Exogenous opsins and cells expressing the rhodopsin.

Abstract

The present invention provides exogenous opsins and cells expressing said rhodopsin. [Solution] A prokaryotic opsin having a specific amino acid sequence is provided. Also provided are cells containing a gene encoding an exogenous opsin, and a method for culturing cells containing the exogenous opsin gene under light irradiation conditions.

Description

[Technical Field]

[0001] This invention relates to exogenous opsins and cells expressing said rhodopsins. The invention also relates to a method for culturing said cells. [Background technology]

[0002] The production of substances using microorganisms, cells, and other living organisms is a field that is attracting attention in the chemical production process. Patent Document 1 discloses yeast expressing deltarhodopsin (dR) derived from Haloterigena turkmenica in the inner mitochondrial membrane.

[0003] Patent Document 1 shows that the resulting yeast exhibits light-dependent proton transport ability, promotes yeast energy production, and improves the production of glutathione, trehalose, and succinic acid. Patent Document 2 discloses Escherichia coli expressing deltarhodopsin (dR) and proteorhodopsin (pR) derived from marine picoplankton.

[0004] Patent Document 2 shows that the resulting E. coli exhibits light-dependent proton transport ability, promotes energy production in E. coli, and improves isoprenol production. Patent Document 3 discloses that introducing exogenous opsins to photoautotrophic organisms such as cyanobacteria provides energy and improves doubling time, CO2 fixation rate, and / or carbon-based product formation. Non-Patent Document 1 shows that opsin expression in mitochondria of mammalian cells such as CHO cells can supply energy to the cells. For example, it has been shown that this can suppress cell death caused by rotenone-mediated inhibition of respiratory chain complex I. Energy supply to cells is expected to be applied to light-driven biological production. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] WO2015 / 170609A [Patent Document 2] WO2020 / 050113A [Patent Document 3] WO2009 / 062190A [Non-patent literature]

[0006] [Non-Patent Document 1] Hara et la., Sci. Rep., 3:1635, 2013 [Overview of the project]

[0007] The present invention provides exogenous opsins and cells expressing said rhodopsins. The present invention also provides a method for culturing said cells.

[0008] The inventors analyzed the genomic information of various microorganisms obtained through metagenomic analysis and identified novel opsin genes. By expressing these newly identified opsin genes in other cells, they found that some of them exhibited proton transport capability. Opsin genes capable of conferring proton transport capability to cells activated cell proliferation under light irradiation conditions, promoted ATP production, enhanced substance production, and resulted in low pH tolerance and high temperature tolerance. This invention is based on these findings. The present invention provides the following: [1] Prokaryotic opsins that satisfy one or more or all of the following conditions C {but not deltarhodopsin and proteorhodopsin}: The amino acid corresponding to the 88th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is Y, I, A, or F; The amino acid corresponding to the 90th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is Y; The amino acid corresponding to the 91st position in the amino acid sequence (OP13) described in Sequence ID No. 9 is V, I, or A; The amino acid corresponding to the 94th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is either L or V; The amino acid corresponding to the 95th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is V, I, F, or L; The amino acid corresponding to the 97th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is either V or T; The amino acid corresponding to the 98th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is P; The amino acid corresponding to the 99th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is L; The amino acid corresponding to the 100th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is L, Q, or M; The amino acid corresponding to the 134th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is either Y or A; The amino acid corresponding to position 135 of the amino acid sequence (OP13) described in Sequence ID No. 9 is A, M, F, P, I, or L; The amino acid corresponding to the 136th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is either G or A; The amino acids corresponding to positions 194 to 195 in the amino acid sequence (OP13) described in Sequence ID No. 9 are AI, AV, GV, FL, or CF. [1A] The opsin described in [1] above, wherein the amino acids corresponding to positions 109 through 112 of the amino acid sequence (OP13) described in Sequence ID No. 9 are not RAIA, AAIA, AAAT, RAVG, KVAG, or AAVT. [2] opsin, (1) Having an amino acid sequence selected from the group consisting of SEQ ID NOs. 2, SEQ ID NOs. 9, SEQ ID NOs. 11, SEQ ID NOs. 13, SEQ ID NOs. 16, SEQ ID NOs. 21, SEQ ID NOs. 22, SEQ ID NOs. 23, SEQ ID NOs. 31, SEQ ID NOs. 34, and SEQ ID NOs. 38, or (2) The opsin according to claim 1, having an amino acid sequence having 80% or more identity with the above amino acid sequence {except deltarhodopsin and proteorhodopsin}. [3] An opsin as described in [1] above, having any of the amino acid sequences of SEQ ID NOs. 41 to 43. [4] Opsins as described in [1] or [2] above, derived from at least one species selected from the group consisting of Gloeobacter violaceus, Roseiflexus sp., Octadecabacter antarcticus, Photobacterium sp. SKA34, Exiguobacterium sibiricum, Uncultured marine bacterium 66A03, Rhodobacterales bacterium, Uncultured bacterium MedeBAC35C06, Uncultured marine bacterium HF1019P19, and Psychroflexus torquis. [5] Cells as described in [4] above, derived from at least one species selected from the group consisting of Roseiflexus sp., Photobacterium sp. SKA34, and Rhodobacterales bacterium. [6] A gene encoding an opsin as described in any of [1] to [4] above, or a gene encoding a functional opsin that can hybridize to said gene under stringent conditions. [7] Includes a gene encoding an exogenous opsin operably linked to a regulatory sequence, The gene encoding the exogenous opsin is the gene described in [6] above. And, If the cell is a prokaryotic cell, opsins are expressed at least on the cell membrane. If the cell is a eukaryotic cell, opsins are expressed at least in the inner mitochondrial membrane. [8] The cell described in [7] above, wherein the cell is a prokaryotic cell selected from the group consisting of actinomycetes, cyanobacteria, bacteria, and archaea. [9] The cell described in [7] above, wherein the cell is a eukaryotic cell selected from the group consisting of animals, plants, algae and fungi.

[10] A cell according to any of [7] to [9] above, further having a series of genes encoding the retinal synthesis system.

[11] A method for culturing cells, To provide cells containing a gene encoding an exogenous opsin operably linked to a control sequence, wherein the gene encoding the exogenous opsin is the gene described in [6] above. A method comprising culturing the cells under light irradiation conditions.

[12] The culture method according to

[11] above, wherein the culture is carried out under conditions that satisfy either or both of the following selected from the group consisting of pH conditions lower than pH 5 and temperature conditions higher than 30 degrees. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 shows the phylogenetic trees of various opsins identified by metagenomic analysis, and the proton transport capacity (proton pump activity) per cell in cells expressing each opsin. [Figure 2] Figure 2 is a graph comparing the rhodopsin transport capacity per cell of deltarhodopsin (dR), OP13, OP17, and OP27, and the average transport capacity of the various rhodopsins shown in Figure 1. [Figure 3A] Figure 3A shows the amino acid sequence alignment data for deltarhodopsin (dR), OP13, OP17, and OP27. Helix A through G represent the amino acids corresponding to the seven helices of the opsin. Underlined amino acids indicate those present in the retinal binding pocket. [Figure 3B] Figures 3B-3D show the alignment data of the displayed opsin amino acid sequence. The numbers displayed below the alignment data correspond to the amino acid numbers in the OP13 sequence. The protein amino acid sequence alignment data is displayed from the N-terminus to the C-terminus in the order of Figure 3B, then Figure 3C, and then Figure 3D. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 4A] Figure 4A shows the retinal synthesis pathway. IPP represents isopentenyl diphosphate, idi represents IPPδ-isomerase, DMAPP represents dimethylaryl diphosphate, ispA represents FPP synthase, GPP represents geranyl diphosphate, FPP represents farnesyl diphosphate, crtE represents geranylgeranyl pyrophosphate synthase, GGPP represents geranylgeranyl pyrophosphate, and crtB represents phytoene compound The terms represent enzymes: phytoene represents phytoene, crtI represents phytoene dehydrogenase, lycopene represents lycopene, crtY represents lycopene cyclase, β-carotene represents β-carotene, BCM(D)O represents 15,15'-β-carotene dioxygenase (blh), and all-trans-retinal represents all-trans retinal. [Figure 4B] Figure 4B shows the color of strains with and without retinal in the presence or absence of retinal, for strains possessing a retinal synthesis system (ATR(+) strain) and strains without a retinal synthesis system (ATR(-) strain). If a strain is colored, it possesses retinal. [Figure 4C] Figure 4C shows that the proton concentration gradient across the cell membrane is enhanced under light conditions. [Figure 4D] Figure 4D shows the proton transport capacity per cell of a strain possessing a retinal synthesis system (ATR(+) strain). [Figure 5] Figure 5 shows the relationship between glycerol concentration and cell concentration (OD600) (top left), glutathione production at the time marked with an asterisk (middle left and right), extracellular solution pH (bottom left), and intracellular ATP concentration (bottom right) in cells into which the retinal synthesis system and opsin were introduced. No additional retinal was added in this experiment. The experiment was conducted under both light and dark conditions. [Figure 6]Figure 6 shows the cell concentration (OD600) (top left), glucose consumption (top right), glutathione production per cell (bottom left), and glutathione production (bottom right) in eukaryotic cells transfected with opsin. Irradiation was performed under 50 μE irradiation conditions. E is the unit representing mol photons m-2s-1. Square symbols represent results for non-opsin transfected cells (Con), and circular symbols represent results for opsin transfected cells (OP13). White symbols represent results at pH 5.0, and black symbols represent results at pH 3.5. [Figure 7] Figure 7 shows the cell concentration (OD600) (top left), glucose concentration (top right), glutathione production per cell (bottom left), and glutathione production (bottom right) in eukaryotic cells transfected with opsin. Irradiation was performed under 0.05 μE irradiation conditions. Square symbols represent results for non-opsin transfected cells (Con), and circular symbols represent results for opsin transfected cells (OP13). White symbols represent results at pH 5.0, and black symbols represent results at pH 3.5. [Figure 8] Figure 8 shows the temperature and low pH tolerance of eukaryotic cells transfected with opsin. The tolerances were as follows: proliferative capacity (upper left for pH and upper right for temperature), intracellular ATP concentration (middle left for pH and middle right for temperature), and glutathione production per cell (lower left for pH and lower right for temperature). Gray bars represent results for non-opsin-transfected cells, while white bars represent results for opsin-transfected cells. [Figure 9] Figure 9 shows the low pH tolerance of eukaryotic cells with and without opsin introduction (+). The low pH tolerance was as shown in the proliferation capacity (top left), glucose consumption (top right), intracellular ATP concentration (bottom left), glutathione production (bottom center), and glutathione production per cell (bottom right). In the proliferation capacity (top left) and glucose consumption (top right), the solid black line represents pH 6.0 (+), the dashed black line represents pH 3.5 (+), the solid gray line represents pH 3.5 (-), and the dashed gray line represents pH 6.5 (-). [Figure 10]Figure 10 shows the high-temperature tolerance of eukaryotic cells into which opsins were introduced. The temperature tolerance was as shown in terms of proliferative capacity (upper left), glucose consumption (upper right), intracellular ATP concentration (lower left), and glutathione production per cell (lower right). [Figure 11] Figure 11 shows the total fatty acid production and cell concentration of Aspergillus oryzae introduced with OP13 under light irradiation conditions. As a negative control, the same species of Aspergillus oryzae introduced with an empty vector was used. Specific description of the invention

[0010] In this specification, "rhodopsin" refers to a complex of the protein opsin and retinal. Examples of rhodopsins include deltarhodopsin (dR), bacteriorhodopsin (bR) and bR-type rhodopsins (e.g., archaealhodopsin, crooksrhodopsin), sensoryrhodopsin I, sensoryrhodopsin II, proteorhodopsin (pR) and pR-type rhodopsins, channelrhodopsin I, channelrhodopsin II, halorhodopsin, xanthrodopsin, sodium pump rhodopsin, and heliorhodopsin. For example, deltarhodopsin, bacteriorhodopsin, and proteorhodopsin are localized in the cell membrane of prokaryotes and have the function of pumping protons from the inside to the outside of the prokaryote under light conditions (light irradiation). In prokaryotes, the proton concentration gradient across the cell membrane can be utilized for ATP production. In eukaryotes, the inner mitochondrial membrane has an oxidative phosphorylation pathway that transports protons from the inside to the outside of the membrane, forming a proton concentration gradient. This proton concentration gradient is utilized for ATP production. Therefore, in eukaryotes, expressing opsins in the inner mitochondrial membrane can create a light-dependent proton concentration gradient, thereby promoting ATP production.

[0011] In this specification, "opsin" refers to a membrane protein having a seven-transmembrane structure. It acquires photoreceptor ability by binding to retinal, a vitamin A derivative. Examples of opsins include those listed in Table 1. However, not all of these exhibit proton transport ability in different organisms. Opsins are not particularly limited, but include opsins from at least one species selected from the group consisting of Gloeobacter violaceus, Roseiflexus sp., Octadecabacter antarcticus, Photobacterium sp. SKA34, Exiguobacterium sibiricum, Uncultured marine bacterium 66A03, Rhodobacterales bacterium, Uncultured bacterium MedeBAC35C06, Uncultured marine bacterium HF1019P19, and Psychroflexus torquis. These opsins possess significant proton transport ability. Opsins also include functional variants of opsins that can form a light-dependent proton concentration gradient.

[0012] In this specification, "opsin-coding gene" refers to an opsin-coding gene operably linked to a regulatory sequence (e.g., a promoter). The regulatory sequence can induce transcription of the opsin in the host (the cell into which the gene has been introduced). The opsin-coding gene may be codon-optimized for the host. Codon optimization is preferable, especially when introducing heterologous opsins, because codon usage differs depending on the host. The opsin-coding gene may be incorporated into a vector (e.g., a plasmid) that has or does not have an origin of replication suitable for replication in the host. The vector may have nutritional requirement markers and / or drug selection markers, and if it has nutritional requirement markers and / or drug selection markers, the host into which the vector has been introduced can be selected based on the nutrition and / or drug. The regulatory sequence may be a constitutive promoter or an inductive promoter. Opsins are expressed in cells and, when bound to retinal in the membrane, are called rhodopsins. Expressing rhodopsin means having opsin in the form bound to retinal in the membrane.

[0013] In this specification, living organisms are broadly classified into prokaryotes and eukaryotes. Furthermore, living organisms are also broadly classified into unicellular and multicellular organisms. Generally, prokaryotes are unicellular, while eukaryotes include both unicellular and multicellular organisms. Examples of prokaryotes include bacteria, archaea, cyanobacteria, and actinomycetes. Specifically, examples include Escherichia coli (e.g., Escherichia coli), Bacillus subtilis (e.g., Bacillus subtilis), Lactobacillus (e.g., Lactobacillus), Acetobacteraceae (e.g., Acetobacteraceae), Corynebacterium, Pseudomonas bacteria, Methanobacterium (e.g., Methanobacterium, Methanosarcina), Streptomyces, Actinomyces, and Agrobacterium. Eukaryotes include animals, plants, and fungi (mycetes).Fungi include, for example, fission yeasts or budding yeasts, such as the Saccharomyces genus (e.g., Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces fragilis, Saccharomyces rouxii), the Schizosaccharomyces genus (e.g., Schizosaccharomyces pombe), the Candida genus (e.g., Candida utilis, Candida tropicalis), and Xanthophyllomyces dendrobium. Examples of yeasts include those belonging to the genera Xanthophyllomyces (such as dendrorhous), Pichia, Kluyveromyces, Yarrowia, Hansenula, Endomyces, and Rhodotorula. Among these, yeasts of the genera Saccharomyces, Schizosaccharomyces, Candida, Xanthophyllomyces, or Pichia are preferred, with Saccharomyces cerevisiae of the Saccharomyces genus, Schizosaccharomyces pombe of the Schizosaccharomyces genus, and Candida utilis of the Candida genus being more preferred, and most preferably S. cerevisiae.Examples include filamentous fungi (e.g., Aspergillus, Trichoderma, Humicola, Acremonium, Fusarium, Blakeslea trispora, and Penicillium species), insect cells (e.g., cells derived from Spodoptera frugiperda and Trichoplusia ni), silkworms, nematodes, plant cells, algae (macroalgae and microalgae), plants (e.g., Arabidopsis thaliana and tobacco), and mammalian cultured cells (e.g., Chinese hamster ovary cells (CHO cells) and human cells). The cells may also be algae (macroalgae and microalgae). The cell or organism may be a photosynthetic cell or organism (photosynthetic cell or photosynthetic organism) or a non-photosynthetic cell or organism (non-photosynthetic cell or non-photosynthetic organism). This is because the introduction of opsins promotes the formation of a proton concentration gradient and thus promotes energy production in any case. The organism or cell into which the gene encoding the opsin is introduced may be referred to herein as the “host.”

[0014] In this specification, "exogenous" means originating from an organism other than the host. Typically, a regulatory sequence can be exogenous. The gene being expressed can also be exogenous. Therefore, in a gene operably ligated to a regulatory sequence, both the regulatory sequence and the gene itself may be exogenous. "Endogenous" means possessed by the host itself.

[0015] In this specification, the "retinal synthesis system" refers to a multi-step route for synthesizing all-trans retinal (ATR) from isopentenyl diphosphate (IPP). In the retinal synthesis system, ATR is synthesized from IPP by the following [1] to [7]. [1] IPP is converted to dimethylaryl diphosphate (DMAPP) by IPPδ-isomerase. [2] DMAPP is converted to farnesyl diphosphate (FPP) by FPP synthase (ispA). [3] FPP is converted to geranylgeranyl pyrophosphate (GGPP) by geranylgeranyl pyrophosphate synthase (crtE). [4]GGPP is converted to phytoene by phytoene synthase (crtB). [5] Phytoenes are converted to lycopene by phytoene dehydrogenase (crtI). [6] Lycopene is converted to β-carotene by lycopene cyclase (crtY). [7]β-carotene is cleaved by 15,15'-β-carotene dioxygenase (blh) and converted to two all-trans retinal (ATR) molecules. IPP can be produced via the mevalonate pathway or the non-mevalonate pathway.

[0016] In this specification, “the set of enzymes in the retinal synthesis system” means the set of enzymes described in [1] to [7] above. Some cells may have endogenous enzymes. For example, Escherichia coli and yeast have IPPδ-isomerase and ispA. Therefore, adding crtE, crtB, crtI, and crtY to Escherichia coli will cause the Escherichia coli and yeast to synthesize β-carotene. Further adding blh to Escherichia coli and yeast will cause the Escherichia coli and yeast to synthesize all-trans retinal. Thus, having the set of enzymes in the retinal synthesis system includes having all the enzymes in the retinal synthesis system, including both exogenous and endogenous enzymes. Of course, all the enzymes in the retinal synthesis system may be supplied exogenously. A person skilled in the art can appropriately determine which enzymes a cell has by analyzing the presence or absence of genes or by analyzing the gene products. In one embodiment, the host expresses all enzymes selected from the group consisting of IPPδ-isomerase, FPP synthase (ispA), geranylgeranyl pyrophosphate synthase (crtE), phytoene synthase (crtB), phytoene dehydrogenase (crtI), lycopene cyclase (crtY), and 15,15'-β-carotene dioxygenase (blh). In another embodiment, the host has exogenous genes encoding some or all of the enzymes selected from the group consisting of IPPδ-isomerase, FPP synthase (ispA), geranylgeranyl pyrophosphate synthase (crtE), phytoene synthase (crtB), phytoene dehydrogenase (crtI), lycopene cyclase (crtY), and 15,15'-β-carotene dioxygenase (blh) {however, the host does not have to have endogenous enzymes or genes}.

[0017] The inventors have now obtained newly discovered opsin genes with strong proton transport ability (see Table 1). These opsin genes do not need to be codon-optimized if their expression is confirmed in the host, but they may be codon-optimized for the host. In Table 1, the proton efflux rate indicates the ability to transport protons from inside the cell to outside the cell. In Table 1, opsin genes showing a proton efflux rate (μmol / gDCW / min) of 0.1 or higher, 0.2 or higher, 0.270 or higher, 0.3 or higher, 0.4 or higher, 0.5 or higher, 0.6 or higher, 0.7 or higher, 0.8 or higher, 0.806 or higher, 0.9 or higher, or 1.0 or higher have proton transport ability and can be preferably used in the present invention. A higher proton transport ability is preferable; for example, opsin genes with a proton efflux rate of 0.270 (μmol / gDCW / min) or higher can be preferably used. In particular, OP13, OP17, and OP27 in Table 1 exhibit excellent proton transport capabilities and can be used especially favorably.

[0018] In some embodiments, opsins are (1) Having an amino acid sequence selected from the group consisting of SEQ ID NOs. 2, SEQ ID NOs. 9, SEQ ID NOs. 11, SEQ ID NOs. 13, SEQ ID NOs. 16, SEQ ID NOs. 21, SEQ ID NOs. 22, SEQ ID NOs. 23, SEQ ID NOs. 31, SEQ ID NOs. 34, and SEQ ID NOs. 38, or (2) Opsins having an amino acid sequence that is 80% or more (or 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identical to the above amino acid sequence. It is possible.

[0019] Therefore, in the present invention, It is opsin, (1) Having an amino acid sequence selected from the group consisting of SEQ ID NOs. 2, SEQ ID NOs. 9, SEQ ID NOs. 11, SEQ ID NOs. 13, SEQ ID NOs. 16, SEQ ID NOs. 21, SEQ ID NOs. 22, SEQ ID NOs. 23, SEQ ID NOs. 31, SEQ ID NOs. 34, and SEQ ID NOs. 38, or (2) Provided are an opsin having an amino acid sequence that is 80% or more (or 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identical to the above amino acid sequence, or a gene encoding the above opsin. The opsin and the gene encoding it may be isolated.

[0020] The opsin-coding gene may be a functional opsin-coding gene that can hybridize with the opsin-coding gene under stringent conditions. Here, stringent conditions include normal stringent conditions and conditions with higher stringency. Under normal stringent conditions, hybridization can be performed for approximately 16 hours at a temperature of 50-60°C in a hybridization solution of 6×SSC or equivalent salt concentration, followed by pre-washing as needed with a solution of 6×SSC or equivalent salt concentration, and then washing in a solution of 1×SSC or equivalent salt concentration. Under conditions with higher stringency (high stringent conditions), the washing can be performed in a solution of 0.1×SSC or equivalent salt concentration. "SSC" is an aqueous solution with a pH of 7 containing 15 mM sodium citrate and 150 mM sodium chloride. "n × SSC" (where n is a positive real number) means an aqueous solution at pH 7 containing n times the concentration of sodium citrate and sodium chloride contained in the SSC. The SSC may be DNase-free and / or RNase-free and may be autoclaved.

[0021] The present invention provides genes encoding opsins shown in Table 1. According to the present invention, the genes encoding opsins shown in Table 1 may be codon-optimized to enhance their expression in a host. The opsin-encoding genes may be incorporated into a vector, and a vector containing opsin-encoding genes is provided. The vector may be a gene expression vector. The gene expression vector contains, in an expression cassette, a gene encoding opsin operably linked to a control sequence. The control sequence is, for example, a promoter sequence, which can be appropriately selected by those skilled in the art depending on the host expressing the opsin. The gene expression vector may further have nutritional requirement markers and / or drug selection markers, allowing for the selection or maintenance of a host containing the vector in the absence of such nutrients and / or in the presence of such drugs.

[0022] The present invention also provides isolated opsins shown in Table 1. The present invention also provides cells or organisms containing the opsins shown in Table 1. In cells containing opsins, the opsins can bind to retinal to form rhodopsin. Thus, the present invention provides cells or organisms containing rhodopsin as a complex of opsins and retinal shown in Table 1. In some aspects of the present invention, the opsins may be exogenous opsins. That is, the opsins may be transcribed and translated from a gene encoding an exogenous opsin operably linked to a regulatory sequence. Thus, cells or organisms containing the opsins shown in Table 1 may have a gene encoding an exogenous opsin operably linked to a regulatory sequence.

[0023] In one embodiment, the opsin may have amino acids R / K, L, X, and I at positions 121 through 124 of the amino acid sequence (OP13) described in Sequence ID No. 9, respectively. This sequence belongs to helix D, as shown in Figure 3. These amino acids are located in the retinal binding pocket that determines the binding of the opsin to retinal, and are common to the highly active OP13, OP17, and OP27, and can enhance the activity of the opsin.

[0024] When various opsins are aligned, the amino acid that aligns to the same position as a particular amino acid is the amino acid corresponding to that particular amino acid. Alignment can be performed using commercially available software such as Clustal W with default parameters.

[0025] In some embodiments, opsins may have common sequences between or before / after retinal binding pockets. For example, in some embodiments, opsins may satisfy 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or all of the following conditions selected from group A: In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 88 is F, I, Y, or A; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 89 is R; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 90 is Y; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 91 is I, V, or A; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acids corresponding to positions 92 and 93 are D and W, respectively; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 94 is either L or V; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 95 is I, L, V, or F, preferably I, L, or V; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 96 is T; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 97 is either V or T; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 98 is P; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 99 is L; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 100 is L, M, or Q; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 103 is either E or D; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 133 is G; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 134 is either Y or A; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 135 is A, M, F, P, I, or L; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 136 is either G or A; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 137 is E; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 149 is G, L, V, or W; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 157 is F, V, I, L, G, or A; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 193 is W; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 194 is A, G, C, or F; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 195 is I, V, F, or L; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 196 is Y; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 197 is P; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 200 is Y; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 224 is D; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 228 is K.

[0026] For example, in one embodiment, an opsin may satisfy one of the following conditions selected from group B, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or all of them, as a common characteristic of opsins with high proton transport activity: In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 88 is F, I, Y, or A; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 89 is R; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 90 is Y; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 91 is I, V, or A; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acids corresponding to positions 92 and 93 are D and W, respectively; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 94 is either L or V; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 95 is I, L, V, or F, preferably I, L, or V; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 96 is T; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 97 is either V or T; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 98 is P; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 99 is L; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 100 is L, M, or Q; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 103 is either E or D; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 133 is G; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 134 is either Y or A; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 135 is A, M, F, P, I, or L; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 136 is either G or A; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 137 is E; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 193 is W; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 194 is A, G, C, or F; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 195 is I, V, F, or L; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 196 is Y; In the amino acid sequence (OP13) described in Sequence ID No. 9, the amino acid corresponding to position 197 is P.

[0027] For example, in one embodiment, the opsin may satisfy 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all of the following conditions C {provided that the amino acids corresponding to positions 109 through 112 of the amino acid sequence (OP13) described in SEQ ID NO: 9 are not RAIA, AAIA, AAAT, RAVG, KVAG, or AAVT}: The amino acid corresponding to the 88th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is Y, I, A, or F; The amino acid corresponding to the 90th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is Y; The amino acid corresponding to the 91st position in the amino acid sequence (OP13) described in Sequence ID No. 9 is V, I, or A; The amino acid corresponding to the 94th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is either L or V; The amino acid corresponding to the 95th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is V, I, F, or L; The amino acid corresponding to the 97th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is either V or T; The amino acid corresponding to the 98th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is P; The amino acid corresponding to the 99th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is L; The amino acid corresponding to the 100th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is L, Q, or M; The amino acid corresponding to the 134th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is either Y or A; The amino acid corresponding to position 135 of the amino acid sequence (OP13) described in Sequence ID No. 9 is A, M, F, P, I, or L; The amino acid corresponding to the 136th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is either G or A; The amino acids corresponding to positions 194 to 195 of the amino acid sequence (OP13) described in Sequence ID No. 9 are AI, AV, GV, FL, or CF. Condition group C above is the group of amino acids that opsins with proton transport activity above the average value in Table 1 have, and that opsins with proton transport activity below the average value do not have.

[0028] Opsins are neither dR nor pR.

[0029] In one embodiment, the opsin may have an amino acid sequence that is 80% or more (or 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 34, and SEQ ID NO: 38.

[0030] In one embodiment, the opsin has an amino acid sequence that is 80% or more (or 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 34, and SEQ ID NO: 38, and the amino acids corresponding to positions 121 to 124 of the amino acid sequence (OP13) described in SEQ ID NO: 9 may be R / K, L, X, and I, respectively.

[0031] In one embodiment, the opsin has an amino acid sequence that is 80% or more (or 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 34, and SEQ ID NO: 38, and can satisfy one or more or all of the conditions selected from the group consisting of condition group A.

[0032] In one embodiment, the opsin has an amino acid sequence that is 80% or more (or 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 34, and SEQ ID NO: 38, and can satisfy one or more or all of the conditions selected from the group consisting of condition group B.

[0033] In one embodiment, the opsin has an amino acid sequence that is 80% or more (or 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 34, and SEQ ID NO: 38, and can satisfy one or more or all of the conditions selected from the group consisting of condition group C.

[0034] In one embodiment, the opsin may have the amino acid sequence of SEQ ID NO: 41.

[0035] In one embodiment, the opsin may have the amino acid sequence of SEQ ID NO: 42.

[0036] In one embodiment, the opsin may have the amino acid sequence of SEQ ID NO: 43.

[0037] According to the present invention, the isolated opsins shown in Table 1 may also be linked to mitochondrial localization signals. Therefore, according to the present invention, isolated opsins shown in Table 1 linked to mitochondrial localization signals, and the genes encoding said opsins are provided. Opsins linked to mitochondrial localization signals, when expressed in eukaryotic cells or eukaryotes, translocate to mitochondria. In this way, eukaryotic cells or eukaryotes having rhodopsin expressed in mitochondria can be obtained. Rhodopsin expressed in mitochondria can form a proton concentration gradient across the inner mitochondrial membrane and promote ATP synthesis. Therefore, opsins linked to mitochondrial localization signals are suitable for expression in eukaryotic cells or eukaryotes. As mitochondrial localization signals, those well known to those skilled in the art can be used, such as ALA synthase Hem1, S2 peptide, or oligoarginine (e.g., octaarginine), peroxiredoxin PRX1, pirubate dehydrogenase β subunit, heat shock protein HSP60, ATP synthase γ subunit, ATP synthase α subunit, cytochrome c oxidase subunits 2 and 4, subunit 6, succinate dehydrogenase cytochrome b small subunit, citrate synthase 1 (CIT1), acetolactates synthase catalytic subunit (ILV2), keto l-acid reductoisomerase, mitfilin, cytochrome c degrading enzyme (Sue), etc. Even without a mitochondrial localization signal, opsins can be localized to mitochondria by translating them in mitochondria. For example, if the tryptophan codon is changed to TAA, such an opsin can be translated only in mitochondria. TGA is a stop codon in cytoplasmic translation and encodes tryptophan in mitochondrial translation. TAA can be used as the stop codon in mitochondrial translation.Therefore, in some embodiments, the gene encoding the opsin may include TGA as a codon encoding tryptophan.

[0038] The host may be a prokaryote or a eukaryote. The host may also be a unicellular or multicellular organism. The host may be a microorganism. The host may be a photosynthetic cell or a photosynthetic organism. The host may be a non-photosynthetic cell or a non-photosynthetic organism. The host may also be an organism selected from the group consisting of bacteria, archaea, cyanobacteria, and actinomycetes. In some aspects, the host may be an organism selected from the group consisting of Escherichia coli (e.g., Escherichia coli), Bacillus subtilis (e.g., Bacillus subtilis), Lactobacillus (e.g., Lactobacillus), Acetobacteraceae (e.g., Acetobacteraceae), Corynebacterium, Pseudomonas bacteria, Methanobacterium (e.g., Methanobacterium, Methanosarcina), Streptomyces, Actinomyces, and Agrobacterium. In some aspects, the host may be an organism selected from the group consisting of animals, plants, and fungi (molds). In some aspects, the host may be a yeast. In some cases, the host may be a fission yeast or a budding yeast.In some cases, the host species include the genera Saccharomyces (such as Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces fragilis, and Saccharomyces rouxii), the genera Schizosaccharomyces (such as Shizosaccharomyces pombe), the genera Candida (such as Candida utilis and Candida tropicalis), and Xanthophyllomyces dendrobium. It may be a yeast selected from the group consisting of the genera Xanthophyllomyces, Pichia, Kluyveromyces, Yarrowia, Hansenula, Rhodotorula, and Endomyces, such as *Dendrorhous*. In some aspects, the host may be selected from a group consisting of filamentous fungi (e.g., Aspergillus, e.g., Aspergillusoryzae, Trichoderma, Humicola, Acremonium, Fusarium, Blakeslea trispora, and Penicillium species), insect cells (e.g., cells derived from Spodoptera frugiperda, cells derived from Trichoplusia ni), nematodes, silkworms, plant cells, algae (macroalgae, microalgae), plants (Arabidopsis thaliana, tobacco), and mammalian cultured cells (e.g., Chinese hamster ovary cells (CHO cells), human cells). The host may also be algae (macroalgae, microalgae). The opsins introduced into these hosts may be exogenous. The host and the opsins may be heterogeneous. The animals may be non-human.When the host is a prokaryote, the opsin can be expressed on the cell membrane. When the host is a eukaryote, the opsin can be expressed on the inner mitochondrial membrane. The method of expression can use techniques well known to those skilled in the art.

[0039] In addition to the gene encoding the exogenous opsin, the host can have all of a series of enzymes in the retinal synthesis system. Thus, when the host can synthesize retinal by itself, the amount of retinal added to the medium can be reduced. When the amount of retinal synthesized by the host is sufficient, retinal does not need to be added to the medium. When the host does not have some of the series of enzymes in the retinal synthesis system, an exogenous gene encoding the enzyme that the host does not have can be introduced into the host. The exogenous gene encoding the enzyme that the host does not have can be operably linked to a control sequence and introduced into an expression cassette in a vector. Whether the host has the enzyme or not can be appropriately determined by those skilled in the art through biochemical analysis (e.g., Western blot), genetic analysis (e.g., sequencing, or PCR).

[0040] The host expressing rhodopsin can be cultured under culture conditions suitable for the host. The culture conditions can be appropriately determined by those skilled in the art. The host expressing rhodopsin can be cultured under light irradiation conditions. By culturing under light irradiation, in the host expressing rhodopsin, ATP synthesis is promoted and energy is supplied by light irradiation. The light irradiation is not particularly limited as long as it is irradiation to the extent that rhodopsin can receive light. For example, 0.01 μmol photons m -2 s -1 or more, 0.02 μmol photons m -2 s -1 or more, 0.03 μmol photons m -2 s -1 or more, 0.04 μmol photons m -2 s -1 or more, or 0.05 μmol photons m -2 s -1The strength of the above is the same. Light irradiation, 0.1μmol photons m -2 s -1 The following, 0.2 μ photons molm -2 s -1 The following, 0.3 μmol photons m -2 s -1 The following, 0.4 μmol photons m -2 s -1 Below, 0.5 μmol photons m -2 s -1 Below, 0.6 μmol photons m -2 s -1 Below, 0.7 μmol photons m -2 s -1 The following, 0.8 μmol photons m -2 s -1 Below, 0.9 μ photons molm -2 s -1 The following, 1.0 μmol photons m -2 s -1 The following, 1.1 μmol photons m -2 s -1 The following, 1.2 μmol photons m -2 s -1 The following, 1.3 μmol photons m -2 s -1 The following, 1.4 μmol photons m -2 s -1 The following, 1.5 μmol photons m -2 s -1 The following, 1.6 μmol photons m -2 s -1 The following, 1.7 μmol photons m -2 s -1 The following, 1.8 μmol photons m -2 s -1 The following, 1.9 μmol photons m -2 s -1 The following, 2μmol photons m -2 s -1 The following, 3μmol photons m -2 s -1The following, 4μmol photons m -2 s -1 The following, 5μmol photons m -2 s -1 The following, 6μmol photons m -2 s -1 The following, 7μmol photons m -2 s -1 The following, 8μmol photons m -2 s -1 The following, 9μmol photons m -2 s -1 The following, 10 μmol photons m -2 s -1 The following, 15 μmol photons m -2 s -1 The following, 20 μmol photons m -2 s -1 The following, 20 μmol photons m -2 s -1 The following, 30 μmol photons m -2 s -1 The following, 40 μmol photons m -2 s -1 The following, 50 μmol photons m -2 s -1 The following, 60 μmol photons m -2 s -1 The following, 70 μmol photons m -2 s -1 The following, 80 μmol photons m -2 s -1 The following, 90 μmol photons m -2 s -1 The following, 100 μmol photons m -2 s -1 The following, 110 μmol photons m -2 s -1 The following, 120 μmol photons m -2 s -1 The following, 130 μmol photons m -2 s -1 The following, 140 μmol photons m -2 s-1 Below, 150 μmol photons m -2 s -1 Below, 160 μmol photons m -2 s -1 Below, 170 μmol photons m -2 s -1 Below, 180 μmol photons m -2 s -1 Below, 190 μmol photons m -2 s -1 Below, 200 μmol photons m -2 s -1 Below, 300 μmol photons m -2 s -1 Below, 400 μmol photons m -2 s -1 Below, 500 μmol photons m -2 s -1 Below, 600 μmol photons m -2 s -1 Below, 700 μmol photons m -2 s -1 Below, 800 μmol photons m -2 s -1 Below, 900 μmol photons m -2 s -1 Below, 1000 μmol photons m -2 s -1 Below, 1500 μmol photons m -2 s -1 Below, or 2000 μmol photons m -2 s -1 It may be below. The light to be irradiated may be any of a light bulb (e.g., an incandescent bulb, a halogen bulb, etc.), a fluorescent lamp, a high-pressure discharge lamp, a low-pressure discharge lamp, an LED, an EL, chemiluminescence, bioluminescence, and sunlight. For example, light having a wavelength of 300 to 800 nm, 450 to 650 nm, for example, 550 nm can be used. The light irradiation may be performed continuously or intermittently. <00­00441> When energy is supplied to the host by light irradiation, the host can reduce its consumption of carbon sources used for energy production. When energy is supplied to the host by light irradiation, the host can use more raw materials such as carbon sources in pathways involved in substance production (e.g., the pentose phosphate pathway). Therefore, light irradiation puts the host in a state suitable for substance production. Thus, by supplying raw materials such as carbon sources to the host, substance production can be preferably carried out. Microorganisms expressing rhodopsin may exhibit low pH tolerance and / or high temperature tolerance under light irradiation conditions.

[0042] Low pH tolerance means that, under low pH conditions (e.g., pH less than 5.0) where the host's capabilities (one or more capabilities selected from the group consisting of growth capacity, ATP synthesis capacity, and substance production capacity) are reduced, one or more capabilities selected from the group consisting of growth capacity, ATP synthesis capacity, and substance production capacity are increased under light irradiation conditions compared to non-irradiated conditions, and / or under rhodopsin expression conditions compared to non-expression conditions. Low pH tolerance means, for example, in the case of yeast, that under pH conditions of less than 5.0, pH 4.5 or lower, pH 4.0 or lower, for example, pH 3.2 to 3.7 (e.g., pH 3.5), one or more capabilities selected from the group consisting of growth capacity, ATP synthesis capacity, and substance production capacity are higher than the corresponding capabilities under non-irradiated conditions and / or rhodopsin non-expression conditions. A host that expresses rhodopsin and possesses low pH tolerance can be preferably cultured even under low pH conditions. Under low pH conditions, one or more abilities selected from the group consisting of growth, ATP synthesis, and substance production of other contaminating microorganisms are suppressed, making it possible to selectively cultivate the host.

[0043] High-temperature tolerance means that, under high-temperature environments where the host's capabilities (one or more capabilities selected from the group consisting of growth ability, ATP synthesis ability, and substance production ability) are reduced, one or more capabilities selected from the group consisting of growth ability, ATP synthesis ability, and substance production ability are increased compared to non-irradiated conditions under light irradiation conditions, and / or rhodopsin expression conditions compared to non-expression conditions. For example, in the case of yeast, high-temperature tolerance means that one or more capabilities selected from the group consisting of growth ability, ATP synthesis ability, and substance production ability are increased under conditions of 32°C or higher, 33°C or higher, 34°C or higher, for example, 34°C to 36°C (for example, 35°C) or higher, compared to the corresponding capabilities under non-irradiated conditions and / or rhodopsin non-expression conditions. Under high-temperature conditions, the growth of other contaminating microorganisms is suppressed, and it may be possible to selectively cultivate the host.

[0044] Microorganisms expressing rhodopsin may possess low pH tolerance and high temperature tolerance under light irradiation conditions. In this case, a host expressing rhodopsin and possessing low pH tolerance and high temperature tolerance can be preferably cultured under low pH and high temperature conditions. Under low pH and high temperature conditions, one or more abilities selected from the group consisting of growth, ATP synthesis ability, and substance production ability of other contaminating microorganisms may be suppressed, making it possible to selectively culture the host.

[0045] The production of the substance may preferably be carried out by culturing cells or organisms expressing rhodopsin under the above conditions. Cells or organisms expressing rhodopsin may be cultured under conditions suitable for the production of a particular substance in order to increase the production of that substance. The culture conditions may satisfy either or both of low pH and high temperature conditions. Further modifications to the cells or organisms expressing rhodopsin are permitted in order to increase the production of a particular substance. Such modifications include, for example, mutations and / or introduction of enzymes related to the production pathway of the substance; disruption, reduction of expression, elimination or reduction of activity of enzymes related to the degradation of the substance; and / or disruption, reduction of expression, and / or elimination or reduction of activity of enzymes related to competing pathways.

[0046] Substances produced through this process include, for example, organic acids, peptides, amino acids, proteins, nucleosides, vitamins, sugars, sugar alcohols, alcohols, isoprenoids, and lipids. More specifically, these include: Organic acids include acetic acid, lactic acid, succinic acid, and α-keto acids (2-oxo acids). Peptides include glutathione, alanylglutamine, and γ-glutamylvalylglycine, while polypeptides include polylysine and polyglutamic acid. Examples of amino acids include L-alanine, glycine, L-glutamine, L-glutamic acid, L-asparagine, L-aspartic acid, L-lysine, L-methionine, L-threonine, L-leucine, L-valine, L-isoleucine, L-proline, L-histidine, L-arginine, L-tyrosine, L-tryptophan, L-phenylalanine, L-serine, L-cysteine, L-3-hydroxyproline, L-4-hydroxyproline, and 5-aminolevulinic acid. Examples of proteins include luciferase, inosine kinase, glutamate 5-kinase (EC 2.7.2.11), glutamate-5-semialdehyde dehydrogenase (EC 1.2.1.41), pyrroline-5-carboxylate reductase (EC 1.5.1.2), γ-glutamylcysteine ​​synthase (EC 6.3.2.2), glutathione synthase (EC 6.3.2.3), human granulocyte colony-stimulating factor, xylose reductase, and P450. Examples of nucleosides include inosine, guanosine, inosinic acid, guanylic acid, and adenylic acid. Examples of vitamins include riboflavin, thiamine, and ascorbic acid. Examples of sugars include xylose and mannose, examples of sugar alcohols include xylitol and mannitol, and examples of alcohols include ethanol. Examples of isoprenoids include mevalonate (MVA), isoprenol, astaxanthin, isoprene, isopentenol, limonene, pinene, farnesene, and bisabolene.Examples of lipids include propionic acid, hydroxypropionic acid, EPA (eicosapentaenoic acid), and DHA (docosahexaenoic acid). Among the useful substances in this invention, acetic acid is particularly preferred among organic acids, glutathione among peptides, mevalonic acid and isoprenol among isoprenoids, and hydroxypropionic acid among lipids. Examples of substances produced include ATP, glutathione, and isoprenoids. [Examples]

[0047] Example 1: Preparation of exogenous rhodopsin-expressing cells Forty-three opsin sequences were obtained through metagenomic analysis using the genome sequences of acquired microorganisms. Escherichia coli was used as the host cell. To express opsins in E. coli, NdeI / KpnI restriction enzyme sequences were introduced, and the codons were optimized for E. coli (see Table 1). The codon-optimized opsins were introduced into the pCDF-Duet1 vector, and E. coli strain MG1655(DE3) was transformed to obtain E. coli expressing one of the 43 rhodopsins.

[0048] Rhodopsin-expressing E. coli was inoculated from glycerol stocks or single colonies into 4 mL of LB medium and grown overnight at 37°C with shaking at 150 rpm to serve as the preculture. The resulting preculture was then added to 50 mL of M9 medium containing 10 g / L glucose for initial OD (Oral Dissociation). 600 The culture was inoculated to a concentration of 0.05 and cultured with agitation in a shaking incubator (BR-43FL, Taitec, Japan) at 37°C and 150 rpm. After 4 hours, 20 μM IPTG and 10 μM all-trans retinal (hereinafter simply referred to as "retinal") were added and cultured with agitation at 37°C and 150 rpm.

[0049] After 24 hours of incubation, the culture medium was collected and centrifuged at 5,000 g at 25°C for 10 minutes to collect rhodopsin-expressing E. coli. The collected rhodopsin-expressing E. coli was washed three times with 100 mM NaCl solution and OD (Oxygen-Draining). 600The rhodopsin-expressing E. coli suspension was suspended in 100 mM NaCl solution so that the ratio was 2. The suspension was irradiated with light using a 300 W halogen projector lamp (JCD100V-300W). The light intensity was measured using a photoanalyzer LA-105 (NK System, Osaka, Japan). The time course of the pH change of the rhodopsin-expressing E. coli suspension was recorded using a pH meter (F-72, Horiba, Japan) connected to a PC. The proton pump activity of the rhodopsin-expressing E. coli was determined from the initial gradient of the pH change. The amount of proton transport per cell was determined by dividing the proton pump activity by the dry cell weight. In the system without retinal supply, no proton transport ability was observed. The results are shown in Table 1, Figure 1, and Figure 2.

[0050] [Table 1] JPEG2026098057000002.jpg229122 JPEG2026098057000003.jpg143115

[0051] As shown in Table 1, many rhodopsins showed no activity or very low activity, with an average value of 0.270 (μmol / gDCW / min). In contrast, as shown in Table 1, Figure 1, and Figure 2, dR showed 0.806 (μmol / gDCW / min), OP13 showed 1.865 (μmol / gDCW / min), OP17 showed 1.022 (μmol / gDCW / min), and OP27 showed 1.447 (μmol / gDCW / min). OP13, OP17, and OP27 are rhodopsins that have the function of light-dependent proton efflux when introduced into cells, and they have a higher proton efflux rate than the conventionally known dR.

[0052] The amino acid sequences of these rhodopsins, which showed particularly high activity, were aligned. The results are shown in Figures 3A to 3D. As shown in Figures 3A to 3D, rhodopsins showed high similarity in amino acid sequences at the retinal pocket (underlined), which is the site where retinal binds. Important amino acid sequences from these amino acid sequences were extracted as condition groups A to C. Condition groups A to C are as described above.

[0053] In addition to the above, as shown in Table 1, dR, GR, OP15, OP20, OP25, OP26, OP35, OP38, and OP42 were identified as rhodopsins with activity exceeding the average value, revealing them to be highly active and useful rhodopsins. Of these, dR, GR, OP15, and OP38 showed activity more than twice the average value. These rhodopsins confer light-dependent proton transport ability to microorganisms.

[0054] In prokaryotes, the proton concentration gradient across the cell membrane drives FoF1 ATP synthase, which is the driving force behind intracellular ATP synthesis. Therefore, it is understood that rhodopsin-expressing microorganisms have an enhanced ability to produce energy in a light-dependent manner.

[0055] Example 2: Introduction of an exogenous retinal synthesis system into cells

[0056] All-trans retinal incorporated into opsin isomerizes to 13-cis-retinal upon exposure to light. In this process, rhodopsin releases protons from inside the cell to outside, lowering the extracellular pH. In Example 1, retinal was supplied into the culture medium, but in this example, we attempted to create a microorganism that exhibits light-dependent proton transport ability even without external retinal supply by incorporating a retinal synthesis system.

[0057] The crt promoter / operon described in Appl Environ Microbiol. 2007, 73: 1355-61 (crtE, crtB, crtI, and crtY in Figure 4A are operably linked under the crt promoter) was cloned using the XbaI and HindIII sites of the pD874 vector (ATUM) to construct the β-carotene synthesis plasmid pD874_CrtP-EBIY. The blh gene (66A03) derived from a marine bacterium with a metagenomic sequence described in Microb Cell Fact. 2011, 10: 59 was codon-optimized for E. coli and amplified by PCR using in-fusion cloning (Takara Bio, Japan). The amplified blh gene was subcloned into pD874_CrtP-EBIY to obtain the retinal autosynthesis plasmid pD874_CrtP-EBIY-blh. Using this pD874_CrtP-EBIY-blh, rhodopsin-expressing E. coli was transformed to obtain E. coli expressing retinal autosynthesis rhodopsin. Below, ATR(+) indicates a microorganism that has incorporated an exogenous retinal synthesis system, and ATR(-) indicates a microorganism that has not incorporated an exogenous retinal synthesis system.

[0058] Retinal autosynthetic rhodopsin-expressing E. coli was inoculated from glycerol stocks or single colonies into 4 mL of LB medium and grown overnight at 37°C with shaking at 150 rpm to serve as the preculture medium. The preculture medium was used as the initial OD medium. 600 The cultures were inoculated into 20 mL of autoclaved M9 medium containing 4 g / L glucose or 2 g / L glycerol to a ratio of 0.1, and incubated at 200 rpm and 37°C. Light conditions were used for 100 μmol photons m -2 s -1 The cells were cultured under white LED light irradiation, and under dark conditions, they were shielded from light using aluminum foil. To retain the plasmids within the cells, an appropriate antibiotic for plasmid maintenance was added to the culture medium at the appropriate concentration.

[0059] As shown in Figure 4B, the obtained Escherichia coli strain with an exogenous retinal synthesis system (ATR(+) strain) was colored regardless of whether retinal was added, while the Escherichia coli strain without an exogenous retinal synthesis system (ATR(-) strain) was colored only when retinal was added. Furthermore, the obtained Escherichia coli expressing retinal-autosynthesizing rhodopsin performed proton transport in response to light (Figure 4C). This clearly demonstrates that the introduction of an exogenous retinal synthesis system eliminates the need for external retinal addition in the expression of light-dependent proton transport ability by rhodopsin-expressing microorganisms. In this example, the dR-introduced strain of Escherichia coli (MG1655(DE3) / pCDFDuet1-dR, pD874-CrtP-EBIY-blh) was used, but it is clear that similar results would be obtained using strains with other introduced rhodopsins.

[0060] The effects of adding retinal were compared with the effects of introducing an exogenous retinal synthesis system without adding retinal. The results are shown in Figure 4D. As shown in Figure 4D, the ATR(+) strain showed proton transport ability comparable to that of the ATR(-) strain to which retinal was added from an external source.

[0061] In Examples 1 and 2, cells capable of light-dependent energy production were created. In such cells, the amount of carbon source consumed for energy production is thought to be reduced, and therefore, more carbon source can be used for material production.

[0062] Example 3: Substance production by rhodopsin-expressing cells In this example, we attempted to produce a substance using rhodopsin-expressing cells.

[0063] E. coli expressing retinal autosynthetic rhodopsin (OP13) was inoculated into 4 mL of LB medium from glycerol stock or single colonies, and grown overnight at 37°C with shaking at 200 rpm to serve as the preculture medium. The preculture medium was used as the initial OD (Oral Production) medium. 600 The molecules were inoculated into 20 mL of autoclaved M9 medium containing 0.2% glycerol to a ratio of 0.1, and cultured at 200 rpm and 37°C (n=3). Under light conditions, 50 μmol m³-2 s -1 The cells were cultured under white LED light irradiation, and under dark conditions, they were shielded from light with aluminum foil. Appropriate antibiotics were added to the culture medium at appropriate concentrations to retain the plasmids within the cells. Culture samples were taken at 18, 23, 42, and 69 hours after incubation, and the pH and OD of the obtained samples were measured. 600 We measured it.

[0064] Cells were separated from the culture medium collected by sampling after centrifugation at 7,000 xg for 2 minutes. The cells were suspended in 50 mM Tris-HCl buffer (pH 8.0) and disrupted using a cell disruption device (Shake Master NEO, Bio Medical Sciences) at 1500 rpm for 10 minutes. Cell debris was removed by centrifugation at 16,000 xg at 4°C for 10 minutes. The glutathione (GSH) concentration of the cell extract was measured using the 5,5'-dithiobis(2-nitrobenzoic acid)-glutathione reductase cycling assay described in Hara et al, 2009. Glutathione production per cell was determined by dividing the glutathione concentration by the cell concentration.

[0065] Intracellular ATP concentration was measured using the luciferin / luciferase assay method described in Hara 2006; Hara 2009, modified as follows: A 150 μl assay mixture containing 50 mM Tris-HCl (pH 8.0), 1.3 μg / ml luciferase, 0.5 mM D-luciferin, 2 mM DTT, 10 mM MgSO4, and 0.2 M EDTA was prepared and added to 50 μl of cells. Luminescence was measured using a Gene light GL-210S series (Microtech Nichion).

[0066] The carbon source in the culture medium of retinal-autosynthesizing rhodopsin-expressing Escherichia coli was measured as follows. The concentrations of glycerol and glucose, which are carbon sources in the culture medium, were measured after the culture medium was collected at an appropriate incubation time, the precipitate was removed by centrifugation, and the concentrations were measured using commercially available F-Kit Glycerol (JK International, Japan) and GlucoseC-II test Kit (Wako Pure Chemical Industries).

[0067] The results are shown in Figure 5. As shown in Figure 5, OD 600 The following showed a good increase in both light and dark conditions. As shown in Figure 5, glycerol content decreased at a similar rate in both light and dark conditions, but consumption tended to be faster in the dark. Also, as shown in Figure 5, 42 hours after glycerol depletion, rhodopsin-expressing cells showed a faster increase in OD values ​​than non-expressing cells. Glutathione production increased statistically significantly under light conditions compared to dark conditions, as shown in Figure 5. The pH of the culture medium (extracellular pH; pH decreases as proton efflux capacity increases) decreased statistically significantly under light conditions compared to dark conditions. No difference was observed in intracellular ATP concentration between light and dark conditions. In rhodopsin-expressing cells, both ATP production and consumption increased, which is thought to be a result of accelerated cell turnover.

[0068] This suggests that rhodopsin-expressing cells produced energy in a light-dependent manner, expelled protons to the extracellular space to create a pH gradient across the cell membrane, and increased glutathione production. It is believed that they converted light energy into glutathione production.

[0069] Furthermore, we attempted to produce isoprenol. Escherichia coli was transformed using the pMevB plasmid containing mevalonate kinase, phosphomevalonate kinase, and mevalonate pyrophosphate decarboxylase genes from Saccharomyces cerevisiae (Martin et al. 2003). A DNA fragment of nudF containing NcoI and BamHI restriction sites was amplified from the Bacillus subtilis genome. The amplified DNA was digested with NcoI and BamHI and inserted into the multicloning site of pCOLADuet-mvaES. Using the obtained pCOLADuet-mvaES-nudF and pMevB, dR-expressing, OP13-expressing, and OP27-expressing Escherichia coli were transformed to obtain dR-expressing isoprenol-producing, OP13-expressing isoprenol-producing, and OP27-expressing isoprenol-producing Escherichia coli, respectively.

[0070] Rhodopsin-expressing isoprenol-producing E. coli was inoculated from glycerol stocks or single colonies into 4 mL of LB medium and grown overnight at 37°C with shaking at 150 rpm to serve as the preculture medium. The preculture medium of rhodopsin-expressing isoprenol-producing E. coli was then added to 100 mL of autoclaved LB medium for initial OD (Oral Dissociation). 600 The cells were inoculated to a concentration of 0.05 and cultured in a shaking incubator (BR-43FL, Taitec, Japan) at 165 rpm and 37°C. After 18 hours, the rhodopsin-expressing isoprenol-producing E. coli was collected by centrifugation at 7,000 × g and 25°C for 4 minutes and washed with glucose-free M9 medium. The washed rhodopsin-expressing isoprenol-producing E. coli was then added to 20 mL of M9 medium containing 8 g / L glucose and incubated with initial OD. 600 The solution was inoculated to a ratio of =5 and cultured in a shaking incubator at 165 rpm and 37°C. Under light conditions, 50 μmol photons m³ were cultured after 1 hour of incubation. -2 s -1 The culture was performed under both light and dark conditions, with the cells shielded from light using aluminum foil. Under both light and dark conditions, all-trans retinal was added to the culture medium after 3 hours of incubation to a final concentration of 5 μM.

[0071] Rhodopsin-expressing isoprenol-producing Escherichia coli was centrifuged at 15,000 rpm for 5 minutes at 4°C after initial culture. The isoprenol concentration was measured using gas chromatography (Agilent Technologies, USA) with a Stabilwax column (Restek, USA) and a flame ionization detector.

[0072] As a result, the isoprenol concentration in cells cultured under dark conditions was slightly less than 0.5 mM, while the isoprenol concentration in cells cultured under light conditions increased to approximately 0.6 mM.

[0073] Example 4: Production of rhodopsin-expressing eukaryotic cells

[0074] In the above example, rhodopsin was expressed in prokaryotes (specifically E. coli) as cells. In this example, rhodopsin was expressed in eukaryotes (specifically yeast).

[0075] Rhodopsin was introduced into yeast in conjunction with a mitochondrial localization signal. Specifically, as a mitochondrial localization signal for S. cerevisiae, nucleic acid encoding rhodopsin (OP13) derived from Roseiflexus sp., with the nucleic acid (DNA) encoding ALA synthase HemI attached to the N-terminus, was synthesized in budding yeast by codon optimization. This synthesized OP13 gene was introduced into a pGK426 vector to create pK426-HemI-OP13. S. cerevisiae BY4741 (ATCC201388) strain was transformed using pK426-HemI-OP13 by lithium acetate. As a control, a strain of S. cerevisiae BY4741 introduced with the pGK426 vector was used. The samples were stored as glycerol stocks at -80°C until immediately before use. The resulting S. cerevisiae BY4741 transformants and controls are referred to as "OP13-expressing yeast" and "control yeast," respectively.

[0076] Rhodopsin-expressing yeast was cultured as follows: Rhodopsin-expressing yeast and control yeast were cultured statically for 3 days at 30°C on plates of synthetic dextrose (SD) (with added Leu, His, and Met) from glycerol stock. Three colonies were picked from the resulting cultures and pre-cultured overnight at 30°C at 175 rpm in 5 mL of YPD medium. The pre-cultured OP13-expressing yeast and control yeast were then infused in 20 mL of SD medium containing amino acids and 10 μM all-trans retinal with initial OD. 600 Inoculation was performed so that the ratio was 0.15. The culture medium used was SD medium with the pH adjusted to 3.5 or 5 with citrate-phosphate buffer (McIlvaine 1921). 10 μM all-trans retinal was added to the medium. All subsequent experiments with eukaryotic cells were performed in the same manner. ±50 μmol photons m using LC-LED 450W (TAITEC, Japan) -2 s -1 The cells were cultured in a shaking incubator at 175 rpm and 30°C using light irradiation.

[0077] Rhodopsin-expressing yeast and control yeast were harvested after appropriate incubation time. Glucose concentration, glutathione production per cell, and ATP content in the culture medium were measured in the same manner as for E. coli. The experiment was repeated three times for each experimental condition.

[0078] The results are shown in Figure 6. As shown in Figure 6, the light intensity was 50 μmol photons m -2 s -1 Under irradiation conditions (E), the cell proliferation rate was not affected by differences in pH or the presence or absence of OP13 insertion. The glucose residue in the culture medium was measured over time, and as shown in Figure 6, glutathione production was higher in the OP13-introduced strain than in the control (Con). Glutathione concentration peaked 24 hours after the start of culture and then decreased, which is thought to be due to the metabolism of glutathione by yeast in SD medium.

[0079] The results above are summarized in the table below.

[0080] [Table 2]

[0081] [Table 3]

[0082] As shown in Tables 2 and 3 above, it was revealed that expressing rhodopsin (OP13) improves glutathione production and also improves the yield relative to glucose. The effects of improving glutathione production and the yield relative to glucose were also observed under pH 3.5 conditions. Lowering the pH can be an effective method to prevent contamination and growth of unwanted bacteria, and it was shown that rhodopsin expression also improves substance production and the yield relative to glucose even under the pH 3.5 conditions used in such cases.

[0083] The above experiment was conducted under identical conditions, except that the light intensity was set to 0.05 μE. The results are shown in Figure 7. The amount of glutathione produced is shown in the table below.

[0084] [Table 4]

[0085] As shown in Table 4, even when the light intensity was reduced to 1 / 100, the effect of rhodopsin introduction on increasing substance production was confirmed.

[0086] Furthermore, we investigated the temperature tolerance and low pH tolerance of rhodopsin-expressing yeast. The experiment was conducted in the same manner as above, except that dR was used as the rhodopsin.

[0087] As shown in Figure 8, yeast to which rhodopsin was introduced (white bars) showed a higher OD (Oxygen Demand) when the pH decreased compared to yeast without rhodopsin (gray bars). 600(Cell concentration) was achieved (see upper left panel). Also, as shown in Figure 8, when the culture temperature was increased, the growth capacity of all yeasts decreased, but no improvement in growth capacity was observed in the rhodopsin-introduced yeast (white bars) compared to the unintroduced yeast (gray bars) (see upper right panel). On the other hand, regarding intracellular ATP concentration, under both low pH conditions (see middle left panel) and high temperatures (see middle right panel), the rhodopsin-introduced yeast (white bars) was higher than that of the unintroduced yeast (gray bars). Similarly, glutathione production per cell was also higher under both low pH conditions (see lower left panel) and high temperatures (see lower right panel) in the rhodopsin-introduced yeast (white bars) compared to the unintroduced yeast (gray bars).

[0088] Thus, it was revealed that in eukaryotic cells, the introduction of rhodopsin and light irradiation increases the pH tolerance of cells and increases energy production and substance production under low pH conditions. Furthermore, it was revealed that in eukaryotic cells, the introduction of rhodopsin and light irradiation also increases energy production and substance production under high temperature conditions.

[0089] The above was further tested by changing the pH conditions. The pH was set to 3.5 and 6.0, and the relationship with the presence or absence of rhodopsin introduction was investigated. As shown in Figure 9, at a low pH (pH 3.5), yeast with rhodopsin introduction (+) had a higher OD than yeast without rhodopsin introduction (-). 600 (Cell concentration) was achieved (see upper left panel). Glucose in the culture medium was consumed most rapidly in rhodopsin-transformed yeast (+) at low pH (pH 3.5), followed by yeast without rhodopsin at low pH (-), and then under high pH conditions. ATP and glutathione (GSH) synthesis were significantly increased in rhodopsin-transformed yeast even under low pH conditions (see the three panels below). This effect was particularly pronounced with longer culture times (see 48 hours and 72 hours).

[0090] Further tests were conducted by changing the temperature conditions. The pH was set to 6, and the temperature conditions were the same as shown in the diagram. The relationship between the presence or absence of rhodopsin introduction and temperature changes was examined 24 hours after the start of culture. As shown in Figure 10, the results showed that in the rhodopsin introduction group, glucose consumption decreased particularly at higher temperatures, and an increase in intracellular ATP concentration and glutathione production was observed.

[0091] The glucose yield was calculated from the data in Figure 10. The glucose yield was defined as the amount of glutathione produced per gram of glucose (mg). The results are shown in Table 5 below.

[0092] [Table 5]

[0093] As shown in Table 5, the glutathione yield relative to glucose was significantly improved in rhodopsin-introduced cells (Rhod (+)) compared with non-introduced cells (Rhod (-)). This indicates that rhodopsin introduction enhances the cell's tolerance to low pH, improves its tolerance to high temperatures, increases its ability to produce substances, reduces glucose consumption, and improves the yield relative to glucose.

[0094] The capabilities of rhodopsin are thought to be related to the rate of proton efflux (i.e., ATP production capacity) mediated by rhodopsin in both eukaryotic and prokaryotic cells. Therefore, by exogenously expressing rhodopsins with proton efflux activity as shown in Table 1, it is possible to confer at least one of the above functions to cells, and it is thought that opsins are more useful the higher their proton transport capacity.

[0095] Furthermore, koji mold is commonly used in the fermentation industry. Aspergillus oryzae OP13 was expressed in the strain. The resulting OP13-expressing strain and a negative control strain expressing only the vector were cultured under light irradiation conditions using a culture medium commonly used for culturing Aspergillus oryzae. Specifically, as for koji mold, Aspergillus oryzaeNS4 ΔligD was used (see Mizutani et al., Fungal Genet. Biol., 45:878-889, 2008). ΔligD relates to the disruption of ligases involved in non-homologous recombination to avoid insertion of nonspecific transgenes into the genome. The pUND vector was used as the expression vector for OP13. An empty pUND vector was used in the negative control group. Czapek Dox liquid medium was used to culture Aspergillus oryzae. Czapek Dox liquid medium contains 20 g glucose, 6 g NaNO3, 1.52 g KH2PO4, 0.52 g KCl, 1 mL of 2 M MgSO4·7H2O (final concentration 2 mM), and 1 mL of trace elements per liter. Trace elements are present per liter: 8.5g ZnSO4·7H2O, 1g FeSO4·7H2O, 0.4g CuSO4·5H2O, 0.1g Na2B4O7·10H2O, 0.05g (NH4)6Mo7O 24 • Provided as a solution containing 4H2O. The vector transformation of Aspergillus oryzae was performed as follows. 1A) The bacterial cells were scraped off with a platinum loop and inoculated into 500 mL of sterile Dpy (2% dextrin, 1% polypeptone, 0.5% yeast extract, 0.5% KH2PO4, 0.05% MgSO4·7H2O) liquid medium in a 500 mL baffled Erlenmeyer flask. After suspension, the cells were cultured with shaking for 18-24 hours at 30°C and 120-150 rpm. 2A) After culturing, the bacterial cells were filtered using a Miracloth, washed with sterile water, and then suspended in TF solution 1 in a sterile L-shaped tube. Protoplasts were formed by shaking at 30°C and 50 strokes / min for 3 hours. 3A) Spores of the transformed strain were obtained using the protoplast-PEG method described in Non-Patent Literature 2 (Katsuya Gomi, "Transformation Method and Host-Vector System of Aspergillus oryzae," Journal of the Japan Society for Bioscience, Biotechnology, and Agricultural Chemistry, 1997, Vol. 71, No. 10, pp. 1013-1017). Culturing was carried out as follows. 5 mL of the above Czapek Dox liquid medium was placed in a 50 mL Erlenmeyer flask (without fins), and incubated with repeated shaking (120 rpm) at 40°C for 7 days (168 hours). Specifically, 1B) Add 7.5 μL (OD) spores of each transformed strain to 5 mL of liquid medium. 600 (Prepared to a concentration of 25%) and added. 2B) All samples were incubated in the dark for 72 hours. During this time, 7.5 μL each of ethanol solution (7 mM) containing all-trans retinal was added at 24 hours and 48 hours. 3B) Subsequently, the cells were cultured at 40°C under 50 μE light irradiation conditions. 4B) After 168 hours, the bacterial cells were collected, the cell mass was measured, and fatty acids were extracted to measure the total fatty acid production. The results are shown in Figure 11. As shown in Figure 11, the total fatty acid production (mg / L) in the OP13-expressing strain increased by approximately 1.65 times compared to the vector control strain. When fatty acid synthesis is enhanced, acetyl-CoA is consumed for fatty acid synthesis, which may reduce the supply of acetyl-CoA to the TCA cycle and potentially adversely affect cell proliferation. However, as shown in Figure 11, cell concentration did not decrease in the OP13-introduced strain, but rather tended to increase. From this, it was considered that forced expression of opsins can improve, restore, or increase the proliferative capacity of cells during substance production, and as a result contribute to an increase in total biomass and the amount of produced substances.

Claims

1. A prokaryotic opsin, the opsin protein, can satisfy one or more or all of the following conditions C {but not deltarhodopsin and proteorhodopsin}: The amino acid corresponding to the 88th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is Y, I, A, or F; The amino acid corresponding to the 90th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is Y; The amino acid corresponding to the 91st position in the amino acid sequence (OP13) described in Sequence ID No. 9 is V, I, or A; The amino acid corresponding to the 94th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is either L or V; The amino acid corresponding to the 95th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is V, I, F, or L; The amino acid corresponding to the 97th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is either V or T; The amino acid corresponding to the 98th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is P; The amino acid corresponding to the 99th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is L; The amino acid corresponding to the 100th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is L, Q, or M; The amino acid corresponding to the 134th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is either Y or A; The amino acid corresponding to position 135 of the amino acid sequence (OP13) described in Sequence ID No. 9 is A, M, F, P, I, or L; The amino acid corresponding to the 136th position in the amino acid sequence (OP13) described in Sequence ID No. 9 is either G or A; The amino acids corresponding to positions 194 to 195 in the amino acid sequence (OP13) described in Sequence ID No. 9 are AI, AV, GV, FL, or CF.

2. It is opsin, (1) Having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 9, 11, 13, 16, 21, 22, 23, 31, 34, and 38, or (2) The opsin according to claim 1, having an amino acid sequence that is 80% or more identical to the above amino acid sequence {however, not dR and pR}.

3. An opsin according to claim 1, having the amino acid sequence of any of SEQ ID NOs: 41 to 43.

4. The opsin according to claim 1 or 2, derived from at least one species selected from the group consisting of Gloebacter violaceus, Roseiflexus sp., Octadecabacter antarcticus, Photobacterium sp. SKA34, Exiguobacterium sibiricum, Uncultured 66A03, Rhodobacterales bacterium, Uncultured MedebAC35C06, Uncultured HF1019P19, and Psychroflexus torquis.

5. The opsin according to claim 4, derived from at least one species selected from the group consisting of Roseiflexus sp., Photobacterium sp. SKA34, and Rhodobacterales bacterium.

6. A gene encoding an opsin according to any one of claims 1 to 4, or a gene encoding a functional opsin that can be hybridized to said gene under stringent conditions.

7. It includes a gene encoding an exogenous opsin operably linked to a regulatory sequence, The gene encoding the exogenous opsin is the gene described in claim 6, If the cell is a prokaryotic cell, opsins are expressed at least on the cell membrane. If the cell is a eukaryotic cell, opsins are expressed at least in the inner mitochondrial membrane. cell.

8. The cell according to claim 7, wherein the cell is a prokaryotic cell selected from the group consisting of actinomycetes, filamentous fungi, microalgae, bacteria, and archaea.

9. The cell according to claim 7, wherein the cell is a cell of a eukaryote selected from the group consisting of animals, plants, and fungi.

10. The cell according to any one of claims 7 to 9, further comprising a series of genes encoding a retinal synthesis system.

11. A method for culturing cells, To provide a cell containing a gene encoding an exogenous opsin operably linked to a control sequence, wherein the gene encoding the exogenous opsin is the gene described in claim 6. A method comprising culturing the cells under light irradiation conditions.

12. The culture method according to claim 11, wherein the culture is carried out under conditions that satisfy either or both of the following selected from the group consisting of pH conditions lower than pH 5 and temperature conditions higher than 30 degrees.

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