WO2022250074A1

WO2022250074A1 - Photosynthetic organism transformant and use thereof

Info

Publication number: WO2022250074A1
Application number: PCT/JP2022/021364
Authority: WO
Inventors: 雄気須藤; 慧一小島
Original assignee: 国立大学法人岡山大学
Priority date: 2021-05-28
Filing date: 2022-05-25
Publication date: 2022-12-01
Also published as: JPWO2022250074A1

Abstract

The present invention addresses the problem of providing a novel method that can control the growth of a photosynthetic organism and providing a use of said method. This problem is solved by providing a photosynthetic organism transformant that expresses a proton pump rhodopsin, and providing a method for controlling the growth of a photosynthetic organism, the method comprising (1) a step of obtaining a transformant that expresses a proton pump rhodopsin and (2) a step of driving the proton pump rhodopsin by exposing the transformant to light.

Description

Transformant of photosynthetic organism and use thereof

The present invention relates to transformants of photosynthetic organisms and uses thereof.

Biomass resources derived from photosynthetic organisms such as plants and algae are attracting attention as renewable resources, especially as renewable energy sources to replace fossil fuels. For example, starch obtained from plants such as sugar cane and corn. Bioethanol, which is obtained by alcohol fermentation, and biodiesel, which is obtained by converting oils and fats obtained from plants such as corn and rapeseed, are used as main raw materials for biofuels. However, many of the plants that have traditionally been used as raw materials for biofuels, such as corn and sugarcane, are also mainly consumed as food. From the point of view, it is not necessarily preferable to use food crops as raw materials for biofuels.

On the other hand, like plants, algae, which are a type of photosynthetic organism, produce various useful substances such as triacylglycerol and starch, which are expected to be used as biofuels. Unlike this, it is not mainly used as food, so it is expected to be a powerful source of biomass. For example, Chlamydomonas, a type of algae, is known to accumulate oils and fats (triacylglycerols) that are raw materials for biofuels when cultured under nitrogen-deficient or phosphorus-deficient conditions. On the other hand, as seen in algae such as spirulina, chlorella, and euglena, it has become clear that some algae produce physiologically active substances that exert various physiological functions, and algae can be used as biomass resources. The use is increasing in importance not only in the energy field but also in the health food field and the cosmetic field.

However, many algae do not accumulate a large amount of biomass such as oils, starches, and bioactive substances per unit cell, and their growth rate is slow, so the biomass production efficiency cannot be said to be high. Therefore, various attempts have been made to increase the efficiency of biomass production in algae, and in particular, breeding of algae is frequently carried out. For example, Patent Document 1 and Non-Patent Document 1 disclose a transformant of Chlamydomonas, a kind of green algae. The transformant contains a gene in which the promoter region of the DGTT4 gene, which is a triacylglycerol synthase, is added with the promoter of the SQD2 gene, another gene whose expression is known to increase under phosphorus-deficient conditions. It is a transformant and has been reported to accumulate more triacylglycerols than ordinary Chlamydomonas under phosphorus-deficient conditions. On the other hand, Non-Patent Document 2 discloses a Chlamydomonas transformant lacking ADP-glucose pyrophosphorylase, which is an enzyme involved in starch biosynthesis, which is also a Chlamydomonas transformant. It is disclosed that the transformant produces a high amount of triacylglycerol per unit cell under nitrogen-deficient conditions.

The above-described transformants are intended to increase biomass production efficiency per unit cell by increasing biomass production efficiency. It does not raise the upper limit of the number of viable cells per volume, ie the saturation cell density. The time and space that can be used for biomass production is limited, and in order to increase the efficiency of biomass production by photosynthetic organisms, it is necessary not only to increase the amount of biomass produced per unit cell, but also to It is extremely important to develop a technology that can cultivate or culture as many photosynthetic organisms as possible in a limited space as quickly as possible.

In response to such problems, various attempts have been made to increase the growth rate of cells and increase the upper limit of the number of viable cells per unit volume, that is, the saturation cell density. For example, in Non-Patent Document 3, by adjusting the composition of trace metal ions in the medium for culturing Chlamydomonas, the rate of increase in the number of Chlamydomonas cells and the increase in the number of Chlamydomonas cells reached a steady state. It has been reported that the number of cells per cell, ie the saturated cell density, is increased. However, the addition of substances, particularly metal ions, to the medium raises concerns about the impact on waste disposal, environmental problems, etc., and also leads to an increase in cost.

By the way, not only photosynthetic organisms, but also the smallest unit constituting organisms is cells, and the inside and outside of cells are separated by cell membranes. The concentrations of substances and ions inside and outside the cell are strictly controlled by proteins embedded in the membrane (membrane proteins), and if the ion concentration balance is disrupted, the homeostasis of the cell (division, development, growth, etc.) will be greatly affected. Change. Among a wide variety of ions, protons (hydrogen ions: H ⁺ ) in particular are the most abundant ions on earth, determine the pH inside and outside cells and inside organelles, and play a vital role in almost all living organisms. It plays an important role in maintenance and expression.

One of the photoreceptive membrane proteins involved in proton transport through the cell membrane is rhodopsin, which is distributed in vertebrates such as humans and microorganisms such as bacteria. Among them, rhodopsins that are activated by receiving light and serve to unidirectionally transport protons from the inside to the outside of the cell or from the outside to the inside of the cell are called proton pump rhodopsins. A proton pump rhodopsin that transports protons from the inside to the outside of the cell is called outward proton pump rhodopsin, and a proton pump rhodopsin that transports protons from the outside to the inside of the cell is called inward proton pump rhodopsin. For example, Archidopsin 3 (AR3), which is a membrane protein derived from highly halophilic archaea, and Rubricoccus marinus xenorhodopsin (RmXeR), which is a membrane protein derived from marine eubacteria, use retinal as a chromophore and have a wavelength of around 550 nm. When receiving green light with an absorption maximum at , it functions as a proton pump that transports protons from the inside to the outside of the cell (when using AR3) or from the outside to the inside of the cell (when using RmXeR), respectively ( Non-Patent Documents 4, 5).

The present applicant and the present inventors, based on the idea that intracellular pH control by proton transport mediated by proton pump rhodopsin, may be able to control the life function of cells, cancer expressing proton pump rhodopsin We have found that the cell death of cancer cells is induced or suppressed by irradiating light to drive proton pump rhodopsin in cells, and disclosed this finding in Japanese Patent Application No. 2020-196718. However, nothing is known about how proton pump prodopsin affects the growth of photosynthetic organisms such as plants and algae.

In addition, as mentioned above, many of the proton-pump rhodopsins are membrane proteins that are activated by receiving green light and function to transport protons unidirectionally from the inside to the outside of the cell or from the outside to the inside of the cell. However, many photosynthetic organisms such as plants and algae are green in appearance, so they mainly emit light other than green, more specifically, blue (wavelength 400-500 nm) and red (wavelength 400-500 nm). 600 nm to 700 nm) and uses it for biological activities. Based on such findings, for example, as seen in Patent Documents 2 to 4, many attempts have been made to selectively irradiate photosynthetic organisms with blue or red light to promote their growth. However, as far as the present inventors know, almost no attempts have been made to promote or control the growth of photosynthetic organisms using green light (wavelength 500 nm to 600 nm).

JP 2014-68638 A JP-A-2001-86860 Japanese Patent Application Laid-Open No. 2020-18210 JP 2015-128448 A

The present invention has been made in order to solve the above-mentioned problems of the prior art, and an object of the present invention is to provide a new method for controlling the growth of photosynthetic organisms and its use.

In the process of earnestly researching to solve the above problems, the present inventors discovered Rubricoccus marinus xenorhodopsin (RmXeR), which is a type of inward proton-pumping rhodopsin that functions to transport protons from the outside to the inside of cells. was expressed in the chloroplast of Chlamydomonas, which is widely used in the field as a model of photosynthetic organisms, and was irradiated with light that drives RmXeR. found to be Therefore, the present inventors expressed archilodopsin 3 (AR3), a type of outward proton pump rhodopsin that transports protons from the inside of the cell to the outside of the cell, in the chloroplasts of Chlamydomonas, contrary to RmXeR. and irradiated with light that drives AR3, the growth of Chlamydomonas expressing AR3 was remarkably suppressed, contrary to the case of expressing RmXeR. Even more surprisingly, in any Chlamydomonas expressing RmXeR or AR3, the number of cells per unit volume in the stationary growth phase when its growth reached a steady state, that is, the saturated cell density, increased proton pump rhodopsin. The level was much higher than that of normal Chlamydomonas not expressing Chlamydomonas cultured under the same conditions. These results suggest that by expressing a proton pump rhodopsin in a photosynthetic organism and irradiating light that drives the proton pump rhodopsin to manipulate the intracellular pH environment, more preferably the pH environment within the chloroplast, the photosynthetic organism This is an epoch-making finding that has never been seen before.

That is, the present invention is based on the findings newly found by the present inventors as described above, and provides a transformant of a photosynthetic organism that expresses a light-driven proton pump rhodopsin. The above problem is solved by providing. The growth of a transformant of a photosynthetic organism expressing proton pump rhodopsin can be controlled by irradiating the transformant with light that drives proton pump rhodopsin.

In a preferred embodiment, the proton pump rhodopsin expressed by the transformant is an inward proton pump rhodopsin that transports protons from the outside to the inside of the cell. As shown in the experimental examples described later, when a transformant of a photosynthetic organism expressing inward proton pump rhodopsin is irradiated with light that drives the inward proton pump rhodopsin to drive the inward proton pump rhodopsin, the transformant It can promote body growth, that is, increase the growth rate. In addition, the upper limit of the number of viable cells per unit volume, that is, the saturation cell density can be increased.

On the other hand, in another preferred embodiment of the present invention, the proton pump rhodopsin is an outward proton pump rhodopsin that transports protons from intracellular to extracellular. As shown in the experimental examples described later, when a transformant of a photosynthetic organism expressing outward proton pump rhodopsin is irradiated with light that drives the outward proton pump rhodopsin to drive the outward proton pump rhodopsin, the transformant It is possible to suppress the growth of the body, that is, to slow down the growth rate. In addition, the upper limit of the number of viable cells per unit volume, that is, the saturation cell density can be increased.

In still another preferred embodiment, the transformant contains an outward proton pump rhodopsin that transports protons from the inside to the outside of the cell and an inward proton pump rhodopsin that transports protons from the outside to the inside of the cell. A transformant expressing both. When both the inward proton pump rhodopsin and the outward proton pump rhodopsin are expressed in the transformant, the growth of the transformant can be freely promoted or suppressed by appropriately selecting the wavelength of light to be irradiated. You get the advantage of getting That is, irradiation with light of a wavelength that drives inward proton-pump rhodopsin promotes the growth of the transformant, while irradiation of light with a wavelength that drives outward proton-pump rhodopsin promotes the growth of the transformant. growth can be suppressed.

As described above, the growth of the photosynthetic organism can be controlled by transforming the photosynthetic organism so that it can express the proton pump rhodopsin and irradiating it with light that drives the proton pump rhodopsin. That is, in another aspect of the present invention, there is provided a method for controlling the growth of photosynthetic organisms, comprising:
(1) obtaining a transformant of a photosynthetic organism that expresses proton pump prodopsin;
(2) driving the proton pump rhodopsin by irradiating the transformant with light;
The above problem is solved by providing a method characterized by comprising:

In addition, according to the transformant or the method for controlling the growth of photosynthetic organisms according to the present invention, the growth of photosynthetic organisms that produce useful biomass can be promoted or suppressed, and high-density culture of photosynthetic organisms is possible. Therefore, it can be expected to improve the efficiency of biomass production using photosynthetic organisms. That is, in still another aspect of the present invention, there is provided a method for producing biomass,
(A) culturing or cultivating a photosynthetic organism using the method described above for controlling the growth of the photosynthetic organism; and (B) recovering biomass from the photosynthetic organism;
A method for producing biomass is also provided.

According to the transformant or the method of controlling the growth of photosynthetic organisms of the present invention, the growth of photosynthetic organisms can be promoted or suppressed by light, and high-density culture of photosynthetic organisms is possible. Moreover, according to the biomass production method of the present invention, biomass derived from photosynthetic organisms can be efficiently produced.

FIG. 2 is a schematic diagram of a chloroplast genome modification method for proton pump rhodopsin gene transfer in Chlamydomonas cells used in Examples. FIG. 2 shows the expression of each rhodopsin in Chlamydomonas cells. FIG. 2 is a diagram showing the number of Chlamydomonas cells dependent on visible light and the time-dependent change thereof. FIG. 2 shows the results of examining the effect of visible light irradiation on the morphology of Chlamydomonas cells. Note that the scale bar in FIG. 4A indicates 12.5 μm. FIG. 2 is a diagram showing the results of examining the effect of visible light irradiation on the dry weight of Chlamydomonas. FIG. 2 is a diagram showing the results of examining the effect of visible light irradiation on the ability of chlamydomonas to form oil droplets (lipid droplets). Note that the scale bar in FIG. 6A indicates 12.5 μm. FIG. 2 shows the amino acid sequence of RmXeR derived from the marine eubacterium Rubricoccus marinus and the base sequence optimized for the codon usage of the chloroplast genome of Chlamydomonas that encodes the RmXeR. FIG. 2 shows the amino acid sequence of AR3 derived from the archaebacterium Halorubrum sodomense and the base sequence optimized for the codon usage of the chloroplast genome of Chlamydomonas that encodes the AR3. FIG. 4 shows amino acid sequences of AR3 mutants having a maximum absorption wavelength around 500 nm. Underlined amino acids in the amino acid sequence indicate amino acids mutated from wild-type AR3 having the amino acid sequence shown in FIG. 8 and SEQ ID NO: 2 in the sequence listing.

The present invention will be described in more detail below.

A transformant according to the present invention is a transformant of a photosynthetic organism and a transformant that expresses a light-driven proton pump rhodopsin. First, the proton pump rhodopsin expressed by the transformant of the present invention will be described.

As used herein, "proton-pump rhodopsin" is a membrane protein present in the cell membrane, driven by receiving light, and unidirectionally pumps protons from the inside to the outside of the cell or from the outside to the inside of the cell. It is a membrane protein that serves to transport. Among the proton pump rhodopsins, the proton pump rhodopsin that transports protons from the inside to the outside of the cell is called outward proton pump rhodopsin, and conversely, the proton pump rhodopsin that transports protons from the outside to the inside of the cell. is called inward proton pump rhodopsin. In the present specification, the term "proton pump rhodopsin" means both "outward proton pump rhodopsin" and "inward proton pump rhodopsin" unless otherwise specified.

As shown in the experimental examples described later, photosynthetic organisms, preferably, in a state in which inward proton pump rhodopsin is expressed in the chloroplast of the photosynthetic organism, are irradiated with light that drives the inward proton pump rhodopsin. By doing so, the growth of the photosynthetic organism can be promoted, that is, the growth rate can be increased, and the upper limit of the number of viable cells per unit volume, that is, the saturated cell density can be increased. Although there is no particular limitation on the type of inward proton-pump rhodopsin that can be used in the present invention, it is considered to be less absorbed and/or utilized by photosynthetic organisms at wavelengths of 450 nm to 650 nm, preferably wavelengths of 470 nm to 620 nm, more preferably wavelengths of 470 nm to 620 nm. It is preferably an inward proton-pump rhodopsin having an absorption maximum wavelength in the wavelength range of 490 nm to 590 nm. As such an inward proton pump rhodopsin, for example, Rubricoccus marinus xenorhodopsin (RmXeR) derived from the marine eubacterium Rubricoccus marinus can be suitably used.

As used herein, Rubricoccus marinus xenorhodopsin (RmXeR) refers to a protein having the amino acid sequence represented by SEQ ID NO: 1 and FIG. A protein having an amino acid sequence substantially identical to the amino acid sequence described herein. Here, the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 and FIG. 7 of the sequence listing is the amino acid sequence represented by SEQ ID NO: 1 and FIG. Similar to the protein having the amino acid sequence represented by SEQ ID NO: 1 and FIG. 7 in the sequence listing, retinal is a chromophore and the maximum absorption wavelength is visible light. It means a protein that exists in the region (approximately 400 to 600 nm), is driven by receiving such visible light, and exhibits the property of transporting protons from the outside to the inside of the cell. For example, an amino acid sequence mutated by substitution, deletion, insertion, and/or addition of one or more amino acids among all amino acids constituting the amino acid sequences of SEQ ID NO: 1 in the sequence listing and FIG. A protein that has Here, the plural number is, for example, a number within 10% of the total number of amino acids constituting the amino acid sequence, more preferably a number within 5%, still more preferably a number within 3%, and even more preferably 1%. The number is within Substitutions, deletions, insertions and/or additions of the above amino acid sequences may be mutations that originally existed in the nucleic acid encoding the protein, or new mutations by modifying the nucleic acid by methods known in the art. may be introduced into

On the other hand, as shown in the experimental examples described later, in a state in which the outward proton pump rhodopsin is expressed in the photosynthetic organism, more preferably in the chloroplast of the photosynthetic organism, light that drives the outward proton pump rhodopsin is applied to the photosynthetic organism. By irradiating with, the growth of the photosynthetic organism can be suppressed, i.e., the growth rate can be slowed down, and the upper limit of the number of viable cells per unit volume, i.e., the saturated cell density can be increased. . Although there is no particular limitation on the type of outward proton-pump rhodopsin that can be used in the present invention, It is preferably an outward proton-pump rhodopsin having an absorption maximum wavelength in the wavelength range of 490 nm to 590 nm. As such an outward proton-pump rhodopsin, for example, archidopsin 3 (AR3) of the archaebacterium Halorubrum sodomense can be suitably used.

As used herein, "archilodopsin 3 (AR3)" refers to a protein having the amino acid sequence represented by SEQ ID NO: 2 and FIG. A protein having an amino acid sequence substantially identical to the amino acid sequence described herein. Here, the protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 and FIG. 8 of the sequence listing is the amino acid sequence represented by SEQ ID NO: 2 and FIG. A protein having an amino acid sequence with relatively few points of difference, similar to the protein having the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing and FIG. (approximately 400 to 600 nm), is driven by receiving visible light in such a wavelength range, and exhibits the property of transporting protons from the inside of the cell to the outside of the cell. For example, among all the amino acids constituting the amino acid sequences represented by SEQ ID NO: 2 and FIG. A protein having an amino acid sequence. Here, the plural number is, for example, a number within 10% of the total number of amino acids constituting the amino acid sequence, more preferably a number within 5%, still more preferably a number within 3%, still more preferably 1 It is a number within %. Substitutions, deletions, insertions and/or additions of the above amino acid sequences may be mutations that originally existed in the nucleic acid encoding the protein, or new mutations by modifying the nucleic acid by methods known in the art. may be introduced into

As a protein having an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 and FIG. 8 in the sequence listing, for example, Yuki Sudo et al., Journal of Biological Chemistry 2013, 288(28), 20624-20632 and having the amino acid sequence represented by SEQ ID NO: 5 in the sequence listing and FIG. 9 (“the mutant AR3” in the same document). The AR3 mutant is an outward proton-pump-rhodopsin with a maximum absorption wavelength around 500 nm, and is said to be driven by light with a shorter wavelength than wild-type AR3 and RmXeR, which have a maximum absorption wavelength around 550 nm. It has characteristics.

On the other hand, as used herein, the term “photosynthetic organism” means an organism that performs photosynthesis, that is, an organism that performs photosynthesis that converts light energy into biologically usable energy, and mainly performs oxygenic photosynthesis. Refers to photosynthetic organisms. Photosynthetic organisms that perform oxygenic photosynthesis are specifically terrestrial plants and algae. Contains plants. On the other hand, algae is a general term for photosynthetic organisms that perform oxygenic photosynthesis, excluding land plants, and most of them are aquatic photosynthetic organisms. Algae include eukaryotic algae having a nucleus and chloroplasts and prokaryotic algae having no nucleus and chloroplasts. Dinoflagellates, gray algae, cryptophytes, haptophytes, chlorarachnion algae, and the like. On the other hand, prokaryotic algae include, for example, cyanobacteria. It is believed that chloroplasts originated from blue-green algae (cyanobacteria) that were taken up by eukaryotes, and that eukaryotic photosynthetic organisms including eukaryotic algae were born. As described above, eukaryotic algae and prokaryotic algae have common properties although they are classified differently.

The photosynthetic organism used in the present invention may basically be any photosynthetic organism as described above, and the type is not particularly limited. - From the viewpoint of easiness of culturing, microalgae are particularly preferably used. Here, the term “microalgae” refers to small algae having a size of several μm to several tens of μm among the algae described above, and mainly refers to unicellular algae. Many microalgae grow quickly and are easy to culture. In addition, since many plants produce biomass at a higher efficiency than higher plants, they are particularly suitable for biomass production.

Specific examples of microalgae belonging to green algae include Chlamydomonas such as Chlamydomonas reinhardtii, Desmodesmus Scenedesmus and Scenedesmus, so-called squid moth, Volvox, Microalgae belonging to the genera Tetraselmis, Chlorococcum, Dunalliella, Neochloris, and Trebouxiophyceae include, but are not limited to. Specific examples of microalgae belonging to the Treboxia algae include the genus Botryococcus such as Botryococcus braunii, the genus Chlorella, the genus Coccomyxa, the genus Pseudococcomyxa ), and microalgae belonging to the genus Trebouxia.

On the other hand, examples of microalgae belonging to Heterochontophytes include microalgae belonging to diatoms and eutectic algae. Specific examples of microalgae belonging to diatoms include the genus Cyclotella, the genus Cylindrotheca, the genus Fistulifera, the genus Mayamaea, the genus Phaeodactylum, the genus Skeletonema, and Examples include, but are not limited to, microalgae belonging to the genus Thalassiosira and the like. On the other hand, specific examples of microalgae belonging to eutectic algae include, but are not limited to, microalgae belonging to the genus Nannochloropsis.

Further, specific examples of microalgae belonging to dinoflagellates include the genus Amphidinium, microalgae belonging to the genus Symbiodinium, and specific examples of microalgae belonging to red algae include the genus Cyanidioschizon. (Cyanidioschyzon), microalgae belonging to the genus Phorphyridium, and microalgae belonging to the euglenid algae include microalgae belonging to the genus Euglena such as Euglena gracilis. It is not limited to these.

Specific examples of microalgae classified as blue-green algae (cyanobacteria) include the genus Anabaena, the genus Arthrospira, the genus Gloeobacter, the genus Microcystis, the genus Nostoc, Microalgae include, but are not limited to, Prochlorococcus, Synechocystis, Spirulina, Synechococcus, Thermosynechococcus, and the like.

The transformant of the present invention is a transformant obtained by transforming a photosynthetic organism as described above, and expresses a light-driven proton pump rhodopsin, that is, encodes a light-driven proton pump rhodopsin. A transformant containing the gene. Incidentally, the term "transformation" refers to a change in the original traits of a host caused by the expression in the host of a gene that the host normally does not express, and the term "transformant" means a transformed host. As described above, photosynthetic organisms such as land plants and algae are used as hosts for transformants of the present invention. For these hosts, proton pump rhodopsin, which is mainly distributed in vertebrates and bacteria, is a foreign gene that is not originally expressed.

There is no particular limitation on the method for obtaining a transformant of a photosynthetic organism that expresses proton pump rhodopsin, and a person skilled in the art can use an appropriate method. can be introduced into a photosynthetic organism. A nucleic acid construct capable of expressing proton pump rhodopsin in a photosynthetic organism is specifically a nucleic acid construct comprising a sequence (DNA sequence or RNA sequence) encoding all or part of proton pump rhodopsin, For example, it may be an expression vector containing a DNA or RNA sequence encoding all or part of the proton pump prodopsin. By introducing such a nucleic acid construct into a photosynthetic organism, proton pump rhodopsin can be expressed in the photosynthetic organism.

A nucleic acid construct used for transformation may contain, in addition to a sequence encoding proton pump rhodopsin, various sequences necessary for expressing proton pump rhodopsin in a host into which the nucleic acid construct is introduced. Such sequences include, for example, promoter sequences, terminator sequences, sequences encoding selectable marker genes, and the like. In addition to the gene sequence encoding the target protein to be expressed in the host, a gene fragment in which a promoter sequence and a terminator sequence are ligated to the 5' end side and 3' end side of the sequence, respectively, is called an expression cassette. The nucleic acid construct can be an expression vector containing one or more expression cassettes. For example, the sequence encoding the target protein proton pump rhodopsin to be expressed in the host and the sequence encoding the selectable marker gene are combined into one It may be contained in an expression vector as a bicistronic expression cassette placed between a promoter sequence and a terminator sequence. Alternatively, it may be contained in an expression vector as two monocistronic expression cassettes arranged between separate promoter and terminator sequences.

A promoter sequence is usually present in a coding region, that is, an untranslated region (5'UTR) at the 5' end of a sequence encoding a target protein to be expressed in a host, and initiates transcription initiation reaction of the sequence encoding the target protein. is a sequence that participates in The promoter sequence may be any promoter sequence as long as it functions in the host into which the nucleic acid construct is introduced. On the other hand, when the host is a eukaryotic photosynthetic organism having a nucleus and a chloroplast, it is preferable to introduce the target gene into the chloroplast genome of the host from the viewpoint of ease of obtaining a stable transformant. In such cases, the promoter sequence possessed by the nucleic acid construct is preferably a promoter sequence that functions in the chloroplast of the host. As the promoter sequence that functions in the chloroplast, a promoter sequence present in the 5' untranslated region of the chloroplast gene can be used, but it does not necessarily have to be derived from the chloroplast gene. It does not have to be derived from photosynthetic organisms. Promoter sequences of chloroplast genes that can be used in nucleic acid constructs include, for example, photosystem I reaction center protein genes (psaA, psaB), photosystem II reaction center protein gene (psbA), 16S ribosomal RNA gene (16S rDNA) , the ribulose 1,5-bisphosphate carboxylase gene (rbcL), and the chloroplast ATP synthase gene (atpA). A person skilled in the art can select an appropriate promoter sequence according to the type of photosynthetic organism or organelle into which the nucleic acid construct is to be introduced.

A terminator sequence is usually present in a coding region, that is, an untranslated region (3'UTR) at the 3' end of a gene sequence encoding a target protein to be expressed in a host, and terminates transcription of the target protein-encoding sequence. A sequence that participates in a reaction. The terminator sequence may basically be anything as long as it is capable of terminating transcription initiated with the participation of the above-mentioned promoter sequence. On the other hand, when the host is a eukaryotic photosynthetic organism having a nucleus and a chloroplast, it is preferable to introduce the target gene into the chloroplast genome of the host from the viewpoint of ease of obtaining a stable transformant. In such a case, the terminator sequence possessed by the nucleic acid construct, like the promoter sequence, is preferably a terminator sequence that functions in the chloroplast of the host. As the terminator sequence that functions in the chloroplast, the terminator sequence of the chloroplast gene can be used, but it does not necessarily have to be derived from the chloroplast gene or from photosynthetic organisms. . Terminator sequences of chloroplast genes that can be used in nucleic acid constructs include, but are not limited to, the sequence of the 3′ untranslated region of the ribulose 1,5-bisphosphate carboxylase gene (rbcL). A person skilled in the art can select an appropriate terminator sequence according to the type of photosynthetic organism or organelle into which the nucleic acid construct is to be introduced.

In addition, the nucleic acid construct may contain a selectable marker gene. Any selectable marker gene can be used as long as it can distinguish individuals in which the selectable marker gene is expressed (i.e., transformed) from non-transformed individuals (i.e., non-transformants). , genes encoding fluorescent proteins such as green fluorescent protein (GFP) and red fluorescent protein (RFP), genes encoding luciferases such as luciferase, genes resistant to selected drugs, and the like are preferably used. be done. For example, when a resistance gene to a selection drug is used as a selection marker gene, a transformant transformed by introducing a nucleic acid construct containing the selection marker gene shows resistance to the selection drug. By culturing a population containing both transformants and non-transformants in a medium or the like containing the selection drug, non-transformants can be killed and transformed transformants can be efficiently selected. can. There is no particular limitation on the resistance gene to the selection drug used as the selection marker gene, but if I dare to exemplify it, for example, the aminoglycoside adenyltransferase gene (aadA) (Z. Svab and P. Maliga, Proc. Natl. Acad. Sci. USA 1993, 90, 913-917.), spectinomycin and streptomycin resistance genes, aminoglycoside phosphotransferase gene (aphA-6) (JM Bateman and S. Purton, Molecular and General Genetics 2000, 263, 404-410.) and kanamycin-resistant genes such as the neomycin phosphotransferase gene (nptII) (H. Carrer et al., Molecular and General Genetics 1993, 241, 49-56.) can be used. A person skilled in the art can select an appropriate selectable marker gene according to the type of photosynthetic organism, organelle, etc. into which the nucleic acid construct is to be introduced.

The selection marker gene may be removed from the transformant after transformation. A method for removing the selectable marker gene from the transformant after transformation is described, for example, in Anil Day et. al. (Plant Biotechnology Journal 2011, 9, 540-553.) and JP-A-2015-171358, but are not limited thereto.

In addition, from the viewpoint of obtaining a transformant that stably expresses proton pump prodopsin, it is preferable that the sequence encoding proton pump prodopsin is integrated into the genome of the host photosynthetic organism by homologous recombination. Therefore, in a preferred embodiment, the nucleic acid construct comprises a sequence to be integrated into the genome of the host by homologous recombination, that is, a sequence encoding the target protein proton pump rhodopsin, or the 5′ end of the expression cassette containing the sequence. It has a pair of homologous recombination sequences flanking the sequence or expression cassette on the 3' end side. A pair of homologous recombination sequences is a pair of base sequences having homology with a base sequence of a predetermined region of the host genome, specifically, a base sequence present upstream of the predetermined region of the host genome A pair of homologous recombination sequences can be a combination of a sequence having homology and a sequence having homology to a base sequence existing downstream of the same region. By crossing a pair of homologous recombination sequences and a sequence having homology to the pair of homologous recombination sequences in the genome of the host, the target protein present between the pair of homologous recombination sequences is encoded. A sequence, in the present invention, a sequence encoding proton pump rhodopsin or an expression cassette comprising said sequence is integrated into the genome of the host.

In addition, when the host photosynthetic organism is a eukaryotic photosynthetic organism having a nucleus and chloroplast, the chloroplast genome possessed by the host has a higher homologous recombination efficiency than the nuclear genome. By targeting the genome, the target gene can be introduced more efficiently by homologous recombination. Therefore, in a preferred embodiment, the pair of homologous recombination sequences is a pair of sequences capable of homologous recombination with a part of the chloroplast genome, i.e., a base sequence of a predetermined region of the chloroplast genome of the host and It is preferably a pair of nucleotide sequences having homology. Specifically, a sequence having homology with a nucleotide sequence existing upstream of a predetermined region of the chloroplast genome of the host and a sequence existing downstream of the same region A combination of a sequence having homology with the base sequence that is used can be used as a pair of homologous recombination sequences. From the viewpoint of efficiently causing homologous recombination, the length of each pair of homologous recombination sequences is preferably 500 bp or more, and usually about 1 kb to 3 kb is used.

The nucleic acid constructs described above can be introduced by an appropriate method, regardless of physical, chemical or biological methods, depending on the host to be transformed. Methods for introducing nucleic acid constructs include, for example, the particle gun method, the Agrobacterium method, and the protoplast method, but are not limited to these methods, and those skilled in the art can use appropriate introduction methods. For example, using viral vectors such as alfalfa mosaic virus (AIMV), tobacco mosaic virus (TMV), plumpox virus (PPV), potato X virus (PVX), cucumber mosaic virus (CMV), zucchini yellow mosaic virus (ZYMV) Alternatively, a cell membrane permeable peptide may be used. For introduction of nucleic acid constructs into photosynthetic organisms using cell membrane penetrating peptides, see, for example, Keiji Numata et al. (Scientific Reports 8, Article number: 10966 (2018)). The Agrobacterium method and the particle gun method are generally used from the viewpoint of the efficiency of introduction of nucleic acid constructs and the efficiency of transformants.

The particle gun method, also called the particle gun method, is a method of introducing a nucleic acid construct by directly shooting microparticles (tungsten particles or gold particles) coated with the nucleic acid construct and having a particle size of about 0.1 to 2.0 μm into the host. (JE Boynton et al., Science 1988, 240, 1534-1538.). Since the particle gun method can directly introduce nucleic acid constructs into intracellular organelles such as chloroplasts and mitochondria, it is particularly suitable for gene introduction into chloroplast genomes.

On the other hand, the Agrobacterium method involves infecting plant cells with bacteria of the genus Agrobacterium, which are soil bacteria that infect plants, such as A. tumefaciens and A. rhizogenes. It is a transformation method that utilizes the property of introducing its own DNA into plant cells when it transforms. The target gene can be introduced into the host by introducing the target gene to be introduced into the transformant into Agrobacterium and infecting the host with the resulting Agrobacterium.

The protoplast method is a method in which cells are treated with a cell wall-degrading enzyme solution (for example, a mixture of cellulase and pectinase), etc., the cell walls are removed to form protoplasts, and then the target gene is introduced into the protoplasts. Electroporation, microinjection, polyethylene glycol, glass beads, and the like are known methods for introducing a target gene into protoplasts. Electroporation is a method of introducing a target gene into protoplasts by applying an electric pulse to the protoplasts to instantaneously create holes in the cell membrane. On the other hand, the microinjection method is a method of directly introducing a target gene into a protoplast using a micro glass tube or the like. The polyethylene glycol method is a method of introducing a target gene into protoplasts by allowing polyethylene glycol to act on the protoplasts. The glass bead method is a method in which glass beads with a diameter of about 0.5 mm and a nucleic acid construct are added to a suspension of protoplasts, and the mixture is stirred and mixed with a vortex mixer or the like to introduce a target gene into protoplasts.

After introducing the nucleic acid construct using an appropriate introduction method as exemplified above, transformants expressing proton pump prodopsin are selected according to the type of selection marker gene contained in the introduced nucleic acid construct. It may be selected by an appropriate method. On the other hand, when the introduced nucleic acid construct does not contain a selection marker gene, transformants may be selected by directly confirming the introduction of the target gene by, for example, the PCR method.

When preparing a transformant of a multicellular photosynthetic organism such as a multicellular plant or algae, the nucleic acid construct is introduced into one or more cells that constitute the multicellular photosynthetic organism. Then, the cell may be differentiated to regenerate the whole photosynthetic organism. More specifically, for example, after introducing a nucleic acid construct into cells constituting plants or algae by an appropriate method, dedifferentiation (callus transformation), and the obtained callus is cultured in a medium containing a predetermined amount of a predetermined plant hormone (auxin or cytokinin) to differentiate and form plants or algae. Transformed multicellular plants or algae can also be obtained by introducing the nucleic acid construct into cells previously callusized and allowing the resulting callus to differentiate. However, the method for preparing a transformant of a multicellular photosynthetic organism is not limited to the above, and a person skilled in the art can obtain a transformant of a multicellular photosynthetic organism using an appropriate method. Other methods include, for example, the in planta method of transforming part of the plant tissue.

As described above, a transformant of a photosynthetic organism that expresses proton pump rhodopsin can be obtained by introducing a nucleic acid construct into a photosynthetic organism, appropriately selecting a transformant, and dedifferentiating/differentiating if necessary. However, specific transformation methods are not limited to those described above, and those skilled in the art can prepare transformants by appropriate methods. The transformation method of photosynthetic organisms is also described, for example, in "The Chemical Society of Japan, Experimental Chemistry Course 29 Basic Technology of Biotechnology 5th Edition, Maruzen Co., Ltd., published on July 25, 2006". .

The method for controlling the growth of photosynthetic organisms of the present invention will be described below.

As used herein, "controlling the growth" of photosynthetic organisms means promoting or suppressing the growth of photosynthetic organisms, and increasing the upper limit of the number of viable cells per unit volume, i.e., increasing the saturated cell density. Or it is a concept that means including reducing. As used herein, the term “growth” of a photosynthetic organism means that the number of cells of the photosynthetic organism increases. , respectively, mean that the rate of increase in cell number is decreased. In addition, when the photosynthetic organism is microalgae, the promotion or inhibition of growth can be observed as an increase or decrease in cell growth rate during the logarithmic growth phase. In addition, an increase or decrease in the upper limit of the number of viable cells per unit volume is observed as an increase or decrease in the number of cells per unit volume in the stationary growth phase when cell growth reaches a steady state, that is, the saturated cell density. can be

As described above, according to the findings of the present inventors, when a transformant of a photosynthetic organism that has been transformed to be able to express proton pump rhodopsin is irradiated with light to drive proton pump rhodopsin, the transformant Body growth can be promoted or inhibited and the upper limit of the number of viable cells per unit volume can be increased. That is, the method for controlling the growth of photosynthetic organisms according to the present invention includes:
(1) obtaining a transformant of a photosynthetic organism that expresses proton pump prodopsin;
(2) driving the proton pump rhodopsin by irradiating the transformant with light;
including. Each step will be described in more detail below.

Step (1) Step of obtaining a transformant of a photosynthetic organism This is a step of obtaining a transformant of a photosynthetic organism expressing proton pump prodopsin by transforming the photosynthetic organism. The transformant and its preparation method are as described above. A nucleic acid construct containing a sequence encoding proton pump prodopsin is introduced into a photosynthetic organism, and then a transformed individual (individual expressing proton pump prodopsin). A transformant expressing proton pump rhodopsin can be obtained by selecting . That is, in a preferred embodiment, the step (1) comprises introducing a nucleic acid construct containing a sequence encoding a proton pump rhodopsin into a photosynthetic organism, and selecting a transformant expressing the proton pump rhodopsin. can include Needless to say, when transformants of photosynthetic organisms expressing proton pump rhodopsin are commercially available, the commercially available transformants may be used.

As long as a transformant that expresses the proton pump prodopsin can be obtained, basically any method can be used to introduce the nucleic acid construct encoding the proton pump prodopsin into the photosynthetic organism. For example, protoplast method, particle gun method, Agrobacterium method, viral vector method, etc. can be used to introduce nucleic acid constructs into photosynthetic organisms.

On the other hand, the method for selecting transformants expressing proton pump rhodopsin is not particularly limited, and transformants expressing proton pump rhodopsin may be selected using an appropriate method. Transformants can be easily selected by transforming using a nucleic acid construct containing a gene. For example, when a nucleic acid construct containing a fluorescent protein is used as a selection marker gene, a transformant transformed by introducing the nucleic acid construct emits a predetermined fluorescence. Transformants can be selected. On the other hand, when a nucleic acid construct containing a sequence encoding a resistance gene to a selection drug is used as a selection marker gene, a transformant transformed by introducing the nucleic acid construct has a resistance gene to a predetermined selection drug. on the other hand, non-transformed individuals do not express the resistance gene. , only transformed individuals can be selectively obtained. Any selection marker gene may be used as long as the transformant can be selected. In addition, it is not always necessary to use a selectable marker gene, and transformants may be selected by directly confirming introduction of the target gene by PCR, for example.

In addition, when the photosynthetic organism is a multicellular photosynthetic organism, for example, a multicellular plant or algae, the nucleic acid construct is introduced into one or more cells of the multicellular photosynthetic organism, and then the cell callus and redifferentiation to regenerate photosynthetic organisms. That is, the step (1) includes a step of introducing a nucleic acid construct containing a sequence encoding proton pump rhodopsin into a photosynthetic organism, a step of obtaining callus from the photosynthetic organism into which the nucleic acid construct has been introduced, and a step of differentiating the obtained callus. to obtain transformants. A callus is a dedifferentiated plant or algae cell, and is typically formed by culturing a portion of a plant or algae cell in the presence of a given plant hormone. By adjusting the concentration and type of plant hormones brought into contact with the callus, the callus once formed can be redifferentiated into individual plants or algae, thus obtaining transformed individual plants or algae. be able to. Needless to say, after obtaining callus, a nucleic acid construct containing a proton pump prodopsin-encoding sequence may be introduced, and the callus introduced with the nucleic acid construct may be redifferentiated.

Step (2) Driving proton pump rhodopsin A step of irradiating the transformant of the photosynthetic organism obtained in step (1) with light to drive the proton pump rhodopsin expressed in the transformant. . As shown in the experimental examples described later, in a state in which proton pump rhodopsin is expressed in photosynthetic organisms, preferably in the cell membrane of photosynthetic organisms, more preferably in a state in which proton pump rhodopsin is expressed in chloroplasts of photosynthetic organisms, proton When pumprhodopsin is driven, the growth of photosynthetic organisms is promoted or inhibited. In addition, by cultivating or culturing in an environment irradiated with light that drives proton pump rhodopsin, the upper limit of the number of cells that can be grown per unit volume, that is, the saturated cell density can be increased.

The wavelength of the light irradiated to the transformant to drive the proton pump rhodopsin is not particularly limited as long as it contains light of a wavelength capable of activating the proton pump rhodopsin and driving the proton pump rhodopsin. Instead, it may be appropriately selected according to the absorption wavelength of the proton pump rhodopsin, the growth environment of the photosynthetic organism to be used, and the like. In addition, proton pump rhodopsin binds retinal as a chromophore, and typically has a maximum absorption wavelength in the wavelength range of about 400 nm to 600 nm. It is activated through isomerization, resulting in proton transport across the cell membrane. Therefore, although it depends on the type of proton-pump-rhodopsin used, the wavelength of light irradiated to the transformant preferably includes light with a wavelength of about 400 nm to 600 nm. Light having such wavelengths includes green light having a wavelength of approximately 500 nm to 600 nm. Thus, the method of controlling the growth of photosynthetic organisms according to the present invention mainly uses green light, which has not been widely used for the growth control of photosynthetic organisms up to now. Of course, light of various wavelengths may be used in combination. For example, green light may be combined with blue light (wavelength 400 to 500 nm) or red light (wavelength 600 nm to 700 nm). can be

The light source for the emitted light is not particularly limited. For example, artificial light sources such as LED lamps, fluorescent lamps, high-pressure sodium lamps, and metal halide lamps may be used, or natural light such as sunlight may be used. Moreover, it may be a combination thereof. Note that the emitted light may be flashing light, continuous light, or a combination thereof.

On the other hand, in still another aspect of the present invention, there is provided a method for producing biomass,
(A) culturing or cultivating a photosynthetic organism using the method described above for controlling the growth of the photosynthetic organism; and (B) recovering biomass from the photosynthetic organism;
A method for producing biomass is provided. According to the transformant according to the present invention and the method for controlling the growth of photosynthetic organisms according to the present invention, the growth of photosynthetic organisms can be promoted or suppressed, and the number of viable cells per unit volume Since the upper limit, that is, the saturated cell density can be increased, various biomass derived from photosynthetic organisms can be efficiently produced.

In this specification, "biomass" means materials derived from photosynthetic organisms, and includes photosynthetic organisms themselves and processed products of photosynthetic organisms. The treated products of photosynthetic organisms include useful substances obtained from photosynthetic organisms and residues of photosynthetic organisms remaining after collecting the useful substances. Examples of useful substances obtained from photosynthetic organisms include, but are not limited to, fats and oils, fatty acids, hydrocarbons, and starches. Chlamydomonas, a type of green algae, is known to produce starch and triacylglycerol, which are raw materials for biofuels.

There are no particular restrictions on the method of collecting biomass, and an appropriate method can be used according to the type of photosynthetic organisms and the type of biomass to be collected. To give an example, for example, when the biomass is a substance soluble in an organic solvent such as fat or hydrocarbon, the photosynthetic organisms are dried and crushed, and then recovered by extraction with an organic solvent. be able to. The collected material may be further purified by operations such as chromatography and distillation.

The disclosure contents of all documents cited in this specification are incorporated by reference into the specification as a whole. Also, throughout this specification, where the singular forms of the words “a,” “an,” and “the” are included, the singular as well as the plural unless the context clearly indicates otherwise. shall include things.

The present invention will be further described below with reference to examples, but the examples are merely illustrations of embodiments of the present invention and do not limit the scope of the present invention.

Experiment 1. Preparation of Plasmid for Chlamydomonas Cell Expression A transforming plasmid for expression in Chlamydomonas reinhardtii (hereinafter simply referred to as "Chlamydomonas"), a kind of green alga, was prepared. A specific configuration is as shown in FIG. At the restriction enzyme SbfI site of pSXY246A, a vector for Chlamydomonas chloroplast transformation, a gene encoding AR3 having a sequence optimized for the codon usage of the chloroplast genome of Chlamydomonas (Genbank accession number: WP_092921078) or RmXeR A gene encoding a gene (Genbank accession number: WP_094549673) is inserted, and a gene encoding an HA (hemagglutinin) tag sequence for detecting the expression of AR3 or RmXeR is inserted at the 5' end or 3' end. is added. In FIG. 1, "psaA" and "rbcL" indicate the promoter sequence of the psaA gene and the terminator sequence of the rbcL gene, respectively, which are located before and after the sequence encoding the proton pump rhodopsin gene (AR3 gene or RmXeR gene). For homologous recombination, the gene sequence of the chloroplast protein psbA (the gene encoding the D1 protein that is the reaction center of photosystem II and 5 On the other hand, the rrn5 (ribosome synthesis gene) gene sequence and the rrnL (ribosome synthesis gene) gene sequence are arranged on the 3′ end side, and these are the present It corresponds to a pair of homologous recombination sequences as referred to in the specification. In addition, E. coli gene (aadA) encoding aminoglycoside adenyltransferase (AAD) that confers resistance to spectinomycin and streptomycin and ATPaseCF1 gene (atpA) are encoded as selectable marker genes for selection/selection of transformants. It contains an array that In the chloroplast genome of the Fud7 strain, which is a cell strain of Chlamydomonas reinhardtii used for transformation, the sequences arranged at the 5′ end side and the 3′ end side of the expression cassette of the above transforming plasmid are By homologous recombination occurring via these sequences, the proton pump rhodopsin gene is incorporated into the chloroplast genome of the host Chlamydomonas reinhardtii. As described above, the promoter sequence of the psaA gene, which encodes the reaction center protein of photosystem I, is used as the promoter sequence that functions in the chloroplast. That is, the plasmid is integrated into the chloroplast genome by homologous recombination, and AR3 or RmXeR encoded by the plasmid is expressed in the chloroplast.
The vector pSXY246A for the Chlamydomonas chloroplast transformant used in this experiment was given by Dr. Yuichiro Takahashi of Okayama University, and was prepared by Yuichiro Takahashi et al., Plant Cell Physiol 37. (2): 161-168 (1996), or Michelet Laure et al. , Plant Biotechnology Journal, 9(5): 565-574 (2011).

Experiment 2. Chloroplast genome recombination of Chlamydomonas cells Fud7 strain, a cell strain of Chlamydomonas reinhardtii, was recombined with the chloroplast genome according to the standard method. The specific procedure is as follows. Chlamydomonas reinhardtii, which is a unicellular eukaryotic photosynthetic organism and a kind of green algae, is widely used in the field of life sciences including the field of botany as the simplest model of photosynthetic organisms.

First, for Chlamydomonas Fud7 suspended in TAP medium having the composition shown in Table 1 below, the AR3 plasmid or RmXeR plasmid prepared in Experiment 1 was added to carrier particles (tungsten particles with a particle size of 1.0 to 1.3 μm). ) and injected into Chlamydomonas using a particle gene introduction device (product name: IDERA, model: GIE-III, Tanaka Co., Ltd.) to introduce the gene into the chloroplast. The cells after gene introduction were applied to a TAP plate containing spectinomycin (final concentration: 150 μg mL ⁻¹ ) according to a conventional method, and placed in an incubator for plant culture (product name “Mini plant incubator 3 in 1 LED lighting growing shelf equipped type”). , Nihon Ika Kikai Seisakusho Co., Ltd.) under the conditions of a temperature of 25 ^° C., an irradiation light wavelength of 660 nm, and a photon amount of 85 μmol photons m ⁻² sec ⁻¹ for 19 days.

Experiment 3. Confirmation of Expression of AR3 or RmXeR in Chlamydomonas Cells Expression of AR3 or RmXeR in Chlamydomonas cells was confirmed by Western blotting for the HA tag sequence added to the N-terminus of AR3 or the C-terminus of RmXeR. Specifically, the colonies of Chlamydomonas cells prepared in Experiment 2 above were transferred to TAP medium and placed in a plant culture incubator at a temperature of 25 ^° C, an irradiation light wavelength of 660 nm, and a photon amount of 20 to 30 µmol photons m ^-2 sec ^-1. , shaking culture at 100 rpm. After that, Chlamydomonas cells were recovered by centrifugation, and a thylakoid membrane fraction was obtained using sucrose density gradient centrifugation. The obtained thylakoid membrane fraction was appropriately diluted so that the amount of chlorophyll was 1 μg (hereinafter referred to as “1 μg Chl”), and electrophoresis was performed using a 5% acrylamide gel as a concentration gel and a 12% acrylamide gel as a separation gel. Applied to a gel for electrophoresis. After electrophoresis, transfer to Immunobilon (R)-FL PVDF membrane (Merck KGaA, Germany), which is a transfer membrane, anti-HA tag antibody (Anti-HA-tag mAb monoclonal, isotype: Mouse IgG2bκ, Medical Biological Research Co., Ltd. place) was used as the primary antibody, and an immunization antibody reaction was performed for 3 hours. Next, HRP-labeled anti-mouse IgG (GE Healthcare Japan Co., Ltd.) diluted 6,000-fold with PBS-Tween (Tween concentration: 0.1%) was used as a secondary antibody, and an immunization antibody reaction was performed for 1 hour. After that, chemiluminescence was detected using ECL prime (GE Healthcare Japan Co., Ltd.). Chemiluminescence was detected using a commercially available image analyzer (product name: "ImageQuant LAS 4000mini", GE Healthcare Japan Co., Ltd.).

The results obtained are shown in Figure 2. As shown in FIG. 2, compared with the non-transformant (“control” in FIG. 2), both AR3 gene-introduced cells (“AR3” in FIG. 2) or RmXeR gene-introduced cells (“RmXeR” in FIG. 2) A strong signal was observed around the putative mass of rhodopsin, 28.8 kDa (AR3) or 26.1 kDa (RmXeR) (Fig. 2). Also, in the chloroplast thylakoid membrane fraction, a band with strong signal intensity was observed at the same position (Fig. 2). These results confirmed that both rhodopsins were mainly expressed in the chloroplast thylakoid membrane of Chlamydomonas cells.

Experiment 4. Observation of Growth Behavior of Chlamydomonas Cells The growth behavior of the Chlamydomonas transformant obtained in Experiment 2 under visible light irradiation was observed. Specific procedures are as follows. First, a transformant expressing AR3 or RmXeR and, as a control, Chlamydomonas (rhodopsin-non-expressing cells) not expressing AR3 or RmXeR were 0.50× in 5 mL of HSM medium having the composition shown in Table 5 below. The cells were suspended at 10 ⁶ cells/mL and cultured in an incubator (product name: tabletop artificial weather device <3in1LED lighting type>, model number: LH-80LED-DT, Nihon Ika Kikai Seisakusho Co., Ltd.). The conditions for light irradiation during culture are as follows: irradiation light wavelength 660 nm (red LED), photon amount 20 μmol photons m ⁻² sec ⁻¹ , irradiation light wavelength 520 nm (green LED), photon amount 10 μmol photons m. ⁻² sec ⁻¹ , irradiation light wavelength 450 nm (blue LED), photon quantity 20 μmol photons m ⁻² sec ⁻¹ . After a predetermined culture time had passed, the number of cells was measured using a cell analyzer (product name “Countess II FL automatic cell counter”, Thermo Fisher Scientific K.K.).

The results obtained are shown in FIG. After 72 hours of cell proliferation (late logarithmic growth phase), the number of cells per unit volume was 8.40±0.341×10 ⁶ cells/mL in the rhodopsin non-expressing cells (“control” in FIG. 3). On the other hand, AR3-expressing cells express in the chloroplast AR3, an outward proton-pump rhodopsin that is thought to transport protons from the inside to the outside of the cell, thereby promoting intracellular alkalinization (Fig. 3 In "AR3"), the number of cells per unit volume was 6.52±0.427×10 ⁶ cells/mL, which was approximately 0.78 times that of the control (FIG. 3). On the other hand, RmXeR-expressing cells (“RmXeR” in FIG. 3) expressing in chloroplasts RmXeR, which is an inward proton pump rhodopsin that is thought to transport protons from the outside to the inside of cells and thus promote intracellular acidification. ), the number of cells per unit volume was 10.9±0.836×10 ⁶ cells/mL, which was approximately 1.3 times that of the control (FIG. 3). Thus, by expressing AR3, which is an outward proton-pump rhodopsin, or RmXeR, which is an inward proton-pump rhodopsin, in Chlamydomonas cells and irradiating light that drives the proton pump rhodopsin, the growth of Chlamydomonas cells in the logarithmic growth phase was achieved. can be promoted or suppressed, respectively.

On the other hand, 168 hours after the cell proliferation reached the stationary phase, the number of rhodopsin non-expressing cells (“control” in FIG. 3) was 8.32±0.978×10 ⁶ cells/mL. On the other hand, AR3-expressing cells expressing in chloroplasts AR3, which is an outward proton pump rhodopsin that is thought to transport protons from the inside to the outside of the cells and thereby promote intracellular alkalinization (“AR3” in FIG. 3). In , the number of cells per unit volume, that is, the cell density was 16.1±0.881×10 ⁶ cells/mL, approximately 1.9 times higher than the control (FIG. 3). On the other hand, RmXeR-expressing cells (“RmXeR” in FIG. 3) expressing in chloroplasts RmXeR, which is an inward proton pump rhodopsin that is thought to transport protons from the outside to the inside of cells and thus promote intracellular acidification. ), the number of cells per unit volume, that is, the cell density was 16.5±1.24×10 ⁶ cells/mL, approximately 2.0 times higher than the control (FIG. 3). Thus, in Chlamydomonas cells, by expressing AR3, which is an outward proton-pump rhodopsin, or RmXeR, which is an inward proton-pump rhodopsin, and growing them in an environment irradiated with light that drives the proton pump rhodopsin, cell proliferation of Chlamydomonas It was shown that the number of cells per unit volume in stationary phase, ie the upper limit of saturation cell density, is increased.

Experiment 5. Simultaneously with observation experiment 4 on the morphology of Chlamydomonas cells , the effect of visible light irradiation on the morphology of Chlamydomonas cells was observed. Morphological changes of cells are observed using an upright microscope (model: CX41N-31, Olympus Corporation). More specifically, cells are set on the stage of an upright microscope, and a microscope CCD camera (FULL HD HDMI Photographs of cell appearance were taken using a Camera TrueChrome II Plus high-precision monitor set, Biotools Inc.). The above observations were made at room temperature (approximately 15 to 25 ^° C). On the other hand, the cell area is measured by taking a photograph of the cell using a cell analyzer (product name "Countess II FL automatic cell counter", Thermo Fisher Scientific K.K.) and analyzing the obtained image by software (ImageJ). I asked for it.

The results are shown in Figure 4. As shown in FIG. 4, the cell area and cell morphology in the logarithmic growth phase 24 hours and 72 hours after the start of culture, and in the stationary growth phase 168 hours after the start of the culture, were observed in AR3-expressing cells or RmXeR-expressing cells expressing proton pump rhodopsin. and a control that did not express proton pump rhodopsin. This result indicates that the growth of transformants expressing proton pump rhodopsin is promoted or suppressed without impairing normal cell functions.

Experiment 6. Measurement of Dry Weight of Chlamydomonas Cells Simultaneously with Experiment 4, the effect of visible light irradiation on the dry weight of the Chlamydomonas transformants was evaluated. Specific procedures are as follows. That is, in Experiment 4, after 168 hours from the start of culture, 5 mL of the culture medium was collected, centrifuged, and then the supernatant was removed. Then, 1 mL of sterilized water was added, and after sufficiently suspending with a vortex mixer (product name “TUBE MIXER TRIO”, manufactured by AS ONE Co., Ltd.), centrifugation was performed and the supernatant was removed. As described above, the washing operation using sterilized water was performed three times in total to remove the medium components. Then, the cells were transferred to an incubator heated to 80° C. and allowed to stand for 4 hours to dry. After that, the dry mass was measured using an analytical electronic balance (product name “HR-100AZ”, manufactured by A&D Co., Ltd.).

The results are shown in Figure 5. As shown in FIG. 5, the dry weight in the stationary growth phase 168 hours after the start of culture was higher in the AR3-expressing cells and RmXeR-expressing cells, which express proton pump rhodopsin, compared to the control that did not express proton pump rhodopsin. increased 2.0-fold and 1.3-fold. The results showed that in transformants expressing proton pump rhodopsin, the total mass of cells in the culture increased as the number of cells in the culture increased, without loss of normal carbon fixation capacity. It is shown that.

Experiment 7. Evaluation of oil droplet (lipid droplet) forming ability of Chlamydomonas cells Simultaneously with Experiment 4, the Chlamydomonas transformant obtained in Experiment 2 was evaluated for oil droplet (lipid droplet) forming ability under visible light irradiation. Oil droplet forming ability was assessed based on Nile Red staining of the oil droplets formed. Specifically, in Experiment 4, after 168 hours from the start of the culture, 1 mL of the culture medium was collected, and Nile Red (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) was added to the collected culture medium at a final concentration of 1.5 mL. Added to 0 μg/mL. Then, this culture solution was dropped onto a slide glass, and a cover glass was placed thereon. Cells were observed using an inverted research microscope (model: IX71, manufactured by Olympus Corporation) equipped with a mercury lamp U-LH100HGAPO (manufactured by Olympus Corporation). The fluorescence emitted by Nile Red was observed by irradiating excitation light with a wavelength of 480±10 nm and detecting fluorescence in a wavelength region of 550±10 nm. The above observations were made at room temperature (about 15 to 25 ^° C). Fluorescence images were acquired and analyzed using a CCD camera (ORCA-AG, Hamamatsu Photonics Co., Ltd.) and Meta Vue software (Version 7.5.6.0, Molecular Devices, USA).

The results are shown in Figure 6. As shown in FIG. 6(A), in the stationary phase of growth 168 hours after the start of culture, the AR3-expressing cells or RmXeR-expressing cells expressing proton pump rhodopsin showed oil growth in the same way as the control not expressing proton pump rhodopsin. A fluorescent signal from Nile Red staining indicating droplet formation could be observed. In addition, as an indicator of the amount of oil droplets formed, the fluorescence intensity of the fluorescence signal derived from Nile Red staining in each cell was compared. As shown in FIG. increased by 1.7 times. On the other hand, no change in fluorescence intensity was observed in RmXeR-expressing cells compared to the control. This result shows that the normal oil droplet formation ability is not impaired in transformants expressing proton pump rhodopsin, and that transformants expressing AR3, which is an outward proton pump rhodopsin (AR3-expressing cells ) rather shows that the oil droplet forming ability is increased.

[Description of Sequence Listing]
SEQ ID NO: 1: Amino acid sequence of RmXeR from the marine eubacterium Rubricoccus marinus SEQ ID NO: 2: Amino acid sequence of AR3 from the archaebacterium Halorubrum sodomense SEQ ID NO: 3: Chlamydomonas chloroplast encoding RmXeR from the marine eubacterium Rubricoccus marinus Nucleotide sequence optimized for the codon usage of the genome SEQ ID NO: 4: Nucleotide sequence optimized for the codon usage of the chloroplast genome of Chlamydomonas, encoding AR3 derived from the archaebacteria Halorubrum sodomense SEQ ID NO: 5: Around a wavelength of 500 nm Amino acid sequences of AR3 mutants with absorption maximum wavelengths

According to the transformant or the method for controlling the growth of photosynthetic organisms of the present invention, the growth of photosynthetic organisms is promoted or suppressed by means of light, which is easily controllable, and the number of photosynthetic organisms capable of growing per unit volume is increased. The upper limit of the number of cells, ie, the saturation cell density, can be increased, enabling high-density cultivation of photosynthetic organisms. Such transformants of photosynthetic organisms and methods of controlling the growth of photosynthetic organisms contribute to the efficiency of various biomass production, and are particularly useful in a wide range of industrial fields toward the construction of a sustainable society. application is expected.

Claims

A transformant of a photosynthetic organism that expresses a proton pump rhodopsin driven by light.
　The transformant according to claim 1, wherein the proton pump rhodopsin is an inward proton pump rhodopsin that transports protons from the outside to the inside of the cell.
　The transformant according to claim 1, wherein the proton pump rhodopsin is an outward proton pump rhodopsin that transports protons from inside the cell to outside the cell.
3. The proton pump rhodopsin is both an inward proton pump rhodopsin that transports protons from the outside to the inside of the cell and an outward proton pump rhodopsin that transports the protons from the inside to the outside of the cell. 1. The transformant according to 1.
　The transformant according to claim 2 or 4, wherein the inward proton pump rhodopsin is Rubricoccus marinus xenorhodopsin (RmXeR).
　The transformant according to claim 3 or 4, wherein the outward proton pump rhodopsin is archilodopsin 3 (AR3).
The transformant according to any one of claims 1 to 6, wherein the photosynthetic organism is microalgae.
The microalgae are microalgae belonging to any one of green algae, red algae, heterochelous algae, euglenoid algae, dinoflagellates, gray algae, cryptophytes, haptophytes, chlorarachniophytes, and cyanobacteria. The transformant according to claim 7.
The transformant according to claim 8, wherein the microalgae are microalgae belonging to green algae.
A method for controlling the growth of a photosynthetic organism, comprising:
(1) obtaining a transformant of the photosynthetic organism according to any one of claims 1 to 9;
(2) driving the proton pump rhodopsin by irradiating the transformant with light;
A method comprising:
A biomass production method comprising:
(A) culturing or cultivating a photosynthetic organism using the method of claim 10; and
(B) recovering biomass from the photosynthetic organism;
A method of producing biomass, comprising: