Method for killing alga at desired time

Abstract

The present disclosure provides: a recombinant alga; and a method for killing an alga at a desired time.

Description

A method for killing algae at a desired time

[0001] The present disclosure relates to recombinant algae and methods for killing algae.

[0002] Algae are promising as an environmentally friendly means of producing biofuels and other useful substances. The production of useful substances using algae is typically carried out by mass cultivation in liquid media. After culturing the algae, sterilization is required before disposal of the media and other residues, which requires time and energy. Furthermore, there are concerns that the algae may impact the existing environment and biodiversity when cultivated outdoors.

[0003] Sterilization treatment after algae cultivation is carried out using a sterilization device such as an autoclave or by adding a chlorine bleach containing sodium hypochlorite as the main component. However, autoclave sterilization requires large, dedicated equipment, and sodium hypochlorite is required to be added in large amounts. Furthermore, sodium hypochlorite is an environmentally hazardous chemical substance, which is problematic (Non-Patent Document 1). These problems become particularly pronounced in large-scale cultivation. Other methods include the use of antibiotics (Non-Patent Documents 2 and 3) or herbicides (Non-Patent Document 4) that act on algae, but these methods have the problem of being very expensive.

[0004] Inactivation of Bacteria by Hypochlorous Acid Water, Bulletin of the Institute of Building and Equipment Engineering No. 34, March 2011, p. 3-11; Fujiwara et al., Development of a Double Nuclear Gene-Targeting Method by Two-Step Transformation Based on a Newly Established Chloramphenicol-Selection System in the Red Alga Cyanidioschyzon merolae, Front. Plant Sci. 8:343. doi: 10.3389 / fpls.2017.00343; Zhang, et al., Genetic Transformation of Tribonema minus, a Eukaryotic Filamentous Oleaginous Yellow-Green Alga, Int. J. Mol. Sci. 2020, 21(6), 2106; https: / / doi.org / 10.3390 / ijms21062106; Shirakawa et al., Studies on a Herbicide, 2-(α-naphtoxy)-N,N-diethylpropionamide (R-7465) (2) The mixing effect of R-7465 with simetryne, Weed Research Vol. 20, 1975, p34-38

[0005] The present disclosure has been made to solve the above-mentioned problems, and aims to provide recombinant algae and a method for killing algae, in particular a method for killing algae at a desired timing.

[0006] In one aspect, recombinant algae are provided, comprising a recombinant gene operably linked to an inducible promoter, wherein a polypeptide encoded by said recombinant gene has at least 90% amino acid sequence identity to SEQ ID NO:1 or SEQ ID NO:2, and when induced by said inducible promoter, inhibits survival or growth of said algae.

[0007] In one embodiment, recombinant algae are provided that are transformed with a vector comprising a sequence consisting of SEQ ID NO:7 or SEQ ID NO:8.

[0008] According to the present disclosure, recombinant algae and methods for killing algae, particularly methods for killing algae at a desired time, can be provided.

[0009] Growth curves of the unicellular red algae schizonts transformed with pNITE-CMJ216C and pNITE in a medium containing ammonia as the sole nitrogen source (A), and photographs of the culture broth on day 9 of cultivation (B). Growth curves of the unicellular red algae schizonts transformed with pNITE-CMJ216C and pNITE in a medium containing ammonia as the sole nitrogen source (A), and photographs of the culture broth on day 9 of cultivation (B). Growth curves of the unicellular red algae schizonts transformed with pNITE-CMJ216C and pNITE in a medium containing nitrate as the sole nitrogen source (A), and photographs of the culture broth on day 9 of cultivation (B). Growth curves of the unicellular red algae schizonts transformed with pNITE-CMJ216C and pNITE in a medium containing nitrate as the sole nitrogen source (A), and photographs of the culture broth on day 9 of cultivation (B).

[0010] Recombinant Algae In one embodiment, recombinant algae are provided, comprising a recombinant gene operably linked to an inducible promoter, wherein the polypeptide encoded by the recombinant gene has at least 90% amino acid sequence identity to SEQ ID NO:1 or SEQ ID NO:2, and when induced by the inducible promoter, inhibits survival or growth of the algae.

[0011] 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.

[0012] 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.

[0013] In this disclosure, recombinant algae refers to algae containing an exogenous recombinant gene. The exogenous recombinant gene may contain nucleic acid of the algal 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 may contain nucleic acid derived from a heterologous organism. The recombinant gene of this embodiment encodes a polypeptide having at least 90% amino acid sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2. SEQ ID NOs: 1 and 2 are polypeptide sequences encoded by the genes CMJ216C and CMF138C, respectively, of Cyanidioschyzon merolae. The amino acid sequence identity of a polypeptide having at least 90% amino acid sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 may be at least 95%, at least 98%, at least 99%, or even 100%. The nucleotide sequences of SEQ ID NOs: 3 and 4, which encode the polypeptide sequences corresponding to SEQ ID NOs: 1 and 2, respectively, can be used to construct the recombinant gene.

[0014] The recombinant algae of the present disclosure include a recombinant gene operably linked to an inducible promoter. A promoter, as used herein, can refer to a regulatory DNA sequence that can drive the transcription or expression of a gene linked thereto, either directly or via another sequence, in an individual or cell of the algae of the present disclosure. As will be understood by those skilled in the art, the term "operably linked" as used herein can 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.

[0015] 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: 5, or a nitrate-inducible promoter having at least 90%, at least 95%, at least 98%, at least 99%, or 100% nucleotide sequence identity to SEQ ID NO: 5. 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.

[0016] The polypeptide encoded by the recombinant gene of this embodiment is a polypeptide that inhibits the survival or growth of the algae when induced by the inducible promoter. In the present disclosure, "inhibiting the survival or growth of algae" means killing the algae or making their growth unobservable for at least two weeks. When the algae are killed, they typically lose their green appearance and become bleached or transparent. Such changes in color or appearance can be determined by observing the algae culture with the naked eye or a microscope. The algae growth level, which can be used to determine the inhibition of algae growth, can be monitored, for example, by measuring the absorbance of the culture medium at 750 nm.

[0017] 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: 6) (Imamura et al., The Plant Journal (2018) 94, 327-339). pNITE contains a nitrite reductase promoter (SEQ ID NO: 5) that induces expression of a gene cloned into the multicloning site (corresponding to positions 3978-4001 of SEQ ID NO: 6) in algae cultured in an environment containing nitrate as the sole nitrogen source.

[0018] In embodiments, the recombinant gene may be provided in the form of a recombinant vector into which a nucleic acid encoding a polypeptide having at least 90% amino acid sequence identity to SEQ ID NO:1 or SEQ ID NO:2, or a nucleic acid comprising the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4, has been cloned. As a more specific example, the recombinant gene of the present disclosure may be provided in the form of a vector (pNITE-CMF216C) comprising the sequence of SEQ ID NO:7, in which a sequence consisting of a gene encoding CMF216C (SEQ ID NO:3) has been cloned into the pNITE vector backbone. Alternatively, the recombinant gene of the present disclosure may be provided in the form of a vector (pNITE-CMF138C) comprising the sequence of SEQ ID NO:8, in which a sequence consisting of a gene encoding CMF138C (SEQ ID NO:4) has been cloned into the pNITE vector backbone. The recombinant algae of the embodiments can be produced by transforming algae with these recombinant genes.

[0019] Recombinant algae of the present disclosure may be produced by transformation. Transformation in the present disclosure 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. Transformation in 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 URA5.3, 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).

[0020] Methods for Killing Algae at Desired Times In one embodiment, methods for killing algae at desired times are provided, comprising subjecting recombinant algae described in the Recombinant Algae section to conditions in which the inducible promoter is induced at the desired times.

[0021] The conditions under which the inducible promoter of this embodiment is induced are conditions under which the expression of the gene to which it is linked 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, these conditions can 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, they can be culture conditions under which the medium is heated. 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.

[0022] In embodiments, the medium for culturing algae can be any aqueous liquid capable of growing natural algae, or a defined aqueous algae medium can be used. The medium can be any medium for algae culture known to those skilled in the art, including, for example, a medium containing nutrients, carbon sources, rare metals, etc. Specific examples of aqueous media 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 contains 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, and 3.6 g / L MnCl. 2・The MA2 medium (pH 2.5) consists of 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 water. When used directly for algal culture, MA2 medium suppresses nitrite reductase promoter activity. Furthermore, by replacing the 40 mM (NH4)2SO4 in MA2 medium with 5 mM NaNO3 and 20 mM Na2SO4, nitrate ions can be used as the sole 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.

[0023] In this embodiment, "placing at the desired time" can include placing the algae under conditions in which the inducible promoter is induced at the desired time when the algae begin to die. Placing at the desired time when the inducible promoter is induced can include starting culture in a medium, such as a medium containing nitrate as the sole nitrogen source, in which a nitrate-inducible promoter, such as a nitrite reductase promoter, is activated. This can be achieved, for example, by transferring algae in a medium or medium that does not contain nitrate as the sole nitrogen source to a medium containing nitrate as the sole nitrogen source, for example, by exchanging the medium or medium. Alternatively, algae can be cultured in a medium containing ammonia and nitrate as nitrogen sources, and the "desired time" can be determined as the time when ammonia is depleted. In such cases, ammonia in the medium is typically preferentially consumed as a nitrogen source by the algae, resulting in the activation of the nitrate-inducible promoter, thereby allowing the algae to die when ammonia is depleted.

[0024] <Method of Killing Algae> In one embodiment, a method of killing algae is provided, comprising culturing recombinant algae described in the <Recombinant Algae> section in a medium containing nitrate as the sole nitrogen source, or in a medium containing ammonia and nitrate as nitrogen sources. Elements of the embodiment methods (e.g., recombinant algae, recombinant genes and their induction conditions, algae cultivation, etc.) can be those described in the <Recombinant Algae> and <Method of Killing Algae at Desired Times> sections. Killing the algae can include halting the growth of the algae culture.

[0025] The recombinant algae, the method for killing algae, and the method for killing algae at a desired time disclosed herein can kill only the targeted algae by simply increasing the expression of genes contained in the algae themselves, thereby providing a method for killing algae that is low in cost and environmental impact.

[0026] Examples of the present disclosure are described below, but the present disclosure is not limited to the examples described below.

[0027] <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 750Activation of the nitrite reductase promoter was achieved by replacing 40 mM (NH4)2SO4 in MA2 medium with 5 mM NaNO3 and 20 mM Na2SO4.

[0028] The vectors used in this example (pNITE-CMJ216C and pNITE-CMF138C) were constructed using nucleic acid sequences corresponding to CMJ216C, CMF138C, and pNITE, which are SEQ ID NOs: 3, 4, and 6, respectively, by a vector construction method using DNA recombination technology and polymerase chain reaction (PCR) known to those skilled in the art.

[0029] Recombinant algae were produced by transforming the wild-type algae with pNITE-CMJ216C, pNITE-CMF138C, and pNITE using the PEG method (Imamura et al., 2013, Biochem. Biophys. Res. Commun., 439, 264-269).

[0030] <Results and Discussion> In previous studies, the inventors identified CMJ216C and CMF138C as candidate genes whose increased expression could kill algae. To demonstrate the effect of CMJ216C and CMF138C expression on algal growth, the coding sequences of these genes were cloned (i.e., operably linked) downstream of the nitrite reductase promoter in the expression vector pNITE to construct expression vectors (pNITE-CMJ216C and pNITE-CMF138C). pNITE-CMJ216C, pNITE-CMF138C, and pNITE (control) were transformed into the unicellular red alga Schizon truncatula and used in the following culture experiments.

[0031] 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.

[0032] Reference Example 1: Growth of the red alga Schizon transformed with pNITE-CMJ216C and pNITE in a medium containing ammonium ions as the sole nitrogen source. Unicellular red alga Schizon transformed with pNITE-CMJ216C 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 CMJ216C protein from pNITE-CMJ216C. Growth was observed in both strains (Figure 1A). Cultures of both strains also exhibited a normal green appearance (Figure 1B).

[0033] Reference Example 2: Growth of the red alga Schizon transformed with pNITE-CMF138C and pNITE in a medium containing ammonium ions as the sole nitrogen source. Unicellular red alga Schizon transformed with pNITE-CMF138C 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, CMF138C protein expression from pNITE-CMF138C. Growth was observed in both strains (Figure 2A). Furthermore, the cultures of both strains exhibited a normal green appearance (Figure 2B).

[0034] Example 1 Growth of red algae schizonts transformed with pNITE-CMJ216C and pNITE in a medium containing nitrate as the sole nitrogen source Unicellular red algae schizonts transformed with pNITE-CMJ216C and pNITE were cultured in modified MA2 medium in which 40 mM (NH4)2SO4 was replaced with 5 mM NaNO3 and 20 mM Na2SO4. Under these conditions, nitrate became the sole nitrogen source, activating the nitrite reductase promoter and thus inducing expression of the CMJ216C protein from pNITE-CMJ216C. Red algae schizonts transformed with pNITE-CMJ216C had a higher OD than red algae schizonts transformed with pNITE. 750The cells of the red alga Schizon transformed with pNITE-CMJ216C were found to have turned bleached and died.

[0035] Example 2 Growth of red algae schizonts transformed with pNITE-CMF138C and pNITE in a medium containing nitrate as the sole nitrogen source Unicellular red algae schizonts transformed with pNITE-CMF138C and pNITE were cultured in modified MA2 medium in which 40 mM (NH4)2SO4 was replaced with 5 mM NaNO3 and 20 mM Na2SO4. Under these conditions, nitrate became the sole nitrogen source, activating the nitrite reductase promoter and thus inducing expression of the CMF138C protein from pNITE-CMF138C. The red algae schizonts transformed with pNITE-CMF138C had a higher OD than the red algae schizonts transformed with pNITE. 750 The cells of the red alga Schizon transformed with pNITE-CMF138C were found to have turned bleached and died.

[0036] The above results demonstrate that expression of the CMJ216C polypeptide or the CMF138C polypeptide in algal cells stops the growth of the algae and causes them to die.

[0037] The present disclosure includes the following embodiments: (Item 1) Recombinant algae comprising a recombinant gene operably linked to an inducible promoter, wherein a polypeptide encoded by the recombinant gene has at least 90% amino acid sequence identity to SEQ ID NO:1 or SEQ ID NO:2 and inhibits survival or growth of the algae when induced by the inducible promoter. (Item 2) The recombinant algae of Item 1, wherein the inducible promoter is a promoter for nitrite reductase. (Item 3) The recombinant algae of Item 1 or 2, wherein the inducible promoter is a nitrite-inducible promoter comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:5. (Item 4) The recombinant algae of any one of Items 1 to 3, transformed with a recombinant vector comprising the sequence of SEQ ID NO:6, into which (i) a nucleic acid encoding the polypeptide, or (ii) a nucleic acid comprising the nucleotide sequence consisting of SEQ ID NO:3 or SEQ ID NO:4 has been cloned at a position corresponding to positions 3978-4001 of SEQ ID NO:6. (Item 5) Recombinant algae transformed with a vector comprising the sequence of SEQ ID NO:7 or SEQ ID NO:8. (Item 6) A method for killing algae at a desired time, comprising placing the recombinant algae of any one of Items 1 to 5 under conditions under which the inducible promoter is induced at the desired time. (Item 7) The method of Item 6, wherein the recombinant algae is the recombinant algae of any one of Items 1 to 5, and the conditions under which the inducible promoter is induced include culturing in a medium containing nitrate as the sole nitrogen source. (Item 8) A method for killing algae, comprising culturing the recombinant algae of any one of Items 1 to 5 in (i) a medium containing nitrate as the sole nitrogen source, or (ii) a medium containing ammonia and nitrate as nitrogen sources.

[0038] 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 an inducible promoter, wherein the polypeptide encoded by the recombinant gene has at least 90% amino acid sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 and inhibits the survival or growth of the alga when induced by the inducible promoter.

2. The recombinant alga according to claim 1, wherein the inducible promoter is a promoter of nitrite reductase.

3. The recombinant alga according to claim 1, wherein the inducible promoter is a nitrate-inducible promoter comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:

5.

4. The recombinant alga according to claim 1, which is transformed by a recombinant vector in which a nucleic acid encoding the polypeptide or a nucleic acid comprising a nucleotide sequence consisting of SEQ ID NO: 3 or SEQ ID NO: 4 is cloned at a position corresponding to positions 3978-4001 of SEQ ID NO: 6 in a vector comprising the sequence of SEQ ID NO:

6.

5. A recombinant alga transformed by a vector comprising a sequence consisting of SEQ ID NO: 7 or SEQ ID NO:

8.

6. A method for killing an alga at a desired timing, the method comprising placing the recombinant alga according to any one of claims 1 to 4 under conditions in which the inducible promoter is induced at the desired timing.

7. The method according to claim 6, wherein the inducible promoter is a nitrate-inducible promoter comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 5, and the conditions for inducing the inducible promoter include culturing in a medium having nitrate as the sole nitrogen source.

8. A method for killing an alga, the method comprising culturing the recombinant alga according to claim 3 (i) in a medium containing nitrate as the sole nitrogen source or (ii) in a medium containing ammonia and nitrate as nitrogen sources.

Images