Alkamides (amides linked to fatty acids) are novel plant secondary compounds that are poorly characterized, but that accumulate to significant levels in Echinacea, making this plant of choice to study these compounds and their biosynthesis. Our understanding of alkamide function in plants is in its infancy, but recent data suggest these compounds may have novel signaling functions in addition to insecticidal properties. This work will lay the foundation for further studies of the importance of these compounds in plant biology as well as open up research on this plant genus which has been used medicinally for hundreds of years. Because the pathways in alkamide biosynthesis involve amino acid metabolism and novel chemical linkages, there are also multiple future biotechnological applications for the enzymes involved. The proposed research will strategically apply high-throughput global profiling technologies to elucidate this natural product biosynthetic pathway. Alkamides appear to be biologically assembled via a modular metabolic pathway that may be an adaptation of amino acid and fatty acid metabolism. Experiments that combine metabolite profiling, metabolic flux studies and transcriptomics will be used to identify genes and enzymes that assemble a diverse collage of alkamides. Defining the alkamide pathway offers the potential of discovering new metabolic processes that generate novel combinations of chemical functionalities, which have wide-ranging applications (e.g., lubrication and detergent industries). In addition, this research project outlines a general methodology that should be broadly applicable to discovering how primary and specialized plant metabolism are juxtaposed and evolve to generate the physiochemical phenotypic differences among plant taxonomic groups. The multilayered bio-prospecting to be used offers the opportunity to browse the metabolic repertoire of an organism, and the system-wide knowledge of the involved biochemical processes should translate to the creation of novel bio-derived compounds relevant to the chemical industries, as well as strategies for pest resistance.
Broader Impacts: This project will facilitate a multidisciplinary partnership between IUPUI, ISU researchers and scientists at the USDA North Central Regional Plant Introduction Station. The collaboration established to conduct this research will mentor young scientists at the increasingly uncommon intersection of organic chemistry, mechanistic biochemistry and functional genomics through the coeducation of undergraduates, graduate students and post-doctoral fellows. Students at IUPUI, a large, urban university, will be drawn from a trainee pool rich in underrepresented minority and first generation students in the McNair and Diversity Scholars STEM research programs. These interactions will allow the project to operate synergistically with IUPUI campus initiatives to mentor undergraduate students toward graduate education. As an outreach activity specific to IUPUI, high school teachers will experience hands-on research. In conjunction with the graduate students, they will engage in experiments leading to biochemical lessons suitable for use at their home schools. This effort dovetails with ongoing NSF GK-12 participation in IUPUI's research programs. At ISU, undergraduates will translate the proposed research into a module in a new course in biotechnological biochemistry.
Interplay between the genetic program of an organism and the regulatory mechanisms that control the biochemical expression of the genome, define the small molecule chemical constituents that constitute the metabolome. The metabolomes of organisms can be divided into the fraction that is common to a wide swath of taxa, which traditionally has been called the products of "primary metabolism", and a unique fraction of that is distinct in isolated taxa; this latter fraction is the product of "specialized metabolism". This project investigated the biosynthesis of the alkamides of Echinacea as a model for discovering new genes and enzymes involved in specialized metabolism. The project integrated new global experimental strategies, which comprehensively profiled the transcriptome of Echinacea organs, with metabolomics data. Novel bioinformatic strategies were implemented within the PMR database (http://metnetdb.org/PMR), which led to the identification of candidate genes that are involved in specialized metabolism. The functionality of these genes in specialized metabolism was validated by a combination of experiments including isotopic tracer experiments, heterologous expression of target genes and biochemical characterization of the catalytic properties of the gene products. This strategy tested and validated the model we had originally proposed that the alkamides of Echinacea are produced by the confluence of amino acid and fatty acid metabolism, integrating a combinatorial, modular assembly of amino acid-derived amines and oxidatively generated polyunsaturated/acetylenic fatty acids. Specific outcomes of the collaborative project with Dr. Robert Minto (Indiana University Purdue University Indianapolis; IUPUI) includes: (1) the discovery of a branched-chain decarboxylase and alkamide ligase, (2) the unexpected mitochondrial origin of fatty acid chains, which has implications in the control of alkamide chain lengths and the initial desaturation, and (3) that the determination that odd chain-length acetylenic alkamides are formed by omega-chain cleavage. This multi-disciplinary collaboration between IUPUI and Iowa State University addressed one of the critical challenges in studying non-model plant systems, and it has contributed a strategy for overcoming challenges associated with the lack of accurately annotated genomic data. The discoveries of new metabolic processes and the isolation of genetic elements that catalyze novel chemical conversions may enable the production of biorenewable products. A broader, long-term impact of the project was its contribution to the training of high school students and teachers, undergraduate students, graduate students and post-doctoral trainees.
