General statement
The long-term objective of my research program is to understand biochemical and genetic principle governing the biosynthesis of plant metabolites applicable to human wellness and chemical industry. My research program ultimately aims at replacing the majority of petrochemicals and pharmaceuticals with more sustainable bio-based materials. My laboratory uses interdisciplinary approaches, such as molecular genetics, genomics, and biochemical tools, to advance the knowledge of metabolic biology. Current specific research activities are as follow.

1. Chemical diversity and evolution of sesquiterpene lactone in lettuce, sunflower, and related plants in Asteraceae family.
Asteraceae is the most successful plant family on earth with more than 23,000 species. Interestingly, a small subclass of terpenoid, sesquiterpene lactone, is found in all most all of the plant species in Asteraceae, indicating a strong retention pressure for this class of terpenoids. It is believed that sesquiterpene lactone played important roles in plant defense against various insects and animals. Sesquiterpene lactone also serves as critical pharmaceuticals, such as anti-malarial drug (artemisinin) in Artemisia annua. We have used the comparative analysis of sesquiterpene lactone metabolisms in Asteraceae. Biochemical and genomics work have resulted in publications which demonstrated that i) the three-step oxidation reactions by germacrene A oxidase (GAO) have emerged at the beginning of the Asteraceae evolution and ii) the enzyme (GAO) for this activity is highly promiscuous, allowing rapid biochemical adaptation to acquire novel activities (Nguyen et al, 2010, J. Biol. Chem.). In addition, using lettuce genomics data, we were able to synthesize the simplest sesquiterpene lactone, costunolide, for the first time using regio- and stereoselective cytochrome P450 enzyme (costunolide synthase) (Ikezawa et al, 2011, J. Biol. Chem.). Costunolide synthase is widely conserved in three major sub-families of Asteraceae but is missing in Heliantheae tribe where the closest homolog has evolved to catalyze a different regio- and stereoselective hydroxylation. We consider this chemical and biochemical diversification for sesquiterpene lactone is a good model system to gain insights into how plant secondary metabolisms have evolved and adapted in different niches. We are designing and performing more sophisticated experiments to understand sesquiterpene evolution in Asteraceae. Funded by NSERC Discovery Grant and Discovery Accelerator Supplement Grant (2007 - ongoing)

2. Molecular genetics and biochemistry of natural rubber in lettuce.
Lettuce (Lactuca sativa) can synthesize a high quality rubber in its specialized organ, laticifer. We are using lettuce as a model to advance our knowledge of natural rubber metabolism. We have used proteomics and genomics approach to identify several genes that have possible implications in rubber biosynthesis in lettuce. Subsequently, their functions have been examined by RNAi-silencing. Also, metabolic genes were expressed in microbial systems to reconstitute natural rubber biosynthesis in vitro. This integrative approach has shown that some proteins proposed to be important in natural rubber biosynthesis (e.g., rubber-elongation factor and small rubber particles proteins) have insignificant roles in natural rubber biosynthesis. Although cis-prenyltransferase (CPT) has shown cis-polyisoprenoid biosynthetic activities in some reports, we could not find the catalytic activity of CPT by expressing it alone in different heterologous systems, but surprisingly lettuce CPT interacts with its inactive version of homolog (CPT-like protein or human Nogo-B receptor homolog) to show cis-polyisoprenoid activities. This hetero-protein complex localizes on endoplasmic reticulum. Nonetheless, high molecular weight cis-polyisoprenoids could not be synthesized in in vitro. This is a non-traditional view of natural rubber biosynthesis, and time will tell us if we are on the right track! These data were published in Qu Y. and Chakrabarty R. et al (J. Biol. Chem, 2015) and Chakrabarty R. and Qu Y. et al (Phytochemistry, 2015). Funded by Alberta Ingenuity New Faculty Award

3. Proanthocyaninin biochemistry in pea
Pea (Pisum sativum) is a commercially important crop in Alberta, Canada, and we have conducted a project to investigate proanthocyanidin (tannin) metabolism in the seed coats of five pea cultivars. Analytical chemistry was used for qualitative and quantitative profiling of tannins in five different cultivars, and key biosynthetic genes were characterized (Ferraro and Jin et al, 2014, BMC Plant Biol.). Furthermore, comparative transcriptomics analysis by 454 sequencing enabled us to identify several novel genes which may be directly or indirectly involved in tannin metabolism in pea. We are currently testing the biological functions of these new genes in pea and their orthologues in Arabidopsis. Funded by Alberta Bio Solution, Collaboration with Jocelyn Ozga at the University of Alberta

4. Synthetic Biosystems for the Production of High Value Plant Metabolites
My group participated in the Genome Canada project to generate next-generation sequencing (NGS) transcript data from 75 medicinal plant species. The cDNAs encoding useful catalysts have been identified and plugged into the yeast implemented with synthetic metabolic pathways. This technology-driven approach has provided opportunities to exercise forefront research highly applicable for modern biotechnology. Using this plug-in system combined with classical analytical biochemistry and chemistry, we have identified a number of novel terpene synthases essential for the biosynthesis of many bioactive terpenes (Attia et al, 2012, Arch. Biochem. Biophysics; Pyle et al, 2012, FEBS J.; Pickel et al., 2012, Biochem. J.). Our gene-mining efforts are being continued to expand the catalogues of biologically beneficial catalysts from plants. Examples of new catalysts discovered from this project are listed below. Mild sedaitve sesquiterpene, Valerenic acid: Valerenadiene synthase from valerian plant (Valeriana officinalis); Natural sweetener, Hernandulcin: (+)-epi-alpha-bisabolol synthase from Lippia dulcis. Anti-protstate cancer drug, Thapsigargin: kunzeaol synthase from Thapsia garganica; Daily heath and cosmetic product, (-)-alpha-bisabolol: (-)-alpha-bisabolol synthase from chamomile; Anti-fungal compound, polygodial: (-)-drimenol synthase from valerian plant Funded by the Genome Canada and Genome Alberta