Method for promoting growth of algae under high co2 conditions

Abstract

The present disclosure provides: a recombinant algae containing a recombinant gene; a method for promoting growth of an algae under high CO2 conditions; and a method for producing an algae culture product.

Description

Method for promoting algae growth under high CO2 concentration conditions

[0001] The present disclosure relates to recombinant algae containing recombinant genes, methods for enhancing algae growth under elevated CO2 conditions, and methods for producing algal cultures.

[0002] Algae have recently attracted attention as an environmentally friendly means for producing biofuels and other useful substances. Various attempts have been made to improve the productivity of algal cultures in order to utilize algae. One method for promoting algal growth is to aerate CO2 into the algal culture medium. For example, Non-Patent Document 1 reports that CO2 promotes the growth of Chlorella.

[0003] On the other hand, it is known that hybrid Rubisco composed of RbcL from rice (a C3 plant) and RbcS from sorghum (a C4 plant) can enhance catalytic activity by approximately twice that of the native rice Rubisco, and that application of this technique improves the nitrogen use efficiency (photosynthetic rate and growth per unit of nitrogen) of plants in high CO2 conditions (Non-Patent Document 2). However, only examples of the application of this technology to plants have been reported, and its applicability to algae has not been verified.

[0004] Rubisco (ribulose-1,5-bisphosphate carboxylase / oxygenase) is an enzyme responsible for CO2 fixation. Rubisco activase is an enzyme that activates Rubisco. It is known that enhancing (e.g., overexpressing) the function of Rubisco activase in algae under atmospheric CO2 concentrations can activate Rubisco, which is normally barely active at those CO2 concentrations, thereby improving biomass production (Non-Patent Document 3). However, at high CO2 concentrations (CO2 concentrations higher than atmospheric), Rubisco is already activated, so enhancing the function of Rubisco activase does not have any effect on increasing biomass production (Non-Patent Document 3).

[0005] Therefore, there is a need for technology that can further improve algae growth when high concentrations of CO2 are aerated into the algae culture solution.

[0006] Koh et al. Transcriptional insights into Chlorella sp. ABC-001: a comparative study of carbon fxation and lipid synthesis under diferent CO2 conditions. Biotechnology for Biofuels and Bioproducts (2023) 16:113Sakoda et al. (2021) Effects of introduction of sorghum RbcS with rice RbcSknockdown by RNAi on photosynthetic activity and dry weight in rice, Plant Production Science,24:3, 346-353, DOI: 10.1080 / 1343943X.2020.1847669Wei et al. Enhancing photosynthetic biomass productivity of industrial oleaginous microalgae by overexpression of RuBisCO activase. Algal Research 27 (2017) 366‐375

[0007] The present disclosure has been made to solve the above-mentioned problems, and aims to provide recombinant algae containing recombinant genes, a method for promoting algae growth under high CO2 conditions, and a method for producing algae cultures.

[0008] In one embodiment, a recombinant alga comprises a recombinant gene operably linked to a promoter, wherein the recombinant gene encodes a protein comprising a polypeptide having 90% or more amino acid sequence identity to SEQ ID NO: 1, wherein the protein, when expressed in the recombinant alga, is at least partially localized in chloroplasts and is capable of stimulating the growth of high CO2. ２Recombinant algae are provided, wherein conditions can be used to promote the growth of the recombinant algae.

[0009] According to the present disclosure, it is possible to provide recombinant algae containing recombinant genes, methods for promoting algae growth under elevated CO2 conditions, and methods for producing algae cultures.

[0010] Growth curves of the unicellular red alga Cyanidioschyzon merolae transformed with pNITE-CTSP-CMV015C or pNITE in a medium containing ammonium ions as the sole nitrogen source. Growth curves of the unicellular red alga Cyanidioschyzon merolae transformed with pNITE-CTSP-CMV015C or pNITE in a medium containing nitrate ions as the sole nitrogen source.

[0011] Non-limiting embodiments of the present disclosure will be described below. The present disclosure is not limited to the examples in the following embodiments.

[0012] <Recombinant Algae> In one embodiment, a recombinant alga comprises a recombinant gene operably linked to a promoter, wherein the recombinant gene encodes a protein comprising a polypeptide having 90% or more amino acid sequence identity to SEQ ID NO: 1, and wherein the protein, when expressed in the recombinant alga, is at least partially localized in chloroplasts and exhibits high CO2 production. ２ In an embodiment, the recombinant algae can be a recombinant gene encoding a fusion protein containing a polypeptide having 90% or more amino acid sequence identity to SEQ ID NO: 1 and a chloroplast targeting signal peptide linked to the polypeptide.

[0013] The type of algae in the present disclosure is not limited. The algae may be non-sessile algae. The algae may be, for example, unicellular algae. Examples of algae that may be used include Aurantiochytrium, Chlamydomonas, Chlorella, Schizon algae, Spirulina, Botryococcus, Euglena, green algae, brown algae, red algae, cyanobacteria, diatoms, xanthophytes, gold algae, dinoflagellates, and seaweed. Schizon algae is a particularly preferred example. The recombinant algae in the present disclosure may be a single species of algae or a population containing multiple species of algae. A genetically homogeneous population of algae (pure line) of the same species may be used, or a population of algae with genetic variation may be used. Algae for transformation may be strains isolated from the wild, or mutant strains generated by mutation from such strains. Furthermore, strains created by genome editing, transduction, transformation, etc. may be used for additional transformation.

[0014] In an embodiment, "red algae" refers to at least algae belonging to the genus Cyanidioschyzon. Examples of algae belonging to Cyanidioschyzon include the unicellular red alga Cyanidioschyzon merolae. In an embodiment, "spirulina" refers to at least algae belonging to the genus Arthrospira. Examples of spirulina include Arthrospira ardissonei, Arthrospira erdosensis, Arthrospira fusiformis, Arthrospira indica, Arthrospira innermongoliensis, Arthrospira jenneri, Arthrospira massartii, Arthrospira maxima, and Arthrospira platensis. In an embodiment, "aurantiochytrium" refers to at least algae belonging to the genus Aurantiochytrium. Examples of Aurantiochytrium include Aurantiochytrium limacinum and Aurantiochytrium mangrovei. In an embodiment, "Botryococcus" includes at least algae belonging to the genus Botryococcus. Examples of Botryococcus include Botryococcus australis, Botryococcus balkachicus, Botryococcus braunii, Botryococcus calcareus, Botryococcus canadensis, Botryococcus comperei, Botryococcus fernandoi, Botryococcus neglectus, Botryococcus pila, Botryococcus protuberans, Botryococcus pusillus, Botryococcus terribilis, and Botryococcus terricola. In an embodiment, "Chlamydomonas" includes at least algae belonging to the genus Chlamydomonas.Examples of Chlamydomonas include Chlamydomonas acidophila, Chlamydomonas caudata, Chlamydomonas ehrenbergii, Chlamydomonas elegans, Chlamydomonas moewusii, Chlamydomonas muriella, Chlamydomonas nivalis, Chlamydomonas ovoidae, Chlamydomonas priscuii, Chlamydomonas smithii, Chlamydomonas reinhardtii, etc. In an embodiment, "chlorella" includes at least algae belonging to the genus Chlorella. Examples of Chlorella include Chlorella autotrophica, Chlorella coloniales, Chlorella lewinii, Chlorella minutissima, Chlorella pituita, Chlorella pulchelloides, Chlorella pyrenoidosa, Chlorella rotunda, Chlorella singularis, Chlorella sorokiniana, Chlorella variabilis, Chlorella volutis, and Chlorella vulgaris. In an embodiment, "Euglena" includes at least algae belonging to the genus Euglena. Examples of Euglena include Euglena chadefaudii, Euglena deses, Euglena gracilis, Euglena granulata, Euglena mutabilis, Euglena proxima, Euglena spirogyra, and Euglena viridis.

[0015] In this disclosure, recombinant algae refers to algae containing an exogenous recombinant gene. The exogenous recombinant gene may be a nucleic acid containing a nucleic acid of the algae species (e.g., an additional copy of a gene nucleic acid introduced from an exogenous source in addition to the same gene naturally present in the algae), or a nucleic acid containing a nucleic acid derived from a heterologous organism. The recombinant gene of this embodiment may encode a fusion protein containing a polypeptide with 90% or more amino acid sequence identity to SEQ ID NO: 1 and a chloroplast targeting signal peptide (CTSP) linked to the polypeptide. Alternatively, a recombinant gene encoding the protein lacking the CTSP may be directly introduced into chloroplasts using a chloroplast transformation method known to those skilled in the art. The possibility that the polypeptide itself possesses chloroplast targeting activity is not excluded. SEQ ID NO: 1 is a polypeptide sequence encoded by the chloroplast DNA gene CMV015C of Cyanidioschyzon merolae and shows only limited amino acid sequence similarity to known Rubisco activators. A polypeptide having 90% or more amino acid sequence identity to SEQ ID NO: 1 may have 95% or more, 98% or more, 99% or more, or even 100% amino acid sequence identity. When expressed in algae as part of the protein under aerated culture conditions with air containing 2% (v / v) CO2, the polypeptide can improve the growth rate (e.g., measured based on the weight of algal biomass grown by 6, 9, or 13 days after the start of aerated culture) compared to corresponding non-expressing algae. The improvement in growth rate can result in an algal biomass weight increase of, for example, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, or 1.4-fold or more. As known to those skilled in the art, a chloroplast targeting signal peptide refers to a peptide sequence that has the activity of targeting a polypeptide fused thereto to intracellular chloroplasts. Specific examples of chloroplast targeting signal peptides are known to those skilled in the art and are described, for example, in J. Gen. Appl. Microbiol., 57, 69-72 (2011).

[0016] In an embodiment, the recombinant gene encodes a protein comprising a polypeptide having 90% or more amino acid sequence identity to SEQ ID NO: 1, and at least a portion of the protein is localized to chloroplasts when expressed in the recombinant alga. The protein may be localized to chloroplasts as a fusion protein with a chloroplast targeting signal peptide. Alternatively, the protein may be localized to chloroplasts as a result of chloroplast transformation with a nucleic acid comprising the recombinant gene.

[0017] In an embodiment, the amino acid sequence of SEQ ID NO: 5 may be used as the chloroplast targeting signal peptide. In the fusion protein of the present disclosure, the polypeptide having 90% or more amino acid sequence identity to SEQ ID NO: 1 and the chloroplast targeting signal peptide may be linked directly or via another sequence such as a linker sequence. In an embodiment, the polypeptide of SEQ ID NO: 2 may be used as the fusion protein comprising a polypeptide having 90% or more amino acid sequence identity to SEQ ID NO: 1 and a chloroplast targeting signal peptide linked to the polypeptide. SEQ ID NO: 2 is the polypeptide sequence of a fusion protein in which the chloroplast targeting signal peptide corresponding to SEQ ID NO: 5 (Watanabe et al. J. Gen. Appl. Microbiol. 57, 69-72 (2011)) is added to the amino terminus of SEQ ID NO: 1. In constructing a recombinant gene, the nucleotide sequences of SEQ ID NOs: 3, 4, and 6, which encode the polypeptide sequences corresponding to SEQ ID NOs: 1, 2, and 5, respectively, may be used.

[0018] The recombinant gene may optionally be accompanied by vector-derived sequences such as an origin of replication, an antibiotic resistance gene or other selectable marker, and a multicloning site. In other words, recombinant algae can be transformed using such a vector. An example of such a vector sequence is pNITE (SEQ ID NO: 8) (Imamura et al., The Plant Journal (2018) 94, 327-339). pNITE contains a nitrite reductase promoter (SEQ ID NO: 7) that induces expression of a gene cloned into the multicloning site (corresponding to positions 3978-4001 of SEQ ID NO: 8) in algae cultured in an environment containing nitrate as the sole nitrogen source.

[0019] The polypeptides encoded by the recombinant genes of the present disclosure, when expressed in the recombinant algal cells, can be used to treat high CO ２ The recombinant algae is a polypeptide capable of promoting growth of the recombinant algae under conditions, i.e., imparting the ability to promote growth. In this disclosure, "promoting algal growth" can mean improving the growth rate compared to corresponding algae lacking expression of the protein or fusion protein (e.g., lacking expression induction or lacking the recombinant gene) but under the same growth conditions. Growth rate can be measured, for example, based on the weight of algal biomass grown up to 6, 9, or 13 days after the start of culture under elevated CO2 and promoter-inducing conditions.

[0020] The recombinant gene in the embodiment can be provided in the form of a recombinant vector into which a nucleic acid encoding the protein or fusion protein, or a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 4, has been cloned. As a more specific example, the recombinant gene in the present disclosure can be provided in the form of a vector (pNITE-CTSP-CMV015C) comprising the sequence of SEQ ID NO: 9, which is obtained by cloning the sequence of SEQ ID NO: 4, which is obtained by adding a sequence encoding a chloroplast targeting signal peptide (CTSP) to the 5' end of the gene encoding CMV015C (SEQ ID NO: 3), into the pNITE vector backbone. The recombinant algae of the embodiment can be produced by transforming algae with these recombinant genes.

[0021] Recombinant algae of the present disclosure can be produced by transformation. Transformation, as used herein, includes introducing a gene from an external source to increase the expression or expression potential of the gene compared to a corresponding non-transformed individual. Transformation can be performed by a variety of techniques known to those skilled in the art, including various genome editing techniques such as the Agrobacterium method, the gene gun (particle gun) method, electroporation, PEG, homologous recombination, and CRISPR / CAS9, as well as combinations thereof. For example, the gene gun method and electroporation method have also been used for chloroplast transformation. Transformation in embodiments of the present disclosure is preferably performed by the PEG method. Transformants can be selected by transforming a URA5.3-deficient strain maintained in a medium supplemented with uracil and 5-fluoroorotic acid with a plasmid expressing the URA5.3 gene, followed by selection in MA2 medium lacking uracil and 5-fluoroorotic acid (Taki et al., 2015, J. Gen. Appl. Microbiol., 61, 211-214; Imamura et al., 2013, Biochem. Biophys. Res. Commun., 439, 264-269).

[0022] The recombinant algae of the present disclosure include a recombinant gene operably linked to a promoter. A promoter in this disclosure may refer to a regulatory DNA sequence that can drive the transcription or expression of a gene linked thereto directly or via another sequence in an individual or cell of the algae of the present disclosure. The driving of transcription or expression may be constitutive or conditional. A promoter that causes conditional driving of transcription or expression may be an inducible promoter. As will be understood by those skilled in the art, the term "operably linked" in this disclosure may mean that the promoter and the gene linked thereto are linked in a manner that is functionally linked, or that the promoter is linked in a manner that allows it to drive the transcription or expression of the gene linked thereto.

[0023] As understood by those skilled in the art, the term "inducible promoter" in the present disclosure refers to a promoter that can initiate or enhance the transcription or expression of a gene linked thereto when an organism containing the promoter is placed under a specific environment. Examples of inducible promoters in embodiments include, but are not limited to, nitrate-inducible promoters and heat shock promoters. Nitrate-inducible promoters are preferred. Nitrate-inducible promoters in the present disclosure refer to promoters whose expression is induced when nitrate ions are the sole nitrogen source, as exemplified by Imamura et al., The Plant Journal (2018) 94, 327-339. Nitrate-inducible promoters include nitrate reductase promoters, nitrite reductase promoters, and nitrate / nitrite reductase promoters. However, it is preferred to use a nitrite reductase promoter such as SEQ ID NO: 7, or a nitrate-inducible promoter having a sequence with 90% or more, 95% or more, 98% or more, 99% or more, or 100% nucleotide sequence identity to SEQ ID NO: 7. The expression-inducing activity of the nitrite reductase promoter is increased in a medium containing nitrate ions as the sole nitrogen source, but is suppressed in a medium containing ammonium ions as the sole nitrogen source.

[0024] <Method for Promoting Algal Growth Under High CO2 Conditions> In one embodiment, a method for promoting algal growth under high CO2 conditions is provided, comprising placing recombinant algae described in the <Recombinant Algae> section under conditions in which the inducible promoter is induced under the high CO2 conditions. In this context, "promoting growth" can mean improving the growth rate compared to corresponding algae lacking expression of the protein or fusion protein (e.g., lacking expression induction or lacking the recombinant gene) but under the same growth conditions. Growth rate can be measured, for example, based on the weight of algal biomass grown up to 6, 9, or 13 days after initiation of culture under high CO2 conditions and promoter-inducing conditions. In an embodiment, the inducible promoter can be a nitrate-inducible promoter, such as a nitrate-inducible promoter comprising a nucleotide sequence having 90% or greater sequence identity to SEQ ID NO:7.

[0025] In this disclosure, "high CO2 conditions" refers to conditions in which the environment is exposed to CO2 concentrations exceeding atmospheric CO2 concentrations. The CO2 concentration under high CO2 conditions may be a CO2 concentration exceeding 278 ppm, which is the pre-industrial atmospheric average concentration (Chapter 2 of the Intergovernmental Panel on Climate Change (IPCC) Sixth Assessment Report, Part I Working Group Report (2021)), preferably exceeding 400 ppm, and more preferably exceeding the global average concentration as of 2022 of 417.9 ppm (World Data Centre for Greenhouse Gases, https: / / gaw.kishou.go.jp / jp). The CO2 concentration (v / v) under high CO2 conditions may preferably be 0.5% or more, 1% or more, 2% or more, or 5% or more. For example, the ability to promote the growth of recombinant algae can be confirmed by comparing the growth rate under high CO2 conditions, in which the algae are cultured under aeration with air at a CO2 concentration of 2%, with that of corresponding algae that lack expression of the protein or fusion protein (e.g., lack expression induction or lack the recombinant gene) but are otherwise grown under the same conditions. The CO2 concentration used for high CO2 conditions can be 100% or less, for example, 50% or less, 20% or less, or 10% or less. Cultivation under high CO2 conditions can be performed by aerating the medium with a CO2-containing gas having the above concentration (e.g., exhaust gas from industrial facilities, incineration facilities, engines, etc., or a gas diluted with air) using methods known to those skilled in the art. Aeration typically involves exposing the liquid medium to the gas by supplying the gas below the surface of the liquid medium.

[0026] In the embodiment, the medium for culturing algae can be any aqueous liquid capable of growing natural algae, or an aqueous algae culture medium with a defined composition can be used. The medium can be any medium for culturing algae known to those skilled in the art, such as a medium containing nutrients, carbon sources, rare metals, etc. Specific examples of the aqueous medium include Koren-Hutner (KH) medium, Cramer-Myers (CM) medium, modified Allen's (MA)2 medium (Ohmura et al., Plant and Cell Physiology, Volume 49, Issue 1, January 2008, Pages 117-120), and mixtures thereof. MA2 medium (pH 2.5) is composed of 40 mM (NH4)2SO4, 4 mM MgSO4, 8 mM KH2PO4, 1 mM CaCl2·2H2O, 0.1 mM FeCl3, 0.075 mM EDTA·2Na, 5.7 g / L H2BO3, 3.6 g / L MnCl2·4H2O, 0.210 g / L ZnCl2, 0.78 g / L Na2MoO4·2H2O, 0.08 g / L CoCl2·6H2O, 0.086 g / L CuCl2, and the remainder is water. When MA2 medium is used directly for algae cultivation, the activity of the nitrite reductase promoter is repressed. Furthermore, by replacing 40 mM (NH4)2SO4 in MA2 medium with 5 mM NaNO3 and 20 mM Na2SO4, nitrate ions can be used as the only nitrogen source, and under these conditions, the nitrite reductase promoter is activated (Imamura et al. Plant Cell Physiol. 51(5): 707-717 (2010)). The algae of the present disclosure may be cultured under natural and / or artificial light.

[0027] The conditions under which an inducible promoter of an embodiment is induced are conditions under which the expression of a gene linked to the inducible promoter is initiated or enhanced by the inducible promoter, and will be understood by those skilled in the art depending on the type of inducible promoter. For example, when a nitrate-inducible promoter is used as the inducible promoter, the conditions may be culture conditions in a medium containing nitrate as the sole nitrogen source, or when a heat shock promoter is used as the inducible promoter, the conditions may be culture conditions in a medium heated with nitrate. The nitrite reductase promoter is preferably used as the nitrate-inducible promoter. A modified MA2 medium in which (NH)SO is replaced with NaNO and NaSO can be used as a medium containing nitrate as the sole nitrogen source.

[0028] <Method for Producing an Algal Culture> In one embodiment, a method for producing an algal culture is provided, comprising culturing the recombinant algae described in the <Recombinant Algae> section under elevated CO2 conditions. Elements of the method for producing an algal culture of the embodiment (e.g., recombinant algae, driving of recombinant genes, elevated CO2 conditions, algae cultivation) can be those described in the <Recombinant Algae> and <Method for Promoting Algal Growth Under High CO2 Conditions> sections.

[0029] The recombinant algae containing recombinant genes, the method for promoting algal growth under high CO2 conditions, and the method for producing algal cultures disclosed herein can further enhance the promotion of algal growth under high CO2 conditions, and can be widely applied to improving CO2 absorption (fixation amount) by algae and productivity of useful substances.

[0030] Examples of the present disclosure are described below, but the present disclosure is not limited to the examples described below.

[0031] <Materials and Methods> The unicellular red alga Cyanidioschyzon merolae was grown in liquid MA2 medium (pH 2.5) aerated with 2% (v / v) CO2 under continuous white light (50 μmol m -1 sec -1) and cultured at 40°C. Transformants were selected by transforming a URA5.3-deficient strain (C. merolae T1 strain) maintained in MA2 medium supplemented with uracil and 5-fluoroorotic acid (0.5 mg / ml final concentration) with a vector expressing URA5.3 and culturing the transformed strain in MA2 medium without uracil or 5-fluoroorotic acid (Taki et al., 2015, J. Gen. Appl. Microbiol., 61, 211-214; Imamura et al., 2013, Biochem. Biophys. Res. Commun., 439, 264-269). Algal growth was monitored by measuring the optical density at 750 nm (OD 750 Activation of the nitrite reductase promoter was achieved by replacing 40 mM (NH4)2SO4 in MA2 medium with 5 mM NaNO3 and 20 mM Na2SO4.

[0032] The vector (pNITE-CTSP-CMV015C) used in this example was constructed by a vector construction method using DNA recombination technology and polymerase chain reaction (PCR) known to those skilled in the art, using the nucleic acid sequences corresponding to CMV015C, CTSP, and pNITE, which are SEQ ID NOs: 3, 6, and 8, respectively.

[0033] Recombinant algae were produced by transforming wild-type algae with pNITE and pNITE-CTSP-CMV015C using the PEG method (Imamura et al., 2013, Biochem. Biophys. Res. Commun., 439, 264-269).

[0034] <Results and Discussion> In a previous study, we identified CMV015C as a candidate gene that could improve algal growth under elevated CO2 conditions. CMV015C was a gene encoded by chloroplast DNA. To demonstrate the effect of CMV015C expression on algal growth, we added the coding sequence for the chloroplast targeting signal peptide (CTSP) in frame to the 5' end of CMV015C and cloned it downstream of the nitrite reductase promoter in the expression vector pNITE to construct an expression vector (pNITE-CTSP-CMV015C). The unicellular red alga Schizon spp. was transformed with pNITE-CTSP-CMV015C and pNITE (control) and used in the following culture experiments.

[0035] In all of the following experiments, the algae were first pre-cultured in MA2 medium containing ammonium ions as the sole nitrogen source. The pre-cultured algae were collected by centrifugation (3,500 rpm, 5 min) and the initial OD 750 MA2 or modified MA2 medium was added so that the β-amylindrical ratio was 0.1. The culture medium was aerated with air containing 2% (v / v) CO2, and the culture medium was stirred.

[0036] Comparative Example 1: Growth of the red alga Schizon transformed with pNITE-CTSP-CMV015C and pNITE in a medium containing ammonium ions as the sole nitrogen source. Unicellular red alga Schizon transformed with pNITE-CTSP-CMV015C and pNITE were cultured in MA2 medium containing ammonium ions as the sole nitrogen source. Under these conditions, ammonium served as the sole nitrogen source, repressing the nitrite reductase promoter and, therefore, expression of the CTSP-CMV015C fusion protein from pNITE-CTSP-CMV015C. No difference in growth rate was observed between the two strains (Figure 1).

[0037] Example 1: Growth of Red Algal Schizon Transformed with pNITE-CTSP-CMV015C and pNITE in Medium with Nitrate as the Sole Nitrogen Source. Unicellular red algal Schizon transformed with pNITE-CTSP-CMV015C and pNITE were cultured in modified MA2 medium, in which 40 mM (NH)SO was replaced with 5 mM NaNO and 20 mM NaSO. Under these conditions, nitrate became the sole nitrogen source, activating the nitrite reductase promoter and thus inducing expression of the CTSP-CMV015C fusion protein from pNITE-CTSP-CMV015C. Red algal Schizon transformed with pNITE-CTSP-CMV015C grew faster and reached higher density cultures than schizont transformed with pNITE (Figure 2).

[0038] The above results demonstrate that expression of the chloroplast-targeted CMV015C polypeptide in algal cells promotes algal growth under high CO2 conditions.

[0039] The present disclosure includes the following embodiments: (Item 1) A recombinant alga comprising a recombinant gene operably linked to a promoter, the recombinant gene encoding a protein comprising a polypeptide having 90% or more amino acid sequence identity to SEQ ID NO: 1, wherein the protein, when expressed in the recombinant alga, is at least partially localized in chloroplasts and is capable of producing high CO2. ２Item 2: The recombinant algae of Item 1, wherein the recombinant gene encodes a fusion protein comprising a polypeptide having 90% or more amino acid sequence identity to SEQ ID NO: 1 and a chloroplast targeting signal peptide linked to the polypeptide. Item 3: The recombinant algae of Item 1 or 2, wherein the promoter is an inducible promoter. Item 4: The recombinant algae of any one of Items 1 to 3, wherein the promoter is a nitrate-inducible promoter comprising a nucleotide sequence having 90% or more sequence identity to SEQ ID NO: 7. Item 5: The recombinant algae of any one of Items 1 to 4, which has been transformed with a recombinant vector comprising the sequence of SEQ ID NO: 8, into which (i) a nucleic acid encoding the fusion protein, or (ii) a nucleic acid comprising the nucleotide sequence consisting of SEQ ID NO: 4 has been cloned at positions corresponding to positions 3978-4001 of SEQ ID NO: 8. (Item 6) A method for promoting algal growth under high CO2 conditions, comprising placing the recombinant algae described in any one of Items 3 to 5 under conditions in which the inducible promoter is induced under the high CO2 conditions. (Item 7) The method of item 6, wherein the promoter is a nitrate-inducible promoter comprising a nucleotide sequence having 90% or more sequence identity to SEQ ID NO: 7, and the conditions in which the inducible promoter is induced include culturing in a medium containing nitrate ions as the sole nitrogen source. (Item 8) A method for producing an algal culture, comprising culturing the recombinant algae described in any one of Items 1 to 5 under high CO2 conditions. (Item 9) Recombinant algae comprising a recombinant gene operably linked to a promoter, wherein the recombinant gene encodes a fusion protein comprising a polypeptide having 90% or more amino acid sequence identity to SEQ ID NO: 1 and a chloroplast targeting signal peptide linked to the polypeptide. (Item 10) Recombinant algae transformed with a vector comprising a sequence consisting of SEQ ID NO: 9.(Item 11) Recombinant algae comprising a recombinant gene operably linked to a promoter, wherein the recombinant gene encodes a protein comprising a polypeptide having 90% or more amino acid sequence identity to SEQ ID NO: 1, wherein the protein, when expressed in the recombinant algae, is at least partially localized to chloroplasts. (Item 12) Recombinant algae comprising a recombinant gene operably linked to a promoter, wherein the recombinant gene encodes a protein comprising a polypeptide having 90% or more amino acid sequence identity to SEQ ID NO: 1, wherein the protein, when expressed in the recombinant algae, is at least partially localized to chloroplasts. ２ A recombinant alga, wherein conditions are capable of promoting the growth of said recombinant alga.

[0040] Although the present disclosure has been described with reference to the above several embodiments, the present disclosure is not limited to the above several embodiments. Various changes can be made to the configuration and details of the present invention within the scope of the present disclosure.

Claims

1. A recombinant alga comprising a recombinant gene operably linked to a promoter, wherein the recombinant gene encodes a protein comprising a polypeptide having an amino acid sequence identity of 90% or more with respect to SEQ ID NO: 1, and when the protein is expressed in the recombinant alga, at least a part thereof is localized in chloroplasts and promotes the growth of the recombinant alga under high CO ２ conditions. The recombinant alga.

2. The recombinant algae according to claim 1, wherein the recombinant gene encodes a polypeptide having an amino acid sequence identity of 90% or more with SEQ ID NO: 1 and a fusion protein comprising a chloroplast transit signal peptide linked to the polypeptide.

3. The recombinant algae according to claim 2, wherein the promoter is an inducible promoter.

4. The recombinant algae according to claim 3, wherein the promoter is a nitrate-inducible promoter comprising a nucleotide sequence having a sequence identity of 90% or more with SEQ ID NO:

7.

5. The recombinant algae according to claim 4, which is transformed with a recombinant vector in which a nucleic acid encoding the fusion protein or (ii) a nucleic acid comprising the nucleotide sequence consisting of SEQ ID NO: 4 is cloned at a position corresponding to positions 3978-4001 of SEQ ID NO: 8 in a vector comprising the sequence of SEQ ID NO:

8.

6. A method for promoting the growth of algae under high CO2 conditions, the method comprising placing the recombinant algae according to any one of claims 3 to 5 under the high CO2 conditions and under conditions in which the inducible promoter is induced.

7. The method according to claim 6, wherein the promoter is a nitrate-inducible promoter comprising a nucleotide sequence having a sequence identity of 90% or more with SEQ ID NO: 7, and the conditions under which the inducible promoter is induced include culturing in a medium using nitrate ions as the sole nitrogen source.

8. A method for producing an algal culture, the method comprising culturing the recombinant algae according to claim 1 or 2 under high CO2 conditions.

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