JP2017012135A - Vector, genetically engineered marine diatom, and method for producing fucoxanthin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a vector that improves production ability of fucoxanthin, a genetically engineered marine diatom with improved production of fucoxanthin, and a method for producing fucoxanthin.SOLUTION: The vector comprises: a PtfcpA (Phaeodactylum tricornutum fucoxanthin chlorophyll a/c-binding protein A gene) promoter derived from a marine diatom; and a gene encoding a phytoene synthase derived from the marine diatom on the downstream side of this promoter. The vector further comprises a gene for antibiotic selection and a gene of high sensitivity green fluorescent protein at the C-terminal side of the gene encoding the phytoene synthase. This vector is a genetically engineered marine diatom for producing fucoxanthin incorporated in the marine diatom as a host.SELECTED DRAWING: Figure 4

Description

  The present invention relates to a vector for improving fucoxanthin-producing ability in marine diatoms, a genetically modified marine diatom, and a method for producing fucoxanthin.

Marine diatoms are considered to be promising producers of useful substances for the future because they are a group of organisms that grow continuously using solar energy and seawater, which can be said to be inexhaustible, and have a high growth rate.
As a substance contained in marine diatoms, fucoxanthin, a kind of carotenoid, has an antioxidant effect (Non-patent Document 1), an anti-obesity action / anti-diabetic action (Non-Patent Documents 2 and 3), an anti-cancer action ( Non-patent literature 4) has been reported and has been attracting attention in recent years.

Regarding anti-obesity effects, there was a report that humans (obese white women) significantly reduced body fat and body weight when taking fucoxanthin 2.4 mg daily for 16 days compared to placebo (control). Yes (Diabet Obesity Met 12: 72-81, 2010), fucoxanthin is a highly useful substance that exerts its effect even in trace amounts.
Fucoxanthin is known to be produced by brown algae including diatoms, wakame and kombu, but its content is only about 0.1% per dry weight.
Thus, since the production amount of fucoxanthin by the wild strain is small, the application of fucoxanthin has not been realized yet.
Moreover, unlike other carotenoids such as astaxanthin supplied by chemical synthesis (for example, Patent Document 1), fucoxanthin has a complex molecular structure, and thus examples of successful chemical synthesis are known. Not. In addition, there are no known examples of successful supply and yield improvement by genetic recombination.

Sachindra et al., 2007, Radical scavenging and singlet oxygen quenching activity of marine carotenoid fucoxanthin and its metabolites. J. Agric. Food Chem. 55 (21): 8516-8522. Hosokawa et al., 2010, Fucoxanthin regulates adipocytokine mRNA expression in white adipose tissue of diabetic / obese KK-Ay mice. Arch. Biochem. Biophys. 504 (1): 17-25. Woo et al., 2010, Fucoxanthin supplementation improves plasma and hepatic lipid metabolism and blood glucose concentration in high-fat fed C57BL / 6N mice. Chem. Biol. Interact. 186: 316-322. Mikami and Hosokawa, 2013, Biosynthetic pathway and health benefits of fucoxanthin, an algae-specific xanthophyll in brown seaweeds. Int. J. Mol. Sci. 14 (7): 13763-13781.

JP 2010-172293 A (paragraph 0003)

  The present invention has been made in view of the above problems, and an object of the present invention is to generate a vector that improves fucoxanthin production ability, a genetically modified marine diatom with improved fucoxanthin production ability, and fucoxanthin production. It is to provide a method.

The present inventors tried to introduce a gene into marine diatoms having fucoxanthin-producing ability using various promoters, but many promoters did not improve fucoxanthin-producing ability. However, a gene encoding a phytoene synthase derived from the marine diatom was introduced into the marine diatom using the endogenous PtfcpA ( Phafactylum tricornutum fucoxanthin chlorophyll a / c-binding protein A gene) promoter derived from the marine diatom. Surprisingly, it has been found that the fucoxanthin-producing ability of the marine diatom is improved, and the present invention has been completed.
That is, according to the present invention, the object is to provide a PtfcpA ( Phafactylum tricornutum fucoxanthin chlorophyll a / c-binding protein A gene) promoter derived from a marine diatom and a phytoene synthase derived from the marine diatom on the downstream side of the promoter. It is solved by a vector having a gene to encode.

Since the vector is configured in this manner, fucoxanthin production ability in marine diatoms by gene transfer can be improved.
Although fucoxanthin is recognized as a useful substance, it has no successful examples of yield improvement by chemical synthesis or genetic recombination, except for extracting trace amounts contained in wild strains of brown algae and diatoms. However, according to the present invention, fucoxanthin, which is a useful substance, can be produced more efficiently than before.

At this time, it is preferable that a gene for antibiotic selection is provided, and a highly sensitive green fluorescent protein gene is provided on the C-terminal side of the gene encoding the phytoene synthase.
The target gene is not necessarily integrated into the genome of all transformant clones selected with antibiotics, and the target gene protein is expressed even in transformant clones that have been confirmed to be integrated. Therefore, transformant clones cannot be sufficiently selected with only antibiotic selection genes. However, in the present invention, an antibiotic selection gene is provided, and the gene encoding the phytoene synthase is selected. Cultivating transformant clones selected with antibiotics because it has a gene for high-sensitivity green fluorescent protein on the C-terminal side, and selecting clones having fluorescence derived from high-sensitivity green fluorescent protein using a fluorescence microscope Thus, a transformant clone expressing the protein can be easily selected.

Moreover, the said vector is good in the genetically modified marine diatom for producing | generating fucoxanthin integrated in the said marine diatom as a host.
Thus, since the vector which can improve the fucoxanthin production ability in marine diatoms is incorporated, a genetically modified marine diatom with enhanced fucoxanthin production ability can be constructed.

The marine diatom may have fucoxanthin producing ability.
Thus, since the marine diatom having the fucoxanthin-producing ability is used, a genetically modified marine diatom having an enhanced fucoxanthin-producing ability can be constructed.
The marine diatom may be Phaeodactylum tricornutum Bohlin, which is a marine flora .

A method for producing fucoxanthin comprising the step of extracting the fucoxanthin from a medium produced by culturing the genetically modified marine diatom in a medium is preferable.
By producing fucoxanthin using the genetically modified marine diatom of the present invention, fucoxanthin can be efficiently produced with higher productivity than before, and the time and cost required for production of fucoxanthin are reduced. Can be

According to the present invention, it is possible to improve fucoxanthin-producing ability in marine diatoms by gene transfer.
Although fucoxanthin is recognized as a useful substance, it has no successful chemical synthesis or genetic recombination, and production methods other than extraction of trace amounts contained in wild strains of brown algae and diatoms However, according to the present invention, fucoxanthin, which is a useful substance, can be produced more efficiently than before.

It is a flowchart which shows the biosynthetic pathway of fucoxanthin expected about diatom, (A) shows a MEP pathway, (B) shows the anticipated carotenoid biosynthetic pathway. P. tricornutum is a flow diagram showing the connection and isolation from the PSY gene, (A), the amplification and purification of Ptpsy fragment, (B), the coupling of PCR Ptpsy fragments and egfp fragment, (C) is, is an explanatory diagram showing the purification joining gene (Ptpsy :: egfp) fragments Ptpsy and egfp. Ptpsy fragment is a photograph showing the results of agarose gel electrophoretic analysis of DNA fragments joining gene Ptpsy :: egfp of egfp fragment and Ptpsy and egfp. It is a figure which shows the structure of the transformation vector constructed | assembled in the Example. It is a typical photograph of the wild type strain | stump | stock using a confocal laser microscope, and the transformant clone of an Example. It is a figure which shows the position of primer PtfcpAPro- (attB1) F and primer EGFP (qPCR) -R which were used for genomic PCR. It is a photograph which shows the electrophoresis result of a transformant clone. It is a graph which shows the result of the statistical analysis of the carotenoid total amount of a wild strain and a transformant clone, Comprising: (A) is the total amount of carotenoid per cell, (B) is the total amount of carotenoid per culture solution. It is a graph which shows the result of the analysis of the carotenoid component using HPLC in a wild strain and a transformant clone, and (A) shows the result of the wild strain (WT), and (B) shows the transformant clone (# 2). -12). 2 is a graph showing the results of analysis of fucoxanthin content and β-carotene content of wild strains and transformant clones using HPLC, wherein (A) is the amount of β-carotene per cell, and (B) is the culture solution. Β-carotene per unit, (C) is fucoxanthin per cell, and (D) is fucoxanthin per culture.

<Gene-modified diatom>
Hereinafter, the genetically modified diatom of the present invention will be described.
The genetically modified diatom of the present invention is a genetically modified diatom for producing fucoxanthin, in which a gene encoding an enzyme derived from the same marine diatom strain as the host is introduced into a marine diatom serving as a host.
Fucoxanthin is a kind of carotenoid of non-provitamin A and is a component belonging to xanthophyll. In recent years, fucoxanthin has been reported to have functions such as anti-obesity / anti-diabetic action, anti-cancer action, in-vivo antioxidant action, anti-angiogenic action, and anti-inflammatory action.

As a marine diatom serving as a host, a marine diatom having an ability to produce fucoxanthin can be used.
For example, Phaeodactylum tricornutum Trichoderma Lunar Tam (Phaeodactylum tricornutum) unicellular diatoms belonging to Phaeodactylum tricornutum genus such as (Phaeodactylum), and unicellular diatoms belonging to Thalassiosira Shudonana (Thalassiosira pseudonana) Thalassiosira genus, such as (Thalassiosira), Shikurotera Kuriputika (Cyclotella cryptica), Shikurotera Meneginiana (Cyclotella meneghiniana), Sukerutonema Kosutatsumu (Skeletonemacostatum), Nitzschia laevis (Nitzschia laevis), include Chaetoceros gracilis (Chaetocerosgracilis).
In the present invention, Phaeodactylum tricornutum Bohlin (National Research Institute of Aquaculture, Fisheries Research Agency, Japan, Strain NRIA-0065) (hereinafter referred to as “ P. tricornutum NRIA-0065 strain”) is used. be able to.

As a gene encoding an enzyme derived from a marine diatom, a phytoene synthase (PSY) gene isolated from a marine diatom, particularly from the same marine diatom strain as the host, can be used.
In the present invention, it is simply derived from Phaeodactylum tricornutum Bohlin (National Research Institute of Aquaculture, Fisheries Research Agency, Japan, Strain NRIA-0065) (hereinafter referred to as “ P. tricornutum NRIA-0065 strain”), which is a marine arachnoid diatom. A separated phytoene synthase (PSY) gene, the gene represented by SEQ ID NO: 1 (the amino acid sequence of phytoene synthase (PSY) encoded by the gene is represented by SEQ ID NO: 2) can be used.
The phytoene synthase is an enzyme indicated by PSY in the carotenoid biosynthetic pathway of FIG. 1B, and is an enzyme for biosynthesis reaction from geranylgeranyl diphosphate (C ₂₀ ) to phytoene.

Fig. 1 is a biosynthetic pathway of fucoxanthin expected for the diatom Phaeodactylumtricornutum (Coesel et al., (2008) Evolutionary origins and functions of the carotenoid biosynthetic pathway in marine diatoms. PLoS One 3 (8 ): e2896.), (A) shows the MEP pathway, and (B) shows the expected carotenoid biosynthesis pathway.
The MEP pathway is one of the IPP (isopentenyl diphosphate) biosynthetic pathways, and glyceraldehyde 3-phosphate and pyruvic acid are converted to 2-C-methyl-D-erythritol-4-phosphate ( This pathway biosynthesizes dimethylanil diphosphate (DMAPP) and isopentenyl diphosphate (IPP) via MEP.
In FIG. 1, the dashed box indicates the predicted carotenoid biosynthesis pathway of the central diatom Thalassiosira pseudonana , Bertrand, (2010), Coesel et al., (2008), Dambek et al., (2012), Hemmerlin ( 2013), Lohr et al., (2012), Mikami and Hosokawa, (2013), and Vranova et al., (2013).
The definitions of abbreviations in Fig. 1 are as follows: DXS: 1-deoxy-D-xylulose 5-phosphate synthase, DXR: 1-deoxy-D-xylulose 5-phosphate reductoisomerase, MCT: 2 C- methyl-D -erythritol 4-phosphate cytidyltransferase, CMK: 4-diphosphocytidyl-2 C -methyl-D-erythritol kinase, MDS: 2 C -methyl-D-erythritol 2,4-cyclodiphosphate synthase, HDS: 1-hydroxy-2-methyl- 2- ( E ) -butenyl 4-diphosphate synthase, HDR: 1-hydroxy-2-methyl-2- ( E ) -butenyl 4-diphosphate reductase, IDI: isopentenyl diphosphate: dimethylallyl diphosphate isomerase, GGDS: geranylgeranyl diphosphate synthase, PSY : phytoene synthase, PDS: phytoene desaturase, Z-ISO: 15- cis -ζ-carotene isomerase, ZDS: ζ-carotene desaturase, CRTISO: carotenoid isomerase, LCYB: lycopene β-cyclase, LUT-like: lutein deficient-like, ZEP: zeaxanthin epoxidase, VDE: violaxanthin de-epoxidase, VDL: violaxanthin de-epoxidase-like, NXS: neoxanthin synthase.

As the phytoene synthase of the present invention, a synthetic enzyme derived from the P. tricornutum NRIA-0065 strain consisting of the amino acid sequence shown in SEQ ID NO: 2, or the amino acid sequence and 70%, preferably 80%, more preferably 90% or more, More preferably 95% or more, particularly preferably 98% or more of a synthase having an amino acid sequence, or one or several amino acids in the amino acid sequence shown in SEQ ID NO: 2 are deleted, substituted, inserted and / or Alternatively, a synthetic enzyme consisting of an added amino acid sequence can be mentioned.

In the present invention, “a gene corresponding to a phytoene synthase-related gene of marine diatoms” refers to a gene functionally equivalent to the above-mentioned synthase-related gene derived from P. tricornutum NRIA-0065 strain.
Examples of the gene corresponding to the phytoene synthase-related gene of marine diatom include any of the following genes (1) to (4).

(1) It consists of a base sequence having 90% or more, preferably 95% or more, more preferably 97% or more, more preferably 99% or more identity with the base sequence represented by SEQ ID NO: 1, and SEQ ID NO: 2. A gene comprising DNA encoding a protein functionally equivalent to a protein comprising the amino acid sequence shown.
In the present invention, a protein functionally equivalent to the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 has substantially the same function as a protein encoded by a phytoene synthase-related gene of marine diatom, and phytoene synthesis Refers to proteins involved in
In the present invention, the identity of the amino acid sequence and base sequence is calculated by the Lipman-Pearson method (Science, 227, 1435, (1985)).

(2) A protein that is hybridized under stringent conditions with a DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 1 and functionally equivalent to a protein comprising an amino acid sequence represented by SEQ ID NO: 2 A gene consisting of DNA encoding.
Here, as the “stringent conditions”, for example, Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W., et al. Russell., Cold Spring Harbor Laboratory Press], for example, 6 × SSC (composition of 1 × SSC: 0.15M sodium chloride, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5 × Examples include conditions in which a solution containing Denhart and 100 mg / mL herring sperm DNA is incubated with a probe at 65 ° C. for 8 to 16 hours and hybridized.

  (3) It consists of an amino acid sequence having 90% or more, preferably 95% or more, more preferably 97% or more, and still more preferably 99% or more identity with the amino acid sequence represented by SEQ ID NO: 2, A gene comprising DNA encoding a protein functionally equivalent to a protein comprising the amino acid sequence shown.

(4) a protein comprising the amino acid sequence represented by SEQ ID NO: 2 consisting of an amino acid sequence in which one to several amino acids are deleted, substituted, inserted and / or added, and comprising the amino acid sequence represented by SEQ ID NO: 2 A gene consisting of DNA encoding a functionally equivalent protein.
Here, 1 to several amino acids are deleted, substituted, inserted and / or added is 1 to 25 amino acids, preferably 1 to 10 amino acids, more preferably 1 to 5 amino acids. Amino acids, more preferably 1 to 2 amino acids. The addition includes addition of 1 to several amino acids at both ends.

  As the “gene encoding phytoene synthase of marine diatom”, a gene corresponding to a phytoene synthase-related gene of marine diatom can be used. Specifically, in the marine diatom having the above-mentioned fucoxanthin-producing ability, Include phytoene synthase homologous genes identified by genome analysis, and these are preferably used in the present invention.

Introduction of a gene corresponding to the phytoene synthase-related gene of marine diatoms, which is such a target protein or target polypeptide, can be performed, for example, by introduction with a vector or insertion into a genome.
When introduced by a vector, a vector containing a gene corresponding to a phytoene synthase-related gene of marine diatoms should be introduced by an appropriate transformation method such as a competent cell transformation method, a protoplast transformation method, or an electroporation method. That's fine. Here, the vector is not particularly limited as long as it is an appropriate carrier nucleic acid molecule for introducing a target gene into a host, allowing it to proliferate and express, and not only a plasmid but also a vector using an artificial chromosome or transposon. , Cosmids.

It is preferable that the vector has a gene for antibiotic selection and has a highly sensitive green fluorescent protein gene on the C-terminal side of the gene encoding phytoene synthase.
That is, the vector of this embodiment is constructed as a transformation vector into which two cassettes for the target gene and the antibiotic selection cassette are incorporated. In order to simplify the selection of transformants in which the transgene is integrated into the genome and the transgene is expressed at the translation level (protein), a high-sensitivity green fluorescent protein EGFP is present at the C-terminus of the target gene. Genes are linked.

In contrast, conventional, in a transformant producing the Phaeodactylum tricornutum, pPha-T1 plasmid (Zaslavskaia, LA, Lippmeier, JC , Kroth, PG, Grossman, AR and Apt, KE (2000) Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) The technology that constructs and uses vectors that incorporate only the target gene into the multicloning site of Journal of Phycology 36, 379-386) is known (Radakovits, R, Eduafo, PM, with a variety of selectable marker and reportergenes. Posewitz, MC (2011) Geneticengineering offattyacidchainlengthin Phaeodactylumtricornutum.Metabolic Engineering 13, 89-95., Yoshinaga, R, Niwa-Kubota, M., Matsui, H., Matsuda, Y. (2014) Characterization of iron-responsive promoters in the marine diatom Phaeodactylum tricornutum. Marine Genomics 16, 55-62).
The pPha-T1 plasmid described in Zaslavskaia et al., (2000) also contains only two cassettes for the target gene and antibiotics.

In the prior art described in these documents, when only the target gene is introduced, transformant clones selected with antibiotics are cultured, genomic DNA is extracted, and the transgene fragment is amplified by PCR. Confirm integration into the genome. In order to examine the expression of the transgene protein, it is necessary to produce an antibody against the transgene protein.
Not all of the transformant clones selected with antibiotics have the target gene integrated into the genome, and even in transformant clones that have been confirmed to be integrated, the protein of the target gene is expressed. Not necessarily.
Therefore, in this embodiment, transformant clones that are expressing proteins are cultured by culturing transformant clones selected with antibiotics and selecting clones having fluorescence derived from EGFP using a fluorescence microscope. It is configured so that it can be easily sorted.

  Further, for example, a method by homologous recombination may be used for insertion into the genome. That is, a DNA fragment in which a part of a chromosomal region that causes introduction into a gene encoding a target protein or polypeptide is combined is introduced into a microbial cell, and homologous recombination occurs in a part of the chromosomal region. Can be integrated into the genome. Here, the chromosomal region causing the introduction is not particularly limited, but a non-essential gene region or a non-gene region upstream of the non-essential gene region is preferable.

By the above method, the genetically modified diatom of the present invention can be constructed.
The genetically modified diatoms of the present invention have improved fucoxanthin productivity compared to marine diatoms, which are wild-type parental microorganisms that have not been modified for the expression of phytoene synthase-related genes.

<Method for producing fucoxanthin using genetically modified marine diatoms>
In the method for producing fucoxanthin of the present invention, a culture step of culturing genetically modified marine diatoms in a medium and an extraction step of extracting fucoxanthin from the medium are performed.
In the culturing step, any medium can be used as long as it is suitable for growing marine diatoms. For example, artificial seawater or seawater to which nutrient salts such as nitrogen and phosphoric acid, vitamins, and trace elements are added can be used. f / 2 medium, BG11 medium, IMK medium, and the like are preferably used.

f / 2 medium is, for example, NaNO ₃ 7.5 mg, NaH ₂ PO ₄・ 2H ₂ O 0.6 mg, vitamin B ₁₂ 0.05 μg, biotin 0.05 μg, thiamine HCl 10 μg, Na ₂ SiO3 ・ 9H ₂ O 1 mg, f / 2 metal (Na ₂ EDTA · 2H ₂ O 440 mg, FeCl ₃ · ₂ O 316 mg, CoSO ₄ · 7H ₂ O 1.2 mg, ZnSO ₄ · 7H ₂ O 2.1 mg, MnCl ₂ · 4H ₂ O 18 mg, CuSO _4. H ₂ O 0.7 mg, Na ₂ MoO ₄ · 2H ₂ O 0.7 mg, distilled water 100 mL) 0.1 mL and seawater 99.9 mL can be prepared. Further, 0.1292 μg of H ₂ SeO ₃ may be added.

BG11 medium is, for example, NaNO ₃ 150 mg, K ₂ HPO ₄・ 3H ₂ O 4 mg, MgSO4 ・ 7H ₂ O 7.5 mg, CaCl ₂・ 2H ₂ O 3.6 mg, citric acid 0.6 mg, ferric ammonium citrate 0.6 mg, Na ₂ EDTA-Mg 0.1 mg, Na ₂ CO ₃ 2 mg, trace metal mixture A ₅ + Co (H ₃ BO ₃ 286 mg, MnCl ₂ · 4H ₂ O 181 mg, ZnSO ₄ · 7H ₂ O 22.2 mg, Na ₂ MoO ₄ · 2H ₂ O 39 mg, CuSO ₄ · 5H ₂ O 7.9 mg, Co (NO ₃ ) ₂ · 6H ₂ O 4.9 mg, distilled water 100 mL) 0.1 mL, distilled water 99.9 mL Then, the pH can be adjusted to 7.4, and 1.5 g of agar is added to 100 mL of the medium to prepare a solid medium.
The IMK medium is prepared, for example, by dissolving 25.2 mg of Daigo IMK powder medium (Nippon Pharmaceutical Co., Ltd.) in 100 mL of seawater. You may autoclave at 121 degreeC for 20 minutes.

The culturing step is preferably performed by a method with improved aeration such as aeration culture, shaking culture, agitation culture, and aeration agitation culture.
Culture is performed under light irradiation conditions, and the irradiation method can be performed by continuous irradiation or photoperiodic irradiation.

  Fucoxanthin is extracted from the algae thus cultured according to a conventional method. Fucoxanthin can be collected by extraction methods known in the art. For example, marine diatoms are collected on a filter such as glass fiber, dried and then extracted with an organic solvent, or cells are crushed by a general method such as a French press or a homogenizer, and then the organic solvent is used. Extraction methods can be applied.

  The solvent for extracting fucoxanthin from marine diatoms is not particularly limited as long as fucoxanthin can be extracted, but methyl acetate, ethyl acetate, acetone, chloroform, toluene and the like can be used. For example, alcohols such as methanol, ethanol, propanol, isopropanol, n-butanol, ketones such as methyl ethyl ketone and acetone, esters such as methyl acetate and ethyl acetate, organochlorine hydrocarbons such as chloroform, and aliphatic carbonization such as hexane Organic solvents such as hydrogen, aromatic hydrocarbons such as benzene and toluene can be used alone or in combination of two or more.

  This organic solvent can be used in a ratio of 1: 100 to 100: 1, preferably 1:50 to 50: 1, with respect to the dried marine diatom. The extraction using the organic solvent can be performed by a conventional method. For example, when ethanol is used as the organic solvent, the temperature is 0 ° C. to 60 ° C., preferably 5 ° C. to 40 ° C., for 0.5 hours to The extraction may be performed for 100 hours, preferably 12 hours to 72 hours. In extraction, if necessary, stirring may be performed with an ultrasonic wave, a stirrer, or the like. Fucoxanthin can be separated and purified by using the organic solvent extract thus obtained as it is or after removing the residue, followed by ion exchange chromatography or reverse phase chromatography using a liquid high performance chromatograph.

  By producing fucoxanthin using the genetically modified marine diatom of the present invention, fucoxanthin can be efficiently produced with higher productivity than before, and the time and cost required for production of fucoxanthin are reduced. Can be

Hereinafter, the present invention will be described with reference to specific examples. However, the present invention is not limited to the following examples.
<Culture of microalgae>
Phaeodactylum tricornutum Bohlin (National Research Institute of Aquaculture, Fisheries Research Agency, Japan, Strain NRIA-0065) (hereinafter referred to as “ P. tricornutum NRIA-0065”) 0065 strain ").
The P. tricornutum NRIA-0065 strain was obtained from an f / 2 liquid medium based on Muroto Deep Sea Water (Guillard, 1975, Culture of phytoplankton for feeding marine invertebrates. In: Culture of Marine Invertebrate Animals (eds Smith, W., Chanley , M., and Guillard, RL). Using Springer US. Pp 29-60.) At 20 ° C, 12 hours light period, 12 hours dark period, under light conditions of about 90 μmol photons / m ² / s Static culture was performed.

<RNA extraction for phytoene synthase gene cloning>
In order to isolate the phytoene synthase (PSY) gene: psy (hereinafter referred to as “ Ptpsy ”) from the P. tricornutum NRIA-0065 strain, the P. tricornutum NRIA-0065 strain in the growth phase and stationary phase RNA was extracted from the resulting cDNA. RNA extraction and DNaseI treatment were performed according to each protocol using RNeasy Plant Mini Kit (QIAGEN Inc., CA) and RNase-Free DNase Set (QIAGEN Inc., CA). The concentration of the extracted RNA was measured using a spectrophotometer DU730 (Beckman Coulter, Inc., CA) and an ultra-trace measurement cuvette nanoVette (Beckman Coulter, Inc., CA). After measuring the concentration, the P. tricornutum NRIA-0065 strain was analyzed with about 100 ng of total RNA using a reverse transcription kit (PrimeScript ^™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Bio Inc., Otsu, Japan). A cDNA was prepared according to the protocol from 1. The obtained cDNA solution was stored at -20 ° C.

<Ploning of PSY gene and ligation of probe gene to 3 ′ end>
Genome analysis and transcriptome analysis (RNA-seq) of P. tricornutum NRIA-0065 strain were performed to obtain a database.
Meanwhile, the genome analysis database of the P. tricornutum CCAP1055 / 1 strain of the Joint Genome Institute (JGI) established by the United States Department of Energy (DOE) (http://genome.jgi-psf.org/Phatr2 /Phatr2.home.html) was searched for PSY gene by keyword search. Based on this PSY gene sequence obtained by searching, BLAST search is performed on the database obtained by genome analysis and transcriptome analysis (RNA-seq) of P. tricornutum NRIA-0065 strain, and PSY candidates The gene sequence and amino acid sequence thereof were obtained. Using ClustalW Multiple aligment of BioEdit Sequence Aligment Editor (www.mbio.ncsu.edu/bioedit/bioedit.html), alignment with PSY amino acid sequences of other organisms was performed, and homology and domain were confirmed. . After confirmation, primers were designed to amplify the coding region of the PSY candidate mRNA sequence. As shown in FIG. 2, an att sequence or a linking sequence described later is added to the primer. The primers used for cloning of the PSY gene are shown in Table 1 and SEQ ID NOs: 3-6.

As shown in FIG. 2 (A), the PSY gene ( Ptpsy ) (SEQ ID NO: 1) derived from the P. tricornutum NRIA-0065 strain and the high-sensitivity green fluorescent protein (enhanced green fluorescent protein) serving as the probe gene An EGFP gene (hereinafter referred to as “ egfp ”) was amplified and purified.
Thereafter, as shown in FIG. 2 (B), Ptpsy has a linker sequence (Gx5 linker) (Ali et al, 2010, Genetic characterization of HIV type 1 Nef-induced vesicle secretion. AIDS Res. Hum. Retroviruses on its 3 terminal side. 26 (2):. 173-192) was ligated to the probe gene egfp through.
As shown in the upper part of FIG. 2 (B), the forward primer (PtPSY- (attB4r) F) of SEQ ID NO: 3 adds the att sequence of the Gateway system (MultiSite Gateway (registered trademark) Pro, Life Technologies Corporation, CA) to the 5 ′ end. a) 3 'end preoccupied removing a sequence of codons, to design reverse primers SEQ ID NO: 4 that addition of a linker sequence linking the egfp (PtPSY- (Gx5) R) , object by PCR from the growth phase of cDNA The DNA fragment was amplified and purified.

Figure 2 (B) as the lower, fragments of egfp linking with Ptpsy, 'forward primer of SEQ ID NO: 5 at the terminal to add the linker sequence (Gx5 linker) (EGFP- (Gx5 ) F) and 3' 5 end It was prepared using the reverse primer (EGFP- (B3r) R) of SEQ ID NO: 6 which adds an att sequence to
Two of Ptpsy fragments and egfp with a linker sequence as a template, PCR was carried out using a forward primer of SEQ ID NO: 3 (PtPSY- (attB4r) F) and reverse primer of SEQ ID NO: 6 (EGFP- (B3r) R) concatenates two DNA fragments, to obtain a joining gene Ptpsy :: egfp of Ptpsy and egfp a PCR product of expected size as shown in FIG. 2 (C).

PCR was performed by using PrimeSTAR (registered trademark) GXL DNA Polymerase (Takara Bio Inc., Otsu, Japan) according to the protocol and making the reaction system 25 μL. Each target DNA fragment was purified after amplification by PCR. The PCR product of the desired size was separated from the reaction solution after PCR by agarose electrophoresis, and the site containing the target DNA fragment was excised.
Figure 2 (A) Ptpsy fragment and egfp fragment and the results of agarose gel electrophoretic analysis of DNA fragments joining gene Ptpsy :: egfp of Ptpsy and egfp of (C) of FIG. 3.
Thereafter, DNA fragments were purified using QIAquick (registered trademark) Gel Extraction Kit (QIAGEN Inc., CA).

<Construction of transformation vector>
Transformation vectors were constructed using the Gateway system (MultiSite Gateway® Pro, Life Technologies Corporation, CA). The constructed transformation vector is shown in FIG.
As a promoter for inducing expression of Ptpsy , the endogenous PtfcpA ( Phafactylum tricornutum fucoxanthin chlorophyll a / c-binding protein A gene) promoter (PtfcpA pro.) Derived from the P. tricornutum UTEX 646 strain was used. As the terminator, a CffcpA terminator of SEQ ID NO: 8 derived from marine diatoms was used. Each entry clone and pCfcp- ble (Poulsen and Kroger, 2005), into which the antibiotic Zeocin ^™ resistance gene Sh ble is incorporated, used a basic destination vector to construct a transformation vector.
Ptpsy is linked to the EGFP gene as a reporter gene via a Gx5 linker.

<Transformation by particle gun method>
Particle gun method using PDS-1000 / He Biolistic system (Biolistic PDS-1000 / He Particle Delivery System, Bio-Rad, CA) against P. tricornutum NRIA-0065 strain in the early stationary phase of less than 2 weeks in culture The transformation was attempted.
According to the protocol of PDS-1000 / He Biolistic system, the transformation vector of FIG. 4 was attached to tungsten particles (Tungsten particle M17, Bio-Rad, CA) having a diameter of 1.1 μm. The host P. tricornutum NRIA-0065 strain to be transformed was adjusted to 1 x 10 ⁸ cells in 100 μL of f / 2 liquid medium, and placed in a plastic petri dish (diameter 9 cm, height 1.5 cm). It was transferred to the center of f / 2 agar medium (0.5% agarose HGS, Nacalai Tesque, Kyoto, Japan) prepared in a volume of 20 mL, and spread to a diameter of about 3 cm using a glass spreader. The conditions for implanting tungsten particles were two 650 psi rupture disks, the He gas pressure condition was set to about 1,300 psi, the distance between the rupture disk and the macro carrier was about 2 cm, the stopping screen and the target. The distance from the P. tricornutum NRIA-0065 strain was about 6 cm. The P. tricornutum NRIA-0065 strain prepared from the same culture immediately before transformation was transformed into 4 petri dishes for each treatment group.

After introducing the gene by the particle gun method, collect the P. tricornutum NRIA-0065 strain cells on the agar medium using 2 mL of the f / 2 liquid medium from the f / 2 agar medium, transfer to a 24-well plate, and 24 hours Was cultured. After culturing for 24 hours, the cells were cultured on an f / 2 agar selective medium (0.5% agarose HGS) containing 500 μg / mL Zeocin ^™ (InvivoGen, CA), and transformants were selected. After 4 weeks of culture, colonies formed on the agar selective medium.
60 clone transformants were obtained on an agar selective medium. The number of positive clones of the transformant in this screening stage is shown in Table 2.

  A colony formed on the agar medium was used as a colony derived from the transformant in subsequent experiments.

<Confirmation and culture of transformants on agar selective medium>
Transformant-derived colonies obtained on f / 2 agar medium were passaged to f / 2 agar selective medium (0.5% agarose HGS) containing 300 μg / mL Zeocin ^™ and 300 for microscopic observation. Algae was planted on a 48-well plate (MICROPLATE 48 Well / Flat Bottom, AGC TECHNO GLASS CO., LTD., Shizuoka, Japan) containing 500 μL of f / 2 liquid medium containing μg / mL Zeocin ^™ .

<Expression analysis of transgene using fluorescence microscope>
In order to confirm the expression of the introduced Ptpsy :: egfp , EGFP fluorescence was used. Transformant clones in a culture system using a 48-well plate were observed using a fluorescence microscope (IX70, Olympus Corporation, Tokyo, Japan). EGFP fluorescence, which is thought to be derived from the transgene ( Ptpsy :: egfp ), is detected using NIBA filters (excitation filter 470-490nm, dichroic mirror 505nm, fluorescence filter 510-550nm, Olympus Corporation, Tokyo, Japan). The expression of the transgene (P tpsy :: egfp ) was confirmed.
Observation was performed multiple times. As a result, as shown in b of Table 2, EGFP fluorescence was observed in 17 clones.

A confocal laser microscope (FV-1000D, Olympus Corporation, Tokyo, Japan) was used for confirmation of the localization of EGFP fluorescence and photography.
FIG. 5 shows typical photographs of a wild strain and a transformant clone of this example using a confocal laser microscope. In FIG. 5, WT indicates a wild strain, and #number indicates a transformant clone. Also, from the left column, a differential interference image, EGFP fluorescence, chlorophyll fluorescence, and a superposition of EGFP fluorescence and chlorophyll fluorescence are shown.
In the wild strain, only a black background appears in the EGFP fluorescence photograph, and red appears on the black background in the chlorophyll fluorescence photograph.
In the transformant clone, green fluorescence appears on the black background in the EGFP fluorescence photograph, and red fluorescence appears on the black background in the chlorophyll fluorescence photograph.
In the photograph of superposition of EGFP fluorescence and chlorophyll fluorescence, red fluorescence appears slightly yellowish on a black background in the transformant clone. On the other hand, in the wild type, the superposed photograph of EGFP fluorescence and chlorophyll fluorescence is only red fluorescence of chlorophyll fluorescence.
As a result of observation using a confocal laser microscope, the localization of the EGFP fluorescence of the transformant clone was consistent with the localization of the chlorophyll fluorescence as shown in FIG.

<Confirmation of transformants by genomic PCR>
Using the transformant clone cells in which fluorescence derived from EGFP was confirmed as a template, genomic PCR was performed to confirm the insertion of the promoter and the transgene ( Ptpsy :: egfp ) into the genome. 300 μL of a culture solution of a transformant clone in a culture system using a 48-well plate was dispensed into a 1.5 mL tube. After obtaining a cell pellet by centrifugation (9,730 × g, 3 min), add 300 μL of sterile Milli-Q water to the cell pellet, centrifuge, and remove the supernatant to remove the salt that inhibits PCR Went. The salt was removed twice with sterile Milli-Q water, and 20 μL of sterile Milli-Q water was added to the cell pellet obtained after the second washing, suspended, and stored at −80 ° C. Genomic PCR was performed using 1 μL of the cell solution of P. tricornutum NRIA-0065 strain as a template. PCR was performed according to the protocol of Tks Gflex (registered trademark) DNA Polymerase (Takara Bio Inc., Otsu, Japan) with the reaction system being 20 μL. 5 μL of the reaction solution after PCR was analyzed by agarose electrophoresis. For confirmation, primers PtfcpAPro- (attB1) F and EGFP (qPCR) -R shown in Table 3 and SEQ ID NOs: 7 and 8, respectively, were used. The positions of the primer PtfcpAPro- (attB1) F of SEQ ID NO: 9 and the primer EGFP (qPCR) -R of SEQ ID NO: 10 used for genomic PCR are shown in FIG.

The result of electrophoresis is shown in FIG. The # numbers in FIG. 7 indicate transformant clones. Transformant clones with #numbers enclosed in squares were confirmed for PCR amplification products of the expected size and were used for carotenoid extraction. NC showed a negative control, and the PCR was carried out by adding the same amount of sterile Milli-Q water instead of adding the template cells.
As a result of genomic PCR, as shown in c of Table 2, insertion of the promoter and Ptpsy :: egfp into the genome was confirmed in 6 of the transformant clones in which EGFP fluorescence was observed.

<Carotenoid extraction>
Carotenoid analysis was performed using clones in which the insertion of the transgene into the genome was confirmed.
Steady phase in a 6-well plate (MICROPLATE 6 Well / Flat Bottom, AGC TECHNO GLASS CO., LTD., Shizuoka, Japan) containing 6 mL of f / 2 liquid medium containing 300 μg / mL Zeocin ^TM Transformant clones (5 mL, approx. 3 x 10 ⁷ cells) in the early stage of 15 mL tubes (15 mL / Centrifuge Tube with Triple Seal Cap, AGC TECHNO GLASS CO., LTD., Shizuoka, Japan), and 1.5 mL Algae was collected by repeated centrifugation (3,000 × g, 10 min) using a tube (REACTION TUBE, greiner bio-one, Kremsmunster, Austria), and the cell pellet was frozen using liquid nitrogen. The cell pellet was stored at −80 ° C. until carotenoid extraction.

  After the cell pellet was freeze-dried, 150 μL of acetone was added and left at room temperature in the dark for 1 hour. After centrifugation (19,060 × g, 3 min), the supernatant is collected in a solvent evaporation tube (Microtube No.1, Maruemu corporation, Osaka, Japan), and 100 μL of acetone is added to the residue for 30 minutes at room temperature in the dark. Left to stand. After centrifugation (19,060 xg, 3 min), the supernatant is collected in a tube for solvent evaporation. Extraction with 100 μL of acetone was repeated three times until the supernatant became transparent. 100 μL of methanol was added to the residue after 4 times of acetone extraction, and the mixture was allowed to stand at room temperature in the dark for 30 minutes.

  After centrifugation (19,060 × g, 3 min), the supernatant was collected in a tube for solvent evaporation, 100 μL of methanol was added to the residue, and the mixture was allowed to stand for 30 minutes in the dark at room temperature. Methanol extraction was repeated 4 times until the supernatant was clear. The solvent evaporation tube was placed in a heat block heated to 40 ° C., and the solvent was evaporated while blowing nitrogen gas. The residue was suspended in 400 μL of methanol and the solution was transferred to a 1.5 mL tube. After centrifugation (19,060 × g, 5 min), 350 μL of supernatant was collected into a 2 mL brown screw vial (Agilent Technologies, CA) and used for analysis.

<Analysis of total amount of carotenoids by spectrophotometry>
The carotenoid extraction solution is diluted with methanol, and a spectrophotometer DU730 (Beckman Coulter, Inc., CA) is used to calculate the previously reported carotenoid total amount (1) (Ryckebosch at al., 2011, Influence of drying and storage on J. Agric. Food Chem. 59 (20): 11063-11069.) was used to compare the total amount of carotenoids in the wild and the transformant clones using lipid and carotenoid stability of the microalga Phaeodactylum tricornutum.

In the formula (1), C _a represents the amount of chlorophyll a, A _665, A ₆₅₂ and A _470, respectively show the values of Abs _665, Abs ₆₅₂ and Abs _470. X _Carotenoids indicates the total amount of carotenoid (μg), DF indicates the dilution rate, and V indicates the amount of the extract. Each sample was measured three times, and the obtained value was converted into the amount per cell and the amount per culture medium, and the total amount of carotenoids of the wild strain and the transformant clone was compared.

The results of statistical analysis are shown in FIG.
In FIG. 8, WT is a wild strain, and #numbers indicate transformant clones. FIG. 8A shows a graph of values obtained by converting the total amount of carotenoids per cell, and FIG. 8B shows a graph of values obtained by converting the total amount of carotenoids per amount of culture solution. The broken line indicates the average value of WT.
As a result of one-way analysis of variance and pooled variance t-test, the #numbers of transformant clones that showed a significant difference from WT are shown enclosed in ellipses. Other combinations are not shown in the figure even though significant differences are observed.
From the results of FIG. 8, it was suggested that the total amount of carotenoid per culture broth in the two clones (# 2-4 and # 2-12) was the same as that of the wild strain or more than that of the wild strain. About these 2 clones, the carotenoid component was analyzed using HPLC.

<Analysis of carotenoid components by HPLC>
The carotenoid extract solution was diluted with methanol, and the carotenoid component was analyzed by HPLC. The HPLC system used for the analysis was CrestPack C18T-5 (4.6 mm ID × 250 mm L, JASCO Corporation, Tokyo, Japan) for the column, Prostar 335 PDA Detector (Agilent Technologies, CA) for the photodiode array detector, pump Prostar 230 (Agilent Technologies, CA) was used.

  As a mobile phase, liquid A consisting of acetonitrile / methanol / distilled water = 75: 15: 10 (containing 0.1% (w / v) ammonium acetate) and ethyl acetate / methanol = 30: 70 (0.1% (w / v) Using B liquid consisting of ammonium acetate), changing the ratio of A liquid / B liquid from 100: 0 to 0: 100 over 20 minutes, and then developing the carotenoid component by flowing B liquid for 15 minutes It was. The flow rate of the developing solution was 1 mL / min, and the column temperature was 40 degrees.

  The settings of the photodiode array detector were a detection wavelength: 450 nm, an acquisition wavelength: 200-900 nm, and a slit width: 4 nm. Using an autosampler 460-LC (Agilent Technologies, CA), 10 μL of the extract was inhaled by the method of μL Pickup injections, and component analysis was performed. From the results of the samples of fucoxanthin and β-carotene, analysis was performed with the peak near retention time 6.5 as fucoxanthin and the peak near 28.5 as β-carotene. A calibration curve was created from the peak area ratio of the sample, and the fucoxanthin content and β-carotene content in the extracted solution were determined. Each sample was measured three times, and the obtained value was converted into the amount per cell and the amount per culture medium, and the amounts of the carotenoid components of the wild type and the transformant clones were compared.

The results of the analysis of carotenoid components using HPLC are shown in FIG. FIG. 9 is a chromatogram of the carotenoid extraction solution obtained by HPLC. FIG. 9 (A) shows the result of the wild type strain (WT), and FIG. 9 (B) shows the result of the transformant clone (# 2-12). The peak around the retention time of 6.5 minutes is fucoxanthin, and the peak around 28.5 minutes is β-carotene. Although not yet identified, the peak near 9.5 minutes is considered to be diazinoxanthine.
As shown in FIG. 9, fucoxanthin and β-carotene were confirmed in the wild strain and the transformant clone. Although not yet identified, P.I. From the relative relationship of retention time with fucoxanthin in the literature (Dambek et al., 2012) that examined carotenoids contained in tricornutum , a peak likely to be diazinoxanthin was confirmed around 9.5 minutes.

<Statistical analysis>
FIG. 10 is a graph showing the results of analysis of carotenoid content using HPLC, and the amount of fucoxanthin and β-carotene of wild-type strains and transformant clones are shown as the amount per cell and the amount per culture solution. The results of statistical analysis are shown.
10A and 10B show the amount of β-carotene, and FIGS. 10C and 10D show the amount of fucoxanthin. FIGS. 10B and 10D show the amount per 10 ⁷ cells, and FIGS. 10A and 10C show the amounts per culture solution.
In FIG. 10, WT is a wild strain, and #numbers indicate transformant clones.

In FIG. 10, one-way analysis of variance and pooled variance t test are used when normality and equal variance are obtained using College Analysis Ver. 5.4. If either of them is not obtained, Kruskal-Wallis test and joint are used. ranking Wilcoxon rank sum test was performed.
For β-carotene, the #numbers of transformant clones that showed a significant difference from WT as a result of one-way analysis of variance and pooled variance t test are shown enclosed in ellipses. For fucoxanthin, the #numbers of transformant clones that showed a significant difference from WT as a result of the Kruskal-Wallis test and the joint ranking Wilcoxon rank sum test are enclosed in ellipses. Other combinations are not shown in the figure even though significant differences are observed.
In the transformant clone # 2-12 in FIG. 10, the amount of fucoxanthin and β-carotene per cell was significantly increased as compared to the wild type, which was 1.29 times and 1.36 times that of the wild type, respectively. It was. In addition, the amount of β-carotene per culture broth was significantly increased to 1.19 times. The amount of fucoxanthin per culture broth was 1.13 times, although no significant difference was observed.
As described above, according to the transformant of this example, it was found that fucoxanthin producing ability was improved as compared with the wild type.

Claims (6)

It has a PtfcpA ( Phafactylum tricornutum fucoxanthin chlorophyll a / c-binding protein A gene) promoter derived from a marine diatom and a gene encoding the phytoene synthase derived from the marine diatom on the downstream side of the promoter A vector. In addition, it has a gene for antibiotic selection,
The vector according to claim 1, which has a highly sensitive green fluorescent protein gene on the C-terminal side of the gene encoding phytoene synthase.   A genetically modified marine diatom for producing fucoxanthin, wherein the vector according to claim 1 or 2 is incorporated into the marine diatom as a host.   The genetically modified marine diatom according to claim 3, wherein the marine diatom has fucoxanthin-producing ability. The genetically modified marine diatom according to claim 3 or 4, wherein the marine diatom is Phaeodactylum tricornutum Bohlin, which is a marine phytodiatom .   A method for producing fucoxanthin, comprising the step of extracting the fucoxanthin from a medium produced by culturing the genetically modified marine diatom according to any one of claims 3 to 5 in a medium.