KSEF RDE: Glucan Kinases: a Key to Designer Starches and Plant Energy Storage

Starch is a key component in many aspects of daily life, including nutrition, biofuel production, and industrial processing: 50-80% of daily caloric intake comes from starch; >20% of corn starch produced in the U.S. is converted to ethanol; and starch is a cheap and renewable industrial feedstock for producing paper, textiles, plastics, and pharmaceuticals. Starch is comprised of intertwined glucose chains that form double helices and make starch both water-insoluble and inaccessible to most enzymes, including amylases that degrade starch. Starch-based feedstocks are generated by modifying starch using physical, chemical and enzymatic methods. Physical modifications include cyclic heating/cooling between 50°C and >100°C. The addition of acids and bases both catalyzes glucose chain hydrolysis and converts insoluble starch to a structure improving the accessibility of glucose chains to amylases. Alternatively, plants utilize an orchestrated reversible phosphorylation system: 1) starch kinases phosphorylate starch to solubilize the starch outer surface, 2) amylases then cleave glucose to release the energy cache, and 3) starch phosphatases dephosphorylate starch to reset the cycle. We discovered starch phosphatases nearly 8 years ago and pioneered the field by determining the x-ray structure of multiple starch phosphatases. While much progress has been made in protein engineering of starch phosphatases, similar work has not progressed for the starch kinases. This deficiency has been due to the difficulty of purifying active starch kinases. We recently overcame this hurdle by generating a potato starch kinase expression system. The objective of this proposal is to determine the molecular mechanisms of starch kinases with a three-prong approach focused on: 1) defining the enzymology of starch kinases. 2) determining the structure of starch kinases. 3) utilizing starch modifying enzymes to enhance starch degradation.