Research
Proof of success is in the results
(Project)
There are several benefits to using plants rather than conventional methods that rely on cultured animal or human cells. (1) Production costs are calculated to be around 1/40 compared to using animal cells in the best case. Although productivity depends on the compatibility between the target protein molecules and host cells, proteins are usually produced in greater amounts in plants than in cultured cells. (2) The scalability is characteristic of the culturing of plants. When proteins are produced in cultured animal cells or microorganisms, it is crucial to estimate the required facility and culture tank sizes for commercialization. On the other hand, plants can be sown in fields or in plant factories, depending on the desired commercial scale. (3) Proteins derived from humans and produced in chloroplasts in plants are not glycosylated, resulting in a lower potential for allergic reactions when ingested by humans. This technology is based on a discovery made in Arabidopsis and published in Proc. Natl. Acad. Sci. USA, 2010, patented as “Constructs for Light-Switch for expressing genes of interest,” Japanese patent 6385644.
We focus on producing biologics such as the world’s best-selling antibody drug, adalimumab, whose trade name is "Humira." This drug is used to treat rheumatoid arthritis; the patents have expired. We have succeeded in the production of the single-chain variable fragment (scFv) in tobacco chloroplasts with a yield of 35 mg/kg tobacco (fresh weight) and activity of Kd = 9.5 ×10⁻⁹ M against human tumor necrosis factor α (TNFα). Using this method to produce adalimumab gives a commercial value of approximately US$400/m²/year.
The proposal entitled “Production of biologics with lower costs by chloroplast engineering with Light-Switch to express foreign genes specifically” received the “Yamaha Motor Corp. Prize” at the Grand Prix of First Shizuoka Tech Planters sponsored by Leave a Nest, Inc., on July 7, 2018, in Hamamatsu, Japan. Another proposal, “Does Light-Switch serve mankind for a sustainable society?”, was nominated as a finalist at the Agri Grand Prix 2018 on September 15, 2018, in Tokyo, Japan.
2. Creation of drought-tolerant grasses by genetic modification to green building roofs and road slope faces (Project)
Salt accumulated on soil surfaces is a serious problem. Globally, salt-affected areas are currently the size of the United States and continue to expand. Because there is no rain in those areas, it has become impossible for farmers to continue growing crops once salt accumulates. It is thought that salt-tolerant genes could enable plant cultivation in such areas, and that agricultural products and biofuels could be produced. This technology is enabled by plant genetic engineering, and although genetically modified organisms are not well received by society, in January 2017, the United States Department of Agriculture (USDA) ruled that GM grasses (Agrostis spp.) are not subject to GM plant regulations.
The salt-tolerant ability of plants is generally associated with drought tolerance. The use of genes for salt-tolerant callus (stc) and photoautotrophic salt tolerance (pst), as revealed using Arabidopsis (published as two papers in PLOS ONE, 2015, and one in Plant Cell, 1999, respectively) can be applied to generate drought-tolerant grasses, which can then be used for greening building roofs and road slope faces. This technology has been patented as “Gene for ABC-transporter conferring salt tolerance on plants,” Japanese patent 5871222, as well as “A method for making transformant plants salt tolerant,” Japanese patent application 2007-065450, and “Polynucleotides imparting environmental stress-tolerance to plants,” Pub. No. WO/2006/098423 and International Application No. PCT/JP2006/305328.
Fig. 2. Phenotype of Arabidopsis with a mutated PST2 gene
Plants were transferred to a 200 mM NaCl (nearly half the concentration of sea) medium and maintained for 3 weeks after growing on a standard medium for 3 weeks.
Fig. 3. Phenotypes of Arabidopsis ectopically expressing SUG genes
Enhancement of photosynthetic activity by degrading SUG genes was also observed.
3. Creation of productivity-enhanced plants by genome editing (Project)
“Genome editing” is the technology of editing the DNA of organisms to a status equivalent to that occurring in nature by specifically targeting genes, for example, disrupting genes. It marks the beginning of a new era in Japan, as knockout (KO)-type genome-edited foods can now be sold once they have been reported to the Ministry of Health, Labour and Welfare (MHLW) and, thereafter, to the Consumer Affairs Agency (CAA). Although genome editing is a form of genetic modification, genome-edited plants are not subject to the same regulatory requirements as other GM plants.
We identified genes responsible for suppressed greening (sug) in Arabidopsis, and knockout of these genes enhances photosynthetic activity. It can be used to elevate the productivity of nutraceuticals and biofuels in plants. This technology has been patented as “Genes for suppressing photosynthetic activity and their application,” Japanese patent 5780506, and “A method for producing green callus cells,” Japanese patent application 2006-069547.
4. Breeding improvement of tea using KO-Type genome editing, a safe breeding technology (Project, Related achievements)
Among “genome editing” methods, the KO-type can rapidly introduce mutations that could occur naturally. Cultivation and sale are now permitted upon notification to the Consumer Affairs Agency (CAA) in Japan. CRISPR/Cas9, the most widely used genome-editing system, is being adapted for tea plants. Stable genetic transformation generally introduces guide RNA (gRNA) and the Cas9 enzyme (gRNA-Cas9) into the target organism's cells. However, crops such as tea plants, in which stable genetic transformation is complex, pose challenges for the application of genome editing.
Fig. 4. Breeding technologies
Advantages and disadvantages are mentioned in mutagenesis achieved through radiation, such as gamma rays, genetic engineering, and KO-type genome editing.
Fig. 5. Development of decaffeinated and immune-boosting tea leaves using KO-type genome editing in tea plants
5. Biofortification of pharmaceutics and nutraceuticals in plants by light-emitted diode (LED) irradiation (Project)
Plants have evolved to use environmental factors such as sunlight, water, and air to adapt to environmental change. Plants respond to different wavelengths of light. We found that blue and red LED light enhanced the content of polyphenols and saponins in Brassicaceae. This discovery can be applied in the biofortification of pharmaceuticals and nutraceuticals in plants.
We have three patent applications related to LED irradiation: “A method for enhancing expression of genes for saponin biosynthesis by exposure of plants to light illumination,” Japanese patent application 2007-087278, “A method for enhancing expression of genes for biosynthesis of lignans and lignins by exposure of plants to light illumination,” Japanese patent application 2008-089093, and “A method for enhancing expression of genes for flavonoid biosynthesis by exposure of plants to light illumination,” Japanese patent application 2008-089111.
