As the major energy cache in plants and algae, starch is a central component of human and animal food and a key constituent in many manufacturing processes. Additionally, starch is both a first-generation biofuel and is vital to future efforts focused on microalgal hydrogen and oil production. Therefore, elucidation of pathways controlling starch metabolism is needed in order to develop novel strategies that manipulate them and satisfy the growing starch demand. A key pathway regulating starch metabolism - and one that is required for starch degradation - is reversible phosphorylation of glucose residues in starch outer glucans, rendering the granule surface accessible to glucan hydrolyzing enzymes. This sequential cycle begins when two glucan dikinases solubilize starch outer glucan chains by phosphorylating glucose units. Starch phosphorylation allows amylases to bind the starch surface and release the stored energy in the form of glucose and maltose; however, amylases do not proceed past the phosphate groups. Two glucan phosphatases reset the cycle by removing the phosphate groups and allowing processive glucan hydrolysis. Plants lacking the glucan phosphatases exhibit excess amounts of starch, impaired growth, starch with increased phosphorylation and accumulation of starch breakdown intermediates. While progress has been made concerning the biology of reversible starch phosphorylation little is known about the molecular mechanisms regulating glucan phosphatase function. This project addresses critical information gaps of this essential pathway by employing a variety of biochemical and biophysical techniques. This research will define the function, dynamics, structures, and regulation of glucan phosphatases as well as generate and evaluate engineered glucan phosphatases. Completion of this work will define the role of glucan phosphatases in starch modification and degradation, providing the needed insights for biotechnological exploitation of these enzymes.
Broader Impacts: Increasing demand has led to competition for starch among food, biofuels, and industrial manufacturers. An additional concern is that starch processing utilizes hazardous chemicals to modify it for industrial application. Therefore, innovative strategies are needed to increase starch production and to modify starch biophysical properties using less hazardous methods. This project contains integrated and synergistic career goals that will impact the ability to increase starch production and generate designer starches. This project involves multidisciplinary training at several levels. 1) Graduate students will receive national (Kentucky and California) and international (Switzerland) training in laboratory skills, scientific ethics, protein purification, enzyme kinetics, crystallography, DXMS methods, and in data analysis and presentation. In addition, they will be mentored in career options. 2) Undergraduate students, including NSF REU students and those from the Appalachia region, will be trained in basic laboratory skills and in the techniques employed in the lab. They will also receive mentoring in career options. 3) The PI will enhance formal undergraduate courses by integrating research on starch metabolism into an upper level undergraduate course. 4) The importance of basic research on the societal and environmental issues of food production, biofuels, and global warming will be presented to high school and undergraduate students, as well as the general public, at state government events. In addition, results will be published in peer-reviewed journals and presented at local, regional, national, and international scientific interdisciplinary meetings.
