Research Areas
Gene Discovery and Natural Products
Introduction
There is a long history of the use of plant materials and extracts for medicinal and commercial purposes. A classic example of a plant-derived drug is salicylic acid, found in willow bark, which is the basis for the use of acetylsalicylic acid, or aspirin, for relief of pain, fever and inflammation. In the past, the development of plant-derived drugs involved identifying plants with relevant medicinal properties and the corresponding active compounds. After testing and finding compounds with the right pharmacological properties, the question of production then arose. With aspirin, the answer is straightforward. It is a simple molecule with no chirality, so it is produced by chemical synthesis on an industrial scale.
At the other end of the spectrum is a drug like Taxol. Taxol is an isoprenoid compound found in the bark of the Pacific yew tree. It is highly successful in cancer treatment, in particular, of ovarian and breast cancers. The total synthesis of Taxol was a chemical tour de force reported in 1994 and involves over 40 steps. With an overall yield of 2% it is clearly not commercially viable. On the other hand, yew bark contains only 0.01% taxol, so supply has been a problem.
So, what is the solution to the supply problem for certain plant-derived drugs and chemicals? If chemical synthesis is impractical and plants sources are not abundant, then there is an additional approach to try - the application of plant molecular biology and biotechnology. This is based on the idea that with the knowledge of the genes involved in the biosynthesis of plant natural products, their production may be enhanced in plants, plant cell cultures or alternative hosts. As part of the National Research Council's Plant Products for Health and Sustainability at the PBI, we are applying molecular genetic approaches plant-derived drugs with supply limitations. Our current focus is on bioactive cyclic peptides, the antimalarial artemisinin, and bioactive triterpenoids. We are participating in the Genome Canada-funded project called "Synthetic Biosystems for Production of High Value Plant Metabolites".
A Systematic Approach to Natural Product Gene Discovery
How do we find genes relevant to natural product biosynthesis? Well, we start by making cDNA preparations to represent the genes in the tissue which makes our compound of interest. We sequence hundreds of thousands of cDNAs to generate what are called expressed sequence tag (EST) collections. Such ESTs are compared against a large sequence database to give tentative identifications. Using our biochemical guesses and perhaps additional data such as knowledge of gene expression in different tissues, we can identify candidate cDNA clones which may encode enzymes of interest. These candidates are then tested by heterologous expression - the genes are introduced into E. coli or yeast, for example, and a test for the enzyme activity of interest is made. The enzyme assays involved require good biochemical and analytical expertise because different and often unusual substrates and products are required for each assay.
What will we do with the genes when we find them? If it is possible to identify all of the genes in a pathway from the point of one or more common metabolites, then it should be possible to metabolically engineer an appropriate host organism. The appropriateness of the host organism is related to concerns about feasibility and containment. Since plant genes are involved a plant host may be the most feasible, although concerns about the production of drugs in plants may make other hosts such as microorganisms which can be easily contained more attractive. Although one might wonder about the difficulty of introducing a number of genes at once into a plant, this is becoming more commonplace. For example, three genes were introduced into rice for the production of beta-carotene and up to 12 have been successfully introduced in soybean.
What if we don't find all of the genes of interest? Individual genes may be put to use in a few ways. One is to open metabolic bottlenecks in plants or cultures; the other possibility is the metabolic engineering of intermediate compounds with potential as improved drugs or drug precursors.
The Antimalarial Artemisinin from Artemisia annua
Artemisinin is the most important compound in the treatment of malaria. It is produced in the glandular trichomes of Artemisia annua, a plant used in traditional Chinese medicine. Using an EST-based approach, we have recently explored the genes involved in artemisinin biosynthesis and discovered four genes involved in artemisinin biosynthesis (see Teoh et al. 2006, Covello et al. 2007, Zhang et al. 2008, Covello, 2008, Teoh, et al. 2009). We have collaborated with Amyris Biotechnologies on the use of two A. annua genes in their yeast synthetic biology systems. NRC has recently announced an agreement with The Institute of One World Health and sanofi-aventis for the use of A. annua genes in efforts towards commercial-scale production of artemsisnin (National Post story).
We have succeeded in producing dihydroartemisinic acid, a late precursor of artemisinin, in yeast (Zhang et al. 2008). We have also investigated the possibility of producing artemisinin or its precursors in genetically-modified plants (see Zhang et al. 2011).
Cyclic Peptides from Saponaria vaccaria
Cyclic peptides have a wide range of interesting bioactivites which include anti-fungal effects. The carnation relative Saponaria vaccaria, a plant used in Traditional Chinese Medicine, contains a number of cyclic peptides with 5-9 of the common amino acids (see Morita, Bioorg. Med. Chem. Lett. 16:4458). Some of these have shown interesting effects, for example on mammalian blood pressure. We are currently investigating the biosynthesis of S. vaccaria cyclic pepitdes in an EST-based approach, in collaboration with John Balsevich and others (Condie et al. 2011).
Bioactive Triterpenoids
We are investigating the genes involved in the biosynthesis of ten plant species under the Genome Canada-funded project called "Synthetic Biosystems for Production of High Value Plant Metabolites".
Tropane Alkaloids
Henbane produces tropane alkaloids such as atropine and scopolamine which are used medicinally. In collaboration with Jon Page, we investigated the genes involved in tropane alkaloid biosynthesis and identified an unusual cytochrome P450 (CYP80F1) using a combined EST and virus-induced gene silencing (VIGS) approach (see Li et al. 2006). We recently collaborated with David O'Hagan to investigate the mechanism of the carbon skeleton rearrangement catalyzed by CYP80F1 (see Nasomjai et al. 2009).