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.

Project Report

Interplay between biochemical pathways leads to many important natural products. The alkamides, a group of compounds from the heavily consumed medicinal plant Echinacea were believed to be produced by the confluence of amino acid and fatty acid metabolism, although this required many transformations for which there was scant precedence or many conceptually possible biosynthetic paths. We performed metabolomic, transcriptomic, and isotopic tracer experiments largely with Echinacea seedlings that were found to support our central hypothesis that alkamide diversity in plants results from a combinatorial, modular assembly of amino acid-derived amines and oxidatively generated polyunsaturated/acetylenic fatty acids. This project’s success is demonstrated through our establishment of an experimentally founded metabolic framework for alkamide biosynthesis. Specific outcomes of our research include: (1) the discovery of a branched-chain decarboxylase and determination that free alkylamines are potential intermediary metabolites, (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) the determination that odd chain-length acetylenic alkamides are formed by omega-chain cleavage. Together with the knowledge that mitochondria tend to generate medium chain acyl groups, our studies define new lines of inquiry into lipid channeling and the sequence of lipid modification steps. The identification and functional characterization of two new Echinacea acetylenases with implied roles in this pathway were investigated by quantitative PCR and transcriptome analysis that linked the activity of a single locus to alkamide biosynthesis. Additional enzymes involved in lipid modification were evaluated and the completion of the genome of Fistulina hepatica uncovered a trifunctional fungal acyl-phosphaditdylcholine diacetylenase activity that lays the groundwork for elucidating the remaining transformations that produce these important specialized metabolites. This multi-disciplinary collaboration between IUPUI and Iowa State University addressed one of the critical challenges in studying non-model plant biosynthetic pathways, the dearth of annotated genomic data, by using the correlations between metabolite and transcript levels as guided by biochemical probing of the underlying fundamental processes to identify gene candidates. The discovery of new metabolic processes and understanding the integration of this pathway may enable the production of biorenewable products. A broader, long-term impact of the project was its contribution to the training of 2 high school, 5 undergraduate, 4 graduate and 1 post-doctoral students in modern organic chemistry and biological techniques. Through the enthusiasm of a graduate student, who was also an NSF GK-12 teaching fellow, Indianapolis Public School middle and high school students carried out experiments looking at the plant biochemistry. Through this project, we additionally provided access to students from the American Chemical Society Project SEED, McNair Scholars, and NSF-funded URM programs to research opportunities.

National Science Foundation (NSF)
Division of Molecular and Cellular Biosciences (MCB)
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Kamal Shukla
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Indiana University
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