LVDS 3 increased (Fig. 3).} It was confirmed that Euglena can effectively produce organic acids (succinic acid, lactic acid, acetic acid, citric acid) by adjusting the LVDS _{3 concentration.}

(Example 3) Production of organic acid by microalgae (Chlamydomonas) In this example, Chlamydomonas reinhardtii strain was used as a microbial species of microalgae. Hereinafter referred to as "C. reinhardtii".

(1) Culture before before
C. reinhardtii colonies grown on TAP agar medium (TAP medium containing 1.5% Agar) were taken with a loop loop, added to an aqueous medium (70 mL), and 100 μmol photons m under _{CO 2 aeration conditions at pH 7.8.} Incubate at 30 ° C for 4-5 days at ^-2 s- ^1. Here, MB6N medium was used as the aqueous medium. Post-culture OD ₇₅₀ was 2.5-3.5. The compositions of TAP medium and MB6N medium are shown in Table 3.

(2) Pre-culture Add C. reinhardtii pre-cultured in (1) above to an aqueous medium (150 mL) so _{that the OD 750 becomes 0.1, and under CO 2} aeration conditions at pH 7.8, 120 μmol photons m. Incubate at 30 ° C for 3 days at ^-2 s- ^1. Here, MB6N medium was used as the aqueous medium. Post-culture OD ₇₅₀ was 2.5-3.5.

(3) Main culture (light independent use)
C. reinhardtii precultured in (2) above was added to an aqueous medium (70 mL) so that _{OD 750 became 0.4, and under CO 2} aeration conditions at pH 7.8, at 120 μmol photons m ^-2 s- ¹ . The cells were cultured at 30 ° C. for 5 days. Here, MB6N medium was used as the aqueous medium. Post-culture OD ₇₅₀ was 3.5-4.5. After culturing, C. reinhardtii was recovered by centrifugation (14000 g, 5 minutes, 4 ° C).

(4) Main culture (anaerobic / dark place)
The C. reinhardtii recovered in (3) above was added to an aqueous medium (10 mL) so _{that the OD 750 became 20.} Here, 50 mM Hepes-KOH (pH 7.8) was used as the aqueous medium. _{In addition, LVDS 3} was added so as to be 0 and 100 mM, and _{after N 2} aeration for 10 minutes, the culture was started at 30 ° C. with stirring at 170 rpm under anaerobic conditions, and the cells were cultured for 72 hours. The aqueous medium containing C. reinhardtii was recovered while maintaining the sealed state.

The aqueous medium recovered in (4) above was centrifuged at 14000 g and 4 ° C for 5 minutes, respectively, and the supernatant was recovered by the same method as in Example 1. 0.45 μm pore size Mini-UniPrep (GE Healthcare Japan Co., Ltd.) Filtered using (manufactured by the company). With respect to the filtrate, the production amounts of succinic acid, lactic acid and acetic acid were further measured by the same method as in Example 1. As a result, the production amounts of succinic acid, lactic acid, and acetic acid increased when _{the LVDS 3 concentration was 100 mM (Fig. 4).} It was confirmed that Chlamydomonas can effectively produce organic acids (succinic acid, lactic acid, acetic acid) by adjusting the LVDS _{3 concentration.}

(Example 4) Production of organic acid by microalgae (Synechocystis) In this example, the production of organic acid when _{microalgae were cultured under saturated CO 2 conditions was confirmed.} In this example, PCC6803 (GT) was used as in Example 1.

(1) Culture before before
PCC6803 (GT) colonies grown on BG-11 agar medium (BG-11 containing 1.5% Agar) were taken with a loop loop, added to an aqueous medium (70 mL), and 50 μmol under aeration conditions at pH 7.8. The cells ^{were cultured in photons m -2} s- ¹ at 30 ° C. for 4 to 5 days. _{Here, as an aqueous medium, a culture medium containing 17.6 mM NaNO 3} , 20 mM Hepes-KOH in a BG-11 liquid medium (Rippka R et al., 1J Gen Microbiol 111: 1-61 (1979)) was used. The algae density was measured by _{OD 750} using a Shimadzu UV mini spectrophotometer (ultraviolet-visible spectrophotometer: manufactured by Shimadzu Corporation). Post-culture OD ₇₅₀ ranged from 1 to 1.5. Ventilation means ventilation by air unless otherwise specified. The same applies to the following examples.

(2) pre-culture above the (1) PCC6803 (GT) was pre-preculture in, OD ₇₅₀ is added to the aqueous medium (0.99 mL) such that 0.1, aeration at pH 7.8, 50 [mu] mol photons m ^{- The} cells were cultured at 30 ° C. for 4 to 5 days at 2 s- ^1. Here, as an aqueous medium, a culture medium containing 17.6 mM NaNO ₃ , 20 mM Hepes-KOH in a BG-11 liquid medium was used. Post-culture OD ₇₅₀ ranged from 1 to 1.5.

(3) Main culture (light independent use)
PCC6803 (GT) pre-cultured in (2) above was added to an aqueous medium (70 mL) so that _{OD 750} became 0.4, and 120 μmol photons m ^-2 s- ¹ _{at pH 7.8 under CO 2 aeration conditions.} Incubated at 30 ° C. for 3 days. Here, as an aqueous medium, a culture medium containing 5 mM NH ₄ Cl and 50 mM Hepes-KOH in a BG-11 liquid medium was used. Post-culture OD ₇₅₀ was 6-7. After culturing, PCC6803 (GT) was recovered by filtration through a filter (polytetrafluoroethylene membrane, pore size 1 μm).

(4) Main culture (anaerobic / dark place)
The PCC6803 (GT) recovered in (3) above was added to an aqueous medium (10 mL) so _{that the OD 750 became 20.} Here, 100 mM Hepes-KOH (pH 7.8) was used as the aqueous medium. The _{cells were cultured under conditions of aeration of N 2} for 72 hours and CO ₂ for 72 hours, and an aqueous medium containing PCC6803 (GT) was recovered. Under these conditions, N ₂ and CO ₂ were saturated in the liquid medium.

The aqueous medium recovered in (4) above was centrifuged at 14000 g and 4 ° C for 5 minutes, respectively, and the supernatant was recovered and filtered using a 0.45 μm pore size Mini-UniPrep (manufactured by GE Healthcare Japan Co., Ltd.). did. For the filtrate, the amount of succinic acid, lactic acid, acetic acid and glycogen produced was measured by high performance liquid chromatography (HPLC) column (Aminex HPX-87H; manufactured by Bio-Rad) and refractive index detector (RID-10A; Shimadzu Corporation). Measured by HPLC equipped with (manufactured by Mfg. Co., Ltd.). The production of fumalic acid, 2-ketoglutaric acid, malic acid, and citrate is described in Non-Patent Document 2 by extracting intracellular metabolites and capillary electrophoresis / mass spectrometry (CE / MS) system (CE: Liquid Chromatography-Triple Quadrupole Mass Spectrometry (LC / QqQ-MS) Controlled by Agilent G7100; MS, Agilent G6224AA LC / MSD TOF; Agilent Technologies) and Mass Hunter Workstation Data Acquisition Software (Agilent Technologies) It was analyzed and measured using a system (LC: Agilent 1200 series; MS, Agilent 6460 with Jet Stream Technology; manufactured by Agilent Technologies). As a result, the production amounts of succinic acid and acetic acid tended to increase under the culture conditions of _{CO 2 (Fig. 5).}

(Example 5) Production of organic acid by PEP carboxylase overexpressing microalgae In this example, production of organic acid by PEP carboxylase overexpressing microalgae was confirmed. In this example, PCC6803 (GT) shown in Example 1 was used to prepare a PEP carboxylase overexpressing strain PCC6803 (PEPox) by a gene recombination operation. The method for producing PCC6803 (PEPox) is as follows. The production pathway of succinic acid by microalgae is shown in FIG. 6, and the PEP carboxylase action point is shown.

5-1. The rbcL terminator and a part of the coding region downstream of the slr0168 region use the oligonucleotides shown in SEQ ID NOs: 1 and 2 and the oligonucleotides shown in SEQ ID NOs: 3 and 4 as primer sets from the genomic DNA extracted from PCC6803 (GT). And amplified by PCR. The obtained amplified fragment was inserted into PstI and HindIII digestion pBluescript II SK (+) (Agilent Technologies, Palo Alto, CA) using the In-Fusion HD Cloning Kit (obtained from Takara Bio Inc., manufactured by Clonetech). , PBluescript-TrbcL-slr0168 was obtained.

SEQ ID NO: 3: 5'-CCTCTAGAGTCGACCTGCAGGTTACAGTTTTGGCAATTAC-3'
SEQ ID NO: 4: 5'-GCCAGCCCCAACACCTGACGCGTTTCCCCACTTAGATAAAAAATCC-3'
SEQ ID NO: 5: 5'-TCTAAGTGGGGAAACGCGTCAGGTGTTGGGGCTGGC-3'
SEQ ID NO: 6: 5'-TGATTACGCCAAGCTTCTAAGTCAGCGTAAATCTGACAATG-3'

5-2. The canamycin resistance cassette and rbcL promoter used the oligonucleotides shown in SEQ ID NOs: 5 and 6 and the oligonucleotides shown in SEQ ID NOs: 7 and 8 as primer sets from the genomic DNA of pCRII-TOPO (Invitrogen, Carlsbad, CA) and PCC6803 (GT). And amplified by PCR. The obtained amplified fragment was inserted into XhoI and XbaI digestion pBluescript-TrbcL-slr0168 using the In-Fusion HD Cloning Kit (obtained from Takara Bio Inc., manufactured by Clonetech), and pBluescript-Km ^r -PrbcL-TrbcL- Obtained slr0168.

SEQ ID NO: 7: 5'-CGGGCCCCCCCCTCGAGCCGGAATTGCCAGCTGGGGC-3'
SEQ ID NO: 8: 5'-TGGACTTTCTAATTAGAGCGGCCGCTCAGAAGAACTCGTCAAGA-3'
SEQ ID NO: 9: 5'-TCTTGACGAGTTCTTCTGAGCGGCCGCTCTAATTAGAAAGTCCA-3'
SEQ ID NO: 10: 5'-CCGGGGATCCTCTAGACATATGGGTCAGTCCTCCAT-3'

5-3. A part of the coding region upstream of the slr0168 region was amplified by PCR using the oligonucleotides shown in SEQ ID NOs: 9 and 10 as a primer set from the genomic DNA extracted from PCC6803 (GT). ^{The obtained amplified fragment was inserted into KpnI and XhoI digestion pBluescript-Km r} -PrbcL-TrbcL-slr0168 using the In-Fusion HD Cloning Kit (obtained from Takara Bio Inc., manufactured by Clonetech), and pBluescript-slr0168- Km ^r -PrbcL-TrbcL-slr0168 was obtained.

SEQ ID NO: 11: 5'-TATAGGGCGAATTGGGTACCATGACTATTCAATACACCCCCCTAG-3'
SEQ ID NO: 12: 5'-TACCGTCGACCTCGAGCACCAGACCAAAGCCGGGAATTTC-3'

5-4. CACATG digested with AatII and EcoRI was replaced at the NdeI site (CATATG) of pUC19 (Takara Bio), and synthetic DNA was inserted. After digesting pBluescript-slr0168-KMR-PrbcL-TrbcL-slr0168 with KpnI and HindIII, the fragment containing slr0168 was inserted into the KpnI / HindIII site of the prepared pUC19 vector to prepare pSKrbcL-slr0168.

5-5. Ppc (sll0920) encoding PEP carboxylase was amplified by PCR from genomic DNA extracted from PCC6803 (GT) using the oligonucleotides shown in SEQ ID NO: 11 and SEQ ID NO: 12 as primer sets. The obtained amplified fragment was inserted into NdeI / SalI digestion pSKtrc-slr0168 using an In-Fusion HD Cloning Kit (manufactured by Clontech, obtained from Takara Bio Inc.) to obtain pSKtrc-slr0168 / sll0920.

SEQ ID NO: 13: 5'-AGGAAACAGACCCATATGAACTTGGCAGTTCCTGC -3'
SEQ ID NO: 14: 5'-AACCTGCAGGTCGACTCAACCAGTATTACGCA -3'

5-6. PCC6803 (GT) was transformed with the obtained plasmid pSKtrc-slr0168 / sll0920 vector (including the sll0920 coding region). As a control, transformation was performed with an empty vector (plasmid pSKtrc-slr0168 vector containing no sll0920 coding region). PCC6803 (GT) transformed by the above and prepared to overexpress sll0920 is referred to as PCC6803 (PEPox). The untransformed wild-type PCC6803 (GT) is referred to as PCC6803 (VC).

5-7. The prepared PCC6803 (PEPox) and PCC6803 (VC) were cultured based on the same culture conditions as in Examples 1 (1) to (3). In the main culture under the anaerobic and dark conditions of (4), 0 or 100 mM LVDS ₃ was added to the aqueous medium, N _{2 was} aerated for 10 minutes, the culture was started under the anaerobic conditions, and 72 hours later. Aqueous media containing PCC6803 (PEPox) or PCC6803 (VC) were recovered. With respect to the recovered aqueous medium, the amounts of succinic acid, lactic acid and acetic acid produced were measured by the same method as in Example 1. As a result, succinic acid and acetic acid, with or without NaHCO ₃ addition, PEP carboxylase over to expressing strain PCC6803 of it is showed high ability to produce (PEPox), producing ability by the presence or absence of lactic acid NaHCO ₃ added It was confirmed that it fluctuates to (Fig. 7).

(Example 6) Production of organic acid by PGM overexpressing microalgae In this example, the production of organic acid by PGM overexpressing microalgae was confirmed. In this example, PCC6803 (GT) shown in Example 1 was used to prepare a PGM overexpressing strain PCC6803 (PGMox) by a gene recombination operation. The method for producing PCC6803 (PGMox) is shown in FIG. The production pathway of succinic acid by microalgae is shown in FIG. 6, and the PGM action point is shown.

5-1. Of Example 5 above. ~ 5-3. PBluescript-slr0168-Km ^r -PrbcL-TrbcL-slr0168 was prepared by the same method as above. PCC6803 (GT) prepared by transforming PCC6803 (GT) with pSKtrc-slr0168 / sll0726 so that sll0726 is overexpressed is referred to as PCC6803 (PGMox) in this example. In addition, untransformed wild-type PCC6803 (GT) was used in Examples 5-6. It is called PCC6803 (VC) as well.

The prepared PCC6803 (PGMox) and PCC6803 (VC) were cultured based on the same culture conditions as in Examples 1 (1) to (3). In the main culture under the anaerobic and dark conditions of (4), _{after 10 minutes of N 2} aeration, the culture was started under anaerobic conditions, and 72 hours later, an aqueous medium containing PCC6803 (PGMox) or PCC6803 (VC) was used. Recovered. With respect to the recovered aqueous medium, the amounts of succinic acid, lactic acid and acetic acid produced were measured by the same method as in Example 1. As a result, the PGM overexpressing strain PCC6803 (PGMox) showed higher production ability in all cases (Fig. 9).

(Example 7) Production of organic acid by PFOR overexpressing microalgae In this example, the production of organic acid by PFOR overexpressing microalgae was confirmed. In this example, using the PCC6803 (GT) shown in Example 1, a strain PCC6803 (PFORox) overexpressing the Synechococcus sp. PCC7002-derived PFOR gene (A1443) was prepared by genetic recombination. The method for producing PCC6803 (PFORox) is shown in FIG. The production pathway of succinic acid by microalgae is shown in FIG. 6, and the action point of PFO is shown.

5-1. Of Example 5 above. ~ 5-3. PBluescript-slr0168-Km ^r -PrbcL-TrbcL-slr0168 was prepared by the same method as above. PCC6803 (GT) was transformed with pSKtrc-PFOR (A1443). PCC6803 (GT) prepared to overexpress PFOR (A1443) is referred to as PCC6803 (PFORox) in this example. In addition, untransformed wild-type PCC6803 (GT) was used in Examples 5-6. It is called PCC6803 (VC) as well.

The prepared PCC6803 (PFORox) and PCC6803 (VC) were cultured based on the same culture conditions as in Examples 1 (1) to (3). In the main culture under the anaerobic and dark conditions of (4), 0 or 100 mM LVDS ₃ was added to the aqueous medium, N _{2 was} aerated for 10 minutes, the culture was started under the anaerobic conditions, and 72 hours later. Aqueous media containing PCC6803 (PFORox) or PCC6803 (VC) were recovered. With respect to the recovered aqueous medium, the amounts of succinic acid, lactic acid and acetic acid produced were measured by the same method as in Example 1. As a result, succinate NaHCO ₃ shows the production ability higher PFOR or without added overexpressing strain PCC6803 (PFORox), to be produced higher production than by the addition of NaHCO ₃ was confirmed. High production capacity of lactic acid _{was confirmed by the addition of LVDS 3} , and the PFOR overexpressing strain PCC6803 (PFORox) showed higher production capacity (Fig. 11A). Cell concentration was also measured by _{OD 750 72 hours after photoculture.} As a result, there was almost no difference in cell density (Fig. 11B).

(Example 8) Production of organic acid by Flv3 overexpressing microalgae 8-1. Preparation of Flv3 overexpressing microalgae In this example, the Flv3 overexpressing strain PCC6803 (Flv3ox) was prepared by gene recombination using the PCC6803 (GT) shown in Example 1. The manufacturing method of PCC6803 (Flv3ox) is as follows.

Flv3 (sll0550) from genomic DNA extracted from PCC6803 (GT) using a forward primer (5'-GGAATTATAACCATAATGTTCACTACCCCCCTCCC-3': SEQ ID NO: 15) and a reverse primer (5'-GGCATGGAGGACATATTAGTAATAATTGCCGACTT-3': SEQ ID NO: 16). The coding region was amplified by PCR.

The obtained 1.7 kb amplified fragment was inserted into an NdeI digested pTCP2031V vector using an In-Fusion Cloning Kit (obtained from Takara Bio Inc., manufactured by Clonetech). The pTCP2031V vector is a recombinant plasmid containing the psbA2 (slr1311) promoter and part of the coding region of slr2030 and slr2031 (as a platform for homologous recombination), and a chloramphenicol-resistant cassette (Satoh S et al., 2001, J. Biol. Chem. 276, 4293-4297; Horiuchi M et al., 2010, Biochem. J. 431, 135-140).

PCC6803 (GT) was transformed with the obtained plasmid pTCP2031V vector (including the flv3 coding region) to prepare PCC6803 (Flv3ox). As a control, transformation with an empty vector (plasmid pTCP2031V vector containing no flv3 coding region) was performed. The untransformed wild-type PCC6803 (GT) is referred to as PCC6803 (VC).

8-2. Examination of organic acid production process In this example, the organic acid production process was examined using PCC6803 (Flv3ox).

(1) Culture before before
PCC6803 (Flv3ox) colonies grown on BG11 agar medium (BG11 containing 1.5% Agar) were taken with a loop loop, added to an aqueous medium (70 mL), and 50 μmol photons m ^{-2 under aeration conditions at pH 7.8. Incubate} at 30 ° C. for 4-5 days at s-1. _{Here, as an aqueous medium, a culture medium containing 17.6 mM NaNO 3} , 20 mM Hepes-KOH in a BG-11 liquid medium (Rippka R et al., 1979, J Gen Microbiol 111: 1-61) was used. Cell density was measured by _{OD 750} using a Shimadzu UV mini spectrophotometer (ultraviolet-visible spectrophotometer: manufactured by Shimadzu Corporation). Post-culture OD ₇₅₀ ranged from 1 to 1.5.

(2) pre-culture above the (1) PCC6803 were pre-preculture in (Flv3ox), OD ₇₅₀ is added to the aqueous medium (0.99 mL) such that 0.1, aeration at pH 7.8, 50 [mu] mol photons m ^{- Incubate} at 30 ° C for 4-5 days at ² s-1. Here, as an aqueous medium, a culture medium containing 17.6 mM NaNO ₃ , 20 mM Hepes-KOH in a G-11 liquid medium was used. Post-culture OD ₇₅₀ ranged from 1 to 1.5.

(3) Main culture (light independent use)
PCC6803 (Flv3ox) pre-cultured in (2) above was added to an aqueous medium (70 mL) so that _{OD 750} became 0.4, and 120 μmol photons m ^-2 s _{under 1% CO 2 aeration conditions at pH 7.8.} ^Incubate at -1 at 30 ° C for 3 days. Here, as an aqueous medium, a culture medium containing 5 mM NH ₄ Cl / NaNO ₃ , 50 mM Hepes-KOH in a BG-11 liquid medium was used. Post-culture OD ₇₅₀ was 6-7. After culturing, the cells were collected by centrifugation (14000 g, 5 minutes, 4 ° C).

(4) Main culture (anaerobic / dark place)
Add PCC6803 (Flv3ox) cultured in (3) above to an aqueous medium (10 mL) so that _{OD 750} becomes 20, stir at 170 rpm after 10 minutes under anaerobic conditions with _{N 2 aeration, and incubate at 30 ° C.} Started. Here, 50 mM Hepes-KOH was used as the aqueous medium. The aqueous medium was recovered while maintaining the sealed state, OD / pH was measured, and organic acid analysis, metabolome analysis, and glycogen analysis were performed.

8-3. Production of organic acids by culturing PCC6803 (Flv3ox) The production amounts of succinic acid and lactic acid were confirmed for PCC6803 (Flv3ox) cultured under the anaerobic and dark conditions of 8-2 above and PCC6803 (VC) as a comparative example. .. _{Ammonium chloride (NH 4} Cl) was used as the nitrogen source in the aqueous medium.
The PCC6803 (Flv3ox) culture solution and the culture solution of the comparative example cultured in 8-2 (4) above were centrifuged at 14000 g and 4 ° C. for 5 minutes, respectively, and the supernatant was collected. Filtered using a 0.45 μm pore size Mini-uniPrep (manufactured by GE Healthcare Japan Co., Ltd.), HPLC column (Aminex HPX-87H; manufactured by Bio-Rad) and refractive index detector (RID-10A; Shimadzu Corporation) The production amounts of succinic acid and lactic acid were measured by using high performance liquid chromatography (HPLC) (manufactured by Shimadzu Corporation) equipped with (manufactured by Shimadzu Corporation). As a result, PCC6803 (Flv3ox) overexpressing Flv3 showed high production of both succinic acid and lactic acid (Fig. 12).

(Example 9) Intracellular glycogen and ATP obtained by culturing PCC6803 (Flv3ox)
The amount of intracellular glycogen and ATP accumulated was measured for PCC6803 (Flv3ox) cultured under the anaerobic and dark conditions of Example 8 (8-2) and PCC6803 (VC) as a comparative example. _{Ammonium chloride (NH 4} Cl) was used as the nitrogen source in the aqueous medium. As a result, PCC6803 (Flv3ox) overexpressing Flv3 showed a high accumulation amount for both glycogen and ATP (Fig. 13).

(1) Glycogen quantification
A 5 mg dry weight PCC6803 (Flv3ox) was collected using a 1 μm pore size polytetrafluoroethylene (PTEE) membrane filter (Omnipore; manufactured by Millipore). Immediately after filtration, cells were washed with 20 mM precooled ammonium carbonate. Immediately, PCC6803 (Flv3ox) on the membrane filter was frozen in liquid nitrogen and lyophilized in a lyophilizer (Labconco). As previously reported (Izumi Y et al., 2013, J Chromatogr B Analyt Technol Biomed Life Sci 930: 90-97), after extracting glycogen from cells, size exclusion HPLC column (OHpak SB-806M HQ; manufactured by Showa Denko Co., Ltd.) The glycogen content was measured using high performance liquid chromatography (HPLC) (manufactured by Shimadzu Corporation) equipped with a refractive index detector (RID-10A; manufactured by Shimadzu Corporation).

(2) ATP quantification
As described in Non-Patent Document 2, the amount of ATP is determined by extracting intracellular metabolites and capillary electrophoresis / mass spectrometry (CE / MS) system (CE: Agilent G7100; MS, Agilent G6224AA LC / MSD TOF; Agilent. Liquid Chromatography-Triple Quadrupole Mass Spectrometry (LC / QqQ-MS) System (LC: Agilent 1200 series; MS, Agilent 6460 with) controlled by Technologies) and Mass Hunter Workstation Data Acquisition software (Agilent Technologies) It was analyzed and measured using Jet Stream Technology (manufactured by Agilent Technologies).

(Reference Example 1) Confirmation of intracellular metabolites in microalgae (Synechocystis) In this reference example, PCC6803 (GT) shown in Example 1 was used as microalgae, and the ¹³ C labeling rate of intracellular metabolites changed over time. It was confirmed. PCC6803 (GT) was suspended in 100 mM Hepes-KOH aqueous medium (pH 7.8) containing 100 mM ^{NaH 13} CO ₃ , and cultured under anaerobic conditions after _{N 2 aeration.} Twenty-four hours after labeling, a 5 mg dry weight PCC6803 (GT) was collected using a 1 μm pore size polytetrafluoroethylene (PTEE) membrane filter (Omnipore; manufactured by Millipore). Immediately after filtration, cells were washed with 20 mM precooled ammonium carbonate. Immediately, PCC6803 (GT) on the membrane filter was frozen in liquid nitrogen and lyophilized in a lyophilizer (Labconco). As described in Non-Patent Document 2, intracellular metabolites are extracted, and a capillary electrophoresis / mass spectrometry (CE / MS) system (CE: Agilent G7100; MS, Agilent G6224AA LC / MSD TOF; manufactured by Agilent Technologies) And Liquid Chromatography Controlled by Mass Hunter Workstation Data Acquisition Software (Agilent Technologies) -Triple Quadruple Mass Spectrometry (LC / QqQ-MS) System (LC: Agilent 1200 series; MS, Agilent 6460 with Jet Stream Technology; It was analyzed and measured using Agilent Technologies. The result is shown in FIG. The intracellular metabolic pathway is shown in FIG. _{From the above results, it was confirmed that LVDS 3} incorporated into microalgae was catabolized into organic acids by anaerobic culture of PCC6803 (GT).

(Reference Example 2) Confirmation of cell density and glycogen accumulation amount by culturing microalgae In this reference example, PCC6803 (GT) was used as the microalgae, and the cell density when cultured under the anaerobic and dark conditions of Example 8. Changes and glycogen accumulation were confirmed. _{Ammonium chloride (NH 4} Cl) or sodium nitrate (NaNO ₃ ) was used as the nitrogen source in the aqueous medium.

(1) Cell Density The cultured PCC6803 (GT) was measured for cell density by _{OD 750} using a Shimadzu UV mini spectrophotometer (ultraviolet visible spectrophotometer: manufactured by Shimadzu Corporation).
As a result, there was almost no difference in cell density due to the difference in nitrogen source (Fig. 16A).

(2) Glycogen Quantification Glycogen was measured by the same method as in Example 9). As a result, there was almost no difference in the intracellular accumulation amount of glycogen due to the difference in nitrogen source (Fig. 16B).

(Reference Example 3) Quantification of organic acids by culturing microalgae In this reference example, the production of acetic acid, lactic acid and succinic acid was confirmed when PCC6803 (GT) was cultured by the same method as in Reference Example 2. For the production of lactic acid and succinic acid, as described in Non-Patent Document 2, intracellular metabolites are extracted, and the capillary electrophoresis / mass spectrometry (CE / MS) system (CE: Agilent G7100; MS, Agilent G6224AA LC) / MSD TOF; Liquid Chromatography-Triple Quadrupole Mass Spectrometry (LC / QqQ-MS) System (LC: Agilent 1200 series) controlled by Agilent Technologies and MassHunter Workstation Data Acquisition Software (Agilent Technologies) It was analyzed and measured using MS, Agilent 6460 with Jet Stream Technology; manufactured by Agilent Technologies.

As a result, the intracellular accumulation amounts of acetic acid, lactic acid and succinic acid in self-fermentation increased or tended to increase with the culturing time. Although there was almost no difference in the intracellular accumulation amount of acetic acid due to the difference in nitrogen source (Fig. 17A), it was observed that the accumulation amount of lactic acid and succinic acid was larger when ammonium chloride was used as the nitrogen source. (FIGS. 17B, C).

(Reference Example 4) Intracellular metabolism profile by culturing microalgae In this reference example, the intracellular metabolism profile when PCC6803 (GT) was cultured by the same method as in Reference Example 2 was investigated. As for the intracellular metabolism profile, as described in Non-Patent Document 2, the intracellular metabolites are extracted, and the capillary electrophoresis / mass spectrometry (CE / MS) system (CE: Agilent G7100; MS, Agilent G6224AA LC / MSD TOF) Liquid chromatography controlled by Agilent Technologies) and MassHunter Workstation Data Acquisition software (Agilent Technologies) -Triple Quadruple Mass Spectrometry (LC / QqQ-MS) System (LC: Agilent 1200 series; MS, Agilent It was analyzed and measured using 6460 with Jet Stream Technology (manufactured by Agilent Technologies).

As a result, intracellular accumulation of organic acids such as succinic acid, lactic acid and malic acid was confirmed by the above culture (FIGS. 18 and 19). In particular, it was observed that the accumulation of succinic acid, lactic acid and malic acid peaked in the microalgae between 6 and 24 hours after the start of culture.

(Reference Example 5) Utilization of ATP by culturing microalgae In this reference example, the amount of intracellular ATP when PCC6803 (GT) was cultured by the same method as in Reference Example 2 was examined. That is, the use of ATP associated with the production of organic acids such as succinic acid, lactic acid and malic acid was confirmed. As a result, it was confirmed that ATP decomposes into ADP and AMP with the production of organic acid (Fig. 20). That is, it was confirmed that organic acids such as succinic acid, lactic acid and malic acid are effectively produced by effectively utilizing the ATP accumulated in the cells. That is, according to PCC6803 (Flv3ox) prepared to overexpress Flv3 of the present invention, it was suggested that organic acids are produced more effectively.

As described in detail above, biomass can be effectively utilized by culturing microalgae by the method of the present invention. Organic acids can be produced from biomass by photosynthesis of microalgae and carbon sources incorporated into microalgae. In addition, _{according to microalgae with enhanced NADPH-O 2} oxidoreductase function and / or rate-determining enzyme function in glycolysis from glycogen to the citric acid cycle, organics, which are intracellular metabolites, are more effective. The acid can be recovered. The supply of carbonate ion and / or bicarbonate ion to the aqueous medium can be effectively utilized by utilizing, for example, _{CO 2 in} the atmosphere industrially emitted in the manufacturing process of electricity, steel and the like. Conventionally, among organic acids, for example, succinic acid and the like were produced from petroleum and the like as raw materials, but CO ₂ was emitted at that time, whereas according to the method of the present invention, the organic acid production process. Not only does it not emit CO ₂ , but it also has an excellent effect on the natural environment in that it can effectively utilize _{CO 2 in the atmosphere.} Further, according to the microalgae of the present invention, the aqueous medium used as biomass can utilize not only fresh water but also seawater, and can be stably and effectively utilized regardless of the depletion of water resources and the limit of cultivated land. be able to.

The produced organic acid is effectively used in foods, pharmaceuticals, and various other fields. For example, succinic acid is also used as an excipient for pharmaceuticals, a pH adjuster, a seasoning as a food, other food additives, and industrially for plating and the like. It is also used as a component of bath salts that foam carbon dioxide gas.

Claims (8)

A method for producing an organic acid from Synechocystis , which comprises the following steps (A) and (B):
(A) Phosphoenol pyruvate carboxylase (PEP carboxylase), pyruvate ferredoxin oxidoreductase, and pyruvate ferredoxin oxidoreductase in an aqueous medium containing 0 to 500 mM carbonate ion and / or bicarbonate ion under dark and anaerobic culture conditions. A step of culturing one or more enzyme-enhanced cinecocystis selected from phosphoglucomase;
(B) A step of recovering the organic acid produced from Synechocystis of (A). An aqueous medium with a carbonate ion and / or bicarbonate ion content of 0-500 mM is (1) filled with carbon dioxide and / or (2) sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, carbonate. The production method according to claim 1, which is an aqueous medium by adding any one or more carbonates selected from magnesium. The production method according to claim 2 , wherein the filling of carbon dioxide is carried out until it becomes saturated in an aqueous medium. The method for producing an organic acid according to any one of claims 1 to 3 , wherein the organic acid is an aliphatic dicarboxylic acid. The method for producing an organic acid according to any one of claims 1 to 3 , wherein the organic acid is any one or more selected from succinic acid, lactic acid and acetic acid. A method for producing any one or more organic acids selected from succinic acid, acetic acid and lactic acid from Synechocystis , which comprises the following steps (a) and (b):
(A) A step of culturing Synechocystis with enhanced PEP carboxylase function in an aqueous medium containing 100 to 500 mM carbonate ion and / or bicarbonate ion in a dark place and under anaerobic culture conditions;
(B) A step of recovering any one or more organic acids selected from succinic acid, acetic acid and lactic acid produced from Synechocystis of the above (a). A method for producing any one or more organic acids selected from succinic acid, lactic acid and acetic acid from Synechocystis , which comprises the following steps (c) and (d):
(C) A step of culturing Synechocystis with enhanced pyruvate ferredoxin oxidoreductase function in an aqueous medium containing 100 to 500 mM carbonate ion and / or bicarbonate ion in a dark place and under anaerobic culture conditions;
(D) A step of recovering any one or more organic acids selected from succinic acid, lactic acid and acetic acid produced from Synechocystis of the above (c). A method for producing any one or more organic acids selected from succinic acid, lactic acid and acetic acid from Synechocystis , which comprises the following steps (e) and (f):
(E) A step of culturing Synechocystis with enhanced phosphoglucomutase function in an aqueous medium under dark and anaerobic culture conditions;
(F) A step of recovering any one or more organic acids selected from succinic acid, lactic acid and acetic acid produced from Synechocystis of the above (e).

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