Black boxes in plant biosynthesis
“Do you remember this commercial?” asks Muranaka pointing to a poster in his office. “This is an advertisement for ‘BioLip’, a biotechnology-based rouge makeup launched in 1984. The theme song for the advertisement, 'Rock'n Rouge' sung by Seiko Matsuda, was a great hit. The rouge itself was groundbreaking.
The dyes used in the rouge contained shikonin, a natural product derived from the rare herbaceous plant Lithospermum erythrorhizon, commonly known as stoneweed. Shikonin is difficult to synthesize chemically, and there had been no way before that time to obtain the chemical except by extraction from the harvested plant. In the 1980s, however, Mitsui Petrochemical Industries Ltd (now Mitsui Chemicals Inc.) succeeded in the large-scale production of cell cultures from the plant, allowing shikonin to be produced in culture systems. That was the world's first successful industrialization of material production using plant cell cultures. The shikonin thus produced was used in the BioLip rouge.
In 1985, Muranaka joined Sumitomo Chemical Co. Ltd, where he attempted to produce scopolamine, a compound that is used as an active ingredient in analgesic medicines, using plant tissue cultures. In those days, however, it was still unknown how the desired compound is biosynthesized in the plant. So other than changing the various culture conditions by trial and error, there was no way to determine the conditions under which the compound could be produced in large amounts.
His attempts to produce scopolamine on an industrial scale encountered problems of low productivity. At the laboratory scale, I succeeded in producing scopolamine with the highest efficiency in the world. However, the productivity was still too low after taking into account the estimated cost of industrial production. A number of other companies in various fields have attempted to produce useful compounds from plant tissues and cells using cultures, but productivity sufficient for industrial application has rarely been achieved. The situation has not changed much. There are only a few cases of successful industrialization, such as the production of ginseng tissue culture.
Mining the genes in action
In 2001, Muranaka was appointed team leader at the RIKEN Plant Science Center. Here at RIKEN, I wanted to retry my attempt that had not succeeded 15 years earlier. He selected as the theme of his work glycyrrhizin, a naturally occurring sweetener compound found in the roots and underground stems (rhizomes) of the leguminous licorice plant. Glycyrrhizin is 150 to 300 times sweeter than sucrose, and it has many applications as a natural sweetener with a low calorific value. It also known to enhance liver function and to exhibit antiviral activity. Glycyrrhizin is formulated in many kampo (traditional) medicines.
Licorice rhizome is thus in high demand, although supplies remain almost exclusively limited to wild indigenous species of the licorice plant found in arid regions of China, the Middle and Near East, and elsewhere. Wild licorice has become endangered as a result of overharvesting. Another problem arises in relation to the deep rooting of the plant; the grasslands forming the natural habitats of licorice are often destroyed during harvesting, which in turn accelerates desertification. Regulations on the export of licorice have been put into effect in some producing countries. In Japan and China, the licorice plant is cultivated experimentally, but the cultivated varieties contain much less glycyrrhizin than the wild varieties, which remains an unresolved problem.
I decided to try and produce glycyrrhizin in a more efficient way by means of a biosynthetic mechanism, rather than by collection from wild licorice plants. Glycyrrhizin is biosynthesized from â-amylin, a compound produced by many plants. However, the biosynthetic process was completely unknown, a black box. In 2003, we began working to elucidate the process. A difference from the 1980s is that it is now possible to obtain genetic information, such as DNA sequences, much more quickly and at lower cost, says Muranaka. This time, using a technology that could not have been devised 15 years earlier, Muranaka identified the genes involved in the biosynthesis of glycyrrhizin by systematically mining all the DNA sequences of the active genes in the cells of the licorice rhizome.
Major projects with almost the same research theme had in fact begun at Chiba University, Tokiwa Phytochemical Co. Ltd, and elsewhere at nearly the same time as our project. We decided to cooperate with these other projects to form an all-Japan system. Through this cooperation, about 56,000 active genes in the cells of licorice rhizomes were reported. After discarding overlapping results, these studies successfully identified 10,372 genes.
Discovery of the genes involved in biosynthesis
The pathway for the biosynthesis of glycyrrhizin from â-amylin seems to be complicated, yet there is only a small difference between the chemical structures of these two compounds. Glycyrrhizin is the product of the oxidation of â-amylin at two sites and the binding of a sugar at one site.
Given these processes, it is the genes for the enzymes oxidase and glycosyltransferase that are important in the biosynthesis of glycyrrhizin. The search for candidate genes can be performed automatically using a computer on the basis of DNA sequence profiles. However, as such genes are sometimes overlooked, visiting researcher Hikaru Seki painstakingly checked the functions predicted from the DNA sequences for all of the 10,372 genes one by one. In the end, he identified 37 genes for oxidase and 33 for glycosyltransferase.
Which of these genes function in glycyrrhizin biosynthesis? In plants, the genes necessary for the biosynthesis of a compound are mostly active in the parts of the plant where the compound is produced. Glycyrrhizin is produced and accumulated in the roots and rhizomes of the licorice plant, and is not found in the top stems or leaves at all. We first identified oxidase genes that function only in the roots and rhizomes, and not in the top stems or leaves.
The 37 oxidase genes were thus narrowed down to five candidates. Oxidase was then produced using each of the five genes and mixed with â-amylin. It was found that only one enzyme among the five oxidized one particular site in â-amylin. Eventually, the oxidase gene involved in the biosynthesis of glycyrrhizin was discovered. Muranaka and others named the gene CYP88D6, and announced the finding in September 2008. They also discovered an enzyme that oxidizes the other site.
The process of transferring a sugar to the triterpene backbone, the other process in the synthesis of glycyrrhizin, is thought to progress in two steps. Researchers have found the glycosyltransferase enzyme that advances the second step of the reaction, and are now searching for the gene associated with the first step.
In carrying out this kind of experiment, it is necessary to chemically synthesize the intermediate compounds produced during biosynthesis. Cooperation with researchers in phytochemistry and pharmacognosy is therefore essential. Research Associate Kiyoshi Ohyama has made a significant contribution in this regard. Although Japan is strong in phytochemistry, researchers in that field and researchers in molecular biology traditionally work separately. In our team, researchers from both fields have cooperated successfully to identify the enzyme genes involved in the biosynthesis of glycyrrhizin The only gene remaining to be identified is that for the glycosyltransferase enzyme that catalyzes the first step of the reaction.
Opening new frontiers in synthetic biology
If the genes involved in the biosynthesis of glycyrrhizin can be identified, what will this achieve? "For example, this will allow the cultivation conditions to be optimized so as to enhance the function of the gene, leading to the cultivation of a variety of the licorice plant that produces glycyrrhizin at higher yields. Furthermore, it will become possible to induce other plants to produce glycyrrhizin," says Muranaka. "Since licorice is a leguminous plant, 'sweet soybeans' , for example, could be produced by introducing the necessary gene into the soybean, another member of the legume family. Furthermore, it may also be possible to produce glycyrrhizin by introducing the gene into yeast, rather than into a plant."
Synthetic biology is a discipline concerned with the design of biosynthetic processes by switching on and off the functioning of particular genes in a manner essentially identical to that in organisms. Such research has become feasible as a result of the identification of the genes involved in biosynthesis.
With advances in synthetic biology, the variety of plants that will become useful to society will increase dramatically. For example, intermediate compounds formed during biosynthesis occur in trace amounts, so the functions of most of these compounds have yet to be investigated. Synthesizing many intermediates and examining their functions will certainly lead to the discovery of compounds that could be selected as candidates for new drugs. Designing biosynthetic pathways will make it possible to create new compounds with excellent functions that are not found in nature.
International cooperative research toward overcoming malaria
There are high expectations for efforts aimed at resolving global issues, such as those associated with the environment, energy and infectious diseases, through international cooperation based on Japan's science and technology. Muranaka and others are also conducting a pioneering study on malaria. "In 2003, South Africa and Japan entered into an agreement concerning cooperation in the fields of science and technology. Motivated by this, I visited South Africa for the first time in 2004, and established an international cooperative research project in 2006."
The project aims to overcome malaria, which is one of three infectious diseases (in addition to HIV/AIDS and tuberculosis) prioritized by the World Health Organization. As many as 200 to 300 million people suffer from malaria at present, with most cases concentrated in sub-Saharan Africa. Although there exists a highly effective medicine, quinine, for the treatment of malaria, malarial protozoa that are resistant to quinine have emerged in recent years, with dire implications for public health. The World Health Organization now recommends treatment with artemisinin, a natural compound produced by the Asian Artemisia annua plant.
In South Africa, a plant of the same genus known as Artemisia afra occurs indigenously, and is already used as a traditional medicine. However, it does not produce artemisinin. We investigated these two plants to compare the functional genes, and found that introducing into A. afra those absent genes found in A. annua may lead to the production of the intermediate that precedes the biosynthesis of artemisinin. Through further investigation, it may be possible to synthesize artemisinin using the South African A. afra, and to develop a remedy for malaria that is more effective than naturally occurring artemisinin. South Africa is a treasure of indigenous plants. I hope that the international collaboration will continue.
Advancing the work of Dr Kihara
In April 2007, Muranaka was inaugurated as professor of the Kihara Institute at Yokohama City University. The institute was founded by Hitoshi Kihara (1893–1986), a scientist of world fame for his research into the origins of wheat and his advocacy of the concept of the genome.
A wide variety of wheat research resources left by Dr Kihara are preserved here. Making use of them using up-to-date approaches will allow us to conduct interesting research. I have just begun working in collaboration with the Metabolic Diversity Research Team, to which I now belong, on a project examining the production of useful compounds using wheat.
By extending the diverse cooperation among RIKEN, research organizations and universities both in Japan and abroad, the project is expected to provide new insights into plant science.
About the Researcher
Toshiya Muranaka was born in Osaka, Japan, in 1960. He graduated from the Department of Agricultural Chemistry at Kyoto University in 1983, and obtained a master degree at the Graduate School of Agriculture at the same university in 1985. In the same year, he joined Sumitomo Chemical Co. Ltd and began research on the production of secondary metabolites by hairy root cultures. From 1988 to 1990, he conducted research as a visiting scientist at the Department of Biology, Nagoya University on the protein kinase, which is involved in sugar and lipid metabolites in plants. He obtained his PhD in agriculture from Kyoto University in 1993, and subsequently served as senior researcher at the Research Institute of Innovative Technology for the Earth (RITE) from 1995 to 2000, where he conducted research on the molecular biology of microalgae. In 1997, he pursued research on the posttranscriptional regulation of plastid genes in green algae at the University of California, Berkeley as a NEDO Fellow. He left Sumitomo Chemical Co. Ltd in March 2001 to join the RIKEN Plant Science Center as team leader in April of the same year, and in 2008 served as Senior Visiting Scientist. In 2007, he attained the post of professor at Yokohama City University.
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