Method for producing useful organic matter utilizing photosynthetic microorganism

Abstract

To provide a method for producing a useful organic matter utilizing a photosynthetic microorganism which has an excellent production amount or yield of an objective useful organic matter (such as organic acid).SOLUTION: A method for producing a useful organic matter includes: a photosynthetic microorganism culture step of culturing a photosynthetic microorganism, where the photosynthetic microorganism emits an organic matter outside a cell during culture; and a heterotrophic microorganism culture step of culturing a heterotrophic microorganism, where the heterotrophic microorganism metabolizes an organic matter emitted from the photosynthetic microorganism during culture to produce an objective useful organic matter.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing useful organic matter by utilizing photosynthetic microorganisms. [Background technology]

[0002] In modern society, the production of many materials, including plastics, fibers, rubber, solvents, paints, detergents, and liquid fuels, is dependent on the petrochemical industry. However, further expansion of petroleum use will exacerbate resource depletion and environmental burdens, so hopes are high for a break from petroleum dependency and the production of fuels and chemicals from starch, molasses, cellulose, and other materials obtained from plant biomass.

[0003] Organic acids such as lactic acid and succinic acid are widely used as synthetic raw materials for food, pharmaceuticals, and other chemical products. It is expected that these organic acids can also be produced from carbohydrate-based biomass such as starch and cellulose. For example, there is a trend to switch the raw material succinic acid for biodegradable plastics such as polybutylene succinate (PBS), which is widely used in agricultural mulch films, packaging materials, agricultural and civil engineering materials, etc., from petroleum-derived succinic acid to biomass-derived succinic acid.

[0004] Photosynthetic microorganisms, which are microorganisms capable of photosynthesis and growing photoautotrophically, such as microalgae and cyanobacteria (blue-green algae), can directly absorb CO2 in a light environment and produce oil, starch, glycogen, organic acids, and other functional substances (pigments, functional lipids, etc.). For this reason, a method for producing useful substances using these photosynthetic microorganisms has been attracting attention as a method for producing useful substances that does not rely on petroleum. In particular, in recent years, various methods for improving productivity in organic acid production using photosynthetic microorganisms have been actively developed toward practical use. For example, Patent Document 1 describes a method for culturing microalgae at a higher temperature condition than before, 35°C to 40°C, for more efficient production in organic acid production using photosynthetic microorganisms.

[0005] However, the productivity of useful organic substances (such as organic acids) using the methods developed so far is still insufficient, and there is a strong demand for the development of production methods that are superior in terms of production amount and yield of useful organic substances. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2018 / 051916 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made in consideration of such circumstances, and an objective of the present invention is to provide a method for producing useful organic matter using photosynthetic microorganisms that are superior in production amount and yield of useful organic matter (such as organic acids). [Means for solving the problem]

[0008] As a result of intensive research conducted by the present inventors to solve the above problems, they discovered that in the production of useful organic matter (such as organic acids) using photosynthetic microorganisms, the production amount and yield of the desired useful organic matter can be increased by further using heterotrophic microorganisms in combination, and thus completed the present invention. The gist of the present invention is as follows.

[0009] [1] A method for producing useful organic matter, comprising: a photosynthetic microorganism culture step of culturing photosynthetic microorganisms, wherein the photosynthetic microorganisms release organic matter outside the cells during culture; and a heterotrophic microorganism culture step of culturing heterotrophic microorganisms, wherein the heterotrophic microorganisms metabolize the organic matter released by the photosynthetic microorganisms during culture to produce the desired useful organic matter. [2] The method according to [1], wherein the photosynthetic microorganism culture step and the heterotrophic microorganism culture step are carried out in the same medium. [3] The method according to [2], wherein the photosynthetic microorganism culture step and the heterotrophic microorganism culture step are carried out simultaneously in the same medium. [4] The method according to any one of [1] to [3], wherein the organic matter released from the photosynthetic microorganism is an organic acid. [5] The method according to [4], wherein the organic matter released from the photosynthetic microorganism is a C4 dicarboxylic acid. [6] The method according to any one of [1] to [5], wherein the photosynthetic microorganism is at least one species selected from microalgae and cyanobacteria. [7] The method according to [6], wherein the photosynthetic microorganism is a cyanobacterium. [8] The method according to any one of [1] to [7], wherein the photosynthetic microorganism is a recombinant organism. [9] The method according to any one of [1] to [8], wherein the photosynthetic microorganism culture step is carried out in a dark anaerobic environment.

[10] The method according to any one of [1] to [9], wherein the heterotrophic microorganism produces a C4 dicarboxylic acid.

[11] The method according to any one of [1] to

[10] , wherein the heterotrophic microorganism produces succinic acid.

[12] The method according to any one of [1] to

[11] , wherein the heterotrophic microorganism releases the desired useful organic matter outside the cells.

[13] The production method described in

[12] , wherein the heterotrophic microorganism has a Dcu system.

[14] The method according to

[13] , wherein the heterotrophic microorganism is at least one species selected from the group consisting of Enterobacter bacteria and Escherichia bacteria.

[15] The method according to any one of [1] to

[14] , wherein the heterotrophic microorganism culture step is carried out in an anaerobic environment.

[16] The method according to any one of [1] to

[15] , wherein the photosynthetic microorganisms are cultured in a medium containing corn steep liquor.

[17] The method according to any one of [1] to

[16] , further comprising a photosynthetic step of culturing a photosynthetic microorganism under photoautotrophic conditions.

[18] The method according to any one of [1] to

[17] , further comprising a useful organic matter recovery step of recovering useful organic matter after the heterotrophic microorganism culture step.

[19] The method according to any one of [1] to

[18] , wherein the useful organic matter is succinic acid.

[20] The method according to any one of [1] to

[19] , wherein the heterotrophic microorganism is an isolated microorganism.

[21] A composition for producing useful organic matter, comprising heterotrophic microorganisms, characterized in that it is used in combination with photosynthetic microorganisms.

[22] A composition for producing useful organic matter, comprising photosynthetic microorganisms and heterotrophic microorganisms.

[23] A kit for producing useful organic matter, comprising photosynthetic microorganisms and heterotrophic microorganisms.

[24] A group of microorganisms that produce useful organic matter, consisting of photosynthetic microorganisms and heterotrophic microorganisms.

[0010] In addition, any two or more of the configurations [1] to

[24] above may be selected and combined. Effect of the Invention

[0011] According to the present invention, it is possible to provide a method for producing useful organic substances using photosynthetic microorganisms, which is excellent in the production amount and yield of the target useful organic substances (such as organic acids). [Brief description of the drawings]

[0012] [Figure 1]Targeted gene disruption by homologous recombination. (A) Recombinant strains were generated separately from each host strain by homologous recombination. Gene cassettes of the psbA2 promoter and chloramphenicol resistance gene with or without the A. succinogenes pckA gene were introduced into the slr2030 locus of the parental Synechocystis 6803 Ppc-ox strain to obtain strains PCCK or Ppc-ox / CT, respectively. A gene cassette containing the pckA gene was introduced into the slr0646 locus of the Ppc-ox / ΔackA strain to obtain strain PCCK-ox / ΔackA. Integration was confirmed by PCR (the positions of primer annealing sites for PCR are indicated by arrows). Abbreviations: Spr: spectinomycin resistance gene; Ptrc: trc promoter; PpsbA2: psbA2 promoter; TrbcL: rbcL terminator; Chlr: chloramphenicol resistance gene. (B) PCR analysis of the DNA region containing each gene cassette in the host strain and each recombinant strain. The sizes of the PCR fragments obtained from the corresponding primer pairs are shown in (A). DNA size markers are shown in lane M. [Diagram 2] C4-dicarboxylic acid, lactate, and acetate concentrations after fermentation of Ppc-ox, Ppc-ox / CT, and PCCK cells in the presence of 4 g-DCW / L of NaHCO3 for 72 h under dark anaerobic conditions. Values ​​represent the mean (± standard deviation) of three biological replicates. Statistical significance relative to PCCK cells at each NaHCO3 concentration was determined using the Tukey-Kramer test (*P<0.01). [Diagram 3] Time course of C4-dicarboxylic acid, lactate, acetate, and glycogen concentrations during dark anaerobic fermentation of 4 g-DCW / L of Ppc-ox (open circles) and PCCK (closed circles) in the presence of 100 mM NaHCO3. Values ​​represent the mean (± standard deviation) of three biological replicates. Statistical significance of differences between Ppc-ox and PCCK strains at each sampling point was determined using Student's t-test (*P<0.01). [Figure 4]Time course of metabolites of hexose, pentose, triose phosphate (A), TCA (B), AMP, ADP, and ATP (C) during dark anaerobic fermentation of Ppc-ox (open circles) and PCCK (closed circles) in the presence of 100 mM NaHCO3. Values ​​represent the mean (± standard deviation) of three biological replicates. Statistical significance of differences between Ppc-ox and PCCK strains at each sampling point was determined using Student's t-test (*P<0.01). [Diagram 5] C4-dicarboxylic acid, lactate and acetate concentrations after fermentation of Ppc-ox, Ppc-ox / CT and PCCK cells with 4 g-DCW / L of raw CSL in the presence of various concentrations of NaHCO3 for 72 h under dark anaerobic conditions. Values ​​represent the mean (± standard deviation) of three biological replicates. Their statistical significance in PCCK cells was determined using the Tukey-Kramer test (*P<0.01). [Figure 6] Time course of C4-dicarboxylic acid, lactate, acetate, and glycogen concentrations during dark anaerobic fermentation of 4 g-DCW / L of Ppc-ox (open circles) and PCCK (closed circles) with raw CSL (A) or sterile CSL (B) in the presence of 300 mM NaHCO3. Values ​​represent the mean (± standard deviation) of three biological replicates. Statistical significance of differences between Ppc-ox and PCCK strains at each sampling point was determined using Student's t-test (*P<0.01). [Figure 7] (A) Succinate and malate concentrations after fermentation of 4 g-DCW / L PCCK cells with 300 mM NaHCO3 for 72 h in the presence of live CSL pretreated with CSL suspension at each temperature. Values ​​represent the mean (± standard deviation) of three biological replicates. (B) Colony isolation from live CSL. 50 μl of 1 g / L CSL suspension incubated at 37 °C was streaked onto LB agar medium and cultured at 37 °C for 24 h. 16S rDNA from each colony was amplified with the universal primer set 27F / 1492R and analyzed by Sanger sequencing, identifying strains related to the following species; (a) Acinetobacter radioresistens; (b) Enterobacter hormaechei; (c) Escherichia hermannii. [Figure 8] C4-dicarboxylic acid, lactate, and acetate concentrations after 72 h of fermentation of 4 g-DCW / L Ppc-ox and PCCK strains in the absence or presence of 1 g / L yeast extract (Bacto™ Yeast Extract, ThermoFisher Scientific) in a dark anaerobic environment. Values ​​represent the mean (± standard deviation) of three biological replicates. Statistical significance of differences in the absence and presence of yeast extract was determined using Student's t-test (*P<0.01). [Figure 9] Specific activity of malate dehydrogenase (MDH) in PCCK crude extracts in the presence or absence of sterile CSL (A). PEPck activity in crude extracts of PCCK and PCCK-ox / ΔackA strains (B). Crude extracts were prepared by sonication from each strain fermented for 24 h in dark anaerobic conditions. Values ​​represent the mean (± standard deviation) of three biological replicates. Statistical significance between PCCK and PCCK-ox / ΔackA strains was determined using Student's t test (*P<0.01). [Figure 10] Identification and analysis of succinate-producing microorganisms from live CSL. Production of C4-dicarboxylic acids during dark anaerobic fermentation of PCCK strains in the presence or absence of live CSL or in the presence of sterile CSL and each strain. All tested vials were supplemented with 300 mM NaHCO3. Values ​​represent the mean (± standard deviation) of three biological replicates. Statistical significance of differences between strains without and with live CSL was determined using Student's t-test (*P<0.01). In the bar graphs with sterile CSL, statistical significance of differences between E. hormaechei, A. radiosistens, E. hermannii, E. coli BW25113 and no strain, and between ΔdcuA and ΔdcuB and E. coli BW25113 was determined using Tukey-Kramer test (*P<0.01), respectively. [Figure 11]Identification and analysis of succinic acid producing microorganisms from raw CSL. Production of C4-dicarboxylic acids during dark anaerobic fermentation of PCCK strain in the presence of sterile CSL and each strain, or in the presence of sterile CSL alone or each strain alone. All vials tested were supplemented with 300 mM NaHCO3. [Figure 12] Production of C4-dicarboxylic acids during dark anaerobic fermentation of strain PCCK or strain PCCK-ox / ΔackA in the presence of 300 mM NaHCO3 for 72 h at different initial cell concentrations in the presence of sterile or live CSL. Values ​​represent the mean (± standard deviation) of three biological replicates. Statistical significance between strain PCCK and strain PCCK-ox / ΔackA was determined using Student's t-test (*P<0.01). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The method for producing useful organic matter, the composition for producing useful organic matter, the kit for producing useful organic matter, and the microorganisms for producing useful organic matter of the present invention will be described in detail below. In addition, the method for using microorganisms and the experimental method (e.g., molecular biology methods such as DNA and vector preparation, etc.) in this specification can be performed by the method described in a general experimental book known to those skilled in the art or a method equivalent thereto, unless otherwise specified. In addition, the terms used in this specification are interpreted as being normally used in the technical field, unless otherwise specified.

[0014] [Production method of useful organic matter] The method for producing useful organic matter of the present invention includes a photosynthetic microorganism culturing step of culturing photosynthetic microorganisms, and a heterotrophic microorganism culturing step of culturing heterotrophic microorganisms. In the photosynthetic microorganism culturing step, the photosynthetic microorganisms release organic matter outside the cells during culturing, and in the heterotrophic microorganism culturing step, the heterotrophic microorganisms metabolize the organic matter released by the photosynthetic microorganisms during culturing to produce the desired useful organic matter. The method for producing useful organic matter of the present invention is characterized by including these steps, and other configurations and conditions are not particularly limited.

[0015] The method for producing useful organic matter of the present invention is based on the following findings (here, a method for producing organic acid is taken as an example). That is, first, in the conventional method for producing organic acid using photosynthetic microorganisms, many attempts have been made to increase the amount of organic acid produced and improve productivity by focusing on the organic acid production pathway of the photosynthetic microorganisms themselves and modifying the reaction enzymes and culturing conditions. However, photosynthetic microorganisms do not grow by producing only the target organic acid during culture, but naturally produce and release organic acids other than the target organic acid. These organic acids are by-products and cause a decrease in purity, but it is very difficult to control the amount of these by-products produced while maintaining the amount of the target organic acid produced at a high level, and the purity and yield of the target organic acid were not increased, and the production amount was not increased, merely by modifying the reaction enzymes and culturing conditions.

[0016] The present inventors conducted a detailed analysis of the conventional organic acid production process using photosynthetic microorganisms and unexpectedly found for the first time that heterotrophic microorganisms may be present in the culture medium during the cultivation of photosynthetic microorganisms, and discovered that in such cases, the heterotrophic microorganisms metabolize the organic acid (by-product organic acid) released extracellularly by the photosynthetic microorganisms to produce the desired organic acid, thereby improving the purity and yield of the organic acid. The present invention is based on such findings, and is characterized in that, during the production of useful organic matter using photosynthetic microorganisms, the organic matter released extracellularly by the photosynthetic microorganisms is metabolized by the heterotrophic microorganisms to produce the desired useful organic matter.

[0017] <Photosynthetic microorganism culture process> In the present invention, the photosynthetic microorganism culturing step is a step of culturing photosynthetic microorganisms as described above. In this step, the photosynthetic microorganisms are cultured under specific culture conditions, whereby the photosynthetic microorganisms release organic matter outside the cells.

[0018] When photosynthetic microorganisms are cultured under photoautotrophic conditions, they can perform photosynthesis and grow by producing complex organic matter (carbohydrates, fats, proteins, etc.) using carbon dioxide, bicarbonate, etc. as a carbon source. On the other hand, when photosynthetic microorganisms are cultured under specific culture conditions (for example, in a dark anaerobic environment), they grow heterotrophically using the organic matter stored under the photoautotrophic conditions, and at that time, the photosynthetic microorganisms produce organic matter such as malic acid, succinic acid, fumaric acid, lactic acid, glycerol, formic acid, acetic acid, and ethanol, and partially release these organic matter outside the cells. The photosynthetic microorganism culture step in the present invention is thus a step of culturing photosynthetic microorganisms under specific culture conditions so as to grow them heterotrophically and produce and release the above-mentioned organic matter.

[0019] As described above, photosynthetic microorganisms release organic matter outside the cells during the photosynthetic microorganism culture process, and the organic matter released from these photosynthetic microorganisms is preferably an organic matter suitable for the heterotrophic microorganism to metabolize and produce the desired useful organic matter in the heterotrophic microorganism culture process described below. The organic matter released from such photosynthetic microorganisms is, for example, malic acid, succinic acid, fumaric acid, lactic acid, glycerol, formic acid, acetic acid, ethanol, etc., and is preferably an organic acid, more preferably a C4 dicarboxylic acid, and particularly preferably malic acid and / or fumaric acid. Aspartic acid is also preferred. Here, in the technical field, photosynthetic microorganisms that release these various organic matters and their culture conditions are each known, and appropriate photosynthetic microorganisms and culture conditions can be selected and used according to the purpose of the invention based on the technical common knowledge in the technical field. It should be noted that when photosynthetic microorganisms release the above-mentioned specific organic matter, there is no restriction on them releasing other organic matters as well.

[0020] In addition, in the photosynthetic microorganism culturing step of the present invention, it is preferable that the photosynthetic microorganism releases the useful organic matter to be produced, more preferable that the useful organic matter to be produced is released in greater amounts than other useful organic matters, and it is particularly preferable that the useful organic matter to be produced is released in the greatest amount compared to other useful organic matters. In such a case, the productivity of useful organic matter by photosynthetic microorganisms alone is high to begin with, and in the present invention, the productivity can be further improved by the heterotrophic microorganism culturing step described below, making it possible to provide a production method with higher productivity with greater certainty.

[0021] (Photosynthetic microorganisms) In the present invention, "photosynthetic microorganisms" refers to all microorganisms capable of photosynthetic growth, including, for example, microalgae (eukaryotic algae such as diatoms and Euglena) and prokaryotic algae (cyanobacteria) that perform oxygenic photosynthesis, as well as non-oxygenic photosynthetic bacteria. In the present invention, photosynthetic microorganisms can be arbitrarily selected and used. Note that, in the present invention, one type of photosynthetic microorganism can be used alone, or two or more types can be used in combination.

[0022] Microalgae and cyanobacteria are microorganisms that have chlorophyll and perform photosynthesis. Microalgae and cyanobacteria fix CO2 in the atmosphere through photosynthesis to synthesize sugars (e.g., glycogen), and can also generate oxygen (O2) from water (H2O) (also called "oxygenic photosynthesis"). In the present invention, microalgae and cyanobacteria may have a unicellular form or a colony form (e.g., a filament, sheet, or ball). Microalgae and cyanobacteria may grow in either the ocean or freshwater.

[0023] In the present invention, examples of cyanobacteria (blue-green algae) include the genera Synechocystis, Arthrospira, Spirulina, Anabaena, Synechococcus, Thermosynechococcus, Nostoc, Prochlorococcus, Microcystis, and Gloeobacter. For example, microbial species of cyanobacteria (blue-green algae) include Synechocystis sp. PCC6803, Synechococcus sp. PCC7002, Arthrospira platensis (also known as "Spirulina"), Spirulina maxima, Spirulina subsalsa, and Anabaena sp. PCC7120.

[0024] In the present invention, the microalgae may be any of, for example, green algae, diatoms, dinoflagellates, red algae, prasinophytes, euglenids, and true eyespot algae. Specifically, for example, green algae such as Chlamydomonas, Chlorella, Dunaliella, Hematococcus, Volvox, and Botryococcus; Rhizosolenia, Chaetoceros, Cyclotella, Cylindrotheca, Navicula, Phaeodactylum, and the like; These include diatoms such as those of the genera Thalassiosira and Fistulifera; dinoflagellates such as those of the genera Amphidinium and Symbiodinium; red algae such as those of the genera Cyanidioschyzon and Porphyridium; prasinophytes such as those of the genera Ostreococcus; euglenae such as those of the genus Euglena; and true eyespot algae such as those of the genus Nannochloropsis.For example, microalgae microbial species include Chlamydomonas reinhardtii, Chlamydomonas sp., Chlorella vulgaris, Chlorella pyrenoidosa, Dunaliella salina, Dunaliella sp., Hematococcus pluvialis, Volvox carteri, Botryococcus braunii, Cyclotella cryptica, Cylindrotheca fusiformis, Navicula saprofila, and the like. saprophila, Phaeodactylum tricornutum, Thalassiosira pseudonana, Fistulifera sp., Amphidinium sp., Symbiodinium microadriaticum, Cyanidioschyzon merolae, Porphyridium sp., Ostreococcus tauri, Euglena gracilis, Nannochloropsis oculata, etc.

[0025] In the present invention, anoxygenic photosynthetic bacteria include purple bacteria, green filamentous bacteria, green sulfur bacteria, heliobacteria, chloracidobacteria, and the like. Examples of purple bacteria include Rhodobacter sphaeroides and Rhodospirillum rubrum. Examples of green filamentous bacteria include Roseiflexus castenholzii. Examples of green sulfur bacteria include Chlorobaculum tepidum. Examples of Heliobacteria include Heliophilum fasciatum and Heliobacterium modesticaldum. Examples of Chloracidobacteria include Chloracidobacterium thermophilum.

[0026] Of these, in the present invention, the photosynthetic microorganism is preferably at least one selected from cyanobacteria (blue-green algae) and microalgae, and more preferably cyanobacteria (blue-green algae). Among cyanobacteria, the genus Synechocystis is more preferred, and Synechocystis sp. PCC6803 is particularly preferred. These can be preferably used as photosynthetic microorganisms that release, for example, C4 dicarboxylic acids. In addition, the microalga Chlamydomonas reinhardtii can be preferably used as photosynthetic microorganisms that release, for example, glycerol, formic acid, etc.

[0027] (Recombinant photosynthetic microorganisms) In the present invention, the photosynthetic microorganism may be a recombinant organism modified to effectively produce an organic substance (e.g., an organic acid) according to the purpose of the invention. The modification method may be any method known in the art or any method to be developed in the future, for example, by a technique such as genetic recombination.

[0028] As an example, an example of a recombinant cyanobacterium is given below, but based on common technical knowledge in the art, any photosynthetic microorganism can be modified and used, not limited to cyanobacteria, so as to enhance the effects of the present invention.

[0029] In the present invention, the recombinant cyanobacteria can be genetically engineered to express or enhance the expression of, for example, phosphoenolpyruvate carboxykinase (Pepck) and / or pyruvate carboxylase (Pyc) in order to enhance the ability to produce organic acids. It is also preferred that the recombinant cyanobacteria be genetically engineered to express or enhance the expression of phosphoenolpyruvate carboxylase (Ppc). It is also preferred that the recombinant cyanobacteria be genetically engineered to delete or reduce the expression of acetate kinase (AK).

[0030] In the present invention, "genetically engineered to express or enhance expression" refers to introducing a gene of interest into an organism to be recombined, or genetically modifying the gene to enhance the expression of the gene, etc. In addition, "genetically engineered to delete or reduce expression" refers to deleting a gene of interest (gene disruption) or genetically modifying the gene to reduce (suppress) the expression of the gene, etc. In the present specification, the form of introducing a specific gene or genetically modifying the gene to enhance the expression of the gene is not particularly limited as long as it is confirmed that the amount of production or activity of a protein related to each gene is increased in the recombinant microorganism compared to before the introduction or modification. In the present specification, the form of deleting a specific gene (gene disruption) or genetically modifying the gene to reduce the expression of the gene is not particularly limited as long as it is confirmed that the amount of production or activity of a protein related to each gene is decreased in the recombinant microorganism compared to before the deletion or modification. The introduction of a gene, modification to enhance its expression, gene deletion, and modification to reduce (suppress) its expression may be performed by a conventionally known method or any method developed in the future.

[0031] Phosphoenolpyruvate carboxykinase (Pepck), pyruvate carboxylase (Pyc), and phosphoenolpyruvate carboxylase will be explained below.

[0032] (Phosphoenolpyruvate carboxykinase (Pepck)) In the present invention, phosphoenolpyruvate carboxykinase (Pepck) is an enzyme that reversibly catalyzes the reaction of producing oxaloacetate (OAA) from phosphoenolpyruvate (Pep) by carbon fixation. In the present invention, Pepck activity refers to the activity of catalyzing the reaction of producing OAA from this Pep. The Pepck used in the present invention is preferably one in which the reaction equilibrium is inclined toward the production of Pep or OAA. The enzyme activity can be determined, for example, by a method of measuring the amount of ATP produced at 37°C using a Sigma Diagnostics ATP Kit (Pil, Kim., et.al., Applied and Enviromental Microbiology, Feb. 2004, p.1238-1241). By using a recombinant cyanobacterium in which Pepck is expressed in the cyanobacterium, it is possible to produce oxaloacetate (OAA) by carbon fixation from PEP derived from sugars (glycogen, etc.) accumulated in the cyanobacterium by culturing under photoautotrophic conditions, by culturing under a dark anaerobic environment.

[0033] The increase in Pepck activity compared to the parent strain can be confirmed by measuring the enzyme activity using the above method, or by comparing the amount of mRNA of the gene encoding Pepck or the amount of the expressed Pepck protein with that of the parent strain. The increase in enzyme activity may be sufficient as long as it is increased compared to the parent strain, and is preferably 1.5 times or more, more preferably 2 times or more, even more preferably 3 times or more, and particularly preferably 5 times or more, compared to the parent strain.

[0034] The Pepck to be introduced into cyanobacteria is not particularly limited as long as it is capable of more efficiently catalyzing the reaction of producing oxaloacetic acid (OAA) through carbon dioxide fixation by culturing in a dark anaerobic environment. For example, enzymes derived from Actinobacillus succinogenes, Mannheimia succiniciproducens, Anaerobiospirillum succiniciproducens, Selenomonas ruminantium, etc., which are a group of bacteria capable of producing succinic acid under high carbon dioxide concentrations, as well as enzymes derived from Escherichia coli, Corynebacterium glutamicum, and the like, and enzymes derived from other bacteria such as Escherichia coli, Corynebacterium glutamicum, and the like, which are capable of producing succinic acid under high carbon dioxide concentrations, as well as ... Examples of suitable Pepck include enzymes derived from Actinobacillus succinogenes and the like, and the enzymes disclosed in "https: / / www.brenda-enzymes.org / enzyme.php?ecno=4.1.1.32". Even if a name other than Pepck is used, it can be used as Pepck in the present invention as long as it has the above-mentioned catalytic activity. As the Pepck to be introduced into cyanobacteria, an enzyme derived from Actinobacillus succinogenes is more preferable.

[0035] Since several sequences of the Pepck gene have already been elucidated, it can be obtained using primers prepared based on those sequences. For example, the coding region of Pepck of Actinobacillus succinogenes and adjacent regions including its control region can be obtained by PCR using the prepared primers and the chromosomal DNA of Actinobacillus succinogenes as a template. Alternatively, sequences designed in a codon-optimized form for the base sequences corresponding to the wild type (SEQ ID NO: 1) can be totally synthesized (SEQ ID NO: 2). Pepck gene homologs of other microorganisms can be obtained in a similar manner. A Pepck gene homolog refers to a gene derived from another microorganism, which shows high homology to the Pepck gene of Actinobacillus succinogenes and codes for a protein having Pepck activity.

[0036] Since there may be differences in the base sequence of the Pepck gene depending on the species or strain of bacteria belonging to the Enterobacteriaceae family, the Pepck gene is not limited to SEQ ID NO: 2, and may be a mutant or artificially modified form encoding a protein having a sequence containing one or several amino acid substitutions, deletions, insertions, or additions at one or more positions in the amino acid sequence encoded by the polynucleotide of SEQ ID NO: 2, as long as the expression of the gene can be enhanced in the cyanobacteria to improve the organic acid (e.g., succinic acid) producing ability of the cyanobacteria. Here, the term "several" varies depending on the position and type of the amino acid residue in the three-dimensional structure of the protein, and is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5. In addition, such amino acid substitutions, deletions, insertions, additions, inversions, etc. also include those that occur due to natural mutations, such as those based on individual differences and species differences in microorganisms carrying the Pepck gene. A polynucleotide encoding a Pepck protein (peptide) is a protein (and a polynucleotide encoding the same) that has at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and particularly preferably 97% or more identity to the above-mentioned specific sequence, and a protein (and a polynucleotide encoding the same) that has Pepck activity (phosphoenolpyruvate carboxykinase activity) is understood to be usable in the present invention.

[0037] Since the degeneracy of the Pepck gene varies depending on the host into which it is introduced, it may be replaced with codons that are easily used in the host into which it is introduced. Similarly, the Pepck gene may be a gene encoding a protein with an extended or truncated N-terminus or C-terminus, as long as it has the function of improving the organic acid producing ability of cyanobacteria by enhancing expression.

[0038] (Pyruvate carboxylase (Pyc)) In the present invention, pyruvate carboxylase (Pyc) is an enzyme of the ligase group that irreversibly carboxylates pyruvate to oxaloacetate. By using a recombinant cyanobacterium expressing Pyc, it becomes possible to produce oxaloacetate (OAA) by carbon fixation from sugar-derived pyruvate accumulated in the cyanobacterium by culturing under photoautotrophic conditions.

[0039] The increase in Pyc activity compared to the parent strain can be confirmed by measuring the enzyme activity by the above-mentioned method, or by comparing the amount of mRNA of the gene encoding Pyc or the amount of the expressed Pyc protein with that of the parent strain. The increase in enzyme activity may be sufficient as long as it is increased compared to the parent strain, and is preferably, for example, 1.5-fold or more, more preferably 2-fold or more, even more preferably 3-fold or more, and particularly preferably 5-fold or more compared to the parent strain.

[0040] The Pyc to be introduced into cyanobacteria is not particularly limited as long as it catalyzes the reaction of producing oxaloacetic acid (OAA) from pyruvate more efficiently, and examples thereof include enzymes derived from Corynebacterium glutamicum, Bacillus subtilis, Saccharomyces cerevisiae, Pichia pastoris, Escherichia coli, etc., and enzymes disclosed in "https: / / www.brenda-enzymes.org / enzyme.php?ecno=6.4.1.1". Note that even if a name other than Pyc is used, it can be used as Pyc in the present invention as long as it has the above-mentioned catalytic activity. In the present invention, among these, Pyc derived from Corynebacterium glutamicum is preferred as the Pyc to be introduced into cyanobacteria.

[0041] Since several sequences of the Pyc gene have already been elucidated, it can be obtained using primers prepared based on those sequences. For example, the coding region of Pyc of Corynebacterium glutamicum (SEQ ID NO: 3) and adjacent regions including its control region can be obtained by PCR using the prepared primers and the chromosomal DNA of Corynebacterium glutamicum as a template. The amino acid sequence encoded by SEQ ID NO: 3 is shown in SEQ ID NO: 4. Alternatively, sequences designed in a codon-optimized form for those base sequences can be totally synthesized. Pyc gene homologs of other microorganisms can be obtained in the same manner. A Pyc gene homolog refers to a gene derived from another microorganism, which shows high homology to the Pyc gene of Corynebacterium glutamicum and codes for a protein having Pyc activity.

[0042] Since there may be differences in the base sequence of the Pyc gene depending on the bacterial strain, etc., the Pyc gene used in the present invention is not limited to SEQ ID NO: 3, and may be a mutant or artificially modified product encoding a protein having a sequence containing one or several amino acid substitutions, deletions, insertions, or additions at one or several positions in the amino acid sequence encoded by the polynucleotide of SEQ ID NO: 3, as long as the expression of the Pyc gene can be enhanced in the cyanobacteria to improve the organic acid (e.g., succinic acid) production ability of the cyanobacteria. Here, the term "several" varies depending on the position and type of the amino acid residue in the three-dimensional structure of the protein, and is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5. In addition, such amino acid substitutions, deletions, insertions, additions, inversions, etc. also include those that occur due to natural mutations such as those based on individual differences and species differences in microorganisms carrying the Pepck gene. It is understood that a polynucleotide encoding a Pyc protein (peptide) is a protein (and a polynucleotide encoding the same) that has at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and particularly preferably 97% or more identity to the above-mentioned specific sequence, and that has Pyc activity (pyruvate carboxylase activity) (and a polynucleotide encoding the same) can be used in the present invention.

[0043] Since the degeneracy of the Pyc gene varies depending on the host into which it is introduced, it may be replaced with codons that are easily used in the host into which it is introduced. Similarly, the Pyc gene may be a gene encoding a protein with an extended or truncated N-terminus or C-terminus, so long as it has the function of improving the organic acid producing ability of cyanobacteria by enhancing expression.

[0044] (Phosphoenolpyruvate carboxylase (Ppc)) In the present invention, phosphoenolpyruvate carboxylase (PEP carboxylase; Ppc) is an enzyme that reversibly catalyzes the reaction of producing oxaloacetate (OAA) from phosphoenolpyruvate (Pep) by carbon fixation. In the present invention, Ppc activity refers to the activity of catalyzing the reaction of producing OAA from this Pep. The Ppc used in the present invention is preferably one in which the reaction equilibrium is inclined toward the production of Pep or OAA. By using a recombinant cyanobacterium in which Ppc expression in cyanobacteria is enhanced, it is possible to efficiently produce oxaloacetate (OAA) by carbon fixation from sugar-derived PEP accumulated in the cyanobacteria by culturing under photoautotrophic conditions, by culturing in a dark anaerobic environment.

[0045] The confirmation that the activity of Ppc is enhanced compared to the parent strain can be confirmed by measuring the Ppc enzyme activity, or by comparing the amount of mRNA of the gene encoding Ppc or the amount of the expressed Ppc protein with that of the parent strain. The increase in enzyme activity may be sufficient as long as it is increased compared to the parent strain, and is preferably, for example, 1.5 times or more, more preferably 2 times or more, even more preferably 3 times or more, and particularly preferably 5 times or more, compared to the parent strain.

[0046] The Ppc to be introduced into cyanobacteria and used to enhance expression is not particularly limited as long as it is capable of more efficiently catalyzing the reaction of producing oxaloacetic acid (OAA) by carbon fixation when cultured in a dark anaerobic environment, but is preferably a Ppc of cyanobacteria (blue-green algae), such as Synechocystis, Arthrospira, Spirulina, Anabaena, Synechococcus, Thermosynechococcus, Nostoc, Prochlorococcus, Microcystis, Gloeobacter, etc. Among them, the Synechocystis genus is more preferable, and specifically, Synechocystis sp. PCC6803 is particularly preferable. In addition to Ppc of cyanobacteria (blue-green algae), the enzymes disclosed in https: / / www.brenda-enzymes.org / enzyme.php?ecno=4.1.1.31 are also listed as candidates for enzymes that can be used in the present invention. Even if a name other than Ppc is used, it can be used as Ppc in the present invention as long as it has the above-mentioned catalytic activity.

[0047] Since some sequences of the Ppc gene have already been elucidated, it can be obtained using primers prepared based on those base sequences. For example, the coding region of Ppc of Synechocystis (SEQ ID NO: 5) and adjacent regions including its control region can be obtained by PCR using the prepared primers and the chromosomal DNA of the cyanobacterium Synechocystis as a template. The amino acid sequence encoded by SEQ ID NO: 5 is shown as SEQ ID NO: 6. A specific example of the cyanobacterium Synechocystis is the PCC6803 species (Synechocystis sp. PCC6803). Alternatively, sequences designed in a codon-optimized form for those base sequences can be totally synthesized. Ppc gene homologs of other microorganisms can be obtained in the same manner. The Ppc gene homolog refers to a gene derived from another microorganism, which shows high homology to the Ppc gene of the cyanobacterium Synechocystis, and which codes for a protein having Ppc activity.

[0048] Since there may be differences in the base sequence of the Ppc gene depending on the species or strain of bacteria belonging to the Enterobacteriaceae family, the Ppc gene used in the present invention is not limited to SEQ ID NO: 5, and may be a mutant or artificially modified product encoding a protein having a sequence containing one or several amino acid substitutions, deletions, insertions, or additions at one or several positions in the amino acid sequence encoded by the polynucleotide of SEQ ID NO: 5, as long as its expression in the cyanobacteria can be enhanced to improve the organic acid (e.g., succinic acid) producing ability of the cyanobacteria. Here, the term "several" varies depending on the position and type of the amino acid residue in the three-dimensional structure of the protein, and is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5. In addition, such amino acid substitutions, deletions, insertions, additions, inversions, etc. also include those that occur due to natural mutations such as those based on individual differences and species differences in microorganisms carrying the Ppc gene. A polynucleotide encoding a Ppc protein (peptide) is understood to be a protein (and a polynucleotide encoding the same) that has at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and particularly preferably 97% or more identity to the specific sequence described above, and that has Ppc activity (pyruvate carboxylase activity) (and a polynucleotide encoding the same) that can be used in the present invention.

[0049] Since the degeneracy of the Ppc gene varies depending on the host into which it is introduced, it may be replaced with codons that are easily used in the host into which it is introduced. Similarly, the Ppc gene may be a gene encoding a protein with an extended or truncated N-terminus or C-terminus, as long as it has the function of improving the organic acid production ability of cyanobacteria by enhancing expression.

[0050] (Acetate kinase (AK)) In the present invention, acetate kinase (AK) is an enzyme that reversibly catalyzes the transfer reaction of a phosphate group between acetate and adenosine diphosphate (ADP). In the present invention, AK activity refers to the activity of catalyzing the reaction of producing acetate from acetyl phosphate. By using a recombinant cyanobacterium in which AK is deleted or expression is reduced, acetate production in a dark anaerobic environment is suppressed, and instead, organic acids such as fumaric acid and succinic acid can be efficiently produced.

[0051] The confirmation that the activity of AK is suppressed compared to the parent strain can be confirmed by measuring the AK enzyme activity, or by comparing the amount of mRNA of the gene encoding AK or the amount of the expressed AK protein with that of the parent strain. The reduction in enzyme activity may be sufficient as long as it is reduced compared to the parent strain, but for example, it is preferably 10 times or less than that of the parent strain, more preferably 50 times or less, even more preferably 100 times or less, and particularly preferably below the detection limit.

[0052] (Culture conditions) The photosynthetic microorganism culture step can be carried out under any culture conditions suitable for the photosynthetic microorganism to release organic matter outside the cells, but is typically carried out in a dark anaerobic environment. Further, more detailed culture conditions can be appropriately determined depending on the photosynthetic microorganism used and the organic matter to be released.

[0053] In the present invention, a "dark" environment refers to a state where light is not irradiated. An "anaerobic" environment refers to a state where the dissolved oxygen concentration in the solution is kept low, specifically, for example, an O2 concentration of 1% or less. To create this anaerobic environment, for example, a method can be used in which the container is sealed and the reaction is carried out without ventilation, an inert gas such as nitrogen gas (N2) is supplied and the reaction is carried out, or an inert gas containing CO2 is passed through.

[0054] In the photosynthetic microorganism culturing step, the pH of the medium can be adjusted to any pH suitable for the photosynthetic microorganism to release organic matter outside the cells, for example, pH 5 to 10, preferably pH 6 to 9, more preferably pH 6 to 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, or the like to the medium.

[0055] In the photosynthetic microorganism culturing step, the temperature condition for the culture can be any temperature suitable for the photosynthetic microorganism to release organic matter outside the cells. The temperature is usually in the range of 25°C to 45°C, preferably 30°C to 40°C, and more preferably 35°C to 40°C.

[0056] In the photosynthetic microorganism culturing step, the culture time can be, for example, from 24 hours to 5 days, and preferably from 3 days to 4 days.

[0057] (Culture medium) The medium for culturing the photosynthetic microorganism in the photosynthetic microorganism culturing step can be appropriately selected and used depending on the photosynthetic microorganism used, the organic matter to be released, etc. For example, BG-11 medium can be used as an inorganic medium. When the photosynthetic microorganism to be cultured is a microalga or a cyanobacteria, an aqueous solution containing a nitrogen source, an inorganic salt, etc. can be used as the culture medium. Specifically, depending on the microalga or cyanobacteria, artificial or natural seawater or fresh water (e.g., distilled water) can be used. 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. can be used.

[0058] In addition, in order to efficiently carry out the organic matter production reaction of the photosynthetic microorganism, for example, an organic raw material may be added to the medium as a carbon source. The organic raw material used for these cultures is not particularly limited as long as it can be assimilated and grown by the photosynthetic microorganism, but usually, fermentable sugars such as carbohydrates such as galactose, lactose, glucose, fructose, sucrose, saccharose, starch, and cellulose; and polyalcohols such as glycerol, mannitol, xylitol, and ribitol are used, and can be selected according to the target organic matter and can be selected from general organic raw materials. For example, glucose, sucrose, or fructose is preferred, and glucose or sucrose is particularly preferred. In addition, starch saccharification liquid, molasses, etc. containing the above-mentioned fermentable sugars can be used, and the fermentable sugars may be sugar liquid extracted from plants such as sugar cane, sugar beet, and sugar maple. These organic raw materials can be used alone or in combination. The medium can contain carbonate ions, bicarbonate ions, or CO2.

[0059] It is also preferable that the medium contains carbonate ions, bicarbonate ions and / or CO2. The concentration of carbonate ions and bicarbonate ions is 5 to 2,000 mM, preferably 10 to 1,000 mM, more preferably 20 to 500 mM, even more preferably 50 mM to 400 mM, and particularly preferably 100 mM to 300 mM. Carbonate ions and / or bicarbonate ions can be introduced into the medium by filling with CO2 or adding at least one carbonate selected from sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, and magnesium carbonate. When filling with CO2, it can be filled until it reaches a saturated state. When CO2 reaches a saturated state, the carbonate ion concentration becomes 20 to 2,000 mM.

[0060] For example, by adding a buffer such as HEPES to the medium for culturing photosynthetic microorganisms, the pH of the medium can be stabilized and the efficiency of organic matter production can be improved. In the photosynthetic microorganism culturing step, when HEPES-KOH is used, the concentration of the buffer in the medium for culturing photosynthetic microorganisms is in the range of 20 mM to 500 mM, preferably 50 mM to 400 mM, more preferably 100 mM to 300 mM, and even more preferably 100 mM to 200 mM.

[0061] The medium for culturing photosynthetic microorganisms may contain, for example, corn steep liquor. By using such a medium, the efficiency of organic matter production by photosynthetic microorganisms can be increased. Here, corn steep liquor refers to corn steep liquor (CSL), which is a liquid obtained by concentrating a soaking liquid containing soluble components dissolved from corn and components produced by lactic acid fermentation in the soaking process of corn wet milling, which is one of the methods for refining corn starch, or a solid (powdered) product obtained by drying the soaking liquid. In other words, it corresponds to the residue when starch is extracted from corn. The corn steep liquor that can be used in the present invention is commercially available as corn steep liquor, and is not particularly limited as long as it can increase the efficiency of organic matter production. Examples of corn steep liquor that can be used include, but are not limited to, those manufactured by Oriental Yeast Co., Ltd. and Spectrum Chemical Co., Ltd.

[0062] Corn steep liquor can also be used by suspending it in water, incubating it at a predetermined temperature (e.g., 37°C) for a predetermined time (e.g., 24 hours), and then adding only the supernatant to the medium. Corn steep liquor sterilized by autoclave or the supernatant of corn steep liquor sterilized by autoclave can also be used in the present invention. In the present invention, the medium containing corn steep liquor includes the case where the medium contains corn steep liquor powder and the case where the medium contains corn steep liquor supernatant. The concentration of corn steep liquor in the medium in the present invention is 10 mg / L to 100 g / L, preferably 100 mg / L to 20 g / L, more preferably 300 mg / L to 10 g / L, further preferably 500 mg / L to 5 g / L, and particularly preferably 500 mg / L to 4 g / L. When photosynthetic microorganisms are cultured in a medium containing corn steep liquor, metabolism is promoted, so it is preferable to use corn steep liquor. Generally, it is believed that the production of succinic acid, fumaric acid, malic acid, etc. is promoted, but the production of other organic substances can also be promoted by modifying the metabolic pathway. When the photosynthetic microorganism is a microalgae or a cyanobacteria, it is preferable to use corn steep liquor since the production of malic acid and fumaric acid by the microalgae or the cyanobacteria is particularly promoted when the microalgae or the cyanobacteria are cultured in a medium containing corn steep liquor.

[0063] (Cell density at the start of culture) In the photosynthetic microorganism culturing process, the cell density at the start of photosynthetic microorganism culturing can affect the productivity of organic matter. Generally, it is preferable to increase the cell density at the start of photosynthetic microorganism culturing, and a cell density of 4 g dry cell weight or more per 1 L of medium is preferable, and 20 g dry cell weight or more per 1 L of medium is more preferable. The upper limit of the cell density at the start of photosynthetic microorganism culturing is preferably 75 g dry cell weight or less per 1 L of medium.

[0064] <Heterotrophic microorganism cultivation process> In the present invention, the heterotrophic microorganism culturing step is a step of culturing heterotrophic microorganisms as described above. In this step, the heterotrophic microorganisms are cultured under specific culture conditions, whereby the heterotrophic microorganisms metabolize the organic matter released by the photosynthetic microorganisms to produce the desired useful organic matter.

[0065] Here, the heterotrophic microorganisms grow by metabolizing the organic matter produced and released by the specific photosynthetic microorganisms described above. During this metabolism, the heterotrophic microorganisms produce useful organic matter (organic acids such as C4 dicarboxylic acids, amino acids, alcohols such as isobutanol, aromatic compounds such as coumaric acid, diamines, diols, biopolymers such as polyhydroxyalkanoic acid, etc.). The heterotrophic microorganism culturing step of the present invention is thus a step of culturing heterotrophic microorganisms so that they grow by metabolizing the organic matter produced and released by the photosynthetic microorganisms, thereby producing the above-mentioned useful organic matter.

[0066] In the heterotrophic microorganism culturing step, as described above, the heterotrophic microorganism metabolizes the organic matter released by the photosynthetic microorganism to produce the desired useful organic matter, and such heterotrophic microorganisms can be appropriately selected and used depending on the desired useful organic matter. For example, in the present invention, the heterotrophic microorganism is preferably a heterotrophic microorganism that produces C4 dicarboxylic acid, and more preferably a heterotrophic microorganism that produces succinic acid. Here, heterotrophic microorganisms that release these various useful organic matters are each known in the art, and appropriate heterotrophic microorganisms and culture conditions can be selected and used according to the purpose of the invention based on the technical common knowledge in the art.

[0067] (Heterotrophic microorganisms) In the present invention, "heterotrophic microorganisms" refers to all microorganisms that cannot directly produce organic matter themselves, but grow heterotrophically by utilizing organic matter synthesized by autotrophic organisms, but the heterotrophic microorganisms actually used are selected and used in accordance with the object of the invention, as described above. Here, the heterotrophic microorganisms may be recombinant organisms that have been metabolically modified to favor the production of the desired useful organic matter. In the present invention, one type of heterotrophic microorganism may be used alone, or two or more types may be used in combination.

[0068] In the present invention, examples of the heterotrophic microorganisms used include heterotrophic microorganisms having a Dcu system when the target useful organic matter is succinic acid.

[0069] The Dcu system is a transport system involved in the uptake of C4-dicarboxylic acids, and functions in an intracellular / extracellular exchange reaction that takes in aspartic acid, malic acid, and fumaric acid and excretes succinic acid. Examples of heterotrophic microorganisms having the Dcu system include Enterobacter bacteria, Escherichia bacteria, Actinobacillus bacteria, and Pseudomonas bacteria. Among them, Enterobacter bacteria and Escherichia bacteria are preferably used because they produce a large amount of succinic acid, as described in the Examples below, and Escherichia bacteria are particularly preferably used. In addition, Escherichia coli and Actinobacillus succinogenes are preferably used because their genome sequences are known, gene recombination systems have been established, and metabolic modification is easy.

[0070] Examples of bacteria belonging to the genus Enterobacter include Enterobacter hormaechei, Enterobacter cloacae, and Enterobacter kobei.

[0071] Examples of bacteria belonging to the genus Escherichia include Escherichia hermannii and Escherichia coli.

[0072] Examples of the Actinobacillus bacteria include Actinobacillus succinogenes, Actinobacillus seminis, and Actinobacillus porcinus.

[0073] Examples of Pseudomonas bacteria include Pseudomonas aeruginosa and Pseudomonas fluorescens.

[0074] In the present invention, examples of heterotrophic microorganisms used when the target useful organic matter is malic acid include Ustilago trichophora, Saccharomyces cerevisiae, Aspergillus niger, Aspergillus oryzae, and Escherichia coli.

[0075] In the present invention, examples of heterotrophic microorganisms used when the target useful organic matter is fumaric acid include Rhizopus arrhizus, Rhizopus oryzae, Lactobacillus plantarum, Escherichia coli, and the like.

[0076] (Culture conditions) The heterotrophic microorganism culturing step can be carried out under any culture conditions (light conditions, dark conditions, aerobic conditions, anaerobic conditions, etc.) as long as the heterotrophic microorganisms are suitable for metabolizing the organic matter released by the photosynthetic microorganisms in the photosynthetic microorganism culturing step and producing the desired useful organic matter, and more detailed conditions can be appropriately determined depending on the heterotrophic microorganisms used and the useful organic matter to be produced. The heterotrophic microorganism culturing step can be carried out, for example, in an anaerobic environment. Furthermore, for example, when the photosynthetic microorganism culturing step and the heterotrophic microorganism culturing step are carried out simultaneously in the same medium, they are carried out under the same culture conditions as the photosynthetic microorganism culturing step, typically in a dark anaerobic environment.

[0077] In addition, the heterotrophic microorganism cultivation process may be carried out under light-activated conditions. In this case, many heterotrophic microorganisms can utilize the organic substances (malic acid, succinic acid, fumaric acid, lactic acid, glycerol, formic acid, acetic acid, ethanol, etc.) released by the photosynthetic microorganisms in the photosynthetic microorganism cultivation process under aerobic conditions, and produce a wide variety of useful organic substances.

[0078] In the present invention, in the heterotrophic microorganism culturing step, it is sufficient that the heterotrophic microorganism metabolizes the organic matter released by the photosynthetic microorganism in the photosynthetic microorganism culturing step, and the photosynthetic microorganism culturing step and the heterotrophic microorganism culturing step may be performed under the same culture conditions or different culture conditions. In addition, the photosynthetic microorganism culturing step and the heterotrophic microorganism culturing step may be performed in the same medium (i.e., heterotrophic microorganisms are cultured in a medium containing photosynthetic microorganisms), or may be performed in different media (i.e., heterotrophic microorganisms are cultured in a medium not containing photosynthetic microorganisms). When the photosynthetic microorganism culturing step and the heterotrophic microorganism culturing step are performed in the same medium, for example, after the culture step of culturing the photosynthetic microorganisms, a culture step of culturing the heterotrophic microorganisms in the same medium is performed. Alternatively, the culture step of culturing the photosynthetic microorganisms and the culture step of culturing the heterotrophic microorganisms are simultaneously performed in the same medium. When the photosynthetic microorganism culturing step and the heterotrophic microorganism culturing step are performed in separate media, for example, after or during the culturing step of culturing the photosynthetic microorganisms, organic matter is collected from the medium, and a new medium containing the organic matter is used to culture the heterotrophic microorganisms. Alternatively, after or during the culturing step of culturing the photosynthetic microorganisms, a culturing step is performed in which the photosynthetic microorganisms are separated from the medium, and the heterotrophic microorganisms are cultured using the new medium. Here, the photosynthetic microorganism culturing step and the heterotrophic microorganism culturing step are preferably performed in the same medium, and more preferably performed simultaneously in the same medium, since this can simplify the production process.

[0079] When the heterotrophic microorganism culturing step is carried out under different culture conditions from the photosynthetic microorganism culturing step, more appropriate culture conditions can be selected depending on the heterotrophic microorganism used and the useful organic matter to be produced. For example, the culture conditions include a pH of approximately neutral (pH 6 to 9), a temperature of 25 to 40 degrees, and a culture time of 24 to 48 hours.

[0080] (Culture medium) The medium for culturing heterotrophic microorganisms in the heterotrophic microorganism culturing step can basically be the same as the medium for culturing photosynthetic microorganisms, and as described above, the medium in which photosynthetic microorganisms are cultured or the medium in which photosynthetic microorganisms are cultured can also be used. However, when the photosynthetic microorganism culturing step and the heterotrophic microorganism culturing step are performed separately, a medium for culturing heterotrophic microorganisms different from the medium for culturing photosynthetic microorganisms may be used. In this case, the medium can be appropriately selected and used depending on the heterotrophic microorganisms used and the useful organic matter to be produced. For example, a rich nutrient medium for bacteria that is widely used, such as LB medium, can be used.

[0081] <Photosynthesis process> The method for producing useful organic matter of the present invention may further include, for example, a photosynthesis step in which photosynthetic microorganisms are cultured under photoautotrophic conditions. This photosynthesis step is a step in which photosynthetic microorganisms are cultured under photoautotrophic conditions and photosynthetically grown. In this step, the photosynthetic microorganisms fix carbon using carbon dioxide, bicarbonate, or the like as a carbon source by culturing under photoautotrophic conditions. By carrying out the photosynthesis step before the photosynthetic microorganism culture step described above, the photosynthetic microorganisms can generate and release organic acids by utilizing the organic matter stored in the cells in this photosynthetic microorganism culture step, so it is preferable to carry out the photosynthesis step before the photosynthetic microorganism culture step.

[0082] In the present invention, the "photoautotrophic" condition refers to a state in which photosynthetic microorganisms produce sugar from CO2 and water by photosynthesis and grow using this as an energy source. The light irradiation conditions during photoautotrophy may be either natural light or artificial light, and the intensity of the light irradiation can be appropriately adjusted depending on the density of the photosynthetic microorganisms in the medium and the depth of the culture tank, etc. For example, 30 to 2,000 μmol photons m -2 s -1 , preferably 30 to 1,000 μmol photons m -2 s -1 , more preferably 50 to 600 μmol photons m -2 s -1Natural or artificial light of about 1000 nm may be used. When the light intensity is within the above range, photosynthetic microorganisms can photosynthesize and grow smoothly. The light irradiation may be continuous or periodic. For large-scale outdoor cultivation, a light / dark cycle may be provided to minimize costs and avoid additional costs for artificial lighting (i.e., natural light such as sunlight with a light / dark cycle may be used).

[0083] The culture conditions and medium in the photosynthesis step other than the photoautotrophic conditions can be appropriately determined in more detail depending on the photosynthetic microorganism used. Typically, the culture conditions and medium of the photosynthetic microorganism in the photosynthetic microorganism culture step may be similar to those in the photosynthetic microorganism culture step, and the above description can be referred to.

[0084] <Useful organic substance recovery process> The method for producing useful organic matter of the present invention may further include a useful organic matter recovery step of recovering useful organic matter after the heterotrophic microorganism culture step. This useful organic matter recovery step is a step of recovering useful organic matter produced in the photosynthetic microorganism culture step and the heterotrophic microorganism culture step. In this step, the useful organic matter can be separated and purified from the cultured microorganism and / or culture medium by any separation and purification method that may be developed in the future, as necessary. When recovering useful organic matter from the medium, specifically, the cultured microorganism and its product are separated by ultrafiltration membrane separation, centrifugation, concentration, etc., and then the desired useful organic matter is purified by a known method such as a column method or a crystallization method, and then dried to recover it as crystals.

[0085] <Production of useful organic matter through a semi-permanent cycle> The method for producing useful organic matter of the present invention can produce useful organic matter in a semi-permanent cycle by repeating the above-mentioned photosynthesis step, photosynthetic microorganism culturing step, heterotrophic microorganism culturing step, and useful organic matter recovery step. The present invention also includes a method for producing useful organic matter in such a semi-permanent cycle. That is, in the photosynthesis step, photosynthetic microorganisms are cultured under photoautotrophic conditions, and the photosynthetic microorganisms fix CO2 and the like in the atmosphere to synthesize organic matter and grow and multiply. Next, in the photosynthetic microorganism culturing step, the photosynthetic microorganisms are cultured under specific culture conditions (for example, in a dark anaerobic environment), and the photosynthetic microorganisms release organic matter outside the cells. Then, in the heterotrophic microorganism culturing step, the heterotrophic microorganisms are cultured under specific culture conditions (for example, in an anaerobic environment), and the heterotrophic microorganisms metabolize the organic matter released by the photosynthetic microorganisms to produce the desired useful organic matter. Thereafter, in the useful organic matter recovery process, the produced useful organic matter is recovered from the medium, and then the photosynthetic microorganisms are suspended in a new medium, and the photosynthesis process, photosynthetic microorganism culture process, heterotrophic microorganism culture process, and useful organic matter recovery process can be continuously carried out again. This method for producing organic acid using a semi-permanent cycle makes it possible to efficiently and continuously produce large amounts of useful organic matter. In this method for producing useful organic matter using a semi-permanent cycle, as described above, it is preferable that the photosynthetic microorganism culture process and the heterotrophic microorganism culture process are simultaneously carried out in the same medium.

[0086] <Useful organic matter to be produced> The useful organic matter (target useful organic matter) to be produced in the present invention is not particularly limited, but includes organic acids such as C4 dicarboxylic acids, amino acids, alcohols such as isobutanol, aromatic compounds such as coumaric acid, diamines, diols, biopolymers such as polyhydroxyalkanoic acid, etc. Examples of organic acids include C4 dicarboxylic acids and citric acid. Examples of C4 dicarboxylic acids include succinic acid, fumaric acid, malic acid, etc. Examples of amino acids include glutamic acid and tyrosine, etc. Examples of alcohols include isobutanol and isopropanol, etc. Examples of aromatic compounds include coumaric acid, ferulic acid, resveratrol, naringenin, etc. Examples of diamines include putrescine and cadaverine, etc. Examples of diols include 1,4-butanediol and 1,3-propanediol, etc. Examples of biopolymers include polyhydroxyalkanoic acid and polylactic acid, etc.

[0087] In the present invention, a combination of photosynthetic microorganisms and heterotrophic microorganisms appropriate for the production of useful organic matter can be selected depending on the useful organic matter to be produced. The type and efficiency of organic matter released during culture differs depending on the type of photosynthetic microorganism, and the organic matter metabolized and useful organic matter produced differ depending on the type of heterotrophic microorganism. Therefore, in order to produce the desired useful organic matter, it is important to select an appropriate combination of photosynthetic microorganisms and heterotrophic microorganisms. These combinations are appropriately selected based on the above description.

[0088] Such combinations of photosynthetic microorganisms and heterotrophic microorganisms include the following:

[0089] When the target useful organic matter is succinic acid, it is preferable to use microalgae, cyanobacteria, etc. (preferably cyanobacteria) as the photosynthetic microorganism, and to use a microorganism having a Dcu system (preferably Enterobacter bacteria or Escherichia bacteria) as the heterotrophic microorganism.

[0090] It is also preferable to use a photosynthetic microorganism capable of anaerobic fermentation as the photosynthetic microorganism, and to select and use a heterotrophic microorganism capable of producing useful organic matter (e.g., organic acids, amino acids, alcohols, aromatic compounds, diamines, diols, biopolymers, etc.) using organic matter (e.g., organic acids, glycerol, etc.) produced by the photosynthetic microorganism through anaerobic fermentation as a carbon source. Here, the heterotrophic microorganism may also be a genetically modified microorganism (e.g., Escherichia coli, Pseudomonas putida, Corynebacterium, hydrogen-oxidizing bacteria, budding yeast, fission yeast, Pichia yeast, actinomycetes, filamentous fungi, etc.) that has been metabolically modified to produce the desired useful organic matter. For example, when the useful organic matter is malic acid, the microalgae Chlamydomonas reinhardtii can be used as the photosynthetic microorganism, and the heterotrophic microorganisms can be Ustilago trichophora, Saccharomyces cerevisiae, Aspargillus niger, Aspargillus oryzae, Escherichia coli, etc. When the useful organic matter is fumaric acid, the microalgae Chlamydomonas reinhardtii can be used as the photosynthetic microorganism, and the heterotrophic microorganisms can be Rhizopus alitus, Rhizopus oryzae, Lactobacillus plantarum, Escherichia coli, etc. For example, when the useful organic matter is an aromatic compound (resveratrol, naringenin, etc.), the photosynthetic microorganism can be the microalga Chlamydomonas reinhardtii, and the heterotrophic microorganism can be a metabolically engineered Pichia yeast (see Kumokita et al., ACS Synth. Biol. 2022, 11, 6, 2098-2107). For example, when the useful organic matter is a biopolymer (polyhydroxyalkanoic acid, etc.), the photosynthetic microorganism can be the microalga Chlamydomonas reinhardtii, and the heterotrophic microorganism can be Cupriavidus necator.

[0091] <Isolated microorganisms> The microorganisms used in the present invention may be, for example, isolated microorganisms. When using isolated microorganisms in the present invention, for example, isolated microorganisms may be used only for photosynthetic microorganisms, isolated microorganisms may be used only for heterotrophic microorganisms, or isolated microorganisms may be used for both photosynthetic microorganisms and heterotrophic microorganisms.

[0092] In the present invention, "isolated" when referring to a microorganism means that the strain of the microorganism is taken out from the environment in which it naturally exists. For example, an isolated microorganism can be obtained by isolating and culturing (pure culture) one strain from among microorganisms that are mixed in nature. In addition, "isolated" means that it has been isolated at least once in the past. Therefore, in the present invention, a plurality of microorganisms may be mixed in one medium, and the microorganism in this state can also be called an isolated microorganism if it is a microorganism that has been isolated and cultured (pure culture) in the past.

[0093] In the present invention, the isolated microorganism is advantageous in that it is easy to genetically modify, for example. Since metabolic modification can be easily performed based on design in the microorganism to be used, it is possible to try various metabolic modifications in the hope of improving the production of the desired useful organic matter.

[0094] Furthermore, since isolated microorganisms can be used in the present invention as described above, the method for producing useful organic matter of the present invention can further include, for example, a photosynthetic microorganism preparation step of isolating and culturing (pure culture) photosynthetic microorganisms, and / or a heterotrophic microorganism preparation step of isolating and culturing (pure culture) heterotrophic microorganisms. These steps can typically be carried out before the cultivation steps of photosynthetic microorganisms and heterotrophic microorganisms, respectively. Furthermore, the microorganisms obtained in the preparation step can be used in the cultivation steps of photosynthetic microorganisms and heterotrophic microorganisms, respectively.

[0095] [Composition for producing useful organic substances] The first useful organic matter producing composition of the present invention is a composition for producing useful organic matter, comprising heterotrophic microorganisms, characterized in that it is used in combination with photosynthetic microorganisms. This first useful organic matter producing composition comprises heterotrophic microorganisms and is characterized in that it is used in combination with photosynthetic microorganisms, and other components, conditions, etc. are not limited. And, as described in the method for producing useful organic matter of the present invention above, this useful organic matter producing composition is characterized in that it combines photosynthetic microorganisms and heterotrophic microorganisms, and is therefore suitable for use in producing useful organic matter.

[0096] The second useful organic matter producing composition of the present invention is a useful organic matter producing composition containing photosynthetic microorganisms and heterotrophic microorganisms. This second useful organic matter producing composition is characterized by containing photosynthetic microorganisms and heterotrophic microorganisms, and other components, conditions, etc. are not limited. And, as described in the useful organic matter producing method of the present invention described above, this useful organic matter producing composition is characterized by combining photosynthetic microorganisms and heterotrophic microorganisms, and is suitable for use in producing useful organic matter. Note that the explanation of the useful organic matter producing method of the present invention described above can be used for these first and second useful organic matter producing compositions.

[0097] [Kit for producing useful organic substances] The useful organic matter producing kit of the present invention is a useful organic matter producing kit containing photosynthetic microorganisms and heterotrophic microorganisms. This useful organic matter producing kit is characterized by containing photosynthetic microorganisms and heterotrophic microorganisms, and other components, conditions, etc. are not limited. As described in the useful organic matter producing method of the present invention described above, this useful organic matter producing kit is characterized by combining photosynthetic microorganisms and heterotrophic microorganisms, and is therefore suitable for use in producing useful organic matter. Note that the description of the useful organic matter producing method of the present invention described above can be used for this useful organic matter producing kit.

[0098] [Microorganism group for producing useful organic substances] The useful organic matter producing microbial community of the present invention is a useful organic matter producing microbial community consisting of photosynthetic microorganisms and heterotrophic microorganisms. This useful organic matter producing microbial community is characterized by being composed of photosynthetic microorganisms and heterotrophic microorganisms, and other conditions are not limited. As described in the useful organic matter producing method of the present invention described above, this useful organic matter producing microbial community is characterized by combining photosynthetic microorganisms and heterotrophic microorganisms, and is therefore suitable for use in producing useful organic matter. Note that the description of the useful organic matter producing method of the present invention described above can be used for this useful organic matter producing microbial community. EXAMPLES

[0099] The present invention will be specifically described in the following examples, but the present invention is not limited to these examples.

[0100] 1. Materials and Methods 1.1. Strains and culture conditions The recombinant strains used in this study were constructed based on the glucose-tolerant (GT) strain Synechocystis sp. PCC6803 (see Williams, JGK Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803. Methods Enzymol. 1988, 167, 766-778. DOI: 10.1016 / 0076-6879(88)67088-1). The recombinant Synechocystis sp. PCC6803 strain was inoculated into modified BG11 medium containing 5 mM ammonium chloride and 0.1 M HEPES-KOH (pH 7.8) in the presence or absence of 50 μg / mL kanamycin and / or 34 μg / mL chloramphenicol. Cultures were grown at 105–115 μmol / m 2 The experiment was carried out at 30°C under continuous light irradiation with a photon flux density of 1 / s and 1% (v / v) CO2. The cell density was measured at 750 nm (OD 750Dry cell weight (DCW) was determined after harvesting the cells by filtration, washing with 20 mM NH4HCO3, and lyophilization. All chemicals were of analytical grade.

[0101] 1.2. Construction of recombinant strains The Escherichia coli DH5α strain (Takara Bio Inc.) was used as a host for gene cloning and plasmid amplification. The Actinobacillus succinogenes pckA gene (UniPro ID: Q6W6X5) was obtained by gene synthesis (ThermoFisher Scientific, Waltham, MA), and the synthesized gene was PCR amplified using the primer pair PEPCK-Fw1 and PEPCK-Rv1. The amplified fragment was cloned into the NdeI site of plasmid pTCP2031 (see Osanai, T.; Shirai, T.; Iijima, H.; Nakaya, Y.; Okamoto, M.; Kondo A.; Hirai, M. Y. Genetic manipulation of a metabolic enzyme and a transcriptional regulator increasing succinate excretion from unicellular cyanobacterium. Front Microbiol. 2015, 6, 1064. DOI: 10.3389 / fmicb.2015.01064) using the In-Fusion® HD Cloning Kit (Takara Bio Inc.) according to the manufacturer's instructions, resulting in pTCP2031-PEPck. To construct the integrative vector, the upstream and downstream regions of slr0646 (1000 bp each) were amplified by PCR using the primer pair uslr0646-Fw and uslr0646-Rv for the upstream region and the primer pair dslr0646-Fw and dslr0646-Rv for the downstream region.The amplified PCR fragment was cloned into the MluI / HindIII and EcoRI / XhoI sites of plasmid pSStrc-slr1556 (see Hasunuma, T.; Matsuda M.; Kondo, A. Improved sugar-free succinate production by Synechocystis sp. PCC 6803 following identification of the limiting steps in glycogen catabolism. Metab. Eng. Commun. 2016, 3, 130-141. DOI: 10.1016 / j.meteno.2016.04.003) using the In-Fusion® HD Cloning Kit, resulting in plasmid pSStrc-0646. The pckA gene was amplified by PCR using the primer pair PEPCK-Fw2 and PEPCK-Rv2, and the resulting fragment was cloned into the NdeI / SalI sites of pSStrc-0646, resulting in pSStrc-PEPCK.

[0102] The principles underlying specific gene integration or disruption in Synechocystis 6803 are described in Hidese, R.; Matsuda, M.; Osanai, T.; Hasunuma T.; Kondo, A. Malic enzyme facilitates d-lactate production through increased pyruvate supply during anoxic dark fermentation in Synechocystis sp. PCC 6803. ACS Synth. Biol. 2020, 9, 260-268. doi: 10.1021 / acssynbio.9b00281. The plasmid pTCP2031-Pepck was used to integrate genes into the host strain Synechocystis Ppc-ox (see Metab. Eng. Commun. 2016, supra), resulting in PCCK. The plasmid pSStrc-PEPck was introduced into the host strain Synechocystis Ppc-ox / ΔackA (see Metab. Eng. Commun. 2016, supra) to obtain the Synechocystis PCCK-ox / ΔackA strain. Integration of each gene cassette was confirmed by PCR. All primer sequences are shown in Table 1 below.

[0103] [Table 1]

[0104] 1.3.Enzyme Assays Photoautotrophically grown cyanobacterial cells (4 g-DCW / L) were fermented at 37°C under dark anoxic conditions, and a crude extract of the fermented cells was obtained using a method previously described (see Metab. Eng. Commun. 2016, supra). Briefly, cells suspended in 50 mM potassium phosphate buffer were disrupted by sonication and centrifuged, and the resulting supernatant was obtained as the crude extract. PEPck activity was measured using a V730-Bio spectrophotometer (Jasco Co., Ltd., Tokyo, Japan) in 0.1 M Mes (pH 6.6), 10 mM MgCl2、 5.0 mM MnCl 2、 1.0 mM DTT, 10 mM adenosine diphosphate (ADP), 75 mM NaHCO 3、 It was measured by monitoring the change in absorbance at 340 nm accompanying the oxidation of NADH in a reaction mixture containing 0.1 mM NADH, 20 U malate dehydrogenase, and crude extract (see Van der Werf, MJ; Guettler, MV; Jain MK; Zeikus, JG Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z. Arch. Microbiol. 1997, 167, 332-342. DOI: 10.1007 / s002030050452.), where the reaction was initiated by the addition of 10 mM phosphoenolpyruvate (PEP) followed by incubation at 30°C for 10 min. To exclude ADP-independent carboxylation of PEP, the same mixture without ADP was used as a control. MDH activity of crude extracts was assayed and measured in a reaction mixture containing 100 mM potassium phosphate buffer (pH 8.0), 0.1 mM NADH, and 1 mM oxaloacetate at 30° C. Protein concentrations were determined by Bradford dye-binding assay using bovine serum albumin for standard curve generation (see Bradford, MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248-254. DOI: 10.1006 / abio.1976.9999.).

[0105] 1.4. Production of organic acids through fermentation Unless otherwise stated, autofermentation was performed as follows: Recombinant Synechocystis sp. strain PCC6803 was cultivated at 30°C under photoautotrophic conditions at an optical density of 0.1 at 750 nm for 3 days. The cultured cells were inoculated at an initial cell concentration of 4 g-DCW / L in 10 mL of 0.1 M HEPES-KOH (pH 7.8) containing 100 mM or 300 mM NaHCO3 and fermented at 37°C for 4 days under dark anoxic conditions (wrapped in foil and bubbled with 100% N2). The accumulation of extracellular organic acids during fermentation was quantified using a high-performance liquid chromatography (HPLC) system (Shimadzu Corporation, Kyoto, Japan) equipped with an Aminex HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and a RID-10A refractive index detector. The HPLC system was operated at 50°C with 5 mM H2SO4 at a flow rate of 0.6 mL / min. Glycogen concentrations were determined by measuring the glucose released from glycogen by enzymatic hydrolysis as previously described (see Metab. Eng. Commun. 2016, supra). Yields were calculated as the ratio of the corresponding amount of C4 dicarboxylic acid to the amount of glucose consumed and the enzymatic hydrolysis of stored intracellular glycogen.

[0106] 1.5.Analysis of intracellular metabolites At each sampling time (6, 24, 48, 72, and 96 h), 5 mg of DCW of cyanobacterial cells in the fermentation culture was obtained by filtration using a 1 μm pore size polytetrafluoroethylene (PTFE) disk (Millipore, Billerica, MA) and immediately washed with 20 mM (NH4)2CO3 pre-cooled to 4 °C. Intracellular metabolites were extracted and analyzed by capillary electrophoresis-mass spectrometry (CE-MS) (Agilent G7100; MS, Agilent G6224AA LC / MSD TOF; Agilent Technologies, Palo Alto, CA) according to a method previously described (see Metab. Eng. Commun. 2016, supra).

[0107] 1.6. Pretreatment of CSL solution for fermentation and isolation of CSL-settling microorganisms Powdered corn steep liquor (CSL) (Sollis 095E, Catalog No.: C42166) was purchased from Oriental Yeast Co., Ltd. (Osaka, Japan). Other CSLs were obtained from Spectrum Chemicals Australia (Cat. No.: C3848; Astral Scientific Pty Ltd., NSW, Australia) and Sigma-Aldrich Co. LLC (Cat. No.: C4648-500G; St. Louis, MO, USA). Unless otherwise stated, powdered CSL was suspended in purified water (concentration 1 g / L) and incubated at 37°C for 24 h. Supernatants were obtained from the CSL suspensions by centrifugation (20000g, 10 min). Sterile CSL was obtained by autoclaving the suspensions and then centrifugation. Five mL of the resulting supernatant was mixed with an equal volume of Synechocystis solution (0.1 M HEPES-KOH (pH 7.8) containing NaHCO3, initial cell concentration 8 g-DCW / L).

[0108] For isolation of CSL-settling microorganisms, the supernatant of a 1 g / L CSL suspension was streaked onto Luria-Bertani agar medium and incubated at 37°C for 24 h. Individual colonies grown on LB agar medium were randomly picked and inoculated into LB liquid medium based on colony morphology. 16S rDNA of isolated microorganisms was amplified by PCR using the universal primer set 27F / 1492R, and partial DNA sequences were analyzed by Sanger sequencing. 27F 5'-AGAGAGTTTGATCCTGCTCAG-3' (SEQ ID NO: 19) 1492R 5'-GGTTACCTTGTTACGTTACGACTT-3' (SEQ ID NO: 20)

[0109] Three related strains, Enterobacter hormaechei (NBRC 105718), Escherichia hermannii (NBRC 105704), and Acinetobacter radioresistens (NBRC 102413), identified by BLAST-guided search, and E. coli strain BW25113 from the KEIO collection, as well as knockout mutants E. coli ΔdcuA and E. coli ΔdcuB, were purchased from the National Institute of Technology and Evaluation (NITE) Biological Resource Center (Japan). Each strain was grown at an optical density of 2.5 × 10- 5 Five mL of the sterilized CSL supernatant suspended in was mixed with an equal volume of Synechocystis solution (0.1 M HEPES-KOH (pH 7.8) containing NaHCO3, initial cell concentration 8 g-DCW / L).

[0110] 2.Results 2.1.Effect of overexpression of phosphoenolpyruvate carboxykinase on organic acid production To overexpress the pckA gene, the gene was introduced into the slr0618 locus of Synechocystis Ppc-ox strain by homologous recombination. A control strain (CT) was created by integrating a chloramphenicol resistance gene into the locus (Figure 1). PEPck activity was observed in the resulting PCCK strains, but not in the Ppc-ox strain. Autofermentation of the resulting PCCK strains was performed in the presence of various concentrations of NaHCO3 (100-500 mM) as a substrate for Ppc, at 37 °C for 72 h in dark anoxic conditions to initiate organic acid secretion (Figure 2). The highest succinic and fumaric acid production was observed in the PCCK strains treated with 100 mM NaHCO3. Lactic acid levels increased in a dose-dependent manner in all strains. The lactate level of the Ppc-ox strain was more than 1.5-fold higher than that of the PCCK and Ppc-ox / CT strains, suggesting that the addition of chloramphenicol contributed to the decrease in lactate level. The concentrations of malate (557 mg / L), fumarate (843 mg / L), and succinate (368 mg / L) of the PCCK strain were quantitatively higher than those of the Ppc-ox strain, but the concentration of acetate (446 mg / L) was lower than that of the Ppc-ox strain. Expression of PEPck increased the concentrations of malate and fumarate by more than 1.3- and 1.4-fold, respectively, by 72 h of fermentation in 100 mM NaHCO3. The concentrations of malate and succinate increased in a time-dependent manner during fermentation, whereas the concentration of fumarate peaked at 72 h of fermentation (Fig. 3). This is probably because the initial glycogen content before fermentation was similar (approximately 45%) between the Ppc-ox and PCCK-ox strains. The rates of glycogen consumption were comparable (18% remained after 72 h). Overall, expression of PEPck reduced acetate levels and enhanced the TCA reductive pathway.

[0111] 2.2.Metabolomic analysis of PCCK strain To investigate the effect of PEPck on the primary metabolism of recombinant Synchocystis 6803, intracellular metabolites were analyzed after 3, 6, 24, 48, 72, and 96 h of fermentation. The pool sizes of hexose, pentose, triose phosphates, organic acids, and acetyl-CoA were obtained by calculating the respective peak areas using CE-MS analysis. The pool sizes of hexose phosphates (G1P, G6P, F6P, and FBP), pentose phosphates (Ru5P and RuBP), and other Calvin-Benson-Bassham intermediates (6PG and S7P) in PCCK cells were significantly lower than those of the Ppc-ox strain during fermentation (Figure 4A). In the Ppc-ox strain, triose phosphates PEP, 2PGA, and 3PGA increased from 48 to 96 h of fermentation and peaked at 96 h. Also, intracellular fumarate and malate accumulated in the PCCK strain during fermentation, whereas the levels of iso-citrate, citrate, and cis-aconitrate, which are metabolites of the TCA oxidative pathway, were relatively low during 0–24 h of fermentation (Figure 4B). Citrate transiently accumulated in the Ppc-ox strain during 0–6 h of fermentation. No pyruvate pool was observed in fermenting PCCK cells; however, the pool size of acetyl-CoA was significantly higher than in the Ppc-ox strain. Oxaloacetate was not quantified due to its low abundance by mass spectrometry. Low adenosine phosphate levels were observed in the PCCK strain. However, the cellular energy charge, calculated as the ratio of ATP to adenosine 5′-diphosphate (ADP) + ATP, was lower in the PCCK strain (0.46 at 3 h and 0.45 at 6 h) than in the Ppc-ox strain (0.62 at 3 h and 0.68 at 6 h) during the early phase of fermentation (0–6 h) and was associated with lower acetate levels, but was comparable to each other during the middle and late phases of fermentation (24–96 h) ( Fig. 4C ).

[0112] 2.3. Corn steep liquor promotes the production of C4 dicarboxylic acids Expression of PEPck contributed to the improvement of C4 dicarboxylic acid production by debottlenecking PEP metabolism to the reductive TCA pathway. However, it was accompanied by high glycogen consumption, and further improvements are needed to increase the production of C4 dicarboxylic acids. The glycogen consumption rate could be increased by increasing the amount of NaHCO3. However, carbon from glycogen is diverted to lactate production due to insufficient inflow into the open TCA cycle of cyanobacteria. Nutrient supplementation is thought to promote carbon flow to the TCA cycle and promote the synthesis of amino acids and proteins, but the effect of nutrient supplementation on cyanobacterial fermentation has not been studied. Among nutritional supplements, CSL, commonly known as "concentrated fermented corn extract," which is rich in organic acids, amino acids, sugars, minerals, and vitamins, has been utilized as a fermentation nutritional supplement due to its high nutritional value and low cost. First, we investigated the effect of non-sterile CSL (raw CSL) (Sollis 095E) treated at 37 °C on fermentation by recombinant Synechocystis with different concentrations of NaHCO3 for 72 h. CE-MS analysis revealed that 1 g / L of Sollys CSL contained 138.3 mg / L lactate, 11.7 mg / L gluconate, 19.6 mg / L L-alanine, 16.0 mg / L L-leucine, and 6.8 mg / L succinate as major organic acids. As shown in Figure 5, succinate production was highest with 300 mM NaHCO3, while lactate and acetate levels remained low at 100–400 mM NaHCO3. The time course of C4 dicarboxylic acids produced by the recombinant Synechocystis strain fermented with 300 mM NaHCO3 showed that succinate levels reached 1621 mg / L at 96 h, whereas malic acid and fumaric acid were not observed after 48 h of fermentation (Figure 6A). Next, because CSL components are not completely solubilized in water, we investigated pretreatment conditions to prepare CSL suspensions by changing the treatment temperature (30, 40, 50, 60, and 80 °C) to extract the active components. Surprisingly, in the autofermentation of PCCK, malic acid increased and succinic acid decreased in a temperature-dependent manner (Figure 7A). Accumulation of malate was observed at high temperatures.Although succinic acid was increased in dark autofermentations using other commercial CSLs, no increasing effect on malic, fumaric, or succinic acids was observed when 1 g / L of yeast extract was tested (Figure 8), indicating that the supplementation of nitrogen source was not the main reason for the increase in C4 dicarboxylic acids.

[0113] We analyzed the production of C4 dicarboxylic acids by autofermentation of the PCCK strain with 300 mM NaHCO3 in the presence of autoclaved CSL. As expected, malic acid accumulation was observed in the PCCK strain compared to the Ppc-ox strain, but succinic acid levels in the PCCK and Ppc-ox strains were similar (Figure 6B), suggesting that unknown active components promoting succinic acid production were lost during sterilization. Acetic acid and lactic acid production were significantly suppressed in the PCCK strain compared to the Ppc-ox strain. In contrast to the change in malic acid level, the fumaric acid level in the PCCK strain was comparable to that in the PCCK strain with 100 mM NaHCO3 in the absence of sterilized CSL. Furthermore, the glycogen consumption rate of the Ppc-ox strain (3.07% after 48 h) was higher than that of the PCCK strain (3.25% after 72 h). This likely reflects the overproduction of lactate and acetate as the final output of energy metabolism in the Ppc-ox strain. In the presence of sterile CSL, the activity of malate dehydrogenase (MDH), which catalyzes the NADH-dependent reduction of oxaloacetate to malate, in the PCCK strain was similar to that in the absence of sterile CSL (FIG. 9A).

[0114] 2.4. Identification of the microorganism responsible for succinic acid production in CSL As mentioned above, some active ingredients contributing to succinic acid production were susceptible to heat treatment, suggesting the possibility that succinic acid-producing microorganisms may be present in the CSL suspension. The CSL suspension treated at 37 °C was streaked on an LB agar plate to isolate microorganisms contributing to the increase in succinic acid. Three colonies that appeared on the LB agar plate at 37 °C were analyzed by 16S rDNA analysis to identify related bacteria (Figure 7B). As a result of BLAST search, each colony was derived from a strain closely related to the enterobacteria Enterobacter hormaechei, Escherichia hermannii, and Acinetobacter radioresistens, with a similarity score of 99% or more. Next, we investigated the effect of adding standard microorganisms distributed by the Public Biological Resources Agency (National Institute of Technology and Evaluation, Biological Resources Center) on Synechocystis fermentation using sterilized CSL (Figure 10). In addition to the contribution of E. hormaechei to succinic acid production (814 mg / L), the addition of E. hermannii was the most effective for succinic acid production (1410 mg / L) without producing malic acid or fumaric acid, suggesting that some microbial strains in the raw CSL, including E. hormaechei, contributed to succinic acid production (1788 mg / L). The addition of A. radioesistens had no effect on succinic acid production or consumption of malic acid and fumaric acid. A similar tendency was also observed in the absence of sterile CSL (addition of bacteria only) (Figure 11). In other words, it was suggested that malic acid and fumaric acid excreted by cyanobacteria were consumed by E. hormaechei and E. hermannii, increasing succinic acid production.

[0115] It has been reported that E. coli K-12 can catabolize malate or fumarate to succinate through the action of the fumarate / succinate antiporter system DcuABC under fermentation conditions. In this study, when E. coli K-12 BW25113 was added to Synechocystis fermentation, succinate was produced similarly to that of E. hermannii (Figure 10). Meanwhile, deletion of the dcuB gene encoding the DcuB protein, which plays a major role as a component of the DcuABC complex in the uptake of malate and fumarate, in E. coli resulted in a decrease in succinate levels. However, malate and fumarate did not disappear completely, and approximately 1 / 5 or 1 / 20 of the amount remained when sterilized CSL was used without the addition of the strain, indicating that the uptake ability of malate and fumarate was partially impaired.

[0116] 2.5. Evaluation of High-Density Fermentation for C4 Dicarboxylic Acid Production Previous reports have clearly demonstrated that the initial concentration of Synechocystis cells is important for increasing succinate titers. However, acetate levels usually increase with increasing initial concentration. Prior to high-density fermentation of Synechocystis strains, the pckA gene was introduced into the slr0646 locus of the Ppc-ox / ΔackA strain, which has a genetic background with a deletion of the endogenous ackA (sll1299) gene encoding acetate kinase in the Ppc-ox strain. The constructed PEPck overexpression system was able to efficiently express P trc Controlled by P psbA2The PEPck activity of PCCK-ox / ΔackA cells was approximately 3.5-fold higher than that of PCCK cells (Figure 9B). We next evaluated the production of C4 dicarboxylic acids, lactate and acetate, in PCCK-ox / ΔackA cells and PCCK cells after 72 h of fermentation in the presence of sterile or live CSL at different initial cell concentrations (4, 10, and 20 g dry cell weight / L) (Figure 12). As expected, deletion of ackA led to a significant decrease in acetate concentration at any initial cell concentration. And instead, fumarate (2286 mg / L) or succinate (5639 mg / L) peaked with sterile or live CSL at 20 g DCW / L of PCCK-ox / ΔackA, respectively. With sterile CSL, malate concentration was very similar between both strains (approximately 3100 mg / L). In fermentation in the presence of live CSL, malic acid and fumaric acid were not observed due to the fermentation action of CSL-settling microorganisms. [Industrial Applicability]

[0117] According to the present invention, it is possible to provide a method for producing useful organic substances using photosynthetic microorganisms, which is excellent in the production amount and yield of the target useful organic substances (such as organic acids).

Claims

1. A photosynthetic microorganism culturing step of culturing photosynthetic microorganisms, wherein the photosynthetic microorganisms release organic matter outside the cells during the culturing step; and A heterotrophic microorganism culturing step for culturing heterotrophic microorganisms, wherein the heterotrophic microorganisms metabolize the organic matter released by the photosynthetic microorganisms during the culturing process to produce the desired useful organic matter. A method for producing useful organic matter, comprising:

2. The method according to claim 1 , wherein the photosynthetic microorganism culturing step and the heterotrophic microorganism culturing step are carried out in the same medium.

3. 3. The method according to claim 2, wherein the photosynthetic microorganism culturing step and the heterotrophic microorganism culturing step are carried out simultaneously in the same medium.

4. The method according to claim 1, wherein the organic substance released from the photosynthetic microorganism is an organic acid.

5. The method according to claim 4, wherein the organic matter released from the photosynthetic microorganism is a C4 dicarboxylic acid.

6. The method according to claim 1, wherein the photosynthetic microorganism is at least one selected from microalgae and cyanobacteria.

7. The method according to claim 6, wherein the photosynthetic microorganism is a cyanobacterium.

8. The method of claim 1 , wherein the photosynthetic microorganism is a recombinant organism.

9. The method according to claim 1 , wherein the photosynthetic microorganism culturing step is carried out in a dark anaerobic environment.

10. 2. The method of claim 1, wherein the heterotrophic microorganism produces a C4 dicarboxylic acid.

11. The method of claim 1 , wherein the heterotrophic microorganism produces succinic acid.

12. The method according to claim 1, wherein the heterotrophic microorganism releases the desired useful organic matter outside the cell.

13. The method according to claim 12, wherein the heterotrophic microorganism has a Dcu system.

14. The method according to claim 13, wherein the heterotrophic microorganism is at least one selected from the group consisting of bacteria of the genus Enterobacter and bacteria of the genus Escherichia.

15. The method according to claim 1 , wherein the heterotrophic microorganism culturing step is carried out in an anaerobic environment.

16. 2. The method according to claim 1, wherein the photosynthetic microorganisms are cultured in a medium containing corn steep liquor.

17. The method according to claim 1 , further comprising a photosynthesis step of culturing a photosynthetic microorganism under photoautotrophic conditions.

18. The method according to claim 1, further comprising a useful organic matter recovery step of recovering useful organic matter after the heterotrophic microorganism cultivation step.

19. 2. The method according to claim 1, wherein the target useful organic substance is succinic acid.

20. 2. The method of claim 1, wherein the heterotrophic microorganism is an isolated microorganism.

21. A composition for producing useful organic matter, comprising heterotrophic microorganisms, which is used in combination with photosynthetic microorganisms.

22. A composition for producing useful organic matter, comprising photosynthetic microorganisms and heterotrophic microorganisms.

23. A kit for producing useful organic matter, comprising photosynthetic microorganisms and heterotrophic microorganisms.

24. A group of microorganisms for producing useful organic matter, consisting of photosynthetic microorganisms and heterotrophic microorganisms.

Images