Method for producing lipid using microorganism having chlorophyll and culture system therefor

Abstract

A method according to the present invention is for producing lipids by biosynthesis, and comprises photoautotrophically culturing a microorganism having chlorophyll in the presence of light and water, thereby producing the lipids, wherein the microorganism is resistant to photoinhibition action.

Description

Method for producing lipids using microorganisms containing chlorophyll and culture system therefor 【0001】 The present invention relates to a method for producing lipid compounds by biosynthesis using chlorophyll-containing microorganisms such as cyanobacteria, which can serve as a next-generation biofuel that can replace petroleum-derived diesel fuel and is carbon-neutral, and to a culture system suitable for said method. This application claims priority based on Japanese Patent Application No. 2024-218911, filed in Japan on December 13, 2024, the contents of which are incorporated herein by reference. 【0002】 Given the depletion of fossil fuels, the SDGs, and the realization of a decarbonized society, developing carbon-neutral next-generation biofuel production technologies has become one of the most important issues to address. Known methods for producing such next-generation biofuels include gasifying woody cellulose to synthesize liquid fuel and refining oils derived from microalgae. For example, research and development is underway to produce free fatty acids by controlling the expression of fatty acid synthesis genes in photosynthetic microorganisms. 【0003】 However, when using oil-producing algae such as Botryococcus, Aurantiochytrium, Pseudochoricystis, Euglena, and Nannochloropsis, it is necessary to add carbon compounds such as sugars as nutrients during cultivation, which poses a problem from a carbon neutrality perspective. 【0004】On the other hand, cyanobacteria are a group of eubacteria that perform photosynthesis using chlorophyll, and are known as photosynthetic microorganisms that produce oxygen and fix carbon dioxide through photosynthesis. It is thought that cyanobacteria entered into intracellular symbiosis (primary symbiosis) with eukaryotes over a billion years ago, which is the origin of chloroplasts, and they are widely used in photosynthesis research as the ancestors of chloroplasts. Among these cyanobacteria, attempts have been made to synthesize fatty acids by controlling the expression of fatty acid synthase genes using the mesophilic cyanobacteria Synechococcus PCC7942 and PCC6803 (for example, Non-Patent Documents 2-3). 【0005】 However, existing methods using mesophilic cyanobacteria show a slight increase in free fatty acids, but have the fatal problem of preventing cell proliferation and leading to lethality. Recent studies have revealed that increased free fatty acids within cells induce photoinhibition of photosynthetic proteins, destroying them and causing cell death, suggesting that photosynthetic microorganisms regulate the amount of free fatty acid synthesized in accordance with their photosynthetic function (e.g., Non-Patent Document 1). Therefore, to date, no method has been reported for efficiently synthesizing free fatty acids using cyanobacteria without inducing photoinhibition of photosynthetic proteins. 【0006】 Jimbo, H., Takagi,K.,Hirashima, T.,Nishiyama, Y., Wada, H., Int.J. Mal. Sci.(2020) 21, 7509Ruffing AM and Jones HD, Biotechnol Bioeng.(2012) 109, 2190-2199Santos-Merino M., Garcillan-Barcia, M,P., de la Cruz, F., Biotechnol. Biofuels. (2018) 【0007】 Therefore, the object of the present invention is to provide a novel method for efficiently producing lipid compounds that can be used as next-generation biofuels by biosynthesis using chlorophyll-containing microorganisms such as cyanobacteria. 【0008】As a result of diligent research to solve the aforementioned problems, the present inventors have found that by using photosynthetic microorganisms such as cyanobacteria whose resistance to photoinhibition has been enhanced by modifying photosynthetic protein genes, and culturing such microorganisms, lipid compounds that can be used as next-generation biofuels can be efficiently produced by biosynthesis. Furthermore, they have also found that this production method does not require the addition of carbon compounds such as sugars as nutrients, can be cultured photoautotrophically using light and water, and can be used in a wide temperature range including high temperatures above room temperature. Based on these findings, the present invention has been completed. 【0009】In other words, the present invention relates in one embodiment to a method for producing lipids using photosynthetic microorganisms resistant to photoinhibition, more specifically: <1> A method for producing lipids by biosynthesis, comprising the steps of: culturing a microorganism having chlorophyll photoautotrophically in the presence of light and water; and producing the lipids thereby, wherein the microorganism is resistant to photoinhibition, the cultivation is carried out in a temperature range of 20 to 90°C, and the production is carried out in a temperature range of 40 to 90°C; <2> The method according to <1>, wherein the microorganism is a transformant that is resistant to photoinhibition by modification of photosynthetic protein genes; <3> The method according to <1>, wherein the cultivation and production are carried out in a temperature range of 40 to 90°C; <3a> The method according to <1>, wherein the cultivation is carried out in a temperature range of 20 to 90°C; <3b> The method according to <1>, wherein the cultivation is carried out in a temperature range of 20 to 90°C and the production is carried out in a temperature range of 40 to 90°C; <3c> The method according to <1>, wherein the culture is carried out in a temperature range of 20 to 90°C and the production is carried out in a temperature range of 45 to 90°C; <4> The method according to <1>, wherein the photoexcitation rate in the photosystem of the microorganism is 200 fs or more and the charge separation rate is 8 ps or more.<5> The method according to <1> above, wherein the cultivation and production are carried out under conditions of a carbon dioxide concentration of 0.03 to 90%; <6> The method according to <1> above, wherein the cultivation and production are carried out under conditions of a carbon dioxide concentration of 3 to 50%; <7> The method according to <1> above, wherein the cultivation is carried out photoautotrophically without the addition of a substance containing hydrocarbons as a nutrient source; <8> The method according to <1> above, wherein the microorganism is a cyanobacterium; <9> The method according to <8> above, wherein the microorganism is a thermophilic cyanobacterium; <10> The method according to <9> above, wherein the microorganism is a thermophilic cyanobacterium belonging to the genus Thermosynechococcus; <11> The method according to <1> above, wherein the microorganism is an alga; <12> The method according to <1> above, wherein the microorganism is a mutant having a structure in which the threonine residue (T) located at positions 177 to 182 in the D1 protein is replaced with a histidine residue (H); <13> The method according to <1>, wherein the microorganism further has a structure in which the isoleucine residue (I) located at positions 175 to 181 in the D2 protein is replaced with a histidine residue (H); and <14> The method according to <7>, wherein the lipid is a free fatty acid, glycolipid, phospholipid, or any combination thereof. 【0010】In another embodiment, the present invention also relates to a culture system suitable for the above-mentioned production method, specifically: <15> A culture system for producing lipids by biosynthesis, comprising: a) a chlorophyll-containing microorganism resistant to photoinhibition, b) a culture medium, and c) a culture facility; <16> The culture system according to <15>, wherein the culture medium does not contain a substance containing hydrocarbons as a nutrient source; <17> The culture system according to <15>, wherein the microorganism is a transformant resistant to photoinhibition due to modification of photosynthetic protein genes; <18> The culture system according to <15>, wherein the microorganism is a thermophilic cyanobacterium belonging to the genus Thermosynechococcus; <19> The culture system according to <15>, wherein the microorganism is an alga; <20> The culture system according to <15>, wherein the microorganism is a mutant having a structure in which threonine residues (T) located at positions 177 to 182 in the D1 protein are replaced with histidine residues (H); and <21> The culture system described in <15> further comprises a microorganism having a structure in which the isoleucine residue (I) located at positions 175 to 181 in the D2 protein is replaced with a histidine residue (H). 【0011】 According to the manufacturing method of the present invention, the addition of carbon compounds such as sugars as nutrients is unnecessary, and lipid compounds that can be used as next-generation biofuels can be efficiently produced by biosynthesis through photoautotrophic cultivation using light and water. In other words, since energy is synthesized using water and light through photosynthesis and carbon dioxide from the air is taken in and metabolized into sugar, it has the advantage of being a carbon-neutral method that allows lipid synthesis using only clean materials that do not require organic matter, and can contribute to solving both energy problems and environmental problems. 【0012】 Furthermore, as shown in the examples described later, the manufacturing method of the present invention exhibits a lipid production efficiency more than 30 times greater than that of wild-type microorganisms. In addition, it can be used in a wide temperature range, including high temperatures above room temperature, making it a highly practical method with excellent environmental adaptability. 【0013】Figure 1 shows the arrangement of electron transfer cofactors that bind to reaction center proteins D1 and D2 of the photosystem II complex, which carries out the initial reaction of photosynthesis, as well as the photoexcitation, charge separation, and electron transfer pathways. Ｄ１ and Chl Ｄ２ This is a schematic diagram showing the overall picture including the following. Figure 2 shows the Chl of thermophilic cyanobacteria (Thermosynechococcus elongatus). Ｄ１ and Chl Ｄ2Figure 3 shows the molecular structures of the transformants "TH mutant," "IH mutant," and "TH+IH mutant" prepared in Example 1 for the Mg ligand. Figure 4 shows the amino acid sequence (1-40) of the D1 protein in representative cyanobacteria and other algae. Figure 5 shows the amino acid sequence (81-120) of the D1 protein in representative cyanobacteria and other algae. Figure 6 shows the amino acid sequence (121-160) of the D1 protein in representative cyanobacteria and other algae. Figure 7 shows the amino acid sequence (161-200) of the D1 protein in representative cyanobacteria and other algae. Figure 8 shows the amino acid sequence (201-240) of the D1 protein in representative cyanobacteria and other algae. Figure 9 shows the amino acid sequence (241-280) of the D1 protein in representative cyanobacteria and other algae. Figure 10 shows the amino acid sequences (281-320) of the D1 protein in representative cyanobacteria and other algae. Figure 11 shows the amino acid sequences (321-344) of the D1 protein in representative cyanobacteria and other algae. Figure 12 shows the analysis results of the ratio of fatty acids produced using the "TH mutant," "IH mutant," and "TH+IH mutant," which are transformants of the thermophilic cyanobacterium (Thermosynechococcus elongatus) prepared in Example 1, to the amount produced by the wild strain (non-transformed). Figure 13 shows the analysis results of the ratio of oils and fats produced using the "TH mutant," "IH mutant," and "TH+IH mutant," which are transformants of the thermophilic cyanobacterium (Thermosynechococcus elongatus) prepared in Example 1, to the amount produced by the wild strain (non-transformed). Figure 14 shows the results of an analysis of the ratio of phospholipids produced using the "TH mutant," "IH mutant," and "TH+IH mutant," which are transformants of the thermophilic cyanobacterium (Thermosynechococcus elongatus) prepared in Example 1, to the amount produced by the wild-type strain (non-transformed).Figure 15 shows the results of an analysis of the ratio of glycerophospholipids produced using the "TH mutant," "IH mutant," and "TH+IH mutant," which are transformants of the thermophilic cyanobacterium (Thermosynechococcus elongatus) prepared in Example 1, to the amount produced by the wild-type strain (non-transformed). Figure 16 is a graph showing the cell proliferation curve (calculated in terms of chlorophyll concentration) when wild-type thermophilic cyanobacterium (Thermosynechococcus elongatus) was cultured under conditions of carbon dioxide concentration from 0.03 to 90%. 【0014】 Embodiments of the present invention will be described below. The scope of the present invention is not limited to these descriptions, and other embodiments may be modified and implemented as appropriate, without impairing the spirit of the invention. 【0015】 1. Method for Manufacturing the Present Invention The method for manufacturing the present invention produces lipids by biosynthesis and comprises: 1) a step of culturing a microorganism having chlorophyll in a photoautotrophic manner in the presence of light and water; and 2) a step of producing the lipids thereby, characterized in that the microorganism has resistance to photoinhibition. 【0016】 The microorganisms used in step 1) are those that possess chlorophyll, and are so-called photosynthetic microorganisms. Such photosynthetic microorganisms are microorganisms that possess chlorophyll and perform oxygen-evolving photosynthesis, and through photosynthesis, they release CO from the atmosphere. ２ By immobilizing it, organic matter (e.g., starch) is synthesized, and on the other hand, water (H ２ O) to oxygen (O) ２ It has the characteristic of generating ). 【0017】 The microorganisms containing chlorophyll used in the present invention are preferably algae. In this specification, algae broadly include prokaryotes such as cyanobacteria (blue-green algae) and eukaryotes such as green algae, diatoms, dinoflagellates, red algae, prasinophytes, Euglena algae, and true eyespot algae. 【0018】There are no particular restrictions on the types of cyanobacteria; all kinds can be included. Preferably, the cyanobacteria belong to the genera Synechocystis, Synechococcus, Thermosynechococcus, Trichodesmium, Acaryochloris, Crocosphaera, and Anabaena, and more preferably, cyanobacteria belonging to the genera Synechocystis, Synechococcus, Thermosynechococcus, or Anabaena. More preferably, Synechocystis sp. PCC6803, Synechocystis sp. PCC7509, Synechocystis sp. PCC6714, Synechococcus elongatas PCC7942, Synechococcus elongatas UTEX 2973, Synechococcus sp. PCC7002, Thermosynechococcus elongatas BP-1 (also known as Thermosynechococcus bestitas BP-1), Thermosynechococcus bulcanus, Trichodesmium erythraeum IMS101, Acariochloris mariana MBIC11017, Crocosphaera watsonii WH8501, and Anabaena sp. PCC7120 can be used, but are not limited to these. Preferably, thermophilic cyanobacteria belonging to the genus Thermosynechococcus can be used. 【0019】Furthermore, eukaryotes include, for example, green algae such as Chlamydomonas, Chlorella, Dunaliella, Hematococcus, Volvox, and Botryococcus; and genera such as Rhizosolenia, Chaetoceros, Cyclotella, Cylindrotheca, Navicula, and Phaeodactylum. Examples include diatoms such as Thalassia and Fistulifera; dinoflagellates such as Amphidinium and Symbiodynum; red algae such as Cyanidioschyzon and Porphyridium; prasinophytes such as Ostreococcus; Euglena algae such as Euglena; and true eyespot algae such as Nannochloropsis. For example, microalgal microbial species include Synechocystis sp. PCC6803, Synechococcus sp. PCC7002, Synechococcus elongatus PCC7942, Arthrospira platensis (also known as "Spirulina"), Spirulina maxima, Spirulina subsalsa, and Anabaena PCC7120. Chlamydomonas sp. PCC7120), Chlamydomonas reinhardtii, Chlamydomonas sp., Chlorella vulgaris, Chlorella pyrenoidosa,Dunaliella salina, Dunaliella sp., Hematococcus pluvialis, Volvox carteri, Botryococcus braunii, Cyclotella cryptica, Cylindrotheca fusiformis, Navicula saprophylla, Phaeodactylum tricornutum tricornutum, Thalassia pseudonana, Fistulifera sp., Amphidinium sp., Cymbiodynum microadriaticum, Cyanidioschyzon merolae, Porphyridium sp., Ostreococcus tauri, Euglena gracilis, Nannochloropsis occulata Examples include *Oculata*, but this is not an exhaustive list. 【0020】 The microorganism used in step 1) of the present invention is one that is resistant to or has enhanced resistance to photoinhibition. Typically, it can be a transformant in which the photosynthetic protein gene is modified to confer or enhance photoinhibition resistance, without altering the lipid synthase gene in the photosynthetic microorganism exemplified above. This allows the microorganism to maintain a high level of its original photosynthetic function while suppressing the effect of lipid production on photoinhibition of photosynthetic proteins, thereby destroying the photosynthetic proteins and causing cell death. If the microorganism is resistant to photoinhibition, a wild-type strain can also be used. 【0021】In this specification, for photosynthetic microorganisms, whether they have resistance to photoinhibition is determined by, for example, the method described below. When the photosynthetic microorganism is cultured for 240 hours under strong light irradiation conditions (2,000 μmol photons·m -2 ·s -1 ), if the cell amount in terms of chlorophyll is 95% or more of the cell amount when cultured for 240 hours under normal optimal conditions, it is determined that the photosynthetic microorganism has resistance to photoinhibition. 【0022】 The photoexcitation rate in the photosynthetic system (photosynthetic system I or II or both photosynthetic systems I and II) of the microorganism used in step 1) of the present invention may be 200 fs or more, may be 300 fs or more, or may be 400 fs or more. Although not particularly limited, the upper limit of the photoexcitation rate may be (4) ps or less. 【0023】 The charge separation rate in the photosynthetic system (photosynthetic system I or II or both photosynthetic systems I and II) of the microorganism used in step 1) of the present invention may be 8 ps or more, may be 12 ps or more, or may be 15 ps or more. Although not particularly limited, the upper limit of the charge separation rate may be (20) ns or less. 【0024】 Specifically, a preferred microorganism in the present invention is a transformant in which the gene of the photosynthetic protein around chlorophyll (P ６８０ chlorophyll), an important cofactor involved in the light response of photosynthesis, has been modified. An overall view including Chl Ｄ１ for excitation / charge separation and electron transfer in photosynthesis is shown in FIG. 1. In this specification, those in which the expression of the gene encoding the target protein is promoted are referred to as "transformants" or "mutants", and those in which the expression of the gene encoding the target protein is not promoted are also referred to as "hosts" or "wild strains". Ｄ１ and Chl Ｄ２ is shown in FIG. 1. 【0025】 More specifically, the microorganism used in the present invention is Chl Ｄ１It is preferable that the mutant has a structure in which a threonine (T) that hydrogen bonds with a water molecule, which is the ligand for the central metal (Mg), is substituted, and it is particularly preferable that the mutant has a structure in which the threonine residue (T) is substituted with a histidine residue (H). The threonine residue is generally located around position 179 of the D1 protein, typically between positions 177 and 182, and in many cases, at position 179. Chl used in the present invention Ｄ１ Figure 2 shows a schematic diagram of the molecular structure in which the threonine residue in the ligand of the central metal (Mg) is replaced with a histidine residue. 【0026】 While it is known that the D1 protein in many cyanobacteria consists of 344 amino acids, in typical forms, some species may have D1 proteins with other amino acids added before these 344 amino acid sequences, or D1 proteins with missing amino acids. The amino acid sequences of D1 proteins in representative cyanobacteria and other algae are shown in Figures 3 to 11. 【0027】 In a typical embodiment, the microorganism used in the present invention is preferably a mutant having a structure in which the threonine residue (T) at position 179 in the D1 protein, which consists of 344 amino acids, is substituted, and is particularly preferably a mutant having a structure in which the T residue is substituted with a histidine residue (H). 【0028】 In a more preferred embodiment, the microorganism used in the present invention is similarly Chl Ｄ２It is preferable that the mutant has a structure in which an isoleucine (I) located near the water molecule, which is the ligand for the central metal (Mg), is substituted, and it is particularly preferable that the mutant has a structure in which the isoleucine residue (I) is substituted with a histidine residue (H). The isoleucine residue is generally located around position 178 of the D2 protein, typically between positions 175 and 181, and in many cases, at position 178. Therefore, it is preferable that the microorganism used in the present invention is a mutant having a structure in which the isoleucine residue (I) located at position 178 of the cofactor D2 protein is substituted, and it is particularly preferable that the mutant has a structure in which the isoleucine residue is substituted with a histidine residue. Chl used in the present invention Ｄ２ Figure 2 also shows a schematic diagram of the molecular structure in which the isoleucine residue in the ligand of the central metal (Mg) is replaced with a histidine residue. 【0029】 Although not necessarily bound by theory, such transformations result in Chl Ｄ１ and / or Chl Ｄ２ It is thought that the central metal (Mg) directly coordinates to the histidine residue without the need for water molecules, thereby conferring or enhancing photoinhibition resistance. 【0030】 In the present invention, it is preferable that the microorganisms used are transformants whose excitation and charge separation rates have been further delayed by modifying the photosynthetic protein genes. 【0031】In the introduction of genomic genes into the microorganisms used in the present invention, for example, vectors such as plasmid vectors can be temporarily used. This plasmid vector is temporarily introduced into the target cells, and genomic genes are introduced by homologous recombination between the genome and the plasmid during DNA replication. The plasmid vector does not remain in the target cells and is decomposed during cell division. The vector is preferably an expression vector. For example, an expression vector containing DNA fragments of a heterologous carotenoid oxidative cleavage enzyme gene and a β-carotene-9-isomerase gene and a promoter for expressing them is constructed. As the promoter, the lac, tac or trc promoter, a promoter related to an inducer that can be induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG), or promoters such as the Rubisco operon (rbc), the PSI reaction center protein (psaAB), and the D1 protein of PSII (psbA) can be used, but it is not limited to these, and various promoters that function in the target microorganisms can be used. The introduction of the gene encoding the target protein into the vector can be carried out by conventional methods such as restriction enzyme treatment and ligation. 【0032】 Further, the above expression vector may further incorporate a marker gene (for example, a drug resistance gene such as kanamycin, chloramphenicol, spectinomycin, erythromycin, etc.) for selecting a host into which the vector has been appropriately introduced. The above expression vector is introduced into microorganisms by known means and transformed. As the method for introducing the vector, it can be appropriately selected from conventional methods according to the type of host to be used. For example, natural transformation method, transformation method using calcium ions, general competent cell transformation method, protoplast transformation method, electroporation method, LP transformation method, method using Agrobacterium, particle gun method, and conjugation method can be used. If the microorganisms after the transformation treatment are cultured in a selection medium, for example, a medium containing an antibiotic, transformants having the desired traits can be selected. 【0033】Furthermore, the type of selection marker used to confirm that the gene encoding the target protein has been incorporated into the genome can be appropriately selected depending on the type of host used. Preferred selection markers include drug resistance genes such as ampicillin resistance gene, chloramphenicol resistance gene, erythromycin resistance gene, neomycin resistance gene, kanamycin resistance gene, spectinomycin resistance gene, streptomycin resistance gene, tetracycline resistance gene, blastosidine S resistance gene, biafos resistance gene, zeosin resistance gene, paromomycin resistance gene, gentamicin resistance gene, and hygromycin resistance gene. In addition, deletions of genes related to nutritional requirements can also be used as selection marker genes. 【0034】 In addition to the gene transfer methods described above, genes can also be introduced into target cells using genome editing technologies such as CRISPR / Cas9. 【0035】 In step 1) of the present invention, the above-mentioned microorganisms are cultured photoautotrophically in the presence of light and water. In this specification, "photoautotrophic" means conditions under which photosynthesis is performed in the presence of water and light (and optionally minerals) without the addition of hydrocarbons (carbohydrates) such as sugars from outside, and bioenergy is obtained by obtaining electrons and hydrogen ions from water in the photosynthetic electron transport system using the driving force of light, and this energy is used to convert carbon dioxide into organic matter. Therefore, in a preferred embodiment, the culture in step 1) is carried out photoautotrophically without the addition of substances containing carbohydrates as a nutrient source. Examples of carbohydrates include monosaccharides, such as glucose, fructose, mannose, galactose, xylose, and arabinose; oligosaccharides, such as fructooligosaccharides and galactooligosaccharides; polysaccharides, such as starch, cellulose, pectin, and xylan; and disaccharides, such as sucrose, maltose, cellobiose, and turanose. 【0036】In the production method of the present invention, such lipid can be produced in step 2) by culturing photoautotrophically. The lipid can be free fatty acid, glycolipid, phospholipid, or any combination thereof. Such lipid molecules can have 16 or more carbon atoms, preferably 18 or more carbon atoms, more preferably 20 or more carbon atoms, and include, for example, arachidic acid, docosahexaenoic acid (DHA), eicosanoic acid, lecithin, phosphatidylethanolamine, etc. 【0037】 Further, as described above, the production method of the present invention can be used in a wide temperature range including a high temperature exceeding room temperature, for example, in the temperature range of 20 to 90 ° C, preferably 30 to 70 ° C. Although not particularly limited, one or both of growth and production may be carried out at 40 to 90 ° C, 40 to 70 ° C, 45 to 90 ° C, 45 to 70 ° C, 50 to 90 ° C, or 50 to 70 ° C. From the viewpoint of culturing within such a temperature range, as the microorganism, it is preferable to use a thermophilic cyanobacterium belonging to the genus Thermosynechococcus, and typical examples of such thermophilic cyanobacteria include Thermosynechococcus elongatus or Thermosynechococcus vestitus. 【0038】 The carbon dioxide concentration in step 1 and / or step 2 may be 0.03 to 90%. The lower limit value of this carbon dioxide concentration may be 0.3%, 3%, 5%, or 7.5%. The upper limit value of this carbon dioxide concentration may be 75%, 50%, 25%, 15%, or 12.5%. 【0039】 2. In another aspect of the culture system of the present invention, the present invention also relates to a culture system suitable for the above production method. The culture system includes a) a microorganism having chlorophyll and having resistance to photo-inhibition, b) a culture medium, and c) culture equipment. 【0040】The above description and examples apply similarly to the microorganisms in a) above. 【0041】 The culture medium in (b) above is not particularly limited and any culture medium known in the art can be used, for example, DTN medium, D medium, BG11 medium, etc. As described above, in the production method of the present invention, microorganisms are cultured photoautotrophically in the presence of light and water, and it is preferable that the culture medium does not contain substances containing hydrocarbons as a nutrient source. 【0042】 The culture equipment in c) above can be one known in the relevant technical field, such as a constant temperature bath equipped with light illumination or CO2. ２ CO2 in incubators and culture vessels ２ It can be equipped with gas injection and bubbling devices, among other things. 【0043】 The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto. 【0044】 1. Production of Cyanobacterial Transformants Resistant to Photoinhibition For the thermophilic cyanobacterium Thermosynechococcus elongatus (wild-type deposit number (National Institute of Technology and Evaluation): NBRC 108920), transformants were produced in which the threonine residue (T) at position 179 in the D1 protein was replaced with a histidine residue (H) ("TH mutant"). Furthermore, transformants were produced in which the isoleucine residue (I) at position 178 in the D2 protein was replaced with a histidine residue (H) ("IH mutant"). In addition, transformants in which both the D1 and D2 proteins had the above substitutions ("TH+IH mutant") were also produced. 【0045】 These transformants were generated by gene mutations in genomic DNA according to the methods described in Takegawa, Y. et al., Biochim. Biophys. Acta, 1860 (2019) and Sugiura, M. et al., Biochim. Biophys. Acta, 1865 (2024). 【0046】Specifically, Thermosynechococcus elongatus BP-1 (also known as Thermosynechococcus vestitus BP-1), which lacked two D1 genes (psbA1 and psbA2) and one D2 gene (psbD2) from the multiple D1 and / or D2 genes present in the Thermosynechococcus elongatus genome, was used as the host cell. A plasmid vector containing the target mutation in psbA3 and / or psbD1 was introduced by electroporation. Selection was repeated until clones were obtained in which all genomic copies of the cells were replaced with the target sequence. 【0047】 2. Lipid synthesis using cyanobacteria transformants resistant to photoinhibition. Lipid synthesis was performed by photoautotrophically culturing the TH mutant, IH mutant, and TH+IH mutant prepared in Example 1. 【0048】 The culture conditions were as follows: DTN medium was used, and the light intensity was 25–100 μmol photosm －２ sec －１ Under illumination of blue and red enhanced light, or white light, 5-7% CO2 ２ The culture temperatures are 45°C and 65°C. 【0049】 Table 1 shows the results of analyzing the obtained products as a ratio to the amount of products of the wild-type (untransformed) strain. In the table, "D1 / T179H" refers to the TH mutant prepared in Example 1, "D1 / T179H-D2 / I178H" refers to the TH+IH mutant prepared in Example 1, and "D2 / I178H" refers to the IH mutant prepared in Example 1. The values ​​in the table also indicate the rate of increase (times) in the production of each fatty acid and oil in the wild-type Thermosynechococcus elongatus strain without the introduction of mutations, compared to the wild-type strain. 【0050】 【0051】As a result, it was demonstrated that both the TH mutant and the TH+IH mutant could produce free fatty acids and phospholipids more efficiently than the wild-type strain. The results from Table 1 above are graphed in Figures 12 to 15, categorized by fatty acid production (1-5), lipid production (6-15), phospholipid production (16-35), and glycerophospholipid production (36-47). 【0052】 1) Regarding the fatty acids of D1 / T179H (cultured at 45°C): The 20:1 fatty acid (presumably eicoseic acid) increased 12.25 times, while the 20:0 fatty acid (presumably arachidic acid) increased only 1.63 times. Furthermore, it was found that the long-chain 22:6 fatty acid (docosahexaenoic acid, DHA) increased 17.24 times. Compared to the comparative example without genetic modification, the fatty acids in T179H increased by approximately 30 times in total. 【0053】 2) It was found that the amount of lipids (neutral fats: triglycerides) in D1 / T179H (cultured at 45°C) increased, particularly lipids with a very high number of carbon atoms (C56, C60). 【0054】 3) Regarding the complex lipids of D1 / T179H (cultured at 45°C), it was found that there was a very high amount of phospholipids with very long-chain fatty acids (30 or more carbon atoms) (the largest being a 34:1 phospholipid, which was 1286 times higher). In addition, it was found that 40:4 phospholipids and 38:4 glycerophospholipids, which are not present in the wild strain, were produced (these cannot be quantified as they are not present in the strain, so for convenience, they are represented as 100 times higher in the table). 【0055】 4) Regarding D1 / T179H-D2 / I178H (45°C culture): To demonstrate that substituting D1 / T179 with histidine (H) is key to increasing lipid synthesis, we analyzed a "double mutant" in which, in addition to this mutation, D2 / I178 of the D2 protein was also replaced with histidine (H). The results showed similar trends in increased fatty acid synthesis and increased levels of long-chain fatty acids in lipids and phospholipids, although there were differences in the numerical values. 【0056】5) Regarding D2 / I178H (cultured at 45°C): We analyzed a mutant in which only D2 / I178 was replaced with histidine (H). Although there were some phospholipids with increased synthesis, the results were close to those of the wild type. From this, it can be concluded that D2 / I178H contributes little to the increase in lipid synthesis. Therefore, the increase in fatty acid and lipid synthesis in the D1 / T179H-D2 / I178H mutant in 4) is due to the D1 / T179H mutation, and the D2 / I178H mutation has little effect. 【0057】 6) To examine the effect of culture temperature on the three mutants cultured at 65°C, cells cultured at 65°C were also analyzed. Regarding fatty acid synthesis, D1 / T179H did not show the same increase as in 45°C culture, but D1 / T179H-D2 / I178H showed a 55.5-fold increase in eicothane. D1 / T179H and D1 / T179HD2 / I178H showed a similar trend to that in 45°C culture, but glycerophospholipid levels were lower than in the 45°C culture. 【0058】 7) We analyzed how carbon dioxide concentration affects the growth of thermophilic cyanobacteria (Thermosynechococcus elongatus) under different carbon dioxide concentrations during cultivation. Figure 16 shows the cell proliferation curves (calculated based on chlorophyll concentration) of wild-type thermophilic cyanobacteria (Thermosynechococcus elongatus) when cultured at 45°C under carbon dioxide concentrations of 0.03–90%. 【0059】 Thermophilic cyanobacteria grew sufficiently at atmospheric carbon dioxide concentrations. When the carbon dioxide concentration was increased to 10%, the cell proliferation rate increased significantly compared to 0.03%. When the carbon dioxide concentration was increased to 90%, the proliferation rate was faster than at 10% and did not hinder cell growth. 【0060】8) Photoexcitation rate and charge separation rate were measured for wild-type, D1 / T179H, D2 / I178H, and D1 / T179H+D2 / I178H in photosystem II. The measurement conditions followed the method described in Non-Patent Literature 4 (Yoneda, Y., Nagasawa, Y., Umena, Y., Miyasaka, H., J. Phys. Chem. Lett. (2019) 10, 3710-3714). 【0061】 【0062】 In the wild type, the photoexcitation rate was 98 fs, while in D1 / T179H it was 508 fs. In D2 / I178H it was 143 fs. In D1 / T179HD2 / I178H it was 680 fs. 【0063】 In the wild type, the charge separation rate was 3.9 ps, while in D1 / T179H it was 17.7 ps. In D2 / I178H it was 9.0 ps. In D1 / T179HD2 / I178H it was 9.0 ps. 【0064】 The lipid production method using chlorophyll-containing microorganisms described in each embodiment, and the culture system therefor, can be understood, for example, as follows. 【0065】(1) A method for producing lipids by biosynthesis, comprising the steps of: culturing a microorganism having chlorophyll photoautotrophically in the presence of light and water; and producing the lipids thereby, wherein the microorganism is resistant to photoinhibition. (2) The method according to (1), wherein the microorganism is a transformant that is resistant to photoinhibition due to modification of photosynthetic protein genes. (3) The method according to (1), wherein the cultivation is carried out in a temperature range of 20 to 90°C. (4) The method according to (1), wherein the cultivation is carried out photoautotrophically without adding a substance containing hydrocarbons as a nutrient source. (5) The method according to (1), wherein the microorganism is an alga. (6) The method according to (1), wherein the microorganism is a thermophilic cyanobacterium belonging to the genus Thermosynechococcus. (7) The method according to (1), wherein the microorganism is a mutant having a structure in which threonine residues (T) located at positions 177 to 182 in the D1 protein are replaced with histidine residues (H). (8) The method according to (7), wherein the microorganism further has a structure in which the isoleucine residue (I) located at positions 175 to 181 in the D2 protein is replaced with a histidine residue (H). (9) The method according to (1), wherein the lipid is a free fatty acid, a glycolipid, a phospholipid, or any combination thereof. (10) A culture system for producing lipids by biosynthesis, comprising a) a chlorophyll-containing microorganism resistant to photoinhibition, b) a culture medium, and c) culture equipment. (11) The culture system according to (10), wherein the culture medium does not contain a substance containing hydrocarbons as a nutrient source. (12) The culture system according to (10), wherein the microorganism is a transformant resistant to photoinhibition due to modification of a photosynthetic protein gene. (13) The culture system according to (10), wherein the microorganism is an alga. (14) The culture system according to (10), wherein the microorganism is a thermophilic cyanobacterium belonging to the genus Thermosynechococcus. (15) The culture system according to (10), wherein the microorganism is a mutant having a structure in which the threonine residue (T) located at positions 177 to 182 in the D1 protein is replaced with a histidine residue (H).(16) The culture system according to (10), wherein the microorganism further has a structure in which the isoleucine residue (I) located at positions 175 to 181 in the D2 protein is replaced with a histidine residue (H). 【0066】 Lipid compounds that can be used as next-generation biofuels can be produced more efficiently through biosynthesis compared to conventional technologies.

Claims

1. A method for producing lipids by biosynthesis, comprising the steps of: culturing a microorganism having chlorophyll in a photoautotrophic manner in the presence of light and water; and producing the lipids thereby, wherein the microorganism is resistant to photoinhibition, the cultivation is carried out in a temperature range of 20 to 90°C, and the production is carried out in a temperature range of 40 to 90°C.

2. The method according to claim 1, wherein the microorganism is a transformant that has resistance to photoinhibition due to modification of photosynthetic protein genes.

3. The method according to claim 1, wherein the cultivation and production are carried out in a temperature range of 40 to 90°C.

4. The method according to claim 1, wherein the photoexcitation rate in the photochemical system of the microorganism is 200 fs or more, and the charge separation rate is 8 ps or more.

5. The method according to claim 1, wherein the cultivation and production are carried out under conditions of a carbon dioxide concentration of 0.03 to 90%.

6. The method according to claim 1, wherein the cultivation and production are carried out under conditions of a carbon dioxide concentration of 3 to 50%.

7. The method according to claim 1, wherein the culture is carried out photoautotrophically without adding a substance containing hydrocarbons as a nutrient source.

8. The method according to claim 1, wherein the microorganism is cyanobacteria.

9. The method according to claim 8, wherein the microorganism is a thermophilic cyanobacterium.

10. The method according to claim 9, wherein the microorganism is a thermophilic cyanobacterium belonging to the genus Thermosynechococcus.

11. The method according to claim 1, wherein the microorganism is an algae.

12. The method according to claim 1, wherein the microorganism is a mutant having a structure in which the threonine residue (T) located at positions 177 to 182 in the D1 protein is replaced with a histidine residue (H).

13. The method according to claim 7, wherein the microorganism further has a structure in which the isoleucine residue (I) located at positions 175 to 181 in the D2 protein is replaced with a histidine residue (H).

14. The method according to claim 1, wherein the lipid is a free fatty acid, a glycolipid, a phospholipid, or any combination thereof.

15. A culture system for producing lipids by biosynthesis, comprising: a) a chlorophyll-containing microorganism resistant to photoinhibition; b) a culture medium; and c) a culture facility.

16. The culture system according to claim 15, wherein the culture medium does not contain a substance containing hydrocarbons as a nutrient source.

17. The culture system according to claim 15, wherein the microorganism is a transformant that has resistance to photoinhibition due to modification of photosynthetic protein genes.

18. The culture system according to claim 15, wherein the microorganism is a thermophilic cyanobacterium belonging to the genus Thermosynechococcus.

19. The culture system according to claim 15, wherein the microorganism is an algae.

20. The culture system according to claim 15, wherein the microorganism is a mutant having a structure in which the threonine residue (T) located at positions 177 to 182 in the D1 protein is replaced with a histidine residue (H).

21. The culture system according to claim 15, wherein the microorganism further has a structure in which the isoleucine residue (I) located at positions 175 to 181 in the D2 protein is replaced with a histidine residue (H).

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