Difference between revisions of "Maeda Lab:Research"

Research Interests in Maeda Lab

Plants produce a diverse array of secondary (specialized) metabolites in order to survive in various ecological niches. In contrast to the well-documented diversification of plant secondary metabolism, primary metabolism, which provides precursors of secondary metabolites, is generally thought to be conserved across different plant species. However, it is not understood to what extent primary metabolic pathways of plants have evolved in different lineages to support the downstream synthesis of diverse secondary metabolites. Such basic knowledge of the interface between plant primary and secondary metabolism will be also crucial for future metabolic engineering and breeding to improve production of plant natural products. To address this question, we are investigating in different plant species the biosynthetic pathways of aromatic amino acids.

• Tyrosine (Tyr) is an aromatic amino acid required for protein biosynthesis in all living cells and, due to the absence of Tyr biosynthesis in animals, is an essential nutrient in human diets. In plants, Tyr also serves as a precursor of numerous natural products, which include tocopherols (vitamin E), cyanogenic glycosides, and isoquinoline alkaloids (e.g., analgesic morphine and codeine, Figure 1). These Tyr-derived plant metabolites have a remarkable structural complexity and a variety of pharmacological and biological activities, making them effective nutritional compounds and pharmaceutical drugs. However, often the low yields of these compounds in plant tissues hamper their commercial production in plants, and there is a growing need to rationally engineer the plant Tyr pathway.

• Although the Tyr biosynthetic pathway has been extensively studied in microbes (e.g., E. coli, yeast), we still do not know how and where Tyr are synthesized in plants. To investigate Tyr biosynthetic pathways, their localization and regulation in different plant species, we use two contrasting model plants, Arabidopsis thaliana and Medicago truncatula, which appear to use different pathways for Tyr synthesis (see our paper, Schenck et al., 2014). We particularly focus on prephenate dehydrogenase (TyrAp/PDH) and arogenate dehydrogenase (TyrAa/ADH) enzymes (Figure 1), which compete prephenate and arogenate substrates, respectively, against phenylalanine biosynthesis. Accurate understanding of the Tyr pathways may provide an opportunity to dramatically increase Tyr availability in plants by redirecting carbon flow from phenylalanine biosynthesis, which consumes major carbon flow for lignin biosynthesis in vascular plants.

Role of Tyrosine Pathway Regulation in the Formation of Tyr-Derived Natural Products

• Towards improving the production of Tyr-derived plant natural products, we also address the question how does the regulation of the Tyr pathway contribute to the formation of downstream Tyr-derived compounds. In this project, we use table beet (Beta vulgaris) as a novel model system, which produces high levels of Tyr-derived pigments, betalains (Figure 2). Using functional genomics approach, we aim to isolate and characterize of B. vulgaris ADH (or PDH) enzymes involved in Tyr biosynthesis. In collaboration with Dr. Irwin Goldman at UW Madison, we will investigate biochemical and genetic changes that has been introduced in the Tyr and betalain pathways during breeding of B. vulgaris germplasm. Finally, we will further engineer the pathway in order to improve the production of Tyr and betalains in table beets as a proof of concept, which can be applied to other plant species for enhanced production of Tyr-derived compounds with nutritional and medicinal values.

Regulation of the Shikimate Pathway in Plants

• The shikimate pathway provides chorismate, a common precursor of all three aromatic amino acids (phenylalanine, tyrosine, and tryptophan). In microbes, the first enzyme of the shipmate pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS), is tightly regulated by aromatic amino acids and controls carbon flux through the shikimate pathway. In plants, previous biochemical studies showed that the plant DAHPS enzymes are not sensitive to aromatic amino acids, suggesting that plants have a different mechanism regulating the shikimate pathway. However, the underlying regulatory mechanism of the plant shikimate pathway is poorly understood, due to limited knowledge of the plant enzymes involved in the early steps of the shipmate pathway. We apply integrated approaches of bioinformatics, enzymology, forward/reverse genetics, cell biology, and analytical chemistry, including stable isotope-assisted metabolic flux analyses, to investigate the enzyme organization and regulatory mechanisms of the plant shikimate pathway.

Evolution of Plant Phenylalanine Biosynthesis

• In plants, phenylalanine (Phe) is a precursor of abundant and diverse phenylpropanoid compounds, such as flavonoids, tannins, and lignin. Plants synthesize Phe predominantly via the arogenate pathway ( Maeda et al., 2010; 2011) in contrast to model microbes (e.g. E. coli) that use exclusively the phenylpyruvate pathway for Phe biosynthesis (see Figure 1). Together with Dr. John Jelesko at Virginia Tech, we conducted phylogenetically-informed biochemical characterization of prephenate aminotransferase (PPA-AT), which catalyzes the first committed step of the arogenate pathway, and uncovered a unique evolutionary history of the arogenate Phe pathway (Figure 3). Our results revealed that prephenate-specific aminotransferase containing Thr84 and Lys169 was transferred from a Chloribi/Bacteroidetes ancestor to a eukaryotic ancestor of the Plantae. This ancient lateral gene transfer likely led to efficient Phe production via the arogenate pathway, which currently supports the production of abundant phenylpropanoid natural products such as lignin. (Dornfeld et al., 2014)

If you are interested in joining our lab, please send email to maeda2@wisc.edu.

Our studies are supported by UW-Madison and NSF IOS.