Succeeded in developing a method to selectively breed a variant of Euglena that produces a large amount of oil

Euglena Co., Ltd.
University of Tokyo
National Research and Development Corporation RIKEN
National Research and Development, Japan Science and Technology Agency
Cabinet Office Policy Officer (in charge of science and technology / innovation)

~ Point ~
○ A method for observing the fat content of Euglena
○ We succeeded in selecting and acquiring Euglena mutants with a high fat content.
○ It is expected to develop into biofuel research using oils and fats produced by Euglena

　As part of the research and development program of Keisuke Goda, Program Manager of the Innovative Research and Development Promotion Program (ImPACT) led by the Council for Science, Technology and Innovation of the Cabinet Office, Osamu Iwata, Senior Researcher (ImPACT Team Leader) of Euglena Professor Keisuke Goda of the Graduate School of Science has developed a variety improvement method (Note 1) that efficiently Euglena
Euglena is being applied to foods and biofuels. Nutritious than so far Euglena high and fuel production efficiency Euglena but is also expected the discovery of a huge number of slightly different features for each individual Euglena from, with the features of the purpose Euglena to singles Was difficult and required the development of efficient methods.
　This research group has developed a method for efficiently Euglena Euglena containing more fats and oils than before from the group of Euglena with various characteristics created by irradiating a heavy ion beam (Note 2) at the Nishina Accelerator Research Center of RIKEN. Was successfully obtained.
　By combining this selection technology with the Serendipitter (Note 3) currently under development, it will be possible to obtain useful features from a Euglena This Euglena 's research on high-efficiency biofuels.
　The results of this research will be published in the electronic journal "Scientific Reports" of the Nature Publishing Group (NPG) at 10:00 (UK time) on May 23, 2016.

This result was obtained through the following programs and R & D issues.

Cabinet Office Innovative Research and Development Promotion Program (ImPACT)
http://www.jst.go.jp/impact/
Program Manager: Keisuke Goda
R & D program: Creating new value through the planned creation of serendipity
R & D subject: Establishing a method for acquiring genetically diverse cell populations
R & D manager: Kengo Suzuki (Euglena Co., Ltd. R & D Department)
Team leader: Osamu Iwata (Euglena Co., Ltd. R & D Department)
Research period: November 2014-March 2017

In this R & D project, we are working on the development of basic technology that stimulates cells quickly and accurately with the resolution of a single cell. Among them, the Iwata team is working on the development of a "method for acquiring genetically diverse cell populations" that creates genetically diverse cell populations containing cells that are worth sorting with serendipita. is.
■ Comment from Keisuke Goda Program Manager ■
This result is due to the research team of Euglena Co., Ltd., which participates in the Cabinet Office Innovative Research and Development Promotion Program (ImPACT) "Creation of New Value by Planned Creation of Serendipity". This team is developing technology to stimulate a huge number of cell populations, create useful cells to be sorted, and identify them. Almost no reports have been made on the improvement of Euglena varieties for industrial use, but the result of this time is that it is possible to Euglena It shows the possibility of getting more useful Euglena In the future, we will promote the program so that we can find Euglena which produces ultra-high fats and oils.

<Announcement content>
1) Background and background of the research
　Microorganisms such as microalgae and yeast can develop cells with various characteristics different from those of wild strains by performing mutagen treatment (Note 4), and among them, those with useful characteristics can be developed. If there is a selection technology, it is possible to acquire strains (breeding) that will lead to industrial application. Euglena, which was the subject of this research, is the scientific name of the organism known as "Euglena" and is a type of microalgae that mainly inhabits freshwater areas around the world (Fig. 1). However, compared to other well-studied microalgaes (eg Chlamydomonas), efficient mutagenic treatment and individual selection techniques have not been established, so acquisition of Euglena with the desired characteristics Was difficult. In addition, from this, it has not been sufficiently verified whether a mutant showing the desired trait can be obtained. Euglena has already been industrially applied as a food material, but if it becomes possible to improve varieties as efficiently as plants such as rice and corn, it can be expected to be used more widely.

2) Contents of research
　A research group consisting of Euglena Co., Ltd., the University of Tokyo, and RIKEN creates a genetically diverse population of Euglena by combining cell stimulation (mutagen treatment and fluorescent staining), and efficiently from there. We have succeeded in selecting Euglena, which contains a large amount of fat and oil, which was the target of this study.
　When cells are irradiated with heavy ion beams as a mutagen treatment, cells with various characteristics appear in the population. In this study, Euglena is irradiated with this to create a population containing Euglena with various characteristics, and in addition to this, a method of observing and selecting individual cells of Euglena by using a cell sorter (Note 5). Was developed (Fig. 2). In particular, it is considered that it was effective in the selection to find the condition that Euglena is less likely to be damaged at the time of selection from various condition examinations. After that, we visualized intracellular fats and oils by BODIPY staining (Note 6), and verified whether Euglena, which is particularly rich in fats and oils, can be selected from the various cell populations created by utilizing the difference in fluorescence intensity. .. As a result, we succeeded in obtaining the Euglena mutant containing about 40% more fat and oil than the wild strain (Figs. 3 and 4). Since this Euglena mutant produces a large amount of fats and oils, it is excellent as a raw material for Euglena biofuel and is expected to contribute to the improvement of productivity. In addition, from the above results, it was demonstrated that the target Euglena can be selected by using the method verified in this study.

3) Future development
　We used an existing cell sorter to select Euglena In the future, it is envisioned that the process will be carried out using the Serendipita under development in this ImPACT program. By using Serendipitter to obtain more high-quality information from Euglena Euglena containing a large amount of fats and oils may be selected more quickly and accurately. In addition, by devising a visualization method, it will be possible to select Euglena which has industrially useful characteristics such as Euglena has a high vitamin content in addition to the oil and fat content. Furthermore, Euglena, it is expected that research on functional ingredients and biofuels will be accelerated.

　Euglena breeding in this presentation is the Euglena Co., Ltd. in the ImPACT program (Aida Program Manager), and the breeding technology by heavy ion beam irradiation is a strategic innovation of the Cabinet Office. It is being developed at the Nishina Accelerator Research Center, RIKEN as part of the Creative Program (SIP).

<Reference diagram>

Figure 1. Visualization of fats and oils accumulated in Euglena

Figure 2. Creation of populations containing diverse Euglena and selection of mutants

Figure 3. BODIPY stained original Euglena (wild strain) and mutant Euglena microscope image
The stronger the fluorescence, the more fat and oil.

Figure 4. Comparison of fat content

<Glossary>
* 1 Creation, selection, breeding ... By Euglena, a population containing mutants with various characteristics is created. After evaluating the characteristics of each individual in the group, we select individuals that match the purpose with a cell sorter and improve the variety.
* 2 Heavy ion beam irradiation: Some electrons are stripped from an atom and charged with electricity is called an "ion", and an ion of an element heavier than helium is called a "heavy ion". Since it can cleave DNA double strands and induce base deletion, it is also used for efficient breeding of plants. In this study, we irradiate accelerated high-energy iron ions at RIKEN Nishina Accelerator Research Center to induce mutations Euglena
* 3 Serendipita: A device that quickly, accurately, low-cost, and minimally invasively discovers and thoroughly analyzes rare but high-impact cells from a huge number (1 trillion or more) of cell populations.
* 4 Mutagen treatment: A treatment that induces gene mutations and causes mutants to appear by exposing organisms to mutagens (ultraviolet rays, chemical mutagens, etc.).
* 5 Cell sorter: A device that observes cells in the flow path using the fluorescence intensity emitted by the cells as an index and sorts the target cells.
* 6 BODIPY staining: A staining method that uses the fluorescent dye BODIPY to stain intracellular lipids. BODIPY is also characterized by its non-cytotoxicity.

<Announcement magazine>
Magazine name: Scientific Reports
Article title: Efficient selective breeding of live oil-rich Euglena gracilis with fluorescence-activated cell
sorting
(Efficient breeding of Euglena containing high fats and oils using FACS)
Authors: Koji Yamada, Hideyuki Suzuki, Takuto Takeuchi, Yusuke Kazama, Sharbanee Mitra, Tomoko Abe,
Keisuke Goda, Kengo Suzuki, Osamu Iwata

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