Fusion proteins of organelle transport sequences and opsin

Abstract

To provide a fusion protein of an organelle transit sequence and an opsin, a nucleic acid encoding the fusion protein, and a cell comprising the nucleic acid.SOLUTION: The present invention discloses a fusion protein of an acidic organelle transit sequence and an opsin, wherein the acidic organelle transit sequence is a transit sequence included in a sub unit of V-ATPase, and the organelle transit sequence is Vph1 protein or its portion retaining a transit ability to an acidic organelle. Also disclosed are a nucleic acid encoding the fusion protein, and a cell comprising the nucleic acid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure provides a fusion protein of an organelle transport sequence and an opsin, a nucleic acid encoding the protein, and a cell containing the nucleic acid. The present disclosure also provides a method for culturing the cell. The culturing can be preferably performed in a medium containing all-trans-retinal under light irradiation conditions. [Background technology]

[0002] Substance production using microorganisms, cells, and living organisms is a field that is attracting attention in the chemical production process. Patent document 1 discloses a yeast that expresses deltarhodopsin (dR) derived from Haloterigena turcumenica in the inner mitochondrial membrane.

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

[0004] Patent Document 2 shows that the resulting E. coli exhibits light-dependent proton transport, promotes energy production in E. coli, and improves isoprenol production. Thus, it has been shown that for biological use in substance production, in addition to the supply of nutrients from the culture medium, utilization of light energy in the plasma membrane or mitochondrial inner membrane using rhodopsin is beneficial. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2015 / 170609A [Patent Document 2] WO2020 / 050113A Summary of the Invention

[0006] The present disclosure provides a fusion protein of an organelle transport sequence and an opsin, a nucleic acid encoding the protein, and a cell containing the nucleic acid. The present disclosure also provides a method for culturing the cell. The culturing can be preferably performed in a medium containing all-trans-retinal under light irradiation conditions.

[0007] The present inventors have found that by expressing microbial rhodopsin on the membranes of acidic organelles and culturing them under light irradiation, the ATP used to maintain the pH of acidic organelles can be utilized for other cellular activities. For example, this technology may be useful for causing cells to produce substances. The present invention was made based on this finding.

[0008] According to the present disclosure, for example, the following inventions are provided: (1) A fusion protein of an acidic organelle transport sequence (e.g., the full-length Vph1 protein or a portion thereof that retains the ability to transport to acidic organelles) with opsin. (2) The fusion protein according to (1) above, wherein the acidic organelle translocation sequence is a translocation sequence contained in a subunit of a V-type ATPase. (3) The fusion protein according to (1) or (2) above, wherein the organelle targeting sequence is a Vph1 protein or a portion thereof that retains the ability to target an acidic organelle. (4) A nucleic acid encoding the fusion protein according to any one of (1) to (3) above. (5) A gene expression vector comprising the nucleic acid according to (4) above operably linked to a regulatory sequence. (6) A cell comprising the nucleic acid described in (4) above operably linked to a control sequence. (7) A method comprising culturing the cell according to (6) above in a medium under conditions suitable for its culture. (8) The method according to (7) above, wherein the medium contains all-trans retinal. (9) The method according to (7) or (8) above, wherein the culture is carried out under light irradiation conditions. (10) A method for reducing the ATP requirement of a eukaryotic cell or the ATP consumption in an acidic organelle of a eukaryotic cell, the method comprising expressing an opsin (or rhodopsin) in the acidic organelle.

[0009] (20) A method for selecting an amino acid sequence of a fusion protein between an organelle transport sequence and opsin, the method comprising: providing an amino acid sequence of a fusion protein between a candidate organelle transport sequence and opsin; and selecting an amino acid sequence that localizes to an acidic organelle when a fusion protein having the amino acid sequence is expressed in a eukaryotic cell. (21) The method according to (20) above, further comprising obtaining a nucleic acid encoding the fusion protein. (22) A method for selecting cells, comprising providing an amino acid sequence of a fusion protein between a candidate organelle transport sequence and opsin, expressing the fusion protein having the amino acid sequence in a eukaryotic cell, and selecting cells having the fusion protein in an acidic organelle. (23) The method according to (22) above, further comprising culturing the cells. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows the relative cell concentration (upper panel) and relative glucose consumption (lower panel) of budding yeast expressing rhodopsin on the membrane of an acidic organelle after culturing under light irradiation conditions. [Figure 2] Figure 2 shows the relative glutathione concentration per culture medium (upper panel) and per cell (lower panel) after culturing budding yeast expressing rhodopsin on the membrane of acidic organelles under light irradiation conditions. Specific Description of the Invention

[0011] As used herein, "rhodopsin" refers to a complex of a protein called opsin and retinal. Examples of rhodopsin include deltarhodopsin (dR), bacteriorhodopsin (bR), and bR-type rhodopsins (e.g., archaealhodopsin and cruxrhodopsin), sensory rhodopsin I, sensory rhodopsin II, proteorhodopsin (pR), and pR-type rhodopsin, channelrhodopsin I, channelrhodopsin II, halorhodopsin, xanthorhodopsin, sodium pump rhodopsin, and heliorhodopsin. For example, deltarhodopsin, bacteriorhodopsin, and proteorhodopsin are localized in the cell membrane of prokaryotes and function to pump protons from the inside to the outside of prokaryotes under light conditions (light irradiation). In prokaryotes, the proton concentration gradient across the cell membrane can be used to produce ATP. In eukaryotes, the inner mitochondrial membrane has an oxidative phosphorylation pathway that transports protons from the inner membrane to the outer membrane, creating a proton concentration gradient. This proton concentration gradient is used to produce ATP. Therefore, in eukaryotes, expressing opsin in the inner mitochondrial membrane can create a light-dependent proton concentration gradient and promote ATP production.

[0012] As used herein, "opsin" refers to a membrane protein with a seven-transmembrane structure. Photoreceptor activity is acquired by binding retinal, a vitamin A derivative. Examples of opsins include those listed in Table 1. However, not all of these exhibit proton transport activity in different organisms. Opsins include, but are not limited to, at least one opsin 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 activity. Opsins also include functional variants of opsins that can generate a light-dependent proton concentration gradient.

[0013] As used herein, an "opsin-encoding nucleic acid" refers to a nucleic acid encoding an opsin operably linked to a control sequence (e.g., a promoter). The control sequence can induce transcription of the opsin in a host (a cell into which the nucleic acid has been introduced). The opsin-encoding nucleic acid may be codon-optimized for the host. Because codon usage differs depending on the host, codon optimization is preferable, particularly when introducing a nucleic acid encoding a heterologous opsin. The opsin-encoding nucleic acid may be incorporated into a vector (e.g., a plasmid) with or without an origin of replication suitable for replication in the host. The vector may have an auxotrophic marker and / or a drug selection marker. If the vector has an auxotrophic marker and / or a drug selection marker, the host into which the vector has been introduced can be selected with the nutrient and / or drug. The control sequence may be a constitutive or inducible promoter. Opsin is expressed in cells and is called rhodopsin when bound to retinal on the membrane. Expressing rhodopsin means having opsin in a form bound to retinal on the membrane.

[0014] In this specification, organisms are broadly classified into prokaryotes and eukaryotes. Organisms are also broadly classified into unicellular organisms and multicellular organisms. Typically, prokaryotes are unicellular, while eukaryotes include both unicellular and multicellular organisms. Examples of prokaryotes include bacteria, archaea, cyanobacteria, and actinomycetes. Specific examples include Escherichia coli (e.g., Escherichia coli), Bacillus subtilis (e.g., Bacillus subtilis), lactic acid bacteria (e.g., Lactobacillus), acetic acid bacteria (e.g., Acetobacteraceae), Corynebacterium, Pseudomonas bacteria, methanogens (e.g., Methanobacterium, Methanosarcina), Streptomyces, Actinomyces, and Agrobacterium. Eukaryotic organisms include animals, plants, and fungi.Examples of fungi include fission yeasts or budding yeasts, for example, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces fragilis, Saccharomyces rouxii, and other Saccharomyces species, Schizosaccharomyces pombe, and other Candida species, such as Candida utilis and Candida tropicalis, and Xanthophyllomyces dendrorhous. Examples of yeasts include those of the genus Xanthophyllomyces, such as Saccharomyces dendrorhous, Pichia, Kluyveromyces, Yarrowia, Hansenula, Endomyces, and Rhodotorula. Among these, yeasts of the genus Saccharomyces, Schizosaccharomyces, Candida, Xanthophyllomyces, or Pichia are preferred, with Saccharomyces cerevisiae (S. cerevisiae), Schizosaccharomyces pombe (S. pombe), and Candida utilis (C. utilis) being more preferred, and S. cerevisiae being the most preferred.Examples of suitable cells 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 cultured mammalian cells (e.g., Chinese hamster ovary cells (CHO cells) and human cells). The cells may also be algae (macroalgae and microalgae). The cells or organisms may be photosynthetic cells or organisms (photosynthetic cells or organisms) or non-photosynthetic cells or organisms (non-photosynthetic cells or organisms). The introduction of opsin promotes the formation of a proton concentration gradient at least within the acidic organelle, suppressing the energy consumption by V-ATPase, which is necessary for the formation of this concentration gradient. The organism or cell into which the opsin-encoding gene is introduced may be referred to herein as a "host." The cells may be, for example, eukaryotic cells, or cells of the above organisms or the above cells.

[0015] As used herein, "exogenous" means derived from an organism other than the host. Typically, the regulatory sequence may be exogenous. The gene to be expressed may also be exogenous. Thus, in the case of a gene operably linked to a regulatory sequence, both the regulatory sequence and the gene may be exogenous. "Endogenous" means that it is contained in the host itself.

[0016] As used herein, the term "retinal synthesis pathway" refers to a multi-step pathway for synthesizing all-trans-retinal (ATR) from isopentenyl diphosphate (IPP). In the retinal synthesis pathway, ATR is synthesized from IPP according to the following steps [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] Phytoene is 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 into two all-trans-retinals (ATR). IPP can be produced by the mevalonate pathway or the non-mevalonate pathway.

[0017] As used herein, the term "a series of enzymes in the retinal biosynthesis system" refers to the complete set of enzymes [1] to [7] above. Some cells possess endogenous enzymes. For example, Escherichia coli and yeast possess IPPδ-isomerase and ispA. Therefore, adding crtE, crtB, crtI, and crtY to Escherichia coli causes the Escherichia coli and yeast to synthesize β-carotene. Adding blh to Escherichia coli and yeast causes the Escherichia coli and yeast to synthesize all-trans-retinal. Thus, "having a series of enzymes in the retinal biosynthesis system" includes having all of the enzymes in the retinal biosynthesis system, including exogenous and endogenous enzymes. Of course, all of the enzymes in the retinal biosynthesis system may also be supplied exogenously. Those skilled in the art can appropriately determine which enzymes a cell possesses by analyzing the presence or absence of genes or gene products. In one embodiment, the host expresses all of the enzymes selected from the group consisting of IPP δ-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 one embodiment, the host contains exogenous genes encoding some or all of the enzymes selected from the group consisting of IPP δ-isomerase, FPP synthase (ispA), geranylgeranyl pyrophosphate synthase (crtE), phytoene synthase (crtB), phytoene dehydrogenase (crtI), lycopene cyclase (crtY), and 15,15'-β-carotene dioxygenase (blh), although the host may not contain endogenous enzymes or genes. Once bacterial opsin binds to all-trans-retinal to form rhodopsin, it can then actively transport protons simply by receiving light.

[0018] According to the present disclosure, a fusion protein of an organelle transport sequence and opsin is provided. The organelle transport sequence can be a sequence that can transport a protein to an acidic organelle. Examples of acidic organelles include organelles located in the secretory pathway and endocytic pathway, such as the Golgi apparatus, secretory vesicles, secretory granules, endosomes, and lysosomes. The lumen side of an acidic organelle is maintained at a weakly acidic pH of approximately 4.5 to 6.5. Vacuolar ATPase (V-type ATPase) is expressed in acidic organelles, and consumes ATP to transport protons (H) into the organelle. + The active transport of rhodopsin maintains a constant pH within acidic organelles. The inventors believe that ATP consumption by V-ATPase can be suppressed by expressing rhodopsin on the membrane of at least acidic organelles and irradiating them with light. Therefore, a fusion protein of an organelle transport sequence with opsin may be useful for this purpose.

[0019] The organelle targeting sequence may be an organelle targeting sequence of the same species or a different species as the host cell, as long as it can target a protein to an organelle in the host cell. Preferably, the organelle targeting sequence is an organelle targeting sequence of the same species as the host cell. The organelle targeting sequence can target a protein to at least an acidic organelle. Examples of organelle targeting sequences include Atg27, Cpy, Vps34, Vps73, Vps68, Vps62, NHX1 (VPS44), Vps68, Vps69, Vps73, Vps69, Vps73, Vps68 ... AM7 (VPS43), Vps41, VAM6 (VPS39), Vps35, VPS33, PEP3 (VPS18), VPS16, PEP5 (VPS11), VPS1, Rho8 (ALP), Pib2, Ivy1, Sna4, Avt1 , Avt3, Avt4, Avt6, Avt7, Vba1, Vba2, Vba3, Vba4, Atg22, Ypq1, Ypq2, Uga4, Vma1, Vma2, Vma13, Vma5, Vma8, Vma4, Vma6, Vma7, Vm Proteins selected from the group consisting of a10, Vma11, Vma16, Vps23, Vps28, Vps37, Vps22, Vps25, Vps36, Vps2, Vps20, Vps24, Vps32, Pep4, Vam7, Vam3, Nyv1, Vti1, Ypt7, Vps11, Vps16, Vps18, Vps33, Vps39, and Vps41, or portions thereof containing an organelle localization sequence (or portions thereof that retain organelle localization ability) can be used. From the perspective of inhibiting ATP consumption by V-ATPase, organelle localization sequences that achieve intracellular distribution similar to that of V-ATPase are preferably used. From this perspective, preferred organelle localization sequences for V-ATPase include, for example, the acidic organelle localization sequence of a V-ATPase subunit and the Vph1 protein, one of the subunits. Thus, in a preferred embodiment, the organelle translocation sequence may be an acidic organelle translocation sequence of a V-ATPase subunit homologous to the host cell, and a Vph1 protein, or a fragment or mutant thereof having acidic organelle translocation ability.

[0020] V-type ATPases consist of V1 (subunits A through H), which is responsible for ATPase activity, and Vo (subunits a, d, c, c', and c''), which couples with V1 to transport protons. The Vph1 protein is one of the subunits of V-type ATPases. Vph1 is also known as V-type proton ATPase subunit a, V-ATPase a subunit, vacuolar ATPase 91 kDa subunit, vacuolar proton pump a subunit, and vacuolar proton transport ATPase subunit a. Vph1 plays an important role in the assembly and activity of V-type ATPases and is involved in the localization of V-type ATPases to acidic organelles. Examples of Vph1 from budding yeast include those with the amino acid sequence of SEQ ID NO: 7 (e.g., GenBank Accession No. NC_001147.6). Orthologs of Vph1 from various species can be used. For example, any of the Vph1 proteins listed in Table 1 can be used as Vph1. Table 1 lists the biological species and the GenBank Accession No. of the Vph1 protein for that species. Amino acid sequence information can be obtained from the GenBank Accession No. Furthermore, Vph1 proteins also include Vph1 proteins that retain the ability to localize to acidic organelles and have amino acid sequences with 90% or more sequence identity to these amino acid sequences. Furthermore, Vph1 proteins also include Vph1 proteins that retain the ability to localize to acidic organelles and have one to several (e.g., 1 to 7, 1 to 5, 1 to 4, 1 to 3, or 1 to 2) mutations selected from the group consisting of insertions, deletions, substitutions, additions, and deletions from these amino acid sequences. The full-length of any of the above Vph1 proteins or a portion thereof (particularly the translocation sequence portion) that retains the ability to localize to organelles can be linked to opsin as an organelle translocation sequence.

[0021] [Table 1] JPEG0007787512000002.jpg240157JPEG0007787512000003.jpg240156JPEG0007787512000004.jpg242157JPEG0007787512000005.jpg225157

[0022] Opsin complexes with all-trans retinal to form rhodopsin. Examples of rhodopsin include deltarhodopsin (dR), bacteriorhodopsin (including archaealhodopsin and cruxrhodopsin), sensory rhodopsin I, sensory rhodopsin II, proteorhodopsin (pR), channelrhodopsin I, channelrhodopsin II, xanthorhodopsin, sodium pump rhodopsin, and heliorhodopsin. For example, deltarhodopsin and bacteriorhodopsin function to pump protons from the inside to the outside of prokaryotes under bright conditions (light irradiation). The rhodopsin of the present invention is preferably deltarhodopsin, bacteriorhodopsin, or proteorhodopsin, and more preferably deltarhodopsin. Examples of deltarhodopsin include those derived from Haloterrigena turkmenica (Haloterrigena sp. Arg-4). In one embodiment, dR having the amino acid sequence of SEQ ID NO: 6 can be used. Examples of opsin-encoding nucleic acids (also referred to as "rhodopsin-encoding genes") include nucleic acids encoding the opsins that make up these rhodopsins. In one embodiment, a nucleic acid having the sequence of SEQ ID NO: 5 can be used.

[0023] The opsin-encoding nucleic acid may be a nucleic acid encoding a functional opsin capable of hybridizing with the opsin-encoding gene under stringent conditions. Stringent conditions include normal stringency conditions and conditions with higher stringency. Under normal stringency conditions, hybridization can be performed in a hybridization solution with 6xSSC or an equivalent salt concentration at 50-60°C for approximately 16 hours, followed by pre-washing, if necessary, with 6xSSC or a solution with an equivalent salt concentration, followed by washing in 1xSSC or a solution with an equivalent salt concentration. Higher stringency conditions (high stringency conditions) can be achieved by washing in 0.1xSSC or a solution with an equivalent salt concentration. "SSC" refers to an aqueous solution containing 15 mM sodium citrate and 150 mM sodium chloride at pH 7. "n x SSC" (where n is a positive real number) refers to an aqueous solution of pH 7 containing n times the concentrations of sodium citrate and sodium chloride contained in the SSC. The SSC may be DNase-free and / or RNase-free, and may be autoclave-sterilized.

[0024] The nucleic acid encoding the fusion protein of the organelle transport sequence and opsin may be DNA or mRNA. The DNA encoding the fusion protein may be operably linked to a regulatory sequence (e.g., a promoter suitable for expression in a host). The DNA encoding the fusion protein may be incorporated into an expression cassette of a protein expression vector. According to the present disclosure, a gene expression vector is provided, comprising DNA encoding the fusion protein operably linked to a regulatory sequence. The gene expression vector may further comprise an auxotrophic marker and / or a drug selection marker, allowing hosts containing the vector to be selected or maintained in the absence of the nutrient and / or the presence of the drug. The mRNA encoding the fusion protein has a structure suitable for translation, for example, preferably having a cap structure at the 5' end and preferably having a polyA sequence at the 3' end.

[0025] In addition to the gene encoding the fusion protein, the host can have all of the enzymes in the retinal synthesis system. This allows the amount of retinal added to the culture medium to be reduced if the host can self-synthesize retinal. If the amount of retinal synthesized by the host is sufficient, retinal does not need to be added to the culture medium. If the host does not have some of the 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 regulatory sequence and introduced into an expression cassette in a vector. Whether or not a host has an enzyme can be determined appropriately by those skilled in the art using biochemical analysis (e.g., Western blot) or genetic analysis (e.g., sequencing or PCR).

[0026] The present disclosure provides a cell comprising a nucleic acid encoding a fusion protein operably linked to a regulatory sequence. According to the invention, the cell may comprise an mRNA encoding the fusion protein operably linked to a regulatory sequence. According to the invention, the cell may comprise the fusion protein. In the cell, the fusion protein may be localized to the membrane of an acidic organelle.

[0027] The present disclosure provides a method for reducing the ATP requirement of a eukaryotic cell or ATP consumption in an acidic organelle of a eukaryotic cell, the method comprising expressing opsin (or rhodopsin) in the acidic organelle. The method may further comprise culturing the eukaryotic cell expressing rhodopsin in the acidic organelle under light irradiation. According to the present invention, reducing ATP consumption in the acidic organelle may result in a stoichiometric excess of ATP remaining that the cell can use for maintenance or growth.

[0028] The present disclosure provides a method for selecting an amino acid sequence of a fusion protein of an organelle transport sequence and opsin, the method comprising: providing an amino acid sequence of a fusion protein of a candidate organelle transport sequence and opsin; and selecting an amino acid sequence that localizes to an acidic organelle when the fusion protein having the amino acid sequence is expressed in a eukaryotic cell. This method may further comprise obtaining a nucleic acid encoding the fusion protein. The localization of the fusion protein can be detected using an antibody against the fusion protein, or by labeling the fusion protein and using various techniques for detecting the label. This method may be useful for obtaining a protein that more strongly promotes organelle transport. Host cells are preferably used as the cells. The method of the present disclosure also provides a method for selecting cells, which may comprise providing an amino acid sequence of a fusion protein of a candidate organelle transport sequence and opsin, expressing the fusion protein having the amino acid sequence in a eukaryotic cell, and selecting cells that have the fusion protein in an acidic organelle. This method may further comprise culturing the cells. The culturing may be performed under light or non-light conditions. This method can be useful when it is unclear whether the organelle transport sequence functions in a specific host cell (particularly in the case of a heterologous host cell). Preferably, host cells can be used as the cells.

[0029] The present disclosure provides a method for culturing cells. The cells contain a nucleic acid encoding a fusion protein operably linked to a regulatory sequence. The cells may also contain the fusion protein. In the cells, the fusion protein may be localized to the membrane of an acidic organelle. When cultured under light irradiation, such cells can supply protons to the acidic organelle, thereby reducing the amount of ATP used to maintain the pH of the acidic organelle. In the cells, the fusion protein binds all-trans-retinal and exerts light-driven proton pump activity. Thus, the cells contain the fusion protein bound to all-trans-retinal at least in the membrane of the acidic organelle. If the cells have a retinal synthesis system, it is not necessary to add all-trans-retinal to the culture medium (although it may be added). However, all-trans-retinal may be added to the culture medium to supply all-trans-retinal to opsin in the fusion protein in the cells. When the purpose of culturing is simply to grow the cells, the cells may be cultured in the absence of all-trans-retinal (under light irradiation or non-light irradiation conditions). However, for the purpose of more efficient cell growth, cells containing a fusion protein containing opsin bound to all-trans-retinal on the membrane of an acidic organelle can be cultured under light irradiation conditions.

[0030] A host expressing a fusion protein (the host is a cell) can be cultured under culture conditions suitable for the host. Those skilled in the art would be able to determine the culture conditions as appropriate. A host expressing a fusion protein can be cultured under light irradiation conditions. By culturing under light irradiation, ATP synthesis is promoted in the host expressing the fusion protein, and energy is supplied by light irradiation. The light irradiation is not particularly limited as long as it is at a level at which rhodopsin in the fusion protein can receive light, but for example, 0.01 μmol photons m -2 s -1 More than 0.02 μmol photons m -2 s -1 More than 0.03 μmol photons m -2 s-1 Above, 0.04 μmol photons m -2 s -1 Above, または0.05μmol photons m -2 s -1 The 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 -1 The 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 -1The 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 The following, 150 μmol photons m -2 s -1 The following, 160 μmol photons m -2 s -1 The following, 170 μmol photons m -2 s -1 The following, 180 μmol photons m -2 s -1 The following, 190 μmol photons m -2 s -1 The following, 200 μmol photons m -2 s -1 The following, 300 μmol photons m -2 s -1 The following, 400 μmol photons m -2 s -1 Below, 500 μmol photons m -2 s -1 The following, 600 μmol photons m -2 s -1 The following, 700 μmol photons m -2 s -1 The following, 800 μmol photons m -2 s -1 The following, 900 μmol photons m -2 s -1 The following, 1000 μmol photons m -2 s -1 The following, 1500 μmol photons m -2 s -1 Below, または2000μmol photons m -2 s -1The light to be irradiated may be any of light bulbs (e.g., incandescent bulbs, halogen bulbs, etc.), fluorescent lamps, high-pressure discharge lamps, low-pressure discharge lamps, LEDs, electroluminescent (EL), chemiluminescence, bioluminescence, and sunlight, and light having a wavelength of, for example, 300 to 800 nm, 450 to 650 nm, e.g., 550 nm, can be used. Light irradiation may be carried out continuously or intermittently.

[0031] When light irradiation provides energy to the host, the host can reduce the amount of ATP consumed to maintain the pH of acidic organelles. When light irradiation reduces the amount of ATP consumed by the host in acidic organelles, the host can use more raw materials, such as carbon sources, in pathways involved in substance production (e.g., the pentose phosphate pathway) and energy production pathways. Therefore, light irradiation puts the host in a state suitable for substance production. Therefore, substance production can be preferably carried out by supplying raw materials, such as carbon sources, to the host. The microorganism expressing the fusion protein may exhibit low pH tolerance and / or high temperature tolerance under light irradiation conditions.

[0032] Low pH tolerance means that, in a low pH environment (e.g., below pH 5.0) where host 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 enhanced under light irradiation conditions compared to non-irradiation and / or under fusion protein expression conditions compared to non-expression. In the case of yeast, for example, low pH tolerance means that one or more capabilities selected from the group consisting of growth ability, ATP synthesis ability, and substance production ability are enhanced under pH conditions below pH 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), compared to the corresponding capabilities under non-irradiation conditions and / or under conditions where the fusion protein is not expressed. A host expressing a fusion protein and having low pH tolerance can be favorably cultured even under low pH conditions. Under low pH conditions, one or more abilities selected from the group consisting of the growth, ATP synthesis ability, and substance production ability of other contaminating microorganisms are suppressed, making it possible to selectively culture the host.

[0033] "High-temperature tolerance" means that, under high-temperature conditions where the host's capabilities (one or more abilities selected from the group consisting of growth ability, ATP synthesis ability, and substance production ability) are reduced, one or more abilities selected from the group consisting of growth ability, ATP synthesis ability, and substance production ability are enhanced under light irradiation conditions compared to non-irradiation and / or under fusion protein expression conditions compared to non-expression. In the case of yeast, for example, "high-temperature tolerance" means that, under conditions of 32°C or higher, 33°C or higher, 34°C or higher, e.g., 34°C to 36°C (e.g., 35°C) or higher, one or more abilities selected from the group consisting of growth ability, ATP synthesis ability, and substance production ability are enhanced compared to the corresponding abilities under non-irradiation conditions and / or under conditions where the fusion protein is not expressed. Under high-temperature conditions, the growth of other contaminating microorganisms is suppressed, potentially enabling the host to be selectively cultured.

[0034] The microorganism expressing the fusion protein may be resistant to low pH and high temperature under light irradiation conditions. In this case, the host expressing the fusion protein and having resistance to low pH and high temperature 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 are suppressed, making it possible to selectively culture the host.

[0035] Substance production can be preferably carried out by culturing cells or organisms expressing the fusion protein under the above conditions. To increase the production of a particular substance, cells or organisms expressing the fusion protein can be cultured under conditions suitable for the production of the substance. The culture conditions can be either or both of low pH and high temperature. To increase the production of a particular substance, further modifications can be made to cells or organisms expressing the fusion protein. Such modifications include, for example, mutation and / or introduction of enzymes involved in the production pathway of the substance; disruption, reduction in expression, or deletion or reduction in activity of enzymes involved in the degradation of the substance; and / or disruption, reduction in expression, and / or deletion or reduction in activity of enzymes involved in competing pathways.

[0036] Substances produced by substance production include, for example, organic acids, peptides, amino acids, proteins, nucleosides, vitamins, sugars, sugar alcohols, alcohols, isoprenoids, and lipids. More specifically, the following can be mentioned: organic acids include acetic acid, lactic acid, succinic acid, and α-keto acids (2-oxo acids); peptides include glutathione, alanylglutamine, and γ-glutamylvalylglycine; and 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. 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), gamma-glutamylcysteine ​​synthetase (EC 6.3.2.2), glutathione synthetase (EC 6.3.2.3), human granulocyte colony-stimulating factor, xylose reductase, and P450. Nucleosides include inosine, guanosine, inosinic acid, guanylic acid, and adenylic acid. Vitamins include riboflavin, thiamine, and ascorbic acid. Sugars include xylose and mannose. Sugar alcohols include xylitol and mannitol. 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 of the present invention, acetic acid is a particularly preferred organic acid, glutathione is a particularly preferred peptide, mevalonic acid and isoprenol are particularly preferred isoprenoids, and hydroxypropionic acid is a particularly preferred lipid. Examples of substances produced include ATP, glutathione, and isoprenoids. In the present disclosure, the method of the present invention can be used to produce, for example, glutathione. production This can be promoted.

[0037] In all aspects of the present disclosure, the protein introduced into the host cell may preferably be a protein from an organism of the same species as the host cell, and / or the nucleic acid may be codon-optimized for expression in the host cell. Codon optimization can be performed appropriately by those skilled in the art based on well-known conventional techniques. [Example]

[0038] Example 1: Proton pump delivery to acidic organelles Intracellular organelles maintain their internal environment by consuming intracellular ATP. For example, acidic organelles actively transport cytoplasmic protons into the organelle by consuming ATP using ATPases (particularly V-type ATPases), maintaining an acidic pH within the organelle. In this example, by expressing an ATP-independent proton pump in the organelle membrane, we succeeded in suppressing intracellular ATP consumption while maintaining the organelle and its pH. Specifically, this is as follows.

[0039] In this example, budding yeast was used as the cell, and rhodopsin was used as the proton pump expressed in the intracellular organelles of budding yeast. Rhodopsin is expressed on the membrane, receives light, and can actively transport protons through the membrane using the energy. In this example, rhodopsin was specifically expressed in the extremely halophilic bacteria Haloterrigena turkmenicaDeltarhodopsin (dR) derived from the yeast Saccharomyces cerevisiae was used. An organelle targeting sequence was added to dR to construct a system for expressing dR on the membrane of acidic organelles. The full-length Vph1 (see SEQ ID NO: 7), one of the subunits of the V-type ATPase of budding yeast, was used as the organelle targeting sequence (see J Biol Chem. 2001; 276(50): 47411-20).

[0040] The gene encoding Vph1 having the amino acid sequence of SEQ ID NO: 7 was amplified by PCR using the primers shown below. [ka]

[0041] H. turkmenica The amino acid sequence of dR (SEQ ID NO: 6) derived from (JCM 9743, Riken BRC) was optimized taking into account the codon usage of budding yeast to obtain a nucleic acid encoding dR having the sequence of SEQ ID NO: 5. [ka]

[0042] A nucleic acid having the sequence of SEQ ID NO: 5 was amplified by PCR using the following primers. [ka]

[0043] The two resulting amplification products were ligated by the in fusion method and introduced into the pGK426 vector to obtain pGK426-Vph1-dR.

[0044] pGK426-Vph1-dR was introduced into the budding yeast BY4741 (ATCC201388) by the lithium acetate method (Ito H. et al. (1983) J. Bacteriol., 153:163-168) to obtain a dR-expressing strain. As a negative control, a dR-nonexpressing strain obtained by introducing the empty vector of pGK426 was used.

[0045] The resulting dR-expressing strains (two budding yeast clones, dR+_3 and dR+_5) and the dR-non-expressing strain were pre-cultured overnight in YPD medium. All subsequent cultures were performed at 30°C under stirring at 175 rpm. The pre-culture solution was added to the synthetic dextrose (SD) medium, which was the main culture medium, and the OD 600 The yeast was cultured in this culture medium for 22 hours, and 10 μM all-trans retinal was added to the medium. This resulted in budding yeast strains with rhodopsin on the membranes of at least the acidic organelles. 22 hours after the start of culture, the concentration of rhodopsin reached 50 μmol m -2 s -1 The culture was continued while irradiating the cells with light.

[0046] After 30, 46, 54, and 70 hours of incubation, a portion of the medium was collected to measure the growth status of the budding yeast and the amount of glucose consumed in the culture medium. The growth of the budding yeast was measured by measuring the OD 600 The cell concentration was calculated based on the values, and the relative cell concentration of each strain was calculated by setting the cell concentration of the dR non-expressing strain at 100. The glucose concentration in the culture medium was determined by obtaining the culture supernatant after centrifugation and measuring the glucose concentration in the culture supernatant using a Glucose C-II Test Kit (Wako Pure Chemical Industries). The glucose consumption was calculated from the initial glucose concentration and the measured value, and the relative glucose consumption of each strain was calculated by setting the glucose consumption of the dR non-expressing strain at 100. The results are shown in Figure 1. As shown in Figure 1, the cell concentration and relative glucose consumption of the dR-expressing strain were at similar levels to those of the dR non-expressing strain. In dR+_5, glucose consumption was reduced by approximately 10%.

[0047] To confirm the decrease in ATP consumption by organelles, we examined the production of glutathione, whose synthesis is promoted by increased intracellular ATP. The glutathione biosynthesis pathway is as follows: In the first step, γ-L-glutamyl-L-cysteine ​​is synthesized from cysteine, glutamate, and ATP. In the second step, glutathione is synthesized from γ-L-glutamyl-L-cysteine, glycine, and ATP. Therefore, two molecules of ATP are used for glutathione biosynthesis. The dR+_3, dR+_5, and dR-nonexpressing strains were cultured in SD medium containing 10 μM all-trans retinal. Portions of the culture were harvested 30, 46, 54, and 70 hours after the start of culture. The culture was centrifuged, and the precipitate was collected to prepare a cell suspension. The cell concentration of the cell suspension was measured by OD using a spectrophotometer. 600 The glutathione concentration was calculated based on the values ​​obtained. The cell suspension was then heated at 95°C for 5 minutes, centrifuged to remove cell debris, and the supernatant was subjected to glutathione concentration measurement. Glutathione concentration was measured using a GSH assay kit (Dojindo Laboratories) with DTNB (5-5'-dithiobis[2-nitrobenzoic acid]) colorimetric assay. The results are shown in Figure 2. As shown in Figure 2, glutathione production increased approximately threefold in the dR-expressing strain. This demonstrates that expression of a proton pump in acidic organelles can reduce ATP consumption by V-type ATPase. It also demonstrates that intracellular ATP can be used in substance production systems. Furthermore, despite the improved substance production capacity, such as glutathione production, there was little effect on cell growth and no increase in glucose consumption in the medium, indicating that providing a proton pump (rhodopsin) to organelles (cytoplasmic organelles other than mitochondria) could be a useful cellular modification for constructing substance production systems.

Claims

1. A fusion protein of an acidic organelle transport sequence and opsin, wherein the acidic organelle transport sequence includes the acidic organelle transport sequence of a Vph1 protein.

2. The fusion protein of claim 1 , wherein the acidic organelle transport sequence is a Vph1 protein.

3. A nucleic acid encoding the fusion protein of claim 1 or 2.

4. A gene expression vector comprising the nucleic acid of claim 3 operably linked to a regulatory sequence.

5. A cell comprising the nucleic acid of claim 3 operably linked to a regulatory sequence.

6. A method comprising culturing the cell according to claim 5 in a medium under conditions suitable for its culture.

7. The method of claim 6 , wherein the medium contains all-trans retinal.

8. The method according to claim 6 or 7, wherein the culture is carried out under light irradiation conditions.

9. A method for reducing the ATP requirement of a eukaryotic cell or the ATP consumption in an acidic organelle of a eukaryotic cell, the method comprising expressing an opsin (or rhodopsin) containing an organelle translocation sequence of a Vph1 protein in the acidic organelle.

10. The method of claim 9, wherein the opsin (or rhodopsin) linked to the organelle transport sequence of a Vph1 protein is an opsin (or rhodopsin) linked to a Vph1 protein.

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